Environ. Sci. Technol. 2002, 36, 989-995
Uptake and Efflux of Cd and Zn by the Green Mussel Perna viridis after Metal Preexposure GRAHAM BLACKMORE* AND WEN-XIONG WANG Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, People’s Republic of China
Cadmium and zinc uptake from the dissolved phase, assimilation efficiency from the dietary phase, efflux rate constants, and body burden as well as clearance rate were measured in the green mussel Perna viridis with or without laboratory preexposure to Cd or Zn. Efflux rate constants and clearance rates were little affected by preexposure to either Cd or Zn. In contrast, the assimilation of Cd increased by 1.2-1.6× in mussels preexposed to Cd (subsequent Cd concentrations 10.2-25.9 µg g-1) as compared to controls (0.19-0.39 µg g-1). This increase corresponded to an elevation in the proportion of Cd associated with the metallothionein-like proteins (MTLPs) in the mussels, suggesting that exposure to Cd and subsequent induction of MTLPs affected Cd accumulation. Exposure to Zn only resulted in elevated body concentrations following 7-d exposure to 250 µg L-1, although Zn and Cd uptake from the dissolved phase were reduced by 2447% by exposure to a lower concentration (100 µg L-1) for 7 and 21 d. Despite the lack of an increase in body Zn concentration, the subcellular distribution was altered such that the proportion of Zn associated with the metal-rich granules increased. This study indicates the importance of the subcellular distribution of metals in affecting the biokinetics and thus the toxic effects of metals on aquatic animals. Cd preexposure has potential effects on its influx from the dietary phase, e.g., increasing the importance of dietary uptake and further increasing the body burdens. In contrast, preexposure to Zn has a negative effect on Cd and Zn influx from the dissolved phase, suggesting the mechanism of Zn regulation but also potentially reducing Cd uptake and body concentrations over the long-term exposure. Such effects may have implications for biomonitoring studies involving a single species that modifies physiological processes affecting metal uptake (and hence bioavailability). Caution is needed in extrapolating data to species not capable of making such changes, particularly for Cd, which is not regulated and for which the effects of an elevated body burden are most obvious.
Introduction Metal bioaccumulation by marine bivalves such as mussels has been studied extensively, and their use as biomonitors * Corresponding author phone +852-2358-7349; fax: +852-23581559; e-mail:
[email protected]. 10.1021/es0155534 CCC: $22.00 Published on Web 01/24/2002
2002 American Chemical Society
is well-established. Mussel Watch Programs have been implemented in many areas of the world, e.g., United States, U.K., Australia, China, and Japan. More recent attention has focused on modeling metal uptake in an attempt to better explain the observed metal concentrations and to allow delineation of exposure pathways (1, 2). The sensitive radiotracer techniques employed in these studies have allowed workers to investigate in greater detail the factors affecting metal accumulation, such as food composition (3), chemical composition of diatom food (4), and body size (5) in addition to temperature, salinity, sex, and reproductive state. More subtle effects on metal accumulation have also been considered. For example, we have recently examined the interpopulation differences in Cd, Cr, Se, and Zn accumulation in the green mussel Perna viridis from contrasting estuarine and coastal environments (Blackmore and Wang, manuscript in preparation). Green mussels collected from a low salinity site accumulated metals from the dissolved phase at rates slower than conspecifics from a high salinity site when measured at the same salinity due to their lower apparent membrane permeability. Care is needed therefore if a biomonitoring study involving a single species that has the capability of making physiological changes affecting metal uptake (and hence bioavailability) is used to draw conclusions on local metal bioavailabilities to other local organisms not making such changes. In this study, we further expand this concept by examining the influence of metal preexposure on metal uptake and loss by the mussels. Little is known on the effects of preexposure (and resultant body metal concentrations) on metal uptake and loss in aquatic invertebrates. Boisson et al. (6) suggested that the past history of metal contamination should be taken into account when evaluating the susceptibility of Macoma balthica to heavy metal exposure. Rainbow et al. (7) investigated the trace metal uptake in crustaceans collected from coastal sites differentially enriched with trace metals. Mean metal uptake rates by amphipods and crabs did not show consistent significant differences between metal-rich and control sites, indicating that exposure was not sufficient to select for a reduction in metal uptake in populations collected from grossly contaminated sites. However, it is possible that several physiological processes facilitating metal tolerance were used to different degrees by the crustaceans from metal-rich habitats (7). A complementary study (8), however, found that metallothionein concentration in crabs was largely related to the variation in natural factors rather than a metal contamination gradient in the Gironde Estuary. These studies did not consider metal uptake via the dietary route. Selck et al. (9) investigated the effects of chronic metal exposure on the Cd assimilation efficiency by a depositfeeding polychaete Capitella. Cd assimilation efficiency was positively related to gut passage time in unexposed worms, but this pattern was reversed following Cd exposure. In this study, we quantified the effect of differential preexposure to Cd and Zn on metal uptake and loss in the green mussel P. viridis. This species has been used extensively as a biomonitor (10) and as a model organism for bioenergetic-based kinetic models (11). It is therefore important to understand any factors affecting body metal concentrations in the mussels. We measured the assimilation efficiency from ingested food, the efflux rate constants, the influx from the dissolved phase, and the subcellular distribution of metals in mussels that were previously exposed to Cd and Zn and had elevated body concentrations of these metals. These physiological parameters are critical for determining body VOL. 36, NO. 5, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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metal concentration. To our knowledge, no previous study has examined the influence of metal preexposure on metal kinetics in marine bivalves.
