The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain

Journal of Experimental Marine Biology and Ecology 307 (2004) 91 – 110 www.elsevier.com/locate/jembe

The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain Graham Blackmore, Wen-Xiong Wang * Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, PR China Received 16 October 2003; received in revised form 19 January 2004; accepted 28 January 2004

Abstract We examined the transfer of cadmium (Cd), inorganic mercury [Hg(II)], methylmercury (MeHg), and zinc (Zn) in an intertidal rocky shore food chain, namely from marine phytoplankton to suspension-feeding rock oysters (Saccostrea cucullata) and finally to predatory whelks Thais clavigera. The uptake of metals from the dissolved phase was also concurrently quantified in the oysters and the whelks. Metal uptake by the oysters was not directly proportional, whereas metal uptake by the whelks was directly proportional to metal concentration in the water. The order of uptake was MeHg>Hg(II)>Zn>Cd, and was much higher in the oysters than in the whelks. The relative uptake of Zn and Cd was comparable between oysters and whelks, whereas MeHg and Hg(II) showed disproportionally higher uptake in oysters than in whelks as compared to Zn and Cd. The assimilation efficiencies (AEs) were in the order of MeHg>Zn>Cd = Hg(II) in oysters, whereas the AEs were highest for MeHg and comparable for Zn, Cd, and Hg(II) in the whelks. Pre-exposure of the oysters to different dissolved concentrations of Cd significantly elevated the AEs of Cd and Hg(II) but not of Zn, in association with the induction of metallothioneins in the oysters. The whelks significantly assimilated Cd and Zn from various prey (barnacles, oysters, mussels, and snails) with contrasting strageties of metal sequestration and storage. There was no significant relationship between the metal AE and the metal partitioning in the soluble fraction (including metallothionein-like proteins, heat stable protein, and organelles). The insoluble fraction of metals was also available for metal assimilation. Our calculations show that the dietary uptake of metals can be dominant in the overall bioaccumulation in the oysters and whelks, and the trophic transfer factor was >1 for all metals. Thus, the four metals have a high potential of being biomagnified in the intertidal rocky shore food chain. MeHg possessed the highest and Hg(II) and Cd the lowest potential of trophic transfer among the four metals considered. D 2004 Elsevier B.V. All rights reserved. Keywords: Trophic transfer; Neogastropod; Oyster; Cadmium; Zinc; Mercury; Methylmercury

* Corresponding author. Tel.: +852-2358-7346; fax: +852-2358-1559. E-mail address: [email protected] (W.-X. Wang). 0022-0981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2004.01.021

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1. Introduction Over the past decades, there has been substantial interest in the potential transfer of metal contaminants in different marine food chains, largely stemming from the recognition of the significance of dietary exposure as a major route for metal bioaccumulation in aquatic animals (Wang, 2002). Despite the general perception that trace metal biomagnification, namely, increasing metal concentration with increasing trophic position along food chain, occurs only for Hg and possibly Cs, more recent studies have shown that potential biomagnification may occur for a few essential metals/metalloids such as Se and Zn along specific food chains (Suedel et al., 1994; Wang, 2002). Contrasting marine food chains possess different potential for biomagnification, largely depending on the strategies of metal handling and storage by the animals concerned. This is especially true for benthic invertebrates with very diverse patterns of metal sequestration and storage. Clearly, there is a need to examine specific aquatic food chains before a general paradigm on metal transfer in various marine food chains emerges. Many studies on the trophic transfer of metals have focused on the control of physicochemical species of metals in the prey organisms, whereas the physiological and biochemical controls on metal transfer remain much less well investigated (Wang, 2002). It has been shown that metal distribution in phytoplankton cytoplasm, which can be considered as a ‘physical’ species of metals, can critically affect assimilation by marine herbivores such as copepods, bivalves, and barnacles as characterized by a relatively short gut passage of metals through their digestive tracts (Fisher and Reinfelder, 1995; Wang and Fisher, 1999; Rainbow and Wang, 2001). In predatory animals, a few studies have also demonstrated that the cytosolic fraction of metals in the prey determine the assimilation by predatory animals such as shrimp (Wallace and Lopez, 1997). The control of metal physico-chemical species on metal assimilation by marine fish is somewhat variable (Ni et al., 2000). Metals are stored and detoxified in diverse forms in the animals. A few limited studies have shown that metals stored in a chemically inert detoxified form (e.g., granules) may not be available to the next trophic level (Nott and Nicolaidou, 1990), but strong experimental evidence is lacking from these previous studies. Recently there has been considerable interest in the potential transfer of metals bound with different subcellular fractions in prey to predators (Wallace and Luoma, 2003). The bioavailability of metals in marine invertebrates has been extensively quantified for a few metals such as Cd, Ag, Se, and Zn, largely due to the availability of radiotracers and their environmental impacts regarding these elements. The pathways of Cd and Zn bioaccumulation in marine bivalves have been comprehensively examined (Wang et al., 1996; Chong and Wang, 2001; Ke and Wang, 2001), whereas the exposure pathways of Hg(II) and methylmercury (MeHg) are mostly unknown even in bivalves that are frequently employed as biomonitors of coastal contamination (O’Connor, 1992; Rainbow, 1993). Furthermore, whereas most trophic transfer studies have focused on marine herbivores grazing on phytoplankton, fewer have considered transfer to higher trophic levels such as top benthic predators, e.g., seastars and gastropods (Fowler and Teyssie, 1997; Wang and Ke, 2002). The trophic transfer of Hg(II) and MeHg in benthic food chain also remains little studied (Kennish, 1997).

