Mercury Methylation in the Epilithon of Boreal Shield Aquatic ...

Environ. Sci. Technol. 2006, 40, 1540-1546

Mercury Methylation in the Epilithon of Boreal Shield Aquatic Ecosystems M EÄ L A N I E D E S R O S I E R S , † D O L O R S P L A N A S , * ,† A N D ALFONSO MUCCI‡ GEÄ OTOP/UQAM/McGill Universite´ du Que´bec a` Montreál, C. P. 8888, Succursale Centre Ville, Montreál, Que´bec, Canada H3C 3P8

Methylation rates by periphyton growing on the rocky shore of a remote boreal shield lake were measured over diurnal cycles at temperatures representative of summer and fall conditions. The measurements were carried out in vitro with natural communities grown on artificial Teflon substrates submerged along the lake’s shore for 1-2 years. At temperatures above 20 °C, epilithon Hg methylation rates were fast and reached a steady state within 12 h upon exposure to 2 ng L-1 of inorganic mercury. A variety of inhibitors were used to identify which microorganisms in the epilithic biofilm are responsible for the methylation. The addition of molybdate, which is believed to suppress the activity of sulfate-reducing bacteria, decreased methylmercury production rates by 60% in both light and dark experiments. The prokaryote inhibitor chloramphenicol reduced the methylation rate by 40% only during dark periods whereas an algal inhibitor (DCMU), which suppresses photosynthesis, decreased the methylation rate by 60% during light periods. Results of this study reveal that epilithon communities may be a significant source of MeHg to higher aquatic organisms in lakes and that the integrity of the epilithic biofilm is important for its ability to methylate Hg.

Introduction In the boreal forest, the soil is rich in organic matter (1) and, thus, Hg deposited from the atmosphere is readily adsorbed within the superficial soil horizons (i.e., O, A, and B) and bound to humic acid functional groups (2, 3). Because organic matter in boreal forest soils decomposes slowly, these watersheds serve as large reservoirs of Hg that can ultimately be exported to aquatic systems by surface and subsurface runoff (4, 5). Mercury methylation is very slow in forested soils (6, 7), and Hg delivered to aquatic systems from the watersheds or by atmospheric deposition is mainly inorganic (i.e., Hg(0) or Hg(II)). Part of this Hg is methylated in the aquatic environment (8) and the methylmercury (MeHg) transferred and biomagnified through the food web (9, 10). In many remote boreal shield lakes, isolated from any identifiable Hg point sources and without a history of human disturbance, MeHg concentrations in predatory fish are often higher than the advisory limit for human consumption of 0.5 ppm (11, 12). Despite a fair knowledge of the sources and * Corresponding author phone: (514) 987-3000 ext (6187); fax: (514) 987-3635; e-mail: [email protected]. † GE Ä OTOP/UQAM/McGill Universite´ du Que´bec a` Montreál. ‡ Present address: Department of Earth and Planetary Sciences, McGill University, 3450 Universite´, Montreál, Que´bec, Canada H3A 2A7. 1540

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speciation of the mercury delivered to aquatic environments, the locus of Hg methylation remains contentious. In lakes, it is generally admitted that Hg methylation takes place in deep (13-15) and littoral sediments (15, 16) and is ascribed to the activity of anoxic bacteria, mainly to sulfatereducing bacteria (SRB) (17, 18). On the rocky shores of Canadian shield lakes, MeHg was detected in the periphyton biofilm (epilithon) (19). Given that periphyton biofilm grow in the littoral zone, where they retain and recycle elements such as Hg delivered from the watershed via runoff, we propose that these communities may be important Hg methylators. To our knowledge, few studies have demonstrated the ability of periphyton, growing on submerged or floating macrophytes (epiphytes), to methylate Hg (20-22). The ability of periphyton biofilm to methylate Hg is not surprising given their community structure. Periphyton is constituted of a mixture of algae, bacteria, fungi, microinvertebrates, and detritus (23, 24). The periphyton biofilm is a highly dynamic microhabitat more or less isolated from the surrounding water by a microboundary layer, and thus, within the biofilm, strong vertical spatial and temporal (daynight) redox gradients can develop, as revealed by in-situ microelectrode measurements (25). The reducing microenvironment may support SRB or other microorganisms capable of Hg methylation. In addition, periphytic algae may produce photosynthesis byproducts (e.g., reductants) that promote redox reactions in the medium and may also excrete organic substrates (26) that fuel bacterial growth as well as stimulate bacterial methylation. In boreal Canadian shield lakes, the littoral zone is the main feeding habitat for many fish (27, 28) and macroinvertebrates, grazers, or detritivors. We propose that, in boreal shield lakes where fish have been found to have high MeHg concentrations, periphyton may play a role in Hg methylation and serve as an important vector of MeHg to higher organisms. Since, to our knowledge, Hg methylation by periphyton, living on rocky substrates in boreal lakes, has never been documented, we initiated a study to evaluate their methylation potential in an oligotrophic boreal shield lake. Hg methylation rates were measured over a 48-h period, through two day/night diurnal cycles and at different temperatures corresponding to the ice-free season. We measured methylation rates by the whole epilithon community, in the presence and absence of inhibitors specific to SRB, prokaryotes other than SRB, and algae, to determine which microorganisms contribute to the Hg methylation.

