Dissolved organic matter thiol concentrations determine methylmercury bioavailability across the terrestrial-marine aquatic continuum

Domain: earth_science
Published: 2023-10-23
Editorial outcome: accept

Abstract
The most critical step for methylmercury (MeHg) bioaccumulation in aquatic food webs is phytoplankton uptake of dissolved MeHg. Dissolved organic matter (DOM) has been known to influence MeHg uptake, but the mechanisms have remained unclear. Here we show that the concentration of DOM-associated thiol functional groups (DOM-RSH) varies substantially across contrasting aquatic systems and dictates MeHg speciation and bioavailability to phytoplankton. Across our 20 study sites, DOM-RSH concentrations decrease 40-fold from terrestrial to marine environments whereas dissolved organic carbon (DOC), the typical proxy for MeHg binding sites in DOM, only has a 5-fold decrease. MeHg accumulation into phytoplankton is shown to be directly linked to the concentration of specific MeHg binding sites (DOM-RSH), rather than DOC. Therefore, MeHg bioavailability increases systematically across the terrestrial-marine aquatic continuum as the DOM-RSH concentration decreases. Our results strongly suggest that measuring DOM-RSH concentrations will improve empirical models in phytoplankton uptake studies and will form a refined basis for modeling MeHg incorporation in aquatic food webs under various environmental conditions.

Introduction
It is estimated that anthropogenic mercury (Hg) emissions have caused a three-fold increase in Hg content in surface marine waters compared to pre-industrial conditions1, increasing the threat of Hg to ecosystem viability and human health2. Methylmercury (MeHg) is of particular concern for human health as it is a neurotoxin that bioaccumulates and biomagnifies in the aquatic food web3, potentially causing high human exposure via fish consumption4,5. The largest enrichment step of MeHg into the aquatic food web occurs in the processes of cellular uptake of MeHg from water by seston. For this step, bioconcentration factors (BCF) range from 3 to 7 log units6, which is several orders of magnitude greater than the approximate ten-fold increase in MeHg concentration with each successive trophic level3,7. Uptake by unicellular organisms such as small algae and bacteria has been shown to control MeHg concentrations in fish6 and thereby the potential exposure of MeHg to humans.

Dissolved organic matter (DOM) is ubiquitous in aquatic systems and plays several critical roles in the biogeochemical cycling of Hg8. Here, DOM refers to all dissolved organic compounds in a natural system, while dissolved organic carbon (DOC) refers to a quantitative measure of the C content of DOM. At environmentally relevant concentrations, MeHg associated with DOM or particulate OM is bound to thiol (RSH) groups present in the matter9,10, but challenges in RSH measurements have led to DOC being the most common proxy used to represent the concentration of dissolved organic MeHg-binding ligands. In laboratory experiments, the cellular uptake of MeHg by phytoplankton generally decreases with increased concentrations of DOC11,12, and several field studies have shown a negative correlation between DOC and MeHg BCF6,13. Such relationships indicate that DOC controls MeHg BCFs in natural systems6 by reducing MeHg’s bioavailability to phytoplankton14. However, the effect of DOC on MeHg uptake tends to vary with DOM source and properties11,15–20 and exceptions have been reported where naturally low DOC waters led to lower accumulation of MeHg in plankton than naturally high DOC waters21. Particularly large variability in MeHg bioconcentration has been reported for different DOM types at DOC concentrations less than ~5 mg/L15. Primarily, these differences have been attributed to unique DOM characteristics including binding site abundances, structure, and aromaticity11,12,16.

Studies more closely focused on the uptake of specific MeHg complexes have shown how the chemical speciation of dissolved MeHg can influence its cellular uptake12,22,23 in phytoplankton cultures. Through such studies, both passive uptake of MeHgCl and active uptake of MeHg complexes with low molecular mass thiols12,23 have been supported, and the uptake rate is generally faster for inorganic MeHg complexes, and particularly MeHgCl, relative to MeHg bound to organic ligands including DOM24. Thermodynamic stability and sterical hindrance effects have been identified as factors controlling the uptake rate and accumulation efficiency of specific MeHg complexes by phytoplankton12,23. Translating uptake potentials from relatively simple laboratory-based studies to complex natural conditions, however, is challenging and requires a mechanistic understanding of how DOM controls MeHg speciation between available ligands in unique aquatic systems. The differences in uptake rates and bioaccumlation mechanisms for MeHg bound to DOM versus inorganic ligands heightens the importance of adequately calculating MeHg speciation when predicting MeHg uptake into phytoplankton. An improved understanding of these processes is necessary for the prediction of biotic MeHg exposure now and under future environmental change scenarios20,25.

In this study, we addressed critical aspects of MeHg bioavailability at two different scales using DOM isolated from a diverse range of coastal waters. First, we evaluated MeHg bioavailability in the presence of DOM based on a generalizable understanding of MeHg–DOM interactions. Second, we elucidated how and why MeHg bioavailability changes across four distinct regions of the terrestrial-marine aquatic continuum based on measured DOM properties. The goals of this study were based on the hypotheses that the DOM (i) binding capacity and (ii) binding affinity decrease, while (iii) the rate of ligand exchange for MeHg–DOM complexes increase, across the terrestrial-marine continuum. The bases for these hypotheses are outlined in the results and discussion. If true, such changes in DOM properties would contribute to an increased availability for cellular uptake and accumulation of MeHg by phytoplankton in marine compared to upstream waters. We evaluated how natural DOM controls MeHg bioavailability by a comprehensive characterization of thermodynamic (binding capacity and binding affinity) and kinetic (rate of ligand exchange reactions) properties of MeHg—DOM interactions. The DOM principles we highlight as driving MeHg uptake were then tested in a phytoplankton uptake experiment. Finally, we elucidated site-specific MeHg bioavailability using updated speciation models. Our results all point to the concentration of thiol ligands associated with DOM as the main control on MeHg bioavailability in natural waters.

Results
CharacteristicsofthestudysitesandDOMproperties
Watercolumnsampleswerecollectedfrom12uniqueestuariesandtwooffshorelocations,20sitesintotal,alongthenortheastcoastoftheUnitedStates(38.9–44.7°N,Fig.1,Fig.S1).Thesiteswerechosenalongestuarinesalinitygradients(0.5–36practicalsalinityunits(psu),median=21psu)withDOCconcentrationsrangingfrom116–590µM(median=235µM)(Table1).Onehighlycontaminatedsitewasincluded(Berry’sCreek,NJ)leadingtoalargerangeindissolvedMeHg(0.001–2.78pM,median=0.067pM)andtotalHg(0.61–45pM,median=1.7pM)concentrationsinthesampledwater(TableS1).DOMwasextractedfrom20–40LofsurfacewaterateachsitefollowingDittmar et al.26.Thissolid-phaseextractionmethod(basedonastyrenedivinylbenzenepolymer,so-calledPPL-SPE)isthemostcommonapproachforDOMenrichmentanddesalting,whichisaprerequisiteformosttypesofmolecularcharacterizations27,includingtheonesinthisstudy.

Ingeneral,PPL-SPEcapturesabroadrangeofcompoundsandretainsoveralldifferencesinthemolecularcompositionoftheDOMbetweensamples28–31. However,sinceexternal factorssuch assample salinity impact the carbon yield of the extraction, we normalized relevant parameterstoDOCconcentrationstoavoidquantitativebiases.TheDOMsampleswerecharacterizedbybulkC,N,andScomposition,fluorescencespectralanalyses17,32,sulfurspeciation(determinedbySK-edgeX-rayabsorptionnearedge(XANES)spectroscopy),andtotalthiolcontent(RSH/DOC)(determinedbyliquidchromatography-tandemmassspectrometry,LC-MS/MS).Forcomparison,thesiteswereseparatedintofourclasses:marsh,river,estuary,andshelf(Table1).SimilaritiesanddifferencesinDOMpropertiesamongthesiteclasseswereevaluatedbyprincipalcomponentanalysis(PCA).

OnePCAfocuseduponthefluorescencespectraldata(Fig.2a,b).ThefirstprincipalcomponentofthisPCAdescribed61%ofthedatavariabilityandseparatedsamplescontainingmorerecentlyproducedfluorescentmaterial(C360)fromthosewithrelativelyoldermaterial(C450).Thesecondprincipalcomponentdescribed27%ofthedatavariabilityanddiscriminatedfluorescentDOMbyorigin(C390bacteriallyderivedmaterial,C520terrestriallydominatedmaterial).TheshelfsiteDOMsamplesstoodoutwithhigherproportionsofrecentlyproducedmaterialoflowermolecularweight(inferredfromthespectralsloperatio)thantheothersiteclasses.Classseparationforthenon-shelfsiteswaslessobviousandsuggeststhemainDOMsourceintheseregionsisterrestrialinput.AslightwatershedeffectwasobservedinthePCAassitesfromthesamegeographicalsystemtendedtogrouptogether(comparesitelocationsinFig.1withsitegroupinginFig.2a).