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In this study, we examined the transfer of four metals/organometals (cadmium, zinc, inorganic mercury, and methylmercury) in an intertidal marine benthic food chain. The whelk Thais clavigera is the top predator in the intertidal rocky shore community, often preying upon the rock oyster Saccostrea cucullata, the mussel Perna viridis, the barnacles Balanus amphitrite, and the mesogastropod snail Monodonta labio (Blackmore, 2001). The composition of ingested prey is dependent on the relative availability and abundance of each prey. Rock oysters, mussels and barnacles are all suspension feeders grazing on the seston (including phytoplankton) available in the water column. The mesogastropod snail (herbivores) grazes on the macroalgae on the rocky surfaces. Our recent studies in two marine predatory gastropods indicated that Cd and Zn may potentially be biomagnified in the top predators because of the very efficient assimilation and an extremely low efflux from the gastropods (Wang and Ke, 2002). Furthermore, field evidence suggests that dietary exposure is the dominant route by which the whelks accumulate metals (Blackmore, 2000, 2001; Blackmore and Morton, 2001). We specifically focused on the transfer of metals from the oysters to the whelks because the oysters constitute an important diet for the predator. In addition, we conducted an experiment by pre-exposing the rock oysters to different concentrations of Cd with subsequent measurements of metallothionein induction and metal assimilation. A recent study has indicated that metal binding with MT may potentially have an effect on dietary metal accumulation (Blackmore and Wang, submitted for publication). Both metal assimilation efficiency (AE) from the dietary source and the dissolved uptake were examined in this study as an index to quantify metal bioavailability from the food and aqueous phases.

2. Materials and methods 2.1. Field collection Whelks, T. clavigera (shell length 25 – 30 mm, dry-tissue weight f 0.2 g), were collected from Starfish Bay, Tolo Harbour, Hong Kong. This site has been shown to be relatively uncontaminated by metals (Blackmore and Morton, 2001), and the metallothionein (MT) concentration in the whelk was low (Blackmore and Wang, submitted for publication). Similarly, the barnacle B. amphitrite, the rock oyster S. cucullata, the mesogastropod snail M. labio, and the green mussel P. viridis were all collected from the same site. During the acclimation and experimental periods, the whelks and their prey were maintained in aerated seawater, and kept at a constant temperature of 20 jC and salinity of 28 psu. The whelks were fed ad libatum with the barnacle B. amphitrite. The mussel P. viridis and the oyster S. cucullata were fed the diatom Thalassiosira pseudonana (clone 3H), the snails M. labio were fed small pieces of macroalgae Ulva sp., and the barnacles were fed the diatom Thalassiosira weissflogii. The diatoms were cultured in f/2 nutrient medium and were fed daily to the suspension feeders (oysters, mussels, and barnacles). Following each acclimation, Cd, Hg(II), MeHg and Zn influx from the dissolved phase and AE from the ingested food source were determined in the whelks and oysters using methods described below.

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2.2. Laboratory pre-exposure of oysters with Cd A series of dissolved Cd pre-exposures was conducted on the oyster S. cucullata in order to investigate metal assimilation following MT induction. The oysters were exposed to dissolved Cd at 5, 20 or 100 Ag l 1 for 14 days. A control group without the Cd spike was also included. The oysters were maintained in aerated seawater at 20 jC and were fed the diatom T. pseudonana (clone 3H). Following the experimental exposure, the Cd, Zn and Hg(II) AEs and the MT concentrations in the digestive gland of the oysters were determined in each group, using methods described below. 2.3. Dissolved metal uptake Eight T. clavigera or S. cucullata from each group were placed individually into 200 ml of 0.22 Am filtered seawater spiked with stable metals and the radioisotopes, 109 Cd 203Hg(II), Me203Hg and 65Zn. 109Cd (in 0.1 N HCl) was purchased from New England Nuclear, Boston, USA, and 203Hg(II) and 65Zn were purchased from Riso National Lab, Denmark. Me203Hg was synthesized from 203Hg(II) using an established method (Rouleau and Block, 1997). Cd, Hg(II) and Zn exposures were combined whereas a separate experiment was conducted for MeHg. Furthermore, for 203Hg(II) and Me203Hg exposure, only the radioisotopes were spiked since the specific activity of the radioisotope was low and the uptake was high. The dissolved concentrations for Cd and Zn used in the exposure were 0.5, 2, 8, and 20 Ag l 1, and 2, 8, 20, and 100 Ag l 1, respectively. Radioisotope additions were 1.85 kBq l 1 for Cd and 3.7 kBq l 1 for Zn. Following radioactive additions, 0.5 N Suprapure NaOH was added to the seawater to maintain the pH (8.0) because the metals were carried in 0.1 N HCl solution. The radioisotopes and the stable metals were equilibrated overnight before the uptake experiments. For MeHg and Hg(II), the dissolved nominal concentrations used in the exposure medium were 0.008, 0.034, 0.172, 0.686, and 3.430 Ag l 1, and 0.034, 0.172, 0.686, 3.430, and 17.15 Ag l 1, respectively. A range of metal concentrations was used in the metal uptake experiments to allow calculation of the uptake rate constant. T. clavigera was exposed to metals for 24 h since previous experiments with snails showed that dissolved metal uptake was much slower as compared to the bivalves (Ke and Wang, 2001). Oysters have a much higher uptake rate and were thus exposed for 1 h (for an explanation see Wang et al., 1996). During the exposure period, the water was regularly stirred to homogenize the metal gradient due to metal uptake by the animals. Care was also taken to ensure the submergence of whelks in the water during the exposure period. Following exposure, the snails and oysters were dissected and the soft tissues radioassayed. The tissues were then dried at 80 jC and dry weights determined. Uptake rates were calculated as the amount of metal accumulated by the soft tissues divided by the exposure duration and standardized to Ag g 1 dry weight day 1. Uptake rate constants (ku) were calculated from the equation Iw = kuCwb, where Iw is the uptake rate from the dissolved phase, Cw is the metal concentration in the dissolved phase, and b is the power coefficient.

