Controls of Dissolved Organic Matter and Chloride on Mercury Uptake

Oct 27, 2009 - the uptake of Hg(II) or MeHg, DOC resulting from diatom decomposition ..... mercury content against uptake time (0-1 h). .... and HgCl4...
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Environ. Sci. Technol. 2009 43, 8998–9003

Controls of Dissolved Organic Matter and Chloride on Mercury Uptake by a Marine Diatom HUAN ZHONG AND WEN-XIONG WANG* Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

Received June 4, 2009. Revised manuscript received October 12, 2009. Accepted October 14, 2009.

The effects of natural dissolved organic carbon (DOC) from different origins (estuarine, coastal, and diatom decomposed) and chloride (Cl) on the uptake of inorganic mercury [Hg(II)] and methylated mercury (MeHg) by the marine diatom Thalassiosira pseudonana was investigated using radiotracer techniques. We first developed a new method to remove the surface adsorbed mercury and quantified the intracellular mercury uptake by the diatoms. The dominant mercury species (DOC or chloride complexes, based on the mercury speciation phase diagrams) was controlled by the concentrations of DOC and Cl-, which could explain the effects of DOC and Cl- on mercury uptake. DOC complexes dominated Hg(II)’s speciation and reduced its uptake in most seawater examined. DOC complexes dominated MeHg’s speciation only at relatively high DOC levels (>100 µM), but it could affect MeHg uptake even when MeHg-Cl complexes dominated. In a mercury-DOC complex dominated system, both the origin and quantity of DOC greatly influenced mercury uptake by the diatoms. Although DOC generally inhibited the uptake of Hg(II) or MeHg, DOC resulting from diatom decomposition enhanced Hg(II) uptake. Under conditions dominated by chloride complexation, neutral mercury chloride species (HgCl2 or MeHgCl) may control the uptake.

Introduction Mercury pollution is a significant global environmental problem, and one of the greatest concerns about mercury is its trophic transfer along the aquatic food chain, especially in its methylated form. This can lead to a significant risk to human health through seafood consumption. Marine phytoplankton are at the bottom of many food chains, and their mercury accumulation could greatly affect its trophic transfer and thus the mercury accumulation at higher trophic levels, including in fish. It is thus important to understand the bioavailability of mercury to marine phytoplankton, which often represents the greatest leap of mercury accumulation in a marine food chain. For example, methylmercury concentration increased by 104.2 times from water to microseston (including phytoplankton and bacterioplankton), 2.2 times from microseston to zooplankton, and 24.5 times from zooplankton to fish in Long Island Sound, U.S. (1). Due to its strong binding with thiol groups (2, 3), mercury is usually complexed with dissolved organic carbon (DOC) in oxic waters (4), and DOC complexes become the dominant mercury species at high DOC levels (2, 3). Chloride is the most abundant inorganic ligand in seawater and could form * Corresponding author e-mail: [email protected]. 8998

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strong complexes with Hg(II) in saline water (5). Such chloride complexes could be the dominating mercury species in high chloride but low DOC conditions (4, 5). A previous study compared the effects of chloride and pH on the speciation and accumulation of mercury in a marine diatom (6). Given the significant complexing of mercury with both DOC and chloride, it might be informative to explore the relative roles of DOC and chloride in the uptake of Hg(II) or MeHg by marine phytoplankton. A few studies have reported limited and contradictory results on the effects of DOC on the uptake of Hg(II) or MeHg by freshwater diatoms, but these studies generally did not differentiate surface adsorption from intracellular uptake. DOC was found to have no effect on Hg(II) uptake (7), decrease (8, 9), or increase (7) the uptake of MeHg. In marine waters, the influence of DOC may be affected by chloride complexation, but there has been no study directly comparing the respective effects of DOC and chloride on mercury uptake. Studies of Hg uptake by marine phytoplankton are often impeded by difficulties in quantifying the true internalization rate excluding surface adsorption. There is no known method able to remove adsorbed mercury from the surface of phytoplankton, although indirect comparison between surface adsorption and intracellular uptake has been made by comparing the uptake by live and dead cells (7). EDTA has previously been used to remove adsorbed MeHg from the surface of freshwater algae, but the removal efficiency was not specifically quantified (8). EDTA’s affinity may not be strong enough to compete with Hg for binding sites (e.g., thiol groups) on the surface of phytoplankton and to remove mercury, especially for Hg(II) with its high affinity with thiol groups. Just recently, a washing technique using glutathione as the main complexing agent to remove adsorbed Hg(II) from bacteria was developed, and high washing efficiencies were demonstrated (10). It thus remains to develop an appropriate method for removing adsorbed mercury from marine phytoplankton before accurately quantifying the intracellular uptake. In this study, a method to remove surface adsorbed Hg(II) or MeHg from diatoms was first developed. This was then applied to quantify the intracellular uptake using a radioisotope technique. The effects of the origin and level of DOC on the uptake of Hg(II) and MeHg were then investigated using DOC from natural seawater and from decomposed diatoms. The different roles of DOC and chloride in controlling mercury uptake were compared by modifying the chloride concentrations in artificial seawater with different DOC levels. The effects of DOC and chloride were explained by the changes of dominant mercury species (DOC or chloride complexes assessed by mercury speciation diagrams) as a function of DOC and Cl- levels.

