Accumulation of heavy metals in tilapia fish species and related histopathological changes in muscles, gills and liver of Oreochromis niloticus occurring in the area of Qahr El-Bahr, Lake Al-Manzalah, Egypt

Abstract

Accumulation of metals (Fe, Zn, Cu, Mn, Pb, Cd and Ni) in the muscles and gills of Oreochromis niloticus, Oreochromis aureus and Tilapia zillii was assessed based on seven locations in the Qahr El-Bahr area in Lake Al-Manzalah. The average accumulation of metals was in the following order: Tilapia zillii > Oreochromis aureus > Oreochromis niloticus. To determine the risk of fish consumption, the estimated daily intake, carcinogenic, non-carcinogenic and relative risk indices were calculated. The results indicate that the intake of individual metals through the consumption of fish is almost safe for human health, whereas the intake of combined metals poses a high potential health risk to consumers. Assessment of carcinogenic risk of Cd and Ni from the consumption of tilapia species indicates that consumers are at risk of cancer. The contribution of Pb and Cd to the overall relative risk index ranged from 34% to 41%. Of the three species studied, Oreochromis niloticus is relatively safe for consumption as it poses the least health hazard, while Tilapia zillii is more predisposed to accumulate metals in its tissues. Histopathological changes were observed in the muscles, gills and liver of Oreochromis niloticus as a result of heavy metal accumulation in these organs.

1Introduction

Lake Al-Manzalah is considered one of the most important outlets for inland fisheries in Egypt and is estimated to account for about 38.02% of the total fish catch from the Nile River Delta lakes. It is considered to be the second major source of fish after Lake El-Burollus. The importance of Lake Al-Manzalah fishery relies on two main targets, i.e. it is a source of protein for human consumption and a provider of employment (El-Bokhty 2010). There are five coastal lakes along the northern coast of Egypt, connected with the Mediterranean. They represent important fishing resources in Egypt. Due to human activity, these lakes are severely environmentally degraded (Abdel-Satar et al. 2017).

Lake Al-Manzalah is considered the largest and most productive lake in Egypt. Due to the increase in agricultural, municipal and industrial wastewater discharge, the fish production and water quality status of the lake have degraded (Ahmed et al. 2006; Goher et al. 2017).

Since the early 20th century, there have been continuous changes in hydrological, chemical and biological characteristics of the lake resulting from increased freshwater supplies associated with municipal and agricultural wastewater disposal (Abdel-Satar & Goher 2009). Lake Al-Manzalah is connected with five drains through its western and southern shores. These drains discharge their effluent into the lake, which affects its water quality (Abdel-Satar 2001, 2008; Ali 2008). Approximately 7500 million cubic meters of untreated industrial, domestic and agricultural drainage waters are discharged into the lake annually through the drains such as Bahr El-Baqer (industrial and domestic wastewater), Ramsis, Hadous, Faraskour and El-Serw (agricultural wastewater; Abu Khatita et al. 2015). The pollutants and wastes discharged from the drains affect the entire area of the lake (El-Naggar et al. 2016).

Several studies have addressed the ecology of Lake Al-Manzalah. These studies covered the lake water quality, hydrological system, geological aspects, benthic invertebrates, phytoplankton composition, bacterial indices, and fishery status of the lake (Yacoub et al. 2005; Abdel-Satar 2008; Abdel-Satar & Goher 2009; El-Refaie 2010; Hamed et al. 2013; Mehanna et al. 2014; Zahran et al. 2015; Orabi & Osman 2015; Hegazy et al. 2016; El-Shafei 2016).

Contamination with metals in aquatic systems has drawn particular attention due to their persistence, toxicity and biological accumulation (Zahran et al. 2015). Heavy metals enter the human body through different paths, such as the food chain, and pose both non-carcinogenic and carcinogenic health risks (Mohanta et al. 2020). Fatal diseases such as renal tumor, nephritis, osteoporosis, cancer, nasopharyngeal congestion, increased blood pressure associated with cardiovascular diseases, and malfunctions of different body systems are caused by heavy metals (Mohanta et al. 2020). Determination of heavy metal levels in environmental biota is an essential process in assessing the human health risk resulting from the presence of these contaminants in food. Pollutants can be categorized as non-carcinogens and carcinogens, and are found mainly in fish (Yu et al. 2014). Risk assessment is one of the fastest processes required to evaluate the impact of hazards on humans. The risks can be divided into non-carcinogenic and carcinogenic effects. The non-carcinogenic risk is based on the Hazard Quotient (HQ), while the carcinogenic risk is based on the Target Cancer Risk (TCR; Markmanuel & Horsfall Jnr. 2016).

Fish are considered one of the most important biomonitors in the aquatic ecosystem for assessing heavy metal pollution (Abou El-Gheit et al. 2012). Furthermore, fish are at the top of the food chain and can accumulate metals that are transferred to humans through consumption of fish, causing acute or chronic diseases (Al-Yousuf et al. 2000).

