Document jgLRBkm3eL0KXDBZdypQ26BD5

4R2Z6'009<\ PFOS: . A SEMI-STATIC LIFE-CYCLE TOXICITY TEST WITH THE CLADOCERAN (Daphnia magna) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A -109 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects OPPTS Number 850.1300 OECD Guideline 211 AUTHORS: Kurt R. Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: July 2,1999 STUDY COMPLETION DATE: February 28,2000 AMENDED REPORT DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 77 001091 AMENDED W il d l if e In t e r n a t io n a l ltd. 2- - PROJECT NO.: 454A-109 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-109 STUDY COMPLETION: February 28,2000 AMENDED REPORT: April 26,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792,17 August 1989; OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM(98) 17); and Japan MAFF, 59 NohSan, NotificationNo. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance is being recharacterized in accordance with GLP. The stability o f the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Senior Biologist SPONSOR APPROVAL: DATE 001092 AMENDED W il d l if e In t e r n a t io n a l Ltd. -3 - PROJECT NO.: 454A-109 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792,17 August 1989; OECD Principles o f 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 Test Initiation Observations Draft Report and Data Analytical Data and Draft Report Final Report Amended Report DATE CONDUCTED: July 9,1999 July 12,1999 July 27,1999 October 12 and 13,1999 October 12 and 13,1999 February 28,2000 April 25 and 26,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: July 9,1999 February 23,2000 July 12,1999 July 13,1999 July 27,1999 July 28,1999 October 13,1999 October 13,1999 October 14,1999 October 14,1999 February 28,2000 February 28,2000 April 26,2000 April 26,2000 James H. Coleman Quality Assurance Representative 00 DATE 001093 AMENDED W il d l if e In t e r n a t io n a l ltd. -4 - PROJECT NO.: 454A-109 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran {Daphnia magna) WILDLIFE INTERNATIONAL L Y . PROJECT NUMBER: 454A-109 STUDY DIRECTOR: Kurt R. Drottar Senior Biologist MANAGEMENT: Henry O. Krueger, Director, Aquatic Toxicology and Non-Target Plants DATE - . 001094 AMENDED W il d l if e In t e r n a t io n a l lto. -5 - PROJECT NO.: 454A-109 TABLE OF CONTENTS Title/Cover Page................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement........................................................................................ 2 Quality Assurance Statement...............................................................................................................................3 Report Approval................................................................................................................................................... 4 Table o f Contents.........................'......................................................................................................................5 Summary............................................................................................................................................................... 7 Introduction........................................................................................................................................................... 8 Objective............................................................................................................................................................... 8 Experimental D esign............................................................................................................................................8 Materials and M ethods................................................................................................................ 9 Results and Discussion............................................................................................ Conclusions......................................................................................................................................... References.............................................:.................................................................. ........................................ 15 14 TABLES Table 1 - Summary o f Analytical Chemistry D ata.................................................. 16 Table 2 - Temperature o f Water in the Test Cham bers................................................. ................................. 17 Table 3 - Dissolved Oxygen Content o f Water in the Test Chambers.............................................................19 Table 4 - pH o f Water in the Test Chambers.................................................................................................... 21 001095 W il d l if e In t e r n a t io n a l Ltd. 6- - PROJECT NO.: 454A-109 TABLE OF CONTENTS - Continued - TABLES (Continued) Table 5 - Specific Conductance, Hardness and Alkalinity in the Negative Control and Highest Treatment Group Test Chambers................................................................................ 23 Table 6 - Summary o f Cumulative Percent M ortality and Treatment-Related Effects for First-Generation Daphnids.......................................................................................................... 24 Table 7 - Summary of Daphnia magna Reproduction During the Life-Cycle Toxicity T est..................... 25 Table 8 - Summary of Length and Dry W eight o f Surviving Individually-Exposed First-Generation Daphnids...................................................... 26 Table 9 - Cumulative Percent Mortality/Immobility and Treatment-Related Effects for Second-Generation Daphnids.......................................................................................................... : 27 APPENDICES Appendix I - Specific Conductance, Hardness, Alkalinity and pH o f Well W ater Measured During the 4-Week Period Immediately Preceding the Test..........................28 Appendix II - Analyses o f Pesticides, Organics, M etals and Other Inorganics in Wildlife International Ltd. Well W ater.........................................................................29 Appendix IQ - The Analysis o f PFOS in Freshwater in Support of Wildlife International Ltd. Project No.: 454A-109...................... 30 Appendix IV - Cumulative Percent M ortality and Treatment-Related Effects First-Generation Daphnids................................................................................................ 49 Appendix V - Neonate Production............................................................................................................. 70 Appendix VI - Length and Dry W eight o f Surviving First-Generation D aphnids............................ 72 Appendix VII - Changes to the Protocol....................................................................................................... 74 Appendix VIH - Personnel Involved in the Study..........................................................................................75 Appendix IX - Report Amendment............................................................................................................. 76 AMENDED 001096 W il d l if e In t e r n a t io n a l ltd. -7 - PROJECT NO.: 454A-109 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: f LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: NOEC: LOEC: MATC: 4 54A -109 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran {Daphnia magna) Negative Control, 1.4,2.9,5.7,11,23 and 46 mg a.i./L Negative Control, 1.5,2.9 ,5 .6 ,1 2 ,2 4 and 48 mg a.i./L Experimental Start - July 12,1999 Biological Termination - August 2,-1999 Experimental Termination - August 4,1999 21 Days Neonate Cladocerans {Daphnia magna) Wildlife International Ltd. cultures Easton, Maryland 21601 Juveniles < 24 hours old 12 mg a.i./L 24 mg a.i./L 17 mg a.i./L AMENDED 001097 W il d l if e In t e r n a t io n a l ltd. - 8- PROJECT NO.: 454A-109 INTRODUCTION This study was conducted by W ildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f the test was conducted from July 12,1999 to August 2,1999. Raw data generated by Wildlife International Ltd. and a copy o f the final report are filed under ProjectNumber 454A-109 in archives located on the Wildlife International Ltd. site. OBJECTIVE The objective o f this study was to determine the effects o f Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) on the survival, growth and reproduction o f the cladoceran (Daphnia magna) during a 21-day exposure period under semi static test conditions. EXPERIMENTAL DESIGN Daphnids were exposed to a geometric series o f 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 o f an exploratory range finding toxicity test. Nominal test concentrations were 1.4,2.9, 5 .7 ,1 1 ,2 3 and 46 mg a.i./L. Mean measured concentrations were determined from new and corresponding old solutions during each week o fthe 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 o f reproduction, and clinical signs o f toxicity. With the onset o f reproduction, the number o f second-generation neonates were counted (number o f live neonates, number o f 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. A t 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 o f the mortality, growth and reproduction data. The maximum acceptable toxicant concentration (MATC)was calculated as the geometric mean o f the NOEC and LOEC. AMENDED 001098 W il d l if e In t e r n a t io n a l ltd. -9 - PROJECT NO.: 454A-109 MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: 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); StandardEvaluation Procedure, Daphnia magna Life-Cycle (21-DayRenewal) Chronic Toxicity Test (3); and ASTM Standard E 1193-87, Standard Guidefo r ConductingRenewalLife-Cycle Toxicity Tests with Daphnia magna (4). Test Substance The test substance was received from 3M Corporation on October 29,1998 and was assigned Wildlife International Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity o f 98.9%, and an expiration date o f2008. The test substance was reanalyzed by the Sponsor and the Certificate ofAnalysis dated March 9,2000 indicated a purity of 90.49%. The test substance was stored at ambient room temperature. Preparation o f Test Concentrations A primary stock solution was prepared by adding the test substance to UV sterilized dilution water at a concentration o f 46 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 the other five test concentrations. New solutions were prepared for each renewal day. After mixing, all test solutions appeared clear and colorless. All reported test concentrations were adjusted for the purity o f the active ingredient in the test substance (90.49%). Test Organism The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group o f aquatic invertebrates and were selected for use in the test based upon 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 W ildlife International Ltd., Easton, Maryland. 01099 AMENDED W il d l if e In t e r n a t io n a l ltd. -10- PROJECT NO.: 454A-109 Adult daphnids were cultured at approximately the same temperature as used during the test in Wildlife TntRmatinnai Ltd. well water. Daphnids in the cultures were held for at least 27 days prior to collection o f the juveniles for testing The adults showed no signs o f disease or stress during the holding period. During the 14-day holding period preceding the test, water temperatures ranged from 20.0 to 20.3C. The pH o f the water ranged from 8.3 to 8.5 and dissolved oxygen ranged from 8.2 to 8.8 mg/L. Instrumentation and methods used for water measurements are described in the Environmental Conditions section of this report. x/ Neonate daphnids (<24 hours old) were obtained for testing from six 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 o f yeast, Cerophyll, and trout chow (YCT, 1800 mg TSS/L), as well as a suspension of the freshwater green alga, Selenastrum capricomutum (3.5 x 107cells/mL). During the test, each test chamberwas fed 0.3 mL of YCT and 0.60 mL of Selenastrum once daily. Test Apparatus Test chambers were 250-mL plastic (Nalgene) beakers containing approximately200 mL oftest solution. The depth of test solution in a representative test chamber was approximately 5 cm. Test chambers were randomly positioned by treatment group in an environmental chamber set to maintain a temperature of 20 1C. The water bath was enclosed in a plexiglass hood to minimize the potential for cross-contamination. The test chambers were labelled with the projectnumber, 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 o f the well water during the four-week period immediately preceding the test are presented in Appendix I. The well water was passed through a sand filter to remove particles greater than approximately 25 ^m , 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 o o iio o W il d l if e In t e r n a t io n a l ltd. - 11- PROJECT NO.: 454A-109 0.45 urn and passed through a UV sterilizer to remove microorganisms and fine particles. The results o fperiodic analyses performed to measure the concentrations o f selected contaminants in well water used by Wildlife International Ltd. are presented in Appendix II. 