MethodArticle
Cleaning and sampling protocol for analysis of mercury and dissolved organic matter in
freshwater systems
AndreaG. Bravoa,*,1,Dolly N.Kothawalab,1,
KatrinAttermeyerb,EmmanuelTessierc,PascalBodmerd,e, DavidAmourouxc
aDepartmentofEnvironmentalChemistry,InstituteofEnvironmentalAssessmentandWaterResearch (IDAEA),SpanishNationalResearchCouncil(CSIC),Barcelona,Spain
bLimnology/DepartmentofEcologyandGenetics,UppsalaUniversity,Uppsala,Sweden
cCNRS/UNIVPAU&PAYSADOUR,InstitutdesSciencesAnalytiquesetdePhysico-Chimiepour l’EnvironnementetlesMateriaux,UMR5254,MIRA,Pau,France
dInstituteforEnvironmentalSciences,UniversityofKoblenz-Landau,Landau,Germany
eChemicalAnalyticsandBiogeochemistry,Leibniz-InstituteofFreshwaterEcologyandInlandFisheries,Berlin, Germany
ABSTRACT
Mercury(Hg),andinparticularitsmethylatedform(methylmercury,MeHg),isahazardoussubstancewiththe potentialtoproducesignificantadverseneurologicalandotherhealtheffects.Enhancedanthropogenicemissions andlong-rangetransportofatmosphericHghaveincreasedHgconcentrationsabovebackgroundlevelsinaquatic systems.Inthiscontext,theMinamataConvention,agloballegallybindingagreementthatseekstoprevent humanexposuretoHg,wassignedandenforcedby128countries,andtodaymorethan90Partieshaveratifiedit.
All these Parties have committed to develop Hg monitoring programs toreport the effectiveness of the convention. For this purpose, we provide a standardized cleaning and water sampling protocol for the determinationoftotal-HgandMeHginfreshwatersatambientlevels.AsHgandorganicmatteraretightlybound, the protocol also describes sample collection for dissolved organic carbon (DOC) concentration and characterizationofdissolvedorganicmatter(DOM)compositionbyfluorescencespectroscopy.Thisprotocol ishighlyusefultonon-expertswithoutapriorbackgroundinHgsamplingandanalysis,andcanserveasauseful basisfornationalmonitoringprograms.Furthermore,thisprotocolshouldhelpincreasequantitativeinventories ofDOC,inorganic-Hg(IHg)andMeHgconcentrationsandDOMcompositioninfreshwater,whichareseverely lackingataglobalscale.
*Correspondingauthor.
E-mailaddress:andrea.garcia@ebc.uu.se(A.G. Bravo).
1 Theseauthorscontributedequallytothiswork.
https://doi.org/10.1016/j.mex.2018.08.002
2215-0161/©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
ContentslistsavailableatScienceDirect
MethodsX
journal homepage:www.elsevier.com/locate/mex
ProvidesastandardizedmethodtocollectwatersamplesforIHg,MeHg,DOCandDOMcompositionfromfreshwater ecosystems.
©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
ARTICLE INFO
Methodname:Samplingprotocolfortotalandmethylmercuryaswellasdissolvedorganicmatteranalysesinfreshwater ecosystems
Keywords:Sampling,Preservation,Mercury,Methylmercury,Water,DOC,Fluorescence Articlehistory:Received1July2018;Accepted2August2018;Availableonline23August2018
SpecificationsTable Subjectarea Chemistry
EnvironmentalScience Morespecific
subjectarea
MercuryBiogeochemistryinAquaticEcosystems
Methodname Samplingprotocolfortotalandmethylmercuryaswellasdissolvedorganicmatteranalysesin freshwaterecosystems
Methodsdetails
Globalstandardizedquantitativeinventoriesofinorganic-Hg(IHg)andmethylmercury(MeHg) concentrationsintheaquaticnetworkarecurrentlylackingbutareundoubtedlyneededtoreportthe effectivenessoftheMinamataConvention.However,determiningIHgandMeHgconcentrationsat low,but environmentallyrelevant levelsremain challenging. Ambient low-levelHg samplesare susceptibletocontaminationfrommanysources,includingimproperlycleanedequipment,improper sample-collectiontechniques,contaminatedreagents,andatmosphericinputsfromdust,dirt,and rain.Cleanproceduresarethusnecessarytominimizecontaminationofsamplesatatypicalambient Hg concentration, which commonly is at the nanogram-per-liter level. Because contamination problemsduringsamplingandstoragehavebeenreportedwidely,muchattentionhasbeenpaidto decontaminationofthematerial,cleaningprocedures,andappropriatestorageofthesamples[1,2].
