Sulfide and iron control on mercury speciation in anoxic estuarinesediment slurries

Seunghee Han a,⁎, Anna Obraztsova a,b, Patrizia Pretto c, Dimitri D. Deheyn a,Joris Gieskes a, Bradley M. Tebo a,d

a Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202, USAb Department of Earth Sciences, University of Southern California, Los Angeles, California 90089, USAc Department of Histology, Microbiology and Medical Biotechnology, University of Padova, Via A. Gabelli 63, 35121 Padova, Italyd Department of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, Beaverton, Oregon 97006, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 September 2007Received in revised form 8 May 2008Accepted 20 May 2008Available online 9 July 2008

In order to understand the role of sulfate and Fe(III) reduction processes in the net production ofmonomethylmercury (MMHg), we amended anoxic sediment slurries collected from the VeniceLagoon, Italy, with inorganic Hg and either potential electron acceptors or metabolicbyproducts of sulfate and Fe(III) reduction processes, gradually changing theirconcentrations. Addition of sulfide (final concentration: 0.2–6.3 mM) resulted in anexponential decrease in the sulfate reduction rate and MMHg concentration with increasingconcentrations of sulfide. Based on this result, we argue that the concentration of dissolvedsulfide is a critical factor controlling the sulfate reduction rate, and in turn, the net MMHgproduction at steady state. Addition of either Fe(II) (added concentration: 0–6.1 mM) or Fe(III)(added concentration: 0–3.5 mM) resulted in similar trends in the MMHg concentration, anincrease with low levels of Fe additions and a subsequent decrease with high levels of Feadditions. The limited availability of dissolved Hg, associated with sulfide removal byprecipitation of FeS, appears to inhibit the net MMHg production in high levels of Feadditions. There was a noticeable reduction in the net MMHg production in Fe(III)-amendedslurries as compared to Fe(II)-amended ones, which could be caused by a decrease in the sulfatereduction rate. This agrees with the results of Hg methylation assays using the enrichmentcultures of anaerobic bacteria: whereas the enrichment cultures of sulfate reducers showedsignificant production of MMHg (4.6% of amended Hg), those of Fe(III), Mn(IV), and nitratereducers showed no production of MMHg. It appears that enhanced Fe(III)-reduction activitiessuppress the formation of MMHg in high sulfate estuarine sediments.

© 2008 Elsevier B.V. All rights reserved.

Keywords:MercuryMethyl mercurySulfate reductionSedimentsItaly, Venice Lagoon

1. Introduction

Monomethylmercury (MMHg), a highly toxic and bioaccu-mulating form of Hg, is produced from inorganic Hg by micro-organisms, primarily sulfate-reducing bacteria (SRB; Compeauand Bartha,1985; King et al., 2000). Anoxic sediments appear to

be significant sources of MMHg to food webs in coastal zonesbecause of the presence of large amounts of Hg derived fromanthropogenic activities, aswell as the occurrence of active SRB(Fitzgerald et al., 2007). Thus,MMHgproduction, coupled to thebiogeochemical conditions of sediments, has been investigatedinnumerousestuarine and coastal sediments (Hammerschmidtand Fitzgerald, 2004; Han et al., 2007b; Mason and Lawrence,1999; Niessen et al., 2003; Sunderland et al., 2006). Thesestudies commonly reported that the total Hg concentration isnot a proper predictor of the MMHg concentration and thatbiogeochemical factors should be considered. In particular, the

Marine Chemistry 111 (2008) 214–220

⁎ Corresponding author. Tel.: +1 858 822 3140.E-mail address: s7han@ucsd.edu (S. Han).

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bioavailability of Hg, which is affected by the solubility andchemical speciation of Hg, has been suggested as a majorcontrolling factor for MMHg production in anoxic sediments(Benoit et al., 1999a,b; Drott et al., 2007; Hammerschmidt andFitzgerald, 2004).

