Development and comparison of two multi-residue methods for the analysis of select pesticides in honey bees, pollen, and wax by gas chromatography–quadrupole mass spectrometry

doi:10.1016/j.talanta.2015.03.031

Talanta

Volume 140, 1 August 2015, Pages 81-87

https://doi.org/10.1016/j.talanta.2015.03.031 Get rights and content

Highlights

•
Pesticide exposure has been linked with honey bee population losses.
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Reliable multi-residue analytical methods are needed for trace pesticide analysis.
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Two sample preparation methods were tested: GPC and d-SPE with Z-Sep.
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Z-Sep cleanup was the preferred method and was successful with field-collected media.

Abstract

One of the hypotheses that may help explain the loss of honey bee colonies worldwide is the increasing potential for exposure of honey bees to complex mixtures of pesticides. To better understand this phenomenon, two multi-residue methods based on different extraction and cleanup procedures have been developed, and compared for the determination of 11 relevant pesticides in honey bees, pollen, and wax by gas chromatography–quadrupole mass spectrometry. Sample preparatory methods included solvent extraction followed by gel permeation chromatography (GPC) cleanup and cleanup using a dispersive solid-phase extraction with zirconium-based sorbents (Z-Sep). Matrix effects, method detection limits, recoveries, and reproducibility were evaluated and compared. Method detection limits (MDL) of the pesticides for the GPC method in honey bees, pollen, and wax ranged from 0.65 to 5.92 ng/g dw, 0.56 to 6.61 ng/g dw, and 0.40 to 8.30 ng/g dw, respectively, while MDLs for the Z-Sep method were from 0.33 to 4.47 ng/g dw, 0.42 to 5.37 ng/g dw, and 0.51 to 5.34 ng/g dw, respectively. The mean recoveries in all matrices and at three spiking concentrations ranged from 64.4% to 149.5% and 71.9% to 126.2% for the GPC and Z-Sep methods, with relative standard deviation between 1.5–25.3% and 1.3–15.9%, respectively. The results showed that the Z-Sep method was more suitable for the determination of the target pesticides, especially chlorothalonil, in bee hive samples. The Z-Sep method was then validated using a series of field-collected bee hive samples taken from honey bee colonies in Virginia.

Graphical abstract

Introduction

The honey bee, Apis mellifera, plays a critical role in agriculture and the global ecosystem by pollinating plants, while at the same time producing bee products with high economic value [1], [2]. Globally, this value has been estimated to be approximately $210 billion, thus honey bees are an essential target for conservation [3]. However, in recent years, honey bee populations have been in a worldwide decline, which has been referred to as colony collapse disorder (CCD) and colony weakening [4], [5]. Multiple causes of colony losses have been proposed, such as exposure to pesticides, pathogens, parasites, and natural habitat degradation [6], [7]. Among these factors, pesticides are suspected by the scientific and beekeeping communities to have a strong impact on honey bee mortality and colony weakening [8], [9]. In modern farming systems, honey bees are readily exposed to pesticides when they gather nectar and pollen from blooming crops, which are routinely treated with pesticides [10], [11]. For example, researchers have demonstrated that low levels of pesticides, such as pyrethroid and neonicotinoid insecticides, may induce adverse sublethal effects in honey bees [8], [12], [13], [14], [15]. Honey bees are also exposed to miticides, like coumaphos and tau-fluvalinate, which are intentionally introduced to the hives to control the parasitic mite, Varroa destructor [16]. However, the relative contribution that pesticides have in colony losses remains unknown. Thus, to better understand the potential involvement that pesticides may have in colony losses, it is essential to develop reliable and sensitive analytical methods for the quantitation of pesticides in honey bees as well as in bee products, including pollen and wax.

In the past few years, several methods have been developed for the detection of pesticides in bee products like honey, pollen, wax, and honey bees [3], [17], [18], [19], [20], [21], [22], [23], [24], [25]. However, most of the reported methods have focused on one or two matrices. To date, there have been very few multi-residue methods described in the literature for the simultaneous analysis of pesticide residues in honey bees, pollen, and wax. Since honey bees are most likely exposed to pesticides in both pollen and wax, it is important to be able to simultaneously quantify pesticide residues from these relevant matrices in one study. In view of these concerns, the aim of the current study was to develop a fast and reliable multi-residue analytical method for the trace analysis of relevant pesticides in honey bees, pollen, and wax. A total of 11 pesticides were selected for this study including the pyrethroid insecticides bifenthrin, lambda-cyhalothrin, permethrin, cyfluthrin, cypermethrin, and tau-fluvalinate, the organophosphate insecticides chlorpyrifos, coumaphos and coralox, the organochlorine fungicide chlorothalonil and the triazine herbicide atrazine (Table 1). These target analytes were chosen based on their potential toxicity to honey bees at low environmental concentrations and their widespread use in plant protection or in the bee hive directly. Two sample preparation methods, based on cleanup with gel permeation chromatography (GPC) and dispersive solid-phase extraction (d-SPE) with a new zirconium-based sorbent (Z-Sep) were compared with subsequent determination by gas chromatography coupled to a quadrupole mass spectrometry (GC–MS). Finally, the Z-Sep method was applied to bee hive samples collected in Virginia to validate this method as well as obtain preliminary data on the pesticides present in the hive.

Section snippets

Chemicals and reagents

Pesticides analyzed in the current study were purchased from ChemService (West Chester, PA, USA), and their purities were >97.0% as certified by the manufacturer. Decachlorobiphenyl (DCBP) and 4,4′-dibromooctafluorobiphenyl (DBOFB) were used as surrogates and were purchased from Supelco (Bellefonte, PA, USA), and had purities >99%. The internal standards, PCB 204, chlorpyrifos d10, coumaphos d10, flucynthrinate, and atrazine d5 (AccuStandard, New Haven, CT, USA) were added to the solutions

Matrix effects

Previous analysis of honey bee and pollen extracts revealed large amounts of lipids and pigments in honey bees, and lipids and proteins in pollen, which proved to be the most serious interferences during the analysis [1]. Wax is a very complex mixture of lipophilic compounds, including esters of long-chain aliphatic alcohols with fatty acids or hydroxy-fatty acids, long-chain hydrocarbons, free long-chain fatty acids, and carotenoids [22]. High molecular weight compounds that are lipid-like and

Conclusions

Honey bee hive matrices are a challenge to cleanup and quantify due to matrix issues created by the large number of fatty compounds, pigments, carbohydrates, and other complex components [39]. Two cleanup methods were compared and contrasted in the current study to quantify 11 target pesticides in honey bees, pollen and wax: an older established GPC method and a newly developed zirconia-based sorbent (Z-Sep) method. Overall, the Z-Sep method had less matrix interference, higher sensitivity,

Acknowledgments

This project was wholly (or partially) funded by a grant (MOA 2013-001) from the Virginia Department of Agriculture and Consumer Services, United States. Funding for this project came from registration fees paid by pesticide companies.

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Development and comparison of two multi-residue methods for the analysis of select pesticides in honey bees, pollen, and wax by gas chromatography–quadrupole mass spectrometry

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals and reagents

Matrix effects

Conclusions

Acknowledgments

J. Food Prot.

J. Chromatogr. A

Pharmacol. Biochem. Behav.

Anal. Chim. Acta

J. Chromatogr. A

Food Chem.

J. Chromatogr. A

Talanta

J. Chromatogr. A

J. Chromatogr. A

Food Chem.

Talanta

Talanta

J. Chromatogr. A

J. Chromatogr. A

PLoS One

J. Apic. Res.

PLoS One

Science