A test method for assessing chronic oral toxicity of a pesticide to solitary nesting orchard bees, Osmia spp. (Hymenoptera: Megachilidae)

Orchard bees of the genus Osmia 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, O. cornifrons, O. bicornis, and O. 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 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 four 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/d, respectively. The methodology described here was aligned as much as possible to the available honey bee and bumble bee standard methods to facilitate comparison of chronic toxicity profiles among bee species.

Methods
Evaluation of a mating step

The consumption and survival were compared among presumed unmated (hereafter ‘unmated’) and presumed mated (hereafter ‘mated’) female bees. This step was included in this evaluation because a mating step was included in the acute oral test protocol for Osmia spp. bees organized by ICPPR (Roessink et al. 2020), to promote feeding of the study bees, and thus it was considered as a potential protocol element in the chronic oral test design development. The emerged female bees were split into two groups, one was held in a ventilated cage (24 cm × 24 cm × 24 cm) without food for 24 h and without male bees, and another group of female bees was held under similar conditions, but male Osmia bees of their corresponding species were included at a 2:1 male:female ratio. After this mating period, one female bee was introduced per cage, 30 bees per mating status group (‘unmated’, ‘mated’). This step was evaluated with O. lignaria and O. cornifrons in 2021. Bee weights and food consumption were measured following methods described in Section 2.2.

Dose-response chronic oral test with dimethoate

Dimethoate (Pestanal ™) was sourced from Sigma-Aldrich (Batch No. BCCF3993, St. Louis, MO) and used in all dose-response tests. Dimethoate is a highly bee toxic insecticide used for the development of bee toxicity protocols and included as a toxic reference treatment group in regulatory guidelines (i.e. OECD 213, OECD 214, OECD 246, OECD 247). Dose-response tests with dimethoate were conducted with O. lignaria (2022), O. cornifrons (2022), O. bicornis (2023) and O. cornuta (2022, 2023), for a total of five dose-response tests with four orchard bee species. The two tests with O. cornuta were conducted to generate information on replicability of the test results. The selected dimethoate concentrations in sucrose solution were 0.125, 0.25, 0.5, 1.0 and 2.0 mg dimethoate/kg of diet or parts per million (ppm), informed from range finder tests conducted in 2021 with O. lignaria and O. cornifrons. A stock solution was first prepared with dimethoate dissolved in water without the aid of a solvent, and then this stock solution was used to prepare the feeding solutions. There were 30 bees (replicates) per control and in each dimethoate test level, with daily food exchange, consumption measurements, sublethal (i.e. affected, moribund) and survival observations. Bee weights and food consumption were measured following methods described in Section 2.2. . Tests with O. lignaria and O. cornifrons were conducted in Chesterfield, MO, USA, using two environmentally controlled chambers. The O. bicornis and O. cornuta tests were conducted in Monheim am Rhein, Germany.

Evaluation of one solvent and one suspension agent

Potential test items hard to solubilize in sucrose solution may benefit from the addition of a solvent or suspension agent to maintain a homogeneous distribution between feeding solution exchanges. Acetone (solvent) and xanthan gum (suspension agent) are commonly used in these situations with honey bee and bumble bee tests. These test materials were evaluated with O. lignaria and O. cornifrons in 2023, but not with O. bicornis or O. cornuta. The test design with each bee species included one control (referred as untreated or UTC) group, acetone at 2.5% v/v (AC_2.5), acetone at 5% v/v (AC_5), xanthan gum at 0.05% w/w (XA_0.05) and xanthan gum at 0.1% w/w (XA_0.1) concentration in the sucrose solution. These levels were selected based on concentrations levels safe for honey bees (Tomé et al. 2023). There were 30 bees (replicates) per control and in each acetone and xanthan gum test level, with daily food exchange, consumption estimate, and survival observations. Bee weights and food consumption were measured following methods described in Section 2.2. . Three evaporation controls with 50% w/v sucrose solution, three with 5% acetone, and three with 0.1% xanthan gum were included in the test to correct daily consumption estimates for their corresponding treatment groups (control, acetone, xanthan gum). Consumption, survival and other sublethal effects were evaluated daily, for ten days.

Environmental Conditions 

All orchard bee tests were maintained in controlled growth chambers (USA – Thermo ScientificTM, Forma 3900 Series; Germany – RUMED, Rubarth Apparate GmbH, CPS-P1060). Environmental conditions were set and maintained at 22 ± 2 ºC, 60 ± 10 % Relative Humidity and a 16:8 h Light:Dark cycle. The only source of light was that emitted by the light bulbs in the growth chamber models (USA – Osram Biolux L 18w/965; Germany – Rhenac-Greentec). The bees were not exposed to UV light.

Statistical Analysis

All statistical analyses were conducted using the statistical software R, version 4.2.2 (R Core Team 2022), with packages ‘multcomp’, ‘tidyverse’, ‘car’, ‘clinfun’ and ‘drc’. For continuous endpoints (food consumption and body weight), significant departures from either normality (Shapiro Wilk, α = 0.01) or variance homogeneity (Levene, α = 0.05) were routinely observed. Therefore, these endpoints were evaluated using non-parametric statistical methods.

Mating step evaluation – The mean daily consumption (mg sucrose solution/bee/d) of the mated and unmated bees was compared via a two-sided Wilcoxon rank sum exact test in each of the two tests, conducted with O. lignaria and O. cornifrons. Differences in survival between treatment groups was evaluated using a Fisher exact test.

Dimethoate dose-response chronic oral tests – The weight of bees randomly assigned to the treatment groups was analyzed via a two-tailed Kruskal Wallis to evaluate for potential bias. The mean daily sucrose solution consumption for each Osmia test was analyzed via a one-tailed Jonkheere-Terpstra trend test.  Survival-response data was fit with a 3-parameter log-logistic model using either nominal treatment concentrations or daily doses (i.e. median Lethal Concentration [LC50] or media Lethal Dietary Dose [LDD50]). The daily doses were estimated by factoring the daily consumption estimate for each individual bee and the nominal concentration of dimethoate in the feeding solution. The no observable effect concentration (NOEC) and the no observed effect dietary dose (NOEDD) were identified via Cochran-Armitage Trend tests (OECD 2006). The dose-based endpoints for each Osmia species are reported as μg dimethoate/bee/d and normalized by weight as μg dimethoate/g bee/d using the average bee weight for each Osmia species.

Chronic oral tests with acetone and xanthan – Mean daily food consumption was first rank transformed and treatments were compared to the control using a Dunnett’s t-test. Starting body weights amongst treatments were compared using a Kruskal Wallace test. Survival in treatments compared to the control were evaluated using a Fisher exact test.