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Environ. Sci. Technol. 2010, 44, 6151–6156

Binding Strength of Methylmercury to Aquatic NOM ABDUL R. KHWAJA,† PAUL R. BLOOM,‡ A N D P A T R I C K L . B R E Z O N I K * ,§ Water Resources Science Graduate Program and Department of Soil, Water and Climate, University of Minnesota, St. Paul, Minnesota 55108, and Department of Civil Engineering, University of Minnesota, Minneapolis, Minnesota 55455

Received April 7, 2010. Revised manuscript received July 15, 2010. Accepted July 16, 2010.

A competitive-ligand, equilibrium-dialysis technique using bromide measured methylmercury (MeHg+) binding to Suwannee River fulvic acid (SRFA) and NOM from a lake and a bog in Minnesota. Distribution coefficients (KOC) and stability constants (K′) varied only slightly over a range of [Br-] and ratios of MeHg+ to reduced sulfur, Sre, the putative NOM binding site. For SRFA at pH 3.0, KOC ranged from 107.7 to 108.2 and K′ ranged from 1015.5 to 1016.0 over MeHg+:Sre ratios from 1:1220 to 1:12 200 (well below Sre saturation). The importance of pH depends on the calculation model for binding constants. Over pH 2.98-7.62, KOC had little pH dependence (slope ) 0.2; r2 ) 0.4; range 107.7-109.1), but K′ calculated using thiol ligands with pKa ) 9.96 had an inverse relationship (slope ) -0.8; r2 ) 0.9; range 1015.6-1012.3). A pH-independent model was obtained only with thiol pKa e ∼4. The mean K′4 for SRFA (K′ with thiol pKa ) 4.2) was 109.8 (range 109.11-1010.27) and small slope (0.02). Similar values were found for Spring Lake NOM; bog S2 NOM had values one-tenth as large. These constants are generally similar to published values; differences reflect variations in methods, pH, types of NOM, and calculation models. Methylmercury, a neurotoxin that bioaccumulates in organisms, is thought to be formed in natural waters and sediments primarily by sulfate-reducing bacteria. Methylmercury uptake by and bioavailability to aquatic organisms, such as algae (1), eggs of yellow perch (2), midge larvae (3), Chaoborus larvae (4) and Daphnia (5), is influenced by natural organic matter (NOM). NOM is a heterogeneous mixture of mostly macromolecular humic substances that play important roles in the chemistry of many metals. Abiotic transformation of methylmercury, e.g., demethylation (6), also is influenced by NOM. Transport of methylmercury to lakes through watersheds has been correlated with dissolved organic carbon concentrations (7, 8), but such correlations are not always observed (9). Methylmercuric ion (MeHg+) is a soft Lewis acid, and according to Pearson’s Soft and Hard Acid Base theory, it forms strong complexes with reduced sulfur-containing ligands. Using X-ray absorption near-edge structure spectroscopy (XANES), Xia et al. (10) showed that up to 50% of the total sulfur in aquatic and soil humic substances is in * Corresponding author e-mail: [email protected]; phone: (612) 625-0866; fax: (612) 626-7750. † Water Resources Science Graduate Program; current address GE Infrastructure Water & Process Technologies, Trevose, PA. ‡ Department of Soil, Water and Climate. § Department of Civil Engineering. 10.1021/es101088k

 2010 American Chemical Society

Published on Web 07/28/2010

chemically reduced forms (Sre): thiols (RSH), thioethers (RSR) or disulfides (RSSR). Qian et al. (11) and Yoon et al. (12) used extended X-ray absorption fine structure spectroscopy (EXAFS) to show that Sre groups in soil and aquatic organic matter form complexes with MeHg+. NOM forms strong complexes with MeHg+, and several studies have reported high values of binding constants of MeHg+ with humic substances. Hintelmann et al. (13, 14) used a Scatchard analysis to obtain constants at pH 6.5 for a strong site (logK ) 13.02-14.48) and weak site (log K ) 12.15-13.07) for MeHg+ with aquatic humic acid (HA) and fulvic acid (FA). They concluded that MeHg+ is bound to “sulfidic” sites in NOM. Amirbahman et al. (15) used a discrete log K spectrum approach to model MeHg+ binding with peat and aquatic humic substances at pH 3.5-9.2. Their model assumed three binding sites, which they characterized as thiols with acidity constants (pKa) of 4, 7, and 10. Calculated MeHg+ binding constants were in the ranges 1010.4-1010.5, 1012.4-1014.8, and 1014.5-1014.8 for these sites. Karlsson and Skyllberg (16) determined MeHg+ binding in an organic soil. They relied on native MeHg+ in the soil and used Br- and Cl- as competitive ligands to obtain measurable MeHg+ in solution. When they assumed one thiol binding site with a pKa ) 9.95 or two sites with pKa values of 8.5 and 9.96, they found K decreased with increasing pH. The two-site model gave log K ) 17.2 at pH 2.0 and log K ) 15.6 at pH 5.1. The negative dependence of K on pH contrasts with a small positive pH dependence of log K for the strong bidentate binding of Hg2+ to peat HA in our previous study (17). Here we describe MeHg+ binding constants determined by a competitive ligand-exchange technique on a fulvic acid reference material (IHSS Suwannee River FA [SRFA]) and aquatic NOM that was isolated from a bog and lake as part of a larger study on mercury cycling in humic-rich waters (18); the humic fraction in aquatic NOM is largely FA in nature. Our objective was to measure MeHg+ binding to aquatic NOM under conditions relevant to natural environmental systems. Consequently, we used low additions of MeHg+ and determined the effects of pH, MeHg+:Sre molar ratios and concentration of the competitive ligand (Br-) on binding constants. We determined the effects of different assumed pKa values for FA and NOM thiols and the number of binding sites on the calculated constants and evaluated our results in the context of previously published values for MeHg+NOM binding.

