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Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of Duttaphrynus melanostictus embryos
Accepted Manuscript Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of Duttaphrynus melanostictus embryos
Mattilang Kharkongor, Rupa Nylla K. Hooroo, Sudip Dey PII:
S0045-6535(18)31361-4
DOI:
10.1016/j.chemosphere.2018.07.097
Reference:
CHEM 21812
To appear in:
Chemosphere
Received Date:
26 April 2018
Accepted Date:
17 July 2018
Please cite this article as: Mattilang Kharkongor, Rupa Nylla K. Hooroo, Sudip Dey, Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of Duttaphrynus melanostictus embryos, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.07.097
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ACCEPTED MANUSCRIPT
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Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of
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Duttaphrynus melanostictus embryos Mattilang Kharkongora*, Rupa Nylla K. Hoorooa and Sudip Deyb
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a Department b Electron
of Zoology, North Eastern Hill University, Shillong-793022, Meghalaya, India.
Microscope Division, Sophisticated Analytical Instrument Facility, North Eastern Hill University, Shillong-793022, India.
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*Corresponding author E-mail: [email protected]
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a [email protected]; b [email protected]
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Abstract
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In an attempt to assess the effects of chlorpyrifos [O,O-diethyl O-(3,5,6-
11
trichloropyridin-2-yl) phosphorothioate], the second largest selling insecticide in India, studies
12
were made with reference to some non-target organisms. The present study was undertaken to
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evaluate the effects in the embryos of Duttaphrynus melanostictus caused by the commercial
14
formulations of chlorpyrifos (Tricel, chlorpyrifos, 20 % EC). The LC50 value for Duttaphrynus
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melanostictus embryos after 48 hour (h) of treatment with chlorpyrifos was found to be 57.525
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ppm. The mortality of the embryo was significantly affected by different concentrations of
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chlorpyrifos when compared with the control groups. An increase in concentration of
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chlorpyrifos resulted in the simultaneous decrease of the hatching percentage and an increase in
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the morphological abnormalities such as compression of the embryo, reduced body size and
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curling of tail.
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Keywords: chlorpyrifos; Duttaphrynus melanostictus; embryos; LC50; SEM
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1. Introduction
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Excessive use of pesticides has not only resulted in unusual chemical pollution but also
24
has a secondary detrimental effect on non-target organisms (Kumar et al., 2009; Matsumura,
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1975). Pesticides are transported to the water bodies through surface run off which then enter the
26
organisms through contact and food web (Brown and Casida, 1987; Edwards, 1973; Murty,
27
1986). In recent years, organophosphates has been widely used as pesticide to help control a
28
variety of sucking, chewing and boring insects of various commercial and food crops (Kanekar
29
et al., 2004).
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Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloropyridin-2-yl) phosphorothioate] is a broad
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spectrum organophosphate insecticide which is used to control varieties of pests (Deb and Das,
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2013). The insecticide acts on insect pests through direct contact, ingestion, and inhalation
33
(Tomlin, 2009). It is known to cause adverse effects not only on the target pests but also on some
34
non-target organism e.g. amphibians (Bernabo et al., 2011)
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The effect of agricultural intensification on amphibians has drawn considerable
36
attention in recent years (Relyea et al., 2005). It was reported that species richness and
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abundance of amphibians was lower in agricultural sites where pesticides are often used than the
38
adjacent non-agricultural sites (Bonin et al., 1997). The early life cycle of most species of the
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amphibians occurs in ponds, temporary pools and streams near the agricultural fields receiving
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pesticide applications. Hence, it is very likely that the eggs and tadpoles are exposed to pesticide
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residues present in water bodies since most of the time pesticide application activity coincides
42
with the breeding and larval development (Hayes et al., 2003; Peltzer et al., 2008; Vertucci and
43
Corn, 1996).
44
In the context of the state of Meghalaya, a Northeastern state in India, 81% of the
45
population depends on agriculture especially in the rural areas and varieties of crops are grown in
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the state. The use of Chemical by farmers is one of the improved and modern agricultural
47
methods that have contributed to the increase in the production of food grains
48
(http://www.megagriculture.gov.in). It is estimated that the consumption of technical grade
49
pesticides in 2014-2015, the state of Meghalaya, consumes about 28 Metric tons of pesticides
50
(http://ppqs.gov.in/divisions/pesticides-monitoring-documentation).
51
available on the extent of impact, if any, on biological entities present in water bodies adjacent to
52
agricultural fields where pesticides are used in the state. In the present study, effects of
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chlorpyrifos on the hatching, mortality and morphological deformities at sub-lethal
54
concentrations in Duttaphrynus melanostictus embryos has been investigated. This study may be
55
relevant from the amphibian conservation point of view.
