Hazardous Effects of Heavy Metal Pollution on Nile Tilapia in the aquatic ecosystem of the Eastern Delta in Egypt

doi:10.21203/rs.3.rs-4983142/v1

Introduction: Heavy metal pollution threatens the biodiversity and ecological equilibrium of the Nile River. This study investigates the impact of heavy metal pollution on aquatic animal as Nile tilapia (Oreochromis niloticus) in the Damietta branch of the River Nile and El-Rayah El-Tawfeeky in Benha city in Egypt.

Methods:Fish and water samples were subsequently analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-OES) revealing significantly higher concentrations of Mg, Cd, Hg, Cr, Cu, Ni, Pb, and Zn in fish muscle tissues collected from Damietta branch compared to El-Rayah El-Tawfeeky samples.

Results: Histopathological examinations revealed noteworthy alterations in tilapia gill, liver, spleen, and muscle tissues, suggesting potential health risks. Gene expression analysis using Real time polymerase chain reaction (RT-qPCR indicated) significant changes in genes related to muscle growth (MyoD, IGF-1) and immune response (TNFa, IL6) in fish from Damietta branch relative to fish of El-Rayah El-Tawfeeky.

Conclusion: The findings raise concerns about heavy metals bioaccumulation and potential health implications for consumers. The study underscores the significance of continuous monitoring, utilizing chemical, histopathological, and molecular tools as bioindicators for environmental protection measures against aquatic pollution.

heavy metals

gene expression

River Nile

Oreochromis niloticus

The increased heavy metals pollution is alarming and threatens the worldwide aquatic ecosystem (Hama Aziz et al., 2023). Heavy metals can come to the water from a variety of sources including idol immersion, dumping hospital, emptying of sewers, recreational activities, etc. However, the natural sources of heavy metals are through ore-bearing rocks, forest fires, vegetation, and windblown dust (Hama Aziz et al., 2023). The River Nile is the main resource of water along Egypt. After Cairo the Nile tracks the westnorth direction and then bifurcates into two main branches at El-kanater El-khayriya. The two branches are the Damietta branch and Rossetta branch that enclose the Delta in between. There is another canal called El-Rayah El-Twfeeky that was constructed in 1889 which starts from the Damietta branch at El-kanater El-khayrya as described in (Fig. 1). The Nile River and its branches face serious ecological challenges due to water pollution (El-Saadani et al., 2022).

Numerous discharges from point and non-point sources have contaminated the Nile River (AbouelFadl et al., 2016). According to (Authman et al., 2013), The biodiversity of aquatic fish can be adversely affected by metal pollution in the Nile River and its branches, which can alter the natural equilibrium of the river environment. Although the heavy metals Cu, Co, Fe, and Zn are essential micronutrients for living things, excessive concentrations of them can be harmful. Cr, Pb, Hg, As, and Cd heavy metals are microelements that are carcinogenic and does not have any beneficial biological impact on living animals (Sadak, 2023). Even at lower concentrations, cadmium, lead, tin, and chromium show significant toxicity (Jaishankar et al., 2014). Heavy metals are known to be non-biodegradable and to be deposited, integrated, and bioaccumulated in aquatic habitats, which in turn affects aquatic creatures (Briffa et al., 2020). The bioaccumulation level of heavy metals in fish tissues is affected by biotic and abiotic factors, such as water temperature, pH of water, concentration of dissolved oxygen, fish biological habitat, body mass, and physiologic conditions (Castro-González & Méndez-Armenta, 2008). Fish health and physiological processes are negatively affected by the bioaccumulation of heavy metals in fish (Malik & Maurya, 2014). The type of fish, the concentration of the metals, and the length of exposure all have a substantial impact on the degree of metal toxicity that may be carcinogenic, teratogenic, and/or mutagenic (Ngo et al., 2011). Heavy metals have adverse effects on the nervous system of the fish (Jamil Emon et al., 2023). Fish exposed to heavy metal pollution have decreased gonadosomatic indexes (GSI), fecundities, fertilization rates, and hatching rates, which all have an adverse effect on fish growth and reproductive activity (Gárriz et al., 2019). Therefore, heavy metals may have a negative impact on several metabolic processes in developing embryos, which may lead to morphological and functional abnormalities, developmental retardation, or even death in the most vulnerable cases. Furthermore, heavy metals trigger energy-intensive detoxification procedures, which means that less energy may be used for growth in inebriated fish (Sfakianakis et al., 2015).

