**1. Introduction**

In recent decades, many beekeepers from all over the world have seen a large number of their honeybee colonies dying every year [1,2]. These deaths pose a threat to global food security because honeybees, along with numerous other insect species, provide a fundamental agricultural pollination service [3,4].

Honeybees can be considered a living monitoring system of various aspects of the ecosystem. Their state of health is in fact influenced by di fferent environmental factors, both natural and induced by human activity, such as climate trends, bee diseases, phytosanitary treatments, and beekeeping practices [5]. Honeybees commonly forage within 1.5 km of their hive (equal to an area of about 7 km<sup>2</sup> around the hive) and exceptionally as far as 10 or 12 km, depending on their need for food and its availability [6]. Their body is covered with hairs that can capture atmospheric residues, and they can be contaminated via food resources when gathering pollen and nectar from flowers or through water [7,8]. Consequently, during foraging flights, bees collect pollen, nectar, plant resins and water, and thus are also valid "samplers" of organic and inorganic chemicals in the environment, which are often taken back to the colony.

In Italy, since 2003, significant honeybee mortality has been recorded in springtime, mainly related to the side-e ffects of maize seed dressed with neonicotinoid insecticides [9]. These events increased significantly in 2008 leading to the creation, in 2009, of a nationwide surveillance network to monitor the health status of beehives and to properly report bee death incidents and their possible causes. The monitoring network, named ApeNet (2009–2010), initially included about 100 apiaries distributed throughout most of Italy, increasing to 300 and a total of approximately 3000 beehives in 2011 with the BeeNet project (2011–2014), both funded by the Italian ministry of Agriculture, Food and Forestry Policies [10]. These projects have made it possible to assess the health status of hives in Italy, through field observations, surveys, and laboratory analyses aimed at identifying specific pathogens and chemicals, and to study episodes of honeybee colony mortality. A bee emergency service team (BEST) has been created, in charge of receiving beekeepers' reports, assessing severity, organizing and participating in investigations, or coordinating the technicians recruited to deploy them in agreemen<sup>t</sup> with the competent authorities [11]. During the five-year monitoring studies, annual and regional variations were observed in pathogens responsible for infection (Deformed Wing Virus, Acute Bee Paralysis Virus, Chronic Bee Paralysis Virus, *Nosema ceranae*), and in *Varroa* mite prevalence [2]. Bee bread was often contaminated with at least one pesticide and the number of detected pesticides was positively related to the size of the agricultural area surrounding the apiaries [12]. Of the honeybee samples received following the application of the BEST protocols, 126 were analyzed, of which approximately 50% were positive for at least one active ingredient. The most frequently detected pesticides were imidacloprid, chlorpyrifos, thiachloprid, chlothianidin, and thiametoxam.

In 2014, the Italian ministry of Health implemented the following regulations: (1) Regulation (EC) No. 1107/2009 [13] concerning the placing of plant protection products (PPPs) on the market (repealing Council Directives 79/117/EEC and 91/414/EEC); (2) Directive 2009/128/EC [14] establishing a framework for Community action to achieve the sustainable use of pesticides; (3) Commission Directive 2010/21/EU amending Annex I to Council Directive 91/414/EEC regarding specific provisions relating to clothianidin, thiamethoxam, fipronil and imidacloprid [15]. Commission Directive 2010/21/EU indicates that member states shall ensure monitoring programs are initiated where and as appropriate to verify the real exposure of honeybees to the aforementioned neonicotinoids in areas extensively used by bees for foraging or by beekeepers. Furthermore, in view of the still frequent beekeeper reports of honeybee death at certain times of the year, and the high degree of public attention paid to this problem, it was considered appropriate for the various Italian regions to adopt a more systematic approach to the managemen<sup>t</sup> (notification to the competent authority, epidemiological and clinical investigation in the apiary, sampling, laboratory investigation) of bee mortality incidents where pesticide poisoning was suspected. In fact, the application of heterogeneous procedures in the managemen<sup>t</sup> of bee killing incidents could result in data that may not be representative or exhaustive. Consequently, the General Directorate of Animal Health and Veterinary Medicinal Products of the Italian ministry of Health, with note number 0016168 dated 31 July 2014 [16] issued the "Linee guida per la gestione delle segnalazioni di moria o spopolamento degli alveari connesse all'utilizzo di agrofarmaci" (Guidelines for the managemen<sup>t</sup> of reports of death or depopulation of bee colonies related to the use of plant protection products). These guidelines provide operational directives for managing these events with the aim of helping to protect beekeeping heritage from poisoning by plant protection products (PPPs), gathering information on the possible causes of death and/or depopulation of beehives, standardizing investigations in terms of the procedures adopted both in the field and at the laboratories responsible for analyzing the sampled dead bees.

The aim of the present study was to investigate the presence of pesticide residues in dead honeybees submitted to our laboratory following the guidelines for managing reports of death or depopulation of bee colonies related to the use of PPPs in Italy, from 2015 to 2019. Sample extraction was based on the QuECheRS technique followed by liquid or gas chromatography, both coupled with mass spectrometry (LC-MS/MS and GC-MS/MS), to analyze the selected active substances.

#### **2. Materials and Methods**
