SEARCH

SEARCH BY CITATION

Keywords:

  • Methylmercury;
  • Dissolved organic matter;
  • Phytoplankton;
  • Bioaccumulation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

Dissolved organic matter (DOM) significantly decreased accumulation of methylmercury (MeHg) by the diatom Cyclotella meneghiniana in laboratory experiments. Live diatom cells accumulated two to four times more MeHg than dead cells, indicating that accumulation may be partially an energy-requiring process. Methylmercury enrichment in diatoms relative to ambient water was measured by a volume concentration factor (VCF). Without added DOM, the maximum VCF was 32 × 104, and the average VCF (from 10 to 72 h) over all experiments was 12.6 × 104. At very low (1.5 mg/L) added DOM, VCFs dropped by approximately half. At very high (20 mg/L) added DOM, VCFs dropped 10-fold. Presumably, MeHg was bound to a variety of reduced sulfur sites on the DOM, making it unavailable for uptake. Diatoms accumulated significantly more MeHg when exposed to transphilic DOM extracts than hydrophobic ones. However, algal lysate, a labile type of DOM created by resuspending a marine diatom in freshwater, behaved similarly to a refractory DOM isolate from San Francisco Bay. Addition of 67 µM L-cysteine resulted in the largest drop in VCFs, to 0.28 × 104. Although the DOM composition influenced the availability of MeHg to some extent, total DOM concentration was the most important factor in determining algal bioaccumulation of MeHg. Environ. Toxicol. Chem. 2012; 31: 1712–1719. © 2012 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

Dissolved organic matter (DOM) shapes the fate of Hg in the environment by binding to both inorganic Hg and methylmercury (MeHg) 1, 2. As a result of this binding, DOM can mobilize and transport Hg within aquatic systems 3, 4 and can affect how Hg enters both microbial cells 5 and algal cells 6. On a macro-scale, DOM has been positively correlated with fish Hg concentrations in a national database 7. However, the effects of DOM on specific processes vary in magnitude and direction. For example, research on the effect of DOM on microbial uptake has been of particular interest because once inorganic Hg2+ enters the cell, some microbes (e.g., sulfate and iron reducers) can transform it into MeHg, the form that biomagnifies up the food chain 8. This research has shown that the effect of DOM on microbial uptake of Hg2+ is species-dependent. For example, although cysteine enhanced the accumulation of Hg2+ by Geobacter sulfurreducens 8, thiol-containing amino acids did not affect the accumulation of Hg2+ by Desulfovibrio desulfuricans 5. Given the variable effects of DOM, this research sought to evaluate the effect of natural DOM on the accumulation of MeHg by algal cells. Algal cells at the base of the food chain are the critical link between aqueous MeHg and dietary MeHg and are the means by which fish acquire most of their MeHg from dietary exposure 9.

Conflicting studies have been conducted on the effects of DOM on MeHg accumulation by phytoplankton. In culture studies with water collected from two sites in the San Francisco Bay Delta, Pickhardt and Fisher 6 found that eukaryotic phytoplankton grown in water with 280 µM dissolved organic carbon (DOC) accumulated at least twice as much MeHg as phytoplankton grown in water with 177 µM DOC. They hypothesized that the phytoplankton actively took up some components of the DOC, such as amino acids, and inadvertently acquired the MeHg associated with that organic matter. In contrast, when Gorski et al. 10 compared water from a variety of field sites, including the San Francisco Bay Delta, river water, and lake water, they found that phytoplankton grown in water with low DOC concentrations, particularly rainwater, had the highest accumulation of MeHg. They hypothesized that MeHg binding to DOC reduced MeHg bioavailability. Field studies have also found variable results for the effects of DOC on MeHg bioavailability, including negative correlations 11 and concentration dependence 12. The inconsistent results may be attributable to variations in the composition of the organic matter and differences in other water chemistry variables between field sites 13.

Part of the uncertainty in understanding the effects of DOM is attributable to its inhomogeneity. Dissolved organic matter consists largely (∼80%) of humic material from the reworking of plant and animal material, of both aquatic and terrestrial origin. Only a small fraction (∼20%) is identifiable as individual carbohydrates, fatty acids, lignin monomers, amino acids, hydrocarbons, or other biochemicals 1. Because of the difficulty in characterizing DOM, it is often measured with respect to the amount of carbon present, as DOC. Approximately 50% of DOM is carbon by weight 1. Only a small portion (<2%) of the DOM is sulfur, and an even smaller percentage is reduced sulfur, which is the portion of the DOM that actually binds Hg most effectively 1, 2.

The goal of the present study was to assess how the concentration and composition of organic matter influenced MeHg bioavailability to phytoplankton. We used the amino acid cysteine, fresh algal lysate, and organic matter isolates collected from four different types of sites in the San Francisco Bay Delta (Fig. 1) 14. The DOM isolates were reconstituted in the laboratory immediately before use, allowing us to vary the concentration and composition of the organic matter while keeping all other parameters (e.g., pH) constant.

thumbnail image

Figure 1. Organic matter isolates were collected from four sites in the Delta: (1) the Sacramento River, which can provide up to 80% of the freshwater input to the Delta 31; (2) Mandeville Tip, a natural freshwater marsh in the Central Delta; (3) Twitchell Island Drain, a below-sea-level island drain on peat soil, and; (4) Shag Slough, which drains the Yolo Bypass, a floodway that can carry high concentrations of Hg into the Delta 4, 31.

Download figure to PowerPoint

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

General approach

We conducted a series of radiotracer experiments using Me203Hg to track the movement of MeHg from the water into algal cells. To investigate the effects of DOM concentration, we varied the amount of added organic matter. To examine the effect of equilibration time, we allowed the Me203Hg to equilibrate (0 vs 16 h) with organic matter (from two different San Francisco Bay sites) before the start of the experiment. To compare DOM from San Francisco Bay with other types of organic material, we created a treatment with cysteine and another with a marine diatom that was lysed in freshwater. Finally, to determine whether algal accumulation was an active (e.g., energy requiring) process, we compared accumulation from live versus dead cells in both the transphilic and the hydrophobic fractions of the field-collected organic matter isolates.

