Article pubs.acs.org/est
Facilitated Bioaccumulation of Cadmium and Copper in the Oyster Crassostrea hongkongensis Solely Exposed to Zinc Fengjie Liu and Wen-Xiong Wang* State Key Laboratory of Marine Pollution, Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong ABSTRACT: Exposure to one metal might have significant effects on the bioaccumulation of other metals. In the present study, we examined the possible effects of Zn exposure on the bioaccumulation of Cd and Cu in three populations of the oyster Crassostrea hongkongensis. We found that Zn exposure significantly enhanced the tissue concentrations of Cd and Cu in all populations, and the tissue concentrations of Cd and Cu were highly and positively related to the tissue Zn concentration. Furthermore, the enhanced bioaccumulation of Cd and Cu resulted mainly from their increasing accumulation and distribution in two subcellular fractions (i.e., metallothionein-like proteins and metal-rich granules). Tissue concentrations of Cd and Cu in the natural Zn-contaminated oysters also covaried with tissue Zn concentration, and prediction analyses revealed that Zn exposure was a significant contributor to tissue Cd and Cu concentrations. Therefore, we concluded that the increased Zn bioavailability in ambient waters not only increased the tissue Zn concentration but also enhanced the overall bioaccumulation of Cd and Cu. This study strongly demonstrates that contamination of metals in oysters may result from concurrent exposure to other metals. Thus, environmental managers should consider the possible exposure to other metals such as Zn in order to interpret/predict the tissue concentrations of toxic metals in oysters.
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INTRODUCTION Zinc is an essential trace metal of exceptional biological and public health importance. Its deficiency is associated with many diseases such as growth retardation, delayed maturation, and infection susceptibility.1 Oysters are an excellent source of Zn and other nutrients and are considered an important dietary Zn supplement.2 In contrast, Cd, chemically similar to Zn, is a nonessential toxic metal and overexposure to Cd can cause damage to human bones, liver, and kidneys.3 The current maximum permitted concentration of Cd in shellfish is 1 μg Cd g−1 wet weight (European Community and mainland China) and 2 μg Cd g−1 wet weight (Hong Kong, Australia, and New Zealand).4 Contamination of seafood by Cd and its health effects have caused great concern in Southern China in recent years, since concentrations as high as 7.7 μg Cd g−1 wet weight have been reported in oysters collected from a metal-contaminated estuary of Fujian, China.5,6 Such high Cd concentrations might be partly attributed to the increasing inputs of anthropogenic pollutants in the rapidly developing areas, although at present little information is available on the sources of pollutants in these areas.5 Among the many toxic metals, Zn is of particular concern in the Chinese estuarine and coastal waters due to releases of substantial quantities of untreated industrial effluents.7 Unfortunately, the oysters are the hyper-accumulators of Zn.8,9 In preliminary surveys conducted in South China, we found that the highest Zn concentrations in oysters were up to 6% of dry tissue weight (Tan and Wang, © 2013 American Chemical Society
unpublished data). Such high tissue Zn concentrations would have probably affected the biochemistry/physiology of oysters, such as induction of metallothionein-like proteins (MTLP) and formation of metal-rich granules (MRG).10−14 Elevated metal sequestration in MTLP or/and MRG is one of the well-known metal detoxification mechanisms in aquatic animals.14−16 However, MTLP induction by Zn exposure might have substantial impacts on the subcellular distribution and bioaccumulation of Cd, which has an extremely high affinity for thiol (−SH) groups of MTLP.14,17 A few laboratory studies have demonstrated that preexposure to one metal can significantly affect the assimilation of the metal.18 For instance, it was observed that Cd assimilation efficiency increased by 1.2- to 1.6-fold in the mussel Perna viridis following Cd exposure,19 and the natural metal-contaminated clam Ruditapes philippinarum could assimilate Cd and Zn more efficiently,20 compared to clean individuals. The enhanced assimilation of the metals may be attributed to MTLP induction. Recently, a piece of evidence has also pointed out the importance of MTLP induction in reducing Cd efflux rate.21 The balance of uptake and efflux determines the overall bioaccumulation of the metals. We found that tissue Zn and Cu concentrations in four marine Received: Revised: Accepted: Published: 1670
October 15, 2012 December 20, 2012 January 2, 2013 January 2, 2013 dx.doi.org/10.1021/es304198h | Environ. Sci. Technol. 2013, 47, 1670−1677
Environmental Science & Technology
Article
bivalves were consistently increased by Cd exposure.22 These findings have important implications for risk assessment and management of metal pollution, since the increased tissue concentrations of one metal may have resulted from preexposure to the metal or contamination by other metals. Unfortunately, few efforts have been made to investigate the possible effect of one metal exposure on bioaccumulation of other metals by aquatic organisms, although it is well-known that metals generally do not occur alone.23,24 In the laboratory, we first examined whether and how Zn exposure could affect the overall bioaccumulation of Cd and Cu in three populations of the oyster Crassostrea hongkongensis, an economically and ecologically important species in Southern China. And we explored the subcellular distributions of Cd and Cu and their relationships with tissue Zn bioaccumulation, in an attempt to identify the possible underlying mechanisms responsible for the possible effects. We hypothesize that Zn exposure alone can enhance the overall bioaccumulation of Cd and Cu by potentially increasing the availability of binding sites for these metals (e.g., thiol (−SH) groups of MTLP). On the other hand, we measured concentrations of Zn, Cd, and Cu in two populations of natural Zn-contaminated oysters and then compared the predicted concentrations of Cd and Cu based on the relationships derived from the laboratory results with the measured values. Taken together, the quantitative contribution of Zn exposure in raising tissue concentration of Cd and Cu in oysters collected from the field can be estimated.