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2. Materials and Methods
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2.1.
Scanty
information
is
Collection and maintenance of eggs
58
Duttaphrynus melanostictus egg strings covered in jelly coat which were used in the
59
experiment were collected from a stream at Mawpat, Shillong, Meghalaya (altitude of 1,407 m
60
ASL) from the month of February to May. The eggs were brought to the laboratory and kept in
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plastic trays filled with pond water at room temperature (22±2 ºC).
62
2.2.
Selection and preparation of insecticide concentrations
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Commercially available chlorpyrifos (Tricel, chlorpyrifos, 20 % EC, Excel Crop Care
64
limited, Gujarat, India) was diluted with acetone (Dimitrie and Sparling, 2014) to prepare a stock
65
solution (1 ml of chlorpyrifos in 1000 ml of acetone). Different concentrations of this stock
66
solution were added to glass bowls (150 mm diameter) containing 500 ml of dechlorinated tap
67
water (Jayawardena et al., 2011). All experiments were conducted at ambient temperature (20±2
68
ºC) and under natural light conditions. The control was prepared with different concentrations of
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acetone in 500 ml of dechlorinated tap water to make the resulting solution 0.1, 0.5, 1, 5, 10, 15,
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20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ppm.
71
2.3. Determination of LC50 values
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The lethal concentration, LC50 of chlorpyrifos was determined by exposing 10
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embryos, Gosner stage 9 (Gosner, 1960) of Duttaphrynus melanostictus in each bowls containing
74
different concentrations of chlorpyrifos (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70
75
ppm) concentrations mixed with 500 ml of dechlorinated tap water for 48 h which was taken in 5
76
replicates.
77
2.4. Sub-lethal exposure
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The embryos were also divided into 4 groups with 10 embryos in each group and in 5
79
replicates. They were then exposed to sub-lethal concentrations (0.1, 0.5 and 1 ppm) of
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chlorpyrifos which was prepared using appropriate volume of dechlorinated tap water as it had
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been estimated that concentration of chlorpyrifos in small water bodies were in the range of 0.1
82
ppm to 1 ppm (Mazanti et al., 2003; Moore et al., 2002). The embryos were maintained till they
83
hatched and the hatching percentage and mortality were recorded. The hatched embryos were
84
then processed for scanning electron microscopy to observe any change in the body morphology.
85
The mean body length and mean body width of control and treated embryos were also recorded
86
by using a dial calliper (Mitutoyo series No. 505-671).
87
2.5.
Scanning Electron Microscopy
88
In order to carry the scanning electron microscopic study, the embryos, Gosner stage
89
18 were fixed in 2.5 % gluteraldehyde in 0.1 M sodium Cacodylate buffer (pH 7.2) for 4 h at 4ºC
90
and then washed in sodium Cacodylate buffer three times for 15 minutes each. The specimens
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were then post-fixed in 1% osmium tetraoxide in the same buffer at 4ºC. The samples were
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dehydrated in increasing concentration of acetone with two changes of 15 to 30 minutes in each
93
grade. The dehydrated samples were then dried with TMS (Tetra Methyl Silane) drying
94
technique of Dey et.al. (1989). The samples were secured to brass stubs (10 mm diameter X 30
95
mm height) with the use of double adhesive tape. Coating of the sample with gold was carried
96
out by using JFC 1100 (Jeol) ion sputter in a relatively low vacuum for ionization of the air
97
particles followed by the application of high voltage. Gold coating prevents sample from
98
damaging due to radiation and also increases conductivity. Observations had been done with
99
scanning electron microscopy JSM 6360 (Jeol) in the secondary electron emission mode at an
100
accelerating voltage of 20 KV with the working distance of 12 mm.
101
2.6.
Statistical analysis
102
LC50 values were determined using Finney’s Probit Analysis (1971). The LC50
103
determination method makes use of the SPSS computer software (Finney, 1971). All data are
104
represented as mean ± SEM and then subjected to one way ANOVA followed by Bonferroni's
105
Multiple Comparison Test. The statistical analysis was performed using Graph Pad Prism
106
version
107
www.graphpad.com.
108
3. Results
109
3.1. LC50 value
4.00
for
windows,
Graph
pad
Software,
San
Diego
California,
USA,
110
According to Finney’s Probit Analysis, the LC50 value of chlorpyrifos for Duttaphrynus
111
melanostictus embryos after 48 h of exposure was found to be 57.525 ppm which represents the
112
relation between chlorpyrifos concentration and mortality rate. The availability of chlorpyrifos
113
to embryos was synchronised with the concentration of the pesticide applied and the LC50 value
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was also an inclusion of other ingredients. No mortality was observed in the controls and sub-
115
lethal concentrations (0.1, 0.5 and 1 ppm) of chlorpyrifos treated groups.
