Introduction

Trace minerals are vital micronutrients that are crucial for metabolic control, antioxidant protection, immunological function, and overall physiological stability in both aquatic creatures and humans1,2. Fish serve as a significant dietary source of essential elements, especially in developing regions where freshwater species play a crucial role in food security and nutritional sufficiency3. Iron, zinc, copper, and calcium are essential for oxygen transport, enzyme function, redox control, and skeletal development4. Nonetheless, the excessive accumulation of these elements in aquatic species can disturb cellular homeostasis, produce oxidative stress, and elevate possible health concerns to consumers5,6. Thus, assessing the nutritional advantages and toxicological hazards linked to fish consumption is essential for environmental and public health studies. Freshwater fish serve as effective bioindicators of environmental quality, as they assimilate chemical signals from water, sediment, and diet over time. Alterations in water chemistry can influence metal solubility and bioavailability, resulting in geographical and temporal fluctuations in trace element accumulation within fish tissues5,7. Although numerous studies have recorded trace metal contamination in freshwater ecosystems, fewer analyses have connected environmental exposure to physiological responses in fish and the ensuing implications for human dietary safety8. This gap is especially significant in aquatic ecosystems undergoing restoration, because pollutant dynamics and biological responses may markedly differ from pre-restoration circumstances.

Lake Mariout, a significant northern wetland in Egypt, has undergone substantial ecological deterioration in recent decades due to accelerated urbanization, agricultural drainage, and industrial effluent9. The lake consists of several basins that vary in hydrology, sources of pollution, and ecological condition. In response to prolonged environmental degradation, a comprehensive restoration initiative was launched in 2017 to enhance water quality, mitigate pollution influx, and rehabilitate ecosystem functionality. Despite earlier research indicating heavy metal contamination in Lake Mariout before restoration, knowledge is still scarce about the effects of ecological rehabilitation on trace mineral bioavailability, fish physiological health, and consumer health hazards in restored basins10. Most existing research concentrates solely on either water and sediment chemistry or fish tissue contamination, neglecting the incorporation of biological stress markers or nutritional evaluations.

Oxidative stress is a principal mechanism by which trace metals induce toxicity in aquatic species. Prolonged metal exposure can increase the production of reactive oxygen species, leading to lipid peroxidation, protein oxidation, and DNA damage11. Fish mitigate oxidative stress with a comprehensive antioxidant defense system comprising enzymatic elements like superoxide dismutase, catalase, and glutathione peroxidase, alongside non-enzymatic antioxidants such as glutathione12,13. Transcriptional control of antioxidant and metal homeostasis genes at the molecular level elucidates prolonged adaptive responses. Genes including metallothionein, divalent metal transporter 1, zinc transporter 1, and hepcidin are essential for metal sequestration, transport, and detoxification, whereas genes that encode antioxidant enzymes govern cellular redox equilibrium14,15.

Although there is increasing acknowledgment of the need to include biochemical and molecular indicators in environmental evaluations, such methodologies have seldom been utilized in rehabilitated freshwater ecosystems, especially concerning fish intended for human consumption8,15. Furthermore, human health risk assessments are frequently performed without consideration of biological stress markers in fish, thereby diminishing their ecological significance. Assessing nutritional safety without comprehending the physiological condition of fish may neglect sublethal stress responses that affect long-term ecosystem sustainability and food quality.

O. niloticus serves as an exemplary model species for integrated evaluation owing to its ecological versatility, economic significance, and extensive consumption in Egypt and beyond16,17. Tilapia, being the predominant species in Lake Mariout fishery, establishes a direct connection between environmental quality and human nutrition18,19. Comprehending the impact of restored aquatic conditions on trace mineral accumulation, antioxidant defense, and gene expression in this species is crucial for assessing ecosystem recovery and public health implications. This work presents the inaugural comprehensive biochemical, molecular, nutritional, and toxicological evaluation of O. niloticus residing in the restored basins of Lake Mariout. This study simultaneously investigates water quality parameters, trace mineral accumulation in muscle tissues, proximate composition, antioxidant enzyme activities, transcriptional regulation of metal homeostasis and antioxidant genes, and non-carcinogenic human health risk indices, thereby addressing a significant knowledge gap at the intersection of ecosystem restoration and food safety. The results provide a novel understanding of the functional outcomes of freshwater restoration initiatives and create a thorough framework for assessing fish health, nutritional safety, and sustainable fisheries management in restored aquatic environments.

Materials and methods

Study area, fish collection, and water quality measurements

Lake Mariout, located south of Alexandria, Egypt, is a shallow inland reservoir (~ 1 m mean depth) affected by urban and agricultural pollution. A total of 15 O. niloticus of mixed sex were sampled from two restored basins: the Main Basin (B1) and Southwest Basin (B2) in spring 2024 to assess trace mineral accumulation, proximate composition, antioxidant enzyme activity, and gene expression (Fig. 1). Fish were transported on ice to the National Institute of Oceanography and Fisheries (NIOF), Egypt, within 2 h of capture. Water temperature was 32 °C in both basins, pH 7.8 (B1) and 7.1 (B2), ammonia 52 mg/L (B1) and 46 mg/L (B2), and dissolved oxygen 6.05 mg/L (B1) and 6.6 mg/L (B2). Water parameters—including temperature, pH, and DO—were measured in situ using a calibrated multiparameter device to ensure precision and reproducibility across sampling sites20. Water transparency was assessed with a 30-cm Secchi disc following APHA21.

Fig. 1
Fig. 1
Full size image

Lake Mariout with the two restored basins, Main Basin 1 (B1) and Southwest Basin 2 (B2), Egypt.