The general design for the experiments consisted of inoculating phytoplankton cells into water containing various DOM treatments, adding microliter quantities of a Me203Hg solution to each treatment, and measuring Me203Hg concentrations in water and cells over time. Each treatment consisted of a unique combination of the type of DOM and the concentration of DOM. Experiments consisted of 12 to 24 flasks containing Me203Hg. All experiments included control flasks without phytoplankton, which were used to correct for adsorption of Me203Hg onto filters and flask walls.

The DOM concentrations were varied by adjusting the amount of dehydrated organic material added to a given volume of Milli-Q deionized water. The amount of DOM (in mg/L) added to each treatment was broadly classified as none (0), very low (∼1.5), low (∼3), medium (∼5), high (∼10), and very high (∼20). The amount of organic matter in each treatment was verified by measuring DOC. Dissolved organic carbon was measured according to established methods using a Shimadzu TOC-5000 analyzer 6, 14. Accordingly, the experimental design is described in terms of DOM classifications (none, very low, low, etc.), and the amount of added organic matter is reported as DOC concentrations, which facilitates comparison with literature values.

The Me203Hg Synthesis

We synthesized radiolabeled methylmercury (CHmath imageHg+) from 203HgCl2, following established methods 6, 15. Briefly, we received 203HgCl2 in 1 N HCl, with specific activities ranging from 149 to 270 kBq/µg. We methylated 203HgCl2 by dissolving 25 mg methylcobalamin (C63H91CoN13O14P) in 12 ml 2 M acetate buffer, adding 2 ml methylcobalamin solution to the 203HgCl2 and allowing it to react for 18 to 23 h. The next day, we performed a series of five extractions with methylene chloride (CH2Cl2) followed by rinses with Milli-Q water to separate lipophilic CHmath imageHg+ from 203Hg2+. Previously, Rouleau and Block 15 used thin-layer chromatography to demonstrate that after organic extraction, the product had only one spot, which corresponded to an MeHg standard. After extraction, we added Milli-Q water to the CHmath imageHg+ in methylene chloride and evaporated the methylene chloride, leaving the CHmath imageHg+ in Milli-Q water. The product was stored in the dark until use.

Conversion efficiencies ranged from 68 to 85%, typical of other applications of this method 6 and reflecting the facts that not all 203Hg2+ is methylated and some CHmath imageHg+ is lost to the walls of the reaction vessels. Nonmethylated 203Hg2+ partitions into the aqueous waste and is discarded. All borosilicate glass and Teflon bottles used to prepare reagents and conduct experiments were acid washed to ensure that no trace metal contamination was present.

Dissolved organic matter collection and isolate preparation

Water was collected from four sites (Fig. 1), with DOC concentrations ranging from 131 to 1,470 µM (Supplemental Data, Table S1). Two fractions of organic matter were isolated from the water within 24 h as described by Kraus et al. 14. The hydrophobic fraction was isolated using an XAD-8 resin to adsorb humic and fulvic acids at acidic pH, separating them from the hydrophilic fraction, which would have passed through both columns 16. The transphilic fraction, which is so named because it captures compounds of intermediate polarity between the hydrophobic and hydrophilic fractions 17, was collected with an XAD-4 resin. Depending on the site, 43 to 59% of the DOC was captured in the hydrophobic fraction and 17 to 24% was captured by the transphilic fraction (Supplemental Data, Table S1). Despite differences in composition between the two fractions (Supplemental Data, Table S1), they both contained relatively refractory material. After extraction, organic matter isolates were freeze-dried and stored in a desiccator until use.

In the laboratory, organic matter isolates were reconstituted by dissolving the organic matter in 0.01 N Suprapur NaOH made with Milli-Q (18.2 MΩ) deionized water and sonicating for 15 min to ensure solution. Corresponding deionized water treatments (no added organic matter) also used 0.01 N NaOH. All treatments were supplemented with nutrients (Na2SiO3, NaH2PO4, NaNO3, NaHCO3, MgSO4, CaCl2, Na2B4O7, NH4Cl, KCl) to produce WCL-1 growth medium 18. Finally, treatments were neutralized to pH 7. In the first experiment, HCl was used for neutralization, bringing the calculated [Cl] in all treatments to 10.9 mM. Thereafter, HNO3 was used for neutralization so that [Cl] was 603 to 888 µM, as measured by ion chromatography. Those concentrations were representative of environmental concentrations in the Delta; porewater concentrations of [Cl] previously reported for the Delta were 3,890 µM for Frank's Tract and 200 for Consumnes River 6.

Concentrations of MeHg and DOM in the experiments

At the start of each experiment, algal cells were resuspended into the flasks containing Milli-Q water and organic matter treatments. Each flask then received microliter quantities of Me203Hg dissolved in Milli-Q water. In the experimental flasks, Me203Hg concentrations ranged from 0.44 to 0.78 nM, corresponding to Me203Hg radioactivity from 4.83 to 16.7 kBq/L. The amount of Me203Hg was determined by the amount of radioactivity present because measuring MeHg directly would have contaminated any Hg analyzer with radioactivity. For comparison, 0.478 pM has been used as an average concentration of MeHg in San Francisco Bay waters 19, although concentrations have been reported up to an order of magnitude lower and higher 3, 4.

