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Honeybee: Chronic Feeding Tests

Honeybees may be exposed to plant protection products after spray application directly by contact or by oral consumption of contami­nated pollen and nectar. Therefore the laboratory tests with adult honeybee workers for acute oral and contact toxicity (OECD 213 and 214) are important parts of the environmental risk assessments for the european registration process. But single acute exposure scenarios are not representative of the exposure of in-hive honeybees to products which may persist in nectar and/or pollen. A chronic oral uptake of a product may cause unacceptable effects to the honey bees in much lower concentrations as an acute single exposition. Therefore the chronic feeding test is an additional laboratory test to consider the effects of a 10 day oral exposition of adult honeybees to a plant protection product.

Study Design

Test organisms

The test is carried out with freshly emerged Apis mellifera worker honeybees from ibacon’s own apiary with disease-free and queen-right colonies. Brood frames are taken out of the hives and bees are swept out. Afterwards the combs are brought to the laboratory and are placed in a hatching box in an incubator. After a hatching period of at least one day, the bees will be taken out from the hatching box in the incubator and will be transferred into test units. The bees will then be acclimatized under test conditions and fed with 50 % w/v sucrose solution for one further day.

Course of the test

Freshly hatched worker honeybees are fed ad libitum with treated sugar solutions for 10 days via feeders. Aim of the test is the determination of a median lethal concentration (LC50) (full dose response test) or the detection of a NOEC (no observed effect concentration) (limit test) for an oral 10 days exposure to a test item.

The test item will be dissolved in 50 % w/v sucrose solution or it will be dissolved in a suitable solvent (e.g. acetone; for test items of low water solubility) and will be mixed with 50 % w/v sucrose solution. The reference item dimethoate will be dissolved in 50 % w/v sucrose solution. Appropriate control treatment groups (e.g. 50 % w/v sucrose solution and a solvent control, if necessary) will be used in the test.

Preliminary non-GLP tests can be carried out in order to determine the choice of appropriate test item solvent and concentrations.

The collected bees are randomly distributed in groups of 10 individuals into stainless steel test cages with a removable glass sheet as front side. The cages are stored in darkness in an incubator at a temperature of 33 ± 2°C.

The above mentioned treated and untreated sugar feeding solutions are administered to the bees ad libitum via weighed feeders. Every day the feeders are replaced by fresh ones to ensure that the bees have a continuous access to treated food throughout the study. The replaced feeders are weighed again for a calculation of individual daily food consumption per bee.

In order to adjust food uptake for possible evaporation of test solutions from the feeders 3 similar replicate cages are set up. These cages will contain no bees, only pre-weighed feeders containing diet of untreated or solvent control. These will be placed in the test environment alongside the test units. At the daily feeder exchange the feeders will be re-weighed and replaced with new feeders. This evaporation figure can then be subtracted from the calculated uptake to give the real uptake accounting the loss by evaporation.

Assessments

The following test parameters are assessed:

  • Mortality: daily counts of dead bees during the entire exposure phase. If possible a LC50 and/or a NOEC are calculated.
  • Behavioural abnormalities: any signs of intoxication are recorded throughout the trial (e.g. movement coordination problems, apathy).
  • Food uptake: every day the actual food consumption per bee is measured and recorded.

Validity Criteria

Control and reference item groups ensure that the test results show high accuracy and trueness. Each test is reliably validated by the following criteria:

  • Mortality in the control group: ≤ 15 % on day 10
  • Mortality in the reference item group: ≥ 50 % on day 10

Endpoints

LC50/LD50; NOEC/NOED

Guidelines and Literature

  • OECD 213: OECD Guideline for the Testing of Chemicals on Honeybee, Acute Oral Toxicity Test, (adopted 21st September 1998)
  • CEB (Commission des Essais Biologiques), 2012. Méthode d’évaluation des effets de toxicité aiguȅ et á court terme des préparations phytopharmaceutiques sur l’abeille domestique (Apis mellifera L.). AFPP method n°230
  • Kling, A. & Schmitzer, S. (2015): Proposal for a new OECD guideline for the testing of chemicals on adult honey bees (Apis mellifera L.) in a 10 day chronic feeding test in the laboratory and results of the recent ring test 2014. Hazards of pesticides to bees - 12th International Symposium of the ICP-PR Bee Protection Group, Ghent (Belgium), 15-17 September 2014. Julius-Kühn-Archiv, 450, pp. 69-74.