PROGRAMME

Over the recent decades, there is great interest from the scientific community in emerging contaminants, such as pharmaceuticals. Previous studies have demonstrated that they are ubiquitous in aquatic ecosystems, mainly due to insufficient removal by wastewater treatment plants (WWTPs). In light of their potential risk to the aquatic environment and organisms, immediate action is required. According to a recent study, many pharmaceuticals belong to the group of ionizable organic compounds (IOC) and they can change their partition coefficient depending on the pH of the surrounding medium. Ionizable organic compounds (IOCs), such as Fluoxetine, are substances that can exist in an aqueous phase in both the ionic and/or neutral form. The ambient pH value can influence this proportion. Although slight variation in the pH can cause considerable changes in the uptake and the toxicity, until now, the pH-effect has not rigorously been considered in risk assessment. The pharmaceutical Fluoxetine (FLX), is a selective serotonin reuptake inhibitor (SSRI). It is one of the most widely prescribed antidepressants and it has been recognized as one of the most toxic human pharmaceuticals in the aquatic environment. Therefore, it is necessary to assess its potentially toxic effects on aquatic organisms. Zebrafish (Danio rerio) embryo (ZFE) has been considered as 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. Hence, it is extensively used as model organism in ecotoxicology research studies, in order to investigate the toxic potency of drugs and chemicals. The aim of this study was to assess the influence of different pH values of the test medium on the acute toxicity, bioaccumulation, and biotransformation of the antidepressant FLX in zebrafish embryos. Furthermore, it was examined if biotransformation data could be used in a complementary way to the internal concentration 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 Guidelines at three different pH values. The toxicity assay was used for the calculation of internal concentrations and LC50 values of FLX. Regarding the sample preparation, the extraction and homogenization were performed by bead-beating technology, using the Cryolis Evolution homogenizer (Bertin Technologies, France). The water samples from exposure experiments and the ZFE extracts were analyzed by RPLC and HILIC in both positive and negative ionization mode using LC-ESI-QTOF-MS. Regarding the identification, target screening approach was followed for the identification of the parent compound and the biotransformation products were identified through an in-house developed suspect and non-target screening workflows. Moreover, the internal concentrations (Cint) in ZFE were determined and the potential bioaccumulation of FLX was evaluated by determining the bioconcentration factors (BCFs). Different biotransformation products (bio-TPs) were detected in the ZFE extracts. The biotransformation of FLX 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 FLX in zebrafish embryos was proposed.