The heavy metals in the aquatic ecosystem can enter the food chain beginning from the fish gills, digestive system, and skin. Then most of them were distributed into the fish body through the blood stream until reach the fillet that can be consumed by human (Jamil Emon et al., 2023). The nutritional value of fish to human is its high-quality protein and inclusion of 2 types of Omega-3 unsaturated fatty acid (Saini et al., 2021). Omega-3 can make protection from different heart disease, thrombosis, reduction of blood clotting (Rodriguez et al., 2022). However, the presence of heavy metal in fish fillet can adverse the benefits of omega-3 in fish and its beneficial effect on heart health (Downer et al., 2017). In addition, heavy metals can be accumulated in food chain and have a major negative impact on human health, including cancer, by increasing their biomagnification over time (Mitra et al., 2022).

The native Egyptian species The Nile tilapia (Oreochromis niloticus), that has expanded worldwide, mostly due to its value as it is easy to raise and reproduce in a variety of fish culture methods (Authman et al., 2012). O. niloticus is a widespread species of freshwater fish utilized in toxicological investigations, primary field research, and laboratory research (Abidemi-Iromini et al., 2022). To ensure that a sustainable ecosystem continues to function well into the future, it is crucial to monitor the concentration of heavy metals and evaluate the degrees of contamination in the aquatic system. Monitoring heavy metal pollution in aquatic environments using fish tissues facilitates evaluating aquatic ecosystem’s quality. Fish tissue contamination can serve as a crucial early warning indicator of sediment pollution and/or related water quality issues (Authman et al., 2015). Fish tissue contamination can be evaluated for pollution's effects on fish, metal concentrations in fish that are dangerous for human consumption can be found, and appropriate action can be taken for the preservation of the environment, public health, and socioeconomic reasons (Panda et al., 2023).

The goal of the current study is to assess the levels of heavy metals in the muscle, liver, spleen, and gill tissues of Nile tilapia (Oreochromis niloticus) fish as well as in water samples taken from the same Damitta branch study locations in Benha City. determining the level of pollution, evaluating the use of tilapia fish as a bioindicator for heavy metal pollution, and creating a model for environmental safety in locations with comparable pollution.

Field study and sample collection

Fish (Oreochromis niloticus) and water samples were collected from Damietta branch of River Nile and the El-Rayah El-Twfeeky, in Benha city as shown in Fig. 1. Samples of water were taken between 10 and 20 cm below the water's surface. Using a drag net and assistance from local fisherman, 120 (60/site) adult fish total with average body lengths of 18.19 ± 0.46, 16.51 ± 0.35 cm and average body weights of 68.85 ± 3.26, 62.54 ± 2.45 g were caught from the two study sites. The animals were handled and collected according to the guidelines of the Animal Ethics Committee of Faculty of Science, Benha University, Egypt, approval (BUFS-REC-2024-114Zoo). An overdose of MS222 (Syndel USA, Ferndale, WA) was used to euthanize fish samples. Samples of muscle were taken in liquid nitrogen and immediately kept at -80°C.

Measuring heavy metals in in water and muscle samples

Inductively coupled plasma optical emission spectrometry (ICP-OES) (Perkin Elmer, Optima 4100DV) was used (Didukh-Shadrina et al., 2019) to examine the muscle tissue and water samples after they had been treated with nitric acid and filtered through a glass filter (0.45 ml pore diameter) in accordance with the procedure outlined in APHA (2005). For the muscle samples, they were prepared for measuring heavy metals according to FAO (1993). Using deionized water, serial dilution of 5, 10, 15, 20, 25, and 30 mg/L were prepared from a stock solution of 1000 mg/L of Aluminum, Arsenic, Cadmium, Cobalt, Chromium, Copper, Nickel, Lead & Strontium. To create the Standard curve, these standards were measured using ICP. Subsequently, WINLAB32 software measured the samples' concentration using this curve. The results were expressed in terms of micrograms per kilogram dry weight.

