Toxic effects of two pesticides, Imazalil and Triadimefon, on the early development of the ascidian Phallusia mammillata (Chordata, Ascidiacea)

Abstract

Azole compounds are fungicides used in agriculture and in clinical area and are suspected to produce craniofacial malformations in vertebrates. Toxicity tests on sperm viability, fertilization and embryogenesis of the solitary ascidian Phallusia mammillata were performed to evaluate the effects of two azole derivatives, Imazalil and Triadimefon. Ascidian (Chordata, Ascidiacea) embryos and larvae could provide biological criteria for seawater quality standards because the larvae are lecitotrophic and have a short pelagic period, allowing to run the larval toxicity tests over a short period of time. Imazalil and Triadimefon proved to have strong consequences on P. mammillata. They could influence the reproductive rate of the animal exerting their effects at different levels: acting as spermiotoxic agents, inhibiting fertilization and impairing embryological development. Fertilization rate significantly decreased after 30 min exposure of sperm to 25 μM Imazalil ( P < 0.0001) and after exposure of both gametes to 50 μM Imazalil ( P < 0.05) and 1 mM Triadimefon ( P < 0.0001) as compared to controls. Malformations caused by exposure of embryos to both substances were dose dependent. Imazalil median teratogenic concentration (TC 50 concentration, the concentration that resulted in 50% malformed larvae) value was 0.67 μM and median lethal concentration (LC 50, the concentration that resulted in 50% embryos dead before completing the development) value was 10.23 μM while for Triadimefon TC 50 value was 29.56 and LC 50 value was 173.7 μM. Larvae developed from embryos treated with Imazalil and Triadimefon showed alterations of the anterior structures of the trunk: papillary nerves and the anterior central nervous system failed to correctly differentiate, as showed by immunostaining with anti-β-tubulin antibody. Comparing the anomalies caused by retinoic acid, reported in a previous study, it was possible to hypothesize that malformations induced by Imazalil and Triadimefon could be due to a perturbation of the endogenous retinoid content, as it has been proposed for mammals. Ascidians proved to be good models to study the toxic effects of pesticides since they offered both the convenience of working with an invertebrate species and the tissue sensitivity to chemical compound comparable to vertebrates.