Biology Safety - Cryogenic materials. The risks posed by using them

Introduction

Liquid nitrogen, oxygen, argon and helium and solid carbon dioxide are used in laboratory environments. In this Department we use only liquid nitrogen (LN) and solid carbon dioxide ('Cardice' or 'dry-ice'). They offer low temperature and a convenient means of storing volumes of gas that would otherwise be compressed into conventional cylinders. There are safety-related issues arising from the storage, transport and use of these compounds within the Department.
We also use compressed carbon dioxide in cylinders and there is a potential asphyxiant risk where these are used (e.g. in tissue culture rooms).

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	Cryogenic materials. The risks posed by using them
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Introduction Liquid nitrogen, oxygen, argon and helium and solid carbon dioxide are used in laboratory environments. In this Department we use only liquid nitrogen (LN) and solid carbon dioxide ('Cardice' or 'dry-ice'). They offer low temperature and a convenient means of storing volumes of gas that would otherwise be compressed into conventional cylinders. There are safety-related issues arising from the storage, transport and use of these compounds within the Department. We also use compressed carbon dioxide in cylinders and there is a potential asphyxiant risk where these are used (e.g. in tissue culture rooms).
Properties		Liquid nitrogen (N₂)	Solid carbon dioxide (CO₂)
Boiling/sublimation * point		-196^oC	-78.5^oC
Expansion ratio **		682	845
Relative density of gas (air=1)		0.967	1.48
* Liquid nitrogen boils to release gas, solid carbon dioxide sublimes directly to gas. ** Expansion ratio is the relative increase in volume when evaporating to gas (from liquid nitrogen or solid carbon dioxide)
Non-contact safety issues The air we breathe normally contains 20.9% oxygen by volume. When liquid nitrogen boils or carbon dioxide sublimes, the increase in the concentration of these gases will reduce the concentration of oxygen in the air. It is this fact which is often the major hazard when dealing with these materials, particularly when used in a confined space. There is a simple formula for calculating the oxygen concentration in a confined space (such as a cold-room or lift) during a worst-case spillage scenario. Breathing even moderately elevated carbon dioxide levels (2-4%) is extremely unpleasant and initiates the “gasp for air” reflex even in the presence of elevated oxygen. Conversely, depleted oxygen in the absence of an increase in carbon dioxide causes hypoxia which is extremely dangerous because the victim floats off into a euphoric sleepy state. We have evolved to react to increasing levels of carbon dioxide proportionately to oxygen depletion – we have rapid detection of increased CO₂ directly from the brain tissue itself. Oxygen receptors in the carotid bodies are extremely slow and only evoke physiological changes, i.e. no panic attack or asphyxiation response. Anesthetic effects only occur at about 14% CO₂ – you would need a massive sudden release into a small volume room, e.g. from a cylinder plug blowing – in effect anyone in there would run out gasping for air in a state of total panic before they were in any danger. We all have the best CO₂ detectors invented to date as a by product of being air breathing mammals. The use of inert gases in a confined space is quite another matter if oxygen is depleted in the absence of an increased CO₂ it is potentially fatal. If the available oxygen drops below 40mmHg (about 8% at normal barometric pressure) death would occur within 4 minutes. Having said that concern about leakage from LN Dewar flasks is often vastly overstated. The stuff does not boil off all at once – you would have to be in a well sealed room with very poor ventilation for a single 25l flask to cause any real problems." However, in October 1999 a lab technician died in an Edinburgh laboratory apparently as a result of oxygen deprivation. Read the BBC news report of the incident. Although in the Department we do not use liquid nitrogen on such a large scale the incident highlights the danger posed by asphyxiant gases. From Laboratory News August 2000; "MRC fined £25,000 in LN2 death. The MRC has been fined £25,000 after admitting responsibility for the death of an experienced laboratory worker. Mr James Graham had been using liquid nitrogen to freeze biological samples. Seven hundred litres leaked into the laboratory and evaporated, asphyxiating him. The MRC admitted that there was inadequate ventilation, that there was no safety device on the storage tank and that a warning alarm was not switched on. Ms Suffolk, a colleague, told Edinburgh Sheriff Court that she had gone into the room and heard a hissing noise. Mr Graham was collapsed on the floor, unconscious and frozen. Liquid nitrogen was streaming from a hose attached to the wall. She was able to turn off the supply and summon help before she too was overcome. MRC executive director Nick Winterton said: "We took professional advice in designing the liquid nitrogen store in 1997. Clearly the ventilation system was inadequate. I think at the time the dangers associated with a serious leakage of liquid nitrogen were not fully appreciated and the facility was not properly designed." The laboratory had no windows, apart form one set in the door, and the ventilation system was inadequate to cope with any big leak. There was no safety valve on the external tank to prevent excess LN2 from entering the room. There was a low oxygen monitor, but this was routinely switched off when transferring liquid nitrogen - otherwise, the court was told, it would have sounded continuously. The monitor could not be seen from outside the laboratory as it was blocked form view by freezers. The Sheriff, Iain MacPhail, had the power to levy an unlimited fine, but said that because the MRC was a non-commercial, state-funded body, it was not necessary to inflict a fine punitive to directors and shareholders. "
Effects and symptoms of oxygen depletion. In general, oxygen deficiency leads to a loss of mental alertness and a distortion of judgement and performance. THIS HAPPENS WITHIN A RELATIVELY SHORT TIME, WITHOUT THE PERSON'S KNOWLEDGE AND WITHOUT PRIOR WARNING.
