ORIGINAL ARTICLE

Comparative Study of Biochemical Responses in ThreeSpecies of Earthworms Exposed to Pesticide and MetalContaminated Soil

Venkadapathi Jeyanthi1,2 & James Arockia John Paul3 &

Balan Karunai Selvi4 & Natchimuthu Karmegam5

Received: 24 August 2015 /Accepted: 14 January 2016 /Published online: 26 January 2016# Springer International Publishing Switzerland 2016

Abstract The aim of this study was to investigate the biochemical responses in three earthwormspecies, Eudrilus eugeniae, Perionyx ceylanensis and Perionyx excavatus induced by exposureto pesticide (carbaryl) and metal (lead) in soil. Earthworms were exposed to increasing concen-trations of carbaryl (12, 25 and 50 mg kg −1) and Pb (75, 150 and 300 mg kg −1) in the soil fordifferent periods of time and the biochemical changes were determined. The protein content wasslightly increased in E. eugeniae, P. ceylanensis when exposed to the lower concentration of Pb(75 mg kg−1). But decreased protein content was observed in P. excavatus at the higherconcentration of carbaryl (50 mg kg−1), and with exposure to 300 mg kg−1 Pb in P. ceylanensis.The changes in the levels of antioxidant enzymes glutathione-S-transferase (GST), glutathioneperoxidase (GPx), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT)were used as functional biomarkers to determine the effect of pesticides and metals on earth-worms. Among these, GST exhibited more sensitive response to both carbaryl and Pb ascompared to the control. Other antioxidant enzymes showed little variation at the time ofexposure. The results of our study suggested that both carbaryl and Pb induced the production

Environ. Process. (2016) 3:167–178DOI 10.1007/s40710-016-0131-9

* Venkadapathi Jeyanthijeyanthi.j1@gmail.com

James Arockia John Pauljajpaul@gmail.com

1 Department of Biotechnology, Sri Kaliswari College, Sivakasi, Tamil Nadu 626 130, India2 Present address: Department of Biotechnology, School of Bioengineering, SRM University,

Kattankulathur, Chennai, Tamil Nadu 603 203, India3 Department of Zoology, Arumugam Pillai Seethai Ammal College, Tiruppattur, Tamil Nadu 630 211,

India4 Department of Botany, V. V. Vanniaperumal College for Women, Virudhunagar, Tamil Nadu 626 001,

India5 Department of Botany, Government Arts College, Salem, Tamil Nadu 636 007, India

of Reactive oxygen species (ROS) by causing oxidative damage to cells. Due to an adaptiveresponse of earthworms in contaminated soils, they could be used as significant biomarkers toasses the toxicity in the environment of the soil ecosystem. This is the first report on thecomparative study of antioxidant enzyme activities in three earthworm species under normaland stress induced conditions.

Keywords Antioxidant enzymes . Carbaryl . Coelomic fluid . Earthworms . Heavymetals

1 Introduction

Earthworms are important components of terrestrial ecosystems and they represent approxi-mately 60–80 % of the total soil biomass. They play an important role in soil processes such asimprovement of soil structure, nutrient cycling and decomposition of organic matter (Yang etal. 2012; Gomez-Eyles et al. 2009). Some major physiological functions such as nutrition,immunity, survival, growth and reproduction have been shown to be disrupted by exposure toenvironmental contaminants (Bernard et al. 2014). However, their successful survival understress conditions is supported by efficient innate immune mechanisms based on cellularactivities of coelomycetes and humoral immune proteins, both components of the earthwormcoelomic fluid (Kauschke et al. 2007). Earthworms have been widely used as an indicatororganism to evaluate the soil ecological toxicity according to the American Society for Testingand Materials (ASTM), the Organization for Economic Cooperation and Development(OECD), and the International Standards Organization (ISO) (Zhang et al. 2014).

