Environ. Sci. Technol. 2002, 36, 5124-5129
Factors Controlling the Bioavailability of Ingested Methylmercury to Channel Catfish and Atlantic Sturgeon JOY J. LEANER AND ROBERT P. MASON* Chesapeake Biological Laboratory, University of Maryland, Center for Environmental Science, P.O. Box 38, Solomons, Maryland 20688
The bioavailability of ingested methylmercury (CH3Hg(II)) was investigated in vitro using the gastric and intestinal fluids of channel catfish, Ictalurus punctatus, and Atlantic sturgeon, Acipenser oxyrinchus. Gastric fluid collected from each species was incubated with CH3Hg(II)-spiked sediment or bloodworms, after which the intestinal fluid of each species was added and incubated further. The proportion of CH3Hg(II) solubilized from bloodworms and sediment appeared to be controlled by complexation to amino acids in both the stomach and the intestinal fluids during the digestive process, with the more thorough digestion of bloodworm organic material enhancing CH3Hg(II) solubilization. A greater proportion of CH3Hg(II) was solubilized by the sturgeon fluids compared to the catfish fluids, especially for the sediment incubations. These differences corresponded to the relative amount of amino acids in the fluids of these fish. A comparison of the catfish gastrointestinal solubilization incubations and a CH3Hg(II) bioaccumulation experiment with bloodworms revealed that the solubilization incubations may be a reasonable surrogate measurement of the bioavailability of CH3Hg(II) to fish. Overall, it appears that digestive processes is the most important controlling factor in the bioavailability of CH3Hg(II) to fish.
Introduction In the aquatic environment, mercury (Hg) poses a health risk once it becomes part of the aquatic organisms’ tissue. Food is the dominant uptake pathway for CH3Hg(II) accumulation in fish (1). Studies using benthic invertebrates (2, 3) have reported that CH3Hg(II) bioavailability may be controlled by intestinal solubilization, i.e., by the digestive processes of the organism rather than due to the limitation of transfer of CH3Hg(II) across the biological barriers, such as the gill, skin or intestinal epithelium (1, 4). Similar studies on aquatic vertebrates are lacking. Overall, there is a limited understanding of the factors that control the bioavailability of ingested CH3Hg(II) to fish and further investigation is needed. Metals accumulated from food are associated with a considerable solubilization process during passage through the digestive systems of organisms and as such become bioavailable to the organism (5, 6). Teleosts exhibit a wide array of digestive mechanisms to exploit their food successfully (7), and it is likely that these mechanisms * Corresponding author phone: (410)326-7387; fax: (410)326-7341; e-mail:
[email protected]. 5124
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affect the extent of metal solubilization and its subsequent bioavailability from food or sediment that is ingested. To investigate this, we utilized the digestive solubilization incubation approach (2, 3, 5, 8-10) to determine the factors that control the bioavailability of CH3Hg(II) to fish. Many of the fish inhabiting the benthic environment feed on benthic invertebrates (7) and may inadvertently take in sediment and organic debris (11, 12) during feeding. Since sediment and benthic invertebrates represent repositories for metals and because they are potential sites from which CH3Hg(II) can be bioaccumulated, we used these agents to determine the impact of gastrointestinal solubilization on the bioavailability of CH3Hg(II) to benthivorous fish. In this study, we incubated the gastric and intestinal fluids of channel catfish, Ictalurus punctatus, and Atlantic sturgeon, Acipenser oxyrinchus, separately, with CH3Hg(II)-spiked sediment or bloodworms, Glycera americana. In addition, we fed a known amount of CH3Hg(II)-spiked bloodworms to catfish to determine the overall bioavailability of CH3Hg(II) to the fish for comparison.
