Document Bvvwx91vZ2N6MdmzwLMgavVo4

m n < -o o ^ PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE CLADOCERAN (.Daphnia magna) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-104 3M LAB REQUEST NO. U2723 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 and OECD Guideline 202 AUTHORS: Kurt R Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: December 10,1998 STUDY COMPLETION DATE: June 4,1999 AMENDED REPORT DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, M innesota 55144 Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410)822-8600 Page 1 o f 43 0006S6 AMENDED W il d l if e International ltd. 2- - PROJECT NO.: 454A-104 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-104 STUDY COMPLETION DATE: June 4,1999 AMENDED REPORT DATE:' 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 MonographNo. 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: Senior Biologist DATE 000687 AMENDED W il d l if e In tern atio na l ltd. -3 - PROJECT NO.: 454A-104 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles 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. 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 REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: Test Substance Preparation February 15,1999 February 15,1999 February 16,1999 Analytical Sample Preparation February 16,1999 February 17,1999 February 19,1999 Water Chemistry Measurements February 17,1999 February 17,1999 February 19,1999 Biological Data and Draft Report . March 17 and 18, 1999 March 18,1999 March 29,1999 Analytical Data and Draft Report March 2 3 -2 5 ,1 9 9 9 March 25,1999 March 26,1999 Final Report Amended Report June 4,1999 April 20 and 25,2000 June 4,1999 April 25,2000 June 4,1999 April 25,2000 ratios H. Coleman Quality Assurance Representative DATE 0006S8 AMENDED W il d l if e In ter n a tio n a l ltd. -4 - REPORT APPROVAL PROJECT NO.: 454A-104 SPONSOR: 3M Corporation TITLE: PFOS: A 48-Hour Static Acute Toxicity Test with the Cladoceran (.Daphnia magna) V' WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-104 ' STUDY DIRECTOR: MANAGEMENT: Non-Target Plants DATE DATE ' 000689 AMENDED W il d l if e In t e r n a t io n a l ltd. -5 - PROJECT NO.: 454A-104 TABLE OF CONTENTS Title/Cover Page........................................................................................................................................................1 Good Laboratory Practice Compliance Statement.......................................................................................... 2 Quality Assurance Statement...................................................................................................................................3 Report Approval.................................................................................................................................. >* f Table o f Contents..................................................................................................................................................... 5 Summary......................................................................................... Introduction.............................................................................................................................................................. 8 O bjective...................................................................................................................................................................8 Experimental Design............................................................................................................................ 8 Materials and Methods.............................................................................................................................................9 Results and Discussion............................................................................................................. 12 Conclusions........................................................................................................... ......i........................................ 13 References........................................................................................................J-.................................................. 14 4 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 Chambers.......................................16 Table 3 - Cumulative Percent Mortality/Immobilily and Treatment-Related Effects........................................ 17 Table 4 - EC10, EC50 and EC90 Values..................................................................................................... 18 000690 W il d l if e In te r n a tio n a l ltd. - 6- PROJECT NO.: 454A-104 TABLE OF CONTENTS - Continued FIGURE Figure 1. Concentration-Response Curve (48 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, Metals and Other Inorganics in Wildlife International Ltd. Well W ater...................................................................................21 Appendix HI - The Analysis o f PFOS in Freshwater in Support o f Wildlife International Ltd. Project No.: 454A -104..............................................................22 Appendix IV - Changes to Protocol.............................................................................. .................................. 40 Appendix V - Personnel Involved in the Study........................................... .................................................. 41 Appendix V I- Report Amendment.................................................................................................................. 42 000G91 AMENDED W il d l if e In t e r n a t io n a l ltd. -7 - PROJECT NO.: 454A-104 spo n so r : SPONSOR'S REPPRESENTATTVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL V LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: SUMMARY 3M Corporation Ms. Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 454A-104 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 48-Hour Static Acute Toxicity Test with the Cladoceran (.Daphnia magna) Negative Control, 11,2 0 ,3 3 ,5 6 and 91 mg a/L Experimental Start - February 16,1999 Biological Term ination-February 18,1999 Experimental Term ination-February 18,1999 48 Hours Neonate cladocerans (.Daphnia magna) W ildlife International Ltd. Cultures Easton, Maryland 21601 < 24 hours at test initiation AMENDED 000692 W il d l if e In ter n a tio n a l ltd. 8- - PROJECT NO.: 454A-104 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 o f the test was conducted from February 16,1999 to February 18,1999. Raw data generated by Wildlife International Ltd. and a copy of the final report are filed under Project Number 454A-104 in archives located on the Wildlife International Ltd. site. OBJECTIVE The objective of this study was to evaluate the acute toxicity of PFOS to the cladoceran, Daphnia magna, during a 48-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Daphnids 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 daphnids in each test chamber for a total of 20 daphnids per test concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results o f an exploratory rangefinding toxicity test. Nominal test concentrations selected were 12,20,33, 55 and 91 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples o f test water collected from each treatment and control group at the beginning of the test, at approximately 24 hours, and at test termination. Daphnids were indiscriminately assigned to exposure chambers at test initiation. Observations o f mortality/immobility and other clinical signs o f toxicity were made at approximately 3,24 and 48 hours after test initiation. Cumulative percent mortality and immobility observed in the treatment groups were used to estimate or calculate EC 10, EC50 and EC90 values at 24 and 48 hours. The no mortality/immobility concentration and the no-observed-effect-concentration (NOEC) were determined by visual interpretation o f the mortality, immobility and clinical observation data. AMENDED 000693 Wild life International ltd. -9 - PROJECT NO.: 454A-104 MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, PFOS: A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna). The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 (1), OECD Guideline for Testing of Chemicals, 202: Daphnia sp. Acute Immobilization Test and Reproduction Test (2) and ASTM Standard E-729-88a, Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebraies and Amphibians (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998 was assigned Wildlife International Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity o f 98.9% and an expiration date o f2008. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated March 9, 2000 indicated a purity o f 90.49%. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 12, 20, 33, 55 and 91 mg a.i./L, based on a test substance purity o f 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 91 mg a.i./L. The primary stock solution was mixed with an electric drum mixer with a stainless steel paddle for approximately 19.5 hours to aid in the solubilization o f the test substance. A fter 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 Organism The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group o f aquatic invertebrates and were selected for use in the test based upon past history o fuse and ease o f culturing in die laboratory. Daphnid neonates used in the test were less than 24-hours old and were obtained from cultures maintained by Wildlife International Ltd., Easton, Maryland. Identification o fthe original brood stock was verified by the Academy o f Natural Sciences, Philadelphia, PA. AMENDED 000694 W il d l if e In te r n a tio n a l ltd. - 10- PROJECT NO.: 454A-104 Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. Adult daphnids in the cultures were held for at least 28 days prior to collection o f the juveniles for testing. The adults showed no signs o f disease or stress during the holding period. During the 14-day period preceding the test, water temperatures ranged from 19.9 to 20.4C. The pH o f the water ranged from 8.1 to 8.3, and dissolved oxygen ranged from 8.3 to 8.8 mg/L. Instrumentation used for water measurements are described in the Environmental Conditions section o f this report. Neonate daphnids were obtained for testing from individual adult daphnids which were observed to have no neonates present less than 24 hours prior to test initiation. The progeny from 10 adults were used in the test. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred to 10-mL glass beakers. The daphnids then were transferred from the beakers to the test compartments. All transfers w oe performed below the air/water interface using a wide-bore pipet. Daphnids in the cultures were fed a mixture of yeast, Cerophyll, and trout chow, as well as a suspension of the freshwater green alga, Selenastrum capricomutum. The adults ware fed prior to test initiation, but neonates were not fed during the test. Test Apparatus Test chambers consisted of250-m L plastic (Nalgene) beakers containing 240 mL o f test solutioa The depth o f test solution in a representative test chamber was 6.4 cm. Test chambers were indiscriminately placed in a temperature-controlled water bath set to maintain a temperature o f 20 1G; Test chambers were labelledwith 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 L td site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, and pH o f the well water during the four-week period immediately preceding the test are presented in Appendix I. The well water was passed through a sand filter to remove particles greater than approximately 25 jm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water again was filtered (0.45 pm) to remove microorganisms and particles. The results o fperiodic analyses performed 000695 Wil d l if e Internatio nal ltd. - 11- PROJECT NO.: 454A-104 to measure the concentrations of selected contaminants in well water used by Wildlife International Ltd. are ELpresented in Appendix Environmental Conditions Lighting used to illuminate the cultures and test chambers during culturing and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod o f 16 hours o f light and 8 hours o fdarkness was controlled with an automatic timer. A 30-minute transition period v .j o f low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was approximately 359 lux at the surface of the water. Light intensity was measured using a SPER Scientific Ltd. light meter. Temperature was measured in each test chamber at the beginning and end o fthe test 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 201C. The pH anddissolved oxygen content of the water were measured in each test chamber at test initiation, at approximately 24 hours after test initiation and at the end o f the test. Hardness, alkalinity, specific conductance and total organic carbon (TOC) were measured in the dilution water at test initiation and at test termination. 