Developing and applying state of the art molecular technologies to discover mechanism of ZnO nanoparticle perturbation in Daphnia-algae chemical signal transfer system

In light of rapid industrial progress of the 21st century, the number of chemicals, including nanomaterials, used on a daily basis and introduced into the environment is constantly increasing. Far less chemicals, however, undergo appropriate risk assessment procedures, to ensure that they are not toxic or hazardous. Therefore, there is a necessity for new high-throughput techniques and tools in order to boost risk assessment. In this study it was attempted to develop a novel risk assessment paradigm, incorporating automated sample preparation for metabolomics toxicity testing, multi-trophic level ZnO nanoparticle (NP) toxicity assessment and an adverse outcome pathway (AOP) in Daphnia-algae information transfer system. In order to enhance metabolomics toxicity screening, several extraction solvent systems were tested (using direct infusion mass spectrometry (DIMS) and liquid chromatography (LC)-MS), ultimately selecting more robotics-compatible Bligh and Dyer method for further automation (even though modified Matyash method provided higher yield and reproducibility [1]). The low-mass tissue (Daphnia) extraction employing Biomek NXp platform was then automated (and assessed) to be integrated with existing high-throughput DIMS into the metabolomics pipeline. The optimised method was capable of a fully-automated extraction for polar and non-polar metabolites (24 Daphnia samples per batch), requiring 90 minutes, with no significant contamination or sample carry-over. Furthermore, multi-trophic toxicity was assessed by studying ZnO NP-induced changes in sulfonated lipids (SLs, participate as kairomones in signalling between Daphnia and algae, inducing defences) and an early molecular mechanism leading to those changes (via multi-omics approach – RNA-seq and LC-MS). It was shown that SLs decrease rapidly after exposure (at 0.3 h) and are not preceded by the changes in sulfur/sulfonation/glutathione metabolism (unlike hypothesized), while the mechanism likely involves activation of TNF/IL1B (tumor necrosis factor/interleukin 1 beta). Ultimately, a putative AOP featuring SL-mediated perturbation in Daphnia and algae (caused by ZnO NPs) was developed to aid the risk assessment of nanomaterials in aquatic food webs, however further research is required to fill in the existing knowledge gaps.