Materials and Methods Atlantic sturgeon, Acipenser oxyrinchus (U.S. Fish and Wildlife Services, Lamar, PA), and channel catfish, Ictalurus punctatus (Zetts Hatchery, Inwood, WV), weighing between 150 and 200 g, were held in 1000-gallon aquariums at the Chesapeake Biological Laboratory (CBL). The fish were maintained at a temperature of 20 ( 1 °C, a 12 h light:12 h dark cycle, and received aerated, filtered brackish water (8‰) in a flowthrough system. All fish were fed daily a commercial pellet diet at 10% their body weight per day. Feeding was terminated 3 days prior to the experiments to allow for gut evacuation, thereby minimizing food interferences with the experiment. Gastrointestinal Solubilization Incubations. Sediment previously collected at Fishing Bay (MD), an unpolluted reference site (13), was mixed with sand collected from the mouth of the Patuxent River, MD. Prior to mixing, the sand was sieved to less than 250 µm and muffled at 550 °C for 24 h to remove any organic matter that was present. The sediment-sand mixture was spiked with methylmercury chloride (CH3HgCl) (Sigma, St. Louis, MO) to obtain a concentration of 560 ng/g-wet weight and was stored at 4 °C until use. The organic matter in the sediment mixture was 1%. Bloodworms, Glycera americana, obtained from a local bait supplier, were maintained in a seawater aquarium at CBL, until they were transferred to a CH3Hg(II)-spiked microcosm (viz. a 4-L beaker, containing a 3 cm layer of the CH3Hg(II)-spiked sediment detailed above and filtered seawater). The bloodworms were maintained in the CH3Hg(II)-spiked microcosm for 5 days, after which they were transferred to clean seawater and allowed to depurate for 1 day to excrete undigested materials. The exposed bloodworm CH3Hg(II) concentration was 120 ng/g wet weight tissue. The organic matter in the bloodworms was 88%. After routine maintenance and termination of feeding as described above, each fish was sacrificed by a blow to the head, followed by decapitation to minimize pain. Thereafter, each fish was dissected, and the stomach and intestine were carefully excised and blotted dry. The stomach and intestine of individual fish were drained of their fluids into acid-cleaned vials, and the fluids were stored on ice for no longer than 15 min. The fluids of catfish (∼0.1 to 0.15 mL per fish) and sturgeon (∼0.25 to 0.5 mL per fish) were pooled separately into acid-cleaned centrifuge vials and centrifuged at 2700g for 30 min at 4 °C to remove any particulate matter. The 10.1021/es011331u CCC: $22.00
2002 American Chemical Society Published on Web 10/26/2002
supernatant in each vial was collected separately and stored at -80 °C for the solubilization incubation experiments. The gastric and intestinal fluids were thawed at 4 °C on the day of the experiment. The gastrointestinal solubilization incubation protocol was adapted from Lawrence (13) and Ruby et al. (14) and was performed in two stages. First, 0.5 mL of the gastric fluid collected from each species was pipetted separately into 24 acid-cleaned Teflon centrifuge tubes (i.e., 12 for catfish fluids; 12 for sturgeon fluids) and incubated with either 0.2 g-wet weight of CH3Hg(II)-spiked sediment or CH3Hg(II)-contaminated bloodworms. A portion of the incubations were terminated after 0.5 and 3 h (n ) 3 per time point) and processed as described below. Thereafter, 0.5 mL of the intestinal fluid was separately added to six incubations of each treatment. Thus, these incubations contained both gastric and intestinal fluids to further simulate the movement of gastric fluid into the intestine. The gastrointestinal incubations were stopped after a further 1 and 2 h (n ) 3 per time point), and samples were processed as described below. Therefore, the latter six incubations in each treatment contained gastric fluid that had been incubating with CH3Hg(II)-spiked material for 3 h, mixed with the intestinal fluids added subsequently. The solid