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 on procedures in StandardMethodsfor the Examination o f Water and Wastewater (4). Total organic carbon was measured using a Shimadzu Model 5000 TOC analyzer. Observations Observations were made to determine the numbers o fmortalities and immobile organisms. Thenumbers of individuals exhibiting clinical signs o f toxicity or abnormal behavior also were evaluated. Observations were made approximately 3 ,2 4 , and 48 hours after test initiation. 000696 W il d l if e In ter n a tio n a l ltd. -12- PROJECT NO.: 454A-104 Statistical Analyses The 24 and 48-hour EC50 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). The no mortality/immobility concentration and NOEC were determined by inspection o f the mortality, immobility and clinical observation data. In this study, EC 10 and EC90 values were calculated when possible using the Bruce-Versteeg Method (9) because there were less than two concentrations with partial mortality or immobility (i.e., the probit method could not be used). Analytical Chemistry Samples were collected from mid-depth o f each replicate test chamber at the beginning o f the test, at approximately 24 hours 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 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 IE). Nominal concentration selected foruse in this study were 12,20,33,55 and 91 mg a.i./L. Samples collected at test initiation (0 Hours) had measured values that ranged from 85 to 112% o f nominal. Samples collected at 24 hours had measured values that ranged from 92 to 113% o fnominal, whereas measured values for samples taken at 48 hours ranged from 92 to 106% of nominal When measured concentrations o f the samples collected at test initiation, 24 hours, and at test termination were averaged, the mean measured concentrations for this study were 11,20,33,56 and 91 mg a.i./L. Mean measured concentrations were used in the estimation or calculation o f EC 10, EC50 and EC90 values. AMENDED 000697 W il d l if e In tern atio na l ltd. -13- PROJECT NO.: 454A-104 Observations and Measurements Measurements o ftemperature, dissolved oxygen and pH are presented in Table 2. Temperatures remained within the 20 1C range established for the test. Dissolved oxygen concentrations remained > 8.6 mg/L (95% of saturation) throughout the test. Measurements o f pH ranged from 8.2 to 8.6 during the test. Daily observations o f mortality, immobility and other clinical signs observed during the test are shown in Table 3. Daphnids in the negative control appeared healthy and normal throughout the test, although 5% mortality was observed at 48 hours. Daphnids in the 11 and 20 mg a.i./L treatment groups also appeared normal with no mortality/immobility or overt clinical signs o f toxicity. After 48 hours o f exposure, mortality/immobility in the 33, 56 and 91 mg ai./L treatment groups was 0, 35 and 100%, respectively. Although no mortality/immobility occurred in the 33 mg a.i./L treatment group, one daphnid was observed to be lethargic. Based on the 5% control mortality observed, this lethargy was not considered to be treatment-related. EC 10, EC50 and EC90 values and 95% confidence limits at 24 and 48 hours were estimated or calculated from the mortality/immobility data, and are shown in Table 4. CONCLUSIONS The 48-hour EC50 value for Daphnia magna exposed to PFOS was 61 mg a.i./L with 95% confidence limits o f 33 and 91 mg a.i./L. The no mortality/immobility concentration and NOEC were 33 mg a.i./L. AMENDED 000698 W il d l if e In ter n a tio n a l ltd. -14- PROJECT NO.: 454A-104 REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.1010. Aquatic invertebrate Acute Toxicity Test, Freshwater Daphnids. 2 O rganisation for Economic C ooperation and Development. 1984. Daphnia sp. Acute Immobilization Test and Reproduction Test. OECD Guideline for Testing o f Chemicals. Guideline 202. Paris. 3 ASTM Standard E729-88a. 1994. Standard Guidefo r ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. 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. W ater Pollution Control Federation, New York. 5 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. 6 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 7 Thom pson, W .R. 1947. Bacteriological Reviews. Vol. 13, No. 2. Pp. 115-145. 8 Finney, D .J. 1971. StatisticalMethods in Biological Assay. Second edition. Griffin Press, London. 9 Bruce, R.D. and DJ . Y ersteeg. 1992. A statistical procedure for modeling continuous toxicity data. Environmental Toxicology and Chemistry, Vol. 11, pp 1485-1494. 000699 W il d l if e In ter n a tio n a l ltd. -15- PROJECT NO.: 454A-104 Table 1 Summaiy o f Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Test Concentration (mg a.i./L) Negative Control 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Replicate Sampling Time (Hours) A ,' 0 B0 A 24 B 24 A 48 B 48 12 A 0 B0 A 24 B 24 A 48 B 48 20 A 0 B0 A 24 B 24 A 48 B 48 33 A 0 B A 24 B 24 A 48 B 48 55 A 0 B0 A 24 B 24 A 48 B 48 91 A 0 B0 A 24 B 24 A 48 B 48 1 The limit of quantitation (LOQ) was 4.58 mg a.i./L. Measured Concentration (mg a.i./L) <LOQ` <LOQ <LOQ <LOQ <LOQ <LOQ 10.5 10.6 11.5 12.5 10.9 12.0 17.2 18.1 22.8 21.6 21.4 18.8 30.2 34.1 34.0 36.1 31.3 34.0 50.5 49.9 57.0 63.0 56.8 56.4 87.6 102 90.1 84.4 88.7 92.4 Mean Measured Concentration (mg a.i./L) <LOQ 11 20 33 .' 