Impact of landscape composition on honey bee pollen contamination by pesticides: A multi-residue analysis.

The honey bee is the most common and important managed pollinator of crops. In recent years, honey bee colonies faced high mortality for multiple causes, including land-use change and the use of plant protection products (hereafter pesticides). This work aimed to explore how contamination by pesticides of pollen collected by honey bees was modulated by landscape composition and seasonality. We placed two honey bee colonies in 13 locations in Northern Italy in contrasting landscapes, from which we collected pollen samples monthly during the whole flowering season in 2019 and 2020. We searched for almost 400 compounds, including fungicides, herbicides, insecticides, and acaricides. We then calculated for each pollen sample the Pollen Hazard Quotient (PHQ), an index that provides a measure of multi-residue toxicity of contaminated pollen. Almost all pollen samples were contaminated by at least one compound. We detected 97 compounds, mainly fungicides, but insecticides and acaricides showed the highest toxicity. Fifteen % of the pollen samples had medium-high or high levels of PHQ, which could pose serious threats to honey bees. Fungicides showed a nearly constant PHQ throughout the season, while herbicides and insecticides and acaricides showed higher PHQ values in spring and early summer. Also, PHQ increased with increasing cover of agricultural and urban areas from April to July, while it was low and independent of landscape composition at the end of the season. The cover of perennial crops, i.e., fruit trees and vineyards, but not of annual crops, increased PHQ of pollen samples. Our work highlighted that the potential toxicity of pollen collected by honey bees was modulated by complex interactions among pesticide category, seasonality, and landscape composition. Due to the large number of compounds detected, our study should be complemented with additional experimental research on the potential interactive effects of multiple compounds on honey bee health.

Ferric chloride assisted QuEChERS method for separate detection of bifenazate and bifenazate-diazene in citrus fruits and its field validation.

Bifenazate is widely recognized as an effective acaricide for citrus production in various regions. Detecting both the parent compound of bifenazate and its metabolite, bifenazate-diazene, simultaneously can be challenging owing to their tendency to undergo chemical interconversion. Current methods developed for detecting bifenazate or bifenazate-diazene residues often involve lengthy incubation periods and may not effectively separate the two compounds. In this study, we developed a convenient and fast method based on a modified QuEChERS method assisted by oxidants to concurrently detect bifenazate and bifenazate-diazene. Based on preliminary analysis, it appears that ferric chloride has the ability to react with a reducing substance present in citrus, which may prevent the reduction of bifenazate-diazene. The method was validated and applied in a field trial. This work reports a novel strategy to establish a balanced 'neutral' condition to create a potential method for efficient determination of bifenazate acaricide residues in fruit matrices.

Acaricide residues in beeswax. Implications in honey, brood and honeybee.

For beekeeping to be sustainable, the management of colonies for the production of bee products must be economically viable without endangering the lives of bees, and must include acceptable practices such as the treatment of hives with appropriate products. Occasionally, the use of acaricides to treat the hives against varroosis is uncontrolled and can accumulate in the hives, putting the colonies at risk. In this work, a screening of seven acaricides was carried out in different apiaries in Andalusia (Spain). Their distribution in beeswax, brood, honey, and bees from colonies in different surroundings was evaluated at different times. It was found that beeswax was highly contaminated but honey, brood and bees had acceptable levels, below their respective MRL or LD50, after a certain period following varrocide treatments. Acaricides banned for their use against Varroa, such as chlorfenvinphos, cypermethrin and especially acrinathrin, were found in the hives analysed.

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

An 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 (

[Surveillance of Acaricides in Honey].

By using the LC-MS/MS method developed by us, we determined the residual amounts of acaricides in honey samples commercially available in Tokyo from April 2015 to March 2021. The results of analyzing 127 honey samples, amitraz was detected in 85 samples at the level of 1.1-34.1 µg/kg. Propargite was detected in 3 samples at 2.4-3.8 µg/kg. None of them was beyond the Japanese MRLs or uniform limits. In these survey for 6 years, amitraz was detected in high rate throughout the year. But, the present results imply that amitraz has been used properly in actual bee-keeping because of no violation of MRL and less fluctuation in the detected levels. On the other hand, propargite was detected at the levels over LOQ in domestic honey samples for the first time in 2020, which may suggest a new trend of acaricide use in apiculture in Japan.