116
3.2. Hatching
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The embryos of Duttaphrynus melanostictus hatched at Gosner stage 18 after 96 h. The
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hatching was observed in both the control and all treated groups. The hatching percentage was
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found to be 100% in the controls. However, the hatching percentage in the treated groups
120
decreased due to the increase in the concentration of chlorpyrifos (Table A). The hatching
121
percentage of Duttaphrynus melanostictus was found to be extremely low at 0.5 and 1 ppm
122
(F3,36= 28.8; p<0.0001; Fig. A). Mean body Concentration
SD (ppm)
Mean body
Hatching % length (mm) ±
SD
width (mm) ±
SD
(96 h) SE
SE
Control
100
0.00
3.213 ± 0.07
0.22
1.113 ± 0.03
0.08
0.1
96
5.16
2.803 ± 0.07
0.23
0.912 ± 0.03
0.09
0.5
83
9.49
2.55 ± 0.08
0.27
0.873 ± 0.02
0.07
1
77
6.75
2.188 ± 0.05
0.16
0.674 ± 0.03
0.09
123
Table A The hatching percentage, mean body length and mean body width of Duttaphrynus
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melanostictus embryos (Gosner stage 18) of control and treated groups at sub-lethal
125
concentrations of chlorpyrifos after 96 h of exposure. All values are given Mean ± SE.
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Fig.A. Hatching percentage of Duttaphrynus melanostictus embryos (Gosner stage 18) of control
128
and treated groups exposed to sub-lethal concentrations of chlorpyrifos after 96 h of exposure.
129
All values are given Mean ± Standard error (SE); N=4.
130
a,b,c Differ
131
d Differ
132
3.3. Body length and body width
significantly from the control groups: p<0.05, 0.01 and 0.001respectively.
significantly from 0.1 ppm treated groups: p< 0.001.
133
It was found that in the chlorpyrifos treated group there was a decrease in the mean
134
body length and mean body width of the embryo (Gosner stage 18) when being compared with
135
the control group (Table A). An increase in the concentration of chlorpyrifos in the treated
136
group, the body length decreased significantly (F3, 36= 36.3; p<0.0001; Fig. B) and a marked
137
decrease in body width too (F3, 36= 46.3; p<0.0001; Fig. C).
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Fig.B. Mean body length (mm) in Duttaphrynus melanostictus embryos (Gosner stage 18) of
140
control and treated groups exposed to sub-lethal concentrations of chlorpyrifos.
141
All values are given Mean ± Standard error (SE); N=4.
142
a,b,c Differ
143
d Differ
significantly from 0.1 ppm treated groups: p< 0.001.
144
e Differ
significantly from 0.5 ppm treated groups: p< 0.001.
significantly from the control groups: p<0.05, 0.01 and 0.001respectively.
145
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Fig.C. Mean body width (mm) in Duttaphrynus melanostictus embryos (Gosner stage 18) of
147
control and treated groups exposed to different sub-lethal concentrations of chlorpyrifos after 96
148
h.
149
All values are given Mean ± Standard error (SE); N=4.
150
a,b,c Differ
151
d Differ
significantly from 0.1 ppm treated groups: p< 0.001.
152
e Differ
significantly from 0.5 ppm treated groups: p< 0.001.
153
3.4. Scanning Electron Microscopy (SEM)
significantly from the control groups: p<0.05, 0.01 and 0.001respectively.
154
The embryo in control groups did not show any malformations in the surface
155
morphological features. Scanning electron microscopy (SEM) of Duttaphrynus melanostictus
156
embryos (Gosner stage 18) showed normal morphology in the control (Fig. D.1) but in the
157
chlorpyrifos treated groups, considerable morphological changes such as compression of the
158
embryo, curling and porosity , poked mark on the tail and reduced body length and width were
159
observed (Fig. D.2, D.3, D.4). The embryo treated with 0.1 ppm showed curling of the tail.
160
However, this was not observed at higher concentrations (0.5 and 1 ppm).
161 162
Besides these morphological changes, mucous secretion was also observed in the oral suckers of embryos exposed to higher concentration of chlorpyrifos (0.5 and 1 ppm).
163
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Fig.D.1. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
165
of the control showing normal morphology (scale bar = 500 µm).
166 167
Fig.D.2. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
168
treated with chlorpyrifos at 0.1 ppm sub-lethal concentration showing curling of tail (scale bar =
169
500 µm).
170 171
Fig.D.3. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
172
treated with chlorpyrifos at 0.5 ppm sub-lethal concentration showing reduced body size and
173
mucous secretion (scale bar = 500 µm).
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Fig.D.4. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
176
treated with chlorpyrifos at 1 ppm sub-lethal concentration showing reduced body size and
177
mucous secretion (scale bar = 500 µm).
