Document LoXy3VMnZYa8dLjqbQnjoxJk3

AR1U-00^3 PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW {Pimephales promelas) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-102 3M LAB REQUEST NO. U2723 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 and OECD Guideline 203 AUTHORS: Kurt R. Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: December 4,1998 STUDY COMPLETION DATE: July 14, 1999 AMENDED REPORT DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 43 000543 AMENDED W il d l if e In t e r n a t io n a l ltd. 2- - PROJECT NO.: 454A-102 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-102 STUDY COMPLETION: July 14,1999 AMENDED REPORT: April 26,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792,17 August 1989; OECD Principles of Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data -are being maintained in the 3M archives. The test substance is beipg recharacterized in accordance with GLP. The stability o f the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R. Drottar Senior Biologist CA DATE SPONSOR APPROVAL: A~&-. (LpdJk_____ Sponsor 7) o DATE 000544 AMENDED W i l d l i f e In t e r n a t i o n a l Ltd . -3 - PROJECT NO.: 454A-102 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Parts 160 and 792,17 August 1989; OECD Principles ofGood Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Test Initiation M atrix Fortification Preparation W ater Chemistry M easurem ents Biological Data and Draft Report Analytical Data and Draft Report Final Report Amended Report DATE CONDUCTED: February 11,1999 February 15,1999 February 15,1999 February 17,1999 M arch 17,1999 M arch 2 2 -2 4 ,1 9 9 9 July 14,1999 April 19 and 20,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 11,1999 February 11,1999 February 15,1999 February 16,1999 February 15,1999 February 18,1999 February 17,1999 February 19,1999 M arch 17,1999 M arch 24,1999 M arch 2 4 ,1 9 9 9 ' July 14,1999 A pril 20,2000 M arch 24,1999 July 14,1999 A pril 24,2000 Timothy A. Springer, Ph.D. Manager, Regulatoiy and Technical Support DATE 000545 AMENDED W il d l if e In t e r n a t io n a l ltd. -4 - REPORT APPROVAL PROJECT NO.: 454A-102 SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas) v WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-102 STUDY DIRECTOR Kurt R Drottar Senior Biologist MANAGEMENT: Henry O.vKmeger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants DATE M e? DAT- 000546 AMENDED W il d l if e In t e r n a t io n a l ltd. -5 - PROJECT NO.: 454A-102 TABLE OF CONTENTS Title/Cover Page......................................................................................................................................................1 Good Laboratoiy Practice Compliance Statement................................................................................................ 2 Quality Assurance Statement.................................................................................................................................3 Report Approval............................................................................................................. .'...................................... 4 Table o f Contents................................................................................................................................................... 5 Summary...................................................................................................................................... Introduction.............................................................................................................................................................8 O bjective.................................................................................................................................................................8 Experimental Design............................................................................................................................ ......:..........8 Materials and Methods........................................................................................................................................... 9 Results and Discussion........................................................................................................................... C o n clu sio n s........................................................................................................................................................... 13 R eferen ces................................................................................................................................... 12 1 TABLES Table 1 - Summary o f Analytical Chemistry D ata............................................................................................. 15 Table 2 - Temperature, Dissolved Oxygen and pH o f Water in the Test Chambers........................................ 16 Table 3 - Cumulative Percent Mortality and Treatment-Related Effects................................................... 17 Table 4 -LC 50 Values................................................................................................................................... 18 000547 W il d l if e In t e r n a t io n a l ltd. 6- - PROJECT NO.: 454A-102 TABLE OF CONTENTS - Continued - FIGURE Figure 1. Concentration-Response Curve (96-Hour D ata)............................................................................. 19 APPENDICES Appendix I - Specific Conductance, Hardness, Alkalinity, and pH o f Well Water Measured During the 4-Week Period Immediately Preceding the T est..................................20 Appendix II - Analyses o f Pesticides, Organics, M etals and Other Inorganics in W ildlife International Ltd. Well W ater.................................................... .......................... 21 Appendix H I- The Analysis ofPFOS in Freshwater in Support o f Wildlife International Ltd. Project No.: 454A -102............................................... ............................. 22 Appendix IV - Changes to Protocol...............................................'.................................................................. 40 Appendix V - Personnel Involved in the Study.............................................................. . .............................. 41 Appendix VI - Report Amendment................................................................ ..................................................42 000548 AMENDED W i l d l i f e In t e r n a t io n a l ltd. -7- PROJECT NO.: 454A-102 B SPONSOR: SPONSOR'S r e p r e s e n t a t iv e : LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation M s. Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: I LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: 4 54A -102 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas) Negative Control, 3 .3 ,5 .6 ,9 .5 ,1 7 and 28 mg a.i./L Experimental Start - February 15,1999 Biological Termination - February 19,1999 Experimental Termination - February 19,1999 96 Hours Fathead Minnow (.Pimephales promelas) Wildlife International Ltd. cultures Easton, Maryland 21601 AGE OF TEST ORGANISMS: MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: w e ig h t (g): t o t a l LENGTH (m m ): Juveniles Mean = 0 .3 6 ; Mean = 35 ; Range = 0.21 to 0.49 Range = 30 to 38 96-h o u r LC50: 95% CONFIDENCE LIMITS: NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: 9.5 mg a.i./L 8.0 and 11 mg a.i./L 3.3 mg a.i./L 3.3 mg a.i./L 000549 AMENDED W il d l if e In t e r n a t io n a l ltd. - 8- PROJECT NO.: 454A-102 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from February 15 to February 19,1999. Raw data generated by Wildlife International Ltd. and a copy o f the final report are filed under Project Number 454A-102 in archives located on the Wildlife International Ltd. site. OBJECTIVE The objective o f this study was to evaluate the acute toxicity o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the fathead minnow, Pimephalespromelas, during a 96-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Fathead minnows were exposed to a geometric series o f five test concentrations and a negative (dilution water) control. Two replicate test chambers were maintained in each treatment and control group, with 10 fathead minnows in each test chamber for a total of 20 fathead minnows per test concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results o f an exploratory range finding toxicity test. Nominal test concentrations selected were 3.6, 5.9, 9.9, 16 and 27 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples oftest watercollected from each treatment and the control group at the beginning o f the test, at approximately 48 hours, and at test term inatioa Fathead minnows were indiscriminately assigned to exposure chambers at test initiation. Observations of mortality and other clinical signs o f toxicity were made at approximately 2.5,24 ,4 8 ,7 2 and 96 hours after test initiatioa Cumulative percent mortality observed in the treatment groups was used to estimate or calculateLC50 values at 2 4 ,4 8 , 72 and 96 hours. The no mortality concentration and the no-observed-effect-concentration (NOEC) were determined by visual interpretation of the mortality and clinical observation data. 000550 AMENDED W il d l if e In t e r n a t io n a l ltd. -9 - PROJECT NO.: 454A-102 MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Static Acute Toxicily Test with the Fathead Minnow (Pimephalesprometas)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1075 (1): OECD Guideline for Testing o f Chemicals 203: Fish, Acute Toxicity Test (2); and ASTM Standard E729-88a,v Standard Guide fo r Conducting Acute Toxicity Tests with Fishes, M acroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on October 29,1998 and was assigned Wildlife International Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity o f 98.9%, and an expiration data o f2008. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated March 9,2000 indicated a purity o f 90.49%. The test substance was stored at ambient room temperature. Preparation o f Test Concentrations Nominal test concentrations were 3 .6 ,5 .9 ,9 .9 ,1 6 and 27 mg a.i./L, based on a test substance purity of 90.49%. All materials which came into contact with the test substance during preparation o f test concentrations were constructed o f plastic or stainless steel. A primary stock solution was prepared in dilution water at a concentration o f 27 mg a.i./L. The primary stock solution was mixed with an electric mixer for approximately 22 hours to aid in the solubilization o f the test substance. After mixing, the primary stock solution was proportionally diluted with dilution water to prepare the four additional test concentrations. All test solutions appeared clear and colorless. T est Organism The fathead minnow, Pimephales prom etas, was selected as the test species for this study. The fathead minnow is representative o f an important group o f aquatic vertebrates and was selected for use in the test based upon past history o f use in the laboratory. Fathead minnows used in the test were obtained from cultures at Wildlife International Ltd., Easton, Maryland. 000551 AMENDED W il d l if e In t e r n a t io n a l ltd. -10- PROJECT NO.: 454A-102 Fathead minnows were held at approximately the same temperature as used during the test. The fish were held for approximately 126 days prior to testing. The fish were acclimated to test conditions for approximately 48 hours prior to test initiation. During the holding and acclimation periods, the fish showed no signs o f disease or stress. During the 14-day holding period preceding the test, water temperatures ranged from 22.0 to 22.8C. The pH o f the water ranged from 8.2 to 8.5 and dissolved oxygen ranged from 7.7 to 8.4 mg/L. Instrumentation and methods used for water measurements are described in the Environmental Conditions section of this report. A t test initiation, the fathead minnows were collected from the acclimation tank and transferred to the test chambers. During the holding period, fathead minnows were fed a commercially-prepared diet. The fish were not fed during the acclimation period (at least 48 hours prior to the test) or during the te st All fish used in the test were from the same source and year class, and the total length o f the longest fish was no more than twice the length o f the shortest. The average total length o f 10 negative control fish measured ' at the end o f the test was 35 mm with a range o f 30 to 38 mm. The average wet weight (blotted dry) of 10 negative control fish at the end o f the test was 0.36 grams with a range o f 0.21 to 0.49 grams. Loading was 0.24 g fish/L o f test water present in the test chambers at any given time. Test Apparatus Test chambers were 25-L polyethylene aquaria containing approximately 15 L o f test solution. The depth o f water in a representative test chamber was approximately 17.6 cm. Test chambers were impartially positioned in an environmental chamber set to maintain a temperature o f 222C. The test chambers were labeled with the project number, test concentration and replicate. Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International Ltd. site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, and pH measurements o f the well water during the four-week period immediately preceding the test are presented in Appendix I. 000552 W il d l if e In t e r n a t io n a l ltd. - 11- PROJECT NO.: 454A-102 The well water was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water again was filtered (0.45 pm) to remove microorganisms and particles. The results o f periodic analyses performed to measure the concentrations o f selected contaminants in well water used by Wildlife International Ltd. are presented in Appendix DL Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod o f 16 hours o f light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period o f low light intensity was provided when lights went on and off to avoid sudden changes in fighting. Light intensity at test initiation was approximately 391 lux at the surface o fthe water. Light intensity was measured using a SPER Scientific Ltd. fight meter. Temperature was measured in each test chamber at the beginning o f the test and at approximately 24-hour intervals thereafter using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ER/C Recorder. The target test temperature during the study was 222C. Dissolved oxygen and pH measurements were made on water samples from all replicate test chambers o f each treatment and control at test initiation and at approximately 24-hour intervals thereafter. Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation. Measurements o f pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based cmprocedures in StandardM ethodsfo r the Examination o f Water and Wastewater (4). Observations Observations were made to determine the number o fmortalities. The number o f individuals exhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 2.5, 2 4 ,4 8 ,7 2 and 96 hours after test initiation. 