Reconstructing the Biokinetic Processes of Oysters to Counteract the

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Reconstructing the Biokinetic Processes of Oysters to Counteract the Metal Challenges: Physiological Acclimation Ke Pan and Wen-Xiong Wang* Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong S Supporting Information *

ABSTRACT: Oyster Crassostrea hongkongensis, a widely cultivated oyster species in Southern China, can accumulate metals (especially for Cu and Zn) to extraordinarily high concentrations (up to 3% of body dry weight). It remains unknown how they were acclimated to contaminated environment and built up such high metal concentrations in their bodies. A seven month transplantation experiment was conducted to rebuild the physiological process of acclimation in oysters to illustrate how they cope with increasing metal bioavailability. The metal concentrations increased substantially in the transplanted oysters from a reference site to a contaminated site. Our results showed that metal biokinetics in the oysters changed dramatically after suffering from metal stress. The clearance rate, dissolved uptake rate (for Cd and Zn), and metal assimilation efficiency (for Zn) was depressed, while the metal efflux rate (for Zn) was enhanced in the contaminated oysters. Beside the change of metal homeostasis, the oysters were able to sequester metals into subcellular nontoxic forms and maintain a low portion of metals distributing in the metal-sensitive fraction. This comparative bioaccumulation study of C. hongkongensis suggested that adjustment of metal biokinetics played an important role in the survival of oysters in metal contaminated environment.

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INTRODUCTION Aquatic invertebrates can develop a variety of resistance mechanisms, allowing their survival in metal contaminated habitats.1−3 The species polychaete Nereis diversicolor, crab Carcinus maenas, bivalve Scrobicularia plana, and amphipod Corophim volutator found in the metal-rich Restronguet Creek of England provided robust evidence of defense and acquisition of tolerance to metals in aquatic invertebrates.4−6 The recent report of blue oyster incident by Wang et al. is another example how the aquatic invertebrates counteract the metal challenges.7 The metals in the blue oysters Crassostrea hongkongensis from a metal contaminated site in Jiulong River Estuary, southeast China, contained extraordinarily high concentrations of metals (Cu levels of 14,000 μg g−1 and Zn levels of 24 000 μg g−1).7 Oysters are considered as the hyperaccumultors of Cu and Zn, and also have high potentials to accumulate other metals such as Cd. They however can well adapt to high levels of metal exposure and survive in metal contaminated environments,8−10 indicating that oysters may have well established strategies to acclimate to increasing metal bioavailability. However, the mechanisms underlying this acclimation process remained largely unexplored. Intracellular storage as detoxified forms (metallothioneinMT, lysosomal derivatives of MT, phosphate-based granules, sulphide complexes) is generally regarded as the physiological basis for metal tolerance in aquatic invertebrates.2,12 Evidences suggested that Cu increased its partitioning into insoluble form © 2012 American Chemical Society

(cellular debris), whereas Zn increased its partitioning into metallothionein-like protein (MTLP) in response to increased metal levels.7 However, increasing intracellular detoxification of metals may be only one of the multiple processes for oysters to acclimate to metal challenges. The role of alteration of metal homeostasis triggered by metal stress in building up the metal tolerance in oysters cannot be ruled out. It is increasingly recognized that the influx rate of metals is important for determining the metal toxicity.13−15 Toxicity is triggered when the rate of uptake of a trace metal (combined uptake from solution and diet in the case of aquatic invertebrates) exceeds the combined rates of excretion and detoxification of that metal.14 Thus, change of metal biokinetics can be another way for organisms to combat with the metals. There are however few field data to test this hypothesis, although many studies found changes of metal biokinetics in organisms after laboratory metal exposure .16 There is also little consistency in the literatures on the processes involved in the manifestation of the tolerance.17 Earlier studies indicated reduced accumulation rate of metals in aquatic organisms from metal-rich sites,4,18 but whether such reduced accumulation was caused by reduced uptake or by increased excretion Received: Revised: Accepted: Published: 10765

