CARBON DIOXIDE
Room air contains 0.04% carbon dioxide (CO2), which is heavier than air and nearly odorless. Inhalation of CO2 at a concentration of 7.5% increases the pain threshold, and higher concentrations of CO2 have a rapid anesthetic effect.53-58
Time to loss of consciousness is decreased by use of higher concentrations of CO2 with an 80 to 100% concentration providing anesthesia in 12 to 33 seconds in rats and 70% CO2 in O2 inducing anesthesia in 40 to 50 seconds.61,62 Time to loss of consciousness will be longer if the concentration is increased slowly rather than immersing the animal in the full concentration immediately.
Several investigators have suggested that inhalation of high concentrations of CO2 may be distressing to animals,63-66 because the gas dissolves in moisture on the nasal mucosa. The resulting product, carbonic acid, may stimulate nociceptors in the nasal mucosa. Some humans exposed to concentrations of around 50% CO2 report that inhaling the gas is unpleasant and that higher concentrations are noxious.67,68 For rats, exposure to increasing concentrations of CO2 (33% achieved after 1 minute) in their home cage produced no evident stress as measured by behavior and ACTH, glucose, and corticosterone concentrations in 70 serum.
Carbon dioxide has been used to euthanatize groups of small laboratory animals, including mice, rats, guinea pigs, chickens, and rabbits,5,71-76 and to render swine unconscious before humane slaughter. 22,63,64
Advantages—(1) The rapid depressant, analgesic, and anesthetic effects of CO2 are well established. (2) Carbon dioxide is readily available and can be purchased in compressed gas cylinders. (3) Carbon dioxide is inexpensive, nonflammable, nonexplosive, and poses minimal hazard to personnel when used with properly designed equipment. (4) Carbon dioxide does not result in accumulation of tissue residues in food-producing animals. (5) Carbon dioxide euthanasia does not distort murine cholinergic markers82 or corticosterone concentrations.83
Disadvantages—(1) Because CO2 is heavier than air, incomplete filling of a chamber may permit animals to climb or raise their heads above the higher concentrations and avoid exposure. (2) Some species, such as fish and burrowing and diving mammals, may have extraordinary tolerance for CO2. (3) Reptiles and amphibians may breathe too slowly for the use of CO2. (4) Euthanasia by exposure to CO2 may take longer than euthanasia by other means.61 (5) Induction of loss of consciousness at lower concentrations (< 80%) may produce pulmonary and upper respiratory tract lesions.67,84 (6) High concentrations of CO2 may be distressful to some animals.
Recommendations—Carbon dioxide is acceptable for euthanasia in appropriate species (Appendices 1 and 2). Compressed CO2 gas in cylinders is the only recommended source of carbon dioxide because the inflow to the chamber can be regulated precisely. Carbon dioxide generated by other methods such as from dry ice, fire extinguishers, or chemical means (eg, antacids) is unacceptable. Species should be separated and chambers should not be overcrowded. With an animal in the chamber, an optimal flow rate should displace at least 20% of the chamber volume per minute.85 Loss of consciousness may be induced more rapidly by exposing animals to a CO2 concentration of 70% or more by pre-filling the chamber for species in which this has not been shown to cause distress. Gas flow should be maintained for at least 1 minute after apparent clinical death.86 It is important to verify that an animal is dead before removing it from the chamber. If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia. Adding O2 to the CO2 may or may not preclude signs of distress.67,87 Additional O2 will, however, prolong time to death and may complicate determination of consciousness. There appears to be no advantage to combining O2 with carbon dioxide for euthanasia.87