Simple SummaryThe World Organization for Animal Health recommends the use of stunning methods before the slaughter of farmed fish destined for human consumption. The use of carbon dioxide (CO2) is not recommended by the World Organisation for Animal Health for the stunning of farmed fish at pre-slaughter. However, its use continues to be common in the world. CO2stimulates chemoreceptors, causing aversion and escape attempts when fish are immersed in CO2-saturated water. The use of CO2in seawater with oxygenated water is allowed for pre-slaughter of halibut in Norway. Hence, there are no studies assessing loss of consciousness in halibut exposed to this stunning method. The best approach to determine unconsciousness is by measuring brain electrical activity through EEG.AbstractAs fish welfare becomes a growing concern, it is important to ensure humane treatment during slaughter. This study aimed to assess the onset of unconsciousness in Atlantic halibut immersed in CO2-saturated seawater through electroencephalography (EEG). Of the 29 fish studied, 10 exhibited escape attempts, indicating aversion to CO2-saturated water despite its oxygenation. EEG signals showed four distinct phases: transitional, excitation (high amplitude–high frequency), suppressed, and iso-electric phases. The onset of the suppressed phase, indicative of unconsciousness, occurred on average 258.8 ± 46.2 s after immersion. The spectral analysis of the EEG signals showed a progressive decrease in median frequency, spectral edge frequency, and high frequency contribution, which corresponded to the gradual loss of consciousness. The study concludes that CO2-saturated water is not recommended for pre-slaughter handling of halibut due to the extended time required for the onset of unconsciousness and the observed aversive behaviour. Ensuring humane treatment during slaughter is important for addressing public concern and safeguarding fish welfare in all stages of production.