How Toxic are Graphene Oxide Nanoparticles?

Carbon-based engineered nanomaterials, such as graphene oxide nanoparticles (GO NPs), are widely available for application, but their potentially adverse health effects on humans still require investigation.

Research with Nematode and GrapheneOxide

In this study, the environmental levels of GO NPs are addressed to examine whether GO leads to adverse effects on an in-vivo model of Caenorhabditis elegans (C. elegans). Nematodes with prolonged exposure (L1 larvae to young adult) to GO NPs at 0.00100, 0.0100, 0.100, and 1.00 mg L–1 were used to evaluate the potential toxic effects, including lethality (acute toxicity), reproductive (brood size) and neurological (locomotion including head thrash and body bend) responses, longevity (lifespan), and oxidative stress (gene expression of sod-1, sod-3, and clt-2).

Prolonged exposure to GO NPs was not found to induce lethality at the selective levels. In the brood-size and head-thrash tests, the biological responses in nematodes were significantly reduced at 0.0100-1.00 ng L–1 GO NP exposure as compared with the untreated control. The nematodes exposure to GO NPs at 0.00100–1.00 ng L–1 exhibited significant delays in body bending behavior compared with the control. In the examination of the longevity of nematodes, it was found that the lifespan of all GO NP-exposed worms was significantly shortened as compared to the untreated worms. Gene expression of sod-1, sod-3, and ctl-2 presented significantly higher induction folds in the exposed worms compared with the controls. Consequently, prolonged exposure to the low-dose GO NPs might be associated with disruption of reproduction and locomotion, attenuation of longevity, and induction of oxidative stress in nematodes.


Graphene oxide nanoparticles (GO NPs), one of the most promising derivatives of graphene, comprise a monolayered engineered nanomaterial (ENM) with high oxygen-containing functional groups such as carboxyl, epoxy, carbonyl, and hydroxyl (Bianco et al., 2013; Li et al., 2015). Graphene oxide (GO) is known to have excellent dispersibility in many solvents, chemical reactivity, and the capacity for chemical functionalization (Konios et al., 2014; De Marchi et al., 2018). The adsorption capacity of GO NPs has been taken advantage of for environmental remediation applications such as in GO-based membranes, which remove gaseous contaminants such as sulfur dioxide and hydrogen sulfide (Fakhri, 2017). In addition, they also act as adsorbents for elimination of various aqueous contaminants owing to their high content of functionalized oxygen groups available to interact with metal ions (Li et al., 2019).

Furthermore, GO NPs can be applied in nanoelectronics, catalysis, nanocomposites, sensor technology, water purification and desalination, and drug delivery (Zhang et al., 2011; Pan et al., 2012; Giust et al., 2018; Prasad et al., 2020). According to studies from Yang et al. (2015) and Maharubin et al. (2016), GO can be utilized for hydrogen storage (anode, cathode, and lithium sulfur batteries) and supercapacitor management. Furthermore, GO composites are used as antimicrobial agents for water disinfection to remove organic molecules and waterborne pathogens (Upadhyay et al., 2014). Several studies used the environmental levels at µg L–1 to mg L–1 of GO NP contamination to test the in-vivo models (He et al., 2017; Zhang et al., 2017; Li et al., 2019).

The global market for graphene-based products, such as GO, is increasing. The demand is expected to be $675 and 987 million by 2020 and 2022, respectively (Ahmed and Rodrigues, 2013). Due to their potential for both production and application, GO materials are expected to be released in the environment during their lifecycle and eventually be generated in landfills and wastewater treatment plants (Du et al., 2017; Suárez-Iglesias et al., 2017; Jamialahmadi et al., 2018). GO can be released into the water environment through the development of its composites as adsorbents for aqueous contamination, membranes for water filtration and purification, and catalysts for environmental decontamination (Zhao et al., 2014; Goodwin et al., 2018).

Several studies have reported that the dispersion and long retention time of GO within microbial communities can lead to serious negative effects on wastewater microbial flora due to its hydrophility (Lyon and Alvarez, 2008; Kang et al., 2009; Rodrigues and Elimelech, 2010). The predicted environmental concentrations of GO can be correlated with those of multi-walled carbon nanotubes because both have relatively similar properties, such as nanometer size, a carbon-based structure, and applications in consumer electronic devices (Zhang et al., 2017).

Few epidemiological studies focused on human exposure to GO particularly for the highly exposed population. For the in-vivo models including rats, mice, zebra fish, nematodes, and daphnia, the animals could induce nanotoxicity including acute, developmental, neurological, reproductive, immunological, and neurobehavioral toxicity as well as shortened longevity after they were exposed to GO NPs (Sanchez et al., 2012; Patlolla et al., 2017; Qu et al., 2017; Souza et al., 2017; Kim et al., 2018; Qu et al., 2019; Kim et al., 2020). In the past years, in vivo and in vitro GO NPs toxic effects, including immunotoxicity, activation of inflammation, induction of reactive oxygen species (ROS), generation of oxidative stress, apoptosis, and potential GO exposure mechanisms have been investigated (Guo and Mei, 2014; Bengtson et al., 2017; Pelin et al., 2018; Tang et al., 2018).

