Chemosphere 88 (2012) 484491

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Chemosphere
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Toxicity assessment of 45 pesticides to the epigeic earthworm Eisenia fetida

Yanhua Wang, Shenggan Wu, Liping Chen, Changxing Wu, Ruixian Yu, Qiang Wang, Xueping Zhao
State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences,
Hangzhou 310021, Zhejiang, China
Key Laboratory for Pesticide Residue Detection of Ministry of Agriculture, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences,
Hangzhou 310021, Zhejiang, China

a r t i c l e

i n f o

Article history:
Received 26 September 2011
Received in revised form 27 February 2012
Accepted 29 February 2012
Available online 28 March 2012
Keywords:
Soil invertebrate
Ecotoxicology
Fungicides
Insecticides
Herbicides

a b s t r a c t
This study was conducted to investigate comparative toxicity of 45 pesticides, including insecticides, acaricides, fungicides, and herbicides, toward the epigeic earthworm Eisenia fetida. Results from a 48-h lter
paper contact test indicated that clothianidin, fenpyroximate, and pyridaben were supertoxic to E. fetida
with LC50 values ranging from 0.28 (0.240.35) to 0.72 (0.600.94) lg cm 2, followed by carbaryl, pyridaphenthion, azoxystrobin, cyproconazole, and picoxystrobin with LC50 values ranging from 2.72 (2.22
0.3.19) to 8.48 (7.3810.21) lg cm 2, while the other pesticides ranged from being relatively nontoxic to
very toxic to the worms. When tested in articial soil for 14 d, clothianidin and picoxystrobin showed the
highest intrinsic toxicity against E. fetida, and their LC50 values were 6.06 (5.606.77) and 7.22 (5.29
8.68) mg kg 1, respectively, followed by fenpyroximate with an LC50 of 75.52 (68.2186.57) mg kg 1.
However, the herbicides uoroglycofen, paraquat, and pyraufen-ethyl exhibited the lowest toxicities
with LC50 values > 1000 mg kg 1. In contrast, the other pesticides exhibited relatively low toxicities with
LC50 values ranging from 133.5 (124.5150.5) to 895.2 (754.21198.0) mg kg 1. The data presented in
this paper provided useful information for evaluating the potential risk of these chemicals to soil
invertebrates.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Earthworms are an important component of decomposer communities and contribute signicantly to organic matter decomposition, nutrient cycling, and soil formation (Coleman and Ingham,
1988; Edwards and Bohlen, 1992). Located near the bottom of
the terrestrial trophic level, earthworms use the sensitive receptors
on their body surfaces to sense chemicals in the soil (Bouch,
1992). Their ecological importance, high biomass in soil, and frequently observed sensitivity to relatively low concentrations of
environmental toxins make them one of the most suitable bioindicator organisms for risk assessment in the soil (Landrum et al.,
2006). The abundance of earthworms in soil represents the health
of soil ecosystems and the level of environmental safety (Xiao
et al., 2004).
In order to increase the crop yield in agricultural areas, synthetic pesticides including insecticides, acaricides, fungicides, and
herbicides were widely applied to control harmful organisms
(Liang and Zhou, 2003; Xiao et al., 2006a; Zhou et al., 2011).
Corresponding author at: Key Laboratory for Pesticide Residue Detection of
Ministry of Agriculture, Institute of Quality and Standard for Agro-products,
Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China.
Tel.: +86 571 86404229; fax: +86 571 86402186.
E-mail address: zhaoxueping@tom.com (X. Zhao).
0045-6535/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2012.02.086

Pesticides are either directly applied to the soil to control soilborne pests or are deposited on soil as runoff from foliar applications (Hans et al., 1990; Wahanthaswamy and Patil, 2004; Gupta
et al., 2011). Pesticide residues impair the physiological functions
of earthworms, leading to their mortality, but there are insufcient
data from eld or laboratory assays to make accurate assessments
of their relative toxicities (Ahmed, 1991; Maboeta et al., 2004). It is
possible that the chemicals will affect not only target species but
also non-target organisms in and adjacent to the target areas
(Schulz et al., 2001). In agricultural areas worldwide, there is an
increasing concern about soil contamination due to the widespread
use of pesticides (Reinecke and Reinecke, 2007). Several researchers have advocated the use of earthworms as an ecotoxicological
model for risk assessment and pesticide bioassay (Edwards and
Bohlen, 1992; Gupta et al., 2011).
Extensive toxicological tests have been conducted to elucidate
the toxicities of heavy metals, polychlorinated biphenyl, and certain conventional pesticide categories to earthworms (Diercxsens
et al., 1985; Kamitani and Kaneko, 2007; Reinecke and Reinecke,
2007; Hackenberger et al., 2008). However, novel modes of pesticides that have recently been introduced are more specic for
the target organism, and there is a paucity of information on their
ecotoxicological effects, including those on earthworms (Damalas
and Eleftherohorinos, 2011). In the present study, experiments
were performed to evaluate the toxic effects of 45 pesticides from

