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AR226-2730 DuPont EMSER Report No. 97-03 Study Title A ctivated Sludge Respiration Inhibition Test of Z onyl^ B ^ ) (OECD 209) Test Guideline(s) OECD (1984) Guideline for Testing of Chemicals, Section 2, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted April 4, 1984 Author(s) William R. Berti, Ph.D. Study Completion Date 30-August-2004 Test Facility E. I. du Pont de Nemours and Company Environmental and Microbiological Sciences & Engineering Biochemical Science and Engineering Central Research & Development Glasgow, Building 300, P.O. Box 6101 Newark, DE 19714-6101 Submitter E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 USA EMSER Study /Project Number 97-03/ 4842 Report Number EMSER 97-03 97-03/4842 Company Sanitized. Does not contain TSC CB1 Page 1 of 19 DuPont EMSER Report No. 97-03 Page Reserved for Specific Country Requirements 97-03/4842 Company Sanitized. Does not contain TSCA CBI Page 2 of 19 DuPont EMSER Report No. 97-03 Good Laboratory Practice Compliance Statement The study described in this report, with the exception o f the items listed below was conducted in compliance with the following GLP Standards: United States Environmental Protection Agency, (FIFRA), Title 40 Code o f Federal Regulations Part 160 (effective October 16, 1989) which are consistent with: The OECD Principles o f Good Laboratory Practice (as revised 1997), ENV/MC/CHEM/98(17), OECD, Paris, 1998 MAFF Japan Good Laboratory Practice Standards (11 NohSan Number 6283). The following items are exceptions to the GLP Standards. These items do not impact the validity o f the study. The test chemical was not characterized under GLP. It is a commercially available material that was provided by the submitter. The certificate o f analysis (COA) was provided by the manufacturer and the accuracy o f the data is considered sufficient for the purposes o f this study. The reference chemical was not characterized under GLP. It is a commercially available material. The COA was provided by the supplier and the accuracy o f the data is considered sufficient for the purposes o f this study. The Study Protocol was not signed by the study director prior to the start o f the study. The study director gave approval to start the study by an electronic message. Analysis to confirm concentration and uniformity o f the test solutions was not performed. This is believed not to affect the validity o f the study because the test chemical was accurately measured and added to the test vessels. Study Director William R. Berti, Ph.D. Date Senior Research Biologist DuPont Environmental and Microbiological Sciences & Engineering Submitter 97-03/4842 Company Sanitized. Does not contain TSCA CBI Page 3 of 19 DuPont EMSER Report No. 97-03 Q uality Assurance Statement Study Number 97-03/4842 Study Title Activated Sludge Respiration Inhibition Test o f Zonyl 'OECD 209) / The conduct o f this study was subjected to periodic Quality Assurance inspections. The dates o f inspection are indicated below: Study Phase Inspected Study Conduct Report Inspection/Audit Dates 14 Nov 2003 07-08 Jul 2004 Dates Findings Reported to Study Director 19 Nov 2003 12 Jul 2004 Dates Findings Reported to Management 19 Nov 2003 12 Jul 2004 Staff Quality Assurance Auditor DuPont Haskell Laboratory for Health & Environmental Services 2 - 7 /Qi/QL/j*-? 2 - 0 0 ^ Date 'te r 97-03/4842 Company Sanitized. Doss not contain TSCftESS Page 4 of 19 DuPont EMSER Report No. 97-03 Certification of A uthenticity Activated Sludge Respiration Inhibition Test of Zonyl ^B 1^(O EC D 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: William R. Berti, Ph.D. Senior Research Biologist Approved by: Study Initiation Date: 17-NOV-2003 Date Study Completed: 30-August-2004 Submitter: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Date Q S r-xo- Date 97-03/4842 Company Sanitized. Does not contain'TSCAGBI Page 5 of 19 DuPont EMSER Report No. 97-03 Table of Contents Page Reserved for Specific Country Requirements................................................................................. 2 Good Laboratory Practice Compliance Statement....................................................................................3 Quality Assurance Statement........................................................................................................................4 Certification o f Authenticity......................................................................................................................... 5 Table o f Contents............................................................................................................................................ 