Importance of Speciation in Understanding Mercury Bioaccumulation

Sep 17, 2010 - Suchrelationshipwasfurther confirmed by the uptake experiments conducted over a lower salinity range (0-6 psu), suggesting that HgCl2...
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Environ. Sci. Technol. 2010, 44, 7964–7969

Importance of Speciation in Understanding Mercury Bioaccumulation in Tilapia Controlled by Salinity and Dissolved Organic Matter RUI WANG AND WEN-XIONG WANG* Section of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

Received April 9, 2010. Revised manuscript received August 30, 2010. Accepted September 9, 2010.

We explored the roles of mercury speciation on the bioaccumulation (both aqueous and dietary uptake and elimination) of inorganic mercury (Hg[II]) and methylmercury (MeHg) in tilapia (Oreochromis niloticus) by controlling the mercury binding to inorganic and organic ligands. For the aqueous uptake, we showed that the uptake rates of Hg(II) were significantly higher at 0 psu compared with those at 10 psu and 28 psu. Based on the mercury-Cl complexes distribution, we found a positive relationship between the Hg(II) aqueous uptake rate and the abundance of neutral HgCl2. Such relationship was further confirmed by the uptake experiments conducted over a lower salinity range (0-6 psu), suggesting that HgCl20 was the predominant species taken up by tilapia. In the presence of dissolved organic carbon (DOC) from different sources (Suwannee River and natural local waters), mercury uptake rates all decreased dramatically over a wide range of salinity, especially for Hg(II), indicating the overwhelming influence of DOC as opposed to the single effect of salinity. Using the mercury-ClDOC model, we demonstrated for the first time that the inhibition of DOC was dependent on the Cl-, which was less significantatmiddlesalinitylevelforbothmercuryforms.Incontrast to the complex influence of water conditions on dissolved uptake, we found no significant influence of acclimated salinity on the dietary assimilation and elimination of both mercury species in tilapia. Our results demonstrated the importance of speciation in understanding the mercury bioaccumulation in various natural systems and its broad biogeochemical cycling.

Introduction The bioaccumulation of mercury in fish has raised great concerns over the past decades since the Minamata disaster took place in Japan in the 1950s. It has been well established that fish can accumulate mercury either directly from the water phase or from dietary food source (i.e., trophic transfer), and the biomagnification of organic mercury via food chain transfer can cause high risk to humans through fish consumption. A number of previous studies focused on the mercury accumulation of fish in various natural systems, and correlations have been found between mercury accumulation and several environmental factors, such as pH, * Corresponding author e-mail: [email protected]. 7964

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dissolved organic carbon (DOC), temperature, salinity, and competitive ions (1-3). However, the underlying mechanisms of such influences remain unclear at present. The water physicochemical properties can directly influence mercury speciation (4), thus further affecting the bioaccumulation process in aquatic organisms. Such influence might be largely due to changes of the bioavailability of dissolved mercury, which could directly affect the aqueous uptake at all trophic levels and even further affect the dietary uptake in higher level consumers via food chain transfer. Besides, some possible physiological changes of biota may also influence the accumulation process by affecting the dietary assimilation and elimination, which are usually less obvious and seldom studied (5). Although dietary uptake is usually considered as the major accumulation pathway for fish (6), the dissolved uptake can be more important than the dietary uptake under some circumstances (e.g., at a low feeding rate of fish (7)), especially for Hg(II). It has been predicted that aqueous uptake represents 18-68% of the Hg(II) accumulation in freshwater tilapia (7), and may be more important to lower trophic levels of biota such as freshwater zooplankton (31-96%) (8). Therefore, understanding the influence of mercury speciation on the whole bioaccumulation process (both aqueous and dietary uptake and elimination) is essential for the prediction of mercury levels in various biota from different aquatic systems. Under normal conditions, dissolved mercury exists as various complexes, either binding with inorganic or organic ligands. The inorganic complexation of mercury is dominated by chloride (Cl) either in freshwater or seawater systems, and the distribution of speciation (such as HgCl2, HgCl3-, HgCl42-) changes with salinity, especially at the low salinity range (4). Besides, DOC has been suspected to be the most important organic ligand for mercury in natural waters due to its strong binding with the reactive thiol group (9, 10), and the influence is complicated and highly related to its molecular size and structure. The bioavailability of mercury (both inorganic Hg[II] and organic MeHg) is then highly controlled by the distribution of dissolved complexes due to the specific uptake mechanisms of different species. For example, mercury-Cl can be taken up via passive diffusion as well as active transport (2, 3, 11), whereas mercury-DOC has a very low bioavailability and the possible uptake mechanism likely involves ligand exchange processes (2, 12). Therefore, the uptake of mercury is greatly dependent on its speciation in aquatic environments. Previous studies examined the effects of salinity and pH on the bioaccumulation in a marine diatom and in shore crab (1, 13), emphasizing the importance of neutral Hg species. The effects of DOC were complicated and highly dependent on its origin, concentration, and size. A decrease (2, 3), an increase (11, 14), and no effect (14) of DOC on Hg(II) uptake have all been found. Tilapia (Oreochromis niloticus) is an important commercial fish species widely cultured around the world. Its production has grown rapidly in recent years, especially in China (15). One of the main reasons underlying such rapid increase in tilapia farming is their high tolerance to salinity, making it possible for the fish to be cultured either in freshwater farms or coastal ponds. It has been reported that the tilapia O. niloticus can survive over a wide range of salinity (16), ranging from freshwater (0 psu) to seawater (30 psu), and therefore is an excellent model organism for investigating mercury bioaccumulation in euryhaline species. In this study, we first investigated the role of Hg speciation on its waterborne uptake by tilapia. Changes in Hg speciation 10.1021/es1011274