21 ® 14%	Increasing pulse rate, tiredness
14 ® 11%	Physical movement and intellectual performance becomes difficult
11 ® 8%	Possibility of headaches, dizziness and fainting after a fairly short period of time
8 ® 6%	Fainting within a few minutes, resuscitation possible if carried out immediately
6 ® 0%	Fainting almost immediate, death or severe brain damage
Effects and symptoms of carbon dioxide enrichment. The UK has assigned an occupational exposure limit of 5,000 ppm (0.5%) over 8 hours and 15,000 ppm (1.5%) for 10 minutes. Carbon dioxide vapour is not truly inert. It is mildly toxic.
1%	Slight, and un-noticeable, increase in breathing rate
2%	Breathing becomes deeper, rate increase to 50% above normal. Prolonged exposure (several hours) may cause headache and a feeling of exhaustion
3%	Breathing becomes laboured, rate increases to 100% normal. Hearing ability reduced, headache experienced with increase in blood pressure and pulse rate
4 ® 5%	Symptoms as above, with signs of intoxication after 30 minute exposure and slight choking feeling
5 ® 10%	Characteristic pungent smell noticeable. Breathing very laboured, leading to physical exhaustion. Headache, visual disturbance, ringing in the ears, confusion probably leading to loss of consciousness within minutes
12%	Characteristic taste
10 ® 100%	Loss of consciousness more rapid, with risk of death from respiratory failure. Hazard to life increased with concentration, even if no oxygen depletion. Concentrations of 20-30% and above are immediately hazardous to life.
The gasping reflex is triggered by excess carbon dioxide and not by shortage of oxygen.
First aid. If there is any concern that rescuers could become overcome the fire brigade must be summoned immediately. Without placing yourself at risk, remove the affected person to fresh air and treat accordingly. If loss of consciousness has occurred at any time refer the person for medical attention.
Oxygen depletion calculation for a typical spillage scenario Calculate the volume (Vr m³) of the confined space Calculate the volume of the released gas (Vg m³) by multiplying the volume of the liquid nitrogen (in m³) or weight of solid carbon dioxide (in Kg) by the expansion ratio (682 for LN and 845 for CO₂). (1,000 litres = 1 m³) Calculate the volume of available oxygen (Vo m³) as 0.2095x(Vr-Vg) Calculate the % oxygen available to breathe as 100xVo/Vr Examples; The cold-rooms opening onto the corridors in Building 4 South have a nominal volume of 26.3 m³ (Vr). If 10 Kg of solid CO₂ (Vg=8,450 litres or 8.45 m³) evaporates slowly into this sealed room, then the oxygen concentration will be depleted (from 20.9%) to; 20.95x(26.3-8.450)/26.3 =14.2% Building 4 South lift has a nominal volume of 3.9 m³ (Vr) If 2 litres of LN (Vg=1,364 litres or 1.4 m³) is suddenly released from a pressurised vessel, the oxygen would be depleted to; 20.95x(3.9-1.4)/3.9 =13.4% (This calculation assumes instantaneous evaporation and mixing with the air.) Using this calculation, if the oxygen concentration can fall to, or below, 18% then action needs taking to minimise risks. As a consequence of this risk assessment we have prohibited the use or storage of liquid nitrogen or solid carbon dioxide in cold rooms or growth rooms and we have prohibited the accompanied transport of pressurised LN₂ containers in the lift. Additional risk factors for consideration The above calculation assumes good mixing so that the oxygen concentration is uniform throughout the room. This may well be the case in a room where the air is vigorously mixed, such as our cold rooms. However, where the oxygen deficiency comes from displacement by evaporated cryogenic materials in a room where air mixing is less vigorous there might be quite a marked vertical concentration gradient due to the temperature gradient (and the gas density in the case of CO₂). If someone collapses, for whatever reason, there could be a much higher concentration of the (colder) nitrogen or (colder and denser) carbon dioxide near the floor, meaning a lower oxygen concentration, and, therefore anyone unconscious on the floor could rapidly asphyxiate. Liquid oxygen may condense in containers of liquid nitrogen or vessels cooled by liquid nitrogen. This can be extremely hazardous because of the pressure rise on the slightest degree of warming above the boiling point of oxygen (-183°C) and the possibility of explosive reaction with oxidisable material.
