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

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<ul><li><p>disproportionally higher uptake in oysters than in whelks as compared to Zn and Cd. The assimilation</p><p>efficiencies (AEs) were in the order of MeHg&gt;Zn&gt;Cd=Hg(II) in oysters, whereas the AEs werehighest for MeHg and comparable for Zn, Cd, and Hg(II) in the whelks. Pre-exposure of the oysters to</p><p>different dissolved concentrations of Cd significantly elevated the AEs of Cd and Hg(II) but not of Zn,</p><p>in association with the induction of metallothioneins in the oysters. The whelks significantly assi-</p><p>milated Cd and Zn from various prey (barnacles, oysters, mussels, and snails) with contrasting stra-</p><p>geties of metal sequestration and storage. There was no significant relationship between the metal AE</p><p>and the metal partitioning in the soluble fraction (including metallothionein-like proteins, heat stable</p><p>protein, and organelles). The insoluble fraction of metals was also available for metal assimilation. Our</p><p>calculations show that the dietary uptake of metals can be dominant in the overall bioaccumulation in</p><p>the oysters and whelks, and the trophic transfer factor was &gt;1 for all metals. Thus, the four metals have</p><p>a high potential of being biomagnified in the intertidal rocky shore food chain. MeHg possessed theThe transfer of cadmium, mercury, methylmercury,</p><p>and zinc in an intertidal rocky shore food chain</p><p>Graham Blackmore, Wen-Xiong Wang*</p><p>Department of Biology, The Hong Kong University of Science and Technology (HKUST),</p><p>Clear Water Bay, Kowloon, Hong Kong, PR China</p><p>Received 16 October 2003; received in revised form 19 January 2004; accepted 28 January 2004</p><p>Abstract</p><p>We examined the transfer of cadmium (Cd), inorganic mercury [Hg(II)], methylmercury (MeHg),</p><p>and zinc (Zn) in an intertidal rocky shore food chain, namely from marine phytoplankton to</p><p>suspension-feeding rock oysters (Saccostrea cucullata) and finally to predatory whelks Thais</p><p>clavigera. The uptake of metals from the dissolved phase was also concurrently quantified in the</p><p>oysters and the whelks. Metal uptake by the oysters was not directly proportional, whereas metal</p><p>uptake by the whelks was directly proportional to metal concentration in the water. The order of uptake</p><p>wasMeHg&gt;Hg(II)&gt;Zn&gt;Cd, and was much higher in the oysters than in the whelks. The relative uptake</p><p>of Zn and Cd was comparable between oysters and whelks, whereas MeHg and Hg(II) showed</p><p>www.elsevier.com/locate/jembe</p><p>Journal of Experimental Marine Biology and Ecology</p><p>307 (2004) 91110highest and Hg(II) and Cd the lowest potential of trophic transfer among the four metals considered.</p><p>D 2004 Elsevier B.V. All rights reserved.</p><p>Keywords: Trophic transfer; Neogastropod; Oyster; Cadmium; Zinc; Mercury; Methylmercury</p><p>0022-0981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.</p><p>doi:10.1016/j.jembe.2004.01.021</p><p>* Corresponding author. Tel.: +852-2358-7346; fax: +852-2358-1559.</p><p>E-mail address: wwang@ust.hk (W.-X. Wang).</p></li><li><p>G. Blackmore, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 91110921. Introduction</p><p>Over the past decades, there has been substantial interest in the potential transfer of</p><p>metal contaminants in different marine food chains, largely stemming from the recognition</p><p>of the significance of dietary exposure as a major route for metal bioaccumulation in</p><p>aquatic animals (Wang, 2002). Despite the general perception that trace metal biomagni-</p><p>fication, namely, increasing metal concentration with increasing trophic position along</p><p>food chain, occurs only for Hg and possibly Cs, more recent studies have shown that</p><p>potential biomagnification may occur for a few essential metals/metalloids such as Se and</p><p>Zn along specific food chains (Suedel et al., 1994; Wang, 2002). Contrasting marine food</p><p>chains possess different potential for biomagnification, largely depending on the strategies</p><p>of metal handling and storage by the animals concerned. This is especially true for benthic</p><p>invertebrates with very diverse patterns of metal sequestration and storage. Clearly, there is</p><p>a need to examine specific aquatic food chains before a general paradigm on metal transfer</p><p>in various marine food chains emerges.