Association of Methylmercury with Dissolved Humic Acids

Association of Methylmercury with Dissolved Humic Acids ... The modeling parameters, which were the equilibrium binding constants and the total bindin...
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Environ. Sci. Technol. 2002, 36, 690-695

Association of Methylmercury with Dissolved Humic Acids A R I A A M I R B A H M A N , * ,† A N D R E W L . R E I D , †,⊥ T E R R Y A . H A I N E S , ‡ J . S T E V E N K A H L , | A N D C EÄ D R I C A R N O L D § Department of Civil and Environmental Engineering, and Senator George J. Mitchell Center for Environmental & Watershed Research, University of Maine, Orono, Maine 04469, U.S. Geological Survey, Orono Field Station, Orono, Maine 04469, and BMG Engineering Ltd., CH-8952 Schlieren, Switzerland

Sorption of methylmercury (MeHg) to three different humic acids was investigated as a function of pH and humic concentration. The extent of sorption did not show a strong pH dependence within the pH range of 5-9. Below pH 5, a decrease in adsorption for all humic samples was observed. The experimental data for equilibrium sorption of MeHg were modeled using a discrete log K spectrum approach with three weakly acidic functional groups. The modeling parameters, which were the equilibrium binding constants and the total binding capacities, represented the data well at all MeHg and humic concentrations and pH values for a given humic sample. The estimated binding constants for complexes of MeHg with humic acids were similar in magnitude to those of MeHg with thiol-containing compounds, suggesting that binding of MeHg involves the thiol groups of humic acids. The results show that only a small fraction of the reduced sulfur species in humic substances may take part in binding MeHg, but in most natural systems, this subfraction is considerably higher in concentration than ambient MeHg. The model developed here can be incorporated into speciation models to assess the bioavailability of MeHg in the presence of dissolved organic matter and competing ligands such as chloride and sulfide.

Introduction Methylmercury (MeHg) has been generally implicated as the species responsible for the toxicity of mercury due to its greater trophic transfer than inorganic mercury in higher organisms (1, 2). Methylation of mercury may take place under both aerobic and anaerobic conditions (3, 4). In most natural waters, however, MeHg is formed by biological methylation carried out by the sulfate-reducing microorganisms (3). Environmental fate (i.e., bioaccumulation, and sorption to soils and sediments) of MeHg depends on the dissolved organic matter (DOM) content. However, the factors governing the binding of MeHg to DOM are poorly understood. * Corresponding author phone: (207)581-1277; fax: (207)581-3888; e-mail: [email protected]. † Department of Civil and Environmental Engineering, University of Maine. ⊥ Present address: Earth Tech Inc., Concord, MA. ‡ U.S. Geological Survey, Orono Field Station. | The Mitchell Center, University of Maine. § BMG Engineering Ltd. 690