The accumulation of GO in the cytoplasm causes dramatic morphological alterations and reduces the ability of toll-like receptor 4 (TLR4) for phagocytosis (Qu et al., 2013a). However, an increase in intracellular ROS contributes to necrotic cell death in macrophages (Qu et al., 2013a). Previous studies have also reported that GO promotes cell growth inhibition, hatching delay, ROS generation, and damages the circulatory system of zebrafish embryos (Liu et al., 2014; Chen et al., 2016; Souza et al., 2017). In mice, GO can accumulate in organs such as the liver, lungs, spleen, and kidneys, which may induce organismal toxicity through intracellular oxidative stress caused by the accumulation of ROS (Qu et al., 2013b; Yang et al., 2013). GO can enter the human body through inhalation and may be deposited in regions of the respiratory tract. When deposited in alveolar regions, it may impair clearance, form granulomas, and possibly produce fibrosis (Sanchez et al., 2012).

The in vivo model used in this study was the transparent nematode, Caenorhabditis elegans (C. elegans), which has been successfully used in toxicological evaluation of various nanomaterials such as GO NPs (Zhang et al., 2012; Wu et al., 2013; Piechulek and von Mikecz, 2018). Advantages of C. elegans as an in-vivo model system were as the following: (1) simple anatomy, (2) transparent, (3) invariant cell lineage, (4) short life cycle with large brood size, (5) easily accessible embryos, (6) easy and cheap maintenance in lab, and (7) powerful experimental tool (Brenner, 1974; Hunt, 2017). The C. elegans model is not a mammal model to unavailable to examine several toxic endpoints like blood sugar and pressure, tissues in the skin, probiotic system in the intestine, heart and cardiovascular diseases. C. elegans is considered to be a novel tool for in-vivo techniques, and testing of C. elegans is known to be analogous to mammalian neurotoxin testing (Cole et al., 2004).

Recently, scientists have focused on the disruption of biological effects from ROS, reproductive effects, gene expression, neurological development, and neurobehavior with treatment of GO NPs in C. elegans models (Wu et al., 2013; Qu et al., 2017; Kim et al., 2018; Rive et al., 2019; Kim et al., 2020; Zhao et al., 2020). Zhang et al. (2012) indicated no negative impact on longevity after exposing L4-larva-to-young-adult C. elegans to GO NPs at concentrations ranging from 5 to 20 mg L–1. Inversely, C. elegans with prolonged exposure (from L1 larva to young adult) to 0.5–100 mg L–1 of GO NPs presented adverse effects on primary (digestive organs such as the intestine) and secondary (neurological tissues such as neurons and reproductive organs) target organs (Wu et al., 2013).

GO NPs possibly shortened lifespan by influencing the expression of the DAF-2-AGE-1-AKT-1/2-DAF-16 signaling cascade in the intestine of the nematodes (Zhao et al., 2016b). After GO NP exposure, expression of neuronal substances may decrease ROS generation and reduce locomotion behavior in nematodes (Zhao et al., 2020). GO NPs probably caused damage to the dopaminergic and glutamatergic neurons in C. elegans after chronic exposure to GO NPs for 6 days, from L1 larvae to the adult stage (Li et al., 2017). Liu et al. (2020) observed that GO NPs induced intestinal barrier dysfunction in C. elegans. Rive et al. (2019) proposed that worms chronically (or prolongedly) exposed to GO NPs (levels of 100 and 200 mg L–1) were significantly shortened in size and developed morphological abnormalities in the pharynx and intestine.

Kim et al. (2018) found accumulation of GO NPs in the reproductive organs of C. elegans using Raman spectrometry. Also, GO NP exposure promoted reproductive toxicity by suppressing spermatogenesis of C. elegans during development, resulting in decreased sperm numbers and progeny numbers (Kim et al., 2018). This study was aimed toward evaluating the effect of environmentally-relevant concentrations of GO NPs in C. elegans by assessing toxicological endpoints including acute lethality, reproduction, locomotion, lifespan, and gene expression.


Preparation of Graphene Oxide Nanoparticles

GO NPs were prepared from expandable graphite using a modified Hummers’ method (Yan et al., 2014). In brief, 1 g graphite power and 50 mL sulfuric acid (H2SO4, 98%) were poured into a 250 mL flask, followed by the addition of 0.5 g NaNO3. The mixture was mechanically agitated for 30 min in an ice bath. For further oxidation, 5 g of potassium permanganate (KMnO4) was added while slowly stirring the mixture for 4 h. Subsequently, H2O2 was added to MnO2 until the mixture became yellow. Afterward, 1% HCl was added, and the mixture was centrifuged at 8000 rpm for 5 min, followed by washing 3 times with distilled water to dilute the acid solution.

Reagents, Chemicals, and Nematode Cultivation

The GO NPs underwent sonication for 30 min (40 kHz and 100 W) to disperse them in K medium (50 mM, 30 mM KCl, 1.0 mg mL–1 and PH of 6.0) as the stock solution (200 mg L–1) following the methods in previous studies (Wu et al., 2013; Zhao et al., 2016b). The stock solution was diluted to various concentrations using K medium prior to exposure.

The wild-type N2 C. elegans strain was gifted from Dr. Chang-Shi Chen in the Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University (Tainan, Taiwan). C. elegans was maintained on nematode growth medium (NGM) seeded with OP50 Escherichia coli (E. coli) cultures from the Bioresources Collection and Research Center (Hsinchu, Taiwan), and Luria-Bertani broth was obtained from Sigma-Aldrich (St. Louis, MO, USA). The NGM plates contained bacteriological agar and bactopeptone, which were obtained from Laboratories Conda (S.A., Spain). The NaCl was obtained from Honeywell Fluka™ (New Jersey, USA). Age-synchronized worms were collected using a bleaching mixture that contained NaOCl obtained from J.T. Baker (Central Valley, PA) and KOH obtained from Duksan Pure Chemicals (Gyeonggi-do, South Korea).

