Document n9eyK2wXQM2jgmeL2Mw10BMwa

A R n Q'oofy PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A -101 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1035 AUTHORS: Kurt R Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: December 4,1998 STUDY COMPLETION DATE: June 24, 1999 AMENDED REPORT DATE: April 26, 2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, MN 55144 Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410)822-8600 Page 1 o f 41 000934 AMENDED W il d l if e In te r n a tio n a l ltd. 2- - PROJECT NO.: 454A-101 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-101 STUDY COMPLETION: June 24,1999 AMENDED REPORT: April 26,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance is being recharacterized in accordance with GLP. The stability o f the test substance under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Senior Biologist SPONSOR APPROVAL: DATE 000935 AMENDED W il d l if e In t e r n a t io n a l Ltd . -3 - PROJECT NO.: 454A-101 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: Test Substance Preparation Matrix Fortification and Sample Preparation Biological Data and Draft Report Analytical Data and Draft Report Final Report Amended Report DATE CONDUCTED: March 5,1999 March 10,1999 March 25,1999 March 24 and 25,1999 June 3,1999 April 19,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: March 8,1999 March 9,1999 March 10,1999 March 12,1999 March 25,1999 March 26,1999 March 25,1999 June 3,1999 April 19,2000 March 25,1999 June 3,1999 April 20,2000 James H. Coleman Quality Assurance Representative " DATE 000936 AMENDED W il d l if e In ter n a tio n a l ltd. -4 - REPORT APPROVAL PROJECT NO.: 454A-101 SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-101 STUDY DIRECTOR: Kurt R. Drottar Senior Biologist MANAGEMENT: Henry O. Krueger, Ph?D. Director, Aquatic Toxicology and Non-Target Plants DATE DATE 7 000937 AMENDED W il d l if e In te r n a tio n a l ltd. -5 - PROJECT NO.: 454A-101 TABLE OF CONTENTS Title/Cover Page............................................................. 1 Good Laboratory Practice Compliance Statement................................................................................................2 Quality Assurance Statement................................................................................................................. :.............3 Report Approval..................................................................................................................................................... 4 Table o f Contents............................................................................................................... 5 Summary.................................................................. 7 Introduction................. 8 O bjective......................................................................................................................... 8 Experimental Design.............................................................................................................................................. 8 Materials and Methods................................................................ 9 Results and Discussion......................................................................................................................................... 12 Conclusions...................................... :................................................................................................................. 12 R efere n ces.............................................................................................................................................................13 TABLES Table 1 - Summary o f Analytical Chemistry D ata.............................................................................................14 Table 2 - Temperature, Dissolved Oxygen and pH o f W ater in the Test Cham bers................................. -- 15 Table 3 - Cumulative Percent M ortality and Treatment-Related Effects.......................................................... 16 Table 4 - LC50 Values......................................................................................................................................... 17 000938 W il d l if e In te r n a tio n a l ltd. 6- - TABLE OF CONTENTS - Continued - PROJECT NO.: 454A-101 A PP E N D IC E S Appendix I - Salinity and pH o f Saltwater Measured During the 4-Week Period Immediately Preceding the T est..............................................................................................18 Appendix II - Analyses o f Pesticides, Organics, Metals and Other Inorganics in Wildlife International Ltd. Saltw ater.................................................................................19 Appendix H I- The Analysis ofPFO S in Saltwater in Support o f Wildlife International Ltd. Project No.: 454A -101........................................................................... 20 Appendix IV - Changes to Protocol................................................................................................................38 Appendix V - Personnel Involved in the Study............................................................................................ 39 Appendix VI - Report Amendment................................................................................................................. 40 000939 AMENDED W il d l if e Internatio nal ltd. -7 - PROJECT NO.: 454A-101 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATAAND A COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: I TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTHOF TEST: SUMMARY 3M Corporation Ms. Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 454A-101 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) Negative Control; 0 .5 7 ,1 .1 ,1 .9 ,3 .0 and 5.4 mg a.i./L Experimental Start - March 8,1999 Biological Term ination-M arch 12,1999 Experimental Termination - March 12,1999 96 Hours AMENDED W il d l if e In t e r n a t io n a l ltd. - 8- PROJECT NO.: 454A-101 IN T R O D U C T IO N This study was conducted by W ildlife International Ltd. for 3M Corporation at the W ildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f die test was conducted from March 8, 1999 to March 12,1999. Raw data generated by Wildlife International Ltd. and a copy o f the final report are filed under Project Number 454A-101 in archives located on the Wildlife International Ltd. site. O B JE C T IV E The objective o f this study was to evaluate the acute toxicity o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the saltwater mysid, Mysidopsis bahia, during a 96-hour exposure period under static test conditions. EXPERIM ENTAL DESIGN Saltwater mysids were exposed to a geometric series o f five test concentrations and a negative (saltwater) control. Two replicate test chambers were maintained in each treatment and control group, with 10 mysids in each test chamber for a total o f 20 mysids per test concentratioa Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results o f an exploratory rangefinding toxicity te st Nominal test concentrations selected were 1.1,1.8,3.0,4.9 and 8.2 mg active ingredient (a.i.)/L. Meanmeasured test concentrations were determined from samples o f test water collected from each treatmentand control group at the beginning o f the test, at approximately 48 hours and at test termination. The mysids were indiscriminately assigned to exposure chambers at test initiation. Observations of mortality and other clinical signs were made at approximately 4 .5 ,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 and no-observed-effect-concentration (NOEC) were determined by visual interpretation o f the mortality and clinical observation data. 000941 AMENDED W il d l if e Internatio nal ltd. -9 - PROJECT NO.: 454A-101 M ATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsis bahiaf. The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 Ecological Effects Test Guidelines, OPPTS Number 850.1035 (1); U.S. Environmental Protection Agency, StandardEvaluation Procedure, Acute Toxicity Testfor Estuarine andMarine O rganism (2); and ASTM Standard E729-88a StandardGuidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned W ildlife 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 1 .1 ,1 .8 ,3 .0 ,4 .9 and 8.2 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 ftest concentrations were constructed o f plastic or stainless steel. A primary stock solution was prepared in dilution water at a concentration o f 8.2 mg a.i./L. The primary stock solution was mixed with an electric mixer for approximately 22 hours to aid in the solubilization o f the test substance. After mixing, the primary stock solution was proportionally diluted with dilution water to prepare the four additional test concentrations. All test solutions appeared clear and colorless. Test Organism The saltwater mysid,Mysidopsis bahia, was selected as the test species for this study. The saltwatermysid is representative o f an important group o f aquatic marine invertebrates and was selected for use in the test based upon past history o f use and ease o f culturing in the laboratory. Mysids used in the test were obtained as juveniles from cultures maintained by Wildlife International Ltd., Easton, Maryland. 000942 AMENDED W il d l if e In t e r n a t io n a l ltd. -10- PROJECT NO.: 454A-101 Adult mysids were held at approximately the same temperature as used during the te st During the holding and acclimation periods, the adults showed no signs o f disease or stress. During the 14-day holding period preceding the te s t water temperatures ranged from 25.0 to 25.5C. The pH o f the water ranged from 7.9 to 8.1, salinity was maintained at 20%o (parts per thousand) and dissolved oxygen ranged from 7.0 to 7.2 mg/L. Instrumentation used for water measurements is described in the Environmental Conditions section o fthis report A t test initiation, thejuvenile mysids were carefully collected from the cultures and transferred to glass beakers. Mysids were transferred from the beakers to the test compartments using a wide-bore pipette. Juvenile mysids are relatively fragile and were, therefore, handled as little as possible. The juvenile mysids were fed live brine shrimp (Artemia sp.) nauplii daily during die test to prevent cannibalism. Test Apparatus Test chambers were 2-L polyethylene buckets filled with approximately 1000 mL o ftestwater. The depth o f the test water in a representative test chamber was approximately 6.6 cm Test chambers were impartially positioned in an environmental chamber designed to maintain a temperature o f 252C. The test chambers were labeled with the project number, test concentration and replicate and were covered with plexiglass throughoutthe te s t Dilution W ater The water used for holding, acclimation and testing was natural seawater collected at Indian River M et, Delaware, and diluted to a salinity o f approximately 20%# with well water. Salinity and pH measurements taken during the four-week period immediately preceding the test are presented in Appendix I. