INTRODUCTION

Background of the Study

Pollution is the introduction of a contaminant into the environment. Pollution has

a detrimental effect on any living organism in an environment, making it virtually

impossible to sustain life. Pollution is caused by industrial and commercial waste,

agricultural practices, everyday human activities and most notably, modes of

transportation.

The Philippines is an archipelago, which means it is made up of a group of islands

and is rich in bodies of water such as the oceans, rivers, and lakes. However, according to

the data released by Water Environment Partnership in Asia (WEPA) in 2005, 42.89% of

the waters in the Philippines are contaminated. (Study Mode. 06.2013) This

contamination is known as water pollution. It occurs when water running over the land

surface and through the ground collects natural or human-made pollutants and deposits to

the water bodies. Sources of water pollution are either from point and non-point sources.

Point sources of pollution come from precise locations or identifiable sources live

industrial discharges, municipal wastewater effluents, manufacturing processing

operations, septic systems and raw sewage. On the other hand, non-point sources are

diverse sources and origins like agricultural run-off, oil grease and toxic chemicals run-

off, leachate from municipal solid waste, oil spills and illegal dumping. (National State of

the Brown Environment Report, 2009)

Water pollution can sway the balance of an ecosystem resulting in danger to

natural wildlife of aquatic environments. It has a negative effect on the delicate balance

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of the ecosystem. It destroys organisms that provide foods for other animals. It can cause

water shortage due to polluted water supplies. Harmful substances in water cause human

illnesses and diseases. Cholera and typhoid fever are caused by eating shellfish taken

from polluted water. Serious damage to the brain, kidneys, and liver may result from

drinking water that contains lead or mercury. (Fears, 2009), (Tolentino, et.al, 2008)

Globally, the effluents that are discharged from wastewater treatment systems

represent one of the largest sources of pollution. The negative impacts of these effluents

to aquatic ecosystems and to humans, from harmful substances found in them have been

documented both at national and international levels Some of these impacts can include

death of aquatic life, algal blooms, habitat destruction from sedimentation, debris, and

increased water flow and other short and long term toxicity from chemical contaminants;

in combination with chemical accumulation and magnification at higher levels of the

food chain. (Akpor O. B., 2011 as cited in Canada Gazzette, 2010).

Bioassay test determines the relative strength of a substance by comparing its

effect on a test organism with that of standard preparation. It also examine the toxicity of

a medium, usually water, soil, or sediment, by evaluating exposure effects on a variety of

organisms. Typically an organism is exposed to the medium of interest for a pre-

determined period. Observations and measurements made during, and after the test are

called endpoints. Endpoints determine if the test organism was negatively impacted from

exposure to the medium. Bioassays may also be called toxicity tests. (EIM, 2013)

Hence, this research was conducted to address some problems in water

pollution in the country and to determine possible effects of wastewater to the living

organisms in bodies water by conducting a bioassay test.

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Objectives of the Study

Generally, this study aimed to determine the effects of effluents from selected

creeks in Pinsao Pilot Project, Baguio City. Specifically, it aims to:

1. Determine if EC50 will be established by any treatment from the two sources of

wastewater.

2. Determine if there are significant difference in the EC50 or mortality means between

and among the treatments (after 48 hours) by the sources of wastewater.

3. Determine which treatment will have the highest and lowest mortality for Daphnia

magna (after 48 hours).

4. Determine which source will have higher and lower mortality for Daphnia magna

(after 48 hours).

Significance of the Study

Bodies of water are one of the most significant resources for human being. Most

of the residents in the Cordillera Administrative Region and in the whole Philippines are

dependent on the water bodies for their domestic activities like using water for washing

clothes, bathing and others. People also use water for economic related activities like

source of irrigation and the like. Studies therefore are very significant in order to provide

relevant and useful data regarding the resource under investigation.

A bioassay was conducted to determine the toxic effects of effluent to the

mortality of Daphnia magna. This study is important because it provides the complete

response of the test organism to all compounds present in wastewater. The test organisms

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represent the living organisms (fauna) in the bodies of water because of their

psychological similarities.

Wastewater coming from the different residents will be carried and disposed to

the bodies of water, thus contaminating it. Therefore, the findings of this study will

provide benchmark information for those interested in finding out the useful value of

Daphnia magna as well as the people, for them to be aware about the water quality of the

wastewater from the selected creeks of the community. With this it will eliminate the

chance of the extinction of living organisms in bodies of water.

Scope and Limitation of the Study

Generally, this study aimed to determine the effects of effluents from selected

creeks in Pinsao Pilot Project, Baguio City. Specifically, it aims to determine if EC 50 will

be reached by any treatment from two sources of wastewater, determine if there are

significant difference in the EC50 or mortality means between and among treatments

(after 48 hours), and to determine which source and treatment will have the highest and

lowest mortality for Daphnia magna (after 48 hours).

A bioassay test was conducted by preparing different treatments with varying

concentrations of effluent. The set-up is in laboratory conditions and considered as

controlled experiment.

Water sample testing of the effluent’s possible contaminants was not conducted

but was identified through citations from acceptable and local references with similar

conditions. These were mentioned in the Review of Literature of this research paper.

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Locale and Time of the Study

The study was conducted from July to August, 2013 in the Science Laboratory of

Pinsao National High School, Pinsao Pilot Project, Baguio City.

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 REVIEW OF LITERATURE

Nature of Bioassay

A bioassay is a procedure that uses living organisms to determine the toxicity of a

chemical. (Cahill, 2006) It is one method of assessing the presence of potentially harmful

compounds. (http://pulse.pharmacy.arizona.edu)

In a bioassay, living organisms are exposed to different concentrations of a

chemical that might be toxicant (harmful chemical). Observations of the effects on the

organisms’ behavior and survival allow researchers to determine if, or at what

concentration, a chemical has harmful effects. Typically researchers test the effects of

toxicants on model organisms that are small and easy to maintain in a laboratory setting.

(Cahill, 2006)

For environmental testing, bioassays provide an integrated picture of overall

toxicity of an effluent or a sample of water, sediment, or soil from a contaminated site.

Fathead minnows, various aquatic invertebrates, earthworms, protozoan, and seed all are

used for bioassay of aquatic samples. The idea behind this bioassay is that the test

organism will react in a predictable way to various types of environmental contaminants.

