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Study Plan aerobicac soil/sedim fluorochert m --. -- StudyDesigns- 3M MicrobiaMletabolismProgram Goal:To optimizeconditiontshatpermitenrichmentofmicrobial populationcsapableofmetabolizinfgluorochemicals. A. Introduction The programand testsystemsdescribebdelowoffear widerangeofconditions designed toenrichformicrobialpopulationsthatcan metabolizefluorochemicals. Even though these systems are fairlycomplex and requiresignificant maintenance,they are designed primary as "screening"systems to assess if microbialmetabolism of individuaflluorochemicalcsan occur. At the screening phase, allpossiblecontrolsystems, multiplereplicatese,tc.can not (and should not)be includedbecause ofthe number of analyticaslamples generated and the effortrequiredto serviceeach testsystem. Because of the complexityof many fluorochemicalsm,etabolism may be limitedor notoccur inmost ofthe systems. Ifmetabolism is seen in screeningsystems,they willserve to guide more detailedmetabolism studiesunder a wider range oftestconditions. The key to a successfulmicrobialselectionor enrichmentprogram (i.eo.ne that maximizes the chance for enrichment of those organisms capable of metabolizingfluorochemicalsi)softenbased on providingthe following: - Use ofnaturalecosystems as the basisforenrichment - Use of naturalnutrientsfrom those ecosystems with supplemental traceminerals,co-factorasnd vitamins - Continualintroductioonfnew microbesfrom differenntaturalsources - Periodicreplenishmentof naturalnutrientswithoutdiluting-outthe speciesbeing enriched. - Providinga realistitcimeframe to permitenrichmentand acclimation - Protectionofmicroorganisms from toxicproductsor metabolitesby use of low substrateconcentrationsr,eplenishmentof nutrientsb,alanced medium (C:N:P,etc.)p,roperpH and provisionof a protectivseurface for growth (vermiculate,sand, soil,activated C, diatomaceous earth, etc.) - Enrichmentinmore concentrate(dhigherbiomass& testmaterial conc.)systems and examinationof biodegradationin more dilute systems. - Separationof systems selectiveforfastgrowing (zymogenous) and slow growing (autochthonous)species. Note that throughout the scheme outlinedbelow, we have attempted to incorporatethe principleosutlinedabove for enrichment. Most of what is describedhas been gained from our background in microbialecology and practicaelxperiencewith isolatioannd cultureofunique microorganisms. 3M Biodeg.Program B. AerobicEnrichmentSystems- GeneralDiscussion Three aerobicsystemsare proposed:a sewage treatmenbtased systemto selectforfastergrowing species (Zymogenous), a soilbased system to selectfor slower growing species (Autothonous)anda pure culturesystem forexamining specificmetaboliccapabilitie(sCytochrome P-450 monooxygenase). 1. Sewage treatment based system Secondary aerobicsewage treatmentsystems includeprimarilayctivatedsludge (AS), rotatingbiologicaclontacters(RBC) and tricklinfgilte(rTF) wastewater treatmentplants. We also includeas partof sewage treatmentsystems the immediate receivingwater and top-most floc(sediment)on the banks of the receivingstream or pond (tertiartyreatment!)T.wo sources of activatedsludge willbe used, Wareham and New Bedford,and one RBC plant,Bridgewater,will be sampled. Wareham and Bridgewaterare primarildyomestic wastewater treatmentplants(VVRP)ofabout 1MGPD flow. New Bedford isa