Advances in Environmental Biology, 9(3) February 2015, Pages: 281-285
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Toxicity of the Chlorfenapyr: Growth inhibition and induction of oxidative stress
on a freshwater protozoan: Paramecium sp.
H. Benbouzid, H. Berrebbah, M.R. Djebar
Laboratory of Cell Toxicology, General Direction of Scientific Research and Technological Development, Annaba University, Algeria
ARTICLE INFO
Article history:
Received 12 October 2014
Received in revised form 26 December
2014
Accepted 1 January 2015
Available online 20 February 2015
Keywords:
Chlorfenapyr, detoxification oxidative
enzymes, Paramecium sp
ABSTRACT
The toxicological impacts of the increasing number of synthetic compounds present in
the aquatic environment are assessed predominantly in laboratory studies where test
organisms are exposed to a range of concentrations of single compounds. The bioindicator Paramecium sp., characterized by a short life cycle, rapid multiplication and
normal behavior that may be affected by the presence of pollutants. We therefore
investigated the inhibitory effect of a newly synthesized acaricide: the chlorfenapyr
tested at concentrations of 250, 300 and 350 µM on a pure culture of Paramecium sp.
during 6 day. Paramecia treated with different concentrations of Chlorfenapyr illustrate
strong inhibition of cell growth from the second day of treatment. Low levels of
glutathione, increased glutathione S-transferase and the decrease in respiratory
metabolism, recorded in the presence of different concentrations of Chlorfenapyr,
involve the activation of detoxification system.
© 2015 AENSI Publisher All rights reserved.
To Cite This Article: H. Benbouzid, H. Berrebbah, M.R. Djebar, Toxicity of the Chlorfenapyr: Growth inhibition and induction of
oxidative stress on a freshwater protozoan: Paramecium sp. Adv. Environ. Biol., 9(3), 281-285, 2015
INTRODUCTION
The extensive use of insecticides, during the past decades has led to a number of negative effects on
terrestrial and aquatic organisms. Chlorfenapyr is a member of a new class of chemicals, namely, pyrroles
(chemical name: 4-bromo-2-(4-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-1H-pyrrole-3carbonitrile;
trade name: Pylon miticide-insecticide). Chlorfenapyr uncouples oxidative phosphorylation in the mitochondria,
resulting in disrupted ATP production, cellular death, and ultimately, death of the organism [8].
Chlorfenapyr, which is a member of the pyrrole class of chemicals, is a proinsecticide compound; that is, its
biological activity depends on its activation by another chemical. Oxidative removal of the N-ethoxymethyl
group of Chlorfenapyr by mixed function oxidases forms uncouples oxidative phosphorylation at the
mitochondria, resulting in disruption of production of ATP, cellular death, and ultimately organism mortality
[3].
Monitoring of aquatic ecosystem pollution represents one of the major activities involved in measures
aimed at environmental protection. Usage of non-targeted organisms in environmental toxicology is needed to
understand the wide range of toxic effects caused by the pesticides on different organisms [23]. Fish and other
aquatic biota that were commonly used as bioindicators of persistent organic pollutants [22] have been replaced
in recent years successfully by ciliates [12]. Protozoan cells are often used as bioindicators of chemical
pollution, especially in aqueous environment. The application of unicellular organisms to study the toxic effects
of pesticides from contaminated wastewater is relatively new throughout the world. Hence, in the present paper,
we have studied the toxic impacts of Chlorfenapyr a novel broad-spectrum insecticide-miticide, stable and
persistent in the environment on oxidative stress in a pure culture of Paramecium sp.
MATERIALS AND METHODS
The biological material used is the typical representative of Protozoa: Paramecium sp. We kept a
continuous pure colony in the laboratory at the University of Annaba from infusions of lettuce [1].
Corresponding Author: H. Benbouzid, Laboratory of Cell Toxicology, General Direction of Scientific Research and
Technological Development, Annaba University, Algeria.
