Study of pyrethroid resistance mutations in populations of Varroa destructor across Spain.

The Varroa destructor mite is a serious worldwide pest of honeybees that is usually controlled with pyrethroid-based acaricides. However, the intensive use of these substances over the past decades has led to the development of resistance in these mites. Here, Varroa samples collected between 2006 and 2021 from apiaries across Spain were studied to evaluate the presence of mutations producing pyrethroid resistance, particularly those in the gene encoding the voltage-gated sodium channel (VGSC). Genotyping of the IIS4-IIS5 region of this gene detected the L925V (Leucine 'CTG' to valine 'GTG') mutation at position 925 and confirmed the presence of the M918L (Methionine 'ATG' to Leucine 'TTG') mutation at position 918 in these Spanish Varroa mites. Interestingly, the M918L mutation was always found in combination with L925V, both of which were always homozygous. Over and above the high frequency of pyrethroid-resistant mutations in Spanish Varroa populations, this apparently recent association of the M918L and L925V point mutations is a combination that appears to trigger greater resistance than that produced by L925V alone.

Biosecurity measures in European beekeeping.

Emerging pathogens of honey bees represent an important threat to the development of the beekeeping sector. The implementation of biosecurity measures in beekeeping (BMBs) plays an essential role in supporting honey bee health within the beekeeping sector. A group of experts, in collaboration with the BPRACTICES (Grant Agreement No. 696231, European Research Area on Sustainable Animal Production Systems [ERA-Net SusAn]) project partners, has provided the definition of BMBs. Thus, BMBs are all those operational activities implemented to control the risk of introduction and spread of specific honey bee disease agents. In this paper, the BMBs in the European beekeeping context are identified for the most relevant honey bee diseases in Europe: varroosis, American foulbrood (AFB), European foulbrood (EFB), nosemosis and aethinosis. Moreover, BMBs were classified in â€Ëœcategories' adapted to consider productivity and the â€ËœOne Health' approach: human health, honey bee health and protection of the environment. The 84 BMBs described by the panel of experts were ranked according to the priority score attributed. The implementation of BMBs represents an essential step forwards to increase the resilience and sustainability of European beekeeping.

L'émergence de nouveaux agents pathogènes chez les abeilles mellifères représente une menace importante pour le développement du secteur apicole. La mise en oeuvre de mesures de biosécurité en apiculture est déterminante pour préserver la santé des abeilles mellifères dans les élevages. En collaboration avec des partenaires du projet BPRACTICES (convention de subvention n° 696231, programme ERA-Net SusAn [European Research Area on Sustainable Animal Production Systems]), un groupe d'experts a élaboré un cadre définissant ces mesures de biosécurité. Sont définies comme mesures de biosécurité en apiculture toutes les activités opérationnelles mises en oeuvre pour contrôler le risque d'introduction et de propagation d'agents pathogènes affectant spécifiquement les abeilles mellifères. Les auteurs décrivent les mesures de biosécurité applicables, dans le contexte apicole européen, aux maladies les plus importantes en Europe : la varroose, la loque américaine, la loque européenne, la nosémose et l'infestation par Aethina tumida. En outre, les mesures de biosécurité en apiculture ont été regroupées en « catégories ¼ afin de prendre en compte la productivité et l'approche « Une seule santé ¼ : santé humaine, santé des abeilles mellifères et protection de l'environnement. Les 84 mesures de biosécurité en apiculture décrites par le groupe d'experts ont été classées en fonction du niveau de priorité qui leur a été attribué. La mise en oeuvre de ces mesures représente une étape cruciale pour accroître la résilience et la durabilité de l'apiculture européenne.

