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Declines in insect pollinators have been linked to a range of causative factors such as disease, loss of habitats, the quality and availability of food, and exposure to pesticides. Here, we analysed an extensive dataset generated from pesticide screening of foraging insects, pollen-nectar stores/beebread, pollen and ingested nectar across three species of bees collected at 128 European sites set in two types of crop. In this paper, we aimed to (i) derive a new index to summarise key aspects of complex pesticide exposure data and (ii) understand the links between pesticide exposures depicted by the different matrices, bee species and apple orchards versus oilseed rape crops. We found that summary indices were highly correlated with the number of pesticides detected in the related matrix but not with which pesticides were present. Matrices collected from apple orchards generally contained a higher number of pesticides (7.6 pesticides per site) than matrices from sites collected from oilseed rape crops (3.5 pesticides), with fungicides being highly represented in apple crops. A greater number of pesticides were found in pollen-nectar stores/beebread and pollen matrices compared with nectar and bee body matrices. Our results show that for a complete assessment of pollinator pesticide exposure, it is necessary to consider several different exposure routes and multiple species of bees across different agricultural systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marie-Pierre CHAUZAT reports financial support was provided by European Commission. Marion LAURENT reports financial support was provided by European Commission. Matthias ALBRECHT reports financial support was provided by European Commission. Mark JF BROWN reports financial support was provided by European Commission. Joachim DE MIRANDA reports financial support was provided by European Commission. Reet KARISE reports financial support was provided by European Commission. Jessica KNAPP reports financial support was provided by European Commission. Jose SERRANO MARINO reports financial support was provided by European Commission. Simon G. POTTS reports financial support was provided by European Commission. Maj RUNDLOF reports financial support was provided by European Commission. Janine SCHWARZ reports financial support was provided by European Commission. Eleanor ATTRIDGE reports financial support was provided by European Commission. Irene BOTTERO reports financial support was provided by European Commission. Elena CINI reports financial support was provided by European Commission. Maria Pilar DE LA RUA reports financial support was provided by European Commission. Gennaro DI PRISCO reports financial support was provided by European Commission. Christophe DOMINIK reports financial support was provided by European Commission. Daniel DZUL reports financial support was provided by European Commission. Andres GARCIA REINA reports financial support was provided by European Commission. Simon HODGE reports financial support was provided by European Commission. Alexandra M KLEIN reports financial support was provided by European Commission. Anina KNAUER reports financial support was provided by European Commission. Marika MAND reports financial support was provided by European Commission. Vicente MARTINEZ LOPEZ reports financial support was provided by European Commission. Giorgia SERRA reports financial support was provided by European Commission. Helena PEREIRA-PEIXOTO reports financial support was provided by European Commission. Risto RAIMETS reports financial support was provided by European Commission. Oliver SCHWEIGER reports financial support was provided by European Commission. Deepa SENAPATHI reports financial support was provided by European Commission. Jane C. STOUT reports financial support was provided by European Commission. Giovanni TAMBURINI reports financial support was provided by European Commission. Cecilia COSTA reports financial support was provided by European Commission. Tomasz KILJANEK reports financial support was provided by European Commission. Anne-Claire MARTEL reports financial support was provided by European Commission. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sustainable agriculture requires balancing crop yields with the effects of pesticides on non-target organisms, such as bees and other crop pollinators. Field studies demonstrated that agricultural use of neonicotinoid insecticides can negatively affect wild bee species 1,2 , leading to restrictions on these compounds 3 . However, besides neonicotinoids, field-based evidence of the effects of landscape pesticide exposure on wild bees is lacking. Bees encounter many pesticides in agricultural landscapes 4-9 and the effects of this landscape exposure on colony growth and development of any bee species remains unknown. Here we show that the many pesticides found in bumble bee-collected pollen are associated with reduced colony performance during crop bloom, especially in simplified landscapes with intensive agricultural practices. Our results from 316 Bombus terrestris colonies at 106 agricultural sites across eight European countries confirm that the regulatory system fails to sufficiently prevent pesticide-related impacts on non-target organisms, even for a eusocial pollinator species in which colony size may buffer against such impacts 10,11 . These findings support the need for postapproval monitoring of both pesticide exposure and effects to confirm that the regulatory process is sufficiently protective in limiting the collateral environmental damage of agricultural pesticide use., (© 2023. The Author(s).)