000553 W il d l if e In t e r n a t io n a l ltd. -12- PROJECT NO.: 454A-102 Statistical Analyses The 2 4 ,4 8 ,7 2 and 96-hour LC50 values and the 95% confidence intervals werecalculated whenpossible by probit analysis, the moving average method or binomial probability with non-linear interpolation (5 ,6 ,7 ) using the computer software o f C.E. Stephan (8). In this study, LC50 values could not be calculated at 24 and 48 hours due to the lack o f an adequate concentration-response pattern, however, the probit method was used to evaluate mortality at 72 hours and the moving average method was used to evaluate mortality at 96 hours. The no mortality concentration and IsTOEC were determined by visual interpretation of the mortality and clinical observation data. Analytical Chemistry W ater samples were collected at mid-depth from each replicate test chamber o f each treatment and control group at the beginning o f the test, at 48 hours and at test termination to measure concentrations o fthe test substance. The samples were collected in plastic (Nalgene) vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis o f the samples are provided in Appendix HI. RESULTS AND DISCUSSION Measurement o f Test Concentrations Results o f analyses to measure concentrations o f PFOS in water samples collected during the test are presented in Table 1 and in the analytical chemistry report (Appendix HI). Nominal concentrations selected for use in this study w ere3.6,5.9,9.9,16 and 27 mg a.i./L. Samples collected at test initiation had measured values that ranged from 85 to 117% o f nominal values. Measured values for samples taken at 48 hours ranged from 86 to 101% o f nominal. Measured values for samples taken at 96 hours ranged from 88 to 98% o f nominal. When measured concentrations o f the samples analyzed at test initiation, approximately 48 hours and at test termination were averaged, the mean measured concentrations for this study were 3 .3 ,5 .6 ,9 .5 ,1 7 and 28 mg a.i./L. Mean measured concentrations were used in the estimation or calculation o f LC50 values. Observations and Measurements Measurements o f temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were within the 222C range established for the test. Dissolved oxygen concentrations remained 7.7 mg/L (88% of saturation) throughout the test. Measurements o f pH ranged from 8.3 to 8.6 during the test. 000554 AMENDED W il d l if e In t e r n a t io n a l ltd. -13- PROJECT NO.: 454A-102 Daily observations ofmortality and other clinical signs o f toxicity observed during the test are shown in Table 3. Fathead minnows in the negative control and the 3.3 mg a.i./L treatment group appeared normal and healthy during the test. After 96-hours o f exposure, mortality in the 5.6, 9.5, 17 and 28 mg a.i./L treatment groups was 2 0 ,5 0 ,8 0 and 100%, respectively. LC50 values and 95% confidence limits at 2 4 ,4 8 ,7 2 and 96 hours were estimated or calculated from the mortality data, and are shown in Table 4. A graph o f the concentration-response curve is presented in Figure 1. CONCLUSIONS .The 96-hour LC50 value for fathead minnows exposed to PFOS was 9.5 mg a.i./L with 95% confidence limits o f 8.0 and 11 mg a.i./L. The 96-hour no mortality concentration and the NOEC were 3.3 mg a.i./L. 000555 AMENDED W il d l if e In t e r n a t io n a l ltd. -14- PROJECT NO.: 454A-102 REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater andM arine. 2 O rganisation for Economic C ooperation and Development. 1993. OECD Guidelines for Testing of Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992. 3 ASTM S tandard E729-88a. 1994. Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 APHA, AWWA, W PCF. 1985. StandardM ethodsfo r the Examination o f Water and Wastewater. ,16th Edition. American Public Health Associatioa American W ater Works Association. Water Pollution Control Federation, New York. 5 Stephan, CJE. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 6 Finney, D .J. 1971. StatisticalM ethods in Biological Assay. Second editioa Griffin Press, London. 7 Thom pson, W .R. 1947. Bacteriological Reviews. Vol. n,N o. 2. Pp. 115-145. 8 Stephan, CJE. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and H azard Evaluations, American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 000556 W i l d l i f e In t e r n a t io n a l ltd. -15- PROJECT NO.: 454A-102 Table 1 Summary o f Analytical Chemistry Data Sponsor: T est Substance: T est Organism : 3M Corporation PFOS Fathead M innow, Pimephales promelas N om inal Test C o n cen tratio n N egative Control R eplicate A B A B A B Tim e (H ours) 0 0 48 48 96 96 3.6 A 0 B0 A 48 B 48 A 96 B 96 5.9 A 0 B0 A 48 B 48 A 96 B 96 9.9 A B A B A B 0 0 48 48 96 96 16 A 0 B0 A 48 B 48 A 96 B 96 27 A 0 B0 A 48 B 48 A 96 B 96 1 The lim it o f quantitation (LOQ) w as 0.458 m g a.i./L. C o n cen tratio n (mg a.i./L) <LOQ` <LOQ <LOQ <LOQ <LOQ <LOQ 3.16 3.53 3.08 3.22 3.46 3.13 6.05 5.07 5.48 5.89 5.70 5.55 8.99 9.47 9.88 9.33 9.70 9.52 18.2 19.3 15.0 15.6 14.8 16.2 28.5 28.5 27.0 27.8 26.8 26.6 M ean M easured C o n cen tratio n .i./L ) <LOQ 3.3 5.6 9.5 17 28 P ercen t of N om inal 92 95 96 106 104 000557 AMENDED PROJECT NO.: 454A-102 - 16Table 2 Temperature, Dissolved Oxygen and pH o f Water in the Test Chambers Sponsor: 3M Corporation Test Substance: Test Organism: PFOS Fathead Minnow, Pimephalespromelas D ilution Water: W ell Water Mean Measured OHour1 Test Concentration Temp2 DO3 (mg a.i./L) Replicate (C) (mg/L) pH Negative Control A 20.5 8.8 8.6 B 20.4 8.8 8.6 24 Hours Temp DO (C) (mg/L) 21.7 7.8 21.7 7.8 pH 8.5 8.5 48 Hours Temp (C ) 21.9 21.4 DO (mg/L) 8.4 8.2 pH 8.4 8.4 ' 72 Hours Temp DO (C) (mg/L) 22.1 8.2 21.6 8.0 pH 8.4 8.4 96 Hours Temp DO (C) (mg/L) 22.0 7.8 21.6 7.8 pH 8.3 8.4 3.3 A 20.5 8.8 8.6 21.7 7.8 8.5 21.3 8.6 8.5 21.6 8.0 8.4 21.4 7.8 8.4 B 20.4 8.8 8.6 21.6 7.9 8.5 21.3 8.4 8.5 21.5 8.0 8.4 21.4 7.8 8.4 5.6 A 20.5 8.8 8.6 21.4 7.8 8.5 21.2 8.6 8.4 21.5 8.0 8.4 21.3 7.8 8.4 B 20.3 8.8 8.6 21.1 7.8 8.5 21.0 8.4 8.4 21.3 8.0 8.4 21.1 7.8 8.4 9.5 A 20.5 9.0 8.5 21.2 7.9 8.5 21.0 8.4 8.4 21.2 8.0 8.4 21.1 7.8 8.4 B 20.7 9.0 8.5 21.2 7.9 8.5 ' 21.0 8.4 8.5 21.2 8.0 8.4 21.2 7.8 8.4 17 A 21.2 9.0 8.5 21.3 7.8 8.5 21.1 8.6 8.4 21.4 8.0 8.4 21.2 7.9 8.4 B 21.4 9.0 8.5 21.4 7.9 8.5 21.2 8.5 8.4 21.5 8.0 8.4 21.3 7.8 8.4 28 A 22.2 9.0 8.5 21.4 7.7 8.5 21.3 8.6 8.4 21.6 8.0 8.4 21.5 7.8 B 22.3 9.0 8.5 21.7 7.7 8.4 21.7 8.4 8.4 21.9 8.0 8.4 21.8 7.8 1 The O-hour dilution water measurements for hardness, alkalinity and specific conductance were 136 mg/L as CaC03, 178 mg/L as CaC03and 315 pmhos/cm, respectively. 2 Temperature measured continuously during the test ranged from approximately 20.5 to 22.0C. 3 A dissolved oxygen concentration o f5 .2 mg/L represents 60% saturation at 22C in freshwater. 8.4 8.4 ooo cn cn AMENDED on -17T ab le3 Cumulative Percent M ortality and Treatment-Related Effects Sponsor: Test Substance: Test Organism: Dilution W ater 3M Corporation PFOS Fathead Minnow,Plmephalespromelas Well Water_______________ _______ Mean Measured Test Concentration (mg a.L/L) Replicate No. Exposed 2.5 Hours No. Dead* Effects2 Negative Control A B 10 0 10 AN 10 0 10 AN 24 Hours No. Dead Effects 0 10 AN 0 10 AN 48 Hours No. Dead Effects 0 10 AN 0 10 AN 72 Hours No. Dead Effects 0 10 AN 0 10 AN Cumulative Peroit Mortality 0 3.3 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 5.6 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 9.5 A 10 0 10 AN 0 10 AN 0 ' 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 17 A 10 0 10 AN 0 10 AN 0 10 AN 1 2AN;7E 15 B 10 0 10 AN 0 10 AN 0 10 AN 2 3AN;5E 28 A 10 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 1 Cumulative number o fdead fish. 2 Observed Effects: AN = Appears N om ai; E = Erratic swimming; N = Loss ofequilibrium. 0 10 AN 4 5E;1N 0 10 AN 6 3E;1N 50 ooo C/l m CO PROJECT NO.: 481A-104 96 Hours No. Dead Effects 0 10 AN 0 10 AN Cumulative Peroit Mortality 0 0 10 AN 0 10 AN 0 3 6AN;1E 20 1 6AN;3E 5 5 E 50 5 5E 9 IE 80 7 3E 10 10 - 100 AMENDED W il d l if e In t e r n a t io n a l ltd. -18- PROJECT NO.: 454A-102 Table4 LC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Lower 95% Confidence LC50 (mg a.i./L) >28 Limits (mg a.i./L) __i > 2 8 __i Upper 95% Confidence Limits (mg a.i./L) __i __i Statistical Method Visual Interpretation Visual Interpretation 72 Hours2 27 22 41 Probit 96 Hours 9.5 8.0 11 Moving Average 1Confidence limit; could not be calculated with the mortality data obtained. 