Detection of Tannery Effluents Induced DNA Damage in Mung ...downloads.hindawi.com/journals/isrn.biotechnology/2014/727623.pdf · Detection of Tannery Effluents Induced DNA Damage

Research ArticleDetection of Tannery Effluents Induced DNA Damage in MungBean by Use of Random Amplified Polymorphic DNA Markers

Abhay Raj1 Sharad Kumar1 Izharul Haq1 and Mahadeo Kumar2

1 Environmental Microbiology Section CSIR-Indian Institute of Toxicology Research MG Marg Post Box No 80Lucknow 226 001 India

2 Animal Facility CSIR-Indian Institute of Toxicology Research MG Marg Post Box No 80 Lucknow 226 001 India

Correspondence should be addressed to Abhay Raj arajiitrresin

Received 19 November 2013 Accepted 29 January 2014 Published 11 March 2014

Academic Editors A DrsquoAnnibale C Lucas C D Murphy and A Tiessen

Copyright copy 2014 Abhay Raj et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Common effluent treatment plant (CETP) is employed for treatment of tannery effluent However the performance of CETP forreducing the genotoxic substances from the raw effluent is not known In this study phytotoxic and genotoxic effects of tanneryeffluents were investigated in mung bean (Vigna radiata (L) Wilczek) For this purpose untreated and treated tannery effluentswere collected from CETP Unnao (UP) India Seeds of mung bean were grown in soil irrigated with various concentrations oftannery effluents (0 25 50 75 and 100) for 15 days Inhibition of seed germination was 90 by 25 untreated effluent and 75treated effluent compared to the control Plant growth was inhibited by 51 and 41 when irrigated with untreated and treatedeffluents at 25 concentration RAPD technique was used to evaluate the genotoxic effect of tannery effluents (untreated andtreated) irrigation on the mung bean The RAPD profiles obtained showed that both untreated and treated were having genotoxiceffects onmung bean plantsThis was discernible with appearancedisappearance of bands in the treatments compared with controlplants A total of 87 RAPD bands were obtained using eight primers and 42 (48) of these showed polymorphism Irrigating plantswith untreated effluent caused 12 new bands to appear and 18 to disappear Treated effluent caused 8 new bands and the loss of15 bands The genetic distances shown on the dendrogram revealed that control plants and those irrigated with treated effluentwere clustered in one group (joined at distance of 028) whereas those irrigated with untreated effluent were separated in anothercluster at larger distance (joined at distance of 042) This indicates that treated effluent is less genotoxic than the untreated Neirsquosgenetic similarity indices calculated between the treatments and the control plants showed that the control and the plants irrigatedwith treated tannery effluent had a similarity index of 075 the control and plants irrigated with untreated 065 and between thetreatments 068 We conclude that both untreated and treated effluents contain genotoxic substances that caused DNA damageto mung beans CETP Unnao removes some but not all genotoxic substances from tannery effluent Consequently use of bothuntreated and treated wastewater for irrigation poses health hazard to human and the environment

1 Introduction

Large amount of water and chemicals like chromium sodiumchloride sulphate calcium salts ammonium salts sodiumsulphide alkali acids fat liquor and organic dyes are usedin tannery during processing of raw hideskin into leatherThe resultant effluents contain excess amount of dissolvedsalts chloride sulphide chromium and high BOD andCOD in the effluent [1] India is the third largest producerof leather in the world having about 3000 tanneries withannual processing capacity of 07 million tonnes of hides and

skin [2] Nearly 80 of the industries in India are engaged inthe chrome-tanning process Residual chromium (trivalentCr III and hexavalent Cr VI) thus is discharged in solid orliquid effluents Soluble Cr VI is extremely toxic and showsmutagenic and carcinogenic effects on biological systemsdue to its strong oxidizing nature Cr III is less toxic andbioavailable than Cr VI as it readily forms insoluble oxidesand hydroxides above pH 5 In case of plants tannery effluentis known to inhibit seed germination and root growth [3 4]

Higher plant provides a useful genetic system for screen-ing andmonitoring of environmental pollutants [5]They are

Hindawi Publishing CorporationISRN BiotechnologyVolume 2014 Article ID 727623 8 pageshttpdxdoiorg1011552014727623

2 ISRN Biotechnology

good indicators of cytogenetic and mutagenic effect whichcan be applied both indoors and outdoors Several studieshave used the chromosome aberration micronucleus orcomet assay to measure the effects of tannery effluent onplants [2 6] The micronucleus test in organism assays isnot a sensitive method for determining pollution levels ofheavy metals in terrestrial ecosystems and another difficultylinked to the comet assay is the necessity to experiment onisolated cells and their inability to provide information onthe effects of toxic metals at the DNA level [7] Advantageof measuring effect of genotoxic chemicals directly on DNAis mainly related to the sensitivity and short time responseRecently advances in molecular biology have led to devel-opment of a number of selective and sensitive assays forDNA analysis in ecogenotoxicology DNA based techniques(RFLP QTL RAPD AFLP SSR and VNTR) are used toevaluate the variation at the DNA sequence level Randomamplified polymorphic DNA (RAPD) of these techniques isa PCR-based method that amplifies random DNA fragmentswith the use of single short primers of arbitrary nucleotidesequence under low annealing conditions RAPD can beused to detect genotoxicity and differences can be clearlyshownwhen comparingDNAfingerprint fromuntreated andtreated individuals to genotoxic agents [8ndash14]

