Document NGX8m9zYEbq8YRB25pDRbzqjD

15) OECD 211-OPPTS 850.1350, Chronic toxicity test (daphnia magna), 454A-130 @ I PFBS: - A SEMI-STATIC _LIFE-CYCLETOXICITY TEST WITH THE CLADOCERAN (Daphnia magna) FINAL REPORT SAN ITlZEO WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A- 130 3M Environmental Lab Project No. E00-1429 U.S. Environmental Protection Agency Series 850 - Ecological Effects OPPTS Nimber 850.1300 OECD Guideline 2 11 DEC 0 9 2003 AUTHORS: Kurt R Drottar Raymond L. Van Hoven, Ph.D. Henry 0. Krueger, Ph.D. STUDY INITIATION DATE: October 6,2000 STUDY COMPLETION DATE: April 11,2001 Submitted to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55 144 WildlifeIntemutional, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 80 wildlife International, Ltd. -2- OEC 20Q3 Project Number 4j4A-130 SANITIZED GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Coqoration TITLE: PFBS: A Semi-static Life-Cycle 'Toxicity Test with the Cladoceran (Daphnia mugnu) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 130 STUDY COMPLETION: April 11,2001 This study was .conducted in compliancewith Good LaboratoryPracticeStandardsaspublished by the U.S. Environmental ProtectionAgency in 40 CFR. Parts 160 and 792,17 August 1989;OECD Principles of Good LaboratoryPractice(ENV/MC/CHEM (98) 17);and Japan MAFF, 59NohSan, NohficationNo.3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The test substancewas not characterizedin compliance with Good LaboratoryPractices prior to its use in the study. However, subsequent GLP compliantcharacterizationresulted in a purity similarto the original characterization purity. The stability of the test substance under cclnditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standmar&. STUDY DIRECTOR: Kurt R Drottar Senior Biologist SPONSOR'S REPRESENTATIVE: DATE W iW t f e International, Ltd. -3- Proiect Number 454A-130 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental ProtectionAgency in 40 CFR. Part 160 and 792, 17August 1989; OECD Principlesof Good Laboratory Practice (ENVMCKHEM (98) 17; and Japan MAFF, 59 NohSaqNotificationNo. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTMTY: Test Substance Preparation Analytical Sample Preparation Exposure Initiation,Offspring Observations, Length Measurements and Offspring Counts Analytical Sampling Biological Data and Draft Report Analytical Data and Draft Report Final Report DATE CONDUCTED: February 7,2001 February 9,2001 February 21,2001 February 28,2001 February 28,2001 March 21 - 23 and 26,2001 March 26 and 29,2001 April 10 and 11,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 7,2001 February 9,2001 February 9,2001 February 14,2001 February 21,2001 February 22,2001 February 28,2001 February 28,2001 March 26,2001 March 29,2001 April 11,2001 March 2,200 1 March 2,2001 March 27,2001 March 29,2001 April, 11,2001 Kimbefy A. Hox& Quality Assurance Representative W i l d l i f eInternational, Ltd. -4- Project Number 454A- 130 mpom APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A Semi-static Life-Cyclc: Toxicity Test with the Cladoceran (Daphniamagna) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A- 130 STUDY DIRECTOR: Kurt R. Drottar Senior Biologist MANAGEMENT: I Henry%. &eger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants DATE DATE wildlife International. Ltd. -5- Proiect Number 454A-130 TABLE OF CONTENTS TitleKover Page ........................................................................................................................................... I Good Laboratory Practice Compliance Statement ....................................................................................... .2 Quality Assurance Statement.......................................... ............................................... 3 Report Approval ........................................................................................................ ...........4. Table of Contents.................................................................................... ....................... 5 summary ............ ................................ ................................................................ 7 ................................................................................................................................ Objective............. ...................................................................................................................................... 8 ExperimentalDesign...................... .......................................... 8 Materials and Methods................... .................................. 9 Results and Discussion .................................. .... .............................................. 13 Conclusions......................................................................................................................................... References ................................................................................................................................................... 16 TABLES Table 1 - Summary of Analytical Chemistry Data .............................. Table 2 - Temperature of Water in the Test Chambers ....................... Table 3 - Dissolved Oxygen Content of Water in the Test Chambers. Table 4 - pH of Water in the Test Chambers........................ ............................................... 17 ............................................... 18 ................................ 20 ..................................... 22 wildlife International, Ltd. -6- Project Number 454A-130 TABLE OF CONTENTS - Continued - TABLES (Continued) Table 5 - Specific Conductance, Hardness and Alkalinity in the Negative Control and Highest Treatment Group ............. ..................................................................................... 24 Table 6 - Summary of Cumulative Percent Mortality and Treatment-RelatedEffects for First-Generation Daphnids ............. ..................................................................................... 25 Table 7 - Summary of Daphnia magna Reproduction During the Life-Cycle Toxicity Test.................. 26 Table 8 - Summary of Length and Dry Weight of SurvivingFirst-GenerationDaphnids....................... 27 Table 9 - Cumulative Percent Mortality/Immobil.ity and Treatment-RelatedEffects for Second-Generation Daphnids ..............,..................................................................................... 28 Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Appendix 7 Appendix 8 APPENDICES - Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week: Period Immediately Preceding the Test ..................... 29 - Analyses of Pesticides, Orgarucs and Metals in Wildlife International, Ltd. Well Water ................................................................. 30 - The Analysis of PFBS in Freshwater in Support of Wildlife International, Ltd. Project No.: 454A-130 .................................................. 32 - Cumulative Percent Mortality and Treatment-RelatedEffects - First-GenerationDaphnids ....................._................................................................... 52 - Neonate Production..................................................................................................... 73 - Length and Dry Weight of SurvivingFirst-GenerationD a p h d s ............................. 77 - Changes to the Protocol.............................................................................................. 79 - Personnel Involved in the Study ................................................................................. 80 ~~~~ WildlifeInternational, Ltd. SANITIZED - 7 - DEC 0 9 2003 Proiect Number 454A-130 1 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL LTD. PROJECT N T J ~ B E R : TEST SUBSTANCE: STUDY: NOMTNAL TEST CONCENTRATIONS MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: SIJMMARY 3M Corporation Wildlife International, Ltd. Easton, Maryland 2 1601 454A- 130 Perfluorobutanesulfonate, Potassium Salt (PFBS) PFBS: A Semi-static Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) Negative Control, 63, 125,250,500, 1000 and 2000 mg a.i./L Negative Control, 60, 121,247,502,995 and 1876 mg a.i./L Experimental Start - February 7,200 1 Biological Termination - February 28,2001 Experimental Termination - March 2,200 1 2 1 Days Neonate Cladocerans (Daphnia magna) Wildlife International, Ltd. cultures Easton, Maryland 21601 Juveniles c 24 hours old MATC : 707 mg a.i./L wildlife International, Ltd. -8- Project Number 454A-130 INTRODUCTION T h s study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from February 7,200 1to February28,200 1. Raw data generatedby Wildlife International,Ltd. and a copy of the final report are filed under Project Number 454A-130 in archves located on the Wildlife International Ltd. site. OB,JECTIVE The objective of thls study was to determinethe effects of Perfluorobutanesulfonate,Potassium Salt (PFBS) on the survival, growth and reproduction of the cladoceran (Daphnia magna) during a 21-day exposure period under semi-static test conditions. EXPERIMENTAL DESIGN Daphmds were exposed to a geometric series of six test concentrationsand a negative (dilutionwater) control. Ten replicatetest chambers were maintainedin each treatment and control group with one daphmd in each test chamber. Nominal test concentrationswere selectedin consultationwith the Sponsor,and were based upon the results of an acute toxicitytest. Nominal test concentrationswere 63, 125,250,500, 1000 and 2000 mg active ingredient (a.i.)