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Evaluation of Carbon Dioxide Euthanasia of Female Sprague Dawley Rats Alone or With Unfamiliar Conspecifics

by Debra L Hickman   View article here




Considerations for Implementing a Facility-Wide Policy on Carbon Dioxide Euthanasia for Laboratory Rodents

What to think about in order to facilitate safe and humane euthanasia.

by Les Anderson

The proper use of CO2 as a euthanasia method is generally considered humane for rodents and safe for personnel. The AVMA guidelines on euthanasia recommend properly used CO2 as a viable methodology. The selection of equipment and development of protocols for CO2 euthanasia need to ensure the humane treatment of the animals and the physical safety and psychological welfare of the technicians performing euthanasia. The protocols also need to consider workflow, possible impact on research parameters, and the cost of implementing and enforcing a facility-wide policy on CO2 humane euthanasia.

Welfare Considerations

Improper use of CO2 causes distress and possibly pain to the animal. To minimize that possibility, there are a number of factors to consider when developing a humane CO2 euthanasia protocol.

Source of the CO2 Gas
The only acceptable source of CO2 for euthanasia is a compressed gas cylinder or in house gas line. Dry ice or chemical means of generating CO2 are universally considered unacceptable.

Euthanasia Chamber
Ideally, animals should be euthanized in their home cage. This eliminates additional handling and introduction into an unfamiliar environment that can cause distress. The cage can either be treated individually with cage tops that allow direct CO2 input or multiple cages can be treated in a euthanasia chamber. Larger euthanasia chambers should ensure even distribution of gas throughout the chamber via a gas distribution manifold.

If euthanasia in the home cage is not possible or practical, the chamber should have space large enough to allow the animals normal postural adjustments. Avoid obstructions that may interfere with the proper input of CO2 or the animal’s exposure to the gas. The chamber should be easy to clean and disinfect. When performing euthanasia outside of the home cage, grouping animals from different cages into a euthanasia chamber is widely considered to be unacceptable.

Inputting CO2 Gas

According to the 2007 AVMA guidelines on euthanasia, operators should introduce 100% CO2 into the euthanasia chamber at a rate of at least 20% of the chamber’s volume per minute. To comply with these guidelines, a device that meters CO2 gas by volume is required. Typically, a flow meter that outputs in liters per minute (lpm) or cubic feet per hour (cfh) is used for this purpose. Using a CO2 regulator that does not include a flow meter makes it impossible to ensure that the ideal rate of input is being used. Determining the proper inflow is simple:

Multiply the length, width, and height of the chamber in inches and divide by 61 to determine the volume in liters

Multiply the volume by 20% to determine the proper flow rate (lpm)

At a fill rate of 20%, gas should input into the chamber for several minutes to ensure that the CO2 concentration reaches a level high enough for euthanasia. The exact flow rates and times should ultimately be chosen based on close observation of the animals, with the goal of choosing parameters that ensure euthanasia of every animal with the least possible distress.

High Flow Rates/Extensive Use
CO2 is stored in cylinders in liquid form. At high rates of flow or prolonged periods of use, the phase change from liquid to gas absorbs a great deal of energy and can result in the freezing of the gas supply equipment and in extreme cases the entire euthanasia system. This freezing shortens the life of the equipment, negatively impacts animal welfare, and can create a dangerous situation for equipment users. With high flow rates or prolonged periods of use, a heated gas source is required.


At one time it was believed that introducing a combination of oxygen and COwould offset the aversive nature of high concentration CO2, but this has largely been debunked. Adding pure oxygen to the incoming CO2 has been demonstrated to prolong the time to unconsciousness, thereby increasing distress. This could also result in the revival of animals that appeared to be euthanized.

Pre-Filling vs Slow Fill
Another practice common at one time was the filling of the euthanasia chamber with CO2 prior to the introduction of animals. This was demonstrated to induce unconsciousness rapidly and thus thought to be more humane. However, a number of studies have determined that such high CO2 concentrations cause increased animal distress. On the contrary, current ‘slow-fill’ methods are designed to induce unconsciousness prior to CO2 concentrations that are high enough to cause pain.

Alternatives or Secondary Methods

There are circumstances that warrant an alternative or secondary method of euthanasia. Neonatal mice and rats, as well as mouse and rat fetuses, are resistant to hypoxia and require prolonged exposures to CO2 to ensure euthanasia. When neonates are to be euthanized with CO2, exposure times of upwards of 60 minutes may be required to ensure euthanasia. Euthanasia must be verified after exposure in each case. The AVMA guidelines on euthanasia recommend that for neonates, a secondary method of euthanasia be used in conjunction with CO2. A policy specific to neonates must be devised and should be based upon close observation of the animals.

Inhalation of CO2 affects specific physiological parameters and can negatively impact certain types of research. A list of the parameters impacted by CO2 is beyond the scope of this article, but a useful resource is the Report of the ACLAM Task Force on Rodent Euthanasia.

