In recent decades, many beekeepers from all over the world have seen a large number of their honeybee colonies dying every year [1
]. These deaths pose a threat to global food security because honeybees, along with numerous other insect species, provide a fundamental agricultural pollination service [3
Honeybees can be considered a living monitoring system of various aspects of the ecosystem. Their state of health is in fact influenced by different 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 km2
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
]. 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-effects 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 agreement 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 management (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 management 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 management 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.
presents a summary of the pesticides detected in the samples analyzed within 5 years of monitoring. Table 2
and Table 3
summarize the main findings by survey year for honeybees and other matrices, respectively. Figure 1
shows the location of the positive and negative samples for each officially reported honeybee mortality event.
In total, 63 different active ingredients were found in honeybee samples, with concentrations ranging from 0.1 to 134,665 ng/bee, and 51 different active ingredients in the other analyzed matrices (beeswax, bee bread, honey and vegetable matrices), ranging from 0.01 to 359.5 mg/kg. Most investigated samples were positive for at least one active ingredient (53%) and contaminated by more than one residue: 53% of the samples were contaminated by at least two different residues, 32% by at least three, while as many as nine active ingredients were detected in one extreme case, coming from Udine province (North-Eastern Italy).
Insecticides were the most frequently detected active substances (49.2%) in honeybees (Table 1
and Table 2
), the most prevalent being the acaricide tau-fluvalinate (38.2%). Pyrethroid permethrin, the second most frequently found active substance, had a prevalence of 13.3%. Chlorpyrifos was the third most commonly determined pesticide (12.9%).
Globally, most of the other matrices analyzed (67%) were positive for at least one active ingredient (Table 3
). Again, the acaricide tau-fluvalinate was the most commonly found active ingredient, with a prevalence of 53.4%, followed by the insecticide methiocarb with a prevalence of 13.6% (also considering the metabolite methiocarb sulfoxide), and the synergist piperonyl butoxide (11.0%).
The geographical distribution of the honeybee death events (Figure 1
) coincides with the areas in Italy in which intensive agriculture is mainly practiced (such as apple and citrus orchards and vineyards mainly other than maize cultivations).
In honeybees (Table 1
and Table 2
), the most frequently detected active substances were insecticides with tau-fluvalinate having the highest prevalence. Tau-fluvalinate is a pyrethroid insecticide authorized both as a PPP and for the control of Varroa
mite infestation of honeybees in Italy. Miticides have already been found by different studies [2
] to be the most frequent residues in honeybee samples around Europe. The pyrethroid permethrin, the second most frequently identified active ingredient, also has the highest detected concentration. It is a contact insecticide which has not been approved for use in the EU as a PPP, due particularly to its acute toxicity to aquatic organisms. Chlorpyrifos was the third most commonly determined pesticide in honeybees and, being an active ingredient highly toxic to bees, it could represent an important factor affecting colony health. Chlorpyrifos has already been identified as one of the most commonly detected insecticides in bees [20
]. Neonicotinoids, mainly imidacloprid, were also frequently identified. In Italy, the use of three neonicotinoids, namely imidacloprid, clothianidin, thiamethoxam, and fipronil, was restricted in 2008 due to evidence of their negative effects on honeybee health. In 2013 the EU definitively banned the use of these active ingredients for seed treatment, soil application and foliar treatment of plants and cereals attractive to bees (but use in greenhouses is allowed) [23
]. However, fipronil and all three neonicotinoids (including the restricted ones) screened in our study were detected in our samples. Therefore, despite the current limitations on the use of PPPs containing these active ingredients, according to the present monitoring results, honeybees are still exposed to potentially harmful levels of these pesticides, as already observed in previous studies [25
]. Fungicides were also often detected (39.3%) with a wide variety of active ingredients, the most frequently found being penconazole and pyrimethanil. Although there are no restrictions on the use of fungicides on crops during blooming, various studies have shown that the impact of fungicides on honeybee health can be harmful, both due to their direct negative effects on honeybee health [28
], and through a synergistic action between fungicides and other types of pesticide [30
]. Our results partially agree with those obtained in a previous study carried out by our laboratory [33
], which assessed the presence of pesticides and viruses in dead honeybees following mortality incidents in northeastern Italy in 2014. Compared to this study, in which imidacloprid was the most frequently detected active ingredient, there has now been a reduction in the presence of neonicotinoids, probably due to limitations imposed on their use by the European Commission [23
]. Tau-fluvalinate and chlorpyrifos were instead confirmed to be among the most frequently identified active ingredients.
