Determination of Dissolve Oxygen by Winkler’s Method using Azide

Modifications

Objectives

Objectives of this lab includes:

 To get knowledge of dissolved oxygen in water

 To understand the concept and phenomenon about dissolved oxygen
 To learn about the role of dissolved oxygen in water quality
 To understand the precautions and procedures involved in the determination of
dissolved oxygen in water

Dissolve Oxygen

Dissolved Oxygen is the amount of gaseous oxygen (O2) dissolved in the water. Oxygen
enters the water by direct absorption from the atmosphere, by rapid movement, or as a waste
product of plant photosynthesis.  Water temperature and the volume of moving water can
affect dissolved oxygen levels. Oxygen dissolves easier in cooler water than warmer water.

Dissolved oxygen refers to the level of free, non-compound oxygen present in water or other
liquids. It is an important parameter in assessing water quality because of its influence on the
organisms living within a body of water. In limnology (the study of lakes), dissolved oxygen
is an essential factor second only to water itself

Dissolved oxygen enters water through the air or as a plant byproduct. From the air, oxygen
can slowly diffuse across the water’s surface from the surrounding atmosphere, or be mixed
in quickly through aeration. The aeration of water can be caused by wind (creating waves),
rapids, waterfalls, ground water discharge or other forms of running water. Man-made causes
of aeration vary from an aquarium air pump to a hand-turned waterwheel to a large dam.

Environmental Significance/Impacts

Aquatic Life

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

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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.

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.

Fish kill / Winterkill

A fish kill occurs when a large number of fish in an area of water die off. It can be species-
based or a water-wide mortality. Fish kills can occur for a number of reasons, but low
dissolved oxygen is often a factor. A winterkill is a fish kill caused by prolonged reduction in
dissolved oxygen due to ice or snow cover on a lake or pond. When a body of water is over
productive, the oxygen in the water may get used up faster than it can be replenished.  This
occurs when a body of water is overstocked with organisms or if there is a large algal bloom
die-off. Fish kills are more common in eutrophic lakes: lakes with high concentrations of
nutrients (particularly phosphorus and nitrogen).

Stratification by Bacteria

Microbes such as bacteria and fungi also require dissolved oxygen. These organisms use DO
to decompose organic material at the bottom of a body of water. Microbial decomposition is
an important contributor to nutrient recycling. However, if there is an excess of decaying
organic material (from dying algae and other organisms), in a body of water with infrequent
or no turnover (also known as stratification), the oxygen at lower water levels will get used
up quicker.

Water Supplies

A high DO level in a community water supply is good because it makes drinking water taste
better. However, high DO levels speed up corrosion in water pipes. For this reason, industries

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use water with the least possible amount of dissolved oxygen. Water used in very low
pressure boilers have no more than 2.0 ppm of DO, but most boiler plant operators try to keep
oxygen levels to 0.007 ppm or less.

Indicator to type of aquatic life in water

The amount of dissolved oxygen often determines the number and types of organisms living
in that body of water. For example, fish like trout are sensitive to low DO levels (less than
eight parts per million) and cannot survive in warm, slow-moving streams or rivers. Decay of
organic material in water caused by either chemical processes or microbial action on
untreated sewage or dead vegetation can severely reduce dissolved oxygen concentration.
This is the most common cause of fish kills, especially in summer months when warm water
holds less oxygen anyway.

Toxicity in water

Numerous studies have confirmed that a pH range of 6.5 to 9 is most appropriate for the
maintenance of fish communities. Low concentrations of dissolved oxygen, when combined
with the presence of toxic substances may lead to stress responses in aquatic ecosystems
because the toxicity of certain elements, such as zinc, lead and copper, is increased by low
concentrations of dissolved oxygen.

Guidelines for Dissolved water

According to WHO, depending on the water temperature requirements for particular aquatic
species at various life stages, the criteria values range from 5 to 9.5 mg l-1, i.e. a minimum
dissolved oxygen concentration of 5-6 mg l-1 for warm-water biota and 6.5-9.5 mg l-1 for
cold-water biota. Higher oxygen concentrations are also relevant for early life stages.

DO Measurement

We will measure the DO using Winkler method with Azide modifications in this lab session.

Working principle

Winkler method is used to measure level of DO in the water samples. Samples are treated
with manganese sulfate and alkaline iodide azide solution (NaOH+KI+NaNO 3) to form

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orange brown flock to precipitate. If the solution doesn’t show the brown flocks, it means
water sample has no DO.
MnSO4 + 2Na+ + 2OH- Mn(O)2 + 2Na+ + SO2-4
Mn(O)2 + ½ O2 (DO sample) MnO2 (orange brown flocks) + H2O
Upon acidification, the flocks react with the iodide to produce free iodine from KI in
proportion to the oxygen concentration.
MnO2 + 2KI + H+ Mn2+ + I2 + 2H2O
The liberated iodine is then titrated with standard sodium thiosulfate using starch solution as
an indicator which brings blue color in solution.
2Na2S2O3 + I2 S4O2-2 + 2I- + 4Na+
The end point is reached when blue color is disappeared.

