NOTE: Nitric oxide fumigation of fresh products starts by establishing ultralow oxygen conditions in fumigation chambers, followed by injection of NO and holding the fumigation chambers at certain temperatures for the duration of a specific treatment, and then is terminated by flushing with N₂ to dilute NO prior to opening the fumigation chambers as illustrated (Figure 3). For measurements of NO₂ in the head space of fumigation chambers and nitrate and nitrite in liquid samples using the Model 405 nm NO₂/NO/NOx monitor and NOA nitric oxide analyzer, please refer to the user manuals from the manufacturers for detailed operation procedures.

Caution: Nitric oxide is a strong oxidizing agent and will react with oxygen spontaneously to produce nitrogen dioxide. Both nitric oxide and nitrogen dioxide are toxic. Please refer to their MSDS for safe handling and use. For personal safety, all steps of small scale fumigation experiments involving handling and potential exposure to NO or NO₂ should be carried out in a fume hood. A personal NO₂ alarm should be used to conduct large scale NO fumigation experiments.

1. Preparation of Materials and Instruments

1. Instruments, parts, and materials needed for NO fumigation
   1. Make a foil bag with a tubing outlet for NO.
      1. Seal the opening of a foil bag around a Polytetrafluoroethylene (PTFE) tube using a heat sealer.
      2. Then use epoxy glue to seal the seams and joint around the Polytetrafluoroethylene (PTFE) tube to produce the foil bag.
      3. Add a stopcock at the end of the tube.
         NOTE: Foil bags with tubing are not commercially available. But they can be made easily in the lab with foil bags from commercial sources using a heat sealer.
         NOTE: Nitrogen: Regular industry nitrogen in compressed cylinders has a purity of ≥99.99% and is suitable for NO fumigation. Two or more cylinders with regulators can be set to have different outlet pressures and connected together. The cylinder with the higher outlet pressure will be used up first before the cylinder with the lower outlet pressure will be used. This will be useful in large fumigation tests.
   2. Airtight fumigation chambers.
      NOTE: Airtightness is critical to NO fumigation because NO will react with O₂ leaked into the chamber. This will reduce available NO for pest control and also produce NO₂ which may damage fresh products.
      1. Glass jar chambers: Grease the rim of the lid lightly with petroleum jelly. Then seal the jar with the lid that has two outlets after loading objects such as insect infested products into the jar.
         NOTE: Each lid of the jar has two outlets and one of the outlets has a plastic tube extended to the bottom of the jar to increase the efficiency of air replacement.
      2. Chambers made of pressure cookers: Grease the rim of the chamber with petroleum jelly. Load products and insects in the chamber and seal it with the lid.
      3. Large fumigation chambers: Grease the gasket lightly with petroleum jelly. Then load products in the chamber. Close the door. Tighten the clamps if necessary to maintain an airtight seal.
         NOTE: NO gas is highly volatile, so there is no need to have a fan in a fumigation chamber to keep air in the chamber mixed.

2. Establishment of ULO Conditions in Fumigation Chambers

1. Connect a chamber to the N₂ line and an O₂ analyzer.
   NOTE: A T-connector with one end goes to the analyzer and the other end equipped with a one-way check valve can be used to release air to avoid high flow to the O₂ analyzer.
2. Release N₂ through a flowmeter to flush the chamber to remove oxygen.
3. Reduce N₂ flow rate to 0.5 – 1 L/min when O₂ level is close to 30 ppm.

3. Injection of NO Gas

1. Fill the foil bag with NO gas.
   1. Fill the bag with N₂ first and then vacuum the air out to wash out O₂ from the bag.
   2. Then release NO gas into the bag in a fume hood.
   3. Hang the bag in a fume hood to be used for NO fumigation.
      NOTE: After prolonged usage, the bag can become degraded and the tubing can become brittle due to the corrosive effects of NO₂. So, the bags will need to be replaced periodically.
2. Inject NO into fumigation chambers.
   1. Wash the syringe and attached tubing with N₂ to flush out O₂.
   2. Take a NO sample from the NO foil bag and inject it into fumigation chambers.
   3. After injection, flush the syringe and attached tubing with N₂.
   4. Place fumigation chambers at 2 °C for the duration of the fumigation treatment.

4. Measure NO Concentration in a Fumigation Chamber

NOTE: NO concentrations in fumigation for pest control may range from 2,000 ppm (0.2%) to 50,000 ppm (5%). This range is "out of range" of current NO monitors. But, NO levels can still be measured in diluted samples or by using a dilution device.

1. Small chamber fumigations
   1. Dilute air samples from treatment jars at the end of fumigation:
      1. Establish ULO conditions at ≤30 ppm O₂ in jars.
      2. Take gas samples from treatment jars to inject them into the ULO jars.
   2. Measure NO and NO₂ levels in the diluted samples by circulating the air through a flue gas monitor.
2. Large chamber fumigations: The procedures are illustrated in Figure 4.
   1. Set up a dilution system.
   2. Measure NO and NO₂ levels.
      1. Turn on the flue gas monitor and flush it with N₂.
      2. Turn on the sample gas flow to measure NO and NO₂ levels.
      3. Finish the measurement by turning off the sample gas flow.

