Environ. Sci. Technol. 2007, 41, 7376-7382
Evidence for Elevated Production of
Methylmercury in Salt Marshes
J O Ã O C A N AÄ R I O , * M I G U E L C A E T A N O
C A R L O S V A L E A N D R U T E C E S AÄ R I O
Instituto Nacional de Recursos Biológicos (INRB/IPIMAR),
Av. de Brasilia, 1449-006 Lisboa, Portugal
Depth variations of total mercury (Hg) and methylmercury
(MeHg) concentrations were obtained in cores from
nonvegetated sediments, sediments colonized by Sarcocornia
fruticosa, Halimione portulacoides, and Spartina maritima
and below-ground biomass in three Portuguese estuaries.
Similar analyses were also performed on the above-ground
plant tissues. Concentrations in below-ground biomass
exceeded up to 9 (Hg) and 44 (MeHg) times the levels in
sediments. Mercury and MeHg in below-ground biomass
were up to 400 (Hg) and 4700 (MeHg) times higher than
those found in above-ground parts, indicating a weak upward
translocation. Methylmercury in colonized sediments
reached 18% of the total Hg, which was 70 times above
the maximum values found in nonvegetated sediments.
Concentrations of MeHg in vegetated sediments were not
related to plant type but were linearly proportional to
the total mercury levels. The analysis of below-ground
biomass at high depth resolution (2 cm) provided evidence
that Hg and MeHg were elevated. The higher enrichment
factors were found where the shifting of redox conditions
suggested high microbial activity. Mercury and MeHg in
below-ground tissues were a function of total levels in
sediments and again were not plant-specific. These results
suggest that the bioremediation of mercury-contaminated
sediments is likely to increase the formation of methylmercury.
Introduction
Marshes are considered ideal areas for the phytoremediation
of metals (1-3). The attractiveness for this low-cost option
comes from the relevance of halophytes in the circulation of
elements, immobilizing and storing them in below-ground
biomass and/or soil or in above-ground tissues (4).
Mercury studies on salt marshes in the past decades (58) showed high concentrations in the rhizosphere (9, 10),
translocation into the aerial parts of plants (11), release from
leaves during plant transpiration (12), and a minor incorporation into leaves via atmospheric mercury deposition (13).
Genetically engineered plants, which are more effective in
removing Hg and MeHg from soils and sediments to the
above-ground tissues than wild species, have been proposed
for phytoremediation of mercury-contaminated sites (3, 18).
However, in spite of the well-known toxicity of methylmercury (MeHg), the conversion of inorganic Hg into MeHg
in salt marshes is poorly documented. The MeHg concentration results from the balance between methylation and
demethylation processes (14). The higher methylation rates
* Corresponding author phone: + 351.21.3027000; fax: +351.
21.3015948; e-mail: [email protected].
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in the rhizosphere have been attributed to microorganisms
attached to roots and associated solids (7, 11). Besides the
availability of inorganic mercury and the specific microbial
community present (15), there are several other important
factors that influence biological methylation such as temperature, pH, salinity, organic carbon, and redox potential
(14, 16, 17).
This work reports the concentration profiles of Hg and
MeHg in sediments colonized by Sarcocornia fruticosa,
Halimione portulacoides, and Spartina maritima. We also
report Hg and MeHg in the respective nonvegetated sediments, as well as in below- and above-ground plant tissues.
Three salt marshes in Portugal with high, moderate, and low
Hg contamination were considered. On the basis of the highresolution concentration profiles, both in sediments and in
below-ground tissues, the stocks of Hg and MeHg in the
colonized and nonvegetated areas were calculated. To our
knowledge this is the first time that MeHg stocks in salt marsh
plants have been estimated.
Experimental Section
Environmental Settings. The study was carried out in
marshes of the Tagus, Sado, and Guadiana estuaries located
in the southwestern Iberian Peninsula (see Supporting
Information Figure SI-1). The upper sediment layers of the
Tagus marsh incorporate large quantities of mercury discharged by nearby industries and urban areas (19, 20). The
Sado estuary marsh is also located near an industrial area
but contains lower Hg concentrations in sediments (21). The
Guadiana marsh sediments contain low levels of anthropogenic mercury, although the Guadiana river crosses the
Iberian Pyrite Belt which has had mining and extraction of
metals since the Roman Age (22, 23). The three salt marshes
are inundated by the tide on a semidiurnal scale and
colonized by Sarcocornia fruticosa, Halimione portulacoides,
and Spartina maritima.