The sulfate reduction rate, an indication of the activity ofSRB, in surface (0–2 cm) estuarine sediments is variable,ranging between 0.02 and 3 μmol cm!3 d!1 (King et al., 2001;Marvin-DiPasquale and Capone, 1998; Pallud and van Cap-pellen, 2006). The large range of bacterial activity isattributable to the variation in quantity and quality of organicmatter, SRB abundance, temperature, and sulfate availability(Pallud and van Cappellen, 2006). A recent study in theScheldt estuary (Belgium, the Netherlands) has shown thatthe sulfate reduction rate in low-salinity sediments is as highas that in high-salinity sediments, despite low sulfateavailability and a smaller SRB density, because of theavailability of labile organic matter (Pallud and van Cappellen,2006). The reliance of the sulfate reduction rate on sulfateavailability has been demonstrated in the vertical profiles ofsulfate reduction rates in marine sediments: the sulfatereduction rate decreases in deep sediments where sulfate islimited (Leloup et al., 2007; Roden and Tuttle,1993). However,this is likely a minor factor in the surface of marine sediment.

If the metabolic activity of SRB remains constant, thebioavailability of Hg could be the primary factor controllingMMHg production. The role of solid organic matter indissolved Hg availability has been reported in numerousestuarine and coastal sediments (Hammerschmidt andFitzgerald, 2004; Mason and Lawrence, 1999; Sunderlandet al., 2006). However, the role of solid FeS in controlling thedissolved Hg availability has not been widely investigated,despite its widespread occurrence in estuarine sedimentsand its well-known importance for the bioavailability of B-type metals (e.g., Zn2+, Cd2+, Pb2+, and Hg2+) in anoxicsediments (Ankley et al., 1996; Morse and Luther, 1999). Insediments from the Venice Lagoon (Italy), there is a negativeexponential relationship between dissolved Fe and Hg, whichmay be attributable to the association of Hg with solid FeS(Han et al., 2007b). In sediments of the Penobscot Riverestuary (Maine, USA), Hg associated with acid volatile sulfiderepresented a consistent fraction of solid phase Hg within thezone of Hg consumption, suggesting that dissolved Hg lost islikely associated with solid FeS (Merritt and Amirbahman,2007).

In our previous literature, we reported that Fe(III) andsulfate reductions are dominant electron-accepting processesin the surface of Venice Lagoon sediments (0–20 cm) andvariation of Hg methylation rate is attributable to thevariation of these two processes (Han et al., 2007b). In thepresent study, we measured MMHg concentration, the sulfatereduction rate, and dissolved Hg, sulfide, sulfate and Feconcentrations in geochemically amended anoxic sedimentslurries, in order to understand the control of Fe(III) andsulfate reduction processes in the net production of MMHg.Anoxic sediments obtained from the Venice Lagoon wereamended with either potential electron acceptors (sulfate orhydrous ferric oxide) or metabolic byproducts of sulfate andFe(III) reduction processes (sulfide or ferrous Fe). Our resultswill illuminate how sedimentary biogeochemistry andMMHgformation are interconnected.

2. Materials and methods

2.1. Sediment sampling

Sediment samples were obtained from Sites SS0 and V1 ofthe Venice Lagoon, Italy, using 30 cm polypropylene cores inJuly 2006, as part of a periodic sampling activity of the SIOSED(Scripps Institution of Oceanography SEDiment researchgroup) project. Sites SS0, a dredged channel site, and V1, anexperimental bank site constructed with dredged SS0 sedi-ments, are located approximately 3 and 1 km north ofMalamocco Inlet, respectively (see the map in Han et al.,2007b). Salinities in the sampling areas were 36 and 34 at V1and SS0, respectively.

Collected sediment cores were extruded and sectioned in0–2.5, 2.5–5, 5–7.5, 7.5–10, 10–15, and 15–20 cm intervalswithin 12 h in a N2-filled glove box. After sectioning cores,approximately 10 g of sediments were stored frozen formeasurement of Hg and MMHg, and approximately 100 g ofsediments were used for pore water extraction by means ofcentrifugation at 5000 rpm. Leftover sediments were storedin 4 °C for slurry experiments. Following centrifugation,filtration of pore water samples was performed underanaerobic conditions and filtered samples were stored formeasurement of dissolved Fe, sulfide and sulfate. Dissolved Fesamples were treated with HCl (0.5%) and dissolved sulfidesamples were treated with Zn acetate solution (20 mM).

