Table 2:

GPN toxicity in zebrafish and other fish species.

Species	Developmental stage	Method of application	Graphene based nanomaterials	Concentration and duration	Effects	References
Zebrafish wild-type (AB strain)	Embryos [2-cell stage]	Microinjection (10 nL total volume)	graphene oxide (GO; area= 40–60 nm; thickness 1–3 nm) Multi-functional graphene (MFG; size 40–60 nm, thickness 4–6 nm)	0.05 and 0.1 ng/nl; [2 cell stage-72 hpf]	GO induced yolk sac edema in 4–6% larvae 6–12% showed tail or spinal cord flexure 2% showed cardiac malfunction MFG Induced yolksac edema in 9–12 % 8% showed tail or spinal cord flexure	Gollavelli and Ling 2012.
Zebrafish wild-type (AB strain) and endothelial cell-specific transgenic [Tg(kdrl:egfp] embryos	1–2 cell stage embryos	Microinjection; (1 nL volume by single/ two/three injections of 0.25 mg/L);	Nanographene oxide [NGO; lateral size=100–200 nm; height 1–1.5 nm] Polyethelene glycol-coated NGO (NGO-PEG) [hydrodynamic diameter: 310.3±87.23 nm] NGO-Alexa568 (NGO-568) [hydrodynamic diameter: 184.3±8.13 nm]	250 pg; 500 pg 750 pg; [30 hpf and 52 hpf ]	NGO: (i) curved spine (ii) shortened body stature (iii) pericardial edema (iv) lowered yolk consumption (v) underdeveloped brain and retinas (vi) occasional disrupted circulation (30 hpf) Apoptotic positive cells in the head region (500 pg, 30 hpf) Blood vessel sprouting with irregular positioning of growing intersomitic vessel (ISV) trunk vasculature of transgenic [Tg(kdrl:egfp)] zebrafish (500pg; 52 hpf) Vascular endothelial growth factor A (vegfaa) gene expression was upregulated. Notch-regulated Ankyrin repeat protein a and (nrarpa/nrarpb) gene expression was downregulated NGO-PEG Toxic effects by NGO only were ameliorated by NGO-PG. NGO-A568 Toxic effects by NGO only were ameliorated by NGO-A568. Elicited the angionic defects	Jeong et al. 2015.
Zebrafish wild-type (AB strain)	2-cell stage embryos (1–4 cell stage)	microinjection;	Graphene (used 0.3% DMSO as solvent) [particle size: 1456.8±16.1 nm for 1μg/mL; 2012.0±18.5 nm for 10 μg/mL; 5808.2±50.2 nm for 50 μg/mL]	1, 10, 50 μg/mL [2 cell stage-4 and 4.5 dpf]	No significant effect on waking and rest activity on 4 dpf larvae Regulator of hypocretin system genes, such as hcrt, hcrtr,aanat2 remained unaltered.	Lu et al. 2017
Zebrafish embryos	4hpf	Immersion (10 embryos in 2 mL) in E3 medium (5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulphate, pH 7.4); media renewed every 24h	Pristine graphene (pG) [size 170–390 nm]	1, 5, 10, 15, 20, 25, 30, 35, 40,45, 50 μg/L [4–96 hpf]	Induce embryo mortality; All the embryos exposed to 30 μg/L pG or above shows 100% mortality of the embryos within 30 min-2h of exposure. Delayed hatching Pericardial disorder Bradycardia Yolk sac and pericardial edema	Manjunatha et al., 2018
Zebrafish embryos (AB strain)	(4 hpf-120 hpf)	Immersion in E3 medium containing 10–15% methylene blue.	Graphene quantum dots (GQD) size: 2.3–6.4 nm; average lateral dimension 3.4 nm (n=200)	12.5, 25, 50, 100, 200 μg/mL	(i) Distributed on myocardial cell cytoplasm (100–200 μg/mL) as observed in 96 hpf (ii) Reduction in heart rate in a concentration-dependent manner (50–200 μg/mL) from 48 hpf-120hpf.	Jiang et al. 2015.