Human risk assessment

The evaluation of fish exposed to the reported doses of heavy metals was done by calculating the average daily human consumption dose (ADD, mg/kg/day) (Khallaf et al., 2018) of specific each metal according to the following equation : \(\:\text{A}\text{D}\text{D}=\frac{\text{C}\:\times\:\text{I}\text{R}\:\times\:\text{E}\text{F}\:\times\:\text{E}\text{D}}{\text{B}\text{W}\:\times\:\text{A}\text{T}}\)

where IR is the average intake rate, which is 0.1424 kg day − 1 for habitual fish eaters and 0.0312 kg day − 1 for normal people, and C is the concentration of heavy metals in fish muscle (mg kg − 1). The factors denoted by EF, ED, BW, and AT stand for exposure frequency (365 days per year), exposure duration (70 years), body weight (70 kg for average individuals), and mean lifespan (70 years × 365 days per year).

Hazard index (HI) was calculate for risk assessment, according to the equation presented by (Omar et al., 2013) using the RfD (the heavy metals' oral reference dose, expressed in mg/kg per day) that is the upper limit of human metal consumption with average body weight of 70 kg. The upper limits for the studied heavy metals Mg, Cd, Hg, Cr, Cu, Ni, Pb, Zn are 0.06, 0.001, 0.006, 2.5, 0.14, 0.012, 0.025, and 0.214 according to ("WHO Guidelines Approved by the Guidelines Review Committee," 2022):

Hazard index (HI) = ADD/oral RfD

It was reported risk effects to human, when the HI is ≥ 1.0 (Omar et al., 2013).

Histological studies analysis

Samples of small muscle, gill, liver, and spleen tissue (10/site) were taken and preserved immediately in 10% neutral buffered formalin. Hematoxylin and eosin (H&E stain) was applied to sections that had been cut into 5 µm thickness using the technique outlined by Bancroft and Gamble (2008).

RNA extraction and investigation of gene expression

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from each fish (eight fish per location, randomly chosen) and reverse transcribed in accordance with the manufacturer's instructions of the reverse transcriptase kit (Shaalan & Iskandar, 2022). Real-time quantitative PCR (RT-PCR) was performed using SYBR green, and the fold change of the target genes between the two studied sites was determined by using the 2^-ΔΔCT method with B-actin acting as the internal control (Shaalan et al., 2019). The primers were designed using primer3 software as listed in table1. The data was presented as mean ± standard deviation (SD). Microsoft Excel was used to compute and report the fold change that represents the gene expression and the significance difference between the groups were calculated using t.test (Rieu & Powers, 2009).

Heavy metals level in water and tissue samples

The concentration of Mg, Cd, Hg, Cr, Cu, Ni, Pb, and Zn in muscle tissue were significantly increased in El-Ryah El-Tawfeek River samples compared to the River Nile samples (p < 0.05) and compared to the reference’s doses given by (Edition, 2011) as presented in Table 2. The concentration of the measured metals are Zn, Mg, Cr, Cu, Pb, Ni, Hg, and Cd from high to low concentration. The concentrations of heavy metals in the Dammietta River fish were significantly increased compared to the reference published doses. The habitual fish eater from the Damietta River branch showed prediction for risk effect from Ni, Mg, and Pb metals. While the normal fish eater from the same site does not show any risk effects. On the other hand, habitual fish eaters were at risk from all the tested metals except Cu and Cr measured in fish muscle collected from El-Rayah El-Tawfeeky River. While the Normal fish eaters are in risk from the exposure to cd and Ni metals. All water samples collected the two studied sites were significant increase compared to the reference dose reported by (Edition, 2011). However, there is a significant increase in Mg, Cr, Cu, and Zn metals (Table 3) for water samples collected from El-Rayah El-Tawfeeky River compared to these collected from The Damietta River Nile (p < 0.05).

Histopathological analysis of gill, liver, spleen, and muscle in Damietta River Nile