/L. Mean measured concentrations were determined from new and correspondingold solutions during each week of the test. Daphnids were transferred to new solutionsevery Monday, Wednesday and Friday during the test. First-generationdaphnidswere observeddaily for survival,the onset of reproduction, and clinical signs of toxicity. With the onset of reproduction,the number of second-generation neonates were counted (number of live neonates, number of dead or immobile neonates and number of aborted eggs) every Monday, Wednesday and Friday duringthe test. If available,twenty second-generation neonatesfrom each treatment and the controlwere exposed under static test conditionsfor 48 hours. At test termination, the length and dry weight of each first-generationdaphmd was determined. The no-observed-effect-concentration(NOEC)and lowest-observed-effect-concentration(LOEC) were determined by examinationof the mortality, growth and reproductiondata. The maximum acceptabletoxicant concentration(MATC)was calculatedas the geometric mean of the NOEC and LOEC. WildlifeInternational, Ltd. SANITIZED - 9 - DEC 0 9 2003 Project Number 454A-130 MATERI.ALSA N D METHODS The study was conducted based on the procedures outlined in the protocol,"PFBS: A Semi-staticLifeCycle Toxicity Test with the Cladoceran (Daphnia magna)".The protocol was based onprocedures outhed in the U.S. Environmental Protection Agency Sleries850 - Ecolog~calEffects Test Guidelines OPPTS Number 850.1300 (1); OECD Guideline 21 1. Daphnia magna Reproduction Test (2); Standard Evaluation Procedure, Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test (3); and ASTM Standard E 1193-96, Standard Guide jbr Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna (4)... Test Substance The test substance was received from 3M Corporation on June 28, 2000 and was assigned Wildlife International, Ltd. identification number 5292. The test substance, a whte powder, was identified as Potassium Perfluoro Butane Sulfonate, AKA Developmental Product, AKA PFBS, lot 2, CAS Number 29420-49-3. Information provided by the Sponsor indicated a purity of 97.90%. A subsequent revision of the certificate of analysis inchcated a l~urityof 97.3% and an Expiratiofleassessment Date of January 17,2002. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 63, 125,250,500, 1000 and 2000 mg a.i./L. A 4-L primarystock solution was prepared by adding the test substance to W sterilized dilution water at a concentrationof 2000 mg a.i./L. The test solution was stirred with an electric stamless steel top-down mixer until all the test substance was dissolved. The primary stock solut.ionwas proportionally diluted with W sterilized dilution water to prepare 2-L of the other five test concentrations. All ddutions were inverted to mix. M e r mixing, 200 mL of test solution was added to the 10 replicate test chambers for each treatment group. New solutions were prepared for each renewal day. After mixing, all test solutions appeared clear and colorless. All test concentrations were adjusted for the original reported purity of the active ingredient in the test substance (97.9%). Test Organism The cladoceran, Daphnia magna, was selected as the test species for thls study. Daphmds are representative of an important group of aquatic invertebrates and were selected for use in the test based upon wildlifeInternational, Ltd. - 10- Proiect Number 454A- 130 past hstory of use and ease of culturingin the lab'oratory. D a p h d neonates used in the test were less than 24-hours old and were obtained from culturesmaintainedby Wildlife International,Ltd., Easton, Maryland. Adult daphnidswere cultured at approximatelythe same temperature as used duringthe test in Wildhfe International,Ltd. well water. Daphnids in the cultures were held for at least 15 days prior to collection of the juveniles for testing. The adults showed no s i p s of disease or stress during the holdingperiod. During the 14-day holding period preceding the test, wate:r temperaturesranged from 19.8to 20.1"C. ThepH ofthe water ranged from 8.1 to 8.3 and dissolved oxygen ranged from 8.3 to 8.6 mg/L. Instrumentation and methods used for water measurements are describedin the Environmental Conditions sectionof h s report. Neonate daphnids ( e 4 hours old) were obtained for testing from four individual adult d a p h d s . At test initiation,thejuvenile d a p h d s were collected from the culturesand placed in glass beakers oneto 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 aidwater interface. Daphnids in the cultures and those used in the test were fed a mixture of yeast, Cerophylla, and trout chow (YCT, 1800 mg TSSL), as well as a suspension of the freshwater green alga, Selenastrum capricornutum (approximately3.5 r: lo7cells/mL). Duringthe test, eachtest chamberwas fed 0.5 mL of YCT and 1.0 mL of Selenastrum once daily. Test Apparatus Test chambers were 250-mL plastic (Nalgenb) beakers containing200 mLof test solution. The depth of test solution in a representative test chamber was approximately 5 cm. Test chambers were indiscriminatelypositionedby treatmentgroup in an environmental chamber set to maintain a temperatureof 20+1C. The test chambers were labelled with the project number, test concentration and replicate. Dilution Water The water used for culturingand testing was freshwaterobtainedfrom a well approximately40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water. The specificconductance, hardness, alkalinity,and pH of the well water duringthe four-week period immediately preceding the test are presented in Appendix 1. wildlife International, Ltd. -11- Project Number 454A- 130 The well water was passed through a sand filter to remove particles greater than approximately25 tun, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water again was filtered to 0.45 pm and passed through a UV sterilizer to remove microorganisms and fine particles. The results of periodic analysesperformed to measure the concentrationsof selected contaminants in well water used by Wildlife International,Ltd. are presented in Appendix 2. Environmental Conditions Lightingused to illuminate the cultures and test chambers duringculturing andtesting was providedby fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone0 50). A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensitywas measured at test initiation and at weekly intervalsthereafterwith a SPER Scientific Ltd. light meter. Light intensity ranged from 357 to 512 lux at the surface of the water (Negative Control, Replicate A). Temperature was measured in alternating replicates of each treatment and the control group at test initiation, in old and new test solutions on renewal days, and at test termination using a liquid-in-glass thermometer. Temperature was also measured continuously in one negative control replicate using a Fulscope EWC Recorder. Recorder measurements were verified with a liquid-in-glassthermometerprior to test initiation and at least weekly thereafter. The target test temperaturewas 20k 1C. Dssolved oqgen 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 speclfic conductance were measured in water samples colltxted from alternatingreplicates of the negativecontrol and the highest test concentration at test initiation and at approximately weekly intervals thereafter (new solutions). Measurements of pH were made using a Fisher Accumet Model 915 pH meter, and dissolvedoxygen was measured using a Yellow Springs Instrument Model 5 1Bdissolvedoxygen meter. Specificconductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperaturemeter. WildlifeInternational, Ltd. - 12- Proiect Number 454A-130 Hardness and alkalinitymeasurements were made by titration based on procedures in StandardMethoakfor the Examination of Water and Wastewater (5). Biological Observations and Measurements The first generation daphmds were observed daily during the test for survival, the onset of reproduction,and clinical signs of toxicity. The criteria for death included whte opaque coloration,lack of movement of appendages,absenceof heartbeat and lack of responseto gentleprodding. Immobilization was defined as lack of movement except for minor activity of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia were also recorded. With the onset of reproduction,the number of second-generation neonates were counted (number of live neonates, number of dead or immobile neonates and number of aborted eggs) and recorded every Monday, Wednesday and Friday during the test. At each observation period, the live first-generation daphnids were retained and the second-generation daphnids were discarded. On Day 14, the secondgenerationneonateswere collected and used for the second-generation acute exposure. At test termination, the length (measured from the apex of the head to the base of the spine) and dry weight were determined for each survivingfirst-generationdaphmd. The individualdaphnidswere removed from the test chambers and placed into tared~aiuminumdrymg pans. The length of each daphmd was determinedusing a caliper. The pans were then placed in a drymg oven at approximately60C for approximately46 hours. The pans were removed from the dryingoven and placed in a desiccatorto cool. When cool, the pans were weighed using a Mettler AE-240 analytical balance. Statistical Analyses Statistical analyses were performed on survivalof first-generationdaphmds, the number of liveyoung and the length and dry weight of the survivingfirst-generation daphnids. Survivaldata were analyzed using Fisher's exact test. Treatment groups with sigmficant survival effects were excluded from the analyses of reproduction and growth. Reproduction and growth (length and dry weight) data were evaluated for normality and homogeneity of varianceusing the chi-squaretest and Bartlett'stest, respectively. If the data met the assumptions of normality and homogeneity, the Bonferroni t-test was used to identify treatment groups that were statisticallyreduced in comparison to the negative control (p I; 0.05). If the data failed the assumptions of normality or homogeneity, transformationswere appliedto the data in an attemptto correct wildlife International, Ltd. - 13 - Project Number 454A-130 the condhon. If transformations failed, Wilcoxon's rank sum test with the Bonferroni