Materials and Methods Diatom and Isotopes. A common coastal diatom Thalassiosira pseudonana (clone 3H) was obtained from the Provasoli-Guillard phytoplankton collection, Maine, maintained in f/2 medium (11) at 18 °C and used for the uptake experiments. The radioactive isotopes 203Hg(II) as 203HgCl2 and Me203Hg as CH3203HgCl were used in the uptake experiments. The γ-emitting radioisotope 203Hg (t1/2 ) 46.6 days, 203 HgCl2 in 0.1 N HCl, with a specific activity of 53 GBq/g) was purchased from Isotope Products Laboratories (Valencia, CA). Me203Hg was synthesized using the method of Rouleau and Block (12). All seawater (natural or artificial) was filtered through 0.22 µm nitrocellulose membranes (Millipore, U.S.) before use. 10.1021/es901646k CCC: $40.75

 2009 American Chemical Society

Published on Web 10/27/2009

TABLE 1. Treatments in the Hg(II) and MeHg Uptake Experiments and the Possible Dominant Complexes (Mercury-DOC Complexes or Mercury-Cl Complexes)a possible dominant speciesb treatment

pH

DOC origin and quantity CW CWUV TH THUV YL YLUV DOC-Cl DOC1 DOC2 DOC1-L DOC2-L

8.32 8.21 7.98 8.37 6.92 7.88 8.21 8.21 8.21 8.21

-

seawater origin DOC origin DOC conc. (µM) Cl conc. (M) CW CW TH TH YL YL artificial artificial artificial artificial

CW CW TH TH YL YL diatom diatom diatom diatom

77 0 106 38 200 0 1 100 1 100

0.52 0.52 0.48 0.48 0.31 0.31 0.52 0.52 0.2 0.2

Hg

MeHg

DOC Cl DOC DOC/Clc DOC Cl Cl DOC DOC/Clc DOC

Cl Cl Cl Cl DOC/Clc Cl Cl DOC/Clc

a CW: Clearwater Bay seawater; TH: Tolo Harbor seawater; YL: Yuen Long seawater; UV: UV oxidation. b Estimation based on the established speciation phase diagrams (3, 17) with the assumption that the ligand equivalent to the DOC ratio was 5 × 10-6 to 50 × 10-6, and the conditional stability constants log K’ was 25 for Hg(II) and log K’ ) 13 for MeHg. c The dominance of mercury-DOC complexes and mercury-Cl- complexes could be comparable.