 2010 American Chemical Society

Published on Web 09/17/2010

were made by controlling the salinity (in terms of chloride) and DOC in the medium. We hypothesized that the influences of water chemistries (salinity and DOC) on mercury uptake were due to the changes in Hg speciation, especially the mercury-Cl and mercury-DOC. We first tested the influences of salinity without the presence of DOC by altering the distribution of inorganic mercury-Cl complexes. Subsequently, we examined the combined effects of inorganic mercury-Cl and organic mercury-DOC complexes on aqueous uptake as explained by a mercury-Cl-DOC model. Moreover, the influences of physiological changes caused by various water conditions on the dietary assimilation and elimination were also quantified.

Materials and Methods Fish and Radioisotopes. The tilapia (Oreochromis niloticus, 3-10 g wet weight and 5-8 cm long) were collected from a local fish farm in Yuen Long, Hong Kong. After they were transported to the laboratory, the fish were first maintained in 180 L of circulating dechlorinated tap water with continuous aeration and fed with commercial fish food for more than one week. Then approximately 150 individuals were removed and placed into another two 180-L freshwater tanks with continuous aeration. The salinity of each tank was increased gradually at the rate of about 2-3 psu per day by adding seawater every 2 days until 10 psu was reached in one tank and 28 psu was reached in the other. Fish were then acclimated under these salinities for one more month at 25 °C. Water salinities were measured and adjusted periodically. There was no mortality during the acclimation period. Radiotracer techniques with nondestructive measurements were used in this study. The radioisotope 203Hg(II) (t1/2 ) 46.6 d, in 1 M HCl, specific activity )45.7-152 GBq g-1) was purchased from Riso National Laboratory, Denmark. Me203Hg was synthesized from the 203Hg(II) following a well-established method (17), and stored in 0.005 M Na2CO3 solution. Waterborne Uptake of Hg(II) and MeHg. To control the metal speciation (including mercury-Cl and mercuryDOC complexes) precisely, we conducted three different uptake experiments using various water media. A standard synthetic freshwater (USEPA (18)) and an artificial seawater (19) were prepared using analytical grade chemicals dissolved in Milli-Q water (18.2 mΩ). Three salinities (0, 10, and 28 psu, corresponding to 0, 160, and 448 mM NaCl, respectively) were then obtained by mixing these two artificial waters. Uptake experiments were first conducted in these water media to specifically determine the effect of inorganic mercury-Cl complexes (in the absence of DOC) over a wide range of salinity, in an attempt to identify the most bioavailable mercury form. According to the diagram of mercury-Cl complexes distribution (4), the speciation of Hg(II) changes rapidly at low salinity values, thus we further tested the Hg(II) uptake at 0, 2, and 6 psu. The combined effects of salinity and DOC were investigated by adding standard humid acid (0.30 mg C/L, Suwannee River) into those artificial waters (henceforth modified artificial waters), to specifically quantify the roles of both inorganic mercuryCl and organic-DOC complexes on the aqueous uptake. Furthermore, we tested the aqueous uptake using natural waters to see whether the effects of DOC were similar regardless of the origin. Three different salinities (0, 10, and 28 psu), representing the typical natural water conditions in freshwater, estuary, and marine systems, respectively, were prepared by mixing 0.22 µm of filtered pond water (collected from a mountain spring on the campus of the Hong Kong University of Science and Technology, N 22.3367°, E 114.2667°) and 0.22 µm of filtered natural seawater (collected from Clear Water Bay, Kowloon, Hong Kong). The related physiochemical parameters of the pond water were as follows:

pH, 8.2; Ca2+, 24 mg L-1; background dissolved Hg concentrations, 0.08-0.18 ng L-1 (8, 20). The DOC concentrations of these three mixed water media (0, 10, and 28 psu) were determined as 2.21, 1.95, and 1.48 mg C L-1, respectively. All the tested waters were aerated overnight before the experiments with their pH values adjusted to 7.3-7.5 (standard synthetic freshwater pH value) for the 0 psu treatment and 8.3-8.5 (typical pH value in artificial seawater) for the other two treatments using 1 N HNO3 or 1 N KOH. Previous studies showed that the Hg uptake rates would not be influenced by these small variations of pH (7). Tilapia of similar sizes (5-7 g) were chosen for the uptake experiments. The fish acclimated at different salinities (0, 10, and 28 psu) were exposed to the water media described above with spiked radioisotopes 203Hg(II) and Me203Hg at certain concentrations (60 ng L-1 for Hg[II] and 20 ng L-1 for MeHg) for 8 h to quantify the dissolved uptake rates. In the low salinity treatments (0, 2, and 6 psu), freshwateracclimated fish were used to minimize the possible physiological changes under different acclimation conditions, because the freshwater tilapia can survive in a wide range of salinity conditions (16) without acclimation. The test waters were prepared one night before each experiment by adding specific amounts of isotopes to reach the equilibrium. The fish were acclimated in the same water media (without isotope spike) one night before the experiments to evacuate their gut contents and were then added randomly into each 1-L beaker following 8 h exposure without food. Our preliminary studies showed that the walls of the beakers adsorbed Hg(II) but not MeHg. Thus, to minimize the potential adsorption, we used Teflon beakers for the Hg(II) uptake experiments and polypropene beakers (21) for the MeHg uptake experiments. During the 8-h exposure period, the waters were not aerated and the fish were removed individually at 2-h intervals, and rinsed in nonradioactive water medium for 1-2 min to remove the weakly adsorbed radioisotopes. The radioactivity of the whole fish body was measured nondestructively and the radioactivity of the tested water medium was also measured at each time point. During the short exposure period, the mercury concentration in the water medium decreased by less than 20%. The uptake rate (ng g-1 h-1) was calculated as the slope of a linear regression between the accumulated metal concentrations in the whole fish body and the exposure time. All the calculations were based on dry tissue weight. Water Permeability. The water permeability of the tilapia was measured under different salinities (0, 10, and 28 psu) by using the artificial waters (without adding humic acid) as described above. Ten microcuries of 3H2O was spiked into each beaker containing 1 L of water medium. The fish acclimated at different salinities were then placed into their corresponding water medium for 2 h without aeration. The tilapia were then removed, rinsed, and dissected into four parts (gills, viscera, head, and the remaining carcass). The tissues were weighed and digested using 5 mL of tissue solubilizer (SOLUENE-350, Perkin-Elmer) at 55 °C overnight in 20-mL liquid scintillation vials. The total volumes of clear tissue solutions were quantified and 2 mL was sampled for measurements. The waters were also sampled at the beginning and end of the uptake period. The water and tissue solution samples were thoroughly mixed with 18 mL of liquid scintillation cocktail (OptiPhase Hisafe 3, Perkin-Elmer Life Science, Turku, Finland) by mechanical shaking. The radioactivity of all the samples was measured with a Beckman LS-6500 Liquid Scintillation Counter. A series of standard water samples were also measured following the same method mentioned above, and were used for quantification and data correction. Each treatment had five replicates. The counting times were adjusted to ensure any propagated counting errors were kept to less than 5%. VOL. 44, NO. 20, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Dietborne Assimilation Efficiency and Efflux. To quantify the assimilation efficiency (AE) of mercury in fish, the oligochaete worm Tubifex tubifex was chosen as the fish diet. A well developed pulse-chase technique was applied (22). Briefly, prey was radiolabeled for two days and pulse-fed to tilapia, followed by a 48-h depuration period in nonradioactive pond water. To radiolabel T. tubifex, about 1000 healthy individuals were selected and maintained in 500 mL of gently aerated pond water containing 100 ng L-1 of spiked 203Hg(II) or 30 ng L-1 of spiked Me203Hg. The worms were radiolabeled for two days, then removed and rinsed in fresh pond water to remove the weakly bound radioisotope before being fed to fish. Tilapia of similar body sizes were picked and kept in pond water to clear their guts one day before experiments. The fish were then placed individually in beakers, and fed with radiolabeled T. tubifex for 30 min at a food concentration of 100 mg L-1. After the pulse feeding, fish were removed, rinsed, and radioassayed for the ingested mercury individually. Each individual was then placed in 1 L of natural pond water and depurated for 48 h. During the depuration period, the remaining radioactivity in the fish was periodically measured and the water media were renewed every 12 h to minimize the possible releasing of radioisotopes. The fish were fed with nonradioactive T. tubifex at 24 h. In the efflux experiment, the worm T. tubifex was radiolabeled with Hg[II] and MeHg for 2 days using the method described above. The feeding period lasted for 7 days with a daily feeding rate of about 10% of fish body weight. After that, the fish were placed in 1 L of pond water individually for 12 h to evacuate their gut content and then radioassayed, followed by a 30-day depuration period. During the depuration time, the fish were fed with nonradioactive fresh T. tubifex every day, and the radioactivity of each individual was measured periodically. The water medium was renewed every 3-5 days.