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2.4. Metal AE in the oysters and in whelks The AE of metals in the rock oysters was determined using a pulse-chase radiotracer technique, as described by Wang and Fisher (1999). The diatom T. pseudonana was radiolabeled with 37 kBq of 109Cd and 74 kBq of 203Hg(II) and 65Zn, or 74 kBq of Me203Hg in 200 ml 0.22 Am filtered seawater. The cells were considered uniformly labeled after they had undergone >4 divisions (4 days) and were collected on a 3-Am polycarbonate membrane, washed and resuspended in 0.22 Am water before being added to the feeding beakers. Oysters from each group were individually placed in 500 ml of filtered seawater and diatoms were added to yield a cell density of 4 –5  104 cells ml 1. Further additions were made at 10-min intervals to maintain this density. Following 30 min of radioactive feeding, the oysters were rinsed in seawater and radioassayed. The oysters were then placed into separate polypropylene beakers (180 ml seawater) held in a 20l enclosed recirculating flow-through aquarium containing seawater. Non-radioactive T. pseudonana was fed twice daily at a ration of f 2% dry weight per day. Fecal pellets were collected regularly to minimize desorption of radiotracers into the surrounding water. The radioactivity remaining in the oysters was measured at 3 –12 h intervals over the 72 h depuration period. AEs were determined as the percentage of initial radioactivity retained in the oysters after 12 h. The assimilation of Cd and Zn was quantified in the whelks by feeding on the prey B. amphitrite, S. cucullata, M. labio, and P. viridis. These prey were radiolabeled following 14 days of aqueous exposure to 37 kBq of 109Cd and 74 kBq of 65Zn, after which time the prey were assumed uniformly labeled. Previous work has shown that assimilation from a bivalve prey by the predatory gastropods varied little following prey labeling either by aqueous or food (algae) exposure (Wang and Ke, 2002). The digestive glands of oysters, snails, and mussels were subsequently used as the diets for whelks because the animals fed readily on these tissues. Moreover, the digestive gland is selectively preyed upon in the field (Black, 1978). The small size of the barnacle allowed the whole bodies to be used as the prey. Digestive glands were dissected out of the radiolabeled oysters, snails, and mussels and the whole barnacle bodies were removed from the shell. They were placed individually in a tray of 50 ml of seawater. Whelks were then added individually and allowed to feed on the prey for 1 h, after which they were rinsed in seawater and radioassayed. Whelks were tagged and ten were placed in 3 l of seawater. Fecal pellets were collected regularly and water was changed at least two times per day to minimize desorption of radiotracers. During the depuration period, whelks were fed ad libatum on the barnacle Balanus amphtrite, which was chosen as the food because of the barnacle’s small size. Any unconsumed tissue would not foul the water. The whelks were radioassayed at 3 – 12 h intervals over the 72 h depuration period. The AEs were determined as the percentage of initial radioactivity retained in the whelks after 72 h. Following the initial Cd and Zn AE experiments with the four prey types, the AEs of Hg(II) and MeHg were investigated in a subsequent experiment using the digestive glands of the oysters. Briefly, S. cucullata were radiolabeled following 14 days of aqueous exposure to 19 kBq Me203Hg or 37 kBq of 203Hg(II), after which the prey was considered uniformly labeled. The digestive gland was then dissected out and the AE determined as