Materials and Methods Sample Collection and NOM Isolation. IHSS SRFA, IR101F, was purchased from the International Humic Substance Society (IHSS), St. Paul, MN. Surface water was collected in acid-cleaned carboys from wetland S2 and Spring Lake in the Marcell Experimental Forest in north-central Minnesota and stored at 4 °C. Wetland S2 is an acidic, ombrotrophic bog. Spring Lake is fed by surrounding wetlands and is moderately colored. DEAE-cellulose, a hydrophilic extractant and weak anion exchanger (19), was used to extract the humic substances. Filtered bog and lake water (0.2-0.6 µm Millipore AP15 glass fiber membranes) was passed through Whatman DEAE-cellulose columns, and material retained on the column was eluted with 0.1 M NaOH. The eluant was passed through an H+ Amberlite strong acid cation exchange column, and the recovered NOM was freeze-dried (Virtis FreezeDryer). Analytical Methods. Ash content of NOM samples was determined by summing results from inductively coupled plasma emission spectrometry for cations (Na+, K+, Mg2+, VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Ca2+) and ion chromatographic analysis for anions (Cl-, SO42-, NO3-). Total carbon in samples was determined on a Carlo Erba CNS combustion analyzer interfaced with a Fisons stable isotope ratio mass spectrometer. NOM samples were analyzed for total S by Huffman laboratories (Golden, CO) using a combustion method. S2 NOM had 21.3% ash, 30.9% C, and 0.37% S; Spring Lake NOM had 5.8% ash, 45.2% C, and 1.84% S. IHSS SRFA has 1.0% ash, 53.0% C, and 0.47% S (20). Dissolved organic carbon (DOC) was determined using U.S. EPA method 415.1 (21) with UV-persulfate oxidation on a Tekmar Dohrmann Phoenix 8000 analyzer. A Beckman PHI 40 meter calibrated with standard buffers (pH 4.0, 7.0, 10.0) was used to measure pH. Methylmercury was determined by the method of Bloom (22) for total Hg after distillation (23) to separate methylmercury. Data Analysis. Visual MINTEQ (24) was used to calculate free MeHg+ from input data consisting of pH, MeHg+ bound to bromide, and Br-, K+, and Na+ concentrations, and MINEQL+ (25) was used to calculate MeHg+ equilibrium distributions in a two-site thiol-NOM model. MINITAB v12.23 was used to generate box and whisker plots and run homogeneity of variance and t tests. Sulfur X-ray Absorption near Edge Spectroscopy (XANES). The S K-edge (2472 eV) XANES spectra of S2 and Spring Lake NOM were collected on bending magnet beamline X-19A of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (Upton, NY). Scans ranged from 2452 to 2522 eV (relative energy of -20 to +50 eV). Five scans were collected for each sample to maximize the signal/noise ratio. The data were processed with MacXAFS (26) and the nonlinear least-squares fitting routine in the SOLVER function of Microsoft Excel. Percentages of sulfur in six oxidation states were calculated assuming a Gaussian shape for absorption peaks of the S species using procedures of Xia et al. (10). The six peaks represent oxidation states of possible sulfur species: thiol (R-SH); S(-I) and S(0); sulfide (R-S-R) and disulfides (R-S-S-R), S(0); sulfoxide (R-SO-R), S(II); sulfone (R-SO2-R), S(IV); sulfonate (R-SO3H), S(V); and sulfate (R-OSO3H), S(VI) (10, 27). The percent S in an oxidation state was determined from ratio of the area of a given peak to the total area of all peaks; the quantity of S in each oxidation state was determined by multiplying the fraction of S in an oxidation state by the total S content. The peaks for S(-I) and S(0) are difficult to separate with XANES; they were summed and reported as reduced sulfur, Sre. Binding Experiments of MeHg+ with IHSS SRFA and NOM Extracts. Studies were conducted to determine the effects of KBr concentration, Hg:Sre molar ratio, and pH on binding constants of MeHg+ with IHSS SRFA. A dialysis membrane method and competitive ligand-exchange technique with Br- was used to determine binding constants. Spectra/Por Cellulose ester membranes of nominal pore size 100 molecular weight cut off (MWCO) used in the equilibrium dialysis separations were purchased from Spectrum Laboratories, Rancho Dominguez, CA. Prior to experiments, membranes were soaked in ultrapure water for 72 h to remove sodium azide preservative. Experiments were carried out in the dark. MeHg+ recoveries and mass balances were performed. All results except one value each in Tables 1 and 3 had >80% recovery. Quality control procedures included the use of acid-cleaned glassware, clean-room techniques, and mass balance checks. The effect of KBr concentration on binding constant values was determined as follows. A 10-mL solution containing 40 mg of SRFA was spiked with MeHg+ to obtain a MeHg+:Sre molar ratio of 1:2460. MeHg+ was equilibrated with SRFA for 48 h, after which the solution was spiked with varying amounts of KBr solution and diluted to 25 mL to obtain Brconcentrations of 1.0, 2.5, 5.0, 10, and 30 mM. The solutions containing MeHg+, SRFA, and Br- were transferred into 100 6152

