Chemosphere 88 (2012) 484–491

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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, ChinaKey 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 2011Received in revised form 27 February 2012Accepted 29 February 2012Available online 28 March 2012

Keywords:Soil invertebrateEcotoxicologyFungicidesInsecticidesHerbicides

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.02.086

⇑ Corresponding author at: Key Laboratory for PeMinistry of Agriculture, Institute of Quality andZhejiang Academy of Agricultural Sciences, HangzhTel.: +86 571 86404229; fax: +86 571 86402186.

E-mail address: zhaoxueping@tom.com (X. Zhao).

a b s t r a c t

This study was conducted to investigate comparative toxicity of 45 pesticides, including insecticides, aca-ricides, fungicides, and herbicides, toward the epigeic earthworm Eisenia fetida. Results from a 48-h filterpaper contact test indicated that clothianidin, fenpyroximate, and pyridaben were supertoxic to E. fetidawith LC50 values ranging from 0.28 (0.24–0.35) to 0.72 (0.60–0.94) lg cm�2, followed by carbaryl, pyri-daphenthion, azoxystrobin, cyproconazole, and picoxystrobin with LC50 values ranging from 2.72 (2.22–0.3.19) to 8.48 (7.38–10.21) lg cm�2, while the other pesticides ranged from being relatively nontoxic tovery toxic to the worms. When tested in artificial soil for 14 d, clothianidin and picoxystrobin showed thehighest intrinsic toxicity against E. fetida, and their LC50 values were 6.06 (5.60–6.77) and 7.22 (5.29–8.68) mg kg�1, respectively, followed by fenpyroximate with an LC50 of 75.52 (68.21–86.57) mg kg�1.However, the herbicides fluoroglycofen, paraquat, and pyraflufen-ethyl exhibited the lowest toxicitieswith LC50 values > 1000 mg kg�1. In contrast, the other pesticides exhibited relatively low toxicities withLC50 values ranging from 133.5 (124.5–150.5) to 895.2 (754.2–1198.0) mg kg�1. The data presented inthis paper provided useful information for evaluating the potential risk of these chemicals to soilinvertebrates.

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1. Introduction

Earthworms are an important component of decomposer com-munities and contribute significantly to organic matter decompo-sition, nutrient cycling, and soil formation (Coleman and Ingham,1988; Edwards and Bohlen, 1992). Located near the bottom ofthe terrestrial trophic level, earthworms use the sensitive receptorson their body surfaces to sense chemicals in the soil (Bouché,1992). Their ecological importance, high biomass in soil, and fre-quently observed sensitivity to relatively low concentrations ofenvironmental toxins make them one of the most suitable bioindi-cator organisms for risk assessment in the soil (Landrum et al.,2006). The abundance of earthworms in soil represents the healthof soil ecosystems and the level of environmental safety (Xiaoet al., 2004).

In order to increase the crop yield in agricultural areas, syn-thetic pesticides including insecticides, acaricides, fungicides, andherbicides were widely applied to control harmful organisms(Liang and Zhou, 2003; Xiao et al., 2006a; Zhou et al., 2011).

ll rights reserved.

sticide Residue Detection ofStandard for Agro-products,ou 310021, Zhejiang, China.

Pesticides are either directly applied to the soil to control soil-borne pests or are deposited on soil as runoff from foliar applica-tions (Hans et al., 1990; Wahanthaswamy and Patil, 2004; Guptaet al., 2011). Pesticide residues impair the physiological functionsof earthworms, leading to their mortality, but there are insufficientdata from field or laboratory assays to make accurate assessmentsof their relative toxicities (Ahmed, 1991; Maboeta et al., 2004). It ispossible that the chemicals will affect not only target species butalso non-target organisms in and adjacent to the target areas(Schulz et al., 2001). In agricultural areas worldwide, there is anincreasing concern about soil contamination due to the widespreaduse of pesticides (Reinecke and Reinecke, 2007). Several research-ers have advocated the use of earthworms as an ecotoxicologicalmodel for risk assessment and pesticide bioassay (Edwards andBohlen, 1992; Gupta et al., 2011).

Extensive toxicological tests have been conducted to elucidatethe toxicities of heavy metals, polychlorinated biphenyl, and cer-tain conventional pesticide categories to earthworms (Diercxsenset al., 1985; Kamitani and Kaneko, 2007; Reinecke and Reinecke,2007; Hackenberger et al., 2008). However, novel modes of pesti-cides that have recently been introduced are more specific forthe target organism, and there is a paucity of information on theirecotoxicological effects, including those on earthworms (Damalasand Eleftherohorinos, 2011). In the present study, experimentswere performed to evaluate the toxic effects of 45 pesticides from

Y. Wang et al. / Chemosphere 88 (2012) 484–491 485

four important pesticide categories—including insecticides, acari-cides, herbicides, and fungicides—on the epigeic earthworm Eiseniafetida. The main objective of this study was to determine the com-parative toxicity of these pesticides on E. fetida and their potentialto damage soil-dwelling invertebrates.