178
4. Discussion
179
Toxicants are reported to have a detrimental and negative impact on the physiology,
180
behaviour, morphology, growth and development in amphibians (Carey and Bryant, 1995).
181
Literature survey reveals that the toxicity of various pesticides varies from species to species and
182
at different developmental stages (Berrill et al., 1998; Bridges and Semlitsch, 2000). However,
183
sufficient information is unavailable in the existing literature on the toxic effects of insecticide
184
on amphibian embryo with special reference to water bodies in the hill areas. Our experimental
185
observation reveals that the median lethal concentration (LC50) of chlorpyrifos to Duttaphrynus
186
melanostictus at the embryonic stage was higher when compared with the hatchlings and the
187
tadpole stages. This shows that at the embryonic stages, the embryos were more tolerant towards
188
the insecticide which is in concordance with the reported literature (Bonfanti et al., 2004). This
189
observation may be due to the fact that the eggs are enclosed in the jelly coat that protects them
190
from direct exposure (Berrill et al., 1998; Pauli et al., 1999). In this context, it is to be noted that
191
the developing embryo is protected by an envelope composed of proteins and glycoprotein’s 11
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which is a coat just beneath the jelly coat (Hedrick and Nishihara, 1991). The glycoprotein sugar
193
residues, act as a hydrophilic shield protecting the embryo from the lipophilic chlorpyrifos
194
(Richards and Kendall, 2002).
195
It is worthwhile observation to mention that in the early stages, chlorpyrifos was reported
196
to cause curvature in the dorsal region, accumulation of cells in the fertilization membrane and
197
change of the jelly coat consistency (Sotomayor et al., 2012). The current observation on dorsal
198
curvature caused by chlorpyrifos supports the earlier work. It may also be concluded that the
199
poor hatching percentage which is observed in the current study may be due to the alteration of
200
the consistency of the jelly coat.
201
The deformities such as dorsal curvature and smaller size of the hatchlings may result
202
from the perturbation of synthesis of collagen as reported by Snawder and Chambers (1990). It
203
may be noted here that collagen, which is a scaffold for all types of connective tissues, forms an
204
integral part in the development of anuran larvae. Organophosphorus pesticides are linked to
205
acetylcholinesterase (AChE) inhibition (Fulton and Key, 2001) which induces axial tail curvature
206
and body tremor. This is usually caused by hydrolysis impairment of acetylcholine, a
207
neurotransmitter that sustains the stimuli before the desensitisation of the receptor (Behra et al.,
208
2002; John et al., 2003; Karalliedde and Henry, 1993; Sotomayor et al., 2012). Our experimental
209
observation reveals that the length of the tail reduced when the concentrations of the pesticide
210
was increased. At higher concentrations, there was an increase in the thickness of the embryo
211
and due to this the possibility of curling through thin regions did not exist. Appearance of
212
mucous in the oral suckers of chlorpyrifos treated embryos is associated with the first mechanism
213
of defense which was also observed in the gills of R. dalmatina when exposed to chlorpyrifos
214
(Bernabo et al., 2011).
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5. Conclusion
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The study clearly reveals that the commercially available chlorpyrifos (Tricel,
217
chlorpyrifos 20 % EC) at sub-lethal doses showed morphological abnormalities in the body of
218
the embryo which can be lethal to the animal. Hence, it is recommended that the use of
219
chlorpyrifos should be restricted for the good health of the ecosystem and conservation of
220
biological entities including anurans found in different water bodies.
221
Acknowledgements
222
The authors acknowledged Sankardev College, Shillong and UGCs for the award of
223
teacher fellowship, under the Faculty Development Programme to M. Kharkongor. The authors
224
would also like to thank the Department of Zoology, North-Eastern Hill University, Shillong, for
225
providing the necessary facilities to carry out the work. Authors also gratefully acknowledge the
226
facilities and assistance provided by the Scanning Electron Microscope unit, SAIF, NEHU,
227
Shillong.
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Sotomayor, V., Lascano, C., De D’Angelo Pechen, A.M., Venturino, A., 2012. Developmental
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and polyamine metabolism alterations in Rhinella arenarum embryos exposed to the
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organophosphate chlorpyrifos. Environmental Toxicology and Chemistry 31, 2052-2058.
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Tomlin, C.D.S., 2009. The Pesticide Manual, a World Compendium, 15th ed.; British Crop
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Production Council: Alton, UK. Vertucci, F.A., Corn, P.S., 1996. Evaluation of episodic acidification and amphibian declines in the Rocky Mountains. Ecological Application 6, 449-457.
