56 0975 - 9573
ISSN
ANU J. Nat. Sci. 1(1), 56-60 (2010)
TOXICITY ASSESSMENT OF MONOCROTOPHOS USING
A SINGLE CELL PROTOZOAN CILIATE PARAMECIUM
CAUDATUM AS AN ALTERNATIVE MODEL
Nageswara Rao Amanchi*
Protozoology and Environmental Toxicology lab, Dept of Zoology, Nizam College (Autonomous),
Basheer bagh, Hyderabad - 500 001, Andhra Pradesh, India.
Abstract: In the present study experiments were conducted to evaluate the toxic effects of monocrotophos on
ciliate model Paramecium caudatum, which can be considered as ideal organism for investigating water
quality fluctuations and risk assessment of pesticides. Cell viability, cell morphology, phagocytosis, contractile
vacuole activity and macronucleus tests were performed using Paramecium caudatum to different
concentrations of monocrotophos. The calculated LC50 value of monocrotophos against mortality curve for
3hrs exposure to Paramecium caudatum was 332.284±57.52ppm. It was observed that the Paramecium
caudatum were highly responsive to monocrotophos. After a short period of exposure (20min to 30 min), there
was an increase in the number of necrotic cells with typical features like blackening of cytoplasm, blebbing,
leaking of internal contents and macronuclear changes leading to cell lysis. Changes in the contractile vacuole
activity, reduction in phagocytosis and morphological changes occurred in the shape of macronucleus. The
present findings indicate a possible necrotic and ecogenotoxic effect of monocrotophos to Paramecium
caudatum and such type of assays suggest the potential of above organism for ecotoxicological studies to
certain pesticides.
Key words: Paramecium caudatum, monocrotophos, acute toxicity, water quality, macronuclear changes.
Introduction
The attitude of scientists changing rapidly,
first, for technical reasons; genome sequencing
programmes have been extended to all organisms including paramecia and this knowledge allows new
experimental approaches to these organisms. So that
these organisms are now model organisms to
scientific community (Narasimhan 1999).The study
of ciliate sensitivity to a wide number of toxic
substances may provide a yardstick for identifying
the intensity and potential for ecological damage
caused by anthropogenic pollutants. Moreover,
ciliates respond more quickly to environmental
contamination than other organisms because of their
faster generation time, ubiquitous nature, and variety
of trophic niches. For various technical reasons - small
size, sensitivity, high reproduction rate and size of
genetic material - these models were best suited to
screen toxic effects of various pollutants (Dias et al
2003; Morange 2006; Masood Hussain et al 2008).
However, despite their acknowledged importance,
* Corresponding author : [email protected]
ANU Journal of Natural Sciences w June 2010 w
little work has been performed to identify suitable
test species among the ciliates. In this context, the
ciliate assay has become a valuable tool for detection
of environmental disturbance and for assessment of
the trophic state.
Materials and methods
Paramecium caudatum was selected as test
species for the present studies. Paramecia were
collected and isolated from freshwater pond within
the vicinity of Osmania University, Hyderabad, India.
Hay infusion was used as the culture medium for
rearing ciliates and it has been widely used as a basic
and most appropriate culture medium for ciliates
(Shiny et al 2005). The commercial grade sample of
monocrotophos (Hycrophos) was used for toxicity
assessment. Stock solution and experimental
concentrations of monocrotophos were prepared as
recommended by APHA (1995). In acute toxicity
experiments 0.5 ml of known concentration of
pesticide solution was added to 4.5 ml of culture
Toxicity Assessment of Monocrotophos using ...
medium to achieve desired concentration of pesticide.
50 organisms were introduced in each cavity block.