new 30 MGPD WTP which receiveslightindustryand domestic waste (approximately20:80). The New Bedford planthas been shown to have greatermetaboliccapabilities forcertainclassesof chemicals. The receivingwater system, siteforsediment collectionw,illbe downstream from the Wareham STP outfalls,ince New Bedford dischargesfarout intothe Bay. There are no TF plantsinthe nearby area,but microbialgrowth from the BridgewaterRBC plantshould mimic attachedgrowth from a TF plant.Fresh raw sewage from the Wareham plantwillbe collectedon a weekly basisand used as the primarygrowth medium. DOC from each of these plantsistypicallbyelow 40 mg/L, so supplementationwithactivatedsludge extractto achieve levelsof -100 mg C/L willbe done. AS extractis prepared from driedBridgewaterAS by mixing one partdriedsludge with4 partswater and autoclavingfor30 min. Aftercoolingthe mixtureissettledand filteretdhrough a fiberglasfsilterT.OC ofthisextractisinthe range of3500 mg/L. Test vesselsforacclimationwillbe baffled300-mL Erlenmeyer (DeLong neck) flaskscontaining-100 mL ofenrichment medium and sealed witha polyurethane plug. Naturalsewage willbe supplemented with OECD mineralsaltsat the concentrationsspecifiedforthe OECD 301 B test,traceminerals(Trace Minerals Corp. 0.01 mUlOO mL) and yeastextract(5 mg/100 mL). A sandy soilwillalso be added (19/100 mL) to providea surfaceformicrobialgrowth as wellas an additionaslource of microorganisms. This soilwas collectedby American Ag Services,Inc.on 10/28/98and isclassifieads a loamy sand with77% sand, 21% silatnd 2% clay.Itcontains2.5% organicmatterand has a pH of4.9. Activated sludge,RBC sludge and receivingwater sediment (1 g/L each d. wt. basis)will be the primarysource of microorganisms. Test materialwillbe added at a concentrationof 20 mg a.i./L.A blank controlflaskcontaininginoculum but no testsubstance willalso be included.Once per week, media from acclimation systems willbe centrifugedand two-thirdosf the supernatantand 10% of the biomass willbe removed. Fresh medium and testmaterialwillbe added. Additionalfresh biomass (activatesdludge,RBC sludge and sediment at a 3M Biodeg. Program 2 concentrationof 10% of the originacloncentrationw)illbe added and the pH adjustedto -7.0,ifnecessary. Weekly additionof -100 mg C/L from natural sewage and AS extractshould allowthe microbialpopulationto increaseon a weekly basis(perhapsby 50 mg cells/bLased on a 50% growth yieldfrom C). On a weekly basis,testmedium andlo% of the centrifugedbiomass willbe extractedand analyzed for DOC, parent materialand potentialmetabolites. Depending on the testsubstance,foam stoppersmay also be extractedand analyzed. The acclimationprocess willcontinueuntilevidence of testmaterial degradationhas occurred or for a maximum of 8-10 weeks. If microbial metabolism isseen, a more definitivaessay willbe conducted. Assay forratesof degradationand metaboliteformationwillbe conducted in sealed 20 or 40-mL serum bottlescontainingtwo-thirdsof the volume as nutrientmedium, inoculum and testmaterial(Oiomass concentrationsinthe 10-50 mg/L range). Bottle headspace willperiodicallbye assayed for carbon dioxideand volatiloerganics witha Thermoglas 1200 TOC analyzer.Medium willalso be assayed forparent materialand metabolitesvia appropriateLC-MS technology. Priorto initiatioofntesting,each fluorocarbonwillbe examined foritsmicrobial toxicityT.he assay