alexa Effect of El-Sail Drain Wastewater on Nile Tilapia (Oreochromis niloticus)from River Nile at Aswan, Egypt | Open Access Journals
ISSN: 2155-9546
Journal of Aquaculture Research & Development
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Effect of El-Sail Drain Wastewater on Nile Tilapia (Oreochromis niloticus)from River Nile at Aswan, Egypt

Sayeda MA1, Eman MY2, Amany MK3, Taghreed BI3 and Wafaa TA3*
1Microbiology Department, National Institute of Oceanography and Fisheries, Aswan Research Station, Egypt
2Fish Diseases Lab, National Institute of Oceanography and Fisheries, Alexandria, Egypt
3Hydrobiology Department, Veterinary Research Division, National Research Center, Dokki, Giza, Egypt
Corresponding Author : Wafaa T Abbas
Department of Hydrobiology
National Research Centre, El-Bohooth Street
Dokki 12622, Giza, Egypt
Tel: 989163128034
E-mail: [email protected]
Received: November 10, 2014; Accepted: November 17, 2014; Published: January 20, 2015
Citation: Sayeda MA, Eman MY, Amany MK, Taghreed BI, Wafaa TA (2015) Effect of El-Sail Drain Wastewater on Nile Tilapia (Oreochromis niloticus) from River Nile at Aswan, Egypt. J Aquac Res Development 6:294. doi:10.4172/2155-9546.1000294
Copyright: © 2015 Sayeda MA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

This study demonstrates the impact of wastewater of El-Sail Drain on the health of Oreochromis niloticus collected from two sites of River Nile at Aswan Governorate. One of these sites is before (I) and the other is after (II) the disposal point of El-Sail drain. The physicochemical parameters of water (pH, electric conductivity, total dissolved solids, dissolved oxygen, biological and chemical oxygen demands, nitrite, nitrate and ammonia) were determined. Heavy metals (Cu, Pb, Cd and Ni) concentrations in water and fish tissues (gills, muscles, liver and gonads) were detected. The microbiological, parasitological and pathological conditions of fish were also investigated. Higher values of pH, EC, BOD and COD were detected in site II than from site I. In contrast to DO, nitrite, nitrate and ammonia which were lower in site II. Heavy metals concentrations in water of both sites, especially Ni, Pb and Cd exceeded the permissible limits and its abundance followed the order: Pb>Ni>Cd>Cu. Total bacterial count, total coliform, Salmonella sp., Shigella sp. and E. coli were detected in higher numbers in water samples from site II. Moreover, the fish caught from that site revealed higher bacterial and parasitic infection. The bioaccumulation of Ni and Pb exceeded the maximum permissible limit; however, Cu and Cd concentrations were below the permissible limit in different tissues. The bioaccumulation factor of Cu showed its highest value in liver. The histopathological lesions were more prominent in fish collected from site II. So, consuming fish caught from the studied sites around El-sail drain disposal point represents serious hazard on human health.

Keywords
El-Sail drain; Waste water; Oreochromis niloticus; River nile; Aswan
Introduction
River Nile is the main source of fresh water in Egypt. It receives a lot of pollutants. Nevertheless, the river is still able to recover in virtually all the locations, with very little exceptions [1].
El Sail drain (Kima drain) is considered as one of the major sources of pollution of the River Nile at Aswan governorate. It is used for the disposal of either treated or not sewage wastewater, household solid waste, as well as industrial wastewater (Kima factory). These pollutants can elevate some water parameters, increase levels of BOD and COD in River Nile water and also increase the incidence of pathogenic bacteria, toxic organic compounds and heavy metals; El-Sail drain discharges high amount of organic matter estimated by 10.1 tons/day COD, 3.2 tons/day BOD, 0.03 tons/day heavy metals and 3.25 E+04 MPN/100 ml faecal Coliform bacteria [2]. Kima drain wastewaters exhibit high concentrations of dissolved salts, particularly close to where the waste of the Kima factory enters and decrease substantially near the end of the Kima drain [3].
Heavy metals are the most-active polluting substances; they affect the quality of the environment, with its long-term impact on living organisms. Determination of trace metals concentration in natural water system has received increasing attention for monitoring the environmental pollution, due to the fact that some metals are not biodegradable and accumulated in different organs of animals and human [4]. They can cause serious impairment to circulatory, metabolic, physiological and even structural systems when high concentrations are present in aquatic ecosystems [5].
