This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Mutation Research 747 (2012) 14– 21

Contents lists available at SciVerse ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

j o ur nal homep ag e: www.elsev ier .com/ locate /gentoxCo mm uni t y add re ss : www.elsev ier .com/ locate /mutres

In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNAstrand-breakage and apoptosis in Oreochromis niloticus niloticus (Linnaeus, 1758)and Clarias gariepinus (Burchell, 1822)

Alaa G.M. Osmana,b,∗, Khaled Y. Abuel-Fadlc, Werner Kloasb,d

a Department of Zoology, Faculty of Science, Al-Azhar University (Assiut Branch), 71524 Assiut, Egyptb Department of Eco-physiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, 12587 Berlin, Germanyc The Egyptian Environmental Affairs Agency (EEAA), Assiut, Egyptd Department of Endocrinology, Humboldt University, Berlin, Germany

a r t i c l e i n f o

Article history:Received 1 June 2011Received in revised form17 November 2011Accepted 25 February 2012Available online 13 April 2012

Keywords:GenotoxicityComet assayDiffusion assayDNA damageApoptosisRiver NileNile tilapiaAfrican catfish

a b s t r a c t

This work is part of a wider eco-toxicological study proposed to evaluate the biological impact of con-taminants along the whole course of the river Nile, Egypt. Here we present data on the presence of DNAstrand-breaks and apoptotic cells assessed by use of comet and diffusion assays in erythrocytes of Niletilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus). The results showed highdegrees of DNA damage and increased frequencies of apoptotic nuclei in blood of fish collected fromdownstream compared with those sampled from upstream river Nile. Qualitative analysis revealed ashift in the frequency of DNA-damage classes towards higher damage levels correlating with the increas-ing pollution gradient. The degree of DNA damage measured by use of comet assay and diffusion assayexhibited seasonal variations. Both fish species showed significant increases in DNA damage during thesummer. The results of our study indicated that the alkaline comet assay seems to be a useful techniquefor in situ genotoxic monitoring. At the same time the diffusion assay is sensitive enough to detect lowfrequencies of apoptotic nuclei. The results reveal species-specific differences in sensitivities, suggestingthat Nile tilapia may serve as a more sensitive test species compared with the African catfish. Based onthe outcome of the comet and diffusion assays, it can be concluded that the water quality of the river Nilewith respect to the presence of genotoxic compounds needs to be improved, especially in its estuaries. Asfar as we know this is the first time that the comet and diffusion assays are used for genotoxic monitoringof the river Nile.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The river Nile is the principal fresh-water resource for Egypt,meeting nearly all demands for drinking-water, irrigation, andindustry [1,2]. For this reason, continuous monitoring for qualityparameters is necessary. Despite the existence of relevant legis-lation, the pollution of the river Nile continues consequence ofincreasing agricultural, industrial and domestic effluents. Whilethe quality of most of the Nile’s water is within acceptable lev-els, there are several hot-spots of pollution, mostly found at certainsites along its course. Compounds present in polluted water arecapable of causing biological alterations that can affect particu-lar populations and entire ecosystems [3]. Some pollutants are

∗ Corresponding author at: Department of Eco-physiology and Aquaculture,Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 301,12587 Berlin, Germany. Tel.: +49 3064181629; fax: +49 3064181682.

E-mail address: osman@igb-berlin.de (A.G.M. Osman).

highly persistent and have mutagenic and/or clastogenic prop-erties. Because of the continued production and release of thesepollutants into the aquatic environment, the investigation of thegenotoxic potential of inland and coastal waters has become amajor task in the monitoring of environmental pollution [4]. Theinteraction of genotoxic contaminants with DNA causes variousgenetic disturbances, which are often irreversible and can be trans-mitted to the next generations [5–7]. The loss of DNA integrity maydetermine the induction of mutations, chromosomal aberrations,birth defects and cancer in vertebrates [8,9]. Therefore, there isa great interest in assessing the impact of genotoxic compoundsreleased into the aquatic environment.

The assessment of genotoxic potential in surface water is oneof the main tasks of environmental monitoring to control pollu-tion [3]. The analysis of environmental genotoxicity provides earlywarning signals of adverse long-term effects of the contamination[7]. DNA damage, such as strand breaks, has been proposed as asensitive indicator of genotoxicity and an effective biomarker inenvironmental bio-monitoring studies [9,10]. The comet assay is

1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.mrgentox.2012.02.013

Author's personal copy

A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21 15

currently the most widely employed method in eco-toxicology todetect DNA lesions. The alkaline comet assay is capable of detect-ing a wide variety of DNA damage, such as DNA single-strandbreaks, double-strand breaks, oxidatively induced base damages,alkali-labile sites, and sites undergoing DNA repair [11–13]. Thepopularity of this test is a result of its sensitivity, relatively lowcosts, simplicity and time efficiency due to automatic scoring of thecomets by use of image-analysis software [14–16]. It also has beenemployed to visualize DNA degradation due to apoptosis [17–19].If the damage produced reaches a high level, it can finally lead tocell apoptosis [20]. As the frequency of apoptosis increases, the per-centage of DNA in the tail comet increases until it disappears fromthe gel [13,21]. The DNA-diffusion assay, a modified version of thecomet assay, allows the detection of apoptosis in single cells [19].The diffusion assay described here is a simple, sensitive, and rapidmethod for estimating apoptosis in single cells.

In eco-toxicological studies it is essential to assess the toxicresponse of indigenous fauna as indicator of environmental pollu-tion [3,22]. Fish are often used as bio-indicators because they play aseveral roles in the trophic web, bio-accumulate toxic substances,and respond to low concentrations of contaminants [22–24]. Esti-mation of DNA damage in fish can be carried out with a varietyof tissue samples. Erythrocytes are the first choice because fishblood ensures great homogeneity of cells for comet studies [25,26].Nile tilapia and African catfish are representative species of riverNile with high economic value. Both species can move along theentire water column, being a versatile indicator species. The eco-logical significance of the selected species could confirm their valueas indicator species, and the wide zoogeographic distribution ofNile tilapia and African catfish could enable the comparison amongdifferent rivers by means of in situ monitoring.

In contrast to controlled laboratory studies, assessment of geno-toxicity in aquatic organisms in their natural environment is acomplicated task, mainly because of the relatively low levels ofgenotoxicants and the existence of multiple potentially genotoxicpollutants, often encountered as complex mixtures [27]. Althoughthe comet assay has been successfully applied on several fishspecies exposed to genotoxic agents in vivo and in vitro [11,28–30],there are only a few aquatic bio-monitoring studies that use thecomet assay in field-sampled fish [31,32]. To our knowledge, thegenotoxicity of water from the river Nile has never been studied.Our previous work [2] made a first attempt to assess genotoxic-ity of the river Nile. Therefore, as a follow-up, we here present aninvestigation of DNA strand-breakage and apoptosis in fish, andassess the feasibility of using the comet and diffusion assays asindicators of genotoxins under actual field conditions. Assessmentof DNA damage was performed in peripheral blood erythrocytes ofAfrican catfish and Nile tilapia collected from whole course of theriver Nile. The information generated here will contribute to theutilization of the selected species for genotoxic bio-monitoring inpolluted water.