E-mail: [email protected]
282
H. Benbouzid et al, 2015
Advances in Environmental Biology, 9(3) February 2015, Pages: 281-285
The pyrrole 4-Bromo-2-(4-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-1H-pyrrol-3-carbonitrile is
the common name of Chlorfenapyr that is used as an acaricide/insecticide and developed by Dow AgroSciences. This synthetic molecule was tested at the following concentrations: 250, 300 and 350 µM. These
concentrations were selected after a series of preliminary finding range toxicity tests.
In accordance with Lavergne [9] and Sauvant, Pepin and Piccini [13], the growth kinetics of Paramecium
sp. were studied by measuring the optical density OD at λ = 600 nm in function of time.
The response in percentage was calculated to evaluate the toxicity of xenobiotics via the inhibition of cell
growth of protists. The percentage of positive values indicates an inhibition of growth, while the negative values
indicate a stimulation of growth [26].
The polarographic method enables measurement of production or consumption of oxygen with an oxygen
electrode, type HANSATECH [11].
The determination of glutathione (GSH) was performed as described by Weeckbeker and Cory [24] and
glutathione S-transferase (GST) by Habig, Pabst and Jakoby [7]. These biomarkers were chosen for their role in
cell protection and in cellular metabolism of xenobiotics. Measurements were made during the exponential
phase of growth (between 48 and 96 h).
All the experiments were repeated three times, and the results were expressed as mean and standard error
(SE) values. Statistical analysis was performed using a two-way ANOVA and the test of Dunnett for
comparison between the control and treated cells. The α-level for significant differences was set at p<0.05 [5].
Results:
Effect of Chlorfenapyr on the growth of Paramecium sp:
The impact of Chlorfenapyr concentrations on the population growth of Paramecium sp. is shown in
(Figure 1). The Chlorfenapyr has an inhibitory effect on cell growth of Paramecium (p<0.001), and this effect
was already visible at 2nd day of the treatment.
Fig. 1: Effect of Chlorfepyr at 250, 300 and 350 µM on the growth of Paramecium sp. Each data point
represents the mean of three independent assays ± standard error. Values are significant from control
values at p < 0.001.
Percentage of response in Chlorfenapyr treated Paramecium sp:
The response percentage measurement results are presented in (Figure 2). It can be said that the positive
evolution of the response percentages confirm the growth inhibition of the treated paramecia and this regardless
of the cell concentration.
Fig. 2: Percentage of response in Chlorfenapyr-treated Paramecium sp. Each value is mean ± standard error of
three replicates.
283
H. Benbouzid et al, 2015
Advances in Environmental Biology, 9(3) February 2015, Pages: 281-285
Effect of Chlorfenapyr on GSH level in Paramecium sp:
As shown in figure 3, the GSH level is lower at the concentrations of 250 and 300 µM than in the nontreated Paramecium. In contrast, with the highest concentration (350 µM), the GSH level was increased
significantly (p <0.001). We hypothesize that this increase results from the started detoxification/metabolism
process.
Fig. 3: Effect of Chlorfenapyr on the rate of GSH in Paramecium sp. Each value is mean ± standard error of
three replicates.
Effect of Chlorfenapyr on GST activity in Paramecium sp:
As demonstrated in figure 4, that GST activity increased significantly in cells treated with different
concentrations of Chlorfenapyr compared to controls (p <0.001).
Fig. 4: Impact of Chlorfenapyr (250, 300 and 350 µM) on the GST activity in Paramecium sp. Each value is
average±standard error of three independent observations.
Effect of Chlorfenapyr on the respiratory metabolism of Paramecium sp:
The O2 consumption of paramecia was significantly affect (p˂0.001) by the action of Chlorfenapyr
concentrations (Figure 5). It should be noted that after 5 min of exposure, cell cultures treated with strongest
concentration (350 µM) present a significantly deceleration of their respiratory activity.