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Dissolved organic matter is known to form strong complexes with both Hg(II) and MeHg (5, 6). Studies of the role of water chemistry in fish mercury concentration nearly always identify DOM as an important variable but have produced conflicting results concerning the nature of the interaction. Review of the existing MeHg bioaccumulation field data suggests a complex relationship involving different chemical and hydrological parameters (7). Dissolved organic matter most likely affects the supply of MeHg that is available for uptake to algae; it is less likely that DOM acts as a direct transfer agent through the food chain (8-12). Hintelmann et al. (6, 13) have studied the equilibrium association of MeHg with DOM. They have conducted equilibrium dialysis experiments at a neutral pH and a DOM concentration of 1 ppm to determine the conditional stability constants for complexation of MeHg by humic and fulvic acids isolated from two lakes in Ontario. Their analysis indicates log formation constants ranging between 12.5 and 13.5. However, these constants are conditional and, as such, are likely to vary under different pH, ionic strength, and DOM concentrations. In one set of experiments, a general decrease in the binding of MeHg to DOM with decreasing pH below 7 was observed (13). Such a characteristic underscores the importance of the acid-base interactions in this system and, therefore, necessitates a pH-dependent study of MeHg-DOM association. Due to the high polarizability of mercury atom, MeHg is characterized as a “soft” Lewis acid that forms strong covalent bonds with other “soft” Lewis bases, notably with reduced sulfur. Accordingly, reduced sulfur ligands in DOM are expected to be important binding sites. This assumption is also supported by recent spectroscopic data (14). Exchange reactions of MeHg involving thiol-containing compounds can be classified as associative and are diffusion-controlled due to their extremely high rates (15, 16). Rabenstein (17) studied the pH dependence of complexation of MeHg with several reduced sulfur-containing organic compounds using proton NMR spectroscopy. Compounds that contain a thiol group such as glutathione and cysteine were shown to form strong complexes with MeHg. The conditional formation constants for these complexes are the largest in the pH range between the pKa values of the thiol group and MeHg and decrease at the pH extremes. Based on the type of ligand, the formation constants of MeHg cover a wide range, with thiol-containing compounds possessing the largest values. These constants are in general smaller than those for complexation of Hg(II) with most organic and inorganic ligands, including the reduced sulfur (17). This has been attributed to the destabilizing effect that two “soft” ligands in mutual trans position would have on each other (18); i.e., the CH3 ligand in MeHg reduces the affinity of Hg for other ligands. This study is a systematic investigation of sorption of MeHg to three humic acids at various pH values. A simple equilibrium model is developed by considering binding to the weakly acidic functional groups of humic substances. The equilibrium formation constants and binding capacities for MeHg-humic interaction are estimated. This model may be used to calculate speciation under different MeHg and ligand concentrations and pH. Model for the Association of MeHg with Humic Substances. Recent spectroscopic evidence with synchrotronbased X-ray absorption spectroscopy (XAS) for interaction between Hg(II) and a soil humic acid suggests the involvement of thiol and disulfide (RSSR′)/disulfane (RSSH) functional groups (14). The presence of disulfide/disulfane groups 10.1021/es011044q CCC: $22.00

 2002 American Chemical Society Published on Web 01/18/2002

of humics in binding Hg(II) was supported by the presence of a second sulfur atom in the second coordination shell. Since the experiments in our study were conducted with MeHg concentrations below the total estimated concentrations of the reduced sulfur species in humic substances (19), we expect the same functional groups be responsible for binding MeHg. We have used a discrete log K spectrum approach (20, 21) to model the experimental data for the association of MeHg with humic substances at different MeHg and humic concentrations and at different pH values. Westall et al. (20) have shown that a discrete log K spectrum model may be used to represent proton and metal complexation of humic substances. This approach has the advantage of circumventing the use of electrostatic correction term for modeling of the stability constants. In the discrete log K spectrum model, humic substances are represented as monoprotic acids that form 1:1 complexes with MeHg. Complexes of MeHg almost always possess a coordination number of one (22). The reactive thiol functional groups of humics may be modeled as multisite acids +

H +

RS(i)

S RS(i)H;

Ka(i)

Ks(i)

(2)

where Ks(i) is the equilibrium formation constant. All relevant reactions involving MeHg used in modeling our experimental data are listed in Table 1. Species (MeHg)2OH+ was neglected due to its very low concentration at typical concentrations of MeHg studied here (22). Also, complexation of Na+ by humic substances was not considered, because Na+ reacts weakly with the thiol groups and does not compete with MeHg for the same binding sites. The total concentration of MeHg may be written as

[MeHg]T ) [MeHg+] + [MeHgOH] +

log Ka H+ + OH- S H2O MeHg+ + H2O S MeHgOH + H+ MeHg+ + Cl- S MeHgCl H+ + Ac-e S HAc MeHg+ + Ac- S MeHgAc H+ + HPO24 S H2PO4 MeHg+ + HPO2S MeHgHPO 4 4 H+ + RS(1) S RS(1)H H+ + RS(2) S RS(2)H H+ + RS(3) S RS(3)H MeHg+ + RS(-i) S RS(i)HgMe

