1.0 Abstract/Summary

Regarding to the objective of this experiment which are to determine the value of

Biochemical Oxygen Demand (BOD) in the water sample and to investigate the amount of

heavy metal in the sample water, we involved the use of Dissolved Oxygen meter (DO meter)

and Spectrophotometer. DO meter is equipment that measures the amount of oxygen

available in dissolved form in the water while Spectrophotometer is used to measure the

composition of heavy metals in it.

In the first experiment, we prepare dilution water and mix it with 50 ml of water sample

and we measured the amount of dissolved oxygen is by using DO meter. Then, the sample

water is left for five days (BOD5) in air incubator at 20 °C before we reread the amount of

dissolved oxygen. While in the second experiment, the amount of Chlorine, Sulphate, Iron,

Phosphorous and Chromium Hexavalent was determine by using Spectrophotometer with

some addition of reagents

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2.0 Introduction

2.1 BASIC WATER QUALITY 1: DETERMINATION OF BOD

Microorganisms such as bacteria are responsible for decomposing organic waste. When

organic matter such as dead plants, leaves, grass clippings, manure, sewage, or even food

waste is present in a water supply, the bacteria will begin the process of breaking down

this waste. When this happens, much of the available dissolved oxygen is consumed by

aerobic bacteria, robbing other aquatic organisms of the oxygen they need to live.

Biological Oxygen Demand (BOD) is a measure of the oxygen used by

microorganisms to decompose this waste. If there is a large quantity of organic waste in

the water supply, there will also be a lot of bacteria present working to decompose this

waste. In this case, the demand for oxygen will be high (due to all the bacteria) so the

BOD level will be high. As the waste is consumed or dispersed through the water, BOD

levels will begin to decline.

Nitrates and phosphates in a body of water can contribute to high BOD levels.

Nitrates and phosphates are plant nutrients and can cause plant life and algae to grow

quickly. When plants grow quickly, they also die quickly. This contributes to the organic

waste in the water, which is then decomposed by bacteria. This results in a high BOD

level. The temperature of the water can also contribute to high BOD levels. For example,

warmer water usually will have higher BOD level than colder water. As water

temperature increases, the rate of photosynthesis by algae and other plant life in the water

also increases. When this happens, plants grow faster and also die faster. When the plants

die, they fall to the bottom where they are decomposed by bacteria. The bacteria require

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oxygen for this process, so the BOD is high at this location. Therefore, increased water

temperatures will speed up bacterial decomposition and result in higher BOD levels.

When BOD levels are high, dissolved oxygen (DO) levels decrease because the

oxygen that is available in the water is being consumed by the bacteria. Since less

dissolved oxygen is available in the water, fish and other aquatic organisms may not

survive.

2.2 BASIC WATER QUALITY 2: DETERMINATION OF HEAVY METAL

CONTENTS IN WATER SAMPLE

Toxic metals can be present in industrial, municipal, and urban runoff, which can be

harmful to humans and aquatic life. Increased urbanization and industrialization are to

blame for an increased level of trace metals, especially heavy metals, in our waterways.

There are over 50 elements that can be classified as heavy metals, 17 which are

considered to be both very toxic and relatively accessible. Toxicity levels depend on the

type of metal, it’s biological role, and the type of organisms that are exposed to it.

The heavy metals linked most often to human poisoning are lead, mercury, arsenic

and cadmium. Other heavy metals, including copper, zinc, and chromium, are required by

the body in small amounts, but can be toxic in larger doses.

Heavy metals in the environment are caused by air emissions from coal-burning

plants, smelters, and other industrial facilities; waste incinerators; process wastes from

mining and industry; and lead in the household plumbing and old house paints. Industry

is not totally to blame, as heavy metals can sometimes enter the environment through

natural processes, in some parts, naturally occurring geologic deposits of arsenic can 3

dissolve into ground water, potentially resulting in unsafe levels of this heavy metal in

drinking water supplies in the area. Once released to the environment, metals can remain

for decades or centuries, increasing the likelihood of human exposure.

In addition to drinking water, we can be exposed to heavy metals through inhalations

of air pollutants, exposure to contaminated soils or industrial waste, or consumption of

contaminated food. Because of contaminated water, food sources such as vegetables,

grains, fruits, fish and shellfish can also become contaminated by accumulating metals

from the very soil and water it grows from.

3.0 Aims/Objectives

3.1 Determination of BOD

To determine the value of Biochemical Oxygen Demand (BOD) in the water

sample.

3.2 Determination of heavy metal contents

To investigates the amount of heavy metal in the waste water.