With the exception of 2018, the year in which we observed close correspondence between honeybees positive to pesticides and bee kill incident reports, percentage positivity stood at around 44% in the other monitoring years: a value probably influenced by various factors, as the speed of reporting and the subsequent sampling intervention. The concentration of pesticides in dead honeybees can rapidly decrease within just hours of the poisoning event and, if not properly stored at −20 °C, samples can reach a level close to environmental residue before being analyzed in the laboratory [20
]. The analysis results may be also affected by the severity of the poisoning event (in terms of the active ingredients involved, their concentration, method of administration) and the presence of other bee parasites or stressors (such as viruses and Varroa
mite) that can contribute to the weakening of colonies and predisposition to mortality events, even with sublethal concentrations of pesticides [28
The honeybee is certainly the most important matrix to be analyzed in case of honeybee mortality incidents, as residues detected in honeybees reflect their exposure both to direct contact with PPPs, biocides, or even veterinary drugs, and to the consumption of contaminated nectar and pollen. However, the analysis of other matrices related to the same incident can help us to better understand the mortality event. For example, bee bread can supply useful data on any PPPs application occurring in the areas surrounding the beehive, while beeswax comb can provide information on exposure over a period of time. Unlike other beehive products, beeswax can remain in the hive for many years, thus resulting in an accumulation of various non-polar active ingredients applied in beekeeping and agriculture [19
]. In the present study, most other analyzed matrices (67%) were positive for at least one active ingredient (Table 3
), and again the acaricide tau-fluvalinate represented the most commonly found active substance, followed by the insecticide methiocarb and the synergist piperonyl butoxide. The most represented matrix was beeswax, with an average of 72% (70 out of a total of 97 samples) of the samples proving to be contaminated with pesticides, mainly tau-fluvalinate. Bee bread showed 74% positivity (25/34), and in this case too, the most commonly detected active ingredient was tau-fluvalinate. Being stored inside the beehive, bee bread can be affected by both beekeeper and agricultural activity. For these reasons, however, in the case of a honeybee killing event, we cannot rely on toxicological information provided by beeswax. Vegetable matrices (most frequently leaves, corn seedlings, maize) were contaminated in 70% of cases (12/17), with the widest variety of active ingredients (27), despite being by far the least numerous matrix received. Honey was also received as a matrix related to honeybee incidents and proved to be contaminated with pesticides in only 20% of cases; but in three samples the detected pesticide concentration exceeded the limits imposed by the EU (methiocarb 0.05 and 0.7 mg/kg and tau-fluvalinate 0.05 mg/kg) [36
]. These results should draw attention to the fact that mortality events are harmful to honeybees, but consumers’ health should also be considered. The risk of contamination of edible beehive products, as honey and pollen, but also beeswax, which can then be reused and lead to the transfer of contaminants to honey, cannot be ruled out [37
It is also worth mentioning the detection of some active ingredients that are no longer authorized but in the past were present in both PPPs and veterinary medicinal products. Authorized active ingredients used against varroosis [38
] were among the main sources of honeybee and hive matrices contamination, but so were old apicultural and agricultural acaricides that are now banned, such as bromopropylate (both), chlorfenvinphos, and rotenone (agricultural). The pyrethroid insecticide permethrin, which is highly toxic to honeybees and authorized as a biocide [39
], was frequently detected, even in high concentrations, in both honeybees and other matrices. The same applies to the potent multi-purpose pyrethroid insecticide tetramethrin, registered in 1968 and often used to control insects presenting risks to public health, but which is highly toxic for honeybees and has never been authorized for use in crop protection. The insecticide thiodicarb was detected in a few honeybee samples. This insecticide and molluscicide is used to control Lepidoptera, Coleoptera, slugs, other pests of fruit, vegetables, and many other crops, with moderate or high toxicity to honeybees, depending on whether the administration is contact or oral [36
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 affected by various factors, from meteorological aspects to beekeeper reporting times and consequently the intervention of the official veterinarian responsible for sampling. This could potentially result in an underestimation of the problem. As a consequence, beekeepers and official 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 management 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, great 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.