Current chemical analysis approaches for contaminants have failed to reveal their biotoxicity. Moreover, conventional bioassays are time consuming and exhibit poor repeatability. In this study, we performed the acute toxicity detection of various contaminants (chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), tin (Sn), nickel (Ni), and bisphenol A (BPA)) with four bioluminescent bacteria ( Q67, ,  T3, and  502) using a rapid, flexible, and low-cost bioassay. We found that the temperature affected the bacterial luminescence, and freeze-dried cells exhibited sensitive toxic responses to contaminants. Indeed, the optimized protectants containing 12% (w/v) trehalose, 4% sucrose, and 2% sorbitol displayed better luminescence and toxic sensitivity. Furthermore, freeze-dried powders of these strains were prepared and subjected to acute toxicity detection. The results showed that all contaminants exhibited acute toxicity toward Q67, but the other strains did not show obvious response to nickel and tin. The relative half-maximal effective concentration (EC) values of BPA, Cr, Cd, Pb, As, Hg, Ni, and Sn to Q67 were 0.674, 1.313, 11.137, 5.921, 4.674, 0.911, 5.941, and 54.077 mg/L, respectively. In addition, the EC values of contaminants toward different strains were suggested to be statistically significant. Freeze-dried Q67 exhibited toxic responses to more contaminants than the other bioluminescent strains; therefore, Q67 was selected to be more suitable than the other strains for single and mixture toxicity detection tests. Compared with other strains, Q67 was more appropriate for the rapid screening of the mixture toxicity of contaminants in samples as a nonspecific screening sensor before the use of standard analysis approaches.

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