Verifying the geographical origin of honey has become a key issue for food quality assurance, consumer safeguards and regulatory enforcement, particularly following recent European regulation mandating verifiable origin labeling. This task is particularly challenging for multifloral honey, as its chemical composition is shaped by local biodiversity, environmental factors, and agricultural activities. In this work, an integrated multi-analytical strategy was employed to characterize Italian multifloral honeys and identify indicators linked to their origin. Seventy-five multifloral honey samples collected from Northern, Central, and Southern Italy were examined using a combination of analytical techniques, i.e., the untargeted profiling of volatile organic compounds (VOCs) via dielectric barrier discharge ionization high-resolution mass spectrometry (DBDI-HRMS), quantification of sugars and amino acids by quantitative nuclear magnetic resonance (q-NMR), elemental analysis by total reflection X-ray fluorescence (TXRF), and and targeted assessment of pesticide residues. In parallel, several thousand melissopalynological analyses performed on multifloral Italian honey collected during 2015-2024 were statistically analyzed to retrieve the pollen species that characterize the Northern, Central and Southern Italian macro-areas. The statistical evaluation revealed clear regional patterns in pollen counts, VOCs, mineral composition, and specific sugars and amino acids, highlighting the influence of local flora, geological background, and environmental conditions. DBDI-HRMS exhibited a high ability to differentiate between Italian macro-regions, with cresol, santene, and acetoaminophenone having a pivotal function in demonstrating the bees' collection of chestnut, rhododendron, and linden nectars in the honey produced in Northern Italy, which is in alignment with the results of our pollen counts. Moreover, our DBDI-HRMS findings indicate that methylsyringate is a typical marker of Central and Southern honeys which had the highest counts of citrus pollen. Similarly, our melissopalynological assessment revealed that Italian Southern honey is distinguished by its high prevalence of citrus pollen. Q-NMR analysis identified distinctive trends in minor carbohydrates and amino acids. Honey from Southern Italy exhibited high proline, leucine, and isoleucine concentrations. These are quality indicators related to specific botanical origins, such as citrus and eucalyptus, which are prevalent in this region as demonstrated by our melissopalynological data. Honey samples originated from central and southern Italy presented significantly elevated bromine levels, probably due to the contribution of marine aerosol, and strontium concentrations revealed moderate increase along the north-south gradient. Within this integrated framework, pesticide residues were interpreted as indicators of environmental exposure and agricultural context rather than as direct geographical markers. Levels of pesticide residues, were generally minimal and largely compliant with regulatory thresholds, confirming the overall high quality of Italian honey. Overall, 62 out of 75 honeys were either completely residue-free or contained only trace amounts of pesticides. The low concentrations of acaricides, such as amitraz, fluvalinate, and coumaphos, reflects controlled use in hive treatments. The occasional detection of agricultural pesticides, such as propamocarb and chlorphenvinphos, suggests localized crop-related inputs and environmental drift consistent with regional land use. Collectively, the combination of diverse analytical datasets provides a solid approach for the geographical authentication of multifloral honey, supporting origin verification and the development of future classification models.

SummaryTwo analytical methods for the quantification of pyrrolizidine (PAs) and tropane alkaloids (TAs) in honey bee‐collected pollen, teas and herbal infusions have been in‐house validated using the UHPLC‐MS/MS technique. These methods showed good linearity, good accuracy and robustness. The methods have been applied to a preliminary investigation of forty‐seven pollen samples and thirty‐three packs of tea‐bags in light of Commission Regulations (EU) 2020/2040 and 2021/1408 regarding maximum limits of PAs and TAs in foodstuffs, respectively. About half of the pollen samples contained searched PAs but in concentrations significantly lower than the maximum limit established; two samples were contaminated with TAs. One third of teas or herbal infusions contained PAs or TAs, one sample exceeded the EU limits for PAs and one for TAs. Despite the final consumer’s health does not seem threatened, these results recommend accurate analytical methods and broader investigations for PAs and TAs in foods.