2. Materials and methods

2.1. Earthworm

The oligochaete E. fetida, one of the favorite worm species forcomposting and organic gardening, is frequently used as a biolog-ical monitor for testing the effects of contaminants on soil biota. Itis also the Organisation for Economic Co-operation and Develop-ment (OECD)-recommended earthworm test species (OECD,1984; Edwards and Coulson, 1992; Yasmin and D’Souza, 2007).Adult earthworms (weighing 350–500 mg) with well-developedclitella were purchased from the College of Animal Sciences, Zhe-jiang University, China, and cultured in the laboratory in artificialsoil according to OECD guidelines (OECD, 1984). Soils were mixedwith decayed leaves and decomposed pig manure, and kept atroom temperature (20 ± 1 �C). Soil water content was measuredevery week and a 35% maximum water-holding capacity wasmaintained by the addition of distilled water as needed. Additionalcontrol tests were carried out with chloracetamide as a toxic refer-ence standard.

2.2. Pesticides

Forty-five pesticides including 9 insecticides, 2 acaricides, 23herbicides, 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 ofthe chemicals on mortality but not the effects of the adjuvantsadded to the commercial products.

2.3. Toxicity test methods

2.3.1. Contact toxicity testWe performed a modified contact filter paper test (OECD, 1984).

A piece of filter paper was placed in a 9-cm petri dish, and treatedwith the test substance dissolved in 2 mL of acetone. After the sol-vent evaporated, the piece of filter paper was remoistened with2 mL of distilled water and 1 earthworm was placed on it. The dishwas incubated in the dark at 20 ± 1 �C for 48 h and mortality wasrecorded. An earthworm was considered dead if it failed to respondto a gentle mechanical touch on the front end.

Earthworms were held on wet filter paper for 24 h at 20 ± 1 �Cin the dark for purging of the gut contents. A preliminary testwas conducted to determine the concentration range of the testchemicals in which 0–100% mortality of the earthworm was ob-tained. To establish the concentration-mortality relationship,earthworms were exposed to at least five different concentrationsin a geometric series and a control for each chemical. Ten replica-tions were used for each concentration. Acetone was used as thecontrol. Treated earthworms were maintained at 20 ± 1 �C under80–85% relative humidity in the dark. Moreover, the mortality inthe controls should not exceed 10% at the end of either test.

2.3.2. Soil toxicity testThe artificial soil consisted of 10% ground sphagnum peat

(<0.5 mm), 20% kaolinite clay (>50% kaolinite), and 70% fine sand(OECD, 1984, 2004). A small amount of calcium carbonate wasadded to adjust the pH to 6.0 ± 0.5. In the toxicity tests, the water

content was adjusted to 35% of the dry weight. For each tested con-centration, the desired amount of pesticide was dissolved in 10 mLacetone and mixed with a small quantity of fine quartz sand. Thesand was mixed for least 1 h to evaporate the acetone and wasthen mixed thoroughly with the pre-moistened artificial soil in ahousehold mixer. The final moisture contents of the artificial soilwere adjusted to the described level by the addition of distilledwater. A total of 0.65 kg of soil (equivalent to 0.5 kg dry artificialsoil) was placed in a 500 mL glass jar (surface area, 63.6 cm2)and 10 adult earthworms were added to each jar. Controls wereprepared similarly but only with 10 ml acetone and no insecticide.The jars were loosely covered with polypropylene lids to allow forair exchange and stored at 20 ± 1 �C with 80–85% relative humidityunder 400–800 lux of constant light. Mortality was assessed at 7and 14 days after treatment. Besides, the mortality in the controlsshould not exceed 10% at the end of either test.

A range of concentrations including 0, 0.1, 1.0, 10, 100, and1000 mg kg�1 dry soil were used in the pre-trials to determinethe concentrations that produced 0–100% mortality. To obtainLC50, 5–6 test concentrations in a geometric series and a controlwere used for each pesticide. Three jars, each containing 10 adultearthworms, were used for each concentration. The earthwormswere preconditioned for 24 h under the same conditions describedabove in the untreated soil before the dose–response test.

2.4. Statistical analysis

A probit analysis was conducted to assess the acute toxicity ofpesticides to E. fetida using a program developed by Chi (Chi,1997). The significant level of mean separation (P < 0.05) detectedwas based on the lack of overlap between the 95% confidencelimits of 2 LC50 values (Prabhaker et al., 2011). In the contact filterpaper test method, based on the resulting LC50 values, the pesti-cides were classified as being supertoxic (<1.0 lg cm�2), extremelytoxic (1–10 lg cm�2), very toxic (10–100 lg cm�2), moderatelytoxic (100–1000 lg cm�2), or relatively nontoxic (>1000 lg cm�2)(Roberts and Dorough, 1984).

3. Results

3.1. Contact toxicity

The acute toxicities of 45 pesticides to E. fetida from the contactfilter paper test are shown in Table 2. The results demonstratedthat the different pesticides varied widely in their contact toxici-ties, while different pesticides within the same category had differ-ent toxicities to E. fetida. Two acaricides (fenpyroximate andpyridaben) and one insecticide clothianidin showed the highestintrinsic toxicity, followed by the insecticide pyridaphenthionand three fungicides (azoxystrobin, cyproconazole, and picoxyst-robin) compared with the other tested pesticides. Overall, amongthe four pesticide categories evaluated, acaricides, several insecti-cides, and fungicides showed great toxicities, while herbicidesexhibited relatively low toxicity against E. fetida (Table 2).

