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PFOS: 2f R % A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL (Unio complamatus) 0 0' l I FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-105 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: May 3,1999 STUDY COMPLETION DATE: August 11,1999 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 000505 AMENDED W il d l if e Internatio nal ltd. 2- - PROJECT NO.: 454A-105 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Freshwater Mussel (Unio complamatus) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-105 STUDY COMPLETION: August 11,1999 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 o f 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 being recharacterized in accordance with GLP. The stability o f the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R. Drottar Senior Biologist SPONSOR APPROVAL: DATE DATE z> 000S06 AMENDED W il d l if e In ter n a tio n a l ltd. -3 - PROJECT NO.: 454A-105 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: DATE CONDUCTED: Test Substance Preparation May 27,1999 Analytical Sample Preparation and Observations May 28,1999 Biological Data and Draft Report June 29,1999 Analytical Data and Draft Report June 29 and 30,1999 Final Report August 11,1999 Amended Report April 20,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: May 27,1999 June 1,1999 May 28,1999 June 3,1999 June 29,1999 June 30,1999 June 30,1999 August 11,1999 April 20,2000 Jun 30,1999 August 11,1999 April 24, 2000 '^ L s ^ Timothy A. Springer, Ph.D. Manager, Regulatory and Technical Support DATE 000807 AMENDED W il d l if e Inter n a tio n a l ltd. -4 - REPORT APPROVAL PROJECT NO.: 454A-105 SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Freshwater Mussel (Unio complamatus) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-105 STUDY DIRECTOR: MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants DATE 000E08 AMENDED W il d l if e In t e r n a t io n a l Ltd . -5- PROJECT NO.: 454A-105 TABLE OF CONTENTS Title/Cover Page.................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement.......................................................................................... 2 Quality Assurance Statement.................................................................................................................................. 3 Report Approval.......................................................................................................................................................4 Table o f Contents..................................................................................... Summary................................................................................................................................................................... 7 Introduction.................................................................................................................. O bjective.................................................................................................................................................................. 8 Experimental Design...................................................................................................................................... 8 Materials and Methods............................................................................................................................ 9 Results and D iscussioa..................................................................... ?..................................... ......................... 12 Conclusions........................................................................................................................................ 13 R eferen ces................................................................................................... TABLES Table 1 - Summary o f Analytical Chemistry D ata...............................................................................................15 Table 2 - Temperature, Dissolved Oxygen and pH o f W ater in the Test Cham bras....................................... 16 Table 3 - Hardness, Alkalinity and Conductivity o f W ater in the Test Chambers.............................................17 Table 4 - Cumulative Percent Mortality and Treatment-Related Effects........................................................... 18 Table 5 -LC 50 Values...........................................................................................................................................19 000S09 W il d l if e In te r n a tio n a l ltd. - 6- PROJECT NO.: 454A-105 TABLE OF CONTENTS - Continued - FIGURE Figure 1. Concentration-Response Curve (96-Hour Data)............................................................................. 20 APPENDICES Appendix I - Specific Conductance, Hardness, Alkalinity, and pH o f Well Water Measured During the 4-Week Period Immediately Preceding the T est.................................. 21 Appendix II - Analyses o f Pesticides, Organics, Metals and Other Inorganics in Wildlife International Ltd. Well W ater................................................................................22 Appendix in - The Analysis of PFOS in Freshwater in Support of Wildlife International Ltd. Project No.: 454A -105................................................................... ..........23 Appendix IV - Changes to Protocol..................................................................................................................40 Appendix V - Personnel Involved in the Study...............................................................................................41 Appendix VI - Report Amendment.................................................................................................................. 