DOI: 10.5061/dryad.wh70rxwxk
title: A test method for assessing chronic oral toxicity of a pesticide to solitary nesting orchard bees, Osmia spp. (Hymenoptera: Megachilidae)

description: 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.
description: <strong>Evaluation of a mating
 step</strong> 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
 <em>Osmia</em> 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 <em>Osmia</em> 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
 <em>O. lignaria</em> and <em>O. cornifrons</em> in
 2021. Bee weights and food consumption were measured following methods
 described in Section 2.2. <strong>Dose-response
 chronic oral test with dimethoate</strong>
 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 <em>O. lignaria
 </em>(2022), <em>O. cornifrons </em>(2022)<em>, O.
 bicornis </em>(2023) and <em>O. cornuta </em>(2022,
 2023), for a total of five dose-response tests with four orchard bee
 species. The two tests with <em>O. cornuta</em> 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 <em>O. lignaria</em>
 and <em>O. cornifrons</em>. 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 <em>O. lignaria</em> and <em>O.
 cornifrons</em> were conducted in Chesterfield, MO, USA, using two
 environmentally controlled chambers. The <em>O. bicornis</em>
 and <em>O. cornuta</em> tests were conducted in Monheim am
 Rhein, Germany. <strong>Evaluation of one solvent
 and one suspension agent</strong> 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
 <em>O. lignaria </em>and <em>O. cornifrons
 </em>in<em> </em>2023, but not with <em>O.
 bicornis</em> or <em>O. cornuta</em>. 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. <strong>Environmental
 Conditions </strong> All orchard bee tests were
 maintained in controlled growth chambers (USA – Thermo
 Scientific<sup>TM</sup>, 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.
 <strong>Statistical Analysis</strong>
 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. <em>Mating
 step evaluation </em>– 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
 <em>O. lignaria</em> and <em>O. cornifrons</em>.
 Differences in survival between treatment groups was evaluated using a
 Fisher exact test. <em>Dimethoate dose-response
 chronic oral tests</em> – 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 <em>Osmia</em> 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
 [LC<sub>50</sub>] or media Lethal Dietary Dose
 [LDD<sub>50</sub>]). 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 <em>Osmia</em> 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
 <em>Osmia</em> species. <em>Chronic
 oral tests with acetone and xanthan</em> – 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.
description: # A test method for assessing chronic oral toxicity of a pesticide to
 solitary nesting orchard bees, Osmia spp. (Hymenoptera: Megachilidae)
 [https://doi.org/10.5061/dryad.wh70rxwxk](https://doi.org/10.5061/dryad.wh70rxwxk) ## Description of the data and file structure The manuscript **"A test method for assessing chronic oral toxicity of a pesticide to solitary nesting orchard bees, *Osmia* spp. (Hymenoptera: Megachilidae)"** describes the method development of a chronic oral test for orchard (mason) bees of the genus *Osmia*. A cage selection and mating step were conducted to develop a setting that would enable the exposure of orchard bees to dimethoate-treated sucrose solution, quantify daily consumption and make observations of survival and sublethal effects during a 10-d test. Dose-response tests were conducted using the insecticide dimethoate with four different species, *Osmia lignaria*, *Osmia cornifrons*, *Osmia bicornis*, and *Osmia cornuta*. Methods were harmonized to those available for honey bees and bumble bees (i.e. number of bees per treatment, test duration). The test design allowed the observation of chronic toxicity endpoints including the No Observed Effect Concentration and dose (NOEC, NOED), Lowest Observed Effect Concentration and Dose (LOEC, LOED), Median Lethal Concentration (LC50), Median Lethal Daily Dose (LDD50). Separate tests were conducted to evaluate potential effects of acetone (solvent) and xanthan gum (suspension agent) to consider them in toxicity evaluations when the test substance has difficulties to get into solution.  ### Files and variables #### File: Osmia\_Data\_MASTER.csv **Description:**  ##### Variables * study_no: Study Number, identification of separate datasets. * study: Category of the study (i.e. mating step, dose-response test with dimethoate, solvent evaluation) * species: Osmia species. * treatment: Treatment group in the corresponding study. * Concentration: Concentration of the test material (treatment). For dose-response tests with dimethoate, units are parts per million (ppm). For evaluation of solvent and suspension agent (acetone, xanthan gum), units are percentage (%). * replicate: Study bee identification in the corresponding study. * incubator: replicates (caged bees) were split into two incubators, for the Osmia lignaria and Osmia cornifrons tests, due to limited incubator capacity. * day: Day of test, in a 10-d test. * weight: weight of individual (study) bee in milligrams (mg). * alive: Observation of survival of the bees.  * abnormal: Any noted sublethal effect. * start: weight of the syringe (in grams, g) containing the feeding solution at the start of a 24 h exposure period. * end: wight of the syringe (in grams, g) containing the feeding solution at the end of a 24 h exposure period. * evaporation: average daily evaporation (in grams, g) from evaporation control syringes, to correct daily consumption estimates.  * diff: the difference between the start weight and end weight of a particular syringe containing feeding solution. This is a daily consumption estimate prior to correction from subtracting the evaporation estimate. Units are expressed as grams (g). * food_intake: Daily consumption estimate corrected for evaporation. Units are expressed as grams (g).  ## Code/software 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’.