Trace-level determination of pyrethroid, neonicotinoid and carboxamide pesticides in beeswax using dispersive solid-phase extraction followed by ultra-high-performance liquid chromatography-tandem mass spectrometry

<div><p>The aim of the work was to develop an analytical procedure able to quantify traces of 13 neonicotinoids and pyrethroids as well as carboxamide in beeswax at low levels (ng g⁻¹) to evaluate the contamination. For this purpose, an efficient sample preparation procedure was developed based on solid–liquid extraction using dispersive diatomaceous earth and acetonitrile. This step was followed by a selective and sensitive analysis based on ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (ESI-MS/MS). This analytical procedure was validated based on International Conference on Harmonization guidelines. The limits of quantification ranged from 1 ng g⁻¹ (thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiacloprid and boscalid) to 40 ng g⁻¹ (lambda-cyhalothrin). The method was then successfully applied to 60 samples of beeswax collected in several areas of France. The presence of thiacloprid, boscalid, imidacloprid and deltamethrin in beeswax was confirmed. The most frequently quantified pesticide was boscalid.</p></div

3D representation of a bee from different angles.

<p>Bees were scanned using X-ray tomography. A: Left lateral view; B: Ventral view; C: Right lateral view; D: Dorsal view.</p

Summary of commercial products and their application rates used for the spraying treatment.

<p>g a.s./ha: gram of active substance per hectare.</p><p>Summary of commercial products and their application rates used for the spraying treatment.</p

Determination of the exposure surface area per bee for each active substance.

<p>S.D.: Standard Deviation (n = 3).</p><p>g a.s./ha: gram of active substance per hectare.</p><p>ng a.s./ha: nanogram of active substance per hectare.</p><p>ng a.s./bee: nanogram of active substance per bee.</p>a<p>The application rate was corrected with the measured concentration (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113728#pone-0113728-t003" target="_blank">Table 3</a>) and the experimental control deposit.</p>b<p>This corresponds to the concentration of active substances found in the bees frozen just after contamination.</p>c<p>The exposure surface area is the ratio between the residues (ng a.s./bee) and the corrected application rate (ng a.s./cm²).</p><p>For each treatment, the bees from the 6 replicates were counted, pooled, and weighed; the mean bee weight was determined by dividing the weight of bees by the actual number of bees.</p><p>Determination of the exposure surface area per bee for each active substance.</p

Distribution of the deposit during preliminary calibrations and experimental controls.

<p>The preliminary calibrations were performed before the experiment, and the experimental calibrations were performed during the experiment. The deposit was expressed in µL/cm² of the disc. T-test preliminary vs. experimental calibrations; (t = 0.0739, df = 148, p-value = 0.9412).</p

Comparison of the commercial and measured concentrations.

<p>S.D.: Standard Deviation.</p><p>The concentrations of commercial products are expressed according to their nature, liquid or solid.</p><p>The quantification of the active substance was performed in triplicate from the phytopharmaceutical preparation used for the spray application.</p><p>The differences were expressed as percentages of the commercial concentrations.</p><p>Comparison of the commercial and measured concentrations.</p

Correlation between mortality induced by spraying and mortality induced by topical treatment.

<p>Each dot represents one substance among the 15 tested on bees by thorax topical contact and spraying. The mortality elicited at 48 hours by spraying was given for the chosen field application rate (g a.s./ha) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113728#pone-0113728-t001" target="_blank">Table 1</a>). On the basis of the mean exposure surface area (1.05 cm²/bee), the application rate (g a.s./ha) was converted into an individual dose (ng a.s./bee) to treat the bees topically. The dose-mortality relationship at 48 hours was modeled for each of the 15 active substances, which enabled assessing the mortality that could be induced at a given field exposure.</p

Comparison of the exposure and physical surface area of a bee.

<p>Mean exposure surface area (± S.D.) of a bee (average of the 20 commercial products) and mean physical surface area (± S.D.) of a bee (sum of the mean body and wing surface area).</p

Determination and comparison of the HQ and the revisited HQ.

<p>g a.s./ha: mass of active substance per hectare expressed in grams.</p><p>µg a.s./bee: mass of active substance per bee expressed in micrograms.</p><p>ng a.s./bee: nanogram of active substance per bee.</p><p>E.D.: Experimental Data.</p><p>LD₅₀: Median Lethal Dose.</p><p>HQ: Hazard Quotient (field rate (g/ha)/LD₅₀ (µg/bee)).</p><p>Revisited HQ (exposure (ng/bee)/LD₅₀ (µg/bee)).</p><p>N.C.: Not Calculated because of the low toxicity of the active substance.</p><p>DAR EFSA: Draft Assessment Report of the European Food Safety Authority.</p><p>PED US EPA: Pesticides Ecotoxicity Database of the United States Environmental Protection Agency.</p>a<p>Time at which the LD₅₀ was determined. The LD₅₀ values resulting from the experimental data were calculated at the time corresponding to a stabilized mortality.</p>b<p>For each active substance, 2 scenarios of exposure are presented: the lowest and the highest homologated application rate.</p>c<p>For each active substance, the highest and the lowest known LD₅₀ values were compared to the lowest and highest homologated application rates, respectively.</p>d<p>HQ is the ratio between the application rate (g a.s./ha) and the LD₅₀ (µg a.s./bee).</p>e<p>The exposure was calculated from the application rate (ng a.s./cm²) and the mean exposure surface area determined with the 20 active substances (1.05 cm²/bee).</p>f<p>The LD₅₀ values from the experimental data were calculated with the BMD software from the US EPA.</p>g<p>The revisited HQ is the ratio between the exposure (ng a.s./bee) and the LD₅₀ (ng a.s./bee).</p>h<p>For each active substance, the dose-mortality relationship was modeled at the time corresponding to a stabilized mortality.</p><p>Determination and comparison of the HQ and the revisited HQ.</p

Comparison of preliminary calibrations and experimental controls of the deposit.

<p>S.D.: Standard Deviation.</p><p>Nc: Number of calibrations.</p><p>C.I. 95%: Confidence Interval at 95%.</p>a<p>Sprayed volume corresponds to the deposit in field conditions.</p><p>Comparison of preliminary calibrations and experimental controls of the deposit.</p