Recently there has been interest in isoflurane as an anesthetizing agent to be used prior to euthanizing with CO2. A newly published study has demonstrated that introducing isoflurane into the process does not lead to less stress for the animal. It, in fact, significantly increases behavioral and neuromolecular signs of stress compared to a pure 20% CO2 induction.

Workflow and Efficiency
An advantage of CO2 is its potential to be applied to multiple small laboratory animals at a time with minimal labor. With a well-designed system and good technique, it is possible to humanely and efficiently euthanize hundreds of animals in one treatment cycle. Large-scale systems allow technicians to spend less time performing euthanasia. This not only frees them to spend time on other important functions, but also reduces the negative emotional and psychological impact of performing euthanasia. This emotional component of euthanasia has been linked to increased employee absenteeism and turnover.


Even when proper euthanasia techniques are incorporated into a well-designed protocol, the responsibility is on the person performing euthanasia to execute proper technique.

When euthanasia is performed in individual labs or in multiple areas throughout the facility, it can be extremely difficult for managers to ensure that euthanasia is being performed properly. Increasing the flow of gas to save time is an especially widespread problem, which negatively impacts animal welfare and is a waste of CO2 that can add up quickly. Even on a small scale, mistakes can turn into major problems for the facility. An operator that neglects to turn off the gas once finished can drain entire tanks and cause gross pollution of the room. In facilities that have piped in gas throughout the building, errors can result in draining the entire building’s supply of CO2.

When euthanasia is centralized, overseeing the process becomes more manageable, but the potential problems are greater. Mistakes made are now applied to tens or hundreds or animals at a time. If large volumes of gas are used, safety can be seriously compromised, even by minor mishaps.

Eliminating as many user-controlled variables as possible is the most effective method of ensuring that proper technique is followed. Using factory pre-set cylinder regulators and flow controls, electronic timers and standardizing the size and shape of the euthanasia chamber used throughout the facility are solid steps to achieving this goal. However, the ultimate solution to ensuring compliance is total automation. Advances in equipment design now offer complete systems that automate the entire euthanasia process. Whether euthanasia is decentralized or offered as a core service, automated euthanasia systems ensure compliance with the mandated protocol. Such systems precisely control the input of gas at the correct flow rate for the correct amount of time and if required, exhaust the gas. Most important, a standardized, automated euthanasia system assures facility management that the process is being done the same way every time.

User Safety

The Occupational Safety and Health Administration (OSHA), American Conference of Governmental Industrial Hygienists (ACGIH), and the National Institute of Occupational Safety and Health (NIOSH) all have a long-term (8-hour workday) CO2 exposure limit of 5,000 ppm and a short-term (10-15 minutes) exposure limit of 30,000 ppm. A value of 40,000 ppm is considered immediately dangerous to life and health. Typical indoor CO2 concentrations can range from 500-1,000 ppm. When CO2 euthanasia is properly performed on a small scale e.g. in individual cages a few times a day, CO2 levels in the room should never deviate far from normal, even if the CO2 is allowed to exhaust into the room.

When large numbers of animals are to be euthanized and a large volume of gas is required, an exhaust system needs to be in place to handle the removal of CO2 from the room. In this case the euthanasia chamber, or chambers, should be hermetically sealed and the exhaust carefully controlled. A vacuum source, or exhaust blower, should be used to remove CO2 from the chamber after euthanasia is completed. Ducting directly to the facility exhaust is ideal. But as an alternative, CO2 exhaust can be ported to a fume hood or similar air handling system that does not return air to the room. Filtration of CO2 from the exhaust is generally impractical. It would require constant filter replacement of large amounts of CO2 specific filter media.

CO2 monitoring should be considered when doing large-scale euthanasia. Many air quality monitors are commercially available that are easily installed in the room where euthanasia is performed.

Advancement of CO2 Euthanasia

The evolution of CO2 euthanasia methodology has advanced to the point of high capacity automated systems that can ensure humane treatment of animals with maximum efficiency and minimal labor. Facilities now have the option of treating animals with assurance that protocols are precisely followed. Human error variables are virtually eliminated.

Additional Reading

•Artwohl, J., et. al. (2005, August). Public statements: Report of the aclam task force on rodent euthanasia.
•Guidelines on euthanasia of rodents. (2001, February).
•Hawkins, P., et. al. (2006, August 9). Newcastle consensus meeting on carbon dioxide on carbon dioxide euthanasia of laboratory animals.
•Valentine, H., Williams, W., and Maurer, K. Sedation or Inhalant Anesthesia before Euthanasia with CO2 Does Not Reduce Behavioral or Physiologic Signs of Pain and Stress in Mice. Journal of the American Association for Laboratory Animal Science (JAALAS), January 2012.

www MAY|JUNE • 2012