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Research Article

Received: 24 June 2008, Revised: 25 July 2008, Accepted: 25 July 2008 Published online in Wiley Interscience: 2008

(www.interscience.wiley.com) DOI 10.1002/jat.1381

J. Appl. Toxicol. 2008; 28: 000–000 Copyright © 2008 John Wiley & Sons, Ltd.

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John Wiley & Sons, Ltd.The assessment of genotoxic effects of wastewater from a fertilizer factoryGenotoxic effects of wastewater from a fertilizer factoryKsenija Durgo,a* Višnja Orešcanin,b Stipe Luliç,c Nevenka Kopjar,d Davor Želježiçd and Jasna Franekiç Coliça

ABSTRACT: In this study we investigated cytotoxic, mutagenic and genotoxic effects of different concentrations of wastewaterfrom the phosphoric gypsum depot near the factory for fertilizing agents ‘INA Petrokemija’ (Kutina, Croatia). The Ames test wasperformed on Salmonella typhimurium TA98 and TA100 strains, in the presence of S9 mix, glutathione and buffer, respectively.Cytotoxicity was studied on human laryngeal carcinoma cells (HEp2) and human cervical cells (HeLa). The level of lipid peroxida-tion in these two cell lines was evaluated in parallel. To establish the levels of primary DNA damage, the alkaline comet assay wasperformed on treated human peripheral blood leukocytes. No mutagenic effects of phosphoric gypsum on Salmonella typhimuriumstrains in the presence of S9 mix, GSH or PBS were observed. However, strong cytotoxic effect was observed on both humancell lines when they were treated with different concentrations of wastewater. Lipid peroxidation was induced and increasedby prolonged time of incubation, highlighting that the damage was not repaired, but increased with the time of incubation.The results of the alkaline comet assay indicate significant DNA damaging potential of wastewater for human leukocytes.Since phosphoric gypsum transport water in its present composition and acidity is highly toxic and acts as prooxidant, caus-ing free radicals formation and DNA damage, urgent neutralization/purification of the wastewater to a level acceptable fordisposal into the environment is mandatory. Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: phosphogypsum; Ames assay; lipid peroxidation; DNA damage; comet assay

Introduction

A growing problem in many European countries is environmen-tal pollution from municipal and industry wastewaters. Pollutedwastewaters often connect through hydrographic systems,generating diffuse pollution, endangering plants and animals ofthe contaminated ecosystem and jeopardizing public health(Horvat et al., 2007).

Phosphogypsum is primary byproduct from phosphoric acidproduction. Separation of the phosphoric acid from the calciumsulfate dihydrate is done by either pressure or vacuum filtration.The filter cake is slurried with the water and pumped to specialstorage areas (ponds) where the solid part is settled and coveredwith highly acidic transport water (pH 2–3) containing high con-centrations of fluoride and elevated amounts of heavy metals.Phosphate rock, which is the origin of phosphogypsum, con-tains many impurities, including heavy and trace metals like Pb,V, Cr (VI), Mn, Fe, Ni, Cu and Zn (Carbonell-Barachina et al., 2002).

DNA damaging effects of the pollutants contained in phosph-ogypsum have been reported previously. Fluoride can combinestably with DNA by covalent bonding, affecting the normalstructure of DNA. It can induce the production of free radicals andmay depress enzyme activity, such as DNA polymerase affectingthe process of DNA replication or repair (Wang et al., 1997;Hayaschi and Tsutsui, 1993; Li et al., 1987). The excessive con-centrations of copper and iron induce reactive oxygen species(ROS) formation, leading to DNA damage and lipid peroxidation,which appears to be important for rapid growth of cancer cells(Valko et al., 2006). Chromium (VI) is potentially toxic and carci-nogenic at high doses. All soluble chromates can actively enterthe cells through channels for the transfer of isoelectric and iso-structural anions such as those for SO4

2− and HPO42− (Valko et al.,

2006). The ultimate product of intracellular reduction of hexa-valent chromium is Cr (III), which causes a wide variety of DNAlesions, including Cr–DNA adducts, DNA–protein cross links,DNA–DNA cross links and oxidative damage. Vanadium also mayparticipate in reactions involving the formation of free radicals.As reported earlier, pentavalent vanadium is an effective genotoxicagent in vitro, inducing DNA damage and chromosome malseg-regation at low doses (Rojas et al., 1996). Nickel is a human carcin-ogen that can alter gene expression by enhanced DNA methylationand compaction, rather than via mutagenic mechanisms. It mayinterfere with DNA repair processes and toxic doses of nickel arefound to induce lipid peroxidation and protein carbonyl forma-tion in animals (Valko et al., 2006; Misra et al., 1993; Werfel et al.,1998; Chakrabarti et al., 2001; Kawanishi et al., 2001). It alsoreacts easily with thiols and generates free radicals, which couldinactivate the repair enzymes (Shi et al., 1993).

Determination of the chemical composition and the potentialgenotoxic potential of wastewaters is crucial for environmental

* Correspondence to: K. Durgo, Faculty of Food Technology and Biotechno-logy, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia. E-mail: kdurgo@pbf.hr

a Faculty of Food Technology and Biotechnology, University of Zagreb, Pierot-tijeva 6, 10000 Zagreb, Croatia.

b USKNI, Laboratory for applied nuclear analytics, A. Jaksica 30, Zagreb,Croatia.

c Ruder Boškoviç Institute, Bijenicka 54, 10000 Zagreb, Croatia.

d Institute for Medical Research and Occupational Health, Ksaverska 2, 10000Zagreb, Croatia.