2The usefulness o f this LC50 is questionable because a concentration-effectrelationship was not demonstrated __ over a reasonable range (e.g., <37 to >63) of percent dead. 000560 AMENDED W i l d l i f e In t e r n a t i o n a l Ltd . -19- Figure 1. Concentration-Response Curve (96-Hour Data) PROJECT NO.: 454A-102 000561 AMENDED W il d l if e In t e r n a t io n a l ltd. -20- PROJECT NO.: 454A-102 APPENDEXI Specific Conductance, Hardness, Alkalinity and pH o f W ell W ater Measured During the 4-Week Period Immediately Preceding the Test aE sassaM K B n^B sssaasaasanaai^H aB B B aR i^B B aB C S snB gti^aH aaniH B ^B H R O T nnaasB B S sanaE a Sponsor. 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water: Well Water ____ Specific Conductance Cumhos/cm) Mean 311 (N = 4) Range 310-315 Hardness (mg/L as CaC03) 131 (N = 4) 128-136 Alkalinity (mg/L as CaC03) 177 (N = 4) 176-178 pH 8.3 (N = 4) 8.3 000562 W il d l if e In t e r n a t io n a l ltd. -21- PROJECT NO.: 454A-102 APPENDIXII Analyses o f Pesticides, Organics, Metals and Other Inorganics in Wildlife International Ltd. Well Water1 ANALYSIS MEASURED CONCENTRATION M iscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon Total Cyanide 286 < 0.050 < 1.0 < 10.0 mg/L mg/L mg/L Mg/L Organochlorines and PCBs Aldrin Alpha BHC BetaBHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' D D T .pp1 Dieldnn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.006 < 0.025 < 0.006 Mg/L Mg/L Mg/L < 0.005 Mg/L < 0.008 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.018 Mg/L < 0.010 . Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.007 Mg/L < 0.005 Mg/L < 0.500 Mg/L < 0.260 Mg/L < 0.260 Mg/L < 0.260 Mg/L < 0.720 Mg/L < 0.720 Mg/L < 0.720 Mg/L < 0.720 Mg/L M etals and Other Inorganics Aluminum3 Arsenic Beryllium: Cadmium3 Calcium3 Chromium Cobalt3 Lead3 Magnesium Manganese Mercury Molybdenum Nickel3 3 Selenium' Silver3 , Sodium3 Zinc3 < 100 < 25.0 < 0.50 < 1.0 35.0 < 2.0 < 1.0 < 20.0 < 100 < 10.0 13.5 < 1.0 < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1.0 21.3 < 20.0 Mg/L Mg/L Mg/L Mg/L mg/L Mg/L Mg/L Mg/L Mg/L Mg/L mg/L Mg/L Mg/L Mg/L Mg/L mg/L Mg/L Mg/L mg/L Mg/L Analyses performed by Environmental, Cainesville, Florida for samples collected on November 3 through kovember 1 ,1997. Analyses performed by Wildlife International Ltd. for the sample collected on November 5,1997. Analyses performed by Wildlife International Ltd. for samples collected on November 5 through 7,1997. 000563 W il d l if e In t e r n a t io n a l ltd. -22- APPENDIX m PROJECT NO.: 454A-102 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 000564 W il d l if e In t e r n a t io n a l ltd. -23- PROJECT NO.: 454A-102 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales propjelas) WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 PRINCIPAL INVESTIGATOR. Raymond L. Van Hoven, Ph.D. Scientist MANAGEMENT: W illard B. Nixon, Ph.IX Manager, Analytical Chemistry DATE DATE^ 0C0565 AMENDED W il d l if e In t e r n a t io n a l ltd. -24- PROJECT NO.: 454A-102 Introduction Freshwater samples were collected from a static acute aquatic toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the fathead minnow (Pimephales promelas). This study was conducted by W ildlife International Ltd. and identified as Project No.: 454A102. The analyses o f these water samples were performed at W ildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on February 15,17 and 19,1999 and were analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for the analytical portion o f this study was W ildlife International Ltd. identification number 4675. The test substance was used to prepare calibration and m atrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical A bstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the freshwater samples was developed at Wildlife International Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II o f W ildlife International Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. Concentrations o f the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- ocogeG W il d l if e In t e r n a t io n a l ltd. -25- PROJECT NO.: 454A-102 Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cu analytical column (100 mm x 2 mm I.D., 3 pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit o f Quantitation Calibration standards o fP F O S prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/!, and 0.05% formic acid (v/v), ranging in concentration from 0.00915 to 0.0457 mg a.i./L were analyzed with the samples. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentrations o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method lim it o f quantitation (LOQ) for these analyses was set at 0.458 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). M atrix Blank and Fortification Samples Three m atrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative chromatogram o f a matrix blank is presented in Figure 5. Freshwater was fortified at 0.915, 9.15 and 45.7 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery o f 97.9%. A representative chromatogram o f a matrix fortification is presented in Figure 6. 000567 AMENDED W i l d l i f e In t e r n a t i o n a l Ltd. -26- PROJECT NO.: 454A-102 Example Calculations Sample number 454A-102-27, nominal concentration o f 3.6 mg a.i./L in freshwater. Initial Volume: 0.250 mL Final Volume: 25.0 mL Dilution Factor: 100 v PFOS Peak Area: 359685 Internal Standard Peak Area: 415648 Peak Area Ratio: 0.865 Calibration curve equation. Slope: 0.023 Intercept: 0.062 Curve is linear. PFOS (pg a.i./L) at instrument = Peak area ratio - (Y-intercept) Slope PFOS (mg a.i./L) in sample = PFOS ip e a.i./L) at instrument x Dilution Factor 1000 Calculated concentration: 3.46 mg a.i./L Note: manual calculations may differ. Percent o f Nominal Cone. = PFOS /me a.i./Ll in sample x 100 PFOS (mg a.i./L) nominal Calculated recovery: 96.9% Note: manual calculation may differ. 000568 AMENDED W il d l if e In t e r n a t io n a l ltd. -27- PROJECT NO.: 454A-102 RESULTS Sam ple Analysis Freshwater samples were collected from the acute toxicity study with the fathead minnow (Pimephales promelas) at test initiation, February 15, 1999 (Hour 0), on February 17, 1999 (Hour 48), and at test termination, February 19, 1999 (Hour 96). The measured concentrations o f PFOS in the samples collected at initiation o f exposure o f the test organisms (Hour 0) ranged from 85.3 to 117% o f the nominal concentrations. Samples collected at Hour 48 had a measured concentration range o f 86.3 to 101% o f nominal values. Samples collected at test termination (Hour 96) had a measured concentration range o f 87.6% to 98.3% o f nominal values (Table 4). A representative chromatogram o f a test sample is shown in Figure 7. 