Materials and Methods Epilithon Field Sampling. Epilithon communities used in laboratory determinations of Hg methylation rates were grown in situ on artificial substrates in a boreal shield lake, Lake Croche (0.19 km2; 45°59′N, 74°01′W). A total of 12 Teflon mesh disks (70 µm mesh size; 9.6 cm2 surface area) were inserted in four paired machined Plexiglas plates mounted at 45 degrees to each other and secured to the arms of a PVC cross. The structure was immersed in the lake and anchored with a clay brick, whereas a floating Nylon rope tied to the top of the cross facilitated its recovery (29). The substrates were submerged near shore at 1 m depth for 1-2 years so that a community comparable (i.e., biomass, chlorophyll-a (Chl a), community structure and metabolism) to the natural rock biofilm would colonize them (30). Lake Croche is an oligotrophic lake with low nutrient, sulfate, and phytoplankton biomass concentrations (Table 1). The Teflon mesh is an inert substrate that does not absorb Hg and allows chemical exchange between the colonizing community and its sur10.1021/es0508828 CCC: $33.50

 2006 American Chemical Society Published on Web 01/31/2006

TABLE 1. Water Chemical and Biological Properties of the Oligotrophic Boreal Study Lake, Lake Croche tot. phosphorus (TP; µg L-1)a tot. nitrogen (TN; µg L-1)a nitrate (NO3; µg L-1)b colored dissolved organic carbon (CDOC; mg L-1) sulfate (SO42-; mg L-1)b phytoplankton biomass (Chl a; µg L-1)a a Data from Carignan et al. (31). communication)