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank. The filtered seawater then was diluted with freshwater from a well on the W ildlife International Ltd. site 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 are presented in Appendix II. Environmental Conditions Lighting used to illuminate the cultures and test chambers during culturing and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colartone 50). A photoperiod of 16 hours of light and 8 hours o f darkness was controlled with an automatic timer. A 30-minute transition period 000943 W il d l if e In t e r n a t io n a l ltd. - 11- PROJECT NO.: 454A-101 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 437 lux at the surface o f 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 252C. Dissolved oxygen and pH measurements were made on water samples collected from all replicate test chambers o f each treatment and control at test initiation, and at approximately 24-hour intervals thereafter. Salinity was measured in the dilution water at test initiatioa Measurements o fpH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Salinity was measured using a Bio-marine, Inc. Aquafauna refractometer. Observations Observations were made to determine the numbero f mortalities. The number o f individuals exhibiting clinical signs o f toxicity or abnormal behavior also were evaluated. Observations were made approximately 4.5, 2 4 ,4 8 ,7 2 and 96 hours after test initiation. Statistical Analyses The 2 4 ,48,72 and 96-hour LC50 values and the 95% confidence intervals were calculatedwhoapossible by probit analysis, the moving average method or binomial probability with non-linear interpolation (4,5,6) using the computer software o f C.E. Stephan (7). In this study, LC50 values could not be calculated at 24 and 48 hours due to the lack o f an adequate concentration-response pattern, however, the probit method was used to evaluate mortality at 72 and 96 hours. The no mortality concentration andNOEC were determinedby visual interpretation o f the mortality and clinical observation 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 and at test termination to measure concentrations o fthe test substance. The samples were collected in plastic (Nalgene) vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix IE. 000944 W il d l if e In te r n a tio n a l ltd. -12- PROJECT NO.: 454A-101 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 1 .1 ,1.8,3.0,4.9and8.2m ga.i./L . Samples collected at test initiation had measured values that ranged from 52 to 71% o f nominal values. After 22 hours o f mixing, the primary stock solution was measured to be 70 to 71% o f nominal and was probably at the limit o f solubility in filtered saltwater. The recoveries in the other test concentrations were similar because they were proportional dilutions o f the primary stock. Measured values for samples taken at 48 hours ranged from 44 to 71% o f nominal. Measured values for samples taken at 96 hours ranged from 36 to 71% 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 forthisstucfy were 0 .5 7 ,1 .1 ,1.9,3.0and5.4m ga.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 252C range established for the te st Dissolved oxygen concentrations remained > 6.6 mg/L (90% of saturation) throughout the te st Measurements o f pH ranged from 8.1 to 8.2 during the test. Daily observations o f mortality and other clinical signs o f toxicity observed during the test are shown in Table 3. Mysids in the negative control, and the 0.57 and 1.1 mg a.i./L treatment groups appeared normal and healthy during the te st After 96-hours o f exposure, mortality in the 1.9,3.0 and 5.4 mg a.i./L treatment groups was 10,40 and 75%, respectively. LC50 values and 95% confidence limits at 2 4 ,4 8 ,7 2 and 96 hours were estimated or calculated from the mortality data, and are shown in Table 4. CONCLUSIONS The 96-hour LC50 value for saltwater mysids exposed to PFOS was 3.6 mg a.i./L. The 95% confidence lim its were 3.0 and 4.6 mg a.i./L, and the slope o f the concentration-response curve was 4.5. The 96-hour no mortality concentration and the NOEC were 1.1 mg a.i./L. 000945 AMENDED W il d l if e In ter n a tio n a l ltd. -13- PROJECT NO.: 454A-101 REFERENCES 1 U.S. Environm ental P rotection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), OPPTS Number 850.1035: M ysid Acute Toxicity Test. 2 U.S. Environm ental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Test fo r Estuarine and Marine Organisms (Shrimp 96-Hour Acute Toxicity Test). Hazard Evaluation Division. Office o f Pesticide Programs. EPA 540/9-85-010. Washington, D.C. 3 ASTM S tandard E729-88a. 1994. Standard Guidefo r ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and M aterials. 4 Thom pson, W .R. 1947. Bacteriological Reviews, Vol. II, No. 2. Pp. 115-145. 5 Stephan, CJE. 1977. "Methods for Calculating an l,5Qn, Aquatic Toxicology cmdHazardEvaluations. American Society for Testing and M aterials. Publication Number STP 634, pp 65-84. 6 Finney, D .J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. 7 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 000946 W il d l if e In te r n a tio n a l ltd. -14- PROJECT NO.: 454A-101 Table 1 Summary o f Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Test Concentration (mg a.i./L) Negative Control 3M Corporation PFOS Saltwater Mysid, Mysidopsis baha Filtered Saltwater fours) B0 A 48 B 48 A 96 B 96 1.1 A 0 B0 A 48 B 48 A 96 B 96 1.8 A 0 B0 A 48 B 48 A 96 B 96 3.0 A 0 B0 A 48 B 48 A 96 B 96 4.9 A 0 B0 A 48 B 48 A 96 B 96 8.2 A 0 B0 A 48 B 48 A 96 B 96 The limit of quantitation (LOQ) was 0.115 mg a.ij.L. Measured Concentration (mga.i./L <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.575 0.622 0.605 0.640 0.391 0.580 1.12 1.19 1.10 1.09 1.04 1.13 1.92 1.99 1.92 1.91 1.79 1.91 3.05 2.66 2.96 3.35 3.11 3.11 5.82 5.78 3.58 5.85 5.22 5.86 Concentration ta g a.i./L) <LOQ 0.57 1.1 1.9 3.0 5.4 Percent Of Nominal 52 61 63 61 66 000947 AMENDED -15- Table 2 Temperature, Dissolved Oxygen and pH o f Water in the Test Chambers Sponsor; Test Substance; Test Organism: Dilution W ater Mean Measured Test Concentration (mg a.i7L) Negative Control 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater OHour1 Temp3 DO3 Replicate (C) (mg/L) A 24.2 7.4 B 25.4 72 pH 8.1 8.1 24 Hours DO (mg/L) pH 7.1 8.1 7.0 8.1 48 Hours DO (mg/L) pH 6.9 82 6.8 82 0.57 A 24.1 7.4 8.1 7.0 8.1 6.8 82 B 25.3 72 82 7.0 8.1 6.8 82 1.1 A 24.0 7.4 82 7.0 8.1 6.8 82 B 24.4 13 82 7.0 8.1 8.8 82 1.9 A 24.0 1A 82 7.1 8.1 6.9 8 2 B 23.8 1 2 82 7.0 8.1 6.8 8 2 3.0 A 23.6 7.3 82 7.0 8.1 6.8 82 B 23.5 7.4 8 2 7.0 8.1 6.9 8.2 5.4 A 23.8 13 82 7.1 8.1 6.8 8.2 B 24.0 1 2 82 7.0 8.1 6.9 8.2 The 0-hour dilution water (negative control) measurement for salinity 20%o. 1 Temperature measured continuously during the test ranged from approximately 23.3 to 26.0'C . 