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described above. Oysters were chosen primarily because the snails fed favorably on this prey type. 2.5. Metal distribution in prey The subcellular 109Cd and 65Zn distributions in the prey used in the whelk AE experiments, i.e., B. amphitrite bodies, S. cucullata, M. labio, and P. viridis digestive glands, were determined after exposure to radioisotopes (during the radiolabeling period) using differential centrifugation and tissue digestion procedures modified from Wallace et al. (1998). Tissues that had been stored at 80 jC were thawed, homogenised in buffer solution and centrifuged at 1450  g for 15 min at 4 jC. The pellet contained tissue fragments and other cellular debris (i.e., membranes and metal rich granules— MRG). This fraction was defined as the insoluble fraction (or the trophically unavailable fraction as defined by Wallace and Luoma, 2003). The 1450  g supernant containing the organelles, cytosol and proteins was defined as the soluble fraction (or the trophically available fraction as defined by Wallace and Luoma, 2003). No attempt was made to further separate the different subcellular fractions. Both fractions were radioassayed for 109Cd and 65Zn to allow estimation of the distribution of these metals in the subcellular fractions. 2.6. MT determination The MT concentrations in the digestive glands of the oysters pre-exposed to Cd for 2 weeks at different dissolved concentrations were determined using a modified silver saturation method (Scheuhammer and Cherian, 1986; Leung and Furness, 1999). Briefly, the digestive glands were dissected and, after the measurement of the wet weights, the tissues were homogenized and centrifuged (at 20,000  g for 20 min). The supernatant was incubated with 0.5 M glycine buffer and 0.5 ml of 20 Ag 110mAg ml 1 to saturate the MT. Excess Ag was removed with the addition of 0.1 ml rabbit red blood cell haemolysate. The amount of Ag present in the final supernatant was proportional to the MT concentration and was determined following radioassayed for 110mAg. MT concentrations (as Ag g 1 wet tissue weight) were calculated as 3.54  the Ag concentrations (Scheuhammer and Cherian, 1986). 2.7. Analytical measurements and statistical analysis Radioactivity was measured using a Wallac gamma counter. Spillover (from the higher energy window to the low energy window) of radioisotopes was corrected and all counts were related to standards for each isotope and corrected for radioactive decay. Individual animals were directly placed in counting tubes without water and sample geometry was maintained the same at each counting point. The gamma emissions of 110mAg were determined at 658 keV, 109Cd at 88 keV, 203Hg at 279 keV, and 65Zn at 1115 keV. Counting times in all samples were adjusted so that the propagated counting errors were typically < 5%. Data were tested for the assumptions of parametric tests and investigated using analysis of variance (ANOVA) and appropriate post hoc tests using SAS 6.12.

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3. Results 3.1. Dissolved metal uptake The influx rates of Cd, Hg(II), MeHg, and Zn into the oysters at different ambient concentrations are shown in Fig. 1. For all four metals, there was a significant ( p < 0.01) log – log linear relationship between the metal influx rate (Iw) and the metal concentration (Cw), such that the metal influx increased with an increase in concentration. The relationships between Iw and Cw are also shown in Fig. 1. The calculated b coefficients of the power relationship between the Iw and the Cw were significantly lower than 1.0 for Cd, Zn and MeHg, but were much higher than 1.0 (1.269) for Hg(II). It appears that the oysters slightly reduced their rates of uptake of Cd, Zn, MeHg in response to increasing dissolved concentrations. The uptake rate constant, ku (intercept of the log –log regression, Wang et al., 1996), was the highest for MeHg and the lowest for Cd. The relative magnitude of the ku among the four metals was MeHg (10.0 )>Hg(II) (6.0 )>Zn (2.2 )>Cd (1 ). The influx rates of Cd, Hg(II), MeHg, and Zn into the whelks at different Cw are shown in Fig. 2. In agreement with the results for the oysters, there was a significant ( p < 0.01) log – log linear relationship between the Iw and the Cw, such that the metal influx increased with an increase in metal concentration. The calculated b coefficients of the power relationship were about 1.0, thus the metal uptake was directly proportional to the Cw.

Fig. 1. The influx rate of metals in the rock oyster S. cucullata at different ambient concentrations. Mean F SD (n = 8).

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Fig. 2. The influx rate of metals in the whelk T. clavigera at different ambient concentrations. Mean F SD (n = 8).

Similar to results for the oysters, the ku was the highest for MeHg and the lowest for Cd. The relative magnitude of ku among the four metals was MeHg (3.6 )>Hg(II) (2.6 )>Zn (2.3 )>Cd (1 ). The MeHg influx uptake was 1.6  greater when compared to Hg(II) at comparable dissolved concentration. The magnitude of difference between MeHg and Hg(II) was however smaller in the whelks than in the oysters (1.9 –5.3 ). The calculated ku was 32 , 26 , 11 , and 11  higher in the oysters than in the whelks for MeHg,

Fig. 3. The retention of metals in the rock oyster S. cucullata following a pulse ingestion of radiolabeled diatoms T. pseudonana. Mean F SD (n = 5).

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Hg(II), Zn, and Cd, respectively. Thus the oysters had a much higher uptake of Hg(II) and MeHg than the whelks as compared to Zn and Cd. 3.2. Metal assimilation by oysters and whelks There were considerable differences in the depuration of ingested metals by the oysters following the initial pulse feeding of radiolabeled diatoms (Fig. 3). Very little MeHg, Cd, and Zn was depurated after the initial loss of unassimilated metals, whereas Hg(II) was lost continuously from the oysters. After 12 h of depuration, the calculated AEs (defined as % metals retained in oysters after 12 h of depuration) were 30.2 F 1.7%, 31.0 F 1.9%, 90.6 F 3.1%, and 52.9 F 2.8% (mean F SD, n = 5), for Cd, Hg(II), MeHg, and Zn, respectively.

