Bull Environ Contam Toxicol (2014) 92:190195
DOI 10.1007/s00128-013-1155-8
Assessment of Toxicity and Potential Risk of Butene-fipronil Using
Drosophila melanogaster, in Comparison to Nine Conventional
Insecticides
Muhammad Shahid Arain Xing-Xing Hu
Guo-Qing Li
Received: 20 June 2013 / Accepted: 6 November 2013 / Published online: 14 November 2013
Springer Science+Business Media New York 2013
Abstract The toxicities of butene-fipronil to Drosophila
melanogaster were evaluated, in comparison to nine con-
ventional insecticides. According to the mean LD50 values of
the larvae from CS, w1118 and Oregan strains, butene-fipro-
nil, abamectin, spinosad and chlorpyrifos exhibited high
levels of toxicity. Imidacloprid, a-cypermethrin and clothi-
anidin showed middle levels of toxicity. And acephate,
methomyl and acetamiprid had low levels of toxicity. To the
adults, butene-fipronil, a-cypermethrin, spinosad, and
chlorpyrifos were relatively more toxic, whereas acephate,
methomyl, acetamiprid, imidacloprid, clothianidin and
abamectin were relatively less toxic. Butene-fipronil and
abamectin were more toxic to larvae. In contrast, spinosad,
a-cypermethrin, and clothianidin were more toxic to adults.
Moreover, the toxic risk assessment using a risk quotient
value revealed that butene-fipronil was safe to D. melano-
gaster adults. Thus, our results suggested that butene-fipronil
is harmful to D. melanogaster larvae, but it is relatively safe
to the adults.
Keywords Butene-fipronil
Drosophila
Toxicity