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MWCO membranes, and the ends were twist-tied with unwaxed dental floss. The membranes were placed in 50mL borosilicate test tubes and immersed in 25 mL of a KBr solution having the same concentration as the inner solution. The solutions were equilibrated for ∼120 h, after which aliquots were collected for analysis of pH and MeHg+. Controls containing SRFA and KNO3 were run to determine DOC leakage from the membranes. Controls were needed because isopropanol preservative in the MeHg stock solution interfered with DOC analysis. The effect of varying MeHg+:Sre molar ratios on binding constants was determined using 10-mL solutions containing 40 mg of SRFA spiked with MeHg+ to obtain MeHg+:Sre molar ratios of 1:1,220, 1:1,530, 1:2,040, 1:3,060, 1:6,120, and 1:12,200. MeHg+ was pre-equilibrated with SRFA for 48 h, after which the solutions were spiked with KBr solution and diluted to 25 mL to obtain a 5 mM KBr solution. Equilibration and analysis of samples and controls were performed as described above. The effect of pH on binding constant values was determined similarly using 10-mL solutions containing 40 mg of SRFA spiked with MeHg+ to obtain a MeHg+:Sre molar ratio of 1:1540. MeHg+ was pre-equilibrated with the SRFA for 48 h, after which the solution was spiked to obtain 5-500 mM Br- concentrations; the higher values were to compensate for anticipated stronger binding at higher pH. Solution pH was adjusted with 0.1 M NaOH after adding KBr to the SRFA-MeHg+ solutions. Equilibration and analysis was performed as described above. Calculation of Stability Constants. A small amount of DOC leakage was observed from the membranes. To correct for MeHg+ bound to this organic matter, we used the following procedure. MeHg+ bound to NOM inside the membrane was estimated from an algorithm assuming that MeHgBr concentrations were the same inside and outside the membrane. This value was used to estimate the amount of NOM-bound MeHg+ that leaked to the outer solution. Subtracting the NOM-bound MeHg+ in the outer solution from the total MeHg+ in the solution yielded the Br--bound MeHg+. This information was inserted into Visual MINTEQ, and the free MeHg+ concentration was calculated. For this calculation, we inserted the stability constant of MeHgBr into Visual MINTEQ: K ) 106.49 (25 °C; ionic strength, I ) 0.5) (28). The value was corrected to I ) 0 by the Davies equation before insertion into the database. RSHT was calculated from ST and XANES data. RSHT is assumed to be the entire Sre. In modeling MeHg+ binding, we first calculated a distribution coefficient, KOC: [MeHg(RS)T]

KOC )

{MeHg+}

(1)

where [MeHg(RS)T] has units of moles MeHg+ per kg C. This definition of KOC is similar to KDOM defined by Haitzer et al. (29) for Hg2+ binding to NOM. We calculated a complexation constant assuming one-to-one binding of thiol RS- to MeHg+: Ka

RSH y\z RS- + H+ KMeHgSR

RS- + MeHg+ y\z MeHg(RS)

(2)

(3)

For NOM samples we assumed RSH ) RSHT, RS- ) RST-, and MeHg(SR) ) MeHg(SR)T. The ionization constant of RSH is Ka )

[RST-][H+] [RSHT]

(4)

TABLE 1. MeHg+ Bound to IHSS SRFA at Different Br- Concentrationsa equilibrium KBr (M)

