1

Water Hyacinth (Eichhornia crassipes), Fingernail

clam (Sphaerium corneum) and Water Cabbage
(Pistia stratiotes): A Natural Approach to Sewage
Waste Water Treatment

An Investigatory Project Submitted

as an Entry to the 2013
Regional ASEP Competition

Researchers:

IVY JOY S. OBTIAL

JANN THROI M. DIMAYUGA
CHRISTIAN P. NARIO

BAYORBOR NATIONAL HIGH SCHOOL

Mataasnakahoy, Batangas

OLIVER R. GUEVARRA
Research Adviser

ANNABELLE E. DE ROXAS
Principal II
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Part II

Table of Contents

I. Title Page 1
II. Table of Contents 2
III. Abstract 3
IV. Research Plan 4-13
a. Experimental design
b. Experimental procedure
c. Analysis of study
V. Introduction 14-25
a. Background of the study
b. Statement of the problem
1. General Objective
2. Specific Objectives
c. Significance of the Study
d. Scope and Limitations of the Study
e. Review of Related Literature
VI. Results and Discussions 26-29
Analysis of Variables
VII. Conclusions and Recommendations 30
VIII. References/Bibliography 31
IX. Acknowledgements 32
X. Appendices 33-49
Tables
Laboratory testing of dissolved oxygen
Chemical oxygen demand test
Others
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Part III

ABSTRACT

This study was conducted to develop a Natural Approach in Treating Sewage Wastewater

using Water hyacinth, Fingernail clam and Water cabbage.

Wastewater was taken from Barangay Lumanglipa, Mataasnakahoy, Batangas and was

tested for physical-chemical and Bacteriological analysis. The variables were collected and

prepared for identification. The data collected were subjected to Student t-test.

The Experimental Design consists of experimental and controlled set-up with fifteen pail

containers for three trials. Each Trial has five treatments one for the controlled set-up and four

for the experimental set-up. The experiment was conducted in three replicates. The containers

contain 3L of freshwater sample and placed in the same area. The set-up was done for seven

days. The controlled set-up is composed of 3L freshwater samples labeled 1A, 2A and 3A.

After seven days, it was found out that the combination of 200 pieces of fingernail clam,

20 pieces of water cabbage and 20 pieces of water hyacinth is the most effective in treating

sewage wastewater. It was concluded that the higher the number of variables the greater the

possibilities in treating sewage wastewater. The researchers recommend that further study on

other parameters should be made in water sample to determine its effectiveness as biofilters.
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Part IV
Research Plan

This part discussed the material and methods used as well as the entire procedure of the

study.

A. Experimental Design

The experimental design consists of experimental set – up and controlled set up with

fifteen pale containers for three trials. Each trial has five treatments, one for the controlled set-up

and four for the experimental set –up of water hyacinth (Eichhornia crassipes), fingernail clam

(Sphaerium corneum) and water cabbage (Pistia stratiotes) and the combination of the three

variables. The experiment was conducted in three replicates. The pail containers contains same

amount of freshwater samples from the lake and placed in a same area. The set-up was done for

seven days.

The controlled set up for three trials is composed of 3L freshwater samples labelled 1A,

2A and 3A.

The following is true in three trials:

In Trial One, the experimental set ups are composed of four pail containers. Container

labelled 1B has 100 pieces of Sphaerium corneum and 3L of freshwater samples. Container

labelled 1C has 5 pieces of Pistia stratiotes plant and 3L of freshwater samples. Container

labelled 1D has 5 pieces of Eichhornia crassipes plant and 3L of freshwater samples. Container

labelled 1E has the combination of 100 pieces Sphaerium corneum, 5 pieces of Pistia stratiotes

and Eichhornia crassipes plant.

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On Trial Two, the experimental set ups are composed of four pail containers. Container

labelled 2B has 150 pieces of Sphaerium corneum and 3L of freshwater samples. Container

labelled 2C has 10 pieces of Pistia stratiotes plant and 3L of freshwater samples. Container

labelled 2D has 10 pieces of Eichhornia crassipes plant and 3L of freshwater samples. Container

labelled 2E has the combination of 100 pieces Sphaerium corneum, 10 pieces of Pistia stratiotes

and Eichhornia crassipes plant.

On Trial Three, the experimental set ups are composed of four pail containers. Container

labelled 3B has 200 pieces of Sphaerium corneum and 3L of freshwater samples. Container

labelled 3C has 15 pieces of Pistia stratiotes plant and 3L of freshwater samples. Container

labelled 3D has 15 pieces of Eichhornia crassipes plant and 3L of freshwater samples. Container

labelled 3E has the combination of 100 pieces Sphaerium corneum, 15 pieces of Pistia stratiotes

and Eichhornia crassipes plant.

Similar trials and set up was done on the second and third replicates.

B. Experimental Procedure

Preparation of Water Samples

The water sample was taken from the lake at Lumanglipa Mataasnakahoy,

Batangas. This area was chosen for the reason that water pollution was greatly observable

in this area due to greater number of population is present there. The water sample was

then placed in a sterilized container and brought to the testing laboratory.

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Preparation / Collection of Materials

 Gathering/Collection of water hyacinth, fingernail clam and water cabbage- The

water hyacinth, water cabbage and fingernail clam were collected at the coastal area

of Brgy. Nangkaan, Mataasnakahoy, Batangas wherein they are abundant. The

materials were brought to the school laboratory for washing, soaking and identifying.

 Washing- The water hyacinth and water cabbage were washed thoroughly with

running water to remove all the unwanted impurities, adhering sand particles and

debris not necessary in the conduct of the experiment.

 Soaking- The fingernail clam was soaked in water overnight to remove the unwanted

impurities and to release and clean the dirt inside the clam.

 Identifying- The species of water lily were delivered at the School laboratory for

identification a day after the specimens were gathered. The identification last for 3

days.

Determining Chemical Content of Polluted Water

Pre-treated and post treated water samples were brought to the Optimal

Laboratory Incorporated to test its physical-chemical test particularly pH, biochemical

Oxygen Demand (BOD), Chemical Oxygen Demand (COD), oil and grease, surfactants,

ammonia, TSS and color and to the Lipa City Water District for biological test

particularly total coliform, fecal coliform and Heterotrophic Plate Count (HPC).
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Analysis of Variables

To ensure the effectivity and reliability of results, the following test were done by

the researchers:

Bacteriological Test

*Methods for Detection and estimation of Coliform Organisms

1. Heterotrophic Plate Count (HPC)

1.1 Procedure. Mix the sample by making 25 complete up and down or back

and forth movements, or about 0.3m (1ft.) in 7 seconds. Pipette 1 mL or

.01 mL of the sample into a sterile Petri dish. Pour aseptically 10-12 mL

meted agar medium that has been allowed to cool in a water bath 42- 450

C. Rotate a plate over a smooth surface first in one direction, then in the

opposite direction or by rotating and tilting the dish. Take care not to

splash mixture over the edge of the dish. Allow the plates to solidify

(within 10 minutes) on a level surface. Invert the plates and incubate at 35

± 0.50 C for 48 ± 3 hours. Check sterility of medium and dilution water

blanks by having control plates for each series of samples.

1.2 Counting and Recording

HPC= Total number of colonies or average number of colonies (if

duplicate plate of the same dilution) per plate times the reciprocal of the

dilution used.
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In preparing plates, volume of samples that will give from 30 to 300

colonies per plate should be planted. If there are less than 30 colonies per

plate count the actual number of colonies.

If there is no plate with 30 to 300 colonies and one and more than

colonies, use the plate(s) having a count nearest to 300 colonies

1.2.1 If there is no plate with 30 to 300 colonies and one

and more than 300 colonies, use the plate(s) having

a count nearest 300 colonies

1.2.2 If plates from all dilutions have no colonies, report

the count as zero or no growth.