Sampling and Storage

There is no holding time for the DO sample and should be tested at the testing site as soon as
possible. But if the water sample could not be tested immediately then it can be stored for 4-8
hours in specified DO/BOD bottles.
 To store the DO sample, take water sample in DO bottle and make the water overflow
using stopper of bottle. This will ensure that no air bubble is present in the water
sample.
 Then add 2ml of manganese sulfate and 2ml of alkaline iodide azide solution by
holding the tip of pipette below the liquid surface.
 Replace the stopper avoiding the entrapment of air bubbles and shake well by gentle
inversion. Repeat shaking after the flock have settled halfway. Allow the flock to
settle again.
After this procedure store the water sample in a dark room at 10-20 degree Celsius. A single
DO sample test is rarely representative of the whole water body so take multiple number of
samples at different locations in water sample.
Note: do not let the sample remain in contact with sunlight or in agitation because This can
cause change in the value of DO.

Interferences in DO testing

Nitrite ions cause maximum interference. Nitrate ions cause most frequent interference in
determination of dissolved oxygen. It occurs principally in the effluents from waste water

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treatment plant that employ biological processes in river water and in BOD samples. Nitrite
oxidizes iodide to iodine under acidic conditions making DO measurements inaccurate, this
nitrite interference is eliminated by azide in reagent.
NaN3 +H+ HN3 + Na+
HN3 + NO2- + H+ H2O +N2O+N2
Some reducing agents like Mg will convert iodine to iodide and cause negative interference.
Cr+3, Mn+2, Fe+2, Ni+2, Cu+2 don’t interfere up to a level of 10 mg/l.

Apparatus

 BOD bottles (300ml)

 Volumetric flask
 Beakers
 Pipette
 Burette
 Graduated Cylinders

Reagents

 MnSO4.4H2O (manganese sulfate solution)

Dissolve 48g of MnSO4.4H2O in 60ml of distilled water then filter and dilute filtrate
to 100ml with distilled water.
 Alkali Iodide Azide Reagent:
Dissolve 50g NaOH, in 60ml of distilled water. In a separate container, dissolve 15g
KI in approximately 25ml of distilled water, when cool, mix both solutions in a large
beaker. Dissolve 1g of sodium azide NaN3 solution with constant stirring to the cool
solution of alkali iodide. Now transfer this alkali iodide azide reagent to 100ml
volumetric flask and dilute up to the mark.
 Concentrated H2SO4
 Starch Solution:
Make a paste from 1g laboratory grade soluble starch with little water, pour the paste
with stirring into 100ml boiling water and boil for 1 minute. Allow the solution to
cool and add 2-3g of KI. Keep the solution in the stopper bottle.
 Sodium Thiosulphate Solution N2S2O3.5H2O (0.025 N)

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 Dissolve 6.205g of sodium thiosulphate in distilled water. For preservation add 0.4g
of NaOH and dilute to 1L. Standardize against a primary standard K 2Cr2O7 (potassium
dichromate solution). Secondary solution is compared with primary solution.
 Standard Potassium Dichromate Solution (0.025N)
Dissolve 1.226g of anhydrous K2Cr2O7 (potassium dichromate) in distilled water and
dilute to 1l to yield a 0.025N solution. Then store in a glass stopper bottle.

Procedure

 Collect the water sample in clean 300ml BOD bottle with glass stopper by allowing
the water sample to overflow. This ensures that no air is entrapped in the bottle.
 By holding the tip of the pipette below the surface of liquid and add 2ml of
MnSO4.2H2O and 2 ml of alkaline iodide azide reagent.
 Replace the stopper and shake well by gentle inversion.
 Repeat shaking after the flocks have settled halfway. Then allow the flocks to settle
down.
 Remove the stopper and add 2ml of concentrated sulfuric acid and replace stopper.
Then shake by gentle inversion until no flock is visible and colour of solution
becomes yellow. Then allow standing for 5min but not in direct sunlight.
 After the above procedure, take 203ml of solution in titration flask and titrate it
against the sodium thiosulfate until the colour of solution becomes light yellow from
yellow colour.
 Then add 1ml of starch solution as an indicator and continue titration until blue colour
just disappears.
 Record the volume of sodium thiosulfate used for titration.
Note: only record the volume that is used for discharging the blue colour of solution.
 DO is expressed in mg/L.
DO = vol. in ml of N2S2O3.5H2O used in titration (used for discharge of blue colour)

Observations and Calculations:

Volume used for light yellow colour appearance = V1 = final reading – initial reading
=30.8 – 0 = 30.8
Volume used for blue colour discharge = V2 = final reading – initial reading = 34.4 - 30.8
V2 = 3.4ml

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 As,
DO = vol. in ml of N2S2O3.5H2O used in titration (used for discharge of blue colour)
DO = V2
DO = 3.4mg/L

Results

The DO of the given water sample is 3.4mg/L.

Comments

In this lab we have acknowledged the importance of dissolve oxygen in water quality and in
marine water. We have learned different concepts about DO. We have performed the Winkler
method for determination of DO with azide modifications.

It was quite interesting and informative activity which will be helpful in study of behavior of
marine water and its fauna.

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