Zebrafish embryos	4–96 hpf	immersion	GQD	12.5, 25, 50, 100 and 200 μg/mL	(i) fluorescence intensity was mainly localized in intestines and heart (ii) heart rate decreased with the increase in GQD concentrations Hatching rates decreased with increasing concentrations of GQD Pericardial edema, vitelline cyst, bent spine and bent tail was observed in the larvae exposed to 200 μg/mL GQD. Spontaneous movement decreased significantly at GQD concentrations 50, 100, and 200 μg/mL. Visible light test (behavior) indicate that the total swimming distance and speed decreased depending on GQD concentration; Embryos exposed to 12.5 μg/mL GQD shows hyper activity and those exposed to 25, 50, 100 and 200 μg/mL shows hypoactivity in visible light (light-dark) test.	Wang et al. 2015
Zebrafish (wild type; locally breed and reared short-fin)	embryos (0–7 dpf)	Immersion; in 2 mL of 30% Danieau’s solution [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM Hepes, pH 7.4]	Thiolated GQD (SH-GQD)	0.02, 0.05, 0.1,0.2, 0.3, 0.6, 0.8, and 1 mg/mL	(i) High concentration of SH-GQD (1 mg/mL) enhanced mortality, however, low concentration of SH-GQD (0.1 mg/mL) did not. (ii) SH-GQD has shown protective effects against oxidative stress SH-GQD induced head deformation, vent tail, pericardial edema, and yolk sac edema in a concentration-dependent manner SH-GQD did not affect the hatching rates, however, 46% of the larvae died on 7 dpf exposed to 1 mg/mL SH-GQD	Oh et al. 2017
Zebrafish (wild-type)	4 hpf	Immersion in E3 medium; [media refreshed every 12 h]	GO (Particle size: 600 nm); Graphenea, New York, USA	5, 10, 50, 100 μg/mL [4–120 hpf]	The survival rate exhibited a time and concentration-dependent behavior between 72–120 hpf with 50–100 μg/mL showed significant effects. Significant reduction in hatching rates between 72–120 hpf in 50–100 μg/mL groups. The heart rates at 72 hpf were found to be significantly reduced in larvae exposed to 50–100 μg/mL GO. The frequency of movement in 96 hpf embryos were significantly reduced in larvae exposed to 50–100μg/mL GO.	d’Amora et al., 2017.
Zebrafish	embryo 4hpf-96 hpf;	Immersion; 10 embryos in 5 mL freshwater/well in 24 well culture plates. Media renewed every 24h.	GO flakes	40 and 80 mg/L [every 24 h]	No embryo mortality was observed. No alteration in growth, brain morphology, pharyngeal arches and jaws, heart, fins, notochord, somites, body shape, cardiovascular function, yolk sac and locomotion. Increase in heme oxygenase 1 (HO-1) mRNA and protein expression by GO Increase in inducible Nitric Oxide synthases (iNOS) mRNA and protein expression by GO.	Pecoraro et al. 2018.
Zebrafish (wild type)	Embryos/larvae	Immersion in system water; buffer containing 1% pluronic F 68; Media changed every 24h until 6 days	GO. Single layer; thickness 5.3nm. [suspended in 1% pluronic F 68 water suspension]	5, 10, 50, 100 mg/L [2–168 hpf]	No change in mortality, hatching rate, and spontaneous movement Heart rates remained unaltered (increased in 10 mg/L dose) No change in eye areas in 120 hpf larvae. Reduction in body length (100 mg/L) in 6 dpf larvae No change in gap43, gfap, nestin gene transcriptions (genes associated with nervous system development) in 6 dpf larvae. Neurogenin1 and dat gene transcriptions (genes associated with nervous system development) were increased at 10 mg/L in 6 dpf larvae Upregulation of synapsinIIa and down regulation of dat genes (genes associated with nervous system development) in 5 mg/L groups in 6 dpf larvae Increase in distance travel, the speed, and the turn angle of the 6 dpf larvae exposed to 10 mg/L. AChE activity remained unaltered in 6 dpf larvae. Dopamine level was reduced in 6 dpf larvae exposed to 10 mg/L bcl2 and caspase3 genes were increased in 6 dpf larvae exposed to 10 mg/L Autophagosome formation, vacuoles, and partial loss of nuclear membrane architecture at specific regions of ventral diencephalon in 6 dpf larvae exposed to 10 mg/L.	Soares et al. 2017.