6 1.0 Summary................................................................................................................................................ 7 2.0 General Study Infonnation................................................................................................................. 7 3.0 Materials and M ethods.........................................................................................................................8 3.1 Test Guidelines..................................................................................................................................8 3.2 Test System........................................................................................................................................ 9 3.2.1 Chemical System .................................................................................................................... 9 3.2.2 Biological System............................. 10 3.2.3 Physical System.....................................................................................................................10 3.3 Test Conduct.................................................................................................................................... 10 3.4 Parameters Observed................................................................................................................... 11 3.4.1 Analysis o f Test Systems.....................................................................................................11 3.5 Result Analysis................................................................................................................................11 3.6 Validity Criteria o f the Study....................................................................................................... 12 4.0 Results and Discussion....................................................................................................................... 12 5.0 Conclusions..........................................................................................................................................12 6.0 Retention o f Records.......................................................................................................................... 13 7.0 Disposal o f Test Item ......................................................................................................................... 13 8.0 References............................................................................................................................................ 13 Table sx Table 1: Influence o f Test Chemical Z o n y l |^ |a n d Reference Chemical 3, 5-dichlorophenol on the O2 Consumption o f Activated Sludge................................................ 14 Figures Figure 1: Influence o f the Reference Chemical 3,5-dichlorophenol on the Respiration Rate o f Aerobic Wastewater Microorganisms after 30 Minutes o f Exposure........................15 Figure 2: Influence o f the Test Chemical Z onyl ((B ^ on the Respiration Rate o f Aerobic Wastewater Microorganisms after 30 Minutes o f Exposure....................................... 16 Appendix ' Appendix A: Data Tables and Calculations for Positive Controls, Reference and Test Chemicals............................................................... ............................................................ v/ 97-03/4842 Company Sanitized. Does not contain TSCA CBI Page 6 of 19 DuPont EMSER Report No. 97-03 Activated Sludge Respiration Inhibition Test of Zonyl(f c c ) (OECD 209) 1.0 Summary Test System: Zonyl(FCC)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 chemical over a range of 10 to 1000 mg L'1. After 30 minutes of incubation, the respiration rate of the activated sludge was determined and compared to the respiration rate of the controls. V Findings: Under the conditions of the test, the highest test concentration of the test substance, 1000 mg L"1, exhibited <20% inhibition of the respiration rate (the detection limit of the system). Conclusions: The EC50of the test chemical could not be quantified because the highest nominal test concentration of 1000 mg/L resulted in less than 20% inhibition after 30 m of contact time. The EC50of the test chemical is estimated to be greater than 1000 mg L"1. 