1) The design includes 4 treatment groups:
- the 2 sunflower varieties,
- the untreated control
- toxic standard (positive reference with dimethoate at 400g AI/ha)
2) The untreated control and the toxic standard are kept in an open space close to the laboratory.
3) The control group receives no treatment. There is no “dusted” toxic reference; thus to ensure bee sensitivity and to validate the design, the toxic reference is treated with a liquid spray of dimethoate (i.e. Dimezyl 1 l/ha = 400 g AI/ha).
4) In this method, the four treatment groups do not have the same route of exposure; the two varieties with coated dressings are tested from dust issued from agricultural practices whereas the toxic standard is a spray and the control is untreated or water treated.
5) Assessments are conducted under controlled conditions where bees are exposed to foliage in hoarding cages similar to LD50 tests (see Williams et al., 2013 and section 220.127.116.11.2. of this manuscript).Sentinel plant foliage is collected 2 hours after seed sowing to look for acute toxicity effects on bees.
6) The surface in each hoarding cage is covered with foliage taken from sentinel plants. The surface of foliage is exactly adapted in number of cm². Twenty honey bees are introduced into all hoarding cages and are allowed to contact the leaves from the sentinel plants. Bees are taken from one single and healthy beehive and dispatched in the 4 groups and containers at random and per Williams et al., 2013.
7) The foliage from the sentinel plants is removed after 24 hours but bees are left in boxes for 2 additional days; thus the test duration is 72 h. Then the laboratory part of this methodology is very similar to standardized LD50 test: CEB 230 (CEB, 2011), EPPO 170 (EPPO, 2010), OECD 214 (OECD, 1998b), with mortality assessments at 4 hours, 24, 48 and 72 h after exposure.
8) From the raw data, the average mortalities are calculated in three (3) replicates of each treatment group using usual formulas in statistical analysis (see section 8.4.1.).
9) These results are validated by mortality at 24 hours of 0% in the control and over 90% in the toxic standard.
10) Item modalities induce intermediate mortalities close to the control or higher according to the amount of dust in contact with bees.
11) Assuming no cross contamination is possible, some lethal effects are observed on bees following the use of one treated seed, and absolutely no effect for the other one.