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protection and public health. Short-term bioassays can detecta wide range of substances that can cause genetic damageand enable quantification of mutagenic hazard even whenthere are no sufficient data about identity and physico-chemicalproperties of compounds present in wastewater (Valko et al.,2006).

The present study aimed at the examination of the chemicalcomposition of the phosphoric gypsum transport water collectedfrom the phosphoric gypsum depot near the factory for fertilizingagents, ‘INA Petrokemija’ (Kutina, Croatia). The established levelsof heavy metals were compared with the maximum allowedlevel that can be contained in wastewaters (Regulation on limitvalues of parameters of hazardous and other substances inwastewater, OG 40/99; 6/01; 14/01). Moreover, genotoxic effectsof wastewater were examined using a baterry of bioassays. Toobtain relevant information regarding the mechanisms underly-ing the toxicity of the wastewater investigated, test systems withdifferent sensitivity, specificity and cellular organization were used.As expected, the results of the present study might be poten-tially useful in implementation of intervention measures aimingto eliminate or significantly reduce groundwater pollution andprevent untoward biological effects in the environment.

Materials and Methods

Bacterial Test Strains

The test strains used were the Salmonella typhimurium, strainsTA98 (his D3052, rfa, uvrB, pKM101) and TA100 (his G46, rfa, uvrB,pKM101) (kindly provided by Maron and Ames, University of Cal-ifornia, CA, USA), grown overnight for 14 h in complete medium(OXOID, UK).

Human Cell Lines

HEp2 and HeLa cells of laryngeal and human cervical carcinoma,respectively (kindly provided by M. Osmak, Ruder Boškoviç Insti-tute, Croatia), were grown as monolayer cultures in DMEM medium(GIBCO, BRL), supplemented with 10% fetal bovine serum (GIBCO),4500 mg l−1 of glucose and 1% penicillin–streptomycin (Osmakand Eljuga, 1993).

Human Leukocytes

Venous blood was drawn from two healthy volunteers; both werenon-smokers (female, 26 years; male, 33 years), who gave informedconsent for participation in the study. Donors had not been exposedto diagnostic or therapeutic irradiation or to known genotoxicchemicals for one year prior to blood sampling. Blood was col-lected under sterile conditions in heparinized vacutainer tubes(Becton Dickinson) containing lithium heparin as anticoagulant.

Phosphogypsum Transport Water

Phosphogypsum transport water was collected from the wasteponds near the factory, INA Petrokemija, Kutina, Croatia. For theanalysis of V, Cr, Mn, Fe, Ni, Cu, Zn and Pb, 100 ml of phosphoricgypsum transport water was adjusted to pH 3 and 11, respectively,by addition of concentrated HNO3/NH4OH and preconcentratedby 2 ml of freshly prepared 1% w/v solution of ammonium-pyroloidinedithiocarbamate (APDC) (Orescanin et al., 2004). Afterthe complexation, which lasted 20 min, the suspension was fil-

tered through a Millipore filter (0.45 μm) and irradiated for 10 000 s.Analysis was carried out in triplicate. In order to obtain a linearrange for the analysis of zinc and iron, the original sample ofwastewater was diluted 20 times and then prepared the analysisas described above. All other elements were determined fromoriginal sample.

Elemental concentrations were determined by EDXRF method(Orescanin et al., 2004). Samples were irradiated by X-rays gener-ated from a 109Cd annular source. The incident angle was 49.76°.Detection of characteristic X-ray radiation from the sample wasconducted with a Si (Li) detector (Canberra) cooled with liquidnitrogen with the following characteristics: detector size = 30 mm2,Si thickness = 3 mm, Be window = 25 μm, FWHM for 5.9 keV, 55Fe,165 eV. The emerging angle was 74.05°, and the distance was1.5 cm. Spectra were collected using Genie–2000 software (Canberra,Meriden, CT, USA). Spectral data were analyzed using WinAxilsoftware (Canberra, Meriden, CT, USA). Calibration file was createdon the basis of the measurements of the standard solutions (Merck)prepared and analyzed in the same way as unknown samples.Elemental concentrations were calculated with the ‘comparedmethod’ from the WinFund package (Canberra, Meriden, CT, USA).Calibration was checked on the basis of measurement of knownconcentrations of the standard solutions (Merck) prepared andanalyzed in the same way as unknown samples. Multielementaltargets were prepared for the concentrations of 0.025; 0.050,0.100, 0.150, 0.200 and 0.300 mg l−1 of the elements V, Cr, Mn, Fe,Ni, Cu, Zn and Pb.

Ion selective electrode (Orion 720A+, 96-09BN) was used forfluoride determination. The atomic absorption spectrometerPerkin Elmer 3110 was used for the analysis of the elements Na,K and Ca. Activities of radionuclides in phosphoric gypsum trans-port water were analyzed by gamma spectrometer equippedwith a BE3830 model High Purity Ge detector (Canberra Indus-tries Inc., USA) with 30% relative efficiency and 2.1 keV resolutionat 1.332 MeV 60Co line. The detector was placed inside a low-background lead shield (Canberra Industries Inc., USA). Thedetector characteristics were as follows: active diameter, 70 mm;active area, 3800 mm2; thickness, 30 mm; outside distance fromthe window, 5 mm; window thickness, 0.5 mm; window material,carbon epoxy. Spectra were recorded during 80.000 secondsand analyzed using Genie 2K Canberra software. All pH mea-surements were made with a Mettler Toledo digital pH meter.

