Nanotechnology has advanced exponentially over the past decade, and nanoscale materials being exploited in several applications (Tsuzuki, 2013). This growth of nanotechnology has not advanced without concerns regarding their potential adverse environmental impacts (Colvin, 2003, 2004; Dowling, 2004; Royal Society Royal Academy of Engineering., 2004; Warheit, 2004) and several nanotoxicology studies have been made in fact to e-value the toxicity of various nanoparticles (NPs) (Moore, 2006).

Engineered nanoparticles (NPs) represent an intermediate supramolecular state of matter between bulk and molecular material (Hoet et al., 2004). NPs biocompatibility surface properties depend on the charges carried by the particle and its chemical reactivity and size that giving a very large surface to volume ratio, The NPs size can be an extremely important factor for toxicity, and biodegradability (Brown et al., 2001; Hoet et al., 2004).

Because of the nanoscale nature of nanoscience and nanotechnology, they already bridge many fields including medicine, pharmaceuticals, manufacturing technologies, electronics and telecommunications (Perkel, 2003; Royal Society Royal Academy of Engineering., 2004; Kim et al., 2005).

The recent biomedical applications of graphene and derivatives have determined a rapid increase of the studies related to the biological interactions of these materials (Sanchez et al., 2012); for example graphene dispersed in air might represent a danger to people daily handling these materials, either by contact or inhalation, and studies on this topic are therefore necessary. Nevertheless, accidental spills and effluent discharges can determine an increased risk of release of these exogenous nanoparticles (NPs) into aquatic environment and even though emissions of graphene oxide (GO) to aquatic environment should be low (if any), their expected low degradability requires adequate investigation.

For graphene use are important to know the level of toxicity that it might reach in a biological system and the degree of safety; unfortunately, potential toxicity of graphene is little studied compared with that of other carbon nanostructures, such as carbon nanotubes (Seabra et al., 2014).

Nanoparticles in terrestrial organisms can be absorbed throughby inhalation or ingestion (Brigger et al., 2002; Moore, 2002; Colvin, 2003, 2004; Dowling, 2004; Warheit, 2004). Instead, in aquatic animals there are other routes of entry which gills and external surface epithelia (Moore, 2002). After absorption, nanoparticles are internalized occur via endocytosis (Na et al., 2003; Panyam and Labhasetwar, 2003; Panyam et al., 2003).

The toxic effects of Engineered Nanoparticles (ENPs) essentially depend on several key factors such as their intrinsic nature and capacity to form larger aggregations, the route of exposure, dose response, exposure time, the response of the receptor organisms to the lack of biocompatibility of ENPs, and the interactions in the mechanisms involved in the physiological process of uptake.

ENPs enter the environment via different exposure routes (Moore, 2002; Daughton, 2004; Moore et al., 2004; Royal Society Royal Academy of Engineering., 2004) and in natural water ecosystems ENPs can be degraded, transformed, carried and accumulated in a variety of ways. ENPs can form colloidal suspensions or can undergo processes of agglomeration or self-aggregation (Lapresta-Fernández et al., 2012), important factors that may influence their toxicity.

One of the most important factors related to the toxicity of ENPs is oxidative stress, with production of reactive oxygen species (ROS) (Lushchak, 2011).

In aquatic organisms, the effect of ROS on lipids can be measured by monitoring the intermediate species of lipid peroxidation and end products, that are tightly associated with exposure to NPs ROS-induced DNA damage may bring physiological consequences that impair reproduction (Guerriero et al., 2004, 2014), influences steroid-regulated physiology (Guerriero et al., 2005, 2017), inhibit growth, and damage both lysosomes and mitochondria. DNA damage leads to the liberation of toxic cations (Guo et al., 2008), induces ultrastructural alteration (Bartiromo et al., 2013), inhibits algal photosynthesis and affects electron and/or ion transport. The disruption of ion transport changes membrane permeability and increases the probability of NPs gaining entry inside the cell, which may lead ultimately to cell death.

The effects of ENPs on aquatic ecosystems are produced essentially by oxidative damage (internalization by cells) and interaction between ENPs and the cell membrane (without internalization) (Reyman et al., 2004). The properties of the NPs affect the uptake of NPs, which are bioaccumulated predominantly in the gills, intestine, liver (for larger NPs) (Scown et al., 2010), kidneys and blood. The latter three organs seem to be mainly due to internalization through the gills and the intestine. The main target of the toxicity mechanism is to disrupt the osmoregulatory function. Thus, the main adverse effects are related to an increase in the diffusive permeability of the membrane leading to a disturbed ion transport, as well as changes in cellular morphology, mitochondrial function, mitochondrial membrane potential and DNA damage-related gene expression that induce potential inflammation, necrosis and apoptosis of cells.

According to these results, ecotoxicology research is urgently required to explain and clarify the behavior of ENPs in their different forms and their environmental impact on the ecosystems. Fish Embryo Toxicity (FET) testis a modern non-animal test representing an effective alternative to acute test with adult fish (Embry et al., 2010; Pecoraro et al., 2017a). Fish embryo-larval assays, in fact, provide an investigative model that can be used for investigation of developmental toxicity mechanisms (Asharani et al., 2010; George et al., 2011; Ong et al., 2013; Brundo et al., 2016; Buccheri et al., 2016; Salvaggio et al., 2016; Xu et al., 2017).

