UPLC-HRESI-MS and GC-MS analysis of the leaves of Nicotiana glauca.

The alkaloid-rich fraction obtained by fractionation of the crude methanolic extract of the leaves of wild tobacco tree Nicotiana glauca Graham (Solanaceae) was analyzed using UPLC-MS and GC-MS. Anabasine, a piperidine alkaloid, was identified as the major constituent with approximately 60 % (m/m) of the alkaloid-rich fraction. In addition to anabasine, six secondary metabolites were identified using high-resolution UPLC-MS. Anabasine was quantified in the leaves to be 1 mg g-1 dry plant material. The GC-MS analysis revealed five compounds with anabasine as the major component, while nicotine was not detected. Moreover, GC-MS was used for the analysis of the volatile oil that was obtained by hydro-distillation from the leaves of N. glauca. The volatile plant oil was found to be rich in oxygenated sesquiterpenes (e.g., ß-bisabolol) and carboxylic acids and esters (e.g., ethyl linoleate and hexadecanoic acid), whereas anabasine was not detected.

Herbivory and Time Since Flowering Shape Floral Rewards and Pollinator-Pathogen Interactions.

Herbivory can induce chemical changes throughout plant tissues including flowers, which could affect pollinator-pathogen interactions. Pollen is highly defended compared to nectar, but no study has examined whether herbivory affects pollen chemistry. We assessed the effects of leaf herbivory on nectar and pollen alkaloids in Nicotiana tabacum, and how herbivory-induced changes in nectar and pollen affect pollinator-pathogen interactions. We damaged leaves of Nicotiana tabacum using the specialist herbivore Manduca sexta and compared nicotine and anabasine concentrations in nectar and pollen. We then pooled nectar and pollen by collection periods (within and after one month of flowering), fed them in separate experiments to bumble bees (Bombus impatiens) infected with the gut pathogen Crithidia bombi, and assessed infections after seven days. We did not detect alkaloids in nectar, and leaf damage did not alter the effect of nectar on Crithidia counts. In pollen, herbivory induced higher concentrations of anabasine but not nicotine, and alkaloid concentrations rose and then fell as a function of days since flowering. Bees fed pollen from damaged plants had Crithidia counts 15 times higher than bees fed pollen from undamaged plants, but only when pollen was collected after one month of flowering, indicating that both damage and time since flowering affected interaction outcomes. Within undamaged treatments, bees fed late-collected pollen had Crithidia counts 10 times lower than bees fed early-collected pollen, also indicating the importance of time since flowering. Our results emphasize the role of herbivores in shaping pollen chemistry, with consequences for interactions between pollinators and their pathogens.

Fast determination of neonicotinoid insecticides in bee pollen using QuEChERS and ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

In this study, a new method has been developed to determine seven neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) in bee pollen using ultra-high performance liquid chromatography coupled to a selective MS detector (qTOF). An efficient sample treatment involving an optimized quick, easy, cheap, effective, rugged and safe method was proposed. In all cases, average analyte recoveries were between 91 and 105%, and no matrix effect was observed. Chromatographic analysis (6.5 min) was performed on a core-shell technology based column (Kinetex® EVO C18 , 50×2.1 mm, 1.7 µm, 100 Å). The mobile phase consisted of 0.1% formic acid in water and 0.1% of formic acid in ACN, with a flow rate of 0.3 mL/min in gradient elution mode. The fully validated method was selective, linear from LOQ to 500 µg/kg, precise and accurate; relative standard deviation and relative error values were below 8%. Low limits LODs and LOQs were obtained, ranging from 0.6 to 1.3 µg/kg (LODs) and 2.1 to 4.0 µg/kg (LOQs). The method was applied to neonicotinoid analysis in several commercial bee pollen samples from different Spanish regions.

High-throughput HPLC-MS/MS determination of the persistence of neonicotinoid insecticide residues of regulatory interest in dietary bee pollen.

