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International Biodeterioration & Biodegradation 77 (2013) 39e44

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Biodegradation of cypermethrin by Bacillus sp. in soil microcosm and in-vitrotoxicity evaluation on human cell line

Smita Sundaram, Mihir Tanay Das, Indu Shekhar Thakur*

School of Environmental Sciences, Jawaharlal Nehru University, JNU Campus, New Delhi 110 067, India

a r t i c l e i n f o

Article history:Received 5 October 2012Received in revised form8 November 2012Accepted 9 November 2012Available online 23 December 2012

Keywords:Bacillus sp.BiodegradationDetoxificationSoil microcosmMTT assayComet assay

* Corresponding author. Tel.: þ91 11 26704321.E-mail addresses: smitasundaram1@gmail.com

hotmail.com (I.S. Thakur).

0964-8305/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2012.11.008

a b s t r a c t

Soil microcosms were set up with cypermethrin (100 mg/kg of dry soil) to assess the biodegradation byBacillus sp. strain ISTDS2. Soil samples from the microcosms were collected at regular intervals during thebiodegradation of cypermethrin and metabolites were extracted with hexane/Acetone (1:1v/v). GCeMSanalysis of the soil extract showed formation of degraded compounds like cyclopropane, carboxylic acid,hydroxyacetonitrile and benzene ethanamine. The toxic effects of degraded product were studied ona human hepatocarcinoma cell line HuH7 before and after bacterial treatment. Methyltetrazolium (MTT)assay for cytotoxicity and alkaline comet assay for genotoxicity were carried for toxicity evaluation. Thebacterium reduced toxicity as shownbya 105-fold increase in LC50 value, and a 3-fold reduction inOlive TailMoment after 30 days of treatment. Biodegradation ability of the strain without toxic by-products revealsits potential for further study as a biological agent for the remediation of soil contaminated withcypermethrin.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Earlier, the synthetic pyrethroid insecticides were considered asthe safest insecticides because of their quick insecticidal capabil-ities and generally low mammalian toxicity (Dorman and Beasley,1991). Recently, many studies showed a lot of environmental andhealth problems due to continuous and excessive use of syntheticpyrethroid insecticides like cypermethrin (Cuthbertson andMurchie, 2010). It is the most toxic type II synthetic pyrethroidwith a a-cyano group and is widely used in agricultural formulationthroughout the world to control a wide range of pests. Cyper-methrin was frequently detected in the environment, includingwater and soil sediments, due to run-off from agricultural fields(McKinlay et al., 2008). It was also reported that cypermethrinresidues are toxic to aquatic life and ultimately to human healththrough bioaccumulation and biomagnification in the food chain.The carcinogenic potential of pyrethroids has also been discussedand cypermethrin has been classified as a possible human carcin-ogen by US Environmental Protection Agency. Moreover, increasingevidences reveal that cypermethrin has endocrine toxicity, geno-toxic effect, neurotoxicological effects and also regarded as

(S. Sundaram), isthakur@

All rights reserved.

a tumour promoter (Chen et al., 2011). Some studies also indicatethat cypermethrin induces cell stress that alters antioxidantenzyme of human cell line. The cleavage of cypermethrin metab-olites releases cyanohydrin, gets decomposed into cyanide andaldehydes, which produce free radical in animal cell and alter themitochondrial dehydrogenase. Cypermethrin also causes inductionof DNA damage and micronucleus in-vitro in human lymphocytesand cultured mouse lymphocytes (Patel et al., 2007).

All these factors together make cypermethrin potentially harmfulto human health and ecosystem. Thus, an effective and environmentfriendly method is required to prevent environmental pollutioncaused by pyrethroid waste. Microbial biodegradation is one of themost influential and significant cost effective processes for pesticideremoval and environmental cleanup (Horne et al., 2002). It is animportant process which influences environmental degradation ofcypermethrin in soil (Lin et al., 2011). Hydrolysis of the ester linkage,whichproduces3-phenoxybenzaldehydeandcyclopropanecarboxylicderivatives, is the main degradation pathway of cypermethrin in soil.However, 3-phenoxybenzaldehyde is more persistent than cyper-methrin in soils, and their presence poses an environmental riskgreater than the parent compound (Garoiaz et al., 2011).