Zebrafish AB strain	2 hpf-96 hpf	Immersion; E3 medium; Media replaced every day	GO nanosheets 0.8–1 nm; diameter 101–258 nm	0.01, 0.1, 1.0, 10, 100 mg/L [48, 72 and 96h]	Mortality did not increase significantly Accumulation of GO in the chorion and severe hatching delay in embryos (both at 72 and 96 hpf) exposed to 100 mg/mL GO. Heart rate significantly decreased in 48 hpf embryos exposed to 1 mg/L GO, however, significantly increased in embryos exposed to 100 mg/L Spontaneous movement of the embryos exposed to 100 mg/L GO were significantly reduced at 48 hpf Incidence of yolk sac edema in 96 hpf embryos were more pronounced and found to be concentration-dependent. Significantly high incidence of Tail/spinal cord flexure was evident in larvae at 96 hpf exposed to 1 mg/mL GO Significantly higher percentage of 96 hpf larvae without eyes/head were observed when the embryos were exposed to 10 and 100 mg/L GO. ROS generation in the whole body of the 96 hpf larvae was increased by GO in a concentration-dependent manner. GO down-regulated SOD activity in a concentration-dependent manner and increased the MDA concentration. GO at 1mg/L suppressed the level of global DNA methylation, while at 100 mg/L promoted methylation	Chen et al. 2015a.
Zebrafish (wild- type)	Blastula	Immersion	GO nanosheets (height ~1.5 nm; lateral size: 1.5μm	5, 25, 50 μg/mL [12, 24, 48 and 72 hpf]	Survival rates were decreased with the increase in exposure time (12–24 h); remained constant 48–72h. Hatching rates slightly decreased (5 mg/mL) and increased at higher concentrations (25 and 50 μg/mL; not significant) Up regulation of apex1, ogg1, polb, creb1 (genes involved in base excision repair pathways) in embryos exposed to GO (50 μg/mL) for 24 h	Lu et al. 2017.
Zebrafish (AB strain);	embryo (2.5hpf–168hpf (7dpf);	Immersion in E3 medium	GO nanosheets (thickness1.01±0.05 nm; lateral length: 0.3–2.6 μm) dispersed in E3 medium	1μg (0.001 mg) /L-100 mg/L,	The embryomortality was not concentration-dependent; so the LC50 for GO in zebrafish embryyos was remained undermined. heart rate increased (100 μg/L) at 96 hpf; heart rate decreased, hatching delayed (1–100 mg/L) spontaneous movement inhibited (10 μg/L; 100 μg/L; 100 mg/L) trunk curved, tail malformed, pericardial edema, yolk sac edema, craniofacial malformation, reduced body length (10 μg/L, 100 μg/L, 1 mg/L, 100 mg/L) ROS intensity (8-OHDG) increased (100 μg/L) Carbonyl protein content increased (1, 10, 100 μg/L) Super oxide radical, MDA, increased (1, 10 and 100 μg/L) Catalase, glutathione peroxidase type I, copper/zinc superoxide dismutase, SOD, GST were down regulated Downregulated (100 μg/L) col1a1a, col11a2, col2a1a, and col11a1a genes. ( these genes played key roles in the development of cartilage and notochord) Inhibition of parasphenoid development (100 μg/L); affects Meckel’s cartilage; reduced the length of lower jaw and intercranial distance (cranial deformities) 87.18% decrease in erythrocyte content (100 μg/L) Cardiac output declined, heart rate increased (100 μg/L)	Zhang et al. 2017b.
Zebrafish AB-strain	embryos 2.5 hpf-72hpf	Immersion in E3 medium; media replaced every day	GO nanosheets (thick ness 0.8–1.2 nm) Humic acid (diameter 2–12 nm)	(i) GO = 0–100 mg/L in E3 medium (ii) HA= 0.01–100 mg/L in E3 medium (iii) GO-HA= 100 mg/L GO+10 mg HA	GO100 (100 mg/L GO): hatching rate reduced; rate of pericardial edema enhanced; heart beat increased HA (0.01–100 mg/L) did not induce adverse effects on embryo development GO-HA (100 mg/L GO + HA (10 mg/L) recovered hatching rate, pericardial edema, heart beats. Mitochondria became swollen and loose, and the integrity of the membrane and cristae was damaged by GO100. GO100 significantly increased GSH, MDA, ROS and inhibited SOD. HA reduced the lipid protein, and DNA damage induced by GO	Chen et al. 2015b.