Several studies have compared the sensitivities of various types of seeds to common

pollutants. (Keddy, 1995)

Toxicity testing has grown steadily in recent years. It is an important parameter in

wastewater quality monitoring as it provides the complete response of test organisms to

all compounds in wastewater. They are useful because they integrate the toxicity of all

factors associated with the medium such as interactive effects between chemicals. For

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example, chemical A might be toxic at a high concentration, but if in the presence of

chemical B, chemical A becomes toxic at a much lower concentration. Conversely,

chemical C might be toxic at a low concentration, but in the presence of chemical D,

chemical C becomes much less toxic and requires much greater concentrations to cause

an effect. In addition, different organisms are sensitive to different chemical

concentrations and mixtures. (EIM, 2013)

Water Pollution in Urban Areas

Urban areas have the potential to pollute water in many ways. Run-off from

streets carries oil, rubber, heavy metals, and other contaminants from automobiles.

Untreated or poorly treated sewage can be low in dissolved oxygen and high in pollutants

such as fecal coli form bacteria, nitrates, phosphorus, chemicals, and other bacteria.

Treated sewage can still be high in nitrates. Groundwater and surface water can be

contaminated from many sources such as garbage dumps, toxic waste and chemical

storage and use areas, leaking fuel storage tanks, and intentional dumping of hazardous

substances. Pollution can lead to acid rain, nitrate deposition, and ammonium deposition,

which can alter the water chemistry of lakes. (Living Lakes Partnership)

Uncontrolled or treated run-off from the urban environment and from construction

activities can run-off the landscape into surface waters. This runoff can include such

pollutants as sediments, pathogens, fertilizers/nutrients, hydrocarbons, and metals.

Pavement and compacted areas, roofs, and reduced tree canopy and open space increase

runoff volumes that rapidly flow into our waters. This increase in volume and velocity of

runoff often causes stream bank erosion, channel incision and sediment deposition in

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stream channels. In addition, runoff from these developed areas can increase stream

temperatures that along with the increase in flow rate and pollutant loads negatively

affect water quality and aquatic life. (Environmental Protection Agency, 2005)

Other common sources of urban pollution include improperly sited, designed and

maintained onsite wastewater treatment (septic) systems, pet wastes, lawn and garden

fertilizers and pesticides, household chemicals that are improperly disposed of,

automobile fluids, road deicing/anti-icing chemicals, and vehicle emissions.

(Environmental Protection Agency, 2005)

An international environmental group is again raising the alarm over pollution in

drinking water in the Philippines, as it recently discovered nitrate contamination of

several water sources in areas in Benguet and Bulacan (PhilSTAR.com, 2007 )

In its new report entitled “Nitrates in drinking water in the Philippines and

Thailand,” Greenpeace examined the nitrate levels in drinking water sources like artesian

wells and creeks in Benguet and Bulacan and their relation to nitrogen fertilizer use in

farming areas in the two provinces. (PhilSTAR.com, 2007 )

Greenpeace has been sending out warning signals that the quality of our

freshwater source is declining, and the study is yet another shocking example of how

water protection measures are inadequate. The study showed that five out of the 18 water

sources where they took samples from, have nitrate contamination that are “alarmingly

well above the safety limits” of the World Health Organization, whose standard is 50

mg/l of nitrates in drinking water. Communities think that the water they drink every day

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is clean because physically, it doesn’t smell bad or look bad, but it is actually laced with

nitrates from fertilizers which people don’t normally associate with pollution.

(PhilSTAR.com, 2007 )

Based on the study, the result of Greenpeace Water Patrol’s weeklong

investigation during the last week of September, drinking water from two artesian wells

in Buguias, Benguet, and three others in Angat, Bulacan were found to have nitrate levels

well above the WHO standard.The highest nitrate level was found in groundwater in

Buguias at 50 percent higher than the WHO standard, the study said. Greenpeace warned

that the pollution could have serious health implications for the local population,

especially since groundwater is the main source of their drinking water. (PhilSTAR.com,

2007 )

The report’s author, Reyes Tirado, of the Greenpeace Science Unit in the

University of Exeter in the United Kingdom, said nitrate pollution of drinking water from

artesian wells correlates with intensive farming practices in the affected areas where

nitrogen fertilizers are applied in excess.

Nitrogen fertilizer consumption in Asia has grown dramatically, increasing

approximately 17-fold in the last 40 years. Fertilizer application rates are increasing

rapidly in some developing countries, reaching excess amounts that can no longer be used

by crops and which cause problems for human health and the environment.Excess

application of nitrogen fertilizers in intensive crop production leads to nitrate pollution of

the artesian wells that people use for drinking water. (Tirado, 2007)

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 There’s really a problem on how the government makes or implements policies

against water pollution. The government is either not doing things properly or doing it

excessively. It’s about time that the government really looks into this and takes concrete

action to ensure non-contamination of clean water sources for the people. (Baconguis,

2007)

Daphnia Magna as Test Organisms in Bioassays

The water flea Daphnia Magna is the most commonly used zooplankton in

toxicological tests in wastewater treatment, due to short doubling time, high sensitivity,

and simplicity; therefore, it was used as an indicator. (APHA, AWWA, WEF, 1992;

Official Gazette, 1996; USEPA, 2000)

This species is found in freshwater and brackish (up to 8 ppt salinity) habitats

including lakes, rivers, and temporary pools. Although they prefer temperatures between

18-22°C, they can tolerate a much broader range. ( Elenbaas, M. 2013 as cited in Ebert,

2005; Haney, 2010; Vanoverbeke, et al., 2007)

These water fleas are very small, usually 2-5 mm long, with an overall shape

similar to a kidney bean. The body is enclosed by a transparent shell-like structure, called

a carapace that is mostly made of chitin. Due to its transparent carapace, this species

tends to be the color of what it is currently eating. The carapace extends into the head

shields, an important diagnostic characteristic for this species. They have two sets of

long, doubly branched antennae and six thoracic appendages that are held inside of the

carapace and help to produce a current of water, carrying food and oxygen to their

mouths and gills. They also have two large claws, used mainly for cleaning the carapace.

