126.96.36.199. Discriminating dose bioassay
The simplest experiment involves treating bees with a single dose, termed the discriminating dose, in the presence and absence of another agent. It is important that an appropriate discriminating dose is chosen that will allow for any changes in toxicity to be detected. Discriminating dose experiments have been extensively conducted in Varroa destructor to determine acaricide resistance (Elzen et al., 1998), and have been used in honey bees as well (Hawthorne and Dively, 2011). A significant drawback to the discriminating dose approach is that the full dose-response curve is not explored and it is impossible to differentiate between interactions affecting the slope and the intercept of the dose-response curve.
1) Preliminary toxicity bioassays are performed singly on both agents to be tested. This bioassay can use adults treated through oral exposure (section 3.2.1.), topical exposure (section 3.2.2.) or exposure on foliage (section 3.2.3.).
2) The dose of the first, less toxic, agent should be chosen using the dose-response curve generated in step 1. Either this “non-killing” dose should be chosen so that it is the maximum dose that can be delivered that does not cause mortality different from control, or it should be an environmentally relevant dose determined through chemical analysis or predicted exposure.
3) The discriminating dose of the second, “killing” agent is chosen using the dose-response curves generated in step 1. The appropriate discriminating dose depends on the expected outcome of the interaction between the two agents – if antagonism is expected, then the LD90 or LC90 of the more toxic agent should be used. If synergism is expected, then the LD10 or LC10 is appropriate. If there are no a priori expectations the LD50 or LC50 should be used. An environmentally relevant dose, based results of chemical analysis or predicted exposure, may also be used.
4) To test for interactions bees are treated as recommended for oral, topical or foliage exposure (sections 3.2.1.-3.2.3.), except that only four groups of bees are used. Bees are then exposed to either the “non-killing” dose of the first agent (Step 2) or a control in combination with, or followed by, the discriminating dose of the second “killing” agent (Step 3), or a control. If the two agents cannot be delivered in combination (e.g. an oral “non-killing” agent and a topical “killing” agent) then the “non-killing” agent should be administered 1 h (topical or foliage) or 24 h (oral) prior to administration of the “killing” agent.
5) Testing in Step 4 is repeated to produce 5 replicates. The proportion of bees dying is transformed using the arcsine square root method, then a simple t-test or ANOVA is used to determine the statistical significance of observed differences in mortality (Hawthorne and Dively, 2011).