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AR226-2687 9 AR226-2687 DuPont EMSE Report No. 18-02 FINAL REPORT Study Title ACTIVATED SLUDGE RESPIRATION INHIBITION TEST OF TELOMER B 8-2 ALCOHOL|QECD 209) ;'., ./'...; Test Guideline OECD (1984) Guideline for Testing of Chemicals, Section 2, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted April 4,1984 Author William R. Berti, Ph.D. Study Completion Date July 31,2002 .. ..,, Test Facility E. I. du Pont de Nemours and Company Environmental and Microbiological Sciences & Corporate Center for Engineering Research Central Research & Development Glasgow, Building 300, P.O. Box 6101 Newark, DE 19714-6101 Engineering Submitter E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. EMSE Study /Project Number T0105/4842 >/'(" ssrnfH"' Report Number EMSER 18-02 T0105/4842 Page 1 of 27 DuPont EMSE Report No. 18-02 PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS T0105/4842 Page 2 of 27 DuPont EMSE Report No. 18-02 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT The study described in this report was conducted in compliance with the following GLP principles with the following exception as noted below: United States Environmental Protection Agency, (FIFRA), Title 40 Code of Federal Regulations Part 160 (effective October 16, 1989) which are consistent with: The OECD Principles on Good Laboratory Practice (ENV/MC/CHEM(98)17) (Paris, 1998) Japan Ministry of Agriculture, Forestry and Fisheries, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, (Tokyo, 1984). The test substance was characterized by the sponsor prior to the initiation of this study. A certificate of analysis was provided. Although the characterization was not performed under Good Laboratory Practice Standards, the accuracy of the data is considered sufficient for the purposes of this study. The reference substance is a commercially available material. The certificate of analysis (COA) was provided by the supplier and the accuracy of the data is considered sufficient for the purposes of this study. The stock test solutions were analyzed; however, the analysis was not performed in compliance with GLP. Analysis to confirm concentration and uniformity of the test solutions was not performed. This is believed not to affect the validity of the study because the test and reference substances were accurately measured and added to the test vessels. Study Director -w ^ X ^ A ^ _ _ _ _ William R. Berti, Ph.D. Senior Research Biologist ^X^oa Date DuPont Environmental and Microbiological Sciences & Engineering Sponsor/Submitter Date TO 105/4842 Page 3 of 27 DuPont EMSE Report No. 18-02 QUALITY ASSURANCE STATEMENT Study Number T0105/4842 Study Title Activated Sludge Respiration Inhibition Test ofGlycolic Acid Technical, 70% Solution (OECD 209) The conduct of this study was inspected periodically. Audit dates are given below: Study Phase Inspected Study Protocol Study Conduct Inspection/Audit Dates ll-Feb-2002 12-Feb-2002 Dates Findings Reported to Study Director 16-Feb-2002 16-Feb-2002 Report_____________1-July-2002______23-July-2002 Dates Findings Reported to Management 16-Feb-2002 16-Feb-2002 26-July-2002 These inspections confirm that the methods, procedures, and observations .are accurately and completely described in this report. The reported results accurately and eompletelyTeflect the raw data of this study. ' '-1 kfjLntbvUnJE&)iUMAT Kimberly B. Brebn^r QA ^ A*--u--diJt;Ao*r- DuPont Co. 5i-'^Ui-^0^ Date T0105/4842 Page 4 of 27 DuPont EMSE Report No. 18-02 CERTIFICATION OF AUTHENTICITY ACTIVATED SLUDGE RESPIRATION INHIBITION TEST OF TELOMER B 8-2 ALCOHOL (OECD 209) We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record of the procedures and results. Report by: ? a ^ ^ e William R. Berti, Ph.D. Senior Research Biologist Approved by: W ^ John?. Cannon, Ph.D. ch Manager 3i4^Fte^ Date''-7 K) 0 9 / ? / / ^ o l --Da--te -----i----./.-------- T0105/4842 Page 5 of 27 DuPont EMSE Report No. 18-02 TABLE OF CONTENTS Page Reserved for Specific Country Requirements...............................................--......................^ Good Laboratory Practice Compliance Statement..........................................................................3 Quality Assurance Statement...........................................................................................................4 Certification of Authenticity............................................................................................................5 Table of Contents.............................................................................................................................6 1.0 Summary.................................................................................................................................8 2.0 General Study Information......................................................................................................8 3.0 Materials and Methods............................................................................................................9 3.1 Test Guidelines...................................................................................................................