3 A dissolved oxygen concentration o f4.4 mg/L represents 60% saturation at 25*C in saltwater with a salinity o f20%o. 72 Hours DO (mg/L) _ PH 6.8 8.1 6.8 8.1 6.8 8.1 6.8 8.2 6.8 8.1 6.8 8.2 6.8 8.2 6.8 8.1 6.7 8.2 6.8 8.2 6.8 82 6.8 82 PROJECT NO.: 454A-101 Temp rc ) 24.7 24.7 96 Hours DO (mg/L) 6.8 6.8 24.7 6.7 24.7 6.7 24.7 6.8 24.8 6.7 24.7 6.6 24.6 6.6 24.5 6.8 24.4 6.8 24.5 6.9 24.5 6.8 PH 8.2 8.2 8.2 8.2 8.2 82 82 82 82 8.2 8.2 8.2 ooo oo AMENDED -16- PROJECT NO.: 454A-101 Table 3 Cumulative Percent M ortality and Treatment-Related Effects Sponsor 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid,Mysidopsis bahla Dilution W ater______Filtered Saltwater Mean Measured Test Concentration (m ga.L/L ) Replicate No. Exposed 4.5 Hours No. Dead1 Effects1 Negative Control A B 10 0 10 AN 10 0 10 AN % Dead 0 0.57 A 10 0 10 AN 0 B 10 0 10 AN 1.1 A 10 0 10 AN 0 B 10 0 10 AN 1.9 A 10 0 10 AN 0 B 10 0 10 AN 3.0 A 10 0 10 AN 0 B 10 0 10 AN 5.4 A 10 0 10 AN 0 B 10 0 10 AN Cumulative number ofdead mysids. 1 Observed Effects: AN " Appears Normal. No. Dead 0 0 24 Hours Effects 10 AN 10 AN % Dead 0 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 5 1 9 AN 2 8 AN 15 1 9 AN No. Dead 0 0 48 Hours Effects 10 AN 10 AN % Dead 0 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 2 8 AN 15 1 9 AN 4 6 AN 45 5 5 AN No. Dead 0 0 72 Hours Effects 10 AN 10 AN % Dead 0 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 5 1 9 AN 4 6 AN 30 2 8 AN 6 4 AN 60 6 4 AN 961lours No. Dead Effects 0 10 AN 0 10 AN Cumulative Percent Mortality 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 2 8 AN 10 5 5 AN 3 7 AN 40 8 2 AN 7 3 AN 75 AMENDED W il d l if e In te r n a tio n a l ltd. -17- PROJECT NO.: 454A-101 Table 4 LC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 3M Corporation PFOS Saltwater Mysid,Mysidopsis bahia Filtered Saltwater Lower 95% Confidence LC50 (mg a.i./L) Limits (m gal/L ) >5.4 >5.4 __i Upper 95% Confidence Limits (mg a.i./L) __i _i 72 Hours 4.4 3.6 6.2 96 Hours 3.6 3.0 4.6 1Confidence limits could not be calculated with the mortality data obtained. Statistical M ethod Visual Interpretation Visual Interpretation Probit Probit 000950 AMENDED W il d l if e International ltd. -18- PROJECT NO.: 454A-101 A PP E N D IX I Salinity and pH o f Saltwater Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: Salinity (%o) 3M Corporation PFOS Saltwater Mysid,Mysidopsis bahia Filtered Saltwater Mean 20 (N = 4) Range 20-20 pH 8.2 (N = 4) 8. 1 - 8.2 000951 W il d l if e International ltd. -19- PROJECT NO.: 454A-101 ANALYSIS APPENDEXn Analyses o f Pesticides, Organics, Metals and Other Inorganics in W ildlife International Ltd. Saltwater1 MEASURED CONCENTRATION M iscellaneous M easurem ents Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon2 Total Cyanide 23,500 < 0.050 < 1.0 < 10.0 m g/L mg/L mg/L Mg/L O rganochlorines and PCBs Aldrin Alpha BHC BetaBH C Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' DDT.pp* D ieldnn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde H eptachlor M ethoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.006 Mg/L < 0.025 Mg/L < 0.006 Mg/L < 0.005 Mg/L < 0.008 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.018 Mg/L < 0.010 Mg/L < 0.005 Mg/L < 0.005 Mg/L < 0.007 Mg/L < 0.005 Mg/L < 0.500 Mg/L < 0.260 Mg/L < 0.260 Mg/L < 0.260 Mg/L < 0.720 Mg/L < 0.720 Mg/L < 0.720 Mg/L < 0.720 Mg/L M etals an d O th er Inorganics A lum inum 3 Arsenic3 B eryllium 3 Cadmium3 Calcium3 C hrom ium 3 Cobalt3 . Lead3 M agnesium 3 M anganese3 M ercury M olybdenum 3 N ickel3 P otassium 3 S elenium 3 S ilv e r Sodium3 Zinc3 < 100 Mg/L < 25.0 Mg/L < 0.50 Mg/L < 1.0 Mg/L 235 mg/L < 2.0 Mg/L < 1.0 Mg/L < 20.0 Mg/L < 100 Mg/L < 10.0 Mg/L 760 mg/L < 4.0 Mg/L < 0.20 Mg/L < 2.0 Mg/L < 20.0 Mg/L 277 mg/L < 25.0 Mg/L < 1.0 Mg/L 6,010 mg/L < 20.0 Mg/L `Analyses performed by OST Environmental, Gainesville, Florida for samples collected on November3 through November 7,1997. 2Analyses performed by Wildlife International Ltd. for the sample collected on November 5,1997. 3Analyses performed by Wildlife International Ltd. for samples collected on November 5 through 7,1997. 000952 W il d l if e In t e r n a t io n a l ltd. -20- A P P E N D E X in PROJECT NO.: 454A-101 THE ANALYSIS OF PFOS IN FILTERED SALTWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-101 000953 W il d l if e In te r n a t io n a l Ltd . -21 - PROJECT NO.: 454A-101 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) W ILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-101 PRINCIPAL INVESTIGATOR: Scientist MANAGEMENT: Manager, Analytical Chemistry 000954 AMENDED W il d l if e In t e r n a t io n a l ltd. -22- PROJECT NO.: 454A-101 Introduction Saltwater samples were collected from a static acute aquatic toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the saltwater mysid (Aiysidopsis baha). This study was conducted by W ildlife International Ltd. and identified as Project N o.: 454A-101. 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 M arch 8,10 and 12, 1999 and were analyzed on each sample receipt day. Test. Substance and Infamai 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 m aterial, was identified as: IH , IH , 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 saltwater samples was developed a t 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 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 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 000955 W il d l if e In t e r n a t io n a l ltd. -23- PROJECT NO.: 454A-101 Chromatograph (HPLC) with a Perkm-Elmer API 100LC Mass Spectrometer equipped w ith a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C18 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% m ethanol: 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 0.00229 to 0.0457 mg a.i./L, were analyzed with the samples. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentrations o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method lim it o f quantitation (LOQ) for these analyses was set at 0.115 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 matrix blank samples (50.0). M atrix Blank and Fortification Samples Three matrix blank samples were analyzed to determine possible interference. N o 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. Saltwater was fortified at 0.457, 4.57 and 9.15 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery o f 97.4%. A representative chromatogram o f a m atrix fortification is presented in Figure 6. 000956 AMENDED W il d l if e International ltd. -24- PROJECT NO.: 454A-101 Example Calculations Sample number 454A-101-3, nominal concentration o f 1.1 mg a.i./L in saltwater. Initial Volume: 0.500 mL Final Volume: 25.0 mL Dilution Factor: 50.0 PFOS Peak Area: 235466 Internal Standard Peak Area: 449012 Peak Area Ratio: 0.5244 Calibration curve equation. Slope: 0.04044 Intercept: 0.05941 Curve is weighted (1/x). PFOS (pg a.iTL) a t instrument Peak area ratio - (Y-intercept) Slope 0.5244 - 0.05941 0.04044 = 11.5 PFOS (mg a.i./L) in sample PFOS (pg a.i./L) at instrument x Dilution Factor -- 11.5 x 50.0 1000 = 0.575 000957 AMENDED W il d l if e In t e r n a t io n a l Ltd. -25 - PROJECT NO.: 454A-101 Percent o f Nominal Concentration PFOS (mg a.i./L) in sample = ------------------------------------ x 100 PFOS (mg a.i./L) nominal Calculated recovery: 52.4% Note: manual calculation may differ. RESULTS Sample Analysis Saltwater samples were collected from the static acute toxicity study with the saltw ater mysid (Mysidopsis bahia) at test initiation, M arch 8, 1999 (Hour 0), on March 10, 1999 (Hour 48), and at test termination, March 12,1999 (Hour 96). The measured concentrations o f PFOS in the samples collected at initiation o f exposure o f the test organisms (Hour 0) ranged from 52.4 to 70.7% o f the nominal concentrations. Samples collected at Hour 48 had a measured concentration range o f 43.5 to 71.0% o f nominal values. Samples collected at test termination (Hour 96) had a measured concentration range o f 35.5 to 71.1% o f nominal values (Table 4). A representative chromatogram o f a test sample is shown in Figure 7. 000958 AMENDED W il d l if e In t e r n a t io n a l lto. -26- PROJECT NO.: 454A-101 IN ST R U M EN T: Table 1 Typical HPLC Operational Parameters 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). ANALYTICAL COLUMN: Keystone Betasil Cm 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 7.2 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.9 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 000959 W il d l if e In t e r n a t io n a l ltd. -27- PROJECT NO.: 454A-101 Table 2 M atrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-101-) MAB-1 Sample Type M atrix Blank Measured Concentration o f PFOS1 (mg a.i./L) < LOQ MAB-2 M atrix Blank <LOQ MAB-3 M atrix Blank <LOO 1 The limit o f quantitation (LOQ) was 0.115 mg 2lX.IL 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). 000960 AMENDED W il d l if e In ter n a tio n a l ltd. -28- PROJECT NO.: 454A-101 Table 3 M atrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-101-) MAS-1 MAS-4 MAS-7 Concentrations o f PFOS (mg a.i./L) Fortified M easured 0.457 0.457 0.457 0.537 _i 0.396 Percent Recovered 117 -- 86.5 MAS-2 MAS-5 MAS-8 4.57 4.57 4.57 4.27 4.43 4.36 93.3 96.8 95.2 MAS-3 MAS-6 MAS-9 9.15 9.15 9.15 8.18 9.42 8.93 89.4 103 97.6 Mean = 97.4 Standard Deviation = 9.41 CV = 9.67 _________________________________________________________________________ N = 8_________ 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. 1Result was 0.942 mg a.i./L or 205% o f the nominal concentration. This result was not used to determine due to probable fortification and/or dilution error. 000961 AMENDED W il d l if e Internatio nal ltd. -29- PROJECTNO.: 454A-101 Table 4 Measured Concentrations o f PFOS in Saltwater Samples from a Saltwater Mysid Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-101-) 1 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 -- -- -- -- -- " 1.1 3 0 40 15 48 16 48 27 96 28 96 0.575 0.622 0.605 0.640 0.391 0.580 52.4 56.6 55.1 58.2 35.5 52.9 1.8 5 0 60 17 48 18 48 29 96 30 96 1.12 64.4 1.19 68.3 1.10 63.2 1.09 62.7 1.04 59.8 1.13 64.5 3.0 7 0 80 19 48 20 48 1.92 65.7 1.99 67.9 1.92 65.8 1.91 65.2 31 96 1.79 61.2 32 96 1.91 65.4 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. 'T he limit o f quantitation (LOQ) was 0.115 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 m atrix blank samples (50.0). 2M ean result o f duplicate redilutions o f the original sample. Original result (12.0 mg a.i./L) was not included in the statistical analysis o f the data. Duplicate results for 454A-101-23 are 3.55 and 3.61. 000962 AMENDED W il d l if e In ter n a tio n a l ltd. -30- PROJECT NO.: 454A-101 Table 4 (Continued) M easured Concentrations o f PFOS in Saltwater Samples from a Saltwater Mysid Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 4.9 Sample Number (454A -101-) 9 10 21 22 . 33 34 Sampling Time (Hours) 0 0 48 48 96 96 PFOS Measured Concentration1 (mg a.i./L) 3.05 2.66 2.96 3.35 3.11 3.11 Percent of Nominal 61.6 53.8 59.9 67.8 63.0 62.9 8.2 11 0 12 0 23 48 24 48 35 96 36 96 5.82 5.78 3.582 5.85 5.22 5.86 70.7 70.2 43.5 71.0 63.3 71.1 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. 'T he limit o f quantitation (LOQ) was 0.115 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). 2M ean result o f duplicate redilutions o f the original sample. Original result (12.0 mg a.i./L) was not included in the statistical analysis o f the data. Duplicate results for 454A-101-23 are 3,55 and 3.61._______ ' 000963 AMENDED W il d l if e In ter n a tio n a l ltd. -31- PROJECT NO.: 454A-101 M ETH O D O U TLIN E FO R TH E ANALYSIS O F PFOS IN SALTW ATER Prepare m atrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into filtered saltwater 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 o f sample and bring the flask to volume with the dilution solvent. Process the m atrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. M ix well by several repeat inversions. Ampulate samples and submit for LCMS analysis. F igure 1. Analytical method flow chart for the analysis o f PFOS in saltwater. 