Accumulation of pollutants disrupts the physiology of fish tissues. The endpoint in assessing the risk of pollutants in the environment is the microscopic examination of target tissues through histopathological parameters (Fatima et al. 2015). Histopathological changes can be used as indicators of the impact of various anthropogenic pollutants on organisms and as a measure of the overall health of the entire aquatic ecosystem (Saad et al. 2011). Harmful effects of pollutants can be manifested in fish tissues before consequential changes in the external appearance and behavior of fish (Mahboob et al. 2020). The exposure of fish living in Lake Al-Manzalah to different types of wastes (industrial, agricultural, and sewage) resulted in several pathological changes in different fish organs (Tayel et al. 2014; Mahmoud & Abd El Rahman 2017).

The objectives of this research were: a) to determine the level of metals in the muscles and gills of Oreochromis niloticus, Oreochromis aureus and Tilapia zillii collected from Qahr El-Bahr in Lake Al-Manzalah, b) to assess the non-carcinogenic and carcinogenic risks associated with the consumption of tilapia fish by humans, and c) to evaluate the histopathological changes in the muscles, gills and liver of Oreochromis niloticus.

2Materials and methods2.1Site description

Lake Al-Manzalah is located between longitudes 31°45′E and 32°22′E and latitudes 31°00′N and 31°35′N (Fig. 1). The lake is bordered by the Mediterranean Sea to the north and northeast, by Dakahlia and Sharkia to the south, by the Suez Canal to the east and by the Nile Branch of Damietta to the west (Hossen & Negm 2016). Three outlets connect Lake Al-Manzalah with the Mediterranean Sea allowing the exchange of water and biota between the lake and the sea: El-Boughdady, El-Gamil, and new El-Gamil (Elewa et al. 2007). The lake is also connected with the Suez Canal at El-Qabouti, a few kilometers south of Port Said, and with the Nile's Damietta Branch by El-Inaniya, El-Ratma and Souffara canals (Sallam & Elsayed 2018).

The area of Qahr El-Bahr, located in the city of Port Said, is a semi-isolated area of Lake Al-Manzalah by the International Ring Road and 30 June road. It has an area of about 12.6 km² and is fed through the El-Qabouti Channel, a narrow opening that connects the seawater with the study area, which is considered one of the deepest part of the lake. Fish samples were collected at seven sites in September 2018. The distribution of heavy metals in the water and sediment of Lake Al-Manzalah is presented in Table 1.

Table 1 Distribution of heavy metals in water and sediment of Lake Al-Manzalah (Goher et al. 2017)

Metal	Lake water		Lake sediment
	Unit	Value	Unit	Value
Fe	μg l−1	216.7–862.4	mg g−1	8.50–15.36
Mn		8.7–34.6	μg g−1	96.4–362.4
Zn		22.2–58.4		15.3–108.3
Cu		4.3–15.0		5.6–19.0
Pb		7.9–74.7		5.7–63.5
Cd		1.0–3.7		1.09–4.10
Ni*		13.99–51.54		10.01–62.73

* after Elmorsi et al. (2015)

2.2Collection of samples

Samples of three tilapia species (Oreochromis niloticus, Oreochromis aureus and Tilapia zillii) were collected at seven sites in the area of Qahr El-Bahr, Lake Al-Manzalah. Every site was represented by 20 fish specimens of each species (Oreochromis niloticus, Oreochromis aureus and Tilapia zillii). The detailed information on the collected fish is listed in Table 2.

Table 2 List of fish species, total length and size of tilapia fishes caught in Qahr El-Bahr, Lake Al-Manzalah

Species	Total length (cm) Mean ± SD	Body weight (g) Mean ± SD
Oreochromis niloticus (O. niloticus)	23.51 ± 1.12	265.2 ± 52.6
Oreochromis aureus (O. aureus)	22.36 ± 0.98	252.4 ± 48.3
Tilapia zillii (T. zillii)	13.11 ± 1.45	95.5 ± 15.6

2.3Analysis of heavy metals

Specimens of muscles and gills of different fish species were dried in an oven at 105°C for about 24 h. The dried specimens were then ground to a fine powder. A representative sample of 1 g dry weight of muscles or gills was taken from fish specimens. The samples were digested according to the method described by Goldberg et al. (1983), during which concentrated nitric and perchloric acid (AR grade) in the 5:5 ratio was used in Teflon beakers on a hot plate at 50°C for about 5 h till complete decomposition of organic matter. The digested solutions were cooled to room temperature, filtered and diluted to a final volume of 50 ml with deionized distilled water. The concentrations of Fe, Pb, Mn, Zn, Cu, Cd and Ni were measured by an Australian GBC atomic absorption reader (model Savant AA-AAS) with a GF 5000 graphite furnace and expressed in mg kg⁻¹ dry weight.