Field Work and Sample Preparation. Salt marsh plants
and sediment cores were sampled in summer 2006 from pure
stands of S. fruticosa (Sado and Guadiana), H. portulacoides
(Tagus and Sado), and S. maritima (Guadiana). Sediment
cores were collected from colonized and nonvegetated
areas at each marsh and sliced in layers of 2-5 cm sediment
thickness. Before slicing dissolved oxygen was measured
using a Diamond Electro-Tech needle electrode following
the method described in Caetano and Vale (24). Measurements were done within 5 min after collection in order to
avoid alterations from the air exposure. Plant above-ground
parts were removed at ground level and stored in plastic
bags. At the laboratory they were washed with ultrapure
Milli-Q water (18.2 MΩ) to remove dust and sediment and
were oven dried at 40 °C. The below-ground material of each
layer was separated from the sediment carefully under a flux
of Milli-Q water using a 212 µm mesh size sieve to remove
any adhering particulate matter. Sediments and roots were
oven dried at 40 °C and weighed to determine below-ground
biomass (25, 26). Sediment and biological samples were
homogenized with an agate mortar for chemical analysis.
Analysis of Sediments. Sediment water content was
estimated by weight loss at 105 °C. Total determinations of
Al, Fe, and Mn were performed by mineralization of the
sediment samples with a mixture of acids (HF, HNO3, and
HCl) according to the method described by Rantala and
Loring (27). Metal concentrations were obtained by flameAAS (Perkin-Elmer AAnalyst 100) using direct aspiration into
a N2O-acetylene flame (Al) or air-acetylene flame (Fe and
Mn). Mercury was determined by atomic absorption spec10.1021/es071078j CCC: $37.00
 2007 American Chemical Society
Published on Web 10/04/2007
trometry using a silicon UV diode detector LECO AMA-254
after pyrolysis of each sample in a combustion tube at
750 °C under an oxygen atmosphere and collection on a gold
amalgamator (28). Methylmercury was determined in dry
sediments by alkaline digestion (KOH/MeOH), preconcentration in dithizone/toluene solution, and quantification by
GC-ECD (29). Recoveries and the possible MeHg artifact
formation were evaluated by spiking several samples with
Hg(II) and MeHg standard solutions with different concentrations. Recoveries varied between 97% and 103%, and no
artifact MeHg formation was observed during our procedure.
In all our metal analysis, precision, expressed as relative
standard deviation (RSD) of four replicate samples, was less
then 4% (p < 0.05). International certified standards (MESS2, PACS-1, IAEA-405, and BCR-580) were used to ensure the
accuracy of our procedure. For all metals investigated,
obtained values were consistently within the ranges of
certified values (p < 0.05). Total sulfur and carbon sediment
content were measured in homogenized and dried sediments,
using a CHN Fissons NA 1500 analyzer. The calibration
standard used was sulfanilamide. Procedural blanks were
obtained by running several empty ashed tin capsules.
Organic carbon was estimated by the difference between
total carbon and inorganic carbon after heating samples at
450 °C during 2 h in order to remove the organic carbon
from the sediment. In all carbon and sulfur analysis, precision
expressed as RSD was less than 1% (p < 0.05) of five replicate
samples.
Analysis of Plant Tissues. Total mercury analysis in aboveand below-ground tissues was performed with the LECOAMA 254 equipment. For MeHg determinations, a recently
developed method was used (30). Dried tissues were digested
with a concentrated HBr (Merck suprapur) solution saturated
with CuSO4. Methylmercury in the digested solution was then
extracted in a dithizone/toluene solution preconcentrated
in a slightly alkaline H2S solution, back-extracted into toluene,
and quantified by GC-ECD. As for the sediment analysis,
recoveries and the possible MeHg artifact formation were
evaluated by spiking several samples with Hg(II) and MeHg
standard solutions with different concentrations. Recoveries
varied between 96% and 104% for all plant tissues investigated, and no artifact MeHg formation was observed. For all
the analysis, precision expressed as the relative standard
deviation of three replicate samples was less than 2.5% (p <
0.05). International certified standards CRM-60 (Lagarosiphon major, aquatic plant), CRM-61 (Plantihypnidium
riparioides, aquatic moss), and IAEA-140/TM (Fucus sea plant
homogenate) were used to ensure the accuracy of the
procedure. Mercury and MeHg concentrations were consistently within the ranges of certified values (p < 0.05).