2.2. Sediment slurry experiments

In order to prepare the stock of sediment slurries,approximately 200 g of sediment, from either Site SS0 or V1(0–20 cm), was mixed with 800 cm3 of filtered coastal water,collected off Scripps Pier in La Jolla, California (salinity 35), inan anaerobic glove box. Seawater was purged with N2 gas for30 min prior to the sediment addition. The redox indicator,resazurin, was added to aliquots of the sediment slurries. Eachset of experiments (sulfate, sulfide, ferric and ferrous Feadditions) was performed after resazurin became clear.Sulfide and sulfate addition experiments were performedsimultaneously with SS0 slurries, and ferric and ferrous Feaddition experiments were performed simultaneously withV1 slurries.

Sulfide (Na2S·9H2O), sulfate (Na2SO4), and ferrous Fe(FeCl2·4H2O) solutions were prepared to final concentrationsof 0.05 and 1 M with N2-purged and filtered seawater.Hydrous ferric oxide (HFO) slurries (0.05 and 1 M) wereprepared as described in Miller (2006). Briefly, Fe(NO3)3 wasadded to a 0.1 M NaNO3 solution followed by the addition ofNaOH to adjust the solution to pH 7. Then this mixture wasequilibrated for several hours and degassed before it wasadded to the sediment slurries. Concentrations of ferric andferrous Fe solutions were determined spectrophotometrically(Gieskes et al., 1991). A working solution of HgCl2 wasprepared in N2-purged and filtered seawater to final con-centration of 50 μM and allowed to equilibrate with naturalligands for 24 h.

From stock slurries, six aliquots of 30 cm3 of the sedimentslurries were placed in six 50 cm3 Teflon vials, then each vialwas amended with 1 M TRIS buffer (pH 8, 1.5 cm3), 50 μMHgCl2 solution (3 cm3), and either sulfate, sulfide, ferric, or

215S. Han et al. / Marine Chemistry 111 (2008) 214–220

ferrous Fe solutions (0.05 and 1 M), with an increase inconcentration (sulfate 0–20 mM, sulfide 0–8.7 mM, Fe(III) 0–3.5 mM, and Fe(II) 0–6.1 mM). In slurries where highconcentrations of sulfide (N3 mM) were added, the final pHwas higher than 8, thus HCl solution was added to maintainpH 8. For the same reason, NaOH solution was added in highlevels of Fe(II)- and Fe(III)-amended slurries. After the sampleslurries had equilibrated for 48 h at room temperature (24 °C),4 cm3 aliquots of the slurries were placed in serum bottles forthe measurement of the sulfate reduction rate. Solid andaqueous phases of the remaining sediment slurries wereseparated by centrifugation (20 min, 3500 rpm), and thesolution phases were collected and filtered using 0.45 μmsyringe filters. Dissolved Hg and Fe samples were treatedwithHCl (0.5%), and dissolved sulfide samples were treated withZn acetate (20 mM). Solution preparation, sediment slurryincubation, and filtration processes were all carried out in anN2-filled glove box.

2.3. Mercury methylation assays in enrichment cultures

In order to develop enrichment cultures of anaerobicbacteria, 0.2 g of sediments, collected from the Venice Lagoon,were inoculated in 20 cm3 of anoxic seawater medium(Widdel and Bak, 1992). The medium was amended with aformate solution (20 mM) as an electron-donor and eitherNa2SO4 (10 mM), NaNO3 (20 mM), FePO4 (40 mM), amor-phous Fe oxide (40 mM), or amorphous Mn oxide solutions(40 mM) as electron acceptors. Growth of bacteria was testedby measuring bacterial byproducts, such as Mn(II), Fe(II) andHS!, or measuring optical densities of bacterial cultures. ForHg methylation assays, each bacterial culture (20 cm3) wasspiked with inorganic HgCl2 solution to a final concentrationof 5 nM. For time 0 controls, aliquots of cultures werecollected immediately after the Hg(II) additions, and frozen at!80 °C after acidification to 0.2% HCl. All assays wereincubated for 6 h at room temperature, and stopped byadding HCl solution (0.2%) and freezing at !80 °C.

2.4. Sample analysis

The methods for the determination of dissolved iron andsulfate have been described by Gieskes et al. (1991). Dissolvedsulfide was determined by a modification of the spectro-photometric method described in Strickland and Parsons(1972).

