Toxicological characterization of the landfill leachate prior/after chemical and electrochemical treatment: A study on human and plant cells

Chemosphere xxx (2013) xxx–xxx

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Chemosphere

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Toxicological characterization of the landfill leachate prior/afterchemical and electrochemical treatment: A study on human and plantcells

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.05.059

Abbreviations: AO, acridine orange; APDC, ammonium-pyrolidinedithiocarbamate; BOD, biochemical oxygen demand; CBMN, cytokinesis-block micronucleus asschemical oxygen demand; EC, electrical conductivity; EtBr, ethidium bromide; HPBL, human peripheral blood lymphocyte; LPO, lipid peroxidation; MAV, maximalvalues; MN, micronucleus; MNi, micronuclei; NBUD, nuclear bud; NPB, nucleoplasmic bridge; ROS, reactive oxidative species; SCGE, single cell gel electrophoresis asuspended solids; TDS, total dissolved solids; TOC, total organic carbon.⇑ Corresponding author. Address: Institute for Medical Research and Occupational Health, Mutagenesis Unit, Ksaverska cesta 2, 10000 Zagreb, Croatia. Tel.: +38

188; fax: +385 1 4673 303.E-mail address: [email protected] (V. Garaj-Vrhovac).

Please cite this article in press as: Garaj-Vrhovac, V., et al. Toxicological characterization of the landfill leachate prior/after chemical and electrochtreatment: A study on human and plant cells. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.059

Vera Garaj-Vrhovac a,⇑, Višnja Orešcanin b, Goran Gajski a, Marko Geric a, Damir Ruk c, Robert Kollar b,Sandra Radic Brkanac d, Petra Cvjetko d

a Institute for Medical Research and Occupational Health, Mutagenesis Unit, 10000 Zagreb, Croatiab Advanced Energy Ltd., 10000 Zagreb, Croatiac Municipal Company Komunalac, 48000 Koprivnica, Croatiad University of Zagreb, Faculty of Science, Department of Biology, 10000 Zagreb, Croatia

h i g h l i g h t s

� The efficiency of two methods forlandfill leachate purification wasinvestigated.� Untreated leachate proved to be both

cyto- and genotoxic to human orplant cells.� Treated leachate did not cause

increase in cyto- and genotoxicdamage.� Both methods have high removal

efficiency and provide toxicologicalsafety.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 October 2012Received in revised form 6 May 2013Accepted 25 May 2013Available online xxxx

Keywords:Landfill leachateChemical treatmentElectrochemical treatmentCytogenotoxicityHuman lymphocytesAllium cepa

a b s t r a c t

In this research, toxicological safety of two newly developed methods for the treatment of landfill leach-ate from the Piškornica (Croatia) sanitary landfill was investigated. Chemical treatment procedure com-bined chemical precipitation with CaO followed by coagulation with ferric chloride and final adsorptionby clinoptilolite. Electrochemical treatment approach included pretreatment with ozone followed byelectrooxidation/electrocoagulation and final polishing by microwave irradiation. Cell viability ofuntreated/treated landfill leachate was examined using fluorescence microscopy. Cytotoxic effect ofthe original leachate was obtained for both exposure periods (4 and 24 h) while treated samples showedno cytotoxic effect even after prolonged exposure time. The potential DNA damage of the untreated/trea-ted landfill leachate was evaluated by the comet assay and cytokinesis-block micronucleus (CBMN) assayusing either human or plant cells. The original leachate exhibited significantly higher comet assay param-eters compared to negative control after 24 h exposure. On the contrary, there was no significant differ-ence between negative control and chemically/electrochemically treated leachate for any of theparameters tested. There was also no significant increase in either CBMN assay parameter comparedto the negative control following the exposure of the lymphocytes to the chemically or electrochemicallytreated landfill leachate for both exposure periods while the original sample showed significantly higher

ay; COD,allowedssay; SS,

5 1 4673

emical

2 V. Garaj-Vrhovac et al. / Chemosphere xxx (2013) xxx–xxx

Please cite this article in press as: Garaj-Vrhovatreatment: A study on human and plant cells. C

number of micronuclei, nucleoplasmic bridges and nuclear buds for both exposure times. Results suggestthat both methods are suitable for the treatment of such complex waste effluent due to high removal effi-ciency of all measured parameters and toxicological safety of the treated effluent.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

United Nations (UN) predicts that the production of solid(including hazardous) waste could rise from 2–4.9 billion tonsannually in 2006 to 2.4–5.9 billion tons annually in 2025. Thesenegative trends in waste production demand consideration of sev-eral issues. Among them there are three endpoints that should behighlighted: public health, impact on environment, and wastemanagement (UN, 2010).

