Optimization of chick embryotoxicity bioassay for testing toxicity potential of fungal metabolites

The optimization of a simple, sensitive procedure using a chick embryotoxicity screening test (CHEST) bioassay for detection of toxic compounds is presented. Dosing protocols of eggs, using several mycotoxins (aflatoxin B1, deoxynivalenol, T-2 toxin) and appropriate controls, were evaluated for embryonic sensitivity, overall practicality of the procedure, and consistency of results. It was found that both type of carrier solvent and volume injected could significantly affect overall embryonic mortality. The chick embryo was most sensitive to the effects of toxins and solvents after 1 or 2 days of incubation; a rapid decrease in response was observed as the age of the embryo at dosing increased. Following administration of the toxins just below the shell membrane by way of a small hole (less than 0.5 mm diameter) punched in the shell, a good dose-response (% mortality) could be obtained regardless of the site of injection (except directly into the yolk), although dosing via the air sac position resulted in a slightly better statistical outcome. Although some variations in calculated LD50 values were found among repeated assays, statistical analyses showed that the differences were not due to dosing protocol but to the variations in embryo sensitivities among batches of eggs. Thus, if standard reference toxins for comparison are run concurrently, the CHEST assay can prove to be a very satisfactory model, as well as having considerable flexibility to be adapted to the needs and resources of many laboratories.