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was measured using a Wallac 1480 NaI (T1) gamma counter (Wallac, Turku, Finland). All counts were related to standards and spillover. The gamma emission of 109Cd was determined at 88 keV and that of 65Zn at 1115 keV. Counting times were adjusted to yield a propagated counting error of less than 5%. To study the metal uptake from the dissolved phase, individual oyster was exposed to 400 mL of 0.22 μm filtered seawater containing 2 μg L−1 Cd and 10 μg L−1 Zn in a 500 mL polypropylene beaker. The exposure medium was prepared by mixing filtered seawater and a stock metal solution (2 μg mL−1 for Cd and 10 μg mL−1 for Zn) containing 7.4 kBq mL−1 radioactive tracers (109Cd and 65Zn). During the exposure period, the radioactivity in the seawater was monitored by taking an aliquot of 3 mL medium for counting. The metal concentrations in the medium were maintained by addition of the stock solution based on the decrease of radioactivity. Slight aeration was provided to avoid oxygen depletion and helped to mix the newly added metals. The level of metal concentrations did not drop beyond 20% throughout the exposure period. At 2, 3, 4, 5, 6 h, five individual oysters were randomly sampled and rinsed with nonradioactive seawater. The oysters were dissected for soft tissues which were dried at 80 °C after the radioactivity measurement. The dissolved uptake constant (ku) of Cd and Zn for individual oyster was calculated from

(or both) remained unknown. It is still arguable whether tolerant species can reduce uptake and enhance efflux when challenged with raised metal bioavailability. For example, Rainbow et al. reported that the mean metal uptake rates of amphipods Orchestia gammarellus, crabs Carcinus maenas and Pachygrapsus marmoratus from Restronguet Creek, Southwest England, were not lower than those of the same crustaceans from a control site.6 In a recent study on the worm N. diversicolor, there was no evidence of decreased Zn uptake or enhanced Zn efflux.17 In this study, we investigated the role of metal biokinetics in the acclimation of oyster C. hongkongensis to a metal contaminated site. We hypothesize that the metal biokinetics in the oysters may change in response to increasing metal bioavailability. To test this hypothesis, we carried out a seven month transplantation experiment to rebuild the physiological acclimation process how the oysters counteracted the metal challenges after translocation to contaminated environment. A batch of oysters was transplanted from a reference site to a contaminated site, and metal accumulation and subcellular distribution were monitored on a monthly basis. We then compared the metal biokinetics including the dissolved uptake, dietary assimilation efficiency, and efflux between the transplanted and reference populations.

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MATERIALS AND METHODS Field Sampling of Oysters and Transplantation. To study the metal accumulation and its related effects in oysters C. hongkongensis between reference and contaminated sites, a 7month transplantation experiment was conducted from March to September, 2011. A batch of clean juvenile oysters (about 500 individuals, 2−3 cm shell heights, transplanted oysters) was transplanted from a reference site (Jiuzhen, JZ) to a highly contaminated site (Bai Jiao, BJ) in Jiulong River Estuary. Early study reported an incidence of blue coloring oysters (resident ́ , 24°13− ́ ́ 118° 02E oysters) found at this site (116°47− ́ ), which was caused by multiple metal contaminations 25°51N (especially Cu and Zn).7 The reference site is about 100 km away from the contaminated site and is far from the industrial area. Another batch of juvenile oysters of similar size was kept in the reference site as the control group (reference oysters). Each month, about 30 oysters were brought back from the reference and contaminated site to the laboratory to defecate gut contents for at least 48 h, among which 15 oysters were randomly sampled and frozen at −80 °C prior to analysis. For each group of oysters, ten individuals were dissected for measurements of total metal concentrations, and five oysters were subjected to subcellular metal analysis as described below. To compare the dissolved metal concentrations at each site in every other month (March, May, July, and September), the diffusive gradients in a thin film (DGT) units were deployed. The deployment, retrieve, and analysis of the DGT units were described as elsewhere (also see Supporting Information (SI)).19 Biokinetics of Cd and Zn in Oysters from Reference and Contaminated Sites. To study the difference of metal biokinetics in reference and transplanted oysters, C. hongkongensis of 2−3 cm shell length prior to and after the transplantation experiment were brought back to the laboratory and acclimated in circulating seawater for 5 days (temperature: 20 °C; salinity: 25 psu) with addition of diatoms Chaetoceros gracilis as food. Due to the availability of radioisotopes, only the biokinetics of Cd and Zn were studied here. The radioactivity

