Environ. Sci. Technol. 2007, 41, 7376-7382
Evidence for Elevated Production of Methylmercury in Salt Marshes JOA ˜ 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 Biolo´gicos (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]. 7376
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 21, 2007
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 (
