Histopathological biomarkers in gills and liver of Oreochromis niloticus from polluted wetland environments, Saudi Arabia

Chemosphere 88 (2012) 1028–1035

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Histopathological biomarkers in gills and liver of Oreochromis niloticus from polluted wetland environments, Saudi Arabia Ashraf M. Abdel-Moneim a,b,⇑, Mohamed A. Al-Kahtani a, Omar M. Elmenshawy a,c a

Department of Biological Sciences, Faculty of Science, King Faisal University, Al Hassa 31982, Saudi Arabia Department of Zoology, Faculty of Science, Alexandria University, Alexandria 21511, Egypt c Department of Zoology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt b

a r t i c l e

i n f o

Article history: Received 29 August 2011 Received in revised form 30 January 2012 Accepted 3 April 2012 Available online 29 April 2012 Keywords: Fish Gills Liver Biomarkers Pollution Histopathology

a b s t r a c t Fish live in direct contact with their immediate external environment and, therefore, are highly vulnerable to aquatic pollutants. In this study, Oreochromis niloticus were caught at three different sites in AlHassa irrigation channels, namely Al-Jawhariya, Um-Sabah and Al-Khadoud. The histological changes in gills and liver were detected microscopically and evaluated with semi-quantitative analyses. Also, heavy metals have been determined in the water samples in these sites. Results showed that all sites were polluted by different kinds of heavy metals. Cd and Pb were mostly detected at concentrations above the WHO reference values. Meanwhile, various histopathological abnormalities were observed in gills and liver of fish specimens. In the gill filaments, cell proliferation, lamellar cell hyperplasia, lamellar fusion, lifting of the respiratory epithelium, and the presence of aneurysmal areas were observed. In the liver, there was vacuolization of the hepatocytes, sinusoidal congestion, necrosis of the parenchyma tissue, nuclear pyknosis, eosinophilic hepatocellular degeneration, pigment accumulation, an increase in the number and size of melanomacrophage centers. Liver tumors with severe chronic inflammation were occasionally found in fish at Al-Khadoud area (first-time report). The histological lesions were comparatively most severe in the liver. Despite heavy metals assessment did not show marked differences among sites, histopathological biomarkers indicated that the surveyed fish are living under stressful environmental conditions. So, we suggest use those biomarkers in future monitoring of aquatic systems. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Due to urban, industrial and agricultural activities, freshwater sources are dumped with different kinds of chemicals that affect the inhabiting biota. In order to evaluate the adverse effects of these complex chemical mixtures on aquatic organism, there is a worldwide trend to complement chemical and physical parameters with biomarkers in aquatic pollution monitoring (Van der Oost et al., 2003; Au, 2004). Fish are important vehicles for the transfer of contaminants to human populations and may indicate the potential exposure to pollutants (Al-Sabti and Metcalfe, 1995). Histopathological studies, in laboratory and in field experiments, have proved to be a sensitive tool to detect direct toxic effects of chemical compounds within target organs of fish (Schwaiger et al., 1997; Au, 2004; Ayas et al., 2007; Camargo and Matinez, 2007; Costa et al., 2009, 2011; Leonardi et al., 2009; Yasser and Naser, 2011). The gills of freshwater fish are the larg⇑ Corresponding author. Address: Department of Zoology, Faculty of Science, Alexandria University, Alexandria 21511, Egypt. Tel.: +966 566828160. E-mail address: [email protected] (A.M. Abdel-Moneim). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.04.001

est fraction of the total body surface area, which is in direct contact with the water (Hughes, 1984). The complexity and constant contact with the surrounding water make the gill the first target to waterborne pollutants (Perry and Laurent, 1993). In fact, pollutants enter the organism through the gills and exert their primary toxic effects on the branchial epithelium (Playle et al., 1992). Thus, changes in fish gills are among the most commonly recognized responses to environmental stressors and are indicative of physical and chemical stress (Mallat, 1985; Au, 2004). On the other hand, liver plays an important role in vital functions in basic metabolism and it is the major organ of accumulation, biotransformation, and excretion of contaminants in fish (Moon et al., 1985; Triebskorn et al., 1997; Figueiredo-Fernandes et al., 2006). Histo-cytopathological changes in livers of fish exposed to a wide range of organic compounds and heavy metals have been reported (Rabitto et al., 2005; Sarkar et al., 2005; Roy and Bhattacharya, 2006; Mela et al., 2007). Al-Hassa springs on the Eastern part of Saudi Arabia play a crucial role on the ecology of the ecosystem. These springs are of great economic importance to the surrounding areas, enabling cultivation of crops and animal farming by various irrigation and drainage