5. Terminate NO Fumigation

1. Fumigation of insects only
   1. Place fumigation chambers in a fume hood.
   2. Open the chambers.
   3. Retrieve insects for mortality evaluation.
      NOTE: Insects are typically held in an environmental chamber overnight after fumigation to allow all live insects to recover before being scored for mortality.
2. Fumigation of fresh products
   1. Move fumigation chambers into a fume hood (for small chambers).
   2. Flush the fumigation chambers with N₂ to allow a specific number air exchange.
   3. Monitor NO level at the exhaust port.
      NOTE: The flue gas monitor can be used to monitor NO levels during the N₂ flush. Typically, we flush fumigation chambers to reduce the NO level below 200 ppm before opening the chambers to ambient air.
   4. Retrieve insects for mortality evaluation (if insects are included).
   5. Store fumigated products for residue analysis and post-treatment quality evaluation.
      NOTE: Allow fumigated products enough time in the fume hood for NO and NO₂ to dissipate before moving them for storage. Fumigated products are usually stored at a low temperature together with controls in a cooler for a certain period before being evaluated for postharvest quality and possible injuries.

6. Residue Analysis

1. Nitrogen dioxide (NO₂) measurement using a 405 nm NO₂/NO/NO_X monitor
   1. Turn on and allow the 405 nm NO₂/NO/NO_X monitor to warm up for 20 – 30 min.
   2. Close the fumigation chamber containing the product.
      NOTE: After fumigation, fumigation chambers were open and placed at a certain temperature to allow NO₂ to dissipate. At the time when NO₂ release is measured, seal the chamber airtight with a lid with two ports equipped with stopcocks. A temperature of 20 °C was used in the procedure demonstration.
   3. Connect the NO₂ monitor to the chamber to circulate the air through the NO₂ monitor.
   4. Immediately start logging data on the NO₂ monitor and collect data for 1 min.
   5. Disconnect the chamber from the monitor and keep the chamber sealed.
      NOTE: Data logging can be started either via MENU -> Dat -> Log on the NO₂ monitor, or via the graphing Software on a computer.
   6. Keep the sealed chambers at 20 °C for 1 h, then repeat the data collection step.
      NOTE: The time interval can be adjusted depending on the release rate of NO₂ from fumigated products.
   7. Calculate the difference between the two NO₂ concentrations and convert the data to mg/kg/h.
2. Nitrate and nitrite measurements with a GE Sievers 280i NO Analyzer
   NOTE: Please refer to the manual of the manufacturer and the paper²¹ by Yang and Liu (2017) for detailed information.
   1. Sample preparation
      1. Homogenize product samples in a blender.
      2. Transfer 15 g of the homogenized sample from the blender into a vial.
      3. Add 100 mL of distilled H₂O to settle for 10 mins in the vial.
      4. Filter the sample and store the filtered solution at 2 °C until use.
   2. Reducing agent preparation for nitrate measurement with nitric oxide analyzer
      NOTE: Please refer to the manual of the manufacturer for detailed information.
      1. Add 0.8 g of vanadium chloride (VCl₃) in a flask.
      2. Slowly add 100 mL of 1 M hydrochloride acid (HCl) in the flask with the VCl₃, cap the flask, and swirl several times.
      3. Filter the solution using filter paper and a funnel and seal the filtered solution bottle with aluminum foil and store it in a refrigerator.
   3. Measurement of both nitrate and nitrite with nitric oxide analyzer
      NOTE: Please refer to the manual of the manufacturer for detailed information.
      1. Preheat the water bath to 95 °C. Add 4 – 6 mL of nitrate reducing agent into a purge vessel and adjust the inert gas flow rate to a proper level.
         NOTE: The inert gas was He. N₂ gas can also be used.
      2. Inject 5 µL of sample solution using a syringe into the purge vessel.
      3. Proceed to the next sample injection when the sample peak is finished.
   4. Nitrite measurement with nitric oxide analyzer
      NOTE: Please refer to the manual of the manufacturer for detailed information.
      1. Adjust the valve on the purge vessel to have 1 – 2 psi pressure for the inert gas.
      2. Add 4 – 6 mL of concentrated acetic acid to fill the first bulb of the purge vessel.
      3. Weigh 50 mg of sodium iodide (NaI) and dissolve it in 1 – 2 mL H₂O.
      4. Add the NaI solution to the purge vessel and allow mixing for 1 – 2 minutes.
      5. Increase the inert gas flow rate to a proper level.
      6. Inject 5 µL of sample solution using a syringe into the purge vessel.
      7. Proceed to the next sample injection when sample peak is finished.

7. Postharvest Quality Evaluation of Fruit and Vegetables

NOTE: Product injuries from NO fumigation may show up immediately after fumigation (Figure 5). However, product quality is usually evaluated after 1 – 2 weeks of post-treatment cold storage. Symptoms of injuries will progress over time and can be better identified in quality evaluation. Procedures for evaluating different fresh products may differ substantially. Only procedures for evaluating lettuce quality are demonstrated here as an example using established procedures⁵.

1. Remove lettuce from cold storage two weeks after fumigation. Remove wraps and inspect surfaces for stains and discoloration for all treatments including controls.
2. Score and record external visual quality for all treatments based on established procedures⁸.
3. Cut lettuce into halves and inspect any stains and discolorations for all treatments.
4. Score and record internal visual quality scores for all treatments.