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Effects on Honey Bee Brood (Apis mellifera) - Brood Feeding Test -

This test system is designed to assess the effects of plant protection products to the honey bee brood. The method of investigating the development of the honey bee brood is based on the method of Oomen et al. (1992). Ontogenesis of eggs, young and old larvae of honey bees are observed as well as mortality of the adult honey bees and sublethal effects, such as changes in behaviour.

Study Design

Test organisms

Honey bee colonies, disease-free and queen-right, bred by ibacon according to normal beekeeping practice. Honey bees are free flying with access to natural nectar sources. At least 3 colonies are used per treatment group.

Course of the test

Application:

The test item is mixed into a ready-to-use sugar solution. 1 L of the spiked sugar solution is offered in a feeding trough per colony. The feeding troughs are removed after complete uptake of the food or at the earliest after 24 hours. If food uptake is incomplete 24 hours after the start of dosing, the food will be left in the hive until food uptake is complete.

Mortality and behavioural abnormalities are recorded each day until the end of the test, typically 21 days after application.

Assessment of bee brood development (photo method):

For bee brood assessment several brood combs are taken out of each colony and a digital picture of each comb is taken. An area with at least 100 cells containing eggs, 100 cells containing young larvae and 100 cells containing old larvae are selected and marked on the first assessment date (= BFD 0) by using a computer assisted digital program (e.g. Honeybee Brood Logger, Supplier: WSC Scientific GmbH, www.wsc-regexperts.com). On each subsequent assessment date, the combs are taken out of the hive again and another digital photo is taken in order to follow up the progress of the initial marked cells. This procedure is repeated in regular intervals (BFDn = 5, 10, 16, 21 days ± 1 day) until a full period of bee development is completed (i.e. 21 days, see table 1). This allows a continuous photo-documentation following the first brood fixing day (BFD0) until the end of the assessment period.

Assessment Date Expected Brood Stage
1 - 2 days before application (=BFD0) eggs young larvae old larvae
+ 5 days (± 1 day) after BFD0 larvae or capped cells old larvae or capped cells capped cells
+ 10 days (± 1 day) after BFD0 capped cells capped cells capped cells
+ 16 days (± 1 day) after BFD0 capped cells capped cells or empty cells empty cells
+ 21 days (± 1 day) after BFD0 empty cells all development stages, empty or food not necessary

Because of biological variances (e.g. an accelerated or delayed development) the definite development pattern should be adopted.

Endpoints

The Endpoint of this test design is the Brood Termination rate, which is the number of unsuccessfully developed cells divided by the total number of marked cells per brood stage (eggs, young larvae and old larvae, respectively). Results obtained from the colonies treated with test item are compared to those obtained from the reference item and the untreated control. If indicated appropriate statistical methods are applied.

Guidelines and Literature

  • Oomen P.A., d Ruijter & J. van der Steen 1992: Method for honey bee brood feeding tests with insect growth-regulating insecticides, OEPP/EPPO Bulletin 22:613-616 (1992)
  • Schmitzer S., Lückmann J. (2013), Evaluation and improvement of the Oomen bee brood test; Poster Presentation at the SETAC GLB Conference in Essen, Germany, 2013 and the 8th SETAC Europe Special Science Symposium, Brussels, Belgium, 2013.