Pesticides and heavy metals are common contaminants of the soil environment as a result ofvarious industrial and agricultural activities (Wang et al. 2010; Vandana and Keshav 2015).One pesticide of particular concern is Carbaryl (1-naphthyl-N-methyl carbamate), a carbamateinsecticide widely used in agricultural applications. The insecticide acts as a potentialneurotoxicant on non-target species and it inhibits the essential enzyme, acetylcholinesterase(AChE) in the nervous system of insects and other animal species (Gambi et al. 2007; Caselliet al. 2006). Some studies have been carried out to determine the toxicological effect ofcarbaryl in different earthworm species such as Eisenia andrei, P. excavatus, Pheretimaposthuma and Metaphire posthuma (Lima et al. 2015; Saxena et al. 2014). Ribera et al.(2001) studied the effects of carbaryl at different concentrations and exposure times to evaluatethe antioxidant reponses in earthworm Eisenia fetida andrei. Pollution due to heavy metals hasalso become a serious problem and hence, the release of such metals to the environment shouldbe restricted due to their non-biodegradable property. Heavy metals have been shown to causelysosomal membrane instability, changes in gene expression, growth reduction, cocoonproduction and slow down of sexual development that affects the population size and speciesdiversity of earthworms (Spurgeon et al. 2005; Zheng et al. 2013). Especially, Lead (Pb)affects earthworms survival, food uptake, behavior and disturbs almost every metabolicfunction in body chemistry (Langdon et al. 2005). Studies on the earthworm Lampito mauritiiexposed to Pb and Zn was carried out by Maity et al. (2008) to evaluate its antioxidant defencesystems. Zheng et al. (2013) reported the effect of multi metal contaminated soils (Cd, Cr, Cu,Ni, Pb, and Zn) to earthworm, Eisenia fetida inorder to evaluate the changes in antioxidantenzymes.

Many investigators have used E. fetida as the standard test species for ecotoxicity studiesdue to its tolerance to high concentration of pesticides (Ribera et al. 2001). Only few reports

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are available on toxicity assessment studies in E. eugeniae and P. excavatus but no reports areavailable for P. ceylanensis. However, this is the first report on the comparative study ofantioxidant responses in three earthworm species, E. eugeniae, P. excavatus and P. ceylanensisunder normal and stress conditions. The living organisms exposed to environmental pollutioncan produce Reactive oxygen species (ROS) and the accumulation of such ROS causes damageto cellular components such as proteins, nucleic acids, and lipids (Halliwell and Gutteridge 1999;Kammenga et al. 2000; Zhang et al. 2014). The antioxidant enzymes play an important role inprotecting the cells against reactive oxygen species (ROS) (Feng et al. 2015). Many enzymaticactivities have been considered as biomarkers of environmental pollution. Herein, the effects ofcarbaryl and Pb on antioxidant enzymes glutathione S-transferase (GST), glutathione peroxidase(GPx), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) wereinvestigated. Overall, an antioxidant response study helps us to understand the toxic effects onthree species of earthworms under normal and stress conditions.

2 Materials and Methods

2.1 Chemicals

All the chemicals used were of analytical grade and of highest purity purchased from SiscoResearch Laboratories (India) and Sigma Chemical Company (USA).

2.2 Experimental Animals

The pre-clitellate earthworms of Eudrilus eugeniae (African night crawler), Perionyxexcavatus (Asiatic species) and recently explored epigeic species, Perionyx ceylanensis(Indigenous species) (Paul et al. 2011) were selected as the test animals. Earthworms wereobtained from the Department of Biology, Gandhigram Rural University, Gandhigram,Dindigul District, Tamil Nadu, India. Animals were carefully transported to the laboratoryand mass multiplied in culture tanks by mixing cowdung powder with water and maintained ata temperature of 28 ± 2 °C.

