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Laboratory Safety Manual

May 2012  

Appendix F
Decontamination & Destruction of Ethidium Bromide (EB)


Ethidium Bromide is also known as:

  • 2,7-DIAMINO-10-ETHYL-PHENYLPHENANTHRIDINIUM BROMIDE

  • 3,8-DIAMINO-5-ETHYL-6-PHENYLPHENANTHRIDINIUM BROMIDE

  • 2,7-DIAMINO-9-PHENYL-10-ETHYLPHENANTHRIDINIUM BROMIDE

  • 2,7-DIAMINO-9-PHENYLPHENANTHRIDINE ETHOBROMIDE

  • DROMILAC

  • HOMIDIUM BROMIDE

  • RD 1572

  • NOVIDIUM BROMIDE

  • BABIDIUM BROMIDE


A. Principles of Decontamination and Destruction

Ethidium Bromide is a powerful mutagen that is widely used in laboratories for visualizing nucleic acids. It may be harmful by inhalation, ingestion, and skin absorption and should be handled only when wearing rubber gloves and non-vented chemical goggles with work being done in a chemical fume hood. It is also irritating to the mucous membranes and respiratory tract.

Several methods have been published for the chemical destruction of EB involving treatment with bleach, hypophosphorus acid and sodium nitrate, and potassium nitrate. Decontamination products from these procedures, however, can also be mutagenic. For example, the projects of bleach decontamination show 20% mutagenic activity of the original EB, products of the H3PO4/NaNO2 procedure show 0.6% mutagenic activity, and KMnO4 procedure shows 0.03%. This information clearly demonstrates that the commonly used technique of bleach decontamination is not the best choice. Because potassium nitrate will stain most materials it contacts, the best choice for destruction or decontamination is the sodium nitrate procedure. The reagents (sodium nitrate and a dilute solution of hypophosphorus acid) are inexpensive and relatively non-toxic.

In the laboratory, EB might be encountered in a variety of solvents including EB dissolved in water, TBE buffer, MOPS buffer, and cesium chloride solution. The method to destroy EB in these solvents is based on a decontamination procedure that was previously published. Lunn and Sansone (1989) conclude that the recommended procedure removes the amino groups from the EB and it may cleave the N-containing ring. The only problem encountered was the failure to add enough hypophosphorus acid to reduce the pH sufficiently (pH<3, approximately) which caused the reaction to fail. Although the procedure was tested only for the solvents listed above, Lunn and Sansone did not see a reason why the method should not work for other buffers, provided that sufficient hypophosphorus acid is added. Validation of the destruction, however, by lack of fluorescence and, preferably, lack of mutagenic activity is, of course, essential.

Hypophosphorus acid is corrosive and should be handled carefully. If your laboratory uses any of these chemicals, call Environmental Health & Safety (EHS) for copies of the Material Safety Data Sheets if you do not have them.

Any towels, gloves, or other materials that are used during the handling of EB should be disposed of carefully as solid waste. Empty containers which once held EB are also considered to be solid waste when empty. Only paper towels or other materials that are grossly contaminated, such as from a spill clean-up, should be decontaminated. The decontamination should be validated through the use of a fluorescent lamp. It should be noted that the fluorescent lamp procedure only determines fluorescent compounds (such as EB) but that the EB is readily changed into non-fluorescent but still toxic compounds. Accordingly, Lunn and Sansone recommend periodic testing of reactions for mutagenicity, if possible. If a source is located either to test the mutagenicity of the decontaminated solutions, or to further validate decontamination procedures, EHS will notify laboratories.

EB can also be determined by thin-layer chromatography using silica gel plates eluted with 1-butanol:acetic acid:H2O (4:1:1).


B. Safety Considerations

The decontamination mixture gives off a small amount of nitrogen dioxide when initially mixed, and should be used in a chemical fume hood. A full description of the recommended procedures for destruction and decontamination of EB follows. Please call EHS at extension 4-7241 with questions regarding these procedures.

The following descriptions are designed to give a sufficiently complete guide to the destruction methods available in order to allow one to implement them successfully. The user may wish to consult other sources cited in the literature to determine the exact reaction conditions, limitations, and hazards. In some cases, more than one procedure is listed. In these instances, all the procedures should be regarded as equally valid unless restrictions on applicability are noted.


C. Destruction and Decontamination Procedures

The following information is largely taken from Destruction of Hazardous Chemicals in the Laboratory:

  1. Destruction of EB in Aqueous Solution

    • Dilute the solution, if necessary, so that the concentration of EB does not exceed 0.5 mg/mL. For each 100 mL of EB in H2O, MOPS buffer (see below), or 1 g/mL cesium chloride solution, add 20 mL of 5% hypophosphorus acid solution and 12 mL of 0.5 M sodium nitrite solution. Stir briefly and allow to stand for 20 hours. Neutralize with sodium bicarbonate (NaCO3), check for completeness of destruction, and discard the solution.
    • The hypophosphorus acid solution is prepared by adding 10 ml of the commercially available 50% solution to 90 ml of water and stirring briefly. It is advisable to prepare the hypophosphorus acid solution and the sodium nitrite solution (34.5 g/liter) fresh each day. Even if less concentrated solutions are to be decontaminated, it is best to add the same volume of hypophosphorus acid and sodium nitrite solutions to ensure the completeness of destruction. For more concentrated solutions, the volumes should be increased pro rata.
    • Caution! Ozone is an irritant. This reaction should be carried out in a properly functioning chemical fume hood. Dilute the solution, if necessary, so that the concentration of EB in H2O, Tris buffer, MOPS buffer, or cesium chloride solution does not exceed 0.4 mg/mL. Add hydrogen peroxide (H2O2) solution so that the concentration of H2O2 in the solution to be decontaminated is 1%. Pass air containing 300-400 ppm of ozone (from an ozone generator) through the solution at a rate of 2L/min. The red solution will turn light yellow. The destruction process typically takes 1 hour. Check the reaction mixture for completeness of destruction and discard it. Degrade residual ozone by making the reaction 1 M in sodium hydroxide.

