Investigating Uptake and Translocation of Mercury ... - ACS Publications

‡Department of Chemistry & Biochemistry and Southeast Environmental Research Center, and ⊥Department of Biological Science, Florida International ...
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Investigating Uptake and Translocation of Mercury Species by Sawgrass (Cladium jamaicense) Using a Stable Isotope Tracer Technique Yuxiang Mao,*,†,‡ Yanbin Li,§,‡ Jennifer Richards,⊥ and Yong Cai*,‡ †

Institute of Resources and Environment, Henan Polytechnic University, Jiaozuo, 454000, China Department of Chemistry & Biochemistry and Southeast Environmental Research Center, and ⊥Department of Biological Science, Florida International University, Miami, Florida 33199, United States § Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao, 266100, China ‡

ABSTRACT: The role of macrophytes in mercury (Hg) cycling in the Florida Everglades ecosystem has not been fully understood. In this study, a stable isotope (199Hg2+) addition technique was used to trace the methylation, uptake, and translocation of Hg by sawgrass (Cladium jamaicense) and quantitatively evaluate the contribution of atmospheric and soil Hg to Hg in sawgrass leaves and belowground biomass. The results showed that spiked 199Hg2+ could be rapidly methylated to monomethylmercury (Me199Hg) in the soil of the sawgrass pots. Only small portions of total Hg (THg) and monomethylmercury (MeHg) in the soil could be taken up by sawgrass, indicated by the ratios of T199Hg and Me199Hg (tracer) concentrations in the sawgrass below-ground biomass (BGBM) over that in the soil (6.50 ± 1.9% and 12.8 ± 3.6% for THg and MeHg, respectively). Concentrations of T199Hg (tracer) and Me199Hg (tracer) in sawgrass leaves only accounted for 5.50 ± 2.8% and 15.6 ± 4.0%, respectively, of that in the BGBM, implying that the fractions of mercury species transported upward by sawgrass were also small. Statistical analysis (t test) showed that sawgrass preferred MeHg over THg in both uptake and upward translocation. The majority (>90%) of THg in sawgrass leaves were estimated to be obtained from atmospheric Hg, rather than from soil, suggesting that assimilation of atmospheric Hg could increase the overall Hg stock in the Florida Everglades ecosystem. The finding about foliar uptake of Hg is especially important for a better understanding of mercury cycling in the Everglades, given the large amount of sawgrass biomass in this ecosystem.

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of inorganic Hg and 0.09−0.26 ng/g of MeHg.1,23 Thus, macrophytes were recognized to be a significant mass storage compartment for both inorganic Hg and MeHg in the Everglades.8,11 In a mass budget study, Liu et al. suggested that approximately 0.2−0.5% of the THg deposited into the Everglades entered into the macrophytes.8 Hg stored in plant rhizosphere/roots generally has low mobility, thus triggering the “phytostabilization” approach for wetland remediation.17 On the other hand, plant roots have exhibited the ability to enhance Hg methylation by altering the physicochemical and biological conditions of the rhizosphere, consequently increasing the ecotoxicity and bioavailability of Hg.19,20 Other plant tissues may also play important roles in Hg cycling. Plant foliage can either uptake atmospheric Hg through the stomata18,21,24 or remove Hg from the plant during transpiration.25−27 These dynamic exchange processes on foliar

INTRODUCTION Mercury contamination has been a concern for decades in the Florida Everglades. Elevated levels of mercury, especially the highly toxic monomethylmercury (MeHg), have been detected in fish, wading birds, and other biota, posing severe health risks to humans and Everglades wildlife.1 Fish consumption advisories have been issued throughout the Everglades. Health concerns stimulated a broad interest in the study of the sources, transport, methylation/demethylation, and bioaccumulation of Hg in this ecosystem.2−12 These previous studies have greatly improved our understanding of the fate of Hg in this wetland ecosystem, but there are still many questions that remain unanswered. An especially important one is the role of vegetation in the biogeochemical cycling of Hg. Macrophytes are an important component of wetland ecosystems and, along with soils and hydrology, define wetland structure.13 They could also play an important role in Hg cycling in wetland ecosystems. Hg species can be absorbed and accumulated in macrophyte roots and then translocated to other tissues.14−22 Both below-ground and above-ground biomass of vegetation in the Everglades contained 5−10 ng/g © 2013 American Chemical Society

