In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes

Abstract

In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.

Introduction

For several decades, shellfish culture has developed in a significant way around the world and particularly in France. The most economically important bivalve species is the Pacific oyster, Crassostrea gigas. This species is frequently reared in disturbed ecosystems subject to pollutants which affect the environmental quality of coastal waters.

Among environmental pollutants, heavy metals may have major ecological consequences. Some of the pollutants introduced in land and aquatic ecosystems may possess an important toxic potential [1]. Metals are not degradable and may thus persist for long periods in ecosystems. Moreover, physical, chemical and biological processes have a greater tendency to concentrate than to dilute heavy metals [2]. In this way, aquatic animals including marine bivalves are likely to be exposed to elevated concentrations of pollutants.

Oysters and other bivalve molluscs have been postulated as ideal indicator organisms for the assessment of environmental pollution [3], [4]. They are already used in different programs monitoring pollution in estuarine ecosystems. Through their ubiquitous, benthic and sedentary mode of life, they are exposed to environmental modifications (temperature, salinity, pollutants, etc.) with no possibility of escaping [5], [6]. Their suspensivore mode of nutrition allows them to concentrate xenobiotics within their tissues by filtration of large volumes of seawater [7]. Pollutant bioconcentration varies according to each pollutant and animal species but it may reach considerable levels. The reactions of animals faced with pollutants depend on the particular type of pollutant, nevertheless bioaccumulation constitutes a common response. Such a bioaccumulation may be the consequence either of a direct contamination by water or an indirect contamination by food [8].

Although numerous studies have been made concerning the accumulation and toxic effects of heavy metals in bivalve molluscs, little effort has been directed at determining their toxic effects at a cellular level [9]. Molluscs possess an open circulatory system which is continually exposed to the fluctuations of environmental factors including contaminants [10]. The cellular defence system of bivalves comprises haemocytes which have various activities including phagocytosis, intracellular degradation of pathogens by means of hydrolytic enzymes, production of reactive oxygen metabolites and phenoloxidase activity [11], [12], [13]. The effects of environmental contaminants may correspond to a direct toxic action on tissues or cells or to more subtle alterations in homeostatic mechanisms including the immune system [14], [15].

Most studies on immunotoxicity consist in the collection of animals located on polluted sites and the determination of immune activity variations during a long period of time [16], [17], [18], [19], [20], [21]. However, in the present work, in vitro effects of different compounds in direct contact with C. gigas haemocytes were investigated. This approach may confirm results obtained from previous in vivo or in situ experiments. Moreover, this approach is suitable for developing immunotoxicity models in order to determine the mode of action of a particular pollutant. The actual aim of the present study was to work in acute toxicity conditions in order to obtain a rapid effect of heavy metals on immune cellular and plasmatic activities. Two heavy metals were selected (mercury and cadmium) and tested at different concentrations. Two forms of mercury were used: mercury chloride, which is the form rejected by industries, and methylmercury, potentially more toxic. The presence of these pollutants was reported in different shellfish culture areas at episodic high concentrations [22]. The effects on haemocytes were studied using flow cytometry. Flow cytometry allows rapid analyses of the morphological and functional characteristics of free cell suspensions [23]. The resulting data were based on autofluorescence light scattering parameters related to cell size and complexity (granularity). Cellular functions were also investigated using fluorescent markers: cell viability, presence of hydrolytic enzymes, and phagocytic activity. Phenoloxidase-like activity was also studied in the plasma fraction of haemolymph by spectrophotometry.