Formation of trichloromethane in chlorinated and fresh-cut as a result of reaction with citric acid

PostharvestBiologyandTechnology109(2015)65–72ContentslistsavailableatScienceDirectPostharvestBiologyandTechnologyjournalhomepage:www.elsevier.com/locate/postharvbioFormationoftrichloromethaneinchlorinatedwaterandfresh-cutproduceandasaresultofreactionwithcitricacid$XuetongFan*,KimberlyJ.SokoraiU.SDepartmentofAgriculture,AgriculturalResearchService,EasternRegionalResearchCenter,600E.MermaidLane,Wyndmoor,PA19038,USA

ARTICLEINFOABSTRACTArticlehistory:Received12November2014Receivedinrevisedform8June2015Accepted11June2015Availableonline21June2015Keywords:TrichloromethaneChlorineby-productChlorineCitricacidLettuceOnionsChlorine(sodiumhypochlorite)iscommonlyusedbythefreshproduceindustryintheU.S.tosanitizewashwater,freshandfresh-cutfruitsandvegetables.However,possibleformationofharmfulchlorineby-productsisaconcern.Theobjectivesofthisstudyweretocomparechlorineandchlorinedioxideintrichloromethaneformation,determinetrichloromethanelevelsinchlorinatedwashwater,cut-lettuceanddicedonions,andevaluatethereactionofchlorinewithcitricacid,achemicaloftenusedtoadjustpHofchlorinesolution.Resultsshowedthatlittletrichloromethane( 3mgLà1)wasproducedfromchlorinedioxidesolutionevenatconcentrationsupto200mgLà1comparedwiththetrichloromethanelevel($40mgLà1)insolutionsofchlorinemixedwithlettuceextract.Theformationoftrichloromethanein1Lof100mgLà1chlorinewashwaterincreasedfrom155to284mgLà1afterrepeateduseofthewashwatertowashsixbatchesof100gcutlettuce.Levelsoftrichloromethaneinthewashedcutlettucewereintherangeof14–22mgkgà1,andwerereducedtolessthan8mgkgà1afterbeingrinsedwithwater.Chlorinesolutionusedtowashdicedonionsproducedmuchlesstrichloromethane(32mgLà1)comparedwiththatforwashingcutlettucedespitehigherchemicaloxygendemandandturbidityinthewashwater.Citricacidreactedwithchlorineandproducedtrichloromethane.Over1000mgLà1trichloromethanewasproducedafter30minreactionwithchlorineat22??C,whilelessthan35mgLà1trichloromethanewasproducedwhenNa-phosphatewasusedtoadjustpHofchlorine.Theamountoftrichloromethaneincreasedwithreactiontimeandconcentrationofcitrate.Ourresultsdemonstratedthatformationoftrichloromethaneinwashwaterdependedontypeofcut-vegetables,andcitricacidcontributedtoasigni?cantamountoftrichloromethaneformation.PublishedbyElsevierB.V.1.IntroductionAnumberofoutbreaksoffoodbornediseaseshavebeenlinkedwithfreshandfresh-cutfruitsandvegetablesinrecentyears,promptingconcernsaboutthemicrobialsafetyoftheseproducts(AFF(AllianceforFoodandFarming),2010;Gouldetal.,2013).Washing(withorwithoutsolutionsofsanitizers)iscommonlyusedbytheproduceindustrytoremovedirtandmicroorganismsaswellasforrapidcoolingoftheproducts(Giletal.,2009).Tofurtherreducethepopulationofmicroorganismsintheproducts/waterandtominimizecrosscontamination,disinfectantsMentionoftradenamesorcommercialproductsinthispublicationissolelyforthepurposeofprovidingspeci?cinformationanddoesnotimplyrecommendationorendorsementbytheU.S.DepartmentofAgriculture.USDAisanequalopportunityemployerandprovider.*Correspondingauthor.Fax:+12152336445.E-mailaddress:xuetong.fan@ars.usda.gov(X.Fan).