000964 W il d l if e In t e r n a t io n a l Ltd. -32- PROJECT NO.: 454A-101 Figure 2. A typical calibration curve for PFOS. Slope = 0.04044; Intercept = 0.05941; r = 0.9930. Curveisweighted(1/x). 000965 AMENDED W il d l if e In te r n a t io n a l ltd. -33- PROJECT NO.: 454A-101 intensity: 3215 cps Figure 3. A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard. 000966 AMENDED W il d l if e Internatio nal ltd. -34- PROJECT NO.: 454A-101 Intenslty: 32743 cps Figure 4. A representative ion chromatogram o f a high-level (0.0457 mg a.i./L) PFOS standard. 000967 AMENDED W il d l if e Internatio nal ltd. -35- PROJECT NO.: 454A-101 100 90 29 80 70 60 50 40 30 20 10 10 --1 I 31 1.30 69 62 99 `A I 'i I ' I "I 61 91 2.56 3.81 132 159 \h 122 121 5.07 151 6.32 intensity: 1287 cps 194 214- 2SO- 181 7.58 211 8.83 Scan Time Figure 5. A representative chromatogram of a matrix blank sample (454A-101-MAB-l). The arrow indicates the retention time o f PFOS. C00968 W il d l if e Intern atio na l ltd. -36- PROJECT NO.: 454A-101 intensity: 11119 ops 173 Figure 6. A representative chromatogram o f a matrix fortification sample (454A-101-MAS-1). 0009^9 W il d l if e In t e r n a t io n a l ltd. -37- PROJECT NO.: 454A-101 intensity: 12998 cps 173 Figure 7. A representative chromatogram of a test sample (454A-101-3). 000970 W il d l if e In ter n a tio n a l ltd. -38- PROJECT NO.: 454A-101 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 substance information and test concentrations. 2. The protocol was amended to remove annual feed analysis. 3. The protocol was amended to specify the analytical method to be used. 4. The test chambers were plastic buckets. 5. pH was measured daily in all test chambers. 000971 W il d l if e In t e r n a t io n a l ltd. -39- PROJECT NO.: 454A-101 APPENDIX V Personnel Involved in the Study The following key Wildlife International Ltd. personnel were involved inthe conduct or management ofthis study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Mark A. Mank, Laboratory Supervisor 4. Timothy Z. Kendall, Laboratory Supervisor 5. Kurt R. Drottar, Senior Biologist 6. Raymond L. VanHoven, Ph.D., Scientist 000972 W il d l if e In t e r n a t io n a l ltd. -40- PROJECT NO.: 454A-101 APPENDIX VI Report Amendment 1. Original Report: Pages 1-4,6 and 21 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 o f analysis dated March 9,2000 indicated a purity o f90.49%. Therefore, all test substance concentrations, including nominal concentrations, measured concentrations, and LC50 values, were recalculated and reported as mg a.i./L based on the 90.49% purity. Reason: To report the results o f the test based on the test substance purity o f 90.49% at the request o f the Sponsor. 5. Original Report: Page 7 Amendment: Experimental Termination - March 12,1999 Reason: To report the experimental termination date for the study. OOO9 V3 AMENDED W il d l if e Internatio nal ltd. -41 - PROJECT NO.: 454A-101 AMENDMENT SIGNATURES: APPENDIX VI -Continued- Report Amendment DATE 2=aHenif 0.cKrueger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: L Jai^ejl H. Coleman Quality Assurance Vg DA' ij~X& -<5(j DATE 000974 AMENDED W il d l if e Internatio nal ltd . PROJECT NO.: 454A-101 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STUDY T ITL E: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST W ITH THE SALTW A TER MYSED (M ysidopsis bahia) P R O T O C O L NO.: 454/110998/MYS-96H1/SUB454 A M EN D M EN T NO.: 1 SPON SOR: 3M Corporation P R O JE C T N O.: 454A-101 E FFEC TIV E DATE: December 21, 1998 AM ENDM ENT: Page 2 Add: Experimental Start Date: 3/8/99 Experimental Termination Date: 3/12/99 Test Concentrations: Negative Control, 1.2, 1.9, 3.2, 5.4 and 9.0 mg a.i./L Test Substance No.: 4675 REA 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 provided to the mysids 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 EA 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 w ith the study. A M E N D M E N T : A PPE N D IX II, P age 13 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 EA SO N : To add the analytical method to be used in the study. 000975 a454\102\amendl RA/i'ejuecI & A r (^lS^3~4'9? W il d l if e International ltd. STUDY DIRECTOR LABORATORY MANAGEMENT a . &dJL SPONSOR'S REPRESENTATIVE PROJECT NO.: 454A-101 Page 2 of 2 3/ r m DATE D DATE a454\l 02\amendl 000976 il d l if e Internatio nal ltd. PROJECT NO.: 454A-101 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454A-101 DATE OF DEFACTO DEVIATION: March 8,1999 DEVIATION: The protocol states that the test chambers will consist of polyethylene bottles. The test chambers actually consisted of plastic buckets. 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 DATE DATE 000977 PROJECT NO.: 454A-101 Page 1 of 1 W il d l if e In t e r n a t io n a l l t d ,_________________________________________________ DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454A-101 DATE OF DEFACTO DEVIATION: March 8,1999 DEVIATION: The protocol states that measurements of pH will be measured in alternate replicates of each treatment and control group. Measurements of pH were actually made in all test chambers. 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 ^ J 3DATE LABORATORY MANAGEMENT 4S4a\101\pnU DATE 000978 PROTOCOL PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1035 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory P.O. Box 33331 St. Paul, Minnesota 55133 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 November 9,1998 000979 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd . -2- PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) 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 0 . Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental Experimental Start Date: ____________________________ Termination Date: ______ Project No.: . -lo t_________________ Test Concentrations: ______________________________________________ Test Substance N o .:__________ Reference Substance No. (if applicable): PROTOCOL APPROVAL STUDY DIRECTOR DATE DATE 7 DATE 000980 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 A/il d l if e In t e r n a t io n a l l t d . -3 - INTRODUCTION Wildlife International Ltd. will conduct a static acute toxicity test with the saltwater mysid (Mysidopsis bahia) for the Sponsor at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on the guidelines provided in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1035 (1); U.S. Environmental Protection Agency, Standard Evaluation Procedure, Acute Toxicity Testfo r Estuarine and Marine Organisms (2); and ASTM Standard E729-88a Standard Guidefo r ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). Raw data for all work performed at Wildlife International Ltd. and a copy o f the final report will be filed by project number in archives located on the Wildlife International Ltd. site, or at an alternative location to be specified in the final report. PTJRJPQSE The purpose o f this study is to determine the acute effects o f a test substance on the saltwater mysid (Mysidopsis bahia) during a 96-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Saltwater mysids 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 mysids in each chamber for a total of 20 mysids per test concentra tion. Nominal test concentrations will be selected in consultation with the Sponsor, and will be based upon the results o f exploratory range-finding toxicity data, known toxicity data, physical/chemical properties of the test substance or other relevant information. Target concentrations need not exceed 1000 mg/L or the solubility limit o f the test substance in water (whichever is lower). Generally, each test substance concentration used in the definitive test will be 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. PROTOCOL NO. : 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 000981 W il d l if e In te r n a tio n a l l t d . -4 - To control bias, juvenile mysids will be impartially assigned to exposure chambers at test initiation. No other potential sources of bias are expected to affect the results of the study. Each group of 10 will then be transferred to the test chamber. Observations of mortality and other clinical signs will be made throughout the 96-hour test period. Cumulative percent mortality observed in the treatment groups will be used to calculate, when possible, LC50 values at 2 4 ,48,72 and 96 hour intervals. The no-mortality concentration and the no-observed-effect concentration (NOEC) will be determined. MATERIALS AND METHODS Test Substance Information on the characterization o f 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 OF 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 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 saltwater mysid (Mysidopsis bahia) has been selected as the test species for this study. Mysids are representative of an important group of aquatic marine invertebrates, and have been selected for use in the test based upon past use history and ease of culturing in the laboratory. Mysids to be used in the test will be less than 24 hours old and will be obtained from cultures maintained at Wildlife 000932 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e Internatio nal ltd . -5 - International Ltd., Easton, Maryland. The identity of the species will be verified by the supplier o f the original culture or by Wildlife International Ltd. personnel using appropriate taxonomic keys such as Price (4). Mysids will be cultured in water from the same source and at approximately the same temperature as will be used during the test. Mysids that show signs of disease or stress will not be used as test organisms. Mysids in the cultures and during testing will be fed at least once daily. The diet will be live brine shrimp nauplii (Artemia sp.). Feeding during culturing may be supplemented with saltwater algae and rotifers as needed. Feed provided to mysids 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 mysid 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. Dilution Water Natural seawater collected at Indian River Inlet, Delaware will be the source of the dilution water used in the culture facility and test system. The seawater will be filtered through a sand filter prior to its delivery to a 37,800-L holding tank. The salinity of the seawater will be diluted to approximately 20 7 0<J (parts per thousand) with Wildlife International Ltd. well water in the holding tank. The 20 7^ saltwater will be aerated using spray nozzles and filtered (0.45 /mi) prior to its delivery to the test system. Salinity and pH will be measured weekly to monitor the consistency of the saltwater. 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 seawater and results of the analyses will be summarized in the final report. 000933 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e Internatio nal ltd . 6- - Test Apparatus Test chambers will consist of polyethylene bottles. The size of bottle and the volume o f test solution will be sufficient to meet the requirements of 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 holding 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 of dark will be controlled with an automatic timer. A 30minute 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. The target test temperature will be 25 2C. Temperature will be measured to each test chamber at the beginning and end o f the test using a liquid-in-glass thermometer. Temperature also will be measured with a continuous recorder in one negative control replicate. Recorder measurements will be verified with a liquid-in-glass thermometer prior to test initiation. 