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 Université du Québec à Montréal,
C. P. 8888, Succursale Centre Ville, Montréal,
Québec, 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 Université du Québec à Montréal.
‡ Present address: Department of Earth and Planetary Sciences,
McGill University, 3450 Université, Montréal, Québec, Canada H3A
2A7.
1540
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 5, 2006
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 (<0.01% methylation of the total Hg).
Loss of Hg by volatilization to the headspace and/or by
sorption onto the container walls was tested during our
experiments. Polycarbonate bottles filled with Hg-spiked
filtered lake water were exposed to the same experimental
conditions, and the water was subsampled every 12 h. Less
than 10% of the 2 ng L-1 Hg spike was lost in the first 24 h,
but the loss increased to 20% after 48 h. In the inhibitor
experiments, Hg loss doubled upon the addition of molybdate
or chloramphenicol but was unaffected in the presence of
DCMU. A modification of the dissolved Hg(II) speciation
resulting from changes in solution chemistry (i.e, redox
potential, nature of the inhibitor) may explain these observations. The loss of Hg radioisotopes was accounted for in
the Hg methylation rate calculations.
Statistical Analysis. One-way and two-way ANOVA (general linear model, GLM) were performed, using JMP 4
statistical packages (SAS Institute Inc.). When significant
differences were found, a Dunnett’s one-tailed t-test for two
groups or a Tukey-Kramer HSD (honestly significant difference) test for multiple comparisons was applied.
Results and Discussion
Epilithon Substrate Characteristics. The mean epilithon
biomass during the three sampling periods (i.e. July-August,
September, and October) were 45 mg m-2, 47 g m-2, and 22
g m-2 for Chl a, DW, and AFDW, respectively (Table 2) and
were not significantly different between sampling periods
(one-way ANOVA; p > 0.05). These biomass concentrations
VOL. 40, NO. 5, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1541
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
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 5, 2006
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
9
1543
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
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 5, 2006
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
Québécois de la Recherche sur la nature et les technologies)
and COMERN. We address special thanks to Prof. R. Carignan
(GRIL, Université de Montréal, C.P. 6128, Montréal, 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.
Literature Cited
(1) Clayton, J. S.; Ehrkich, W. A.; Cann, D. B.; Day, J. H.; Marshall,
I. B. Soils of Canada; Research Branch Department of Agriculture: Ottawa, Canada, 1978; Vols. 1 and 2.
(2) Meili, M. The coupling of mercury and organic matter in the
biogeochemical cycle-towards a mechanistic model for the
boreal forest zone. Water, Air, Soil Pollut. 1991, 56, 333.
(3) Mierle, G.; Ingram, R. The role of humic substance in the
mobilisation of mercury from watershed. Water, Air, Soil Pollut.
1991, 56, 349.
(4) Aastrup, M.; Johnson, J.; Bringmark, E.; Iverfeldt, A. Occurrence
and transport of mercury within a small catchment area. Water,
Air, Soil Pollut. 1991, 56, 155.
(5) Bishop, K.; Lee, Y.-H.; Pettersson, C.; Allard, B. Terrestrial sources
of methylmercury in surface waters: The importance of the
riparian zone on the Svatberget catchment. Water, Air, Soil
Pollut. 1995, 80, 435.
(6) Matilainen, T.; Verta, M.; Korhonen, H.; Uusi-Rauva, A.; Niemi,
M. Behavior of mercury in soil profiles: impact of increased
precipitation, acidity, and fertilization on mercury methylation.
Water, Air, Soil Pollut. 2001, 125, 105.
(7) Hintelmann, H.; Harris, R.; Heyes, A.; Hurley, J. P.; Kelly, C. A.;
Krabbenhoft, D. P.; Lindberg, S.; Rudd, J. W. M.; Scott, K. J.; St.
Louis, V. L. Reactivity and mobility of new and old mercury
deposition in a boreal forest ecosystem during the first year of
the METAALICUS study. Environ. Sci. Technol. 2002, 36, 5034.
(8) St. Louis, V. L.; Rudd, J. W. M.; Kelly, C. A.; Hall, B. D.; Rolfhus,
K. R.; Scott, K. J.; Lindberg, S. E.; Dong, W. Importance of the
forest canopy to fluxes of methyl mercury and total mercury to
boreal ecosystems. Environ. Sci. Technol. 2001, 35, 3089.