1.2.3 If the number of colonies per plate far exceeds 300,

do not report the result as too numerous to count

(TNTC).

1.2.4 If there are fewer than 10 colonies per square

centimetre (co/cm2), count colonies in 13 squares (

of colony counter) having representative colony

distribution. If possible, select 7 consecutive

squares horizontally across plate and 6 consecutive

colonies x5 to the compute the estimated colonies

per plate when the area of the plate is 65 cm2.

1.2.5 When there are more than 10 colonies per cm2

count 4 representative squares, take the average

count per square cm and multiply by appropriate

 9

factor to estimate the colonies per plate usually

about 65.

1.2.6 When bacterial counts crowded plates are greater

than 100 colonies/cm2, report the result as greater

than (>) 6,500 times the highest dilution plated.

1.2.4.1 Heterotrophic plate counts can be carried out in dilution of

1:10, 1: 100, 1: 1000, 1;10,000

a. Place 9mL of sterile buffered water in each of the sterile test

tubes.

b. Mix sample by making 25 complete up and down or back and

forth movements, or about 0.3m (1ft) in seven seconds.

c. To the first test tube, pipette 1 mL of water sample and mix.

d. With the same pipette, transfer 1 mL from the first tube to the

second and mix.

Continue the same procedure until the water sample is transferred

from the third to the fourth and the contents are well mixed. This

procedure gives 1:10, 1:100, 1:1000, 1:10,000 dilution of the

original sample.

1.3 Reporting the Result

Report Heterotrophic Plate Count as colonies per mL (cfu/mL)

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2. Multiple Tube Fermentation Technique (MTFT) for members of the Coliform

group of organism

2.1 Presumptive Test. Mix sample by making 25 complete up and down or

back and forth movements, or about 0.3m (1ft) in 7 seconds. With a sterile

10 mL pipette, inoculate 20 mL of water sample into each 5 tubes of

primary lauryl tyrptose broth. It is advisable to shake gently to distribute

the sample uniformly throughout the medium. Incubate the tubes at 35 ±

0.50C for 24 hours. After the end of the 24 hour incubation period,

examine each tube for the presence of gas. If present, gas can be seen in

the Durham tube; if none is visible, gently shake the tube, if any

effervescence (stream of tiny bubbles) is observed, the tube should be

considered positive. Enter the number tubes in a table. If no gas has

developed, reincubate tubes for further 24-hr period. At the end of this

period check the tube again for gas production as no.4 above. Enter the

number of positive tubes in the table. Use pure culture of E.coli and

Staphylococcus aureus for positive and negative controls. Include method

blank per batch of analysis.

2.2 Confirmatory test. Gently shake or rotate primary fermentation tube

showing gas. Using sterile loop, transfer one or two drops each

presumptive tube into EC medium broth and BGLB. Mix by gently

shaking. To confirm the presence of total coliform, incubate BGLB from

each presumptive positive tube for 24 hours at 35 ± 0.50C. After 24 hours,

enter the number of positive tubes in a table. Re-incubate negative tubes

 11

for a further 24-hour period. At the end of this period, check the tubes

again for gas production. Enter the number of positive in a table. Gas

production at the end of either 24 or 48 hours incubation is confirmed to

be due to the presence of Coliform the sample. Incubate EC tube at 44.5 ±

0.20C for 24 hours. If the end of 24 hour gas is present in tubes, the

presence of thermo tolerant (fecal) Coliform is confirmed. Record the

number of positive tubes in the table.

3. Preparation of Media

3.1 Presumptive Phase

3.1.1 Lauryl tyrptose broth (LTB) dehydrated. Weigh 106.8 g

dehydrated medium (refer table 2). Dissolve in 100 mL of distilled

water. Warm slightly to dissolve completely. Distribute into test

tubes with fermentation vials (10mL each). Sterilized for 15

minutes at 15 lbs. pressure. Final pH= 6.8 ± 0.2 after sterilizing.

Dehydrated
Amount of Volume of
LTB required
Inoculum Medium in Medium +
(g/L)
(mL) Tube (mL) Inoculum (mL)
1 10 or more 11 or more 35.6
10 10 20 71.2
10 20 30 53.4
20 10 30 106.8
100 50 150 106.8
100 35 135 137.1
100 20 120 213.6

3.2 Confirmed Phase

3.2.1 Brilliant green Lactose Bile Broth (BGLB), dehydrated. Weigh

40g BGLB broth. Dissolve in 1000 mL of distilled water. Warm

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slightly to dissolve completely. Distribute into test tubes with

fermentation vials (10mL each). Sterilized for 15 minutes at 15 lbs

pressure pH=7.2 ± 0.2.

3.2.2 EC Medium. Weigh 37 grams of EC medium. Dissolve in 1000

mL of distilled water. Distribute into test tubes with fermentation

vials (10mL each). Sterilize for 15 minutes lbs pressure. Final pH=

6.9 ± 0.1.

3.3 Completed Phase

3.3.1 HPC

3.3.1.1 Nutrient Agar Dehydrated. Weigh 23 grams dehydrated

nutrient agar medium. Dissolve in 1000 mL distilled water.

Heat to boiling to dissolve completely. Sterilized for 15

minutes at 15 lbs pressure. Final pH = 6.8 ± 0.2. Distribute into

test tubes (5mL each).

3.3.1.2 Plate Count Agar. Same as nutrient agar but pH is 7.0 ± 0.2.

3.3.2 Dilution Water

3.3.2.1 Buffered Water. Stock phosphate buffer solution. Dissolve 34g

potassium dihydrogen phosphate (KH2PO4) in 500 mL reagent

grade water, adjust to pH 7.2 ± -0.5w/ 1 N sodium hydroxide

(NaOH) and dilute 1L reagent grade water. Add 1.25 mL stock

phosphate buffer solution and 5.0 mL magnesium chloride

solution (81.1g Mg Cl2.6H2O/L reagent.

*SOURCE: National Reference Laboratory.East Avenue Medical Center.Department of Health.East Avenue, Diliman Quezon City.
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Physical Chemical Test

Testing the Effectivity of the Materials

In order to evaluate the effective used of Fingernail clam (Sphaerium corneum),

Water cabbage (Pistia stratiotes) and Water hyacinth (Eichhornia crassipes), the samples

were tested in polluted water from the source.

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Part V

Introduction

Pollution happened everywhere. The increasing number of people living along the lake or

bay cause pollution because many of them thrown their garbage along the lake specifically toxic

waste or chemical from factories, human waste and other chemicals that leads into sickness

because of contamination and water pollution. On the other hand, sewage water treatment plants

have been to be ineffective in removing household products and other water pollutants.

Our government as well as other non-government organization did their best to make a

solution to prevent this kind of pollution and until now they are still trying and searching for the

best prevention.

As a concerned citizen of our country, the researchers are also trying to make an

alternative solution to save our environment particularly the bodies of water. The researchers

want to prove that the Fingernail clam (Sphaerium corneum), Water cabbage (Pistia stratiotes)

and Water hyacinth (Eichhornia crassipes) can be used to remove impurities or pollutants in

bodies of water. It is a natural approach to sewage treatment waste water, it serve as a Biofilter to

our lake because it will avoid a Nitrate and ammonia Levels from sewage treatment waste water

and pollution will be controlled.

Through the cooperation of the government, support of the responsible citizen and these

Fingernail clam, Water Cabbage and Water lilies used as an alternative solution to our polluted

bay or water there is a possibility of reducing water pollution in our environment.

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A. Background of the Study

Waste Water

Wastewater, also written as waste water, is any water that has been adversely affected in

quality by anthropogenic influence. Municipal waste water is usually conveyed in a combined

sewer or sanitary sewer, and treated at a waste water treatment plant or septic tank. Treated

waste water is discharge into receiving water via effluent sewer.

