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1st Rule of Lab Safety
Haz-Waste No-No
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Updated January 2003
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
Services (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:
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.
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.
Decontamination of Equipment Contaminated with EB
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.
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.
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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