Although the MeHg concentrations used in the present study are on the high end of the natural range, the experiments are still representative of environmental conditions, based on the ratio of reduced sulfur sites to MeHg concentrations. As demonstrated by Haitzer et al. 2, binding of Hg and DOM is concentration dependent, with Hg2+ first binding to reduced sulfur groups at Hg2+/DOM ratios below 1 µg Hg2+/mg DOM and then binding to oxygen groups at ratios above 10 µg Hg2+/mg DOM, a situation that would not be representative of most natural waters 2. At the ratios used in the present study (our highest was 0.5 ng MeHg/mg DOM), all MeHg was presumably bound to reduced sulfur, not to oxygen groups. For example, at Shag Slough, we measured 1.32% sulfur in the hydrophobic fraction and 1.84% in the transphilic fraction of the DOM, consistent with the range (0.5–2.0%) previously reported for sulfur in DOM 1. Assuming that approximately 60% of the total sulfur was in the reduced form (i.e., RSH or RSR) 2, our very high (20 mg/L) hydrophobic DOM treatment for Shag Slough would have contained 4.9 × 10-6 M reduced sulfur. Our very low (1.5 mg/L) hydrophobic DOM treatment contained 3.7 × 10-7 M reduced sulfur. Therefore, given the range (0.5–2.0%) of sulfur concentrations in DOM, our samples likely contained between 0.1 and 7 × 10-6 M of reduced sulfur, far in excess of the concentration (0.44–0.78 × 10-9 M) of MeHg.

Even when the amount of MeHg in the isolates was considered, the reduced sulfur sites should have exceeded the MeHg concentrations. For example, DOM from Twitchell in the year 2000 had 1.17 nmol MeHg per gram DOM (T. Kraus, U.S. Geological Survey, California Water Science Center, Sacramento, CA, personal communication). Thus, for a treatment with 20 mg/L DOM, the isolate would have contributed 2.3 × 10-11 mol Hg, keeping the MeHg levels far below the number of reduced sulfur sites.

Phytoplankton procedures

For all experiments, we used the centric diatom Cyclotella meneghiniana because it is present in the Delta, is easy to grow in laboratory cultures, and has been used in past studies of MeHg accumulation 6. Before the experiments, C. meneghiniana was grown in WCL-1 medium without ethylenediaminetetra-acetic acid or trace metal additions for 7 to 10 d, until cells reached stationary phase, as described in Pickhardt and Fisher 6. The cultures used for inocula and during experiments were grown on a 14:10 h light:dark cycle at 18°C. We used axenic techniques to handle the cultures, but no attempts were made to check cultures for bacterial contamination. To follow the concentrations of Me203Hg in the water and phytoplankton, we collected water and algal samples over time. Algal cells were obtained by filtering 10 ml water through 1.0-µm polycarbonate filters. We corrected for sorption of MeHg onto the filters (typically <5%) by subtracting counts from corresponding control treatments in which no algal cells had been added. Both water and algal samples were analyzed using an LKB Pharmacia Wallac 1282 Compugamma with a well-type NaI(Tl) detector. Samples were analyzed at 279 keV and counted for 10 min to achieve propagated counting errors less than 5%. Cell counts and volumes were assessed for each time point with a Coulter Multisizer Counter.

To characterize the enrichment in cells relative to the surrounding waters, we calculated volume concentration factors (VCFs), according to

  • equation image

Volume concentration factor is one of the partition coefficients used to express the concentration of a chemical in an organism relative to an aqueous phase. Volume concentration factors are reported here because it is appropriate to think about enrichment in phytoplankton on a volume basis 20. Volume concentration factors are similar to bioconcentration factors in that both factors express the cellular concentration (including both adsorbed and absorbed forms) relative to that in the aqueous phase 21. However, bioconcentration factors (essentially identical to Kd values) generally express the concentration of a chemical in the tissues of an organism on a dry weight basis, whereas VCFs express the concentration of a chemical on a volume basis 22. Given the cellular volume and dry weight of C. meneghiniana 6, bioconcentration factors would be roughly double the reported VCFs.

Description of experiments

In the experiment on the effects of DOM concentration, we measured algal uptake of MeHg at zero, medium, high, and very high concentrations of added DOM (two replicates per DOM concentration). We also had four corresponding control flasks that we used to account for adsorption onto filters and flask walls. Between 45 and 48% of the added DOM was DOC, which was consistent with the composition of the DOM (Supplemental Data, Table S1). All the organic matter in this experiment was the hydrophobic fraction from Mandeville Tip, the natural freshwater marsh site.

In the experiment on equilibration time, we varied the amount of time (0 vs 16 h) for which the Me203Hg was equilibrated with the DOM before addition of the algal cells. This length of time should have been sufficient to allow equilibration; previous studies on the kinetics of Hg2+ and DOM binding have found it takes a few hours (e.g., 1–7 h 23; 6 h 24) for Hg2+ to equilibrate with DOM. We looked at the effects of equilibration across two different sites (Mandeville Tip and Twitchell Island Drain) and four different levels of DOM (none, low, medium, and very high). The resulting experimental design had two equilibration treatments (equilibrated and non-equilibrated) for each unique site/DOM concentration. We also had corresponding control flasks.

In the experiment to compare different types of DOM, we compared the refractory DOM isolates from San Francisco Bay, labile DOM, DOM that was rich in reduced sulfur, and water without any added DOM. To create labile DOM, we lysed a marine diatom, Thalassiosira pseudonana. We resuspended 3 × 108 T. pseudonana cells in 400 ml Milli-Q water, which caused osmotic bursting of the cells. Microscopic observations confirmed that all cells had been lysed. The 400 ml lysed algal material was then filtered through a combusted glass fiber filter, and the filtered water was used in the experiment. The fresh algal lysate had DOC concentrations of 217 µM. For comparison, the San Francisco Bay treatments had 114 µM DOC, which consisted of the hydrophobic fraction of the organic matter from Mandeville Tip. To create a treatment that was rich in reduced sulfur, we used 67 µM of the amino acid L-cysteine. Cysteine (C3H7NO2S) contains three carbons for every mole of cysteine, and thus this treatment had a measured DOC concentration of 204 µM. For this experiment, we ran each type of DOM in duplicate, and we had controls (no added algal cells) for each DOM type.