Sulfate reduction ratemeasurement involves incubation of35SO4

2! with sample sediments, extraction of 35S-sulfideproduced, and quantification of the 35S-sulfide (Ulrich et al.,1997). Four cm3 of sediment slurry sample was placed intoserum bottles under anaerobic conditions and Na235SO4 wasinjected. Serum bottles were incubated for 2 days at roomtemperature, after which a test tube containing 2.5 cm3 of 10%(w/v) anoxic zinc acetate solution was placed into the serumbottles, and the bottles were injected with anoxic 1 M Cr(II)-HCl (8 cm3) and 12 N HCl (4 cm3) solution and gently agitatedfor 2 days. The liberated H2S precipitated as ZnS in thetrapping solution, and 35S-ZnS was quantified by scintillationcounting.

For the analysis of total Hg in sediments, approximately0.5–1 g of sediment was digested overnight in Teflon® bottles

with 8 cm3 of HCl (12 M) and 2 cm3 of HNO3 (14 M) anddiluted with 500 cm3 of Milli-Q water. For the analysis ofdissolved Hg in slurry water, acidified (0.5% HCl) sampleswere treated with ultraviolet irradiation prior to analysis(Choe et al., 2004). Monomethylmercury in sediments (0.5–1 g) was extracted into the organic phase following a three-hour reaction with 5 cm3 of acidic KBr solution, 1 cm3 of 1 MCuSO4 solution, and 10 cm3 of CH2Cl2. Back-extraction ofMMHg from the organic phase into the aqueous phase wascarried out using the distillation method described in Choeet al. (2004). For the MMHg analysis in enriched cultures,5 cm3 of samples were treated with the aqueous-phasedistillation prior to instrumental analysis (Choe et al., 2004).Total Hg and MMHg were quantified by cold vapor atomicfluorescence spectrometry. The water content in sedimentswas determined separately to transform the concentrationsof Hg and MMHg in g-wet sediment to g-dry sediment.

3. Results and discussion

3.1. Geochemical parameters of sampling sites

Vertical profiles of sulfate, sulfide, and Fe in pore waters of the V1 coreshow that active sulfate reduction occurs during organic matter oxidation(Fig. 1). Distribution of dissolved Fe appears to be restricted by sulfide,possibly due to the precipitation of FeS. Total Hg in dry sediment, rangingbetween 3.0 and 5.4 nmol g!1, agrees to the typical Hg contents incontaminated sites of the northern Venice Lagoon (Han et al., 2007b). Thefraction of MMHg in total Hg shows relatively constant vertical distributionwith the exception of the surface. Enhanced activity of SRB and/or increasedbioavailability of Hg may cause the higher production of MMHg at thesurface. It is noted that %MMHg/total Hg is generally lower in Site V1 whencompared to other estuarine sediments (0.06–0.74%; Fitzgerald et al., 2007)and other sites in the Venice Lagoon sediments (0.08–0.3%; Han et al.,2007b). Throughout the SIOSED project (from June 2005 to February 2007),Site V1 consistently showed low solubility of Hg in surface 20 cm: thelogarithm of the particle–water partition coefficient, Kd=Cs (mol kg!1)/Cw(mol dm!3), averaged 5.2±0.3 (n=20) in Site V1, whereas those of othercoastal sediments range between 3.2 and 4.8 (Hammerschmidt andFitzgerald, 2004). In addition to this, extremely high sulfide concentrationscommonly found in V1 cores may inhibit active metabolism of SRB. Althoughgeochemical parameters were not collected from the SS0 Site, similarmicrobial and geochemical conditions are expected, since the V1 bank hasbeen built with SS0 material.

