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ACTIVATED SLUDGE RESPIRATION INHIBITION TEST TEST SUBSTANCE Identity:N-Ethylperfluorooctanesulfonamidmea;y alsobe referredtoas U1464, ETFOSA, ETPOSA, F-6309,or FX-12. (1Octanesulfonamide,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-N-ethylC-A,S # 4151-50-2) Remarks: Materialisa whitesolid.Sample purityand lotnumber were not recorded. METHOD Method: OECD 209 Test type: Staticacute GLP: No Year Completed: 1998 Analyticalmonitoring:Dissolvedoxygen concentrations. Test organism source:Activatedsludgecollectefdrom theWestern Lake SuperiorSanitarydistricDtu,luth,MN Test conditions: Dilutionwater: Carbon-treateddechlorinatedCityof Duluth,MN, tap water Synthetic Sewage: 1 liteDrI water 16.0 g Bacto.peptone 11.0 g Beef extract 3.0 g'urea 0.7g NaCi 0.4 g CaC12 -2H20 0.2 g MgS04 -7H20 2.8 g K2HP04 Reference and testsolutionpreparation:A 500 mg/L stock solutionofthe referencesubstance 3,5-dichlorophenowlas prepared using Milliporweater. Concentrationstestedwere 4, 16, and 32 mg/L, prepared by dilutintghe stocksolutionwithMillipore@) water. ETFOSA was added directltyotestvesselsratherthan by volumetricadditionofa stocksolution. Test vessels: Mixtureswere prepared (500 mL finalvolume) and aerated in1000 mL borosilicatgelassbeakers and then transferred into300 mL BiochemicalOxygen Demand (BOD) bottlesto obtain oxygen uptake rates. Number of concentrations: One, induplicateplusthreereference substance concentrationsand two Blank controls Temperature: 20.1-21.80C TotalSuspended Solids of sludge on day of testing:6.5 g/L (TSS intestsolutions= 3.3g/L).Sludge was used one day after collection. Element Basis: Respirationinhibitioans determined by dissolved oxygen concentration. Test mixtureswere prepared at7-minute intervalasnd aerated. After30-minutesand 3-hoursofcontacttime,dissolvedoxygen was measured every 0.5 minutes forup to 5.5 minutes. RESULTS Nominal concentrations: Two blank controlst,hree reference substance concentrations1,000mg/L (induplicatet)estmaterialsolutions StatisticaAlnalyses: EC50 valueswere calculatedforthe reference materialby theTrimmed Spearman-Karber Method. An EC50 value could not be calculatedforthe testsubstance. AnalyticalMethodology: Analysisof DO concentrationsinalltest solutionswere performed atAScl Corporationusing a YSI Model 54A dissolvedoxygen meter. Dissolvedoxygen readingswere recordedevery 30 seconds for5.5 minutes. Respirationrateswere normalizedfor200C priorto calculatinpge*rcentinhibition. Respiration Rates and Percent Inhibitionsafter3-Hours Exposure Treatment Control1 Control2 Testsubstance 1000mg/L Testsubstance 1000 mg/L (duplicate) Respiration Rate Mg 02/Llmin. (@ 20-C) 0.2714 0.3134 0.3981 1 0.4081 1 Percent Inhibition NA NA -36 -40 Control response: satisfactoryd;ifferencebetween controlsranged from 6.8% at 30-minutesto 14.4% after3-hoursofexposure. ReferenceSubstance: 3-hrEC50= 18.4mg/L indicatitnhgesludge had normal sensitivity. CONCLUSIONS The testsubstance exhibiteda no inhibitoreyffecton respiratioanta nominal testsubstance concentrationof 1000 mg/L after3-hours of exposure. The EC50 (respiratioinnhibitioins)thereforegreaterthan 1000 mg/L. Submifter: 3M Company, Environmental Laboratory,P.O. Box 33331, St. Paul,Minnesota, 55133 DATA QUALITY Reliability:Klimischranking2.This study,whilewellconducted usingthe methodology acceptableatthe time,lacksanalyticadlatafor determinationofthe testsubstance concentrationinthe testsolutionsand determinationofthe sample purity.Testingwas alsodone wellabove the solubilitlyimiotfthe testsubstance REFERENCES This study was conducted atASci Corporationof Duluth,Minnesota atthe requestof the 3M Company. OTHER Last changed: 5/18/00 Lt ASci