The Ascidian Embryo Teratogenicity assay in Ciona intestinalis as a new teratological screening to test the mixture effect of the co-exposure to ethanol and fluconazole

The aim of this work was to evaluate the Ascidian Embryo Teratogenicity assay (AET) as new alternative invertebrate model to test the developmental effects of the co-exposure to ethanol and fluconazole. Ciona intestinalis embryos were exposed to the azolic fungicide fluconazole, (FLUCO, 7.8–250 μM), to ethanol (Eth, 0.01–0.5%) and to their mixture (0.01% Eth + FLUCO 7.8–250 μM) from neurula to larval stage. At the end of the exposure period, larvae were morphologically evaluated and benchmark analysis performed by using the PROAST modelling software. Both compounds were teratogenic in a concentration-related manner, particularly affecting the pigmented organs. The co-exposure to Eth enhanced the effects of FLUCO, the additive hypothesis was not rejected by the modelling. The results demonstrated that AET could be considered a good vertebrate-free alternative model for toxicological investigation in embryos.