EFFECT OF CRUDE OIL FRACTION ON THE DEVELOPMENT OF Radix

QUADRASI
A research proposal submitted in partial fulfilment of the requirements for
ZOOLOGY 117: Developmental Biology under the supervision of Dr.
Emmanuel Ryan C. de Chavez

ZOO 117 CD-1L

Group 2
Katrina L. Magbojos
Renline B. Oliva
Joanna Celeste M. Quintana
Gellie Ann D. Valencia
Princess Collin Borromeo

INTRODUCTION
Background of the Study
Oil or oil products are considered as one of the most widespread
organic pollutant globally (Shesterin, n.d.). Oil pollution is a major problem in
the aquatic ecosystem as it causes serious threats to the habitat and health
productivity of various aquatic organisms (Enujiugha and Nwanna, 2004). Oil
spill is considered detrimental to wildlife from the organisms of the bottom to
the top of the food chain (U.S. Fish and Wildlife Service, 2010). It may result
to death or serious damage right after the organisms are exposed to the
toxicants released while some other organisms are slowly being poisoned
even after a long-term contact to the toxic substances (US Environmental
Protection Agency, n.d.).
According to U.S. Fish and Wildlife Service (1998), oils are subdivided
into five types: Type I includes jet fuel and gasoline; Type II includes diesel,
no.2 fuel oil, light crude and home heating; Type III includes most crude oils;
Type IV includes heavy crude oils, no. 6 fuel oil and Bunker C and; Type V
includes very heavy oils. The studies on the effect of crude oil pollution on
different aquatic organisms are becoming an area of concern of different
scientists (Loya & Rinkevich, 1980; Incardona et. al., 2005; Brannon et al.,
2006). A study conducted by Farid, Al-Saad, and Al-Adhub (2009) involves
crude and refined oil products as toxicants to different mollusc species and it
was found out that the level of toxicity varies on different types of oils (Farid,
Al-Saad, & Al-Adhub, 2009).

This study focuses on the effect of fraction of crude oil (gasoline and
diesel) on the development of Radix quadrasi. Diesel and gasoline are both
light oils which are considered as having higher toxicity levels than those
that are heavy oils Farid, Al-Saad, & Al-Adhub, 2009). Radix quadrasi will be
the target species since it is commonly prese t in freshwater bodies in the
Philippines and may be considered as a bioindicator of pollution (de Lara &
Enriquez, 1981 as cited in Factor & de Chavez, 2012).
Significance of the study
Human activities through time have had put negative influences on the
balance

of

freshwater

organisms.

Some

of

which

are

caused

by

contaminants, effects brought about by alterations of watershed hydrology,

habitat modifications and exploitation of energy sources upon which aquatic
life depends. The effect of pollutions to organisms serves as bioindicators of
the state of a natural ecosystem. The population, activities or status of these
species can be assessed to evaluate the degree of health of an ecosystem
(Karr, 2011). In this study, developmental stages will be used as
bioindicators which will be exposed with the water soluble fraction of gas and
diesel to induce the toxicant levels in the natural freshwater ecosystem.
Objectives of the study

This study generally aims to determine the effect of toxicity of the

crude oil fraction of gas and diesel combination on the developmental stages
of Radix quadrasi.
Specifically, the objectives of the study are:
1. To evaluate the acute toxicity properties of the different concentrations at
LC50 and LC90 of gas and diesel combined.
2. To assess the comparable level of induced concentration of crude oil fraction
to the levels of detected toxicants in polluted freshwater ecosystems in the
Philippines.
Time and Place of study
The test organisms will be collected from the University of the
Philippines Los Baos (UPLB), Limnological Research Station, which will then
be acclimatized and reared at the Animal Biology Research Laboratory,
Institute of Biological Sciences, UPLB using the same method used by de
Lara and Enriquez (1981) and de Chavez and de Lara (2003). The crude oil
fractionation of gas and diesel will be done at the Microbiology Research
Laboratory, IBS, UPLB.
Scope and Limitation of the study
This study will only be focusing on the effect of toxicity of both gasoline
and diesel on the different developmental stages of the test organism, R.
quadrasi. The individual effects of each oil used will not be investigated
further.