The precision of the analyzed metal values was controlled by including triplicate samples in analytical batches. Standard deviations for mean values of triplicate measurements were up to 5%, which was regarded as an acceptable precision. Practical quantitation limits for the analysis of heavy metals were in the range of 0.02–0.05 μg g⁻¹.

2.4Health risk assessment

In the present study, the muscles of tilapia species were specifically selected for heavy metal analysis because they are the only edible tissue, therefore the level of toxicants present in this tissue is particularly relevant.

2.5Estimated daily intakes (EDIs)

The estimated daily intake (mg kg⁻¹ body weight) for heavy metals was calculated in fish muscle samples using the following formula (Shaheen et al. 2016):

$$\mathit{EDI}=\frac{(\mathit{FIR}\times \mathit{Cm})}{\mathit{BW}}$$EDI = {{(FIR \times Cm)} \over {BW}}where FIR is the food ingestion rate (g person⁻¹ day⁻¹; 57 g in Egypt, FAO 2014), Cm is the metal concentration in fish muscle samples (mg kg⁻¹), and BW is the body weight of an adult assumed to be 70 kg.

2.6Non-carcinogenic risk

The non-carcinogenic risk for each metal in the muscles was assessed by the hazard quotient (HQ) (USEPA 2000) using the following equation:

$$\mathit{HQ}=\left\{\frac{\mathit{EF}\times \mathit{ED}\times \mathit{FIR}\times \mathit{Cm}}{\mathit{RfD}\times \mathit{BW}\times \mathit{AT}}\right\}\times {10}^{-3}$$HQ = \left\{{{{EF \times ED \times FIR \times Cm} \over {RfD \times BW \times AT}}} \right\} \times {10^{- 3}}where EF is the exposure frequency, ED is the lifetime exposure duration (70 years), RfD is the oral reference dose (mg kg⁻¹ day⁻¹), AT is the averaging time for non-carcinogens (365 days year⁻¹ × 70 years) and 10⁻³ is the unit conversion factor. If the HQ is higher than 1, there is a potential health risk (Islam et al. 2017).

The overall potential for non-carcinogenic effects from all studied metals was assessed using the hazard index (HI). The hazard index was expressed as the sum of the hazard quotients (USEPA 2000) using the following equation:

$$\mathit{HI}=\sum _{i=1}^n\mathit{HQi}$$HI = \sum\limits_{i = 1}^n {HQi}

2.7Carcinogenic risk

The carcinogen risk was estimated using the target carcinogenic risk (TCR; USEPA 2000), which was derived from the intake of Pb and Cd using the following equation:

$$\mathit{TCR}=\frac{\mathit{EF}\times \mathit{ED}\times \mathit{FIR}\times \mathit{Cm}\times \mathit{CP}{S}_o}{\mathit{BW}\times \mathit{AT}}\times {10}^{-3}$$TCR = {{EF \times ED \times FIR \times Cm \times CP{S_o}} \over {BW \times AT}} \times {10^{- 3}}where CPS_o is the oral carcinogenic potency slope (mg kg⁻¹ day⁻¹). The CPS_o values for Pb, Cd and Ni were 0.0085 (OEHHA 2009), 0.38 and 1.7 (USEPA 2011), respectively. The US Environmental Protection Agency set acceptable risk levels for carcinogens ranging from 10⁻⁴ to 10⁻⁶ (Atique Ullah et al. 2017; Miri et al. 2017).

2.8Relative risk

The relative risk (RR) of pollutants, defined by Yu et al. (2014), can be helpful in identifying the most harmful metals. The RR was calculated according to the following equation:

$$\mathit{RR}=\frac{\mathit{Cm}}{\mathit{RfD}}$$RR = {{Cm} \over {RfD}}

Human health risk from fish consumption should increase as the relative risk values increase (Yu et al. 2014).

2.9Histological studies

Muscles, gills and liver of the fish Oreochromis niloticus collected from the area of Qahr El-Bahr were carefully removed and immediately fixed in 10% formalin for 48 h, dehydrated in ascending grades of alcohol and cleared in xylene. The fixed tissues were embedded in paraffin wax and cut with an Euromex Holland microtome into 4–6 microns. Sections were stained using the Harris haematoxylin and eosin (H&E) method (Saad et al. 2011). They were then examined under a microscope and photos were taken by a microscope camera.

2.10Statistical analysis

The results were tested for significant differences for metals between different sites and species using one-way ANOVA, and p < 0.05 was considered statistically significant. In addition, the relationships between the analyzed metals were determined by calculating the Pearson correlation index.

