Evaluation of the potential migration of acaricides from stamped beeswax to honey simulating beehive conditions: a pilot study

Producción CientíficaThere is an issue concerning the migration of pesticides from stamped beeswax to other bee products, mainly, honey. This implies that honey and other beehive-based products could be contaminated, which would represent a potential risk to consumers and food safety. To determine the degree to which acaricides can be transferred from the stamped beeswax to the honey, a pilot experiment was designed consisting of placing beeswax that contained acaricides (endogenous content, τ-fluvalinate, chlorfenvinphos, and coumaphos; spiked samples, coumaphos) in contact with multifloral honey in an incubator. Temperature and shaking conditions were settled simulating beehive conditions. The isolation of the analytes from beeswax involved the use of a QuEChERS (quick, easy, cheap, effective, rugged & safe) method, and honey was analyzed through a solvent extraction procedure. Acaricides were determined in the resulting extracts by gas chromatography with mass spectrometry detection. Coumaphos was the only acaricide transferred into honey in beeswaxes with endogenous acaricide content; meanwhile, results of the experiments with spiked beeswax samples showed that the initial concentration present in the contaminated beeswax significantly influences the transfer to honey, as it was observed that in the spiked beeswax sheets with high levels of coumaphos, the migration from beeswax to honey arose.Plan Nacional de Investigación e Innovación Científica y Técnica 2013–2016, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria-INIA–FEDER (RTA2017-00004-C02-02

Trace analysis of flubendiamide in bee pollen using enhanced matrix removal-lipid sorbent clean-up and liquid chromatography-electrospray ionization mass spectrometry

Producción CientíficaIn this work, a new method has been proposed with the aim of determining flubendiamide, a recently commercialized insecticide, in bee pollen by using liquid chromatography coupled to electrospray ionization mass spectrometry. For this purpose, a novel sample treatment has been proposed that has proven efficient in terms of recovery (average analyte recoveries were between 90% and 102%) and absence of matrix effect, and one which is effective, fast and selective. This involved a solvent extraction using an acetonitrile and water mixture, and a clean-up stage where, in addition to freezing, an enhanced matrix removal-lipid sorbent was successfully used for the first time with this matrix and analyte. The chromatographic conditions were also optimized, by selecting a C18 based column (Gemini® C18) and acetic acid (1 mM) in water and methanol as mobile phase components, allowing elution of flubendiamide in<4 min, with a total analysis time of 14 min. Validation was carried out, with the result that all the parameters studied complied with existing European legislation. It should be noted that the sensitivity of the method was excellent, with a quantification limit (4 μg/kg) well below the maximum residue level established for this insecticide in bee products (50 μg/kg). Finally, several bee pollen samples were analyzed, and flubendiamide residues were not found in any of the cases.Este trabajo forma parte de los proyectos de investigación financiados por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2013-00042-C10-03 y 06)

Fast determination of neonicotinoid insecticides in beeswax by ultra-high performance liquid chromatography-tandem mass spectrometry using an enhanced matrix removal-lipid sorbent for clean-up

Producción CientíficaThis study is concerned with the development of a simple, rapid analytical method for simultaneously determining seven neonicotinoid insecticides (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid and thiacloprid) in beeswax, by means of ultra-high performance liquid chromatography coupled to a quadrupole-time-of-flight mass detector. An efficient sample treatment is proposed (with average analyte recoveries between 93% and 106%) involving solvent extraction with a methanol and ethyl acetate mixture (70:30, v/v), centrifugation, freezing with dry ice, a dispersive solid phase extraction (enhanced matrix removallipid) followed by evaporation. Chromatographic analysis (10.5 min) was performed on a core-shell technologybased column (Kinetex® EVO C18). The method was fully validated in terms of selectivity, ruggedness, limits of detection (LODs) and quantification (LOQs), matrix effect, linearity, uncertainty, trueness and precision. Low LOQs, ranging from 1.2 to 4.9 μg/kg, were obtained in all cases. Finally, the proposed methods were applied to neonicotinoid analysis of twenty one beeswax samples from different Spanish regions, only one of the pesticides (imidacloprid) being detected at low levels (< LOQ).Este trabajo forma parte de los proyectos de investigación financiados por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2013-00042-C10-03 y 06)

Development and validation of UHPLC–MS/MS methods for determination of neonicotinoid insecticides in royal jelly-based products

Producción CientíficaIn this study, new methods have been proposed to determine seven neonicotinoid insecticides (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid and thiacloprid) in royal jelly-based products, by means of ultra-high-performance liquid chromatography coupled to a quadrupole-time-of-flight mass detector. Efficient sample treatments (with average analyte recoveries between 83% and 109%) involving solid phase extraction (polymeric sorbent, Strata® X) and a dispersive liquid–liquid microextraction, were proposed to determine these neonicotinoids in liquid dietary supplements containing freeze-dried royal jelly and fresh royal jelly, respectively. Chromatographic analysis (8 min) was performed on a core-shell technology-based column (Kinetex® EVO C18). Both methods were fully validated and the data demonstrated that they are consistent, reliable and have a wide linear range of applicability. Low limits of quantification, ranging from 2.5 to 9.5 μg/kg, were obtained in all cases, and it was necessary to employ matrix-matched standards for correct quantification of three of the compounds in each of the royal jelly-based products. Finally, the proposed methods were applied to neonicotinoid analysis of royal jelly-based products from different Spanish regions.Este trabajo forma parte de los proyectos de investigación financiados por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2013-00042-C10-03 y 06)