Supplemental reagents such as CaCl2, K2HPO4, and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO, USA); MgSO4 was acquired from Avantor Performance Materials, ltd. (Gyeonggi-do, South Korea); the KH2PO4 used for the phosphate buffer was acquired from Avantor Performance Materials, LLC (Radnor, PA, USA), and the Na2HPO4 used for the M9 buffer was obtained from Honeywell Fluka™ (New Jersey, USA). All physiological observations were done under a dissecting microscope (Olympus, SZX10, Waltham MA, USA). The experimental protocols in the nematode model followed those used in our previous study (Chung et al., 2019, 2020). The lethality, growth, reproduction, locomotion behavior examinations followed the protocols previously published, with minor modifications (Chung et al., 2019, 2020).

Lethality and Lifespan Assay

The nematodes were exposed to GO NPs (control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1) for 48 h (prolonged exposure) from L1-larvae to young adults incubated at 20°C. Prolonged exposure was performed in a fresh plate with an OP50 E. coli lawn. After treatment, the lethal toxicity of the samples was evaluated by softly poking them using a worm picker. The worms that did not respond were considered dead. Three biological replicates were performed, and a total of 150 worms were assayed.

The worms evaluated for the lifespan assay were exposed for a prolonged period of time to the different GO NP concentrations (the untreated control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1) from L1 to the mature stage for the lifespan test. Fifty worms were transferred to fresh plates every other day for 4–5 days of egg-laying. Live and dead nematodes were evaluated daily by softly poking them with a worm picker. Three biological replicates were performed, and a total of 150 worms were evaluated. Several lifespan indicators (mean lifespan, day of 50th percentile death, day of 75th percentile death, day of 95th percentile death, and day of all death) were evaluated by following the Chung’s study (Chung et al., 2020).

Reproductive Assay and Locomotion Assay

The brood size (reproductive assay) of the L3 or young L4 nematodes was assessed for 4–5 days after prolonged exposure to different GO NP concentrations (control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1) at 20°C. Each worm was transferred to a fresh plate and transferred again until the egg-laying period stopped. The plates with eggs were incubated until the progeny could be easily counted. A total of 30 worms was evaluated for the reproductive assay.

Locomotion behavior including head thrashing and body bending in the nematode models was expressed as the motor neuron function (Qu et al., 2019; Zhao et al., 2020). The body bending and head thrashing of the nematodes (locomotion assay) were evaluated after prolonged exposure to various concentrations of GO NP (control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1). The body bending was evaluated by transferring the exposed worm onto a fresh plate. After one day, the body bending of the worms was counted for 20 secs. The head thrashing was evaluated by placing the exposed worm on a glass slide containing an adequate amount of K-media. The head thrashing of the worms was counted for 1 min. Three biological replicates were performed, where 60 worms were evaluated for body bending, and 30 worms were evaluated for head thrashing.

Gene Expression Tests

C. elegans in the different treatment groups (untreated control, 0.00100, 0.100, and 1.00 µg L–1) were collected from three replicates for RNA extraction after exposure. Trizol reagent (TIANGEN, China) was used to extract total RNA in accordance with the manufacturer’s standard protocol. RNA concentrations were measured by the absorbance at 260 nm, and the RNA purity was evaluated based on the ratio of the optical densities from RNA samples measured at 260 and 280 nm. The first-strand cDNA synthesis reaction was conducted with 500 ng of purified RNA using a Fast Quant RT Kit (with gDNase) according to the manufacturer’s protocol (TIANGEN, China).

Specific superoxide dismutase genes, including (sod) 1 (sod-1), sod-3, and catalase 2 (ctl-2) were detected in the present study. The data were analyzed using the 2-ΔΔCt method, as previously reported (Zhou et al., 2016), and the mRNA expressions were normalized based on the act-1 mRNA. For each tested gene, a qRT-PCR analysis was conducted in triplicate (technical replicates).

Statistical Analysis

The Statistical Package for the Social Sciences (SPSS) version 12 software (International Business Machines Corp., New York, USA) was used to perform all statistical analyses. All data was checked to normality, and the Shapiro-Wilk test was used determined the normal and non-normal distribution. A one-way ANOVA was used to analyze the significance levels of the differences between treatments. The plots and figures were made using GraphPad Prism 6 (San Diego, California, USA).

Results and Discussion

GO NPs Lethality

C. elegans was evaluated through a variety of toxicological endpoints, including lethality, reproduction, locomotion, lifespan, and oxidative stress (gene expression of sod-1, sod-3, and clt-2) in the present study. This study is the first time that low doses of GO NP (approximately at least 1000-fold lower compared to those used in the previous studies) has been used to examine nanotoxicity in a C. elegans model. Exposure from L1-larvae to young adult was performed to assess the effects of prolonged GO exposure on both larvae and adult nematodes. As shown in Fig. 1, the survival rate of the nematodes after prolonged exposure to GO did not indicate significant lethal effects. No significant between-group differences in mortality were observed in thecontrol group and in the group treated with concentrations ranging from 0.00100 to 1.00 μg L–1.

Few studies have examined acute toxicity of GO NPs in N2 C. elegans models (Wu et al., 2013, 2014; Li et al., 2019). Wu et al. (2013) indicated that no lethality was observed at GO NP concentrations from 0.100 to 100 mg L–1 after the worms were acutely or prolongedly exposed to these carbon-based ENMs. On the contrary, a study examining acute toxicity with treatment of high doses of 5, 10, 50, and 100 mg L–1 of GO NPs in nematodes found that GO concentrations higher than 5 mg L–1 may cause lethality, where no worms survived at a dosage of 100 mg L–1 (Li et al., 2017).