Unfavorable environmental conditions are the main contributors to stress phenomenon that languish fish immunity and opens the pathway to pathogens and parasites [6]. Many previous studies have stated the presence of Salmonella, Shigella and Escherchia coli in fish harvested from water polluted with human and animal wastes. Also, representatives of family Enterobacteriaceae are usually found among the most prevalent bacteria on the fresh water fish. These microorganisms adsorbed on the surfaces of fish and may be found also in their intestinal contents [7].
Fish frequently serve as intermediate or transport host for larval parasites of many animals, including humans. Most of fish parasites are believed to cause little or no harm to their host under the natural environmental conditions. However, their mere presence often renders fish undesirable by consumers [8]. While; severe parasitic infection is becoming a threat for fish health management and production throughout the world. It cause decrease in growth rate, weight loss, spread human and animal diseases, postpone sexual maturity of fish and increase fish mortalities [9]. Moreover, the parasitic infection in fish may be detrimental to the fish industry because it lowers the quality, quantity and the economic value of fish [10].
Fish can be used as a monitoring tool for the quality of the aquatic environment and fish histopathology, with a broad range of causes, is increasingly being used as indicator of environmental stress since it provides a definitive biological end-point of historical exposure [11]. As well as histopathology can be used as indicators for the effects of various anthropogenic pollutants on organisms and are a reflection of the overall health of the entire population in the ecosystem [12].
Although, Aswan is fortunate in having good quality fresh water from River Nile comparing to the upward Cairo Province, but it receives domestic, agriculture and industrial wastewaters from different drains. Therefore, this work was planned to determine the effect of El-Sail drain discharge on River Nile water at Aswan and its impact on heavy metals concentrations, microbial and parasitological infection and also the histopathological alterations in different tissues of Oreochromis niloticus fish.
Materials and Methods
The experimental area
The studied area is selected to be under the influence of El-Sail drain effluent at Aswan city during autumn 2013. Water and fish samples were collected from two sites of the River Nile which were 141 m before the disposal point of El-Sail drain (site I, N:24° 06` 54.94"; E: 32° 53` 54.74") and 245 m after the disposal point (site II, N:24º 07` 05.14"; E: 32° 53` 50.33") as shown in Figure 1. Water samples were collected from the surface water, (ca. <1 m ashore), in sterile brown bottles (200 ml capacity) for microbiological analysis, and in one liter polyethylene bottle for chemical analysis. Samples of Nile Tilapia (Oreochromis niloticus) were collected from each site (80 fish/site). Fish were transposed a live back after catching to the laboratory for subsequent analysis. Means of fish total lengths and total weights were 16.98 ± 1.94 cm and 80.15 ± 16.05 g respectively.
Physicochemical analysis of water
Water samples have been subjected to various analyses including pH value, electric conductivity (EC) and total dissolved solids (TDS) by using portable devices (pH meter model HI 8314 and digital conductivity meter HI2300 Hanna Ins. Romania). Dissolved oxygen (DO) was measured using the modified Winkler method and biological oxygen demand (BOD) with the five-day incubation method [13]. Chemical oxygen demand (COD) was carried out using the potassium permanganate method [14]. Colorimetric methods were used to determine ammonia and nitrite [13] and nitrate [15].
Heavy metals analysis
Heavy metals (Cd, Pb, Cu and Ni) in water samples and Nile tilapia organs (gills, muscles, liver and gonads) were determined using atomic absorption spectrometry (Perkin-Elmer 3110, USA) with graphite atomizer HGA-600, after using the digestion technique by nitric acid according to the standard methods for examination of water and wastewater [16]. The bioaccumulation factor was estimated according to Authman and Abbas [17] as the following equation: Bioaccumulation factor (BAF)=(Pollutant concentration in fish organ (mg/kg)/Pollutant in water (mg/l)).
