Chemosphere xxx (2011) xxx–xxx

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Chemosphere

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A protocol for the evaluation of genotoxicity in bile of carp (Cyprinus carpio)exposed to lake water treated with different disinfectants

Milena Villarini a, Massimo Moretti a,⇑, Luca Dominici a, Cristina Fatigoni a, Ambrosius Josef Martin Dörr b,Antonia Concetta Elia b, Silvano Monarca a

a Department of Medical–Surgical Specialties and Public Health, University of Perugia, Via del Giochetto, I-06122 Perugia, Italyb Department of Cellular and Environmental Biology, University of Perugia, Via Elce di Sotto, I-06124 Perugia, Italy

a r t i c l e i n f o

Article history:Received 22 November 2010Received in revised form 31 March 2011Accepted 7 April 2011Available online xxxx

Keywords:Disinfection by-productsSurface water disinfectionCyprinus carpioBileGenotoxicityComet assay

0045-6535/$ - see front matter � 2011 Published bydoi:10.1016/j.chemosphere.2011.04.031

⇑ Corresponding author. Tel.: +39 075 5857363; faxE-mail address: massimo.moretti@unipg.it (M. Mo

Please cite this article in press as: Villarini, M.,treated with different disinfectants. Chemosphe

a b s t r a c t

A sensitive and rapid method to evaluate toxic and genotoxic properties of drinking water supplied fromLake Trasimeno (Umbria, Central Italy) was worked out analysing bile in Cyprinus carpio exposed for 20 dto lake water treated with 3 different disinfectants, sodium hypochlorite (NaClO), chlorine dioxide (ClO2)and peracetic acid (PAA). Fish were sacrificed at 0, 10 and 20 d in order to investigate the time course ofthese endpoints. An aliquot of bile samples was fractionated by adsorption on C18 silica cartridges and thegenotoxic potential of whole bile and of bile fractions was evaluated by the single-cell microgel-electro-phoresis (comet) assay on human colonic adenocarcinoma cells (Caco-2). Bile (both whole and fraction-ated) from specimens exposed to the three disinfectants always showed a genotoxic activity as comparedto the control group. The results of this study provide evidence that all three disinfectants cause anincrease in bile genotoxicity of chronically exposed fish.

� 2011 Published by Elsevier Ltd.

1. Introduction

A growing interest in biological effects caused by hazardousenvironmental pollutants has led to the development of the ‘‘bio-marker approach’’ concerning various groups of aquatic organisms.Fish and mussels are often considered as sentinel organisms inhealth assessment studies. In this context, bile analysis has beenused to assess the exposure to various substances in fish, e.g. poly-cyclic aromatic hydrocarbons (PAH), chlorinated phenols andestrogenic substances (Pettersson et al., 2006). The analysis of bilemetabolites is considered a convenient and relatively rapid methodfor monitoring PAH contamination (Ruddock et al., 2003; Blahovaet al., 2008). Bile from rainbow trout caged downstream of sewagetreatment plants in Sweden showed to have contaminants (estro-gens, bisphenol A and 4-nonylphenol) at levels that could be linkedto their water concentrations. Therefore, analysis of fish bile fluidcan be used as an indicator of exposure and uptake of xenobiotics(Pettersson et al., 2006). Only few studies investigated the geno-toxicity on fish bile. De Flora et al. (1993) and Viganò et al.(2002) found mutagenic activity, as evaluated with the Ames test,in bile of fish caught from polluted areas of the Po river.

