Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor

Abstract

Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg⁻¹ sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. ⁶⁷ZnO NPs (DLS: 21–34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of ⁶⁷ZnO NPs in sediment, ⁶⁷Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to ⁶⁷ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, ⁶⁷Zn concentrations in sediment showed higher levels in the upper section (1 cm: 2.59 mg kg⁻¹) decreasing progressively (2 cm: 1.63 mg kg⁻¹, 3 cm: 0.90 mg kg⁻¹, 4 cm: 0.67 mg kg⁻¹) to a minimum value at the bottom (5 cm: 0.31 mg kg−1). ⁶⁷Zn bioaccumulation was observed in both organisms exposed to ⁶⁷ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, ⁶⁷ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to ⁶⁷ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to ⁶⁷ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of ⁶⁷ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone.

Introduction

The field of nanotechnology has shown a huge expansion during the last decade. With an increasing range of nanomaterials under production, questions arise concerning environmental and human health (Auffan et al., 2009, Klaine et al., 2008). Since aquatic environments are the final destination of contaminants, ecotoxicological studies on coastal and marine environment are required.

ZnO is one of the most commonly used types of metal-based nanoparticles in fields like electronics, personal care and UV protection (Kool et al., 2011) using their specific optic and photonic properties. ZnO nanoparticles (ZnO NPs) are also used as antimicrobial agents in paints, plastics and toothpastes (Hooper et al., 2011). At the present time, there are many papers concerning ZnO NPs but no consensus about the role of nanosize property on their toxicity. Some recent works suggest that risk assessments do not need to go beyond considering the metal component of ZnO NPs in soils (Hooper et al., 2011) or that toxicity is associated with the solubilized Zn in aqueous systems (Auffan et al., 2009, Heinlaan et al., 2008, Mortimer et al., 2010). On the other hand, other scientists showed that the toxicity of nanosized metal oxides is at least partly due to the size effect and not explained by dissolution of the particles alone (Wang et al., 2009).

According to Studart et al. (2007), suspensions with a high concentration of nanoparticles (>40 vol%) can be very advantageous, in many technologies for device fabrication but need to be stabilized. In most cases, the toxicity of the stabilizing agent is not investigated. Despite a lot of work on toxicity of nanoparticles on seawater and freshwater species, there is a little number of studies dealing with sediment (Mühling et al., 2009, Pang et al., 2012). Several authors underline the need to carry out experiment using soil matrix (Hooper et al., 2011, Kool et al., 2011) or sediments (Keller et al., 2010) to well understand complex mechanisms which can modify NPs bioavailability in this particular compartment. In the environment, metals naturally present are associated with sediment and biota at relatively high concentrations. Techniques using isotopes are recommended to follow the uptake of metals added in experiments and their bioavailability (Croteau et al., 2011, Rodgers et al., 2011). In addition, in seawater, nanoparticles become rapidly agglomerated favouring deposition onto the sediment surface; thus benthic organisms may be at higher risk of exposure (Keller et al., 2010).

With the main objective of investigating putative ecological impairments caused by engineered nanoparticles (ENPs), it is needed to select species which have a key role in the structure and functioning of ecosystems. In this way, marine invertebrates such as the ragworm Hediste diversicolor and the bivalve mollusc Scrobicularia plana (Berthet et al., 2011, Solé et al., 2009) are recognized as good models for biomonitoring purposes.

Biomarkers of defence are part of the very first mechanisms of protection against pollutants in the environment. Metallothionein (MT) is a core biomarker involved in the regulation and detoxification of metals in organisms (Amiard et al., 2006) and may be involved in the defence against oxidative stress (Buico et al., 2008). Catalase (CAT), glutathione-S-transferase (GST), superoxide dismutase (SOD) are also able to cope with oxidative processus (Regoli et al., 2011). These enzymes are of great interest since oxidative stress is recognized as a main effect of NPs on biota (Klaine et al., 2008, Moore, 2006).

Defence capacities of animals are limited because of the energy cost of protection against pollutants. LDH activity is particularly important when a considerable amount of energy is rapidly required (Diamantino et al., 2001, Moreira et al., 2006) under adverse conditions. When defences are not sufficient to cope with toxicity, damages can appear. Thiobarbituric acid reactive substances (TBARS) can reveal the state of peroxidation of lipids from membranes (Knight et al., 1988). Cholinesterases (ChE) are commonly used in environmental assessment and are core biomarkers promising for future implementations into the OSPAR coordinated environmental monitoring program (CEMP) as neurotoxicity marker (Burgeot et al., 2010). In addition, behavioural biomarkers are sensitive tools to assess the impact of contaminants at concentrations far below lethal effects (Amiard-Triquet, 2009) particularly in S. plana (Byrne and O'Halloran, 2001) and H. diversicolor (Moreira et al., 2006).

The aims of the present study were: (i) to evaluate the fate of ZnO NPs in sediment and bioaccumulation in endobenthic species (S. plana, H. diversicolor) using purpose-made and well characterised isotopically modified nanoparticles (⁶⁷ZnO NPs); (ii) to investigate ZnO NPs toxicity using a set of biochemical and behavioural biomarkers taking into account the role of the stabilizing solution (diethylene glycol, DEG) in the toxicity of ZnO suspension.