42 000810 AMENDED W il d l if e In t e r n a t io n a l Ltd. PROJECT NO.: 454A-105 SPONSOR: SPONSOR'S REPRESENTATIVE: 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: LENGTH OF TEST: B B B K B S S S B B S aK S X I TEST ORGANISM: SOURCE OF TEST ORGANISMS: 454A-105 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 96-Hour Static Acute Toxicity Test with the Freshwater Mussel (Unio complamatus) Negative Control, 5 .3 ,1 2 ,2 0 ,4 1 and 79 mg a.i./L Experimental Start - May 28,1999 Biological Termination - June 1,1999 Experimental Termination - June 3,1999 96 Hours Freshwater Mussel (Unio complamatus) Carolina Biological Supply Company Burlington, North Carolina 27215 AGE OF TEST ORGANISMS: MEASUREMENTS OF 2 0 REPRESENTATIVE MUSSELS: WET WEIGHT (g ): TOTAL LENGTH (m m ): Unknown Mean = 76.5; Mean = 48.7; Range = 57.5 to 93.5 Range = 44.6 to 54.9 96-H O U R LOSO: 9 5 % CONFIDENCE LIMITS: NO-M ORTAUTY CONCENTRATION: 5 9 mg a.i./L 51 and 68 mg a.i./L 2 0 m g a.i./L 0C0811 AMENDED W il d l if e In ter n a tio n a l ltd. - 8- PROJECT NO.: 454A-105 INTRODUCTION This study was conducted by W ildlife International Ltd. for 3M Corporation atthe Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f the test was conducted from May 28 to June 1, 1999. Raw data generated by W ildlife International Ltd. and a copy o f the final report are filed under Project Number 454A-105 in archives located on the W ildlife 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 freshwater mussel, Unio complamatus, during a 96-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Freshwater mussels 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 freshwater mussels in each test chamber for a total o f 20 freshwater mussels per test concentration Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an exploratory range finding toxicity test. Nominal test concentrations selected were 5.7,11,23,46 and 91 mg active ingredient (a.i.)/L. The test solutions were renewed at approximately 48 hours o f the test. New test solutions wereprepared and the mussels were transferred from the old to the new solutions. Mean measured test concentrations were determined from samples o ftest water collected from each treatment and the control group at the beginning ofthe test, at approximately 48 hours, and at test termination. Freshwater mussels were indiscriminately assigned to exposure chambers at test initiation. Observations o f mortality and other clinical signs o f toxicity were made at approximately 6 ,2 4 ,4 8 ,7 2 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to estimate or calculate LC50 values at 24,48,72 and 96 hours. The no-mortality concentration was determined by visual interpretation ofthe mrtaIitydata' 0C0S12 AMENDED Wil d l if e In te r n a tio n a l ltd. -9 - PROJECT NO.: 454A-105 MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Static Acute Toxicity Test with the Freshwater Mussel". 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 E72988a, Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates andAmphibians (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 of 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 5 .7 ,1 1 ,2 3 ,4 6 and 91 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 ftest concentrations were constructed o f plastic or stainless steel. A primary stock solution was prepared in dilution water at a concentration o f 91 mg a.i./L. The primary stock solution was mixed with an electric paddle mixer for approximately 24 hours to aid in the solubilization o f file 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. Test solutions were renewed on Day 2 o f the test with freshly prepared solutions. Test Organism The freshwater mussel, Unio complamatus, was selected as the test species for this study. The freshwater mussel is representative o f an important group o f aquatic invertebrates and was selected far use in the test based upon commercial availability. Freshwater mussels used in the test were obtained from Carolina Biological Supply Company, Burlington, North Carolina. The identification o f the species was provided by the supplier. AMENDED 0G0S13 W il d l if e International ltd. -10- PROJECT NO.: 454A-105 Freshwater mussels were held at approximately the same temperature as used during the test. The mussels were held for approximately 15 days prior to testing. During the holding periods, the mussels showed no signs o f disease or stress. The mussels were not fed during the holding period or during the test. During the 14day holding period preceding the test, water temperatures ranged from 20.3 to 22.2 C. The pH o f the water ranged from 8.2 to 8.6 and dissolved oxygen ranged from 7.6 to 8.8 mg/L. Instrumentation and methods used for water measurements are described in the Environmental Conditions section o f this report. A t test initiation, the mussels were collected from the holding tank and transferred to the test chambers. The average total length (distance from the tip o f the umbo to the distal valve edge) o f 20 indiscriminately selected mussels from the same lot o ftest organisms was 48.7 mm with a range o f 44.6 to 54.9 mm. The average wet weight was 76.5 grams with a range o f 57.5 to 93.5 grams. Test Apparatus Test chambers were 25-L polyethylene aquaria containing approximately 20 L o f test solution. The depth ofwater in a representative test chamber was approximately 23.2 cm. Test chambers were indiscriminately positioned in an environmental chamber set to maintain a temperature of 222C. The test chambers were labeled with the project number, test concentration and replicate. Dilution Water The water used for holding 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. The well water was passed through a sand filter to remove particles greater than approximately 25 urn, 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 n. 000814 W il d l if e Internatio nal ltd. - 11- PROJECT NO.: 454A-105 Environmental Conditions Lighting used to illuminate the holding tanks and test chambers during holding and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16 hours o f light and 8 hours o f darkness was controlled with an automatic timer. A 30-minute transition period o f low tight intensity was provided when tights went on and offto avoid sudden changes in lighting. Light intensity at test initiation was approximately 369 lux at the surface o f the water. Light intensity was measured using a SPER Scientific Ltd. tight meter. Temperature was measured in each test chamber at the beginning and end o f the test using a liquid-inglass 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 measurements were made on water samples from all replicate test chambers o f each treatment and the control at test initiation and at approximately 24-hour intervals thereafter. Measurements of pH were made in one alternating replicate o f each treatment group and the control at test initiation and at approximately 24 hour intervals thereafter. Hardness, alkalinity and specific conductance were measured in the dilution water and low, middle and high test substance concentrations at test initiation and at test termination. Measurements o f pH were made using a Fisher Accumet Model 915 pH meter, and dissolvedoxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in StandardMethodsfo r the Examination o f Water and Wastewater (4). Observations Observations were made to determine the number o f mortalities. Mussels with open shells and not responding to gentle prodding were considered dead The number of individuals exhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 6,24,48,72 and 96 hours after test initiation. 