system willconsistof 125-mL Erienmeyer flaskstowhich 50- mL OECD mineralsaltsmedium containing20 mg/L testsubstance and 10 mg/L 14 C-labelledsodium benzoate willbe added. Blank controlswillbe includedand containallingredientesxcept testsubstance. The flaskswillhave a reservoir above the testmedium thatcontains750 @iLO.lN KOH. The assay willbe initiatebdy adding 1.0 mL of medium from the blank controlflask,described above, and sealingthe vesselwitha siliconsetopper. Assay for14C 02 willbe conducted at 4-6 hours and for severaldays (e.g.at days 1, 2 and 4) by removing 100 @tLfrom the KOH reservoirand countingon the LSC. Toxicitywill of 14 be assessed by comparing the rate C02 evolvedfrom thetestsubstanceand controlsystems. 2. Soil/sediment based system Soil/sedimentstudieswillbe conducted in 50-ml testvessels containing20 g of the soil/sedimenmtixture.Individuatlestvesselswillbe sacrificeadt each time interval.Three soilsand two sediments,freshlycollectedw,illbe used as the source ofmicroorganisms.Soilswillbe from severaldiversesources (hardwood forestp,ineforesatnd a rivebrank soil/sedimfernotmBridgewaterm)i,xedand screenedthrougha 2-mm mesh screen.The two sedimentswillbe collected from the Agawam River from areas below the Wareham WTP and at low tide from a brackishsitebehindthetown of Wareham. These sedimentswillbe combined inequalvolumes based on dryweight. Equalamounts of each soil and theone sedimentmixturewilble combined(drywt.basis)supplementedwith traceminerals(1.0mukg, yeastextract(50 mg/kg) and soilextract(100 mg C/kg).Soilextractispreparedfroma richorganicsoilor humus by mixingone partwithfourpartswaterand autoclavinfgor30 minutes.Aftercoolingand filtrattihornougha glassfiberfilteTrO,C isintherangeof500-800 mg C/L. Test 3M Biodeg.Program 3 materialwillbe added at 20 mg/kg. Blank controlsoils/sedimenstystems will alsobe setup and willcontainallingredientesxcept testsubstance. On a weekly basis,individuatlestvesselswillbe sacrificeadnd analyzed forthe particulafrluorocarbon.Soilextractand yeast extractwillbe added as fresh nutrientsand to adjustsoilmoisture. 3. Pure culturesystem Springbornmaintainsa bank ofbacteria,actinomycetesand fungithathave been demonstrated to metabolizecomplex molecules in a manner very similarto mammalian (microsomes)and plantspecies.These specieshave been isolated from naturalecosystems (soilsprimarilya)nd containthe cytochrome P-450 monooxygenase enzyme system. Mammalian systems (liverhepatocytes)are known to rapidlymetabolizeET-FOSE-OH and there is potentialfor these microorganisms todo the same. We have reviewed a portionofour data base consistingof nineteenspecies and ten complex molecules and have selectedthree fungi and one actinomycete based on theirabilittyo metabolizethe greatestrange of molecules. These species are: Cunninghamella echinulataM,ucor circinelloidePsh,anerochaete chrysosporium and Streptomyces griseus. These cultureswillbe grown in complex media in 250-mL shake flasksin the presence of the chemical of interesftorseven days. Followinggrowth,cellswillbe separated from brothand both willbe extractedand examined forthe parent materialand metabolites. Also, since certainculturesmay not readilymetabolizedifficutlot degrade substances,restingcellsuspensions (washed to remove complex organics growth nutrientsm)ay be examined for theirabilittyo transformthe test