Interaction of Insecticides and Fungicides in Bees.

Honeybees and wild bees are among the most important pollinators of both wild and cultivated landscapes. In recent years, however, a significant decline in these pollinators has been recorded. This decrease can have many causes including the heavy use of biocidal plant protection products in agriculture. The most frequent residues in bee products originate from fungicides, while neonicotinoids and, to a lesser extent, pyrethroids are among the most popular insecticides detected in bee products. There is abundant evidence of toxic side effects on honeybees and wild bees produced by neonicotinoids, but only few studies have investigated side effects of fungicides, because they are generally regarded as not being harmful for bees. In the field, a variety of substances are taken up by bees including mixtures of insecticides and fungicides, and their combinations can be lethal for these pollinators, depending on the specific group of insecticide or fungicide. This review discusses the different combinations of major insecticide and fungicide classes and their effects on honeybees and wild bees. Fungicides inhibiting the sterol biosynthesis pathway can strongly increase the toxicity of neonicotinoids and pyrethroids. Other fungicides, in contrast, do not appear to enhance toxicity when combined with neonicotinoid or pyrethroid insecticides. But the knowledge on possible interactions of fungicides not inhibiting the sterol biosynthesis pathway and insecticides is poor, particularly in wild bees, emphasizing the need for further studies on possible effects of insecticide-fungicide interactions in bees.

Determination of the [15N]-Nitrate/[14N]-Nitrate Ratio in Plant Feeding Studies by GC⁻MS.

Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [15N], has been frequently used. In plants, it is usually applied in form of [15N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [15N]-nitrate/[14N]-nitrate ratio in leaves are useful for the planning and evaluation of feeding and pulse-chase experiments. Here we describe a simple and sensitive method for determining the [15N]-nitrate to [14N]-nitrate ratio in leaves. Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were sufficient for analysis, allowing a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was analyzed by gas chromatography-mass spectrometry with electron ionization. Separation of the derivatized samples required only 6 min. The method shows excellent repeatability with intraday and interday standard deviations of less than 0.9 mol%. Using the method, we show that [15N]-nitrate declines in leaves of hydroponically grown Crassocephalum crepidioides, an African orphan crop, with a biological half-life of 4.5 days after transfer to medium containing [14N]-nitrate as the sole nitrogen source.