Impact of exposure time



Impact of exposure time, particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus galloprovincialis

Younes Bouallegui, Ridha Ben Younes, Faten Turki and Ridha Oueslati

Research Unit for Immuno-Microbiology Environmental and Cancerogenesis, Sciences Faculty of Bizerte, University of Carthage, Bizerte, Tunisia

ABSTRACT Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanopar- ticles (AgNP: <50 nm and <100nm) on the frequency of hemocytes subpopulations as immunomodula- tion biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over dif- ferent durations of exposure (3, 6 and 12 h). Differential hemocyte counts (DHC) revealed significant varia- tions in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e. no significant effects after 3 h with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and duration of exposure dependent. This study highlighted how not only was NP size important, but that dif- fering internalization mechanisms could be key factors impacting on the potential for NP in the environ- ment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.

ARTICLE HISTORY Received 13 February 2017 Revised 6 May 2017 Accepted 24 May 2017

KEYWORDS Silver nanoparticles; endocytosis; hyalinocytes; granulocytes; Pappenheim panoptical staining


Nanoparticles (NP) are defined as materials with all dimensions in nanoscale [1–100 nm] (Luoma 2008). Silver nanoparticles (AgNP) have become the fastest growing product category in nanotechnology due to their thermoelectrical conductivity, cata- lytic activity and nonlinear optical behavior and have great value in the formulation of inks, microelectronic products and biomed- ical facilities (i.e. imaging devices) (Tiede et al. 2009; Katsumiti et al. 2015). Their exceptional broad-spectrum bactericidal prop- erties and biocompatibility (i.e. as drug delivery agent) have also made AgNP extremely useful in a diverse range of consumer goods (Luoma 2008; Rainville et al. 2014; Cozzari et al. 2015; Katsumiti et al. 2015; Marisa et al. 2016).

Worldwide AgNP production is estimated at � 55 tonne/yr (Piccinno et al. 2012). However, release of AgNP into aquatic environs can happen through wastewaters generated during AgNP synthesis and/or incorporation into goods and consumer products (Canesi et al. 2012; Matranga & Corsi 2012; Katsumiti et al. 2015; Marisa et al. 2016). As such, AgNP have emerged as potential stressors that might enter marine environment (Luoma 2008). A lack of appropriate tools to evaluate effective NP (of AgNP in particular) levels in aquatic environments make selection of appropriate testing levels a major problem in risk assessment of engineered NP. As a result, predicted environmen- tal concentrations for AgNP are often set at a level of � 0.01 lg/L (Tiede et al. 2009; Katsumiti et al. 2015). Even so,

levels much lower than that have commonly been used in aquatic species ecotoxicity tests (1–100 lg/L) (Tiede et al. 2009; Canesi & Corsi 2016), including those with mollusk models.

In the mussel Mytilus galloprovincialis (filter-feeding organ- ism), hemocytes are hemolymph cells responsible for immune defence and serve as a first line of defence against foreign substan- ces (Gosling 2003; Parisi et al. 2008; Giron-Perez 2010; Matozzo & Bailo 2015). Immune defences carried out by hemocytes constitute important targets for potential NP toxicity (Canesi et al. 2012; Canesi & Prochazkova 2013; Katsumiti et al. 2015).

Several studies have shown that different NP types, that is, car- bon black, C60 fullerenes, TiO2, SiO2, ZnO, CeO2, Cd-based, Au- based and Ag-based, are rapidly taken up by hemocytes. Internalization of these NP subsequently impacted on morpho- logic/functional characteristics including immune responses (Canesi et al. 2008, 2010a, b, 2012; Katsumiti et al. 2015; Marisa et al. 2016). Various mussel hemocyte parameters, including total hemocyte count (THC), differential hemocyte count (DHC), hemocyte viability, phagocytic activity and lysosomal membrane stability, have been used as a tool for screening of immunomodu- latory effects of differing NP (Matozzo et al. 2007; Parisi et al. 2008; Hoher et al. 2013; Matozzo & Bailo 2015; Canesi & Corsi, 2016; Marisa et al. 2016). Specifically, hyalinocytes and granulo- cytes have been assessed for morphological changes among hemo- cytes in Mytilus galloprovincialis (Pipe et al. 1997; Chang et al. 2005; Garcia-Garcia et al. 2008).

CONTACT Younes Bouallegui Research Unit of Immuno-Microbiology Environmental and Cancerogenesis, Sciences Faculty of Bizerte, Zarzouna 7021, Bizerte, Tunisia � 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.




While granulocytes are large ovoid-shaped cells with a small eccentric nucleus and granulated cytoplasm (low nucleus/cyto- plasm [N/C] ratio) that are able to spread out and produce pseudo- podia), hyalinocytes are small round cells with an agranular (zero- few granules) small cytoplasm surrounding a large nucleus (high N/C ratio) (Carballal et al. 1997; Parisi et al. 2008; Cima 2010; Matozzo & Bailo 2015). Overall, hemocytes can be classified into two types, granulocytes and hyalinocytes (so-called agranulocytes), based on morphological characteristics (the presence/absence of granules in cytoplasm). Staining of the cytoplasm by certain dyes allows for sub-distinguishing of acidophils from basophils among the granulocytes. Ultimately, the basophils of M. edulis appear as granulocytes with small granules, while acidophilic granulocytes contain large granules. In comparison to the granulocytes, hyalino- cytes in bivalve have only basophilic properties. Thus, in earlier studies that described hemocyte subpopulations, the author indi- cated that basophilic cells (hyalinocytes þ basophils) made up about 40% of the total hemocyte pool in bivalves/mussels while eosinophils accounted for the remaining � 60% of all hemocytes (Chang et al. 2005; Garcia-Garcia et al. 2008).

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