In the experiment on active versus passive uptake, we compared MeHg accumulation in live and dead cells across two different fractions of organic matter (transphilic vs hydrophobic) and four different DOM concentrations (none, very low, low, and high). Both the transphilic and hydrophobic DOM fractions were from Shag Slough, which drains the Yolo Bypass. Dead cells were heat-killed by submerging small volumes of cells in a water bath of 55°C for 10 min; preliminary experiments demonstrated that no cell growth occurred after this treatment and that the heat-killed cells stayed intact for the duration of the experiment. The resulting experimental design had 16 experimental treatments (2 fractions of organic matter × 4 concentrations × 2 live and dead). We ran eight corresponding controls (no added algal cells), one for each fraction/DOM concentration.

Data analysis

To evaluate how the DOM concentration affected VCFs, we performed statistical analysis with Systat (ver 12.02). For each experiment, we used a repeated-measures type design because VCFs were measured repeatedly in the treatments at 2, 5, 10, 24, 48, and 72 h. In experiments in which we varied both the DOM concentration and another parameter of interest (e.g., equilibration time), we checked for interactions, or the possibility that the parameter of interest would have a different effect at low DOM concentrations than at high DOM concentrations. To analyze experiments with multiple dependent variables (time points) and multiple independent variables (e.g., DOM and equilibration time), we ran a repeated-measures multivariate analysis of variance for each experiment (see Supplemental Data).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

In the experiment on the effects of DOM concentration from a single site (Fig. 2), VCFs were inversely related to the DOM concentration. The highest VCFs across all time points (7.1–32 × 104) were found in the treatment with no added DOM. Volume concentration factors were lower (1.1–6.3 × 104) in the treatment with medium added DOM, even lower (0.89–2.7 × 104) in the treatment with high added DOM, and lowest (0.66 to 1.9 × 104) in treatment with very high added DOM.

thumbnail image

Figure 2. Effects of dissolved organic matter (DOM) concentration on cell densities, percentage of MeHg associated with cells, and volume concentration factors (VCFs) for the diatom Cyclotella meneghiniana. Replicate treatments (plotted individually) were run for each of the four DOM levels. All DOM was from Mandeville Tip (hydrophobic fraction). The amount of DOM added was none (0 dissolved organic carbon [DOC]), medium (185 µM DOC), high (374 µM DOC), or very high (724 µM DOC). Pairwise post hoc tests showed that all four treatments had significantly (p < 0.05, Supplemental Data, Table S2) different VCFs.

Download figure to PowerPoint

Phytoplankton in water without added DOM accumulated MeHg at a much faster rate, followed by a more pronounced decline, than phytoplankton in water with added organic matter (Fig. 2), as indicated by the significant interaction between time and the amount of added DOM (Supplemental Data, Table S2). This interaction was not attributable to differences in growth among the cultures (Fig. 2). In all experiments with live cells, cell numbers increased over time (e.g., Fig. 2), and a corresponding increase occurred in the percentage of MeHg associated with cells (Fig. 2). The inverse relationship between VCF and DOM was observed in all of the remaining experiments, although we continued to use a range of DOM concentrations to look for interactions between DOM and other parameters of interest.

There was no significant (p > 0.05) difference between treatments in which the MeHg was equilibrated with the DOM for 16 h before adding cells and treatments in which MeHg was not equilibrated with DOM (e.g., MeHg, DOM, and cells were added at the same time). Table 1 presents a summary of the VCFs for MeHg in the diatoms (equilibrated and nonequilibrated) for varying DOC concentrations. As the amount of DOM increased, VCFs decreased for both the Mandeville Tip and Twitchell Island sites (Supplemental Data, Fig. S1).

Table 1. Effects of equilibration time on volume concentration factors (VCFs × 104) for MeHg in Cyclotella meneghiniana when Me203Hg was equilibrated with the DOM for 16 h before adding cells (equilibrated) and when Me203Hg was added to the DOM and cells immediately before the start of the experiment (nonequilibrated)
Type of DOMaAmount of added DOMDOC (µM)VCFb × 104 equilibratedcVCFb × 104 nonequilibratedc
  • a

    The types of DOM are as follows: none (Milli-Q water without added DOM but with nutrients), the hydrophobic fraction of organic matter from Twitchell Island Drain, and the hydrophobic fraction of organic matter from Mandeville Tip.

  • b

    VCFs are the average VCFs from the 10-, 24-, 48-, and 72-h time points.

  • c

    There were no significant (p > 0.05, Supplemental Data, Table S2) effects of equilibration time on VCFs when all time points were included in the statistical model (Supplemental Data, Table S2, Fig. S1).

    VCF = volume concentration factor; DOM = dissolved organic matter; DOC = dissolved organic carbon.

NoneNone1523.515.1
TwitchellLow1385.277.50
MandevilleLow5314.913.9
TwitchellMedium2304.154.94
MandevilleMedium2133.353.56
TwitchellHigh7681.712.10
MandevilleHigh6542.121.95

In the experiment comparing different types of DOM, treatments with DOM derived from freshly lysed algal cells had average VCFs (from 10 to 72 h) of 4.4 × 104. Those VCFs were comparable to average VCFs (4.7 × 104) from treatments with low concentrations of the hydrophobic isolate from Mandeville (Fig. 3). Adding L-cysteine to some treatments dramatically lowered average VCFs (0.28 ×104) (Fig. 3).

thumbnail image

Figure 3. Effects of dissolved organic matter (DOM) type on the percentage of MeHg associated with cells and volume concentration factors (VCFs) for Cyclotella meneghiniana cells. Replicate treatments (plotted individually) were run for each of the four types of organic matter, which were no added DOM, organic matter from freshly lysed algal cells, the hydrophobic fraction of organic matter from Mandeville, and the amino acid L-cysteine. Corresponding dissolved organic carbon (DOC) concentrations were 22, 217, 114, and 204 µM, respectively.