Los agentes patógenos emergentes que afectan a la abeja melífera suponen una importante amenaza para el desarrollo del sector apícola. La aplicación de medidas de seguridad biológica dentro de este sector cumple una función esencial para proteger la salud de las abejas. En colaboración con asociados en el proyecto BPRACTICES (acuerdo de subvención nº 696231, programa ERA-Net SusAn [Espacio Europeo de Investigación - «Sistemas sostenibles de producción animal¼]), un grupo de expertos definió las «medidas de seguridad biológica en apicultura¼ como todas aquellas acciones realizadas para controlar el riesgo de penetración y propagación de agentes patógenos de la abeja melífera. Los autores, situándose en el contexto de la apicultura europea, exponen las medidas de seguridad biológica que ayudan a controlar las principales enfermedades de la abeja melífera en Europa: varroosis, loque americana, loque europea, nosemosis y aethinosis (infestación por el escarabajo de las colmenas). Por otra parte, estas medidas fueron divididas en diferentes «categorías¼ para poder tener en cuenta las cuestiones de productividad y el enfoque de «Una sola salud¼: salud humana, salud de la abeja melífera y protección del medio ambiente. Las 84 medidas de seguridad biológica en apicultura que describió el cuadro de expertos fueron jerarquizadas en función de una puntuación atribuida por su nivel de prioridad. La aplicación de este tipo de medidas representa un crucial paso adelante para conferir más resiliencia a la apicultura europea y hacerla más sostenible.

Good farming practices in apiculture.

Modern European beekeeping is facing numerous challenges due to a variety of factors, mainly related to globalisation, agrochemical pollution and environmental changes. In addition to this, new pathogens threaten the health of European honey bees. In that context, correct colony management should encompass a wider vision, where productivity aspects are linked to a One Health approach in order to protect honey bees, humans and the environment. This paper describes a novel tool to be applied in beekeeping operations: good beekeeping practices (GBPs). The authors ranked a list of GBPs scored against their importance and validated by an international team, including researchers, national animal health authorities and international beekeepers' associations. These activities were carried out in the project 'BPRACTICES', approved within the transnational call of the European Research Area Network on Sustainable Animal Production (ERA-NET SusAn) in the Horizon 2020 Research and Innovation Programme of the European Union. This study, created through an international collaboration, aims to present an innovative and implementable approach, similar to applications already adopted in other livestock production systems.

L'apiculture moderne européenne est confrontée à de nombreuses difficultés dues à divers facteurs, pour la plupart liés à la mondialisation, à la pollution agrochimique et à la modification de l'environnement. À ces facteurs s'ajoute l'émergence de nouveaux agents pathogènes qui menacent la santé des abeilles mellifères d'Europe. Dans ce contexte, une gestion appropriée des colonies d'abeilles devrait reposer sur une vision plus large, dans laquelle les aspects relevant de la productivité sont examinés suivant une approche « Une seule santé ¼ afin de protéger les abeilles mellifères, les humains et l'environnement. Les auteurs décrivent un nouvel outil destiné à l'apiculture : les bonnes pratiques apicoles. Ils ont évalué et classé par ordre d'importance une liste de bonnes pratiques apicoles validées par une équipe internationale composée de chercheurs, d'autorités nationales de la santé animale et d'associations internationales d'apiculteurs. Ces activités ont été conduites dans le cadre du projet « BPRACTICES ¼, proposition retenue suite à l'appel à projets transnationaux du réseau ERA­NET SusAn (European Research Area Network on Sustainable Animal Production) au sein du Programme Horizon 2020 de l'Union européenne pour la recherche et l'innovation. Conçue sous forme de collaboration internationale, cette étude vise à proposer une approche innovante et pratique, similaire aux applications précédemment adoptées dans d'autres systèmes de production animale.

La apicultura europea hace frente a numerosos problemas resultantes de diversos factores, relacionados principalmente con la mundialización, la contaminación agroquímica y los cambios ambientales, a todo lo cual se suman nuevos patógenos que amenazan la salud de las abejas melíferas europeas. En este contexto, una correcta gestión de las colonias debe traer aparejada una visión más global, en la que las cuestiones de productividad se consideren en clave de «Una sola salud¼ con objeto de proteger tanto a las abejas melíferas como a las personas y el medio ambiente. En este artículo se describe una novedosa herramienta aplicable a la actividad apícola: las buenas prácticas de apicultura. Los autores jerarquizaron una serie de buenas prácticas de apicultura seleccionadas, validadas y puntuadas según su importancia por un equipo internacional que incluía a investigadores, autoridades nacionales de sanidad animal y asociaciones internacionales de apicultores. Este trabajo formaba parte del proyecto «BPRACTICES¼, aprobado con ocasión de la convocatoria internacional abierta por la Red del espacio europeo de investigación en sanidad animal sostenible (ERA­NET SusAn), inscrita a su vez en Horizonte 2020, el programa de investigación e innovación de la Unión Europea. El estudio aquí descrito, fruto de la colaboración internacional, tiene por objeto presentar un planteamiento novedoso y viable, parecido a las aplicaciones ya implantadas en otros sistemas de producción animal.

Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae.

The honeybee disease nosemosis type C is a serious problem since its causative agent, microsporidium Nosema ceranae, is widespread among adult honey bees. Some of the feasible alternative treatments that are used to control this disease are plant extracts. The aim of the present work was to evaluate the effects of essential oils of Chilean plant species, such as Cryptocarya alba, which is used against N. ceranae, and to identify and quantify the majority active compounds in the EO as well as their potential use for the control of nosemosis. Essential oils were obtained using the stripping steam technique with Clevenger equipment and were subsequently analyzed by Gas chromatography-mass spectrometry. Mortality was recorded daily over at least 8days as worker honeybees were exposed to a range of doses of EO dispersed in a sucrose solution. C. alba oil appears to be nontoxic to A. mellifera adults at the tested concentration (the same concentration inhibits the growth of N. ceranae), showing that this oil can be used for the treatment of nosemosis. EO effectiveness was demonstrated against N. ceranae by calculating the percentage of decrease in infected bees from untreated infected groups vs infected groups treated with EO or the reference drug fumagillin. It was determined that a dose of 4µg EO/bee was most effective in controlling N. ceranae development. We determined innocuous doses of C. alba essential oil for honeybees. We demonstrated the antifungal activity of C. alba EO at 4µg/bee against N. ceranae and compared it to its major monoterpenes, such as ß-phellandrene (20µg/bee), eucalyptol (20µg/bee) and α-terpineol (20µg/bee). The major compounds of C. alba EO, α-terpineol, eucalyptol and ß-phellandrene, had significant effects against Apis mellifera infection by N. ceranae, but the antifungal effect of the complete essential oil on N. ceranae was larger than the effect of α-terpineol, eucalyptol or ß- phellandrene separately, showing that C. alba oil may be a candidate for the treatment or prevention of nosemosis.

Characterization of Nosema ceranae Genetic Variants from Different Geographic Origins.

In recent years, large-scale colony losses of honey bees (Apis mellifera) have been reported and the infection with the microsporidia Nosema ceranae has been involved. However, the effect of N. ceranae at the colony level and its role in colony losses vary in different geographic areas. This difference may be related to the presence of multiple N. ceranae genetic variants resulting in different biological consequences. In this study, we analyzed the genetic diversity of 75 N. ceranae samples obtained from 13 countries and Hawaii through inter-sequence single repetition (ISSR) and evaluated if two of these genetic variants triggered different immune responses when infecting Apis mellifera iberiensis. The genetic diversity analysis showed that 41% of the samples had the same DNA amplification pattern, including samples from most European countries except Spain, while the remaining samples showed high variability. Infection assays were performed to analyze the infection levels and the immune response of bees infected with N. ceranae from Spain and Uruguay. The infected bees presented similar infection levels, and both isolates downregulated the expression of abaecin, confirming the ability of the microsporidia to depress the immune response. Only N. ceranae from Uruguay downregulated the expression level of imd compared to control bees. On the other hand, both genetic variants triggered different expression levels of lysozyme. As imd and lysozyme play important roles in the response to pathogens, these results could reflect differences in the biological consequences of N. ceranae variants in A. mellifera infection.

High prevalence and infection levels of Nosema ceranae in bumblebees Bombus atratus and Bombus bellicosus from Uruguay.

Nosema ceranae is one of the most prevalent pathogens in Apis mellifera and has recently been found in multiple host species including several species of bumblebees. Prevalence and infection intensity of N. ceranae was determined in two species of native bumblebees from Uruguay. Nosema ceranae was the only microsporidia identified and mean prevalence was 72% in Bombus atratus and 63% in Bombus bellicosus, values much higher than those reported elsewhere. The presence of this pathogen in bumblebees may be threatening not only for bumblebee populations, but also to the rest of the native pollinator community and to honeybees.

Recent worldwide expansion of Nosema ceranae (Microsporidia) in Apis mellifera populations inferred from multilocus patterns of genetic variation.