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channels, as well as providing sufficient quantities of fish and shrimps. However, as a result of industrial, agricultural, and other anthropogenic activities, this aquatic ecosystem has been degraded over the last decades. The heavy metal contamination has been an important factor to the decline of water and sediment quality and may adversely affect fish health (Al-Kahtani, 2009). A previous study at Al-Khadoud spring, one of the most important water resources in Al-Hassa region showed that its water had an obvious increase in electrical conductivity, chemical oxygen demand (COD), total alkalinity, nitrates, phosphorus, chloride, and potassium (Fathi and Al-Kahtani, 2009). These features indicated pollution with organic wastes, increased salinity and deteriorated oxygenated state. The main objectives of the current study were, to (1) to assess the concentrations of some toxic metals in water samples from AlHassa irrigation channels. This can serve as an indicator for the extent of pollution in these wetland water bodies, (2) to characterize the histopathological effects of pollution on liver and gills of fish (Oreochromis niloticus) captured from these sites, (3) to expand our knowledge on the toxicity of sublethal concentrations of heavy metals in fish in their natural habitats, (4) to provide a set of baseline data for the potential use (either qualitatively or quantitatively) as indicators of a changing health status in fish (i.e. as biomarkers). This work is important due to the fact that it is the first comprehensive study in Al-Hassa springs for determining the pollution levels using quantitative histopathological data. 2. Materials and methods 2.1. Study area The springs in the study area are located in three groups along the Western edge of Al-Hassa Oasis (Fig. 1). Among the largest and most important springs are: Al-Khadoud, Um Sabah, and Al-Jawhariya. To increase water for irrigation, the water pumped into the spring canals so as to increase the amount of water used for local irrigation. Over the years, the spring and its connected canals have become a place of fish and other aquatic organisms’ growth. In fact, this water body has attracted the attention of local people for fishery. Due to the extended domestic activities and urbanization as well as the continuous agricultural growth of the region, water quality of Al-Hassa springs is potentially changing as the case of Al-Khadoud spring (see Al-Kahtani, 2009; Fathi and AlKahtani, 2009). Some of untreated domestic and agricultural sewage may elapse into spring water. Such contamination must be an important issue regarding the health of the aquatic ecosystem and its animals and in turn, to human’s health. 2.2. Sampling strategy From March to December 2010, samples of O. niloticus (body weight: 90–130 g; total length: 18–22 cm; N = 40 per site) were collected at three sites, Al-Jawhariya (site #1), Um-Sabah (site #2) and Al-Khadoud (site #3), using bottom trap net. Fish were dissected and samples of gills and liver were fixed for histopathological studies. Simultaneously with fish collection, 2 L of water samples were taken for each station for chemical analyses of heavy metals; water samples were transported to the laboratory at 4 °C in clean plastic bottles and analyzed according to standard procedures (APHA, AWWA, WEF, 1998) using Varian Spectra-AA 20 instrument with Flame Ionizing Detector (FID). All the glasswares used in heavy metal estimation were cleaned with chromic acid and finally rinsed with deionized water before use in order to avoid metal contamination. Water samples containing particulate or organic material generally require pretreatment before spectroscopic analysis. 20 mL of 1:1 HNO3 was mixed with 100 mL water sample;