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Highlights Chlorpyrifos as serious toxicant against anuran embryo. LC50 value for Duttaphrynus melanostictus embryo after 48 h was found to be 57.525 ppm. Survival and hatching percentage of the embryo was significantly effected by variation in concentration of chlorpyrifos. SEM revealed morphological abnormalities by application of chlorpyrifos.
Mattilang Kharkongor, Rupa Nylla K. Hooroo, Sudip Dey PII:
S0045-6535(18)31361-4
DOI:
10.1016/j.chemosphere.2018.07.097
Reference:
CHEM 21812
To appear in:
Chemosphere
Received Date:
26 April 2018
Accepted Date:
17 July 2018
Please cite this article as: Mattilang Kharkongor, Rupa Nylla K. Hooroo, Sudip Dey, Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of Duttaphrynus melanostictus embryos, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.07.097
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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1
Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of
2
Duttaphrynus melanostictus embryos Mattilang Kharkongora*, Rupa Nylla K. Hoorooa and Sudip Deyb
3 4 5
a Department b Electron
of Zoology, North Eastern Hill University, Shillong-793022, Meghalaya, India.
Microscope Division, Sophisticated Analytical Instrument Facility, North Eastern Hill University, Shillong-793022, India.
6 7
*Corresponding author E-mail: [email protected]
8
a [email protected]; b [email protected]
9
Abstract
10
In an attempt to assess the effects of chlorpyrifos [O,O-diethyl O-(3,5,6-
11
trichloropyridin-2-yl) phosphorothioate], the second largest selling insecticide in India, studies
12
were made with reference to some non-target organisms. The present study was undertaken to
13
evaluate the effects in the embryos of Duttaphrynus melanostictus caused by the commercial
14
formulations of chlorpyrifos (Tricel, chlorpyrifos, 20 % EC). The LC50 value for Duttaphrynus
15
melanostictus embryos after 48 hour (h) of treatment with chlorpyrifos was found to be 57.525
16
ppm. The mortality of the embryo was significantly affected by different concentrations of
17
chlorpyrifos when compared with the control groups. An increase in concentration of
18
chlorpyrifos resulted in the simultaneous decrease of the hatching percentage and an increase in
19
the morphological abnormalities such as compression of the embryo, reduced body size and
20
curling of tail.
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Keywords: chlorpyrifos; Duttaphrynus melanostictus; embryos; LC50; SEM
22
1. Introduction
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Excessive use of pesticides has not only resulted in unusual chemical pollution but also
24
has a secondary detrimental effect on non-target organisms (Kumar et al., 2009; Matsumura,
25
1975). Pesticides are transported to the water bodies through surface run off which then enter the
26
organisms through contact and food web (Brown and Casida, 1987; Edwards, 1973; Murty,
27
1986). In recent years, organophosphates has been widely used as pesticide to help control a
28
variety of sucking, chewing and boring insects of various commercial and food crops (Kanekar
29
et al., 2004).
30
Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloropyridin-2-yl) phosphorothioate] is a broad
31
spectrum organophosphate insecticide which is used to control varieties of pests (Deb and Das,
32
2013). The insecticide acts on insect pests through direct contact, ingestion, and inhalation
33
(Tomlin, 2009). It is known to cause adverse effects not only on the target pests but also on some
34
non-target organism e.g. amphibians (Bernabo et al., 2011)
35
The effect of agricultural intensification on amphibians has drawn considerable
36
attention in recent years (Relyea et al., 2005). It was reported that species richness and
37
abundance of amphibians was lower in agricultural sites where pesticides are often used than the
38
adjacent non-agricultural sites (Bonin et al., 1997). The early life cycle of most species of the
39
amphibians occurs in ponds, temporary pools and streams near the agricultural fields receiving
40
pesticide applications. Hence, it is very likely that the eggs and tadpoles are exposed to pesticide
41
residues present in water bodies since most of the time pesticide application activity coincides
42
with the breeding and larval development (Hayes et al., 2003; Peltzer et al., 2008; Vertucci and
43
Corn, 1996).
44
In the context of the state of Meghalaya, a Northeastern state in India, 81% of the
45
population depends on agriculture especially in the rural areas and varieties of crops are grown in
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the state. The use of Chemical by farmers is one of the improved and modern agricultural
47
methods that have contributed to the increase in the production of food grains
48
(http://www.megagriculture.gov.in). It is estimated that the consumption of technical grade
49
pesticides in 2014-2015, the state of Meghalaya, consumes about 28 Metric tons of pesticides
50
(http://ppqs.gov.in/divisions/pesticides-monitoring-documentation).
51
available on the extent of impact, if any, on biological entities present in water bodies adjacent to
52
agricultural fields where pesticides are used in the state. In the present study, effects of
53
chlorpyrifos on the hatching, mortality and morphological deformities at sub-lethal
54
concentrations in Duttaphrynus melanostictus embryos has been investigated. This study may be
55
relevant from the amphibian conservation point of view.
