APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1997, p. 4267–4271 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 63, No. 11
Effects of Dissolved Organic Carbon and Salinity on Bioavailability of Mercury TAMAR BARKAY,1* MARK GILLMAN,2†
AND
RALPH R. TURNER1‡
Gulf Ecology Division, National Health and Environmental Effects Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Florida 32561,1 and Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, Florida 325142 Received 28 April 1997/Accepted 3 September 1997
Hypotheses that dissolved organic carbon (DOC) and electrochemical charge affect the rate of methylmercury [CH3Hg(I)] synthesis by modulating the availability of ionic mercury [Hg(II)] to bacteria were tested by using a mer-lux bioindicator (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083– 3090, 1993). A decline in Hg(II)-dependent light production was observed in the presence of increasing concentrations of DOC, and this decline was more pronounced at pH 7 than at pH 5, suggesting that DOC is a factor controlling the bioavailability of Hg(II). A thermodynamic model (MINTEQA2) was used to select assay conditions that clearly distinguished among various Hg(II) species. By using this approach, it was shown that negatively charged forms of mercuric chloride (HgCl32/HgCl422) induced less light production than the electrochemically neutral form (HgCl2), and no difference was observed between the two neutral forms, HgCl2 and Hg(OH)2. These results suggest that the negative charge of Hg(II) species reduces their availability to bacteria and may be one reason why accumulation of CH3Hg(I) is more often reported to occur in freshwater than in estuarine and marine biota. The effects of environmental factors on the availability of inorganic mercury [Hg(II)] to bacteria in aquatic environments are central to human and ecological health concerns with mercury contamination. A major cause of mercury toxicosis is exposure to edible fish and shellfish that contain hazardous levels of methylmercury [CH3Hg(I)] (40). Because fish do not methylate mercury (20, 28), the source of CH3Hg(I) in their bodies is soluble CH3Hg(I) that is accumulated and biomagnified through the aquatic food chain (25, 38). Methylmercury is formed by microbial (8, 14) as well as abiotic (39) processes that transfer methyl groups to Hg(II). Thus, environmental factors that control the bioavailability of Hg(II) to methylating bacteria regulate the rate of CH3Hg(I) synthesis and consequently of its accumulation in edible biota. To date, the effects of environmental factors on the production of CH3Hg(I) have been deduced by relating CH3Hg(I) concentrations in water and aquatic biota to changing environmental parameters (13, 21, 42). As a result of this approach, it was proposed that enhanced accumulation rates were correlated with low pH (13, 42), low alkalinity (41), low selenium concentration (12, 33), the presence of sulfate (13, 42), low levels of dissolved organic carbon (DOC) in seepage lakes (15), high levels of DOC in drainage lakes (10, 12), and decomposable organic matter (22). One of the mechanisms by which these factors might influence CH3Hg(I) accumulation is by controlling the availability of Hg(II), the substrate for methylation, to methylating microbes. Investigating how environmental factors affect CH3Hg(I) synthesis requires the measurement of bioavailable Hg(II). This task could only be accomplished by bioindicators that respond exclusively to bioavailable Hg(II) (31, 34, 37), because distinguishing avail-
able Hg(II) from other forms of Hg(II) in environmental samples is presently beyond the capabilities of analytical methods. Here we used a previously described molecular fusion (31) between the regulatory region of the mercury resistance (mer) operon and the luminescence (lux) gene from Vibrio fischeri. Bacteria harboring the fusion emit light in proportion to the quantity of bioavailable Hg(II) in their environment (2). By varying assay conditions, we showed that DOC and high NaCl concentrations inhibited expression of mer-lux, suggesting a reduction in the availability of Hg(II) to bacteria. MATERIALS AND METHODS Bacterial cultures and growth conditions. Escherichia coli HMS174(pRB28) was used as an indicator for the presence of bioavailable Hg(II) as described by Selifonova et al. (31). E. coli HMS174(pRB27) is an isogenic strain of HMS174(pRB28), except that in this strain lux is expressed constitutively. Restriction enzyme analysis of pRB27 showed a small deletion upstream of the merRo/pT9 insertion that created pRB28 that might be responsible for the constitutive phenotype (32). Strain HMS174(pRB27) was used as a control to distinguish the effects of various conditions on the light-emitting reaction from the effects of various conditions on the Hg(II)-dependent induction of the mer-lux gene fusion. The strains were grown and prepared for mer-lux assays as described by Selifonova et al. (31). mer-lux bioassays. The effect of DOC