Development and Validation of a Gas Chromatography-Mass Spectrometry Method for Determining Acaricides in Bee Pollen.

Pesticides can be found in beehives for several reasons, including contamination from surrounding crops or for their use by beekeepers, which poses a risk to bee ecosystems and consumers. Therefore, efficient and sensitive methods are needed for determining pesticide residues in bee products. In this study, a new analytical method has been developed and validated to determine seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, α-endosulfan, bromopropylate, coumaphos, and τ-fluvalinate) in bee pollen using gas chromatography coupled to mass spectrometry. After an optimization study, the best sample treatment was obtained when using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method employing an ethyl acetate and cyclohexane as the extractant mixture, and a mixture of salts for the clean-up step. A chromatographic analysis (<21 min) was performed in an Agilent DB-5MS column, and it was operated under programmed temperature conditions. The method was fully validated in terms of selectivity, limits of detection (0.2-3.1 µg kg-1) and quantification (0.6-9.7 µg kg-1), linearity, matrix effect (<20% in all cases), trueness (recoveries between 80% and 108%), and precision. Finally, the proposed method was applied to analyze commercial bee pollen samples, and some of the target pesticides (chlorfenvinphos, α-endosulfan, coumaphos, and τ-fluvalinate) were detected.

Glutathione-modified graphene quantum dots as fluorescent probes for detecting organophosphorus pesticide residues in Radix Angelica Sinensis.

A novel fluorescent sensor was developed in this study based on glutathione-functionalized graphene quantum dots (GQDs@GSH) to detect organophosphorus pesticide residues in Radix Angelica Sinensis. GQDs@GSH was synthesized by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.9% and its structure was characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. GQDs@GSH exhibited excellent fluorescence property showing strong blue fluorescence under UV irradiation. The fluorescence of GQDs@GSH could be quenched by Fe3+ by electron transfer and the quenched fluorescence could be recovered due to the strong chelating and reducing ability of phytic acid (PA). Under the catalyzation of acetylcholinesterase (AChE) and choline oxidase (ChOx), acetylcholine (ACh) could be decomposed to H2O2, which could further oxidize Fe2+ to Fe3+ thus quenching the fluorescence of GQDs@GSH once again. Coumaphos, a kind of organophosphorus pesticide, could inhibit AChE activity, thus making the quenched fluorescence turn on again. Several parameters influencing the fluorescence response such as Fe3+, PA, ACh and coumaphos concentration, pH value and reaction time were optimized. Based on such a fluorescence "off-on-off-on" ngkmechanism, GQDs@GSH was successfully applied to the detection of coumaphos in Radix Angelica Sinensis. A good linear relationship between the fluorescence intensity and coumaphos concentration was obtained in the range of 0.1-10.0 µmol·L-1. By a standard addition method, the recoveries were measured to be 101.44-117.90% with RSDs lower than 1.98%. The biosensor system is simple, sensitive and accurate. It has a good application prospect in the detection of organophosphorus pesticide residues in traditional Chinese medicine and agricultural products, and also expanded the application scope for glutathione as a highly selective biological molecule.

Concentration- and time-dependent toxicity of commonly encountered pesticides and pesticide mixtures to honeybees (Apis mellifera L.).

Honeybees are exposed to a wide range of pesticides for long periods via contaminated water, pollen and nectar. Some of those pesticides might constitute health hazards in a time- and dose-dependent manner. Time-dependent toxicity profiles for many applied pesticides are lacking, despite the fact that such profiles are crucial for toxicological evaluations. Therefore, we sought to determine the time-dependent toxicities of pesticides/pesticide metabolites frequently found in Israeli beehives, namely, amitraz metabolites, N'-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) and N-(2,4-dimethylphenyl)-formamide (DMF), coumaphos, imidacloprid, thiacloprid, acetamiprid and dimethoate (toxic reference). By applying accepted methodological approaches such as the modified Haber's rule (product of concentration and exposure duration leads to a constant effect) and comparisons between cumulative doses at different time points, we determined the time-dependent toxicities of these pesticides. We also studied the mixture toxicities of frequently occurring pesticide combinations and estimated their potential contributions to the overall toxicities of neonicotinoids. Thiacloprid was the only pesticide that complied with Haber's rule. DMPF, dimethoate and imidacloprid exhibited time-diminished -toxicities. In contrast, DMF and acetamiprid exhibited time-reinforced toxicities. Neither the binary mixtures nor the tertiary mixtures of DMF, DMPF and coumaphos at 10 times their environmentally relevant concentrations potentiated the neonicotinoids' toxicities. DMPF and imidacloprid were found to present the greatest hazard to honeybees, based on their 50% lethal cumulative dose and 50% lethal time. Amitraz's instability, its low detection frequency and high toxicity profile of its metabolite, DMPF, lead us to the conclusion that DMPF constitutes the actual toxic entity responsible for amitraz's toxic effect.