(9) Watras, C. J.; Back, R. C.; Halvorsen, S.; Hudson, R. J. M.;
Morrison, K. A.; Wente, S. P. Bioaccumulation of mercury in
pelagic freshwater food webs. Sci. Total. Environ. 1998, 219,
183.
(10) Morel, F. M. M.; Kraepiel, A. M. L.; Amyot, M. The chemical
cycle and bioaccumulation of mercury. Annu. Rev. Ecol. Syst.
1998, 29, 543.
(11) Bodaly, R. A.; Rudd, J. W. M.; Fudge, R. J. P.; Kelly, C. A. Mercury
concentrations in fish related to size of remote Canadian shield
lakes. Can. J. Fish. Aquat. Sci. 1993, 50, 980.
(12) Garcia, E.; Carignan R. Mercury concentrations in fish from
forest harvesting and fire-impacted Canadian boreal lakes
compared using stable isotopes of nitrogen. Environ. Toxicol.
Chem. 2005, 24, 685.
(13) Furutani, A.; Rudd, J. W. M. Measurement of mercury methylation in lake water and sediment samples. Appl. Environ.
Microbiol. 1980, 40, 770.
(14) Korthals, E. T.; Winfrey, M. R. Seasonal and spatial variations
in mercury methylation and demethylation in an oligotrophic
lake. Appl. Environ. Microbiol. 1987, 53, 2397.
(15) Matilainen, T.; Verta, M.; Niemi, M.; Uusi-Rauva, A. Specific
rates of net methylmercury production in lake sediments. Water,
Air, Soil Pollut. 1991, 56, 595.
(16) Krabbenhoft, D. P.; Gilmour, C. C.; Benoit, J. M.; Babiarz, C. L.;
Andren, A. W.; Hurley, J. P. Methyl mercury dynamics in littoral
sediments of a temperate seepage lake. Can. J. Fish. Aquat. Sci.
1998, 55, 835.
(17) Kerry, A.; Welbourne, P. M.; Prucha, N.; Mierle, G. Mercury
methylation by sulphate-reducing bacteria from sediments of
an acid stressed lake. Water, Air, Soil Pollut. 1991, 56, 565.
(18) Gilmour, C. C.; Henry, E. A.; Mitchell, R. Sulfate stimulation of
mercury methylation in freshwater sediments. Environ. Sci.
Technol. 1992, 26, 2281.
(19) Desrosiers, M.; Planas, D.; Mucci, A. Total mercury and
methylmercury accumulation in periphyton of boreal shield
lakes: influence of watershed physiographic characteristics. Sci.
Total Environ. 2006, 355, 247.
(20) Guimarães, J. R. D.; Meili, M.; Malm, O.; Brito, E. Hg methylation
in sediments and floting meadows of a tropical lake in the
Pantanal flood plain, Brazil. Sci. Total Environ. 1998, 213, 165.
(21) Cleckner, L. B.; Gilmour, C. C.; Hurley, J. P.; Krabbenholf, D. P.
Mercury methylation in periphyton of the Florida Everglades.
Limnol. Oceanogr. 1999, 44, 1815.
(22) Guimarães, J. R. D.; Meili, M.; Hylander, L. D.; Silva, E.; Roulet,
M.; Mauro, J. B. N.; de Lemos, R. A. Mercury net methylation
in five tropical flood plain regions of Brazil: high in the root
zone of floating macrophyte mats but low in surface sediments
and flooded soils. Sci. Total Environ. 2000, 261, 99.
(23) Sand-Jensen, K. In Periphyton of freshwater: Proceeding of the
First International Workshop on periphyton of freshwater
ecosystems; Wetzel, R. G., Ed.; Dr. W. Junk Publisher: Boston,
MA, 1983; pp 63-71.
(24) Wetzel, R. G. Limnology, 2nd ed.; Saunders College Publishing
& Harcourt Brace College Publishers: New York, 1983.
(25) Jørgensen, B. B.; Revsbech, N. D.; Blackburn, T. H.; Cohen, Y.
Diurnal cycle of oxygen and sulfide microgradients and
microbial photosynthesis in a cyanobacterial mat sediment.
Appl. Environ. Microbiol. 1979, 38, 46.
(26) Price, N. M.; Morel, F. M. M. Role of extracellular enzymatic
reactions in natural waters. In Aquatic chemical kinetics; Stumm,
W., Ed.; Wiley: New York, 1990; p 235.
(27) France, R. Differentiation between littoral and pelagic food web
in lakes using stable isotopes. Limnol. Oceanogr. 1995, 40, 1310.