The aim of this research was toxicological characterization ofthe landfill leachate prior/after the purification treatment usingtwo different approaches. Landfill leachate was taken from thePiškornica (Koprivnica, Croatia) sanitary landfill which is situatedapproximately 9 km north of Koprivnica city (Fig. 1). The municipalas well as industrial waste was deposited since 1982 at the Piškor-nica landfill. Until 2005 the waste material was loaded directly onthe ground without: preliminary foundation of the basic leak prooflayer, daily cover of the deposited waste, and leachate collectionand treatment. Today, the estimated production of the leachate is25 m3 d�1. As the result of the completed fermentation and wasteaging processes, landfill leachate is characterized by very low bio-chemical oxygen demand/chemical oxygen demand (BOD5/COD)ratio and high amount of bio-refractory compounds (Orešcaninet al., 2011).

The first attempts of the treatment of landfill leachate from thePiškornica landfill were done by Orešcanin et al. (2011,2012) andresulted in the removal efficiencies of color, turbidity, suspendedsolids (SS), ammonia, COD, Fe and Zn for up to 99.48%. Although,treatment methods gave satisfactory results concerning the chem-ical composition of the final effluent, suitability and safety of themethods prior to their wide range applications must be confirmedby toxicological characterization using various test systems.

Consequently, in this work we examined the cytotoxic andgenotoxic potential of treated leachates as well as untreated leach-ate on human and plant cells. As each test organism/cell type canbe sensitive to different toxic substances, in toxicity assessmentof environmental samples, it is generally recommended to use atleast two species belonging to different trophic groups (Georgeet al., 1995; Wenzel et al., 1997). Application of biotests with dif-ferent model systems gives the opportunity to treat data fromthe tests as the information about the whole ecosystem, whichafterwards makes it easier to assess a real hazard in the environ-ment (Wenzel et al., 1997). Conventionally, Allium cepa as a plantmodel system has been used to evaluate DNA damage in termsof chromosome aberrations and disturbances in the mitotic cycle.However, in the present study, A. cepa was used in the comet assayin order to obtain better comparison with the results of the cometassay performed on human cells.

Fig. 1. The position of Piškornica landfill situated approximately 10 km north fromKoprivnica city which is the capital of Koprivnica-Krizevci County in the north-eastof Croatia.

2. Materials and methods

2.1. Sampling and sample handling

A 100 L of landfill leachate was collected from the lagoon ofPiškornica sanitary landfill in five polyethylene containers andtransported to the laboratory. In order to obtain homogeneoussample the effluent was combined into the single tank and mixed

c, V., et al. Toxicological charachemosphere (2013), http://dx.

for 10 min (600 rpm) before analysis or each purificationexperiment.

2.2. Purification experiments

2.2.1. Chemical treatmentChemical treatment of landfill leachate consisted of three steps

procedure as described in our previous paper (Orešcanin et al.,2011). Briefly, 1 L of landfill leachate was mixed with 25 g of cal-cium oxide on a magnetic stirrer for 30 min and subjected to a fur-ther treatment by 0.570 mg Fe3+ L�1 added in the form ofFeCl3 � 6H2O and mixed on a magnetic stirrer for another 30 minfollowed by 30 min of settling. After the settlement the liquid part

terization of the landfill leachate prior/after chemical and electrochemicaldoi.org/10.1016/j.chemosphere.2013.05.059

V. Garaj-Vrhovac et al. / Chemosphere xxx (2013) xxx–xxx 3

was decanted and mixed with 25 g L�1 powdered clinoptilolite for4 h followed by 2 h of settling. The liquid part was decanted andanalyzed.