Determination of acaricides in honeys from different botanical origins by gas chromatography-mass spectrometry

Producción CientíficaAn analytical method has been proposed and validated to determine seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, α-endosulfan, bromopropylate, coumaphos, and τ-fluvalinate) in honeys from different botanical origins (multifloral, heather and rosemary) by means of gas chromatography-mass spectrometry. An efficient and simple sample treatment was proposed that involved a solvent extraction with an ethyl acetate and cyclohexane (50:50, v/v) mixture. Chromatographic analysis (<25 min) was performed in a DB-5MS column under programmed temperature conditions. The method was validated in terms of selectivity, limits of detection (0.2–2.0 μg kg-1) and quantification (0.5–7.6 μg kg-1), linearity (limit of quantification-700 (heather) or 800 (multifloral and rosemary) μg kg-1), matrix effect (<20 % in most cases), trueness (recoveries between 81 % and 108 %), and precision (relative standard deviation < 15 %). Finally, of the seven acaricides investigated in several honey samples only τ-fluvalinate residues (<limit of quantification - 23 μg kg-1) were found.Este trabajo forma parte del proyecto de investigación financiado por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2017-00004-C02-02)

An optimized extraction procedure for determining acaricide residues in foundation sheets of beeswax by using gas chromatography-mass spectrometry

Producción CientíficaPesticides can be found in beehives for several reasons, including contamination from surrounding cultivars; yet one of the most pertinent is related to the fact that beekeepers employ acaricides to control various types of mites, which may accumulate in beeswax due to their lipophilic nature. In the present study, foundation sheets of different origins, collected over a period of three years, were analyzed to detect the residues of seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, alpha-endosulfan, bromopropylate, coumaphos, tau-fluvalinate) by gas chromatography with mass spectrometric detection. An efficient sample treatment (recoveries between 90% and 108%) is proposed, involving solvent extraction with 1% acetic acid in acetonitrile mixture followed by dispersive solid-phase extraction (enhanced matrix removal lipid) and a polishing step. An evaluation was made of the analytical performance of the proposed method. It was shown to be selective, linear from a limit of quantification to 5000 µg/kg, precise (relative standard deviation values were below 6%), and with a goo sensitivity (limit of quantification ranging from 5 to 10 µg/kg). Finally, results showed that a large majority of the sheets analyzed (>90%) contained residues of at least one of these compounds. Coumaphos and tau-fluvalinate residues were the most common, with chlorpyrifos and chlorfenvinphos detected to a lesser extent.Plan Nacional de Investigación Científica y Técnica y Innovación 2013–2016, Instituto Nacional de Investigación y Tecnología Agroalimentaria (INIA) y Fondo Europeo de Desarrollo Regional (FEDER) - (grant RTA2017-00004-C02-01 and 02

Residual tau-fluvalinate in honey bee colonies is coupled with evidence for selection for Varroa destructor resistance to pyrethroids

Producción CientíficaVarroa destructor is one of the most prevalent honey bee (Apis mellifera) pathogens worldwide. Nowadays, the main method to control this parasite involves the application of different acaricidal treatments, among which the pyrethroid tau-fluvalinate is one of the most widely used. However, the intensive and repetitive application of these chemicals generates a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies. Here we analysed the presence of residual tau-fluvalinate and the patterns of genetic resistance to this acaricide in Varroa mites collected from tau-fluvalinate untreated honey bee colonies. Our results show the widespread and persistent pyrethroid contamination of beeswax and beebread in the hives, along with an excess of pyrethroid-resistant genotypes and an overall increase in the frequency of the pyrethroid-resistant allele in the mite population over time. Persistent contamination of the hives likely compromises the efficacy of tau-fluvalinate treatments and, therefore, may have serious long-term consequences for the control of varroosis.Varroa destructor is considered one of the most devastating parasites of the honey bee, Apis mellifera, and a major problem for the beekeeping industry. Currently, the main method to control Varroa mites is the application of drugs that contain different acaricides as active ingredients. The pyrethroid tau-fluvalinate is one of the acaricides most widely used in beekeeping due to its efficacy and low toxicity to bees. However, the intensive and repetitive application of this compound produces a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies, compromising the acaricidal treatments efficacy. Here we studied the presence of tau-fluvalinate residues in hives and the evolution of genetic resistance to this acaricide in Varroa mites from honey bee colonies that received no pyrethroid treatment in the previous four years. Our data revealed the widespread and persistent tau-fluvalinate contamination of beeswax and beebread in hives, an overall increase of the pyrethroid resistance allele frequency and a generalized excess of resistant mites relative to Hardy–Weinberg equilibrium expectations. These results suggest that tau-fluvalinate contamination in the hives may seriously compromise the efficacy of pyrethroid-based mite control methods.Plan Nacional de Investigaciones Científicas y Técnicas e Innovación 2013-2016, Instituto Nacional de Investigación Agraria y Alimentaria y Tecnología (INIA) y Fondo Europeo de Desarrollo Regional (FEDER) - (Projets RTA2017-00004-C02-01 and RTA2017-00004-CO2-02

PID_19_20_012 Anexo 4

Examen de la convocatoria ordinaria.Departamento de Química Analític

pid_18_19_14_anexo 4.pdf

Exámenes realizados a los estudiantes de la asignatura Química Analítica II durante el curso 2018-2019.Departamento de Química Analític

pid_18_19_14_anexo 2.pdf

Poster enviado a la XXII reunión de la Sociedad Española de Química Analític