DOI: 10.5061/dryad.wwpzgmssv
title: Development of a test design for a semi-field, colony feeding study for the common eastern bumble bee (Bombus impatiens [Hymenoptera:Apidae])

description: Ecological risk assessment is a key component of the regulatory process
 required for registration of crop protection products around the world.
 The western honey bee (Apis mellifera) is the model organism for pesticide
 risk assessments for bees, but there are uncertainties over whether it is
 predictive of risks to other bees. Consequently, efforts are underway to
 develop test methodologies for other non-Apis bees. We conducted a
 semi-field colony feeding study with Bombus impatiens colonies to develop
 a colony-level methodology for bumble bees. We exposed commercially
 available bumble bee colonies to diets consisting of four concentration
 treatments of dimethoate insecticide (0.05, 0.19, 0.75, and 3.0 mg a.i./L)
 via supplemental sugar solution for six weeks and compared exposed
 colonies to untreated controls. Each treatment group had 10 replicate
 colonies, with one replicate per treatment group represented at each of
 the 10 study rural locations. We collected data on various colony-level
 endpoints including production of female reproductive (gyne) offspring,
 colony weight, foraging activity, and consumption of provisioned sugar
 solution. Our results indicated that the test design could be used to
 derive concentration-response relationships for several endpoints
 including the most sensitive, colony mass (No Observed Adverse Effect
 Concentration = 0.05 mg a.i./L). Overall, our study provides the
 foundation for a semi-field, colony feeding study test design for bumble
 bees, thus adding to the growing body of studies that may be used to
 assess the protectiveness of the honey bee risk assessment framework for
 non-Apis bees exposed to pesticides.
description: <strong>Test Material</strong>
 We used pure Pestanal<sup>TM</sup> dimethoate (Sigma
 Aldrich, St. Louis, MO; Lot # BCBS9338, purity ≥98%) as the test
 material. <strong>Bumble Bee
 Colonies</strong> On 16 June 2020, we obtained 80
 Excel-type <em>B. impatiens</em> colonies from Koppert
 Biological Systems Inc. (Michigan, USA). Excel-type commercial colonies as
 described by the vendor contained a minimum of 70 worker-caste females and
 a queen nesting on a plastic foundation within a cardboard hive box. The
 plastic foundation has a hive entry of limited size that acts as a queen
 excluder, allowing the traffic of worker and drones, but not queens. Each
 hive was fitted a plexiglass observation lid and without any cover to the
 nest by request to the supplier. Bees were fed <em>ad
 libitum</em> with a sugar syrup solution and honey bee-collected
 pollen for one week. Upon receipt, the weight of each colony was measured.
 Sixty queenright study colonies were selected for field deployment by
 removing the most extreme by weight colonies, i.e., the lightest and
 heaviest colonies. A quick visual assessment of the brood area was also
 conducted and those that had queen pupae were removed from
 consideration. <strong>Experimental
 Design</strong> The colonies were moved to the
 field on 26 June 2020. Colonies were randomly assigned to treatment group
 and site. We conducted the study at 10 sites established along the margins
 of hayfields (organic-certified or managed without pesticides) on working
 dairy farms in central Vermont. The sites were clustered at three farms
 (hereafter, “locations”) that were separated by &gt;
 500 m. In May 2020, an area that was approximately 3 × 65 meters
 in size was mowed at each site in preparation for the relocation of
 colonies from the test facility to the field. Each mowed area was
 surrounded by a solar-powered electric fence for protection from bears. We
 performed maintenance on fencing weekly and mowed fence lines as necessary
 during the season. At each site, six colonies were deployed in one row
 with a 10 m spacing.  Each bumble bee colony was placed
 in an open-sided plastic box to prevent rain damage. Each hive was
 balanced on a stand in a second plastic box filled with 5 to 10 cm of
 water to prevent ants from entering the hive. Each hive was stabilized
 with a pair of grade stakes pounded into the ground.
