PROGRAMME

The release of emerging contaminants (such as pharmaceuticals) into aquatic ecosystems has become an issue of concern among scientists. Most of them as well as their transformation products (TPs) are frequently detected in the aquatic environment mainly due to insufficient removal by wastewater treatment plants (WWTPs) and may bioaccumulate in aquatic organisms causing adverse effects. Most pharmaceuticals belong to the group of ionizable organic compounds (IOCs), and their mode of action depends on the pH values of the surrounding exposure medium. IOCs are substances that can exist in an aqueous phase in both the ionic and/or neutral form. Neutral species permeate more easily through membranes, so the neutral form of a substance increases uptake and bioaccumulation and thereby toxicity. The ratio of neutral or ionic form is determined by pH value. Although slight variations in the pH can cause considerable changes in the uptake and the toxicity, until now, the pH-effect has not been rigorously considered in risk assessment. Propranolol (PRO) is an IOC, while it is the most common nonselective lipophilic β-blocker. Propranolol is a weak base and is utilized for cardiovascular disease treatments. Propranolol has been detected widely in effluents of WWTPs (due to inefficient removal), in rivers in low concentrations, and in aquatic organisms. Different aquatic organisms are reported to respond to the concentrations of propranolol in the range of ng L-1, such as daphnids, marine mussels, and zebrafish. Zebrafish (Danio rerio) embryo (ZFE) has been considered a well-characterized experimental model organism in the fields of molecular genetics and developmental biology. It is also a very promising model organism for toxicokinetic experiments since it poses similar biotransformation systems to mammals. The objectives of this study were to study the toxicity of the β-blocker Propranolol in zebrafish embryos, and to assess the potential pH-effect on the acute toxicity, uptake, and bioaccumulation of PRO. In addition, the identification of biotransformation products (bio-TPs) of PRO in ZFE was a major objective. Finally, we aimed to examine if biotransformation data could be used in a complementary way to the internal concentration (Cint) of the parent compound, for a holistic interpretation of toxicity at different pH values. For this purpose, the fish embryo toxicity test (FET) with ZFE was conducted according to the OECD 236 Guideline at three different pH values, and the LC50 values of PRO were determined. For the extraction of PRO in ZFE samples, organic solvents were added, and a bead-beating homogenization process using the Cryolis Evolution homogenizer (Bertin Technologies, France) was followed. The ZFE extracts were analyzed by RPLC and HILIC in both positive and negative ionization modes using LC-HRMS. A target-screening approach was followed for the identification of PRO, whereas identification of tentative bio-TPs was performed through in-house developed suspect and non-target screening workflows. Through this study, the toxicity of PRO was investigated. The internal concentrations (Cint) in ZFE were determined and the potential bioaccumulation of PRO was evaluated by determining the bioconcentration factors (BCFs). Moreover, different biotransformation products (bio-TPs) were detected in the ZFE extracts. The biotransformation of PRO by zebrafish embryos, as well as the contribution of bio-TPs to the concentration of the parent compound was extensively studied. Finally, a potential biotransformation pathway of PRO in zebrafish embryos was proposed.