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Biochemical oxygen demand, or BOD, measures the amount of oxygen consumed by

microorganisms in decomposing organic matter in stream water. BOD also measures the
chemical oxidation of inorganic matter for example the extraction of oxygen from water via
chemical reaction. The rate of oxygen consumption in a stream is affected by a number of
variables: temperature, pH, the presence of certain kinds of microorganisms, and the type of
organic and inorganic material in the water.

Azide Modification of Winkler Method is one simple way in determining amount of Dissolve
Oxygen (DO) in water. This multi step chemical method involves the adding of chemicals such
as ManganousSulfate Powder Pillow and Alkaline Iodide-Azide Reagent Powder Pillow
followed bySulfamic Powder Pillow , that react with water and fixes it. Certain reagents will
be placed in sample water that is taken from a water supply. Any observation during these
periods shows the presence or absence of DO in water. The adding of these reagents will produce
a prepared sample which will be titrated with sodium thiosulfate using a titrator body with
titration cartridge until a pale yellow solution is formed. A simple calculation later on will
determine the exact amount of DO in the sample water.


The stream system both produces and consumes oxygen. It gains oxygen from the atmosphere
and from plants as a result of photosynthesis. Running water, because of its churning, dissolves
more oxygen than still water, such as that in a reservoir behind a dam. Respiration by aquatic
animals, decomposition, and various chemical reactions consume oxygen.

Wastewater from sewage treatment plants often contains organic materials that are decomposed
by microorganisms, which use oxygen in the process. (The amount of oxygen consumed by these
organisms in breaking down the waste is known as the biochemical oxygen demand or BOD.
Other sources of oxygen-consuming waste include stormwater runoff from farmland or urban
streets, feedlots, and failing septic systems.

Oxygen is measured in its dissolved form as dissolved oxygen (DO). If more oxygen is
consumed than is produced, dissolved oxygen levels decline and some sensitive animals may
move away, weaken, or die.

DO levels fluctuate seasonally and over a 24-hour period. They vary with water temperature and
altitude. Cold water holds more oxygen than warm water and water holds less oxygen at higher
altitudes. Thermal discharges, such as water used to cool machinery in a manufacturing plant or a
power plant, raise the temperature of water and lower its oxygen content. Aquatic animals are
most vulnerable to lowered DO levels in the early morning on hot summer days when stream
flows are low, water temperatures are high, and aquatic plants have not been producing oxygen
since sunset.

Total dissolved gas concentrations in water should not exceed 110 percent. Concentrations above
this level can be harmful to aquatic life. Fish in waters containing excessive dissolved gases may
suffer from "gas bubble disease"; however, this is a very rare occurrence. The bubbles or emboli
block the flow of blood through blood vessels causing death. External bubbles (emphysema) can
also occur and be seen on fins, on skin and on other tissue. Aquatic invertebrates are also affected
by gas bubble disease but at levels higher than those lethal to fish.

Adequate dissolved oxygen is necessary for good water quality. Oxygen is a necessary element
to all forms of life. Natural stream purification processes require adequate oxygen levels in order

to provide for aerobic life forms. As dissolved oxygen levels in water drop below 5.0 mg/l,
aquatic life is put under stress. The lower the concentration, the greater the stress. Oxygen levels
that remain below 1-2 mg/l for a few hours can result in large fish kills.

The concentration of dissolved oxygen in water is affected by many factors including ambient
temperature, atmospheric pressure, and ion activity. Accurate data on the concentration of
dissolved oxygen (DO) in environmental water resources are essential for documenting changes
that result from natural phenomena and human activities. Many chemical and biological
reactions in ground water and surface water depend directly or indirectly on the amount of
available oxygen. Dissolved oxygen is necessary in aquatic systems for the survival and growth
of many aquatic organisms and is used as an indicator of the health of surface-water bodies.


The main purpose for doing this experiment are :

To determine the dissolved oxygen in various water samples (lake water, tap water, drain
water and deionized water samples )

To ascertain whether the quality complies with the Malaysian Water Standards

To complete this experiment successfully


In the Winkler method, , the dissolved oxygen is made to react with Iodide ion to yield Iodine
which will be titrated afterwards with thiosulfate.

