Innovative lab-on-a-chip technologies for real-time analysis of sub-lethal endpoints in aquatic macroinvertebrates

Currently there is a profound lack of automated and sub-lethal toxicity testing within the field of ecotoxicology. In order to meet future needs regarding high-throughput toxicity testing and rapid analysis of novel chemical compounds, there is a pressing requirement to adopt new methodologies. Manual procedures for conducting traditional mortality-based endpoints are slow, labour-intensive, low resolution and prone to unintentional bias or error. In this thesis, I present for the first time, new proof-of-concept methodologies for caging aquatic invertebrates as well as a novel approach to generating ultra-thick SU-8 photoresist layers for generating high-aspect ratio features in a soft-lithography mould.<br><br>These Lab-on-a-Chip (LOC) systems involved designing novel caging environments that could effectively hold the test organisms within a central toxicity chamber for the purpose of testing traditional mortality-based endpoints as well as sub-lethal behavioural assays. In order to achieve this, the caging environments were specifically designed with the intention of keeping the test organisms within a shallow x,y arena such that their movements would be easily recorded with a miniature USB camera. Additionally, each system incorporated a perfusion-based environment specifically calibrated for the optimal survival of the test organisms. With the assistance of automated tracking algorithms, this thesis illustrates how LOC perfusion-based systems can be effectively leveraged to track aquatic invertebrates and quantify sub-lethal changes as a surrogate for traditional testing methods.<br><br>The first chapter explores an innovative LOC system for caging the marine amphipod Allorchests compressa. The research examines various aspects of the system that are critical for optimal survival of A. compressa within a chip-based environment including chamber design and flowrate. Subsequently, comparative analysis between mortality data in traditional glass jar environments and the newly posited chip-based environment were performed. While metal exposures produced excellent agreement between test environments, there was a strong divergence between results when testing with petroleum hydrocarbons. Chemical analysis later revealed that the reduction in observed toxicity was due to sorption of the test hydrocarbons out of solution.<br><br>Behavioural analysis of A. compressa in the chip-based environment was very successful with minor perturbations in swimming patterns becoming evident at concentrations an order of magnitude lower than the known LC10 values for copper. Such sensitivity in detection was only made possible by the custom enclosures designed to optimise video recording of A. compressa’s movements and resulting behavioural analysis.<br>A similar approach was conducted with the freshwater rotifer Brachionus calyciflorus by caging it within a perfusion-based LOC environment. As with A. compressa, the test environment was specifically tuned to suit the test organism. Mortality testing identified excellent agreement between results in conventional multi-well plate environments when compared with the custom designed perfusion-based chip device. Moreover, behavioural analysis of swimming patterns again proved a significantly more sensitive endpoint than LC10 values for many of the toxicants tested.<br><br>This thesis also compares existing soft lithography based methods for creating high aspect ratio structures suitable for caging the smallest aquatic invertebrates as well as presenting an innovative, yet intuitive, approach for fabricating them in traditional cleanroom facilities. The newly described method is capable of rapidly and reliably creating photoresist layers between 100 and 1500 μm thick with almost no photoresist waste. This novel methodology enables a new suite of structures to be fabricated specifically to suit ecotoxicological applications for trapping and caging aquatic invertebrates within an LOC environment.<br>