Effects of nanosilver on Mytilus galloprovincialis hemocytes and early embryo development
Introduction
Silver nanoparticles (AgNPs) have a large number of applications because of their chemico-physical characteristics and, above all, because of their biocidal action. The widespread utilization of AgNPs in a large range of consumer products, including textiles, care products and food packaging, will inevitably lead to their release in the environment (reviewed in Pulit-Prociak and Banach, 2016; McGillicuddy et al., 2017). According to Gottschalk et al. (2009) Predicted Environmental Concentrations (PECs) of AgNP for surface waters in Europe are expected to be in the low ng/L-range (0.76 ng/L), but AGNPs are expected to be released in larger quantities within the next decades (Fabrega et al., 2011; McGillicuddy et al., 2017). Once in the aquatic environment, AgNPs can undergo several transformation processes (agglomeration/aggregation, oxidation, dissolution, adsorption with soluble and particulate organic matter); in particular, release of Ag+ ions may represent an additional source of silver in the environment (Levard et al., 2012; Sendra et al., 2017). With regards to ecotoxicity, it is widely acknowledged that the impact of AgNPs mainly depend on the Ag+ released from the nanomaterial (Jemec et al., 2016). However, AgNPs may also exhibit a particle-specific toxicity, possibly mediated by physical interactions of the nanoparticulate form with biological systems (Li et al., 2014; Fabrega et al., 2011; Magesky and Pelletier, 2018).
The impact of AgNPs has been widely investigated in marine invertebrates; these studies showed that in vivo exposure leads to silver accumulation, and to different types of responses at molecular, cellular and tissue level (reviewed in Magesky and Pelletier, 2018). The bivalve Mytilus spp. is considered a suitable model for studying the effects and mechanisms of action of different types of NPs (Canesi et al., 2012; Canesi and Procházová, 2013; Rocha et al., 2015; Canesi and Corsi, 2016; Beyer et al., 2017; Faggio et al., 2018). In vivo exposures to AgNPs have previously shown silver accumulation, induction of oxidative stress and damage to several cell components, including DNA (Zuykov et al., 2011; Gomes et al., 2013; McCarthy et al., 2013; Jimeno-Romero et al., 2017). These data provided valuable information at the whole organism level, also considering different potential pathway of exposure.
With regards to in vitro data, the application of a battery of functional tests on Mytilus immune cells, the hemocytes, has been proven as a powerful tool for the rapid in vitro screening of the immunomodulatory effects and identification of the mechanisms of action of different types of NPs (Canesi et al., 2012; Canesi and Procházová, 2013; Canesi and Corsi, 2016; Canesi et al., 2016). These studies also underlined the importance of exposure medium in determining particle behaviour and interactions with target cells in a physiological environment (Balbi et al., 2017a; Canesi et al., 2017). However, information on the impact of AgNPs in bivalves at the cellular level is scarce, with only one study available to date. Exposure of M. galloprovincialis hemocytes and gill cells to several types of maltose-coated AgNPs of different sizes revealed higher toxicity of smaller size NPs, damages to cell components and activation of cellular defences after 24 h (Katsumiti et al., 2015). These data underlined the importance to further investigate the in vitro effects of AgNPs in mussel cells, in order to better understand their mechanism of action and possible toxicity.
In marine invertebrates, NPs generally do not have lethal effects at environmental concentrations, but can induce changes in their life cycle, growth or anatomical deformities that can lead to a diminished biological performance of wild populations (Canesi and Corsi, 2016). The application of developmental assays, involving exposure during the most sensitive stages of the organisms to environmental contaminants, would greatly help in the fast screening of NP toxicity (Fabbri et al., 2014). Most data are available on the sea urchin model, where exposure to nanoparticulate and ionic silver induced distinct effects depending on the life stage (Šiller et al., 2013; Magesky and Pelletier, 2018). Developmental effects were also reported for AgNPs in oysters (Ringwood et al., 2010). However, no information is available on the effects of AgNPs on Mytilus embryos.
In the present study, the effects of bare AgNPs were investigated at the cellular level in short term in vitro experiments of M. galloprovincialis hemocytes; the impact on mussel early embryo development was also evaluated. Parallel experiments were carried out using AgNO3 in order to compare the effects of ionic silver. Hemocytes were exposed in vitro for 30 min to different concentrations of AgNPs (0.1–1000 μg/mL) or AgNO3 (0.1–10 μg/mL) and Lysosomal Membrane Stability (LMS) was first evaluated as a marker of cellular stress. Functional immune parameters were also evaluated (extracellular reactive oxygen species-ROS production, phagocytosis). Experiments with AgNPs were carried out in either artificial sea water (ASW) or hemolymph serum (HS) to evaluate the influence of exposure medium. Moreover, at selected concentrations of AgNPs and AgNO3, the effects on mitochondrial membrane potential and on actin cytoskeleton were evaluated by Confocal Laser Scanning Microscopy (CLSM).
The developmental effects of AgNPs were evaluated by the 48 h embryotoxicity test; fertilized eggs were exposed to AgNP (0.001–1000 μg/L) or AgNO3 (0.1–25 μg/L). At the end of the assay, both percentage of normal D-larvae and the type of effect (malformations, delayed development, death) were evaluated.
Section snippets
Characterization of NPs
AgNPs (47MN-03) were purchased from Advanced Materials Inframat. The sample is a silver, black and ultrafine nanopowder with no coating. Characterization of primary particles and AgNP suspensions were performed as previously described (Brunelli et al., 2013). The average size of particle distribution of primary particles was evaluated by HR-TEM (High Resolution Transmission Microscopy) using a JEOL (Tokyo, Japan) 3010 microscope operating at 300 kV. Specific surface area was evaluated using BET
AgNP characterization
In Fig. 1 the data on physico-chemical characterization of AgNP are reported. Primary particle characterization showed that AgNPs are irregular elongated polyhedrons and spherical particles with rounded and smooth edges (Fig. 1A, TEM images). The Z-average particle size obtained was 61 nm, with values ranging between 41 and 81 nm (64% of particles between 35 and 65 nm) (Fig. 1A distribution graph by frequency), very similar to the declared value (i.e. 40–90 nm). The sample presented little
Discussion
Marine invertebrates can represent a significant target to nanosilver (Magesky and Pelletier, 2018), one of the most widespread NP types (Pulit-Prociak and Banach, 2016). In this work, data are presented on the effects of bare AgNPs (47 nm) in the marine bivalve M. galloprovincialis evaluated at the cellular and whole organism level using two model systems i.e. short term in vitro exposure of hemocytes and the 48 h embryotoxicity assay. The effects were compared with those of AgNO3.
Conclusions
The data reported in the present study represent a first attempt to compare the possible effects and mechanism of action of AgNPs and soluble Ag+ in mussels at the cellular and organism level. In both experimental settings, AgNPs was effective at much higher concentrations that those of AgNO3, indicating little toxicity; moreover, the mechanisms of action of AgNPs appeared to be distinct from those of Ag+ in both hemocytes and embryos. The lower toxicity of AgNPs may be partly due to
Funding
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement PANDORA N°671,881.
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