Many chemical contaminants have been identified in surfacedrinking water deriving from industrial, agricultural practices

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et al. A protocol for the evaluare (2011), doi:10.1016/j.chemo

and disinfection treatments (WHO, 1996; Calderon, 2000; Hoferand Shuker, 2000; Monarca et al., 2002). It has been shown thatnatural organic substances (e.g. humic and fulvic acids) in surfacewaters may react with disinfectants to produce volatile andnon-volatile disinfection by-products (DBP) that are potentiallyharmful to human and aquatic organisms. In particular, it has beendemonstrated that chlorination, the most widely used method forwater disinfection, leads to the formation of DBP with mutagenicand/or carcinogenic activity (Sujbert et al., 2006; Umbuzeiro Gdeet al., 2006; Schenck et al., 2009). Moreover, several epidemiolog-ical studies revealed a positive association between the use ofchlorinated drinking water obtained from surface sources withthe incidence of cancer (e.g. cancer of urinary and gastrointestinaltracts) (Richardson et al., 2007) as well as with small for gesta-tional age/intrauterine growth retardation and preterm delivery(Nieuwenhuijsen et al., 2009). To reduce potential health risks,disinfectant with reduced capability of DBP formation are neededas an alternative to sodium hypochlorite (NaOCl). Chlorine dioxide(ClO2) has replaced NaOCl for the disinfection of drinking water inItaly and elsewhere in Europe. Even though ClO2 produces lowerlevels of trihalomethanes than NaOCl, it produces inorganicby-products such as chlorite and chlorate that recently havebeen shown to be genotoxic (Feretti et al., 2008). Peracetic acid(CH3–COO2H, PAA) is a water disinfectant used mainly for waste-water tertiary treatment. However, if used for disinfection ofdrinking water PAA gives rise to a very low level of genotoxicity

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and produces only non-mutagenic carboxylic acids (Monarca et al.,2002).

The present study is part of a research project supported by theItalian Ministry of Education, University and Scientific Research(MIUR), which aim is to evaluate the potential genotoxic risks aris-ing from the disinfection of surface water for human consumption.The objective of the project was also to evaluate the applicability ofa new combined approach for the assessment of DBP formation indisinfected drinking water and to compare old and new disinfec-tants by performing chemical and microbiological analyses concur-rently with a complex battery of in vivo and in vitro tests todetermine toxicity and genotoxicity of disinfected surface water(Elia et al., 2006). For these purposes, a pilot plant was constructedto treat lake drinking water with different disinfectants (NaClO,ClO2 or PAA), and to compare their bactericidal activity and poten-tial DBP formation (Monarca et al., 2004).

The aim of the present research was to propose and evaluate anexperimental protocol to study the toxic and genotoxic propertiesof drinking water supplied from Lake Trasimeno (Umbria, CentralItaly) by analysing bile in Cyprinus carpio exposed in situ. The LakeTrasimeno water was disinfected with 3 different chemicals (i.e.NaClO, ClO2 and PAA). To this purpose, the pilot plant was suppliedwith water from Lake Trasimeno characterized by high concentra-tions of total organic carbon (TOC) and bromides, that could besimilar to other surface water suppliers. The genotoxicity of thefish bile was evaluated in vitro in human colonic adenocarcinomacells (Caco-2) with the single-cell microgel-electrophoresis (co-met) assay. Caco-2 cells were chosen as target since bile acidsare discussed to be involved in colon carcinogenesis (Rosignoliet al., 2008).

The comet assay is a sensitive and rapid technique to detectDNA single- and double-strand breaks, alkali-labile sites, andsingle-strand breaks associated with incomplete excision repair.

This assay has previously been used to investigate the genotox-icity of the surface drinking water from the mentioned pilot plantboth in in vitro and in vivo studies in circulating erythrocytes of C.carpio and in Zebra mussel cells (Bolognesi et al., 2004; Buschiniet al., 2004; Maffei et al., 2005).

