Environmental risk assessment approach based on ecotoxicological tools applied in industrialized and agricultural areas

Environmental pollution is one of the most serious problems facing humanity and other life forms on our planet today. The characterization of chemicals spread in the environment, through the standard procedure of Environmental Risks Assessment (ERA) still evaluates the risk substance-by-substance. However, it is known that in the environment we are exposed to a mixture of chemicals coming from several different sources. In order to overcome such limitations of current ERA approach, more recently ERA highly recommended the usage of bioanalytical tools which is still emerging and not established in each European Member State. Within this thesis the suitability of bioanalytical tools in the ERA of industrial and agricultural areas, where mixtures of contaminants are present, has been demonstrated. In particular, the suitability of the in vitro cellular bioassay DR CALUX® (Dioxin Responsive, Chemically Activated LUciferase gene eXpression) has been evaluated. DR CALUX® were able to detect the biological toxicity of topsoils collected from Taranto municipality (Apulia Region). Through a combined approach of bioassay and chemical analysis, the occurrence and spatial distribution of polyhalogenated dioxins, furans and dl-PCBs in topsoil samples were assessed. This approach not only confirmed already known sources (e.g. industrial air emissions) but also new ones (e.g. illegal disposal of electric transformers containing Apirolio® and asbestos cement-fiber plates). Considering the extension of illegal landfills in the Taranto municipality area, it is an urgent need to investigate potential sources and impact of dioxin-like compounds. Here we evaluated the suitability of DR CALUX® to support their application in future monitoring surveys. The risk represented by the extensive use of soils for agricultural and food purposes in Taranto area, further underlines the need to apply monitoring tools that quickly and extensively highlight the hazard posed by dioxin-like compounds in terms of human and environmental health. The high costs associated to extensive monitoring surveys find in the application of bioassays a successful alternative, able to identify new and unexpected sources and their route of exposure that would require long times and huge investments if only based on analytical chemical analysis. The possibility to couple proteomics to the DR CALUX® cells bioassay was also investigated in the present thesis. A case study was thus designed and tested by using selected topsoil samples to extrapolate differentially abundant proteins by proteomics and thus monitor other chemicals including those of emerging concerns (CECs) present in the soil sample able to activate specific cellular pathways. Moreover, differently abundant proteins spots found will subjected to functional and pathway analysis, in order to highlight potentially altered molecular pathways and suggesting potential link to human pathologies. The differential proteins identified were related to an altered unfolded protein response (UPR) inducing Endoplasmic Reticulum (ER) stress, oxidative stress, and altered signal transduction, transforming growth factor beta (TGF-β) signaling alteration. All these mechanisms are reported to be related to carcinogenic mechanisms. The combined approach leads to estimate ecological and human risk by legacy and CECs as pharmaceuticals. The suitability of combining bioanalytical tools with chemical analyses in order to evaluate potential risks for both humans and environment, was demonstrated also in sewage sludge (SL) and hydrochar (HC) samples. A case study was designed to test urban SL and relative HC from six wastewater treatment plants (WWTPs), located in the Tuscany territory. DR CALUX® bioassay was able to detect the presence of dioxins and dioxin-like compounds in both SL and HC underlying differences probably related to hydrothermal carbonization process (HTC). Chemical analysis show that highly chlorinated congeners prevailed in SL compared to HC samples, in agreement with previous studies. HTC process seems responsible for such changes in the level of chlorinated congeners from high to low ones, which are more toxic. It can be assumed that the contamination of the HC is mainly due to a previous contamination of SL feedstock. The increase in the biological toxicity observed by DR CALUX® can be related to the de-chlorination of dioxins and dioxin-like compounds and the high loss of water which might affect their levels. DR CALUX® bioassay was able to screen the presence of dioxins and dioxin-like compounds in SL and HC, raising concern due to their possible applications as soil amendments. In the light of the above findings, it is possible to state that the DR CALUX® bioassay has been proven to be an effective and sensitive tool able to screen the presence of dioxins and dioxin-like compounds in various environmental matrices such as topsoil, sludge and hydrochar. Moreover, it has been proven to be an integrated state-of-the-art tool capable of assessing the real toxicity of complex mixtures of contaminants that occur in environmental samples. The combined application of DR CALUX® bioassay with chemical analysis and proteomics, opens new paths to the future of the Environmental Risk Assessment.