Rigorouscleaningprocedureshavebeenestablishedforalllaboratorywareincludingsampling vialsandotherequipmentthatcomesintocontactwithsamples.Therearevariousdifferentcleaning proceduresavailable,buttheyallgenerallyincludeseveralacidbathsandintermediaterinseswith Hg-free deionized water or double distilled water, with storage in a designated Hg-free area, preferablysealedincleanplasticbags[1].Theimportanceofthechoiceofthematerialforsampling storage has been previously discussed [2]. For example, water samples can be stored in polytetrafluoroethylene(PTFE,Teflon1),glass orpolyethyleneterephthalate (PET)bottles(see ref [1]andreferencestherein).QuartzorborosilicateglassbottlesminimizethelossofHg(II)[3]andare suitableforMeHgsolutionstorage[4].Forextremelylowconcentrations,asfoundinseawater,PTFE presentsthelowestriskforcontamination[1].However,PTFEbottlesaremoreexpensivethanglass bottlesandnotalllaboratoriescanaffordthem.
Here,weprovideacompletelistofmaterialsneededtoachieveeffectivesamplingofthetypicallylow ambientHglevelsfoundinfreshwaters.Topreventcontamination,wedescribeaprotocolforequipment cleaningandsampleprocessing.AsthereisnoconsensusonreportingIHgandMeHgconcentrationfrom filteredor unfiltered water fractions, we give details for collecting both water fractions:filtered (dissolved)andunfiltered(total).AlthoughdetailedprotocolsforcollectionofwaterwithlowHglevels are available (e.g. https://water.usgs.gov/owq/FieldManual/chapter5/pdf/5.6.4.B_v1.0.pdf), here we describeanupdated,simpleanddidacticprotocolforcollectingsamplesfromfreshwaterecosystemsfor
Hganalyses.Furthermore,asHgbindsstronglytoorganicmatter,weincludeaproceduretocollect samplesfor determiningdissolvedandtotalorganiccarbonconcentration(DOCandTOC)aswellasDOM composition.Consequently,thisprotocolcanbeextremelyhelpfulfornon-expertsinHgsamplingand analysis,aswellasforallHgresearchfocusingontheroleoforganicmattercompositionrelatedtoHg concentrationsinthewater[5–9].It isnoteworthytomentionthatthisprotocoldoesnotprovide guidelinesonthesamplingdesign.Theusersofthisprotocolareencouragedtoplantheirsampling consideringtheoptimalsitelocation(e.g.deepestpointoflake,distancefromshoreline,etc.),depth(e.g.
hypolimnion,epilimnion,etc), number ofreplicates,numberoftimesthe samplingwillbeperformedina year,etc.Itisalsoimportanttohighlightthatbecausethechoiceofmaterialusedtosamplewithisglass (insteadofPTFE),thisprotocolisnotsuitableforseawaters andwaterwithultratraceHglevels (i.e.below 0.01ngL 1forIHgand0.002ngL 1forMeHg).
Fourdifferentwatersampleswillbecollectedfollowingtheprotocoldescribedhere:
11 Total-Hg,IHgandMeHg(unfiltered):250mLamberborosilicateglassbottle.
2 Total-Hg,IHgandMeHg(filtered):250mLamberborosilicateglassbottle.
3 TOC(unfiltered):60mLglassvial.
4 DOC,fluorescencespectroscopy,anionsandcations(filtered):60mLglassvial.
Listofmaterials(Fig.1)
60mL glass vials (EPA, VWR, ref: 548-0156) and screw white caps in polypropylene and polytetrafluoroethylene(PTFE,VWR,ref:548-0871).Halfofthebottlesshouldbelabeledasfiltered, theotherhalfasunfiltered.
250mL amber borosilicate bottles (Boston Round Glass Bottles, Unlined, Standard, Thomas Scientific,ref: D0154-8). (NotethatTeflon1bottles maybealso used,although theyare more expensive).Halfofthebottlesshouldbelabeledasfiltered,theotherhalfasunfiltered.
