Internal distribution of uranium and associated genotoxic damages in the chronically exposed bivalve Corbicula fluminea

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Abstract

Uranium (U) internal distribution and involved effects in the bivalve Corbicula fluminea have been studied after direct chronic exposure (90 d, 10 μg.L-1). U distribution was assessed at the subcellular level (Metal Rich Granules -MRG-, pellets and cytosol fractions) in two main organs of the bivalve (gills and visceral mass). Micro-localisation was investigated by TEM-EDX analysis in the gills epithelium. DNA damage in gill and hemolymph samples was measured by the Comet assay. The 90-d exposure period led to a significant increase of U concentration in gills over time (×5) and a large U quantity in subcellular granules in gills. Finally, a significant increase (×2) in DNA damage was noted in exposed gills and haemocytes. This study shows that the accumulation levels and consequently the potential toxicity cannot be successfully predicted only on the basis of concentration in water or in tissues and subcellular fractions after chronic exposure.

Highlights

► Relevant information concerning the chronic impact of uranium on biota is scarce. ► We study its biological speciation to explain bioavailability, accumulation, toxicity. ► 80% of U accumulated was measured in the pellet fraction (organelles + granules/MRG). ► Chronic exposure to U induced genetic damage in gill and haemolymph cells of the bivalve.

Introduction

In addition to understanding the uptake of pollutants and their depuration mechanisms in non-human species, the study of pollutant distribution in tissues and cells provides data which are useful for exposure and effect analyses within any ecological risk assessment. For a given organism, this can be achieved by linking the internal exposure in the whole body, tissue or cell to the occurrence and intensity of the induced effect (Escher and Hermens, 2004). The internal concentration at a tissue/cellular level results from both uptake and detoxification mechanisms. Among the latter, sequestration in non-toxic forms can be achieved using metal-binding proteins (metallothioneins), by internalization in lysosomes and/or by metal association to granules (Metal Rich Granules, MRGs) (Baudrimont et al., 1997, Bonneris et al., 2005, Olsson et al., 1998, Vijver et al., 2004, Vesk and Byrne, 1999, Rainbow and Smith, 2010; Wallace and Luoma, 2003). MRGs play a role in the storage and excretion of metals in molluscs (Viarengo, 1989, Bustamante et al., 2002). Uranium accumulation in MRGs has been observed in bivalves and crustaceans (Chassard-Bouchaud, 1996, Markich et al., 2001, Marigomez et al., 2002).

Natural uranium (U) in freshwater ecosystems varies from below 12 ng L−1 to more than 2 mg L−1 (Betcher et al., 1988), and is considered to be both a radiological and a chemical hazard (Labrot et al., 1999, Cooley et al., 2000, Kuhne et al., 2002). The World Health Organisation recommends that U in drinking water should not exceed 2 μg L−1.

Current knowledge of uranium uptake is limited to the understanding of the metal’s bioavailability according to its aqueous speciation (Denison, 2004), and attempts have been made to link its toxicity to some designated chemical species (Hogan et al., 2005, Fortin et al., 2007). However, few studies have focused on its distribution in various organs and in subcellular fractions even though this knowledge is crucial to understanding the effects of uranium.

As uranium is considered to be a heavy metal and a radioelement (emission of α particles) with an ability for reactive oxygen species (ROS) formation (Linares et al., 2007), the genotoxic effect of U in fish was recently studied (Barillet, 2007). The authors showed that genotoxic assays revealed a significant effect of waterborne U on the DNA integrity of fish erythrocytes, and oxidative stress (catalase, superoxide dismutase activities) was induced by U exposure (Barillet et al., 2007). Genotoxicity can be assessed by the level of damage to genetic material such as DNA strand breaks (Siu et al., 2004). The Comet assay is frequently used for the detection of DNA strand breakage and alkaline labile sites after laboratory or in situ exposures (Wilson et al., 1998, Rank and Jensen, 2003, Rigonato et al., 2005, Rocher et al., 2006). Moreover, stable metals and/or ionising radiations can generate DNA damage (Emmanouil et al., 2007). In addition, haemocytes have been shown to be sensitive to metals and reference genotoxicants (Rigonato et al., 2005).

For this study, we selected the Asiatic clam, Corbicula fluminea, as a biological model and applied chemical exposure conditions known to favour uranium bioavailability for this species (Denison, 2004, Simon and Garnier-Laplace, 2004). Molluscs are widely distributed in freshwaters, are easy to collect and can bioaccumulate contaminants, even at low exposure concentrations. Thus, bivalves, in particular C. fluminea, are valuable organisms for environmental monitoring and, as such, have been used for many years (Rigonato et al., 2005).

The aim of this study was three-fold: (1) to test the influence of chronic and low level waterborne uranium exposure (i.e., a 90-d exposure period at 10 μg L−1 – an environmental level) on U tissue distribution (mainly in gills and visceral mass) in the bivalve compared to the distribution observed after acute waterborne exposures (i.e., a 10-d exposure period at 20, 100 and 500 μg L−1), (2) to study the dominant sequestration mechanisms after chronic exposure, on the basis of TEM observations and (3) to examine the relationship between genotoxic effect and U accumulation in gills and haemolymph.

Section snippets

Collection of the organism

C. fluminea was collected manually from the bank of the river Moselle (Metz, France). The background uranium level in the collected bivalves was below the detection limit of the ICP-AES equipment under our analytical conditions. The bivalves were acclimatized to laboratory conditions for at least one month prior to the beginning of the experiments. The bivalves were kept in a storage tank containing quartz sand (grain size: 0.8–4.0 mm, Silaq, Gironde, France) and aerated water (20 °C), renewed

Accumulation

The mortality rate did not exceed 5% over time, irrespective of the experimental conditions.

For the chronic exposure treatment, the average U concentrations (n = 134) in water measured over 90 days were close to the nominal concentration (14.3 ± 7; 12.8 ± 6.8; 16.8 ± 6.9 μg L−1) for each replicate. The mass and relative mass of the 4 organs remained constant over time (ANOVA, P < 0.05, n = 90). The average whole body mass (g, fw) of the bivalves was 0.26 ± 0.05 and the relative mass (%) of the

Discussion

Significant differences in accumulation levels and relative distribution were observed in the gills and in the visceral mass following chronic U exposure (10 μg L−1, 90 d) (ANOVA, P < 0.05, 3 independent replicates of 10 individuals, result not shown). Chronic exposure demonstrated a low and linear accumulation in gills as a function of time in comparison with the visceral mass. In the case of U gill uptake, exposure duration and U exposure levels strongly influenced the accumulation level.

Conclusion

U accumulation in the visceral mass was found not to be linearly linked to levels of U exposure. Accumulation in gills was low and linked to waterborne exposure. At least 80% of the total burden accumulated in the animals studied was measured in the pellet fraction (organelles + granules/MRG) which represented the main site of U accumulation.

This study indicates that chronic exposure to U induced genetic damage in gill and haemolymph cells of the bivalve, C. fluminea. As previously demonstrated

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