Removal of Surface Adsorbed Hg(II) and MeHg. Different washing procedures were tested for removing the surface adsorbed mercury. In order to minimize intracellular mercury uptake, dead diatoms were used in most experiments to quantify the surface adsorbed mercury and thus the removal efficiency of different washing procedures, while live cells were only used in a few treatments for comparison. Diatom cells in the exponential growth phase were filtered gently onto a 3 µm polycarbonate membrane, rinsed with filtered seawater and then resuspended in filtered seawater at an approximate cell density of 105 cells/ml. Diatoms were heat killed at 50 °C for 10 min while the cells remained intact (13). The dead cells were filtered onto a 3 µm polycarbonate membrane and resuspended in seawater spiked with 203Hg(II) or Me203Hg for 5 min. After this, a 1 mL sample was transferred to a ultracentrifuge tube with 0.22 µm membrane (Millipore, U.S.), centrifuged at 2267g for 1 min, rinsed with the same uptake medium without Hg isotopes and centrifuged again, after which the radioactivity in the diatoms was measured as the amount of surface adsorbed Hg(II) and MeHg. In other treatments, the radiolabeled dead cells were resuspended in seawater containing 0.08, 0.8, or 8 mM of freshly prepared cysteine (Sigma-Aldrich, U.S.) for 1, 5, or 10 min. The mixtures were then removed into the 0.22 µm ultracentrifuge tubes, centrifuged at 2267g for 1 min, rinsed with the same uptake medium without Hg isotopes and centrifuged again. The radioactivity remaining in the diatoms was measured and the washing efficiency was calculated. Two replicates were conducted for all treatments. Besides cysteine, 8 mM glutathione (GSH) (Sigma-Aldrich, U.S.) was also used for mercury washing for 1 min. A similar washing procedure (0.8 mM cysteine, 1 min) was applied to live cells for comparison. Uptake Experiments. Two different experiments were conducted to determine the influence of DOC quantity and quality and the relative importance of DOC and chloride on the internalization of Hg(II) or MeHg by the diatoms (Table 1). The first experiment was designed to quantify the influences of DOC origin and level on Hg uptake. Natural seawater was collected from Clearwater Bay (CW), Tolo Harbor (TH), and Yuen Long (YL) in Hong Kong. Clearwater Bay is heavily influenced by Kuroshio ocean current, while Yuen Long is heavily influenced by the Pearl River estuary. Difference of Cl concentration is shown in Table 1. Tolo Harbor is an enclosed harbor with high nutrient levels and high phytoplankton concentrations. A portion of each seawater sample was photo-oxidized (ACE UV lamp, model

7480) for 48 h to eliminate any DOC. (After irradiation, these materials were designated CWUV, THUV, and YLUV respectively.) In a second experiment, Hg(II) uptake by the diatoms was quantified at different DOC and Cl- concentrations. DOC from diatom decomposition was used in this experiment. The diatoms were first grown to their stationary phase and then decomposed in CWUV seawater for 4 weeks until the decomposition reached steady-state (14). The seawater was then filtered and diluted with artificial seawater to reach two different DOC concentrations and two chloride concentrations (DOC1: 1 µM DOC, 0.52 M Cl-; DOC2: 100 µM DOC, 0.52 M Cl-; DOC1-L, 1 µM DOC, 0.2 M Cl-; DOC2-L: 100 µM DOC, 0.2 M Cl-). The original artificial seawater recipe included 0.32 M NaCl, 22.5 mM Na2SO4, 7.3 mM KC1, 1.87 mM NaHCO3, 42.4 mM MgC12, and 0.34 mM H3BO3, with the concentrations of NaCl, KCl, and MgCl2 decreased proportionally to reach the lower chloride concentration (0.2 M Cl-) in our artificial seawaters (DOC1-L and DOC2-L). The pH was adjusted to 8.21 with sodium hydroxide or hydrogen chloride. No MeHg uptake experiment was conducted at the lower chloride concentration, since no data about the affinity of MeHg with DOC was available to make dependable MeHg speciation predictions. The various uptake media (Table 1) were spiked with radioactive Hg(II) or MeHg (at 400 ng/L for Hg(II) and 200 ng/L for MeHg). The radioisotopes were equilibrated at 18 °C for 2 h before the uptake experiments. Based on our preliminary experiments, further increase of equilibration time did not change the mercury uptake significantly but increased the adsorption of mercury onto walls of containers. To measure the Hg(II) or MeHg uptake, diatom cells in the exponential growth phase were filtered gently onto a 3 µm polycarbonate membrane, rinsed with the appropriate uptake medium and then resuspended in the medium at an approximate cell density of 105 cells/mL. At 15, 30, 45, and 60 min, a 1 mL sample was removed, with which 8 mM cysteine solution was reacted for 5 min (for the Hg-YL treatment) or 1 min (for all other treatments) to remove surface adsorbed Hg(II) or MeHg. The cells were then centrifuged at 2267g in the 0.22 µm ultracentrifuge tube for 1 min. A 5 min wash was used for the YL treatment mainly because early runs showed that removing surface absorbed Hg took longer than in the other experiments (1 min). The diatom cells were rinsed with the same uptake medium and centrifuged again in the 0.22 µm ultracentrifuge tube for 1 min. VOL. 43, NO. 23, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Washing efficiencies of different reagents for removing Hg(II) or MeHg from the surface of diatom cells (heat-killed except the column labeled Live) exposed to seawater collected from Clearwater Bay. Cys: Cysteine; GSH: Glutathione; a: 0.08 mM; b: 0.8 mM; c: 8 mM; 1 or 10 min denotes the washing time. Mean + semirange (n ) 2). The uptake experiments lasted for 1 h at 18 °C to guarantee a linear increase of uptake with time. During the hour, the cell density remained unchanged, while the mercury concentration in the medium decreased by 90%) of the Hg(II) or MeHg from the dead cells, while the unwashed mercury could either be due to the diffusion of mercury into cells during the 5 min contact or strong binding between mercury and cell membrane. Such high removal efficiency was probably due to the competitive binding of mercury with cysteine in the medium and cysteine on the biological membrane. Seawater DOC played a minor role in removing mercury since seawater itself removed little of the adsorbed mercury from cells according to our preliminary 9000

