ACKNOWLEDGEMENTS

This study would not have been made possible without the full support, guidance

and assistance of the following individuals who extended their untiring efforts that

inspired the researchers to finish this research paper. The researchers wish to extend their

heartfelt gratitude to all those who contributed to the success of this study;

To their supportive adviser, Zybrinskie T. Bangcado, for his guidance, financial

support, patience, provisions, reminders, corrections and comments done to make this

project of best quality;

To Ma’am Wilen Mina-Galvan of University of the Philippines- Baguio (Biology

Department) for her generosity in giving Daphnia Magna to the researchers and to her

valuable advices for culturing the test organisms;

To the faculty and staffs of Pinsao National High School for their unending

support to this project;

Special thanks to their parents, Mr. Benson Calawen and Mrs. Naty Calawen; Mr

Jose Alalag and Ellen Alalag ; Mr. Godfrey Ayan and Mrs. Carmelita Ayan for their

unending love and affections, pieces of advice, guidance, efforts, sacrifices, and financial

and moral support;

To their siblings and relatives for their guidance and love, financial and moral

support;

To their friends and classmates who cheered them during the tough times of the

research.
 And most of all, to the Almighty Father, who is the source of all knowledge and

that, lent the researchers the thinking power to this research, for his countless blessings,

love and guidance from whom everything was made possible.

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. Water pollution is the introduction of chemical; biological and physical

matter into large bodies matter into large bodies of water that degrade the quality of life

that lives in it and consumes it.

Water pollution 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 life 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 come as a result of natural or unnatural compounds being

added to water source. These can sway the balance of an ecosystem resulting in danger to

natural wildlife of aquatic environments. Amphibians tend to be very pollution sensitive

due to the fact that they absorb chemicals in the water through their skins; this is one

reason why many amphibian populations are in distress today. With addition to the
detergents to the ecosystem, insects that normally have the ability to walk on water will

be unable to. Younger animals also have the greater sensitivity to chemical compounds,

especially those that mimic organic compounds, since they are still undergoing the

physiological changes associated with maturation. (Fears, 2009)

Water pollution has a negative effect on the delicate balance of the ecosystem. It

destroys organisms that provide foods for other animals. Another effect of water

pollution is 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. (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).

Wastewater pollution has always been a major problem throughout the world.

These pollutants can be divided into two broad categories: biological and chemical (Kris,

2007).

The major chemical pollutants in wastewater are nitrogen, phosphorus, heavy

metals, detergents, pesticides and hydrocarbons. Of these chemicals, the two commonest
nutrient limiting ones are nitrogen and phosphorus (Larsdotter, 2006). The presence of

nitrogen in wastewater discharge can be undesirable because it has ecological impacts

and also affect public health. The principal forms of nitrogen are organic nitrogen,

ammonium (NH4+ or NH3), nitrite (NO2-) and nitrate (NO3-) (Hurse & Connor, 1999).

These occurrences are generally associated with disposal of municipal sewage and

fertilizer application to agricultural crops.

The majority of waterborne microorganisms that cause human disease come from animal

and human fecal wastes. These contain a wide variety of viruses, bacteria, and protozoa that may

get washed into drinking water supplies or receiving water bodies (Kris, 2007). Microbial

pathogens are considered to be critical factors contributing to numerous waterborne outbreaks.

Diseases caused by bacteria, viruses and protozoa are the most common health hazards associated

with untreated drinking and recreational waters. Contaminated water is a vehicle for several

waterborne diseases, such as cholera, typhoid fever, shigellosis, salmonellosis,

campylobacteriosis, giardiasis, cryptosporidiosis and Hepatitis A (WHO, 2004)

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

effect on a test organism with that of standard preparation. Most commonly, effluent

from industrial or municipal wastewater discharges are tested. Acute tests measure how

well organisms survive, while chronic tests measure survival and sub-lethal effects, such

as sample’s effect on the organism growth, reproduction, or fecundity. (EIM, 2013)

Bioassays 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)

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, 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)

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.

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 the effects of the different treatments with varying concentrations of

effluent on the mortality rate means of Daphnia Magna.

2. Identify which source of effluent is more prone to water pollution.

3. Determine if there are significant differences between the different treatments with

varying concentrations of effluent using Analysis of Variance (ANOVA).

Significance of the Study

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

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 rate of daphnia magna and to assess the water quality of the effluent collected.