adjustment was used to evaluate the data (p I 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 2nd old solutions each week during the test. Duplicate samples of new solutions were collected from the freshly prepared solutions. Duplicate samples of the old solutions were collected from a composite of the remaining replicate test chambers. Samples were collected in plastic scintillation vials and were analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3. RESULTS A N D DISCUSSION Verification of Test Concentrations Results of analyses to measure concentrations of PFBS in water samples collected during the test are presented in Table 1 and the analytical chemistryreport (Appendix3). Nominal concentrationsselected for use in the study were 63, 125, 250, 500, 1000 and 2000 mg a.i./L. Measured values of new (freshly prepared) samples ranged from 85 to 103% of nominal values, whereas measured values from the .ol.d solutionsranged from 86 to 106%of nominal values. Consequently,PFBS was stablethroughouttherenewal periods. When measured concentrationsofsamples collected during each week ofthe test were averaged,the mean measured concentrations were 60,121,247,502,995 and 1876 mg a.i./L, which represented 95,97, 99, 100, 100 and 94% of nominal, respectively. The results of the study were based on mean measured concentrations. Physical and Chemical Measurements of Water Measurements of temperature, dissolved oxygen and pH are presented in Tables 2, 3 and 4, respectively. Manual water temperature measurements were within the range of 20&l0Cestablishedfor the test. However, there were two instances when the continuous temperature monitor reached 18C. This occurred duringrenewal of test solutions and was probably the result of the temperatureprobebeingexposed to air. Dissolved oxygen concentrations remained 27.9 mg/L (87% of saturation) throughout the test. Measurements of pH ranged from 7.9 to 8.7. wildlifeInternational, Ltd. - 14- Project Number 454A- 130 Measurements of specificconductance, hardness and alkalinity in the negative control and the hghest treatment group are presented in Table 5. No apparent dlfferences were observed for hardness or alkalinity throughoutthe exposure period. However, condmtivitywas elevatedin the 1876mg a.i./L treatment group in comparison to the negative control. Survival and Biological Observations A s u m m a r y of the results of the observations of mortality and sublethal signs of toxicity for first- generationdaphnids are shown in Table 6. Daily observationsare provided in Appendix 4. ARer 21 days of exposure, survival in the negative control group was 90%. Survival in the 60, 121,247,502, 995 and 1876 mg a.i./L treatment groups was 100, 100, 100, 100, 70 and 20%, respectively. Fisher's exact test showed that survivalwas significantlydifferent in the 1876 mg a.i./Ltreatment group in comparison to the negative control (p 5 0.05). Reproduction A s u m m a r y of the mean number of liveyoung produced by the first-generationd a p h d s is presented in Table 7. A complete listing of the number of neonatesproduced is provided in Appendix 5. Daphds in the control and PFBS treatment groups ~ 9 9 m5 g a.i./L started producing neonates on Day 8. There were no observations of aborted eggs in any treatment group. Reproduction averaged 86 young per adult in the negative control. Reproduction in the 60,121,247,502 and 995 mg a.i./Ltreatment groups averaged 173, 149, 167, 89 and 43 young per adult, respectively. The Bonfenoni t-test showed that reproduction was significantlyreduced in the 995 mg a.i./Ltreatmentgroup in comparison to the negativecontrol (JJ I 0.05). The 1876mg a.i./Ltreatment group was not included in the statistical analysis of reproductionor growthdata due to a statistically significant effect on survival. Growth A s u m m a r y of the mean lengths and mean d r y weights of the surviving first-generationd a p h d s are presented in Table 8. Individual measurements are provided in Appendix 6. The mean length and dry weight of first-generationdaphnids in the negative control was 4.18 mm and 0.80mg, respectively. Wilcoxon'srank sum test showed that mean length was significimtly reduced in the 995 mg a.i./L treatment group in comparison to the negative control (JJ r; 0.05). wiId1(fe I nternationa1, Ltd. - 15 - Project Number 454A-130 Second-Generation Acute Exposure The results of the second-generation acute: exposure are presented in Table 9. After 48-hours of exposure, survival in the negative control was 100%. Survival in the 60, 121,247,502 and 995 mg a.i./L treatment groups was 100, 100, 100, 100 and loo%, respectively. A second-generation acute exposurewas not performed for the 1876 mg a.i./L treatment group due to a lack of neonates. CONCLUSIONS There were no adverse effects on survival, reproduction or growth of Daphnia magna exposed to PFBS at concentrations g502 mg a.i./L. Daphnia magna exposed to 1876 mg a.i./L had sigtllficantly reduced survival. Daphnia magna exposed to 995 mg a.i./L had sipficantly reduced reproduction and length. Consequently, the LOEC for this study, klased on reproduction and length was 995 mg a.i./L. The NOEC was 502 mg a.i./L and the MATC was calculated to be 707 mg a.i./L. The results of the secondgeneration acute exposure indicated a NOEC of 995 mg a.i./L. wildlifeInternational, Ltd. - 16- Project Number 454A- 130 REFERENCES 1 U.S. Environmental 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 2 11: Daphnia magna Reproduction Test. 3 US.Environmental Protection Agency. 1986. Standard Evaluation Procedure, Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test. Hazard EvaluationDivision. Officeof Pesticide Programs, EPA 540/9-86-141. 4 American Society for Testing and Materials. 1997. Standard Guidefor Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna, ASTM E 1193-96. Phladelpha, PA. 5 APHA, AWWA, WPCF. 1985. StandardMethodsfor the Examination of Waterand Wastewater. 16th Edition, American Public Health Association. American water Works Association. Water Pollution Control Federation, New York. '. 6 West, Inc. and D.D. Gulley. 1996. TOXSTAT, Version 3.5. Western Ecosystems Technology,Inc. Cheyenne, Wyoming. - 17- Project Number 454A-130 Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Nominal Concentration (mg a.i./L) Day 0 (New) Day 2 (New) Day 5 (Old) Negative Control 63 <LoQ' <LOQ 57.1 59.4 <LOQ <LOQ 63.4 62.2 <LOQ <LoQ 63.7 64.5 125 117 121 127 118 125 132 250 239 251 265 237 249 262 500 490 498 544 484 498 537 1000 973 1004 1055 980 1007 1067 2000 1893 1879 2014 1955 1908 2078 'The limit of quantitation (LOQ) was 20.0 mg a.i./L. Day 9 (New) <LOQ <LOQ 58.3 59.3 115 116 24 1 230 474 482 956 946 1774 1835 Day 12 (Old) <LOQ <LOQ 58.2 58.7 116 117 235 238 507 516 999 1002 1894 1962 Day 16 (New) <LOQ <LOQ 54.5 53.2 114 119 257 248 497 477 973 960 1805 1836 Day 19 (Old) <LOQ <LOQ 63.9 60.0 121 122 250 248 503 507 1014 1022 1841 1885 D~~ 2 1 (Old) <LoQ <LoQ 63.8 64.6 127 127 25 1 250 525 500 964 991 1719 1734 Mean Measured Concentration (mg a.i./L) <LOQ 60 121 247 502 995 1876 Percent of Nominal -- 95 97 99 100 100 94 - 18 - Table 2 Temperature of Water in the Test Chambers' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration (mg a.i.4,) Negative Control Day 0 New 20.4 Day 2 Old New 20.9 21.0 Day 5 Old New 21.0 19.4 Temperature ( O C) Day 7 Old New 20.8 20.4 60 19.9 20.9 20.5 21.0 19.0 20.7 20.0 121 19.8 20.8 20.8 20.8 9.0 20.8 19.7 247 19.9 21.0 20.9 21.0 9.0 20.9 19.8 502 19.9 20.9 20.9 21.3 9.0 20.9 19.8 995 20.0 21.0 21.0 20.9 19.0 21.0 19.9 1876 20.2 21.0 21.0 21.4 19.2 21.0 20.1 'Temperature measured continuously during the test ranged from 18.0"C to 21.O"C. Project Number 454A-130 Day 9 Old New 20.6 19.9 20.4 19.7 20.6 19.9 20.8 19.4 20.5 20.8 20.4 20.8 20.6 20.8 Day 12 Old New 19.5 19.8 19.3 19.6 9.5 19.6 9.4 20.0 9.3 20.0 20.8 20.1 19.6 20.1 - 19- Project Number 454A-130 Table 2 (Continued) Temperature of Water in the Test Chambers' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a i /L) Negative Control Dav 14 ~ Old New 20 9 20 9 Temperature ( O C) Dav 16 Old New 20 5 19.8 60 20.3 20.5 20.4 19.4 121 20.8 20.2 19.9 19.3 247 20.9 20.0 19.6 19.3 502 20.8 20.1 20.7 19.7 995 21.3 20.1 19.8 19.7 1876 21.1 20.3 20.6 19.6 'Temperature measured continuously during the test ranged ffom 18.0"C to 21.O"C. Dav 19 Old New 20.7 20 3 21.0 19.7 21.0 19.5 20.9 19.5 20.5 19.6 21.0 19.7 21.0 19.9 Dav 21 Old 21 0 20.8 19.9 21.0 20.8 21.0 21.0 - 20 - Project Number 454A-130 Table 3 Dissolved Oxygen Content of Water in the Test Chambers' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control Day0 New 7.9 Day 1 Old 8.2 Day 2 Old New 8.6 8.4 Dissolved Oxygen Concentration (rng/L) Day 3 Day 4 Day 5 Old Old Old New 8.6 8.4 8.4 8.6 60 8.2 8.2 8.6 8.5 8.6 8.4 8.4 8.6 1.2 9 1 8.0 8.2 8.6 8.5 8.6 a .? a.4 8.6 247 8.0 8.2 8.6 8.5 8.6 8.4 8.4 8.6 502 7.9 8.2 8.6 8.6 8.6 8.4 8.4 8.6 995 8.1 8.2 8.6 8.4 8.6 8.4 8.4 8.6 1876 8.2 8.2 8.6 8.5 8.6 8.4 'A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20C in freshwater 8.4 8.6 Day 6 Old 8.5 8.5 8.5 8.5 8.5 8.5 8.6 Day 7 Old New 8.2 81 8.2 8.3 8.2 8.3 8.2 8.2 8.2 8.2 8.2 8.5 8.2 8.8 -21 - Project Number 454A-130 Table 3 (Continued) Dissolved Oxygen Content of Water in the Test Chambers' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control Day 9 Old New 8.3 8.4 Dissolved Oxygen Concentration (mg/L) Day 12 Day 14 Day 16 Old New Old New Old New 8.7 8.0 8.2 8.6 8.3 8.4 60 8.3 8.5 8.6 8.7 8.3 8.4 8.2 8.6 121 8.2 8.6 8.6 8.6 8.2 8.4 8.2 8.7 247 8.2 8.6 8.6 8.6 8.2 8.4 8.4 8.6 502 8.3 8.6 8.6 8.6 8.3 8.4 8.2 8.7 995 8.4 8.8 8.6 8.8 8.2 8.4 8.4 8.9 1876 8.3 9.0 8.8 9.0 8.4 9.0 'A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20C in freshwater. 8.3 9.0 Day 19 Old New 8.4 8.1 8.2 8.1 n.4 8.! 