b

3.8 ( 0.8 211 ( 10 22 ( 3 5.3 ( 0.3 1.31 ( 0.01 1.4 ( 1.0

Data from R. Carignan (personal

rounding environment. The use of such artificial substrates minimizes perturbations of the community structure during field sampling and laboratory manipulations. The integrity of the community is essential to avoid changes in matrix structure and redox zonation within the biofilm. Upon recovery in the field, the colonized mesh disks were removed from the supporting structure and transferred to 60-mL (clear, acid-washed, Nanopure rinsed) polycarbonate bottles containing 10 mL of filtered lake water (0.2 µm Whatman polycarbonate membrane filters). The average biomass carried by each substrate was on the order of 0.6 g wet weight (WW). The polycarbonate bottles were transported immediately to the laboratory and maintained at the lake temperature. To minimize loss and epilithon manipulations, the incubations were carried out in the sampling bottles. Polycarbonate bottles were used because this material does not alter visible light transmission and absorbs minimal amounts of Hg and MeHg (21). In addition, three epilithon artificial substrates were sampled for each of the biomass (Chl a), dry weight (DW), ash free dry weight (AFDW), and background Hg (THg, MeHg) measurements. Laboratory incubations of Hg-spiked samples were always started within 2 h of field sampling. Methylation Incubation Design. Once in the laboratory, the volume of the polycarbonate sample bottles was brought up to 60 mL with filtered lake water (0.2 µm), leaving a 15 mL headspace. The solution was then spiked with 203HgCl2 or 194HgNO3 (Oak Ridge National Laboratory) at specific activities of 7.296 and 3.69 kBq mg-1 respectively. The spiked water solution was allowed to sit for 12 h so the speciation of the added Hg could equilibrate to near natural conditions. The final concentration of the radioactive tracer was ∼2 ng L-1 (or 115 pg/60 mL) corresponding to an exposure of 3 ng Hg g-1 DW of epilithon. Methylation rates measured at 20 °C using the two individual radioisotopes (203Hg and 194Hg) were not significantly different (two-way ANOVA; p > 0.05). The incubations were carried out in a thermostated water bath at the following seasonal lake temperatures: 25, 20, and 15 °C ((1 °C), corresponding to respectively July-August, September, and October. A photoperiod cycle (12 h light/12 h dark) was achieved with an artificial daylight lamp (MH175, 175 W, metal halide lamp; General Electric Inc.), simulating the complete visible light spectrum (400-800 nm). The lamp does not emit in the UV range, but in Lake Croche, a DOCcolored lake, less than 1% of UVA and/or UVB penetrate to a depth of 1 m (32). The light intensity was set at 700 µE m-2 s-1, corresponding to the incident light at which the artificial substrates were exposed in the lake at 1 m depth during the study period (762 ( 370 µE m-2 s-1). Every 12 h, at the end of the light or dark periods, three replicates bottles were sampled, over two diurnal cycles (i.e., 48 h). During the incubations, periphyton total and algal biomass did not vary significantly (T-test; p > 0.05). MeHg was extracted from the epilithon using the multiple back-extraction protocol described by Gilmour and Riendel (33). Each sample (i.e., Teflon disk) was placed in a 50 mL Teflon tube, and reagents were

added sequentially following vortex mixing and centrifugation (250 rpm; 30 min) after each extraction. The final toluene extract was transferred to a glass scintillation vial. This extraction method was tested on a MeHg standard (DORM1; 0.73 ( 0.06 mg Hg kg-1; National Research Council of Canada (NRC)) and yielded a 93 ( 7% (n ) 6) recovery. Unlike the KOH/methanol (34) extraction method, this protocol does not carry over inorganic Hg (i.e., Hg0, Hg(II)) in the final extract. MeHg concentrations in the original, unamended epilithon were measured by cold-vapor atomic fluorescence spectroscopy (CVFAS; 19, 34) following its ethylation and thermal decomposition. Radioisotope activities (DPM; decay/ min) were counted with a highly sensitive Germanium Well detector γ counter (Canberra model 2000) whose counting geometry approaches 4π, a linear quenching between 0.1 and 6.1 pCi (r2 ) 0.98; p < 0,0001), and a counting efficiency of 77 ( 3% (n ) 11) for radioactive 194Hg and 76 ( 4% (n ) 7) for 203Hg. The detection limit of Hg methylation was lower than 0.01% of the total Hg (or 10-5 DPM). The counting time varied between 4 h and 1 to 5 days depending on the Me203,194Hg concentrations (10-1, 10-3, or 10-5 DPM, respectively). The inhibitor experiments were carried out in mid to late summer when temperatures were between 20 and 25 °C. Sodium molybdate (20 mM), chloramphenicol (0.2 mM), and ∆3-(3,4-dichlorophenyl)-1,1 dimethyl urea (10 µM; DCMU) were used as inhibitors of SRB, broad-spectrum prokaryotic bacteria, and algae, respectively. Since no significant differences were observed between the inhibition measurements carried out at 20 °C and 25 °C (two-way ANOVA; p > 0.05; data not shown), results of these incubations were combined to increase the number of observations and, hence, the statistical power of interinhibitor comparisons. For each set of measurements, we used acid-killed controls (1 mL of 4 N HCl), carried through the same conditions and protocols (i.e., growth, exposure, and analytical) as the experimental substrates, to assess the contribution of abiotic methylation. The algal biomass (Chl a) decreased from 34.8 ( 1.0 to 2.2 ( 0.3 µg/bottles upon acidification (p < 0.0001), reflecting the efficiency of the treatment. In our acid-killed blanks, the formation of radioactive MeHg was always under the detection limit ( 0.05). These biomass concentrations VOL. 40, NO. 5, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Epilithon Biomass (Chl a, DW, and AFDW; Means ( Standard Error) and Background Mercury Concentrations (THg and MeHg) in Lake Croche before Each Set of Experiments (n ) 3) season (temp, °C) July-August (25) September (20) October (15) 47.9 ( 4.5 46 ( 3 23 ( 2 88 ( 2 3.61 ( 0.08 4.1