2.2.2. Electrochemical treatmentFor each experiment 10 L of water was subjected to ozonation

for 2 h (flow rate of 2.5 mL min�1) followed by simultaneous ozon-ation and electrochemical treatment by stainless steel electrode set(I = 12 A; U = 6 V; reaction time 5 h), simultaneous electrocoagula-tion and ozonation using followed to electrocoagulation/ozonationby aluminum electrode set (I = 12 A; U = 6 V; reaction time 1 h),slow mixing with ozone bubbles for additional 30 min, while addi-tional 30 min was needed for flock’s settlement (Orešcanin et al.,2012). Clear water was subjected to final treatment with micro-waves supplied by magnetron for 20 min.

2.2.3. Chemical analysis of heavy metalsFor the analysis of heavy metals in the samples of original and

purified landfill leachate the samples were digested according tothe protocol described in our previous papers (Orešcanin et al.,2011, 2012). Pretreated samples were adjusted to pH 3 and precon-centrated to 1% (w/v) solution of ammonium-pyrolidinedithiocar-bamate (APDC) (Merck, Schuchardt, Germany). After thecomplexation lasted for 20 min, the suspension was filteredthrough a Millipore HAWP filter (pore size 0.45 lm; diameter25 mm). The prepared thin targets were air-dried, protected witha thin mylar foil (2 lm) and analyzed by X-ray spectrometer (Oreš-canin et al., 2011, 2012).

2.2.4. Determination of other parametersColor, turbidity, SS, NHþ4 , COD and total organic carbon (TOC)

were determined by HACH DR890 colorimeter (Hach Company,Loveland, CO). For sample digestion DRB 200 reactor (Hach Com-pany) was used (Orešcanin et al., 2011, 2012). BOD5 was deter-mined by OxiTop system (WTW, Weilheim, Germany). pH value,electrical conductivity (EC) and total dissolved solids (TDS) weredetermined by PHT-027 – water quality multiparameter monitor(Kelilong Electron Co., Ltd., Fuan Fujian, China).

2.3. Toxicity tests

2.3.1. Blood sampling and treatmentThe effects of the leachate samples were evaluated in human

peripheral blood lymphocytes (HPBLs) obtained from a healthymale, non-smoking donor who had not been exposed to ionizingradiation for diagnostic or therapeutic purposes or to known geno-toxic chemicals that might have interfered with the results of thetesting for a year before blood sampling. The study was approvedby the institutional ethics committee and observed the ethicalprinciples of the Declaration of Helsinki. Blood was drawn by ante-cubital venipuncture into heparinised vacutainers containing lith-ium heparin as anticoagulant (Becton Dickinson, Franklin Lakes,NJ) under aseptic conditions.

For toxicological testing 100 lL of the leachate was added into900 lL of peripheral blood to make the final concentration andkept for 4 and 24 h period. In each experiment, a nontreated con-trol was included.

2.3.2. Cell viability (cytotoxicity) assayAfter the treatment, lymphocytes were isolated by Histopaque

(Sigma, St. Louis, MO) density gradient centrifugation method(Singh, 2000). Cytotoxicity was determined by differential stainingwith acridine orange (AO) and ethidium bromide (EtBr) and byfluorescence microscopy (Duke and Cohen, 1992). A total of 100cells per repetition were examined with an Olympus BX51 micro-scope (Tokyo, Japan). The cells were divided in two categories: live

Please cite this article in press as: Garaj-Vrhovac, V., et al. Toxicological charactreatment: A study on human and plant cells. Chemosphere (2013), http://dx.

cells with a functional membrane and with uniform green stainingof the nucleus, and dead cells with uniform red staining of thenucleus.

2.3.3. Human lymphocytes comet (SCGE) assayThe comet assay was performed according to Singh et al. (1988)

with slight modifications (Gajski et al., 2008) using peripheralblood lymphocytes as human sample. Sides were composed ofthree agarose layers and lysed overnight at 4 �C. After the lysis,the slides were placed into alkaline solution for 20 min at 4 �Cand subsequently electrophoresed for 20 min at 1 V cm�1. Finally,the slides were neutralized, stained with EtBr and analyzed at250� magnification of epifluorescence microscope (Zeiss, Göttin-gen, Germany) using image analysis system (Comet Assay II; Per-ceptive Instruments Ltd., Haverhill, Suffolk, UK). One hundredrandomly captured comets from each slide were examined.

