Effect-directed analysis for identification of river basin specific pollutants. Ph.D. Dissertation Zuzana Toušová Masaryk University, Faculty of Science, Centre RECETOX Brno, Czech Republic 2020 Supervisor: Dr. Ing. Jaroslav Slobodník 2 BIBLIOGRAPHIC ENTRY Author: Mgr. Zuzana Toušová Faculty of Science, Masaryk University Centre RECETOX Title of Dissertation: Effect-directed analysis for identification of river basin specific pollutants Degree Programme: Environmental Health Sciences Specialization: Environmental Chemistry and Toxicology Supervisor: Dr. Ing. Jaroslav Slobodník Supervisor specialist: prof. RNDr. Luděk Bláha, Ph.D. Year: 2020 Keywords: contaminants of emerging concern, effect directed analysis, in-vitro bioassays, prioritization, non- target screening. 3 BIBLIOGRAFICKÝ ZÁZNAM Autor: Mgr. Zuzana Toušová Přírodovědecká fakulta, Masarykova univerzita Centrum RECETOX Název práce: Užití efektem řízené analýzy pro identifikaci specifických znečišťujících látek v povodích Studijní program: Životní prostředí a zdraví Specializace: Environmentální chemie a toxikologie Školitel: Dr. Ing. Jaroslav Slobodník Školitel specialista: prof. RNDr. Luděk Bláha, Ph.D. Rok: 2020 Klíčová slova: nově se objevující znečišťující látky, efektem řízená analýza, biotesty in-vitro, prioritizace, necílená analýza. 4 © Zuzana Toušová, Masaryk University, 2020 5 ACKNOWLEDGEMENTS I would like to thank my supervisor Dr.. Jaroslav Slobodník for his great guidance, encouragement, support and patience during my postgraduate study. My deep gratitude also goes to prof. RNDr. Luděk Bláha, Ph.D., who carefully co- supervised my work, inspired me, provided me with great scientific training and contributed to the direction and richness of this research. Prof. Bláha was always ready to give advice and discuss immediate problems, which helped me greatly to progress smoothly with both laboratory work and writing papers. I am also very grateful to doc. Mgr. Klára Hilscherová, PhD., who largely contributed to the study design and results’ interpretation in publications II and III. Doc. Hilscherová kindly shared her rich practical experience with me and devoted a lot of her time to help me finalize the publications. My thanks also go to all co-authors of our common publications, for their great work on the research and writing papers. Namely, I would like to thank Dr. Tobias Schulze and doc. Ing. Branislav Vrana, PhD. for their great work on the sampling part of this research. I gratefully acknowledge Dr. Peter Oswald and Dr. Natalia Glowacka., colleagues from the EI, who contributed on the analytical part and helped me with lots of practical issues. I extend my thanks to all members of the EDA EMERGE Project for all those inspirational discussions, training courses, hospitality and fun. My thanks go to all RECETOX colleagues for making such a friendly environment at the workplace and being helpful at all times. I express my deep gratitude to all my family. I would like to thank my parents and grandparents for their huge support throughout my studies. Great thanks go to my husband for his encouragement, support, patience and time with the kids. I am very grateful to my mother and especially to my late mother- in-law for being such good grandmas and all the baby-sitting, without which this thesis would never had taken shape. The PhD training programme was funded by the European Union under the Marie Curie Actions—Initial Training Networks, FP7-PEOPLE-2011-ITN, EDA-EMERGE project, Grant Agreement No. 290100. 6 ABSTRACT Pollution of surface waters with contaminants of emerging concern (CECs) has become a pressing environmental issue of global importance. CECs is a group of organic compounds of diverse structure, physicochemical properties and usage patterns comprising pesticides, pharmaceuticals, personal care products, flame retardants and others. CECs can be found in surface and waste waters, they are not monitored or regulated and raise concerns of the research community and public due to their extensive occurrence and possible adverse effects on biota, which are mostly unknown. Innovative approaches to monitor surface waters, with regard to CECs, have been proposed. These include novel sampling techniques, effect assessment with sensitive bioassays, non-target chemical screening and use of advanced software tools for data interpretation. Further, a powerful technique to identify bioactive compounds in complex environmental mixtures, known as effect-directed analysis (EDA), is based on the combination of bioassays, fractionation and chemical analysis. The objective of this thesis was to contribute to the identification of relevant toxicity drivers in surface waters by advancing the EDA utilization. The specific objectives, addressed by