Results and Discussion
Sediment Characteristics. Aluminum concentrations varied
within narrower intervals throughout the cores of Tagus and
Guadiana (8-11%) than those of Sado (5-10%). Since Al is
a proxy of clay fraction (31, 32), the lower Al percentage
registered in the cores of Sado suggest the presence of coarser
material, and the levels in Tagus and Guadiana suggest the
prevalence of fine particles. At each salt marsh the concentration profiles of Al were similar in colonized and nonvegetated areas. Organic carbon content in sediments from the
Sado varied in a broader range (0.23-5.0%) than those from
Tagus and Guadiana (1.3-2.5% and 0.72-2.3%, respectively).
The colonized sediments from Sado and Guadiana contained
significantly (p < 0.05) higher organic carbon than the
respective nonvegetated sediments, but no significant differences (p < 0.05) were observed in the sediments colonized
by the different plants at each marsh. Otherwise, organic
carbon in Tagus sediments does not differ significantly
between colonized and nonvegetated areas. Higher total
sulfur concentrations in sediments were found in Tagus
(0.02-2.7%) than in Sado (0.08-1.7%) and Guadiana (0.021.3%). The colonized sediments of Tagus and Guadiana
contained significantly (p < 0.05) higher sulfur concentrations
than the respective nonvegetated sediments, while an inverse
pattern was observed for Sado.
Below-Ground Biomass and Dissolved Oxygen. The
whole below-ground biomass was higher in the areas
colonized by H. portulacoides (4200 g m-2) and S. fruticosa
(2200 g m-2) of Sado than in areas of Tagus (H. portulacoides:
1400 g m-2) and Guadiana (S. martitima: 1020 g m-2; S.
fruticosa: 470 g m-2). The biomass varied with the depth:
in Sado the roots being concentrated in the first 24 cm, while
in Tagus and Guadiana they were distributed until 35 cm
depth. The difference on below-ground biomass and root
penetration depth usually reflects adaptive responses of
plants to the environment (33) like nutrients or the nature
of sediment particles. Figure SI-2 (see Supporting Information) shows the vertical profiles of below-ground biomass of
H. portulacoides and dissolved oxygen concentration in the
Tagus marsh that are illustrative of the conditions observed
in other marshes. The dissolved oxygen in the colonized
sediments was found until 35 cm depth contrasting with the
nonvegetated areas (<1 cm). Similar profiles of dissolved O2
concentrations have been measured in previous studies (34)
and interpreted as the delivery of oxygen by roots to the
surrounding sediments (24). It should be noted that the
presence of oxygen in the rooting sediments means that
delivery of O2 exceeds its consumption by the oxidation of
organic matter and other associated redox reactions.
Iron and Manganese. The Fe and Mn concentrations in
solid sediments were normalized to Al to correct the
considerable variation of grain size. Depth profiles of Fe/
Al and Mn/Al ratios were relatively uniform in the nonvegetated areas: Fe/Al ratios were 0.29 ( 0.07, 0.58 ( 0.06,
and 0.51 ( 0.03 and Mn(10-4)/Al ratios were 46 ( 3.9, 38 (
3.1, and 56 ( 9.8 for Tagus, Sado, and Guadiana, respectively.
The Fe/Al and Mn/Al ratios in colonized sediments exceeded
the values measured in the nonvegetated areas: the enrichment was 1.1-1.6 for Fe and 1.7-4.2 for Mn. These increases
result from the intense mobilization of Fe and Mn in the
vicinity of roots as O2 is released into anoxic sediments (34).
Previous studies reported the formation of iron plaques (3538) and Fe-oxide concretions (39, 40) around the roots, as
a consequence of the oxidation of iron sulfides and rapid
precipitation of Fe(III) (41).
Total Mercury in Sediments. The concentrations of total
Hg were more variable in sediments where roots are present
than in nonvegetated sediments at the three marshes: 0.502.37 µg g-1 and 0.77-1.92 µg g-1 (Tagus), 0.70-2.79 µg g-1
and 0.58-1.07 µg g-1 (Sado), and 0.27-1.07 µg g-1 and 0.161.29 µg g-1 (Guadiana). Elevated levels in colonized sediments
have been observed in previous works (8, 42-45) and are
interpreted as the result of Hg uptake by roots and subsequent
retention in the buried litter (44, 45), as well as incorporation
in the abundant organic matter accumulated in these
sediments (44).
