Endosulfan Exposure Disrupts Pheromonal Systems in the Red-Spotted Newt: A Mechanism for Subtle Effects of Environmental Chemicals

Environmental Health Perspectives, July, 2001 by Daesik Park, Steven Hempleman, C., Catherine R. Propper

General behavioral tests. We measured behavioral activity and food uptake just before (0 h) and at 24, 48, and 96 hr after the onset of endosulfan exposure. The activity was measured by calculating the number of squares crossed in an open field box (30 x 15 x 9 cm) for 10 min. We also recorded the number of air gulps each animal took during a 10-min observation period as an indication of breathing rate. To measure the amount of food consumed, five young crickets (less than 1 week old) were randomly selected and supplied to each individual tank at 2200 hr. The next morning (0800-0900 hr), we counted the remaining crickets. Data for the behavioral activity were analyzed by two-way analysis of variance (ANOVA) (23). For the amount of food consumed and breathing rates, we used Kruskal-Wallis tests (23).

Visual mate selection. To determine if endosulfan impairs visual aspects of mate choice, visual selection tests were conducted following Verrell's method (16). An aquarium measuring 80 x 40 x 50 cm was divided lengthwise into two halves by drawing a vertical line. Two transparent plastic boxes measuring 12 x 10 x 15 cm were submerged, one in the center of each half of the aquarium, and each box had either a control or an endosulfan-exposed female. The two females were paired by weight ([is less than] 0.5 g). A test male was then released into the center of the aquarium. We recorded which half of the aquarium the male was in at the end of each minute for 30 min. During selection tests, the boxes were watertight to prevent female pheromones from reaching the test males. Therefore, the males could show mate preferences based only on female visual cues. After each experiment, the aquarium and test boxes were rinsed with aged tap water. Data obtained were analyzed by Wilcoxon signed-rank tests (23).

Olfactory mate selection. After the endosulfan treatment, we placed females in fresh water for 12 hr to remove potential endosulfan residues from the skin. Olfactory selection tests were conducted using a standard Y-maze olfactometer (16). Two arms of the Y-maze, containing either experimental or control individuals, who were paired by weight ([is less than]0.5 g), each received 150 mL/min aged tap water from a reservoir. Each chamber of the arm could reserve 10 mL water until overflowing. The amount of water was controlled by a flow meter (Gilmont Instruments, Barrington, IL). At this flow rate, water flowing through the two arms remained laminar to a drain at the end of the Y-maze. Test newts were placed at the end of the maze behind a start gate for 5 min to allow them to adjust to the test situation. After 5 min, we slowly raised the gate when the newt was facing away from the gate. Each newt was allowed 20 min to crawl or swim up the main body of the maze. A choice was considered to be made when the test newt moved more than one-third of the length of a given arm. After each trial, the Y-maze was rinsed with aged tap water. Data obtained were analyzed by binomial tests (23).