Polytechnic University of the Philippines – Sta.

Mesa, Manila

Senior High School Department

Science, Technology, Engineering, and Mathematics

Analysis of Physico-chemical and Microbial Properties of Lagoon Water in

Polytechnic University of the Philippines – Manila

Research Proponents

Almeria, Wilmalyn Claire S.

Garcia, Ana Mari Leoni M.

Nabus, Aldrich G.

Parane, Pauline S.

Piaduche, Andrey Marie V.

Research Adviser

Mr. Jose Carlos Basilio L. Taroy

 Chapter 1

INTRODUCTION

1.1 Background of the Study

Water, also known as the “universal solvent”, is an odorless, colorless, tasteless,

formless substance wherein every molecule contains two (2) atoms of Hydrogen (H)

and one (1) atom of Oxygen (O). It is a vital constituent of most living organisms along

with Carbon (C), Oxygen (O), Nitrogen (N), and Sulfur (S). Water is a necessity in the

lives of all human beings for a reason that it makes up approximately 50% - 75% of the

human body. It aids in regulating most bodily functions such as metabolizing proteins

and carbohydrates,oxygen and nutrients transportation, and waste excretion

(Helmenstine, 2019).It also covers more than 70% of the Earth’s surface. This 70%

water on Earth’s crust is further divided into three (3) classes: 1) 97% saline or ocean

waters, 2) 2.06% glaciers and ice caps, and 3) 0.94% surface and ground water (Bice,

D. & Bralower, T., n.d.).

Consequently, humans are surrounded by different bodies of water.These

bodieshave been crucial for the survival of human beings considering thatthey serve as

an avenue for livelihoodand is a great source of food. These bodies of water range from

oceans, seas, and falls to streams, lakes, rivers, marshes, swamps, and lagoons.

National Ocean Service (2018) described lagoons as shallow bodies of water

separated from a larger body of water, usually an ocean or sea, by reefs, sandbars, or

other natural barriers. The term “lagoon” was derived from the Italian word laguna,
which means “pond” or “lake”. There are two (2) types of lagoon namely: 1) Atoll

lagoons and 2) Coastal lagoons. Complete recession of an island beneath the water

that leaves a ring of coral that tends to continue to develop upwards results to the

formation of Atoll lagoons. This process may take about 300,000 years to occur.

Meanwhile, Coastal lagoons arise along gently sloping coasts and are generally

shallower than Atoll lagoons. Most of the time, these are connected to the ocean

through an inlet (Sawe, 2017). However, lagoons can also be man-made. These man-

made lagoons are usually made for recreation,landscape beautification,and wastewater

treatment.

Established in 1904, the Polytechnic University of the Philippines was a pioneer

in business education in the country. Among the famous landmarks found inside its

main campus is the PUP Lagoon.This lagoon was built to add aesthetic means to the

university, however, PUP Lagoon has showed numerous signs of eutrophication for the

past years. Mohammed Khalid, part of the The Borgen Project (2018), notes that water

pollution in the Philippines and a lack of proper sewage kills 55 people every day.The

need for a good water quality is a critical attribute for all organisms in an ecosystem,

hence, BS Biology students of PUP annually conduct different tests to acquire

quantitative data on the current status of the lagoon. There are varioustests that can be

done to assess the quality of a certain water body – ranging from physical to biological

analysis.

Physico-chemical analysis is a method used to determine the physical and

chemical characteristics, and their correlation, of a certain water body. Physical tests

include parameters such as Total Suspended Solids (TSS), temperature, odor, color,
pH level, and turbidity. On the other hand, chemical tests involve analysis of

Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Dissolved

Oxygen (DO), alkalinity, heavy metal concentration, and others (Deshmukh, et al.

2012).

Another water quality assessment is microbial or bacteriological analysis.

Microbial water analysis is a method used for estimating quantitative data of bacteria

present in a certain water sample and, if necessary, used for finding out what sort of

bacteria these are (Hassan, 2018). Measurement of the total coliforms, fecal coliforms,

and fecal streptococci are the most common parameter used in microbial water

analysis.

This study intends to analyze the physico-chemical and microbial properties of

the water in PUP Lagoon.

1.2 Statement of the Problem

The researchers of the study aim to: (1) obtain quantitative information on the

physical, chemical, and microbial characteristics of the water in PUP Lagoon, (2)

investigate the viable antecedents and adverse effects of the physico-chemical and

microbial properties of the lagoon water to its environment, and; (3) determine the

possibility of survival of life forms with regards to the existing state of the water in

lagoon.

1.3 Objectives of the Study

This study generally aims to identify the current status and water quality of the

PUP lagoon through the use of different parameters. To achieve this general objective,
the following specific objectives are: (1) assess the water quality of PUP Lagoon

through the analyzation of the possible heavy metal content in the form of Cadmium

(Cd) and Chromium (Cr), Biochemical Oxygen Demand (BOD), Chemical Oxygen

Demand (COD), Total Suspended Solids (TSS), pH level, temperature, color, oil and

grease, nutrient (phosphate and nitrate) concentration, total coliform, fecal coliform, and

fecal streptococci, (2) to assure the safety of water with regards to the physical,

chemical, and microbial parameters and improve its quality, and; (3) align the results of

this study to the standards set by the Department of Environment and Natural

Resources (DENR) to conceive information on the capability of lagoon water in holding

different life forms.

1.4 Significance of the Study

In the development of this study, it gave accessible data on the current status of

the water quality of the PUP Lagoon. The results that will be obtained in this research

might be useful in the devising of methods for the treatment and restoration of the water

in PUP lagoon. It is also significant in raising awareness and disseminating information

on the importance of good water quality in its environment. Furthermore, this study can

be used as a reference for researches in line with water quality.

1.5 Scope and Limitation

The scope of this study focuses on the water in the lagoon of the Polytechnic

University of the Philippines – Manila, located in the northern bank of the Pasig River.

For the physico-chemical properties, the researchers will only assess the following

parameters: Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD),

Dissolved Oxygen (DO), Total Suspended Solids (TSS), pH level, temperature, color,

nutrient (phosphate and nitrate) concentration, and oil and grease.

