Methylmercury Oxidative Degradation Potentials in Contaminated and ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1995, p. 2745–2753 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 61, No. 7

Methylmercury Oxidative Degradation Potentials in Contaminated and Pristine Sediments of the Carson River, Nevada† RONALD S. OREMLAND,1* LAURENCE G. MILLER,1 PHILIP DOWDLE,1 TRACY CONNELL,1 AND TAMAR BARKAY2 U.S. Geological Survey, Menlo Park, California 94025,1 and U.S. Environmental Protection Agency Environmental Research Laboratory, Sabine Island, Gulf Breeze, Florida 325612 Received 6 March 1995/Accepted 8 May 1995

Sediments from mercury-contaminated and uncontaminated reaches of the Carson River, Nevada, were assayed for sulfate reduction, methanogenesis, denitrification, and monomethylmercury (MeHg) degradation. Demethylation of [14C]MeHg was detected at all sites as indicated by the formation of 14CO2 and 14CH4. Oxidative demethylation was indicated by the formation of 14CO2 and was present at significant levels in all samples. Oxidized/reduced demethylation product ratios (i.e., 14CO2/14CH4 ratios) generally ranged from 4.0 in surface layers to as low as 0.5 at depth. Production of 14CO2 was most pronounced at sediment surfaces which were zones of active denitrification and sulfate reduction but was also significant within zones of methanogenesis. In a core taken from an uncontaminated site having a high proportion of oxidized, coarsegrain sediments, sulfate reduction and methanogenic activity levels were very low and 14CO2 accounted for 98% of the product formed from [14C]MeHg. There was no apparent relationship between the degree of mercury contamination of the sediments and the occurrence of oxidative demethylation. However, sediments from Fort Churchill, the most contaminated site, were most active in terms of demethylation potentials. Inhibition of sulfate reduction with molybdate resulted in significantly depressed oxidized/reduced demethylation product ratios, but overall demethylation rates of inhibited and uninhibited samples were comparable. Addition of sulfate to sediment slurries stimulated production of 14CO2 from [14C]MeHg, while 2-bromoethanesulfonic acid blocked production of 14CH4. These results reveal the importance of sulfate-reducing and methanogenic bacteria in oxidative demethylation of MeHg in anoxic environments. mercurial lyase cleaves the carbon-mercury bond, resulting in the accumulation of methane (4, 18). A number of studies have focused on the presence of mer genes in natural populations and the volatilization of elemental mercury from Hg(II) and MeHg (6, 7, 25, 34, 53) or have exploited this phenomenon to develop biosensors for the detection of available Hg(II) (47). The discovery that anoxic sediments and bacterial cultures could also form 14CO2 as well as 14CH4 when incubated with [14C]MeHg revealed that another bacterial mechanism besides the lyase reaction for demethylation exists (38). This phenomenon, termed oxidative demethylation, is theorized to result from usage of methylmercury as a substrate analog, which because it is present at low concentrations is metabolized by pathways established for the dismutation of normal electron donors. Inhibitor manipulations of sediments suggested the involvement of both sulfate-respiring bacteria and methanogenic flora in oxidative demethylation (38). However, although both 14CH4 and 14CO2 were detected in cultures of methanogens, sulfate reducers formed only 14CH4 from [14C]MeHg and oxidative demethylation in sulfate-reducing bacterium cultures remains to be demonstrated (38). A non-lyase-associated formation of methane from MeHg was recently reported to exist in methylmercury-resistant cultures of Desulfovibrio desulfuricans (5). The methane was believed to have resulted from the breakdown of dimethylmercury sulfide which accumulated during growth. These reports have underscored the complex bacterial dynamics of methylation-demethylation reactions in anoxic sediments and have stressed the need for more data concerning the occurrence of oxidative demethylation in nature. To this end, we undertook a study of oxidative demethylation in mercury-contaminated as well as pristine sediments of the Carson River to determine if the process was a common feature of such systems and whether it correlated with down-

Factors affecting the chemical speciation, toxicity, and bioavailability of mercury in contaminated environments are of a complex nature and require interdisciplinary approaches to understand the workings of individual ecosystems (58). Additionally, great care must be taken in determining the chemical abundance and speciation of mercury in environmental samples (20). An important focus of mercury biogeochemistry investigations concerns its most toxic forms, namely, monomethylmercury (MeHg) and dimethylmercury (Me2Hg), and hence the biological mechanism(s) for and extent of methylation reactions in nature have been the subject of much attention. A complication of this phenomenon is the fact that some microbes have the ability to degrade methylmercury. Thus, a number of field and laboratory investigations have conducted concurrent measures of rates of methylation and demethylation in an attempt to derive estimates of net rates (13, 28, 29, 43, 50). With regard to mechanistic studies, recent work on methylation has revealed the primary involvement of sulfate-respiring bacteria rather than methanogens (30, 57) in both marine and freshwater systems (14, 22). Apparently, sulfate-respiring bacteria enzymatically methylate inorganic mercury via cobalamin-mediated reactions (9–11). For demethylation, attention has been drawn primarily to the workings of the mer operon as a model for methylmercury detoxification (via the merBspecified lyase) and volatilization (via the merA-specified mercuric reductase) in the environment (45, 51, 52). The organo* Corresponding author. Mailing address: U.S. Geological Survey, ms 465, 345 Middlefield Rd., Menlo Park, CA 94025. Phone: (415) 329-4482. Fax: (415) 329-4463. Electronic mail address: roremlan@ mprcamnl.wr.usgs.gov. † In memory of Murray Oremland, 1903–1994. 2745