Unnao district (UP) India is one of the major industrialareas especially for leather tannery industry Most of thetanneries in this area are in the small scale and cannot affordexpensive treatment plant on their ownTherefore tomitigatethe pollution from tanneries a common effluent treatmentplant (CETP) was established in 1996 having treating capacityof 215million liters per day (MLD)This is an activated sludgeprocess-based plant which receives 19 million liters per day(MLD) of tannery wastewater from a cluster of 21 tanneriesprocessing approximately 475 tons average daily raw hidesThe treated effluent from CETP is finally discharged in theGanga River through drain This wastewater is used forirrigation purposes in agricultural field on both sides ofdrain before entering in the Ganga River Ramteke et al[15] assessed the efficiency of CETP and found a significantreduction in COD and BOD levels during the course oftreatment in CETP However the impact of CETP treatmentfor reducing genotoxic substances from raw effluent is notknown Therefore in the present study the impact of CETPon genotoxic activity of tannery effluent was assessed byplant genotoxicity analysis The present study uses RAPDtechnology to evaluate the genotoxic effects in mung beanplant irrigated with untreated and treated tannery effluents

2 Materials and Methods

21 Effluent Samples and Physicochemical Analysis Theuntreated (UT) and treated (T) effluents were collected frominlet and outlet points respectively from common effluenttreatment plant (CETP) Unnao (2648∘N 8043∘) UP stateIndia during the month of February 2013 Samples weretaken in 5-litre Jerry cane and transported to laboratoryfor analysis The effluents were filtered through Whatmanfilter paper number 1 to remove suspended particles and

stored at 4∘C till completion of the analysis The followingparameters were determined based on standard methods[16] pH total dissolved solids (TDS) total hardness totalalkalinity chemical oxygen demand (COD) biochemicaloxygen demand (BOD) and total chromium (total Cr)Total Cr was analyzed with atomic absorption spectrometer(Perkin Elmer model A Analyst-300) after digestion ofsamples (100mL) in digestion mixture of (5 1) of nitric-perchloric acid [16] Electrical conductivity was determinedby conductivity meter (Thermo Orion model-162A USA)

22 Plant Growth Experiment Themung beanVigna radiataL Wilczek (var K-851) a most important pulse crops plantgrown in India especially in Indo-Gangetic plains was usedin this study Bean is a diploid (2119899 = 22) and it has beenused in physiological biochemical and molecular analysesof toxicology [11 13] The selected mung bean seeds weresurface-sterilized with 75 (vv) ethanol for 5min followedby 10 (vv) sodium hypochlorite for 10min and washedthoroughly with distilled water [13] Seed germination andplant growth were studied in plastic beakers (size 75mm times90mm) filled with 250 g of oven-dried powdered gardensoil Four treatment concentrations namely 25 50 75 and100 vv prepared by diluting tannery effluents with distilledwater were used to irrigate pots Each treatment was run intriplicate with 10 seedsbeaker Beakers irrigated with onlydistilled water were taken as control Seeds were allowed togerminate and grow at room temperature Proper moisturewas maintained in pots using equal volume (50mL) of testsolutions after 3 days of time interval After 15 days shootsfrom plants were taken from each group and pooled for DNAextraction Seed germination percentages and plant growthwere also determined from the same experimental slots

23 DNA Extraction Shoots were thoroughly washed withdouble distilled water and DNA was extracted from theshoots using theGeneiPure Plant GenomicDNAPurificationKit (Merck India) following manufacturerrsquos procedure asfollows about 03 g of fresh plant shoots was homogenizedin a cold mortar with 400 120583L of lysis buffer and lysate wastransferred into the 15mL microcentrifuge tubes Ten 120583LRNase A solution (10mgL) was added to the lysates andmixed well before incubation at 65∘C for 30min It was thenadded with 75 120583L of precipitation buffer mixed thoroughlyand incubated for 5min on ice The lysate was transferredintoGenePure column (violet ring) and centrifuged for 2minat 11000 rpm to remove cellular debris The obtained flow-through was mixed with 450120583L binding buffer loaded ontothe GenePure column (green ring) and centrifuged for 1minat 11000 rpm Flow-through was discarded and column waswashedwith washing buffer 1 (400120583L) and subsequently withwashing buffer 2 (700120583L) to remove the residual debris Thebound DNA was eluted with 40 120583L of prewarmed (70∘C)elution buffer and stored at minus20∘C DNA yield was calcu-lated by spectrophotometer (Techcomp Korea) at 260 nmThe index of DNA purity (OD260280) was found to be180 The integrity of the extracted DNA was analyzed byelectrophoresis through 08 agarose gel visualized under

ISRN Biotechnology 3

Table 1 Physicochemical characteristics of tannery effluents from CETP Unnao Values are mean plusmn standard deviation (119899 = 3)