Y. Wang et al. / Chemosphere 88 (2012) 484491

four important pesticide categoriesincluding insecticides, acaricides, herbicides, and fungicideson the epigeic earthworm Eisenia
fetida. The main objective of this study was to determine the comparative toxicity of these pesticides on E. fetida and their potential
to damage soil-dwelling invertebrates.
2. Materials and methods
2.1. Earthworm
The oligochaete E. fetida, one of the favorite worm species for
composting and organic gardening, is frequently used as a biological monitor for testing the effects of contaminants on soil biota. It
is also the Organisation for Economic Co-operation and Development (OECD)-recommended earthworm test species (OECD,
1984; Edwards and Coulson, 1992; Yasmin and DSouza, 2007).
Adult earthworms (weighing 350500 mg) with well-developed
clitella were purchased from the College of Animal Sciences, Zhejiang University, China, and cultured in the laboratory in articial
soil according to OECD guidelines (OECD, 1984). Soils were mixed
with decayed leaves and decomposed pig manure, and kept at
room temperature (20 1 C). Soil water content was measured
every week and a 35% maximum water-holding capacity was
maintained by the addition of distilled water as needed. Additional
control tests were carried out with chloracetamide as a toxic reference standard.
2.2. Pesticides
Forty-ve pesticides including 9 insecticides, 2 acaricides, 23
herbicides, and 11 fungicides were tested in this study (Table 1).
The selected pesticides are widely used in agriculture worldwide.
Active ingredients were used instead of commercial formulations,
aiming to document the effects of the neurotoxic molecules of
the chemicals on mortality but not the effects of the adjuvants
added to the commercial products.

485

content was adjusted to 35% of the dry weight. For each tested concentration, the desired amount of pesticide was dissolved in 10 mL
acetone and mixed with a small quantity of ne quartz sand. The
sand was mixed for least 1 h to evaporate the acetone and was
then mixed thoroughly with the pre-moistened articial soil in a
household mixer. The nal moisture contents of the articial soil
were adjusted to the described level by the addition of distilled
water. A total of 0.65 kg of soil (equivalent to 0.5 kg dry articial
soil) was placed in a 500 mL glass jar (surface area, 63.6 cm2)
and 10 adult earthworms were added to each jar. Controls were
prepared similarly but only with 10 ml acetone and no insecticide.
The jars were loosely covered with polypropylene lids to allow for
air exchange and stored at 20 1 C with 8085% relative humidity
under 400800 lux of constant light. Mortality was assessed at 7
and 14 days after treatment. Besides, the mortality in the controls
should not exceed 10% at the end of either test.
A range of concentrations including 0, 0.1, 1.0, 10, 100, and
1000 mg kg 1 dry soil were used in the pre-trials to determine
the concentrations that produced 0100% mortality. To obtain
LC50, 56 test concentrations in a geometric series and a control
were used for each pesticide. Three jars, each containing 10 adult
earthworms, were used for each concentration. The earthworms
were preconditioned for 24 h under the same conditions described
above in the untreated soil before the doseresponse test.
2.4. Statistical analysis
A probit analysis was conducted to assess the acute toxicity of
pesticides to E. fetida using a program developed by Chi (Chi,
1997). The signicant level of mean separation (P < 0.05) detected
was based on the lack of overlap between the 95% condence
limits of 2 LC50 values (Prabhaker et al., 2011). In the contact lter
paper test method, based on the resulting LC50 values, the pesticides were classied as being supertoxic (<1.0 lg cm 2), extremely
toxic (110 lg cm 2), very toxic (10100 lg cm 2), moderately
toxic (1001000 lg cm 2), or relatively nontoxic (>1000 lg cm 2)
(Roberts and Dorough, 1984).

2.3. Toxicity test methods

3. Results
2.3.1. Contact toxicity test
We performed a modied contact lter paper test (OECD, 1984).
A piece of lter paper was placed in a 9-cm petri dish, and treated
with the test substance dissolved in 2 mL of acetone. After the solvent evaporated, the piece of lter paper was remoistened with
2 mL of distilled water and 1 earthworm was placed on it. The dish
was incubated in the dark at 20 1 C for 48 h and mortality was
recorded. An earthworm was considered dead if it failed to respond
to a gentle mechanical touch on the front end.
Earthworms were held on wet lter paper for 24 h at 20 1 C
in the dark for purging of the gut contents. A preliminary test
was conducted to determine the concentration range of the test
chemicals in which 0100% mortality of the earthworm was obtained. To establish the concentration-mortality relationship,
earthworms were exposed to at least ve different concentrations
in a geometric series and a control for each chemical. Ten replications were used for each concentration. Acetone was used as the
control. Treated earthworms were maintained at 20 1 C under
8085% relative humidity in the dark. Moreover, the mortality in
the controls should not exceed 10% at the end of either test.
2.3.2. Soil toxicity test
The articial soil consisted of 10% ground sphagnum peat
(<0.5 mm), 20% kaolinite clay (>50% kaolinite), and 70% ne sand
(OECD, 1984, 2004). A small amount of calcium carbonate was
added to adjust the pH to 6.0 0.5. In the toxicity tests, the water

3.1. Contact toxicity

The acute toxicities of 45 pesticides to E. fetida from the contact
lter paper test are shown in Table 2. The results demonstrated
that the different pesticides varied widely in their contact toxicities, while different pesticides within the same category had different toxicities to E. fetida. Two acaricides (fenpyroximate and
pyridaben) and one insecticide clothianidin showed the highest
intrinsic toxicity, followed by the insecticide pyridaphenthion
and three fungicides (azoxystrobin, cyproconazole, and picoxystrobin) compared with the other tested pesticides. Overall, among
the four pesticide categories evaluated, acaricides, several insecticides, and fungicides showed great toxicities, while herbicides
exhibited relatively low toxicity against E. fetida (Table 2).
3.1.1. Insecticides and acaricides
The toxicities of nine insecticides to E. fetida from the four
chemical groups (Table 1) were evaluated in this study. The various
insecticides exhibited a wide range of contact toxicities. The neonicotinoid insecticide clothianidin was supertoxic to E. fetida with
an LC50 value of 0.28 (0.240.35) lg cm 2, and the organophosphate insecticides pyridaphenthion and carbamate insecticide carbaryl were extremely toxic with LC50 values of 3.84 (2.876.18)
and 4.21 (2.695.57) lg cm 2, respectively. In contrast, four insect
growth regulators (IGRs; buprofezin, onicamid, fufenozide, and