Sewage is the subset of waste that is contaminated with feces and urine, but is often used

to mean any waste water. Sewage includes domestic, municipal or industrial liquid waste

products disposed of, usually via a pipe or sewer (sanitary or combined), sometimes in a

cesspool emptier.

Sewage is the physical infrastructure, including pipes, pumps, screens, channels, etc. used

to convey sewage from its origin to the point of eventual treatment or disposal. It is found in all

types of sewage treatment, with the exception of septic system , which treat sewage on – site.

Waste water or sewage can come from human waste (used toilet paper or wipes, urine, or

the other bodily fluids), also known as black water; cesspit leakage; septic tank discharge;

sewage treatment plant discharge; industrial waste; etc.

Sphaerium corneum,, Also known as the European Fingernail clam is very small

freshwater clam, an aquatic bivalve mollusk in the family Sphaeriidae, the fingernail clams.

These small clams are found in shallow, freshwater habitats with slow moving waters,

including freshwater lakes, rivers and creeks. As with most bivalves, S. corneum is mainly a

filter feeder and thus prefers more eutrophic waters that provide a greater food source. These

clams have exhibited a unique ability to climb up plants and structures around their habitat to

find more optional locations for feeding.

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Pistia stratiotes

Pistia is a genus of aquatic plant in the arum family, Araceae. The single species it

comprises, P. stratiotes, is often called water Pistia is a genus of aquatic plant in

the arum family, Araceae. The single species it comprises, P. stratiotes, is often called water

cabbage, water lettuce, Nile cabbage, or shellflower. Its native distribution is uncertain, but

probably pan tropical; It is now present, either naturally or through human introduction, in nearly

all tropical and subtropical fresh waterways. The genus name is derived from the Greek word

pistos, meaning "water," and refers to the aquatic nature of the plants.

Based from the study conducted, Pista stratiotes may be used for phytoremediation of

water bodies polluted by heavy metals, chromium and cobalt, in a sustainable way.

Eichhornia crassipes

Water hyacinth is a free-floating perennial aquatic plant or hydrophyte with broad, thick,

glossy and ovate leaves. Water hyacinth may rise above the surface of the water as much as one

meter in height. The leaves are ten to twenty centimeters across, and float above the water

surface. They have long, spongy and bulbous stalks. The feathery, freely hanging roots are

purple black. An erect stalk supports a single spike of 8-15 conspicuously attractive flowers,

mostly lavender to pink in color with six petals. One of the fastest growing plants known, water

hyacinth reproduces primarily by way of runners or stolons, which eventually form daughter

plants. Each plant can produce thousands of seeds each year, and this seeds can remain viable

for more than 28 years. The common water hyacinth is vigorous growers known to double their

population in two weeks.

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Water hyacinth removes arsenic from arsenic contaminated drinking water. It may be

useful tool in removing arsenic from tube well water.

Water hyacinth is also observed to enhance nitrification in waste water treatment cells of

living technology. Their roots zones are super micro-sites for bacterial communities.

Water hyacinth is a common fodder plant in the third world especially Africa though

excessive use can be toxic. It is high in protein (nitrogen) and trace minerals and the goat feces

are a good source of fertilizer as well.

Reports showed that water hyacinth can be used to aid the process of water purification

either for drinking water or for liquid effluent from sewage systems. In a drinking water,

treatment plant water hyacinths have been used as part of the pre-treatment purification step.

Clean, healthy plants have been incorporated into water clarifiers and help with the removal of

small flocs that remain after initial coagulation and floc removal or settling. The result is a

significant decrease in turbidity due to the removal of flocs and also slight reduction in organic

matter in the water (Haider, 1989).

Based from the information and data, the researchers have decided to conduct a scientific

study of using water hyacinth, fingernail clam and water cabbage as a natural approach to

sewage waste water treatment.

B. Statement of the Problem

This study entitled “Water Hyacinth (Eichhornia crassipes), Fingernail clam

(Sphaerium corneum) and Water Cabbage (Pistia stratiotes): A Natural Approach to

Sewage Waste Water Treatment” will answer the following objectives generally:
 18

To develop a natural sewage waste water treatment using water hyacinth

(Eichhornia crassipes), fingernail clam (Sphaerium corneum) and water cabbage (Pistia

stratiotes).

Specifically it aims answer the following:

1. Which among the water hyacinth (Eichhornia crassipes), fingernail clam (Sphaerium

corneum) and water cabbage (Pistia stratiotes) has the ability to reduce water

contamination?

2. What is the effect of the combination of water hyacinth (Eichhornia crassipes),

fingernail clam (Sphaerium corneum) and water cabbage (Pistia stratiotes) in treating

sewage waste water to eliminate water pollutants such nitrates, ammonium,

phosphates and other water pollutants?

3. How much number of water hyacinth (Eichhornia crassipes), fingernail clam

(Sphaerium corneum) and water cabbage (Pistia stratiotes) are needed for the

reduction of water pollutants?

Null Hypothesis

The combination of water hyacinth (Eichhornia crassipes), fingernail clam

(Sphaerium corneum) and water cabbage (Pistia stratiotes) is the best for the natural

approach in treating waste water.

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C. Significance of the Study

This study is important because it will reveal the importance of Hyacinth

(Eichhornia crassipes), Fingernail clam (Sphaerium corneum) and Water Cabbage (Pistia

stratiotes) as a biofilter to reduce water pollution.

Likewise, this project could give fishermen an idea on how to use water hyacinth

(Eichhornia crassipes), fingernail clam (Sphaerium corneum) and water cabbage (Pistia

stratiotes) to clean the bodies of water in each locality at the same time gaining a means

of income in cultivating fingernail clam.

Furthermore, this will also add to the knowledge of other researchers of what

possible species of clams and lilies will be used in treating sewage waste water.

Lastly, this study aims to contribute in the field of science and technology in such

a way of determining a natural biofilters of local lake pollutants.

D. Scope and Limitations

This study entitled “Water Hyacinth (Eichhornia crassipes), Fingernail clam

(Sphaerium corneum) and Water Cabbage (Pistia stratiotes): A Natural Approach To

Sewage Waste Water Treatment” focuses mainly on the use of water hyacinth

(Eichhornia crassipes), fingernail clam (Sphaerium corneum) and water cabbage (Pistia

stratiotes) in treating sewage waste water.

This study conducted at Bayorbor National High School, Bayorbor

Mataasnakahoy, Batangas.

Considering that the scope and limited then the result should be tested or

validated in other areas in order to prove if it can be duplicated.

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E. Review of Related Literature

For the basis of accurate results and acceptance of conclusions to be drawn the

following literature and studies are evidently laid.

FOREIGN STUDIES

Gopal (1987) stated that in sewage systems, the root structures of water hyacinth

(and other aquatic plants) provide a suitable environment for aerobic bacteria to function.

Aerobic bacteria feed on nutrients and produce inorganic compounds which in turn

provide food for the plants. The plants grow quickly and can be harvested to provide rich

and valuable compost. Water hyacinth has also been used for the removal or reduction of

nutrients, heavy metals, organic compounds and pathogens from water.

Haider (1989) reported that water hyacinth can be used to aid the process of

water purification either for drinking water or for liquid effluent from sewage systems. In

a drinking water treatment plant water hyacinth have been used as part of the

pretreatment purification step. Clean, healthy plants have been incorporated into water

clarifiers and help with the removal of small flocs that remain after initial coagulation and

floc removal or settling. The result is a significant decrease in turbidity due to the

removal of flocs and also slight reduction in organic matter in the water.

Williams et al. (1993) estimated that 55 percent of the nearly 300 species of

freshwater mussels are endanger of extinction and only 25 percent are considered stable.