Fig. 4. The retention of metals in Cd pre-exposed rock oyster S. cucullata following a pulse ingestion of radiolabeled diatoms T. pseudonana. The oysters had been pre-exposed to different dissolved Cd concentrations for 2 weeks before the assimilation measurements. Mean F SD (n = 5).

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Table 1 Metallothionein (MT) concentrations in the rock oyster S. cucullata following two weeks pre-exposure to dissolved Cd and the quantified assimilation efficiency (AE) of Cd, Hg(II) and Zn by the oysters Treatment

MT concentration (Ag g 1 wet weight)

Cd AE (%)

Hg(II) AE (%)

Zn AE (%)

Control Diss Cd (5 Ag l 1) Diss Cd (20 Ag l 1) Diss Cd (100 Ag l 1) Post hoc

12.0 F 5.0 15.0 F 3.6 42.6 F 13.3 53.5 F 9.4 p < 0.001 100 = 20>5 = C

30.2 F 1.7 36.5 F 3.9 39.2 F 6.2 43.3 F 4.5 p = 0.002 100 = 20 = 5>C

31.0 F 1.9 36.4 F 4.4 37.8 F 3.1 35.4 F 2.2 p = 0.017 100 = 20 = 5>C

51.2 F 3.0 55.0 F 8.1 46.0 F 3.2 38.7 F 3.5 p = 0.15 C = 5>20>100

Data are means F SD (n = 5).

Cd, Hg(II) and Zn depuration by the oyster S. cucullata pre-exposed to different dissolved Cd concentrations is shown in Fig. 4. In this experiment, the MT concentration increased by a factor of 1.2 , 3.6 , and 4.5  in the dissolved Cd concentrations of 5, 20, and 100 Ag l 1 treatment, respectively (Table 1), as compared to the control treatment without Cd pre-exposure. In agreement with Fig. 3, there was very little loss of Cd and Zn after the first 12 h of depuration, whereas Hg(II) was also continuously lost from the

Fig. 5. The retention of metals in the whelk T. clavigera following a pulse ingestion of radiolabeled oyster S. cucullata, mussel P. viridis, gastropod Mondonta labio digestive glands (DG), or whole barnacle bodies B. amphitrite. Mean F SD (n = 5).

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oysters. The calculated AEs of Cd and Hg(II) increased with increasing concentrations of Cd pre-exposure (Table 1), whereas the Zn AEs were lower (although not significant, p = 0.15) with increasing Cd concentrations. The tissue Cd concentrations in the preexposed oysters were not determined in this study. The retention of ingested Cd and Zn by the whelks following ingestion of different radiolabeled prey is shown in Fig. 5. There were some variations in the depuration of metals by the whelks. In general, there was an initial loss of unassimilated metals from the whelks within the first 12 h, after which the metals were efficiently retained by the animals. The AEs determined after 72 h of depuration were 71.0 F 4.1%, 85.7 F 3.2%, 71.2 F 4.9%, and 75.0 F 4.6% (mean F SD, n = 5) for Cd, and 75.6 F 5.7%, 90.0 F 4.5%, 86.4 F 6.1%, and 68.2 F 3.7% for Zn, in animals fed upon barnacles, snails, mussels and oysters, respectively. Metals bound in the digestive glands of the snails were assimilated at the highest efficiency, whereas metals bound in the other prey tissues were assimilated at a somewhat comparable efficiency by the whelks. The prey tissues were further fractionated to determine the soluble metal fraction (including the cytosol, heat stable proteins, and metallothionein-like proteins) and the insoluble fraction (including the debris and the metal rich granules, Fig. 6). In general, a relatively small fraction of Cd and Zn was found in the soluble fraction in the barnacles and oysters. Only 9% and 14% of Zn, and 37% and 28% of Cd were associated with this fraction in the barnacle and oyster prey, respectively.

Fig. 6. The relationship between Cd and Zn AEs by the whelk T. clavigera and their percentages in the soluble fraction (proteins and organelles) of the four prey. Mean F SD (n = 5).

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Fig. 7. The retention of Hg(II) and MeHg in the whelk T. clavigera following a pulse ingestion of radiolabeled oyster S. cucullata digestive glands. Mean F SD (n = 5).

About 30 –40% of Zn was in this fraction for the mussel and snail prey, while for Cd, this fraction was somewhat higher (60 – 70%). No relationship was found between the Cd AE and its distribution in the soluble fraction (Fig. 6). For Zn, there was a general trend that AEs were higher with increasing distribution in this fraction, although the regression was not statistically significant given that only four prey types were considered (Fig. 6). The depuration of ingested Hg(II) and MeHg by the whelks following the ingestion of the radiolabeled oyster digestive glands is shown in Fig. 7. Very little ingested MeHg was lost from the animals and the calculated AE was 94.7 F 3.6% (n = 5). Hg(II) had a somewhat lower AE (69.7 F 11.0%) than MeHg, but its AE was comparable to Cd and Zn from the same prey (the oyster digestive gland).