Received: Revised: Accepted: Published: 9678

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surfaces might be regulated partially by atmospheric Hg0 concentrations.28 Lindberg et al.25,26 and Marsik and Keeler27 investigated the flux of gaseous elemental Hg (Hg0) over vegetation stands in the Florida Everglades. Their studies suggested that transpiration by macrophytes might be an important pathway for Hg removal and that the transpired Hg likely originated from the sediment.25,26 Previous studies showed that Hg0 evasion flux on the water surface in the presence of vegetation was much larger than that without vegetation, implying that macrophytes contribute significantly to the evasion of Hg0 in the Everglades.25 In addition, decomposition of senescent plant tissues can release Hg stored in plants back into the environment, increasing the amount of Hg available for methylation and uptake by other biota.29−31 Although macrophytes are expected to play an important role in Hg cycling in wetlands, it is still unknown how and to what extent different species of Hg (i.e., inorganic Hg and MeHg) are taken up by macrophytes and how Hg translocation occurs within plant tissues after being taken up. Such information is needed for a better understanding of the biogeochemical cycling of Hg in the Everglades. Sawgrass (Cladium jamaicense) is the dominant emergent macrophyte species in the Everglades, covering 50 to 70% of the landscape.32−35 Sawgrass grows in both peat and marl soils and in hydrologic conditions ranging from shallow, short hydroperiod marshes to deeper long hydroperiod marshes.32−36 Sawgrass is a sedge (Cyperaceae) and grows as a rhizomatous perennial plant with stiff, erect, strap-shaped leaves that can vary in length from 1 to >3 m in adult plants.33,37 Sawgrass biomass estimates vary with plant age, density, time since last burn, nutrients and hydrology, and whether live or live and standing dead parts are considered,34,38−46 but above-ground biomass estimates for Everglades field samples range from 14046 to 4517 g/m2,38 while below-ground to above-ground biomass is on the order of 0.8.37 The stable isotope tracer technique has been successfully applied to the studies of Hg biogeochemistry, such as methylation and demethylation in the past decades.10,12,47−49 The primary objectives of this study were (1) to elucidate the uptake efficiency of Hg species by sawgrass and determine the allocation patterns of Hg species within the plant tissues and (2) to assess the relative importance of assimilation of atmospheric Hg and uptake of soil Hg in Hg levels in sawgrass.

TN (mg/g, N = 11), and total carbon, TC (mg/g, N = 11) of 162 ± 36, 7.9 ± 1.5, and 146.5 ± 42.8, respectively.50 In order to simulate natural growing conditions, the sawgrass pots were kept in a polyethylene pool outdoors in a fenced yard at Florida International University. Tap water was added to the tank until it reached the soil surface in the pots. During the dry season, tap water was added to the tank twice per week to maintain an adequate water level; during the rainy season, the water vaporized from the tank was naturally balanced by precipitation. After allowing the plants to stabilize for 2 months, 199Hg tracer solution in deionized water (1.00 mg/L) was injected into the soil of sawgrass pots (10 mL of 199Hg solution was injected into each of the five positions evenly distributed in each pot), forming a final 199Hg concentration of approximately 200−300 ng/g as Hg, comparable to the ambient THg levels in Everglades soil. After introduction of 199Hg, sawgrass was cultured under the above-mentioned conditions from November 15th, 2008, to September 10th, 2009. At this time, plants had grown from seedlings to plants with leaves 50−70 cm in length. Sawgrass seedlings grown under similar conditions from 2011 to 2012 and for a similar amount of time had a final biomass of 11.3 ± 4.0 g DW (N = 9), which was partitioned 48.6 ± 6.7% into leaves and 51.4 ± 6.7% into roots and rhizomes (42.0% roots vs 9.5% rhizomes) (unpublished data). Sample Preparation and Analysis. At the end of the culture experiment, soil samples were collected from five positions evenly distributed in each pot and homogenized to make a composite soil sample for each pot. The composite samples were stored at −20 °C in a freezer and analyzed within 28 days. The sawgrass plants were divided into leaves and below-ground biomass (BGBM). The plant samples were collected on-site in the field. Soil associated with the plants was first removed with tap water before being brought into the laboratory to avoid contamination. DI water and 1% HCl were subsequently used in the laboratory to remove the remaining soil and mercury adsorbed on the surface of plants.51 The leaves and BGBM were cut into 2 cm pieces and ground into fine powder with mortar and pestle under liquid nitrogen. The finely powdered samples were doubly bagged with polyethylene zip-lock bags and kept at −20 °C (USEPA method 1631) in a freezer. The samples for THg analysis were digested using a method reported previously.8 Briefly, 0.2 g of sawgrass or soil samples were weighed into 10 mL glass ampules, followed by the addition of 1 mL deionized water and 2 mL concentrated HNO3. The ampules were then sealed and heated to 105 °C in an autoclave for 1 h. After digestion, samples were introduced into a FIAS400 system (PerkinElmer, Waltham, MA), where inorganic Hg was reduced by stannous chloride. The elemental Hg generated was then purged out of the gas−liquid separator of the FIAS400 system and carried into an Elan DRC-e ICPMS (PerkinElmer, Waltham, MA), where 202Hg and 199Hg were analyzed. With respect to MeHg, sample preparation and analysis were conducted using the aqueous phenylation followed by purgeand-trap preconcentration procedures reported previously.52 Briefly, samples were digested with acidic KBr/CuSO4 and extracted with CH2Cl2. MeHg present in CH2Cl2 was back extracted into 1% HCl via volatilization of the CH2Cl2 with a gentle flow of N2 (100 ml min−1), followed by aqueous phase phenylation and purge-and-trap. The Tenax traps retaining the phenylation products were then inserted into the GC injection