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FIGURE 2. Uptake of Hg(II) or MeHg by the diatom Thalassiosira pseudonana exposed to seawater collected from Clearwater Bay over time. experiments. Washing efficiencies increased with cysteine concentration from 0.08 to 0.8 mM, but further increase to 8 mM did not result in higher efficiency. When the washing time increased from 1 to 10 min, there was only a small increase in washing efficiency. Washing with 8 mM cysteine for 1 min was enough to ensure a washing efficiency of >90% in most treatments. The only exception was the YL seawater, in which a 5 min of washing was required to obtain >90% efficiency for Hg(II). This may have been due to a higher DOC concentration and different DOC origin in this medium. One interesting observation was that in YL seawater little MeHg was adsorbed on the cells (below the detection limit). Nevertheless, the washing procedure (8 mM Cysteine, 1 min) was still applied for consistency. Hg(II) washing efficiencies with dead and live cells using the same washing procedures were comparable, indicating that little Hg(II) entered the cells within 5 min and heat treatment of cells may not affect much the diffusion of Hg(II) into cells. For MeHg, a lower washing efficiency was obtained for the live cells (74% compared to 95% for the dead cells) using the same washing procedure, suggesting much faster intracellular uptake of MeHg than Hg(II). Recently, Schaefer and Morel (10) have reported that cysteine increases the uptake and methylation rates of Hg(II) by bacteria at low concentrations (1-100 µM), but these rates decreased rapidly at higher cysteine levels (>100 µM). In this study, the much higher cysteine concentration (8 mM) should not have contributed to intracellular uptake, as evidenced by the very high washing efficiencies. In addition, when the live diatoms were exposed to mercury-cysteine complexes (prepared by reacting 400 ng/L radioactive Hg(II) or 200 ng/L radioactive MeHg, the same as those used in the uptake experiments, with 8 mM cysteine for 1 h), no radioactivity was detected in either the intracellular or adsorbed pool within 1 h, further suggesting that cysteine washing did not affect the intracellular uptake of mercury in this study. Influences of DOC Origin and Level on Hg(II) or MeHg Uptake. Intracellular mercury in diatom cells increased linearly (r2 > 0.9 and p < 0.05) with exposure time (0-1 h) in most treatments (Figure 2). The uptake rate was therefore calculated as the slope of the linear regression between intracellular accumulation and exposure time. The calculated uptake rates of Hg(II) or MeHg in different coastal or estuarine seawater before or after the removal of DOC are shown in Figure 3. Photo-oxidization by UV eliminated nearly all DOC in the seawater except for the TH seawater (Table 1), in which about 76% of the DOC was removed by photo-oxidation.

FIGURE 4. The percentage of intracellular Hg(II) or MeHg in the diatom Thalassiosira pseudonana after 1 h of exposure. The treatment abbreviations are defined in Table 1. Mean + semirange (n ) 2).

