Periodic Reporting for period 1 - EXPOZOL (Exposure of aquatic ecosystems to antifungal azoles : assessment of occurence and fate in sediment, water and aquatic organisms) Berichtszeitraum: 2017-09-15 bis 2019-09-14 Zusammenfassung vom Kontext und den Gesamtzielen des Projekts Antifungal azoles englobe both the triazole and imidazoles chemical classes. They are widely used as agricultural fungicides or pharmaceuticals to treat mycoses, parasitic infections and cancer in humans. Initially designed to inhibit CYP51 enzymes, responsible for the biosynthesis of the ergosterol in fungi, they also disrupt a broad range of other CYPs involved in steroidogenesis (e.g. CYP19) and xenobiotic detoxification (e.g. CYP1A) in mammals and aquatic species. Thus, they have the capacity to act as endocrine disruptors and to affect survival, development, growth, reproduction and behaviour of non-target organisms. Although a few of these compounds are routinely investigated and detected, an accurate exposure assessment of most of them was still lacking to evaluate the associated environmental risk. The project aimed to fill the gap of knowledge regarding the occurrence, the fate and effect(s) of antifungal azoles in aquatic ecosystems in order to improve environmental risk assessment. First, we studied the distribution of antifungal azoles in various environmental matrices and biota using availability HRMS raw data. Second, we investigated the toxicokinetic (TK) of azoles (i.e. prochloraz, propiconazole, tebuconazole) and their metabolites and the associated effect(s) (TD) in invertebrates and fish cells. Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse Based on the literature and usage in both Switzerland and Germany, we selected 61 antifungal azoles and investigated them retrospectively by using digitally stored HR-MS/MS data from various projects at Eawag. These data were from 95 sites widely distributed on the Swiss plateau, including 150 surface water, 46 municipal effluent, 33 groundwater, 67 sediment, 15 soil, 24 biofilm, 3 leave, 41 gammarid and 49 fish samples. Overall, the results showed the wide occurrence of antifungal-azoles in all the investigated compartments at most sites. Among the investigated chemicals, 21 could be detected in at least 2 samples and were further evaluated.For this dataset we compared the actual concentrations to the predicted one by a fugacity model level I. The results highlighted that the partitioning differ among the azoles in accordance with their various physico-chemical properties. Discrepancies between the predicted and the actual concentration level may reflect an incorrect representation of biotransformation and/or persistence by the model. Based on quality standards (QS) available in the literature and those calculated in this study, we evaluated the risk (ΣRQ) associated with the mixtures of antifungal azoles in all the samples and their respective sites. Our results showed that the risk can differ depending on the environmental compartment and the corresponding QS value used to calculate the ΣRQ. Although the risk associated with antifungal azoles seem to be limited in Switzerland for both human and aquatic ecosystems, the fact that some sites appeared at risk raised the question of adverse impacts in countries where the use of these chemicals is higher or the dilution in the surface water lower.In the second part of the project we implemented TK models predicting uptake and elimination of chemicals over time and TD models predicting the development of effects over time as a function of the modeled or measured internal chemical concentrations. This was done in the benthic organism Chironomus riparius and in rainbow trout cell lines, as an alternative to whole fish experiment.We first investigated the TK of tebuconazole in chironomids through exposure to spiked sediment. Three tentative candidates were identified, including an oxidation product already referenced in the literature as TEB_M324a and two new metabolites. Then, a full TK experiment was implemented through exposure of chironomids to sediment spiked with tebuconazole at 200 ng/g with a 24h uptake and 96h depuration phase. The results showed very fast uptake and depuration of tebuconazole, as reported in the literature for another invertebrate. TD experiments showed a decrease of the survival probability with increasing exposure time and concentration as well as a decrease of the growth with increasing exposure time and concentration. The effect of the tebuconazole on the growth of chironomids raises the question of a potential common mechanism across species (e.g. CYP51) since some evidence recently showed the ability of some azole to reduce the growth of fish and proliferation of fish gill cells.As we did with chironomids, we investigated TK and TD of azoles in rainbow trout cell lines. From the biotransformation experiments, we proposed six tentative bioTPs for prochloraz whereas no one could be observed for both propiconazole and tebuconazole. Among them, three have been previously reported in invertebrates and are registred in MassBank (PRZ_M282, PRZ_M325 and PRZ_M352). Overall, our results were in accordance with those in gammarids where higher number of bioTPs were found with prochloraz than with the other azoles. In our case, no conjugation products were observed meaning that these enzymatic pathways might be non functional in these cell lines or that we lack sensitivity to detect them. A last experiment was performed in order to characterize accurately the TK of prochloraz and its metabolites in the RTL-W1 cells. The in vitro data collected will be used to implement a PBTK model in order to extrapolate biotransformation rate and growth rate at the whole organism level. The results described above were presented in various congress (SETAC Europe 2018, 2019, EUSSAT) and three publications were or will be written. Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts) The project brings new knowledge regarding exposure, bioaccumulation, biotransformation and effects of antifungal azoles. Distribution of azoles- The innovative retrospective analysis of digital archives of HRMS/MS data allowed a comprehensive spatial and temporal analysis of the exposure.- Antifungal azole are widely distributed into aquatic compartments including biota in accordance to their broad range of physico-chemical properties. - Of the 60 pre-selected azoles, 21 were detected (LOQ of 1 μg/L in water and LOQ of 50 ng/g in sediment or biota). - More pesticides were detected than pharmaceuticals. Also, pesticides were detected in all the compartments whereas pharmaceuticals were mainly found in WWTP effluents demonstrating their urban origin. - Risk quotient calculations revealed risk of exposure especially if some of the investigated rivers and streams are used for drinking water production.Bioaccumulation, biotransformation and effects of azoles- The detection of antifungal azoles in biota confirms the actual exposure of aquatic organism.- Chironomids share at least one common bioTPs of the tebuconazole with gammarids- Tebuconazole follows a fast uptake and elimination in chironomids, as reported for other antifungal azoles in this species - Tebuconazole led to an inhibition of the growth of the chironomids (IEC50 values of 7 ug/g d.w.) raising the question of the molecular mechanism(s) involved.- Prochloraz, propiconazole and tebuconazole can trigger cytotoxicity in rainbow trout cell lines. Prochoraz is the most cytotoxic- Antifungal azoles are biotransformed by rainbow trout cell lines. - Prochloraz is the most metabolized with six candidates transformation products identified, among those, three are shared with gammarids.