REVIEW OF RELATED LITERATURE

There are several experiments which tested and proved the
detrimental effects of gasoline in organisms particularly in mollusks. One
example is the study of Fedato et al,. in which they examined the genotoxic
and mutagenic effects of acute exposures (6, 24, and 96 h) to gasoline
water-soluble fraction (GWSF), diluted to 5%, on the Asian clam Corbicula
fluminea. The comet assay and the micronucleus (MN) test were performed
on hemocytes and gill cells of C. fluminea. For the three different times
tested, the comet assay indicated DNA damage in hemocytes and gill cells
of C. fluminea exposed to GWSF. The MN test detected significant damage in
the genetic material of the hemocytes only after 96 h of exposure to GWSF.
Overall, the findings indicate that GWSF has genotoxic and mutagenic effects
on C. fluminea.
Another study by Popov et al., examined the effect of various toxic
compounds

(phenol,

gasoline,

detergents,

halogenated benzenes,

and

copper salts) on the activity and multiple forms of acid DNase. It was
investigated in the liver of the widespread freshwater snail species Viviparus
viviparus L. It was shown in the results that toxic compounds has negative
effects

on

the

snail

and

that

the

pattern

of DNase isoforms

in V.

viviparus could be an index of water pollution.

Sinmonato et al., evaluated biochemical biomarkers in the
neotropical freshwater fish Prochilodus lineatus following acute exposures to

the water-soluble fraction of gasoline (WSFG). Fish were exposed to the

WSFG diluted to 5% in water (WSFG group) or only to water (Control group)
for 6, 24 and 96 h and the gills and liver were removed for the biochemical
analyses. Fish exposed to WSFG for 24 and 96 h showed significant increase
in the activity of 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-Stransferase (GST) both in liver and gills, pointing toward phase I and phase II
biotransformation of the compounds present in the WSFG. The results also
indicated the activation of antioxidant defenses in both the liver and gills
after fish exposure to WSFG.
The effects of diesel oil on burying and crawling behaviour in the
intertidal gastropod Polinices incei were examined by Chapman et al. Both
activities were reduced in the presence of diesel oil. Concentrations required
to produce a significant response in terms of burial after 30 min and of
crawling activity were greater than the 96-h LD50. Only the burying response
after 24 h paralleled the 96-h LD50. It has the potential of becoming an
indicator by means of the lethal effects it has on the snails.
Millemann et al. tested the water-soluble fractions (WSFs) of coal- and
shale-derived crude oils and a No. 2 diesel fuel oil for acute and chronic
toxicity to the pulmonate snails Physa gyrina and Helisoma trivolvis. Fullstrength diesel fuel oil WSF did not kill adult snails of either species in 48 h.
In the chronic toxicity tests, snail embryos were exposed continuously to
sublethal WSF concentrations from the time of oviposition through 1 week

post hatching (1123 days). The lowest concentrations of coal oil, shale oil
and

diesel

fuel

oil

WSFs

that

significantly

reduced

hatching

of H.

trivolvis were 0125%, 05% and 32%, respectively, and for P. gyrina they
were 11%, 25% and 8%. Some of the high WSF concentrations significantly
delayed hatching of both snail species by about 2 days as compared with the
controls. There were no effects of the WSFs on survival of hatched snails. The
data suggest that the WSFs may be teratogenic for snail embryos.

MATERIALS & METHODS

Test Organism
Adult R. quadrasi

will be collected from the University of the

Philippines Los Baos (UPLB), Limnological Research Station and will be

acclimatized and reared at the Animal Biology Research Laboratory, Institute
of Biological Science, UPLB following the method of de Lara and Enriquez
(1981) and de Chavez and de Lara (2003). Five to six snails will be placed in
a basin with 3-L tap water at room temperature. Snails will be fed with fresh
lettuce leaves ad libitum. Snails excreta and unconsumed food will be
removed regularly and the water will be changed twice a week. Air supply
will be constantly supplied to prevent from fouling. Laid egg masses will be
placed in Petri dishes where embryos will be allowed to reach trochopore
stage.
Preparation of Water-soluble Fraction
LC50 and LC90 will be the concentrations that will be prepared for the
experiment. Water-soluble fraction of the mixture of gasoline and diesel will
be extracted using a shaker and will be done for 24 hours. After shaking, the
liquid that will be separated from the mixture will be the water-soluble
fraction.
Acute Toxicity Test

Water-soluble fraction with its specific concentrations will be induced

on the snail embryos. Each experimental set-up has five replicates (20-30
embryos) and will be conducted three times. The viability of embryos will be
checked and recorded every 24 hours. To determine dead embryos,
coagulation, immobility and loss of heartbeat will be observed. Individual
embryos will be observed and checked for its abnormalities and deformities.
Samples with abnormalities will be photographed using photo microscope.
Hatchability and incubation period will also be determined.
Statistical Analysis
Data will be presented as mean SD and will be tested for normality
and homogeneity of variance. One-way analysis of variance (ANOVA) will be
used to determine any significant differences in developmental abnormalities
and incubation period among treatments. Statistical Package for Social
Sciences (SPSS) for Windows will be used in all statistical analyses.

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