FIGURE 3. Calculated internalization rates of Hg(II) or MeHg by the diatom Thalassiosira pseudonana in different natural seawaters before or after UV oxidation. The treatment abbreviations are defined in Table 1. Mean + semirange (n ) 2). The uptake of Hg(II) or MeHg was generally inhibited by the presence of DOC, especially for Hg(II), depending on the quantity of DOC. Eliminating DOC with UV light greatly increased the uptake of Hg(II) (by 3.6-9.2 times, p < 0.05 for CW and YL, p < 0.1 for TH). For MeHg, the uptake increased by 10-30% for coastal seawater (CW or TH) and 13.2 times (p < 0.05) for estuarine seawater (YL), showing that the MeHg uptake was less influenced by DOC than that of Hg(II) at higher chloride concentrations, perhaps due to the relatively lower affinity of MeHg for DOC (17) and thus the dominance of MeHg-Cl complexes (discussed below). Furthermore, the uptake of MeHg decreased (from 36.6 to 2.7 µg/g/h) as DOC concentrations increased from 77 to 200 µM, further indicating the inhibitory effects of DOC on the bioavailability of MeHg. Such a trend was not observed in the Hg(II) data. The inhibitory effects of DOC on the uptake of Hg(II) or MeHg have been well documented for different organisms including freshwater phytoplankton (8, 9) and invertebrates (17). In an earlier study, Pickhardt and Fisher (7) observed insignificant effects of DOC on the uptake of Hg(II) by freshwater phytoplankton, which may have been due to the different origin of the DOC examined. They also observed that MeHg uptake increased with DOC concentration, presumably because of the higher chloride level in the higher DOC water, resulting in higher percentage of permeable neutral MeHg chloride complex. As in an earlier study comparing mercury uptake by dead and living phytoplankton (7), a higher percentage of MeHg than of Hg(II) was observed in the intracellular pool with these diatoms (Figure 4). After 1 h of uptake, 21-57% (average 38%) of the total Hg(II) and 29-74% (average 57%) of the total MeHg was detected in the intracellular pool. This may explain the higher efficiency of MeHg transfer in marine food chains, since mercury in diatom cytoplasm would be more bioavailable (6). The speciation of mercury as affected by both the DOC and chloride (18) may help explain these observations. According to the mercury-Cl-DOC ligand complexation model (4, 18), the DOC complexes of Hg(II) dominate in low chloride and high DOC conditions, whereas the chloride complexes dominate in high chloride and low DOC conditions. When the chloride complexes dominate, it is as a progression of chloride complexes from HgOHCl to HgCl2 and then to HgCl3- and HgCl42- (6, 18). For MeHg, its speciation follows a similar pattern dominated by chloride complexes at low DOC levels and DOC complexes at high DOC levels. The dominance of mercury complexation in these

