2.3.2. Experimental protocol
Since P. hypophthalmus survived forced submergence in normoxic
water for 6 days, we measured standard and routine metabolic rates
(SMR and RMR, respectively) in 13 fish (136±24 g) denied access to
air in normoxic water. SMR was determined as the average of the 5
lowest MO2 determinations during the 24-h period. The 10% quantile
was also used as a measure of SMR for comparison. RMR was
calculated as the average MO2 during the entire measurement period.
In a further 7 fish (117±21 g), we measured SMR and RMR during
mild hypoxia (12–13 kPa) since preliminary studies showed that mild
hypoxia reduced the variability in MO2, presumably as a result of
reduced spontaneous activity, and therefore allowed for a more reliable
determination of SMR. This oxygen level was well above the critical
oxygen partial pressure (Pcrit) that was measured after 8 h of mild
hypoxia (12–13 kPa) during preliminary trials. Pcrit was determined as
the PO2w, where MO2 was reduced below the SMR determined prior to
hypoxia (Affonso and Rantin, 2005; Thuy et al., 2010). This methodwas
chosen because fish were seldomat restwhen themeasurement of Pcrit
was initiated, in which case the point at which SMR cannot be
maintained was not revealed through the measurement of MO2 with
decreasing PO2 alone. The fish were then allowed to recover for 15 h at
12–13 kPa before being exposed to an oxygen level close to the Pcrit of
the individual fish for 6 h (6–8 kPa), followed by an additional 15-h
recovery. These studies were performed to investigate whether MO2
increased immediately after hypoxia as indication of an oxygen debt
when confined to aquatic breathing in severe hypoxia.