3.2. Effect of sul!de addition

Fig. 2 depicts the effects of sulfide on the net MMHg production,dissolved Hg concentration, and the sulfate reduction rate. The MMHgconcentration increased from 0.009 to 0.24 nmol g!1 (from 0.01 to 0.41% oftotal Hg) during the 48 h of incubation, following the addition of inorganic Hg(Fig. 2a). A notable increase of MMHg following the addition of inorganic Hgdemonstrates that MMHg was mainly produced from the newly addedinorganic Hg. Kinetic studies were not performed in the present study.However, time-dependent observations of MMHg formation in marinesediment slurries have demonstrated that Hg methylation and demethyla-tion reach equilibrium conditions in 12 to 24 h at room temperature (Kinget al., 1999, 2000). Consequently, we consider that MMHg reached steady-state conditions after 48 h of incubation. In situ MMHg concentration isdetermined by the relative rate of two processes: methylation anddemethylation. Although our data do not provide information on the initialrates of methylation or demethylation, we argue that the steady-state MMHgconcentration is primarily controlled by the Hg methylation rate. A numberof studies have reported that Hg methylation overruled demethylation in thesurface sediments of various environments, hence the steady-state MMHgconcentration (or %MMHg) can be used as a proxy for the in situ rate of Hgmethylation (Drott et al., 2008; Heyes et al., 2006; Lambertsson and Nilsson,2006). In fact, in freshwater wetland sediments, a consistent demethylationrate was observed under sulfate- and Fe(III)-reducing, and methanogenicconditions (Warner et al., 2003).

216 S. Han et al. / Marine Chemistry 111 (2008) 214–220

The net MMHg concentration, ranging from 0.03 to 0.24 nmol g!1,exponentially decreasedwith an increase in dissolved sulfide of between 0.23and 6.3mM(Fig. 2a). This trendwas consistent in two sets of sediment slurriescollected from different sites. The sulfate reduction rate, ranging between 1.2and 6.4 μmol g!1 d!1, also decreased exponentially with an increase indissolved sulfide, indicating that the natural SRB communities in sedimentslurries are sensitive to sulfide toxicity at these levels (Fig. 2b). This resultagrees with other reports, showing that the activity and growth of SRB areinhibited by the added and produced sulfide, although SRB possess a hightolerance to sulfide (Icgen and Harrison, 2006; Okabe et al., 1995; Reis et al.,1991). Laboratory experiments using cultures of sulfate reducers have provedthat the inhibition of SRB by produced sulfide is due to a direct inhibition ofsulfate uptake, and is not due to the unavailability of dissolved Fe as a result ofFeS precipitation (Reis et al., 1991).

The dissolved Hg concentration increased, and then decreased, as afunction of dissolved sulfide (Fig. 2a). The early increase in Hg solubility at low

levels of sulfide addition may have been caused by the formation of Hg–polysulfide complexes. It has been shown that the addition of dissolvedsulfide enhances the short-term reactivity of Hg in the presence of Fe(III) byproducing S0 and polysulfide, and formingHg–polysulfide complexes (Sloweyand Brown, 2007). A decrease in dissolved sulfide during 48 h of incubation(from 0.8 to 0.2 mM), without the addition of further sulfide, supports thispossibility. We hypothesize that solid FeS formation is responsible for thedecrease in dissolved Hg, with the continuous addition of dissolved sulfide(sulfide N0.4 mM). The saturation quotient of amorphous FeS becomes N1when the dissolved sulfide concentration is N10!3 M, assuming 10!7 Mdissolved Fe(II) (Ksp of FeS=102.95, I=1, Davison, 1991; pKa of H2S=6.55, I=0.7,Millero et al., 1988). In fact, dissolved Fe was not detected in the fullexperimental range (detection limit: 10 μM). Mercury shows the highestDTMP (degree of tracemetal pyritization) amongmanymetals, even though itis expected to form solid HgS based on kinetic considerations: Hg has fasterreaction kinetics with sulfide than Fe2+ does (Morse and Luther, 1999). Morseand Luther (1999) suggested that Hg undergoes significant chloride andhydroxide complexation in seawater conditions, which can retard the reactionwith sulfide and allow the incorporation of Hg into FeS. In addition, theformation of HgS(s) was shown to be inhibited by the presence of lowconcentrations of dissolved organic matter (DOM; Ravichandran et al., 1999).

3.3. Effect of sulfate addition

When dissolved sulfate concentrations were amended to reach twicethat of the initial sulfate concentration, from 27 mM to 54 mM, the netconcentrations of MMHg, dissolved Hg and sulfide, and the sulfate reductionrate showed little variation over the range of sulfate concentrations (data notshown). This is because the concentration of sulfate in SS0 sediments farexceeds the saturation concentration of sulfate required for the maximumsulfate reduction rate. Kinetic studies of sulfate reduction rate havedemonstrated that half saturation concentrations of sulfate, using theMichaelis–Menten equation, fall between 0.1 and 0.3 mM in freshwaterand marine sediments (Pallud and van Cappellen, 2006).

