Table 1.

Summary of graphene and graphene oxide (GO) toxicity in aquatic animals.

Animal	Route of Graphene Exposure	Adverse Outcome	Dosage Concentration and Time	Ref.
Tested in invertebrate species
Crassostrea virginica	Waterborne exposure	Short-term GO exposures can induce oxidative stress, epithelial inflammation, and adversely affect overall Crassostrea virginica health.	1 and 10 mg/L 72 h static renewal.	[77]
Crassostrea virginica	Waterborne exposure	Elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities were observed in gills and digestive gland tissues of the GO-exposed oysters. Oxidative damage, stress signaling leading to adverse effects on cellular health.	2.5 and 5 mg/L 14 days	[78]
Diopatra neapolitana	Waterborne exposure	GO induced negative effects on the regenerative capacity, altered energy-related responses, especially glycogen content, and decrease in metabolism, cellular damage in Diopatra neapolitana.	0.01, 0.10 and 1.00 mg/L 28 days	[79]
Daphnia magna	Waterborne exposure	GO induced significant toxicity to Daphnia magna. 21 days LC50 chronic toxicity 3.3 mg L−1. In the presence of HA, the decreased toxicity of GO was attributed to the alleviation of oxidative damage by HA.	50.0, 65.0, 84.5, 110.0 and 143.0 mg/L 21 days	[80]
Daphnia magna	Waterborne exposure	14C-labeled graphene accumulated 1% of the organism dry mass. Excretion of graphene at constant phase in depuration. Addition of algae and humic acid to water during the depuration period resulted in release of a significant fraction (~90%) of the accumulated graphene, some remained in the organism. Accumulated graphene in adult Daphnia was likely transferred to the neonates.	250, 100, 50 and 25 µg/L 48 h Depuration 24 h	[81]
Ceriodaphnia dubia	Waterborne exposure	GO induced lethality, reproduction inhibition, ROS generation, reduction on feeding rates and accumulation on gut tract. There was a shift in the available energy for self-maintenance rather than feeding or reproduction activities.	Acute exposure: 0.1; 0.2; 0.4; 0.8; 1.6 and 3.2 mg/ L, 48 h Chronic exposure: 0.05; 0.1; 0.2; 0.4 and 0.8 mg/ L 7 days	[82]
Palaemon pandaliformis	Waterborne exposure	GO did not present acute ecotoxicity at concentrations up to 5.0 mg/L. The 96 h LC50 of Cd associated with GO was 1.7 times less than the 96 h LC50 of Cd alone and the 96 h LC50 of Zn associated with GO was 1.8 times less than the 96 h LC50 of Zn alone. The co-exposure of GO with trace elements impaired the routine metabolism of Palaemon pandaliformis.	GO - 0.1; 1.0; 2.5 and 5.0 mg/L 96 h Co-exposure of GO 1.0 mg/L with trace elements Cd 1.0 mg/L and Zn 1.0 mg/L	[83]
Cyprinus carpio L.	Waterborne exposure	Significant decrease in RBC count. No significant effect on WBC, PCV, and Hb.	0, 10, 20 mg/L, 10 days	[109]
Tested in fish cell lines
PLHC-1	Co-exposure - increasing concentration of AhR agonist alone or in presence of GO and CXYG. Pre and post exposure – increasing concentration of BKF and α-MEM, to α-MEM + 4 mg/mL CXYG or to α-MEM + 16 mg/mL CXYG for another 24 h	GO and CXYG had potentiating effect on PAH- and PCB-induced Cyp1A expression at both the transcriptional and the enzymatic levels. It suggested surface chemistry of GO and CXYG did not had influence on the direct or indirect interaction with the selected AhR agonists. The obtained results suggest that a preceding and/or simultaneous exposure to GO or CXYG nanoplatelets may modify the toxicokinetics of aromatic environmental pollutants such as PAHs and PCBs.	GO and carboxyl graphene (CXYG) at 16 µg/mL, AhR agonist.	[84]
PLHC-1	α-MEM medium	PLHC-1 cells demonstrated significantly reduced mitochondrial membrane potential (MMP) and increased ROS levels at 16 μg/mL GO and CXYG (72 h), but barely any decrease in cell viability. The observation of intracellular graphene accumulations not enclosed by membranes suggests that GO and CXYG internalization in fish hepatoma cells occurs through an endocytosis-independent mechanism.	GO: 0.125–16 µg/mL; CXYG: 0.25–32 µg/mL	[85]
BF2	GO in milli Q water (stock solution) + Eagle’s medium	GO caused mitochondrial and lysosomal damage to BF-2 cells, oxidative stress, and morphological changes by GO through ROS, as indicated by the evaluated biomarkers LPO, GSH, SOD, CAT, and 8-OHdG.	0, 10, 20, 40, 60, 80 and 100 μg/mL for 24 h	[86]
PLHC-1 and CLC	GRMs – Carbon nanofibers (CNFs) and graphene oxide (GO)	GO sheets were present within vesicles as well as free in the cytoplasm of both cell types. CNFs toxicity was inversely related to the graphitization degree.	0–200 μg /mL of GRMs for 24 and 72 h	[87]
Tested at embryonic or larvae stages of fish
Danio rerio	Waterborne exposure	Hatching delay of zebrafish embryos at a high dosage of 50 mg/L. Embryos exposed to GO exhibited significant cellular apoptosis only in the forehead and eye region, and no aggravation of cellular apoptosis was observed with increasing concentration of GO.	0, 3.4, 7.6, 12.5, 25 and 50 mg/L 96 h post fertilization	[95]
