Elsevier

Environmental Pollution

Volume 225, June 2017, Pages 118-128
Environmental Pollution

Nanotoxicity of graphene oxide: Assessing the influence of oxidation debris in the presence of humic acid

https://doi.org/10.1016/j.envpol.2017.03.033Get rights and content

Highlights

  • GO reduced overall length and acetylcholinesterase activity in zebrafish larvae.

  • Humic acid enhance GO stability in reconstituted water and its toxicological effects.

  • Oxidative debris removal decreased GO stability and toxicological effects.

Abstract

This study sought to evaluate the toxicological effects of graphene oxide (GO) through tests with Danio rerio (zebrafish) embryos, considering the influence of the base washing treatment and the interaction with natural organic matter (i.e., humic acid, HA). A commercial sample of GO was refluxed with NaOH to remove oxidation debris (OD) byproducts, which resulted in a base washed GO sample (bw-GO). This process decreased the total oxygenated groups in bw-GO and its stability in water compared to GO. When tested in the presence of HA, both GO and bw-GO stabilities were enhanced in water. Although the embryo exposure showed no acute toxicity or malformation, the larvae exposed to GO showed a reduction in their overall length and acetylcholinesterase activity. In the presence of HA, GO also inhibited acid phosphatase activity. Our findings indicate a mitigation of material toxicity after OD removal. The difference in the biological effects may be related to the materials’ bioavailability and biophysicochemical interactions. This study reports for the first time the critical influence of OD on the GO material biological reactivity and HA interaction, providing new data for nanomaterial environmental risk assessment and sustainable nanotechnology.

Introduction

Graphene is a promising nanomaterial (NM) in several areas, e.g., electronics, composites, energy, biosensors, drug-delivery, and environmental applications such as the removal of metals, organics pollutants and microorganisms from both water and air (Zhao et al., 2011, Georgakilas et al., 2012, Faria et al., 2014). Many applications are still in the research phase, but the promising uses of NMs have raised concerns about their risks to human health and the environment. The environmental presence of NMs may be due to their industrial use or the improper disposal of waste or its products.

Considering the graphene material family (Enoki et al., 2013), graphene oxide (GO) is used more than graphene, mainly due to its oxygen reactive groups (e.g., -COOH, -OH, =O) and good dispersion in water (Hu and Zhou, 2013, Faria et al., 2012). The presence of reactive oxygenated groups in GO facilitates the interaction with several proteins, leading to the irreversible denaturation (or activation) of proteins and potential long-term toxicity (Feng and Liu, 2011). In addition, binding to metals may affect the organism and change the availability of enzyme cofactors (Wintterlin and Bocquet, 2009, Batzill, 2012). Furthermore, several studies have shown that oxidative stress is an omnipresent cytotoxicity mechanism among NMs, including carbon-based NMs (Chen et al., 2016, Riebeling et al., 2016, Vale et al., 2016).

The biomedical application and toxicological effects of GO are still not well defined because contrasting results have been exposed in the literature. In addition to differences in the experimental design, differences in the material physical-chemical properties of GO could have led to the contrasting results. In 2011, Rourke et al. (2011). proposed a two-component structural model for GO. According to this model, GO consists of large sheets and oxidized debris deposits, which are formed by humic and fulvic-like fragments (Rodrigues-Pastor et al., 2015). Oxidation debris (OD) is a term that is used to describe small carbonaceous fragments (byproducts, typically < 1 nm) (Rourke and Wilson, 2016) that originate after the oxidation of nanocarbons (e.g., nanotubes and graphene) during chemical treatments with oxidant acids (e.g., H2SO4, HNO3) at high temperatures. These oxidized fragments remain strongly adsorbed on the carbon nanomaterial surface through π−π stacking interactions or van der Waals interactions and can be removed when the GO suspension is heated with a base, such as NaOH. These groups promote the electrostatic stabilization of oxidized carbon nanotubes and GO in water and are used to anchor functional groups (Faria et al., 2012, Rodrigues-Pastor et al., 2015, Rourke and Wilson, 2016, Paula et al., 2011, Rourke et al., 2011).

However, the two-component structural model for GO is contested by some authors (Dimiev and Polson, 2015), who consider that OD is in fact derived from GO during the base treatment via C–C bond cleavage reactions. According to them, the base washing deoxygenates and leads to a new functional group, i.e., carboxylate ions, in addition to promoting structural changes. Thus, this model has been the subject of one of the main discussions in GO chemistry over the last several years (Naumov et al., 2016).