</p><p>Many studies on the trophic transfer of metals have focused on the control of</p><p>physicochemical species of metals in the prey organisms, whereas the physiological and</p><p>biochemical controls on metal transfer remain much less well investigated (Wang,</p><p>2002). It has been shown that metal distribution in phytoplankton cytoplasm, which can</p><p>be considered as a physical species of metals, can critically affect assimilation by</p><p>marine herbivores such as copepods, bivalves, and barnacles as characterized by a</p><p>relatively short gut passage of metals through their digestive tracts (Fisher and</p><p>Reinfelder, 1995; Wang and Fisher, 1999; Rainbow and Wang, 2001). In predatory</p><p>animals, a few studies have also demonstrated that the cytosolic fraction of metals in</p><p>the prey determine the assimilation by predatory animals such as shrimp (Wallace and</p><p>Lopez, 1997). The control of metal physico-chemical species on metal assimilation by</p><p>marine fish is somewhat variable (Ni et al., 2000). Metals are stored and detoxified in</p><p>diverse forms in the animals. A few limited studies have shown that metals stored in a</p><p>chemically inert detoxified form (e.g., granules) may not be available to the next trophic</p><p>level (Nott and Nicolaidou, 1990), but strong experimental evidence is lacking from</p><p>these previous studies. Recently there has been considerable interest in the potential</p><p>transfer of metals bound with different subcellular fractions in prey to predators</p><p>(Wallace and Luoma, 2003).</p><p>The bioavailability of metals in marine invertebrates has been extensively quantified for</p><p>a few metals such as Cd, Ag, Se, and Zn, largely due to the availability of radiotracers and</p><p>their environmental impacts regarding these elements. The pathways of Cd and Zn</p><p>bioaccumulation in marine bivalves have been comprehensively examined (Wang et al.,</p><p>1996; Chong and Wang, 2001; Ke and Wang, 2001), whereas the exposure pathways of</p><p>Hg(II) and methylmercury (MeHg) are mostly unknown even in bivalves that are</p><p>frequently employed as biomonitors of coastal contamination (OConnor, 1992; Rainbow,</p><p>1993). Furthermore, whereas most trophic transfer studies have focused on marine</p><p>herbivores grazing on phytoplankton, fewer have considered transfer to higher trophic</p><p>levels such as top benthic predators, e.g., seastars and gastropods (Fowler and Teyssie,</p><p>1997; Wang and Ke, 2002). The trophic transfer of Hg(II) and MeHg in benthic food chainalso remains little studied (Kennish, 1997).</p></li><li><p>and the dissolved uptake were examined in this study as an index to quantify metal</p><p>bioavailability from the food and aqueous phases.</p><p>G. Blackmore, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 91110 932. Materials and methods</p><p>2.1. Field collection</p><p>Whelks, T. clavigera (shell length 2530 mm, dry-tissue weight f 0.2 g), werecollected from Starfish Bay, Tolo Harbour, Hong Kong. This site has been shown to be</p><p>relatively uncontaminated by metals (Blackmore and Morton, 2001), and the metal-</p><p>lothionein (MT) concentration in the whelk was low (Blackmore and Wang, submitted for</p><p>publication). Similarly, the barnacle B. amphitrite, the rock oyster S. cucullata, the</p><p>mesogastropod snail M. labio, and the green mussel P. viridis were all collected from</p><p>the same site.