International amphibian micronucleus standardized procedure (ISO 21427-1) for in vivo evaluation of double-walled carbon nanotubes toxicity and genotoxicity in water.

Considering the important production of carbon nanotubes (CNTs), it is likely that some of them will contaminate the environment during each step of their life cycle. Nevertheless, there is little known about their potential ecotoxicity. Consequently, the impact of CNTs on the environment must be taken into consideration. This work evaluates the potential impact of well characterized double-walled carbon nanotubes (DWNTs) in the amphibian larvae Xenopus laevis under normalized laboratory conditions according to the International Standard micronucleus assay ISO 21427-1:2006 for 12 days of half-static exposure to 0.1-1-10 and 50 mg L(-1) of DWNTs in water. Two different endpoints were carried out: (i) toxicity (mortality and growth of larvae) and (ii) genotoxicity (induction of micronucleated erythrocytes). Moreover, intestine of larvae were analyzed using Raman spectroscopy. The DWNTs synthetized by catalytic chemical vapor deposition (CCVD) were used as produce (experiment I) and the addition of Gum Arabic (GA) was investigated to improve the stability of the aqueous suspensions (experiment II). The results show growth inhibition in larvae exposed to 10 and 50 mg L(-1) of DWNTs with or without GA. No genotoxicity was evidenced in erythrocytes of larvae exposed to DWNTs, except to 1 mg L(-1) of DWNTs with GA suggesting its potential effect in association with DWNTs at the first nonacutely toxic concentration. The Raman analysis confirmed the presence of DWNTs into the lumen of intestine but not in intestinal tissues and cells, nor in the circulating blood of exposed larvae.