Parameter Untreated Treated Reduction in pollutants () ISI standardlowast

pH 851 plusmn 04 828 plusmn 04 3 55ndash90Electrical conductivity (mScm) 782 plusmn 11 761 plusmn 11 3 NSTotal dissolved solids (mgL) 15200 plusmn 760 14100 plusmn 705 7 2100Total hardness (mgL) 1360 plusmn 68 940 plusmn 47 31 600Total alkalinity (mgL) 2400 plusmn 120 1900 plusmn 95 21 NSChemical oxygen demand (mgL) 2848 plusmn 142 1280 plusmn 64 55 250Biochemical oxygen demand (mgL) 1375 plusmn 69 650 plusmn 33 53 30Total chromium (mgL) 448 plusmn 012 381 plusmn 012 3 20lowastISI standards number 2490 (1974) NS not specified

UV transilluminator and photographed and recorded usingthe Gel Documentation system (GBOX Syngene UK)

24 RAPD Analysis Ten decamer plant specific primers inthis study (RPi-C1 to RPi-C10 cat number 610692700101730)purchased from Merck India were used Sequences(51015840 rarr 31015840) from 1 to 10 utilized are AAAGCTGCGG (RPi-C1) AACGCGTCGG (RPi-C2) AAGCGACCTG (RPi-C3)AATCGCGCTG (RPi-C4) AATCGGGCTG (RPi-C5) ACA-CACGCTG (RPi-C6) ACATCGCCCA (RPi-C7) ACC-ACCCACC (RPi-C8) ACCGCCTATG (RPi-C9) ACG-ATGAGCG (RPi-C10) respectively PCR reactions wereperformed in reaction mixture of 25 120583L containing 125 120583Lof 2X GeNei HotStart PCR Master Mix (Merck) 1 120583Ltemplate DNA (25 ng) 1 120583L (25 pmol) primer and 105 120583Lnuclease-free water (Merck) Amplification was performedat following PCR cycle program initial denaturation for5min at 94∘C followed by 38 cycles each consisting of 1minat 94∘C (denaturing) 1min at 36∘C (annealing) 1min at72∘C (extension) and followed by 1 cycle for 5min at 72∘C(final extension step) Before running the RAPD analysisoptimization of PCR conditions was performed to checkreproducibility and consistency Negative controls withwater replacing the template DNA were always included tomonitor the contamination RAPD amplified products wereelectrophoresed on 18 agarose gel stained with ethidiumbromide in 1x TAE buffer GeNei low range DNA ruler plus(100ndash3000 bp) was used as molecular weight DNA standardThe size of each amplified product was approximated usingGeneTools gel image analyzer software available with the GelDocumentation system

25 Band Scoring and Data Analysis PCR products werescored for appearance and disappearance of band for eachprimer analyzed Only reproducible bands were scoredScoring and matching of bands in different gels were done byGenesnap and Genetool software (Syngene UK) along withthe GeNei low range DNA rulers (100 bp and 3000 bp) Thepresence and absence of RAPDbandwere recorded as ldquo1rdquo andldquo0rdquo respectivelyThe binary coded characters (1 0) were usedfor the genetic analysisThe scores obtained using all primersin the RAPD analysis were then pooled for constructing asingle data matrix The genetic similarity among the plantsirrigated with untreated effluent treated effluent and water

(control) was estimated from Neirsquos unbiased measure [17] inPOPGENE version 131 software Cluster analysis was per-formed and a dendrogram generated using the unweightedpair group method with the arithmetic means (UPGMA)algorithm of POPGENE [18]

3 Results and Discussion

31 Characteristics of Tannery Effluents Physicochemicalanalysis of both effluents is indicated by high pH EC TDSBOD COD and chromium which is a clear reflectionof the presence of high amount of organic and inorganiccompounds (Table 1) However treated effluent had com-paratively low amount of pollutants due to treatment effectof CETP The values of TDS COD and BOD recordedin the treated effluent were 67 51 and 217 times higherthan the standard limits as recommended by ISI [19] forindustrial wastewater discharge The results of comparativephysicochemical analysis indicated that CETP treatmentremoves COD and BOD by 55 and 53 respectively butnot EC TDS and chromium significantly The high EC andTDS are due to use of large quantities of salts Salts are verymuch soluble in water and chemically stable under aquaticconditions making it effectively impossible to remove themeasily from effluent Chromium concentration in treatedeffluent was 381mgL which is higher than the dischargelimit of 2mgL [19] Continuous discharge of chromium ineffluent even at low concentration can cause toxic effect onaquatic life by disrupting food chain [20]

32 Effect of Tannery Effluents on Germination and Growth ofMung Bean Seeds Figure 1 shows an example of the germi-nation and growth of mung bean seeds in plastic pots andrecorded data are given in Table 2 When irrigated withuntreated effluent germination was inhibited by 90 at 50concentration and prevented by 75 concentration Whenirrigatedwith treated effluent 90germinationwas inhibitedat both 75 and 100 concentrations It is clear that untreatedeffluent above 25 and treated effluent above 50 had toxiceffect on seed germination (Figure 1) This indicated thatuntreated tannery effluent was more toxic than the treatedone The total length of plant reduced with increasing efflu-ents concentration as compared to control and it was more


Cont 25 50 75 100

(a)

Cont 25 50 75 100

(b)

Figure 1 Effect of untreated (a) and treated (b) tannery effluents on seedling growth of mung bean at different concentrations