2.3.4. Cytokinesis-block micronucleus (CBMN) assayThe CBMN assay was carried out basically as described by Fen-

ech and Morley (1985) with minor modifications (Gajski et al.,2008). After the exposure to the different leachate samples thewhole blood (500 lL) was incubated in a Euroclone medium (Chro-mosome kit P, Euroclone, Milano, Italy) at 37 �C in an atmosphereof 5% CO2. Cytochalasin-B (Sigma) was added at a final concentra-tion of 3 lg mL�1 44 h after the culture was started. The cultureswere harvested at 72 h, fixed in the methanol–acetic acid solution,air-dried and stained with 5% Giemsa solution (Merck, Darmstadt,Germany). One thousand binuclear lymphocytes were analyzedunder a light microscope (Olympus CX41, Tokyo, Japan) at 400�magnification using parameters according to the HUMN projectcriteria published by Fenech et al. (2003).

2.3.5. Allium cepa growth and treatmentPrior to initiating the test, the outer scales of the bulbs (1.5–

2.0 cm in diameter) of the common onion, A. cepa and the dry bot-tom plate were removed without destroying the root primordia.For each sample, series of six bulbs were placed in distilled waterand rotted for 48 h at room temperature (22 ± 2 �C). The bulbs withsatisfactory root lengths (2–2.5 cm) were used in the study, whilethose with exceptionally long or short roots were discarded (onaverage 2–3 bulbs). Therefore, individual sets of three bulbs wereexposed to each water sample. Apart from the original (100%leachate), landfill leachate was diluted to 5%, 25% and 50% withdH2O. Comet assay on onion root cells was performed after 24 hof the exposure to original and diluted leachate as well as purifiedwater samples. Distilled water was used as a negative control.

2.3.6. Allium cepa root cells comet assayThe comet assay was performed under alkaline conditions

according to Gichner et al. (2004) with slight modifications(10 min denaturation, 15 min electrophoresis at 1 V cm�1,300 mA) using A. cepa as a plant sample. Three slides were evalu-ated per water sample. For each slide, 50 randomly chosen nucleiwere analyzed using a fluorescence microscope with an excitationfilter of BP 520/09 nm and a barrier filter of 610 nm. A computer-ized image-analysis system (Komet version 5, Kinetic ImagingLtd., Liverpool, UK) was employed.

2.4. Statistical analysis

For statistical evaluation Statistica 6.0 software package wasused. The difference in the cell viability and CBMN assay parame-ters between control and tested samples was done by v2 test. Thedifference between control and tested samples as well as betweentreated samples for the comet assay parameters was assessed by

terization of the landfill leachate prior/after chemical and electrochemicaldoi.org/10.1016/j.chemosphere.2013.05.059

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Fig. 2. The effect of landfill leachate on the viability of human peripheral bloodlymphocytes (HPBLs). The viability was determined by differential staining withacridine orange (AO) and ethidium bromide (EtBr) and by fluorescence microscopyafter different exposure periods (4 and 24 h) to original or purified landfill leachateor negative control. C – Negative control; L – original (untreated) landfill leachate;P-EC – landfill leachate electrochemically purified; P-C – landfill leachate chem-ically purified. �Statistically significant P < 0.05.

4 V. Garaj-Vrhovac et al. / Chemosphere xxx (2013) xxx–xxx

Newman-Keuls test (human blood cells) or Duncan test (A. ceparoot cells). The level of statistical significance was set at P < 0.05.

3. Results

3.1. Chemical analysis

The values of physico-chemical parameters of the landfill leach-ate from the Piškornica landfill prior/after the treatment are pre-sented in Table 1. Results showed that it was stable leachatewith slightly alkaline reaction, dark brown to black color (causedby the presence of high molecular weight humic substances), highturbidity and SS. Several exceeding of permissible limits were ob-served and following the purification treatment, all measuredparameters, except ammonia, decreased significantly. Both treat-ment methods resulted in clear, colorless and odorless effluent.

