J Bangladesh Agril Univ 16(3): 411–416, 2018 https://doi.org/10.3329/jbau.v16i3.

39410

ISSN 1810-3030 (Print) 2408-8684 (Online)

Journal of Bangladesh Agricultural University
Journal home page: http://baures.bau.edu.bd/jbau, www.banglajol.info/index.php/JBAU

Determination of toxicity of spinosad against the pulse beetle, Callosobruchus

chinensis L.
Pronita Mondal, Mohammad Mahir Uddin and Mohammad Tofazzal Hossain Howlader
Department of Entomology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

ARTICLE INFO Abstract

Article history: Pulses serve as one of the main sources of protein and minerals as well as play a vital socio-economic role
Received: 12 September 2018 in the diet of common people of Bangladesh but these pulses suffer enormous losses due to infestation of a
Accepted: 07 December 2018 serious insect pest pulse beetle, Callosobruchus chinensis L. As a part of eco-friendly management of this
Published: 31 December 2018 detrimental pest, an experiment was conducted at the laboratory to determine the toxicity of spinosad
against the pulse beetle, C. chinensis L. directly by using dry film contact toxicity method and indirectly
Keywords: by treating chickpea seeds. In the direct method spinosad showed contact toxicity and the lethal
Spinosad; toxicity; chickpea; concentration of spinosad was 117.46, 76.05 and 37.45 ppm at 24, 48 and 72 hours after treatment,
dry film; Callosobruchus respectively. On the other hand, the LC50 values were 990.34, 301.35 and 95.08 ppm after 24, 48 and 72
chinensis HAT, respectively as per indirect method. A dose-dependent relation with the mortality was observed by
Correspondence: both methods. Spinosad was also significantly effective in reducing the number of eggs (68.00 to 98.60%
Mohammad Mahir Uddin inhibition over control), adult emergence (56.70 to 100% inhibition over control), percent seed infestation
: [email protected] (62.79 to 100% inhibition over control) and seed weight loss (50.81 to 100% inhibition over control). The
present results indicated that spinosad could be suggested for the efficient management of C. chinensis in
storage.
Copyright:

©2018 by authors and BAURES. This work is licensed under the Creative Commons Attribution International License (CC By 4.0).