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 alternate replicates of each treatment and control group 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. Dilution water salinity will be measured at test initiation using a Bio-Marine, Inc. Aquafauna refractometer, or equivalent. 0009S4 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e International ltd. -7 - Biological Measurements Observations ofmortality and clinical signs o f toxicity will be made between 0-24 hours, and at 24, 48, 72 and 96 hours 1 hour. The criteria for death include lack of movement, absence of respiratory movements, and lack of reaction to gentle prodding. Sublethal effects, such as abnormal locomotion, will also be noted. Dead organisms will be removed from the test chambers at each observation time. 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 of the test to determine concentrations of the test substance. In the event that 100% mortality occurs in any treatment, then sampling o f that treatment will terminate following the next sampling interval. Samples will be collected at mid-depth from each test chamber, analyzed immediately or placed in an appropriate storage container (e.g., polyethylene or polypropylene bottle) and stored under refrigeration until analyzed. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group OHour 48 Hours 96 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 2 2 2 2 2 2 2 Totals 14 14 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. 0009S5 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l l t d . 8- - Analvtical 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 dose-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 and the no-observed-effect concentration (NOEC) will be determined by visually interpreting the clinical observation data. 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 o f the signed protocol. 2. Identification and characterization of the test substance, if provided by the Sponsor. 3. Dates of initiation and termination o f the test. 4. Mysid history and culture records. 5. Stock solution calculation and preparation, if applicable. 6. Observations. 7. Water chemistry results (e.g., dissolved oxygen). 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. 0C09S6 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e Internatio nal ltd. -9 - 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 o f 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 o f the test organism, including the source, scientific name, age or life stage, feed types, light intensity and photoperiod. 9. A description o f 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 o f the test. 11. A description of circumstances that may have affected the quality or integrity o f 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 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. 14. Statistical methods used to evaluate the data. 15. The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 16. The location where raw data and final report are to be stored. 0009S7 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l ltd . - 10- 17. 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. 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 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 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. 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 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. 000938 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e International ltd . - 11 - REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number 850.1035: Mysid Acute Toxicity Test. 2 U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Test for Estuarine and Marine Organisms (Shrimp 96-Hour Acute Toxicity Test). Hazard Evaluation Division. Office of Pesticide Programs. EPA-540/9-85-010. Washington, DC. 3 ASTM Standard E729-88a 1994. Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 Price, W.W. 1982. Key to the Shallow Water Mysidacea o f the Texas Coast with Notes on their Ecology. Hydrobiologia 93, 9-21. Pr. W Juk Publishers, The Hague. Printed in The Netherlands. 5 Stephan, C.E. U.S. EPA. 1978. Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 6 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. 7 Thompson, W .R 1947. Bacteriological Reviews. Vol. II, 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 6584. PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 0009S9 3M LAB REQUEST NO. U2723 W il d l if e Internatio nal ltd . - 12- 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 me test substance and reference standard been determined prior to its use in this study in accordance with GLP Standards? Y es_____No X HI. Test Substance Storage Conditions Please indicate the recommended storage conditions at Wildlife International Ltd. Ambient room temperature _______________________________________________________ Has the stability of the test substance under these storage conditions been determined in accordance with GLP Standards? Y es_____ No X Other pertinent stability information: ------------------------------------------------------------------------------------- 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 fne material AS IS. V. Toxicity Information: Mammalian: Rat LD50 251 mg/ke Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphnia magna: 27 mg/L__________ Rainbow Trout: 11 mg/L______ Daphnia magna: 50 me/L__________ Fathead Minnow: 38 mg/L Other Toxicity Information (including findings of chronic and subchronic tests): Please see MSDS------------------------------------------------------------------------------------------------------ 000990 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e International ltd. - 13- APPENDIXn Analytical Method to be Provided by Sponsor 000991 PROTOCOL NO.: 454/110998/MYS-96H1/SUB454 3M LAB REQUEST NO. U2723