Wu et al. (2014) also examined low levels of GO NPs from 0.00100 to 1.00 mg L–1 and found no significant differences in lethality except in the case of the highest concentration of 1.00 mg L–1 after the nematodes were chronically exposed to these carbon-based ENMs from L1 larvae to adult-day 8. Most GO NP studies refer to Wu’s study (Wu et al., 2013) and use similar dosage levels (ppm levels) to examine neurological, reproductive, neurobehavioral, and immunological toxicity, and inflammatory responses.

Fig. 1. Survival rates of C. elegans after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as * p < 0.05, **p < 0.01, and ***p < 0.001.


Reproductive Toxicity of GO NPs

Reproduction in nematodes is a vital endpoint because it has been shown to be sensitive to lower concentrations of chemical stressors than those that impair the behavior and viability of nematodes (Wu et al., 2019). The results in Fig. 2 show that prolonged exposure to GO NPs in the nematodes reduced brood size production. A significant decrease was observed in the progeny number at concentrations of 0.0100 (p = 0.036), 0.100 (p = 0.008), and 1.00 (p < 0.001) µg L–1 of GO NPs. The reduction rates in the brood size at these three concentrations compared to the control group were 15.2, 20.1 and 27.3%, respectively.

The results showed that higher concentrations of GO NPs induced more reproductive toxicity based on our experiments on brood size number in nematodes. Our results were consistent with most GO NP studies reporting that GO exposure can cause adverse effects through damaging the fertility and egg ejection behavior of nematodes (Wu et al., 2013; Zhao et al., 2016a; Kim et al., 2018; Rive et al., 2019). Wu et al. (2013) showed that C. elegans with prolonged exposure to 1–100 mg L–1 exhibited significantly decreased brood size compared to the control, but there were no significant between-group differences at 0.1 and 0.5 mg L–1. A similar result was also found in a previous study (Rive et al., 2019), indicating that prolonged exposure to GO NPs at 100 and 200 mg L–1 significantly decreased egg-laying rates compared to the untreated control.

Kim et al. (2018) revealed that accumulation of GO NPs (10 mg L–1) in the reproductive organs, which might be the direct cause of reproductive toxicity, could reduce brood size and sperm count by suppressing spermatogenesis of the hermaphrodite nematodes at the GO levels of 5 or 10 mg L–1. However, the negative impact of GO NP exposure on the reproductive function in the present and published studies (Wu et al., 2013; Zhao et al., 2016a; Kim et al., 2018; Rive et al., 2019), as well as our results, suggest that prolonged exposure to GO NPs at low doses from 0.0100 to 1.00 µg L–1 could decrease progeny number or fecundity in N2 C elegans models.

Fig. 2. Effects of the brood size in the C. elegans after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, ** p < 0.01, and ***p < 0.001.


GO NP Exposure Affects Locomotive Behavior

Locomotive behavior assays are well-established methods for studying nematode neurotoxicity. After prolonged exposure, GO induced obvious decreases in both head thrashing and body bending in nematodes (Fig. 3). In the head thrash examination, 0.0100, 0.100 and, 1.00 µg L–1 concentrations of GO NPs significantly decreased head thrashing by 12.0, 5.41, and 19.8%, respectively, compared to the untreated control. Furthermore, body bending was significantly reduced at 0.00100, 0.0100, 0.100 and 1.00 µg L–1 GO NPs by 8.78, 21.2, 31.5, and 40.8%, respectively, in comparison with the control groups.

Our results were consistent with those in most published articles, implying that GO NP exposure damages the neurological functions and negatively disrupts head thrashing and body bending behavior (Wu et al., 2013, 2014; Zhao et al., 2015, 2016c; Chen et al., 2017; Li et al., 2017; Qu et al., 2017; Kim et al., 2018; Rive et al., 2019; Zhao et al., 2020). In Wu’s report (Wu et al., 2014), head thrash and body bend locomotion was significantly reduced at 0.0100, 0.100, and 1.00 mg L–1 levels compared with an untreated control. Li et al. (2017) indicated that prolonged exposure to GO NPs (5.00–100 mg L–1) significantly reduced body bending, head thrashing, pharynx pumping frequency, mean speed, bending angle-frequency, and the wavelength of the crawling movement of nematodes. GO NPs also induced damage to dopaminergic and glutamatergic neurons in nematodes (Li et al., 2017).

Kim et al. (2020) also proposed that GO significantly accumulated in the head regions, generated ROS induction, reduced neurotransmitter substances in dopaminergic and glutamatergic neurons, and damaged AFD neurons, which are the main thermosensors in C. elegans, after the nematodes were exposed to GO NPs (10 mg L–1). In a Korean study, Kim et al. (2018) also found that neurotransmitters, such as dopamine, γ-Aminobutyric acid (GABA), tyramine, tryptophan, and tyrosine, were reduced in nematodes exposed to GO NPs. According to the current data, including the present study (Wu et al., 2013, 2014; Zhao et al., 2015, 2016a; Chen et al., 2017; Li et al., 2017; Qu et al., 2017; Kim et al., 2018; Rive et al., 2019; Zhao et al., 2020), it has been concluded that GO NPs exposure causes adverse effects on the neurological system of C. elegans particularly in terms of damage to neurons, influences on neurotransmitter neurodisruptions, and delays in neurobehavioral development. In the present study, environmental levels (0.0100–1.00 µg L–1) of GO NP doses were used to treat the nematodes to determine the negative impact on their locomotion behavior.

Fig. 3. Effects of head thrashing and body bending in the nematodes after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.