Br--bound MeHg+(M)

pH

NOM-bound MeHg+(mol/kg C)

calculated free MeHg+(M)

KOC

K′ for MeHg- SRFA

0.0010 0.0025 0.0050 0.010 0.030

2.9 × 10-9 1.0 × 10-8 8.1 × 10-9 7.6 × 10-9 1.4 × 10-8

2.81 2.80 2.80 2.81 2.87

5.0 × 10-5 3.3 × 10-5 3.6 × 10-5 2.5 × 10-5 2.7 × 10-5

7.7 × 10-13 1.1 × 10-12 4.6 × 10-13 2.3 × 10-13 1.6 × 10-13

6.5 × 107 2.9 × 107 7.9 × 107 1.1 × 108 1.7 × 108

6.7 × 1015 3.1 × 1015 8.2 × 1015 1.1 × 1016 1.5 × 1016

a K calculated assuming all Sre sites are RSH with pKa ) 9.96. NOM ) 615 mg C L-1; MeHg+ ) 5410 ng L-1; MeHg:Sre molar ratio ) 1:2460.

TABLE 2. MeHg+ Bound to IHSS SRFA at Various MeHg+:Sre Molar Ratiosa MeHg:Sre molar ratio 1:12200 1:6120 1:3060 1:2040 1:1530 1:1220 a

MeHg added (ng L-1) 1080 2160 4330 6500 8660 10800

pH 2.97 2.99 3.02 3.01 2.98 3.01

Br--bound MegH+(M)

NOM-bound MeHg+(mol/kg C)

-9

-5

1.4 × 10 5.6 × 10-9 7.4 × 10-9 1.7 × 10-8 1.8 × 10-8 1.6 × 10-8

1.1 × 10 2.0 × 10-5 4.0 × 10-5 5.6 × 10-5 4.8 × 10-5 7.8 × 10-5

calculated free MeHg+(M) -14

7.7 × 10 3.1 × 10-13 4.2 × 10-13 9.4 × 10-13 1.0 × 10-12 9.1 × 10-13

KOC

K′

1.5 × 10 6.3 × 107 9.5 × 107 5.9 × 107 4.7 × 107 8.6 × 107 8

1.1 × 1016 4.6 × 1015 6.5 × 1015 4.1 × 1015 3.5 × 1015 6.0 × 1015

K′ calculated assuming all Sre sites are RSH with pKa ) 9.96. NOM ) 615 mg C L-1.

Ka initially was assumed to have a value of 10-9.96, representative of compounds like mercaptoacetic acid (30). The conditional stability constant (K′) of MeHg+ bound to NOM was calculated from: K′ )

[MeHg(SR)T] [RST-]{MeHg+}

(5)

where {MeHg+}, the activity of MeHg+, ) γMeHg[MeHg+].

Results and Discussion Sulfur Content of NOM. Total organic sulfur was 1.95% (w/ w) for Spring Lake NOM and 0.47% (w/w) for S2 NOM; values are corrected for ash content. Spring Lake NOM had 5.8% ash and S2 NOM had 21.5% ash. The ash primarily was NaCl, a product of the cation-exchange regenerant, HCl, and DEAEcellulose eluant, NaOH. The difference in S content may reflect hydrogeochemical differences between the sites. Bog S2 has no groundwater input; Spring Lake has contiguous wetlands and receives organic carbon from terrestrial sources. The S XANES spectrum of Spring Lake and S2 NOM showed three broad peaks and one shoulder that were deconvoluted into six Gaussian peaks representing sulfur oxidation states. The fraction of ST in reduced states was 20% for Spring Lake NOM and 27% for S2 NOM. The total S in reduced states (Sre) thus was 0.39% (w/w) for Spring Lake NOM and 0.13% (w/w) for S2. In contrast, IHSS SRFA has a total S of 0.47% (w/w), of which 47% is in reduced states (10); its Sre thus is 0.24% (w/w). MeHg+ Binding with IHSS SRFA. Preliminary Studies. A preliminary study with SRFA and 500- and 100-MWCO Spectra/Por dialysis membranes evaluated DOC leakage from the membranes. Almost 6% of the organic carbon leaked from 500-MWCO membranes, but leakage from 100-MWCO membranes was 4 (Table 4) but significant dependency below this pH. The KOC values (Table 4) are >10× greater than those of our aquatic samples (Table 3). The former data were obtained at a very high Sre:MeHg+ molar ratio (2 × 106). Although we did not see a dependency of KOC on the Sre: MeHg+ ratio (Table 2), the very low MeHg+ loadings of Karlsson and Skyllberg may have accessed a small population of RS- groups that form stronger bonds with MeHg+, but the differences also could simply reflect sample and method differences. The effect of pH on stability constants of MeHg+ bound to SRFA was evaluated using several assumptions about the nature of Sre. When all Sre was assumed to be in the form of thiol groups with pKa ) 9.96 (representative of compounds like mercaptoacetic acid), K′ decreased from 1015.6 at pH 2.98 to 1012.3 at pH 7.62 (Figure 1). Decreasing the pKa of the thiol binding sites to 8.5 lowered K′ to the range 1014.3 (pH 2.98) to 1011.3 (pH 7.62) (Figure 1), but the overall trend did not change. A similar trend was found by Karlsson and Skyllberg (16). They tried to account for the pH dependence by assuming that the RSH groups in NOM are a mixture of two sites, 90% with pKa ) 9.95 and 10% with pKa ) 8.5, but this had little effect on the pH dependence. The decrease in K′ with increasing pH for the thiol binding model is counterintuitive. Normally, such values are pHinvariant, but an increase in conditional stability constants with pH could be explained for macromolecular ligands like SRFA and NOM by electrostatic considerations (increasing 6154