∑[RS HgMe] + ∑[MeHgL ] (3) (i)

i

(j)

j

where L(j) represents other ligands in this study that form complexes with MeHg such as chloride, acetate, and phosphate. For the range of MeHg concentrations studied here, three weakly acidic functional groups were sufficient to adequately model the adsorption of MeHg to humics; i.e., i ) 3 in eq 1. We have assumed fixed log Ka(i) values of 4, 7, and 10 for these functional groups (eq 1). These values cover the typical range for the acidity constants of thiol functional groups in organic acids. For example, the thiol groups in amino acids such as cysteine, glutathione, and penicillamine have log Ka values close to 8, and other thiol-containing organic acids such as mercaptoacetic acid and 2-mercaptoehtanol have log Ka values close to 10. Application of Taft equation indicates that log Ka of the disulfane group may be close to 4 (25). Humic acid carboxylic and amino groups may also contribute to a small extent to the binding of MeHg. The selected log Ka values also cover the range of acidity constants of these

13.97b -4.63c 5.22d 4.73b 2.95b 7.14b 5.41b 4.0 f 7.0 f 10.0 f f

a Equilibrium constants given for I ) 1 mM using Davies formula. Reference 23. c Reference 24. d Reference 22. e Ac is abbreviation for acetate ion used in experiments at pH 5.2 only. f This work. See Table 3 for the log Ks values for adsorption of MeHg to humic substances used here. b

TABLE 2. Elemental Analysis of Humic Substancesa

(1)

where RS(i) and RS(i)H represent the deprotonated and protonated forms of the ith thiol functional group of humics, respectively, and Ka(i) is the corresponding apparent acidity constant. The subscript i indicates a continuous spectrum of acidic ligand sites with different Ka(i) values. In the absence of binding cations other than H+, the total concentration of the ith thiol group, RST(i), is equal to the summation of the concentrations of RS(i)H and RS(i) species. The interaction between MeHg and the humic functional group RS(i) may be written as

MeHg+ + RS(i) S RS(i)HgMe;

TABLE 1. Equilibrium Constants for MeHg and Ligands

SRHAb

PHAb

BBHAc

52.55 4.40 42.53 1.19 0.58 3.10 0.61

56.37 3.82 37.34 3.69 0.71 1.12 0.50

49.82 5.06 40.27 1.9 0.61 3.26 0.61

C H O N S ash atomic O/C

a Values in the table are given in % w/w. b Data provided by The International Humic Substances Society. c Data provided by Huffman Laboratories, Inc., Golden, CO.

groups. Due to their relatively small concentrations in humic substances, the densities of thiol groups cannot be determined using simple acid-base titration (19). Instead in this study, total concentrations of the ligands, RST(i), as well as their corresponding equilibrium formation constants, Ks(i), have been estimated from the results of the MeHg-humic binding experiments.

