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/IR 21G -0093 PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454E-101 3M LAB REQUEST NUMBER: U2723 AUTHORS: Edward C. Schaefer R. Scott Flaggs STUDY INITIATION DATE: December 11, 1998 STUDY COMPLETION DATE: March 23, 1999 AMENDED REPORT DATE: April 28, 2000 SUBMITTED TO: 3M Environmental Laboratory Bldg 2-3E-09 St. Paul, Minnesota 55133 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 49 000367 AMENDED W il d l if e In ter n a tio n a l l t d . 2- - PROJECT NO.: 454E-101 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: An Activated Sludge, Respiration Inhibition Test 3M LAB REQUEST NUMBER: U2723 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454E-101 STUDY COMPLETION: March 23,1999 REPORT AMENDED: April 28, 2000 This study was conducted in compliance with the U.S. Environmental Protection Agency in 40 CFR Part 160; 17 August 1989; OECD Principles of Good Laboratory Practices [OCDE/GD(92)32] Environment Monograph No. 45; Paris 1992; and Japan MAFF 59 NohSan, Notification No, 3850, Agricultural Production Bureau, with the following exceptions: The reference substance, obtained from Aldrich Chemical Company (Milwaukee, WI), was not characterized in compliance with Good Laboratory Practice Standards. A certificate of analysis has been supplied by Aldrich. Stability, homogeneity and verification of the test and reference substances in the mixtures were not determined. The test substance was not characterized in accordance with lull GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance is being recharacterized in accordance with GLP. The stability o f the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Manager, Biodegradation DATE 000S68 AMENDED W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 -3 - QUALITY ASSURANCE STATEMENT This study was examined for compliance with the U.S. Environmental Protection Agency in 40 CFR Part 160, 17 August 1989; OECD Principles of Good Laboratory Practices [OCDE/GD(92)32] Environment Monograph No. 45 [OCDE/GD(92)32] and Japan MAFF, 59 NohSan, Notification No, 3850, Agricultural Production Bureau; Wildlife International, Ltd. Standard Operating Procedures and the study protocol. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Chamber Preparation DO Measurements Raw Data and Draft Report Final Report Amended Final Report DATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: December 22,1998 December 22,1998 December 23,1998 December 22,1998 December 22,1999 December 22,1998 January 27 and 28,1999 January 28,1999 February 3,1999 March 23, 1999 April 28,2000 March 23,1999 April 28,2000 March 23,1999 April 28,2000 T. Hynson Assurance Program S 000869 AMENDED W il d l if e Internatio nal l t d . PROJECT NO.: 454E-101 -4 - REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: An Activated Sludge, Respiration Inhibition Test 3M LAB REQUEST NUMBER: U2723 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454E-101 STUDY DIRECTOR: Edward C. Schaefer, B.S. Manager, Biodegradation MANAGEMENT: L Henry OrKrueger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants SPONSOR'S REPRESENTATIVE: Susan A. Beach 3M Corporation Cx. JDATE DATE 7 5 AIn) DATE 000870 AMENDED W il d l if e Internatio nal ltd . -5 STUDY INFORMATION Study Initiation Date: Experimental Start Date: Experimental Termination Date: Study Completion Date: Amended Final Report Date: December 11,1998 December 22,1998 December 22,1998 March 23,1999 April 28,2000 PROJECT NO.: 454E-101 Study Director: Sponsor: Sponsor's Representative: Study Personnel: Edward C. Schaefer B.S. 3M Environmental Laboratory St. Paul, Minnesota 55133 Susan A. Beach Edward C. Schaefer, B.S., Manager, Biodegradation Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants Abul Siddiqui, B.S., Scientist, Biodegradation Sheri Trumbull, Technologist, Biodegradation R. Scott Flaggs, B.S., Biologist, Biodegradation 000571 AMENDED W il d l if e In te r n a tio n a l l t d . -6 TABLE OF CONTENTS PROJECT NO.: 454E-101 Titie P a g e ................................................................................................................................................... Page 1 Good Laboratory Practice Compliance Statement ..................................................................................Page 2 Quality Assurance Statement .................................................................................................................. Page 3 Report Approval .......................................................................................................................................Page 4 Study Information .....................................................................................................................................Page 5 Table of Contents .....................................................................................................................................Page 6 A b stract..................................................................................................................................................... Page 8 Introduction............................................................................................................................................... Page 9 O bjective................................................................................................................................................... Page 9 Experimental D esig n ........................................................................... Page 9 Materials and Methods ............................................................................................................................ Page 9 Test Substance............................................................................................................................ Page 9 Reference Substance.................................................................................................................... Page 10 Route of Exposure and D uration................................................................................................Page 11 Test System ............ Page 11 Test Conditions and A pparatus..................................................................................................Page 11 Test Inoculum.............................................................................................................................. Page 11 Procedure .....................................................................................................................................Page 12 Sample A nalysis................................................................................................................................Page12 Calculations ................................................................................................................................ Page 12 Statistical Analyses .................................................................................................................... Page 13 Results and Discussion .............................................................................................................................Page 13 Conclusion...................................................................................................................................................... Page14 References ................................................................................................................................................. Page 15 000372 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454E-101 -7 - TABLE OF CONTENTS - Continued - TABLE Table 1. Respiration Rates and Percent Inhibitions............................................................................ Page 16 FIGURES Figure 1. Dissolved Oxygen Concentration Over Time, Control 1 ......................................................... Page17 Figure 2. Dissolved Oxygen Concentration Over Time, Control 2 ......................................................... Page18 Figure 3. Dissolved Oxygen Concentration Over Time, 3,5-Dichlorophenol, 3 m g /L .......................... Page19 Figure 4. Dissolved Oxygen Concentration Over Time, 3,5-Dichlorophenol, 15 m g /L ........................ Page20 Figure 5. Dissolved Oxygen Concentration Over Time, 3,5-Dichlorophenol, 50 m g /L ........................Page21 Figure 6. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 0.90 mg a.i./L.............................................................................. Page 22 Figure 7. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 2.7 mg a.i./L................................................................................ Page 23 Figure 8. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 9.0 mg a .i./L .............................................................................. Page 24 Figure 9. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 27 mg a .i./L ................................................................................ Page 25 Figure 10. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 90 mg a .i./L ................................................................................ Page 26 Figure 11. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 271 mg a .i./L .............................................................................. Page 27 Figure 12. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 905 mg a .i./L .............................................................................. Page 28 APPENDICES Appendix I. Measured Dissolved Oxygen (DO) Concentrations (mg 0 2/L ) ................................. Page 29 Appendix II. Graphical Presentation of Percent Inhibition and Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Concentration........................................................................ Page 31 Appendix IE. Protocol, Amendment and D eviation........................................................................... Page 33 Appendix IV. Report Amendment ......................................................................................................Page 48 AMENDED 000S73 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454E-101 8- - ABSTRACT The effect of the test substance on activated sludge microorganisms was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained a control, reference and treatment groups. The control group was used to determine the background respiration rate of the sludge and was not dosed with the test or reference substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3,15 and 50 mg/L. The test substance was dosed at 0.90,2.7, 9.0, 27, 90, 271 and 905 mg a.i./L. After an exposure period of 3 hours, the respiration rates o f the test solutions were measured using a dissolved oxygen meter. The respiration rates in the two controls were 39.8 and 41.1 mg Oj/L/hr, or a difference of 3.2%. The difference in control respiration rates was within the 15% difference limit established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference group, which resulted in an EC50 o f 9.0 mg/L. The EC50 was within the 5 to 30 mg/L range considered acceptable for the test. Differences in 0 2respiration rates between the control group and test substance treatments were observed at all test substance concentrations. Following is a summary o f the results. Treatment Control 1 Control 2 3,5-dichlorophenol 3 mg/L 3,5-dichlorophenol 15 mg/L 3,5-dichlorophenol 50 mg/L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 0.90 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 2.7 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 9.0 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 27 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 90 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 271 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 905 mg a.i./L Respiration Rate Percent mg O/L/hour Inhibition 39.6 NA 41.1 NA 31.3 22.9 14.6 63.8 5.1 87.4 38.9 3.6 35.1 13.0 33.5 17.0 37.9 6.1 32.7 19.0 28.1 30.4 24.7 38.8 000S74 AMENDED W il d l if e In t e r n a t io n a l l t d . -9 - PROJECT NO.: 454E-101 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. biodegradation facility in Easton, Maryland. Original raw data generated by Wildlife International Ltd. and a copy of the original final report are filed undo-Project Number 454E-101 in the archives located on the Wildlife International Ltd. site. OBJECTIVE The objective of this study was to assess the effects of the test substance on activated sludge microorganisms by measuring the respiration rate. EXPERIMENTAL DESIGN The test contained control, reference, and treatment groups. The control group was used to determine the background respiration rate of the sludge and was not exposed to the test or reference substances. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor o f respiration, at concentrations o f 3, 15, and 50 mg/L. The test substance was tested at 0.90,2.7,9.0,27,90,271 and 905 mg a.i./L. MATERIALS AND METHODS This study was conducted according to the procedures outlined in the protocol, "PFOS: An ActivatedSludge, Respiration Inhibition Test," (Appendix IV). The protocol was based on the procedures specified in the OECD Guideline for Testing of Chemicals, Method 209 (1) and Council of the European Communities, Guideline C. 11, Activated Sludge, Respiration Inhibition Test (2). Test Substance The test substance, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), was received at Wildlife International Ltd. on October 29,1998 and was assigned Wildlife International Ltd. identification number 4675. AMENDED 000875 W il d l if e In te r n a tio n a l l t d . - 10- PROJECT NO.: 454E-101 The test substance was stored under ambient conditions. Following is a description of the test substance used in this study: Name: Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Lot Number: 217 Physical Description: White powder Expiration Date: 2008 Purity: 90.49% Water Solubility: Slight Storage Conditions: Ambient Room temperature On the day of administration, the test substance was added to the appropriate test flasks via direct weight addition. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated March 9,2000 indicated a purity of 90.49%. Reference Substance A stock solution of the reference substance, 3,5-dichIorophenol was prepared by dissolving 500 mg in 10 mL of IN NaOH and then diluting to 30 mL with NANOpure water. While stirring, enough IN H2S 0 4was added to reach the point o f incipient precipitation. The solution of 3,5-dichlorophenol then was diluted to 1 L with NANOpure water. The pH of an aliquot of the reference substance stock solution was measured (pH = 7.18). Following is a description of reference substance used in this study. Name: Manufacturer: Lot Number: Physical Description: Handling Precautions: Expiration Date: Purity: Water Solubility: Storage Conditions: CAS Number: 3,5-dichlorophenol Aldrich Chemical Co., Milwaukee, WI 00410KZ White crystal Standard laboratory precautions March 13, 2000 97% 7.39 g/L Ambient 591-35-5 000S76 AMENDED W il d l if e In te r n a tio n a l l t d . - 11 - PROJECT NO.: 454E-101 Route of Exposure and Duration The reference substance was administered by volumetric addition. Volumetric addition is the most f appropriate route of administration of a substance in an aqueous solution. The test substance was administered by direct weight addition. Direct weight addition was selected as the most appropriate route of administration based on the observed solubility of the test substance in water. Stock solutions that were prepared at nominal concentrations of lg/L and 0.5 g/L in NANOpure water and sonicated for approximately 20 minutes contained test material that was not in solution. The duration of the exposure was 3 hours. Test System The biological test system was a consortium of microorganisms common to the activated sludge treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic, and facultative bacteria. The test system was chosen because it is representative of a treatment process that may receive the test substance. Test Conditions and Apparatus Control, reference, and treatment test mixtures were incubated at 20 2C and aerated for 3 hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension. The mixtures were prepared and aerated in 500 mL Erlenmeyer flasks and then transferred into 300 mL Biochemical Oxygen Demand (BOD) bottles to conduct the dissolved oxygen (DO) measurements. Test mixtures were identified by project number, test substance ID, test concentration and bottle number, when appropriate. Test Inoculum Activated sludge was collected from the Prospect Bay Wastewater Treatment Facility, Grasonville, Maryland on December 21, 1998. The Prospect Bay facility receives wastes from predominately domestic sources. The sludge was sieved using a 2 mm screen and then settled for approximately 30 minutes. The supernatant above the settled solids was drained and the total suspended solids (TSS) concentration of the settled sludge was determined. Based on the result, the concentration of the sludge was adjusted to 4000 mg/L ( 10%) by diluting with municipal water. Approximately 50 mL of synthetic sewage (Protocol, Appendix IV) was added to each liter o f activated sludge and the sludge was aerated overnight at 20 2C. Before use, the pH and total suspended solids concentration of the activated sludge were determined. 