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Y. Wang et al. / Chemosphere 88 (2012) 484491

Table 1
Detailed information about the pesticides used in this study.

a
b
c
d

Chemical group

Pesticides

Technical grade (a.i.) (%)

Manufacturer

Insecticides
Carbamate
IGRsa
IGRsa
IGRsa
IGRsa
Neonicotinoid
Organophosphate
Organophosphate
Organophosphate

Carbaryl
Buprofezin
Flonicamid
Fufenozide
Lufenuron
Clothianidin
Acephate
Malathion
Pyridaphenthion

98
98.2
98.7
96.5
98
96.5
94.9
96
95

Jiangsu Changlong Chemical Co., Ltd.

Jiangsu Changlong Chemical Co., Ltd.
Ishihara Sangyo Kaisha Ltd.
Jiangsu Pesticide Institute Co., Ltd.
Zhejiang shangying Fine Chemical Co., Ltd.
Hunan Bide Biochemical Co., Ltd.
Zhejiang Ruite Chemical Co., Ltd.
Jiangsu Xinyin Taisong Chemical Co., Ltd.
Anhui Xinsaide Chemical Co., Ltd

Acaricides
Heterocyclic
Heterocyclic
Herbicides
Acetanilide
Acetanilide
Acetanilide
APPc
APPc
APPc
Bipyridylium
Diphenylether
Dinitroaniline
Diphenylether
Dinitroaniline
Imidazolinone
Imidazolinone
Organophosphorus
Organophosphorus
Organophosphorus
PAb
Sulfonylurea
Sulfonylurea
Sulfonylurea
Triazine
Triazine
Triazine

Fenpyroximate
Pyridaben

97
95

Xinyin Yongcheng Chemical Co., Ltd.

Nanjing Red-Sun Group Co., Ltd.

Acetochlor
Pretilachlor
S-metolachlor
Fenoxaprop-p-ethyl
Fluazifop-p-butyl
Quizalofop-p-ethyl
Paraquat
Fluoroglycofen
Prodiamine
Pyraufen-ethyl
Triuralin
Flutamone
Mesotrione
Anilofos
Glufosinate-ammonium
Glyphosate
2,4-D Na Salt
Metsulfuron-methyl
Rimsulfuron
Sulfometuron-methyl
Atrazine
Metamitron
Terbutryn

93
98.8
96
95
90
95
42
95
97
95
97.2
98
95
95
96
95
87
97
99
95
95
98
97

Shandong Qiaochang Chemical Co., Ltd.

Hangzhou Qingfeng Agrochemical Co., Ltd.
Zhongnong Minchang Chemical Co., Ltd.
Jiangsu Tianrong Chemical Co., Ltd.
Zhejiang Yongnong Co., Ltd.
Jiangsu Tianyong Group Chemical Co., Ltd.
Shandong Lvfeng Pesticide Co., Ltd.
Lianyungong Liben Chemical Co., Ltd.
Sichuan Luzhou Orient Chemical Co., Ltd.
Ishihara Sangyo Kaisha Ltd.
Dongyang Dongnong Chemical Co., Ltd.
Shanghai Henong Pesticide Co., Ltd.
Liaoning Dandong Pesticide Co., Ltd.
Shandong Binnong Technology Co., Ltd.
Sichuan Lier Chemical Co., Ltd.
Jiangsu Nantong Taihe Chemical Co., Ltd.
Jiangsu Biochemical Co., Ltd.
Jiangsu Ruidong Pesticide Co., Ltd.
DuPont Company of USA
DuPont Company of USA
Zhejiang Changxing Chemical Co., Ltd.
Zhejiang Leshi Chemical Co., Ltd
Shandong Binnong Technology Co., Ltd.

Fungicides
CAAd
Heterocyclic
Strobilurin
Sulfonyl imidazole
Strobilurin
Strobilurin
Triazole
Triazole
Triazole
Triazole
Triazole

Flutolanil
Isoprothiolane
Azoxystrobin
Cyazofamid
Picoxystrobin
Trioxystrobin
Cyproconazole
Epoxiconazole
Hexaconazole
Ipconazole
Tebuconazole

98
95
98
96.9
99
96
95
96
95
97.5
96

Jiangsu Taizhou Baili Chemical Co., Ltd.

Jiangsu Zhongqi Chemical Co., Ltd.
Zhejiang Shangyin Fine Chemical Co., Ltd.
Ishihara Sangyo Kaisha Ltd.
DuPont Company of USA
Qinghai Lvyuan Biotechnical Co., Ltd.
Jiangsu Fengdeng Pesticide Co., Ltd.
Sichuan Lier Chemical Co., Ltd.
Jiangsu Fengdeng Pesticide Co., Ltd.
Chemtura Corporation of USA
Jiangsu Fengdeng Pesticide Co., Ltd.

IGRs, insect growth regulators.