Material and Methods Humic Substances. Three different humic acid samples representing aquatic and organic soil environments were used. Suwannee River humic acid (SRHA) and peat humic acid (PHA) were obtained in freeze-dried form from the International Humic Substances Society (IHSS). Both samples were dissolved at a basic pH and quickly passed through a column of Amberlite IR-120 cation-exchange resin in H+ form (Aldrich). The humic acid fraction from Baker Brook (BBHA), a stream located in east central Maine that drains a wetland, was also isolated (26, 27). Baker Brook had a dissolved organic carbon concentration of 35 ppm at the time of sampling. Details of the isolation procedure are given elsewhere (28). The results from the elemental analysis of the humic substances in this study are presented in Table 2. Dialysis Experiments. Equilibrium dialysis technique was used to study the extent of association of MeHg with humic substances. These experiments were performed in dialysis cells with two 15 mL glass chambers separated by a cellulose ester asymmetric dialysis membrane sheet (Spectrum Laboratories, Inc., Laguna Hills, CA) with a molecular size cutoff of 500 Da (21, 29). The dialysis experiments were conducted by adding a known concentration of MeHg to one cell only. A known concentration of the humic substance of interest was added to the other cell. The solution in each cell had an ionic strength of 1 mM that was generally maintained by the pH buffer. A low ionic strength was used to minimize the diffusion of the VOL. 36, NO. 4, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Diffusion kinetics of MeHg through the 500 Da dialysis membrane. Only one dialysis cell contained IHSS peat humic acid. MeHg at a concentration of 1.46 nM was initially added to the dialysis cell that did not contain the humic acid. The pH was 5.2 in both cells. Solid and open circles represent MeHg concentrations in the cell with and without PHA, respectively. humic substances across the membrane. Buffer solutions used in the dialysis experiments were acetate, phosphate, and borate for pH values of 5.2, 7.1, and 9.2, respectively. These buffers were chosen because of their relatively low affinities to MeHg. Also, organic pH buffers interfered with the measurement of MeHg. For the experiments at pH 3.5 and 4.6, the pH was adjusted by adding aliquots of 0.01 M Optima grade HNO3 (Fisher Scientific). The ionic strength was adjusted to 1 mM with NaClO4 when necessary. The pH was measured at the beginning and end of each experiment using a Metrohm pH electrode (Brinkmann Instruments, Inc., Westbury, NY). The pH was also only measured in one reaction cell per sample in order to limit MeHg cross contamination. Equilibrium for sorption of MeHg to the humic substances at typical concentrations studied here was reached in approximately 12 h, as illustrated in Figure 1 for PHA. In a separate experiment conducted to study the diffusion of MeHg across the membrane in the absence of any humics a similar equilibrium time was observed. This indicates that the rate-limiting step was indeed diffusion across the dialysis membrane and not complex formation. To ensure equilibrium, the cell pairs were shaken on a rotary shaker at a rate of 15 ( 5 rpm for 24 h at 21 ( 2 °C in the dark. Measurement of MeHg. Concentration of MeHg was measured in both cells. The method used to analyze MeHg was adopted from EPA method 1631, which is for measurement of total mercury using cold vapor atomic fluorescence spectrophotometer (CVAFS). Following the initial oxidation of the organic matter in solution using BrCl, all mercury species in the system are reduced to Hg(0) with SnCl2. The reduced mercury is purged with N2 gas and captured on a gold trap. The captured mercury is then desorbed by heating the gold trap and carried to a detector (Brooks-Rand CVAFS Model 2) by Ar gas. Dialysis experiments in the absence of humic acids showed a 89 ( 3% recovery of MeHg. For experiments in the presence of humic acids, MeHg recovery was 80-90%. Total background mercury concentration in a 1 mM solution of the selected buffers did not exceed 10 pM. Total background mercury concentrations for a 1 ppm solution of humic acids ranged between 10 and 40 pM. More details of the dialysis experiments and MeHg measurement procedure are presented elsewhere (28). 692

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Reagents. All chemicals used in this study were reagent grade. Optima grades of HNO3 and HCl were used in the experiments and for cleaning the glassware and Teflon bottles. MeHgOH working standards containing 100 ppb in a 0.005% KOH matrix were obtained on a monthly basis from Brooks-Rand (Seattle, WA). This solution was checked against a secondary standard obtained from Frontier Geosciences (North Vancouver, BC). Modeling of the Experimental Data using FITEQL. We used the computer program FITEQL (30) to model the association of MeHg with humic substances at equilibrium. FITEQL is a speciation program that can be used to determine the unknown thermodynamic parameters including equilibrium constants and binding capacities. This program uses as input total or free concentrations of chemical components and species and the known equilibrium constants. In this study, experimental results from the dialysis experiments for varying concentrations of MeHg and 1 ppm of humic substances at a fixed pH were used as the serial input data. Other input data were the pH, the relevant equilibrium reactions, and their corresponding constants listed in Table 1, and the total added concentrations of MeHg and ligands other than humic substances such as chloride and acetate. At pH 7.1, phosphate had a negligible effect on speciation of MeHg. The fitting parameters were equilibrium binding constants for the MeHg-humic acid complexes, Ks(i), and the total concentrations of the thiol groups, RST(i). To validate the model, simulation results were compared to the experimental results outside the calibration range for other pH values, chloride, and total humic concentrations.