Filters:Sterivex-HV;0.45mm(PVDF,ref:SVHV01015,Merck).
Syringes:Single-usesyringes,2-piece,NORM-JECT1(50mLVWR).
Hydrochloricacid(HCl,33–36%,UltrexIIultrapurereagent,J.T.Baker,Phillipsburg,NJ,USA).Prepare alsoa2Msolutionwiththisacid.
Tips:50 1000mL(VWR,ref:612–5756).
Nitrilegloves:(VWR,forreferencenumbercheckyoursize).
Coolingbox/Styrofoamboxwithcoolingelements.
Plasticbags(zipsealed)forsamplestorage(Minigrip1bags,PE,VWR,ref:MINIITM024243).
Pipette(50 1000mL)
The abovelist of materialsaccounts for materialsneeded for one sample replicate. Multiply accordinglytosamplesize,basedonthenumberofstudysitesandanadditionalminimumof3blanks.
Cleaning
Ultracleanproceduresarerequiredtopreventcontaminationofsamples.Wearcleanglovesand avoidbreathingdirectlyoverthesamples.
Thecleaningprocedurefortheamberborosilicatebottlesusedtostorewaterandanalysemercury speciesshouldbecarriedoutinaseriesofthreebaths:
AsoapExtran1bathfor1hundersonification*andthenrinsedwithMilliQwater.
2-hsonification*ina10%(v:v)HNO3(Emsure1,Merck,ref:1004562500)bath,conductedasecond timeafterchangingtheacidbathandrinsingwithMilliQwater.
2-hsonification*ina10%(v:v)HCl(Emsure1,Merck,ref:1003172500)bathfollowedbyaMilliQ waterrinse.
*Ifanultrasonicbathsonicatorisnotavailable,leavethelaboratorywareineachofthebathsfor48h.
60mLglassvialsusedtostorewaterandanalyseDOCandTOCshouldbemuffledat550Cfor1h.
Thecleaningprocedureforthescrewcapsusedtostorewaterandanalysemercuryspeciesand DOC/TOCconcentrationshouldfollowthesameprocedureasforthebottles,butthesoapandtheacid bathsshouldbemoredilutedinordertoavoidanydamage:
A soap Extran1 bath diluted 10 times (100mL of soap and 900mL of MilliQ) for 1h under sonificationandthenrinsedwithMilliQwater.
2-hsonification*ina3%(v:v)HNO3(Emsure1,Merck,ref:1004562500)bath,conductedasecond timeafterchangingtheacidandrinsingwithMilliQwater.
Fig.1.Summaryofthematerialneededforonesamplereplicate.
2-hsonification*ina3%HCl(Emsure1,Merck,ref:1003172500)bathfollowedbyaMilliQwater rinse.
*Ifanultrasonicbathsonicatorisnotavailable,leavethescrewcapsineachofthebathsfor48h.
Samplingprocedure
Thisprotocolwasprovidedto30earlycareerresearchers,whowerenon-expertsinHgsampling andanalysis,tocollectsurfaceriverwatersacrossawidegeographicalscaleinEurope[9].
1Putonnitrilegloves.
2Labelallbottles.
3ConditionbottlestocollectUN-filteredwatersamplesforTOCandtotal-Hg,IHgandMeHg:
a Takethe250mLbrownglassbottlelabeled“YoursamplenameMercuryunfiltered”andthe60mL glassvial“YoursamplenameTOCunfiltered”.TheseareyourUN-filteredwatersamples.
b BottleConditioning:fillabitmorethanhalfofthebrownglassbottlelabeled“Yoursamplename Mercuryunfiltered”withsitewatersample.Thecollectionofthesurfacemicrolayershouldbe avoidedbyimmersingthebottleclosed.Shakevigorouslyanddiscardthewaterdownstreamor awayfromthesamplingsite.Repeatthreetimes.Dothesameprocedurewiththe60mLglassvial labeled“YoursamplenameTOCunfiltered”.
4Collectunfilteredtotal-HgandMeHgsample:Fillthepre-conditionedbrownglassbottle“samplename Mercury unfiltered” (not completely, leave about 0.5cm without water). Add 250mL of HCl
(concentrated)acid(UltrexIIultrapurereagent,J.T.Baker).Closethebottlewiththescrewcaplidand shakegently.YouhavenowcollectedUN-filteredwaterfortotal-Hg,IHgandMeHgmeasurements.