mass: fluid mass ratio was 1:2.5 for the gastric fluid incubations (0.5 mL gastric fluid) and 1:5 for the intestinal fluid incubations (0.5 mL gastric fluid + 0.5 mL intestinal fluid). The mixtures were vigorously shaken on an orbital shaker held at room temperature, and samples were centrifuged at 2700g for 30 min at 4 °C, when taken. The supernatant was collected, and both supernatant and pellet was stored at -80 °C until further CH3Hg(II) analysis. In an additional experiment to determine the effect of storage on solubilization, the digestive fluids of an individual sturgeon was frozen immediately, while a subsample of the fresh fluids was kept at room temperature for 2 days. The fresh fluids held at room temperature for 2 days, in conjunction with the thawed fluids, were incubated with CH3Hg(II)-spiked sediment and the solubilization compared. Exposure of Whole Body Channel Catfish to CH3Hg(II)-Contaminated Bloodworms. After routine maintenance and feeding, as described above, a total of six catfish were transferred individually to 10-L experimental microcosms and were allowed to acclimate to the experimental conditions, viz. salinity of 8‰, temperature of 23 ( 1 °C. On the day of the experiment, the fish were anesthetized with 100 mg/L tricaine (MS-222, Sigma, St. Louis, MO) for 5 min, after which ∼1.0 g of CH3Hg(II)-contaminated bloodworms was forcefed to the fish (n ) 3). The force-feeding procedure performed was similar to the technique outlined in Rouleau et al. (15). All fish kept their food after force-feeding. The water was changed and collected at 3, 16, 24, 36 h after oral dosage to prevent potential recontamination from any CH3Hg(II) released in the feces. Water and feces samples were stored in Teflon bottles and vials at -20 °C until further analysis to estimate the excretion of CH3Hg(II) by the fish. This allowed for estimation of the overall assimilation efficiency of CH3Hg(II) in each fish. The experiment was terminated at 36 h after feeding, and the fish were stored frozen at -20 °C until further CH3Hg(II) analysis. Analytical Techniques. All samples were thawed on the day of analysis and were digested with 50% H2SO4 and 20% KCl and distilled for CH3Hg(II) as previously described (1618). After ethylation derivitization, the volatile gaseous methylethylmercury was trapped onto Tenax-filled quartz columns, followed by gas chromatography separation and quantification by CVAFS, as previously described (17, 18). Routine quality assurance: quality control procedures were included in each analysis. Sample blanks were significantly lower than all sample concentrations; standard reference material (IAEA-142) samples were within the certified range
of the International Atomic Energy Agency (Monaco); sample duplicates were not significantly different; and between 90 and 120% of the CH3Hg(II) from CH3Hg(II)-spiked samples was recovered. The organic content of the sediment mixture and bloodworms was determined as the loss on ignition at 550 °C (13). The dissolved organic carbon (DOC) content of the gastric and intestinal fluids of the fish was determined following the method of Sugimura and Suzuki (19). The total hydrolyzable amino acids in the gastrointestinal fluids were determined by capillary gas chromatography with flame ionization detection (GC-FID) (20). Briefly, all samples were hydrolyzed (6 N HCl; 150 °C; 2 h), followed by esterification of the carboxyl groups with acidified 2-propanol (110 °C; 1 h). The amino, hydroxyl and thiol groups were derivatized by trifluoroacetic anhydride (110 °C; 10 min), dried under N2, dissolved in CH2Cl2, and analyzed by GC-FID. Statistical Analyses. A one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) means comparison test was used to verify differences (P < 0.05) between the CH3Hg(II)-spiked sediment and bloodworm incubations of both fish species and between the CH3Hg(II)-spiked and unspiked treatments in the bioaccumulation experiment.
