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Abstract
INTRODUCTION
Rationale
their inherent characteristics. These include: (a) their dual lifestyle allows
they have very thin skin that readily absorbs toxins and; (c) they are highly
1
Important toxins introduced to the environment are pesticides, which
are chemicals used in agriculture and households to remove pests from crops
and many insects. Organophosphate pesticides are the most widely used
introduced in 1950 and has been used to kill insects on many types of crops
since this time (Hunter and Barker, 2003). In the Philippines, malathion is the
second most common pesticides used for crops (Dioquino, 2002). Malathion
(ectoparasites) and head and body lice. However, studies have shown that
species, such as frogs and toads, found in areas where pesticides spraying
usually occur (Fordham et al. 2001; Gilbertson et al. 2003; Taylor et al. 1999;
to 0.6 mg/L. Although, malathion does not persist in the environment with its
light. Despite malathion’s rapid degradation, even brief exposure can alter the
2
malathion degradation also results to compounds more toxic than malathion
focuses on the acute toxicity of absorbed malathion on Bufo sp. larvae, which
is the most common anuran species found in the Philippines. Acute toxicity
rodents. Amphibian toxicity studies were more on Gosner stage 25, where
mouthparts are prominent and the spiracle is visible. This study followed the
limb bud formation under field stage 2 or Gosner stages 26-30. This study
involved field stages 2 and 3, which includes Gosner stages 26-30 and 31-35,
Review of Literature
What is Malathion?
3
Malathion is a manufactured product (molecular weight: 330.3503) that
structure containing a P=S bond and another S attached with an alkyl group
(Masicotte, 2001).
concentrate, wettable powder, dustable powder and ultra low volume liquid
malathion.
4
Malathion toxicity
slightly toxic compound. Malathion interferes with the nervous system function
signals so the next nerve impulse can be transmitted across the synaptic gap,
thereby paralyzing and killing insects. Studies have shown that malathion is
Degradation of malathion
5
degradation (Racke, 1992; Massachusetts Department of Agricultural
P=S to P=O oxon intermediate) can produce two metabolites, malaoxon and
times more toxic than malathion. Malaoxon, however, has lower lyphophilic
al., 1997).
where they are found (Wallace, 1992; Taylor 1999a,b; Johnson et al. 2000;
absorption through transport involving hair follicles and other adnexa. This
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pesticides in amphibian skin (Hall and Kolbe, 1980; Ling, 1990). The
involving hair follicles and other adnexa, which are appendages of an organ
e.g hair follicle of skin. However, amphibians do not possess hair follicles
2001).
interior. This is due to the depolarization of the exterior cell surface of skin
epithelium and not in the basal surface, which therefore results to a higher
Hypotheses
control group.
as, deformed body axis and increase liver size) in Bufo sp. larvae.
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5) The increase of degree of deformities (body axis and liver size) will
Objectives
Specifically it aimed:
the control.
5) to determine if the degree of deformity (body axis and liver size) will
8
MATERIALS AND METHODS
The acute toxicity test for malathion to tadpoles were performed within
the Biology laboratory of the University of the Philippines Cebu College – Arts
Sample Collection
Bufo sp. larvae were collected from a clean permanent pond located in
performed for all treatments. Tadpoles collected were of similar sizes and
that new eggs were not laid before tadpoles fully developed to adult frogs.
tadpoles. Therefore, samples collected were of similar age and come from the
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same egg clusters. Sample collected belonged to field stage 4 larval stage
[36th – 40th Gosner (1960) stages]. Prior to experimentation, the samples were
of the larvae (starved or fed) and type of vessel (petri dish or 5-L jar) for the
malathion, 80 g/L emulsifier and 350 g/L xylene). Distilled water was preferred
over natal water as medium since the latter could contain dissolved
substances that could potentially affect the results of the experiment. The
volume of the solution used was 300 ml, since previous test runs had shown
that higher volumes with the same concentration contain higher amounts of
limit available oxygen required for respiration. The vessel found appropriate
for the each set-up was a 5-L jar, which had greater volume-capacity and
bigger diameter at the bottom than Petri dish, in order to allow more space for
Three set-ups were made per treatment with 10 tadpoles, which served
10
cases were used per treatment including the control groups, which only
were turned on and off with a 12:12 hour ratio on a daily basis for
gut, which was found to be more lethal and biological factors such as
degradation of food also result to mortality of the larvae in both treated and in
The static toxicity test was patterned after Sayim et al. (2005).
