2.2. Hypoxia challenge test (HCT)
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.