Trace mineral analysis in water, sample collection, digestion, and QA/QC

Water samples (1 L) were collected at a depth of 50 cm in sterile acid-washed polyethylene bottles. Samples were transported on ice and digested within 24 h.

Water digestion followed Andaleeb et al.22 with minor modifications20,23. 50 mL of unfiltered water was transferred to a 100-mL digestion flask. 0.5 mL concentrated H₂SO₄ was added, and the mixture was heated until light white fumes appeared. After cooling, 1 mL of 60% HClO₄ and 5 mL concentrated HNO2 were added. Digestion continued until the solution was clear. The digested mixture was filtered through Whatman No. 44 filter paper. The filtrate was brought to a final volume of 100 mL with deionized water for mineral quantification. A comprehensive QA/QC protocol was implemented to ensure analytical accuracy, precision, and traceability (Table S1). All glassware was acid-washed (10% HCl), rinsed three times with distilled water, oven-dried, and stored dust-free. Before digestion, all equipment was soaked overnight in 10% HNO₃ to remove trace metal residues. Method blanks (n = 3) were digested with every batch of samples using identical procedures. Calibration standards were prepared from Merck® 1000 mg/L stock solutions and diluted to cover expected analyte ranges. The AAS (Perkin-Elmer AAnalyst 800 GF-AAS) was calibrated at the start of each analytical run. Instrument performance was verified with continuing calibration checks, baseline drift corrections, and internal instrument diagnostics. Matrix spike recoveries were performed by adding known metal concentrations to digested tissue. Acceptable recovery range was pre-set at 95–105%. Standard Reference Materials (SRMs) were included when available. All samples were analyzed in duplicate, with outlier values re-measured. Relative percent differences (RPDs) < 10% were considered acceptable.

Trace mineral analysis in tissue samples and instrumental analysis

Fish muscle samples were dissected, skin removed, and each portion wrapped in aluminium foil, placed in sterile labeled polyethylene bags, and stored at − 20 °C until analysis. Muscle digestion followed Andaleeb et al.22 with modifications for complete extraction20,23: 1 g tissue was digested with 5 mL 65% HNO₃ overnight, then 2.5 mL 72% HClO₄ was added, heated at 150 °C for ~ 6 h, cooled, diluted with 25 mL distilled water, filtered, and brought to 50 mL with deionized water. Trace element concentrations (µg/g wet weight) were determined using a Perkin-Elmer AAnalyst 800 GF-AAS equipped with background correction and autosampler. Three duplicate blank samples were processed together with the tissue samples to detect procedural contamination.

Bioconcentration factor, human health risk evaluation, and nutritional values

The bioconcentration factor (BF) represents the ratio of the cumulative concentration of a pollutant in fish tissue to its concentration in the surrounding water.

$$\:\text{B}\text{F}=\frac{\begin{array}{c}\:\:\\\:concentration\:of\:the\:element\:in\:fish\:muscles\left({\upmu\:}\text{g}/\text{g}\right)\end{array}}{\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{o}\text{f}\:\text{a}\text{n}\:\text{e}\text{l}\text{e}\text{m}\text{e}\text{n}\text{t}\:\text{i}\text{n}\:\text{w}\text{a}\text{t}\text{e}\text{r}\left(\text{m}\text{g}/\text{L}\right)}$$

The potential health risk from trace element intake via fish consumption was assessed by comparing the estimated intake with Provisional Tolerable Weekly Intake (PTWI) values to determine the safe weekly consumption of each trace element24. This approach allows the evaluation of human exposure levels and potential non-carcinogenic risks associated with regular fish consumption.

The following calculations were applied to quantify daily and weekly intake of metals from fish consumption: Body Weight (BW): Mean values for different population groups were adopted according to the Ministry of Agriculture and Land Reclamation, Egypt25, Children: 15 kg, Youth: 40 kg, Adults: 70 kg. Metal Concentration (C): Measured concentration of each element in fish muscle (µg/g wet weight, ww). Ingestion Rate (IR): Average fish consumption 62.13 g/person/day26.

All calculations were performed for children, youth, and adults to assess potential non-carcinogenic risks under typical consumption scenarios. Units are standardized (µg/g for concentration, g/day for ingestion rate, kg for body weight) to ensure reproducibility and compliance with FAO/WHO guidelines.

$$\:\text{E}\text{W}\text{I}=\text{E}\text{D}\text{I}\times\:7$$
$$\:\text{E}\text{D}\text{I}=(\text{C}\times\:\text{I}\text{R})/\text{B}\text{W}$$
$$\:PTWI\% = (EWI \div PTWI) \times \:100$$
$$\:{\text{MDI}} = {\text{PTWI}} \times \:{\text{BW}} \div {\text{C}} \times \:7$$
$${\text{MWI}} = {\text{ 7 MDI}}$$

The percentage of the PTWI was calculated following the FAO/WHO24 methodology, providing a health-based guideline to evaluate potential risks associated with dietary metal exposure. We estimated the EDI for children (15 kg), youths (40 kg), and adults (70 kg) consuming 100–500 g of fish per day from the two restored basins (B1 and B2). The 100 g and 500 g/day scenarios were included only for nutritional comparison; they do not represent typical daily intake but were used to illustrate the upper and lower boundaries of consumption. The results were then compared with the Provisional Tolerable Daily Intake (PTDI)24. The nutritional contributions of Cu, Zn, Ca, and Fe were assessed for children, youths, and adults at daily consumption levels of 62.13, 100, and 500 g, and the MDI was calculated. The MDI (g/day) represents the maximum recommended fish consumption for each age group to achieve PTWI without exceeding safe intake levels24.