The analysis of total coliform count, fecal coliform, and fecal streptococci through

Membrane Filtration Method are placed on microbial or bacteriological properties of

water. The researchers used Escherichia Coli (E. coli), Salmonella spp., and

Pseudomonas aeruginosa as the indicator species.

For heavy metal content, the researchers will just focus for the presence of

Chromium (Hexavalent) and Cadmium (Cd). Traces of these heavy metals will be

analyzed using Atomic Absorption Spectrophotometry (AAS). All the parameters will be

conducted through laboratory tests and experiments. The data will be analyzed based

on the Water Quality Guidelines and Effluent Standards set by the Department of

Environment and Natural Resources – Philippines.

1.6 Definition of Terms

 Atomic Absorption Spectrophotometry (AAS) – is a technique in which free

gaseous atoms absorb electromagnetic radiation in a specific wavelength to

produce a measurable signal.

 Biochemical Oxygen Demand (BOD) – amount of dissolved oxygen needed by

an aquatic organism to breakdown organic material.

 Chemical Oxygen Demand (COD) – amount of oxygen needed to oxidize

organic material into Carbon Dioxide (CO2) and Water (H2O).

 Coliform – gram-negative, rod-shaped bacteria normally present in the

environment, in the intestines, and in the feces of all warm-blooded animals and

humans.

 Colorimetric Analysis – is a technique used to determine the concentration of a

chemical compound in a solution using a color reagent.

 Dissolved Oxygen (DO) – refers to the level of free oxygen present in the water

bodies and essential for the survival of aquatic organisms. Dissolved oxygen is

one of the most important indicators of water quality.

 Escherichia coli (E. coli) – a bacterium usually found in the intestines of

humans and other animals, some strains of which can cause severe food

poisoning.

 Eutrophication – a process by which the level of dissolved oxygen in water

bodies is reduced because of an increase in mineral and organic nutrients.

 Fecal Coliform – also known as “thermotolerant coliform”. These are rod-shaped

bacteria which can grow without oxygen and at temperatures 44o or 44.5o C.

They can produce acid and gas by fermenting lactose.

 Fecal Streptococci –bacteria that are found within the intestines of humans and

animals. Presence of these bacteria indicates fecal pathogensand an evidence of

fecal contamination in water. Among the three (3) types of coliform bacteria, fecal

streptococci tend to persist longer in the environment.

 Heavy Metal – metals that can be highly poisonous if present in large quantities.

 Membrane Filtration Method – is a technique used for testing fluid samples for

microbiologicalcontamination.
 Nitrate – chemical compound that includes Nitrogen (N) and Oxygen (O). It is

generally formed by the action of bacteria on Ammonia (NH 3) and other nitrogen-

containing compounds.

 pH – stands for “potential of Hydrogen”. It refers to the amount of Hydrogen (H)

found in a substance. It is the scale used for the measurement the acidity or the

alkalinity of water.

 Phosphate – is a chemical compound that contains Phosphorus (P) and is often

used in fertilizers. High levels of phosphate in water speeds up eutrophication.

 Physico-chemical –property measurement for testing for liquids and solids

providing essential information needed in industry.

 Pseudomonasaeruginosa – a “blue-greenpusbacterium” opportunistically

infecting people, especially those who are immunocompromised.

 Salmonellae - bacteria that occur mainly in the intestine, especially a serotype

causing food poisoning.

 Total Coliform – group of bacteria that are not generally harmful. They are

usually found in drinking water.

 Total Suspended Solids – refers to the particles that are larger than 2 microns

(the average size for filters).

 Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

2.1 Related Literatures

2.1.1 Polytechnic University of the Philippines (PUP) Lagoon

These are smaller water bodies separated from larger bodies of water by

sandbars, barrier reefs, coral reefs, or other natural barriers. The word "lagoon" was

derived from the Italian word laguna, which means "pond" or "lake”. Although lagoons

are well defined geographically, the word “lagoon” is sometimes used as a name for a

larger region that contains one or more lagoons. Lagoons are more than just scenic

water bodies; they are often important habitats for aquatic life (National Oceanic and

Atmospheric Administration, 2018). Artificial or human-made lagoons are also existent.

Lagoons of this kind are created for the treatment of wastewater, sometimes even for

recreation and leisure.

The Polytechnic University of the Philippines (PUP) – Main Campus contains

parks inside its vicinity, one of which is the Luntiang Pilipinas Forest Park. This park

houses a man-made lagoon sealed by walls imitating those at Fort Santiago,

Intramuros.

2.1.2 Water Quality

Water quality is described as the condition of the water pertaining to its physical,

chemical, and biological characteristics, usually with respect to its suitability for a
specific purpose. The bodies of water can be used for recreation, drinking, fisheries,

agriculture, and industry. Each of these specified uses has different defined chemical,

physical and biological standards necessary to support the purpose that a certain water

body serves (Daniels, et. al, n.d.). As per the National Marine Sanctuaries of Florida,

water quality is measured by several factors, such as the concentration of dissolved

oxygen, bacteria levels, the amount of salt (salinity), and the amount of materials

suspended in the water (total suspended solids). In some bodies of water, the

concentration of microscopic algae and quantities of pesticides, herbicides, heavy

metals, and other contaminants may also be measured to determine its quality.

2.1.3 Water Quality Assessment

Water quality assessment refers to the comprehensive process of the evaluation

of biological, physical, and chemical properties of the water in relation to natural quality,

human effects, and intended uses – particularly, uses which may affect human health

and the water body itself (Helmer and Meybeck, 1992). It provides the threshold

information on the safety and quality of a certain water body. The data obtained in the

assessment is used to determine the water’s status and manage the different factors

that influence in the deterioration or enhancement of its quality.