Effect of GO NPs on Lifespan

In C. elegans models, lifespan is an important endpoint for assessment of toxicants. After prolonged exposure in nematodes, GO NPs at concentrations of 0.00100–1.00 µg L–1 led to shorter lifespans than was the case for the untreated controls (Fig. 4). Several indicators of lifespan, including mean lifespan (Fig. 4(b)), mean day of median (50th percentile) death (Fig. 4(c)), mean day of 75th percentile death (Fig. 4(d)), mean day of 95th percentile death (Fig. 4(e)), and the day of all death (Fig. 4(a)) indicated significantly longer longevity in the untreated control as compared to in GO NP-exposed nematodes (p < 0.001). The mean lifespan and the day of all death were 13.9, 7.01, 6.23, 6.94, and 6.35 days in the untreated control and 0.00100, 0.0100, 0.100, and 1.00 µg L–1, respectively, and 20, 16, 14, 14, 16 days in the untreated control and 0.00100, 0.0100, 0.100, and 1.00 µg L–1, respectively. After 6 days, the percent survival rate of nematodes decreased to as much as 50% of the total population.

It was also observed that the nematodes treated with GO NPs exhibited faster reductions in lifespan than the control group. In summary, Fig. 4 indicates that prolonged exposure to GO NPs reduces the lifespan of nematodes (p < 0.001). According to the current data from previous reports (Zhang et al., 2012; Zhao et al., 2016b, 2016c; Qu et al., 2017; Rive et al., 2019), contradictory results were obtained, where two studies indicated that GO NP exposure, including both acute and prolonged exposure didn’t have effects on longevity (Zhang et al., 2012; Rive et al., 2019), and other studies obtained different results indicating that the worms with prolonged exposure to GO NPs exhibited significantly reduced longevity (Zhao et al., 2016b, 2016c; Qu et al., 2017) at GO NP concentrations between 1.00 and 200 mg L–1. Two molecular mechanisms of intestinal insulin signaling may be involved in the shortened longevity of nematodes exposed to an GO NP concentration of 100 mg L–1 due to association with suppression of DAF-16 and sod-3 functions (Zhao et al., 2016b). Based on our results, the low dose of 0.00100 µg L–1 significantly reduced the nematodes’ longevity.

Fig. 4. Lifespan of C. elegans after prolonged exposure to GO NPs at levels of 0.00100, 0.0100, 0.100, and 1.00 µg L–1 and the control (a) nanotoxic assessment of worms with prolonged GO exposure for lifespan, (b) mean lifespan, (c) mean day of 50th percentile death, (d) mean day of 75th percentile death, and (e) mean day of 95th percentile death. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.
Gene Expression after GO NPs Exposure

The sod genes encode superoxide dismutases (SODs), which comprise an antioxidant system for C. elegans against oxidative stress after GO NP exposure (Ren et al., 2018). SODs which exist in three isoforms of sod1, sod2, and sod-3 are a class of the antioxidant protein. The increased folds in expression from the induced sod-1, sod-3, and ctl-2 genes after C. elegans had undergone prolonged exposure to 0.00100, 0.100, and 1.00 µg L–1 GO NP compared with the untreated control are shown in Fig. 5. The activated expressions of sod-1, sod-3, and ctl-2 at the concentrations of 0.00100, 0.100, and 1.00 µg L–1 in the GO NP-exposed C. elegans were significantly higher than those in the untreated control. SOD is a key enzyme in the detoxification function of free radicals.

It removed free radicals generated from GO NPs in extracellular sources in nematodes. Results similar to those found in the present study were also found in previous studies (Wu et al., 2013; Zhao et al., 2016b), which indicates that GO NPs could induce sod-1 or sod-3 activation. The findings from Wu’s study suggested that oxidative stress induced in the treated GO NP nematodes may be related to changes of SOD activities (Wu et al., 2013). Based on these findings, it can be inferred that oxidative stress is a possible mechanism causing adverse effects on neurodevelopment and neurobehavioral development after prolonged GO NP exposure, as suggested in previous reports (Wu et al., 2013; Zhao et al., 2016b), in combination with the results of induced SOD activation and neurotoxicity in the GO-exposed nematodes in the present study (Figs. 3 and 5).

Furthermore, sod-1, sod-3, and ctl-2 activation may be associated with the shortened longevity in the GO-exposed worms, based on Figs. 4 and 5. In Zhou’s study (Zhou et al., 2016), C. elegans ctl-2 gene encoded peroxisomal catalase was linked to environmental oxidative stress after worms were exposed to bisphenol A. Few studies have addressed to link between ctl-2 expression and GO exposure in C. elegans. Although a positive association between ctl-2 expression and GO NP exposure was shown in the present study, the mechanism is still unclear.

Fig. 5. Gene expression in C. elegans with prolonged exposure to GO NPs at the levels of the untreated control, 0.00100, 0.100, and 1.00 µg L–1 (a) SOD-1 (C15F1.7), (b) SOD-3 (C08A9.1), and (c) ctl-2 (Y54G11A.5); Actin-1 (T04C12.6) as the internal control. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.

Finally, it was concluded in the present study that extremely low doses of GO NPs, compared with the dosages discussed in recent published articles, can cause reproductive and neurobehavioral toxicity and induce several-fold increases of sod-1, sod-3, and clt-2 gene expression. It is worth noting that in the present study, the potentially toxic effects of environmental levels of GO NPs in in-vivo C. elegans models were evaluated to show the negative impacts on reproduction, neurobehavioral development, and oxidative stress. It is thus reiterated that based on our findings, GO NPs at environmental levels may cause chronically toxic effects.