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heated in oven at 80 °C until 10 or 5 mL was left. Then it was filtered with Whatman filter paper No. 41 to make up the volume up to 100 mL with dH2O. The sample was then analyzed by AAS. For blank preparation, the same procedure was followed except taking deionized water in lieu of sample water. 2.3. Light microscopic study Tissue specimens of gills and liver were cut into small pieces and fixed in 4% neutral buffered formaldehyde for 24 h. Fixed tissues were rinsed in tap water, dehydrated through a graded series of ethanol, infiltrated with xylene and then embedded in paraffin. Fourmicron thick sections were cut in the microtome from the tissue blocks and picked up on glass slides. The sections were deparaffinized in xylene, rehydrated through decreasing concentrations of ethanol, stained with hematoxylin and eosin, and then examined by light microscopy. The presence of histological alterations for each organ was evaluated semi-quantitatively by the degree of tissue change (DTC), which is based on the severity of the lesions. For DTC calculation, the alterations in each organ were classified in progressive stages of damage to the tissue: stage I alterations, which do not alter the normal functioning of the tissue; stage II, which are more severe and impair the normal functioning of the tissue; and stage III, which are very severe and cause irreparable damage. A value of DTC was calculated for each animal by the formula: DTC = (1  SI) + (10  SII) + (100  SIII) where I, II and III correspond to the number of alterations of stages I, II and III, respectively. The DTC value obtained for each fish was used to calculate the average index for each sampling site. DTC values between 0 and 10 indicate normal functioning of the organ; values between 11 and 20 indicate slight damage to the organ; values between 21 and 50 indicate moderate changes in the organ; values between 51 and 100 indicate severe lesions and values above 100 indicate irreversible damage to the organ (Poleksic and Mitrovic-Tutundzic, 1994). 2.4. Statistical analysis Mean values of DTC obtained for each sampling site were compared with each other, using one-way ANOVA and multiple comparison tests (LSD), with a level of significance of P 6 0.05. 3. Results 3.1. Water quality The mean concentrations of the heavy metals in water samples at the selected sites together with World Health Organization (WHO, 2006) limits are presented in Table 1. Most heavy metals recorded in water in this study were generally low, when compared to WHO standards except for Cd and Pb. In water samples, according to the analysis results, the following findings were obtained for the concentration ranges of metals: Fe: 0.087–0.096 mg L 1; Mn: 0.001– 0.008 mg L 1; Zn: 0.016–0.027 mg L 1; Cu: 0.014–0.033 mg L 1; Ni: 0.005–0.011 mg L 1; Cd: 0.003–0.006 mg L 1; Pb:0.020– 0.025 mg L 1; Cr: 0.002–0.009 mg L 1; Co: 0.003–0.004 mg L 1; Ba: 0.049–0.635 mg L 1; Hg: 0.0004–0.0007 mg L 1; Mo: 0.003– 0.007 mg L 1. Site #1 was appreciably higher in Fe, Mn, Zn, Cu, Cd, Pb, Co and Hg concentrations. The highest value of Ni were reported at site #2 while the highest values of Cr, Ba and Mo were reported at location #3. 3.2. Histopathological observations Fish from the three sampling sites showed a series of histological alterations in the gills and liver (Table 2). The occurrence of

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Fig. 1. Map showing Al-Hassa springs and irrigation channels; site #1 (Al-Jawhariya), site # 2 (Am- Sabah), and site # 3 (Al-Khadoud).

lesions in site #1 individuals was low. An increase of lesion gradient in site #2 and #3-tested fish was clearly discernible. The most relevant branchial changes were: vasodilation (Fig. 2B), hyperplasia, occasionally resulting in lamellar fusion (Fig. 2B and C), epithelial and lamellar capillary aneurysms (Fig. 2C), and epithelial lifting (Fig. 2D). The estimated gill DTC values were

6.44 ± 2.52 at site #1, 13.57 ± 3.85 at site #2 and 12.33 ± 2.97 at site #3, indicating normal functioning of the organ (Table 3). The vacuolization of hepatocytes was more common than other liver pathologies (Fig. 3B). At the initial stage of this disturbance, several small vacuoles appear in the cellular cytoplasm, and they subsequently fuse to form a large vacuole. The

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A.M. Abdel-Moneim et al. / Chemosphere 88 (2012) 1028–1035 Table 1 Mean metal concentrations (mg L Sampling sites Al-Jawhariya Um-Sabah Al-Khadoud WHO (2006)

1

) of water samples from irrigation channels in Al-Hassa (Saudi Arabia) as compared to WHO permissible limits. Fe