at 20 and 40 min. The rate was then calculated by the following equation (14):
CR ) vol × [ln(C1) - ln(C2)]/t
Materials and Methods The green mussels Perna viridis with a shell length of 3-3.5 cm and tissue dry weight of 0.069-0.291 g were collected from Wu Kai Sha, Tolo Harbour. This site is considered relatively unpolluted by both Cd and Zn, as reflected in bioavailabilities to both barnacles and mussels (12, 13). During the acclimation and experimental period, the mussels were maintained in aerated seawater and kept at a constant temperature of 23 °C. The mussels were fed with the diatom Thalassiosira pseudonana (clone 3H) at a ration of about 1-2% of their body tissue dry weight per day. Metal Exposure Treatments. Cd and Zn body burdens were elevated in the mussels following the dissolved exposure. Three experiments were conducted by exposing the mussels separately to different concentrations of Cd or Zn: (i) low metal for a short period, i.e., 50 µg L-1 Cd and 100 µg L-1 Zn for 7 d; (ii) high metal for a short period, i.e., 100 µg L-1 Cd and 250 µg L-1 Zn for 7 d; and (iii) low metal for a long period, i.e., 20 µg L-1 Cd and 100 µg L-1 Zn for 21 d. Cd and Zn were spiked into separate groups of mussels. These metal concentrations, while environmentally unrealistic, were required in our experiments to ensure induction of physiological processes that might affect subsequent metal uptake and loss. Furthermore, the preexposure at these concentrations resulted in environmentally realistic body concentrations of Cd and Zn (see Results and Discussion). During the metal exposure, mussels were fed daily with the diatoms T. pseudonana. A control group without metal spiking was run simultaneously in each experiment. Following each exposure, the clearance rate, influx from the dissolved phase, assimilation efficiency from ingested food source, efflux rate, subcellular distribution, and stable metal concentrations were determined in each group using the methods described below. Trace Metal Influx from the Dissolved Phase. The influx rate from the dissolved phase describes the rate at which the mussels take up the metals from the dissolved phase. A short exposure time (1 h) was chosen to measure the influx rate (1). Eight mussels from each treatment were placed individually into 200 mL of the 0.22-µm filtered seawater spiked with the stable metals and radioisotopes (109Cd and 65Zn) for 1 h. Stable metals and radiotracers were previously added and allowed to equilibrate overnight so that the concentration was about 5× its typical concentration in uncontaminated coastal waters, i.e., 2 µg L-1 for Cd (in CdCl2) and 5 µg L-1 for Zn (in ZnCl2). Radioisotope additions were 1.85 kBq L-1 for Cd and 3.7 kBq L-1 for Zn. Following these additions, microliter amounts of 0.5 N Suprapure NaOH were added to the seawater to maintain pH. After 1-h exposure, the mussels were dissected, and the radioactivities of soft tissues were measured. The tissues were then dried at 80 °C overnight, and dry weights were determined. The influx rate was calculated as the amount of metal accumulated by the soft tissues of mussels and was standardized as nanogram dry weight per day (ng g-1 h-1). Clearance Rate of the Mussels. Eight individual mussels from each treatment were individually placed in 1.5 L of filtered seawater within a polypropylene beaker. The mussels were allowed to open and pump normally, usually within 10 min. Diatoms T. pseudonana were filtered from their growth medium and added to each beaker at a concentration of 104 cell mL-1, and this suspension was homogenized by a magnetic stirrer. Immediately after addition of the algae, a 10-mL aliquot water sample was taken and the cell density counted using a Coulter Counter. Further samples were taken 990
