Document npOzjNgaXknbXoN929OJwLMp8

bm lr< X > & PFOS: *' A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrea virginica) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-106 3M LAB REQUEST NO. U2723 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1025 AUTHORS: Kurt R Drottar Henry 0 . Krueger, Ph.D. STUDY INITIATION DATE: May 3,1999 STUDY COMPLETION DATE: August 11,1999 AMENDED REPORT DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 40 000745 AMENDED W il d l if e Internatio nal ltd. 2- - PROJECT NO.: 454A-106 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Shell Deposition Test with the Eastern Oyster (Crassostrea virginica) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-106 STUDY COMPLETION: August 11,1999 AMENDED REPORT: ApriL26,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792,17 August 1989; OECD Principles o f Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the followingexceptions: 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 stQrage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R Drottar Senior Biologist SPONSOR APPROVAL: DATE DATE 000746 AMENDED W il d l if e Inter n a tio n a l ltd. -3 - PROJECT NO.: 454A-106 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFRParts 160 and 792,17 August 1989; OECD Principles o fGood Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Test Initiation and Matrix Fortification Preparation Biological Data and Draft Report Analytical Data and Draft Report Final Report Amended Report DATE CONDUCTED: May 21,1999 May 24,1999 July 1,1999 July 1,1999 August 11,1999 April 25,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: May 21,1999 May 21,1999 May 24,1999 May 26,1999 July 1,1999 July 2,1999 July 1,1999 August 11,1999 April 25,2000 July 2,1999 August 11,1999 April 26,2000 j- t. C c 6 L/yv/v-e-^ JanresH. Coleman Quality Assurance Representative _________________ Lj-'X& r-'O O DATE 000747 AMENDED W il d l if e In te r n a tio n a l ltd. -4 - REPORT APPROVAL PROJECT NO.: 454A-106 SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Shell Deposition Test with the Eastern Oyster (Crassostrea virginica) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-106 STUDY DIRECTOR: MANAGEMENT: Henry OTCrueger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants DATE DAT 000748 AMENDED W il d l if e In te r n a tio n a l ltd. -5 - PROJECT NO.: 454A-106 TABLE OF CONTENTS Title/Cover Page...................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement.................................................................................................2 Quality Assurance Statement................................................................................................. Report Approval..................................................................................................................................................... 4 Table o f Contents................................................................................................................................ Summary.................................................................................................................................................................. 7 Introduction.............................................................................................................................................................8 O bjective................................................................................................................................................................. 8 Experimental Design.......................................................................................................................... Materials and Methods........................................................................................................................................... 9 Results and Discussion............................................................................................................... 12 Conclusions..........................................................................................................................*.............................. 13 R eferen ces.............................................................................................................. 14 5 8 TABLES Table 1 - Summary o f Analytical Chemistry D ata....................................................................... Table 2 - Temperature, Dissolved Oxygen and pH o f W ater in the Test Chambers.......................................16 Table 3 - Shell Growth and Shell Growth Inhibition at Test Termination......................................................... 17 Table 4 - Individual Oyster Shell M easurements................................................................................................18 15 000749 W ild life International ltd. 6- - TABLE OF CONTENTS - C ontinued - PROJECT NO.: 454A-106 A PP E N D IC E S Appendix I - Salinity and pH of Unfiltered Saltwater Measured During the 4-Week Period Immediately Preceding the T est.................................. 19 Appendix II- Analyses o f Pesticides, Organics, M etals and Other Inorganics in Wildlife International Ltd. Saltw ater...................................................................................20 Appendix HI - The Analysis of PFOS in Unfiltered Saltwater in Support o f Wildlife International Ltd. Project No.: 454A -106..............................................................................21 Appendix IV - Changes to Protocol..................................................................................................................37 Appendix V - Personnel Involved in the Study.......................................................... .................................... 38 Appendix VI - Report Amendment...................................................................................................................39 000750 AMENDED W il d l if e In te r n a tio n a l ltd. -7 - PROJECT NO.: 454A-106 sponsor: SPONSOR'S REPRESENTATIVE: LOCATIONOF STUDY, RAW DATAANDA COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: V/ TEST SUBSTANCE: study: MEANMEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: OYSTER LENGTH (N = 20): 96-h o u r e c 50: 9 5 % CONFIDENCE l im it s : N O -O B S E R V E D -E F F E C T CONCENTRATION: LO W EST-O B SER V ED -EFFEC T CONCENTRATION: SUMMARY 3M Corporation Ms. Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 454A-106 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 96-Hour Shell Deposition Test with the Eastern Oyster (Crassostrea virginica) Negative Control, 0.36,0.40,1.3,1.9 and 3.0 mg a.i./L Experimental Start - May 24,1999 Biological Termination - May 28,1999 Experimental Term ination-June 14,1999 96 Hours ' - Eastern Oyster (Crassostrea virginica) P. Cummins Oyster Company, Inc. 618 W est 33ri Street Baltimore, Maryland 21211 Mean = 33.8 mm, Range = 27.8 to 41.5 mm >3.0 mg a.i./L Not Calculable 1.9m ga.i./L 3.0 mg a.i./L | 000751 AMENDED W il d l if e In t e r n a t io n a l ltd. - 8- IN T R O D U C T IO N PROJECT NO.: 454A-106 This study was conducted by W ildlife International Ltd. for 3M Corporation atthe Wildlife TntpmaHnnai Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f the test was conducted from May 24 to May 28,1999. Raw data generated by W ildlife International Ltd. and a copy o f the final report are filed under Project Number 454A-106 in archives located on the W ildlife International Ltd. site. 