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3.1 Deactivation Rules

  1. Understand the physical properties and toxicological characteristics of chemicals used in the deactivation procedure. Refer to the appropriate Material Safety Data Sheets (MSDSs), which are available from EH&S.
  2. Perform all deactivations in a chemical fume hood.
  3. Wear gloves, lab coat, and eye protection.
  4. Read deactivation procedures thoroughly before starting.
  5. Do not attempt a deactivation if you do not have the experience or equipment a procedure requires, or if the volume exceeds one liter. Instead, prepare the chemical waste for EH&S pickup.
  6. Do not use substitutes for chemicals in the procedures.

3.2 Ethidium Bromide

Ethidium bromide is a strong mutagen and EH&S recommends that it be deactivated before being sewered.

There are two options for treatment of ethidium bromide for sewer disposal: deactivation or filtration. The following procedure permits the destruction of 100 ml of 0.5 mg/ml solution of ethidium bromide in TBE buffer, MOPPS buffer or cesium chloride. If higher concentrations or volumes of ethidium bromide are to be destroyed, the procedure should be adjusted proportionally. Alternatively, ethidium bromide solutions may be filtered according to the procedure in Section 3.2.3.

3.2.1 Preparation of Reagents for Deactivation

It is recommended that these solutions be made up and used within the same day:

  • 5% hypophosphorous acid: 10 ml of a 50% stock solution is added to 90 ml of water
  • 0.5M sodium nitrite: 34.5 gm of solid material is put into solution with one liter of water
  • 5% sodium hydroxide: 10 ml of a 50% stock solution is added to 90 ml of water

3.2.2 Ethidium Bromide Deactivation Procedure

To 100 ml of ethidium bromide waste, add 12 ml of 0.5M sodium nitrite and 20 ml of the hypophosphorous acid. This mixture should be stirred thoroughly and the pH should be tested. If the pH is not less than 3, then the reaction may not go to completion. Extra acid may be added to achieve this pH.

Once the proper pH is reached, the mixture should be left to react for 20 hours. During the 20-hour reaction, the mixture will change from the characteristic pink of ethidium bromide to a yellowish color, indicating that the desired reaction has taken place.

To confirm complete deactivation, mix a small sample of the reaction mixture with an equal volume of DNA (20 μg/ml). This sample should be viewed under a long wave UV light as a rough check for unreacted ethidium bromide, which fluoresces strongly and is easily detected if present.

The completely reacted mixture must be neutralized prior to disposal into the sanitary sewer. It will take approximately 25-30 ml of 5% sodium hydroxide to neutralize 100 ml of the ethidium bromide mixture.

The amount of sodium hydroxide may vary according to the buffer used. Therefore, it is suggested that the pH be checked often during the neutralization so that the desired pH range is not passed.

Once the pH is between 6 and 8, the mixture can be sewered with copious amounts of water.

3.2.3 Ethidium Bromide Filtration

the drain. The process is safe, effective, and simplified by use of commercially available filtration systems such as FluorAway, S&S Extractor, and The Green Bag Kit.

  1. Filter the ethidium bromide solution through the charcoal filter.
  2. Pour the filtrate down the drain.
  3. Place the charcoal filter in a sealed bag and collect for hazardous waste disposal as described in Section 4.

Note: if using a house vacuum to speed filtration, use a flask capable of withstanding the vacuum to prevent implosion (do not use a standard side-arm filtering flask).

3.3 3,3'-Diaminobenzidine (DAB) Deactivation

Although not regulated by the state as a hazardous waste, 3,3'-diaminobenzidine is not permitted to be discharged into the King County sewer system due to its mutagenic properties. It must first be detoxified prior to discharge into the sanitary sewer. DAB must not be detoxified using bleach, as the end products remain toxic. Instead, use the procedure that follows.

3.3.1 Preparation of Reagents for DAB Deactivation

  • 0.2 M potassium permanganate solution: 31.6 g KMnO4 per liter of solution with water
  • 2.0 M sulfuric acid solution: 112 ml concentrated H2SO4 per liter of solution with water

3.3.2 DAB Deactivation Procedure

  1. Dilute the DAB until its concentration does not exceed 0.9 mg/ml.
  2. For each 10 ml of DAB solution, add:
    1. 5 ml of the KMnO4solution;
    2. 5 ml of the H2SO4 solution.
  3. Allow the mixture to stand for at least 10 hours.
  4. Test the pH of the solution. If the pH is between 6 and 9, the mixture may be poured down the drain. If not, refer to Section 2.1.2 for the neutralization procedure prior to sewer disposal.

3.4 Formalin Deactivation

When formaldehyde makes up 1% or more of a formalin solution, the solution must be collected as hazardous waste as described in Section 4. Formalin solutions containing less than 1% formaldehyde are not state-regulated hazardous wastes, but King County does not allow sewer disposal unless formaldehyde concentration is lower than 0.1%. Dilution with water to meet the 0.1% limit is not allowed. However, the following commercially available products will reduce the concentration of a treated sample to below 0.1% so that the solution may be disposed of in the sewer:

  • Scigen/Tissue Tek Neutralex
  • Baxter Healthcare VYTAC 10F
  • Waste & Compliance Management Aldex (solidifies formalin solution for disposal as hazardous waste)
  • S&S SASCO Formalex

If your lab generates large volumes of formalin waste containing greater than 1% formaldehyde, these products may also work to allow sewer disposal; however, you must first contact EH&S for approval and additional instructions.

Note that many of these products will dilute formalin to well below the 0.1% sewer limit, and therefore the treatment compounds may be diluted before using them to treat the waste solution. For example, one packet of Neutralex is described as treating one gallon of buffered formalin to 15 ppm. Since the sewer limit is 1000 ppm, one packet can actually treat 50 times as much formalin and still meet the sewer limit of 0.1%.