The aim of this work is the development of a simple, fast, quantitative, and economic method for the determination of neonicotinoid insecticide residues in dietary bee pollen. Several parameters of the method, such as extraction solvent, extraction time, and solid-phase extraction sorbents for purification [silica, C18, primary-secondary amine (PSA), and Envi-Carb II/PSA], were studied. The final proposed method based on solid-liquid extraction with hexane, cleanup with Supelclean™ Envi-Carb II/PSA cartridges, and subsequent analysis by high-performance liquid chromatography with tandem mass spectrometry was validated and applied to the analysis of commercial bee pollen samples from different geographical zones. Method performance was assessed by the evaluation of several quality parameters of the method, such as recovery values, repeatability, reproducibility, linearity, and limits of detection and quantification. Matrix effects on the chromatographic signal were also studied. The quality parameters of the method were equivalent to or better than those obtained with previously published methods, with recoveries between 81 and 99% and repeatabilities lower than 8.8%. The detection and quantification limits were in the ranges 0.2-2.2 µg kg(-1) and 0.4-4.3 µg kg(-1), respectively.

Environmental fate of soil applied neonicotinoid insecticides in an irrigated potato agroecosystem.

Since 1995, neonicotinoid insecticides have been a critical component of arthropod management in potato, Solanum tuberosum L. Recent detections of neonicotinoids in groundwater have generated questions about the sources of these contaminants and the relative contribution from commodities in U.S. agriculture. Delivery of neonicotinoids to crops typically occurs as a seed or in-furrow treatment to manage early season insect herbivores. Applied in this way, these insecticides become systemically mobile in the plant and provide control of key pest species. An outcome of this project links these soil insecticide application strategies in crop plants with neonicotinoid contamination of water leaching from the application zone. In 2011 and 2012, our objectives were to document the temporal patterns of neonicotinoid leachate below the planting furrow following common insecticide delivery methods in potato. Leaching loss of thiamethoxam from potato was measured using pan lysimeters from three at-plant treatments and one foliar application treatment. Insecticide concentration in leachate was assessed for six consecutive months using liquid chromatography-tandem mass spectrometry. Findings from this study suggest leaching of neonicotinoids from potato may be greater following crop harvest in comparison to other times during the growing season. Furthermore, this study documented recycling of neonicotinoid insecticides from contaminated groundwater back onto the crop via high capacity irrigation wells. These results document interactions between cultivated potato, different neonicotinoid delivery methods, and the potential for subsurface water contamination via leaching.

Insecticide residues in pollen and nectar of a cucurbit crop and their potential exposure to pollinators.

Neonicotinoids are systemic insecticides widely used on many pollinated agricultural crops, and increasing evidence indicates that they move to some extent into pollen and nectar. This study measured levels of neonicotinoid residues in pollen and nectar from a pumpkin crop treated with formulated products containing imidacloprid, dinotefuran, and thiamethoxam using different timings and application methods. Environmental conditions have a significant effect on overall residue levels; nectar residues were 73.5-88.8% less than pollen residues, and metabolites accounted for 15.5-27.2% of the total residue amounts. Foliar-applied treatments and chemigated insecticides applied through drip irrigation during flowering resulted in the highest residues of parent insecticide and metabolites, which may reach average levels up to 122 ng/g in pollen and 17.6 ng/g in nectar. The lowest levels of residues were detected in treatment regimens involving applications of insecticides at planting, as either seed dressing, bedding tray drench, or transplant water treatment.

Refined methodology for the determination of neonicotinoid pesticides and their metabolites in honey bees and bee products by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

An analytical method was refined for the extraction and determination of neonicotinoid pesticide residues and their metabolites in honey bees and bee products. Samples were extracted with 2% triethylamine (TEA) in acetonitrile (ACN) followed by salting out, solid phase extraction (SPE) cleanup, and detection using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated in triplicate at three fortification concentrations in each matrix. Good recoveries were observed for most analytes and ranged between 70 and 120% with relative standard deviations between replicates of <20% in most cases. The method limits of detection were 0.2 ng/g for the parent neonicotinoid pesticides and ranged between 0.2 and 15 ng/g for the neonicotinoid metabolites. This refined method provides lower detection limits and improved recovery of neonicotinoids and their metabolites, which will help researchers evaluate subchronic effects of these pesticides, address data gaps related to colony collapse disorder (CCD), and determine the role of pesticides in pollinator decline.

Another death due to ingestion of Nicotiana glauca.

Deaths attributed to ingestion of Nicotiana glauca are extremely rare. We report here a case where a 43-year-old man was found dead after apparently drinking a water extract of Nicotiana glauca. The primary alkaloid in the plant is anabasine. Toxicological analysis by capillary gas chromatography showed the deceased had a blood anabasine concentration of 2.2 mg/L. Clinically, the features of poisoning are nicotine-like and if death occurs it results from respiratory paralysis. The case further supports the view that, in the human, anabasine is considerably more toxic than nicotine.