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).
Solid carbon dioxide (CO2)
Expansion ratio **
Relative density of gas (air=1)
* 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
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
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
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,
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
"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.
|Increasing pulse rate, tiredness
|Physical movement and intellectual
performance becomes difficult
|Possibility of headaches, dizziness
and fainting after a fairly short period of time
|Fainting within a few minutes,
resuscitation possible if carried out immediately
|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.
|Slight, and un-noticeable, increase in breathing
|Breathing becomes deeper, rate
increase to 50% above normal. Prolonged exposure (several hours) may cause
headache and a feeling of exhaustion
|Breathing becomes laboured, rate
increases to 100% normal. Hearing ability reduced, headache experienced
with increase in blood pressure and pulse rate
|Symptoms as above, with signs
of intoxication after 30 minute exposure and slight choking feeling
|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
|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
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
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;
Building 4 South lift has a nominal volume of 3.9
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
(This calculation assumes instantaneous evaporation and mixing with the
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 +35oC to
- 75oC or -196oC (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.
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 (<40oC)
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.
The main issue to consider in the safe storage of cryogenic gases is
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
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
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.
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
- 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
- Liquid nitrogen is extremely cold and there is a risk of freezing
your fingers should you handle ultra-cold items. LN boils at -196oC
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.