Danio rerio	Waterborne exposure	GO impaired DNA modification, protein carbonylation, ROS generation (also superoxide radical)	1–100 µg/L 2.5 hpf-7 dpf	[60]
Danio rerio (adult and embryo)	Waterborne exposure	GO translocated from the water to the brains of parental and offspring fish with a significant loss of claudin5a. GO did not trigger obvious neurotoxicity in parental zebrafish, whereas remarkable neurotoxicity occurred in the offspring, which exhibited a loss of dopaminergic neurons and reductions in acetylcholinesterase activity.	GO exposed to parental zebrafish 24 h prior mating 0.01–1 μg/L	[97]
Danio rerio	Waterborne exposure	Regardless of the presence of HA, larvae exposed to GO for 5 days showed an increase in locomotor activity, reduction in the yolk sac size, and total length and inhibition of AChE activity, but there was no difference in enzyme expression. Results indicated that HA is associated with the toxicity risk modulation by GO.	GO-100 mg/L & HA 20 mg/L alone or together for 5–7 days	[99]
Danio rerio	Waterborne exposure	GO adhered to and enveloped the chorion of zebrafish embryos mainly via hydroxyl group interactions, blocked the pore canals of the chorionic membrane, and caused marked hypoxia and hatching delay. GO induced excessive generation of reactive oxygen species and increased oxidative stress, DNA damage, and apoptosis	0, 0.01, 0.1, 1, 100 mg/L for 24, 48 and 96 hpf	[101]
Danio rerio	Waterborne exposure of GO, Humic Acid (HA) and GO-HA	GO induced significant cardiac edema and hatching delay. HA decreased the interaction between GO and chorion, mitigated chorion damage by regulating morphology, structures, and surface negative charges of GO	GO 0–100 mg/L HA 0–100 mg/L 2.5 hpf-72 hpf	[100]
Danio rerio (larvae and adult)	Injections at ventral end of larvae	GO induced hepatic dysfunction through the ROS and PPAR-α mediated innate immune signaling in zebrafish	0, 0.25, 0.5, and 1 mg/L for 72 h	[98]
Danio rerio	Waterborne exposure of GO and reduced graphene (rGO)	GO had significant effects on the heart rate, while rGO affected the embryos hatching and the length of larvae in a dose-dependent manner	1, 5, 10, 50, 100 mg/L for 96 h	[96]
Danio rerio	Waterborne exposure	GO induced cardiac and dopaminergic alterations, as well as neuronal gene expression and morphology modifications. Altered locomotion in terms of increase of turn angle suggesting parkinsonian-like motor symptoms (at low concentrations).	5, 10, 50 or 100 mg/L for 6 days	[102]
Danio rerio	Waterborne exposure	GOs induced oxidative stress and apoptosis. In particular, the immune cell number, pro-inflammatory iNOS activity, and AChE activity (a neural development indicator) were found to be induced to some extent after GO exposure, suggesting the presence of both immunomodulatory and neurotoxic effects in zebrafish larvae. The waterborne-GO exposure on zebrafish during early development was not merely dependent on GO concentration but also the associated GO sizes.	GO particles (50–200 nm, <500 nm, and >500 nm) at 0.1, 1, 10, and 100 mg/L for 4–124 h post-fertilization	[103]
Danio rerio	Microinjection (4 nL/embryo)	Graphene induced no significant locomotion alterations, sleep behavior, and gene expression patterns.	Graphene at 1, 10, 50 µg/mL	[60]
Tested at adult stage of fish
Danio rerio	Waterborne exposure	GO caused toxicity-Oxidative stress and tissue damage induced in fish by GO through ROS, indicated by the biomarkers of MDA, GSH, SOD, and CAT; GO caused immunotoxicity in fish indicated by increased expression of inflammatory cytokines, TNF-, IL-1, and IL-6.	0, 1, 5, 10 or 50 mg/L GO for 14 days	[104]
Danio rerio	Carbon 14 labeled few-layered graphene (FLG)	At 48 h larger FLG (L-FLG) at 250 µg/L the amount of graphene was close to 48 mg/kg fish dry mass, 170-fold greater than body burden of the same concentration of smaller FLG (S-FLG). L-FLG accumulated in gut and S-FLG accumulated in gut and liver. L-FLG and S-FLG had significantly different impact on intestinal microbial community structure.	L-FLG- 300–700 nm S-FLG-30–70 nm 50 μg/L, 75 μg/L and 250 μg/L 4, 12, 24, 48, 72 h	[105]
Danio rerio	Graphene family materials (GFMs), Monolayer graphene powder (GR), graphene oxide nanosheet (GO), reduced graphene oxide powder (rGO)	GFMs led to different inflammatory responses and significantly altered the relative composition of the gut bacterial species. GFMs altered the intestinal morphology and antioxidant enzyme activities.	1 µg in fish diet for 21 days	[106]
Danio rerio	Waterborne exposure	GO caused increase in oxidative stress, increase in lipid peroxidation, changes in SOD, CAT, GPx. After 168 h: GO toxic effects decreased, but homeostasis not fully recovered.	2, 10, and 20 mg/L 48 h, recovery period 168 h	[107]
Anabas testudineus	Injection at base of caudal fin	GO induced oxidative stress in cell and mitochondria in fish	200 µL from 1 g/L 100 mg/L, 10 mg/L of GO in aqueous solution 24 h	[108]