</p><p>During the acclimation and experimental periods, the whelks and their prey were</p><p>maintained in aerated seawater, and kept at a constant temperature of 20 jC and salinity of28 psu. The whelks were fed ad libatum with the barnacle B. amphitrite. The mussel P.</p><p>viridis and the oyster S. cucullata were fed the diatom Thalassiosira pseudonana (clone</p><p>3H), the snails M. labio were fed small pieces of macroalgae Ulva sp., and the barnacles</p><p>were fed the diatom Thalassiosira weissflogii. The diatoms were cultured in f/2 nutrient</p><p>medium and were fed daily to the suspension feeders (oysters, mussels, and barnacles).</p><p>Following each acclimation, Cd, Hg(II), MeHg and Zn influx from the dissolved phase</p><p>and AE from the ingested food source were determined in the whelks and oysters usingIn this study, we examined the transfer of four metals/organometals (cadmium, zinc,</p><p>inorganic mercury, and methylmercury) in an intertidal marine benthic food chain. The</p><p>whelk Thais clavigera is the top predator in the intertidal rocky shore community, often</p><p>preying upon the rock oyster Saccostrea cucullata, the mussel Perna viridis, the barnacles</p><p>Balanus amphitrite, and the mesogastropod snail Monodonta labio (Blackmore, 2001).</p><p>The composition of ingested prey is dependent on the relative availability and abundance</p><p>of each prey. Rock oysters, mussels and barnacles are all suspension feeders grazing on the</p><p>seston (including phytoplankton) available in the water column. The mesogastropod snail</p><p>(herbivores) grazes on the macroalgae on the rocky surfaces. Our recent studies in two</p><p>marine predatory gastropods indicated that Cd and Zn may potentially be biomagnified in</p><p>the top predators because of the very efficient assimilation and an extremely low efflux</p><p>from the gastropods (Wang and Ke, 2002). Furthermore, field evidence suggests that</p><p>dietary exposure is the dominant route by which the whelks accumulate metals (Black-</p><p>more, 2000, 2001; Blackmore and Morton, 2001). We specifically focused on the transfer</p><p>of metals from the oysters to the whelks because the oysters constitute an important diet</p><p>for the predator. In addition, we conducted an experiment by pre-exposing the rock oysters</p><p>to different concentrations of Cd with subsequent measurements of metallothionein</p><p>induction and metal assimilation. A recent study has indicated that metal binding with</p><p>MT may potentially have an effect on dietary metal accumulation (Blackmore and Wang,</p><p>submitted for publication). Both metal assimilation efficiency (AE) from the dietary sourcemethods described below.</p></li><li><p>G. Blackmore, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 91110942.2. Laboratory pre-exposure of oysters with Cd</p><p>A series of dissolved Cd pre-exposures was conducted on the oyster S. cucullata in</p><p>order to investigate metal assimilation following MT induction. The oysters were</p><p>exposed to dissolved Cd at 5, 20 or 100 Ag l 1 for 14 days. A control group withoutthe Cd spike was also included. The oysters were maintained in aerated seawater at</p><p>20 jC and were fed the diatom T. pseudonana (clone 3H). Following the experi-mental exposure, the Cd, Zn and Hg(II) AEs and the MT concentrations in the</p><p>digestive gland of the oysters were determined in each group, using methods</p><p>described below.</p><p>2.3. Dissolved metal uptake</p><p>Eight T. clavigera or S. cucullata from each group were placed individually into</p><p>200 ml of 0.22 Am filtered seawater spiked with stable metals and the radioisotopes,109Cd 203Hg(II), Me203Hg and 65Zn. 109Cd (in 0.1 N HCl) was purchased from New</p><p>England Nuclear, Boston, USA, and 203Hg(II) and 65Zn were purchased from Riso</p><p>National Lab, Denmark. Me203Hg was synthesized from 203Hg(II) using an established</p><p>method (Rouleau and Block, 1997). Cd, Hg(II) and Zn exposures were combined</p><p>whereas a separate experiment was conducted for MeHg. Furthermore, for 203Hg(II)</p><p>and