AnotherPCAwasmadetovisualizethechemicalspeciationofdissolvedorganicsulfurbylocationgivenitsroleinMeHgbinding(Fig.2c,d).Thefirsttwoprincipalcomponentsdescribed79%ofthedatavariability(with52%inthefirstprincipalcomponent)andthesitesweremorestronglyseparatedinthisPCAcomparedtotheonebasedonfluorescencespectraldata.Thegreatestcontributionstosite separationcamefromtheredoxstateofdissolvedorganicsulfurwherethemostoxidizedforms,sulfateandsulfonate,andthemostreducedforms,organicdisulfides(RSSR)andRSH+organicmonosulfides(RSR),loadedontooppositesidesoftheplot.TheXANESresultsdemonstratedacharacteristicallyhighproportionofthethreereducedorganicsulfurforms(i.e.,thesumofRSH,RSR,andRSSR)attheriverandmarshsites,ahighproportionoforganicsulfateandsulfonateatshelfsites,andalargevariabilityamongtheestuarinesiteslikelyexplainedasamixinggradientbetweentheterrestrialandmarineDOMsources(Fig.2c,d).Overall,thefluorescenceanalysisanddissolvedorganicsulfurspeciationresultsoftheextractedDOMsamplessupportedtheinitialgeographicalgroupingofsitesintothefourclasses:marsh,river,estuary,shelf,andshowthatthesitesrepresentarangeinkeyparameterscoveringtheterrestrial-marineaquaticcontinuum.

ThermodynamicsoftheMeHginteractionwithDOM—bindingcapacityandbindingaffinity
ThebindingcapacityandbindingaffinityofMeHgligandsareimportantthermodynamicparametersinfluencingthechemicalspeciationandbioavailabilityofdissolvedMeHginnaturalwaters.Here,wedefinetheMeHg-bindingcapacityastheconcentrationofstrongbindingligands,i.e.,thethiolfunctionalgroups.TheMeHg-bindingcapacityoftheextractedDOMisexpressedasthethiolcontentpermolcarbonoftheDOM(RSH/DOC,µmol/mol;hereforwardreferredtoastheDOMbindingcapacity),andtheinsituMeHg-bindingcapacityinwateratagivensiteisexpressedasthetotalmolarconcentrationofDOM-associatedthiols(DOM-RSH,nmol/L;hereforwardreferredtoastheinsitubindingcapacity).TheinsitubindingcapacitywasdeterminedbymultiplyingtheDOMbindingcapacitybythelocalDOCconcentration(mol/L).WequantifiedtheDOMbindingaffinityastheformationconstant(logK)ofMeHg(DOM-RS)complexesforthedifferentextractedDOM.Thetwothermodynamicparameters(RSH/DOCandlogK)weredeterminedusingindependentexperiments.TheDOMbindingcapacitywasquantifiedusingarecentlydevelopedthiol-specificmethodbasedonLC-MS/MS33.Bindingaffinitywasdeterminedwithacompetingligandexchangereaction(adaptedfromLiem-Nguyenetal.34)usingN-Acetyl-Penicillamine(Nacpen)asthecompetingligandandspecificallyquantifyingtheMeHg(Nacpen)complexbyLC-inductivelycoupledplasmaMS(LC-ICPMS).

TheDOMbindingcapacityvariedbyafactorof~10amongthesites(range39–418µmolRSH/molDOC)(Table1)withthemarshsiteshavingthehighestRSH/DOCandmarinesitesthelowest,althoughthemarinesiteswerenotstatisticallydifferentfromtheriverandestuarinesites.ThetrendinRSH/DOCagreedwiththeorganicreducedsulfurpoolofthedissolvedorganicsulfurasmeasuredbySXANES,(RSH+RSSR)/DOC,andthetwoindependentmeasurementscorroborateageneraldecreaseinreduceddissolvedorganicsulfurcompounds,includingthiols,frommarshesandriversintomarinesystems.ThevariabilityintheRSH/DOCratioshowedthatDOCconcentrationaloneisapoorproxyfortheinsituMeHg-bindingcapacityacrossdifferentaquaticecosystems.Indeed,theinsituDOCconcentrationsvariedbyonlyafactoroffiveamongsites(116–590µM),buttheinsituDOM-RSHconcentrationvariedbyafactorof40(5−215nM).TheDOM-RSHconcentrationvariedsystematicallywiththetypeofsitewheretheaveragevaluedecreasedintheorder:marsh>river≈estuary>shelf(Fig.3a).ThesubstantialrangeandsystematictrendinDOM-RSHconcentrationacrossourstudysitesdenoteinsitubindingcapacityasakeyfactorcontrollingMeHgspeciationandbioavailabilityacrossterrestrial-marineaquaticenvironments.

ThethiolcontentofnaturalDOMhasnotbeenreportedinmanyfieldorexperimentallabstudies,inpartbecausetheirisolationandquantificationareanalyticallychallenging.Tocomparewiththefewpublishedstudies,wedividedourindependentlydeterminedRSH(byLC-MS/MS)bytheorganicreducedsulfur(RSSR+RSR+RSHbySXANES).Theresultsrangedfrom1.3%to5.5%(average2.8%),withthelowestfractionsfoundatthemarineshelfsitesandthehighestatmarsh-impactedsites.ThisislowerthanSuwanneeRiverDOM(2R101N,fromtheInternationalHumicSubstancesSociety),whichwasdeterminedtohave~15%RSHoforganicreducedsulfurintwopriorindependentstudies33,35,aswellassolidpeatandwetlandsoilswhichhave15–30%RSHoforganicreducedsulfur9,34–36.Indirect,operationallydefinedproceduresthatquantifyundefined“strongbindingsites”forMeHgandHg2+inDOM(e.g.,37,38)arelesscertainthanthedirectmeasuresusedinthisstudysincetheyare sensitivetotheexperimentalconditions39 andspecificligandtypesarenotdefined.Evenso,thegeneralpatternsofHg2+-bindingsitespergDOCobservedbyLamborgetal.40aresimilartothetrendsinDOMbindingcapacityfoundhere,withratiosdecreasingfromterrestrialtomarineenvironments.TheconsistenttrendsacrosssitesmeansthattheRSH/DOCresultsinTable1provideameanstoestimateinsituMeHg-bindingcapacitiesincoastalwatersforwhichlocalDOCconcentrationsandsitecharacteristicsareknown.Furthermore,therecentlydevelopedLC-MS/MSmethodusedheretodeterminetotalthiolconcentrations33ismoreaccessible,andrequireslesssampleamounts,thanextendedX-rayabsorptionfinestructureandXANEStechniquesandwillfacilitateanexpansionofsuchmeasurementsinfuturestudies.

RegardingMeHg-bindingaffinities(logK)oftheDOMsamples,wehypothesizedthattheywouldreflectthestrongbondbetweenMeHgandthiolligands(e.g.9,37).EventhoughMeHgdominantlybindswithreducedthiolfunctionalgroups,thestrengthoftheMeHg-Sbondcanbeinfluencedbystructuralelectrostaticeffects,asdemonstratedbyReidandRabenstein41.Forinstance,protonationofaminefunctionalgroupslocatedclosetothethiolmoietycanreducethelogKofMeHg-thiolcomplexesbyredistributingchargefromthethiolmoiety41.BecausetheC/NratioisgenerallylowerformarinealgalDOMcomparedtoterrestrialhumicDOM42,wehypothesizedthatthelogKwouldslightly,butsystematically,decreaseacrosstheterrestrialtomarinecontinuum.

WedeterminedMeHg–DOMstabilityconstantsbythecompetingligandexchangeexperimentsbasedonspecificquantificationoftheMeHg(Nacpen)complexbyLC-ICPMS,thetotalDOM-RSHconcentration,andthepreviouslydeterminedstabilityconstantfortheMeHg(Nacpen)complex41.ThelogKforMeHgcomplexeswiththeuniqueDOMextractsvariedfrom16.1±0.19to17.4±0.19(Eq.2,Table1,Fig.3b).ThisrangeagreeswellwithpreviousstudiesonMeHgbindingtoreducedsulfurgroups(presumedtobethiols)inaquatic39andsoil9,10,43,44organicmatter(TableS2).TheaveragelogKof16.5isalsoclosetothatoftheMeHgcomplexwithNacpen(logK=16.76),thecompetingligandintheseexperiments,andtootherspecificthiolcompounds41,45.ThisresultsupportsthehypothesisthatMeHgbindssolelytothiolgroupsintheDOM.Akeyfinding,however,isthattherewasnosystematictrendamongthesiteclasseseventhoughlogKvariedamongtheDOMextracts(Fig.3b),andtherewasnocorrelationbetweenlogKandtheC/Nratioincontrasttothehypothesis.WethusconcludethatMeHg-bindingaffinityislikelynotaprimaryfactorthatsystematicallycontrolsMeHgbioavailabilityacrosstheterrestrialtomarineaquaticcontinuum.

KineticsofMeHgligandexchangewithDOM
Inadditiontothermodynamics,thechemicalspeciationandbioavailabilityofMeHgcanbekineticallycontrolledbystericalhindranceeffectsinducedbythe3DstructureofMeHgcomplexes12,23.Inthisway,largeor“bulky”chemicalstructuresmayleadtoaslowerexchangerateofMeHgbetweendissolvedandparticulatephases,includingcellsandotherligands.IthasbeenshownthatsubstantialfractionsofinorganicHg(II)boundtoDOMandmineralphasescanformpoolsthatexchangeligandsatverylowrates(so-called“non-exchangeable”pools46).IfMeHgformedsuchpoolswithnaturalDOM,bulkMeHgmeasurementswouldoverestimatethebioavailablepoolandunderestimateMeHgBCFs.MoststudiessuggestfastligandexchangekineticsfordissolvedMeHgcomplexes45,47,48,includingwithDOM11,buthavelackedtheabilitytodeciphernon-exchangeablepools.WeusedacompetingligandexchangeexperimentwhereNacpenandDOM-RSHwerepre-equilibratedwithseparateenrichedstableisotopesofMeHg,i.e.,Me204Hg(Nacpen)andMe200Hg(DOM-RS).Thetwoequilibratedisotopesweremixed,andtheconcentrationandHgisotopiccompositionofMeHg(Nacpen)determinedstartingatfiveminutesaftermixinguntilsteady-statewasreached.Ultimately,thenon-exchangeablepoolofMeHgwassmall(<10%,Fig.S2)foralltheinvestigatedDOMsamplesandnotsignificantlydifferentunderdifferentDOMandcompetingligandratios.Wethusconcludethatformationofnon-exchangeableMeHgpoolsisnotaquantitativelyimportantprocessfortheinteractionofMeHgwiththehighlycontrastingDOMtypesinvestigatedinthisstudy.

