Exploring sources of inaccuracy and irreproducibility in the CDC bottle bioassay through direct insecticide quantification.

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 have 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 effectiveness of cleaning bottles by autoclaving because this method is commonly used in insecticide assessment laboratories. The two insecticides used in this study were chlorpyrifos and lambda-cyhalothrin (λ-cyhalothrin). Insecticides were removed from glass bioassay bottles by rinsing with ethyl-acetate and n-hexane and then quantified using GC-MS/MS. RESULTS: The CDC bottle bioassay cleaning methods did not sufficiently remove both insecticides from the glass bottles. The cleaning methods removed chlorpyrifos, which has higher water solubility, more effectively than λ-cyhalothrin. 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 for 6 h 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 the bottle during the 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.

Investigating the Effects of Temperature, Relative Humidity, Leaf Collection Date, and Foliar Penetration on Leaf-Air Partitioning of Chlorpyrifos.

Leaf-air partition coefficient (Kleaf-air) values are needed to understand and predict pesticide volatilization and persistence in agroecosystems. The objectives of this work were to measure Kleaf-air values and foliar penetration for the insecticide chlorpyrifos (as an active ingredient alone and in a pesticide formulation) on alfalfa (lucerne) leaves at a range of temperatures and relative humidities and when using leaves collected in different summer months. Kleaf-air values were measured using a solid-phase fugacity meter. A portion of the leaves were also used for foliar penetration experiments. Kleaf-air values for chlorpyrifos as an active ingredient alone decreased with temperature, while the effects of temperature on chlorpyrifos in the formulation were negligible. No correlations between Kleaf-air values and relative humidity were observed. Foliar penetration increased with temperature for chlorpyrifos both as an active ingredient and in the formulation. Increasing foliar penetration with temperature is attributed to increasing diffusion into inner leaf layers. Both volatilization and foliar penetration affect the measured Kleaf-air values, so understanding the link between these processes is necessary to predict Kleaf-air values. The leaf collection date had a substantial effect on the measured Kleaf-air values, highlighting the need for a better understanding of the role of leaf properties on Kleaf-air.

Synthesis of C2 -Symmetric Dimeric Re(I) Peptide Complexes.

The reactions of [Re(CO)3(H2O)3]Br or Re(CO)5Cl with two peptides, glycylglycine or glycylalanine, were investigated. Each reaction produced a unique, well-defined product. Structural elucidation showed the formation of chiral compounds with the formula [Re(CO)3(Gly-Xxx-O)]2, Xxx = Gly 1, Ala 2. Each dimer displays C2 -symmetry and a nearly rectangular shape. The ligands are bound via the amine and amide carbonyl at a rhenium center and via the pendant carboxylate to the adjacent rhenium center. Both products are fully characterized, via X-ray structure elucidation.