Sensitive determination of mixtures of neonicotinoid and fungicide residues in pollen and single bumblebees for exposure assessment

Limit=250 words To accurately estimate exposure of bees to pesticides, analytical methods are needed to enable quantification of ng/g (ppb) levels of contaminants in small samples of pollen or the individual insects. A modified QuEChERS extraction method coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was tested to quantify residues of 19 commonly used neonicotinoids and fungicides and the synergist, piperonyl butoxide, in 100 mg samples of pollen and in samples of individual bumblebees (Bombus terrestris). Recoveries of many fungicide analytes were significantly improved by reducing the mass of dispersant used in the QuEChERS and an additional acetonitrile/toluene extraction step. Final recoveries ranged from 71 to 102 % for most compounds with a repeatability of below 20 % for both pollen and bumblebee matrices. The method enables the detection of all compounds at sub ppb levels in both matrices; the method detection limits (MDL) ranged from 0.01 to 0.84 ppb in pollen and 0.01 to 0.96 ppb in individual bumblebees. Using this method, mixtures of neonicotinoids (thiamethoxam, clothianidin, imidacloprid and thiacloprid) and fungicides (carbendazim, spiroxamine, trifloxystrabin) were detected in pollens of field bean, strawberry and raspberry at concentrations ranging from <MDL to 67.1 ppb for neonicotinoids and from <MDL to 14.1 ppb for fungicides. In wild bumblebees, the insecticides thiamethoxam and thiacloprid were detected while fungicides such as carbendazim, boscalid, tebuconazole, flusilasole and metconazole were quantified at ppb levels in some bees. This first method developed to analyse mixtures of neonicotinoids and fungicides in small quantities of pollen and individual bumblebees is sensitive enough for the detection of these pesticides at trace levels.