4. Discussion 4.1. Metal accumulation in the oysters The dissolved uptake rate constants, ku, of Cd (0.343 l g 1 day 1) and Zn (0.745 l g 1 day 1) measured for the rock oyster S. cucullata in this study were lower than those measured for two other oysters Saccostrea glomerata and Crassostrea rivularis (0.534 – 0.719 l g 1 day 1 for Cd, and 1.206 – 2.050 l g 1 day 1 for Zn; Ke and Wang, 2001). In general, the ku in the oysters were higher than those of other species of marine bivalves such as mussels and clams, due to the high filtration rates of the oysters (Wang, 2001). Consistent with these two oyster species, S. cucullata reduced their rates of uptake of Cd and Zn uptake in response to increasing metal concentration, and the b coefficients of the power relationship between the influx rate and the dissolved metal concentration were < 1.0 (0.767 – 0.870). There is no reported ku of Hg(II) and MeHg by oysters to compare with our measurements. Our data indicated that the ku in the oysters were MeHg (10.0 )>Hg(II) (6.0 )>Zn (2.2 )>Cd (1 ). The uptake of Hg(II) increased disproportionally with increasing Hg(II) concentration in the water, whereas the uptake of MeHg

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was reduced with increasing concentration in the water. Passive diffusion may be the dominant pathway for the uptake of MeHg by the oysters and facilitated uptake may be the primary pathway for the accumulation of Hg(II), Zn, and Cd. The order of their respective uptake rates was consistent with the binding affinity of these metals with the sulfur ligand, further demonstrating that facilitated transport by binding with sulfur containing ligands may be a major pathway for their uptake. MeHg is highly lipophilic and its rapid passive diffusion may account for its highest uptake rate among the different metals examined in this study. The assimilation of metals from ingested marine phytoplankton has been quantified in a few oyster species, including C. virginica, C. rivularis, and S. glomerata (Reinfelder et al., 1997, Ke and Wang, 2001) using similar pulse-chase feeding techniques. In this study, the AEs of metals in the oysters (30% for Cd and 51% for Zn) were only quantified for one single algal diet, the diatom T. pseudonana, and were lower than those quantified for the oysters C. rivularis and S. glomerata (67 – 75% for Cd, and 60– 72% for Zn) feeding on the same diatom diet (Ke and Wang, 2001). The much higher AE of MeHg as compared to Hg(II) is consistent with many studies in other groups of animals (Jackson, 1998; Mason, 2002). The very efficient assimilation of MeHg (91%) by the oysters may have been due to its rapid desorption within the digestive tract from ingested particles, the desorbed MeHg being then rapidly assimilated due to its high lipophilicity. Among the four metals examined, Hg(II) was continuously lost from the oysters after the initial digestion, whereas there was essentially no loss of Cd, Zn and MeHg from the oysters during this period. It is unknown whether protein turnover may explain the loss of Hg(II). A few studies have considered metal accumulation in marine bivalves after metal preexposure (Blackmore and Wang, 2002; Shi et al., 2003). The AEs of Cd and Hg(II) increased significantly following Cd pre-exposure, consistent with our previous study on green mussel P. viridis (Blackmore and Wang, 2002). In green mussels, Cd AEs increased in association with increasing Cd binding with metallothionein-like proteins as a result of Cd pre-exposure. Similarly, the increasing AEs of Cd and Hg(II) in the oysters were accompanied by an increase in the concentrations of MTs that may act as specific ligands in binding these metals. In marine bivalves, Cd is a much more potent inducer of MT than Zn (Langston et al., 1998), and MT concentrations in S. cucullata increased by 4.4x following exposure to dissolved Cd 100 Ag l 1 for 2 weeks. Cd AE was more affected by Cd pre-exposure than the Hg(II) AE. At the highest Cd concentration (100 Ag l 1), its AE increased from 30% to 43%, as compared to the increase from 31% to 35% in Hg(II) AE. These data imply that the induction of MT may play an important role in the assimilation of Cd and Hg(II) by the oysters, consistent with other recent experimental studies (Blackmore and Wang, 2002, submitted for publication). Decreases in Zn AE with increasing Cd pre-exposure concentrations and the concomitant increase in MT induction is rather unexpected from this study. Zn has much smaller affinity for MT as compared with other metals such as Cd, Ag, Cu, and Hg(II) (Langston et al., 1998). One possible mechanism for the underlying decrease (albeit insignificant) was the saturation of MT by Cd during pre-exposure, perhaps reducing the ligands available for the binding of Zn, leading to a lower Zn AE. This however remains to be examined further. The relative significance of the dietary vs. dissolved exposure of metals can be assessed by a simple kinetic equation (Ke and Wang, 2001) by comparing the relative magnitude of