Risk assessment
Insects are major biotic factors that cause losses in agricul-
tural production. Continuous development of new insecti-
cides with selective properties and specific action is
necessary for their control (Hainzl and Casida 1996).
Butene-fipronil is a novel compound, and has lower toxicity
M. S. Arain
X.-X. Hu
G.-Q. Li (&)
Education Ministry Key Laboratory of Integrated Management
of Crop Diseases and Pests, College of Plant Protection, Nanjing
Agricultural University, Nanjing 210095, China
e-mail: ligq@njau.edu.cn
123
to fish than other phenylpyrazole chemicals such as fipronil
(Niu et al. 2008). It is an excellent insecticide for the effec-
tive control of many pest species in Lepidoptera, Hemiptera,
Coleoptera and Orthopteran (Niu et al. 2008; Liu et al. 2009;
Yuan et al. 2009). Moreover, butene-fipronil is safe to par-
asitoid Trichogramma confusum Viggiani (Wang et al. 2013)
and non-target insect Bombyx mori (Yu et al. 2012), but not
to other two parasitoids T. nubilale (Wang et al. 2012a) and
T. ostriniae (Pang and Chen) (Wang et al. 2012b). However,
the toxicity of butene-fipronil to dipterans remains
undetermined.
Since Drosophila melanogaster is easy to care for,
breeds quickly, and lays many eggs, it is an excellent
model system to assess toxicities of insecticides (Scharf
et al. 2006; Karatas and Bahceci 2009), chemical products
and contamination substances (Shayela et al. 1988;
Yes ilada 1999; Ouchi et al. 2007; Babczynska and Migula
2002; Goldstein and Babich 1989; Mutlu 2012), and the
products from other organisms (Uysal and Kaya 2004;
Altun et al. 2011). Because of the inadequate empirical
information in the literature, the toxicities of butene-fi-
pronil to the larvae and the adults of D. melanogaster were
evaluated here, in comparison to nine conventional insec-
ticides. Since D. melanogaster is an important non-target
arthropod in environment and the adults are more likely to
be exposed to sprayed butene-fipronil during foraging and/
or oviposition, the potential risk of butene-fipronil to the
adults was also estimated.
Materials and Methods
In the tests we carried out, D. melanogaster Canton-S (CS),
w1118 and Oregan flies were used. The flies were raised on
standard cornmeal/molasses/agar medium under controlled
Bull Environ Contam Toxicol (2014) 92:190195
temperature (25 1C), photoperiod (12 h light/12 h
dark) and relative humidity (about 50 %).
In the experiment, technical grade butene-fipronil
(90.0 % pure, field recommended rate (FR) 1530 g AI
ha-1), abamectin (93 % B1a pure, FR 21.632.4 g AI
ha-1), acephate (96.5 % pure, FR 5401,080 g AI ha-1),
chlorpyrifos (97.4 % pure, FR 450600 g AI ha-1),
methomyl (95.0 % pure, FR 90180 g AI ha-1), a-cyper-
methrin (94.3 % pure, FR 4590 g AI ha-1), imidacloprid
(96.4 % pure, FR 1530 g AI ha-1), acetamiprid (96.2 %
pure, FR 1822.5 g AI ha-1), clothianidin (96.4 % pure,
FR 1530 g AI ha-1), and spinosad (94.2 % pure, FR
1530 g AI ha-1) were obtained from Chinese manufac-
tures. The purity of butene-fipronil was confirmed by
injected into a Hewlett Packard 5988A gas chromatograph
fitted with an FID, a split/splitless injector and a fused
silica capillary column (DB-35 ms, 30 m 9 0.25 mm i.d.,
film thickness 0.25 mm, J&W Scientific, Folsom, CA),
with the temperature of 200C in column, of 250C in
vaporizing chamber and in detector. The purity of nine
conventional insecticides was individually tested by an
Agilent 1100 HPLC system with auto sampler. Among
them abamectin, imidacloprid, spinosad, a-cypermethrin
were analyzed on a 200 9 5 mm2 reverse phase C18 col-
umn (HYPERSIL-ODS) with hexane/ethanol/diethyl ether
(85/10/5) at a flow rate of 1.0 mL/min, with methanol/
acetonitrile/water (40:20:40) at a flow rate of 1.0 mL/min,
with methanol/acetonitrile/water (45:50:5) at a flow rate of
1.0 mL/min, and with petroleum ether/diethyl ether/ethyl
acetate (99.5:0.3:0.2) at a flow rate of 0.2 mL/min.
Whereas acetamiprid, clothianidin, acephate, chlorpyrifos
and methomyl were tested on a 200 9 5 mm2 stainless
steel column (YWG-C18) with methanol/water at the ratio
of 50/50, 45/55, 15/85, 90/10 and 90/10 respectively at a
flow rate of 1.0 mL/min. The quantity of each insecticide
was evaluated using external standard method. These
chemicals were kept in a refrigerator between the experi-
mental sessions.
A method described previously (Wang et al. 2012c) was
used to determine insecticide toxicity to the larvae. Insec-
ticides were dissolved in and serially diluted twofold with
acetone to obtain several solutions. An aliquot of 200 lL of
test solution (treatment) was mixed with 10 g of hot con-
ventional artificial diet to prepare at least four toxic arti-
ficial diets within a mortality range of approximately 0 %
100 % based on preliminary assays. The concentration
ranges for butene-fipronil were 0.0110.00 mg AI per gram
diet. Those for abamectin, acephate, chlorpyrifos, meth-
omyl, a-cypermethrin, imidacloprid, acetamiprid, clothi-
anidin, and spinosad were 0.0115.00, 0.50160.00,
0.013.65, 0.5015.00, 0.105.00, 0.0515.00, 0.1015.00,
0.102.00, and 0.011.00 mg AI per gram diet, respec-
tively. In all bioassays, acetone was used as negative
191
control. Thirty second-instar larvae were transferred into a
vial (12 cm in length and 3 cm in diameter) containing
10 g of one of the toxic artificial diets or acetone control
diet and sealed with a cotton plug. The larvae (n = 90) in
three replicates were kept at 25C and the mortality was
recorded after adult emergence.
For the adult flies, bioassays were performed using a
method described previously (Grant 2002; Watson et al.
2010). Briefly, test solutions (in 2:1 acetone:water/10 %
sucrose solution) were applied to an agarose substrate in
128-well assay trays. After drying, adult flies were placed
in the wells, 35 flies per well, four wells per dose. Typi-
cally each test was replicated at least three times with each
dose response line composed of five to six doses with an
average (total) of 236305 flies. Flies were held at 25C
and examined for mortality (inability to translocate) at 24 h
post-treatment. Controls for each assay included solvent-
only and untreated wells.
Abbotts formula was used to correct the data for control
mortality (Abbott 1925). Probit analysis was used to esti-
mate the concentrations needed to cause 50 % mortality
(LD50), their fiducial limits and the slope of the line
relating probit mortality to the log dose by POLO Plus logit
probit software (LeOra Software Company, Petaluma, CA,
USA).
To assess the potential risk to D. melanogaster adults,
the risk quotient method was used (Peterson 2006; Schleier
and Peterson 2010). Risk quotients for the insecticides
were calculated according to the formula: risk quo-
tient = recommended field rate (g AI per ha)/LD50 of
insect (lg AI per gram). A risk quotient value \50 for a
pesticide was considered safe; 502,500, slightly to mod-
erately toxic; and [2,500, dangerously toxic (Preetha et al.
2010).
Results and Discussion
In this study, 10 insecticides were selected. Among these
insecticides, butene-fipronil is a noncompetitive antagonist
and abamectin is an activator of glutamate receptor excit-
atory synapse. Acephate and chlorpyrifos belong to orga-
nophosphates and methomyl is a methylcarbamate, the
toxicity of the three is attributable to inhibition of acetyl-
cholinesterase (AchE). Cypermethrin is a modulator of
voltage-dependent Na? channel. Neonicotinoids imida-
cloprid, acetamiprid and clothianidin act as agonists
whereas spinosad is the nAChR allosteric activators of
nicotinic Ach receptors (Casida and Durkin 2013).
The toxicities of insecticides to the larvae vary widely
among different insecticides and among different strains
(Table 1). The LD50 values to the larvae ranged from 0.06
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192 Bull Environ Contam Toxicol (2014) 92:190195