EventhoughpracticallyallMeHgwasexchangeable,bioavailabilityandcellularuptakecanstillbeinfluencedbytherateofligandexchange.Fortheinvestigatedshelf(LIS),estuary(NB)andriver(PAW)DOM,MeHgexchangedbetweenbindingligandsfasterthancouldbemeasuredwiththecompetingligandexchangemethod,i.e.,steady-statewasreachedbefore5min(Fig.S2).ThetwomarshDOM(SIandBI)exchangeratesweresomewhatslower,butstillbarelymeasurable.AslowerligandexchangerateforthemarshDOMisconsistentwithitslargermolecularsizedistributioncomparedtotheothersiteclasses(Fig.S3).ThelargermolecularstructureofmarshDOM,assumingtheDOM-RSHcompoundsarealsolarger,couldintheorycontributetoalowercellularuptakerateofMeHgboundtomarsh-typeDOM-RSHcompounds.However,theexchangerateofMeHgbetweenthecompetingligandNacpenandalltheinvestigatedDOMsamples(i.e.,betweenMeHg(Nacpen)andMeHg(DOM-RS))wasconsiderablyfasterthantheexchangeratebetweenMeHg(Nacpen)andphytoplanktoncellsdeterminedinapreviousstudywithgreenalgae23.Applyingthesamepseudo-first-orderratemodelasSkrobonjaetal.23,theturnoverforMeHgexchangebetweenNacpenandDOM-RSHwas2–3minformarshsitesand<1minfortheotherDOMtypesinourstudy,whichismuchfasterthanthe~30minturnoverforMeHgexchangebetweenNacpenandgreenalgaecells23atsimilarconcentrations(100–300nM)ofNacpen.TheseresultsshowthattheMeHgligandexchangereactionsamongDOM-RSHcompoundsinsolutionarenotamajorprocesscontrollingdifferencesinMeHgbioavailabilityacrosstheterrestrialtomarineaquaticcontinuum.

Hypothetically,theresultsofthekineticexchangetestscouldbeinfluencedbytheuseofextractedDOMifthemolecularcompositionofawatersampleimpactstheextractionyieldsofspecificcompounds.WhilethePPL-SPEusedinthisstudycapturesabroadrangeofcompoundsandretainsoveralldifferencesinthemolecularcompositionoftheDOM,recentstudiessuggestthatsmallhydrophilicmoleculesarepartlylostfromthesorbentduringtheextractionprocess31.SuchcompoundsformthemostbioavailableandeasilyexchangedMeHg-thiolcomplexes12,23.Intheory,iftherecoveryofsuchcompoundswereincompleteintheDOMextraction,theinsituturnoverforMeHgligandexchangemightbeevenfasterthanthe<1–3minwecalculated.Therefore,ourconclusionthatMeHgligandexchangekineticsamongDOM-RSHcompoundsisfastandnotamajorcontrolofMeHgbioavailabilitywouldstillbevalid,orevenamplified,iftherewerelossesofsmallhydrophilicthiolsinthePPL-SPEisolation.

BioavailabilityandVCFofMeHgdependonthetotalconcentrationofDOM-associatedthiols
ThisstudyaimedtoprovideamolecularunderstandingforhowDOMdecreasesMeHgbioavailabilitytophytoplankton.WeshowedthroughaseriesofthermodynamicandkineticreactionexperimentsthattheinsituMeHg-bindingcapacityofDOMvariedsignificantlyandsystematicallyacrosstheterrestrial-marineaquaticcontinuum,whereasbindingaffinityvariedbutnotsignificantlyorsystematically,andligandexchangekineticswerefastforalltypesofDOM.TotestwhethertheabsoluteDOM-RSHconcentrationinwaterdictatesMeHgbioaccumulation,andwhetherDOM-RSHconcentrationisastrongerpredictorforMeHgBCFsthanDOCconcentration(ascommonlyusedinpreviousstudies),anuptakeexperimentwasconducted.ThediatomThalassiosirapseudonanawasexposedtoMeHgpre-equilibratedwithvariedconcentrationsofDOMfromonemarsh(SI)andoneriver(OR)sitewithhighlycontrastingRSH/DOCratiosandoverallsulfurspeciation(Table1andFig.2b).ThesesiteswerechosenfortheirsimilarMeHg(DOM-RS)stabilityconstants(logK=16.3and16.4,respectively)tospecificallytestthehypothesizedcausalrelationshipbetweenDOM-RSHconcentrationandMeHguptake.Theresultsarepresentedasvolumeconcentrationfactors(VCFs,whichareBCFsnormalizedtothevolumeofthecells)after4hoursofuptake(Fig.4).

ThemeasuredVCFsareofsimilarmagnitudetopriorstudies11,49anddepictsimilartrendswithDOCtothosemeasuredacrossacreatedDOCgradientbyLuengenetal.11(Fig.S4).Ashypothesized,theresultsshowedaconsiderablystrongerrelationshipbetweenMeHgVCFandDOM-RSHconcentration(Fig.4a)comparedtotheDOCconcentration(Fig.4b).ThefitbasedonDOM-RSHwasparticularlyimprovedatlowDOM-RSHconcentrationsof~2–50nM,correspondingto~50–150µMDOC.TheempiricalrelationshipbetweenMeHgVCFandDOM-RSHconcentrationwasbestfitbyapowerfunction(VCF=0.67×[DOM-RSH]−0.4,R2=0.96,p<0.001)insteadofThemorecommonlyusedexponentialrelationshipforVCFvsDOC11,25.

TheresultssupportthatthetotalDOM-RSHconcentrationisaprimarycontrollingfactorofMeHgbioavailabilityandcellularaccumulationbyphytoplankton.ThesignificantandstrongrelationshipbetweenDOM-RSHandMeHgVCFcorroboratesthefindingthatotherfactors,likekineticconstraints,areoflessimportanceincontrollingMeHguptakethanspeciationwithDOM-RSH.TherelationshipfurtherpointstodifferencesinDOMthiolconcentrationsasalikelyprincipalfactorbehindthehigh,previouslyunexplainedvariabilityinMeHgVCFamongdifferenttypesofDOMatlowDOCconcentrationsobservedinpreviousstudies15,49.Site-specificDOM-RSHconcentrationsaloneareamuchbetterpredictorofMeHgVCFthanDOCconcentrationsandshouldbequantifiedinexperimental,modeling,andfield-orientedresearchonMeHgspeciationandbioaccumulation.

Basedoncurrenttheory,twocasescanbeinvokedtoexplainthepowerfunctionrelationshipbetweenMeHgVCFsandDOM-RSHconcentrations(Fig.S5).TheDOM-RSHconcentrationmaycontroltheequilibriumpartitioningbetweenMeHgboundtoDOM-RSHandaccumulatedinplanktoncellsviareversibleuptakemechanisms(caseA).Alternatively,itmaycontroltheequilibriumdistributionbetweenMeHg(DOM-RS)andinorganicMeHgcomplexes(e.g.MeHgCl)insolutionwhichareaccumulatedincellswithspecies-specificratesviaquasi-irreversiblemechanisms(caseB).CaseApresentsMeHgcellularuptakeasathermodynamicequilibriumfunctionbetweenoneMeHgspeciesinsolution(MeHg(DOM-RS))andoneassociatedwithcells(MeHg(cell)n+1)withaconditionalconstantK (equation(S1d)).RearrangingtheequilibriumexpressionofMeHgbetweencellularligandsandDOM-RSresultsintheMeHgVCF(i.e.[MeHg(cell)n+1]/[MeHg(DOM-RS)])beingproportionalto1/[DOM-RSH]assumingthetermK [H+][cell]isconstantundertheexperimentalconditions(fullequationsinSI).Withthelimiteddatageneratedforthisstudy,theempiricalrelationshipbetweenVCFand1/[DOM-RSH]isnotlinearinthespaceaswasexpected(Fig.S5),thustheequilibriummodellikelydoesnotfullycapturethecomplexprocessofMeHguptake.

CaseBaccountsforasimplified,simultaneousuptakeofthedominantorganicandinorganicformsofMeHgintheexperimentalassays(MeHg(DOM-RS)andMeHgCl,respectively)basedontheiruniqueuptakerateconstantsandspecificconcentrations.PriorstudiesdenoteafasteruptakeofMeHgClduetopassivediffusionacrossthecellmembrane22whileuptakeofMeHg(DOM-RS)complexesarethoughttobeslowedbyinteractionsatthecellsurfacepriortointernalization23.TheuptakerateconstantofMeHg(DOM-RS)wasestimatedbasedonthehighestDOMexposureconcentrationusedinthisstudyat0.004amolµm−3hr−1nM−1.TheMeHgCluptakerateconstantwassettofitThedata,resultinginavalueof0.5amolµm−3hr−1nM−1;atthehighendofpublishedvalues(e.g.,0.17512and0.549).TheCaseBmodelingresultsareplottedinFig.S5againsttheRSHconcentrationoftheexposuremedium.Themodelfittheresultswell,butfurthermechanisticstudiesoncellularinternalizationofMeHgarerequiredtobetterparameterizeuptakeasadirectfunctionofMeHgspeciation,cellularabundance,andcellsize.Basedonourmodelingwedo,however,suggestthatbioaccumulationofMeHgintophytoplanktonneedstobedeterminedusingacombinedthermodynamic-kineticmodelthatconsiderswatercolumnMeHgspeciationandthedifferencesintheuptakerateconstantsforthedifferentMeHgcomplexes.