Sample Preparation for Genotoxic Investigations

The sample of phosphogypsum wastewater had a pH of below 2,so prior to genotoxicity testing it was sterilized through a Miliporefilter (0.22 μM) and corrected to pH 7 by adding 10 M NaOH.

Chemicals and Reagents

The S9 fraction was prepared from the liver of Sparague–Dawleymale rats, using Arochlor 1254 as the inducer (Ames et al., 1975)and used for the metabolic activation of test compounds; theS9 mix was prepared as described by Maron and Ames (1983).NADPH, glucose-6-phosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), malondyaldehyde (MDA),NADH and thiobarbituric acid (TBA), were purchased fromSigma Chemicals (Germany). All other chemical used were ofanalytical grade. Chemicals and reagents used in the alkalinecomet assay were purchased from Sigma Chemical Co. (St Louis,MO, USA), if not otherwise specified.

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Mutagenicity Assay — Salmonella typhimurium Strains

Mutagenicity assay was performed as follows: 100 μL of Salmonellatyphimurim strain (TA98 or TA100) was incubated with differentvolumes of wastewater (50–100 μL; the pH value was correctedto 7) and 500 μL of PBS (phosphate buffer), GSH (glutathione,5 mM) and S9 mix (microsome fraction), respectively, for 20 minat 37 °C. The modified standard plate incorporation procedure–preincubation assay was used. Following the incubation of bac-terial strains with tested water samples, the top agar (V = 2 ml;kept in a 45 °C water bath) was added, mixed and poured intominimal Vogel–Bonner plates. Plates were incubated for 72 h at37 °C. Induction of mutation was obtained after division of thenumber of induced revertants with the number of spontaneousrevertants. Methyl methane sulfonate (MMS) served as positivecontrol without metabolic activator and 2-aminoanthracene(2-AA) with S9 activation. Each experiment was repeated thriceand two plates were taken for each concentration/volume of thetest sample (Ames et al., 1975; Maron and Ames, 1983).

Cytotoxicity Assay

Cytotoxicity of fosfogypsum was determined by MTT assay, asdescribed by Mickisch et al. (1990). HEp2 and HeLa cells wereseeded in wells (5.4 × 103 per well) of 96-well plates and treatedwith different dilutions of wastewater for 72 h. The final concen-trations of wastewater in growth medium corresponded to 2.5,5, 7.5 and 10%, respectively. The intensity of absorbance wasmeasured at 540 nm in a multititer reader spectrophotometer(Cecil Instruments Ltd, Technical Centre Cambridge, UK). Eachconcentration was tested in quadruplicate, and each experimentwas repeated three times.

Measurement of Lipid Peroxidation

Lipid peroxidation was measured in cells during the exponentialphase of growth. Cells were seeded in 10 cm Petri dishes, treatedwith different wastewater dilutions and incubated for 24, 48 and72 h. As a positive control, hydrogen peroxide (80 μM) was used.Thereafter, the cells were scraped, washed twice with PBS, lysedon ice in 220 μL of potassium chloride (1.15%) and 400 μL of trichlo-roacetic acid (10%) for 10 min, and centrifuged at 5000 rpm. A600 μL aliquot of supernatant was incubated with 600 μL of TBA(thiobarbituric acid) (0.8%) for 15 min at 100 °C, cooled at roomtemperature, and centrifuged at 1000 rpm/10 min. After coolingin tap water, the absorbance of the samples was determined atboth 532 and 600 nm (the latter concerns non-specific absorbance).The concentration of MDA–TBA (malondyalehyde–thiobarbituricacid) complex, serving as an indicator of lipid peroxidation, wascalculated from standard curves (Cereser et al., 2001; McCarthyet al., 2004). The results are expressed as concentrations of TBA–MDA complex (nμ/106 cells). Each experiment was repeated threetimes.

Alkaline Comet Assay

The comet assay was carried out under alkaline conditions, prin-cipally as described by Singh et al. (1988), with some modifica-tions. Two replicate slides per sample per blood donor wereprepared. Agarose gels were prepared on fully frosted slidescoated with 1 and 0.6% normal melting point (NMP) agarose.Whole blood (5 μl) was mixed with 0.5% low melting point

(LMP) agarose, placed on slides and covered with a layer of 0.5%LMP agarose. Slides were immersed for 1 h in freshly preparedice-cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl,1% Na-sarcosinate, pH 10) with 1% Triton X-100 and 10% dime-thyl sulfoxide (Kemika). Alkaline denaturation and electrophore-sis were carried out at 4 °C under dim light in freshly preparedelectrophoretic buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13.0).After 20 min of denaturation, the slides were randomly placedside by side in the horizontal gel-electrophoresis tank, facing theanode. Electrophoresis at 25 V lasted another 20 min. After elec-trophoresis, the slides were gently washed with a neutralizationbuffer (0.4 M Tris–HCl, pH 7.5) three times at 5 min intervals. Slideswere stained with ethidium bromide (20 μg ml−1) and stored at4 °C in humidified sealed containers until analysis.

