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. 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

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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. (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,

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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

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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.

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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.

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 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.

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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. 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)

Perhaps the oldest and most commonly known example is the canary in the coal

mine. Traditionally, coal miners have taken caged canaries down into the mines to help

ensure a safe air supply. Canaries are more sensitive than humans to methane, an odorless

gas released during the mining process, so they were used to provide an advanced

warning of when methane was building up to dangerous levels in the mines. If the canary

died, it meant the miners should leave the mine as quickly as possible.

Another sort of bioassay is used to test the effects of compounds being considered

for use in drugs or skin care products. Before a chemical compound receives FDA (Food

and Drugs Administration) approval as an ingredient in products for human use, it must

be thoroughly tested on laboratory animals.

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

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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)

Water Pollution in Urban Areas

Urban areas have the potential to pollute water in many ways. Runoff 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. (Source: Living Lakes Partnership)

Uncontrolled or treated runoff 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

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

affect water quality and aquatic life.

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.

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.

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. According to Ocampo, a study indicated

“worrisome” levels of nitrate pollution in water sources in farming areas in Benguet and

Bulacan. 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.

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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.

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. ( Ocampo, 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)

Specifically, the group urged the Department of Agriculture to phase out its

subsidies for fertilizers, including the “dangerous practice” of over-using fertilizers in

intensive agriculture, and the Department of Environment and Natural Resources to

seriously reassess its programs on the protection of clean water sources. Greenpeace said

the experience of other countries shows that fertilizer reduction policies significantly

contribute to a cleaner and safer water supply. The greatest risk of nitrate poisoning, it

said, is the “blue baby syndrome” or methemoglobinemia, which occurs in infants given

nitrate-laden water and particularly affects babies under four months old. The blue baby

syndrome can cause cyanosis, headache, stupor, fatigue, tachycardia, coma, convulsions,

asphyxia, and ultimately death. Drinking water contaminated with nitrates has a potential

role in developing cancers of the digestive tract, and has also been associated with other

types of cancer such as non-Hodgkin’s lymphoma, bladder, and ovarian cancer. (Balabo,

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,

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

Official Gazette, 1996; USEPA, 2000)

Daphnia are planktonic crustaceans that belong to the Phyllopoda (sometimes

called Branchiopoda), which are characterized by flattened leaf-like legs used to produce

a water current for the filtering apparatus. Within the branchiopods, Daphnia belong to

the Cladocera, whose bodies are enclosed by an uncalcified shell known as the carapace.

It has a double wall, between which hem lymph flows and which is part of the body

cavity. The carapace is largely made of chitin, a polysaccharide. Cladocera have up to 10

pairs of appendages, which are (from front to back): antennules, antennae (the second

antennae, used for swimming); maxillae; and mandibles; followed by 5 (as in Daphnia) or

6 limbs on the trunk. The limbs form an apparatus for feeding and respiration. At the end

of the abdomen is a pair of claws. The body length of Cladocera ranges from less than 0.5

mm to more than 6 mm. Males are distinguished from females by their smaller size,

larger antennules, modified post-abdomen, and first legs, which are armed with a hook

used in clasping.

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

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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.

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.

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-

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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.

Daphnia populations can be found in a range of water bodies, from huge lakes

down to very small temporary pools, such as rock pools and vernal pools (seasonally

flooded depressions). Often they are the dominant zooplankton and form, as such, an

essential part of the food web in lakes and ponds. In many lakes, Daphnia are the

predominant food for planktivorous fish, at least at times. As a consequence, the Daphnia

species distribution and life history are closely linked with the occurrence of predators.

Typically, Daphnia species found in lakes with planktivorous fish are smaller and more

transparent than species found in fishless water bodies. Large species such as D. magna

and D. pulex usually cannot survive under intensive fish predation, whereas small species

such as D. galeata, D. cucullata, and D. hyalina are usually not found in fishless water

bodies. A number of invertebrates are known to prey on Daphnia, the best investigated

probably being the larvae of the phantom midge Chaoborus and the water boatman

Notonecta and related genera. Although visually-hunting fish usually show a preference

for larger prey items, invertebrates may prefer smaller prey or even a specific size class.

These differences in size-specific mortality rates are believed to be a key factor in the

evolution of Daphnia body size. High juvenile mortality caused by Chaoborus has been

suggested as the cause for evolution of larger sizes at birth and to phenotypic ally plastic

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adjustments of birth size and growth rates. In contrast, predation by fish has led to

smaller sizes and earlier maturation age.

Other Researches on Bioassay Using Daphnia Magna

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.