'V O BJECTIV E The objective o f this study was to evaluate the acute toxicity o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) on shell deposition o f the eastern oyster, Crassostrea virginica, during a 96-hour exposure period under static test conditions. EXPERIM ENTAL DESIGN Eastern oysters were exposed to a geometric series o f five test concentrations and a negative (dilution water) control. One replicate test chamber was maintained in each treatment and control group,with 20 oysters in each test chamber. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results o f an exploratory range finding toxicity test. Nominal test concentrations selected were 1.2,2.0, 3.3,5.5 and9.1 mg active ingredient (a.i.)/L.. Mean measured test concentrations were determined from samples o ftest water collected from each treatment and the control group at the beginning o f the test, at approximately 48 hours, and at test termination. The oysters were indiscriminately assigned to exposure chambers at test initiation. Algal cells {Thalassiosira pseudonana, Skeletonema sp., Chaetoceros sp., and Isochrysis sp.) were provided to supplement naturally occurring algae and to maximize oyster growth rates during the test. Measurements o f shell deposition for each oyster were made at 96 hours and were used to estimate the EC50 value, the no-observed-effect-concentration (NOEC) and the lowest- observed-effect-concentration (LOEC). The EC50 is the concentration o f test substance in water that is estimated to inhibit shell deposition by 50%, relative to the control. 000752 AMENDED W il d l if e In t e r n a t io n a l Ltd. -9 - PROJECT NO.: 454A-106 M ATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Shell Deposition Test with the Eastern Oyster (Crassostrea virginica)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1025 (1); Standard Evaluation Procedure, Acute Toxicity Test fo r Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study) (2) and A SIM Standard E729-88a V' Standard Guidefo r 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 Wildlife International Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 98.9%, and an expiration 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 o f Test Concentrations Nominal test concentrations were 1.2,2.0,3.3,5.5 and 9.1 mg a.i./L, based on a test substance purity of 90.49%. All materials which came into contact with the test substance during preparation of test concentrations were constructed o f plastic or stainless steel. A primary stock solution was prepared in dilution water at a concentration o f 9.1 mg a.i./L. The primary stock solution was mixed with an electric paddle mixer for approximately 24 hours to aid in the solubilization o f the test substance. After mixing, the primary stock solution appeared clear and colorless with some white particulate material suspended throughout the solution. The primary stock was proportionally diluted with dilution water to prepare the four additional test concentrations. All test solutions appeared clear and colorless. Test Organism The eastern oyster, Crassostrea virginica, was selected as the test species for this study. The Eastern Oyster is representative o f an important group o f aquatic organisms and was selected for use in the test based upon past history o f use and ease o f handling in the laboratory. Eastern oysters used in the test were obtained 000753 AMENDED W il d l if e In ter n a tio n a l ltd. - 10- PROJECT NO.: 454A-106 from P. Cummins Oyster Company, Baltimore, Maryland. The oysters were held in water from the same source as used during the test. The oysters were supplied unfiltered natural seawater during holding and were held for 12 days prior to test initiation. To supplement the diet o f the oysters and enhance their condition and growth, the oysters were provided with an algal suspension ofThalassiosira sp., Skeletonema sp., Chaetoceros sp., and Isochrysis sp. continuously during holding and testing. During the 12-day holding period preceding the test, the oysters showed no signs o f disease or stress. During the holding period, watertemperatures ranged from 21.8 to22.9C. The pH o f the waterranged from 7.9 v.' to 8.1, salinity remained at 20 %o (parts per thousand) and dissolved oxygen ranged from 7.0 to 7.6 mg/L. Instrumentation used for water measurements is described in the Environmental Conditions section o fthis report Prior to test initiation, recently deposited shell at the rounded (ventral) end was removed using a small electric grinder. Care was taken to remove the shell rim uniformly to produce a smooth, rounded, blunt profile. The lengths o f 20 impartially-selected oysters were determined by measuring the longest distance from the umbo to the edge o f the shell. Measurement was made to the nearest 0.05 mm to confirm that the oysters fell within the 25 to 50 mm size criterion. T heaveragelengthoftheoystersw as33.8m m w itharangeof27.8to41.5m m . A t -test initiation, the oysters were collected from the holding tank jand indiscriminately transferred to the test chambers. Dilution W ater The water used for holding and testing was natural seawater collected at Indian River Inlet, Delaware, and diluted to a salinity o f approximately 20%o with well water. The dilution water was stored in a 19,000-L tank where it was aerated by recirculation. Salinity and pH measurements taken during the four-week period immediately preceding the test are presented in Appendix I. The results o f analyses performed to measured the concentrations o f selected contaminants in saltwater used by Wildlife International Ltd. are presented in Appendix n. Test Apparatus Test chambers were 52-L polyethylene aquaria containing approximately 40 L o ftest solution. Each test chamber was continuously stirred to circulate the supplemental algae using an electric paddle mixer. In addition, each test chamber was gently aerated. The depth o f water in a representative test chamber was approximately 21 cm. Test chambers were impartially positioned in an environmental chamber set to maintain a temperature of 221C. The test chambers were labeled with the project number and test concentration. 