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FIG. 1. Map showing the location of Carson River sites sampled at Gardnerville (GV), Fort Churchill (FC), and Lahontan Reservoir (LR).

core measures of denitrification, sulfate reduction, and methanogenesis. Because our use of [14C]MeHg in this investigation raised the pool size of MeHg by 2 to 3 orders of magnitude above the ambient level, we report rates as potential demethylations. Nonetheless, we observed that oxidative demethylation is the dominant demethylation process in these sediments. MATERIALS AND METHODS Study site. The Carson River is located in western, central Nevada and drains the eastern slope of the Sierra Nevada mountains (Fig. 1). Nineteenth century precious metal mining and milling activities associated with the Comstock Lode located near Virginia City, Nev., resulted in the release of an estimated 6.8 3 106 kg of mercury into the surrounding environment (2, 48). A reconnaissance study of the Carson River identified low levels (0.1 ppm) of total mercury in upstream sediments a few kilometers west of Fort Churchill, but the mercury content of streambed sediments at Fort Churchill and locations east was 2 to 3 orders of magnitude higher and was associated mostly with fine-grain silts and clays (54). Subsequent studies of mercury in fish tissues found elevated levels at downstream sites (below Fort Churchill), with values of 1.0 ppm or higher common in fishes from Lahontan Reservoir (15, 16). Sampling. Sediments were collected by taking core samples from three locations just above dammed impoundments (Fig. 1), including a pristine location near the town of Gardnerville (site GV), a gauging station about 2 km west of Fort Churchill (site FC), and a site 1 km east of the Lahontan Reservoir dam (site LR). We selected these dam sites because we assumed they would accumulate fine-grain, anoxic sediments. Sediments from site LR were recovered by gravity core, and hand cores were used by divers to recover sediment at sites GV and FC. Sediments were sampled during July 1994 (water temperature of 24 to 288C) and November 1994 (water temperature of ;108C). Cores were processed within 24 h of collection at the U.S. Geological Survey District Office in Carson City, Nev. Cores were vertically extruded in 4-cm horizons from which subcores were taken. Subcores were fashioned from plastic syringes (with hub ends removed) and were used for bioassays and determination of methane content (as described below). The sediment sections remaining after subsampling were placed in N2-pressured squeezers to extrude interstitial pore fluids for the determination of ammonium, sulfate, dissolved inorganic carbon (S CO2), and sulfide (39, 40, 44). The remaining squeezed sediment was analyzed for total mercury content (as described below). Bulk sediment for laboratory investigations was also collected from all the locations, by bottom-grab or hand-held cores, and stored at 48C within completely filled mason jars. Activity assays. Sediment was subsampled in cut-off plastic syringes having volumes of 3.0 cm3 (demethylation and methanogenesis assays; volume of sediment, 2.5 cm3) or 10 cm3 (sulfate reduction assays; volume of sediment, 5.0 cm3). The syringes were plugged with black rubber stoppers and injected with [14C]MeHg (0.1 mCi/100 ml; specific activity [sp. act.], 3.7 mCi/mmol; Amersham