$(sanitizers)areoftenusedinthewashwater.Washingwithsanitizersisoneofthecriticalprocessingstepsforfreshandfresh-cutproduce.ThemostcommonsanitizersusedbythefreshproduceindustryintheU.S.arechlorine-basedcompounds,suchassodiumhypochlorite(chlorine)andchlorinedioxide(ClO2),eventhoughtheef?cacyofthechlorine-basedsanitizersinreducingpopulationofmicroorganismsonfreshproduceisverylimited(1–2logsatthemost)(Beuchatetal.,2004).Easeofuseandrelativelylowcostmakechlorineaverycommonwaterdisinfec-tantthroughoutthefreshproduceindustryintheU.S.Chlorineisusedintheconcentrationrangeof50–200mgLà1,althoughrecentstudies(Luoetal.,2011;Shenetal.,2012)indicatedthatlowerconcentrationsoffreechlorineweresuf?cienttoinactivatepathogenicbacteriainwaterandtoavoidcross-contamination.However,thereisaconcernabouttheuseofchlorineinthefreshproduceindustryandotherindustriesduetopotentialenviron-mentalandhealthrisksassociatedwiththeformationofcarcinogenichalogenateddisinfectionby-products(DBPs)http://dx.doi.org/10.1016/j.postharvbio.2015.06.0090925-5214/PublishedbyElsevierB.V.66X.Fan,K.J.Sokorai/PostharvestBiologyandTechnology109(2015)65–72(?lmezandKretzschmar,2009).ChlorinereactswithorganicmatterandformscarcinogenichalogenatedDBPs,suchastrichloromethane(HuaandReckhow,2007;Singer,1994).PartlyduetothepossiblegenerationofhalogenatedDBPsinthewater,theuseofchlorineinfresh-cutproducewashingisprohibitedinEuropeancountries(VanHauteetal.,2013).ClO2producesfewerpotentiallycarcinogenichalogenatedDBPsandislesscorrosivethanchlorine(HuaandReckhow,2007),andisallowedatamaximumconcentrationof3mgLà1intheU.S.(CFR(CodeofFederalRegulations),2007).Theantimicrobialef?cacyofchlorinelargelydependsonthepHandtheamountoforganicmaterialinthewater,andtoalimitedextent,onthetemperatureofwater(Suslow,2001).AtpHvaluesabove7.5,averylowpercentageofchlorineexistsintheactiveHClOform.AtpHvaluesbelow6.0,off-gas(probablyasdi-andtri-chloramines)mayoccurandirritateworkers(Black&VeatchCorporation,2009).Inaddition,lowpHmayincreaseequipmentcorrosion.Therefore,itisdesirableinthemanagementofchlorinethatthepHofwaterbeadjustedandmaintainedatvaluesof6.5–7.0tomaximizetheHClOlevelandminimizeoff-gas(Suslow,2001).Chlorine(sodiumhypochlorite)solutionatpH6.5iscurrentlythemostcommonsanitizerusedinthefresh-cutproduceindustryintheU.S.(Shenetal.,2012).ToadjustthepHofchlorinesolution,citricacidiscommonlyusedbytheindustry(Suslow,1997;HerdtandFeng,2009;Shenetal.,2012).Maintainingappropriatelevelsofeffectivechlorineinwashingsolutionswithfresh-cutproduceisachallengeduetothereleaseofcopiousamountofjuicesfromfresh-cutproductsintowashwater(Shenetal.,2012).Inaddition,otherorganicmaterialsfromsoilandmicroorganismsmayaccumulateduringproducewashing(Allendeetal.,2008).Replenishingchlorineincludingcontinual?xeddosing,automateddemand-basedinjectionsystems,andmanualdemand-basedperiodicdosingisacommonpracticeinfresh-produceprocessing.Withincreasingorganicloadsoriginat-edfromfreshproduceintherecirculatedwater,andtheneedtomaintainacertainlevelofchlorine,thelevelsofchlorine-by-productsinthewaterwouldincreaseover-time.Asaresult,theproductionoflargeamountsofwastewaterwithhighlevelsofbiologicaloxygendemandandchlorine-byproductsmaybecomeaconcern(?lmezandKretzschmar,2009).Toreducewaterconsumptionandconserveenergyassociatedwithcoolingorheatingduringprocessingoffresh-cutproduce,mostpostharvestprocessesrecirculateusedwater.Theamountofwaterlostduringtransferandtransportoftheproductsisreplacedwithfreshwater,andchlorineconcentrationisconstantlyadjustedtotargetedlevels.Whileinformationaboutchlorineby-productformationindrinkingwaterisavailable(Richardsonetal.,2000;Gopaletal.,2007;HuaandReckhow,2007),thereislimitedresearchabouttrichloromethaneformationinprocesswaterandinthefreshproducethathavebeenwashedwithchlorine(Klaiberetal.,2005;COT(CommitteeonToxicityofChemicalsinFood,ConsumerProductsandtheEnvironment,2006;López-Gálvezetal.,2010;Gómez-Lópezetal.,2013).Klaiberetal.