000754 W il d l if e Intern atio na l ltd. - 11- PROJECT NO.: 454A-106 Environmental Conditions Lighting used to illuminate the holding tanks and test chambers during holding and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod o f 16 hours of light and 8 hours o f darkness was controlled with an automatic timer. A 30-minute transition period o f low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was approximately 498 lux at the surface o f the water. Light intensity was measured using a SPER Scientific Ltd. light meter. v.' Temperature was measured in each test chamber at the beginning and end o f the test using a liquid-in glass thermometer. Temperature also was measured continuously in the negative control test chamber using a Fulscope ER/C Recorder. The target test temperature during the study was 221C. Dissolved oxygen was measured in each test chamber daily. Measurements o f pH were made in each test chamber at test initiation, after approximately 48 hours and at the end o fthe test. Dilution water salinity was measured at test initiation andtest term inatioa 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. Salinity was measuredusing a Bio-Marine, Inc. Aquafauna refractometer. Observations Oysters were observed daily during the test for mortality and clinical signs o ftoxicity. A t the end o fthe test, the longest finger o f new shell growth was measured to the nearest 0.05 mm. Statistical Analyses Shell growth inhibition was calculated for each treatment group as the percent reduction in shell growth relative to mean shell growth in the negative control. The following formula was used: Mean Shell GrowWoi - Mean Shell Growthreataent % Inhibition = Mean Shell Growthcoonoi 000755 W il d l if e In t e r n a tio n a l ltd. -12- PROJECT NO.: 454A-106 The EC50 value was estimated by visual inspection o fthe shell growth inhibition data. The shell growth data was evaluated for normality and homogeneity o f variances using the Chi-Square test and Bartlett's test, respectively. Dunnett's test was then used to identify treatment groups which had a statistically significant (< 0.05) reduction in shell growth as compared to the control (4). Analytical Chemistry Water samples were collected at mid-depth from each treatment and the control group at the beginning of the test, at approximately 48 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. Afterreview of the analytical results, the samples were reanalyzed in duplicate. Analytical procedures used in the analysis o fthe samples are provided in Appendix ID. 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 EH). Nominal concentrations selected for use in this study were 1.2,2.0,3.3,5.5 and9.1 mga.i./L. Samples collected at test initiation had measured values that ranged from 28 to 46% o f nominal values. After 24 hours o f mixing, the primary stock solution was measured to be 32 to 38% o f nominal and was probably at the limit o f solubility in unfiltered 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 15 to 41% o f nominal. Measured values for samples taken at 96 hours ranged from <LOQ to 52% o fnominal. When measured concentrations o fthe samples analyzed at test initiation, approximately 48 hours and at test termination were averaged, the mean measured concentrations for this study were 0 .3 6 ,0.40,1.3,1.9 and 3.0 mg a.i./L. Mean measured concentrations were used in the estimation o f the EC50 value. Observations and Measurements Measurements o f temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were within the 221C range established for the test. Dissolved oxygen concentrations remained >6.1 mg/L (79 percent of saturation) throughout the test and pH ranged from 7.5 to 8.1. Dilution water salinity measured at test initiation and test termination was 20 and 2 l%o, respectively. 000756 AMENDED W il d l if e In te r n a tio n a l ltd. -13- PROJECT NO.: 454A-106 Oysters in the negative control and all PFOS treatment groups appeared normal and healthy throughout the test period. After 96 hours o f exposure, oyster shell growth in the negative control was 2.67 mm (Table 3). Mean shell growth in the 0.36,0.40,1.3,1.9 and 3.0 mg a.i./L treatment groups was 2.50,2.40,2.51,2.13 and 1.91 mm, respectively. When compared to the negative control group, shell growth inhibition ranged from 6.4% in the 0.36 mg a.i./L treatment group to 28% in the 3.0 mg a.i./L treatment group. Dunnett's test showed that oyster shell growth was significantly reduced in the 3.0 mg a.i./L treatment group in comparison to the negative control. Individual measurements o f oyster shell growth are presented in Table 4. V' C O N C L U S IO N S The 96-hour EC50 value for eastern oysters (Crassostrea virginica) exposed to PFOS was >3.0 mg a.i./L, the highest concentration tested and the practical limit o f solubility in unfiltered saltwater. Based on a statistically significant reduction in shell deposition in the 3.0 mg a.i./L treatment group, the LOEC was 3.0 mg a.i./L and the NOEC was 1.9 mg a.i./L. 000757 AMENDED W il d l if e In t e r n a t io n a l ltd. -14- REFERENCES PROJECT NO.: 454A-106 1 U.S. Environm ental Protection Agency. 1996. Series 850-Ecological Effects Test Guidelines (draft), OPPTS Number 850.1025: Oyster Acute Toxicity Test (Shell Deposition). 2 U.S. Environm ental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Test fo r Estuarine andMarine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study). EPA 540/9-85-011. 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 Materials. 4 W est, Inc. and D. D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western EcoSystems Technology, Inc., Cheyenne, Wyoming. 000758 W il d l if e In te r n a tio n a l ltd. -15- PROJECT NO.: 454A-106 Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Test Concentration (mg a,i,/L) Negative Control Table 1 Summary o f Analytical Chemistry Data 3M Corporation PFOS Eastern Oyster, Crassostrea virginica Sampling Time (Hours) 0 0 48/ 48 96 96 Measured Concentration <LOQ <LOQ <LOQ <LOQ <LOQ Mean Measured Concentration 1.2 0 0 48 48 96 96 0.331 0.353 0.341 0.429 <LOQ <LOQ 0.36l2 2.0 0 0 48 48 96 96 0.622 0.633 0.299 0.313 0.249 0.257 0.40 0 1.36 1.3 0 1.15 48 0.924 48 0.878 96 1.58 96 1.72 0 2.42 1.9 0 2.53 48 2.02 48 2.24 96 1.45 96 0.970 0 3.44 0 3.01 48 3.74 48 3.57 96 1.99 96 2.19 lThe limit of quantitation (LOQ) was 0.115 mg a.i./L. 2Mean measured concentration does not include values <LOQ. 3.0 Percent Of Nominal 30 20 39 35 33 000759 AMENDED -16- Table 2 Temperature, Dissolved Oxygen and pH o f W ater in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Eastern Oyster, Crassostrea virginica Unfiltered Saltwater Mean Measured OHour Test Concentration Temp1 DO2 (mg a.i./L) (C) (mg/L) pH 24 Hours DO (mg/L) Negative Control 22.3 7.6 8.1 6.8 48 Hours DO (mg/L) pH 6.9 7.7 0.36 22.3 7.6 8.1 6.6 6.7 7.7 0.40 22.4 7.6 8.1 6.4 6.6 7.7 1.3 22.6 7.6 8.1 6.4 6.6 7.7 1.9 22.6 7.6 8.1 6.4 6.7 7.7 3.0 22.7 7.7 8.1 6.4 6.$ 7.7 1 Temperature measured continuously during the test ranged from approximately 22.0 to 22.5'SC. 2 A dissolved oxygen concentration of 4.7 mg/L represents 60% saturation. 72 Hours DO (mg/L) 6.6 6.5 6.3 6.3 6.3 6.1 PROJECT NO:: 481A-105 Temp (C) 22.2 22.1 22.1 22.1 22.0 21.8 96 Hours DO (mg/L) 6.8 6.7 6.7 6.6 6.3 6.4 pH 7.6 7.6 7.6 7.6 7.5 7.6 ooo ^5 o AMENDED W il d l if e In ter n a tio n a l ltd. - 17- PROJECT NO.: 454A-106 Table 3 Shell Deposition and Shell Growth Inhibition at Test Termination Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Eastern Oyster, Crassostrea virginica Dilution Water: Unfiltered Saltwater Mean Measured Concentration (mg a.i./L) Shell Deposition (mm) Negative Control 2.67 0.824 Shell Growth Inhibition (%) 0.36 2.500.933 6.4 0.40 2.40 1 0.820 10 1.3 2 .5110.919 6.0 1.9 2.1310.804 20 3.0 1.91*210.591 28 The 96-hour EC50 value was >3.0 mg a.i./L. 