This study is important because it provides the complete response of the test organism to

all compounds present in wastewater. The test organisms represent the living organisms

(fauna and flora) in the bodies of water because of their similarities. This study also

wants to assess the water quality of effluent by identifying the contaminants present.

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

the bodies of water. Most of the residents in the Cordillera Administrative Region and in

the whole Philippines are dependent on the water bodies for their domestic and economic

related activities.

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. Thus, 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 in terms of mortality rate as compared to the

positive control. A bioassay test was conducted by preparing different treatments with

varying concentrations of effluent. The sit-up is in laboratory conditions. This study also

aims to assess the water quality of effluent from the selected creeks in Pinsao Pilot

Project based on its pH and identifying the contaminants found. Lastly, it aims determine

any significant differences between and among the different treatments with varying

concentrations of effluent using Analysis of Variance (ANOVA).

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

but was identified through citation from acceptable and valid references and mentioned in

the Review of Related 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.

MATERIALS AND METHODS

Effluent Collection site

Figure 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.

Daphnia Magna as Test Organism

Plate 1 shows a daphnia magna used as a test organism

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

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

termed as "water fleas". These are predominantly small crustaceans, and 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)

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

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 Magna was used as a test organism because of their high-sensitivity to

toxic substances, ease of identification, simplicity, ease of handling, ease of cultivation,

and 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.

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 researchers

provided the optimal condition for Daphnia. Observations were recorded by the

researchers in their project data book.

Methodology

Experiment1. Effect of Effluent to the Mortality rate of Daphnia Magna

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

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

The test bottles were filled with appropriate test solutions. Juvenile 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 with plastic

cover to avoid insects or any organisms to enter the bottle on the time of the experiment.

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 rate- number of dead daphnids were counted. Daphnia is identified as dead if

it’s immobile.

2. pH- the pH of the solutions was measured using a pH paper.

3. Temperature- temperature of the solutions were measured using a laboratory

thermometer.

Statistical Analysis

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

replications per treatment and no replication for the control. Raw data from Source 1 and

2 in the form of mortality rate means were subjected to Analysis of Variance (ANOVA)

to determine any significant difference between and among treatments.

 RESULTS AND DISCUSSION

Table 1. Effect of Effluent from Source 1 on Mortality Rate Means of Daphnia Magna

Treatment O R1 R2 Total Mean

T0- (as positive control with pure 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 1 shows that the effluent from source 1 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. 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 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.

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

Source of Variation Degrees of Sum of Mean Computed Tabular F

Freedom Squares Square F 5% 1%
Between Groups 2 0.18 0.0922 0.0475 4.74 9.55
(Treatment)
Within Groups 7 13.58 1.9395
(Experimental Error)
Total 9 13.76
Decision= not significant at 5% and 1 % level

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.

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.

Table 3. Effect of Effluent from Source 2 on Mortality Rate Means of Daphnia Magna

Treatment O R1 R2 Total Mean

T0- (as positive control with pure 1 1 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= 19

Table 2 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. It might have been a good result because only

one daphnia died in Treatment 0. 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.

Based on the results, the creek in Purok 3 (Source 2) is more prone to water

pollution because the total number of dead daphnia is higher which is 19 compared to the

total number of dead daphnia in Source 1 which is 18.

Table 4. 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 1.25 0.6247 0.4278 4.74 9.55
(Treatment)
Within Groups 7 10.22 1.4603
(Experimental Error)
Total 9 11.47
Decision= not significant at 5% and 1 % level

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.

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.
 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.

The effluent from source 1 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. 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.

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. It might have been a good result because only one daphnia died in

Treatment 0. 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.

Based on the statistical analysis, results show that there are no significant

differences between the treatments with varying concentrations of effluent from source 1

and source 2
Conclusions

Based on the results, the following conclusions were drawn:

1. Effluents from source 1 and 2 increases the mortality rate of Daphnia.

2. Highest mortality rate mean was obtained in Treatment 1 in both of the Sources

1 and 2.

3. Results proved that creek in Purok 3 is more prone to water pollution because

of the higher total of dead daphnids.

3. Statistical analysis showed that there are no significant differences between the

treatments with varying concentrations of effluent from source 1 and 2.

Recommendations

Based on the findings, the following recommendations were drawn:

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

obtain better results.

2. Another study using other test substance can be conducted for future research.

3. Another study using other test organisms can be conducted.