56
2. Materials and Methods
57
2.1.
Scanty
information
is
Collection and maintenance of eggs
58
Duttaphrynus melanostictus egg strings covered in jelly coat which were used in the
59
experiment were collected from a stream at Mawpat, Shillong, Meghalaya (altitude of 1,407 m
60
ASL) from the month of February to May. The eggs were brought to the laboratory and kept in
61
plastic trays filled with pond water at room temperature (22±2 ºC).
62
2.2.
Selection and preparation of insecticide concentrations
63
Commercially available chlorpyrifos (Tricel, chlorpyrifos, 20 % EC, Excel Crop Care
64
limited, Gujarat, India) was diluted with acetone (Dimitrie and Sparling, 2014) to prepare a stock
65
solution (1 ml of chlorpyrifos in 1000 ml of acetone). Different concentrations of this stock
66
solution were added to glass bowls (150 mm diameter) containing 500 ml of dechlorinated tap
67
water (Jayawardena et al., 2011). All experiments were conducted at ambient temperature (20±2
68
ºC) and under natural light conditions. The control was prepared with different concentrations of
3
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69
acetone in 500 ml of dechlorinated tap water to make the resulting solution 0.1, 0.5, 1, 5, 10, 15,
70
20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ppm.
71
2.3. Determination of LC50 values
72
The lethal concentration, LC50 of chlorpyrifos was determined by exposing 10
73
embryos, Gosner stage 9 (Gosner, 1960) of Duttaphrynus melanostictus in each bowls containing
74
different concentrations of chlorpyrifos (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70
75
ppm) concentrations mixed with 500 ml of dechlorinated tap water for 48 h which was taken in 5
76
replicates.
77
2.4. Sub-lethal exposure
78
The embryos were also divided into 4 groups with 10 embryos in each group and in 5
79
replicates. They were then exposed to sub-lethal concentrations (0.1, 0.5 and 1 ppm) of
80
chlorpyrifos which was prepared using appropriate volume of dechlorinated tap water as it had
81
been estimated that concentration of chlorpyrifos in small water bodies were in the range of 0.1
82
ppm to 1 ppm (Mazanti et al., 2003; Moore et al., 2002). The embryos were maintained till they
83
hatched and the hatching percentage and mortality were recorded. The hatched embryos were
84
then processed for scanning electron microscopy to observe any change in the body morphology.
85
The mean body length and mean body width of control and treated embryos were also recorded
86
by using a dial calliper (Mitutoyo series No. 505-671).
87
2.5.
Scanning Electron Microscopy
88
In order to carry the scanning electron microscopic study, the embryos, Gosner stage
89
18 were fixed in 2.5 % gluteraldehyde in 0.1 M sodium Cacodylate buffer (pH 7.2) for 4 h at 4ºC
90
and then washed in sodium Cacodylate buffer three times for 15 minutes each. The specimens
91
were then post-fixed in 1% osmium tetraoxide in the same buffer at 4ºC. The samples were
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dehydrated in increasing concentration of acetone with two changes of 15 to 30 minutes in each
93
grade. The dehydrated samples were then dried with TMS (Tetra Methyl Silane) drying
94
technique of Dey et.al. (1989). The samples were secured to brass stubs (10 mm diameter X 30
95
mm height) with the use of double adhesive tape. Coating of the sample with gold was carried
96
out by using JFC 1100 (Jeol) ion sputter in a relatively low vacuum for ionization of the air
97
particles followed by the application of high voltage. Gold coating prevents sample from
98
damaging due to radiation and also increases conductivity. Observations had been done with
99
scanning electron microscopy JSM 6360 (Jeol) in the secondary electron emission mode at an
100
accelerating voltage of 20 KV with the working distance of 12 mm.
101
2.6.
Statistical analysis
102
LC50 values were determined using Finney’s Probit Analysis (1971). The LC50
103
determination method makes use of the SPSS computer software (Finney, 1971). All data are
104
represented as mean ± SEM and then subjected to one way ANOVA followed by Bonferroni's
105
Multiple Comparison Test. The statistical analysis was performed using Graph Pad Prism
106
version
107
www.graphpad.com.
108
3. Results
109
3.1. LC50 value
4.00
for
windows,
Graph
pad
Software,
San
Diego
California,
USA,
110
According to Finney’s Probit Analysis, the LC50 value of chlorpyrifos for Duttaphrynus
111
melanostictus embryos after 48 h of exposure was found to be 57.525 ppm which represents the
112
relation between chlorpyrifos concentration and mortality rate. The availability of chlorpyrifos
113
to embryos was synchronised with the concentration of the pesticide applied and the LC50 value
5
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114
was also an inclusion of other ingredients. No mortality was observed in the controls and sub-
115
lethal concentrations (0.1, 0.5 and 1 ppm) of chlorpyrifos treated groups.
