Liquid Nitrogen Handling | Environmental Health and Safety

Purpose and Applicability

Liquid nitrogen is one of the cryogenic liquids commonly used in research labs.  As “cryogenic” means related to very low temperature, it is an extremely cold material.  It is liquefied under high pressure condition and can expand to a very large volume of gas.  This generic chemical safety guidance describes basic prudent safety practice for handling this chemical in research labs. The principal investigator (PI) or the lab manager is responsible for developing and implementing standard operating procedures (SOPs) for the purchase, storage, and safe handling of this chemical that are specific to the PI’s research.

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Hazards

Extreme Cold

The vapor of liquid nitrogen can rapidly freeze skin tissue and eye fluid, resulting in cold burns, frostbite, and permanent eye damage even by brief exposure.

Asphyxiation

Liquid nitrogen expands 695 times in volume when it vaporizes and has no warning properties such as odor or color.  Hence, if sufficient liquid nitrogen is vaporized so as to reduce the oxygen percentage to below 19.5%, there is a risk of oxygen deficiency which may cause unconsciousness. Death may result if oxygen deficiency is extreme.  To prevent asphyxiation hazards, handlers have to make sure that the room is well ventilated when using cryogens indoors.

Oxygen Enrichment

When transferring liquid nitrogen, oxygen in the air surrounding a cryogen containment system can dissolve and create an oxygen-enriched environment as the system returns to ambient temperatures. Since the boiling point of nitrogen is lower than oxygen’s, liquid oxygen evaporates slower than nitrogen and may build up to levels which can increase the flammability of materials such as clothing near the system. Equipment containing cryogenic fluids must be kept clear of combustible materials in order to minimize the fire hazard potential.  Condensed oxygen in a cold trap may combine with organic material in the trap to create an explosive mixture.

Pressure Buildup and Explosions

Without adequate venting or pressure-relief devices on the containers, enormous pressures can build upon cryogen evaporation. Users must make sure that cryogenic liquids are never contained in a closed system. Use a pressure relief vessel or a venting lid to protect against pressure build-up.

Handling

Prudent Safety Practices

• Liquid nitrogen should be handled in well-ventilated areas. 
• Handle the liquid slowly to minimize boiling and splashing. Use tongs to withdraw objects immersed in a cryogenic liquid - Boiling and splashing always occur when charging or filling a warm container with cryogenic liquid or when inserting objects into these liquids. 
• Do not transport liquid nitrogen in wide-mouthed glass Dewars or Dewars not protected with safety tape.
• Use only approved containers. Impact resistant containers that can withstand the extremely low temperatures should be used.  Materials such as carbon steel, plastic and rubber become brittle at these temperatures.  
• Only store liquid nitrogen in containers with loose fitting lids (Never seal liquid nitrogen in a container).  A tightly sealed container will build up pressure as the liquid boils and may explode after a short time. 
• Never touch non-insulated vessels containing cryogenic liquids. Flesh will stick to extremely cold materials. Even nonmetallic materials are dangerous to touch at low temperatures. 
• Never tamper or modify safety devices such as cylinder valve or regulator of the tank 
• Liquid nitrogen should only be stored in well-ventilated areas (do not store in a confined space). 
• Do not store liquid nitrogen for long periods in an uncovered container.   
• Cylinders and Dewars should not be filled to more than 80% of capacity, since expansion of gases during warming may cause excessive pressure buildup.

Personal Protective Equipment

Eye/face protection 

A full face shield over safety glasses or chemical splash goggles are recommended during transfer and handling of cryogenic liquids to minimize injuries associated with splash or explosion. 

Skin protection

Loose-fitting thermal insulated or leather gloves, long sleeve shirts, and trousers without cuffs should be worn while handling liquid nitrogen.  Safety shoes are also recommended while handling containers.
 
A special note on insulated gloves: Gloves should be loose-fitting so they are able to be quickly removed if cryogenic liquid is spilled on them. Insulated gloves are not made to permit the hands to be put into a cryogenic liquid. They will only provide short-term protection from accidental contact with the liquid.

Training

The PI is responsible for SOPs specific to use of this chemical in their lab. The PI/Lab Manger is responsible for the site specific and hands-on training for the use of this chemical in their lab. Training should be directly documented in the researcher’s lab notebook. On each day of training, both trainer and trainee should sign the lab notebook. 

Initially, researchers should perform the procedures with the PI or senior researcher present to observe the safe handling of this chemical. Review the reagent-specific safety data sheets (SDSs). Evaluate the hazards associated with the chemical procedure and experimental setup.

External Links

Part Three of our A Practical Guide to Planning a Cryogenic Storage Facility Series

For more LN2 Storage Tankinformation, please contact us. We will provide professional answers.

In our previous blog post, we uncovered key safety considerations and why monitoring your LN2 facility and storage units is imperative in ensuring the safety of your facility. In this part of our blog series, we lay out the key factors to consider when installing and maintaining LN2 equipment.

