Sample Collection Guide for

Laboratory Testing of Dissovled Oxygen by Titration (Winkler Method).

Use the BOD bottle. BOD bottles have a ground glass stopper that fits tightly into ground glass neck of the bottle. When closed, these are air tight bottles would not allow any exchange of atmospheric oxygen. This same bottle is used in the laboratory to incubate prepared solutions in the BOD test process, hence the name. Note that samples for BOD need not be collected in BOD bottle. But samples for laboratory testing of dissolved oxygen must be collected in BOD bottle, fixed on the spot, and the closed BOD bottle transported to the laboratory. Remember that the water sample must be collected in such a way that you can replace the bottle stopper while it is still submerged. That means that you must be able to reach into the water with both arms and the water must be deeper than the sample bottle. Remove the stopper of the BOD bottle. Slowly lower the bottle into the water, pointing it downstream, until the lower lip of the opening is submerged in the grab sample intake zone. Allow the water to fill the bottle very gradually, avoiding any turbulence. Turbulence would add oxygen to the sample and adversely affect test result. Hence, gradual filling and avoidance of any turbulence is very important. When the water level in the bottle has stabilized (it won't be full because the bottle is tilted), slowly turn the bottle upright and fill it completely. Keep the bottle under water and allow it to overflow for 2 or 3 minutes to ensure that no air bubbles are trapped. Replace the stopper while the bottle is still submerged. Lift it out of the water and look around the "collar" of the bottle just below the bottom of the stopper. If you see an air bubble, pour out the sample and try again.

Fixing the dissolved oxygen sample:

1. Remove the stopper and first add 2 ml of the first fixing reagent labelled – ‘M’ (Manganese sulfate) to the sample. Use the calibrated dropper provided with the reagent bottle. This reagent must be added slowly by touching the dropper tip to inside of the BOD bottle neck, so that the reagent flows in and mixes with sample without introducing any air bubble.
2. Then, add 2 mL of the second fixing reagent labelled ‘I’ (alkaline iodide-azide solution) to the sample contained in the BOD bottle. This reagent must also be added gently by touching the reagent dropper to inside of sample bottle-neck and gradually releasing the reagent.
3. Replace stopper of the BOD bottle immediately and mix the contents thoroughly.
4. An orange-brown flocculent precipitate will form if oxygen is present.
5. Wait a few minutes until the floc in the solution has settled.
6. Place the bottle in cool box for preservation and transportation to the laboratory.

Caution about handling of the fixing reagents:

1. Both fixing reagents [manganous sulfate solution (labelled – ‘M’) and alkaline-iodide-azide (labelled ‘I’)] are;
   1. Harmful in contact with skin;
   2. Can cause severe burns and eye damage; and
   3. Harmful to fish in lake.
2. So, handle the reagent bottles carefully and comply with following safety precautions:
   1. Wear protective glass and hand gloves.
   2. Place the reagent bottles in a firmly seated tray and do not spill the reagent.
   3. Draw required volume, inject into the sample bottle, replace the calibrated dropper with cap and close the cap as soon as possible.
   4. First aid in case of skin contact: rinse and wash with plenty of soap and water; Take off contaminated clothing, if any and wash in water.
   5. First aid in case of eye contact: Rinse cautiously with bottled water for several minutes. Remove contact lenses, if any, and continue rinsing with bottled water.

Updated on 26-01-2025