Many reports have shown detoxification efficiency by per-forming phytotoxicity bioassays like seed germination tests androot elongation tests (Jadhav et al., 2010). These bioassays are time-consuming, non-specific and more qualitative than quantitative. Inthis context In-vitro models using human cancer cell lines have

S. Sundaram et al. / International Biodeterioration & Biodegradation 77 (2013) 39e4440

become well-established tools for rapid and accurate evaluation oftoxicity at acute, chronic and sub chronic levels with fair repro-ducibility (Tai et al., 1994; Chang et al., 2007). Hepatocytes expressmany nuclear receptor proteins that regulate the expression ofxenobiotic metabolizing enzymes, including cytochromeP450 3A4(CYP3A4) responsible for the metabolism of multiple endogenousand exogenous chemicals making these cells ideal in-vitro modelsfor toxicological studies. It was reported that CYP3A4 was inducedmost powerfully by pyrethroids and 50 mM cypermethrin increasedCYP3A4 mRNA 35-fold (Khaled et al., 2012). In this regard, thehuman hepato-carcinoma HuH7 cell line has been shown to bepromising for toxicity evaluation under in-vitro condition (Daset al., 2012). Therefore, in the present investigation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)and alkaline comet assays were carried out with the HuH7 cell linefor comparative toxicity evaluation of cypermethrin toxicity beforeand after bacterial biodegradation. The MTT assay is an overallindicator of cytotoxicity and is based on the ability of living cells toreduce dissolved MTT (yellow) into insoluble formazan (blue) inthe presence of mitochondrial succinate dehydrogenase(Mosmann, 1983). While single cell gel electrophoresis (Cometassay) is a technique for quantifying DNA damage and repair underin vivo and in-vitro conditions. It measures, double strand breaks(DSBs), single strand breaks (SSBs), alkali labile sites, oxidative DNAbase damage, DNAeDNA/DNAeprotein/DNAedrug crosslinkingand DNA repair (Patel et al., 2007).

The present investigation was designed to study the degrada-tion of cypermethrin in soil microcosm by Bacillus sp. strain ISTS2.The study simultaneously aims to reveal howMTT assay along withalkaline comet assay in HuH7 cell lines can be used to evaluatebiodegradation and detoxification efficiency.

2. Materials and methods

Analytical grade cypermethrin (Catalogue no. 36128) wasprocured from Sigma Aldrich (St. Louis, MO, USA). All otherchemicals were purchased from Sigma Aldrich or Merck (Darm-stadt, Germany) unless stated otherwise, and were used withoutfurther purification.

2.1. Enrichment and screening of degrading strains

The bacterial strain ISTDS2 (gene bank accession numberJX473586) was isolated from Alwar marble mining site (27.56 N,76.62 E) Rajasthan, India and was identified to be Bacillus sp. by 16SrDNA sequencing method as described earlier by Mishra andThakur (2010). The bacteria was shown to sequester CO2 chemo-lithotrophically in minimal salt medium (MSM) through contin-uous culture in chemostat (paper communicated). In the presentstudy the bacteria was evaluated for its potential to grow inpesticide contaminated soil so that it can be used in agriculturalfields for CO2 sequestration and pesticide degradation. Theenriched bacterium of the chemostat was further grown in shakeflask culture in MSM containing (g/l), Na2HPO4$2H2O, 7.8; KH2PO4,6.8; MgSO4, 0.2, ammonium ferric citrate, 0.01; Ca(NO3)2$4H2O,0.05; NaNO3, 0.085, trace element solution, and cypermethrin(50 mg/l) as carbon source, adjusted to pH 7.0, stirring at 150 rpmand temperature 30 �C (Thakur, 1995). Culture medium wasremoved after different time intervals for determination of thegrowth and utilization of cypermethrin by GC. Percent utilization ofcypermethrin was determined by area of the peak obtained by GC.The strain utilized cypermethrin as sole carbon source for growthand was further selected for degrading studies in soil microcosm.Enriched bacterial cells were harvested fromMSM culture mediumand were grown in LB broth for inoculation in soil microcosm.