3.1.1. Insecticides and acaricidesThe toxicities of nine insecticides to E. fetida from the four

chemical groups (Table 1) were evaluated in this study. The variousinsecticides exhibited a wide range of contact toxicities. The neon-icotinoid insecticide clothianidin was supertoxic to E. fetida withan LC50 value of 0.28 (0.24–0.35) lg cm�2, and the organophos-phate insecticides pyridaphenthion and carbamate insecticide car-baryl were extremely toxic with LC50 values of 3.84 (2.87–6.18)and 4.21 (2.69–5.57) lg cm�2, respectively. In contrast, four insectgrowth regulators (IGRs; buprofezin, flonicamid, fufenozide, and

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

Chemical group Pesticides Technical grade (a.i.) (%) Manufacturer

InsecticidesCarbamate Carbaryl 98 Jiangsu Changlong Chemical Co., Ltd.IGRsa Buprofezin 98.2 Jiangsu Changlong Chemical Co., Ltd.IGRsa Flonicamid 98.7 Ishihara Sangyo Kaisha Ltd.IGRsa Fufenozide 96.5 Jiangsu Pesticide Institute Co., Ltd.IGRsa Lufenuron 98 Zhejiang shangying Fine Chemical Co., Ltd.Neonicotinoid Clothianidin 96.5 Hunan Bide Biochemical Co., Ltd.Organophosphate Acephate 94.9 Zhejiang Ruite Chemical Co., Ltd.Organophosphate Malathion 96 Jiangsu Xinyin Taisong Chemical Co., Ltd.Organophosphate Pyridaphenthion 95 Anhui Xinsaide Chemical Co., Ltd

AcaricidesHeterocyclic Fenpyroximate 97 Xinyin Yongcheng Chemical Co., Ltd.Heterocyclic Pyridaben 95 Nanjing Red-Sun Group Co., Ltd.HerbicidesAcetanilide Acetochlor 93 Shandong Qiaochang Chemical Co., Ltd.Acetanilide Pretilachlor 98.8 Hangzhou Qingfeng Agrochemical Co., Ltd.Acetanilide S-metolachlor 96 Zhongnong Minchang Chemical Co., Ltd.APPc Fenoxaprop-p-ethyl 95 Jiangsu Tianrong Chemical Co., Ltd.APPc Fluazifop-p-butyl 90 Zhejiang Yongnong Co., Ltd.APPc Quizalofop-p-ethyl 95 Jiangsu Tianyong Group Chemical Co., Ltd.Bipyridylium Paraquat 42 Shandong Lvfeng Pesticide Co., Ltd.Diphenylether Fluoroglycofen 95 Lianyungong Liben Chemical Co., Ltd.Dinitroaniline Prodiamine 97 Sichuan Luzhou Orient Chemical Co., Ltd.Diphenylether Pyraflufen-ethyl 95 Ishihara Sangyo Kaisha Ltd.Dinitroaniline Trifluralin 97.2 Dongyang Dongnong Chemical Co., Ltd.Imidazolinone Flutamone 98 Shanghai Henong Pesticide Co., Ltd.Imidazolinone Mesotrione 95 Liaoning Dandong Pesticide Co., Ltd.Organophosphorus Anilofos 95 Shandong Binnong Technology Co., Ltd.Organophosphorus Glufosinate-ammonium 96 Sichuan Lier Chemical Co., Ltd.Organophosphorus Glyphosate 95 Jiangsu Nantong Taihe Chemical Co., Ltd.PAb 2,4-D Na Salt 87 Jiangsu Biochemical Co., Ltd.Sulfonylurea Metsulfuron-methyl 97 Jiangsu Ruidong Pesticide Co., Ltd.Sulfonylurea Rimsulfuron 99 DuPont Company of USASulfonylurea Sulfometuron-methyl 95 DuPont Company of USATriazine Atrazine 95 Zhejiang Changxing Chemical Co., Ltd.Triazine Metamitron 98 Zhejiang Leshi Chemical Co., LtdTriazine Terbutryn 97 Shandong Binnong Technology Co., Ltd.

FungicidesCAAd Flutolanil 98 Jiangsu Taizhou Baili Chemical Co., Ltd.Heterocyclic Isoprothiolane 95 Jiangsu Zhongqi Chemical Co., Ltd.Strobilurin Azoxystrobin 98 Zhejiang Shangyin Fine Chemical Co., Ltd.Sulfonyl imidazole Cyazofamid 96.9 Ishihara Sangyo Kaisha Ltd.Strobilurin Picoxystrobin 99 DuPont Company of USAStrobilurin Trifloxystrobin 96 Qinghai Lvyuan Biotechnical Co., Ltd.Triazole Cyproconazole 95 Jiangsu Fengdeng Pesticide Co., Ltd.Triazole Epoxiconazole 96 Sichuan Lier Chemical Co., Ltd.Triazole Hexaconazole 95 Jiangsu Fengdeng Pesticide Co., Ltd.Triazole Ipconazole 97.5 Chemtura Corporation of USATriazole Tebuconazole 96 Jiangsu Fengdeng Pesticide Co., Ltd.

a IGRs, insect growth regulators.b PA, phenoxyalkanoic acid.c APP, aryloxyphenoxy propionic acid.d CAA, carboxylic acid amide.