^ 3.2 Test Components...............................................................................................................10 3.2.1 Chemical System .....................................................................................................10 3.2.2 Biological System.....................................................................................................ll 3.2.3 Physical System .....................................................................................................12 3.3 TestConduct......................................................................................................................l2 3.4 Parameters Observed.........................................................................................................12 3.4.1 Analysis of Test Systems.......................................................................................... 12 3.4.2 Analysis of the Test Substance Stock Solutions....................................................... 13 3.5 Result Analysis..................................................................................................................13 3.5.1 Respiration Rate .....................................................................................................13 3.5.2 Inhibitory effect .....................................................................................................14 3.6 Validity Criteria of the Study ............................................................................................14 3.7 Protocol Deviations ...........................................................................................................14 4.0 Results and Discussion..........................................................................................................14 5.0 Conclusions...........................................................................................................................15 6.0 Retention of Records.............................................................................................................16 7.0 Disposal of Test Substance...................................................................................................16 8.0 References.............................................................................................................................16 Tables Table 1: Influence of Test Substance Telomer B 8-2 Alcohol and Reference Substance 3, 5-dichlorophenol on the Oz consumption of activated sludge..............................................17 Table 2: concentrations of the test substance stock solutions........................................................18 Figures Figure 1: Influence of the Reference Substance 3,5-dichlorophenol on the respiration rate of aerobic wastewater microorganisms after 30 minutes of exposure..................................19 Figure 2: Influence of the Test Substance Telomer B 8-2 Alcohol on the respiration rate of aerobic wastewater microorganisms after 30 minutes of exposure..................................20 dmx T0105/4842 Page 6 of 27 DuPont EMSE Report No. 18-02 Appendix Appendix A: Data tables and calculations for positive solvent controls, reference and test substances, and negative control samples.............................................................................21 Positive Control 1.................................................................................................................21 Positive Control 2.........................................................--.....................................................21 Solvent Control 2.................................................................................................................22 Reference 1: 16 mg L'1 3,5-dichlorophenol ........................................................................23 Reference 2: 5 mg L'1 3,5-dichlorophenol ..........................................................................23 Reference 3: 1.6 mg L'1 3,5-dichlorophenol ......................................................................24 Test 1: 10 000 ug L'1 Telomer B 8-2 Alcohol ....................................................................24 Test 2: 3 200 u,g L"1 Telomer B 8-2 Alcohol ......................................................................25 Test 3: 1 000 |ig L'1 Telomer B 8-2 Alcohol ......................................................................25 Test 4: 320 |^g L'1 Telomer B 8-2 Alcohol .........................................................................26 Test 5: 100 ug L'1 Telomer B 8-2 Alcohol .........................................................................26 Negative control: Negative control with 10 000 |^g L'1 Telomer B 8-2 Alcohol...............27 TO 105/4842 Page 7 of 27 1.0 2.0 DuPont EMSE Report No. 18-02 ACTIVATED SLUDGE RESPIRATION INHIBITION TEST OF TELOMER B 8-2 ALCOHOL (OECD 209) William R. Berti, Ph.D. SUMMARY Test System Telomer B 8-2 Alcohol was tested for toxicity towards activated sludge according to OECD Guideline 209 in the version dated 4-April-1984. For the determination of the toxic behavior of the test substance, activated sludge from the aeration tank of a municipal sewage treatment plant was exposed to the test substance at 100,320,1000,3200, and 10000 u,g L'1 