APPL. ENVIRON. MICROBIOL. Corp., Arlington Heights, Ill.), NaH14CO3 (5 mCi/100 ml; sp. act., 54.4 mCi/ mmol; ICN Biomedicals, Irvine, Calif.), or Na35SO4 (7.25 mCi/250 ml; sp. act., 1,017 mCi/mmol; ICN Biomedicals) from stock dilutions kept in serum bottles containing an N2 headspace. The final MeHg concentration achieved by addition of this amount of [14C]MeHg to high-porosity (.90% water content) sediments was ;12 mM. Cores were incubated at room temperature (308C in July and 168C in November) for 24 h (for methanogenesis and sulfate reduction) and 46 to 70 h for the July demethylation studies and 40 h for the November demethylation studies. Microbial activity was arrested by freezing the samples at 2608C. In the November experiments, the demethylation and methanogenesis experiments were modified by immediately extruding the 2.5 cm3 of subsample into small serum bottles (13 ml) while simultaneously flushing the serum vial with N2 emanating from a cannula, after which they were capped with black butyl rubber stoppers, crimped, and flushed with N2 for 5 min before the radioisotope was added. In the demethylation and methanogenesis assays, frozen subcores were extruded into 13-cm3 serum bottles which contained 2.5 ml of NaCl-saturated water (July experiments) or the water was injected directly into the sealed bottles (November experiments). The bottles were capped, crimped and sealed, injected with 0.5 ml of 6 N HCl (demethylation assays only), and shaken for 18 h (200 rpm). The headspace gas was subsampled by syringe, and 14CH4 and 14CO2 levels were determined by gas proportional counting in sequence with gas chromatography (17). The pressure in the bottles was measured by deflection of wetted glass syringes. The ratio of 14CO2 to 14CH4 is referred to herein as the oxidized/ reduced demethylation product ratio (ORDP ratio) and is indicative of the overall importance of oxidative demethylation. Extraction of H235S from samples was done by heated distillations of sediment in 10-ml volumes of 1.0 M CrCl2 solution in 1.0 N HCl during continuous flushing with N2. The exiting gases were bubbled through two sequential scintillation vial traps containing 5 ml of 2% zinc acetate, and after 20 min the distillation traps were removed and counted by liquid scintillation spectrometry (26). Rates of sulfate reduction, demethylation, and methane production were calculated from the following equation: rate (micromoles liter21 day21) 5 kCF, where k is the rate constant determined from the percent isotope reacting per day; C is the micromolar concentration of sulfate, bicarbonate, or added MeHg; and F is the porosity. Potential denitrification was measured by the acetylene block assay (3) by employing 10-ml subsamples of sediment extruded into 141-ml conical flasks. The flasks contained 50 ml of deionized water with 1 mM NaNO3. Flasks were sealed with black rubber stoppers, flushed with N2, and injected with acetylene (14 ml). Samples were incubated at 208C with constant rotary shaking, during which time the headspace gas was sampled by syringe for determination of N2O by electron capture gas chromatography (31, 40). Rates were calculated from linear regressions of the headspace N2O production data, with a correction factor of 1.47 applied to account for dissolved N2O and a 1.26 Henry’s law constant applied to the partitioning equation (46). Rates were expressed as millimoles per kilogram (dry weight) of sediment per day. Potentials for Hg(II) methylation were determined only for the July 1994 experiments after a delay of 72 h from the time of collection to allow for transport. Subsamples contained in 3-ml (LR) or 10-ml (FC and GV) plastic syringes (see description above) were injected with 203Hg(NO3)2 [sp. act., 2.5 Ci/g of Hg(II) for LR and 71 mCi/g of Hg(II) for FC and GV]. Substrate 203 Hg(NO3)2 was prepared by mixing high-sp.-act. 203HgCl2 (5.5 Ci/g; Buffalo Materials Research Center, Buffalo, N.Y.) with cold Hg(NO3)2. A total of 2 ng (;20 pmol) of Hg(II) was injected into each syringe, and each core section was assayed in triplicate. Samples were incubated at 298C for 24 h, after which Me203Hg was extracted (19, 28) and quantified with a Tri-Carb 2500 liquid scintillation counter (Packard Instruments Co. Inc., Meriden, Conn.). Laboratory investigations. To determine if the production rates of 14CO2 and 14 CH4 were linear over the time course of the incubation, freshly collected sediment (the upper 4 cm) from site LR was subsampled by syringe, injected with [14C]MeHg, and incubated as indicated above. At various time intervals, biological activity was arrested by freezing triplicate sets of subsamples at 2608C. Subsequent quantification of radiolabelled gases was achieved after extrusion into serum bottles and acidification by methods already stated. The effect of molybdate and 2-bromoethanesulfonic acid (BES), compounds which, respectively, inhibit sulfate reduction and methanogenesis (37), upon the production of radiolabel products was investigated. Sediment from LR and FC (3 cm3) was placed in serum bottles as indicated previously, but in addition 0.2 ml of 20 mM Na2MoO4 was included. Sediment samples incubated without inhibitors and controls consisting of heat-killed sediments (autoclaved at 1218C and 250 kPa for 1 h) were also run. The samples were flushed with O2-free N2 (passed through a reduced, hot copper column) and incubated statically at 208C for several days, during which time the evolved headspace gases were analyzed. The experiments were terminated by injection of 0.5 ml of 6 N HCl, and the samples were shaken overnight and then analyzed for S 14CO2 (see the description above). A second experiment using slurries generated from FC sediments which were homogenized in a blender with an equal volume of deionized water was performed, and the resulting homogenate (10 ml) was transferred to serum bottles (57-ml volume) which contained an additional 10 ml of deionized water amended with sodium sulfate (final concentration, 2 mM) or BES (final concentration, 20 mM). The bottles were crimp sealed with butyl rubber stoppers and flushed for 5 min with O2-free N2. The addition of 1 ml of [14C]MeHg solution was done by syringe

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METHYLMERCURY DEGRADATION IN SEDIMENTS

(0.44 mCi added per bottle). Slurries were incubated at 208C with constant rotary shaking (200 rpm). Headspace samples were analyzed for 14C-labelled gases as indicated above. The experiments were terminated by injection of 2 ml of 6 N HCl, and the bottles were shaken for an additional 12 h before determination of S 14CO2. Analysis. The porosity of sediments was determined by weight loss upon oven drying at 858C for 5 days. The methane content of sediments was determined by extruding 5-cm3 subcores into 145-ml conical flasks which contained 60 ml of NaCl-saturated water. The flasks were sealed with black rubber stoppers and shaken continuously (200 rpm) for 12 h. The headspace gas was sampled by syringe, and the methane content was quantified by flame ionization gas chromatography (31). Ammonium content was determined by the method of Soloranzano (49), and sulfide content was determined by the method of Cline (12) after preservation with zinc acetate (32). Dissolved inorganic carbon (S CO2) was quantified by acidifying 0.5 ml of pore water with 0.1 ml of 6 N HCl in a sealed 37-ml serum bottle. After an overnight shaking, headspace CO2 was measured by gas chromatography with a thermal conductivity detector (17). The total mercury content in sediments was determined with a PS200 Automated Mercury Analyzer (Leeman Labs, Inc., Lowell, Mass.) after digestion of 0.2 to 0.3 g of wet sediment in 2.4% KMnO4 for 45 min at 958C and reduction of excess KMnO4 with 3.1% NH2OH z HCl. The dry weight of the digested samples was determined by multiplying the weight of the analyzed samples by the percentage of solids that was obtained after drying of the sediments to completeness at 1508C. Sediment size fractionations were conducted by wet sieving of predried fractions from each depth horizon through a series of sieves of decreasing mesh sizes. Collected sediment grains retained by the individual meshes were redried and weighed, and data are reported as percentages of the total weight of the cumulative size fractions. Sediment organic carbon content was analyzed commercially (Huffman Laboratories, Golden, Colo.) as total carbon by combustion under O2 at 1,5008C followed by infrared detection of the evolved CO2. The inorganic carbon content, determined colorimetrically, was below the detection limit (0.02% by weight) in all samples.

