4.11. Application of test substances

Substances (toxins or pathogens) can be tested in chronic or acute conditions. In chronic testing, a substance is mixed into the diets at a constant concentration and provided to the larvae at each feeding day. Acute exposure can occur at every feeding day by mixing the tested substance into the diet once. The testing compound is dissolved in water. If it is not soluble in water at the experimental concentrations, other solvents such as acetone can be used. In that case, it is required to have a second control group fed with diet containing the solvent at the same concentration as the treated samples. The proportion of the solvent must not exceed 10 % of the final volume. A different rate has to be justified. In all cases, a constant volume for the different dilutions must be used in order to maintain a constant concentration between the control diet and the test substance diet. In order to assess the adequate LD50 or LC50 range, it is recommended to run a preliminary experiment where doses of the test substance may vary according to a geometrical ratio from 5 to 10 (for examples, see the BEEBOOK paper on toxicology methods (Medrzycki et al., 2013)).