000877 W il d l if e In te r n a tio n a l l t d . -12- PROJECT NO.: 454E-101 Procedure Test mixtures were prepared at 15 minute intervals starting with the first control. The control contained 9.6 mL of synthetic sewage, 120 mL of inoculum, and enough municipal water to bring the total volume up to 300 mL. The mixture was promptly aerated at a rate sufficient to provide aerobic conditions and keep the solids in suspension. Subsequent mixtures contained 9.6 mL of synthetic sewage, 120 mL of inoculum, the appropriate amount of test substance or volume of reference substance stock solution, and enough municipal water to bring the total volume up to 300 mL. Finally, a second control was prepared. All mixtures were aerated for three hours. Sample Analysis After three hours of aeration, the contents of the first vessel were transferred to a BOD bottle and the respiration rate was measured ova-a period of up to 10 minutes. Dissolved oxygen readings were recorded every 10 seconds for 10 minutes or until the DO dropped below 1.0 mg/L, whichever came first using a YSI Model 50B Dissolved Oxygen Meter. The respiration rate in subsequent vessels was determined in an identical manner at 15 minute intervals so that the contact time of the test substance with the activated sludge was three hours. Calculations A respiration rate was calculated for each test mixture and expressed in mg 0 2/L/hour. The rate was calculated using DO values between approximately 6.5 mg 0 2/L and 2.5 mg 0 2/L, or over a 10 minute period if the DO did not reach approximately 2.5 mg 0 2/L. The respiration rate was calculated using the following equation: Respiration rate (mg O/L/hour) = Oxygen uptake over the calculation period/duration of calculation period The percent inhibition for each test substance concentration was calculated using the following equation and plotted against the logarithm of test substance concentration (Appendix III): 000S78 W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 -13- Percent In h ib itio n 2R 1 ----- ---- X 100 RC + RC where: R, RQ RC2 = = = oxygen consumption rate at a given concentration of the test substance oxygen consumption rate, Control 1 oxygen consumption rate, Control 2 Statistical Analyses When the dose response pattern allows for the calculation of ah EC50 value, the data are analyzed using the computer program of C.E. Stephan (3). The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average, or binomial probability with nonlinear interpolation (4, 5, 6). In this study, probit analysis was used to evaluate inhibition in the reference group. An EC50 value could not be calculated for the test substance. RESULTS AND DISCUSSION The pH of the reference stock solution was 7.18. Respiration rates and percent inhibitions are presented in Table 1. Dissolved oxygen readings recorded during the test are presented in tabular and graphical forms in Appendices I and II, respectively. The temperature range recorded the day of the test was 19-21 C and was within the limits specified in the test guideline. The measured total suspended solids (TSS) concentration and pH of the sludge on the day of testing was 4380 mg/L and 7.87, respectively. The respiration rates in the two controls were 39.6 and 41.1 mg 0 2/L/hr, or a difference of 3.7%. The validity of the test was further supported by the results from the 3,5-dichlorophenoI reference group, which resulted in an EC50 of 9.0 mg/L. The EC50 was within the 5 to 30 mg/L range considered acceptable for the test. The 95% confidence limits for the EC50 were 6.9 and 11.4 mg/L. 000879 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454E-101 -14- The inhibitory effects upon respiration at test substance concentrations analyzed (0.90,2.7, 9.0,27,90, 271 and 905 mg a.i./L) ranged from 3.6% to 38.8%. Respiration rates of the test substance concentrations evaluated ranged from 24.7 mg 0 2/L/hr (at 905 mg a.i./L) to 38.9 mg 0 2/L/hr (at 1 mg/L). CONCLUSION The maximum inhibitory effect upon respiration from Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) was observed at a test substance concentration of 905 mg a.i./L, but only reached 38.8%. The EC50 is therefore greater than 905 mg a.i./L. 000880 AMENDED W il d l if e In te r n a tio n a l l t d . - 15REFERENCES PROJECT NO.: 454E-101 1. O rganisation for Economic Cooperation and Development. 19B9. Activated Sludge Respiration Inhibition Test. OECD Guideline 209. 2. Council of the European Communities. Directive 67/548/EEC. Annex V. Guideline C. 11, Activated Sludge Respiration Inhibition Test. 3. Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 4. Finney, D.J. 1971. Statistical Methods in Biological Assay, second edition. Griffin Press, London. 5. Thompson, W.R. 1947. Bacteriological Reviews, Vol. II, No. 2: 115-145. 6. Stephan, C.E. 1977. "Methods for Calculating an l,5Q"Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 000881 W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 -16- Table 1 Respiration Rates and Percent Inhibitions Treatment Control 1 Control 2 3,5-dichlorophenol 3 mg/L 3,5-dichlorophenol 15 mg/L 3,5-dichlorophenol 50 mg/L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 0.90 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 2.7 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 9.0 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 27 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 90 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 271 mg a.i./L Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 905 mg a.i./L Respiration Rate mg Oj/L/hour 39.6 41.1 31.1 14.6 5.1 38.9 35.1 33.5 37.9 32.7 28.1 24.7 Percent Inhibition NA NA 22.9% 63.8% 87.4% 3.6% 13.0% 17.0% 6.1% 19.0% 30.4% 38.8% 000332 AMENDED -17- Control 1 PROJECTNO.: 454E-101 Time (seconds) Figure 1. Dissolved Oxygen Concentration Over Time, Control 1. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED ESSOOO -18- PROJECTNO.: 454E-101 Control 2 -j D) wE c o 0) > X O D 0 > (A M0H) Time (seconds) Figure 2. Dissolved Oxygen Concentration Over Time, Control 2. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 000SS4 -19- 3,5-Dichlorophenol 3mg/L PROJECTNO.: 454E-101 Time (seconds) Figure 3. Dissolved Oxygen Concentration Over Time, 3,5-Dichlorophenol, 3 mg/L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 000SS5 -20I 3,5-Dichlorophenol 15mg/L PROJECT NO.: 454E-101 Time (seconds) Figure 4. Dissolved Oxygen Concentration Over Time, 3,5-Dichlorophenol, 15 mg/L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 0008S6 -21- 3,5-Dichlorophenol 50mg/L PROJECTNO.: 454E-101 Tim e (seconds) Figure 5. Dissolved Oxygen Concentration Over Time, 3,5-Dichlorophenol, 50 mg/L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 000SS7 -22- PFOS 0.90 mg a.i./L PR0JECTN 0.: 454E-101 Time (seconds) Figure 6. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 0.90 mg a.i./L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 000888 -23- PFOS 2.7 mg a.i./L PROJECTNO.: 454E-101 Time (seconds) Figure 7. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 2.7 mg a.i./L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 000SS9 -24- PFOS 9.0 mg a.i./L PR0JECTN 0.: 454E-101 Time (seconds) Figure 8. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 9.0 mg a.i./L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 068000 -25- PFO S 27 mg a.i./L PROJECTNO.: 454E-101 T im e (seconds) Figure 9. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 27 mg a.i./L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED T 6S000 -26- PFOS 90mg a.i./L PR0JECTN 0.: 454E-101 Tim e (seconds) Figure 10. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 90 mg a.i./L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 000892 -27- PFO S 271 m g/ a.i.L PROJECTNO.: 454E-101 T im e (seco n ds) Figure 11. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 271 mg a.i./L. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration rate. AMENDED 0C0S93 -28- PFOS 905 mg a.i./L PR0JECTN 0.: 454E-101 Time (seconds) Figure 12. Dissolved Oxygen Concentration Over Time, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), 905 mg &X.IL. Arrows indicate measured dissolved oxygen concentrations used to calculate respiration. AMENDED 000394 W il d l if e Internatio nal ltd . PROJECT NO.: 454E-101 -29- APPENDIXI Measured Dissolved Oxygen (DO) Concentrations (mg 0 2/L) Treatment Dissolved Oxygen (mg/L) (min.) 3,5-Dichlorophenol 3,5-Dichlorophenol 3,5-Dichlorophenol Control 1 3 mg/L 15 mg/L 50 mg/L 00:10 00:20 00:30 00:40 00:50 01:00 01:10 01:20 01:30 01:40 01:50 02:00 02:10 02:20 02:30 02:40 02:50 03:00 03:10 03:20 03:30 03:40 03:50 04:00 04:10 04:20 04.30 04.40 04.50 05:00 05:10 05:20 05:30 05:40 05:50 06:00 06:10 06:20 06:30 06:40 06:50 07:00 07:10 0 1 -2 0 0 7 :3 0 07:40 07:50 08:00 08:10 08:20 08:30 08:40 08:50 09:00 09:10 09:20 09:30 09:40 09:50 10:00 7.3 6.5 6.0 5.8 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 5.0 4.8 4.8 4.5 4.3 4.2 4.1 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.5 1.5 1.3 1.3 1.2 1.1 0.9 _ -- -- _ _ _ -- _ -- - 8.0 7.8 7.6 7.5 7.4 7.3 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.7 8.3 8.2 8.2 8.4 8.1 8.4 8.0 8.5 8.0 8.5 7.9 8.5 7.9 8.5 7.9 8.5 7.8 8.5 7.8 8.5 7.8 8.5 7.7 8.4 7.7 8.4 7.7 8.4 7.6 8.4 7.6 8.4 7.5 8.4 7.5 8.4 7.5 8.4 7.4 8.3 7.4 8.3 7.3 8.3 7.3 8.3 7.3 8.3 7.2 8.3 7.2 8.2 7.1 - 8.2 7.1 8.2 7.1 8.2 7.0 8.2 7.0 8.1 6.9 8.1 6.9 8.1 6.9 8.1 6.8 8.1 6.8 8.1 6.7 8.0 6.7 8.0 6.7 8.0 6.6 8.0 6.6 8.0 6.6 8.0 6.5 8.0 6.5 7.9 6.4 7.9 6.4 7.9 6.4 7.9 6.3 7.9 6.3 7.9 6.3 7.8 6.2 7.8 6.2 7.8 6.1 7.8 6.1 7.8 6.1 7.7 6.0 7.7 6.0 7.7 6.0 7.7 5.9 7.7 5.9 7.7 Bold numbers indicate dissolved oxygen concentrations used to calculate respiration rates. PFOS 0.90 mg a.i./L 7.6 6.9 6.6 6.5 6.3 6.2 6.1 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 .0 0.9 -- -- - PFOS 2.7 mg a.i./L 7.3 7.0 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.1 2.9 2.8 2.7 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 AMEo S&895 W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 -30- APPENDIXI (Continued) Measured Dissolved Oxygen (DO) Concentrations (mg 0 2/L) lime (min.) PFOS 9.0 mg a.i./L PFOS 27 mg a.i./L Treatment Dissolved Oxygen (mg/L) PFOS 90 mg a.i./L PFOS 271 mg a.i./L 00:10 00:20 00:30 00:40 00:50 01:00 01:10 01:20 01:30 01:40 01:50 02:00 02:10 02:20 02:30 02:40 02:50 03:00 03:10 03:20 03:30 03:40 03:50 04:00 04:10 04:20 04.30 04.40 04.50 05:00 05:10 05:20 05:30 05:40 05:50 06:00 06:10 06:20 06:30 06:40 06:50 07:00 07:10 07:20 07:30 07:40 07:50 08:00 08:10 08:20 08:30 08:40 08:50 09:00 09:10 09:20 09:30 09:40 09:50 10:00 7.3 6.8 6.5 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.8 3.6 3.6 3.5 3.4 3.3 3.2 3.2 3.1 3.0 2.9 2.8 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 7.4 6.7 6.4 6.2 6.1 6.0 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 Z7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 -- -- _ -- 7.5 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.2 2.1 2.0 1.9 1.8 1.7 7.6 7.3 7.1 7.0 6.9 6.8 6.7 6.7 6.6 6.5 6.4 6.4 6.3 6.2 6.1 6.0 6.0 5.9 5.8 5.7 5.7 5.6 5.5 5.4 5.3 5.3 5.2 5.1 5.1 5.0 4.9 4.8 4.7 4.7 4.6 4.5 4.4 4.3 4.3 4.2 4.1 4.0 3.9 3.9 3.8 3.7 3.6 3.5 3.5 3.4 3.3 3.2 3.2 3.1 3.0 3.0 2.9 2.8 2.7 2.6 Bold numbers indicate dissolved oxygen concentrations used to calculate respiration rates. PFOS 905 mg a.i./L 7.3 6.9 6.8 6.7 6.6 6.5 6.5 6.4 6.3 6.3 6.2 6.1 6.0 6.0 5.9 5.9 5.8 5.7 5.7 5.6 5.5 5.5 5.4 5.3 5.2 5.2 5.1 5.0 5.0 4.9 4.8 4.8 4.7 4.6 4.6 4.5 4.4 4.4 4.3 4.2 4.1 4.1 4.0 3.9 3.9 3.8 3.7 3.7 3.6 3.5 3.5 3.4 3.3 3.2 3.2 3.1 3.0 3.0 2.9 2.8 Control 2 7.9 7.4 7.1 7.0 6.9 6.7 6.6 6.5 6.4 6.3 6.1 6.1 6.0 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.0 0.9 - AMENDED 000896 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454E-101 -31APPENDIX II Graphical Presentation of Percent Inhibition and Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Concentration 000897 W il d l if e Inter n a tio n a l l t d . -32APPENDIXII PROJECT NO.: 454E-101 Percent Inhibition versus PFOS Concentration (mg/L) Figure 1. Percent Inhibition versus Perfluorooctane Sulfonic Acid, Potassium Sait (PFOS) Concentration 000898 AMENDED W il d l if e Internatio nal l td . -33APPENDIX III Protocol, Amendment and Deviation PROJECT NO.: 454E-101 000899 W il d l if e In ter n a tio n a l l t d . -34APPENDIX III PROJECT NO.: 454E-101 PROTOCOL PFO S: A N ACTIVATED SLUDG E, RESPIRATION INHIBITION TEST O rganisation for Econom ic C ooperation and D evelopm ent OECD G uideline 209 and C ouncil o f European Communitie s D irective 67/548/E E C A nnex V , G uideline C. 11 3M Lab Request N o. U 2723 Subm itted to 3M Corporation Environmental Laboratory P .O .B o x 33331 S t Paul, M innesota 55133 W il d l if e I n t e r n a t io n a l ltd. 8598 Commerce D rive Easton, M aryland 21601 (4 1 0 )8 2 2 -8 6 0 0 December 1 ,1 9 9 8 PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 000900 W il d l if e In t e r n a t io n a l l t d . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l ltd. -35 APPENDIX in 2- - PFO S: A N ACTIVATED SLUDG E, RESPIRATION INHIBITION TEST SPONSOR: 3M Corporation Environm ental Laboratory P .O .B o x 3 3 3 3 1 S t P su l, M innesota SS133 SPO NSO R'S REPRESENTATIVE: M s. Susan A . B each TESTING FACILITY: W ildlife International Ltd. 8598 Com m erce D rive Easton, M aryland 21601 ST U D Y DIRECTOR- Ed Schaefer LABORATORY MANAGEMENT: Henry 0 . Krueger, P h D . M anager o f A quatic T oxicology & N on-Target Plants FOR LABORATORY USE ONLY Proposed D ates: Experim ental Start Date: ___ n In h f ProjectN o.: m e - t o t T est Concentrations: */*, T est Substance N .: b i s Experim ental #. Term ination D a te:. J V J llf J - Study Room : - V O J J tl---------- Int/D atc: <foS> -- R eceipt D ate: . M J 3 ' ? 1 PROTOCOL APPROVAL c"SLjr.JU.L 7ST U D Y DIRECTOR "-/"h r DATE d tp fit ATE / 7 PROTOCOLNO.: 454/120198/ASRTT/SUB454 3M LAB REQUEST NO. U 2723 OCOSOl W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 W i l d l if e In t e r n a t io n a l lt d . -36APPENDIX III -3 - IN TRO DUC TIO N T he purpose o f this test is to provide a screening m ethod to identify substances that m ay adversely affect aerobic microbial treatment plants and to indicate suitable non-inhibitoty test substance concentrations for u se in biodegradability tests. O BJEC TIV E The objective o f the study w ill b e to assess the effects o f th e test substance on activated sludge m icroorganism s b y m easuring the respiration rate. A n E C 50 w ill b e calculated, i f possible. EXPERIMENTALDESIGN T he test w ill contain a control, reference, and treatm ent group. T he control group is used to determ ine fo e background respiration rate o f foe sludge and w ill n ot b e exp osed to fo e test substance. T he reference groiq> w ill b e dosed w ife 3,5-dichloropbenol, a know n inhibitor o f respiration, at concentrations o f 3 ,1 5 , a n d 3 0 m g /L T he test substance w ill b e tested at 1 ,3 ,1 0 ,3 0 ,1 0 0 ,3 0 0 , and 1000 m g/L. MATERIALSANDMhlllQDS T est m ethods are based o n the procedures sp ecified in d ie OECD Guideline for T esting o f C h em icals, M ethod 209 ( I ) and C ouncil o f th e European C om m unities, Guideline C .l l, A ctivated Sludge, R espiration Inhibition T est (2 ). T est Substance Information on the characterisation o f test, control or reference substances is required b y G ood Labontory Practice Standards (G LP), 4 0 C FR Part 160.31. T he Sponsor is responsible for providing W ildlife International Ltd. written verification that the test substance h as been characterized according to G LPs prior to using in the te st T he attached form ID E N T IF IC A T IO N O F T E S T SU B ST A N C E B Y SP O N SO R (A ppendixII) is to b e used to provide inform ation necessary for GLP com pliance. I f writtenverification ofG L P test substance d ia ra cta iza tk n is n ot provided to W ild life International L td., it w ill b e noted in the com pliance statem ent o f the final report. PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 000902 W ild life International ltd . