Abstract

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)
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)
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 water bodies 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 if EC50 will be established by any treatment from two
sources of wastewater from Pinsao Pilot Project creeks.
2. Determine any significant difference in the EC50 or mortality
means (after 48 hours) of the two sources of wastewater from the
selected creeks of Pinsao Pilot Project.
3. 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.
4. Determine which treatment will have the highest and lowest
mortality for Daphnia magna (after 48 hours).
5. Determine which source will have the highest and lowest mortality
for Daphnia magna (after 48 hours).

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 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) 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. Specifically, it
aims to determine if EC50 will be reached by any treatment from two
sources of wastewater from Pinsao Pilot Project creeks, determine any
significant difference in the EC50 or mortality means (after 48 hours)
of the two sources of wastewater, determine any significant difference
in the EC50 or mortality means between treatments (after 48 hours) by
sources of wastewater, determine any significant difference in the
EC50 means or mortality (after 48 hours) of the two sources of
wastewater from the selected creeks of Pinsao Pilot Project, and to
determine which source and treatment will have the highest and
lowest mortality for Daphnia magna (after 48 hours).
A bioassay test was conducted by preparing different treatments with
varying concentrations of effluent. The set-up is in laboratory
conditions and considered as controlled experiment.
Water sample testing of the effluent’s possible contaminants was not
conducted but was identified through citations from acceptable and
local references with similar conditions. These were mentioned in the
Review of Literature of this research paper.
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

Daphnia magna as Test Organism

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

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.
Methodology
Experiment1. Effect of Effluent to the Mortality of Daphnia Magna
Acute toxicity test was the procedure used for the experiment.
Acute to
The procedures were adopted from the standardized protocol for
conducting Daphnia magna Toxicity Bioassays by Biesinger,
Williams and Van der Schalie (1987) with slight modifications.
Twenty-four (300 ml) clear glasses that can contain 80-ml solution
were washed with tap water, and then distilled water. The bottles were
properly labelled. The effluents from Source 1 and 2 were filtered
separately using filter paper and test solutions were prepared by
making appropriate dilutions. The treatments were:
T0- (as positive control with pure distilled water)
T1 (100%) - (80 ml wastewater + 0 ml distilled water)
T2 (50%) – (40 ml wastewater + 40 ml distilled water)
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. 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 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 did’nt touch the rim of the bottle leaving a space for the
oxygen needed by the test organisms.
Then observations, pH and temperature were recorded after 5
minutes, 10 minutes, 20 minutes, 1 hour, 2 hours, 3 hours, 24 hours
and 48 hours.
Data Collection Parameters
1. Mortality - number of dead daphnids were counted. Daphnia is
identified as dead if it’s immobile.
2. 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.
2.pH- the pH of the solutions was measured using a pH paper.
3. Temperature- temperature of the solutions was measured using a
laboratory thermometer.

Statistical Analysis
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. 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.