Seasonal and geographical variations in insecticide and miticide residues in hop samples produced across three hop-growing regions in the Czech Republic during the years 2018-2020.

The aim of this study was to determine the presence of zoocide (insecticide and miticide) residues in hops collected in three hop-growing regions located in the Czech Republic, and to assess their zoocide profiles and residue levels in terms of variability in temperature and precipitation across the 2018-2020 seasons. Furthermore, the weather factors that influenced the occurrence of hop pests are described and discussed. During our 3-year survey, a total of 120 samples of whole-cone hops samples harvested in three hop-growing regions were analysed for the presence of 29 insecticides and miticides using the modified QuEChERS extraction method, followed by liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry. A majority, 119 of 120 samples, contained a residue of at least one of the active substances surveyed in this study, and 34 analysed samples contained multiple residues with three to four zoocides presented. Concerning the most frequently detected zoocide residues, spirotetramat and/or its metabolites were found in 94.2% of the samples at levels ranging from 0.02 to 1.08 mg/kg. Of the other zoocides surveyed, residues of fenpyroximate, hexythiazox, bifenazate and lambda-cyhalothrin were routinely found in hop cone samples. Obtained data were then used for evaluating seasonal and geographical variations in the profile of zoocide residues among the hop-growing regions in the years 2018-2020, and the compliance with legal regulations concerning the use of zoocides on hops was ascertained. The results showed that (1) the profile and levels of zoocide residues found in the samples reflected seasonal prevalence of pest infestation on hop plants; (2) the strategy to control pests (especially aphids) used in most of hop yards was consistent across the seasons; and (3) a concentration of spirotetramat residues less than 1 mg/kg was typical for hops grown in the Czech Republic.

Long-term monitoring of lipophilic acaricide residues in commercial Swiss beeswax.

BACKGROUND: A national survey on pesticides in recycled beeswax originating from beekeeping has been conducted in Switzerland for almost three decades. It allowed obtaining a good overview of the lipophilic products used for beekeeping within the last 30 years. RESULTS: The use of the veterinary drugs containing bromopropylate or tau-fluvalinate two decades ago led to substantial residues in commercial beeswax. These contaminants are still detectable although in Switzerland the corresponding products have been out of use for many years. The level of coumaphos substantially increased in 2015 up to an annual value of 3.25 mg·kg-1 , suggesting that at least a few beekeepers used coumaphos-containing products. Consequently, an information campaign was launched, and the annual value decreased again. Maximal levels of thymol up to an annual value of 87.5 mg·kg-1 were measured in 2009. Since that time, a steady decrease of thymol residues suggests that beekeepers less frequently use thymol-containing products. Twenty-five years ago, 1,4-dichlorobenzene (PDCB) was widely used for the control of the wax moth, resulting in residues in beeswax up to an annual value of 10.9 mg·kg-1 whereas nowadays, PDCB residues are rarely detected in Swiss beeswax. CONCLUSIONS: Our survey illustrates that several beekeeping-associated pesticides persist in recycled beeswax for many years. Most recent analyses show lower residue levels in Swiss beeswax as compared to previous years. Nowadays Swiss beekeepers mostly use hydrophilic substances for treatment against the Varroa destructor that do not accumulate in beeswax, thus reducing exposure of the honey bees to lipophilic contaminants.

Identification of a non-host semiochemical from miniature pinscher, Canis lupus familiaris, that repels Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P > 0.05) and higher than the means observed for cocker spaniels (P < 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P < 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineus s.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm-2, P < 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineus s.l. ticks.

Evaluation of potential miticide toxicity to Varroa destructor and honey bees, Apis mellifera, under laboratory conditions.

The honey bee, Apis mellifera L., is the world's most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

Residues and dissipation kinetic of abamectin, chlorfenapyr and pyridaben acaricides in green beans (Phaseolus vulgaris L.) under field conditions using QuEChERS method and HPLC.