Member states of the European Union are required to ensure the initiation of monitoring programs to verify honey bee exposure to pesticides, where and as appropriate. Based on 620 samples of dead honey bees—42 of pollen, 183 of honey and 32 of vegetables—we highlighted the presence, as analyzed by liquid and gas chromatography coupled with tandem mass spectrometric detection, of many active substances, mainly tau-fluvalinate, piperonyl butoxide, chlorpyrifos and chlorpyrifos-methyl, permethrin and imidacloprid. Among the active substances found in analyzed matrices linked to honey bee killing incidents, 38 belong to hazard classes I and II, as methiocarb, methomyl, chlorpyrifos, cypermethrin and permethrin, thus representing a potential risk for human health. We have shown that, at different times between 2015 and 2020, during implementation of the Italian national guidelines for managing reports of bee colony mortality or depopulation associated with pesticide use, pesticide pollution events occurred that could raise concern for human health. Competent authorities could, as part of a One Health approach, exploit the information provided by existing reporting programs on honey bees and their products, in view of the close correlation to human health, animal health and ecosystem health.

Honeybee health can be compromised not only by infectious and infesting diseases, but also by the acute or chronic action of certain pesticides. In recent years, there have been numerous reports of colony mortality by Italian beekeepers, but the investigations of these losses have been inconsistent, both in relation to the type of personnel involved (beekeepers, official veterinarians, members of the police force, etc.) and the procedures utilized. It was therefore deemed necessary to draw up national guidelines with the aim of standardizing sampling active ties. In this paper, we present the results of a survey carried out in Italy from 2015 to 2019, following these guidelines. Residues of 150 pesticides in 696 samples were analyzed by LC-MS/MS and GC-MS/MS. On average, 50% of the honeybee samples were positive for one or more pesticides with an average of 2 different pesticides per sample and a maximum of seven active ingredients, some of which had been banned in Europe or were not authorized in Italy. Insecticides were the most frequently detected, mainly belonging to the pyrethroid group (49%, above all tau-fluvalinate), followed by organophosphates (chlorpyrifos, 18%) and neonicotinoids (imidacloprid, 7%). This work provides further evidence of the possible relationship between complex pesticide exposure and honeybee mortality and/or depopulation of hives.

In spring there is often a rise in honey bee mortality incidents. The aim of this study was to investigate the potential correlation, in the reported incidents, between exposure to pesticide treatments and virus infections. Here we summarize the situation in northeastern Italy during spring 2014, evaluated by monitoring 150 active ingredients and three honey bee viruses in dead honey bees and other matrices. At least one active ingredient was found in 72.2% of the 79 dead honey bee samples, with the most abundant (59.4%) being insecticides, mainly belonging to the class of neonicotinoids (41.8%), followed by fungicides (40.6%). Imidacloprid, chlorpyrifos, tau-fluvalinate, and cyprodinil were the most frequently detected active ingredients. Multiple virus infections were monitored, revealing a high prevalence of chronic bee paralysis virus (CBPV) and deformed wing virus (DWV), detected in all samples except one. 71 and 37% of the CBPV- and DWV positive samples, respectively, showed a high number of viral copies per bee (>107). This work emphasizes the possible relationship between spring mortality in honey bees and pesticide treatments. Honey bee viruses could synergistically exacerbate the negative impact of pesticides on honey bee health, endangering the survival of colonies.

Lead is a naturally occurring element but largely originating from human activities. Food is the major source of exposure to lead for humans and the publication of a scientific opinion from the European Food Safety Authority on the risks to human health related to the presence of lead in foodstuffs, led European Union to establish more restrictive limits for this contaminant in food from 1 January 2016. In particular, a maximum level of 0.10mgkg(-1) was established for honey. The retrospective evaluation of 995 honey samples analysed since 2005, revealed a progressive reduction in the concentration of lead, with a mean value of 0.045mgkg(-1) in 2015. Total 1.5% of honeys analysed in 2015 exceeded the maximum lead level and therefore will no longer be marketable. Interested beekeepers should clarify the causes of honey contamination and adopt corrective actions to keep their honey production within the legal levels of lead.

The aim of the present study was to evaluate the quality of 36 samples of different honey type supplied by local producers from Algeria in order to verify its compliance with the standards of Codex Alimentarius and European Union (EU). For that, five physicochemical parameters were analyzed using the HPLC method: hydroxyl-methyl furfural (HMF), sugars, diastase activity and search of antibiotic contamination with streptomycin and tetracycline. The physicochemical analyses of the Algerian honeys show that 56% of samples correspond to Codex standards and 44% not in conformity with the standards required by the Codex Alimentarius and EU, because part of the samples had one or more defects. The percentage not in conformity was due to the high rates of hydroxyl-methyl furfural, sucrose and also to the low enzyme level. Analysis performed by the laboratory to detect residues of tetracycline and streptomycin in honey have revealed insignificant traces of oxytetracycline in two samples of honey (0.03 ppb). From the present study, it is observed that the Algerian honey samples is not completely in agreement with the requirements of international honey standards which could be caused by inappropriate actions during processing and storage steps. Key words: Honey quality, sugar, diastase activity, hydroxymethylfurfural, antibiotic residues.