2.1.4 Water Quality Guidelines and General Effluent Standards

Water Quality Guidelines and General Effluent Standards (WQG-GES) is a

guideline issued by the Department of Environment and Natural Resources (DENR) of

the Philippines pursuant to Section 19e and 19f of the Republic Act 9275, also known

as the “Philippine Clean Water Act of 2004”, and Executive Order 192. It is mandated to
protect, preserve, and revive the quality of different bodies of water; provide guidelines

for the classification of water bodies; determine time trends; evaluate stages of

deterioration or enhancement in water quality; and serve as a basis in preventing,

controlling or abating water pollution. Specifically, Water Quality Guidelines (WQG) refer

to the numerical values of physical, chemical, biological, and bacteriological or

radiological parameters which are used to categorize water resources and their use.

Moreover, General Effluent Standards (GES) are the restriction on quantities, rates, and

concentrations of physical, chemical, or biological parameters of effluent which a source

can discharge into a body of water as per the classification of the receiving water body.

2.1.5 Physico-chemical Analysis

2.1.5.1 Dissolved Oxygen

Dissolved oxygen is the measurement of the level of free, non-compound

oxygen present in a water body. It is one of the most important parameters to

analyze water quality because it is needed for the aquatic organisms’ survival.

The oxygen can enter the water through aeration, oxygen diffusion across the

water’s surface, or as a product of photosynthesis from phytoplankton, algae, or

other aquatic plants (Dissolved Oxygen, 2013). Dissolved oxygen analysis can

be used to measure the health, amount and type of biomass, and amount of

decomposition happening in a water body (Bruckner, n.d.).

2.1.5.2 Biochemical Oxygen Demand

The Biochemical Oxygen Demand is the measurement of the dissolved

oxygen required for microorganisms, mainly bacteria, to oxidize organic materials

(Hocking, 2005). It requires some kind of empirical test to determine the

molecular oxygen given during a specified incubation period or time (usually five

days), for the biochemical deterioration of organic matter (carbonaceous

demand) and the oxygen used to oxidize inorganic matter (e.g., sulphides and

ferrous iron) (Scholz, 2006). BOD denotes the amount of putrefiable organic

matter that can be found in water (Robson, n.d.). Therefore, in order to have a

good quality of water you need to have a low BOD, because a high BOD

indicates polluted water for a reason that a high BOD gives a greater amount of

organic matter or “food” available for oxygen absorbing bacteria. If the rate of DO

consumption by bacteria exceeds the supply of DO from aquatic plants, algae

photosynthesis or diffusing from air, unfavorable conditions occur. (Biochemical

Oxygen Demand, 2017).

2.1.5.3 Chemical Oxygen Demand

Chemical Oxygen Demand (COD) is the measure of the capacity of water

to consume oxygen during the time of decomposition of the organic matter and

the oxidation of the inorganic chemicals (COD or Chemical Oxygen Demand

Definition, 2009). COD is an important in the water quality parameter similar to

Biochemical Oxygen Demand (BOD) because it provides index to assess the

effect of discharged wastewater present in the environment (COD in Wastewater,

n.d.).

2.1.5.4 Oil and Grease

 American Public Health Association defined oil and grease as “any

material recovered as a substance soluble in the solvent. It includes other

material extracted by the solvent from an acidified sample (such as sulfur

compounds, certain organic dyes, and chlorophyll) and not volatilized during the

test.” The concentration of dispersed oil and grease is an important water quality

parameter (Pisal, 2009). It can decrease the level of dissolved oxygen and

increase BOD in the water body due to the formation of oil layer on the surface of

the water that prevents oxygen diffusion (Katiyar et al., 2007). This reduces the

biological activity of treatment process where oil forms around microbes in

suspended matter and water (El-Gawad, 2014).

2.1.5.5 pH Level

pH, potential of Hydrogen, is the measure of the acidity and the alkalinity

of the water-soluble substances. It is usually used to check the water to make

sure it is not too alkaline or acidic (pH Scale, 2009). Determining the pH level is

important because it will also determine the acidity of the water sample

concerning to its water quality (pH in Water, n.d.).

2.1.5.6 Nutrient Concentration (Nitrate and Phosphate)

According to the Minnesota Pollution Control Agency, Phosphorus (P) and

Nitrogen (N) are the primary nutrients that in excessive amounts pollute different

water bodies (lakes, streams, etc.). Nitrogen is essential to the production of

plant and animal tissue. It is used primarily by plants and animals to synthesize

protein. Nitrogen enters the ecosystem in several chemical forms and occurs in
other dissolved or particulate forms, such as tissues of living and non-living

organisms. Nitrate, a compound containing nitrogen, exists as dissolved gas in

water, and at elevated levels can have harmful effects on humans and animals.

Nitrates in water can cause severe illness in infants and domestic animals.

Common sources of excess nitrate reaching lakes and streams include septic

systems, animal feed lots, agricultural fertilizers, manure, industrial waste waters,

sanitary landfills, and garbage dumps.

According to the United States Geological Survey, Phosphorus is a

common constituent of agricultural fertilizers, manure, and organic wastes in

sewage and industrial effluent. It is an essential element for plant life, but when

there is too much of it in water, it can speed up eutrophication of rivers and lakes.

Under natural conditions phosphorus is typically scarce in water. In the late

1960s, scientists discovered Phosphorus contributed by human activity to be a

major cause of excessive algae growth and degraded lake water quality.

Phosphates, the inorganic form, are preferred for plant growth, but other forms

can be used when phosphates are unavailable. Phosphorus builds up in the

sediments of a lake. When it remains in the sediments it is generally not available

for use by algae; however, various chemical and biological processes can allow

sediment phosphorus to be released back into the water. An example is bottom-

feeding rough fish, such as Carp, can stir up bottom sediments, releasing

phosphorus back into the water. (Nutrients: Phosphorus, Nitrogen Sources,

Impact on Water Quality, 2008)

 In the 1997 Clean Water Action Plan, the U.S. Environmental Protection

Agency identified high level of nutrients as a significant national problem

contributing to water pollution. States reported that more than half of all lakes

were affected. Water bodies require some nutrients to be healthy, but too much

can be harmful. When lakes receive an overabundance of nutrients, they can

become polluted by excessive amounts of algae. Die-off and decomposition of

algal blooms can reduce dissolved oxygen and suffocate fish and other aquatic

life. Some forms of algae (blue green) may produce toxins that can be harmful if

ingested by humans and animals. Also, the severe nuisance algal blooms yield

unpleasant odor and appearance that reduce the aesthetic appeal of lakes.

