**5. Conclusions**

The data collected following the five-year monitoring survey showed that the application of ministerial guidelines allows the gathering of data on honeybee mortality incidents at national level in a consistent and reliable manner. We have shown that honeybee mortality events are still occurring and widespread, and that honeybees and beehive products are widely exposed to a large number of substances used legally and illegally, in agricultural practices and in beekeeping. In the honeybee matrix, 50% of the samples were found to be positive, while a greater proportion of the other matrices were contaminated. The honeybee is certainly the most interesting matrix for this study but also the most delicate from an analytical point of view, considering that laboratory results may be a ffected by various factors, from meteorological aspects to beekeeper reporting times and consequently the intervention of the o fficial veterinarian responsible for sampling. This could potentially result in an underestimation of the problem. As a consequence, beekeepers and o fficial veterinarians need to be highly aware and well informed of this problem to ensure that reporting and samplings are as punctual and prompt as possible. It is also important for the laboratory assigned to sample analysis to be aware of the problems linked to the possibility of pesticide concentration decreasing rapidly in dead bees and therefore of the best ways to conserve the samples before analysis. The pesticide

panel must also be kept up to date, based on the continuous evolution of the pesticides available on the market. Furthermore, the notification of honeybee killing incidents to the competent national and regional authorities could contribute to increase the awareness of farmers about the possible impact on honeybees of PPPs application. Moreover, this awareness could lead to a more reasonable application of the mitigation measures (established at regional level), such as proper maintenance of PPP application machines together with the use of deflectors to reduce the drifting of active ingredients during treatment, as well as to cut the grass on the orchard or vineyard surface when blossoms are present. The latter measure could strongly reduce the risk of exposure of honeybees to contaminated sources of nectar and pollen, even when the orchard is not blooming.

Our results, based on the appropriate managemen<sup>t</sup> of bee killing events, as described above, together with laboratory investigations, could contribute to a better understanding of the influence of pesticide mixtures on honeybee health, even at sublethal concentrations. The application, for example, of otherwise sublethal doses of miticides when tau-fluvalinate and coumaphos are simultaneously present in the hive could lead to honeybee mortality [41]. Likewise, grea<sup>t</sup> synergy is observed in the laboratory between EBI fungicides at field application rates and pyrethroids used as varroacides [42]. The present type of forensic study cannot demonstrate a direct link between honeybee mortality and pesticide mixtures but does provide us with valid indications of the interactions between active ingredients and therefore the pesticides that warrant further study in the future.

**Author Contributions:** Conceptualization, F.M., M.M.; Methodology, M.M., A.G.; Validation, M.M. and A.G.; Investigation, M.M., C.M., A.B., L.E.A., N.D., I.G.; Data Curation, C.M.; Writing—Original Draft Preparation, M.M.; Writing—Review & Editing, F.M., M.M.; Supervision, F.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors wish to thank Claudia Casarotto, GIS O ffice of Istituto Zooprofilattico Sperimentale delle Venezie, for preparing the maps and Andrea Maroni Ponti, Italian ministry of Health, Directorate General of animal health and veterinary medicines for supporting the program.

**Conflicts of Interest:** The authors declare no conflict of interest.
