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Assessing the chronic toxicity of a compound to developing honey bees (Apis mellifera L.) has proven to be a challenge since the mid-2000s. Such data are requested by global regulatory authorities so they can evaluate the risk of compounds to larval honey bees when exposure is likely to occur in the environment. Poor test performance has led to frequent study failures and data uncertainty. Here we highlight a recent effort by the Pollinator Research Task Force (PRTF)1 to validate the use of a method for evaluating the chronic toxicity of a compound (e.g., a pesticide) to an immature honey bee for use in a risk assessment. A ring test protocol was selected and based upon the current OECD guidance document No. 2392 with amendments developed at the University of Florida (Schmehl et al. 2016)3. Fifteen independent laboratories on three continents representing government, academia, and industry followed the same testing protocol to: 1) determine if test performance is robust across different geographic regions and different laboratory personnel and 2) identify limitations associated with the methodology. The control performance criteria for a valid test according to OECD GD 239 is ≥ 85% survival at the end of the larval development and ≥ 70% survival through adult emergence. Thirteen trials (81.3%) satisfied the validity criteria and the test design’s performance was determined adequate for regulatory testing. The toxic reference chemical (dimethoate) had a consistent response with a 22-day EC 50 range of 8-22 μg active substance (a.s.)/g diet. An acetone concentration at the maximum concentration allowed by the OECD GD 239 (2% acetone) was observed to be problematic to test performance. In conclusion, the ring test methods based upon the OECD GD 239 demonstrated that the repeat (chronic) exposure of a compound on developing bees can be successfully conducted. A copy of the full study report4 can be accessed here.

The assessment of pesticide risks to bees in North America currently relies in part on Tier 1 honey bee laboratory toxicity studies to support the registration and registration review processes for crop protection chemicals. For immature stages, the studies follow two standardized test designs recommended by the Organization for Economic Cooperation (OECD), evaluating acute (seven‐day single‐dose, TG OECD 237) and chronic (22‐day repeated‐dose, GD OECD 239) toxicity in bee larvae. In this article, we aim to evaluate the current approach for generating and interpreting honey bee larval toxicity data, enhancing pesticide risk assessment for pollinators. First, by considering that the repeated‐dose larval study covers all stages of honey bee brood development up to adult emergence, we compared endpoints (larval LD/ED50 and LC/EC50 values) from seven‐day acute exposure studies with the 22‐day chronic exposure studies. Our goal was to identify the study design offering greater sensitivity in assessing pesticide toxicity to immature bees. Our second objective involved analyzing available weight data from emerged adults and comparing it to survival endpoints (e.g., NOEL and LD50) to determine if the weight after adult emergence would accurately represent a sensitive indicator of pesticide effects on developing honey bees. Our analysis determined that the use of a single 22‐day chronic exposure study adequately covers all immature stages and that the toxicity values based on cumulative dose are more accurate and representative measures of exposure for immature bees than using endpoints based on estimated daily doses. Furthermore, our analysis suggests that measuring the weight of emerged adults was a more sensitive indicator than mortality of treatment‐related effects in 22% of the compounds included in our analysis. Here we also discuss the importance of standardized protocols for proper collection of weight after emergence and the need for further discussion on the relevance of this parameter at risk assessment scheme. Integr Environ Assess Manag 2024;20:2283–2293. © 2024 Pollinator Research Task Force. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Key Points: A meta‐data assessment was conducted to evaluate the current approach for generating and interpreting honey bee larval toxicity data.The chronic larval study tends to provide a more robust screening‐level risk determination than the acute larval study design.The weight after adult emergence from chronic exposure larval study may be an indicator of pesticide effects on growth.Standardized protocols for measuring the weight of emerged adults are needed. [ABSTRACT FROM AUTHOR]