(28) Sierszen, M. E.; McDonald, M. E.; Jensen, D. A. Benthos as the
basis for arctic lake food webs. Aquat. Ecol. 2003, 37, 437.
(29) Groulx, S.-R. Réponse comparative des algues périphytiques
aux perturbations du bassin versant: coupe forestière et feu de
forêt. (Comparative responses of watershed disturbances in
periphytic algae: forest logging and wildfires). M.S. Thesis,
Université du Québec à Montréal, 2002.
(30) Hamelin, S. Comparaison du métabolisme et de la structure
des communautées périphytiques colonisant les roches et les
substrats artificiels de Nitex dans les lacs du bouclier canadien.
(Comparison of metabolism and community structure of
periphyton growing in natural rocky shore and Nitex artificial
substrata in boreal Canadian shield lakes). M.S. Thesis, Université du Québec à Montréal, 2002.
(31) Carignan, R.; Planas, D.; Vis, C. Planktonic production and
respiration in oligotrophic shield lakes. Limnol. Oceanogr. 2000,
45, 189.
(32) Huovinen, P. S.; Penttilä, H.; Soimasuo, M. R. Spectral attenuation of solar ultraviolet radiation in humic lakes in Central
Finland. Chemosphere 2003, 51, 205.
(33) Gilmour, C. C.; Riedel, G. S. Measurement of Hg methylation
in sediments using high specific-activity 203Hg and ambient
incubation. Water, Air, Soil Pollut. 1995, 80, 747.
(34) Bloom, N. Determination of picogram levels of methylmercury
by aqueous phase ethylation, followed by cryogenic gas chromatography with cold vapor atomic fluorescence detection. Can.
J. Fish. Aquat. Sci. 1989, 49, 1131.
(35) Hintelmann, H.; Keppel-Jones, K.; Evans, R. D. Constant of
mercury methylation and demethylation rates in sediments and
comparison of tracer and ambient mercury availability. Environ.
Toxicol. Chem. 2000, 19, 2204.
(36) Gilmour, C. C.; Riedel, G. S.; Ederington, M. C.; Bell, J. T.; Benoit,
J. M.; Gill, G. A.; Stordal, M. C. Methylmercury concentrations
and production rates across a trophic gradient in the northern
Everglades. Biogeochemistry 1998, 40, 327.
(37) Compeau, G.; Bartha, R. Sulfate-reducing bacteria: principal
methylators of mercury in anoxic estuarine sediment. Appl.
Environ. Microbiol. 1985, 50, 498.
(38) Hintelmann, H.; Evans, R. D.; Villeneuve, J. Y. Measurement of
mercury in sediments by using enriched stable mercury isotopes
combined with methylmercury determination by gas chromatography-inductively coupled plasma mass spectrometry. J.
Anal. Atom. Spectrom. 1995, 10, 619.
(39) Pak, K.-R.; Bartha, R. Mercury methylation and demethylation
in anoxic lake sediments and by strictly anaerobic bacteria.
Appl. Environ. Microbiol. 1998, 64, 1013.
(40) Mauro, J. B. N.; Guimarães, J. R. D.; Melamed, R. Mercury
methylation in macrophyte roots of a tropical lake. Water, Air,
Soil Pollut. 2001, 127, 271.
(41) Ramlal, P. S.; Kelly, C. A.; Rudd, J. W. M.; Furutani, A. Sites of
methyl mercury production in remote Canadian shield lakes.
Can. J. Fish. Aquat. Sci. 1993, 50, 972.
(42) Mauro, J. B. N.; Guimarães, J. R. D.; Melaned, R. Mercury
methylation in a tropical macrophyte: influence of abiotic
parameters. Appl. Organomet. Chem. 1999, 13, 631.
(43) Callister, S. M.; Winfrey, M. R. Microbial methylation of mercury
in upper Wisconsin river sediments. Water, Air, Soil Pollut. 1986,
29, 453.
(44) Oremland, R. S.; Capone, D. G. Use specific inhibitors in
biochemistry and microbial ecology. Adv. Microb. Ecol. 1988,
10, 285.
(45) Ben-Bassat, D.; Mayer, A. M. Light-induced Hg volatilization
and O2 evolution in Chlorella and the effect of DCMU and
methylamine. Physiol. Plant. 1978, 42, 33.