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l l t d . -37APPENDIX III -4 - The Spoosor is responsible for all inform ation related to the test substance and agrees to accept any unused test substance nd/or test substance containers rem aining at th e end o f the study. Stock Solutio n Preparation A stock solu tion o f the test substance w ill b e prepared at a nom inal concentration o f 1000 m g a ctiv e/L in high quality water (c.g.N aaopu rc). In addition, a stock solution o f 3,5-dichlorophcnoI w ill prepared b y d isso lv in g 50 0 m g in 10 m L o f IN N aO H and then diluting to 3 0 m L w ith high quality w ater. W hile stirring, enough IN H^SO (approx imately 8 m L) w ill b e added to reach the poin t o f incipient precipitation. T he solution o f 3,5-dichlorophenol then w ill b e diluted to 1 L w ith high quality w ater. T est C onditions and Apparatus C ontrol, reference, and treatm ent test m ixtures w ill b e incubated at 2 0 2C and aerated for 3 hours at a rate su fficien t to m aintain solid s in suspension. T he m ixtures w ill b e prepared and aerated in 5 0 0 m L plastic Erienm eyer flask s and then transferred into a 30 0 m L B iochem ical O xygen Dem and (B O D ) b ottle to the conduct d issolved oxygen (D O ) m easurem ents. lest Inocuhm Activated sludge from fe e ProspectB y W astewater TreatmentPlant w ill b e used as the inoculum fo r th e t e s t T he Prospect B ay F acility receives w astes from predom inantly dom estic sources. The slu d g e w ill b e siev ed nsin g a 2 mm screen and then settlod for approx im ately 3 0 m inutes. The supernatant above the settled solid s w ill b e drained and the total suspended so lid s (T S S ) concentration o f th e settled sludge w ill b e determ ined. B ased o n fe e result, the concentratio n o f d ie sludge w ill b e adjusted t o 4 0 0 0 m g /L ( 10% ) b y dihitm g w ith m unicipal water. I f the sludge cannot b e used on the day o f collection or i f th e sam e batch is required to b e used o n subsequent days (m axim um four d ays), 5 0 m L o f synthetic sew age (A ppendix II) w ill b e added to each liter o f activated sludge at the end o f each w orking day. T he sludge w ill b e aerated overnight at 20 2 C . B efore u se, the pH and total suspended so lid s concentration o f the activated sludge w ill be determined and, if necessary, adjusted to pH 6 .0 - 8 .0 and a so lid s concentration o f 4 0 0 0 m g/L ( 10%). PROTOCOL NO.: 454/120198/ASRTOSUB454 3M LAB REQUEST NO. U2723 000903 W il d l if e Internatio nal l t d . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l l t d . -38APPENDIX III -5 - Proccdicc T est mixtures w ill be prepared at 15 m inute intervals starting w ith the first control. T he control w ill contain 9.6 m L o f synthetic sew age, 120 mL o f inoculum and enough m unicipal w ater to bring the total volum e up to 300 m L The m ixture w ill b e prom ptly aerated at a rate su fficien t to keep the solid s in suspension. Subsequent m ixtures w ill contain 9 .6 m L o f synthetic sew age, 120 m L o f inoculum , the appropriate volum e o f test or reference substance stock solution , and enough m unicipal w ater to bring the total volum e up to 300 mL. F inally, a second control w ill b e prepared. A ll m ixtures w ill be aerated for three hours. Sam ple A nalysis A fter three hours o f aeration, the contents o f the first v essel w ill b e transferred to a B O D bottle and the respiration rate w ill be m easured over a period o f up to 10 m inutes. D issolved oxygen readings w ill b e recorded every 10 seconds for 10 m itm tes or until the D O drops below 1.0 m g/L , w hich ever com es first T he respiration rate in subsequent v essels w ill b e determ ined in an identical m anner at 13 m inute intervals so that the contact tim e o fth e test substance w ith the activated sludge is three hours. Calculations A respiration rate w ill b e calculated for each te st m ixture and expressed in m g OJUbxx. The rate w ill b e calculated using D O values betw een approxim ately 6 .5 m g OJLwaAUSmgOJL, or over a 10 m hm te period i f the D O d oes n ot reach approxim ately 2 3 m g OJL. T he percent inhibition fix each test substance concentration w ill b e calculated u sin g the follow ing equation and plotted against concentration on lo g paper: P ercen t In h ib itio n - 1 - X 100 KCX + RC2 PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 000904 W ild life International ltd . PROJECT NO.: 454E-101 -39- APPENDIX III W i l d l i f e In t e r n a t i o n a l l t o ._________________________ 6- - wherc R , = ox y g ea consum ption rate at a given concentration o f the test substance RCj " ox y g ea consum ption rate. C ontrol 1 R C j - ox y g ea consum ption rate, Control 2 A n EC50 value w ill b e derived, i f possible, b ased on the percent inhibition versus test substance concentration- C onfidence lim its (95% ) for the E C 50 w ill b e determ ined usin g standard statistical procedures (3). Quality Control T he test is considered valid only i f the follow ing criteria arc m et: the tw o control respiration rates are w ithin 15% o f each other; tbe EC50 (3 hours) o f 3,5-dichlonopheaol is in the accepted nm ge o f 5 to 3 0 m g/L. RECORDS T O B E M A IN TA IN ED R ecords to b e m aintained vriH include, bu t n ot lim ited to , th e follow ing: 1. A copy o f th e signed protocol. 2. Mcwfificarinn and riiarartcriartinn ofthe test mhstance as provided by Sponsor. 3 . . T est in itiatioa and tenninatioa dates. 4 . Experim ental initiation and term ination dates. 5 . Stock aohition concentration calculations and solution preparation. 6 . A ctivated slud ge source and pretrcatment details. 7 . T est tem perature and duration. 8. R eference substance results. 9 . A ll d issolved oxygen m easurem ents. 10. Tem perature range recorded during test period. 11. Inhibition curve and m ethod for calculation o f E C 50. 12. I f calculated, E C 50 and 95% confidence lim its. 13. A copy o f the final report PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 000905 W il d l if e Internatio nal l t d . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l l t d . -40APPENDIX III -7 - ki NAT. REPORT A final report o fth e results o f the study w ill be prepared by W ildlife International L td T he report is to include, but is not lim ited to, the follow in g w hen applicable: 1. N am e and address o f facility perform ing the study. 2 . D ates on w hich th e study w as initiated and com pleted. 3 . A statem ent o f com pliance signed b y th e Study Director addressing any exceptions to G ood Laboratory Practice Standards. 4 . Objectives and procedures stated in the approved protocol, including a ry changes in the original p r o to c o l 5. and characterization ofthe test substance as provided by Sponsor mduding name, C A S number, percent active, and other characteristics, i f provided b y th e Sponsor. 6 . A description o f th e transform ations and calculations perform ed on th e data. 7 . A description o f th e m ethods used and reference to any standard m ethod em ployed. 8 . A description o f th e test system . 9 . A description o f the preparation o f the test solutions, the testing coPccatratioa(s), the route o f administration, and d ie duration o f the te st 10. A description rtfa ll circum stances that m ay o f affected the quality or integrity o f th e data. 11. The nam e o f th e study director, the nam es o f other scien tists o r professionals, and th e nam es o f a ll supervisory personnel, involved in the study. 12. The signed and dated reports o f each o f the individual scien tists or other professionals involved in the study, i f applicable. 13. T he location w h en the raw data and fin al report are to be stored. 14. A staten*nt prepared b y th e Q uality A ssurance U n it listin g th e dates that the study inspections and aud its w ere m ade and d ie dates o f any findings w ere reported to the Study D irector and M anagem ent 13. I f it is necessary to make corrections or additions to a final report after it has been accepted, such changes w ill b e m ade in the form o f an amendmentissued by d ie Study D irector. T he amendment w in dearly identify the part o f d ie final report that is bring amended and the reasons for the amendment., and w ill b e signed by the Study Director. PROTOCOL NO.: 454/120198/ASRTT/SUB454 3M LAB REQUEST NO. U2723 OGOSOb W il d l if e Intern atio na l l t d . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l l t d . -41 APPENDIX III 8- - CH ANGING O F PRO TOCOL Planned changes to the protocol w ill be in the form o f w ritten amendments signed b y the Study Director and the Sponsors R epresentative. Am endm ents w ill b e considered as part o f the protocol and w in b e attached to the final protocol Any other changes w ill b e in the form o f w ritten deviations signed b y the Study Director and filed w ith tbe raw data. A ll changes to the protocol w ill be indicated in the final report G OO D LABO RATO RY PRAC TICES T his study w ill b e conducted in accordance w ith G ood Laboratory Practice Standards for EPA (40 CFRPart 160); OECD Principles o f G ood Laboratory Practices (O C D E/G D (9 2 ) 3 2 , Environm ent M onograph N o . 