2. Materials and methods

2.1. Chemicals and reagents

ClO2 was produced on site from an 8% NaClO2 solution and a10% HCl solution through an automated generator (Tecme, Gardolodi Trento, Italy). NaClO was obtained from Solvay (Rosignano,Italy). PAA was obtained from Promox (Leggiuno, Italy). C18 car-tridges (Sep-Pak Plus tC18 Environmental Cartridges) were sup-plied by Waters Chromatography (Milford, MA, USA). Gibcoculture media and buffered solutions were purchased from Invitro-gen (Milan, Italy): Dulbecco’s modified Eagle medium (DMEM),foetal calf serum (FCS), L-glutamine, non-essential aminoacids,antibiotics (penicillin and streptomycin), Dulbecco’s phosphatebuffered saline pH 7.4 (PBS), 0.5% trypsin-EDTA. All general labora-tory chemicals and all reagents for electrophoresis used were ofanalytical grade. Ethylacetate (ETA), dichloromethane (DCM),methanol (MET), dimethylsulphoxide (DMSO), ethylenediamin-etetracetic acid disodium salt (Na2EDTA), sodium chloride (NaCl)and sodium hydroxide (NaOH) were purchased from Carlo Erba(Milan, Italy). Low- and normal-melting temperature agarose(LMA and NMA, respectively), fluorescein diacetate (FDA), propidi-um iodide (PI), ethidium bromide (EB), tris-(hydroxymethyl)-aminomethane (Tris–HCl), Triton X-100 and 4-nitroquinoline-N-oxide(4NQO) were purchased from Sigma–Aldrich (Milan, Italy). Dis-tilled water was used throughout the experiment.

Please cite this article in press as: Villarini, M., et al. A protocol for the evaluatreated with different disinfectants. Chemosphere (2011), doi:10.1016/j.chemo

2.2. Pilot disinfection plant

The pilot plant, which was supplied with water from lake Trasi-meno (Umbria, Italy), consisted of the following units (Monarcaet al., 2004):

(1) One pump to capture raw lake water.(2) One large and high sedimentation system connected with

two additional and subsequent tanks (1 m3 each) to clarifyraw lake water.

(3) Two filtration systems, a 50 lm stainless filter followed by a25 lm filter cloth to remove suspended particles.

(4) One pumping system adding sulphuric acid to neutralize thebasic pH value of the Trasimeno lake water.

(5) One main pipeline from which the water flow wasdivided into four secondary pipelines to supply the contacttanks.

(6) Four 300 L stainless contact tanks; three supplied with thedifferently disinfected water and one with untreated water(negative control).

(7) Four exposure basins (1 m3 stainless tanks) receiving thewater flow from their contact tanks for the in situ exposureof carps.

The scheme of the pilot plant is shown in Fig. 1.

2.3. Disinfectants

The raw lake water was treated in the three contact tanks withNaClO, ClO2 or PAA, respectively. Disinfectant concentrations werechosen on the basis of preliminary experiments aimed to deter-mine water disinfectant demands. Furthermore, disinfectant con-centrations were adjusted to produce a disinfectant residuallower than 0.2 mg L�1 to avoid toxicity to the exposed organ-isms.The mean disinfectant concentrations used during the exper-iment were: NaClO, 0.7 mg L�1; ClO2, 1.6 mg L�1; PAA, 0.6 mg L�1

(Monarca et al., 2004).

2.4. Animal exposure

Specimens of C. carpio (n = 64), provided from the Fish BreedingCentre of Lake Trasimeno (Sant’ Arcangelo, Umbria, Italy) wereacclimated for 20 d in four 1 m3 tanks supplied with clarifiedsuperficial lake water, at pH 7 before starting the flow-through testfrom June to July 2001 (Elia et al., 2006). Immature, 14 months oldcarps were chosen in order to exclude differences between sexes.

Before starting the experiment (t0), a sample of eight specimensfrom untreated water was sacrificed. Then three tanks were sup-plied with the disinfectants, NaClO, ClO2 and PAA, respectively,while the fourth (untreated water) was employed as negativecontrol.

The number, weight and total length for specimens collocatedin each tank were: controls (n = 14) 91.1 ± 16.8 g, 17.4 ± 1.0 cm;NaClO (n = 14) 94.2 ± 19.1 g, 17.8 ± 1.5 cm; ClO2 (n = 14)86.3 ± 21.3 g, 16.7 ± 1.1 cm; PAA (n = 14) 86.2 ± 13.2 g, 17.1 ±1.1 cm. Fish were fed with a specific commercial diet of 1% bodyweight/day, and no mortality occurred during the experiment.

