Graphene can be transferred from wheat roots to shoots and enter the cytoplasm and chloroplasts (Hu et al., 2014c); however, GO accumulation was not observed in the root cells of wheat (Chen et al., 2018a). Furthermore, GO did not accumulate in the seedlings of spinach and chive from if their seeds were treated with 50 mg/L GO (He et al., 2018). In another study, Zhao et al. (2015) found that GO in the range of μg/L accumulated in root hair and root parenchyma cells; however, it did not translocate into the stem or leaves of Arabidopsis thaliana. This finding was supported by Chen et al. (2017); GO was readily absorbed by the plant roots, but the absorbed GO showed limited upward translocation. Different quantitative and distributional trends between the two graphene materials in pea plants were observed by Chen et al. (2019). Their study showed that rGO was translocated into leaves after being absorbed by the roots. The uptake amount in the root tended to stabilize at 15-day exposure, and the cumulative amount in the leaves was higher than that in the roots at 20-day exposure, reaching accumulation amounts up to mg level. Conversely, GO mainly accumulated in the roots and low levels in leaves. Besides root accumulation and translocation from root to shoot, Huang et al. (2018) further discovered that ~9% of the accumulated FLG was degraded to CO₂ in the rice plant, and that the hydroxyl radical in the leaf played an important role in degrading FLG. Earlier studies have demonstrated that CO₂ was the final product of the complete enzymatic catalyzed oxidation of GO (Kotchey et al., 2011), and H₂O₂ was a key component of this degradation process (Xing et al., 2014). In plants, H₂O₂ plays an important role in regulating biotic and abiotic stress responses (Sun et al., 2018). Thus, given the widespread presence of H₂O₂ in plants, they can potentially eliminate accumulated GFNs and could be used as phytoremediation agents for environmental clean-up.

Generally, GFNs produce a delaying effect in plant germination. For example, in rice seeds treated with 50 mg/L of graphene, germination started 3 days after the control group (Nair et al., 2012). A later study confirmed that the increase in graphene stress (≤ 200 mg/L) induced a delay in the initiation of the germination process in rice, but had no negative effects on the final germination percentage (Liu et al., 2015). A similar delay occurred in the appearance of the cotyledons and the root system of tomato, cabbage, and red spinach seeds treated with graphene (Begurn et al., 2011). However, tomato seeds exposed to graphene at concentrations as low as 40 mg/L obtained rapid seed germination and higher germination rates, which was attributed to the penetration of the seed coat by the graphene, thus facilitating water uptake (Zhang et al., 2015b). GO also significantly stimulated plant germination at 50 mg/L because its hydrophobic sp² domains transported more water to the seed in the soil (He et al., 2018). GO at concentrations in the order of μg/L (10–1000 μg/L) had no obvious influence on the germination of Arabidopsis seeds (Zhao et al., 2015). When GO concentration was up to 100 mg/L, the rice germination percentage insignificantly decreased; the effect became significant at 500 mg/L GO (Liu et al., 2015). Similar to rice, the germination of wheat seeds was inhibited owing to GO concentrations exceeding 400 mg/L (Chen et al., 2017).

The common toxicity symptoms observed in plants exposed to GFNs are a severe loss of morphology and decreases in growth parameters, such as root and shoot length, root number, root diameter, and biomass production (Table 1). For instance, the morphology of rice seedlings was significantly inhibited if graphene concentration reached 100 mg/L (Liu et al., 2015). GO also adversely affected biomass accumulation and stem elongation in wheat seedlings (Chen et al., 2017). After exposure to 50 and 500 mg/kg rGO, negative effects on the shoot height and root length of rice seedlings were observed by Hao et al. (2018); in addition, the root diameter and the number of cells in the transverse section significantly decreased. However, GO in the range of μg/L did not cause significant changes in shoot and root development of Arabidopsis seedlings, or flowering time (Zhao et al., 2015). These apparently contrasting results suggest that the toxic effects of GFNs are associated with exposure concentrations.