Table 1 Susceptibilities of CS, w1118 and Oregan stocks of D. melanogaster 2nd-instar larvae to ten insecticides
Strain Insecticide Na X2 dfb Slope (SE) LD50 (lg/g) (95 %FL) RTIc

CS Acephate 450 2.7 4 1.38 (0.12) 6.20 (4.688.21) 77.50

Chlorpyrifos 630 4.2 6 3.61 (0.21) 0.11 (0.100.12) 1.38
Methomyl 450 2.4 4 3.22 (0.21) 1.36 (1.211.52) 17.00
a-Cypermethrin 540 2.5 5 1.24 (0.24) 0.97 (0.731.27) 12.13
Imidacloprid 450 2.6 4 1.13 (0.28) 0.41 (0.310.56) 5.13
Acetamiprid 540 2.6 5 2.36 (0.16) 0.65 (0.440.80) 8.13
Clothianidin 540 3.3 5 2.01 (0.12) 0.56 (0.330.81) 7.00
Butene fipronil 540 4.4 5 1.86 (0.13) 0.08 (0.070.10) 1.00
Abamectin 450 3.3 4 1.15 (0.21) 0.74 (0.531.04) 9.25
Spinosad 540 2.8 5 3.41 (0.10) 0.24 (0.220.27) 3.00
w1118 Acephate 540 3.1 5 1.88 (0.06) 11.18 (9.2613.49) 74.53
Chlorpyrifos 540 2.7 5 1.19 (0.10) 0.06 (0.050.07) 0.40
Methomyl 450 2.4 4 2.98 (0.66) 1.33 (1.131.52) 8.87
a-Cypermethrin 540 2.5 5 1.57 (0.08) 1.00 (0.771.30) 6.67
Imidacloprid 450 2.4 4 1.20 (0.18) 1.39 (1.031.86) 9.27
Acetamiprid 630 4.0 6 2.45 (0.20) 1.71 (1.132.28) 11.40
Clothianidin 720 2.8 7 1.98 (0.18) 0.91 (0.521.31) 6.07
Butene fipronil 450 2.5 4 1.72 (0.04) 0.15 (0.120.19) 1.00
Abamectin 540 3.5 5 1.11 (0.04) 0.10 (0.070.14) 0.67
Spinosad 540 4.1 5 2.49 (0.27) 0.15 (0.130.19) 1.00
Oregan Acephate 450 3.1 4 2.15 (0.17) 9.38 (7.5411.45) 85.27
Chlorpyrifos 540 3.0 5 2.84 (0.21) 0.12 (0.100.13) 1.09
Methomyl 540 3.7 5 3.11 (0.27) 2.42 (2.182.73) 22.00
a-Cypermethrin 540 3.3 5 3.42 (0.19) 0.54 (0.320.72) 4.91
Imidacloprid 540 2.9 5 2.15 (0.19) 0.81 (0.521.11) 7.36
Acetamiprid 540 2.2 5 1.91 (0.17) 1.26 (1.001.73) 11.45
Clothianidin 540 3.4 5 2.25 (0.20) 0.64 (0.510.90) 5.82
Butene fipronil 540 3.7 5 2.05 (0.21) 0.11 (0.080.14) 1.00
Abamectin 450 3.3 4 2.15 (0.17) 0.30 (0.130.61) 2.73
Spinosad 540 2.8 5 2.18 (0.16) 0.20 (0.190.24) 1.82
a
Number of total tested individuals from 4 to 7 concentrations
b
Degree of freedom
c
Relative toxicity index (RTI) was determined by comparing butane fipronil LD50 value with the LD50 value of other insecticide. The same as
Table 2