8.2 8.2 8.4 8.4 8.5 8.6 8.5 9.0 Day21 Old 8.3 8.2 8.3 8.0 8.2 8. I 8.4 - 22 - Project Number 454A-130 Table 4 pH of Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control 60 121 247 502 995 - 1876 Day 0 New 7.9 8.0 8.0 8.0 8.1 8.1 8.2 Day 2 Old New 8.5 8.0 8.5 8.2 8.6 8.2 8.6 8.2 8.6 8.3 8.6 8.3 8.7 8.4 PH Day 5 Old New 8.5 8.1 Day 7 Old New 8.4 8.2 8.6 8.1 8.4 8.2 8.6 8.1 8.4 8.2 8.6 8.1 8.4 8.3 8.6 8.1 8.5 8.3 8.7 8.2 8.5 8.4 8.7 8.2 8.6 8.5 Day 9 Old New 8.4 8.0 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.2 Day 12 Old New 8.5 7.9 8.5 8.0 8.5 8.0 8.5 8.0 8.6 8.1 8.6 8.1 8.7 8.3 - 23 - Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Table 4 (Continued) pH of Water in the Test Chambers Mean Measured Test Concentration (mg a.i./L) Negative Control 60 121 247 502 995 1876 Day 14 Old New 8.3 8.2 8.3 8.1 8.4 8.1 8.4 8.2 8.5 8.2 8.5 8.2 8.6 8.2 Day 16 Old New 8.3 8.0 8.3 8.1 8.4 8.1 8.4 8.1 8.5 8.2 8.5 8.3 8.6 8.4 Project Number 454A-130 Day 19 Old New 8.3 8.0 8.3 8.0 8.3 8.0 8.4 8.0 8.4 8.1 8.5 8.2 8.6 8.4 Day 21 Old 8.5 8.5 8.6 8.5 8.6 8.6 8.6 wildlife International, Ltd. - 24 - Project Number 454A-130 Table 5 Specific Conductance, Hardness and Alkalinity in the Negative Control and Highest Treatment Group Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Negative Control . Time Parameter Specific Conductance (pmhos/cm) Day 0 325 Day 7 335 Day 14 340 Day 21 330 Hardness 128 128 132 128 ( m a as CaC03) Alkalinity 178 180 182 184 ( m a as CaC03) 1876 mg a.i./L Parameter Specific Conductance (pmhos/cm) Hardness (mgL as CaC03) Alkalinity (mg/L as CaC03) Day 0 800 140 178 Day 7 800 140 180 Day 14 800 128 176 Day 21 850 132 184 - 25 - Project Number 454A-130 Table 6 Summary of Cumulative Percent Mortality and Treatment-Related Effects' for First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Cumulative Observations to Day 7 Test Concentration YO YO (mg a.i./L) Mortality Immobile Effects Negative Control 0 0 10 AN Cumulative Observations to Day 14 YO YO Mortality Immobile Effects 0 0 10 AN 60 0 0 10 AN 0 0 10 AN 121 0 0 10 AN 0 0 10 AN 247 0 0 10 AN 0 0 10 AN 502 0 0 10 AN 0 0 10 AN 995 0 0 6AN, 4D 30 0 5AN, lD, 1D,G 1876 20 0 8G 80 0 2G 'Observed Effects: AN = Appears Normal, D = Discoloration, G = Appears Smaller *Indicates a si@icant difference fiom the negative control on Day 21 using Fisher's exact test (p 5 0.05). Cumulative Observations to Day 2 1 % Mortality YO Immobile Effects 10 0 8AN, 1D 0 0 10 AN 0 0 10 AN 0 0 10 AN 0 0 10 AN 30 0 6AN, 1D 80* 0 2 D,G - 26 - Table 7 Summary of Daphnia magna Reproduction During the Life-Cycle Toxicity Test Project Number 454A-130 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Number Of Daphnids Negative Control 9 Mean Live Young/ Surviving Adult Daphnid (k SD) 86 (+27) First Day of Reproduction 8 Total Number of Dead/Immobile Neonates 0 60 10 173 (+34) 8 0 121 10 l l n l "-I\ 14Y (k4l) 8 0 247 10 167 (+30) 8 0 502 10 89 (+26) 8 0 995 7 43* (+28) 8 0 1876' 2 14 (k 14) 11 3 `Ths treatment was excluded from the statistical analyses of reproduction due to a statistically significant survival effect. *Indicates a statistically significant difference from the control using the Bonferroni t-test (p < 0.05). Total Number of Aborted Eggs 0 0 0 0 0 0 0 wildlife International, Ltd. - 27 - Proiect Number 454A-130 Ta.ble 8 Summary of Length and Dry Weight of Surviving First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Number of Surviving Daphnids Negative Control 9 60 10 121 10 247 10 5 02 10 995 7 Total Length (mm) Mean (kSD) 4.18 (k0.182) 4.89 (kO.111) 4.78 (k0.207) 4.77 (k0.206) 4.26 (k0.480) 3.69* (k0.352) Dry Weight (mg) Mean (k SD) 0.80 (k0.29) 1.22 (k0.31) 1.21 (k0.34) 0.96 (k0.16) 0.88 (k0.16) 0.51 (k0.16) 1876' 2 2.68 (k0.106) 0.10 (kO.0) `This treatment was excluded from the statistical analyses of growth due to a statistically significant survival effect. *Inlcates a sipficant lfference from the negative control using Wilcoxon's rank sum test (p s 0.05). - 28 - Project Number 454A-130 Table 9 Cumulative Percent Mortalityhnrnobility and Treatment-Related Effects' for Second-Generation Daphmds Sponsor Test Substance: Test Organism: 3M Corporation PFBS Cladoceran, Daphnia magna Mean Measured Concentration (mg a.i./L) Negative Control Replicate A B Daphnia/ Replicate 10 10 Number Dead 0 0 60 A 10 0 B 10 0 .-- 1'41 A .^ 1u 0 B 10 0 247 A 10 0 B 10 0 502 A 10 0 B 10 0 995 A 10 0 ' B 10 0 Observed Effects: AN = Appears Normal 2 Hours Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 Effects 10 AN 10 AN 10 AN 10 AN i o AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN Number Dead 0 0 0 0 0 0 0 0 0 0 0 0 24 Hours Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN Percent Immobile and Dead 0 0 0 0 0 0 Number Dead 0 0 0 0 0 0 0 0 0 0 0 0 48 Hours Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN Percent Immobile and Dead 0 0 0 0 0 0 wildlifeInternational, Ltd. - 29 - Project Number 454A-130 Appendix 1 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Irnmediately Preceding the Test Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Cladoceran,Daphnia magna Dilution Water: Well Water Specific Conductance , (pmhos/cm) Mean 309 (N = 4) Range 305 - 3 10 Hardness (mgL as CaC03) Alkalinity (mgLas CaC03) 131 (N=4) 178 (N= 4) 124 - 136 176 - 182 PH 8.0 (N = 4) 8.0-8.1 wildlifeInternational, Ltd. - 30 - Proiect Number 454A-130 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Component Measured Concentration Component Measured Concentration Pesticides and Organics Aclonifen Alachlor Ametryn Atrazin Azinphos-ethyl Azinphos-methyl ' Azoxystrobin Bifenthrin Bioallethrin Bitertanol Brornacil Bromophos Brornophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos C hloridazon C hlorpropham Chlorpyriphos C hlorpyriphos-methyl Chlorthalonil Coumaphos Cyanazin Cytluthrin Cypermethrin C yproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desisopropylatrazin Desrnetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid ~ 0 . 0 p3 g 5 CO.01 pg/L <0.01 pg/L <0.01 pg/L ~ 0 . 0 4pg/L ~ 0 . 0 8pg/L ~ 0 . 2 p5g/L ~ 0 . 0 5pg/L ~ 0 . 0 5pg/L ~ 0 . 0 5pg/L ~ 0 . 0 5pg/L (0.02 pg/L C0.02 pg/L C0.02 pg/L <0.05 pg/L 4 . 0 5 pg/L ~ 0 . 0 3pg/L C0.02 pg/L C0.02 pg/L ~ 0 . 0 5pg/L 4 . 0 2 pg/L CO.01 pg/L <0.01 pg/L -4.04 pg/L <0.02 pg/L <0.05 pg/L ~ 0 . 0 5pg/L ~ 0 . 2 5pg/L ~ 0 . 0 5ppjL C0.02 pg/L C0.02 pg/L 10.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L CO.01 pg/L <0.03 pg/L C0.02 pg/L CO.01 pg/L <0.03 pg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan-P Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifop-butyl Fluoroglycofen-ethyl F1uroxypyr:meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 p a <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L 4 . 0 2 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L C0.02 pg/L <0.05 pg/L C0.05 pg/L CO.01 pg/L <0.03 pg/L <00.03pg/L ~ 0 . 0 5pg/L ~ 0 . 2 5pg/L <0.01 pg/L CO.01 pg/L <0.02 p g n C0.02 pgL. <0.02 pg/L <0.05 pg/L <0.02 pg/L CO.01 pg/L <0.02 pg/L <0.02 pa <0.01 p a 10.05 pg/L <0.02 pg/L <0.05 pg/L C0.02 pg/L C0.02 pg/L ~ 0 . 0 5pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. wildlifeInternational, Ltd. - 31 - Project Number 454A- 130 Appendix 12 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, ~ t dW. ell Water' ~~ Pesticides and Organics (Page 2) Component Measured Concentration Component Measured Concentration Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pyrazophos <0.01 pg/L ~ 0 . 2 5pg/L <0.02 pg/L 4 . 0 3 pa C0.02 pg/L <0.05 pg/L C0.01 pg/L 4 . 0 1 pg/L ~ 0 . 0 5pg/L <0.03 pg/L co.01 pg/L CO.01 pg/L <0.05 pg/L <0.02 pg/L ~ 0 . 0 5pg/L <0.01 pg/L <0.01 pg/L CO.01 pg/L C0.02 pa <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L -4.02 p& co.05 pgrL ~ 0 . 0 3pg/L <0.02 pg/L <0.02 pg/L ~ 0 . 0 3p& - Methoxychlor Metolachlor Metri buzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl Tolylfluanid Triadimefon Triadimenol Triallaat Triazophos Trifluralin Vamidothion Vinchlozolin CO.01 pg/L ~ 0 . 0 1pg/L <0.02 pg/L CO.01 pg/L ~ 0 . 0 5pg/L 4 . 0 1 pg/L CO.01 pg/L <0.01 pg/L C0.02 pg/L CO.01 pg/L 4 . 0 2 pg/L <0.05 pg/L ~ 0 . 0 5pg/L CO.01 pg/L <0.01 pg/L CO.01 pg/L ~ 0 . 0 5pg/L C0.01 pg/L <0.05 pg/L ~ 0 . 0 4pg/L <0.01 pg/L ~ 0 . 0 4pg/L <0.05 pg/L -4.05pg/L 4 . 0 2 pg/L C0.02 pglL c0.02 pg/L <0.01 pg/L CO.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mg/L 29 mg/L ~ 0 . 0 1 5mg/L 1.1 mg/L ~ 0 . 0 2mg/L CO.1 pg/L C0.2 pg/L < O S pg/L C0.2 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 4 . 1 pg/L ~ 0 . 7pg/L <00.25pg/L ~ 0 . p3g/L 4 . 2 Pg/L CO.1 pg/L <OS pg/L <0.025 pg/L < O S pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. wildlife International, Ltd. - 312 - Proiect Number 454A-130 Appendix 3 THE ANALYSIS OF PFBS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A- 130 wildltfe International, Ltd. - 33 - Project Number 454A- 130 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A SEMI-STATIC LIFE-CYCLE TOXICITY TEST WITH THE CLADOCERAN (Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-130 3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429 PRINCIPAL INVESTIGATOR: Raw&Van Hoven, Ph.D. Scientist MANAGEMENT: Willard'B. Nixon, Ph.D. Director, Analytical Chemistry DATE Wildl5feInternational, Ltd. .