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EXPERIMENTAL SECTION Chemicals and Materials. All mercury standards were purchased from Ultra Scientific (N. Kingstown, RI, USA). A standard solution of methylmercury chloride (MeHgCl) was prepared by dissolving the standard in methanol. Enriched 199 HgO (atomic percentage 91.09 ± 0.05) was purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA) and dissolved in 10% HCl. Other reagents used were of reagent grade or higher. Experimental Design. Sawgrass seedlings were obtained from the sawgrass culture laboratory at Florida Atlantic University. Seedlings had 3−4 leaves, and leaves were 10−30 cm in length. They were repotted into 4 parallel polyethylene pots containing approximately 2 kg of Everglades soil collected in the southern Everglades (N 25° 41.771′, W 80° 34.801′). This soil was a peaty marl. Soil collected over 3 years at four sites within 3−8 km had 18.8 ± 7.2% organic matter (N = 43), 0.35 ± 0.10 gDW/cm3 field bulk density (N = 43), pH 7.6 ± 0.3 and total phosphorus, TP (μg/g, N = 43), total nitrogen, 9679

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port, and Me202Hg and Me199Hg were determined by GCICPMS. To ensure the quality of analysis, triplicates of each sample were analyzed. Two procedure blanks, matrix spikes, and certified reference material (CRM, IAEA-405) analysis were performed for each batch of samples. All procedure blanks determined were below the method detection limits (0.2 ng/g for THg and 0.01 ng/g for MeHg in soil and sawgrass tissues), and recoveries of 80−120% for both THg and MeHg were obtained for all matrix spikes and CRM. Total mercury concentrations in tap water and precipitation were approximately 70 and 15 ng/L, respectively. Methylmercury was 0.1 ng/L in tap water and below the method detection limit (0.02 ng/L) in precipitation. Data Analysis. The transient signals recorded by ICPMS were processed by Origin 6.0 (Microcal Software Inc., MA). Quantification of Hg isotopes was carried out using peak areas of the corresponding species. Statistical analysis (two-tail t test) was performed using Microsoft Office Excel 2003. Concentrations of MeHg and THg originated from the ambient Hg (CMeHg(ambient) and CTHg(ambient)) were calculated by the concentrations of T202Hg and Me202Hg and natural abundance of 202Hg (eq 1 and 2). Both ambient Hg and spiked 199Hg contributed to the final concentrations of T199Hg and Me199Hg in samples. Contributions of ambient Hg, including both MeHg and THg, to the measured 199Hg could be calculated using the concentrations of 202Hg and the ratio of 199Hg over 202Hg (P199/P202) in natural samples. Then, concentrations of 199Hg originated from the spiked 199Hg tracer (CMeHg(tracer) and CTHg(tracer)) could be calculated by subtracting the 199Hg originated from ambient Hg from the measured 199Hg (eq 3 and 4). C THg(ambient) = CMeHg(ambient) =