Bacteriological analyses
Ten ml of each water sample was subjected to serially dilutions (10-1 to 10-5) with sterile physiological saline (0.85% wt/vol. NaCl) in deionized water. The pour plate technique and the nutrient agar were used for the enumeration of total bacterial counts at both 22°C and 37°C incubation temperatures [13]. For total spore-forming bacteria, water samples and its successive dilutions were pasteurized for 15 min at 80°C, prior to plating in nutrient medium and incubating at 30°C. MPN technique was used for enumerated total and faecal coliforms (using MacConky broth medium) and faecal streptococci (using Azide dextrose broth medium), then incubated at 37°C for 24-48 h [13]. Eosin Methylene Blue (EMB) was used for enumerated Escherichia coli and Violet Red Bile agar (VRB) for enumerated Enterobacterease. As well as Salmonella Shigella agar (SS agar) was used for enumerated Salmonella sp. and Shigella sp. [16].
Also the fish body surface was wiped with 70% ethanol, and parts of gills, skin, dorsal muscles, liver, gut and gonads were taken from each fish. 10 g of each organ was aseptically transferred in to 90 ml of sterilized 0.85% normal saline, homogenized and centrifuged for 2.5 min at 14000 rpm and then allowed to stand for about five min. Tenfold serial dilutions up to 107 were done. Nutrient agar was used for total plate count at 22 Co & 37 Co, EMB for Escherichia coli, VRB for Enterobacterease spp., and SS agar for Salmonella sp., and Shigella sp. [16].
Parasitological examination
Examination of gills and branchial cavity: Branchial cavity was dissected and examined by naked eye for the presence of large cysts. The gill arches were isolated and put in a saline solution; monogenean worms and crustacean parasites were collected and permanently mounted un-stained in glycerol jelly [6].
Examination of muscles: Small snips of muscles (about one gram) were taken from fish samples, compressed between 2 large glass slides (compressorium) and examined under the binocular dissecting microscope for the presence of metacercariae. Isolated metacercariae were withdrawn by fine long tipped pipette into 0.5% saline solution, stained by acetic acid alum carmine stain, mounted and examined under the microscope [18].
Examination of gastrointestinal tract: The alimentary canal of each fish was separated, dissected and divided into small pieces, washed with physiological saline for several times to get rid of mucus and coarse particles that may be adherent to the parasites, then each part was opened and examined in a Petri dish under binocular dissecting microscope, the helminthes were collected by Pasteur pipette, stained with carmine stain, mounted and examined under the microscope [6].
Histopathological examination
After dissecting the fish, gills, skin, liver, spleen and gonads were carefully removed and small pieces were fixed in 10% formalin, dehydrated in ascending grades of alcohol and cleared in xylene. The fixed tissues were embedded in paraffin wax and sectioned at 5 microns. Sections were stained according to Harris Hematoxylin and Eosin method [19], examined microscopically and photographed by using a microscopic camera.
Histopathological examination
Data were statistically analyzed using analysis of variance [20], using the STATISTICA (6.0) computer programs.
Results
Determination of water quality
The physicochemical, heavy metal and microbiological analysis of the water samples collected from two sites in River Nile; after and before El-Sail drain were showed in Table 1.
The physicochemical analysis revealed that, pH value, EC, COD and BOD were significantly higher in site II (8.01, 259 μm/l 2.4 mg/l and 1.06 mg/l) than site I (7.82, 253 μm/l, 1.2 mg/l and 0.83 mg/l), respectively. On the other hand, the lower DO value was recorded at site II (1.92 mg/l) and also the soluble forms of nitrogen; nitrite, nitrate and ammonia showed significant reduction in site II (0.009, 0.097 and 0.043 mg/l) comparing to site I (0.012, 0.128 and 0.091 mg/l), respectively.
In respect to the heavy metal analysis, in general, the abundance of tested heavy metals in water followed the order: Pb>Ni>Cd>Cu. Site II had lower Cd concentration and higher Cu concentration comparing to site I, while, Ni and Pb had convergent concentrations in both studied sites. Ni, Pb and Cd concentrations in water of both sites were exceeded the allowable limits.
The microbiological analysis of water samples showed increase in total bacterial counts in site II than Site I, while there was no difference between the two sites in the spore-forming bacterial count. Coliform bacteria were detected in the two sites with four fold increases in site II than site I, similarly the faecal coliform increased to about five folds in site II than site I. On the other hand, there was no significant difference in fecal Streptococcus count in both sites. The FC: FS ratio was 0.5 in site I and 2.5 for site II. Pathogenic bacteria (Salmonella sp., Shigella sp., and E. coli) were detected in water samples of both sites with higher values in site II than site I.