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They have one compound eye, which appears as an anterior dark spot, and one simple

eye (ocellus). Males are smaller than females (typically only 2 mm long while females

are 3-5 mm long) but have longer antennules and modified, hook-like first appendages

used for clasping females during mating. (Elenbaas, M. 2013)

The life cycle begins when a female produces a clutch of eggs (usually 6-10) that

are released into her brood chamber, located under her carapace. Eggs hatch into

juveniles within this brood chamber and are released when their mother molts, typically

within 2-3 days. Juveniles, which already resemble adults, go through a series of molts

and instars. Females are considered sexually mature after developing brood pouches,

usually after 4-6 instars, usually 6-10 days. (Elenbaas, M. 2013)

Lifespan of these water fleas depends heavily on environmental conditions such

as oxygen levels, food availability, and temperature. In general, as temperature decreases,

lifespan increases, with averages of 40 days at 25°C and 56 days at 20°C. Unstable

environmental conditions tend to lead to shorter lifespans. While it has been suggested

that males of this species have shorter lifespans than females, recent research shows

evidence that this is likely not the case. (Clare, 2002; Grzesiuk, et al., 2010; Pietrazak, et

al., 2010)

Daphnia feed on small, suspended particles in the water. They are suspension

feeders (filter feeders). The food is gathered with the help of a filtering apparatus,

consisting of the phylopods, which are flattened leaf-like legs that produce water current.

As the current flows anterior to posterior, the Daphnia collect particles that are

transferred into the food groove by special setae. Although the feeding apparatus is so

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efficient that even bacteria can be collected, the food is usually made up of plank tonic

algae. Green algae are among the best food, and most laboratory experiments are done

with either Scenedesmus or Chlamydomonas, both of which are easy to culture in

monoclonal chemo stats. Daphnia usually consume particles from around 1 μm up to 50

μm, although particles of up to 70 μm in diameter may be found in the gut content of

large individuals. (Ebert,2005)

The gut is more or less tubular with three parts: the esophagus, the midgut, and

the hindgut. There are two small digestive ceca (diverticula) that are easily seen in the

head section of the midgut. The midgut is lined with an epithelium and bears microvilli.

Peristaltic contractions of the gut wall pass food through the gut, but a peritrophic

membrane contains the food and prevents it from entering the ceca. Epithelial cells do not

phagocytose particles but absorb molecules. The pH is 6 to 6.8 in the anterior part of the

midgut and 6.6 to 7.2 in the posterior part. Food is expelled from the hindgut by

peristaltic movement but also requires the pressure of more recently acquired food

particles. The color of Daphnia adapts to the food that is predominant in their diet.

Daphnia feeding on green algae will be transparent with a tint of green or yellow,

whereas those feeding on bacteria will be white or salmon-pink. Well-fed animals are

more strongly colored than starved animals. (Ebert,2005)

Daphnia have an open blood circulation. The heart is located dorsally and anterior

from the brood chamber. At 20ºC, it beats about 200 times per minute, slowing down at

lower temperatures. Blood cells are easily visible through the transparent body as they

flow rapidly through the body cavity. To support oxygen transport, Daphnia have the

extracellular respiratory protein hemoglobin (Hb), a multi-subunit, multi-domain

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macromolecule. There are at least four Hb genes. Daphnia tend to develop more Hb to

increase oxygen uptake from the water. In response to environmental changes (oxygen

concentration, temperature), the Hb concentration varies up to about 20-fold. Oxy-

hemoglobin, the form that is loaded with oxygen, is red and gives the transparent animals

a reddish appearance. Because certain parasites also cause the hem lymph to become red,

one cannot easily determine the cause of the red color from sight alone. However, low

oxygen usually affects an entire population, coloring all animals reddish, whereas

parasites usually infect only a portion of the population. (Ebert,2005)

Other Organisms Used in Bioassay

Duckweed it is a small aquatic plant that floats on the surface of ponds, wetlands,

nutrient rich lakes. Worldwide, there are over 40 species of duckweed (Family

Lemnaceae), with 20 species found in the United States. Each plant consists of one or

more fronds. The frond look like little leaves but actually rootlet that dangles down in

water. Although duckweed reproduces through budding- new fronds grow from their

roots and break off to become independent plant.

(http://ei.cornell.edu/toxicology/bioassays/Duckweed/)

Duckweed is useful for conducting bioassay experiments with water samples

because it can be measured by growth rate by counting how many new fronds develop

over a five-day period. By measuring the number of new fronds of duckweed plants

growing in a test solution and comparing that to the number of new fronds in control

solution, you can test the sensitivity of duckweed to different compounds, or various

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concentrations of a single compound.

(http://ei.cornell.edu/toxicology/bioassays/Duckweed/)

Elodea is a rooted multi-branched perennial plant but can survive and grow as

floating fragments. The dark green blade-like leaves (3/5 inch long and 1/5 inch wide) are

in whorls of three with finely toothed margins. The flowers of Elodea have three white

petals with a waxy coating that makes them float. (http://aquaplant.tamu.edu/plant-

identification/alphabetical-index/elodea/)

Submerged portions of all aquatic plants provide habitats for many micro and

macro invertebrates. These invertebrates in turn are used as food by fish and other

wildlife species (e.g. amphibians, reptiles, ducks, etc.). After aquatic plants die, their

decomposition by bacteria and fungi provides food (called “detritus”) for many aquatic

invertebrates. Elodea has no known direct food value to wildlife but is used extensively

by insects and invertebrates. (http://aquaplant.tamu.edu/plant-identification/alphabetical-

index/elodea/)

Elodea is often confused with Hydrilla and Egeria. Elodea has only 3 leaves in the

whorl and no midrib teeth. (Department of Wildlife & Fisheries Sciences Texas A&M

AgriLife Extension Service)

Planarian is any of several free-living (non-parasitic) worms. The planarian is the

simplest of all flatworms. Most planarianare found in freshwater or salt water, but a few

live in moist soils on land. (http://animal.discovery.com/worms/planarian-info.html)

The planarian has a soft, flat, wedge-shaped body that may be black, brown, gray,

or white and is about a half inch (1.3 cm) long. The blunt, triangular head has two ocelli

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(eyespots), pigmented areas that are sensitive to light. There are two auricles (earlike

projections) at the base of the head, which are sensitive to touch and the presence of

certain chemicals. The mouth is located in the middle of the underside of the body, which

is covered with cilia (hair like projections). The nervous system consists of a simple brain

from which two nerve cords extend the length of the body. Other nerves connect these

cords, forming a ladder like structure. There are no circulatory or respiratory systems;

oxygen entering and carbon dioxide leaving the planarian's body diffuses through the

body wall. (http://animal.discovery.com/worms/planarian-info.html).

Scientists have used the planarian for studies on regeneration and for research on

learning and behavior. The planarian can learn to respond to a particular stimulus. When

such a planarian is cut into several pieces, the new planarians regenerated from the pieces

in many cases “remember” the learned response of the original planarian.