Table 2 Effect of untreated (UT) and treated (T) tannery effluent on germination and growth ofmung bean seeds Values aremeanplusmn standarddeviation (119899 = 3)

Effluent conc () Germination () Total length (cm) Root length (cm) Shoot length (cm)UT T UT T UT T UT T

0 100 plusmn 0 100 plusmn 0 128 plusmn 10 128 plusmn 10 24 plusmn 08 24 plusmn 08 89 plusmn 07 89 plusmn 07



75 0 10 plusmn 0 0 65 plusmn 05 0 15 plusmn 01 0 35 plusmn 02


affected by untreated than the treated with the same con-centrations However the root length of plant irrigated with25 treated effluent was increased by 30 cm as comparedto control 24 cm (Table 2) On the other hand shoot lengthof plants showed a decreasing trend with increasing con-centration of both untreated and treated tannery effluentsThus the reduction in seed germination percentage and plantgrowth at higher concentration of tannery effluents may beattributed to high salts and various ingredients of effluent likesulphide or chromium as reported by several workers [3 2122] The salt content outside the seed causes less absorptionof water by osmosis and inhibits the germination of seedswhile chromium in addition to germination also affects plantgrowth by inhibition of root cell division and elongation Onthe other hand excess amount of sulphide present in effluentmay induce deficiency of cationic micronutrient such as ZnCu Fe and Mn Significant reduction in plant growth wasobserved at 25 and therefore plant from this concentrationwas used to extract DNA for RAPD analysis

33 RAPD Profile of the Control and Treated Plants RAPDtechnique has shown promising result in the detection ofpollutant-induced DNA effects due to its rapidity appli-cability to any organism (since no information on thenucleotide sequence cell cycle or chromosome complementis required) and sensitivity to detect a wide range of DNAdamage andmutations [9ndash11]However RAPD is a qualitativemethod and the nature and amount of DNA effects can onlybe speculated At the same time this technique has beeninvariably used to detectDNAdamage andmutation in plantsinduced by heavy metals [11 13 14] and wastewaters [23ndash25]In this study RAPD analysis was performed on three pooledgenomic DNA extracted from shoots of 25 of untreated andtreated and control plants after 15 daysThe reason for poolinggenomic DNA from three individuals was to avoid the

intrapopulation genetic polymorphism potentially revealedby RAPD analysis [14] Ten decamer plant specific primers(RPi-C1ndashRPi-C10) of 60ndash70 GC content were utilized forscreening of the mung bean genome for changes Amongthem 8 primers (RPi-C1ndashRPi-C8) gave clear and stablebands RAPD profiles generated by these primers revealeddifferences between control and treated plants with visiblechange in the number and size of amplified DNA fragmentsA total of 87 RAPD bands were obtained by using 8 primersand 42 (48) of these bands showed polymorphism Otherstudies obtained similar percentage of polymorphic bands48 [11] and 46 [25] However some other studies [26]found much higher percentage (8356) of polymorphicbands Further all bands generated were ranged between160 and 1114 bp The band size obtained in this study iswithin the range of other studies Enan [11] analyzed thegenotoxic effect of heavy metals in kidney bean (Phaseolusvulgaris) using RAPD primers RAPD profiles obtained fromsix primers exhibited bands between 200 and 1600 bp inlength Swaileh et al [25] obtained bands ranging from 150 bpto 2000 bp from 15 primers used to detect the wastewater-induced genotoxicity in oat plants (Avena sativa)

Figure 2 shows an example of the change in the bandingpatterns between control and treatments The change inRAPD profile of the control plants and treatments wasdiscernible from appearancedisappearance of some bandswhen treatments were comparedwith control (Table 3) Eightprimers generated a total of 34 RAPD bands ranging from160 to 1170 bp in control plants All primers generated 4RAPD bands with different size except RPi-C2 which gave 6bands Comparedwith the control plants those irrigatedwithuntreated effluent recorded the appearance of 12 new bandsand disappearance of 18 bands Plants irrigated with treatedeffluent yielded 8 new bands and 15 bands disappearedThe total number of bands that appeared and disappeared


Table 3 The number of bands in control and molecular sizes (base pair bp) of disappearance (minus) andor appearance (+) of DNA bands inuntreated and treated effluent irrigated mung bean plants using Gene tool software

Primers Control Untreated effluent (25) Treated effluent (25)

RPi-C1 4 (283ndash800 bp) minus 800 560 400 283 800+ 0 430

RPi-C2 6 (390ndash1170 bp) minus 970 390 970 790 670+ 1114 413 580 300 275 200

RPi-C3 4 (551ndash842 bp) minus 842 740 640 551 842 740+ 0 970430


RPi-C5 4 (650ndash955 bp) minus 955 850 704 650+ 0 0

RPi-C6 4 (172ndash516 bp) minus 0 516 244 172+ 650 350 220 620

RPi-C7 4 (160ndash1026 bp) minus 1026 0+ 850 540 260 180 0


Total 34 18 (minus) 12 (+) 15 (minus) 8 (+)

RPi-C1 RPi-C2 RPi-C3

3000

2000

1000

(bp)