000515 W il d l if e In ter n a tio n a l ltd. -12- PROJECT NO.: 454A-105 Statistical Analyses The 2 4 ,48,72 and 96-hour LC50 values and the 95% confidence intervals were calculated 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 of an adequate concentration-response pattern. However, the binomial method was used to evaluate mortality at 72 hours and the probit method was used to evaluate mortality at 96 hours. The no-mortality concentration was determined by visual interpretation o f the mortality data. Analytical Chemistry Water 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 (old solutions) and at test termination to measure concentrations o f the test substance. The samples were collected in plastic (Nalgene) bottles and analyzed immediately without storage. Analytical procedures used in the analysis o f the samples are provided in Appendix III. A t test termination, all remaining live mussels were sacrificed for collection o f tissue samples. The tissue samples were composited by replicate, frozen and shipped to the Sponsor for possible analysis. 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 El). Nominal concentrations selected for use in this study were 5.7, ll ,2 3 ,4 6 a n d 9 1 m ga.i./L. Samples collected at test initiation had measured values that ranged from 74 to 96% o f nominal values. Measured values for samples taken at 48 hours ranged from 81 to 99% o f nominal. Measured values for samples taken at 96 hours ranged from 89 to 130% 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 5.3,12,20,41 and 79 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 22 2C range established for the test. Measurements ofpH ranged from 7.9 to 8.5 duringthe test On AMENDED 000S16 W ild life International ltd. -13- PROJECT NO.: 454A-105 Day 1 of the test, dissolved oxygen concentrations had dropped as low as 5.0 mg/L (57% o f saturation). Consequently, mild aeration was initiated to all test chambers. After aeration, dissolved oxygen concentrations remained >5.9 mg/L (68% o f saturation) throughout the test. Measurements o f hardness, alkalinity and conductivity are presented in Table 3. Daily observations o f mortality and other clinical signs o f toxicity observed during the test are shown in Table 4. Freshwater mussels in'the negative control, 5.3,12 and 20 mg a.i./L treatment groups appeared normal and healthy during the test. After 96-hours o f exposure, mortality in the 41 and 79 mg a.i./L treatment groups was 5 and 90%, respectively. LC50 values and 95% confidence limits at 24,48,72 and 96 hours were estimated or calculated from the mortality data, and are shown in Table 5. A graph o f the concentration-response curve is presented in Figure 1. CONCLUSIONS The 96-hour LC50 value for freshwater mussels (Unio cojnplamatus) exposed to PFOS was 59 mg a.i./L. The 95% confidence limits were 51 and 68 mg a.i./L and the slope o f the concentration-response curve was 10. The 96-hour no-mortality concentration was 20 mg a.i./L. 000S17 AMENDED W il d l if e In ter n a tio n a l ltd. -14- PROJECT NO.: 454A-105 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 andMarine. 2 O rganisation for Economic Cooperation 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 ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates, andAmphibians. American Society for Testing and Materials. 4 APHA, AWWA, W PCF. 1985. StandardMethodsfo r the Examination o f Water and Wastewater. 16th Edition. American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 6 Finney, D .J. 1971. StatisticalMethods in Biological Assay. Second edition. Griffin Press, London. -7 Thom pson, W .R. 1947. Bacteriological Reviews. Vol.-H, No. 2. Pp. 115-145. 8 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 0C0S18 W il d l if e In te r n a tio n a l ltd. -15- PROJECT NO.: 454A-105 Table 1 Summary o f Analytical Chemistry Data Sponsor: Test Substance: Test Organism: DutionWater: Nominal Test Concentration Negative Control 3M Corporation PFOS Freshwater Mussel, Unto complamatus Well Water Sampling Time lours) Concentration A0 B0 A 48 B 48 A 96 <LOQ` <LOQ <LOQ <LOQ <LOQ B 96 <LOQ A0 B0 A 48 B 48 A 96 B 96 5.47 4.93 5.18 5.70 5.24 5.26 11 A 0 B0 A 48 B 48 A 96 B 96 11.4 10.1 112 10.5 10.9 15.4 A0 B0 A 48 B 48 A 96 B 96 19.0 16.8 18.7 18.7 22.9 22.4 A0 B0 A 48 B 48 A 96 B 96 37.2 40.6 37.1 39.5 48.2 40.5 A0 B0 A 48 B 48 A 96 B 96 1 The limit of quantitation (LOQ) was 0.115 mg a.i./L. 69.0 74.7 81.3 77.6 88.2 85.7 Mean Measured Concentration (mg at./, <LOQ 5.3 12 20 41 79 Percent of Nominal 93 109 87 89 87 000S19 AMENDED -16Table 2 Temperature, Dissolved Oxygen and pH o f Water in the Test Chambers Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Freshwater Mussel, Unio complamatus Dilution Water: Well W a t e r ____________ Mean Measured OHour Test Concentration Temp1 DO2 (mg a.i./L) Replicate (C) (mg/L) pH Negative Control A 21.4 8.5 8.4 B 21.4 8.5 - 24 Hours DO3 . (mg/L) pH 5.8 -- 6.1 8.0 48 Hours (Old) DO (mg/L) 8.2 8.1 pH 8.3 5.3 A 21.5 8.5 8.5 5.7 B 21.5 8.5 - 6.1 8.0 8.2 8.3 8.2 - 12 A 21.7 8.5 8.4 6.0 _ B 21.8 8.5 - 5.6 8.0 8.2 8.3 8.1 - 20 A 21.8 8.5 8.5 5.8 _ 8.1 8.3 B 21.8 8.5 - 5.7 8.0 ` 8.2 - 41 A 22.5 8.5 8.5 5.4 B 22.5 8.5 - 5.6. 8.0 79 A 23.7 8.6 8.5 5.0 __ B 23.6 8.5 -- 5.2 8.1 1 Temperature measured continuously during the test ranged from approximately 21.0 to 21,5C. 2 A dissolved oxygen concentration of S.2 mg/L represents 60% saturation at 22C in freshwater. 3 Mild aeration was initiated to all test chambers. 