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

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).

Figure 1

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.

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.

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.

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.

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): EDI=(FIR×Cm)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.

The non-carcinogenic risk for each metal in the muscles was assessed by the hazard quotient (HQ) (USEPA 2000) using the following equation: HQ={EF×ED×FIR×CmRfD×BW×AT}×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: HI=∑i=1nHQi HI = \sum\limits_{i = 1}^n {HQi}

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: TCR=EF×ED×FIR×Cm×CPSoBW×AT×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).

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: RR=CmRfD RR = {{Cm} \over {RfD}}

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

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.

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.

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).

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.

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.

Figure 2

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).

Figure 3

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.

Figure 4

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).

Figure 5

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).