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 o f 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 329 to 383 lux at the surface ofthe water. 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 Fulscope ER/C Recorder. The target test temperature during the test 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 the negative control and the highest test concentration with surviving daphnids at test initiation and at approximately weekly intervals thereafter. Measurements o fpH 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. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methodsfo r 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 o f reproduction, and clinical signs o f toxicity. The criteria for death included white opaque coloration, lack o f movement o f appendages, absence of heartbeat and lack o f response to gentle prodding. Immobilization was defined as lack o f movement except for minor 001101 W il d l if e In t e r n a t io n a l ltd. - 12- PROJECT NO.: 454A-109 spontaneous random movement o f 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 o f second-generation neonates were counted (number o f live neonates, number o f dead or immobile neonates and number o f aborted eggs) and recorded every Monday, Wednesday and Friday during the test. A t each observation period, the live first-generation daphnids were retained and the second-generation daphnids were discarded. On Day 14, the second-generation neonates were collected and used for the second-generation acute exposure. A t test termination, the length (measured from the apex o f the head to the base o f 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 o f each daphnid was determined using a caliper. The pans were then placed in a drying oven at approximately 60C for approximately 43 hours. The pans were removed from the drying oven and placed in a desiccator to cool. When cool, the pans were weighed using a M ettler AE-240 analytical balance. Statistical Analyses Statistical analyses were performed on survival o f first-generation daphnids, the number o f live young and die length and dry weight o f the surviving first-generation daphnids. Survival data were analyzed using Fisher's exact test. Reproduction and growth (length and dry weight) data were evaluated for normality and homogeneity o fvariance using the Shapiro-Wilk's test and Bartlett's test, respectively. Dunnett's test was then used to identify treatment groups that were statistically significant in comparison to the negative control (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 o f the old solutions were collected from a composite o f the remaining replicate test chambers. Samples were collected in plastic (Nalgene) bottles and were analyzed as soon as possible without storage. Analytical procedures used in the analysis o f the samples are provided in Appendix m. 001102 W il d l if e In t e r n a t io n a l ltd. -13- PROJECT NO.: 454A-109 RESULTS AND DISCUSSION Verification of Test Concentrations Results o f analyses to measure concentrations o f PFOS in water samples collected during the test are presented in Table 1 and the analytical chemistry report (Appendix IQ). Nominal concentrations used in the study were 1.4,2.9,5.7,11, 23 and 46 mg a.i./L. Measured values o fnew samples ranged from 94 to 121% o fnominal values, whereas measured values from the old solutions ranged from 90 to 108% o f nominal values. Consequently, PFOS was stable throughoutthe renewal periods. When measured concentrations o f samples collected during each week o f the test were averaged, the mean measured concentrations were 1 .5 ,2 .9 ,5 .6 ,1 2 ,2 4 and 48 mg a.i./L, which represented 107,100,98,109,104 and 104% of nominal, respectively. The results o f the studjy were based on mean measured concentrations. Physical and Chemical Measurements o f W ater Measurements o f temperature, dissolved oxygen and pH are presented in Tables 2, 3 and 4, respectively. Water temperature measurements were within the range o f 20 1C established for the test. Dissolved oxygen concentrations remained * 8.3 mg/L (91% o f saturation) throughout the test. Measurements o f pH ranged from 8.1 to 8.5. Measurements o f specific conductance, hardness and alkalinity in the negative control and the highesttreatment group are presented in Table 5. No apparent differences were observed for any o f these parameters throughout the exposure period. Survival and Biological Observations A summary o f the results o fthe observations o f mortality and sublethal signs o ftoxicity for first-generation daphnids are shown in Table 6. Daily observations are provided in Appendix V. After 21 days o f exposure, survival in the negative control group was 100%. Survival in the 1 .5 ,2 .9 ,5 .6 ,1 2 ,2 4 and 48 mg a.i./L treatment groups was 9 0 ,9 0 ,9 0 ,9 0 ,0 and 0%, respectively. A t test termination, all surviving daphnids exposed to PFOS appeared normal. Fisher's exact test showed that survival in the 24 and 48 mg a.i./L treatment groups was statistically different when compared to the negative control {p <; 0.05). C01103 AMENDED W il d l if e In t e r n a t io n a l ltd. - 14- PROJECT NO.: 454A-109 Reproduction A summary of the mean number o f 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 V. Daphnids in the control and PFOS treatment groups s 12 mg a.i./L started producing neonates on Day 9. There were no observations o f aborted eggs in any treatment group. Reproduction averaged 122 young per adult in the negative control. Reproduction in the 1.5,2.9,5.6 and 12 mg a.i./L treatment groups averaged 142,136,132 and 119 young per adult, respectively. The Bonferroni t-test showed that reproduction was not significantly reduced in any treatment group s 12 mg a.i./L (p > 0.05). The 24 and 48 mg a.i./L treatment groups were not included in the statistical analysis o f reproduction due to a statistically significant effect on survival. Growth A summary o f the mean lengths and mean diy weights of the surviving first-generation daphnids are presented in Table 8. Individual measurements are provided in Appendix VI. The mean length and dry weight o f first-generation daphnids in the negative control was 4.65 mm and 0.695 mg, respectively. The Bonferroni t-test showed that mean length and dry weight in the PFOS treatment groups s 12 mg a.i./L were not significantly reduced in comparison to the negative control {p > 0.05). Second-Generation Acute Exposure The results of the second-generation acute exposure are presented in Table 9. After 48-hours o fexposure, survival in the negative control was 95%. Survival in the 1.5,2.9,5.6,12 and 24 mg a.i./L treatment groups was 100,100,100,90 and 0%, respectively. Fisher's exact test showed that survival in the 24 mg a.i./L treatment group was significantly different from the negative control (p z 0.05). CONCLUSIONS There were no adverse effects on survival, reproduction or growth of Daphnia magna exposed to PFOS at concentrations s 12 mg a.i./L for 21 days. Daphnia magna exposed to 24 and 48 mg a.i./L had significantly reduced survival. Consequently, the LOEC for this study, based on survival was 24 mg a.i./L. The NOEC was 12 mg a.i./L and the MATC was calculated to be 17 mg a.i./L. The results o fthe second-generation acute exposure also indicated aNOEC of 12 mg a.i./L. 001104 AMENDED W il d l if e In t e r n a t io n a l ltd. -15- PROJECT NO.: 454A-109 REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), 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. Environm ental P rotection Agency. 1986. Standard Evaluation Procedure, Daphnia magnaLife-Cycle (21Day Renewal) Chronic Toxicity Test. Hazard Evaluation Division. Office of Pesticide Programs, EPA 540/9-86141. 4 Am erican Society for Testing and M aterials. 1991. Standard Guide fo r Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna, ASTM E 1193-87. Philadelphia, PA. 5 APHA, AWWA, W PCF. 1985. StandardMethodsfo r the Examination ofW ater and Wastewater. 16th Edition, American Public Health Association. American water Works Association. W ater Pollution Control Federation, New York. 6 W est, Inc. and D.D. Gulley. 1996. TOXSTAT, Version 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming. 0011.05 -16- PROJECTNO.: 454A-109 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna W ell W ater Nominal Test Concentration (mg a.i./L) Day 0 (New) Day 2 (O ld) Negative Control 1.4 2.9 5.7 11 23 46 <lo q ` <LOQ 1.78 1.72 3.20 3.05 5.97 5.87 11.5 11.5 24.2 23.1 47.3 48.0 <LOQ <LOQ 1.58 1.56 3.01 3.07 5.65 5.72 11.6 11.8 24.0 24.6 49.1 49.4 `The lim it o f quantitation (LOQ) was 0.458 mg a.i./L. 'Measurements discontinue due to 100% mortality. Table 1 Summaiy o f Analytical Chemistry Data Day 11 (New) <l o q <LOQ 1.38 1.47 2.75 2.77 5.63 5.59 11.3 11.3 22.8 22.5 _2 Day 14 (O ld) <LOQ <LOQ 1.36 1.32 2.85 2.71 5.36 5.39 11.2 11.6 23.6 23.1 _ Day 18 (New) <l o q <LOQ 1.38 1.43 2.79 2.81 5.58 5.75 11.8 11.6 24.8 25.0 Day 21 (O ld) <LOQ <LOQ 1.50 1.45 2.81 2.82 5.24 5.37 11.5 11.3 __2 -- / M ean M easured Concentration (mg a.i./L) <LOQ 1.5 2.9 5.6 12 24 48 Percent of Nominal 107. 100 98 109 104 104 SGTTOO AMENDED -17- PROJECT NO.: 454A-109 Table 2 Temperature o f Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control DayO New 19.4 Day 2 Old New 19.6 19.5 Day 4 Old New 19.5 19.6 Temperature (C) Day 7 Old New 19.8 19.8 1.5 19.4 19.6 19.5 19.5 19.6 19.8 19.8 2.9 19.4 19.6 19.5 19.5 19.6 19.8 19.8 5.6 19.4 19.6 19.5 19.5 19.6 19.8 19.8 12 19.4 19.5 19.5 19.5 19.6 19.8 19.8 24 19.4 19.5 19.5 19.5 19.7 48 19.4 19.5 19.5 --2 _ tem perature measured continuously during the test ranged from 19.0C to 20.0C. 2No measurement taken due to 100% mortality. 19.8 19.8 Day 9 Old New 20.0 20.1 20.0 20.1 20.0 20.1 20.1 20.1 20.1 20.1 20.1 20.1 __ Day 11 Old New 20.1 19.8 20.0 19.8 20.0 19.8 20.0 19.8 20.0 19.8 20.1 19.9 _ __ 001107 AMENDED - 18- PROJECT NO.: 454A-109 Table 2 (Continued) Temperature o f Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control Day 14 Old 19.9 New 19.8 Temperature (C) Day 16 Old New 19.9 19.8 Day 18 Old New 20.1 20.1 1.5 19.8 19.8 19.9 19.8 20.1 20.1 2.9 19.8 19.8 19.9 19.9 20.0 20.1 5.6 19.9 19.9 19.9 19.9 20.1 20.1 12 19.9 19.9 19.9 24 19.9 19.9 20.0 48 __ _ __ 'Temperature measured continuously during the test ranged from 19.0C to 20.0C. 2Measurements discontinued due to 100% mortality. 19.9 19.9 __ 20.1 20.1 __ 20.1 _2 _ Day 21 Old 20.1 20.0 20.0 20.0 20.0 -_ conos AMENDED -19- PROJECT NO.: 454A-109 Table3 Dissolved Oxygen Content o f Water in the Test Chambers' Sponsor. Test Substance: Test Organism: Dilution W ater 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Dissolved Oxygen Concentration (mg/L) _ _ _ _ _ _ ^_ _ _ =======1======_ _ _ _ _ _ DayO Day 1 Day 2 Day 3 Day 4 New Old Old New Old Old New Negative Control 8.3 8.3 8.6 8.9 8.7 8.6 8.6 1.5 8.3 8.3 8.6 8.9 8.8 8.6 8.8 2.9 8.3 8.3 8.6 8.9 8.8 8.6 8.8 5.6 8.3 8.3 8.6 8.9 8.8 8.6 8.8 12 8.3 8.3 8.6 9.0 8.8 8.6 8.8 24 8.4 8.3 8.6 8.9 8.8 8.6 48 8.4 8.4 8.6 8.9 J2 'A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20C in freshwater ^Measurements discontinued due to 100% mortality 8.8 Day 5 Old 8.7 8.8 8.8 8.8 8.8 8.8 Day 6 Old 8.8 8.8 8.9 8.9 8.8 8.9 Day 7 Old New 8.7 8.8 8.7 8.8 8.7 8.8 8.7 8.8 8.7 8.8 8.7 8.8 6GTT00 AMENDED -20- PROJECTNO.: 454A-109 Table 3 (Continued) Dissolved Oxygen Content o f Water in the Test Chambers1 Sponsor 3M Corporation Test Substance: PFOS Test Organism: Cladoceran, Daphnia magna M u t io n W a te r ^ Mean Measured Test Concentration Negative Control Dayj Old New 8.7 8.8 Day 11 Old New 8.4 8.7 Day 14 Old New 8.4 8.8 1.5 8.7 8.9 8.4 8.8 8.4 8.8 2.9 8.7 8.9 8.4 8.8 8.4 8.8 5.6 8.7 8.8 8.5 8.8 8.4 8.8 12 8.7 8.8 8.4 8.8 8.4 8.8 24 8.7 8.9 8.4 8.8 8.5 8.9 48 __ __ __ _ __ 1A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20C in freshwater. Day 16 Old New 8.8 8.8 8.8 8.8 8.8 8.8 8.8 8.8 8.8 8.8 8.9 8.9 .. .. Day 18 Old New 8.4 8.7 8.4 8.7 8.5 8.8 8.4 8.7 8.4 8.7 8.4 _2 ---- Day 21 Old 8.6 8.5 8.5 8.6 8.5 -- 0TTTO o AMENDED -21- PROJECT NO.: 454A-109 Table4 pH o f Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control DayO New 8.3 Day 2 Old New 8.3 8.1 1.5 8.4 8.4 8.2 2.9 8.4 8.4 8.2 5.6 8.4 8.4 8.2 12 8.4 8.4 8.3 24 8.4 8.4 8.3 48 8.5 8.4 'Measurements discontinued due to 100% mortality. 