Bees stung by insecticides in pollen

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  • Published: Mar 12, 2007
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Bees stung by insecticides in pollen

There are many pesticides which are highly successful in culling their target populations but which have a detrimental effect on other, more friendly forms of wildlife. One such agent is the insecticide fipronil which is used to control pests in crops, including locusts. It is also effective against domestic insect pests such as ants and cockroaches, as well as ticks, mites and fleas carried on animals.

Much of the success of fipronil relies on the fact that it is slow acting. When it is mixed with bait, it is taken back to the colonies and spread about, accelerating the rate of kill. The predicted rate of kill after 3 days is about 95% for ants and cockroaches. It acts by attacking the central nervous system, disrupting the chloride channel that is regulated by gamma-aminobutyric acid (GABA).

Unfortunately, fipronil, a phenylpyrazole type of insecticide, is also highly toxic to bees with a low 50% lethal dose (LD50) of 0.004-0.006 µg/bee. Its long half-life on treated vegetation of 3-7 months ensures prolonged exposure for the bees, which can come into contact via the vegetation itself and through pollen and nectar. Even at sub-lethal doses, the insecticide adversely affects the feeding activity of bees and the ratio of inactive to active bees.

It is the combination of this high apiarian toxicity and widespread use that has prompted a team of Spanish scientists at the University of Valladolid to ask if fipronil is responsible for the current decline observed in the population of honey bees. Furthermore, if bees inadvertently collect fipronil and take it back to the hive, it will end up in hive-based products such as honey and royal jelly. While human health is not a primary concern because the World Health Organisation classifies fipronil as a moderately hazardous compound, extensive human testing has not been carried out.

The research team noted that there are several published methods for measuring pesticide residues in pollen but found only two specifically for fipronil. Recognising that extraction is an essential part of the analytical process, they set out to compare various sample preparation protocols for removing fipronil from pollen for subsequent analysis by gas chromatography with electron capture and mass spectrometric detection.

Three different generalised techniques were explored - extraction with organic solvents, solid-phase extraction (SPE) on cartridges or a Florisil column, and matrix-solid-phase dispersion (MSPD).

The solid-liquid extractions were carried out with a group of solvents that covered a wide polarity range - methanol, ethyl acetate, acetonitrile, acetone, dichloromethane and hexane. After two-fold extraction, the best recoveries measured by GC/ECD were close to 66% for acetonitrile and ethyl acetate. Subsequent liquid-liquid extraction removed co-extracted impurities to give cleaner chromatograms. The GC/MS recoveries were artificially high (up to 200%) due to the presence of co-extracted matrix components.

For SPE, the acetonitrile or methanol extracts were cleaned up on 5 types of cartridge or the Florisil column. The best SPE performances were obtained by adding water to the extracts then using C18 or polymeric (styrene-divinylbenzene) cartridges, with recoveries of more than 80% measured by GC/ECD. With Florisil, the figure was more than 75%. Once again, matrix effects distorted the yields in GC/MS.

MSPD using Florisil mixed with pollen gave comparable recoveries but the chromatograms showed an irregular baseline and the presence of many co-extracted compounds which obscured the fipronil peak at lower concentrations.

The researchers recommended the use of SPE or Florisil chromatography of acetonitrile extracts as the best recovery methods. Although GC/ECD was able to determine fipronil at trace concentrations, they also advocated the use of GC/MS because it has the added benefit of being able to identify the insecticide peak. The use of matrix-matched calibrations using spiked samples corrected the abnormally high recoveries observed.

With this technique, detection limits of 0.07 µg/kg pollen were achieved, about 12-19-fold above the LD50 value of fipronil in bees. The proposed method could enable an attempted correlation between pollen levels of the insecticide and honey bee populations.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.



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