PA, phenoxyalkanoic acid.
APP, aryloxyphenoxy propionic acid.
CAA, carboxylic acid amide.

lufenuron) and one organophosphate, acephate, were relatively

nontoxic (i.e., all LC50 values > 1000 lg cm 2). Moreover, different
insecticides within the same chemical group had different
toxicities against E. fetida, e.g., the organophosphate insecticide
pyridaphenthion was 29.8  more toxic than malathion against E.
fetida. The order of toxicity to E. fetida based on LC50 values was
as follows: clothianidin > pyridaphenthion and carbaryl > malathion > acephate, buprofezin, onicamid, fufenozide, and lufenuron. The 2 acaricides fenpyroximate and pyridaben showed
similar supertoxicities to E. fetida with LC50 values of 0.72 (0.60
0.94) and 0.55 (0.420.65) lg cm 2, respectively (Table 2).
3.1.2. Herbicides
Similar to the insecticides, the toxicity results of the 23 herbicides belonging to the 10 chemical groups induced variable toxicity
responses against E. fetida. The acetanilide herbicides acetochlor,

pretilachlor, and S-metolachlor were very toxic to E. fetida with

LC50 values of 24.02 (19.7231.76), 19.23 (11.5425.92), and 20.63
(17.7049.76) lg cm 2, respectively, as well as the organophosphorus herbicide anilofos, the triazine herbicide terbutryn, and the imidazolinone herbicide utamone were also very toxic to E. fetida with
LC50 values of 34.69 (29.2044.01), 10.31 (8.3112.23), and 26.98
(21.9437.14) lg cm 2, respectively, while diphenylether herbicides (uoroglycofen and pyraufen-ethyl), sulfonylurea herbicides
(metsulfuron-methyl and rimsulfuron), dinitroaniline herbicides
(prodiamine and triuralin), and aryloxyphenoxy propionic acid
herbicide (fenoxaprop-p-ethyl) were the least toxic against E. fetida
and had LC50 values > 1000 lg cm 2. In contrast, all of the other
tested herbicides (atrazine, 2,4-D sodium salt, uazifop-p-butyl,
glufosinate-ammonium, glyphosate, mesotrione, metamitron, and
paraquat) were moderately toxic with LC50 values ranging from
102.5 (66.57138.7) to 566.1 (437.4905.4) lg cm 2.

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Y. Wang et al. / Chemosphere 88 (2012) 484491

Table 2
Summary of parameter estimates for the acute toxicity with contact lter paper test of 45 pesticides to Eisenia fetida.
Pesticide
Insecticides
Carbaryl
Buprofezin
Flonicamid
Fufenozide
Lufenuron
Clothianidin
Acephate
Malathion
Pyridaphenthion
Acaricides
Fenpyroximate
Pyridaben
Herbicides
Acetochlor
Pretilachlor
S-metolachlor
Fenoxaprop-p-ethyl
Fluazifop-p-butyl
Quizalofop-p-ethyl
Paraquat
Fluoroglycofen
Prodiamine
Pyraufen-ethyl
Triuralin
Flutamone
Mesotrione
Anilofos
Glufosinate ammonium
Glyphosate
2,4-D sodium salt
Metsulfuron-methyl
Rimsulfuron
Sulfometuron-methyl
Atrazine
Metamitron
Terbutryn
Fungicides
Flutolanil
Isoprothiolane
Azoxystrobin
Cyazofamid
Picoxystrobin
Trioxystrobin
Cyproconazole
Epoxiconazole
Hexaconazole
Ipconazole
Tebuconazole

Slope (SE)

LC50 (95% CI) lg cm

3.15 (0.46)

3.17 (0.66)
3.10 (0.46)

4.21 (2.695.57) c
>1000
>1000
>1000
>1000
0.28 (0.240.35) a
>1000
114.4 (68.6368.1) f
3.84 (2.876.18) c

Extremely toxic
Relatively nontoxic
Relatively nontoxic
Relatively nontoxic
Relatively nontoxic
Supertoxic
Relatively nontoxic
Moderately toxic
Extremely toxic

4.38 (0.62)
5.30 (0.82)

0.72 (0.600.94) b
0.55 (0.420.65) b

Supertoxic
Supertoxic

3.76 (0.51)
3.25 (0.49)
4.88 (0.73)

24.02 (19.7231.76) de
19.23 (11.5425.92) d
20.63 (17.7049.76) de
>1000
518.0 (408.6782.3) h
>1000
235.0 (199.4279.5) fg
>1000
>1000
>1000
>1000
26.98 (21.9437.14) de
545.1 (422.2893.3) h
34.69 (29.2044.0) de
557.2 (417.5897.1) h
566.1 (437.4905.4) h
422.7 (344.7577.3) h
>1000
>1000
>1000
102.5 (66.57138.7) f
407.5 (328.6550.7) h
10.31 (8.3112.23) d

Very toxic
Very toxic
Very toxic
Relatively nontoxic
Moderately toxic
Relatively nontoxic
Moderately toxic
Relatively nontoxic
Relatively nontoxic
Relatively nontoxic
Relatively nontoxic
Very toxic
Moderately toxic
Very toxic
Moderately toxic
Moderately toxic
Moderately toxic
Relatively nontoxic
Relatively nontoxic
Relatively nontoxic
Moderately toxic
Moderately toxic
Very toxic