Iodine is a moderately strong oxidizing agent making it useful for titrating strong reducing agents
like dissolved oxygen. Iodide ion, on the other hand, is a weak reducing agent, and is the basis of
determining the amount of the strong oxidizing agents like the oxygen in the sample pond water.
This is done by determining the amount of I3 produced, which is equivalent to the dissolved
oxygen using titration with thiosulfate.

Thiosulfate may be oxidized by strong oxidation agents or form complexes with some, making it
inadvisable to titrate the water sample directly with thiosulfate. By adding iodide ions first before
titration, the strong oxidizing agent is destroyed and an equivalent amount of Iodine is
produced. This iodine will be converted into I3- which will react stoichiometrically with the

The thiosulfate to be used for titration was first standardized using KIO 3 as the primary standard.
KI crystals and sulfuric acid was added to the KIO 3 solution one after the other. KI crystals
should be added first or the solution may produce more O 2 and add to the analyzed dissolved
oxygen of the sample:

+ O2 + HIO
+ H

The resulting solution was titrated right away with Na2S2O3 as the titrant and starch as the
indicator. The first step of the process resulted to the following reaction:

+ 3 I 2+ 3 H 2 O
+ 6 H
+5 I

Like other halogens, iodine reacts with water, yielding the reaction:

++ I
I 2 + H 2 O H

To prevent the forward reaction, and produce more of the desired I2, the solution was made acidic
by adding H2SO4 which will cause the equilibrium to shift to the left due to the common-ion
effect. This is also the same reason why excess KI was added. Unreacted iodide does not
interfere with the reaction but may be oxidized by air if not titrated immediately.

But the iodine has a low solubility in water, so it is first converted to a complex
consisting of I3- which is very soluble. This is done by dissolving I 2 in a potassium iodide

I 3
I 2 + I

The I2 produced in this reaction is then titrated with Na2S2O3 which yields the reaction:

+S4 O6
2 2 I
I 2 +2 S 2 O3

From this titration, the molarity of thiosulfate can be calculated using the knowledge that:

=3 moles I 2=6 moles S 2 O3
1 mole IO3

The endpoint of the reaction is indicated using starch, which does not dissolve in solutions but
exists as colloid and is only suspended in the analyte. The starch indicator should be added just
prior to the endpoint or when the yellow iodine color is beginning to fade. Adding starch at the
start of titration when the iodine concentration is still high will form an iodine-starch complex,
producing a diffuse endpoint and a reddish brown color which will not be readily discharged at
the endpoint because of the excess iodine still present due to the slow dissociation of the

complex. Also, starch may hydrolyze and interfere with the reaction in acidic solutions, which is
the case of the analyte in this experiment. However, if iodine was the titrant, starch can be added
at any point since iodine is not in excess until the endpoint.

It exhibits a deep blue color when it absorbs iodine. When the iodine is converted into iodide
ion, the blue color disappears and the complete disappearance of the color indicates the endpoint.
The presence of strong solutions of electrolytes, organic materials like alcohol and heat may
interfere with the absorption of iodine and will not produce a good color, thus these conditions
should be avoided. Starch should also not be kept suspended for too long since long exposure to
water can make it susceptible to bacterial action, causing it to decompose and become less

For the sample analysis, the same concepts were used to determine the amount of dissolved
oxygen. The dissolved oxygen was first added with NH 4HCO3 to form a more reactive oxygen-
sensitive carbonate. If NH4HCO3 was not added, the dissolved oxygen will not react or will react
very slowly with the solution, even though MnSO4 was already added. Then, a basic KI solution
(NaOH with KI crystals) is added to react with the Mn2+ ions and t an OH- salt with Mn:

2++ SO 4
MnSO4 Mn

Mn ( OH )2
2+ +2OH

The Mn(OH)2 produced will then react with the oxygen. There are two possible reactions that
may occur:

Mn ( OH )2 +2 H 2 O+2 O2 4 Mn (OH )3

which will produce a brownish inorganic oxide floc, the fluffy mass seen suspended in the pond
water sample, or

4 Mn (OH )2 +O2 4 MnO ( OH )+ 2 H 2 O

which will also produce a brown solution.

