Neonicotinoids in excretion product of phloem-feeding insects kill beneficial insects.

Pest control in agriculture is mainly based on the application of insecticides, which may impact nontarget beneficial organisms leading to undesirable ecological effects. Neonicotinoids are among the most widely used insecticides. However, they have important negative side effects, especially for pollinators and other beneficial insects feeding on nectar. Here, we identify a more accessible exposure route: Neonicotinoids reach and kill beneficial insects that feed on the most abundant carbohydrate source for insects in agroecosystems, honeydew. Honeydew is the excretion product of phloem-feeding hemipteran insects such as aphids, mealybugs, whiteflies, and psyllids. We allowed parasitic wasps and pollinating hoverflies to feed on honeydew from hemipterans feeding on trees treated with thiamethoxam or imidacloprid, the most commonly used neonicotinoids. LC-MS/MS analyses demonstrated that both neonicotinoids were present in honeydew. Honeydew with thiamethoxam was highly toxic to both species of beneficial insects, and honeydew with imidacloprid was moderately toxic to hoverflies. Collectively, our data provide strong evidence for honeydew as a route of insecticide exposure that may cause acute or chronic deleterious effects on nontarget organisms. This route should be considered in future environmental risk assessments of neonicotinoid applications.

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.

Beeswax cleaning by solvent extraction of pesticides.

We set out to test if the methodology used to clean sheep wool wax (Lanolin) from pesticides could be used to clean beeswax as well. We first made an aggregate sample of brood comb wax from three different US beekeepers. Sub-samples of these aggregate wax samples were analyzed for pesticide contamination. The remaining wax, was then dissolved into hexane solution and run through four N, N-Dimethylformamide (DMF) washes. During these extractions, the pesticides partitioned into the DMF, and so were removed from the beeswax. Following the solvent extractions, the beeswax was tested again for pesticides. An average of 95% of the pesticide contamination was removed by the chemical wash procedure. •Beeswax is the beekeeping matrix with the highest pesticide content.•This study developed methodology for solvent-based removal of pesticides from beeswax (>95%).•Of 24 pesticides detected in beeswax samples before to the solvent extraction, only 3 pesticides were detected after the extraction with DMF.

Pesticide residues in honey bees, pollen and beeswax: Assessing beehive exposure.

In order to study the distribution of pesticide residues in beekeeping matrices, samples of live in-hive worker honey bees (Apis mellifera), fresh stored pollen and beeswax were collected during 2016-2017 from 45 apiaries located in different landscape contexts in Spain. A total of 133 samples were screened for 63 pesticides or their degradation products to estimate the pesticide exposure to honey bee health through the calculation of the hazard quotient (HQ). The influence of the surrounding environment on the content of pesticides in pollen was assessed by comparing the concentrations of pesticide residues found in apiaries from intensive farming landscapes to those found in apiaries located in mountainous, grassland and urban contexts. Beeswax revealed high levels of miticides used in beekeeping such as coumaphos, chlorfenvinphos, fluvalinate and acrinathrin, which were detected in more than 75% of samples. Pollen was predominantly contaminated by miticides but also by insecticides used in agriculture such as chlorpyrifos and acetamiprid, which showed concentrations significantly higher in apiaries located in intensive farming contexts. Pesticides residues were less frequent and at lower concentrations in live honey bees. Beeswax showed the highest average hazard scores (HQ > 5000) to honey bees. Pollen samples contained the largest number of pesticide residues and relevant hazard (HQ > 50) to bees. Acrinathrin was the most important contributor to the hazard quotient scores in wax and pollen samples. The contributions of the pesticides dimethoate and chlorpyrifos to HQ were considered relevant in samples.

Pesticide analysis in coffee leaves using a quick, easy, cheap, effective, rugged and safe approach and liquid chromatography tandem mass spectrometry: Optimization of the clean-up step.

