Exploring Sources of Inaccuracy and Irreproducibility in the CDC Bottle Bioassay Through Direct Insecticide Quantification

Abstract Background The Centers for Disease Control and Prevention (CDC) bottle bioassay is a commonly used susceptibility test for measuring insect response to insecticide exposure. However, inconsistencies and high variability in insect response when conducting CDC bottle bioassays has been reported in previous publications. We hypothesized that the CDC bottle bioassay results may be compromised when expected and actual insecticide concentrations in the bottles are not equivalent, and that inadequate bottle cleaning and/or loss during insecticide introduction and bottle storage steps could be responsible. We explored this hypothesis by quantifying insecticides using gas chromatography tandem mass spectrometry (GC-MS/MS) in bottles that had been cleaned, prepared, and stored according to the CDC guidelines.Methods We investigated the bottle cleaning, preparation, and storage methods outlined in the CDC bottle bioassay procedure to identify sources of irreproducibility. We also investigated the cleaning effectiveness of autoclaving bottles because this method is commonly used in insecticide assessment labs. The two insecticides used in this study were chlorpyrifos and lambda-cyhalothrin (λ-cyhalothrin). Insecticide was removed from glass bioassay bottles by rinsing with ethyl-acetate and n-hexane and quantified using GC-MS/MS.Results The CDC bottle bioassay cleaning methods did not sufficiently remove insecticide from the glass bottles. The cleaning methods removed chlorpyrifos, which has higher water solubility, more effectively than λ-cyhalothrin, which is less water soluble. Chlorpyrifos experienced significant loss during the bottle coating process whereas λ-cyhalothrin did not. As for bottle storage, no significant decreases in insecticide concentrations were observed during six hours following the initial drying period for either insecticide.Conclusions The CDC bottle bioassay protocol is susceptible to producing inaccurate results since its recommended bottle cleaning method is not sufficient and semi-volatile insecticides can volatilize from glass during the bottle coating process. This can lead to the CDC bottle bioassay producing erroneous LC50 values. High levels of random variation were also observed in our experiments, as others have previously reported. We have outlined several steps that CDC bottle bioassay users could consider that would lead to improved accuracy and reproducibility when acquiring toxicity data.