The study was designed to test the feasibility of using 3D-printed pollen traps for long-term monitoring of Bombus terrestris colonies' exposure to pesticide residues in pollen loads collected by them, along with an assessment of the resulting risks to the bumblebee's adults, larvae, and queens. Bumblebee colonies were placed in the vicinity of flowering orchards, winter oilseed rape, allotments, or home gardens for 6 weeks of the experiment. Pollen traps printed in 3D technology were installed in the hive inlets. The weight of bumblebee pollen loads obtained using pollen traps was in the range of 0.036-5.83 g. Pollen load samples were analyzed for residues of up to 261 pesticides and their metabolites by liquid and gas chromatography techniques coupled to tandem mass spectrometry (LC-MS/MS and GC-MS/MS). Residues of 18 fungicides, 12 herbicides, 6 insecticides, and an acaricide were detected. Herbicide - pendimethalin, fungicide - thiophanate-methyl, and insecticide - chlorpyrifos-ethyl were the most commonly detected pesticides. Chlorpyrifos and thiacloprid residues were detected in pollen load samples in the next year after their ban from use as plant protection products in the European Union. The risk of acute or chronic effects was assessed as negligible or low, although the chronic risk of bumblebee queens to insecticide chlorpyrifos and the acute risk of larvae exposed to acaricide fenpyroximate could be interpreted as moderate. The risk of sublethal effects related to chronic exposure of adult bumblebees and queens to pollen loads contaminated by chlorpyrifos-ethyl and cypermethrin cannot be excluded. The risk of chronic toxicity or sublethal effects may be particularly relevant for bumblebee queens, especially during their foraging in the initial period of establishing a new colony., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Biogenic amines (BAs) are organic, basic nitrogenous compounds formed during the decarboxylation of amino acids. A method for the determination of eight biogenic amines (tryptamine, 2-phenyletylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine) in ripened cheeses was developed and validated. Cheese samples with the addition of internal standards were extracted with 0.2 M perchloric acid and pre-column derivatized with dansyl chloride at 60 °C for 15 min, purified with toluene and dried under a stream of nitrogen. The samples were analyzed using high performance liquid chromatography with diode array detector (HPLC-DAD). The method was validated with the BAs at three concentration levels: 50, 100, and 200 mg/kg, respectively. The obtained values of correlation coefficient (R2) ranged at 0.9997−0.9998 for all of compounds. The limits of detection (LOD) and quantification (LOQ) were in ranges 1.53−1.88 and 5.13−6.28 mg/kg, respectively. The recovery for all of biogenic amines ranged from 70 to 120% and the precision (RSDr) value were <20%. The validated method was applied to analysis of 35 real ripened cheese samples purchased in Poland.

Pesticide exposure and food stress are major threats to bees, but their potential synergistic impacts under field-realistic conditions remain poorly understood and are not considered in current pesticide risk assessments. We conducted a semi-field experiment to examine the single and interactive effects of the novel insecticide flupyradifurone (FPF) and nutritional stress on fitness proxies in the solitary bee Osmia bicornis . Individually marked bees were released into flight cages with monocultures of buckwheat, wild mustard or purple tansy, which were assigned to an insecticide treatment (FPF or control) in a crossed design. Nutritional stress, which was high in bees foraging on buckwheat, intermediate on wild mustard and low on purple tansy, modulated the impact of insecticide exposure. Within the first day after application of FPF, mortality of bees feeding on buckwheat was 29 times higher compared with control treatments, while mortality of FPF exposed and control bees was similar in the other two plant species. Moreover, we found negative synergistic impacts of FPF and nutritional stress on offspring production, flight activity, flight duration and flower visitation frequency. These results reveal that environmental policies and risk assessment schemes that ignore interactions among anthropogenic stressors will fail to adequately protect bees and the pollination services they provide.