At exposure times t10 (after 10 d) and t20 (after 20 d), treatedand untreated C. carpio were sacrificed, gall bladder removed andstored at �80 �C until used.

The experiments were conducted in accordance with theEuropean and national guidelines (European Commission, Direc-tive 86/609//EC and Italian Directive 116/1992, respectively).

tion of genotoxicity in bile of carp (Cyprinus carpio) exposed to lake watersphere.2011.04.031

Fig. 1. Schematic diagram of the pilot plant for in situ exposure of fish to untreated lake water or lake water treated with different disinfectants (ClO2, NaClO, and PAA).

M. Villarini et al. / Chemosphere xxx (2011) xxx–xxx 3

2.5. Samples preparation

Because of the small size of the gall bladder in juvenile speci-mens of C. carpio the bile samples of each treatment group werepooled.

The C18 silica sorbent columns were activated in sequence withETA, DCM, MET and distilled water (5 mL each). Aliquots of 250 lLof bile samples were diluted 1:100 with distilled water, acidified topH 2 with sulphuric acid and then pumped through the cartridges(25 mL per cartridge) placed in a Vac Elut SPS 24 multisample con-centration system (Varian, Palo Alto, CA, USA) using a vacuumwater pump. The compounds retained on the C18 cartridges wereeluted sequentially from the column using ETA, DCM and MET(5 mL each). The eluates were collected separately in differentvials, reduced to a small volume by means of a rotating vacuumevaporator and dried under a nitrogen flow. Each of the residueswas then dissolved in 500 lL of DMSO (10 lL of fish bilecorresponded to 20 lL of extract) to give the correspondingETA, DCM and MET fractions. For quality control, a blank cartridge(control cartridge) was performed by sucking distilled waterthrough a C18 column that was subsequently processed exactlyas described for bile samples. All fractions were stored at �20 �Cuntil analyzed.

The three bile fractions (ETA, DCM and MET), the pooled frac-tions (ETA + DCM + MET) and the whole bile were analysed inCaco-2 cell line for their citotoxic and genotoxic activity. All sam-ples were assayed in triplicate.

2.6. Caco-2 cell line

The human colonic adenocarcinoma Caco-2 cells (ATCC HTB 37)were obtained from the Istituto Zooprofilattico Sperimentale dellaLombardia e dell’Emilia Romagna (Brescia, Italy). Caco-2 cells weresub-cultured weekly in DMEM supplemented with 10% FCS, 2%L-glutamine, 1% non-essential aminoacids, 100 U mL�1 penicillinand 100 mg mL�1 streptomycin, at 37 �C in air containing 5% CO2.For the experiments, monolayer cells in exponential growthwere harvested using 0.5% trypsin–EDTA, seeded in 6-well plates(2 mL DMEM, 1 � 106 cells/well) and allowed to adhere for 18 hat 37 �C.

Please cite this article in press as: Villarini, M., et al. A protocol for the evaluatreated with different disinfectants. Chemosphere (2011), doi:10.1016/j.chemo

2.7. Fluorochrome-mediated viability test

Preliminarily, the non-cytotoxic range was defined in controlfish only. In order to identify sub-toxic sample doses, cytotoxicity ofbile fractions and whole bile was assessed by the fluorochrome-mediated viability test (Jones and Senft, 1985) with minor modifi-cations (Moretti et al., 2005). This assay is based on the simulta-neous determination of live and dead cells with the detection ofintracellular esterase activity by FDA and of plasma membraneintegrity by PI, respectively. FDA penetrates freely into intact cellsand it is hydrolyzed to its fluorochrome, fluorescein, which is re-tained in the cell and fluoresces green. PI, an indicator of irrevers-ible loss of membrane integrity, stains only nucleic acids ofmembrane-damaged cells and fluoresces red.