nominal concentration. After 30 minutes of incubation period the inhibition of the respiration rate of the activated sludge was determined in comparison to a test solution without any test substance. Findings Under the conditions of the test, the highest test concentration with 10000 ug test substance in 1 liter tapwater exhibited less than 20% inhibition of the respiration rate, which is the detection imit of the system. Conclusions The EC5o of the test substance is estimated to be greater than 10000 ug L'1 (10 mg L"1). GENERAL STUDY INFORMATION ,-NIJ-P^ w^w Study Objectives The aim of this study was the determination of the acute toxic behaviour of Telomer B 8-2 Alcohol towards the microorganisms of activated sludge according to OECD guideline 209 in the version of April 4,1984. The objectives of this study were to determine the: Effect of the test substance, Telomer B 8-2 Alcohol, on microorganisms from municipal sewage sludge and Suitable non-inhibitory concentrations of the test substance to be used in biodegradability tests using a microbial inoculum and an artificial sewage feed and measuring the respiration rate of the test system over a 30 minute time period under controlled laboratory conditions. The purpose of the study was to assess the potential of the test substance to adversely affect aerobic municipal sewage treatment plants. Test System Justification The test system is outlined by the OECD guideline 209 and was requested by the submitter. T0105/4842 Page 8 of 27 3.0 3.1 DuPont EMSE Report No. 18-02 Study Personnel E. I. du Font de Nemours and Company Environmental and Microbiological Sciences & Engineering and the Corporate Center for Analytical Sciences Management: Study Director: Analytical Chemist: Technical Personnel: John T. Gannon, Ph.D. William R. Berti, Ph.D. E. I. du Pont de Nemours and Company Environmental and Microbiological Sciences & Engineering Central Research & Development, Glasgow Building 300 P.O. Box 6101 Newark, DE 19714-6101 USA Daryl P. Cobranchi, Ph.D. E. I. du Pont de Nemours and Company Corporate Center for Analytical Research Central Research & Development, Glasgow Building 300 P.O. Box 6101 Newark, DE 19714-6101 USA Mark Stair Patrick W. Folsom Study Execution Dates A Study Initiation Date: 11 -Feb-2002 Experimental Start Date: 11 -Feb-2002 Experimental Completion Date: 25-Mar-2002 Study Completion Date: 31 -July-2002 MATERIALS AND METHODS Test Guidelines The purpose of the procedure is to provide a rapid screening method whereby substances that may adversely affect aerobic microorganisms from municipal sewage treatment plants can be identified and to indicate the suitable non-inhibitory concentrations of test substances to be used in biodegradability tests. The test uses a microbial inoculum and a synthetic sewage feed. The respiration rate of the test system is measured after an exposure period of 30 minutes under controlled laboratory conditions. The inhibitory effect of the test substance at the particular concentrations is expressed as percentage of the mean respiration rate of two controls. TO 105/4842 Page 9 of 27 3.2 3.2.1 3.2.1.1 Test Components Chemical System Test Substance Name: Synonym: Active substance(s) CAS Name: CAS Number(s): Structure: DuPont EMSE Report No. 18-02 Telomer B 8-2 Alcohol (Perfluorooctyl)ethanol Telomer B 8-2 Alcohol, 99% 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro678-39-7 H Number Lot Number: EMSE Sample Number: Concentration ofa.s., nominal: Concentration ofa.s., analyzed: Certificate of Analysis Date: Date Received: Solubility at 25C: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: H-24691 P.00/001 E93386-63 99% 99.2% 13-Sept-2001 26-Mar-2002 -140 ug L-1 0.023 mm Hg Stable at ambient room temperature White solid Room temperature; keep tightly closed Wear lab coat, protective gloves, and safety glasses T0105/4842 Page 10 of 27 DuPont EMSE Report No. 18-02 3.2.7.2 3.2.1.3 3.2.1.4 Reference Substance Toxic Reference Name: Active substance(s) CAS Number(s): Product Number: Lot Number: EMSE Sample No. Concentration ofa.s., nominal: Concentration ofa.s., analyzed: Certificate of Analysis Date: Expiry Date: Date Received: Appearance/Color: Storage Conditions: Safety Precautions: Manufacturer: Manufacturer Address: 3,5-dichlorophenol 3,5-dichlorophenol 591-35-5 AldrichD70600 15809KI M57657 97% 99.7% August 2000 August 2005 January 2002 White Powder Store in a cool dry place; keep tightly closed Irritant; wear suitable gloves and eye/face protection. Aldrich Chemical Co., Inc. 1001 West St. Paul Milwaukee. WI 53233 USA Test Vehicle The test substance was dissolved in a stock solution ofethanol at 10000, 3000, 1000, 300, andlOOmgL"'. Application Information The test substance was added to the test vessels at the following concentrations: 10000,3200, 1000,320 and 100 ugL''. 3.2.2 Biological System Secondary activated sludge from Wilmington, DE USA Publically Owned Treatment Works (POTW) was used as the microbial inoculum. The activated sludge was aerated and used within 24 h of collection. The sludge was washed as follows: first centrifuge the sludge for about 10 min at approximately 10,000 m s"2 and discard the supernatant. Resuspend the sludge in tap water, remove this by recentrifuging, and then repeat twice more. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in tap water to yield a concentration equivalent to about 2 to 4 g per liter (on dry weight basis). T0105/4842 Page 11 of 27 DuPont EMSE Report No. 18-02 3.2.3 3.2.3.1 3.2.3.2 Physical System Test Units All test vessels are 300-mL Biological Oxygen Demand (BOD) glass bottles for incubation and Dissolved Oxygen (DO) determinations. Test Conditions Test solutions were kept closed and not aerated during a 30-minute incubation period at room temperature. 3.3 3.4 3.4.1 Test Conduct Sufficient microbial solution for all the test vessels except the negative control was made. The microbial solution contained 32 mL of synthetic sewage feed, 400 mL microbial inoculum (containing 2 to 4 g dry weight/L), and about 568 mL tap water to make one L. The microbial solution was aerated at about 0.5 to 1 liter per minute until used. The test was initiated with the first positive control by adding 300 mL of the microbial solution to the test vessel and the test vessel stoppered. Aeration was stopped. The test vessels were continuously stirred using a stir bar and stir plate. After approximately 15 minutes (an arbitrary but convenient interval) the above procedure was repeated, except that the solvent was added to the test vessel at the concentration that was used with the test substance (first solvent control) and the was test initiated by adding 300 mL of the microbial solution to the test vessel. The test vessel was immediately capped. Again, after approximately 15 minutes this procedure was repeated, this time with a known amount of the reference substance with no solvent first added to the test vessel and the test initiated by adding 300 mL of the microbial solution to the test vessel and capping it. The procedure was repeated at approximately 15-minute intervals with the reference substance and then the test substance in solvent to give a series of test vessels containing different concentrations of the test substance. The negative control (sec. 12.7) test was performed next and then the second solvent control was performed. The final flask was a second positive control, prepared exactly as the first. The test was conducted so that the reference and test substances were bracketed by the two controls, for example: 1. first positive control 2. first solvent control 3. reference substance (no solvent) at 3 concentrations 4. test substance (with solvent) at 5 concentrations 5. negative (abiotic) control at highest concentration of test substance 6. second solvent 7. second positive control Parameters Observed Analysis of Test Systems The respiration rate of a well-mixed sample of each treatment was determined after exactly 30 minutes incubation time with no further aeration from the beginning of the 30-minute incubation period once the microbial solution had been added to the test vessel. The oxygen T0105/4842 Page 12 of 27 3.4.2 3.5 3.5.1 DuPont EMSE Report No. 18-02 concentration was measured with an oxygen electrode and recorded over a period of about 10 minutes. During measurement, the samples were continuously stirred with the built-in stirrer and measurements recorded using the attached printer. The rate of rate of oxygen consumption (in mg O-i L"' h'') was determined from the most linear part of the respiration curve over a period of about 10 minute. The water temperature was measured in the positive control medium at the start and the end of the incubation period. Analysis of the Test Substance Stock Solutions Concentrations of the test substance stock solutions were estimated by GC/MS. GC/MS Conditions for Telomer B 8-2 Alcohol Analysis Gas Chromotography: Dectector: GC Column: Mobile Phase: Flow Rate: Column Temperature: Injection Volume: lonization mode: Data acquisition mode: Hewlett Packard (HP) 5 890 Series II HP 5972 Series Mass Selective Detector Restek Rtx-200; 60 m, 0.25 mm ID, 1 urn film thickness Helium 1.4 mL/min (constant flow mode) 80C for 1 min. 15C/min to 300C. Hold 10 minutes 1 uL splitless injection Electron lonization selected ion monitoring, acquiring m/z of 31, 69,95, 131, 463 Result Analysis "^; Respiration Rate The respiration rate was calculated from the recorder output as mg Oz L'1 h'1 over a 10 min period. This can be done by graphing mg Oz L'1 on the y-axis and time in hours on the x-axis, drawing the line of best fit, and then determining the slope of the line. The slope of the line is the extent to which the y-axis changes for 1 unit of change in the x-axis. Alternatively, the respiration rate can be calculated as follows: ^_]>>-(1>)(1>) ny^-x^-x^)x2r where b = Respiration Rate n = pairs ofx and y values x = time in hours y = mg Oz L"1 at time = x The portion of the respiration curve over which the respiration rate is measured should be linear. T0105/4842 Page 13 of 27 3.5.2 3.6 3.7 4.0 DuPont EMSE Report No. 18-02 Inhibitory effect The inhibitory effects was determined by comparing the rates at each concentration of either test or reference substance to the rates in the controls. The results are expressed as percentage of the mean value of the respiration rates of the two controls according to: 77? 