Monitoring of neonicotinoid pesticides in beekeeping

Abstract The decline of pollinating species is correlated to the extensive use of neonicotinoids against pest insects for crop protection. In this study, the concentrations of neonicotinoid insecticides were determined in honeybees, honeycomb and honey samples, collected in Spring 2015 (blooming period) from different areas in Sicily (IT), to carry out an evaluation of bees products’ safety and an overview of neonicotinoid contamination in beekeeping. The results obtained showed only the presence of clothianidin in bee samples and these concentrations don’t represent a risk for bees’ vitality and safety. The absence of residue in all honey samples, instead, showed the quality of bee products.


Introduction
The need for more selective insecticides acting on pests has led to a large employment of neonicotinoids, a new group of compounds derived from nicotine. Used since 1990 to control pests, actually they are largely employed about 30% by value of the global insecticide (Jeschke et al. 2011), in agricultural crops and particularly pollinated crops, for high economic value of honeybee (Apis mellifera L.) production.
Neonicotinoids are a class of neuroactive compounds including imidacloprid, thiamethoxam and thiacloprid with a chloro-substituted heterocyclic group (chlorpyridinyl or chlorthiazolyl), joined to a second heterocyclic ring; instead acetamiprid, nitenpyram, clothianidin and dinotefuran with an acyclic group, in place of the second heterocyclic ring (Kamel 2010) ( Figure 1S).
Neonicotinoids are neurotoxic compounds acting on ion channels of nervous system (Aliouane et al. 2009). They are agonists of nicotinic acetylcholine receptors (Buckingham et al. 2005) and their different properties are due to the compositions of α and β subunits of these receptors. They showed a good activity against pest sucking insects, some heteroptera, Coleoptera, Lepidoptera and insects resistant to organophosphates, carbamates, pyrethroids, chlorinated hydrocarbon and several other compounds (Nauen et al. 2003) and are less toxic in birds and mammals. however, the extensive use of neonicotinoids has been correlated to a decline of pollinating species as A. mellifera L., exposed to these chemicals when visiting melliferous plants (halm et al. 2006;Samson-robert et al. 2014;Williams et al. 2015). The neonicotinoids contained in pollen e nectar of treated plants can be accumulated in the hives, and the young honeybees can be exposed to repeated sublethal doses of pesticides during their early life (Aliouane et al. 2009). The honey bee's mortality is due to multi-factorial causes involving parasites, pathogens and pesticide stressors (Sánchez-Bayo et al. 2016). honeybees are considered as environmental indicators  used to assess the ecotoxicological impacts of pesticides, though they are more resilient to neonicotinoids than wild bees (Cresswell et al. 2012). The study of honey bees exposure to neonicotinoid pesticides is of great interest for their effects on bees' life and honey production (halm et al. 2006;Jones & Turnbull 2016).
Previous studies documented the acute toxicity of neonicotinoids, thiamethoxam and acetamiprid, in honeybees after oral and topical exposure (el hassani et al. 2008;Aliouane et al. 2009), and also subchronic exposure (Aliouane et al. 2009). Particularly, the sub-lethal doses of neonicotinoids modify honey bee behaviours, reproduction activity, larval development, adult emergence, longevity (di Prisco et al. 2013;Fairbrother et al. 2014), and inhibit bees' immune defence, increasing susceptibility to infection (Abbo et al. 2016). Considering honeybees' behaviour, acetamiprid acts increasing sucrose responsiveness, locomotor displacements, and inducing long-term memory impairment (el hassani et al. 2008). The exposure to sub-lethal doses of imidacloprid affects significantly the survival of honeybees, acting on energy homeostasis, instead, clothianidin adversely affects bees' immunity and promotes replication of viral pathogens (di Prisco et al. 2013).
Actually, the european Union has restricted the use of certain neonicotinoids, particularly imidacloprid and clothianidin, confirming a high acute risk to honey bees exposed via dust drift for the seed treatment uses in maize, oilseed rape and cereals and via residues in nectar and/or pollen (eFSA 2015).
Therefore, it is important to carry out regular monitoring to assess the environment contaminants, considering the different inorganic (Lo Turco et al. 2012;Salvo et al. 2014Salvo et al. , 2015di Bella et al. 2015;Naccari et al. 2015;Bua et al. 2016) and organic polluting substances, (Wågbø et al. 2012;Arukwe et al. 2014;di Stefano et al. 2014;Ferrante et al. 2014) which can affect agricultural production and the life of some insects including bees.
The aim of this study was to monitor the use of neonicotinoids in apiculture in Sicily trough residual analysis in honeybees, honeycomb and honey, for an evaluation of bee products safety and quality of beekeeping.

Results and discussion
The results obtained show that in honeycomb and honey samples all neonecotinoids were under the detection limit; instead in samples of dead bees only the presence of Clothianidin (57.2 M.V. μg/kg) was observed . Considering the area of sampling, only in seven provinces there were positive samples and the higher concentrations were found in province of Marsala, Gela and Palermo, where the agricultural activity is very developed (Table 1).
These data confirm the importance of monitoring on clothianidin, as specified by eFSA, for the greater risk to honey bees (eFSA 2015). The residues of clothianidin found, in fact, could be due to the use of neonicotinoids in agricultural practice; however the levels found don't affect bees' vitality and safety, as confirmed also by the absence of clothianidin in honeycomb, analysed for a more complete ecotoxicological monitoring (Smith & Wilcox 1990) These data are in accordance with those of Krupke et al. (2012), who found the presence of clothianidin in samples of dead bees, collected near hive entrances. Kasiotis et al. (2014), instead, found in bees sample residues of clothianidin (50%), thiamethoxam (9%) and imidacloprid (4.5%). At the same time, the absence of residue in all honey samples showed the quality of bee products in this area.
other authors, instead, found also in honey samples only the presence of neonicotinoids, particularly Clothianidin, Thiamethoxam (Jones & Turnbull 2016) and Imidacloprid (Sánchezhernández et al. 2016) in concentrations higher than detection limits. however, it's possible admit that the absence of neonicotinoids in honey samples from Sicily is due to a filtering effect of bees ) but also to a traditional agricultural practice in this region.

Experimental
See Supplementary materials for: reagents, sampling and analysis.

Conclusions
This study confirms that honey bees living and foraging near agricultural fields could be exposed to pesticides as neonicotinoids, particularly Clothianidin found in bee samples. A regular monitoring is need to evaluate the effects of these insecticides on bees behavior and their survival, considering that these pesticides are one of the possible responsible of the progressive decline of pollinating species, and of honey production. data obtained, however, showed that there isn't a direct relationship between the presence of neonecotinoids in bees and honey, as confirmed by the absence of residues also in honeycomb. In addition, it's possible to confirm the quality of honey produced in the areas studied and consequently not significant effect on Sicilian apiculture.

Disclosure statement
No potential conflict of interest was reported by the authors.