substance. Ifcertainculturesare observed to metabolizethe chemical of interestd,efinitivsetudieswillbe conducted to establishkineticsof degradation and to identifmyetabolites. C. Aerobic Enrichment Systems - Test System Summaries 1. Sewage Treatment Acclimation System (Zymogenous sp.) a. Test vessels - 300-mL baffledErlenmeyer flasks,polyurethane & s.s. stoppers. b. Test medium - Naturalsewage from one A.S. plant+ OECD mineralsalts+ traceminerals& yeastextract(50 mg/L). Supplementationwillbe done withA.S. extractto achieve-100 mgll C. Also, an appropriatesurfacefor microbial growth (sandy soil)T.otalvolume 100 muflask; incubationat220C. c. lnoculum - A.S from Wareham & New Bedford,attachedmicrobialgrowth (periphytonf)rom BridgewaterRBC STP, receivingwater sediment (flocf)rom Wareham STP (each@1000 mg/L d.wt.solids). 3M Biodeg.Program 4 d. TestMateria-lTestsystemwilble dosedat20 a.im.g/L.Blankcontrolwsith no testmaterialwillalso be includedand flaskswillbe somewhat larger(5001000 mL) to accommodate analyticanleeds forfivetestsubstances. Additional flasks,identicailn every way except thatthey containno biomass willalso be included as analyticalcontrols. Potentialtoxicitoyf testmaterialwillbe establishedvia respirometry(3-4day study ina miniaturizetdestsystem using 14C-benzoate).Sterilecontrolsmay be added based on analyticarlequirements. e. Enrichment Conditions - Once per week, testmedium willbe centrifuged and 2/3rdof the supernatantand 10% ofthe biomass willbe removed. Medium removed willbe replacedwith freshmedium and testmaterial.An additional amount of A.S.,RBC microbes and sediment equivalentto 10% of the original amount willalso be added weekly. Medium pH willbe adjustedto-7.0 withO.lN HCI or NAOH ifnecessary. Enrichmentwillcontinueforat least8-10 wks. f. Analysis - On a weekly basis,test medium andlo% of the centrifuged biomass willbe extracted(ifappropriatea)nd analyzed forparent& metabolites. Samples not assayed immediatelywillbe refrigeratedC.arbon analysiswillbe conducted on supernatants.Analysisof foam plugsmay be includedbased on the testmaterial.Analyticalresultswilltriggerneed fordefinitivtestingas indicatedbelow. g. Test Material Requirements - Enrichment systems willrequire16.0 mg of activeingredien(ta.i.[)2.0mg day 0, 1.5 mg weekly for9 weeks, and 1.0mg for toxicitayssay]. Definitivaessays willrequireabout 10 mg [setup 16 vialswith20 mL medium]. h. DefinitiveAssays - For enrichment systems showing positivesigns of biodegradation,metabolism profilewsillbe developed in 20 or 40-mL serum vials.Vialswillcontain2/3ravolume as nutrientmedium and inoculum at a concentrationfrom the acclimatedenrichment medium (10-50mg solids/Lp)lus testmaterial.Headspace willbe assayed on days 0, 1,3, 7, 14, 21 and 28 for C02, volatiloerganicsand perhaps 02. Medium willbe assayed forparentand metabolites.ondays 0,7, 14,21 & 28. 