on the availability of Hg(II) was tested by the assay method of Selifonova et al. (31) with 107 cells ml21, to which increasing amounts of a DOC concentrate (see below) were added. Cell preparation and assays were as described previously (31) except that the assay medium was modified to contain 50 mM Na2HPO4 buffer, 5 mM pyruvate, and 7.6 mM (NH4)2SO4. Induction was achieved with 50 nM Hg(NO3)2. Assays were performed at pH 7 (adjusted with 19.5 mM NaH2PO4 and 30.5 mM K2HPO4), pH 6 (adjusted with 43.9 mM NaH2PO4 and 6.1 mM K2HPO4), and pH 5 (adjusted with citrate-phosphate buffer [24.3 mM citrate, 51.4 mM Na2HPO4]). The pHs of DOC-supplemented assay solutions were measured with a Beckman F40 pH meter after the assays were terminated. To ensure that interactions between Hg(II) and DOC reached equilibrium prior to initiation of the assays, Hg(NO3)2 was incubated for 20 min in the assay buffer before the addition of bioindicator cell suspensions. Assay conditions were modified in experiments testing the effect of Hg(II) speciation on bioavailability. Cells were prepared as described by Selifonova et al. (31). The assay medium contained 105 cells ml21, 75 mM (NH4)2SO4, 5 mM pyruvate, and 67 mM PO4. Hg(NO3)2 (0.1 nM) was used for induction. mer-lux assays performed at pH 6.0 contained 8.2 mM K2HPO4 and 58.8 mM NaH2PO4, and those performed at pH 5 contained 32 mM citric acid and 67 mM Na2HPO4. To express the results of mer-lux assays, expression factors (the maximal
* Corresponding author. Present address: Dept. of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel. Phone: 972-3-640 8712. E-mail:
[email protected]. † Present address: Florida Dept. of Environmental Protection, Pensacola, FL 32501-5794. ‡ Present address: Frontier Geosciences, Seattle, WA 98106. 4267
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TABLE 1. Concentrations of DOC and major cations during preparation of a DOC fraction from a freshwater sample collected from Ft. Pickens, Santa Rosa Island, Fla. Concna Substance
Untreated water
After rotovaporation
After cation exchange
DOCb
18.5 6 0.2
346.0 6 1.5
340 6 9.6
39 6 29 2.7 0.25
790 140 730 66 5.1
2.4 1.9 22 68 0.13
Cac Mg Na Si Sr
a Concentrations are given in milligrams of C per liter (DOC) and milligrams per liter (cations). b Means of three replicate analyses 6 standard deviations. c Only cations that were present at .0.1 mg per liter are reported.
increase in light output per minute) in log quanta per minute were calculated from data of light production (L, in quanta) with time (t, in minutes) after induction as follows: (dL/dt)max 5 (log light output at t2 2 log light output at t1)/t2 2 t1. t1 and t2 were selected in the area of the curve where light production (on a log scale) increased linearly with time. We found this parameter of the light-emitting reaction to most consistently discriminate among various induction conditions (2). The relative standard deviations of the means of expression factors were calculated for various time intervals when the range of a linear increase in light production consisted of more than two measurement intervals. In such cases, the relative standard deviations were mostly in the 10% range and never exceeded 40%. Production of light by the constitutive control, strain HMS174(pRB27), was expressed as yield of light output (in quanta). Preparation of DOC. A freshwater sample was collected from a stream in Ft. Pickens on Santa Rosa Island, Fla. Solutes were concentrated 32-fold from an initial volume of 3.5 liters by rotovaporation (with an RE 121 Rotovapor (Bu ¨chi, Laboratorums-technik AG, Flawel, Switzerland) at 55°C. Cations were removed from the concentrated fraction by mixing with AG MP-50 cation exchange resin, 100/200 mesh, hydrogen form (Bio-Rad Laboratories, Richmond, Calif.), 20:1 (vol/wt), for 1 h at room temperature followed by vacuum filtration through a Whatman no. 1 filter. Concentrated samples were stored at 220°C. To evaluate the efficiency of the DOC concentration procedure, samples for DOC and ion content analyses were removed before and after rotovaporation and after the cation exchange step. DOC was analyzed with a Shimadzu (Kyoto, Japan) TIC5000 analyzer as described by Kroer (24), and cations were measured by inductively coupled plasma optical emission spectroscopy (36) at the analytical facility at Oak Ridge National Laboratory. Results (Table 1) indicated that 57% of the original DOC was recovered in the concentrated fraction and that, with the exception of silicon, all cations were present in this fraction at concentrations lower than those in the original sample. Modeling of Hg(II) speciation. The geochemical equilibrium speciation model, MINTEQA2 (1), was used to calculate the aqueous speciation of Hg(II) in various assay solutions. Thermodynamic data for Hg(II) complexes with hydroxide, chloride, and ammonia included with the model were used in all calculations. No thermodynamic data for complexes of Hg(II) with pyruvate are known to exist.