3Results and discussion

Salts of heavy metals constitute a very serious form of pollution. They can bioaccumulate in fish tissues and the extent of bioaccumulation depends on species, age and trophic transfer (Islam et al. 2017). It was difficult to compare the contamination with trace metals in all fish species at different sites. Therefore, tilapia species were selected as indicators to account for the degree of pollution by heavy metals in the area of Qahr El-Bahr in Lake Al-Manzalah, where they were successfully obtained from all sampling sites. The concentrations of metals (Fe, Zn, Cu, Mn, Pb, Cd and Ni) in different fish species are listed in Table 3.

Table 3 Metal concentration (mg kg⁻¹) in the muscles and gills of fish species collected from Qahr El-Bahr

Fish species (muscle)		Fe	Zn	Cu	Mn	Pb	Cd	Ni
O. niloticus	RangeMean ± SD	36.4–83.953.3 ± 21.0	28.5–41.535.7 ± 4.8	2.0–4.22.6 ± 0.8	2.9–6.94.4 ± 1.7	1.5–3.32.2 ± 0.7	0.4–1.10.7 ± 0.3	4.39–6.015.51 ± 0.5
O. aureus	RangeMean ± SD	37.9–84.352.1 ± 15.5	30.7–45.637.3 ± 5.4	2.3–6.43.7 ± 1.4	2.9–7.54.9 ± 1.5	1.6–4.83.1 ± 1.2	0.4–1.41.0 ± 0.3	4.68–6.655.54 ± 0.6
T. zillii	RangeMean ± SD	60.9–98.378.1 ± 11.9	39.8–50.145.5 ± 3.7	3.3–4.84.1 ± 0.5	4.7–8.16.1 ± 1.1	2.2–4.93.8 ± 1.1	1.0–1.41.2 ± 0.2	5.52–6.736.05 ± 0.5
Fish species (gills)		Fe	Zn	Cu	Mn	Pb	Cd	Ni
O. niloticus	RangeMean ± SD	119.3–220.0163.5 ± 41.7	55.0–76.665.4 ± 10.2	4.8–7.76.1 ± 1.2	21.3–54.232.3 ± 15.1	3.6–6.04.8 ± 1.1	1.0–1.41.3 ± 0.2	11.6–13.812.5 ± 1.0
O. aureus	RangeMean ± SD	131.5–221.5176.5 ± 36.3	52.6–105.273.4 ± 18.5	5.9–10.17.4 ± 1.6	30.7–61.346.8 ± 13.3	5.5–7.26.3 ± 0.6	1.4–2.11.8 ± 0.3	11.4–14.012.5 ± 1.0
T. zillii	RangeMean ± SD	189.5–291.2220.4 ± 42.7	66.9–105.287.7 ± 15.9	4.7–8.26.0 ± 1.4	35.9–68.949.2 ± 12.5	4.3–7.26.0 ± 1.1	1.4–2.31.8 ± 0.4	12.5–15.914.0 ± 1.2

There were no differences in the order of heavy metal levels in the muscles and gills of the three fish species. Fe accumulated the most in the muscles and gills of the three tilapia species, and was followed by Zn, Ni, Mn, Cu, Pb, and Cd. The levels of essential elements (Fe, Zn, Ni, Mn, Cu) were higher than those of the non-essential ones (Pb, and Cd). The overall concentrations of the studied metals in the muscles and gills of the fish species were in the following descending order: Tilapia zillii > Oreochromis aureus > Oreochromis niloticus, and the metal concentrations showed distinct differences between different species (Elnabris et al. 2013; Islam et al. 2017). The present results indicate significant differences between the distribution of Fe, Zn, Pb, Cu, and Cd in different species (p < 0.05). However, there was no clear spatial variation in the contamination with heavy metals (p > 0.05) for the three species. In addition to species differences, differences in metal concentrations depend on the types of tissues analyzed (Abarshi et al. 2017), with gills containing a higher level of the studied metals compared to the muscles in all tilapia species (Table 3). The tissue of gills in all fish has the ability to accumulate significant levels of metals compared to other tissues and their surface has a negative charge and therefore provides a possible site for positively charged elements (Shovon et al. 2017).

Iron is the most important metal for biological life. It plays a greater biological role than any other heavy metal. Its toxicity causes diarrhea, hemorrhagic gastroenteritis, liver necrosis and leads to death by hepatic coma (Clarke et al. 1981). According to WHO (1989), the permissible limit of Fe concentrations in fish species is about 100 mg kg⁻¹. The concentrations of Fe in tissues of tilapia species are still below this limit and the fish can be considered as uncontaminated (< 100; Table 3).