2.3 Pesticide and Metal Exposure of Earthworms

All the experiments were carried out under laboratory conditions. Earthworms were exposed toincreasing concentrations of carbaryl (12, 25 and 50 mg kg −1 artificial soil) and Pb (75, 150and 300 mg kg −1 artificial soil) for 10 days and 14 days, respectively, using standard soiltesting methods (Maity et al. 2010; OECD 2002). For each species, six replicates of eighteensets of artificial soil substrate were prepared in plastic container by mixing cow dung and soil(1:1) with water at a ratio of 1:1 (w/v). Three types of artificial soil substrate were prepared asfollows: (i) control (soil + cow dung powder +10 earthworms) (ii) Metal treated (soil + cowdung powder + Pb +10 earthworms) and (iii) Pesticide treated (soil + cow dung powder +carbaryl +10 earthworms). The cow dung powder was amended with the appropriate amountof Pb and carbaryl solution made with distilled water. The same procedure using distilled waterwas applied to prepare a set of three control substrates without Pb and carbaryl. The averageheight of each substrate was 10 cm and initial pH of the substrate was 6.8 ± 0.05. Thetemperature was maintained at 28 ± 2 °C throughout the study period.

Responses of Earthworms Exposed to Pesticides and Metals in Soil 169

2.4 Collection of Earthworm Coelomic Fluid

After the 7th and 14th day of metal exposure and the 5th and 10th day of pesticide exposure,coelomic fluid was collected from exposed and control worms. The fluid sample was obtainedby puncturing post clitellum segments of the coelomic cavity with a Pasteur micropipetteand kept at 4 °C. Coelomic fluid suspension from each treated earthworms were pooledand centrifuged at 500 g for 10 min at 4 °C. Then, the supernatant was again centrifugedat 7000 g for 10 min at 4 °C and the resultant suspension was stored at −20 °C for further study(Kohlerova et al. 2004).

2.5 Determination of Total Protein Contents

The acetone precipitation method was used to isolate the proteins from the crude coelomicfluid sample. The coelomic fluid was added with four times the sample volume of cold(−20 °C) acetone. Then the samples were vortexed for ten seconds and kept at −20 °C for60 min. The samples were then centrifuged at 14,000 g for 10 min at 4 °C. The supernatantwas decanted and the precipitated protein pellet was dissolved in 20mMTris hydrochloride (Tris-HCl) for further analysis. Total crude coelomic fluid protein concentration for samples collectedfrom control and exposed organisms was determined according to the Lowry’s method usingbovine serum albumin as standard (Lowry et al. 1951).

2.6 Enzyme Bioassays

2.6.1 Sample Preparation

The gut cleaned earthworm tissue samples were homogenized in 0.1 M cold phosphate buffer(pH 7.2) and then centrifuged at 10,000 g for 20 min at 4 °C. The clear supernatants were usedfor the determination of SOD, GSH, GPx, GSTand CATactivities or stored at −80 °C for futurework (Song et al. 2009).

2.6.2 SOD

The activity of SOD was determined by measuring its ability to inhibit the photochemicalreduction of nitroblue tetrazolium chloride (NBT), as described by Giannopolitis and Ries(1977). The assay mixture contained the sample extract, 50 mM phosphate buffer (pH 7.8),100 mM ethylenediaminetetraacetic acid (EDTA), 130 mM methionine, 750 mM NBT and20 mM riboflavin. The reaction was allowed to proceed for 15 min. Absorbance of the reactionmixture was read at 560 nm. One unit of enzyme activity (U) was defined as the amount ofenzyme exhibiting 50 % inhibition of the auto-oxidation rate of NBT and the results wereexpressed as U/mg protein.

2.6.3 GSH

Reduced glutathione was measured according to the method of Anderson (1996). The reactionmixture contained the sample extract, 125 mM sodium phosphate, 6.3 mM disodium EDTA,0.3 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) (pH 7.5) and5,5’dithiobis-2-nitrobenzoic acid (DTNB) solution. One unit of GSH reductase was added to

170 Jeyanthi V. et al.

the assay mixture to convert all GSSG (oxidized glutathione) into GSH. After 5 min ofincubation, GSH content was measured at absorbance of 412 nm and the results were expressedas mg/100 g of tissue.