284
H. Benbouzid et al, 2015
Advances in Environmental Biology, 9(3) February 2015, Pages: 281-285
Fig. 5: Impact of Chlorfenapyr (250, 300 and 350 µM) on respiratory metabolism of Paramecium sp. Each
value is mean± standard error of three independent observations.
Discussion and conclusions:
Paramecium that has long been a model organism for cellular aging and clonal lifespan [18,17,16,15,20,21].
In the present study, we showed that Chlorfenapyr concentrations molecule caused a dose-dependent
growth inhibition of Paramecium sp. population. In addition, for the three concentrations tested, it can be said
that the evolution of the response percentages confirm the growth inhibition of the treated Paramecium and this
regardless of the cell concentration.
Low level of GSH and high GST activities recorded in the presence of xenobiotics may be indicative of
activation of the detoxification system. On the other hand, as reported before [14,27,10,6] the direct capture of
oxygen free radicals caused by xenobiotics is carried out by radical compounds as traps or enzyme systems
located nearby the initial glutathione production system.
Chlorfenapyr is a member of a new class of chemicals -- the pyrroles. The compound is a pro-insecticide,
i.e. the biological activity depends on its activation to another chemical. Oxidative removal of the Nethoxymethyl group of Chlorfenapyr by mixed function oxidases forms uncouples oxidative phosphorylation at
the mitochondria, resulting in disruption of production of ATP, cellular death, and ultimately organism mortality
[3]. This inhibitory effect is reflected in our work by a metabolic disorder in the cellular respiration generated by
an oxidative stress [4,25].
The results suggest that the antiproliferative effect of Chlorfenapyr may be mediated by reducing the chitin
and cuticle of cells, also by the effect on mitochondria and lashes, our results allow us to conclude that the tested
Chlorfenapyr inhibits the growth of Paramecium, and that this effect is mediated by a disruption of its cellular
metabolism.
After considering all the experimental data obtained throughout the study, it appears that the ciliate protists
used in our work is a material of choice for studies in toxicology and occupies a privileged position in aquatic
ecosystems because it is one of the basic elements of food chain, hence the need for a deep study of the impact
of pollution on our environment.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
Beaumont R. and C. Cassier, 1998. Travaux Pratiques de Biologie Animale, Zoologie, Embryologie,
Histologie, 3ème édition. Dunod Ed., pp: 123-143.
Chan, K.M. and E.A. Decker, 1994. Endogenous skeletal muscle antioxidants. Crit. Rev. Food Sci. Nutr.
34: 403-426.
Choi, U.T., G.H. Kang, Y.S. Jang, H.C. Ahn, J.Y. Seo and Y.D. Sohn, 2010. Fatality from acute
chlorfenapyr poisoning, Clinical Toxicology, 48(5): 458–459.
Csaba, G. and P. Kovács, 1999. Localization of $-endorphin in Tetrahymena by confocal microscopy.
Induction of the prolonged production of the hormone by hormonal imprinting. Cell Biology International,
23 : 695-702.
Dagnelie, P., 1999. Statistique théorique et appliquée. Tome 1. Statistique descriptive et bases de
l'inférence statistique. Paris et Bruxelles, De Boeck et Larcier, pp: 508.
Ha, H., N.S. Sirisoma, P. Kuppusamy, J.L. Zweier, P.M. Woster and R.A.JR. Casero, 1998. The natural
polyamine spermine functions directly as a free radical scavenger. Proc. Natl. Acad. Sci. USA, 95: 1114011145.
285
H. Benbouzid et al, 2015
Advances in Environmental Biology, 9(3) February 2015, Pages: 281-285
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Habig, W.H., M.J. Pabst and W.B. Jakoby, 1974. Gluthation-S-transferases: the first enzymatic step in
mercapturic acid formation. Journal of Biological Chemistry, 249: 7130-7139.