43); and Japan M AFF (3 9 N ohSan, N otification N o . 3 8 5 0 , A gricultural Production B ureau). Each study conducted by W ildlife International Ltd. is routinely exam ined b y th e W ildlife International Ltd. Q uality A ssurance U n it for com pliance w ith G ood Laboratory P ractices, Standard O perating Procedures and the sp ecified protocol. A statem ent o f com pliance w ith G ood Laboratory Practices w ill b e prepared for a ll portions o f die study conducted b y W ild life International Ltd. Raw data for all work performed at W ildlife International Ltd, and a copy o f the final report w ill b e filed b y project num ber in archives located o n the W ild life International Ltd, site, or at an alternative location to be sp ecified in the final report PROTOCOL NO.: 454/120198/ASR IT/SU B454 3M LAB REQUEST NO. U2723 000907 W il d l if e In t e r n a tio n a l l t d . W i l d l i f e In t e r n a t i o n a l l t d . -42APPENDIX III -9 - REFERENCES PROJECT NO.: 454E-101 1 O rgan isation fo r E conom ic C oop eration and D ev elo p m en t 1989. Activated Sludge Respiration Inhibition Test. OECD G uideline 209. 2 C ou ncil o f th e E uropean C om m unities. D irective 67/548/E E C . A nnex V . G u id d in e C .il, ActivatedSludgeRespiration Inhibition Test. 3 S tep h a n , C E . 1977. "M ethods for Calculating an LC50," Aquatic Toxicology andHazard Evaluations. American S o d d y fix T esting and M aterials. Publication Num ber STP 6 3 4 , pp 6 5 - 84. PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U 2723 000908 W il d l if e In t e r n a t io n a l l t d . PROJECT NO.: 454E-101 W i l d l if e In t e r n a t io n a l lt d . -43APPENDIX III - 10- APPENDDCI IDENTIFICATION OF TEST SU BSTA N C E B Y SPO N SO R T o b e Com pleted b y Sponsor L T est Substance Identity (nam e to b e used in the report): Birooctane Sulfonic A d d . Potassium Salt Sam ple C ode or B atch N um ber Lot 217_____________ Purity (% A ctiv e Ingredient): 98.9% Expiration D ate: 2008 IL T est Substance Characterization H ave the identity, stren gth purity and com position or other characteristics w hich ropropnately define fire test substance been determhKd prior to th e s t u t or th is stud^m a cco o sn ce w ith GLP Standards? Y e s____ N o X EL T est Substance Storage Conditions P lease indicate tbe recom m ended storage conditions at W ildlife International Ltd. Ambientroomtempgatms,--------------------------------------------------------------------- c o n d itio n s Yes No X IV . T oxicity Inform ation: M am m alian: RatLDSO 2 5 1 m g /kg M o u se L D 5 0 N /A A quatic: Invertebrate T oxicity (EC /LC 50) F ish T oxicity (L C 50) Duphniamayna: 27me/L___________ RamhOfftTOUt: llm g /L ----- Daphnia magna: SOme/L_________ FgthCHd mimOW:J m / L - O tiier T o x icity Inform ation (m duding findings o f chronic and subchronic tests): SecMSDS____ __________________________________________ PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 000909 W il d l if e Internatio nal l t d . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l l t d . -44APPENDIX III - 11- APPENDIX il SYNTHETIC SEW AGE T he syn th etic sew age provides the necessary nutrients required for bacterial m etabolism . It is prepared b y dissolving d ie follow in g amounts o f substances in 1 liter o f m unicipal water. 16.0 g peptone 11.0 g m eat extract 3.0 g urea 0.7 g N aC l 0.4 g C a O ^ O 0 .2 g MgSO< 7H zO 2 .8 g K*HPO< Reagent grade chem icals or better w ill be used when available. T he constituents o f the synthetic sew age are n ot know n to con tain any contam inants O ut are reasonable expected to b e present and are know n to b e capable o f interfering w ith d ie study. PROTOCOL NO.: 454/120198/ASR IT/SU B454 3M LAB REQUEST NO. U2723 000910 W il d l if e Intern atio na l l t d . W i l d l i f e In t e r n a t i o n a l lto. -45APPENDIX III PROJECT NO.: 454E-101 PROJECT NO.: 454^ 101 Page t o f 2 AMENDMENT TO STUDY PROTOCOL ST U D Y T IT L E : PFOS: A N ACTIVATED SLU DG E, RESPIRATION INHIBITION TEST PR O TO C O L N O .: 454/I20198/A SR IT /SU B 4S4 A M EN D M EN T N O .: 1 SP O N SO R : 3M Corporation P R O JE C T N O .: 454E -101 3M L A B R E Q U E ST N O .: U 2723 A M EN D M EN T: Page 3; T est Substance Add: T he te st substance w ill b e adm inistered b y direct w eight addition. D irect w eight addition is the m ost appropriate route o f adm inistration o f test substances that are relatively insoluble in water. R E A SO N : R oute o f adm inistration w as changed from volum etric addition o f an aqueous stock so lu tio n to direct w eight addition because o f the low w ater solub ility o f d ie test substance. EFFEC TIV E D A TE: 21 Decem ber 1998 A M EN D M EN T: Page 4; Stock S olution Prcpartioa D elete: A stock solution o f the test substance w ill b e prepared at a nom inal concentration o f 1000 m g active/L in high quality w ater (e.g . N anopure). R E A SO N : R ou te o f adm inistration w as changed from volum etric addition o f an aqueous stock so lu tio n to direct w eight addition because o f th e low w ater solub ility o f d ie test su b sta n ce. EFFEC TIVE D A TE: 21 Decem ber 1998 TufV ' 17-ZKw 000911 W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 W i l d l i f e In t e r n a t i o n a l l t d . -46APPENDIX III PROJECT NO.: 454E-101 Page 2 o f 2 A M EN D M EN T: Page S; Procedure Change: T est m ixtures w ill be prepared at 15 m inute intervals starting w ith the first control. The control w ill contain 9 .$ m L o f synthetic sew age, 120 mL o f inoculum and enough m unicipal water to bring the total volum e up to 30 0 m L. The m ixture w ill be prom ptly aerated at a rate su fficien t to keep the solid s in suspension. Subsequent m ixtures w ill contain 9 .6 m L o f synthetic sew age, 120 m L o f inoculum , the appropriate volum e o f test or reference substance stock solution , and enough m unicipal water to bring the total volum e up to 30 0 m L. F in ally, a second control w ill b e prepared. A ll m ixtures w ill be aerated for three hours. To: T est m ixtures w ill be prepared at 15 m inute intervals starting w ith the first control. The control w ill contain 9 .6 m L o f synthetic sew age, 120 m L o f inoculum and enough m unicipal water to bring the total volum e up to 30 0 m L The m ixture w ill be prom ptly aerated at a rate su fficien t to keep th e solid s in suspension. Subsequent m ixtures w ill contain 9 .6 m L o f synthetic sew age, 120 m L o f inoculum , th e appropriate amount o f te st substance or volum e o f reference substance stock solution, and enough m unicipal w ater to bring d ie total volum e up to 3 00 m L. F inally, a second control w ill be prepared. A ll m ixtures w ill b e aerated for three hours. R EA SO N : R oute o f adm inistration w as changed from volum etric addition o f an aqueous stock solu tion to direct w eight addition because o f the low water solubility o f the test substance. EFFE C T IV E DA TE: 21 December 1998 f i . r . J j . L . _____________ ST U D Y DIRECTOR' n /a h * DATE A-- ( X if L ju c h SPO NSO R'S REPRESENTATIVE 'ffle eovCvoed - 000912 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454E-101 -47- W i l d l i f e In t e r n a t i o n a l l t d . APPENDIX III DEVIATIO N TO STU DY PR O TO C O L PROJECT NO.: 454E-I01 Page 1 o f 1 ST U D Y T IT L E : PFOS: A N ACTIVATED SLUDG E, RESPIRATIO N INHIBITION TEST PR O T O C O L N O .: 454/120198/A SR IT /SU B 454 D E V IA T IO N N O .: 1 SPO N SO R : 3M Corporation P R O JE C T N O .: 454E -101 D A T E O F D EFA C TO D EV IA TIO N : December 1 2 ,1 9 9 8 D EV IA TIO N : Procedure, Page -5-: The 3 m g/L 3,5-dichlorophenol m ixture w as aerated for three hours and one m inute. REASON: Inadvertent error by study personnel. IM PA C T: T his deviation did n ot im pact the integrity o f the study. STU D Y DIRECTOR 91 DATE L DAT 7 000913 W il d l if e In ter n a tio n a l l t d . PROJECT NO.: 454E-101 -48APPENDIXIV Report Amendment 1. Original Report: Pages 1-5 and 7 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 IV. Reason: To reflect the issuing of an amended report. 2. Original Report: Page 2 Amendment: Compliance statement was revised. Reason: To clarify how the test substance was characterized. 