3.4. Effect of ferrous Fe addition

In the Fe(II) addition experiments, V1 and SS0 slurries were amendedwith 1 mM dissolved sulfide in order to stimulate increases in MMHg at lowlevels of Fe(II) additions. Indeed, the net MMHg concentration in V1 (Fig. 3a)and SS0 (Fig. 3d) increased at lower Fe(II) additions, and then decreasedwhen the added Fe(II) was higher than the dissolved sulfide concentration(2.2 mM in V1 and 0.99 mM in SS0). Similar observations have been made inanoxic wetland sediment slurries amended with Fe(II) (Mehrotra and Sedlak,2005).

The sulfate reduction rate, ranging between 6.6 and 10 μmol g!1 d!1,gradually increasedwith an increase in Fe(II) concentration (Fig. 3b). This agreeswith the trend in the sulfate reduction rate shown in Fig. 2. If the sulfatereduction rate in Fig. 3b is plotted as a function of dissolved sulfide, the sulfatereduction rate decreases along with increasing sulfide, indicating that sulfide

Fig. 2. Effect of sulfide addition on the net production of monomethylmer-cury (MMHg); (a) MMHg in sediment and dissolved Hg in aqueous phase, and(b) sulfate reduction rate (SRR) and pH, shown as a function of the measuredsulfide concentration. Each parameter was measured at 48 h after theaddition of inorganic Hg in SS0 slurries.

Fig. 1. For V1 core in July 2006, vertical profiles in pore water of dissolved sulfate, sulfide and Fe, and in sediments of total Hg, monomethylmercury (MMHg) andfraction of MMHg in total Hg.

217S. Han et al. / Marine Chemistry 111 (2008) 214–220

toxicity could be the reason for the trend in the sulfate reduction rate.Interestingly, the pH gradually droppedwith the addition of Fe(II). This may bedue to FeS precipitation and simultaneous acid generation (Fe2++HS!=FeS+H+)that exceeds the buffering capacity of the system.

The role of FeS in controlling the Hg solubility is evident in Fig. 3a and c.At low Fe(II) additions, dissolved Hg increased, but at high Fe(II) additions,dissolved Hg decreased markedly, which could be caused by sulfide removal.This may be the reasonwhy the MMHg concentration decreased significantlywhen the added Fe(II) was higher than that of the dissolved sulfide, asituation that results in the removal of dissolved sulfide by FeS precipitation,thus no production of S0 and polysulfide that forms Hg–polysulfidecomplexes. Overall, it is plausible to argue that the limited availability ofdissolved Hg inhibits MMHg accumulation in the high sulfate reduction raterange.

3.5. Effect of ferric Fe addition

Fig. 4 depicts the effects of hydrous ferric oxide (HFO) additions on thenet production of MMHg, dissolved Hg concentration, and on the sulfatereduction rate. An increase in MMHg at low Fe(III) additions seems to becaused by an increase in dissolved Hg, and the subsequent decrease could bethe result of a low availability of dissolved Hg and a low SRR. The reductivedissolution of HFO by sulfide has been shown to be a major control ondissolved sulfide and associated trace metals in organic-rich sediments(Morse, 1994). This coupling of HFO reduction with sulfide oxidation mayexplain the increases of dissolved Hg at low levels of HFO addition (Fig. 4a). Itseems that additions of higher concentrations of HFO decreased Hg solubilityas a result of FeS precipitation, a similar situation with high levels of Fe(II)additions.

The marked differences between Fe(II) and HFO additions are shown in thesulfate reduction rate: the sulfate reduction rate was suppressed with HFOadditions, which may have been attributable to enhanced competition betweenSRB and Fe(III)-reducing bacteria for organic substrates. A decrease in the sulfatereduction rate with increasing Fe(III) reduction is reasonable, because organicmatter oxidation in sediment proceeds in the sequence of energy yield from eachof the electron-accepting processes: O2NnitrateNMn oxideNFe oxideNsulfate(Libes, 1992).