FIGURE 1. Aqueous uptake rates of Hg(II) and MeHg in O. niloticus measured over 8-h exposure in artificial waters and natural waters with different salinity and DOC conditions. A: artificial waters (without DOC); B: modified artificial waters (with added standard humic acid, 0.30 mg C L-1, Suwannee River); C: natural waters (2.21, 1.95, and 1.48 mg C L-1 for the 0, 10, and 28 psu, respectively). Data are mean ( standard deviations (n ) 4). Bars with different letters are significantly different (p < 0.05).

Results and Discussion Hg Aqueous Uptake in Hg-Cl Complexes System. The uptake experiments were first conducted over a wide range of salinity (0, 10, and 28 psu), and various distributions of mercury-Cl complexes were obtained by altering the salinity of artificial waters without adding DOC. Figure 1A shows that the uptake rates at 0 psu for both mercury species were significantly higher than those at 10 and 28 psu. The uptake at 28 psu was slightly higher than that at 10 psu, which might be due to the fish drinking up some of the seawater to compensate for osmotic water loss (23). Besides mercury, the aqueous uptake of other metals (such as Ag, Cd, Se, Zn) by fish exhibited similar responses to salinity, e.g., decreased with increasing salinity (24-26). The significant decrease in mercury uptake from freshwater to saline water may be due to the changes in the Hg speciation distribution as well as some possible physiological changes of the fish during longterm acclimation. For inorganic Hg(II), the mercury speciation changes greatly as the chloride concentration increases (4), especially within the low chloride range (0-0.1 M chloride). The neutral HgCl2 is the dominant species at low salinity, and increases initially with salinity until it peaks at 2 psu (0.03 M chloride) and then plunges to a constant level. With the increase in salinity, the negatively charged complexes (HgCl3-, HgCl42-) become more dominant. Among the different mercury-Cl complexes, we found that only the decrease in neutral HgCl2 was consistent with our uptake results, implying that neutral HgCl2 was probably the most bioavailable mercury species. To further confirm this hypothesis, we measured the uptake rates at very low salinity values (0, 2, and 6 psu), at which the abundance of neutral HgCl2 changes rapidly. The same acclimated batch of tilapia was used to minimize the influences of physiological changes during the acclimation. 7966