Zebrafish (AB strain)	embryos 2–120 hpf;	Immersion in E3 medium; Media changed every 24 h	GO nanosheets (thickness: 0.87±0.157 nm; lateral length 50–200 nm) CysGO (thickness 1.42±0.234 nm; lateral length 78–590 nm	GO (0.01–10 mg/L) CysGO (0.01 –10 mg/L+ Arsenic 1mg/L) in E3 medium;	Pericardial edema, tail flexure, eye malformation in 5–9% larvae (120 hpf) exposed to GO (0.01–10 mg/L);No toxic effects were observed with CysGO Hatching rates were 10–30% in embryos exposed to GO at 72 hpf in contrast to 75% in control embryos; no hatching delay for CsyGO (0.01–1 mg/mL) were observed; however, embryos exposed to 10 mg/mL CysGO only 70% of the embryos hatched at 72 hpf. At 96 hpf all embryos were hatched (control and treated) 5–8% death occurred in GO-exposed larvae; however, CysGO did not induce embryonic death. GO suppressed cell nucleus development in the inner eye tissue while CysGO did not. Pericardium development in embryos exposed to GO were ill -developed; but pericardial development is normal in embryos exposed to CysGO. No notable oxidative damage to DNA occurred in larvae exposed either to GO or CysGO at concentrations 0.01–10 mg/L. GO reduced the Na+K+ATPase activity in a concentration-dependent manner; CysGO (0.01–10 mg/L) did not significantly alter the activity. Mitochondrial membrane polarization lost by GO; however, the damage was relatively less in larvae exposed to CsyGO CysGO protects the embryos from arsenic poisoning.	Mu et al., 2015.
Zebrafish	embryos (2 hpf-120 hpf)	Immersion in E3 medium; media replaced every 24h until 120 hpf	GO nanosheets in biological secretions (GOBS) (thickness 10 nm; lateral length 19.5–282 nm) GO nanosheets (GONS) (thickness 0.83±0.12 nm; lateral length is 0.5μm-several microns)	exposed to 0.01, 0.1,1 mg/L)	GONS is more readily taken up by the embryos than GOBS. GOBS showed increased mortality, malformation, faster heart beats, upregulation of β-galactosidase and loss of mitochondrial membrane potential than GONS; Both GOBS and GONS induced stronger adverse effects than controls (embryos exposed to bulk-activated carbon powders with an average diameter of 147±41 μm) Pericardial edema and tail flexture were also observed in juvenile fish, especially in larvae exposed to GOBS. Embryonic death and faster heart beat was more pronounced in larvae exposed to GOBS than GONS. No alteration in ROS DNA methylation enhanced by both GOBS and GONS. Both GOBS and GONS enhanced β-galactosidase levels Both GOBS and GONS inhibited calcium exchange in the embryos	Mu et al., 2016.
Zebrafish	embryos 1hpf-96 hpf	Immersion	GO [width 394.21±215.05 nm; Height 0.89±0.01 nm] base washed GO (bw GO) [width 286.53 ±104.42 nm Height 0.94±0.02 nm GO+ 20 mg/L humic acid (HA) bwGO+ 20 mg/L HA	GO=1, 10, 100 mg/L in medium (96 mg/L NaHCO3; 60 mg/L MgSO4; 4 mg/L KCl; 60 mg/L CaSO4, 2H2O; pH 7.4) at 27 oC bwGO= 100 mg/L in medium HA=20 mg/L in medium	Mortality below 17% independent of exposure conditions. Reduced body length in GO (100 mg/L), GO (100 mg/mL)+HA (20 mg/mL), and in bwGO (100 mg/mL)+ HA (20 mg/mL) No alteration in Catalase enzyme activity by GO, bwGO either alone or in presence of HA GST remained unaltered by GO, GO+HA20 mg/L, but significantly decreased by bwGO (100 mg/L) and increased by bwGO (100 mg/L)+HA (20 mg/L) Acid phosphatase (AP) remained unaltered by GO (1,10, or 100 mg/L); HA (20 mg/L) increased AP; GO+HA reduced AP in a concentration-dependent manner; bwGA alone was unable to alter AP, however, in presence of HA (20 mg/mL), AP enzyme activities reduced significantly Significant reduction in AChE activity by GO (100 mg/L GO) alone or in presence of 20 mg/mL HA (GO+HA)	Clemente et al 2017.