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TABLE 1. Depth profiles of sediment porosity, sulfate, sulfide, ammonia in pore water, and methane and mercury contents for three study sites on the Carson River during November 1994 Porosity (% water)

SO45 (mM)

S5 (mM)

NH41 (mM)

CH4 (mmol/g)

Hg (mg/g)

Lahontan Reservoir 0–4 4–8 8–12 12–16 16–20 20–24

82 71 71 65 63 66

1.23 0.15 0.11 0.10 0.05 0.03

1.1 0.4 2.4 2.4 2.1 4.6

0.41 1.11 1.33 1.47 1.44 1.53

1.34 5.85 3.51 4.18 2.94 3.03

3.14 3.21 4.05 3.24 4.07 5.78

Fort Churchill 0–4 4–8 8–12 12–16

57 52 61 24

0.22 0.02 0.09 0.06

5.5 5.5 5.3 5.3

0.30 0.93 1.44 1.22

3.77 3.52 4.71 1.50

Gardnerville 0–4 4–8 8–12 12–16 16–20

69 30 32 36 42

0.61 5.0 0.47 4.7 0.49 5.4 0.46 6.7 0.52 BDLa

0.26 0.005 0.005 0.005 ND b

0.003 0.001 0.002 0.005 0.001

Site and depth (cm)

a b

26.5 33.2 44.3 15.8 0.047 0.087 1.460 0.255 0.110

BDL, below detection limit. ND, not determined.

RESULTS Sediment characteristics. The fine-grain sediments of the LR site had the highest values of porosity, while the values for stream sediments from the FC and GV sites were lower, indicating a smaller water-holding capacity (i.e., greater proportions of sand-sized sediments) (Table 1). We observed that the LR sediments were uniformly silty in appearance and texture, while those from the FC and GV sites were composed of coarser, sandy material which was unevenly distributed downcore. These observations were borne out by size fractionation analysis. The percentages, relative to the total dry weight, of the silt-and-clay size fraction (,0.063-mm diameter) as determined for all depth horizons (means 6 standard deviations [SD], with n representing the number of 4-cm depth horizons sampled), were as follows: for GV, 11.9 6 7 and n 5 5; for FC, 26.6 6 5.9 and n 5 4; and for LR, 98.3 6 0.2 and n 5 6. In contrast, the percentages of the medium-to-coarse-grain sand size fraction (0.25 to 2.0 mm) were 36.2 6 19.3 for GV, 26.7 6 11.1 for FC, and 0 6 0 for LR. The sediments from the FC site also had pieces of woody debris as well as gravel present in the .2-mm size fraction (mean, 4.2%). Indeed, the FC site itself had a considerable amount of woody material present as entrapped logs and branches whose dislodgement from the sediments released abundant bubbles with a 10% methane content as determined by gas chromatography. The percent organic carbon contents in the sediments from the three sites (mean 6 standard error) averaged over the upper 16 cm of the sediment column were 2.85 6 3.39 for GV, 2.69 6 1.61 for FC, and 1.61 6 0.08 for LR. Sediments from LR and FC contained comparable levels of methane and ammonia, while those from GV had values lower by about 2 orders of magnitude (Table 1). The LR down-core profiles of decreasing sulfate with increasing sulfide and ammonia are typical of lake sediments, while the stream profiles were more erratic (especially that for GV) and indicative of a higher-energy, changing environment. Free sulfide was detected at all sites (#6.7 mM). Dissolved inorganic carbon values at LR and FC were comparable, increasing, respectively,

from 2.8 and 3.1 mM at the surface to 7.2 mM at a $16-cm depth (data not shown). In contrast, the GV dissolved inorganic carbon concentration was considerably lower, ranging from 0.05 to 0.56 mM, and no trends were apparently connected with depth (data not shown). Total sediment mercury concentrations were highest at FC and displayed an increase with depth down to 12 cm and a decrease at 12 to 16 cm. The Hg values at the FC site were generally comparable to those reported for Japan’s Minamata Bay (33). Similar down-core increases were observed in the LR core, although absolute values of total mercury were almost an order of magnitude lower than the FC values. Mercury levels at the uncontaminated GV site were at least 30-fold lower than those at the FC site and within the concentration range recognized as background abundance (1). Sediments investigated in previous studies of mercury methylation contained much lower levels of S Hg than the FC and LR sites, with freshwaters having 0.015 to 0.045 mg/g (22) and San Francisco Bay muds having 0.1 to 1.3 mg/g (35). Sediment assays. Preliminary time course demethylation experiments conducted during July 1994 with LR surficial sediments (0 to 4 cm) indicated increasing production of 14CH4 and 14CO2, achieving an ORDP ratio of ;5.5 with ;16% mineralization of the added [14C]MeHg after 46 h of incubation (Fig. 2). However, a slight decline in the level of recovered 14 CH4 as well as that of unlabelled CH4 (data not shown) was observed after 46 h of incubation, which implied outward diffusion of entrapped methane. Therefore, these incubations may have produced underestimations of the demethylation rates and subsequent assays were done with serum bottles, which yielded linear production rates for both gases (as discussed below). Nonetheless, significant 14CO2 production from [14C]MeHg was noted at all three sites studied and for every depth assayed. The highest ORDP ratios and activities were achieved in the top layer (0 to 4 cm) of assayed sediments (1.5, 5.4, and 3.1 for LR, FC, and GV, respectively), which repre-