2.6.4 GPx

The GPx activity was measured as described by Clair and Chow (1996). The reaction mixturecontained the sample extract, 2 mM disodium EDTA, 1 mM sodium azide, 1 mM GSH,0.2 mMNADPH and 1 unit of glutathione reductase (GR) in 50 mM Tris-HCl buffer (pH 7.5).The reaction was started by addition of 1.2 mM cumene hydroperoxide and GPx was measuredbased on the decrease in absorbance at 340 nm. The results of enzyme activities were expressedas mg/100 g of tissue.

2.6.5 GST

The activity of GST was determined according to the method of Habig et al. (1974). Thereaction mixture contained 100 mM potassium phosphate buffer (6.9), 1 mM GSH, 1 mM1-chloro-2,4-dinitrobenzene (CDNB) and the sample extract. The rate of change in opticaldensity was measured at absorbance of 340 nm and the results were expressed as mg/100 g oftissue.

2.6.6 CAT

Catalase activity was determined according to the method of Claiborne (1985). The reactionmixture contained the sample extract, 66 mM phosphate buffer (pH 7.0) and 3 % hydrogenperoxide (H2O2). The decomposition of H2O2 by catalase present in the sample was deter-mined at absorbance of 240 nm and the results are expressed as U/mg of protein.

2.7 Statistical Analysis

The statistical difference of the growth rate and antioxidant enzymes in the coelomic fluid ofearthworms exposed to pesticide and metal stress with respect to the control sets weredetermined by the use of one-way analysis of variance (ANOVA). A significant differencewas indicated as P < 0.05. All values are presented as mean ± SD.

3 Results

3.1 Survival and Growth

During the present investigation, earthworms survival was 100 % in E. eugeniae and P.excavatus exposed to different concentrations of carbaryl and Pb at different exposure periods.But in the case of P. ceylanensis, the survival rate was 93.6 % and 95 % when exposed to thecarbaryl at 50 mg kg−1 for 10 days and Pb at 300 mg kg−1 for 14 days. No avoidance behaviorwere noted in all three species exposed to Pb and carbaryl at different concentrations. The bodyweight was not decreased significantly in all three earthworms species from 0 to 7 days of Pbtreatment (75 mg kg−1) and 0–5 days of carbaryl treatment (12 mg kg−1). But, when compared

Responses of Earthworms Exposed to Pesticides and Metals in Soil 171

to the control, the rate of body weight of earthworms was significantly reduced (P < 0.05) in allthree species when treated with increasing concentration of Pb after 7 days (150 and300 mg kg−1) and carbaryl (25 and 50 mg kg−1) after 5 days. Especially, in P. ceylanensis,there was a significant reduction (68.9 %) in body weight at 50 mg kg−1 of carbaryl on 10th day(Fig. 1a) and 71 % at 300 mg of Pb on 14th day (Fig. 1b). In E. eugeniae and P. excavatus, thegrowth rate was about 72.5% and 79.96% respectively at a concentration of 50mg kg1 carbarylon 10th day (Fig. 1a). The same two earthworm species showed a growth rate of 75.4 % and78.3% respectively with the Pb concentration of 300 mg kg−1 on 14th day (Fig. 1b). Our resultsrevealed that the low concentrations of carbaryl and Pb enhanced the growth of earthwormswhereas the gradual increase in concentration decreased the growth rate of earthworms.

3.2 Effect of Carbaryl and Lead on Protein Content

When treated with 75mg kg−1 of Pb, the protein content was slightly increased inP. ceylanensisand E. eugeniae as compared to the control. But at the concentration of 25 mg kg−1 of carbaryland 150 mg kg−1 of Pb, the protein contents were slightly decreased in all the test species ascompared to the control. When the concentration of carbaryl was increased at 50 mg kg−1,significant protein reduction was observed in P. excavatus as 1.20 mg mL−1 (Fig. 2a).Simultaneously, in P. ceylanensis, the protein content was reduced (2.22 mg mL−1) aftertreatment with the 300 mg kg−1 of Pb (Fig. 2b). In E. eugeniae, no significant protein contentreduction was observed under both carbaryl and Pb with increasing concentrations.