Zebrafish	Embryos [2–96 hpf]	suspension	GO and rGO	1,5,10, 50, 100 mg/L [ 2–96 hpf]	rGO inhibited hatching rGO decreased the length of the hatched larvae at 96 hpf no mortality or morphological malformation were induced by GO and rGO	Liu et al. 2014.
Zebrafish (wild-type and transgenic Tg (cyp1a:gfp);	4 hpf -168 hpf (7 dpf)	Immersion in deionized water (DI); media renewed everyday	Reduced graphene oxide quantum dots (rGOQDs) (10 nm lateral size; 1 nm height)	25, 50, 100 μg/mL	No effect on hatching rate (72 hpf), body length, and mortality in wild-type fish Heart beats (96 hpf) reduced (100μg/mL) in wild-type larvae Pericardial edema, vitelline cyst, bent spine (100 μg/mL) observed in wild-type larvae Upregulation of cyp1a, cyp1c, cyp7a1, hsp70 (100 μg/mL) (7 dpf) in wild-type larvae Green fluorescence protein expression was significantly increased in wild-type and transgenic Tg(cyp1a:gfp) larvae exposed to 50 and 100 μg/mL rGOQD on 7 dpf.	Zhang et al., 2017a
Japanese medaka embryo	(1 dpf-7 dpf);	suspension in embryo rearing meium (17.1 mM NaCl, 272 mM CaCl2,2H2O, 402 mM KCl, 661 mMMgSO4, 7H2O; pH 6.3); Media replaced every alternate day	Sonicated or unsonicated oxidized Graphene nanoribbons (O-GNR); Diameter: 250–400 nm; Average length 744±178 nm (bath sonicated); 323±50nm (sonicated for 1 min); 201±28 nm (sonicated for 5 min); 100±10 nm (sonicated for 10 min)	20 μg/mL	Probe sonicated O-GNR increased embryo-larval mortality depending upon the sonication time; however, no significant effects on mortality was observed in embryos exposed to bath-sonicated o-GNR (20 min) O-GNR is able to enter inside the chorion Bath-sonicated O-GNR induced hatching of the embryos 2 days earlier than control embryos.	Mullick Chowdhury et al 2014.
Zebrafish	larvae (72 hpf-96 hpf)	Cultured in E3 medium	GO (thickness 1.02±0.15 nm; lateral length 0.5μm-several microns)	0.01, 0.1 and 1μg/L GO in E3 medium for 24 h	(i) Larval zebrafish incubated with 0.01 μg/L GO (72hpf-96 hpf) at 120 hpf exhibited tail flexure and spinal curvature; Pericardial edema seen in larval zebrafish exposed to 0.1 μg/mL GO; pericardial and yolk edema coexists in larval zebrafish exposed to 1μg/mL GO. Distribution of dopaminergic neurons in the diencephalon was reduced by ~70% in larvae exposed to GO (0.01–1.0 μg/L) at 96 hpf. 222–522% increase in α-synuclein and 69–179% increase in ubiquitin occurred in 96 hpf larvae exposed to 0.01–1.0μg/mL GO (72–96 hpf) The swim speed of 7 dpf larvae was decreased by 19–57% following GO exposure (0.01–1 μg/L; 72–96 hpf) Other movement-related disorders such as nearest neighbor distance (distance between a given fish and its nearest neighbor, NND) decreased by 22–49%, and inter individual distance (average distance of a given fish from its nearest neighbor, IID) increased by 31–91% in 7 dpf larvae exposed to GO (0.01–1μg/L; 72–96 hpf) Upregulation of caspase 8 (38–152%) protein was occurred in larvae exposed to GO (0.01–1.0 μg/L; 72–96 hpf) Increase in β-galactosidase activity (41–83%) was observed in larvae exposed to GO (0.01–1.0μg/L; 72–96 hpf). GO exposure resulted in metabolic disturbance in 96 hpf larvae.	Ren et al. 2016.