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FIG. 2. Production of 14CO2 (F) and 14CH4 (E) from [14C]MeHg (0.1 mCi) during incubation of subcores taken from Lahontan Reservoir during July 1994. The results represent the means for three individual sediment samples, and the bars indicate 61 SD. An absence of bars indicates that the error margin was smaller than the symbol.

sented mineralization of 19% (46 h), 33% (52 h), and 15% (70 h) of the added [14C]MeHg. ORDP ratios declined with depth at all the sites, achieving near equivalency between 14CH4 and 14 CO2. For example, LR samples achieved an ORDP ratio of 1.0 over the depth interval of 8 to 25 cm (four assayed depths), and similar results were observed at site FC for the 8-to-16-cm interval (two assayed depths). Only one deep sample could be assayed in the GV core (4 to 8 cm), and its ORDP ratio was 1.4. Potential methylation was detected in all core sections from GV and FC but not from LR (limit of detection, 4 pg/g of sediment). Specific methylation rates of FC samples ranged between (0.7 6 0.09)% and (1.1 6 0.3)% per day and peaked 8 to 12 cm below the water-sediment interface (data not shown). Higher methylation rates were observed at GV than at the other sites, with 14.2% per day in the 0-to-4-cm horizon and 7.5% per day in the 4-to-8-cm horizon. In November 1994, sediments from LR (Fig. 3) exhibited the highest rates of sulfate reduction at the core surface (;125 mmol liter21 day21), and the rates declined steadily with depth, reaching values reduced by nearly fourfold at 22 cm (Fig. 3A). In contrast, methanogenic activity exhibited a generally increasing trend with depth, but overall mean rates ranged between 1.0 and 4.0 mmol liter21 day21 and were therefore about

APPL. ENVIRON. MICROBIOL.

10- to 100-fold lower than those for sulfate reduction. A clear decreasing trend in denitrification potentials was also observed over the length of the core, with values at the surface about eightfold higher than those at the bottom (Fig. 3C). Denitrification was not detected in sediment incubated without the addition of nitrate (data not shown). Total demethylation activity was also highest at the top of the core, with a combined rate of ;1.6 mmol liter21 day21 and an ORDP ratio of 4.4 (Fig. 3B). Production of 14CO2 decreased with depth, reaching steady values by midcore, while there was a slightly increasing trend in 14CH4 production. This change resulted in a downcore decrease of ORDP ratios, with values at or near 1.0 at depths below 10 cm. The overall demethylation rate at the core bottom was ;0.9 mmol liter21 day21. By h 39 of incubation, demethylation mineralized ;21% of the added [14C]MeHg in the surface sediments, with a decreasing down-core trend which reached ;12% mineralization by a 24-cm depth. The Hg-contaminated stream site at FC demonstrated clearly different trends (Fig. 4). First, sulfate reduction rates exhibited an irregular pattern, while methanogenesis rates generally increased with depth (Fig. 4A). Sulfate reduction was quantitatively about the same as that at the LR site and generally exceeded the methanogenesis rate. However, values measured for the two processes at 6 and 14 cm were comparable. In addition, methanogenic activity at the FC site was considerably higher (4- to 10-fold) than that measured at LR, with mean values of 9 to 26 mmol liter21 day21. Overall demethylation activity was greater at FC than at LR, with rates ranging from 1.7 to 2.7 mmol liter21 day21 and ORDP ratios dropping from 2.8 at the surface to 0.6 at lower depths (Fig. 4B). The down-core pattern of methanogenesis (Fig. 4A) closely followed that of 14CH4 production from [14C]MeHg (Fig. 4B). The quantities of [14C]MeHg mineralized during the incubations ranged from 24 to 44% (48-h incubation). Denitrification was not measured at the FC or GV site. The uncontaminated GV site exhibited a third pattern of activity (Fig. 5). Neither sulfate reduction nor methanogenesis was a quantitatively significant process, with each discernible only at the top of the core and decreasing to zero or barely detectable values with depth (Fig. 5A). The mean values for individual depths were #1.0 mmol liter21 day21 for sulfate reduction and #0.01 mmol liter21 day21 for methanogenesis. In contrast to the other sites, demethylation activity at GV was characterized by 14CO2 being the only product detected at four of five depths (Fig. 5B). The overall rates of demethylation

FIG. 3. Activity profiles of Lahontan Reservoir sediments taken during November 1994. (A) Sulfate reduction (å) and methanogenesis (E); (B) MeHg demethylation products CH4 (E) and CO2 (F); (C) denitrification. The results represent the means for three samples, and the bars indicate 61 SD. An absence of bars indicates that the error margin was smaller than the symbols. Denitrification measurements were obtained from time course experiments conducted with single samples.