Results and Discussion Adsorption experiments were performed at pH values ranging from 3.5 to 9.2 for all three humic substances. Results for the sorption of MeHg onto SRHA and PHA are shown in Figures 2 and 3, respectively. The sorption isotherms for BBHA also followed a similar trend (results not shown here). Among all humic acids studied here, MeHg has the highest adsorption affinity for SRHA and the lowest adsorption affinity for PHA. All humics exhibit similar trend of pH dependence. This pH dependence is characterized by relatively constant adsorption at pH values from 5.2 to 9.2. Below pH 5.2, the extent of adsorption decreases primarily due to the competition of MeHg+ with H+ for binding to the functional groups, which are Brønsted bases. Under more alkaline conditions than pH 9.2, we expect the adsorption to decrease due to the competition of OH- ions with the functional groups for MeHg+. Experiments at pH values higher than 9.2 were not conducted due to the lack of stability of the dialysis membranes. The model in this work was developed from the reactions listed in Table 1. The experimental data at all pH values were adequately modeled by considering three discrete functional groups (eqs 1 and 2). The log acidity constants for these functional groups were fixed at 4, 7, and 10. The equilibrium formation constants and the corresponding binding capacities were initially estimated by fitting the model to the experimental data at pH 7.1 and 9.2. Since at these pH values adsorption of MeHg at low concentrations is largely due to functional groups with log Ka(i) of 7 and 10, the formation constants and adsorption capacities for these groups only were initially used as the fitting parameters. The adsorption data at pH 3.5 were then used to estimate KS(1) and RST(1). Given these initial estimates, the binding constants and adsorption capacities for the three functional groups were evaluated for the combined adsorption data at all pH values, except for pH 5.2 due to the binding of MeHg to acetate that has to be included in chemical speciation (Table 1). The fitting parameters are shown in Table 3. The modeling results, shown as solid lines in Figures 2 and 3, indicate that our choice of functional group can adequately account for the adsorption of MeHg onto all humic acids and at all pH values

FIGURE 3. Sorption isotherms of MeHg to IHSS peat humic acid at pH values of (a) 3.5 and 5.2 and (b) 7.1 and 9.2. Total chloride concentration was 8.6 µM for all experiments as measured with the ion chromatograph. The solid line represents the model fits using eqs 1 and 2 and the constants listed in Table 3. Error bars represent one standard deviation from three separate dialysis experiments. FIGURE 2. Sorption isotherms of MeHg to IHSS Suwannee River humic acid at pH values of (a) 3.5 and 4.6, (b) 5.2, and (c) 7.1 and 9.2. Total chloride concentration was 14.5 µM for all experiments, except as noted, as measured with the ion chromatograph. The solid line represents the model fits using eqs 1 and 2 and the constants listed in Table 3. Error bars represent one standard deviation from three separate dialysis experiments. and MeHg concentrations used here. The weighted sum of squares over degree of freedom, as calculated by FITEQL, ranged from approximately 7 to 14 for all the adsorption data. Figure 4 shows the pH dependence for the association of MeHg with SRHA for a total MeHg concentration of 0.4 nM. This plot illustrates the relative contribution of each binding site at different pH values. At all pH values, the RST(2) and RST(3) functional groups dominate the adsorption. Omission of the RST(1) functional group, however, leads to underestimation of the extent of adsorption at low pH. Maximum adsorption takes place at a pH where the product of the activities of the two reacting species in eq 2, MeHg+ and RS(i) , is at a maximum. This pH, therefore, corresponds to the average of the log acidity constants of the two reacting species. Depending on the dominance of each of the two thiol groups, RS(2) and RS(3), which in turn depends on the pH, the maximum adsorption could vary between pH 6.3 and 7.3. To validate our model assumptions, the parameters listed in Table 3 were used to simulate the association of MeHg with humic substances at pH and total humic concentrations outside the range of our model calibration. To this end, we simulated the adsorption of MeHg onto all humic samples at pH 5.2. Due to the presence of 1 mM acetate in pH 5.2

TABLE 3. Equilibrium Binding Constants and Binding Capacities for Formation of MeHg Complexes with Humic Substancesa,b humic substances

log Ks(1)

RST(1)

log Ks(2)

RST(2)

log Ks(3)

RST(3)

Suwannee River HA 10.39 0.15 14.74 0.24 14.84 1.44 Peat HA 10.42 0.43 12.39 0.13 14.47 1.51 Baker Brook HA 10.54 0.25 14.77 0.10 14.96 0.76 a See eq 2 in the text for the reactions. b Binding capacities of humic substances are denoted as RST(i) and given in nmol mg-1.

