Environ. Sci. Technol. 2006, 40, 6181-6186
Sediment-Bound Inorganic Hg Extraction Mechanisms in the Gut Fluids of Marine Deposit Feeders HUAN ZHONG AND WEN-XIONG WANG* Atmospheric, Marine and Coastal Environment Program and Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong
The contributions of different free amino acids and proteins to the overall extraction of sediment-bound inorganic mercury (Hg) by gut fluids collected from depositfeeding sipunculans and sea cucumbers were evaluated. The organic content and the Hg concentration in the sediment were modified to investigate their effects on Hg extraction. Cysteine was the key free amino acid in complexing Hg while proteins in the gut fluids also contributed significantly to the extraction. Different size fractions of gut fluids had different bindings with Hg at different Hg concentrations. Hg first bound with the 100 kD fraction when the Hg concentration was increased. Removing the organic matter (OM) from the sediments enhanced Hg extraction, indicating that competition for Hg binding between the strong binding sites in sediments (the organic matter) and gut fluids (cysteine) may control the extraction. However, Hg complexation with weak binding sites (e.g., Fe/Mn oxides) in sediments should not be ignored. We identified two sediment Hg pools with different mobilities based on Hg binding, which was influenced by the Hg concentration in the sediment and the ratio of binding sites between gut fluids and sediments. Our results help to explain the variations in gut fluid extraction and Hg bioaccumulation in different marine deposit feeders.
Introduction Mercury (Hg) is a toxic metal and sediments are considered the major repository for Hg in aquatic environments (1). Relatively high Hg concentrations in sediments are frequently reported in the literature, i.e., up to 19 µg/g in sediments in Baltimore Harbor, U.S. (2) and an average of 3.6 µg/g in the floodplain of the Elbe transect in Germany (3). Inorganic Hg [Hg(II)] is generally the dominant Hg species in sediments (4, 5), thus there is a considerable interest in understanding the bioavailability of Hg(II) to sediment deposit feeders. Although organic Hg species such as methylated mercury (MeHg) was preferably accumulated by the organisms, many methylation processes may occur inside the organisms (6). The assimilation of inorganic Hg (all Hg mentioned below refers to inorganic Hg) from sediments could also be important in studying the accumulation of methylated mercury. The digestive solubilization of sediment-bound metals in the gut of deposit feeders is considered a key process in * Corresponding author phone: +(852) 2358 7346; fax: (852) 2358 1559; e-mail:
[email protected]. 10.1021/es061195z CCC: $33.50 Published on Web 09/02/2006
2006 American Chemical Society
metal accumulation (6). Gut fluid extraction (7) has been used widely to assess the bioavailability of sediment-bound metals and organic pollutants as well as in risk assessments (6, 8-10). A few previous studies investigated the mechanisms of metal extraction by gut fluids, which are generally considered as a complexation process instead of an enzymatic one (8, 11-14). Several complexation agents have been identified, including histidine, as the main binding sites for copper in gut fluid and proteinous materials (11, 13). SulfurHg bond was also more stable than most metal-organic bonds (15). Bovine serum albumin (BSA) has been used to mimic real gut fluids in both metal and polycyclic aromatic hydrocarbons extraction (8, 10, 11). Earlier studies established that extraction increases with increasing total amino acid (TAA) concentration, but little has been found on the contribution of different proteinous materials to overall extraction. In addition to these complexation processes, the geochemistry of sediments, such as the organic carbon and Hg concentration, may also be important in controlling digestive gut solubilization. In our previous study (16), we investigated the Hg geochemical speciation in sediment controlling Hg bioavailability to benthic invertebrates. In this study, we examined the effects of both gut fluid composition (free amino acids and proteins) and sediment geochemistry (organic content and Hg concentration) on gut fluid extraction of sedimentbound Hg. We first identified the key amino acids in complexing Hg in gut fluids and quantified the contributions of both free amino acids (AA) and proteins to the overall extraction. We then investigated how the organic content and Hg concentration of the sediments affected the extraction. Our aims were to understand the mechanisms of inorganic Hg solubilization by digestive gut fluids, which may provide useful information to explain the differences in Hg assimilation and bioaccumulation in benthic invertebrates. We used two deposit feeders (sea cucumbers and sipunculans) in our experiments, both of which are common in sandy mud sediments and are known to process significant amounts of sediments for nutritional purposes.