Figure 1. Concentrations of THg (ambient) (A) and THg (tracer) (B) in pot soil and different parts of sawgrass plants. S1, S2, S3, and S4 stand for the samples taken from the four sawgrass pots tested in the study. Error bars represent 1 standard deviation (n = 3).

with an average of 232.8 ± 61.2 ng/g (Figure 1B). Average Hg/199Hg ratios of THg (including both ambient and tracer Hg) ranged from 0.26 to 0.32 (average 0.28 ± 0.02) in the soil (Figure 3A). Large variations in soil THg concentration among the four pots could be attributed to the differences in Hg processing in pots during the experimental period, such as the difference in individual plant, methylation and demethylation, and oxidation and reduction of Hg among the pots. This is particularly true for MeHg since its concentration is determined by extra factors controlling the methylation/demethylation of Hg. These differences in processes seem to have similar effects on the concentrations of both 202Hg and trace 199Hg, as evidenced by a relatively small relative standard deviation of the 202 Hg/199Hg ratios in the soil of the four pots (see Figure 3). Average concentrations of MeHg (ambient) in the soil of the four sawgrass pots ranged from 1.35 to 2.72 ng/g (average of 2.00 ± 0.57 ng/g) (Figure 2A), and average CMeHg(tracer) was 202

C T202Hg P202

(1)

C Me202Hg P202

C THg(tracer) = C T199Hg −

(2)

P199C T202Hg

CMeHg(tracer) = C Me199Hg −

P202

(3)

P199C Me202Hg P202

(4)

where CT202Hg and CT199Hg are the measured concentrations of 202 Hg and 199Hg isotopes of THg at the end of sawgrass culture (ng/g); CMe202Hg and CMe199Hg are the measured concentrations of 202Hg and 199Hg isotopes of MeHg (ng/g); P202 and P199 are the natural isotopic abundances of 202 Hg and 199 Hg. CMeHg(ambient) and CTHg(ambient) are the concentrations of MeHg and THg in samples originated from the ambient Hg (ng/g); CMeHg(tracer) and CTHg(tracer) are the concentrations of MeHg and THg in samples originated from the spiked 199Hg tracer (ng/g).

Figure 2. Concentrations of MeHg (ambient) (A) and MeHg (tracer) (B) in pot soil and different parts of sawgrass plants. S1, S2, S3, and S4 stand for the samples taken from the four sawgrass pots tested in the study. Error bars represent 1 standard deviation (n = 3).

in the range of 5.32−7.56 ng/g (average 6.75 ± 1.11 ng/g) (Figure 2B). In the soil of four sawgrass pots, average 202 Hg/199Hg ratios of MeHg were from 0.09 to 0.14, with an average of 0.12 ± 0.04 (Figure 3B). The 202Hg/199Hg ratio of ambient THg in the pot without the addition of 199Hg2+ was measured to be 1.78 ± 0.03 for both Everglades soil and sawgrass biomass. This ratio for MeHg was 1.77 ± 0.03 and 1.76 ± 0.01 for Everglades soil and sawgrass biomass, respectively. These results indicate that there

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RESULTS AND DISCUSSION Mercury Species in Soil of the Sawgrass Pots. Average concentrations of THg (ambient) in the soil of four sawgrass pots were determined to be from 162.2 to 322.5 ng/g, with an average of 265.5 ± 73.7 ng/g (average of the four pots ± one standard deviation) (Figure 1A). Average concentrations of T199Hg (tracer) in the soil were between 145.6 and 310.2 ng/g, 9680

