Experiments were conducted between April and May 2019. Twenty-five commercial colonies of B. terrestris were purchased from Koppert Agricultural Co., Ltd. (Beijing, China). Each colony contained about 200 workers, a brood at all developmental stages, and a laying queen. The bumblebees were reared on a diet that included pollen and nectar and were provided by the company in an incubator with continuous darkness, at a temperature of 25 ± 1 ℃ and a relative humidity of 60 ± 10%.
Chlorpyrifos (CAS No. 2921-88-2, 96% technical material(TC)) was supplied by the Hunan Research Institute of Chemical Industry (Hunan, China). Imidacloprid (CAS No. 138261-41-3, 96% TC) was supplied by Shandong Zhongnong United Biological Technology Co., Ltd. (Shandong, China). Thiamethoxam (CAS No. 153719–23-4, 97% TC) was obtained from the Hailier Pesticides and Chemicals Group (Shandong, China). Each insecticide was dissolved in dimethyl sulfoxide (DMSO) and diluted in a 50% (w/w) sugar solution as the Organization for Economic Co-operation and Development (OECD) guideline37 and Yue et al.35 described, and where the volume ratio of DMSO to sugar solution was 1:500 (v:v). The data of our preliminary experiment in this study showed that there was no significant difference between blank control and DMSO control in mortality (the average mortality for the blank control and DMSO control group is 3.33% and 2.22%, respectively, 90 workers were used for each treatment, triplicate). Each stock solution was diluted to six test concentrations by using a calibrated micropipette and volumetric flasks.
The acute oral toxicity of the insecticides to the worker was tested according to the method recommended by the OECD37 (Organization for Economic Co-operation and Development). Briefly, one leg of the workers with same size was clamped gently with a forceps, and the bees were quickly transferred to a thermostat-controlled wooden box (dimensions 12 cm × 8 cm × 8 cm; Fig. 1). Fifteen workers were placed in each wooden box in the dark at room temperature (25 ± 1 ℃) and a relative humidity of 60 ± 10% with a sufficient amount of noncontaminated 50% sugar solution (w/w). The bees were left alone for at least 8 h for adaptation. The experiment was conducted when the mortality rate of bumblebees in the wooden box did not exceed 10%. A 300 μL of quantity of the 50% sugar solution was then either contaminated with an insecticide or fed uncontaminated to the worker bumblebees via a 5 mL syringe with the tip removed (Fig. 2) for 6 h, followed by 2 h of starvation. The sugar solution was immediately replaced with a sufficient amount of uncontaminated sugar solution once the 300 μL of sugar solution had been consumed over the 6 h. The mass of each test solution was weighed and recorded before and after each feeding.
Figure 1Bumblebees in the wooden for toxicity assessment.
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Figure 2A 5 mL syringe with the tip removed.
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A preliminary experiment suggested that evaporation of the sugar solution in the syringe did not significantly affect the mass change (a loss of about 0.001 g). Therefore, the consumption of the sugar solution could be inferred from the differences before and after insecticide exposure. The mixtures were then converted from concentrations into doses in micrograms of the active ingredient per worker. The LD50 values were calculated by probit analysis using POLO-PC software38.
The individual and combined toxic effects of insecticides on bumblebees were assessed using the median-effect equation described by Liu et al.37 and Chou and Talalay39:
$$f_{a} /f_{u} = \, \left( {D/D_{m} } \right)^{m}$$
(1)
where D is the dose of an insecticide, Dm is the dose for a 50% effect, fa is the mortality influenced by D (percentage of mortality), fu is the survival rate uninfluenced by D (percentage of survival, fu = 1 − fa), and m is the coefficient determining the shape of the dose–effect relationship.
By rearranging Eq. (1), we can obtain the following equations:
$$f_{a} = { 1}/\left[ {{1 } + \, \left( {D_{m} /D} \right)^{m} } \right]$$
(2)
$$D = D_{m} [f_{a} /({1} - f_{a} )]^{{{1}/m}}$$
(3)
Therefore, if we know the values for m and Dm, we can easily assess the effect (fa) for any given dose (D) in Eq. (2). In the same way, the dose (D) can easily be calculated by the effect (fa) given in Eq. (3). In addition, if we take the logarithm of both sides of Eq. (1) and assume that x = log(D) and y = log(fa/fu), we can obtain the following middle-effect diagram:
$${\text{log}}\left( {f_{a} /f_{u} } \right) \, = m{\text{log}}\left( D \right) - m{\text{log}}\left( {D_{m} } \right)$$
(4)
In the median-effect plot in Eq. (4), we can easily determine the Dm, where m for the Dm means the antilog of the x-intercept and m is the slope. Here, m > 1, m = 1, and m < 1 signify sigmoidal, hyperbolic, and flat sigmoidal dose–effect curves, respectively. In addition, the linear correlation coefficient (r) of the median-effect plot can reveal how the data conform to the median-effect plot, where r = 1 shows excellent conformity.
Therefore, we can easily calculate the combination index (CI) values by using the CI equation for a combination of n insecticides, which is given as
$$n(\mathrm{CI})x={\sum }_{j=1}^{n}\frac{(D)j}{({D}_{x})j}={\sum }_{j=1}^{n}\frac{({D}_{x}{)}_{1-{\text{n}}}\{[D]j/{\sum }_{i}^{n}[D]\}}{({D}_{m}{)}_{j}\{({f}_{ax})j/[1-({f}_{ax})j{]}^{1/mj}}$$
(5)
where (CI)x is the combination index for n insecticides at x% effect (fa); (Dx)1−n is the sum of the dose of n insecticides causing x% effect (fa) in combination; [D]j/\({\sum }_{1}^{n}[D]\) is the proportionality of the dose of n individual insecticides causing x% effect (fa) in combination; (Dm)j{(fax)j/[1 − (fax)j]1/mj} is the dose of individual insecticides causing x% effect (fa); and fax is the fractional effect (fa) at x% effect (fa), where CI > 1, CI < 1, and CI = 1 indicate an antagonistic, synergistic, and an additive effect, respectively.