Results and Discussion The Role of Enzymatic Reactions in CH3Hg(II) Bioavailability. There were no significant differences (P > 0.05, student t-test) between the total solubilization of CH3Hg(II) after incubation with fresh or thawed sturgeon gastric plus intestinal fluids (6.1% and 5.3%, respectively) suggesting that denaturing of the enzymes and other bioactive chemicals in the fluid due to the 2-d holding time had no impact on solubilization potential. Chen and Mayer (9) similarly reported that the extent of Cu solubilization was not different between untreated or heat-treated digestive fluids of deposit feeders in their solubilization incubations and suggested that solubilization was controlled by complexing agents in the digestive fluids, as any enzymatic processes would be destroyed by heating. The similarity in the results presented here suggests that solubilization is due to the presence of stable organic ligands in the fluid that complex and solubilize the CH3Hg(II) from the substrate and not due to enzymemediated processes. Effect of pH and Complexing Agents on Gastrointestinal Solubilization of CH3Hg(II). The percent CH3Hg(II) solubilized from sediment after addition of the gastric fluid to the incubation was low and not significantly different (P > 0.05) between the time points and the two species and ranged between 0.73 and 1.34%, overall (Figure 1a). The low solubility of CH3Hg(II) from sediment contrasts with the much greater solubility of CH3Hg(II) from bloodworms. Overall, the percent CH3Hg(II) solubilized from bloodworms in the gastric fluid incubations of both species was approximately 30- to 40fold higher than from sediment and ranged between 24 and 58%, overall (Figure 1b). In absolute terms, 5-10 times more CH3Hg(II) was released from the bloodworms even though the CH3Hg(II) concentration in the bloodworms was about five times less than that of the sediment. In general, the CH3Hg(II) solubilized within each treatment in the gastric fluid incubations, irrespective of species, was similar (P > 0.05), except for the percent CH3Hg(II) solubilized from bloodworms at 3 h (P < 0.05). The differences in the CH3Hg(II) solubilized in the bloodworm incubations at 3 h may be attributed to differences in the complexing agents of the two species or to different degrees of digestion of the tissue between species, as discussed below. Addition of the intestinal fluid to the gastric fluid incubations after 3 h resulted in a significant increase (P < VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Values of Stability Constants for the Species Used in the Speciation Calculations ligand Cl-
OHRSRCOORNH2
FIGURE 1. The percent methylmercury (CH3Hg(II)) solubilized from a) sediment and b) bloodworms, after incubation with Atlantic sturgeon or channel catfish gastric and intestinal fluids over time. Bars represent the mean ( SD (n ) 3); asterisks indicate significant differences between species in the same substrate, determined by ANOVA, followed by LSD (P < 0.05). 0.05) in the extent of CH3Hg(II) solubilized from sediment (Figure 1a), but only in a slight increase, which was not statistically significant (P > 0.05) from bloodworms (Figure 1b), for both species. After 5 h, the extent of CH3Hg(II) solubilized from bloodworms was 4.0-fold higher in sturgeons and 11.5-fold higher in catfish compared to sediment and indicate substantial differences in CH3Hg(II) solubilization from different substrates. Differences in the extent of CH3Hg(II) solubilization between the two species was also apparent, and overall, sturgeon fluids solubilized more CH3Hg(II) (P < 0.05) (Figure 1b). Mass balance was achieved in these experiments, within the error of measurement (10%), with all the CH3Hg(II) recovered either in the fluid or solid fractions. In this study, the measured pH in the gastric fluid incubations of sturgeon and catfish ranged between 1 and 2 and increased to 8 after addition of the intestinal fluids. The digestion of substrates (food) in the stomach of teleosts occurs mainly by the action of pepsin produced by the gastric glands (7), although the HCl produced in the stomach (21) determines the stomach acidity. Assuming that complexation controls solubilization, it