ThereareadditionalfactorsthatcontributetodifferencesinMeHgspeciationandbioavailabilityinnaturalsystems
IdentifyingandquantifyingtheDOMpropertiesthatdictateMeHgspeciationandbioavailability,thecoreresultsofthisstudy,providea basistosubstantiallyrefineMeHgspeciationanduptakemodelsincoastalwaters.WecalculatedtheinsituMeHgspeciationfortheconditionsateachsiteatthetimeofsampling(TableS4,SITextModel2).Themodelingpredictedthat>99%ofMeHgexistsasMeHg(DOM-RS)complexesacrosstheentirecontinuumdespitethattheCl−concentrationvariedfrom8to562mM.AtDOM-RSHconcentrationsbetween5nM(thelowestinourstudy)and35nM,thefractionofMeHg(DOM-RS)complexesvariedinanarrowrangebetween99.09%and99.99%,andwas≥99.99%whenDOM-RSHwas>35nM(Fig.S6).TheremainingMeHgfractionsweredominantlyMeHgClandminorlyMeHgOH.Inmarinesystems,priorspeciationmodelsoftenpredictedCl−asthedominantMeHg-bindingligand.Forinstance,ZhongandWang18proposedthatMeHgClwoulddominateoverMeHgcomplexeswithDOMinhighsalinity(300–500mMCl−),lowDOC(<100µM)waters,conditionsmetintheopenocean.UsinganupdatedMeHg(DOM-RS)averagestabilityconstantof16.7andRSH/DOCratiosfromtheshelfsites(Table1),ourmodelingpredictsthatCl−isthedominantbindingligandat500mMCl−onlywhenDOCislessthan~0.05µM,conditionsunlikelytobemetintheglobalocean50.

ThedominanceofMeHg(DOM-RS)speciesdoesnotnegatethepotentialforsignificantMeHgCluptake.GoingbacktocaseBdescribedfortheuptakeexperiment,thefastuptakeofMeHgClmeansthatevenarelativelysmallfractionmaydriveMeHguptakeincertainnaturalwatersandthisspeciesshouldnotbeignored.TheuniqueuptakeratesofMeHgClandMeHg(DOM-RS)meanthattherelativeproportionofMeHgcomplexesinasystemissignificantforunderstandingitsincorporationintocells.BasedonthespeciationmodelinTableS3,themolarratioofthecomplexesMeHgClandMeHg(DOM-RS)isrelatedto[Cl-],1/[DOM-RS],K,andpH.OurresultsshowthattheMeHgCl/MeHg(DOM-RS)molarratioacrosstheterrestrial-marineaquaticcontinuumwassignificantly,positivelycorrelatedwith1/[DOM-RSH](r2=0.50,p=0.001),butnotwith[Cl-],logK,orpH.ThesmallvariabilityinMeHgspeciation(includingthemagnitudeoftheMeHgClfraction)wasthusprimarilydrivenbydifferencesintheDOM-RSHconcentrationamongsitesandnotbyCl−.ThisresulthighlightsDOM-RSHconcentrationasthemajorcontrolofMeHgbioavailabilityforcellularuptake,regardlessoftheexactuptakemechanism.

Extrapolatingourexperimentalresultsontostudieswithconditionsoutsideofwhatwasexaminedhereisassociatedwithincreaseduncertainty,butisdonetoillustratehowexpectedgradientsinDOM-RSHthiolconcentrationsacrossandwithinsystemsmayclarifypreviousobservations.Forexample,observedDOM-RSHconcentrationsintheNorthPacificOceanwerelowercomparedtoouroffshoreNorthAtlanticandnearshoreLongIslandSoundsites;1–5nM51,~5nM,and13–35nMrespectively.SestonMeHgBCFsweremeasuredtobegreatestinthePacific(4.33–6.12forthe0.2–5µmfraction52)comparedtooffshoreNorthAtlantic(2.67–4.41forthesamesizefraction53)andLongIslandSound(2.55–3.25)53,thusdecreasingwithincreasingDOM-RSHconcentrations.Asimilarapproachcanbeextendedtoriverinesystems.Luengenetal.11andPickhardtandFisher21bothmeasuredMeHgVCFsinlaboratoryexperimentsusingwatercollectedupstreamoftheSanFranciscoBay.PickhardtfoundacuriousrelationshipwhereuptakewashigherintheirhighDOCsitecomparedtotheirlowDOCsite:asituationnotreadilyapparentintheLuengenstudy.AwatershedanalysisofthesitesinbothstudiessuggeststhatPickhardtandFisher’sanomalouslowDOC,lowuptakesitewaslocatednearamarshsystem.Basedonourresults,itcanbehypothesizedthatthemarshsitehadelevatedDOM-RSHandthereforesuppresseduptaketoagreatherextentthanDOCconcentrationsalonewouldpredict.Thiseffect,however,remainstobetestedforthespecificsites.

ThereareadditionalfactorsthatcontributetodifferencesinMeHgBCFacrosssystemssuchasphytoplanktonabundance,communitycomposition,cellularsizedistribution,anddissolvedMeHgconcentrations12,25.Further,laboratorystudieshaveshownthatthecellularuptakerateofMeHg-thiolcomplexesbyphytoplankton12,23(andsimilarlytheuptakeofinorganicHg-thiolcomplexesbybacteria54,55candifferdependingonthechemicalstructureoftheDOM-RSHcompound(e.g.molecularweightandpotentialforchelationeffects).MoreworkisneededtoclarifytowhatextentthemolecularcompositionofthiolsvariesinnaturalDOM,andifthisisacontributingcontrollingfactorforMeHgbioavailabilityintheenvironment56.OurresultshighlightthesignificantimprovementthatquantifyingDOM-RSHcanhavewhenparameterizingMeHgBCFresultsfromnaturalsystemsinsteadofrelyingonDOCasthebindingligandproxy.

Implications
ThisstudyprovidesthermodynamicandkineticinformationontheinteractionsbetweenMeHgandDOMextractedfromarangeofcoastallocations,andaquantitativeassessmentofhowDOMcontrolsMeHgbioavailabilitytophytoplankton.ThekeyresultsareconceptuallyvisualizedinFig.5.ThefigureillustrateshowDOM-RSHconcentrationcontrolsMeHgbioavailability(Fig.5a),andhowdecreasingtrendsinDOM-RSHconcentrationsacrosstheterrestrialtomarineaquaticcontinuum(Fig.5b)leadstohigherMeHgavailabilityandplanktonicuptakeinsystemsdominatedbymarineDOMrelativetothoseimpactedbymorehumic,terrestrialDOM(Fig.5c).DecreasingDOM-RSHtrendsaredrivenbydecreasingDOCconcentrationsandRSH/DOCratios,thelatterofwhichareimpactedbytheDOMsource.Acomparisonoftheresultsfromfieldobservationsinterrestrial19,estuarine,and/ormarineaquaticsystems17,52,53showhigherMeHgconcentrationsinsestonfromsystemswithhigherlevelsofmarineDOMoverterrestrialDOM.Suchtrendshavealsobeenobservedinlaboratoryuptakeexperiments18.Wherethesepreviousstudiesinvokevariousprinciplesinanefforttoexplaindifferencesinuptake,suchashigheraromaticity19,largermolecularweight17,andspeciationwithinorganicligands14,18wecannowmoreclearlylinkMeHguptaketotheDOM-RSHconcentration.Specifically,ourresults,includingtheuptakeexperiment,suggestthatchangesinbioavailabilityaredrivenbyMeHgspeciationwithDOM-RSH,whichisnotwellpredictedbylocalDOCconcentrations.

UnderstandinghowDOMcontrolsMeHgbioavailabilityiscriticallyimportanttopredictMeHgexposuretohightrophicbiota,includinghumans,nowandunderfuturescenarios6.EnvironmentalchangescenariosareexpectedtodirectlyimpactDOMloadingoaaquaticecosystems,andthereforeMeHgbioavailability57.Forinstance,eutrophicationleadstotheincreasedproductionofautochthonousDOM58,59whichtendstoproduceDOMwithlowRSH/DOC.Allochthonous,terrestrialDOMinputstoaquaticsystemsareimpactedbychangesinwatershedrunoffduetoe.g.climatechangeorlanduseandtendtodeliverDOMwithhighRSH/DOCtocoastalsystems.Basedonourresults,increasedproportionsoffresh,marineDOMwouldleadtodisproportionatelysmallerincreasesinlocalDOM-RSHthanifterrestriallyderivedDOMincreasedinasystem.TheuseofDOCasaproxyforMeHg-bindingcapacitywouldthusunderestimateMeHgbioavailabilityinalgalbloomsandoverestimateitforincreasedterrestrialrunoff.