2.1.5.7 Heavy Metals (Cadmium and Chromium)

Heavy metals can enter water by industrial and consumer waste, and

even from acidic rain releasing heavy metals into streams, lakes, rivers and

groundwater. The usual heavy metals found on the water are the Cadmium (Cd)

and Chromium (Hexavalent). (Heavy Metals, 2019) Heavy metals that are found

in drinking water are considered harmful to health (Health Risks of Heavy Metals,

n.d.). Some of the heavy metals are Cadmium (Cd) and Chromium (Hexavalent)

that has a high degree of toxicity (Patlolla et al., 2014).

2.1.5.8 Temperature

Temperature is one of the key factors of an aquatic system. It affects the

chemical and biological processes in a body of water. It can be used in the

determination of salinity, in colorimetric analyses, and in general laboratory

activities. This parameter can be measured using any type of thermometer, but

the use of mercury-filled thermometer is not advisable as it poses environmental

risks (Baird et al., 2017).

2.1.5.9 Color

Although color is not a toxic characteristic, it is considered by the EPA as

a secondary parameter affecting the appearance and palatability of the water

(Oram, n.d.) Highly colored water has significant effects on aquatic life and

growth. Light is very critical for the growth of aquatic plants and colored water

can limit the penetration of light. Thus, a highly colored body of water could not

sustain aquatic life which could lead to the long-term impairment of the

ecosystem. Very high algal growth that stays suspended in a water body can

prevent light penetration as well as use up the dissolved oxygen in the water

body, causing a eutrophic condition that can drastically reduce all life in the water

body (Color and Drinking water, n.d.).

Color analysis is any technique by which an unknown color is evaluated in

terms of known colors. Color analysis is widely used in scientific studies involving

the appearance of objects and lights and is of great importance for the analysis

of raw materials and finished products of industry. Color analysis is also

important for monitoring process liquids and as a measurement of water quality

for distribution or discharge. Color in water or wastewater may result from the

presence of metals, organic acids, microbiological matter and/or industrial wastes

(Color Analysis, n.d.).

 2.1.5.10 Total Suspended Solids (TSS)

Solids are particles dissolved or suspended in different water bodies, as

well as waste-waters, which can pose adverse impacts on the water quality.

Waters with highly-concentrated solids can induce negative physiological

reaction in the consumer thus, a limit of 500 mg dissolved solids/L was set as a

guideline for drinking water. Consequently, waters of this kind may be

unappealing and not be favorable for bathing and other recreational activities

(APHA, 2017).

Total Suspended Solids (TSS) refers to the retained portion of materials

(total solids) when an aqueous sample is filtered. These are any particles

suspended in any water body that measures larger than two (2) microns. These

are mostly made up of inorganic materials. High concentration of suspended

solids may affect aquatic photosynthesis, increase water temperatures, and

decrease the level of dissolved oxygen (Fondriest Environmental Inc., n.d.).

2.1.6 Microbiological/Bacteriological Analysis

2.1.6.1 Total Coliform

Total Coliform refers to a group of related bacteria which are not generally

harmful to humans. Coliform bacteria are found, especially, in the feces of all

humans and warm-blooded animals (Coliform Bacteria and Drinking Water, n.d.).

Although total coliform bacteria are unlikely to be harmful, this group of bacteria

can indicate presence of pathogens in water bodies. Pathogens are a group or

variety of parasites, viruses, and harmful bacteria and ingestion of these

biological and non-biological microorganisms can cause health risks in humans.

According to the Environmental Protection Agency (EPA), total coliforms

are used to determine the acceptability of water treatment and the integrity of the

distribution system (Revised Total Coliform Rule and Total Coliform Rule, 2017).

Regular testing of coliform bacteria is one of the most essential factors to assess

and monitor possible fecal contamination in water bodies.

2.1.6.2 Fecal Coliform

According to the Washington State – Department of Ecology, Fecal

coliforms are a group of bacteria found in the feces of warm-blooded animals

such as people, livestock, pets, and wildlife. The amount of fecal coliform in a

stream or lake increases with the amount of sewage waste and/or manure. High

levels of fecal coliform can cause other problems as well. Sewage and manure

contain Nitrogen and Phosphorus, which act as fertilizer for algae and other

aquatic plants. An overgrowth of plants can deplete Oxygen (O 2) in the water that

is needed by fish and other aquatic animals, affect the natural acidic/alkaline (pH)

balance of water, interfere with recreational activities such as boating, fishing,

skiing, and swimming, and create odor problems and unpleasant views (Focus

on Fecal Coliform Bacteria, n.d.).

2.1.6.3 Fecal Streptococci

According to the United States Environmental Protection Agency, Fecal

Streptococci (Enterococcus) is a subgroup of the genus streptococcus. Fecal

 streptococci can be found within stomachs and intestines of humans and animal.

They can be distinguished because of its great resistance (Fecal Coli Bacteria

and Streptococci, 2019). Although they are generally not harmful themselves,

fecal streptococci indicate the possible presence of pathogenic bacteria, viruses,

and protozoans that also live in human and animal digestive systems. Therefore,

their presence in different bodies of water indicate that pathogenic

microorganisms might also be present and that activities relating the specific

body of water might be a health risk. Since it is difficult, time-consuming, and

expensive to test directly for the presence of a large variety of pathogens, water

is usually tested for coliforms and fecal streptococci instead. Sources of fecal

contamination to surface waters include wastewater treatment plants, on-site

septic systems, domestic and wild animal manure, and storm runoff. In addition

to the possible health risk associated with the presence of elevated levels of fecal

bacteria, they can also cause cloudy water, unpleasant odors, and an increased

oxygen demand (Water: Monitoring & Assessment-5.11 Fecal Bacteria, 2012).