would superficially appear that the extent of CH3Hg(II) solubilization in these fluids would be controlled by CH3Hg(II) binding to Cl- ions (K ) 105.25) under acidic conditions and to OH- ions (K ) 109.37) at the neutral pH of the intestine (Table 1; 22-24). However, the role of organic ligands needs to be considered. Based on the pKa values of the amino acids (25), most of the side chain functional groups for the amino acids present in the gastric fluids of both species would be protonated at pH 1-2 (see pKa values Table 2). The pKa of the thiolate (-SH) moiety of cysteine indicates that this binding site would be largely 5126
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species
log K
CH3HgCl CH3HgOH CH3Hg-SR CH3Hg-(OOCR) CH3Hg-NH2R+
5.25 (24) 9.37 (24) 15.7 (24) 4.5 (26) 7.3 (27)
deprotonated at pH 8, as would be the carboxylate (-COO-) moieties (see pKa values Table 2). Model calculations for CH3Hg(II) speciation in the gastric fluid incubations were done using the species and stability constants listed in Table 1 and the acid dissociation constants and concentrations in Table 2, assuming a pH of 2 and [Cl-] ) 10-2 M. The stability constants for RCOO- and RNH2 were estimated from Dyrssen and Wedborg (26) and Lindqvist (27), respectively. The following ligand concentrations for the dissociated forms of the amino acid groups were estimated, [RS-] ) 10-10.7 M; [RCOO-] ) 10-2.7 M; [RNH2] ) 10-10.8 M, assuming that only side chain ligands of the amino acids are available for complexation (Table 2). Contrary to initial expectation, our speciation calculations demonstrate that CH3Hg-SR is the most important dissolved species in the gastric fluid incubations of both species, i.e., even though the pH is 2, complexation of CH3Hg(II) to the thiol ligand of cysteine would dominate over complexation to chloride. At a pH of 8 (i.e., that of the intestinal fluids) and with [Cl-] ) 5 × 10- 3 M (due to dilution effects), we estimated the dissociated ligand concentrations to be [RS-] ) 10-4.8 M; [RCOO-] ) 10-2.7 M; [RNH2] ) 10-4.8 M, and calculations indicate that essentially all of the CH3Hg(II) in the dissolved form would be bound to thiols (CH3Hg-SR). Therefore, in both the stomach and intestine, dissolved speciation of CH3Hg(II) is not due to inorganic complexation, as suggested by others (28), but due to complexation to thiols. In addition, any digestion of protein would obviously increase the thiol concentrations and the resultant complexation. The lack of difference between the 3 to 5 h time points in the bloodworm incubations for each species was possibly due to the rapid digestion of the tissue and the release of CH3Hg(II) bound to amino acids of the tissue into the dissolved phase. The overall DOC concentrations in the digestive fluids of the two species were initially low (Table 2) and would have little influence on CH3Hg(II) complexation in the bloodworm incubations if substantial amounts of biochemicals (i.e., peptides, amino acids) were released as a result of tissue digestion. Generally, food is digested within a few hours of ingestion by the fish (29). Since bloodworms are composed primarily of soft tissue, they would be easily digested through chemical attack under acidic conditions. Therefore, the higher solubilization of CH3Hg(II) from bloodworms is most likely due to both “co-transport” of CH3Hg(II) with ligands released from the solid phase by tissue digestion and solubilization via complexation with gastrointestinal fluid compounds. As thiol groups are not part of the backbone structure of proteins, the molecular size (i.e., protein, peptide or free amino acids) has a small effect on their binding capacity. The higher CH3Hg(II) solubilized in bloodworms compared to sediment therefore probably reflects the easier digestion of bloodworms compared to sediment, although other factors may be involved. As mentioned above, the same effectiveness of dissolving the bloodworms was not seen in the sediment incubations. Sediment resists chemical attack except for the strongest chemicals (e.g., H2O2; HNO3) (30) and is more refractory and would not lose particle-associated CH3Hg(II) easily. Our model calculations predict that CH3Hg-SR complexes would