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FIGURE 1. Binding constants of MeHg to IHSS SRFA at different pH values. Diamonds represent binding with RSH sites with a pKa ) 9.96; circles represent binding with RSH sites with pKa ) 8.5; open triangles represent binding with proton-independent RSSR or RSR sites. MeHg:Sre molar ratios were 1:1540; Br- concentrations varied from 0.005 to 0.5 M. Dark square represents Spring Lake NOM and dark triangle represents S2 NOM (RSH sites with assumed pKa ) 9.96 in both cases). deprotonation of NOM at higher pH should increase its attraction for cations). Thiol-based binding constants that are independent of pH can be derived only if we use a thiol pKa of ∼4.0 or less, which is lower than usually reported for thiol groups. Karlsson and Skyllberg (16) expressed reservations about such models, but thiols with low pKa values are known. Metal-binding proteins containing clustered cysteine groups with low pKa (4.5 or less) are thought to be common in plant metallothionines (31), and a pKa of 3.8 was measured for cysteine sites in enzymes (32). We searched the NIST database of critical stability constants (33) and found several thiophenols containing electrophilic substituents with pKa values of 4.7-6.8; in addition, thionitrobenzoic acid has a pKa of 4.4 (34). Using SPARC (35), we calculated a pKa of 3.94 for 3-acetyl-5-hydroxy-2-mercaptobenzaldehyde, which is structurally consistent with models of aromatic components in humic substances. Thiophenols can form readily in anoxic sediments by the addition of HS- to NOM (36, 37). By assuming that MeHg+ in SRFA and the organic soil of Karlsson and Skyllberg (16) is bound to thiols with a pKa of ∼4 we were able to generate pH-invariant stability constants, hereafter referred to as K′4. We used pKa ) 4.2 to generate such stability constants for SRFA (Table 3) and pKa ) 3.7 for the stream bank organic soil (Table 4). The mean of K′4 for SRFA is 6.36 × 109 (109.80), and the K′4 for the organic soil is 7.52 × 1010 (1010.88).