8.4 pH Day 4 Day 7 Old New Old New 8.4 8.2 8.4 8.4 8.4 8.2 8.5 8.3 8.4 8.2 8.5 8.3 8.5 8.2 8.5 8.4 8.5 8.3 8.5 8.4 8.5 8.4 8.5 8.5 _i __ __ __ Day 9 Old New 8.3 8.2 8.4 8.2 8.4 8.3 8.4 8.3 8.4 8.3 8.5 8.4 __ Day 11 Old New 8.3 8.1 8.3 8.2 8.2 8.2 8.3 8.2 8.3 8.2 8.3 8.3 --,m _ com i AMENDED -22- Table 4 (Continued) pH o f Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control .... Old 8.3 Day 14 New 8.2 1.5 8.3 8.2 2.9 8.3 8.2 5.6 8.3 8.3 12 8.4 8.3 24 8.4 48 . . 'Measurements discontinued due to 100% mortality. 8.3 _ pH Day 16 Old New 8.3 8.1 8.3 8.1 8.3 8.1 8.3 8.1 8.3 8.2 8.3 8.3 ---- PROJECT NO.: 454A-109 Day 18 Old New 8.3 8.2 8.3 8.2 8.3 8.2 8.3 8.2 8.3 8.3 8.4 __1 ---- Day 21 Old 8.3 8.3 8.3 8.3 8.3 __ com 2 AMENDED W il d l if e In t e r n a t io n a l ltd. -23- PROJECT NO.: 454A-109 Table 5 Specific Conductance, Hardness and Alkalinity in the Negative Control and Highest Treatment Group Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Parameter Specific Conductance (mhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) Day 0 310 136 178 Day 7 320 Time Day 14 310 128 132 176 172 Day 21 315 128 170 Specific Conductance (umhos/cm) 310 Hardness (mg/L as CaC03) 136 Alkalinity (mg/L as CaC03) 178 48 mg ai./L 1/ 24 mg a.i./L2/ 12 mg a.i./L3 Time 320 310 128 132 176 174 320 128 174 AMENDED 001113 -24- PROJECT NO.: 454A-109 Table 6 Summary o f Cumulative Percent Mortality and Treatment-Related Effects1for First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Cumulative Observations toiDay 7 %% Mortality Immobile Effects Negative Control 0 0 10 AN Cumulative Observations to Day 14 % Mortality % Immobile Effects 0 0 10 AN 1.5 0 0 10 AN 0 0 10 AN 2.9 0 0 10N 0 0 10 AN 5.6 0 0 10 AN 0 0 10 AN 12 0 0 10 AN 10 0 9 AN 24 70 0 3 AN 90 0 IA N 48 . 100 0 100 0 __ 1 Observed Effects: AN = Appears Normal. * Indicates a significant difference form the negative control on Day 21 using Fisher's exact test (p <, 0.05), Cumulative Observations to Day 21 % Mortality % Immobile Effects 0 0 10 AN 10 0 9 AN 10 0 9 AN 10 0 9 AN 10 0 9 AN 100* 0 -- 100* 0 f'TTTOO AMENDED -25- PROJECT NO.: 454A-109 Table 7 Summary o f Daphnia magna Reproduction During the Life-Cycle Toxicity Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Number o f Surviving Individually-Exposed DaphnidS Negative Control 10 Mean Live Young/ Surviving Adult Daphnid (SD) 122 (19.2) First,Day of Reproduction 9 Total Number of Dead/Immobile Neonates 0 1.5 9 142 (24.7) 9 0 2.9 9 136 (17.9) 9 0 5.6 9 132 (19.5) 9 0 12 9 119 (26.5) 9 1 241 0 -- 11 10 481 0 None 1This treatment was excluded from the statistical analyses o f reproduction due to a statistically significant survival effect. Total Number of Aborted Eggs 0 0 0 0 0 0 ,, STTTOO AMENDED W il d l if e In t e r n a t io n a l ltd. -26- PROJECT NO.: 454A-109 Table 8 Summary of Length and Dry W eight o f Surviving Individually-Exposed First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Number of v Individually-Exposed Surviving Daphnids Negative Control 10 1.5 9 2.9 9 5.6 9 12 9 24 0 48 0 Total Length (mm) Mean (SD) 4.65 (0.111) Dry W eight (mg) M ean (SD) 0.695 (0.100) 4.66 (0.118) 0.669 (0.0623) 4.62 (0.100) 0.724 (0.110) 4.61 (0.124) 0.727 (0.0665) 4.59 (0.102) 0.723 (0.0661) ---- - ---- 001116 AMENDED -27- PR0JECTN0.: 454A-109 Table 9 Cumulative Percent Mortality/Immobility and Treatment-Related Effects1for Second-Generation Daphnids Sponsor 3M Corporation Test Substance: PFOS Test Organism: Cladoceran,Daphnia m affia Dilution W ater Well Water Mean Measured Concentration (mg a.L/L) Daphnia/ Number Replicate Replicate Dead Negative Control A B 10 0 10 0 5 Hours Number Immobile Effects 0 10 AN 0 10 AN Number Dead 0 0 24 Hours Number Immobile 0 0 ______ Effects /Percent Immobile and Dead 10 AN 10 AN 0 1.5 A 10 0 0 10 AN 0 0 10 AN 0 B 10 0 0 10 AN 0 0 10 AN 2.9 A 10 0 0 10 AN 0 0 10 AN 0 B 10 0 0 10 AN 0 0 10 AN 5.6 A 10 0 0 10 AN 0 0 10 AN 0 B 10 0 0 10 AN 0 0 10 AN 12 A 10 0 0 10 AN 0 0 10 AN 0 B 10 0 0 10 AN 0 0 10 AN 24 A 4 0 0 B 40 0 1 Observed Effects: AN = Appears Normal, C = Lethargic 4 AN 4 AN 11 20 * Indicates a significant difference from the negative control at 48 hours using Fisher's exact test (p <,0.05). IAN,1C 1AN.1C 50 ______ 48 Hours Number Number Dead Immobile 00 10 00 00 00 00 00 00 10 10 40 40 Effects 10 AN 9 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 9 AN 9 AN _ - Percent Immobile and Dead 5 0 0 0 10 100* 001117 AMENDED W il d l if e In t e r n a t io n a l ltd. -28- PROJECT NO.: 454A-109 APPENDIXI Specific Conductance, Hardness, Alkalinity and pH o f Well W ater Measured During the 4-Week Period Immediately Preceding the Test Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Cladoceran, Daphnia magna Dilution Water: Well Water Specific Conductance Gumhos/cm) Mean 329 (N = 4) 3 1 5 -3 4 0 Hardness (mg/L as CaC03) 124 (N = 4) 120 - 128 Alkalinity (mg/L as CaC03) pH 169 (N = 4) 8.2 (N = 4) 164 - 172 8.0 - 8.3 c o in s W il d l if e In t e r n a t io n a l ltd. -29- PROJECT NO.: 454A-109 APPENDEXH Analyses o f Pesticides, Organics, Metals and Other Inorganics in W ildlife International Ltd. Well W ater1 ANALYSIS MEASURED CONCENTRATION Miscellaneous M easurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon2 Total Cyanide <<< 286 0.015.00 10.0 mg/L mg/L m gL M g/L inochlorines and PCBs Aldrin S' Alpha BHC BetaBHC Delta BHC Gamma BHC (Lindane) Chloidane DDD, pp' DDE, pp' DDT, pp' Dieldnn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-124S PCB-1254 PCB-1260 <<<<<<<<<<<<<<<<<<<<<<<<<< 0.005 0.005 0.005 0.005 0.006 0.025 0.006 0.005 0.008 0.005 0.005 0.005 0.018 0.010 0.005 0.005 0.007 0.005 0.500 0.260 0.260 0.260 0.720 01720 0.720 0.720 M g Mg/L Mg/L Mg/L M g M g/L M g/L Mg/L M g/L Mg/L M g/L M g/L Mg Mg M g/L M g/L M g/L M g/L Mg Mg/L Mg/L Mg Mg/L Mg/L Mg/L Mg/L Metals and O ther Inorganics Aluminum3 Arsenic3 Beryllium3 Cadmium3 Calcium3 Chromium3 Cobalt* Lead3 Magnesium3 Manganese3 MM eerrcury Molybdenum Nickel Potassium < 100 < 25.0 < 0.50 < 1.0 35.0 < 2.0 < 1.0 < 20.0 < 100 < 10.0 13.5 < 1.0 < 0.20 < 2.0 < 2.0 6.62 Mg/L Mg/L Mg/L Mg/L mg/L Mg/L Mg/L Mg/L Mg/L Mg/L mg/L Mg/L Mg/L Mg/L Mg/L mg/L Sele;nimumr 3 Silvier , < 25.0 < 1.0 Mg/L Mg/L Sodium 21.3 mg/L Zinc3 < 20.0 Mg/L T 2 Analyses performed by QST Environmental, Gainesville,Florida for samples collected on November 3 through November 7,1997. Analyses performed by Wildlife International Ltd. for the sample collected on November 5,1997. 3 Analyses performed by Wildlife International Ltd. for samples collected on November S through 7,1997. 001119 W il d l if e In t e r n a t io n a l ltd. -30- PROJECT NO.: 454A-109 APPENDIX m THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDUFE INTERNATIONAL LTD, PROJECT NO.: 454A-109 001120 W il d l if e In t e r n a t io n a l ltd. -31 - PROJECT NO.: 454A-109 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) W ILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-109 PRINCIPAL INVESTIGATOR: Jo t/k. MacGregor, B.S Scientist MANAGEMENT: W illard B. Nixon, P h D / Manager, Analytical Chemistry YA & /*e> DATE j 001121 AMENDED W il d l if e In t e r n a t io n a l ltd. -32- PROJECT NO.: 454A-109 Introduction Freshwater samples were collected from a semi-static life-cycle toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the cladoceran (Daphnia magna). This study was conducted by Wildlife International Ltd. and identified as Project No.: 454A-109. The analyses o f these water samples were performed at W ildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis between July 12,1999 and August 2, 1999 and were analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for this study was W ildlife International Ltd. identification number 467S. The test substance was used to prepare calibration and m atrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned. W ildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular m aterial, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic A dd, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the freshwater samples was developed at W ildlife International Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II o f W ildlife International Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. Concentrations o f the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- 001122 W il d l if e In t e r n a t io n a l ltd. -33- PROJECT NO.: 454A-109 Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Ci8 analytical column (50 mm x 2 mm I.D ., 3 pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit o f Quantitation Calibration standards o f PFOS prepared in a 50% methanol : 50% NANOpure w ater solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00915 to 0.0915 mg a.i./L, were analyzed w ith the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis o f individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit o f quantitation (LOQ) for these analyses was set a t 0.458 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50). Matrix Blank and Fortification Samples Six 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 o f a matrix blank is presented in Figure 5. Freshwater was fortified at 0.915, 9.15 and 54.9 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery o f 104%. A representative ion chromatogram o f a m atrix fortification is presented in Figure 6. AMENDED 001123 W il d l if e In t e r n a t io n a l ltd. ! -34- PROJECT NO.: 454A-109 Example Calculations Sample number 454A-109-3, nominal concentration o f 1.4 mg a.i./L in freshwater. Peak Area Ratio = Analyte Peak Area/Intemal Standard Peak Area Concentration Ratio = Concentration o f Analyte/Concentration o f Internal Standard Internal Standard Concentration: 0.100 mg/L Initial Volume: 0.500 mL Final Volume: 25.0 mL Dilution Factor: 50.0 PFOS Peak Area: 3091181 Internal Standard Peak Area: 3169689 Peak Area Ratio: 0.97523 Calibration curve equation. Slope: 2.42974 Intercept: 0.11482 Curve is weighted (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at Instrument = Slope x Internal Standard Concentration PFOS (mg a.iVL) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor Calculated concentration: 1.78 mg a.i./L Note: manual calculations may differ. PFOS (me a.i./L) in sample Percent o f Nominal Concentration = pF 0 S ( 4 a.i./L) nominal x 100 Calculated recovery: 121% Note: manual calculation may differ. AMENDED 001124 W il d l if e In t e r n a t io n a l ltd. -35- PROJECT NO.: 454A-109 RESULTS Sample Analysis Freshwater samples were collected from the semi-static life-cycle toxicity study with the cladoceran (Daphnia magna) at test initiation, July 12, 1999 (Day 0), on July 14,1999 (Day 2 old solutions), on July 23, 1999 (Day 11 - new solutions), on July 26,1999 (Day 14 - old solutions), on July 30, 1999 (Day 18 - new solutions) and at test termination on August 2,1999 (Day 21 old solutions). The measured concentrations o f PFOS in the samples collected at initiation o f exposure o f the test organisms (Hour 0) ranged from 96.5 to 121% o f the nominal concentrations (Table 4). Samples collected at Day 2, Day 11, Day 14, and Day 18 had measured concentration ranges o f 97.8 to 108%, 94.3 to 101%, 89.8 to 103% and 94.5 to 109% o f nominal values, respectively. Samples collected at test termination (Day 21) had a measured concentration range o f 91.0 to 102% o f nominal values. A representative ion chromatogram o f a test sample is shown in Figure 7. AMENDED 001125 W il d l if e In t e r n a t io n a l ltd. -36- PROJECT NO.: 454A-109 Table 1 Typical HPLC/MS Operational Parameters INSTRUMENT: 'Sr/ ANALYTICAL COLUMN: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perldn-Elmer API 100LC Mass Spectrometer equipped w ith a Perldn-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). Keystone Betasil C column (50 mm x 2 mm I.D ., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOWRATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure W ater containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 4.3 minutes INTERNAL STANDARD RETENTION TIME: Approximately 3.0 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 001126 W il d l if e In t e r n a t io n a l ltd. -37- PROJECT NO.: 454A-109 Table 2 M atrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-109-) MAB-1 Sample Type M atrix Blank M easured Concentration o f PFOS1 (mg a.i./L) < LOQ MAB-2 M atrix Blank < LOQ MAB-3 M atrix Blank < LOQ . MAB-4 M atrix Blank < LOQ MAB-5 M atrix Blank < LOQ A S O MAB-6 M atrix Blank 1 The limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the m atrix blank samples (50). AMENDED 0 0 1 3 -2 7 W il d l if e In t e r n a t io n a l ltd. -38- PROJECT NO.: 454A-109 Table 3 M atrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-109-) MAS-1 MAS-4 MAS-7 M A S-10 MAS-13 M A S-16 MAS-2 MAS-5 MAS-8 MAS-11 MAS-14 M A S-17 Concentrations o f PFOS (mg a.i./L) Fortified Measured 0.915 V . ' 0.915 0.915 0.915 0.915 0.915 0.988 1.02 0.9521 0.942 0.997 0.979 9.15 9.33 9.15 9.33 9.15 9.33 9.15 9.15 9.15 9.15 9.15 9.61 Percent Recovered 108 111 104 103 109 107 102 102 102 100 . 