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in the BGBM (0.11−0.19, average 0.16 ± 0.03) was much smaller than that of the natural MeHg (1.78 ± 0.03). The results of both ambient and tracer THg and MeHg in the BGBM of sawgrass clearly reveal that sawgrass can uptake Hg from soil. On average (average of the four pots ± one standard deviation), concentrations of THg (ambient) and T199Hg (tracer) in the BGBM of sawgrass were 5.25 ± 3.20% and 6.50 ± 1.91% of their counterparts in the corresponding soil. Average concentrations of MeHg (ambient) and Me199Hg (tracer) in BGBM accounted for 18.8 ± 3.5% and 12.8 ± 3.6% of that in the corresponding soil. It seems that sawgrass BGBM could uptake but could not accumulate either THg or MeHg from the surrounding soil. Statistical analysis (t-test) showed that ratios of MeHg in BGBM over MeHg in soil was significantly higher than THg(BGBM)/THg(soil) (for ambient Hg, p < 0.01; and for tracer Hg, p < 0.05), suggesting that MeHg is preferred over THg with respect to uptake by sawgrass from soil. Thus, methylation of inorganic Hg to MeHg in Florida Everglades soil may enhance its uptake availability by sawgrass. On the other hand, ambient Hg and tracer Hg showed no significant difference on their uptake by sawgrass (for THg, p > 0.1; for MeHg, p > 0.05). Translocation of Hg Species within Sawgrass. Average THg (ambient) and T199Hg (tracer) in sawgrass leaves were determined to be 6.4−12.4 (average 9.2 ± 2.4) ng/g and 0.5− 1.3 (average 0.8 ± 0.3) ng/g, respectively. With respect to MeHg (ambient) and Me199Hg (tracer), the corresponding data were 51.4−104.8 (average 70.2 ± 22.6) × 10−3 ng/g and 66.4−166.1 (average 135.9 ± 59.6) × 10−3 ng/g, respectively. Detection of T199Hg (tracer) and Me199Hg (tracer) in the leaf samples suggests that both Hg species taken up through the roots can be translocated to the above ground tissues of sawgrass. On average (average of the four pots ± one standard deviation), T199Hg (tracer) and Me199Hg (tracer) in the leaf samples accounted for 5.50 ± 2.75% and 15.6 ± 4.0%, respectively, of that in the BGBM, indicating that only small portions of T199Hg (tracer) (mainly inorganic Hg) and Me199Hg (tracer) in the BGBM were transported to the leaves of sawgrass. This observation is in good agreement with several previous findings.16,17,19,20,23,58 It should be noted that the ratio of Me199Hg (tracer) in leaves over Me199Hg (tracer) in BGBM (15.6 ± 4.0% on average) was significantly higher than the ratio of T199Hg (tracer) in leaves over T199Hg (tracer) in BGBM (5.50 ± 2.75% on average) (p < 0.01). In addition, Me199Hg (tracer)/T199Hg (tracer) ratios in leaf samples (Figure 4) were significantly higher than that in BGBM (p < 0.01). Previously, there was no evidence on the occurrence of within plant Hg methylation in the leaf tissues of any plant species, it is reasonable to speculate that Me199Hg (tracer) found in sawgrass tissues originated from soil. This finding implies that MeHg, rather than inorganic Hg (the major part of THg), is preferentially translocated upward by sawgrass. Similar results have been reported for rice and ground vegetation.14,22 Moreover, differentiation of MeHg and inorganic Hg by rice might be attributed to the phytochelatins, which could selectively sequester inorganic Hg but not MeHg.59 MeHg (ambient) seems to be translocated from BGBM to leaves at a similar efficiency as Me199Hg (tracer) (p > 0.05), with an average concentration in the leaves amounting to ∼19.4 ± 4.9% of that in the BGBM (Figure 2A). However, THg (ambient) showed a different distribution pattern in comparison to T199Hg (tracer). Ambient THg in the leaves of sawgrass

Figure 3. 202Hg/199Hg ratios of THg (A) and MeHg (B) in pot soil and different parts of sawgrass plants. S1, S2, S3, and S4 stand for the samples taken from the four sawgrass pots tested in the study. Error bars represent 1 standard deviation (n = 3).