Contact safety issues A simple temperature change across the skin from +35^oC to - 75^oC or -196^oC (or lower) is likely to cause damage. The enthalpy of vaporisation for ultracold gases is far lower than that of water and so a cryogenic burn may have a similar effect to that of a scald or, at worst, a fat or oil burn. However, first aid treatment of more severe cryogenic burns is different. Effects on the body As the living skin tissue is rapidly cooled local, transient, pain may be experienced. Affected areas can become pale yellow and waxy because local blood circulation closes down and skin lipids solidify. When cold burns thaw, intense pain can occur and, if the area affected is large, the injured person may go into shock. Contact with surfaces at cryogenic temperatures tends to cause the flesh to 'stick', and removal can cause the flesh to tear off. First aid The aim is to slowly raise the temperature of the affected area back to normal. For minor burns do not pull clothing away from the area but loosen any clothing that may restrict blood circulation and make the person comfortable. Place the affected area in tepid (<40^oC) water. The skin should gradually change colour, via blue, back to pink. Use a sterile wound dressing to protect the injury and get the patient to the casualty department of the Royal United Hospital in Bath. For major injuries send for an ambulance. Apply first aid measures as far as possible.
Storage The main issue to consider in the safe storage of cryogenic gases is ventilation. Due to the potential asphyxiant risks, liquid nitrogen and solid carbon dioxide must not be stored in the cold rooms or growth rooms in Building 4 South. Liquid nitrogen. Store only in a purpose-designed vessel. 'Dewar' is the generic term used for such a vacuum insulated non-pressurised vessel. They vary in volume and the type used here for decanting from are 25 litre containers known as 'onions'. Culture storage vessels are often 50 litre volume. Liquid nitrogen is delivered twice weekly and decanted directly into the 'onions' and pressurised containers. Temporary storage can be in small Dewar vessels of 1 or 2 litre capacity. However, any lid must be vented to avoid the build-up of pressure which would happen in a sealed vessel. Pressurised vessels are used where dispensing via a flexible hose is needed. Carbon dioxide. Pellets of solid CO₂ (or 'Cardice') are delivered in cardboard boxes. They are transferred to insulated containers. Transport Pressurised containers of liquid nitrogen must not be accompanied by anyone when being transported in the lift due to the potential risk of sudden release if a bursting disc failed. The 25 litre unpressurised containers can be moved either with the decanting trolley (if the 'onion' is fitted with side lugs, or trunions) or with a set of small wheels. Unpressurised containers of LN should not be carried up, or down, stairs. In the Building 4 South it is deemed safe to accompany them in the lift BUT ONLY DURING NORMAL WORKING HOURS.
Use Do not decant from liquid nitrogen storage vessels you are not confident in handling. Seek help from the technical staff. Only decant into dedicated insulated liquid nitrogen containers (Dewar or vacuum flasks). Do not seal the lids. Do not use other types of container (such as polystyrene 'igloos' or plastic ice buckets). Liquid nitrogen is extremely cold and there is a risk of freezing your fingers should you handle ultra-cold items. LN boils at -196^oC and the liquid is even colder. Personal protective equipment Safety spectacles are a minimum requirement when decanting LN₂. A full-face visor may be needed if vigorous boiling and consequent spalshing can be anticipated. Gloves. There is dispute over the advisability of wearing gloves while handling liquid nitrogen because there is a belief that gloves could fill with liquid and therefore prolong hand contact which would make burns more severe. If gloves are worn they should be loose fitting and easily removable. Cryogloves must be worn if you will be handling ultra-cold items. Lab coat or overalls are advisable to minimise skin contact, also, wear trousers over shoe/boot tops to prevent shoes filling in the event of a spillage.