Pestanal

100 mg/L stock solutions of thiamethoxam (99.6% purity; 37924, PESTANAL®, Sigma-Aldrich, Oakville, Ontario), imidacloprid (99.9% purity; 37894, PESTANAL®, Sigma-Aldrich, Oakville, Ontario), and clothianidin (99.9% purity; 33589, PESTANAL®, Sigma-Aldrich, Oakville, Ontario) were prepared in distilled water. Pollen patties were prepared from a mixture of 1 kg soybean flour and brewer’s yeast (3:2 ratio), 1.3–2 liters 2:1 (w:w) sucrose syrup and 0.5–2 kg (15.8–39.5% final patty weight) pollen obtained from pollen traps placed on colonies in 2015 and 2016. The quantity of pollen in the patties was increased over the course of the trial to satisfy the nutritional requirements of growing colonies. The neonicotinoid stock solutions were diluted in sucrose syrup (1:1 w:w) to a concentration of either 20 nmol/L (~5 ng/g) or 80 nmol/L (~20 ng/g). Neonicotinoid concentrations were calculated in molarity to obtain an equal number of molecules of each neonicotinoid in the experimental diet. Aliquots of the pollen patties and sugar solutions from treatment and control groups were submitted for measurement of neonicotinoid concentration by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) (Government of Alberta Agriculture and Forestry Chemistry Laboratory, Edmonton, Alberta).

The chemicals used in the toxicity assays were the commercial formulation Winner Top® (Selectis®, Portugal), terbuthylazine (Pestanal®, Sigma-Aldrich®, Steinheim) and nicosulfuron (Pestanal®, Sigma-Aldrich®, Steinheim). Winner-Top is a two-way herbicide formulating terbuthylazine (250 g/L) and nicosulfuron (16.75 g/L). Stock solutions were prepared immediately before each assay by dissolving the a.s. or diluting the commercial formulation in culture medium or in tap water for testing with L. minor and P. oleracea, respectively. No solvent carriers were used.

We adopted a simplified knockdown assay^{51 (link)} to evaluate susceptibility to the knockdown effect to type I (permethrin) and type II (deltamethrin) pyrethroids in larvae. We prepared solutions with technical grades permethrin (Pestanal, Sigma-Aldrich) and deltamethrin (Pestanal, Sigma-Aldrich) by dissolving each in 1 mL acetone and then ethanol to make 250 ppm stock solutions. From this, we prepared a 20 mL solution for each pyrethroid, with 10 replicas for two concentrations (0.1 ppm and 0.4 ppm) in H₂O. Two negative control cups with ethanol (32 µL) in 20 mL of H₂O were run in parallel. We used one L4 larva per replica and registered the knockdown every 5 min, for half an hour. The KdT₅₀, i.e., the time when 50% of the larvae were knocked down, was scored according to these categories: 1 (0–5 min), 2 (6–10 min), 3 (11–15 min), 4 (16–20 min), 5 (21–30 min) or 6 (> 30 min). The susceptibility index (SI) for each species against deltamethrin and permethrin was obtained by multiplicating the KdT₅₀ categories of both concentrations (0.1 and 0.4 ppm). Population with the lower SI is considered more sensitive. The obtained SI values were the mean of three independent assays, with Rock in parallel as an internal control in all of them.