Installing and maintaining liquid nitrogen equipment can be a daunting task, but it’s important to understand the basics if you want your facility to run smoothly.

Installation and Setup

Installation and setup should be verified using manufacturer recommendations for electrical and LN2 supply as well as facility requirements. Your storage unit should undergo a set of qualification procedures to ensure that it is installed correctly and meets all specifications:

• Installation Qualification (IQ): Oversight and verification of every physical aspect of the equipment (materials, dimensions, pressure ratings) and components (operational parameters, accuracy, voltage)
• Operational Qualification (OQ): Testing each individual component, feature, and physical specification of the equipment
• Performance Qualification (PQ) or IQ/OQ/PQ: Testing the operational requirements of the equipment under real-world conditions

Electrical Power

While LN2-based cryogenic storage units are not dependent on electrical power for active cooling, it is required for alarms and monitoring. A backup power system with sufficient monitoring and alarm capabilities should also be in place for when the facility is unattended.

• LN2 Supply: The LN2 supply system should contain enough LN2 for at least 21 days of normal usage and replenishment intervals. It’s a good idea to have a backup supply system in case the off-site provider is unable to re-supply within this time frame. In addition, your bulk tanks or cylinders as well as LN2 supply volume and pressure should be verified regularly, and the LN2 connections should be routinely checked.
• Secondary Monitoring: When using secondary systems for monitoring temperature and LN2 levels, make sure to install, calibrate, and verify before introducing samples.
• Pressure Relief Valves: These devices are essential when working with LN2. Because nitrogen gas buildup can lead to an explosion, a risk mitigation plan must be in place. Relief valves must be present on all storage units and components that could trap LN2, and pressure relief valves should be piped to the outside, verified, and replaced regularly.
• Initial Fill: LN2 storage units must be filled with liquid nitrogen per manufacturer recommendations and stabilized up to 48 hours prior to introducing specimens. An inventory rack system should also be installed at this time.

Calibration

Instrument calibration is a key step in ensuring accurate readings. Calibrated instruments are more reliable than those without any adjustments, and they also provide better protection against errors due to changes over time. Records should include readings taken both before and after calibration. In addition, a record that includes calibration date, name of who is performing the calibration, name/serial number of the device used, and reference to the SOP should be included.

Verification

The installation and operation of cryogenic storage systems should be verified per manufacturer recommendations. For LN2-based systems, this includes installation qualification and components.

• Documentation: User manuals, certificates, maintenance logs, and factory acceptance documentation needs to be kept in a central location that is available to staff.
• Personnel: Staff contact information should be made available and kept in a known location.
• Training and SOPs: A training program that includes safety procedures, routine verification process, and emergency response plans should be in place for all staff members.
• Temperature Mapping: Temperature mapping of the storage unit is typically conducted by the manufacturer as part of the design qualification. To confirm the temperature within the unit, a secondary sensor should be used at the highest level that specimens can be stored.

Routine Maintenance

The importance of performing system maintenance cannot be underestimated. It is essential to be in close communication with manufacturers and service providers to develop appropriate maintenance and verification schedules.

• LN2 Storage Units: Daily or weekly manual verification of storage temperature, LN2 level, and usage is suggested. Be sure to keep records of manual verifications to watch for trends or changes in system performance.
• Condensation, Frost, and Ice Buildup: Maintaining a dry environment can reduce condensation, frost, and ice accumulation, which impacts performance and usability. To improve efficiencies and equipment life, be sure to routinely wipe surfaces and remove buildup.
• Thaw, Clean, and Dry: Periodic rounds of thawing, cleaning, and drying may be required to maximize the efficiency and lifespan of storage units. Precautions should be taken to ensure that samples are safely removed and stored securely in another unit during the process.
• Backup Power: Given that batteries have a limited shelf-life and should be replaced per manufacturer’s recommendations, it is important to verify backup power.
• Alarms and Alerts: All alarm systems should be routinely tested and verified. The SOP should outline the process for conducting system verification, and results should be documented and made available for audits.
• Steps, Handles, and Lids: Interface components should be routinely examined for deterioration and verified to ensure functionality. Lid gaskets should be inspected for sealing and venting and replaced if damaged, as this can result in inefficiencies.
• Plumbing: For auto-fill systems, plumbing should be regularly verified and replaced per manufacturer’s recommendations.

Summary

As the need for cryogenic storage grows in an organization, it’s important that stakeholders understand the value of proper installation and maintenance of LN2-based systems. By following the appropriate guidelines, repositories can ensure that these systems are operated safely and efficiently.

Interested in learning more about cryogenic storage and how to set up your own facility? Download our full guide.

References

1. Best Practices: Recommendations for Repositories (4th ed), Addendum 1: Liquid Nitrogen-Based Cryogenic Storage of Specimens. ISBER. .

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