8.2 8.2 8.1 8.0 8.3 - 8.2 -- "' 72 Hours DO (mg/L) pH 8.2 8.1 8.2 8.0 -- 8.1 8.2 8.1 _ 8.1 8.2 8.2 __ 8.2 8.2 8.1 _ 8.2 8.2 7.8 -- 7.7 8.1 PROJECT NO.: 454A-105 96 Hours Temp DO (C) (mg/L) 21.8 8.0 21.6 7.9 pH 8.3 -- 21.6 7.9 8.2 21.7 7.8 -- 21.7 8.1 8.2 21.7 7.9 - 21.7 7.9 8.2 21.7 8.0 - 21.9 8.1 8.3 21.9 8.1 21.8 5.9 7.9 21.8 6.3 -- ooo 00 o AMENDED W il d l if e Internatio nal ltd. -17- PROJECT NO.: 454A-105 Table 3 Hardness, Alkalinity and Conductivity o f Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Concentration Negative Control 5.3 20 79 3M Corporation PFOS Freshwater Mussel, Unio complamatus Well Water Sampling Time Hardness (mg/L as CaC03) A0 B 96 A0 B 96 A0 B 96 A0 B0 140 132 136 136 144 136 144 136 Alkalinity Conductivity (mg/L as CaC03) (umhos/cm) 172 300 174 330 174 300 174 330 174 320 178 340 178 340 176 350 000821 AMENDED * - 18- Table4 Cumulative Percent Mortality and Treatment-Related Effects Sponsor. Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Freshwater Mussel, Unio complamatus Well Water Mean Measured Test Concentration (mga.L/L) Replicate No. Exposed 6 Hours No. Dead1 Effects1 Negative Control A B 10 10 0 10 AN 0 10 AN 24 Hours No. Dead 0 0 Effects 10 AN 10 AN 48 Hours No. Dead 0 0 Effects 10 AN 10 AN 72 Hours No. Dead 0 0 Effects 10 AN 10 AN 53 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 12 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 20 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 41 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 79 A 10 B 10 1 Cumulative number ofdead mussels. 1 Observed Effects: AN "Appears Normal 0 0 10 AN 10 AN 3 7 AN 3 7 AN 3 7 AN 5 5 AN 4 6 AN 6 4 AN ooo 05 M N PROJECT NO.: 454A-105 96 Hours No. Dead 0 0 Effects 10 AN 10 AN Cumulative Percent Mortality 0 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 1 9 AN 0 10 AN 5 9 IAN 90 9 IAN AMENDED W ild life Internatio nal ltd. -19- PROJECT NO.: 454A-105 Table 5 LC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 3M Corporation PFOS Freshwater Mussel Unio complamatus Well W ater Lower 95% Confidence LC50 Limits (mg a.i./L) (mg a.i./L) >79 --1 > 7 9 mm\ 72 Hours2 79 >41 Upper 95% Confidence Limits (mg a.i./L) J __i Statistical M ethod Visual Interpretation Visual Interpretation Binomial 96 Hours 59 51 68 Probit ` 2The usefulness o fthis LC50 is questionable because a concentration-effectrelationship was not demonstrated over a reasonable range (e.g., <37 to >63) o f percent dead. 000S23 AMENDED W il d l if e In te r n a t io n a l ltd. -20- Figure 1. Concentration-Response Curve (96-Hour Data) PROJECT NO.: 454A-105 0C054 AMENDED W il d l if e In ter n a tio n a l ltd. -21- PROJECT NO.: 454A-105 APPENDIXI Specific Conductance, Hardness, Alkalinity and pH o f Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Mussel Unio complamatus W ell Water Specific Conductance (pmhos/cm) Mean 321 (N = 4) Range 310-330 Hardness (mg/L as CaC03) 126 (N = 4) 120-132 Alkalinity (mg/L as CaC03) 174 (N = 4) 170-178 pH 8.3 (N = 4) 8.1-8.5 000825 Wil d l if e Internatio nal ltd. -22- PROJECT NO.: 454A-105 APPENDIX 0 Analyses o f Pesticides, Organics, Metals and Other Inorganics in W ildlife International Ltd. Well Water1 ANALYSIS MEASURED CONCENTRATION Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon2 Total Cyanide < < < 286 0.050 1.0 10.0 mg/L mg/L mg/L Pg/L Organochlorines and PCBs Aldrin Alpha BHC BetaBHC Delta BHC Gamma BHC (Lindane) Chlordane DDD.ppDDE, pp' DDT, pp' Dieldnn Endosulfan,A Endosulfen, 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 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0360 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 P&L Mg Pg Hg PL P&L pgA \i%/L Pg P&L V&L P&L V&L Pg lig/L (x&L Pg V%/L P&L P&L P&L WL Metals and Other Inorganics Aluminum3 Arsenic3 Beryllium3 Cadmium3 Calcium3 Chromium3 Cobalt3. Lead3 Magncsiunr Manganese3 Mercuiy Molybdenum3 Nickel3 Potassium3 Selenium3 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 1.0 213 20.0 P&L P#L WL WL mg/L MgAlig/L P&L Pg PgAmg/L JigA. V&L 1% mg/L P&L P&L mgA, JJ^g/L Analyses performed byQST Environmental, Gainesville, Floridafor samples collected on November 3 through November 7,1997. Analyses performed by Wildlife International Ltd. for the sample collected on November 5,1997. Analyses performed by Wildlife International Ltd. for samples collected on November 5 through 7,1997. 000826 W il d l if e Inter n a tio n a l ltd. -23- A P P E N D IX m PROJECT NO.: 454A-105 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-105 000827 W il d l if e In ter n a tio n a l ltd. -24- PROJECT NO.: 454A-105 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Freshwater Mussel W ILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-105 PRINCIPAL INVESTIGATOR MANAGEMENT: W illard B. Nixon, P h .D / Manager, Analytical Chemistry 4-QJl-OO DATE 000S28 AMENDED W il d l if e In te r n a tio n a l ltd. -25 - PROJECT NO.: 454A-105 Introduction Freshwater samples were collected from an acute toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the freshwater mussel (Unio complamatus). This study was conducted by W ildlife International Ltd. and identified as Project No.: 454A-105. 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 v May 28, 30 and June 1, 1999. ' Samples 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 standards and matrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned W ildlife International Ltd. identification number 4526 upon receipt. The-intemal 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 W ildlife International Ltd. and entitled "Analytical Method Validation for the Determination o f PFOS in Freshwater, Saltwater, and Algal Media". 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 w ater 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 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- 000829 W il d l if e In ter n a tio n a l ltd. -26- PROJECT NO.: 454A-105 Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Ci* analytical column (100 mm x 2 mm I.D ., 3 pm particle size). The instrument param eters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit o f Quantitation Calibration standards o f PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00229 to 0.0274 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 concentration ratios (PFOS : internal standard) o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit o f quantitation (LOQ) for these analyses was set at 0.115 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the m atrix blank samples (50.0). M atrix Blank and Fortification Samples Three 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 chromatogram o f a m atrix blank is presented in Figure 5. Freshwater was fortified at 0.915, 45.7 and 110 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 94.7%. A representative chromatogram o f a matrix fortification is presented in Figure 6. 