Similar toxicity mechanisms between graphene oxide and oxidized multi-walled carbon nanotubes in Microcystis aeruginosa
Graphical abstract
Introduction
Carbon nanomaterials (CNMs) are a family of carbon nanostructures that include the 0-dimensional fullerene, 1-dimensional carbon nanotube (CNTs), and 2-dimensional graphene (Mauter and Elimelech, 2008; Perreault et al., 2015a,b). The exceptional mechanical, electrical, and thermal properties of this class of nanomaterials (NMs) have led to their applications in a wide range of commercial and industrial applications in fields as diverse as electronics (Jariwala et al., 2013), sensors (Peña-Bahamonde et al., 2018), medicine (Loh et al., 2018), photovoltaics (Jariwala et al., 2013), construction (Sanchez and Sobolev, 2010), or water treatment (Perreault et al., 2015a,b; Smith and Rodrigues, 2015). However, this widespread use may ultimately lead to an increased release of CNMs in the environment (Gottschalk et al., 2013). To mitigate the potential risks associated with CNM exposure, a fundamental understanding of the interactions of CNMs with biological systems is needed to guide a safer, more sustainable CNMs development in nano-enabled products (Du et al., 2013; Falinski et al., 2018; Gilbertson et al., 2015).
For CNTs, toxic effects have been shown for a range of organisms, including bacteria, microalgae, invertebrates, and fishes (Falinski et al., 2019; Petersen et al., 2009; Sanchez et al., 2012). In aquatic photosynthetic microorganisms, which are commonly used for ecotoxicological assessment due to their sensitivity, ease of maintenance, and relevance in the aquatic trophic chain, CNTs have been shown to induce growth inhibition and cell death through a variety of mechanisms. Youn et al. showed that gum arabic-stabilized single-walled CNTs inhibit the growth of the green alga Pseudokirchneriella subcapitata at concentrations > 0.5 mg/L in a 96 h exposure assay (Youn et al., 2012). Wei et al. showed that, at concentrations ranging between 1 and 10 mg/L, oxidized multi-walled CNTs induced oxidative stress and inhibited photochemical processes at the photosystem II (PSII) level in the green alga Dunaliella tertiolecta (Wei et al., 2010). On the other hand, Schwab et al. observed a 96 h EC50 value of 1.8 mg/L and 20 mg/L for Chlorella vulgaris and P. subcapitata exposed to CNTs; however, growth inhibition was primarily (>85%) attributed to a self-shading effect due to light absorption by CNTs, which limited photosynthetic activity (Schwab et al., 2011). Similarly, Long et al. showed that in green alga, Chlorella sp., exposed to a concentration inducing a 50% decrease in cell growth (EC50) after 96 h, physical interactions associated with agglomeration and self-shading explained ∼50% of the growth inhibition. These results highlight the complexity and variability of CNTs’ toxicity in photosynthetic microorganisms, which is attributed to the intrinsic variability in CNTs’ properties, such as tube length, diameter, purity, and chirality, differences in the dispersion and exposure conditions, as well as the sensitivity of the different biological models (Bennett et al., 2013; Jiang et al., 2020; Liu et al., 2009).
Similarly, graphene and graphene oxide (GO) have been shown to induce toxicity to multiple biological models (Ahmed and Rodrigues, 2013; Barrios et al., 2019; Falinski et al., 2019; Li et al., 2019). Like CNTs, their toxicity in microalgae appears to be driven by mechanisms associated with oxidative stress, inhibition of photosynthesis, and physical interactions leading to cell death. Tang et al. reported growth inhibition of Microcystis aeruginosa at GO concentrations above 10 mg/L, an effect that was associated with the adhesion of GO sheets on the cell surface, the induction of oxidative stress, and the inhibition of the photosynthetic electron transport (Tang et al., 2015). In Raphidocelis subcapitata, the growth inhibition 96 h EC50 value was found to be ∼20 mg/L and was characterized by cell membrane damage, oxidative stress, chlorosis, and physical interactions between the GO sheets and algal cells (Nogueira et al., 2015). Likewise, reduced GO (rGO) exposure in Scenedesmus obliquus led to growth inhibition characterized by the cellular deposition of rGO sheets, the inhibition of PSII electron transport, oxidative stress, and lipid peroxidation (Du et al., 2016). However, this effect was observed at much higher concentration than the oxidized form (i.e. GO), with a 72 h EC50 value of 148 mg/L. Like for CNTs, differences in the GO properties (Barrios et al., 2019; Faria et al., 2018), exposure conditions, and biological models led to a high variability in the measured toxicity thresholds reported in the literature.
The similarities in toxicological interactions between the 1-D and 2-D forms of CNMs can be explained by their similarities in chemical structure. Indeed, CNTs are essentially rolled-up graphene sheets. However, this morphological change results in important differences in physicochemical properties (Biswas and Lee, 2011; Kauffman and Star, 2010), particularly in water or when the oxidized forms of these materials, such as GO, are considered. For example, GO is an insulating, hydrophilic material that is highly stable in water while CNTs are typically more conductive with reduced stability in water (Dreyer et al., 2010; Qi et al., 2016). Aggregation in aqueous conditions will lead to aggregates of different density, with GO having a more open house-of-cards structure (Ersan et al., 2017). For toxicological interactions, edges and defects, which are found all along the edges in 2-D materials but mainly at the tubes’ tip in 1-D CNTs, have been shown to be the main reactive sites for oxidative interactions (Faria et al., 2018; Liu et al., 2011). The distribution of edge sites was also shown to change how CNMs interact with cell membranes, with the penetration of graphene and GO into cell membranes being facilitated by the abundance of sharp irregular edges in the 2-D form (Li et al., 2013; Shi et al., 2011). However, no side-by-side comparison of the toxicity of 1-D CNT and 2-D GO was made for photosynthetic organisms. The variability in doses, materials, and organisms used in toxicity studies makes it hard to determine if the two carbon allotropes share the same mechanisms of toxicity. Similarities can be expected based on their composition, but important differences are also likely due to their different morphologies and physicochemical properties.
