This article was published in an Elsevier journal. The attached copy
is furnished to the author for non-commercial research and
education use, including for instruction at the author’s institution,
sharing with colleagues and providing to institution administration.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
Author's personal copy
Available online at www.sciencedirect.com
PESTICIDE
Biochemistry & Physiology
Pesticide Biochemistry and Physiology 90 (2008) 66–70
www.elsevier.com/locate/ypest
Low cost microbioassay test for assessing cytopathological
and physiological responses of ciliate model Paramecium caudatum
to carbofuran pesticide
Mohd. Masood Hussain *, Nageswara Rao Amanchi,
Venkata Ramanaiah Solanki, Mohan Bhagavathi
Protozoology and Environmental Toxicology Laboratory, Department of Zoology, University College of Science,
Osmania University, Hyderabad 500007, Andhra Pradesh, India
Received 31 March 2007; accepted 23 July 2007
Available online 1 August 2007
Abstract
One of the major changes that have occurred over the past few years is the re-examination of conventional methods and the global
demand for innovative low cost bioassay tests for assessing water quality, toxicity evaluation and bioremediation. In the present study
acute toxicity, physiological and cytotoxic impact of carbofuran to a commonly occurring fresh water ciliate was measured. Intrinsic
cytotoxicity was evident on macro nuclear apparatus that exhibited deformities such as fragmentation, deep incision, vacuolization
and degenerative macronucleus. Depletion in the food vacuole formation of Paramecia was evident. Changes in the pulsatory vacuole
activity of Paramecium caudatum, highlighted dose dependent response by the test compound. The simplicity of handling this ciliate
makes unicellular eukaryote, an alternative organism for the toxicity assessment of pollutants. The tests carried in this study are simple
and fast, a bioassay that gives overall information about the physiological and cytotoxic effects of carbofuran to P. caudatum. Such bioassay tests using ciliates are more suitable for risk assessment of water quality, early detection of water pollution and possible role of
ciliates as bioindicators and strategic tools in the bioremediation of water bodies.
2007 Elsevier Inc. All rights reserved.
Keywords: Carbofuran; Paramecium caudatum; Bioremediation; Cytotoxicity; Macronucleus; Food vacuole; Pulsatory vacuole
1. Introduction
A better understanding of indicator potential and physiological responses of ciliates to toxicant stress could provide more information for gauging ecosystem viability
and stress recovery. Microorganisms represent one of the
links of which pesticides could be transmitted through
the food chain to man [1]. In the present study attempt
has been made to evaluate possible toxicity of carbofuran
on an organism other than the target. Because of the
absence of the relative data on the environmental toxicity
*
Corresponding author.
E-mail address: [email protected] (M.M. Hussain).
0048-3575/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.pestbp.2007.07.006
of this pesticide, the cellular model Paramecium sps. is chosen for evaluation. With the change from an organism
based society to an industrial one and the consequent concentration of population, the problem of wastewater has
become acute [2]. Protozoa, algae and bacteria form the
broad base of food chains and protozoan cells are often
used as bioindicators of chemical pollution, especially in
aqueous environment [3].
Dias et al. reported morphological and physiological
changes in another ciliate Tetrahymena pyriformis exposed
to Triton X-100 [4]. Morange reported Paramecium is a
model for molecular biologists has been suggested in [5].
The characterization of telomerase enzyme that opposes
the permanent reduction in length of the extremities of
chromosomes, the telomeres at each generation was done
Author's personal copy
M.M. Hussain et al. / Pesticide Biochemistry and Physiology 90 (2008) 66–70
in Tetrahymena [6]. Ciliates are also major concern as they
are more important as nutrient regenerators particularly of
nitrogen and phosphorus than bacteria. These unicellular
organisms offer the possibility of direct study of independent cells possessing specific features of single cells and of
whole organisms at the same time [7]. Bick produced an
illustrated guide based on the KLM system or the well
known ‘saprobian system’ and its indicator species concept,
a list of 84 ciliates to be used as indicators in fresh water
bodies. He has given the saprobiology, ecology, and saprobian classification [8]. This study is of immense use not only
for assessing the quality of water bodies subjected to
organic pollution, animal waste and sewage influx, but also
it throws light on the responses and toxic effects of chemical pollutants in the water bodies.