000830 AMENDED W il d l if e Inter n a tio n a l lTM. -27- PROJECT NO.: 454A-105 Example Calculations Sample number 454A-105-16, nominal concentration o f 5.7 mg a.i./L in freshwater. Initial Volume: 0.100 mL Final Volume: 25.0 mL Dilution Factor: 250 PFOS Peak Area: 596872 Internal Standard Peak Area: 401298 Peak Area Ratio: 1.4874 Calibration curve equation. Slope: 6.30785 Intercept: 0.04889 PFOS (pg a.i./L) at instrument (Peak area ratio - (Y-intercept)) x I.S. Concentration Slope (1.4874 - 0.04889) x 100 pg a.i./L 6.30785 = 22.8 pg a.i./L Note: I.S. = internal standard. PFOS (mg a.i./L) in sample PFOS (pg a.i./L) at instrument x Dilution Factor 1000 pg/mg 22.8 x 250 T . 000821. = 5.70 mg a.i./L AMENDED Wil d l if e In t e r n a t io n a l ltd. -28- PROJECT NO.: 454A-105 Percent o f Nominal Concentration PFOS (mg a.i./L) in sample = ------------------------------------ x 100 PFOS (mg a.i./L) nominal Calculated recovery: 98.9% Note: manual calculation may differ. RESULTS Sample Analysis Freshwater samples were collected from the acute toxicity study with the freshwater mussel (Unio complamatus) at test initiation, May 28, 1999 (Hour 0), on May 30, 1999 (Hour 48), and at test termination, June 1, 1999 (Hour 96). The measured concentrations o f PFOS in the samples collected at initiation o f exposure o f the test organisms (Horn 0) ranged from 73.7 to 96.0% o f the nominal concentrations. Samples collected at Hour 48 had a measured-concentration range o f 81.2 to 98.9% o f nominal values. Samples collected at test termination (Hour 96) had a measured concentration range o f 88.5 to 130% o f nominal values (Table 4). A representative chromatogram o f a test sample is shown in Figure 7. 000S32 AMENDED W ild life International ltd. -29- PROJECT NO.: 454A-105 Table 1 Typical LC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC M ass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). V* ANALYTICAL COLUMN: Keystone Betasil C ^ 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% Methanol : 28.0% NANOpure W ater containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 6.4 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.4 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 000833 W il d l if e Intern atio na l ltd. -30- PROJECT NO.: 454A-105 Table 2 M atrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-105-) MAB-4 MAB-2 Sample Type M atrix Blank v.J M atrix Blank Measured Concentration o f PFOS1 (mg a.i./L) . <LOQ <LOQ A O O MAB-3 M atrix Blank 1 The limit o f quantitation (LOQ) was 0.1 IS mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). 000S34 AMENDED W il d l if e In ter n a tio n a l ltd. -31 - PROJECT NO.: 454A-105 Table 3 M atrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-105-) M A S-10 MAS-4 MAS-7 MAS-11 MAS-5 MAS-8 Concentrations o f PFOS (mg a.i./L) Fortified M easured 0.915 0.915 0.915 0.780 0.952 0.858 45.7 43.7 45.7 43.5 45.7 42.7 Percent Recovered 85.2 104 93.8 95.7 95.0 93.4 MAS-12 110 94.2 85.5 MAS-6 110 113 103 MAS-9 110 106 96.7 ' Mean = 94.7 Standard Deviation = 6.50 CV =6.86 - 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. 000835 AMENDED W il d l if e In t e r n a t io n a l ltd. -32- PROJECT NO.: 454A-105 Table 4 Measured Concentrations o f PFOS in Freshwater Samples from a Freshwater Mussel Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-105-) 1 s'2 13 14 25 26 Sampling Time (Hours) 0 0 48 48 96 96 PFOS Measured Concentration1 (mg a.i./L) < LOQ < LOQ < LOQ <LOQ < LOQ < LOQ Percent of Nominal -- - -- -- -- 5.7 3 0 40 15 48 16 48 27 96 28 96 5.47 94.9 4.93 85.5 5.18 89.8 5.70 98.9 5.24 90.9 5.26 91,2 11 5 0 60 17 48 18 48 29 96 30 96 11.4 96.0 10.1 84.3 11.2 94.1 10.5 88.2 10.9 91.3 15.4 130 23 7 0 80 19 48 20 48 31 96 32 96 19.0 83.3 16.8 73.7 18.7 81.8 18.7 81.8 22.9 100 22.4 98.0 46 9 0 10 0 21 48 22 48 33 96 34 96 37.2 81.4 40.6 88.8 37.1 81.2 39.5 86.4 48.2 105 40.5 88.5 91 11 0 69.0 75.4 12 0 23 48 74.7 81.6 81.3 88.9 24 48 77.6 84.8 35 96 36 96 88.2 96.4 85.7 93.7 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. 1 The limit of quantitation (LOQ) was 0.115 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (50,0), AMENDED 000S36 W il d l if e In ter n a tio n a l ltd. -33- PROJECT NO.: 454A-105 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER 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. i Dilute matrix 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 of sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. i Ampulate samples and submit for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in freshwater. - 000837 W il d l if e In te r n a tio n a l ltd. -34- PROJECT NO.: 454A-105 Figure2. A typical calibration curve for PFOS. Slope = 6.30785; Intercept = 0.04889; r 0.9986. 000838 AMENDED Wil d l if e In t e r n a t io n a l ltd. -35- PROJECT NO.: 454A-105 Figure 3. A representative ion chromatogram o f a low-level (0.00229 mg a.i./L) PFOS standard. 000839 AMENDED Wil d l if e International ltd. -36- PROJECT NO.: 454A-105 intensity: 50611 cps 152 Figure 4. A representative ion chromatogram o f a high-level (0.0274 mg a.i./L) PFOS standard. 000840 AMENDED W il d l if e In ter n a tio n a l ltd. -37- PROJECT NO.: 454A-105 intensity: 118 cps Figure 5. A representative chromatogram o f a matrix blank sample (454A-105-MAB-2). The arrow indicates the retention time o f PFOS. 000841 W il d l if e In ter n a tio n a l ltd. -38- PROJECT NO.: 454A-105 Intensity: 42545 cps 152 Figure 6. A representative chromatogram o f a 1.00 mg a.i./L matrix fortification sample (454A-105-MAS-4). 000S42 W il d l if e In te r n a tio n a l ltd. -39- PROJECT NO.: 454A-105 lOO- OO- 80- 70 60- 50- 40- 30- 20- 10OH I I25~i 41r I ' i i 31 61 1.30 2.55 i 9l1 4106i 126 hi47rai 91 121 3.61 5.06 intensfty: 39233 cps 174 I 20Ia I I I 211 Scan 8.83 Time Figure 7. A representative chromatogram o f a 6.3 mg a.i./L test sample (454A-105-16). 