Nosema ceranae has been found infecting Apismellifera colonies with increasing frequency and it now represents a major threat to the health and long-term survival of these honeybees worldwide. However, so far little is known about the population genetics of this parasite. Here, we describe the patterns of genetic variation at three genomic loci in a collection of isolates from all over the world. Our main findings are: (i) the levels of genetic polymorphism (πS≈1%) do not vary significantly across its distribution range, (ii) there is substantial evidence for recombination among haplotypes, (iii) the best part of the observed genetic variance corresponds to differences within bee colonies (up to 88% of the total variance), (iv) parasites collected from Asian honeybees (Apis cerana and Apis florea) display significant differentiation from those obtained from Apismellifera (8-16% of the total variance, p<0.01) and (v) there is a significant excess of low frequency variants over neutral expectations among samples obtained from A. mellifera, but not from Asian honeybees. Overall these results are consistent with a recent colonization and rapid expansion of N. ceranae throughout A. mellifera colonies.

Is Acarapis woodi a single species? A new PCR protocol to evaluate its prevalence.

Acarapisosis is a disease of the adult honey bee Apis mellifera L., caused by the tracheal mite Acarapis woodi (Rennie), that affects the prothoracic tracheas of worker honey bees. Although it is not usually considered a real problem for honey bee colonies in southern Europe (mainly Spain and Greece), where the majority of professional beekeepers are located in Europe, recent works have reported the constant presence of this mite in this area, making it a potential cofactor for colony losses. In this study, we developed a specific PCR diagnostic tool that improves the techniques used so far and allowed us to confirm the presence of this parasite in Spain, urging the need to monitor its prevalence and implications in the health of the colonies. Indeed, in a total of 635 apiaries analysed, the prevalence of A. woodi in 2010 was 8.3 and 4 % in 2011. The mite is present in bee colonies over time and should not be underestimated as a possible cofactor in the collapse of bee colonies. Additionally, some positive samples were cloned so a genetic analysis on the diversity within A. woodi isolates was also approached. This allowed us to identify different genetic variants within an isolate, even when they were present at low frequencies. And this genetic analysis revealed the existence of a different clade of Acarapis sequences that could represent a new species or subspecies, although more research is required to verify the identity of this novel lineage at genetic and morphological level.

Flow cytometry analysis of Nosema species to assess spore viability and longevity.

Nosema apis and Nosema ceranae are microsporidia which present resistant spores for the transmission stage (environmental spores) that play an important role for epidemiology and for laboratory studies of honey bee microsporidiosis. In this study, the long-term longevity of N. apis and N. ceranae spores exposed to 4 °C, room temperature (mean 25 °C) and 35 °C for 6-month long and to -20 °C for 10-month long has been assessed by flow cytometry. Storage temperature and the length of storage duration had adverse effects on spore viability of both Nosema spores, with significant differences between the two species. The greatest increase in spore mortality was observed in N. apis spores stored at 33 °C (64, 89%) and in N. ceranae spores at -20 °C (53.55%) and at 33 °C (51.97%). For N. ceranae spores at -20 °C, the loss in viability was very quick, getting an increase over 20% just after 6 days of exposure. Results on viability were confirmed by the infectivity tests where the lowest infectivity for N. ceranae was observed with spores stored for 10 months at -20 °C (79%; P < 0.05) and for N. apis with spores stored at 33 °C (71%; P < 0.05). For both Nosema species, the best storage temperatures were 25 and 4 °C, especially for N. apis that was almost unaffected at those temperatures.

Low prevalence of honeybee viruses in Spain during 2006 and 2007.

RNA viruses that affect honeybees have been involved in colony losses reported around the world. The aim of the present work was to evaluate the prevalence and distribution of honeybee viruses during 2006-2007 in Spanish professional apiaries, and their association with colony losses. Four hundred and fifty-six samples from apiaries located in different geographic regions of Spain were analyzed. Thirty-seven percent of the samples had viral presence. Most (80%) had one virus and 20% two different viruses. All the analyzed viruses, Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Kashmir Bee Virus (KBV) were detected, but detection rates were lower than expected. According to these results and considering the high prevalence of other honeybee pathogens in Spain, the role of viruses in colony losses in Spain may be discussed.