100 105 MAS-3 MAS-6 MAS-9 M A S-12 MAS-15 MAS-18 54.9 54.9 54.9 54.9 54.9 54.9 59.9 56.7 55.3 55.9 57.3 55.7 109 103 101 102 104 102 Mean = 104 Standard Deviation = 3.32 CV =3.19% N = 18 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and m anual calculations. Values have been rounded for reporting purposes. 1 Reported value represents mean o f duplicate re-analyses (0.970 and 0.933 mg a.i./L), 001128 AMENDED W il d l if e In t e r n a t io n a l ltd. -39- PROJECT NO.: 454A-109 Table 4 Measured Concentrations o f PFOS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test BBIH3HBBB3nBnSES3BBBBI^^B^B^9BSBB89SB9MKB^^^^^^BSBBIBMBDSBBXSHBBS8BnB^S89S^^^^BIB8SXEB8^^^BBBBBB39BBnBSBBa!IBSSBBaSSSBSBBSSBB9nBSSS3SSSSS8SS^nSSBSSSBSBSS9BSSB^BS8 Nominal Test Sample Sampling PFOS Measured Percent Concentration Number Time Concentration1 of (454A-109-) ^ fo g a .i./L ) Nominal 0.0 . 1 0 <LOQ -- 20 <LOQ -- 15 2 <LOQ -- 16 2 < LOQ -- 29 11 < LOQ -- 30 11 < LOQ -- 41 14 <LOQ -- 42 14 <LOQ -- 53 18 <LOQ -- 54 18 < LOQ -- 65 21 < LOQ -- 66 21 < LOQ -- 1.4 3 0 1.78 121 40 1.72 117 17 2 18 2 1.58 108 1.56 107 31 ' 11 1.38 94.3 32 11 1.47 101 43 14 1.36 93.1 44 14 55 18 56 18 1.32 89.8 1.38 94.2 1.43 97.4 67 21 1.50 102 68 21 1.45 98.5 Note: Results and corrections for new test substance purity were generated using M acQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 The limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00915 mg a.iVL) and the dilution factor o f the m atrix blank samples (50)._______ 001129 AMENDED W il d l if e In t e r n a t io n a l ltd. -40- PROJECT NO.: 454A-109 Table 4 (continued) Measured Concentrations o f PFOS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Concentration ==(m y7L)na= 2.9 Sample Number (454A-109-) 5 6 19 20 33 34 45 46 57 58 69 70 Sampling Time )a\ 0 0 2 2 11 11 14 14 18 18 21 21 PFOS Measured Concentration1 : a.i./ 3.20 3.05 3.01 3.07 2.75 2.77 2.85 2.71 2.79 2.81 2.81 2.82 Percent of 113 107 106 108 96.9 97.6 101 95.5 98.3 99.1 99.0 99.2 5.7 7 0 80 21 2 22 2 35 ` 11 36 11 47 14 48 14 59 18 5.97 104 5.87 102 5.65 98.1 5.72 99.1 5.63 97.6 5.59 96.9 5.36 93.0 5.39 93.5 5.58 96.8 60 18 5.75 99.6 71 21 72 21 5.24 91.0 5.37 93.2 Note: Results and corrections for new test substance purity were generated using M acQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 The limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50). 001130 AMENDED W il d l if e In t e r n a t io n a l ltd. -41- PROJECT NO.: 454A-109 Table 4 (continued) Measured Concentrations o f PFOS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Concentration ; a .i./ 11 Sample Sampling Number Time (454A -109-)_________ (D ay) 90 TO 0 23 2 24 2 37 11 38 11 49 14 50 14 61 18 62 18 73 21 74 21 PFOS Measured Concentration1 11.5 11.5 11.6 11.8 11.3 11.3 11.2 11.6 11.8 11.6 11.5 11.3 Percent of 96.5 97.0 97.8 99.3 95.0 94.5 94.0 97.5 99.4 97.4 97.1 94.5 11 0 12 0 25 2 26 2 39 ' 11 40 11 51 14 52 14 63 18 64 18 24.2 106 23.1 101 24.0 105 24.6 108 22.8 99.6 22.5 98.3 23.6 103 23.1 101 24.8 108 25.0 109 46 13 0 14 0 47.3 103 48.0 105 27 2 49.1 107 28 2 49.4 108 Note: Results and corrections for new test substance purity were generated using M acQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 The limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50). 001131. AMENDED W il d l if e In t e r n a t io n a l ltd. -42- PROJECT NO.: 454A-109 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Prepare m atrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into filtered freshwater using gas-tight syringes and Class A volumetric flasks. Dilute matrix fortification and test samples into the range o f the calibration standards by partially filling Class A volumetric flasks w ith 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the Appropriate volume o f sample and bring the flask to volume with the dilution solvent. Process the m atrix blank san ale using foe same dilution and aliquot volume as for the lowest fortification level. M ix well by several repeat inversions. i Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for foe analysis o f PFOS in freshwater. 001132 W il d l if e In t e r n a t io n a l ltd. -43- PROJECT NO.: 454A-109 Figure 2. A typical calibration curve for PFOS. Slope = 2.42974; Intercept = 0.11482; r = 0.9974. Curve is weighted (1/x). 001133 AMENDED W il d l if e In t e r n a t io n a l ltd -44- P lU ll Uan. M 1*. 1M9 19:27 T*iHinrt~n F ir C tt PPOS 12JULM Pa*t API 190LC HDMasaCtaoin 1.c4C4Al09_l9JULM: OommMr 4MA-1M a IM0M Uto CydB Oqr 0 1SJULM PFO3_101 STD 04100 497SA004C-1 Mon. M 12, 1990 1 2 4 ) PROJECT NO.: 454A-109 VMriW IJ fcIfan, Ad 1t. 1M9 1947 a w n r C i PPOS tXJUUt Pate API 1MU5 HMMCrnIjMOUMOOJUM: ate 4S4A-10 a magm Ufe Oyda - Day 12JULM rFwOnS*,,oi*ol4C-< to*100 4 J 0 In 1 p alM pros >-- nmmoiOL n r r o Uh Am M M U i hmhomi n m 1: 4 * 0 0 1 * . W ocono 499.9 NotMltaM. 10.0 Quart D u m . 19ft. VMh IM .V M S B M .U b u r m iM m oo* BVMMNT .9 S 9 40 9 1 4.17 Ai m 1 0 4 1 9 9 2 HrtoM 9 7 4 9 im iim end Urn MngnSM tfMh IWwrtonTHM MtgraSon iyp# til 4M asr 441 ABB Hon. JO I I , 1000 1 S P w l.a MoraMy: CMOS cp . Figure 3. A representative ion chromatogram o f a low-level (0.00915 mg a.i./L) PFOS standard. AMENDED 001134 W il d l if e In t e r n a t io n a l ltd -45- PROJECT NO.: 454A-109 Mm Oum , ventea1.S M ate* Mm , M 11. 1 IN 1fc*7 C lIlllllf lt C M P W H IJ U W M teA * 10 0 lC MD*4wCNem 1A4S4A1W .tAM; Oemmentr 4MA-1M 0 . m ens Uto tycto *0 t* 0 13JUIM P F O S .1 0 S STD .100 4675A404C-5 Men. Ju l 12, 1900 M M 1: 4 J0 Q l M . 207 scene 420.7 Notes Tim e. 2 .0 Q uaniTtm e. 0.2 M n. WWH 3 M IL Wktth Bees. M dth 40 R TVfln. (secs) 20 BmoeSt 1 Expected RT LOS Aim 3 2 1 4 1 Height 2S321I Start Time End Time MegreSoa W Mh Retenten Timo Integretoo Type 2 J1 3 .4 0X7 2X6 A -B 8 Mm Oum . ventea t A m m Mo, m it , m m ie srr o f t e M M O I I U I N M M AH MOCO 1 Uto Orte Dtp 1UULM M enetty; 25321 cpe mmi n Figure 4. A representative ion chromatogram o f a high-level (0.0915 mg a.i./L) PFOS standard. AMENDED 001135 W il d l if e In t e r n a t io n a l ltd -46- PROJECT NO.: 454A-109 i 1 i i l M . M ll 1*0* 127 H H k C tt P 202 1 S JU M N k API 100LC HOrll-- Ctwcw 1.0c4&4Al00_l2JULt*: a: A -i o . M Q M l n O m b o*y o iij u l m P9* IS M 2F02LW 7 4 5 4 A -1 0 9 - M AS-1 4 J2 M 1 period 4 ffQ 8 Mon. Ju l 12, 1026 1121 \ 1: 4 J2 Q l U , 227 aoma 422.7 Nota* Tim s. 2 .0 Q uantum *. 0 .2 M n. WMOi MuH. W M h Baa*.VMdlh RTVMn. fooca) flm ooti 9 2 40 20 1 Ev m M R T 2 JS Aim 9114702 Height 242240 Start Timo 2 J1 En* Tim* M ograaon W Mh flalanlon Tim# Intagraion Typo 9.42 0 J7 901 * >BB lO O i 00 90 70 20 SO 40 10 20 10 o- W w 246440 eps 23 > 122 148 21 21 21 121 161 L U 1 J9 1 J ) t M W 227 4 78 121 211 241 1 UJ a U 4 M A W .ttJU L N : MUH P FO S .W 7 4 S 4 A -1 0 2 - M AS-1 Mon, Ju l 12, 1229 I t t i 4HPF06 Figure 5. A representative ion chromatogram o f a matrix blank sample (454A-109-M AB-1). The arrow indicates the retention time o f PFOS. 001136 W il d l if e In t e r n a t io n a l ltd -47- PROJECT NO.: 454A-109 MaeQuan. v antarti. Malato Mon. Jto IX. tm 1ST a fOK Cal PR3SISJULM MM: A * fL C tOMm a Uto Ojwto Oaf U A U * PPO_tO 4S4A -100- MAS-1 Men. JU IS. ISM 1S7 htoanoSy: SS49S1 epe MaeOaan, vantoa 1J Malato Man, A i i t. iee iw ? -----m im P*k CM*MO IXJUUt M feAM teetC M M taaO m n lJE4S4A1ML1 J U : a U a Oycte * (toy e UJULN m ijM 4S4A-100* M AS-1 4 JS to parted p ro s IIP K U U e e A ie e Abeblula M e n to n U n e M C M Q l IM , SV7 eoane est. N olle U m . Qum* Three. M W M ut. V M S 10.0 t.0 s s B ate. VM M KT\M n.4aeea) Swaopi Fip iceed H T 40 to 1 4.17 A iee 1SS01S0 Height 11SP44 Start TMm COI GndU m 4 JS mtagrePon M d li 047 M e n to n Thee M egraton Type 444 A -B 6 Men. M 12. IS M 12*7 1001 SO 10 70 so so 40 SO 20 10 11 *? 11 0 J2 Papaie alee M enaty: 11M44 epe Figure 6. A representative ion chromatogram o f a matrix fortification sample (454A-109-MAS-1). 001137 W il d l if e In t e r n a t io n a l ltd -48- PROJECT NO.: 454A-109 cAPMMLC H O d iw O w i 1A 44A1.1*JUUf; Day 0 UJULM rP O t.1 1 4 3 Uon, <M 12. 1090 14:08 4S4A .1M . 4 M In t parted s *im**m**MmmI8MM/ 1: 4 J3 Q 1 M .8 07 acans 43*.7 N e lM lM M . 8.0 Q u M tlte e e . 0J8 M feW M i 3 IM L V M ti 4 B ttS .V M B t 40 nrw * n.(M G < t o fcnoeOi 1 BVKM Rr 1M Area 31004O9 HelgM 874304 Start Time M TU engranen m n neunten Tim e toUgrafton Type US 3LS0 047 SM A -B B 100 00 00 70 40 SO 40 30 80 10 o- iH K O r iM S t t U M Ante API 10QLC K M te ft 4MA>MI & a n p e U OpeM- Ctar 1AMM W O*_114 484A-100- 3 Mon. Jul I t, 1090 144* Intensity: 878394 cpa 181 PepiMelee Figure 7. A representative ion chromatogram o f a test sample (454A-109-3). C0113S - 49!- PROJECT NO.: 454A-109 APPENDIXIV - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphmds Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation FFOS Cladoceran, Daphnia magna Well Water_____________________________ _ Mean Measured Test Concentration (mg a.iiL) Replicate Initial _________ Number Number Exposed2 immobile Negative Control A B C D E F G H I J 10 10 10 10 10 10 10 10 10 10 Day 1 Cumulative Dead_____ Effects 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN A 10 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J V/ 0 A 10 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C = Lethargic. 2 All organisms on Day 0 appeared n o r m a l . __________ 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 2 Cumulative Dead______ Effects 0 IAN ' 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 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 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 IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Cumulative Dead ^ E ffe c ts 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 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 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 IAN AMENDED -50- PROJECT NO.: 454A-109 APPENDIXIV (Continued) Cumulative Percent M ortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor 3M Corporation Test Substance: PFOS Test Organism: Cladoceran, Daphnia magna Dilution Water______ Well Water_______________ Mean Measured Test Concentration (mg a.i) Replicate Initial Number Exposed2 5.6 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0 0 D ayl Cumulative 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 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 2 Cumulative 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 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 IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N 12 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 1 >' 0 J 10 24 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C = Lethargie. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 '0 0 0 0 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 IA N IA N 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 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 1C IA N 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 1 __ 0 1 IA- N 0 IAN 1- 1- 0 IAN 0 IAN 1- 1 AMENDED -51- PROJECT NO.: 454A-109 APPENDIXIV (Continued) Cumulative Percent Mortality and Treatntnt-Related Effects1- First-Generation Dapbnids Sponsor. Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed7 Number Immobile 48 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN " Appeared Normal; C = Lethargic. 