(2005)determinedthattheby-productformationduetochlorinationofminimallyprocessedcarrotswithtapwatercontaining200mgLà1freechlorinewasnegligible(<0.2mgLà1).Therehavebeennostudiesdealingwithaccumulationofchorineby-productsinre-usedchlorinewaterwitha?xedchlorineconcentrationduringwashing.Itisunknownwhethercitricacid,whichiscommonlyusedforpHadjustment,wouldaffecttheformationoftrihalomethanes.Theobjectivesofthepresentstudywerethereforetoinvestigatetheformationoftrichloromethaneinwashwater,cutlettuceanddicedonions,tocomparesodiumhypochloritewithchlorinedioxideinproducingtrichloromethane,andtoevaluatetheimpactofcitricacidontrichloromethaneformation.2.Materialsandmethods2.1.ChemicalsTrichloromethane(chloroform,highpurity)waspurchasedfromAmericanBurdickandJackson(Muskegon,MI).Citricacid(99%),1-bromo-3-chloropropane(99%)andsodiumphosphatewerefromSigma–Aldrich(St.Lois,MO).Sodiumcitrate,NaOHandhydrochloricacid(36.5–38%)werefromJ.T.Baker(Phillipsburg,NJ).UltrapurewaterfromaBarnsteadE-pure(Dubuque,IA)waterpuri?cationsystemwasusedaswashwaterandforpreparingsolutions.Thedeionizedwaterinthelaboratorywasfoundtocontainabout2mgLà1trichloromethane.Thepuri?edwatercontainedlessthan0.5mgLà1trichloromethane.Solutionsofsodiumhypochloritewerepreparedbydiluting8.25%sodiumhypochlorite(Clorox,Oakland,CA).ThepHofthesolutionswasmaintainedat$6.5using50mmolLà1Na-phosphatebufferunlessotherwisestated.2.2.PlantmaterialsWholeheadsoficeberglettuce,onions,andfresh-cutproduceincludingiceberglettuce,freshsalsa,coleslaw,shreddedcarrotsandshreddedredcabbagewerepurchasedfromlocalsuper-markets.Theingredientsforfreshsalsaweretomato,cucumber,greenpepper,redpepper,redonion,cilantro,limejuice,tomatojuice,garlic,poblanopepper,serranopepperandsalt.2.3.EstablishmentoftrichloromethanestandardandanalysisoftrichloromethaneAseriesoftrichloromethaneand1-bromo-3-chloropropane(asaninternalstandard)concentrations(0–500mgLà1),preparedin7mLglassvials,wereequilibratedat35??Conaheatingblockfor25minbeforethecarboxen/polydimethylsiloxanesolidphasemicroextraction(SPME)?ber(85mmcoating,Stable?ex,Sigma–Aldrich)wasinsertedintotheheadspaceofthevialsthroughPTFE-facedsepta.Previousstudiessuggestedthatcarboxen/polydime-thylsiloxane?berwasthebestoptionforanalysisoftrihalometh-ane,particularlyfortrichloromethane(Allardetal.,2012;Antoniouetal.,2006;SanJuanetal.,2007).TheSPME?berwasloweredintotheheadspaceofthevialsfor20min.Duringincubationandabsorptionperiod,thesamplesinthevialsweremixedusingaminimagnetstirbarinsideofthevialonastirrer/heaterataspeedof11.7sà1(modelnumberSP133835,BarnsteadIntern.,Dubuque,IA).The?berwasretrievedfromthevialsandinsertedintotheinjectionport(temperature:250??C)ofgaschromatograph(GC)withadesorptiontimeof4min.SeparationwasachievedusingDB-5MS(30m,0.32mmIDand1mm?lmthickness)capillarycolumn(AgilentTechnology,SantaClara,CA)coupledwithanAgilent6890GCand5973MSD.Column?owwassetataconstant?owof1mL/min.Temperature??programforoven??was40??Cfor1min,increasedto70Cat5??C/min,thento