Each individual was subjected to three different test conditions: Boat noise, Gaussian noise, and no-noise control.
In each test condition, a single fish was placed in a plastic bucket (21 cm height, 22 cm diameter, 12 cm water depth) containing 3 l of water for 30 min. The water in the bucket originated from a 200-l storage water tank, that did not contain any fish.
Cortisol levels were measured in 1 l samples of the storage tank water to control for the potential presence of waterborne
substances that could bind to the cortisol antisera used.
The levels of immuno-reactive cortisol in these samples were below the detection limit of the assay. The mean temperature during experiments was 22 ± 1 C