- 34 - Project Number 454A-130 Introduction Freshwater samples were collected from a sc:mi-static life-cycle aquatic toxicity study designed to determine the effects of PFBS (Perfluorobutane Sulfonate, Potassium Salt) to the cladoceran (Daphnia magna). T h ~ sstudy was conducted by Wildlife International, Ltd. and identified as Project Number 454A -130. The analyses of these water samples were performed at Wildlife International, Ltd. using hgh performance Liquid chromatography with mass spectrometric detection (HPLCIMS). Samples were received for analysis between February 7, 2001 and February 28, 2001 and were prepared for analysis on .. each sample receipt day. Analytical Standard The analytical standard was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International:, Ltd. Identification number 52 16, and stored under ambient condltions. The analylcal standard, a whte powder, was identified as: Potassium Perfluorobutane Sulfonate 1, Lot 2), expiration date: March 2010. The analytical standard was further identified with the 3M Environmental Laboratory test control and reference number TCR . The test substance had a reported purity of 97.90%. A subsequent revision of the certificate of analysis indlcated a purity of 97.3% and an Expiratiofieassessment Date of January 17, 2002. The analytical standard was the same material and lot number as the test substance (Wildlife International, Ltd. Identification number 5292). The analytical standard was used to prepare calibration standards and matrix fortification samples. Analytical Method Water samples were analyzed accordmg to the method entitled "Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater" (Wildlife International, Ltd. Project No. 454C-115). Samples were diluted in a 50% methanol : 50% NANOpure@water solution so that they fell withm the calibration range of the PFBS methodology. Aliquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in freshwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 N g h Performance Liquid Chromatograph ("LC) interfaced with a Perkin- W i l d l @ International, Ltd. - 35 - Proiect Number 454A-130 Elmer API IOOLC mass spectrometer (single quadrupole) operated in selective ion monitoring (STM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographc separations were achieved using a Keystone PRISM RP column (30 nun x 1.5 mm, 3 prn particle size) fitted with a Keystone Javelin C ~GSuard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1. Primary and Secondary Stock Solutions All primary and secondary stock preparations were adjusted for the purity of the analytical standard (97.90%). A 10.0 mg a.i./mL primary stock solu.tion of PFBS in methanol was prepared by weighmg 1.0243 g of the analytical standard and bringing to final volume with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) of I'FBS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 5050 methanol: NANOpure@water by appropriate dilutions of the 10.0 mg a.i./L stock solution of PFBS in methanol. The calibration standards of PFBS, ranging in concentration from 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end of each run,and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms of low and hgh calibration standards are presented in Figures 3 and 4, respectively. Limit of Quantitation The method limit of quantitation (LOQ) for these analyses was set at 20.0 mg a.i./L calculated as the product of the lowest calibration standard analyzed (:0.0100mg a.i./L) and the dilution factor of the matrix blank samples (2000). WildlifeInternational, Ltd. - 36 - Project Number 454A-130 Matrix Blank and Fortification Samples Eight matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5 . Freshwater was either fortified with the appropriate PFBS stock solution in methanol (30.0 mg a.i./L), or directly fortified (i.e. without use of camer solvent) with PFBS (400 and 2500 mg a,i./L). Direct fortifications were required for the higher fortificatioin levels due to the limitation in preparing a sufficiently concentrated methanol stock of PFBS. The fortified samples were analyzed concurrently with the test samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean overall procedural recovery of 96.394. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-130-9, nominal concentration of 500 mg a.i./L in freshwater. First Initial Volume: 0.100 mL Calibration curve equation: First Final Volume: 10.0 mL Slope: 10184392 Second Initial Volume: 0.100 mL Intercept: 21932.54 Second Final Volume: 25.0 mL Curve regression weighted llx Dilution Factor: 25000 PFBS Peak Area: 221526 eak area - (y-intercept) PFBS (mg a.i./L) measured at ins-trument= slope PFBS (mg a.i./L) in sample = PFBS measured at instrument (mg a.i./L) x dilution factor -- 221526 - 21932.54 25000 10 184392 = 489.9 Wildlife International. Ltd. -37- Project Number 454A-130 Percent of Nominal Concentration = - PPFFBBSS((mmggaa.i..i/.L/L))innosmaminpalle - - 48-9.9 x 100=98.0% - 500 Calculated with HPLCNS instrument s o h a r e : MacQuan, version 1.6. RESULTS Sample Analysis Freshwater samples were collected from a senu-static life-cycle toxicity study with the cladoceran (Daphnia magna) at test initiation, February 7, 2001 (Day 0), on February 9, 2001 (Day 2 - new solutions), on February 12, 2001 (Day 5 - old solutions), on February 16, 2001 (Day 9 - new solutions), on February 19, 2001 (Day 12 - old solutions), on February 23, 2001 (Day 16 - new solutions), on February 26, 2001 (Day 19 - old solutions) and at test termination on February 28, 2001 (Day 21 - old solutions). The measured concentrations of PFBS in the samples collected at initiation of exposure of the test organisms (Hour 0) ranged from 90.7 to 98.0% of the nominal concentrations (Table 4). Samples collected at Day 2, Day 5, Day 9, Day 12, Day 16 and Day 19 had measured concentration ranges of 94.0 to 101%, 101 to 109%, 88.7 to 96.5%, 92.4 to 103%, 84.5 to 103% and 92.0 to 102% of nominal values, respectively. Samples collected at test termination (Day 2 1) had a measured concentration range of 85.9 to 105% of nominal values. A representative ion chromatogram of a test sample is shown in Figure 7. W i l d l i f eInternational, Ltd. - 38 - Project Number 454A-330 Table 1 Typical HPLCMS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perlun-Elmer API IOOLC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perlun-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone PRISM RP (30 nun x 1.5nun,3-pm particle size) GUARD COLUMN: Keystone Javelin CIScartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 200 j.lL/min 25% NANOpzlreO Water with 0.1% Ammonium Formate: 75% Methanol 5.0 pL PFBS PEAK RETENTION TIME: Approximately2.1 minutes PFBS MONITORED MASS: 299.0 a m u w i l d l i f e International, Ltd. - 39 - Project Number 454A-130 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-130-) MAB- 1 Sample ' Preparation Diite (Day) 0 Measured Concentration of PFBS (mg a.i./L) < LOQ MAB-2 2 < LOQ MAB-3 5 < LOQ MAB-4 9 < LOQ MAB-5 12 < LOQ MAB-6 16 < LOQ MAB-7 19 < LOQ MAB-8 21 < LOQ 1 Preparation Date refers to the Day during the defbtive test that these quality control samples were prepared concurrent with sampling of the test samples. 2 The limit of quantitation (LOQ) was 20.0 mg a.i../Lbased upon the product of the lowest calibration standard analyzed (0.0100 m a ) and the cl~lutionfactor of the matrix blank samples (2000). Wildlife I nternational, Ltd. -40- Project Number 454A-130 Talble 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A- 130-) MAS-1 MAS-4 MAS-7 MAS-10 MAS-13 MAS-16 MAS-19 MAS-22 Preparation Date (Day)' 0 2 5 9 12 16 19 21 Concentrations of PFBS (mg a..i./L) Fortified Measured2 30.0 31.6 30.0 31.0 30.0 29.8 30.0 30.6 30.0 30.0 30.0 29.5 30.0 31.2 30.0 31.1 Percent of Nominal2 105 104 99.3 102 100 98.4 104 104 MAS-2 0 400 390 97.5 MAS-5 2 400 376 94.0 MAS-8 5 400 348 87.1 MAS-1 1 9 400 36 1 90.3 MAS- 14 12 400 350 87.6 MAS-17 16 400 361 90.2 MAS-20 19 400 372 93.0 MAS-23 21 400 4 14 103 MAS-3 0 2500 2355 94.2 MAS-6 2 2500 2346 93.8 MAS-9 5 2500 2275 91.0 MAS-12 9 2500 2346 93.8 MAS-15 12 2500 2212 88.5 MAS-18 16 2500 2409 96.4 MAS-2 1 19 2500 2356 94.2 MAS-24 21 2500 248 1 99.2 Mean = 96.3 Standard Deviation = 5.63 CV = 5.84% N = 24 ' Preparation Date refers to the Day during the defhtive test that these quality control samples were prepared concurrent with sampling of the test samples. 2 Measured and Percent of Nominal values wen: calculated using MacQuan, version 1.6 software Manual calculations may vary slightly. Mean of duplicate redilutions (2399 and 2419 mg a.i./L) of original fortification. A dilution error occurred during original sample preparation. wild llfe I nternationaI, Ltd. .-41 - Project Number 454A-130 Table 4 Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-static Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-130-) 1 2 15 16 29 30 43 44 57 58 71 72 85 86 99 100 Sampling Time (Day) 0 0 2 2 5 5 9 9 12 12 16 16 19 19 21 21 PFBS Measured Concentration' (mg a.i./L) <LOQ' <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Percent o-f- Nominal` -_ 63 3 0 4 0 17 2 57.1 90.7 59.4 94.3 63.4 101 18 2 62.2 98.7 31 5 63.7 101 32 5 64.5 102 45 9 58.3 92.6 46 9 59.3 94.1 59 12 58.2 92.4 60 12 58.7 93.2 73 16 54.5 86.5 74 16 53.2 84.5 87 19 63.9 101 88 19 60.0 95.2 101 21 63.8 101 102 21 64.6 103 I Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. f i f i 2 The limit of quantitation (LOQ) was 20.0 mg a.i./L based upon the product of the lowest calibration w i l d l i f e International, Ltd. - 42 - Project Number 454A-130 Table 4 (Continued) Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-static Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i.L) 125 Sample Number (454A-130-) 5 6 ' 19 20 33 34 47 48 61 62 75 76 89 90 103 104 Sampling Time: (Day) 0 0 2 2 5 5 9 9 12 12 16 16 19 19 21 21 PFBS Measured Concentration' (mg a.i.L) 117 118 121 125 127 132 115 116 116 117 114 119 121 122 127 127 Percent of Nominal' 93.3 94.8 96.7 99.9 102 105 92.4 93.0 92.5 93.7 91.2 95.5 96.6 97.7 102 101 250 7 0 8 0 239 95.7 237 94.8 21 2 25 1 101 22 2 249 99.7 35 5 265 106 36 5 262 105 49 9 24 1 96.5 50 9 230 92.1 63 12 235 94.1 64 12 23 8 95.0 77 16 25 7 103 78 16 248 99.1 91 19 250 100 92 19 248 99.3 105 21 25 1 100 106 21 250 100 1 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. w i l dIlfe InternationaI, Ltd. - 43 - Proiect Number 454A-130 Table 4 (:Continued) Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-static, Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) 500 Sample Number (454A-130-) 9 10 ' 23 24 37 38 51 52 65 66 79 80 93 94 107 108 Sampllng Time (Day 1 0 0 2 2 5 5 9 9 12 12 16 16 19 19 21 21 PFBS Measured Concentration' (mg a.i./L) 490 484 498 498 544 537 474 482 507 5 16 497 477 503 507 525 5 00 