No other group of animals in North America is in such grave danger.

Bogan, (1993); Master (1990); Williams &Neves (1995) stated that the

freshwater mussels (Unionidae) are some of the most imperiled fauna in North America –
 21

from 43% to 72% of the native species have been classified as extinct, endangered,

threatened, or vulnerable.

Eden (1994) considered the requirements for large-scale production of charcoal

briquettes from water hyacinth. He stated that with an energy density of 8.3 GJ/m3 this

would be comparable with the energy density of charcoal at 9.6 GJ/m3. However, for a

plant to produce 40 tons per day of briquettes an area of 12 hectares would be required

for drying the water hyacinth, 1,300 tonnes of wet hyacinth would be required daily and

the climate would need to be one of low humidity and relatively high temperature.

Saelthun (1994) suggested that the rate of water loss due to evapotranspiration

can be as much as 1.8 times that of evaporation from the same surface but free of plants.

This has great implications where water is already scarce. It is estimated that the flow of

water in the Nile could be reduced by up to one tenth due to increased losses in Lake

Victoria from water hyacinth.

Nguyen Van Thu and Nguyen Thi Kim Dong (April 1998) -conducted a study

entitled “Water Hyacinth (Eichhornia Crassipes)as a feed resource for feeding growing

rabbits” which proves that water hyacinths can be used for feeding growing rabbits for

improving nutrient digestibility and Nitrogen retention”. The replacement of water

hyacinth from 40 to 60% (DM basis) to para grass improved the feed utilization, growth

performance and economic returns.

Water Services (Amendment) Act 2012 – provides for the introduction of a

registration and inspection system for domestic wastewater treatment system, including

septic tanks and similar systems. It was introduced to address the European Court of

Justice ruling against Ireland in October 2009, and even more important, to project
 22

ground and surface water quality (particularly drinking water sources) from the risks

posed by malfunctioning system.

Vanden Wymelenberg et al. (2006) reported a large number of genes involved

in breakdown of lignin from the fungus Phanerochaetechrysosporium genome. Thus,

there is also the possibility of expressing such genes in water hyacinth by a transgenic

approach which may further reduce the cost of biofuel production. The use of transgenes

could also underpin conventional breeding to modify biofuel production, in view of

growing public concern over potential environmental damage and health hazards by the

use of fossil fuel, and the limited availability of suitable genes to modify synthesis of

sugars, lignin and lipids in plants, by adopting classical breeding approaches.

Ralph et al. (2006) found that flexible polymerization is possible in plants,

whereby monolignins can be actively substituted by polyphenols. This is turn may not

affect the development process of plants, but will facilitate the extraction of saccharides

needed for biofuel production from the plants. This same strategy can be applied to water

hyacinth, which is naturally low in lignin and thus it will be more effective to extract

fermentable saccharides.

Kidd et al. (2007) concern is growing over the possible effects of exposure to

these contaminants in surface water, regardless of the source. Effect studies have shown

that exposure to only 5 ppt of 17α-ethinylestradiol can result in the collapse of fathead

minnow populations.

Gunnarsson and Petersen (2007) estimated the presence of lignin in aquatic

plants at less than 10%. This would enable them to be processed efficiently into biofuels.
 23

The major hurdle to producing biofuel is the high cost of conversion of cellulose and

hemicellulose into ethanol.

Barber et al. (2007); Lee et al. (2008); Schoenfuss et al. (2008) It is not known

what particular chemicals in the effluent or mixtures of them induced these changes in

fish, although alkylphenols have been demonstrated to cause similar responses under

laboratory conditions

Lee et al. (2008) conducted a prior study on three rivers in Minnesota that

showed thatalkylphenols and other contaminants were present in river water upstream of

WWTPs.

Ferrey et al. (2008) Writer et al. (2010), Statewide EDC Study, revealed that

several pharmaceuticals, alkylphenols, and hormones were present in surface water and

sediment of urban and rural lakes as well as in remote lakes with limited or no

development.

MA Al-Batanony, MK El-Shafie(October 2011) have found that the antibody

level against both HAV and HEV was significantly higher among Waste Water

Treatment Plants workers than the comparison group that is in keeping with other studies

which revealed that HAV and HEV were significantly more prevalent among workers

occupationally exposed to sewage and reported a high prevalence of abdominal pain and

body ache among them.

National Urban Waste Water Infrastracture Study – involves the collection,

collation,mapping and analysis of urban drainage system including waste water treatment

facilities and an assessment of future waste water requirements.

 24

House and Building Research Institute in Dhakahas carried out experimental

work on the production of fibre boards from water hyacinth fibre and other indigenous

materials. They have developed a locally manufactured production plant for producing

fibreboard for general-purpose use and also a bituminised board for use as a low cost

roofing material.

Mennonite Central Committee of Bangladesh established two projects that

make paper from water hyacinth stems. The water hyacinth fibre alone does not make a

particularly good paper but when the fibre is blended with waste paper or jute the result is

good. The pulp is dosed with bleaching powder, calcium carbonate and sodium carbonate

before being heated. The first project is quite large with 120 producers involved in paper

manufacture. The equipment for pulping is relatively sophisticated and the end product is

of reasonable quality. The second project involves 25 - 30 people and uses a modified

rice mill to produce pulp. The quality of the paper is low and is used for making folders,

boxes, etc.

Mashewat Lenka, Kamal K.Panda, Brahma B. Panda – proved the water

hyacinth (eichhornia Crassipes) plants were employed to assess bioconcentration and

genetoxity of aquatic mercury. Plants were exposed to water contaminated with mercuric

chlorine (MC) or phenyl Mercuric Acetate (PMA) at 0.001 to 1.0 mg liter -1 , or mercury

contaminated effluent from chloralkali plant for various periods of 4 to 96 h. Root

samples taken after 4, 8, 12, 24, 48, 72 and 96 h of exposure were analyzed for

bioconcentration of mercury spectrophotometrically, and the root meristem were fixed in

aceto-ethanol for cytological analysis to determine the frequencies of cells with

micronuclei (MNC). Ethyl methane sulfonate and tap water served as positive and
 25

negative control, respectively. The results indicated that bioconcentration of mercury in

root tissue was both time- and concentration-dependent, proving evidence that water

hyacinth is a good absorbant of aquatic mercury. The frequency of root meristematic cells

with MNC followed a concentration-response. The findings indicate the potential of

water hyacinth plants for monitoring and for mitigation of aquated mercury pollution.

Wilson et al – stated that population of fingernail clams declined significantly in

five of eight navigation pools on the upper Mississippi River for which historical data

existed, and warned that these decreases could signal a large scale deterioration in the

health of this ecosystem. Result of research on clams declines in other rivers, such as the

Upper Mississippi River.

 26

Part VI
Results and Discussions

This chapter presents and discusses the results of the study.

A. Analysis of Samples
Table 1. Bacteriological Examination of Water Samples

Results Analysis
Heterotrophic Plate Count (HPC) Remarks
Total Fecal
Pour Plate method 350C/48 hr
Coliform Coliform
Plate Count Agar
More than More than
8.0 8.0 More than 6,500 cfu/mL Failed

Based from the results of the test, it was found out that the water sample is high in total

coliform, fecal coliform and HPC. This explains that the water pollutants or microorganism

present is in high risks.