In this study, we aimed to determine if the mechanisms of interaction between CNMs and aquatic photosynthetic organisms differ between 1-D and 2-D CNMs. The cyanobacterium M. aeruginosa was used as a model organism for toxicity assays due to its ease of culture, sensitivity to environmental contaminants, and frequent use as a model for nano-ecotoxicological studies (Luo et al., 2018; Tang et al., 2015; Wang et al., 2011; Yang et al., 2018). Cyanobacterial cells were exposed to different concentrations of the oxidized forms of two CNMs, oxidized multi-walled CNTs and GO, to provide a better stability in aqueous media. The two CNMs were evaluated at the same biological endpoint, the EC50 value, and compared on the basis of change in pigment content, photosynthetic activity, membrane integrity, and oxidative stress in order to distinguish each CNMs’ respective mechanism of toxicity. The results of this work indicate that, despite important differences in how they interact with cells, both GO and CNTs have similar impacts on the physiology of M. aeruginosa.
Section snippets
Chemicals and reagents
A modified Hummer’s powdered single layer GO (∼99% pure) was purchased from ACS Materials LLC (Medford, MA, USA) and used as received. Multi-walled CNTs (>95% pure) were purchased from CheapTubes (Cambridgeport, VT, USA). The pristine material was acid-treated with nitric acid (HNO3, 70%) for 4 h under reflux to increase surface oxygen concentrations (Falinski et al., 2019). The fluorescent dyes fluorescein diacetate (FDA), BODIPY™ 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s
Characterization of carbon nanomaterials
Thorough NM characterization in nanotoxicology studies is essential to understand how the properties of the material can influence its toxicity as well as to make the toxicological data generated relevant for other researchers and regulators (Fadeel et al., 2015; Petersen and Henry, 2012). Therefore, the composition, morphology, and size of the GO and CNTs used in this work were characterized using SEM, TEM, and XPS analyses (Fig. 1). SEM imaging of GO showed a material with a typical
Conclusion
Despite different physicochemical properties, GO and CNTs appeared to have similar level of toxicity and mechanisms of interaction with the cyanobacterium M. aeruginosa. Toxicity of both CNMs was characterized by a decrease in photosynthetic electron transport rate and a decrease in FDA fluorescence, suggesting a reduction in cell metabolic activity. The absence of CNMs-induced oxidative stress and membrane damage in cells exposed to CNMs support the hypothesis of physical interactions leading
Credit author statement
E. Cruces: Conceptualization, Supervision, Investigation, Validation, Visualization, Writing- Original draft preparation, Writing- Reviewing and Editing. A.C. Barrios: Conceptualization, Supervision, Investigation, Validation, Visualization, Writing- Original draft preparation, Writing- Reviewing and Editing. Y.P. Cahue: Investigation, Validation, Writing- Reviewing and Editing. B. Januszewski : Investigation, Validation, Writing- Reviewing and Editing. L.M. Gilbertson: Conceptualization,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was partially funded by the CBET-1708681/1709031 and EEC-1449500 awards of the National Science Foundation, and the Fondo Nacional de Desarrollo Científico y Tecnológico through the FONDECYT award no 11171079. We acknowledge David Lowry for his assistance in sample preparation and acquisition of micrographs and the use of facilities within the Life Science Electron Microscopy Lab and the Eyring Materials Center at Arizona State University, supported in part by NNCI-ECCS-1542160. We
References (91)
- et al.
Investigation of acute effects of graphene oxide on wastewater microbial community : a case study
J. Hazard Mater.
(2013) - et al.
Stability , metal leaching , photoactivity and toxicity in freshwater systems of commercial single wall carbon nanotubes
Water Res.
(2013) - et al.
Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA)
Biochim. Biophys. Acta Bioenerg.
(2004) - et al.
EC50 estimation of antioxidant activity in DPPH∗ assay using several statistical programs
Food Chem.
(2013) - et al.
Chemical oxidation of multiwalled carbon nanotubes
Carbon
(2008) - et al.
Inhibitory effects of silver nanoparticles on photosystem II performance in Lemna gibba probed by chlorophyll fluorescence
Curr. Plant Biol.
(2018) - et al.
Understanding the toxicity of carbon nanotubes in the environment is crucial to the control of nanomaterials in producing and processing and the assessment of health risk for human: a review
Environ. Toxicol. Pharmacol.
(2013) - et al.
Reduced graphene oxide induces cytotoxicity and inhibits photosynthetic performance of the green alga Scenedesmus obliquus
Chemosphere
(2016) - et al.
Adsorption of organic contaminants by graphene nanosheets: a review
Water Res.
(2017) - et al.
Keeping it real: the importance of material characterization in nanotoxicology
Biochem. Biophys. Res. Commun.
(2015)