ku =

A tissue SA × W × C w × t

where Atissue is the radioactivity in the whole soft tissue after exposure (ccpm), SA is the specific activity of the metals in the seawater (ccpm μg−1), W is the dry weight of the soft tissue (g), Cw is the exposure concentration, and t is the exposure time (day). To study metal uptake from the dietary phase in oysters, the log phase diatom C. gracilis was radiolabeled with 74 kBq L−1 109Cd and 65Zn in f/2 medium with N, P, Si, vitamins and metals minus Zn, Cu, and EDTA for three days. After radiolabeling, the algae were resuspended using nonradioactive filtered seawater to minimize any desorption of radiotracers during the radioactive feeding. The radiolabeled algae was fed to oysters at a concentration of 5 × 104 cells mL−1 for 30 min, after which the oysters were rinsed thoroughly with filtered seawater and assayed for their initial radioactivity. The radioactivity in each oyster was counted at frequent time intervals over 48 h. The metal’s AE was determined as the percentage of the initial radioactivity retained in the oysters after 48 h of depuration. To measure the efflux rates of Cd and Zn in oysters, the oysters were placed in 2 L beaker containing 1.5 L seawater. Radioisotopes 109Cd and 65Zn were added at 37 kBq L−1, and radiolabeled diatoms C. gracilis obtained as mentioned above were added to the beaker simultaneously. The exposure medium was changed twice a day and the radioactive diatoms were added likewise at each time. The labeling procedure was repeated for six days. On the seventh day, all oysters were rinsed thoroughly, after which the initial radioactivity of oyster was measured. The oysters were then transferred to a 10 L recirculating seawater system containing nonradioactive seawater with aeration and depurated for 27 days. The radioactivity in the oysters was assayed daily throughout the experiment. The radioactivity remained on the shells only represented a small portion (5−10%) of the overall radioactivity and had little effect on the calculation of efflux rate. The efflux rate constant (ke, d−1) was calculated from the slope of 10766

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differences (p < 0.01) between the reference and transplanted/ resident oysters.

the regression between the natural log of the percentage of metal retained in the slow exchanging compartment in the oysters and the time of depuration. An additional experiment was conducted to measure the clearance rate in oysters from the reference and contaminated sites. This experiment was conducted to examine if there was any difference in the filtration ability between the reference oysters and the resident oysters (blue oysters). To exclude the body size effect, resident oysters with similar size (7−10 cm) to reference oysters were selected to conduct the experiment. Reference oysters from JZ and resident oysters were quickly transported to the laboratory and acclimated in the laboratory as described above. Twelve individuals of reference oysters or resident oysters were randomly selected for the assay. The clearance rate (L h−1 g−1) was determined by placing individual oyster in a beaker containing 4 L of 0.22 μm filtered seawater positioned on stirrer plates to keep the water thoroughly mixed and oxygenated. Diatom C. gracilis was added to generate an approximate food concentration of 5× 104 cell mL−1 after all oysters resumed pumping water. Decline of algal concentration was monitored by a Beckman Coulter Counter. The clearance rate was calculated as follows:

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RESULTS Metal Accumulation in Transplanted Oysters. The labile dissolved metal concentrations of Cd, Cu, Zn, and Cr in the reference site and the contaminated site as recorded by DGT units are shown in SI Table SI-1. The contaminated site had higher metal concentrations than the reference site (p < 0.01). Typically, BJ had much higher Zn and Cu concentrations (7.7−57.2 μg L−1 for Zn and 9.1−74.9 μg L−1 for Cu) than those in JZ (0.68−3.6 μg L−1 for Zn and 0.20−1.0 μg L−1 for Cu). The resident oysters C. hongkongensis grown in the contaminated site contained high concentrations of metals than those reference oysters. The Cd, Zn, Cu, and Cr concentrations in the resident oysters were 15.8 ± 3.7 μg g−1, 26094 ± 7492.6 μg g−1, 6283.1 ± 1755.7 μg g−1, 8.8 ± 2.9 μg g−1, respectively, and were far higher (p < 0.01) than those observed in reference oysters (2.6 ± 0.5 μg g−1, 5491.1 ± 1499.2 μg g−1, 375.8 ± 85.1 μg g−1, 0.9 ± 0.3 μg g−1 for Cd, Zn, Cu, and Cr, respectively). The accumulation of the four metals in the reference and the transplanted oysters is shown in Figure 1. As expected, the

