Abstract: This research aimed to clarify the effects of fungicides applied in Medicago sativa production on the safety of an important pollinating insect, the honeybee (Apis mellifera), One-day-old adult honeybees were fed a diet containing dimetachlone, prochloraz, iprodione, at a range of concentrations (diluted with water 1:500, 1:1000, 1:1500, 1:2000 and 1:2500) or no fungicide (Control). Subsequently, the activities of three protective enzymes, superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and three detoxifying enzymes, carboxylesterase (CarE), glutathione S-transferase (GST), cytochrome P450 (P450) in the honeybees were measured. It was found that different concentrations of all the three fungicides could induce increased activities of SOD and POD in the honeybees. In particular, treatment with prochloraz solution at the 1:1500 dilution (0.17 mg·L^-1) induced a 1.82-fold SOD activity and a 5.40-fold POD activity compared to the Control treatment. All three fungicides showed low concentration induction and high concentration inhibition of CAT, CarE and GST activities, and showed inhibition (prochloraz) or induction (dimetachlone and iprodione) of P450 activity. With extended treatment time, the 1:1000 dilution of all three fungicides (0.40, 0.25 and 0.50 mg·L^-1 of dimetachlone, prochloraz, and iprodione, respectively) showed an overall induction effect on the activities of the three protective enzymes in Italian bees, but a variable effect on the activities of detoxifying enzymes. Specifically, dimetachlone initially increased the activities of the three protective enzymes and then inhibited activity after 6 h; iprodione induced P450 activity at all times, but inhibited the activities of CarE and GST in the first 12 h, then induced their activity thereafter; prochloraz elicited a gradual decrease in P450 activity with time, but initially induced then later inhibited the activities of CarE and GST. The results indicate that Italian bees can reduce the negative effects of fungicides by regulating the activities of protective and detoxifying enzymes in their bodies, but the fungicides have an impact on the normal physiology and metabolism of bees, even so. Therefore, care should be taken when applying fungicides to protect bees and ensure their safety while pollinating agricultural crops.

Key words: Medicago sativa, fungicides, Apis mellifera, detoxifying enzymes, protective enzyme