Methylmercury in Sediments. The differences of MeHg
concentrations between colonized and nonvegetated sediments reached two orders of magnitude. The percentages of
MeHg in nonvegetated sediments were lower than 0.6% of
the total Hg, while in the sediments colonized by the three
plants the values were up to 18%. These differences reflect
a higher methylation in colonized sediments, which is
consistent with previous studies in other marshes (7, 8, 42)
and with values reported in soils planted with rice and tobacco
plants (18, 45).
Effect of Sediment Depth. Plotting the percentage of
MeHg (relative to the total Hg) with sediment depth (Figure
1a) allows for a better understanding of the effect of plant
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FIGURE 1. Relationships between MeHg proportion (% total Hg) and depth (a) and between MeHg (ng g-1) and Hg (µg g-1) concentrations
(b) in sediments colonized by H. portulacoides, S. fruticosa, and S. maritima in the Tagus, Sado, and Guadiana marshes.
roots on mercury methylation. It was observed that MeHg
in colonized sediments could be quantified to a depth of 27
cm in the presence of S. fruticosa and to a depth of 44 cm
in the presence of S. maritima and H. portulacoides. This is
in clear contrast with the thin MeHg layer (usually less than
5 cm) observed in the nonvegetated sediments. The network
of roots, distributed heterogeneously throughout the sediment, creates a correspondingly heterogeneous pattern of
sediment redox properties (34). This shifting of redox
conditions appears to be ideal for high microbial activity,
which when coupled with the reservoirs for bacterial
respiration (e.g., organic carbon and sulfate) favors Hg
methylation (14, 46). Consequently, the presence of high
MeHg concentrations at certain depths indicates that mercury
methylation should occur at preferential layers.
Effect of Plant Species. Furthermore, the percentages of
MeHg were higher in sediments colonized by H. portulacoides
in both Tagus and Sado marshes than in those occupied by
S. fruticosa and S. maritima (Figure 1a). The specific area of
roots may be the major reason for the observed differences.
In fact, for the same below-ground biomass, H. portulacoides
has a larger root surface area than the other two plant species,
which increases the root-sediment interface where redox
reactions and methylation occur. Roots of H. portulacoides
were thinner than the majority of S. maritima roots (47).
Effect of Total Hg in Sediments. Figure 1b presents the
relationships between the concentrations of MeHg and total
Hg in root-bearing sediments. The linear relationships
obtained for each marsh containing plants showed that the
slope for Guadiana (18 ( 2.3) is statistically different (p <
0.05) from those obtained for Tagus (244 ( 26) and Sado (181
( 22). These differences may be interpreted as a higher
methylation occurring in Tagus and Sado than in Guadiana
for similar increases of Hg concentration in sediments. The
higher methylation in Tagus and Sado marsh sediments may
result from the increased proportion of anthropogenic Hg in
sediments. Otherwise, at Guadiana Hg is mainly from
geogenic origin and presumably less available for methylation. In contrast, a similar relationship was not observed
in the nonvegetated sediments since adequate conditions
for methylation are predictably restricted to the topmost
layers (14, 46, 48-50). This is consistent with the lack of
correlations between MeHg and Hg in surface sediments
from each aquatic system (20). The complexity and interdependence among several variables influencing methylation
contribute to this variability.
Partitioning of Total Mercury in Plants. The mercury
concentrations in below-ground biomass of the three
analyzed plants (0.17-13 µg g-1) was 2 orders of magnitude
higher than the levels found in above-ground biomass
(0.018-0.22 µg g-1). The difference in below- and aboveground Hg levels suggests a poor translocation of mercury
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from roots to the above plant parts, which is consistent with
several studies examining the existence of a blocking mechanism in below-ground tissues of wild species (9, 10, 18, 42).
Nevertheless, Hg concentrations in above-ground parts of
H. portulacoides from Tagussthe polluted siteswere 1 order
of magnitude higher (0.12-0.22 µg g-1) than those at Sado
(0.033-0.076 µg g-1) and Guadiana (0.018-0.056 µg g-1).