116
3.2. Hatching
117
The embryos of Duttaphrynus melanostictus hatched at Gosner stage 18 after 96 h. The
118
hatching was observed in both the control and all treated groups. The hatching percentage was
119
found to be 100% in the controls. However, the hatching percentage in the treated groups
120
decreased due to the increase in the concentration of chlorpyrifos (Table A). The hatching
121
percentage of Duttaphrynus melanostictus was found to be extremely low at 0.5 and 1 ppm
122
(F3,36= 28.8; p<0.0001; Fig. A). Mean body Concentration
SD (ppm)
Mean body
Hatching % length (mm) ±
SD
width (mm) ±
SD
(96 h) SE
SE
Control
100
0.00
3.213 ± 0.07
0.22
1.113 ± 0.03
0.08
0.1
96
5.16
2.803 ± 0.07
0.23
0.912 ± 0.03
0.09
0.5
83
9.49
2.55 ± 0.08
0.27
0.873 ± 0.02
0.07
1
77
6.75
2.188 ± 0.05
0.16
0.674 ± 0.03
0.09
123
Table A The hatching percentage, mean body length and mean body width of Duttaphrynus
124
melanostictus embryos (Gosner stage 18) of control and treated groups at sub-lethal
125
concentrations of chlorpyrifos after 96 h of exposure. All values are given Mean ± SE.
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126 127
Fig.A. Hatching percentage of Duttaphrynus melanostictus embryos (Gosner stage 18) of control
128
and treated groups exposed to sub-lethal concentrations of chlorpyrifos after 96 h of exposure.
129
All values are given Mean ± Standard error (SE); N=4.
130
a,b,c Differ
131
d Differ
132
3.3. Body length and body width
significantly from the control groups: p<0.05, 0.01 and 0.001respectively.
significantly from 0.1 ppm treated groups: p< 0.001.
133
It was found that in the chlorpyrifos treated group there was a decrease in the mean
134
body length and mean body width of the embryo (Gosner stage 18) when being compared with
135
the control group (Table A). An increase in the concentration of chlorpyrifos in the treated
136
group, the body length decreased significantly (F3, 36= 36.3; p<0.0001; Fig. B) and a marked
137
decrease in body width too (F3, 36= 46.3; p<0.0001; Fig. C).
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138 139
Fig.B. Mean body length (mm) in Duttaphrynus melanostictus embryos (Gosner stage 18) of
140
control and treated groups exposed to sub-lethal concentrations of chlorpyrifos.
141
All values are given Mean ± Standard error (SE); N=4.
142
a,b,c Differ
143
d Differ
significantly from 0.1 ppm treated groups: p< 0.001.
144
e Differ
significantly from 0.5 ppm treated groups: p< 0.001.
significantly from the control groups: p<0.05, 0.01 and 0.001respectively.
145
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146
Fig.C. Mean body width (mm) in Duttaphrynus melanostictus embryos (Gosner stage 18) of
147
control and treated groups exposed to different sub-lethal concentrations of chlorpyrifos after 96
148
h.
149
All values are given Mean ± Standard error (SE); N=4.
150
a,b,c Differ
151
d Differ
significantly from 0.1 ppm treated groups: p< 0.001.
152
e Differ
significantly from 0.5 ppm treated groups: p< 0.001.
153
3.4. Scanning Electron Microscopy (SEM)
significantly from the control groups: p<0.05, 0.01 and 0.001respectively.
154
The embryo in control groups did not show any malformations in the surface
155
morphological features. Scanning electron microscopy (SEM) of Duttaphrynus melanostictus
156
embryos (Gosner stage 18) showed normal morphology in the control (Fig. D.1) but in the
157
chlorpyrifos treated groups, considerable morphological changes such as compression of the
158
embryo, curling and porosity , poked mark on the tail and reduced body length and width were
159
observed (Fig. D.2, D.3, D.4). The embryo treated with 0.1 ppm showed curling of the tail.
160
However, this was not observed at higher concentrations (0.5 and 1 ppm).
161 162
Besides these morphological changes, mucous secretion was also observed in the oral suckers of embryos exposed to higher concentration of chlorpyrifos (0.5 and 1 ppm).
163
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Fig.D.1. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
165
of the control showing normal morphology (scale bar = 500 µm).
166 167
Fig.D.2. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
168
treated with chlorpyrifos at 0.1 ppm sub-lethal concentration showing curling of tail (scale bar =
169
500 µm).