TABLE 2. Amino Acid Composition and Concentration in Gastric and Intestinal Fluids of Channel Catfish and Atlantic Sturgeon channel catfish amino acid alanine glycine threonine serine valine leucine isoleucine cysteine proline aspartate methionine glutamate phenylalanine tyrosine lysine arginine cystine total amino acids DOC (g/L)
pK1 r-COOH (25) 2.35 2.35 2.09 2.19 2.29 2.33 2.32 1.92 1.95 1.99 2.13 2.10 2.20 2.20 2.16 1.82 1.92
pK2 r-NH3+ (25) 9.87 9.78 9.10 9.21 9.74 9.74 9.76 10.70 10.64 9.90 9.28 9.47 9.31 9.21 9.06 8.99 10.70
pKR -SH (25)
gastric (M) 10-3
8.37
8.37
outcompete all other dissolved complexes in the gastric fluid incubations, but the relative distribution between the dissolved and the particulate phase would be controlled by the relative amount and strength of the binding sites in solution compared to that in the sediment matrix. This would control the degree to which organic material would be solubilized from sediment during the experiment. Clearly, the low pH and thiol content of the gastric juices is not sufficient to “dissolve” the CH3Hg(II) from the organic matter in the sediment. Under the higher pH of the intestine, the relative complexation strength of the ligands in solution is increased by about 6 orders of magnitude, based on our calculations, and this leads to more CH3Hg(II) being released from the solid phase under these conditions. The total amino acid (TAA) content of the gastric and intestinal fluids of sturgeon were, respectively, 1.5- and 1.3fold higher than in the fluids of catfish (Table 2), and the concentration of cysteine was relatively enriched in the gastric and intestinal fluids of sturgeon. In concert, at the end of the 5 h incubation, sturgeon digestive fluids displayed a greater ability to extract CH3Hg(II) from both substrates, viz. the percent CH3Hg(II) solubilized by the sturgeon digestive fluids was 3.7- and 1.3-fold higher (P < 0.05) in the sediment and bloodworm incubations, respectively (Figure 1). The difference in the amount of CH3Hg(II) solubilized in the gastric fluid incubations using sediment compared to that of the gastric-intestinal fluid mixture supports the notion that the availability of thiol groups to extract CH3Hg(II) from the substrate (i.e., sediment) most likely controls the bioavailability of CH3Hg(II) to the fish. That is, complexation is more important in solubilization of complex matrixes such as sediment compared to more easily digestible biological tissue. The percent CH3Hg(II) extracted from the sediment (∼3.6 to 17%) in this study is low compared to the percent CH3Hg(II) extracted from sediment (∼2 to 38%) using invertebrate digestive fluids (2, 3), at similar organic matter content (∼1%). This difference can be attributed to the differences in the amino acid concentrations in the fluids of the different organisms. The amino acid concentrations in the gastric and intestinal fluids of both catfish and sturgeon were at least an order of magnitude lower than found in invertebrate digestive fluids (3, 13). Sediment composition plays an important role in regulating the degree of CH3Hg(II) solubilization from sediment. Gagnon and Fisher (31) reported that the organic coatings on sediment and the binding strength of CH3Hg(II) to the
1.01 × 6.54 × 10-4 5.97 × 10-4 1.55 × 10-4 1.46 × 10-3 2.51 × 10-3 8.76 × 10-3 2.05 × 10-5 1.12 × 10-3 1.95 × 10-3 7.62 × 10-9 1.79 × 10-3 1.56 × 10-3 3.04 × 10-5 5.84 × 10-4 6.40 × 10-9 4.85 × 10-9 1.43 × 10-2 7.53
Atlantic sturgeon
intestine (M) 10-3
1.88 × 1.94 × 10-3 1.79 × 10-3 1.37 × 10-3 3.04 × 10-3 4.18 × 10-3 1.94 × 10-3 9.77 × 10-5 2.21 × 10-3 3.49 × 10-3 7.62 × 10-9 3.75 × 10-3 2.51 × 10-3 9.60 × 10-4 2.01 × 10-3 3.11 × 10-6 1.75 × 10-5 3.12 × 10-2 4.31
gastric (M) 10-3
1.25 × 1.00 × 10-3 1.39 × 10-3 1.13 × 10-3 1.76 × 10-3 2.80 × 10-3 1.11 × 10-3 5.89 × 10-5 1.80 × 10-3 2.87 × 10-3 3.73 × 10-6 2.67 × 10-3 1.92 × 10-3 6.44 × 10-4 1.05 × 10-3 5.10 × 10-6 4.90 × 10-6 2.15 × 10-2 2.34
intestine (M) 2.64 × 10-3 2.87 × 10-3 2.85 × 10-3 1.74 × 10-3 4.07 × 10-3 5.46 × 10-3 2.67 × 10-3 2.26 × 10-4 3.46 × 10-3 4.60 × 10-3 7.01 × 10-6 4.95 × 10-3 3.16 × 10-3 6.63 × 10-4 2.16 × 10-3 4.87 × 10-6 1.60 × 10-5 4.16 × 10-2 10.64
sediment contributed to the lower (
