OECD 237: Honey Bee Larval Toxicity Test, Single Exposure

The honey bee (Apis mellifera) larval toxicity test OECD TG 237 is a completion to the acute oral and acute contact toxicity tests OECD TG 213/214. The purpose of this test design is to determine the acute toxicity of a plant protection product or chemical to honey bee larvae (Apis mellifera) after a single exposure for a period of 72 hours.

Study Design

Test organisms

The larvae of honey bees are collected from adequately fed and healthy colonies originated from the ibacon apiary to guarantee well known history and physiological status. The tests are performed during the egg laying period of the queen. Three days before grafting the queens of minimum three colonies, each representing a replicate, are confined in their own colonies in an exclusion cage containing an empty comb. The exclusion cage is placed close to combs containing brood. The time of the isolated queens should be held as short as possible in order to minimise the variability in size and age between larvae. After release of the queens the combs containing the eggs are left in the cages inside the hives until hatching (D1).

Course of the test

The usual test duration is ten days in total: D-3 to D1: in hive; D1 to D4: pre exposure; D4 to D7: exposure phase. During the lab phase the larvae are fed with special diets prepared from royal jelly and aqueous solutions of yeast extract glucose and fructose. Diet A, B and C are composed according to the needs of the larvae at different stages of development. Find a schematic representation of the important steps of the larval toxicity test in the following picture.

On D4, a minimum of twelve well-fed larvae from each of the three colonies are selected and treated with 30µl of the diet C containing the test solution or reference item at the suitable concentration.

The larvae are kept in plexiglas containers placed in an incubator at 34 – 35 °C. A dish filled with saturated K2SO4-solution is placed in the plexiglas containers in order to keep a water saturated atmosphere.

At D5, D6 and D7, mortalities are checked and counted. An immobile larva is noted as dead. Dead larvae are removed systematically for sanitary reasons. At D7 the test is terminated by freezing the plates at ≤ -10°C.

Endpoints

Mortality of the larvae is used as the toxic endpoint. The LD50 (i.e. 72 hours) with 95 % confidence limits of the test item is estimated with suitable statistical methods, if indicated. Other endpoints like a NOEC / NOED can be determined, if appropriate.

Guidelines and Literature