170 171
Fig.D.3. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
172
treated with chlorpyrifos at 0.5 ppm sub-lethal concentration showing reduced body size and
173
mucous secretion (scale bar = 500 µm).
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174 175
Fig.D.4. Scanning electron micrograph of Duttaphrynus melanostictus embryo (Gosner stage 18)
176
treated with chlorpyrifos at 1 ppm sub-lethal concentration showing reduced body size and
177
mucous secretion (scale bar = 500 µm).
178
4. Discussion
179
Toxicants are reported to have a detrimental and negative impact on the physiology,
180
behaviour, morphology, growth and development in amphibians (Carey and Bryant, 1995).
181
Literature survey reveals that the toxicity of various pesticides varies from species to species and
182
at different developmental stages (Berrill et al., 1998; Bridges and Semlitsch, 2000). However,
183
sufficient information is unavailable in the existing literature on the toxic effects of insecticide
184
on amphibian embryo with special reference to water bodies in the hill areas. Our experimental
185
observation reveals that the median lethal concentration (LC50) of chlorpyrifos to Duttaphrynus
186
melanostictus at the embryonic stage was higher when compared with the hatchlings and the
187
tadpole stages. This shows that at the embryonic stages, the embryos were more tolerant towards
188
the insecticide which is in concordance with the reported literature (Bonfanti et al., 2004). This
189
observation may be due to the fact that the eggs are enclosed in the jelly coat that protects them
190
from direct exposure (Berrill et al., 1998; Pauli et al., 1999). In this context, it is to be noted that
191
the developing embryo is protected by an envelope composed of proteins and glycoprotein’s 11
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192
which is a coat just beneath the jelly coat (Hedrick and Nishihara, 1991). The glycoprotein sugar
193
residues, act as a hydrophilic shield protecting the embryo from the lipophilic chlorpyrifos
194
(Richards and Kendall, 2002).
195
It is worthwhile observation to mention that in the early stages, chlorpyrifos was reported
196
to cause curvature in the dorsal region, accumulation of cells in the fertilization membrane and
197
change of the jelly coat consistency (Sotomayor et al., 2012). The current observation on dorsal
198
curvature caused by chlorpyrifos supports the earlier work. It may also be concluded that the
199
poor hatching percentage which is observed in the current study may be due to the alteration of
200
the consistency of the jelly coat.
201
The deformities such as dorsal curvature and smaller size of the hatchlings may result
202
from the perturbation of synthesis of collagen as reported by Snawder and Chambers (1990). It
203
may be noted here that collagen, which is a scaffold for all types of connective tissues, forms an
204
integral part in the development of anuran larvae. Organophosphorus pesticides are linked to
205
acetylcholinesterase (AChE) inhibition (Fulton and Key, 2001) which induces axial tail curvature
206
and body tremor. This is usually caused by hydrolysis impairment of acetylcholine, a
207
neurotransmitter that sustains the stimuli before the desensitisation of the receptor (Behra et al.,
208
2002; John et al., 2003; Karalliedde and Henry, 1993; Sotomayor et al., 2012). Our experimental
209
observation reveals that the length of the tail reduced when the concentrations of the pesticide
210
was increased. At higher concentrations, there was an increase in the thickness of the embryo
211
and due to this the possibility of curling through thin regions did not exist. Appearance of
212
mucous in the oral suckers of chlorpyrifos treated embryos is associated with the first mechanism
213
of defense which was also observed in the gills of R. dalmatina when exposed to chlorpyrifos
214
(Bernabo et al., 2011).
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215
5. Conclusion
216
The study clearly reveals that the commercially available chlorpyrifos (Tricel,
217
chlorpyrifos 20 % EC) at sub-lethal doses showed morphological abnormalities in the body of
218
the embryo which can be lethal to the animal. Hence, it is recommended that the use of
219
chlorpyrifos should be restricted for the good health of the ecosystem and conservation of
220
biological entities including anurans found in different water bodies.
221
Acknowledgements
222
The authors acknowledged Sankardev College, Shillong and UGCs for the award of
223
teacher fellowship, under the Faculty Development Programme to M. Kharkongor. The authors
224
would also like to thank the Department of Zoology, North-Eastern Hill University, Shillong, for
225
providing the necessary facilities to carry out the work. Authors also gratefully acknowledge the
226
facilities and assistance provided by the Scanning Electron Microscope unit, SAIF, NEHU,
227
Shillong.
228
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Highlights Chlorpyrifos as serious toxicant against anuran embryo. LC50 value for Duttaphrynus melanostictus embryo after 48 h was found to be 57.525 ppm. Survival and hatching percentage of the embryo was significantly effected by variation in concentration of chlorpyrifos. SEM revealed morphological abnormalities by application of chlorpyrifos.