56 91 Percent of Nominal 92 100 100 102 100 AMEoNoDEoDvoo W il d l if e In t e r n a t io n a l ltd. -16- PROJECT NO.: 454A-104 T ab le2 Temperature, Dissolved Oxygen and pH o f Water in the Test Chambers Sponsor 3M Corporation Test Substance: PFOS Test Organism: Cladoceran, Daphnia magna Dilution Water: _____ Well Water_______________________________________ Mean Measured Concentration (mg a.i7L) Negative Control Replicate A B 0 Hour1 Temp1234 ( C> ' 19.6 19.5 (mg/L) 8.9 8.9 pH 8.2 8.3 24 Hours DO (mg/L) pH 8.6 8.4 8.6 8.5 Temp (C) 20.2 19.9 48 Hours4 DO (mg/L) 8.6 8.6 pH 8.5 8.5 11 A 19.5 8.9 8.3 8.6 8.5 20.1 8.7 8.5 B 19.4 8.9 8.3 8.6 8.5 20.1 8.6 8.5 20 A 19.4 8.9 8.3 8.6 8.5 20.1 8.6 8.5 B 19.4 8.9 8.3 8.6 8.5 20.0 8.7 8.6 33 A 19.4 9.0 8.3 8.6 8.5 20.0 8.6 8.6 B 19.4 9.0 8.3 8.6 8.5 20.1 8.6 8.6 56 A 19.3 9.1 8.4 8.6 8.5 20.0 8.7 8.6 B 19.3 9.1 8.4 8.6 8.5 '.2 0 .0 8.7 8.6 91 A 19.4 9.1 8.5 8.6 8.5 20.1 8.6 8.6 B 19.3 9.1 8.5 8.6 8.5 20.0 8.7 8.6 1 The O-hourdilution water measurements for hardness, alkalinity, specific conductance and TOC were 136 mg/LasCaCOj, 178 mg/Las CaCOj, 315 mhos/cm and < 1 mgC/L, respectively. 2 Temperature measured continuously during the test ranged from approximately 19.5 to 20.5"C. 3 A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20C in freshwater. 4 The test termination dilution water measurements for hardness, alkalinity, specific conductance and TOC were 140 mg/L as CaCOj, 180 mg/L as CaCOs, 315 imhos/cm and < 1 mgC/L, respectively. AMENDED 000701 W il d l if e Internatio nal ltd. -17- PROJECT NO.: 454A-104 Table 3 Cumulative Percent Mortality/Immobility and Treatment-Related Effects1 Sponsor Test Substance: Test Organism: Dilution Waten 3M Corporation PFOS Cladoceran,Daphnia magna Well Waler ___________ Mean Measured Concentration (mg a.iVL) Replicate Daphnia/ Number Replicate Dead Negative Control A B 10 0 10 0 3 Hours Number Immobile 0 0 Effects 10 AN 10 AN 11 A 10 0 0 10 AN B 10 0 0 10 AN 20 A 10 0 0 10 AN B 10 0 0 10 AN 33 A 10 0 0 10 AN B 10 0 0 10 AN 56 A 10 0 0 10 AN B 10 0 0 . JO A N 91 A 10 0 0 . B 10 0 0 1 Observed Effects: AN = Appears Normal, C = Lethargy. 10AN 10 AN Number Dead 0 0 24 Hours Number Immobile 0 0 Effects 10 AN 10 AN 0 0 10 AN 0 0 10 AN 0 0 10 AN 0 0 10 AN 0 0 10 AN 0 0 10 AN 0 0 9AN,1C 0 0 10 AN 3 0 5AN.2C 4 0 4AN.2C Percent Immobile and Dead 0 0 0 0 0 35 Number Dead 1 0 48 Hours Number Immobile 0 0 Effects 9 AN 10 AN Percent Immobile and Dead 5 0 0 10 AN 0 0 0 10 AN 0 0 10 AN 0 0 0 10 AN 0 0 10 AN 0 0 0 9AN.1C 5 0 2A N JC 35 2 0 4AN,4C 10 0 10 0 -- 100 - ................ 000702 AMENDED W il d l if e In ter n a tio n a l ltd. -18- PROJECT NO.: 454A-104 Table 4 EC10, EC50 and EC90 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 3M Corporation PFOS Cladoceran, Daphnia magna Well Water Lower 95% EC10 Confidence (mg a.i./L) Limits 82 81 Upper 95% Confidence Limits 83 Statistical Method Bruce-Versteeg 48 Hours Time 24 Hours 53 EC50 (mg a.i./L) >91 <11 Lower 95% Confidence Limits __i >91 Upper 95% Confidence Limits __t Bruce-Versteeg Statistical Method Visual Inspection 48 Hours Time 24 Hours 61 EC90 (mg a.i./L) >91 33 Lower 95% Confidence Limits 91 Upper 95% Confidence Limits __i Binomial Statistical Method - Visual Inspection 48 Hours 63 <11 >91 Bruce-Versteeg 1Confidence limits could not be calculated with the mortality/immobility data obtained. 000703 AMENDED W il d l if e In t e r n a t io n a l ltd. - 19- Figure 1. Concentration-Response Curve (48 Hour Data) PROJECT NO.: 454A-104 000704 AMENDED W il d l if e In te r n a tio n a l ltd. -20- PROJECT NO.: 454A-104 APPENDEXI Specific Conductance, Hardness, Alkalinity and pH o f Well W ater Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: 3M Corporation PFOS Cladoceran, Daphnia magna Specific Conductance (/mhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) pH Mean 313 (N = 4) 132 (N = 4) 178 (N = 4) 8.3 (N = 4) Range 3 1 0 -3 1 5 128 -1 3 6 176-178 8 .2 -8 .3 000705 W il d l if e In t e r n a t io n a l Ltd . - 21- PROJECT NO.: 454A-104 APPENDEXn Analyses o f Pesticides, Organics, Metals and Other Inorganics in Wildlife International Ltd. Well W ater1 ANALYSIS MEASURED CONCENTRATION Miscellaneous M easurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon2 Total Cyanide O rganochlorines and PCBs Aldrin Alpha BHC BetaBHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' DDT, pp' Dieldnn Endosulfen, A Endosulfen, B Endosulfen Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 M etals and O ther Inorganics Aluminum3 Arsenic3 Beryllium3 Cadmium3 Calcium3 Chromium3 Cobalt3 Lead3 Magnesium3 Manganese3 Mercury Molybdenum3 Nickel3 3 Selenium Sve?" Sodium3 Zinc3 286 < 0.050 < 1.0 < 10.0 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < , 0.720 < 0.720 < 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 Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L g/L Mg/L Mg/L mg/L MMgg/L Mg Mg/L Mg/L mg/L Mg/L g/L g/L g/L mg. g/L Mg/L mg/L Mg/L * Analyses performed by OST Environmental, GaincsviUe,Florida for samples collected on November3 through November 7,1997. Analyses performed by Wildlife International Ltd. for the sample collected on November S, 1997. Analyses performed by Wildlife International Ltd. for samples collected on November 5 through 7,1997. 000706 W il d l if e In te r n a tio n a l ltd. -22- A PP E N D IX m PROJECT NO.: 454A-104 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-104 000707 W il d l if e International ltd. -23- PROJECT NO.: 454A-104 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 48-Hour Static Acute Toxicity Test with the Cladoceran (.Daphnia magna) v, f WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-104 PRINCIPAL INVESTIGATOR: MANAGEMENT: W illard B. Nixon, Ph.D/ Manager, Analytical Chemistry ' 4- 35-00 DATE DATE 000708 AMENDED W il d l if e Intern atio na l ltd. -24- PROJECT NO.: 454A-104 Introduction Freshwater samples were collected from a static acute aquatic toxicity study designed to determine the effects of PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the cladoceran (Daphnia magna). This study was conducted by Wildlife International Ltd. and identified as Project No.: 454A-104. 