486 Y. Wang et al. / Chemosphere 88 (2012) 484–491

lufenuron) and one organophosphate, acephate, were relativelynontoxic (i.e., all LC50 values > 1000 lg cm�2). Moreover, differentinsecticides within the same chemical group had differenttoxicities against E. fetida, e.g., the organophosphate insecticidepyridaphenthion was 29.8 �more toxic than malathion against E.fetida. The order of toxicity to E. fetida based on LC50 values wasas follows: clothianidin > pyridaphenthion and carbaryl > mala-thion > acephate, buprofezin, flonicamid, fufenozide, and lufenu-ron. The 2 acaricides fenpyroximate and pyridaben showedsimilar supertoxicities to E. fetida with LC50 values of 0.72 (0.60–0.94) and 0.55 (0.42–0.65) lg cm�2, respectively (Table 2).

3.1.2. HerbicidesSimilar to the insecticides, the toxicity results of the 23 herbi-

cides belonging to the 10 chemical groups induced variable toxicityresponses against E. fetida. The acetanilide herbicides acetochlor,

pretilachlor, and S-metolachlor were very toxic to E. fetida withLC50 values of 24.02 (19.72–31.76), 19.23 (11.54–25.92), and 20.63(17.70–49.76) lg cm�2, respectively, as well as the organophospho-rus herbicide anilofos, the triazine herbicide terbutryn, and the imi-dazolinone herbicide flutamone were also very toxic to E. fetida withLC50 values of 34.69 (29.20–44.01), 10.31 (8.31–12.23), and 26.98(21.94–37.14) lg cm�2, respectively, while diphenylether herbi-cides (fluoroglycofen and pyraflufen-ethyl), sulfonylurea herbicides(metsulfuron-methyl and rimsulfuron), dinitroaniline herbicides(prodiamine and trifluralin), and aryloxyphenoxy propionic acidherbicide (fenoxaprop-p-ethyl) were the least toxic against E. fetidaand had LC50 values > 1000 lg cm�2. In contrast, all of the othertested herbicides (atrazine, 2,4-D sodium salt, fluazifop-p-butyl,glufosinate-ammonium, glyphosate, mesotrione, metamitron, andparaquat) were moderately toxic with LC50 values ranging from102.5 (66.57–138.7) to 566.1 (437.4–905.4) lg cm�2.

Table 2Summary of parameter estimates for the acute toxicity with contact filter paper test of 45 pesticides to Eisenia fetida.

Pesticide Slope (SE) LC50 (95% CI) lg cm�2 Toxicity grade

InsecticidesCarbaryl 3.15 (0.46) 4.21 (2.69–5.57) c Extremely toxicBuprofezin >1000 Relatively nontoxicFlonicamid >1000 Relatively nontoxicFufenozide >1000 Relatively nontoxicLufenuron >1000 Relatively nontoxicClothianidin 4.12 (0.57) 0.28 (0.24–0.35) a SupertoxicAcephate >1000 Relatively nontoxicMalathion 3.17 (0.66) 114.4 (68.6–368.1) f Moderately toxicPyridaphenthion 3.10 (0.46) 3.84 (2.87–6.18) c Extremely toxic

AcaricidesFenpyroximate 4.38 (0.62) 0.72 (0.60–0.94) b SupertoxicPyridaben 5.30 (0.82) 0.55 (0.42–0.65) b Supertoxic

HerbicidesAcetochlor 3.76 (0.51) 24.02 (19.72–31.76) de Very toxicPretilachlor 3.25 (0.49) 19.23 (11.54–25.92) d Very toxicS-metolachlor 4.88 (0.73) 20.63 (17.70–49.76) de Very toxicFenoxaprop-p-ethyl >1000 Relatively nontoxicFluazifop-p-butyl 5.24 (0.82) 518.0 (408.6–782.3) h Moderately toxicQuizalofop-p-ethyl >1000 Relatively nontoxicParaquat 4.34 (0.61) 235.0 (199.4–279.5) fg Moderately toxicFluoroglycofen >1000 Relatively nontoxicProdiamine >1000 Relatively nontoxicPyraflufen-ethyl >1000 Relatively nontoxicTrifluralin >1000 Relatively nontoxicFlutamone 4.33 (0.62) 26.98 (21.94–37.14) de Very toxicMesotrione 6.65 (1.16) 545.1 (422.2–893.3) h Moderately toxicAnilofos 4.26 (0.60) 34.69 (29.20–44.0) de Very toxicGlufosinate ammonium 5.76 (0.94) 557.2 (417.5–897.1) h Moderately toxicGlyphosate 5.74 (0.93) 566.1 (437.4–905.4) h Moderately toxic2,4-D sodium salt 4.24 (0.60) 422.7 (344.7–577.3) h Moderately toxicMetsulfuron-methyl >1000 Relatively nontoxicRimsulfuron >1000 Relatively nontoxicSulfometuron-methyl >1000 Relatively nontoxicAtrazine 1.86 (0.26) 102.5 (66.57–138.7) f Moderately toxicMetamitron 3.20 (0.42) 407.5 (328.6–550.7) h Moderately toxicTerbutryn 4.07 (0.56) 10.31 (8.31–12.23) d Very toxic