Percent of Nominal' 98.0 96.7 99.5 99.7 109 107 94.9 96.4 101 103 99.5 95.4 101 101 105 100 1000 11 0 973 97.3 12 0 980 98.0 25 2 1004 100 26 2 1007 101 39 5 1055 106 40 5 1067 107 53 9 956 95.6 54 9 946 94.6 67 12 999 99.9 68 12 1002 100 81 16 973 97.3 82 16 960 96.0 95 19 1014 101 96 19 1022 102 109 21 964 96.4 110 21 99 1 99.1 1 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations m w g h t l y. wiIdI(fe I nternationaI, Ltd. - 44 - Project Number 454A-130 Table 4 (Continued) Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-Static:Life-Cycle Toxicity Test Nominal Test Sample Sampling PFBS Measured Percent Concentration Number Time Concentration' of (mg a.i./L) (454A-130-) (Day) (mg a.i./L) Nominal' 2000 13 0 1893 94.7 14 0 1955 97.8 27 2 1879 94.0 28 2 1908 95.4 41 5 2014 101 42 5 2078 104 55 9 1774 88.7 56 9 1835 91.8 69 12 1894 94.7 70 12 1962 98.1 83 16 1805 90.2 84 16 1836 91.8 97 19 1841 92.0 98 19 1885 94.2 111 21 1719 85.9 112 21 1734 86.7 1 Measured and Percent of Nominal values werl: calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. W i l d l t f eInternational, Ltd. - 45 - Proiect Number 454A-130 METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHWATER Prepare matrix fortification samples in freshwater as follows: For target PFBS concentrations I 200 mg a.i./L, fortify freshwater with the appropriate stock solution of PFBS. For target PFBS concentrations > 200 mg a.i./L, prepare by weighing the requisite amount of PFBS test substance on an analytical balance and transferring lrectly into a Class A volumetric flask partially filled with freshwater. Rinse weighing paper and the sides of the flask with repeat freshwater rinses. Swirl the flask to dissolve the test substance and then bring to final volume with freshwater. Sonicate as appropriate and mix with several repeat inversions. The matrix blank is unfortified freshwater. Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFBS LCMS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure@ water dilution solvent. Add the appropriate volume: of sample and bring to volume with ddution solvent. Perform secondary dilutions as necessary. Process matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. .L Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFBS in freshwater. WildlifeInternational, Ltd. - 46 - Proiect Number 454A-130 Area 50000040000P 30000020000 10000 Concentration pg a.i./L Figure 2. A typical ealibration curve for PFBS. Slope = 10184392; Intercept = 21932.54; r = 0.99818, w i l d 1(fe I ntemational, L td. - 47 - Proiect Number 454A-130 PFBS-1 STD 0.0100 mg r.lJL 5216-002C-6 2.97 in 1 period pws No Internal Standard Use Area 1: 2.96 Q1 MI, 178 scans 299.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 20.0 20.0 3 6 100 10 1 2.16 Area 124515 Height 14822 Start Time 2.03 End Time 2.43 integration Width 0.40 Retention Time Integration Type -2.16 A 88 Thu, Feb 8, 2001 19:12 'OOl intensity: 120000 cps 129 A 0 :I 1 61 91 121 151 scan 0.52 1.02 1.52 2.03 2.53 Time Figure 3. A representative ion chromatogram of a low-level (0.0100 mg a.i.L) PFBS standard. wildlife International. Ltd. - 48 - Project Number 454A-130 PFBS-5 STD 0.0500 mg a.l.lL 521 6-002C-10 2.97 in 1 period RBS No Internal Standard Use Area 1: 2.96 Q1 MI, 299.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 178 scans 20.0 20.0 3 6 100 10 1 2.16 Area 548302 Height 5721 2 Start Time End Time Integration Width Retention Time Integration Type 2.01 2.68 0.67 -2.16 A BE Thu, Feb E, 2001 19:28 0 9 ~~ ~ intensily: 120000 cps 4;;, 0 24 129 30 20 10 0 :3 1 61 91 121 151 scan '3.52 1.02 1.52 2.03 2.53 Time Figure 4. A representative ion chromatogram of a high-level (0.0500 mg a.i./L) PFBS standard. wild lcfe I ntemationa1, L td. - 49 - Project Number 454A-130 PFBS-7 454A-130- MAB-1 Thu, Feb 8, 2001 19:37 2.97 in 1 period pws No Internal Standard Use Area 1: 2.96 Q1 MI, 178 s a n s 299.0 Noise Thres. 20.0 Quanl Thres. Min. Width Mult. Width Base. Width RT Win. ( s e a ) Smooth 20.0 3 6 100 10 1 Expected RT 2.1 6 Area 0 Height 0 Start Time 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type :! 2 0J, 9,16, 38 31 0.52 56 61 1.02 80 91 1.52 intensity: 120000 cps 1 115 129 121 2.03 162 151 2.53 Scan Time Figure 5. A representative ion chromatogram oFa matrix blank sample (454A-130-MAJ3-1). The arrow indicates the retention time of PFBS. WiId 1(fe I nternationaI, L td. -50- Proiect Number 454A-130 PFBS-9 454A-130- MAS-2 Thu, Feb 8, 2001 19:45 2.97 in 1 period pws No Internal Standard Use Area 1: 2.96 ai MI, 178 scans 299.0 Noise Thres. 20.0 Quant Thres. Min. Width 20.0 3 Mull. Width 6 Base. Width 100 FIT Win. (sea) 10 Smooth 1 Expecled FIT 2.16 Area 419242 Height 43383 Start Time 2.03 End Time 2.63 Integration Width 0.60 Retention Time Integration Type 2.18 A - EB intensity: 120000 cps 120 n 0.52 1.02 1.52 2.03 2.53 Time Figure 6 . A representative ion chromatogram of a matrix fortification sample (454A-130-MAS-2, nominal PFBS concentration of 400 mg a.i./L, dilution factor = 10000~). WildlifeInternational, Ltd. -51 - Project Number 454A-130 PFBS-21 9 454A-130- Thu, Feb 8, 2001 20:34 2.97 in 1 period pws No Internal Standard Use Area 1: 2.96 Q1 MI, 178 scans 299.0 Noise Thres. 20.0 Quant Thres. 20.0 Min. Width 3 Mutt. Width 6 Base. Width 100 RT Win. (sea) 1 0 Smooth 1 Expected RT 2.16 Area 221526 Height 24774 Start Time 2.04 End Time 2.50 Integration Width 0.45 Retention Time Integration Type 2.18 A - BB g1100'1 8 intensity: 120000 cps 130 A Figure 7. A representative ion chromatogram of a test sample (454A-130-9, nominal PFBS concentration of 500 mg a.i./L, dlution factor = 25000~). - 52 - Project Number 454A-130 Appendix 4 Cumul tive Percent Mortalit. and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Initial Test Concentration Number Number (mg a.i.k) EXPOSed Immobile Negative Control A 1 0 B 1 0 C 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 I 1 0 J 1 0 Day 0 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN 1AN IAN IAN IAN IAN IAN IAN 60 A 1 0 0 IAN B 1 0 0 1" C 1 0 0 IAN D 1 0 0 1AN E 1 0 0 1AN F 1 0 0 IAN G 1 0 0 IAN H 1 0 0 IAN I 1 0 0 IAN J 1 0 0 IAN 121 A 1 0 0 IAN B 1 0 0 IAN C 1 0 0 1AN D 1 0 0 IAN E 1 0 0 IAN F 1 0 0 1" G 1 0 0 1AN H 1 0 0 IAN I 1 0 0 IAN ' J 1 0 0 IAN Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 1 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1AN 1AN 1" IAN 1AN IAN IAN IAN IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 1" 0 0 1" 0 0 IAN 0 0 1" 0 0 IAN 0 0 1AN 0 0 IAN 0 0 1" 0 0 1AN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 IAN 0 0 IAN . Number Immobile 0 0 0 0 0 0 0 0 0 0 Day2 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN 1AN 1AN IAN IAN IAN 1" IAN 1AN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 1AN 0 0 1" 0 0 1AN 0 0 1" 0 0 1AN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 1AN 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 1AN 1AN IAN 1AN IAN IAN IAN IAN 1" 1AN 0 0 1AN 0 0 1AN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1" 0 0 1AN 0 0 IAN 0 0 1" 0 0 1AN 0 0 1AN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN - 53 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Initial Replicate Number Exposed A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 1 60 A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 1 121 Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 4 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ' J 1 0 0 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects IAN IAN 1AN 1AN IAN IAN 1AN IAN IAN IAN 1AN IAN IAN 1" IAN IAN 1AN 1AN 1AN IAN IAN 1AN 1AN 1AN IAN 1AN IAN IAN IAN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day5 . Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1AN IAN IAN 1AN IAN 1AN 1AN IAN IAN IAN 1AN 1AN IAN IAN 1AN 1AN IAN IAN IAN IAN 1AN 1AN 1AN 1AN IAN 1AN 1AN IAN 1AN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 6 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1AN 1AN 1AN 1AN IAN 1AN IAN IAN IAN IAN 1AN IAN IAN IAN 1AN IAN 1" IAN 1AN IAN IAN 1AN 1AN IAN IAN 1AN 1AN IAN IAN - 54 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Initial Test Concentration (mg a.i./L) Replicate Number Exposed Negative Control A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 1 60 A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 1 121 Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 7 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 J 1 0 0 Observed Effects: AN =Appeared Normal, D = Discoloration (Pale), G = Small Effects 1AN IAN 1" 1AN 1" IAN 1" 1AN 1AN IAN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1AN IAN 1AN 1AN 1AN IAN 1AN IAN 1AN 1AN IAN 1AN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 8 Cumulative Dead 0 0 0 0 0 0 '0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1AN IAN 1AN 1" 1AN 1" IAN IAN 1AN IAN 1AN 1AN IAN IAN IAN IAN IAN IAN IAN 1AN IAN 1AN 1AN 1AN IAN 1AN 1AN 1AN 1" IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 9 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN 1AN 1AN IAN IAN 1AN 1AN IAN IAN IAN IAN 1AN 1AN IAN 1AN IAN IAN IAN 1" 1AN 1AN 1AN IAN 1" IAN IAN IAN IAN IAN - 55 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Initial Test Concentration (mg a.i./L) Replicate Number Exposed Negative Control A 1 Number Immobile 0 Day 10 Cumulative Dead 0 60 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 121 ' Observed Effects A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 AN = Appeared Normal, D = Discoloration(Pale), G = Small Effects IAN IAN IAN IAN IAN IAN 1" IAN IAN IAN IAN IAN IAN IAN 1" 1AN IAN 1AN 1" 1AN 1AN 1AN IAN 1" IAN 1AN 1AN 1" 1AN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 0 0 0 0 0 0 0 0 0 .O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN 1AN 1AN IAN IAN 1" IAN IAN IAN 1AN IAN 1" 1AN IAN IAN 1" 1AN 1" IAN IAN IAN IAN 1AN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 12 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1AN IAN IAN IAN IAN IAN 1AN IAN IAN IAN 1AN IAN 1" IAN IAN IAN 1AN IAN 1AN 1AN IAN IAN IAN IAN IAN 1AN 1AN IAN IAN IAN -56- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Initial Replicate Number Exposed A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 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 60 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 121 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' J 1 0 0 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects IAN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1AN 1AN IAN 1AN IAN 1AN IAN 1AN 1AN IAN IAN IAN 1AN 1AN IAN 1AN 1AN IAN IAN 1AN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day14 . Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN 1AN 1AN IAN IAN IAN IAN 1AN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN 1AN 1" IAN 1AN IAN IAN IAN 1AN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN ~ 1D 1AN IAN IAN IAN IAN IAN IAN 1AN IAN IAN 1AN IAN IAN IAN IAN IAN 1AN IAN 1AN 1AN 1AN 1AN 1AN IAN IAN IAN 1AN IAN - 57 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Oreanism: DilutiolWater: 3M Corporation PFBS Cladoceran.DaDhnia ma"ma Well Water' ' Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed Negative Control A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 16 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 60 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 121 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' J 1 0 0 Observed Effects: AN = Appeared Normal, D = Discoloration(Pale), G = Small Effects 1"-- 1AN 1AN 1" 1AN IAN 1AN IAN IAN 1AN 1AN 1AN 1AN IAN IAN 1AN 1AN 1" 1" IAN IAN 1AN 1" IAN 1AN 1AN 1AN 1AN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 17 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects I A-N IAN 1AN IAN 1" 1" 1" IAN IAN IAN 1AN IAN IAN IAN IAN IAN IAN IAN 1AN IAN IAN IAN 1AN 1" IAN 1AN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects I A--N IAN 1AN IAN IAN IAN IAN IAN 1" 1AN IAN IAN 1AN 1AN 1AN IAN IAN 1" 1" 1AN IAN IAN IAN IAN IAN IAN IAN IAN 1AN Project Number 454A-100 -58- Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationD a p h d s Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Initial Replicate Number Exposed A 1 Number Immobile 0 0 0 0 0 0 0 0 0' 0 Day 19 Cumulative Dead 0 60 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 121 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' J 1 0 0 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1AN - 1AN 1AN IAN IAN IAN IAN IAN 1AN 1AN IAN IAN IAN 1AN 1" 1AN IAN 1AN 1AN 1AN 1" 1" 1AN IAN 1AN 1" 1" 1AN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day20. Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN -- IAN 1" IAN 1" IAN IAN 1AN IAN IAN IAN 1AN 1AN IAN IAN 1AN 1AN 1AN 1AN 1AN 1" 1AN IAN IAN IAN 1" 1AN 1AN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 21 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN -- IAN 1AN 1" IAN 1AN IAN 1D 1" 1" 1AN IAN IAN IAN IAN IAN 1AN 1AN 1" IAN IAN IAN IAN IAN IAN 1AN IAN IAN IAN - 59 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-Generation Daphnids Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Cladoceran,Daphnia magna Dilution Water: Well Water Mean Measured Initial Test Concentration (mg a.i.n) Number Number Immobile 247 A 1 0 B 1 0 C 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 I 1 0 J 1 0 Day 0 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN 502 A 1 0 0 IAN B 1 0 0 IAN C 1 0 0 IAN D 1 0 0 IAN E 1 0 0 IAN F 1 0 0 IAN G 1 0 0 IAN H 1 0 0 IAN I 1 0 0 IAN J 1 0 0 IAN 995 A 1 0 0 IAN B 1 0 0 IAN C 1 0 0 IAN D 1 0 0 IAN E 1 0 0 IAN F 1 0 0 IAN G 1 0 0 IAN H 1 0 0 IAN I 1 0 0 IAN ' J 1 0 0 1 AN Observed Effects. AN = Appeared Normal, D = Discoloration (Pale), G = Small Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 1 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1" 1" 1AN IAN IAN IAN IAN 1" IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 1" 0 0 1AN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 IAN 0 0 1" 0 0 IAN 0 0 IAN ~ , Number Immobile 0 0 0 0 0 0 0 0 0 0 Day2 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN 1AN IAN IAN 1AN 0 0 IAN 0 0 1AN 0 0 1" 0 0 IAN 0 0 IAN 0 0 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 1AN 0 0 IAN 0 0 1" 0 0 IAN 0 0 IAN 0 0 1 AN ~ 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 1" IAN IAN IAN IAN 1" 1AN 1AN IAN IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 1" 0 0 1AN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 IAN - 60 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed 247 A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 4 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 502 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 995 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' Observed Effects: J 1 0 0 AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects IAN 1AN 1AN 1AN 1AN 1AN IAN 1AN IAN IAN 1AN 1AN 1AN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1AN 1" 1AN IAN 1AN IAN 1AN 1AN 1AN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day5 . Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN 1AN IAN 1AN IAN IAN 1AN 1AN 1" 1AN IAN IAN 1AN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1AN 1AN 1AN 1AN IAN IAN 1AN IAN IAN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 6 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN 1AN 1" 1AN IAN IAN IAN 1" IAN IAN 1AN IAN IAN IAN IAN IAN 1AN 1AN 1AN 1AN 1D 1D 1AN IAN 1D 1D IAN 1AN I A.-N ~ IAN -61 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Cladoceran, Daphnia magna Dilution Water: Well Water Mean Measured Initial Test Concentration (mg a.i./L) Replicate Number Exposed 247 A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 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 0 502 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 995 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 ' I 1 0 0 J 1 0 n Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects IAN IAN IAN 1AN 1AN 1" 1AN IAN IAN IAN 1AN 1AN 1" 1AN 1AN 1AN 1AN IAN IAN 1" 1D 1D 1" 1AN 1D 1D IAN 1AN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day8 , Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 EfFects 1AN 1" 1AN 1AN IAN 1AN 1AN 1AN IAN IAN 1AN 1" IAN 1AN 1AN 1AN 1AN 1AN 1" IAN 1D 1D IAN 1" 1D 1D IAN 1AN 1AN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 9 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 Effects IAN IAN IAN IAN 1AN IAN 1AN 1AN 1AN IAN IAN 1AN IAN 1AN IAN 1" IAN 1AN IAN 1AN __ 1D IAN 1AN 1D 1D IAN IAN IAN - - 62 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationD a p h d s Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Initial Test Concentration (mg a.i./L) Replicate Number Exposed 241 A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 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 0 502 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 995 A 1 0 1 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 1 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' J 1 0 1 Observed Effects: AN =Appeared Normal, D = Discoloration (Pale), G = Small Effects 1AN 1AN IAN IAN IAN IAN IAN 1AN IAN 1AN IAN 1AN 1AN IAN 1AN 1AN 1AN 1AN IAN 1AN -- 1D IAN IAN -_ 1 D,G 1" IAN 1AN -- Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects 1AN 1" 1AN 1AN IAN IAN 1AN 1AN IAN IAN 1AN IAN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1AN __ 1D IAN 1"-- 1 D,G IAN IAN IAN -- Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 12 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects IAN IAN IAN 1AN IAN IAN IAN IAN IAN IAN IAN IAN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1" - 1D IAN 1 _A_ N 1 D,G IAN IAN IAN - - 63 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i.L) 247 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Initial Replicate Number Exposed A 1 B 1 C 1 D 1 E 1 F 1 G 1 H 1 I 1 J 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 0 502 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 995 A 1 0 1 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 1 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' J 1 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1AN 1AN 1" 1AN IAN 1" IAN 1AN IAN IAN 1AN 1AN 1AN 1" 1AN 1" 1AN IAN IAN 1AN -- 1D 1AN 1AN -- 1 D,G 1AN IAN I A__ N Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day14. Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects 1AN IAN IAN 1AN 1AN 1AN 1AN 1AN IAN IAN 1AN 1AN 1AN 1AN 1AN 1AN 1AN IAN 1AN 1AN -- 1D 1AN 1AN -- 1 D,G IAN IAN IAN __ Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects 1AN 1AN 1AN 1AN IAN 1AN 1AN 1AN 1AN IAN 1" 1AN 1" 1AN 1AN 1AN IAN IAN 1AN 1" -- 1D 1AN 1AN -- 1 D,G IAN 1AN ID -- - 64 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Oreanism: 3M Corporation PFBS Cladoceran. DaDhnia m a m a Mean Measured Test Concentration (me a.i./L) 241 Replicate A Initial Number Exposed 1 Number Immobile 0 Day 16 Cumulative Dead 0 502 A 1 0 0 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 0 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 J 1 0 0 995 A 1 0 1 B 1 0 0 C 1 0 0 D 1 0 0 E 1 0 1 F 1 0 0 G 1 0 0 H 1 0 0 I 1 0 0 ' J 1 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration(Pale), G = Small Effects 1AN 1AN 1AN 1AN IAN IAN 1AN 1AN 1AN 1AN 1AN IAN 1AN 1AN IAN 1AN 1AN 1AN 1AN IAN I 1AN 1AN I A--N 1D 1AN 1AN 1D -- Number Immobile 0 Day 17 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 Effects 1AN 1AN IAN IAN 1AN 1AN IAN 1" IAN IAN IAN IAN IAN 1AN 1AN 1AN 1AN IAN 1AN IAN -- IAN 1AN I A__N IAN IAN IAN IAN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects 1.. A_ N 1" 1AN 1AN IAN IAN IAN IAN IAN IAN 1AN 1AN IAN IAN IAN IAN IAN IAN IAN 1AN - IAN 1AN IAN -_ IAN IAN IAN IAN - - 65 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) ~ ~~~~ 247 3M Corporation PFBS Cladoceran,Daphnia mugnu Well Water Initial Replicate ~~ A B C D E F G H I J Number Exposed 1 1 1 1 1 1 1 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 502 A 1 0 B 1 0 C 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 I 1 0 J 1 0 995 Day 19 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ' J 1 0 1 Observed Effects: AN = Appeared Normal, D = DiscolorationPale), G = Small Effects IAN IAN IAN IAN IAN 1" 1AN 1AN IAN IAN IAN IAN IAN IAN 1AN IAN IAN 1" 1AN 1" -- 1" IAN I A_-N 1D 1AN IAN I A__N 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 1" 1" IAN IAN IAN IAN 1AN IAN 1AN IAN 0 0 1" 0 0 1AN 0 0 1" 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 lAN 0 0 1AN 0 0 IAN - IAN 1AN IAN -- ID 1AN 1" IAN 0 1 - 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 1AN 1AN IAN 1AN IAN 1" 1" 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 IAN 0 0 IAN - IAN IAN IAN _- 1D 1" IAN 0 1 I A-N - 66 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Initial Day 0 Test Concentration (mg a.i./L) Number Number Cumulative Immobile Dead Effects 1876 A 1 0 0 IAN B 1 0 0 IAN C 1 0 0 IAN D 1 0 0 1" E 1 0 0 IAN F 1 0 0 1AN G 1 0 0 1AN H 1 0 0 1AN I 1 0 0 1AN ' J 1 0 0 IAN Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Number Immobile 0 0 0 0 0 0 . 