OurstudyhighlightsthatanyenvironmentalprocesscausinganincreaseinthemolarconcentrationofDOM-RSHwilldecreaseMeHgbioavailability.Thiseffectisexpectedtobelargeatlowtomoderatenanomolar-levelconcentrationsofDOM-RSH(≤~25nMinouruptakeexperiment,Fig.4)andminorathigherconcentrations.TheprincipalinteractionsbetweenMeHgandDOM-RSHpresentedinthestudyprovidearefinedframeworkforhowtoconsiderdissolvedMeHgspeciationinMeHguptakemodels.Whilemoreworkisneededtobetterunderstandthemechanismsforuptake,morefrequentDOM-RSHmeasurementsintandemwithVCForuptakeexperimentswillleadtostrongerempiricalfitsbetweendissolvedMeHganduptakeacrossarangeofsites.Together,thisandfuturestudieswillbecomeanimportantfeaturetorefinemodelspredictingMeHgincorporationinaquaticfoodwebsforcurrentandfutureHgemissionscenariosundervariousenvironmentalchangescenarios.

Methods
Samplecollectionandpreparation
Watersampleswerecollectedusingcleantechniquesbyhandfromshoreatallbuttheshelfsites.TheGulfofMainesamplewascollectedbyloweringabucketoverthesideofasmallboat,andtheshelfbreaksamplewascollectingfrom25mdepthusingaNiskinbottle.DOMwasextractedfrom20–40L(pendingwatercolorandexpectedDOCconcentration)ofeachwatersamplewithin24hoursofcollectionusingthesolid-phaseextractiontechniquedescribedbyDittmar et al.26 taking care to load less than 10L of sample or 2mmolDOCpergramofsorbent.Briefly,theseawaterwasfilteredto<0.2μmusingapre-cleaned0.45μmMeissnercartridgefilterfollowedbya0.2μmglassfibercapsulefilter,andthenacidifiedtopH2usingtracemetalgradehydrochloricacidbeforeloadingontoacleanmodifiedbenzenestyrenepolymercartridgeataflowrateof<40mL/minunderthelimitationssetbyDittmaretal.26.Theloadedcartridgewasrinsedwith0.01MhydrochloricacidanddriedunderArbeforeelutingwithmethanolandacetone.Theorganicsolventsweredriedundernitrogen(N-EVAP111)at40°Candtheresiduewasfreeze-dried.

DOMcharacterization
TheextractedDOMwascharacterizedbytotalC,N,andScontent,aswellasSspeciationandspectralindices.DissolvedorganicCandtotalNwerequantifiedusingaShimadzuTOC-V+TNM-1analyzerattheSwedishUniversityofAgriculturalSciences(SLU;Umeå,Sweden)afteracidificationofthesample(Triplicateanalyticalpercentrelativestandarddeviationwas0.85and2.36forDOCandtotalN,respectively).TotalsulfurwasdeterminedviaICPMS60.Briefly,1mLof10Mhydrochloricacidwasaddedtoadry,weighedDOMsamplefollowedby3mLof12Mnitricacid.Afteraseriesofsonicationandheatingsteps,1mLof30%(w/w)hydrogenperoxidewasslowlyaddedtothesolution.Whenthereactionterminated,thesampleswereheatedagainbeforedilutingforanalysisviaICPMSusingmagnesiumsulfateforthestandardcurve.TheCRM129(HayPowder)wasusedtoverifySrecovery(102.3±9.6%).

Between24and40µgCoffreeze-driedDOMwerereconstitutedin4mL0.1Mphosphatebuffer(pH7)todetermineopticalproperties.OpticalmeasurementswerecarriedoutwithaShimadzuUV2600(absorbance)andaHoribaAquaLog(fluorescence)withquartzcuvettes(10mmpathlength).Phosphatebufferwasusedasthemeasurementblank.Absorbancewasdeterminedbetween200and800nm(1nmincrement)andfluorescenceemissionwasdeterminedbetween250and820nm(increment~4.5nm)atexcitationwavelengthsbetween240and480nm(3nmincrement).FurtherdataprocessingwascarriedoutwiththedrEEMtoolbox(0.5.161).Inner-filtereffectswerecorrectedusingtheabsorbance-basedmethod62andRayleighandRamanscatterwerereplacedwithmissingnumbersandnotinterpolated.TherawfluorescencesignalsinarbitraryunitswerenormalizedtotheareaoftheRamanpeakat350nm.Humificationindex,fluorescenceindex,freshnessindex,andspectralsloperatioweredeterminedwiththedrEEMtoolboxasdescribedelsewhere63–66(Fig.S7).Parallelfactoranalysisofthefluorescence(PARAFAC)datawascarriedoutinconjunctionwiththeN-waytoolbox67(Fig.S8).Allmodelswereinitializedwithorthogonalizedrandomvalues,constrainedtononnegative scoresandloadings,andconsideredconvergedwhentherelativeimprovementinfitbetweeniterationswassmallerthan10−6.Betweenthreeandsixcomponentswereconsidered.Ultimately,afour-componentmodelexplained99.9%ofthedatasetwithacoreconsistencyof55%wassplithalf-validated.Itshouldbenotedthatthedatasetverylikelycontainedmorecomponents,butthesamplesizepreventedtherobustidentificationoffurthercomponents.ThefourPARAFACfluorescencecomponentswerenamedaccordingtotheiremissionpeakposition(C360,C390,C450,andC520).Duetothedifficultyofquantitativelyassessingthefluorescenceintensitiesinsolid-phaseextracts,allcomponentscores(i.e.,therelativecomponentabundances)werenormalizedtothesumofthefluorescencescoresineachsample.

SulfurK-edgeXANESdatawerecollectedonbeamline4B7AequippedwithaSi(111)double-crystalmonochromatoratthemid-energyX-raystationintheBeijingSynchrotronRadiationFacilities,China.Thestorageringwas2.2GeVandringcurrent100mA.ThedryDOMsampleswererubbedontosulfur-freetapeinathinlayerfortheanalysis.ThesamplewasthenmountedtothesamplecellandflushedwithHe.Measurementswereperformedunderhighvacuum(10−6to10−8mbar)atambienttemperatureandtheincidentX-rayenergyrangescannedwas2462to2500eVwithastepsizeof0.2eV.Thereferencecompoundsincludedsodiumsulfate(Na2SO4),sodiumsulfite(Na2SO3),elementalsulfur(S0),cysteine(-SH),methionine(−S=),sodiummethanesulfonate(CH3SO3Na),andironsulfide(FeS).AllchemicalswereanalysisgradepurchasedfromSigma-AldrichexceptforFeSwhichwaspurchasedfromAlfaAeser.TheresultingsamplespectrawereprocessedfollowingthemethodofYektaetal.68(Fig.S9).First,apolynomialpre-edgefunctionwassubtractedfromthespectrum.Thedatawerethennormalizedbetween2460and2490evusingthesoftwareWinXAS97.MicrosoftExcelwasusedtodeconvolutethecorrectedspectrumintosulfurpseudo-components(representingtypicalreferencecompounds)usingaleast-squarefittingprocedure.TheresultsaresummarizedinTableS5.

ThetotalconcentrationofDOM-RSHwasdeterminedontheDOMextractbytitrationwithmonobromo(trimethylammonio)bimanebromide(qBBr)andquantificationofexcessqBBrbyLC-ESIMS/MS33.

ThePCAplotsweregeneratedinRStudioversion1.4.1103usingtheprcomp{stats}function(statsversion4.0.3)69.Thedatawerecenteredandscaledusingargumentswithinthefunction.TheSspeciationdatainFig.2c,dincludedsevenSXANESpseudo-componentsaspercentagesoftotalsulfur,andtheindependentlyquantifiedtotalDOM-RSHconcentrationasapercentofmeasuredtotalS.AllboxplotswerealsogeneratedinRStudio.Thetopandbottomedgeofeachboxcorrespondtothefirstandthirddataquartiles,whilethecenterlinerepresentsthedatamean.Thewhiskersextendtothefurthestdatapointsnotconsideredoutliers,definedasvaluesbeyond1.5timestheinterquartilerange.

LC-ICPMSmethods
TheMeHg(Nacpen)complexwasmeasuredusingLC-ICPMS(PerkinElmerElanDRC)(adaptedfromLiem-Nguyenetal.70).TheLCcolumnusedwasaPhenomenexKinetexBiphenylcolumn(150mm×3mm×5mm)witha4×3mmPhenomenexguardcolumn.Thecolumnoventemperaturewassetto25°C.Themobilephasewas7%(v/v)1-propanolin0.5mMphosphatebuffer.Theliquidflowratewassetto0.4mL/minwitha0.1mL/minpostcolumnflowrateofasolutioncontainingthallium(10ng/mL)toassessthesensitivityoftheICPMSdaytodayandoverthecourseoftheexperiment.AMiraMistnebulizerandacyclonespraychambercooledto+4°Cwasused.Thenebulizerandauxiliarygasflowratesweresetto0.6and1.2L/min,respectively.ExternalcalibrationwasusedforcalculatingthesampleconcentrationsusingarangeofMeHg(Nacpen)standardconcentrationsin0.5mMphosphatebuffer,forwhichtheconcentrationwasverifiedusingadirectmercuryanalyzer.TheLC-ICPMSpeakswereintegratedmanuallyusingOriginPro9softwareandnormalizedtothethalliumsignalpriortocalculatingtheMeHg(Nacpen)concentrationsforboththestandardsandthesamples.Further,theMeHg(Nacpen)concentrationoftheMeHg(Nacpen)standardsusedforcompetingligandexchangeexperimentswasquantifiedforeachindividualsamplebatchbyICPMSwithanaveragerecoveryof116%.TherecoverywasaccountedforinthelogKcalculations(describedbelow).Moreinformationonblanksandrecoveriesisdetailedinthe‘CompetingLigandExchangeExperiments’sectionsbelow.