2. Soil Based System (Autochthonous sp.) a. Test vessels - 50-mL culturetubes withpolyurethanestoppers. b. lnoculum - Three soilsand two sedimentscollectefdrom varioussitesinthe Cape Cod area willbe used. Soilswillbe sievedthrougha 2 mm screen and mixed. Sediments willbe collectedat low tideand mixed on an equal d.wt.basis withthe soils.Ten grams ofthe soilmixturewillbe added toeach culturetube. c. Test Material- Test materialwillbe added at20 mg/kg. Blank controlswith no testmaterialwillalsobe included.Need forabioticontrolswillbe dependant 3M Biodeg.Program 5 on testmaterial.Potentialtoxicitoyf testmaterialwould not need to be reestablishedviarespirometrysinceitwillbe done inthe sewage system. d. Enrichment Conditions - Soils willbe adjusted to approximately 75% moisture holdingcapacity(MHC) withsoilextractprepared from a richorganic soilor peat. MHC isdetermined on driedsoilsamples placed ina column and passing water through the column untilbreak-through.Additionasloilextract, tracemineralsand yeast extractwillbe added on a weekly basis.The enrichment willcontinueforat least8-10 wks. e. Analysis - On a weekly basis,blank tubes and tubes containingtestmaterial willbe sacrificede,xtractedand analyzed forparent& metabolites.Based on analyticarlesultsf,reshsamples of the same soil/sedimenmtixturewillbe redosed withtestmaterialand kineticsofdegradationwillbe monitoredover time. f. Test MaterialRequirements - Enrichment systems willrequire6 mg [0.4mg foreach tube times enough tubes for 10 weeks, and 2 mg fortoxicitayssay]. Definitivaessays willrequireabout another6 mg. 3. Pure Culture Systems (Cytochrome P-450 Enzyme System) a. Test vessels - 250-mL Erienmeyerflasks,polyurethane& ss stoppers. b. Test medium - Growth willbe conducted in complex microbiologicamledia (soybean grits-glucosfeo)rallspecies.Totalvolume -60 mL /flaski;ncubationat 260C. C. Microorganisms - Four species [Cunninghamella echinulata, Mucor circinelloides, Phanerochaete chrysosporium and Streptomyces griseus] will be evaluatedfortheirabilittyo metabolizetestmaterials. d. Test Material- Test materialwillbe added at 20 mg/L. Blank controlswith no testmaterialwillalsobe included. e. Growth Conditions - Activelygrowing cultureswillbe transferrefdrom agar slantsto freshmedium (Stage 1). Stage 1 cultureswillbe grown for3-6 days and 6.0 mL willbe transferretdo 60-mL offreshmedium containingtestmaterial (Stage 2). Stage 2 cultureswillbe grown forseven days untilthe earlyto midstationaryphase of growth. Cells willbe separatedfrom broth and each examined analyticallyB.ased on resultsfrom growing cellsuspensions,cells may also be grown in complex medium withouttest materialto the early stationaryphase, centrifugedand washed. Washed cellswould then be resuspended in mineralsaltsmedium containingtrace minerals,vitaminsand testmaterial(restingcellsuspensions). After72 hr,cellsand brothwillbe separated foranalysis. 3M Biodeg.Program 6 f.Analysis- Cellsand brothareextracte(dattimeszeroand aftesrevendays) and analyzed forparent& metabolites.Foam stoppersmay be analyzed based on potentiavlolatiliotfytestmaterialsor degradationproducts. g. Test MaterialRequirements - Assays willrequire9.0 mg forgrowing cells and an additiona9l.0 mg ifrestingcellsare studied.. h. DefinitiveAssays - Establishkineticsof degradation and metabolite identification. D. Test matrix Tables 1 and 2 summarize the proposed testsampling matrix.Table 1 displays the samples under study and those to be assayed on a weekly basis. Fivetest substances (Group 1) willbe examined first.Additionatlestsubstances (Group 2) as wellas testsystems willbe added over time. Table 2 presentsmore detail of the actualnumbers of samples foreach testsubstance on a weekly basis.A "0"inTable 2 indicatesthe day of initiatifoonrthatparticulatrestmatrixand that no sample was taken. Pure culturestudiesare sampled on