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uptake from each source. For dietary exposure, the relative uptake can be calculated as the AE times the ingestion rate (IR) and the bioconcentration factor in the ingested food (BCF) (Wang et al., 1996; Ke and Wang, 2001). This can then be compared with the relative uptake ku from the dissolved phase. Using our experimentally determined values for ku and AE and the mean IR of the oysters (0.45 g g 1 day 1, Ke and Wang, 2001) (Table 2), our calculations show that the relative importance of dietary vs. aqueous uptake of the four metals is greatly dependent on the BCF of these metals in the diets (Fig. 8). At typical BCFs for these metals (>2  103 for Cd, >104 for Zn, 104 for Hg(II), and 5  104 for MeHg; IAEA, 2000; Watras et al., 1998), however, dietary uptake was the predominant pathway for the accumulation of Cd, MeHg, and Zn by the oysters, primarily because of the high AE, IR, and BCF, even though the ku of these metals were high when compared to other bivalve species (e.g., mussels and clams). The AEs of Cd and Hg(II) was the lowest among the four metals examined. The relative importance of their dietary uptake was less than for the other two metals. Only the mean values were used in these calculations, although it is recognized that these parameters (such as IR, AE, BCF) are not likely to be constant in the field conditions. 4.2. Metal accumulation in whelks Metal uptake from the dissolved phase by the whelks was much lower than that observed in the oysters, as a result of the very low ventilation rate of the animals. Wang and Ke (2002) documented a ku of 0.029 – 0.056 l g 1 day 1 for Cd and 0.057– 0.122 l g 1 day 1 for Zn in two gastropods Nassarius teretiusculus and Babylonia formasae habei, as compared to 0.030 l g 1 day 1 for Cd and 0.069 l g 1 day 1 for Zn in the whelks T. clavigera. Similar to the results for the oysters, the ku was the highest for MeHg (3.6 )>Hg(II) (2.6 )>Zn (2.3 )>Cd (1 ). However, the relative magnitude of the difference between these two species was somewhat different among the metals. The difference in MeHg and Hg(II) uptake between these two species was much greater (about 3 ) than the difference in Cd and Zn uptake, suggesting that the oysters had a much higher uptake of Hg(II) and MeHg than the whelks as compared to Zn and Cd. It is likely

Table 2 Numeric values of parameters used in the calculation of the exposure pathways and the trophic transfer potentials of metals in the oyster S. cucullata and the whelk T. clavigera Parameters

Hg(II)

MeHg

Cd

Zn

Saccostrea cucullata AE (%) IR (g g 1 day 1) ku (L g 1 day 1)

30 0.45 2.604

90 0.45 3.445

30 0.45 0.343

50 0.45 0.745

Thais clavigera AE (%) IR (g g 1 day 1) ku (L g 1 day 1)

70 0.05 0.079

95 0.05 0.108

75 0.05 0.030

80 0.05 0.069

AE: assimilation efficiency, IR: ingestion rate, ku: uptake rate constant from the dissolved phase.

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Fig. 8. The model predicted relative importance of dietary uptake in the rock oyster S. cucullata and the whelk T. clavigera as a function of metal concentration factor (BCF) in the ingested food. Note the log scale in the x-axis.

that the transport of Cd and Zn may be controlled by the same mechanism in both oysters and whelks. In contrast to the oysters, metal uptake by the whelks was directly proportional to metal concentration in ambient water. A few studies have examined the transfer of metals from prey to predators (Nott and Nicolaidou, 1990; Wallace and Lopez, 1996; Wang and Ke, 2002). Nott and Nicolaidou (1990) fed the carnivorous gastropod Murex trunculus with gastropod Cerithium vulgatum containing detoxified metals in insoluble granules in the digestive gland, the kidney of scallops (Chlamys opercularis) and the bodies of barnacles (Balanus balanoides). They found that the metal granules passed directly through the gut into the feces and, thereby, concluded that there was no metal assimilation from them, although the metal AE was not directly quantified in this study. Wallace and Lopez (1996, 1997) separated cytosolic and granule-bound Cd from the oligochaete worm Limnodrilus hoffmeisteri and fed them to the shrimp Palaemonetes pugio. They demonstrated that there was a 1:1 relationship between the amount and the percentage of Cd in the oligochaete cytosol and the amount and percentage of Cd absorbed by the shrimp, indicating that only metal bound to the soluble fraction of prey (e.g., MTLP) is available to the higher trophic level. Cd bound with metal-rich granules was relatively unavailable to the shrimp.