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where CR is clearance rate (L h-1), C1 is the cell density (cell mL-1) at time 1, C2 is the cell density at time 2, t is time of measurement increment (i.e., t1 - t2, in h), and vol is the volume of water (L). The clearance rate for each individual was calculated from the mean of the two consecutive measurements. Metal Assimilation Efficiencies. The diatom T. pseudonana was radiolabeled with γ-emitting 109Cd and 65Zn. Radioactivity additions were 74 kBq for 109Cd and 65Zn. After 4 d of inoculation, the cells had undergone about four divisions and were considered uniformly labeled. These cells were collected on a 3-µm polycarbonate membrane and resuspended in 12 mL of water before adding to the feeding beakers. Five individual mussels from each treatment (exposed to Cd or Zn and a control) were placed in 500 mL of filtered seawater. Algae were added to each beaker to yield a cell density of (4-5) × 104 cells mL-1. Further additions were made at 10-min intervals to maintain this density. After 30 min of radioactive feeding, the mussels were rinsed in seawater and analyzed for their radioactivity. Individuals were then placed into separate polypropylene beakers (180 mL of seawater) held with a 20-L enclosed recirculating flowthrough aquarium containing seawater of appropriate salinity. Nonradioactive T. pseudonana was fed twice daily at a ration of about 2% dry weight per day. Radioactivity retained in the mussels was measured over a 72-h depuration period at intervals from 1 to 12 h. Fecal pellets were collected frequently to minimize desorption of radiotracers from the fecal materials to the surrounding water. Assimilation efficiencies were determined as the percentage of initial radioactivity retained in the mussels after 60 h of depuration. P. viridis completed digestion and assimilation of trace elements within 60 h (15). Metal Efflux. To determine the efflux rate constant of Cd and Zn, mussels were radiolabeled with either 109Cd or 65Zn during their exposure to stable metals from the dissolved phase (see above). In the control treatment, only radioisotopes were added. The water was changed once every 2 days. A total of 18.5 kBq of 109Cd and 37 kBq of 65Zn was added in increments over the course of the exposure. Following radiolabeling, the mussels were removed and rinsed with nonradioactive water, and radioactivity was measured. They were subsequently placed in an enclosed 20-L recirculating seawater aquarium (water changed twice weekly), as described above, to depurate for at least 22 d. On day 0, 16, and the last day of the experiment, two individuals from each treatment were dissected, and the radioactivity associated with the shell, digestive gland and other soft tissues was counted. Subcellular Cd and Zn Distributions. The subcellular 109Cd and 65Zn distribution in mussel soft tissues after exposure to stable metals and radioisotopes (described above) was determined by subjecting the mussel tissues to differential centrifugation and tissue digestion procedures, using a modified method of Wallace et al. (16). Mussels that had been stored at -80 °C were thawed and homogenized in 8 mL of distilled water, and the homogenate was centrifuged at 1450g for 15 min at 4 °C. The pellet contained tissue fragments and other cellular debris (i.e., membranes and metal-rich granules; MRG). MRG were isolated from the other cellular debris by resuspending the pellet in 1 mL of distilled water and heating at 100 °C for 2 min. Subsequently an equal volume of 1 N NaOH was added, followed by heating at 70 °C for 1 h. This dissolved the tissue,