day "0". E. Additional test matrices Additionatlestmatriceswillincludeanaerobicsludgesand sedimemts as wellas soiland sewage sources providedby 3M. These willbe added tothistextinthe near future.Also,the need toincorporatesterilceontrolmatriceswillbe evaluatedas analyticarlesultsare received. Anaerobic Enrichment Systems A. Sewage Treatment Acclimation and Assay Systems Test vessels - 160-mL serum boftleswithcrimped butylrubber or Teflon@ stoppers.Assay based on OPPTS 835-3400 and subsequent modificationbsy Birchet aL At leastthreeboftleswillbe set up foreach testmaterial. Test medium - OECD mineralsalts+ traceminerals& vitaminsand 0.0002% resazurinwillbe added to A.S. extractT.otalvolume -100 mL/boftleafter inoculum addition. Inoculum - Mixtureofanaerobic digestersludge from Wareham & New Bedford STP's willbe mixed equallyunder conditionsto maintainanaerobicityand added tothe testmedium (10% v/v).Incubationwillbe at35'C. Test Material- Additioninthe 30-50 mg/L (carbonbasis)range. Blank controls withno testmaterialalso included. 3M Biodeg.Program 7 EnrichmentCondition-s On a weeklybasisa,dd additionaacltivatseldudge extractthroughthe septum whilemaintaininganaerobicconditions.Continue enrichment forat least8 wks. Analysis - On a weekly basis,remove 5 mL ofmixed liquorfrom the test vessels,centrifugetestmedium, extractand analyze forparent& metabolites. Methane and carbon dioxidewillalsobe followedinthe headspace viaGC. Test MaterialRequirements - Enrichment systems willrequire22.5 mg [50 mg/L C - assume 75 mg/L testmaterial,300 mL medium = 7.5 mg per vessel]. DefinitiveAssays - Definitivteestsystems willbe used toevaluatemetabolic ratesinmore detailand willbe designed based on resultsfrom the inititaelst bottles.Definitivaessays willprobablycontainlessbiomass tofacilitate analyticamleasurements. B. Soil/Sediment Acclimation and Assay Systems Test vessels - 160-mL serum bottleswithcrimped butylrubberor Teflon@ stoppers.Assay based on OPPTS 835-3400 and work ofSufliteataL At least three bottleswillbe setup foreach testmaterial. Test medium - OECD mineralsalts+ traceminerals& vitaminsand 0.0002% resazurinwillbe added to an aquifersediment extractT.otalvolume -100 muboffle afterinoculumaddition. lnoculum - Inoculumwillbe obtainedfrom an anoxicreceivingwater or aquifer. Water and sedimentwillbe collectedunder conditionsto maintainanaerobicity and added to thetestmedium (-5 g d.wt.basis).Incubationwillbe at220C. Test Material- Additioninthe 30-50 mg/L (carbonbasis)range. Blankcontrols with no testmaterialalsoincluded. Enrichment Conditions - On a weekly basis,add additionaalquiferextract(5 mL) throughthe septum while maintaininganaerobicconditions.Continue enrichmentforatleast8 wks. Analysis- On a weeklybasis,remove 5 mL ofmixed liquofrromthetestvessels (anaerobicallyc)e,ntrifugteestmedium, extractand analyzeforparent& metabolites.Methane and carbon dioxidewillalsobe followedinthe headspace viaGC. Test MaterialRequirements - Enrichmentsystemswillrequire22.5 mg [50 mg/L C - assume 75 mg/L testmaterial3,00 mL medium = 7.5mg pervessel]. DefinitivAessays - Definititveestsystemswillbe used toevaluatemetabolic ratesinmore detaialnd willbe designedbased on resultfsromtheinititaelst 3M Biodeg.Program 8 I bottles.Definitivaessays willprobablycontainlessbiomass tofacilitate analyticamleasurements. I 3M Biodeg.Progrwn 9 Table1. 