2.0 General Study Information Study Objectives v The aim o f this study was the determination of the acute toxic behaviour of Z o n y l (^ ^ ) 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, Z o n y l ^ on microorganisms from municipal sewage sludge and Assess the potential of the test chemical to adversely affect aerobic municipal sewage treatment plants The testing protocol used a microbial inoculum derived from a municipal wastewater treatment plant and an artificial sewage feed. The respiration rate of the test system was determined after a 30 m time period under controlled laboratory conditions. Test System Justification The test system is outlined by the OECD guideline 209 and was requested by the submitter. 97-03/4842 IJelfipsny 'Sanitized. Does not contain TSCA CBl Page 7 of 19 DuPont EMSER Report No. 97-03 Study Personnel E. I. du Pont de Nemours and Company Environmental and Microbiological Sciences & Engineering Management: Study Director: Principle Investigator: 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 William R. Berti, Ph.D. Same as above Study Phase(s): All Lynn Ann Dekleva Patrick W. Folsom Mark Starr Lisa M. Sulecki Study Execution Dates Study Initiation Date: Experimental Start Date: Experimental Termination Date: Study Completion Date: 17-NOV-2003 13- Nov-2003 14- Nov-2003 30-Aug-2004 3.0 Materials and Methods 3.1 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 chemical at the particular concentrations is expressed as percentage of the mean respiration rate of the controls. 97-03/4842 Company Sanitized. Does not contain TSCA CB Page 8 of 19 3.2 Test System 3.2.1 3.2.1.1 Chemical System Test Item Name: Synonyms: Active substance: CAS Name: CAS Number: EMSE Sample Number: Supplier Submitter Sample: Lot Number: DuPont EMSER Report No. 97-03 ZonyKS^B^ ^ NA Zonyl(j^t } ^ NA NA CRD 12005 Scott Krenzel DuPont Haskell Laboratory Newark, DE H-24616 436 3.2.1.2 Reference Item Toxic Reference Name: Active substance(s): CAS Number(s): Product Number: Lot Number: EMSE Sample No. Concentration of a.s., nominal: Concentration of a.s., analyzed: Certificate of Analysis Date: Expiry Date: Date Received: Appearance/Color: Storage Conditions: Safety Precautions: Manufacturer: 3.5- dichlorophenol 3.5- dichlorophenol 591-35-5 Aldrich D70600 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 97-03/4842 .CoiSpany Sanitized. Does not contain TSCA OBI Page 9 of 19 DuPont EMSER Report No. 97-03 3.2.1.3 3.2.1.4 Test Vehicle No test vehicle was used in this study for the test substance. The test chemical was mixed with synthetic sewage feed, water, and inoculum before addition to the test vessel. Application Information The test chemical was added to the test vessels from a stock solution at the following concentrations: 1000, 320, 100, 32 and 10 mg L"1. 3.2.2 Biological System Secondary activated sludge from Cecil County Fletchwood Facility, Maryland (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 solids were separated by coarse filtration, centrifuged at 10,000 m s"2 for approximately 10 minutes, washed twice, and resuspended in tap water. The solids concentration was determined by dry weight and adjusted to yield an inoculum concentration equivalent to about 4 g per liter (on dry weight basis). 3.2.3 3.2.3.1 3.2.3.2 Physical System Test Units The test vessels used in the incubation period were glass flasks of approximately 1-liter. For the measurement of the respiration rate, samples were transferred to 300-mL Biological Oxygen Demand (BOD) glass bottles. Test Conditions Test solutions were aerated for a 30-minute contact period at room temperature. 3.3 Test Conduct All test vessels were 1-liter glass flasks and contained a final volume of 500 mL. The test solution contained 16 mL of synthetic sewage feed, 200 mL microbial inoculum (containing approximately 4 g dry weight/L), the test chemical at the appropriate test solution concentration, and tap water to a final test volume of 500 mL. The test was initiated with the first positive control by combining the sewage feed, tap water, and inoculum in a 500 mL graduated cylinder and transferring it to the appropriately labeled test vessel. Aeration at 0.5 to 1.0 liter per minute initiated the test. The test vessel was continuously stirred using a stir bar and stir plate. The procedure was repeated at approximately 15-minute intervals with the reference chemical (at three concentrations) and then the test chemical to give a series of test vessels containing different concentrations of the test substance. A negative (abiotic) control at the highest test concentration was initiated by combining sewage feed, tap water, and test substance stock solution at the highest concentration in a 500 mL graduated cylinder. The solution was transferred to the test vessel to begin aeration. 