Chronic larval and adult honey bee laboratory testing: Which dietary additive should be considered when a test substance is not solubilized in acetone?

Chronic toxicity tests on adult and larval honey bees (Apis mellifera) can require the use of dietary additives (solvents, emulsifiers, adjuvants and viscosifier agents) when the active ingredient of plant protection products cannot be dissolved or does not remain stable and homogeneous within the test diets. Acetone is the widely used and accepted solvent allowed within the international regulatory guidelines, but it can be ineffective in keeping certain compounds in solution and can cause toxicity to adults and larvae. In this publication, we present an evaluation of alternative additives in adult and larval diets. Six dietary additives including five solvents (ethanol, isopropanol, n-propanol, propylene glycol and triethylene glycol) and a viscosifier agent (xanthan gum) at five concentrations along with a negative control and a solvent control (acetone) were investigated at seven laboratories. The safe levels for bees were determined for each of the additives used in the 10-day chronic adult and 22-day chronic larval tests. In the 10-day chronic adult study, ethanol and isopropanol were found to be safe at concentrations ≤ 5.0 %, while xanthan gum can be reliably used at concentrations ≤ 0.1 %. Greater variability across laboratories was observed for N-propanol, propylene glycol, and triethylene glycol and these agents may cause mortality when added to diets at concentrations above 0.25-0.5 %. The safe levels of additives to larval diet in the 22-day chronic larval test had a greater variability and were generally lower than what were observed for adult diet. Our results do not recommend the inclusion of ethanol or n-propanol into the larval diet, and isopropanol, propylene glycol, and triethylene glycol may cause mortality at concentrations above 0.25-0.5 %. Safe levels for xanthan gum were more variable than what was observed for adults, but it can be used reliably at concentrations ≤ 0.05 %. Our analyses conclude that several additives can be integrated successfully in honey bee laboratory bioassays at levels that cause low mortality to adults and larvae.