Maintaining a stable, well-mixed, well-characterized drug or toxicant concentration is essential for inhalation studies. The gas phase concentration is a major parameter for any exposure that attempts to calculate the inhaled dose, to establish dose–response relationships, to estimate such parameters as the LD50, and for conducting comparative studies across species or interventional strategies, for example. Gas phase concentrations should be measured by acquiring samples in the breathing zone of animals through a sampling port in the WB chamber or through an animal port in a NO-HO chamber. There are two techniques for concentration monitoring, a time-averaged sample and continuous real-time monitoring.
A time-averaged sample provides the average concentration during a given exposure period but it does not provide information on the stability of chamber concentrations. The oxidant gas is usually collected with impingers or bubblers, and the collected samples are then analyzed usually for a product formed (either directly or indirectly) from a relatively facile chemical reaction. For chlorine, a gas sample may be passed through two midget impingers in series, each containing 10 ml of 1mM sodium hydroxide. The collected fluid was then analyzed spectrophotometrically for total chlorine content (17
). Similarly, chlorine gas in a WB exposure chamber (range, ~200 to 300 ppm) was sampled by passing through a fritted glass bubbler containing 100 ml of 1% sulfamic acid solution at a flow rate of 1 L/minute. Aliquots of the collected samples were then analyzed using a modified ASTM method for airborne chlorine (4
The continuous monitoring of a chamber atmosphere provides a real-time concentration profile that can be used to determine the stability of gas generation/delivery or provide a warning when there is problem in the generation and/or delivery system that affect the exposure conditions. Continuous monitoring is usually achieved by using real-time monitors, which operate using a variety of principles. For example, in a WB exposure system, an electrochemical reduction detector was used to monitor the chlorine concentration (5
), while ozone has been monitored via ultraviolet photometers (7
) and NO2
concentration via chemiluminescent NO2
/NO detectors (7
). The monitor output signal can also be used to regulate generation and delivery of the toxicant to achieve a desired exposure concentration within the chamber.
shows chlorine concentration monitored during a 30-minute inhalation exposure of chlorine gas in a small glass WB exposure chamber (5
). When the exposure starts there is a rapid rise of Cl2
concentration, and in a few minutes it reaches a quasi–steady-state concentration. When the generation is stopped at 30 minutes, Cl2
concentration decreases within a few minutes to the background concentration. The rise and fall of chamber concentrations can be expressed by the theoretical Equation 1
where C is the chamber concentration at time, t, Co
is the steady-state concentration, Q is the flow rate, V is the chamber volume, and t is time. When toxicant inflow stops, the concentration decreases exponentially from Co
following the equation:
Cl2 concentration in the 3.5-L glass chambers (shown in ) with two rats.
The concentration–time characteristics of a chamber are most usefully expressed by stating the time, t99
, required to attain 99% of the steady-state concentration by
These equations are derived on the basis of assumptions of constant gas concentration and flow rate entering the chamber (which can either be the high concentration during exposure initiation or zero when exposures are ended), and a single well-mixed compartment. Also, the volume occupied by the animals and gas losses due to the presence of animals are ignored. In practice, t99 is often obtained from the real-time monitoring such as that shown in . The total exposure time for animals is then equal to T (duration of the test material delivered to the chamber) + t99.