In the present study attempt has been made to investigate the physiological, cytotoxic and ecological effects of
carbofuran pesticide on Paramecium caudatum and to
explore the possibility of using them as bioindicators of
water quality and consequentially as tools for bioremediation. There is an international consensus that in vitro data
serve better purpose for screening of toxicants and that
they are indirectly suited for conclusions regarding risk
assessment [9]. Moreover the use of large animals in the
laboratory have been put to question internationally by
the societies pleading for good treatment and prevention
of harassment and cruelty to animals. Hence it is the present trend of using P. caudatum as one of the suitable model
for screening toxicity of chemicals [10]. The technical grade
sample of 75% carbofuran (2,3,dihydro-2,2-dimethyl-7benzofuranyl methyl carbamate) pesticide was made available by Rallis India Limited. Carbofuran is a systemic carbamate insecticide of broad spectrum utility applied to
control wide variety of insects and pests.
67
solution, again rinsed in water, dehydrated in alcohols,
cleared in xylol and mounted with DPX. Food and pulsatory vacuoles of P. caudatum were studied by exposure to
sub-lethal concentrations of the pesticides.
2.1. Observation of food and pulsatory vacuoles
The food vacuole studies were made on 25 test organisms which were exposed to concentrations of 87, 100,
115 and 135 ppm of carbofuran pesticide separately for
30 min duration. The cells were divided into two groups
for experimental studies one is exposed and group two is
control. Twenty-five treated cells from each concentration
after 30 min exposure time in the toxicant were picked with
the help of micropipette mixed with carmine suspension
and kept for 10 min. Ten organisms from each concentration were taken, immobilized on protamine coated slides
and number of food vacuoles was counted. Paramecia
under control study were kept devoid of carbofuran pesticide but mixed with the carmine suspension. Immobilization and counting of number of food vacuoles formed
were done after 10 min in the similar manner. Preparation
of carmine suspension and counting of food vacuoles was
done by the method initially suggested by Brutkowska [11].
Pulsatory vacuole activity was studied by immobilizing
paramecia as per the method suggested by Marsot [12],
after the 25 animals were exposed to each concentrations
of 87, 100, 115, 135 ppm for 10 and 20 min. Single individuals normal in every respect were picked and the rate of
pulsation of one vacuole was determined. Rate of pulsation
is the time required for one complete pulsation (from the
beginning of one contraction to the beginning of the next).
Observations were made on the cells in each concentration.
The rate of pulsation for each individual is calculated and
compared with the controlled cells.
2. Materials and methods
3. Results and discussion
Stock cultures of P. caudatum were cultured in the laboratory in the hay infusion medium at room temperature.
Meat extract was supplemented to the culture medium to
boost bacterial multiplication. Acute toxicity was conducted for 3 h duration and chronic toxicity was done for
144 h to find the growth inhibition of ciliates. Morphological, behavioral responses have been observed during the
exposure time. Cytochemical studies were conducted to
demonstrate general cytology, nuclear morphology of P.
caudatum on exposure to sub-lethal concentrations of the
pesticide. Cytotoxicity was done by staining the cells by
Feulgen fast green method. In the procedure, Paramecium
cells were initially fixed in Carnoy’s fixative (ethyl alcohol
and acetic acid in the ratio of 3:1, respectively) and prior
hydrolysis was done first briefly in 1 N HCl maintained
at room temperature and then at 60 C for exactly 8 min.
Then the slides are washed with 1 N HCl at room temperature. The hydrolysis is followed by transferring the slides
to Schiff’s reagent and incubating for 1 h. Then the cells
were immersed in three changes of sulphurous acid salt
Concentration of carbofuran 320 ppm and above caused
lethality immediately in all the exposed paramecia within
5 min of exposure time. A sudden change in mobility was
among the first observable modification to occur after the
organisms were exposed to 135 ppm whereas lower concentration of 100 ppm caused no abnormal change in motility.
When the paramecia were exposed to 240 ppm, the movements progressively retarded and in another 10 min ciliary
movements were totally arrested. Immediate cytotoxic
effects included darkening of cytoplasm, deformation of
cell body, and ultimately lysis of cells. Median tolerance
limit was 210 ppm, a concentration at which 50% of paramecia in the experimental set died, the value being obtained
from the survival curve plotted against the concentrations.
