Removal of micropollutants by fungal laccases in model solution and municipal wastewater: evaluation of estrogenic activity and ecotoxicity

This study describes a multidisciplinary approach that investigates the breakdown potential of a laccase mediated system from Trametes pubescens MUT 2400 against several micropollutants including already recognized endocrine disrupting chemicals at their natural residual concentrations (from μg/L up to ng/L). In model solution, the chemical speciation focused on a mixture of 18 analytes and adopted stir bar sorptive extraction with directed in-situ derivatization followed by gas chromatography and mass spectroscopy analysis. The method's key performance parameters were evaluated in consideration of the chemical peculiarities and complexity of real wastewaters: precision, accuracy, estimated working range extended to a wide residual concentration interval (10 ng/L to100 μg/L) indicated its fitness for purpose. Laccases were extremely active towards all the target compounds, both in term of removal yields and rate. The maximal percentage of removal was obtained for 4-t-butylphenol, 2-hydroxybiphenyl, 4-n-octylphenol, salicylic acid and estrone (percentage of removal above 90%). Enzymes concentration played a central role and in most of the cases, the catalyzed reactions were very fast: the initial concentration of 9 compounds was halved within the first 3 h. The laccase-mediated treatment was then applied to a municipal wastewater collected in a real wastewater treatment plant, containing at least 9 xenobiotics as drugs, pesticides, plasticizers and personal care products. Although the harsh chemical and biological conditions of the effluent influenced enzyme stability, the reaction took place, and above 70% transformation was obtained for most analytes during the 24 h experiment. Bioassays were carried out to estimate the estrogenic activity (the E-screen test and the MELN gene-reporter luciferase assay) and the ecotoxicity (Lepidium sativum, Pseudokirchneriella subcapitata and Vibrio fischeri), demonstrating the capability of laccases to mediate an effective detoxification of the wastewater and a decrease of the estrogenic activity.