(0.050.07) to 11.18 (9.2613.49) lg AI g-1. According to
the relative toxicity index (RTI), the ten tested insecticides
could be classified into three groups with relatively least
(RTI [ 10.0), middle (5.0 \ RTI \ 10.0) and most (RTI \
5.0) toxicity to the larvae. From the mean LD50 values in CS,
w1118 and Oregan strains, acephate, methomyl, and ace-
tamiprid fell into the first category, imidacloprid, a-cyper-
methrin and clothianidin belonged to the second group,
whereas butene-fipronil, abamectin, spinosad and chlor-
pyrifos were placed into the third group (Table 1). Since D.
melanogaster is physiologically, biochemically and
genetically similar to dipterans (Scharf et al. 2006; Karatas
and Bahceci 2009), our results suggested that butene-fipronil
is also toxic to other dipteran larvae.
The toxicities of insecticides to the adults also differ
widely among tested insecticides, and among different
strains. For ten tested insecticides, the LD50 values to the
adults ranged from 0.05 (0.040.07) to 40.44 (36.1343.24)
lg AI g-1 (Table 2). From the mean LD50 values in CS,
w1118 and Oregan strains, acephate, methomyl, acetami-
prid, imidacloprid, clothianidin and abamectin were least
toxic, with the RTI [ 1.01. In contrast, butene-fipronil,

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Bull Environ Contam Toxicol (2014) 92:190195 193

Table 2 Susceptibilities of CS, w1118 and Oregan stocks of D. melanogaster adults to ten insecticides
Strain Insecticide Na X2 df Slope (SE) LD50 (lg/g) (95 %FL) RTI