7 All organisms on Day 0 appeared normal. Cumulative 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 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 2 Cumulative Dead 1 1 1 I 1 1 1 1 1 1 Effects -- - -- - -- -- -- -- -- -- Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 3 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects - - - - - - - -- - 001141 AMENDED - 52 - PROJECT NO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: 3M Corporation PFOS Cladoceran, Daphnia magna Mean Measured Test Concentration (m ga.i ) Negative Control Replicate A B C D E F G H I J Initial Number Exposed2 1 1 1 1 1 1 1 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day4 Cumulative 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 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day5 Cumulative 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 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 6 Cumulative 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 IA N IA N 1.5 A 1 0 0 IAN 0 0 IAN 0 0 IAN B 1 0 0 IAN 0 0 IAN 0 0 IAN C 10 0 IAN 0 0 IAN 0 0 IAN D 1 0 0 IAN 0 0 IAN 0 0 IAN E 1 0 0 IAN 0 0 IAN 0 0 IAN F 10 0 IAN 0 0 IAN 0 0 IAN G 10 0 IAN 0 0 IA N 0 0 IAN H 1 0 0 IAN 0 0 IAN 0 0 IAN I 1/ 0 0 IAN 0 0 IAN 0 0 IAN J 1 0 0 IAN 0 0 IAN 0 0 IAN 2.9 A 1 0 0 IA N 0 0 IA N B 1 0 0 IAN 0 0 IAN C 1 0 0 IAN 0 0 IAN D 1 0 0 IAN 0 0 IAN E 1 0 0 IAN 0 0 IAN F 1 0 0 IAN 0 0 IAN G 1 0 0 IAN 0 0 IAN O H 1 0 0 IAN 0 0 IAN O I 1 0 0 IAN 0 0 IAN P J 1 0 0 IAN 0 0 IAN P 1 Observed Effects: AN = Appeared Normal; C = Lethargic. 2 All organisms on Day 0 appeared normal. N 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 AMENDED -53- PROJECT NO.: 454A-109 APPENDIXIV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water. Mean Measured Test Concentration (mg a.i7L) 5.6 3M Corporation PFOS 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 Day4 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1 AN 1 AN 1 AN 1 AN 1 AN 1AN 1AN 1 AN 1 AN 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 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN Number Immobile 0 0 0 0 0 0 0 0 0 0 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 12 A 10 0 1AN 0 0 1AN 0 0 1 AN B 10 0 1 AN 0 0 1 AN 0 0 1AN C 10 0 1 AN 0 0 1 AN 0 0 1 AN D 10 0 1 AN 0 0 1AN 0 0 1AN E 10 0 1 AN 0 0 1 AN 0 0 1 AN F 10 0 1 AN 0 0 1 AN 0 0 1 AN G 10 0 1AN 0 0 1 AN 0 0 1AN H 10 0 1 AN 0 0 1 AN 0 0 1AN I J 1y 0 ' 0 0 1 AN 0 1AN 0 0 0 1 AN 0 1AN 0 0 1 AN 0 0 1AN 2412 A 1 0 B 0 C 0 D 0 E 0 F 0 G 0 H 0 n I 0 O J 0 P 1 Observed Effects: AN = Appeared Normal; C = Lethargic. P 2 All organisms on Day 0 appeared n o rm al._____________ K* co 1 1-- 1-- 0 1AN 1-- 1-- 0 1AN 0 1 AN 1-- 1- 0 0 0 0 0 0 0 0 0 0 1 110 1AN 110 1AN 0 1 AN 1- 1- 0 1-- 0 1-- 0 10 0 1AN 0 10 10 0 1AN 0 0 1 AN 0 10 1-- AMENDED -54- PROJECT NO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water1 Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 Number Immobile 48 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C = Lethargic. Day 4 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- - -- -- -- -- -- -- Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 5 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- - - - -- -- - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 6 Cumulative Dead 1 1 1 1 1 1 I 1 1 1 Effects - - oo p h* AMENDED - 55 - PROJECT NO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: 3M Corporation PFOS Cladoceran, Daphnia magna Mean Measured Test Concentration (mg a.i./L) Negative Control Replicate A B C D E F G H I J Initial Number Exposed2 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 IAN IAN IAN IA N IAN IAN IAN IA N IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 8 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IAN IAN IAN IAN IAN IAN IA N IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 9 Cumulative Dead ^ I if le c ts 0 IAN 0 1AN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 1.5 A1 0 0 IAN 0 0 IAN 0 0 IAN B1 0 0 IAN 0 0 IAN 0 0 IAN C 1 0 0 IAN 0 0 IAN 0 0 IAN D1 0 0 IAN 0 0 IAN 0 0 IAN E1 0 0 IAN 0 0 IAN 0 0 IAN F 1 0 0 IAN 0 0 IAN 0 0 IAN G1 0 0 IAN 0 0 IAN 0 0 IAN H1 0 0 IAN 0 0 IAN 0 0 IAN I 1> 0 J10 0 IAN 0 IAN 0 0 IAN 0 0 IAN 0 0 0 IAN 0 IAN 2.9 A1 0 0 B1 0 0 C1 0 0 D1 0 0 E1 0 0 F1 0 0 G1 0 0 H1 0 0 O I10 0 O J10 0 I-* 1 Observed Effects: AN = AppearedNormal; C - Lethargic. 2 All organisms on Day 0 appeared normal._________________________ W IAN IAN IAN IAN IAN IAN IAN IA N IAN 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 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 IAN _________________________________________ AMENDED -56- PROJECTNO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor. Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoccran, Daphnia magna Well Water______________ Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 5.6 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 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 IA N IA N IAN IA N IAN IA N IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 8 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 9 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N IAN IA N IAN IAN IAN IA N IA N IA N 12 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 1 >' 0 J 10 24 A 1 0 B 10 C 10 D 10 E 10 c F 10 p G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C " Lethargic. & 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 1_ 1-- 1-- 0 IAN 1-- 1-- 0 IAN 0 IAN 1-- 1- 00 00 00 00 00 00 0. 0 00 00 00 01 01 01 00 01 01 00 00 01 01 IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN 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 IAN 0 1-- 0 10 10 0 IAN 0 10 1-- 0 0 IAN 10 0 10 1- AMENDED -57- PROJECT NO.: 454A-109 APPENDIXIV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed12 Number Immobile 48 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN " Appeared Normal; C **Lethargic. 2 All organisms on Day 0 appeared normal.______________ Day 7 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- -- -- -- - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 8 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- - - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 9 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects - o a p p p AMENDED -58- PR 0JEC TN 0.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatmnt-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna Well Water______________ Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 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 10 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IA N IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 12 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN 1.5 A 1 0 0 IAN 0 0 IAN 0 0 IAN B 1 0 0 IAN 0 0 IAN 0 0 IAN C 1 0 0 IAN 0 0 IAN 0 0 IAN D 1 0 0 IAN 0 0 IAN 0 0 IAN E 1 0 0 IAN 0 0 IAN 0 0 IAN F 1 0 0 IAN 0 0 IAN 0 0 IAN G 1 0 0 IAN 0 0 IAN 0 0 IAN H 1 0 0 IAN 0 0 IAN 0 0 IAN I 1> 0 0 IAN 0 0 IAN 0 0 IAN J 1' 0 0 IAN 0 0 IAN 0 0 IAN 001148 2.9 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C = Lethargie. 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 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 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 IAN AMENDED -59- PR 0JEC TN 0.: 454A-109 APPENDIX IV (Continued) . Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Waten 3M Corporation PFOS Ciadoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 5.6 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 Effects IAN IA N IAN IA N IAN IA N IA N IAN IA N IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IA N IAN IA N IAN IAN IA N IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 12 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN 1 AN IA N IA N IAN IAN IA N IAN IA N IA N 12 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 1> 0 J 1' 0 24 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN " Appeared Normal; C = Lethargic. 2 All organisms on Day 0 appeared normal. 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 1_ 1-- 1-- 0 IAN 1-- 1-- 0 IAN 1-- 1_ 1- 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 01 01 01 -- 0 0 IAN 01 010 0 IAN 01 01 01 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1C 0 0 IAN 0 0 IAN 0 1-- 0 1-- 0 10 0 IAN 0 10 101 0 10 10 1-- AMENDED 001149 - 60 - PROJECT NO.: 454A-109 APPENDIX IV (Continued) -Cumulative Percent Mortality and Treatmeht-Related Effects1- First-Generation Daphnids Sponsor. Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.iVL) Replicate Initial Number Exposed2 Number Immobile 48 A1 0 B1 0 C1 0 D1 0 E1 0 F1 0 G1 0 H1 0 I10 J1 0 1 Observed Effects: AN " Appeared Normal; C = Lethargie. 2 All organisms on Day 0 appeared normal.______________ Day 10 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- -- -- - - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- - - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 12 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- - - oo p p otn AMENDED - 61- PROJECT NO.: 454A-109 APPENDIXIV (Continued) . Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation FFOS Cladoceran, Dapknia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 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 IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 14 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 0, 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IA N IAN IAN IA N 1.5 A 10 0 IAN 0 0 IAN 0 0 IAN B 1 0 0 IAN 0 0 IAN 0 0 IAN C 1 0 0 IAN 0 0 IAN 0 0 IAN D 10 0 IAN 0 0 IAN 0 0 IAN E 1 0 0 IAN 0 0 IAN 0 0 IAN F 1 0 0 IAN 0 0 IAN 0 0 IAN G 10 0 IAN 0. 0 IAN 0 0 IAN H 10 0 IAN 0 0 IAN 0 0 IAN I J 1> 0 1' 0 0 IAN 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 2.9 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Nbrmair C = Lethargic. 2 All organisms on Day 0 appeared normal._ _________ 0 0 0 0 0 0 *0 0 0 0 IAN IAN IAN IA N IAN IAN IAN IAN IAN 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 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 IAN AMENDED -62- PROJECT NO.: 454A-109 APPENDIXIV (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor 3M Corporation Test Substance: PFOS Test Organism: Ciadoceran, Daphnia magna Mean Measured Test Concentration (mga.i./L) 5.6 Replicate A B C D E F G H I J Initial Number Exposed2 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 IAN IAN IAN IAN IAN IAN IAN IAN IA N IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 14 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IAN IA N IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IA N IA N IA N IAN 12 A 10 0 IAN 0 0 IAN 0 0 IAN B 10 0 IAN 0 0 IAN 0 0 IAN C 10 0 IAN 0 0 IAN 0 0 IAN D 10 0 IAN 0 0 IAN 0 0 IAN E 10 0 IAN 0 0 IAN 0 0 IAN F 10 0 IAN 0 0 IAN 0 0 IAN G 10 0 IAN 0. 0 IA N 0 0 IAN H 10 1-- 0 1- 0 1- I 1> 0 0 IAN 0 0 IAN 0 0 IAN J 17 0 0 IAN 0 0 IAN 0 0 IAN 24 A 1 0 B 10 C 10 D 10 E 10 O F 10 O G 10 H 10 enP I 1 0 J 10 o 1 Observed Effects: AN = Appeared Normal; C " Lethargic. 2 All organisms on Day 0 appeared normal. 1 1 1 0 1 1 1 1 1 1 _ -- -- IAN -- -- --' -- -- - 01 0 1-- 0 1- 0 0 IAN 0 1- 0 10 10 10 1- 0 1- 0 1-- 0 1-- 0 10 0 IAN 0 I0 1" 0 10 1-- 0 10 1- AMENDED -63- PROJECTNO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magrta Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 Number Immobile 48 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN " Appeared Normal; C - Lethargic. 2 All organisms on Day 0 appeared normal. Day 13 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- '-- -- - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 14 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- - - - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects - 6 P w co AMENDED -64- PROJECT NO.: 454A-109 APPENDIXIV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Cladoceran, Daphnia magna Dilution Water Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 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 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 17 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IA N IAN IA N IAN IAN 1.5 - A B C D E F G H I J 10 10 10 10 10 10 10 10 10 10 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 IAN IAN IAN IAN IAN IAN IAN IAN IAN 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 IAN 2.9 O O h* cn A 10 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C = Lethargic. 2 All oreanisms on Day 0 appeared normal. 