Planarian belongs to the class Turbellaria of the phylum Platyhelminthes. There

are many families and genera. (Animal Planet)

Of all the possible water quality bioassay organisms, lettuce seeds might be one of

the last you would think of using. Lettuce seed bioassays have proven to be an easy and

inexpensive means of testing the toxicity of some types of contaminants of concern in

water and sediments, including heavy metals and some pesticides and other organic

toxicants. (Animal Planet)

Lettuce seeds provide distinct advantages over most other test organisms: they are

inexpensive, easy to culture, and require no upkeep between experiments. Although any

variety of lettuce might work, Lactucasativa L. var. Buttercrunch is the standard species

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recommended for bioassays by the U.S. Environmental Protection Agency, the Food and

Drug Administration, and the Organization for Economic Cooperation and Development.

(Animal Planet)

So for future studies, test organisms like duckweed, elodea , planaria and lettuce

seeds may be used for bioassay studies.

Water Quality of the Cordillera Administrative Region

Sources of pollution for the Cordillera Administrative Region includes the

following:

Domestic Source

In the previous reports of Department of Health (DOH), majority of the surveyed

household population had access to safe water. Households which had sanitary toilets

have a higher percentage compared to the satisfactory garbage disposal.

On the contrary, NSCB-CAR reports that based on the 263,851 household

population in year 2000, 61.13% have access to safe water or through a community water

system, with 32,392 households (12.28%) using water bodies as their domestic water

source. Majority of these were from Benguet province and Baguio City, followed by the

provinces of Ifugao, Apayao and Kalinga. The provinces of Abra and Mountain Province

were the least users.

Since household source is considered as a major source of water pollution, these

figures are vital in monitoring the water quality of the water bodies and in identifying the

kind of efforts provided to protect these sources.

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 Industrial Source

Although the Region does not cater large industrial firms, waste water discharges

from small industries may still threaten the water quality of the receiving water bodies.

Most industries have Pollution Control Officers (PCOs) who ensure compliance to

environmental laws and observance of cleaner production and pollution prevention.

Types of projects being monitored in the Region include mining, hotel and

restaurant, semiconductor, power plant, mall, hospitals, among others.

Agricultural Source

This sector identifies residues from farm inputs such as pesticides, fertilizers,

fungicides and herbicides, including rotten agricultural wastes as the pollutants. These are

carried down to the water bodies through surface run-off and/or infiltration/percolation

and may affect the water quality of the receiving water body.

Unfortunately, the contribution of agricultural activities/inputs in polluting water

bodies has not been monitored as the Office’s laboratory is not yet capable of analyzing

the presence of pesticides and chemical fertilizers. Monitoring the number of farmers

associations in the Region using agricultural inputs will help establish situations which

eventually would lead to policy review and/or formulation to address the matter.

Solid Waste Discharge and Leachate

Decades ago, inhabitants used to enjoy water bodies close to their abode. In our

present era, these are now the receivers of all types of solid wastes from various sources,

especially from households and industries. Notably, as urban migration and natural

population increase through time, the volume of wastes generation increases and

improper disposal practices result to water pollution. Total suspended solids and the

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leachate from rotting garbage alter the water quality of a water body, eventually affecting

aquatic and human lives.

(Environmental Management Beaureu-CAR)

Other Researches on Using Daphnia Magna to Evaluate Toxicity

Several researches were also conducted using Daphnia magna as bio indicator.

Villegas Navaro et al. (1999) reported the use of Daphnia Magna as a toxicity

indicator for textile industrial effluents to show that the toxicity tests combined with

physicochemical analysis are essential in the evaluation of effluent quality and also in the

assessment of treatment plant efficiency in Mexico.

The toxicity removal efficiency of different units of Istahan Wastewater

Treatment Plant (IWTP) was evaluated and the validity of regular physicochemical

parameters as limits for discharge to receiving waters. Daphnia was collected from a

natural park. Initially one of the isolated daphnia was cultured. In the next step, the

recultured daphnids are used to prepare the final culture. For this reason, 100 ml of the

final culture was poured into special bottles. Then, one single Daphnia was added to each

bottle. To support the growth of Daphnia during the day after initial culture one mg of

yeast was added to each bottle, every other day. Identification of Daphnia was carried out

according to US-EPA (2000). Thirteen samples were taken from four different points of

IWTP. Samples were taken from raw wastewater influent, and also from preliminary,

primary and secondary sedimentation tanks effluents. The samples were diluted by 3, 4.5,

7, 15, 10, 23, 34, 51, 77, and 100% (v/v). Ten daphnids were added to each dilution and

the results of the daphnid mortality rate were recorded after 48 hours (LC50). The result of

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experiments was acceptable only in cases where daphnids in the blank tubes were

observed to have a mortality rate of less than 10%. Totally, 520 samples were tested. It

should be noted that temperature was checked regularly using a thermometer in the

culture medium. At the end of the experiment, Acute Toxicity Unit (ATU), efficiency of

each unit and totally efficiency values were determined. (Movahedian, Bina and Asghari,

2005)

The result obtained for the influent to the effluent from each unit showing 48h-

LC50 and ATU in the raw wastewater and in preliminary, primary, and secondary

treatment effluents. The results were analyzed using SPSS software and Probit facility.

The toxicity results obtained from IWTP shows 48h-LC50 for raw wastewater as the

effluent of the plant was 30% (v/v) and 3.3 as ATU (Acute Toxicity Unit). The highest

and lowest levels (95% confidence limit) were 48.5 and 21, respectively. Similar results

were reported by Blinova (2000) which reported 48h-LC50 up to 34% (v/v) for raw

wastewater. However, it should not be noted that the quality and quantity of raw

wastewater could be quite different due to culture, custom, nutrition, health and education

(Metcalf and Eddy, 2003). As the result of both studies were found to be in the same

range of 95% confidence limit, therefore, the accuracy of the results is acceptable. The

48h-LC50 for wastewater effluent after preliminary treatment was 32% (v/v) with the

highest and lowest levels being 53 and 22, respectively. The confidence limit was 95%.

