Effects of nanoparticle treatments on the development of the water frog, Pelophylax sinkl esculentus.

The increasing use of nanoparticles (NP) in industrial and medical applications renders necessary to understand how they affect both biological and environmental systems. Therefore, we tested the effects of three NP (cobalt, nickel, iron) on developing embryos of the water frog Pelophylax sinkl. esculentus. This frog is commonly found in Italian wetlands and is regarded as a good biomarker. Samples came from an artificial tank in the University Botanic Garden in Bari. Embryos at the developmental stage 10 (earliest involution of blastopore dorsal lip) were treated with iron, nickel or cobalt NP from IoLiTec (Heilbronn, Germany). A control group and three treatments per NP at increasing concentrations (LC50/2, LC50, and 2xLC50, according to literature) were considered, for a total of ten groups. Each group included about 20 individuals. Groups were monitored for the following ten days. A low mortality was observed and it was similar throughout the groups. Total length (LT) and eye diameter (LO) were significantly higher in the control (mean LT in control = 7.8 mm, mean LT in treatments = 6.3 mm; mean LO in control = 0.3 mm, mean LO in treatments = 0.2mm). Significant differences in proportions were observed between controls and treatments in developmental stages (30% of controls reached stage 23 and 66% stage 21, lower values were observed in treatments). Malformations were observed in about 30% of controls, whereas in treatments they reached 60%. The most common malformations observed were abnormally large ventral mass and bent body axe, followed by abnormal development of the head and eye malformations. Investigations of integument at light, SEM and TEM microscopy revealed that in the epidermis of the treatments muciparous cells were hypertrophic, ciliated cells showed a higher number of cilia in respect to controls and ionocytes presented several swelled mitochondria, indicating a stressed condition. Besides, treatments were infested by the chytrid fungus Batrachochytrium dendrobatidis, as indicated by the several zoospores and zoosporangia observed in the epidermis. It is concluded that NP treatments are responsible of reduced growth, developmental delay and malformations, increase of secretion and oxidative stress in the cells of the integument, as well as reduced resistance to fungal infections.