The biological impacts of nanomaterials are dependent on their size, chemical composition, surface structure, solubility, shape, and aggregation. Of these properties, size and surface area are important characteristics from a toxicological perspective; a small size and a large surface area increase the uptake and interaction with biological tissues, thus increasing the probability of generating adverse biological effects in living cells (Nel et al., 2006).

Because the large size of GO sheets hinders its translocation from the roots to the stem and leaves, GO bioaccumulation was much lower than that of fullerenol in wheat roots (Chen et al., 2017). Using freshwater algae as a test plant, Zhao et al. (2017) investigated the toxicity of GFNs based on their different physicochemical properties and colloidal behaviors. They found that GO with abundant functional groups could adsorb more macronutrients (N, P, Mg, and Ca) from the culture medium than rGO, thus leading to stronger nutrient depletion-induced indirect toxicity; rGO could directly penetrate into algal cells, but GO, with more flexible sheets, could not. In addition, GO had a significant shading effect on algal growth due to its good dispersibility and transformation. Compared to graphene and GO, hydrated graphene ribbon (HGR) not only promoted the germination rate of aged seed, but also increased root differentiation; the disordered layer structures of HGR played a key role in this process (Hu and Zhou, 2014). The observations of Chen et al. (2018a) showed that GO induced obvious toxic symptoms in wheat, while amine-functionalized GO was non-toxic and enhanced plant growth. They inferred that the introduction of amines could decrease the surface electrical resistivity of GO, creating higher electronic conductivity, and activating bioactivity in plant cells.

The general effect of GFNs on plant growth is dose-dependent. Graphene at 5 mg/L promoted the number of adventitious roots, and increased the root and shoot fresh weight of rice seedlings; however, at a concentration of 50 mg/L, it significantly inhibited the stem length and fresh shoot weight (Liu et al., 2015). Graphene at 500 mg/L resulted in only a slight decrease in root and shoot length of tomato, cabbage, and red spinach, whereas a marked inhibition was induced by graphene at concentrations up to 2,000 mg/L (Begurn et al., 2011). After a 10-day exposure to GO, the lower concentrations (5 and 10 mg/L) had no significant effect on root length and fresh weight, but the higher concentrations (50 and 100 mg/l) showed inhibited root growth (Cheng et al., 2016). However, this effect is not completely concentration-dependent. Anjum et al. (2014) found that GO at 1600, 200, and 100 mg/L significantly inhibited the germination rate and root length of the faba bean (Vicia faba), while the health status of the plant was improved with exposure to GO at 400 and 800 mg/L. Their previous study assessed the tolerance of faba bean to GO, in which the plant showed a significantly higher sensitivity to GO at 1,600, 200, and 100 mg/L, and its tolerance increased when exposed to 400 and 800 mg/L concentrations (Anjum et al., 2013). Their further investigations indicated that the sensitivity and/or tolerance of the plant to GO depended on the cellular GSH redox system. Additionally, the concentration-dependent toxicity of GFNs is also related to exposure time. Zhang et al. (2016) found that long-term graphene exposure (30 d) caused wheat leaf deformities and yellowing, whereas no distinct alterations in leaf elongation were found after short-term exposures (24 or 48 h). This exposure time-dependent toxicity was also observed by Zhao et al. (2017). In their investigation, the growth inhibition of GFNs to freshwater algae highly increased with increasing exposure times (24–96 h).

Heretofore, only a few studies have been designed to compare the toxicity of GFNs to different plants. The hydroponics experiments conducted by Begurn et al. (2011) indicated that graphene had little or no significant toxic effect on lettuce seedlings, but significantly inhibited the growth and biomass of tomato, cabbage, and red spinach seedlings under the same conditions. Among these selected vegetable species, tomato seedlings exhibited the highest sensitivity to graphene, according to the root and shoot weight data.