The Mn(OH)3 precipitates formed was then dissolved with phosphoric acid while the MnO(OH)
precipitated will react with the iodide ions from the NaOH solution with KI:

2+ + I 2+ 4 H 2 O
2 Mn
++ MnO(OH )(s) +2 I
6 H

The solution was then closed tightly and kept in a dark locker. This is done to prevent the
accelerating effect of light to the decomposition of hypoiodous acid:

+ O2
++2 I
2 HIO 2 H

and the oxidation of iodide ion caused by air:

++O2 2 I 2 +2 H 2 O
+4 H
4 I

The I2 produced in the desired reaction will be equivalent to the amount of dissolved oxygen in
the pond water sample:

1 mole O2=4 moles S 2 O3

To be able to determine the exact amount of I 2, and also the dissolved oxygen, the solution was
titrated with thiosulfate, again, with the reaction:

+S4 O6
2 2 I
I 2 +2 S 2 O3

This experiment is conducted to calculate the present of dissolved oxygen in the water sample
and to determine the level of water pollution in each water sample. The dissolved oxygen contain
in lake water is determined to be 6.4 mg/L, tap water 6.2mg/L, drain water 8.6 mg/L, deionized
water 7.4mg/L. The lake water and tap water is determined to be polluted.


1. 300mL BOD bottle

2. Glove

3. Googles

4. Dissolved oxygen field kit

5. Sodium Thiosulfate Titration Cartridge

6. Delivery tube

7. Graduated cylinder

8. 250 mL Erlenmeyer flask

9. Pipette

10. DO Meter calibration


1. Manganous Sulfate Powder Pillow

2. Alkaline Iodide-Azide Reagent Powder Pillow

3. Sulfamic Acid Powder Pillow

4. Sodium Thiosulfate

5. Starch indicator solution


1. A water sample in a clean 300 mL BOD bottle was collected. The sample was allowed to
overflow the bottle for 2-3 minutes to ensure the air bubbles were not trapped.

2. Next the sample was added with one Manganous Sulfate Powder Pillow and one Alkaline
Iodide-Azide Reagent Powder Pillow.

3. A stopper was inserted immediately without trapping air inside the bottle. The bottle was
inverted several times to mix the solution.

4. After the bottle was inverted several times,waited until the floc settles and a clear
solution were obtained.

5. The stopper was removed and the content were added with one Sulfamic Powder Pillow.
The stopper was replaced without trapping the air inside the bottle and then again the
prepared sample was inverted several times.

6. A sample volume and Sodium Thiosulfate Titration Cartridge were selected

corresponding with expected dissolved oxygen (DO) concentration.

7. Then a clean delivery tube was inserted into the titration cartridge to the titrator body.
After that, the delivery knob was turned to inject a few drops of titrant. The counter
isresetted to zero and the tip was wiped.

8. A graduated cylinder was used to measure the sample volume and the sample was
transferred to 25mL Erienmeyer flask.

9. The delivery tube was placed into the solution and the flask is swirled while the solution
was titrated with sodium thiosulfate to a pale yellow colour.

10. Next, two 1 mL dropper of Starch Indicator solution were added and the mixture was

11. The titration was continued to get a colourless end point. The number of digits required

12. Finally, dissolved oxygen were calculated and being tabulated in a table.


Types of water samples Calculations Dissolved oxygen (mg/L)

Lake 320 0.02 = 6.4 6.4

Tap 310 0.02 = 6.2 6.2

Drain 860 0.01 = 86 8.6

Deionized 370 0.02 = 7.4 7.4

Solution Observation/conclusion

A=Water sample + 1 Produce orange-brown flocculent. Indicates that oxygen

ManganousSulfate Alkaline is present in sample water.
Iodide + 1 Azide Reagent Powder

B=A + 1 Sulfamic Acid Powder Flocculent dissolved and leaves yellow coloured
Pillow solution. Indicates that oxygen is present in sample

C=100mL of B + Sodium Drop by drop until solution turns pale yellow.

Thiosulfate (titration)

D=C + two 1mL droppers of Solution turns dark blue. Continued titration until
Starch Indicator Solution solution turns colourless. Number of digits required is


Number of digits required x digit multiplier = mg/L dissolved oxygen

Calculation :

-Lake water : 320 0.02

: 6.4

-Tap water : 310 0.02

: 6.2

-Drain water : 860 0.01

: 8.6

-Deionized water : 370 0.02

: 7.4


This experiment was conducted to verify the amount of dissolve oxygen contain in various
sample water using Azide Modification of Winkler Method.