1 - ------s-- * 100 == Percent inhibition ^ci + ^c2. where Rs = oxygen consumption rate at tested concentration of test substance Rci = oxygen consumption rate of positive control 1 Rc2 = oxygen consumption rate of positive control 2 The 30-minute ECso (Effective Concentration of the substance giving a calculated or interpolated inhibition of oxygen consumption of 50% compared with a blank control) and their 95%-confidence limits are calculated by standard statistical methods. If possible, the EClo and the ECgo also will be calculated and reported for the test substance. The percent inhibition is calculated at each test concentration as above. For the test substance, the percent inhibition is plotted against concentration as a log-normal (or log-probability) graph and an EC5o value derived from the graph. For the reference substance, the percent inhibition is plotted against concentration as a normal-normal graph and an ECso value derived directly from the graph. Validity Criteria of the Study ' The test results are valid if: The two positive control respiration rates (PCRR) and the two solvent control respiration rates are within 15 percent of each other: The EC5o (30 minutes) of3,5-dichlorophenol is in the accepted range of 5 to 30 mg L'1, which is determined by graphing the percent inhibition on the y-axis and the concentration of the reference substance at which that level of inhibition was measured on the x-axis. The ECso can be determined directly from the graph. Protocol Deviations The test systems were not maintained at a temperature of 23 2C. This did not affect the test, however, because the validity criteria of the test were met, as stated in section 3.6. RESULTS AND DISCUSSION EC5o of the test substance at 30 min was greater than 10000 ug test substance per liter ECzo of the test substance at 30 min was greater than 10000 ug test substance per liter ECgo of the test substance at 30 min was greater than 10000 ug test substance per liter T0105/4842 Page 14 of 27 5.0 DuPont EMSE Report No. 18-02 The test substance showed no acute toxic effect towards the microbial inoculum (activated sludge) at an oxygen consumption inhibition level >, 20% at the highest concentration of 10000 ug L"1 that was tested. EC5o of the reference substance at 30 min was 12 mg reference substance per liter, which was within the acceptable range of from 5 to 30 mg L"1 The two positive control respiration rates were within 40% of each other and not within 15% as required for a valid test. The test is valid, however, because the reference substance has an ECso of 12 mg L'1, which is within the acceptable range. Additionally, if the first positive control values is not used to calculate the respiration inhibition because of the low temperature at which it was studied, the ECso of the reference substance would be 19 mg L"1, which is also within the acceptable range. All microbial inoculum was refrigerated overnight and was not allowed to completely warm to room temperature prior to the start of the experiment. This resulted in the temperature of the first positive control of 15.0C, which was signficantly lower than the temperature of the second positive control solution of20.2C. On the other hand, the respiration rates of the two solvent control solutions, which were used to calculate the respiration inhibition of the test substance, were within 9% of each other. This is less than 15% as required for a valid test. Temperatures in the positive control medium at the beginning and end of the incubation period were 15.0 and 20.2C, respectively. The concentrations of the test substance stock solutions were determined to be 10910, 3450, 1284,320, and 100 mg L'' (Table 2). This compares favorably to the target concentrations of 10000, 3000, 1000, 300, and 100 mg L'', respectively, for test substance in the stock solutions. CONCLUSIONS The EC5o of the test substance is estimated to be greater than 10000 u.g L'' (10 mg L"') The ECso of the reference substance is 12 mg L"1 The test is valid. TO 105/4842 Page 15 of 27 6.0 7.0 8.0 DuPont EMSE Report No. 18-02 RETENTION OF RECORDS For the periods demanded by GLP guidelines and specific country requirements, study documents and materials will be archived at DuPont CR&D Environmental & Microbiological Sciences and Engineering (EMSE) and/or Iron Mountain, Wilmington, Delaware USA, including but not limited to: study protocol; any protocol and/or report amendments or addenda or SOP deviations; all raw data; one original signed copy of the final report; laboratory-specific or site-specific raw data such as personnel files, instrument, equipment, refrigerator, and/or freezer raw data. Documents and materials are archived according to the principles of Good Laboratory Practice in the organization of the testing facility. DISPOSAL OF TEST SUBSTANCE After issuance of the final report, the remaining test substance will be stored at the DuPont EMSE laboratory until testing is complete and then destroyed by incineration, unless other arrangements are made between the submitter and the Test Facility. REFERENCES 1. OECD (1984) Guideline for Testing of Chemicals, Section 2, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted April 04, 1984. 