PROGRAMME

Recently, the release of emerging contaminants into the aquatic ecosystems has become an issue of concern among scientists since some of them are frequently detected in the environment at levels that may cause harm. One of the main categories of emerging contaminants is the human and veterinary pharmaceuticals. Metoprolol (MET) is a cardioselective b-blocker widely used in the treatment of various cardiovascular disorders. It has been frequently reported in aquatic environments with mean concentrations ranging from ng L-1 to μg L-1 range, mainly due to its inefficient elimination by the conventional wastewater treatment plants (WWTPs). As a result, MET and its metabolites may bioaccumulate in aquatic organisms causing adverse effects. Thus, it is urgent to evaluate its potential toxic effects in the aquatic forms. Furthermore, MET is an ionizable organic compound (IOC). IOCs are substances that can exist in an aqueous phase both in ionic and/or neutral form. The chemical speciation, neutral or ionized form, of the IOCs (such as MET) depends on the pH values of the ambient medium. Neutral species, which are less polar than their ionized counterparts, permeate more easily through membranes and thus may be more toxic for the organisms. Therefore, slight shifts in the ambient pH values can cause considerable changes in the uptake and thus in the toxicity of the IOCs. However, the influence of pH in the toxicity and the bioaccumulation of MET has not been extensively investigated so far. For environmental toxicity tests many organisms, such as phytoplankton, zooplankton, invertebrates, and fish are used. Zebrafish (Danio rerio) is a powerful model organism, which is widely used in ecotoxicology research studies for assessing the potential effects of xenobiotics on aquatic organisms due to the numerous beneficial traits over other model organisms. Its main advantages are its rapid development, the short generation time and its similar biotransformation systems to mammals. The objective of the current study was to evaluate the extent of the influence of different pH values on the uptake, bioaccumulation, biotransformation as well as the toxicity of the b-blocker MET in Zebrafish Embryos (ZFE). Another goal was to determine the potential biotransformation products (bio-TPs) of MET and to assess whether the bio-TPs could contribute to the toxicity of the parent compound. For this purpose, the fish embryo toxicity test (FET) with ZFE was conducted according to the OECD 236 Guidelines at different pH values. The LC50 values of MET at 3 different environmentally relevant pH values (6, 8, and 9) were determined. Subsequently, these LC50 values were used for the toxicokinetic experiments of MET. The extraction of the MET in ZFE samples was performed with the Cryolis Evolution homogenizer (Bertin Technologies, France) operating at 8200 rpm at 4 ˚C (3 cycles of 15 s with a 60 s break between each cycle). Exposure water samples and ZFE extracts were analyzed by RPLC and HILIC in both positive and negative ionization mode using LC-ESI-QTOF-MS. A target screening approach was followed for the identification of the parent compound (MET), whereas the detection and identification of tentative bio-TPs were performed through in-house developed suspect and non-target screening workflows. The internal concentrations (Cint) of MET in ZFE extracts were determined. The bioaccumulation of MET was evaluated and the bioconcentration factors (BCF) of MET were calculated. Finally, the biotransformation of MET in ZFE was studied and a potential biotransformation pathway was proposed.