11.03 (9.0212.97) d
91.95 (75.97128.2) f
2.72 (2.223.19) c
>1000
3.15 (2.173.96) c
>1000
8.48 (7.3810.21) d
>1000
12.07 (10.2614.53) d
21.76 (17.4426.32) de
31.57 (25.6636.62) de

Very toxic
Very toxic
Extremely toxic
Relatively nontoxic
Extremely toxic
Relatively nontoxic
Extremely toxic
Relatively nontoxic
Very toxic
Very toxic
Very toxic

4.12 (0.57)

5.24 (0.82)
4.34 (0.61)

4.33
6.65
4.26
5.76
5.74
4.24

(0.62)
(1.16)
(0.60)
(0.94)
(0.93)
(0.60)

1.86 (0.26)
3.20 (0.42)
4.07 (0.56)
4.35 (0.61)
3.84 (0.52)
4.59 (0.66)
4.49 (0.66)
5.96 (0.98)
4.31 (0.60)
3.17 (0.39)
4.22 (0.61)

Toxicity grade

Means followed by the same letters are not signicantly different at P = 0.05 within a column.

The different herbicides within the same chemical group had

different toxicities to E. fetida, e.g., the organophosphorus herbicide
anilofos was 16.3- and 15.7-fold more toxic than glufosinateammonium and glyphosate, respectively, and the triazine herbicide
terbutryn was 9.94- and 39.5-fold more toxic than atrazine and
metamitron, respectively. However, some exceptions were seen in
the case of acetanilide herbicides, e.g., acetochlor, pretilachlor,
and S-metolachlor have the same toxicity level against E. fetida
(Table 2).
3.1.3. Fungicides
The toxicity results of 11 fungicides from 5 chemical groups to
E. fetida are shown in Table 2. The strobilurin fungicides (azoxystrobin and picoxystrobin) and triazole fungicide (cyproconazole)
were extremely toxic; their LC50 values ranged from 2.72 (2.22
3.19) to 8.48 (7.3810.21) lg cm 2. Triazole fungicides (hexaconazole, ipconazole, and tebuconazole), carboxylic acid amide
fungicide (utolanil), and heterocyclic fungicide (isoprothiolane)

were very toxic; their LC50 values ranged from 11.03 (9.02
12.97) to 91.95 (75.97128.2) lg cm 2. In contrast, the other strobiluron fungicide trioxystrobin, the sulfonyl imidazole fungicide
cyazofamid, and the triazole fungicide epoxiconazole were relatively nontoxic; all of their LC50 values were > 1000 lg cm 2. A
high degree of toxicity variation was found among fungicides of
the same chemical group, e.g., the toxicities of the strobilurin fungicides azoxystrobin and picoxystrobin were similar but were signicantly greater than that of trioxystrobin against E. fetida, while
the toxicities of triazole fungicides hexaconazole, ipconazole, and
tebuconazole were apparently greater than that of epoxiconazole
but less than that of cyproconazole (Table 2).
3.2. Soil toxicity
The acute toxicities of 45 pesticides from the articial soil test
of E. fetida are shown in Table 3. The data exhibited a clear concentration-dependent relationship and the mortality increased when

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Y. Wang et al. / Chemosphere 88 (2012) 484491

Table 3
Summary of parameter estimates for the acute toxicity with articial soil test of 45 pesticides to E. fetida.
Pesticides

7-day interval

14-day interval

Slope (SE)

LC50 (95% CI) mg kg

Insecticides
Carbaryl
Buprofezin
Flonicamid
Fufenozide
Lufenuron
Clothianidin
Acephate
Malathion
Pyridaphenthion

16.65 (2.90)
10.14 (1.50)
16.73 (13.2)
9.69 (1.44)
19.25 (1.80)
12.29 (1.93)
7.82 (1.67)
10.25 (1.04)
10.14 (1.45)

152.2 (138.4182.5)
425.2 (383.0505.4)
350.8 (235.1436.8)
217.4 (194.9261.0)
450.7 (345.6769.3)
7.44 (6.659.06) a
582.8 (468.8969.7)
425.8 (314.6614.8)
297.1 (271.3342.4)

c
ef
de
cd
ef

Acaricides
Fenpyroximate
Pyridaben

6.45 (0.95)
8.45 (1.43)

97.06 (84.67119.7) b
516.8 (423.1755.5) ef

8.09 (1.33)
6.30 (0.92)

75.52(68.2186.57) b
362.5 (317.3442.1) ef

5.93 (0.86)
2.92 (0.62)
3.62 (0.44)
16.96 (13.2)
10.79 (1.88)
8.63 (1.51)

6.75 (1.02)
3.35 (0.62)
3.66 (0.44)
12.08 (2.27)
11.59 (2.22)
8.30 (1.42)

7.45 (1.20)
17.26 (13.2)
4.68 (0.65)
5.40 (0.64)
22.68 (14.9)
3.86 (0.53)
5.13 (0.75)
6.21 (1.02)
5.91 (0.92)
3.08 (0.59)
5.01 (0.65)
5.29 (0.71)
4.55 (0.57)

334.1 (284.3432.0) de
662.6 (423.31856.4) ef
187.8 (132.9268.9) c
347.9 (289.6458.1) de
456.4 (349.6776.6) ef
413.1 (264.9687.6) de
>1000
>1000
231.0 (205.8271.5) cd
>1000
601.1 (488.7877.5) ef
344.0 (294.3469.1) de
307.4 (220.7801.8) de
204.6 (173.0276.0) c
167.2 (94.51259.4) c
345.8 (241.3920.6) de
335.5 (276.3461.6) de
408.6 (329.4543.7) de
369.4 (298.3538.4) de
560.7 (385.81211.5) ef
204.8 (179.9236.4) c
228.3 (163.6547.9) cd
177.9 (155.1205.6) c