2. International Standard ISO 8192: "Water quality - Test for inhibition of oxygen consumption by activated sludge". First edition 1986-07-15. Ref. No. ISO 8192-1986 (E). T0105/4842 Page 16 of 27 DuPont EMSE Report No. 18-02 TABLE 1 INFLUENCE OF TEST SUBSTANCE TELOMER B 8-2 ALCOHOL AND REFERENCE SUBSTANCE 3, 5-DICHLOROPHENOL ON THE 02 CONSUMPTION OF ACTIVATED SLUDGE 0, 0; Consumption No. Treatment Concentration Concisntrationt Inhibition:!: Start End 1 Positive Control 1 13 Positive Control 2 mg/L 0 0 mgOz/L 7.17 6.75 6.51 5.80 mgOs/Uh 2.89 4.33 % NAK NA Mean 3.61 Standard deviation 1.02 2 Solvent Control 1 0 6.74 5.80 5.91 NA 12 Solvent Control 2 0 5.62 4.61 6.46 NA Mean 6.18 Standard deviation 0.39 3 3,5-dicholorophenol 16 4 3,5-dicholorophenol 5 5 3,5-dicholorophenol 1.6 8.90 8.07 7.68 8.73 7.67 7.11 1.12 2.66 3.78 69.0 26.2 -0.85 6 Telomer B 8-2 Alcohol 7 Telomer B 8-2 Alcohol 8 Telomer B 8-2 Alcohol 9 Telomer B 8-2 Alcohol 10 Telomer B 8-2 Alcohol 11 Telomer B 8-2 Alcohol negative control 10 3.2 1.0 0.32 0.10 10 5.96 5.79 5.68 5.71 5.64 9.86 4.82 4.62 4.50 4.65 4.57 9.80 7.16 7.46 7.23 6.72 7.70 0.51 -15.8 -19.7 -16.9 -8.71 -24.5 91.7 "jThe readings of the Dissolved Oz meter at start and end of 10-minute period over which data was collected after 30 min of aeration ^Inhibition of the reference substance was calculated using the average value of the positive control solutions; the inhibition of the test substance was calculated using an average value of the solvent control solutions. ITNA= Not Applicable. T0105/4842 Page 17 of 27 DuPont EMSE Report No. 18-02 TABLE 2 CONCENTRATIONS OF THE TEST SUBSTANCE STOCK SOLUTIONS EMSE Number M57777 M57778 M57779 M57780 M57781 Desired Stock Solution Concentrations mg L 10000 3 000 1000 300 100 Measured Stock Solution Concentrations mg L 10910 3450 1284 320 100 TO 105/4842 Page 18 of 27 DuPont EMSE Report No. 18-02 FIGURE 1 INFLUENCE OF THE REFERENCE SUBSTANCE 3,5-DICHLOROPHENOL ON THE RESPIRATION RATE OF AEROBIC WASTEWATER MICROORGANISMS AFTER 30 MINUTES OF EXPOSURE Concentration, mg/L T0105/4842 Page 19 of 27 DuPont EMSE Report No. 18-02 FIGURE 2 INFLUENCE OF THE TEST SUBSTANCE TELOMER B 8-2 ALCOHOL ON THE RESPIRATION RATE OF AEROBIC WASTEWATER MICROORGANISMS AFTER 30 MINUTES OF EXPOSURE 100 1000 10000 Concentration, pg/L TO 105/4842 Page 20 of 27 DuPont EMSE Report No. 18-02 APPENDIX A DATA TABLES AND CALCULATIONS FOR POSITIVE CONTROLS, SOLVENT CONTROLS, REFERENCE AND TEST SUBSTANCES, AND NEGATIVE CONTROL SAMPLES Positive Control 1 Positive Contro 11 Reading Time of Number Day h:mm:ss 1 9:03:05 2 9:03:35 3 9:04:04 4 9:04:35 5 9:05:05 6 9:05:34 7 9:06:04 8 9:06:35 9 9:07:04 10 9:07:34 11 9:08:05 12 9:08:35 13 9:09:04 14 9:09:34 15 9:10:04 16 9:10:34 17 9:11:05 18 9:11:34 19 9:12:04 20 9:12:34 DO Reading mg/L 7.17 7.20 7.17 7.14 7.13 7.10 7.08 7.05 7.02 7.00 6.97 6.95 6.93 6.89 6.87 6.85 6.83 6.81 6.77 6.75 Time h:mm:ss 0:00:00 0:00:30 0:00:59 01:30.0 0:02:00 0:02:29 0:02:59 0:03:30 0:03:59 0:04:29 0:05:00 0:05:30 0:05:59 0:06:29 0:06:59 0:07:29 0:08:00 0:08:29 0:08:59 0:09:29 Total Time s 0 30 59 90 120 149 179 210 239 269 300 330 359 389 419 449 480 509 539 569 Total Time 171 0.00 0.50 0.98 1.50 2.00 2.48 2.98 3.50 3.98 4.48 5.00 5.50 5.98 6.48 6.98 7.48 8.00 8.48 8.98 9.48 Total Time h 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.07 0.08 0.09 0.10 0.11 0.12 0.12 0.13 0.14 0.15 0.16 Rate of RespiraUon (Rci) = 2.894351 mg O; / L / h Positive Control 2 Positive Control 2 Reading Time of Number Day /i.'/n/n.'ss 1 12:01:32 2 12:02:03 3 12:02:32 4 12:03:02 5 12:03:33 6 12:04:03 7 12:04:32 8 12:05:03 9 12:05:33 10 12:06:02 11 12:06:33 12 12:07:03 13 12:07:32 14 12:08:02 15 12:08:32 16 12:09:02 17 12:09:32 18 12:10:03 19 12:10:33 20 12:11:03 DO Reading mgIL 6.51 6.44 6.41 6.37 6.34 6.33 6.26 6.24 6.20 6.15 6.12 6.08 6.05 6.01 5.98 5.95 5.92 5.88 5.84 5.80 Time h:mm:ss 0:00:00 0:00:31 0:01:00 0:01:30 0:02:01 0:02:31 0:03:00 0:03:31 0:04:01 0:04:30 0:05:01 0:05:31 0:06:00 0:06:30 0:07:00 0:07:30 0:08:00 0:08:31 0:09:01 0:09:31 Total Time s 0 31 60 90 121 151 180 211 241 270 301 331 360 390 420 450 480 511 541 571 Total Time m 0.00 0.52 1.00 1.50 2.02 2.52 3.00 3.52 4.02 4.50 5.02 5.52 6.00 6.50 7.00 7.50 8.00 8.52 9.02 9.52 Total Time ft 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Rate of RespiraUon (Rifi) = 4.331447 mg Oa / L /h T0105/4842 Page 21 of 27 DuPont EMSE Report No. 18-02 Solvent Con\trol 1 Solvent Control 1 Reading Time of Number 1 Day ;T.mm'.ss 9:18:34 2 9:19:05 3 9:19:34 4 9:20:04 5 9:20:35 6 9:21:05 7 9:21:35 8 9:22:04 9 9:22:34 10 9:23:04 11 9:23:35 12 9:24:04 13 9:24:35 14 9:25:04 15 9:25:34 16 9:26:04 17 9:26:35 18 9:27:04 19 9:27:35 20 9:28:04 DO Read Ing ing/I. 6.74 6.70 6.65 6.59 6.55 6.50 6.46 6.40 6.35 6.30 6.26 6.20 6.15 6.10 6.05 6.00 5.96 5.94 5.85 5.80 Time h:mni:ss 0:00:00 0:00:31 0:01:00 0:01:30 0:02:01 0:02:31 0:03:01 0:03:30 0:04:00 0:04:30 0:05:01 0:05:30 0:06:01 0:06:30 0:07:00 0:07:30 0:08:01 0:08:30 0:09:01 0:09:30 Time s 0 31 60 90 121 151 181 210 240 270 301 330 361 390 420 450 481 510 541 570 Time m 0.00 0.52 1.00 1.50 2.02 2.52 3.02 3.50 4.00 4.50 5.02 5.50 6.02 6.50 7.00 7.50 8.02 8.50 9.02 9.50 Time h 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Rate of Respiration Q= 5.911737 mg 0; / L ft) Solvent Cor,itrol2 Solvent Coiltrol2 ReadI ng Time of