Site #1 Site #2 Site #3

0.096 0.092 0.087 1

Mn 0.008 0.001 0.005 0.5

Zn 0.027 0.016 0.018 5

Cu 0.033 0.015 0.014 2

Ni

Cd

Pb a

0.005 0.011 0.007 0.02

0.006 0.003 0.004a 0.003

a

0.025 0.020a 0.023a 0.01

Cr

Co

Ba

Hg

Mo

0.006 0.002 0.009 0.05

0.004 0.003 0.003 –

0.049 0.410 0.635 –

0.0007 0.0005 0.0004 0.001

0.003 0.003 0.007 –

N = 9; a: Values are higher than the recommended WHO (2006) drinking water quality guidelines.

Table 2 Histological alterations found in the gills and liver of Oreochromis niloticus collected from three sites in Al-Hassa irrigation tributaries, their respective stages of damage to the tissue and frequency of occurrence. Lesion

Stage

Site #1

Site #2

Site #3

Gills

Hyperplasia of the gill epithelium Hypertrophy of the gill epithelium Blood congestion Dilation of the marginal channel Epithelial lifting of the lamellae Lamellar fusion Lamellar disorganization Lamellar aneurysm Rupture of epithelial cells with hemorrhage Complete fusion of all the lamellae Rupture of pillar cells Rupture of the lamellar epithelium

I I I I I I I II II II II II

+ + + ++ + + ++ 0+ 0 + 0+ 0

++ ++ ++ +++ ++ ++ +++ + 0+ ++ + 0+

++ ++ ++ +++ ++ ++ +++ + 0+ ++ + 0+

Liver

Melanomacrophages aggregates Eosinophilic granules in the cytoplasm Cellular hypertrophy Nuclear hypertrophy Irregular shaped cells Irregular shaped nucleus Nucleus in a lateral position Cytoplasmic vacuolation Eosinophilic granules in the cytoplasm Nuclear vacuolation Cytoplasmic degeneration Cell rupture Hyperemia Nuclear degeneration Pyknotic nucleus Bile stagnation Necrosis Hepatic neoplasms

I I I I I I I I I II II II II II II II III III

+ + + + + + + + + 0+ + + + + + 0+ + 0

++ ++ ++ ++ ++ ++ ++ +++ ++ + ++ ++ +++ ++ ++ + ++ 0

++ ++ ++ ++ ++ ++ ++ +++ ++ + ++ ++ +++ ++ ++ + ++ 0+

Note: 0, absent; 0+, rare; +, low frequency; ++, frequent; +++, very frequent.

cytoplasm and nucleus are forced to the periphery of the cell. The nucleus is also subject to changes: the chromatin is condensed, and the optical density of the nucleus increases. The terminal stage of vacuolization, when a single vacuole is observed in a cell, is called hypervacuolization. Two types of vacuolization were encountered, diffuse and focal vacuolization. The latter, occurring much less frequently than the former, was characterized by vacuolated cells forming foci and often lying perivascularly. The diffuse vacuolization affected the whole hepatic parenchyma. We observed various patterns of diffuse liver vacuolization. Another pathology observed in the liver of tilapia studied was hyperemia, a blood circulation disturbance characterized by congestion (Fig. 3C) and occasionally accompanied by erythrocyte destruction, giving an indication of hematological anomalies. In a few cases, small punctate bleedings into surrounding tissues were observed. Some liver areas showed focal necrosis (Fig. 3C and D) and contained severe infiltration of leukocytes. The foci of local hepatic tissue necrosis were characterized by entirely destroyed hepatic tubules and, in most cases, displayed no cellular structure. They contained lysed hepatocytes remnants. With diffuse necrosis, only separate dead hepatocytes were observed, identified by their pyknotic nuclei. Cellular alteration foci as preneoplastic