Chl a (mg m-2) DW (g m-2) AFDW (g m-2) THg (ng g-1 of DW) MeHg (ng g-1 of DW) MeHg/THg × 100 (%)

41.4 ( 0.6 51 ( 6 27 ( 5 116 ( 5 2.39 ( 0.19 2.1

44.4 ( 3.6 43 ( 4 15 ( 4 229 ( 4 1.99 ( 0.10 0.9

FIGURE 1. Kinetics of Hg methylation (means ( standard error) by epilithon at different temperatures. are similar to those we determined in oligotrophic, unperturbed boreal shield lakes (19). The mean Hg concentrations in the epilithon biofilm growing on artificial substrates were also within the range observed in other unperturbed boreal lakes (19) but varied significantly between sampling periods. Whereas THg concentrations in epilithon increased, MeHg levels decreased with decreasing in-lake temperatures (oneway ANOVA; p < 0.001; Table 2). Methylation Rate Estimates. Results of the Hg methylation experiments are presented in Figure 1 as the percent ratio of the activity (or concentration) of the radioactive MeHg produced to the amount of radioactive 203,194Hg(II) added to the system ([Me203,194Hg]/[203,194THg] × 100) as a function of time. At 20 and 25 °C, the ratio levels off and remains constant after 12 h. This result can be interpreted as either an inhibition of the metabolic activity as the MeHg concentration increases in the system or, as is more commonly proposed, a steady state determined by the relative rates of the methylation and demethylation reactions (35). To compare results of our incubations with those reported in the literature, we assume that the kinetics can be represented by a pseudo-first-order, reversible reaction written as follows: 203,194

kr

Hg(II) w\ x Me203,194Hg k f

Given that the initial Me203,194Hg concentration is small, as would be the rate of the reverse reaction, the initial stage of the reaction can be modeled as a pseudo-first-order, irreversible reaction: 203,194

kf

Hg(II) 98 Me203,194Hg

Here the rate, R, is given by R ) d[Me203,194Hg]/dt ) kf[203,194Hg(II)], where kf is the pseudo-first-order rate constant for the methylation reaction. A plot of ln(1 + ([Me203,194Hg]/ [203,194Hg])) as a function of time in the initial stages of the 1542

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reaction will yield a straight line whose slope is equal to the rate constant for the methylation reaction. All the data from the 15 °C experiment, including the origin (i.e., [Me203,194Hg] ) 0 at t ) 0), define this line (r2 ) 0.975). At 20 and 25 °C, the steady state was reached by the time of the first measurement (i.e., 12 h), and thus, the line that represents the methylation-dominated portion of the reaction can only be defined by two points (i.e., the origin and the first data points). The rate constant derived from this slope is therefore a minimal value. The estimated rate constants at the three experimental temperatures are presented in Table 3. The MeHg production (([Me203,194Hg]/[203,194Hg])([Hg]ambient/ incubation time)) and the specific methylation rates were normalized to the growth surface area (m2) (36) or to the dry weight of epilithon. Methylation Rates. The Hg methylation rates measured in epilithon in this study are faster than rates typically measured in sediments or in biofilm growing on roots of macrophytes (21, 22). The net Hg methylation rates measured in vitro in sediments generally increase rapidly over the first 24 h, slow thereafter, and stabilize after 4-8 days (37-39), whereas methylation rates measured in biofilm roots are usually very slow and steady state is commonly reached on day 5 (40). The specific rate constants derived in this study were not significantly different between 20 and 25 °C (t-test; p > 0.05) and were significantly higher than the one measured at 15 °C (Table 3; one-way ANOVA; p ) 0.0007). A temperature dependence of the Hg methylation rates was reported previously from studies carried out using sediments from remote Canadian shield lakes (41) and biofilm growing on free-floating macrophyte roots in tropical waters (42) as well as in other lakes or river sediments (14, 43). In tropical regions, the highest methylation rates were measured at ≈30 °C in sediments or macrophytes biofilm roots (20, 42), but rates decreased significantly below 10 °C (42). A sharp decrease of the methylation rates coinciding with the fall lake turnover was reported in the littoral and deep sediments of a temperate Northern Wisconsin oligotrophic lake (14). In our study, the 26-fold decrease in the methylation rate between 20 and 25 °C and 15 °C also coincides with the beginning of the fall turnover in Lake Croche. The lower methylation rates at temperatures below 15 °C could result from a lower microbial metabolism. The methylation ratio estimated in this study at the higher temperatures (i.e., 20 and 25 °C) is comparable to the specific methylation rates observed, at similar Hg exposures, in the epiphytes of the least eutrophic Everglades wetlands (21). Influence of Inhibitors on Methylation Rates. All the inhibitors tested in this study significantly slowed (by 4060%) the methylation rates measured at either 20 or 25 °C (Figure 2). The extent of inhibition varied according to the diurnal cycle, whereas no significant difference in methylation rates was observed between day and night controls in the absence of inhibitors (T-test; p > 0.05). DCMU additions had no effect on methylation rates during dark periods but decreased the rates by 60% during light periods (Figure 2). In contrast, the addition of chloramphenicol inhibited methylation rates by 40% during the dark periods but was ineffective during the light periods. Finally, molybdate was equally effective during light and dark periods, decreasing methylation rates by 60% (Figure 2). The effect of these inhibitors on methylation rates by epilithon of a boreal lake reveals that microorganisms other than SRB may contribute directly or indirectly to inorganic Hg methylation. To our knowledge, no other study has ever considered the role of epilithon algae on Hg methylation in the littoral zone of lakes. The potential contribution or influence of algae on Hg methylation is revealed by our observations of the inhibitory effect of DCMU on methylation rates during the