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varied between 0.5 to 0.7 mmol liter21 day21, which accounted for 7 to 11% mineralization of the added [14C]MeHg, and were therefore significantly lower than those at the FC and LR sites. An intersite comparison of the total demethylation and the relative contributions of 14C-product gases to this total was made by integrating activities recorded for the top 16 cm of the sediment columns from the three locations during November 1994 (Table 2). Total demethylation rates were lowest at GV and increased by a factor of about 2 going from GV to LR and again by a factor of 2 from LR to FC. There was a correspondingly large drop in the ORDP ratio, which declined from 51 at GV to 0.6 at FC. Production of 14CO2 from [14C]MeHg also increased progressively from GV to LR and from LR to FC; however, the corresponding increase in 14CH4 production was larger than the decline in the ORDP ratio (Table 2). Methanogenic activity at FC was over eightfold greater than that at LR, while sulfate reduction rates at these two sites were comparable and of more overall significance than methanogenesis. Sulfate reduction and methanogenesis at GV, although detectable, were unimportant. Preliminary measurements made during July 1994 agreed with this data set, with total demethylation at LR and FC calculated as 101 6 25 and 212 6 24 mmol m22 day21, respectively. A 16-cm-long core could not be retrieved from GV during July, and demethylation rates for the top 8 cm were calculated to be 27 6 11 mmol m22 day21. Denitrification measurements were conducted only on LR sediments, and the integrated potential rate in November (presented as nitrate consumed) was 112,860 mmol m22 day21. Laboratory incubations. Production of 14CH4 and 14CO2 from [14C]MeHg proceeded at nearly linear rates during incubation of LR and FC sediments (data not shown). Sediments from FC mineralized [14C]MeHg more rapidly than the LR sediments, with FC radiolabel gas production leveling off by day 6 of incubation and achieving ;100% mineralization of the [14C]MeHg (data not shown). Table 3 indicates the final levels of radiolabelled gases achieved after 12-day incubations of FC and LR material with and without molybdate. Final ORDP ratios for FC were 0.89 for the uninhibited sediments. The addition of molybdate to FC sediments strongly inhibited the production of 14CO2 (62% inhibition), and final ORDP ratios decreased to 0.23. Nonetheless, the overall mineralization rate of [14C]MeHg was 95% and was therefore comparable to that of the uninhibited sediments. For LR, the final ORDP ratio observed after acidification was 1.5 and only 50% of the added 14 C-MeHg was mineralized by day 12. The addition of molybdate to these sediments also altered the pattern of radiolabelled gas evolution by shifting production to 14CH4 and away from 14CO2, which was inhibited by 40%. The final ORDP ratios in the molybdate-inhibited sediments were 0.66, but the amount of [14C]MeHg mineralized (44%) was comparable to that in the uninhibited sediments. In FC sediments, formation rates of CH4 were linear (data not shown) and nearly identical under both incubation conditions and final levels of accumulated CH4 (in micromoles) were comparable, being 1,363 6 35 and 1,334 6 121 (means of three slurries 6 SDs) for the uninhibited and molybdate inhibited conditions, respectively. About 10-fold less methanogenic activity was noted in the LR samples than in the FC sediments (data not shown). Uninhibited and molybdate-containing LR sediments, respectively, accumulated 145 6 41 and 211 6 10 mmol of CH4 by day 11. The rates of production of CO2 were roughly equivalent for all incubation conditions at both sites, with values ranging between 147 and 189 mmol by the end of the incubation periods (data not shown). Heatkilled controls did not produce any radiolabelled gases, CH4 or CO2 (data not shown).


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FIG. 4. Activity profiles from Fort Churchill samples collected during November 1994. (A) Sulfate reduction (å) and methanogenesis (E); (B) MeHg demethylation products CH4 (E) and CO2 (F). The symbols represent the means for three samples, and the bars indicate 61 SD. An absence of bars indicates that the error margin was smaller than the symbols.

Unamended sediment slurries from site FC produced 14CO2 and 14CH4, yielding a final ORDP ratio of 0.38 (Fig. 6A). Addition of sulfate decreased the amount of CH4 and 14CH4 formed while increasing the production of 14CO2 (Fig. 6B), thereby achieving a final ORDP ratio of 1.4. Sulfate amendment also lowered the final level of accumulated CH4 from 714 6 214 mmol (uninhibited) to 425 6 41 mmol (plus sulfate). Methanogenesis was strongly inhibited (95%) in slurries incubated with BES and did not form 14CH4 over the course of the live incubation (Fig. 6C). After acidification, a large amount of 14 CH4 was evident in the gas phase. The final level of 14CO2 present in BES-inhibited slurries (74 6 3 nCi) was slightly lower than those in the uninhibited controls (89 6 17 nCi). DISCUSSION These experiments extend our observations on the occurrence of oxidative demethylation of MeHg in nature. Previous work had found oxidative demethylation during prolonged incubation (2 to 4 weeks) of anaerobic sediment slurries from estuarine (San Francisco Bay), freshwater (Searsville Lake)

FIG. 5. Activity profiles from Gardnerville samples collected during November 1994. (A) Sulfate reduction (å) and methanogenesis (E); (B) MeHg demethylation products CH4 (E) and CO2 (F). The symbols represent the means of three samples, and the bars indicate 61 SD. An absence of bars indicates that the error margin was smaller than symbols.

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TABLE 2. Areal rates of demethylation, methanogenesis, and sulfate reduction calculated for the top 16 cm of sediment columns from three study sites during November 1994 Activity (mmol m22 day21) Site

GV LR FC

CH4a

14

1.4 6 0.05 43.0 6 6 194 6 30

CO2a

14

71 6 11 105 6 16 124 6 20

ORDPb

DM ratec

SR ratec

MP ratec

51.0 2.4 0.64

72 6 11 148 6 17 318 6 36

51 6 35 15,362 6 2,393 12,990 6 2,244

0.15 6 0.05 306 6 127 2,625 6 722

Mean production of 14CH4 or 14CO2 from [14C]MeHg 6 SD. ORDP ratio, 14CO2/14CH4. c DM rate, mean total demethylation rate (14CO2 1 14CH4); SR rate, mean sulfate reduction rate; MP rate, mean methane production rate (6 SD). a b