(46) Poulain, A. J.; Amyot, M.; Findlay, D.; Telor, S.; Barkay, T.;
Hintelmann, H. Biological and photochemical production of
dissolved gaseous mercury in a boreal lake. Limnol. Oceanogr.
2004, 49, 2265.
(47) Weber, J. H. Review of possible paths for abiotic methylation
of mercury(II) in the aquatic environment. Chemosphere 1993,
26, 2063.
(48) Porter, S. K.; Scheckel, K. G.; Impellitteri, C. A.; Ryan, J. A. Toxic
metals in the environment: thermodynamics considerations
for possible immobilization strategies for Pb, Cd, As, and Hg.
Crit. Rev. Environ. Sci. Technol. 2004, 34, 495.
(49) Vazquez, D. Inhibitors of protein synthesis. FEBS Lett. 1974, 40,
S63.
(50) Henry, E. The role of sulfate-reducing bacteria in environmental
mercury methylation. Ph.D. Dissertation, Harvard University,
1992.
(51) Nielsen, L. P.; Christensen, P. B.; Revsbech, N. P. Denitrification
and photosynthesis in stream sediment studied with microsensor and whole-core techniques. Limnol. Oceanogr. 1990, 35,
1135.
VOL. 40, NO. 5, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1545
(52) Sanz, J. L.; Rodriguez, N.; Amils, R. The action of antibiotics on
the anaerobic digestion process. Appl. Microbiol. Biotechnol.
1996, 46, 587.
(53) Landner, L. Biochemical model for the biological methylation
of mercury suggested from methylation studies in vitro with
Neurospora crassa. Nature 1971, 230, 452.
(54) Oremland, R. S.; Culbertson, C. W.; Winfrey, M. R. Methylmercury
decomposition in sediments and bacterial culture: Involvement
of methanogens and sulfate reducers in oxidative demethylation.
Appl. Environ. Microbiol. 1991, 57, 130.
(55) Pak, K. R.; Bartha, R. Mercury methylation by interspecies
hydrogen and acetate transfer between sulfidogens and methanogens. Appl. Environ. Microbiol. 1998, 64, 1987.
(56) Froelich, P. N.; Klinkhammer, G. P.; Bender, M. L.; Luedtre, N.
A.; Heath, G. R.; Cullen, D.; Dauphin, P.; Hammons, D. Early
oxidation of organic matter in pelagic sediments of the eastern
equatorial Atlantic: suboxic diagenesis. Geochim. Cosmochim.
Acta 1979, 43, 1075.
(57) Conrad, R.; Soil microorganisms as controllers of atmospheric
trace gases (H2, CO, CH4, OCS, N2O and NO). Microbiol. Rev.
1996, 60, 609.
(58) Jørgensen, B. B.; Boudreau, B. P., Diagenesis and sedimentwater exchange. In The Benthic Boundary Layer; Transport
processes and biogeochemistry; Boudreau, B. P., Jørgensen, B.
B., Eds.; Oxford University Press: Oxford, U.K., 2001; p 211.
1546
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 5, 2006
(59) Gagnon, C.; Pelletier, EÄ .; Mucci, A.; Fitzgerald, W. F. Diagenetic
behavior of methylmercury in organic-rich coastal sediments.
Limnol. Oceanogr. 1996, 41, 428.
(60) Mucci, A.; Lucotte, M.; Montgomery, S.; Plourde, Y.; Pichet, P.;
Van Tra, H. Mercury remobilization from flooded soils in a
hydroelectric reservoir of northern Quebec, La Grande-2: results
of a soil resuspension experiment. Can. J. Fish. Aquat. Sci. 1995,
52, 2507.
(61) Neiff, A. P.; Neiff, P. P.; Orfeo, O.; Carignan, R. Quantitative
importance of particulate matter retention by roots of Eichhornia
crassipes in the Parana flood plain. Aquat. Bot. 1994, 47, 213.
(62) Rask, M.; Metsälä, T.-R.; Salonen, K. Mercury in the food chains
of a small polyhumic forest lake in southern Finland. In Mercury
pollution: integration and synthesis; Watras, C. J., Huckabee,
J., Eds.; Lewis Publishers: Boca Raton, FL, 1994; p 409.
(63) Hecky, R.; Hesslein, R. Contributions of benthic algae to lake
food webs as revealed by stable isotope analysis. J. N. Am.
Benthol. Soc. 1995, 14, 631.
Received for review May 9, 2005. Revised manuscript received December 13, 2005. Accepted December 23, 2005.
ES0508828