184.108.40.206.3. Substance administration
1) Starve the bees for 1-2 hours before the test so that all bees will feed once the study begins.
2) All bees in a cage are exposed to one of the test substances dispersed in a sucrose solution by being allowed to feed ad libitum. The sucrose solution is mixed at 500 g sugar to 1 l distilled water.
3) The number of doses and replicates tested should meet the statistical requirements for determination of LD50 with 95% confidence limits. A preliminary test (range finder) is usually conducted with a dose range of factor 10 in order to determine the appropriate doses for the formal test (1, 10, 100, 1000, etc.). Secondly the acute toxicity test is conducted with five doses in a geometric series with a factor 2 in order to cover the range for the LD50 (ex; 100, 200, 400, 800, etc.).
4) Bees are provided with 10 µl/per bee of the sucrose solution containing the test substance at the different concentrations. In each test group, the feeder is removed from the box when empty (within 2-4 hours) and replaced with another one containing untreated sucrose solution.
5) In all groups, the eventual remaining treated diet is weighed and replaced with untreated sucrose solution after 6 hours; the amount of treated diet consumed per group is recorded.