The Azide used in this experiment inhibits the interference of nitrogen ion during the redox
reaction by oxygen and iodine.

The addition of ManganousSulfateamd Alkaline Iodide Reagent Powder Pillow produce an

orange-brown precipitate due to the reduction of oxygen by Mn.

2 Mn2+ + 4 OH- + O2 2MnO2(s) + 2 H2O

(brown precipitate)

Sulfamic Acid Powder Pillow, HNSO was added and causeMnO to oxidise I. Therefore, the
precipitate dissolve and leave an orange-brown precipitate. The orange-brown precipitate
indicates the presence of oxygen in the sample water.

MnO2(s) + 4 H+ + 2 I- I2 + Mn2+ + 2 H2O

(orange-brown solution)

A definite amount of this sample is measured and selected to be titrated with Sodium
thiosulphate solution. Before titrating, a few drops were ejected from the tube to ensure there are
no bubbles traped that may cause an error. The titration tube is immersed directly into the
solution to ensure full reaction. The container of the solution is swirl throughout the titration to
speed up the rate of reaction.

The I which present as triiodide ion, I is titrated with Sodium Thiosuphate until it turns pale

4 Na2S2O3+ 2I2 2Na2S4O6+ 4NaI

(pale yellow)

Starch indicator is added and solution turns dark blue because of the presence of iodine in the
solution. As known, the MnO formed earlier s directly proportional to the amount of oxygen

present in the sample water. Now, the amount of iodine present is directly proportional to the
MnO present. Therefore, by titrating iodide with sodium thiosulphateuntilm the solution turns
colourless will indicates the amount of DO. The amount of DO was calculated using this value,
which are 6.4 mg/L, tap water 6.2mg/L, drain water 8.6 mg/L, deionized water 7.4mg/L.


Using Azide Modification of Winkler Method, the DO of various sample water is known.
Addition of Azide will inhibit the interference of nitrogen ion during the redox reaction of
reagents in the water sample. Compared to the National Water Quality Standards for Malaysian,
the sample water has the rate of DO among the III class.

The means that contribute as class III is:

Defined with the primary objective of protecting common and moderately tolerant aquatic
species of economic value. Water under this classification may be used for water supply with
extensive / advance treatment. This class of water is also defined to suit livestock drinking
needs.The dissolved oxygen contain in lake water is determined to be 6.4 mg/L, tap water
6.2mg/L, drain water 8.6 mg/L, deionized water 7.4mg/L. The lake water and tap water is
determined to be polluted.


Here are some of the recommendations regarding to this study, before conducting experiment
students should put on gloves and goggles. This is to avoid contamination. Always hold the
dropper perpendicular to and approximately one inch above the sample bottle so that drop size
will be consistent. Next, avoid the issue of bubble formation by using the dissolution medium
after equilibration at 37C for 24 hour in a water bath. Logic behind this approach is when
medium is equilibrated for sufficiently long time at 37C and when transfer to dissolution bath, no
temperature changes, no change in dissolved gasses solubility, thus no bubble formation. The
titrator plunger has a tendancy to stick at the bottom or wherever it has been stored. To help
avoid this, always store the titrator with plunger not fully depressed. Before use, push on the
plunger to loosen it before pulling up. Besides, it is suggested to use digital titrator to get

accurate and precise result. Experiment can be quite variable so collect sufficient field of water
and split replicates (10 to 20 percent) to provide an estimate of method variability


1.Brown, Theodore L., H. Eugene LeMay, Jr., and Bruce E. Burston. (2006)Chemistry: The
Central Science. 10th ed. Upper Saddle River, NJ: Pearson Education, Inc.,

2.Lide, D. R. (Ed.) (1990). CRC Handbook of Chemistry and Physics (70th Edn.). Boca Raton
(FL):CRC Press.

3.Sawyer, C. N., McCarty, P. L., and Parkin, G. F.(2003) Chemistry for Environmental
Engineering, 5th ed., McGraw Hill

4. Water Quality Criteria,(1972) Environmental Studies Board, National Academy of Sciences,

5. Study and Interpretation of the Chemical Characteristics of Natural Water,(1970) United States
Geological Survey, Water Supply Paper 1473,

6. Quality Criteria for Water, (Last updated on Sunday, November 06, 2011)