The ATU was found as 3.1. the LC50 obtained in this step was not significantly compared

with that of raw wastewater. Preliminary treatment consisted of screening and grit

removal. It was assumed that some of the toxic materials, which were toxic to Daphnia,

might be absorbed by grit and large suspended solids and, thus, removed. However, the

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results of this study showed that these units were not efficient in removing these

materials. The toxicity removal efficiency of preliminary treatment was found to be

8%.(Movahedian, Bina and Asghari, 2005)

An approach to compare the toxicities employing the whole effluent toxicity

(WET) test, using Daphnia magna and chemical analysis with GC/MS and ICP/MS, was

conducted to the nine South Korean wastewater treatment plants (WWTPs). From the

chemical analysis and bioassay experiments, heavy metals (i.e., Cu and Zn) were found

to be the major compounds causing toxic effects toward D. magna. In the whole effluent

toxicity (WET) tests using D. magna, toxicities were observed in 34% of the effluent

samples. However, the biological toxic unit (TU) value showed a non-toxic response (i.e.,

0 TU) in many samples despite the response indicated by the chemical TU values. This

may be due to the species sensitivity, environmental parameters, mixture effects, and

limitation of the chemical analyses. (Department of Environmental Science and

Engineering, Gwangju Institute of Science and Technology, Buk-Gu, Gwangju, South

Korea, 2008 )

Whole effluent toxicity (WET) tests, with Daphnia magna and Selenastrum

capricornutum, were introduced to evaluate the biological toxicities of effluents from the

wastewater treatment plants (WWTPs) in Korea. In WET tests of WWTPs effluents,

33.3% (33/99) for D. magna and 92.6% (75/81) for S. capricornutum revealed greater

than 1 toxic unit (TU), even though all the treatment plants investigated were operating in

compliance with the regulations, as assessed using conventional monitoring methods (i.e.,

BOD and total concentration of N or P, etc). There were only minor differences in

toxicities according to the types of influents (municipal and agro-industrial) in all

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treatment plants. However, the effluents treated by an activated sludge treatment process

were found to exhibit significantly lower toxicity than those treated by rotating biological

contactor (RBC) and extended aeration processes. The seasonal variations in the toxicity

were lower in the summer compared to winter, which may have been due to the rainfall

received to the sewage intake system during the former period. (Environmental

Monitoring & Assessment,2007)

Another study entitled, “Evaluation of water treatment sludges toxicity using

the Daphnia bioassay” was conducted. Alum and ferric chloride sludges from two water

treatment plants (WTPs) were analyzed regarding their physicochemical characteristics

and toxicity to Daphnia similis. Experiments were carried out in the dry and rainy

seasons. Acute and chronic toxicity was measured using survival and reproduction as

measurement endpoints. No acute toxicity of the sludge was observed in 48 h exposure.

Ferric chloride sludge caused chronic toxicity, demonstrated by low fecundity and some

mortality, while alum sludge caused chronic toxicity characterized by low fecundity.

Some sludge characteristics varied between samplings, including turbidity, solids

contents, N, P and metal (Al and Fe) concentrations. These variables and the increase of

chemical oxygen demand (COD) were identified as the main cause of degradation of the

receiving waters. However, no relationship was observed between these variables and

degree of toxicity. It is apparent from these results waste water treatment sludge may be

toxic and therefore may impair receiving waters. Aluhat sludge was less toxic than ferric

chloride sludge. (Santos, 2005)

In their study entitled,” Toxicity assessment of a complex industrial wastewater

using aquatic and terrestrial bioassays Daphnia pulex and Lactuca sativa,” aquatic and

21
terrestrial bioassays were used to assess toxicity at several stages in an industrial

wastewater treatment plant that processes 400 L/s from a complex influent formed by

wastewater from 135 industries. Daphnia pulex and Lactuca sativa were used to assess

and compare toxicity between the influent wastewater and effluent wastewater from an

activated sludge process, and compare their relationship with physicochemical

parameters of Biological Oxygen Demand (BOD); Chemical Oxygen Demand (COD);

Total Suspended Solids (TSS); total Nitrogen (N (N-total)), and ammonia Nitrogen (N (N

- NH3)). Samples from the primary clarifiers (PC), mix liquor stage (ML) and secondary

clarifiers (SC) were processed using physicochemical and bioassay test. Toxicity results

with Daphnia pulex showed decreased mean values of acute Toxic Units (a.T.U.)

between PC (2.1 a.T.U.) and SC (1,25 a.T.U.). Lactuca sativa showed high values of

toxicity between PC and SC (3.37 and 3.32 a.T.U. respectively). Some samples exhibited

higher toxicity values at the effluent stage (SC) than the influent stage (PC). The highest

correlations of physicochemical properties with toxicity were obtained with COD and

nitrogen compounds in effluent samples (SC), but not with influent samples (PC).

(Sánchez-Meza JC, 2007)

22
 MATERIALS AND METHODS

Daphnia magna as Test Organism

Plate 1. Daphnia magna under a compound microscope (50 x magnification)

Daphnia belong to a group known as the Daphniidae (which in turn is part of the

Cladocera, relatives of the freshwater shrimp, Gammarus et al, and the brine shrimp,

Artemia spp).

Daphnia magna (or Daphnids) are members of a collection of animals that are

broadly termed as "water fleas". These are predominantly small crustaceans, and live in

fresh water such as ponds, lakes, and streams. They serve as an important source of food

for fish and other aquatic organisms. Other information about Daphnia magna can be

found in the Review of Literature of this research paper.

23
 Daphnia are excellent organisms to use in bioassays because they are sensitive to

changes in water chemistry and are simple and inexpensive to raise in an aquarium. They

mature in just a few days, so it does not take long to grow a culture of test organisms.

They provide ease of identification, ease of handling, ease of extensive use in toxicity

testing.

Propagation and Culture of Daphnia

Daphnia Magna were acquired from the University of the Philippines-Baguio.

The acquired organisms were cultured by the researchers for a month to attain the

population needed.

The culture water was collected from Wright Park, Baguio City. The culture water

was a pond water to ensure that there are algae present for food source

In culturing the test organisms, the researchers started by filling the containers (6

liters bottle) with culture water. Next, two daphnids were transferred in each bottle using

a dropper having an inside diameter of 1.5 times the size of the Daphnia. Care was taken

not to bruise the daphnids while transferring them to new media. The organisms were

introduced below the surface of the new medium to avoid trapping air under their

carapaces. The researchers provided the optimal condition for Daphnia. Observations

were recorded by the researchers in their project data book.

Effluent

24
 Effluent generally liquid waste flowing out of a factory,

farm, commercial establishment, or a household into a water body such as a creek, river,

lake, or lagoon, a sewer system or reservoir.

Wastewater or sewage can come from human waste, usually from lavatories,

cesspit leakage, septic tank discharge, washing water (personal, clothes, floors, dishes,

etc.), rainfall collected on roofs, yards, hard- standings, groundwater infiltrated into

sewage, surplus manufactured liquids from domestic sources (drinks, cooking

oil, pesticides, lubricating oil, paint, cleaning liquids, Urban rainfall runoff from roads,

carparks, roofs, sidewalks, or pavements (contains oils, animal

feces, litter, gasoline, diesel or rubber residues, soapscum, metals from vehicle exhausts,

etc.)