FungicidesFlutolanil 4.35 (0.61) 11.03 (9.02–12.97) d Very toxicIsoprothiolane 3.84 (0.52) 91.95 (75.97–128.2) f Very toxicAzoxystrobin 4.59 (0.66) 2.72 (2.22–3.19) c Extremely toxicCyazofamid >1000 Relatively nontoxicPicoxystrobin 4.49 (0.66) 3.15 (2.17–3.96) c Extremely toxicTrifloxystrobin >1000 Relatively nontoxicCyproconazole 5.96 (0.98) 8.48 (7.38–10.21) d Extremely toxicEpoxiconazole >1000 Relatively nontoxicHexaconazole 4.31 (0.60) 12.07 (10.26–14.53) d Very toxicIpconazole 3.17 (0.39) 21.76 (17.44–26.32) de Very toxicTebuconazole 4.22 (0.61) 31.57 (25.66–36.62) de Very toxic

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

Y. Wang et al. / Chemosphere 88 (2012) 484–491 487

The different herbicides within the same chemical group haddifferent toxicities to E. fetida, e.g., the organophosphorus herbicideanilofos was 16.3- and 15.7-fold more toxic than glufosinate-ammonium and glyphosate, respectively, and the triazine herbicideterbutryn was 9.94- and 39.5-fold more toxic than atrazine andmetamitron, respectively. However, some exceptions were seen inthe case of acetanilide herbicides, e.g., acetochlor, pretilachlor,and S-metolachlor have the same toxicity level against E. fetida(Table 2).

3.1.3. FungicidesThe toxicity results of 11 fungicides from 5 chemical groups to

E. fetida are shown in Table 2. The strobilurin fungicides (azoxyst-robin and picoxystrobin) and triazole fungicide (cyproconazole)were extremely toxic; their LC50 values ranged from 2.72 (2.22–3.19) to 8.48 (7.38–10.21) lg cm�2. Triazole fungicides (hexaco-nazole, ipconazole, and tebuconazole), carboxylic acid amidefungicide (flutolanil), and heterocyclic fungicide (isoprothiolane)

were very toxic; their LC50 values ranged from 11.03 (9.02–12.97) to 91.95 (75.97–128.2) lg cm�2. In contrast, the other stro-biluron fungicide trifloxystrobin, the sulfonyl imidazole fungicidecyazofamid, and the triazole fungicide epoxiconazole were rela-tively nontoxic; all of their LC50 values were > 1000 lg cm�2. Ahigh degree of toxicity variation was found among fungicides ofthe same chemical group, e.g., the toxicities of the strobilurin fun-gicides azoxystrobin and picoxystrobin were similar but were sig-nificantly greater than that of trifloxystrobin against E. fetida, whilethe toxicities of triazole fungicides hexaconazole, ipconazole, andtebuconazole were apparently greater than that of epoxiconazolebut less than that of cyproconazole (Table 2).

3.2. Soil toxicity

The acute toxicities of 45 pesticides from the artificial soil testof E. fetida are shown in Table 3. The data exhibited a clear concen-tration-dependent relationship and the mortality increased when

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

Pesticides 7-day interval 14-day interval

Slope (SE) LC50 (95% CI) mg kg�1 Slope (SE) LC50 (95% CI) mg kg�1

InsecticidesCarbaryl 16.65 (2.90) 152.2 (138.4–182.5) c 15.2 (2.60) 133.5 (124.5–150.5) cBuprofezin 10.14 (1.50) 425.2 (383.0–505.4) ef 8.03 (1.05) 363.3 (333.9–407.4) efFlonicamid 16.73 (13.2) 350.8 (235.1–436.8) de 9.32 (1.62) 297.7 (251.5–410.0) deFufenozide 9.69 (1.44) 217.4 (194.9–261.0) cd 10.5 (1.50) 192.6 (176.8–220.0) cdLufenuron 19.25 (1.80) 450.7 (345.6–769.3) ef 19.2 (12.4) 399.9 (307.8–513.4) efClothianidin 12.29 (1.93) 7.44 (6.65–9.06) a 9.25 (1.25) 6.06 (5.60–6.77) aAcephate 7.82 (1.67) 582.8 (468.8–969.7) ef 8.56 (1.13) 360.4 (332.2–402.9) efMalathion 10.25 (1.04) 425.8 (314.6–614.8) ef 9.32 (3.12) 351.7 (279.4–495.1) dePyridaphenthion 10.14 (1.45) 297.1 (271.3–342.4) de 9.26 (1.25) 270.6 (249.9–302.3) de

AcaricidesFenpyroximate 6.45 (0.95) 97.06 (84.67–119.7) b 8.09 (1.33) 75.52(68.21–86.57) bPyridaben 8.45 (1.43) 516.8 (423.1–755.5) ef 6.30 (0.92) 362.5 (317.3–442.1) ef

