Research Communications Ethylmercury in the Soils and Sediments of the Florida Everglades Y O N G C A I , † R U D O L F J A F F EÄ , ‡ , § A N D R O N A L D J O N E S * ,†,| Southeast Environmental Research Program, Department of Chemistry, Drinking Water Research Center, and Department of Biological Sciences, Florida International University, University Park, Miami, Florida 33199
Introduction Widespread reports concerning the presence and ecotoxicological importance of mercury in a variety of environmental and biological samples have resulted in increased interest in studies concerning the environmental sources and biogeochemical processes of mercury (1-5). It is well known that the various chemical forms (organic and inorganic) of mercury in the natural environment can behave differently, thereby affecting its biogeochemical behavior and toxicity to organisms. As far as organic mercury is concerned, virtually all literature reports have focused on the presence of methylmercury compounds, especially monomethylmercury (MeHg+), in environmental and biological samples (1-6). Studies of the occurrence of other organic mercury compounds in the environment have so far been largely ignored, and reports of their presence in environmental and biological samples are scarce (7-11). However, Jernelo¨v and Wennergren have reported ethylmercury (EtHg+) in sediments from St. Clair River (9), the occurrence of EtHg+ in Florida Everglades soils was observed during the development of an analytical technique that combined capillary gas chromatography with atomic fluorescence spectroscopy (GC-AFS) (10), and more recently, the presence of organic mercury species other than MeHg+ at mercury contaminated industrial sites in Germany and at a mining area in Slovenia have been reported (11). The presence of the non-MeHg+ organic mercury species in the environment has important implications for the understanding of the biogeochemical cycling of this toxic metal. The lack of more observations on the occurrence of EtHg+ in environmental and biological samples could be partly attributed to the analytical methods presently used, including sample preparation and detection, since so far these have mainly been developed for the analysis of MeHg+ (6, 12). Traditionally, gas chromatography with electron-capture detection (GC-ECD) was widely used for the determination and speciation studies of organomercury compounds in many environmental and biological samples. However, the nonspecificity of the ECD detector and the possibility of co-elution with other interfering compounds would diminish the speciation information gained from the analysis, even if other organic mercury compounds were present in the sample (6, 12). Another technique that is widely used at present for MeHg+ and Hg2+ analysis is aqueous ethylation followed by * Corresponding author fax: (305)348-4096; e-mail: serp@servax. fiu.edu. † Southeast Environmental Research Program. ‡ Department of Chemistry. § Drinking Water Research Center. | Department of Biological Sciences.
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purge-and-trap of the volatile alkylmercury derivatives with subsequent GC separation and atomic absorption spectrometry (AAS) (13, 14) or AFS (15) detection. Although this method has some advantages in terms of the practical convenience of the aqueous derivatization reaction, reduced analysis time, and organic solvent usage, it is not capable of measuring ethylmercury species because both Hg2+ and EtHg+ are converted to diethylmercury during the derivatization procedure. Therefore, an alternative method has been developed for the analysis of MeHg+ and EtHg+ by employing a modified solvent extraction procedure followed by GC-AFS detection (10). This sensitive method, with a limit of detection (LOD) of 0.01 ng of Hg/g dry wt, is an ideal tool for the investigation of trace levels of organomercury in the aquatic environment, in this particular case, the Florida Everglades system. Since 1989, mercury has been found in elevated concentrations in various biota from the Florida Everglades (16). However, the sources, fate, transport, and transformation pathways of mercury in south Florida ecosystems need to be better understood. In this paper, we present evidence for the occurrence of another important and abundant organomercury compound, namely, ethylmercury, in soils and sediments from the Florida Everglades.