25
Effluent Collection site

Map 1: Map representation of the sources of wastewater

Effluent samples were collected from the two selected creeks of Pinsao Pilot

Project, Baguio City. The two sources were located at Purok 3 and 7 of the community.

The creek in Purok 3 was narrow, wastewater coming from the residents was freely

flowing, little pieces garbages were found, and the wastewater has a slightly brown color.

The creek in Purok 7 was wide, bubbles were found on the flowing wastewater, foul odor

was smelled, and weeds were growing on the soil near the creek. Effluent samples were

taken and placed in 1.5 bottles.

26
 Methodology

Experimental Design

The experiment was arranged in a completely randomized design (CRD) with 2

replications per treatment. The treatments are randomly assigned to the experimental

subject without restriction. The experimental subjects are assumed to be homogenous

with respect to the factors that could affect the treatments being compared. Daphnids

were transferred randomly from the acclimation stock to bottles containing the

appropriate experimental conditions.

Bioassay Experiment

Acute toxicity test was the procedure used for the experiment. Acute toxicity is

ashort term lethal or other effect, usually defined as occurring within 48 hours for

Daphnia. The procedures were adopted from the standardized protocol for conducting

Daphnia magna Toxicity Bioassays by Biesinger, Williams and Van der Schalie (1987)

with slight modifications. Twenty-four (300 ml) clear glasses that can contain 80-ml

solution were washed with tap water, and then distilled water. The bottles were properly

labelled. The effluents from Source 1 and 2 were filtered separately using filter paper and

test solutions were prepared by making appropriate dilutions.

The treatments were:

T0- (as positive control with pure distilled water)

T1 (100%) - (80 ml wastewater + 0 ml distilled water)

T2 (50%) – (40 ml wastewater + 40 ml distilled water)

27
T3 (25%) – (20 ml wastewater + 60 ml distilled water)

The test bottles were filled with appropriate test solutions. Daphnia (24-hour-old)

should be transferred with a medicine dropper (with an inside diameter about 1.5 times

the size of the daphnids) into the bottles. The daphnids were randomly added into each

test bottle until each bottle contained 4 daphnids. The procedure was accomplished in less

than one hour.

The bottles were covered to avoid insects or any organisms to enter the bottle on

the time of the experiment and to minimize the evaporation of the test solutions. More

importantly, the contamination of volatile compounds and air particulates should be

avoided because these are harmful to the test organisms and might cause experimental

errors. This will not deprive the daphnids of oxygen because the test solution didn’t touch

the rim of the bottle leaving a space for the oxygen needed by the test organisms.

The test organisms were not fed because in this experiment, the death of the

Daphnia were expected that it is due to the contaminants that are found in wastewater

and not because of the food itself.

Then observations, pH and temperature were recorded after 5 minutes, 10

minutes, 20 minutes, 1 hour, 2 hours, 3 hours, 24 hours and 48 hours.

Data Collection Parameters

1. Mortality - number of dead daphnids were counted. Daphnia is identified as dead if it’s

immobile. Immobilization means that no visible movement of appendages when gently

prodded.

28
2. EC50- a statistically estimated toxicant concentration at which 50 percent of exposed

organisms would be killed a specific time of the observation (48 hours)

2.pH- is the measure of acidity or basicity of the solution. The pH of the solutions was

measured using a pH paper. pH of the solutions were gathered after 20 minutes, 2 hours,

1 day and 2 days. The data gathered were recorded in the logbook

3. Temperature- defined as the hotness or coldness of the body. Temperature of the

solutions was measured using a laboratory thermometer. Temperature was gathered after

20 minutes, 2 hours, 1 day and 2 days. The data gathered were recorded in the logbook.

Statistical Analysis

Raw data from Source 1 and 2 in the form of mortality means were subjected to

Analysis of Variance (ANOVA) to determine any significant difference between and

among treatments.

29
 RESULTS AND DISCUSSION

Table 1. Mortality or EC 50 of dead Daphnia per treatment in the 2 Sources of Effluent

Treatments Total Number of dead Total Number of dead

Daphnia Daphnia

(Source 1) (Source 2)

T0- (as positive control with 0 3

pure distilled water)
T1 (100%) - (80 ml 11 (EC50) 10(EC50)
wastewater
+ 0 ml distilled water)
T2 (50%) – (40 ml 5 5
wastewater
+ 40 ml distilled water)
T3 (25%) – (20 ml 2 3
wastewater
+ 60 ml distilled water)
Total 18 21

Table 1 shows the mortality or EC 50 of dead Daphnia per treatment in Source 1

(Purok 7) and source 2. T0, T2 and T3 in both of the sources did not establish an EC 50 .

An acceptable EC50 test will have at least two test concentrations where the number of

immobile (dead) animals bracket 50 percent unless there is less than 50 percent response

in the 100 percent solution at the solubility limit of the toxicant in water.

T1 in both of the sources established an EC50 because above 50% of the daphnids

subjected in this treatment died.

It was observed also that as the concentration of wastewater increases, the death

of the Daphnia magna increases, too. Therefore, the two variables are directly related.

30
Table 2. One- Way Analysis of Variance on the Mortality of Daphnia magna in Source 1

Source of Degrees of Sum of Mean Computed Tabular F

Variation Freedom Squares Square F 5% 1%

Between Groups 2 0.5 0.2500 0.1401 4.26 8.02

(Treatment)

Within Groups 9 16.0625 1.7847

(Experimental Error)

Total 11 16.5625

Decision= not significant at 5% and 1 % level

Table 2 shows the One- Way Analysis of Variance on the Mortality Rate of Daphnia in

Source 2.

Based on the result of statistical analysis even though there are slight differences

with the mortality means of Daphnia magna applied with different treatments with

varying concentrations of effluent, there is no significant difference between and among

them.

Sources of error might include the intervening variables like the dissolved oxygen

and others.

Temperature and the pH of the solution are not the source of experimental error

because the two parameters were monitored in the time of the experiment.

31
Table 3. One- Way Analysis of Variance on the Mortality Rate of Daphnia in Source 2

Source of Variation Degrees of Sum of Mean Computed Tabular F

Freedom Squares Square F 5% 1%

Between Groups 2 .6668 0.3334 0.2308 4.26 8.02

(Treatment)

Within Groups 9 13 1.4444

(Experimental Error)

Total 11 13.6668

Decision= not significant at 5% and 1 % level

Table 3 shows the One- Way Analysis of Variance on the Mortality Rate of Daphnia in

Source 2.