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 0 IAN 00 00 0 .0 00 00 00 00 00 00 00 IAN IAN IAN IAN IAN IAN IAN IAN IAN 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 IAN AMENDED -65- PROJECT NO.: 454A-109 APPENDIXIV (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphmds Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 5.6 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 16 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN , Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 17 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 0 0 0 0 0 0 0 0 0 1 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN ~ .t 12 A 1 0 0 IAN 0 0 IAN 0 0 IAN B 1 0 0 IAN 0 0 IAN 0 0 IAN C 1 0 0 IAN 0 0 IAN 0 0 IAN D 1 0 0 IAN 0 0 IAN 0 0 IAN E 1 0 0 IAN 0 0 IAN 0 0 IAN F 1 0 0 IAN 0 0 IAN 0 0 IAN G 1 0 0 IAN 0. 0 IAN 0 0 IAN H 10 1-- 0 1-- 0 1- I 1 ,, 0 0 IAN 0 0 IAN 0 0 IAN J 1' 0 0 IAN 0 0 IAN 0 0 IAN 24 A 10 1 0 1 mm B 10 1-- 0 1- C 10 1-- 0 1- D 1 0 0 IAN 0 1 - E 10 1-- 0 1- n O F 10 1-- 0 1- G 10 1 --' 0 1- H 10 1-- 0 1- P I 10 1-- 0 1- I-* J 1 0 in sss= ^= ssssa ssm ^B S S S S S S S ^^^^^^^B sssssss= ^sss^^B B S S S ^^^ssssa a B 1 Observed Effects: AN " Appeared Normal; C = Lethargic. cn 2 All organisms on Day 0 appeared normal. 1- 0 1- 01 0 1 0 1-- 0 10 10 10 10 10 101 AMENDED -66- PROJECT NO.: 454A-109 APPENDIXIV (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 Number Immobile 48 A 1 0 B 10 C 10 D 10 E 1 0, F 10 G 10 H 10 I 10 J 10 1, . . . ------------Observed Effects: AN = Appeared Normal; C = Lethargic. 2 All organisms on Day 0 appeared normal.______________ Day 16 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- -- -- -- Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 17 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- -- -- -- Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects - - d O Cl AMENDED Ci -67- PROJECTNO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphmds Sponsor Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Ciadoceran, Daphnia magna Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 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 19 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 20 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 21 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IA N IAN IAN IA N IA N IAN IA N 1.5 A 1 0 0 IAN 0 0 IAN 0 0 IAN B 1 0 0 IAN 0 0 IAN 0 0 IAN C 1 0 0 IAN 0 0 IAN 0 0 IAN D 1 0 0 IAN 0 0 IAN 0 1 -- E 1 0 0 IAN 0 0 IAN 0 0 IAN F 1 0 0 IAN 0 0 IAN 0 0 IAN G 1 0 0 IAN o 0 IAN 0 0 IAN H 1 0 0 IAN 0 0 IAN 0 0 IAN I 1 0 0 IAN 0 0 IAN 0 0 IAN J 1 0 0 IAN 0 0 IAN 0 0 IAN 2.9 A 10 0 IAN 0 0 IAN 0 1 B 10 0 IAN 0 0 IAN 0 0 IAN C 10 0 IAN 0 0 IAN 0 0 IAN D 10 0 IAN 0 0 IAN 0 0 IAN E 10 0 IAN 0 0 IAN 0 0 IAN O O P F 10 0 IAN G 1 0 .' 0 IAN H 10 0 IAN I 10 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 J 1 0 . 0 IAN 0 0 IAN 0 0 IAN Cl si 1 Observed Effects: AN = Appeared Normal; C = Lethargic. 2 All organisms on Day 0 appeared normal. AMENDED -68- PROJECT NO.: 454A-109 APPENDIX IV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water______________ Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed2 5.6 A 1 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 0 0 0 0 0 0 0 0 1 Effects IAN IAN IA N IAN IAN IAN IA N IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 20 Cumulative Dead 0 0 0 0 0 0 0 0 0 1 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 21 Cumulative Dead 0 0 .0 0 0 0 0 0 0 1 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN 12 A 10 0 IAN 0 0 IAN 0 0 IAN B 10 0 IAN 0 0 IAN 0 0 IAN C 10 0 IAN 0 0 IAN 0 0 IAN D 10 0 IAN 0 0 IAN 0 0 IAN E 10 0 IAN 0 0 IAN 0 0 IAN F 10 0 IAN 0 0 IAN 0 0 IAN G 10 0 IAN 0 0 IAN 0 0 IAN H 1 0 1-- 0 1- 0 1- I J 1 / 0 10 0 IAN 0 IAN 0 0 0 IAN 0 IAN 0 0 IAN 0 0 IAN 24 O A 10 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C = Lethargic. 05 2 All organisms on Day 0 appeared normal. 1 1 1 1 1 1 '1 1 1 1 -- -- -- -- -- -- -- -- - 0 1 mm 0 1- 0 10 10 10 10 1- 0 10 1- 0 1- 01 0 10 10 10 10 10 10 10 10 1- AMENDED -69- PROJECT NO.: 454A-109 APPENDIXIV (Continued) - Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) plicate Initial Number Exposed*1 Number Immobile 48 A 1 0 B 10 C 10 D 10 E 10 F 10 G 10 H 10 I 10 J 10 1 Observed Effects: AN = Appeared Normal; C ~ Lethargic. 1 All organisms on Day 0 appeared normal. Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- -- -- -- Number Immobile 0 0 0 0 0 0 0 0 0 0 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects -- -- -- -- -- -- -- -- Number Immobile 0 0 0 0 0 0 0 0 0 0 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effec -- -- -- -- / o 0 01 C> AMENDED W il d l if e In t e r n a t io n a l ltd PROJECT NO.: 454A-109 -70- APPENDIX V Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Cladoceran, Daphnia magna WeU Water Mean Measured Test Concentration (mg a.i./L) 2 5 7 9 11 14 16 18 21 Replicate Total Negative Control A 0 0 0 0 27 40 0 25 26 118 B 0 0 0 9 41 0 45 4 14 113 C 0 0 0 0 14 43 0 27 10 94 D 0 0 0 3 29 48 0 38 25 143 E 0 0 0 8 40 41 0 35 27 151 F 0 0 0 0 24 37 0 34 31 126 G -0 0 0 14 0 38 39 18 20 129 H 0 0 0 0 21 29 0. 21 29 I 0 0 0 9 32 45 0 31 23 100 140 J 0 0 0 0 20 33 34 0 18 105 X( SD) =122 (19.2) 1.5 A 0 0 0 0 21 43 0 36 31 131 B 0 0 0 2 0 39 39 0 26 106 C 0 0 0 6 37 47 0 38 35 163 D 0 0 0 0 23 44 0 0 32 99* E 0 0 0 0 20 33 35 0 31 119 F 0 0 0 5 38 51 0 36 30 160 G 0 0 0 0 25 48 31 0 22 126 H 0 0 0 16 46 44 1 41 34 182 1 0 0 0 10 37 27 12 40 33 159 J 0 0 0 3 28 41 0 37 27 136 5? ( SD) =142 (24.7) 2.9 A 0 0 0 0 13 37 35 7 17 109* B 0 0 0 7 38 41 4 41 36 167 C 0 0 0 11 37 38 0 42 34 162 D 0 0 0 0 25 45 33 0 20 123 E 0 0 0 0 24 43 34 3 23 127 F 0 0 0 19 0 47 36 'D 14 G 0 0 0 0 19 44 35 0 24 116 122 H 0 0 0 0 17 40 43 0 35 I 0 0 0 15 41 0 38 33 15 135 142 J 0 0 0 0 26 46 29 0 28 129 x ( SD) =136 (17.9) 1 This value excluded from calculation of the mean due to adult death. 001160 AMENDED W il d l if e In t e r n a t io n a l ltd. PROJECT NO.: 454A-109 -71- APPENDIX V (Continued) Neonate Production Sponsor Test Substance: Test Organism: Dilution Water Mean Measured Test Concentration 5.6 3M Corporation PFOS Cladoceran, Daphnia magna Well Water plicate 2 5 A 00 B 00 C 00 D 00 E 00 F 00 G 00 H 00 I 00 J 00 7 0 0 0 0 0 0 0 0 0 0 9 0 5 0 0 0 11 0 5 0 0 11 21 34 22 27 18 36 17 29 14 19 14 16 38 31 43 0 37 19 41 29 43 38 52 0 38 4 46 0 39 40 35 19 12 A 0 0 0 0 19 39 0 B - 0 0 0 0 16 40 34 C 0 0 0 0 24 36 35 D 0 0 0 18 0 38 34 E 0 0 0 0 26 36 37 F 0 0 0 10 37 0 40 G 0 0 0 10 34 41 0 H 0 0 0 0 20 ~ -- I 0 0 0 0 11 30 32 J 0 0 0 0 13 27 29 24 A0 ---- .. -- B 0 ---- -- -- ---- C 0 ---- -- -- w -- D 00 0 0 0 8 24 E 0 ---- -- -- ---- F 0 ---- -- -- __ _ G 00 0 0 9 -- -- H 00 0 0 -- -- -- I 0- - - - -- J0 48 A --- - -- -- B ---- -- -- -- C --- -- -- -- D ---- -- -- -- E ---- -- -- -- F -- -- -- --- _ G --_ -- -- -- H ---- -- -- -- I _ --- -- J ---- -- -- -- This value excluded from calculation ofdie mean due to adult death. ---- ---- -- ---- ---- ---- --~ ---- ---- ---- 18 21 0 35 31 33 0 26 0 20 0 29 37 34 34 32 38 30 0 34 0 28 17 20 0 34 19 0 0 34 37 15 39 41 ---- 0 30 0 14 -- ---- ---- --- ---- -- __ ---- -- /v ---- ---- --- ---- ---- ---- ---- ---- ------ Total 125 146 104 117 128 170 125 148 127 731 X( SD) = 132 (19.5) 86 127 129 109 133 139 165 201 103 83 S( SD) = 119 (26.5) 0 0 0 321 0 0 9* 0 0 0 * =0 0 0 0 0 0 0 0 0 0 0 C01161 AMENDED W il d l if e In t e r n a t io n a l ltd. PROJECT NO.: 454A-109 -72- APPENDIX VI Length and Dry Weight of Surviving First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dili^onW aten 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L' Individual Length (mm) Mean Length (mm SD) Negative Control 4.70 4.65 4.55 4.65 4.55 4.80 4.55 4.50 4.75 4.80 4.65 ( 0.111) 1.5 4.45 4.66 ( 0.118) 4.60 4.70 4.80 4.55 4.75 4.80 4.60 4.65 2.9 . . 4.62 ( 0.100) 4.75 4.50 4.65 4.70 4.55 4.45 4.70 4.60 4.65 Individual Dry Weights fog) 0.73 0.68 0.87 0.65 0.60 0.69 0.75 ' 0.61 0.55 0.82 0.68 0.70 0.63 0.81 0.61 0.67 0.63 0.68 0.61 .. 0.63 0.68 0.70 0.94 O.?* 0.80 0.82 0.60 0.63 Mean Dry Weights fog * sp ) 0.695( 0.100) 0.669 ( 0.0623) 0.724 ( 0.110) C011G2 AMENDED W il d l if e In t e r n a t io n a l ltd. PROJECT NO.: 454A-109 -73- APPENDIX VI (Continued) Length and Dry Weight o f Surviving First-Generation Daphnids Sponsor: Test Substance: Test Organism: 3M Corporation PFOS Cladoceran, Daphnia magna Mean Measured Test Concentration (mg a.i./L) 5.6 Individual Length (mm) 4.45 4.70 4.5 4.70 4.65 4.80 4.45 4.55 4.65 Mean Length (mm SD 4.61 ( 0.124) Individual Dry Weights (mg) 0.71 0.69 0.83 0.75 0.82 0.68 0.62 ` 0.73 0.71 Mean Dry Weights (ingjJSD) 0.727 ( 0.0665) 12 4.70 4.59 ( 0.102) 0.76 0.723 ( 0.0661) 4.60 0.64 4.75 0.76 4.65 0.65 4.45 0.82 4.60 0.65 4.55 0.70 4.45 0.74 4.55 0.79 001163 AMENDED W il d l if e In t e r n a t io n a l ltd. -74APPENDIX VE Changes to Protocol PROJECT NO.: 454A-109 This study was conducted in accordance with the approved Protocol with the following changes: 1. The proposed experimental start and termination dates were amended to the protocol. 2. During the second generation exposure, neonates were observed at 5 ,2 4 and 48 hours. /v 001164 W il d l if e In t e r n a t io n a l lto . -75APPENDEXVm Personnel Involved in the Study PROJECT NO.: 454A-109 The following key W ildlife International Ltd. personnel were involved in the conduct or management ofthis study: 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. W illard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Jon A. MacGregor, Scientist 4. Mark A. Mank, Laboratory Supervisor 5. Kurt R Drottar, Senior Biologist 0011G5 W il d l if e In t e r n a t io n a l ltd. -76APPENDIXIX Report Amendment PROJECT NO.: 454A-109 1. Original Report: Pages 1-4,6 and 31 Amendment: The pages were changed to include the amended report date, revised page numbers, and new signatures and dates due to the addition o f the report amendment as Appendix IX. Reason: To reflect the issuing o f an amended report. 2. Original Report: Page 2 Amendment: The compliance statement was revised. Reason: To clarify how the test substance was characterized. 3. Original Report: Page 9 Amendment: Information provided by the Sponsor reflecting the reanalysis o f the test substance, including the reanalvsis date and the puritv. was added to the Test Substance section. Reason: To reflect the current test substance information provided by the Sponsor. 4. Original Report: Entire report Amendment: All test substance concentrations were changed to reflect the purity o fthe test substance as determined by the Sponsor in a reanalysis o f the test substance (FC-95, Lot 217). Test concentrations originally were based on the reported purity o f 98.9%. The certifcate o f analysis dated March 9,2000 indicated a purity o f90.49%. Therefore, all test substance concentrations, including nominal concentrations, measured concentrations, and NOEC, LOEC and MATC values, were recalculated and reported as mg a.i./L based on the 90.49% purity. 'N Reason: To report the results o f the test based on the test substance purity o f 90.49% at the request o f the Sponsor. 0011G6 AMENDED W il d l if e In t e r n a t io n a l l t d . -77A P P E N D IX IX - -ContinuedReport Amendment AMENDMENT SIGNATURES: DATE PROJECT NO.: 454A-109 Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: Ja^bs H. Coleman Quality. Assurance DATE 0011G7 AMENDED l* PROTOCOL PFOS: A SEMI-STATIC LIFE-CYCLE TOXICITY TEST W ITH THE CLADOCERAN (Daphnia magna) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1300 OECD Guideline 211 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, M innesota 55144 W ild life In tern ation al ltd. 8598 Commerce Drive Easton, M aryland 21601 (410) 822-8600 June 10,1999 CQ11S3 W il d l if e In t e r n a t io n a l , ltd 2- PFOS: A SEMI-STATIC LIFE-CYCLE TOXICITY TEST W ITH THE CLADOCERAN (Daphnia magna) SPONSOR: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: W ildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Kurt Drottar Senior Aquatic Biologist LABORATORY MANAGEMENT: Henry O. Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants ____________________________ FO R LABORATORY USE ONLY_________ Proposed Dates: E xperim ental Start Date: _________________________ E xperim ental Termination Date: _________ Project No.: ________________________ _ Test Concentrations: Negative Control. 