Nillson observed changes in proliferation and motility in
Tetrahymena by a calcium channel blocker Lanthanum
[13]. He observed in higher concentration of Lanthanum,
the cells became motion less with rocking movements, however, most cells regained motility after some hours. Usually
Author's personal copy
68
M.M. Hussain et al. / Pesticide Biochemistry and Physiology 90 (2008) 66–70
Table 1
Carbofuran versus Paramecium caudatum — macronuclear abnormal forms
Conc. in ppm
Percent abnormal
forms (%)
Unevenly
divided
Vacuolated
Fragmented
Diffusion of
macronucleus
Rod shaped
Other
deformities
100
210
280
16
53
47
1
8
7
1
35
30
7
1
4
2
—
—
2
7
3
3
2
3
proliferating ciliates respond to addition of xenobiotics by
exhibiting a 2–3 h lag before proliferation is resumed. Nillson further reported that high ethanol concentrations
induced loss of shape in Tetrahymena and they showed
spherical deformity with an enlarged contractile vacuole.
Swelling of cells was also observed in some of them and
he found small refractive granules appeared in all ethanol
treated cells as typically seen in Tetrahymena during stress
condition [14]. He could distinguish two populations one of
normal size and another of small mostly spherical cells. The
small round and spherical cells were lying at the bottom of
the culture dish looking motion less appearing dead.
The genotoxic effect of carbofuran pesticide on the macronuclear apparatus in P. caudatum was analyzed, which
was basically intended to ascertain weather the pesticide
has any damaging effect on DNA. The concentration
100 ppm induced 16% deformities ranging from fragmented nuclei, rod shaped deformity and diffusion of macronucleus (Table 1 and Fig. 3). The higher concentrations
210 and 180 ppm caused 53% and 47% deformities such
as vacuolization of the macronucleus, unevenly division
of macronucleus and rod shaped deformities. Other deformities included extrusion of bodies from macronucleus,
degenerating macronucleus and marginalization of
nucleus.
It is evident from findings that carbofuran acts as potential genotoxic substance at certain concentrations to ciliate
models. Marginalization of nuclear apparatus and dislocation of nucleus are considered to be the transforming characteristics of normal cells towards malignancy.
Carbofuran induced inhibitory effect on phagocytosis in
P. caudatum and caused 19.71% reduction in food vacuole
formation at 135 ppm, concentrations 115 and 87 ppm
could not bring any change in the food vacuole formation
(Graph 1 and Fig. 2). The mean number of food vacuoles
in controlled cells was 7.1 as compared to that of exposed
10
Foodvacuole activity in Paramecium caudatum exposed to Carbofuran
Fig. 1. Contractile vacuole activity in Paramecium caudatum exposed to
carbofuran.
No of Food vacuoles
8
6
4
2
0
Control
87 ppm
115 ppm
135 ppm
Concentration of Carbofuran
Mean ± S.D Values are significantly different at P< 0.05 level.
Graph 1. Food vacuole activity in Paramecium caudatum exposed to
carbofuran means ± SD. Values are significantly different at P < 0.05
level.
Fig. 2. Inhibition in phagocytosis in Paramecium caudatum exposed to
carbofuran.
Author's personal copy
M.M. Hussain et al. / Pesticide Biochemistry and Physiology 90 (2008) 66–70
69
Carbofuran Vs Contractile vacuole activity in
Paramecium caudatum
3.5
Exposure time 10 min
Exposure time 20 min
pulsation per minute
3
2.5
2
1.5
1
0.5
0
0 ppm
87 ppm
100 ppm
115 ppm
135 ppm
Concentration in (ppm)
Graph 2. Carbofuran vs. contractile vacuole activity in Paramecium
caudatum.
Fig. 3. Various macronuclear aberrations in Paramecium caudatum
exposed to carbofuran. (A) Macronucleus in control Paramecium
(400·). (B) Fragmentation of macronucleus in Paramecium (400·). (C)
Marginalization of macronucleus in Paramecium (400·). (D) Stretching of
macronucleus in Paramecium (400·). (E) Vacuolization of macronucleus
in Paramecium (400·). (F) Different macronuclear aberrations in Paramecium (100·).
which was 6.9. The values show there is no significant
decrease in phagocytosis in both the controlled and
exposed and were nearly equivocal. Fournier reported a
significant decrease in phagocytic activity of hemocytes in
clams exposed to mercuric chloride. Furthermore he established a correlation between the body burden of mercury
and affects on phagocytic activity of hemocytes [15].
Decrease in pulsatory vacuole activity was the common
observation recorded after addition of various concentrations of carbofuran in P. caudatum. A concentration of
135 ppm caused maximum decrease in water expulsion
activity and the pulsations per minute were 1.2 as compared to 3.2 in the controls. Concentrations 115 and
87 ppm showed increased vacuolar output with increase
exposure time as compared to the controls (Graph 2 and
Fig. 1). Okada et al. opined that the fundamental property
of animal cells is the ability to regulate their own cell volume and hypotonic stress cells readjust their volume after
transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD) [16].
