Background
Ethidium bromide (3,8 diamino-5-ethyl-6-phenyl phenanthridinium bromide, dromilac, CAS #1239-45-8) , is a compound used in many Columbia University laboratories Ethidium bromide is available as a dark red, crystalline, non-volatile solid and is moderately soluble in water.  Since it fluoresces readily with a reddish-brown color when exposed to ultraviolet (UV) light and with increased brightness when bound to double stranded-DNA and single-stranded RNA, it is commonly used in gel electrophoresis applications for visualization of these molecules.  For these applications, liquid ethidium bromide solutions are incorporated into the electrophoresis gel as a dye for the DNA, RNA, or other molecules to be visualized.

Safety Considerations.
Ethidium bromide is mutagenic and moderately toxic and must be handled with care.  The powder form is considered an irritant to the upper respiratory tract, eyes, and skin.  Ethidium bromide is thought to act as a mutagen because it bonds in double stranded DNA, thereby deforming the molecule. This is believed to block or trip biological processes occurring on DNA.  Preparation of stock solutions and any operations capable of generating ethidium bromide dust or aerosols should be conducted in a fume hood to prevent inhalation. Nitrile gloves, a lab coat, and eye protection should be worn at all times, as with working with any hazardous material.

When working with ethidium bromide, minimize the potential for spills. Where practical, purchase
pre-mixed stock solutions from chemical manufacturers in lieu of preparing solutions. If solutions of ethidium bromide must be prepared, consider performing this process in a fumehood. Perform all processes that generate ethidium bromide dusts or mists inside the fume hood to minimize inhalation exposures.  Prevent accidents by transporting small quantities of ethidium bromide in secondary containment.

When an ultraviolet light source is used in work with ethidium bromide, added caution is required.
As a general rule, avoid exposing unprotected skin and eyes to intense UV sources.

Spill Response Procedures.
See “Surface Decontamination Procedures” below, for both liquid and solid material spills on surfaces.  Proper personal protective equipment (nitrile gloves, protective eyewear, and lab coat) must be worn during any spill response. 

Disposal Procedures.
Surprisingly, ethidium bromide is not specifically classified as a hazardous waste per EPA or NYSDEC.   however due to its potential to cause adverse health effects Columbia University requires ethidium bromide wastes, including contaminated personal protective equipment and spill clean-up debris, to be properly handled and disposed through the University’s Hazardous Waste Management program. Ethidium bromide disposal containers The following sections outline handling and disposal procedures for electrophoresis gels and buffer solutions containing or contaminated with ethidium bromide.

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Electrophoresis Gel Disposal
Gels containing ethidium bromide are a common waste stream at Columbia University.  All gels containing ethidium bromide are collected and disposed of through EH&S as chemical waste. EH&S prefers all gels to be collected in 5-gallon, open head buckets provided by EH&S.  The containers, when provided by EH&S, are pre-labeled as “NonHazardous Waste – Ethidium Bromide Gels & Debris”.  If you choose to collect gels in a different container, please inform EH&S so we can provide the proper label.  When the container is approximately 75 % full, submit an online Chemical Waste Pickup Request. 



Ethidium Bromide Solution Decontamination/Destruction
Buffer solutions containing ethidium bromide should be filtered, decontaminated, or destroyed using one of the methods below or collected as chemical waste.  Users are required to verify that buffer solutions do not fluoresce prior to drain disposal.

Aqueous buffer solutions containing ethidium bromide can be released down the drain after decontamination or destruction.   Any solution containing heavy metals, organic solvents, cyanides, sulfides, acids (pH<2), or bases (pH>12) must be disposed of as hazardous chemical waste. As with all hazardous waste, waste solutions cannot be diluted or neutralized for drain disposal.

Methods of Decontamination/Destruction

Commercially Available Filtration Kits
Filtering buffer solutions through a bed of activated carbon is a simple and effective method of removing ethidium bromide. The filtrate can then be drain disposed. One such kit is available from Q-Biogene, who offers the EtBr GreenBag Disposal Kit. The Green Bag is essentially a charcoal “teabag” which is placed in the ethidium bromide solution over night and removes up to 10 mg EtBr/bag. One kit (50 bags) has the capacity to remove 500 mg of ethidium bromide from solutions. After the solution has been treated, it may be poured down the drain and the bag can be disposed of as hazardous waste through EH&S.

Decontamination of Dilute Solutions (Up to 100 ug/ml)

Method 1 (Lunn and Sansone 1987)
1. Add 2.9 g of Amberlite XAD-16 for each 100 ml of solution up to 100 ug/ml ethidium bromide. Amberlite XAD-16, a nonionic, polymeric absorbent, is available from Rohm and Haas and sold by Sigma Chemical Company.
2. Store the solution for 12 hours at room temperate, shaking it intermittently.
3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain.
4. Seal the filter and Amberlite in a plastic bag, apply a completed Chemical/hazardous Waste label, and dispose of the bag as hazardous waste through EH&S.

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Method 2 (Bensaude 1988)
1. Add 300mg of powdered activated charcoal for each 100 ml of up to 10 ug/ml solution of ethidium bromide.
2. Store the solution for 1 hour at room temperature, shaking it intermittently.
3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain.
4. Seal the filter and activated charcoal in a plastic bag, apply a completed Chemical/hazardous Waste label and dispose of the bag as hazardous waste through EH&S.

Destruction of Concentrated Solutions (0.5 mg/ml) (Lunn and Sansone 1987)

1. Add sufficient water to reduce the concentration of ethidium bromide to 0.5mg/ml or less.
2. Prepare a fresh 5% hypophosphorous acid solution. The solution must be freshly diluted before use: 10 ml of hypophosphorous acid into 90 ml of water, stirred briefly. Use a chemical fume hood to prepare this solution.  Important: Hypophosphorous acid is usually supplied as a 50% solution, which is corrosive.  When working diluting any hazardous chemical, the appropriate personal protective equipment (eyewear, gloves, lab coat) must be worn.
3. Sodium nitrate solution, 0.5 molar, should be freshly prepared by dissolving 3.45 grams of sodium nitrite in water to a final volume of 100 ml. Use a chemical fume hood to prepare this solution.
4. For each 100 ml of ethidium bromide solution, add 20 ml of fresh 5% hypophosphorous acid and 12 ml of fresh 0.5 molar sodium nitrite.
5. Decontamination solution must be stirred for at least 20 hours at room temperature.
6. Bring the pH to neutral (pH = 7) with sodium bicarbonate. Discard the solution to the drain. As per RCNY Title 15, Chapter 19 Section 3 , no solution with a pH lower than 5 or greater than 11 can be drain disposed.

*Do Not Use hypochlorite (bleach) to treat ethidium bromide. Bleach treatment can produce mutagenic products and leave behind up to 20% of the original ethidium bromide.*

Surface Decontamination of Ethidium Bromide (Bensaude, 1988)

• Wipe up excess liquid with paper towels. Using fresh paper towels, soak the paper towels in ethanol then place paper towels over contaminated surface area.   Then sprinkle activated charcoal on the surface.
• Wipe up the charcoal/ethanol mixture with additional paper towels. Place the contaminated spill debris (including PPE) into a clear, plastic bag.  Seal the  debris bag, and affix a completed hazardous waste label.
• Submit for a hazardous waste pickup request online through EH&S.

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