Each slide was examined using a 250× magnification fluores-cence microscope (Zeiss) equipped with an excitation filter of515–560 nm and a barrier filter of 590 nm. Comets were randomlycaptured at a constant depth of the gel, avoiding the edges ofthe gel, occasional dead cells and superimposed comets. Using ablack and white camera, the microscope image was transferredto a computer-based image analysis system (Comet Assay II, Per-ceptive Instruments Ltd). To avoid variability, one well-trainedscorer scored all comets. The comet parameters analyzed weretail length, tail DNA% and tail moment. A total of 100 cometsper blood donor (50 from each of two replicate slides) werescored and afterwards data obtained for two blood donors werepooled.

Experimental procedure. Using the whole blood, samples ofboth donors’ standard microgels for the alkaline comet assaywere prepared. Slides were immersed into test-solutions andtreated for 30 min at 37 °C. Following the treatment, slides werequickly rinsed with bidistilled water and then subjected to lysisand other steps of the alkaline comet assay.

The range of wastewater concentrations tested correspondedto 1, 5, respectively 20% solution of the original water, withoutpH adjustment. Also, original water and its 20% solution afteradjustment of pH to 7.00 were studied. In order to obtain test-solutions, the original wastewater was diluted with bidistilledwater. Bidistilled water was used as negative control. As thepositive control, hydrogen peroxide (1 mM) was used since it isable to produce an excess of single and induce double strandbreaks even at millimolar concentrations (Olive and Banáth,2006); the treatment of cells lasted for 10 min on ice.

Statistical Analyses

Statistical analyses were performed using the SPSS version 8.0(SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA)was employed to determine whether the means obtained withvarious groups significantly differed from each other. The signifi-cance was established using the Dunnett post-hoc test. Theprobability level of P < 0.05 was considered significant. All dataare expressed as means ± standard deviations (SD) of the valuesobtained by three independent measurements.

Statistical analyses considering the alkaline comet assaywere carried out with the commercial programme Statistica 5.0(StatSoft, Tulsa, USA). The extent of DNA damage, as recordedby the alkaline comet assay, was analyzed considering the mean(± standard error or standard deviation of the mean), median andrange of the comet parameters. In order to normalize distribu-tion and to equalize the variances, a logarithmic transformation

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of data was applied. The comparisons between samples weredone using ANOVA and subsequently the post-hoc Scheffé testwas used for the calculations concerning pair-wise comparisons.

Results

Phosphogypsum – Chemical Analysis

Compared with permissible limits (Table 1), phosphogypsumtransport water (PGTW) did not satisfy the regulations for dis-charge into a natural recipient due to the heavy metal content,high concentration of fluoride and low pH value (Regulation onlimit values of parameters of hazardous and other substances inwastewater, OG 40/99; 6/01; 14/01). Among determined ele-ments in the wastewater, fluorine acounted for more than 48%of the total content, while all determined heavy metals acountedfor only 1.23% of the total content (Table 2).

Mutagenicity Assay – Salmonella typhimurium Strains

PGTW did not cause point mutations in the presence or absenceof metabolic activator or GSH (Fig. 1). PGTW caused a slightdecrease in the number of TA98 revertants at the highest con-centration without metabolic activator and in the presence ofGSH, indicating its possible toxic effect. A certain decrease in thenumber of revertants was observed when S9 was added, butthis event was not statistically significant.

Morphological Changes of HEp2 and HeLa Cells after 24, 48 and 72 h Exposure to Phosphoric Gypsum

Microscopic analysis showed that PGTW caused severe morpho-logical changes on treated human cells in culture. Similar changes

were observed in both cell lines exposed to original wastewateradded to growth medium (Fig. 2).

Cytotoxicity Assay

PGTW significantly reduced the survival of cells during standardtime of incubation (72 h) expressed as mitochondrial dehydro-genases activity (Table 3). A 2.5% dilution was shown to be nottoxic for both cell lines.

Measurement of Lipid Peroxidation

Nontoxic concentration of PGTW (2.5%) induced strong oxidationof cellular lipids, which led to cell death at the concentrationat which it is present at the depot (Fig. 3). The basal level of TBA-MDA increased with time and was statistically significant com-pared with the control.

The Alkaline Comet Assay

Results of the alkaline comet assay are summarized in Table 4.Data corresponding to the tail length, tail intensity (DNA%) andtail moment are reported. Since the original PGTW tested with-out pH adjustment, even after 30 min exposure, caused total dis-integration of human peripheral blood leukocytes, we decidedto test its diluted solutions: 20, 5 and 1%. In addition, original

Table 1. Concentration of macro, micro and trace elementsand activity of radionuclides determined in phosphoric gyp-sum transport water (PGTW) and maximum allowed valuesfor wastewaters suitable for the discharge into the environ-ment (MAV)

Element WWBP ± SD MAVa

F (mg l−1) 1540 ± 3.7 5Pb (mg l−1) 0.720 ± 0.005 0.2V (mg l−1) 1.725 ± 0.090 0.05Cr (VI) (mg l−1) 0.360 ± 0.018 0.05Mn (mg l−1) 2.747 ± 0.143 2Fe (mg l−1) 25.820 ± 0.593 2Ni (mg l−1) 1.090 ± 0.052 1Cu (mg l−1) 0.580 ± 0.025 0.1Zn (mg l−1) 5.746 ± 0.018 1K (mg l−1) 71 ± 4 —Na (mg l−1) 1012 ± 51 —Ca (mg l−1) 503 ± 25 —K-40 (Bq l−1) 4.11 ± 0.28 —Pb-214 (Bq l−1) 7.43 ± 0.56 —Bi-214 (Bq l−1) 8.01 ± 0.57 —pH 1.79 6.5–8aRegulation on limit values of parameters of hazardous andother substances in wastewater, OG 40/99; 6/01; 14/01.