2.2. Soil microcosm

Microcosms were set up in sealed plastic jars according toGautam et al. (2003). In microcosm, 500 g of small grits were placedat the bottom followed by 500 g sand and 1 kg of garden soil (loamysoil, pH 7.5 and not treated with any pesticide for at least 10 years)at the surface. Soil was dried at 50 �C for 3 days, sieved througha 0.4 cm sieve and sterilized in small paper packets by autoclavingthree times on consecutive days at 121 �C for 60 min. Steriledistilled water was added to the soil to reach final moisture contentof 40% (volume/weight). Cypermethrin was added to the finalconcentration 100 mg per kilogram of dry soil in acetone solution.After mixing and solvent evaporation, the microbial suspensionwas inoculated into soils to make the final concentration of5.0� 107 cells/g and then incubated at 30 �C. Five microcosms wereprepared with duplicates to facilitate sample collection at fivedifferent time intervals from designated microcosms. Four micro-cosms were inoculated with bacterial culture and one was kept assuch and was taken as 0 h control. Microcosms were incubated at30 �C. After 0, 5,15 and 30 days, 500 g of soil samples were collectedfrom microcosms for extraction and analysis of metabolites.

2.3. Extraction of compounds from sediment and GCeMS analysis

500 g soil samples were removed at various intervals aftertreatment with cypermethrin. Extraction of organic compoundsfrom the soil samples was carried out according to Das et al. (2012)with some modifications. Briefly, soil samples were air dried andpassed through a 2 mm screen before Soxhlet extraction (US EPAmethod 3500C, 2007) with hexane/acetone (1:1, v/v) for 24 h.The filtered extracts were divided into two parts (4:1) and bothparts were evaporated to dryness at room temperature usinga vacuum rotary evaporator. First part was reconstituted with 1 mlof DMSO for toxicological analysis while the other was dissolved in1 ml acetone for GCeMS analysis.

The analysis was done using a GCeMS instrument (ShimadzuQP2010 Plus) equipped with a capillary column (DB-5 MS; 0.25 mmfilm thickness, 0.25 mm i.d., 30 m in length). One mL of each samplewas analysed by GCeMS at conditions: split less mode; initialtemperature 80 �C held for 2 min; temperature increased from 80to 200 �C at a rate of 10 �Cmin�1 held for 6 min on reaching 200 �C;temperature further increased from 200 to 300 �C at a rate of5 �Cmin�1 held for 15min on reaching 300 �C. The head pressure ofthe helium carrier gas was 81.7 kPa; helium flow rate 1.21 ml/min.Data were compared with the inbuilt standard mass spectra librarysystem (NIST-05 and Wiley-8) of GCeMS (Lin et al., 2011).

2.4. Toxicological analysis

2.4.1. Cell cultureHuH7 cells were maintained in Dulbecco’s Modified Eagle’s

Medium (DMEM) supplemented with 10% foetal bovine serum, 1%antibiotic antimycotic solution (final concentrations: penicillin,100 units/ml; streptomycin, 0.1 mg/ml; amphotericin B, 2.5 ng/ml)in a humidified atmosphere of 5% CO2 at 37 �C.

For MTT assay, cells were seeded in 96-well surface treatedpolystyrene tissue culture plates (Corning 3596) whereas foralkaline comet assay cells were seeded in 12-well plates (Costar,Corning 3513) both at a density of approximately 2.5�105 cells/ml.After 24 h, at 90% confluence, cells were treated with 0.5% DMSO,with positive control chemical [Benzo (a) Pyrene (BaP), 50 mM],with reference chemical [cypermethrin (CYP), 1 mM] or with testsamples for another 24 h. All positive control chemicals and testsamples were dissolved in DMSO and were added to the cellcultures in different dilutions to work out the dose response

Time (h)

0 20 40 60 80 100 120 140 160 180

Abso

rban

ce 6

00nm

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Perc

enta

ge u

tilis

atio

n of

cyp

erm

ethr

in

0

20

40

60

80

100

120Bacterial GrowthCypermethrin utilization

Fig. 1. Growth and utilization of cypermethrin by Bacillus sp. strain ISTS2 in minimalsalt medium (MSM) containing cypermethrin (50 mg/l) as a sole source of energy.Percentage utilisation of cypermethrin was determined by GCeMS peak area. 100%presence of cypermethrin was considered at 0 h sample.