Within the great variety of nanomaterials used for environmental applications, titania and graphene-based materials are extensively investigated (Scuderi et al., 2014). Graphene oxide (GO) and GO-based nanocomposites were recently proposed for the removal of pollutants (Yeh et al., 2013) and for adsorption of organics dyes from water (Sharma et al., 2013) and they are increasingly used for wastewater treatment in hybrid nanocomposite membranes (Filice et al., 2015). GO is highly dispersible in water and it shows semiconducting properties. The energy gap of GO can be tuned by a reduction of the oxygen functional groups making the valence and conduction band of GO suitable, respectively, for O₂ and H₂ evolution from water decomposition. The reduction of the oxygen content can be achieved by several ways, such as chemical or thermal treatments or UV light irradiation. Moreover, visible laser irradiation of GO flakes in water solution causes a modification of the oxygen content (D'Angelo et al., 2015) and size of the GO flakes. The conductive properties of reduced graphene oxide (RGO) become similar to that of pure graphene but with lower electron mobility (Mattevi et al., 2009). Graphene, GO and RGO showed also antibacterial properties (Liu et al., 2011; Buccheri et al., 2016).

Titania shows photocatalytic activity under UV light irradiation and TiO₂ NPs are extensively used in the degradation of organic contaminant from water (Fujishima et al., 2000). Graphene Oxide (GO) flakes present an high capability to adsorb metal ions, can interact with microorganisms and therefore GO can be used in the wastewater treatments (Zhao et al., 2011; Buccheri et al., 2016). Recently, we have demonstrated (Filice et al., 2015) that Nafion-TiO₂ under irradiation shows photocatalytic activity for degradation of Methyl Orange (MO) azo-dye with the formation of phenolic by-products. Nafion-GO_SULF is efficient like the Nafion-TiO₂ membrane in the dye removal, without leaving the toxic by-products in the MO solution. Moreover, Nafion can be used as a polymer matrix in which TiO₂, GO, and GO_SULF can be incorporated as nanoadditives, without any significant reduction of the photocatalytic efficiency with respect to the same fillers dispersed directly in solution (Filice et al., 2015).

In our paper we have evaluated the toxicity of Nafion based nanocomposites by zebrafish embryo toxicity test (ZFET), an alternative method to animal testing (Council Directive 86/609/EEC, 1986), that is considered a excellen test for the assessment of toxicity of nanocomposites (Brundo et al., 2016; Pecoraro et al., 2017a,b). As biomarkers of exposure, we analyzed Heme-Oxygenase 1 (HO1) and inducible Nitric Oxide Synthases (iNOS).

The materials tested in the present work are nanocomposite membranes prepared by dispersing various fillers, such as anatase-type TiO₂ nanoparticles and graphene oxide (GO) in Nafion polymer. Furthermore, the ZFET results performed on the nanocomposite materials, were compared with results obtained for free TiO₂ nanostructures and GO flakes dispersed as powder in the water solution.

In Figures 1a,c we report, respectively, the photos of the materials used for the present study both as fillers or as free particles: TiO₂ nanoparticles and GO dispersed in water solution. SEM images of some aggregates of TiO₂ nanoparticles and GO flakes deposited on silicon substrates are shown in Figures 1b,d.

Figures 2a,c show the nanocomposite membranes (Nafion-TiO₂ and Nafion-GO, respectively) prepared by casting and Figures 2b,d report the same membranes observed by SEM in cross section. The fillers are homogeneously dispersed in the polymer, as also verified by energy dispersive X-ray analysis in a previous work (Filice et al., 2015).

Environmental safety of the nanocomposite materials and fillers was investigated through the ZTET, a modern toxicity test that representing an effective alternative to an acute test with adult fish. Zebrafish is considered an excellent animal model for the investigation of developmental toxicity mechanisms in environmental studies. ZFET revealed neither mortality nor sublethal effects caused by the different nanocomposites and free nanoparticles tested. In particular, no one of the evaluated endpoints (viability, growth, brain morphology, pharyngeal arches and jaw, heart, fins, notochord, somites, body shape, cardiovascular function, yolk sac and locomotor function) were satisfied. Significant differences were detected, conversely, in the expression of biomarkers in larvae exposed to the nanocomposites or to the free nanoparticles. Immunohistochemical analysis, in fact, performed in larvae exposed to nanocomposite membranes, did not show the presence of biomarkers, as well as control samples. Vice versa, the larvae exposed to GO flakes and TiO₂ NPs showed a positive response to anti-HO1 and iNOS in the whole body (Figures 3a,b). These results were confirmed by gene expression analysis (Figures 4, 5).

In this work, we evaluated the toxicity of nanocomposite membranes prepared using Nafion polymer combined with anatase-type TiO₂ nanoparticles and graphene oxide. The analyses were also carried out with free TiO₂ nanoparticles and GO flakes.

The results confirmed the non-toxicity of these nanocomposites, already shown to have great potential in eco-friendly water/wastewater purification in our previous studies (Filice et al., 2015; Buccheri et al., 2016; Scalese et al., 2016), was established by testing the effect of the different materials on Danio rerio larvae, with FET test that is a new non-animal test.

MB, RP, and FM have carried out the planning of experiments, have elaborated the data and have drafted the manuscript; FC and CI take care fish facilities; RP, ES, and DT have carried out experiments of immunohistochemical and gene expression analysis; AS and GG have revised the manuscript; DD, SS, SF, and IN have realized NPs/nanocomposite membranes and have revised the manuscript.