Table 2. Indicants of Water quality in a controlled set-up and the Limits of the each indicant

Indicants *Limit Indicants Result

pH 6.5-9.0 pH 8.39
BOD 50 mg/L BOD 25 mg/L
COD 33 mg/L COD 44 mg/L
Oil and Grease 5 mg/L Oil and Grease 6 mg/L
Surfactants 5.0 mg/L Surfactants 0.0275 mg/L
Ammonia 0.0606 mg/L Ammonia 0.1515 mg/L
TSS 70 mg/L TSS 92 mg/L
Color 150 pcu Color 63 pcu

Remarks: * DENR Administrative Order No. 35. Table 2 A Effluent Standards: Conventional and other Pollutants in Inland
Waters Class C, NPI.
Reference: Standard Method for the Examination of Water and Wastewater, 21 st Edition, 2005.
 27

Indicants of Water Quality tested in 100

pcs., 150 pcs., and 200 pcs. of Sphaerium
corneum

30
25
20
15
10 Trial 1
5
0 Trial 2
Trial 3

Indicants of Water Quality tested in 5

pcs., 10 pcs., and 20 pcs. Of Pistia
Stratiotes plant
30
25
20
15
10 Series 1
5
0 Series 2
Series 3
 28

Indicants of Water Quality tested in 5

pcs., 10 pcs., and 20 pcs. Of Eichhornia
crassipes plant
30
25
20
15
10 Trial 1
5
0 Trial 2
Trial 3

Indicants of Water Quality tested in the combination of

100 pcs. S. corneum, 5 pcs. P. stratiotes and 5 pcs. E.
crassipes; 150 pcs. S. corneum, 10 pcs. P. stratiotes and
10 pcs. E. crassipes; and 200 pcs. S. corneum, 20 pcs. P.
stratiotes and 20 pcs. E. c

30
25
20
15
10 Trial 1
5
0 Trial 2
Trial 3
 29

Summary:

Based from the results, it was found out that the combination of 200 pcs S.

corneum, 20 pcs of P. stratiotes plant and 20 pcs of E. crassipes plant had the lowest

indicants for each trials and replicates. The pH of water turns neutral with 7.0 level. The

biochemical oxygen demand also decreases from 25 mg/L – 2 mg/L, it was below 50 mg/L

margin. The chemical oxygen demand also decreases from 44 mg/L – 13 mg/L. It was found

out that the variables were effective in treating waste water. Surfactant also decreases from

0.0275 mg/L – 0.0207 mg/L. Ammonia also decreases from 0.1515 mg/L – 0.0557 mg/L.

Aside from that, oil and grease, and TSS also decreases and becomes low. Color changes to

little darker – whitish and bacteriological components also decreases.

 30

Part VI
CONCLUSIONS AND RECOMMENDATIONS

Conclusions

Based from the results of the experiment the following conclusions were drawn:

1. Fingernail clam (Sphaerium corneum), water cabbage (Pistia stratiotes) and water

hyacinth (Eichhornia crassipes) are effective in treating sewage waste water.

2. The combination of 200 fingernail clam (Sphaerium corneum), 20 pieces water cabbage

(Pistia stratiotes) and 20 pieces water hyacinth (Eichhornia crassipes) is the most

effective in treating sewage waste water.

3. The greater the number of fingernail clam (Sphaerium corneum), water cabbage (Pistia

stratiotes) and water hyacinth (Eichhornia crassipes) the higher possibilities of treating

pollution on sewage waste water.

Recommendations

Based on the result of the experiment and conclusions, the following recommendations

are hereby made:

1. To conduct further investigations on other parameters that is present in water samples

used in sewage waste water treatment.

2. To improve further the study of using fingernail clam (Sphaerium corneum), water

cabbage (Pistia stratiotes) and water hyacinth (Eichhornia crassipes) in treating sewage

waste water.
 31

Part VIII

REFERENCES/BIBLIOGRAPHY

Hayat, Yousaf, Mahmood, Qaisar, Zheng, Ping - Anatomical studies on water hyacinth
(Eichhornia crassipes (Mart.) Solms) under the influence of textile wastewater*
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1390441/

Work-Related Health Effects among Wastewater Treatment Plants Workers Vol. 2 Number
4; (October, 2011)

http://www.theijoem.com/ijoem/index.php/ijoem/article/viewFile/104/211

Wastewater Treatment Plant Endocrine Disrupting Chemical Monitoring Study (February

2011)

http://www.pca.state.mn.us/index.php/view-document.html?gid=15610

Bhattacharya, Anjanabha and Kumar, Pawan - WATER HYACINTH AS A POTENTIAL

BIOFUEL CRO, National Environmental Sound Production Agriculture Laboratory, University
of Georgia, Tifton, GA 31794, USA

http://www.cabi.org/isc/fulltextpdf/2010/20103065885.pdf

WATER HYACINTH CONTROL AND POSSIBLE USES

http://www.solucionespracticas.org.pe/fichastecnicas/pdf/water_hyacinth_control.pdf

EFFECTS OF ENVIRONMENTAL AND SPATIAL FEATURES ON CLAM

POPULATIONS AND COMMUNITIES IN A NORTH AMERICAN RIVER

http://ny.water.usgs.gov/pubs/jrn/ny0160/jrn06-r32100a.pdf

United States Environmental Protection Agency

http://water.epa.gov/type/rsl/monitoring/vms52.cfm
 32

Part IX

ACKNOWLEDGEMENTS

Foremost the researchers would like to acknowledge Almighty God for His endeavour that all
things come from HIM.

The researchers would like to express their gratitude and appreciation to the following people
who in one way or another helped in the accomplishment of this study.

Mrs. Aida M. Platon, Registered Medical Technologist, Laboratory Head at Metro Lipa Water
District Laboratory for her expertise in conducting water analysis of the samples.

Rene P. Addatu, Manager-Water Division, Grand Aces Ventures Inc., Angono Rizal for his
expertise in conducting chemical analysis of water samples.

Jobel Tono, Optimal Laboratories Inc., Lipa City, Batangas, for her assistance in performing
chemical test of water samples.

Mrs. Annabelle E. De Roxas, Principal II of the school, for her inspiration and support.

Mrs. Corazon D. Bariuan, Master Teacher I, for her valuable assistance and support to the
researchers.

Mrs. Romana Glenda S. Lagmay, Science Coordinator of the school, for her wisdom which
contributed much in the accomplishments of this project.

Mr. Christopher Kenneth N. Tionko and Ms. Analyn D. Katimbang, for their assistance rendered
to the researchers.

Mr. Oliver R. Guevarra, Teacher III of the school and the research adviser, for his wisdom,
encouragement and inspiration given to the researchers to undertake this project.

To all teachers of the school for their valuable assistance.

To our beloved family, friends and relatives who have given their whole hearted supports for this
project to be a success.
 33

Appendices

Table 3. Indicants of water quality tested in 100 pcs. of S. corneum, 5 pcs P. stratiotes
plant, 5 pcs E. crassipes plant and the combination of 100 pcs . of S. corneum, 5 pcs of P. stratiotes and E. crassipes
plant (T1-R1)

Combination of 100
5 pcs P. pcs. S corneum, 5
100 pcs S.
Indicants stratiotes 5 pcs E. crassipes plant pcs of P. stratiotes
corneum
plant plant and 5 pcs E.
crassipes plant

pH 8.10 7.99 8.09 8.11

BOD 23 mg/L 24 mg/L 25 mg/L 20 mg/L

COD 29 mg/L 28 mg/L 28 mg/L 25.2 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 4.8mg/L

Surfactants 0.0263 mg/L 0.0258 mg/L 0.0259 mg/L 0.0245 mg/L

Ammonia 0.0620 mg/L 0.0623 mg/L 0.0623 mg/L 0.0613 mg/L

TSS 20 mg/L 21 mg/L 18 mg/L 13 mg/L

Color 28 pcu 18 pcu 13 pcu 10 pcu

Below 6,500
Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Table 4. Indicants of water quality tested in 150 pcs. of S. corneum, 10 pcs P. stratiotes plant, 10 pcs E. crassipes
plant and the combination of 150 pcs . of S. corneum, 10 pcs of P. stratiotes and E. crassipes plant (T2-R1)
Combination of 150
10 pcs P. pcs. S corneum, 10
150 pcs S. 10 pcs E. crassipes
Indicants stratiotes pcs of P. stratiotes
corneum plant
plant plant and 10 pcs E.
crassipes plant

pH 7.85 7.83 7.8 7.79

BOD 21 mg/L 19 mg/L 15 mg/L 12 mg/L

COD 23 mg/L 22 mg/L 20 mg/L 5 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 4.6 mg/L