Nevertheless, the literature has been exploring the role that the presence of OD plays in GO biotechnological applications and toxicity. Oxidation debris could be a crucial component of GO surface chemistry because the carbon nanostructures interact with the environment through its surface (Heister et al., 2010, Wang et al., 2010, Georgakilas et al., 2016, Okobiah and Reidy, 2016). For example, Coluci et al. (2014). demonstrated that OD has a critical influence on the non-covalent interactions of graphene oxide with environmental pollutants. Ma et al. (2016). discovered that the surface adsorption of organic molecules (i.e., 1,10-phenanthroline-5,6-dione) on graphene oxide was improved greatly after the elimination of OD. Recently, Pattammattel et al. (2015). suggested that OD plays a major role in controlling the chemical and biological nature of the protein-GO interface. In their in vitro study, GO without OD reduced the biological compatibility compared to GO with OD.

The behavior of NMs in aqueous media changes depending on environmental and exposure conditions. One of the factors that has been receiving a lot of attention in nanosciences is that the presence of humic acid (HA) in natural waters can improve the NM dispersion (Grillo et al., 2015, Gunsolus et al., 2015, Li et al., 2015, Hyung et al., 2007). HA can reduce NM agglomeration by overlaying them and increasing the magnitude of the negative surface charge (zeta potential) (Hyung et al., 2007, Zhang et al., 2009). Additionally, some studies have demonstrated that the presence of HA in the exposure medium alters the toxicity of NMs (Hu et al., 2011, Dasari and Hwang, 2013, Kim et al., 2013, Yang et al., 2013). Deng et al. (2016). hypothesized that a decrease in toxicity could be ascribed to HA acting both as a physical barrier between the nanocomposite and the organism and as an antioxidant. The results reported by Cai et al. (2015). suggested that π-π interactions between aromatic groups significantly participate in natural organic matter binding but that electrostatic interactions may also influence the sorption capacity, depending on the solution pH and graphene surface charge.

Despite some studies that have evaluated GO through in vivo and in vitro tests (Chen et al., 2016, Seabra et al., 2014), no study has investigated the influence of its structural composition on its biological effects (i.e., toxicity to zebrafish) and colloidal aspects after interacting with humic substances in water. In this study, the two-component structural model for GO (Rourke et al., 2011) was considered to explore the biological implication of a base washing treatment of GO. Thus, the aim of this study was to evaluate the ecotoxicological effects of GO through tests with Danio rerio (zebrafish) embryos, considering the influence of OD removal and the presence of HA in the medium. To accomplish this, we evaluated lethal and sublethal parameters, such as the larvae's total length, classical biochemical markers of metabolism (i.e., glutathione S-transferase and acid phosphatase activities), oxidative stress (i.e., catalase activity) and neurotoxicity (i.e., acetylcholinesterase activity). Advances in the nanoecotoxicity testing of GO, especially considering the influence of OD removal and interactions with humic substances, will reduce the uncertainty in NM environmental risk assessment and lead to the sustainable development of nanotechnology (Selck et al., 2016).

Section snippets

Reconstituted water

D. rerio embryos were kept in reconstituted water (rw), prepared as moderately hard water described by the USEPA (US.EPA, 2002). The characteristics of the water used for the embryo exposures were NaHCO3 96 mg/L, MgSO4 60 mg/L, KCl 4 mg/L, CaSO4·2H2O 60 mg/L, pH 7.0 ± 0.5, conductivity 350 ± 100 μS/cm, and temperature 27.0 ± 1 °C. Humic acid was purchased from Sigma Aldrich (HA, sodium salt, lot STBCS468V, cod. H16752) and added to reconstituted water to establish a concentration of 20 mg/L in

Characterization

Characterization measurements and AFM images of the materials are presented in Table 1 and Fig. 1.

The surface microchemical environment of GO and bw-GO was assessed through X-ray photoelectron spectroscopy (XPS) measurements. Table 1 shows the chemical compositions of the samples obtained from the survey XPS spectra (full-energy range, 0–1300 eV), where it is possible to confirm the purity of the samples. The base washing process led to a decrease in the oxygen content. To obtain detailed

Discussion

The chorion is an acellular membrane that protects the embryo from exposure to external agents, and its loss seems to make the organism more susceptible. In the present study, no significant malformation or mortality was observed during the embryonic period, but there were adverse effects on the larvae, depending on the concentration, presence of oxidation debris and presence of humic acid in the exposure medium. As reported in other studies (Clemente et al., 2014, Chen et al., 2011), we

Acknowledgments

The study was supported by grants 2014/01995-9 and 2014/12891-0, São Paulo Research Foundation. The authors are grateful to CNPEM facilities (LCS, LAM, and LMN), CAPES, National Institute for Science, Technology, and Innovation on Complex Functional Materials (INCT-Inomat), Brazilian Nanotoxicology Network (Cigenanotox), Brazilian Network of Nanotechnology Applied to Agrobusiness (AgroNano), and National System of Laboratories on Nanotechnologies (SisNANO). The authors also extend gratitude to

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