18.104.22.168. Mortality assessment
1) In all treated and control groups, mortality (see section 2) is recorded at 4, 24 and 48 h post exposure. Data should be summarised in tabular form, showing for each treatment group, as well as control and toxic standard groups, the number of bees used, mortality at each observation time, and number of bees with adverse behaviour (Table 2). Any abnormal effects observed during the test are recorded in order to inform about possible subletal effects (Table 1). When mortality continues to increase after 48h, it is appropriate to extend the duration of the test up to 72 or 96 hours.
2) For the validity of the test, mortality in the negative (untreated) reference should be < 10% (OECD, 1998a; CEB, 2011) or 15% (EPPO, 2010b) and the mortality of the toxic standard dimethoate (positive reference) should meet the specified range: almost 50% with the lower dose (0.10 µg AI/bee) to 80-100% for the higher dose (0.35 µg AI/bee). Data from tests failing to meet these standard criteria should not be used.
3) Mortality data are submitted to a statistical
analysis. The LD50 has to be calculated (see 22.214.171.124.) for each
recommended observation time (i.e. 24h, 48h and if relevant, 72h, 96h) based on
mortality data corrected for control mortality using Abbott’s formula (see
Table 2. Example of data sheet: both mortality and number of living bees, abnormal behaviour of living bees are recorded simultaneously. For behavioural effects see Table 1.