end of every 24 hour period throughout the course of the experiment. Dead
were measured using the profile projector. Behaviors, including swimming and
Data Analysis
11
Mortality data from the replicate samples from each malathion
intervals. The 96-hour LC50 and 95% confidence interval were determined
Mean of tail curvature was compared per treatment and with the control
if they varied significantly using ANOVA SPSS version 10.0 for windows. The
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RESULTS
Mortality
144 hours (6 days) of exposure to 15 ppm malathion and after 264 days (11
the lower concentrations but did not reach to 50% of the population at the end
13
120
100
Mortality (%)
80
60
40
20
0
0 1 5 10 15 20
Concentration (ppm)
malathion.
Linear regression, the R2 value obtained was 0.9194, signifying that the
concentration of malathion.
120
R2 = 0.9194
100
Mortality (%)
80
60
40
20
0
0 5 10 15 20 25
Concentration (ppm)
Figure 3. Bufo sp. field stage 4 larvae mortality throughout 12-day exposure
period to malathion.
14
The calculated LC50 values for malathion for 9-12 days of exposure is
Table 1. Lethal concentrations (LC10, LC50, and LC90) in ppm for Buffo sp.
larvae exposed to malathion
LC10 LC50 LC90
Day P value
(95% CI) (95% CI) (95% CI)
5.96889 13.47137 20.97385
9 0.026
(-52.98967-10.72630) (8.67738-70.22988) (14.53161-185.54629)
3.39752 12.13965 20.88179
10 0.086
(-15.83596-7.56189) (8.01651-27.44735) (14.57982-64.62197)
2.04066 9.58642 17.13217
11 0.477
(-0.70704-3.83990) (8.13695-11.35248) (14.70630-21.13970)
0.03329 6.42104 12.80879
12 0.141
(-8.43267-3.12100) (3.40900-10.03471) (9.41705-22.78203)
resembling the structure of a golf club and a bulge in the right abdominal
ppm of malathion, only tail curvature was found in tadpoles that were exposed
values were found to differ significantly among treatment groups and from the
treatment groups. However, the degree of tail curvature at 20 ppm did not
15
ppm, 5 ppm and 10 ppm) and the control. Degree of tail curvature in 15 ppm
The R2 value of 0.9431 for tail curvature suggests that degree of tail
Axis bending at the head region was found to occur with increasing
length of exposure such that no axis bending at the head occurred at 20 ppm
after 48 hours of exposure. The mean values for axis bending at the head
were 2° at 5 ppm, 8° at 10 ppm and 7° at 15 ppm. These values did not differ
showed that the increase in degree of axis bending at the head is attributed to
The bulge at the lower right region of the abdomen was found to be a
result from liver edema. Mean measurements in liver size ranged from 1.5
mm at 1 ppm to 1.7 mm at 15 ppm, while the mean size in the control was
also 1.7 ppm. Measurements of the liver did not differ between the highest
concentration and the control but only differed significantly between 10 ppm
tadpoles in the control group also established liver edema (Figure 7).
Figure 4. Buffo larvae in deformed state: A-B) bent body axis at the head and
C.) base of the tail; D-E.) bulge at the lower right abdominal region and; F.)
curved
A at the tip of tail. B C
D E F 16
14
2
12 R = 0.9431
Angle (degrees)
10
8
6
4
2
0
0 5 10 15 20 25
Concentration (ppm)
Figure 5. Tail curvature of Bufo sp. field stage 4 larvae after malathion
exposure.