TABLE 3. Methylation Rate Constant (kf; Means ( Standard Error), Epilithon Maximum Hg Methylation (%), MeHg Production (ng h-1), Areal MeHg Production (ng h-1 m-2), and Specific MeHg Production on a Dry Weight Basis (ng h-1 g-1DW) at 25, 20, and 15 °C for a 2 ng L-1 Spike temp (°C) param

25

20

15

kf (10-4 h-1) Hg methylation (%) MeHg productn (ng h-1) areal MeHg productn (ng m-2 h-1) specific MeHg productn (ng g-1of DW h-1)

5.9 ( 0.9 0.74 ( 0.13 0.051 ( 0.008 68 ( 10 1.7 ( 0.2

4.4 ( 0.6 0.66 ( 0.06 0.051 ( 0.007 51 ( 7 1.0 ( 0.2

0.16 ( 0.04 0.04 ( 0.016 0.004 ( 0.001 4.2 ( 0.8 0.10 ( 0.02

FIGURE 2. Mean percentage (%) of inhibition of methylation rates (means ( standard error) in the presence of a photosynthesis inhibitor (DCMU), a bacterial inhibitor (chloramphenicol), and a SRB inhibitor (molybdate): filled/black columns ) dark period (oneway ANOVA p ) 0.0017); white/open columns ) light period (oneway ANOVA p ) 0.0009). Double asterisks indicate significantly different than the control. light period (60%; Figure 2). DCMU is a specific inhibitor in photosystem II as it blocks the electron transport in the photosynthesis chain reaction (44). The influence of photosynthesis on Hg speciation/bioavailability has already been documented, as Hg(II) may serve as an electron acceptor within the cell (45, 46). This process could also promote Hg0 excretion outside the algal cell (45, 46). On the other hand, given that strong redox and pH gradients that develop within the first mm of the biofilm (25), oxidation of this Hg0 and its bioavailability to other microorganisms inside the biofilm may stimulate Hg methylation. It seems that the role of algae may not be limited to intracellular processes since photosynthetic byproducts excreted to the surroundings become a source of reductants for chemically and biologically mediated reactions (26). These exudates can not only bind to the metals but also promote redox reactions in the medium (26) and, thus, may contribute to inorganic Hg methylation. If photosynthesis is inhibited, as upon the addition of DCMU, the production of these reductants may be limited within and/or outside the cells and translate into a decrease in the Hg methylation rate during the light period. The epilithon community is composed of a large proportion of photoautotrophs, and their contribution to the methylation process may not only be limited to the production of reductants but also to the excretion of fresh carbon used by bacteria that are capable of methylating Hg. More studies will be required to elucidate the relationship between algal activity and Hg methylation. Our results contrast with those of Cleckner et al. (21), who reported that the addition of DCMU did not significantly decrease the Hg methylation rates by epiphytes in a wetland. It is important to note, however, that their measurements were not carried out through a day cycle but