Based on the result of statistical analysis even though there are slight differences

with the mortality rate means of Daphnia Magna applied with different treatments with

varying concentrations of effluent, there is no significant difference among them.

Sources of error might include the intervening variables like the dissolved oxygen

and others.

Temperature and the pH of the solution are not the source of experimental error

because the two parameters were monitored in the time of the experiment.

To obtain a better data, the addition of replicates per treatment should be done in

future research and also if possible, reduce the value of the experimental error

32
Table 4. Mortality of Daphnia magna in Source 1 (Purok 7)

Treatment O R1 R2 Total Mean

T0- (as positive control with pure 0 0 0 0 0

distilled water)
T1 (100%) - (80 ml wastewater 4 4 3 11 3.66
+ 0 ml distilled water)
T2 (50%) – (40 ml wastewater 2 1 2 5 1.66
+ 40 ml distilled water)
T3 (25%) – (20 ml wastewater 1 0 1 2 .66
+ 60 ml distilled water)
Total= 18

Table 4 shows that the effluent from source 1 affects the mortality of Daphnia

magna applied with Treatment 1, 2, and 3 which might have been caused by the toxic

contaminants found in the effluent. It might have been a good result because there are no

dead daphnia in Treatment 0. Treatment 1 has the highest total of dead daphnia which is

11 with 80 ml wastewater and 0 ml distilled water solution. Treatment 2 and treatment 3

has a total of 5 and 2 dead Daphnia, respectively.

It is worth pointing out that the mean value of the mortality in Treatment 1 is the

highest among all the treatments. Daphnids in Treatment 1 were expected to be severely

affected because of greater quantity of toxic contaminants in the effluent. During the

collection of effluents from the creek in Purok 7, it was observed that bubbles were found

on the flowing wastewater, foul odor was smelled, and weeds were growing on the soil

near it. Therefore, contaminants like phosphates (due to the occurrence of the bubbles)

33
might be found in the wastewater where the Daphnia were subjected and caused the

death of the test organisms.

Table 5. Mortality of Daphnia magna in Source 2 (Purok 3)

Treatment O R1 R2 Total Mean

T0- (as positive control with pure 1 1 1 3 1

distilled water)
T1 (100%) - (80 ml wastewater 2 5 3 10 3.33
+ 0 ml distilled water)
T2 (50%) – (40 ml wastewater 2 2 1 5 1.67
+ 40 ml distilled water)
T3 (25%) – (20 ml wastewater 1 0 2 3 1
+ 60 ml distilled water)
Total= 21

Table 5 shows that the effluent from source 2 affects the mortality rate of

Daphnia Magna applied with Treatment 1, 2, and 3 which might have been caused by the

toxic contaminants found in the effluent. Treatment 1 has the highest total of dead

daphnia which is 10 with 80 ml wastewater and 0 ml distilled water solution. Treatment 2

and treatment 3 has a total of 5 and 3 dead daphnia, respectively.

It is worth pointing out that the mean value of the mortality rate in Treatment 1 is

the highest among all the treatments. Daphnids in Treatment 3 were expected to be

severely affected because of greater quantity of toxic contaminants in the effluent.

During the collection of effluent sample, it was observed that the creek in Purok 3 was

narrow, wastewater coming from the residents was freely flowing, little pieces garbages

were found, and the wastewater has a slightly brown color. Contaminants like oil and

34
grease (due to the occurrence of brown color) might be found in the wastewater where

the Daphnia were subjected to.

Lowest total of dead Dapnia was obtained in T3 and in the control. This is

because of the idea that smaller quantity of toxic contaminants was introduced to the test

organisms in these treatments.

Table 6: Total number of dead Daphnia in Source 1 and Source 2

Source Total Dead Daphnia

1 (Purok 7) 18

2 (Purok 3) 21

Table 6 shows the total number of Daphnia in Source 1 and Source 2. Higher total

of mortality was obtained in source 2 with a total of 21 dead Daphnia. Lower total of

mortality was obtained in source 2 with a total of 21 dead Daphnia.

With these results, the researchers may infer that most of the residents in Purok 3

produce more pollutants that may cause pollution to the creeks that flows going to the

bodies of water. Furthermore, these results should be presented to the barangay officials

for them to implement rules and regulations to mitigate water pollution and lessen the

extinction of living fauna in the bodies of water.

35
 SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary

The experiment conducted a bioassay test to observe the effects of the effluent

from the selected creeks of Pinsao Pilot Project, Baguio City with the idea that the death

of the test organisms could mean the occurrence of toxic contaminants in the effluent.

Based on the results, the following are the salient findings from this research

1. T0, T2 and T3 of Sources 1 and 2 did not establish an EC50 . T1 in both of the

sources established an EC50 because above 50% of the daphnids subjected in this

treatment died.

2. Based on the result of statistical analysis even though there are slight

differences with the mortality means of Daphnia magna applied with different treatments

with varying concentrations of effluent, there is no significant difference between and

among them.

3. Treatment 1 in both of the sources has the highest total of dead daphnia which

is 18 and 21 for Source 1 and 2, respectively. It is worth pointing out that the mean value

of the mortality in Treatment 1 is the highest among all the treatments. Daphnids in

Treatment 1 were expected to be severely affected because of greater quantity of toxic

contaminants in the effluent.

4. Higher total of mortality was obtained in source 2 with a total of 21 dead

Daphnia and lower mortality was obtained in source 1 with a total of 18 dead daphnids.

36
Conclusions

Based on the results, the following conclusions were drawn:

1. When the concentration of wastewater was increased, the mortality of Daphnia

increases, too.

2. There are no significant differences between and among the treatments.

3. Treatment 1 in both sources have the highest total of dead Daphnia.

4. Higher total of mortality was obtained in source 2 with a total of 21 dead

Daphnia and lower mortality was obtained in source 1 with a total of 18 dead daphnids.

5. Wastewater coming from the selected creeks of Pinsao can’t severely affect the

test organisms since results prove that EC50 was not obtained in all of the treatments.

Recommendations

Based on the findings, the following recommendations were drawn:

1. Since EC50 was established in one of the treatments, this research must be

presented to the residents of Pinsao Pilot Project and the barangay officials as well for

them to be aware about the quality of water in the creeks and for them to find ways to

lessen water pollution in the community.