Risk assessment for chemicals in the United States relies upon the honey bee (Apis meliffera L. [Hymenoptera: Apidae]) as a surrogate for other bee species. There is uncertainty in extrapolating honey bee toxicity data to bumble bees due to differences in life history strategies, food consumption, and nest structure. Here we evaluated the design of a queenless bumble bee microcolony test that could be considered for generating larval toxicity data. Three microcolony studies were conducted with Bombus impatiens to evaluate the effects of exposure to 1) diflubenzuron in pollen, 2) dimethoate in pollen, and 3) dimethoate in sucrose. Immature drone bee emergence, worker survival, pollen, and sucrose utilization were measured throughout the study duration. For dimethoate, a 10-d chronic adult bumble bee study was also conducted to compare microcolony endpoints to toxicity endpoints on individual adults. Microcolonies exposed to 10 mg diflubenzuron/kg pollen produced fewer adult drones despite no effects on worker survival. Microcolonies treated with dimethoate at ≥3 mg a.i./kg pollen and ≥0.1 mg a.i./kg sucrose solution produced fewer drones. Exposure to dimethoate in the 10-d chronic adult study resulted in direct mortality to the adult workers at ≥0.1 mg a.i./kg diet. Results from the 10-d study suggest direct effects of dimethoate on workers in the microcolony will alter provisioning of diet to the brood, resulting in lower drone production in the microcolony. Our data suggest that the microcolony study is only appropriate to assess brood effects to bumble bees for substances with low toxicity to adults, as demonstrated with diflubenzuron. [ABSTRACT FROM AUTHOR]

Beekeepers commonly supplement honey bee (Apis mellifera L.) colonies' nutrition with commercial pollen and nectar substitutes in an effort to encourage growth and reduce colony losses. However, there is a broad lack of understanding regarding the extent to which supplemental protein feeding affects honey bee colony health. We conducted a field study to determine if feeding protein substitutes affected colony strength and Nosema spp. spore intensity in commercially managed honey bee colonies. Seventy-five honey bee colonies were randomly assigned to one of six treatments (no supplemental protein, one of four commercially available protein supplements, or wildflower pollen supplement). The number of adult bees, the number of capped brood cells, and Nosema intensity were assessed prior to-, 4 wk post-, and 8 wk post-treatment. There was an overall decrease in Nosema intensity across all treatments over time. However, there were no statistically detectable differences in colony strength or Nosema intensity between any of the pollen feeding treatments and those of the negative control treatment. Thus far, multiple investigations regarding supplemental protein feeding have failed to provide a clear consensus on the impact that this practice has on honey bee colony strength or productivity. Additional research is needed to determine the impact, if any, that diet supplementation, including microbial and nutritional supplements, has on colony health, to better inform beekeepers' management decisions. [ABSTRACT FROM AUTHOR]

For over twenty years, the honey bee has been relied upon as the representative pollinator surrogate species for a pesticide risk assessment due to its global geographic distribution, ease of management, and validated test methods. More recently there have been questions on whether the risk of a chemical to the honey bee is truly representative for the other ~20,000 bee species globally. Honey bees have a eusocial life history comprised of tens of thousands of individuals, which is in contrast with the majority of bees that are semi-social or solitary. Bumble bees are a well known group of over 250 species that are important in agriculture and being considered as a representative semi-social bee in risk assessments. The majority of method development has been conducted in Europe on the buffedtailed bumble bee (Bombus terrestris). While this species is used reliably for acute (OECD guidelines 246 and 247) and chronic toxicity bioassays, its performance is less predictive in a microcolony (brood test) or colony-level study. Here we present toxicity data for the Common Eastern Bumble Bee (Bombus impatiens), the commercially-available species of bumble bee in North America. We demonstrated consistent and predictive performance as individuals and in groups across the laboratory and field levels. Exposure of B. impatiens to the reference toxicant dimethoate yielded a toxicity profile that is comparable to B. terrestris, suggesting that B. impatiens endpoints are suitable and valid in cases when bumble bee data are required for use in a pesticide risk assessment. [ABSTRACT FROM AUTHOR]