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 16, 17 and 18, 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 W ildlife 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 W ildlife International Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II o f W ildlife International Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. Concentrations o f the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- 000709 W il d l if e Inter n a tio n a l ltd. -25- PROJECT NO.: 454A-104 Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C u 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 w ater solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00915 to 0.0457 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 limit o f quantitation (LOQ) for these analyses was set at 4.58 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 (500). 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 matrix blank is presented in Figure 5. Freshwater was fortified at 9.15, 45.7 and 91.5 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 96.2%. A representative chromatogram o f a m atrix fortification is presented in Figure 6. 000710 AMENDED W il d l if e In t e r n a t io n a l ltd. -26- PROJECT NO.: 454A-104 Example Calculations Sample number 454A-104-27, nominal concentration o f 12 mg a.i./L in freshwater. Initial Volume: 0.100 mL Final Volume: 50.0 mL Dilution Factor: 500 PFOS Peak Area: 302788 Internal Standard Peak Area: 585991 Peak Area Ratio: 0.5167 Calibration curve equation. Slope: 0.02117 Intercept: 0.05525 Curve is linear. PFOS (pg a.i./L) at instrument PFOS (mg a.i./L) in sample Peak area ratio - (Y-intercept) Slope 0 .5 1 6 7 -0 .0 5 5 2 5 0.02117 21.8 PFOS (pg a.i./L) at instrument x Dilution Factor 1000 21.8 x 500 1000 10.9 000711 AMENDED W il d l if e Internatio nal ltd. -27- PROJECT NO.: 454A-104 Percent o f Nominal Concentration PFOS (mg a.i./L) in sample = ----------------------------------- x 100 PFOS (mg a.i./L) nominal Calculated recovery: 91.6% Note: manual calculation may differ. RESULTS Sample Analysis Freshwater samples were collected from the acute toxicity study with the cladoceran (Daphnia magna) at test initiation, February 16, 1999 (Hour 0), on February 17, 1999 (Hour 24), and at test termination, February 18, 1999 (Hour 48). The measured concentrations o f PFOS in the samples collected at initiation o f exposure o f the test organisms (Hour 0) ranged from 85.5 to 112% o f the nominal concentrations. Samples collected at Hour 24 had a measured concentration range o f 92.2 to 115% o f nominal values. Samples collected at test termination (Hour 48) had a measured concentration range o f 91.6 to 106% of nominal values (Table 4). A representative chromatogram o f a test sample is shown in Figure 7. 000712 AMENDED W il d l if e International ltd. -28- PROJECT NO.: 454A-104 Table 1 Typical LC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). v ANALYTICAL COLUMN: Keystone Betasil Q g 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: 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 000713 Wil d l if e In ter n a tio n a l ltd. -29- PROJECT NO.: 454A-104 Table 2 M atrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-104-) MAB-1 MAB-2 MAB-3 Type M atrix Blank M atrix Blank M atrix Blank a o V Measured Concentration o f PFOS1 (mg a.i./L) <LOQ <LOQ :samples (500). 000714 AMENDED W il d l if e In ter n a tio n a l ltd. -30- PROJECT NO.: 454A-104 Table 3 M atrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-104-) MAS-1 MAS-4 MAS-7 Concentrations o f PFOS (mg a.i./L) Fortified M easured 9.15 v 9.15 9.15 8.42 9.721 9.15 Percent Recovered 92.0 106 100 MAS-2 MAS-5 MAS-8 45.7 45.7 45.7 40.3 48.1 45.1 88.1 105 98.7 MAS-3 MAS-6 MAS-9 91.5 91.5 91.5 78.7 84.0 89.7 86.0 91.8 98.0 . Mean = 96.2 Standard Deviation =7.11 CV = 7.39 ______________________________________________________________________ N = 9__________ Note: Results and corrections for new test substance purity were generated using M acQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1Mean result of duplicate injections o f the rediluted original sample. Original result (5.48 mg a.i./L) was not included in the statistical analysis o f the data. Duplicate results for 454A-104-MAS-4 are 9.24 and 10.2. 000715 AMENDED W il d l if e In te r n a tio n a l ltd. -31 - PROJECT NO.: 454A-104 Table 4 Measured Concentrations o f PFOS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-104-) V1 2 13 14 25 26 Sampling Time (Hours) 0 0 24 24 48 48 PFOS M easured C oncentration1 (mg a.i./L) <LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal -- -- -- -- -- -- 12 - 20 30 40 15 24 16 24 27 48 28 48 50 60 17 24 18 24 29 48 30 48 10.5 88.5 10.6 89.5 11.5 96..8 12.5 105 10.9 91.6 12.0 101 17.2 85.5 18.1 90.2 22.8 113 21.6 107 21.4 106 18.8 93.6 33 7 0 30.2 91.7 80 19 24 20 24 31 48 32 48 34.1 104 34.0 103 36.1 110 31.3 95.1 34.0 103 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. 1The limit o f quantitation (LOQ) was 4.58 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 (500). 2Mean result o f duplicate injections o f the rediluted original sample. Original result (113 mg a.i./L) was not included in the statistical analysis o f the data. Duplicate results for 454A-104-23 are 90.6 and 89,6, AMENDED 000716 W il d l if e In te r n a tio n a l ltd. -32- PROJECT NO.: 454A-104 Table 4 (Continued) Measured Concentrations o f PFOS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 55 Sample Number (454A-104-) .9 10 21 22 33 34 Sampling Time (Hours) 0 0 24 24 48 48 PFOS Measured Concentration' (mg a.i./L) 50.5 49.9 57.0 63.0 56.8 56.4 Percent of Nominal 92.1 90.9 104 115 104 103 91 11 0 12 0 23 24 24 24 35 48 36 48 87.6 102 9 0 .12 84.4 88.7 92.4 95.7 112 98,5 92.2 96.9 101 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 4.58 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 (500). 2M ean result o f duplicate injections of the rediluted original sample. Original result (113 mg a.i./L) was not included in the statistical analysis o f the data. Duplicate results for 454A-104-23 are 90.6 and 89.6, 000717 AMENDED W il d l if e In ter n a tio n a l ltd. -33- PROJECT NO.: 454A-104 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. 