Evaluation of the Honey Bee Brood Humidity Rearing Protocol in Laboratory Conditions for Ecotoxicological Testing in Apis mellifera iberiensis

Apis mellifera is a key species in ecotoxicological assessments of pest control products. OECD and EFSA guidelines include bee brood toxicity tests, but these protocols are largely based on central and northern European bees. A. mellifera iberiensis, widely used in southern Iberian assays, shows biological differences. We assessed brood survival under different laboratory humidity regimes in assays: (1) OECD 95/80/60% versus 70/70/60% humidity, for days 1–8, 8–15, and 15–22, respectively; (2) the same regimes with or without laboratory paper; (3) laboratory groups versus brood in the original hives. Results show that reducing humidity during in vitro brood rearing significantly improves brood survival. Humidity can be lowered either by using absorbent laboratory paper or by modifying the incubator regime from 95/80/60% to 70/70/60%. Under these conditions, brood survival increased by 32.64–43.26 percentage points, indicating that A. mellifera iberiensis may have distinct in vitro rearing requirements. However, adult emergence remained below the OECD validity threshold, and the proposed rearing regime therefore requires further independent validation before it can be considered for regulatory application. In addition, other factors, including colony of origin and larval diet, may influence brood viability and should be investigated to further optimize the rearing protocol.