10.64 (1.89)
7.27 (1.14)
5.11 (0.69)
6.51 (1.38)
22.99 (15.3)
3.95 (0.53)
5.83 (0.83)
6.23 (0.99)
6.25 (0.94)
3.42 (0.57)
5.52 (0.75)
5.24 (0.70)
7.46 (1.18)

283.0 (247.7347.5) de
516.2 (367.41014.9) fg
184.8 (133.6256.4) cd
306.5 (256.5442.7) de
416.4 (321.3727.2) ef
389.2 (309.6605.3) ef
>1000
>1000
213.7 (191.1246.3) cd
>1000
499.1 (421.5694.3) fg
307.2 (258.7414.6) de
258.3 (198.0464.5) cd
180.1 (158.6221.3) cd
162.2(102.7284.6) c
327.8 (238.8672.2) de
275.4 (236.1349.6) de
382.8 (254.34231.8) ef
330.5 (274.2454.0) de
448.6 (335.8760.0) ef
180.4 (158.4204.5) cd
195.2 (172.1225.7) cd
106.6 (83.91122.9) b

8.70
5.92
9.51
6.92
5.57
21.9
9.61
9.61
6.69
6.46
5.33

184.9 (167.7209.1) c
330.4 (274.9453.6) de
362.4 (302.1517.5) de
673.3 (552.9954.3) fg
9.22 (7.4510.65) a
414.1 (316.75324.7) de
221.6 (199.2266.1) cd
356.8 (286.8561.5) de
840.7 (643.21426.1) fg
281.2 (240.7361.9) cd
1035 (842.41469.9) fg

7.02
6.46
9.53
6.41
5.88
20.3
9.90
7.81
6.01
5.06
6.78

150.4 (132.0169.7) c
281.2 (240.7361.9) de
327.4 (279.9439.0) de
568.7 (483.4742.8) fg
7.22 (5.298.68) a
401.3(309.4539.1) ef
211.8 (191.7249.6) cd
333.1(275.6470.6) de
562.0 (476.5733.4) fg
249.2 (215.5305.7) cd
895.2 (754.21198.0) gh

Herbicides
Acetochlor
Pretilachlor
S-metolachlor
Fenoxaprop-p-ethyl
Fluazifop-p-butyl
Quizalofop-p-ethyl
Paraquat
Fluoroglycofen
Prodiamine
Pyraufen-ethyl
Triuralin
Flutamone
Mesotrione
Anilofos
Glufosinate-ammonium
Glyphosate
2,4-D Na Salt
Metsulfuron-methyl
Rimsulfuron
Sulfometuron-methyl
Atrazine
Metamitron
Terbutryn
Fungicides
Flutolanil
Isoprothiolane
Azoxystrobin
Cyazofamid
Picoxystrobin
Trioxystrobin
Cyproconazole
Epoxiconazole
Hexaconazole
Ipconazole
Tebuconazole

6.94 (1.23)

(1.48)
(0.88)
(1.69)
(1.09)
(0.78)
(11.7)
(1.80)
(1.80)
(1.06)
(0.96)
(0.81)

ef
ef
de

Slope (SE)

LC50 (95% CI) mg kg

15.2
8.03
9.32
10.5
19.2
9.25
8.56
9.32
9.26

133.5 (124.5150.5)
363.3 (333.9407.4)
297.7 (251.5410.0)
192.6 (176.8220.0)
399.9 (307.8513.4)
6.06 (5.606.77) a
360.4 (332.2402.9)
351.7 (279.4495.1)
270.6 (249.9302.3)

c
ef
de
cd
ef

(2.60)
(1.05)
(1.62)
(1.50)
(12.4)
(1.25)
(1.13)
(3.12)
(1.25)

6.79 (1.09)

(1.08)
(0.96)
(1.65)
(0.6)
(0.87)
(12.1)
(1.89)
(1.27)
(1.34)
(0.68)
(1.04)

ef
de
de

Means followed by the same letters are not signicantly different at P = 0.05 within a column.

the exposure period increased for most of the pesticides tested.

Similar to the contact toxicity results, each of the pesticides evaluated in the articial soil test showed a different degree of toxicity
to E. fetida (Table 3).
3.2.1. Insecticides and acaricides
In the 7-day interval, the neonicotinoid insecticide clothianidin
showed the highest intrinsic toxicity with an LC50 value of 7.44
(6.659.06) mg
kg 1, followed by the heterocyclic acaricide fenpyroximate with an LC50 value of 97.06 (84.67119.7) mg kg 1, while
the other insecticides and acaricides exhibited relatively low toxicities to E. fetida with LC50 values ranging from 152.2 (138.4182.5)
to 582.8 (468.8969.7) mg kg 1. In the 14-day interval, among the
nine insecticides and two acaricides tested, the neonicotinoid
insecticide clothianidin still showed the highest intrinsic toxicity
with an LC50 value of 6.06 (5.606.77) mg kg 1, followed by the
heterocyclic acaricide fenpyroximate with an LC50 value of 75.52