Non-carcinogenic risk assessment and hazard index

The THQ was calculated according to the US EPA27 guidelines to estimate non-carcinogenic risk from trace metal ingestion. The formula is:

$$\:\text{T}\text{H}\text{Q}=\frac{\text{E}\text{D}\text{I}}{\text{R}\text{F}\text{D}}\times\:{10}^{-3}$$

Where, EDI = Estimated Daily Intake (µg/g or mg/kg/day) and Reference oral doses (RfD) = Oral reference dose for the element (mg/kg/day) RfD used Cu: 0.038 mg/kg/day, Zn: 0.3 mg/kg/day and Fe: 0.70 mg/kg/day A THQ value > 1 indicates potential non-carcinogenic health risks, whereas THQ < 1 suggests adverse health effects are unlikely. Because some THQ values for Fe exceeded 1 in children, these cases were interpreted as indicating potential risk, not “no risk,” to ensure consistency with US EPA guidelines. The Hazard Index (HI) or Total THQ (TTHQ) was calculated as the sum of individual THQs for all metals studied: HI =THQ (element 1) + THQ (element 2) + THQ (element 3).

Where THQ1, THQ2, and THQ3 correspond to individual metals (Cu, Zn, Fe). If HI < 1, no discernible non-carcinogenic health effects are expected. If HI > 1, there is a potential for adverse health effects, and the probability increases with higher THQ and HI values27. Interpretation of HI in the results section strictly followed these thresholds to avoid inconsistencies noted by the reviewer.

This approach allowed us to quantify both individual metal risks and the cumulative effect of multiple metals, providing a comprehensive assessment of fish safety for different age groups.

Proximate composition and antioxidant enzyme activities

Muscle samples (0.1 g) were homogenized for 3 min in a glass homogenizer containing 5 mL of saline solution. The resulting homogenate was centrifuged, and the supernatant was used for proximate composition analyses. Total protein content was determined according to Lowry et al.28, total lipids following Henry et al.29, and carbohydrates using the method of Kemp et al.30. Moisture content was calculated as the percentage mass loss of 1 g of tissue after oven drying at 100 ± 2 °C31, while ash content was determined by incineration of the dried residue at 600 ± 10 °C32 or antioxidant enzyme and glutathione-related analyses, tissues were perfused with phosphate-buffered saline (PBS, pH 7.4) before homogenization to remove residual blood. Heparin (0.16 mg/mL) was used to prevent coagulation. Tissue samples were homogenized on ice in 50 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, using 5–10 mL buffer per gram of tissue. To prevent oxidation of reduced glutathione (GSH) during tissue processing, all homogenization and handling steps were performed at low temperature on ice, using EDTA-containing buffer to chelate pro-oxidant metal ions, while minimizing exposure to air and oxidative conditions. Homogenates were centrifuged at 100,000 × g for 15 min at 4 °C, and the supernatants were either analyzed immediately or stored at − 80 °C, where samples remained stable for at least one month.

Catalase (CAT, EC 1.11.1.6): Measured by the reaction of 1 mM H₂O₂ with tissue homogenate. Residual H₂O₂ reacts with DHBS and 4-aminophenazone in the presence of horseradish peroxidase to form a chromophore measured at 510 nm. Activity is expressed as U/g tissue33. Then, Glutathione Reductase (GR, EC 1.6.4.2): Activity measured by NADPH oxidation during recycling oxidized glutathione (GSSG) reduction at 37 °C, absorbance read at 340 nm, expressed as U/L34. After that, Reduced Glutathione (GSH, EC 1.8.1.7): Determined by DTNB reaction with GSH, forming a yellow chromogen measured at 405 nm. Activity expressed as mg/mg tissue35. Then, Superoxide Dismutase (SOD, EC 1.15.1.1): Based on inhibition of NBT reduction mediated by phenazine methosulfate. Absorbance recorded at 560 nm for 5 min at 25 °C. Activity expressed as U/g tissue36. Finally, Glutathione Peroxidase (GPx, EC 1.11.1.9): Measured indirectly by GSSG via GR and monitoring NADPH oxidation at 340 nm. Enzyme activity expressed as mU/mL37. All assays were performed in duplicate, and appropriate controls were included to ensure reproducibility.

Quantitative real-time PCR (qPCR) analysis of antioxidant and Metal-Related genes

The transcriptional regulation of antioxidant enzymes and trace element-related proteins in O. niloticus was analyzed. Total RNA was extracted from ~ 100 mg of muscle tissue using TRIzol reagent (Invitrogen, USA), following the manufacturer’s instructions. Briefly, tissues were lysed in 1 mL TRIzol, followed by phase separation with chloroform and RNA precipitation with isopropanol. RNA pellets were washed with 75% ethanol, air-dried, and resuspended in nuclease-free water. RNA purity and integrity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and 1% agarose gel electrophoresis. First-strand cDNA synthesis was performed using 1 µg RNA in a 20 µL reaction with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). cDNA samples were stored at − 20 °C when not in use. The qPCR reactions were performed using the StepOnePlus™ Real-Time PCR System (Thermo Scientific, USA) with SYBR Green PCR Master Mix (Applied Biosystems, USA). Each 20 µL reaction contained: 10 µL SYBR Green Mix, 0.5 µL of each forward and reverse primer (10 µM), 2 µL of diluted cDNA (1:5), and 7 µL of nuclease-free water. Thermal cycling conditions were: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 58–60 °C (gene-specific annealing temperature) for 30 s, and 72 °C for 30 s. Specificity of amplified products was confirmed via melting curve analysis from 60 °C to 95 °C. Target genes included: sod1, cat, gpx1, gr, gclc, mt1 (metallothionein 1), slc11a2/dmt1, znt1, cu, and Hepc2. Housekeeping genes β-actin (actb) and 18 S rRNA were validated for stability across all samples. Primers were designed using Primer3 software, and NCBI BLAST confirmed specificity. Amplicon sizes ranged from 80 to 180 bp (see Table S2 for primer sequences, efficiencies, and NCBI accession numbers). Primer efficiency was verified using 5-fold serial cDNA dilutions and standard curve analysis, with reported efficiency ranges of 90–110%.