The aim of this study was to develop a method for the determination of glyphosate, its metabolite aminomethylphosphonic acid (AMPA), and glufosinate ammonium residues in beebread samples, which could then be used to assess bees' exposure to their residues. The complexity of beebread's matrix, combined with the specific properties of glyphosate itself, required careful selection and optimization of each analysis step. The use of molecularly imprinted solid-phase extraction (MIP-SPE) by AFFINIMIP glyphosate as an initial clean-up step significantly eliminated matrix components and ensured an efficient derivatization step. Colorless beebread extracts were derivatized by the addition of 9-fluorenylmethyl chloroformate (FMOC-Cl). After derivatization, in order to remove FMOC-OH and residual borate buffer, a solid-phase extraction (SPE) clean-up step using Oasis HLB was carried out. Instrumental analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method was validated according to the SANTE/11312/2021 guideline at concentrations of 5, 10, and 100 µg/kg, and satisfactory recovery (trueness) values (76-111%) and precision (RSDr) ≤ 18% were obtained. The limit of quantification (LOQ) was 5 µg/kg for AMPA and glufosinate ammonium and 10 µg/kg for glyphosate. The method was positively verified by the international proficiency test. Analysis of beebread samples showed the method's usefulness in practice. The developed method could be a reliable tool for the assessment of beebread's contamination with residues of glyphosate, its metabolite AMPA, and glufosinate ammonium.

Current work presents developed and validated miniaturized method for residue analysis of 261 pesticides and their metabolites as well as 6 congeners of non-dioxin like polychlorinated biphenyls (ndl-PCB) in a very low mass beebread sample. Sample preparation is based on modified QuEChERS protocol with all steps miniaturized to enable multiresidue analysis of sample with extremely low weight. Sample of beebread (0.3 g) was extracted with 1 mL of acetonitrile containing 5% formic acid and ammonium formate salt were added, then extract was subjected to clean-up by freezing and two-step dispersive solid phase extraction (dSPE) with a Supel QuE Verde sorbents (Supelclean ENVI-Carb Y; Supelclean PSA; Z-Sep+; magnesium sulfate). After 1st step dSPE a portion of extract was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for 200 pesticide residues. Remaining extract was subjected to 2nd step dSPE clean-up by another Supel QuE Verde and then after concentration and solvent exchange it was analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) for another 61 pesticide and 6 ndl-PCB residues. Method enables determination of residues of 101 insecticides, 72 herbicides, 67 fungicides, 10 acaricides, 6 growth regulators, 5 veterinary drugs and 6 ndl-PCB's. Particular attention was paid to the pesticides being active substances of plant protection products recommended for the protection of winter oilseed rape and apple orchards which during their blooming periods are one of the most attractive sources of food for pollinators and could serve as representatives of other economically important crops. Method was validated according to the Guidance document SANTE/12682/2019 at six concentration levels from 0.001 to 0.5 mg kg -1 . The analysis of beebread samples spiked at the level of 0.01 mg kg -1 showed mean recovery (trueness) value of about 98% and RSDr (precision) below 20%. The small weight of the sample did not adversely affect the limits of quantification and 75% of analytes could be quantified at least at concentration of 0.005 mg kg -1 . Developed mini-method was tested in the analysis of beebread samples, each extracted from individual cell of honeycomb. It is the first time when analyses at single comb cell level were possible., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Current work presents a modified QuEChERS method for the determination of 207 pesticide residues in honey by LC-MS/MS and GC-MS/MS. Acetate buffered acetonitrile extraction with Z-Sep+ and PSA dispersive-SPE clean-up were used for sample preparation. Optimised conditions allows determination of neonicotinoids as well as other insecticides, fungicides, herbicides, acaricides, growth regulators and veterinary drugs in honey samples. Validated method enable sensitive analysis at least at concentrations from 0.001, 0.005 or 0.01 mg/kg for 45%, 41% and 14% of pesticides, respectively. Method was utilised for the analysis of 155 honey samples from Poland during 2015-2017. Residues of 21 pesticides were determined in honey. Cyano-substituted neonicotinoids (acetamiprid, thiacloprid) were quantified in 77% of samples and were the most frequently detected pesticides. Concentrations of acetamiprid was from 0.001 to 0.13 mg/kg whilst thiacloprid from 0.001 to 0.2 mg/kg. Fungicides were determined in 50% and amitraz metabolites in 35% of honey samples., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Amitraz