 Each of the six treatment groups, two controls (fed and unfed
 sucrose solution) and four dimethoate concentrations, were represented at
 each site by one experimental unit (colony). The “Fed” control colonies
 received 50% (w/v) supplemental sucrose solution whereas the “Unfed”
 control colonies did not receive supplemental sucrose solution. The four
 dimethoate treatment groups involved exposure to 0.05, 0.19, 0.75, or 3.0
 mg dimethoate/L sucrose solution. In the dimethoate treatments, pure
 Pestanal<sup>TM</sup> dimethoate powder was dissolved in
 measured volumes of 50% sucrose solution. One L of solution per week per
 colony was then provisioned to bees via supplemental nectar feeders. The
 feeding solutions were poured into the plastic bags, which acted as nectar
 feeders, with calibrated cylinders. The exposure period lasted six weeks
 starting on 2 July 2020 and ending 14 August 2020. This exposure period
 was selected to harmonize the test design to the honey bee colony feeding
 study design, and to represent a worst-case scenario exposure to an
 indeterminate blooming crop variety. Following the exposure period,
 colonies remained at the same locations for post-exposure assessments as
 described below. During the field phase encompassing
 the exposure and post-exposure periods, colony condition assessments
 (hereafter “CCA”) were performed weekly starting at dusk after the forager
 bees had returned to the colony for the night. Headlamps provided enough
 light for the conduct of CCAs. Each hive was weighed (including the colony
 and the plastic hive insert box), and observations were made on queen
 survival and production of reproductive offspring (gyne pupae, new gynes,
 males). New gynes were differentiated from the foundress queen by the
 presence of intact wings, intact setae especially in the abdomen, and
 coloration (newly emerged queens have a grey complexion before
 sclerotization is complete). Presence of wax moths (adult and larva
 stages), ants, snails, beetles, wasps, mold, and bumble bees from a
 different species was recorded. At each CCA, the previous sugar solution
 provisions were removed and replaced with a fresh 1 L bag. We calculated
 the amount of solution consumed by each colony by subtracting the initial
 fresh mass of the bag from the mass of the bag remaining after one week in
 the field. Foraging activity at each colony was
 determined on four dates during the exposure period. Hive entrances were
 observed for 10 minutes between the hours of 0900 to 1600, and the number
 of bees leaving and returning to the hive recorded. We also made notes on
 bee behavior and the presence of insects and other invertebrates in/on the
 colonies. After the six-week exposure period, bees in
 the colonies continued to forage freely. Weekly CCAs continued until the
 experiment was terminated on 9 September 2020. At that time, approximately
 75% of the surviving colonies had produced female reproductive offspring
 (hereafter “gynes,” those individuals who will found new colonies the
 following spring). Most other colonies had either died or were incapable
 of reproduction. After the field phase, we placed colonies in a freezer
 (≤-15 ºC) until dissection in the final CCA.
 <strong>Statistical Analysis</strong>
 Statistical analyses were conducted in the R statistical
 computing environment (Version 4.3.1) using tidyverse, DescTools, lme4,
 lmerTest, vegan, FedData, and other R libraries (Bates et al. 2015;
 Kuznetsova et al. 2017; Wickham et al. 2019, R Core Team 2021, Signorell
 2023). To evaluate access of the colonies to floral resources across the
 study, we compared patterns of land use/land cover in a 1 km radius around
 sites. This radius is within the reported bumble bee foraging ranges of
 250 m to 3000 m. The foraging range is affected by species differences in
 colony size and body size (Goulson 2010). Furthermore, workers from
 commercial <em>B. impatiens</em> colonies in lowbush blueberry
 fields foraged up to 400 m from the colony (Desjardins and De Oliveira,
 2006). Using the R library, FedData (Bocinsky et al. 2023), the 2016
 National Land Cover Dataset was downloaded for an area around the study
 site. The R library, ‘Raster’, was used to extract pixels within 1 km of
 each site. Proportional representations of 12 land cover classes at each
 site were then determined. We also explored land use patterns among sites
 using non-metric multi-dimensional scaling (NMDS) implemented with the R
 library, vegan. Dimethoate treatment groups were
 compared to the Fed control for sucrose solution consumption, colony mass,
 numbers of gyne offspring produced, colony survival, and foraging
 activity. To determine the effect of supplementing hives with 50% sucrose
 solution, we compared responses between the two controls (Fed and Unfed).
 Foraging behavior was expressed as the number of bees returning to the
 hive per hour after normalizing from 10-minute counts. Data from one site
 were excluded because all six colonies were destroyed by bears between
 CCAs 2 and 3. In addition, dead colonies were excluded from CCA-specific
 analyses. For some response variables, such as colony mass, analyses were
 precluded at CCA 7 because the treatment colonies exposed to 0.75 and 3.0
 mg/L dimethoate had poor survival. For final counts of gynes, we excluded
 colonies that were lost to bears, but included colonies that died before
 the end of the study. We excluded five hives from the analysis for gyne
 production because their plexiglass lids had failed, possibly affecting
 response variables. The Shapiro-Wilk Test was used to test for normality
 of residuals and Levene’s Test to test for homogeneity of variance among
 treatment groups. Dunnett’s multiple comparison procedure was used to
 determine the NOAEC and LOAEC for response variables that met the
 assumptions of parametric statistical tests. Otherwise, the non-parametric
 Kruskal-Wallis test was used.