One caveat in the HFO experiment is that HFO was prepared with Fe(NO3)3 hence nitratemay have been reduced together with sulfate and Fe(III).While nitrate reduction is not likely to methylate Hg, it may have decreasedthe sulfate reduction rate due to the higher energy yield of nitrate reduction.Nevertheless, similar trends in MMHg and dissolved Hg between Fe(II) and

HFO additions indicate that HFO reduction is involved in electron-acceptingprocesses.

3.6. Limitations of sediment slurry experiments

Whereas the field data do not allow a clear understanding of relationshipbetween sedimentary geochemistry, in particular MMHg production, and Fe(III) and sulfate reductions, slurry methods allow to measure the response ofthe net MMHg production to changes in slurry composition. The standarddeviations of MMHg concentration and other geochemical parametersdetermined in duplicate slurries were generally within the instrumentaluncertainty, confirming reproducibility of the slurry results. However, slurryconditions may not be representative of fieldmicrobial and geochemical onesfor several reasons. First, the spiked inorganic Hg increased sedimentary Hgto an order higher than that found in natural sediments (8000–12,000 ng g!1

in slurries; 600–1400 ng g!1 in SS0 and V1), and pore water Hg was five totwenty times higher than the upper limit of dissolved Hg found in VeniceLagoon pore waters (1–50 nM in slurries; 10–200 pM in Venice Lagoon).Although no literature data provide information on the potential communitychanges by nM Hg additions, it is arguable that the bacterial communitiesmay not be significantly altered in the experimental slurries due to the wideoccurrence of Hg-resistant bacteria in Hg-contaminated sediments (Smalla etal., 2006). Secondly, the sediment structure was destroyed in the slurryexperiments, and the water–sediment ratio was higher than in naturalconditions, which may enhance the availability of dissolved substrates forparticle-bound SRB. This factor could be the reason why fractions MMHg intotal Hg were higher in the experimental slurries than natural V1 sediments.In addition to these, sediments used for slurry preparation may have hadenhanced availability of dissolved Hg possibly due to the oxidation of solidsulfide. Indeed, Kd of Hg decreased from 5.2±0.3 (n=20) in V1 sediments to3.7±0.4 (n=12) in V1 slurries. Therefore, information from the present studyshould be understood within the constraints imposed by the experimentalconditions.

3.7. Implications for MMHg production in anoxic estuarine sediments

The results of sulfide additions suggest that, in organic-rich, sulfidicsediments, accumulated sulfide in pore water may change the sulfatereduction rate, and in turn, the net production of MMHg. This gives aplausible explanation for the MMHg profile in Fig. 1: %MMHg decreased withan increase in sulfide. Actually, the decrease in MMHg associated with anincrease in dissolved sulfide has been reported for a number of freshwater

Fig. 3. Effect of ferrous Fe addition on the net production of monomethylmercury (MMHg); (a) MMHg in sediment and dissolved Hg in aqueous phase, (b) sulfatereduction rate (SRR) and pH, (c) dissolved Fe and sulfide, and (d) MMHg in sediment and dissolved Hg in aqueous phase, shown as a function of the amended Fe(II)concentration. Each parameter was measured at 48 h after the addition of inorganic Hg in V1 (a, b, and c) and SS0 (d) slurries.

218 S. Han et al. / Marine Chemistry 111 (2008) 214–220

and estuarine anoxic sediments (Benoit et al., 1999a,b; Drott et al., 2007;Gilmour et al., 1998). In those studies, the role of chemical speciation hasbeen emphasized, because no correlation between dissolved Hg and MMHghas been observed. Specifically, the trend of the Hg methylation rate as afunction of dissolved sulfide was explained by Hg–sulfide speciation: whendissolved sulfide is lower than 10!5M, dissolved Hg speciation is dominated byHgS0, which has a high bioavailability, and when dissolved sulfide is higher than10!5 M, dissolved Hg speciation is dominated by HgSH2

