Studying Honey Bee Decline with Chromatography

June 21, 2013
E-Separation Solutions

Volume 0, Issue 0

LCGC spoke to Professor Andrea Tapparo, of the University of Padova (Italy) about studying honey bee decline with chromatography.

LCGC spoke to Professor Andrea Tapparo, of the University of Padova (Italy) about studying honey bee decline with chromatography.

Q. What are your main research interests and how did you become interested in declining honeybee populations?

A: In the field of analytical chemistry I’m interested in environmental analysis (micro-pollutants and their metabolites; aerosol characterization) and metal speciation in aqueous solutions of environmental and biological interest.

In 2008, I discussed with my colleague entomologist, Professor Vincenzo Girolami here at the University of Padova, the analytical procedures available for the analysis of environmental samples with possible relevance in the honeybee loss phenomena. At that time, severe colony losses — generally described as colony collapse disorder (CCD) — were observed by beekeepers before corn(maize) seeds coated with neonicotinoids were banned in Italy (September 2008). So we began collaborating, and we hypothesized, and confirmed experimentally by quantitative data, two mechanisms of possible bee exposure and contamination: corn guttations (drops of xylem sap exuded on the tips or edges of leaves of the corn seedling that can contain high concentration of the systemic insecticide) and the direct uptake of particles containing the insecticide by flying bees during the corn sowing. The collaboration between entomologists and analytical chemists at University of Padova is still active and productive.

Q. What is colony collapse disorder exactly?

A: Colony collapse disorder is a generalised term that describes the worldwide crisis of honeybee colonies. Several hypotheses have been proposed to explain CCD but none can be sustained by accepted experimental evidence.

Our attention has been mainly oriented to the massive deaths of bees observed in spring occurring concomitantly with the corn sowing, demonstrating the effective contamination of flying bees, approaching the drilling machine, with the seed coating particles released during the sowing.

Q. What are neonicotinoids and why are there concerns over their use?

A: Neonicotinoid insecticides are active molecules that show high toxicity towards insects (they are neurotoxic, agonists of the nicotinic acetylcholine receptor) but have very low activity toward other organisms, including mammals. In general, they are systemic insecticides – they can translocate from soil or move from the surface of plants into plant fluids and tissues and protect the whole plant, including the radical apparatus – thanks to their good solubility in water. For these properties they are widely used in crop protection around the world through spray and soil treatments, or by the seed coating modality.

All these treatments can be dangerous for bees and other pollinator insects. The seed coating, largely used in maize crops, is of concern for two reasons. Firstly, guttation drops produced by corn seedlings grown from seeds coated with neoniconioids contain high concentrations of the insecticide, lethal for bees that may use these solutions as water source. Secondly, the sowing of coated corn seeds, using pneumatic drilling machines, releases in the air large amounts of powders including fragments of the seed coating that contains the insecticide. Foraging bees can efficiently intercept these particles, with their consequential contamination at lethal doses, if they cross the sowing field to reach areas with flowers where they can collect nectar and pollen for the hive.

Q. What analytical method did you use to determine levels of neonicotinoids in honeybees?

A: The exposure of flying bees to the seed coating particles released in air by the drilling machines during the corn sowing can induce bee contaminations in the range of 50–1200 ng/bee of insecticide, with consequential acute lethal effects. These concentrations have been determined (for the first time in single bees) by an optimized procedure based on a QuEChERS extraction and ultrahigh-performance liquid chromatography method with diode array detection (UHPLC–DAD) instrumental analysis.

Analysis of these insecticides in single bees is of great importance in the study of exposure mechanisms and in the quantification of the consequent contamination of bees in flight over the sowing field and approaching the drilling machine.

Q. How have you optimised sample extraction, preparation and analysis of specimens?

A: All steps of the analytical procedure have been optimized by the computation of the recovery factors and repeatability. Moreover, the comparison of results with those obtained by an independent method (UHPLC with quadrupole time-of-flight mass spectrometry detection) revealed the probable absence of bias in the UHPLC-DAD method.

Q. Did you experience any technical difficulties? What are the limitations of the method?

A: The main advantage of the optimized QuEChERS–UHPLC-DAD procedure is that it makes it possible to perform the analysis on a single bee. Other published methods refer to larger bee samples of 2–15 g (20–150 bees). As a consequence, the main limitation of our method is in terms of the detection limit.

The limits of detection (LODs) were 5 ng per bee for thiamethoxam, N -desmethyl thiamethoxam, and thiacloprid, 7 ng per bee for clothianidin and imidacloprid, and 11 ng per bee for acetamiprid. These LODs are significantly higher than those reported in the literature for HPLC-MS methods , but still adequate for the analysis of single bees exposed to acute levels of neonicotinoid insecticides.

Also compensating for the lower LODs, however, is the fact that the UHPLC-DAD method uses simpler instrumentation that more laboratories have access to.

Q. Are other new analytical methods still needed for the study of colony collapse disorder?

A: Although well established procedures have been developed for the analysis of neonicotinoids in environmental samples relevant to colony collapse disorder (CCD) —such as pollen, honey, bees, and wax —new accurate analytical methods are desired. This is in view of more rapid procedures for extraction and analysis; the possibility of analyzing a single insect; investigating the effects of very low exposure (for instance, sub-lethal doses); investigation into metabolites; more accurate studies on bee exposure; and studies on the effects of neonicotinoids on other insects (pollinators).

Q. What are you going to work on next?

A: We are currently studying selected degradation mechanisms of these insecticides in the environment. Actually, low concentration of neonicotinoids after acute exposure in bee samples could be the result of rapid metabolic conversion in bees or, after death, their biological degradation. In this respect, both the analyses of metabolites and of photodegradation products are of high interest. For these studies, proper UHPLC-ESI/Q-TOF/MS methods will be optimized and validated.

Andrea Tapparo graduated with a degree in Chemistry in 1987 and became an Associated Professor of Analytical Chemistry in 1998, currently based at the University of Padova (Italy), Dept. of Chemical Sciences. The research activities of Professor Andrea Tapparo have been focused on the optimization of new analytical methodologies for the studies of metal ion interactions in aqueous systems of biological and environmental relevance. More recently, he has also developed studies in the field of environmental analytical chemistry, by the optimization of new analytical methodologies for the determination of environmental micro-pollutants and their metabolites. His recent studies on the exposure of honey bees to neonicotinoid insecticides have significantly contributed to clarify the role of seed coating insecticides used in agriculture, in the colony loss phenomena observed worldwide. He is co-author of 61 scientific publications and about 70 communications to Italian and international conferences.