uptake media was assessed using established speciation phase diagrams (6, 18) to help explain the effects of DOC and Cl on mercury uptake. The speciation phase diagrams were based on the mercury-Cl-DOC ligand complexation model, which considered a range of DOC of different origins (e.g., river water, coastal water, city water, sediment pore water, and phytoplankton exudates) with different affinities to mercury and could be used to predict the dominant mercury species (DOC or chloride complexes) based on the relative abundance of DOC and Cl. In a mercury chloride complex dominated system, it is also possible to determine the speciation of Hg(II) or MeHg based on the speciation diagrams of inorganic complexes (6, 18). In view of the DOC or chloride concentrations and pH values in all seawaters used in this study, other inorganic ligands such as hydroxide could not dominate mercury complexing (4). The possible dominant mercury species (DOC or chloride complexes) in each experimental system is listed in Table 1. No data is available about the affinity of MeHg with marine DOC, so the speciation estimate was based on the average conditional stability constants for freshwater humic and fulvic acids (18). In the case of Hg(II), the DOC complexes dominated in all the natural seawaters (CW, TH, and YL). The sharp increase of Hg(II) uptake in natural seawater after photo-oxidation, which was accompanied by a change in the dominant Hg(II) species from DOC complexes to chloride complexes, indicated that the DOC complexes of Hg(II) were less bioavailable than its chloride complexes. DOC complexation appears to have decreased the Hg(II) uptake, probably because of the competitive binding of Hg(II) with DOC in the uptake medium and binding sites on the biological membrane (9). Another possible explanation was the relative large size of Hg(II)-DOC complexes, which could hinder the diffusion of Hg(II) through the biological membrane (17). For MeHg, DOC complexes were the probable dominant species only in the estuarine water (YL) with its lower chloride level (0.31 M) and higher DOC concentration. In fact, it has been reported that MeHg-DOC complexes dominate in freshwater systems (19). Similarly, the decrease of MeHg uptake with increased DOC concentration could be explained by the dominance of MeHgDOC complexes (less bioavailable because of their larger size) at higher DOC levels. The sharp increase of MeHg uptake in estuarine water (YL) after UV-oxidation could be due to a change in dominant species from DOC complexes to chloride complexes. In contrast to Hg(II), the MeHg uptake did not increase after DOC removal in coastal seawater (CW and TH), mainly because chloride complexes could be the dominated species (in view of the much weaker affinity of MeHg to DOC than Hg(II) as well as the mercury speciation diagram) there both before and after the UV oxidation, given the low DOC and high chloride levels. The uptake of Hg(II) was also affected by the origin of the DOC. Although Hg(II)-DOC dominated in all three natural seawaters (CW, TH, and YL) and binding of Hg(II) with DOC VOL. 43, NO. 23, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Calculated internalization rates of Hg(II) or MeHg by the diatoms Thalassiosira pseudonana in artificial seawater containing two different concentrations of DOC or chloride. Mean + semirange (n ) 2). in the uptake medium and binding sites on the cells should have resulted in lower Hg(II) uptake in seawater with higher DOC level, no relationship between DOC concentration and uptake was found. This could be explained by the heterogeneity of DOC from different origins. The YL seawater was greatly influenced by the Pearl River estuary, and its DOC may have been largely of terrestrial origin, while the DOC of the coastal waters may have come largely from biological activity such as phytoplankton decomposition. Note that the Hg(II) uptake rate from YL water was comparable to that from CW water despite of the 2.6 times difference in their DOC concentrations. The Roles of DOC and Chloride in Controlling the Uptake of Hg(II) or MeHg. We determined the relative controls of DOC and Cl on mercury uptake by the diatoms using DOC prepared from diatom decomposition. Mercury uptake rates from an artificial medium with different DOC levels (from diatom decomposition) and chloride levels are shown in Figure 5. Only in seawater with little DOC (1 µM) were lower chloride levels (0.2 M) associated with greater Hg(II) uptake (2 times greater). This may have been due to the dominance of Hg(II)-Cl species and the increase of neutral Hg(II) species (such as HgCl2) with decreasing chloride concentration (see below). Interestingly, the Hg(II) uptake in the high chloride (0.52 M) low DOC (1 µM) artificial seawater was comparable with that in UV-oxidized CW seawater (0.52 M Cl and 0 µM DOC, Figure 3), further confirming that chloride controlled the speciation and uptake of Hg(II) at low DOC levels. At a high DOC concentration (100 µM), the chloride levels showed no relationship with Hg(II) uptake. Thus, in seawater with a higher DOC concentration, chloride concentration changes did not significantly affect the uptake of Hg(II). This result provided another explanation for the comparable Hg(II) uptake from estuarine (YL) and coastal seawater (CW and TH). In addition, even when Hg(II)-Cl complex dominated after the DOC had been removed by UV light, the Hg(II) uptake was still rather comparable, despite the Cl concentration varying from 0.31 to 0.52 M. For MeHg, uptake rates were comparable in two UV-oxidized seawater with contrasting chloride levels (TH and UV), although higher in UVoxidized coastal seawater (CW). Overall, it appears that Hg(II) or MeHg uptake in an estuarine or coastal environment is less affected by the chloride concentration than by the concentration and origin of DOC. The percentage of neutral species (under chloride complex dominated conditions) may help explain the effects of chloride on mercury uptake. Under low DOC level conditions 9002