3M TestMatrix CleanSyste(mM) Product 0 1 2 3 4 6 8 10 Date 4/5 4/12 4/19 4/26 5t3 5/17 5124 5/31 BlankControl 0 3 5 5 5 6 7 8 FC-1 395 001 1 1 11 1 AO Foamer 01 1 1 1 1 1 1 54F 001 1 1 1 1 1 Et.PFOS alc. 0 1 1 1 1 1 1 1 PFOS 0111 111 1 27C 011 1 FX-3539 01 1 PFOSA 01 34E Total 6 10 10 10 12 -f4--f6 Sewage(M+B) Week # 0 1 2 3 4 6 8 10 Date 4/5 4/12 4119 4/26 5/3 -5/17 5/31 6/14 0 6 10 10 10 12 14 16 00222222 02222222 0 022222 0 2222222 0 2 2 2 2 2 2 -2 0222 22 02 2200120200 24 -T8--32 I Soil(S) Week # 0 1 @2 3 4 6 8 10 Date 4/26 5/3 5/10 5117 5124 6f7 6/21 7/5 03556788 01 1 1 11 1 011 1 1 11 1 01 1 1 1 1 1 0111111 1 01111111 01 1 1 1 o111 01 1 0 -6- 1010$12 14 16 16 Product Pure Culture(M+B) G Days #0 & 7 AnaerobicDigestor(B+M) Week # 0 1 2 4 6 8 10 AnaerobicSediment(B+M) Week # 0 1 2 4 6 8 10 BlankControl FC-1 395 AO Foamer 54F Et.PFOS aic. PFOS 027C F2PX7F-C-O33S1653399 PPToFFtOOaSSlAA 34E 4112 4/19 4/26 513 161 321 32116 a8 088 008 8 088 88 88 5t3 &10 5/17 5t3i 6M4 6/28 7/12 0 6 10 101 12 14 14 0 2 21 2 2 2 0 22 2 2 2 2 02 2 2 2 2 0 22 2 2 22 0 22 2 2 2 0222 -2 2 0 5/10 5/17 5/24 6f7 6121 7/5 7/19 0 4 1 101 iO 112 0222 02222 0222 01002 208 2 2 2 00222 02 0 14 14 22 2 -2 22 22 22 2 21 22 a 0- @l2t22040 24 I28 Sterilceontrolmsay be includedbased on analyticarlesults. Legend :M = medium; P = foam plug(optionalB);= biomass/sludgeS;= soilG; 20 20 growingcellsR; = restincgells OtherHighPrioriPtryoducts-Existing(FX-3539,FC-807A,FC-1395,FC-228,FC-845);Now (Ll5655,Ll5656,Ll5660) Standards(PFOSAA, PFOSA, ME.PFOS Aic.) Table2. 3M weeklysamples FTest Mati, 415 Product Group W Th F Clean Blank 1 54F 1 FC-1395 1 AO Foamer 1 ET.PFOS Aic. 1 PFOS 1 Blank 2 27C 2 FX-3539 2 PFOSA 2 35E 2 0* 0 0 0 0 0 4/12 M T W Th F 00 11 1 0 0 Week of: samples) 4/19 M T W Th F 4/26 M.. T W Th F 1 0 5/3 M T W Th F 10 Sewage I Blank 1 54F 1 FC-1395 1 AO Foamer 1 ET.PFOS Aic. 1 PFOS 1 Blank 2 27C 2 FX-3539 2 PFOSA 2 35E 2 0 00 0 0 0 00 222 0 0 2 2 2 22 222 2 2 2 2 2 2 2222 2 2 2 2 2 0 0 -2 2 2 2 2 2 2 2 2 2 20 2 0 Soil itotal ITotal Blank 1 54F 1 FC-1395 1 AO Foamer 1 ET.PFOS Aic. 1 PFOS 1 Blank 2 27C 2 FX-3539 2 PFOSA 2 35E 2 0 0 o 0 0 0 0 3 2 I 1 Fill I Group I I 1 0 1 0 JE 1 0 1 0 1 6 1 6 F-6116 1 6 1 6 1 6T-6 6 6 1 6 F6-T-61 1 6 1121 6 1 1OF67 Group I samples Mon.= FC-1 395, Tues.= 54F, Wed.= PFOS, Thur.= ET.PFOS Aic.,Fri.=AO Foamer I Group 2 1 E@@ 1 0 1 0 10 1-0-F-01 1 0 1 0 1 0 1 0 FO-1 Group 2 samples- Mon.=, Tues.=, Wed.= 27C, Thur.= Fx-3539, Fri.=PFOSA 0 1 0 10 1-0-F-0-1 10 10 16 1 0 = Day zero Table2 (cont.)3M weeklysamples TestMatrix Product Pure Culture Blank 1 54F 1 FC-1395 1 Samples on AO Foamer 1 Day 0 & 7 ET.PFOS Alc. 1 PFOS 1 Blank 2 27C 2 FX-3539 2 PFOSA 2 3SE 2 4/5 W Th F 4/12 m T W Th F 8 8 8 8 Week of: samples) 4/19 M T W Th F 4/26 M T W Th F 888 8 8 -8 8 8 8 88 8 18 88 8 8 5/3 M TW 8 8 Anaerobic Blank 1 Sludge 54F 1 0 FC-1395 1 0 AO Foamer 1 0 ET.PFOS Ale. 1 0 PFOS 1 0 Blank 2 27C 2 FX-3539 2 PFOSA 2 35E 2 Anaerobic Sediment itotal itotal Blank I 54F 1 FC-1395 I AO Foamer 1 ET.PFOS Aic. 1 PFOS 1 Blank 2 27C 2 FX-3539 2 PFOSA 2 35E 2 Group I F161 0 1161 0-F-0@ 116116 IT6-F-Ol-16-1 1 0 1 161 0 11 Group I samples-Mon.= FC-1395,Tues.=54F, Wed.= PFOS, Thur.=ET.PFOS Alc.,Fri.=AO Foamer Group 2 0 1 0 1 0 Fo-To-I Lo-I o I o I-oT--o-IF-0-10 1161 0 1 0 1 Group 2 samples-Mon.=, Tues.=,Wed.= 27C, Thur.=FX-3539,Fri.=PFOSA 1 0 0 120 1__O1 0 1161