2.3. Aquatic oxygen consumption, SMR, and Pcrit
2.3.1. Intermittent-closed respirometry
Oxygen consumption without access to air (MO2)was measured using
intermittent-closed respirometry for at least 20 h at 27.0±0.1 °C
(Steffensen et al., 1984; 1989). The respirometer, which was submerged
in water in a large tank (~30 L), consisted of a chamber containing the
fish and a closed loop where water passed a galvanic oxygen electrode
that continuously measured the oxygen partial pressure of the water
(PO2w) within the respirometer. In addition, an open loop could be
activated to flush the respirometer with fresh water from the tank
containing the system. The opening and closure of the flush pump as
well as the logging of the PO2w measurements were automated by a
software system (Respirometer 2.0) developed at the Zoophysiology
section at Aarhus University. Each individual MO2 measurement lasted
15 min, where the respirometer was closed for 3 min, so the decline in
PO2w could be used to calculate MO2, followed by 12min of flush to
renewoxygen levels. This setup provided independentmeasures ofMO2
at 15-min intervals. MO2 for all points was calculated as:
MO2 =
jΔPO2
Δt
j⋅βO2⋅Vsys
Mb
where ΔPO2/Δt is the rate at which PO2w declined during the closed
period, βO2 is the solubility of oxygen in water at 27 °C
(1.617 μmol mm Hg−1 L−1), Vsys is the volume of water in the system
corrected for the volume of the fish (density assumed to be 1 kg L−1),
and Mb is body mass. Values for MO2 were omitted when the R2 of the
linear regression for the decline in PO2w was less than 0.985. At the
completion of the experiment, measurements were continued for 0.5–
2 h after removing the fish from the respirometer to determine
background MO2. Background MO2 was subtracted from the measured
MO2, to control for bacterial oxygen uptake (7±4 mg O2 kg−1 h
−1). All parts of the respirometer system were carefully cleaned after
each experiment and re-filled with chlorine free tap water, and the
PO2w was allowed to reach equilibrium before proceeding with the
next fish. The O2 electrode was calibrated before each measurement.