1 0 0 0 Day 1 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1AN 1AN 1AN IAN IAN -- 1AN 1AN 1AN . Number Immobile 0 0 0 0 0 0 0 0 0 0 Day2 Cumulative Dead 0 0 0 0 0 0 1 0 0 0 Effects 1AN 1AN 1AN IAN IAN 1AN -- 1" IAN 1AN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 3 Cumulative Dead 0 1 0 0 0 0 1 0 0 0 Effects 1G __ 1G 1G 1G 1G -- 1G 1G 1G - 67 - Project Number 454A- 130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Initial Day 4 Test Concentration (mg a.i./L) Replicate Number Exposed Number hmobile Cumulative Dead 1876 A 1 0 0 1 0 0 0 0 1 0 0 ' J 1 0 0 Observed Effects: AN = Aooeared Normal. D = Discoloration (Pale). G = Small Effects 1-_G 1G 1G 1G 1G __ 1G 1G 1G Number Immobile 0 Day.5 Cumulative Dead 0 0 0 Effects 1__G 1G 1G 1G 1G __ 1G 1G 1G Number Immobile 0 Day 6 Cumulative Dead 0 0 0 Effects 1G -- IG 1G 1G 1G -- 1G 1G 1G - 68 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Initial Test Concentration (mg a.i.L) Replicate Number Exposed 1876 A 1 Number Immobile 0 Day 7 Cumulative Dead 0 ' J 1 0 0 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1G -- 1G 1G 1G 1G -- 1G 1G 1G Number Immobile 0 Day .8 Cumulative Dead 0 0 0 Effects 1G __ 1G 1G 1G 1G _- 1G 1G 1G Number Immobile 0 Day 9 Cumulative Dead 0 0 0 Effects 1G -- 1G 1G 1G 1G -- 1G 1G 1G - 69 - Project Number 454A- 130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed Number Immobile Day 10 Cumulative Dead 1876 A 1 0 1 B 1 1 C 1 0 D 1 0 E 1 0 F 1 0 G 1 1 H 1 0 I 1 0 ' Observed Effects: J 1 0 0 AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects --- 1G 1G 1G 1G -_ 1G 1G 1G Number Immobile 0 Day 11 Cumulative Dead 1 0 0 Effects -_ _- 1G - 1G 1G -- 1G 1G 1G Number Immobile 0 0 Day 12 Cumulative Dead 1 1 0 1 1 1 1 0 0 0 Effects __ -_ 1G -__ __ 1G 1G 1G Project Number 454A-130 - 70 - Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution- Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Initial Test Concentration (mg a.i.L) Number 1876 A 1 Number Immobile 0 Day 13 Cumulative . Dead 1 Effects -- Number Immobile 0 Day 14 Cumulative Dead 1 Effects - Number Immobile 0 Day 15 Cumulative Dead 1 Effects - -- 1G ' 1G J 1 0 0 1 D,G 0 1 -- Observed Effects: AN = Appeared Normal, D = Discoloration(Pale),G = Small - 1G 1G 0 1 - - 71 - Project Number 454A- 130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Duphniu mugnu Well Water Mean Measured Initial Day 16 Test Concentration (mg a.i./L) Replicate Number Exposed Number hmobile Cumulative Dead 1876 A 1 0 1 B 1 0 1 C 1 0 1 D 1 0 1 E 1 0 1 F 1 0 1 G 1 0 1 H 1 0 0 I 1 0 0 ' J 1 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration pale), G = Small Effects -_ --- __ __ -- 1G 1--G Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 1.7 Cumulative Dead 1 1 1 1 1 1 1 0 0 1 Effects --- - ---- 1G 1G - Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 1 1 1 1 1 1 1 0 0 1 Effects -- - - 1G 1-G - 72 - Project Number 454A- 130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-RelatedEffects' - First-GenerationDaphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration Initial (mg a.i./L) Replicate Exposed 1876 A 1 Number Immobile 0 Day 19 Cumulative Dead 1 Effects -- Number Immobile 0 Day 20 Cumulative Dead 1 Effects -_ Number Immobile 0 Day 21 Cumulative Dead 1 Effects -- ' J 1 0 1 -_ Observed Effects: AN = Appeared Normal, D = Discoloration pale), G = Small -- -_ 1 D,G 1 D,G 1 D,G 1 D.G 0 1 -_ 0 1 -- - 73 - Project Number 454A-130 Appendix 5 Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water 3M Corporation PFBS Cladoceran, Daphniamagna Well Water Mean Measured Test Concentration 0 2 5 7 (mg a.i./L) Replicate Negative Control A B 0 0 0 0 0 0 0 0 C 0 0 0 0 D 0 0 0 0 E 0 0 0 0 F 0 0 0 0 G 0 0 0 0 H 0 0 0 0 I 0 0 0 0 J 0 0 0 0 Day 9 12 14 16 19 21 0 31 31 14 19 0 9 27 26 - -- -_ 0 0 16 0 6 0 0 0 22 36 24 0 0 0 21 27 32 0 0 7 26 40 17 0 0 24 44 28 22 0 0 17 38 30 0 16 0 33 20 28 0 25 0 12 35 0 18 15 60 A 0 0 0 0 5 13 57 61 27 0 B 0 0 0 0 4 38 50 62 35 0 C 0 0 0 0 0 18 45 52 28 0 D 0 0 0 0 0 26 44 52 28 0 E 0 0 0 0 3 55 0 47 32 0 F 0 0 0 0 35 57 0 61 54 25 G 0 0 0 0 18 56 0 52 57 30 H 0 0 0 0 2 32 54 44 18 0 I 0 0 0 0 0 31 44 49 25 0 J 0 0 0 0 6 39 15 33 69 45 Total 95 62' 22 82 80 90 118 101 106 80 % (i SD) = 86 (* 27) 163 189 143 150 137 232 213 150 149 - 207 % (t SD) = 173 (* 34) - 74 - Project Number 454A-130 Appendix 5 (Continued) Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water Mean Measured Test 3M Corporation PFBS - Cladoceran,Daphnia magno Well Water Concentration 0 2 5 7 (mg a.i./L) Replicate 121 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0 D 0 0 0 0 E 0 0 0 0 F 0 0 0 0 G 0 0 0 0 H 0 0 0 0 I 0 0 0 0 J 0 0 0 0 Day 9 12 14 16 19 21 0 15 27 20 0 9 0 18 46 58 30 0 0 12 31 30 10 0 0 74 0 48 51 0 5 50 0 57 55 27 4 55 0 43 33 9 0 74 0 53 34 33 4 39 57 57 51 0 0 34 46 45 29 0 0 16 41 0 43 17 247 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0 D 0 0 0 0 E 0 0 0 0 F 0 0 0 0 G 0 0 0 0 H 0 0 0 0 I 0 0 0 0 - J 0 0 0 0 'This value excluded from calculation of the mean due to adult death. 8 11 43 47 46 0 6 35 41 32 35 0 0 17 21 40 28 0 2 24 40 45 43 53 10 48 0 41 43 16 21 35 0 27 40 37 22 29 0 45 50 32 8 54 0 40 50 33 0 24 41 50 40 0 7 39 54 0 57 52 Total 71 152 83 173 194 144 194 208 154 117 2 (t SD) = 149 (* 47) 155 155 106 207 158 160 178 185 155 209 - 75 - Project Number 454A-130 Appendix 5 (Continued) - Sponsor: Test Substance: Test Organism: Dilution Water Mean Measured Test 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Neonate Production Day - Concentration 0 2 5 7 9 12 14 16 19 21 (mg a.i.iL) Replicate 502 A 0 0 0 0 0 12 1 41 42 0 B 0 0 0 0 0 22 40 26 25 0 C 0 0 0 0 0 11 22 27 29 0 D 0 0 0 0 6 25 39 26 41 0 E 0 0 0 0 0 0 0 27 35 0 F 0 0 0 0 0 0 0 10 22 25 G 0 0 0 0 0 4 13 19 25 2 H 0 0 0 0 0 20 35 30 30 0 1 0 0 0 0 0 0 3 6 33 41 J 0 0 0 0 0 10 32 0 8 25 Total 96 113 89 137 62 57 63 115 83 75 X (t SD) = 89 (*26) 995 A 0 0 0 0 -- -- -- -- _- 0' B 0 0 0 0 0 0 0 0 19 35 C 0 0 0 0 0 6 15 9 26 56 D 0 0 0 0 0 4 21 8 18 51 E 0 0 0 0 0 I -- -- - 0' F 0 0 0 0 0 0 0 0 12 12 G 0 0 0 0 0 5 12 7 4 28 H 0 0 0 0 5 13 19 24 34 95 I 0 0 0 0 0 3 0 5 13 21 J 0 0 0 0 -_ 0' c ii(t SD) = 43 (i28) - 76 - Project Number 454A- 130 Appendix 5 (Continued) Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration 0 2 5 7 (mg a.i./L) Replicate 1876 A 0 0 0 0 Day 9 12 14 16 19 21 0 -- 0 0 0 0 -- -- 0 5 0 0 0 0 'This value excluded from calculation of the mean due to adult death. Total 0' 0' 0' 0' O1 0' O1 24 4 0' wiIdlife I nternationaI , Ltd. - 77 - Proiect Number 454A-130 Appendix 6 Length and Dry Weight of Surviving First-Generation D a p h d s Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Individual Length (-1 . 4.25 4.05 4.30 3.95 4.40 4.30 4.40 4.00 4.00 Mean Length (mm SD) 4.18 ( 2 0.182) 60 4.90 4.89 (t 0.111) 4.90 4.90 4.65 4.90 5.00 5.00 4.80 5.00 4.80 121 4.75 4.78 (t 0.207) 4.80 4.50 4.60 4.85 5.00 4.65 4.90 4.55 5.15 Individual Dry Weights (mg) 0.7 1.o 0.9 0.9 1.4 0.5 0.7 0.6 0.5 1.7 1.3 1.4 1.2 1.4 0.9 0.6 1.4 1.3 1 .o 0.9 1.5 1.9 1.6 0.9 1.1 0.9 1.2 1.1 1.o Mean Dry Weights (mg t SD) 0.80 (2 0.29) 1.22 (t 0.31) 1.21 (t 0.34) wildlife International, Ltd. - 78 - Project Number 454A- 130 Appendix 6 (Continued) Length and Dry Weight of Surviving First-Generation D a p h d s Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i.4,) 247 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Individual Length , 4.75 4.75 4.60 4.65 5.00 4.35 4.80 5.00 4.75 5.00 Mean Length (mm ?r SD) 4.77 (2 0.206) 502 4.35 4.26 (k 0.480) 4.40 4.30 4.25 4.45 2.95 4.20 4.65 4.60 4.40 Individual Dry Weights (mg) 1.o 1.2 1.1 0.8 0.9 0.8 0.8 0.9 1.2 0.9 1.o 0.9 1.o 1.1 1.o 0.6 0.9 0.9 0.7 0.7 995 4.10 3.69 (k0.352) 0.7 3.65 0.7 3.60 0.4 3.30 0.4 3.65 0.3 4.20 0.6 3.30 0.5 1876 2.75 2.68 (kO.106) 0.1 2.60 0.1 Mean Dry Weights (mg k SD) 0.96 (k 0.16) 0.88 (2 0.16) 0.51 (t0.16) 0.10 (tO.0) wildlife International. Ltd. - 79 - Proiect Number 454A-130 Appendix 7 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the experimental start and termination dates, test concentrations and test substance number. 2. The protocol was amended to correct the analytical sampling schedule. 3. During the second-generation exposure, neonates were observed at 2 , 2 4 and 48 hours. 4. Treatment groups were indiscriminately positioned. 5. There were two instances where the continuous temperature recorder reached 18C. wiId1(fe Internationa1, Ltd. - 80 - / Project Number 454A-130 Appendix 8 Personnel Involved in the Study The followingkey Wildlife International Ltd. personnel were involved in the conduct or management of ths study: 1. Henry 0.Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Raymond L. Van'Hoven, Ph.D., Scientist 4. Cary A. Sutherland, Laboratory Supervisor 5. Kurt R. Drottar, Senior Biologst 6. Susan T. Platania, Biologist i