Ethidium bromide is strongly mutagenic and is irritating to the eyes, skin, mucous membranes and upper respiratory tract. It is suspected to be carcinogenic and teratogenic because of its mutagenicity, however, there is no direct evidence of either effect. Whenever possible, alternative stains should be used.

The following guidelines are in effect on Missouri State campuses for the deactivation and disposal of solid and liquid ethidium bromide wastes, and for the decontamination of equipment contaminated with ethidium bromide. Alternative protocol may be used if approved by the Director of Environmental Management.

Solid Waste

All solid wastes contaminated with ethidium bromide need to be disposed of through the Environmental Management Department. The wastes will be shipped off-site for incineration.

• Gel waste is to be collected in a bucket lined with two red plastic bio-hazard bags and separated from all other solid waste
• Solid waste including contaminated gloves, equipment and debris, test tubes, paper towels, etc. must be placed in a medical waste bag or red bio-hazard bag for incineration
• No liquids should be placed in the containers with the solid wastes.

Liquid Waste

Containers with liquid ethidium bromide should be kept closed at all times except when adding or removing the waste. Solutions containing less than 5 mg/L ethidium bromide can be poured down the drain with copious amounts of water. Small amounts (100 ml or less) of solutions containing greater than 5 mg/L ethidium bromide may be deactivated, neutralized and poured down the drain with copious amounts of water. Deactivation may be confirmed using UV light to detect fluorescence under both "long" and "short" UV wavelengths. Larger amounts of ethidium bromide solution will be disposed of through the bio-waste disposal company. Missouri State personnel may choose whether or not they wish to treat the solution or collect it for disposal by the Environmental Management Department. If it is disposed of into the sanitary sewers, a Conditionally Acceptable Wastewater Discharge Application form must be completed and submitted to the city through the Director of Environmental Management.

The recommended treatment method is the Lunn and Sansone Method although commercially available products may be used. Using destaining bags and charcoal filtration requires additional equipment and/or generates a material that must be disposed of as a hazardous waste.

Lunn and Sansone Method

For each 100 ml of ethidium bromide solution:

1. Add 20 ml 5% hypophosphorus acid.
2. Add 12 ml of 0.5 M sodium nitrate.
3. Stir briefly and let stand for 20 hours.
4. Adjust pH to 5-9 using sodium hydroxide or sodium bicarbonate.
5. Pour down drain and flush with copious amounts of water.

Armour Method

This is the simplest method, but is somewhat controversial. One study found traces of mutagenic reaction mixtures using this method. This method, however, was recommended by Springfield Public Works personnel.

1. Combine equal amounts of ethidium bromide solution and household bleach.
2. Stir constantly for four hours or let sit for 2-3 days.
3. Adjust pH to 5-9 with sodium hydroxide or sodium bicarbonate.
4. Pour down drain and flush with copious amounts of water.

Commercially available products, like "Destaining Bags" provide an alternative method of treatment for solutions. The destaining bags are simple to use and inexpensive. (Available through companies such as GTS, Inc and Omega Bio-Tek).

1. Drop a destaining bag into your solution,
2. Periodically swirl it around a few times,
3. Let it stand overnight.
4. In the morning, remove the bag and collect for disposal by the Environmental Management Department.
5. Perform UV check of the solution. If it no longer fluoresces and no other hazardous chemicals are present, pour the solution down the drain.

EtBr Greenbag Kit

The EtBr GREENBAG™ Disposal Kit is a commercially-available product that consists of a "teabag" containing activated carbon which is placed into the waste solution to adsorb the ethdium bromide. The solution can then be disposed down the sanitary sewer, and the GREENBAG is to be subsequently disposed of as solid hazardous waste. Contact Environmental Management for assistance in managing this waste. This product is currently available from several suppliers, including VWR and MP Biomedicals.

Charcoal Filtration

Filtering the aqueous ethidium bromide waste solutions, free of other contaminants, through a bed of activated charcoal is a relatively simple and effective method for removal of ethidium bromide. Schleicher and Schuell and VWR supply a commercial filter funnel kit that uses a packaged charcoal disk. This is particularly useful for labs that generate large amounts of solutions at a time.  The kit is available through Schleicher and Schuell or VWR.

1. Filter the ethidium bromide solution through the charcoal filter.
2. Pour filtrate down the drain.
3. Place charcoal filter in a sealed bag (e.g., zip-lock) and place in biohazardous waste container for incineration.

The charcoal disks are graduated for easily tracking the amount of aqueous solution calculated for a fixed quantity of ethidium bromide residue (filtering up to ten liters per filter).

Decontamination of Equipment

Glass, stainless steel, and other equipment may be decontaminated with the following procedure. Proper personal protective equipment, including goggles and latex or nitrile gloves, must be worn.

1. Wash the contaminated equipment with a paper towel soaked in the decontamination solution as described in the Lunn and Sansone Method.

   The pH of the decontamination solutions is 1.8. If this is too corrosive for the surface to be decontaminated, wash with a paper towel soaked in water instead.

2. Wash the surface five times with paper towels soaked in water using a fresh towel each time.
3. Soak all towels 1 hour in decontamination solution.
4. Neutralize the decontamination solution by adding sodium bicarbonate until the evolution of gas ceases.
5. Test the decontamination solution for the presence of ethidium bromide using UV light to detect fluorescence as described in the Liquid Waste section.
6. Discard the decontamination solution as a nonhazardous liquid, pouring it down the drain and flushing with copious amounts of water. The paper towels can be disposed as solid wastes by placing them in a plastic bag other than a red bag, then disposing of them into the trash. Be certain there are no free liquids in the bag. It may be necessary to add additional towels or an absorbent.

Note. The practice of oxidizing ethidium bromide with household bleach has been shown to be inefficient and may produce additional hazardous compounds, so it should be avoided.

For spills, use absorbent or a spill pillow to soak up aqueous ethidium bromide. Carefully clean up solid ethidium bromide to avoid creating dusts. Place in a sealable container and contact the Environmental Management Department for disposal.

The Environmental Management Department should also be contacted for the disposal of unused ethidium bromide (6-8334 or EnvironmentalManagement@missouristate.edu).