3.3 Antioxidant Enzymes Activities

In all the three earthworm species, the antioxidant enzymes activities showed no variationswith the concentrations of carbaryl (12 and 25 mg kg−1) and Pb (75 and 150 mg kg−1) atdifferent exposure times when compared to the control. But at the increasing concentration ofcarbaryl at 50 mg kg−1 and Pb at 150 mg kg−1 showed significant variation with P < 0.05 inantioxidant enzymes (Table 1).

When compared to E. eugeniae and P. excavatus, the activity of GSH was decreasedsignificantly as 7.18 ± 0.07 (mg/100 g) in P.ceylanensis on day 10 exposed to 50 mg kg−1 ofcarbaryl and also reduced as 7.36 ± 0.01 (mg/100 g) on day 14 exposed to 150 mg kg−1 of Pbas compared to the control. Similarly at the same concentration of carbaryl and Pb, the SODactivity was inhibited. Particularly, in E. eugeniae the activity was decreased as 2.95 ± 0.33 onday 14 exposed to Pb. The GPx activity was slightly increased compared to untreated controlin all test species exposed to both Pb and carbaryl.

The effect of maximum concentration of carbaryl (50 mg kg−1) and Pb (150 mg kg−1) onGST activity in three different groups of earthworm species are displayed in Table 1. In E.eugeniae and P. excavatus, there was a significant increase in the GST activity (266.00 ± 1.04and 272.43 ± 1.21 mg/100 g respectively) on 14th day of Pb treatment. When exposed tocarbaryl, the stimulation of same enzyme activity was observed in E. eugeniae and P.ceylanensis (247.00 ± 2.92 and 252.16 ± 1.1421 mg/100 g, respectively) on 10th day. Thesignificant inhibition in CAT activity was observed in E. eugeniae and P. excavatus on bothcarbaryl and Pb treatment. Especially in the case of Pb treated worms, the CAT level was foundas 14.2 ± 0.08 (U/mg) when compared to the control (22.4 ± 0.07 U/mg) in E. eugeniae.Simultaneously, in the case of P. excavatus, the same enzyme level was observed as16.5 ± 0.06 U/mg when compared to the untreated control (23.04 ± 0.09 U/mg).

172 Jeyanthi V. et al.

4 Discussion

Earthworms have long been known for their tolerance to toxic chemicals present in contam-inated soils and hence have been widely used as indicator organisms for ecotoxicity studies.

Fig. 1 Growth rate (%) of earthworms exposed to carbaryl (a) and lead (b)

Responses of Earthworms Exposed to Pesticides and Metals in Soil 173

However, earthworm survival in such stress conditions depends upon several physico-chemical factors such as soil texture, pH of the medium, organic matter, nature and extentof the clay minerals (Maity et al. 2008). According to our knowledge, many researchershave used E. fetida as a model test species for toxicity assessment studies. This is the firstattempt to investigate the effect of both pesticide (carbaryl) andmetal (Pb) on the disturbance of

Fig. 2 Protein concentration (mg/mL) in coelomic fluid from control earthworms and earthworms exposed tocarbaryl (a) and lead (b)

174 Jeyanthi V. et al.

the biochemical system in three different earthworm species, E. eugeniae, P. excavatus and P.ceylanensis.