Experimental Section Sample preparation and analysis of MeHg+ and EtHg+ were performed employing a modified version of the solvent extraction (17, 18) and GC-AFS technique developed previously (10). Briefly, a portion (5-6 g, wet wt) of the homogenized sample was placed in a 20-mL borosilicate glass scintillation vial. Distilled water (5 mL) followed by a copper sulfate (1.0 M; 1.0 mL) and acidic potassium bromide solution (3.0 mL) were added, and the mixture was shaken for 1 h at 330 rpm. Dichloromethane (5 mL) was added, and the mixture was shaken for 12 h and then centrifuged for 10 min at 5000g. A known volume of the dichloromethane layer (3.5 mL) was transferred to a 7.0-mL borosilicate glass scintillation vial, and sodium thiosulfate solution (0.01 M; 1.0 mL) was added. The mixture was shaken for 20 min at 330 rpm and centrifuged at high speed in an IEC clinical centrifuge. The aqueous phase (0.8 mL) was placed in a 2.0-mL microcentrifuge tube, and acidic potassium bromide/copper sulfate (3:1) and dichloromethane (0.2 mL) were added. The contents were shaken for 10 min at 330 rpm and centrifuged for 2 min at high speed in a Hermle centrifuge. The dichloromethane was transferred to an autosampler vial with a low-volume insert and analyzed by GC-AFS. Quantifications of MeHg+ and EtHg+ in the sediments were achieved by a standard addition method. Recovery of 75% was obtained for both MeHg+ and EtHg+ compounds. The organic matter content in the sediment did not influence the recoveries of organomercury species using the extraction procedure described above. Details of the standards and the reagents used in this study have been described elsewhere (10). The identity of ethylmercury was confirmed by converting EtHg+ ion extracted from sediment samples to ethylbutylmercury (EtHgBu) using Grignard derivatization and by chromatographic analysis on two different columns (DB-1 and DB-17). For derivatization with Grignard reagent (butylmagnesium chloride, 2.0 M solution in diethyl ether, Aldrich), four portions of the sample were extracted. The four extracts obtained (dichloromethane layer) were com-
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1996 American Chemical Society
FIGURE 1. Chromatograms of organomercury standards (A), an Everglades soil sample (B), and procedural blank (C). Peak identification: 1, MeHgBr; 2, EtHgBr. GC/AFS conditions: column, DB-1, 15 m, 0.53 mm i.d., 1.5 µm film; injection temperature, 250 °C; injection volume, 5 µL; GC temperature gradient, initially 50 °C for 1 min, 30 °C/min to 140 °C, held there for 3 min, then increased to a final temperature of 200 °C at a rate of 30 °C, and then held for 2 min. bined and placed in a 7.0-mL borosilicate glass scintillation vial. Then 1 mL of Grignard reagent was added slowly, and the vial was sealed and shaken for 1 min and then allowed to react for 15 min. The vial was placed on ice, and sulfuric acid (0.5 M; 4 mL) was added by the drop to decompose the excess Grignard reagent. The organic phase was separated and placed in a glass vial and analyzed by GC-AFS. Organomercury analyses were performed using the P. S. Analytical mercury speciation system, Model PSA 10.723. This consists of an integrated gas chromatography-mercury atomic fluorescence instrument, which is comprised of an Ai (Cambridge, U.K.) Model GC 94 gas chromatograph equipped with a CTC A200S autosampler and an optic injector module and coupled to the PSA Merlin detector via a pyrolysis oven held at 800 °C. This system was similar to the one used previously in our laboratory (10). The details of the GC conditions are described in the figure captions.
Results and Discussion The main objective of this paper is to present evidence for the presence of EtHg+ in Everglades soils and sediments, as will be shown below. Following the extraction procedure described above, the organomercurials were converted into their bromide derivatives by the addition of potassium bromide at the last extraction stage before GC-AFS analysis on a DB-1 column. A GC-AFS chromatogram of standards is shown Figure 1A to illustrate the peak identification, while Figure 1B shows a typical chromatogram from an Everglades
FIGURE 2. Chromatograms of butylated MeHg+ and EtHg+ with DB1: (A) standards; (B) Everglades soil sample. Peak identification: 1, MeHgBu; 2, EtHgBu; 3, Bu2Hg; X, impurity from standards and/or Grignard reagent (unidentified). GC/AFS conditions: column, DB-1, 15 m, 0.53 mm i.d., 1.5 µm film; GC temperature gradient, initially 50 °C for 1 min, 4 °C/min to 120 °C, then increased to a final temperature of 200 °C at a rate of 30 °C, and held there for 2 min. Other conditions as in Figure 1. soil sample. Figure 1C presents the instrument response to a procedural blank, which was generated in the same manner as the environmental samples, except without added soil. This illustrates that the organomercury peaks shown in the chromatogram of the extract (Figure 1B) result from the sample, not from the contaminants of the reagents used. To confirm the presence of EtHg+ in the Everglades soils, the following experiments were done. First, to confirm that peak 2 in Figure 1 is EtHg+, and not caused by spectral interference from the sample matrix, sample extracts were analyzed on both a DB-1 and a DB-5 column, and the presence of EtHg+ in the sample extract was verified based on the coelution with authentic standards on both columns. In addition to the GC-AFS analysis of organomercury bromide compounds directly on DB-1 and DB-5 columns, MeHgBr and EtHgBr were derivatized with Grignard reagent into methylbutylmercury (MeHgBu) and ethylbutylmercury (EtHgBu), followed by chromatographic analysis on a DB-1 column. By comparing the retention times of the derivatized standards on Figure 2A with the derivatized sample extracts shown in Figure 2B, the presence of EtHg+ in the sediment sample extracts was confirmed. The small peak on the chromatogram of standards (peak X) was not identified but is most likely caused by a combined effect of the mercury standards used and the Grignard reaction, since no such peak was apparent in either the chromatograms of MeHgBr and EtHgBr of the standards (Figure 1A) and the real samples (Figure 1B) or the chromatograms of the butylated real sample (Figure 2B). Unfortunately, due to the low concentration of the peak X, this compound could not be identified by other techniques such as GC/MS. Co-injections with authentic standards on a DB-17 capillary column were used to further verify the identities of the derivatives. Typical chromatograms for derivatized standards and Everglades samples are given in Figure 3.