and alkaline-hypersaline (Mono Lake) environments (38). We have now shown that freshwater stream and reservoir sediments, some of which are highly contaminated with mercury, also display oxidative demethylation. In addition, these sediments were assayed with minimal disruption (Fig. 2 to 5) rather than being slurried, which allowed for the calculation of potential demethylation rates. In general, the extent and rates of demethylation we observed in the Carson River system were greater than those of systems investigated previously. In the Carson River system, we observed that the sediments which contained the highest levels of mercury (Table 1) also had the highest rates of MeHg demethylation (Table 2). The uncontaminated control site (GV) had the lowest overall rates of demethylation and the highest ORDP ratios (Fig. 4; Table 2), which may have been a consequence of the chemical properties of these sediments being generally more oxidized than those of the other two sites (Table 1). An elevated potential for demethylation in contaminated sediments has also been reported by Gilmour and Henry (21). Production of 14CO2 from MeHg is by definition indicative of oxidative demethylation rather than the organomercurially specified lyase reactions, and our Carson River investigations allow us to scrutinize this phenomenon more closely than elsewhere. The production of 14CO2 from [14C]MeHg was immediate and steady over time (Fig. 2) and thus was not an artifact of prolonged incubation. Autoclaved sediments produced neither 14 CO2 nor 14CH4, which underscores the biological nature of the process. Formation of 14CO2 via oxidation of lyase-formed 14 CH4 by methanotrophs would not have been possible, because sediments were incubated under anaerobic conditions. The efficacy of the imposed anoxic conditions was confirmed by the detection of oxygen-sensitive respiratory processes (e.g., sulfate reduction, methanogenesis, and denitrification) in our samples (Fig. 3 to 5; Table 2). Oxidative demethylation occurred at all three sites, in all the samples assayed, and for LR and FC was most extensive in the surficial layer (Fig. 3 to 5). The high ORDP ratios observed in the surficial layer could, in part, represent the contributions of respiratory anaerobes employing electron acceptors like nitrate (Fig. 3C) or perhaps metals like Mn41, although mercury demethylation by these types of anaerobes has not been examined. Indeed, these alternate electron acceptors may have contributed substantially to the oxidative demethylation encountered at the GV site, where very little methanogenesis and sulfate reduction were encountered (Fig. 5; Table 2). Clearly, oxidative demethylation was of sufficient magnitude not to be dismissed as a trivial mechanism. It is apparent that oxidative demethylation was the predominant process at the GV and LR sites by virtue of their ORDP ratios exceeding unity (Table 2). Nonetheless, the production of 14CO2 at the FC site was also significant (Table 2), being 62% of that observed for 14CH4. Methanogenic activity was greater at FC than at LR, al-

though the two sites had equivalent rates of sulfate reduction, which was quantitatively more important than methanogenesis (Table 2). The methanogenic rates calculated for Table 2 are probably underestimates, because they account only for CO2 reduction and ignore the contribution of acetoclastic methanogenesis, which is important in freshwater systems (55). The presence of a higher degree of methanogenic activity at FC was also borne out by the laboratory sediment incubation data which indicated about 10-fold more CH4 accumulation than at the LR site over the same incubation period (see Results). Therefore, the data support the concept that the demethylation observed at FC (and the low ORDP ratio of 0.64) was primarily due to oxidative demethylation occurring in an environment with a higher degree of methanogenic activity than observed at other sites. The concept of oxidative demethylation postulates that MeHg acts as a 1-carbon (C1) substrate analog and that the gaseous products formed from MeHg will be reflective of the dominant respiratory end processes operative in the environments studied (38). Hence, the relative importance of 14CH4 production from [14C]MeHg should increase under methanogenic conditions as opposed to those for sulfate reduction or denitrification. This phenomenon was observed in the case of [14C]dimethylselenide degradation by salt marsh sediments in which its metabolism was achieved by either methanogens or sulfate reducers by pathways established for dimethylsulfide (42). If methanol is a viable model of a C1 compound for which MeHg serves as an analog (38), then the ORDP ratio observed under purely methanogenic conditions would be 0.33, that is, 3 mol of CH4 and 1 mol of CO2 formed for every 4 mol of methanol metabolized (36). Sediments from the 4-to-8- and 8to-12-cm sections assayed at FC actually displayed ORDP ratios of ;0.38 (Fig. 4B), which approached this theoretical value for methanol. Under conditions of sulfate reduction or of respiration of other anaerobic electron acceptors, 14CO2 would be the only product and the ORDP ratio would be infinite. In

TABLE 3. Effects of molybdate on levels of 14CH4 and 14CO2 production from [14C]MeHg (100 nCi) added to undiluted sediments from Lahontan Reservoir and Fort Churchill Production level (nCi)a Site and conditions

FC, no additions FC, plus molybdate LR, no additions LR, plus molybdate

14

CH4

53.2 6 4.5 76.8 6 7.7 20.0 6 2.9 26.6 6 4.9

14

CO2

47.4 6 6.2 18.0 6 2.5 29.2 6 4.3 17.5 6 3.2

a Results represent the acidified endpoint values determined after 12 days of static incubation. b The values represent the means 6 SDs for three sediment samples.