Some histopathological changes were recognized in tilapia fish collected from the Damietta River Nile. The microscopic examination of the gills of Nile tilapia revealed mild histopathological changes where the secondary lamellae of these gills were covered by thin layer of single epithelium. The gill filaments were covered by stratified epithelium with aggregation of few mononuclear cells in some of these filaments (Fig. 2A). Partial fusion of some secondary lamellae and aggregation of some inflammatory cells in gill filaments, mainly lymphocytes were also seen (Fig. 2B). Focal areas of hepatocellular degenerative changes in the form of hydropic and vacuolar degeneration of hepatocytes with focal aggregation of few lymphocytes in-between hepatocytes and activation of kupffer cells were the main detectable microscopic changes in the examined Tilapia liver (Fig. 2C). However, aggregation of some melanomacrophages within the hepatopancreas was also recorded (Fig. 2D). The microscopic examination of the spleen showed normal cyto-architecture of the spleen. The examined spleen was covered by a thin capsule and contained red and white pulps with ellipsoids and melanomacrophage centers which formed from aggregation of macrophages and melanin pigments scattered throughout the splenic parenchyma (Fig. 2F). The examined muscles tissue of tilapia showed normal histological structure of these muscles where unbranched and elongated muscle fibers with peripheral flattened nuclei and loose collagenous tissue in-between these muscle fibers were found (Fig. 2E).

Histopathological analysis of gill, liver, spleen, and muscle in El-Rayah El-Tawfeeky

The examined gill of tilapia collected from El-Rayah El-Tawfeeky revealed severe histopathological changes. Mononuclear inflammatory cellular aggregation in the gill filaments with activation of mucous cells were prevalent lesions particularly at the tip of these filaments (Fig. 3A). Fusion of secondary lamellae with extensive inflammatory cellular infiltration of gill filament was also prominent (Fig. 3B). In addition, focal areas of desquamation and necrosis of gill lamellar epithelium were detected (Fig. 3C). Moreover, congestion with aggregation of mononuclear cells and eosinophilic granule cells (EGCs) in the gill arch with proliferation of mucous cells were also recognized (Fig. 3D). Multiple areas of necrosis and degeneration of the hepatocytes with perivascular lymphocytic cellular aggregation were scattered throughout the examined liver of tilapia (Fig. 3E). Mononuclear infiltration of the hepatopancreas and hyperplasia of the bile ductal epithelium were detected (Fig. 3F& G). Moreover, vacuolation of the pancreatic acini was also noticed in the hepatopancreas (Fig. 3H). The examined spleen revealed thickening of the splenic capsule due to fibrous connective tissue proliferation with severe depletion of the splenic hematopoietic tissue and necrosis of the ellipsoidal sheaths (Fig. 3J). In addition, destruction of melanomacrophage centers was prevalent in the examined spleen (Fig. 3K). Moreover, perivascular edema and aggregation of melanomacrophage cells around splenic blood vessels were also recorded (Fig. 3L). Vacuolation and even cavitation of the muscles were the main microscopic changes recorded in the skeletal muscles of tilapia (Fig. 3I)

Relative gene expression of MyoD, IGF-1, TNFa, and IL6

The Expression of some genes that have a critical role in muscle growth and immunity have been measured by RT-qPCR using B-actin gene as a reference gene for normalization. The results of gene expression of MyoD in muscle tissue showed a significant decrease in its expression in the samples of El- Rayah El-Tawfeky relative to that of the Damietta River Nile samples (p ≤ 0.05). In addition to reporting a significant decrease in IGF-1 gene in muscle tissue of the fish from El-Rayah El-Tawfeky relative to the River Nile fish as in (Fig. 4). on studying the gene expression of TNFa and IL6 genes in spleen tissue. It was recorded a significant increase in TNFa gene by 25.69-folds in fish from El-Rayah El-Tawfeeky relative to that from Damietta River Nile as showed in (Fig. 5). IL6 gene was revealed a significant increase by 5.22 folds in fish samples collected from El-Rayah El-Tawffeky relative to that from Damietta River Nile (p ≤ 0.05).

A variety of sources, including the Earth's crust, sewage spills, agricultural practices, and oil extraction, can introduce heavy metals into the aquatic ecosystem. Metals that have entered sediment are accessible to the ecology because they haven't completely dissolved from their solid phase (Yozukmaz & Yabanlı, 2023). Because they are bioavailable, dissolved metals can build up in aquatic life. Subsequently, The fish live in this ecosystem may absorb heavy metals through their gills, gut, or food chain (Rajeshkumar & Li, 2018). The present study reported different concentration of Mg, Cd, Hg, Cr, Cu, Ni, Pb, and Zn metals in water samples collected from El-Rayah El-Tawfeeky River and the Damietta River Nile. The large concentrations of pollution in water systems are caused by the disposal of dead animals and the dumping of sewage into the canal (El-Kowrany et al., 2016). This finding was also reported in Alexandria (Haidar et al., 2006). Previous studies revealed that most sites of River specially when their was low flow seasons have higher concentrations of Mn, Ni, and Cd (Al-Afify & Abdel-Satar, 2020). It was measured high concentrations of Zn, Mg, Cr, Cu, Pb, Ni, Hg, and Cd metals in fish muscles of El-Rayah El-Tawfeeky River compared to the Damietta River Nile branch. Rivers get silt from a variety of point and diffuse sources, which settles at the bottom and may be a source of metal accumulation in the aquatic food chain due to the biomagnification process (Debnath et al., 2021).