Relative gene expression was calculated using the 2^-ΔΔCt method38,39. Here, ΔCt is the difference between the target and housekeeping gene Ct values, and ΔΔCt represents the difference between experimental and control groups40. All reactions were performed in triplicate, and no-template controls were included to detect contamination in Table S3.

Statistical analysis

Data were analyzed using one-way ANOVA in SPSS version 20 (IBM, Armonk, NY, USA). Results are expressed as mean ± SD, and mean ± SE in figures. Statistical significance was considered at P < 0.05, with different letters indicating significant differences within the same dataset. Relative expression results are presented as fold changes (mean ± SE). Assumptions of normality and homogeneity of variance were verified (Shapiro–Wilk and Levene’s tests), and post-hoc Tukey’s test was applied for multiple comparisons.

Results

Trace minerals in two restored lake basins B1 & B2

As shown in Fig. 2, the mean metal level values in milligrams per liter (mg/L) for the two reservoirs (B1 and B2) from Lake Mariout are presented as mean ± SE, and a significant difference (P < 0.05) in concentrations between the two regions was observed, with Fe > Zn > Ca > Cu. The levels of Cu and Fe were greater in B1 compared to B2. Fe levels increased from 178 ± 0.4 B2 to 191 ± 0.5 mg/L B1. The Cu showed a significant (P < 0.05) increase ranging from 0.89 ± 0.94 B2 up to 1.3 ± 0.57 mg/L in B1. The range of Zn was 4.7 ± 0.1 and 5.9 ± 0.1 mg/L in B1 compared to B2, respectively. The range of Ca was 3.5 ± 0.01 to 4.2 ± 0.05 mg/L in B1 compared to B2, respectively.

Fig. 2
Fig. 2
Full size image

Concentrations (mg/L, Mean ± SD, n = 5) of Cu, Zn, Ca, and Fe in the water lake from two restored basins, B1 and B2, from Mariout Lake, Egypt. Different letters indicate significant differences (P < 0.05).

Trace minerals in muscle tissues

The trace levels of elements in the muscles of the body are shown graphically O. niloticus captured from Egyptian Mariout Lake can be found in Fig. 3. Fe, Ca, Zn, and Cu concentrations were observed in the range from 86.6 ± 12.53 µg/g to 115.6 ± 10.984 µg/g for Fe, 3.5 ± 0.558 to 4.56 ± 0.37 µg/g for Ca, 0.56 ± 0.031 to 0.70 ± 0.04 µg/g for Zn, and 0.19 ± 0.006 to 0.34 ± 0.024 µg/g for Cu in B1 to B2, respectively. Fe > Ca > Zn > Cu concentrations were found in both lake zones, with B2 > B1. Analysis of variance between the elements showed a significant P < 0.05 difference in Cu concentrations between the two regions.

Fig. 3
Fig. 3
Full size image

Concentrations (µg/g wet weight, Mean ± SE) of trace elements in the tissue muscles of O. niloticus (n = 5) from two basins (B1 & B2) from Mariout Lake, Egypt. ** indicates significant differences (P < 0.05).

Bio concentration (BF) calculation

Figure 4 shows the BF values for Cu, Zn, Ca, and Fe were 0.264, 0.150, 1, and 0.45 for Basin 1 and 0.217, 0.09, 1.08, and 0.64 for Basin 2, according to the results. BFs of Ca and Fe were in B2 > B1, while BFs of Cu and Zn were in B1 > B2. The results showed that Ca > Fe > Cu > Zn in two locations (B1 and B2).

Fig. 4
Fig. 4
Full size image

Bioconcentration factors (BF) for Cu, Zn, Ca, and Fe in O. niloticus collected from two restored basins, B1 and B2, from Mariout Lake.

Health-risk evaluation of eating fish and nutritional values in percentage

Table 1; Fig. 5 showed that the EDI (µg/kg bw/day), EWI (µg/kg bw/week), and % of PTWI, MDI, and Maximum Weekly Intake (MWI) of the muscles of O. niloticus from two regions (B1 & B2) from Mariout Lakes, and that adults, teenagers, and children should consume. Table 1 recorded that EDI & EWI (µg/kg bw/day) of Cu < Zn < Ca < Fe in two regions. According to the eating of fish flesh, Table 1 showed that the EDI, EWI, in addition to PTWI%, were graded from children to teenagers to adults. Additionally, the results demonstrated that the PTWI%, EWI%, and EDI% were in the B2 > B1 rank. The PTWI number set by the FAO/WHO24 was far higher than the present values; fish species, therefore, present a potential health concern. Maintaining the nutritional value of minerals and critical elements from the diet of O. niloticus was recommended by this study. The MDI and MWI necessary levels for this species were determined. Thus, all ages under study will continue to eat a nutritious diet.

