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.

Recovery of photosynthesis and growth rate in green, blue-green, and diatom algae after exposure to atrazine.

We evaluated the recovery of photosynthesis and growth rate in green (Pseudokirchneriella subcapitata), blue-green (Anabaena flos-aquae), and diatom (Navicula pelliculosa) algae after pulsed exposure to atrazine. Subsequent to a grow-up period of 24 to 72 h to establish requisite cell density for adequate signal strength to measure photosystem II (PSII) quantum yield, algae were exposed to a pulse of atrazine for 48 h followed by a 48-h recovery period in control media. Photosynthesis was measured at 0, 3, 6, 12, 24, and 48 h of the exposure and recovery phases using pulse amplitude modulation fluorometry; growth rate and cell density were also concomitantly measured at these time points. Exposure to atrazine resulted in immediate, but temporary, inhibition of photosynthesis and growth; however, these effects were transient and fully reversible in the tested species of algae. For all three algal species, no statistically significant reductions (p ≤ 0.05) in growth rate or PSII quantum yield were detected at any of the treatment concentrations 48 h after atrazine was removed from the test system. Effects at test levels up to the highest tested exposure levels were consequently determined to be algistatic (reversible). Both biochemically and physiologically, recovery of photosynthesis and growth rate occur immediately, reaching control levels within hours following exposure. Therefore, pulsed exposure profiles of atrazine typically measured in Midwestern U.S. streams are unlikely to result in biologically meaningful changes in primary production given that the effects of atrazine are temporary and fully reversible in species representative of native populations.

Performance of potential non-crop or wild species under OECD 208 testing guideline study conditions for terrestrial non-target plants.

The inclusion of 52 potential non-crop or wild species in new OECD guidelines for terrestrial non-target plant (TNTP) testing led to a ring test conducted by four laboratories experienced in regulatory testing. Species selected had shown potential to meet validity criteria of emergence for TNTP studies in a previous evaluation of the 52 species. OECD 208 guideline conditions were applied, with and without seed pretreatments recommended to enhance germination. These species were Abutilon theophrasti (L.) Medic., Avena fatua L., Fallopia convolvulus (L.) Adans., Galium aparine L., Ipomoea hederacea (L.) Jacq. and Veronica persica Poir. Only I. hederacea met the validity criterion of 70% emergence in all laboratories and showed a low variability in biomass. Of the other species, none led to 70% emergence in all four laboratories. The recommended pretreatments did not have a major impact on emergence. Biomass was also investigated with A. theophrasti, A. fatua, Centaurea cyanus L., I. hederacea and Rumex crispus L. Variability of biomass, a key parameter in TNTP regulatory studies, exceeded normal biomass variability of crop species used for TNTP studies. The addition of a thin layer of quartz sand to the soil surface resulted in improved emergence of C. cyanus, G. aparine and V. persica; however, such a procedure, while routine in screening studies to improve germination, is a deviation from the TNTP guidelines. These initial studies indicate that some species could meet the emergence criteria for TNTP testing. However, there is a need for further studies on seed source, seed quality and conditions for uniform emergence before their use in routine regulatory testing.