000843 W il d l if e In t e r n a t io n a l ltd. -40- PROJECT NO.: 454A-105 APPENDIXIV 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, test concentrations and test substance number. 2. The protocol was amended to renew the test solutions at 48 hours. 3. The protocol was amended to specify that GLP analyses o f well water will be reported. 4. The protocol was amended to reduce the loading rate. 5. The weight and length of the mussels was determined by weighing and measuring 20 individuals from the same lot of test organisms. 6. The mussels were indiscriminately distributed to the test chambers. 000S44 W il d l if e In te r n a tio n a l ltd. -42- PROJECT NO.: 454A-105 APPENDIX VI Report Amendment 1. Original Report: Pages 1-4,6 and 24 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 of 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 thepurity ofthe test substance as determined by the Sponsor in a reanalysis o f the test substance (FC-95, Lot 217). Test concentrations originally were based on the reported purity of 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 of 90.49% at the request o f the Sponsor. 000845 AMENDED W il d l if e In ter n a tio n a l ltd. -43- PROJECT NO.: 454A-105 APPENDIX VI -Continued- Report Amendment Y /& & / DATE Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: Quality Assurance DATE 000846 AMENDED Wil d l if e International ltd. PROJECT NO.: 454A-105 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STUDY TITL E: PFOS. A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL PR O T O C O L NO.: 454/042199/MUS/SUB454 AMENDM ENT NO.: 1 SPONSOR: 3M Corporation PR O JE C T NO.: 454A-105 E F F E C T IV E D ATE: May 20, 1999____________________________________________________ _ AMENDMENT: Page 2 Add: Experimental Start Date: 5/28/99 Experimental Termination Date: 6/1/99 Test Concentrations: Negative Control, 6.3, 13, 25, 50 and 100 mg a.i./L Test Substance No.: 4675 REA SON : The above information was not known when the protocol was signed by the Study Director. AM ENDM ENT: EXPERIMENTAL DESIGN. Page 3 - Add: Test solutions will be renewed at approximately 48 hours. New test solutions will be prepared and the mussels will be transferred from the old to the new test solutions. REA SO N : Test solutions will be renewed at 48 hours to improve the w ater quality. A M ENDM ENT: Dilution Water. Page 5 Change: Analyses will be performed at least once annually to determine the concentrations of selected organic and inorganic constituents o f the well water and the results o f the analyses will be summarized in the final report. To: Analyses will be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents o f the well water and the results o f the most recent GLP compliant analyses will be summarized in the final report. R EA SO N : To specify that GLP analyses will be reported. 000847 a 4 S 4 \l OSXamendl * A' W il d l if e International ltd. PROJECT NO.: 454A-105 Page 2 of 2 AM ENDM ENT: Test Apparatus. Page 5 Change: Test chambers will be 25-L, polyethylene aquaria filled with approximately 15 L o f water To: Test chambers will be 25-L, polyethylene aquaria filled with approximately 20 L o f water. REASON: To reduce the loading rate. DATE h~ ? A 4 DATE ' y li'ih q DATE a454\105\am endl 000848 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454A-105 Page 1 o f 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL PROTOCOL NO.: 454/042199/MUS/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454A-105 DATE OF DEFACTO DEVIATION: June 1,1999 DEVIATION: The protocol states that valve heights and weights of the individual mussels will be measured at the end of the test. The valve heights and weights of 20 representative mussels from the same lot were measured. REASON: Biologist oversight The mussels used in the test were shipped to the Sponsor before they were weighed and measured. The mussels used in the test and the mussels which were weighed and measured were indiscriminately selected from the same lot of test organisms. Consequently, it is the best judgment of the Study Director that this deviation did not adversely affect the results of the study. STUDY DIRECTOR 7 7 LABORATORY MANAGEMENT KRD454a\]05\prdl < //-s"/7 ? DATE DATE 000849 W il d l if e Internatio nal l t d . PROJECT NO.: 454A-105 Page 1 o f 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL PROTOCOL NO.: 454/042199/MUS/SUB454 DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454A-105 DATE OF DEFACTO DEVIATION: May 28,1999 DEVIATION: The protocol states that to control bias, freshwater mussels will be randomly assigned to exposure chambers at test initiation. The mussels were actually indiscriminately distributed (a random number table was not used to distribute the mussels). REASON: Biologist oversight. It is the best judgment of the Study Director that this deviation did not adversely affect the results of the study. STUDY DIRECTOR fe/a* / ? ? DATE LABORATORY MANAGEMENT KRD454a\105\prd2 DATE 000350 PROTOCOL PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL 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 <l r Wildlife 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue . St. Paul,Minnesota 55144 international ltd 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 April 21,1999 000853. W il d l if e In te r n a tio n a l, ltd 2- - PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL SPONSOR: 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 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 MANAGEMENT: Henry 0 . Krueger, Ph.D. Director of Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental Start Date: ___________________________ Experimental Termination Date: _____ Project No.: - / O S " ________________________________ Test Concentrations: Test Substance No.: __________ Reference Substance No. (if applicable): PROTOCOL APPROVAL PROTOCOL NO. 454/042199/MUS/SUB454 DATE _____ DATE x 7 DATE 000852 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -3- INTRODUCTION Wildlife International, Ltd. will conduct a static acute toxicity test with the freshwater mussel 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 Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates andAmphibians (3). Raw data for all work performed at Wildlife International, Ltd