Methods:
This experimental design will incorporate the factors magnitude, frequency and duration of exposure paradigms. All animals will be used only once throughout the experiment and will consist of an equal ratio of male to female crayfish. An artificial stream system consisting of eight flow-through streams (160 × 40.6 × 40.6 cm, interior L x W x H) will be constructed using cinder blocks and 4 mil polyethylene sheeting to expose animals to atrazine. Unfiltered water from the East Branch of the Maple River (Pellston, MI) will be pumped into head tanks placed at the upstream end of the artificial stream system. River water will be delivered to each stream via 1 cm inner diameter garden hosing at an approximate flow of 0.2 L/s. Collimators will be placed within the stream to regulate flow pattern of water entering the exposure arenas. Each stream will have a sand substrate to a depth of approximately 3 cm.
Crayfish will be exposed to atrazine with exposure regimes consisting of incremental increases in concentration, duration, and frequency independently. In addition, paired incremental increases of each characteristic and lastly, incremental increases in all three characteristics simultaneously. Duration of pulses will range from 6 to 48 hours (i.e. continuous exposure). Concentration of atrazine will range from 50 to 200 µg/L, all of which are environmentally relevant concentrations (USEPA, 2014). Levels of frequency will be 1.0 x 10¬-5 to 4.0 x 10¬-5 Hz. Frequency will be manipulated using small valves (Parker General Valve Corporation) powered by a Grass stimulator unit. Animals will be in exposure arenas for a total of 48 hours before a behavioral assay is performed.
After the exposure period, crayfish will be immediately removed and placed within a two-current choice arena (63.5 x 66x 19.1 cm; L x W x H). Two reservoir tanks will feed river water into the two-current flume with one reservoir housing a food odor stimulus. The food stimulus reservoir will consist of sardine gelatin food source held in the reservoir tank via a weighted mesh bag (Jurcak et al., 2015). Side of choice arena initially receiving water from food stimulus reservoir will be randomly assigned. Crayfish will be acclimated for a total of 15 minutes in the center of choice arena and then allowed to roam the choice arena freely for a total of 11.5 minutes. After 5 minutes of the trial period, the side of the choice arena receiving food stimulus source will be switched to prevent side bias. Each trial will be video recorded and Xcitex tracking software will be utilized to digitize and track crayfish movements throughout trial period. Measurements will include time spent in the side of choice arena containing odor from food source, time spent in the side of choice arena without odor from food source, and time to reach the food odor source (location of food odor input). Proportion of time spent in areas of the choice arena will be analyzed to determine relative contribution of magnitude, frequency, and duration of exposure paradigms to impairment in olfactory abilities.