Me203Hg exposure, only the radioisotopes were spiked since the specific activity</p><p>of the radioisotope was low and the uptake was high. The dissolved concentrations for</p><p>Cd and Zn used in the exposure were 0.5, 2, 8, and 20 Ag l 1, and 2, 8, 20, and 100Ag l 1, respectively. Radioisotope additions were 1.85 kBq l 1 for Cd and 3.7 kBql 1 for Zn. Following radioactive additions, 0.5 N Suprapure NaOH was added to theseawater to maintain the pH (8.0) because the metals were carried in 0.1 N HCl</p><p>solution. The radioisotopes and the stable metals were equilibrated overnight before the</p><p>uptake experiments. For MeHg and Hg(II), the dissolved nominal concentrations used</p><p>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 concentrationswas used in the metal uptake experiments to allow calculation of the uptake rate</p><p>constant.</p><p>T. clavigera was exposed to metals for 24 h since previous experiments with snails</p><p>showed that dissolved metal uptake was much slower as compared to the bivalves (Ke and</p><p>Wang, 2001). Oysters have a much higher uptake rate and were thus exposed for 1 h (for</p><p>an explanation see Wang et al., 1996). During the exposure period, the water was regularly</p><p>stirred to homogenize the metal gradient due to metal uptake by the animals. Care was also</p><p>taken to ensure the submergence of whelks in the water during the exposure period.</p><p>Following exposure, the snails and oysters were dissected and the soft tissues radio-</p><p>assayed. The tissues were then dried at 80 jC and dry weights determined. Uptake rateswere calculated as the amount of metal accumulated by the soft tissues divided by the</p><p>exposure duration and standardized to Ag g 1 dry weight day 1. Uptake rate constants(ku) were calculated from the equation Iw = kuCw</p><p>b, where Iw is the uptake rate from the</p><p>dissolved phase, Cw is the metal concentration in the dissolved phase, and b is the power</p><p>coefficient.</p></li><li><p>G. Blackmore, W.-X. Wang / J. Exp. Mar. Biol. Ecol. 307 (2004) 91110 952.4. Metal AE in the oysters and in whelks</p><p>The AE of metals in the rock oysters was determined using a pulse-chase radiotracer</p><p>technique, as described by Wang and Fisher (1999). The diatom T. pseudonana was</p><p>radiolabeled with 37 kBq of 109Cd and 74 kBq of 203Hg(II) and 65Zn, or 74 kBq of</p><p>Me203Hg in 200 ml 0.22 Am filtered seawater. The cells were considered uniformly labeledafter they had undergone &gt;4 divisions (4 days) and were collected on a 3-Am polycar-bonate membrane, washed and resuspended in 0.22 Am water before being added to thefeeding beakers. Oysters from each group were individually placed in 500 ml of filtered</p><p>seawater and diatoms were added to yield a cell density of 45 104 cells ml 1. Furtheradditions were made at 10-min intervals to maintain this density. Following 30 min of</p><p>radioactive feeding, the oysters were rinsed in seawater and radioassayed. The oysters</p><p>were then placed into separate polypropylene beakers (180 ml seawater) held in a 20-</p><p>l enclosed recirculating flow-through aquarium containing seawater. Non-radioactive T.</p><p>pseudonana was fed twice daily at a ration of f 2% dry weight per day. Fecal pellets werecollected regularly to minimize desorption of radiotracers into the surrounding water. The</p><p>radioactivity remaining in the oysters was measured at 312 h intervals over the 72</p><p>h depuration period. AEs were determined as the percentage of initial radioactivity</p><p>retained in the oysters after 12 h.</p><p>The assimilation of Cd and Zn was quantified in the whelks by feeding on the prey</p><p>B. amphitrite, S. cucullata, M. labio, and P. viridis. These prey were radiolabeled</p><p>following 14 days of aqueous exposure to 37 kBq of 109Cd and 74 kBq of 65Zn, after</p><p>which time the prey were assumed uniformly labeled. Previous work has shown that</p><p>assimilation from a bivalve prey by the predator...</p></li></ul>