In brief, 1 � 106 Caco-2 cells spread in 2 mL of DMEM were trea-ted for 4 h with different concentrations of bile fractions and bilepooled fractions (corresponding to 1.25, 2.5, 5.0, 7.5, 25, 50 and75 lL bile/assay), as well as with whole bile (0.5, 1.0 1.25, 2.5,5.0, 7.5, 25, 50 and 75 lL bile/assay) deriving from control fish.

After the treatment cells were harvested by trypsinization andsubjected to simultaneous staining with FDA and PI, with stainingsolutions made immediately prior to their use. Aliquots of cell sus-pensions were incubated with 100 lL of 20 lg mL�1 FDA and 30 lLof 0.4 lg mL�1 PI at room temperature for 5 min. Cell counts werethen performed using ten-chamber disposable microscope slidesprovided with a haemocytometer-like counting grid (Kova, HycorBiomedical, USA). The cells were examined with a standard fluo-rescence microscope (Dialux 20, Leitz, Germany) equipped withepi-illumination provided by a 50 W high-pressure mercury lamp(HBO 50, Osram, Germany). Cell viability was expressed in percent-age: cell survival levels corresponding to 75–85% or >85% wereconsidered as moderately cytotoxic or non-cytotoxic, respectively.

2.8. Comet assay

In order to study the bile genotoxic potential, freshly madeCaco-2 sub-cultures (1 � 106 cells in 2 mL of DMEM) were exposedfor 4 h to the highest non-cytotoxic concentration of each bile frac-tion and whole bile (0.125 lL eq/assay for the extracts and0.50 lL eq/assay for whole bile).

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4 M. Villarini et al. / Chemosphere xxx (2011) xxx–xxx

To check the possible effects of the solvent (DMSO) on Caco-2cells and the performance of the comet assay, negative (1% DMSO)and positive (0.1 mM 4NQO) controls were included in all theexperiments. Immediately after the exposure, Caco-2 cells werecollected by trypsinization and processed in the comet assay underalkaline conditions (alkaline unwinding/alkaline electrophoresis,pH > 13), according to the original procedure (Singh et al., 1988)with slight modifications (Moretti et al., 2002).

Conventional microscope slides were coated with 1.0 % NMAand allowed to dry overnight. Approximately 1 � 105 cells wereembedded in 75 lL of 0.7% LMA, spread on the precoated slidesand protected with a top layer of 75 lL of 0.7% LMA. The slideswere submerged in ice-cold freshly prepared lysing solution(10 mM Tris–HCl, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X-100,and 10% DMSO; pH 10) for at least 1 h to obtain the lysis of cellularand nuclear membranes of the embedded cells. After lysis, slideswere aligned in a horizontal gel electrophoresis tank (HE99, HoeferScientific Instruments, San Francisco, CA, USA) filled with freshlymade alkaline electrophoresis solution (300 mM NaOH, 1 mMNa2EDTA, pH > 13) to a level of 0.25 cm over the slides. Microgelswere equilibrated for 20 min in the alkaline solution and electro-phoresis was then performed for 20 min (1 V cm�1, 300 mA). Afterelectrophoresis, the slides were washed three times with neutral-ising buffer (0.4 M Tris–HCl buffer, pH 7.5) and stained with100 lL EB (2 mg mL�1). The extent of induced DNA damage wasmeasured as % of fluorescence migrated in the comet tail (i.e. tailintensity) by a computer-based image analysis system (Comet As-say III, Perceptive Instruments, Blois Meadow, Suffolk, UK). A totalof 150 randomly selected cells were analysed for each experimen-tal point.

2.9. Statistical analysis

Experimental results were expressed as the mean ± standard er-ror of the mean (SEM) of triplicate assays.

Statistical analysis was aimed to verify correlations between tailintensity in Caco-2 cells treated in vitro and time exposure of fishto different disinfected waters. Pearson’s correlation coefficient(r) was used to examine the association between tail intensity inCaco-2 cells and time exposure of fish to different disinfectedwaters (p < 0.05 was considered statistically significant).The statis-tical analysis was performed using the Statistical Package for SocialScience software (SPSS 10.0; Chicago, IL, USA).