Surfactants 0.0257 mg/L 0.0248 mg/L 0.0250 mg/L 0.0239 mg/L

Ammonia 0.0611 mg/L 0.0613 mg/L 0.0615 mg/L 0.0608 mg/L

TSS 18 mg/L 15 mg/L 16 mg/L 12 mg/L

Color 14 pcu 15 pcu 13 pcu 5 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL
 34

Table 5. Indicants of water quality tested in 200 pcs. of S. corneum, 15 pcs P. stratiotes plant, 15 pcs E. crassipes
plant and the combination of 200 pcs . of S. corneum, 15 pcs of P. stratiotes and E. crassipes plant (T3-R1)
Combination of 200
20 pcs P. pcs. S corneum, 20
200 pcs S. 20 pcs E. crassipes
Indicants stratiotes pcs of P. stratiotes
corneum plant
plant plant and 20 pcs E.
crassipes plant

pH 7.82 7.81 7.8 7.83

BOD 4 mg/L 4 mg/L 3 mg/L 2 mg/L

COD 20 mg/L 18 mg/L 18 mg/L 5 mg/L

Oil and Grease 5.3 mg/L 5.2 mg/L 5.1 mg/L 5 mg/L

Surfactants 0.0223 mg/L 0.0215 mg/L 0.0218 mg/L 0.0207 mg/L

Ammonia 0.0606 mg/L 0.0607 mg/L 0.0608 mg/L 0.0557 mg/L

TSS 15 mg/L 23 mg/L 20 mg/L 12 mg/L

Color 5 pcu 7 pcu 6 pcu 5 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL
Table 6. Indicants of water quality tested in 100 pcs. of S. corneum, 5 pcs P. stratiotes plant, 5 pcs E. crassipes plant
and the combination of 100 pcs . of S. corneum, 5 pcs of P. stratiotes and E. crassipes plant (T1-R2)

Combination of 100
5 pcs P. pcs. S corneum, 5 pcs
100 pcs S.
Indicants stratiotes 5 pcs E. crassipes plant of P. stratiotes plant
corneum
plant and 5 pcs E. crassipes
plant

pH 7.99 8.0 8.01 7.80

BOD 23 mg/L 24 mg/L 25 mg/L 20 mg/L

COD 29 mg/L 28 mg/L 28 mg/L 25.2 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 5 mg/L

Surfactants 0.0263 mg/L 0.0258 mg/L 0.0259 mg/L 0.0245 mg/L

Ammonia 0.0620 mg/L 0.0623 mg/L 0.0623 mg/L 0.0613 mg/L

TSS 20 mg/L 21 mg/L 18 mg/L 13 mg/L

Color 28 pcu 18 pcu 13 pcu 10 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL
 35

Table 7. Indicants of water quality tested in 150 pcs. of S. corneum, 10 pcs P. stratiotes plant, 10 pcs E. crassipes
plant and the combination of 150 pcs . of S. corneum, 10 pcs of P. stratiotes and E. crassipes plant (T2-R2)
Combination of 150
10 pcs P. pcs. S corneum, 10 pcs
150 pcs S. 10 pcs E. crassipes
Indicants stratiotes of P. stratiotes plant
corneum plant
plant and 10 pcs E.
crassipes plant

pH 7.95 7.96 8.86 7.80

BOD 21 mg/L 19 mg/L 15 mg/L 12 mg/L

COD 23 mg/L 22 mg/L 20 mg/L 19 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 5 mg/L

Surfactants 0.0257 mg/L 0.0248 mg/L 0.0250 mg/L 0.0239 mg/L

Ammonia 0.0611 mg/L 0.0613 mg/L 0.0615 mg/L 0.0608 mg/L

TSS 18 mg/L 15 mg/L 16 mg/L 12 mg/L

Color 14 pcu 15 pcu 13 pcu 5 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Table 8. Indicants of water quality tested in 200 pcs. of S. corneum, 20 pcs P. stratiotes plant, 20 pcs E. crassipes
plant and the combination of 200 pcs . of S. corneum, 20 pcs of P. stratiotes and E. crassipes plant (T3-R2)
Combination of 200
20 pcs P. pcs. S corneum, 20
200 pcs S.
Indicants stratiotes 20 pcs E. crassipes plant pcs of P. stratiotes
corneum
plant plant and 20 pcs E.
crassipes plant

pH 7.82 7.85 7.87 7.79

BOD 4 mg/L 4 mg/L 3 mg/L 2 mg/L

COD 20 mg/L 18 mg/L 18 mg/L 13 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 4.5 mg/L

Surfactants 0.0223 mg/L 0.0215 mg/L 0.0218 mg/L 0.0207 mg/L

Ammonia 0.0606 mg/L 0.0607 mg/L 0.0608 mg/L 0.0557 mg/L

TSS 15 mg/L 23 mg/L 20 mg/L 12 mg/L

Color 5 pcu 7 pcu 6 pcu 5 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL
 36

Table 9. Indicants of water quality tested in 100 pcs. of S. corneum, 5 pcs P. stratiotes plant, 5 pcs E. crassipes plant
and the combination of 100 pcs . of S. corneum, 5 pcs of P. stratiotes and E. crassipes plant (T1-R3)
Combination of 100
5 pcs P. pcs. S corneum, 5 pcs
100 pcs S.
Indicants stratiotes 5 pcs E. crassipes plant of P. stratiotes plant
corneum
plant and 5 pcs E.
crassipes plant

pH 6.3 6.5 7.89 8.0

BOD 23 mg/L 24 mg/L 25 mg/L 20 mg/L

COD 29 mg/L 28 mg/L 28 mg/L 25.2 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 5 mg/L

Surfactants 0.0263 mg/L 0.0258 mg/L 0.0259 mg/L 0.0245 mg/L

Ammonia 0.0620 mg/L 0.0623 mg/L 0.0623 mg/L 0.0613 mg/L

TSS 20 mg/L 21 mg/L 18 mg/L 13 mg/L

Color 28 pcu 18 pcu 13 pcu 10 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Table 10. Indicants of water quality tested in 150 pcs. of S. corneum, 10 pcs P. stratiotes plant, 10 pcs E. crassipes
plant and the combination of 150 pcs . of S. corneum, 10 pcs of P. stratiotes and E. crassipes plant (T2-R3)
Combination of 150
10 pcs P. pcs. S corneum, 10
150 pcs S.
Indicants stratiotes 10 pcs E. crassipes plant pcs of P. stratiotes
corneum
plant plant and 10 pcs E.
crassipes plant

pH 6.6 6.6 6.5 6.8

BOD 21 mg/L 19 mg/L 15 mg/L 12 mg/L

COD 23 mg/L 22 mg/L 20 mg/L 19 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 4.9 mg/L

Surfactants 0.0257 mg/L 0.0248 mg/L 0.0250 mg/L 0.0239 mg/L

Ammonia 0.0611 mg/L 0.0613 mg/L 0.0615 mg/L 0.0608 mg/L

TSS 18 mg/L 15 mg/L 16 mg/L 12 mg/L

Color 14 pcu 15 pcu 13 pcu 5 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL
 37