Triplicates were maintained for all concentrations and
control was maintained deprived of test
concentration. The acute toxicity test was conducted
for 3hrs duration. The cavity block, after adding
pesticide was placed under binocular microscope and
direct manual counting was performed (Apostol 1973;
Amanchi and Bhagavathi 2009). Food vacuole
activity was studied in Paramecium caudatum when
cells exposed to monocrotophos for 30 min and 1hr
duration. Preparation of carmine suspension and
counting of food vacuoles was done by the method
suggested by Brutkowska (1967). For contractile
vacuole study the animals were exposed to the
different concentrations for 10 min and 20 min, single
individuals were picked normal in every visible
respect and the rate of pulsation of one vacuole i.e.,
the time required for one complete pulsation was
determined. Observations were made on Paramecium
in each concentration. The rate of pulsation for each
individual is calculated separately by the method as
suggested by Stock et al (2002). DNA localization
was done by Feulgen Fast Green technique and it is
intended to screen possible genotoxic effects of test
pesticide on macronucleus (Rizzo and Nooden.,
1973).
Results and Discussion:
Acute toxicity studies:
High concentrations of 1000ppm and above
caused immediate death of all cells. Concentration
of 200ppm and 250ppm initially caused increased
mobility, longer exposure decreased mobility. After
30min of exposure to 250ppm, cells aggregated at
the corners of cavity block. Concentration of 450ppm
and above immediately decreased the mobility and
cells became immobile. The LC50 value as calculated
against the mortality curve was 332.284±57.52ppm.
The common morphological alterations observed
were swelling, shortening of longitudinal axis,
narrowing of anterior, blackening of cytoplasm,
blebbing and rupturing of cell membrane. First visible
reactions at non lethal concentrations were
irregularities in ciliary beating, which often resulted
in swimming away of the cell. At higher
concentrations of monocrotophos, the movement of
the cilia became weaker and irregular after a while
57
the cell shape became deformated and finally the cell
died.
Food vacuole activity in paramecium exposed to
monocrotophos:
That monocrotophos exerted an obvious
inhibitory effect in Paramecium caudatum reducing
the formation of food vacuoles below the control level
when exposed to 30min, and 1hr at 100ppm
concentration. The highest inhibition occurred at
100ppm concentration after 1hr exposure, where
mean numbers of food vacuoles obtained were 3.5
as compared to 8.6 in the control cells, there by
59.31% inhibition in phagocytosis. The mean number
of food vacuoles was 5.3 obtained with the same
(100ppm) concentration after 30min of exposure,
where 38.83% inhibition was seen. Triplicates were
maintained throughout the study and the values are
significantly different at P < 0.05. Nilsson (2005)
reported a close relationship between the motility of
cell and capacity for phagocytosis. When cell motility
was disturbed or cells exposed to high concentration,
the phagocytic activity was very much reduced.
Changes in the rate of the ciliary movement are
largely responsible for changes in the rate of the food
ingestion. Rebendal and Karpinska (1981) reported
the inhibition of the food vacuole formation in
Tetrahymena on exposure to Colistin and penicillin
“V” and “G”. Similar findings with various pesticides
were reported by Masood Hussain (1984) and Jaleel
(2002) on phagocytosis in ciliates.
Contractile vacuole activity:
When Paramecium were exposed to 100ppm
concentration of monocrotophos for 20min, it resulted
in a drastic retardation in the rate of pulsatory output
where it was recorded as an average 5.5 pulsations
for one min, whereas in control cells it was 8.66
pulsations for one min. This was the maximum
decrease of vacuolar output in paramecium when
exposed to 100ppm of monocrotophos. The lowest
retardation recorded was 7.5 pulsations per one min
of 25ppm at 10min exposure (table 1). From the
results obtained on contractile vacuole activity it was
clear that at lower concentrations, the effect of
monocrotophos is very less when compared to higher
concentrations. To support the findings, there are
certain reports suggesting that in higher
ANU Journal of Natural Sciences w June 2010 w
58
Nageswara Rao
Graph 1: Food vacuole activity in Paramecium caudatum exposed to monocrotophos.
concentrations the contractile vacuole apparatus was
ruptured or disrupted, so that the pulsatory frequency
rate was reduced (Naitoh et al, 1997, Masood Hussain
and M.A. Khan, 1993; Amanchi and Hussain 2008).