In our study, it was found that the carbaryl (50 mg kg−1) stress caused a significant bodyweight reduction in P. ceylanensis and showed a growth rate of 68.9 % on 10th day whereas Pbat 300 mg kg−1 exposure for 14 days showed the 71 % growth rate as compared to E. eugeniaeand P. excavatus. The successful survival of earthworms in contaminated soils was due to itsdetoxification property. The results of our study indicated that the growth rate of earthwormswere not decreased at low concentrations of both carbaryl and Pb whereas the higherconcentration decreased the growth rate. A number of studies have been conducted todetermine the impact of pesticides and metals on the growth of earthworms. Lima et al.(2015) reported that the increasing concentration of carbaryl at 80 mg kg−1 in soils at 7 days ofexposure significantly affected the survival of earthworm, E. andrei. For determining the toxiceffects of acetochlorine and methamidophos, weight loss was considered to be a valuableparameter compared to mortality (Zhou et al. 2006). In addition, it was found that the leadcontamination of about >2000 mg kg−1 in the soils significantly affected the growth of adultearthworm, E. andrei with significant weight loss and complete inhibition of reproduction(Luo et al. 2014). Also, the juveniles of E. fetida were exposed to cadmium (Cd) 296 μg /g andlead (Pb) 911 μg/g (Zaltauskaite and Sodiene 2014) and were found to have significantreduction in weight, prolongation of the time to sexual maturation and reduction of cocoonproduction.

It was previously reported that the proteins from the coelomic fluid of earthworms possesseda wide range of biological activities such as antibacterial, hemolytic, cytotoxic, hemagglutinatingand proteolytic activities (Kobayashi et al. 2004). Zhang et al. (2013) reported that the protein

Table 1 Biochemical responses of earthworms exposed to pesticide (50 mg/kg) and metal stress (300 mg/kg).Values are mean ± SD

Treatments Biochemical parameters

Reducedglutathione(mg/100 g)

Glutathioneperoxidase(mg/100 g)

Glutathione-S-transferase(mg/100 g)

Superoxidedismutase(U/mg)

Catalase (U/mg)

E. eugeniae

Control 8.12 ± 0.06 4.32 ± 0.12 215.06 ± 1.90 4.32 ± 0.35 22.4 ± 0.07

Pesticide stress 7.84 ± 0.08 5.23 ± 0.15 247.00 ± 2.92* 3.54 ± 0.22* 14.6 ± 0.06*

Metal stress 7.65 ± 0.04 6.30 ± 0.09* 266.00 ± 1.04* 2.95 ± 0.33* 14.2 ± 0.08*

P. excavatu s

Control 8.29 ± 0.02 4.65 ± 0.10 218.13 ± 1.62 4.16 ± 0.21 23.04 ± 0.09

Pesticide stress 7.94 ± 0.03 5.83 ± 0.15* 234.16 ± 1.54* 3.12 ± 0.36* 17.2 ± 0.05*

Metal stress 7.73 ± 0.06 5.95 ± 0.12* 272.43 ± 1.21* 3.84 ± 0.27 16.5 ± 0.06*

P. ceylanensis

Control 8.52 ± 0.04 4.16 ± 0.08 224.29 ± 1.84 4.50 ± 0.16 23.12 ± 0.02

Pesticide stress 7.18 ± 0.07 6.08 ± 0.04* 252.16 ± 1.14* 2.66 ± 0.14* 20.30 ± 0.07

Metal stress 7.36 ± 0.01 6.15 ± 0.20* 246.12 ± 1.25* 3.16 ± 0.28* 18.70 ± 0.05*

*Difference in mean values between treatments with respective controls within columns for each species issignificant at p < 0.05 by ANOVA

Responses of Earthworms Exposed to Pesticides and Metals in Soil 175

content was decreased in earthworm, E. fetidawhen treated with aluminium at the concentrationof 300 mg kg−1. The decrease in protein contents were also observed in earthworm E. fetida dueto pesticide and Pb toxicity (Tai et al. 2008). In our study, with increase in the concentration of Pband carbaryl stress, the concentration of protein was either increased or decreased whencompared with the respective controls. But the significant protein reduction was observed inP. excavatus and P. ceylanensis when treated with increased concentration of carbaryl and Pbrespectively.