description: # Development of a test design for a semi-field, colony feeding study for
 the common eastern bumble bee (Bombus impatiens \[Hymenoptera:Apidae])
 [https://doi.org/10.5061/dryad.wwpzgmssv](https://doi.org/10.5061/dryad.wwpzgmssv) We have submitted our raw data (cfs.arrival.csv, Bayer.CFS.basic.information.csv, bayer.cfs.cca.2022.csv, bayer.cfs.2020.foraging.csv), treatment groups (bayer.cfs.trts.csv), locations/sites coordinates (Bayer_CFS_sites.csv), codes for land use from the National Land Cover Dataset (NLCD.codes.csv), and R script (bayer.CFS.2020.R). **Descriptions** cfs.arrival.csv - Contains the observations of 80 bumble bee (*Bombus impatiens*) commercial colonies at the time of receipt in the field facility and from which 60 colonies were selected for the study. The variables recorded were: * Hive.no: Assigned number from 1 to 80, to identify the colony. * Date: Date of observations * Hive.mass: The mass (weight) of the commercial colony including the bumble bee nest, brood, adults, and plastic foundation (box) containing the colony. Units are grams (g). * Notes: Observations made by personnel. * Excluded: X denotes colonies that were excluded from the study. Sixty study colonies were selected from 80 initially aquired. The excluded colonies were due to the presence of queen pupae, and by colony mass on both ends of the distribution tail. * CCA.no: Colony Condition Assessment number. This assessment was done at arrival of the colonies to the field facility. * Hive.dead: To note if any colony was dead or alive at the time of receipt at the field facility.   Bayer.CFS.basic.information – Lists the basic information regarding the experimental design including the location, site number, site name, position of the study colony within each site, treatment assignment, and hive number (identification assigned at time of arrival into the field facility). * Location: Farm or location identified by name. * Site.no: Site number. Identification of the site within locations. There were four sites within the Codling location, three sites within the Stoney location, and three sites within the Pullman location.  * Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.  * Position: Position of the colony within a site. The colonies were placed in a linear manner within a site, separated by 10 m. The position of the colonies and treatments was randomly allocated.  * trt: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.  * Hive.no: Hive number. Identification made at time of arrival in the field facility.        bayer.cfs.cca.2022.csv - Contains the compiled data and observations at each Colony Condition Assessment (CCA). The variables recorded were: * Sort: Position of the colony within one site (randomly designated). The position was maintained in the site, but was identified with new numbers as CCAs progressed, thus, for CCA 1, the positions were identified from 1-60, for CCA2, positions were identified from 61-120, and so on.  * Loc.sort: Location of the colony. Colonies were distributed within 10 sites, an dthese sites were clustered into three farms or locations. * Date: Date of observation. * CCA.no: Colony Condition Assessment number. * Location: Farm or location identified by name.           * Site.no: Site number. Identification of the site within locations. There were four sites within the Codling location, three sites within the Stoney location, and three sites within the Pullman location.              * Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.       * Position: Position of the colony within a site. The colonies were placed in a linear manner within a site, separated by 10 m. The position of the colonies and treatments was randomly allocated.             * trt: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.              * Gynes.total.09.11.2020: Number of gynes (new bumble bee queens) per surviving colony. During the last CCA the colonies were dissected, alloweing for the quantification of all adult new queens.        * Notes.field: Observations made in the field such as presence of other insects, or dead queens, colonies killed by bears, etc.               * Notes.09.11.2020: Observations made on the last day of the experiment on 9 September 2020. * Dead queens from margin of the hive not collected 09.11.2020: Number of dead queens found at the margin of the hives that were not collected, but their presence was noted. Only two colonies had this scenario, one with one queen and the other with three queens.  * New.bag.no: Identification of the new bag containing the provisioned sucrose solution to each colony upon replacement in the field.    * New.nectar.g: Weight of the new bag containing fresh provisioned sucrose solution. Units are grams (g). * Old.mass.g: Weight of the old bag containing provisioned sucrose solution upon replacement. Units are grams (g).              * Nectar.consumed: Amount of sucrose solution consumed weekly during the six weeks of exposure. Units are grams (g). * nectar.notes.2022: Observations made in regard to not being able to measure the old bag weight due to ants chewing the bag (1 colony) and due to plexiglass in the hive being broken (1 colony).      * Hive.no: Hive number. Identification made at time of arrival in the field facility.              * Hive.mass.g: Mass (weight) of the colony including the nest, brood, adults, and plastic foundation (box) containing the colony. Units are grams (g).   * Hive.dead: Survival observation. Noted if the colony was dead.        * No.queens: Number of queens observed.     * Queen.pupae: Number of queen pupae observed. Queen pupae cells are notably bigger than worker/male pupae cells.              * Gynes.present: Presence or absence of gynes (new queens).             * Queen.dead: Foundress (mother) queen survival observation.   * Gyne.or.pupae: Noted the presence or absence of gynes or gyne pupae.              * Males.present: Noted the presence of males in the colony.             * Date.last.CCA: Date of CCA.             * Days.since.last.CCA: While the initial plan was to conduct CCAs in a weekly basis, due to scheduling and other factors, the intervals ranged from 7 to 13 days.  * hrs.since.last.CCA: Hours since last CCA. This was the results of multiplying the days since the last CCA by 24 hours/day.              * Notes.field.Q.verbatim: Informal records of observations made in the field in regard to the foundress queen or new queens.           * Notes.data.entry: Any notes that were taken into consideration for data entry.              * Gyne.count.exclude: Identification of colonies that were excluded from the analysis.   * Gynes.collected.09.11.2020: Number of gynes (new queens) collected at the end of the experiment.              * unopened.gyne.pupae.09.11.2020: Number of unopened gyne pupae.     bayer.cfs.2020.foraging.csv - Contains the compiled data and observations corresponding to the foraging activity assessment. The variables recorded were: * Location: Farm or location identified by name.            * Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively.          * Position: Position of the colony within a site. The colonies were placed in a linear manner within a site, separated by 10 m. The position of the colonies and treatments was randomly allocated.               * Hive.no: Hive number. Identification made at time of arrival in the field facility.               * Treatment: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.                * Date: Date of observation for purposes of identifying observation events.     * Date.actual: Actual date of observation where bees were recorded entering and exiting their colonies. In some instances this happend -1 day of the date identified for the observation event.      * Start.time: Time of the day when the foraging observation intiated.          * 8am: 8:00      * Noon: 12:00               * Mins.to.noon: The number of minutes before or after 12:00pm.   * Mins.since.8am: The number of minutes after 8:00 am.           * Observer: Initials of personnel.           * Bee.in: Number of bees entering a colony within the 10 minute observation period allocated to each colony.                * Bee.out: Number of bees exiting a colony within the 10 minute observation period allocated to each colony.                  * Ratio.in.out: Ration between the number of bees entering and the number of bees exiting a colony within the 10 minute observation period allocated to each colony.      * Bees.in.per.hr: Number of bees entering the colony per hour. This value was obtained by multiplying the number of Bee.in by 6. * Exclude: Identified colonies excluded from analysis. The reasons are noted in column Notes.1.               * Notes.1: Observations made in regards to the colonies.             * Notes.2: Observation made in one Site (Codling 2).                          bayer.cfs.trts.csv - Lists the treatments and a sorting number from 1-6 to enable the random allocation of colonies to positions within sites. * trt: Treatment. There were six treatment: unfed control, fed control, and dimethoate concentrations of 0.05, 0.19, 0.75 and 3.0 ppm via sucrose solution.    * sort: Numbers 1 to 6, to enable random allocation of the study colonies to a position within a site. Bayer_CFS_sites.csv - Identifies the site, locations, nearest town, latitude, and longitude of the sites. * Site: Site identified by a combination of the location name and a number, from 1-4 in the Codling location, from 1-3 in the Stoney and Pullman locations, respectively. * Location: Farm or location identified by name.   * Town: Nearest town to the farm (location). * latitude: Coordinate. * longitude: Coordinate. NLCD.codes.csv - Contains the code and corresponding description of the land use according to National Land Cover Dataset. * NLCD.code: Numerical code associated with a category of land use.      * NLCD.desc: Short and general description of the land use category.      * NLCD.desc.verbose: Additional details included in the general description of the land use category.               * NLCD.desc.verbose: Accounting for the 'Perennial' nature of the category Ice/snow.   * NLCD.full.desc: Detailed description of the land use category.  bayer.CFS.2020.R - Contains the R code to analyze the data. The script was created using version 4.3.1. Notes included for clarification of certain steps.