The current study estimated the dissipation rates of abamectin, chlorfenapyr and pyridaben acaricides in pods of green beans (Phaseolus vulgaris L.) under field conditions in Egypt. Pesticides were extracted and cleaned-up by QuEChERS method and were analyzed by HPLC. The dissipation of these acaricides followed the first order kinetics model with half-life (t1/2) values 1.00, 3.50 and 1.50 days for abamectin, chlorfenapyr and pyridaben, respectively. The lowest residues, at different time intervals of field application rate of each pesticide, were observed with abamectin followed by pyridaben and then chlorfenapyr. Pre-harvest intervals (PHIs) were 10.00, 13.50 and 6.00 days for abamectin, chlorfenapyr and pyridaben, respectively and were below the established European maximum residue limits (EU MRLs) 10-14, 14-21 and 7-10 days after application, respectively. If the fresh pods will be consumed after harvest, it is expected that the presence of these pesticides in the food will have a negative impact on human health. Therefore, the elimination of the residues of these harmful pesticides must be carried out.

Humic acid functionalized hyperbranched polytriazine based dispersive solid-phase extraction for acaricides determination in tea matrix.

Hyperbranched polytriazine functionalized with humic acid was prepared and developed as new sorbents for dispersive solid-phase extraction of three acaricides (clofentezine, fenpyroximate, and pyridaben) in tea samples combined with high-performance liquid chromatography detection. The sorbents were characterized by scanning electron microscopy, energy dispersive spectroscopy, Zeta-potential, and Fourier transform infrared spectroscopy. The extraction parameters (extraction time, ionic strength, desorption conditions) were optimized. The adsorption mechanism was evaluated utilizing Fourier transform infrared spectra. Under optimum conditions, satisfactory analytical performances were achieved, which included high precision (1.33-9.62%), low limits of detection (0.19-3.54 µg/L), and wide linear range (2.5-500 µg/L) for the analysis of the acaricides. Moreover, the proposed method proved highly effective for the determination of acaricides in tea samples, with the relative recoveries in the range of 65.20-108.13% and relative standard deviations < 9.87%. The method has great application potential for the detection of acaricides in tea samples.

A two-year monitoring of pesticide hazard in-hive: High honey bee mortality rates during insecticide poisoning episodes in apiaries located near agricultural settings.

Pesticide residues in beebread, live and dead honey bees, together with honey bee death rate were monitored from June 2016 to June 2018 in three apiaries, located near agricultural settings and in wildlands. Dead honey bees were only collected and analyzed when significant mortality episodes occurred and pesticide content in beeswax of each experimental apiary was evaluated at the beginning of the study. Samples were extracted by a modified QuEChERS procedure and screened for pesticides residues by liquid chromatography mass spectrometry (LC-MS/MS). Pesticide hazard in the samples was evaluated through the hazard quotient approach (HQ). Beebread was widely contaminated with coumaphos and amitraz degradate 2, 4-dimethylphenylformamide (DMF), miticides detected in 94 and 97% of samples respectively. However, insecticides sprayed during citrus bloom like chlorpyrifos (up to 167 ng g -1) and dimethoate (up to 34 ng g -1) were the main responsible of the relevant pesticide hazard in this matrix. Pesticide levels in live bees were mostly residual, and pesticide hazard was low. Beeswax of the apiaries, contaminated by miticides, revealed a low pesticide hazard to honey bee colonies. Acute mortality episodes occurred only in the two apiaries located near agricultural settings. Dead bees collected during these episodes revealed high levels (up to 2700 ng g -1) of chlorpyrifos, dimethoate, omethoate and imidacloprid. HQ calculated in dead bees exceeded up to 37 times the threshold value considered as elevated hazard to honey bee health.

tau-Fluvalinate and other pesticide residues in honey bees before overwintering.

BACKGROUND: Pesticides have often been linked to honey bee colony losses, which occur mainly over winter. In this study, we investigated residues in nine colonies at a model agricultural research site during the period before wintering. Moreover, we applied the acaricide tau-fluvalinate to the colonies via a strip formulation. The pesticide content was determined by UHPLC-QqQ-MS/MS in bees from brood comb initially collected in mid-September immediately prior to the start of tau-fluvalinate treatment and 30 later at the time of tau-fluvalinate strip removal. RESULTS: In addition to commonly analyzed pesticides, we detected two plant growth regulators, chlormequat and metazachlor, in the bee colonies. Whereas thiacloprid, chlormequat and acetamiprid decreased after 30 days and contributed considerably to differences between sample time points, other pesticides appeared to be rather stable. Interestingly, we identified diazinon, which has been banned in the European Union since 2007. The residues of methiocarb sulfoxide and imidacloprid-urea in the absence of their parent compounds indicate historical environmental contamination that can be identified by the detection of residues in a bee colony. tau-Fluvalinate was detected only after the 30-day treatment at an average (± SD) concentration of 1.29 ± 1.93 ng/bee, ranging from 0.06 to 7.13 ng/bee. CONCLUSION: The multidimensional behavior of pesticides in a bee colony was indicated. Although the research area is used for agriculture, the measured pesticide level was relatively low. The recorded concentrations of tau-fluvalinate should not be dangerous to bees, as the values were ∼ 200-5000-fold lower than the reported median lethal dose (LD50 ) values. © 2019 Society of Chemical Industry.