9
8
Angle (degrees)
7
6
2
5 R = 0.7876
4
3
2
1
0
0 5 10 15 20
Concentration (ppm)
Figure 6. Angle of head bending of Bufo sp. field stage 4 larvae after
malathion exposure.
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Average liver size (mm)
1.75
1.7 2
R = 0.2689
1.65
1.6
1.55
1.5
1.45
0 5 10 15 20
Concentration (ppm)
Figure 7. Liver size of Bufo sp. field stage 4 larvae exposed to malathion at
varying concentrations.
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DISCUSSION
already lethal to Bufo sp. larvae in field stage four since 100% mortality was
attained in a short time of exposure of 48 hours. Bufo sp. larvae were found to
ppm. The result showed that Bufo sp. larvae are quite sensitive to malathion.
Not all Bufo species, however, offer the same sensitivity. For instance,
ppm already caused death after 48 hours but in lower concentrations, deaths
organisms (Hall and Kolbe, 1980). This can be related to their reduce
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Effects of toxicity were manifested through morphological abnormalities
such as curved tail, bent body axis (head) and bulging at the lower right
abdominal region. This result was consistent with the study conducted by
the larvae exposed to 0.25 ppm malathion. In the experiment, however, the
maximum average degree (12.6 degrees) of tail curvature of Bufo sp. was
observed in 15 ppm. In a previous study, Pawar et al. (1983) also found body
not well understood. Chemotti et al. (2006) argued, however, that bending of
axis may be related to the integrity of the extracellular matrix making up the
and hydroxyproline levels necessary for the formation of collagen’s triple helix.
In this study, the direction of tail bending was found to be dorsal suggesting
that bending was possibly caused by deformities of the notochord and not by
contractions of the tail muscles. This was not tested, however, in this study.
Unlike that of tail curvature which was observed after 48 hours, axis deformity
in the head takes time to develop. This means malathion must first be
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head region. This requires exposure at lower concentrations than that which
since it was also found that liver size in the control group had no significant
absorbed, is degraded in the liver and produces products more potent than
investigate further the effects of malathion on liver size and function of Bufo
the lower right abdominal region since the opposite region was found to be
depressed.
circular pattern and constant twitching of the tail during swimming and at the
believed that these behaviors are consequences from the bending of body
axis. Muscle twitching in the tail, on the other hand, was considered to be the
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Abnormal behaviors were usually followed by death as observed in the
axis reduce its ability for normal locomotion, which in turn can limit the ability
to reach food sources and increase the risks of predation and desiccation.
CONCLUSION
ppm in a decreasing pattern, showing that levels of toxicity of Bufo sp. field
Bufo sp. larvae exposed to malathion was dosage dependent (R2 = 0.9194).
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Exposure also produced abnormalities in morphology including: axis
deformities in the head and tail and the presence of a bulge on the lower right
abdominal region. Axis deformities include: tail curvature and head bending
with a golf-like pattern. Tail curvature and head bending was dosage
differed (P<0.05) between the 20 ppm group and the control and the lower
concentrations, except with 15 ppm. Liver size increase due to edema did not
RECOMMENDATION
can be species specific or may vary between species. In addition, the type of
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studies must be pursued regarding the toxicity of malathion and other
edema, however the mechanism on how malathion can cause such result was
This study was designed in the lab to solely define the acute toxicity
effects of malathion only. The experiments done definitely did not replicate the
natural environment of the Bufo sp. larvae. Therefore, the effects observed in
the lab may vary with those present in the real environment due to other
studies should also consider designing set ups that could replicate the true
environment of Bufo sp. or other anuran species and the effects of other
towards organophosphates).
ACKNOWLEDGEMENT
Evacitas, for her patience and guidance in the laboratory and in making the
manuscript. I would also like to thank Miss Ruby Caminade and especially Mr.
Tristan Arvin Jain, who generously gave their assistance in the field and in the
laboratory. Lastly, I would also thank the University of the Philippines in the
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Visayas – Cebu College for the equipment and facilities and as the place to
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