(68.2186.57) mg kg 1, while the other insecticides and acaricides

exhibited relatively low toxicities against E. fetida with LC50 values
ranging from 133.5 (124.5150.5) to 399.9 (307.8513.4) mg kg 1.
The toxicities of the different insecticides and acaricides varied
widely. Based on the LC50 value, the insecticide clothianidin is
66.0- and 60.0-fold more toxic than lufenuron and buprofezin,
respectively, and acaricide fenpyroximate is 4.80-fold more toxic
than pyridaben in the 14-day interval. Moreover, as the exposure
period increased, the toxicity of each insecticide and acaricide also
increased. The toxicity of acephate in the 7-day interval was significantly lower than that in the 14-day interval. However, the toxicities of the other insecticides and acaricides in the 7-day interval
were apparently not lower than those of the 14-day interval: the
LC50 value of the insecticide pyridaphenthion decreased from
297.1 (271.3342.4) mg kg 1 in the 7-day interval to 270.6
(249.9302.3) mg kg 1 in the 14-day interval, while the LC50 value
of the acaricide pyridaben decreased from 516.8 (423.1755.5)

Y. Wang et al. / Chemosphere 88 (2012) 484491

mg kg 1 in the 7-day interval to 362.5 (317.3442.1) mg kg

the 14-day interval (Table 3).

in

3.2.2. Herbicides
Among the 23 evaluated herbicides, the diphenylether herbicides (utamone and pyraufen-ethyl) and bipyridylium herbicide
(paraquat) showed the least toxicity with their LC50 values
being > 1000 mg kg 1, while the LC50 values of other herbicides
ranged from 167.2 (94.51259.4) to 662.6 (423.31856.4) mg kg 1
at the 7-day interval. In the 14-day interval, the diphenylether herbicides (utamone and pyraufen-ethyl) and bipyridylium herbicide (paraquat) still exhibited the least toxicities with their LC50
values being > 1000 mg kg 1, while the LC50 values of other herbicides ranged from 106.6 (83.91122.9) to 516.2 (367.41014.9)
mg kg 1. Similar to the insecticides and acaricides, the toxicities
of the herbicides increased with exposed period, e.g., the toxicity
of terbutryn in the 14-day interval was signicantly higher than
that in the 7-day interval. In contrast, the toxicities of the other
herbicides in the 14-day interval were apparently not higher than
those of the 7-day interval, e.g., the LC50 of triuralin only decreased from 601.1 (488.7877.5) mg kg 1 at the 7-day interval
to 499.4 (421.5694.3) mg kg 1 at the 14-day interval. In addition,
different herbicides within the same chemical group have different
toxicities to E. fetida, e.g., the toxicity of acetanilide herbicide Smetolachlor was signicantly higher than that of pretilachlor,
and the toxicity of triazine herbicide terbutryn was also signicantly higher than that of atrazine in the 14-day interval (Table 3).
3.2.3. Fungicides
The toxicity of 11 fungicides of 5 chemical groups to E. fetida
was assessed in this study. The results demonstrated that different
fungicides varied widely in their soil toxicities and that different
fungicides within the same chemical group have different toxicities. In the 7-day interval, the strobilurin fungicide picoxystrobin
showed the highest toxicity with an LC50 value of 9.22 (7.45
10.65) mg kg 1, while the triazole fungicides hexaconazole and
tebuconazole exhibited the lowest toxicities against E. fetida compared with the other tested fungicides. In the 14-day interval, the
strobilurin fungicide picoxystrobin still showed the highest toxicity and had an LC50 value of 7.22 (5.298.68) mg kg 1, while the
triazole fungicide tebuconazole exhibited the lowest toxicity
among all of the evaluated fungicides. Although the toxicity of each
fungicide increased with treated time, this phenomenon was not
immediately apparent since the LC50 of tebuconazole decreased
from 1035 (842.41469.9) mg kg 1 in the 7-day interval to 895.2
(754.21198.0) mg kg 1 in the 14-day interval. In contrast, the different fungicides within the same chemical group had signicantly
different toxicities against E. fetida, e.g., the toxicity of the strobilurin fungicide picoxystrobin was 55.6- and 45.3-fold more toxic
than that of trioxystrobin and azoxystrobin, respectively, while
the toxicity of the triazole fungicide cyproconazole was 4.23-fold
more toxic than that of tebuconazole (Table 3).

4. Discussion
Laboratory tests play an important role in the risk assessment of
chemicals toward earthworms and are considered valuable if they
predict the effects on earthworms under eld conditions (Heimbach, 1998; Zhou et al., 2008). Among these, acute toxicity tests
are considered the most relevant for laboratory testing, and earthworm mortality has been the main end point (Kula, 1998).
Although considering it to be of low ecological relevance,
Heimbach (1992) found a reasonable correlation between the
results of acute toxicity tests and the effects observed in the eld.