Corporation/ASci-Duluth Environmental Testing Division Submitted to 3M Co. - 5/98 AScl Study ID# 5010-068 R@MBMON STUDY TITLE OF U1464 FOR ACTIVATED SLUDGE RESPIRATION DATASTANDARD OECD GUIDELINE 209 STUDY COMPLETED MAY 15, 1998 TESTING FACILITY ASEI Corporation/AScl-Duluth EnvironmentalTestingDivision 4444 Airpark Boulevard Duluth,MN 55811 Tel.No. (218)722-4040 PROJECT IDENTIFICATION NUMBERS ASCI Study ID# 5010-068 3M Company Lab Request# U1464 Sponsor: 3M Company sponsor Study ID# U1464 ASel Corporation/AScl-Duluth Environmental Testing Division Submitted to 3M Co. - 5/98 ASci Study [D# 5010-068 1.0 GENERAL INFORMATION Study Title Data Standard Sponsor Sponsor's Representative Sponsor's Lab Request# Testing Facility PrincipalInvestigator ProjectDirector TestingFacilityDirector DefinitiveTest Date Data Validationand Report Review Inhibitioonf U1464 For ActivatedSludge Respiration. OECD Guideline209. 3M EnvironmentalTechnology and SafetyServices,Building 2-3E-09,935 Bush Avenue, St.Paul, MN 55133i Tel No. (612)778-7452. RochelleR. Robideau, 3M Environmental Technology and SafetyServices,Building2-3E-09, 935 Bush Avenue, St. Paul,MN 55133; Tel No. (612)778-7065. U 1464. AScl Corporation/AScl-DuluthEnvironmentalTesting Division,4444 AirparkBoulevard,Duluth, MN 55811; Tel. No. (218)722-4040. Joe Dierkes Joe Amato Donald Mount May 1, 1998 Alan Mozol Report Signature: Retentionof Raw Data and Final Five yearsfrom the date studyiscompleted. Report Locationof Raw Data and Final Report AScl Corporation/AScI-DuluthEnvironmental Testing Division,4444 AirparkBoulevard,Duluth, MN 55811; Tel.No. (218)722-4040. Sponsor: 3M Company Sponsor Study ID# U 1464 2 ASel Corporation/ASci-Duluth Environmental Testing Division Submitted to 3M Co. - 5/98 ASel Study ID# 50IG-068 2.0OBJECTIVE To determinief,possiblteh,e30-minutaend3-h(contatcitmesE)C50'S ofthetesstubstance U1464 foractivatedsludgerespiration. This studywas conductedaccordingto the AScl studyplan retainedby theSponsor and OE Guideline209 (OECD 1993). 3.0TEST METHODS TestSubstance Properties:U1464, whitecrystalss,pecifigcravity= 1.7(water 1),stablea,nd negligiblseolubilitiynwater. Reference Substance Testconcentrations0: and 1000 mg/L preparedby directaddition. Name: 3,5-dichlorophenoplu,rity97%, AldrichChemicals,Lot# 09615KG. Test Organisms Testconcentrations:0, 4, 16,and 32 mg/L preparedby direct additionof 500 mg/L stocksolutionin Millipoi-ewOater. The referencetestwas performed concurrentlywith theexposure for U1464. Resultsand controlchartappended toreport. Source: Activatedsludgefrom the Westem Lake SuperiorSanitary DistricDtuluth,Minnesotacollecteodn April30, 1998. SyntheticSewage Feed ActivatedsludgecharacteristicTsh:e sludgewas determinedtohave a totalsuspendedsolidsconcentratioonf 6.5 g/L attesttime. The total suspendedsolidsconcentratioinnthetestsolutionwsas 3.3 g/L. The mixturewas fed syntheticsewage feed and aeratedat21 "C untiltest initiationT.he sludgewas used one day aftercollection. Composition: Chemical p-dissolved/Lof deionizedwater Bacto.peptone Beef extract Urea NaCl CaC12.2H20 MgS04.7H20 K2HP04 16.0 11.0 3.o 0.7 0.4 0.2 2.8 Sp-onsor, 3M Company Sponsor Study ID# U1464 3 AScl Co rporationA/Sel-Du luth Environmental Testing Division Submitted to 3M Co. - 5/98 ASci Study ID# 5010-068 DilutionWater Test Setup Source: Carbon-tTeatdeedchlorinateCdityofDuluth,MN, tapwater. The most recentchemicalanalysisof postcarbon (PC) water isin the Raw Data Package. Fxposure vessels:4, I-L borosilicatgelassbeakers;two inoculum controlsand 1 testconcentrationexposure in duplicate. Order ofpreparation: At 7-minute time intervals(:1) firstinoculum control,(2)testexposureconcentrationreplicatesa,nd (3)second inoculum control.The finalvolume in each