4.0 Theory

4.1 Determination of BOD

Biochemical oxygen demand (BOD) is a chemical procedure for determining is the

amount of dissolved oxygen needed by aerobic biological organisms in a body of water to

break down organic material present in a given water sample at certain temperature over a

specific time period. It is not a precise quantitative test, although it is widely used as an

indication of the quality of water.4

The amount of oxygen consumed during microbial utilization of organics is called the

biochemical oxygen demand (BOD). The BOD is measured by determining the oxygen

consumed from a sample placed in an air-tight container and kept in a controlled

environment for a pre-selected period of time. In the standard test, a 300mL BOD bottle

is used and the sample is incubated at 20°C for 5 days. Light must be excluded from the

incubator to prevent algal growth that may produce oxygen in the bottle. It is used in

water quality management and assessment, ecology and environmental science. BOD is

not an accurate quantitative test, although it could be considered as an indication of the

quality of a water source. To ensure that all other conditions are equal, a very small

amount of micro-organism seed is added to each sample being tested. This seed is

typically generated by diluting activated sludge with de-ionized water.  

The BOD test is carried out by diluting the sample with de-ionized water saturated

with oxygen, inoculating it with a fixed aliquot of seed, measuring the dissolved oxygen

and sealing the sample (to prevent further oxygen dissolving in). The sample is kept at

20°C in the dark to prevent photosynthesis (and thereby the addition of oxygen) for five

days, and the dissolved oxygen is measured again. The difference between the final DO

and initial DO is the BOD. The apparent BOD for the control is subtracted from the

control result to provide the corrected value.

Unseeded BOD, mg/L = [DO initial (D1) –DO final (D2)] / P

Where, P = Volume of sample water / (Volume of dilution water + volume

of sample water)

4.2 Determination of heavy metal contents

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Heavy metals in wastewater come from industries and municipal sewage, and they are

one of the main causes of water and soil pollution. Accumulation of these metals in

wastewater depends on many local factors such as type of industries in the region,

people’s way of life and awareness of the impacts done to the environment by careless

disposal of wastes. As the focal point, wastewater treatment plants are expected to control

the discharge of heavy metals to the environment.

The content of heavy metals in waste is primarily a consequence of the intended use

of heavy metals in industrial products. At the end of their useful life all products will end

up in waste to the extent they are not attractive for recycling. Heavy metals may,

however, also be lost to waste during production and use phases. Losses in the

manufacturing process are often disposed of as manufacturing waste, while products may

be exposed to wear and tear inclusive corrosion during the use phase.

5.0 Apparatus

5.1 Determination of BOD 5.1 Determination of heavy metal

contents

1. Deionised water

2. Water samples (2 different

samples)

3. BOD nutrient

4. Dilution water tank

5. 300 ml BOD bottles

6. Dissolved oxygen meter

7. Air incubator

1. Sample cells

2. Reagents

3. DPD Total Chlorine Powder Pillow

4. DPD Free Chlorine Powder Pillow

5. SulfaVer 4 Reagent Powder Pillow

6. PhosVer 3 Phosphate Powder Pillow

7. FerroVer Iron Reagent Powder

Pillow

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8. Gloves 8. ChromaVer® 3 Reagent Powder

Pillow

9. Spectrophotometer

10. Water samples

6.0 Experimental Procedure

6.1 Determination of BOD

Dilution water preparation

1. 3 L of deionised water is poured into a dilution water tank.

2. Then, the deionised water is added with a sachet of BOD nutrient and shook well.

Preparation of BOD test for sample 1 and 2

1. First, 50ml of water sample is poured into a 300ml BOD bottle and labelled as

Sample 1.

2. Dilution water is then poured into the BOD bottle until it is full enough.

3. Next, the probe of the Dissolved Oxygen meter is soaked into the bottle and the

stirrer is switched on.

4. The diluted sample is left to be stabilised and the DO value for Day 1 is recorded.

5. The BOD bottle is then cap with the ground-glass stopper.

6. Then, dilution water is added to the bottle before it is covered using aluminium

foiled. The BOD bottle is foiled with aluminium foil before stored in the incubator

at standard temperature of 20C.

7. The samples in left incubated for a period of 5 days and the final DO value is

measured and recorded on Day 5.7

8. Step 1 until 7 is then repeated for different water sample and noted as Sample 2.

6.2 Determination of heavy metal contents

1. The spectrophotometer is switched on and the required test is selected.

2. Two sample cells are filled with10ml of water sample 1 each.

3. Preparation and testing for the metals are using spectrophotometer.

4. The content of a DPD Total Chlorine Powder Pillow is added into one of the

sample cell containing water sample 1.Then the sample is swirled to allow mixing.

5. The prepared sample is inserted to the cell holder of the spectrophotometer and

the timer is started. (3 minutes reaction period begun)

6. After 3 minutes, the prepared sample is taken out from cell holder and replaced by

the blank sample (the blank sample cell must be wiped before inserted to cell

holder).

7. As the blank sample is in the cell holder, the instrument is set to ZERO and

displaying 0.00 mg/L Cl2.