!. Further study on the Hgcomplexation under sulfidic conditions revealed that there is an interactionbetween Hg, sulfide and dissolved organic matter, thus the previousthermodynamic model has overestimated the abundance of dissolved Hg–sulfide complexes (Han et al., 2007a; Hsu-Kim and Sedlak, 2005; Milleret al., 2007). The metabolic activity of SRB has not been considered in theseattempts to understand net MMHg production as a response to thedissolved sulfide concentration. Based on the results of the present study,we suggest that dissolved sulfide is a critical factor controlling the sulfatereduction rate. Thus, Hg methylation rate (or %MMHg/Hg) needs to benormalized to the sulfate reduction rate for assessing the role of sulfide inbioavailability of inorganic Hg. The inhibitory effect of sulfide on the sulfatereduction rate has been reported at concentrations as low as 0.25 mMsulfide in culture studies (McCartney, and Oleszkiewicz, 1991; Okabe et al.,1995). The responses of the sulfate reduction andMMHgproduction need tobe investigated in low sulfide (b10!4 M) conditions.

From the results of Fe additions, a net MMHg production appears to behigher in sulfate-reducing environments than in Fe(III)-reducing ones undersimilar dissolved Hg conditions, although recent literature has reported thatFe(III)-reducing bacteria methylate Hg: pure cultures of Geobacter and De-sulfuromonas strains have been shown to methylate Hg at rates comparableto those of SRB (Fleming et al., 2006; Kerin et al., 2006). In order to test thepossibility of Hg methylation by other anaerobic organisms, inorganic Hg(5 nM) was incubated in enrichment cultures of sulfate-, nitrate-, Mn(IV)-,and Fe(III)-reducing bacteria obtained from the Venice Lagoon sediment.After 6 h of incubation, the cultures of sulfate reducers showed significantproduction of MMHg (4.6% of amended Hg), while the other cultures showedno production of MMHg. This result agrees with the report by Warner et al.(2003): with river basin sediments, sulfate consumption was almostcompletely suppressed in Fe(III)-reducing conditions, and in these condi-tions, Hg methylations were close to those in killed controls. It appearsthat enhanced Fe(III) reduction suppresses the formation of MMHg in

high sulfate, estuarine sediments. Obviously, further study is required tounderstand the role of the microbial community in the transformation of Hgin various electron-accepting processes.

Currently, it is generally accepted that the steady-state MMHgconcentration (or %MMHg/Hg) can be used as a proxy for the in situ rateof Hg methylation and this rate is controlled by two factors: the availabilityof inorganic Hg and the activity of SRB. The importance of these two factorsin the prediction of Hg methylation rate is demonstrated in Fig. 5.The fraction of total Hg as MMHg is better explained by the log particle–water distribution coefficient, normalized to the sulfate reduction rate,as compared to the plots which consider only Hg solubility or sulfatereduction rate, indicating that these two factors are equally important forMMHg production in high organic, sulfidic sediments. If the sulfatereduction rate remains constant, the availability of dissolved Hg can bethe main control for Hg methylation. This may be the case in coastal marinesediments, where the dissolved sulfide concentration is relatively lowand constant.

Acknowledgments

We thank the associate editor and the anonymousreviewer for their valuable comments and suggestions toimprove this manuscript. Chiara Castellani, Fabrizio Perin, andAndrea Berton are acknowledged for the sampling assistance.Thismaterial is produced in the framework of SIOSED (ScrippsInstitution of Oceanography SEDiment research group)project, supported by Magistrato alle Acque di Venezia, Italy

Fig. 5. For SS0 slurries, relationship between the fraction of monomethyl-mercury (MMHg) in total Hg and (a) log particle–water partition coefficient(Kd), (b) sulfate reduction rate (SRR), and (c) log Kd normalized to SRR.

Fig. 4. Effect of ferric Fe addition on the net production of monomethylmer-cury (MMHg); (a) MMHg in sediment and dissolved Hg in aqueous phase, and(b) sulfate reduction rate (SRR) and pH, shown as a function of the amendedhydrous ferric oxide concentration. Each parameter was measured at 48 hafter the addition of inorganic Hg in V1 slurries.

219S. Han et al. / Marine Chemistry 111 (2008) 214–220

(Venice Water Authority) throughout Consorzio VeneziaNuova and Thetis S.p.A. Any opinions, findings, and conclu-sions or recommendations expressed in this publication arethose of the author(s) and do not necessarily reflect the viewsof the Magistrato alle Acque di Venezia (Venice WaterAuthority), Consorzio Venezia Nuova or Thetis S.p.A.

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