1 .6 ,3 .1 .6 ,3 .1 3 .2 5 and 50 mg a.i./L Test Substance No.: 4675- Reference Substance No. (if applicable): 4526 PROTOCOL APPROVAL STUDY DIRECTOR DATE LA ORATORY MANAGEMENT 1 SPONSOR'S REPRESENTATIVE DATE Lh o I t f DATE 0011G9 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 3- IN T R O D U C T IO N W ildlife International, Ltd. w ill conduct a semi-static life-cycle toxicity test w ith the cladoceran, Daphnia magna, for the Sponsor at the W ildlife International, Ltd. aquatic toxicology facility in Easton, M aryland. The study w ill be performed 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 E 1193-87 Standard Guide fo r Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna (4). Raw data for all work perform ed at W ildlife International, Ltd. and a copy o f the final report w ill be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in the final report. PURPOSE The purpose o f this study is to determine the effects o f the test substance on the survival, growth, and reproduction o f the cladoceran, Daphnia magna, under semi-static test conditions, for a period o f 21 days. EX PERIM ENTAL DESIGN Daphnids w ill be exposed to a geom etric series o f six test concentrations, and a negative (dilution w ater) control for 21 days. Individual neonate daphnids w ill be placed in ten replicate test chambers im partially assigned to each test concentration and control. Individually exposed daphnids w ill be observed for survival, reproduction and growth. Nom inal test concentrations w ill be selected in consultation w ith the Sponsor, and w ill be based upon inform ation such as the results o f exploratory range-finding toxicity data, known toxicity data, physical/chem ical properties o f the test substance or other relevant information. Each test substance concentration w ill be 50% o f the next higher one. W ater samples w ill be collected at specified intervals for analysis o f the test substance. Results o f the analyses w ill be used to calculate mean m easured test concentrations. New test solutions w ill be prepared three times per week (e.g., M onday, W ednesday, Friday), and the test organisms w ill be transferred from the old to the new solutions. To control bias, neonate daphnids w ill be im partially assigned to exposure cham bers at test initiation. No other potential sources o f bias are expected to affect the results o f the study. Observations o f the survival o f the first-generation daphnids w ill be made daily. The presence o f eggs in the brood patch, males PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 001170 W il d l if e In t e r n a t io n a l , ltd -4- or winter eggs also will be recorded daily. W ith the onset o f reproduction, the number o f young present in each test chamber will be counted every M onday, W ednesday and Friday during the test and at test term ination. An acute exposure w ill also be performed for any treatm ent groups w hich produce young. Twenty neonates from each treatment group and the control will be exposed under static test conditions for 48 hours. The results o f the test will be used to calculate the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC). The NOEC is defined as the highest concentration that produces no statistically-significant effect on survival, reproduction, or growth. The LOEC is defined as the lowest concentration that produces a statistically significant effect oh survival, reproduction, or growth. The maximum acceptable toxicant concentration (MATC) will be calculated as the geom etric m ean o f the NOEC and LOEC. M ATERIALS AND METHODS Test Substance Information on the characterization o f test, control or reference substances is required by OECD Principles o f Good Laboratory Practice. The Sponsor is responsible for providing W ildlife International, Ltd. written verification that the test substance has been characterized according to GLPs. I f w ritten verification o f GLP test substance characterization is not provided to W ildlife International, Ltd., it w ill be noted in the compliance statement o f the final report. The attached form ID EN TIFIC A TIO N O F T E ST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide inform ation necessary for GLP com pliance. The Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end o f the study. Preparation o f Test Concentrations The test substance w ill be mixed directly into dilution water. The solutions w ill be m ixed until the test material is dissolved. Once dissolved, the solution w ill be distributed into test chambers or diluted w ith dilution water to achieve the desired test concentrations. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 001171 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -5 - Test Organism The cladoceran, Daphnia magna, has been selected as the test species for this study. Daphnids are representative o f an im portant group o f aquatic invertebrates, and have been selected for use in the test based upon past use history and ease o f culturing in the laboratory. Daphnid neonates to be used in the test w ill be less than 24 hours old and w ill be obtained from cultures maintained at W ildlife International, Ltd., Easton, M aryland. The identity o f the species w ill be verified using appropriate taxonomic keys such as Pennak (5). Daphnids w ill be cultured in w ater from the same source .and at approximately the same tem perature as w ill be used during the test. Daphnids in the cultures producing neonates for the test w ill be held for at least 10 days prior to collection o f the neonates for testing. Neonates from daphnids that show signs o f disease or stress w ill not be used as test organisms. Daphnids in holding that produce ephippia also, w ill not be used to supply neonates for testing. Daphnids in the cultures w ill be fed once daily. The diet will be a mixture o f yeast, Cerophyll, and trout chow (YCT), supplemented w ith a suspension o f the freshwater green alga Selenastrum capricomutum. Specifications for acceptable levels o f contaminants in daphnid diets have not beenestablished. However, there are no known levels o f contaminants reasonably expected to be present in the diet that are considered to interfere w ith the purpose or conduct o f the test. N eonates w ill be obtained for testing from at least three individual adults. Prior to test initiation, the neonates w ill be collected from cultures and transferred to glass beakers. The daphnids w ill be released into the test compartments below the w ater surface using a wide-bore pipette. Daphnids will be fed daily during the test using the same diet as used in the cultures. Daphnids w ill be fed 0.3 mL o f YCT and 0.6 mL o f Selenastrum once daily. The frequency o f feeding w ill be documented in the raw data and the final report. D ilution W ater W ater used for the culturing and testing o f daphnids will be obtained from a well approximately 40 m eters deep located on the W ildlife International, Ltd. site. The water will be passed through a sand filter and pumped into a 37,800-L storage tank where the w ater w ill be aerated with spray nozzles. Prior to use the w ater w ill be filtered to 0.45 pm and passed through an ultraviolet sterilizer in order to remove PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd 6- - microorganisms and fine particles. W ater used for culturing and testing is characterized as m oderately hard. Typical values for hardness, alkalinity, pH and specific conductance are approxim ately: Hardness, mg/L as C aC 03 Alkalinity, mg/L as C aC 03 pH Specific Conductance, pmhos/cm 145 190 8.1 330 Hardness, alkalinity, pH and specific conductance w ill be measured w eekly to m onitor the consistency o f the well water. Means and ranges o f the measured param eters for the four-week period preceding the test will be provided in the final report. Analyses w ill be performed at least once annually to determine the concentrations of selected organic and inorganic constituents o f the w ell w ater and results o f the most recent GLP compliant analyses will be summarized in the final report. Test Apparatus Test chambers will consist o f 250-mL Nalgene beakers containing 200 mL o f test solution. Test chambers will be randomly positioned by treatm ent group in a tem perature-controlled w ater bath or an environmental chamber. Test chambers will be labeled with the project number, test concentration and replicate. Environmental Conditions Lighting used to illuminate the cultures and test chambers during culturing and testing w ill be provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g., Colortone 50). A photoperiod o f 16 hours o f light and 8 hours o f dark will be controlled with an autom atic tim er. A 30minute transition o f low light intensity will be provided when lights go on and o ff to avoid sudden changes in light intensity. Light intensity w ill be measured ju st prior to test initiation and a t approxim ately w eekly intervals thereafter with a SPER Scientific Ltd. light meter or equivalent. The target test temperature will be 20 1 C. Temperature w ill be m easured in one alternating replicate o f each treatm ent and the control group at test initiation, in the old and new solutions on renewal days, and at test termination using a liquid-in-glass thermometer. Temperature also w ill be m easured with a continuous recorder in one negative control replicate. Recorder measurements w ill be verified w ith a liquid-in-glass thermometer prior to test initiation and at least weekly thereafter. 0011^3 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -7 - Dissolved oxygen w ill be measured in one alternating replicate o f each treatm ent and the control group daily during the first week o f the test, in the old and new solutions on renewal days, and a t test term ination using a Yellow Springs Instruments Model 5 IB dissolved oxygen meter, or equivalent. In the event that dissolved oxygen levels fall below 60% saturation, dissolved oxygen w ill be measured in every test cham ber and appropriate actions w ill be taken after consultation with the Sponsor. M easurements o f pH w ill be made in one alternating replicate o f each treatm ent and the control group at test initiation, in the old and new solutions on renewal days, and at test term ination using a Fisher Accumet M odel 915 pH m eter, or equivalent. I f a treatm ent level reaches 100% mortality, dissolved oxygen, pH and tem perature measurements w ill be taken at that tim e, then discontinued. H ardness, alkalinity, and specific conductance w ill be measured in the negative (dilution water) control and the highest test concentration at test initiation and at approximately weekly intervals thereafter. H ardness and alkalinity measurements w ill be made by titration using procedures based on methods in Standard Methods fo r the Examination o f Water and Wastewater (6). Specific conductance will be measured using a Yellow Springs Instruments Model 33 Salinity-Conductivity-Tem perature meter, or equivalent. Biological Observations The first generation daphnids w ill be observed daily during the test for survival, the onset o f reproduction, and clinical signs o f toxicity. The criteria for death include absence o f heartbeat, white opaque coloration, lack o f movement o f appendages and lack o f response to gentle prodding. Im m obilization is defined as a lack o f m ovement except for m inor spontaneous random movement o f die appendages. Examples o f clinical signs o f toxicity include inability to maintain position in the water column, uncoordinated swimming and cessation o f feeding. The presence o f eggs in the brood pouch, aborted eggs, m ales, or ephippia also w ill be recorded. A fter the onset o f reproduction, tw enty neonates from each treatm ent group and the control will be exposed for 48-hours under static test conditions. The neonates w ill be observed at 24 and 48 hours for mortality/immobility and clinical signs o f toxicity. W ith the onset o f reproduction, the number o f second-generation daphnids w ill be counted and recorded approxim ately three tim es per week. Second generation neonates will be observed for clinical signs o f toxicity. Second-generation daphnids not used for the acute exposure w ill be discarded. The PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 001174 W il d l if e In t e r n a t io n a l , ltd - 8- length and dry weight o f the surviving first-generation daphnids will be measured at the end o f the test. The test w ill not be considered acceptable if control survival is <80% or if the number o f young produced by each surviving daphnid living the full 21 days is less than an average o f 60. Sampling for Analytical Measurements W ater samples will be collected from the new and old solutions during each week o f the test to determine concentrations o f thd test substance. Duplicate samples for the new solutions w ill be collected from the freshly prepared solutions, whereas samples from the old solutions w ill be obtained from a composite o f all replicate test chambers for each control and treatm ent level. In the event th at 100% m ortality occurs in any treatment, then sampling o f that treatm ent w ill term inate follow ing the next sampling interval. Samples will be collected at mid-depth and analyzed immediately. The sample scheme is summarized below: PROPOSED NUMBERS OF SAMPLES Week 1 Week 2 Week 3 Experimental Group New* 01db New* 01db New* Old" Control 22 Level 1-Low Concentration 2 2 Level 2 22 Level 3 22 Level 4 22 Level 5 22 Level 6-High Concentration 2 2 22 22 22 22 22 22 22 22 2 2 22 22 22 22 22 TOTALS 14 14 14 14 14 14 * New solutions (duplicate samples collected from intermediate chambers) b Old solutions (duplicate samples from a composite of all replicate test chambers from each control and test level) The above numbers o f samples represent those collected from the test and do not include quality control (QC) samples such as m atrix blanks and fortifications prepared and analyzed during the analytical chemistry phase o f the study. 