1.ABSTRACT

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 Manganous Sulfate Powder Pillow and Alkaline Iodide-Azide Reagent Powder Pillow

followed by Sulfamic 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.

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2.INTRODUCTION

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

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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.

3.OBJECTIVE

The main purpose for doing this experiment are :

To determine the dissolved oxygen in water sample (lake water)

To ascertain whether the quality complies with the Malaysian Water Standards

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1. THEORY

There are various type of element contained in a water. One of the elements is oxygen. The

stream system both produces and consumes oxygen. Oxygen dissolves in the stream by diffusion

of oxygen from the atmosphere and also by photosynthesis from plants. Running water will

dissolve much more oxygen than a still water such as reservoir. Oxygen is measured in its

dissolved form as dissolved oxygen (DO). DO levels differ seasonally and over a 24-hour period.

They vary with water temperature and altitude. Cold water contains more oxygen than warm

water and water contained less oxygen at higher altitudes.

DO is measured either in milligrams per liter (mg/L) or "percent saturation." Milligrams per

liter is the amount of oxygen in a liter of water. Percent saturation is the amount of oxygen in a

liter of water relative to the total amount of oxygen that the water can hold at that temperature.

DO samples are collected using a special BOD bottle. Dissolved oxygen can be measured either

by using the Winkler method or by using a meter and probe.

The Winkler method involves filling a sample bottle completely with water. The dissolved

oxygen is then added by a series of reagents that form an acid compound that is titrated. Titration

involves the drop by drop addition of a reagent that neutralizes the acid compound and causes a

change in the colour of the solution. The point at which the colour changes is the endpoint of the

titration and is equivalent to the amount of oxygen dissolved in the sample.

Sodium thiosulphate is the reducing agent normally used and starch solution is used to

determine the end point of the titration. All reactions in the determination of oxygen involves

oxidation and reduction. However, starch is used as end point indicator, and forms a starch-

iodine complex with iodine from dilute solutions to produce a blue colour and change to a colour

less form when the iodine is all reduced to iodide ion. The reaction involved in Winkler

procedure is given as follow.

Mn2+ + 2OH- Mn(OH)2

Mn2++ 2OH- + ½ O2 MnO2 + H2O

Mn(OH)2 + ½ O2 MnO2 + H2O

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The oxidation of Mn(II) to MnO2, sometime called fixation of the oxygen, occurs slowly,

particularly at low temperatures. Furthermore, it is necessary to move the flocculated material

throughout the solution to enable all the oxygen to react. Under low pH conditions the MnO2

oxidizes I- to produce I2

MnO2 + 2I- + 4 H+ Mn2++ I2 + 2 H2O

Iodine is the rather insoluble in water, but forms a complex with the excess iodide present to

reversibly form the more soluble tri-iodate, thus preventing escape of I2 from solution

I2 + I- I3-

The sample is then titrated against a standard thiosulphate solution, using starch as indicator

towards the end. From the titer value, results can be interpreted directly in terms of milligrams

per liter of DO.[10]

5.METHODOLOGY

APPARATUS

300mL BOD bottle

Glove

Googles

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Dissolved oxygen field kit

Sodium Thiosulfate Titration Cartridge

Delivery tube

Graduated cylinder

250 mL Erlenmeyer flask

Pipette

DO Meter calibration

MATERIALS

Manganous Sulfate Powder Pillow

Alkaline Iodide-Azide Reagent Powder Pillow

Sulfamic Acid Powder Pillow

Sodium Thiosulfate

Starch indicator solution

6.PROCEDURE

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.

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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 is

resetted 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

swirled.

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

were recorded.

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

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7.RESULTS

Volume

Sample

water

( Lake

water )

Observation Volume

Sodium

Thiosulphate

used (mL)

Dissolve

oxygen

(DO) levels

(mg/L)

Manganous

sulphate +

iodide azide

Sulfamic acid starch

300mL

Brown

orange

flock

*Flock dissolve

*Form yellow

colour solution

Solutions

turns blue

217 4.34

60mL Brown

orange

flock

*Flock dissolve

*Form orange-

yellow colour

solution

Solutions

turns blue

42 4.12

Solution Observation/conclusion

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

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Sulfate Alkaline Iodide + 1 Azide Reagent Powder Pillow.

is present in sample water.

B=A + 1 Sulfamic Acid Powder Pillow

Flocculent dissolved and leaves yellow coloured solution. Indicates that oxygen is present in sample water.

C=100mL of B + Sodium Thiosulfate (titration)

Drop by drop until solution turns pale yellow.

D=C + two 1mL droppers of Starch Indicator Solution

Solution turns dark blue. Continued titration until solution turns colourless. Number of digits required is recorded.

8.CALCULATIONS

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

Volume of water sample ( lake water )

Volume of Sodium Thiosulphate used (mL)

Dissolve Oxygen (DO) level (mg/L)

300 mL 217 = 217 0.02

= 4.34

60 mL 42 = 42 0.1

= 4.2

DISCUSSION

The main objective of this experiment is to determine the dissolved oxygen in water sample

lake water. The dissolved oxygen in water sample is determined by using the winkler azide

method. Based on an article on How To Measure Dissolved Oxygen by Department of Ecology,

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State of Washington and an article produced by an online botanical encyclopaedia, it shows that

winkler method is the most precise and reliable titrimetric procedure for dissolved oxygen

analysis.

