## 2 Standard Methods
### Acute Toxicity Bioassay
\begin{table}
\begin{tabular}{c c} \hline \hline
**Item Letter Corresponds to the First** \\
**Mention of the Item in the Text** & **Item and Description** \\ \hline A & Pollen patties with 4\% pollen (Global Patties, Butte, \\  & MT, USA) \\ \hline B & Analytical Balance (Mettler Toledo, AL 204) \\ \hline C & Plastic wrap (Glad Food Wrap) \\ \hline D & 118.29 mL (4 oz) clear cup with white lid (Uline, \#S-9934) \\ \hline E & \begin{tabular}{c} Binder incubator (Hogentgler, \#BD400 UL-120V), or \\ equivalent. The incubator must maintain temperature \\ within \(\pm\)0.5 \({}^{\circ}\)C. \\ \end{tabular} \\ \hline F & \begin{tabular}{c} 30.5 cm \(\times\) 30.5 cm \(\times\) 45.7 cm desiccators (Thermo \\ Scientific, \#08-642-23C) \\ \end{tabular} \\ \hline G & Data loggers (Onset, HOBO \#UX100-011) \\ \hline H & \begin{tabular}{c} RH/Temperature Monitoring Hygrometer \\ (Traceable, 4154) \\ \end{tabular} \\ \hline I & \begin{tabular}{c} Amber Glass Threaded Vials, 1 dram \\ (Fisherbrand, 03-339-23B) \\ \end{tabular} \\ \hline J & \begin{tabular}{c} Stainless steel laboratory scoopula \\ (Fisherbrand, \#14357Q) \\ \end{tabular} \\ \hline K & 47 mm Petri dish (Fisher Scientific, 09-720-501) \\ \hline L & \
\begin{tabular}{c} Insect collection aspirator, with aluminum intake and \\ outtake valves and plastic tubing \\ (Bartovation, \#0649985933202) \\ \end{tabular} \\ \hline M & Cannister vacuum (Dewalt, DCV580H) \\ \hline N &
\begin{tabular}{c} Maxell CD-365 Slimline Jewel Cases \\ (Office Depot, \#981294) \\ \end{tabular} \\ \hline O & Professional Hot knife (Walnut Hollow, WH29639) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Tools and supplies used for the acute toxicity laboratory bioassay and field trial. 2. Prepare cages with SHBs. 1. Randomly place ten unsexed adult SHBs into each experimental cage using gloved hands. Provide water [we used tap water] on cotton balls ad libitum in each cage and place all cages inside the desicators in the incubator. Note: Prepare at least five cages for each control group (negative, solvent, positive) and concentration of each compound to be tested. It is best to prepare 1-3 extra cages. 2. To ensure SHB health and control for mishandling during experimental establishment, wait 24 h and replace all dead SHBs with those housed in the extra cages. Each cage should begin the study with ten living adult SHBs. 3. After the 24-h survival period, randomly assign cages to treatment groups. At least five replicates are needed for each of the three controls (negative, solvent, positive) and each concentration of all compounds. At least five concentrations are needed for each compound to graph a dose-response curve accurately. The typical replicate schedule: (3 control groups (negative, solvent, positive) + 5 concentration groups of compound X) \(\times\) 5 replicate cages/group = 40 cages (or 400 SHBs = 40 cages \(\times\) 10 SHBs/cage). Every new compound tested adds 25 cages (5 concentration groups \(\times\) 5 replicate cages/group) to the study. 2.4 Clearly label each cage with the date, compound or control group, concentration, and replicate number. 3. Prepare solutions. 3.1 Clearly label amber vials (Table 1) with the date, compound, and concentration of compound that will be added. Figure 1: (**A**) Experimental cage design used for acute toxicity bioassays. Ten adult small hive beetles (SHBs) are placed in a ventilated cage with a 1 g ball of treated pollen. (**B**) Trap placed in the center of the hive between the lid and frames for the field trial. Traps were made from CD cases with three entrances to allow adult SHBs to access 25 g of treated pollen in the center (denoted by arrow 2). All other access to the pollen was prevented by covering small holes in the center of the CD case with tape (denoted by arrow 1). Prepare 50 \(\mu\)L of test solution for each experimental cage. It is best to prepare at least 10% excess to account for evaporation. 1.