2. Addition of replicates in each treatment is recommended for future studies to

obtain better results.

37
 3. Another study using other test substance and other test organisms can be

conducted for future research.

4. Other parameters like dissolved oxygen can be measured for quality research.

5. Other endpoints like increased heart rate and decreased appetite can be

investigated for further research aside from mortality.

6. Water sample testing of the possible contaminants in the wastewater can be don

also.

38
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44
 APPENDICES

Appendix 1: Plates

Plate 2: Collection of Effluent in Purok 7, P.P.P.(Source 1)

Plate 3: Collection of Effluent in Purok 3, P.P.P.(Source 2

45
 Plate 4: Labelling of the Test Bottles

Plate 5: Measuring of the Test Substance

46
 Plate 6: Filtering of the Test Substance

Plate 7: Pouring of the Test substance with a concentration to the test bottle

47
 Plate 8: Transferring of Daphnids to the Test Bottles

Plate 9: Test Bottles with treatments of varying concentrations (Source 1)

48
Plate 10: Test Bottles with treatments of varying concentrations (Source 2)

Plate 11: Taking of Observations

49
Appendix B: Raw data on the Moratlity of Daphnia

Table 7: Raw data on the Moratlity of Daphnia (Source 1-Purok 7)

5 mins 10mins 20 mins 1 hr 2 hrs 1 day 2 days T

O R1 R2 O R1 R2 O R1 R2 O R1 R2 O R1 R2 O R1 R2 O R1 R2

T0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
T1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 3 3 3 11

T2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 2 5

T3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 2

T 18

Table 8: Raw data on the Moratlity of Daphnia (Source 2)

5 mins 10mins 20 mins 1 hr 2 hrs 1 day 2 days T

O R1 R2 O R1 R2 O R1 R2 O R1 R2 O R1 R2 O R1 R2 O R1 R
2

T0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 3

T1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 1 4 1 10

T2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 1 5

T3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 3

T 21

50
APPENDIX C: pH and Temperature of Effluent

Table 9: pH and Temperature of Effluent After 20 minutes ( Source 1)

pH Temperature (Original)
( In degree celcius)

O R1 R2

T0 5 5 5 21

T1 8 7 7 19

T2 8 8 8 19

T3 8 8 8 20

Table 10: pH and Temperature of Effluent After 2 hours ( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 6 6 21

T1 8 8 8 21

T2 8 8 8 21

T3 8 7 7 19

51
Table 11: pH and Temperature of Effluent After 1 day( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 6 6 21

T1 8 8 8 22

T2 7 7 7 21

T3 7 7 7 21

Table 12: pH and Temperature of Effluent After 20 minutes ( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 6 6 22

T1 8 8 8 23

T2 7 8 8 23

T3 7 6 8 23

52
Table 13: pH and Temperature of Effluent After 20 minutes ( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 7 7 7 20

T1 8 8 8 21

T2 8 8 8 21

T3 6 6 6 21

Table 14: pH and Temperature of Effluent After 2 hours ( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 6 6 21

T1 8 8 8 21

T2 8 8 8 21

T3 6 6 6 21

53
Table 15: pH and Temperature of Effluent After 1 day( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 6 6 21

T1 8 8 8 22

T2 7 7 6 23

T3 5 5 6 23

Table 16: pH and Temperature of Effluent After 20 minutes ( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 6 6 24

T1 8 8 7 24

T2 7 5 6 24

T3 7 6 5 24

54
APPENDIX D: Figures

Figure 1: Line Graph on the Mortality or EC 50 of dead Daphnia per treatment in the 2

Sources of Effluent

Mortality or EC 50 of dead Daphnia per treatment in the 2 Sources

of Effluent
12

10
Mortality or EC50

6 Source 1
Source 2
4

0
Control T1 T2 T3

Figure 2: Line Graph on the Mortality of Daphnia magna in Source 1 (Purok 7)

Line Graph on the Mortality of Daphnia magna in Source 1 (Purok 7)

12

10

8
Mortality

6 R2

4 R1
Original
2

0
T1 T2 T3 Control
Treatment

55
Figure 3: Line Graph on the Mortality of Daphnia magna in Source 2 (Purok 3)

Line Graph on the Mortality of Daphnia magna in Source 2 (Purok 3)

4
Mortality

3 Original
R1
2
R2
1

0
Control T1 T2 T3
Treatment

Figure 4 : Line Graph on the total number of dead Daphnia in Source 1 and Source 2

Total number of dead Daphnia in Source 1 and Source 2

21.5
21
20.5
20
19.5
Mortality

19
18.5
18
17.5
17
16.5
Source 1 Source 2
Sources of Effluent

56
57
 Biographical Sketch

Raquel Calawen , a 16 year-old student was born on June 31, 1997 . She was born

in La Trinidad. She lives at # 204-B Upper Pinget , Baguio City. Her analytical parents

were Mr. Benson Calawen and Mrs. Natividad Calawen. As of her educational

background , she took her Pre-school at Pinget Barangay Hall , Kindergarten at Pinget

Elementary School, for Elementary at she graduated at Pinsao Elementary School. At

present she is now studying at Pinsao National High School. She likes listening over the

radio, writing literatures , editing videos , web surfing , and playing with her other sibling

. She also likes learning all the academic subjects. In the future , she wants to become a

Certified Public Accountant. Her Motto in life is “ Education is a lifetime Treasure , it

can’t be brought or stolen.”

Denzel Alalag, 15 year- old student was born on April 3,1998. He was born in

Baguio City. He lives at #30 Purok 7 Pinsao Pilot Project. His parents were Mr. Jose and

Mrs. Ellen Alalag. He took his Kindergarten and Elementary at Pinsao Elementary

School . At present he is studying at Pinsao National High School. He likes fun activities

likes outdoor games , activities , and he is always excited when it comes to Science

Expriments. He always want to do something unique and challenging tasks like solving

difficult problem solving. His Motto is “ It is better to taste the hardship of education than

to taste the bitterness of ignorance.” In the future, he wants to become a Veterinarian.

Wendy Ayan , 15 year-old student was born on February 12 ,1998. She was born

in Baguio City. She lives at #8 Purok 9 Upper Pinget. Her parents were Mr. Godfrey Sr.

and Mrs. Angelita Ayan. She took her Elementary at Pinget Elementary Shool. She likes

58
surfing the web and learning foreign languages. She likes to do experiments and observe

amazing things. Her motto is “ What is beauty if the brain is empty.” In the future she

wants to do many things, and enjoy travelling.

59