Neonicotinoid contamination in wildflowers collected from citrus orchards in a northwestern Mediterranean Region (Spain) after tree foliar treatments.

Ground-cover vegetation attracts and harbors beneficial insects to the agrosystem, playing an important role in conservation biological control. Integrated pest management (IPM) program guidelines recommend the implantation of sowed or resident wild covers in perennial crops. Given the high-quality fruit requirements, even in IPM programs, insecticides can be required in citrus crops. This study presents, over a year, the levels of neonicotinoids (thiamethoxam and imidacloprid) in not-target ground-cover wildflowers growing spontaneously in citrus orchards after foliar treatment of citrus trees. The presence and persistence of these neonicotinoids in different wildflower species were studied. Concentrations of thiamethoxam and imidacloprid in whole wildflowers ranged from < method quantification limit (MQL) to 52.9 ng g-1 and from < MQL to 98.6 ng g-1, respectively. Thiamethoxam was more frequently detected than imidacloprid. Thiamethoxam and imidacloprid were detected up to 336 and 230 days after treatment, respectively. The highest detection frequencies (100%) and highest thiamethoxam and imidacloprid mean concentrations (26.0 ± 7.3 ng g-1 and 11.0 ± 10.6 ng g-1, respectively) occurred in wildflowers collected 9 days after the treatments. Since application, a clear decrease in the concentration of both compounds and differences in the accumulation depending on wildflower species were observed. Cross contamination was detected, indicating a transport from adjacent treated plots. Maintaining a cover crop in citrus orchards may lead to detrimental effects on non-target arthropods if these neonicotinoid compounds are used for pest control since they can entail a chronic exposure during at least 230 days for imidacloprid and 336 days for thiamethoxam.

Microbial Decontamination of Bee Pollen by Direct Ozone Exposure.

The bee pollen is a complete and healthy food with important nutritional properties. Usually, bee pollen is consumed dehydrated, but it is possible to market it as fresh frozen pollen, favoring the maintenance of its properties and greatly increasing its palatability, compared to dried pollen. However, fresh frozen pollen maintains a high microbiological load that can include some pathogenic genus to human health. In this work, ozonation combined with drying is applied to reduce the microbiological load. The lowest timing exposure to ozone (30 min) was chosen together with hot-air drying during 15 min to evaluate the shelf-life of treated bee-pollen under cold storage (4 °C), and initial reductions of 3, 1.5, and 1.7 log cycles were obtained for Enterobacteriaceae, mesophilic aerobes, and molds and yeasts counting, respectively. Six weeks after treatment the microbial load was held at a lower level than initially observed in fresh bee-pollen. In addition, ozone treatment did not have a negative impact on the polyphenols evaluated. Likewise, the sensory profile of the bee pollen under different treatments was studied. For all these assays the results have been favorable, so we can say that ozonation of fresh pollen is safe for human consumption, which maintains its polyphenols composition and organoleptically is better valued than dried pollen.

The toxic unit approach as a risk indicator in honey bees surveillance programmes: A case of study in Apis mellifera iberiensis.

The influence of genetic diversity and exposure to xenobiotics on the prevalence of pathogens was studied within the context of a voluntary epidemiological study in Spanish apiaries of Apis mellifera iberiensis, carried out during the spring season of years 2014 and 2015. As such, the evolutionary lineages of the honey bee colonies were identified, a multiresidue analysis of xenobiotics was carried out in beebread and worker bee samples, and the Toxic Unit (TUm) was estimated for each sampled apiary. The relationship between lineages and the most prevalent pathogens (Nosema ceranae, Varroa destructor, trypanosomatids, Black Queen Cell Virus; and Deformed Wing Virus) was analysed with contingency tables, and the possible relationships between TUm and the prevalence of these pathogens were studied by using a factor analysis. The statistical analysis supported the associations between V. destructor and Deformed Wing Virus (DWV), and between N. ceranae and Black Queen Cell Virus (BQCV), but the association between these pathogens and trypanosomatids was not observed. TUm values varied between 5.5 × 10-6 and 3.65 × 10-1. When TUm < 3.35 × 10-4, it was mainly determined by coumaphos, tau-fluvalinate and/or chlorfenvinphos. At higher values, other insecticides also contributed to TUm, although a clear predominance was not seen up to TUm ≥ 1.83 × 10-2, when it was mainly defined by acrinathrin, spinosad and/or imidacloprid. The possible cumulative effect from the joint action of xenobiotics was >10% in the 63% of the cases. The prevalence of pathogens did not appear to be influenced by the distribution of evolutionary lineages and, while the prevalence of V. destructor was not found to be determined by TUm, there was a trend towards an increasing prevalence of N. ceranae when TUm ≥ 23 10-4. This study is an example of using TUm approach beyond the field of the ecotoxicology.

Evaluation of Residue Levels of Imidacloprid and Thiamethoxam After Foliar Application to the Citrus Varieties Lane Late, Valencia Late, Rohde Summer, and Nules.