Concentration of heavy metals in fish tissues
The bioaccumulation of different heavy metals in fish tissues were in the following order, Cu: liver>gonads>gills>muscles; Ni: gills>liver>gonads>muscles; Pb: gills>liver>muscles>gonads and Cd: gonads>muscles>gills>liver. Generally, the highest metal concentrations were recorded in liver and muscles of fish from site II whereas, site I recorded the highest values of metals in gills and gonads. Moreover, the highest bioaccumulation factor (BAF) was that of Cu in liver, followed by Ni and Pb in gills and then Cd in gonads. However, the lowest values of BAF were Cu and Ni in muscles, followed by Pb in gonads and Cd in liver (Table 2).
Microbial load for various organs of Nile tilapia
In general, the highest bacterial load of various fish organs was recorded at site II, followed the order: gut>gills>skin>muscles. The highest TBC recorded in site II (5-2570 cfu×104/g) compared with that of site I (1-124 cfu×104/g). As well as, the highest count recorded in skin and gut and the lowest recorded in gonads. Similarly, the total sporeforming bacteria (TSF) counts showed increase in site II compared to site I; Gonads and gills of fish from site II recorded the highest TSF count comparing with other organs. Enterobacteriaceae spp. ranged 1–2309 cfu×103/g and its number was the highest in gut organs at site II than other organs. E. coli detected in all studied organs in site II and detected only in 33% from the examined organs in site I. E. coli load in site II was >103, the highest count recorded in gills (3218 cfu/g ) and skin (2356 cfu/g). Salmonella sp detected in 50% of tissue samples collected from site I while, it detected in all tissue samples collected from site II at a count of (17-215 cfu/g) in the following order: gills >gut>gonads>skin>liver>muscles. Shigella sp. ranged from 1-131×102 cfu/g, high count showed in skin of fish collected from site II after El- Sail drain disposal point (Table 3).
Parasitic infection
Different types of external and internal fish parasites were recorded in both studied sites with higher prevalence in site II than site I. The monogenean and crustaceans parasites were recorded in gills with percentages of 70% and 30% in site II and I respectively (Figures 2a and 2b). Regarding the internal parasites, Clinostomum sp. trematodes were detected in gills and branchial cavity of fish in lower infestation, 10% in both sites (Figure 2c). While, nematodes and Acanthocephala sp. were detected in the intestine of fishes with high percentage (40%) in site II and with low rate (10%) in site I (Figures 2d and 2e). Also, Diplostomum sp. encysted metacercareae were recorded in higher number in fish from site II (10/gm) than that in site I (3/gm) (Figure 2f).
Parasitic infection
Fish collected from site II showed much greater damage in their histopathological examination compared to site I which revealed mild changes. The skin of fish collected from site I revealed slight accumulation of melanophores in the epidermal layer (Figure 3a), while severe hyperplasia, hypertrophy and excessive accumulation of melanophores were observed in the epidermal layer of fish collected from site II (Figure 4a). Gills exhibited slight congestion in the lamellar and branchial blood vessels in site I fish (Figure 3b), where gills of site II fish showed edema with epithelial lifting and telangiectasis (Figure 4b) and severe lamellar fusion and epithelial hyperplasia with external parasites were shown in between the gill tissues (Figures 4c and d). Liver in site I fish showed moderate vacuolar degeneration with mononuclear inflammatory cells infiltration in between hepatic parenchyma (Figure 3c) accompanied with slight congestion and widening of the hepatic blood (Figure 3d), on the other hand severe degenerative and necrotic changes in the hepatocytes and pancreatic tissues with aggregation of mononuclear inflammatory cells were observed in site II fish (Figure 4e). Spleen of site II fish had hyper activation of melanomacrophage cells (Figure 4f) accompanied with depletion of the lymphocytic tissues and blood vessels hyperplasia (Figure 4g). Ovary of site I fish revealed normal oocytes with different stages of maturation, stromal edema, (Figure 3e), however site II fish ovary seen in ripe stage with histological changes of oocytes as liquefaction of cytoplasm, nucleus loses and degeneration in oocyte wall with liquefaction of the yolk sphere (Figure 4h). Testis of site I fish exhibited degeneration and necrosis leading to decrease in the number of spermatogenic cells in the numerous siminiferous tubules (++) (Figure 3f), but site II fish testis showed severe degeneration in semniferous tubules, associated with decrease in the number of spermatocytes or spermatids (+++) (Figure 4i).