000569 AMENDED W i l d l i f e In t e r n a t i o n a l Ltd . -28- PROJECT NO.: 454A-102 INSTRUMENT: Table 1 Typical HPLC Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC M ass Spectrometer equipped w ith a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C]8 column (100 mm x 2 mm I.D ., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 10.0 minutes FLOWRATE: 0.220 mL/minute MOBILE PHASE: 72.0% M ethanol: 28.0% NANOpure W ater containing 0.1% Formic Acid INJECTION VOLUME: 50.0 pL PFOS RETENTION TIME: Approximately 7.1 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.9 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 000570 W il d l if e In t e r n a t io n a l ltd. -29- PROJECT NO.: 454A-102 T ab le2 M atrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-102-) MAB-1 Sample Type M atrix Blank M easured Concentration o f PFOS1 (mg a.i./L) <LOQ MAB-2 M atrix Blank < LOQ a o V MAB-3 M atrix Blank 1 The limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0,00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0), 000571 AMENDED W il d l if e In t e r n a t io n a l ltd. -30- PROJECT NO.: 454A-102 Table 3 M atrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-102-) MAS-1 M A S-4 MAS-7 MAS-2 MAS-5 MAS-8 Concentrations o f PFOS (mg a..i./L) Fortified M easured v 0.915 0.915 0.915 1.03 0.842 0.970 9.15 8.65 9.15 8.75 9.15 8.74 Percent Recovered 113 92.0 106 94.5 95.6 95.5 MAS-3 MAS-6 MAS-9 45.7 45.7 45.7 48.3 37.6 44.0 106 82.2 96.2 Mean = 97.9 Standard Deviation = 9.12 CV = 9.32 -N = 9 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 000572 AMENDED W il d l if e In t e r n a t io n a l lto. -31- PROJECT NO.: 454A-102 Table 4 Measured Concentrations o f PFOS in Freshwater Samples from a Fathead Minnow Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 3.6 5.9 Sample Number (454A-102-) 1 2 13 14 25 26 3 4 15 16 27 28 5 6 17 18 29 30 Sampling Time (Hours) 0 0 48 48 96 96 0 0 48 48 96 96 0 0 48 48 96 96 PFOS Measured Concentration1 (mg a.i./L) < LOQ < LOQ <LOQ < LOQ*2 < LOQ < LOQ 3.162 3.53 3.08 3.22 3.46 3.13 6.052 5.07 5.48 5.89 5.70 5.55 Percent of Nominal -- -- -- -- -- -- 88.6 98.9 86.3 90.3 96.9 87.6 102 85.3 92.2 99.0 95.8 93.5 9.9 7 0 8.992 89.3 80 9.472 94.0 19 48 9.88 97.8 20 48 9.33 92.8 31 96 9.70 96.3 32 96 9.52 94.3 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 'T he limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). 2M ean result o f duplicate redilutions o f the original sample. Duplicate results (in parentheses) for the following 454A-102 sample numbers are: 3 (3.07 and 3.25), 5 (6.11 and 5.99), 7 (9.01 and 8.96), 8 (9.70 and 9.24) and 14 (<0.458 and <0.458). The original results for these samples (4.44, 7.98, 21.2, 2.21, and 0.74, respectively) are not included in the statistical analysis o f the data due to suspect dilutions (e.g. contamination).________________________ 00057 AMENDED W i l d l i f e In t e r n a t io n a l Ltd. -32- PROJECT NO.: 454A-102 Table 4 (Continued) Measured Concentrations o f PFOS in Freshwater Samples from a Fathead Minnow Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 16 Sample Number (454A-102-) 9 10 21 22 33 34 Sampling Time (Hours) 0 0 48 48 96 96 PFOS Measured Concentration1 (mg a.i./L) 18.2 19.3 15.0 15.6 14.8 16.2 Percent of Nominal 110 117 90.9 94.5 90.2 98.3 27 11 0 12 0 23. 48 24 48 35 96 36 96 28.5 104 28.5 104 27.0 98.4 27.8 101 26.8 97.6 26.6 97.0 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 'The limit o f quantitation (LOQ) was 0.458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00915 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). 2Mean result o f duplicate redilutions o f the original sample. Duplicate results (in parentheses) for the following 454A-102 sample numbers are: 3 (3.07 and 3.25), 5 (6.11 and 5.99), 7 (9.01 and 8.96), 8 (9.70 and 9.24) and 14 (<0.458 and <0.458). The original results for these samples (4.44, 7.98, 21.2, 2.21, and 0.74, respectively) are not included in the statistical analysis of the data due to suspect dilutions (e.g. contamination). 000574 AMENDED Wild life International ltd. -33- PROJECT NO.: 454A-102 M ETHOD OUTLINE FO R TH E ANALYSIS O F PFOS IN FRESHW ATER Prepare matrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into freshwater using gas-tight syringes and Class A volumetric flasks. Dilute m atrix fortification and test samples into the range o f the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the appropriate volume o f sample and bring the flask to volume with the dilution solvent. Process the m atrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. M ix well by several repeat inversions. 4- Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis o f PFOS in freshwater. 000575 W il d l if e In te r n a tio n a l ltd. -34- PROJECT NO.: 454A-102 Cancentration(mgal/L) Figure 2. A typical calibration curve for PFOS. Slope = 0.023; Intercept = 0.062; r = 0.996 0 0 0 5 7 6 AMENDED W il d l if e In t e r n a t io n a l ltd. -35- PROJECT NO.: 454A-102 intensity: 8361 cps Figure 3. A representative ion chromatogram of a low-level (0.00915 mg a.i./L) PFOS standard. 000577 AMENDED W il d l if e International ltd. -36- PROJECTNO.: 454A-102 Figure 4. A representative ion chromatogram o f a high-level (0.04S7 mg a.i./L) PFOS standard. 000578 AMENDED W il d l if e In te r n a tio n a l ltd. -37- PROJECT NO.: 454A-102 intensity: 426 cps 1 Figure 5. A representative chromatogram o f a matrix blank sample (454A-102-MAB-3). The arrow indicates the retention time o f PFOS. 000579 W il d l if e In te r n a tio n a l ltd. -38- PROJECT NO.: 454A-102 Figure 6. A representative chromatogram o f a matrix fortification sample (454A-102-MAS-7). 000580 W il d l if e In te r n a tio n a l lto. -39- PROJECT NO.: 454A-102 Figure 7. A representative chromatogram o f a test sample (454A-102-27). 000581 W il d l if e In te r n a tio n a l ltd. -40- PROJECT NO.: 454A-102 APPENDEXIV 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 proposed experimental start and termination dates, study room, test substance information aVnd test concentrations. 2. The protocol was amended to remove annual feed analysis. 3. The protocol was amended to remove the calculation o f an incipient LC50 value. 4. The protocol was amended to specify the analytical method. 000582 W il d l if e In ter n a tio n a l ltd. -41- PROJECT NO.: 454A-102 APPENDIX V Personnel Involved in the Study The following key Wildlife International Ltd. personnel were involved in the conductormanagementofthis study: 1. Henry O. Krueger, Ph.D.,Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Mark A. Mank, Laboratory Supervisor 4. Timothy Z. Kendall, Laboratory Supervisor 5. Kurt R. Drottar, Senior Biologist 6. Raymond L. VanHoven, Ph.D., Scientist 000583 W il d l if e In ter n a tio n a l ltd. -42- PROJECT NO.: 454A-102 APPENDIX VI Report Amendment 1. Original Report: Pages 1-4,6 and 23 Amendment: The pages were changed to include the amended report date, revised page numbers, and new signatures and dates due to the addition of the report amendment as Appendix VI. Reason: To reflect the issuing o f an amended report. 2. Original Report: Page 2 Amendment: The compliance statement was revised. Reason: To clarify how the test substance was characterized. 3. Original Report: Page 9 Amendment: Information provided by the Sponsor reflecting the reanalysis o f the test substance, including the reanalvsis date and the puritv. was added to the Test Substance section. Reason: To reflect the current test substance information provided by the Sponsor. 4. Original Report: Entire report Amendment: All test substance concentrations were changed to reflect the purity ofthe test substance as determined by the Sponsor in a reanalysis of the test substance (FC-95, Lot 217). Test concentrations originally were based on the reported purity o f 98.9%. The certificate o f analysis dated March 9,2000 indicated a purity o f90.49%. Therefore, all test substance concentrations, including nominal concentrations, measured concentrations, and LC50 values, were recalculated and reported as mg a.i./L based on the 90.49% purity. Reason: To report the results o f the test based on the test substance purity o f 90.49% at the request of the Sponsor. 