'M ean and standard deviation for 20 oysters. 2Indicates a significant difference from the negative control using Dunnett's test (p<0.05). 000761 AMENDED W ild life International ltd. -18- PROJECT NO.: 454A-106 Table 4 Individual Oyster Shell Measurements Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Eastern Oyster, Crctssostrea virginica Dilution Water: Unfiltered Saltwater Mean Measured Concentrations (mg a.i./L) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean SD Range Negative Control 4.00 3.25 3.00 2.55 2.50 2.75 2.70 2.70 1.85 2.20 4.40 2.05 1.55 3.05 2.45 4.20 1.45 1.75 2.85 2.20 2.67 0.824 1 .4 5 -4 .4 0 0.36 2.05 1.80 1.90 3.65 2.55 1.90 2.40 4.00 1.45 2.55 3.55 1.80 3.35 2.30 3.70 3.75 2.60 0.90 2.85 0.95 2.50 0.933 0 .9 0 -4 .0 0 0.40 3.20 2.25 3.10 0.90 2.35 2.10 2.25 2.80 1.75 2.45 2.85 3.00 4.45 3.05 1.95 1.20 1.70 2.05 1.65 2.90 2.40 0.820 0 .9 0 -4 .5 5 1.3 1.50 2.80 2.65 2.25 2.40 2.70 2.90 4.45 1.80 3.05 1.60 0.85 4.10 2.70 2.45 3.00. 1.85 2.95 0.95 3.30. 2.51 0.919 0 .8 5 -4 .4 5 1.9 2.20 1.45 3.60 1.80 2.25 2.30 3.10 0.65 0.80 2.40 2.70 1.65 1.80 2.20 1.70 3.15 0.90 2.20 2.90 2.85 2.13 0.804 0 .6 5 -3 .6 0 3.0 1.65 0.80 2.20 1.75 3.05 1.60 0.90 2.40 2.25 1.80 2.70 1.10 2.35 1.70 2.15 2.60 1.60 1.45 2.00 2.20 1.91 0.591 0 .8 0 -3 .0 5 000762 AMENDED W il d l if e In te r n a t io n a l ltd. -19- PROJECT NO.: 454A-106 APPENDLXI Salinity and pH o f Unfiltered Saltwater Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: Salinity 3M Corporation PFOS Eastern Oyster, Crassostrea virginica Unfiltered Saltwater Mean 21 (N = 4) Range aBMmmaaaaa 20-21 pH 8.1 (N = 4) 8.0 - 8.2 000763 W il d l if e In t e r n a t io n a l ltd. -20- PROJECT NO.: 454A-106 APPENDIX H A nalyses o f Pesticides, O rganics, M etals and O ther Inorganics in W ildlife International L td. S altw ater1 A N A L Y S IS M EASURED CONCENTRATION Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon1 Total Cyanide Organochlorines and PCBs 23,500 < 0.050 < 1.0 < 10.0 mg/L mg/L mg/L Pg/L Aldrin Alpha BHC BetaBHC Deha BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' DDT, pp' Dieldrin Endosulfan, A Endosulfan, B Endosulfan Sulfide Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 ` < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260" < 0.260 ' < 0.720 < 0.720 < 0.720 < . 0.720 Pg/L Pg/L Pg/L Pg/L Pg/L Pg/L pg/L Pg/L pg/L Pg/L pg/L pg/L Pg/L Pg/L P&L Pg/L Pg/L pg/L Pg/L Pg/L pg/L Pg/L Pg/L Pg/L Pg/L Pg/L Metals and Other Inorganics Aluminum1 Arsenic1 Beryllium1 Cadmium1 Calcium1 Pcntirronnmuinumm^ Cobalt1 Copper1 Iron1 Lead1 Magnesium1 Manganese1 Mercury Molybdenum1 Nickel1 Potassium1 Selenium1 Silver1 Sodium1 Zinc1 < 100 < 25.0 < 0.50 < 1.0 235 < 2.0 < 1.0 < 20.0 < 100 < 10.0 760 < 4.0 < 0.20 < 2.0 < 20.0 277 < 25.0 < 1.0 6,010 < 20.0 Pg/L Pg/L Pg/L pg/L mg/L pg/L pg/L pg/L Pg/L Pg/L mg/L Pg/L Pg/L Pg/L Pg/L mg/L Pg/L pg/L mg/L Pg/L 'Analyses performed by QST Environmental, Gainesville, Florida for samples collected on November 3 through November 7,1997. 'Analyses performed by Wildlife International Ltd. for the sample collected on November 3,1997. 'Analyses performed by Wildlife International Ltd. for samples collected on November 3 through 7,1997. 000764 W il d l if e In ter n a tio n a l ltd. - 21- APPENDEXm PROJECT NO.: 454A-106 THE ANALYSIS OF PFOS IN UNFILTERED SALTWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-106 As 000765 W il d l if e In t e r n a t io n a l ltd. - 22 - REPORT APPROVAL PROJECT NO.: 454A-106 SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Shell Deposition Test with the Eastern Oyster (Crassostrea virginica) W ILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-106 PRINCIPAL INVESTIGATOR: * 2 5 -0 0 DATE MANAGEMENT: _____________ ____ W illard B. Nixon, Ph.D : Manager, Analytical Chemistry M DATE7 / 000766 AMENDED W il d l if e International ltd. -23- PROJECT NO.: 454A-106 Introduction Unfiltered saltwater samples were collected from an acute toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) on the shell deposition o f the eastern oyster (Crassostrea virginica). This study was conducted by W ildlife International Ltd. and identified as Project No.: 454A-106. The analyses o f these w ater 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 May 24, 26 and 28, 1999. Samples were analyzed on each sample receipt day. All samples were reanalyzed on June 6, 1999 to confirm original analyses. The second analysis was reported. Test Substance and Internal Standard The test substance used for the analytical portion o f this study was W ildlife International Ltd. identification number 4675. The test substance was used to prepare calibration standards and 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: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the unfiltered saltwater samples was developed at W ildlife International Ltd. and entitled "Analytical Method Validation for the Determination o f PFOS in Freshwater, Saltwater, and Algal Media". This methodology was included as Appendix II o f W ildlife International Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure w ater solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. 000767 W il d l if e In ter n a tio n a l ltd. - 24 - PROJECT NO.: 454A-106 Concentrations o f PFOS in the standards and samples were determined by reversed-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC M ass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cig analytical column (100 mm x 2 mm I.D ., 3 pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit o f Quantitation Calibration standards o f PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00229 to 0.0274 mg a.i./L were analyzed w ith the samples. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. A typical calibration curve is presented in - Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit o f quantitation (LOQ) for these analyses was set at 0.115 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the m atrix blank samples (50.0). Matrix Blank and Fortification Samples One m atrix blank sample was analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). The matrix blank chromatogram is presented in Figure 5. Unfiltered saltwater was fortified a t 0.457,4.57 and 11.0 mg a.i./L and analyzed concurrently with the samples (Table 2). Sample concentrations were not corrected for mean procedural recovery. High recoveries were obtained for the low-level (0.457 mg a.i./L ) m atrix fortification sample. Previous analyses in saltwater verified that the method is capable o f producing quantitative recoveries at this concentration level. A representative chromatogram o f a matrix fortification is presented in Figure 6. 