and the MRG were collected by centrifugation at 5000g for 10 min at 20 °C. The 1450g supernates were further fractionated at 100000g for 1 h at 4 °C to produce an intracellular pellet containing nuclear, mitochondrial, and microsomal fractions. The supernatants contained the cytosol and proteins and was further fractionated following heat treatment (80 °C for 10 min then ice cooling for 1 h) by centrifugation at 50000g (10 min at 4 °C). This separated the heat-stable fraction or metallothionein-like proteins (MTLP), which remained in the supernate, from the heat-sensitive proteins (HSP) that were denatured by the heat treatment and, therefore, formed the pellet. This differential centrifugation and heat treatment resulted in three pellets [i.e., MRG (P2), organelles (P3), and HSP (P4)] and two supernates [i.e., cellular debris (S4) and MTLPs (S3)]. All fractions were radioassayed for 109Cd and 65Zn to allow estimation of the subcellular distribution of these metals. In addition to Wallace et al. (16), similar differential centrifugation separation techniques have been used by many authors (e.g., refs 17-19) such that it was considered unnecessary to verify each fraction. Stable Metal Body Concentrations. By the end of the exposure, a group of mussels from each treatment was dissected, and the soft tissues were dried at 60 °C to constant weight and then digested at 100 °C in concentrated nitric acid (HNO3, Aristar grade, BDH Ltd.). These digests were made up to a known volume with double-distilled water and diluted to give metal concentrations in the appropriate range for analysis. Digests or dilutions thereof were analyzed for Cd and Zn using ICP-MS (Perkin-Elmer, Elan 6000). Throughout the analyses, random checks were made using aliquots of a certified reference material (Standard Reference Material 1566a, oyster tissue, U.S. Department of Commerce, Technology Administration, National Institute of Standards and Technology, Gaitherburg, MD). Agreement was considered good, i.e., within 10%. All metal concentrations have been expressed as µg g-1 dry wt. Analytical Measurements. Radioactivity was measured using a Wallac γ-counter. Spillover of radioisotopes was corrected, and all counts were related to standards for each isotope and corrected for radioactive decay. The γ emissions of 109Cd were determined at 88 keV and of 65Zn were determined at 1115 keV. Counting times in all samples were adjusted so that the propagated counting errors were typically 50% lower in both the long-term (21 d) and shortterm exposure (7 d) to low concentrations (50 µg of Cd L-1) (10.2 and 11.7 µg g-1, respectively). Zn soft tissue concentrations were similar in both exposed and control mussels (60.1122 µg g-1). There was only a significant difference (p ) 0.0001) between the control (73.2 µg g-1) and following 7-d exposure to high dissolved concentrations (250 µg of Zn L-1) (122 µg g-1). These results agree well with previous laboratory (20) and field (10, 21) studies showing that P. viridis is a partial regulator of Zn. Thus, Zn body concentrations were similar until the exposure level reached 250 µg of Zn L-1 at which regulatory ability was reduced. Cd as a nonessential metal was accumulated in proportion to the exposure levels and time (20). It should be noted that Cd and Zn concentrations
TABLE 1. Soft Tissue Concentrations of Cd and Zn in Green Mussels (Perna viridis) following Laboratory Exposurea treatment
Cd
Zn
Short-Term Low control 0.326 ( 0.095 60.1 ( 10.3 -1 Cd (50 µg L ) 11.7 ( 3.26 Zn (100 µg L-1) 77.2 ( 15.7 p ) 0.0001; Cd > Con p ) 0.0739; Con ) Zn post hoc Short-Term High control 0.187 ( 0.061 73.2 ( 14.7 Cd (100 µg L-1) 25.9 ( 11.8 Zn (250 µg L-1) 122 ( 20.4 post hoc p ) 0.0001; Cd > Con p ) 0.0001; Zn > Con Long-Term Low control 0.391 ( 0.159 80.7 ( 12.8 Cd (20 µg L-1) 10.2 ( 1.73 Zn (100 µg L-1) 69.5 ( 21.0 post hoc p ) 0.0001; Cd > Con p ) 0.2664; Con ) Zn a
Data are mean ( SD (n ) 5).