2. Material and methods

2.1. Sampling sites

Six sites were selected along the whole course of the river Nile from its springat Aswan to its estuaries at Rosetta and Damietta (Fig. 1).

2.2. Water quality assessment

Water-quality criteria [electrical conductivity, pH, water temperature, chemicaloxygen demand (COD), total organic carbon (TOC), total solids (TS), ammonia (NH3),nitrate (NO3), orthophosphate (o-PO4), chloride (Cl), fluoride (F), sulfate (SO4), phe-nolics (Phenol)] of the chosen sites were monitored bimonthly during the periodfrom July 2009 to June 2010. Total Pb, Cu, Cr, Hg, and Cd were measured usinggraphite furnace AA (GFAA) spectroscopy. Sampling and assessment of water qual-ity were done according to the traditional manual methods [33]. Data on the selectedsites are shown in Table 1.

Fig. 1. Map showing the sampling sites along the whole course of the river Nile,from its spring at Aswan to its estuary at Damietta and Rosetta branches.

2.3. Fish sampling

Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepi-nus) were caught bimonthly by gill net from the selected sites during the periodfrom July 2009 to June 2010 (72 specimens from each species; body-weight rangingfrom 240 to 290 g for Nile tilapia and from 280 to 350 g for African catfish). Periph-eral blood was collected by cardiac puncture with heparinized syringes from eachfish as described by Osman et al. [2] for comet and diffusion assays. Blood sampleswere kept on ice and immediately processed for genotoxicity. Erythrocytes wereprocessed for the evaluation of DNA integrity and apoptosis by comet assay anddiffusion assay under yellow light.

2.4. Comet assay

The alkaline comet assay was performed according to the basic procedure ofSingh et al. [34] considering the modification of Osman et al. [30] because of theunique characteristics of the blood. Heparinised blood was immediately diluted50-fold in phosphate-buffered saline (PBS) [35]. Five �l of each diluted blood sam-ple was added to 95 �l of 0.5% (w/v) low-melting agarose and the mixture wasadded onto a frosted microscope slide pre-coated with 1% (w/v) of normal-meltingagarose and covered with a cover slip. The slide was incubated at 4 ◦C for 15 minto allow solidification and was subsequently coated with an additional layer of0.5% low-melting agarose. After solidification at 4 ◦C for 20 min, the embedded cellswere lysed in lysing buffer [2.5 M NaCl, 100 mM NaEDTA, 10 mM TRIS base, pH10, 1% Triton X-100, 10% DMSO] at 4 ◦C for 120 min. After a 30 min incubation inelectrophoresis buffer [300 mM NaOH, 1 mM EDTA, pH ≥ 13] electrophoresis wascarried out at 20 V and 300 mA for 30 min. Subsequently, neutralization was per-formed in three washing steps in 0.4 M Tris–HCl (pH 7.5). To visualize DNA strandbreaks, slides were stained with ethidium-bromide solution [20 �g/ml] for 10 min,and images were captured at 400× magnification by use of an Olympus fluores-cence microscope (VANOX, AHBT3, model BH2-RFCA; Olympus America, Melville,NY, USA) and a color video camera (Olympus DP 20). DNA strand-breakage wasquantified as the amount of fluorescence in the comet tail (% DNA in comet tail;%tail-DNA) with TriTek CometScoreTM (Freeware image-analysis software v1.5). Thepercentage of DNA in the comet tail–assessed as optical density at 515–560 nm –was calculated for each nucleus [36]. Considering 100 nuclei, four replicates persite were analyzed (n = 2400 for each fish species). Calculations are means perreplicate.

For qualitative evaluations, nuclei were categorized, according to the degree ofdamage (using % tail-DNA) based on the criteria of Anderson et al. [37] and Mitchel-more and Chipman [12], into five classes: undamaged nuclei (% tail-DNA ≤ 10%),

Author's personal copy

16 A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21

Table 1Physicochemical parameters of the water samples collected from different sites along the whole course of the river Nile, Egypt (n = 108 samples).

Parameter (unit) Sites

Aswan Kena Assiut Beny-Suef Damietta Rosetta Permissible limitMean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

pH (unit) 7.86 ± 0.25 8.01 ± 0.41 8.15 ± 0.19 8.27 ± 0.27 8.40 ± 0.44 8.23 ± 0.45 7–8.5Conductivity (M s/cm) 0.26 ± 0.03 0.27 ± 0.07 0.29 ± 0.09 0.34 ± 0.07 0.38 ± 0.10 0.58 ± 0.11 –Temperature (◦C) 22.68 ± 2.22 23.72 ± 3.15 23.33 ± 4.67 23.64 ± 4.24 25.29 ± 5.66 24.53 ± 4.44 Over 5 ◦CChemical oxygen demand (ppm) 10.58 ± 3.62 9.17 ± 3.04 10.63 ± 2.18 7.88 ± 2.15 8.59 ± 2.51 18.00 ± 10.38 10Total organic carbon (ppm) 5.65 ± 2.88 5.89 ± 1.93 5.74 ± 0.92 4.93 ± 2.58 5.21 ± 2.64 8.61 ± 6.06 –Total solid (ppm) 198.88 ± 14.09 212.67 ± 23.70 227.75 ± 16.29 259.50 ± 44.33 305.25 ± 55.96 411.25 ± 85.66 500Ammonia (ppm) 0.11 ± 0.16 0.01 ± 0.002 0.02 ± 0.01 0.01 ± 0.01 0.05 ± 0.05 0.15 ± 0.09 0.5Nitrate (ppm) 0.89 ± 0.40 0.77 ± 0.39 0.51 ± 0.21 0.72 ± 0.63 1.13 ± 1.13 2.05 ± 2.27 45Chlorides (ppm) 7.05 ± 1.44 8.56 ± 1.90 10.03 ± 2.58 15.28 ± 5.10 22.42 ± 4.93 40.55 ± 6.72 –Florid (ppm) 0.29 ± 0.15 0.32 ± 0.14 0.39 ± 0.16 0.31 ± 0.13 0.30 ± 0.10 0.37 ± 0.08 0.5Orthophosphate (ppm) 0.01 ± 0.02 0.037 ± 0.04 0.10 ± 0.090 0.03 ± 0.034 0.13 ± 0.07 0.21 ± 0.19 –Sulphate (ppm) 34.16 ± 13.30 45.30 ± 15.37 47.95 ± 14.11 45.25 ± 15.81 51.00 ± 12.76 68.13 ± 11.42 200Phenol (ppm) 0.01 ± 0.02 0.01 ± 0.02 0.01 ± 0.02 0.01 ± 0.01 0.03 ± 0.02 0.04 ± 0.02 0.02Pb (ppm) 0.01 ± 0.02 0.02 ± 0.01 0.02 ± 0.02 0.02 ± 0.01 0.03 ± 0.04 0.06 ± 0.08 0.05Cd (ppm) 0.004 ± 0.004 0.002 ± 0.002 0.01 ± 0.01 0.002 ± 0.002 0.02 ± 0.02 0.01 ± 0.02 0.01Cu (ppm) 0.03 ± 0.03 0.03 ± 0.02 0.03 ± 0.03 0.03 ± 0.03 0.03 ± 0.03 0.0544 ± 0.027 1Cr (ppm) 0.003 ± 0.002 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.05 ± 0.06 0.10 ± 0.15 0.05Hg (ppm) 0.0000 ± 0.001 0.0004 ± 0.001 0.0005 ± 0.001 0.0009 ± 0.001 0.002 ± 0.001 0.003 ± 0.001 0.001

low-damaged nuclei (10% < % tail-DNA ≤ 25%), median-damaged nuclei (25 < % tail-DNA ≤ 50%), highly damaged nuclei (50 < % tail-DNA ≤ 75%), and totally damagednuclei (% tail-DNA > 75%) (Fig. 2).