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FIGURE 2. Aqueous uptake rates of Hg(II) in O. niloticus measured over 8-h exposure in modified artificial waters at low salinity (0, 2, and 6 psu), in the absence of DOC, showing a positive relationship with the abundance of neutral HgCl2. Data are mean ( standard deviations (n ) 4). As we expected, the changes in Hg(II) uptake rates were consistent with the HgCl2 speciation (Figure 2), i.e., initially increased and then decreased with the salinity gradient (0, 2, and 6 psu) and topped out at 2 psu, again indicating the important role of the neutral species. Previous studies have suggested that the neutral HgCl2 favors passive diffusion through biological membranes because of its lipophilicity, and was the main pathway of Hg(II) aqueous uptake in phytoplankton (13). MeHgCl and MeHgOH are the most important MeHg species, with MeHgCl dominating in all saline waters (pCl < 3, equivalent to salinity higher than 0.06 psu; (13)). In our study, MeHgCl was less abundant at 0 psu, and was comparable between 10 and 28 psu (>90%). Some previous studies suggested that MeHgCl was the most bioavailable MeHg species, and was taken up either by passive diffusion (1, 13) due to its higher lipophilicity than MeHgOH or by active uptake (14). In our study, the dissolved uptake rates of MeHg were 1.4-1.9 times higher than those of Hg(II),

TABLE 1. Water Permeability in Different Tissues of O. niloticus, Shown as the 3H2O Uptake Rates (Data are Mean ± Standard Deviations (n = 4)) H2O uptake rates in fish tissues (µL g-1 h-1 wet wt.)

3

treatment 0 psu 10 psu 28 psu

gill

viscera

head

muscle

2.53 ( 0.39 0.85 ( 0.20 0.22 ( 0.05 0.23 ( 0.04 1.58 ( 0.21 0.59 ( 0.13 0.08 ( 0.04 0.19 ( 0.04 0.97 ( 0.12 0.55 ( 0.16 0.10 ( 0.06 0.16 ( 0.06

despite its tested concentration being 3 times lower. However, the increase in MeHgCl could not explain the sharp decrease in MeHg uptake at 10 and 28 psu as compared to that at 0 psu. Thus, MeHg dissolved uptake may also be controlled by other factors in addition to its speciation (discussed below). Besides metal speciation, the potential physiological changes should also be considered. For example, the metal uptake could be influenced by the osmoregulatory changes, such as water permeability, in the organisms. To compensate for osmotic water and ion loss, marine fish drink water and have some accompanying accelerated ion uptake mechanisms, which may affect the dissolved metal uptake. Besides, the morphology and cellular activity of chloride cells change with salinity (27), and mercury uptake may be via the chloride transporters (reported in rat, (28)). In this study, we tested the water permeability in tilapia under various salinities (Table 1). The water uptake of gills after 2-h exposure was significantly higher at lower salinities, and exhibited a good negative correlation with salinity (r2 ) 92%). Interestingly, the water uptake in viscera was found to be even higher in freshwater fish, indicating that the drinking by seawateracclimated fish was negligible compared to the faster water transport in freshwater fish. Furthermore, if the mercury uptake is only via passive diffusion, the uptake rates are expected to have a consistent pattern with water permeability. In our uptake experiments, the uptake rates of seawater were slightly higher than that of middle salinity water, which could not be solely explained by the single passive diffusion of neutral HgCl2 and MeHgCl. This indicates that other uptake mechanisms may also be involved. Some previous studies showed that mercury uptake by isolated organs of blue crab were reduced by decreasing the water temperature and by Na+/K+ ATPase inhibitors (29), and that photosynthetic inhibitors decreased the uptake in algae (30). These suggest that energy was at least partially involved in mercury uptakes. Hg Aqueous Uptake in Hg-DOC System. To explore the role of mercury-DOC complexes, uptake experiments were conducted both in modified artificial waters (by adding humic acid) and natural waters (representing natural DOC). During the 8-h water exposure, the tilapia exhibited a linear uptake of either mercury species in all the test waters. Typically, the uptake of both Hg(II) and MeHg was inhibited by the presence of DOC. After adding standard humic acid (0.30 mg C L-1, Suwannee River) to the artificial waters, the uptake rates of Hg(II) at different salinities decreased by 54-82%, while the uptake rates of MeHg decreased by 7-58% (Figure 1A and B). In natural waters (Figure 1C), we observed much slower uptake rates of Hg(II) and MeHg as compared to those determined in modified artificial waters (over 100 times higher for Hg[II] and 15-27 times higher for MeHg). Besides the 5-7 times difference in DOC concentration, such great difference in the degree of inhibition might be due to the different DOC origin and binding affinity. Because the inhibitory effects of DOC are presumably due to the strong binding with reactive thiol functional groups, which constitutes only a small fraction of DOM molecules (10), the interaction between DOC and mercury could be largely determined by the DOC type (ligand-rich or ligand-poor) (9).