000768 AMENDED W il d l if e In te r n a tio n a l ltd. -25- PROJECT NO.: 454A-106 Example Calculations Sample number 454A-106-5B, nominal concentration o f 5.5 mg a.i./L in unfiltered saltwater. Initial Volume: 0.100 mL Final Volume: 25.0 mL Dilution Factor: 250 PFOS Peak Area: 71220 Internal Standard Peak Area: 74271 Peak Area Ratio: 0.95892 Calibration curve equation. Slope: 7.78372. Intercept: 0.17175 Curve is weighted (1/x). PFOS (mg a.i./L) at instrument (Peak area ratio - (Y-intercept)) x I.S. Concentration Slope (0.95892 - 0.17175) x 0.100 mg a.i./L 7.78372 = 0.0101 mg a.i./L Note: I.S. = internal standard. PFOS (mg a.i./L) in sample PFOS (mg a.i./L) at instrument x Final Volume (mL) Initial Volume (mL) 0.0101 x 25.0 0.100 = 2.53 mg a.i./L 000769 AMENDED W il d l if e In ter n a tio n a l ltd. -26- PROJECT NO.: 454A-106 Percent o f Nominal Concentration PFOS (mg a.i./L) in sample = ------------------------------------ x 100 PFOS (mg 2lX.IL) nominal Calculated recovery: 46.1% Note: manual calculation may differ. RESULTS Sample Analysis Unfiltered saltwater samples were collected from the shell deposition test with the eastern oyster (Crassostrea virginica) at test initiation, M ay 24, 1999 (Hour 0), on M ay 26, 1999 (Hour 48), and at test termination, May 28,1999 (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 27.9 to 46.1% o f the nominal concentrations. Samples collected at Hour 48 had a measured concentration range o f 14.9 to 40.9% o f nominal values. Samples collected at test termination (Hour 96) had a measured concentration range o f < LOQ to 52.2% o f nominal values (Table 3). A representative chromatogram o f a test sample is shown in Figure 7. 000770 AMENDED W il d l if e In t e r n a t io n a l Ltd . -27- PROJECT NO.: 454A-106 IN STR U M EN T: Table 1 Typical LC/MS 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 Cig column (50 mm x 2 mm I.D ., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% M ethanol: 28.0% NANOpure W ater containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 3.6 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.6 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 000771 W il d l if e Internatio nal ltd. -28- PROJECT NO.: 454A-106 Table 2 M atrix Blank and Fortifications Analyzed Concurrently During Sample Analysis Number (454-106-) MAB-4 Sample Type M atrix Blank Concentrations o f PFOS (mg a.i./L) Fortified 0.0 M easured1 <LOQ Percent Recovered - M A S-10 M A S-10A 2 M A S-10B 2 M atrix Fortification M atrix Fortification M atrix Fortification 0.457 0.457 0.457 0.760 0.812 0.769 166 177 168 M A S-11 M atrix Fortification 4.57 4.57 99.8 M A S-12 M atrix Fortification 11.0 13.0 119 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 The limit 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). 2 Sample 454-106-M AS-10 was rediluted in duplicate and analysis confirmed original result; suspect fortification error. __________________ ,__________________________________ 000T7Z AMENDED W il d l if e In te r n a tio n a l ltd. -29- PROJECT NO.: 454A-106 Table 3 Measured Concentrations o f PFOS in Unfiltered Saltwater Samples from an Oyster Shell Deposition Test Nominal Test Concentration (mR a.i./L) 0.0 Sample Number 1A IB 7A 7B 13A 13B Sampling Time 0 0 48 48 96 96 PFOS Measured Concentration1 (mg a.iTL) <LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal -- -- -- -- -- - 1.2 2A 0 0.331 27.9 2B 0 0.353 29.7 8A 48 0.341 28.7 8B 48 0.429 36.1 14A 96 < LOQ -- 14B 96 < LOQ - 2.0 3A 0 3B 0 9A 48 9B 48 15A 96 15B 96 0.622 0.633 0.299 0.313 0.249 0.257 30.9 31.5 14.9 15.6 12.4 12.8 3.3 4A 0 - 1.36 41.4 4B 0 1.15 34.9 10A 48 0.924 28.0 10B 48 0.878 26.7 16A 96 1.58 48.0 16B 96 1.72 52:2 5.5 5A 0 5B 0 11A 48 11B 48 17A 96 17B 96 2.42 2.53 2.02 2.24 1.45 0.970 44.1 46.1 36.9 40.9 26.4 17.6 9.1 6A 0 _2 _ 6B 0 6C 0 3.39 37.1 3.44 37.6 6D 0 3.01 32.9 12A 48 3.74 40.9 12B 48 3.57 39.0 18A 96 1.99 21.7 18B 96 2.19 23.9 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 The limit of quantitation (LOQ) was 0.115 mg a.iTL 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). 2 Result for sample 6A (6.21 mg a.i./L) was not included in the table. Sample 6 was rediluted in duplicate (6C and 6D) and results of these samples confirmed result for sample 6B. 000773 AMENDED W il d l if e International ltd. -30- PROJECT NO.: 454A-106 M ETHOD OUTLINE FO R TH E ANALYSIS O F PFOS IN UNFILTERED SALTW ATER Prepare matrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into unfiltered 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 matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. Amputate samples and submit for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis o f PFOS in unfiltered saltwater. 000774 W il d l if e In t e r n a t io n a l ltd. -31- PROJECT NO.: 454A-106 Figure 2. A typical calibration curve for PFOS. Slope = 7.78372; Intercept = 0.17175; r = 0.9825. Curve is weighted (1/x). 000775 AMENDED W il d l if e In ter n a tio n a l ltd. -32- PROJECT NO.: 454A-106 intensity: 1102 cps. F igure 3. A representative ion chromatogram o f a low-level (0.00229 mg a.i./L) PFOS standard. 000776 AMENDED Wil d l if e Intern atio na l ltd. -33- PROJECTNO.: 454A-106 intensity: 6897 cps Figure 4. A representative ion chromatogram o f a high-level (0.0274 mg a.i./L) PFOS standard. 000777 AMENDED W il d l if e In t e r n a t io n a l Ltd . -34- PROJECT NO.: 454A-106 intensity: 280 cps Figure 5. The matrix blank ion chromatogram (454A-106-MAB-4) The arrow indicates the retention time o f PFOS. 000778 W il d l if e International ltd. -35- PROJECT NO.: 454A-106 intensity: 9001 cps Figure 6. A representative ion chromatogram o f a 4.57 mg a.i./L matrix fortification sample (454A-106-MAS-11). 000779 AMENDED W il d l if e Intern atio na l ltd. -36- PROJECT NO.: 454A-106 intensity: 5239 cps Figure 7. A representative ion chromatogram o f a 5.5 mg a.i./L test sample (454A-106-5B). 000780 AMENDED W il d l if e In t e r n a t io n a l ltx>. -37- PROJECT NO.: 454A-106 APPENDDOV 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 specify that GLP analyses of saltwater will be reported. 3. The protocol was amended to change the analytical sampling schedule. 4. Salinity was measured in the negative control at test initiation and termination. 0007S1 W il d l if e Intern atio na l ltd. -38- PROJECT NO.: 454A-106 APPENDIX V Personnel Involved in the Study The following key Wildlife International Ltd personnel were involved in the conduct or management o fthis study: 1. Henry 0 . 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, PLD., Scientist 0007S2 W il d l if e In t e r n a tio n a l ltd. -39- PROJECT NO.: 454A-106 APPENDIX VI Report Amendment 1. Original Report: Pages 1-4,6 and 22 Amendment: The pages were changed to include the amended report date, revisedpage 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 rcanalysis 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 purify ofthe test substance as determined by the Sponsor in a reanalysis o f the test substance (FC-95, Lot 217). Test concentrations originally were based on the reported purity of 98.9%. The certificate of analysis dated March 9,2000 indicated purify 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% purify. Reason: To report the results of the test based on the test substance purify of 90.49% at the request o f the Sponsor. 