2.5. Diffusion assay

The detailed procedure for estimating apoptosis in single cells by use of the diffu-sion assay has been described elsewhere [19]. The assay involves mixing the diluted

Fig. 2. A photomicrograph of Nile tilapia, Oreochromis niloticus niloticus, (represen-tative species) erythrocyte nuclei showing grades of DNA damage (representativestages) assessed by comet assay (a–e) and diffusion assay (f–j). Magnification: 400×.

erythrocytes (50-fold in PBS) with 0.5% low-melting agarose and making a micro-gelon a frosted microscope slide pre-coated with 1% (w/v) of normal-melting agarose,then lysing the embedded cells in lysing buffer [as described above for the cometassay] at 4 ◦C for 120 min to allow diffusion of low molecular-weight DNA in agarose.After lysis, the slides were immersed for 20 min at room temperature in a freshly pre-pared alkaline solution (300 mM NaOH, 1 mM EDTA, pH ≥ 13). Final visualization ofDNA, not subjected to electrophoresis, was accomplished by addition of an ethidiumbromide solution [20 �g/ml] for 10 min. Images were captured at 400× magnifica-tion with an Olympus fluorescence microscope (VANOX, AHBT3, model BH2-RFCA;Olympus America, Melville, NY, USA) and a color video camera (Olympus DP 20).The diameters of the nuclei were measured in pixel with TriTek CometScoreTM (Free-ware image-analysis software v1.5). Considering 125 nuclei, four replicates per sitewere analyzed (n = 3000 for each fish species). Calculations are means per replicate.

For qualitative evaluations, nuclei were categorized according to the degree ofdamage (by use of nuclear diameter, ND) into five classes: undamaged (ND ≤ 50pixel), low-damaged (50 pixel < ND ≤ 60 pixel), median-damaged (60 pixel < ND ≤ 75pixel), highly damaged (75 pixel < ND ≤ 90 pixel) and apoptotic nuclei (ND > 90 pixel)(Fig. 2). Apoptotic cell nuclei, characterized by high dispersion of DNA (mean nucleusdiameter > 90 pixel) have a hazy or undefined outline without any clear boundarydue to nucleosomal-sized DNA diffusing into agarose [9] (Fig. 2j). The frequency ofapoptotic cells was determined by scoring apoptotic cells and non-apoptotic cells.

2.6. Statistical analysis

DNA damage in each group was expressed with mean ± SD for both cometand diffusion assays. The data obtained were subjected to the one-way analysis ofvariance (non-parametric ANOVA) test with the statistical package for the socialsciences [38]. Means were tested with the least-significant-difference (LSD) test tocompare the degree of DNA damage among different sites. Two levels of significancewere reported: *p < 0.05; **p < 0.01.

3. Results

3.1. Physico-chemical water parameters

Table 1 shows mean ± SD of the measured physico-chemicalparameters of the water samples collected from the selected sitesduring the period of monitoring. Most of these parameters showedthe highest values in the water of river Nile downstream. For nearlyall parameters significant differences (p < 0.05) were observedbetween the selected sites. Mean values of conductivity, chemi-cal oxygen demand (COD), total organic carbon (TOC), ammonia(NH3), nitrate (NO3), total solid (TS), sulphate (SO4), chloride (Cl),and orthophosphate were recorded to be higher in the water ofDamietta and Rosetta sites compared with other sampling sites.Also most of the detected heavy metals showed highest values atDamietta and Rosetta sites. The levels of these parameters weresignificantly (p < 0.05) increased from the spring of the river Nileat Aswan toward its estuaries at the Damietta and Rosetta sites.

Author's personal copy

A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21 17

All measured parameters were below the permissible limits in theriver Nile at its spring. In contrast, the levels of COD, phenol, Pb,Cd, Cr and Hg were above the permissible limits in the water ofdownstream river Nile (Rosetta and/or Damietta sites).

3.2. DNA strand-breakage

DNA damage in the erythrocytes of Nile tilapia and African cat-fish as measured with the comet assay and the diffusion assay areshown in Table 2. The highest degree of DNA damage (% tail-DNA;a parameter correlated to the degree of DNA damage measuredwith the comet assay) was recorded in blood of fish collected fromRosetta sites (30.8% and 31.0%) followed by Damietta site (28.7%and 27.7%) for Nile tilapia and African catfish, respectively. Thedegree of DNA damage was very low in the blood of fish col-lected from Aswan (10.2% and 8.6%) and Kena (10.2% and 9.8%)for both fish species, respectively. With the diffusion assay, thehighest level of DNA damage was detected in the blood of fishcollected from Rosetta sites (78.4 pixel and 75.7 pixel) for Niletilapia and African catfish, respectively and the lowest level wasrecorded in the blood of fish collected from river Nile upstream.For both assays the degree of DNA damage increased in fish ery-throcytes, from low levels in Aswan and Kena via intermediatelevels in Assiut and Beny-Suef to high levels in the mainly pol-luted sites Damietta and Rosetta. Compared with the Aswan site,nearly all other sites exhibited highly significant (p < 0.01) increasesin the amount of DNA damage detected by comet assay (Table 2). Inparallel, the data of the DNA-diffusion assay exhibited such a sig-nificant (p < 0.01) increase only in fish collected from downstreamsites (highly polluted sites) compared with the Aswan site, for bothfish species (Table 2). The levels of DNA damage detected by cometand diffusion assay in erythrocytes of Nile tilapia were higher (notsignificant, p > 0.05) than those detected in erythrocytes of Africancatfish collected from the same sites (excluding Beny-Suef) and atthe same sampling time (Table 2).

The distribution of DNA damage categories measured with thecomet assay in individual erythrocytes is shown in Fig. 3. For bothfish species the number of cells showing high levels of DNA damagewas greater in fish collected from downstream river Nile com-pared with other sites. Similarly, qualitative analysis revealed ashift in the frequency of DNA-damage classes towards higher lev-els, which correlated with the increasing pollution-gradient fromthe upstream to the downstream river Nile. DNA damage increasedtowards higher DNA-damage categories compared with samplescollected at Aswan in which the undamaged nuclei have the high-est frequency (74% and 62% for African catfish and Nile tilapia,respectively) during the whole period of investigation (Fig. 3).Accordingly, in the samples collected from Rosetta and Damietta17–20% of the nuclei were highly damaged and nearly 11.5% of thenuclei were totally damaged for both fish species (Fig. 3).