Zn and Cu are essential micronutrients for all organisms. They are required at considerable levels as constituents of various enzymes in organisms to maintain certain biological functions. Zn was found in high concentrations in samples of some fish species, exceeding the limit (40 mg kg⁻¹) specified by FAO (1983). Zn levels were higher than the permissible limit in 14.2%, 28.4%, and 71.1% of the muscle samples of Oreochromis niloticus, Oreochromis aureus and Tilapia zillii, respectively. The concentration of metals, especially zinc, was more elevated in the gills than in the muscles because the gills are the main entrance for metals into the fish. They are taken up by fish directly from water, especially through mucus and gills (Skidmore 1964). The high concentration of zinc may be due to domestic, sewage and agricultural wastes discharged into the lake through different drains and affecting the entire area of the lake (Abdel-Satar 2008; Abdel-Satar & Geneid 2009; El-Naggar et al. 2016; Elmorsi et al. 2019). FAO (1983) and WHO (1989) proposed a permissible limit of 30 mg kg⁻¹ for Cu concentration. Cu concentrations in all fish muscle samples harvested from the study area were below the recommended level and did not appear to pose a contamination hazard.

Mn deficiency causes reproductive and skeletal abnormalities. Daily intake of small amounts of Mn is recommended for growth and good health of children. However, excess consumption of Mn can lead to neurologic and psychological disorders (Ahmed et al. 2016). Mn concentrations in the muscles of the studied fish were higher than the permissible concentration (1 mg kg⁻¹) recommended by WHO (1989). The increase in Mn levels in fish gills and muscles is related to a large amount of agricultural drainage water entering Lake Al-Manzalah. Our results are consistent with those of Mahmoud & Abd El Rahman (2017) for the fish species Clarias gariepinus and Mugil capito from the same lake. Agrochemicals, such as pesticides and herbicides, release Mn and contribute to its accumulation in fish (Ibrahim & Mahmoud 2005). Fish samples showed a similar accumulation of heavy metals to that observed in another study conducted in Lake Al-Manzalah by Elmorsi et al. (2019), where Fe, Mn, Ni and Zn were the most accumulated metals in fish. This may indicate that the enrichment of biota with heavy metals was affected by their concentrations in water, sediment and plants.

Pb and Cd play no role in biological processes of living organisms and are highly toxic non-essential elements even at low concentrations (Dimari et al. 2008). They are also potent mutagenic and carcinogenic agents (Markmanuel & Horsfall 2016). Pb inhibits impulse conductivity by inhibiting the activity of acetylcholine esterase and monoamine oxidase, leading to pathological changes in organs and tissues (Rubio et al. 1991). It also impairs the larval and embryonic growth of fish species (Dave & Xiu 1991).

Pb and Cd concentrations in the muscles and gills of the three fish species were higher than the permissible limits recommended by FAO (1983) as criteria for human health protection, indicating that Pb and Cd may pose a risk to humans through the consumption of these contaminated fish species. Cd concentrations in fish muscles of the collected species were higher than its permissible limit (0.5 mg kg⁻¹), while Pb concentrations were 3 to 10 times higher than the limit of 0.5 mg kg⁻¹ recommended by FAO (1983) in the muscles of all species.

Ni is a well-known essential metal necessary for enzymes and other cell components with critical functions for living organisms, yet very high intakes can lead to serious health problems (Elnabris et al. 2013), therefore IARC (2012) classified Ni as a human carcinogen. Ni concentrations in the muscles (4.39–6.73 mg kg⁻¹) and gills (11.4–15.6 mg kg⁻¹) of tilapia species were found to be lower than the limit (70–80 mg kg⁻¹) recommended by USFDA (1993).

Correlation coefficients were calculated to clarify the relationships between the analyzed metals. Matrix analysis showed significant positive correlations (n = 7; p < 0.05) for each pair of metals contained in the muscles: Zn/Cu (r = 0.68), Zn/Pb (r = 0.81), Zn/Cd (r = 0.64), Cu/Pb (r = 0.89), Cu/Cd (r = 0.77) and Pb/Cd (r = 0.83) for Oreochromis niloticus, Fe/Zn (r = 0.79), Fe/Cu (r = 0.91), Fe/Ni (r = 0.66) and Zn/Cu (r = 0.91) for Oreochromis aureus and finally between Fe/Ni (r = 0.55) and Pb/Cu (r = 0.56) for Tilapia zillii. Whereas positive correlations (n = 7; p < 0.05) were found for the following pairs of metals contained in the gills: Fe/Zn (r = 0.75), Fe/Pb (r = 0.90), Fe/Cd (r = 0.82), Fe/Ni (r = 0.85), Zn/Ni (r = 0.82), Pb/Ni (r = 0.91) and Pb/Zn (r = 0.90), Pb/Cd (r = 0.83) and Mn/Cu (r = 0.84) for Oreochromis niloticus, Fe/Ni (r = 0.88), Fe/Pb (r = 0.66) and Pb/Ni (r = 0.82) for Oreochromis aureus and Fe/Zn (r = 0.74), Fe/Ni (r = 0.90), Pb/Mn (r = 0.86), Pb/Zn (r = 0.76), Pb/Cu (r = 0.79), Pb/Cd (r = 0.90), Mn/Zn (r = 0.73), Mn/Cu (r = 0.93), Mn/Cd (r = 0.90), Cu/Zn (r = 0.87), Cu/Cd (r = 0.78) for Tilapia zillii. Significant positive correlations between metals indicate their release from the same sources (drainage water) and mutual dependence (Ali et al. 2019).

