Lifestyle and dietary habits are currently mainly assessed using traditional epidemiological methods, such as surveys and sales statistics. While these methods are useful, they have many limitations due to their subjective nature and/or incomplete data. Wastewater-based epidemiology (WBE), sometimes referred to as sewage chemical information mining, is a relatively new, objective, cost-effective and timely approach to obtaining some relevant epidemiologic information at the population level. It is based on the chemical analysis of specific human urinary biomarkers in wastewater. WBE has been successfully applied as a complementary method to assess collective illicit drug use, but municipal wastewater is a potential repository of numerous other chemicals that may reflect population lifestyle, exposure to contaminants, and health status. However, the potential of WBE to assess dietary habits has hardly been investigated and only a few studies can be found in the literature. The aim of this project is to investigate lifestyle and dietary habits in Croatian regions using an innovative WBE approach. The study will include biomarkers of the consumption of alcoholic beverages, tobacco/nicotine, several classes of commonly abused pharmaceuticals, and various biomarkers related to dietary habits. Selected compounds will be analyzed by liquid chromatography ‒ tandem mass spectrometry (LC-MS/MS). Due to the relatively large number of biomarkers with very different physico-chemical properties and concentrations in wastewater, several highly specific analytical methods will be developed and validated. In addition, the stability of the target substances will be evaluated both in the sample and in the sewer system. The applicability of the concept will be tested by analyzing raw municipal wastewater from at least six Croatian cities during different seasons to investigate spatial and temporal differences in lifestyle and diet. The results of the wastewater analysis will be compared with data obtained using conventional epidemiological methods.

The problem of environmental contamination with pharmaceutical chemicals has been widely recognized over the past 20 years. The current literature is strongly dominated by studies focused on assessing the exposure concentrations in abiotic matrices (e.g. water and sediments) while reports on the pharmaceutical concentrations in aquatic organisms remain comparatively scarce. However, when considering the possible biological effects, the aqueous exposure concentrations are only an indirect measure of potential toxicity, while the key parameter which determines the substance's potential for adverse effects is its internal concentration in the organism. Consequently, this project aims to provide new insights into the bioaccumulation and metabolic behaviour of pharmaceuticals in freshwater organisms, which is essential for an improved risk assessment of these contaminants as well as for their scientifically-sound prioritization. The project plan includes several interconnected phases, from method development and laboratory-controlled exposure experiments to field studies. Highly specific analytical methods for quantitative multiresidue determination of trace levels of selected pharmaceutical compounds in different environmental matrices (biota, water, sediment) will be developed and validated. These methods will be applied in carefully planned models and field experiments focusing on the characterization of the pharmaceutical exposome in freshwater fish. The model bioaccumulation experiments will be performed using zebrafish (Danio rerio) as the target organism while considering different exposure scenarios. The biochemical responses in fish will be followed by measuring the induction of the major cytochrome P450 detoxification enzymes. As its final step, the project includes a ground-truthing study in the Sava River to assess the pharmaceutical exposome's environmental relevance and collect the critical data for prioritization of individual pharmaceutical contaminants.

Aquatic ecosystems throughout the world are exposed to an increasing pressure from various anthropogenic sources that release large variety of potentially harmful synthetic organic contaminants, including pharmaceutically active compounds. Most of the environmental studies of pharmaceuticals are focused mainly on the determination of parent compounds and encompass only one environmental compartment. Such simplified approach often underestimates the ecotoxicological relevance of a contaminant class, either by neglecting the contribution of its transformation products or by overlooking some critical mechanisms that may pose a threat to the environmental safety and human health. As opposed to that, this project aims at demonstrating the importance of the comprehensive assessment of aquatic contaminants, based on the study of two important groups of pharmaceuticals, macrolide antibiotics (MAs) and opioid analgesics (OAs), by including their main transformation products (TPs) and addressing the issue of physico-chemical partitioning in the exposure assessment. 

The ECOFUN-MICROBIODIV project aimed at developing and evaluating innovative tools to estimate the ecotoxicological impact of low dose pesticides used in agriculture on soil functional microbial biodiversity. It proposed to estimate the impact of sulfonylurea herbicide on soil microbial biodiversity and functioning. This project is based on an interdisciplinary approach conducted at different scale, from the field plot to the greenhouse, to test in corn crop the impact of chemical weeding compared to mechanical.