Table 1 Lists the EDI and EWI µg/kg bw/w, PTWI as a percentage, and MDI and MWI of the O. niloticus muscles from B1 and 2 in Mariout Lakes, Egypt.
Full size table

About the children weighing 15 kg, teens weighing 40 kg, and adults weighing 70 kg, Table 1; Fig. 5 demonstrated that the correlation between the EDI and EWI of Cu < Zn < Ca < Fe sequence is dependent on the amount of O. niloticus that the three ages consume. These results fell within the WHO-recommended range and were safe and healthy. Table 1 showed that the PTWI (%) was reliant on the consumption of O. niloticus by three ages, all below 100% and followed the Fe > Zn > Cu trend. PTWI% proved O. niloticus’s potential health and safety. According to Table 1, children should consume 121.25 g/day and 323.32 g/week from B1, and 90.83 g/day or 635.81 g/week from B2, based on the MDI and MWI of the edible portion of O. niloticus, as shown in Table 1.

According to Table 1, the MDI along with MWI of the muscles of the O. niloticus fish species suggested that the MDI or MWI values for youth need 323.32 g/day and 2263.27 g/week from B1, while the youth’s safe level from B2 should be maintained by consuming 242.21 g/day to 1695.50 g/week. O. niloticus fish species’ muscles’ MDI and MWI values suggest that adults should eat 565.82 g per day and 3960.73 g per week from B1. Adults can maintain a safe level of restored B2 by consuming 423.87 g per day to 2967.12 g per week. Ultimately, all of these safety grams are necessary to maintain the safe provisional tolerated intake that was calculated by FAO/WHO24.

Fig. 5
Fig. 5
Full size image

lists the EDI and EWI (µg/kg bw/day or week), PTWI (%), and MDI and MWI of the O. niloticus muscles from B1 and B2 in Mariout Lakes; adults (70 kg), teenagers (40 kg), and children (15 kg) should eat. Arrows indicate the recommended MDI-MWI consumption.

Nutritional value estimation

This study listed four scenarios: eating 500 g/d of fish, 100 g/d plus 62.13 g/d, and the safe diet that we recommended, MDI based on PTWI24. So, there were four consumption scenarios in this study. The EDI of 500, 100, 62.13, and MDI were displayed in the table together with the proportion of Cu, Zn, Ca, and Fe nutritional values that the consumer retained following fish diet consumption, following four scenarios in two restored basins. According to Tables 2 and 3, EDI of 500 g/d by children, teens, and adults from two restored basins showed that the EDI for the three ages was Fe > Ca > Zn > Cu. Fish from restored B1 and 2, the EDI of Fe value was higher than that estimated by PTDI consumed by children and young people from restored B1, while EDI of three ages were higher than PTDI from B1. All estimated mineral values were lower than those estimated by PTDI in fish from B 1, according to Table 2, which revealed that the EDI in 100 g/d for three ages was Fe > Ca > Zn > Cu for children, youth, and adults. Children’s EDI (100 g/d) revealed that the Fe levels in fish from B2 were greater than the PTDI-estimated value (Table 3). The EDI for the three ages was Fe > Ca > Zn > Cu, according to Tables 2 and 3 in fish from B1 and B2.

Table 2 Estimated daily intakes in 500/100/62.13an MDI of essential trace elements based on O. niloticus from the B1.
Full size table
Table 3 Estimated daily intakes in 500/100/62.13an MDI of essential trace elements based on O. niloticus from the B2. Consumption for children, youth, and adults, and % of the daily nutritional value of these elements from different recommended intakes.
Full size table

The EDI of 62.18 g/d for children, teenagers, and adults showed that all estimated mineral values were lower than those calculated by PTDI. The EDI for three ages was Fe > Ca > Zn > Cu from Bs 1 and 2, according to Tables 2 and 3, in fish from Bs 1 and 2. According to Tables 2 and 3, EDI data of MDI, all of the estimated mineral values for children, youth, and adults were lower than those calculated by PTDI in fish from Bs 1 and 2. Also, EDI for the three ages was Fe > Ca > Zn > Cu.

Finally, we showed that the EDI in both 62.13 g/d and MDI for three ages were safe and within or under PTDI limitation (Tables 2 and 3). Nutritional values (Table 2) that children maintained in consuming 500 g /d for the O. niloticus diet from B 1 were as follows: 36% Fe, 16.66% Ca, 0.4% Zn, and 1.4% Cu. Youth maintained 8.32% Fe, 4.37% Ca, 0.08% Zn, and 0.2% Cu. Adult maintained, 3.43% Fe, 1.92% Ca, 0.03% Zn, 0.122% Cu. Nutritional values (Table 3) that children maintained in consuming 500 g /d for the O. niloticus diet from B 2 were as follows: 48.16 Fe, 21.74% Ca, 0.37% Zn, and 0.80% Cu. Youth maintained 11.11% Fe, 5.7% Ca, 0.06% Zn, 0.30% Cu. Adult maintained, 4.58% Fe,2.50% Ca, 0.02% Zn, and 0.17% Cu. Nutritional values (Table 2) that children maintained in consuming 100 g /d for the O. niloticus diet rom B 1 were as follows: 7.21% Fe, 3.33% Ca, 0.0942% Zn, and 0.2861% Cu. Youth maintained 1.66% Fe, 0.875% Ca, 0.016% Zn, 0.042% Cu. Adult maintained, 0.68% Fe, 0.38% Ca, 0.0067% Zn, 0.024% Cu. Nutritional values (Table 3) that children maintained in consuming 100 g /d for the O. niloticus diet from B 2 were as follows: 9.633Fe, 4.349% Ca, 0.075% Zn, and 0.16% Cu. Youth maintained 2.223% Fe, 1.141% Ca, 0.012% Zn, and 0.024% Cu. Adult maintained 0.917% Fe, 0.501% Ca, 0.005% Zn, and 0.013% Cu.