beaker was 500 ml. Preparation:(1)Added requiredamount of carbon-treatewdater,(2) added 16 ml of syntheticsewage feed to each beaker,and (3)in test exposuresadded the requiredamounts of the testsubstance,(4) and added 200 ml of thesludge,fora finaltestsolutionvolume of 500 Determinationof Respiration Calculationof RespirationRate Endpoint Calculation Incubationand aeration:20.1' to21.8"C and atan aerationrateof 0.5 to 1.0 literpser minute. Sequence: Same as exposureswere prepared. Determination: At 30-min and 3-h contacttimes: (1) filledone 300ml BOD bottlewith the exposure medium and continuedto aeratethe remainingmedium, (2)usinga YSI 54A oxygen meter with a selfstirrinpgrobe, determinedtheDO concentrationin the exposure medium every 0.5 minutesforup to 5.5 minutes(thefirst determinationwas made after0.5 minutes),and (3)thesub-sample was placedback intothe I-L exposurechamber. DO meter calibratiocnhecks were made between the setsof measurements for each controlor treatment. For each exposure,correlatedDO depletionand time intervala,nd calculatedthe slopeof the regressionlineas the respiratiornate. I EC50: Trimmed Spearman-Karber Analysis (Hamilton, 1977). Sponsor: 3M Company Sponsor Study ID# U1464 4 3.0 RESULTS Test SubstanceToxicity (Pertinendtataare inTable 1) 30-minuteEC50: > 1000 mg/L. 3-h EC,,n: > 1000 mg/L. AScl Corporation/ASci-Duluth EnvironmentalTesting Division Submitted to 3M Co. - 5/98 ASel Study ID# 5010-069 4.0 TEST QA/QC QA/QC Criteria Respirationratesof two inoculum controls must not vary by more than 15 3-h EC50 of referencesubstancemust be between 5-30 mg/L. Test durationmust be 3 h. Data Variationsafter30-minute and 3-h contact timeswere 7% and 14%, respectively. 3-h EC5()was 1.8.4mg/L, indicatingthesludge had normal sensitivity. The testdurationwas 3 h. 5.0 REFERENCES Ham ilton,M.A., R.C. Russo, and R.V. Thurston.1977. Trimmed Spearman-Karber Method for EstimatingMedian LethalConcentrationsin ToxicityBioassays.Environ.Sci.Technol. 11(7): 714-719; Correction12(4):417.1978. Organiza*on forEconomic Cooperationand Development (OECD). 1993. OECD Guidelinesfor Testingo Chemicals.OECD PublicationInformationCenter,Washington, D.C. Sponsor: 3M Company Sponsor Study ID# U1464 5 TABLE 1.0 OECD ACTIVATED SLUDGE RESPIRATION INHIBITION TEST #209 E CALCULATION OF RESULTS Test Substance: U1464 Analyst: DIERKES 30-Minute Exposure Dissolved Ox en Concentrations, /L mg/L 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Tamp,-c R (1) Ra (2) % In BK1 IOOOA -IOOOB 8.5 8.2 7.9 7.7 7.5 7.2 6.8 6.6 6.3 5.9 5.7 8.1 7.8 7.5 7.2 6.9 6.6 6.3 6.0 5.7 5.3 5.0 8.4 8.1, 7.9 7.6 7.3 7.1 6.8 6.5 6.2 5.9 5.6 21.2 21.2 21.2 0.5648 0.6194 0.5600 0.5196 0.5698 10.5152 BK2 8.5 8.2, 7.9 7.6 7.3 7.0 6.6 6.3 6.0 5.7 % diffincontrols(4): 6.8 EC50 (95% Cl): > 1000 mg] 8 0.6303 0.5562 RaCm (5): 0. 3-Hour Exposure Dissolved Oxygen Concentrations,mg/L mg/L 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Tamp, c R (1) Ra (2) %In BK1 1OOOA IOOOB 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 7.9 7.6 7.4 20.2 0.2752 0.2714 8.7 8.6 8.5 8.3 8.1 7.9 7.7 7.4 7.3 7.1 6.8. 20.2 0.4036 0.3981 9.0 8.8, 8.7 8.5 8.2 8.1, 7.9 7.7, 7.4 7.2 7.0 20.1 0.4109 0.4081 BK2 1 8.7, 8.6, 8.4 8.3 8.1 7.9 7.6, 7.5. 7.4 7.31 % diffincontrols4): 14.4 EC 50 (95% Cl): > 1000 ma/ illR- Oxygen uptake rate(mg 02/L-min) (2) Ra- A adjustedto 20*C= (R/o.Exi (3) % Inhibition(=(R&Cm)-RaS/(RaCm)1100 (4) % differenciencontrols:IlRaBkl-RaBk2l/RaCmIlOO(Two controlsmust be within15% of each other.) J5) Mean adjustedA, control-s IRaBk1 + RaBk2f/2 71 21.51 0.3479 10.3134 R aC m (5): 0. 1.072 RaCm- mean adjustedR. controls Bk - blankcontrolS,- sample Tobs = sampletemp JX= Tobs-20*C Sponsor: 3M Company Si-,on-.oSr(tidyID# U1464