Gaschromatography(GC)-ICPMSmethods
SubsamplesforquantificationoftotalMeHgconcentrationwerecollectedafterthecompletionoftheLC-ICPMSanalysisandimmediatelypreservedbyacidificationto0.5%sulfuricacidandstoredcold.TotalMeHgconcentrationsweredeterminedusingisotopedilutionanalysisandmeasuredusinganAgilent7890GC(HP-1column,30m,0.320mmwidebore,1.00μmfilm)coupledtoanAgilent7700xICPMS.AnMe201Hginternalstandardwasaddedtothesamplesandallowedtoequilibratefor24hoursbeforedigestingwith1%(v/v)sulfuricacidforanother>12hours.4.5MacetatebufferwassubsequentlyusedtoadjustthesamplepHandtheMeHgwasethylatedusingsodiumtetraethylborateandextractedintotoluene.Onceallreagentswereadded,thesamplewasmixedforatleast1hourpriortotransferringtoGCvialsforanalysis.Thetoluenesampleswereinjectedtotheinstrumentasa1µLsplitsampleinjection.Theinjectiontemperaturewas150°CandtheGCseparationwasdoneisothermallyat80°C.TheICPradiofrequencypowerwas1350Wandthemonitoredisotopeswere200Hg,201Hg,202Hg,204Hgwithadwelltimeof30ms.Thetotalacquisitiontimewas252s,andthederivatizedMeHgretentiontimewas196s.ThesameMeHg(Nacpen)standardsusedtodeterminetheLC-ICPMPrecoverywerealsoanalyzedfortotalMeHgbyGC-ICMPSwithanaveragerecoveryof110%.Moreinformationontherecoveriesisdetailedinthe‘CompetingLigandExchangeExperiments’sectionsbelow.

CompetingligandexchangeexperimentsanddeterminationoflogKforMeHg(DOM-RS)
Standardcompetingligandexchangetechniqueswereusedintheoveralldesignoftheexperiment71,butwererefinedbythedirectquantificationoftheMeHgcomplexwiththecompetingligand,i.e.,MeHg(Nacpen),usingLC-ICPMS34 asdescribedabove.SolutionsofDOM,MeHg,andNacpen((CH3)2C(SH)CH(NHCOCH3)CO2H)weremadeunderanoxicconditions.Specifically,aknownmassoftheDOMsamplewasreconstitutedinNsparged0.5mMphosphatebuffersuchthatthefinalDOMconcentrationwas500mg/LandthepH7.5.Allsolutionswerekeptinthedarkandwererotatedovernightpriortofilteringto<0.2μmusingsyringefilters.Methylmercury(~100nMMeHgasaClspecies)andNacpenwereaddedsimultaneouslytotheDOM(~85mg/LDOM)solutions,mixed,andlefttorotatefor24hourspriortoanalysis.TheMeHgandDOMconcentrationswerekeptconstantacrosstheexperimentalsampleswhileNacpenwasaddedatsevendifferentconcentrationsbetween~0.05and50μMwithslightvariationsbetweenDOMtypes,andmostsamplesweremadeandanalyzedinduplicate(~14totalexperimentalsamplesperDOM).

Theanalyticalstandarddeviationbetweenexperimentalduplicateswasonaverage6nMfortheLC-ICPMSmeasurementsand3.8nMfortheGC-ICPMSmeasurements.Thecontrolsfortheexperimentincludedamatrixblank(buffer+2μMNacpen;averagefromallruns=1.3±2.5nMMeHg(Nacpen)measuredbyLC-ICPMSand4.9±6.7nMtotalMeHgmeasuredbyGC-ICPMS),amatrixblankwith82mg/LDOMadded(averagefromallruns=5.8±2.5nMMeHg(Nacpen)and6.6±6.8nMtotalMeHg),andaDOMblankwith82mg/LDOMand100nMMeHgadded(averagefromallruns=40±57nMMeHg(Nacpen)and93±11nMtotalMeHg).Intheory,noMeHg(Nacpen)shouldhavebeendetectedintheDOMblanksample.However,wefoundthatmemoryeffectsontheLCcolumnoccasionallycausedelevatedsampleconcentrationsabovewhatwasexpectedbasedonGCmeasurements.Therefore,theDOMblankwassubtractedfromtheMeHg(Nacpen)valueswhenelevatedinrespecttotheGC-ICPMSmeasurement,whichincludedtenDOMexperiments(blankrange=1.2–62nM,mean=15.9nM).AnewstocksolutionofNacpenwasmadeforeachcompetingligandexchangeexperiment.Therefore,astandardwithMeHginexcessofNacpenwasusedtoverifytheNacpenconcentrationofthestocksolutionandusedtocorrectthefinalNacpenconcentrationsinthespeciationcalculations.AsamplewithNacpeninexcessofMeHgwasalsoruntoverifytheMeHgconcentrationoftheworkingsolution,whichwasthawedfromafrozenstockbeforeeachuse(averagefromallruns=105±18.7nMMeHg(Nacpen)afterblankcorrectionand111±11.1nMtotalMeHg).Expectedconcentrationwas100nMMeHg(Nacpen)andtotalMeHg.

ToassureMeHgwasstableoverthetimeframeoftheexperiment,preliminaryexperimentswerecarriedoutthatshowedinsignificantdegradationovertheperiodofsevendays.EffectivelyallMeHgwasassumedtobecomplexedwithNacpenorDOM-RS−basedonspeciationmodelingresults(usingtheWinSGWsoftware).TheconcentrationofMeHg(DOM-RS)complexeswasthuscalculatedasthedifferencebetweenthetotalMeHgconcentrationdeterminedindependentlywithGC-ICPMS,andtheMeHg(Nacpen)concentrationdeterminedbyLC-ICPMS.TheconcentrationoffreeDOM-RSHligandswascalculatedasthetotalDOM-RSHconcentration,determinedbythemassofDOMaddedtothereactionandtheDOM-RSH/gDOMquantifiedbytheqBBr-titration,minustheconcentrationofMeHg(DOM-RS)complexes.

Theoverallcompetingligandexchangereactionisdescribedby:
MeHg(Nacpen) + DOM-RSH = MeHg(DOM-RS) + Nacpen; K1 = (K2/K3) (1)

basedonthefourindividualreactions:
MeHg+ + DOM-RS- = MeHg(DOM-RS); K2 = determinedinthecompetingligandexchangeexperiment (2)
MeHg+ + Nacpen- = MeHg(Nacpen); K3 = 1016.76 (3)
Nacpen-H = Nacpen- + H+; K4 = 10-9.6 (4)
DOM-RSH = DOM-RS- + H+; K5 = 10-9.6 (5)

Reactions4and5describetheaciddissociationreactionofthethiolgroupsinNacpenandDOM,respectively,andthenumericalvaluesofthecorrespondingconstantsweretakenfromLiem-Nguyenetal.70.TheconcentrationsofMeHgspecieswithCl−,PO43−andOH−werenegligibleintheseexperimentalsystemsbutthespecieswereneverthelessincludedinthecompletespeciationmodel(TableS3,Eq.(2)-(12)).ForeachindividualDOMsample,thechemicalspeciationofMeHgwascalculatedforthesevenNacpenadditions(0.05–20μM).Themodelwasbasedontheinputvariables:totalconcentrationsofMeHg,Nacpen,DOM-RSH,Cl−andPO43−,pH,andtheknownstabilityconstantsinTableS3(SITextModel1).ThestabilityconstantforMeHg(DOM-RS),i.e.,logK2,wasvariedbetween15and17.ForeachlogK2tested,arootmeansquareerror(RMSE)wascalculatedbetweentheMeHg(Nacpen)concentrationsdeterminedbythespeciationmodelingandbythedirectLC-ICPMSmeasurements.TheRMSE’swereplottedversusthelogK2values,fitagainstaquadraticformula,andsolvedforthelogK2achievingthelowesterror.ThedeterminedvaluesofK2arethuslinkedtotheconstantsK3–K5andthecompetingligandexchangeexperimentaluncertaintyismainlydeterminedbytheuncertaintyinthemeasuredMeHg(Nacpen)concentration,whichtypicallywasupto20%.WeestimatedtheexperimentaluncertaintyinlogK2bynumericallyvaryinginthecalculationschemetheMeHg(Nacpen)concentrationwithin20%ofthemeasuredvalue.ThisresultedinanaverageuncertaintyinlogK2of±0.19,whichisaverysimilaruncertaintyasinpreviousstudiesdetermininglogKforinorganicHgcomplexeswiththiolcompoundsusingcompetingligandexchange-basedapproaches34,35,72.