2.1.7 Instrumentation

2.1.7.1 Winkler’s Azide Modification Method

Winkler’s Method is the standard test used to measure Dissolved Oxygen

(DO) and Biochemical Oxygen Demand (BOD) in a water body (Bruckner, n.d).

Initially, the quantification of dissolved oxygen was conducted by separating and

collecting the dissolved gases and by analyzing it for oxygen. However, this

method is complicated and takes so much time to perform, paving the way for the

development of the Winkler technique (Kunz, 2009). The Winkler Method uses
titration to extract dissolved oxygen in a water sample. In the analysis, the water

sample is treated with a series of reagents that form an acid compound that is

then titrated with a neutralizing compound that will produce a certain color. This

change in color is called the “endpoint”, which occurs simultaneously with the

dissolved oxygen concentration in the sample. . If a white precipitate appears, no

oxygen was present in the sample. If a brown precipitate appears,

MnS04 + 2NaOH Mn(OH)2 + Na2SO4 White precipitate

2Mn(OH)2 + O2 2MnO(OH)2 Brown precipitate

For every titration of 200 mL sample, 1 mL 0.025M Na2S2O3 = 1 mg DO/L.

This method is ideally conducted in the field immediately after collection as the

sample will be less altered by various factors (Bruckner, n.d)

2.1.7.2 Partition-Gravimetric Method

Partition-Gravimetric Method is the approved method of analysis for oil, grease,

and Total Suspended Solids (TSS) by the DENR. It is the procedure used for the

analysis of oil and grease; and is recommended for liquid samples. This method

relies on the determination of weight as a medium to quantify an analyte (Baird

et. al, 2017). There are four (4) main types of gravimetric method: Precipitation

Gravimetry, Electrogravimetry, Volatilization Gravimetry, and Particulate

Gravimetry (Harvey, 2008).

In determining oil and grease using the Partition-Gravimetric Method, dissolved

or emulsified oil and grease are extracted from the water sample through an

intimate contact with trichlorotrifluoroethane; petroleum ether (40/60) or hexane

(APHA – 2530C, 2017).

In determining the Total Suspended Solids, the sample is filtered and the

residue on the filter is dried to a constant weight at 103 to 105oC. The increase in

the weight of the filter is the TSS value of the sample (APHA – 2540D, 2017).

Calculation of the total suspended solids is given by the equation:

𝑚𝑔 𝑡𝑜𝑡𝑎𝑙 𝑠𝑢𝑠𝑝𝑒𝑛𝑑𝑒𝑑 𝑠𝑜𝑙𝑖𝑑𝑠 (𝐴 − 𝐵) 𝑥 1000

=
𝐿 𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒, 𝑚𝐿

where A is the final weight of the filter with the dried residue in mg, and B

is the weight of the filter in mg.

2.1.7.3 Open Reflux Method

Open Reflux Method is the most preferable method in determining the

Chemical Oxygen Demand in a water sample. This method is the most used

technique among the other standard procedures set by the American Public

Health Association (APHA) due to its suitability for a wide range of wastes where

a large sample size is essential. In this process, most types of organic matter are

oxidized by a boiling chromic and sulfuric acid mixture. The sample is treated in

strongly acid solution with a known excess of potassium dichromate and titrated

with a series of reagents. The higher oxidizing ability of the dichromate reflux

makes the method advantageous to others (Clesceri et al., 1998).

2.1.7.4 Membrane Filtration Method

Microbiological analyses refer to the different methods used for identifying

presence of pathogenic microorganisms in water resources. Membrane Filtration

Method is one of the most common microbiological analyses used to identify

presence of coliform bacteria in water. Membrane Filtration (MF) Method refers

to the technique used in estimating bacterial population in low-turbid waters and

that involves the use of membrane filters (Membrane Filtration Guidelines, n.d.).

These filters are thin porous sheets made up of cellulose esters or polymers of

the like. Membrane filters act as two-dimensional screens wherein all particles,

both biotic and abiotic, exceeding the size of the pore, are retained on the

surface of the filter and separated from the fluid passing through (Aryal, 2018).

Introduced in the late 1950s, the Membrane Filtration (MF) Method is an

alternative technique for Multiple Fermentation Tube (MPN) technique used for

microbiological assessment of water. The use of membrane filtration method is

highly advisable because of the several advantages that it offers compared to

other traditional techniques of microbiological analysis. Some of these

advantages are: (1) MF method allows testing of large sample volumes, (2)

shorter preparation time compared to MPN technique, (3) it allows the isolation

and enumeration of separated colonies of bacteria, and (4) it allows the removal

of bacteriostatic agents that would not be eliminated in MPN technique or other

traditional methods (Membrane Filter Technique, n.d.). However, Membrane

Filtration (MF) method is not advisable for analyzing highly turbid waters that

contain large suspended minerals as these particulates may clog the pores of

membrane filters, inhibiting the passage of water (Aryal, 2018).

The standard unit of measurement for the number of viable bacterial cells

present in water samples is the Colony Forming Unit (CFU).

 The CFU is given by the equation:

𝐶𝐹𝑈 𝐵𝑎𝑐𝑡𝑒𝑟𝑖𝑎𝑙 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

= 𝑥 100
𝑚𝑙 𝑚𝐿 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒

where mL of the sample refers to the actual volume of the sample and not the

volume after the series of dilution.

2.1.7.5 Atomic Absorption Spectrophotometry

Atomic Absorption Spectrophotometry (AAS) refers to an analytical

method of measuring the concentration of elements. It is a sensitive technique

used for the quantitative determination of more than sixty metals. Atomic

Absorption Spectrophotometry is an important method of water quality analysis

because it detects traces of heavy metals in water bodies. Failure in detection of

these heavy metals poses great risk both for the aquatic system and the

organisms that benefit from it, especially if these heavy metals are present in

high levels – wherein they tend to be poisonous.

In Atomic Absorption Spectrophotometry (AAS), wavelengths of light that

are specifically absorbed by the sample heavy metal or element are used.