adsorption experiments, the data at this pH were not modeled simultaneously with the data at other pH values. The results are shown in Figures 2 and 3 for SRHA and PHA. In all cases the model represents the experimental data at pH 5.2 reasonably. Figure 5 shows the bound concentration of MeHg as a function of BBHA concentration at pH 5.2. The concentration of BBHA in this set of experiments varied from 0.5 to 15 ppm and the total MeHg concentration was 1 nM. The background electrolyte concentration was kept at 1 mM by adding acetate buffer. Model simulation using the parameters obtained at a total BBHA concentration of 1 ppm (Table 3) is also shown as a solid line. The model provides a reasonable representation for the data at humic concentrations outside the range of its calibration. The relatively high values of the equilibrium binding constants listed in Table 3 are similar to those for the association of MeHg with thiol-containing compounds, suggesting that at the concentrations studied here, MeHg associates primarily with the thiol groups in humic acids. The range of MeHg concentrations used here was chosen VOL. 36, NO. 4, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Sorption of MeHg to the IHSS Suwannee River humic acid as a function of pH. Total chloride concentration was 14.5 µM for all experiments. Open circles are the experimental data points and are obtained by interpolating from the sorption isotherms (Figure 2) for a total MeHg concentration of 0.4 nM. The dotted lines represent the contributions of the individual sites (eq 2). The solid line represents the summation of the three contributing functional groups. The fitted values of equilibrium constants and binding capacities are listed in Table 3.

FIGURE 5. Sorption of MeHg to Baker Brook humic acid as a function of total humic concentration at pH 5.2. Total initial MeHg concentration in both dialysis vials was 1 nM, and total chloride concentration was 13.7 µmol mg-1 as measured with the ion chromatograph. The solid line represents the model fit (eqs 2 and 3) to the experimental data. such that the maximum bound MeHg concentration would not exceed the total concentration of the reduced sulfur in humic substances as estimated by Xia et al. (19). Higher concentrations of MeHg would also be irrelevant for natural systems. Sulfur speciation in humic substances has been previously examined using X-ray absorption near-edge structure spectroscopy, XANES (19, 31). Both studies indicate the presence of sulfur in various oxidation states. Xia et al. estimated the electronic oxidation states of sulfur in several humic substances, which indicate the actual electronic density in the valence shell of the sulfur atom (19). Depending on the bonding environment of the reduced sulfur, the electronic oxidation states of the sulfur atom in organosulfide groups are between 0 and 0.5. An electronic oxidation state of 0.2 was estimated for sulfur in cysteine and methionine 694