We made several attempts to fit our data to a two-site model, but none yielded a better fit than the simple one-site, pH-independent model described above. For example, we used the measured values in Table 3, assumed binding site pKa values of 4.0 and 9.96, and assumed that the low pKa sites represented 0.1% of Sre. Best results were obtained when we used an assumed binding constant of 1 × 1012 for the weak acid site, but MatLab-fitted values of the binding constant for the strong acid site ranged from 1.3 × 1012 to 8.3 × 1013, much larger than the range of K′4 in the single site model (Table 3). When we used the mean K′4 for SRFA found from the one-site model (109.80) and assumed they constituted 1% Sre and further assumed that a weak-acid thiol site (pKa ) 9.96) with K′ ) 109.30 constituted 99% of Sre, we obtained the results in the last three columns of Table 3 using MINEQL+. The amounts of NOM-bound MeHg+ calculated using this model agree reasonably well over the pH range ∼3.0-7.6 with our measured values; the weak acid binding site starts to become important only at pH > 7. NOM likely contains proton-independent reduced S groups like thioethers (RSR), which occur in methionine, and disulfide (RSSR) groups, which occur in proteins as the amino acid dimer, cystine. MeHg+ binding to these sites is weak, however, and unlikely to account for MeHg+ binding in NOM. Reported logK values range from -1.2 to +1.8 (38). Binding of MeHg+ by methionine is with the carboxyl and amino groups, not the thioether (39), and K for MeHg+-methionine (107.4) is similar to that for MeHg+glycine (107.9) (33). NOM-MeHg+ Binding. The NOM extracts were loaded with MeHg+ at MeHg+:Sre molar ratios of 1:5990 (Spring Lake) and 1:1280 (Bog S2). Partition coefficients (KOC) determined with 0.005 M KBr were 107.9 and 106.9, respectively, and stability constants (K′) at pH 3.0 were 1015.5 for Spring Lake and 1014.5 for S2 when we assumed that all Sre was present as thiol groups with pKa ) 9.96. Using pKa ) 4.0 for thiol groups yielded K′4 values of 109.6 for Spring Lake and 108.52 for S2. The K′ and K′4 values of Spring Lake NOM are similar to those for SRFA at pH 2.98 using the RSH model, but values for S2 NOM are lower by factors of ∼10. Statistical Analyses. Box and whisker plots of the KOC values in Tables 1-3 showed there were no outliers in the data. Homogeneity of variance and two-sample t tests were run on the KOC values. The experiments in Table 1 and 2 were done at the same pH (∼3) under varying bromide concentration or MeHg:Sre ratios and thus were good data sets to test the robustness of the results. The p-values for homogeneity of variance (0.571) and the t tests (0.80) indicate that KOC values in both tables had equal variances and means were statistically similar. Comparison with Other Studies. Comparison of our findings with previous studies is difficult because of differences in analytical conditions and calculation models among the studies. Hintelmann et al. (14) used an equilibrium dialysis membrane (500 MWCO) and Scatchard analysis to determine stability constants of MeHg+ bound to NOM from Canadian lakes and IHSS SRFA. Scatchard plots produce convex curves if two or more binding sites are present; slopes of the steep and shallow asymptotes of the curve are used to obtain strong and weak stability constants. It is not possible to extract more than two constants from Scatchard plots, even if more sites exist in the complexing agent. (A Scatchard plot of our data did not extend far enough on the x-axis to yield evidence of a weak site.) Hintelmann et al. (14) reported binding constants at pH 6.5 of 1013.02-1014.56 for strong sites and 1012.15-1013.07 for weak sites. Their approach differed from ours in that concentrations of sites were generated from the Scatchard plots and the effect of H+ was not considered. Their experiments used solutions with 1 mg L-1 of NOM and 0.5-100 ng L-1 of MeHg. If their NOM was 50% C and 0.5%

ST, of which 50% was Sre (similar to IHSS SRFA), the MeHg+: Sre ratio at their lowest loading was 1:167, much higher than our highest MeHg loading (1:1220). At their highest loading, the MeHg+:Sre ratio approached 1. Amirbahman et al. (15) also used 500-MWCO equilibriumdialysis membranes and a discrete log K spectrum approach to model MeHg+ binding. They assumed pKa values of 4, 7, and 10 for three thiol binding sites and analyzed their data by the program FITEQL. Binding capacities and log K values were varied to obtain a best fit. For IHSS Suwannee River humic acid (SRHA), IHSS Peat HA, and a stream HA from Maine, they obtained binding constants in the ranges 1010.39-1010.54, 1012.39-1014.77 and 1014.47-1014.84 for sites with pKa values of 4, 7 and 10, respectively. They attributed the three sites to disulfane (RSSH), cysteine, and other thiolcontaining organic acid groups, respectively. The site with pKa ) 7 was dominant for MeHg+ binding over most of the pH range 3.5-9.2. Most of their data were obtained at much higher loading of MeHg+ than in our study, and unlike our results (Table 2), their sorption data were not linear. Their highest MeHg+ additions corresponded to MeHg+:Sre ratios >10 times greater than our largest additions. Log KOC values for these additions were e7. The stability constants calculated by Amirbahman et al. (15) are difficult to compare to our results partly because the RST- concentrations they used for eq 5 were calculated from FITEQL, whereas we used total Sre. The measured Sre concentration in SRHA is 550× the concentration of sites with pKa ) 4 determined by Amirbahman et al. and 45× the concentration of the sum of all their sites. If we assumed a similar relative site density for pKa ) 4 sites in SRFA, our pH-independent K′4 would be 1012.5 instead of 109.8; the increase simply reflects a 550-fold decrease for RST- in the denominator of eq 5. It is apparent from the above comparisons that binding constants depend on MeHg:Sre ratios and the model used to determine constants. Important parameters include binding site concentrations and acidity constants of RSH in NOM. At low MeHg+ loadings, the binding strength is pH-independent at pH > ∼4, suggesting that MeHg+ is bonding with thiols having a pKa of ∼4; at higher MeHg+ loadings, thiols with higher pKa values may become important in MeHg+ binding, requiring more complicated multisite models, but our data did not extend to high enough loadings to access these sites. The low-pKa sites may be cysteine thiols clustered in remnants of metal-binding proteins incorporated into NOM or may be highly substituted thiophenols. Our simple one-site approach, which lumps all Sre together and uses pKa as the only fitting parameter, is easy to use and requires only a few data points to estimate K′. The approach probably overestimates the concentration of very acidic thiol groups and oversimplifies the complexity of thiol moieties in NOM, but lacking a more detailed understanding of the chemistry of Sre in NOM, we conclude that it works well to model MeHg+-NOM binding at the very low concentrations of MeHg+ typically found in surface waters and soils.