HerbicidesAcetochlor 5.93 (0.86) 334.1 (284.3–432.0) de 6.75 (1.02) 283.0 (247.7–347.5) dePretilachlor 2.92 (0.62) 662.6 (423.3–1856.4) ef 3.35 (0.62) 516.2 (367.4–1014.9) fgS-metolachlor 3.62 (0.44) 187.8 (132.9–268.9) c 3.66 (0.44) 184.8 (133.6–256.4) cdFenoxaprop-p-ethyl 16.96 (13.2) 347.9 (289.6–458.1) de 12.08 (2.27) 306.5 (256.5–442.7) deFluazifop-p-butyl 10.79 (1.88) 456.4 (349.6–776.6) ef 11.59 (2.22) 416.4 (321.3–727.2) efQuizalofop-p-ethyl 8.63 (1.51) 413.1 (264.9–687.6) de 8.30 (1.42) 389.2 (309.6–605.3) efParaquat >1000 >1000Fluoroglycofen >1000 >1000Prodiamine 6.94 (1.23) 231.0 (205.8–271.5) cd 6.79 (1.09) 213.7 (191.1–246.3) cdPyraflufen-ethyl >1000 >1000Trifluralin 7.45 (1.20) 601.1 (488.7–877.5) ef 10.64 (1.89) 499.1 (421.5–694.3) fgFlutamone 17.26 (13.2) 344.0 (294.3–469.1) de 7.27 (1.14) 307.2 (258.7–414.6) deMesotrione 4.68 (0.65) 307.4 (220.7–801.8) de 5.11 (0.69) 258.3 (198.0–464.5) cdAnilofos 5.40 (0.64) 204.6 (173.0–276.0) c 6.51 (1.38) 180.1 (158.6–221.3) cdGlufosinate-ammonium 22.68 (14.9) 167.2 (94.51–259.4) c 22.99 (15.3) 162.2(102.7–284.6) cGlyphosate 3.86 (0.53) 345.8 (241.3–920.6) de 3.95 (0.53) 327.8 (238.8–672.2) de2,4-D Na Salt 5.13 (0.75) 335.5 (276.3–461.6) de 5.83 (0.83) 275.4 (236.1–349.6) deMetsulfuron-methyl 6.21 (1.02) 408.6 (329.4–543.7) de 6.23 (0.99) 382.8 (254.3–4231.8) efRimsulfuron 5.91 (0.92) 369.4 (298.3–538.4) de 6.25 (0.94) 330.5 (274.2–454.0) deSulfometuron-methyl 3.08 (0.59) 560.7 (385.8–1211.5) ef 3.42 (0.57) 448.6 (335.8–760.0) efAtrazine 5.01 (0.65) 204.8 (179.9–236.4) c 5.52 (0.75) 180.4 (158.4–204.5) cdMetamitron 5.29 (0.71) 228.3 (163.6–547.9) cd 5.24 (0.70) 195.2 (172.1–225.7) cdTerbutryn 4.55 (0.57) 177.9 (155.1–205.6) c 7.46 (1.18) 106.6 (83.91–122.9) b

FungicidesFlutolanil 8.70 (1.48) 184.9 (167.7–209.1) c 7.02 (1.08) 150.4 (132.0–169.7) cIsoprothiolane 5.92 (0.88) 330.4 (274.9–453.6) de 6.46 (0.96) 281.2 (240.7–361.9) deAzoxystrobin 9.51 (1.69) 362.4 (302.1–517.5) de 9.53 (1.65) 327.4 (279.9–439.0) deCyazofamid 6.92 (1.09) 673.3 (552.9–954.3) fg 6.41 (0.6) 568.7 (483.4–742.8) fgPicoxystrobin 5.57 (0.78) 9.22 (7.45–10.65) a 5.88 (0.87) 7.22 (5.29–8.68) aTrifloxystrobin 21.9 (11.7) 414.1 (316.7–5324.7) de 20.3 (12.1) 401.3(309.4–539.1) efCyproconazole 9.61 (1.80) 221.6 (199.2–266.1) cd 9.90 (1.89) 211.8 (191.7–249.6) cdEpoxiconazole 9.61 (1.80) 356.8 (286.8–561.5) de 7.81 (1.27) 333.1(275.6–470.6) deHexaconazole 6.69 (1.06) 840.7 (643.2–1426.1) fg 6.01 (1.34) 562.0 (476.5–733.4) fgIpconazole 6.46 (0.96) 281.2 (240.7–361.9) cd 5.06 (0.68) 249.2 (215.5–305.7) cdTebuconazole 5.33 (0.81) 1035 (842.4–1469.9) fg 6.78 (1.04) 895.2 (754.2–1198.0) gh

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

488 Y. Wang et al. / Chemosphere 88 (2012) 484–491

the exposure period increased for most of the pesticides tested.Similar to the contact toxicity results, each of the pesticides evalu-ated in the artificial soil test showed a different degree of toxicityto E. fetida (Table 3).

3.2.1. Insecticides and acaricidesIn the 7-day interval, the neonicotinoid insecticide clothianidin

showed the highest intrinsic toxicity with an LC50 value of 7.44(6.65–9.06) mg�kg�1, followed by the heterocyclic acaricide fenpy-roximate with an LC50 value of 97.06 (84.67–119.7) mg kg�1, whilethe other insecticides and acaricides exhibited relatively low toxic-ities to E. fetida with LC50 values ranging from 152.2 (138.4–182.5)to 582.8 (468.8–969.7) mg kg�1. In the 14-day interval, among thenine insecticides and two acaricides tested, the neonicotinoidinsecticide clothianidin still showed the highest intrinsic toxicitywith an LC50 value of 6.06 (5.60–6.77) mg kg�1, followed by theheterocyclic acaricide fenpyroximate with an LC50 value of 75.52