The winkler method is a technique which uses titration to determine the dissolved oxygen

in freshwater systems. According to an article by Monica Z. Brukner in Microbial Life

Educational Resource (2011), dissolved oxygen is used as an indicator of the health of a water

body, where higher dissolved oxygen concentrations are correlated with high productivity and

little pollution. The dissolved oxygen analysis can be used to determine the health or cleanliness

of a lake or stream, the amount and type of biomass a freshwater systems can support, and the

amount of decomposition occurring in the lake or stream.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 Manganous Sulfate amd Alkaline Iodide Reagent Powder Pillow produce an orange-brown precipitate due to the reduction of oxygen by Mn²⁺.

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

Sulfamic Acid Powder Pillow, H₃NSO₃ was added and cause MnO₂ 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

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solution to ensure full reaction. The container of the solution is swirl throughout the titration to

speed up the rate of reaction.

The I₂ which present as triiodide ion, I₃⁻ is titrated with Sodium Thiosuphate until it turns pale yellow.

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 MnO₂ 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 MnO₂ present. Therefore, by titrating iodide with sodium thiosulphate until the solution turns colourless will indicates the amount of DO. The amount of DO was calculated using this value, which are 4.34 mg/L.

For the first step in this experiment, all water samples were collected in the Water

Sampling Bottle also known as BOD bottle and stoppered brim full with sample water. The

sample water was allowed to overflow a little before capping as this is crucial to avoid aeration

and additional oxygen into the water samples. Then, Manganous Sulphate Powder Pillow and

Iodide Azide Powder Pillow was added to the water samples and inverted which resulting to

formation of brown orange solution with precipitate called floc indicating the presence of

dissolved oxygen. According to Dissolved Oxygen Water Quality Test Kit Instruction Manual by

La Motte , immediately upon formation of the precipitate, the oxygen in the water oxidizes an

equivalent amount of the manganous hydroxide to brown-coloured manganic hydroxide. For

every molecule of oxygen in the water, four molecules of manganous hydroxide is converted to

manganic hydroxide.

Then a Sulfamic Acid Powder Pillow was added to the water samples and the bottle was

inverted several times. This reagent is added to dissolve the floc and leaving only the orange-

yellow solution in the bottle. The acid converts the manganic hydroxide to manganic sulfate. At

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this point, the sample is fixed and can be stored for up to eight hours if kept in a cool, dry place.

Simultaneously, iodine from Iodide Azide Solution is oxidized by manganic sulfate, releasing

free iodine into the water. Since the manganic sulfate for this reaction comes from the reaction

between the manganous hydroxide and oxygen, the amount of iodine released is directly

proportional to the amount of oxygen present in the original sample. The release of free iodine is

indicated by the sample turning a orange-yellow colour. After that, 100 ml of this water sample

solution was transferred into a conical flask and titrated with sodium thiosulphate solution and

swirled until the yellow colour turns pale. Then, starch was added as an indicator and the

solution turns blue black upon addition. The water sample in the conical flask was further titrated

until the colour turns colourless which indicates the endpoint of the titration. The sodium

thiosulfate reacts with the free iodine to produce sodium iodide. When all the iodine has been

converted the sample changes from yellow to colourless. The reason for titrating until the

solution turns pale earlier is to let the starch stays dark blue right up until it goes clear, unlike

most titrations where the colour gradually moves toward the endpoint. Therefore, it is easy to

become complacent during the titration and add an excess amount of titrant which overshooting

the endpoint, thinking that you are far from the endpoint because the colour is not changing.

Another reason was starch can be partially decomposed by a large amount of iodine. Therefore,

the starch should not be added until the bulk of the iodine has been reduced.

The amount of dissolved oxygen in lake water is calculated to be 4.34 mg/L when using

300 mL of water sample , while 4.2 mg/L when using 60 mL water sample , whereas de-ionized

water contained the most dissolved oxygen which is 4.34 mg/L. Based on the Proposed National

Water Quality Standards for Malaysia prepared by Yayasan Sabah Forest Management Area,

these water samples can be categorized into a few classes which are class I, IIA, IIB, III, IV, and

V based on their Dissolved Oxygen (DO) value (see appendix).

For type I classes the value of DO ranged from 7 and above. Based on this experiment, it

was proven that one of the water sample, which is de-ionized water falls into this category. This

type I class represents the excellent water quality which meets the most stringent requirement for

human health and aquatic life protection.

Nevertheless, our experiment is nowhere near perfect. There are some errors made during the

experiment. In my opinion, the dissolved oxygen value of lake water is not an actual value due to

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the presence of air bubbles inside the water sample in the water sampling bottle. Besides that, the

water sample is not collected at near the middle of the lake or at arm’s length below the water

surface. This is because dissolved oxygen concentrations may change drastically in lakes

depending upon depth and distance from shore. The starch solution added is also not accurately

of 1ml for each drop and there might also be the presence of air bubbles in the delivery tube,

resulting in the accuracy of the results obtained. These problems adds to reasons of my

perspective that the results obtained were not of the precise value.

10.CONCLUSION

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.

11.RECOMMENDATIONS

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

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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

12.REFFERENCES

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. Determination of dissolved oxygen by Winkler titration.

http://www.core.org.cn/NR/rdonlyres/Earth--Atmospheric--and-Planetary-Sciences/12-

097January--IAP--2006/35FAFFC3-135B-4266-93B4-2144F7487004/0/dissolved_oxygen.pdf

search on : 10 may 2013

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

(FL):CRC Press.

4. Sawyer, C. N., McCarty, P. L., and Parkin, G. F.(2003) Chemistry for Environmental

Engineering, 5th ed., McGraw Hill

5. Anonymous (2011), How to Measure Dissolve Oxygen, Department of Ecology State of

Washington retrieved from http://www.ecy.wa.gov

search on : 10 may 2013

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6. Study and Interpretation of the Chemical Characteristics of Natural Water,(1970) United

States Geological Survey, Water Supply Paper 1473,

13.APPENDICES

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