M C UT T M C UT T M C UT T

Figure 2 RAPD profiles generated by RPi-C1ndashRPi-C3 from mungbean plant irrigated with untreated (UT) and treated (T) tanneryeffluents Lane C control and Lane M low range DNA rulers (100ndash3000 bp)

because of both treatments were 20 and 33 recpectively Thisindicates that both untreated and treated effluent inducedDNA change in plants However the number of bands thatdisappeared was higher in plants irrigated with untreatedeffluent as compared to those irrigated with treated effluentwhich revealed that DNA damage was more severe in plantsirrigated with untreated effluent Enan [11] found that 22new fragments appeared and 43 disappeared as result ofusing 350mgL heavy metals to irrigate kidney bean Lessband appearancedisappearance was observed when using150mgL Similarly Cenkci et al [13] found that 21 and 11new bands appeared and 64 and 42 disappeared in root ofPhaseolus vulgaris L seedling exposed to Hg Cr and Zn at350 and 150mgL respectively

The disappearance of control bands may be related tothe DNA damage (eg single- and double-strand breaksmodified bases abasic sites oxidized bases bulky adduct andDNA-protein cross links) point mutations andor complexchromosomal rearrangements induced by genotoxins [1027] When Taq DNA polymerase encounters a DNA adduct

there are number of possible outcomes including blockagebypass and the possible dissociation of the enzymeadductcomplex which will cause the loss of bands [28] Theappearance of new PCR products may reveal a change insome oligonucleotide priming sites due to mutations (newannealing event(s)) large deletions (bringing preexistingannealing site closer) andor homologous combination (jux-taposing two sequences that match the sequence of primer)[9] Atienzar et al [29] reported that mutation can only beresponsible for the appearance of new bands if they occur atsame locus in a sufficient number of cells (a minimum of 10of mutations may be required to get new PCR product visiblein agarose) to be amplified by PCR Briefly new bands couldbe attributed tomutation while the bands which disappearedcould be attributed to DNA damage [28]

The cluster analysis method is considered one of themost effective methods in numerical analysis regarding bandscoring and analysis of RAPD finger printing [25] It cancalculate the distances between every pair of entities andthen summarize the community data sets In the presentstudy cluster analysis was done to estimate the level of DNApolymorphism between the control plants and those irrigatedwith untreated or treated tannery effluent A dendrogramwas constructed using distances matrix by using UPGMAmethod of phylip software (Figure 3)The control and treatedsamples were clustered in one group (join at distance of028) and the untreated were separated in another clusterat larger distance (join at distance of 042) This resultclearly showed that untreated effluent contains much moregenotoxic substances than the treated one Swaileh et al [25]demonstrated that there is larger distance between controland raw wastewater as compared to between control andtreated plants

Genetic diversity of population that is genetic similarityindices is an important parameters in RAPD analysis [27 30]


UT

C

T1

Figure 3 Dendrogram representing genetic distance among threesamples of mung bean plants by UPGMA method based on RAPDanalysis C = control T = treated and UT = untreated

Neirsquos genetic similarity indices which measure the propor-tion of the shared fragments in the amplifications werecalculated and are shown in Figure 4 Mung bean plantsirrigated with distilled water (control) and those irrigatedwith treated tannery effluent showed genetic similarity 75The plant irrigated with untreated tannery effluent showedclearly less genetic similarity (65) to the control ones Inaddition plant irrigated with treated tannery effluent showed68 similarity to those irrigated with untreated tanneryeffluent This is in accordance with the results obtained fromthe cluster analysis where the control and treated tanneryeffluent irrigated plants grouped in one cluster and the plantsirrigated with untreated tannery effluent were grouped in aseparate cluster joined at a larger distance

The DNA damage in mung bean plants is possibly dueto toxic effect of 448 and 381mgL chromium present inuntreated and tannery effluent Chromium (gt2 ppm) hasbeen reported to be inhibitory for plant growth Among thedifferent forms of chromium hexavalent chromium (Cr VI)is more toxic and carcinogenic due to its high solubilityin water rapid permeability through biological membraneand subsequent interaction with intracellular proteins andnucleic acid [31] Genotoxic effect of chromium in plantshas been well documented for instance DNA damage dueto exposure to chromium was observed in Vicia faba [32]Labra et al [33] foundhypermethylation ofDNAand increasein DNA polymorphism in Brassica napus in response to Cr(VI) exposure Cr (VI) also induced genotoxicity detected byAFLP analysis in Arabidopsis thaliana L [34] FurthermoreKnasmuller et al [35] compared Cr (VI) and Cr (III) withrespect to their ability to inducemicronucleus inTradescantiaand found that only in Cr (VI) exposed plants there wasan increase in micronucleus frequencies Previous studieshave showed that tannery effluent (125) and Cr (4mgL)induced various chromosomal abnormalities in plant cellsthereby severely reducing mitotic index and root growth[2 36] To the best of our knowledge there is no reportavailable on plant DNA damage induced by tannery effluentusing RAPD analysis Hence the finding of the present studyincreased our understanding on treatment efficiency of CETPand nature of wastewater which is being discharged intothe environment after treatment In conclusion our datashowed that both untreated and treated tannery effluentshad genotoxic substances causing different levels of genotoxiceffects However untreated effluent is more genotoxic than