3.3. Apoptosis

The frequency distributions of the apoptotic nuclei detected bydiffusion assays are shown in Table 3. A higher frequency distri-bution was detected in blood of fish collected from Damietta andRosetta sites compared with other sites. The frequency of apop-totic nuclei was very low in blood of fish collected from Aswan.The percentage of apoptotic nuclei in erythrocytes of African cat-fish was higher than those in erythrocytes of Nile tilapia collectedfrom nearly all sites and at the same sampling time (Table 3).

3.4. Seasonal variation of DNA strand breaks

For both assays, higher frequencies of DNA strand-breaks wereobserved in blood of fish collected during the summer from the

Fig. 3. Frequency distribution (means ± SD, 400 nuclei corresponding to 100%)of DNA damage categories (undamaged, low-damaged, medium-damaged, highlydamaged, and totally damaged nuclei using % tail-DNA) detected by the comet assayin erythrocytes from Nile tilapia and African catfish, collected from different sitesalong the whole course of the river Nile, Egypt.

whole course of the river Nile (Tables 4 and 5). Significant differ-ences (p < 0.05) among seasons were recorded in the amount ofDNA damage detected by the comet and diffusion assays in bothfish species (Tables 4 and 5).

4. Discussion

The detailed investigation [39] of water-quality assessmentalong the whole course of the river Nile showed higher mean valuesof nearly all the detected physico-chemical parameters in watercollected from sampling sites downstream river Nile comparedwith those collected from upstream river Nile. This increase provesthe presence of large quantities of organic and inorganic pollu-tants in the waters around Rosetta and Damietta. This finding wasexpected due to the fact that the level of contamination is greaterdownstream river Nile, compared with upstream sampling sites,where more domestic and industrial effluents have been releasedinto the Nile without adequate treatment. The river Nile receivesduring its course wastewater discharges from 264 point-sources,of which 121 are agricultural drains, 70 are industrial outfalls, and73 are sewage discharges (unpublished data). Sources of pollutionalong upstream areas are mainly agro-industrial, including sugarcane industries and small private industries. In contrast, differenttypes of agricultural, domestic, and industrial wastes are disposedof in downstream areas. It was estimated that the aquatic envi-ronment of downstream areas receives more than three million

Author's personal copy

18 A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21

Table 2DNA strand-breakage measured with the comet assay (% tail-DNA) and the diffusion assay (ND) in erythrocytes of Nile tilapia, Oreochromis niloticus niloticus (n = 72), andAfrican catfish, Clarias gariepinus (n = 72), collected from along the whole course of the river Nile, Egypt.

Assays Fish species Sites

Aswan, n = 24 Kena, n = 24 Assuit, n = 24 Beny-Suef, n = 24 Damietta, n = 24 Rosetta, n = 24

Comet assay (% tail-DNA) Oreochromis niloticus niloticus 10.23a ± 5.15 10.26a ± 8.78 17.83a ± 11.64** 19.55a ± 10.20** 28.74a ± 8.99** 30.84a ± 9.93**

Clarias gariepinus 8.56 a ± 7.57 9.84a ± 10.19 15.95a ± 11.41** 23.97b ± 12.65** 27.69a ± 10.67** 30.97a ± 11.87**

Diffusion assay (ND) Oreochromis niloticus niloticus 62.86 a ± 5.65 63.66a ± 7.76 65.55a ± 5.36 66.54 ± 11.08* 75.06a ± 6.52** 78.39a ± 7.69**

Clarias gariepinus 59.66 a ± 7.44 60.97a ± 8.57 65.07a ± 8.64 67.25 ± 8.59* 75.05a ± 12.28** 75.67a ± 15.37**

Results are expressed as means ± SD.Values showing similar letters (a, b) are not significantly different within the same site and within the same test (comet or diffusion assay) for both fish species at p < 0.05level (vertical comparison).% tail-DNA = percentage of the damaged DNA in the comet tail.ND = Nuclear diameter in pixel.

* Significant compared to Aswan site at p < 0.05 level (by non-parametric Bonferroni).** Significant compared to Aswan site at p < 0.01 level (by non-parametric Bonferroni).

Table 3Percentage of frequency distribution of apoptotic nuclei in erythrocytes of Nile tilapia, Oreochromis niloticus niloticus (n = 72), and African catfish, Clarias gariepinus (n = 72),collected from along the whole course of the river Nile, Egypt, measured with diffusion assays.

Fish species Sites

Aswan, n = 24 Kena, n = 24 Assiut, n = 24 Beny-Suef, n = 24 Damietta, n = 24 Rosetta, n = 24

Oreochromis niloticus niloticus 4.10% 8.90% 5.90% 14.00% 16.10% 22.60%Clarias gariepinus 3.40% 9.00% 10.60% 15.80% 18.00% 24.40%

Table 4Seasonal variation of DNA strand-breakage measured with the comet assay (% tail-DNA) in erythrocytes of Nile tilapia, Oreochromis niloticus niloticus (n = 72), and Africancatfish, Clarias gariepinus (n = 72), collected from along the whole course of the river Nile, Egypt.

Seasons Sites

Aswan, n = 24 Kena, n = 24 Assuit, n = 24 Beny-Suef, n = 24 Damietta, n = 24 Rosetta, n = 24

Oreochromis niloticus niloticusWinter 5.22 ± 7.23a 6.11 ± 07.95a 14.31 ± 11.82* a 18.25 ± 05.85** a 27.05 ± 11.81** a 28.42 ± 07.23** a

Spring 11.48 ± 12.06b 9.75 ± 07.85b 16.95 ± 10.31a 15.33 ± 10.59b 28.38 ± 10.31** a 31.40 ± 12.06** a

Summer 12.10 ± 07.51b 16.17 ± 10.42c 21.31 ± 15.73* b 22.06 ± 11.38* c 30.99 ± 15.73** a 32.17 ± 07.51** a

Autumn 12.13 ± 12.81b 9.01 ± 06.46b 18.75 ± 08.05b 22.55 ± 11.68* c 28.51 ± 08.05** a 31.37 ± 12.81** a

Clarias gariepinusWinter 5.09 ± 03.42a 5.86 ± 1.99a 15.19 ± 10.39* a 16.13 ± 12.98* a 22.88 ± 13.46** a 32.02 ± 12.67** a

Spring 7.21 ± 05.89a 4.30 ± 2.39a 11.71 ± 10.49b 28.76 ± 8.83** b 27.62 ± 09.95** b 29.61 ± 15.35** b

Summer 14.47 ± 09.48b 17.78 ± 10.01b 22.11 ± 11.01c 32.19 ± 13.48** b 28.67 ± 09.58** b 34.13 ± 10.16** a

Autumn 7.47 ± 07.53a 11.39 ± 14.69c 14.78 ± 12.65a 18.82 ± 08.22* a 31.60 ± 08.80** b 28.13 ± 09.40** b

Results are expressed as mean ± SD.Values showing similar letters (a, b, c) are not significantly different within the same site for each fish species at p < 0.05 level (vertical comparison).%tail-DNA= percentage of the damaged DNA in the comet tail.