Introduction discouraged globally due to many problems such as

Pulse is an important source of protein (approximately resistance, residue problems. A tendency to use safe and
21-25%) (Tiwari and Shing, 2012). It also provides low toxicity insecticides has been increased (Odeyemi et
carbohydrates (50-60%) and several vitamins and al., 2006; Sadeghi et al., 2006).
minerals (Chakraborty and Mondal, 2015). A large
number of pulses are grown in Bangladesh such as lentil, In recent years, spinosad is a safe biopesticide derived
gram, black gram, grasspea, mungbean, cowpea, etc. and from naturally occurring soil actinomycete,
the total production of pulse was 2,03,535 metric tons Saccharopolyspora spinosa Mertz and Yao (Bacteria:
from the area of 5,57,508 acres in 2007–2008 (BBS, Actinobacteridae) (Sparks et al., 1998). Spinosad for its
2009). After harvesting, about 85% of the pulse growers limited non-target and chronic effects, in 1993, spinosad
in Bangladesh store pulses throughout the year in their was labeled as a "reduced risk" compound by the EPA
houses (Ferdowsi, 2013). and in 2005, the EPA registered spinosad at 1 mg/kg
active substance (a.s.) as a grain protectant on
Unfortunately, in storage, pulses suffer enormous losses commodities including wheat, corn, rice, millets, oats,
due to bruchid attack, which starts infestation either in sorghum and barley (Huang and Subramanyam, 2007). It
the field on the maturing pod and is carried to the stores is toxic to insects by ingestion or contact and it acts on
with the harvested crops or it originates in the storage an insect’s nervous system at the nicotinic acetylcholine
itself (Fletcher and Ghosh, 2002). Among the different and gamma-aminobutyric acid (GABA) receptor sites
species of pulse beetle, Callosobruchus chinensis L. is (Salgado and Sparks, 2005; Osorio et al., 2008). The test
the most destructive in Bangladesh which cause losses insect dies within 1 to 2 days after ingesting the active
up to 30% in a short period of time (Raja and John, ingredient.
2008). The post-harvest seed losses due to the beetle can
reach even up to 100% during severe infestation It has been found to be effective against stored pests
(Srinivasan et al., 2010). (Hertlein et al., 2011). It is predicted that the Spinosad
will be used more widely in many countries for the
During storage for grain protection, liquid insecticides management of storage pests (Vayias et al., 2009). In
and gases in the form of phosphine and methyl bromide Bangladesh, spinosad has already been registered for use
have been widely used and Bangladeshi farmers are also on cotton, jute, rice against Lepidopteran pests.
using this synthetic chemical insecticides and fumigants Information on effects of spinosad against the pulse
(Matin, 2003). However, their application is being beetle is very limited in Bangladesh. So, considering the
Cite this article
Mondal P., Uddin M.M. and Howlader M.T.H. 2018. Determination of toxicity of spinosad against the pulse beetle, Callosobruchus chinensis L.
Journal of Bangladesh Agricultural University, 16(3): 411–416.
Biotoxicity of spinosad against the pulse beetle
above situations, the present research was conducted to For direct toxicity assay of spinosad dry film contact
determine the toxicity of spinosad against the pulse method described by Sadat and Asghar (2006) was used
beetle, C. chinensis under laboratory condition. to determine the toxicity of spinosad to adults of pulse
beetle, C. chinensis under laboratory conditions.
Materials and Methods Bioassays were performed on filter paper placed inside
The experiment was conducted at the laboratory of Petri-dishes (90 mm x 14 mm, diameter x height). The
Department of Entomology, Bangladesh Agricultural required dilutions were prepared from the commercial
University (BAU), Mymensingh during March 2015 to formulation of spinosad with distilled water described
May 2016. The experiment was laid out in Completely earlier. For control treatment, filter paper was soaked
Randomized Design (CRD) having four treatments. only in distilled water. Seven concentrations including
Fresh and disease free chickpea (Cicer arietinum L.) one control were used for the bioassay test. Initially,
seeds were used as host for the pulse beetles and filter paper disks on both sides of the Petri-dishes fitted
purchased from the local ‘K & R Market’ of BAU with yellow tape and then they were treated with 2 ml
campus, Mymensingh. Pulse beetle, C. chinensis L. was each of different concentrations of spinosad solutions
used as test insect in this study. The insects were along with control placed in the bottom and the lid of
collected from a stock culture of the Entomology 90-mm Petri-dishes and allowed to dry. After drying of
Department, BAU as well as from Entomology Division the Petri-dishes, 15 newly emerged adults of C.
of Bangladesh Institute of Nuclear Agriculture (BINA) chinensis were separated from cultures and introduced
and then reared in the laboratory as per the following into each dish of different concentrations and control.
protocol. The average temperature and relative humidity The experiment was replicated for four times.
during the study were 27±3°C and 72±3%, respectively. Insect mortality data were recorded at 24, 48 and 72 hrs
Pulse beetle was mass reared in glass jar and in plastic of post release. Insects that were immobile or unable to
box also. Approximately 100 adults of the insect were move were considered as dead insect. The percent
released in each box containing 500 g of chickpea seeds mortality was determined as per following formula:
and the mouth being closed with fine mesh net for
proper ventilation. The beetles were allowed for free No. of dead pulse beetle
Percent mortality =  100
mating followed by oviposition for a maximum period Total no.of released pulse beetle
of 10 days. After oviposition, the beetles were separated
from the seeds through sieving and seeds along with To determine corrected mortality Abbott’s formula was
eggs were left in the containers to maintain a stock used and LC50 values were calculated by probit analysis.
culture of the test insect. The stock culture of test insect p - C
was maintained throughout the experimental period and Abbott’s formula, P =  100
100 - C
this process was repeated regularly to get sufficient
number of adult beetles for conducting the experiments. Where,
In this experiment, the toxicity of spinosad was P = the corrected mortality (%)
evaluated through bioassay procedure. A serial dilution P’ = the observed mortality (%)
of the formulated Spinosad was prepared and toxicity C = the control mortality
was determined against C. chinensis. Different Pexp - Pcont
concentrations of Spinosad were considered as Abbott’s formula, Pcorr =
1 - Pcont
experimental treatment. To prepare them, at first 100 ml
Where,
10,000 ppm of spinosad was prepared as first stock
Pcorr = Mean experimental treatment response corrected
solution by adding 2.22 ml spinosad with 97.78 ml
for control response
distilled water. Then, 100 ml 1000 ppm of spinosad as
Pexp = Mean experimental treatment response
second stock solution (10.00 ml from 1st stock
Pcont = Mean control response
solution+90.00 ml dH2O) was prepared. Then the
experimental treatments were prepared by taking
Toxicity of spinosad was also tested indirectly against
required amount from second stock solution for each
the pulse beetle, C. chinensis indirectly by treating the
experiment as shown below.
pulses and thereafter evaluating toxicity against the
Table 1. Preparation of Spinosad serial dilutions insect. In order to do that five concentrations and a
control with three replications were tested. The five
Treatments Concentration Amount of spinosad Amount of
(in ppm*) (ml/10ml in distilled distilled
serial diluted concentrations (50, 100, 200, 500 and 1000
water) water ppm) of spinosad were prepared following same
T1 0 0 10 protocols mentioned earlier. To assess the toxicity, 50 g
T2 30 0.3 9.7 of chickpea seeds was taken in each Petri-dish and
T3 50 0.5 9.5 treated with one milliliter of prepared diluted
T4 100 1 9.0 concentration of spinosad for each treatment and mixed
T5 150 1.5 8.5
T6 200 2 8.0
thoroughly by shaking. The treated seeds were then kept
T7 500 5 5.5 for few minutes for drying. Five pairs of newly emerged
(*ppm= parts per million)

