Endosulfan acute toxicity in Bufo bufo gills: ultrastructural changes and nitric oxide synthase localization

Endosulfan is an organochlorine pesticide used in agriculture for a wide range of crops. Endosulfan concentrations of up to 0.7 mg/L can be found in ponds and streams near sprayed agricultural fields. We investigated the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles after 24, 48, and 96 h of exposure. Acute toxicity was evaluated at nominal concentrations ranging from 0.01 to 0.6 mg/L: concentrations that could be found after the application of pesticide. Our results show that 0.43 mg/L of endosulfan caused 50% mortality (LC(50)). The effects of a sublethal endosulfan concentration (0.2mg/L) on gill apparatus morphology were evaluated by scanning and transmission electron microscopy. Immunohistochemical methods were also applied to detect the expression pattern of the inducible isoform of nitric oxide synthase (iNOS) in the gills using the confocal laser scanner microscope. Exposure to 0.2mg/L of endosulfan caused an apparent increase in mucus production, the occurrence of secretory vesicles and lamellar bodies, a widening of intercellular spaces and additionally there was evidence of an inflammatory response in the gill apparatus. The morphological alterations occurred after 24h and were more pronounced after 48 and 96 h of exposure. Altered morphology and increased mucus secretion indicate impaired gas exchange and osmoregulation in the gills. In addition, there was an increase of iNOS expression after 24 and 48 h which may reflect hypoxia and inflammation in the gill epithelium. Our results clearly indicate that short-term exposure to a sublethal concentration of endosulfan, near the high end of the environmental range, disrupts gill morphology and function in B. bufo tadpoles.