* Significant compared to Aswan site at p < 0.05 level (horizontal comparison).** Significant compared to Aswan site at p < 0.01 level (horizontal comparison).

Table 5Seasonal variation of DNA damage measured with the diffusion assay (ND) in erythrocytes of Nile tilapia, Oreochromis niloticus niloticus (n = 72), and African catfish, Clariasgariepinus (n = 72), collected from along the whole course of the river Nile.

Seasons Sites

Aswan, n = 24 Kena, n = 24 Assuit, n = 24 Beny-Suef, n = 24 Damietta, n = 24 Rosetta, n = 24

Oreochromis niloticus niloticusWinter 61.83 ± 06.59a 54.79 ± 05.26* 65.35 ± 05.36a 62.17 ± 06.51a 72.58 ± 07.01** a 74.22 ± 07.31** a

Spring 61.58 ± 05.91a 65.36 ± 04.83b 62.22 ± 03.92a 64.57 ± 09.57a 73.05 ± 05.87** a 77.31 ± 09.43** a

Summer 65.27 ± 05.80a 68.39 ± 0 6.41b 69.82 ± 05.74a 75.61 ± 15.05** b 79.17 ± 05.81** a 84.57 ± 05.59** b

Autumn 62.74 ± 04.09a 66.09 ± 06.87b 64.81 ± 03.73a 63.8 ± 06.82a 75.43 ± 06.06** a 77.47 ± 04.35** a

Clarias gariepinusWinter 53.9 ± 06.85a 52.23 ± 06.67a 62.37 ± 10.9* a 60.36 ± 05.76a 80.38 ± 19.71** a 64.22 ± 11.53* a

Spring 61.61 ± 08.16b 63.64 ± 09.72b 65.28 ± 07.01a 67.51 ± 07.58* b 75.02 ± 09.2** a 69.67 ± 05.28a

Summer 61.53 ± 05.27b 65.99 ± 04.69b 70.14 ± 08.69* b 70.61 ± 07.58* b 77.63 ± 07.53** a 89.73 ± 19.71** b

Autumn 61.59 ± 07.06b 61.99 ± 05.93b 62.49 ± 05.96a 70.54 ± 09.72* b 68.76 ± 06.32b 79.06 ± 07.43** c

Results are expressed as mean ± SD.Values showing similar letters (a, b, c) are not significantly different within the same site for each fish species at p < 0.05 level (vertical comparison).ND = Nuclear diameter in pixel.

* Significant compared with Aswan site at p < 0.05 level (horizontal comparison).** Significant compared with Aswan site at p < 0.01 level (horizontal comparison).

Author's personal copy

A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21 19

cubic meters daily of untreated or partially treated domestic andindustrial wastes, and, in addition, agricultural drainage water [40].

Fish are especially useful for assessing biological risk of haz-ardous and toxic wastes in water since they are occupying differenttrophic levels, are in direct contact with the contaminants in theirenvironment and are sensitive to anthropogenic compounds [32].This sensitivity may lead even to genetic damage, and possiblydeath [41,42]. In the present study, we used Nile tilapia and Africancatfish as test organisms because these fish have been shown tobe sufficiently sensitive to anthropogenic compounds in laboratorytests [30,43] and therefore suitable for bio-monitoring studies.

The alkaline comet assay was previously confirmed as an initialindicator of general, nonspecific DNA damage, providing an effec-tive biomarker for environmental monitoring [9,12,44]. Severalparameters including percentage of DNA in comet tail (% tail-DNA),tail length (TL), and tail moment (TM) were used in the past tomonitor DNA strand-breakage with the comet assay. We have pre-viously [36] evaluated the application of these end points to detectDNA strand-breakage in fish. According to these results, TL and TMdid not describe the genotoxic effect in a better way. Osman et al.[36] clearly indicated that % tail-DNA was the most appropriatecriterion to quantify DNA strand-breakage in fish. In line with thatconclusion, other authors also had pointed to the percentage of DNAin tail as the most valid endpoint [45]. Accordingly, we considered% tail-DNA in the current work, as a sensitive end point, to detectpotential genotoxicity in the river Nile.

The amount of DNA damage in blood of fish collected fromthe downstream river Nile was higher than that in blood of fishsampled upstream. The DNA damage was significantly elevatedin peripheral blood erythrocytes of Nile tilapia and African catfishderived from heavily polluted areas. In parallel, qualitative analysisrevealed a shift in the frequency of DNA-damage classes towardshigher levels (from Aswan to Damietta and Rosetta), which corre-lated with the increasing pollution gradient. Aswan and Kena werethe least genotoxic sites and are considered as being negligibly pol-luted [2]. DNA damage in erythrocytes of Nile tilapia (O. niloticusniloticus) and African catfish (C. gariepinus) was shown to be associ-ated with contamination levels in downstream areas. DNA damageis significantly affected by many external factors, particularly theexposure to pollutants [44]. In accordance with the current results,Devaux et al. [46] demonstrated that the levels of DNA damage werehigher in erythrocytes from chub (Leuciscus cephalus) sampled inpolluted sites on a French river compared with those from a refer-ence area. Similarly, greatest DNA damage was detected with thecomet assay in erythrocytes from bullhead fish collected from heav-ily polluted sites in the Great Lakes of Canada [47]. A higher levelof DNA damage was detected in eelpout erythrocytes sampled atcontaminated areas, in comparison with uncontaminated sites [9].Extensive DNA damage was observed in erythrocytes of fish (Cypri-nus carpio) exposed to polluted water samples [3,24]. The simpleprogressive increase in the extent of genotoxicity along a watercourse found for the river Nile is a good example that DNA damageis associated, in the wild, with pollution levels when a gradient ofpollution exists along the water course.

Based on our results, it was not possible to reveal a single chem-ical in the river Nile that causes the DNA damage observed in thepresent study. However, it has been recognized that synergisticeffects from a combination of chemicals may cause DNA damage[44]. This assumption is also supported by the results of physico-chemical analyses, showing increased concentrations of the mostdetected physicochemical parameters in the water of downstreamriver Nile (Rosetta and/or Damietta sites). COD, phenol, Pb, Cd, Crand Hg were above the permissible level in the latter areas. Theheavy metals residues in the tissues of African catfish [39] and Niletilapia (unpublished data) were investigated. The highest concen-trations of the selected heavy metals (Pb, Cd, Cr, Hg, and Cu) were

recorded in the tissues of fish collected from downstream riverNile (Rosetta and/or Damietta). Pb, Cd and Hg were considered tobe the most toxic metals causing genotoxicity in fish [36,48,49].The metabolism of these heavy metals generates reactive oxygenspecies that can attack cellular macromolecules such as DNA, lead-ing to serious damage [50,51]. Therefore, the high levels of DNAdamage observed in blood from Nile tilapia and African catfish maybe attributed to various pollutants (including heavy metals) derivedfrom agricultural, domestic, and industrial effluents released intoand accumulated in downstream river Nile.