Acknowledgments This work was supported by a grant from the 104G national competitive grants program of the Water Resources Research Institutes Program operated by the USGS and by a USDA grant (USDA NRI 98-35107-6515). We thank Chung-Ming Lin for XANES analysis, Neal Hines for help in collecting water samples, and Ulf Skyllberg for sharing his raw data on MeHg binding.

Supporting Information Available Details of MeHg analysis, MeHg mass balances for the experiments, a figure showing the calculation algorithm to compute MeHg+ bound to NOM, a table of literature-based pKa values for substituted thiols, a figure of box and whisker VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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plots, and a table of statistical information. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Moye, H. A.; Miles, C. J.; Philips, E. J.; Sargent, B.; Merritt, K. K. Kinetics and uptake mechanisms for monomethylmercury between freshwater algae and water. Environ. Sci. Technol. 2002, 36, 3550–3555. (2) Hammerschmidt, C. R.; Wiener, J. G.; Frazier, B. E.; Rada, R. G. Methylmercury content of eggs in yellow perch related to maternal exposure in four Wisconsin lakes. Environ. Sci. Technol. 1999, 33, 999–1003. (3) Rouleau, C.; Block, M.; Tja¨lve, H. Kinetics and body distribution of waterborne 65Zn(II), 109Cd(II) and 203Hg(II) and CH3203Hg(II) in phantom midge larvae (Chaoborus americanus) and effects of complexing agents. Environ. Sci. Technol. 1998, 32, 1230– 1236. (4) Sjoblom, A.; Meili, M.; Sundbom, M. The influence of humic substances on the speciation and bioavailability of dissolved mercury and methylmercury, measured as uptake by Chaoborus larvae and loss by volatilization. Sci. Total Environ. 2000, 261, 115–124. (5) Monson, B. A.; Brezonik, P. L. Influence of food, aquatic humus, and alkalinity on methylmercury uptake by Daphnia magna. Environ. Toxicol. Chem. 1999, 18, 560–566. (6) Matilainen, T.; Verta, M. Mercury methylation and demethylation in aerobic surface waters. Can. J. Fish. Aquat. Sci. 1995, 52, 1597–1608. (7) Watras, C. J.; Morrison, K. A.; Host, J. S.; Bloom, N. S. Concentration of mercury species in relationship to other sitespecific factors in the surface waters of northern Wisconsin lakes. Limnol. Oceanogr. 1995, 40, 556–565. (8) Pettersson, C.; Bishop, K.; Lee, Y.-H.; Allard, B. Relations between organic carbon and methylmercury in humic rich surface waters from Svartberget catchment in northern Sweden. Water, Air Soil Pollut. 1995, 80, 971–979. (9) Hurley, J. P.; Krabbenhoft, D. P.; Cleckner, L. B.; Olson, M. L.; Aiken, G. R.; Rawlik, P. S., Jr. System controls on the aqueous distribution of mercury in the northern Florida Everglades. Biogeochemistry 1998, 40, 293–310. (10) Xia, K.; Weesner, F.; Bleam, W. F.; Bloom, P. R.; Skyllberg, U. L.; Helmke, P. A. XANES studies of oxidation states of sulfur in aquatic and soil humic substances. Soil Sci. Soc. Amer. J. 1998, 62, 1240–1246. (11) Qian, J.; Skyllberg, U. L.; Frech, W.; Bleam, W. F.; Bloom, P. R.; Petit, P. E. Bonding of methyl mercury to reduced sulfur groups in soil and stream organic matter as determined by X-ray absorption spectroscopy and binding affinity studies. Geochim. Cosmochim. Acta 2002, 66, 3873–3885. (12) Yoon, S.-J.; Diener, L. M.; Bloom, P. R.; Nater, E. A.; Bleam, W. F. X-ray absorption studies of CH3Hg+-binding sites in humic substances. Geochim. Cosmochim. Acta 2005, 69, 1111–1121. (13) Hintelmann, H.; Welbourne, P. M.; Evans, R. D. Binding of methylmercury compounds by humic and fulvic acids. Water, Air Soil Pollut. 1995, 80, 1031–1034. (14) Hintelmann, H.; Welbourn, P. M.; Evans, D. R. Measurement of complexation of methylmercury(II) compounds by freshwater humic substances using equilibrium dialysis. Environ. Sci. Technol. 1997, 31, 489–495. (15) Amirbahman, A.; Reid, A. L.; Haines, T. A.; Kahl, J. S.; Arnold, C. Association of methylmercury with dissolved humic acids. Environ. Sci. Technol. 2002, 36, 690–695. (16) Karlsson, T.; Skyllberg, U. L. Bonding of ppb levels of methylmercury to reduced sulfur groups in soil organic matter. Environ. Sci. Technol. 2003, 37, 4912–4918. (17) Khwaja, A. R.; Bloom, P. R.; Brezonik, P. L. Binding constants of divalent mercury (Hg2+) in soil humic acids and soil organic matter. Environ. Sci. Technol. 2006, 40, 844–849.