3.1Heath risk assessment

As fish consumption is a possible source of heavy metal accumulation in humans, it is important to consider the daily intake of metals through fish consumption (Elnabris et al. 2013). The EDI was estimated by considering that a 70 kg person consumes 57 g fish per day. The EDI of heavy metals through the consumption of three tilapia fish species by humans is presented in Table 4. The results revealed that Mn, Ni, Cu, Pb, and Cd constituted the lowest daily intake, while Fe and Zn – the highest daily intake. The EDI values of Fe, Pb, and Cd in the selected fish species were higher than the maximum tolerable daily intake (MTDI) values recommended by FAO\WHO (2011) for a 70 kg person (MTDI-70), indicating a high human health risk associated with the consumption of the examined fish. Whereas the estimated daily intake of Zn, Cu, Mn and Ni in the muscles of tilapia species was below the corresponding permissible tolerable daily intake (Table 4).

Table 4 Estimated daily intakes of heavy metals for consumable fish collected from Qahr El-Bahr, the oral reference dose (RfD) and the maximum tolerable daily intake (MTDI)

Estimated Daily Intakes (EDIs; mg kg−1)
Species		Fe	Zn	Cu	Mn	Pb	Cd	Ni
O. niloticus	RangeMean ± SD	29.7–68.341.7 ± 17.1	23.2–33.829.1 ± 3.9	1.7–3.42.2 ± 0.6	2.3–5.63.6 ± 1.3	1.2–2.71.8 ± 0.5	0.3–0.90.5 ± 0.2	3.6–4.94.5 ± 0.4
O. aureus	RangeMean ± SD	30.9–68.642.4 ± 12.6	25.9–37.130.3 ± 4.4	1.9–5.23.0 ± 1.1	2.4–6.14.0 ± 1.2	1.3–3.92.5 ± 1.0	0.3–1.10.8 ± 0.3	3.8–5.44.5 ± 0.5
T. zillii	RangeMean ± SD	49.6–80.063.6 ± 9.7	32.4–40.837.0 ± 3.0	2.7–3.93.4 ± 0.4	3.8–6.65.0 ± 0.9	1.8–4.03.1 ± 0.9	0.8–1.21.0 ± 0.1	4.5–5.54.9 ± 0.4
RfD*		0.7	0.3	0.037	0.14	0.0035	0.001	0.02
MTDI**		0.8	1	0.5	1	0.003	0.0008	5
MTDI-70		56	70	35	70	0.21	0.056	350

* WHO (2018)

** FAO/WHO (2011)

MTDI-70 – maximum tolerable daily intake for a 70 kg person (mg day⁻¹) = MTDI × 70 kg

The health risk assessment was carried out to determine potential risks resulting from the consumption of the three fish species collected from the selected section of Lake Al-Manzalah. The results of the health risk assessment using the hazard quotient index are shown in Table 5. The HQ values for Fe, Zn, Mn, Cu and Ni were below 1 for all tilapia species from all sampling locations, indicating that there is no health risk associated with the exposure to these individual heavy metals. However, the HQ for Pb was above 1 in 14.3% of Oreochromis aureus samples and 42.8% of Tilapia Zillii samples. Whereas the HQ value for Cd was greater than 1 in 28.5% of Oreochromis aureus samples and 42.8% of Tilapia Zillii samples collected from Qahr El-Bahr, indicating that a potential risk may occur upon consumption of fish belonging to these species.

Table 5 Carcinogenic and non-carcinogenic risk of metals due to fish consumption based on fish samples collected from Qahr El-Bahr

