Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

The overall goal of this video is to present the use of nitric oxide fumigation for post-harvest pest control and demonstrate how to measure residues on the fumigated products. Nitric oxide is a new fumigant for post-harvest pest control. However, because of its reactive nature with oxygen, nitric oxide fumigation must be conducted in ultra-low oxygen conditions and has a stringent requirement of fumigation procedures.

Demonstrating the procedures will be Dr.Xiangbing Yang, who is the post doc in my lab, and also Tiffany Masuda, who is a technician in my lab. To begin, load the chambers with insects or products. Grease the gaskets with petroleum jelly and seal the chambers air tight.

It is important during nitric oxide fumigation to keep the chambers sealed air tight, so be sure to properly grease the chamber's gaskets and keep the lids on tight. Next, connect one outlet to an oxygen analyzer via a T-connector with a check valve and the second outlet to a flow controller, which is connected to a nitrogen cylinder. Now, flush the chamber with nitrogen to establish an ultra-low oxygen atmosphere.

Start with a high-flow rate, then when oxygen levels are close to 30 parts per million, reduce the flow rate to between 0.5 and 1 L per minute. When the oxygen drops low enough, close the stop cocks to seal the chamber. Next, prepare the syringe and attached micro-tubing that are used to deliver the nitric oxide to the chamber.

First, flush out the oxygen with nitrogen gas. Then, load the syringe and tubing with nitric oxide from the foil bag. Now, connect the micro tubing to the first chamber and briefly open the stop cock to inject the gas.

Repeat this process for each chamber to be treated. Then, transfer the fumigation to a temperature-controlled cabinet for the duration of the treatment. When fumigating insects without products, open the chamber in a fume hood to terminate the fumigation.

After at least 30 minutes of ventilation, transfer the insects into an environmental chamber overnight before scoring their mortality. To terminate the fumigation of fresh products, first flush the chamber with about 10 air exchanges of nitrogen. Then, open the chamber and ventilate it for an hour or more.

Then, store the products in a refrigerator for future evaluation. Place the large chamber in a walk-in cooler equipped with an exhaust system. Always carry a nitrogen dioxide alarm when working with nitric oxide outside a fume hood.

Grease the gasket and seal it. Then, flush the chamber with nitrogen as before. Establishing ultra-low oxygen conditions in the large chamber can take a long time.

Hypothetically you would need 13 air exchanges of nitrogen to reduce oxygen to below 30 parts per million. You can estimate the time needed based on the flow rate and the volume of the chamber. After injection, connect a foil bag to balance pressure.

Then, set up sample dilution device and gas monitor to measure the nitric oxide concentration. Prepare large nitrogen-loaded foil bag with a strip of connectors, all joined to a single tube. Include a flue gas monitor with a pump and connect this assemblage to the chamber.

Use the monitor to measure the nitric oxide levels in the chamber. Now, conduct the termination process of nitric oxide fumigation. Turn on nitrogen flow to flush the chamber, turn on the exhaust system and open the valve.

Now, follow the described protocol to finish the termination process of nitric oxide fumigation. Once the nitrogen flush is done, which is when the nitric oxide level falls below 200 ppm, open the chamber. To measure nitrogen dioxide release from the fumigated products, first warm up the 405 nanometer nitrogen oxide gas monitor.

Then, close the fumigation chamber with the products inside. Next, connect the monitor to the chamber and circulate chamber air through the monitor. Collect baseline data for one minute.

Then, disconnect the monitor from the chamber and keep the chamber sealed for one hour. An hour later, collect another minute of data to calculate the nitrogen dioxide release rate. To measure residues of fumigated product, first prepare a homogenized product sample, mix 15 grams of homogenized sample into 100 mL of distilled water and let the mixture settle.

Then, vacuum filter the samples and store the filtrate at 2 degrees Celsius. To measure the nitrite concentration of fumigated sample, add 4 to 6 mL of concentrated acetic acid to the first bulb of the nitric oxide analyzer. Next, set up a purge vessel with the inert gas pressure around 1 to 2 psi.

Then, dissolve 50 mg of sodium iodide in 1 to 2 mL of water and add this to the first bulb of the purge vessel. Allow the solutions to mix for a few minutes. Adjust the gas flow rate to make the cells'pressure between 4 and 7 Torr.

Now, inject 5 mcL of sample solution into the purge vessel and then find the nitrite level on the computer screen. Please refer to the text protocol for measuring combined nitrate and nitrite level at 95 degrees Celsius. Three fumigation treatments of various fruits and vegetables demonstrate the damage done without the nitrogen gas flush prior to exposing the product to ambient air.

The top row is the control state. In the second row, nitric oxide fumigation was terminated properly with the nitrogen gas flush. The bottom row shows the results terminating the fumigation with an air flush, which often results in stains or discolorations.

24 hours after a 16-hour fumigation, the products were analyzed by residue analysis. For most products, there were no significant differences in NO3 and NO2 levels between the treatment that was terminated with the nitrogen flush and the control;however, when there was no flush with nitrogen prior to exposure to atmospheric gases, NO3 and NO2 levels increased in fumigated products. The most critical aspects of this fumigation procedures are to keep the fumigation chamber sealed air tight and to establish and maintain ultra-low oxygen condition, and also, the flush chamber with the nitrogen at its end.

It is important to remember that the length of time needed to establish ultra-low oxygen condition can vary drastically, depending on what kind of products and how much products to be fumigated. Different fresh products have different tolerance to nitrogen dioxide. This determines how much the fumigation chamber needs to be flushed with the nitrogen before opening the chamber to expose product to ambient air.

The procedure time here can also be modified. For example, the vacuum can be used in combination with the nitrogen flush to establish ultra-low oxygen conditions more efficiently. Please take in safety precautions in conducting nitric oxide fumigation.

Nitric oxide is not laughing gas. It is a potent fumigant.