estuaries of JZ and ST are the important farming sites for oyster, and seawater salinity ranges from 10 to 35 psu which fluctuates with tide. The shell lengths of the oysters were around 5 cm for JZ, 6 cm for BJ and 10 cm for ST. The ages of the cultured JZ and ST oysters were 1.0−1.5 years and 2.5−3.0 years, respectively, whereas the age of the wild BJ oysters was unknown. The animals were cleaned and reared in aquaria in aerated clean coastal seawater (pH 8.1, 34 psu, concentrations of dissolved trace metals given in Table 1, Clear Water Bay, Hong Kong) at 16 ± 2 °C. The oysters were fed algal powders (Ori Culture, Trouw Nutrition International, concentrations of trace metals given in Table 1) at a rate of approximately 2% of their soft tissue dry weight per day. During the acclimatization and exposure periods, all oysters were maintained in 34 psu seawater except for the ST oysters, which were reared in 17 psu seawater (50% 34 psu seawater +50% distilled water), since the ST oysters were from a site with 10−20 psu salinity. The oysters were acclimatized for two weeks prior to the Zn exposure experiment. Laboratory Dissolved Zn Exposure. Shells of the oysters were thoroughly cleaned again to avoid any organic materials and small organisms. Four oysters from each site were randomly selected to determine the initial concentrations of Zn, Cd, and Cu (Table 1). Then, the oysters were randomly separated and placed in aquaria filled with 15 L of 1 μm filtered water. There were six treatments of different dissolved Zn concentrations (clean seawater, 100, 500, 1000, 5000, and 10 000 μg Zn/L) for each population. To significantly raise the tissue Zn concentrations within a relatively short period (i.e., two months), the high Zn dosage was necessary. The dissolved Zn concentrations were within the range of concentrations in field contaminated waters (e.g., 128−57 230 μg/L).5,24 Each aquarium had 15−25 individuals, and there were two independent experimental replicates. During the exposure period, the waters were regularly sampled, and the measured mean concentrations of dissolved Zn (determined after 0.22 μm-filtration) were 1.7, 104, 464, 893, 4492, and 9705 μg/L. The exposure media were prepared by spiking an appropriate volume (15−1500 μL) of 100 g/L stock solution (as ZnCl2) into 1 μm-filtered water, and the concentration of trace metals in the stock solution were negligible (Table 1). To ensure water quality and keep concentration of dissolved Zn relatively constant, the exposure water was gently aerated and completely changed every one to three days. On average, each day the oysters were exposed to dissolved Zn for 20 h and fed with algal powders for the other 4 h in clean water to avoid any possible influence of the food on uptake of dissolved Zn. The majority of live oysters were collected on the last days of the two-month exposure, while oysters exposed to 5000 and 10 000 μg/L were sampled in the first two weeks since few of them survived until the end of exposure due to acute Zn toxicity. The oysters were collected prior to the feedings to avoid possible gut contamination and were then dissected and rinsed, wetweighed and stored at −80 °C until chemical and subcellular fractionation analyses. Then, the soft tissues were individually homogenized at 4 °C, and one portion of the homogenate was used for determination of total metal concentration, whereas the other portion was used for subcellular analyses. The homogenates for determination of total metal concentration and the algal powders were freeze-dried at −80 °C (FreezeDryer, ilShinBioBase Co. Ltd.) for one week and then microwave digested (speedwavefour, BERGHOF) in concen-
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MATERIALS AND METHODS Oyster Collection for Laboratory Zn Exposure. Three populations of the oyster C. hongkongensis with contrasting metal exposure history were used in the laboratory experiment for assessing effects of Zn exposure (Table 1). The oysters were Table 1. Concentration of Trace Metals in Seawater, Algal Powders, the Zn Stock Solution and Oysters from the Three Sites without Laboratory Zn Exposure Used in the Laboratory Experimenta components
Unit
Zn
Cd
Cu
seawaterb algal powdersc Zn stock solution JZ oyster
μg/L μg/g dry wt. μg/L
1.7 ± 0.59 47.2 ± 5.5
0.018 ± 0.014 0.13 ± 0.01
0.36 ± 0.16 7.6 ± 0.5
100 g/L