lesions or precursors in hepatic neoplasms histogenesis were also found in all test sites. Altered hepatocytes typically presented more eosinophilic (acidophilic) cytoplasm (thus retaining much eosin, an acidic reddish pigment), accompanied by an alteration in shape and size, losing their common polyedric outline and frequently presenting hypertrophy (Fig. 3C and D). These foci were occasionally found to be associated with proliferation and swelling of blood vessels (Fig. 3C). The most damaged livers frequently presented a combination of severe changes like necrosis and eosinophilic hepatocellular alteration. In these cases, Kupffer cells (liver-specialized macrophages) were often observed intruding into the damaged tissue, whereas melanomacrophages were more frequently observed (Fig. 3D), occasionally forming dense centers adjacent to portal veins. A rounded and nonencapsulated tumor presenting a completely differentiated and visible mass of liver cells was only observed in fish samples from site #3 (with incidences 6%). Tilapia hepatic neoplasms were characterized as hepatocellular carcinomas (Fig. 3E), which contained portions of biliary structures revealing variable cellular anaplasia (not shown). Values of liver DTC ranged from 39.35–71.88, indicating that in most cases, the hepatic lesions caused moderate to severe damage to the organ (Table 3).

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Fig. 2. Light micrographs of gills of Oreochromis niloticus. Gill histopathology of fish collected from site #1; (A) The tips of the secondary lamellae exhibit peculiar malformations such as globate structures (arrows) and curved tips at ending (arrowheads). Gill histopathology of fish collected from site #2; (B) High severity of filamentar epithelium hyperplasia that induced complete lamellar fusion (F). High severity vasodilation (V) in the lamellar vascular axis and necrosis with rupture of covering filamentar epithelium (arrow). Gill histopathology of fish collected from site #3; (C) Aneurysm (telangiectasia) with high grade of severity that extends through the entire lamellar vascular axis (arrows). Note complete lamellar fusion, arrowheads point to proliferating mucous cells; (D) High severity degree of lifting of filamentar and lamellar epithelium (arrows). In filament, the lifting might be due to degeneration of epithelial cells and edema. The lifting observed in lamellae is probably due to the high levels of edema. Scale bars: 200 lm, H&E stain.

Table 3 Degree of tissue change (DTC) for gills and liver of Oreochromis niloticus collected from three sites in Al-Hassa irrigation tributaries. Site* Al-Jawhariya Um-Sabah Al-Khadoud *

Site #1 Site #2 Site #3

Gills

Liver

6.44 ± 2.52a 13.57 ± 3.85b 12.33 ± 2.97b

39.35 ± 3.09a 64.44 ± 15.80b 71.88 ± 20.07b

Different letters, within columns indicate significant differences (p < 0.05).

4. Discussion Water quality can be determined using different physical, chemical and biological parameters. From this study, there seems in general a pollution problem caused by Cd and Pb with relatively high concentrations throughout the studied areas when compared to WHO standards. Al-Kahtani (2009) showed that, the fish from Al-Khadoud spring (corresponding to site #3), based on the higher levels of metal bioaccumulation, could be unsafe for human consumption. Fish are at the higher levels of food chain and therefore may biomagnify toxicants from the food. In addition, they can bioaccumulate toxicants from the water. Accumulation patterns of contaminants in fish and other aquatic organisms depend both on uptake and elimination rates (Hakanson, 1984). Difficulties in the field data interpretation may arise from various abiotic factors, such as chemical form of metal in the water, water temperature, pH value, dissolved oxygen concentration, water transparency, all contributing to the availability of different contaminants that

affect fish (Has-Schon et al., 2006). Chemical analyses alone may not suffice to describe the adverse effects of the complex mixtures of chemicals present at contaminated sites. Therefore, the use of a set of biomarkers for assessment of environmental quality has been recommended by many researchers (Cajaraville et al., 2000; Lionetto et al., 2003; Mayon et al., 2006; Fernandes et al., 2007; Sanchez et al., 2007, 2008; Tejeda-Vera et al., 2007; Linde-Arias et al., 2008). In the present study, a wide spectrum of histopathologies was revealed in the gills and liver of fish living in Al-Hassa waterways. Several investigators have reported histopathological changes in the gills of different fish species exposed to pesticides, petroleum hydrocarbon, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals (Heath, 1987; Alazemi et al., 1996; Arellano et al., 1999; Erkmen et al., 2000; Mondon et al., 2001; Cengiz and Ünlu, 2002; Pacheco and Santos, 2002; Moore et al., 2003). These included several alterations similar to those of tilapia confined to contaminated sites in the present study, such as lifting of respiratory epithelium, hyperplasia and hypertrophy of chloride cells and mucous cells, edema of epithelial cells, clubbing of gill filament, and aneurysm. The histopathological changes of the gills likely resulted in hypoxia, respiratory failure problems with ionic and acid–base balance (Alazemi et al., 1996; Yasser and Naser, 2011). Lifting and swelling could be related to a decrease in the gill Na± and K± activated ATPase and/or a decline in blood Na± and Cl concentration (Neiboer and Richardson, 1980). Fusion of secondary lamellae could cause a decrease in free gas exchange thus affecting the general health of fish (Skidmore and Tovell, 1972). Cell proliferation of secondary lamellar filaments