Discussion
River Nile is the donor of life to Egypt; it represents the principle freshwater source that meets nearly all the demands for drinking water and irrigation. In recent years, the problems of pollution in water bodies have become a point of local concern. The water quality released from the High Dam in Aswan shows little degradation and it remains remarkably clean from chemical pollution until it reaches to the Delta [21]. Water quality of the River Nile in Aswan is generally meeting the water quality standards stipulated by the Egyptian governmental law 48/1982 [22]. While there are some polluted drains in Aswan which have very bad effect on the water quality; El Sail (KIMA), El Ganayen and El Berba drains. Many health problems were determined previously especially with the high bacterial counts in that water [3].
The present study indicated that, El Sail drain wastewater caused many changes in water quality of River Nile and consequently affects the fish health. Although physicochemical valuesare within the allowable values stated in the Egyptian law 48/1982 [22] but, there is more or less differences comparing the two studied sites after and before the drain disposal point. In general, El-Sail drain waste water causes oxygen content reduction in site II compared with site I, this may be due to the presence of high load of organic pollutants that consumes the dissolved oxygen during oxidation processes, this result was agree with that revealed by other researchers [23,24]. As well as, the BOD values of water from site II was lower than COD which may reflect the hardly biodegradable of discharge compounds [25]. Also, this study reported a decrease in the soluble nitrogen in water of site II comparing to site I, this may be attributed to the decrease in biological activities of aquatic organisms and nitrification in the water column due to the presence of pollution [26].
Regarding the heavy metal concentration, the present study indicated that the concentration of Cu in the water of two sites (0.26-0.41 mg/l) were lower than the permissible levels (1 mg/l) permitted by the Egyptian Organization for Standardization [27]. On the other hand, a high Pb concentration (2.59–2.63 mg/l) which exceed the Egyptian Standards of the Environmental Laws no. 48/1982 [22] (the maximum Pb concentration in water is 0.05 mg/l), Pb can find its way to the water of the River Nile through the leaching of gasoline from the fishery boats and the tour ships travels from Aswan to Sudan. Ni concentrations of water in the two sites were also exceeded the permissible level of EOS (0.07 mg/l) [27]. The Cd concentrations in the water of tested site (0.13–1.04 mg/l) are higher than the permissible level (0.01 mg/l) recommended by the Egyptian Organization for Standardization. Generally, the increase in heavy metals concentrations at the two studied sites around the drain can be attributed to the huge quantities of sewage and industrial wastes via El-Sail drain. Since metals are regarded as serious pollutants of the aquatic environment because of their environmental persistence and tendency to be concentrated in aquatic organisms; heavy metals in fish tissues can reach concentrations up to 20000 fold higher than its concentrations in the surrounding water environment [28]. The difference in the tendency of different organs to accumulate different metals depends upon the target organ, fish species as well as the metal type [29]. The present results showed that the order of metal concentrations was Pb>Ni>Cu>Cd in the gills and muscles and Cu>Pb>Ni>Cd in the liver and gonads. The present data also showed that liver accumulated higher amounts of Cu and this may be due to its ability to retain and store Cu [30]. Similarly, some previous studies found that Cu exhibited its highest levels in the liver and the lowest values in the muscles [31,32]. The high accumulation of Cu in the liver could be attributed to the specific metabolic processes and enzyme catalyzed reaction involved Cu that taking place in the liver. The sulfur legends in the liver also have a great tendency to coordinate with Cu via oxygen carboxylate amino group nitrogen and/or sulfur of the mercapto group in the metalothionin protein which is in the highest concentration in the liver [33]. The concentrations of Cu in the muscles of the studied fish are still below the permissible level for Cu (30 mg/kg) recommended by the National Health and Medical Research Council [34]. On the other hand, Pb concentrations in all organs of fish are higher than US FDA maximum permissible level (2.0 mg/kg) and its accumulation in the tissues was in the following order: gills>liver>muscles>gonads. This high Pb accumulation was in agreement with its high concentration in water due to the gasoline pollution caused by boat traffic in this location. The concentrations of Cd in all fish organs are still below the WHO permissible level (2.0 mg/ kg) [35].