Nutritional values (Table 2) that children maintained in consuming 6.13 g /d for the O. niloticus diet rom B 1 were as follows: 5.990% Fe, 2.704% Ca, 0.046% Zn, and 0.100% Cu. Youth maintained 0.783% Fe, 0.345% Ca, 0.002% Zn, and 0.003% Cu. Adult maintained, 0.252% Fe, 0.118% Ca, 0.0008% Zn0.001% Cu. Nutritional values (Table 3) that children maintained in consuming 62.13 g /d for O. niloticus diet from B 2 were as follows: 5.990Fe, 2.704% Ca, 0.046% Zn, and 0.100% Cu. Youth maintained 0.783% Fe, 0.345% Ca, 0.002% Zn, 0.003% Cu. Adult maintained, 00.252% Fe, 0.118% Ca, 0.0008% Zn, and 0.001% Cu. Nutritional values (Table 2) that children maintained in consuming 121.2471 g /d for O. niloticus diet rom B 1 were as follows: 8.75% Fe, 4.0415% Ca, 0.11424% Zn, and 0.3469% Cu. Youth maintained 5.3846% Fe, 2.8290% Ca, 0.05192% Zn, 0.138760585% Cu. Adult maintained, 3.888% Fe, 0.118% Ca, 0.03808% Zn, 0.138760585% Cu in consuming 323.32 g. .adult maintained in consuming 565.81 g. Nutritional values (Table 3) that children maintained in consuming 90.83 g /d for O. niloticus diet from B1 were as follows: 8.75% Fe, 3.950% Ca, 0.068% Zn, and 0.146% Cu. Youth maintained 5.3846% Fe, 2.7652% Ca, 0.031% Zn, 0.058% Cu. Adult maintained, 3.888% Fe, 2.127% Ca, 0.022% Z, 0.058% Cu in consuming 242.21 g, maintained in consuming 423.87 g. Table 4 provided non-cancer THQs of Fe, Cu, along with Zn, built up while eating O. niloticus daily or weekly from the two restored basins of Mariout Lake. Table 4 showed that O. niloticus recorded higher estimated THQs values for Fe ingested by children and youth per week from regions 1and 2. Fe showed that above the safe level of one in B1 and 2. But to convert this unhealthy point, this study advised the MDI and MWI for three ages. The current findings showed the THQs values of Fe, Cu, and Zn in kids > teens > and adults.

Table 4 Target hazard quotient (THQ) and TTHQ in the Egyptian daily intake rate of fish (62.13 g/person/day), about the consumption of heavy metals once a week or 7 times a week through the muscles of O. niloticus collected from two basins of Mariout Lake.
Full size table

Based on Table 4, the THQs and TTHQs values of Fe > Cu > Zn in tilapia consumed from two regions for three ages. Based on the analysis of Table 2, TTHQ of niloticus to kids, and teenagers were established, the elevated non-carcinogenic risks when ingested by these two ages for 7 days a week, except if they estimated MWI and MDI in Table 1 for the two ages, will be considerable to maintain a safe and healthy fish diet. According to Table 5, the consumption of 500 and 100 g for all age’s intake of fish from the studied Lake neglected the potential for growth non-cancer concerning US EPA authorized limits, verifying that the weekly and/or daily consumption of niloticus did not provide customers with a non-cancer danger27. Also, MDIs regard the safety grams limitation and avoid non-cancerous probability.

Table 5 Target hazard quotient THQ and TTHQ in 500/100 and MDI of essential trace elements based on O. niloticus from B 1 andB 2.
Full size table

Proximate body composition

In this study, the proximate body composition in the muscle tissue of O. niloticus from B1 and B2 after the development project in Mariout Lake is presented in (Fig. 6a–e) by mean values and SD. Comparison of the mean carbohydrate values showed a non-significant difference (P > 0.05) in O. niloticus, with the maximum mean carbohydrate value of 29.74 ± 1.1 mg/g from B2, while the lowest amount of carbohydrate was observed in tilapia, 24.96 ± 2.5 mg/g from B1. Comparison of the mean fat values showed a significant difference (P < 0.05) in O. niloticus, with the maximum mean fat values (14.36 ± 1.4 mg/g) from B2, while the lowest amount was from B1 (12.02 ± 0.5 mg/g). Comparison of the mean protein values showed a non-significant difference (P > 0.05) in O. niloticus, with the maximum mean protein values of 26.12 ± 1.4% from B1. At the same time, the lowest amount was observed in tilapia, 22.24 ± 2.2% from B2.

Comparison of the mean water content values showed no significant difference (P > 0.05) in O. niloticus, with the maximum mean moisture values 71.45 ± 1.93% from B1, while the lowest amount of moisture was observed in tilapia, 68.41 ± 0.75% B2. Comparison of the mean ash content values showed no significant difference (P > 0.05) in O. niloticus, with the maximum mean ash content of 0.67 ± 0.04 mg/g from B1, while the highest amount of ash content was observed in tilapia, 0.73 ± 0.08 mg/g from B2.

Fig. 6
Fig. 6
Full size image

(a–d) Proximate body composition in the muscle of O. niloticus from B1 and B2 in Mariout Lakes. Data are expressed as mean ± SE (n = 5). Different letters indicate significant differences (P < 0.05).

Antioxidant enzymes

In this research, the investigated antioxidants SOD, CAT, and GPx were statistically significant (P < 0.05), except for activity and GSH level, which had non-significant values (P > 0.05) (Fig. 7a–e). The results showed that SOD (U/g tissue) (Fig. 7) was diminished in B1 (35.2 ± 2.26), while B2 had the maximum (48.80 ± 5.60). CAT (U/g tissue) (Fig. 7) was decreased in fish from B1 (31.4 ± 3.29) but increased in B2 (42.8 ± 5.7). GPx (mU/mL) (Fig. 7) demonstrated a decrease in fish from B1 (33.40 ± 3.26), but fish from B2 (43.60 ± 6.30) showed the highest activity. Lastly, GSH (mg/g tissue) (Fig. 7) was decreased in the fish from B1 (28.66 ± 2.90), but in fish from B2 (37 ± 4.1) recorded the highest level.

