Ethidium Bromide waste disposal in the Department of Biological Sciences :

Ethidium bromide: CAS #1239-45-8

other names include:
2,7-diamino-10-ethyl-phenylphenanthridium bromide
dromilac
homidium bromide
RD1572
novidium bromide
babidium bromide

Background:

Ethidium bromide (EB) is a red cationic fluorescent dye that may be used for, among other things, to visualize DNA and RNA and as a protein synthesis inhibitor. It is considered to be toxic and it is also a powerful mutagen having greater mutagenic activity than both N-methyl-N'-nitro-N-nitrosoguanidine (another mutagen) as well as benzo-(a)-pyrene (a known carcinogen) (ref 3). In addition to the mutagenic properties, it has been shown to have other effects on chromosomes and cell division in a variety of cell systems (ref 5). Because of these characteristics, waste material should not be disposed of in the sewer system or regular garbage. Rather, it should either be sent for incineration or treated to destroy the ethidium bromide. In our department, the greatest usage is probably for staining nucleic acids in electrophoresis gels and waste material includes solutions, gels and solids.

Solids:

• ethidium bromide powder: place container in a plastic bag, seal the top and dispose of through the Office of Environmental Health and Safety (EH&S).
• gloves, pipette tips, paper towels: if items contain only traces of EB, they can be disposed of in regular garbage. For material that is grossly contaminated (e.g. materials used to clean up a spill), place items in a plastic bag, seal, label contents and dispose of via EH&S.

Electrophoresis Gels:

• deposit directly into a yellow plastic garbage pail labelled with a Biohazard sign.
• be sure to label the pail as containing "ethidium bromide waste"
• don't bother lining the pail with a plastic bag as the pail and contents go into the incinerator (a bag just adds to the plastic load of the incinerator)
• do not overfill the pail. Send for disposal when about 90% full.
• leave the lid off during gel accumulation to allow the gels to dry
• pails are sold at the BioStores (Z207) and cost about $3.50 (July 2001)

Solutions:

Option 1 is viable if you only generate very small quantities of solution or have very concentrated solutions that might be difficult to process effectively.
Option 2A: is relatively easy to do but people should be aware that several methods have been proposed and they are not all totally effective. What you are trying to do is get rid of the mutagenic activity of the material, spectroscopic assays for the presence of EB may not accurately reflect destruction of mutagenicity.
Option 2B: is also fairly easy and greatly reduces the volume of material that has to be sent to EH&S.

Other notes:

• do not use any procedure that involves destruction of EB with household bleach as one published method has been reported to leave 20% of the original mutagenicity after the treatment.
• all these procedures have been tested for solutions containing EB in water, physiological buffers or CsCl2 with similar results.
• you can increase the sensitivity of spectroscopic detection of EB by adding DNA (10 µg/mL in final solution) to the sample (increases the fluorescent yield);
• the charcoal methods may be best for dilute solutions (<10 µg/mL) while the chemical destruction can accommodate higher concentrations (<500 µg/mL)
• methods for removal of EB using an ion exchange resin have been published but this is likely to be more costly than using charcoal as an adsorbent.
• the methods here are for treating 100 mL of solution containing EB. Adjust the amounts proportionately for larger volumes but keep the listed values for samples less than 100 mL.
• these procedures should be validated when first used to ensure they are working as expected. EB may be measured spectroscopically in a fluorometer but the mutagenic activity should also be assessed, especially if the procedures are modified.
• if gels are stained in a solution of ethidum bromide after they are run (rather than including EB in the gels and buffer), the volume of liquid that needs to be dealt with is greatly reduced. The stain solution can be reused many times, it can be refreshed by adding more EB, and this practice only adds a few minutes to the procedure (this point contributed by Neil Adames).

Materials and Reagents:

all prices in CDN$ (July 2001)

References:

1. Bensaude, O. 1988. Ethidium bromide and safety-readers suggest alternative solutions. Trends in Genetics 4(4):89-90.
2. Lunn, G. and E. Sansone. 1987. Ethidium bromide: destruction and decontamination of solutions. Analytical Biochemsitry 162: 453-458.
3. Quillardet, P. and M. Hofnung. 1988. Ethidium bromide and safety-readers suggest alternative solutions. Trends in Genetics 4(4):89.
4. Decontamination and destruction of ethidium bromide (EB). Appendix F in: Oklahoma State University Lab Safety Manual.
5. Summary of data for chemical selection ethidium bromide. 1994. National Institute of Health.

original: July26, 2001
revised:
BG McCashin
Department of Biological Sciences