5.3.2. Toxicity testing

1) The experimental unit is the 48-larvae plate. For each test, the following treatments should be used:

   - control without solvent (1 plate),
   - control with solvent (1 plate) if necessary,
   - 5 treatments, i.e. the 5 doses or concentrations to be tested (1 plate per treatment),
   - reference treatment with dimethoate (1 plate).
   - 1 additional plate (totally or partially filled with larvae, according to the number of available, remaining larvae) can
        be used at D4 in the acute toxicity test to replace the larvae which died before D4.

One test has a minimum of three replicates with different larvae origin and new tested solutions for each replicate.

2) The tested pesticide is preferably dissolved in water. If it is not soluble in water at the experimental concentrations, one can use another solvent such as acetone. In that case, it is necessary to prepare a second negative reference fed with diet containing the solvent at the same concentration as in the treated samples.

3) Dilutions of the stock solutions are made with osmosed water, using disposable pipette tips equipped with a filter.

4) The rate of the tested solution in the diet must not exceed 10% of the final volume. In all cases, it is necessary to use a constant volume for the different treatments in order to have a constant rate between the diet and the test pesticide solution.

5) The toxic reference is dimethoate:

   - in acute toxicity test: 3 μg/larva mixed with diet C and provided at D4,
   - in chronic toxicity test: mixed with the three diets at the constant concentration of 20,000 µg/kg diet.

6) In an acute toxicity test, larvae are treated at D4 with diet C containing the preparation to test at the suitable concentration. For a chronic toxicity test, larvae are treated every day (except D2) with the diets containing the preparation to test at a constant concentration.

7) In order to assess the adequate LD50 range, it is recommended to run a preliminary experiment where doses of the test preparation may vary according to a geometrical ratio from 5 to 10.