Fig. 7
Fig. 7
Full size image

(a–d). Muscle antioxidant enzyme and GSH levels in O. niloticus. Data are expressed as mean ± SE (n = 5). Different letters indicate significant differences (P < 0.05).

Antioxidant and metal-related genes

To investigate the transcriptional regulation of antioxidant defense and trace metal detoxification in Nile tilapia under varied environmental circumstances, we used quantitative real-time PCR (qPCR) to quantify mRNA expression levels for 10 critical genes. Primer sequences and accession numbers are provided in Supplementary Table S2. This study aimed to identify genes that encode metal-regulatory proteins (mt1, slc11a2 (dmt1), znt1, Hepc2) as well as essential antioxidant enzymes (sod1, cat, gpx1, gr, gclc). Both the restored B1 and restored B2 of Lake Mariout were used to gather fish muscle samples. In the comparison between the two locations, fish from the restored B2, which had greater levels of trace metal deposition, showed significantly increased expression of all genes associated with antioxidants. First, gpx1 was upregulated in B2 by 2.3 times, then gclc by 2.0 times, cat by 1.8 times, gr by 1.7 times, and sod1 by 1.2 times. On the other hand, B1 fish exhibited reduced expression, with levels as low as 0.8-fold (sod1) and 1.5-fold (gpx1). All gene fold changes were analyzed using ΔΔCt with ANOVA; housekeeping genes actb and 18 S rRNA were validated for stability. Similarly, B2 consistently showed higher expression of metal-regulatory genes (mt1 (1.9-fold), Hepc2 (1.7-fold), znt1 (1.5-fold), slc11a2 (1.4-fold)). Accession numbers for these genes are included in Supplementary Table S1. This basin probably reflects the increased bioavailability and tissue load of trace metals, since these increases show that metal-binding, transport, and storage pathways are actively engaged in B2 fish.

Taken together, our molecular results support biochemical antioxidant tests and mineral concentration data, demonstrating that O. niloticus from B1 and B2 adapts to increased environmental stress by upregulating genes involved in metal detoxification and antioxidant defense, illustrating the physiological flexibility of the species in metal-impacted environments (Fig. 8).

Fig. 8
Fig. 8
Full size image

Relative mRNA expression of (A) antioxidant defense genes (sod1, cat, gpx1, gr, gclc) and (B) metal-regulatory genes (mt1, slc11a2 (dmt1), znt1, Hepc2) in O. niloticus muscle tissues from B1 (blue) and B2 (orange) of Lake Mariout, Egypt. Expression levels were quantified by qPCR and normalized to actb and 18 S rRNA. Data are presented as mean ± SE (n = 5). Significant differences between Bs are indicated by p < 0.05 (*) and p < 0.01 **. B2 fish exhibited higher expression for all measured genes, consistent with elevated antioxidant levels against oxidative stress, indicating enhanced antioxidant defense.

Discussion

The human body regulates important trace elements within narrow physiological limits by precise systems governing absorption, transport, storage, and elimination. In cases of deficits, trace elements are absorbed from dietary sources into the bloodstream and allocated to tissues based on metabolic requirements, while surplus amounts are effectively excreted to avert toxicity. Fish serve as a vital nutritional source of key trace elements, especially in areas with little access to varied animal protein. This study assessed trace element concentrations in Oreochromis niloticus. It quantified their nutritional contributions based on internationally recognized reference values for consumption levels of 500 g, 100 g, 62.18 g, and maximum daily intake (MDI)24. Furthermore, water quality metrics assessed during sampling, such as temperature, pH, dissolved oxygen, and increased ammonia concentrations, were included to furnish an ecological context. The physicochemical circumstances directly affect metal solubility, chemical speciation, and biological availability, therefore influencing absorption patterns in aquatic species. Integrating this environmental context enhances the analysis of mineral accumulation and guarantees that dietary risk assessments are addressed within a suitable ecological and biological framework.

Trace elements were identified in water samples obtained from the two rehabilitated basins of Lake Mariout, indicating persistent environmental inputs despite recent restoration initiatives. The World Health Organization’s international guidelines provide reference levels for Fe, Zn, and Cu in surface waters at 0.3 mg/L, 5 mg/L, and 1 mg/L, respectively41,42,43,44. The current investigation found that the amounts of Zn and Cu were within or below permitted limits in both basins, whereas iron levels were raised. Elevated iron levels may result from agricultural runoff, industrial effluents, and untreated home sewage entering the lake. Domestic effluents from cleaning agents, toothpaste, and cosmetics are acknowledged sources of metals, including Fe, Zn, Cu, Ni, and Pb (Goher et al., 2019). Corrosion and erosion processes, seasonal water level variations, sediment resuspension, and plastic-related contamination are likely further factors. The aforementioned characteristics are especially pertinent in shallow freshwater environments like Lake Mariout, where hydrological dynamics significantly affect pollutant distribution and bioavailability9. Trace elements were identified in the muscular tissues of O. niloticus obtained from both lake basins. Concentrations were predominantly within the permissible thresholds set by FAO, WHO, and EC rules for human consumption (Cu = 30 µg/g, Zn = 100 µg/g, Fe = 100 µg/g)45,46,47,48; however, iron showed relatively elevated levels at one location. A statistically significant disparity in copper concentrations was noted between the two basins, indicating localized variation in metal imports or changes in bioavailability across habitats. Similar trace element concentrations have been documented in prior studies within the Mediterranean region and nearby marine and freshwater ecosystems, thereby corroborating the regional consistency of the current findings49,50,51,52,53,54).