was no significant difference between soil and sawgrass biomass in terms of 202Hg/199Hg of THg and MeHg (p > 0.1). Amendment of the inorganic 199Hg tracer greatly changed the isotopic compositions of THg in the soil, decreasing the 202 Hg/199Hg ratio of THg from 1.78 ± 0.03 to 0.28 ± 0.02. The logic of using an spiked stable isotope tracer of Hg was that, if methylation of the inorganic 199Hg tracer occurred, extra Me199Hg would be produced, thus a decrease of Me202Hg/ Me199Hg ratio in Everglades soil would be detected. In fact, 202 Hg/199Hg ratios of MeHg in the soil of sawgrass pots were determined to be 0.12 ± 0.04 (Figure 3B), much lower than that of ambient MeHg. Thus methylation of the 199Hg tracer occurred in the soil of sawgrass pots. Although stable isotope fractionations of Hg have been observed in a variety of natural processes,53−55 the variation is generally very small, with a Δ value of a few parts per thousand in plants. As large amounts of 199 Hg tracer was added into the soils, the ratio of 202Hg/199Hg in soil was changed to a large extent (from 1.78 to 0.28). Compared with the large change of 202Hg/199Hg ratio in the spiked soil, the fractionation of Hg in both atmospheric and root-induced uptake is negligible. It was calculated that, on average (net methylation), 6.75 ± 1.11 ng/g of Me199Hg (tracer) was produced from the enriched stable isotope tracer (199Hg) spiked into the soil, accounting for 3.25 ± 1.51% of CTHg(tracer) spiked in the soil. In comparison, MeHg (ambient) in the soil of sawgrass pots was at a much lower level, 2.00 ± 0.57 ng/g, which only accounted for 0.86 ± 0.55% of CTHg(ambient). These results suggest that the freshly added Hg isotope tracer showed a higher availability for the methylation process than the “old” ambient mercury (p < 0.05), agreeing with several previous studies.12,56,57 Uptake of Mercury Species by Sawgrass. Average concentrations of THg (ambient) in BGBM of the four pots ranged from 10.1 to 16.9 ng/g (average 12.7 ± 2.9 ng/g; Figure 1A). Average concentrations of T199Hg (tracer) in the BGBM were between 12.2 and 21.4 ng/g (average 16.9 ± 5.1 ng/g; Figure 1B). Average 202Hg/199Hg ratios of THg in BGBM varied from 0.14 to 0.30 (average 0.22 ± 0.06; Figure 3A). In the BGBM, 0.26−0.48 ng/g of MeHg (ambient) were detected, giving an average of 0.37 ± 0.10 ng/g (Figure 2A). With respect to Me199Hg (tracer) in the BGBM, the average concentration varied between 0.52 and 2.03 ng/g, with an average of 1.13 ± 0.65 ng/g (Figure 2B). Isotope ratio measurements (Figure 3B) showed that 202Hg/199Hg of MeHg 9681

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Hg/199Hg ratio in BGBM and that in soil (p > 0.1), indicating that most Hg in sawgrass BGBM was obtained from the soil. However, a significant difference was observed between 202 Hg/199Hg in leaves and that in soil or atmosphere (p < 0.01), suggesting that both sources were important. In order to calculate the contribution of atmosphere and soil to Hg in sawgrass leaves, a new model was developed as follows (eqs 5−8). As all tracer T199Hg originated from soil, the distribution coefficient of T199Hg (tracer) from BGBM to leaf (RTHg(tracer)) could be described as the concentration of THg (tracer) in leaf (Cleaf THg(tracer))/the concentration of THg (tracer) in BGBM (CBGBM THg(tracer)) (eq 5). RTHg(tracer) =

Figure 4. Average ratios of MeHg (ambient)/THg (ambient) and average ratios of MeHg (tracer)/THg (tracer) in the soil, the sawgrass leaf, and below ground biomass. The averages were taken from the samples of the four sawgrass pots tested. Error bars represent one standard deviation (n = 4).

leaf C THg(tracer) BGBM C THg(tracer)

(5)

Assuming that there was no significant difference between the upward translocation of THg (ambient) and T199Hg (tracer) from BGBM to leaf of sawgrass, RTHg(ambient) should be equal to RTHg(tracer) (eq 6).