RESULTS Use of a constitutive lux control. A constitutive derivative of HMS174(pRB28), HMS174(pRB27), was used to ensure that factors tested for their influence on Hg(II) bioavailability affected light production at the Hg(II)-dependent induction level rather than at the light production level. The usefulness of this approach is demonstrated by the results of assays on the effect of DOC on light emission by HMS174(pRB27) at pH 6 (Fig. 1). At low concentrations (2 to 37.4 mg of C ml21) DOC had no effect on light production, but at the two highest concentrations luminescence was reduced by an order of magnitude with 93.8 mg of C ml21 and by two orders of magnitude with 187.3 mg of C ml21. These results indicated that the effect of DOC on the bioavailability of Hg(II) at pH 6 could be studied at concentrations that did not exceed 37.4 mg of C
FIG. 1. The effect of DOC on light production by the constitutive control strain HMS174(pRB27) at pH 6. The graph shows light output in assays with DOC at 0 (■), 2.0 ( ), 3.7 ( ), 37.4 (h), 93.8 ({), and 187.3 (Ç) mg of C ml21 at pH 6.
ml21. The HMS174(pRB27) control was used routinely in this study. Effect of DOC on bioavailability of Hg(II). Light production by the constitutive control, strain HMS174(pRB27), was not affected by DOC at 93.8 mg of C ml21 when assays were performed at pHs 7 and 5. At pH 7 luminescence was stably maintained at 2.6 3 106 6 0.3 3 106 quanta for the no-DOC control and at 2.4 3 106 6 0.2 3 106 quanta in the presence of 93.8 mg of C ml21. The corresponding values at pH 5 were 3.5 3 105 6 0.1 3 105 and 3.8 3 105 6 0.4 3 105 quanta for the 0 and 93.8 mg of C ml21 treatments, respectively. Thus, the effect of DOC on mer-lux induction in strain HMS174(pRB28) could be studied at concentrations as high as 93.8 mg of C ml21 at pH 7 and 5 although similar assays at pH 6 would be invalid (Fig. 1). At pH 7 a gradual reduction in light output was noted with increased DOC concentrations (Fig. 2). When the percent induction of the no-DOC control was calculated by using expression factors obtained for each curve in Fig. 2, only 22% of the activity remained in the presence of 26.3 mg of DOC ml21 (Fig. 3). When mer-lux assays were performed at pH 5, induction was inhibited to a lesser extent, with 71% of the activity remaining in the presence of 93.8 mg of DOC ml21. DOC additions at the reported levels did not alter the pH of assay solutions at either pH 7 or 5 by more than 0.2 pH units. Results are representative of experiments that were performed two to three times and are presented as percent activity to accommodate data collected at both pH values. The luminescence reaction is severely affected by pH, as indicated by almost an order of magnitude reduction in light output by the constitutive control at pH 5 as compared to its output at pH 7 (see above). Thus, the results suggest that DOC reduces the bio-
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FIG. 2. Effect of DOC on mer-lux induction at pH 7. The graph shows light output in assays with strain HMS174(pRB28) containing DOC at 0 ({), 3.7 (h), 26.3 (Ç), 48.8 (E), 74.9 (■), and 94.8 (F) mg of C ml21.