Table 1Identification of metabolites formed at different time intervals after bacterialdegradation.

Peakretentiontime (min)

Present in sample Name of identified compound

T0 T5 T15 T30

11.98 e þ þ þ Benzeneacetic acid16.62 e þ e e 3-Phynoxy benzoic acid18.98 e e e þ Hexadecane20.09 e e e þ Methylicosan23.41 e þ e e 3-(2,2-dichlorovinyl) 2,2-dimethyl

cyclopropane carboxylate (DCVA)23.49 e e e þ 4-Cyclohepta-2,4,6-trienyl-benzoylazide23.65 e þ e e 3-Phynoxy phenyl hydroxyacetonitrile23.74 e þ þ e Cyclopropane carboxylic acid25.08 þ e e e Cypermethrin

S. Sundaram et al. / International Biodeterioration & Biodegradation 77 (2013) 39e44 41

relationships. The final concentration of DMSO in the medium was0.5% and all experiments were carried out in three replicates (Daset al., 2012).

2.4.2. Cell viabilityThe effect of test compounds or samples on cellular viability of

the cell lines was evaluated using the MTT assay (Mosmann, 1983;Laville et al., 2004). Cell viability was expressed as percentage of thecorresponding control (DMSO 0.5%). Further Cypermethrin-equivalents (CYP-Eq) for each sample were determined as the ratioof the LC50 of cypermethrin expressed as g/l to that of the sampleexpressed as g Sed Eq/l. Sigmoid doseeresponse curves for cyper-methrin and different samples along with their LC50 values werederived from the global curve fitting analysis with four parameterlogistic curve equation (Das et al., 2012). F test was carried out fordose response parallelism.

2.4.3. Alkaline single-cell gel electrophoresis e comet assayAlkaline comet assay was performed as described by Tice et al.

(2000). HuH7 cells treated with either test samples, positivecontrol (BaP, 50 mM) or negative control (DMSO 0.5%) for 24 h. Theslides were stained with ethidium bromide (2 mg/ml, 100 ml perslide) and visualised with a 40x lens fitted to a fluorescencemicroscope with an excitation and emission setting of 518/605 nm.The percentage of DNA in tail, tail moment and olive tail moment(OTM) of 40 randomly selected cells were analysed from each slideby using Comet Score Freeware software (www.tritekcorp. com).The comets were divided into five classes on the basis of amount ofDNA in the tail; Class I, less than 1% DNA in tail (intact nucleus);Class II, 1e20% DNA in tail; Class III, 20e50% DNA in tail; Class IV,50e75% DNA in tail and Class V, more than 75% DNA in tail(Miyamae et al., 1998).

2.5. Statistical analysis

All experimental data were expressed as means � standarddeviation of three replicates. All statistical analyses including globalcurve fitting and F test for dose response parallelism were per-formed with sigma plot 11 statistical package (Systat Software, SanJose, CA). Statistical differences between the control and treatedcells were examined with the aid of ANOVA followed by multiplecomparisons (Dunnett’s Method). A value of P < 0.05 was used todetermine significance in statistical analyses.

3. Results and discussion

3.1. Enrichment and utilization of cypermethrin by bacterium inMSM culture

Bacillus sp. strain ISTDS2 utilized cypermethrin (50 mg/L) asa source of energy during the process of enrichment in MSM-chemostat culture. The enriched bacterium of the chemostat wasfurther grown in shake flasks with MSM containing cypermethrin(50 mg/L). In shake flask culture the growth of bacterium and utili-zation of cypermethrin was determined from 0 h to 120 h. Acontinuous enhancement in the growth of the bacteriumwas regis-tered from0 h to 120 h (fifth day) indicating three growth phases i.e.,a short lag phase of 48 h, a log phase up to 120 h and a stationaryphase thereafter. Maximum utilization of cypermethrin coincidedwith the log phase of bacterial growth and cypermethrinwas almostcompletely utilized within 180 h (Fig. 1). The bacterial growthsuggests that Bacillus sp. has potency to grow on cypermethrin asa sole carbon source and utilize it. Earlier studies have reported thatmicrobial consortia may metabolize a particular compound asa single source of carbon and energy (Jilani and Khan, 2006).

