MATERIALS AND METHODS

Daphnia magna as Test Organism

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

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

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

shrimp, Artemia spp).

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

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

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

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

can be found in the Review of Literature of this research paper.

Daphnia are excellent organisms to use in bioassays because they are

sensitive to changes in water chemistry and are simple and inexpensive to raise in an
aquarium. They mature in just a few days, so it does not take long to grow a culture of

test organisms. They provide ease of identification, ease of handling, ease of

extensive use in toxicity testing.

Propagation and Culture of Daphnia

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

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

population needed.

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

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

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

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

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

was taken not to bruise the daphnids while transferring them to new media. The

organisms were introduced below the surface of the new medium to avoid trapping air

under their carapaces. The researchers provided the optimal condition for Daphnia.

Observations were recorded by the researchers in their project data book.

Effluent

Effluent generally liquid waste flowing out of a factory,

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

river, lake, or lagoon, a sewer system or reservoir.

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

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

Experimental Design

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

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

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

homogenous with respect to the factors that could affect the treatments being

compared. Daphnids were transferred randomly from the acclimation stock to bottles

containing the appropriate experimental conditions.

Bioassay Experiment

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

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

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

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

(1987) with slight modifications. Twenty-four (300 ml) clear glasses that can contain

80-ml solution were washed with tap water, and then distilled water. The bottles were

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

filter paper and test solutions were prepared by making appropriate dilutions.
The treatments were:

T0- (as positive control with pure distilled water)

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

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

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

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

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

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

into each test bottle until each bottle contained 4 daphnids. The procedure was

accomplished in less than one hour.

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

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

More importantly, the contamination of volatile compounds and air particulates

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

experimental errors. This will not deprive the daphnids of oxygen because the test

solution didn’t touch the rim of the bottle leaving a space for the oxygen needed by

the test organisms.

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

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

and not because of the food itself.

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

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

Data Collection Parameters

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

it’s immobile. Immobilization means that no visible movement of appendages when

gently prodded.

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

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

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

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

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

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

solutions was measured using a laboratory thermometer. Temperature was gathered

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

logbook.

Statistical Analysis

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

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

and among treatments.