An uncontrolled used of antibiotics and sulphonamides is responsible for the presence of residues in beehive products and contributes to the problem of food safety. The optimisation of analytical methods for the detection of residues of antibacterial drugs (sulphonamides, SPs; tylosin, TL; streptomycin, ST) in honey was pursued and the methods applied to routine samples. ST was investigated by liquid chromatographic technique with a post column reaction and fluorimetric detection. SPs were quantified by HPLC equipped with fluorimetric detector and C8 column after a precolumn derivatisation by fluorescamine. The presence of TL residues in honey was confirmed by liquid chromatography - tandem mass spectrometric detection. Chromatographic separation of TL was performed on a C18 column. The validation parameters obtained with these methods were: 88%, 101% and 95-102% for recovery and 10 ng/g, 5 ng/g and 1 ng/g as quantification limits for ST, TL and SPs, respectively.

Although American Foulbrood is well known, this disease is still a relevant cause of beehive and economic losses to beekeepers. Gamma radiation from a Cobalt-60 source provides an effective means of treating AFB contaminated beekeeping material for its reuse in apiary. Contaminated equipment (hive, frames, wax, honey) was irradiated at three different dosages: 10, 15 and 25 kGray. The infectivity of the spores contained in scales and honey was tested before and after the radiation treatment by culture. Furthermore, the gamma rays effects on beeswax and honey were investigated. None of the irradiated samples was positive to P. larvae irrespective of the radiation level applied. At a 10 kGray radiation level no particular changes in beeswax composition were registered. The main physico-chemical modifications observed in honey were those of enzymatic activity decrease, bubbles formation and leakage of honey out of frames.

The use of antibiotic drugs in the control of American foul brood (AFB) and other honeybee diseases , is still a common practice. Two type of drugs, tetracyclines (TCs) and sulphonamides (SPs), are frequently used, although during the last years other antibiotics like chloramphenicol (CAF), streptomycin (ST) and nitrofuran have been recognised to be administered to colonies by beekeepers. The use of these drugs is illegal because no maximum residue limits (MRL’s) have been established, whereas only OTC is approved in USA. Since antibiotics are active against the vegetative form of bacteria, in the case of AFB the drug administration to infected colonies suppress bacterial replication, but the symptoms readily reappear once the drug is discontinued (Ratnieks, 1992). As a consequence, beekeepers have to repeat drug treatments as preventive method to control of the diseases. This uncontrolled drugs administration determines both an increase of resistant bacteria and the contamination of honey. According to Council Directive 96/23/EC laying down measures to monitor certain substances and residues thereof in live animals and animals residues, honey imported from third countries has to be investigated for antibacterial substances. Honey samples analysed for antibiotics residues from 2001 to 2003 were considered in our laboratory.

Although American Foulbrood is well known, this disease is still a relevant cause of beehive and economic losses to beekeepers. Gamma radiation from a Cobalt-60 source provides an effective means of treating AFB contaminated beekeeping material for its reuse in apiary. Contaminated equipment (hive, frames, wax, honey) was irradiated at three different dosages: 10, 15 and 25 kGray. The infectivity of the spores contained in scales and honey was tested before and after the radiation treatment by culture. Furthermore, the gamma rays effects on beeswax and honey were investigated. None of the irradiated samples was positive to P. larvae irrespective of the radiation level applied. At a 10 kGray radiation level no particular changes in beeswax composition were registered. The main physico-chemical modifications observed in honey were those of enzymatic activity decrease, bubbles formation and leakage of honey out of frames.

National Reference Laboratory for Beekeeping Istituto Zooprofilattico Sperimentale delle Venezie Viale dell’Universita, 10; 35020 Legnaro (PD), Italy; Phone: +39 049 8084259; Fax: +39 049 8084258; email:fmutinelli@izsvenezie.it *Istituto Nazionale di Apicoltura,via di Saliceto, 80, 40128 Bologna, Italy, e-mail: agsabatini@inapicoltura.org **Universitat Hohenheim, Landesanstalt fur Bienenkunde, 70593 Stuttgart, Germany, e-mail: bienewa@uni-hohenheim.de ***Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Via A. Bianchi, 9, 25124 Brescia, Italy, e-mail: rpiro@bs.izs.it