Ishida et al (1993) suggested that, In Paramecium
multimicronucleatum, cells injected with monoclonal
antibody DS-1, the reduction of the expulsion
frequency was dose, time and site dependent, in lower
concentrations the expulsion frequency rate was about
94% of the normal cell, but with higher concentration
the pulsation frequency rate was reduced by half
(50%). The overall fluid output per cell reduced
slightly at 10min, but it was declined to 43% of the
control output by 45min.
Monocrotophos induced macronuclear changes in
Paramecium caudatum:
It is clear from the data that the treated cells
displayed variations with regard to size, shape, and
location of the macronucleus. The present result
shows that the presence of large number of rod shaped
Table 1. Contractile vacuole activity in Paramecium caudatum exposed to monocrotophos.
Concentration
in ppm.
Exposure time
in minutes.
Average time for
one pulsation in
seconds
Pulsations
per min.
100
10
20
9.47
10.90
6.33
5.5
50
10
20
9.0
8.57
6.66
7.0
25
10
20
8.0
8.57
7.5
7.0
Control
____
____
6.92
____
8.66
ANU Journal of Natural Sciences w June 2010 w
Toxicity Assessment of Monocrotophos using ...
59
deformities when exposed to different concentrations
of monocrotophos. Uneven division of macronucleus
was induced in 14 % cells in concentrations of
100ppm, the cells treated with the same concentration
monocrotophos for one hour showed 9 %
vacuolization in the macronucleus. The average
lowest abnormalities recorded were 29%, at 10ppm
concentration, whereas the highest total abnormalities
recorded were 58% at 100ppm concentration of
monocrotophos. In concentrations of 100ppm,
50ppm, 25ppm and 10ppm, the percent abnormalities
recorded were 58%, 44%, 37%, and 29% respectively
(table 2). The occurrences of macronuclear
aberrations were dose dependent. An in vivo study
with fish erythrocytes showed a dose related increase
in DNA strand breaks with monocrotophos using the
comet assay (Saleha Banu et al, 2001). Yong Cui et
al (2006) suggested, the chlorpyrifos did not form
the DNA adducts but the active metabolites of
cypermethrin, instead of the parent cypermethrin,
may have contributed to the formation of DNA
monoadducts and DNA interstrand cross links in the
primary mouse hepatocytes. If the DNA adducts are
not repaired or are mistakenly repaired before DNA
replication, they may lead to gene mutations and
initiate carcinogenesis (Gupta and Spencer-Beach,
1996). Similar findings reported in mice by Sarbani
Giri et al (2002) and in Allium cepa by Topaktas and
Rencuzogullari (1996) when exposed to carbosulfan
and carbamate pesticides respectively. Such changes
could be the result of direct interaction of pesticides
with the nuclear material.
Conclusion:
It may be concluded that ciliates are attractive
models for toxicological studies due to their relative
ease of culturing, shorter life cycle and sensitivity to
environmental changes. It is further concluded that
bioassay tests using ciliates are suitable for risk
assessment of water quality, early detection of water
pollution and possible role of ciliates as strategic tools
in the bioremediation of water bodies and
genotoxicity studies.
Acknowledgements:
The author is thankful to Prof. S. Sabita Raja,
The Head, Department of Zoology, Nizam College
(Autonomous) and Prof. Naidu Ashok, The Principal
of the College, for their constant encouragement and
cooperation to during my work period.
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Table 2: Monocrotophos induced macronuclear changes in Paramecium caudatum exposed for one hour.
VARIOUS
ABNORMAL
FORMS
Conc/ppm
Percent
abnormal
forms
Unevenly
divided
Vacuolated
Fragmented
Rod
shaped
Other
deformities
100
58 %
14
9
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44 %
8
4
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13
12
25
37 %
8
3
6
16
4
10
29 %
6
4
2
14
3
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