Several researchers have observed that above-ground plant
tissue near to Hg-contaminated sites reflect the Hg release
to the atmosphere (51-53). Two explanations may be invoked
for the increased retention of Hg in leaves and stems of plants
from the Tagus marsh: sorption of Hg compounds from
contaminated water during the tidal inundation, and uptake
of gaseous Hg from the atmosphere. Assuming that air
emissions from industries are minor due to recent environmental regulations, uptake of Hg° from the contaminated
intertidal sediments may be the main source. This hypothesis
is supported by a previous field experiment in the Tagus
marsh that proved the release of gaseous Hg to the
atmosphere when sediments are exposed to solar radiation
(54).
Partitioning of Methylmercury in Plants. The MeHg
concentrations in above-ground biomass varied from 0.11
to 0.72 ng g-1, while the below-ground biomass levels reached
941 ng g-1. When comparing the MeHg levels in below- and
above-ground parts of the analyzed plants ([MeHg]blg/
[MeHg]abv on weight basis), the following enrichments were
obtained: 5-4700 times (Tagus), 32-1050 times (Sado), and
1-430 times (Guadiana). The observed partitioning of MeHg
is in agreement with the findings of other authors showing
that roots are accumulators of MeHg in various types of plants
(10, 42). However, such high enrichment factors are rarely
reported in the literature. The levels of MeHg in above-ground
biomass showed no significant differences between plants
and the studied marshes. The lack of difference between the
contaminated marsh (Tagus) and the other sites may be
attributed to the higher ability of MeHg to be translocated
from below-ground to above-ground plant parts.
Mercury and Methylmercury in Below-Ground Biomass.
The vertical profiles of Hg and MeHg concentrations in belowground biomass of H. portulacoides, S. fruticosa, and S.
maritima and in the respective rooting sediments, with a 2
to 5 cm depth resolution, are illustrated in Figure 2. Previous
studies on accumulation of mercury in roots have considered
the below-ground biomass as a whole (11), and therefore the
relationships with the sediment composition taking into
account its depth variation were not possible to establish.
Our results allow for the examination of how the accumulation of Hg and MeHg in below-ground biomass varies with
chemical zonation of the sediments with the depth. Clearly,
both Hg and MeHg were more concentrated in below-ground
biomass than in respective sediments of Tagus and Sado
FIGURE 2. Vertical profiles of Hg (µg g-1) and MeHg (ng g-1) concentrations in roots and corresponding sediments in H. portulacoides
(Tagus marsh), S. fruticosa (Sado marsh), and S. maritima (Guadiana marsh).
marshes, which are those containing a higher proportion of
anthropogenic Hg. At Guadiana the low Hg concentration
and its possible geogenic origin make this trend unclear at
all depths. These results do not clarify whether Hg species
are mainly bound to cell walls of root tissues or are
incorporated in the cell cytoplasm. According to recent works
(11, 45, 55) roots can effectively uptake Hg and MeHg by
sequestration into two classes of cysteine-rich peptides: the
metallothioneins and phytochelatins (55, 56). The mechanism
involves Hg binding with organic sulfur (R-SH) groups on
the cysteine residues in these peptides being glutathionated
and transported into vacuoles for long-term sequestration
(56, 57). The bacteria in the rhizosphere may also contribute
to Hg accumulation by complex processes involving the
production of heat-labile compounds in root exudates, which
enhance Hg accumulation in plants (10), the transformation
of Hg into MeHg more readily taken up into roots (58), and
the production of sulfate-transport protein in the root plasma
membrane (59).
Enrichment Factors. The capability of these plants to
accumulate mercury compounds from sediments was examined through the enrichment factor (EF), calculated as
the ratio between metal concentration on a weight basis in
roots and the respective sediment at each layer. The EF (Hg)
for Tagus and Sado increased with depth, reaching a
maximum 9 below the maximum oxygen layers, while for
Guadiana the EF did not vary significantly with depth and
was lower than 2. The differences in EFs appear to mirror the
amount of phytoavailable Hg in each marsh.
Depth Variation. The levels of both Hg and MeHg varied
with depth for all analyzed plants in the three salt marshes
(Figure 2), which indicates preferential layers of retention or
transfer of Hg and MeHg from sediments to below-ground
tissues. In Tagus and Sado marshes there is a tendency for
this uptake/retention enhancement with the depth. This
pattern may be related with the increase of Hg/MeHg
availability or with root efficiency to remove mercury from
sediments. Because the analyzed below-ground biomass
includes live and dead material we have not excluded the
possibility of passive association of Hg with litter (53, 60).