Materials and Methods Organisms and Chemicals. Oxic sediments were collected from Tathong Channel, Hong Kong, with a background total Hg concentration of 70 ng/g dry weight. In the laboratory, the sediments were dried at 50 °C and passed through 63 µm mesh. The loss on ignition (LOI) of the sediments (expressed as a percentage of dry weight, determined after 4 h of ashing at 450 °C) was 5.4%. The sea cucumbers, Holothuria leucospilota, (15-20 cm in body length) were collected from Clear Water Bay, Hong Kong, and the sipunculans Sipunculus nudus (5-7 cm in body length) were collected from Ting Kok, Hong Kong. The animals were immediately dissected (about 100 sipunculans and 20 sea cucumbers) after being brought to the laboratory to collect their gut fluids. The pooled (from 100 sipunculans and 20 sea cucumbers) gut fluids of each species containing sediments were centrifuged at 2267 g for 1 h at 4 °C to remove the sediments and stored at -80 °C before the extractions were performed within 1 week. About several hundred microliters gut fluids were recovered from each sipuncula individual and several milliliters from each sea cucumber individual. The free L-amino acids (AA), bovine serum albumin (BSA, initial fractionation by heat shock, fraction V), and HgCl2 were purchased from Sigma. The gamma radioactive isotope 203Hg (t1/2 ) 46.6 d, 203HgCl2 in 0.1 N HCl, with a specific activity of 77.3 GBq/g) was purchased from Isotope Products Laboratories (California, VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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U.S.). The seawater was filtered through a 0.22 µm membrane before use. Amino Acids and Proteins. The free amino acid concentrations in the gut fluids were measured by a HITACHI 835-50 amino acid analyzer (Tokyo, Japan) after the gut fluids were deproteinized by 0.5 M HCl. Standard amino acid samples were run before each measurement. The protein concentration was determined by bicinchoninic acid (BCA) protein assay kits obtained from Pierce, and the absorption was read at 562 nm on a UV/vis 8500 double-beam spectrophotometer (Techcomp) using the colorimetric method. Proteins in the gut fluids were separated and purified using cold ethanol precipitation by mixing the gut fluids with icecold 100% ethanol (gut fluid:ethanol ) 1:9 by volume). The mixture was then vortexed for 30 s and centrifuged at 10 000 g for 15 min at 4 °C. Afterward, the precipitated proteins were washed three times with ethanol and dried by N2 gas. The concentrations of the purified proteins were then measured. The recovery rate was calculated as the concentration of the reconstituted protein solution divided by that in the gut fluid. Total hydrolyzed amino acids (TAA) in the gut fluids, which include both the free amino acids and the amino acids hydrolyzed from proteins, were calculated as the sum of the free amino acid and protein concentration in the gut fluid. Extraction of Sediment Hg by the Free Amino Acids, Proteins or Gut Fluids. Radiolabeled sediments were used in all extraction experiments. The sediments (60 mg dry weight) were resuspended in 8 mL of 0.22-µm filtered seawater and spiked with different amounts of 203Hg for 1 h at room temperature to obtain sediments with different Hg concentrations. NaOH was added to adjust the pH to 8.0 since the Hg isotope was carried in 0.1 N HCl. We used a relatively short contact time between Hg and sediments in our experiments in order to magnify the percentage extracted from sediments and facilitate our observation of the Hg extraction by different agents. Our previous study showed that Hg extraction by gut fluid decreased with contact time between Hg and sediments (16). After radiolabeling, the sediments were centrifuged and washed twice in filtered seawater. The partition coefficient (Kd) was calculated as the radioactivity per gram of sediment divided by the radioactivity per mL in the overlying water after radiolabeling. The methylation was negligible (100 kD, 50-100 kD, 10-50 kD and 4 µg/g) due to the gradual saturation of the strong binding sites. More Hg may be partitioned into the weaker binding sites at higher concentrations, leading to an increase of extraction. Consequently, the strong competition for Hg binding between strong binding sites in sediments (e.g., OM) and the gut fluid (e.g., thiol groups) controls the bioavailability of Hg and leads to the different extraction patterns observed at different Hg concentrations. The binding of Hg at different sites in the sediments could hinder extraction and thus explain why there was not a consistent pattern in the amount of Hg extracted as a function of Hg concentration in the sediments.