CS Acephate 167 5.0 5 2.14 (0.33) 32.25 (28.4135.14) 6.14

Chlorpyrifos 151 4.6 5 1.84 (0.22) 0.53 (0.310.75) 0.10
Methomyl 191 3.7 5 1.68 (0.25) 6.15 (4.688.75) 1.17
a-Cypermethrin 182 4.4 5 1.98 (0.21) 0.45 (0.300.61) 0.09
Imidacloprid 148 3.1 5 3.41 (0.14) 4.58 (2.616.68) 0.87
Acetamiprid 184 4.2 5 2.55 (0.36) 7.54 (5.109.99) 1.44
Clothianidin 157 3.6 5 3.13 (0.27) 0.73 (0.540.91) 0.14
Butene fipronil 223 4.3 6 1.75 (0.27) 5.25 (2.996.42) 1.00
Abamectin 203 4.8 6 1.87 (0.18) 7.24 (5.3410.25) 1.37
Spinosad 220 4.6 6 1.84 (0.23) 0.05 (0.040.07) 0.01
w1118 Acephate 164 3.5 5 2.03 (0.25) 30.34 (26.3633.96) 6.40
Chlorpyrifos 203 3.7 6 1.95 (0.17) 0.87 (0.601.15) 0.18
Methomyl 182 3.4 5 2.01 (0.19) 7.25 (5.127.90) 1.53
a-Cypermethrin 176 3.3 5 2.00 (0.34) 0.50 (0.300.64) 0.11
Imidacloprid 187 3.5 5 2.43 (0.11) 4.57 (1.646.73) 0.96
Acetamiprid 205 3.6 5 3.13 (0.22) 7.80 (5.4110.21) 1.65
Clothianidin 214 3.1 6 2.87 (0.23) 0.61 (0.340.95) 0.13
Butene fipronil 216 5.1 6 1.56 (0.20) 4.74 (2.115.83) 1.00
Abamectin 221 4.8 6 2.14 (0.31) 5.34 (3.158.48) 1.17
Spinosad 208 4.9 6 1.58 (0.31) 0.05 (0.030.09) 0.01
Oregan Acephate 191 3.5 5 1.75 (0.23) 40.44 (36.1343.24) 8.06
Chlorpyrifos 207 3.6 6 1.67 (0.20) 0.64 (0.410.92) 0.13
Methomyl 152 3.5 5 2.21 (0.24) 8.45 (6.019.13) 1.68
a-Cypermethrin 176 2.9 5 2.02 (0.17) 0.48 (0.310.64) 0.10
Imidacloprid 184 4.1 5 1.98 (0.12) 4.12 (3.125.41) 0.82
Acetamiprid 167 3.1 5 2.81 (0.14) 6.65 (5.128.28) 1.32
Clothianidin 196 2.8 5 1.94 (0.09) 0.80 (0.561.12) 0.16
Butene fipronil 217 3.8 6 1.88 (0.11) 5.02 (2.646.11) 1.00
Abamectin 178 4.6 5 1.75 (0.19) 3.17 (2.633.79) 0.63
Spinosad 159 4.4 5 1.88 (0.24) 0.05 (0.020.07) 0.01
a
Since some adults escaped from the assay trays, the final number of tested individuals are less than those introduced

a-cypermethrin, spinosad and chlorpyrifos were most
toxic, with the RTI equal or less than 1.00 (Table 2).
The ratios of LD50 values of tested insecticides for
adults to corresponding LD50 values for larvae are shown
in Fig. 1. The ratios of butene-fipronil and abamectin were
more than 10, demonstrating that the two insecticides were
more toxic to larvae. In contrast, spinosad, a-cypermethrin,
and clothianidin had the ratios less than 1.0, demonstrating
that the three insecticides were more toxic to adults
(Fig. 1).
The toxic risk of butene-fipronil to D. melanogaster adults
was estimated from the mean LD50 values in CS, w1118 and
Oregan strains, in comparison to nine conventional insecti-
cides. The tested insecticides were classified into different
groups based on risk quotient values according to Peterson
(2006) and Schleier and Peterson (2010) (Table 3). Chlor-
pyrifos, a-cypermethrin and spinosad were slightly to
moderately toxic to D. melanogaster. In contrast, butene-
fipronil, acephate, methomyl, imidacloprid, acetamiprid,
clothianidin and abamectin were safe to D. melanogaster
(Table 3). Thus, our results suggested that butene-fipronil
is relatively safe to D. melanogaster and other dipteran
adults.
In conclusion, our results revealed that butene-fipronil is
relatively less toxic to D. melanogaster adults than that to
the larvae, as a result it is safe to the adults. Considering
many agricultural pests caused damages in larval stage and
many natural enemy and environmental insect adults are
more likely to be exposed to sprayed butene-fipronil during
foraging and/or oviposition, our results indicate that

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