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(31). Part of the reduced sulfur in dissolved organic matter is due to amino acids that are either associated with or structurally part of the organic matter. The concentration of the associated amino acids is expected to be larger than the structural amino acids (32). Stream and peat humic acids from IHSS have reported methionine concentrations of 1.3 and 4.0 nmol mg-1, respectively. In a forest humic substance, cysteine and methionine were measured at concentrations of 2.74 and 1.71 nmol mg-1, respectively (33). However, these values are perhaps lower end estimates, since isolation of humic substances involves passing the DOM samples through a strong cation-exchange resin, which may cause the amino acids that are associated with the dissolved organic matter to dissociate (32). According to the measurements by Xia et al. (19), the reduced sulfur fraction may vary from nearly 10% for a mineral soil sample to 46% for SRHA supplied by IHSS. Considering a sulfur weight fraction of 0.58% for SRHA (Table 2), the reduced sulfur content with an electronic oxidation state less than 1 in this humic acid is approximately 83 nmol mg-1. This value is nearly 45 times higher than the total binding capacity of 1.83 nmol mg-1 for SRHA estimated in this work (Table 3), suggesting that approximately only 2% of the highly reduced sulfur species in SRHA take part in binding MeHg. This fraction would perhaps be somewhat higher for other humic acids used here. The absence of reactivity of all reduced sulfur species as estimated by Xia et al. (19) toward MeHg may be attributed in part to the available thiol species that constitute only a fraction of the total reduced sulfur in humics. For example, the presence of monosulfide species (RSR′) in humic substances with an approximate electronic oxidation state of 0.5 has been suggested (19). However, we do not expect significant contributions from the monosulfide species in humics to binding of MeHg. Based on previous NMR and X-ray crystal structure determination studies, the monosulfide bridge in amino acid methionine binds MeHg only to a small extent (17, 34). Methionine binds with MeHg+ species to form a positively charged complex, but this complex has a relatively small formation constant that decreases with increasing pH due to the decrease in the concentration of MeHg+ species (17). Other possible explanations for the relative unavailability of all the reactive sulfur functional groups toward MeHg may be the presence of other heavy metals in humics that compete with MeHg by binding strongly to these functional groups, and humic conformations. Given our extant knowledge, it would be difficult to predict the extent of sorption of MeHg based on the type and measurable properties of a DOM sample. In a limited number of samples, the fraction of the reduced sulfur was shown to be the highest for humic substances isolated from the aquatic samples followed by organic and mineral soil samples (19). Among the humic samples examined, SRHA in particular had the largest fraction of reduced sulfur species (19). When considering ∑i(Ks(i) × [RST(i)]) from Table 3, the following sequence is obtained: SRHA > BBHA > PHA. This sequence is not reflected in the total elemental sulfur (Table 2). Compared to the aquatic humic acids studied here, PHA possesses the lowest extent of binding of MeHg. This humic sample has the largest average molecular size among all humics studied here, suggesting that the extent of binding of MeHg to humics does not correlate directly to the molecular size distribution. We have argued that at the MeHg concentration range studied here, binding of MeHg to functional groups other than those containing reduced sulfur is unlikely, due to the higher affinity of MeHg to reduced sulfur than to oxygen- or nitrogen-containing functional groups. At higher MeHg concentrations, however, binding to latter functional groups or other nonspecific interactions may be possible as observed in both model (17, 35) and environmental systems (36).

Literature Cited

FIGURE 6. Speciation of MeHg in the presence of 1 mg L-1 Baker Brook humic acid. Total MeHg concentration ) 0.1 nM, and total chloride concentration ) 1 mM. Simulation is performed using eqs 1 and 2 and the constants in Tables 1 and 3. RSHgMe represents the total concentration of MeHg associated with the humic acid. Binding of Hg(II) by oxygen-containing groups has been previously reported in the presence of low Hg(II)/humic ratios, suggesting either a two-coordinate binding environment with sulfur and oxygen or binding by an oxygencontaining functional group in humic substances (14). Unlike Hg(II), however, a two-coordinate binding environment in MeHg is unlikely (22). The MeHg speciation model presented here has several advantages. The set of parameters developed are capable of adequately modeling the experimental data over the range of MeHg and humic concentrations and pH values of environmental significance and are based on the previous mechanistic observations of Hg(II)-humic interactions. The discrete log K spectrum approach used requires a minimum number of fitting parameters necessary to describe chemical speciation at different concentrations and pH. The effect of competing reactions involving other metals and ligands can be accounted for easily with the binding constants and site densities developed here. Last, this model can easily be coupled to chemical speciation models and be used in MeHg availability and toxicity studies. An example of speciation of MeHg in a typical aqueous environment using the parameters developed here is shown in Figure 6. According to Figure 6, in freshwater systems at low pH and low DOM concentrations, MeHgCl species can dominate the speciation of MeHg for a chloride concentration of 1 mM. This is not surprising given the relatively low density of the available reduced sulfur species in humic substances (typical value of 2 nmol mg-1). The relative concentrations of the competing ligands such as chloride and sulfide compared to humic substances in natural systems can have important implications for the bioavailability and toxicity of MeHg.

Acknowledgments Funding for this work was provided by the U.S. Geological Survey. The authors wish to thank Marty Richards, Philip Ruck, and Andrew Fiske for their help in the analysis of methylmercury. Critical comments of two anonymous reviewers are acknowledged.

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Received for review June 6, 2001. Revised manuscript received October 30, 2001. Accepted November 5, 2001. ES011044Q

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