and a copy of the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. PURPOSE The purpose of this study is to determine the acute effects of a test substance on the freshwater mussel during a 96-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Freshwater mussels 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 freshwater mussels in each chamber for a total of 20 freshwater mussels per test concentration. Nominal test concentrations will be selected in consultation with the Sponsor, and will be based upon information such as the results of 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 of the test substance in water (whichever is lower). Generally, each test substance concentration used in the definitive test will be at least 60% of the next higher concentration unless information concerning the concentration-effect curve indicates that a different dilution factor would be more appropriate. Water samples from each test chamber will be collected at specified intervals for analysis of the test substance. Results of analyses will be used to calculate mean measured test concentrations. To control bias, freshwater mussels will be randomly assigned to exposure chambers at test initiation. No other potential sources of bias are expected to affect the results of the study. Observations of mortality and other clinical signs will be made throughout the 96-hour test period. Cumulative percent mortality 000353 PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l, ltd -4- observed in the treatment groups will be used to calculate, when possible, LC50 values at 24,48, 72 and 96 hour intervals. The no-mortality concentration will be determined. MATERIALS AND METHODS Test Substance 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 in the study. If written verification of 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 IDENTIFICATION O F TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. 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 of the study. Preparation o f Test Concentrations 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 organisms. Test Organism The freshwater mussel has been selected as the test species for this study. Freshwater mussels are representative of an important group of aquatic invertebrates, and have been selected for use in the test at the request o f the Sponsor. Valve height and weights of the individual mussels will be measured at the end of the test. Mussels will be obtained from a commercial supplier or hatchery, and the identity of the species will be verified by the supplier. Freshwater mussels 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 3 C per day. Freshwater mussels will be held at the test temperature for a minimum of 7 days prior to test initiation. Freshwater mussels will not be used in the test if they show signs of disease or stress or if more than 5% die during the 7 days prior to the test. At test initiation, the freshwater mussels will be collected from holding or acclimation tanks and transferred to the test chambers. At test termination all 000354 PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l, ltd -5 - remaining live mussels will be sacrificed for collection o f tissue samples. The tissue samples will be composited by replicate, frozen and shipped to the Sponsor for possible analysis. Dilution Water Water used for the holding and testing of freshwater mussels will be obtained from a well approximately 40 meters deep located on the Wildlife 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 the water will be filtered to 0.45 m in order to remove fine particles. Water 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 CaC03 Alkalinity, mg/L as CaC03 pH Specific Conductance, /mhos/cm 145 190 8.1 330 Hardness, alkalinity, pH and specific conductance will be measured weekly to monitor the consistency of the well water. Means and ranges of 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 of selected organic and inorganic constituents o f the well water and results of the analyses will be summarized in the final report. Test Apparatus Test chambers will be 25-L, polyethylene aquaria filled with approximately 15 L of water. Test chambers will be positioned in an environmental chamber or temperature-controlled water bath to maintain a temperature of 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 of 16 hours of light and 8 hours o f dark will be controlled with an automatic timer. A 30-minute transition period of low light intensity will be provided when lights go on and off to avoid sudden changes in light intensity. Light intensity will be measured at test initiation with a SPER Scientific Ltd. light meter or equivalent 000S55 PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l, ltd 6- - The target test temperature will be 22 2C. Temperature will be measured in all replicates at the beginning and end of the test using a liquid-in-glass thermometer. Temperature also will be measured with a continuous recorder in a beaker adjacent to the test. Recorder measurements will be verified with a liquidin-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 of pH will be made in alternating replicates of 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. Hardness, alkalinity, and specific conductance will be measured in the dilution water and low, middle and high test substance concentrations at test initiation and at test termination. Hardness and alkalinity measurements will be made by titration using procedures based on methods in Standard Methods fo r the Examination o f Water and Wastewater (4). Specific conductance will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter, or equivalent. 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. open or gaping shell) in the mussel after gentle prodding. All clinical observations will be noted. Sampling for Analytical Measurements Water samples will be collected from each test chamber at the beginning of 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% mortality occurs in any treatment, then sampling of 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: 000S56 PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l, ltd -7 - PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group 0 Hours 48 Hours 96 Hours Control Level 1-Low Concentration Level 2 Level 3 Level 4 Level 5-High Concentration 222 222 222 222 222 222 Totals 12 12 12 Total Number of Verification Samples = 36 The above numbers of 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 o f the study. At the discretion of 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. Analytical Chemistry Chemical analysis of the 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 concentration-response pattern allows calculation of an LC50 value, the data will be analyzed using the computer software of C.E. Stephan (5). 