3. Original Report Page 10 Amendment: Information provided by the Sponsor reflecting the reanalysis of 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 o f the test substance as determined by the Sponsor in a reanalysis of the test substance (FC-95, Lot 217). Test concentrations originafly were reported in mg/L. The certificate of analysis Dated March 9, 2000 indicated a purity of 90.49%. Therefore, all test substance concentrations were recalculated and reported as mg a.i./L based on the 90.49% purity. Reason: To report the results of the test based on the substance purity of 90.49% at the request o f the Sponsor. 000914 AMENDED W il d l if e In te r n a tio n a l l t d . -49APPENDIXIV - Continued Report Amendment AMENDMENT SIGNATURES: PROJECT NO.: 454E-101 Edward C. Schaefer Manager, Biodegradation DATE Henry Q. Krueger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants REVIEWED BY: Marshall T. Hynson Quality Assurance Program Sufrrvisor DATE 000915 AMENDED W il d l if e In te r n a tio n a l l t d . PROJECT NO.: 454E-101 Page 1 o f 2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST PROTOCOL NO.: 454/120198/ASRIT/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PR O JEC T NO.: 454E-101 3M LAB REQUEST NO.: U2723 AMENDMENT: Page 3; Test Substance Add: The test substance will be administered by direct weight addition. Direct weight addition is the most appropriate route of administration o f test substances that are relatively insoluble in water. REASON: Route o f administration was changed from volumetric addition of an aqueous stock solution to direct weight addition because of the low water solubility of the test substance. EFFECTIVE DATE: 21 December 1998 AMENDMENT: Page 4; Stock Solution Prepartion Delete: A stock solution of the test substance will be prepared at a nominal concentration of 1000 mg active/L in high quality water (e.g. Nanopure). REASON: Route o f administration was changed from volumetric addition of an aqueous stock solution to direct weight addition because of the low water solubility of the test substance. EFFECTIV E DATE: 21 December 1998 000916 W i l d l i f e In t e r n a t i o n a l ltd. PROJECT NO.: 454E-101 Page 2 of 2 AMENDMENT : Page 5; Procedure Change: Test mixtures will be prepared at 15 minute intervals starting with the first control. The control will contain 9.6 mL of synthetic sewage, 120 mL of inoculum and enough municipal water to bring the total volume up to 300 mL. The mixture will be promptly aerated at a rate sufficient to keep the solids in suspension. Subsequent mixtures will contain 9.6 mL of synthetic sewage, 120 mL o f inoculum, the appropriate volume of test or reference substance stock solution, and enough municipal water to bring the total volume up to 300 mL. Finally, a second control will be prepared. All mixtures will be aerated for three hours. To: Test mixtures will be prepared at 15 minute intervals starting with the first control. The control will contain 9.6 mL of synthetic sewage, 120 mL of inoculum and enough municipal water to bring the total volume up to 300 mL. The mixture will be promptly aerated at a rate sufficient to keep the solids in suspension. Subsequent mixtures will contain 9.6 mL of synthetic sewage, 120 mL of inoculum, the appropriate amount of test substance or volume of reference substance stock solution, and enough municipal water to bring the total volume up to 300 mL. Finally, a second control will be prepared. All mixtures will be aerated for three hours. REASON: Route of administration was changed from volumetric addition of an aqueous stock solution to direct weight addition because of the low water solubility of the test substance. EFFECTIVE DATE: 21 December 1998 STUDY DIRECTOR DATE SPONSOR'S REPRESENTATIVE 000917 i W i l d l i f e In t e r n a t i o n a l ltd . DEVIATION TO STUDY PROTOCOL PROJECT NO.: 454E-101 Page 1 of 1 STUDY TITLE: PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST fl PROTOCOL NO.: 454/120198/ASRIT/SUB454 A SPONSOR: 3M Corporation DEVIATION NO.: 1 PROJECT NO.: 454E-101 DATE OF DEFACTO DEVIATION: December 12, 1998 A DEVIATION: Procedure, Page-5-: d The 3 mg/L 3,5-dichlorophenol mixture was aerated for three hours and one minute. d REASON: Inadvertent error by study personnel. IMPACT: This deviation did not impact the integrity of the study. d d c. d STUDY DIRECTO J '3 -9 1 DATE d LABORATORY MANAGEMENT DAT 000918 PROTOCOL PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST Organisation for Economic Cooperation and Development OECD Guideline 209 and Council o f European Communities Directive 67/548/EEC Annex V, Guideline C. 11 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 December 1,1998 000919 PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 M I W il d l if e In ter n a tio n a l l t d . 2- I PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST I SPONSOR: 3M Corporation 1 Environmental Laboratory P.O.Box 33331 I St. Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach I TESTING FACILITY: I STUDY DIRECTOR: Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 Ed Schaefer d LABORATORY MANAGEMENT: Henry 0 . Krueger, Ph.D. Manager of Aquatic Toxicology & Non-Target Plants I FOR LABORATORY USE ONLY Proposed Dates: 1 Experimental Start Date: __ /I / n h * Experimental >. __________ Termination D ate:. fl' f n d Project No.: 4 * 3 4 k - l o t -- Study Room: ^______ Test Concentrations: lA ,0.7> io o ,v> o ,t fot/Date; . ( f o S >*//* ^ __ d Test Substance No.: *75" Receipt Date: , t O J 7 ' 9 i d PROTOCOL APPROVAL i /<i9 r DATE PROTOCOL NO.: 454/120198/ASRIT/SUB454 DATE 7 a J?/^ DATTTE? 000920 3M LAB REQUEST NO. U2723 i W i l d l i f e In t e r n a t i o n a l ltd . -3 - I INTRODUCTION I The purpose o f this test is to provide a screening method to identify substances that m ay adversely affect aerobic microbial treatment plants and to indicate suitable non-inhibitory test substance I concentrations for use in biodegradability tests. I OBJECTIVE The objective of the study will be to assess the effects o f the test substance on activated sludge I microorganisms by measuring the respiration rate. An EC50 will be calculated, if possible. I EXPERIMENTAL DESIGN The test will contain a control, reference, and treatment group. The control group is used to I determine the background respiration rate of the sludge and will not be exposed to the test substance. The reference group will be dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at I concentrations of 3, 15, and 50 mg/L. The test substance will be tested at 1,3,10,30,100, 300, and 1000 mg/L. I MATERIALS AND METHODS I Test methods are based on the procedures specified in the OECD Guideline for Testing o f Chemicals, Method 209 (1) and Council o f the European Communities, Guideline C .ll, Activated I Sludge, Respiration Inhibition Test (2). I XestSubstan Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP), 40 CFR Part 160.31. The Sponsor is responsible for providing fl Wildlife International Ltd. written verification that the test substance has been characterized according to GLPs prior to using in the test The attached form IDENTIFICATION OF TEST SUBSTANCE f t BY SPONSOR (Appendix II) is to be used to provide information necessary for GLP compliance. If written verification of GLP test substance characterization is not provided to Wildlife International Ltd., f t it will be noted in the compliance statement of the final report. 000921 ft PROTOCOL NO.: 454/120198/ASRIT/SUB454 ft 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l l td . -4 - 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. Stock Solution Preparation A stock solution of the test substance will be prepared at a nominal concentration o f 1000 mg active /L in high quality water (e.g. Nanopure). In addition, a stock solution of 3,5-dichlorophenol will prepared by dissolving 500 mg in 10 mL of IN NaOH and then diluting to 3Q. mL with high quality water. While stirring, enough IN H2S 0 4 (approximately 8 mL) will be added to reach the point of incipient precipitation. The solution of 3,5-dichlorophenol then will be diluted to 1 L with high quality water. Test Conditions and Apparatus Control, reference, and treatment test mixtures will be incubated at 20 2C and aerated for 3 hours at a rate sufficient to maintain solids in suspension. The mixtures will be prepared and aerated in 500 mL plastic Erlenmeyer flasks and then transferred into-a 300 mL Biochemical Oxygen Demand (BOD) bottle to the conduct dissolved oxygen (DO) measurements. Test Inoculum Activated sludge from the Prospect Bay Wastewater Treatment Plant will be used as the inoculum for the test. The Prospect Bay Facility receives wastes from predominantly domestic sources. The sludge will be sieved using a 2 mm screen and then settled for