The extent of DNA damage detected with the comet and diffu-sion assays exhibited seasonal variations. Both fish species showeda significant increase in DNA damage during summer. These resultsare in accordance with our previous work [2] where an increase inmicronucleus frequencies was found in blood of fish collected fromthe river Nile during summer. This is consistent with the results ofde Andrade et al. [29] who reported an increase in baseline DNAdamage associated with high temperature. A strong seasonal influ-ence on the level of DNA damage detected with the comet assayhas been previously observed by Buschini et al. [52], both in termsof baseline levels and also in sensitivity towards pollution. Weobserved that the concentration of metals at all sites reinforcesthe association between warm seasons and high levels of DNAdamage: total metal concentrations were highest in the summercompared with other seasons (unpublished data). Furthermore, itis known that during summer heavy elements are discharged morefrequently into the river Nile by agricultural and other anthro-pogenic activities. These results suggest that the river Nile is morecontaminated during the summer as a result of higher agriculturaland human activities.

In general, significant differences between all sites and alsobetween seasons were recorded with the comet assay. The resultsof our previous work [2] concluded that the frequencies of micronu-clei in the blood of Nile tilapia and African catfish were notsignificantly increased until highly polluted sites at downstreamriver Nile, indicating that the comet assay is more sensitive detec-tion method than the micronucleus test for low-level toxicity. Thesensitivity difference between the comet assay and micronucleustest may be due to their different endpoints [53]: the alkaline cometassay is capable of detecting a wide variety of DNA damage, whilethe micronucleus test only detects micronuclei, which arise fromdisruptions in the chromosomal distribution. Our results confirmedthe high sensitivity of the comet assay for detection of DNA damagein erythrocytes of African catfish and Nile tilapia. Thus the cometassay is a useful tool for in situ bio-monitoring, which is urgentlyrequired in Egypt with regard to the recently observed increasein aquatic pollution. Moreover, if we compare the results of thecomet assay, the diffusion assay and the micronucleus test for bothfish species, it is possible to observe a general correspondence indetection of DNA damage by the three tests. The apoptotic responseevaluated in erythrocytes of fish sampled from the lower river Nileshowed significantly higher frequency distribution in apoptoticnuclei compared with fish from the upper river Nile as expressed bythe diffusion assay. The present diffusion-assay procedure makes itpossible to detect virtually all early and late apoptotic nuclei [19].

The selection of fish species is a critical issue in genotoxicitybio-monitoring assays. Rodriguez-Cea et al. [54] noted that somefish species are more sensitive to genotoxic pollutants than others.Although the majority of fish species avoid long-term exposure topollutants dissolved in water at a particular site by actively swim-ming, some species have a more stationary behavior and thus maybe exposed for a longer time [44]. In the present work, Nile tilapiashowed a higher degree of DNA damage (not significant) and higherdamage-categories than African catfish collected from the samesites and at the same sampling time, proving that Nile tilapia pro-vides a more sensitive bio-indicator for genotoxicity. These results

Author's personal copy

20 A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21

are in line with our previous work [2], where a significant increasewas found in the frequencies of micronuclei in the blood of Niletilapia compared with African catfish collected from the same sitesand at the same times. The difference in sensitivity of Nile tilapiaand African catfish to DNA damage might be attributed to theirfeeding peculiarities [2]. These findings could explain that Africancatfish is more abundant in many polluted sites along the river Nilecompared with Nile tilapia.

Over the past few years, several studies have been conductedthat employ comet and diffusion assays to detect genotoxicity inbio-indicator fish species [9,19,31,47,55,56]. However, there havebeen only few studies employing these methods as field biomarkersin fish sampled from aquatic environments during different seasons[32]. Everaarts et al. [57] demonstrated increased levels of dam-age in hepatic DNA isolated from redbreast sunfish from pollutedsites. Similarly, hard-head catfish exhibited also increased levelsof DNA strand breakage at contaminated sites [11]. Field studieswere also included by Bolognesi et al. [58], who observed higherDNA strand-breakage in gill cells of mussels (M. galloprovincialis)taken from a site polluted with polycyclic aromatic hydrocarbons.In the current in situ investigation, higher degrees of DNA strand-breakage were found in blood of fish collected from heavily pollutedareas. Our results confirm the value of both assays for environmen-tal bio-monitoring. Based on our experience, we propose using thealkaline comet assay as a screening tool for bio-monitoring contam-ination of complex mixtures. Field samples are complex mixturesof organic and inorganic compounds that may interact to produceadditive, synergistic, or antagonistic effects [32,59]. Although nospecific cause-effect relationships were established, the data indi-cate that there might be associations between COD, phenol, Pb, Cd,Cr, Hg, and water temperature with the level of damaged cells inblood of Nile tilapia and African catfish collected along the wholecourse of the river Nile.

5. Conclusions

The comet assay proved to be a useful technique for detec-tion of genotoxic contaminants in the river Nile, indicating thatit can be applied successfully in fish for in situ genotoxic moni-toring of the aquatic environment. At the same time, the diffusionassay is sensitive enough to detect low frequencies of apoptoticnuclei. Nile tilapia and African catfish are useful organisms withsufficient sensitivity to be effective monitors of biological hazards.The results, however, demonstrate differential sensitivity of thetwo fish species, suggesting that Nile tilapia may serve as a moresensitive test organism than African catfish. This study suggeststhat erythrocytes of O. niloticus niloticus and C. gariepinus are goodindicators of genotoxicity as revealed by the comet and diffusionassays. Based on the current results, it can be concluded that thewater quality of the river Nile with respect to the presence of geno-toxic compounds needs to be improved, especially at its estuaries.As far as we know this is the first time that the comet and diffusionassays are used in parallel for genotoxic monitoring of the riverNile.

Conflict of interest

Authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by Science and Technology develop-ment fund (project ID 448). Parts of this study were conductedduring 2010–2012 in the framework of an Alexander von Hum-boldt foundation postdoctoral fellowship for the first author

(3.4-AGY/1134716 STP), hosted by Leibniz-Institute of Freshwa-ter Ecology and Inland Fisheries (IGB), Berlin, Germany. We wouldlike to thank Mr. A. Gad El-Rab, A. Moustafa, M. Nassar, R. Said,Department of Zoology (Al-Azhar University, Egypt) for their sup-port during sampling.

References

[1] A.G.M. Osman, A. Abd El Reheem, K. Abuel-Fadl, A. GadEl-Rab, Enzymatic andhistopathologic biomarkers as indicators of aquatic pollution in fishes, Nat. Sci.2 (2010) 1302–1311.

[2] A.G.M. Osman, A. Abd El Reheem, M.A. Moustafa, U.M. Mahmoud, K.Y. Abuel-Fadl, W. Kloas, In situ evaluation of the genotoxic potential of the river Nile:I.micronucleus and nuclear lesion tests of erythrocytes of Oreochromis niloti-cus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822), Toxicol.Environ. Chem. 93 (2011) 1002–1017.