00

02

04

06

08

10

Untreated Treated

Sim

ilarit

y in

dex

Figure 4 Genetic similarity between mung bean plants irrigatedwith untreated and treated tannery effluents and those irrigatedwithdistilled water (control) Error bars represent plusmn standard deviationof the mean values from 8 primers

the treated one Thus from this study it is clear that CETPUnnao removes some but not all genotoxic substances foundin untreated tannery effluent Consequently use of treatedwastewater for irrigation poses health hazard to both humanand animals of the environment

Conflict of Interests

The authors declare that there is no conflict of interestsregarding publishing this work in any publication house

Acknowledgments

The authors gratefully acknowledge the facilities providedby Director CSIR-Indian Institute of Toxicology ResearchLucknow UP India They are also thankful to Dr A Kannanfor editing the paper Financial support for this study wasprovided by CSIR under Integrated NextGen approachesin health disease and environmental toxicity (INDEPTH)Project (BSC0111)

References

[1] J Jawahar M Chinnadurai J K S Ponselvan and G Annadu-rai ldquoPollution from tanneries and options for treatment of efflu-entrdquo Indian Journal of Environmental Protection vol 18 pp 672ndash672 1998

[2] K Gupta S Gautam and K Mishra ldquoStudies on phyto-geno-toxic assessment of tannery effluent and chromium on Alliumcepardquo Journal of Environmental Biology vol 33 no 1 pp 557ndash563 2012

[3] PMishra andA K Bera ldquoEffect of tannery effluent on seed ger-mination and early seedling growth in wheatrdquo Seed Researchvol 23 pp 129ndash131 1995


[4] G Rao and N V Kumar ldquoImpact of tannery effluent on seedgerminability and chlorophyll contents of Cicer arientinum LrdquoPollution Research vol 2 no 1 pp 33ndash36 1983

[5] H Sandermann Jr ldquoHigher plant metabolism of xenobioticsthe ldquogreen liverrdquo conceptrdquo Pharmacogenetics vol 4 no 5 pp225ndash241 1994

[6] S T Matsumoto M S Mantovani M I A Malaguttii A LDias I C Fonseca and M A Marin-Morales ldquoGenotoxicityand mutagenicity of water contaminated with tannery effluentsas evaluated by the micronucleus test and comet assay usingthe fish Oreochromis niloticus and chromosome aberrations inonion root-tipsrdquo Genetics and Molecular Biology vol 29 no 1pp 148ndash158 2006

[7] S Cambier P Gonzalez G Durrieu and J-P BourdineaudldquoCadmium-induced genotoxicity in zebrafish at environmen-tally relevant dosesrdquo Ecotoxicology and Environmental Safetyvol 73 no 3 pp 312ndash319 2010

[8] D Savva ldquoUse of DNA fingerprinting to detect genotoxic ef-fectsrdquo Ecotoxicology and Environmental Safety vol 41 no 1 pp103ndash106 1998

[9] F A Atienzar M Conradi A J Evenden A N Jha and MH Depledge ldquoQualitative assessment of genotoxicity using ran-dom amplified polymorphic DNA comparison of genomictemplate stability with key fitness parameters inDaphniamagnaexposed to benzo[a]pyrenerdquo Environmental Toxicology andChemistry vol 18 no 10 pp 2275ndash2282 1999

[10] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000

[11] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquoBiotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006

[12] Y-C Lee V C Yang and T-S Wang ldquoUse of RAPD to detectsodium arsenite-induced DNA damage in human lymphoblas-toid cellsrdquo Toxicology vol 239 no 1-2 pp 108ndash115 2007

[13] S Cenkci M Yildiz I H Cigerci M Konuk and A BozdagldquoToxic chemicals-induced genotoxicity detected by randomamplified polymorphic DNA (RAPD) in bean (Phaseolus vul-garis L) seedlingsrdquo Chemosphere vol 76 no 7 pp 900ndash9062009

[14] W Liu L Sun M Zhong et al ldquoCadmium-induced DNAdamage andmutations in Arabidopsis plantlet shoots identifiedby DNA fingerprintingrdquo Chemosphere vol 89 no 9 pp 1048ndash1055 2012

[15] P W Ramteke S Awasthi T Srinath and B Joseph ldquoEfficiencyassessment of Common Effluent Treatment Plant (CETP) treat-ing tannery effluentsrdquo Environmental Monitoring and Assess-ment vol 169 no 1ndash4 pp 125ndash131 2010

[16] APHA (American Public Health Association) Standard Meth-ods for the Examination of Water and Wastewater AmericanPublic Health Association Washington DC USA 21st edition2005

[17] M Nei ldquoEstimation of average heterozygosity and genetic dis-tance from a small number of individualsrdquo Genetics vol 89 no3 pp 583ndash590 1978

[18] F C Yeh R Yang T J Boyle Z Ye and J M Xiyan POPGENE32 Microsoft Window-Based Freeware for Population Genetic

Analysis Version 132 Molecular Biology and BiotechnologyCentre University of Alberta Edmonton Canada 2000

[19] ISI (Indian Standard Institute) ldquoTolerance limits of industrialwastewater discharge into inland surface waterrdquo Tech Rep2490 Indian Standard Institute New Delhi India 1974