Frequently encountered pesticides can cause multiple disorders in developing worker honey bees.

Pesticide exposure is regarded as a contributing factor to the high gross loss rates of managed colonies of Apis mellifera. Pesticides enter the hive through contaminated nectar and pollen carried by returning forager honey bees or placed in the hive by beekeepers when managing hive pests. We used an in vitro rearing method to characterize the effects of seven pesticides on developing brood subjected dietary exposure at worse-case environmental concentrations detected in wax and pollen. The pesticides tested included acaricides (amitraz, coumaphos, fluvalinate), insecticides (chlorpyrifos, imidacloprid), one fungicide (chlorothalonil), and one herbicide (glyphosate). The larvae were exposed chronically for six days of mimicking exposure during the entire larval feeding period, which is the worst possible scenario of larval exposure. Survival, duration of immature development, the weight of newly emerged adult, morphologies of the antenna and the hypopharyngeal gland, and gene expression were recorded. Survival of bees exposed to amitraz, coumaphos, fluvalinate, chlorpyrifos, and chlorothalonil was the most sensitive endpoint despite observed changes in many developmental and physiological parameters across the seven pesticides. Our findings suggest that pesticide exposure during larvae development may affect the survival and health of immature honey bees, thus contributing to overall colony stress or loss. Additionally, pesticide exposure altered gene expression of detoxification enzymes. However, the tested exposure scenario is unlikely to be representative of real-world conditions but emphasizes the importance of proper hive management to minimize pesticide contamination of the hive environment or simulates a future scenario of increased contamination.

Comparative impact of coumaphos, amitraz and plant extract of Ageratum conyzoides on the oogenesis of Rhipicephalus microplus.

The present experiment was conducted to evaluate and compare the impact of Ageratum conyzoides plant extract (ACE) with routinely used synthetic acaricides i.e., amitraz and coumaphos on the oogenesis of engorged adult females of Rhipicephalus microplus tick. On the day of dropping from the host, panoistic ovary of R. microplus appeared white in colour, horseshoe shaped, hollow tubular organ with immature oocytes predominantly in dorsal groove. Different developmental stages of oocytes (I-V) proceed simultaneously and asynchronously. Oocytes showed gradual increase in size, deep brown colored with accumulation of eggs in oviduct during 24-72 hours of development.At LC90 concentration a highly significant (p < 0.001) cessation of egg laying after exposure to amitraz and ACE while significant reduction (p < 0.01) of egg laying in coumaphos treated ticks was observed. Upon dissection of treated ticks, uterus and oviduct packed with eggs, which failed to pass out was observed. The histo-architectural alterations including presence of extensive vacuolation, alteration of oocyte morphology, deformation of chorion and disorganization of yolk granules were observed in the treated ovaries. Histochemically, low level of storage or synthesis of essential elements viz., proteins, polysaccharides and lipids in treated oocytes responsible for reduction of fertility and inhibition of progress of vitellogenesis was observed.

An assessment of pesticide exposures and land use of honey bees in Virginia.

Large-scale honey bee colony loss threatens pollination services throughout the United States. An increase in anthropogenic pressure may influence the exposure of hives to household and agricultural pesticides. The objective of this survey was to provide an assessment of the risk of exposure to commonly used pesticides to honey bee colonies in Virginia in relation to land use. Adult honey bee, pollen, and wax samples from colonies throughout Virginia were evaluated for pyrethroid, organophosphate, organochlorine, and triazine pesticides using gas chromatography-mass spectrometry analysis. Of the 11 pesticides analyzed, nine were detected in one or more hive matrices. The probability of detecting a pesticide in pollen was less in forests than in pasture, agriculture, or urban landscapes. Coumaphos and fluvalinate were significantly more likely to be detected across all matrices with concentrations in wax as high as 15500 and 6970 ng/g (dry weight), respectively, indicating the need for further research on the potential effects of miticide accumulation in wax to larval and adult bees.

Boron and Coumaphos Residues in Hive Materials Following Treatments for the Control of Aethina tumida Murray.