412
 Mondal et al.
adults of C. chinensis were released in each Petri-dish for 30, 50, 100, 150, 200 and 500 ppm of spinosad,
and kept in room temperature for recording of data. respectively. At 72 HAT, 100.00% insects were died at
Mortality data were recorded at 24, 48 and 72 hrs of post 500 ppm concentration whereas at 30, 50, 100, 150, 200
release from each Petri-dish. The percent mortality was ppm, the percent corrected mortality was 42.50, 57.50,
determined as per the formula mentioned earlier. 77.50, 82.50 and 82.50, respectively at the same time
Number of eggs laid by the released beetles, termed as (Fig. 1). It was observed that higher percent mortality
rate of oviposition was recorded after 10 days of post occurred at higher concentration of spinosad and vice-
release. The emerged C. chinensis adults were counted versa, i.e., in a dose-dependent relation. Spinosad may
daily from the beginning of the first insect emergence cause 100% mortality at 24 HAT (Vishwamithra et al.,
started at 20 days after post insect release and continued 2014). Similar results were also reported by
up to 3 weeks. The data was recorded at 25 and 45 days Duraimurugan et al. (2014) and Sadat and Asghar
after insect release (DAIR). Then the percent seed (2006). The present findings are supported by these
infestation was calculated according to the following results.
formula stated by Enobakhare and Law-Ogbomo (2002).
Number of bored seeds
Percent grain damage =  100
Total no. of seeds observed
Data recorded at 45 DAIR and then the percent weight
loss was measured using the following formula as stated
by Lal (1988).
Percent weight loss =
Weight loss seed grains per Petri - dish
 100
Initial weight of seed grains per Petri - dish
Weight loss per Petri-dish = (Initial weight- final
weight) of grains per Petri-dish. Fig. 1. Toxicity of spinosad against pulse beetle, C. chinensis
determined by direct film method
The inhibition of oviposition rate, adult emergence, seed
infestation and seed weight loss was computed by using
The LC50 values of spinosad to C. chinensis were
the following formula as stated by Shukla et al. (2007).
117.46, 76.05 and 37.45 ppm after 24, 48 and 72 HAT,
Control mean - Treatent mean respectively. It was indicated that lower the time higher
Percent inhibition =  100
Control mean amount of spinosad was required and vice-versa. Other
lethal concentrations values followed the same trend
The mortality data were corrected by using Abott’s (Table 2). Duraimurugan et al. (2014) observed that
formula (Abbott, 1925) and the LC50 values were LC50 values of spinosad at 24, 48 and 72 hours post-
calculated by probit analysis (Finney, 1971). All other treatment were 51.05, 11.99 and 1.92 ppm, respectively
collected data were analyzed in Completely Randomized against C. chinensis. Similar toxic effect of spinosad was
Design (CRD) by using Analysis of Variance also reported by various authors (Abd El-Razik and
(ANOVA). The treatment mean values of different Zayed, 2014; Abouelghar et al., 2013; Hameed et al.,
parameters were separated by using the Duncan's 2012; Sadat and Asghar, 2006 and Hussain et al., 2005).
Multiple Range Test (DMRT) (Duncan, 1951) and Least The findings of the present research work are almost
Significant Difference (LSD). Data analysis was carried similar with the previous reports.
out using the computer package MSTAT-C and
graphical works through Microsoft Excel program in a Indirect toxicity of spinosad against pulse beetle, C.
computer. chinensis
Similar to direct film method, it was found that mortality
Results and Discussion of pulse beetle increased proportionately with the
Direct toxicity of spinosad against pulse beetle, C. increase of spinosad and exposure time. The percent
chinensis corrected mortality of C. chinensis was 3.85, 23.08,
The direct toxicity of spinosad against the adults of C. 30.77, 38.46 and 46.15 at 24 HAT; and at 48 HAT, it
chinensis at different hours after treatment (HAT) using was 29.17, 33.33, 41.67, 58.33 and 66.67 for 50, 100,
seven different concentrations viz., 0, 30, 50, 100, 150, 200, 500 and 1000 ppm of spinosad, respectively. At 72
200 and 500 ppm was tested and results showed that the HAT the highest mortality (88.89%) was found at 1000
mortality rates of C. chinensis increased proportionately ppm concentration (Fig. 2). The LC50 values of spinosad
with the increase of spinosad concentration and exposure to C. chinensis were 990.34, 301.35 and 95.08 ppm after
time. The percent corrected mortality of C. chinensis 24, 48 and 72 HAT, respectively (Table 3). Comparing
with different concentrations of spinosad was 28.30, to direct method, it was found that higher amount of
43.40, 49.06, 52.83, 58.49 and 66.04 at 24 HAT and spinosad were required when applied indirectly might be
34.04, 38.30, 59.58, 63.83, 65.96 and 78.72 at 48 HAT due to repellent effect.
413
Biotoxicity of spinosad against the pulse beetle
The present study revealed the reduction of insect exposure even at 0.3 g/kg and did not exceed 20% and
population was due to the use of spinosad and also mortality increased with the increase of exposure
agreed with the previous findings of Khashaveh et al. interval and doses. Rajput et al. (2013), Mirmoayedi et
(2011) where they stated that the mortality of exposed al. (2011) and Hussain et al. (2009) reported similar
individuals in treated commodities was low at 1-day kind of observations.
Table 2. Toxicity of spinosad against pulse beetle, C. chinensis at 24, 48, 72 HAT by direct film method
Time No. of insects Lethal Concentration (ppm)
(hr) tested Slope±SE χ2 (Fiducial Limit)
LC25 LC50 LC90
24 60 0.75±0.17 1.26 14.80 117.46 6016.08
(2.59-30.35) (75.23-185.87) (1688.03-156163.37)
48 60 1.04±0.18 1.15 17.02 76.05 1307.30
(6.23-29.03) (51.54-102.51) (663.88-4851.62)
72 60 1.47±0.26 1.18 13.05 37.45 277.73
(4.87-21.54) (23.19-49.73) (187.60-583.87)