250Cat25??C/minandheldat250??Cfor6min.MSsourcetemperaturewas230??C.Massspectrawererecordedinfullscanmode.Theidenti?cationoftrichloromethaneandtheinternalstandardwasbasedontheretentiontimesoftheauthenticcompoundswiththeassistanceoftheNISTversion02library.Inaddition,them/z83and85ionsandtheratioof83/85wereusedforthecon?rmationoftrichloro-methanewhilem/z158and77ionsandtheratioof158/77wereusedforthecon?rmationof1-bromo-3-chloropropane.Ions83and158wereusedasthequanti?ersfortrichloromethaneand1-bromo-3-chloropropane,respectively.TheeffectofsaltadditionontheSPMEextractionef?ciencyoftrichloromethanewasstudied.Additionofsaltatamountsupto0.5kgLà1didnotsigni?cantlyincreaseextractionef?ciencyofX.Fan,K.J.Sokorai/PostharvestBiologyandTechnology109(2015)65–7267trichloromethanefromaqueoussolutions.Onlyabout11%ofincreasewasobservedat0.5kgLà1NaClcomparedtothatwithoutsalt.Therefore,nosaltwasaddedtothevialforthesimplicityoftheprocedure.Usageofstirringduringextractionincreasedtheextractionef?ciencybyabout7%.Thedetectionlimitoftrichloro-methaneinwaterwas1mgLà1,andinproducewas4mg/mLduetothedilutionofsamples.2.4.ComparisonsoftrichloromethaneformationfromchlorineandchlorinedioxidereactingwithlettucejuiceClO2wasproducedaccordingtoamethodasoutlinedinStandardMethodsfortheExaminationofWaterandWastewater(APHA(AmericanPublicHealthAssociation),1998)withminormodi?cation(Kingsleyetal.,2013).Chlorinedioxidecollectedin1LcoldwaterwasmeasuredusingtheDPDmethod(HachCo.,Loveland,CO)withadditionof0.1mL10%glycinetocompensateforinterferenceofCl2andotherchlorineproducts.Lettucejuice(extract)waspreparedbyhomogenizing40giceberglettucepiecesin200mLpuri?edwaterusingaVirtishearhomogenizer(Virtis,Gardner,NY)ataspeedsettingof70for2min.Thelettucejuicewas?lteredthrough2layersofcheesecloth.Thejuicewasthenstoredatà80??Cforfutureuse.Chemicaloxygendemand(COD)ofjuicewasmeasuredafterdilutionusingtheHach8000digestionmethod(Loveland,CO).TheCODlevelofthejuicewasabout3070mgLà1.Toeachvialcontaining0.9mLlettucejuice,0.1mLchlorinesolutions(pH6.5)wereaddedtoachieve0,50,100,150,200and250mgLà1targetedchlorineconcentrationsinthemixturesifpreparedinwater.Theconcentrationsofchlorinemaybehigherthanthosecommerciallyusedbysomefreshproducecompanies.Similarly,aseriesofClO2concentrations(0–200mgLà1ifinwater)wasprepared.Tobecomparablewithchlorine,weusedthesamelevelsofClO2asforchlorineeventhoughthelevelswereabovethepermittedlevelintheU.S.Internalstandardwasaddedtoeachvialwiththe?nalconcentrationof100mgLà1.Afterreactionof5minat22??C,thetrichloromethaneinthesolutionsweremeasured.ThelevelsoffreechlorineandClO2werealsomeasuredusingtheDPDmethods.2.5.FormationoftrichloromethanefromwashwaterandcutlettuceIceberglettucewascutinto$2.5?2.5cmpieces,and100gofcutlettuce,takenatrandom,wasplacedinto1L100mgLà1freechlorine(pH6.5).Themixturewasstirredfor1minbeforethelettucepiecesweretakenout.Theamountofwaterandthelevelofchlorineinthesolutionwerere-adjustedto1Land100mgLà1byaddingpuri?edwaterandstockchlorinesolution,respectively.Thetrichloromethaneinthelettuceandchlorinewashsolutionafterlettucewashwasmeasured.Then,100gofthesecondbatchofcutlettucewasaddedtotheadjustedchlorinesolution.Theprocedurewasrepeatedsixtimestosimulatethecommercialpracticeinwhichthewashwaterwasusedcontinually(afterwateradditionandchlorineadjustment)totreatlettuce.Trichloromethanelevelwasmeasuredineachbatchoflettuceandwashsolution.Chlorinelevels,CODandturbiditywerealsomeasuredaftereachsampleaddition.Turbidityinthesolutionwasmeasuredusingtheabsorptometricmethod(Hachmethod8237)andreportedasFormazinAttenuationUnit(FAU)equivalent.Afteraportionofthelettuce(40g)thathadbeenwashedwithchlorinesolutionwasusedfortrichloromethanemeasurement,theremainingcutlettucewasrinsedin300mLpuri?edwater.Therinsewaterandlettucewerethenmeasuredfortrichloromethane.Trichloro-methaneinthelettucewasmeasuredbyhomogenizing40glettucein160mLpuri?edwaterusingaStomacher400Circulator(Seward,Worthing,UK)at3.8sà1for2min(López-Gálvezetal.,2010).Theinternalstandards,1-bromo-3-chloropropane(15mgLà1?nalconcentration),wasadded.Thehomogenatewas?lteredthroughthebuilt-in?lterinthebagandanalyzedfortrichloro-methaneusingtheSPME–GC–MSDmethod.2.6.FormationoftrichloromethanefromwashwateranddicedonionsDicedwhiteonions(100g)preparedfreshlyfromwholeonionswereaddedinto0,25,50,100,150and200mgLà1of1Lchlorinesolutions(pH6.5),andstirredfor1minat22??C.Thedicedonionsweretakenoutandmeasuredfortrichloromethane.Trichloro-methane,freechlorinelevelandCODwerealsomeasuredinthechlorinesolutionsafterdicedonionswereremovedfromthesolution.Theprocedurewasrepeatedsixtimesasdescribedforcutlettuce.Internalstandard,1-bromo-3-chloropropane(15mgLà1?nalconcentration),wasaddedfortrichloromethaneanalysis.2.7.TrichloromethaneformationfromreactingwithcitricacidpHofthechlorinesolutions(100mgLà1)wasadjustedtopH6.5withcitricacidorbufferedwith50mmolLà1Na-phosphate.Theamountof1%citricacidneededtoachievepH6.5waspredeterminedinpreliminaryexperiments(?nalcitricacidinthesolutionwasabout0.5mmolLà1).Thechlorinesolutions(0.8mL)bufferedwithcitricacidandNa-phosphateweremixedwith0.1mLlettucejuiceorpuri?edwater.Theinternalstandard,1-bromo-3-chloropropane,wasaddedtoa?nalconcentrationof150mgLà1.After5and30minat22??C,0.1mLof5%sodiumthiosulfatewasaddedtoneutralizechlorine.Duringthe5minincubation,thesolutionswerestirredataspeedsettingof1onastirplate(Model120mR,FisherScienti?c,Nepean,Ontario,Canada).ThetrichloromethaneinthesolutionswasmeasuredusingtheSPME–GC–MSDmethod.Tostudytheeffectofcitrateconcentrationontrichloromethaneformation,differentconcentrations(1,2.5,5.0,7.5and10mmolLà1)ofNa-citratesolutionswithpH6.5wereprepared,andpHwasadjustedwithNaOH.Then0.8mLoftheabovesolutionswasaddedto7mLvialsfollowedbyadditionofstockchlorinesolutiontoreachthe?nalconcentrationof100mgLà1freechlorine.Puri?edwaterwasaddedtoeachvialtoreachthe?nalvolumeof0.9mLineachvial.Thevialsweresealedusingseptaandscrewcaps,andsetonastirplate(Model120mR,FisherScienti?c)ataspeedsettingof1at22??C.After30minreaction,0.1mL5%sodiumthiosulfatewasaddedtoquenchthechlorine.Trichloromethanewasthenanalyzed.Inaseparateexperiment,theeffectofreactiontimewasevaluated.Na-citrate(2.5mmolLà1,0.8mL)wasmixedwith0.1mLchlorine(?nalconcentration:100mgLà1)andincubatedat22??Cfor5,10,20and30minbeforeadding0.1mL5%sodiumthiosulfate.Trichloromethanewasthenmeasured.2.8.SurveyoftrichloromethaneincommercialproductsFortygramsofshreddediceberglettuce,freshsalsa,shreddedredcabbage,shreddedcarrots,andcoleslawpurchasedfromlocalsupermarketsweretakenfromtheirpackagesandanalyzedfortrichloromethaneasdescribedearlier(Section2.5).Theseproductswerechoseneitherbecauseoftheirhighvolumesofconsumptionor?nelycutpiecespermasswhichmayhavehighpotentialofleachingorganicmaterialsfromtheproductsduringprocessing,andhigherchanceoftrichloromethaneaccumulation.2.9.StatisticalanalysisTheexperimentswereacompletelyrandomizeddesign.Forthecutlettuceanddicedonionwashexperiments,eachheadof68X.Fan,K.J.Sokorai/PostharvestBiologyandTechnology109(2015)65–72iceberglettuceor2onionsservedasareplicate.Allexperimentswererepeatedfourtimes.Eachreplicatedexperimentwasconductedonseparatedays.DatawassubjectedtoSASanalysis(version9.2,SASInstitute,Cary,NC)usingthegenerallinearmodel(GLM)procedure.Thesigni?canceoftreatmenteffectwasseparatedusingtheleastsigni?cantdifference(LSD)procedure.Onlysigni?cant(P<0.05)resultswerediscussedunlessstatedotherwise.3.Results3.1.ComparisonoftrichloromethaneformationfromchlorineandchlorinedioxidereactingwithlettucejuiceTrichloromethaneformationfromchlorinereactingwithlettucejuiceincreasedlinearly(R2=0.99)withincreasingchlorineconcentration(Fig.1A).Therewas21mgLà1oftrichloromethaneformationforevery100mgLà1increaseinchlorineconcentration.Lowlevelsoftrichloromethanewereformedinthechlorinedioxidesolution(Fig.1B).Evenat200mgLà1ofchlorinedioxide,onlyabout3mgLà1oftrichloromethanewasproduced.BothfreechlorineandClO2levelsinthemixturefellbelow1mgLà1aftermixingwithlettucejuice,suggestingthattheorganicloadinthelettucejuicedepletedalmostallthechlorineorClO2.3.2.TrichloromethaneformationincutlettuceandchlorinesolutionChlorine(1L,100mgLà1)solutionwasusedtowashsixbatches(100geachbatch)ofcutlettuce.CODinthesolutionsincreasedFig.1.Formationoftrichloromethanefromchlorine(A)orchlorinedioxide(B)reactingwithlettucejuiceasafunctionofchlorineorchlorinedioxideconcentration.Thereaction(5min)tookplaceat22??C.Verticalbarsrepresentstandarddeviations(n=4).500A400)1-L g300m( DOC200100500B40)uaf(30 ytidibru20T1001 2 3 4 5 6Number o f lett uce b atc hFig.2.Changesinchemicaloxygendemand(COD)(A)andturbidity(B)ofchlorinesolutionusedtowashcutlettuce.Uptosixbatchoflettuce(100g)wasaddedto1Lchlorinesolution(100mgLà1).CODandturbidityweremeasuredafterremovalofeachadditionalbatchesoflettuce.Verticalbarsrepresentstandarddeviations(n=4).linearly(R2=0.99)withtherepeateduseofthesolutionforwashinglettuce(Fig.2).Afterwashing6batchesofcutlettuce,theCODlevelreached370mgLà1inthechlorinewashwater.Similarly,theturbidityincreasedlinearly(R2=0.99)withtheamountoflettuce.TheincreaseinCODandturbidityindicatedthatorganicmaterialsuchascellcontentofcutlettuceleakedoutintothechlorinesolution,andtheleakedmaterialreactedwithchlorinetoformtrichloromethane.Thetrichloromethanelevelinthechlorinatedwaterincreasedwithincreasingbatchesofcutlettuce(Fig.3A).Frombatch1to4,thelevelsoftrichloromethaneinthechlorinewaterincreasedfrom155to278mgLà1.Nosigni?cantincreaseintrichloromethanewasfoundafteranadditionalbatchoflettuce(278–284mgLà1).Thetrichloromethanelevelsinthechlorine-washedlettucewerefrom14to22mgkgà1,lessthan1/8ofthelevelsinchlorinesolution(Fig.3B).Afterthechlorine-washedcutlettucewasrinsedwithpuri?edwater,thetrichloromethanelevelwaslessthan8mgkgà1(Fig.3),thetrichloromethanelevelwaslessthan8mgkgà1(Fig.3).Trichloromethanelevelinthewaterusedtorinsethelettucewasintherangeof2–4.5mgkgà1(Fig.3).3.3.FormationoftrichloromethaneinchlorinesolutionanddicedonionsTheCODinthechlorinesolutionsthathadbeenusedtowashdicedonionswasabove1400mgLà1regardlessoftheconcentra-tionofchlorineinthesolution(datanotshown).Freechlorinelevel