Enabling sub-lethal behavioral ecotoxicity biotests using microfluidic Lab-on-a-Chip technology

Dynamic behavioral parameters are increasingly postulated as alternatives to traditional mortality testing. Behavioral changes can precede mortality endpoints at substantially lower concentrations and are therefore considered as a useful indicator of sub-lethal effects. Despite recent acknowledgement of the importance of sub-lethal behavioral biotests, their implementation is profoundly limited by the lack of appropriate and user-friendly laboratory automation. This work outlines development of a proof-of-concept miniaturized Lab-on-a-Chip (LOC) platform for rapid aquatic toxicity tests based on changes in swimming patterns exhibited by a ubiquitous saltwater crustacean Artemia franciscana (Artoxkit M™) commonly used for marine ecotoxicity testing. In contrast to any conventionally performed end-point analysis based on counting numbers of dead/immobile specimens we performed a fully automated time-resolved video data analysis to dynamically assess the effect of a reference toxicant on selected behavioral parameters. Our proof-of-concept system combined innovative microfluidic device designed to keep free-swimming Artemia sp. nauplii under continuous microperfusion with a miniaturized video acquisition system for automated movement analysis of test specimens. For the majority of chemical stressors tested, behavioral changes were very rapid and observed often from the first few minutes of toxicant exposure. Furthermore, we identified distinct behavioral responses such as hyperactivity and hypoactivity syndromes in response to chemical stressors exposure. Both behavioral syndromes occurred in a concentration- and exposure time-dependent manner. This work provides evidence that sub-lethal, behavioral analysis performed under microperfusion can prospectively provide much more sensitive effect end-points as compared to conventional protocols where mortality is used as the main evaluation criterion. Innovative Lab-on-a-Chip technologies open a brand new alternative for inexpensive and rapid aquatic ecotoxicity protocols.