## 3 Results and Discussion
### Honeybee Matrix
Fe showed the highest levels in Pianello (site 3) both in October and June, with similar values of 135.52 and 134.92 mg kg\({}^{-1}\), respectively, exceeding the reference concentration by 1.7 times. Mn showed the highest levels in Pianello (site 3--October), with 81.06 mg kg\({}^{-1}\), exceeding the reference concentration by 1.7 times. Ni had higher levels in Pianello (site 3--June), with 1.44 mg kg\({}^{-1}\), exceeding the reference concentration by 12.0 times. Pb had higher levels in Pianello (site 3--June), with 0.25 mg kg\({}^{-1}\), exceeding the reference concentration by 5.0 times. V had higher levels in Pianello (site 3--October), with 0.09 mg kg\({}^{-1}\), exceeding the reference concentration by 3.8 times. Zn had higher levels in Ponte Rio (site 1--October), with 140.10 mg kg\({}^{-1}\), exceeding the reference concentration by 2.7 times (Table 2). As had higher levels in Ponte Valleceppi (site 2--June), with 0.034 mg kg\({}^{-1}\), comparable to the reference concentration (Perugia control sites) (ratio of 1.2). Hg had higher levels in Ponte Rio (site 1--June), with 0.005 mg kg\({}^{-1}\), exceeding the reference concentration (Perugia control sites) by 2.4 times (Table 2). In summary (Table 1a), the highest concentration values of the 13 elements detected in the honeybee bodies always showed higher levels than the reference, with ratios between 1.2 (As, Ponte Valleceppi, site 2, June) and 12.0 (Zn, Pianello, site 3, June). Overall, within \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Element** & \
\begin{tabular}{c} **Maximum** \\ **Concentration** \\ \end{tabular} & **Locality** & **Site** & **Month** & **Control Site** &
\begin{tabular}{c} **Reference** \\ **Concentration** \\ \end{tabular} & **Ratio** \\ \hline Al & 34.669 & Pianello & 3 & June 2022 & Alviano Lake & 10.705 & 3.2 \\ As & 0.034 & Ponte & 2 & June 2022 & Perugia & 0.028 & 1.2 \\ Cd & 0.146 & Pianello & 3 & October 2022 & Alviano Lake & 0.028 & 5.2 \\ Co & 0.298 & Ponte & 2 & June 2022 & Alviano Lake & 0.092 & 3.2 \\ Cr & 0.106 & Pianello & 3 & June 2022 & Alviano Lake & 0.026 & 4.1 \\ Cu & 17.982 & Ponte Rio & 1 & October 2022 & Alviano Lake & 11.053 & 1.6 \\ Fe & 135.521 & Pianello & 3 & October 2022 & Alviano Lake & 77.822 & 1.7 \\ Hg & 0.005 & Ponte Rio & 1 & June 2022 & Perugia & 0.002 & 2.4 \\ Mn & 81.061 & Pianello & 3 & October 2022 & Alviano Lake & 48.822 & 1.7 \\ Ni & 1.445 & Pianello & 3 & June 2022 & Alviano Lake & 0.120 & 12.0 \\ Pb & 0.252 & Pianello & 3 & June 2022 & Alviano Lake & 0.050 & 5.0 \\ V & 0.088 & Pianello & 3 & June 2022 & Alviano Lake & 0.023 & 3.8 \\ Zn & 140.095 & Ponte Rio & 1 & October 2022 & Alviano Lake & 51.851 & 2.7 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Maximum and reference concentrations (mg kg\({}^{-1}\), dry weight) and ratios of 13 trace elements in whole bodies of honeybees (B) measured at three sites in the study area during June and October 2022 and at the control sites of Alviano Lake and Perugia. the study area, Pianello (site 3) showed, for most of the selected elements, the maximum concentration values (Al, Cd, Cr, Fe, Mn, Ni, Pb, and V), while Ponte Rio (site 1) showed the maximum concentration values for Cu (very similar to the value at site 3), Hg, and Zn, and Ponte Valleceppi (site 2) showed the maximum concentration values for As and Co. The application of the Honeybee Contamination Index for honeybee (body, B) allowed for the assessment of contamination by certain elements, such as Cd, Pb, Cr, and Ni. The HCI showed low levels of contamination by Cd and Pb in both June and October at all investigated sites, while for Cr, an intermediate level of contamination was revealed in all cases except for site 3 in June, where a high level of contamination was reached. Finally, for Ni, an intermediate level of contamination was observed in all cases, except for site 2 and site 3 (both sites in June), where a high level of contamination was reached (Figure 2).