availability of Hg(II) and that this reduction is more pronounced under neutral than under acidic conditions. Effect of Hg(II) speciation on bioavailability. The speciation of Hg(II) in the assay medium was altered by varying NaCl concentrations at pHs 5 and 6. At these pH values the electrochemically neutral species Hg[OH]2 predominated in the absence of NaCl and the electrochemically neutral species
HgCl2 predominated when the medium was supplemented with 1 mM NaCl (Table 2). At pH $7, Hg(OH)2 predominated regardless of the presence or absence of NaCl (not shown). At pH 5 an expression factor of 18 3 1023 6 7 3 1023 (log quanta) min21 was elicited by HgCl2 and a factor of 17 3 1023 6 5 3 1023 (log quanta) min21 was observed for Hg(OH)2, suggesting that both forms of Hg(II) had availabilities similar to that of HMS174(pRB28). This observation was repeated at pH 6 (Table 2). Larger expression factors at pH 6 were due to enhanced light production at this pH compared to that at pH 5, as was indicated by the constitutive lux control, HMS174(pRB27). Assays with the constitutive control also indicated that light production was not affected by NaCl up to 100 min into the assay. After 100 min at pH 5, but not at pH 6, light production was slightly depressed in 1 mM NaCl (data not shown). Expression factors were therefore calculated by using data that were obtained during the first 100 min of the assays. The results clearly show that the availability of the two forms of Hg(II) that are most commonly found in aerated natural waters, the chloride and the hydroxide (21), was not affected by the nature of their counterions.
TABLE 2. Effect of HgCl2 and Hg(OH)2 on expression of mer-lux
FIG. 3. Comparison of the effect of DOC on mer-lux induction at pHs 7 and 5. Induction of mer-lux in strain HMS174(pRB28) is expressed as percentage of (dL dT21)max of a no-DOC control at pH 7 ( ) and pH 5 (■).
pH
NaCl (mM)
5 5 6 6
1 0.001 1 0.001
Speciation (mol%) ofa: HgCl2
HgClOH
97.5
1.6
79.1
12.5
Hg(OH)2
98.5 7.6 99.3
Expression factorb
18 6 7 17 6 5 39 6 7 40 6 8
a Results obtained by MINTEQ for Hg(II) species that were present at $1% in assay media. b Expressed as (log quanta) 3 1023 per minute.
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FIG. 4. Relationships of Hg(II) speciation to mer-lux induction. The graph shows the moles percent values of uncharged Hg(II) ( ) [including Hg(OH)2, HgClOH and HgCl2 at 1 nM NaCl and HgCl2 exclusively at higher NaCl concentrations] and charged Hg(II) ({) (including HgCl32 and HgCl422). Expression factors [(dL dT21)max] calculated for light produced by HMS174(pRB28) are depicted by bars (means 6 standard deviations).
To investigate how negative charge may affect the availability of Hg(II), the concentration of NaCl in assay buffer at pH 6 was gradually increased from 1 to 200 mM, a range covering Cl2 concentrations typical of fresh to marine waters. pH 6 was selected because at that pH the proportion of charged mercuric chloride species (HgCl32/HgCl422) increases gradually as the proportion of HgCl2 decreases (Fig. 4). mer-lux assays showed a corresponding decrease in expression [from 59 3 1023 6 7 3 1023 to 24 3 1023 6 1 3 1023 (log quanta) min21] between 1 and 200 mM NaCl concentrations. The decrease in light production was likely due to the Hg(II)-dependent induction of mer-lux in strain HMS174(pRB28), because the constitutive lux control showed only slight variation under the employed test conditions (data not shown). The results show that uncharged mercuric chloride more readily reaches the bacterial cytoplasm than do electrochemically charged forms. DISCUSSION The results reported here are the first attempt to establish an experimental tool that distinguishes bioavailable Hg(II) from other forms of mercury. The mer-lux bioindicator was used to demonstrate that DOC and the charge of the Hg(II) species affected bioavailability. This approach could be applied to other environmental factors as well as to the measurement of bioavailable Hg(II) (35), and thus, it could contribute significantly to the evaluation of the potential for CH3Hg(I) formation in natural waters. The development and application of a constitutive lux bioindicator were essential to ensure that observed changes in light emission were due the altered Hg(II) availability. Luminescent bacterial indicators, in which the transcription of lux is controlled by environmental change or the presence of pollutants, are widely used in environmental microbiology (3, 6, 17, 23, 30, 31). As the experimental design often implies changing assay conditions, and light emission is highly sensitive to such changes (18, 26), the possibility that variations in detected light are due to effects on lux-specified reactions rather than on induction of lux transcription needs to be ruled out. This goal was achieved in this study by the use of a constitutive derivative of the mer-lux bioindicator. Thus, the effect of a high concen-
APPL. ENVIRON. MICROBIOL.