Compared with studies on the ecotoxicity of single nanomaterials, relatively little research has focused on the interaction of nanoparticles and other contaminants (Zhang et al., 2018). In general, GFNs coexist with other pollutants in the natural environment. Heavy metals and organic pollutants (OPs) are common in the water and soil environment. The interactive toxic effects are different when GFNs are combined with these pollutants; these are termed additive or antagonistic effects. The research of Hu et al. (2014a) found that GO amplified the phytotoxicity of As in wheat. It further revealed the main mechanisms of indirect toxicity of GO: (a) enhancing the uptake of GO and As by damaging cellular structures and electrolyte leakage, and (b) promoting the transformation of As⁵⁺ to high-toxicity As³⁺. Conversely, another investigation found that GO alleviated the inhibitive effects of Cd²⁺ on the seminal root and bud growth of rice, which possibly resulted from Cd²⁺ adsorption in available contact sites or accumulation in the interlayer space of GO (Yin et al., 2018). Early researchers had already noticed the strong attractive forces between Cd²⁺ and GO, and had used GO as a sorbent for heavy metal removal from waste water (Tan et al., 2015). Lingamdinne et al. (2016) found that the adsorption occurred through physical and chemical interactions between heavy metal ions and oxygen-containing surface functional groups, and the π-π bond electrons of GO. Moreover, functionalized GO exhibited significantly higher adsorption capacity (Pirveysian and Ghiaci, 2018). However, once desorption occurs, high adsorption capacity implies the potential release, thus presenting a high risk to public health and the environment (Yang and Xing, 2007). GFNs have also been widely used to remove OPs from the environment (Chowdhury and Balasubramanian, 2014; Amaranatha et al., 2015; Zhang et al., 2015a). Although no direct data currently suggest that interaction of GFNs and OPs enhances phytotoxicity of either, it has been confirmed that GO can serve as an insecticide carrier to enhance contact toxicities (Wang et al., 2019). This synergistic mode of adsorption-delivery-release is most likely equally effective for plants. Moreover, previous research demonstrated that C₆₀ fullerenes significantly increase the bioaccumulation of DDE (dichlorodiphenyldichloroethylene, DDT metabolite) into three selected food crops (De La Torre-Roche et al., 2012). These findings indicate that the carbon nanomaterials can affect the accumulation and bioavailability of co-existing pollutants, and thereby be regarded as a toxic alert to plants.

The main physical mechanisms for the phytotoxicity of GFNs include the shading effect, mechanical injury, and physical blockage. Both shading effect and mechanical injury were observed by Zhao et al. (2017). They found that the dispersed and darkened GO reduced light transmittance, thus decreasing the available light required to support plant growth, resulting in approximately 16% of growth inhibition; and more interestingly, direct penetration into algal cells by graphene materials was discovered for the first time. The physical blockage is closely related to the size of GFNs. If nanoparticle diameter is larger than the diameter of root cell wall pores, particles will accumulate at the root surfaces and form surface layers, thus decreasing the hydraulic conductivity and uptake of nutrients (Asli and Neumann, 2009).

GFNs enhance the generation of reactive oxygen species (ROS) and inhibit antioxidant enzyme activities, resulting in oxidative stress, which has been recognized as one of the most important mechanisms in growth-limiting effects on plants (Hu et al., 2014b; Zhang et al., 2016; Chen et al., 2017). GO also caused metabolic disturbances linked to key biological processes, such as inhibiting carbohydrate and amino acid metabolisms, and increasing the ratios of unsaturated to saturated fatty acids, changing the flux of nitrogen metabolism (Hu et al., 2014b).

It is well-known that photosynthesis is critical for plant survival and growth. Graphene significantly inhibited the biosynthesis of chlorophyll and decreased chlorophyll content in plants, leading to impaired photosynthesis and reduced growth (Hu et al., 2014c; Zhang et al., 2016). Hu et al. (2014c) also found that glyconic acid and aconitic acid were upregulated by graphene, and these metabolites were negatively correlated with the biosynthesis of chlorophyll.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.