3. Results

3.1. Cytotoxicity

The data shown in Table 1 display the effects of the tested con-centrations of bile fractions (1.25–75 lL bile/assay), and whole bile

Table 1Cytotoxicity effect (cell survival %, mean ± SEM of triplicate assays) of various bile extract

Sample (lL bile/assay) Bile extracts

Ethyl acetate Dichloromethane

75 66.66 ± 4.26 70.00 ± 2.8650 68.42 ± 2.85 62.50 ± 1.9225 85.71 ± 1.98 73.68 ± 1.117.5 94.12 ± 1.12 81.82 ± 6.015.0 94.74 ± 1.57 96.23 ± 0.802.5 81.25 ± 3.23 90.53 ± 3.151.25 94.44 ± 2.16 97.30 ± 1.251.0 nd nd0.5 nd ndControl cartridge 97.43 ± 2.54 95.00 ± 2.87

nd: not detected.

Please cite this article in press as: Villarini, M., et al. A protocol for the evaluatreated with different disinfectants. Chemosphere (2011), doi:10.1016/j.chemo

(0.5–75 lL bile/assay) of untreated specimens of C. carpio on viabil-ity on Caco-2 cells after 4 h incubation.

Non-cytotoxic or moderately cytotoxic effects were observedfor the bile fractions at concentrations ranging from 1.25 to5.00 lL bile/assay and for whole bile at concentrations rangingfrom 0.5 to 1.25 lL bile/assay. Higher concentrations of bile frac-tions and whole bile from control fish (non-treated C. carpio, t0)caused an increase in percent of nonviable cells.

After the non-cytotoxic range was defined in control fish, thecell viability after 4 h incubation with bile fractions (1.25, 2.5and 5.0 lL bile/assay) or whole bile (0.5, 1.0 and 1.25 lL bile/assay)from C. carpio treated whit the tested disinfectants at differenttime intervals as been evaluated.

Only the lowest concentration examined (1.25 lL bile/assay and0.50 lL bile/assay for bile fractions and whole bile, respectively)did not affect viability of Caco-2 cells (viability > 85%) (resultsnot shown). Based on these results, these concentrations have beenchosen for genotoxicity testing of bile fluids of control and treatedfishes.

3.2. Genotoxicity

In order to assess DNA damage triggered by the different bilesamples, Caco-2 cells were examined by comet assay after expo-sure to 1.25 lL bile fractions/assay or 0.5 lL whole bile/assay. Ta-ble 2 reports the mean percent of DNA in the tail (tail intensity)± SEM as an indicator of DNA damage.The main results are thefollowing:

(1) no differences (i.e. tail intensity) were found in Caco-2 cellsbetween cells of negative controls (0.1% DMSO), cartridgecontrols and cells treated with bile fractions or whole bileof fish groups housed in untreated lake water at t0 exposure(results not shown);

(2) there were no differences in tail intensity of Caco-2 cellstreated with bile fractions or whole bile of fish populationshoused in untreated lake water at t10 and t20 days withrespect to t0 (data not shown);

(3) with regard to fish housed in ClO2 lake water a strong asso-ciation between DNA damage and treatment time wasobserved for DCM fraction (ETA fraction r = 0.988,p = 0.099; DCM fraction r = 1.000, p < 0.000; MET fractionr = 0.966, p = 0.165; pooled fractions r = 0.995, p = 0.061,whole bile r = 0.954, p = 0.193);

(4) no significant correlation was observed between time oftreatment and bile genotoxicity for fishes housed in NaClOtreated lake water (ETA fraction r = 0.714, p = 0.494; DCMfraction r = 0.929, p = 0.242; MET fraction r = 0.005,p = 0.997; pooled fractions r = 0.711, p = 0.497, whole biler = 0.687, p = 0.518);

s and whole bile concentrations in untreated specimens of Cyprinus carpio.