001175 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd 9- A nalvtical Chemistry Chem ical analysis o f the samples w ill be performed by W ildlife International, Ltd. The analytical m ethod used w ill be based upon methodology provided by the Sponsor and identified in Appendix EL M odifications made to the analytical method w ill be documented in the raw data and described in the final report. Data Analyses Statistical analyses w ill be performed on the survival o f first-generation daphnids, the number o f live young produced by each individually-exposed daphnid, and the length and dry w eight o f the individuallyexposed first-generation daphnids. To evaluate whether or not survival is affected by the treatm ents, 2 X 2 contingency tables and the chi-square test w ill be used on survival data (i.e., numbers o f alive and dead daphnids). Analyses o f reproduction and growth (weight and length) data will be evaluated for normality and homogeneity o f variances. I f the data are deemed normal w ith homogeneous variances, hypothesis testing using analysis o f variance (ANOVA) and m ultiple means tests (e.g., Dunnett's, Bonferroni, Schefife) w ill be used. I f the data foil the tests for norm ality or homogeneity, then data transformations w ill be tried in an attem pt to correct the condition. W hen the data transformations fail to correct for non-normality or heterogeneity o f variances, nonparam etric procedures w ill be used to identify statistically significant differences among the experimental groups. The statistical analyses o f survival, growth and reproduction data w ill aid but may not be exclusively used in the determ ination o f the no observed effect concentration (NOEC) and low est observed effect concentration (LOEC). The maximum acceptable toxicant concentration (MATC) w ill be calculated as the geom etric mean o f the NOEC and LOEC. RECO RD S TO BE M AINTAINED Records to be m aintained for data generated at W ildlife International, Ltd. will include, but not be lim ited to: 1. A copy o f the signed protocol. 2. Identification and characterization o f the test substance, if provided by the Sponsor. 001176 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 10- 3. Dates o f initiation and term ination o f the study. 4. Length and dry weight measurements. 5. Reproduction parameters. 6. Test organism culture records. 7. Results o f rangefinding tests, when applicable. 8. Stock solution calculation and preparation. 9. Daily observations. v 10. W ater chemistry calculations (e.g., hardness and alkalinity). 11. I f applicable, the methods used to analyze test substance concentrations and the results o f analytical m easurem ents. 12. Statistical calculations. 13. Test conditions and physical/chemical measurements. 14. Calculation and preparation o f test concentrations. 15. Copy o f final report. FINAL REPO RT A final report o f the results o f the study w ill be prepared by W ildlife International, Ltd. The report w ill include the following, when applicable: 1. Name and address o f the facility performing the study. 2. Dates upon which the study was initiated and completed. It is the responsibility o f the Sponsor to provide the final date that data are recorded for chem istry, pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement o f compliance signed by die Study D irector addressing any exceptions to G ood Laboratory Practice Standards. 4. Objectives and procedures, as stated in the approved protocol, including changes in the original protocol. 5. The test substance identification, including name, chemical abstract number or code num ber, strength, purity, composition, and other inform ation provided by the Sponsor. 6. Stability and solubility o f the test substance under the conditions o f administration, if provided by the Sponsor or contracted to W ildlife International, Ltd. 7. A description o f the methods used to conduct the test. 001177 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - ii- 8. A description o f the test organism s, including the source, scientific name, age, life stage, feed types, light intensity and photoperiod. 9. A description o f the preparation o f the test solutions, the methods used to allocate organisms to test chambers and begin the test, the num ber o f organisms and chambers per treatment, and the duration o f the test. 10. A description o f circum stances th at may have affected the quality or integrity o f the data. 11. The name o f the S tudyD irector and the names o f other scientists, professionals, and supervisory personnel involved in the study. 12. A description o f the transform ations, calculations, and operations performed on the data, a summary and analysis o f the biological data and analytical chemistry data, and a statement o f the conclusions drawn from the analyses. 13. Statistical methods used to evaluate the data. 14. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study. 15. The location where raw data and final report are to be stored. 16. A statem ent prepared by the Q uality Assurance U nit listing the dates that study inspections and audits were made and findings reported to the Study Director and Management. 17. I f it is necessary to make corrections or additions to a final report after it has been accepted, such changes w ill be made in the form o f an amendment issued by the Study Director. The amendment w ill clearly identify the p art o f the final report that is being amended and the reasons for the amendment, and w ill be signed and dated by the Study Director. CHANGING O F PROTOCOL Planned changes to the protocol w ill be in the form o f w ritten amendments signed by the Study D irector and the Sponsor. Amendments w ill be considered as part o f the protocol and w ill be attached to the final protocol. Any other changes w ill be in the form o f w ritten deviations filed with the raw data. All changes to the protocol w ill be indicated in the final report. C0117S PROTOCOL N O .: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , l td - 12- GOOD LABORATORY PRACTICES This study w ill be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR P art 160 and/or Part 792); OECD (ENV/MC/CHEM (98) 17); and Japan MAFF (59 NohSan, N otification No. 3850, Agricultural Production Bureau). Each study conducted by W ildlife International, Ltd. is routinely examined by the W ildlife International, Ltd. Quality Assurance U nit for-com pliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statem ent o f com pliance with Good Laboratory Practices w ill be prepared for all portions o f the study conducted by W ildlife International, Ltd. The Sponsor w ill be responsible for compliance w ith G ood Laboratory Practices for procedures performed by other laboratories (eg ., residue analyses or pathology). Raw data for all work performed at W ildlife International, Ltd. and a copy o f the final report w ill be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in die final report. PROTOCOL NO.. 454/061099/DAP-LC3/SUB454 C O l^ 7 9 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd 13- REFERENCES 1 U.S. E nvironm ental P rotection A gency. 1996. Series 8S0- Ecological Effects Test Guidelines {draft), OPPTS Number 850.1300: Daphnid Chronic Toxicity Test. 2 O ECD G uideline fo r T esting o f C hem icals. 1997. Guideline 211: Daphnia magna Reproduction Test. V' 3 U.S. E nvironm ental P rotection Agency. 1982. Standard Evaluation Procedure, Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test. EPA 540/9-86-141. Environmental Protection Agency, Office o f Pesticide Programs. W ashington, D.C. 4 A m erican Society fo r T esting an d M aterials S tan d ard E1193-96. 1997. Standard Guide fo r Conducting Life-Cycle Toxicity Tests with Daphnia magna. American Society for Testing and M aterials. Philadelphia, PA. 5 P ennak, R .W . 1978. Freshwater Invertebrates o fthe United States. 2nd Ed. 365 p. 6 A PH A , AW W A, W PC F. 1985. Standard Methodsfo r the Examination o f Water and Wastewater. 16th Edition, Am erican Public H ealth Association. American W ater W orks Association. W ater Pollution Control Federation, New York. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 CG11SO 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 14- APPENDKI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I. Test Substance Identity (name to be used in the report): PFOS (Perfluorooctane Sulfonic A cid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A Internal Standard: 1.1.2.2H.H.H.H Perfluorooctane Sulfonic Acid T est Substance Sample Code or Batch Number: Lot 217 Test Substance Purity (% Active Ingredient): 98.9______ Expiration Date: 2008 II. Test Substance Characterization Have 1he identity, strength, purity and composition or other characteristics which appropriatelydenne the test substance and reference standard been determined prior to its use in this study in accordance with GLP Standards? _____ Yes x No m . Test Substance Storage Conditions Please indicatethe recommended storage conditions at Wildlife International Ltd. Ambient_______________________________________________________________________ Has the stability o fthe test substance underthese storage conditions been determined in accordance with GLP Standards? _____ Yes x No Other pertinent stability information: IV. Test Concentrations: _____ x Adjusttest concentration to 100% a i based upon the purity (%1 given above. Do not adiusttest concentrationto 100% ___________ a i Testthe material AS IS. V. Taxidty Information: Mammalian: RatLD50 251 m e/ke Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphnia m am a: 27 me/L____________ Rainbow Trout: 11 me/L Daphnia maena: 50 me/L____________ Fathead M innow: 38 m e/L Other Toxicity Information (including findings o fchronic and subchronic tests): Please see MSDS_________________________________________ _______________________ PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 001131 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 15- APPENDEXn Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. Liquid Chromatography M ass Spectrometry (LCMS) M ethod for the Determ ination o f Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) In Freshwater, Saltwater and A lgal M edium A copy o f the above method will be maintained in the raw data. The actual methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 C01132 3M LAB REQUEST NO. U2723 07/09/99 13:31 822 8915 AQUATIC BLDG 002/002 W il d l if e In t e r n a t io n a l ltd. PROJECT NO.: 454A-109 Page 1 of I AMENDMENT TO STUDY PROTOCOL STU D Y T IT L E : PFO S: A SEM I-STATIC LIFE-CYCLE TOXICITY TEST W ITH TH E CLADOCERAN (Daphnia magna) P R O T O C O L N O .: 4 5 4/061099/D A P-LC3/SU B454 A M EN D M EN T N O .: 1 SPO N SO R : 3M C orporation P R O JE C T N O .: 454A -109 E F F E C T IV E D A T E : July 1 2 ,1 9 9 9 s; AM ENDM ENT: Page 2 Add: Experim ental S tart D ate: 7/12/99 Experim ental T erm ination D ate: 8/2/99 R E A SO N : T he above inform ation w as not know n when the protocol w as signed by th e Study D irector. STUDY D IRECTOR LABORATORY MANAGEMENT DATE DATE aJ54\109\a*tndl 001153