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TA5 Soil Ecotoxicology and Risk Assessment
() High-throughput sublethal toxicity tests using the nematode Caenorhabditis elegans.
Boyd, Windy1, McBride, Sandra1, Rice, Julie1, Freedman, Jonathan1, 1 Duke University, Durham, NC, USA
ABSTRACT- Regulatory agencies have recognized the need for alternative toxicological methods and models to decrease the time and expense of current toxicity testing protocols. The numerous advantages of using Caenorhabditis elegans as a model organism are well documented. Short life cycles, easy and inexpensive maintenance and culturing, and detailed biological knowledge allow for the development of rapid, low-cost toxicity tests that readily lend themselves to mechanistic studies of toxicant actions. We are developing the means to screen neurological and developmental toxicants rapidly and reproducibly using C. elegans. Sublethal toxicity endpoints including growth, reproduction, movement, and feeding are automated using two robotic laboratory workstations for liquid handling and pipetting; a COPAS BIOSORT for dispensing worms and analyzing worm length and fluorescence; and an imaging workstation for motion tracking and multidimensional image analysis. To increase the rate and efficiency of chemical screens, 96-well plate formats are used for sample preparation, dispensing of test organisms, and quantification of specific toxicological endpoints. As worms are dispensed to exposure wells, the COPAS BIOSORT measures the time of flight, extinction, and fluorescence for each worm. Gravid adult worms and eggs are bleached and the eggs are either transferred directly to plates seeded with E. coli or allowed to hatch overnight in buffer. Worms are used at a specific developmental stage for each test: L1s for 72-h growth, L4s for 48-h reproduction, and 3-day-old adults for 4-h or 24-h movement and feeding assays. After toxicant exposures for growth, reproduction, or feeding, the samples are aspirated with the COPAS REFLX and the same parameters are measured. Movement tracking and image capturing of worms are also performed after toxicant exposures. For each test, the effective concentration that results in a 50% reduction in response relative to controls (EC50) is then calculated. Sample data from several chemicals will be presented as illustrations of the methods and results of each.
Key words: high-throughput, Caenorhabditis elegans, sublethal, COPAS BIOSORT
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