8. All the procedure for chlorine total is repeated but using a different reagent – a

DPD Free Chlorine Powder Pillow for chlorine free, SulfaVer 4 Reagent Powder

Pillow foe sulphate, PhosVer 3 Phosphate Powder Pillow for phosphorus,

FerroVer Iron Reagent Powder Pillow for iron, and ChromaVer® 3 Reagent

Powder Pillow for chromium hexavalent.

9. This overall procedure is then repeated for water sample 2.

10. Results are recorded and tabulated.

7.0 Results

7.1 Determination of BOD8

Sample Water DO Reading (day 1) DO Reading (day 6)

1 9.56 mg O2/l (26.61°C) 8.09 mg O2/l (20.65 °C)

2 9.36 mg O2/l (26.38 °C) 8.42 mg O2/l (20.71°C)

7.2 Determination of heavy metal contents

Heavy Metals Reagent used Sample 1 Sample 2

Chlorine Total DPD Free Chlorine

Powder Pillow

0.02 mg/L Cl2 0.28 mg/L Cl2

Chlorine Free DPD Total Chlorine

Powder Pillow

0.09 mg/L Cl2 0.03 mg/L Cl2

Sulphate SulfaVer 4 Reagent

Powder Pillow

7.00 mg/L SO42- 3.00 mg/L SO4

2-

Phosphorus PhosVer 3 Phosphate

Powder Pillow

0.08 mg/L PO43- 0.13 mg/L PO4

3-

Iron FerroVer Iron Reagent

Powder Pillow

0.08 mg/L Fe 0.06 mg/L Fe

Chromium

Hexavalent

ChromaVer® 3 Reagent

Powder Pillow

0.01 mg/L Cr6+ 0.01 mg/L Cr6+

8.0 Sample Calculations

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The BOD of the water samples is the difference between the final DO and initial DO times

the dilution factor.

BOD, mg/L = (D1 – D2) / P

P = Volume of sample water / (Volume of dilution water + volume of sample

water)

In this experiment, P = 50 mL / 300 mL = 1/6

SAMPLE 1 SAMPLE 2

BOD = (9.56 – 8.09) mg O2/L X 6

= 8.82 mg O2/L

BOD = (9.36 – 8.42) mg O2/L X 6

= 5.64 mg O2/L

9.0 Discussion

9.1 Determination of BOD

Biological Oxygen Demand (BOD) is one of the most common measures of pollutant

organic material in water. BOD indicates the amount of purescible organic matter

present in water. Therefore, a low BOD is an indicator of good quality water, while a

high BOD indicates polluted water. The BOD test serves an important function in

stream pollution-control activities. It is a bioassay procedure that measures the

amount of oxygen consumed (Dissolved Oxygen) by living organisms while they are

utilizing the organic matter present in water. Dissolved Oxygen (DO) is the actual

amount of oxygen available in dissolved form in the water. When the DO drops below

a certain level, the life forms in that water are unable to continue at a normal rate.

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There are two part of experiment in basic water quality. In the first experiment

we have to determine the value of Biochemical Oxygen Demand (BOD) in two

different water samples. For results of the BOD test to be accurate, much care must be

taken in the actual process. For example, additional air cannot be introduced and

therefore, we covered the sample with aluminium foil. Temperature also must be

20°C, which is the usual temperature of bodies of water in nature. However, while

doing this experiment and it can be observed trough result data as we only able to

maintain the temperature at around 26 °C for day 1. Supposed that this experiment

should be done in five (5) day as it indicates five-day BOD (BOD-5) test is used in

environmental monitoring, however we only manage to take the DO Reading on day 6

as the laboratory is closed in day 5. This test is utilized as a means of stating what

level of contamination from pollutants is entering a body of water. In other words, this

test measures the oxygen requirements of the bacteria and other organisms as they

feed upon and bring about the decomposition of organic matter. Time and

temperature, as well as plant life in the water, will have an effect on the test. A high

BOD indicates the presence of a large number of microorganisms, which suggests a

high level of pollution.

9.2 Determination of heavy metal contents

In the second part of this experiment, we have to investigate the amount of heavy

metal in the sample water.

Free chlorine (chlorine gas dissolved in water) is toxic to fish and aquatic

organisms, even in very small amounts. However, its dangers are relatively short-

lived compared to the dangers of most other highly poisonous substances. That is

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because chlorine reacts quickly with other substances in water (and forms combined

chlorine) or dissipates as a gas into the atmosphere. The free chlorine test measures

only the amount of free or dissolved chlorine in water. The total chlorine test

measures both free and combined forms of chlorine.

If water contains a lot of decaying materials, free chlorine can combine with

them to form compounds called trihalomethanes or THMs. Some THMs in high

concentrations are carcinogenic to people. Unlike free chlorine, THMs are persistent

and can pose a health threat to living things for a long time.