Neonicotinoids are used to protect citrus trees against pests. Dissipation and persistence of neonicotinoids in pollen and nectar of citrus trees after foliar applications and their potential exposure to pollinators have not been well characterized. Field studies were conducted using three orange and one mandarin varieties to compare the imidacloprid and thiamethoxam residue levels and their decline in pollen and nectar after treatments in pre-bloom close to flowering period and their persistence 1 yr after treatment. The possible risk to honeybees was assessed. In nectar, thiamethoxam and imidacloprid residues were between 61 and 99% lower than in pollen, depending on the citrus variety or/and the days after treatment when applied close to blooming. At the end of the flowering period, imidacloprid in pollen and nectar was not detected in the mandarin variety after treatment in pre-bloom, whereas for thiamethoxam, no residues were detected in nectar but 10 ng/g was detected in pollen. There were no quantifiable levels of residues for either neonicotinoids in pollen or nectar during the flowering period of the following year. Neonicotinoid residue levels and their decline in nectar and pollen in citrus depended on the timing of applications relative to flowering and on the citrus variety. The absence of neonicotinoid residues 1 yr out after foliar applications in all varieties assayed demonstrated that none of the neonicotinoids tested were persistent. The results could be different in other citrus varieties, and therefore, also the exposure assessment for managed pollinators.

Analysis of pesticide residues in honeybee (Apis mellifera L.) and in corbicular pollen. Exposure in citrus orchard with an integrated pest management system.

In the last years, the honeybee population is facing growing threats such as expansion of pathogens, incorrect use of phytosanitary products and environmental contaminants, loss or fragmentation of habitat, invasive species and climate change. The citrus cropping by Integrated Pest Management (IPM) in Spain combines strategies to reduce pest populations preventing environmental problems and reducing levels of damage by using chemicals only when it is strictly necessary. The goal of this study is to develop a simple analytical method to evaluate pesticide residue levels in honeybees and corbicular pollen when honeybees are exposed to plant protection products (PPPs) used in integrated pest management citrus orchards. The proposed method is based in an ultrasound assisted extraction procedure followed by a dispersive solid phase extraction (d-SPE) clean-up with alumina and LC-MS/MS pesticides determination. The method was validated in samples of honeybee and corbicular pollen for 10 pesticides commonly used in citrus orchards under IPM. This procedure was compared with QuEChERS methodologies for these matrices. The developed method was applied to determine pesticides in both matrices in a two -year study in citrus orchards.

Determination of hexabromocyclododecane isomers in sewage sludge by LC-MS/MS.

A method has been developed to determine alpha, beta and gamma diastereoisomers of hexabromocyclododecane (HBCD), a brominated flame retardant, in sewage sludge, based on the ultrasonic-assisted extraction (UAE) of samples with dichloromethane-ACN (1:1) and the subsequent clean-up of extracts by dispersive solid phase extraction with primary-secondary amine. Levels of HBCD diastereoisomers were determined by LC coupled with ESI MS/MS. Evaluation of the matrix effect showed a high ion suppression for all the diastereoisomers studied, which was counteracted by using a mixture of labelled HBCD diastereoisomers as internal standards. This method yielded recoveries in the range of 79.6-112.5% with SDs equal or lower than 9.1 The limits of detection were 0.3 ng/g for alpha- and beta-HBCD and 0.2 ng/g for gamma-HBCD. The developed method was successfully applied to 19 sludge samples collected from the province of Madrid (Spain). In most of the samples, beta-HBCD was below the method detection limit, whereas alpha- and gamma-HBCD were quantified in all of the sludge samples, and gamma-HBCD was the predominant diastereoisomer in 63% of the analyzed samples and alpha-HBCD predominated in the rest.

A combination of ultrasonic assisted extraction with LC-MS/MS for the determination of organophosphorus pesticides in sludge.

A method has been developed for the analysis of a wide polarity range of the currently used organophosphorus pesticides (OPs) and their metabolites in sewage sludge samples. Extraction was carried out using ultrasonic assisted extraction (UAE) with different solvents. The levels of OPs in sludge were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization (ESI) in positive or negative ion mode. Extraction with acetonitrile containing 1% acetic acid gave the best results with recoveries between 83.2% and 106.4% and RSD < or = 8.7%. Evaluation of matrix effect showed high ion suppression for OPs of intermediate polarity, which decreased to approximately 50% by matrix dilution to an equivalent of 0.5 g of sludge per millilitre; however, for polar OPs, the matrix effect was negligible at the same concentration. Therefore, matrix-matched standards were used for OPs quantification. The limits of quantification were in the range of 1-14 ng g(-1). This method was successfully applied to real sludge samples collected from Madrid Province and chlorpyrifos along with its metabolite TCPY were the OPs found in sludge at the highest levels, 113-344 ng g(-1) and 33-307 ng g(-1), respectively.