In the current study, it was recorded for Zn to be in highest concentration in fish muscle. The same result was reported for fish collected from Southern Caspian Sea, Iran (Tabari et al., 2010). previously It was reported by (Ahmad et al., 2015) that fish species from Rabul River in Pakistan have a concentration of chromium 489–703 mg/kg. In addition, fish species from Calicut in India reported 0.74µg/g of chromium (Sankar et al., 2006). It was measured by (Sankar et al., 2006) that Mn has concentration of 0.5 mg/kg in marine fish species from Kochi Waters. Several fish species collected from Indian waters recorded 2.9 µg/g Mn concentration (Kumar et al., 2011). It was reported that fish collected from Kabul River of Pakistan has 75–135 µg/g of Nickel (Ahmad et al., 2015), while it was measured from those collected from Iskenderum Bay of Turky to have 0.11–12.9 µg/g (Türkmen et al., 2005). In the present study, the concentrations of copper in Tilapia muscle were 54.4 and 38.9 mg/kg in fish from El-Rayah El-Tawfeeky and Damietta River branch, respectively. El Rayah- el tawfeeky reported higher concentration of copper more than that was reported in fish collected from Bangshi River (8.33–43.18 mg/kg) in Bangladesh (Rahman et al., 2012). Previous study in Peral River Delta, China reported (0.02–0.06 µg/g) cadmium (Leung et al., 2014), while it was 0.09–0.89 mg/kg in Bangshi River, Bangladesh (Velusamy et al., 2014). It was recorded a high concentration of Hg in some fish species of Red see (Younis et al., 2021). Lead concentration was reported to be 0.22–0.85 mg/kg in middle Black sea Turky (Tuzen, 2009) and different species from Red sea (Younis et al., 2021). Unusually large metal inputs into the aquatic environment have damaged commercial fisheries, caused significant financial losses, and in certain circumstances, posed health risks to humans (Banerjee, 2003).It was reported that the fish collected from El-Rayah El-Tawfeky River Showed risk effects, from all measured metals to human how mostly have the fish in their meal. Researchers gave a great attention to the heavy metal in aquatic environments due to its great concerns about their accumulation and toxic effects in aquatic organisms and human through the food chain (Tchounwou et al., 2012).

Histopathological study

It was reported that some histopathological changes in tilapia fish collected from the Damietta River Nile (DRN) compared to fish collected from El-Rayah El-Tawfeeky branch (RTB). Gills are the first target for pollution, being in close contact with the surrounding environment and the primary site for heavy metal absorption. Therefore, the histopathological alterations of gills were generally attributed to the toxic effects of heavy metals. It was reported previously that fish exposed to metals have also been observed to exhibit similar gill changes (Ayoola & Alajabo, 2012; Ayoola & Alajabo). It has been demonstrated that the histology of the gills reflects various environmental circumstances for fish and is susceptible to copper exposure (Ayoola & Alajabo, 2012). In the present study there was a fusion of secondary lamellae with extensive inflammatory cellular infiltration of gill filament. The same results were reported for grass carp exposed to heavy metals (Shah et al., 2020).