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(18) Hines, N. A.; Brezonik, P. L. Input-output analysis of mercury forms for a small lake in northern Minnesota. Biogeochemistry 2007, 84, 265–84. (19) Miles, C. J.; Tuschall, J. R., Jr.; Brezonik, P. L. Isolation of aquatic humus with diethylaminoethyl cellulose. Anal. Chem. 1983, 55, 410–411. (20) IHSS Web site. http://www.ihss.gatech.edu/. (21) U.S. EPA. Methods for the Chemical Analysis of Water and Wastes (MCAWW); EPA/600/4-79/020; Washington, DC, 1974. (22) Bloom, N. S. Determination of picogram levels of methylmercury by aqueous phase ethylation, followed by cryogenic gas chromatography with cold vapor atomic fluorescence detection. Can. J. Fish. Aquat. Sci. 1989, 46, 1131–1139. (23) Horvat, M.; Bloom, N. S.; Lang, L. Comparison of distillation with other current isolation methods for the determination of methylmercury compounds in low level environmental samples. Anal. Chim. Acta 1993, 135–152. (24) Visual MINTEQ. Visual MINTEQ, version 2.1, www.lwr.kth.se/ English/OurSoftware/vminteq, 2002. (25) MINEQL+ version 4.6, a chemical equilibrium modeling system. Environmental Research Software, Hallowell, ME, http:// www.mineql.com, 2008. (26) Bouldin, C.; Furenlid, L.; Elam, T. MacXAFS: an EXAFS analysis package for the Macintosh. Phys. B 1995, 208/209, 190–192. (27) Skyllberg, U. L.; Xia, K.; Bloom, P. R.; Nater, E. A.; Bleam, W. F. Binding of mercury(II) to reduced sulfur in soil organic matter along upland-peat soil transects. J. Environ. Qual. 2000, 29, 855–865. (28) Martell, A. E.; Smith, R. M. Critical Stability Constants Vol. 4: Inorganic Complexes, Plenum Press: New York, 1976. (29) Haitzer, M.; Aiken, G. R.; Ryan, J. N. Binding of mercury(II) to dissolved organic matter: the role of the mercury-to-DOM concentration ratio. Environ. Sci. Technol. 2002, 36, 3564–3570. (30) Hilton, B. D.; Man, M.; His, E.; Bryant, R. G. NMR studies of mercury-halogen equilibria. J. Inorg. Nucl. Chem. 1975, 37, 1073– 1077. (31) Freisinger, E. Plant MTsslong neglected members of the metallothionein superfamily. Dalton Trans. 2008, 47, 6663– 6675. (32) Guengerich, F. P.; Fang, Q. M.; Liu, L. P.; Hachey, D. L.; Pegg, A. E. O-6-Alkylguanine-DNA alkyltransferase: low pK(a) and high reactivity of cysteine 145. Biochemistry 2003, 42, 10965– 10970. (33) Martell, A. E.; Smith, R. M.; Motekaitis, R. J. NIST Standard Reference Database 46 Version 7.0. Critically Selected Stability Constants of Metal Complexes; National Institute Standards Technology: Gaithersburg, MD, 1998. (34) Summa, D.; Spiga, O.; Bernini, A.; Venditti, V.; Priora, R.; Frosali, S.; Margaritis, A.; Di Giuseppe, D.; Niccolai, N.; Di Simplicio, P. Protein-thiol substitution or protein dethiolation by thiol/ disulfide exchange reactions: the albumin model. Proteins 2007, 69, 369–378. (35) SPARC (http://ibmlc2.chem.uga.edu/sparc/). (36) Urban, N. R.; Ernst, K.; Bernasconi, S. Addition of sulfur to organic matter during early diagenesis of lake sediments. Geochim. Cosmochim. Acta 1999, 63, 837–853. (37) Perlinger, J. A.; Kalluri, V. M.; Venkatapathy, R.; Angst, W. Addition of hydrogen sulfide to juglone. Environ. Sci. Technol. 2002, 36, 2663–2669. (38) Bach, R. D.; Vardhan, H. B. A Hg-199 NMR-study of the complexation of methylmercury with thio-crown etherssthe absence of a macrocyclic ligand effect. J. Org. Chem. 1986, 51, 1609–1610. (39) Fairhurst, M. T.; Rabenstein, D. L. Nuclear magnetic-resonance studies of solution chemistry of metal-complexes. 12. Binding of methylmercury by methionine. Inorg. Chem. 1975, 14, 1413–1415.

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