3.2. Biodegradation of cypermethrin in soil microcosm and GCeMSanalysis

The soil samples were collected from soil microcosm at intervalsof 0 (T0), 5 (T5), 15 (T15) and 30 days (T30). The zero day sample istaken as control and compared with 5, 10, 30 days of degradedsample by GCeMS. The identified compounds in the chromato-graph extracted from T0, T5 and T30 samples respectively, aresummarised in Table 1. The degradation products mentioned in thetable were identified from the available standards of the authenticcompounds documented in NIST-05 libraries. Comparative analysisof total number of peaks in the GCeMS chromatogram bymicrobialdegradation was evaluated. An increase in the total number ofpeaks from T0 toT30was observed. The total number of peaks at T0was 2, T5 was 16 and T30 was 18. It can be inferred from this datathat initial complex compounds were broken down into simplercompounds which were later on gradually mineralized. Earlierstudies on biodegradation of complex molecules have reported anincrease in the number of peaks during the course of biodegrada-tion (Raj et al., 2007; Kaushik et al., 2010).

After five days, cypermethrin disappeared concomitantly withformation of cypermethrin metabolites. During biodegradation,there was an apparent peak of cypermethrin occurring at a reten-tion time of around 25.08 min in T0 sample. Complete removal of

Table 2MTTassay LC50 values of T0, T5, T15 and T30 samples along with corresponding CYP-Eq and comet assay OTM values.

Treatmentsa MTT LC50b R2(LC50) CYP-Eqc Comet assay: OTM

Cypermethrin (CYP) 3.2745 0.9943 e 4.7177 � 2.8332T0 5.0880 e2 0.9952 6.4357 e�3 6.2718 � 4.3960T5 5.4178 e3 0.9942 6.0440 e�4 4.3077 � 2.6705T15 1.8629 e5 0.9922 1.7578 e�5 2.7540 � 2.3179T30 5.4123 e7 0.9783 6.0501 e�8 1.8137 � 1.8174

a HuH7 cell lines were treated with different test samples (dilutions, 10�3e

104 g Sed Eq L�1) or Cypermethrin (dilutions, 10�5 to 10�2 g L�1) for 24 h for MTTassay. For comet assay only single concentration was considered; 1 mM for CYP and104 g Sed Eq L�1 for test samples.

b LC50 was derived using global curve fitting model with four parameters logisticnon-linear regression equation, expressed in terms of g Sed Eq L�1(test samples) org L�1 (CYP).

c CYP-Eq (g g Sed Eq�1) ¼ LC50 of CYP (g L�1)/LC50 of Sample (g Sed Eq L�1).

0.5%

) 120

Concentration (g Sed Eq L -1)

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

% C

ell V

iabi

lity

(100

% =

DM

SO 0

.5%

)

0

20

40

60

80

100

120

Cypermethrin (g L -1)

T0 (g Sed Eq L -1)

T5 (g Sed Eq L -1)

T15 (g Sed Eq L -1)

T30 (g Sed Eq L -1)

a

b

S. Sundaram et al. / International Biodeterioration & Biodegradation 77 (2013) 39e4442

the cypermethrin peak was observed in the treated sample asconfirmed on comparing with cypermethrin standard. The peakarea around 25.08 min was decreased and diminished completelyafter 5 days of incubation. Complete degradation of cypermethrinwas reported after 30 days. There was no detectable cypermethrindegraded products at T30. The results of the study indicatedcomplete mineralization of cypermethrin by Bacillus sp. within 30days.

