Biological response and developmental toxicity of zebrafish embryo and larvae exposed to multi-walled carbon nanotubes with different dimension

Abstract

The development and use of nanomaterials are increasing significantly. Among nanomaterials, carbon nanotubes are of particular interest due to its distinctive physicochemical properties. This material composed of sheets of graphite has very high thermal conductivity, metallic-type electrical conductivity, stiffness, toughness and unique ability to bond to itself in an extended network with extraordinary strength. Its application in the industry is continuously growing, which could lead to the accumulation in the environment and a consequent impact on both humans and ecosystems. Considering that environmental systems are dynamic, it is difficult to predict the risks associated with the release of nanomaterials to the environment. Bioindicators are useful tools as primary signals of environmental risk, and their responses reveal the organism and ecosystem health. In the present study, we evaluated the impact of multi-walled carbon nanotubes with different dimensions and agglomeration pattern on zebrafish embryo and larvae; mainly, studies were focused on physiological and behavioral responses. In embryos, measurements were hatching rate, morphology changes, and viability. In larvae, locomotor activity, heart rate, innate inflammatory response, general and tissue-specific morphology were measured. MWCNT-S (short, wide and mostly dispersed) caused depression of the locomotor activity of larvae, indicating an alteration of the central nervous system, and depression of neutrophil migration activity. MWCNT-L (long, thin and agglomerated) caused malformations during larval development, a decrease of neutrophil migration and alteration of cardiac rhythm. Results obtained for both carbon nanotubes were different, highlighting the importance of dimensions of the same nanomaterial, and also the kind of agglomeration and shape adopted, for the toxic effects on organisms.

Keywords: Toxicology.

Fig. 1

Experimental design: timeline. Zebrafish embryos of 4 h post-fertilization (hpf) (0 dpf) were incubated with MWCNTs at 0.005, 0.05, 0.5, 5 and 50 ppm final concentrations. Hatching, mortality and morphological changes were studied up to 2 dpf. On the other hand, zebrafish larvae of 5 dpf were incubated with MWCNTs at 0.005, 0.05, 0.5, 5 and 50 ppm final concentrations. Morphological changes, neurotoxicity, cardiotoxicity, hepatotoxicity and immunotoxicity were studied up to 7 dpf.

Fig. 2

SEM micrographs of MWCNTs. MWCNT-S and MWCNT-L at 50 ppm concentration were dispersed in distilled water by sonication and then dried by lyophilization. Representative micrographs of MWCNTs before (A) and after (A and B) sonication of different magnifications are shown.

Fig. 3

TEM micrographs of MWCNTs. MWCNT-S and MWCNT-L at 50 ppm concentration were dispersed in distilled water by sonication and then placed in TEM grids. Representative micrographs of different magnifications are shown.

Fig. 4

Hatching and mortality of embryos exposed to MWCNTs. Zebrafish embryos of 4 hpf were incubated with the 0.005, 0.05, 0.5, 5 and 50 ppm of MWCNT-S or MWCNT-L. (A) Hatching was observed until 48 hpf. Results are expressed as the percentage of hatched embryos respect to total embryos. (B) Representative photographs of hatched and non-hatched zebrafish exposed to 50 ppm of MWCNTs are shown. (C) Cumulative mortality curves were constructed from 0 to 48 hpf. Results are expressed as the percentage of dead embryos respect to total embryos. Values are shown as mean ± SEM. Values are shown as mean ± SEM. Significant differences respect to control were analyzed by TWO-WAY ANOVA test followed by Dunnett's multiple comparisons post-test.

Fig. 5

Neuro and cardiac activity of larvae exposed to MWCNTs. Zebrafish larvae of 5 dpf were incubated with 0.005, 0.05, 0.5, 5 and 50 ppm of MWCNT-S and MWCNT-L for 48 h (hpi). (A) Swimming activity was measured. Results are expressed as the percentage of spontaneous movement respect to control. (B) Cardiac rhythm was quantified. Results are expressed as the percentage of heart rate respect to control. Values are shown as mean ± SEM. Significant differences respect to control were analyzed by ONE-WAY ANOVA test followed by Dunnett's multiple comparisons post-test (*p < 0.05, **p < 0.01).

Fig. 6

Morphology abnormalities of larvae exposed to MWCNTs. Zebrafish larvae of 5 dpf were incubated with 0.5, 5 and 50 ppm of MWCNT-S or MWCNT-L for 48 h; (A) larvae were scored based on the severity of their morphological defects. Scores range for morphological anomalies defects are: 0 for no visible toxic effects, 1 for minor degree (one to two effects), 2 for moderate (three to four effects) and 3 for severe (more than four effects). Sublethal defects studied were bent spine (BS), jaw malformation (JM), opaque head region (OH), opaque liver (OL), opaque yolk salc (OY), uninflated swim bladder (USB), edema (E), small head (SH), tail malformation (TM), yolk not depleted (YND). (B) Representative pictures of endpoints included in the scoring evaluation are shown. (C) Craniofacial parameters were analyzed. Meckel's cartilage length (ML), angle (MA), distance to fins (MD) and Ceratohyal cartilage length (CL), angle (CA), distance to fins (CD) were quantified and expressed as percentage respect to control larvae. (D) Ventral view of representative pharyngeal skeletons of MWCNT-incubated and control larvae are shown. Values are shown as mean ± SEM. Statistical analysis was performed by ONE-WAY ANOVA test followed by Dunnett's multiple comparisons post-test (*p < 0.05).

Fig. 7

Histopathology of larvae exposed to MWCNTs. Zebrafish larvae were incubated with 0.005, 0.05, 0.5, 5, and 50 ppm of MWCNT-S and MWCNT-L for 48 h; brain, heart and liver phenotype (cells size and shape) were analyzed. Representative photomicrographs of hematoxylin and eosin staining of the whole body and tissue sections are shown.

Fig. 8

Migration activity of neutrophils in zebrafish larvae exposed to MWCNTs. Zebrafish larvae were incubated with 5 and 50 ppm of MWCNT-S and MWCNT-L for 48 h, then transection of the tail tip was performed. Immediately and 8 h post-transection, myeloperoxidase histochemical staining of larvae was done. (A) Peroxidize-positive cell were quantified. Results are expressed as the percentage of neutrophil migration respect to control. Values are shown as mean ± SEM. Significant differences respect to control were analyzed by ONE-WAY ANOVA test followed by Dunnett's multiple comparisons post-test (**p < 0.01, ***p < 0.001). Representative photographs of controls (B) and MWCNT-incubated larvae are shown (C). Dash line (--) indicates transection site.