Download figure to PowerPoint

Concentrations of MeHg in live cells were two to four times higher than in dead cells (Fig. 4, Supplemental Data, Table S3). A significant (p = 0.001) effect of isolate type also was seen, with higher VCFs in the transphilic fraction of the organic matter than in the hydrophobic fraction (Fig. 4). Across all experiments, an inverse but nonlinear relationship was found between the amount of DOM and MeHg VCFs, with VCFs decreasing asymptotically to 2.0 × 104 or less at very high DOM concentrations (Fig. 5).

thumbnail image

Figure 4. Effect of active versus passive uptake and organic matter fraction on mean volume concentration factors (averaged over all time points) for Cyclotella meneghiniana cells. We compared two different fractions of organic matter from Shag Slough, three different dissolved organic matter (DOM) concentrations, and live versus dead cells, all of which were significant (p < 0.01, Supplemental Data, Table S2). Error bars show the standard error of the statistical model. Corresponding dissolved organic carbon (DOC) concentrations are given in Supplemental Data, Table S3. Microscopic observations showed that dead cells, created by heat-killing cells in a warm water bath, remained intact for the duration of the experiment.

Download figure to PowerPoint

thumbnail image

Figure 5. Relationship between mean volume concentration factors (VCFs) for the hydrophobic fraction of the organic matter and dissolved organic carbon (DOC) concentrations. The mean VCFs are the average of the 10-, 24-, 48-, and 72-h time points. The highest VCFs were from treatments with no added organic matter. Addition of the hydrophobic fraction of the organic matter, regardless of the site (Mandeville Tip, Sacramento River, Shag Slough, or Twitchell Island Drain), decreased VCFs. Some data are shown for sites that were not discussed in the text. Algal organic matter, created from freshly lysed algal cells, resulted in VCFs comparable to those from the hydrophobic fraction of the organic matter. Addition of 67 µM of L-cysteine dramatically reduced VCFs.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

Additions of small amounts of DOM caused a sharp initial decline in algal accumulation of MeHg, as can be seen in Figure 5. We attributed this decline to DOM complexing MeHg and competing with the phytoplankton for MeHg. Presumably, that complexation was mediated through reduced sulfur atoms, as is the case for Hg2+ 25, 26, as long as DOM is in abundance with respect to MeHg 2. The strength of the complexation would then depend on the specific form of the reduced sulfur 1. For example, binding constants for MeHg bound to freely accessible thiols (e.g., cysteine) are orders of magnitude higher than for MeHg bound to sulfides (e.g., methylcysteine and methionine) where the thiol group is methylated 2. A further variable is the potential for MeHg to coordinate with multiple sulfur ligands 27.

Further additions of DOM caused only a minimal decrease in algal accumulation of MeHg, as can be seen in Figure 5. We attributed that result to a change in the form of reduced sulfur, consistent with previous reports that only a small percentage (2–5%) of the reduced sulfur sites associated with DOM are involved in strong mercury binding 2, 26, 28, 29. If 2.5% (an intermediate number) of the reduced sulfur is involved in strong Hg binding, and low DOM concentrations contain 3.7 × 10-7 M of sulfur (calculated previously), 9.3 × 10-9 M of strong binding sites would be found, which is approaching the concentrations of MeHg (0.44–0.78 × 10-9 M) used. One possibility is that at low DOM concentrations, where the relative number of strong binding sites is small, MeHg is bound to sites across a range of binding strengths (e.g., both thiols and other sulfur-containing species). Thus, the initial drop in VCFs was caused by a combination of strong and weak binding. As DOM concentrations increased, and the number of strong binding sites approached the concentration of MeHg, the slope of the relationship progressively decreased until virtually all of the aqueous MeHg was bound to strong binding sites (e.g., thiols), and additional DOM had little or no effect.

The idea that not all binding was to strong binding sites (e.g., thiols) was further illustrated by the difference between cysteine and other DOM (Fig. 3). Given that 67 × 10-6 M L-cysteine was added, there would have been orders of magnitude more thiols in the cysteine than were naturally present in the DOM (9.3 × 10-9 to 1.2 × 10-7 M), explaining why VCFs for Mandeville Tip and Twitchell Island (1.71–2.12 × 104) were higher than the VCF for cysteine (0.28 × 104). That the thiols alone might not account for all binding was consistent with previous findings that Hg2+ was strongly bound to DOM, even when the DOM lacked sulfide 24.

Despite the range of material used (Fig. 3), DOC concentration alone could be largely used to predict MeHg bioavailability. For example, DOM from freshly lysed algal cells was presumably much more labile than DOM from Mandeville, yet both had similar VCFs. A possible explanation is that different types of DOM with similar VCFs (Fig. 5) had similar numbers of sites involved in active binding. Given that assessing the number of strong sulfur-binding sites would require X-ray absorption near edge structure spectroscopy 1, 27, measuring DOC concentrations and specific ultraviolet absorbance (SUVA) to indicate aromaticity, could provide a much more practical way to evaluate MeHg bioavailability.

Clearly, the composition of the DOM was important to some extent; the hydrophobic fraction resulted in lower VCFs than the transphilic fraction (Supplemental Data, Table S3, Fig. 4). The hydrophobic fraction may have complexed MeHg more readily than other fractions because of its higher aromatic content, as has been previously observed 1. For example, at Shag Slough, the aromatic content was 20.2% of the hydrophobic fraction and 14.5% of the transphilic fraction (Supplemental Data, Table S1). As in previous studies 30, we found a direct relationship between SUVA and aromaticity (Supplemental Data, Table S1); SUVA can be measured far more easily. Elevated SUVA and aromatic content are both consistent with lower bioavailability and a geochemical provenance similar to reduced sulfur species 30, 31, suggesting that it is an important parameter for future studies.

