PROGRAMME

The contamination of the aquatic environment has raised concerns in the scientific community and regulatory authorities, over the last decades. Given the large number of xenobiotics (such as pharmaceuticals), for most of them there is a striking deficit in the literature concerning their adverse effects on aquatic organisms. Tetracaine (TCN) belongs to the category of ionizable organic compounds (IOCs). Therefore, its behavior such us the uptake, bioaccumulation and toxicity can be significantly altered by shifts in the ambient pH values. Zebrafish (Danio rerio) has emerged as a powerful model organism to study various aspects of developmental and cell biology as well as physiology, while it provides a promising alternative model for acute toxicological studies. The fish embryo test (FET) with zebrafish is a well-established and standardized (OECD Guideline 236) in vivo toxicity test that is commonly used to evaluate potential adverse effects on early development of fish. Thus, it is a helpful tool for risk assessment in aquatic environments. However, until now, neither pH nor the particular properties of ionizable organic compounds (IOC), like TCN, have sufficiently been considered in risk assessment. Characteristics of IOC vary, depending on their presence either as ions or as neutral species, in particular in respect to the uptake into organisms. Due to their electrical charge, ions pass poorly through biological membranes, whereas neutral species permeate more easily through membranes and are, thus, potentially of higher toxicity. The pH is one major factor influencing the proportion of dissociated and non-dissociated ions. Shifts in ambient pH cause alterations of the ionic proportions and thus, are key to IOC toxicity. Although many IOC are partly or completely ionized under environmental relevant conditions and already slight variances of pH can cause considerable changes in toxicity, little attention has been paid to pH and its consequences in toxicity testing. The objectives of this study were (1) to assess to what extent TCN induces toxicity to zebrafish embryos. In addition (2), the uptake and biotransformation processes of TCN by zebrafish were evaluated. The following hypothesis was examined; whether biotransformation data could be used complementarily to the concentration of the parent compound to interpret the induced toxicity.. The final goal (3) was to evaluate to which extent the pH is influencing TCN’s uptake, potential bioaccumulation and biotransformation, as well as toxicity. More specifically, the zebrafish embryo toxicity assay was used to calculate the LC50 of TCN, as well as to evaluate potential sub-lethal endpoints (e.g. hatching rate). Exposure experiments were conducted at three different pH values (5, 6 and 8), to assess potential pH-dependent differences in an environmental relevant pH range. Concerning the toxicokinetic part of the study, the extraction was carried out with the tissue homogenizer Cryolis Evolution® (Bertin Technologies, France) operating at 8200 rpm for 5 cycles of 15 sec with a 60 s break at 4ºC. Two different organic solvent mixtures (methanol-water and methanol-dichloromethane) were used for the extraction of zebrafish embryos, in order to cover a very wide range of physicochemical properties. Exposure water samples and zebrafish extracts were analysed by RPLC and HILIC methods, in both positive and negative ionization mode, to cover the widest possible range of polarities, using LC-QTOF HRMS. Internal concentration of parent TCN and its bio-TPs was determined. Potential pH dependent differences of TCN’s uptake and biotransformation were evaluated, as well. Detection and identification of tentative TCN biotransformation products were performed through in-house developed suspect and non-target screening workflows. Finally, the biotransformation pathway of TCN in zebrafish embryos was proposed.