[3] P. Rajaguru, S. Suba, M. Palanivel, K. Kalaiselvi, Genotoxicity of a polluted riversystem measured using the alkaline comet assay on fish and earthworm tissues,Environ. Mol. Mutagen. 41 (2003) 85–91.

[4] P. Rajaguru, L. Vidya, B. Baskarasethupathi, P.A. Kumar, M. Palanivel, K. Kalai-selvi, Genotoxicity evaluation of polluted ground water in human peripheralblood lymphocytes using the comet assay, Mutat. Res. 517 (2002) 29–37.

[5] D.R. Dixon, J.T. Wilson, P.L. Pascoe, J.M. Parry, Anaphase aberrations in theembryos of the marine tubeworm Pomatoceros lamarckii (Polychaeta: Serpul-idae): a new in vivo test assay for detecting aneugens and clastogens in themarine environment, Mutagenesis 14 (1999) 375–383.

[6] A.N. Jha, Genotoxicological studies in aquatic organisms: an overview, Mutat.Res. 552 (2004) 1–17.

[7] A. Rybakovas, J. Barsiene, T. Lang, Environmental genotoxicity and cytotoxicityin the offshore zones of the Baltic and the North Seas, Mar. Environ. Res. 68(2009) 246–256.

[8] M. Nigro, G. Frenzilli, V. Scarcelli, S. Gorbi, F. Regoli, Induction of DNA strandbreakage and apoptosis in the eel Anguilla anguilla, Mar. Environ. Res. 54 (2002)517–520.

[9] G. Frenzilli, V. Scarcelli, I. Del Barga, M. Nigro, L. Foerlin, C. Bolognesi, J. Sturve,DNA damage in eelpout (Zoarces viviparus) from Goeteborg harbour, Mutat.Res. 552 (2004) 187–195.

[10] L. Xu, G.J. Zheng, P.S.K. Lam, B.J. Richardson, Relationship between tissueconcentrations of polycyclic aromatic hydrocarbons and DNA adducts in green-lipped mussels (Perna viridis), Ecotoxicology 8 (1999) 73–82.

[11] C. Mitchelmore, J. Chipman, Detection of DNA strand breaks in brown trout(Salmo trutta) hepatocytes and blood cells using the single cell gel electrophore-sis (comet) assay, Aquat. Toxicol. 41 (1998) 161–182.

[12] C.L. Mitchelmore, J.K. Chipman, DNA strand breakage in aquatic organisms andthe potential value of the comet assay in environmental monitoring, Mutat.Res. 399 (1998) 135–147.

[13] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y.Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelinesfor in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35(2000) 206–221.

[14] G.M. Alink, J.T.K. Quik, E.J.M. Penders, A. Spenkelink, S.G.P. Rotteveel, J.L. Maas,W. Hoogenboezem, Genotoxic effects in the Eastern mudminnow (Umbra pyg-maea L.) after exposure to Rhine water, as assessed by use of the SCE and Cometassays: a comparison between 1978 and 2005, Mutat. Res. 631 (2007) 93–100.

[15] O. Garcia, I. Romero, J.E. Gonzalez, T. Mandina, Measurements of DNA damageon silver stained comets using free Internet software, Mutat. Res. 627 (2007)186–190.

[16] G. Frenzilli, M. Nigro, B.P. Lyons, The comet assay for the evaluation of genotoxicimpact in aquatic environments, Mutat. Res. 681 (2009) 80–92.

[17] P.L. Olive, G. Frazer, J.P. Banath, Radiation-induced apoptosis measured in TK6human B lymphoblast cells using the comet assay, Radiat. Res. 136 (1993)130–136.

[18] N. Kizilian, R.C. Wilkins, P. Reinhardt, C. Ferrarotto, J.R. McLean, J.P. McNamee,Silver-stained comet assay for detection of apoptosis, BioTechniques 27 (1999)926–930.

[19] N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. CellRes. 256 (2000) 328–337.

[20] H. Hao, F. lei, Y. Nancai, S. Wen, O. Xiaojuan, The single-cell gel electrophore-sis assay to determine apoptosis induced by siRNA in Colo 320 cells, Afr. J.Biotechnol. 8 (2009) 3731–3733.

[21] S.P. Cregan, B.P. Smith, D.L. Brown, R.E. Mitchel, Two pathways for the inductionof apoptosis in human lymphocytes, Int. J. Radiat. Biol. 75 (1999) 1069–1086.

[22] K. Al-Sabti, C.D. Metcalfe, Fish micronuclei for assessing genotoxicity in water,Mutat. Res. 343 (1995) 121–135.

[23] B. Ateeq, M. Abul Farah, W. Ahmad, Detection of DNA damage by alkaline singlecell gel electrophoresis in 2,4-dichlorophenoxyacetic-acid- and butachlor-exposed erythrocytes of Clarias batrachus, Ecotoxicol. Environ. Saf. 62 (2005)348–354.

[24] G.I.V. Klobucar, A. Stambuk, M. Pavlica, M. Sertic Peric, B.K. Hackenberger, K.Hylland, Genotoxicity monitoring of freshwater environments using caged carp(Cyprinus carpio), Ecotoxicology 19 (2010) 77–84.

[25] G. Mohanty, J. Mohanty, A.K. Nayak, S. Mohanty, S.K. Dutta, Application of cometassay in the study of DNA damage and recovery in rohu (Labeo rohita) fingerlings

Author's personal copy

A.G.M. Osman et al. / Mutation Research 747 (2012) 14– 21 21

after an exposure to phorate, an organophosphate pesticide, Ecotoxicology 20(2011) 283–292.

[26] A.M. Gontijo, C.M. Green, G. Almouzni, Repairing DNA damage in chromatin,Biochimie 85 (2003) 1133–1147.

[27] N. Avishai, C. Rabinowitz, B. Rinkevich, Use of the comet assay for studyingenvironmental genotoxicity: comparisons between visual and image analyses,Environ. Mol. Mutagen. 42 (2003) 155–165.

[28] D. Nacci, S. Cayula, E. Jackim, Detection of DNA damage in individual cells frommarine organisms using the single cell gel assay, Aquat. Toxicol. 35 (1996)197–210.

[29] V.M. de Andrade, J. da Silva, F.R da Silva, V.D. Heuser, J.F. Dias, M.L. Yoneama,T.R.O. de Freitas, Fish as bioindicators to assess the effects of pollution in twosouthern Brazilian rivers using the Comet assay and micronucleus test, Environ.Mol. Mutagen. 44 (2004) 459–468.

[30] A. Osman, E. Ali, M. Hashem, M. Mostafa, I. Mekkawy, Genotoxicity of twopathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces lae-vis) on erythrocytes of Nile tilapia Oreochromis niloticus niloticus, Ecotoxicol.Environ. Saf. 73 (2010) 24–31.

[31] C. Russo, L. Rocco, M.A. Morescalchi, V. Stingo, Assessment of environmentalstress by the micronucleus test and the Comet assay on the genome of teleostpopulations from two natural environments, Ecotoxicol. Environ. Saf. 57 (2004)168–174.