In our experiments, the inhibitory effects of DOC were dependent on mercury species. The uptake rates of MeHg determined in modified artificial waters (with humic acids) were 15-27 times higher than those tested in natural waters, while over 100 times differences were found for Hg(II), suggesting that the uptake of MeHg was less affected by DOC. This observation was further confirmed by comparing the dissolved uptake rates in artificial waters, where the decrease was greater for Hg(II) (54-82%) than for MeHg (7-58%) with the addition of humic acid (Figure 1B). Similarly, Laporte et al. (2) found that humic acids had a greater inhibition on Hg(II) uptake than on MeHg uptake for blue crabs. The differential inhibition of DOC on Hg(II) and MeHg uptake may also be due to their different uptake mechanisms. It has been shown that Hg(II) and MeHg uptakes were by passive diffusion, but mercury-DOC complexes were unlikely to transport across the biological membrane. A ligand exchange may be required to exchange MeHg from the complex to an uptake site (2, 12). The DOC inhibitory effect could then be explained by the competitive binding of mercury between the external DOC and binding sites on the membrane. MeHg has a relatively lower affinity to DOC (2) but higher permeability to lipophilic biolayers as compared to Hg(II), which could result in reduced competition for DOC and a less inhibited uptake. Hg Aqueous Uptake in Hg-Cl-DOC System. We further explored the control mechanisms of Hg speciation on mercury uptake based on a mercury-Cl-DOC model (4, 9). The hydrophobic complexes such as HgOHCl and MeHgOH were not considered because they were negligible within our tested pH range (7.5-8.5). According to the model, the distribution of mercury complexes is highly dependent on the ligand abundance of DOC as well as the ligand strength. The ligand abundance can be presented as the ligand to DOC molar ratio, and the mercury-DOC is more likely to be the dominant species if the DOC has a high L/DOC molar ratio, indicating that the DOC type could influence the mercury speciation. Typically, the mercury-DOC complex is dominant in high DOC and low Cl conditions, whereas the mercury-Cl complex tends to dominate in high Cl and low DOC conditions. However, this assumption can only be applied to an ideal environment in which the organic ligand strength does not change with salinity. Under realistic conditions, the organic ligand strength K′ is salinity-dependent, and normally increases with salinity (9). This could enhance the importance of mercury-DOC complexes at high chloride level, and even make it the dominant species. Considering the variation in organic ligand strength with salinity, the model predicted a minimum mercury-DOC complex at 15 psu (9). Our experiments showed consistent results with the model prediction described above, that the uptake rates were always the highest at middle salinity in the presence of DOC (10 psu). To better express the inhibitory effects, we compared the uptake rates in the presence and absence of DOC. We assumed that the uptake rates determined in the absence of DOC represented the maximal uptake capability (100%), and then normalized the uptake rates as kn, shown in Table 2. Therefore the inhibition of DOC can be expressed as the decrease in normalized uptake rates (1 - kn). For Hg(II), the uptake rates in modified artificial waters (with humic acid added) decreased to a greater extent at 0 and 28 psu (decreased by 80-82%) as compared to that at 10 psu (by 54%). Although the uptake in natural waters showed a much greater inhibitory effect by DOC, a surprisingly similar trend was still observed, that the dissolved uptake rates determined in modified artificial waters were 104-105 times of that determined in natural waters at all salinities (Table 2). For MeHg, the decrease generally followed a similar trend also. With the presence of additional humic acid (0.30 mg L-1), VOL. 44, NO. 20, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Normalized Dissolved Uptake Rates Calculated under Various Conditions, With or Without the Presence of Organic Ligands (Humic Acid and Natural DOC), Assuming That the Uptake in the Absence of DOC (Conducted in Artificial Waters) Represents the Maximal Uptake Capability normalized uptake rates kn DOC inhibited treatment