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Among the four different prey considered in this study, the barnacles exhibited the smallest fraction of metals in the soluble form (cytosol and proteins), and the majority of metals were associated with the insoluble fraction due to the presence of numerous Zn containing pyrophosphate granules beneath the gut epithelium (Stratum perintestinale and below) serving for detoxification of Zn deposition (Rainbow, 1987; Pullen and Rainbow, 1991). These granule-deposited metals are unavailable for the normal physiological function of the animals (Rainbow, 2002). In the snails, the majority of Cd (68%) was in the soluble form. It is thus likely that Cd was mainly bound with proteins instead of granules (which is also known as a main binding ligand in the gastropod; Langston et al., 1998). Several studies have indicated that Cd was associated with cytosolic metallothioinein-like proteins and mineralised granules in the gastropod Littorina littorea (Langston and Zhou, 1986, 1987; Nott and Langston, 1989; Bebianno et al., 1992). Andersen et al. (1989) identified the presence of a low molecular weight, non-metallothionein-like metal binding protein in the gastropod Nassarius reticulatus. In mussels and oysters, the majority of Cd and Zn were found in the insoluble form and only Cd in the mussels was found predominantly in its soluble form. Our study suggested a very high assimilation of metals (>65%) bound in different prey by the whelks. This is consistent with our previous study on two other predatory gastropods N. teretiusculus and B. formasae habei (>50%, Wang and Ke, 2002). Such high assimilation may be attributed to the efficient sequestration and storage of metals in the gastropods. Furthermore, there was no strong relationship between the metals in the soluble fraction and corresponding AEs. Our results were thus in contrast to results for the shrimp (Wallace and Lopez, 1996; Wallace and Luoma, 2003), suggesting that whelks have a more complicated digestive system as compared with crustaceans. It is clear from our study that insoluble metals in the prey may still serve as an important source for metal transfer to a higher trophic level, instead of acting as a sink for metals during food chain transfer. It is also possible to simulate the exposure pathway of metals in the whelks, using the experimentally determined values for AE, ku, IR, and BCF (Table 2, Fig. 8). Using the mean AE, ku, and IR (0.05 g g 1 day 1; Wang and Ke, 2002), our calculations show that dietary uptake predominated the overall bioaccumulation for all metals in the whelks at typical BCFs for these metals (>2  103 for Cd, >104 for Zn, 104 for Hg(II), and 5  104 for MeHg). This was due to the high AE and the very low ku, despite the IR being much lower in this predatory animal compared to the suspension-feeding oysters. Thus, the trophic transfer of metals plays a particularly important role in this intertidal food chain with whelks as the top predator, which is consistent with other predatory gastropods (Wang and Ke, 2002). 4.3. Trophic transfer potential in the intertidal food chain The potential trophic transfer factor (TTF) can be calculated as the AE  IR/ke, where ke is the metal efflux rate from the consumer/predator (Wang, 2002). Biomagnification can be assumed as taking place between two components of the trophic levels when the TTF>1. Wang (2002) summarized that the TTF in marine bivalves is generally greater than 1 because of high IR and high AEs. We simulated the TTF in the oysters and the whelks using the mean values of AE and IR (Table 2). The ke in the oysters C. rivularis and S.

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glomerata was 0.004 – 0.014 day 1 for Cd and 0.003– 0.014 day 1 for Zn (Ke and Wang, 2001), and ke in the gastropods (B. formasae habei and N. teretiusculus) was 0.001 –0.006 day 1 for Cd and 0.006 –0.014 day 1 for Zn (Wang and Ke, 2002). We thus simulated the TTF as a function of ke (i.e., assuming that the ke is variable, Fig. 9). These calculations clearly demonstrated that the TTFs of all four metals were greater than 1 over the likely range of ke for both oysters and whelks, suggesting that these metals are biomagnified during their transfer in this food chain. MeHg has the highest potential for biomagnification, consistent with numerous studies in the field (Jackson, 1998; Mason, 2002). The high trophic transfer potential of metals in bivalves was mainly due to their high AE and IR, whereas efflux played a small effect (Wang, 2002). In whelks, the high potential of trophic transfer was a result of efficient metal assimilation and low efflux. Of course, the TTF is likely to be variable in the field conditions given that AE, IR and ke are all likely to be influenced by various environmental and biological conditions. However, given the very high concentrations of Zn in the barnacles and oysters (with typical concentrations of a few thousand Ag g 1), the predicted Zn concentration in the whelks will be much higher if the TTF>1. The typical concentration of Zn in the whelks in Hong Kong coastal waters is in the range of 200– 600 Ag g 1 (Blackmore and Wang, submitted for publication), which is at least a few times lower than its concentration in

Fig. 9. The model predicted trophic transfer factor (TTF) in the rock oyster S. cucullata and the whelk T. clavigera as a function of metal efflux rate constant.

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barnacles and oysters. Three reasons may explain the discrepancy between the model prediction and the field observation of the Zn concentration in predators and prey. First, the ingestion rate on the barnacles or oysters may be lower than the value used in the modeling simulation (5% body dry weight per day). The prey selectivity as a function of metal concentration in the prey is unknown at present. Second, the whelks may possess a more efficient efflux system for Zn when the Zn-enriched prey (oysters and barnacles) are ingested. Third, the Zn AE may be lower in the field situation because of the dominance of Zn in the granule forms, which may be less bioavailable than those measurements based on the radiotracer technique. Our calculations would indicate that the TTF is lower than 1 when the Zn ke>0.04 day 1 (or 4% loss from the animals on a daily basis) or lower if the IR is lower than 5% body dry weight per day. It will be interesting to test the variability of the Zn efflux in animals feeding on different prey species containing different Zn body concentrations. Clearly, the diversity of metal handling strategies and cellular storage among different marine benthic invertebrates further adds a huge complexity to the prediction of metal trophic transfer in different marine food chains (Wang, 2002). The specificity of prey needs to be carefully considered in assessing any food chain biomagnification of metals in marine systems.

Acknowledgements We are grateful to Prof. Phil Rainbow and anonymous reviewer for their very constructive comments on this work. This study was supported by a Competitive Earmarked Research Grant from the Hong Kong Research Grants Council (HKUST6097/ 02M) to W.-X. Wang. G.B. was additionally supported by a postdoctoral fund from the Hong Kong University of Science and Technology. [RW]

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