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 (6,7,8). The LC50 value will be calculated, when possible, using mortality data collected at 24,48,72 and 96 hours. The no-mortality concentration will be determined. 000S57 PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd 8- - RECORDS TO BE MAINTAINED Records to be maintained for data generated by Wildlife International, Ltd. will include, but not be limited to: 1. A copy of the signed protocol. 2. Identification and characterization of the test substance, if provided by the Sponsor. 3. Dates of initiation and termination of the test. 4. Test organism history, holding and acclimation records. 5. Stock solution calculation and preparation, if applicable. 6. Observations. 7. Water 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 of test concentrations. 12. Copy of final report. FINAL 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 of 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 statement of compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives 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 of the test substance under the conditions of administration, if provided by the Sponsor. 7. A description of the methods used to conduct the test. 8. A description of the test organisms, including the source, scientific name, age or life stage, lengths and weights of a representative group of test organisms, feed types, light intensity and photoperiod. 9. A description of the preparation of the test solutions. 000S58 PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l, ltd -9 - 10. The methods used to allocate organisms to test chambers and begin the test, the number of organisms and chambers per treatment, and the duration of the test. 11. A description of circumstances that may have affected the quality or integrity of the data. 12. The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study. 13. A description of the transformations, calculations, and operations performed on the data, a summary and analysis of the biological data and analytical chemistry data, and a statement of the conclusions drawn from the analyses. 14. Statistical methods used to evaluate the data. 15. A graph of the concentration-mortality curve at the end of the test. 16. The signed and dated reports of each of 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. 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 the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the final report that is being amended and the reasons for the amendment, and will be signed by the Study Director. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of 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 final report. GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 and/or Part 792); OECD Principles of Good Laboratory Practice (OCDE/GD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International, Ltd. is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement of compliance with Good Laboratory Practices will be prepared for all portions of the study conducted by Wildlife International Ltd. The Sponsor will be 0085 PROTOCOL NO. 454/042199/MUS/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 -10- responsible for compliance with Good Laboratory 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 of the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. PROTOCOL NO. 454/042199/MUS/SUB454 000S60 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a 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 andMarine. 2 Organisation for Economic Cooperation and Development 1993. OECD Guidelines for Testing o f Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992. 3 ASTM Standard E729-88a. 1994. Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 APHA, AWWA,WPCF. 1985. StandardMethodsfor the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 6 Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 7 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 8 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. PROTOCOLNO. 454/042199/MUS/SUB454 000361 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 12- APPENDEXI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I. Test Substance Identity (name to be used in the report): PFOS (Perfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A______________ 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 accordancewith GLP Standards? _____ Yes x No ID. Test Substance Storage Conditions Please indicate the recommended storageconditions at Wildlife International Ltd. _____ Ambient__________________________________________________________________________ Has the stability ofthe test substance underthese storage conditions been determined in accordancewith GLP Standards? , _____ Yes x No Other pertinent stability information: IV. Test Concentrations: _____ x Adjust test concentration to 100% ai. based upon thepurity (%) given above. Do not adjust test concentration to 100% ___________ a i Test thematerial AS IS. V. Toxicity Information: Mammalian: RalLD50 251 mg/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphnia magna: 27 me/L__________ Rainbow Trout: 11 mg/L Daphnia maena: 50 mg/L__________ Fathead Minnow: 38 me/L Other Toxicity Information (includingfindings ofchronic and subchronic tests): Please see MSDS______________________________________________________________ PROTOCOL NO. 454/042199/MUS/SUB454 000S62 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -13- APPENDIXII Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination of Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f the above method will be maintained in the raw data. The actual methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO. 454/042199/MUS/SUB454 000S63 3M LAB REQUEST NO. U2723
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