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. 000718 W il d l if e Intern atio na l ltd. -34- PROJECT NO.: 454A-104 Concentration (mg aL/L) Figure 2. A typical calibration curve for PFOS. Slope = 0.02117; Intercept = 0.05525; r : 0.9961. 000719 AMENDED W il d l if e In t e r n a t io n a l ltd. -37- PROJECT NO.: 454A-104 intensity: 1772 cps 169 Figure 5. A representative chromatogram o f a matrix blank sample (454A -104-MAB-3). The arrow indicates the retention time o f PFOS. 000720 W il d l if e In te r n a tio n a l ltd. -40- PROJECT NO.: 454A-104 APPENDIX IV 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 delete annual feed analyses. 3. The protocol was amended to add the analytical method. 4. The protocol was amended to change the statistical analyses. 000721 W il d l if e Internatio nal ltd. -42- PROJECT NO.: 454A-104 APPENDDC 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 of 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 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 of 98.9%. The certificate of analysis dated March 9,2000 indicated a purity o f90.49%. Therefore, all test substance concentrations, including nominal concentrations, measured concentrations, and EC values, were recalculated and reported as mg a.i./L based cmthe 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 o f the Sponsor. 000722 AMENDED W il d l if e In ter n a tio n a l ltd. -43- APPENDIX VI -Continued- Report Amendment PROJECT NO.: 454A-104 Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: j-\, les H. Coleman Quality Assurance DATE DATE 000723 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-104 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST W ITH THE C LA D OCERAN (D aphnia m agna) P R O T O C O L NO.: 454/120198/DAP-48H1/SUB454 AM END M EN T NO.: 1 SPON SOR: 3M Corporation P R O JE C T NO.: 454A-104 E F F E C T IV E D A TE: December 21, 1998 AM ENDM ENT: Page 2 Add: Experimental Start Date: 2/16/99 Experimental Termination Date: 2/18/99 Test Concentrations: Negative Control, 13, 22, 36, 60 and 100 mg a.i./L T est Substance N o .: 4675 R EA SO N : The above information was not known when the protocol was signed by the Study Director. A M EN D M EN T: Test Organism. Page 5 Delete: Feed (Y CT) provided to daphnids in the cultures 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. R E A SO N : Historical analyses o f Wildlife International Ltd. aquatic feed have shown that no contaminants are present at levels known to be capable o f interfering with the study. A M E N D M E N T : A PPE N D IX II, Page 14 Add: Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination o f Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Filtered Saltwater and Algal Medium. R E A SO N : To add the analytical method to be used in the study. 000724 a 4 5 4 \l 04\am endl Wild life International ltd. y ' u^ j P cX / J ^ \ SPONSOR'S REPRESENTATIVE PROJECT NO.: 454A-104 Page 2 of 2 p li ff DATE DATE a4S4\l04\am endl 000725 W i l d l i f e In t e r n a t i o n a l ltd. PROJECT NO.: 454A-104 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST W ITH THE CLA D O CERA N (D aphnia magnd) P R O T O C O L N O .: 454/120198/DAP-48H1/SUB454 A M EN D M EN T NO.: 2 SPONSOR: 3M Corporation P R O JE C T NO.: 454A-104 E F F E C T IV E D A T E : M arch 9, 1999 A M E N D M E N T : D ata Analysis. Page 8 Change: W hen th e dose-response pattern allows calculation o f EC 10, E C 50 and E C 90 values, the data will be analyzed using the com puter software o f C.E. Stephan (6). To: W hen the dose-response pattern allows calculation o f EC 10, E C 50 and EC 90 values, the data will be analyzed using the computer software o f C.E. Stephan (6), or another appropriate m ethodology. R E A SO N : The com puter software o f C.E. Stephan does not calculate EC 10 or EC90 values. SPONSOR'S REPRESENTATIVE 3 / /il r r DATE DATE v / iI m DATE a 4 S 4 \l 04\am end2 000726 3/joJqO PROTOCOL PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE CLADOCERAN (Daphnia magna) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 and OECD Guideline 202 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory P.O.Box 33331 St. Paul, Minnesota 55133 Wil d l if e In tern atio n al ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 December 1,1998 000727 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd . -2- PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE'CLADOCERAN (Daphnia magna) 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 MANAGEMENT: Henry O. Krueger, Ph.D. Director of Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental Start Date: ____ Project No.: Test Concentrations: Test Substance No.: . - /Q Q Experimental Termination Date: Reference Substance No. (if applicable): PROTOCOL APPROVAL //O DATE SPONSOR'S REPRESENTATIVE VE' ' b fa DATE 000728 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l ltd. -3 - INTRODUCTION Wildlife International Ltd. will conduct a static acute toxicity test with the cladoceran, Daphnia magna, for the Sponsor at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. !I The study will be performed based on procedures in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 (1), in OECD Guideline for Testing of Chemicals, 202: Daphnia sp. Acute Immobilization Test and Reproduction Test (2) and ASTM Standard E-729-88a, Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinver tebrates 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 El OBJECTIVES The objective ofthis study is to determine the acute effects of a test substance on the cladoceran, Daphnia magna, under static test conditions for a period of 48 hours. afl iri El A Btf t K EXPERIMENTAL DESIGN Daphnids will be exposed to a geometric series of at least five test concentrations and a negative (dilution water) control for 48 hours. Two replicate test chambers will be maintained in each treatment and control group, with 10 neonate daphnids in each chamber so that a total of 20 neonate daphnids are exposed in each treatment and control group. Nominal test concentrations will be selected in consultation with the Sponsor, and will be based upon information such as the results of exploratoiy range-finding toxicity data, known toxicity data, physical/chemical properties o f 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 concentration oftest substance used in the definitive test will be at least 60% o f the next higher treatment, 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 o f the test substance. Results o f the analyses will be used to calculate mean measured test concentrations. 