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 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

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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

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organisms 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.

Furthermore, results of this study will also provide basis for the local

government units concerned in formulating rules and regulations on the proper

management of wastewater for better economic endeavours.

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 EC50

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

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with similar conditions. These were mentioned in the Review of Literature of this

research paper.

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 example, chemical A might be toxic at a high concentration,

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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 stream channels. In addition, runoff from these developed

areas can increase stream temperatures that along with the increase in flow rate and

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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 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 )

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 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)

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)

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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. 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

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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 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

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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 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)

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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

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

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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 (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.

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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 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

15
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.

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.

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 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 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.

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 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 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.

18
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 LC 50

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 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

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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 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

20
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 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

21
compounds in effluent samples (SC), but not with influent samples (PC). (Sánchez-

Meza JC, 2007)

Based on the past studies, Villegas Navaro et al. (1999) reported that the

toxicity tests combined with physicochemical analysis are essential in the evaluation

of effluent quality. On the other hand, Movahedian, Bina and Asghari, 2005 said that

based on the obtained results and regarding the improvement of water quality

standards, coupled with public expectations in Iran, it is necessary to consider more

stringent water quality policies for regular monitoring and toxicity assessment.

Meanwhile, Environmental Monitoring & Assessment,2007 said that 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. Santos 2005 said that waste water treatment sludge may be toxic

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

ferric chloride sludge. Sanchez, 2007 said that 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).

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.

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

23
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

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,

24
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.)

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

25
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.

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.

26
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)

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.

27
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.

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.

28
 RESULTS AND DISCUSSION

Table 1. Mortality or EC 50 of dead Daphnia per treatment in Source 1 (Purok 7)

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

EC50 . 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.

29
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.

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.

30
Table 3. One- Way Analysis of Variance on the Mortality Rate of Daphnia in Source 2

Source of Degrees of Sum of Mean Computed Tabular F

Variation 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

31
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) might be found in the wastewater where the Daphnia were

subjected and caused the death of the test organisms.

32
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 grease (due to the occurrence of brown color) might be found in the

wastewater where the Daphnia were subjected to.

33
 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.

34
 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.

35
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.

36
 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.

37
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42
 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

43
 Plate 4: Labelling of the Test Bottles

Plate 5: Measuring of the Test Substance

44
 Plate 6: Filtering of the Test Substance

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

45
 Plate 8: Transferring of Daphnids to the Test Bottles

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

46
 Plate 10: Test Bottles with treatments of varying concentrations (Source 2)

Plate 11: Taking of Observations

Appendix B: Raw data on the Moratlity of Daphnia

47
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

48
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

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

49
 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

50
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

51
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

52
APPENDIX D: Figures

Figure 1: Mortality or EC 50 of dead Daphnia per treatment in Source 1 (Purok 7)

Mortality or EC 50 of dead Daphnia per treatment in Source 1

(Purok 7)

12

10
Mortality or EC50

6 Source 1
Source 2
4

0
Control T1 T2 T3

Figure 2: Mortality of Daphnia magna in Source 1 (Purok 7)

Mortality of Daphnia magna in Source 1 (Purok 7)

4.5
4
3.5
3
Mortality

2.5
R1
2
R2
1.5
1
0.5
0
Control T1 T2 T3

53
54
 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 surfing the web and learning foreign languages. She likes to do

55
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.

56