Microfluidic electric parallel egg-laying assay and application to in-vivo toxicity screening of microplastics using...

Environmental pollutants like microplastics are posing health concerns on aquatic animals and the ecosystem. Microplastic toxicity studies using Caenorhabditis elegans (C. elegans) as a model are evolving but methodologically hindered from obtaining statistically strong data sets, detecting toxicity effects based on microplastics uptake, and correlating physiological and behavioural effects at an individual-worm level. In this paper, we report a novel microfluidic electric egg-laying assay for phenotypical assessment of multiple worms in parallel. The effects of glucose and polystyrene microplastics at two concentrations on the worms' electric egg-laying, length, diameter, and length contraction during exposure to electric signal were studied. The device contained eight parallel worm-dwelling microchannels called electric traps, with equivalent electrical fields, in which the worms were electrically stimulated for egg deposition and fluorescently imaged for assessment of neuronal and microplastic uptake expression. A new bidirectional stimulation technique was developed, and the device design was optimized to achieve a testing efficiency of 91.25%. Exposure of worms to 100 mM glucose resulted in a significant reduction in their egg-laying and size. The effects of 1 μm polystyrene microparticles at concentrations of 100 and 1000 mg/L on the electric egg-laying behaviour, size, and neurodegeneration of N2 and NW1229 (expressing GFP pan-neuronally) worms were also studied. Of the two concentrations, 1000 mg/L caused severe egg-laying deficiency and growth retardation as well as neurodegeneration. Additionally, using single-worm level phenotyping, we noticed intra-population variability in microplastics uptake and correlation with the above physiological and behavioural phenotypes, which was hidden in the population-averaged results. Taken together, these results suggest the appropriateness of our microfluidic assay for toxicological studies and for assessing the phenotypical heterogeneity in response to microplastics.