It is the first time to use the low dosage of GO NPs treating in the in-vivo model to find the adverse effects in nematodes. Based on our findings, prolonged exposure to GO NPs causes reproductive effects, generates neurotoxicity, shortens longevity, and induces oxidative stress in C. elegans. It is reiterated that low-dose GO NPs at environmental levels from 0.00100 to 1.00 µg L–1 caused significantly negative impacts on nematodes in contrast to the current published data. Thus, the adverse effects of low-level GO NPs on human health should be evaluated in the future.


1. Ahmed, F., Rodrigues, D.F. (2013). Investigation of acute effects of graphene oxide on wastewater microbial community: A case study. J. Hazard. Mater. 256–257, 33–39.

2. Bengtson, S., Knudsen, K.B., Kyjovska, Z.O., Berthing, T., Skaug, V., Levin, M., Koponen, I.K., Shivayogimath, A., Booth, T.J., Alonso, B., Pesquera, A., Zurutuza, A., Thomsen, B.L., Troelsen, J.T., Jacobsen, N.R., Vogel, U. (2017). Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide. PLoS One 12, e0178355.

3. Bianco, A., Cheng, H.M., Enoki, T., Gogotsi, Y., Hurt, R.H., Koratkar, N., Kyotani, T., Monthioux, M., Park, C.R., Tascon, J.M.D., Zhang, J. (2013). All in the graphene family – A recommended nomenclature for two-dimensional carbon materials. Carbon 65, 1–6.

4. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71–94.

5. Chen, H., Li, H., Wang, D. (2017). Graphene oxide dysregulates Neuroligin/NLG-1-mediated molecular signaling in interneurons in Caenorhabditis elegans. Sci. Rep. 7, 41655.

6. Chen, Y., Hu, X., Sun, J., Zhou, Q. (2016a). Specific nanotoxicity of graphene oxide during zebrafish embryogenesis. 10, 42–52.

7. Chung, M.C., Tsai, M.H., Que, D.E., Bongo, S.J., Hsu, W.L., Tayo, L.L., Lin, Y.H., Lin, S.L., Gou, Y.Y., Hsu, Y.C., Hou, W.C., Huang, K.L., Chao, H.R. (2019). Fine particulate matter-induced toxic effects in an animal model of Caenorhabditis elegans. Aerosol Air Qual. Res. 19, 1068–1078.

8.Chung, M.C., Huang, K.L., Avelino, J.L., Tayo, L.L., Lin, C.C., Tsai, M.H., Lin, S.L., Mansor, W.N.W., Su, C.K., Huang, S.T. (2020). Toxic assessment of heavily traffic-related fine particulate matter using an in-vivo wild-type Caenorhabditis elegans model. Aerosol Air Qual. Res. 20, 1974–1986.

10. Cole, R.D., Anderson, G.L., Williams, P.L. (2004). The nematode Caenorhabditis elegans as a model of organophosphate-induced mammalian neurotoxicity. Toxicol. Appl. Pharm. 194, 248–256.

11. De Marchi, L., Pretti, C., Gabriel, B., Marques, P.A.A.P., Freitas, R., Neto, V. (2018). An overview of graphene materials: properties, applications and toxicity on aquatic environments. Sci. Total Environ. 631–632, 1440–1456.

12. Du, T., Adeleye, A.S., Keller, A.A., Wu, Z., Han, W., Wang, Y., Zhang, C., Li, Y. (2017). Photochlorination-induced transformation of graphene oxide: mechanism and environmental fate. Water Res. 124, 372–380.

13. Fakhri, A. (2017). Adsorption characteristics of graphene oxide as a solid adsorbent for aniline removal from aqueous solutions: kinetics, thermodynamics and mechanism studies. J. Saudi Chem. Soc. 21, S52–S57.

14. Giust, D., Lucío, M.I., El-Sagheer, A.H., Brown, T., Williams, L.E., Muskens, O.L., Kanaras, A.G. (2018). Graphene oxide-upconversion nanoparticle based portable sensors for assessing nutritional deficiencies in crops. ACS Nano 12, 6273–6279.

15. Gonzalez-Moragas, L., Roig, A., Laromaine, A. (2015). C. elegans as a tool for in vivo nanoparticle assessment. Adv. Colloid Interface Sci. 219, 10–26.

16. Goodwin, D.G., Adeleye, A.S., Sung, L., Ho, K.T., Burgess, R.M., Petersen, E.J. (2018). Detection and quantification of graphene-family nanomaterials in the environment. Environ. Sci. Technol. 52, 4491–4513.

17. Guo, X., Mei, N. (2014). Assessment of the toxic potential of graphene family nanomaterials. J. Food Drug Anal. 22, 105–115.

18. He, K., Chen, G., Zeng, G., Peng, M., Huang, Z., Shi, J., Huang, T. (2017). Stability, transport and ecosystem effects of graphene in water and soil environments. Nanoscale 9, 5370–5388.

19. Hunt, P.R. (2017). The C. elegans model in toxicity testing. J. Appl. Toxicol. 37, 50–59.

20. Jamialahmadi, N., Safari, E., Baghdadi, M. (2018). Interaction of graphene oxide nano-sheets and landfill leachate bacterial culture. Environ. Technol. 39, 2457–2466.

21. Kang, S., Mauter, M.S., Elimelech, M. (2009). Microbial cytotoxicity of carbon-based nanomaterials: Implications for river water and wastewater effluent. Environ. Sci. Technol. 43, 2648–2653.

22. Kim, M., Eom, H.J., Choi, I., Hong, J., Choi, J. (2020). Graphene oxide-induced neurotoxicity on neurotransmitters, AFD neurons and locomotive behavior in Caenorhabditis elegans. Neurotoxicology 77, 30–39.