Regarding the bacterial load in water, the present study indicated an increase in bacterial count in water from site II compared to site I. This may be attributed probably to the sewage disposal from El-Sail drain which spills its untreated waste water directly to river near this site. The ratio of TBC at 22ºC to TBC at 37ºC not exceeded 10 in both two sites, which explain the high pollution from El-Sail drain wastewater. The bacterial load in water increases by increasing the water temperature and the organic matter [36]. The total bacterial count of skin and gut of fish caught from site II was exceeded the permitted limit. Also, the total spore-forming bacteria, Enterobacteriacae, E. coli, Salmonella sp. and Shigella sp. was detected in all fish organs and it was the lowest in fish muscles from the two sites comparing with other organs. Microorganisms adsorbed on the surfaces of the fish and that found in their intestinal contents; They do not affect the fish during life but after death saprophytic and commensally residents invade the flesh and bring about its decomposition [37], as well as it can induce disease to humans when handling and consuming such flesh. The recommended total count of bacteria is106 per gram as a maximum permitted limit for fish, and the fish must be free from Salmonella sp. and Shigella sp. as documented by EGASQC [38]. Faecal coliform bacteria, a subgroup of the total coliform population, had a direct correlation with faecal contamination from warm-blooded animals. This results were emphasizes on a large amounts of sewage disposal directly in the River Nile without any treatments. Fish and fish products have long been considered a vehicle of food-borne bacterial and parasitic infections leading to human illness. Further research is needed to elucidate the behavior of bacterial contaminants in tilapia fish as well as in River Nile ecosystem.
Parasites are attracting increasing interest from parasite ecologists as potential bioindicators of environmental quality due to the variety of ways in which they respond to anthropogenic pollution [39]. Certain parasites can provide valuable information about the chemical state of their environment not only through their presence or absence but also through their ability to concentrate environmental toxins within their tissues in much more concentrations than found in their host. So the present study revealed that the higher percentage of parasitic infestation in site II may be attributed to the water pollution with heavy metals. This is in accordance with other studies which indicated that heavy metal pollution affecting the prevalence of internal parasitic diseases in cultured fishes [40]. In addition to the higher bacterial infection in that site which open the way to the second parasitic infection through weaken the fish and lower its immunity [6].
Concerning the histopathological examinations, fish collected from site II revealed much higher incidence of pathological alterations. The effect of sewage in the present study can be detected using an analysis of pathologies, where the pathological alterations in fish are the net result of adverse biochemical and physiological changes within the organism. Histopathologies are clear symptoms of in situ exposure to pollution in the form of structural alterations [41]. Also such pathological changes in histological structure can substantially impair the function of tissues and organs in fish [42]. A practical advantage of using fish histopathology in environmental assessments is that multiple organs can be examined; this increases the sensitivity at which pollution impacts can be detected. Fish exposed to sewage have more frequent and severe epitheliocystis [43]. Many toxicants (e.g. hydrocarbons, organochlorines and ammonia) cause a wide range of gill pathologies that include telangiectasis and lamellar fusion, epithelial hyperplasia, hypertrophy of chloride and mucus cells, and hyperplasia, as well as higher infestation rates with ectoparasites [44]. The liver is particularly susceptible to damage from a variety of toxicants; it is the major storage site of lipids in fish, liver metabolism is a potential target for the toxic action of chemicals [45]. Fish exposed to contaminated sediments are frequently affected by liver and kidney damage [44,46]. Structural abnormalities can result in the suppression or inhibition of physiological function, irrespective of whether the pathologies are caused by chemical, physical or secondary parasitic irritation. In this study, fish exhibited multifocal pathologies in the gills, liver, kidney, spleen and skeletal muscle tissue, indicating sublethal changes and potential reduction in the functional efficiency of these organs and hence affect the fish health.
Conclusion
This study provides evidence that of water quality and fish health is poorer in the area after disposal point (site II), affected by El-sail drain wastewater which spill the untreated sewage directly to the River Nile at Aswan. Thus, great efforts and cooperation between different authorities are needed to protect the River Nile from pollution and reduce environmental risk at this area which may achieved by treatment of industrial and sewage discharge and regular evaluation of pollutants.
 
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