GB/T 41691-2022 in English

This standard is established based on OECD Test Guideline No. 237. It uses 48-well cell culture plates as the core test device to determine the 72h median lethal dose (72h-LD₅₀) of chemicals to Italian honey bees (Apis mellifera) larvae by artificially feeding spiked feed. The test is divided into three key stages:

1. Larva preparation period (D1-D3): Establishment of standardized feeding foundation
2. Exposure treatment period (D4): Single feeding of the test article
3. Observation and recording period (D5-D7): Mortality statistics

1. Test system verification

Dimethoate technical (CAS 60-51-5) must be used as a procedural control and the mortality rate of the 8.8μg/larvae dose group should be ≥50%. Required to be confirmed before the test:

• Royal jelly contaminant test results (pesticide residue ≤0.01mg/kg)
• Integrity of culture plate sterilization treatment (alcohol immersion ≥30min)
• Calibration certificate of temperature and humidity recording system

2. Special substance treatment plan

For poorly soluble substances:

Compared with the 2008 EU ring test version, this standard has the following major improvements:

1. Precision of feed formula: Clarify the glucose/fructose gradient concentration (12%-18%) in the D1-D3 stage
2. Standardization of bee colony management: Require the queen bee isolation time to be ≤30h to reduce individual differences in larvae
3. Statistical data standardization: Mandatory use of the Abbott formula to correct mortality

Typical case: A test of a neonicotinoid pesticide showed that the larval LD₅₀ was 1/5-1/3 of the adult bee value, proving that the larval stage is more sensitive to specific compounds.