000729 PROTOCOL NO.: 454/120198/DAP-48H1/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. -4 - To control bias, neonate daphnids will be impartially assigned to exposure chambers at test initiation. No other potential sources ofbias are expected to affect the results of the study. EC 10, EC50 and EC90 values will be calculated, when possible, based on the number of dead or immobilized daphnids observed in each test concentration after each 24-hour interval of exposure. The no-mortality concentration and the no-observed-effect concentration (NOEC) 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 o f 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 o f the study. Preparation of 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 of exposure to aquatic organisms. Test Organism The cladoceran, Daphnia magna, has been selected as the test species for this study. Daphnids are representative of an important group of aquatic invertebrates, and have been selected for use in the test based upon past use history and ease of culturing in the laboratory. Daphnid neonates to be used in the test will be less than 24 hours old and will be obtained from cultures maintained at Wildlife International Ltd., Easton, Maryland. The identity of the species will be verified by the supplier of the 000730 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l t d . -5 - original culture or by Wildlife International Ltd. personnel using appropriate taxonomic keys such as Pennak (4). Daphnids will be cultured in water from the same source and at approximately the same temperature as will be used during the test Daphnids in the cultures producing neonates for the test will be held for at least 10 days prior to collection of the neonates for testing. Adult daphnids in the culture will produce an average of at least 3 young per adult per day over the 7 day period prior to the test. Neonates from daphnids that show signs of disease or stress will not be used as test organisms. Daphnids in holding that produce ephippia also will not be used to supply neonates for testing. Daphnids in the cultures will be fed once daily. The diet will be a mixture of yeast, Cerophyll, and trout chow (YCT), supplemented with a suspension of the freshwater green alga Selenastrum capricornutum. Adults are fed during the 24-hour period prior to test initiation, but neonates will not be fed dining the test. Feed (YCT) provided to daphnids in the cultures will be analyzed at least once annually to ensure that there are no contaminants at levels known to be capable of interfering with the study. Specifications for acceptable levels of contaminants in daphnid 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 of the test. Neonates will be obtained for testing from at least three individual adults. Prior to test initiation, the neonates will be collected from cultures and transferred to small containers. The daphnids will be released into the test compartments below the water surface using a wide-bore pipette. Dilution Water The water used for culturing and testing 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 fum 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: 000731 PROTOCOLNO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e International ltd. 6- - 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 in the water and results o f the analyses will be summarized in the final report. Test Apparatus Test chambers will consist of polyethylene bottles. The size of bottle and the volume of test solution will be sufficient to meet the requirements o f sampling for analytical verification o f test substance concentrations and routine water chemistry analyses. Test chambers will be indiscriminately positioned in a temperature-controlled water bath or an environmental chamber. 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 culturing and testing will be provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g., Colortone 50). A photoperiod of 16 horns of light and 8 hours of darkness 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 and at test termination with a SPER Scientific Ltd. light meter or equivalent. The target test temperature will be 20 1C. Temperature will be measured in each test chamber at the beginning and end of the test using a hand-held thermometer. Temperature also will be measured with a continuous recorder in one negative control chamber. Recorder measurements will be verified with a hand-held thermometer prior to test initiation. 000732 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd . -7 - Dissolved oxygen will be measured in all replicates of 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 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, pH, alkalinity, specific conductance and total organic carbon (TOC) will be measured in the dilution water at the beginning and end of the test. 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 (5). Specific conductance will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter, or equivalent. Total organic carbon will be measured on a Shimadzu Model 5000 TOC analyzer, or equivalent. Biological Observations Observations of mortality, immobilization and clinical signs o f toxicity will be made between 0- 24 hours, and at 24 and 48 hours 1 hour. Daphnids not able to swim within 15 seconds after gentle agitation o f the test chamber will be considered immobile. SamplingjprAnalytical Mgasuretpgots Water samples will be collected freon each test chamber at the beginning of the test and at 24 and 48 hours ( 1 hour) to determine concentrations of 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: 000733 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l ltd . 8- - PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group 0 Hours 24 Hours Control Solvent Control (if needed) Level 1-Low Concentration Level 2 Level 3 Level 4 Level 5-High Concentration 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Totals ' 14 14 48 Hours 2 2 2 2 2 2 2 14 Total Number of Verification Samples = 42 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 of the study. 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 n. 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 of EC10, EC50 and EC90 values, the data will be analyzed using the computer software of C.E. Stephan (6). The program was designed to calculate the EC values and the 95% confidence intervals by probit analysis, the moving average method, or binomial probability with nonlinear interpolation (7, 8 and 9). The EC values will be calculated using mortality/immobility data collected at 24 and 48 hours. The no-mortality concentration and the no- observed-effect concentration (NOEC) will be determined. 000734 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e Intern atio na l ltd. -9 - RF.CORPS TO BE MAINTAINED Records to be maintained for data generated by Wildlife 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 of initiation and termination of the test. 4. History o f the test organism, culture and holding records. 5. Stock solution calculation and preparation. 6. ' Daily observations. 7. Water chemistry results (e.g., hardness and alkalinity). 8. If applicable, the methods used to analyze test substance concentrations and the results of analytical measurements. 9. Statistical calculations. 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 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 statement o f 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 p ro v id ^ Q Q ^ 3 5 by the Sponsor. PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd. - 10- 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, feed types, results of latest feed analyses, light intensity and photoperiod. 9. A description of the preparation o f the test solutions. 10. The methods used to allocate organisms to test chambers and begin the test, the number o f organisms and chambers per treatment, and the duration of the test. 11. A description o f circumstances that may have affected the quality or integrity of 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 of the biological data and analytical chemistry data, and a statement o f the conclusions drawn from the analyses. A graph plotting the concentration response curve for each period an EC50 is calculated, when sufficient data exists. If the data is conducive to evaluation by probit analysis, the slope o f the concentration-response curve will be reported. 14. Statistical methods used to evaluate the data. 15. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study. 16. The location where raw data and final report are to be stored. 17. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates o f any findings reported to the Study Director and Management 18. 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 o f 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 of 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. OOO V3 6 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l td . - 11 - 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 o f the study conducted by Wildlife International Ltd. The Sponsor will be 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 o f 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. id PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 000737 3M LAB REQUEST NO. U2723 Wil d l if e Intern atio na l l t d . - 12- REFERENCES 1 UJS. Environmental Protection Agency. 1996. Series 850-Ecological Effects Test Guidelines {draft), OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids. 2 O rganisation for Economic Cooperation and Development 1984. Daphnia sp. Acute Immobilization Test and Reproduction Test. OECD Guideline for Testing o f Chemicals. Guideline 202. Paris. 3 ASTM Standard E729-88a. 1994. Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, andAmphibians. American Society for Testing and Materials. 4 Pennak, R.W. 1978. Freshwater Invertebrates o f the United States. 2nd Ed. 365 p. 5 APHA, AWWA, WPCF. 1985. Standard Methods fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 6 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 7 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. 8 Thompson, W.R. 1974. Bacteriological Reviews. Vol.II, No. 2. Pp. 115-145. 9 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. TT> ATAOrvr \ t a . a e a m* \ f \i r o /r\ A n. a ot t i /O T TT> A C 4 000738 X t a r> t* r ?rxr Tr? c1'T \ x r \ T m o o W il d l if e In t e r n a t io n a l ltd. -13 - APPENDIX i 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 accordance with GLP Standards? Y es____ No X III. Test Substance Storage Conditions Please indicate the recommended storage conditions at Wildlife International Ltd. Ambient room temperature______________________________________________________________ Has the stability o f the test substance under these storage conditions been determined in accordance with GLP Standards? Y es____ No X Other pertinent stability information: ------------------------------------------------------------- ;----------------------- IV. Test Concentrations: 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. V. Toxicity Information: Mammalian: Rat LD50 251 mg/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphnia magna: 27me/L__________ Rainbow Trout: l l mp / L Daphnia magna: 50me/L_________ _ Fathead Minnow; 38mg/L Other Toxicity Information (including findings o f chronic and subchronic tests): Please see MSDS___________________________________________________________ 000739 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd. - 14- APPENDIXII . Analytical Method to be Provided by Sponsor 000740 PROTOCOL NO.: 454/120198/DAP-48H1/SUB454 3M LAB REQUEST NO. U2723