Table 2. Percentage of each element in the tested sample

Element %

F 48.66Pb 0.02V 0.05Cr (VI) 0.01Mn 0.09Fe 0.82Ni 0.03Cu 0.02Zn 0.18K 2.24Na 31.98Ca 15.89

Table 3. Cytotoxic effect of different PGTW dilutions onHEp2 and HeLa cells after 72 h of incubation (MTT assay).Results are shown as mean values obtained with three ex-periments (±SD)

Concentration of PGTW

HEp2 cell line HeLa cell line

Cell survival(%)

SD Cell survival(%)

SD

PGTW 2.5% 79.1 12 71.3 13PGTW 5% 2.21 2 4.96 3PGTW 7.5% 0.18 0.2 0.133 0.11PGTW 10% 0 0 0 0

PGTW, phosphogypsum transport wastewater.

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Figure 1. Number of induced revertants after 20 min of incubation of Salmonella typhimurium strains TA98 (a) and TA100 (b) with different PGTWvolumes (50–100 μl) in comparison to negative control (nc) and in the presence of metabolic activator, buffer and glutathione respectively. The con-centration of glutathione chosen represents the highest concentration of this compound that can be found in the mammalian cells. Results are shownas mean values obtained with three experiments (±SD).

Figure 2. Morphological changes of HEp2 and HeLa cells after 24, 48 and 72 h of exposure to PGTW without dilution. Magnification: 600× .

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wastewater and its 20% solution were also tested for toxicityafter adjustment of pH to 7.00.

The values of comet parameters measured in the negativecontrol indicate the low level of spontaneous damage in lym-phocyte DNA. As expected, the hydrogen peroxide, applied as

positive control, induced a significant response in the assay. Apositive dose-dependent relationship was observed for all cometparameters evaluated in samples treated with solutions of waste-water in the dose range 1–20%. In the samples treated with 1, 5 and20%, respectively, without pH adjustment, significantly increased

Table 4. Results of the alkaline comet assay on human white blood cells exposed in vitro for 30 minutes to PGTW samples.Negative control and positive control (1 mM hydrogen peroxide) were also included.

Sample Alkaline comet assay

Tail length (μm) Tail intensity (DNA %)

Mean ± SE Min Max Mean ± SE Min Max Mean ± SE Min Max

1% solution of the wastewater 14.80 ± 0.17 10.90 25.00 0.43 ± 0.06 0.00 6.01 0.06 ± 0.01 0.00 0.895% solution of the wastewater 25.04 ± 1.05a 11.54 98.07 3.49 ± 0.47b,c,d 0.00 31.72 0.80 ± 0.12a 0.00 8.8420% solution of the wastewater 32.14 ± 0.82a 13.46 70.51 7.26 ± 0.61a 0.00 47.16 1.67 ± 0.17a 0.00 16.3220% solution of the wastewater,pH adjusted to 7.00

14.09 ± 0.14 10.26 26.92 2.27 ± 0.33c,d 0.00 27.12 0.28 ± 0.04c 0.00 3.30

Wastewater, pH adjusted to 7.00 15.08 ± 0.20 10.90 28.85 2.26 ± 0.33b,c,d 0.00 29.27 0.30 ± 0.05c 0.00 4.13Positive control 16.70 ± 0.23a 11.54 41.02 1.09 ± 0.23 0.00 27.28 0.16 ± 0.03 0.00 3.85Negative control 14.66 ± 0.10 11.54 17.95 0.11 ± 0.02 0.00 1.58 0.02 ± 0.002 0.00 0.24

Note. Primary DNA damage was evaluated in 200 comets per sample and statistical evaluation (ANOVA with post-hoc Schéffe test)was done on logarithmically transformed data. Statistically significant differences are: aas compared with all other samples; bascompared with positive control; cas compared with negative control; das compared with sample treated with

Figure 3. Primary cell injury — lipid peroxidation; MDA–TBA (malondyaldehyde–thiobarbituric acid complex) concentration in HEp2 and HeLa cellline after (a) 24, (b) 48 and (c) 72 h of incubation with 2.5% solution of PGTW, which was shown to be non-toxic for both cell lines. Pooled dataobtained with three experiments (the mean at the point ±SD). *Statistically significant difference, obtained with HEp2 and HeLa cells as compared withthe control (cells treated with distilled water).

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values for all comet parameters were observed. Following pHadjustment, we observed a significant reduction of DNA damag-ing potential of wastewater samples. The values of comet taillength were lower compared with those measured in samplestreated with diluted original wastewater. However, mean tailintensity and mean tail moment values in those samples were stillincreased compared with both negative and positive controls.Detailed explanations and the statistical significance of theresults obtained are reported in Table 4.