Identification and measurement of veterinary drug residues in beehive products.

There is an increasing concern about the negative impacts of veterinary drugs in beehive compartments. This study evaluates the presence and distribution of chemical residues in beeswax, bee bread and honey and determinates in what extension honeybees are exposed to them. Samples were analyzed by LC-MS/MS and GC-MS/MS with a wide scope of 322 chemical residues. Samples were collected from apiaries located in rural and forest areas, showing no difference in contamination of phytosanitary applications. Residues of acaricides used for sanitary treatments, coumaphos and two transformation products of amitraz (DMF and DMPF), were quantified at higher levels in wax and bee bread than in honey in most cases. Coumaphos, DMF and DMPF were detected in honey in the range 6-36 µg.kg-1; 45-541 µg.kg-1; 15-107 µg.kg-1, respectively. All, except one sample, were below the EU MRLs, 396/2005 Regulation. Other pesticide residues were detected in beeswax and bee bread at various levels.

Simultaneous determination of nine acaricides and two metabolites in comb honey by LC/MS/MS.

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSDr) ranged from 0.8 to 14.8% and that of within-laboratory reproducibility (RSDWR) ranged from 1.3 to 14.8%. All criteria met Japanese validation guidelines. The LOQ was 1.0 µg kg-1 for all analytes. We applied this method to 10 comb honey and 31 honey samples commercially available in Tokyo. From the results of the analysis of 41 samples, we observed that amitraz remained as N-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) in 9 comb honey and 23 honey samples and that their residual concentrations were less than 20 µg kg-1. Using this new method, we improved recovery and precision, which enabled precise quantitative determination. Furthermore, the residual amitraz value in honey determined by both this new and the previous method were in good agreement.

Non-target evaluation of contaminants in honey bees and pollen samples by gas chromatography time-of-flight mass spectrometry.

This work presents a non-targeted screening approach for the detection and quantitation of contaminants in bees and pollen, collected from the same hive, by GC-EI-ToF-MS. It consists of a spectral library datasets search using a compound database followed by a manual investigation and analytical standard confirmation together with semi-quantitation purposes. Over 20% of the compounds found automatically by the library search could not be confirmed manually. This number of false positive detections was mainly a consequence of an inadequate ion ratio criterion (±30%), not considered in the automatic searching procedure. Eight compounds were detected in bees and pollen. They include insecticides/acaricides (chlorpyrifos, coumaphos, fluvalinate-tau, chlorfenvinphos, pyridaben, and propyl cresol) at a concentration range of 1-1207 µg kg-1, herbicides (oxyfluorfen) at a concentration range of 212-1773 µg kg-1 and a growth regulator hormone (methoprene). Some compounds were detected only in pollen; such as herbicides (clomazone), insecticides/acaricides and fungicides used to control Varroa mites as benzylbenzoate, bufencarb, allethrin, permethrin, eugenol and cyprodinil. Additional compounds were detected only in bees: flamprop-methyl, 2-methylphenol (2-49 µg kg-1) and naphthalene (1-23 µg kg-1). The proposed method presents important advantages as it can avoid the use of an unachievable number of analytical standards considered target compounds "a priori" but not present in the analyzed samples.