3.2. Cytotoxicity of landfill leachate in HPBLs

The results of the cell viability obtained after different exposureperiods (4 and 24 h) of the HPBLs to the negative control, originalor purified landfill leachate were presented in Fig. 2. Significant de-crease in survival was obtained after the treatment of the HPBLswith the original landfill leachate after both exposure periods,while in the case of purified samples there was no significant dif-ference in the cell viability compared to negative control for eitherexposure period.

3.3. Genotoxicity of landfill leachate in HPBLs

Basic statistical parameters for the selected parameters of thecomet assay obtained after different exposure periods of the HPBLsto original or purified landfill leachate or negative control werepresented in Fig. 3A. Newman-Keuls test confirmed that after bothexposure periods to the original (untreated) landfill leachate (L-4 h,L-24 h) resulted in significantly higher values of HPBLs comet assayparameters compared to negative control (C-4 h, C-24 h). Purifica-tion of the leachate either chemically (P-C-4 h, P-C-24 h) or elec-trochemically (P-EC-4 h, P-EC-24 h) resulted with significantdecrease in the genotoxicity. All purified samples showed signifi-cantly lower values compared to untreated leachate, while there

Table 1Composition of untreated landfill leachate from Piškornica old sanitary landfill andpurified effluent treated chemically or electrochemically compared with maximumallowed values for wastewater suitable for discharge into natural recipient.

Measuredparameter

Originalleachate

Treated leachate MAV

Chemically Electrochemically

Color (PtCo) 5600 219 82 –Turbidity (NTU) 1550 4 8 –SS (mg L�1) 576 10 6 35pH 7.56 7.65 8.27 6.5–

9EC (mS cm�1) 4.79 9.42 4.33 –TDS (mg L�1) 3350 6570 3030 –COD (mg L�1) 617 46 36 125BOD (mg L�1) 61 13 5 25NH4-N (mg L�1) 125 23.00 1.5 10Fe (mg L�1) 2.017 0.038 0.029 2Zn (mg L�1) 0.63 0.038 0.16 2Cr (mg L�1) 0.016 <0.001 <0.001 0.1Ni (mg L�1) 0.023 <0.001 <0.001 0.5Cu (mg L�1) 0.047 0.008 0.006 0.5Pb (mg L�1) 0.003 <0.001 <0.001 0.5

BOD – biochemical oxygen demand; COD – chemical oxygen demand; EC – elec-trical conductivity; MAV – maximum allowed value; SS – suspended solids; TDS –total dissolved solids.

Please cite this article in press as: Garaj-Vrhovac, V., et al. Toxicological charactreatment: A study on human and plant cells. Chemosphere (2013), http://dx.

was no significant difference compared to negative control, nor be-tween two treatment methods.

Similar trend was observed for CBMN assay parameters wherethe highest frequencies of micronuclei (MNi), nucleoplasmicbridges (NPBs) and nuclear buds (NBUDs) were obtained in thecase of the exposure of the HPBLs to original landfill leachate forboth exposure periods (Fig. 4). On the other hand, purified samplesturned to be nongenotoxic as mentioned parameters did not statis-tically differ from negative control.

3.4. Genotoxicity of landfill leachate in Allium cepa root cells

Results of the comet assay performed on plant cells showedgood correlation with those obtained using HPBLs. In comparisonwith negative control, the values of the comet assay parameterssignificantly increased (Fig. 3B) in A. cepa roots exposed to serialdilutions of landfill leachate. DNA damage of root nuclei was re-corded even in 5% solution of landfill leachate. After 24 h periodof exposure, there was no significant difference in the values ofthe comet assay parameters between two treatment methods.There was also no significant difference between treated landfillleachate and negative control.

4. Discussion

Dealing with landfill leachates is one of the most important top-ics in waste management because of its composition and ability tointerfere with the environment. The complex composition of theleachate causes multiple mechanisms of toxicity to the cells. Tox-icity can be direct; caused by the presence of the substances likeammonia, pesticides and heavy metals as well as indirect; causedby the metabolic activation of the xenobiotics that leads to the for-mation of the secondary compounds such as reactive oxidativespecies (ROS), i.e. free radicals that could damage cellular biomol-ecules (Renou et al., 2008).