CR = (ln C0 − ln Ct ) × V /(t − t0)

where CR is the clearance rate of oyster (L h−1), Ct is the cell density at time t (cells L−1), C0 is the initial cell density at the beginning (cells L−1), V is the volume of water (L). After the experiment, both groups of oysters were sacrificed for measurement of dried weights and total metal concentrations. Subcellular Fractionation. The subcellular fractionation was carried out according to previous study and five fractions were separated (cellular debris, metal-rich granules-MRG, organelle, heat-sensitive protein [HSP], and metallothioneinlike protein [MTLP]).20 Briefly, 0.5 g fresh tissues were homogenized in 5 mL of 30 mM Tris-NaCl buffer (pH 8.0; 0.15 M sodium chloride; 5 mM freshly prepared antiprotease, 2-mercaptoethanol; 0.1 mM phenylmethylsulfonyl fluoride, PMSF). The homogenate was centrifuged at 1,450 g at 4 °C for 15 min. The separated pellets were digested in 1 N NaOH at 80 °C for 10 min, and centrifuged at 5000g again for 10 min. The resulting pellets were the MRG and the supernatant was the cellular debris fraction. The supernatant from the first centrifugation was further subjected to centrifugation at 100 000g at 4 °C for 1 h. The pellet from this step was the organelle fraction (organelles). After 80 °C treatment of the supernatant from this step, the HSP was denatured and separated by centrifugation at 50 000g and 4 °C. The final supernatant was the MTLP fraction. The metal subcellular distribution was defined as the percentage of metals in each fraction. Measurement of Metals and Statistics. Oysters were dissected and dried at 80 °C to obtain the dry weights. The dried tissues and the fractionated subcellular samples were digested using concentrated HNO3 (70%) with addition of 200 μL H2O2. The mussel standard 2976 (National Institute of Standards and Technology, Gaithersburg, MD) were digested simultaneously. All digested samples and reagent blank samples were quantified for metal concentrations using a Perkin-Elmer ICP-OES (Optima 7000 DV) or a Perkin-Elmer atomic absorption spectrometer (AAS, Analyst 800). The measurements of metals in standard materials all met the requirements of achieving a recovery between 85 and 110%. Regressions were performed using software Sigmaplot 10.0 and statistical analysis was performed using one-way ANOVA to detect the significant

Figure 1. Accumulation of metals (Cd, Cu, Zn, Cr) in reference and transplanted oysters C. hongkongensis. Data are means ± SD (n = 10).

transplanted oysters accumulated much higher metal concentrations in tissues than the reference oysters after 7-month transplantation. For example, the Zn concentrations in transplanted oysters (6000−7000 μg g−1) were almost twice as those in reference ones. The Zn concentrations in reference oysters increased continuously from an original level around 2000 μg g−1 to 4000 μg g−1. The Cu concentrations in transplanted oysters increased steadily throughout the transplantation period and reached around 3000 μg g−1. Green color started to appear in the oyster tissues collected in May when the Cu level reached 2400 μg g−1 (SI Figure SI-1). Contrastingly, the Cu concentrations in reference oysters remained rather constant (∼200 μg g−1) over the seven months of growth. The average Cd and Cr concentrations in the transplanted oysters in each month were 3.7−7.0 μg g−1 and 4.3−30.3 μg g−1, respectively, which were significantly higher (p < 0.01) than those in the reference oysters (2.1−4.0 μg g−1 for Cd, and 1.5−4.3 μg g−1). The growth of two groups of oysters is shown in SI Figure SI-2. The growth of transplanted oysters 10767

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Figure 2. Metal uptake from the dissolved phase (left panels, n = 6), the retention of metals in the oysters following a pulse ingestion of radiolabeled diatoms (middle panels, assimilation experiments, n = 12), and the elimination (right panels, n = 10) of Cd and Zn following 6 days radiolabeling in the reference and transplanted oysters C. hongkongensis. Data are means ± SD.

tissue metal concentrations (Figure 3). The percentages of metals in the MSF maintained at a stable and low level and displayed a decreasing trend with increasing tissue metal concentrations (Figure 3).