approximately 30 minutes. The supernatant above the settled solids will be drained and the total suspended solids (TSS) concentration o f the settled sludge will be determined. Based on the result, the concentration of the sludge will be adjusted to 4000 mg/L ( 10%) by diluting with municipal water. If the sludge cannot be used on the day of collection or if the same batch is required to be used on subsequent days (maximum four days), 50 mL of synthetic sewage (Appendix II) will be added to each liter of activated sludge at the end of each working day. The sludge will be aerated overnight at 20 2C. Before use, the pH and total suspended solids concentration of the activated sludge will be determined and, if necessary, adjusted to pH 6.0 - 8.0 and a solids concentration of 4000 mg/L ( ^ ^ ' q q 0 2 2 PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd . -5 - Procedure Test mixtures will be prepared at 15 minute intervals starting with the first control. The control will contain 9.6 mL of synthetic sewage, 120 mL of inoculum and enough municipal water to bring the total volume up to 300 mL. The mixture will be promptly aerated at a rate sufficient to keep the solids in suspension. Subsequent mixtures will contain 9.6 mL of synthetic sewage, 120 mL o f inoculum, the appropriate volume o f test or reference substance stock solution, and enough municipal water to bring the total volume up to 300 mL. Finally, a second control will be prepared,. All mixtures will be aerated for three hours. Sample Analysis After three hours o f aeration, the contents o f the first vessel will be transferred to a BOD bottle and the respiration rate will be measured over a period o f up to 10 minutes. Dissolved oxygen readings will be recorded every 10 seconds for 10 minutes or until the DO drops below 1.0 mg/L, which ever comes first The respiration rate in subsequent vessels will be determined in an identical manner at 15 minute intervals so that the contact time of the test substance with the activated sludge is three hours. Calculations A respiration rate will be calculated for each test mixture and expressed in mg 02/L/hour. The rate will be calculated using DO values between approximately 6.5 mg OJL and 2.5 mg OJL, or over a 10 minute period if the DO does not reach approximately 2.5 mg OJL. The percent inhibition for each test substance concentration will be calculated using the following equation and plotted against concentration on log paper: P ercent In h ib itio n 2R 1 - X 100 RC, + RC,, PROTOCOL NO.: 454/120198/ASRTT/SUB454 000923 3M LAB REQUEST NO. U2723 W il d l if e Inter n a tio n a l l t d . 6- - where R, = RQ = RC2 = oxygen consumption rate at a given concentration of the test substance oxygen consumption rate, Control 1 oxygen consumption rate, Control 2 An EC50 value will be derived, if possible, based on the percent inhibition versus test substance concentration. Confidence limits (95%) for the EC50 will be determined using standard statistical procedures (3). d Quality Control d The test is considered valid only if the following criteria are met: the two control respiration rates are within 15% of each other; d the EC50 (3 hours) of 3,5-dichlorophenol is in the accepted range of 5 to 30 mg/L. d RECORDS TO BE MAINTAINED Records to be maintained will include, but not limited to, the following: 1. A copy o f the signed protocol. 2. Identification and characterization of the test substance as provided by Sponsor. d 3. Test initiation and termination dates. 4. Experimental initiation and termination dates. d 5. Stock solution concentration calculations and solution preparation. 6. Activated sludge source and pretreatment details. d 7. Test temperature and duration. 8. Reference substance results. d 9. All dissolved oxygen measurements. 10. Temperature range recorded during test period. 11. Inhibition curve and method for calculation o f EC50. t f 12. If calculated, EC50 and 95% confidence limits. d 13. A copy of the final report 000924 d PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l t d . -7 - FINAL REPORT A final report of the results of the study will be prepared by Wildlife International Ltd. The report is to include, but is not limited to, the following when applicable: 1. Name and address of facility performing the study. 2. Dates on which the study was initiated and completed. 3. A statement o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures stated in the approved protocol, including any changes in the original protocol. 5. Identification and characterization of the test substance as provided by Sponsor including name, CAS number, percent active, and other characteristics, if provided by the Sponsor. 6. A description of the transformations and calculations performed on the data. 7. A description of the methods used and reference to any standard method employed. 8. A description o f the test system. 9. A description of the preparation of the test solutions, the testing concentration(s), the route of administration, and the duration of the test. 10. A description of all circumstances that may o f affected the quality or integrity of the data. 11. The name of the study director, the names of other scientists or professionals, and the names of all supervisory personnel, involved in the study. 12. The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 13. The location where the raw data and final report are to be stored. 14. A statement prepared by the Quality Assurance Unit listing the dates that the study inspections and audits were made and the dates of any findings were reported to the Study Director and Management. 15. If it is necessary to make corrections or additions to a final report after it has been accepted, such changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part o f the final report that is being amended and the reasons for the amendment, and will be signed by the Study Director. 000925 PROTOCOL NO.: 454/120198/ASRIT/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- - 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); OECD Principles of Good Laboratory Practices (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. 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.: 454/120198/ASRIT/SUB454 000926 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 - REFERENCES 1 O rganisation for Economic Cooperation and Development. 1989. Activated Sludge Respiration Inhibition Test. OECD Guideline 209. 2 Council of the European Communities. Directive 67/548/EEC. Annex V. Guideline C. 11, Activated Sludge Respiration Inhibition Test. 3 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number SIT 634, pp 6584. PROTOCOL NO.: 454/120198/ASRIT/SUB454 000927 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l t d . -10- APPENDIXI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor L Test Substance Identity (name to be used in the report): P.erfl" orooctane Sulfonic Acid. Potassium Salt Sample Code or Batch Number: Lot 217____________________________________ _______ Purity (% Active Ingredient): 98.9%_____________ Expiration Date: 2008__________ II. Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately define the test substance been determined prior to the start o f this study in accordance with GLP Standards? Yes___No X III. 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. Toxicity Information: Mammalian: RatLD50 251 mg/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) P aphnia magna: 27me/L__________ Rainbow trout: l l m e / L Daphnia magna: 50me/L__________ Fathead minnow: 38mg/L Other Toxicity Information (including findings of chronic and subchronic tests): See MSDS-__________________________________________________________________ PROTOCOL NO. : 454/120198/ASRIT/SUB454 000928 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l l t d . 11 - a a i a a a a a APPENDIX II. SYNTHETIC SEWAGE The synthetic sewage provides the necessary nutrients required for bacterial metabolism. It is prepared by dissolving the following amounts of substances in 1 liter of municipal water: 16.0 g peptone 11.0 g meat extract 3.0 g urea 0.7 g NaCl 0.4 g CaCl22H20 0.2 g MgSO, 7H20 2.8 g KjHPO,, Reagent grade chemicals or better will be used whan available. The constituents o f the synthetic sewage are not known to contain any contaminants that are reasonable expected to be present and are known to be capable of interfering with the study. PROTOCOL NO.: 454/120198/ASRIT/SUB454 000929 3M LAB REQUEST NO. U2723
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