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: 1. first positive control 2. reference chemical at 3 concentrations 3. test chemical at 5 concentrations 97-03/4842 Page 10 of 19 DuPont EMSER Report No. 97-03 3.4 3.4.1 3.5 4. negative (abiotic) control at highest concentration of test substance 5. second positive control Parameters Observed Analysis o f Test Systems The respiration rate of a well-mixed sample of each treatment was determined after exactly 30 minutes of incubation time with aeration. The oxygen concentration was measured with an oxygen electrode and recorded over a period of up to about 10 minutes. During measurement, the samples were continuously stirred with a stirrer built into the oxygen electrode and measurements were recorded using an attached printer. The rate of oxygen consumption (in mg 0 2L'1h'1) was determined from the most linear part of the respiration curve. The temperature was measured in the positive control flasks at the start and the end of the incubation period. Result Analysis The respiration rate was calculated from the recorder output as mg 0 2L'1If1over a period of approximately 10 minutes or less. The portion of the respiration curve over which the respiration rate was determined should be linear. The inhibitory effects were determined by comparing the rates at each concentration of either test or reference chemical 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: 1 2Rs *100= Percent inhibition . *c, + R c2 ( 1) where Rs =oxygen consumption rate at tested concentration of test substance Rci = oxygen consumption rate o f positive control 1 Rc2--oxygen consumption rate of positive control 2 The percent inhibition was calculated at each test concentration as above. A linear regression analysis was performed using the Regression Analysis Tool in Microsoft Excel 2000. Percent inhibition was used as the input y-values. LoglO of the test or reference chemical concentration was used as the input x-values. From this analysis, an equation relating chemical concentration to percent inhibition was determined at the 95% confidence level (p = 0.05) Percent Inhibition = A * [loglO (EC)] + B (2) where: A = X Variable 1, determined by the regression analysis EC = Effective Concentration of the Test or Reference Chemical, in mg/L B = intercept, determined by the regression analysis 97-03/4842 .. oinpan]r Sanitized. Does not containTSCACM Page 11 o f 19 DuPont EMSER Report No. 97-03 To calculate an EC50, equation (2) was solved for [log)0(EC)]. Percent Inhibition was then set equal to 50. Logio(EC50) = (Percent Inhibition - B)/ A (3) For A = 50.933 and B = -5.404 Log10(EC5o) = (50 - (-5.404))/ 50.933) = 1.088 EC50= lO1088= 12 mg/L Similarly, an EC20 and EC80 can be calculated by using a Percent Inhibition equal to 20 or 80, respectively, in equation (3). For example: Log10(EC20) = (20 - (-5.404))/ 50.933) = 0.4988 EC20= 10'4988 = 3 mg/L The percent inhibition also was plotted against concentration as a log-normal (or logprobability) graph, from which an EC50value can be derived graphically. 3.6 Validity Criteria o f the Study The test results are valid if: The two positive control respiration rates (PCRR) rates are within 15 percent of each other. The EC5oof reference chemical 3,5-dichlorophenol is in the accepted range of 5 to 30 mg L '1. 4.0 Results and Discussion The test chemical showed no acute toxic effect towards the microbial inoculum (activated sludge) at the highest concentration of 1000 mg L'1that was tested. ECS0of the reference chemical was 12 mg 3,5-dichlorophenol per liter, which was within the acceptable range of from 5 to 30 mg L'1. The respiration rates of the two positive controls were within 4%. 5.0 Conclusions The EC50of the test chemical Zony estimated to be greater than 1000 mg L'1. The EC50of the reference chemical 3,5-dichlorophenol is 12 mg L'1. The respiration rates of the two positive controls were within 15% of each other. The test is valid. 97-03/4842 Company Sanitized. Does net contain TSCA CBI Page 12 of 19 6.0 7.0 8.0 DuPont EMSER Report No. 97-03 Retention of Records For the periods demanded by GLP guidelines and specific country requirements, study documents and materials will be stored in the archives of the DuPont CR&D Environmental & Microbiological Sciences and Engineering (EMSE) archives and/or Iron Mountain Records Management, Wilmington, Delaware 19802 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; all documentation generated by the Quality Assurance Unit (to be archived by the Quality Assurance Unit, separate from study records); 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 Item uu f - After issuance of the final report, the remaining test chemical will be stored at the DuPont EMSE laboratory until its expiration date and then destroyed by burning, unless other - arrangements are made between the submitter and the Test Facility. ' References cc?..p. 