Due to the paucity of low-concentration and long-term exposure scenarios on volatile organic compounds (VOCs), this study aimed to develop a novel computer vision method to investigate the mortality and behavioural effects of toluene exposure time and concentration on D. melanogaster and their sex differences. An automatic location approach was developed for multiple D. melanogaster locations under an uneven-lighting and a low-contrast background. Combined with survival analysis, three behavioural parameters (occupancy preference, detection ratio, and average movement distance) and one index (cumulative movement distance) were proposed. The results indicated that the toluene-induced additional mortality generally occurred at the later exposure stage and exhibited sex differences over time. Besides, the trends in these behavioural parameters over time also exhibited sex differences, with the T2300 and T3800 groups significantly increasing detection ratios in males at the middle stage of exposure, potentially triggering the hormesis effect. Furthermore, the relationships between the cumulative movement distance and concentration exhibited non-monotonic patterns, demonstrating the potential hormesis effect. This study highlighted the importance of time and sex factors in exposure investigations and revealed the potential stimulatory effects of toluene on D. melanogaster activity. Moreover, computer vision technology is a promising method for evaluating and controlling air quality.
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