Result of the study indicated that at T0 (before biodegradation),peak detected contain cypermethrin significantly. The emergence ofvaried and increased number of peaks at early retention times in theT5 samples were mostly identified as degraded products of cyper-methrinproducedby theactionofhydrolaseenzyme.GCeMSanalysisshowed formation of major biodegraded products of cypermethrinlike 3-phenoxy phenyl hydroxyacetonitrile, carboxylate, 3-(2, 2-dichloroethenyl)-2, 2-dimethylcyclopropanecarboxylate (DCVA) and3-phenoxybenzaldehyde (PBA).Gaoet al., 2010have reported that thecommon initial step inpyrethroidmetabolism in the soil is hydrolysisof the ester linkage leading to the formation of 3-phenoxy phenylhydroxyacetonitrile and DCVA (Fig. 2). Further, 3-phynoxy phenylhydroxyacetonitrile that contains the cyanohydrin functional group isunstable in the presence of water. Thus, cyanohydrin eliminates HCNin a non-enzymatic reaction to produce PBA. DCVA and PBA furtherundergo mineralisation process (Fig. 2). The strain ISTS2 cancompletely degrade100mg/kgof cypermethrin in the soilmicrocosmwithin 30 days. The degradation pathway followed by Bacillus sp. onthe basis of data obtained by GC/MS, involves oxidative as well ashydrolysing process in soil microcosm (Fig. 2). The degradationpathway construct on the basis of biodegradation ability of strainwithout toxic by-products revealed its potential for further study asa biological agent for the remediation of soil contaminated withcypermethrin.

3.3. Cell viability

The cell viability in terms of MTT assay derived LC50 values(Table 2) and the dose response curves (Fig. 3a) showed that thelevel of toxicity decreased with increasing duration of bacterialtreatment. As the data failed F test for dose response parallelismonly the minimum and maximum parameters of four parameterlogistic equation were shared to obtain LC50 values which werefurther used for calculation of biological CYP-Eqs (Table 2). Thelowest value of LC50 was found for the T0 sample and the highest

Fig. 2. Proposed pathway for degradation of cypermethrin by Bacillus sp. strain ISTS2in soil microcosm.

for T30. LC50 values increased about 105 times after 30 days ofbacterial treatment. The highest values of CYP-Eqs were found forthe T0 sample (6.4357 e�3 g g Sed/Eq) and the lowest values werefound for 30 days treated (T30) sample (6.0501 e�8 g g Sed/Eq).

Treatment conditions

BaP 50 µM T0 T5T15

T30

DMSO 0.5%

CYP 1mM

% C

ell V

iabi

lity

(100

% =

DM

SO

0

20

40

60

80

100

Fig. 3. Evaluation of toxicity of cypermethrin degraded by Bacillus sp. strain ISTS2.Acronyms correspond to the different samples to which HuH7 cells were exposed. T0:soil sample extract at 0 h control, T5: metabolites extracted after 5 days, T15:metabolites extracted after 15 days, T30: metabolites extracted after 30 days. Valuesrepresent the mean � SD, n ¼ 3. (a) Cell viability measured after 24 h exposure period;100% cell viability was considered for 0.5% DMSO treatment. Global goodness of fitR2 ¼ 0.9926.

S. Sundaram et al. / International Biodeterioration & Biodegradation 77 (2013) 39e44 43

Thus post biodegradation increase in LC50 value and decrease inCYP-Eq value clearly indicates steady mineralisation of cyper-methrin in soil microcosm.

The relative cell viability against solvent control is shown inFig. 3b. HuH7 cell lines were treated for 24 h with different testsamples at concentration 104 g Sed Eq/l along with positive controlBaP (50 mM), reference chemical cypermethrin (1 mM) and solventcontrol DMSO (0.5% v/v) to find out relative cell viability. Cyper-methrin (1 mM) showed 69.7% cell viability whereas T0 and T30samples showed 25.6% and 78.4% respectively.

