220.127.116.11.3. Toxic reference
1) A toxic (positive) reference should be included in the test series.
2) At least three doses should be selected to cover the expected LD50 value.
3) A minimum of three replicate cages, each containing ten bees, should be used with each test dose.
4) The preferred toxic reference is dimethoate. Gough et al. (1994) evaluated the use of dimethoate as a reference compound for acute toxicity tests on honey bees. The results of 63 contact tests of technical dimethoate were evaluated, using the 95% confidence linear regression of logit transformation on log10 dose (μg/bee), adjustments using Abbott’s correction. The contact LD50 assessed with six concentrations in acetone, control acetone and administration on the thorax was 0.16 (min 0.11, max 0.26) μg AI/bee. LD50 values ranging from 0.075 to 0.30 μl AI/bee in groups of 10 bees should be considered as valid results of the toxic standard Dimethoate. The LD50 (48 h) was similar to 24 h. For the contact LD50 tests, the contact LD50 (24 h) should be in the range of 0.10-0.30 μg AI/bee.
5) A dose range of 0.075 to 1.0 µg/bee is recommended and results falling in this range validate the test.
6) Other toxic standards would be acceptable where sufficient data can be provided to verify the expected dose response (e.g. parathion).