Table 11. Indicants of water quality tested in 200 pcs. of S. corneum, 20 pcs P. stratiotes plant, 20 pcs E. crassipes
plant and the combination of 200 pcs . of S. corneum, 20 pcs of P. stratiotes and E. crassipes plant (T3-R3)
Combination of 200
20 pcs P. pcs. S corneum, 20
200 pcs S.
Indicants stratiotes 20 pcs E. crassipes plant pcs of P. stratiotes
corneum
plant plant and 20 pcs E.
crassipes plant

pH 6.8 7.0 7.0 7.0

BOD 4 mg/L 4 mg/L 3 mg/L 2 mg/L

COD 20 mg/L 18 mg/L 18 mg/L 13 mg/L

Oil and Grease 5 mg/L 5 mg/L 5 mg/L 4.5 mg/L

Surfactants 0.0223 mg/L 0.0215 mg/L 0.0218 mg/L 0.0207 mg/L

Ammonia 0.0606 mg/L 0.0607 mg/L 0.0608 mg/L 0.0557 mg/L

TSS 15 mg/L 23 mg/L 20 mg/L 12 mg/L

Color 5 pcu 7 pcu 6 pcu 5 pcu

Fecal Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

Total Coliform Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

HPC Below 8.0 Below 8.0 Below 6,500 cfu/mL Below 6,500 cfu/mL

*COD Test. According to VLAREM II Basic Environmental Quality Standards for Surface Water, the range of COD should be
< 30 mg/L.
*Laboratory Testing of Dissolved Oxygen

How to collect and analyze samples

The procedures for collecting and analyzing samples for dissolved oxygen consist

of the following tasks:

TASK 1 Prepare before leaving for the sampling site

Refer to section 2.3 - Safety Considerations for details on confirming sampling

date and time, safety considerations, checking supplies, and checking weather and

directions. In addition to the standard sampling equipment and apparel, when sampling

for dissolved oxygen, include the following equipment:

If Using the Winkler Method

Labels for sample bottles

Field kit and instructions for DO testing

 38

Enough reagents for the number of sites to be tested

Kemmerer, Van Dorn, or home-made sampler to collect deep-water samples

A numbered glass BOD bottle with a glass stopper (1 for each site)

Data sheet for dissolved oxygen to record results

If Using a Meter and Probe

DO meter and probe (electrode) (NOTE: Confirm that the meter has been calibrated

according to the manufacturer's instructions.)

Operating manual for the meter and probe

Extra membranes and electrolyte solution for the probe

Extra batteries for the meter

Extension pole

Data sheet for dissolved oxygen to record results

TASK 2 Confirm that you are at the proper location

The directions for sampling should provide specific information about the exact

point in the stream from which you are to sample; e.g., "approximately 6 feet out from

the large boulder downstream from the west side of the bridge." If you are not sure you

are in the exact spot, record a detailed description of where you took the sample so that it

can be compared to the actual site later.

TASK 3 Collect samples and fill out the field data sheet

Winkler Method

Use a BOD bottle to collect the water sample. The most common sizes are 300

milliliters (mL) and 60 mL. Be sure that you are using the correct volume for the titration

method that will be used to determine the amount of DO. There is usually a white label
 39

area on the bottle, and this may already be numbered. If so, be sure to record that number

on the field data sheet. If your bottle is not already numbered, place a label on the bottle

(not on the cap because a cap can be inadvertently placed on a different bottle) and use a

waterproof marker to write in the site number.

If you are collecting duplicate samples, label the duplicate bottle with the correct

code, which should be determined prior to sampling by the lab supplying the bottles. Use

the following procedure for collecting a sample for titration by the Winkler method:

1. Remember that the water sample must be collected in such a way that you

can cap the bottle while it is still submerged. That means that you must be

able to reach into the water with both arms and the water must be deeper

than the sample bottle.

2. Carefully wade into the stream. Stand so that you are facing one of the

banks.

3. Collect the sample so that you are not standing upstream of the bottle.

Remove the cap of the BOD bottle. Slowly lower the bottle into the water,

pointing it downstream, until the lower lip of the opening is just submerged.

Allow the water to fill the bottle very gradually, avoiding any turbulence

(which would add oxygen to the sample). When the water level in the bottle

has stabilized (it won't be full because the bottle is tilted), slowly turn the

bottle upright and fill it completely. Keep the bottle under water and allow it

to overflow for 2 or 3 minutes to ensure that no air bubbles are trapped.

 40

4. Cap the bottle while it is still submerged. Lift it out of the water and look

around the "collar" of the bottle just below the bottom of the stopper. If you

see an air bubble, pour out the sample and try again.

5. "Fix" the sample immediately following the directions in your kit:

6. Remove the stopper and add the fixing reagents to the sample.

7. Immediately insert the stopper so air is not trapped in the bottle and invert

several times to mix. This solution is caustic. Rinse your hands if you get

any solution on them. An orange-brown flocculent precipitate will form if

oxygen is present.

8. Wait a few minutes until the floc in the solution has settled. Again invert

the bottle several times and wait until the floc has settled. This ensures

complete reaction of the sample and reagents. The sample is now fixed,

and atmospheric oxygen can no longer affect it. If you are taking the

sample to the lab for titration, no further action is necessary. You can store

the sample in a cooler for up to 8 hours before titrating it in a lab. If you

are titrating the sample in the field, see Task 4: Analyze the Samples.
 41

Figure 5.7

Taking a water sample for DO analysis

Point the bottle downstream and fill gradually. Cap underwater when full.

Using a DO Meter

If you are using a dissolved oxygen meter, be sure that it is calibrated

immediately prior to use. Check the cable connection between the probe and the meter.

Make sure that the probe is filled with electrolyte solution, that the membrane has no

wrinkles, and that there are no bubbles trapped on the face of the membrane. You can do

a field check of the meter's accuracy by calibrating it in saturated air according to th e

manufacturer's instructions. Or, you can measure a water sample that is saturated with

oxygen, as follows. (NOTE: You can also use this procedure for testing the accuracy of

the Winkler method.)

 42

1. Fill a l-liter beaker or bucket of tap water. (You may want to bring a gallon

jug with water in it for this purpose.) Mark the bottle number as "tap" on the

lab sheet.

2. Pour this water back and forth into another beaker 10 times to saturate the

water with oxygen.

3. Use the meter to measure the water temperature and record it in the water

temperature column on the field data sheet.

4. Find the water temperature of your "tap" sample in Table 5.3. Use the meter

to compare the dissolved oxygen concentration of your sample with the

maximum concentration at that temperature in the table. Your sample should

be within 0.5 mg/L. If it is not, repeat the check and if there is still an error,

check the meter's batteries and follow the troubleshooting procedures in the

manufacturer's manual.

Once the meter is turned on, allow 15 minute equilibration before

calibrating. After calibration, do not turn the meter off until the sample is

analyzed. Once you have verified that the meter is working properly, you are

ready to measure the DO levels at the sampling site. You might need an extension

pole (this can be as simple as a piece of wood) to get the probe to the proper

sampling point. Simply secure the probe to the end of the extension pole. A

golfer's ball retriever works well because it is collapsible and easy to transport. To

use the probe, proceed as follows:

1. Place the probe in the stream below the surface.

 43

2. Set the meter to measure temperature, and allow the temperature reading to

stabilize. Record the temperature on the field data sheet.

3. Switch the meter to read dissolved oxygen.

4. Record the dissolved oxygen level on the field data sheet.

TASK 4 Analyze the samples

Three types of titration apparatus can be used with the Winkler method: droppers,

digital titrators, and burets. The dropper and digital titrator are suited for field use. The

buret is more conveniently used in the lab (Fig. 5.8) Volunteer programs are most likely

to use the dropper or digital titrator. For titration with a dropper or syringe, which is

relatively simple, follow the manufacturer's instructions. The following procedure is for

using a digital titrator to determine the quantity of dissolved oxygen in a fixed sample:
 44

1. Select a sample volume and sodium thiosulfate

titration cartridge for the digital titrator corresponding

to the expected dissolved oxygen concentration

according to Table 5.4. In most cases, you will use the

0.2 N cartridge and the 100-mL sample volume.

2. Insert a clean delivery tube into the titration cartridge.

3. Attach the cartridge to the titrator body.

4. Hold the titrator with the cartridge tip up. Turn the

delivery knob to eject air and a few drops of titrant.