Li et al. (2008) confirmed adverse effect of the landfill leachateon the brain and liver of the mice following per os exposure. Theeffects were manifested at two levels; through the lipid peroxida-tion (LPO) and through the changes in the oxidative status mea-sured through the concentration of thiobarbiturate substances, aswell as increased concentration of antioxidant enzymes (Cu, Zn-superoxide dismutase, Se-dependent glutathione peroxidase andcatalase) that are induced by increased concentrations of free rad-icals. Bhargav et al. (2008) observed expression of the hsp70 as wellas increased activity of the antioxidant enzymes and increasedconcentrations of LPO products after exposure of Drosophila

terization of the landfill leachate prior/after chemical and electrochemicaldoi.org/10.1016/j.chemosphere.2013.05.059

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Fig. 3. The effect of landfill leachate on the alkaline comet assay parameters (tail length and tail moment) in: (A) human peripheral blood lymphocytes (HPBLs) after differentexposure periods (4 and 24 h) to original, purified landfill leachate or negative control. (B) Allium cepa root cells after 24 h exposure period to original and diluted landfillleachate (5%, 25%, 50% and 100%), purified landfill leachate or negative control. C – Negative control; L – original (untreated) landfill leachate; P-EC – landfill leachateelectrochemically purified; P-C – landfill leachate chemically purified. �Statistically significant P < 0.05.

V. Garaj-Vrhovac et al. / Chemosphere xxx (2013) xxx–xxx 5

melanogaster species to the landfill leachate. These findings con-firmed that the production of the free radicals represent the basicmechanism of the toxic effects of the landfill leachate.

According to our results, chemical characterization of testedleachate showed multiple exceeding of maximal allowed values(MAV) in SS, COD, BOD, and ammonium, whereas slight exceedingof iron was observed. Although below MAV it is important toemphasize the presence of metals (Zn, Cr, Ni, Cu, Pb) because oftheir potential toxicity. Chemical composition observed in our re-search is in accordance with leachates investigated in other studies(Sang et al., 2006; Bakare et al., 2007; Li et al., 2008; Amahdar et al.,2009; Gajski et al., 2012).

In order to detect impact of landfill leachate on human health,we have tested its genotoxic potential on HPBLs that are reliablebiomarkers of exposure to toxic substances (Clare et al., 2006).The present study is consistent with other researches that detectedsimilar effects (Bakare et al., 2007; Gajski et al., 2012). Similarly,tested sample proved to be genotoxic to plant biomarker (Chandraet al., 2005) A. cepa root cells, as well.

Several studies dealt with toxicity of compounds that were de-tected in our leachate sample on various model organisms. The ef-fect of organic carbon matter from sewage effluent was observedby Wu et al. (2010) where it proved to be genotoxic to Salmonellatyphimurium. In the same study, authors have tested purified efflu-ent that showed significant decrease in genotoxicity what was alsothe case in our study. Addition of landfill leachate with similarcomposition to animal (Tewari et al., 2005) or plant models re-sulted with increased genotoxicity in Triticum aestivum (Li et al.,2008), Hordeum vulgare (Sang et al., 2006), and Vicia faba (Sanget al., 2004; Feng et al., 2007), leading to the conclusion that land-fill leachates could have negative effect on environment.

Observed negative effects demand better waste managementmethods that will reduce all possible negative risks of landfillleachates. Methods that were used in the present research cer-tainly display adequate properties in reducing chemical composi-tion responsible for the genome damage detected in our study.Chemical and electro-chemical purification lead to decrease inthe measured parameters in HPBLs and A. cepa root cells which

Please cite this article in press as: Garaj-Vrhovac, V., et al. Toxicological charactreatment: A study on human and plant cells. Chemosphere (2013), http://dx.

indicates that landfill leachates must be processed before its re-leased in the environment. These results are consistent with thoseinvestigated by Wu et al. (2010) where the addition of NHþ4 inter-fered and inhibited purification of sewage effluent and restoredgenotoxic effects. Sample tested in the present study had high con-centrations of NHþ4 as well, leading to the conclusion that chemicaland electro-chemical purification could be more effective way ofpurification waste leachates.