Toxicity test were also used for evaluation of domestic and industrial waste water

effluents. It is reported that although the effluent meets all physicochemical requirements

but regarding its toxicity, it may cause considerable negative effects in receiving waters.

Effluent quality evaluation in Iran is based on physicochemical parameters. 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. (Tisler and Zagorc, 1999; Villegas Navaro et al., 1999; Richard et al.,

2000)

Daphnia was collected from a natural park. Initially one of the isolated daphnia

was cultured. In the next step, the recultured daphnids ere 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

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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. An aerator pump was to provide

oxygen. At the end of the experiment, Acute Toxicity Unit (ATU), efficiency of each unit

and totally efficiency values were determined.

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

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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%.

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.

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.

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.

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 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.

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

whorl and no midrib teeth. (Source: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 planarian are found in freshwater or salt water, but a few

live in moist soils on land.

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.

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 The planarian travels with a gliding motion by moving its cilia. It can also travel

short distances by using a rapid crawling motion. The planarian feeds on crustaceans,

larvae, and small worms. To eat, it extends a long, tubular pharynx from its mouth.

Through this tube it secretes digestive juice onto its prey, and then sucks in bits of partly-

digested food.

Planarian is hermaphroditic; that is, they possess both male and female sex

glands. However, a planarian cannot fertilize its own eggs; the eggs must be fertilized by

the sperm of another planarian. In another method of reproduction, a planarian constricts

behind the pharynx and separates itself into two pieces (a process called fragmentation);

then, both pieces grow back their missing parts (a process called regeneration) and

become two new planarians.

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. (Source: Animal Planet)

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

the last you would think of using. Lettuce doesn't live in water, so why would it even be

considered? The answer is that lettuce seed bioassays have proven to be an easy and

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

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

toxicants.

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, Lactuca sativa 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.

Within any one species, individuals respond differently when exposed to any

particular chemical. Between species, there are even greater differences in chemical

sensitivities. You can test this idea by comparing your lettuce seed results with bioassays

using another organism such as water fleas (Daphnia sp.). (Source: Environmental

Inquiry, Cornell University and Penn State University, 2009)

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 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.

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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)

22
 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

23
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

24
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.

25
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.

26
 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.

27
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

28
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.

29
 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.

30
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.

31
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35
 APPENDICES

Appendix 1: Plates

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

Plate 3: Collection of Effluent in Purok 7, P.P.P.(Source 2)

36
 Plate 4: Labelling of the Test Bottles

Plate 5: Measuring of the Test Substance

37
 Plate 6: Transferring of Daphnids to the Test Bottles

Plate 7: Test Bottles with treatments of varying concentrations (Source 1)

38
Plate 8: Test Bottles with treatments of varying concentrations (Source 2)

Plate 9: Taking of Observations

39
Appendix B: Raw data on the Moratlity rate of Daphnia

Table 5: Raw data on the Moratlity rate of Daphnia (Source 1)

After

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

O R1 R2 O R1 R2 O R1 R2 O R R O R1 R2 O R1 R2 O R1
1
R
2 2

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

Table 6: Raw data on the Moratlity rate of Daphnia (Source 2)

After

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

O R1 R2 O R1 R2 O R1 R2 O R R O R1 R2 O R1 R2 O R1
1
R
2 2

T0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1

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

40
APPENDIX C: pH and Temperature of Effluent

Table 7: pH and Temperature of Effluent After 20 minutes ( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 5 21

T1 8 7 7 19

T2 8 8 8 19

T3 8 8 8 20

Table 8: pH and Temperature of Effluent After 2 hours ( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 21

T1 8 8 8 21

T2 8 8 8 21

T3 8 7 7 19

41
Table 9: pH and Temperature of Effluent After 1 day( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 21

T1 8 8 8 22

T2 7 7 7 21

T3 7 7 7 21

Table 10: pH and Temperature of Effluent After 20 minutes ( Source 1)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 22

T1 8 8 8 23

T2 7 8 8 23

T3 7 6 8 23

42
Table 11: pH and Temperature of Effluent After 20 minutes ( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 7 20

T1 8 8 8 21

T2 8 8 8 21

T3 6 6 6 21

Table 12: pH and Temperature of Effluent After 2 hours ( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 21

T1 8 8 8 21

T2 8 8 8 21

T3 6 6 6 21

43
Table 13: pH and Temperature of Effluent After 1 day( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 21

T1 8 8 8 22

T2 7 7 6 23

T3 5 5 6 23

Table 14: pH and Temperature of Effluent After 20 minutes ( Source 2)

pH Temperature (Original)

( In degree celcius)

O R1 R2

T0 6 24

T1 8 8 7 24

T2 7 5 6 24

T3 7 6 5 24

44