used in the preexposure period were much higher than typical coastal water concentrations, and the environmental relevance of the data may be of some concern. However, dissolved concentrations approaching these values may be reached in extremely metal-enriched environments, e.g., inner Tolo Harbour in the 1980s and Restronguet Creek, U.K. (22, 23). Furthermore, such preexposure results in environmentally realistic body concentrations. For example, Zn body concentrations in both exposed and control groups were within those observed in the field in Hong Kong, and Cd body concentrations following exposure were similar to the maximum concentrations recorded (9 µg g-1) in mussels collected from Hong Kong coastal waters (24). The clearance rates of the control and exposed mussels with elevated metal concentrations are shown in Table 2. Clearance rates were not significantly different between the controls and the preexposed mussels with elevated Cd or Zn body concentrations (p > 0.069). Clearance rates were however 3-40% lower in exposed mussels than in the controls (Table 2). Clearance rates have been considered good indicators of toxic effects of contaminants (25); therefore, the lack of a significant difference between control and those mussels preexposed to either Cd or Zn suggests that there was no significant toxic effect (14). Kraak et al. (26) found that Zn (ranging from 40 to 3000 µg L-1) and Pb (ranging from 4 to 400 µg L-1) affected the filtration rate in zebra mussels Dreissena polymorpha. The filtration rates in P. viridis were also reduced following exposure to Cu (25 µg L-1) and Hg (25 µg L-1) (27). The influx of Cd and Zn into preexposed mussels with elevated body concentrations is shown in Table 2. Cd influx was similar in control individuals (26.9-29.5 ng g-1 h-1) and those with elevated body Cd concentrations (25.9-26.5 ng g-1 h-1) as a result of previous metal exposure. In contrast, Cd influx into those individuals preexposed to Zn (now with elevated Zn concentrations) was 24-47% lower (14.4-21.4 ng g-1 h-1) as compared to the controls. This reduction was however only significant (p ) 0.005) for the group that had been exposed to 250 µg of Zn L-1 for 7 d. The influx of Zn into mussels was similar between control groups (171-199 ng g-1 h-1) and those that had been previously exposed to Cd (199-151 ng g-1 h-1). The influx of Zn (138-161 ng g-1 h-1) was reduced by 19-22% following exposure to Zn, but these reductions were not statistically significant (p > 0.08). Previous exposure to Cd with subsequent elevation of body concentrations, therefore, appears to have little effect on the accumulation of either Cd or Zn in P. viridis. In agreement, Rainbow et al. (7) report that Ag, Cd, and Zn uptake in the VOL. 36, NO. 5, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Cd and Zn Uptake and Clearance Rate (CR) by Green Mussels (Perna viridis) with Elevated Body Concentrations of either Cd or Zn (see Table 1)a uptake rate (ng g-1 h-1)
a
prior treatment
CR (L g-1 h-1)
Cd
Zn
control Cd (50 µg L-1) Zn (100 µg L-1) post hoc
9.30 ( 4.40 7.71 ( 4.32 7.89 ( 1.75 p ) 0.0691
Short-Term Low 26.9 ( 7.20 26.5 ( 4.63 20.5 ( 4.59 p ) 0.0568; Con ) Cd ) Zn
199 ( 75.9 199 ( 26.6 161 ( 27.4 p ) 0.2270; Con ) Cd ) Zn
control Cd (100 µg L-1) Zn (250 µg L-1) post hoc
8.38 ( 1.13 6.67 ( 2.93 8.15 ( 3.39 p ) 0.3984
Short-Term High 27.1 ( 11.04 26.1 ( 6.67 14.4 ( 2.46 p ) 0.0048; Con ) Cd > Zn
170 ( 50.2 181 ( 35.1 138 ( 26.1 p ) 0.0854; Cd ) Con ) Zn
control Cd (20 µg L-1) Zn (100 µg L-1) post hoc
10.6 ( 5.43 6.69 ( 2.55 6.53 ( 2.24 p ) 0.6474
Long-Term Low 29.5 ( 5.84 25.9 ( 8.93 21.4 ( 4.40 p ) 0.0739; Con ) Cd ) Zn
196 ( 38.2 151 ( 54.1 154 ( 42.7 p ) 0.1139; Con ) Zn ) Cd
Data are mean ( SD (n ) 8).