AA Adsorption on Sediments. Different metal extraction patterns have been reported in gut fluids over time (8, 9, 12, 18). In this study, we found that extraction by the free amino acid mixture increased within the first hour of incubation and then decreased. In contrast, extraction by gut fluids was relatively steady with time, but at a much lower rate. The rapid decrease in extraction by free amino acids may be due to both amino acid degradation and adsorption. Although degradation may account for the decrease after several hours, adsorption but not degradation should explain most of the decrease within a short time (e.g., the first 2 h) since Hg extraction by the lowest amino acid concentration extractant (AA-1) decreased to a less extent than that of the other extractrants. Complexation and adsorption, therefore, counteracted the amino acid extraction. Proteins were also adsorbed onto the sediments during the extraction process, but their adsorption generally leveled off after 1 h incubation (about 15-20% gut fluid protein was adsorbed). The gut fluid extraction was more stable with increasing incubation time even after 24 h, indicating that gut fluid proteins are more important than free amino acids in extracting Hg after a long incubation time (e.g., 24 h) due to their lower adsorption or decay. Adsorption may modify extraction as a result of partitioning of Hg complexation agents between the sediment and the gut fluid. Lawrence et al. (8) also indicated that changes in the distribution of complexing agents between gut fluids and sediments were responsible for the different kinetics of extraction, and adsorption may account for the decrease of Hg extraction with time. In our previous experiments, we also observed that Hg in the organocomplexed phase increased after gut fluid extraction (16), further implying the transfer of complexing agents, such as free amino acids, between sediments and extractants during extraction. To conclude, free amino acids (especially cysteine) and proteins are the essential Hg binding agents in the gut fluids of two deposit-feeding invertebrates, while cysteine or its residue may be the dominant binding sites for Hg. Hg may first enter the refractory pool, probably the organic matter of sediments, but this also depends on the contact time between organic matters and sediments. Later, Hg is partitioned into the exchangeable pool, probably Fe/Mn oxides that complex Hg in the sediment mineral lattice, when the refractory pool becomes saturated. Competition for Hg binding between binding sites in the sediments and gut fluids eventually determines the Hg extraction by gut fluids. These findings are useful in explaining the different extractions and bioaccumulation observed for different organisms. For example, the more important role of certain amino acids in metal complexation (e.g., cysteine for Hg binding) may explain the absence of a consistent relationship between TAA and the amount of complexed metals (13). Hg accumulation may be low if the ratio between binding sites in the gut fluids and in the sediments are low, when the gut fluids cannot compete for Hg binding even if there is a relatively high Hg concentration in the sediments. It is thus critical to consider the composition of both gut fluids and sediments when assessing the bioavailability of sedimentbound metals using gut fluid extraction. Dissolution of sediment components with associated Hg in the gut fluid could also affect the extraction and thus assimilation.
Acknowledgments We thank the three anonymous reviewers for their helpful comments on this work. This research was supported by a competitive earmarked research grant from the Hong Kong Research Grants Council (HKUST6097/02M) to W.-X.W. VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Supporting Information Available Concentrations of free amino acids and proteins in the gut juices, relationship between Hg(II) extraction and partitioning coefficient, as well as extraction of Hg(II) from radiolabeled intact (LOI ) 5.4) or ashed (LOI ) 0) sediments by the gut juices or cysteine. This material is available free of charge via the internet at http://pubs.acs.org.
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Received for review May 18, 2006. Revised manuscript received July 28, 2006. Accepted August 9, 2006. ES061195Z