was greatly depressed in the contaminated site (no obvious growth), whereas there was substantial growth in the reference oysters (0.007 d−1). Starting from a similar size of 0.2 g dry weight, the reference oysters grew as four times bigger as their original size, reaching around 0.8 g in September. Differences of Biokinetics of Cd and Zn in Oysters. Biokinetic measurements of Cd and Zn in the oysters are shown in Figure 2 and SI Table SI-2. There were substantial differences in the biokinetic parameters between the reference and transplanted oysters. The dissolved uptake rate constants (ku) of Cd and Zn for transplanted oysters (0.08 L g−1 h−1 and 1.15 L g−1 h−1) were significantly lower (p < 0.01) than those for the reference oysters (0.12 L g−1 h−1 and 2.40 L g−1 h−1). The reference oysters absorbed around 50% of Cd and up to 70% of Zn from diet when fed on the diatom C. gracilis. Only the AE of Zn was affected (15% lower) in transplanted oysters in comparison with the reference group, while there was no difference in Cd AE between the two groups of oysters. Similarly, the loss pattern of Cd in two groups of oysters was rather comparable. The efflux rate of Zn in the transplanted oysters (0.014 d−1) was significantly higher than that of the reference oysters (0.006 d−1). The clearance rate of resident oysters (4.1 L g−1 h−1) was significantly lower than that of the reference oysters (9.1 L g−1 h−1). Subcellular Detoxification of Metals. The subcellular distributions of metals in the sampled oysters plotting as a function of metal concentrations in the oyster tissues are shown in SI Figure SI-3. MTLP was the major fraction for binding Cd (20−60%), and its percentage increased significantly with increasing Cd levels in the oyster tissues. Both cellular debris and MTLP were important storage sites for Zn and there were significant correlations between the subcellular distributions and the tissue concentrations. Most Cr was stored in insoluble forms such as MRG, cellular debris and organelles, with MRG as the major binding site (up to 70%). The percentage of insoluble Cr in cellular debris increased with increasing tissue Cr concentrations. The partitioning of Cu in the oysters was complicated and there was no trend with increasing Cu tissue levels. Significant positive relationships were observed between Cd and Zn partitioning in BDM fractions and the respective

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DISCUSSION The metal concentrations in the resident oysters were amazingly high, especially for Cu and Zn, which were comparable to those measured in previous study.7 The bearing of over 3% of body weight of metals suggested that this oyster species had an extremely high capacity for accumulating metals. In a similar transplantation study, Geffard et al. found no cellular pathology in the transplanted oysters from a reference site to a contaminated site, indicating that oysters can well adapt to elevated metal level in the environment.8 Two related questions, therefore, are, how the oysters acclimated to the increasing metal influx when there was raised metal bioavailability, and how the metals were effectively detoxified during this process. Our transplantation experiment shed light on such acclimation process. Starting from a background concentration of 2000 μg g−1 for Zn, the reference oysters accumulated Zn steadily up to 3000 μg g−1 over the 7 month of transplantation, despite of the relatively low ambient Zn concentration (Figure 1). The increasing accumulation pattern was not observed in Cu, Cd, and Cr. The relatively stable Cu level implied that the Cu was well regulated in the oysters under low Cu bioavailability. The transplanted oysters quickly reacted to the raised metal exposure after transplantation, and efficiently accumulated Zn and Cu in their tissues. The contrasting accumulation patterns of different metals may ascribe to the metal-specific biokinetics. For example, the high uptake rate of Zn from water (2.4 L g−1 d−1) and high AE from food (70%) were coupled with an extremely low efflux rate (0.006 d−1), which were the major reasons for the high Zn body burden and for the continuous increasing pattern of Zn concentration in the reference oysters. Ke and Wang reported that the estuarine oysters C. rivularis (a closely related species to C. hongkongensis) had a high ku of 2.0 L g−1 d−1 for Zn and a high AE for Zn from diatom (70%), and only eliminated Zn at a rate of 0.014 d−1.21 Oysters also 10768

