While graphene oxide (GO) has been found to be the most toxic graphene-based nanomaterial, its environmental fate is still unexplored. In this study, the aggregation kinetics and stability of GO were investigated using time-resolved dynamic light scattering over a wide range of aquatic chemistries (pH, salt types (NaCl, MgCl2, CaCl2), ionic strength) relevant to natural and engineered systems. Although pH did not have a notable influence on GO stability from pH 4 to 10, salt type and ionic strength had significant effects on GO stability due to electrical double layer compression, similar to other colloidal particles. The critical coagulation concentration (CCC) values of GO were determined to be 44 mM NaCl, 0.9 mM CaCl2, and 1.3 mM MgCl2. Aggregation and stability of GO in the aquatic environment followed colloidal theory (DLVO and Schulze-Hardy rule), even though GO's shape is not spherical. CCC values of GO were lower than reported fullerene CCC values and higher than reported carbon nanotube CCC values. CaCl2 destabilized GO more aggressively than MgCl2 and NaCl due to the binding capacity of Ca(2+) ions with hydroxyl and carbonyl functional groups of GO. Natural organic matter significantly improved the stability of GO in water primarily due to steric repulsion. Long-term stability studies demonstrated that GO was highly stable in both natural and synthetic surface waters, although it settled quickly in synthetic groundwater. While GO remained stable in synthetic influent wastewater, effluent wastewater collected from a treatment plant rapidly destabilized GO, indicating GO will settle out during the wastewater treatment process and likely accumulate in biosolids and sludge. Overall, our findings indicate that GO nanomaterials will be stable in the natural aquatic environment and that significant aqueous transport of GO is possible.

While two-dimensional graphene oxide (GO) is used increasingly in biomedical applications, there is uncertainty on how specific physicochemical properties relate to biocompatibility in mammalian systems. Although properties such as lateral size and the colloidal properties of the nanosheets are important, the specific material properties that we address here is the oxidation state and reactive surface groups on the planar surface. In this study, we used a GO library, comprising pristine, reduced (rGO), and hydrated GO (hGO), in which quantitative assessment of the hydroxyl, carboxyl, epoxy, and carbon radical contents was used to study the impact on epithelial cells and macrophages, as well as in the murine lung. Strikingly, we observed that hGO, which exhibits the highest carbon radical density, was responsible for the generation of cell death in THP-1 and BEAS-2B cells as a consequence of lipid peroxidation of the surface membrane, membrane lysis, and cell death. In contrast, pristine GO had lesser effects, while rGO showed extensive cellular uptake with minimal effects on viability. In order to see how these in vitro effects relate to adverse outcomes in the lung, mice were exposed to GOs by oropharyngeal aspiration. Animal sacrifice after 40 h demonstrated that hGO was more prone than other materials to generate acute lung inflammation, accompanied by the highest lipid peroxidation in alveolar macrophages, cytokine production (LIX, MCP-1), and LDH release in bronchoalveolar lavage fluid. Pristine GO showed less toxicity, whereas rGO had minimal effects. We demonstrate that the surface oxidation state and carbon radical content play major roles in the induction of toxicity by GO in mammalian cells and the lung.

While the antibacterial properties of graphene oxide (GO) have been demonstrated across a spectrum of bacteria, the critical role of functional groups is unclear. To address this important issue, we utilized reduction and hydration methods to establish a GO library with different oxidation, hydroxyl, and carbon radical (•C) levels that can be used to study the impact on antibacterial activity. Using antibiotic-resistant (AR) bacteria as a test platform, we found that the •C density is most proximately associated with bacterial killing. Accordingly, hydrated GO (hGO), with the highest •C density, had the strongest antibacterial effects through membrane binding and induction of lipid peroxidation. To explore its potential applications, we demonstrated that coating of catheter and glass surfaces with hGO is capable of killing drug-resistant bacteria. In summary, •C is the principle surface moiety that can be utilized for clinical applications of GO-based antibacterial coatings.

Engineered carbonaceous nanomaterials (ECNs), including single-wall carbon nanotubes (SWCNTs), multi-wall carbon nanotubes (MWCNTs), graphene and graphene oxide (GO), are potentially hazardous to the lung. With incremental experience in the use of predictive toxicological approaches, seeking to relate ECN physicochemical properties to adverse outcome pathways (AOPs), it is logical to explore the existence of a common AOP that allows comparative analysis of broad ECN categories. We established an ECN library comprised of three different types of SWCNTs, graphene and graphene oxide (two sizes) for comparative analysis according to a cell-based AOP that also plays a role in the pathogenesis of pulmonary fibrosis. SWCNTs synthesized by Hipco, arc discharge and Co-Mo catalyst (CoMoCAT®) methods were obtained in their as-prepared (AP) state, following which they were further purified (PD) or coated with Pluronic (PF108) or bovine serum albumin (BSA) to improve dispersal and colloidal stability. Graphene oxide (GO) was prepared as two sizes, GO-small (S) and GO-large (L), while the graphene samples were coated with BSA and PF108 to enable dispersion in aqueous solution. In vitro screening showed that AP- and PD-SWCNTs, irrespective of the method of synthesis, as well as graphene (BSA) and GO (S and L) could trigger interleukin 1β (IL-1β) and transforming growth factor (TGF-β1) production in myeloid (THP-1) and epithelial (BEAS-2B) cell lines, respectively. Oropharyngeal aspiration in mice confirmed that AP-Hipco tubes, graphene (BSA), GO-S and GO-L could induce IL-1β and TGF-β1 production in the lung in parallel with lung fibrosis. Notably, GO-L was the most pro-fibrogenic material based on rapid kinetics of pulmonary injury. In contrast, PF108-dispersed SWCNTs and -graphene failed to exert fibrogenic effects. Collectively, these data indicate that the dispersal state and surface reactivity of ECNs play key roles in triggering a pro-fibrogenic AOP, which could prove helpful for hazard ranking and a proposed tiered testing approach for large ECN categories.

The aggregation and stability of graphene oxide (GO) and three successively reduced GO (rGO) nanomaterials were investigated. Reduced GO species were partially reduced GO (rGO-1h), intermediately reduced GO (rGO-2h), and fully reduced GO (rGO-5h). Specifically, influence of pH, ionic strength, ion valence, and presence of natural organic matter (NOM) were studied. Results show that stability of GO in water decreases with successive reduction of functional groups, with pH having the greatest influence on rGO stability. Stability is also dependent on ion valence and the concentration of surface functional groups. While pH did not noticeably affect stability of GO in the presence of 10 mM NaCl, adding 0.1 mM CaCl2 reduced stability of GO with increased pH. This is due to adsorption of Ca(2+) ions on the surface functional groups of GO which reduces the surface charge of GO. As the concentration of rGO functional groups decreased, so did the influence of Ca(2+) ions on rGO stability. Critical coagulation concentrations (CCC) of GO, rGO-1h, and rGO-2h were determined to be ∼ 200 mM, 35 mM, and 30 mM NaCl, respectively. In the presence of CaCl2, CCC values of GO and rGO are quite similar, however. Long-term studies show that a significant amount of rGO-1h and rGO-2h remain stable in Call's Creek surface water, while effluent wastewater readily destabilizes rGO. In the presence NOM and divalent cations (Ca(2+), Mg(2+)), GO aggregates settle from suspension due to GO functional group bridging with NOM and divalent ions. However, rGO-1h and rGO-2h remain suspended due to their lower functional group concentration and resultant reduced NOM-divalent cation bridging. Overall, pH, divalent cations, and NOM can play complex roles in the fate of rGO and GO.