crustaceans Orchestia gammarellus, Carcinus maenas, and Pachygrapsus marmoratus were not significantly different in individuals differentially enriched with trace metals. Boisson et al. (6) found however that, following chronic exposure to Ag in a contaminated estuary, the clam Macoma balthica accumulated Ag at a significantly lower rate than the conspecifics from a clean estuary. Adaptation to Cu in C. maenas resulted in a reduction in Cu uptake and more efficient metal transfer from the haemolymph to the tissues (28). P. viridis is at least a partial regulator of Zn, and the observed reduction in the uptake of this metal may partially explain this ability. Cd and Zn are chemically similar and often share similar uptake pathways (29, 30), thus the uptake of both Cd and Zn are affected when exposed to high Zn concentrations. Depuration of Cd and Zn in mussels following a pulse radioactive feeding is shown in Figure 1. Cd and Zn were rapidly egested within the first 24 h, after which much less was lost from the mussels. Assimilation efficiencies (AE) were calculated as the amount of radioactivity left in the mussels at 60 h divided by the amounts ingested, measured following the 0.5-h pulse radioactive feeding (Table 3). The assimilation of Cd was significantly (ANOVA p < 0.031) affected by the preexposure to Cd. Cd AE was 1.2-1.6× greater (SNK p < 0.05) in those individuals that had elevated Cd body concentrations as compared to those of the controls. Cd assimilation in those individuals that had been exposed for 7 and 21 d to l00 µg of Zn L-1 was similar to the controls but was 1.4× greater (SNK p > 0.05) following 7-d exposure to 250 µg of Zn L-1 than the controls. In contrast, Zn assimilation was largely unaffected by preexposure to Cd or Zn with subsequent elevations in body concentrations. One exception to this was following 7-d exposure to 100 µg of Cd L-1 in which Zn AE was 1.3× greater (SNK p > 0.05) than the control and following 7-d exposure to 250 µg of Zn L-1. To our knowledge, little work has been conducted on the effects of preexposure on metal assimilation efficiencies. Selck et al. (9) reported that Cd AE increased with increasing gut passage time in Capitella sp., but this pattern was reversed in worms following preexposure to Cd. Data presented herein suggest that preexposure to elevated Cd or Zn does affect metal AE, in particular, those of Cd following Cd exposure. These results contrast with the metal influx from the dissolved phase where exposure to Zn appeared to have the greatest effect. Metal AEs are typically variable, and those presented herein are within the normal ranges for marine bivalves (1, 3, 15, 31). 992
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FIGURE 1. Retention of Cd (left panel) and Zn (right panel) in green mussels (Perna viridis) preexposed to either Cd (Cd-exposed) or Zn (Zn-exposed) for 7 or 21 d (with subsequent elevated body concentrations, see Table 1) and controls following a pulse ingestion of the radiolabeled diatom Thalassiosira pseudonana. STL, shortterm low; STH, short-term high; LTL, long-term low. Mean ( SD (n ) 5). The depuration of Cd and Zn in mussels following exposure to radioisotopes and stable metals is shown in Figure 2. There was an initial rapid loss of both metals during the first 4-5 d followed by a second slower loss for the remaining depuration period (5-32 d). The efflux rate constants, calculated from the slope of the natural log of the percent of metal retained in the mussel and the time of depuration (between 5 and 32 d), are shown in Table 4. Cd efflux constants (0.007-0.012 d-1) were not significantly different between the controls and following 7-d exposure to 100 µg of Cd L-1 (p ) 0.0939) or 21-d exposure to 20 µg of Cd L-1 (p ) 0.958). Zn efflux constants (0.034-0.038 d-1) were not significantly different between the control and following 21-d exposure to 100 µg of Zn L-1 (p ) 0.392). The efflux constant (0.035 d-1) was however 21% lower (p ) 0.017) in those individuals exposed to 250 µg of Zn L-1 for 7 d, as compared to the control (0.044 d-1). Metal efflux rates are
TABLE 3. Cd and Zn Assimilation Efficiencies (AE, %) in Green Mussels (Perna viridis) with Elevated Body Concentrations of either Cd or Zn (see Table 1)a prior treatment
Cd
Zn
control Cd (50 µg L-1) Zn (100 µg L-1) post hoc
Short-Term Low 23.3 ( 6.45 30.6 ( 2.79 22.6 ( 3.48 p ) 0.0310; Cd > Con ) Zn
30.0 ( 4.39 30.0 ( 11.9 27.1 ( 7.38 p ) 0.8553; Con ) Cd ) Zn
control Cd (100 µg L-1) Zn (250 µg L-1) post hoc
Short-Term High 32.5 ( 15.1 56.7 ( 10.2 50.0 ( 9.89 p ) 0.0203; Cd ) Zn > Con
26.7 ( 3.25 36.3 ( 5.66 24.6 ( 5.77 p ) 0.0069; Cd > Con ) Zn
control Cd (20 µg L-1) Zn (100 µg L-1) post hoc
Long-Term Low 14.9 ( 3.70 41.1 ( 4.11 17.0 ( 4.07 p ) 0.0001; Cd > Zn ) Con
31.4 ( 6.67 33.1 ( 6.04 29.9 ( 9.76 p ) 0.8059; Cd ) Con ) Zn
a
Data are mean ( SD (n ) 6).