[32] V.M. de Andrade, T.R.O. de Freitas, J. da Silva, Comet assay using mullet (Mugilsp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxicpollutants in aquatic environment, Mutat. Res. 560 (2004) 57–67.

[33] APHA, Standard methods for the examination of water & wastewater, AmericanPublic Health Association, 2005.

[34] N. Singh, M. McCoy, R. Tice, E. Schneider, A simple technique for quantita-tion of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988)184–191.

[35] F. Mouchet, L. Gauthier, C. Mailhes, V. Ferrier, A. Devaux, Comparative study ofthe comet assay and the micronucleus test in amphibian larvae (Xenopus laevis)using benzo(a)pyrene, ethyl methanesulfonate, and methyl methanesulfonate:establishment of a positive control in the amphibian comet assay, Environ.Toxicol. 20 (2005) 74–84.

[36] A.G.M. Osman, I.A. Mekkawy, J. Verreth, S. Wuertz, W. Kloas, F. Kirschbaum,Monitoring of DNA breakage in embryonic stages of the African catfish Clariasgariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline cometassay, Environ. Toxicol. 23 (2008) 679–687.

[37] D. Anderson, T.W. Yu, B.J. Phillips, P. Schmezer, The effect of various antioxi-dants and other modifying agents on oxygen-radical-generated DNA damagein human lymphocytes in the COMET assay, Mutat. Res. 307 (1994) 261–271.

[38] SPSS, SPSS-Inc for Windows Release, Chicago, 10, 1998.[39] A.G.M. Osman, W. Kloas, Water quality and heavy metal monitoring in water,

sediments, and tissues of the African catfish Clarias gariepinus (Burchell, 1822)from the river Nile, Egypt, J. Environ. Prot. 1 (2010) 389–400.

[40] A. El-Naggar, S. Mahmoud, S. Tayel, Bioaccumulation of some heavy metalsand histopathological alterations in liver of Oreochromis niloticus in relation towater quality at different localities along the river Nile, Egypt, World J. FishMar. Sci. 1 (2009) 105–114.

[41] P.A. White, J.B. Rasmussen, The genotoxic hazards of domestic wastes in surfacewaters, Mutat. Res. 410 (1998) 223–236.

[42] J.W. Bickham, S. Sandhu, P.D. Hebert, L. Chikhi, R. Athwal, Effects of chemi-cal contaminants on genetic diversity in natural populations: implications forbiomonitoring and ecotoxicology, Mutat. Res. 463 (2000) 33–51.

[43] A. Osman, A. Harabawy, Hematotoxic and genotoxic potential of ultraviolet-Aradiation on the African catfish Clarias gariepinus (Burchell, 1822), J. Fish Int. 5(2010) 44–53.

[44] N. Kopjar, P. Mustafic, D. Zanella, I. Buj, M. Caleta, Z. Marcic, M. Milic, Z. Dolenec,M. Mrakovcic, Assessment of DNA integrity in erythrocytes of Cobitis elongataaffected by water pollution: the alkaline comet assay study, Folia Zoolog. 57(2008) 120–130.

[45] C.L. Mitchelmore, C. Birmelin, D.R. Livingstone, J.K. Chipman, Detection of DNAstrand breaks in isolated mussel (Mytilus edulis L.) digestive gland cells usingthe Comet assay, Ecotoxicol. Environ. Saf. 41 (1998) 51–58.

[46] A. Devaux, P. Flammarion, V. Bernardon, J. Garric, G. Monod, Monitoring ofthe chemical pollution of the River Rhine through the measurement of DNAdamage and cytochrome P4501A induction in chub (Leuciscus cephalus), Mar.Environ. Res. 46 (1998) 257–262.

[47] R. Pandrangi, M. Petras, S. Ralph, M. Vrzoc, Alkaline single cell gel (comet) assayand genotoxicity monitoring using bullheads and carp, Environ. Mol. Mutagen.26 (1995) 345–356.

[48] F. Ayllon, E. Garcia-Vazquez, Induction of micronuclei and other nuclear abnor-malities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna:an assessment of the fish micronucleus test, Mutat. Res. 467 (2000) 177–186.

[49] C. Risso-de Faverney, A. Devaux, M. Lafaurie, J. Girard, B. Bailly, R. Rahmani,Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytesthrough generation of reactive oxygen species, Aquat. Toxicol. 53 (2001) 65–76.

[50] D.R. Livingstone, Contaminant-stimulated reactive oxygen species produc-tion and oxidative damage in aquatic organisms, Mar. Pollut. Bull. 42 (2001)656–666.

[51] E. Mamaca, R.K. Bechmann, S. Torgrimsen, E. Aas, A. Bjornstad, T. Baussant,S. Le Floch, The neutral red lysosomal retention assay and Comet assay onhaemolymph cells from mussels (Mytilus edulis) and fish (Symphodus melops)exposed to sryrene, Aquat. Toxicol. 75 (2005) 191–201.

[52] A. Buschini, A. Martino, B. Gustavino, M. Monfrinotti, P. Poli, C. Rossi, M. Santoro,A.J.M. Doer, M. Rizzoni, Comet assay and micronucleus test in circulating ery-throcytes of Cyprinus carpio specimens exposed in situ to lake waters treatedwith disinfectants for potabilization, Mutat. Res. 557 (2004) 119–129.

[53] J.L. He, W.L. Chen, L.F. Jin, H.Y. Jin, Comparative evaluation of the in vitromicronucleus test and the comet assay for the detection of genotoxic effects ofX-ray radiation, Mutat. Res. 469 (2000) 223–231.

[54] A. Rodriguez-Cea, F. Ayllon, E. Garcia-Vazquez, Micronucleus test in freshwaterfish species: an evaluation of its sensitivity for application in field surveys,Ecotoxicol. Environ. Saf. 56 (2003) 442–448.

[55] V. Bombail, D. Aw, E. Gordon, J. Batty, Application of the comet and micronu-cleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth,Scotland, Chemosphere 44 (2001) 383–392.

[56] G. Frenzilli, A. Falleni, V. Scarcelli, I. Del Barga, S. Pellegrini, G. Savarino, V.Mariotti, M. Benedetti, D. Fattorini, F. Regoli, M. Nigro, Cellular responses inthe cyprinid Leuciscus cephalus from a contaminated freshwater ecosystem,Aquat. Toxicol. 89 (2008) 188–196.

[57] J.M. Everaarts, L.R. Shugart, M.K. Gustin, W.E. Hawkins, W.W. Walker, Biologicalmarkers in fish: DNA integrity, hematological parameters and liver somaticindex, Mar. Environ. Res. 35 (1993) 101–107.

[58] C. Bolognesi, G. Frenzilli, C. Lasagna, E. Perrone, P. Roggieri, Genotoxicitybiomarkers in Mytilus galloprovincialis: wild versus caged mussels, Mutat. Res.552 (2004) 153–162.

[59] K. Fent, Ecotoxicological problems associated with contaminated sites, Toxicol.Lett. 140–141 (2003) 353–365.