It was observed hepatocellular degeneration and activation of Kupffer cells in the tested DNR tilapia liver. Moreover, necrosis, degeneration of hepatocytes, and hyperplasia of bile ductal epithelium were observed in RTB. Melanomacrophages, hypertrophy, and biological disarray were noted in the fish treated with various aluminum doses by (Alibraheemi, 2019). A high concentration of pollutants that cause hepatocyte loss could be the cause of the necrosis seen in the centrilobular zone (Rajamanickam & Narayanan, 2009). The examined spleen of tilapia fish collected from RTB revealed thickening of splenic capsule, decrease in splenic hematopiotic tissue, necrosis, and edema. The observed data is consistent with the research conducted by (David & Kartheek, 2015), which examined immune suppression in the rainbow trout splenic region after exposure to various chemical toxicant concentrations. Given certain physiological and histological circumstances, might result in changes to the spleen (Garcia-Abiado et al., 2004; Gogal et al., 1999). It was observed vacuolation and cavitation of the muscles of tilapia collected from the Damietta River branch. Lates calcarifer exposed to copper had thickening and separation of muscular bundles with significant oedema (Maharajan et al., 2016). It was previously reported for tilapia fish exposed to heavy elements and microbes contaminants to have degeneration, necrosis, atrophy, and disintegration of muscular bundles in addition to enlargement of the muscle fiber (Dawood et al., 2023).

Gene expression analysis

Understanding the extent of harm is a crucial component of ecotoxicological study, and tissue specific gene expression is considered as biomarker which indicates the kind, degree, and condition of alterations that need to be thoroughly investigated. Numerous researchers have also carried out investigations using tissue-specific expression levels (Kessabi et al., 2023). Different stressors was studied to affect muscle growth like that was for reported for tilapia (Shaalan et al., 2023). After the liver and spleen, muscle exhibits a notable increase in gene expression due to the bioaccumulative nature of heavy metals. The present study revealed a significant decrease in MyoD and IGF-1 gene expression in Tilapia muscle of RNB compared to that from DRN. The myogenic determining factor (MyoD) gene is one of the muscle specific transcription factors that control the development of the muscle, proliferation, myofibril formation (Zammit, 2017). It was reported that the fish MyoD gene was negatively affected by different stressors. It was revealed the low expression of myoD gene in gibel carp fish which was exposed to 168 h of acute thermal stress (Hu et al., 2023). MyoD and Myf5 are two examples of the muscle-specific genes whose transcription is modulated by Wnt signaling during myogenesis via PKA and the transcription factor CREB (Chen et al., 2005). It was reported previously that Wnt signaling pathways are vulnerable to heavy metal exposure in the environment and are essential for regular cellular processes (Khalid et al., 2021). Previous study presented the effects of zinc and cobalt exposure on rainbow trout's IGF-I, IGF-II, and GH expression levels. It was reported that the exposure of rainbow trout for time to zinc and cobalt resulted in significantly decrease of the expressions of IGF-1 gene and that may be due to the interactions between these metals and metal binding proteins (Ekinci et al., 2011). On the other hand, the results showed a significant increase in TNFa and IL6 gene expression in Tilapia spleen of RNB compared to that from DRN. In Asian carp head kidney, there was a positive correlation found between the expression of mir155 and the mRNA levels of proinflammatory cytokines, such as TNF-α (Jing et al., 2020). It was revealed that the biomolecular response to exposure of D. Setosum to Cd heavy metals demonstrated that TNF-α protein expression, activation, and concentration increased as the concentration of Cd-containing heavy metals increase (Rumahlatu et al., 2019). Oxidative stress is one of the basic chemical mechanisms that underlies toxicity caused by metals (Chen et al., 2018). Certain cellular inflammatory factors such as IL-6 and immunological factors are significantly changed when the immune system is repressed (Jantawongsri et al., 2021). It was measured that the IL-6 was significantly increased in carp spleen exposed to pollutant as Difenoconazole (Liu et al., 2022). This implies that heavy metals may target MyoD gen in muscle as well as TNF-a and IL-6 genes in spleen in order to cause harmful consequences. Our findings suggest that exposure to heavy metals causes immunological system malfunction and muscle atrophy in addition to spleen tissue damage. Tissue damage, immunosuppression brought on by heavy metal exposure, oxidative stress, inflammation, and apoptosis are all deeply interrelated.

In the present study, the use of various biomarkers at different levels for identification of the heavy metal pollution in Tilapia fish and the surrounding water. It gives indication about the health condition of the fish and the risk effect to the consumer. The data provided in this study justifies one of the most harmful kinds of pollution that cannot be denied which must affect the environment. These toxins threaten the aquatic fish and the human population. The levels of bioaccumulation of heavy metals in fish muscle were on the verge of exceeding safety thresholds in the studied sites, raising concerns for the health of consumers. As a result, it is important to regularly check the metal levels in fish species found in Benha Damietta Branch and El-Rayah El-Tawfeeky branch. The best way to protect these sites from ongoing pollution is to keep waste out of the watershed, lower environmental risks, and regularly monitor the riverine ecosystem before metal levels rise too high and endanger aquatic life as well as humans. The study's findings might provide decision-makers with new information on how to better safeguard the River Nile, branches and related canals from potentially dangerous hazards.