Several studies have shown that organic material derived
from plants may be a sink for mercury through passive
absorption or physiological mechanisms of microbes (6164). The increase of Hg and MeHg levels with depth was,
however, less clear in the Guadiana marsh, which may be
related to the much lower mercury levels in sediments.
Relation between Levels in Below-Ground Tissues and
Sediments. We hypothesize that accumulation of Hg and
MeHg in roots is related to sediment concentrations. To test
this hypothesis, concentrations of Hg species in biological
material were thus plotted against those in sediments. Two
separated plots were considered since the concentration
ranges in Guadiana are much narrower than those in Tagus
and Sado (Figure 3). A logarithmic relationship was found
for Hg in Tagus and Sado including H. portulacoides and S.
fruticosa. This indicates that Hg in below-ground tissues is
a function of the total levels in sediments, which appear to
reflect its phytoavailability, however not for a specific plant.
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FIGURE 3. Relationships between MeHg (ng g-1) and Hg (µg g-1) concentrations in roots and those in sediments for all analyzed plants
in the Tagus, Sado, and Guadiana marshes (O outliners).
Although a good relationship was found in Guadiana, a
much lower slope (slopeTagus+Sado ) 6.8 ( 0.1; slopeGuadiana )
0.14 ( 0.01) suggests that available Hg is only a minor fraction
of the total Hg. The ore mineral influence in the Guadiana
sediment composition may act as a barrier to the Hg uptake.
The MeHg concentrations in below-ground tissues tend to
increase linearly with the levels in sediments for Tagus/Sado
(four outliners excluded) and Guadiana (Figure 3), suggesting
that MeHg uptake by roots follows their production in
sediments. The uptake was found at two concentration
scales: <20 ng g-1 (Guadiana) and 10-125 ng g-1 (Tagus/
Sado). It was found that above 180 ng g-1 of MeHg in
sediments, the accumulated levels in below-ground biomass
were below the linear relationship. A possible explanation is
that MeHg concentration in sediments surpassed the threshold value, inducing plant stress and thus limiting the MeHg
uptake (55).
Stocks of Mercury and Methylmercury in Marshes. On
the basis of the results obtained for below-ground biomass,
sediment density, water content, and mercury concentration
in sediments and below-ground tissues, we estimated the
stock of Hg and MeHg (g m-2) in vegetated sediments. Stocks
were calculated for a 35 cm thick horizon that constitutes
the rooting zone. Due to the much lower mercury concentrations and low biomass, the small contribution of aboveground stocks were not considered in this study. Note that
due to the higher below-ground biomass of H. portulacoides,
stocks in the Sado (25 g m-2 of Hg and 1.2 g m-2 of MeHg)
exceeded the values calculated for the contaminated Tagus
(5.7 g m-2 and 0.6 g m-2). These elevated stocks were up to
102 (Hg) and 104 (MeHg) times higher than the values reported
by Canário et al. (20-65) in the surface layer (0-5 cm) of the
contaminated Tagus sediments (Hg ) 65 × 10-3 g m-2; MeHg
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) 59 - 81 × 10-6 g m-2) and for the soils contaminated by
chlor-alkali plant (Hg ) 66 - 150 × 10-3 g m-2) in Vosges,
France (66-67). Even stocks of Hg (0.3-0.4 g m-2) and MeHg
(9.7-25 × 10-3 g m-2) in the noncontaminated Guadiana
marsh were 10 and 1000 times higher than those found in
the contaminated Tagus estuary sediments. These results
emphasize the importance of considering stocks of Hg and
MeHg in plant-colonized areas for the budgets in aquatic
ecosystems containing marshes. Furthermore, the biotransformation of mercury to the toxic methylmercury in sediment
increases the toxicity of plant-colonized sediments, which
should be taken into account in the cost-benefit equation
of bioremediation projects for contaminated soils and
sediments.
Acknowledgments
The authors express their appreciation for the financial
support provided by the “Fundação para a Ciência e
Tecnologia” to J.C. (FCT Grant SFRH/BPD/26324/2006) and
to the projects MARE 22-05-01-FDR-00005 and POCTI/CTA/
48386/2002. Special thanks to Dr. Vanda Franco for the carbon
and sulfur analysis.
Supporting Information Available
One figure showing the study areas and another one showing
an oxygen and below-ground biomass vertical profile. This
material is available free of charge via the internet at http://
pubs.acs.org.
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Received for review May 8, 2007. Revised manuscript received August 3, 2007. Accepted August 14, 2007.
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