KineticsoftheMeHgligandexchangereaction
Thekineticligandexchangeexperimentwasset-upinasimilarwayasthecompetingligandexchangeexperiment,butoneisotopeofMeHgwaspre-equilibratedtotheDOM(Me204Hg)andanotherwaspre-equilibratedtoNacpen(Me200Hg)forfourdayspriortomixingandanalyzing.TheMeHgisotopestandardswereaddedata2:1ratiointhefinalmixture(100nMMe204Hg,200nMMe200Hg).FiveDOMsamplesweretested;PAW(riverine),NB(estuarine),BIandSI(marsh),andSB(shelfbreak).Threedifferentcompetingligand(Nacpen)concentrationsweretestedsuchthattheratioofDOM-RSHtoNacpenwasapproximately1:0.1,1:1,and1:10.Immediatelyaftermixing,asamplewasremovedandtheMeHg(Nacpen)concentrationforbothMe204HgandMe200HgwasdeterminedviaLC-ICPMS.Sampleswerethenremovedfromtheanaerobicchamberandanalyzedeverytenminutes(thelengthofoneanalysisrun)forthefirsthourofthereaction,andthenperiodicallyoverthecourseofthefollowingfivedaystoensurethesampleshadreachedequilibrium.The204Hg/200HgisotoperatioforMeHg(Nacpen)(RMeHg(Nacpen),determinedbyLC-ICPMS)wasdividedbythe204Hg/200HgisotoperatiofortotalMeHginthesystem(RtotalMeHg,determinedbyGC-ICPMS)andplottedversustime(Fig.S2).Atequilibrium,thetwoisotoperatiosequal,i.e.,RMeHg(Nacpen)/RtotalMeHg=1.0.Thedeviationfrom1.0atthestartoftheexperimentisameasureof“non-exchangeable”MeHg(Nacpen)toMeHg(DOM-RS)ratioatsteady-state.Suchdeviationwaslessthan10%forallthetestedDOMsamples(Fig.S2).Pseudo-firstorderrateconstantsforMeHgligandexchangebetweenNacpenandDOC-RSHwascalculated(Rsoftware)basedonthemeasuredMe204Hg(Nacpen)molefractionoftotalMe204HgfollowingSkrobonjaetal.23:

f(Me204Hg(Nacpen)) = (kf / (kf + kr)) * (1 - e-(kf+kr)t) (6)

wherekfistherateconstantfortheforwardreaction(Me204Hg(DOM-RS)→Me204Hg(Nacpen))andkrforthereversereaction(Me204Hg(Nacpen)→Me204Hg(DOM-RS)).Theturnover((kf+kr)−1)wasthencalculatedfromthetwoconstants.