The idea that DOM composition may affect MeHg VCFs has also been explored by comparing estuarine versus marine DOM, but those results were confounded with differences in Cl- concentrations 32. More work is needed to compare VCFs from different types of DOM while controlling for other variables. This research shows significant differences between the hydrophobic and transphilic fractions (Supplemental Data, Table S2). However, the effect of concentration was far greater, based on the mean square errors in the statistical models (see Supplemental Data), which were used to assess the contribution of the terms to the model fit.

The inverse relationship of DOM concentration and MeHg bioconcentration was consistent with previous results from Gorski et al. 10 but differed from a previous study by Pickhardt and Fisher 6. The latter study found that VCFs were approximately two times higher for diatoms and chlorophytes grown in water (from the Central Delta), with moderate (280 µM) DOC concentrations versus water (from the Consumnes River, a tributary of the Delta) with low (177 µM) DOC concentrations. Differences between these studies may be attributable to differences in water quality parameters (e.g., pH and Cl are known to affect MeHg uptake 13), which varied between the sites in these studies. Furthermore, field studies have reported both positive and negative relationships between DOC concentrations and MeHg in fish 11, 12, which may be attributable to the dual nature of DOM in mobilizing MeHg in the watershed but also in limiting its accumulation by phytoplankton.

The limited bioavailability of MeHg at high DOM concentrations implied that the mechanism of MeHg accumulation by phytoplankton was not through uptake and metabolism of DOM. This result differed from our original hypothesis that MeHg would bind to L-cysteine and then be transported across the cell membrane, possibly through a methionine uptake pathway. This hypothesis was based on the mechanism of accumulation in vertebrates, where the MeHg L-cysteine complex is carried across membranes by transporters of large amino acids, presumably because it resembles the amino acid L-methionine 21. Previous research has also found that cysteine increases the uptake of Hg2+ by the bacterium G. sulfurreducens 8. However, the mechanism of MeHg uptake by phytoplankton is apparently different from that of heterotrophs.

Although the curve leveled off, VCFs did not reach zero (Fig. 5). Presumably, phytoplankton were able to compete with the DOM for the MeHg. We found that MeHg uptake was an active process in diatoms; VCFs for MeHg in live cells were two to four times greater than the VCFs for dead cells (Fig. 4), consistent with previous research 6, 33. Once MeHg was internalized by cells, it would have been in a different pool, helping the cells to compete against the DOM. This active uptake also could have explained why allowing the Me203Hg to equilibrate with the DOM for 16 h before addition of the cells did not affect VCFs (Table 1). Our results were similar to those of Zhong and Wang 32, who found that equilibrating the Hg2+ or MeHg with the DOM for more than 2 h did not alter accumulation by phytoplankton.

The accumulation of MeHg by living algal cells makes its behavior relatively rare among metals because many metals (e.g., Sn, Am, Pu, Hg2+) bind passively to cell surfaces, as demonstrated by equivalent concentrations in live and dead cells 6, 20, 34. Passive binding also appears to occur with MeHg. However, active accumulation of MeHg into the cytoplasm occurs once it is associated with the cells 6, 33. Volume concentration factors were always higher in live cells than dead cells, allowing us to interpret main effects; we concluded that living cells accumulated significantly (p < 0.001, Supplemental Data, Table S2) more Me203Hg than dead cells. Other examples for active metal/metalloid uptake by autotrophs include strains of the cyanobacterium Synechococcus, which use nickel (Ni) for an Ni-dependent superoxide dismutase. Those cells increase uptake rates at low Ni concentrations, and dead cells take up only 20 to 30% of the Ni found in live cells 35. Similarly, in the diatom Thalassiosira pseuodonana, in which selenium (Se) is required for the enzyme glutathione peroxidase 36, live cells have VCFs six times higher than those of dead cells 34. Although MeHg has no known role in the cellular processes of C. meneghiniana, it is possible that active uptake of MeHg may be a case of mistaken identity, analogous to the mechanism of toxicity for many trace metals (e.g., Cd taken up by Mn transporters 37). Alternatively, MeHg may bind to other molecules acquired by the cells. Additional research is needed to establish the pathway by which cells transport MeHg across membranes.

In conclusion, the major factor determining MeHg bioavailability for the diatom C. meneghiniana was the DOM concentration. One implication is that DOM concentrations could explain variability in bioaccumulation in different ecosystems. For example, DOC concentrations have been inversely correlated with bioaccumulation factors between seston and water and between zooplankton and water in the western Great Lakes 38. Within an ecosystem, species from high DOM environments (e.g., benthic diatoms) may have lower VCFs than their counterparts in low DOM environments. Similarly, total organic carbon in sediments has been inversely related to biota–sediment concentration factors in estuaries in the Gulf of Maine 39, and organic carbon concentrations in sediments were inversely related to MeHg accumulation in amphipods in laboratory microcosm studies 40.

Another implication of this research is that the effects of temporal fluctuations in DOM on MeHg bioavailability need to be evaluated. For example, during a spring bloom in South San Francisco Bay, DOC concentrations can increase from 200 to 700 µM 41. The increase in DOM could initially stimulate MeHg production by providing a carbon source to sulfate-reducing bacteria 3, but later, as the bacteria reduce sulfate to sulfide, the buildup of sulfide could inhibit methylation 42. Dissolved organic matter also could limit the bioavailability of any MeHg that is produced by complexing the MeHg. The overall impact of a DOM pulse is not known.

Evaluating the effects of temporal changes in DOM is also important to understand the implications of long-term changes in organic carbon concentrations in ecosystems. For example, beginning in 1999, baseline concentrations of chlorophyll-a have increased in San Francisco Bay, and a new autumn bloom has been observed 43. Previous research on bloom dilution (e.g., Chen and Folt 44) implies that one possible implication of an increase in phytoplankton biomass is a decrease in MeHg concentrations in fish. However, the net effect of the increase in organic carbon is unknown, given that some processes may act in opposite directions (e.g., stimulation of MeHg production in sediments vs a decrease in the bioavailability of that MeHg through complexation to DOM). The effects of DOM on MeHg bioavailability need to be further addressed for bacteria, phytoplankton, and higher trophic levels.