Fig. 2. Toxicity of spinosad against pulse beetle, C. chinensis determined

by indirect application method

Table 3. Toxicity of spinosad against pulse beetle, C. chinensis at 24, 48, 72 HAT by indirect method
Time No. of insects Lethal Concentration (ppm)
(hr) tested Slope±SE χ2 (Fiducial Limit)
LC25 LC50 LC90
24 60 0.96±0.25 2.94 195.45 990.34 21620.69
(90.12-337.18) (525.89-4679.24) (4609.20-2256880.84)
48 60 0.80±0.23 0.25 42.73 301.35 12331.18
(4.20-92.95) (161.19-739.23) (2695.42-2325034.11)
72 60 0.96±0.24 1.67 18.76 95.08 2076.01
(1.80-45.08) (36.19-159.69) (854.59-22444.10)

Effect of spinosad on oviposition, adult emergence, 1000 ppm of spinosad. The number of eggs at 100, 200
seed infestation (%), seed weight loss (%) and and 500 ppm concentration were 26.33, 17.00, and 8.33,
inhibition rate (%) of C. chinensis respectively (Table 4). The inhibition of oviposition rate
was increased from 68 to 98.60%. The highest inhibition
Effect on oviposition rate of C. chinensis on oviposition rate of C. chinensis was recorded when
The mean number of eggs laid by C. chinensis on seeds were treated with 1000 ppm spinosad (Table 4).
chickpea seeds in different treatments ranged from 2.33 The findings on the oviposition of the present study is
to 166.67 per 250 seeds and differed significantly similar with the report of Vishwamithra et al. (2014)
(p≤0.01). Among the treatments, the highest number of who found 28.67 eggs per 30g seed where they treated
eggs (166.67) was laid in control and the lowest number the seeds with spinosad 45SC @ 4ppm/kg seed. These
of eggs (2.33) was deposited on the seeds treated with findings derive support from who found.

