**4. Discussion**

Multiresidue methods (LC, GC-MS/MS) constitute the mainstay of analytical laboratories involved in pesticide residue analysis in a multitude of matrices. The latter was verified in this work through the detection and quantitation of numerous active substances and metabolites in both pollen beebread and honey. It is important to point out that the presented results are the outcome of the chemical analysis of randomly collected and dispatched honey and pollen samples. Hence, it cannot be viewed under an organized sampling/monitoring scheme in which PPP applications could be straightforwardly connected

to specific crops via concomitant monitoring of residues in apicultural commodities, crops, and soil. Nevertheless, based on the incoming information from individual authorities that sent the samples, it was endeavored to proceed to potential associations of active substances detected with the predominant crops in some of the regions and the timing of sampling. In the same context, honey and pollen (and beebread) samples associated with bees' intoxication incidents (in which deliberate application cannot be excluded) were not included in these interpretations since it would possibly lead to misleading assumptions for PPP applications in the specified areas. A characteristic example stems from one of the few lowland areas of the island region of Chios Island (northern Aegean Sea), in which the cultivation of vegetables and watermelons is documented. From this region, two honey samples were found positive for pesticides. The detection of coumaphos in one sample can be attributed to acaricidal treatments. However, detecting the fungicide active substance penconazole in both honey samples designates the potential uptake of this chemical by bees in the nearby cultivations and subsequent transfer to the beehive, or potential transfer due to pesticides' drift favored by environmental conditions. Penconazole formulations were approved to control the fungi *Sphaerotheca Fuliginea*, *Erysiphe cichoracearum* in watermelon, and other flowering plant species of the Cucurbitaceae family. Plants of this family, such as watermelon (abundant in the region), are pollinated by bees; therefore, this finding can be a logical hypothesis. In the same samples, the organophosphate fumigant insecticide pirimiphos-methyl was also detected. Its presence seems disconnected from the predominant (active) cultivations of the area, yet, this active substance is usually applied to control pests, such as *Sitophilus granarius* and *Oryzaephilus surinamensis*, of stored seeds. Nevertheless, in the past, the specific region had significant production of wheat, which can interplay in the specific finding through the potential application of pirimiphos-methyl in stored wheat areas. Acetamiprid and etofenprox (both insecticides) detection can be attributed to field applications to combat aphids such as *Macrosiphum euphorbiae* in vegetables, or Lepidoptera such as *Pieris brasiccae* in cabbage and European grapevine moth *Lobesia botrana*. In two additional characteristic samples originating from Northern Greece (Chalkidiki region), 11 active substances and metabolites (chlorpyrifos ethyl, carbendazim, dimethoate, omethoate, tebuconazole, trifloxystrobin, acetamiprid, pyraclostrobin, azoxystrobin, pirimiphos-methyl, and fenpropathrin) in total were identified in two samples of pollen (9 and 10 synchronous detections in the two samples correspondingly). The detection of these compounds can be attributed to PPP applications related to crops of the respective area, such as olives, grapes, and cereals. From the above-mentioned substances, dimethoate (and its metabolite omethoate), along with chlorpyrifos, were authorized at the time of the study and widely used for the control of various pests, such as the olive fruit fly (*Dacus oleae*). The strobilurin fungicides detected are also used in the olive crop and grape for the control of mildew or black rot in grapes. Acetamiprid is a neonicotinoid active substance with a wide range of applications, such as the control of *Lobesia botrana* (major threat to grapes) and *Scaphoideus titanus* in grapes. In addition, the documented substances are applied in crops that bees visit for pollen and nectar, such as stone fruits that are cultivated in the specific region (i.e., peach tree). Consequently, it is possible to rationalize the presented results in the context of land use of the specific regions and PPP applications. For coumaphos and amitraz (via its metabolites detection), their prevalence is expected due to their use as veterinary medicinal products to control the parasitic mite *Varroa destructor*. Last decade's literature verifies that these compounds are still detected in hive products [30–34], demonstrating in some cases exceedances of the ascribed MRLs (see indicatively [31]). As regards the other detected substances, the findings are largely in line with the recent bibliography. More specifically, in the recent literature concerning honey, pollen, and beebread [35–38], substances such as boscalid, carbendazim, pyraclostrobin, chlorpyrifos, tau-fluvalinate, cypermethrin, fenpropathrin, and λ-cyhalothrin that were detected in the presented study are also reported. Nevertheless, compounds such as fluopyram and chlorothalonil that are reported in the bibliography were not included in the scope of the presented analytical method.

Regarding the human health risk assessment, the current findings should not be neglected due to the less frequent consumption of pollen/beebread. The last 20-year trend in the consumption of raw, unprocessed food [39], especially of organic origin with proven health benefits, involves apicultural products and is an attitude that steadily increases. The inclination to these products is further strengthened by the scientific reviews on the beneficial effects of the consumption of apicultural commodities on human health [40]. Therefore, it is anticipated that more adults and children are expected to consume such products in all their variations (e.g., pure pollen, beebread, honey, or wax-containing products). Another viewpoint is the consumption of beebread or honeycomb containing it, especially in agricultural communities where the proximity to "raw, unprocessed" food is easier. Honeycombs usually are not devoid of Varroa mites, leading to inevitable applications of coumaphos and other acaricides belonging to the class of organic chemicals. Last but not least, bees' susceptibility to mixtures of chemicals via pollen consumption is also apparent and can affect their longevity and survival. Consequently, any work or report on residue finding is of utmost importance and should be disseminated to increase awareness for the protection of such a pivotal insect.