Analyzing a sample to check if it contains traces of element means using light

from the element itself. At first, sample is atomized, converted into free atoms in

the vapor state, and a beam of electromagnetic radiation containing excited

atoms of tested element is passed through the free atoms. The amount of the

light absorbed and number of atoms of tested element are proportional. The
calculation of concentration of element is done by comparing the calibration

curve and the amount of light absorbed (Atomic Absorption Spectrometry, n.d.).

2.1.7.6 Colorimetric Analysis

Colorimetric Analysis is a technique normally used to determine the

concentration of analyte through the comparison of color changes of the solution

(Trends in Analytical Chemistry, 2018). It is a method or analysis in which

reagents are added to a rock solution to form colored compounds with specific

elements. The intensity of the color, measured on a spectrophotometer, is

proportional to the concentration of the element (Oxford University Press, 1999).

In this method, two (2) fundamental laws are applied: 1) Lambert’s Law, which

relates the distance travelled by the light and the amount of light absorbed and 2)

Beer’s Law, wherein the light absorption and concentration of the absorbing

substance are correlated to one another (Ozase, 2016).

2.1.7.7 Visual Comparison Method (Platinum-Cobalt Scale)

Visual Comparison is the standard quantification method for the color of

the water set by the APHA. From the name itself, color is determined through

differentiation of the sample with known concentrations of colored solutions

(Platinum-Cobalt). The Platinum-Cobalt method is applicable to a wide range

water body ranging from natural waters to industrial waters (APHA, 2017). True

color refers to the color of the water without the particulate matter, while apparent

color is the color of the water from the mixture of true color and any particulate
 matter. The standard unit of measurement for this is the Hazen Unit (HU) or True

Color Units (TCU) (NHRMC, 2013).

The TCU is given by the equation:

𝐴 𝑥 50
𝐶𝑜𝑙𝑜𝑟 =
𝐵

where A is the estimated color of the diluted sample, and B is the volume of

sample (mL) taken for dilution.

2.1.8 Statistical Data Treatment and Evaluation

2.1.8.1 The Mean

The widely and commonly used measure of central tendency is the mean,

represented as X. The mean, also called as the arithmetic mean or the average,

is obtained by dividing the sum of replicate measurements by the number of

measurements in the data set.

∑𝑥𝑖
𝑋 =
𝑛

where Xi represents the individual values of X that makes up the set of N

replicate measurements (Skoog, 2013).

2.1.8.2 Standard Deviation and Relative Standard Deviation

The population standard, σ, is the measure of the precision of the

population or how measurements of the population are spread out from the mean

or expected value.
 ∑(𝑥 − 𝑥̅ )2
σ=√
𝑛

where n is the number of data points that make up the population (Skoog, 2013).

Frequently, standard deviations are given in relative rather than absolute

terms. To calculate the relative standard deviation, the standard deviation is

divided by the mean or average value of the set of data.

𝜎
𝑅𝑆𝐷 = 𝑥 100%
𝑥

where σ is the standard deviation of the population and x is the population mean.

2.1.8.3 Range

The range is the difference between the maximum and minimum values of

a data set. It is the easiest method to measure how the set of data are spread out

(Taylor, 2019).

𝑅𝑎𝑛𝑔𝑒 = 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑉𝑎𝑙𝑢𝑒 − 𝑀𝑖𝑛𝑖𝑚𝑢𝑚 𝑉𝑎𝑙𝑢𝑒

2.2 Related Studies

In the study “Indicative Factors in Deterioration of the Water Quality of Lahug

River, Cebu City, Philippines” conducted by Bensig et al. on 2014, the water quality of

Lahug River in Cebu City was evaluated using coliforms as indicators and its correlation

with physico-chemical parameters which was done once a month from November 2011

to April 2012 in three stations (upstream, midstream, and downstream). The level of the

indicator bacteria increased from the upstream towards the low000er reaches of the
river, especially in the months of February to April; however, there were no significant

difference in values across time that implied that there was a continuous fecal pollution

in the river. Observation suggested a negative relationship between Fecal Coliforms

and Total Coliforms with the pH level, Total Suspended Solids, Dissolved Oxygen, and

nitrate (p>0.05). These results suggest the presence of sewage and organic pollutants

in the river.

The study entitled “Assessment of water quality in Alamein Marina Recreational

Lagoon” conducted by El-Rayis in the year 2012, attempted to evaluate the

environmental impacts of the touristic summer activities on the lake and to determine at

what level this recreational lagoon stands as a healthy resort, among the other resorts

around the Mediterranean Sea Basin. The Mediterranean Sea Basin was established to

contain most of the recreational lakes. Water samples were collected from fourteen (14)

sampling stations. The physical, chemical, and bacteriological characteristics of the

lagoon water were determined. After the analysis, results showed the water pH ranging

between 6.8 and 7.9. Dissolved Oxygen (DO), Biochemical Oxygen Demand, nitrates

and phosphates were 6.5 mg/L, 1 mg/L, 0.5 mg/L, and 3.8 mg/L respectively. Fecal

coliform were not detected in all sampling stations. The study concluded that the

characteristics of water in Alamein Marina Lagoon were complying with the standards

set by Egyptian legislations concerning recreational water, all over the study period. The

water was also complying with the levels set by different international organizations

such as the WHO and Blue Flag. Consequently, by calculating the water quality index

for Alamein Marina Lagoon and its four sub-basins, water was found to be of an

‘excellent’ quality.
 The study entitled “A comparative study of physicochemical and biological water

quality parameters of Iligan Bay, Philippines” conducted by Bugtong et al. in the year

2018, analyzed the physico-chemical and biological water quality parameters of three

selected coastal waters of Iligan Bay, Philippines which has been a major fishing ground

for its rich fishery resources. A total of 11 physico-chemical and 3 biological parameters

were observed. Results showed that selected sites in Iligan Bay generally, did not

comply with the limits of quality set by the DENR especially for surface water

temperature and pH. Moreover, results for bacteriological analysis implied that the three

sites examined exhibited relatively poor water quality during the study period analysis

and stressed that water samples have high concentrations of fecal contamination in

almost all sources studied. In this respect, “poor” water quality is attributed to

anthropogenic activities in the area.

