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Depending on the interactions between water molecules and gas molecules, gases are dissolved in water. Because the natural water resources are in contact with air, gases like oxygen and carbon dioxide are dissolved in water naturally.

When it comes to water quality, dissolved oxygen level (DO) is an important parameter. The dissolved oxygen (DO) levels in natural water bodies and waste waters mainly depend on the physical, chemical, and biochemical activities in the water source. Dissolved oxygen analysis is a fundamental test in water pollution evaluation and waste treatment process control.

Methods of testing dissolved oxygen level in the water 

There are several methods for determining DO levels in water samples. Some methods are titrimetric methods, and some are in situ testing methods.

Here we are discussing three methods.

The iodometric method also known as the Winkler method is a titrimetric method. The membrane method and Optical probe method are in-situ testing methods.

01. Iodometric method – Winkler method

The iodometric method is the most precise method of analyzing DO. Samples are added divalent Manganese solution (MnCl2) and strong alkali solution. In the presence of the base, divalent Manganese ions are oxidized to Mn (iv) resulting in manganese dioxide (MnO2) by Oxygen present in the sample.

The generated MnO2 is reduced to the divalent state (Mn(IV)) by using an iodide solution in an acidic media. liberated iodine (I2) is titrated with thiosulphate solution in the presence of starch as the indicator. The reactions that occurred in the Winkler method can be summarized as follows.

Dissolved Oxygen eq 01

The iodometric method is interfered with by some oxidizing and reducing agents. If there are oxidizing agents, they will oxidize iodide ions and liberate iodine. It is a positive interference. If there are reducing agents, they will reduce iodine into iodide ions and give negative interference. Therefore, some modifications are done to minimize interference.

i. Azide modification

Azide modification is done for samples interfered with Nitrite (NO2-) ions. The Azide modification is done for samples containing Nitrite ions (higher than 50μg) and Fe (II) ions (less or similar than (<=) 1mg). Generally, this method is used to analyze wastewater, effluent, and stream water samples.

  1.  Manganous sulfate solution
    • Manganese sulfate (MnSO4) solution is prepared by dissolving 480 g MnSO4. 4H2O, 400 g MnSO4.2H2O, or 364 g MnSO4.H2O in 1 liter of distilled water.
  2. Alkali-iodide-Azide reagent
    • to get a strong alkali solution, we use strong bases like NaOH or KOH.
    • To prepare alkali iodide Azide solution, first 500 g sodium hydroxide (NaOH) or 700 g potassium hydroxide (KOH) and 135 g sodium iodide (NaI) or 150 g KI are dissolved in distilled water and dilute to 1 L.
    • About 10 g of NaN3 that has been dissolved in 40 mL of distilled water is added to the above solution.
  3. Sulfuric acid, Conc
  4. Starch 
    • An aqueous solution of starch is used as the indicator of titration.
    • This solution is prepared by dissolving 2 g of soluble starch and 0.2 g of salicylic acid in 100 mL of hot distilled water.
  5. Standard sodium thiosulfate (Na2S2O3)
    • Sodium thiosulfate is the titrant. To prepare this solution, at first 6.205 g of Na2S2O3.5H2O is dissolved in distilled water.
    • Then 1.5 mL of 6M NaOH or 0.4 g solid NaOH is added. This solution is diluted up to 1000mL. The prepared solution should be standardized with potassium Bi-iodate [KH(IO3)2] solution.


Samples are collected in 300 mL BOD bottles. When collecting samples, ensure no air bubbles are mixed with the sample.

Just after the collection of the sample, 1 mL MnSO4 and 1 mL alkali-iodide-azide reagent are added to the sample. When adding Manganese sulfate solution and alkali-iodide-azide reagent, a brown color precipitate of Manganese dioxide is formed.

After precipitation is completed, the sample is taken for titration. After taking the sample for titration, 1 mL of conc Sulfuric acid (H2SO4) is added to dissolve the precipitate. In the acidic medium, iodide ions are reducing Mn (IV) into Mn(II) liberating Iodine (I2).

When adding reagents to the sample, some volume of the sample is lost. When titrating we take a volume equivalent to 200 mL of the original sample. This sample is titrated with 0.025M Na2S2O3 solution from blue color to a pale straw color in the presence of starch as the indicator.

Dissolved Oxygen eq 02

We add 2 mL of reagents (MnSO4 and alkali-iodide-azide reagent) to the above sample. Therefore, some volume of the original sample is lost. The volume of the original sample after adding reagents would be as follows.

Dissolved Oxygen eq 03

If we should take a volume equivalent to 200 mL of the original sample, the volume of sample to be taken from the reagent added sample would be,

Dissolved Oxygen eq 04

According to stoichiometry,

Dissolved Oxygen eq 05

V is the endpoint volume of the titration, and it is taken in milliliters (mL).

Dissolved Oxygen eq 06

According to the above relationship, the volume of the Na2S2O3 solution is equal to the DO level of the sample.

Dissolved Oxygen eq 07

ii. Permanganate modification

The permanganate modification method is used only on samples that contain a high level of Fe(II) like several hundred milligrams per liter.

  • E.g. Acid mine water

Permanganate modification is also included the Azide modification. Here we minimize the interference from both Fe(II) and Nitrite (NO2-) ions.