VOL. 61, 1995

FIG. 6. Time courses of [14C]MeHg (0.44 mCi) demethylation during incubation of sediment slurries from Fort Churchill with no additions (A), 2 mM sulfate (B), and 20 mM BES (C). The symbols represent the means for three samples, and the bars indicate 61 SD. An absence of bars indicates that the error margin was smaller than the symbols. Symbols: Ç, CH4; E, 14CH4; å, CO2; F, 14 CO2. The arrows indicate the times of addition of acid.

environments where both methanogenesis and sulfate reduction occur, the ORDP ratio should fall to some numerical value in between. This result occurs when [14C]methanol is metabolized in marine sediments (27). Given the infinite range of values possible for the ORDP ratio, however, assignment of relative degrees of involvement of sulfate reduction versus methanogenesis is a futile exercise, even when modified along the lines of ‘‘respiratory indices’’ used in studies of methanogenesis in sediments (56). This difficulty arises because this line of reasoning ignores the possibility of 14CH4 production from [14C]MeHg being carried out by nonmethanogens, which could include detoxification reactions by certain sulfate reducers (5) as well as by anaerobic sediment organisms possessing the organomercurial lyase enzymes. The results from the laboratory sediment incubations generally reinforce the above conclusion with regard to the involvement of sulfate reduction and methanogenesis in oxidative demethylation. Sediments from both LR and FC sites exhibited depressed ORDP ratios when inhibited with molyb-


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date, and overall demethylation shifted to production of 14CH4 rather than 14CO2 (Table 3). This phenomenon was observed with freshwater sediment slurries with active demethylating populations of sulfate reducers and methanogens (38). Likewise, the addition of sulfate to sediment slurries from FC elevated 14CO2 production over unamended controls (Fig. 6A and B). These types of shifts in end product gases are typical of anaerobic systems in which methanogenesis and sulfate reduction are manipulated either by inhibition with molybdate (37) or by amendment with sulfate (56). BES totally inhibited production of 14CH4 and caused a 95% inhibition of CH4 production by sediment slurries (Fig. 6C). However, the appearance of a large quantity of 14CH4 after biological activity that was stopped by acidification is puzzling and indicates a chemical disruption of the carbon-mercury bond or perhaps some interaction with BES and [14C]MeHg under acid conditions. We did not observe the occurrence of this phenomenon in any of our other samples. Our studies employing [14C]MeHg have one caveat in that the addition of this isotope to sediments raised the MeHg pore water concentration to ;12 mM, a value probably much higher than actual concentrations. Hence, the reported rates for oxidative demethylation in Table 2 must be viewed as demethylation potentials and are valid only at these high concentrations. Indeed, the question of whether oxidative demethylation actually occurs at nanomolar levels of MeHg is a valid criticism of this work and cannot be addressed by the techniques we employed in this study. Values of MeHg for the specific reaches of the Carson River system which we investigated have not been reported, although concentrations of as high as 0.032 nM were detected in surface waters (8) while sediments from the contaminated regions contain ;8 ng of MeHg per g (dry weight) (24), a value which would extrapolate to ;20 nM, assuming a porosity of 0.9 and a sediment dry weight density equivalent to that of quartz (2.4 g/cm3). Hence, our employment of [14C]MeHg may have raised the pool size of MeHg by 500-fold. The caveat of demethylation potentials is a consequence of employing 14C in the study of the kinetics of this phenomenon or its relation to measured rates of mercury methylation in the same system (28, 43, 50). Indeed, mercury methylation had integrated rates for the top 16 cm of the FC sediment column of 0.0022 6 0.0005 mmol m22 day21 (n 5 3), while for the top 8 cm of the GV site the rate was 0.014 6 0.006 mmol m22 day21. The corresponding demethylation measurements were 212 6 24 (FC) and 27 6 11 (GV) mmol m22 day21 (n 5 3). Methylation occurred at a much lower rate in LR sediment than at GV or FC. Thus, the most active methylating sample (GV) was collected at the pristine site where the lowest demethylating rate also occurred, whereas methylation was lower at the contaminated FC site where demethylation was the greatest. This result suggested a reduced potential for MeHg accumulation in the anoxic sediments of the FC site. The point of our investigation, however, was not to assess relative rates of methylation versus demethylation but to determine if oxidative demethylation was a potentially significant process in the cycling of mercury in a contaminated environment and as a secondary point to qualitatively screen the sediments to determine if they also had the ability to methylate inorganic mercury. Clearly, the potential for both processes occurring in these sediments exists, and this phenomenon has been observed by many other workers. The fact that sulfate addition enhanced production of 14CO2 from [14C]MeHg in FC sediment slurries (Fig. 6B) while sulfate additions to freshwater lake sediments enhanced production of MeHg from added HgCl2 (22) underscores a need to put the phenomenon

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OREMLAND ET AL.


of oxidative MeHg demethylation in the context of working at realistic substrate concentrations in conjunction with concurrent assays of mercury methylation. Hence, the currently prevailing paradigm that sediments represent the site of net mercury methylation may have to be reexamined in light of the phenomenon of oxidative demethylation. The recent report of the availability of high-sp.-act. 203Hg21 (23) may afford an opportunity to pursue this course, especially if a high-sp.-act. synthesis of Me203Hg can be achieved. Such a tool may also allow the determination of the fate of the Hg21 after MeHg undergoes oxidative demethylation in anoxic sediments. In addition, the detection of substantial oxidative demethylation at the GV site where sulfate reduction and methanogenesis rates were low implies that respiratory anaerobes other than sulfate reducers and methanogens [e.g., denitrifiers and Fe(III) and Mn(IV) reducers] may also carry out oxidative demethylation.

24. 25.

ACKNOWLEDGMENTS

26.

We are grateful to C. Culbertson, J. Guidetti, V. Nelms, K. Reed, and J. Schaefer for technical assistance; to R. Hoffman, M. Lico, and H. Bevans of the U.S. Geological Survey (USGS) District Office in Carson City for logistical support and discussions; and to G. Taylor for consultation. We thank C. Gilmour and D. Krabbenhoft for their critical reviews of the manuscript. This work was supported by EPA grant DW14936864-01-1, EPA grant DW14936802-01-0, and the USGS National Water Quality Assessment Program.

18. 19. 20. 21. 22. 23.

27. 28. 29. 30.

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