MeHgcellularuptakeexperiment
Artificialseawater(psu=35;19.52g/LCl−,11.94g/LNa+,5.27g/LSO42−,1.33g/LMg2+,0.36g/LCa2+,0.42g/LK+,0.36g/LHCO3−;modifiedfrom73)wasmadeusingacidcleanedglasswareandpre-combustedsaltstomaintainalowDOCcontamination.DOMextractfromsitesSIorORwasaddedtotheartificialseawatertofinalDOCconcentrationsof50,150,and500µM,equatingto17–170and2.2–22nMDOM-RSHforSIandOR,respectively.MeHgwasaddedtotheDOMsolutionstoafinalconcentrationof10pM,allowedtoequilibrateovernight,andthensplitintotriplicate200mLaliquotsforeachDOMtypeandDOCconcentration.Aftertheequilibrationperiod,2.4×107cellsofThalassiosirapseudonanawereaddedtoeach200mLflask(1.2×105cells/mL),determinedbycountingthecelldensityofthestockculturewithaCoulterMultisizerIIe,and30mLsofthedilutedculturewasimmediatelyfilteredtoobtaintheinitialMeHgparticulateanddissolvedconcentrations.Thecellswereincubatedunderfullspectrumlightsfor4hours,then30mLsfromeachflaskwerefiltered.Thefilterswerepreservedbyfreezingwhilethefiltratewaspreservedwith1%sulfuricacid.ThefinalcellcountoftheincubatedculturewasdeterminedusingaCoulterMultisizerIIe.TheMeHgsampleswereprocessedandanalyzedfollowingstandardtechniques74,75. Briefly,thefiltersweredigestedin4.5Nnitricacidovernight.Adigestaliquotwasneutralizedwithpotassiumhydroxideandacetatebufferandethylatedusingsodiumtetraethylborate.ThesamplewasthenmeasuredonaTekran2700AutomatedMethylmercuryAnalysisSystembygaschromatographyandcoldvaporatomicfluorescencedetectionwithcalibrationagainstastandardcurve(AlfaAesarCAS:115–09–3,LOT:1791821).TheMeHgconcentrationwasabovetheanalyticaldetectionlimitof0.004ng/L(range=0.018–0.532ng/L),andanalyticalduplicateshadRSDsof15±6.0%(n=4)foralltheparticledigests.DissolvedMeHgsampleswereanalyzedsimilarlyexcept2.5%L-ascorbicacidwasaddedwhenthesamplewasneutralized75(AlfaAesarCAS:115–09–3,LOT:1791821).Allsampleswereabovetheanalyticaldetectionlimit(0.35–1.05ng/L)butwerenotruninduplicate.Treatmentreplicates,however,hadanaverageRSDof11±5.3%andspikerecoveriesof97±24%.ThesuspendedparticulateMeHgconcentrationswereconvertedtonmolMeHg/cellvolumefortheVCFcalculation.Toso,acelldiameterof8µmwasused(113.1µm3)basedontheaveragecellsizefromtheCoultercounter,andtheinitialMeHgcellularconcentrationsweresubtractedfromthefinalcellularconcentrations.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper addresses a long-standing question in the mercury community about why the effects of DOM on mercury uptake by phytoplankton are so variable. The authors use a variety of techniques to demonstrate that the amount of DOM-associated thiol groups is really key to predicting algal uptake of MeHg. In demonstrating this point, the authors also ruled out a couple of other factors that researchers have considered potentially important (e.g., Cl- concentrations and kinetic control of MeHg partitioning). As such, the paper is an important contribution to the literature. The conceptual model diagramed in Fig. 5 is also a very nice mechanistic contribution to the field, showing how both DOC concentrations and thiol groups associated with the DOM vary across a continuum of sites from marsh to river to estuaries to continental shelves.\nHowever, the ability of this paper to achieve its full impact is hampered by how difficult it is to read. The technical content and methodology are outstanding, but much, much more attention is needed to present the material in a digestible format to the reader. This paper will be of wide interest to the mercury community, not just those who are experts in Hg/DOM interactions so the authors should spell out some of the implications to the reader, including giving an occasional background sentence. In addition to a little more explanation, a fair bit of work is needed to improve the writing, such as grouping material into paragraphs with clear topic sentences. The table and figures also need improvements to make them more understandable. Some of the text also seems like it should be moved from the results to the discussion. To this end, many of the comments below are designed to address the readability of the paper or to point out places where the implications should be discussed in more detail.\nAbstract\nLine 23: Awkward punctuation with the “e.g.” Adding a comma and parentheses would help, as in “. . . in aquatic food webs (e.g., following various Hg emission and environmental change scenarios). But better yet combine into a single sentence, “. . . we formulate a generalizable MeHg bioavailability model to predict MeHg incorporation in aquatic food webs under various emission and environmental change scenarios.”\nTry to work some of the major numbers from the paper into the abstract. The finding on line 141 that the DOM-RSH (nM) thiols varied by a factor of 40 while the DOC only varied by a factor of 5 was a key one for me, but is not really reflected in the abstract.\nThe paper also emphasized that Cl- speciation is not controlling MeHg bioavailability, but this finding is not emphasized in the abstract. Instead, the abstract says that salinity ranged from 0.5 to 36 ppt (check the units here). Stating the salinity in the abstract makes it wound like salinity is important, when from my reading of the paper, it is really about the DOM composition.\nWork on summarizing the results, not just what was studied. For example, line 18 states, “We show that the concentration of DOM-associated thiol functional groups (DOM-RSH) dictates MeHg speciation and bioavailability across the terrestrial to marine aquatic continuum.” It would be better if you gave the direction of the relationship (and maybe even the magnitude), as in, “As the RSH-DOM ratio decreases from terrestrial to marine systems, there is a resulting decrease in MeHg bioavailability to phytoplankton.”\nTo help illustrate how the abstract could be improved, see the classic piece by Kenneth Landes, A Scrutiny of the Abstract. I’m including the link here in case it is of use to graduate students/post docs involved with this project:\nhttps://www.caam.rice.edu/~symes/CAAM600/abstract_scrutiny.pdf\nIntroduction\nThe introduction is very clearly framed and succinctly summarizes the existing literature.\nLine 27. Work on succinctness. For example, “It is estimated that anthropogenic Hg emissions have caused a three-fold increase in Hg content in surface marine waters compared to pre-industrial conditions . . .” could become, “Anthropogenic Hg emission have caused a three-fold increase . . .”\nResults\nLine 86. Remove the comma after et al., in “. . . Dittmar et al., (2008) and . . . ”\nBreak up the long paragraph that begins on line 80. Lines 80 – 89 are a brief summary of methods. Start the results of the PCA analysis in a new paragraph and improve the topic sentence on line 90 so that it is clear that you will have a paragraph just on the spectral data. Use a different paragraph for the S speciation data.\nLines 90 – 96. Were there two separate PCA analyses done here? One on the spectral data and one on the S speciation? Some of the language in the text is confusing. For example, line 95 states, “The second PC described 27% of the data variability and discriminated fluorescent DOM by origin (C390 bacterially derived material, C520 terrestrially dominated material).” I think this is the first PCA analysis, but the second PC, but I had to closely study Fig. 2 to check this. If indeed there are two separate PCA analyses done here, describing each in its own paragraph would help considerably. You may even want to consider subheaders, such as paragraph header.\nOK, as I’m reading along to line 103, I am seeing that there are two PCAs here. Not introducing the second PCA on line 92 would help. Wait and introduce the second PCA and Figure 2c and d on line 103 when you give the results for this PCA.\nLine 112 – the huge variability between the estuarine sites is really interesting here and the explanation about mixing between both terrestrial and marine algal DOM makes sense.\nLines 146 – 168 – break up this long paragraph, perhaps by starting a new summary paragraph on line 161, with “Combined, these results. . .”\nLine 167. Correct punctuation is “. . . experiments are, however, not straightforward. . .”\nLine 201. The two isotopes were a nice approach to determine that the non-exchangeable pool of MeHg was very small.\nLine 244. Can you put the reference to the relevant figure or table at the end of this sentence?\nLine 263. Ah, it is so nice to see all of the variability in MeHg VCFs explained in terms of DOM-RSH.\nLine 270. Oh no, it is hard to even begin to read this giant paragraph. Use paragraphs of 3 – 7 sentences to help your reader! Consider paragraph breaks on line 277 with the thermodynamic modeling. Make line 285 saying that MeHg speciation is not driven by Cl- concentration the topic sentence of a paragraph on your thermodynamic results. The importance of Cl- has been brought up enough in the literature that this result should be easy to find in the topic sentence. Can you move some of this material into the discussion? Some of the interpretation, such as that the role of Cl- in controlling MeHg speciation has been overstated, sounds like it is more discussion than results.\nLine 297. I can’t wait to see your next study, where you include the open ocean. But again, I think this material is discussion, not results.\nLine 309. Remember to offset “however” with commas.\nThe extensive use of acronyms in this manuscript don’t help with readability. For example, N-Acetyl-Penicillamine is abbreviated Nacpen. Even though this acronym is used a lot during the manuscript, it was not one that I knew so it complicated the reading. Completing ligand exchange is CLE. While some of these acronyms (e.g., DOM, BCF, etc.) are quite common in the field, I had to repeatedly refer back to others. Could you systematically go through this manuscript and try to remove a couple of the less common and/or less frequently used acronyms so that the paper reads less like alphabet soup?\nReadability is also hampered by some of the notation. For example, the paper refers both to the RSH/DOC ratio and also to the DOM-RSH, which is not a ratio, but is the concentration of DOM-associated thiol groups (line 19). Is there some clearer way to display DOM-RSH so the poor reader is not getting mixed up between the ratios and the concentration of thiol groups? Maybe just call it thiol concentration?\nDiscussion\nLine 319. It should be “. . . how DOM controls MeHg bioavailability. . .”\nLine 319. I think you are missing the word “of” and the tense is wrong. It should be “. . . a quantitative assessment of how DOM controls MeHg bioavailability. . .”\nLines 344 – 368. Can you separate out your discussion of how MeHg is taken up by phytoplankton cells and implications for Fig. 4 from your discussion of Fig. 5.\nFigure 5 is a very interesting figure and nicely summarizes the paper, but is only mentioned briefly on lines 345 and 351. For such an important visual summary, this figure merits more play. Discuss it in its own paragraph. Also discuss it in light of the literature. For example, do field studies support the idea that shelf systems have the highest MeHg in algal cells? Does this translate up the food chain? And how does it tie in with the common observation that oligotrophic lakes often have very high MeHg concentrations in fish?\nLine 374. Spell out what you mean by “potentially opposite impacts on MeHg formation and bioaccumulation.” Which anthropogenic alteration is likely to have which effect?\nLine 378. Are you anticipating an increase in DOC overall or a change in the RSH/DOC ratio? Would changing the concentration of both RSH and DOC without changing the ratio change VCFs?\nLine 380. How would an algal bloom change the RSH/DOC ratio? Why would terrestrial runoff increase RSH/DOC ratio?\nThe results of the PCA analysis are never mentioned in the discussion. How does the PCA analysis relate to the material in the discussion on MeHg bioavailability to phytoplankton?\nOverall, spend some more time on the implications of the importance of the DOM-associated thiols. I think what I’m hearing is that measuring DOC is not really sufficient to predict bioavailability to phytoplankton. Instead, XANES is needed to quantify the number of thiol groups associated with the DOM. But most researchers don’t have XANES! So what would you recommend for researchers seeking to study global environmental change and for monitoring programs that still use DOC?\nMethods\nLine 418 sounds like it is statistical methods – separate it out into a new paragraph.\nCheck punctuation on line 439.\nLines 479- 515. Break up this long paragraph.\nLine 499. What was the general magnitude of the DOM blank that had to be subtracted?\nLine 564. Salinity units of ppt are not appropriate -see comments under Table 1.\nTable 1.\nSalinity in ppt is a rather old notation and not consistent with the use of a salinometer to measure it. When measured with a meter, salinity is measured as conductivity relative to a standard, which makes it unitless. Those who hate unitless numbers use practical salinity units (psu). In contrast, latitude and longitude do have units, which are not currently shown in the table.\nFigure 1\nPlease add a few place names to the map, to orient people who are not very familiar with the East Coast of the United States. Also consider the legibility of this map when printed in black and white or viewed by people who are color blind. In addition to using colors for habitat type, use different symbols (e.g., triangles, squares, etc.) to distinguish between these habitats.\nFigure 2\nPlease make the legend and axes and site names larger. I can hardly read them, even blown up 150% and when they are published in the journal, they are going to be even smaller.\nLabel some of the individual panels, such as with Fluorescence spectral data and the other with S speciation. The current labeling just says, “variables – PCA” which is not particularly helpful. Improve this heading. Write out the x-axis labels in panels b and d. I’m assuming that Dim1 is dimension 1? You are really making the reader work hard to interpret these panels. Write out axes labels and use the text and caption to help the reader interpret the figure. The addition of the oxidation states on panel d definitely helps, but add a sentence about the dimensions, particularly linking the dimensions, the loadings, and the contributions. I do not understand the scale of the “contributions” on 4'2+06 ( '2* *# & '1 86+* 73 7-.2/.2, '(387 )31432+27 03'*.2", "reviewer_2": "Reviewer #2 Attachment on the following page\nThe manuscript is entitled \"Dissolved organic matter thiol concentrations determine\nmethylmercury bioavailability across the terrestrial-marine aquatic continuum\"\nGeneral comments:\nThis is a manuscript which deals with the role of the DOM-Thiol groups for the\ncomplexation and bioavailability of MeHg across different aquatic systems. The\nauthors determined the thermodynamic and kinetic interactions between MeHg and\nDOM-Thiol groups extracted from varied aquatic sites. The results are quite\ninteresting to me to see such a good relationship between RSH concentrations and\nVCF MeHg in phytoplankton (Anyway, is there any relationship between them if we\nremoved only one data of shelf sea in Fig. 4a). Additionally, the observation that K\ndoesn’changetoomuchisprettyvaluable.However, one big gap is that the DOM\ntheyarestudyingishighmolecularweight…so,thisisonlyapartofthestory.The\nstrategy of the sampling also lacks the shelf data although the trends with that point\nmake sense. Nature Comm is short like Nature. This seems like a pretty long paper,\nfull of small details. I think it’s well done and pretty important. I indeed learned a lot\nfrom this paper related to potential mechanisms How MeHg gets into the cell of\nphytoplankton.\nSome minor comments:\n=============================================================\nLine 118, “the chemical speciation and bioavailability of dissolved MeHg…” This is\nkind of a hypothesis. To be sure, this paper helps support this hypothesis, but this\nstatement sounds too categorical.\nLine 122 “DOM binding capacity” is a little too ambiguous a term in this context.\nSince it is DOC normalized, consider using something like “DOM-specific binding\ncapacity” or something.\nLine 129-132, “binding affinity…” citation for this method?\nLine 133, how do these results compare to total Hg? Wouldn’t we expect that their\nconcentrations would be similar?\nLine 199, the ambient ligands come out looking like Nacpen…this is what happens\nusing CLE. Any comments on analytical “windows” that are created by this approach?\nLine 224, complexation for MeHg sounds faster than for Hg(II)…any comments?\nLine 261, “The results demonstrate that the total DOM-RSH…” The word\n“demonstrate” is a little too strong here. Change to “the results are consistent with\nthe hypothesis that…”\nLine 394, talk about the limitations of Dittmar approach…this is mostly high\nmolecular weight material. By ignoring LMW thiols, the importance of complexation\nis underestimated a bit. But, LMW thiols have been reported to be especially\nbioavailable…so, the MW/LW distinction might be important to discuss.\nLine 463, citation for this method?\nLine 522, citation for the K6 value?\n============================================================="}, "author_response": "We thank the reviewers for their positive overall assessment of our manuscript and for\ntheir valuable specific remarks and constructive criticism. We have carefully revised the\nmanuscript based on the specific comments by the reviewers.\nBoth reviewers made a remark regarding the data point with highest VCF value in figure 4.\nIt turned out that this data point was incorrect due to a calculation error. We are very\ngrateful for the reviewers’ remarks and we have gone through the entire data set of the\nmanuscript to ensure there are no further errors. The rectification of the incorrect VCF\ndata point does not affect the key findings of the manuscript, i.e. that DOM associated thiols\ncontrol MeHg bioavailability and is a much stronger predictor for MeHg VCF than DOC is.\nRevision of the VCF data set, however, affects the theoretical interpretation of the observed\nrelationship between DOM-RSH concentration and VCF. In the revised manuscript, we\npresent one thermodynamic and one kinetic model (Lines 280-308 and Supporting\nInformation Figure S5) describing how DOM-RSH control MeHg uptake and we note that\nthe kinetic model fits our experimental results the best. We have further revised the layout\nof Figure 4 by plotting VCF versus the concentration of DOM-RSH (Fig. 4a) or of DOC\n(Fig. 4b) instead of plotting VCF versus 1/DOM-RSH or 1/DOC as in the original\nmanuscript. We have also revised the relevant text in the Discussion and Abstract to\nharmonize with the revised Figure 4 and MeHg uptake models.\nAdditional major revisions made to the manuscript in response to reviewer comments are\nto merge the Results and Discussions into a joint section and to revise Figure 5 to more\nclearly communicate the three core messages of the manuscript regarding the role of DOM-\nRSH in controlling MeHg bioavailability.\nIn our point-by-point responses below our specified line numbers refer to the revised\nmanuscript without track-changes indicated."}]

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