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the uptake rate of Hg(II) more than doubled (from 5 to 11 µg/g/h) when the percentage of neutral species (HgCl2) increased from 8 to 20%, indicating that the uptake may be largely controlled by the neutral species. In contrast, the smaller difference in chloride concentration between UVoxidized CW and YL seawater (0.52 and 0.31 M) resulted in a smaller change in the percentage of neutral species (8-12%) and thus a comparable uptake. Similarly, Barkay et al. have shown that a neutral Hg(II) species (HgCl2) is more bioavailable to bacteria than charged forms such as HgCl3- and HgCl4- (20). Mason et al. have proposed that the high lipophilicity of neutral chloride complexes favors mercury’s passive diffusion through biological membranes, and that this is the main route of Hg(II) or MeHg uptake by phytoplankton (6). For MeHg, changes of chloride concentration under MeHg-Cl dominated conditions resulted in little change in the neutral species MeHgCl (93-98%) and the uptake rate differences (27-46 µg/g/h) may have been due to the presence of DOC, the binding of which with MeHg could be stronger at lower chloride levels due to less ion shielding and less condensation of hydrophobic functional groups. This suggested that DOC might still affect the uptake of MeHg even when chloride species dominate. Furthermore, the different percentages of neutral species may partly explain the difference in uptake rates between Hg(II) and MeHg. Based on all uptake rates under chloride complex dominated conditions (Table 1), the average MeHg uptake rate constants (calculated from the slope of linear regression of intracellular mercury content divided by the mercury concentration in the uptake medium against uptake time), which were not influenced by mercury concentration, were 11.2 times those of Hg(II), while the average percentage of neutral MeHg species (MeHgCl) was 8.4 times that of HgCl2. The uptake of MeHg at 1 µM DOC (prepared by diatom decomposition) was triple that at 100 µM, consistent with the observation that a decrease in DOC in natural seawater increased MeHg uptake. It is also interesting to note that the uptake of Hg(II) increased significantly with DOC from diatom decomposition at the same Cl level (0.52 M, Figure 5), in contrast to the results obtained with DOC from natural seawater before and after UV-oxidation (Figure 3). The measured uptake rates of Hg(II) with DOC from diatom decomposition were also higher than those for natural seawater and UV-oxidized seawater (dominated by Hg(II)-Cl complexes), further showing that the origin of the DOC can affect Hg(II) uptake. The increased Hg(II) uptake may be explained by increased membrane permeability in the diatoms in the presence of DOC from diatom decomposition (21, 22), which facilitated Hg(II)’s passage through the membrane. It is also possible that Hg(II) was taken up by the diatoms when bound with small organic molecules like amino acids resulting from diatom decomposition, as has been reported for bacteria (10). In natural seawater, Hg(II) may be mainly complexed with larger organic compounds such as humic acids, which could hinder its passage through biological membranes (17). Besides, higher diatom-decomposed DOC may also increase the Hg(II) adsorption on cell membranes and thus facilitate the uptake. We did find that Hg(II) binding on cell membranes in higher DOC level (100 µM) was 1.6 times as that in lower DOC level (1 µM) at 0.52 M Cl after 1 h uptake. In contrast, the MeHg uptake was reduced in the presence of DOC from diatom decomposition, consistent with the results obtained for natural seawater, probably due to the larger size of the MeHg-DOC complexes. To conclude, the data suggest that DOC and chloride control the speciation and thus the uptake of Hg(II) or MeHg by marine diatoms. The relative importance of DOC and chloride depends on their concentrations. When DOC complexes dominate the mercury speciation, DOC inhibits the uptake of Hg(II) or MeHg, but DOC from decomposed

diatoms was observed to increase the Hg(II) uptake rate. When mercury chloride complexes dominated, the chloride concentration controlled the mercury speciation by changing the fraction of neutral Hg(II) or MeHg species and thus the bioavailability of mercury. DOC is more important in controlling the uptake of Hg(II) or MeHg in common estuarine and coastal environments. For Hg(II), DOC complexes dominated under most of these experimental conditions, except when the DOC concentration was very low. For MeHg, DOC complex dominated only when the DOC concentration was relatively high (>100 µM), but DOC could still affect the uptake of MeHg even when chloride complexes dominated. When chloride complexes dominate, passive diffusion of neutral mercury species may be the major route of mercury uptake.

Acknowledgments We thank the anonymous reviewers for their helpful comments. This study was supported by a General Research Fund from the Hong Kong Research Grants Council (663009) to W.-X.W.

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