000584 AMENDED W il d l if e In te r n a tio n a l ltd. -43- APPENDIX VI -Continued- Report Amendment PROJECT NO.: 454A-102 AMENDMENT SIGNATURES: y /^ / DATE Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: Timothy A. Springer, Ph.D. Quality Assurance DATE 000585 AMENDED W i l d l i f e In t e r n a t i o n a l ltd. PROJECT NO.: 454A-102 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STU D Y T IT L E : PFO S: A 96-H O U R STATIC A CUTE TO X IC ITY TEST W ITH THE FA TH EA D M IN N O W (.P im ephales p ro m ela s) P R O T O C O L N O .: 4 5 4 /1 10998/FA T-96H 1/SU B 454 A M E N D M E N T N O .: 1 SPO N SO R : 3M C orporation P R O JE C T N O .: 454A -102 E F F E C T IV E D A T E: Decem ber 21, 1998 AM ENDM ENT: Page 2 Add: Experim ental S tart D ate: 2/15/99 Experim ental Term ination D ate: 2/19/99 T est C oncentrations: N egative C ontrol, 3.9, 6.5, 11, 18 and 30 mg a.i./L T est Substance N o.: 4675 R E A S O N : T he above inform ation w as not know n w hen the protocol w as. signed by the Study D irector. A M E N D M E N T : T est O rganism . P age 5 D elete: F eed provided to the fish w ill be analyzed a t least once annually to ensure th a t th ere are no contam inants at levels know n to be capable o f interfering w ith th e study. R E A S O N : H istorical analyses o f W ildlife International L td. aquatic feed have show n th at no contam inants are present at levels know n to be capable o f interfering w ith the study. A M E N D M E N T : D ata Analysis. P age 8 Change: The LC50 value will be calculated, w hen possible, using m ortality d ata collected a t 24, 48, 72 and 96 hours, as w ell as th e incipient LC 50. To: T he L C 50 v alue will be calculated, w hen possible, using m ortality data collected a t 24, 48, 72 and 96 hours. R E A SO N : The incipient LC 50 cannot be determ ined in a 96-hour static acute test. 000586 a4S4\l 02\amendl W i l d l i f e In t e r n a t i o n a l ltd. PROJECT NO.: 454A-102 Page 2 o f2 A M E N D M E N T : A PPEN D IX II, P age 13 Add: L iquid C hrom atography M ass Spectrom etry (LCM S) M ethod for th e D eterm ination o f Perfluorooctane Sulfonic A cid, Potassium Salt (PFO S) in F reshw ater, F iltered S altw ater and A lgal M edium . R E A S O N : T o add th e analytical m ethod to be used in the study. STUDY D IRECTO R &h M DATE a4S4\102\amendl 000587 PROTOCOL PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales prom elas) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 and OECD Guideline 203 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory P.O. Box 33331 St. Paul, Minnesota 55133 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 November 9,1998 000588 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 Wil d l if e In t e r n a t io n a l ltd. -2- PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) SPONSOR: 3M Corporation Environmental Laboratory P.O. Box 33331 St. Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Kurt Drottar Senior Aquatic Biologist LABORATORY MANAGE!B la u Henry O. Krueger, Ph.D. Director of Aquatic Toxicology & Non-Target Plants Proposed Dates: Experimental Start Date: P ro iectN o .: Test Concentrations: Test Substance No.: FOR LABORATORY USE ONLY - " !0 X - Experimental Termination Date: Reference Substance No. ( if aoolicableV PROTOCOL APPROVAL STUDY DIRECTOR \ /J/*/(9 8 DATE X hLABORATORY MANAGEMENT SPONSOR'S REPRESENTATIVE ~ /V y V / DATE t ill3 o ) j DATE 000589 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 V il d l i f e In t e r n a t i o n a l ltd. -3 - INTRODUCTION W ildlife International Ltd. will conduct a static acute toxicity test with the fathead minnow (Pimephalespromelas) for the Sponsor at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 (1); OECD Guideline for Testing of Chemicals 203: Fish, Acute Toxicity Test (2); and ASTM Standard E729-88a Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, M acroinvertebrates and Amphibians (3). Raw data for all work performed at Wildlife International Ltd. and a copy o f the final report will be filed by project number in archives located on the W ildlife International Ltd. site, or at an alternative location to be specified in the final report. PURPOSE The purpose o f this study is to determine the acute effects o f a test substance on the fathead minnows (.Pimephales promelas) during a 96-hour exposure period under static test conditions. e x pe r im e n t a l d e sig n Fathead minnows will be exposed to a geometric series o f at least five test concentrations and a negative (dilution water) control for 96 hours. Two replicate test chambers will be maintained in each treatment and control group, with 10 fathead minnows in each chamber for a total o f 20 fathead minnows per test concentration. Nominal test concentrations will be selected in consultation with the Sponsor, and will be based upon information such as the results o f exploratory range-finding toxicity data, known toxicity data, physical/chemical properties of the test substance or other relevant information. Target concentrations need not exceed 1000 mg/L or the solubility limit o f the test substance in water (whichever is lower). Generally, each test substance concentration used in the definitive test will be '* higher concentration unless information concerning the concentration-effe different dilution factor would be more appropriate. W ater samples from collected at specified intervals for analysis o f the test substance. Results < calculate mean measured test concentrations. i To control bias, fathead minnows will be impartially assigned to initiation. No other potential sources ofbias are expected to affect the result o f mortality and other clinical signs will be made throughout the 96-hour test PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M -^el -QUEST NO. U2723 000590 j 1 W i l d l i f e In t e r n a t i o n a l ltd. I -4- mortality observed in the treatment groups will be used to calculate, when possible, LC50 values at 24, I 48,72 and 96 hour intervals. The no-mortality concentration and the no-observed-effect concentration a (NOEC) will be determined. a Test Substance M ATERIALS AND METHODS a Information on the characterization of test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International Ltd. written verification that the test substance has been characterized according to GLPs prior to its use a in the study. If written verification o f GLP test substance characterization is not provided to Wildlife International Ltd., it will be noted in the compliance statement of the final report. The attached form a IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. a The Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end o f the study. a PreparationofTest Concentrations a The test substance will be administered to the test organism directly into dilution water. This route o f administration was selected because it represents the most likely route o f exposure to aquatic organism s. a a Test Organism The fathead minnow (Pimephales prom elas) has been selected as the test species for this study. Fathead minnows are representative o f an important group o f aquatic vertebrates, and have been selected a for use in the test based upon past use history in the laboratory. Fish will be from the same source and year class, and the standard length o f the longest fish measured will be no more than twice that o f the a shortest Fish will weigh between 0.1 g and 3.0 g each and the total weight in each test chamber will not exceed 0.8 grams fish/L of solution Total lengths and wet weights o f the individual fish in one negative a control replicate will be measured at the end of the test and will be considered representative of the length and weight o f all fish used in the study. Fish will be obtained from a commercial supplier or hatchery, and the identity of the species will be verified by the supplier or by Wildlife International Ltd. personnel a using appropriate taxonomic keys, such as Eddy (4). 