Aknowledment

Authot would like to thank anomalous refrees for their valuable comments.

Data Availability

All data supporting the findings of this study are available within the paper.

Funding Information

The authors did not receive support from any organization for the submitted work.

Author Contribution

W. M. S. conducted the experiment, analyzed the data, wrote and revised the manuscript.

Conflict of Interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethical Statement

The experiments were approved by Ethics committee of faculty of Science, Benha university, Egypt, protocol (BUFS-REC-2024-114Zoo).

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Table (1) Primers of RT-PCR

Gene name	Forward primer	Reverse primer	Accession number
MyoD	ACGCATGTACTCCAGTCCAA	CAGTGAACACAGCAGTCGTC	XM_025907525.1
IGF-1	CACCCTCTCACTACTGCTGT	CACAGTACATCTCAAGGCGC	NM_001279503.1
TNFa	CATCTTTCCGGGTTGACTGC	CTGCAGAGTACCAGTTCCCA	XM_025902124.1
IL6	TTTCTTGCTACATGGCCACG	CCTCATGATCTCAAACCGCG	XM_005448195.3
B-actin	TCTCGGCTGTGGTGGTGAA	GACCCACACAGTGCCCATCT	XM_003455949

Table (2) the heavy concentrations in muscle tissue of Oreochromis niloticus in mg/kg muscle weight.

	El Rayah El Tawfeeky			Damietta River Nile
	Concentration (mg/kg)	HI (H.)	HI (N.)	Concentration (mg/kg)	HI (H.)	HI (N.)
Mg	74.533±0.075^*a	2.527	0.553	59.146±0.980^*	2.005	0.439
Cd	2.286±0.115^*a	4.651	1.019	0.270±0.053^*	0.549	0.120
Hg	10.360±0.075^*a	3.512	0.7696	1.500±0.030^*	0.508	0.111
Cr	63.020±0.346^*a	0.051	0.011	38.853±0.449^*	0.031	0.006
Cu	54.406±0.116^*a	0.790	0.173	38.986±0.991^*	0.566	0.124
Ni	23.560±0.019^*a	3.993	0.875	20.823±0.106^*	3.530	0.773
Pb	33.403±2.068^*a	2.718	0.595	14.673±0.723^*	1.193	0.261
Zn	212.086±4.328^*a	2.016	0.441	99.930±0.370^*	0.949	0.208

Table (3) the heavy metals concentrations in water samples from the selected sites in mg/L.

	Max allowable conc. (mg/L)	El-Rayah El-Tawfeeky Water (mg/L)	Damietta River Nile Water (mg/L)
Mg	0.4	0.757±0.004^*a	0.589±0.006^*
Cd	0.00001	0.238±0.008^*	0.241±0.011^*
Hg	0.002	0.153±0.006^*	0.135±0.008^*
Cr	0.05	0.437±0.004^*a	0.381±0.015^*
Cu	1.5	0.545±0.012^*a	0.359±0.025^*
Ni	0.07	0.244±0.004^*	0.222±0.023^*
Pb	0.1	0.152±0.002^*	0.146±0.006^*
Zn	0.01	1.044±0.015^*a	0.975±0.022^*

No competing interests reported.

Hazardous Effects of Heavy Metal Pollution on Nile Tilapia in the aquatic ecosystem of the Eastern Delta in Egypt

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Field study and sample collection

Measuring heavy metals in in water and muscle samples

Human risk assessment

Histological studies analysis

RNA extraction and investigation of gene expression

Results

Heavy metals level in water and tissue samples

Histopathological analysis of gill, liver, spleen, and muscle in Damietta River Nile

Histopathological analysis of gill, liver, spleen, and muscle in El-Rayah El-Tawfeeky

Relative gene expression of MyoD, IGF-1, TNFa, and IL6

Discussion

Histopathological study

Gene expression analysis

Conclusion and recommendations

Declarations

References

Tables

Additional Declarations

Status:

Version 1