Keepitcoldanddarkinthecoolingboxinthefield,andstoreitinthefridgeuntilthesamplesget measuredorsenditimmediatelyfordeliveryifnomoresamplesneedtobecollected(Fig.2).
5CollectTOCsample:Fillcompletely(withoutbubbles,headspacefree)the60mLglassvial“Your samplenameTOCunfiltered”oncemore(afterbottleconditioning).ThisisnowthesampleforTOC (unfiltered).Keepitcoldandindarkinthefieldandstoreit inthefridgeinthelaborsendit immediately bymail currierifnomore samplesneedtobecollected.TOC samplesshouldbe acidifiedwithHCl2M(100mL)andpreservedat4Cuntilanalysed(Fig.3).
6CollectDOCsample:
a. Rinsethesyringe:Takethesyringe.Rinseitthreetimeswithsitewater.
b. Rinsethefilter:Fillyoursyringe.ConnecttheSterivexfiltertothesyringe.Startfilteringand wastethefirst20mLofsamplingwater.
c.Rinsethe60mLglassviallabeled“YoursamplenameDOCfiltered”:Puttheremaining30mLin the60mLglassviallabeled“YoursamplenameDOCfiltered”.Shakethevialvigorouslyandwaste thefilteredwater.Repeatthistwomoretimes(foratotalofthree).
d. Collectthesample:Filtersitewaterandfill(withoutbubbles,headspacefree)the60mLglassvial
“YoursamplenameDOCfiltered”thatyouhavepreviouslyrinsed.Youhavenowcollectedfiltered watersampleforDOC,fluorescence,anions,cationsandanyancillaryparameterofthedissolved
Fig.2.Preservingunfilteredwatersampleformercuryspecies(total-Hg,IHgandMeHg)analyses.
fraction.Keepitcoldanddarkinthefieldandstoreitinthefridgeinthelaborsenditimmediately bymailifnomoresamplesneedtobecollected.DOCsamplesshouldbeacidifiedwithHCl2M (100mL)priortoanalysis.Inthelaboratory acidifytoapHof3.5andpreserveat4Cuntilanalysed.
Beforeacidifying,safeasubsampleforfluorescence,anionsandcationsmeasurements.
7 Collectfilteredwaterfortotal-Hg,IHgandMeHgmeasurements:
a. TakeyoursyringeandSterivexfilter,filter60mLofsampleandputitinthebrownglassbottle
“YoursamplenameMercuryfiltered”.Shakethebrownbottlevigorouslyandwastethewater.
Repeattwomoretimes(foratotalofthree).
b.Collectsamplewaterandfilter250mLintothebrownglassbottlelabeled“Yoursamplename Mercuryfiltered”(avoidthesurfacemicrolayerbyimmersingthesyringeatleast10cmbelow thesurface).Add250mLofHClconcentratedacid(33–36%,UltrexIIultrapurereagent,J.T.Baker, Phillipsburg,NJ,USA).(InBravoetal.,[9])thisacidwasprovidedincleanedgaschromatography glassvial).Youhavecollectedthewatersampleformeasurementsoffilteredtotal-Hg,IHgand MeHg.
8 Double-bagallsamplecontainersindividuallyinzip-sealed(Minigrip1)plasticbags.Keepitcold anddarkinthefieldandstoreitinthefridgewhenbacktothelaboratory.Rebagallsamplesuntil processing and preservation. Store and transport all samplingand processing equipment and supplies ina cleanplastic container. Usepackingfoamif necessarytoensurethat bottlesare securelyprotectedduringtransport.
Summaryofthesamplescollected(Fig.4)
Fig.3.PreservingunfilteredwatersampleforTOCanalyses.
Fig.4.Summaryofthesamplescollected.Besidesthese,youneedtocollectyourblanks.
Preparingtheblanks
Collectaninitialfieldblanktoevaluatethepotentialforcontaminationassociatedwiththefield methods,materialsused,andsamplingenvironment.Subsequentfieldblanksshouldbecollectedto addressfield-siteconcerns,thesamplingtimeframe,anddata-qualityrequirements.Fieldblanksare processed inthe samemanner and underthe sameenvironmental conditions as environmental samples.Followsteps1–7butuseMilliQwaterinstead.Repeatthisfivetimespersamplingcampaign inordertohaveseveralsampleblank-replicates(Fig.5).