000591 a PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd. -5 - Fathead minnows will be held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test. Any changes in water temperature will not exceed 3C per day. Fathead minnows will be held at the test temperature for a minimum of 7 days prior to test initiation. Fathead minnows will not be used in the test if they show signs o f disease or stress or if more than 5% die during the 48 hours prior to the test. A t test initiation, the fathead minnows will be collected from holding or acclimation tanks and transferred to the test chambers. During die holding period the test fish will be fed at least once daily. The diet will consist o f live or frozen brine shrimp nauplii (Artemia sp.), and/or commercial food. Fish will not be fed for at least two days prior to the test or during the test. Feed provided to the fish will be analyzed at least once annually to ensure that there are no contaminants at levels known to be capable o f interfering with the study. Specifications for acceptable levels o f contaminants in fish diets have not been established. However, there are no known levels o f contaminants reasonably expected to be present in the diet that are considered to interfere with the purpose or conduct o f the test. DilutionHater W ater used for the culturing and testing o f fathead minnows will be obtained from a well approximately 40 meters deep located on the W ildlife International Ltd. site. The water will be passed through a sand filter and pumped into a 37,800-L storage tank where the water will be aerated with spray nozzles. Prior to use die water will be filtered to 0.45 m xi in order to remove fine particles. W ater used for culturing and testing is characterized as moderately hard. Typical values for hardness, alkalinity, pH and specific conductance are approximately: Hardness, mg/L as C aC 03 Alkalinity, mg/L as CaC03 pH Specific Conductance, mhos/cm 145 190 8.1 330 H ardness, alkalinity, pH and specific conductance will be measured weekly to monitor the consistency o f the well water. Means and ranges o f the measured parameters for the four-week period preceding the test will be provided in the final report. Analyses will be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents of the well water and results o f the analyses will be summarized in the final report. 000592 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 Wil d l if e Intern atio na l ltd . 6- - Test Apparatus Test chambers will be 25-L, polyethylene aquaria filled with approximately 15 L of water. Test cham bers will be positioned in an environmental chamber or temperature-controlled water bath to m aintain a temperature o f 22 2C. Test chambers will be labelled with the project number, test concentration and replicate. Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation, and testing will be provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g., Colortone 50). A photoperiod o f 16 hours of light and 8 hours o f dark will be controlled with an automatic timer. A 30-minute transition period o f low light intensity will be provided when lights go on and o ff to avoid sudden changes in light intensity. Light intensity will be measured at test initiation with a SPER Scientific Ltd. light meter or equivalent. The target test temperature will be 22 2 C. Temperature will be measured all replicates at the beginning o f the test and at approximately 24-hour intervals thereafter using a liquid-in-glass therm ometer. Temperature also will be measured with a continuous recorder in one negative control chamber. Recorder measurements will be verified with a liquid-in-glass thermometer prior to test initiation. Dissolved oxygen will be measured in all replicates o f the treatment and control groups at test initiation and at approximately 24-hour intervals thereafter using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter, or equivalent. In the event that dissolved oxygen levels fall below 60% saturation, appropriate actions will be taken after consultation with the Sponsor. Measurements o f pH will be made in all replicates o f the treatment and control groups at test initiation and at approximately 24-hour intervals thereafter using a Fisher Accumet Model 915 pH meter, or equivalent. If a treatment group reaches 100% mortality, dissolved oxygen, pH, and temperature measurements will be taken at the next sampling interval, then discontinued. H ardness, alkalinity, and specific conductance will be measured in the dilution water at test initiation. Hardness and alkalinity measurements will be made by titration using procedures based on methods in StandardMethodsfo r the Examination o f Water and Wastewater (5). Specific conductance will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter, or equivalent. 000593 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l ltd . -7 - Biological Measurements Observations of mortality and clinical signs of toxicity will be made between 0-24 hours, and at 24,48,72 and 96 hours 1 hour. Lethality is defined as the lack of visible movement (e.g. lack o f fin or opercular movement) in the fish after gentle prodding. All clinical observations including abnormal behavior will be noted. Sampling for Analytical Measurements W ater samples will be collected from each test chamber at the beginning o f the test, during the test, and at the end o f the test to determine concentrations o f the test substance. In the event that 100% m ortality occurs in any treatment, then sampling o f that treatment will terminate following the next sampling interval. Samples will be collected at mid-depth from each test chamber and analyzed immediately or placed in an appropriate storage container (e.g., polyethylene or polypropylene bottle) and stored under refrigeration until analyzed. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group 48 0 Hours Hours 96 Hours Control Solvent Control (if needed) Level 1-Low Concentration Level 2 Level 3 Level 4 Level 5-High Concentration 222 222 222 222 222 222 222 Totals 14 14 14 Total Number o f Verification Samples = 42 The above numbers o f samples represent those collected from the test and do not include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the analytical chemistry phase of the study. At the discretion o f the Study Director, water samples from one or more appropriate test chambers will be collected and analyzed if an analytical error in sampling or analysis is suspected. The reason for the additional samples will be described by the Study Director and documented in the raw data and final report. 000594 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd. 8- - Analvtical Chemistry Chemical analysis ofthe samples will be performed by Wildlife International Ltd. The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix II. The methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. Data Analysis When the dose-response pattern allows calculation o f an LC50 value, the data will be analyzed using the computer software of C.E. Stephan (6). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, or binomial probability with nonlinear interpolation (7,8,9). The LC50 value will be calculated, when possible, using mortality data collected at 24,48,72 and 96 hours, as well as the incipient LC50. The no-mortality concentration and the no-observed-effect concentration (NOEC) will be determined. RECORDS TO BE MAINTAINED Records to be maintained for data generated by W ildlife International Ltd. will include, but not be limited to: 1. A copy o f the signed protocol. 2. Identification and characterization of the test substance, if provided by the Sponsor. 3. Dates o f initiation and termination o f the test. 4. Test organism history, holding and acclimation records. 