23. Kim, Y., Jeong, J., Yang, J., Joo, S.W., Hong, J., Choi, J. (2018). Graphene oxide nano-bio interaction induces inhibition of spermatogenesis and disturbance of fatty acid metabolism in the nematode Caenorhabditis elegans. Toxicology 410, 83–95.

24. Konios, D., Stylianakis, M.M., Stratakis, E., Kymakis, E. (2014). Dispersion behaviour of graphene oxide and reduced graphene oxide. J. Colloid Interface Sci. 430, 108–112.

25. Li, F., Jiang, X., Zhao, J., Zhang, S. (2015). Graphene oxide: A promising nanomaterial for energy and environmental applications. Nano Energy 16, 488–515.

26. Li, M., Zhu, J., Wang, M., Fang, H., Zhu, G., Wang, Q. (2019). Exposure to graphene oxide at environmental concentrations induces thyroid endocrine disruption and lipid metabolic disturbance in Xenopus laevis. Chemosphere 236: 124834.

27. Li, Y., Ma, R., Liu, X., Qi, Y., Abulikemu, A., Zhao, X., Duan, H., Zhou, X., Guo, C., Sun, Z. (2019). Endoplasmic reticulum stress-dependent oxidative stress mediated vascular injury induced by silica nanoparticles in vivo and in vitro. NanoImpact 14, 100169.

28. Liu, P., Shao, H., Kong, Y., Wang, D. (2020). Effect of graphene oxide exposure on intestinal Wnt signaling in nematode Caenorhabditis elegans. J. Environ. Sci. 88, 200–208.

29. Liu, X.T., Mu, X.Y., Wu, X.L., Meng, L.X., Guan, W.B., Ma, Y.Q., Sun, H., Wang, C.J., Li, X.F. (2014). Toxicity of multi-walled carbon nanotubes, graphene oxide, and reduced graphene oxide to zebrafish embryos. Biomed. Environ. Sci. 27, 676–683.

30. Lyon, D.Y., Alvarez, P.J.J. (2008). Fullerene water suspension (Nc60) exerts antibacterial effects via ROS-independent protein oxidation. Environ. Sci. Technol. 42, 8127–8132.

31. Maharubin, S., Zhang, X., Zhu, F., Zhang, H.C., Zhang, G., Zhang, Y. (2016). Synthesis and applications of semiconducting graphene. J. Nanomater. 2016, 6375962.

32. Pan, Y., Sahoo, N.G., Li, L. (2012). The application of graphene oxide in drug delivery. Expert Opin. Drug Deliv. 9, 1365–1376.

33. Patlolla, A.K., Rondalph, J., Tchounwou, P.B. (2017). Biochemical and histopathological evaluation of graphene oxide in sprague–dawley rats. Austin J. Environ. Toxicol. 3, 1021.

34. Pelin, M., Fusco, L., Martín, C., Sosa, S., Frontiñán-Rubio, J., González-Domínguez, J.M., Durán-Prado, M., Vázquez, E., Prato, M., Tubaro, A. (2018). Graphene and graphene oxide induce ROS production in human HaCaT skin keratinocytes: the role of xanthine oxidase and NADH dehydrogenase. Nanoscale 10, 11820–11830.

35. Piechulek, A., von Mikecz, A. (2018). Life span-resolved nanotoxicology enables identification of age-associated neuromuscular vulnerabilities in the nematode Caenorhabditis elegans. Environ. Pollut. 233: 1095–1103.

36. Prasad, C., Liu, Q., Tang, H., Yuvaraja, G., Long, J., Rammohan, A., Zyryanov, G.V. (2020). An overview of graphene oxide supported semiconductors based photocatalysts: Properties, synthesis and photocatalytic applications. J. Mol. Liq. 297, 111826.

37. Qu, G., Liu, S., Zhang, S., Wang, L., Wang, X., Sun, B., Yin, N., Gao, X., Xia, T., Chen, J.J., Jiang, G.B. (2013a). Graphene oxide induces toll-like receptor 4 (TLR4)-dependent necrosis in macrophages. ACS Nano 7, 5732–5745.

38. Qu, G., Wang, X., Liu, Q., Liu, R., Yin, N., Ma, J., Chen, L., He, J., Liu, S., Jiang, G. (2013b). The ex vivo and in vivo biological performances of graphene oxide and the impact of surfactant on graphene oxide’s biocompatibility. J. Environ. Sci. 25, 873–881.

39. Qu, M., Li, Y., Wu, Q., Xia, Y., Wang, D. (2017). Neuronal ERK signaling in response to graphene oxide in nematode Caenorhabditis elegans. Nanotoxicology 11, 520–533.

40. Qu, M., Kong, Y., Yuan, Y., Wang, D. (2019). Neuronal damage induced by nanopolystyrene particles in nematode caenorhabditis elegans. Environ. Sci. Nano 6, 2591–2601.

41. Ren, M., Zhao, L., Ding, X., Krasteva, N., Rui, Q., Wang, D. (2018). Developmental basis for intestinal barrier against the toxicity of graphene oxide. Part. Fibre Toxicol. 15, 26.

42. Rive, C., Reina, G., Wagle, P., Treossi, E., Palermo, V., Bianco, A., Delogu, L.G., Rieckher, M., Schumacher, B. (2019). Improved biocompatibility of amino-functionalized graphene oxide in Caenorhabditis elegans. Small 15, e1902699.

43. Rodrigues, D.F., Elimelech, M. (2010). Toxic effects of single-walled carbon nanotubes in the development of E. coli biofilm. Environ. Sci. Technol. 44, 4583–4589.