Hg(II) MeHg

0 psu 10 psu 28 psu 0 psu 10 psu 28 psu

without DOC

humic acid

natural DOC

100% 100% 100% 100% 100% 100%

18% 46% 20% 69% 93% 42%

0.17% 0.44% 0.19% 2.70% 6.30% 1.50%

the uptake rate at 10 psu was only slightly reduced (by 7%), while the inhibitory effect became more obvious at 0 psu (decreased by 31%) and 28 psu (decreased by 58%). In natural water systems, the uptake rates decreased by 97%, 94%, and 98% at the salinities of 0, 10, and 28 psu, respectively, again indicating that the MeHg uptake was less affected by DOC at middle salinity level. Overall, our experiments demonstrated that DOC could inhibit Hg uptake at all salinities, and could even mask the effect of salinity. Moreover, we found that the influences were less important at the middle level of salinity (10 psu), which was consistent with the prediction by the mercuryCl-DOC model when considering the influences of salinity on organic ligand strength. According to the model, the organic ligand strength under higher salinity is stronger, and the dominance of mercury-DOC will then increase, resulting in more obvious inhibited uptakes. Therefore, this model could help explain the uptake results determined in natural waters, where the apparent uptake rates at 10 psu were found to be similar to or even higher than those for freshwater for both mercury species (Figure 1C). Hg Assimilation and Elimination at Different Acclimated Salinities. We also investigated the influence of water conditions on dietary mercury uptake in fish. To explore whether the acclimated salinity affected the trophic transfer and elimination of Hg, we measured the AE and efflux rate of tilapia. In the AE experiment (Figure 3A and B), the ingested Hg(II) decreased rapidly within the first 36 h of depuration and then stayed almost constant, whereas MeHg was released very slowly throughout the entire 48 h of depuration. The AEs were calculated as the percentage of Hg remaining in fish at 36 h (23-47% for Hg(II) and 83-93% for MeHg). No significant difference was observed among different acclimated salinities for either mercury species. In the efflux experiment, only 9-12% of Hg(II) remained in the fish while 88-91% of MeHg remained after 30 days of depuration (Figure 3C and D). The efflux rate constant (ke) was calculated from the slowest-exchange compartment (12-30 days of depuration). Compared to MeHg, the calculated ke of Hg(II) was 5.2-6.2 times higher, indicating a faster elimination process of Hg(II). However, the difference in efflux rates among different salinities was not significant. Our results suggested that the potential salinity-caused physiological changes did not influence the dietborne mercury uptake and elimination. Similarly, Ni et al. (24) found that the dietary assimilation and elimination of Cd, Se, and Zn in the intertidal mudskipper (Periophthalmus cantonensis) were not influenced by salinity within a range of 10-30 psu. Although food is an important exposure pathway for fish (especially MeHg), the accumulation process was hardly influenced by water conditions but affected significantly by the food conditions (quality and quantity, (31)). In contrast, the Hg dissolved 7968

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FIGURE 3. Dietary assimilation and efflux of Hg(II) and MeHg in O. niloticus in natural waters with different salinity and DOC conditions (2.21, 1.95, and 1.48 mg C L-1 for the 0, 10, and 28 psu, respectively). Assimilation showing as A and B: retention of Hg(II) and MeHg in O. niloticus following a pulse feeding with radiolabeled oligochaete T. tubifex, during 48 h depuration; Efflux showing as C and D: retention of Hg(II) and MeHg in O. niloticus following 7 d of feeding on radiolabeled oligochaete T. tubifex, during 30 days depuration. Data are mean ( standard deviations (n ) 5-6). uptake (especially for Hg[II], for which dissolved uptake contributed 18-68% of its bioaccumulation in O. niloticus, (7)) was largely influenced (over two orders) by water conditions. Therefore, water environmental conditions may contribute to the variations of Hg levels in fish living in various aquatic environments. To conclude, the aqueous uptake of Hg(II) and MeHg by tilapia was greatly influenced by salinity and DOC due to the changes in Hg speciation (mercury-Cl and mercury-DOC complexes), whereas dietary uptake and elimination were not affected by the acclimated salinity. In the absence of DOC, the fish generally had higher uptake in freshwater than at higher salinity as a result of the increasing importance of neutral HgCl2 species, suggesting that mercury uptake was presumably by passive diffusion. In the presence of DOC, the uptake was under the control of both inorganic mercury-Cl and organic mercury-DOC complexes. DOC inhibited the uptake over a wide range of salinity (0-28 psu), especially for Hg(II). This study for the first time demonstrated that the inhibition of DOC is dependent on the chloride, which is consistent with the prediction by the mercury-ClDOC model. Due to the less obvious protection by DOC, the uptake rates at middle salinity levels could be even higher than those in freshwater. Our results also may be applicable in explaining the different mercury levels in other aquatic organisms living under various environments (freshwater, estuary, and marine systems).

Acknowledgments We thank the anonymous reviewers for their comments on this work. This study was supported by a General Research Fund (663009) and a Collaborative Research Fund (HKBU 1/07C) from the Hong Kong Research Grants Council.

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