Many different types of environmental contaminants disturb the normal metabolic processof the cell resulting in the production of reactive oxygen species (ROS) causes damage of cellsand tissues, hence, the presence of antioxidant enzymes could be able to protect the cells (Fenget al. 2015). In the case of carbaryl (12 and 25 mg kg−1) and Pb (75 and 150 mg kg−1)treatment, no significant changes in antioxidant enzyme levels were observed in the threedifferent earthworm species. A significant decrease in SOD activity was observed in E.eugeniae exposed to 150 mg kg−1 of Pb on 14th day. A possible explanation would be thatthe inhibition of SOD by H2O2 (Sandalio et al. 2001). It was also demonstrated that significantdepletion in CAT enzyme was observed in E. eugeniae and P. excavatus exposed to the sameconcentration of Pb. These results are also supported by Liu et al. (2011) who reported theinhibition of SOD and CAT enzymes activities in E. fetida followed by the exposure of1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-c-2-benzopyran (HHCB) at the con-centration of 50–100 mg kg−1 after 7 days of exposure. Du et al. (2015) also indicated that theSOD and CAT levels were significantly decreased in E. fetida exposed to 50–100mg kg−1 of Di-n-butyl phthalates (DBP) for 28 days.

Many studies on the relationship between cellular glutathione level and metal toxicityshowed that the glutathione has a protective function against metal induced toxicity (Saint-Denis et al. 2001). The present investigation clearly demonstrates that the higher dosage of Pbsignificantly affected GSH, GST and GPx activities. The GSH inhibition was observed inP. ceylanensis on day 10 and 14 exposed to 50 mg kg−1 of carbaryl and 150 mg kg−1 of Pb,respectively. This is in agreement with other studies on Lampito mauritii when exposed toincreasing concentrations of Pb (75, 150, 300 mg kg−1) for 7 days, which resulted in depletionof GSH level (Maity et al. 2008). In our study, a significant enhancement in GST level wasobserved in E. eugeniae and P. excavatus exposed to Pb and the same enzyme activity wasincreased in E. eugeniae and P. ceylanensis under carbaryl stress condition. The GPx activitywas also stimulated in all three earthworm species due to both carbaryl and Pb treatment.Maity et al. (2008) also found that GST and GPx levels were increased in L. mauritii exposedto different concentrations of Pb at 75, 150 and 300 mg kg−1 in contaminated soil. In our study,the activity profile of GST, GPx, GSH, SOD and CAT clearly indicated the tolerance capacityof earthworms after long-term exposure of pesticide and metal contaminated soil. Moreover,the antioxidant enzymes in earthworms are directly involved in the adaptive response ofearthworm survival and neutralize the free radicals and ROS.

5 Conclusions

In the present study, measurements of different parameters, such as the earthworm survival,growth rate, protein concentration and antioxidant enzymes, were used to evaluate the toxicity inthree different earthworm species, E. eugeniae, P. excavatus and P. ceylanensis. At the higherconcentration of carbaryl (50 mg kg1) and Pb (300 mg kg−1) treatment, the body weight of

176 Jeyanthi V. et al.

P. ceylanensis was reduced compared to E. eugeniae and P. excavatus. In addition, the proteinsynthesis was also affected in P. excavatus and P. ceylanensis. Antioxidant enzymes, includingGST, GPx, GSH, SOD and CAT, were used as potential biomarkers to assess the ecotoxicolog-ical effects on earthworms. Both carbaryl and Pb exposure induced oxidative stress in all thetest species. The antioxidant profile results showed the earthworm ability to survive atmaximum exposure time and concentration of carbaryl and Pb. GST was more sensitive tothese soil contaminants when compared to other enzyme activities. Our results suggest thatearthworms could be used as important bioindicators for monitoring chemical toxicity in the soilecosystem.

Acknowledgments Authors sincerely acknowledge Prof. (Mrs.) Thilagavathy Daniel, Department of Biology,Gandhigram Rural Institute, Gandhigram for her keen interest in the study and for valuable suggestions.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest.

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