We predict that the decrease in algal VCFs under high DOM concentrations will be passed on to grazers, but this hypothesis needs to be tested both in the laboratory and in the field. The MeHg concentration in phytoplankton is critical because fish and other organisms accumulate most of their MeHg from dietary exposure 9. Overall, the data suggest that variability in DOM concentration could be used to account for spatial and temporal variations in MeHg entry into aquatic food webs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

We thank the members of Fisher and Bergamaschi research groups for their assistance, particularly, S. Zegers for help in the laboratory, T. Kraus for compiling data on the organic matter extracts, and W. Kerlin for analyzing dissolved organic carbon concentrations. We also acknowledge D. Barfuss, Georgia State University, for supplying 203Hg and P. Pickhardt, Lakeland College, for his instruction on 203Hg methylation and other laboratory procedures. We thank P. Raimondi for advice on statistical analyses and three anonymous reviewers for thoughtful comments. The present study was prepared for the California Bay-Delta Authority and funded under California Bay-Delta Authority Agreement No. U-04-SC-005.

SUPPLEMENTAL DATA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

Additional description of the statistical methods.

Table S1. Characteristics of the organic matter isolates from four sites and two fractions.

Table S2. Summary of the MANOVA results for the four experiments.

Table S3. Volume concentration factors (VCFs) in live and dead cells from two fractions of organic matter collected at Shag Slough.