Phosphates stimulate the growth of plankton and water plants that provide

food for fish. This may increase the fish population and improve the waterway’s

quality of life. If too much phosphate is present, algae and water weeds grow wildly,

choke the waterway, and use up large amounts of oxygen. Many fish and aquatic

organisms may die.

Hach Company makes kits to test for the presence of phosphate. You’ll

probably use the cube kit that measures the most common form—orthophosphate—or

the color disk that determines orthophosphate and metaphosphate. A total phosphate

kit measures all three types of phosphates. Some values for total phosphate-

phosphorus are given below.

Most of the synthetic detergents designed for the household applications

contain large amounts of polyphosphates as builders. Many of them contain 12-13%

phosphorous or over 50% poly-phosphates. The organisms involved in the biological

processes of wastewater treatment require phosphorous for reproduction and synthesis

of new cellular material.

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Sulfate is one of the major anions occurring in natural waters. Sulfates form hard

scales in boilers and heat exchangers. Sulfate assumes significance in water and

wastewater, as it is associated with odor and sewer-corrosion problems resulting from

the reduction of sulfate into hydrogen sulfide under anaerobic conditions. Sulfate in

water or wastewater can be estimated by precipitation with barium chloride, acidified

with hydrochloric acid.

Hexavalent chromium is accumulated by aquatic species by passive diffusion.

In general, invertebrate species, such as polychaete worms, insects, and crustaceans

are more sensitive to the toxic effects of chromium than vertebrates such as some fish.

Microorganisms Hexavalent chromium is toxic to microorganisms; a property utilised

in chromium-based biocides. In general, toxicity for most microorganisms occurs in

the range of 0.05 - 5 mg chromium/kg of medium.

10.0 Conclusion

As for conclusion, the BOD for sample 1 is 8.82 mg O2/L and for sample 2 is 5.64 mg

O2/L. Therefore, the water was not badly polluted as D1 – D2 value for both sample 1

and 2 less than 2 mg O2/L. Based on results, it is proven that the temperature of the

water can also contribute to high BOD levels.

Meanwhile, the value of Chlorine Total in sample 1 is 0.02 mg/L Cl2 and sample 2 is

0.28 mg/L Cl2. The value of Chlorine Free in sample 1 is 0.09 mg/L Cl2 and sample 2 is

0.03 mg/L Cl2. The value of Sulphate in sample 1 is 7.00 mg/L SO42- and sample 2 is

3.00 mg/L SO42-. The value of Phosphorus in sample 1 is 0.08 mg/L PO4

3- and sample 2

is 0.13 mg/L PO43-. The value of Iron in sample 1 is 0.08 mg/L Fe and sample 2 is 0.06

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mg/L Fe. Lastly, the value of Chromium Hexavalent in sample 1 is 0.01 mg/L Cr6+ and

sample 2 is 0.01 mg/L Cr6+.

Heavy metal always present in waters but human or any living organisms only need a

little dose of each element. Thus if the amount of heavy metal is overdose, it will cause

harm to health. Heavy metals are a cumulative toxin that the body cannot dispose of and

they accumulate to harmful levels with repeated exposure. In larger concentrations these

metals may have detrimental health effects on man. Thus, the unknown content of water

can make it unsuitable for many uses such as drinking, boiler feed, or process uses

where high degree purities are required.

11.0 Recommendations

Dilution water may be prepared immediately before use.

To avoid dust or dirt contamination while allowing oxygenation, use a proper

equipment to cover the bottle opening.

Care is taken to ensure that dilution water is oxygen saturated.

Each BOD bottle is filled by slowly adding sufficient dilution water so that the

stopper can be inserted without leaving an air bubble but not so much that there is

overflow.

Completely fill two bottles with dilution water to be incubated as blanks

Label each bottle carefully as to sample and volume used.

Ensure that the spectrophotometer is wash with distilled water before taking the DO

reading.

The temperature should be monitor to 20 °C.

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12.0 References

12.1 Reference book:

o Laboratory Manual; Chemical Engineering Laboratory (CHE 315), Faculty of

Chemical Engineering, UiTM Shah Alam.

o Fahid, R. Biological Wastewater Treatment-Lecture Notes in Advanced Sanitary

Engineering, Faculty of Engineering. Islamic University of Gaza (2005-2006.)  

1.2 Online journals:

o http://www.ungiwg.org/openwater/?q=node/98

o http://www.indiana.edu/~bradwood/eagles/bod.htm

o http://watersence/heavymetals.html

o http://www.answers.com/topic/biochemical-oxygen-demand

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13.0 Appendices

13.1 Determination of BOD

Dissolved oxygen meter

13.2 Determination of heavy metal contents

Spectrophotometer

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