Whole bile

Methanol Pooled

0 0 00 0 00 0 00 90.32 ± 4.04 084.46 ± 0.94 77.94 ± 1.64 095.83 ± 2.60 77.47 ± 3.40 088.63 ± 0.96 92.11 ± 1.63 77.94 ± 1.90nd nd 90.77 ± 2.17nd nd 90.24 ± 2.3399.68 ± 1.58 96.80 ± 4.50 –

tion of genotoxicity in bile of carp (Cyprinus carpio) exposed to lake watersphere.2011.04.031

Table 2Tail intensity (mean ± SEM of triplicate assays) in Caco-2 cells treated with bile extracts or whole bile of Cyprinus carpio exposed in situ to untreatedlake water and lake water treated with different disinfectants (ClO2, NaClO, and PAA) at different time exposure (0, 10 and 20 d).

Exposure (days) Untreated lake water Lake water + ClO2 Lake water + NaClO Lake water + PAA

Ethylacetate extract0 2.55 ± 0.24 – – –10 2.23 ± 0.30 5.84 ± 0.70 6.56 ± 0.28 5.73 ± 0.2920 2.80 ± 0.36 7.73 ± 0.99 5.52 ± 0.85 9.50 ± 1.29r* (p-value) 0.437 (0.721) 0.988 (0.099) 0.714 (0.494) 0.999 (0.031)

Dichloromethane extract0 1.61 ± 0.15 – – –10 2.55 ± 0.06 6.24 ± 1.29 4.29 ± 0.54 5.94 ± 0.7220 3.12 ± 0.54 10.87 ± 0.09 4.78 ± 0.31 10.46 ± 1.48r* (p-value) 0.990 (0.089) 1.000 (0.000) 0.929 (0.242) 1.000 (0.008)

Methanol extract0 4.00 ± 0.53 – – –10 3.43 ± 0.29 5.78 ± 1.17 7.64 ± 0.38 7.40 ± 0.3420 3.89 ± 0.24 10.60 ± 0.24 4.02 ± 0.33 14.26 ± 0.89r* (p-value) -0.182 (0.884) 0.966 (0.165) 0.005 (0.997) 0.982 (0.122)

Pooled extract0 2.69 ± 0.11 – – –10 3.59 ± 0.61 6.94 ± 1.48 8.13 ± 2.04 7.27 ± 0.4620 2.92 ± 0.33 12.88 ± 0.80 6.78 ± 1.14 14.72 ± 0.82r* (p-value) 0.246 (0.842) 0.995 (0.061) 0.711 (0.497) 0.991 (0.087)

Whole bile0 3.23 ± 0.44 – – –10 3.36 ± 0.45 6.12 ± 0.50 6.40 ± 0.37 7.90 ± 0.5620 3.36 ± 0.53 15.85 ± 1.29 5.47 ± 0.72 12.67 ± 1.27r* (p-value) 0.866 (0.333) 0.954 (0.193) 0.687 (0.518) 1.000 (0.004)

Negative control: 2.86 ± 0.38; Positive control: 14.23 ± 0.16.Control cartridge: ETA extract, 2.94 ± 0.85; DCM extract, 2.92 ± 0.47; MET extract, 3.35 ± 0.44; pooled extracts, 3.14 ± 0.45.* Pearson’s correlation coefficient.

M. Villarini et al. / Chemosphere xxx (2011) xxx–xxx 5

(5) with regard to fish treated with PAA disinfected lake watersignificant time-dependence was observed for ETA andDCM fractions and for whole bile (ETA fraction r = 0.999,p = 0.031; DCM fraction r = 1.000, p = 0.008; MET fractionr = 0.982, p = 0.122; pooled fractions r = 0.991, p = 0.087,whole bile r = 1.000, p = 0.004).