(68.21–86.57) mg kg�1, while the other insecticides and acaricidesexhibited relatively low toxicities against E. fetida with LC50 valuesranging from 133.5 (124.5–150.5) to 399.9 (307.8–513.4) mg kg�1.The toxicities of the different insecticides and acaricides variedwidely. Based on the LC50 value, the insecticide clothianidin is66.0- and 60.0-fold more toxic than lufenuron and buprofezin,respectively, and acaricide fenpyroximate is 4.80-fold more toxicthan pyridaben in the 14-day interval. Moreover, as the exposureperiod increased, the toxicity of each insecticide and acaricide alsoincreased. The toxicity of acephate in the 7-day interval was signif-icantly lower than that in the 14-day interval. However, the toxic-ities of the other insecticides and acaricides in the 7-day intervalwere apparently not lower than those of the 14-day interval: theLC50 value of the insecticide pyridaphenthion decreased from297.1 (271.3–342.4) mg kg�1 in the 7-day interval to 270.6(249.9–302.3) mg kg�1 in the 14-day interval, while the LC50 valueof the acaricide pyridaben decreased from 516.8 (423.1–755.5)

Y. Wang et al. / Chemosphere 88 (2012) 484–491 489

mg kg�1 in the 7-day interval to 362.5 (317.3–442.1) mg kg�1 inthe 14-day interval (Table 3).

3.2.2. HerbicidesAmong the 23 evaluated herbicides, the diphenylether herbi-

cides (flutamone and pyraflufen-ethyl) and bipyridylium herbicide(paraquat) showed the least toxicity with their LC50 valuesbeing > 1000 mg kg�1, while the LC50 values of other herbicidesranged from 167.2 (94.51–259.4) to 662.6 (423.3–1856.4) mg kg�1

at the 7-day interval. In the 14-day interval, the diphenylether her-bicides (flutamone and pyraflufen-ethyl) and bipyridylium herbi-cide (paraquat) still exhibited the least toxicities with their LC50

values being > 1000 mg kg�1, while the LC50 values of other herbi-cides ranged from 106.6 (83.91–122.9) to 516.2 (367.4–1014.9)mg kg�1. Similar to the insecticides and acaricides, the toxicitiesof the herbicides increased with exposed period, e.g., the toxicityof terbutryn in the 14-day interval was significantly higher thanthat in the 7-day interval. In contrast, the toxicities of the otherherbicides in the 14-day interval were apparently not higher thanthose of the 7-day interval, e.g., the LC50 of trifluralin only de-creased from 601.1 (488.7–877.5) mg kg�1 at the 7-day intervalto 499.4 (421.5–694.3) mg kg�1 at the 14-day interval. In addition,different herbicides within the same chemical group have differenttoxicities to E. fetida, e.g., the toxicity of acetanilide herbicide S-metolachlor was significantly higher than that of pretilachlor,and the toxicity of triazine herbicide terbutryn was also signifi-cantly higher than that of atrazine in the 14-day interval (Table 3).

3.2.3. FungicidesThe toxicity of 11 fungicides of 5 chemical groups to E. fetida

was assessed in this study. The results demonstrated that differentfungicides varied widely in their soil toxicities and that differentfungicides within the same chemical group have different toxici-ties. In the 7-day interval, the strobilurin fungicide picoxystrobinshowed the highest toxicity with an LC50 value of 9.22 (7.45–10.65) mg kg�1, while the triazole fungicides hexaconazole andtebuconazole exhibited the lowest toxicities against E. fetida com-pared with the other tested fungicides. In the 14-day interval, thestrobilurin fungicide picoxystrobin still showed the highest toxic-ity and had an LC50 value of 7.22 (5.29–8.68) mg kg�1, while thetriazole fungicide tebuconazole exhibited the lowest toxicityamong all of the evaluated fungicides. Although the toxicity of eachfungicide increased with treated time, this phenomenon was notimmediately apparent since the LC50 of tebuconazole decreasedfrom 1035 (842.4–1469.9) mg kg�1 in the 7-day interval to 895.2(754.2–1198.0) mg kg�1 in the 14-day interval. In contrast, the dif-ferent fungicides within the same chemical group had significantlydifferent toxicities against E. fetida, e.g., the toxicity of the strobilu-rin fungicide picoxystrobin was 55.6- and 45.3-fold more toxicthan that of trifloxystrobin and azoxystrobin, respectively, whilethe toxicity of the triazole fungicide cyproconazole was 4.23-foldmore toxic than that of tebuconazole (Table 3).

4. Discussion

Laboratory tests play an important role in the risk assessment ofchemicals toward earthworms and are considered valuable if theypredict the effects on earthworms under field conditions (Heim-bach, 1998; Zhou et al., 2008). Among these, acute toxicity testsare considered the most relevant for laboratory testing, and earth-worm 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 theresults of acute toxicity tests and the effects observed in the field.

Several earthworm protocols have been developed for testingthe toxic potential of chemicals and contaminated soil, e.g., theimmersion test, topical application test, injection test, forced feed-ing test, feeding on treated food test, natural soil test, artisol test,contact filter paper test, and artificial soil test (Heimbach, 1984;Luo et al., 1999; Kandalkar and Naik, 2004). Among these, onlycontact filter paper test and artificial soil test exposure protocolsusing mortality (LC50) as the toxic endpoint and E. fetida as the testspecies have received the most attention, with the latter beingadopted by both OECD (1984) and European Economic Community(EEC, 1985) in Europe and the United States Environmental Protec-tion Agency (Greene et al., 1989) in USA. The contact filter papertest is simpler, cheaper, and faster, and is designed in such a waythat the earthworms are exposed to the toxicant both by contactand in the aquatic phase (Tripathi et al., 2010). This test is reportedto be an excellent screening technique to assess relative toxicity(Miyazaki et al., 2002; Grumiaux et al., 2010). In contrast, the arti-ficial soil test is more representative of the natural earthwormenvironment, and the chemicals are absorbed mainly by the gutin this method (Udovic and Lestan, 2010). Thus, the artificial soiltest is more practical when the pesticide toxicities to earthwormsare assessed.