Species		HQ							HI	TCR
		Fe	Zn	Cu	Mn	Pb	Cd	Ni		Pb	Cd	Ni
O. niloticus	Range	0.04–0.10	0.08–0.11	0.05–0.09	0.02–0.04	0.34–0.77	0.33–0.90	0.18–0.25	1.21–2.10	1 × 10−5–2.3 × 10−5	1 × 10−4–3 × 10−4	6 × 10−3–8 × 10−3
	Mean ± SD	0.06 ± 0.02	0.10 ± 0.01	0.06 ± 0.02	0.03 ± 0.01	0.51 ± 0.16	0.53 ± 0.23	0.22 ± 0.01	1.51 ± 0.40	2 × 10−5 ± 5 × 10−6	2 × 10−4 ± 9 × 10−5	8 × 10−3±7 × 10−4
O. aureus	Range	0.04–0.10	0.08–0.12	0.05–0.14	0.02–0.04	0.37–1.11	0.33–1.10	0.19–0.27	1.43–2.69	1 × 10−5–3 × 10−5	1 × 10−4–4 × 10−4	6 × 10−3–9 × 10−3
	Mean ± SD	0.06 ± 0.02	0.10 ± 0.01	0.08 ± 0.03	0.03 ± 0.01	0.71 ± 0.28	0.84 ± 0.27	0.23 ± 0.03	2.05 ± 0.50	2 × 10−5 ± 8 × 10−6	3 × 10−4 ± 1 × 10−4	8 × 10−3 ± 9 × 10−4
T. zillii	Range	0.07–0.11	0.11–0.14	0.07–0.11	0.03–0.05	0.51–1.14	0.84–1.16	0.23–0.27	2.08–2.84	2 × 10−5–3 × 10−5	3 × 10−4–4 × 10−4	8 × 10−3–9 × 10−3
	Mean ± SD	0.09 ± 0.01	0.12 ± 0.01	0.09 ± 0.01	0.04 ± 0.01	0.89 ± 0.26	0.99 ± 0.12	0.25 ± 0.02	2.46 ± 0.27	3 × 10−5±8 × 10−6	4 × 10−4 ± 5 × 10−5	8 × 10−3 ± 7 × 10−4

The health risk assessment of metal exposure from the consumption of tilapia fish species from Lake Al-Manzalah should allow for the combined effects of various heavy metals studied. Therefore, the HI value is quite necessary to assess the health risk associated with fish consumption (Zhu et al. 2016). The HI was above 1 for the three fish species. The minimum HI was observed for Oreochromis niloticus (1.21–2.10), while the highest HI was recorded in Tilapia zillii samples (2.08–2.84). Of all the studied metals, Cd and Pb were the major contributors to the HI values (34.3–40.6% and 33.7–35.5%, respectively), followed by Ni (9.6–15.7%) for the three tilapia species.

Although the HI for all three species falls under the medium non-cancer risk category (1 > HI < 4), the consumption of fish from Qahr El-Bahr was found unsafe when consumed for an extended period of time. Of the three species studied, Oreochromis niloticus is relatively safe for consumption as posing the lowest health hazard, while Tilapia zillii is more predisposed to accumulate heavy metals in its tissues.

According to USEPA (2012), Fe, Mn, Zn, and Cu do not cause any carcinogenic effects as their CPS_o has not yet been established. The average TCR factors for Pb over a lifetime of exposure through the consumption of contaminated Oreochromis niloticus, Oreochromis aureus and Tilapia zillii were 1.5 × 10⁻⁵, 2.1 × 10⁻⁵ and 2.6 × 10⁻⁵, respectively. Since USEPA sets the value of 10⁻⁵ as an acceptable lifetime carcinogenic risk, the TCR of Pb appears to be negligible. On the other hand, all three fish species pose a significant health risk from the intake of Cd and Ni, with TCR ranging from 6 × 10⁻³ to 4 × 10⁻⁴.

Cd poses the highest relative risk (RR) for the three tilapia species, followed by Pb and Ni, while Mn poses the lowest risk. The contribution of Pb and Cd to the overall relative risk index ranged from 34% to 41% (Figure 2). Thus, the consumption of tilapia species should be limited to avoid a potentially harmful exposure to these metals, especially Cd and Pb. Many coastal cities in Egypt rely primarily on fish as a source of protein in their meals, hence fish consumption is relatively high, so their exposure to heavy metal toxicity increases.

3.2Histopathological observations

For field assessment, histopathology is a rapid and cost-effective method of detecting adverse, acute and chronic effects of exposure in various tissues and organs. Oreochromis niloticus is one of the most commercially important fish that can tolerate a wide range of severe environmental conditions and shows little susceptibility to diseases (Tayel et al. 2008).

The muscular system constitutes the largest part of the fish body. Its overall functions include locomotion, pumping of blood, synchronized movement of skeletal components, peristaltic constriction of visceral organs and their related structures (Kadry et al. 2015). Muscles which are mainly composed of segmental myomeres are covered with skin. Each myomere is regarded as a muscle and its fibers are parallel along the body axis (Bayomy & Tayel 2007; Yacoub et al. 2008).

The results of histopathological examination of skin and muscles of Oreochromis niloticus indicate degeneration of the epithelial layer, separation of epidermis from the dermal layer, degeneration of muscle fibers and the hypodermal layer, edema, hemorrhage in muscle fibers and balloon necrosis in hypodermal layers (Fig. 3a–f). These results are consistent with those obtained by El-Serafy et al. (2005), Yacoub et al. (2008) and Saad et al. (2011). These changes in the muscles may be attributed to the accumulation of heavy metals and/or inorganic fertilizers that are discharged from different drains into the lake with a large amount of wastes (Mahmoud & El-Naggar 2007; Yacoub et al. 2008; Tayel et al. 2018) and to parasitic infections (Saad et al. 2011; Abou El-Gheit et al. 2012).