FIGURE 2. Retention of Cd (left panel) and Zn (right panel) in green mussels (Perna viridis) preexposed to either Cd (Cd-exposed) or Zn (Zn-exposed) for 7 or 21 d (with subsequent elevated body concentrations, see Table 1) and controls. STH, short-term high; LTL, long-term low. Mean ( SD (n ) 8).
TABLE 4. Cd and Zn Efflux Rate Constants in Green Mussels (Perna viridis) with Elevated Body Concentrations of either Cd or Zn (see Table 1)a prior treatment control Cd (100 µg L-1) Zn (250 µg L-1) post hoc control Cd (20 µg L-1) Zn (100 µg L-1) post hoc a
Cd
Zn
Short-Term High 0.011 ( 0.0050 0.007 ( 0.0031
0.044 ( 0.0089
p ) 0.0939 Long-Term Low 0.012 ( 0.0039 0.012 ( 0.0056
p ) 0.9581
0.035 ( 0.0037 p ) 0.0173 0.038 ( 0.0086 0.034 ( 0.0096 p ) 0.3918
Data are mean ( SD (n ) 8).
usually relatively constant and less than 0.04 d-1 (1, 32, 33), and these figures agree well with our results. The distribution of Cd and Zn in soft tissue, digestive gland, and shell throughout the depuration period is shown in Figure 3. The shell generally contained negligible amounts of Cd throughout the experiment (1-4%). A larger fraction
FIGURE 3. Distribution of Cd (left panel) and Zn (right panel) in the digestive gland, other soft tissue, and shell of the green mussels (Perna viridis) with elevated body concentrations of either Cd or Zn (see Table 1) following metal exposure (day 0), after 16-d depuration (day 16), and at the end of the depuration (end) in nonradioactive waters. STH, short-term high; LTL, long-term low; Con, control mussels. Mean ( semi-range (n ) 2). of Zn was found on the shell (5-40%), particularly in those groups that were exposed to elevated dissolved concentrations. Most of the Cd and Zn were distributed within the soft tissues of the mussel (>65%). There was no evidence to suggest that an elevated body concentration of either Cd or Zn altered their distribution within the soft tissues. Subcellular Cd and Zn distributions in control and exposed mussels are shown in Figure 4. Control mussels had similar Cd distributions with about 40% being distributed in the tissue fraction, 20%). Much less was found in the intracellular fractions (organelles), i.e., 20% and 50% of Cd in M. edulis is accumulated from the dissolved phase (1). In agreement, uptake in P. viridis is similarly dominated by uptake from the dissolved phase, but trophic transfer becomes more important with increasing metal partition coefficient in the food particles (11). Any increase in efficiency of Cd uptake from diet may be moderated by the comparative unimportance of this route of uptake. Nevertheless, somewhat higher body concentrations may be measured in individuals living in Cd-enriched environments than would otherwise be expected, in particular in areas whether metal partition coefficient in particles is high (e.g., dominated by phytoplankton) (47). In contrast to the effects of Cd on dietary uptake, preexposure to Zn affected dissolved Zn and Cd uptake. The partial Zn regulatory ability of P. viridis is well-documented (20), and our results elucidate a potential mechanism for this ability, i.e., reduced permeability to this metal. In addition, due to the chemical similarity of Cd and Zn, this mechanism reduces Cd uptake. Cd and Zn body concentrations in mussels collected from Zn-enriched environments may thus be lower than otherwise expected. In both M. edulis and P. viridis, Zn bioaccumulation is dominated by uptake from the particulate phase (1, 11); therefore, any factor affecting dissolved uptake is of minor importance. Prior exposure to Zn at levels insufficient to significantly elevate body concentrations but sufficient to increase the proportion of Zn associated with MRG should therefore be considered when interpreting the Cd body concentration in mussels.
Acknowledgments We are grateful to the anonymous reviewers for their constructive and helpful comments on this work. This study was supported by a Competitive Earmarked Research Grant from the Hong Kong Research Grants Council (HKUST6137/ 99M) to W.-X.W. G.B. was additionally supported by a
postdoctoral fund from the Hong Kong University of Science and Technology.
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Received for review June 8, 2001. Revised manuscript received December 10, 2001. Accepted December 13, 2001. ES0155534
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