In another study entitled “The Water Quality of the Pasig River in the City of

Manila, Philippines: Current Status, Management and Future Recovery” conducted by

Gorme et al. in the year 2010, assessed the water quality of Pasig River an important

river in the Metro Manila, Philippines, since it provides food, livelihood and transport to

its residents, and connects two major water bodies; Laguna de Bay and Manila Bay.

Despite the efforts of the government to revive the quality of the Pasig River and its

tributaries, it continues to deteriorate over time due to the large amount of wastes

dumped into the river, such as domestic, industrial and solid wastes. The paper

provided an overview of the current condition of the Pasig River. The existing water

management policies were reviewed, and the issues and challenges hindering the

improvement of its water quality were identified. Moreover, the qualities of different
rivers in Metro Manila were compared to those of the major rivers in South Korea. The

current watershed management system practiced by South Korea has been discussed

to serve as a guideline for future recovery of the water quality of the rivers in the

Philippines.

In a biodiversity assessment conducted in the waters of the Pasig River,

Philippines which was carried out by the DENR in the year 2009 showed that eight

species of fish had already returned to the river after just a decade of rehabilitation. Five

of these were exotic, two were native while the Manila sea catfish or kanduli was

endemic. Despite these findings, analyses showed that the fish remained unsafe for

human consumption. Based on the Pasig River Rehabilitation Commission’s (PRRC)

sampling and tests done earlier this year (2019), the fish caught at different ferry boat

stations around Pasig river had fecal coliform levels that exceeded the standard of 10

most probable number (MPN) per gram, high levels of lead and mercury. Whereas

exposure to and consumption of these toxic chemicals at high amounts pose health

risks. According to the PRRC, this could be due to the wastewater discharged by

nearby medical institutions and a distillery. But while authorities hailed these as new

signs of life in the river once declared as “biologically dead,” the PRRC cautioned the

public against eating the fish taken from its waters. The warning was issued after

laboratory tests conducted by the PRRC on some of the fish that showed alarmingly

high amounts of toxic chemicals. Samples also tested positive for overwhelmingly high

levels of fecal coliform — bacteria from human and animal feces.

 Chapter 3

METHODOLOGY

This study is consisted of five phases: (1) sampling of water in five stations from

Polytechnic University of the Philippines (PUP) Lagoon; (2) physical analysis of water

samples; (3) chemical analysis of water samples; (4) microbial or bacteriological

analysis of water samples; and (5) statistical treatment of data.

Water sampling

Microbiological
Physical Analysis Chemical Analysis
Analysis

Statistical
Treatment of Data

Figure 1. Schematic diagram for the experimental processes and statistical

interpretation of data
All the procedures and methods conducted in this study are aligned with the

Department of Environment and Natural Resources (DENR) – Philippine Standard

Methods for Air and Water, and Standard Methods for the Examination of Water and

Waste Waters set by the American Public Health Association, together with the

American Water Works Association (AWWA) and Water Pollution Control Federation

(WPCF).

3.1 Sampling of Water in Stations from PUP Lagoon

The researchers selected five (5) different sampling sites based on their

accessibility.

Table 1: The Sampling Sites of Water in Lagoon

Sampling Point Location

Station 1 _

Station 2 _

Station 3 _

Station 4 _

Station 5 _

The study area is located inside the walls of the Luntiang Pilipinas Forest Park

within the vicinity of PUP Main Campus; surrounded by different food stalls. Water

samples were collected using bottles pre-treated according to the method with respect

to their parameters; and processed based on their respective quantifying procedures.

3.2 Physical Analysis of Water Samples

The five (5) water samples collected from the sampling sites in the lagoon: (1)

Station 1, (2) Station 2, (3) Station 3, (4) Station 4, and (5) Station 5 were tested for

their physical parameters. Real-time temperature of each sample was determined on-

site using a Thomas Enviro-Safe Pocket Thermometer and the results were recorded

and analyzed.

3.2.1 Visual Comparison Analysis (Platinum-Cobalt Scale)

100 mL sample water from each of the station were stored in acid-washed

amber bottles and kept cold for 24 hours before warming up at room temperature

for analysis. The pH levels of the samples were adjusted to pH 7 by adding

Sodium Hydroxide (NaOH). The 0.22-µM-pore-diam membrane filter was first

washed by passing 50 mL water through the filter. 25 mL of sample water was

filtered, and then the filtrate was discarded. Another 50-mL of same sample

water was filtered in which the filtrate was analyzed. The matched nessler tubes

were filled to the 50-mL mark with the samples and compared to the Platinum-

Cobalt standard.

3.3 Chemical Analysis of Water Samples

3.3.1 Winkler’s Azide Modification Method

3.3.1.1 DO Analysis

2 mL MnSO4 solution were carefully added to the samples collected

in a 300-mL BOD bottle through the pipette followed by 2 mL alkali-iodide-

 azide reagent. The solutions were mixed carefully by inverting several

times. When a brown precipitate had settled, the solution was thoroughly

mixed again, then 2 mL of concentrated H2SO4 were added. In a glass

flask, the 201-mL samples were titrated with a 0.025M Na2S2O3 solution. 2

mL of starch solution was added to the mixture and continuously titrated

through a calibrated pipette until the blue color disappeared. The

dissolved oxygen was measured with a titration of 200 mL sample, 1 mL

0.025M Na2S2O3 = 1 mg DO/L.

3.3.1.2 BOD Analysis

For the treatment of the BOD bottles, the researchers used

detergent and dilute hydrochloric acid (HCN) (3N) then rinsed it thoroughly

with deionized (DI) water. The bottles were incubated 20oC in the dark

until samples were taken. After the samples were taken, the bottles were

sealed with water seal and incubated again 20oC in the dark. The samples

were taken immediately to the laboratory for analysis. For reproducibility

and precise measurements of BOD, the samples were examined

professionally for five (5) days under SGS Philippines, Inc.