in light and dark bottles and over very short periods of time (e4 h). Molybdate is regarded as a narrow-spectrum SRB-specific inhibitor that operates at the level of ATP sulfurylase (44). The presence of this inhibitor should rapidly deplete the ATP in SRB and trigger the death of the cells (44). The inhibition observed (60%; Figure 2) upon the addition of molybdate to our experimental system could be interpreted as evidence of the presence of SRB in our epilithon samples and their contribution to the methylation process. The SRB are believed to account for most of the Hg methylation in natural aquatic systems: about 100% in sediments (17, 18, 36, 37), 95% in the epiphytes in the Everglades’ wetlands (21), and nearly 80% in the biofilm roots of Eichhornia sp. in Brazil (40). In our study, however, molybdate additions partially inhibited the epilithon methylation rates. Given the molybdate inhibitor was equally effective during dark and light periods leads us to believe that the epilithon supports the SRB population and that the microenvironment of the biofilm allows these bacteria to be active not only at night, when photosynthesis is shutdown, but through the daily cycle. Conversely, the inhibition of methylation rates observed in the presence of molybdate may not strictly reflect a shutdown of SRB activity since this compound can modify the redox potential and promote Hg reduction in solution (47), decreasing the bioavailable pool of Hg(II). In addition, other artifacts related to changes in the speciation of Hg in the presence of molybdate cannot be excluded since the oxyanion can bind metals ions (e.g., As, Cu, Fe, Mg, Zn; 48). The significant decrease in methylation rate (40%; Figure 2) upon the addition of chloramphenicol during the dark period is striking. This antibiotic is a broad-spectrum prokaryotic inhibitor that suppresses ribosome protein production (49), but there is evidence that known strains of SRB methylators are not affected by it (21, 50). Consequently, the partial inhibition of Hg methylation by chloramphenicol entails that bacteria other than SRB may participate in the Hg methylation process in epilithon. Results of NO3 amendment and chloramphenicol addition experiments carried out in periphyton of the Everglades led the authors to propose that denitrifying bacteria may participate in Hg methylation processes (21). We do not believe this to be the case in our experiments because whereas bacterial denitrification in periphyton biofilm is more active at night (3-fold) when photosynthesis switches off and the oxygen concentration decreases (51), denitrifiers can also be active at the oxic/anoxic boundary within the biofilm during the light period (51). Accordingly, if denitrifiers play a role in Hg methylation processes by the epilithon biofilm in our experiments, at least some inhibition of the methylation should have been observed during the light period. Chloramphenicol is recognized as a very efficient inhibitor of methanogens, with a total inhibition at a concentration (50 mg L-1) lower than used in this study (52). Only few studies have investigated the role of methanogens in the Hg methylation/demethylation process (53, 54). An in vitro study (55) revealed a strong synergetic effect of methanogens and VOL. 40, NO. 5, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 4. Maximum Percentage of Hg Methylation Production/Unit of Biomass (DW) from Ecosystems Where Periphyton Methylation Rates Were Measured between 20 and 28 °C ref

site

20

tropical lake (Brazil)

21 22

Everglades (U.S.) flood plain tropical lake (Brazil)

40

tropical lake (Brazil)

this study

boreal lake (Canada)

a

compartment

DW (g)

% MeHg

% MeHg g-1 of DW

1 1 20a 2 2 1 1 0.04

10.4 1.2 0.1-10 34 0.6 22.7 5.8 0.87

10.4 1.2 0.005-0.5a 17 0.3 22.7 5.8 21.8

macrophyte root biofilm sediments macrophyte root biofilm macrophyte root biofilm sediments macrophyte root biofilm sediments epilithon

Periphyton biomass is in wet weight (g).