Fig. S1. Volume concentration factors for MeHg in Cyclotella meneghiniana over time at two sites and three concentrations of DOM. (147 KB PDF).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information
  • 1
    Ravichandran M. 2004. Interactions between mercury and dissolved organic matter: A review. Chemosphere 55: 319331.
  • 2
    Haitzer M, Aiken GR, Ryan JN. 2002. Binding of mercury(II) to dissolved organic matter: The role of the mercury-to-DOM concentration ratio. Environ Sci Technol 36: 35643570.
  • 3
    Luengen AC, Flegal AR. 2009. Role of phytoplankton in mercury cycling in the San Francisco Bay estuary. Limnol Oceanogr 54: 2340.
  • 4
    Conaway CH, Squire S, Mason RP, Flegal AR. 2003. Mercury speciation in the San Francisco Bay estuary. Mar Chem 80: 199225.
  • 5
    Gilmour CC, Elias DA, Kucken AM, Brown SD, Palumbo AV, Schadt CW, Wall JD. 2011. Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation. Appl Environ Microbiol 77: 39383951.
  • 6
    Pickhardt PC, Fisher NS. 2007. Accumulation of inorganic and monomethylmercury by freshwater phytoplankton in two contrasting water bodies. Environ Sci Technol 41: 125131.
  • 7
    Krabbenhoft D, Booth N, Fienen MN, Lutz M. 2010. Mapping mercury vulnerability of aquatic ecosystems across the contiguous United States. Abstract. Golschmidt Conference, June 13-18, Knoxville, Tennessee.
  • 8
    Schaefer JK, Morel FMM. 2009. High methylation rates of mercury bound to cysteine by Geobacter sulfurreducens. Nat Geosci 2: 123126.
  • 9
    Mathews T, Fisher NS. 2008. Evaluating the trophic transfer of cadmium, polonium, and methylmercury in an estuarine food chain. Environ Toxicol Chem 27: 10931101.
  • 10
    Gorski PR, Armstrong DE, Hurley JP, Krabbenhoft DP. 2008. Influence of natural dissolved organic carbon on the bioavailability of mercury to a freshwater alga. Environ Pollut 154: 116123.
  • 11
    Watras CJ, Back RC, Halvorsen S, Hudson RJM, Morrison KA, Wente SP. 1998. Bioaccumulation of mercury in pelagic freshwater food webs. Sci Total Environ 219: 183208.
  • 12
    Driscoll CT, Blette V, Yan C, Schofield CL, Munson R, Holsapple J. 1995. The role of dissolved organic carbon in the chemistry and bioavailability of mercury in remote Adirondack lakes. Water Air Soil Pollut 80: 499508.
  • 13
    Mason RP, Reinfelder JR, Morel FMM. 1996. Uptake, toxicity, and trophic transfer of mercury in a coastal diatom. Environ Sci Technol 30: 18351845.
  • 14
    Kraus TEC, Bergamaschi BA, Hernes PJ, Spencer RGM, Stepanauskas R, Kendall C, Losee RF, Fujii R. 2008. Assessing the contribution of wetlands and subsided islands to dissolved organic matter and disinfection byproduct precursors in the Sacramento-San Joaquin River Delta: A geochemical approach. Organic Geochemistry 39: 13021318.
  • 15
    Rouleau C, Block M. 1997. Working methods paper: Fast and high-yield synthesis of radioactive (CH3Hg)-Hg-203(II). Appl Organometallic Chem 11: 751753.
  • 16
    Aiken GR, McKnight DM, Thorn KA, Thurman EM. 1992. Isolation of hydrophilic organic acids from water using nonionic macroporous resins. Organic Geochem 18: 567573.
  • 17
    Croué JP, Debroux JF, Amy GL, Aiken GR, Leenheer JA. 1999. Natural organic matter: Structural characteristics and reactive properties. In Singer PC, ed, Formation and Control of Disinfection By-products in Drinking Water. American Water Works Association, Denver, CO, USA, pp 6593.
  • 18
    Guillard RRL. 1983. Culture of phytoplankton for feeding to invertebrates. In Berg CJ, ed, Culture of Marine Invertebrates: Selected Readings. Hutchinson Ross Publishing Company, Stroudsberg, PA, USA, pp 108132.
  • 19
    Yee D, McKee LJ, Oram JJ. 2011. A regional mass balance of methylmercury in San Francisco Bay, California, USA. Environ Toxicol Chem 30: 8896.
  • 20
    Fisher NS, Bjerregaard P, Fowler SW. 1983. Interactions of marine plankton with transuranic elements. 1. Biokinetics of neptunium, plutonium, americium, and californium in phytoplankton. Limnol Oceanogr 28: 432447.
  • 21
    Luoma SN, Rainbow PS. 2008. Metal Contamination in Aquatic Environments: Science and Lateral Management. Cambridge University Press, New York, NY, USA.
  • 22
    Twiss MR, Twining BS, Fisher NS. 2004. Bioconcentration of inorganic and organic thallium by freshwater phytoplankton. Environ Toxicol Chem 23: 968973.
  • 23
    Miller CL, Southworth G, Brooks S, Liang L, Gu B. 2009. Kinetic controls on the complexation between mercury and dissolved organic matter in a contaminated environment. Environ Sci Technol 43: 85488553.
  • 24
    Gasper JD, Aiken GR, Ryan JN. 2007. A critical review of three methods used for the measurement of mercury (Hg2+)-dissolved organic matter stability constants. Appl Geochem 22: 15831597.
  • 25
    Black FJ, Bruland KW, Flegal AR. 2007. Competing ligand exchange-solid phase extraction method for the determination of the complexation of dissolved inorganic mercury(II) in natural waters. Anal Chim Acta 598: 318333.
  • 26
    Gerbig CA, Kim CS, Stegemeier JP, Ryan JN, Aiken GR. 2011. Formation of nanocolloidal metacinnabar in mercury-DOM-sulfide systems. Environ Sci Technol 45: 91809187.
  • 27
    Hesterberg D, Chou JW, Hutchison KJ, Sayers DE. 2001. Bonding of Hg(II) to reduced organic, sulfur in humic acid as affected by S/Hg ratio. Environ Sci Technol 35: 27412745.
  • 28
    Amirbahman A, Reid AL, Haines TA, Kahl JS, Arnold C. 2002. Association of methylmercury with dissolved humic acids. Environ Sci Technol 36: 690695.
  • 29
    Gerbig CA, Ryan JN, Aiken GR. 2011. The effects of dissolved organic matter on mercury biogeochemistry. In Cai Y, Liu G, O'Driscoll N, eds, Advances in Environmental Chemistry and Toxicology of Mercury. Wiley, New York, NY, USA, pp 259292.
  • 30
    Weishaar JL, Aiken GR, Bergamaschi BA, Fram MS, Fujii R, Mopper K. 2003. Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon. Environ Sci Technol 37: 47024708.
  • 31
    Stepanauskas R, Moran MA, Bergamaschi BA, Hollibaugh JT. 2005. Sources, bioavailability, and photoreactivity of dissolved organic carbon in the Sacramento-San Joaquin River Delta. Biogeochemistry 74: 131149.
  • 32
    Zhong H, Wang WX. 2009. Controls of dissolved organic matter and chloride on mercury uptake by a marine diatom. Environ Sci Technol 43: 89989003.
  • 33
    Moye HA, Miles Carl J, Phlips Edward J, Sargent B, Merritt Kristen K. 2002. Kinetics and uptake mechanisms for monomethylmercury between freshwater algae and water. Environ Sci Technol 36: 35503555.
  • 34
    Fisher NS, Wente M. 1993. The release of trace elements by dying marine phytoplankton. Deep-Sea Res Pt I 40: 671694.
  • 35
    Dupont CL, Barbeau K, Palenik B. 2008. Ni uptake and limitation in marine Synechococcus strains. Appl Environ Microbiol 74: 2331.
  • 36
    Price NM, Harrison PJ. 1988. Specific selenium-containing macromolecules in the marine diatom Thalassiosira pseuodonana. Plant Physiol 86: 192199.
  • 37
    Sunda WG, Huntsman SA. 1998. Processes regulating cellular metal accumulation and physiological effects: Phytoplankton as model systems. Sci Total Environ 219: 165181.
  • 38
    Rolfhus K, Hall B, Monson B, Paterson M, Jeremiason J. 2011. Assessment of mercury bioaccumulation within the pelagic food web of lakes in the western Great Lakes region. Ecotoxicology 20: 15201529.
  • 39
    Chen CY, Dionne M, Mayes BM, Ward DM, Sturup S, Jackson BP. 2009. Mercury bioavailability and bioaccumulation in estuarine food webs in the Gulf of Maine. Environ Sci Tech 43: 18041810.
  • 40
    Lawrence AL, Mason RP. 2001. Factors controlling the bioaccumulation of mercury and methylmercury by the estuarine amphipod Leptocheirus plumulosus. Environ Pollut 111: 217231.
  • 41
    Luengen AC, Raimondi PT, Flegal AR. 2007. Contrasting biogeochemistry of six trace metals during the rise and decay of a spring phytoplankton bloom in San Francisco Bay. Limnol Oceanogr 52: 11121130.
  • 42
    Gilmour CC, Henry EA, Mitchell R. 1992. Sulfate stimulation of mercury methylation in freshwater sediments. Environ Sci Technol 26: 22812287.
  • 43
    Cloern JE, Jassby AD, Thompson JK, Hieb KA. 2007. A cold phase of the East Pacific triggers new phytoplankton blooms in San Francisco Bay. Proc Natl Acad Sci U S A 104: 1856118565.
  • 44
    Chen CY, Folt CL. 2005. High plankton densities reduce mercury biomagnification. Environ Sci Technol 39: 115121.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. SUPPLEMENTAL DATA
  9. REFERENCES
  10. Supporting Information

All Supplemental Data may be found in the online version of this article.

FilenameFormatSizeDescription
etc_1885_sm_SupplInfo.pdf211KSupplementary Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.