The gills are the most delicate structure of the teleost body, having an external location. They are exposed to damage and pathological abnormalities by irritants that are suspended or dissolved in water, thereby reducing their surface area and retarding the respiratory function (Tayel et al. 2014). The importance of gills as a target organ for contaminants results from their large surface area and the fact that they are in constant and direct contact with irritants. Exposure to heavy metals results in respiratory, osmoregulatory and circulatory impairment (Fernandes et al. 2008). In the present study, histopathological changes were observed in the gills of Oreochromis niloticus, including degeneration, necrosis, hemorrhage progressing to hemolysis in the secondary lamellae, edema, curling, lamellar aneurysm as well as hyperplasia progressing to complete fusion of the secondary lamellae (Fig. 4a–f). According to Mallat (1985), the edema of the gill epithelium is one of the major structural changes caused by exposure to heavy metals. The current results confirm that this lesion is due to heavy metal exposure. Alvarado et al. (2006) reported that a significant increase in the number of chloride cells in the gills induces epithelial thickening of the filament, which enhances the migration of chloride cells to the edges of the secondary lamellae and causes hypertrophy and fusion of the secondary lamellae. This may be viewed as a non-specific biomarker response to the exposure to heavy metals and disruption of fish health.

The intralamellar hyperplasia is a consequence of excess mucus production. Penetration of contaminants activates the secondary lamellae epithelium to increase the number of mucus cells. Hyperplasia with excess mucus lamellae causes fusion, which reduces the surface area of the gills and thus affects the ability of the gas exchange (Kumari et al. 2012). The discharge of wastewater into natural water bodies containing a large amount of organic matter results in high levels of organic phosphorus (Marcogliese et al. 2015), which can reduce the gaseous exchange capacity in fish (Dalzochio et al. 2018).

The liver is the main organ of detoxification in fish, with one of the main functions being to cleanse the body of any poisons coming from the intestine (El-Naggar et al. 2009). Any damage to the fish liver ultimately leads to multiple physiological disorders and subsequent fish death (Mahboob et al. 2020). The liver of Oreochromis niloticus caught from Lake Al-Manzalah in Qahr El-Bahr showed many histopathological changes, including degeneration, necrosis, fatty degeneration, hemorrhage and accumulation of hemosiderin in hepatocytes. Hemorrhage, hemosiderin and degeneration in blood vessels were observed in addition to congestion in blood sinusoids (Fig. 5a–f).

These changes may be due to fertilizers, salts and sewage discharged into Lake Al-Manzalah. Tayel et al. (2014) and Mahmoud & Abd El Rahman (2017) found similar histopathological changes in the liver of Mugil species and Clarias gariepinus caught in the same lake. Degeneration and necrosis of hepatocytes may be due to the accumulation of heavy metals. This is consistent with the findings of Authman & Abbas (2007) who stated that the liver is involved in a detoxification of toxins such as heavy metals. Accumulation of hemosiderin in liver cells may contribute to the rapid and continuous destruction of red blood cells (Hashem et al. 2020; Tayel et al. 2018; Ibrahim & Mahmoud 2005). Degeneration of hepatocytes can be caused by oxygen deficiency due to intravascular hemolysis and vascular dilation (Gaber & Gaber 2006). Toxins secreted by microorganisms in sewage water may cause necrosis and hemorrhage (Saad et al. 2011). Fatty degeneration can be caused by an increased rate of utilization of energy reserves or an induced imbalance between fat utilization and production (El-Naggar et al. 2009). The liver is an organ that excretes and binds proteins such as metallothionein. Metal-binding proteins, which are present in the nuclei of hepatocytes, increase the cell damage (Mela et al. 2007).

4Conclusions

The study showed that tilapia fish species caught in the area of Qahr El-Bahr in Lake Al-Manzalah contained varying concentrations of metals and the degree of their accumulation varied among different tilapia species. The average level of heavy metals ranged as follows: Tilapia zillii > Oreochromis aureus > Oreochromis niloticus. The accumulation rate of heavy metals in the muscles and gills of tilapia fishes were in the following order; Fe > Zn > Ni > Mn > Cu > Pb > Cd. The studied metals do not pose carcinogenic health hazards individually but their combined effects are potentially hazardous to the health of consumers (HP > 1). The muscles of Oreochromis niloticus occurring in the Qahr El-Bahr area of Lake Al-Manzalah are almost safe for human consumption. Thus, Oreochromis niloticus can withstand the risk of heavy metals in the study area. In general, a moderate intake of tilapia fish from Lake Al-Manzalah is strongly recommended for consumers to avoid serious health problems, including cancer and kidney malfunctions. Histopathological changes, including degeneration, fatty degeneration, necrosis, edema, hemorrhage, hemolysis, hemosiderin, curling and hyperplasia were found in the muscles, gills and liver of Oreochromis niloticus due to the accumulation of heavy metals in fish organs.

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