Ethidium Bromide Handling and Disposal

34. (2003)

An incident occurred recently where a student who was pouring distilled water over an ethidium bromide (EtBr) gel was splashed on the face with the solution. The student was not wearing safety glasses at the time. The procedure in the laboratory did not take into account the hazardous nature of ethidium bromide and there was uncertainty as to the correct disposal procedure. This situation has highlighted the importance of the correct treatment and disposal of ethidium bromide.

Handling

Ethidium bromide (3,8-diamino-5ethyl-6-phenylphenanthridinium bromide) is moderately toxic and is a mutagen and possible carcinogen and teratogen. It is necessary to wear personal protective clothing (gloves, splash resistant eyewear with side shields, laboratory coat and covered footwear) when handling EtBr.

Disposal

Disposal of EtBr requires a process of decontamination of liquid solutions. The easiest method of decontamination is to adsorb the EtBr onto activated charcoal. This can be done in a couple of ways:

• Add 3 grams of powdered activated charcoal to 1 litre of EtBr liquid waste2. Concentration should be no greater than 10 mg/L. Leave to stir overnight then filter the solution. The activated charcoal and filter are then sent to be incinerated, and the liquid is safe to be poured down the sink.
• Alternately, Bio-101 produces 'tea-bags' of activated charcoal, called the Ethidium Bromide 'Green Bag' Disposal Kit (supplied by Integrated Sciences, catalogue number 2350-200). The kit contains 50 bags, each one filters 10 mg of EtBr, and will be enough to satisfy the requirements of most laboratories for a year.

Each of these methods can be checked for satisfactory decontamination of the solution by adding some DNA, waiting 15 minutes then checking for fluorescence under a UV light. If fluorescence occurs the solution requires further decontamination.

It is required by the University that all EtBr liquid waste, including dilute buffer solutions, be decontaminated before being disposed of to sewer.

Note: EtBr waste should never be autoclaved.

Gels, gloves, pipette tips and other solids contaminated with EtBr should be sent for incineration.

Where possible laboratories should buy in the stock solution already made up, or EtBr in tablet form to avoid handling the chemical in the more hazardous powder form.

The University would expect all areas to have written protocols for the handling and disposal of ethidium bromide waste.

Spills

To clean up a spill of EtBr follow these steps:

1. Absorb the majority of the liquid with paper towel.
2. Wet the surface with ethanol (ethanol dissolves EtBr), after ensuring no ignition sources are present.
3. Sprinkle activated charcoal on the wet surface.
4. Wipe up the paste with paper towel, place in a plastic bag and dispose of to incineration.

Use of Bleach

It has been one practice to inactivate EtBr solutions by treatment with bleach. This method is not recommended as it can result in incomplete destruction of the mutagenic properties of the EtBr, and potentially produces more mutagenic compounds1. There has also been one report of an explosion occurring after inactivation with bleach was carried out in a Winchester bottle containing acid residue4. The lid was closed on the bottle overnight and chlorine gas built up pressure, which caused the bottle to explode. For these reasons, inactivation using bleach is not considered to be an acceptable procedure.

References

1. Lunn, G., and E. Sansone. 1987. Ethidium bromide: destruction and decontamination of solutions. Analytical Biochemistry 162:453-458.
2. Bensaude, O. Ethidium Bromide and Safety- Readers Suggest Alternative Solutions. Trends Genet. 4:89; 1988.
3. Quillardet, P.; Hofnung, M. Ethidium Bromide and Safety ReadersSuggest Alternative Solutions. Trends Genet. 4:89.
4. http://www.safety.uwa.edu.au/hazard_alerts/ethidium_bromide_disposal

For further information contact:
OHS Officer, e-mail: OHS.Officer@anu.edu.au