Numfc>er Day h:mm:ss 1 11:47:46 2 11:48:15 3 11:48:45 4 11:49:15 5 11:49:46 6 11:50:16 7 11:50:46 8 11:51:15 9 11:51:45 10 11:52:15 11 11:52:46 12 11:53:15 13 11:53:45 14 11:54:16 15 11:54:45 16 11:55:15 17 11:55:45 18 11:56:15 19 11:56:46 20 11:57:15 DO Read Ing nig/I. 5.62 5.57 5.52 5.46 5.40 5.35 5.30 5.23 5.19 5.13 5.08 5.03 4.98 4.93 4.87 4.81 4.75 4.70 4.65 4.61 Time /i:nim:ss 0:00:00 0:00:29 0:00:59 0:01:29 0:02:00 0:02:30 0:03:00 0:03:29 0:03:59 0:04:29 0:05:00 0:05:29 0:05:59 0:06:30 0:06:59 0:07:29 0:07:59 0:08:29 0:09:00 0:09:29 Time s 0 29 59 89 120 150 180 209 239 269 300 329 359 390 419 449 479 509 540 569 Time m 0.00 0.48 0.98 1.48 2.00 2.50 3.00 3.48 3.98 4.48 5.00 5.48 5.98 6.50 6.98 7.48 7.98 8.48 9.00 9.48 Time ft 0.00 0.01 0.02 0.02 0.03 0.04 0.05 0.06 0.07 0.07 0.08 0.09 0.10 0.11 0.12 0.12 0.13 0.14 0.15 0.16 Rate of Respiration (2) = 6.458075 mg Oz / L ftl T0105 / 4842 Page 22 of 27 w^wssw&wss mw^^i'sw^i^w'i'Ksa'i DuPont EMSE Report No. 18-02 Reference I: 16 msL'1 3,5-dichlorophenol Reference 1:16 Reading Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 mg/L3,5-djchlorophenol Time of Day DO Reading h:mm:ss mg/l. 9:33:40 8.90 9:34:11 8.91 9:34:40 8.90 9:35:10 8.89 9:35:41 8.88 9:36:10 8.86 9:36:40 8.86 9:37:11 8.85 9:37:40 8.83 9:38:10 8.83 9:38:40 8.83 9:39:10 8.82 9:39:41 8.81 9:40:11 8.80 9:40:41 8.79 9:41:10 8.77 9:41:41 8.76 9:42:11 8.76 9:42:40 8.74 9:43:10 8.73 Time h:mm:ss 0:00:00 0:00:31 0:01:00 0:01:30 0:02:01 0:02:30 0:03:00 0:03:31 0:04:00 0:04:30 0:05:00 0:05:30 0:06:01 0:06:31 0:07:01 0:07:30 0:08:01 0:08:31 0:09:00 0:09:30 Total Time s 0 31 60 90 121 150 180 211 240 270 300 330 361 391 421 450 481 511 540 570 Total Time m 0.00 0.52 1.00 1.50 2.02 2.50 3.00 3.52 4.00 4.50 5.00 5.50 6.02 6.52 7.02 7.50 8.02 8.52 9.00 9.50 Total Time h 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Percent I nhibition = '> V 1 2.89435 + 1 11BO X 100 = 4.3314 Rate of Respiration (R.) = 69.05 % 1.11818 mgOa/L/h Reference 2: 5msL~ 3,5-dichlorophenol Reference 2: 5 Reading Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 mgfL 3,5-dichlorophenol Time of Day DO Reading h:mm:ss 9:47:52 mg/l. 8.07 9:48:23 8.08 9:48:53 8.06 9:49:22 8.04 9:49:52 8.02 9:50:23 9:50:53 9:51:23 9:51:53 9:52:22 9:52:52 7.99 7.98 7.95 7.93 7.90 7.88 9:53:22 7.86 9:53:52 9:54:22 7.83 7.80 9:54:53 7.79 9:55:23 7.76 9:55:53 7.76 9:56:22 9:56:53 7.72 7.70 9:57:23 7.67 Time /i:mm:ss 0:00:00 0:00:31 0:01:01 0:01:30 0:02:00 0:02:31 0:03:01 0:03:31 0:04:01 0:04:30 0:05:00 0:05:30 0:06:00 0:06:30 0:07:01 0:07:31 0:08:01 0:08:30 0:09:01 0:09:31 Total Time s 0 31 61 90 120 151 181 211 241 270 300 330 360 390 421 451 481 510 541 571 Total Time m 0.00 0.52 1.02 1.50 2.00 2.52 3.02 3.52 4.02 4.50 5.00 5.50 6.00 6.50 7.02 7.52 8.02 8.50 9.02 9.52 Total Time A 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Rate of Respiration (R.) = 2.665095.ing.jQ2i/!L Ih "^. >. iiIU; " "}-. (V. Percent I nhibition = 0 v 0 KRK.A 1 - 2.89435 + 4.3314 x100= 26.23 % TO 105/4842 Page 23 of 27 DuPont EMSE Report No. 18-02 Reference 3: 1.6 msL~1 3. 5-dichlorouhenol References: 1.6 Reading Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 mg/L 3,5 -dichlorophenc01 Time of Day fi:mm:ss DO Reading mg/L 10:03:44 7.68 10:04:15 7.67 10:04:45 7.64 10:05:14 7.62 10:05:45 7.59 10:06:15 7.55 10:06:44 7.52 10:07:15 7.49 10:07:44 7.47 10:08:14 7.40 10:08:45 7.39 10:09:14 7.36 10:09:44 7.34 10:10:14 7.31 10:10:45 7.29 10:11:14 7.24 10:11:45 7.22 10:12:14 7.19 10:12:44 7.15 10:13:15 7.11 Time h:mm:ss 0:00:00 0:00:31 0:01:01 0:01:30 0:02:01 0:02:31 0:03:00 0:03:31 0:04:00 0:04:30 0:05:01 0:05:30 0:06:00 0:06:30 0:07:01 0:07:30 0:08:01 0:08:30 0:09:00 0:09:31 Total Time s 0 31 61 90 121 151 180 211 240 270 301 330 360 390 421 450 481 510 540 571 Total Time m 0.00 0.52 1.02 1.50 2.02 2.52 3.00 3.52 4.00 4.50 5.02 5.50 6.00 6.50 7.02 7.50 8.02 8.50 9.00 9.52 Total Time h 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Percent In hibition = 1 2x 2.89435 + 3.6436 X 100 = 4.3314 Rate of Respiration (R.) = 3.643633 mg 02 / L /h -0.85 % Test I: 10 000 /us L~1 Telomer B 8 -2 Alcohol Test 1:10000 miB/LTBA Reading Time of Number Day fi:mm:ss 1 10:17:47 2 10:18:17 3 10:18:46 4 10:19:17 5 10:19:47 6 10:20:17 7 10:20:46 8 10:21:16 9 10:21:46 10 10:22:17 11 10:22:47 12 10:23:17 13 10:23:47 14 10:24:17 15 10:24:47 16 10:25:16 17 10:25:47 18 10:26:16 19 10:26:47 20 10:27:17 DO Reading mg/L 5.96 5.89 5.84 5.77 5.71 5.66 5.60 5.54 5.48 5.42 5.35 5.30 5.24 5.18 5.12 5.06 5.00 4.94 4.88 4.82 Time fi:mm:ss 0:00:00 0:00:30 0:00:59 0:01:30 0:02:00 0:02:30 0:02:59 0:03:29 0:03:59 0:04:30 0:05:00 0:05:30 0:06:00 0:06:30 0:07:00 0:07:29 0:08:00 0:08:29 0:09:00 0:09:30 Total Time s 0 30 59 90 120 150 179 209 239 270 300 330 360 390 420 449 480 509 540 570 Total Time m 0.00 0.50 0.98 1.50 2.00 2.50 2.98 3.48 3.98 4.50 5.00 5.50 6.00 6.50 7.00 7.48 8.00 8.48 9.00 9.50 Total Time h 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.12 0.13 0.14 0.15 0.16 Percent In hibition = 1 2x 5.91174 + 7.1632 X 100 = 6.4581 RateofRespIratton (R.) = 7.163217 mg 0; / L Ih -15.82 % t'*a iat TO 105/4842 Page 24 of 27 DuPontEMSE Report No. 18-02 Test 2: 3 200 us U' Telomer B 8-2 Alcohol - Test 2: 3000 mgfl. TBA Reading Time of Number Day ft-'mnrss 1 