Size dependent bioaccumulation and ecotoxicity of gold nanoparticles in an endobenthic invertebrate: The Tellinid clam Scrobicularia plana

Abstract

Gold nanoparticles (AuNPs) have important technological applications resulting in an increased potential for release to the environment, and a greater possibility of toxicological effects. The marine bivalve Scrobicularia plana was exposed to AuNPs of size 5, 15 and 40 nm during a 16 d laboratory exposure at 100 μg Au L⁻¹. After exposure to AuNPs forming aggregates (>700 nm), the clams accumulated Au in their soft tissues. Biochemical (biomarkers) and behavioral (burrowing and feeding) responses were investigated. Au NPs were responsible of metallothionein induction (5, 40 nm), increased activities of catalase (15, 40 nm) and superoxide dismutase (40 nm) and of glutathione S-transferase by the three sizes of AuNPs indicating defense against oxidative stress. Exposure to AuNPs impaired burrowing behavior. However, it must be underlined that these effects were observed at a dose much higher than expected in the environment.

Introduction

Nanotechnology is a rapidly emerging discipline with a variety of applications. In nanomaterials, the exponential increase in the ratio of surface-to-bulk atoms with decreasing particle size results in greater activity per unit mass compared to identical bulk materials (Oberdörster et al., 2005). AuNPs are of special interest because of their functions in medicine and therapeutics (reviewed in Kunzmann et al., 2011), in electronics, catalysis, cosmetic, and food industries (Daniel and Astruc, 2003). Widespread use of NPs will likely lead to their increased release into the environment (Klaine et al., 2008), especially in coastal environments that are close to human populations, and potentially impact organisms through the release of their component (e.g. ions for metal-containing NPs) or through one of the most common effect of oxidative stress caused by their inherent mechanical, catalytic, optical properties, and electrical conductivity (Klaine et al., 2008, Johnston et al., 2011, Moore, 2006, Nel et al., 2006). AuNPs have been shown to induce reactive oxygen species (ROS) production with (Pan et al., 2007, Pan et al., 2009, Tedesco et al., 2010b) or without (Farkas et al., 2010) consequent toxicity. The large surface area and high surface reactivity of NPs is likely to induce oxidative stress (Tedesco et al., 2010b), thus even particles made of low toxicity material like polystyrene can be deleterious in nano scales (Brown et al., 2001), and this toxicity may be size-dependent (Johnston et al., 2011, Pan et al., 2007).

Catalase (CAT), glutathione S-transferase (GST) and superoxide dismutase (SOD) have been proposed as markers involved in antioxidant systems of defense in various aquatic species such as bivalves (Van der Oost et al., 2005). Metallothioneins are a useful tool to reveal the presence of bioavailable metals (reviewed in Amiard et al., 2006), and may be involved in both metal detoxification and antioxidant defense (Viarengo et al., 1999). Defense against the stress caused by contaminants is energy consuming, and lactate dehydrogenase (LDH) activity is particularly important when a considerable amount of energy is rapidly required (Diamantino et al., 2001) under adverse conditions. When the defense mechanisms in organisms are not sufficient to cope with oxidative stress, changes in the concentrations of thiobarbituric acid reactive substances (TBARS) are able to reflect the state of lipid peroxidation of the membranes (Knight et al., 1988). In addition, behavioral biomarkers (burrowing, feeding rate) are sensitive tools to assess the impact of contaminants at concentrations far below lethal levels (Amiard-Triquet, 2009), as well-documented in the bivalve mollusk Scrobicularia plana (Bonnard et al., 2009, Solé et al., 2009). To date the exact mechanisms concerning the in vivo toxicity of NPs (including AuNPs), and the link between biomarkers at different levels of biological organization (e.g. molecular vs. individual) remain poorly understood.

Sediments are the final sink for most contaminants, including NPs. Ferry et al. (2009) showed recently that Au nanorods passed from the water column to sediment (concentration factor was 33.1 at 12 d) and the marine food web (e.g. in clam concentration factor was 2.28 × 10⁴ at 12 d) using laboratory-constructed estuarine mesocosms. The aquatic benthic bivalve S. plana is a key species in the structure and functioning of estuarine ecosystems and a major link in estuarine food webs, playing an important role in biogeochemical cycles of both nutrients and contaminants as a consequence of its bioturbation activity. It has been shown to be a relevant model organism for the determination of biomarkers (Bonnard et al., 2009, Solé et al., 2009) and was successfully adopted in our recent nanotoxicity studies on CuO NPs (Buffet et al., 2011). In the present study, we carried out in vivo evaluation of biological responses of S. plana to AuNPs of three sizes (5, 15, and 40 nm). The fate of AuNPs of the three sizes tested was characterized in stock solution and experimental media (seawater with or without the presence of bivalves). In this aim, complementary methods were used to assess size distribution, zeta potential and purity. Animal metabolic condition (LDH activities) and the bioaccumulation of Au from NPs, together with tiered biomarker sets of defense (MT, CAT, GST, and SOD) and damage (TBARS and AChE activity) as well as behavioral biomarkers (postexposure feeding and burrowing) were adopted.