The Pollinator Risk Assessment framework in North America and other regions is based on a tiered approach with the honey bee, Apis mellifera, as the representative organism. The protectiveness of the honey bee risk assessment for non-Apis bees has not been extensively validated due to limited availability of standardized methods. We developed a chronic oral test for orchard bees with Osmia lignaria, O. cornifrons, and O. cornuta. Our protocol includes elements from other chronic oral toxicity bee tests including the OECD 245 honey bee guideline and a validated protocol for bumble bees; these elements include the 10-d test duration, replication, and validity criterion for control survival. We measured the daily consumption of the feeding solutions and observed survival and other adverse effects. Evaporation controls were included to correct consumption estimates. On average, O. lignaria, O. cornifrons and O. cornuta body weight was 105 ± 12, 71 ± 8, and 129 ± 16 mg, respectivelly. Consumption in the control group was 49 ± 14, 85 ± 21, 157 ± 35 mg sucrose solution/bee/d for O. lignaria, O. cornifrons, and O. cornuta, respectively. Control survival was ≥ 85% for the three species evaluated. A fourth test was conducted with O. bicornis but outside the typical active season, which may affect the representativity of the results for this species. Dose-response tests with dimethoate, a positive control in bee toxicity tests, were conducted with each Osmia species and comparison of the resulting toxicity endpoints between honey bee and Osmia species will be presented. [ABSTRACT FROM AUTHOR]

The interest in and use of biological materials (e.g. biostimulants, biopesticides) in crop production is increasing globally at a rapid pace. Part of the interest is that these technologies are viewed as safer alternatives to conventional chemicals and provide value in a holistic integrated pest management approach. While establishing the safety of these materials is as important as for conventional chemicals, there are important distinctions between them. For example, micro-organisms need to be evaluated for their pathogenic potential. The current EPA honey bee test guideline for assessing the pathogenicity potential of a microbial pesticide (OCSPP 885.4380) requires a 30-day observation period after dosing, but this test duration is difficult to achieve. A reliance upon shorter 10-day duration studies based upon OECD guideline #245 may not capture signs of pathogenicity, as some known bee pathogens take up to two weeks to elicit signs of an infection. Additionally, microbial-based test material can be difficult to deliver within a syringe feeder due to potential clogging or difficulty in maintaining homogeneity. The goal of the present study is to identify the factors that may increase adult longevity in laboratory cage bioassays, including age, cage type, number of bees, the presence of wax, honey, or water, and time of year were investigated. Factors that led to consistently high survival may inform an optimized test design for assessing the potential pathogenicity of a microbe to honey bee adults. [ABSTRACT FROM AUTHOR]

Orchard bees of the genus Osmia Panzer are important pollinators of fruit trees in various regions of the world, with some species commercially available in the United States and Europe. In addition to their pollination services, Osmia lignaria, Osmia cornifrons, Osmia bicornis, and Osmia cornuta have been identified as potential model species for solitary bees in pesticide risk assessment and have been used for the development of new methods to test acute lethal effects via contact and oral routes of exposure. Our goal was to expand the available methodology to characterize the toxicity of pesticides for these solitary bees through a chronic oral test for adult bees. Chronic oral toxicity of pesticides to orchard bees has been reported, but methods differ among research groups. In our study, O. lignaria, O. cornifrons, O. bicornis, and O. cornuta female bees had access to sucrose solution ad libitum in separate, species-specific 10-day tests. Mean body mass, mean daily consumption, and survival differed among the studied bee species. The dose-response test design was validated with dimethoate, a reference toxic compound, and chronic toxicity endpoints were estimated for the 4 Osmia species. The median lethal daily doses normalized by weight for O. lignaria, O. bicornis, O. cornuta, and O. cornifrons were within the same order of magnitude at 0.23, 0.26, 0.49, and 0.61 µg dimethoate/g bee/day, respectively. The methodology described here was aligned as much as possible with the available honey bee and bumble bee standard methods to facilitate the comparison of chronic toxicity profiles among bee species., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)