DiscussionThe results of the present study obtained by chemical analysisand genotoxicity assays confirmed that most of the componentsfound in the wastewater from the phosphoric gypsum depotnear the factory for fertilizing agents, ‘INA Petrokemija’ (Kutina,Croatia), exceeded permissible limits. The greatest enrichmentcompared with the maximum allowed values was found forfluorine (308 times), followed by vanadium (35 times), iron (13times) chromium (VI) (seven times), cooper and zinc (six times).Therefore, pH adjustment as well as fluorine and heavy metalsremoval prior to discharge and/or reuse is strongly recommended.An overall comparison of our data with the values as proposedby the Council Directive 98/83/EC on the quality of water indentedfor human consumption indicates that in the water samples ana-lyzed in our study there were significantly increased amountsof fluoride, manganese, chromium, iron, nickel and sodium. Con-sidering the maximum allowable value (MAV) values as proposedby the Croatian legislative, the higher amounts of fluorides,manganese, iron and nickel are tolerated, whereas the amountsof lead and copper should be lower than in the EU legislation.The MAV of chromium are the same in both legislatives.

Considering that Croatia is an candidate for the membershipin the EU, it is reasonable to assume that in our country the EUlegislation in the field of water quality will be adopted as well. Asis known, the Water Framework Directive (Directive 2000/60/EC)is a legislative instrument for the protection and improvementof the European Unions water resources. The WFD was imple-mented in December 2000 – charging Member States with itsinclusion in national law by the end of 2003 – with the target ofachieving ‘good’ status for all European waters by 2015.

Although solid phosphoric gypsum is usually characterized bygreatly enhanced activity of naturally occurring radionuclides,especially radium 226Ra (Santos et al., 2006; Othman and Al-Misri,2007; Mazilli et al., 2000), their concentrations in the transportwater were extremely low (Table 1). This could be explained bythe chemical similarity of radium to calcium; it replaces calciumin the crystalline structure of insoluble phosphogypsum andfluorapatite minerals so its release into the transport waterfrom this minerals is negligible. To date, bioassays have typicallybeen used as a research tool rather than a regulatory device.They have proven useful for testing wastewater treatment planteffluents, leachate and hazardous wastes, groundwater andoil drilling waste fluids (Bulich, 1984). In toxicity tests it iscommon to use aquatic organisms, such as invertebrates, algae,luminiscent bacteria, fish or duckweed, because of their manyadvantages (World Health Organization, 1985).

In order to determine mutations, bacteria are the most com-monly used organisms (World Health Organization, 1985). Theyare capable of detecting forward and reverse mutations andDNA repair. To increase sensitivity, more than one strain is ana-lyzed per test. In this study, two strains of Salmonella typhimu-

rium were used: strain TA 98 for point mutation detection and TA100 for detection of base substitution mutations. These strainswere used for determination of the cytotoxic and mutageniceffects of examined water in the presence/absence of the meta-bolic activator as well as in the presence of GSH (the concentra-tion of GSH was chosen at a biomimetic level). Schneider et al. (1999)showed that some contaminants may act as ultimate genotoxi-cants after conjugation with GSH. Since the phosphogypsum is acomplex mixture, formation of reactive GSH conjugates, whichform adducts with nucleobases or DNA, can occur. As phospo-gypsum transport water did not induce histidine revertants, itcould be concluded that it does not cause point mutations withor without metabolic activator. The presence of GSH did notcause induction of histidine revertants, so it can be concludedthat GSH plays no role in mutagenicity of the phosphogypsumwastewater. Nevertheless, it can be noted that the highestconcentration of PGTW tested caused a statistically significantdecrease in the number of the histidine revertants in the strainTA98 when the metabolic activator was not added (Fig. 1). Theslight decrease in the number of revertants was noticed whenmetabolic activator was added, but this event was not statisti-cally significant. The evaluation of characteristics of the finalpopulation on the agar plates after 48 h incubation showed aslight thinning of the background lawn on the plates withoutmetabolic activator. Accompanied by a decrease in the numberof revertant colonies, it can be concluded that PGTW at thehighest concentrations has a toxic effect on strain TA 98. In thereview by Ohe et al. (2004), it can be seen that results obtainedin the Ames assay with different water samples differ signifi-cantly, from non-mutagenic effect to positive results. Lah et al.(2008) investigated water soil leachates and no positive resultswere detected. They concluded that the Ames test does notappear to be sensitive enough for water soil leachate genotoxic-ity evaluations where heavy metal contamination is anticipated.There are several reports on lower sensitivity of this point muta-tion test and disability in detection of genotoxic effects causedby heavy metals (Knasmüller et al., 1998, Monarca et al., 2002,Radetski et al., 2004). Knasmüller et al. (1998) reported thatthe metal amounts in the aqueous leachates were 5- to 10-foldlower than concentrations required to cause detectable effectson Salmonella DNA. Mathur et al. (2007) found that themutagenic activity of chemical sludge was less than that of bio-logical sludge. In the present study, phosphogypsum wastewa-ter represents chemical wastewater so it is also a possible reasonfor inability to determine its possible mutagenic effect using theAmes assay.

Although bacterial strains have many advantages when usedas test system, cellular mechanisms and mechanisms of defensediffer significantly among prokaryotic and eukaryotic cells, makingbacterial cells more resistant to the toxic effect of many compounds,especially heavy metals, which in many cases stimulate cellulardifferentiation and growth. Different cellular defense mechanisms,response to oxidative damage and high tolerance toward muta-tions in prokaryotic cells could be possible reasons for the weakerresponse to the genotoxic effect of PGTW observed in this study.Therefore, to further clarify molecular mechanisms of phospho-gypsum (geno) toxicity, we decided to use epithelial humancells in the culture and human lymphocytes.