Previous in-vitro studies on human blood samples (Amer, 1993)and lymphocyte culture (Chakravarthi et al., 2007) have shown thetoxicological effects of the cypermethrin in human cell lines.According to their data low doses of cypermethrin pesticide couldinduce single strand breaks inDNAandwhen lymphocyte cellsweretreated with varying concentrations of cypermethrin from 3.6 to7.6 mM, the percentage of cells with aberration varied from 1 to 32.However no studies have so far reported any post biodegradation

Treatment conditionsBaP

50 µM T0 T5

T15 T

Tail

Mom

ent

0

10

20

30

40

50

**

*

*

a

b

Fig. 4. Genotoxicity of the contaminants before and after treatment with Bacillus sp. strainplotted against different samples. Tail moments of 40 randomly selected comets are prespercentiles; a solid line in the box presents the median value while dotted line represents moutlying points beyond 5th and 95th percentiles. Olive tail moments of same 50 comets are sfrom the DMSO control group (Dunnett’s Method p < 0.05) for both data sets. (b) Imagesethidium bromide. Roman numerical indicates class of comet.

cell viability assessment with cypermethrin on human cell lineHuH7.

3.4. Comet assay

The outcome of the single cell gel electrophoresis (Comet assay)with biodegraded (T5, T15, T30) and 0 h sample (T0) is shown inFig. 4a andb. The cometsweredivided intofiveclasses on thebasis ofamount of DNA in tail (Fig. 4b). HuH7 cells treated with the T0sample resulted in 67.5% comets that fell under either of the classesIII, IV orV;whereas only 25% comets fell under the same classeswithT30 sample. Tail moments of 40 randomly selected comets arepresented as quantile box plots. The plot shows that distribution ofcomets became more homogenous with lower tail moment(2.2882 � 3.5380) by the T30 sample in comparison to T0 sample(tail moment ¼ 10.6566 � 9.3808). This indicated that the cometclass shifted towards a lower value of tailmoment after the bacterialtreatment. The olive tail moment data showed a decreasing trend

30

DMSO 0.5%

CYP 1mM

Oliv

e Ta

il M

omen

t

0

5

10

15

20

25

30

Tail MomentOlive Tail Moment

*

*

ISTS2. Acronyms as mentioned in Fig. 3. (a) The tail moment and the olive tail momentented as quantile box plots. The edges of the box represent the 25th and the 75thean value. Error bars indicate 90th and 10th percentiles and the black circles indicatehown as the mean � standard deviation. The asterisk denotes the significant differenceof different classes of comets seen under fluorescent microscope after stained with

S. Sundaram et al. / International Biodeterioration & Biodegradation 77 (2013) 39e4444

with increasing duration of bacterial treatment. T30 sample resultedin a 3-fold decrease in DNA migration (OTM ¼ 1.8137 � 1.8174) incomparison to that of the T0 sample (OTM ¼ 6.2718 � 4.3960).Statistically significant DNA damage (Dunnett’s Method p < 0.05)noticed in T0, T5 and T15 samples with respect to negative controlgroup (DMSO 0.05%) confirmed the genotoxic nature of cyper-methrin contaminated soil.

Present study indicates cypermethrin produce cytotoxicity andDNA damage in mammalian cells. From the results of the cometassay, it is clear that bacterial treatment for 30 days was efficient inreducing the genotoxic nature of cypermethrin contaminated soilsediment. The application of this method in different fieldsmakes ita powerful tool in human genotoxic study as well as in the esti-mation of environmental pollution (Fucic, 1997).

4. Conclusion

In the present study Bacillus sp. strain ISTS2was evaluated for itsdegradation and detoxification potential. The bacterium is capableof degrading cypermethrin, a potent genotoxic pesticide. GCeMSanalysis showed complete mineralization of cypermethrin in soilmicrocosm and significant toxicity reduction was shown by MTTand Comet bioassays on human cell line. Novelty of the study lies inthe use of multiple bioassays using human cell lines for properevaluation of biodegradation and detoxification. The degradationpotential of the strain without any toxic by-products revealed itssignificance as a biological agent for the remediation of soilcontaminated with cypermethrin.

Acknowledgements

This paper was supported by the research grants of Sat paulMittal fellowship, New Delhi, India. We thank to Mr. Ajai Kumar ofAdvanced Instrumentation Research Facility (AIRF) JawaharlalNehru University, New Delhi for GCeMS analysis.

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