tration of DOC at pH 6 could not be studied, as it inhibited light production by strain HMS174(pRB27) (Fig. 1). Likewise, attempts to study the effect of low pH, a factor known to increase CH3Hg(I) production and accumulation (13, 42), on the bioavailability of Hg(II) were hindered by dramatic changes in light production by the constitutive control at altered pH values (data not shown). This control was also useful in mer-lux assays in natural waters to verify the existence of conditions suitable for bacterial luminescence (35). The inhibitory effect of DOC on the availability of Hg(II) to methylating bacteria was previously suggested to explain a negative correlation between DOC and CH3Hg(I) in fish from seepage lakes (15) and a reduced production of CH3203Hg(I) when DOC concentrations increased in lake water incubations with 203Hg(II) (27). The results presented here (Fig. 2 and 3) support this hypothesis by showing that DOC controls the availability of the substrate for methylation, Hg(II), to bacteria. The fact that DOC inhibition of Hg(II) availability was less severe at pH 5 than at pH 7 suggests that H1 may compete with Hg(II) for negatively charged binding sites in DOC. As the DOC fraction that was used here was not chemically characterized, the nature of the Hg-DOC interactions remains unknown. The chemistry of the DOC fraction at various pHs might also explain why DOC inhibited light production by the constitutive control more severely at pH 6 (Fig. 1) than at pHs 7 and 5. The mer-lux bioindicator could be used for further and more detailed investigation of how DOC, as well as other ligands, controls bioavailability of Hg(II) to methylating microbes. The strong inhibition of Hg(II) bioavailability at pH 7 may imply that in circumneutral natural waters DOC has a mitigating effect on the production and accumulation of CH3Hg(I), as suggested by Miskimmin et al. (27) and Grieb et al. (15), respectively. Uncharged HgCl2 was more bioavailable than anionic forms of mercuric chloride (Fig. 4), and the counterion associated with the uncharged Hg(II) species did not affect bioavailability (Table 2). It is generally accepted that neutral forms of mercurials permeate biological membranes more readily than charged forms under physiological conditions (4, 5, 21, 29). An observation by Gutknecht (16) that Hg(OH)2 and HgClOH did not permeate lipid bilayer membranes at significant rates is in contrast to our data. As the bacterial cell wall is composed of other components in addition to lipids, the transfer efficiency of various mercurial compounds may vary. Increasing NaCl concentration resulted in a decreased expression from merO/P in the mer-lux fusion in HMS174(pRB28) (Fig. 4). This decrease might be an underestimation of the effect of charge on bioavailability, because increased salinity might have stimulated expression by enhancing promoter activity. Higgins et al. (19) showed that osmotic stress exerted by 50 to 400 mM NaCl increases DNA supercoiling and that supercoiling stimulates transcription from merO/P (9). This observation brings up the possible effects on transcription from merO/P as a consideration when mer-lux expression is used to assess Hg(II) bioavailability. The effect of Cl2 on Hg(II) availability to biological systems is complex. At low Cl2 concentrations increases in membrane permeability (16) and toxicity to bacteria (11) occur. However, a decreased bioavailability of Hg(II) to the bioindicator above 1 mM Cl2 (Fig. 4) and a decline in transfer of Hg(II) through artificial membranes when Cl2 concentrations were increased above 10 mM (4, 16) were attributed to an increased proportion of negatively charged HgCl32/HgCl422 in experimental solutions. These results suggest that the availability of Hg(II) for microbial transformations should be reduced in estuarine and marine environments compared to that in freshwater sys-