Results regarding the cytotoxicity of phosphogypsum trans-port water indicate its strong cytotoxic effect, which increasedwith the time of exposure. Morphological changes like cellularshrinkage, detachment from surface and granulation point out

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that irreversible cellular events occurred after 24, 48 and 72 h ofexposure. Since the pH value in experiments with the cell cultureswas adjusted, we assume that high acidity was not a reason forthe toxicity. There is reason for concern if this wastewater leaksout of the depot, as diffuse pollution could endanger plants andanimals. Such pollution could diminish the biodiversity of aquaticecosystems, contaminate food chain and consequently jeopar-dize human health (Horvat et al., 2007; Khan et al., 2000).

Metals like iron, copper, chromium, vanadium and nickel (whichwere measured in excessive quantities in phopspogypsum trans-port water) generally cause lipid and protein peroxidation, DNAdamage and alteration of gene expression in human cells. Phos-phogypsum was examined for its ability to induce lipid peroxida-tion, as a measure of the prooxidative nature of metals presentin this mixture. It is worth mentioning that lipid peroxidation alsoincreases in control cells with the incubation time, but there isstill significant difference between the LPO level in treated cellsand control ones.

The alkaline comet assay was employed earlier in biomonitoringstudies aiming to determine the potential genotoxicity of waterpollutants (Kosz-Vnenchak and Rokosz, 1997; Reifferscheid andGrummt, 2000; Avishai et al., 2002; Matsumoto et al., 2003; Lemoset al., 2005). The present study used the alkaline comet assay toexplore the levels of primary DNA damage in wastewater-treatedhuman leukocytes. The positive results obtained indicate an increasein DNA damage in a dose-dependent manner. Since the alkalinecomet assay specifically detects single strand breaks and alkali-labile sites in DNA (Valko et al., 2006; Wang et al., 1997), it is likelythat following the treatment this type of lesion was inducedeither directly or indirectly. Our results are in accordance withthe reports of previous alkaline comet assay studies, as well asother studies that investigated the toxicity of wastewater con-taminants. Genotoxic potential of fluorides was reported earlierby Kleinsasser et al. (2001) and Ge et al. (2005). Ivancsits et al.(2002) reported that exposure of human fibroblasts to vanadateeffectively causes DNA strand breaks, and co-exposure of cells toother genotoxic agents may result in persistent DNA damage. Inthe study of Blasiak and Kowalik (2000) the effects of tri- andhexavalent chromium on the DNA damage and repair in humanlymphocytes were compared, using the alkaline comet assay(Stearns et al., 1995). Cr(III) caused greater DNA migration thanCr(VI). Their results suggest that reactive oxygen species andhydrogen peroxide may be involved in the formation of DNAlesions by hexavalent chromium. Saleha Banu et al. (2001) stud-ied DNA damage in leukocytes of mice treated with zinc sulfate.The results indicated a significant DNA damage at all the dosestested when compared with controls showing a clear dose-dependent response. Guecheva et al. (2001), using the alkalinecomet assay, evaluated the genotoxic potential of copper sulfatein freshwater planarians and reported inhibition of DNA repairby copper ions (Saleha Banu et al., 2004). Wozniak and Blasiak(2002), using the comet assay, also showed that free radicalsmay be involved in the formation of strand breaks and/or alkalilabile sites as well as DNA–protein cross-links in human lympho-cytes treated with nickel chloride (Cavallo et al., 2003). Devi et al.(2003, 2004), using the alkaline comet assay, studied single-strandedDNA breakage induced by lead nitrate in mice and occupationallyexposed subjects. In both studies they observed a significantincrease of DNA damage, indicating that lead nitrate causesDNA damage effectively. Beside the genotoxic effects of toxicelements (especially metals), DNA damaging effects of radionu-clides (although their concentrations in the transport water were

extremely low) should not be neglected. The ability of radionu-clides to induce primary DNA damage in terms of single anddouble strand breaks is well documented in earlier in vitro andin vivo studies (Pedraza Lopez et al., 2000; Calini et al., 2002; Kopjarand Garaj-Vrhovac, 2005; Grzesiuk et al., 2006; Morita et al., 1992).Therefore, when discussing the overall levels of damage asrecorded by the alkaline comet assay, possible synergistic effectsof heavy metals and radionuclides should also be anticipated.

We presumed that the low pH of wastewater tested in thiscomet assay study also significantly contributed to the levels ofprimary DNA damage in exposed human blood cells. Severalstudies have suggested that acidic pH may induce DNA damage.Acidic pH induced chromosome aberrations, sister chromatidexchanges and chromatid breaks in CHO cells (Morita, 1995). Previousstudies indicate that cells exposed to low pH had diminishedcapacity for DNA repair and elevated levels of induced mutagen-esis. Moreover, hyper-mutability due to the reduced DNA repaircapacity at low pH was also reported. Possibly the unfavorableenvironment may functionally inactivate or impair the activity ofmany proteins involved in DNA repair. It is also likely that low pHdisturbs protein conformation and folding and disrupts protein–protein interactions, further compromising the ability of cells toperform repair functions when challenged with DNA damage(Xiao et al., 2003). In addition, the results of another in vitro studyindicate that acidic pH acts like a topoisomerase II poison andinduces topoisomerase II-mediated DNA damage. As observedin the present study, adjustment of the pH to 7.00 significantlyreduced the levels of primary DNA damage caused in leukocytesby wastewater, confirming earlier observations on the deleteri-ous effects of acidity.