Determination of tylosins A, B, C and D in bee larvae by liquid chromatography coupled to ion trap-tandem mass spectrometry.

A LC-MS/MS method has been developed to simultaneously quantify tylosins A, B, C and D in bee larvae, compounds currently used to treat one of the most lethal diseases affecting honey bees around the world, American Foulbrood (AFB). The influence of different aqueous media, temperature and light exposure on the stability of these four compounds was studied. The analytes were extracted from bee larvae with methanol and chromatographic separation was achieved on a Luna C(18) (150 × 4.6 mm i.d.) using a ternary gradient composed of a diluted formic acid, methanol and acetonitrile mobile phase. To facilitate sampling, bee larvae were initially dried at 60°C for 4h and afterwards, they were diluted to avoid problems of pressure. MSD-Ion Trap detection was employed with electrospray ionization (ESI). The calibration curves were linear over a wide range of concentrations and the method was validated as sensitive, precise and accurate within the limits of quantification (LOQ, 1.4-4.0 ng/g). The validated method was successfully employed to study bee larvae in field tests of bee hives treated with two formulations containing tylosin. In both cases it was evident that the minimal inhibitory concentration (MIC) had been reached.

Evaluation of large-scale dissemination of Nosema ceranae spores by European bee-eaters Merops apiaster.

Identification of transmission routes and of factors affecting the spatial positions of pathogens, hosts and vectors is basic to an adequate disease management. Nosema ceranae is a Microsporidian recently described as a parasite of Apis mellifera honeybees and is currently considered the aetiological agent of an emergent illness named nosemosis type C. In this article we evaluate the role of a bird species, the European bee-eater, Merops apiaster, as a large-scale dispersive agent of N. ceranae. We found a high prevalence of viable spores of N. ceranae in pellets regurgitated by bee-eaters in different locations in the Iberian Peninsula, Central Europe and central Asia. In contrast, spores of Nosema apis, considered till recently the most common microsporidium infecting honeybees, were detected in a single locality and Nosema bombi spores were not noticed. Since non-viable spores were also found in bee-eater nests from different locations, this bird species could also reduce the fraction of infected insects by withdrawing pathogens from the colonies. We conclude that bee-eater mobility and migration may have played an important role in the transmission of the pathogen N. ceranae.

Overview of pesticide residues in stored pollen and their potential effect on bee colony (Apis mellifera) losses in Spain.

In the last decade, an increase in honey bee (Apis mellifera L.) colony losses has been reported in several countries. The causes of this decline are still not clear. This study was set out to evaluate the pesticide residues in stored pollen from honey bee colonies and their possible impact on honey bee losses in Spain. In total, 1,021 professional apiaries were randomly selected. All pollen samples were subjected to multiresidue analysis by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS; moreover, specific methods were applied for neonicotinoids and fipronil. A palynological analysis also was carried out to confirm the type of foraging crop. Pesticide residues were detected in 42% of samples collected in spring, and only in 31% of samples collected in autumn. Fluvalinate and chlorfenvinphos were the most frequently detected pesticides in the analyzed samples. Fipronil was detected in 3.7% of all the spring samples but never in autumn samples, and neonicotinoid residues were not detected. More than 47.8% of stored pollen samples belonged to wild vegetation, and sunflower (Heliantus spp.) pollen was only detected in 10.4% of the samples. A direct relation between pesticide residues found in stored pollen samples and colony losses was not evident accordingly to the obtained results. Further studies are necessary to determine the possible role of the most frequent and abundant pesticides (such as acaricides) and the synergism among them and with other pathogens more prevalent in Spain.

High-level resistance of Nosema ceranae, a parasite of the honeybee, to temperature and desiccation.

Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4',6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35 degrees C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4 degrees C were also detected.

Trace analysis of fumagillin in honey by liquid chromatography-diode array-electrospray ionization mass spectrometry.