414
 Mondal et al.
Table 4. Toxic effect of spinosad on oviposition, adult emergence, seed infestation (%), seed weight loss (%)
and inhibition rate (%) of C. chinensis
Dose (ppm) Number of Inhibition Number of adults Inhibition Seed Seed Inhibition Seed weight Inhibition
eggs/ 250 rate emerged/250 rate infestation (%) infestation (%) rate (%) loss (%) at rate
seeds (%) seeds (%) at 25 DAIR* at 45 DAIR 45 DAIR (%)
Control 166.67a - 87.00a - 9.33 a 57.33a - 4.33a -
50 53.33b 68.00 37.67b 56.70 0.40 b 21.33b 62.79 2.13b 50.81
100 26.33c 84.20 16.67c 80.84 0.23 b 10.27c 82.09 0.90c 79.21
200 17.00d 89.80 8.67d 90.04 0.10 b 4.93d 91.40 0.30d 93.07
500 8.33e 95.00 2.67e 96.93 0.0 b 1.17e 97.96 0.17de 96.07
1000 2.33f 98.60 0.00e 100 0.0 b 0.00 d 100 0.00e 100
LSD value 4.953 - 5.125 - 0.6504 3.018 - 0.1764 -
Level of
0.01 - 0.01 - 0.01 0.01 - 0.01 -
Significance
CV (%) 8.08 - 4.35 - 15.52 7.64 - 5.42 -

*DAIR= Days after insect release, Different letters in a column differ significantly, p≤0.01

Effect on adult emergence of C. chinensis chickpea seeds treated with spinosad after 45 DAIR. The
The mean number of adults of C. chinensis emerged weight loss of seed in different treatments including the
from the chickpea seeds ranged from 0.0 to 87.00 which control ranged from 0.00 to 4.33%. The highest seed
differed significantly (p≤0.01). Significantly the highest weight loss was recorded in control (4.33%) and no seed
number of adult was emerged from control (87.00) and weight loss was recorded when seeds were treated with
no adult was emerged from the chickpea seeds treated 1000 ppm of spinosad (0.00) which was statistically
with 1000 ppm of spinosad which was statistically almost similar to 500 ppm (0.17) of spinosad. The
similar to 500 ppm spinosad (2.67). When the inhibition inhibition rate followed the similar trend as observed for
rate of adult emergence over control was compared, it oviposition and adult emergence. The result is as well
showed the same trend as observed for oviposition rate concurring with the studies made by Subramanyam et al.
(Table 4) which coincided with Vishwamithra et al., (2003), Sadat and Asghar (2006), Vayias et al. (2009)
(2014) study who found 8.55% adult emergence in seeds and Hertlein et al. (2011) who reported the toxicity of
treated with Spinosad (4ppm/kg seed) and also parallel spinosad to coleopteran stored-grain pests.
with Adel khashaveh et al. (2011) where they reported
that the application of spinosad significantly reduced Conclusion
progeny production. In case of direct toxicity assay through dry film contact
method it was found that spinosad possessed significant
Effect of spinosad on seed infestation (%) contact toxicity against C. chinensis. The corrected
The effect of spinosad on seed damage due to feeding by mortality rate increased proportionately with the
C. chinensis is presented in Table 4. At 25 DAIR increase of spinosad concentration and exposure time.
infestation was negligible in treated seeds but in Comparing to direct method, these values were higher in
untreated seeds 9.33% infestation was recorded. indirect method might be due to the repellent effect. The
Statistically similar results were observed from the results revealed that the biopesticide was effective in
treatments. reducing the number of eggs, number of adults emerged,
seed infestation and seed weight loss over control. From
On the other hand, with the increase of time seed the results of the present investigation, it could be
damage increased and it differed significantly (p≤0.01) concluded that toxicity of spinosad was increased
at 45 DAIR among the treatments and the rate varied significantly with the increasing doses and duration of
from 0.00 to 57.33%. At 45 DAIR, the highest seed exposure. Therefore, spinosad might be an alternative,
infestation was recorded in control (57.33%). No seed safe and eco-friendly tactic for the management of pulse
infestation was recorded when seeds were treated with beetle in the storage.
1000 ppm of spinosad resulting in maximum inhibition
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