[20] H Oliveira ldquoChromium as an environmental pollutant insighton induced plant toxicity a reviewrdquo Journal of Botany vol 2012Article ID 375843 8 pages 2012

[21] LMalla and B KMohanty ldquoEffect of papermill effluent on ger-mination of green gram (Phaseolus aureus Roxb) and growthbehaviour of itrsquos seedlingsrdquo Journal of Environmental Biologyvol 26 no 2 pp 379ndash382 2005

[22] S Karunyal G Renuga andK Paliwal ldquoEffects of tannery efflu-ent on seed germination leaf area biomass andmineral contentof some plantsrdquo Bioresource Technology vol 47 no 3 pp 215ndash218 1994

[23] M H Nielsen and J Rank ldquoScreening of toxicity and genotox-icity in wastewater by the use of the Allium testrdquoHereditas vol121 no 3 pp 249ndash254 1994

[24] C K Grisolia A B B de Oliveira H Bonfim and M NKlautau-Guimaraes ldquoGenotoxicity evaluation of domestic sew-age in a municipal wastewater treatment plantrdquo Genetics andMolecular Biology vol 28 no 2 pp 334ndash338 2005

[25] K M Swaileh R Hussein and A Ezzughayyar ldquoEvaluatingwastewater-induced plant genotoxicity using randomly ampli-fied polymorphic DNArdquo Environmental Toxicology vol 23 no1 pp 117ndash122 2008

[26] P Padmesh K K Sabu S Seeni and P Pushpangadan ldquoThe useof RAPD in assessing genetic variability in Andrographis panic-ulata Nees a hepatoprotective drugrdquo Current Science vol 76no 7 pp 833ndash835 1999

[27] H D Wolf R Blust and T Backeljau ldquoThe use of RAPD inecotoxicologyrdquoMutation Research vol 566 no 3 pp 249ndash2622004

[28] F A Atienzar and A N Jha ldquoThe random amplified polymor-phic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006

[29] F A Atienzar P Venier AN Jha andMHDepledge ldquoEvalua-tion of the random amplified polymorphic DNA (RAPD) assayfor the detection of DNA damage and mutationsrdquo MutationResearch vol 521 no 1-2 pp 151ndash163 2002

[30] M Lynch ldquoThe similarity index and DNA fingerprintingrdquoMolecular Biology and Evolution vol 7 no 5 pp 478ndash484 1990

[31] S P B KamaludeenMMegharaj A L Juhasz N Sethunathanand R Naidu ldquoChromium-microorganism interactions in soilsremediation implicationsrdquo Reviews of Environmental Contami-nation and Toxicology vol 178 pp 93ndash164 2003

[32] G Koppen and L Verschaeve ldquoThe alkaline comet test on plantcells a newgenotoxicity test forDNAstrand breaks inVicia fabaroot cellsrdquoMutation Research vol 360 no 3 pp 193ndash200 1996

[33] M Labra F Grassi S Imazio et al ldquoGenetic and DNA-methylation changes induced by potassium dichromate inBrassica napus Lrdquo Chemosphere vol 54 no 8 pp 1049ndash10582004

[34] M Labra T di Fabio F Grassi et al ldquoAFLP analysis as bio-marker of exposure to organic and inorganic genotoxic sub-stances in plantsrdquo Chemosphere vol 52 no 7 pp 1183ndash11882003


[35] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998

[36] D I Olorunfemi G EOkoloko A A Bakare andAAkinboroldquoCytotoxic and genotoxic effects of cassava effluents using theAllium cepa assayrdquo Research Journal of Mutagenesis vol 1 no 1pp 1ndash9 2011

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good indicators of cytogenetic and mutagenic effect whichcan be applied both indoors and outdoors Several studieshave used the chromosome aberration micronucleus orcomet assay to measure the effects of tannery effluent onplants [2 6] The micronucleus test in organism assays isnot a sensitive method for determining pollution levels ofheavy metals in terrestrial ecosystems and another difficultylinked to the comet assay is the necessity to experiment onisolated cells and their inability to provide information onthe effects of toxic metals at the DNA level [7] Advantageof measuring effect of genotoxic chemicals directly on DNAis mainly related to the sensitivity and short time responseRecently advances in molecular biology have led to devel-opment of a number of selective and sensitive assays forDNA analysis in ecogenotoxicology DNA based techniques(RFLP QTL RAPD AFLP SSR and VNTR) are used toevaluate the variation at the DNA sequence level Randomamplified polymorphic DNA (RAPD) of these techniques isa PCR-based method that amplifies random DNA fragmentswith the use of single short primers of arbitrary nucleotidesequence under low annealing conditions RAPD can beused to detect genotoxicity and differences can be clearlyshownwhen comparingDNAfingerprint fromuntreated andtreated individuals to genotoxic agents [8ndash14]