In this work a new liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) method has been developed for the determination of fumagillin residues in honey. This procedure involves a solid-phase extraction on polymeric cartridges for the isolation of fumagillin from diluted honey. Chromatographic separation of fumagillin was performed in isocratic mode, on a C(18) column (150 mm x 4.60mm i.d., 5 microm), the mobile phase consisted of a mixture of ammonium formate 20mM in water and acetonitrile (61/39, v/v), at 35 degrees C and the flow rate was set at 1.0 mL/min. Average analyte recoveries, influenced by the botanical origin were from 88 to 96% in replica sets of fortified honey samples. The detection limits of the LC-DAD-ESI-MS method were between 24 and 1 microg/kg for clear honeys (rosemary) and between 45 and 4 microg/kg for dark honeys (heather). The developed method has been applied to the analysis of fumagillin residues in honey samples collected from veterinary treated beehives, infected by Nosema ceranae and fed with the technical product at different doses.

Trace analysis of tiamulin in honey by liquid chromatography-diode array-electrospray ionization mass spectrometry detection.

A liquid chromatography with diode array or electrospray ionisation mass spectrometry detection (LC-DAD-ESI-MS) method for the determination of tiamulin residues in honey is presented. The procedure employs a solid-phase extraction (SPE) on polymeric cartridges for the isolation of tiamulin from honey samples diluted in aqueous solution of tartaric acid. Chromatographic separation of the tiamulin is performed, in isocratic mode, on a C18 column using methanol and ammonium carbonate 0.1% in water, in proportion (30:70, v/v). Average analyte recoveries were from 88 to 106% in replica sets of fortified honey samples. The LC-ESI-MS method detection limits differ from 0.5 microg kg(-1) for clear honeys to 1.2 microg kg(-1) for dark honeys. The developed method has been applied to the analysis of tiamulin residues in multifloral honey samples collected from veterinary treated beehives.

Extraction of thymol, eucalyptol, menthol, and camphor residues from honey and beeswax. Determination by gas chromatography with flame ionization detection.

A gas chromatographic method to determine thymol, eucalyptol (cineole), menthol and camphor residues in honey and beeswax is proposed. To isolate the compounds, three methods involving liquid-liquid extraction with methylene chloride, distillation, or solid-phase extraction on octadecylsilica cartridges can be used. The GC separation is carried out on a 60 m x 0.53 mm Stabilwax DA capillary column, using a flame ionization detector. The method is applied to the analysis of natural honey and also honey and beeswax samples from beehives treated with the above compounds.

Determination of oxalate, sulfate and nitrate in honey and honeydew by ion-chromatography.

An ion chromatographic method for determining the anions oxalate, sulfate and nitrate in honey and honeydew samples is described. To prevent matrix interference and to isolate the anions a clean-up step using solid-phase extraction on anionic cartridges and eluting with a 0.01 M chromate solution is recommended. The anions are separated on an anionic column with a mobile phase of borate-gluconate buffer and using conductimetric detection. The method is applied to the analysis of samples from different botanical origin.

Gas chromatographic determination of acrinathrine and 3-phenoxybenzaldehyde residues in honey.

A procedure involving an extraction step and further gas chromatographic analysis with flame ionization detection to determine residues of acrinathrine and its main metabolite, 3-phenoxybenzaldehyde, in honey is proposed. Residues can be isolated from the matrix by means of liquid-liquid extraction with a mixture of benzene-isopropanol, by solid-phase extraction with octadecylsilane cartridges or Florisil packed columns, the latter method giving higher recoveries. Assays on spiked honey samples are carried out to test the procedures that are afterwards applied to honey samples from treated beehives.

Determination of rotenone residues in raw honey by solid-phase extraction and high-performance liquid chromatography.

A method for determining residues of the insecticide rotenone in raw-honey by high-performance liquid chromatography (HPLC) is described. To extract the residues, organic solvents such as ethyl acetate, n-hexane/dichloromethane and solid-phase extraction with octadecylsilane cartridges or Florisil packed columns were tested. Determination was carried out by reversed-phase HPLC using acetonitrile-buffer phosphate (pH 7) (60:40, v/v) as mobile phase and detection at 210 nm. Although the data showed that the two extraction methods were able to isolate the pesticide residues, the extraction on octadecylsilane cartridges was preferred due to its simplicity and higher recovery. Recoveries depended strongly on the fortification level for the two extraction procedures. Practical determination limits of 0.015 mg/kg were obtained. In the analysis of honeys, from beehives treated with rotenone at therapeutical doses for 1 month, residual amounts below 0.2 mg/kg were found.