489

Several earthworm protocols have been developed for testing

the toxic potential of chemicals and contaminated soil, e.g., the
immersion test, topical application test, injection test, forced feeding test, feeding on treated food test, natural soil test, artisol test,
contact lter paper test, and articial soil test (Heimbach, 1984;
Luo et al., 1999; Kandalkar and Naik, 2004). Among these, only
contact lter paper test and articial soil test exposure protocols
using mortality (LC50) as the toxic endpoint and E. fetida as the test
species have received the most attention, with the latter being
adopted by both OECD (1984) and European Economic Community
(EEC, 1985) in Europe and the United States Environmental Protection Agency (Greene et al., 1989) in USA. The contact lter paper
test is simpler, cheaper, and faster, and is designed in such a way
that the earthworms are exposed to the toxicant both by contact
and in the aquatic phase (Tripathi et al., 2010). This test is reported
to be an excellent screening technique to assess relative toxicity
(Miyazaki et al., 2002; Grumiaux et al., 2010). In contrast, the articial soil test is more representative of the natural earthworm
environment, and the chemicals are absorbed mainly by the gut
in this method (Udovic and Lestan, 2010). Thus, the articial soil
test is more practical when the pesticide toxicities to earthworms
are assessed.
Clothianidin, a neonicotinoid insecticide, is particularly effective
against sucking and chewing pests (Jeschke and Nauen, 2008). The
insecticide acts as a competitive inhibitor on the nicotinic acetylcholine receptor in the central nervous system (Tomizawa and
Casida, 2003). Results from this study showed that clothianidin
was the most toxic to E. fetida among all the pesticides tested under
two different testing systems. Although organophosphates and carbamates are structurally different, both inhibit acetylcholinesterase
(AChE) activity (Carmo et al., 2010). IGRs inhibit chitin synthesis
and kill target insects by disturbing exoskeleton formation after
molting, and they are able to specically kill the target insect pests
with minimal effect on non-target organisms (Schneider et al.,
2008). In general, most organophosphates and IGRs have low toxicity to earthworms (Edwards and Bohlen, 1992). In this study, the
toxicities of organophosphates and IGRs to E. fetida were similar
using the articial soil test method and were relatively low compared to those of carbamates. Similar results have also been obtained in other earthworm species (Stenersen et al., 1973;
Stenersen, 1979). It has been demonstrated that many organophosphates such as azinphos-methyl, diazinon, fenitrothion, and malathion are only slightly toxic or not toxic to earthworms (Hopkins
and Kirk, 1957; Grifths et al., 1967; Voronova, 1968). Since organophosphates and carbamates both inhibit acetylcholinesterase
(AChE), the higher toxicity of carbamates to E. fetida than organophosphates may be due to the reversibility of AChE inhibition confers advantages to the former over the latter. The acaricides had
relatively low toxicities to E. fetida in the soil test method, while
they are supertoxic in the contact test system, showing that these
pesticides are more easily absorbed by the skin than by the gut.
Herbicides are by far the most commonly used pesticide category worldwide, especially in the USA (Short and Colborn, 1999;
Muthukaruppan et al., 2005). The issue of herbicide side effects
on earthworms is very important since their use is increasing year
by year (Zhou et al., 2006). Very few herbicides are directly toxic to
earthworms (Edwards, 1989), although they may exert considerable indirect effects due to their inuence on weeds as a source
of supply of organic matter on which earthworms feed in soil
(Edwards, 1989). Several herbicides (acetochlor, anilofos, utamone, pretilachlor, S-metolachlor, and terbutryn) were very toxic
in contact toxicity but were low toxic in soil toxicity testing. Similar results have been reported in other studies (Lydy and Linck,
2003; Mosleh et al., 2003; Xiao et al., 2006a). Therefore, an ecotoxicological assessment of the effects of herbicides on earthworms
should be carefully evaluated.

490

Y. Wang et al. / Chemosphere 88 (2012) 484491

Similar to herbicides, fungicides also comprise a very important

pesticides category in agricultural areas worldwide (Carmo et al.,
2010). The strobilurin fungicide picoxystrobin had both high contact toxicity and high soil toxicity, similar to the neonicotinoid
insecticide clothianidin. In contrast, most of the other fungicides
tested had high contact toxicity and exhibited low soil toxicity,
and their toxicities differed less than 6-fold in the soil testing system. In earlier studies, none of the tested fungicides were toxic to
earthworms, with the exception of the carbamate fungicides such
as benomyl, which is very toxic, and carbendazim, which is moderately toxic (van Gestel, 1992; Leistra and Matser, 2005; Ellis et al.,
2007).
The acute toxicity test has been traditionally used to assess the
toxicity of soil contamination to earthworms (Chen et al., 2011).
The results in the present study are based on acute toxicity, but
the adverse effect that occurred after subchronic or chronic exposure is also important in ecological risk assessments (Jensen et al.,
2007; Liu et al., 2011). Moreover, sublethal endpoints involving
various biomarkers are also used, such as lysosomal membrane
stability measured by neutral red retention time and genetic effects assessed by comet assay (Lock and Janssen, 2002; Xiao
et al., 2006b). The use of mixed pesticides is becoming increasingly
popular in agriculture owing to their high efciency, convenience,
and rapid action. Results from single-pesticide experiments did not
actually reect eld situations in which multiple pesticides or pesticide mixtures are used (Zhou et al., 2011). Therefore, more studies on the long-term effects of insecticides on earthworms are
needed for adequate ecological risk assessment. Furthermore, juvenile earthworms may be more sensitive to pollutants than adults
(Zhou et al., 2008). Estimating ecotoxicological risk using toxicity
data from adults and single-pesticide experiments may lead to
underestimation of the effects of pollutants on soil invertebrate
populations (van Straalen and Denneman, 1989).
Acknowledgments
The authors acknowledge the technical assistance of Xiao Hu
and Weihua Yu (Zhejiang Academy of Agricultural Sciences). This
research was supported by the International Cooperation Fund
and the National High-Tech R&D Program of the Ministry of Science and Technology of China (Grant Nos. S2010GR0905 and
2011AA100806) and the cooperation project of Zhejiang Academy
of Agricultural Sciences and Chinese Academy of Sciences.
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