Quantification of imidacloprid uptake in maize crops.

The systemic imidacloprid is one of the most used insecticides in the world for field and horticultural crops. This neurotoxicant is often used as seed-dressing, especially for maize, sunflower, and rape. Using a LC/MS/MS technique (LOQ = 1 microg/kg and LOD = 0.1 microg/kg), the presence of imidacloprid has been measured in maize from field samples at the time of pollen shed, from less than 0.1 microg/kg up to 33.6 microg/kg. Numerous random samples were collected throughout France from 2000 to 2003. The average levels of imidacloprid measured are 4.1 microg/kg in stems and leaves, 6.6 microg/kg in male flowers (panicles), and 2.1 microg/kg in pollen. These values are similar to those found previously in sunflower and rape. These results permit evaluation of the risk to honeybees by using the PEC/PNEC ratios (probable exposition concentrations/predicted no effect concentration). PEC/PNEC risk ratios were determined and ranged between 500 and 600 for honeybees foraging on maize treated with imidacloprid by seed dressing. Such a high risk factor can be related to one of the main causes of honeybee colony losses.

A method to quantify and analyze the foraging activity of honey bees: relevance to the sublethal effects induced by systemic insecticides.

The assessment of agropharmaceuticals' side effects requires more realistic simulations of field conditions than those deduced from the dose-lethality relation obtained under laboratory conditions. Because the presence of sublethal doses or concentrations may also alter the behavior of foraging insects, we attempted to devise a quantifiable and accurate protocol for evidencing various alterations in free-flying bees. Such a protocol was illustrated by testing new classes of systemic insecticides. The protocol focused on video recording to quantify the foraging activity of small colonies of honey bees confined in insect-proof tunnels. The basis of the protocol was not the colony itself but the change in each colony on a specific day and between days. First, the paradigms of attendance at a safe feeding source were established by observing 8 control colonies at different times of the season during 5 days after the necessary forager training was accomplished. Second, on three different colonies we considered the paradigms on the control day before contamination and during 4 days after the feeding source was contaminated. During the same period, one more colony was exclusively fed with safe food to serve as control. Two plant-systemic insecticides were tested at contamination levels 70 times lower than the 50% of the lethal concentration. Imidacloprid, at 6 microg/kg, clearly induced a decrease in the proportion of active bees. Fipronil, at 2 microg/kg, induced an additional decrease in attendance at the feeder. Such levels are still higher than the corresponding lowest observable effect concentration (LOEC). Our protocol, which provided intermediate conditions between field and laboratory conditions, allowed the quantification, with an enhanced level of sensitivity, of sublethal effects on foraging bees.

A LC/APCI-MS/MS method for analysis of imidacloprid in soils, in plants, and in pollens.

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.