The ciliate P. caudatum is used as a model cell system in
cytotoxicology and is highly adoptable to environmental
stress. The findings also indicate the great potential of the
use of P. caudatum as a biomonitor to toxicant influx faced
by water bodies. It is the kind of richness and diversity presented by ciliates which interests biologists as [5] finds them
as wonderful objects to go beyond genes. Such studies will
definitely add to the existing information on the use of biomonitors and bioindicators in water quality assessment and
provides a scope to develop a strategic role in the abatement of water pollution and also to maintain ecosystem
viability.
Acknowledgments
The authors are thankful to Prof. B. Raghavender Rao,
Head, Department of Zoology, University College of Science, Osmania University for extending lab facilities and
for his encouragement.
References
[1] R. Rouabhi, H. Berrebbah, M.R. Djebar, Toxicity evaluation of
flucycloxuron and diflubenzuron on the cellular model Paramecium
sps., Afr. J. Biotechnol. 5 (1) (2006) 045–048.
[2] K.J. Shiny, K.N. Remani, E. Nirmal, T.K. Jalaja, V.K. Sasidharan,
Biotreatment of wastewater using aquatic invertebrates, Daphnia
magna and Paramecium caudatum, Bioresour. Technol. 96 (2005) 55–
58.
[3] Mohd. M. Hussain, Toxicity evaluation of cypermethrin (cymbush)
using a ciliate protozoan Paramecium caudatum, Bull. Environ. Sci.
xxiv (2) (2006) 177–182.
[4] N. Dias, R.A. Mortare, N. Lima, Morphological and physiological
changes in Tetrahymena pyriformis for the in vitro cytotoxicity
assessment of Triton X-100, Toxicol. In vitro 17 (2003) 357–366.
Author's personal copy
70
M.M. Hussain et al. / Pesticide Biochemistry and Physiology 90 (2008) 66–70
[5] M. Morange, What history tells us IV. Ciliates as models. . .of what?
J. Biol. Sci. 31 (1) (2006) 27–30.
[6] C.W. Greidar, E.H. Blackburn, Identification of a specific telomere terminal
transferase activity in Tetrahymena extracts, Cell 43 (1985) 405–413.
[7] W.G. Landis, M.H. Yu, Introduction to Environmental Toxicology
Impacts of Chemicals upon Ecological Systems, second ed., Lewis,
Boca Raton, FL, 1995.
[8] H. Bick, Ciliated Protozoa. An Illustrated Guide to the Species used
as Biological Indicators in Freshwater Biology, World Health
Organization, Geneva, 1972, p. 198.
[9] M.A. Jaleel, In vitro toxicity evaluation of some pesticides using
certain ciliate protozoa, Ph.D. Thesis, submitted to Osmania
University, 2002.
[10] J. Venkateswara Rao, K. Srikanth, S.K. Arepalli, V.G. Gunda, Toxic
effects of acephate on Paramecium caudatum with special emphasis on
morphology, behaviour and generation time, Pestic. Biochem. Physiol. 86 (2006) 131–137.
[11] M. Brutkowska, The effect of certain salt solutions and osmotic
stimuli on the ciliary movement and food intake in Paramecium
caudatum, Acta Protozool. 4 (1967) 353–364.
[12] P. Marsot, P. Couillard, The use of protamine coated slides for
immobilizing protozoa, J. Protozool. 20 (1) (1973) 105–106.
[13] J.R. Nillson, Lanthanum affects proliferation and motility of Tetrahymena, Eur. J. Protistol. 39 (2003) 468–474.
[14] J.R. Nillson, Ethanol affects endocytosis and proliferation of Tetrahymena pyriformis GL and promotes Encystment, Acta Protozool. 44
(2005) 293–299.
[15] M. Fournier, J. Pellerin, Y. Clermont, Y. Morin, P. Brousseau,
Effects of in vivo exposure of Mya arenaria to organic and inorganic
mercury on phagocytic activity of hemocytes, Toxicology 161 (2001)
201–211.
[16] Y. Okada, E. Maeno, T. Shimizu, K. Dezaki, J. Wang, S. Morishina,
Receptor-mediated control of regulatory volume decrease (RVD) and
apoptotic volume decrease (AVD), J. Physiol. 532 (2001) 3–16.