5. Original Report: Page 15 Amendment: Add footnotes 1 and 2 to Table 1, Summary o f Analytical Chemistry Data. Reason: To clarify the measured concentrations reported. 0007S3 AMENDED Wil d l if e In ter n a tio n a l ltd. -40- PROJECT NO.: 454A-106 APPENDIX VI -Continued- Report Amendment 6. Original Report: Page 17 Amendment: Change the 96-hour EC50 value of >10 mg a.i./L to >3.0 mg a.i./L. Reason: To correct the reported EC50 value to reflect the mean measured concentration, rather than the nominal concentration. AMENDMENT SIGNATURES: Kurt R. Drottar Study Director Henry O. K ru e g e r^ h S Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: Jais H.CCoolleman Quality Assurance y /c 2 > / 0 DATE H- 6 -0 0 DATE 000784 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-106 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrea virginica) PROTOCOL NO.: 454/042199/OYS-DEP/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454A-106 EFFECTIV E DATE: May 20, 1999_______________________________________ AMENDMENT: Page 2 Add: Experimental Start Date: 5/24/99 Experimental Termination Date: 5/28/99 Test Concentrations: Negative Control, 1.3, 2.2, 3.6, 6.0 and 10 mg a.i./L Test Substance No.: 4675 REASON: The above information was not known when the protocol was signed by the Study Director. AMENDMENT: Dilution Water. Page 5 Change: Analyses will be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents o f the seawater and the results of the analyses will be summarized in the final report. To: Analyses will be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents of the seawater and the results of the most recent GLP compliant analyses will be summarized in the final report. REASON: To specify that GLP analyses will be reported. AM ENDM ENT: Sampling for analytical Measurements. Page 7 Change: Water samples will be collected from each test chamber at test initiation and every 24 hours (1 hour) during the test to determine concentrations of the test substance. To: Water samples will be collected from each test chamber at test initiation, at the midpoint o f the test (approximately 48 hours) and at test termination to determine concentrations of the test substance. 000785 REASON: The Sponsor requested a change in the analytical sampling schedule. a454\l 06\amendl Revicuj&d be/ Wil d l if e In t e r n a t io n a l ltd. PROJECT NO.: 454A-106 Page 2 o f2 STUDY DIRECTOR DATE a 4 S 4 \l 06\am endl 000786 M W il d l if e In ter n a tio n a l ltd . PROJECT NO.: 454A-106 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrea virginicd) PROTOCOL NO.: 454/042199/0YS-DEP/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454A-106 DATE OF DEFACTO DEVIATION: May 24,1999 DEVIATION: The protocol states that dilution water salinity will be measured in each test chamber at test initiation and at the end of the test. Dilution water salinity was only measured in the negative control at the beginning and end o f the test. REASON: Biologist oversight. Wildlife International Ltd. stores unfiltered saltwater in a 5,000 gallon tank which is recirculated to maintain constant salinity. Consequently, it is the best judgment of the Study Director that this deviation did not adversely affect the results of the study. STUDY DIRECTOR I ; LABORATORY MANAGEMENT KRD454a\106\prdl I I I - I I n I & a rm DATE 000787 PROTOCOL PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrea virginica) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1025 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 April 21,1999 0007S8 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 SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrea virginica) SPONSOR: 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 SPONSORS REPRESENTATIVE: Ms. Susan A. Beach 0 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 o f Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental Start Date: ___________________________ Experimental Termination Date: _____ Project No.: J S 4 4 - !Q (j> ________________________________ ! Test Concentrations: ______________________________________________ Test Substance No.: __________ Reference Substance No. (if applicable): PROTOCOL APPROVAL r STUDY DIRECTOR ^ - /s M DATE fLABORATORY MANAGEMENT f}- fbduA SPONSOR'S REPRESENTATIVE PROTOCOL NO. 454/042199/OYS-DEP/SUB454 0007S9 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 -3 - INTRODUCTION Wildlife International, Ltd. will conduct a static acute toxicity test with the Eastern oyster (Crassostrea virginica) for the Sponsor at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1025 (1); StandardEvaluation Procedure, Acute Toxicity Testfor Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study) (2) and ASTMStandard E729-88a Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates andAmphibians (3). Raw data for all work performed at Wildlife International, Ltd. and a copy of the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. PURPOSE The purpose of this study is to determine the effects of a test substance on the shell deposition of the eastern oyster (Crassostrea virginica) during a 96-hour exposure under static test conditions. EXPERIMENTAL DESIGN Oysters will be exposed to a geometric series o f at least five test concentrations and a negative (dilution water) control for 96 hours. One test chamber will be maintained for each treatment and control group with 20 oysters in each chamber. Nominal test concentrations will be selected in consultation with the Sponsor, and will be based upon information such as the results o f exploratory range-finding toxicity data, known toxicity data, physical/chemical properties of the test substance or other relevant information. Target concentrations will not exceed 120 mg/L or the solubility limit of the test substance in water (whichever is lower). Generally, each test substance concentration used in the definitive test will be at least 60% of the next higher concentration unless information concerning the concentration-effect curve indicates that a different dilution factor would be more appropriate. Water samples from appropriate test chambers will be collected at specified intervals for analysis of the test substance. Results o f analyses will be used to calculate mean measured test concentrations. Oysters approximately 25 to 50 mm long will be maintained at Wildlife International, Ltd. in unfiltered saltwater for a period of at least 10 days. To control bias, oysters used in the test will be indiscriminately distributed. No other potential sources of bias are expected to affect the results o f the study. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 00079C 3M LAB REQUEST NO. U2723 Wild life In ter n a tio n a l, ltd -4 - Just prior to the initiation of the test, recently deposited shell will be removed by grinding the periphery of the oysters against an electric disc grinder or similar apparatus. The initial shell length (after grinding) of twenty indiscriminately selected oysters will be measured and 20 indiscriminately selected oysters will be placed in each test chamber. At the conclusion of the 96-hour test, deposition o f new shell will be measured for each oyster. Since shell deposition is often asymmetrical, the length of the longest "finger" of new shell will be measured using calipers. Shell growth inhibition for each treatment group will be determined by calculating mean shell deposition as a percentage o f the control. Mean shell growth inhibition in each treatment group will be expressed as a percentage of mean control, shell deposition. An EC50 value will be calculated, when possible, to express the concentration of test substance that induces a 50% reduction in shell deposition. MATERIALS AND METHODS Test Substance Information on the characterization of test, control or reference substances is required by Good Laboratoiy 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 o f GLP test substance characterization is not provided to Wildlife Tntftmatinnal 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 of the study. Preparation o f Test Concentrations The test substance will be administered to the test organism in water. This route of administration was selected because it represents the most likely route o f exposure to aquatic organisms. PROTOCOL NO. 454/042199/0YS-DEP/SUB454 000791 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l, ltd -5 - Test Organism The eastern oyster (Crassostrea virginica) will be used in this test. This species is representative of an important group of organisms and was selected for use in the test based upon past use and ease of handling in the laboratory. Test organisms will be obtained from a commercial supplier or research facility. All organisms will be obtained from the same source and held in the laboratory for at least 10 days prior to testing. Oysters will not be used in the test if they show signs of disease or stress or if more than 5% die during the 7 days prior to the test. Saltwater supplied to the oysters during holding and testing will be unfiltered. A saltwater alga such as Thalassiosira sp., Skeletonema sp., Chaetoceros sp. and/or Isochrysis sp. will be added to the water for supplemental nutrition. Dilution Water Water used for holding and testing will be natural unfiltered seawater collected at Indian River Inlet, Delaware. The seawater will be pumped into a 19,000-L holding tank where the salinity of the seawater will be diluted to approximately 20 /oo (parts per thousand) with freshwater from a well on the Wildlife International, Ltd. site. The saltwater in the holding tank will be aerated by recirculation prior to delivery to the test chambers. Analyses will be performed at least once annually to determine the concentrations of selected organic and inorganic constituents of the seawater and the results of the analyses will be summarized in the final report. Dilution water salinity should be 20 l%o, and the pH should not vary by more than 1 unit during the test. Test Apparatus Test chambers will be 52-L polyethylene chambers filled with approximately 40 L of test solution. Test chambers will be impartially positioned in an environmental chamber or a temperature-controlled water bath to maintain a temperature of 22 1C. An electric mixer will be placed in each test chamber to maintain the suspension of the algal diet hi addition, each test chamber will be mildly aerated to reduce the biological oxygen demand due to the feed. Test chambers will be labeled with the project number and test concentration. 000V 92 PROTOCOL NO. 454/042199/OYS-DEP/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 6- - Environmental Conditions Lighting used to illuminate the holding and test chambers will be provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g., Colortone 50). A photoperiod o f 16 hours of light and 8 hours o f dark will be controlled with an automatic timer. A 30-minute transition period 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 22 1C. Temperature will be measured in each test chamber at the beginning and end of the test using a liquid-in-glass thermometer. Temperature also will be measured with a continuous recorder in the negative control chamber. Recorder measurements will be verified with a liquid-in-glass thermometer prior to test initiation. Dissolved oxygen will be recorded in each test chamber daily. The pH will be measured in each treatment and control group at test initiation, midpoint (approximately 48 hours), and the end of the test. Dissolved oxygen will be measured using a Yellow Springs Instrument Model 5IB dissolved oxygen meter, or equivalent. Measurements of pH will be made using a Fisher Accumet Model 915 pH meter, or equivalent In the event that dissolved oxygen concentrations fall below 60% saturation, dissolved oxygen measurements will be made in every test chamber and appropriate actions will be taken after consultation with the Sponsor. Dilution water salinity will be measured in each chamber at test initiation and at the end o f the test. Salinity will be measured using a Bio-Marine, Inc. AquaFauna refractometer, or equivalent. If a treatment group reaches 100% mortality, dissolved oxygen, pH and temperature measurements will be taken at that time, then discontinued. Biological Measurements Oysters will be inspected visually at approximately 0-24 hours and at 24,48,72, and 96 hours ( 1 hour) after test initiation. Oysters having open shells and not responding to gentle prodding will be considered dead and removed. Any shell abnormalities observed will be recorded. When the test is terminated, shell deposition will be recorded for each oyster by measuring the length of the longest finger of new shell. Shell growth inhibition for each treatment group will be determined by calculating mean shell deposition as a percentage o f the control. 000793 PROTOCOL NO. 454/042199/0YS-DEP/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 Sampling for Analytical Measurements Water samples will be collected from each test chamber at test initiation and every 24 hours ( 1 hour) during the test 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, analyzed immediately or placed in an appropriate storage container (e.g., glass or polypropylene bottle) and stored under refrigeration (~4C) until analyzed. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES 24 48 72 Experimental Group 0 Hours Hours Hours Hours 96 Hours Control 11 111 Level 1-Low Concentration 1 1 1 1 1 Level 2 11 111 Level 3 1 1 1_ 1 1 Level 4 11 111 Level 5 11 111 66 666 Total Number of Samples = 30 The above numbers o f samples represent those collected from the test and do not include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the analytical chemistry phase of the study. Analytical Chemistry Chemical analysis of the samples will be performed by Wildlife International, Ltd. The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix II. The methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 000794 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l , ltd 8- - Data Analysis Mean shell growth inhibition in each treatment group will be expressed as a percentage of control growth deposition. An EC50 value will be calculated when possible to express the concentration o f test substance that induced a 50% inhibition in shell deposition. The following formula will be used: %Inhibition = ^ ' T X 100 C Where C = Mean growth of negative controls T = Mean growth of treated The data will be analyzed by linear interpolation using ICPIN computer software (when possible) (4). The no-observed-effect-concentration (NOEC) and lowest-observed-effect-concentration (LOEC) will be determined based (when possible) on a statistical analysis of the dose-response data and an assessment of the dose-response pattern. RECORDS TO BE MAINTAINED Records to be maintained for data generated by Wildlife International Ltd. will include, but not be limited to: 1. Copy of signed protocol. 2. Identification and characterization of the test substance, if provided by sponsor. 3. Dates o f initiation and termination of the test 4. Holding records. 5. Calculation and preparation of test concentrations. 6. Stock solution calculations and preparation, if applicable. 7. Observations. 8. If applicable, the methods used to analyze test substance concentrations and the results o f analytical measurements. 9. Statistical calculations, if applicable. 10. Test conditions and physical/chemical measurements. 11. Measurements of shell deposition. 12. Copy Qf final report. 000795 PROTOCOL NO. 454/042199/OYS-DEP/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 -9 FINAL REPORT A 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. It is the responsibility of the Sponsor to provide the final date that data are recorded for chemistry, pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement of compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures as stated in the approved protocol, including any changes in the original protocol. 5. The test substance identified by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor. 6. Stability and the solubility of the test substances under the conditions o f administration, if provided by the Sponsor or contracted to Wildlife Internationa^Ltd. 7. A description of the methods used to conduct the test. 8. A description of the test system, including the source of the test organisms, scientific name, life stage, means and ranges o f lengths, observed diseases, treatments and holding procedures. 9. A description of the preparation of the test solutions, methods used to allocate organisms to test chambers and begin the test, numbers of organisms and chambers per treatment, allocation of organisms to test chambers and duration of the test. 10. A description of circumstances that may have affected the quality or integrity of the data. 11. The name o f the Study Director and the names o f other scientists, professionals, and supervisory personnel involved in the study. 12. A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the biological data and analytical chemistry data, and a statement of the conclusions drawn from these analyses. 13. Statistical methods used to evaluate the data, if applicable. 14. The signed and dated reports of each o f the individual scientists or other professionals involved in the study, if applicable. 15. The location where raw data and final report are to be stored. 000796 PROTOCOL NO. 454/042199/OYS-DEP/SUB454 m 3M LAB REQUEST NO. U2723 W ild life In tern atio na l, ltd -10- 16. 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. 17. 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 amendments. Amendments will be signed and dated 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 th 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 o f Good Laboratory Practice (OCDE/GD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International Ltd. is routinely examined by the Wildlife International Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement of compliance with Good Laboratory Practices will be prepared for all portions of the study conducted by Wildlife International Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at Wildlife International Ltd. and a copy of the final report will be filed by project number in archives located on the Wildlife International Ltd. site, or at an alternative location to be specified in the final report. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 000797 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 11 - REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number 850.1025: Oyster Acute Toxicity Test (Shell Deposition). 2 U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Test fo r Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study). EPA-540/9-85-011. 3 ASTM Standard E729-88a. 1994. Standard Guide fo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 Norberg-King, T.J. 1993. A Linear Interpolation Methodfor Sublethal Toxicity: The Inhibition Concentration (ICp) Approach. Version 2.0. U.S. Environmental Protection Agency. National Effluent Toxicity Assessment Center. Duluth, Minnesota. Technical Report 03-93. PROTOCOL NO. 454/042199/0YS-DEP/SUB454 000798 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 12- APPENDKI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I.Test Substance Identity (name to be used in the report): PFOS CPerfluorooctane 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____________ H. Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately definethe test substance and reference standardbeen determinedprior to its use in this study in accordance with GLP Standards? _____ Yes x No HI. Test Substance Storage Conditions Please indicate the recommended storage conditions at Wildlife International Ltd. _____ Ambient________________________________________________________ Has the stabilityofthe test substanceunder these storage conditions been determined in accordancewith GLP Standards? ' _____ Yes x No Other pertinent stability information: IV. Test Concentrations: _____ x Adjusttest concentrationto 100% ai. based upon the purity (%) given above. Do not adjusttest concentrationto 100% ___________ ai. Test tne material AS IS. V. Toxicity Information: Mammalian: RatLD50 251 me/ke Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity(LC50) Daphnia mama: 27 me/L__________ Rainbow Trout: 11 me/L Daphnia maena: 50 mg/L__________ Fathead Minnow: 38 mg/L Other Toxicity Information (including findings ofchronic and subchranic tests): Please see MSDS______________________ _____________________________________ 000799 PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd - 13- APPENDIX II Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination of Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f the above method will be maintained in the raw data. The actual methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 000800 3M LAB REQUEST NO. U2723