Clothianidin, a neonicotinoid insecticide, is particularly effectiveagainst sucking and chewing pests (Jeschke and Nauen, 2008). Theinsecticide acts as a competitive inhibitor on the nicotinic acetyl-choline receptor in the central nervous system (Tomizawa andCasida, 2003). Results from this study showed that clothianidinwas the most toxic to E. fetida among all the pesticides tested undertwo different testing systems. Although organophosphates and car-bamates are structurally different, both inhibit acetylcholinesterase(AChE) activity (Carmo et al., 2010). IGRs inhibit chitin synthesisand kill target insects by disturbing exoskeleton formation aftermolting, and they are able to specifically kill the target insect pestswith minimal effect on non-target organisms (Schneider et al.,2008). In general, most organophosphates and IGRs have low toxic-ity to earthworms (Edwards and Bohlen, 1992). In this study, thetoxicities of organophosphates and IGRs to E. fetida were similarusing the artificial soil test method and were relatively low com-pared to those of carbamates. Similar results have also been ob-tained in other earthworm species (Stenersen et al., 1973;Stenersen, 1979). It has been demonstrated that many organophos-phates such as azinphos-methyl, diazinon, fenitrothion, and mala-thion are only slightly toxic or not toxic to earthworms (Hopkinsand Kirk, 1957; Griffiths et al., 1967; Voronova, 1968). Since orga-nophosphates and carbamates both inhibit acetylcholinesterase(AChE), the higher toxicity of carbamates to E. fetida than organo-phosphates may be due to the reversibility of AChE inhibition con-fers advantages to the former over the latter. The acaricides hadrelatively low toxicities to E. fetida in the soil test method, whilethey are supertoxic in the contact test system, showing that thesepesticides are more easily absorbed by the skin than by the gut.

Herbicides are by far the most commonly used pesticide cate-gory worldwide, especially in the USA (Short and Colborn, 1999;Muthukaruppan et al., 2005). The issue of herbicide side effectson earthworms is very important since their use is increasing yearby year (Zhou et al., 2006). Very few herbicides are directly toxic toearthworms (Edwards, 1989), although they may exert consider-able indirect effects due to their influence on weeds as a sourceof supply of organic matter on which earthworms feed in soil(Edwards, 1989). Several herbicides (acetochlor, anilofos, fluta-mone, pretilachlor, S-metolachlor, and terbutryn) were very toxicin contact toxicity but were low toxic in soil toxicity testing. Sim-ilar results have been reported in other studies (Lydy and Linck,2003; Mosleh et al., 2003; Xiao et al., 2006a). Therefore, an ecotox-icological assessment of the effects of herbicides on earthwormsshould be carefully evaluated.

490 Y. Wang et al. / Chemosphere 88 (2012) 484–491

Similar to herbicides, fungicides also comprise a very importantpesticides category in agricultural areas worldwide (Carmo et al.,2010). The strobilurin fungicide picoxystrobin had both high con-tact toxicity and high soil toxicity, similar to the neonicotinoidinsecticide clothianidin. In contrast, most of the other fungicidestested had high contact toxicity and exhibited low soil toxicity,and their toxicities differed less than 6-fold in the soil testing sys-tem. In earlier studies, none of the tested fungicides were toxic toearthworms, with the exception of the carbamate fungicides suchas benomyl, which is very toxic, and carbendazim, which is moder-ately toxic (van Gestel, 1992; Leistra and Matser, 2005; Ellis et al.,2007).

The acute toxicity test has been traditionally used to assess thetoxicity of soil contamination to earthworms (Chen et al., 2011).The results in the present study are based on acute toxicity, butthe adverse effect that occurred after subchronic or chronic expo-sure is also important in ecological risk assessments (Jensen et al.,2007; Liu et al., 2011). Moreover, sublethal endpoints involvingvarious biomarkers are also used, such as lysosomal membranestability measured by neutral red retention time and genetic ef-fects assessed by comet assay (Lock and Janssen, 2002; Xiaoet al., 2006b). The use of mixed pesticides is becoming increasinglypopular in agriculture owing to their high efficiency, convenience,and rapid action. Results from single-pesticide experiments did notactually reflect field situations in which multiple pesticides or pes-ticide mixtures are used (Zhou et al., 2011). Therefore, more stud-ies on the long-term effects of insecticides on earthworms areneeded for adequate ecological risk assessment. Furthermore, juve-nile earthworms may be more sensitive to pollutants than adults(Zhou et al., 2008). Estimating ecotoxicological risk using toxicitydata from adults and single-pesticide experiments may lead tounderestimation of the effects of pollutants on soil invertebratepopulations (van Straalen and Denneman, 1989).

Acknowledgments

The authors acknowledge the technical assistance of Xiao Huand Weihua Yu (Zhejiang Academy of Agricultural Sciences). Thisresearch was supported by the International Cooperation Fundand the National High-Tech R&D Program of the Ministry of Sci-ence and Technology of China (Grant Nos. S2010GR0905 and2011AA100806) and the cooperation project of Zhejiang Academyof Agricultural Sciences and Chinese Academy of Sciences.

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