SRB on Hg methylation, resulting in a 10-fold increase in MeHg production. The contribution of methanogens to the methylation process is believed to be indirect, through the transfer of acetate (carbon source) produced during methanogenesis to SRB (55). That the synergetic effect of methanogens occurs only at night in epilithon biofilm is consistent with the redox zonation theory according to which electron acceptors (i.e., oxidants) are used sequentially in an order dictated by their free energy yield (56-58). The development of suboxic and anoxic environments as well as conditions conducive to Hg methylation have been described in marine (59) and freshwater sediments (14), as well as flooded forest soils (60). When photosynthesis is shut down at night, the redox potential in epilithon biofilm becomes more negative than during the day (25) as oxygen and other electron acceptors are consumed (56, 57) and methanogens may become active in the deeper layer of the epilithon biofilm. Importance of Epilithon Hg Methylation. The relative amount of inorganic Hg methylated by epilithon in our experiments (approximately 1%; Table 3) was generally lower than measured in biofilm from Brazilian tropical systems at temperatures between 20 and 25 °C (6.5-34%; 22, 40, 42). In the Brazilian methylation studies, 15 g WW of roots was typically used, compared to 0.6 g WW of epilithon in our study. Once normalized to the biomass weight, our Hg methylation rates (18.5 ( 3.25 and 16.62 ( 1.53% g-1 WW, at 20 and 25 °C, respectively) are similar or higher than those reported for the floating macrophyte roots and higher than rates measured in sediments (Table 4). Those studies were carried out in eutrophic systems with biofilm of floating macrophyte roots that often have a very rich organic matter layer and may support high bacterial activities (61). The discrepancy with our Hg methylation efficiencies (i.e., [MeHg]/[Hgtot]) could be ascribed to the different amounts of periphyton biomass used in the Hg methylation incubations. In conclusion, mercury methylation in epilithon may be an important source of MeHg for boreal lake biota, from grazers to fish. The relative contribution of Hg methylation by epilithon to the whole lake inventory will depend on the ratio of the surface area of the littoral zone to the total area of the lake, the dissolved inorganic Hg inputs, and the biomass of epilithon, the latter being related to the trophic status of the lake. Lake Croche is a small and low productivity lake, and thus, the estimated epilithon methylation capacity for this lake is probably conservative. In boreal lakes, transfer to macroinvertebrates from the periphyton leads to a 2- and 10-fold biomagnification in primary producers and secondary consumers, respectively (62). Given the importance of the littoral zone as a feeding habitat for fish, the abundance of macroinvertebrates which graze on periphyton, and the fact that epilithon is at the base of the littoral trophic food web in boreal lakes (as well as in tropical lakes; 27, 28, 63), the transfer of periphyton MeHg to fish via macroinvertebrates may be very important. In future studies of Hg methylation 1544

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in natural aquatic environments, the role of periphyton in regions without punctual Hg or MeHg sources should not be ignored as it could be a key vector of this metal to fish. Given the rapid establishment of steady state MeHg concentrations in our experimental system, future studies should also include shorter time-scale measurements as well as measurements of demethylation rates by periphyton to better quantify the importance of periphyton as a net source of MeHg. In addition, the bioavailability of inorganic Hg inside periphyton and the influence of inhibitors on Hg speciation should be addressed. This study also emphasizes that the integrity of the epilithic biofilm may be critical in determining its ability to methylate Hg. Whereas the inhibitor experiments provided some insights into the interplay between the microorganisms that make up the periphyton matrix, their identification and specific roles need to be confirmed by further studies, through the use of taxonomic and/or genetically tools.

Acknowledgments This project was made possible through funding by COMERN, the Collaborative Mercury Research Network, a research network established in 2001 with the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) to integrate Canadian research efforts toward a better understanding of processes governing mercury exchange and accumulation in a wide range of ecosystems of the North American continent. M.D. also benefited from postgraduate fellowships from FQRNT (Fonds Que´bećois de la Recherche sur la nature et les technologies) and COMERN. We address special thanks to Prof. R. Carignan (GRIL, Universite´ de Montreál, C.P. 6128, Montreál, Qc, Canada) for sharing his nitrate and sulfate Lake Croche data and to Jean-Claude Bonzongo for his valuable comments on the manuscript. Finally, we thank the anonymous journal reviewers and its associate editor, Dr. Suflita, for their critical and incisive comments.

Note Added after ASAP Publication Reference 19 was updated from that in the version published ASAP January 31, 2006. The revised version was published ASAP February 1, 2006.

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Received for review May 9, 2005. Revised manuscript received December 13, 2005. Accepted December 23, 2005. ES0508828