22.214.171.124. Discussion and conclusion
Based on the OECD Guidance Document 75 (OECD, 2007), numerous studies were performed and it became obvious that the brood termination rate (= mortality of bee brood in selected cells on combs) was subject to a certain degree of variation, e.g. resulting in replicates with increased rates up to 100% in the control and reduced rates in the reference item group down to 21% (Pistorius et al., 2011). Additionally, a high variation between replicates within a respective treatment group occurred sometimes. The variability which was distinctly more present under semi-field conditions compared to a field method (Oomen et al., 1992) complicates the interpretation of results regarding potential brood effects of a test item with the outcome that some studies were regarded as invalid. The time between BFD and the following assessment on BFD +5 days turned out to be the most critical for such variations. Due to these variances, no definite conclusions regarding potential brood effects were possible in such cases, and the studies needed to be repeated.
In 2011, possible causes and improvements for the existing method were shown by Pistorius et al. (2011) and at the ICPBR (now ICPPR) meeting in Wageningen. Attempts to improve the methodology were initiated by the Working Group "Honey bee brood" of the German AG Bienenschutz. In 2011, honey bee brood studies adapted to these identified possible improvements, resulting in better results compared to historical data (for details see Pistorius et al., 2011).
Based on the analysed results, the working group recommended to improve the method by using bigger colonies with more brood, using 4 instead of 3 replicates for better interpretation of data, starting the study early in the season, avoiding major modifications of the colonies shortly before application and using larger tunnels with effective crop areas preferably > 80 m². To carry out quicker brood cell assessments to reduce stress for the colonies, it is recommended to use digital photo brood assessment as described in section 5.2.3., which allows marking a higher amount of cells (e.g. 200 to 400 cells).
In the overall outcome of the studies of the German working group, the combination of the suggested improvements showed a reduction in the breakup rate of the brood development in single cells and in the variability of the results in the control group (Pistorius et al., 2011). However, it also showed that even when fulfilling all the described improvements, it may happen that the brood mortality increases to such a high level, that an evaluation of the test product data still is not possible.
Since the bee colonies are kept under semi-field conditions with restriction in their normal collection and flying behaviour, they generally are sensitive to any interference from outside. Therefore, one should avoid stressing the bees too much during the assessments as well as before set-up of the colonies in the tunnels.
For this reason, it is important to analyse the importance of additional factors in the future in order to be able to improve semi-field studies and studies under field conditions, where the detailed brood assessments are integrated into the study design.