Japanese medaka	larvae (24–48 hph)	in moderately hard-constituted water; live brine shrimp was given as food.	Graphene and graphene-TiO2 nanoparticle composites(GNP-TiO2)	2, 5, 8, 10, 14, 17, and 20 mg/L GNP-TiO2 under simulated solar radiation (SSR) exposure; 167 and 500 mg/L GNP under dark conditions.; exposure period is 4–48 h	Graphene exhibited no toxicity in both dark and SSR conditions. Under dark conditions LC50 for GNP-TiO2 was greater than 500 mg/L. Under (SSR) the LC50 for GNP-TiO2 was 11 mg/L.	Liu et al. 2014.
Adult zebrafish	(2-month-old).	10 males and 10 females per tank (6-L glass tanks); fed with brine shrimp naupli. Half of the exposure water was renewed every day.	GO	1, 5, and 10 mg/L for 14 days	No apparent damage to gill histology by GO (1, 5 and 10 mg/L).Vacuolation, loose arrangement of cells, histolysis, and disintegration of cell boundaries were seen in both liver and intestine of fish exposed to GO in a concentration-dependent manner. Number of goblet cells increased with higher GO concentrations. Malondialdehyde (MDA) content in liver was increased in day 1 by 1,5 and 10 mg/L and in day 4 by 1mg/L GO. GSH was decreased in liver in day 1 by by 1, 5, and 10 mg/L and in day 4 by 1 and 10 mg/l GO. SOD and catalase was increased in liver by 1, 5 and 10 mg/L GO only in day 4. Expression of tumor necrosis factor α (TNF-α), interleukin-1 β, and interleukin -6 was increased in the spleen of zebrafish exposed to GO for 14 day in a concentration-dependent manner.entration-depenobserved in a concentration-dependent manner.	Chen et al. 2016.
Adult zebrafish local commercial source	male and female; 6 months old;	Fed with Tetra Color- tropical flakes.	GO Thickness: 1nm; area: 0.58 μm2	2, 10, 20 mg/L; Exposure: short-term: either 24h or 72 h Long-term: 14 days	The number of apoptotic and necrotic cells in gills were increased in both 24 or 72 h exposure to GO (2 or 20 mg/L) ROS increased significantly in gill cells after 24 h of GO exposure (2, 10, or 20 mg/L). No DNA damage in blood cells was observed in any concentration of GO used in this study after 72 h of exposure. Gill morphology with regard to dilated marginal channel, lamellar fusion, clubbed tips, aneurysms, and necrosis was disrupted in fish exposed to 2, 10 and 20 mg/L GO for 14 days (chronic exposure). The liver of fish exposed to 2 mg/L of GO for 14 days showed peripheral nucleus; those with 10 mg/L showed pyknotic nuclei and those with 20 mg/L showed necrosis in the liver. The lumen of the GUT exposed to GO (2, 10, or 20 mg/L; 14 days) filled with unidentified brown spots.	Souza et al. 2017.
Common carp (Cyprynus carpio) obtained from local fish market.	3 months old (Juvenile)	water	GO Thickness 0.7–1.8 nm Surface area: 208.6 m2/g	1mg/L GO+500 ng/L perfluorooctanesulfonate (POFS) [GO+POFS] 1mg/L GO + 500 ng/L perfluorooctanesulfonate (POFS) +2 mg C/L Fulvic acid.[GO+POFS+FA] Exposure period 28 days; recovery period 29, 30, 34, 38, 44, 50, 56, 62, and 82 (considering the date of exposure is 0 day)	The accumulation of POFS was enhanced by GO in blood, intestine, liver, kidney, gill, and muscle tissues of common carp. Large amount of black residues were found in the intestine of common carp exposed to either GO+POFS or GO+POFS+FL. FA reduced the accumulation of PFOS in tissues of common carp by reducing the bioavaiability of GO and PFOS	Qiang et al. 2016.