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FIGURE 3. Chromatograms of butylated MeHg+ and EtHg+ obtained with DB-17: (A) standards; (B) Everglades sediment sample. Peak identification: 1, MeHgBu; 2, EtHgBu; 3, Bu2Hg; X, impurity from standards and/or Grignard reagent (unidentified). GC column, DB17, 30 m, 0.53 mm i.d., 1.5 µm film. Other GC/AFS conditions as in Figure 1. Further steps were taken in order to determine if the EtHgBr peak is a result of the transformation of MeHg+ or inorganic mercury present in the soil during the sample extraction and workup procedure. To do this, two types of sediments with high organic matter content and in which EtHg+ had previously been observed (one from a canal, percent organic matter (%OM) ) 15.9, another from a marsh environment, %OM ) 83.5) were spiked with inorganic mercury (as HgCl2) and followed by the extraction procedure described above. Both spiked and unspiked experiments were performed in triplicate. The spiking raised the HgCl2 concentration in the spiked samples by a factor of about 15 times that found in the unspiked sediments. For the canal sample, the EtHg concentrations were found to be 0.54 ( 0.08 and 0.52 ( 0.04 ng/g as Hg in the unspiked and spiked samples, respectively, while for the marsh sample, they were
0.62 ( 0.04 and 0.58 ( 0.02 ng/g as Hg in the unspiked and spiked samples, respectively. The results showed that the concentrations of EtHg+ in the spiked sediments were not increased because of the addition of inorganic mercury. Similar experiments were performed by spiking methylmercury (as MeHgCl), and the results illustrate that no transformation to EtHg+ occurred during the extraction procedure. The results of these experiments demonstrate that the presence of ethylmercury is not derived from the analytical methodology used. A number of soil and sediment samples were obtained from the Florida Everglades and analyzed using the procedure described above. Both MeHg+ and EtHg+ are widely observed in the samples. The locations from which the marsh and canal samples were collected are shown in Figure 4. The concentrations in these sediments were in the range of the LOD up to 10 ng of Hg/g dry wt for MeHg+ and from the LOD to 5 ng of Hg/g dry wt for EtHg+. In some cases, the concentration of EtHg+ was found to be higher than that for MeHg+. Results from some of the samples together with the percent organic matter (%OM), total phosphorus (TP), bulk density, and total mercury contents are listed in Table 1. All these samples were collected in sites far removed from major industrial areas or other possible point sources of pollution. From the data obtained thus far, conclusions cannot be made concerning the sources and distribution of EtHg+ in the Everglades ecosystems. However, its presence in these samples could be significant. Unlike the occurrence of EtHg+ observed by Hintelmann et al. (11), which was explained as a consequence of industrial activities, the presence of EtHg+ in the Florida Everglades seems to have a more complex explanation. Possible mercury sources in south Florida include natural mineral and peat deposits, atmospheric deposition, fossil fuel-based power plants, municipal waste incinerators, medical laboratories, paint, and agricultural operations (16). However, none of these individual sources appears adequate to explain the widespread presence of EtHg+ in the Everglades. Besides the possibility of anthropogenic sources, natural processes like biotic and abiotic alkylation could be a pathway for the existence of EtHg+ in the environmental samples. Indeed, organomercury compounds were produced when methylcobalamine and propylcobalamine were allowed to react with two individual batches of Hg2+ in mild reducing conditions (19). The products of these reactions were identified as methylmercury and propylmercury, respectively. In addition, the formation of EtHgCl and (Et)3PbCl has been reported when high-octane gasoline containing (Et)4Pb was mixed with an aqueous solution of HgCl2 (9). The presence of EtHg+ in St. Clair River sediments has been attributed to this kind of chemical alkylation (9). Fortmann et al. (8) reported the formation of EtHg+ when
TABLE 1. MeHg+, EtHg+, Total Hg, % Organic Matter (% OM), Total Phosphorus (TP), and Bulk Density in Soils and Sediments of Florida Everglades sample sitea
MeHg+ (ng of Hg/g)
EtHg+ (ng of Hg/g)
total Hg (ng of Hg/g)
% OM
TP (ppb)
bulk density (g/mL)
M-005 M-015 M-025b M-035 M-045 M-065 M-075 M-085 M-105b C-156 C-161 C-167 C-177 C-197
10.18, 8.89 0.86, 0.50 3.51 0.90, 1.10 0.34, 0.26 0.94, 0.76 4.79, 4.50 0.00, 0.10 0.05 0.35, 0.28 0.11, 0.13 0.03, 0.03 0.52, 0.56 0.06, 0.05