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tems. Campeau and Bartha (7) showed that incubating estuarine sediments with increased salinity resulted in a decreased production of CH3Hg(I). Thus, reduced bioavailability of Hg(II) to methylating bacteria may be one reason why CH3Hg(I) accumulation is more common in freshwater than in estuarine and marine biota. The utility of the mer-lux bioindicator as a tool to study factors affecting Hg(II) bioavailability was demonstrated here for DOC and Hg(II) speciation. This approach requires that responses of the light-producing apparatus and of transcription from merO/P are ruled out, leaving bioavailability as the only factor determining expression of mer-lux. Because the public health concern with mercury is with its presence as CH3Hg(I), understanding the processes that determine the availability of Hg(II) for methylation and the development of tools to detect and measure bioavailable Hg(II) could provide a basis for more rational regulatory criteria for mercury in natural waters. ACKNOWLEDGMENTS Gratitude is extended to K. Dillen for DOC analysis. We thank Eliora Ron and three other reviewers, whose reviews have helped us improve the manuscript. This study was partially supported by an agreement (RP3015-04) between the University of West Florida and the Electric Power Research Institute. This work was performed while the third author held a Senior Research Associateship under the auspices of the National Research Council-USEPA/NHEERL at Gulf Breeze, Fla. REFERENCES 1. Allison, J. D., D. S. Brown, and K. J. Novo-Gradac. 1991. MINTEQA2/ PRODEFA2, a geochemical assessment model for environmental systems: version 3.0 user’s manual. U.S. Environmental Protection Agency publication no. EPA/600/3-91/021. U.S. Environmental Protection Agency, Athens, Ga. 2. Barkay, T., R. R. Turner, L. Rasmussen, J. W. D. Rudd, and C. A. Kelly. Lux facilitated detection of bioavailable mercury in natural waters. In R. A. LaRossa (ed.), Bioluminescent protocols. Humana Press, Inc., New York, N.Y., in press. 3. Belkin, S., D. R. Smulski, A. C. Vollmer, T. K. Van Dyk, and R. A. LaRossa. 1996. Oxidative stress detection with Escherichia coli harboring a katG9::lux fusion. Appl. Environ. Microbiol. 62:2252–2256. 4. Bienvenue, E., A. Boudou, J. P. Desmazes, C. Gavach, D. Georgescauld, J. Sandeaux, R. Sandeaux, and P. Seta. 1984. Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration. Chem. Biol. Interactions 48:91–101. 5. Boudou, A., D. Georgescauld, and J. P. Desmazes. 1983. Ecotoxicological role of the membrane barriers in transport and bioaccumulation of mercury compounds, p. 117–136. In J. O. Nriagu (ed.), Aquatic toxicology. John Wiley & Sons, New York, N.Y. 6. Burlage, R. S., and C. T. Kuo. 1994. Living biosensors for management and manipulation of microbial consortia. Annu. Rev. Microbiol. 48:291–309. 7. Compeau, G. C., and R. Bartha. 1987. Effect of salinity on mercury-methylating activity of sulfate-reducing bacteria in estuarine sediments. Appl. Environ. Microbiol. 53:261–265. 8. Compeau, G. C., and R. Bartha. 1985. Sulfate-reducing bacteria: principal methylators of mercury in anoxic estuarine sediment. Appl. Environ. Microbiol. 50:498–502. 9. Condee, C. W., and A. O. Summers. 1992. A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. J. Bacteriol. 174:8094–8101. 10. Driscoll, C. T., C. Yan, C. L. Schofield, R. Munson, and J. Holsapple. 1994. The mercury cycle and fish in the Adirondack lakes. Environ. Sci. Technol. 28:136A–143A. 11. Farrell, R. E., J. J. Germida, and P. M. Huang. 1990. Biotoxicity of mercury as influenced by mercury(II) speciation. Appl. Environ. Microbiol. 56:3006– 3016. 12. Fjeld, E., and S. Rognerud. 1993. Use of path analysis to investigate mercury accumulation in brown trout (Salmo trutta) in Norway and the influence of environmental factors. Can. J. Fish. Aquat. Sci. 50:1158–1167. 13. Gilmour, C. C., and E. A. Henry. 1991. Mercury methylation in aquatic systems affected by acid deposition. Environ. Pollut. 71:131–169. 14. Gilmour, C. C., E. A. Henry, and R. Mitchell. 1992. Sulfate stimulation of mercury methylation in freshwater sediments. Environ. Sci. Technol. 26: 2281–2287.
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