was 73.8 ± 18.0% of THg (ambient) in the BGBM (Figure 1A). This distribution ratio (concentration in leaf/concentration in BGBM) is much higher than that for T199Hg (tracer) (5.50 ± 2.75%). In addition, MeHg (ambient)/THg (ambient) ratios were smaller in leaves than in BGBM (p < 0.01). For the 199 Hg tracer, the opposite trend was observed (Figure 4). These differences were likely caused by the higher THg (ambient) concentration in the leaves, which reached 9.2 ± 2.4 ng/g (average of the four pots), a value much higher than CTHg(tracer) (0.8 ± 0.3 ng/g, average of the four pots) in the leaf samples. It is unlikely that sawgrass tissues selectively transported more ambient THg than tracer T199Hg to its leaves, especially to such a large extent. Thus, there should be other Hg sources contributing ambient THg to sawgrass leaves in addition to the Hg (ambient) transported from sawgrass roots, while the T199Hg (tracer) in the leaves originated solely from the sawgrass BGBM. This additional Hg source most probably is atmospheric Hg, which has the identical natural isotopic composition to ambient THg in the soil of sawgrass pots. The assimilation of atmospheric Hg by sawgrass leaves could increase the THg (ambient) contents and consequently decrease the MeHg/THg ratios of ambient Hg in sawgrass leaves. Foliar Hg is influenced primarily by atmospheric Hg concentrations and to a minor extent by soil Hg concentrations.18,21,24,60,61 It should be pointed out that such conclusions were drawn mainly from correlation analysis of Hg concentrations in foliar and atmospheric/soil Hg contents. In these previous studies, it was difficult to quantitatively assess the relative contributions of atmospheric Hg and soil Hg to plant foliar Hg contents due to the lack of an analytical technique that can distinguish Hg of the two origins. The use of stable Hg isotope tracer technique in this study made such distinction possible. Sources of THg in Sawgrass BGBM and Leaves: Atmospheric Hg vs Soil Hg. Mercury in sawgrass BGBM and leaves may originate from two sources: atmosphere and soil. As there is a significant difference in 199Hg/202Hg ratio between the two sources (because of the spiked 199Hg), 199 Hg/202Hg ratio in sawgrass BGBM or leaves should be significantly different from that in soil or atmosphere if both sources contribute significantly to Hg in sawgrass. The results showed that there was no significant difference between the

RTHg(ambient) = RTHg(tracer)

(6)

Then, the relative contributions of BGBM transport to THg contents in sawgrass leaves (PBGBM THg(ambient)) could be calculated using eq 7. BGBM PTHg(ambient) =

BGBM C THg(ambient) RTHg(ambient)

CBGBM THg(ambient)

leaf C THg(ambient)

× 100% (7)

Cleaf THg(ambient)

Where, and are the respective THg (ambient) concentrations in the BGBM and leaves of sawgrass (ng/g). Then the relative contributions of atmospheric assimilation of ambient Hg to THg contents in sawgrass leaves (Pair THg(ambient)) could be calculated using eq 8. air BGBM PTHg(ambient) + PTHg(ambient) = 100%

(8)

The relative contributions of BGBM transport and leaf assimilation to the THg (ambient) contents of sawgrass leaves are shown in Figure 5. For the four sawgrass pots tested, the portions of THg (ambient) transported from the BGBM only accounted for 5.0−13.0% (average 7.4 ± 3.7%) of the THg

Figure 5. Relative contribution percentages of below ground biomass transport and leaf assimilation to the THg (ambient) in the sawgrass leaves. S1, S2, S3, and S4 stand for the samples taken from the four sawgrass pots tested in the study. Error bars represent one standard deviation (n = 3). 9682

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(ambient) contents in sawgrass leaves. The majority (>90%) of THg (ambient) in sawgrass leaves was obtained from atmospheric Hg. Atmospheric Hg−foliar exchange is expected to be a bidirectional process,18,21,27 meaning that assimilation of Hg by plant leaves and emission of Hg from the leaves occur simultaneously. Depending on the environmental conditions, the plant species, and in particular Hg0 concentrations, one process could overwhelm the other, as reported previously.18,21,25−28 The results of the present study suggest that assimilation of atmospheric Hg could dominate the bidirectional exchange process, resulting in the accumulation of Hg in sawgrass leaves. Considering the vast amount of sawgrass biomass in the Florida Everglades, assimilation of atmospheric Hg into sawgrass leaves could definitely increase the Hg stock in this aquatic ecosystem. When sawgrass leaves senesce and decompose, foliar Hg would be released into other compartments of the Everglades. Therefore, further studies are strongly suggested to reveal how and to what extent foliar assimilation of sawgrass and other macrophyte species is involved in the Hg biogeochemical cycling in the Florida Everglades.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 86-13782761630. Fax: 86-391-3987982. E-mail address: [email protected] (Y.M.). Tel.: 305-348-6210. Fax: 305348-3772. E-mail address: cai@fiu.edu (Y.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Joel Trexler and Aaron Parker at FIU for help with sample collection. This work was financially supported in part by the National Natural Science Foundation of China (21177035, 21120102040), the Open Foundation of the State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (KF2010-10), and the US EPA Region 4, the National Park Service, the Florida Department of Environmental Protection (CA H5297-05-008). This is contribution number 634 of the Southeast Environmental Research Center at FIU.

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REFERENCES

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