1. Towards a sustained bee health: Recent Updates on the in-vitro bee larvae test
Kimmel, Stefan1, Liedtke, Anja1, Winter, Jessica2, and Höger, Stefan1
1: Harlan Laboratories Ltd. 2: Hochschule für Life Sciences FHNW Zelgliweg Werkareal Rosental, Mattenstr Itingen/ Switzerland Basel/Switzerland
Correspondence:
Keywords: Bee protection, bee larvae toxicity, regulation 1107/2009, TIERed testing approach
Introduction & Objectives
The impact of plant protection products and therefore ecotoxicological testing on pollinators in common and on honeybees in special has become an important focus throughout the recent years. Not only since the new regulation 1107/2009, protection and intensive research on bee health, being already an important part, has become an even more important piece of the registration process, especially for, but not only limited to pesticides and insect growth regulators.
In the light of the recently published authority opinion and highly discussed repressive strategy on certain neonicotinoids and their effects on bees, an in-deep and profound research on bee health becomes even more important.
Scope of this presentation is a brief update on the recent proceedings from OECD workshop and seminars on bee larvae toxicity from the CRO perspective as well as adaption and modification of the bee larvae toxicity test protocol introduced by Aupinel et al. (2005, 2007 & 2009):
A worst case approach focussing on larvae mortality after single dose exposure (OECD guideline publication in progress)
An extended and prolonged approach with multiple/daily dose exposure (further validation necessary, intended to proceed with additional ringtests). 
Materials & Methods
The test setup described by Aupinel et al. (1;2;3;4) is predominantly modified by the following details:
Reduced number of larvae per treatment in order to make the design more handy and practically/financially affordable (since it is designed to be a worst case TIER I test)
Dividing the setup into a worst case, single dose 7 day test and a chronic, repetetive dosing test aiming for the full span of bee larvae development
Avoidance of questionable substances such as Glyerol etc.
Refinement on equipment/handling, e.g. use of a warming plate, background information on the used royal jelly, randomisation of larvae right before treatment...
Figure 1: Schedule of the worst case approach testing after single dose exposure
Results & Discussion
Table 1-3: Results of three representative trials on the reference item dimethoate
LC50 at pupation: 2.9 [µg /larva]
LC50 at pupation: 2.6 [µg /larva]
LC50 at pupation: 3.2 [µg /larva]
The here presented results demonstrate a reliable and robust method for TIER I testing on effects of bee larvae. Further on, this setup completes the semi-field and even field testing setup (e.g. Shur et al.) for the risk assessment scheme. It is demonstrated that the upcoming guideline on worst case testing of bee larvae toxicity can be of great importance and is able to fill a so far vacant position of in vitro testing within the field of bee toxicology. Towards a sustained bee health.
Conclusion
The split into these two approaches enables a fast and intense experience of a possible hazard effect on bee health as well as emphasis on different endpoints, e.g. LDx and NOEC. Further on, this testing strategy fills the gap of having no laboratory worst case test system for bee larvae toxicity which can be conducted prior to tunnel, semi-field and field testing of whole honeybee colonies (e.g. OECD GD 75, 2007; Oomen et al., 1992). The presented methods also suit the recently increased governmental authority requests for intense bee health research within the risk assessment for plant protection products (Alix & Lewis, 2010; US EPA, 2011, EFSA, 2012).
Aupinel, P., Fortini, D., Dufour, H., Tasei, J. N., Michaud, B., Odoux, J. F., & Pham-Delegue, M. H. (2005). Improvement of artificial feeding in a standard in vitro method for rearing Apis mellifera larvae. Bulletin of Insectology, 58,
(2) Aupinel, P., Fortini, D., Michaud, B., Marolleau, F., Tasei, J. N., & Odoux, J. F. (2007, Oct 12-14). Toxicity of dimethoate and fenoxycarb to honey bee brood (Apis mellifera), using a new in vitro standardized feeding method. Paper presented at the 9th International Symposium of the ICP-BR-Bee-Protection-Group, York, ENGLAND.
(3) Aupinel, P., Medrzycki, P., Fortini, D., Michaud, B., Tasei, J. N., & Odoux, J. F. (2007). A new larval in vitro rearing method to test effects of pesticides on honey bee brood. Redia, 90,
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(5) United States Environmental Protection Agency (US EPA), Interim Guidance on Honey Bee Data Requirements (2011), available at:
(6) European Food Safety Agency (EFSA), draft Guidance on the Risk Assessment of Plant Protection Products on Bees (2012), available at:
(7) Alix, A., Lewis, G. (2010). Guidance for the assessment of risks to bees from the use of plant protection products under the framework of Council Directive 91⁄414 and Regulation 1107⁄2009. OEPP/EPPO, Bulletin OEPP/EPPO Bulletin, 40, 196–203