  1. Potassium permanganate (KMnO4) solution
    • Prepared by dissolving 6.3 g of KMnO4 in distilled water and diluting it to 1 L.
  2. Potassium oxalate (K2C2O4) solution
    • Prepared by dissolving 2 g of K2C2O4.H2O in 100 mL of distilled water.
  3. Potassium fluoride (KF) solution
    • Prepared by dissolving 40 g KF 2H2O in distilled water and diluting it to 100 mL.
  4. All the reagents required in the Azide modification method


Samples are collected in 300 mL BOD bottles.

First, 1 mL of KMnO4 solution and 1 mL of KF solution were added to the sample. After adding KMnO4 and KF, 0.7 mL of conc H2SO4 is added by using a 1 mL pipette. MnO4- ions will oxidize Fe(II) into Fe(III).

Dissolved Oxygen eq 08

When adding KMnO4 to the sample, the color of MnO4- will immediately disappear because MnO4- ions will be reduced to Mn2+, and Fe2+ will be oxidized to Fe3+. We should add enough KMnO4 to oxidize all the Fe2+ ions in the medium. But make sure not to add large excess of KMnO4. if the violet tinge of the sample persists for about 5 min, that means all the Fe2+ ions have oxidized.

Then K2C2O4 solution was added to the sample. K2C2O4 will reduce excess MnO4- ions in the medium. Therefore, the solution will be discolored. And the Oxalate ions (C2O42-) ions will form a stable complex with Fe3+ ions.

Dissolved Oxygen eq 09

From now, we are continuing the azide modification procedure. But the difference is, here we add 1 mL of MnSO4 instead of 2 mL and 3 mL of iodide azide solution instead of 2 mL.


For the titration, we take a similar volume to 200 mL of the original sample. We have added 1 mL of KMnO4, 1 mL of KF, 0.7 mL of conc H2SO4, 1mL of K2C2O4, 1mL of MnSO4, and 3 mL of alkali iodide azide solution. The total volume of the reagents is 7.7 mL. Therefore, the sample should be taken from the test solution would be,

Dissolved Oxygen eq 10

As same in azide modification, the volume of the 0.025M Na2S2O3 solution is equal to the DO level of the sample.

iii. Alum flocculation modification

Alum flocculation modification is applied for samples with highly suspended solids. Suspended solids will consume a considerable amount of iodine. We remove suspended solids by alum flocculation.

  1. Alum solution
    • Alum solution is prepared by dissolving 10 g of aluminum potassium sulfate [AlK(SO4)2.12H2O] in distilled water and diluting it to 100 mL.
  2. Conc. Ammonium hydroxide (NH4OH)
  3. All the reagents used in azide modification.


The sample is collected into a 500 mL to 1000 mL glass bottle without any air bubbles mixed with the sample.

Then 10 mL of a prepared alum solution and 1-2 mL of conc Ammonium hydroxide solution were added to the sample.

Let the solution about 10 min for the sedimentation of the suspended solids.

After 10 minutes the clear supernatant is transferred to a 300 mL BOD bottle until it overflows. Make sure to avoid aeration when the sample is transferred. Then the azide modification method is taken place for the sample.

iv. Copper Sulfate-Sulfamic Acid Flocculation Modification

This modification method is used for samples with biological flocks like activated sludge mixtures.

  1. Copper sulfate-sulfamic acid inhibitor solution
    • First, dissolve 32 g of technical-grade sulfamic acid (NH2SO2OH) in 475 mL of distilled water.
    • Second, dissolve 50 g copper sulfate (CuSO4.5H2O) in 500 mL distilled water.
    • Then mix the above two solutions together and add 25 mL conc acetic acid.
  2. All the reagents used in the azide modification method.


Add 10 mL of the Copper sulfate-sulfamic acid inhibitor solution to a 1000 mL glass stoppered bottle.

A tube is inserted near the bottom of the above bottle. The test sample is collected in the bottle through that tube. Transfer the sample until it overflows about 25% - 50% of the bottle volume.

Let this solution suspend the solid.

After the suspension, the supernatant is collected in a 300 mL BOD bottle, and azide modification, or a suitable modification method is taken place.

02. Membrane electrode method

Although the Iodometric method or the Winkler method is the most precise method for finding dissolved oxygen level, it cannot be applied to various industrial and domestic wastewater samples. And since it is a titrimetric method, we cannot determine the DO in situ.

The membrane electrode method is a polarographic method where an electrode that has been covered with a membrane is used. The electrode is covered with a membrane because the impurities in the wastewater can cause electrode poisoning or other interferences. Therefore, an oxygen-permeable polymer membrane is used. The membrane is a barrier to impurities.

The electrode is placed in the sample and stirred in the test solution (water sample) to determine the amount of dissolved oxygen. The apparatus will automatically calculate the DO level in the water.

03. Optical probe method

The optical probe method is a luminescence base oxygen sensing method to determine DO in water.

Here, an Oxygen-sensitive optical probe with an appropriate meter and a stirring device is used.

To measure the DO the probe is placed in the sample and started stirring. The apparatus will automatically calculate the DO level. This method is also an in-situ method.

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References and Attributes

Lipps, W.C., Braun-Howland, E.B. and Baxter, T.E. (2022) Standard methods for the examination of water and wastewater. Atlanta: APHA Press.


The cover image was designed using an image by Michal Jarmoluk from Pixabay

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