HCT consisted of a rapid decrease in water oxygenation (from nearly 100% air saturation to 20% air saturation in about 1 h), followed by a much slower descent (approximately 2% air saturation per hour) until the experiment ended (Fig. 1). Ambient oxygenation was controlled by bubbling nitrogen in the input of a submersible pump placed in the tank. Nitrogen flow in the pump was manipulated using a controller and solenoid valve connected to a cylinder (Oxy-REG; Loligo Systems, Tjele, Denmark). As soon as a fish lost its ability to maintain balance i.e., when the incipient lethal level (ILOS) was reached, it was quickly removed from the experimental arena, identified (pit tag reading) and placed in a fully aerated recovery tank. The corresponding time and oxygenation level was also recorded. Challenge ended as the last fish was removed from the experimental arena and it must be noted that less than 1% mortality was observed following HCT.