Hoshiko, M., S. Naito, M. Koga, M. Mori, K. Hara and T. Ishitake, 2007. Case report of acute death on the
7th day due to exposure to the vapor of the insecticide chlorfenapyr, Chudoku Kenkyu, 20(2): 131–136.
Lavergne M., 1986. Etude des effets du diapézan et du médazépan sur un protiste cilié. Mémoires de
D.E.A. de l’université de Paris, VII : 46.
Packer, L., E.H. Witt and H.J. Tritschler, 1995. Alpha-lipoic acid as a biological antioxidant. Free Radic.
Biol. Med., 19: 227-250.
Rouabhi, R., H. Berrebah and M.R. Djebar, 2006. Toxicity evaluation of flucycloxuron and diflubenzuron
on the cellular model, Paramecium sp. Afric. J. Biotechn, 5: 45-48.
Sako, F., N. Taniguchi, N. Kobayashi and E. Takakuwa, 1977. Effects of food dyes on Paramecium
caudatum: toxicity and inhibitory effects on leucine aminopeptidase and acid phosphatase activity,
Toxicol. Appl. Pharmacol, 39: 11–17.
Sauvant, M.P., D. Pepin and E. Piccini, 1999. Tetrahymena pyriformis. A tool for toxicological Sci. USA,
95: 11140-11145.
Sevanian, A., S.F. Muakkassah-Kelly and S. Montestruque, 1983. The influence of phospholipase A2 and
glutathione peroxidase on the elimination of membrane lipid peroxides. Arch. Biochem.
Smith-Sonneborn, J., 1981. Genetics and aging in protozoa. Int. Rev. Cytol, 73: 319–354.
Smith-Sonneborn, J., 1985. Aging in unicellular organisms. In: Finch, C.E., Schneider, C.L. (Eds.),
Handbook of the Biology of Aging, second ed. Van Nostrand Reinhold, New York, pp: 79–104.
Sonneborn, T.M., 1974. Paramecium aurelia. In: Mayr, E.(Ed.),HandbookofGenetics, Plenum, NewYork,
London, 2: 433–467.
Sonneborn, T.M., 1954. The relation of autogamy to senescence and rejuvenescence in Paramecium
aurelia. J.Protozool, 1: 38–53.
Studies. Chemosphere, 38: 1631-1669.
Takagi, Y., 1999. Clonal life cycle of Paramecium in the context of evolutionally acquired
mortality.In:Macieira-Coelho,A.(Ed.),Cell Immortalization.Spring- er,Berlin, pp: 81–101.
Takagi, Y., 1988. Aging.In:Go¨ rtz, H.-D.(Ed.), Paramecium. Springer,Berlin, pp: 131–140.
Van der Oost, R., J. Beyer and N.P.E. Vermeulen, 2003. Fish bioaccumulation and biomarkers in
environmental risk assessment: a review, Environ. Toxicol. Pharm, 13: 57–149.
Wan, M.T., R.G. Watts and D.J. Moul, 1994. Impact of chemigation on selected non-target aquatic
organisms in cranberry bogs of British Columbia, Bull. Environ. Contam. Toxicol, 53: 828–835.
Weckberker, G. and G. Cory, 1988. Ribonucleotidereductase activity abd growth of glutathione depleted
mouse leukemial 1210 cells in vitro. Cacerletters, 40: 257-264.
Who, 2004. Novaluron. (±)-1-[3-chloro-4-(1,1,2- trifluoro-2-rifluoromethoxyethoxy)phenyl]-3-(2,6difluorobenzoyl) urea. Geneva, World Health Organization (WHO Specifications and Evaluations for
Public Health Pesticides.
Wong, C.K., Yo. Cheung and Ming-Ho, 1999. Toxicological assessement of coastal sediments in Hong
Kong using a flagellate Dunalliella tertiolecta. Environmental pollution, 105: 175-183.
Yu, B.P., 1994. Cellular defences against damage from reactive oxygen species. Physiol. Rev., 74: 139162.