Imidacloprid, the most used systemic insecticide, is suspected of having harmful effects on honeybees at nanogram per bee or at microgram per kilogram levels. However, there is a lack of methodology to detect imidacloprid and its metabolites at such low levels. We developed a method for the determination of low amounts of imidacloprid in soils, plants (leaves and flowers), and pollens by using HPLC coupled to tandem mass spectrometry (APCI-MS/MS). Extraction, separation, and detection were performed according to quality assurance criteria, to Good Laboratory Practice, and to criteria from the directive 96/23/EC, which is designed for banned substances. The linear range of application is 0.5-20 microg/kg imidacloprid in soils, in plants, and in pollens, with a relative standard deviation of 2.9% at 1 microg/kg. The limits of detection and of quantification are LOD = 0.1 microg/kg and LOQ = 1 microg/kg, respectively. For the first time, this study permitted us to follow the fate of imidacloprid in the environment. When treated, flowers of sunflower and maize contain average values of approximately 10 microg/kg imidacloprid. This explains that pollens from these crops are contaminated at levels of a few micrograms per kilogram, suggesting probable deleterious effects on honeybees.

Following evidence for the intoxication of bees, the systemic insecticide imidacloprid was suspected from the mid nineties of having harmful effects. Recently, some studies have demonstrated that imidacloprid is toxic for the bees at sub-lethal doses. These doses are evaluated in the range between 1 and 20 μg kg -1 , or less. It appeared thus necessary to study the fate of imidacloprid in the environment at such low levels. Thus, we developed methods for the determination of low amounts, in the μg kg -1 range, of the insecticide imidacloprid in soils, plants and pollens using high pressure liquid chromatography -tandem mass spectrometry (LC/APCI/MS/MS). The extraction and separation methods were performed according to quality assurance criteria, good laboratory practices and the European Community's criteria applicable to banned substances (directive 96/23 EC). The linear concentration range of application was 1-50 μg kg -1 of imidacloprid, with a relative standard deviation of 2.9% at 1 μg kg -1 . The limit of detection and quantification are respectively LOD = 0.1 μg kg -1 and LOQ = 1 μg kg -1 and are suited to the sub-lethal dose range. This technique allows the unambiguous identification and quantification of imidacloprid. The results show the remanence of the insecticide in soils, its ascent into plants during flowering and its bioavailability in pollens.

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