4. Discussions and conclusions

The present study evaluated for the first time the bile genotox-icity of fish exposed to surface water treated with three differentwater disinfectants. In particular, this study provided further evi-dence that two biocides widely used in drinking water disinfection(NaClO and ClO2) and a relatively new disinfectant (PAA) can pro-duce genotoxic DBP that are excreted in bile of C. carpio.

Our current results confirm the potential genotoxic hazards re-lated to the use of disinfected surface waters, previously evidencedin the same pilot plant in C. carpio (Buschini et al., 2004) and inDreissena polymorpha (Bolognesi et al., 2004) exposed to surfacewater disinfected in the same way.

In these previous studies the comet assay and the micronucleustest were applied in circulating erythrocytes of C. carpio and hae-mocytes and gills of D. polymorpha, respectively. Genotoxic dam-age was shown in C. carpio exposed to water disinfected withNaClO and ClO2 that seemed to represent the major source of geno-toxic by-products following superficial water disinfection for pota-bilization. Also in D. polymorpha the results of comet andmicronucleus assays clearly indicate an interaction of NaClO andClO2 by-products with DNA. In both studies PAA did not induceneither clastogenic/aneugenic effects nor DNA damage on thestudied bioindicator. There was a good correspondence amongthe above mentioned data and those obtained in the same pilotplant from in situ mutagenicity tests in plants (Tradescantia spp.,Allium cepa and Vicia faba) (Monarca et al., 2003) and from a bat-

Please cite this article in press as: Villarini, M., et al. A protocol for the evaluatreated with different disinfectants. Chemosphere (2011), doi:10.1016/j.chemo

tery of in vitro tests (Guzzella et al., 2004), which confirmed thestrongest mutagenic effect observed following exposure to NaClO-and ClO2-disinfected waters.

On the contrary, in the present study exposure of fish popula-tions showed evidence for genotoxic effects induced by all thethree disinfectants tested. In fact, primary DNA damage was clearlyvisible without distinction in Caco-2 cells treated with bile samplescollected from fish specimens maintained in water disinfectedwith chlorine compounds or PPA. Throughout the experimentalperiod, it was evident a time-dependent genoxicity induced inCaco-2 cells by bile from fishes housed in ClO2 and PAA waters. Bilefrom fishes housed in NaClO water caused an increase in the extentof DNA damage in Caco-2 cells treated with bile from fishes ex-posed for 10 d, whereas decreased DNA migration extent was ob-served when testing bile from fishes exposed for 20 d. Thisparticular behaviour could be related, at least to some extent, tocytotoxic effects (e.g. loss of heavily damaged cells). A similar trendwas already observed in vivo when the comet assay was performedin circulating erythrocytes of C. carpio specimens exposed in situfor 20 d to water containing disinfectants (Buschini et al., 2004).

The differences of results obtained from the present and theprevious studies seem to support the feasibility of a complemen-tary approach, using comet assay both in fish bile and in circulatingerythrocytes.

In conclusion the exposure protocol presented in this work indi-cates that the formation of genotoxic DBP could be assessed suc-cessfully by analyzing unfractionated (whole) bile of C. carpiodirectly exposed to disinfected drinking water as a rapid screeningtest. However, the fractionation of bile samples could give addi-tional information, especially when coupled with advanced chem-ical analyses (e.g. gas-chromatography/mass spectrometry).

This simple, rapid and sensitive method (based on fish exposedto drinking water complex mixtures and testing whole bile by thecomet assay) could be used also for other water pollutants and

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6 M. Villarini et al. / Chemosphere xxx (2011) xxx–xxx

could be integrated with the traditional standard methods, as anaspecific monitoring method, in order to produce drinking waterof better quality.

These results confirm that routine monitoring of disinfecteddrinking water for genotoxicity and toxicity is recommended, inorder to minimize chronic exposure to noxious pollutants (i.e.DBP) in humans.

Conflict of interest

None declared.

Acknowledgments

This study was supported by the Italian Ministry of Education,University and Scientific Research (MIUR 2001; National Head Prof.S. Monarca).

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