Analyticalmethods
DifferentmethodscanbeusedtomeasureIHgandMeHg[10].IHgandMeHgconcentrations weremeasuredusingspecies-specificisotopedilutionandcapillarygaschromatography(TraceGC Ultra,ThermoFisher,Waltham,MA,USAequippedwithaTriPlusRSHauto-sampler)hyphenatedto ainductivelycoupledplasmamassspectrometer(ThermoXSeries2)[11,12].Thedetectionlimitsof this method are 0.02ng L 1 for IHg and 0.005ng L 1 for MeHg. Total-Hg concentration was calculated by adding IHg and MeHg concentrations. The measurement error (calculated by analyzingeachsamplethreetimes)waslessthan2.6%and3.8%forIHgandMeHgconcentrations, respectively[9](Fig.6).
Qualityassuranceandqualitycontrol(QA/QC)
Besidesthestrictcontroloftheblanks,theprotocolandanalyticalprocedurehastobecoupledto anextensive QualityAssurance andQualityControl(QA/QC) procedure,involving replicatetests, certifiedreferencematerialsand,ifpossible,inter-laboratorycomparisons.
a)Tocalculatethemeasurementerror(precision),analyseeachsamplethreetimes.Besureyouhave lessthan15%forIHgandMeHgconcentrations,respectively.
b)Toestimatetheaccuracy,performrepeatedanalysesofcertifiedreferencematerials(CRM,e.g.
ORMS-5, NRNCC https://www.nrc-cnrc.gc.ca/eng/solutions/advisory/crm/certificates/orms_5.
html)andverifythatyour valuesarealwayswithintherangeofconcentrationgivenforCRM (e.g.ORMS-5,THg=26.21.3pgg 1).
Fig.5.Summaryoftheblanksneededtoensurethequalityofthesampling.
c)Bymeasuringthesamesampleattwodifferentlaboratories,youcanperformaninter-laboratory comparisonandidentifyanypitfallassociatedwithaparticularlaboratoryoroperator.Itisthus recommended to measure at least three samples in two different laboratories (analytical platforms).
Greenanalyticalchemistry
Throughoutthisprotocolweencouragetheuseofgreenanalyticalchemistry.Themaingoalof greenanalyticalchemistryistoachieveamoreeco-friendlyanalysisinlaboratories.Thiscanbedone throughdifferentstrategiesandtechniques,replacetoxicreagents,andmodifyorreplaceanalytical methodsand techniques with saferones,making itpossible toreduce theamounts of reagents consumedandwastegenerated[13].Inthecleaningprotocolheredescribed,weusedthreereagents:
soapExtran1,HNO3andHCl,whichareunavoidabletoensurethatalllaboratorywareisfreefrom contamination.Thesecleaningreagentsneedtobehandledwithcarebecauseoftheirtoxicity(oral, dermal,inhalation),skinirritation,eyeirritationandtheriskofcorrosion(readcarefullythesafety documentationoftheproduct).However,treatedwithcare,thewasteisrelativelyeasytotreatand canbesimplyneutralized[14].Handlingacidbathswithcareandprecautionsextendstheirlife-time andthusreducestheamountofwaste.Therefore,makesurethatallthetubesputinthebathshave beenpreviouslywashedwiththesoapExtran1andrinsedwithMilliQ.Keepthesebathsdesignated forlowlevelHganalysis.Thisreducesthecostsandtheenvironmentalimpactofthestudy.Glass bottlesandplasticsyringesusedforsamplingcanbere-usedseveraltimesuntiltheybreak.The unavoidableuseofplasticpipettetipsandSterivex-HVfiltersarethemainwasteproducts.
Fig.6. Masschromatogramsoftwosampleswith contrastingmethylmercuryandinorganicmercuryconcentrationsand differentblank(Blk)samplesthattested:i)thereagents(Blk-Reagent);ii)thesampling,handling,storageandreagents(Blk- Unfiltered);andiii)thesampling,handling,filtering,storageandreagents(Blk-Filtered).Thelinesrepresenttheaverageof3 replicatesamples(eachofthemcalculatedbythreeanalyticalreplicates).