is a formamide exhibiting both acaricidal and insecticidal activity and is frequently used by beekeepers to protect honeybee colonies against Varroa destructor mites. The aim of this apiary trial was to evaluate the impact of honeybee colony fumigation with amitraz on the level of contamination of honey stored in combs., Material and Methods: Experimental colonies were fumigated four times every four days with one tablet of Apiwarol per treatment. Honey was sampled from combs of brood chambers and combs of supers one day after each amitraz application and from harvested honey. Amitraz marker residues (as a total of amitraz and metabolites containing parts of molecules with properties specific to the 2,4-DMA group, expressed as amitraz) were evaluated in honey., Results: All analysed samples were contaminated with amitraz metabolites. 2,4-DMA and DMPF were the most frequently determined compounds. The average concentration of amitraz marker residue in honey from groups where a smouldering tablet was located directly in beehives was significantly higher than that of residue in honey from groups with indirect smoke generation. No significant effect on the honey contamination deriving from the place where it was exposed to smoke (combs of brood chambers and supers) was noted. Amitraz marker residues exceeded the MRL in 10% of honey samples from combs., Conclusion: Fumigation of beehives with amitraz results in contamination of honey stored in combs., Competing Interests: Conflict of Interests Statement. The authors declare that there is no conflict of interests regarding the publication of this article. Financial Disclosure Statement. This work was financially supported by statutory activity of the National Veterinary Research Institute in Pulawy and Research Institute of Horticulture in Skierniewice. Animal Rights Statement. None required.

Study combines data about the exposure of honeybees to pesticides from plant protection products and veterinary medicinal products. Residues of 200 pesticide and pesticide metabolites in 343 live and 74 poisoned honeybee samples, obtained during the years of 2014-2015, were determined by LC-MS/MS and GC-MS/MS. In 44% of live honeybee 48 different pesticide residues were found, mainly amitraz metabolites (DMF, DMPF) and chlorpyrifos. In 98% of poisoned honeybee 57 pesticides and metabolites were detected, mainly chlorpyrifos, dimethoate and clothianidin. In total 84 different pesticides were detected both in live and poisoned honeybees, they indicate 30 various modes of action. Differences between mean number of pesticide residues detected in live and poisoned honeybees clearly indicate the impact of multiple pesticides on honeybee health. Possible impact of systemic fungicides on the health of honeybees was studied. Applicability of hazard quotient counted as ratio between concentration of pesticides in honeybees and lethal dose in the interpretation whether detected concentration indicates acute toxic effects was shown., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A method for the determination of 200 pesticides and pesticide metabolites in honeybee samples has been developed and validated. Almost 98% of compounds included in this method are approved to use within European Union, as active substances of plant protection products or veterinary medicinal products used by beekeepers to control mites Varroa destructor in hives. Many significant metabolites, like metabolites of imidacloprid, thiacloprid, fipronil, methiocarb and amitraz, are also possible to detect. The sample preparation was based on the buffered QuEChERS method. Samples of bees were extracted with acetonitrile containing 1% acetic acid and then subjected to clean-up by dispersive solid phase extraction (dSPE) using a new Z-Sep+ sorbent and PSA. The majority of pesticides, including neonicotionoids and their metabolites, were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) but some of pesticides, especially pyrethroid insecticides, were analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS). The procedure was validated according to the Guidance document SANCO/12571/2013 at four concentration levels: 1, 5, 10 and 100 ng/g bees and verified in the international proficiency test. The analysis of bee samples spiked at the limit of quantification (LOQ) showed about 98% mean recovery value (trueness) and 97% of analytes showed recovery in the required range of 70-120% and RSDr (precision) below 20%. Linearity and matrix effects were also established. The LOQs of pesticides were in the range of 1-100 ng/g. The developed method allows determination of insecticides at concentrations of 10 ng/g or less, except abamectin and tebufenozide. LOQ values are lower than the median lethal doses LD50 for bees. The method was used to investigate more than 70 honeybee poisoning incidents. Data about detected pesticides and their metabolites are included., (Copyright © 2016 Elsevier B.V. All rights reserved.)