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). 97-03/4842 !5empany Sanitized. Does not contain TSCA CBI Page 13 of 19 DuPont EMSER Report No. 97-03 Table 1: fN Influence of Test Chemical Z onyl ( ( B R and Reference Chemical 3, 5-dichlorqphenol on the 0 2 Consumption of Activated Sludge No. T reatm en t C o n cen tratio n 1 Positive Control 1 11 P o s itiv e C o n tro l 2 m g /L 0 0 o2 C o n c e n tratio n t S tart End m g O 2/L 6 .3 2 2 .7 2 4 .3 7 2.61 o2 C o n sum ption m g O s/Uh 4 4 .0 4 2 .6 In hibition:): % NAH NA M ean S tandard deviation 4 3 .3 1 .0 2 2 3,5-dicholorophenol 3 3,5-dicholorophenol 4 3,5-dicholorophenol 3 .2 10 32 6 .2 6 6 .50 8 .00 3 .1 9 3 .5 4 6 .8 9 3 3 .4 2 3 .5 12.2 2 2 .9 4 5 .7 7 1 .9 5 Z o n y l(s ^ N h 6 Z o n y l ( ^ ^ l 'j 7 Z o n y K S ^ B i^ } 8 Z o n y l ,^ ^ '\ 9 Z o n y i (jH | 10 32 100 320 1000 4 .9 8 5 .1 8 6 .5 2 6 .3 8 6 .35 2 .5 3 2 .7 4 2 .6 4 2 .5 2 2 .3 8 41.8 41.9 42.1 4 1 .9 3 9 .7 3 .5 8 3 .3 1 2 .7 3 3 .2 7 8 .2 5 10 Zo nyl(R | l ^ 0 f | (negative control) 1000 8 .6 9 8.71 -0.11 100 fThe readings of the Dissolved 0 2 meter at start and end of linear portion during the period over which respiration rate was calculated between approximately 6.5 and 2.5 mg 0 2/L or over a 10 min period when the repiration rate was low. ^Inhibition of the reference and test substances was calculated using the average value of the positive control solutions. A negative value indicates that respiration was enhanced, not inhibited. if NA = Not Applicable. ' j 97-03/4842 Company Sanitized. Does not contain TSCA CBI Page 14 of 19 DuPont EMSER Report No. 97-03 Figure 1: Influence of the Reference Chemical 3,5-dichlorophenol on the Respiration Rate of A erobic W astewater Microorganisms after 30 M inutes of Exposure 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Concentration, log10 mg/L 97-03/4842 BteHjpaftf 'Sanitized. Does not contain TSCA CB6 Page 15 of 19 DuPont EMSER Report No. 97-03 Figure 2: x Influence of the T est C hemical Z onyl P B J |on the Respiration Rate of A erobic W astewater M icroorganisms after 30 Minutes of Exposure 100 90 80 70 Test Substance 40 30 20 -- Linear (Test Substance) _y = 1.8578x + 0.5076 R2= 0.4179 10 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Zonyl(FCC^ log10 mg/L 97-03/4842 Company Sanitized Does not contain TSCA CBf Page 16 of 19 DuPont EMSER Report No. 97-03 A ppendix A: Data Tables and Calculations for Positive Controls, Reference and Test Chemicals Positive Controls Total Tim e 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 P o s itiv e P o s itiv e Control 1 C ontrol 2 D issolved Oxygen, m g/L 8.96 8.94 6.69 4.48 6.32 4.37 5.97 4.04 5.63 3.64 5.31 3.29 4.68 2.95 4.34 2.61 4.02 2.24 3.70 1.91 3.35 1.50 3.06 1.20 2.72 0.87 2.40 2.06 1.77 97-03/4842 Company Sanitized. Does not contain TSCA CBf Page 17 of 19 DuPont EMSER Report No. 97-03 Reference Chemical: 3,5-dichIorophenol Total Tim e 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 R eference 1 : 3.2 R eferen ce 2: R eference 3: 32 m g/L 10 m g/L m g/L D issolved O xygen, m g/L 9.07 9.36 8.09 7.89 6.50 8.00 7.65 6.28 7.91 7.36 6.07 7.78 7.16 5.88 7.68 6.83 5.68 7.59 6.55 5.48 7.50 6.26 5.27 7.39 5.96 5.08 7.30 5.68 4.87 7.19 5.38 4.71 7.07 5.10 4.50 6.98 4.83 4.32 6.89 4.56 4.12 4.29 3.93 4.01 3.73 3.71 3.54 3.46 3.19 97-03/4842 eoepanjr Sanitized, Does not contain Ter* mm. Page 18 of 19 DuPont EMSER Report No. 97-03 Test Chemical: Zonyl' T o ta l Tim e h 0 .0 0 0.01 0 .0 2 0 .0 3 0 .0 3 0 .0 4 0.05 0 .0 6 0.07 0.08 0 .0 8 0 .0 9 0 .1 0 0.11 0 .1 2 0 .1 3 0 .1 3 0 .1 4 T est C hem ical, 10 T est C hem ical, 32 m g/L m g/L 8 .5 8 4 .9 8 4 .6 0 4 .2 2 3 .9 0 3.55 3.21 2 .8 6 2 .5 3 2 .2 0 1.88 1.47 1.14 0 .8 3 8 .0 5 5.18 4 .8 3 4 .4 9 4 .1 5 3 .7 9 3.45 3 .0 8 2 .7 4 2.39 2.09 1.75 1.36 1.03 T e s t C hem ical, T e s t C hem ical, 100 m g/L 320 m g/L D isso lved O xygen, m g/L 8.20 8.84 6 .5 2 6.71 6 .1 3 6 .3 8 5.81 6 .0 3 5.44 5.66 5.09 5 .2 6 4 .7 4 4 .9 7 4.37 4 .6 2 4 .0 5 4 .2 6 3 .6 8 3 .9 4 3 .3 7 3 .5 6 2 .9 8 3 .2 2 2 .6 4 2 .8 8 2.31 2 .5 2 2 .0 0 2 .1 9 1.58 1.85 1.25 1.48 1.11 T est C hem ical, 1000 m g/L A b io tic C ontrol, 1000 m g /L 8.64 7 .0 0 6.68 6.35 6 .0 4 5.70 5.36 5.04 4.71 4 .3 9 4.01 3.71 3 .3 9 3.07 2.71 2.38 9 .0 3 8 .6 9 8.71 8 .7 2 8 .7 4 8 .7 4 8 .7 2 8 .7 7 8 .7 3 8 .7 3 8 .7 6 8 .7 2 8 .7 2 8 .7 3 8.71 97-03/4842 Company Sanitized. 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