10:32:40 2 10:33:11 3 10:33:41 4 10:34:11 5 10:34:41 6 10:35:11 7 10:35:41 8 10:36:10 9 10:36:40 10 10:37:11 11 10:37:40 12 10:38:10 13 10:38:40 14 10:39:10 15 10:39:41 16 10:40:11 17 10:40:41 18 10:41:11 19 10:41:41 20 10:42:10 DO Reading mg/L 5.79 5.73 5.68 5.59 5.54 5.48 5.42 5.36 5.29 5.23 5.17 5.10 5.05 4.98 4.92 4.85 4.80 4.74 4.68 4.62 Time ft:mm:ss 0:00:00 0:00:31 0:01:01 0:01:31 0:02:01 0:02:31 0:03:01 0:03:30 0:04:00 0:04:31 0:05:00 0:05:30 0:06:00 0:06:30 0:07:01 0:07:31 0:08:01 0:08:31 0:09:01 0:09:30 Total Time s 0 31 61 91 121 151 181 210 240 271 300 330 360 390 421 451 481 511 541 570 Total Time m 0.00 0.52 1.02 1.52 2.02 2.52 3.02 3.50 4.00 4.52 5.00 5.50 6.00 6.50 7.02 7.52 8.02 8.52 9.02 9.50 Total Time ft 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0,12 0.13 0.13 0.14 0.15 0.16 Percent nhlbitton = 1- 5.91174 2x + 7.4036 X 100 = 6.4581 Rate of Respiration (R.) = 7.403571 mg 0; / L /h -19.70 % Test 3: I 000 usL~1 Telomer B 8-2 Alcohol Test 3:1000mg/LTBA Reading Time of Number Day h:mm:ss 1 10:47:39 2 10:48:09 3 10:48:38 4 10:49:08 5 10:49:38 6 10:50:08 7 10:50:39 8 10:51:08 9 10:51:39 10 10:52:09 11 10:52:38 12 10:53:08 13 10:53:39 14 10:54:09 15 10:54:39 16 10:55:09 17 10:55:39 18 10:56:09 19 10:56:38 20 10:57:09 DO Reading mgll5.68 5.60 5.54 5.47 5.40 5.34 5.28 5.23 5.17 5.11 5.05 4.99 4.93 4.88 4.81 4.75 4.69 4.64 4.58 4.50 Time h:mm:ss 0:00:00 0:00:30 0:00:59 0:01:29 0:01:59 0:02:29 0:03:00 0:03:29 0:04:00 0:04:30 0:04:59 0:05:29 0:06:00 0:06:30 0:07:00 0:07:30 0:08:00 0:08:30 0:08:59 0:09:30 Total Time 5 0 30 59 89 119 149 180 209 240 270 299 329 360 390 420 450 480 510 539 570 Total Time m 0.00 0.50 0.98 1.48 1.98 2.48 3.00 3.48 4.00 4.50 4.98 5.48 6.00 6.50 7.00 7.50 8.00 8.50 8.98 9.50 Total Time ft 0.00 0.01 0.02 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Rate of Respiration(Ra)^ -ss's\^ "i!'^-' 7.231915 mgOa/L/h Percent nhibition = 1- 5.91174 2x + 67..42538119 X 100 = -16.92845 % T0105/4842 Page 25 of 27 DuPont EMSE Report No. 18-02 Test 4: 320 us L'1 Telomer BK-7. Alcohol Test4:300mg/LTBA Reading Time of Number Day 1 11:03:11 2 11:03:40 3 11:04:11 4 11:04:40 5 11:05:10 6 11:05:40 7 11:06:10 8 11:06:40 9 11:07:10 10 11:07:40 11 11:08:10 12 11:08:40 13 11:09:10 14 11:09:41 15 11:10:10 16 11:10:41 17 11:11:11 18 11:11:40 19 11:12:10 20 11:12:41 DO Reading mg/L 5.71 5.65 5.60 5.54 5.47 5.44 5.38 5.32 5.26 5.22 5.15 5.09 5.03 4.98 4.93 4.87 4.81 4.75 4.70 4.65 Time s 0:00:00 0:00:29 0:01:00 0:01:29 0:01:59 0:02:29 0:02:59 0:03:29 0:03:59 0:04:29 0:04:59 0:05:29 0:05:59 0:06:30 0:06:59 0:07:30 0:08:00 0:08:29 0:08:59 0:09:30 Total Time s 0 29 60 89 119 149 179 209 239 269 299 329 359 390 419 450 480 509 539 570 Total Time m 0.00 0.48 1.00 1.48 1.98 2.48 2.98 3.48 3.98 4.48 4.98 5.48 5.98 6.50 6.98 7.50 8.00 8.48 8.98 9.50 Total Time h 0.00 0.01 0.02 0.02 0.03 0.04 0.05 0.06 0.07 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Percent In nibition = 2x 1- 5.91174 + 6.7236 X 100 = 6.4581 Rate of Respiration (R.) = 6.723553 mg 02 / L Ih -8.71 % Test 5: 100 us L'1 Telomer B 8-2 Alcohol Test5:100mg/lLTBA Reading Time of Number Day h:mni:ss 1 11:20:42 2 11:21:12 3 11:21:42 4 11:22:12 5 11:22:41 6 11:23:11 7 11:23:41 8 11:24:11 9 11:24:42 10 11:25:12 11 11:25:41 12 11:26:12 13 11:26:42 14 11:27:11 15 11:27:41 16 11:28:12 17 11:28:42 18 11:29:12 19 11:29:41 20 11:30:11 DO Reading mg/L 5.64 5.32 4.91 4.60 4.36 4.20 4.11 4.02 3.96 3.89 3.84 3.79 3.75 3.72 3.69 3.65 3.61 3.58 4.43 4.57 Time h:mm:ss 0:00:00 0:00:30 0:01:00 0:01:30 0:01:59 0:02:29 0:02:59 0:03:29 0:04:00 0:04:30 0:04:59 0:05:30 0:06:00 0:06:29 0:06:59 0:07:30 0:08:00 0:08:30 0:08:59 0:09:29 Total Time s 0 30 60 90 119 149 179 209 240 270 299 330 360 389 419 450 480 510 539 569 Total Time n? 0.00 0.50 1.00 1.50 1.98 2.48 2.98 3.48 4.00 4.50 4.98 5.50 6.00 6.48 6.98 7.50 8.00 8.50 8.98 9.48 Total Time ft 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Rate of Respiration!,) = ^r^ 'WW 7.70189 mg 02 / L Ih - '-*"< '...; Percent In hibitton = 1- 5.91174 2x + 67..47508119 x100= -24.53 % TO 105/4842 Page 26 of 27 DuPont EMSE Report No. 18-02 Negative control: Negative control with 10 000 usL'1 Telomer B 8-2 Alcohol Negative Control vm'th TBA at 10000 mg/L Reading Time of Number Day DO Reading ft:mm:ss 1 11:34:30 ing/I. 9.86 2 11:35:01 9.87 3 11:35:30 4 11:36:01 5 11:38:30 6 11:37:01 9.88 9.89 9.89 9.88 7 11:37:31 8 11:38:00 9.87 9.87 9 11:38:31 9.86 10 11:39:00 11 11:39:30 9.85 9.85 12 11:40:00 9.85 13 11:40:30 14 11:41:00 9.78 9.85 15 11:41:31 16 11:42:00 9.84 9.82 17 11:42:30 18 11:43:01 9.82 9.81 19 11:43:30 20 11:44:01 9.82 9.80 Time h:mm:ss 0:00:00 0:00:31 0:01:00 0:01:31 0:02:00 0:02:31 0:03:01 0:03:30 0:04:01 0:04:30 0:05:00 0:05:30 0:06:00 0:06:30 0:07:01 0:07:30 0:08:00 0:08:31 0:09:00 0:09:31 Total Time s 0 31 60 91 120 151 181 210 241 270 300 330 360 390 421 450 480 511 540 571 Total Time m 0.00 0.52 1.00 1.52 2.00 2.52 3.02 3.50 4.02 4.50 5.00 5.50 6.00 6.50 7.02 7.50 8.00 8.52 9.00 9.52 Total Time h 0.00 0.01 0.02 0.03 0.03 0.04 0.05 0.06 0.07 0.08 0.08 0.09 0.10 0.11 0.12 0.13 0.13 0.14 0.15 0.16 Rate of Respiration (R,) = 0.51422 mg 0; / L fti Percent in1-'"""" - 1- 5.91174 2x + 06..54518412 Xv ii nunu ---- 0a 1i .fbtfolRoHoQy <Ay>. T0105/4842 Page 27 of 27