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Fig. 3. Light micrographs of liver of Oreochromis niloticus. (A) Nearly normal hepatic parenchyma of fish from site #1, exhibiting well-defined hepatocytes, polyedric in shape. HPV: hepatic portal vein branch with erythrocytes, (s) transversally sectioned sinusoid. Liver histopathology of fish collected from site #2; (B) Hepatocytes with diffuse vacuolization in the cytoplasm (arrows), and nuclear degeneration; (C) Sinusoidal congestion (arrows) is quite evident. Note also eosinophilic hepatocellular degeneration (arrowheads) and necrotic area of liver parenchyma () with invading inflammatory cells. Liver histopathology of fish collected from site #3; (D) Hepatic tissue showing focal necrosis (), melanomacrophages aggregates (arrows), and eosinophilic bodies (hyaline degeneration) (arrowheads). Note: Granulomatous lesion in the liver. The lesion is located around a regressed bile duct (bd) and infiltrates the highly damaged surrounding tissue. pt: pancreatic tissue; (E) Hepatocellular carcinoma (HCA) in Tilapia liver. This lobe of the liver contained a single, relatively well differentiated, but invasive neoplasm composed of densely packed neoplastic hepatocytes arranged in irregular cords that are somewhat thicker than the adjacent, normal hepatocytes (at right). The neoplastic cells have abundant eosinophilic cytoplasm with less vacuolation than normal, mildly increased mitotic figures, and minimal nuclear atypia. Note: Melanomacrophages center (arrow) in tumor mass. Scale bars: 200 lm, H&E stain.

and lamellar cell hypertrophy decreases the space between lamellae and causes fusion. Such lesions would increase the thickness of water–blood barrier and decrease the oxygen uptake. These lesions can cause capillary hemorrhage (Nowak, 1992; Jiraungkoorskul et al., 2002). Generally, separation of lamellar epithelium or hyperplasia of epithelium and tip inflammation could be a defense response of the circulatory system against pollutants (Richmonds and Dutta, 1989; Ortiz et al., 2003). Liver changes in the fish samples were more severe and in some cases irreparable, reflecting the poor water quality of Al-Hassa water systems. Livers are useful to describe and document the effects of pollutants. Necrosis in livers is not necessarily due to

specific pollutants since little evidence links damage to specific organic or inorganic compounds (Chang et al., 1998; Rabitto et al., 2005). Necrosis is strongly associated with oxidative stress where lipid peroxidation is a clear source of membrane bilayer susceptibility (Li et al., 2000; Avci et al., 2005). Pollutants (pesticides – Azzalis et al., 1995, heavy metals – Stohs and Bagghi, 1995, PAHs – Ibuki and Goto, 2002) are associated with increased free radical concentrations within the cytosol. These oxidative forms may increase programmed cell death or disturbed cell homeostasis and cellular necrosis. Of all the liver pathologies, vacuolization of the hepatocytes, resulting from lipid dystrophies, occurred most frequently. Hepatic lipidosis is believed to be a

