**4. Discussion**

The present study aimed to improve the evaluation methodologies for analyzing honeybees' exposure to pesticides in order to better identify and quantify the contamination of honeybees' food resources from the main treated crops, as well as to have a first image of their presence and quantification in Romania in some treated crops. Thus, the most important levels of neonicotinoid residues, in the vegetation conditions of the 2018 season, were identified in oilseed rape honeybees, honey, and pollens, as well as in corn pollen.

In view of these results, the analysis of neonicotinoids in these matrices is very important for establishing a basic exposure level of honeybees to pesticides in a specific area at a certain moment.

The identification and quantification of neonicotinoids in any sample could be a combined result of many factors, but the collection and sample preparation until their analysis, as well as the analysis methodology with the lowest LOD (limit of detection) and the lowest LOQ, are of grea<sup>t</sup> importance.

In order to identify and quantify the neonicotinoids, the obtained results in the two laboratories also showed the importance of the mono-floral sample preparation of honeys and pollens.

One interesting finding concerned the presence of di fferent neonicotinoids in detectable and/or quantifiable amounts in all matrices that were obtained from oilseed rape crops, while some neonicotinoids (acetamiprid, imidacloprid and thiacloprid) were only found in detectable amounts in sunflower honey. The low level of neonicotinoid residues obtained in some sunflower honey samples

can be explained by the fact that sunflower honey is actually a multi-floral honey, so the contamination residues were diluted by di fferent sources of nectars. However, the lack of neonicotinoids in the selected sunflower pollen samples remains questionable, and further research is necessary.

Following the identification of neonicotinoids (imidacloprid) in all the three corn pollen samples and in view of the fact that honeybees collect high quantities of corn pollen in areas with intensive crops, this study can be seen as o ffering an important overview of the importance of this crop for honeybee nutrition.

What is remarkable here, from the field observation, is the fact that corn tassels were intensely visited by honeybees, even when the nearby sunflower crops are in full bloom. This shows that corn tassels are an important source of pollen for honeybee colonies, as is sunflower pollen. For this reason, the corn crops represent important sources of nutrients, not only by guttation water but also by pollen, so its contamination with pesticides could a ffect honeybees during the whole vegetation period.

The obtained results are similar with those found in the literature [20–29,39] that have shown that the residues of neonicotinoids in honeys and pollens are found in the range of a few nanograms per gram. Their lethal or sublethal e ffects on honeybees depend on many factors [28] such as daily consumption, seasonal conditions, the activity and strength of the colony, the age and the duration of exposure, and health state. For example, the literature data show that the lethal toxicity of imidacloprid is in the range of a few picograms if ingestion is repetitive for minimum eight days and of a few nanograms if the ingestion is for one-to-two days [40]; however, its sublethal e ffects, such as learning and orientation ability modifications, could appear at a concentration of 0.1 ng/bee [30], which was the case in our study in real conditions.

Taking into account the toxic and cumulative e ffects of neonicotinoids, based on irreversible bind on the nicotinic acetylcholine receptors (nAChRs), as well as the toxic e ffects of their metabolites proven by research [41–45], the found concentrations pose serious risks to honeybee health in the short and long term. One such study [44] that was done over a short period of time (10–30 days) showed, by extrapolation, that the daily ingestion of about 0.005 ng/day of imidacloprid produced important lethal toxic e ffects (LT50) in 150 days. If a bee consumes around 0.02 g honey per day [28], a concentration of 0.25 ppb in honey (which is not a quantifiable amount (see LOQ, Table 3)) can cause long-term mortalities (over 150 days), as it happens in the winter. These low residue quantities can be consistently supplied by residues in storage pollens (beebread) in the late winter-to-early spring period when colonies begin rearing their brood, and this situation can explain the collapse of colonies due to a long exposure to sublethal e ffects.

Some research has gone even deeper, demonstrating that the use of neonicotinoids can lead to a wider range of sub-lethal e ffects on honeybees as a result of very low concentrations of neonicotinoids. Thus, if lethal acute e ffects can be rapidly noticed by the rapid decrease of the population or mortalities in front of the hive, sub-lethal e ffects can be di fficult to observe, as colonies generally have problems of development or slow mortality for longer periods (autumn, winter, and early spring depopulation), inducing a variety of behavioral dysfunctions. Many of these dysfunctions a ffect orientation, memory, communication [46–48], foraging and flight [49,50], the olfactory sense [51], the glandular system and respiratory rhythm [52,53], reproduction [54,55], global temperature and metabolism [56,57], sensitivity to diseases, and immunity reduction [58–64].

Taking into account the high level of residue of thiacloprid found in one sample (four times more than the maximum residual limit for human consumption), it is important to show the risks that the EFSA mentioned in its report published in 2019 [65], such as: "delayed e ffects or relevant sub-lethal effects on bees at relatively low concentrations cannot be excluded" and "thiacloprid presents important risks for human health."

Honeybees are a very important biosensor that can be managed to obtain information about the environment. Nonetheless, the samples collected by honeybees need to be melissopalinologically analyzed when it comes to the necessity of analyzing a specific crop or plant species.

The collection, preparation and preservation of samples should be done so that to reflect the pesticide residues in nectar and pollens at the time of their collection by honeybees. This is of grea<sup>t</sup> importance in pesticide exposure studies in order to identify the real residues of neonicotinoids at detection or quantification levels and their risks to honeybees.

The monitoring of honey flows by electronic hives, even if not very important for sample collection and preparation, is the first indicator about honey flow, weather conditions, depopulations or other activities of honeybee colonies that give preliminary information on weight gain and honeybee populations (e.g., swarming). This basic electronic system could be completed by a specific electronic device that could measure, with high accuracy, entrance activity in order to highlight any slight modification in the number of foragers and their loss in the field over the normal levels of depopulation. In this sense, a series of new research projects are necessary to quantify the number of outgoing and incoming bees at the entrance and by these data processing measures in order to o ffer important information about abnormal depopulations and to send specific alerts in a useful time. Through this approach, it is possible to collect important information about realistic field depopulations and to collect relevant samples for neonicotinoid residues at depopulation moments in order to better analyze the impact of pesticide field exposure on honeybee health.

Thus, the registration of honey flow monitoring data by electronic means, the detection of general colony dynamic activities by electronic sensors, the collection and preparation of mono-floral honeys or pollens, the use of small samples for analyses in order to facilitate their mono-floral preparation for the further specialized laboratory analyses, and the good preservation of samples from the collection moment to the laboratory analyses all contribute to an e ffective system for neonicotinoid identification.