In the search of alternatives for controlling Aethina tumida Murray, we recently proposed the BAA trap which uses boric acid and an attractant which mimics the process of fermentation caused by Kodamaea ohmeri in the hive. This yeast is excreted in the feces of A. tumida causing the fermentation of pollen and honey of infested hives and releasing compounds that function as aggregation pheromones to A. tumida. Since the boron is the toxic element in boric acid, the aim of this article is to assess the amount of boron residues in honey and beeswax from hives treated with the BAA trap. For this aim, the amount of bioaccumulated boron in products of untreated hives was first determined and then compared with the amount of boron of products from hives treated with the BAA trap in two distinct climatic and soil conditions. The study was conducted in the cities of Padilla, Tamaulipas, and Valladolid, Yucatan (Mexico) from August 2014 to March 2015. The quantity of boron in honey was significantly less in Yucatan than in Tamaulipas; this agrees with the boron deficiency among Luvisol and Leptosol soils found in Yucatan compared to the Vertisol soil found in Tamaulipas. In fact, the honey from Yucatan has lower boron levels than those reported in the literature. The BAA treatment was applied for four months, results show that the BAA trap does not have any residual effect in either honey or wax; i.e., there is no significant difference in boron content before and after treatment. On the other hand, the organophosphate pesticide coumaphos was found in 100% of wax samples and in 64% of honey samples collected from Yucatan. The concentration of coumaphos in honey ranges from 0.005 to 0.040 mg/kg, which are below Maximum Residue Limit (MRL) allowed in the European Union (0.1 mg/kg) but 7.14% of samples exceeded the MRL allowed in Canada (0.02 mg/kg).

Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera.

As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses.

The antioxidant effect of wheat germ oil on subchronic coumaphos exposure in mice.

Forty-eight male Balb/C mice, allocated to 4 equal groups, constituted the material of the study. The first group was maintained as the control group and was administered solely with a vehicle, which was used to dissolve coumaphos in the third and fourth groups. The second group was administered with 1.5 ml/kg.bw/day (∼1400 mg/kg.bw/day) of wheat germ oil. The third group received 5.5mg/kg.bw/day (1/10 LD50(oral)) of coumaphos. Finally, the fourth group was given both coumaphos and wheat germ oil at the doses indicated above. In all groups, the compounds were given directly into the stomach using a gastric tube, and treatment was continued for a period of 45 days. At the end of the 45th day, the liver, lungs, kidneys, brain, heart and spleen were extirpated in all of the animals. Tissue homogenates prepared from the tissue specimens were analysed for malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. In conclusion, it was determined that coumaphos led to adverse alterations in the majority of the oxidative stress markers investigated. The administration of wheat germ oil alleviated the coumpahos-induced adverse effects detected in the tissues examined.

Serial analysis of gene expression in the southern cattle tick following acaricide treatment of larvae from organophosphate resistant and susceptible strains.

Organophosphate resistant and susceptible tick larvae from laboratory strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus were exposed to low doses of the organophosphate (OP) acaricide, coumaphos. Serial analysis of gene expression (SAGE) was used to analyse differential gene expression in response to OP treatment and to compare the responses of OP-treated and -untreated resistant and susceptible tick larvae. An R. microplus Gene Index was used as an EST database to identify genes which corresponded to SAGE tags whose abundance changed in response to acaricide exposure. Relative quantitative RT-PCR was used to confirm the differential expression results from the SAGE experiments. Of particular interest is a SAGE tag which corresponds to a cytochrome P450-like EST in the Gene Index which was more abundant in untreated OP resistant larvae compared to untreated OP susceptible larvae. This SAGE tag was also more abundant in OP resistant larvae treated with OP compared to OP susceptible larvae treated with OP.

Formic acid treatment for control of Varroa destructor (Mesostigmata: Varroidae) and safety to Apis mellifera (Hymenoptera: Apidae) under southern United States conditions.

The efficacy of a formic acid pad formulation was field tested for control of the honey bee parasitic mite Varroa destructor Anderson & Trueman in Florida and Texas. This pad formulation gave 39.8 +/- 11.1% control at the end of a 6-wk treatment period, which did not significantly differ from the initial sample date. Coumaphos treatment provided poor control (38.4 +/- 11.1%) over the 6-wk period, confirming reports of coumaphos resistance in the region. Under relatively warm winter conditions in southern Texas, formic acid caused mortality of developing eggs and brood. If resistance by V. destructor to the two acaricides registered for its control in the United States continues, the formic acid pad could provide an alternative compound to use as part of an integrated pest management approach. Given the low control seen in this trial, however, modifications of application technology would seem necessary.

Determination of malathion, coumaphos, and fluvalinate residues in honey by gas chromatography with nitrogen-phosphorus or electron capture detectors.

A rapid, reliable, and inexpensive extraction method was developed to determine acaricide residues in honey by gas chromatography (GC) with nitrogen-phosphorus (NP) or electron capture (EC) detectors. Because of the high selectivity of the NP detector, no interfering peaks were present and no cleanup was necessary. A simple cleanup step is proposed for the GC-ECD analysis. Recoveries from spiked honey samples ranged from 79 to 94.4%, with coefficients of variation of 0.3-18.5%. The quantitation limit obtained was 0.015 mg/kg for malathion, 0.020 mg/kg for coumaphos, and 0.005 mg/kg for fluvalinate. The method was used to determine the disappearance of malathion and coumaphos residues from honey samples collected from beehives treated with these acaricides. The disappearance of both acaricides was rapid and followed a first-order model for the duration of the experiment.