Acaricidal activity of leaves of Morus nigra against the cattle tick Rhipicephalus microplus / Atividade acaricida das folhas de Morus nigra em carrapato Rhipicephalus microplus

The acaricidal activity of crude ethanolic extract and fractions from the leaves of Morus nigra (Moraceae) was carried out on female cattle ticks Rhipicephalus microplus, using the adult immersion test. The mortality and fertility of females exposed to different concentrations of hexane, chloroform and ethyl acetate fractions, as well as ethanolic extract of M. nigra with concentrations of 5, 10 and 25mg/ml were evaluated using three treatment groups, two control groups and triplicate tests. The study also identified the main phenolic compounds of the extract and fractions of this species by HPLC. The chloroform fraction of leaves of M. nigra (25mg/mL) showed the best results for this species, obtaining 62.6% of inhibition of oviposition, 39.3% of eggs eclosion average and 65.4% of effectiveness. HPLC analysis revealed the presence of phenolic compounds, which may be related to biological activity shown by the extracts, which can be used as an alternative control against R. microplus adult tick.(AU)

A atividade acaricida do extrato etanólico bruto e das frações das folhas de Morus nigra (Moraceae) foi avaliada no carrapato Rhipicephalus microplus, utilizando-se o teste de imersão de fêmeas ingurgitadas. Foram avaliadas a mortalidade e a fertilidade das fêmeas ingurgitadas, expostas a diferentes concentrações (5, 10 e 25 mg/mL) das frações hexânica, clorofórmica e acetato de etila, bem como do extrato etanólico. Os testes foram realizados em triplicata. O estudo também identificou os principais compostos fenólicos do extrato e das frações dessa espécie por HPLC. A fração clorofórmica das folhas de M. nigra (25mg/mL) apresentou o melhor resultado para essa espécie, obtendo-se 62,6% de inibição da oviposição, 39,3% da eclosão dos ovos e 65,4% de eficácia. A análise por HPLC revelou a presença de compostos fenólicos, os quais podem estar relacionados com a atividade biológica demonstrada pelos extratos, que podem ser utilizados como auxiliar no controle contra carrapato adulto de R. microplus.(AU)

An ionic liquid-based nanofluid of titanium dioxide nanoparticles for effervescence-assisted dispersive liquid-liquid extraction for acaricide detection.

Phytophagous mites are usually considered a difficult problem for agricultural planting, and acaricides are applied to control diseases and pests. However, the overdose and misusage of acaricides causes pesticide residues. In this work, a simple and practical ionic liquid-based TiO2 nanofluid, effervescence-assisted, dispersive liquid-liquid microextraction (EA-DLLME) method was developed to detect acaricides in honey and tea by high-performance liquid chromatography (HPLC-DAD). Oleophilic TiO2 nanoparticles were synthesized by a facile solvothermal method to obtain greater stability of the nanofluid. The experimental parameters were optimized by a one-factor-at-a-time approach and included the effervescent tablet composition, ionic liquid selection, extractant composition, nanofluid volume, extraction temperature, extraction time and desorption conditions. Under the optimized conditions, the linear ranges of this proposed method were 0.5-500µgL-1, with correlation coefficients in the range of 0.9985-1.0000. The extraction efficiencies for the target analytes varied from 70.70 to 84.58%. The detection and quantitation limits were in the ranges of 0.04-0.18µgL-1 and 0.13-0.60µgL-1, respectively. The intra- and inter-day relative standard deviations (n=3) were found to range from 2.32 to 5.71%, which showed perfect repeatability. Overall, the EA-DLLME method was time-saving and environmentally friendly, with future potential for microextraction.

The effects of neem oil (Azadirachta indica A. JUSS) enriched with different concentrations of azadirachtin on the integument of semi-engorged Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) females.

Several studies searching for methods to control Rhipicephalus sanguineus s.l., (dog tick) infestations have been developed aiming to minimize the damages caused by these ectoparasites to the hosts and the environment, which is harmed by the indiscriminate use of toxic acaricide products. In this scenario, neem oil has been used as a natural alternative against ticks, once this chemical has repellent properties and interferes in the growth regulation of these ectoparasites, inhibiting ecdysis. The present study evaluated the effects of azadirachtin-enriched neem oil on the integument of semi-engorged R.sanguineus s.l., females through morphohistological techniques. The results showed the occurrence of significant morphological and histochemical alterations, mainly in the females exposed to higher concentrations, which demonstrates the dose-dependent action of the chemical. A decrease in the cuticle thickness was observed, as well as a modification in the distribution of the epithelial cells, which displayed pyknotic and fragmented nuclei, and intensely vacuolated cytoplasm, indicating that these cells would be undergoing death processes. These morphological alterations observed in the integument of the females exposed to the azadirachtin-enriched neem oil encourage the use of this chemical as a strategy to control these ectoparasites.