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prenecrotic stage and has been observed in fish exposed to metals (Arellano et al., 1999; Giari et al., 2007), crude oil extracts (Solangi and Overstreet, 1982) and in feral fish from sites contaminated by mixtures of xenobiotics (Greenfield et al., 2008; Triebskorn et al., 2008). While lipid accumulation may be normal physiological storage, it may also be a mechanism for defence against liposoluble contaminants (Biagianti-Risbourg et al., 1997). The presence of eosinophilic hepatocellular degeneration in highly damaged livers is one of the most conspicuous alteration pattern observed. Its exact biological consequence is unclear but Koehler et al. (2004); for instance, found that the metabolic activity is upregulated in preneoplastic eosinophilic hepatocytes in feral flounders from PCBscontaminated sites. Eosinophilic bodies in flatfish liver and kidney have already been linked to the exposure to xenobiotics (Camargo and Matinez, 2007; Van Dyk et al., 2007). Information on the nature of the substances contained in these inclusions is absent but, considering the affinity of eosin to structural proteins such as actin, it is possible that eosinophilic bodies retain peptide material absorbed from the cytoplasm of degenerating cells. This is supported by data on eosinophilic bodies found in neoplastic areas of human epithelia (Buchner et al., 1976) and liver (Chedid et al., 1999). Considering their correlation to necrosis, eosinophilic bodies may be indicators of severe cirrhosis. An increase in the density of the melanomacrophage aggregates, as observed in the liver of O. niloticus in this study, is generally related to important hepatic lesions (Pacheco and Santos, 2002), such as degenerative and necrotic processes. Several authors have suggested that the involvement of melanomacrophage centers in various disease processes and the changes brought about in them by such factors as starvation (Agius and Roberts, 1981), or chemical exposure (Long et al., 1995; Couillard and Hodson, 1996; Meinelt et al., 1997) indicates that these centers can provide sensitive indicators of stressful conditions in the aquatic environment (Chang et al., 1998). Tumors found in livers of tilapia are results of long-term exposure to a combination of potentially carcinogenic pollutants. Similarly, liver tumors have been reported in Microgadus tomcod from the Hudson River, an area highly contaminated with PAHs, PCBs, pesticides and heavy metals (Dey et al., 1993). It is generally accepted that changes in enzyme activity in the extracellular fluid or plasma is a sensitive indicator of even minor cellular damage, since the level of these enzymes will be higher than normal (Van der Oost et al., 2003). Thus, the measurement of phosphatase and transaminase activities in the circulating fluid is frequently used as a diagnostic tool in water pollution studies (Adham et al., 1997). An increase in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities due to metals (Zn, Cu and Cd) was also found in experimental conditions (Oluah, 1999; Varanka et al., 2001; Zikic et al., 2001), as well as in fish chronically exposed to metals (Levesque et al., 2002). Using enzymatic parameters as biomarker together with histological parameters is a very realistic approach and may be applied in AlHassa water channels for future studies.

5. Conclusions The present study showed that, despite water quality assessment of Al-Hassa irrigation channels did not show a severe anthropogenic impact, the use of non-specific histopathological biomarkers in O. niloticus represented a sensitive and effective tool for reflecting adverse environmental conditions for fish health. However, further studies are crucial to corroborate the presence of xenobiotics or other environmental toxics affecting this species within the studied areas. On the other hand, there is a strong necessity for monitoring the effects of environmental pollution on wild fish in other parts of Saudi Arabia.

Acknowledgments The financial support for this Project from the Deanship of Scientific Research, King Faisal University, Saudi Arabia is gratefully acknowledged (Project No.: 110022). We also thank Mr. Ali Al-Kahtani for assistance with sampling design and field collection, Dr. Magdy Shaheen for helping with the analysis of water samples, and Mr. Ali Al-Shehri for technical support in the histological study. References Adham, K., Khairalla, A., Abu-Shabana, M., Abdel-Maguid, N., Abdel Moneim, A., 1997. Environmental stress in Lake Maryut and physiological response of Tilapia zilli Gerv. J. Environ. Sci. Health Part A 32, 2585–2598. Agius, C., Roberts, R.J., 1981. Effects of starvation on the melano-macrophage centres of fish. J. Fish Biol. 19, 161–169. Alazemi, B.M., Lewis, J.W., Andrews, E.B., 1996. Gill damage in the fresh water fish Gnathonemus ptersii (Family: Mormyridae) exposed to selected pollutants: an ultrastructural study. Environ. Technol. 17, 225–238. Al-Kahtani, M., 2009. 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