particular studies, were to apply a newly developed simplified EDA protocol (study I and IV), identify compounds responsible for algal growth and acetylcholine esterase inhibition in a WWTP effluent extract (study II and V) and characterize contamination of the Bosna River with CECs (study III). Novel sampling device, based on solid phase extraction of large water volumes (LVSPE), was tested and optimized in a sampling campaign spanning across four European river basins (Studies I and IV). In the effect assessment of extracts, the most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity, whereas the major portions of the observed effects could not be explained by analyzed compounds. Simplified risk assessment procedure with analyzed compounds enabled identification and prioritization of 21 candidate compounds for future monitoring efforts. Effect directed analysis was applied to identify compounds contained in a wastewater treatment plant (WWTP) effluent extract that were responsible for growth inhibition of green algae (Study II) and acetylcholinesterase inhibition in vitro (Study V). Our results suggest that pesticides and their transformation products, pharmaceuticals (barbiturate derivatives and macrolide antibiotics e.g. azithromycin), industrial compounds or caffeine and its metabolites were the most likely toxicity drivers for green algae in study II. 7 A combined chemical and effect screening of water quality in the River Bosna was carried out, to characterize, in some detail, its pollution with CECs (Study III). The assessment of cumulative pollutant concentrations and hazard profiles enabled to identify the major source of contamination with CECs and hotspots of biological potency. Simplified risk assessment procedure of detected target compounds suggested that 7 compounds, namely diazinon, diclofenac, 17-β estradiol, estrone, benzo[k]fluoranthene, fluoranthene and benzo[k]fluoranthene, might pose serious risks to aquatic biota in the Bosna River. The presented dissertation demonstrates that combination of novel sampling techniques with effect-based methods and chemical screening is a functional strategy to address CECs in surface waters. This thesis shows that EDA is a useful tool to reduce the complexity of environmental mixtures and to identify relevant toxicity drivers. Risk assessment and prioritization of detected chemicals is a crucial step in the process of identifying river basin specific pollutants. 8 ABSTRAKT Znečištění povrchových vod nově se objevujícími znečišťujícími látkami (Contaminants of emerging concern - CECs) se stalo vážným environmentálním problémem celosvětového rozsahu. CECs jsou velmi rozmanitou skupinou organických látek, co se týče struktury, fyzikálně chemických vlastností a oblastí jejich využití. Mezi CECs řadíme například pesticidy, farmaka, výrobky pro osobní hygienu, zpomalovače hoření a další. CECs se vyskytují v odpadních a povrchových vodách, přičemž nejsou pravidelně monitorovány ani nijak regulovány. Díky častému výskytu a možným nežádoucím účinkům na živé organismy vyvolává skupina CECs znepokojení veřejnosti a velkou pozornost vědecké komunity. Nově navrhované přístupy k monitoringu povrchových vod s ohledem na CECs proto zahrnují speciální vzorkovací techniky, sledování biologických účinků citlivými biotesty, necílený chemický screening a užití pokročilých softwarových nástrojů k interpretaci získaných dat. Dále také efektem řízená analýza (EDA – effect directed analysis), založená na kombinaci biotestů, frakcionace, a chemické analýzy, je účinnou metodou k identifikaci látek zodpovědných za pozorované biologické účinky v komplexních environmentálních směsích. Cílem této disertace bylo přispět k identifikaci látek zodpovědných za toxické účinky sledované v povrchových vodách užitím a dalším vývojem metody EDA. Dílčími cíli pak byly i) využití zjednodušeného protokolu pro efektem řízenou analýzu (studie I a IV); ii) identifikace inhibitorů růstu zelených řas a acetylcholinesterázy v extraktu přečištěné odpadní vody (studie II a V) a iii) charakterizace znečištění řeky Bosny nově se objevujícími znečišťujícími látkami V rámci studie I a IV proběhla vzorkovací kampaň ve čtyřech povodích Evropských řek, přičemž bylo testováno a optimalizováno nové vzorkovací zařízení založené na extrakci velkého objemu vody na pevné fázi. Nejčastěji sledovaným biologickým efektem byla estrogenita, dále toxicita pro zelené řasy a rybí embrya. Převážnou část těchto efektů nebylo možné vysvětlit výskytem cílových analytů. Na základě zjednodušené analýzy rizik cílových analytů bylo identifikováno a prioritizováno 21 látek zajímavých pro budoucí monitoring povrchových vod. K identifikací látek způsobujících toxicitu pro zelené řasy (Studie II) a inhibici acetylcholinesterázy