3.3.2 Partition-Gravimetric Method

For the determination of oil and grease, the samples were first weighed for

determination of the sample volume, and then acidified. The samples were

then transferred to a separatory funnel via liquid funnel. The bottles were

rinsed with 30 mL extracting solvent and solvent washings were also added to
the separatory funnels. The aqueous layers were drained in the original

container and solvent layer through a funnel with a filter paper and 10 g

Na2SO4. The clear solvent was then drained with pre-moistened filter paper

and 10 g Na2SO4. Recombine the aqueous layer with the solids in the funnel.

The steps were repeated but with a 30 mL solvent each time. Finally, the

extracts were combined with distilling flasks and filtered with Na2SO4 and

additional 15 mL solvent. The remaining solvents were distilled in a water bath

at 85oC. The solvent was collected in an ice-bath-cooled receiver then a

vacuum was connected to draw air. The desicator was cooled in until a

constant weight is obtained. The oil and grease were calculated as follows:

𝑚𝑔 𝑜𝑖𝑙 𝑎𝑛𝑑 𝑔𝑟𝑒𝑎𝑠𝑒 𝑊𝑟

=
𝐿 𝑉𝑠

where W r is equal to total weight of flask and residue, minus tare weight of

flask, mg, and Vs is equal to initial sample volume, in L.

In determining the total suspended solids of each sample, 500 mL

of each well-mixed samples were filtered through a commercially prepared

glass-fiber filter. The filters were washed with three successive volumes of

10-mL reagent-grade water, then vacuumed for the complete removal of

water. The filters were removed from the filtration apparatus, dried for two

(2) hours in a 103-105oC oven, cooled in a desiccator to ambient

temperature, and weighed. The procedures were repeated until the weight

change became less than 0.5 mg. The results were calculated and

analyzed.
3.3.3 Open Reflux Method

To obtain the COD of the samples, 50 mL of it were pipetted into a

500-mL refluxing flask. 1 g of HgSO4, 5 mL sulfuric acid reagent, 25 mL

0.04167 M K2Cr2O7 were added to each of the samples respectively. The

flask was attached to condenser then the remaining sulfuric acid reagents

were added slowly. The mixtures were refluxed for 2 hours, cooled at

room temperature then the excess K2Cr2O7 were titrated with Ferrous

Ammonium Sulfate (FAS), using 0.10 to 0.15 mL ferroin indicator. From

the results, the COD levels were calculated and analyzed.

3.3.4 Atomic Absorption Spectrophotometry

The samples were examined for the presence of Cadmium and

Chromium using Atomic Absorption Spectrophotometer (SGS Philippines,

Inc).

3.3.5 Colorimetric Analysis

The samples were examined for the concentration of Nitrate and

Phosphate using Colorimetric Analysis (SGS Philippines, Inc.). A standard

curve was prepared by plotting the absorbance of standards against the

standards of the respective elements involves.

3.4 Microbiological or Bacteriological Analysis of Water Samples

3.4.1 Membrane Filtration Method

100 mL samples from each of the five (5) different stations were

collected to test for the presence of Total Coliform, Fecal Coliform, Fecal

Streptococci, E. coli, and Pseudomonas aeruginosa. The samples were

placed in 60 mm by 15 mm borosilicate glass Petri dishes with flat

bottoms.

3.4.1.1 Dilution

Water samples from non-potable sources must be diluted to

a volume at which bacteria can be measured (Membrane Filtration

Guidelines, n.d.). To create a 10-fold dilution factor, 11 mL of

sample was added into the bottle of buffered dilution water using a

sterile pipette. An 11 mL of the 10-fold diluted sample solution was

added into another dilution bottle thus, creating a 100-fold diluted

sample. Another 11 mL of the 100-fold diluted sample was added

into another bottle of dilution water hereby creating an ideally 1000-

fold diluted sample.

3.1.4.2 Filtration

Using sterile forceps, place a sterile filter over porous plate

of the vacuum support, with the grid side up. Carefully place funnel

unit over the vacuum without touching the inside of the funnel. Pour

the diluted sample on the funnel and with filter still in place, rinse
 funnel by filtering three 20- to 30-mL portions of sterile dilution

water to uniformly spread the bacteria over the filter. Remove the

funnel and immediately remove the membrane filter. Using sterile

forceps, place the filter on a saturated pad with a rolling motion to

avoid entrapment of air.

3.1.4.2 Culture/Incubation

The different samples used to test for specific coliform

bacteria and indicator species were placed in specific culture media

and incubated under prescribed time and temperature. Autoclaved

Erlenmeyer flasks with metal caps were used for the storage of

different culture media.

 For Total Coliform Bacteria, m-Endo medium was used, and the

sample was incubated at 24 ± 2 hours at 35.0 ± 0.5o C.

 For Fecal Coliform Bacteria, m-FC medium was used, and the

sample was incubated at 44.5 ± 0.2o C for 24 ± 2 hours.

 For Fecal Streptococci Bacteria, KF medium was used, and the

sample was incubated at 48 ± 2 hours at 35.0 ± 0.5o C.

 For Escherichia coli (E. coli), m-Tech medium was used for

primary culture, and the sample was incubated at 44.5 ± 0.2 o C for

24 hours after resuscitating for 2 hours at 35.0 ± 0.5 o C. After 22 to

24 hours, transfer the membrane filter to urea substrate broth for 15

to 20 minutes.
  For Pseudomonas aeruginosa, pseudomonas medium was used,

and the sample was incubated for 24 hours at 35 ± 0.5 o C.

3.5 Statistical Treatment and Evaluation

The data gathered from the Winkler’s Azide Modification Method, Partition

Gravimetric Method, Open Reflux Method, Atomic Absorption Spectrophotometry,

Colorimetric Analysis were evaluated using the different statistical treatment. The

results of the assessments were aligned with the Department of Environment and

Natural Resources (DENR) – Water Quality Guidelines and General Effluent Standards.

The number of colonies counted after the Membrane Filtration Method was

treated using the equation for the colony forming units per mL. The results from the

calculation were interpreted and aligned with the Water Quality Guidelines and General

Effluent Standards set by the Department of Environment and Natural Resources

(DENR).
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