Reset the counter to 0 and wipe the tip.

5. Use a graduated cylinder to measure the sample

volume (from the "fixed" sample in the 300-mL BOD

bottle) according to Table 5.4.

6. Transfer the sample into a 250-mL Erlenmeyer flask,

Figure 5.8
and place the flask on a magnetic stirrer with a stir

bar. If you are in the field, you can manually swirl the
Titrating a DO sample using a buret
flask to mix.

7. Place the delivery tube tip into the solution and turn the stirrer on to stir the sample

while you're turning the delivery knob.

8. Titrate to a pale yellow color.

9. Add two dropperfuls of starch indicator solution and swirl to mix. A strong blue color

will develop.
 45

10. Continue to titrate until the sample is clear. Record the number of digits required. (The

color might reappear after standing a few minutes, but this is not a cause for concern.

The "first" disappearance of the blue color is considered the endpoint.)

11. Calculate mg/L of DO = digits required X digit multiplier (from Table 5.4).

12. Record the results in the appropriate column of the data sheet.

Some water quality standards are expressed in terms of percent saturation. To calculate percent

saturation of the sample:

1. Find the temperature of your water sample as measured in the field.

2. Find the maximum concentration of your sample at that temperature as given in Table

5.3.

3. Calculate the percent saturation, by dividing your actual dissolved oxygen by the

maximum concentration at the sample temperature.

4. Record the percent saturation in the appropriate column on the data sheet.

TASK 5 Return the samples and the field data Expected Sample Titration Digit Table 5.4

sheets to the lab/drop-off point Range Volume Cartridge Multiplier

If you are using the Winkler method and 1-5 200 mL 0.2 N 0.01 Sample

delivering the samples to a lab for titration, mg/L volume

double-check to make sure that you have 2-10 100 mL 0.2 N 0.02 selection and

recorded the necessary information for each site mg/L corresponding

on the field data sheet, especially the bottle values for

10+ 200 mL 2.0 N 0.10
number and corresponding site number and the Winkler
mg/L
times the samples were collected. Deliver your titration
 46

samples and field data sheets to the lab. If you have already obtained the dissolved oxygen

results in the field, send the data sheets to your sampling coordinator.

What is biochemical oxygen demand and why is it important?

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 (i.e., the extraction of oxygen from water via chemical

reaction). A test is used to measure the amount of oxygen consumed by these organisms during a

specified period of time (usually 5 days at 20 C). 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.

BOD directly affects the amount of dissolved oxygen in rivers and streams. The greater the

BOD, the more rapidly oxygen is depleted in the stream. This means less oxygen is available to

higher forms of aquatic life. The consequences of high BOD are the same as those for low

dissolved oxygen: aquatic organisms become stressed, suffocate, and die.

Sources of BOD include leaves and woody debris; dead plants and animals; animal manure;

effluents from pulp and paper mills, wastewater treatment plants, feedlots, and food-processing

plants; failing septic systems; and urban stormwater runoff.

Sampling Considerations

BOD is affected by the same factors that affect dissolved oxygen (see above). Aeration of stream

water by rapids and waterfalls, for example will accelerate the decomposition of organic and

inorganic material. Therefore, BOD levels at a sampling site with slower, deeper waters might be

higher for a given volume of organic and inorganic material than the levels for a similar site in

highly aerated waters.

 47

Chlorine can also affect BOD measurement by inhibiting or killing the microorganisms that

decompose the organic and inorganic matter in a sample. If you are sampling in chlorinated

waters, such as those below the effluent from a sewage treatment plant, it is necessary to

neutralize the chlorine with sodium thiosulfate. (See APHA, 1992.)

BOD measurement requires taking two samples at each site. One is tested immediately for

dissolved oxygen, and the second is incubated in the dark at 20 C for 5 days and then tested for

the amount of dissolved oxygen remaining. The difference in oxygen levels between the first test

and the second test, in milligrams per liter (mg/L), is the amount of BOD. This represents the

amount of oxygen consumed by microorganisms to break down the organic matter present in the

sample bottle during the incubation period. Because of the 5-day incubation, the tests should be

conducted in a laboratory.

Sometimes by the end of the 5-day incubation period the dissolved oxygen level is zero. This is

especially true for rivers and streams with a lot of organic pollution. Since it is not known when

the zero point was reached, it is not possible to tell what the BOD level is. In this case it is

necessary to dilute the original sample by a factor that results in a final dissolved oxygen level of

at least 2 mg/L. Special dilution water should be used for the dilutions. (See APHA, 1992.)

It takes some experimentation to determine the appropriate dilution factor for a particular

sampling site. The final result is the difference in dissolved oxygen between the first

measurement and the second after multiplying the second result by the dilution factor. More

details are provided in the following section.

How to Collect and Analyze Samples

The procedures for collecting samples for BOD testing consist of the same steps described for

sampling for dissolved oxygen (see above), with one important difference. At each site a second
 48

sample is collected in a BOD bottle and delivered to the lab for DO testing after the 5-day

incubation period. Follow the same steps used for measuring dissolved oxygen with these

additional considerations:

 Make sure you have two BOD bottles for each site you will sample. The bottles should

be black to prevent photosynthesis. You can wrap a clear bottle with black electrician's

tape if you do not have a bottle with black or brown glass.

 Label the second bottle (the one to be incubated) clearly so that it will not be mistaken

for the first bottle.

 Be sure to record the information for the second bottle on the field data sheet.

The first bottle should be analyzed just prior to storing the second sample bottle in the dark for 5

days at 20 C. After this time, the second bottle is tested for dissolved oxygen using the same

method that was used for the first bottle. The BOD i s expressed in milligrams per liter of DO

using the following equation:

BOD (mg/L)= DO (mg/L) of first bottle - DO (mg/L) of second bottle

*APHA. 1992. Standard methods for the examination of water and wastewater. 18th edition. American Public Health
Association, Washington, DC.

Determining the Chemical Oxygen Demand (COD)

Procedure:

Fifty (50) mL of sample was taken into a refluxing flask and several boiling

stones were added. 0.1g HgSO4 was added to the solution. 5 ml of concentrated H2SO4

was also added to the solution. To ensure that HgSO4 dissolved completely, the solution

was swirled slowly while adding Sulphuric acid. 0.1 g of Ag2SO4 was added to this

solution. Finally Potassium dichromate was added. Thorough mixing of the solution was

ensured by swirling the flask in a water bath to recover any volatile substances that may
 49

have escaped from the liquid state. The flask was then attached to the condenser and

further cooling was done. 20 ml of Sulphuric acid was added to the solution in the flask

continuing cooling and swirling to mix the solution. The solution was refluxed for 1 hour.

A blank run (using 50 ml distilled water instead of sample) was simultaneously

conducted with the same procedure after cooling; the solution was transferred to an

Erlenmeyer flask. The reflux flask was rinsed thrice, pouring the rinsing water to the

Erlenmeyer flask. The solution was diluted to about 300 ml and about 8 drops of

Phenanthroline ferrous sulphate was added to the solution as an indicator. The solution

was titrated against the Mohr’s salt and the titer volume required for the color change

from blue-green to reddish blue was noted. The procedure was repeated for the blank run.

Calculations:

COD = 8000 * (Vbl – Vs)* M/ original volume of sample taken mg/l

Where,

Vbl = Titer volume for the blank

Vs = Titer volume for the sample

M = Molarity of Mohr’s solution