  2. Decontamination of EB in Aqueous Solution

    Dilute the solution, if necessary, so that the concentration of EB does not exceed 0.1 mg/mL. For each 100 mL of EB in H2O, TBE buffer (see below), MOPS buffer (see below), or cesium chloride solution add 2.9 g of Amberlite XAD-16 resin. Stir for 20 hours, then filter the mixture. Place the beads, which do not contain EB, with the hazardous solid waste. Check the liquid for completeness of decontamination and discard it. An alternative procedure for solutions that are more concentrated than 0.1 mg/mL is to increase the relative amount of resin. This procedure should be fully validated before employing it on a routine basis.

  3. Decontamination of Equipment Contaminated with EB

    • Wash the equipment once with a paper towel soaked in a decontamination solution consisting of 4.2 g of sodium nitrite and 20 mL of hypophosphorus acid (50%) in 300 mL of H2O. Then wash five times with wet paper towels using a fresh towel each time. Soak all the towels in decontamination solution for 1 hour. Check for completeness of decontamination and discard it. Make up the decontamination solution just prior to use.
    • If the decontamination solution (pH 1.8) is felt to be too corrosive for the surface to be decontaminated, then use six H2O washes. Again, soak all towels in decontamination solution for 1 hour before disposal. Glass, stainless steel, Formica, floor tile, and the filters of transilluminators have been successfully decontaminated using this technique.
    • No change in the optical properties of the transilluminator filter could be detected even after a number of decontamination cycles using the decontamination solution.

  4. Decontamination of EB in Isopropanol Saturated with Cesium Chloride

    Dilute the solution, if necessary, so that the concentration of EB in the isopropanol saturated with cesium chloride does not exceed 1 mg/mL. For each volume of EB solution, add 4 volumes of a decontamination solution consisting of 4.2 g of sodium nitrite and 20 mL of hypophosphorus acid (50%) in 300 mL of H2O and stir the mixture for 20 hours. Neutralize with NaCO3, test for completeness of destruction, and discard it.

  5. Decontamination of EB in Isoamyl Alcohol and 1-Butanol

    • Dilute the solution, if necessary, so that the concentration of EB in the alcohol does not exceed 1 mg/mL. For each volume of EB solution, add four volumes of a decontamination solution consisting of 4.2 g of sodium nitrite and 20 mL of hypophosphorus acid (50%) in 300 mL of H2O and stir the two-phase mixture rapidly for 72 hours. For each 100 mL of total reaction volume, add 2 g of activated charcoal and stir for another 30 minutes. Filter the reaction mixture, neutralize with NaCO3, and separate the layers. More alcohol may tend to separate from the aqueous layer on standing. Test the layers for completeness of destruction and discard them. It should be noted that the aqueous layer contains 4.6% of 1-butanol or 2.3% of isoamyl alcohol. Discard the activated charcoal with the solid waste.
    • This procedure has been tested in three separate experiments for both isoamyl alcohol and 1-butanol. In one experiment, one plate (TA 1530 with S9 activation) indicated significant mutagenic activity. The number of revertants was 2.6 times background. All the other plates for this experiment and all the other experiments showed no significant mutagenicity. For comparison, untreated EB solutions were between 39 and 122 times background, depending on the solvent and tester strain.

  6. Decontamination of Ethidium Bromide Spills

    Given the wide usage of EB in laboratories, spills may be expected to occur, e.g., on benches, in fume hoods, or on the floor. Another problem that may occur is the build-up of EB residues on the surfaces of equipment such as centrifuges. Transilluminators are used to view the gels which contain DNA stained with EB, and residues of EB may build up on the large UV filter which forms the top surface of these units. Any decontamination method should not, of course, damage the optical surface. The decontamination solution described below is less corrosive than potassium permanganate in hydrochloric acid and does not appear to damage surfaces of transilluminator filters and Formica. Another method using activated charcoal and ethanol may leave activated charcoal on optical surfaces. The method described here does not appear to leave residues on nonporous surfaces.

    Although low levels of mutagenic activity were found when high (10 mg/ml) concentrations of EB in solution were degraded, the sodium nitrite/hypophosphorus acid method does not produce significantly mutagenic solutions when the EB concentration does not exceed 0.5 mg/ml.

    • De-energize all electrical equipment before decontamination and wear appropriate protective equipment, including rubber gloves, lab coat, and chemical goggles. A small amount of nitrogen dioxide is given off when the decontamination solution is mixed. This procedure would be best if carried out in the fume hood.
    • Scrub the contaminated surface or equipment with a paper towel soaked in a freshly prepared decontamination solution (prepared by adding 20 ml of 50% hypophosphorus acid to a solution of 4.2 g of sodium nitrite in 300 ml of water). Scrub another five times with wet paper towels, using a fresh towel each time.
    • Place all towels in a large container and soak them in fresh decontamination solution for at least 1 hour before disposing of them.
    • Test the used decontamination solution and squeezings from the towels used for some of the later washes for fluorescence and/or mutagenicity.
    • Dry off the decontaminated surface or equipment and place it in service again. Neutralize the used decontamination solution with sodium bicarbonate and discard as aqueous waste.

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