Since wastewaters represent complex mixtures composed ofvarious chemical substances, interpretation of comet results iscomplicated. Such a diverse array of constituents can have manydifferent mechanisms of toxicity, and several mechanisms mayexist for a single compound, each contributing to various degreesto the final deleterious effect. Also, there is no simple relationshipbetween the amount of DNA damage caused by specific chemi-cal and the biological impact of that damage. Nevertheless, theresults of the alkaline comet assay the confirmed toxicity of thewastewater collected at the phosphoric gypsum depot and indi-cated that it represents an environmental hazard.

The problem concerning phosphogypsum is a worldwide prob-lem; by the year 2000, it is estimated that nearly 2 billion tons ofphosphogypsum will be occupying storage facilities throughtUSA (Carbonell-Barrachina et al., 2002). The storage of phospho-gypsum has occupied vast areas of land that remain abandonedand subject to leaching and erosion by wind and water. The majorconstraint for phosphogypsum use in the environment is thepresence of several heavy metals. Furthermore, industrial wastedisposal can substantially contaminate ground and surface waterreservoirs. In the case of the contamination of surrounding areawith phosphogypsum wastewater, genotoxic effects of compoundspresent in wastewater could be expressed as well as their directimpact on human health through inhalation of contaminateddust, ingestion of plants that have absorbed the compoundsfrom the contaminated soil and the leaching of the compoundsfrom the soil to ground and surface water used for drinking (Saueiaand Mazzilli, 2006; Santos et al., 2006). The situation is getting worsewhen we know that this wastewater is a complex chemicalmixture which becomes toxic by additive, synergistic or antago-nistic interactions among the chemicals. The higher incidence ofcancer and other adverse health effects can also be attributed

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to the presence of heavy metals in the environment in generaland water in particular (Ohe et al., 2004). Previously it has beenshown that chemical mutagens usually do not affect differentgenetic end-points with the same degree of efficiency (Crebelliet al., 2005). Pavlica et al. (2000) showed that wastewater fromphosphogypsum depot did not cause changes in mitotic activityin common oak that was grown near the depot. In this study itwas shown that, by combining chemical and several differentbioassays, the wastewater is highly toxic and able to causesevere damage to DNA. The present study describes the resultsof genotoxicity testing using various in vitro endpoints on bacte-ria and human cells. Genotoxicity tests are well established asappropriate tools in the first phase of testing of the compoundwith unknown toxicity and their significance could not beneglected. The results obtained on bacteria and cell lines arecrucial for the planning of further studies using the organismswith a higher level of organization. The extrapolation of the resultsobtained in vitro on the level in vivo is sometimes complex, asmany other parameters should be taken into account (metabolicprocesses, the differences in physiological processes among dif-ferent species, the differences, etc.). Nevertheless, if a compoundtested is confirmed as genotoxic in vitro, there is a possibilitythat the same would apply also in vivo. Also, it is well establishedthat there are also some compounds which are not genotoxicin vitro on bacteria, but cause severe toxic effects in vivo due tospecific mechanisms underlying their toxicity. Ecological riskassessment should take into account different levels of organiza-tion since in the environment there is an continuous interplaybetween the microorganisms, plants, animals and human beings.Each of them are affected by a certain pollution, but it is mani-fested in different way. The effects of pollution as described herewith phosphogypsum wastewater might obviously differentiallyaffect the organisms of diverse cellular organization. The resultsof our study indicate a high geno/cytotoxicity of the phospho-gypsum wastewater. Based on the results obtained, we assumethat the first disturbances will occur in the plankton community,then in the aquatic invertebrate and fish communities. Both pro-cesses would lead to the disturbance of food chains, affectingdirectly or indirectly the ecosystem itself. Obviously, to exactlydefine the risk of pollution from phosphogypsum wastewater,further studies on higher organisms are needed. Moreover, besideslaboratory studies, in situ studies using organisms collected atthe polluted area also should be performed. These will be performedin the future.

ConclusionsPhosphoric gypsum is highly toxic for human cells. It does notcause point mutations, but does induce oxidative damage andsevere lipid peroxidation, causing cell death in concentrations inwhich it is present at the depot. PGTW did not satisfy the regula-tions for discharge into natural recipient due to exceeded heavymetal content, low pH value and its biological effect. Based onthe results obtained, pH adjustment as well as heavy metalsremoval prior to discharge and/or reuse of PGTW is stronglyrecommended.

Acknowledgements

This study was supported by the Ministry of Science, Educationand Sports of the Republic of Croatia (grant nos 058-0582261-2246 and 022-0222148-2137).

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Queries From Publisher

Title of Journal: Journal of Applied Toxicology

Paper Ref. No.: jat_1381.fm

During the copyediting of your article the following queries have arisen. Please mark your correctionsand answers to these queries directly on to the proof at the relevant place – do not mark your correc-tions on this query sheet.

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No. Query Remarks

1 Olive and Banáth, 2006 not in Reference list?

2 ‘sample treated with’ — text missing?

3 define LPO?

4 Wozniak and Blasiak not in Reference list?

5 Saueia and Mazzilli, 2006 not in Reference list?

6 Crebelli et al., 2005 not in Reference list?

7 Rajaguru not mentioned in text?

8 Singh 2000 not mentioned in text?