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oysters were still far lower than those in resident oysters after 7 month of transplantation. Such discrepancy implied that the transplanted oysters may develop strategies to counteract the increasing metal influx in the contaminated site. The change of metal biokinetics may be one of the reasons. Significant difference was observed in the metal biokinetics (ku, AE, ke) between the reference and transplanted oysters (Figure 2 and SI Table SI-2). The dissolved uptake rates in transplanted oysters were significantly reduced when compared to the reference ones. The simultaneous reduction of uptake rate of Cd and Zn in transplanted oysters implied that the filtration rates of oysters were depressed. Interestingly, the filtration rates in resident oysters were also lower than those in reference oysters (4.1 L g−1 h−1 compared with 9.1 L g−1 h−1). These results indicated that the reduced filtration rate may be one of the adaptive mechanisms for oysters to counteract the metal challenge. In fact, postexposure feeding depression was frequently reported in aquatic invertebrates exposed to metals or nanometals.23−25 Reduced clearance rate and feeding activity may lead to a decreased contact with waterborne and dietborne metals, and thus reduce the uptake and accumulation. Since the influx rate of metal is one of the important factors determining metal toxicity,13,14 it is valid to consider that the reduced metal uptake may act as a buffer step to encounter the increasing metal bioavailability. To further elucidate the metal accumulation in the reference and transplanted oysters, a biokinetic model can be applied to predict the Cd and Zn concentrations in the field conditions.26,27 Metal bioaccumulation in the oysters can be determined by the equation as follows: Css = Figure 3. Relationship between the metal distribution in the metalsensitive fraction (MSF) and the biologically detoxified metals (BDM) and the metal tissue concentrations in the oysters C. hongkongensis grown in a reference site (JZ, close circles) and a highly contaminated site (BJ, open circles). Each point represented one individual oyster.

(k u × Cw ) + (AE × IR × Cf ) ke + g

where Css is the tissue concentration of metals under steadystate conditions in the oysters, ku is the metal uptake rate constant from the dissolved phase (L g−1 d−1), Cw is the metal concentration in the ambient water (μg L−1), AE is the metal’s assimilation efficiency, IR is the ingestion rate (g g−1 d−1), Cf is the metal concentration in the ingested suspended particles (μg g−1), ke is the metal efflux rate constant (d−1), and g (d−1) is the growth rate. A few assumptions were made in the biokinetic modeling calculation. The ingestion rate of the oysters in the field varied greatly, depending on the food quality and food quantity.28−30 We used two ingestion rates (0.02 and 0.2 g g−1

efficiently absorb Cu both from water (1.27 L g d−1) and diet (85%), but eliminated less Cu (0.03 d−1) than other bivalve species including scallops, clams, and mussels.22 The Zn and Cu biokinetics also well explain the extremely high metal body concentrations of Cu and Zn in the resident oysters. However, metal concentrations (for Cu, Zn, and Cd) in the transplanted

Table 1. Biokinetic Modeling of Metal Accumulation in the Reference and Transplanted Oystersa metal

oyster

ku (L g−1 d−1)

Cwb (μg L−1)

IR (g g−1d−1)

AE (%)

Cf (μg g−1)

ke (d−1)

gc (d−1)

Rd (%)

Css

Cd

reference oysters-low IR reference oysters-high IR transplanted oysters-low IR transplanted oysters -high IR reference oysters-low IR reference oysters-high IR transplanted oysters - low IR transplanted oysters -high IR

0.12 0.12 0.08 0.08 2.4 2.4 1.15 1.15

0.059 0.059 0.31 0.31 2.6 2.6 28 28

0.02 0.2 0.02 0.2 0.02 0.2 0.02 0.2

48.2 48.2 51.5 51.5 70.9 70.9 50.3 50.3

0.59 0.59 3.1 3.1 260 260 2800 2800

0.012 0.012 0.011 0.011 0.006 0.006 0.014 0.014

0.007 0.007 0 0 0.007 0.007 0 0

44.5 88.9 56.3 92.8 37.1 85.5 46.7 89.7

0.7 3.4 5.2 31.3 764 3316 4312 22420

Zn

a

See Text for the Details of the Values for ku, AE, and ke. Cw, average labile dissolved metal concentration; IR, assumed ingestion rate; g, specific growth rate; R, percentage of uptake from dietary phase; Css, predicted metal concentrations in the oysters under steady state conditions. bAverage value of the labile dissolved metal concentration measured by DGT units. cEstimated by using a Kd of 1000 L kg−1 for Cd, and 20000 L kg−1 for Zn, see text. dSpecific growth rate, g = (ln[Wt]-ln[W0])/t; W0 and Wt, average dry weight of oysters at the beginning and ending of the transplantation experiment; t, transplantation period, totally 231 days. No obvious growth was observed for transplanted oysters and the g is assumed to zero. eR = (IR × AE × Cf)/(Cw × ku + IR × AE × Cf) × 100. 10769