Unnao district (UP) India is one of the major industrialareas especially for leather tannery industry Most of thetanneries in this area are in the small scale and cannot affordexpensive treatment plant on their ownTherefore tomitigatethe pollution from tanneries a common effluent treatmentplant (CETP) was established in 1996 having treating capacityof 215million liters per day (MLD)This is an activated sludgeprocess-based plant which receives 19 million liters per day(MLD) of tannery wastewater from a cluster of 21 tanneriesprocessing approximately 475 tons average daily raw hidesThe treated effluent from CETP is finally discharged in theGanga River through drain This wastewater is used forirrigation purposes in agricultural field on both sides ofdrain before entering in the Ganga River Ramteke et al[15] assessed the efficiency of CETP and found a significantreduction in COD and BOD levels during the course oftreatment in CETP However the impact of CETP treatmentfor reducing genotoxic substances from raw effluent is notknown Therefore in the present study the impact of CETPon genotoxic activity of tannery effluent was assessed byplant genotoxicity analysis The present study uses RAPDtechnology to evaluate the genotoxic effects in mung beanplant irrigated with untreated and treated tannery effluents

2 Materials and Methods

21 Effluent Samples and Physicochemical Analysis Theuntreated (UT) and treated (T) effluents were collected frominlet and outlet points respectively from common effluenttreatment plant (CETP) Unnao (2648∘N 8043∘) UP stateIndia during the month of February 2013 Samples weretaken in 5-litre Jerry cane and transported to laboratoryfor analysis The effluents were filtered through Whatmanfilter paper number 1 to remove suspended particles and

stored at 4∘C till completion of the analysis The followingparameters were determined based on standard methods[16] pH total dissolved solids (TDS) total hardness totalalkalinity chemical oxygen demand (COD) biochemicaloxygen demand (BOD) and total chromium (total Cr)Total Cr was analyzed with atomic absorption spectrometer(Perkin Elmer model A Analyst-300) after digestion ofsamples (100mL) in digestion mixture of (5 1) of nitric-perchloric acid [16] Electrical conductivity was determinedby conductivity meter (Thermo Orion model-162A USA)

22 Plant Growth Experiment Themung beanVigna radiataL Wilczek (var K-851) a most important pulse crops plantgrown in India especially in Indo-Gangetic plains was usedin this study Bean is a diploid (2119899 = 22) and it has beenused in physiological biochemical and molecular analysesof toxicology [11 13] The selected mung bean seeds weresurface-sterilized with 75 (vv) ethanol for 5min followedby 10 (vv) sodium hypochlorite for 10min and washedthoroughly with distilled water [13] Seed germination andplant growth were studied in plastic beakers (size 75mm times90mm) filled with 250 g of oven-dried powdered gardensoil Four treatment concentrations namely 25 50 75 and100 vv prepared by diluting tannery effluents with distilledwater were used to irrigate pots Each treatment was run intriplicate with 10 seedsbeaker Beakers irrigated with onlydistilled water were taken as control Seeds were allowed togerminate and grow at room temperature Proper moisturewas maintained in pots using equal volume (50mL) of testsolutions after 3 days of time interval After 15 days shootsfrom plants were taken from each group and pooled for DNAextraction Seed germination percentages and plant growthwere also determined from the same experimental slots

23 DNA Extraction Shoots were thoroughly washed withdouble distilled water and DNA was extracted from theshoots using theGeneiPure Plant GenomicDNAPurificationKit (Merck India) following manufacturerrsquos procedure asfollows about 03 g of fresh plant shoots was homogenizedin a cold mortar with 400 120583L of lysis buffer and lysate wastransferred into the 15mL microcentrifuge tubes Ten 120583LRNase A solution (10mgL) was added to the lysates andmixed well before incubation at 65∘C for 30min It was thenadded with 75 120583L of precipitation buffer mixed thoroughlyand incubated for 5min on ice The lysate was transferredintoGenePure column (violet ring) and centrifuged for 2minat 11000 rpm to remove cellular debris The obtained flow-through was mixed with 450120583L binding buffer loaded ontothe GenePure column (green ring) and centrifuged for 1minat 11000 rpm Flow-through was discarded and column waswashedwith washing buffer 1 (400120583L) and subsequently withwashing buffer 2 (700120583L) to remove the residual debris Thebound DNA was eluted with 40 120583L of prewarmed (70∘C)elution buffer and stored at minus20∘C DNA yield was calcu-lated by spectrophotometer (Techcomp Korea) at 260 nmThe index of DNA purity (OD260280) was found to be180 The integrity of the extracted DNA was analyzed byelectrophoresis through 08 agarose gel visualized under













Cont 25 50 75 100

(a)

Cont 25 50 75 100

(b)


























3000

2000

1000

(bp)









UT

C

T1




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Untreated Treated

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Cont 25 50 75 100

(a)

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(b)


























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(bp)









UT

C

T1




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Microbiology


Cont 25 50 75 100

(a)

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(b)


























3000

2000

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(bp)









UT

C

T1




00

02

04

06

08

10

Untreated Treated

Sim

ilarit

y in

dex





Acknowledgments


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Volume 2014

Zoology






















Advances in

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Volume 2014




Enzyme Research



Microbiology














3000

2000

1000

(bp)









UT

C

T1




00

02

04

06

08

10

Untreated Treated

Sim

ilarit

y in

dex





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


UT

C

T1




00

02

04

06

08

10

Untreated Treated

Sim

ilarit

y in

dex





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Detection of Tannery Effluents Induced DNA Damage in Mung ...downloads.hindawi.com/journals/isrn.biotechnology/2014/727623.pdf · Detection of Tannery Effluents Induced DNA Damage