Whole-Body Nanoparticle Aerosol Inhalation Exposures

The overall goal of the following experiment is to construct a whole body nanoparticle aerosol inhalation exposure facility for nano-sized titanium dioxide inhalation toxicology studies. This is achieved by first generating nanoparticle aerosols with a novel nanoparticle aerosol generator. As a second step, deliver nanoparticle aerosols to a whole body inhalation exposure chamber to create test atmospheres that simulate occupational environmental, or domestic engineered nanomaterial aerosol exposures.

Next, expose lab animals to the well-characterized nano titanium dioxide aerosols monitor and control the test atmospheres in order to achieve the designed particle deposition. In lungs, results are obtained that show the nanoparticle aerosols generated and delivered to the exposure chamber have steady mass concentrations, homogenous composition, free of contaminants and stable particle size distributions. This system reliably and repeatedly creates test atmospheres that simulate occupational environmental, or domestic engineered nanomaterial aerosol exposures.

Visual demonstration of this method is critical because nanoparticle aerosol generation and characterization techniques are very difficult to learn and no formal instruction manual exists. Further, specifically designed instrumentation such as our nanoparticle aerosol generator have multiple variables that must be properly controlled in order to successfully generate aerosols and characterize them in real time. Demonstrating the procedure will be members of my research program, Dr.Jing Hay, a research assistant professor, and Mr.Carol McBride, my chief research assistant Beacon.

Begin conditioning of the titanium dioxide nanoparticle powders at least one day before an inhalation study. Use appropriate personal protective equipment and work in a fume hood. Place 200 grams of the nanoparticle powders in a non-transparent container leaving the container lid open.

Next place the container in a dry desiccate for at least 24 hours for conditioning at least one hour prior to an experiment. Turn on the air monitoring and data acquisition systems. The aerosol monitoring SMPS.

Also turn on the powered ELPI allow these systems to warm under the fume hood and wearing personal protective equipment. Open the cylinder caps on the aerosol generators and replace the filters. Next, measure approximately four grams of conditioned nanoparticle powders and load a cylinder with it.

Repeat for each cylinder. Once this is done. Replace the cylinder caps on the aerosol generators what wipe all areas suspected of nano powder contamination.

Take the aerosol generator to the exposure chamber. Connect all outlets of the aerosol generators via a manifold to a cyclone separator, which is at the inlet of the inhalation exposure chamber. Next, connect compressed air tubing to the Venturi Dispersers in the aerosol generators.

To begin connect sensors for temperature, relative humidity, pressure, oxygen, and carbon dioxide to the atmosphere monitoring ports on the inhalation exposure chamber. Now connect the inlet of an aerosol diluter to one of the aerosol sampling ports on the inhalation exposure chamber. Chamber connect its outlet to the inlet of the electric low pressure impactor.

Next, connect the s and PS to an aerosol sampling port. Also connect the inlet of a particle concentration monitor to a separate aerosol sampling port. At this point, take A-P-T-F-E membrane filter that has been weighed and loaded into a stainless steel filter holder.

Connect the inlet of the filter holder to one of the aerosol sampling ports on the inhalation exposure chamber and connect its outlet to a sampling pump. Now activate the ELPI data acquisition software and check set up parameters. Next turn on the power to the ELPI vacuum pump.

After this zero, the LPI and measure the pre-exposure concentration. Activate the SMPS data acquisition software and record pre-exposure concentration. Also activate the software for monitoring and controlling the chamber environment.

After weighing the experimental animals, mark them and their cages so they can be put back in the same cages after exposure if needed, open the door of the inhalation exposure chamber and load the experimental animals into the wired cages. Water may be provided for the animals close and secure. The door of the inhalation exposure chamber.

Observe the animals through the observation windows of the chamber frequently. The first step is to turn on the exhaust vacuum pump of the inhalation exposure chamber. Use the control and data acquisition software to supply filter dry air to the exposure chamber and establish a slightly negative chamber pressure with a set point of negative two-tenths millibar.

The software should also collect data on the exposure environment's, pressure, temperature, relative humidity, and levels of oxygen and carbon dioxide. Now turn on the aerosol generators. Run the ELPI and SMPS data acquisition software to continuously monitor particle size and relative mass concentration in the inhalation exposure chamber when the aerosol concentration is stable.

Within about 20 minutes, set up the sampling time and turn on the aerosol sampling pump to collect representative samples of nanoparticles. Once the sampling time is reached, wear personal protective equipment and the filters and plug the sampling ports with rubber plugs To prevent test materials from escaping the exposure chamber, weigh the filters and calculate the mean mass concentration in the exposure chamber. If the mean concentration is off the targeted value, manually adjust the airflow in the aerosol generators to ensure the targeted concentration is attained.

Use the information to calculate the particle deposition. In animal lungs. The dose is equal to the mean mass concentration times the minute volume, times the exposure duration times the fraction of the material deposited or absorbed.

Please clean pre weight filters in the filter holders. Return the assembly to the exposure chamber. Input a new sampling time and turn on the sampling pump once again.

At the end of the sampling time, remove the filters and plug the ports. Weigh the filters and calculate the mean mass concentration. Determine the remaining exposure time for the animals.

Using the targeted particle deposition and the information collected, the remaining exposure time is equal to the difference between the targeted and measured dose. Divided by the mean mass concentration, the minute volume, and the fraction of material deposited or absorbed. When the time has elapsed, turn off the aerosol generator.

Flush the inhalation exposure chamber with filtered air until the particle concentration reading is close to the pre-exposure concentration in the chamber. At this point, turn off the chamber exhaust vacuum pump and stop the control and data acquisition software. Remove the experimental animals from the exposure chamber and return them to their holding facility.

Observe the animal frequently and ensure that the experimental animal is comfortable. Shut down the remaining software and equipment as seen in this plot of chamber pressure. As a function of time, the chamber pressure was monitored and maintained at a slightly negative value during the experiment in order to minimize leakage of the test substance into the laboratory.

Shown here is the inlet flow rate in red and the exhaust flow rate in black as a function of time. The difference in flow rates created the desired negative pressure. The whole body chamber was operated on a dynamic flow basis with about 90 liters per minute, continuous flow of air through the chamber.

This provided more than the minimum air changes per hour required by the US Environmental Protection Agency for acute inhalation exposure studies. This plot shows how well the values of temperature and relative humidity were maintained during the experiment. Oxygen and carbon dioxide concentrations were also monitored as shown here.

Oxygen was stable at about 21%Carbon dioxide was stable at 580 parts per million. Particle size was measured with ELPI. The left is that particle size versus time during four hour exposure.

The right is the count. Median aerodynamic diameter D equals 157 nanometers as seen in this plot. The particle size was measured with SMPS.

Here is an example of the real-time mass concentration during a four hour inhalation exposure. Finally, aerosol mass concentrations were found for 29 individual four hour inhalation exposures with the targeted concentration of six milligrams per cubic meter shown in red. This plot suggests the system can provide stable and reproducible titanium dioxide test atmospheres for acute inhalation exposures.

Once mastered, this technique can be completed in eight hours if the exposure period is four hours. While attempting this procedure, it is important to remember to properly monitor and characterize all test atmospheres. Don't forget that working with nano materials can be extremely hazardous and precautions such as engineering controls.

Special practices and personal protective equipment should always be taken while performing this procedure.