5. Stock solution calculation and preparation, if applicable. 6. Observations. 7. W ater chemistry results (e.g., alkalinity and hardness). 8. The methods used to analyze test substance concentrations and the results o f analytical measurements. 9. Statistical calculations, if applicable. 10. Test conditions (light intensity, photoperiod, etc.). 11. Calculation and preparation o f test concentrations. 12. Copy o f final report. PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 000595 3M LAB REQUEST NO. U2723 Wil d l if e Inter n a tio n a l ltd. -9- F1NAL REPORT A final report of the results of the study will be prepared by Wildlife International Ltd. The report will include, but not be limited to, the following, when applicable: 1. Name and address o f the facility performing the study. 2. Dates upon which the study was initiated and completed, and the definitive experimental start and termination dates. 3. A statem ent o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. O bjectives and procedures, as stated in the approved protocol, including all changes to the protocol. 5. The test substance identification including name, chemical abstract number or code number, strength, purity, composition, and other information provided by the Sponsor. 6. Stability and solubility o f the test substance under the conditions o f administration, if provided by the Sponsor. 7. A description o f the methods used to conduct the te st 8. A description o f the test organisms, including the source, scientific name, age or life stage, lengths and weights o f a representative group o f test organisms, feed types, light intensity and photoperiod. 9. A description o f the preparation of the test solutions. 10. The m ethods used to allocate organisms to test chambers and begin the test, the number o f organisms and chambers per treatment, and the duration o f the test. 11. A description o f circumstances that may have affected the quality or integrity o f the data. 12. The name of the Study Director and the names o f other scientists, professionals, and supervisory personnel involved in the study. 13. A description o f the transformations, calculations, and operations performed on the data, a summary and analysis o fdie biological data and analytical chemistry data, and a statement o f the conclusions drawn from the analyses. 14. Statistical methods used to evaluate the data. 15. A graph o f the concentration-mortality curve at the end o f the te st 16. The signed and dated reports of each o f the individual scientists or other professionals involved in the study. 17. The location where raw data and final report are to be stored. 18. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates of any findings reported to the Study Director and Management. 000596 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 I I Wil d l if e In t e r n a tio n a l ltd. I - 10- I 19. If it is necessary to make corrections or additions to a final report after it has been accepted, such changes will be made in die form of an amendment issued by the Study Director. The amendment will clearly identify the part o f the final report that is being amended and the reasons for the I amendment, and will be signed by the Study Director. I CHANGING OF PROTOCOL Planned changes to the protocol will be in the form o f written amendments signed by the Study 1 Director and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the 1 final report. I GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA E (40 CFR Part 160 and/or Part 792); OECD Principles o f Good Laboratory Practice (OCDE/GD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, I Agricultural Production Bureau). Each study conducted by Wildlife International Ltd. is routinely exam ined by the Wildlife International Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement o f I compliance with Good Laboratory Practices will be prepared for all portions o f the study conducted by W ildlife International Ltd. The Sponsor will be responsible for compliance with Good Laboratory I Practices for procedures performed by other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at Wildlife International Ltd. and a copy o f the final report will be filed by project I num ber in archives located on the Wildlife International Ltd. site, or at an alternative location to be specified in the final report. I I I I 000597 K PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e Intern atio na l ltd. - 11 - REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850-Ecological Effects Test Guidelines {draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine. 2 O rganisation fo r Economic Cooperation and Developm ent. 1993. OECD Guidelines for Testing o f Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992. 3 ASTM S tandard E729-88a. 1994. Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, M acroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 Eddy, S. 1974. The Freshwater Fishes. Wm. C. Brown Company Publishers, Dubuque, Iowa. 5 A PH A , AW W A, W PCF. 1985. Standard M ethods fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American W ater Works Association. W ater Pollution Control Federation, New York. 6 S tephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 7 Thom pson, W .R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 8 S tephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and H azard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 6584. 9 Finney, D .J. 1971. Statistical M ethods in Biological Assay. Second edition. Griffin Press, London. PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 000598 3M LAB REQUEST NO. U2723 | W il d l i f e In t e r n a t i o n a l ltd. - 121 APPENDIX I I IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I I. Test Substance Identity (name to be used in the report): PFOS fPerfluorooctane Sulfonic Acid Potassium Salt 1 Reference Standard (if applicable): Analytical Standard: N/A____________________________________ c Internal Standard: 1.1.2.2H.H.H.H Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch Number: Lot 217________ _______________________________ Test Substance Purity (% Active Ingredient): 98.9______ Expiration Date: 2008_______________ II. Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately define the test substance and reference standard been determined prior to its use in this study in accordance with GLP Standards? Y es____ No X HI. Test Substance Storage Conditions Please indicate the recommended storage conditions at W ildlife International Ltd. ___ Ambignt roomtemperature______________________________________________ i Has the stability of the test substance under these storage conditions been determined in accordance with GLP Standards? Y es____ No X Other pertinent stability information: -------------------------------------------------------------------------------------- i IV. Test Concentrations: i V. Toxicity Information: Adjust test concentration to 100% a.i. X based upon the purity (%) given above. Do not adjust test concentration to 100% _____a.i. Test the material AS IS. M am m alian: Rat LD50 251 mg/ke Mouse LD50 N/A i Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphnia maena: 27 me/L__________ Rainbow Trout; 11 m g /l i Daphnia magna: 50m e/L__________ Fathead MmnQWl-3.8 mg/L Other Toxicity Information (including findings o f chronic and subchronic tests): Please see MSDS_________________________________________________________________ PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 000599 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd. -13- APPENDIXII Analytical Method to be Provided by Sponsor PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 000600 3M LAB REQUEST NO. U2723