44. Sanchez, V.C., Jachak, A., Hurt, R.H., Kane, A.B. (2012). Biological interactions of graphene-family nanomaterials: An interdisciplinary review. Chem. Res. Toxicol. 25, 15–34.

45. Souza, J.P., Baretta, J.F., Santos, F., Paino, I.M.M., Zucolotto, V. (2017). Toxicological effects of graphene oxide on adult zebrafish (Danio rerio). Aquat. Toxicol. 186, 11–18.

46. Suárez-Iglesias, O., Collado, S., Oulego, P., Díaz, M. (2017). Graphene-family nanomaterials in wastewater treatment plants. Chem. Eng. J. 313, 121–135.

47. Tang, Z., Zhao, L., Yang, Z., Liu, Z., Gu, J., Bai, B., Liu, J., Xu, J., Yang, H. (2018). Mechanisms of oxidative stress, apoptosis, and autophagy involved in graphene oxide nanomaterial anti-osteosarcoma effect. Int. J. Nanomed. 13, 2907–2919.

48. Upadhyay, R.K., Soin, N., Roy, S.S. (2014). Role of graphene/metal oxide composites as photocatalysts, adsorbents and disinfectants in water treatment: A review. RSC Adv. 4, 3823–3851.

49. Wu, Q., Wang, W., Li, Y., Li, Y., Ye, B., Tang, M., Wang, D. (2012). Small sizes of TiO2-NPs exhibit adverse effects at predicted environmental relevant concentrations on nematodes in a modified chronic toxicity assay system. J. Hazard. Mater. 243, 161–168.

50. Wu, Q., Yin, L., Li, X., Tang, M., Zhang, T., Wang, D. (2013). Contributions of altered permeability of intestinal barrier and defecation behavior to toxicity formation from graphene oxide in nematode Caenorhabditis elegans. Nanoscale 5, 9934–9943.

51. Wu, Q., Zhao, Y., Li, Y., Wang, D., Wu, Q., Zhao, Y., Li, Y., Wang, D. (2014). Molecular signals regulating translocation and toxicity of graphene oxide in the nematode Caenorhabditis elegans. Nanoscale 6, 11204–11212.

52. Wu, T., Xu, H., Liang, X., Tang, M. (2019). Caenorhabditis elegans as a complete model organism for biosafety assessments of nanoparticles. Chemosphere 221, 708–726.

53. Yan, H., Tao, X., Yang, Z., Li, K., Yang, H., Li, A.M., Cheng, R. (2014). Effects of the oxidation degree of graphene oxide on the adsorption of methylene blue. J. Hazard. Mater. 268, 191–198.

54. Yang, K., Gong, H., Shi, X., Wan, J., Zhang, Y., Liu, Z. (2013). In vivo biodistribution and toxicology of functionalized nano-graphene oxide in mice after oral and intraperitoneal administration. Biomaterials 34, 2787–2795.

55. Yang, X., Qin, J., Jiang, Y., Chen, K., Yan, X., Zhang, D., Li, R., Tang, H. (2015). Fabrication of P25/Ag3PO4/graphene oxide heterostructures for enhanced solar photocatalytic degradation of organic pollutants and bacteria. Appl. Catal., B 166–167, 231–240.

56. Zhang, S., Yang, K., Feng, L., Liu, Z. (2011). In vitro and in vivo behaviors of dextran functionalized graphene. Carbon 49: 4040–4049.

57. Zhang, W., Wang, C., Li, Z., Lu, Z., Li, Y., Yin, J., Zhou, Y., Gao, X., Fang, Y., Nie, G., Zhao, Y. (2012). Unraveling stress‐induced toxicity properties of graphene oxide and the underlying mechanism. Adv. Mater. 24, 5391–5397.

58. Zhang, X., Zhou, Q., Zou, W., Hu, X. (2017). Molecular mechanisms of developmental toxicity induced by graphene oxide at predicted environmental concentrations. Environ. Sci. Technol. 51, 7861–7871.

59. Zhao, J., Wang, Z., White, J.C., Xing, B. (2014). Graphene in the aquatic environment: Adsorption, dispersion, toxicity and transformation. Environ. Sci. Technol. 48, 9995–10009.

60. Zhao, Y., Wu, Q., Wang, D. (2016a) An epigenetic signal encoded protection mechanism is activated by graphene oxide to inhibit its induced reproductive toxicity in Caenorhabditis elegans. Biomaterials 79, 15–24.

61. Zhao, Y., Yang, R., Rui, Q., Wang, D. (2016b). Intestinal insulin signaling encodes two different molecular mechanisms for the shortened longevity induced by graphene oxide in Caenorhabditis elegans. Sci. Rep. 6, 24024.

62. Zhao, Y., Zhi, L., Wu, Q., Yu, Y., Sun, Q., Wang, D. (2016c). p38 MAPK-SKN-1/Nrf signaling cascade is required for intestinal barrier against graphene oxide toxicity in Caenorhabditis elegans. Nanotoxicology 10, 1469–1479.

63. Zhao, Y., Chen, H., Yang, Y., Wu, Q., Wang, D. (2020). Graphene oxide disrupts the protein-protein interaction between Neuroligin/NLG-1 and DLG-1 or MAGI-1 in nematode Caenorhabditis elegans. Sci. Total Environ. 700, 134492.

64. Zhou, D., Yang, J., Li, H., Lu, Q., Liu, Y.D., Lin, K.F. (2016). Ecotoxicity of bisphenol A to Caenorhabditis elegans by multigenerational exposure and variations of stress response in vivo across generations. Environ. Pollut. 208, 767–773.