An analytical method using a quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure for multi-residue determination of 52 pesticides in coffee leaf extractshas been developed and validated according to SANTE/11945/2015 guidelines. Different sorbent combinations for dispersive solid phase extraction (d-SPE) clean-up as well as dispersive liquid-liquid microextraction (DLLME) were tested. The relative standard deviations (RSDs) for the recovery of 87-94% of pesticides added to coffee leaf extracts,was ≤20% for samples spiked at concentrations up to 50ng*g-1 depending on the clean-up procedures. However, samples spiked with a 100ng*g-1 pesticide mixture gave RSDs>20% for most pesticides when d-SPE was carried out adding Supelclean ENVI-Carb 120/400. To explain this fact,the secondary metabolic profile was analyzed in all the extraction and clean-up procedures. Only in the clean-up procedure with the addition of Supel QuE Z-Sep+, does caffeine show a constant adsorption between blank and spiked samples. In other clean-up procedures, the amount of caffeine was higher in those samples spiked with pesticides. This indicates competition between caffeine and pesticides for adsorption to the sorbent. Addition of Supel QuE Z-Sep+ to the procedure revealed only a 32% matrix effect, whereas using PSA+ C18 the matrix effect was close to 97%. The process efficiency is up to 54% with the addition of Supel QuE Z-Sep+ and just up to 7% for the other clean-up procedures. The method was successfully tested in coffee leaves from different types of cultivars. Pesticides were not detected in organic coffee leaf extracts, but thiametoxan was clearly detected in 50% of coffee leaf extracts harvested from coffee trees grown under traditional conditions as determined by UHPLC-TOFMSLC/QqTOF-MS/MS.

Occurrence of pesticide residues in Spanish beeswax.

Beeswax from Spain was collected during 2016 to determine pesticide residues incidence. The 35 samples were divided in foundation, old combs, cappings or virgin beeswax to compare pesticide content between groups. Wax was screened for 58 pesticides or their degradation products by QuEChERS extraction and liquid chromatography mass spectrometry (LC-MS/MS). Beeswax was uniformly contaminated with acaricides and, to a much lesser extent, with insecticide and fungicide residues. Virgin followed by cappings were less contaminated than foundation and old combs beeswax. The miticides applied in-hive had a contribution to average pesticide load higher than 95%. Compounds widely used as acaricides, as coumaphos (100%), fluvalinate (86%) and amitraz (83%), were the pesticides most frequently detected with maximum concentrations of 26,858, 3593 and 6884ng·g-1, respectively. Chlorfenvinphos, acrinathrin and flumethrin, also acaricides, were detected in 77, 71 and 54%, respectively. Frequencies of pesticides used in crops were 40% for chlorpyrifos, 29% for dichlofenthion, 9% for malathion, 6% for fenthion-sulfoxide and 3% for azinphos-methyl, carbendazim, ethion, hexythiazox, imazalil and pyriproxyfen. Pesticide assessment in beeswax could be an excellent monitoring tool to establish veterinary treatments applied by beekeepers and environmental contaminants exposure of honey bees.

Efficiency of QuEChERS approach for determining 52 pesticide residues in honey and honey bees.

A comparison between QuEChERS and other pesticide extraction procedures for honey and honey bee matrices is discussed. Honey bee matrix was extracted by solvent based procedure whereas solid phase extraction was the protocol for the honey matrix. The citrate buffered QuEChERS method was used for both matrices. The methods were evaluated regarding cost (equipment and reagents), time, accuracy, precision, sensitivity and versatility. The results proved that the QuEChERS protocol was the most efficient method for the extraction of the selected pesticides in both matrices. •QuEChERS is the most economical and less time-consuming procedure.•SPE and solvent-based extraction procedures show equivalent recoveries to QuEChERS.•QuEChERS can be used to extract pesticide residues from both matrices.

Influence of pesticide use in fruit orchards during blooming on honeybee mortality in 4 experimental apiaries.

Samples of dead honey bees (Apis mellifera L.) were collected periodically from 4 different locations during citrus and stone fruit trees blooming season to evaluate the potential impact of agrochemicals on honey bee death rate. For the determination of mortality, dead honey bee traps were placed in front of the experimental hives entrance located in areas of intensive agriculture in Valencian Community (Spain). A total of 34 bee samples, obtained along the monitoring period, were analyzed by means of QuEChERS extraction method and screened for 58 pesticides or their degradation products by LC-MS/MS. An average of four pesticides per honey bee sample was detected. Coumaphos, an organophosphate acaricide used against varroosis in the experimental hives, was detected in 94% of the samples. However, this acaricide was unlikely to be responsible for honey bee mortality because its constantly low concentration during all the monitoring period, even before and after acute mortality episodes. The organophosphates chlorpyrifos and dimethoate, as well as the neonicotinoid imidacloprid, were the most frequently detected agrochemicals. Almost 80% of the samples had chlorpyrifos, 68% dimethoate, and 32% imidacloprid. Maximum concentrations for these three compounds were 751, 403, 223 ng/g respectively. Influence of these pesticides on acute honey bee mortality was demonstrated by comparing coincidence between death rate and concentrations of chlorpyrifos, dimethoate and imidacloprid.