Ethidium bromide and COSHH
The ethidium bromide problem
Ethidium bromide is very widely used to detect and quantify DNA/RNA, but is an extremely hazardous material. Under COSHH regulations we have a duty as users to eliminate or reduce risks and hazards wherever possible. Ready prepared solutions of Ethidium bromide reduce the risks associated with handling the solid when making up stock solutions, but the fact remains that Ethidium bromide has significant risks associated with it. These risks are:
R 23 Very Toxic by inhalation
R22 Harmful if swallowed
R 68 Possible risks of irreversible effects
Ethidium Bromide is also an intercalating agent and frameshift mutagen.
These risks require that the University has to put in place special measures in handling, containment and disposal of this product and all materials associated with it. These are Schedules 3.2 3.3 and 7.1 of the University hazardous substance policy control measures (UHSP/15/HS/00). (These were relevant in 2005 but may now have been superseded). In essence this means that ethidium bromide waste has to be disposed of by incineration, which is both expensive and environmentally damaging.
What are the advantages of using ethidium bromide?
It is cheap to purchase, but the hidden costs of disposal are very high.
* for costs see below
Are there any alternatives to ethidium bromide?
Yes. Invitrogen sell a DNA stain called SYBR Safe Green. Details can be seen on SYBR Safe Green can be used in exactly the same way as ethidium bromide, but gel running buffers and staining solutions can be disposed of safely down sinks, and gels can be disposed of without the need for incineration.Many people using real-time RT-PCR are already using SYBR Safe green PCR products.
Why change from ethidium bromide to SYBR Safe?
- SYBR Safe Green displays no acute oral toxicity and greatly reduced genotoxicity.
- It is better for the environment as it offers non hazardous waste status and clean water compliance. This product carries no risk phrases and requires no special handling storage or disposal protocols.
- Little or no on costs after purchase.
- Reduces waste disposal costs.
- Compliance with COSHH requirement to use the least hazardous product.
- SYBR Safe stained DNA can be excised from gels under non-UV blue light, which reduces UV-induced nicking and crosslinking of the DNA you want to clone or further manipulate- Invitrogen claim a dramatically improved cloning efficiency when this is done.
Disadvantages of SYBR Safe?
In a wordcost.It is more expensive to buy than ethidium bromide, but still only a relatively small proportion of the overall direct cost of a gel (agarose, running buffer, DNA ladder and gel documentation costs, charcoal filters, operator time to filter the waste running buffer, and bagging of solid waste for incineration). If you add in the incineration costs (which the School pays out of its budget), and the environmental costs of incineration, which are likely to increase, there is a very strong case for changing.
* for costings see below
Conclusions?
Ethidium bromide may appear to be a very inexpensive chemical but the purchase of consumables, the extra space required, together with monitoring, storage and disposal are all hidden costswhich should be acknowledged.
While we can reduce risks in using it by moving to pre-made solutions, ethidium Bromide is still an extremely hazardous chemical.
COSHH puts a burden on us to substitute less hazardous products whenever possible.
As COSHH regulations are updated there could be increased costs for the School for specialist disposal.
Ethidium bromide is widely used in the school at present, but SYBR Safe green is a safer substitute which gives comparable results. SYBR Safe Green in preliminary tests we have conducted has performed as well as ethidium bromide and has none of the hazards associated with ethidium bromide. It should be considered by all users as a safer alternative for general use.
Sceptical?
Massachusets Institute of Technology (MIT) has already made the change from ethidium bromide to Sybr Safe Green. MIT estimated they were spending >$10,000 per year disposing of ethidium bromide contaminated waste in 2005. For their rationale for change and the economic costs of incinerating ethidium bromide contaminated waste see:
Want to try SYBR Safe green?
Contact Barbara Russell in Tower lab 101 (46551). Barbara has samples of SYBR Safe which you can try- for free!
*Notes on comparative costs:
Ethidium bromide
Sigma supplied solution
Cat No E1510 – 10ml (10mg/ml)£ 31.50
This is usually diluted and kept as a stock solution of 0.5 mg/ml (1000 x solution)
10 ml of Sigma stock (10mg/ml) £3.15/ml
Diluted Sigma Stock (0.5mg/ml) 16p/ml
Costs for a 100ml agarose gel (final etbr concentration 0.5ug/ml) 1.6p
100l of 0.5mg/ml stock solution Cost per 100ml staining solution (0.5g/ml) 1.6p
Cost per 100ml staining solution (10g/ml) 32p
SYBR Safe
This product is supplied as
SYBR Safe 10,000 x concentrate in DMSO400l @ £36.10
SYBR Safe in TAE and TBE Buffer 1L @ £ 55.60
4 L@£136.20
Invitrogen recommend a number of protocols for using SYBR safe:
- Stock Concentrated SYBR Safe Green can be diluted from the 10,000 x concentrate to a 1x solution in water. Use 1l of 1x SYBR Safe/ml of stain solution. Stain gels for 30 minutes.
- Stock concentrate SYBR Safe Green can be incorporated into agarose gels (dilute 1: 1000). 1l/ml of the 10x stock is added to the agarose gel.
- The stock 10,000x concentrate SYBR Safe Green can also be added directly to any DNA sample. Dilute 10,000x to 100x (1: 100) in DMSO and then add 1lof the diluted SYBR Safe green to 9l of DNA sample (Clare Chemicals)
Costings below assume that the SYBR Safe Green concentrate is added directlyinto an agarose gel
Invitrogen recommendation
Dilute the 10,000x concentrate (1: 10,000) into gel running buffer before adding to powdered agarose. Mix well before heating. The final concentration of the dye in the gel should be equivalent to10l of SYBR Safe Green 10,000x stock for100ml ofagarose gel.
400l of 10,000xSYBR Safe green concentrate: £36.10 9p/l
Cost per 100ml agarose gel (10l of SYBR Safe Green 10,000x stock for 100ml of agarose gel) 90p
Bringing the cost of using SYBR Safe down
- Test gels using Bio-line Hyperladder 1 indicate that SYBR Safe Green could be used at considerably lower concentrations.
- In Test gels diluting the Sybr Safe Green 10,000 stock by 100 still gave good resolution with Hyper Ladder 1 bands.
- This would bring the cost of each gel down by a factor of 10.
- Invitrogen have discussed the option of Bioscience having a distribution point with considerable reductions if this product were supplied in bulk. This would give further savings
- A filter system can be supplied which gives enhanced signal for this product. Invitrogen representatives have looked at our imaging system and estimated the cost of such a filter system would be approximately £50