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d−1) in the modeling calculation. We also used the partition coefficient (Kd) to calculate the metal concentration in the suspended particles. An average value of Kd of 104 L kg−1 for Cd and 105 L kg−1 for Zn was used in the calculation.31 Table 1 shows that the predicted Cd and Zn concentrations in the transplanted oysters were within the range of measured metal concentrations. The measured Cd and Zn concentrations in reference oysters at the end of transplantation were indeed very close to the predicted Css using high IR (3.4 μg g−1 for Cd, and 3316 μg g−1 for Zn), and those in transplanted oysters were close to Css using the low IR (5.2 μg g−1 for Cd, and 4312 μg g−1 for Zn). Such consistency was in accordance with our hypothesis that transplanted oysters reduced metal uptake in the contaminated environment by lowering the feeding activity. The modeling calculation indicated that up to 80% of Cd and Zn were derived from the dietary pathway when the IR was high (for the reference oysters). Thus a reduction in Zn assimilation from the dietary phase may significantly help to regulate the overall metal uptake. The deceased AE of Zn provided additional evidence of reducing metal uptake from food in the contaminated oysters. Reducing the ingestion of contaminated food may be important for reducing the influx rate of metals into the oysters, but the trade-off is that the food acquisition (energy intake) is reduced simultaneously. The transplanted oysters may have to increase the energy allocation to counteract metal challenges, which would otherwise be used for somatic growth and reproduction. The depressed growth of transplanted oysters may be caused by metal toxicity, such as a decline in mitochondrial capacity to synthesize ATP.32 Combating metals can be an energy costly process in which increased transcription and synthesis of detoxified proteins (such as metallothionein) are required.33 Confounding factors such as low food availability/poor food quality caused by metal pollution may also contribute to the low growth rate in the transplanted oysters. Lower growth rate may increase the metal concentrations by reducing growth dilution, but the intensified metal efflux (up to 100% change for Zn) compensated such adverse effect. Given the extremely high metal concentrations in the contaminated oysters, metals must be effectively detoxified when accumulated. MTLP appeared a subcellular pool sequestering elevated Cd and Zn and storing Cu in the transplanted oysters (SI Figure SI-3), indicating the importance of metallothionein (MT) in the detoxification of these metals. Such observation was in agreement with other studies of the importance of MT for metal detoxification and regulation in oysters.34,35 For example, Crassostrea gigas in the chronically Cd-contaminated Gironde estuary (France) stored 40−60% of Cd in the soluble form and 30−40% of this fraction was associated with the MTs.36 Bioaccumulated metals can be stored as insoluble forms in oysters, such as mineralized lysosomes and granules.8 In our study, cellular debris also appeared an important binding site (40%) for Cu and Zn, and Cr was mainly stored in the insoluble fractions with MRG (20− 60%). Such metal subcellular distribution was similar to those reported in the resident oysters. The metal distributed in the biological detoxified metal (MRG+MTLP) fraction for Cd and Zn increased with increasing metal concentration (Figure 3). Overall, although the metal concentrations increased around 5−10 times in the tissues of oysters, the percentages of metals in the metal sensitive fraction (organelles+HSP) maintained at a constant level or even displayed a decreasing trend (Figure 3),

which may have important implications for the oysters to survive at high metal concentrations in tissues. The present transplantation experiment provided clues for the survival of oysters in a contaminated environment, emphasizing the importance of biokinetics. The physiological acclimation of oysters in contaminated environment was not only due to their effective sequestration of metals into nontoxic forms, but also due to the change of metal homeostasis. The reduced clearance rate with associated reduced uptake rate from both dissolved and dietary phases, and the enhanced efflux of the metal can all be regarded as an enhanced detoxification rate of the metal under conditions of increased bioavailability from solution or diet. The increased metal detoxification rates are crucial for the survival of oysters when challenged with metals. Any physiological changes of the oysters found in the transplantation experiment may represent the early stage of development of metal tolerance in oysters. However, it should not be regarded as that metal biokinetics was superior to the subcellular detoxification, as both processes are interactive and can be considered as a set of mechanisms before genetic adaptation come into effects.

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ASSOCIATED CONTENT

S Supporting Information *

Table SI-1, Table SI-2, and Figure SI(1−3) are included. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (852) 23587346; fax: (852) 23581559; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the support from the colleagues (Dr. GUO Feng, GUO Xiao-yu, TU Ri-wei, WANG Lei) in Xiamen University, for their help in field sampling in this study. This study was supported a Key Project of Natural Science Foundation of China (21237004) and the Hong Kong Research Grants Council (662610).

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REFERENCES

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