Caging of planktonic rotifers in microfluidic environment for sub-lethal aquatic toxicity tests.

Quantification of neuro-behavioural responses of intact small model organisms has been proposed as a sensitive, sub-lethal alternative to conventional toxicity testing. Such bioassays are characterized by a high physiological and ecological relevance, short response times, increased sensitivity, and non-invasive nature. Despite a significant potential for predictive aquatic toxicology analysis of behavioural traits of micro-invertebrates in microfluidic environment has received little attention. In this work, we demonstrate a new Lab-on-a-Chip technology capable of effectively caging freshwater rotifers Brachionus calyciflorus for real-time video-microscopy analysis. We demonstrate that behavioural bioassays performed under microfluidic perfusion can significantly enhance the sensitivity of conventional ecotoxicology test protocols.

Rapid Fabrication of Chip-Based Physiometers for Neurobehavioral Toxicity Assays Using Rotifers Brachionus calyciflorus.

An increased interest in implementations of Lab-on-a-Chip (LOC) technologies for in-situ analysis of multicellular metazoan model organisms and their embryonic stages demands development of new prototyping techniques. Due to size of multicellular organisms the fabrication of soft-lithography molds requires features with high aspect ratios as well as deposition of layers with significant thicknesses. This makes them time consuming and difficult to fabricate using conventional photolithography techniques. In this work we describe development of a rapid technique capable of generating thick films achieved with high viscosity SU-8 and used in fabricating master templates for high aspect ratio micro- and mesofluidic devices. The cost effective and rapid method eliminated the need for multiple spin coating cycles as well as edge bead artifacts while preserving low surface roughness and superior surface uniformity. Due to elimination of spin coating steps, typically constrained to clean room facilities, the new method allows to significantly reduce microfabrication costs. We have utilized the prototyping technique to develop proof-of-concept chip-based devices capable of effectively caging freshwater rotifers Brachionus calyciflorus for high-definition video-microscopy analysis. The combination of time-resolved video-microscopy and chip-based physiometers enabled us to demonstrate new applications for neurobehavioral assays utilizing non-invasive sub-lethal end-points.

Unsuitable use of DMSO for assessing behavioral endpoints in aquatic model species.

Dimethyl sulfoxide (DMSO) is a universally used aprotic solvent with the ability to permeate biological membranes and thus is commonly used to achieve appropriate biological availability of hydrophobic toxicants. While DMSO as a carrier medium has a reportedly low toxicity and is routinely employed in ecotoxicology, very little is known about its effect on dynamic behavioral parameters. This study presents a comparative analysis of the lethal and behavioral effects of exposures to DMSO concentrations of 0.1-10% on several test species such as: neonates of the freshwater crustacean Daphnia magna, nauplii of the marine crustacean Artemia franciscana, the marine crustacean Allorchestes compressa, embryos and larvae of the freshwater fish Danio rerio. The results demonstrated that DMSO did not cause statistically significant mortality even at concentrations close to 1% but induced clear and significant behavioral abnormalities in response to sublethal concentrations on all test species. These included hypoactivity syndrome in A. franciscana, A. compressa, D. magna and zebrafish larvae while a slight time-dependent hyperactivity response was observed in zebrafish embryos. For the majority of test species, behavioral changes such as moving distance, acceleration and burst movement were often observed during the first hours of exposure. These results indicate that caution should be exercised when using DMSO as a carrier solvent in experiments assessing behavioral endpoints.