The presence of metals in landfill leachate could be one of thefactors that contributed to increase of genome damage in testedmodels. Several authors have reported toxicity of iron (Prá et al.,2008), zinc (Orieux et al., 2011), copper (Gabbianelli et al., 2003),chromium (Trzeciak et al., 2000), lead (García-Lestón et al., 2011;Martínez-Alfaro et al., 2012), and nickel (Cavallo et al., 2003; Woz-niak et al., 2004) to animal and human cells and populations. Someof those studies showed that genome damage arises mostly fromproduction of ROS causing oxidative DNA damage. Except causingoxidative stress nickel and lead were reported to impair DNA re-pair (Cavallo et al., 2003; Wozniak et al., 2004; Martínez-Alfaroet al., 2012). Another factor that increases nickel toxicity is pres-ence of suspended and dissolved solids (Cloran et al., 2010) whichexceeded MAV in our sample as well.

Taken together, we can assume that multiple interactions be-tween organic compounds, metals, dissolved and suspended solidscould have additive and/or synergistic effects on DNA molecule.The DNA damage, that was produced when human or A. cepa rootcells were exposed to landfill leachate, could arise from single-and/or double-strand DNA breaks, sites of incomplete repair, alka-li-labile sites and DNA–DNA and DNA–protein crosslinks, and weredetected using the alkaline comet assay (Collins et al., 2008; Kuma-ravel et al., 2009). Such DNA modifications have also been relatedto oxidative stress arisen from higher formation of ROS produced athigher concentration of environmental pollutants (Cunningham,1997; Rucinska et al., 2004; Yildiz et al., 2009). However, otherpossible mechanisms of toxicity are breakage of chromosomes orchromatides, dysfunction in mitotic spindle, DNA miss-repair, telo-mere end-fusion, and over amplified DNA that were detected usingCBMN assay (Fenech et al., 2003).

terization of the landfill leachate prior/after chemical and electrochemicaldoi.org/10.1016/j.chemosphere.2013.05.059

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Fig. 4. The effect of landfill leachate on the cytokinesis-block micronucleus (CBMN)assay parameters (total number of micronuclei, nucleoplasmic bridges and nuclearbuds) in human peripheral blood lymphocytes (HPBLs) after different exposureperiods (4 and 24 h) to original or purified landfill leachate or negative control. MNi– Micronuclei; NPBs – nucleoplasmic bridges; NBUDs – nuclear buds; BNC –bunucleated cells; C – negative control; L – original (untreated) landfill leachate; P-EC – landfill leachate electrochemically purified; P-C – landfill leachate chemicallypurified. �Statistically significant P < 0.05.

6 V. Garaj-Vrhovac et al. / Chemosphere xxx (2013) xxx–xxx

5. Conclusion

Regardless of the cell type, toxicological effects of the originallandfill leachate were confirmed by all three conducted tests. Sucheffluent in case of discharge into the environment without appro-priate treatment could represent a significant source of thepollution of ground and surface water sources and consequently,induce genome damage in the population consuming such contam-inated water. Both, chemical and electrochemical treatment of theleachate significantly reduced genome damage in the humanlymphocytes as well as onion root cells. There was no significantincrease of either comet assay or CBMN assay parameterscompared to the negative control following the exposure of thelymphocytes to the chemically or electrochemically treated landfillleachate even after prolonged exposure of the cells. Treated leach-ate did not induce DNA damage in A. cepa roots according to thecomet assay as well. These results confirmed that both methodsare suitable for the treatment of such complex waste effluentdue to high removal efficiency of all measured parameters. More-over, proposed treatment methods do not produce toxic by-products that could pose toxic effects to the exposed population.

Please cite this article in press as: Garaj-Vrhovac, V., et al. Toxicological charactreatment: A study on human and plant cells. Chemosphere (2013), http://dx.

Acknowledgements

This investigation was supported by the Croatian Ministry ofScience, Education and Sports (Grant Nos. 022-0222148-2125,119-1191196-1202, and 119-0982934-3110).

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