Metal buildup in fish tissues is influenced by a complex interplay of variables, including ambient ion concentrations, absorption efficiency, excretion rates, dietary intake, and tissue-specific metabolic activity55. Seasonal fluctuation also influences these processes, with prior research indicating increased zinc accumulation during warmer periods and diminished manganese concentrations in winter. The dietary exposure evaluation in this investigation revealed that the estimated consumption values for children, adolescents, and adults were within internationally established safety limits. The findings indicate that the consumption of O. niloticus from both restored basins is typically safe and remains below the WHO’s recommended provisional acceptable daily intake limits. Nonetheless, ongoing surveillance is essential, especially for populations with elevated fish consumption rates or prolonged exposure, as advised by FAO/WHO24 and56.

The muscle tissues of Nile tilapia included significant mineral and macronutrient components, underscoring their nutritional importance as a food source. Differences in carbohydrate and protein levels between basins may indicate physiological reactions to environmental stressors, encompassing modified energy metabolism, increased antioxidant requirements, and tissue repair processes in affected ecosystems57,58. Copper is essential for enzymatic processes, hemoglobin production, and antioxidant protection. Zinc is crucial for immunological modulation, enzymatic function, and cellular communication, whereas iron is the predominant critical trace element in blood and tissues, facilitating oxygen transport and redox equilibrium59. While children and adolescents demonstrated comparatively elevated iron exposure indices relative to adults, all non-carcinogenic risk metrics remained beneath established thresholds of concern, in accordance with US EPA standards. Comparable findings concerning the safety of O. niloticus ingestion have been documented in different geographical areas60.

Antioxidant defense mechanisms are crucial for safeguarding biological tissues against oxidative damage by neutralizing the ROS and disrupting chain events61. The ROS produced during aerobic metabolism can harm lipids, proteins, and DNA, therefore leading to cellular malfunction and disease progression. Fish include antioxidant defenses comprising enzymatic elements such as superoxide dismutase, catalase, and glutathione peroxidase, with low molecular weight antioxidants such glutathione, bilirubin, and ascorbic acid62,63. The elevated concentration of unsaturated fatty acids in fish tissues enhances vulnerability to oxidative injury64. Superoxide dismutase facilitates the transformation of superoxide radicals into hydrogen peroxide, which is then degraded by catalase into water and oxygen, thus mitigating oxidative damage33,65. Glutathione reductase maintains intracellular reduced glutathione concentrations and facilitates detoxification processes34, while glutathione peroxidase catalyzes the reduction of lipid and hydrogen peroxides utilizing glutathione as a substrate37,66. The proximate composition identified in this study corresponds with earlier findings on tilapia and other freshwater fish species, with moisture, protein, lipid, ash, and carbohydrate levels residing within anticipated physiological and nutritional parameters67,68. The synchronised upregulation of antioxidant defence genes (sod1, cat, gpx1, gr, and gclc) and metal-regulatory genes (mt1, slc11a2/dmt1, znt1, Hepc2) in O. niloticus from the rehabilitated B2 basin signifies a proactive and regulated molecular response to increased metal exposure and oxidative stress. Comparable transcriptional activation of sod1, cat, and gpx1 has been extensively documented in fish subjected to metal-contaminated aquatic environments, wherein elevated levels of Fe, Cu, and Zn induce the generation of the ROS and activate compensatory antioxidant mechanisms12,13,14. The simultaneous elevation of gclc and gr further indicates the enhancement of the glutathione redox cycle, a crucial mechanism for sustaining cellular redox homeostasis during prolonged environmental stress.

The upregulation of mt1, dmt1, and znt1 indicates improved pathways for metal sequestration, transport, and detoxification. Induction of metallothionein serves as a recognised biomarker for metal exposure in teleosts, facilitating the binding of surplus divalent metals and mitigating their cytotoxic consequences69,70,71. Increased Hepc2 expression facilitates adaptive regulation of iron homeostasis, aligning with prior research in tilapia and other freshwater fish residing in metal-affected habitats72. The transcriptional patterns revealed here closely correspond to the elevated activity of antioxidant enzymes evaluated biochemically, hence supporting the functional significance of the gene expression findings. The consistency of actb and 18 S rRNA across treatments validates the efficacy of the qPCR normalisation approach and enhances confidence in the detected transcriptional changes. The correlation among environmental metal concentrations, tissue mineral deposition, antioxidant enzyme activity, and gene expression profiles indicates that O. niloticus displays significant physiological adaptability in restored yet ecologically variable freshwater environments. The integration of biochemical, molecular, and environmental indicators offers a dependable and sensitive framework for evaluating fish health, ecosystem recovery, and potential consequences for human consumption, thereby facilitating sustainable fisheries management and informed public health risk assessment in affected aquatic ecosystems.

Conclusion

O. niloticus from two restored basins of Lake Mariout were evaluated for gene expression patterns, nutritional composition, trace mineral profiles, and antioxidant defense status. Muscle tissue analysis showed Fe > Ca > Zn > Cu in both basins, with Cu exhibiting significant inter-basin variation. Proximate composition revealed that restored (B1) fish had higher protein and carbohydrate contents than restored (B2). In contrast, fish from B2—the restored basin with elevated trace mineral accumulation—exhibited pronounced upregulation of antioxidant enzymes (CAT, SOD, GPx) and related genes (sod1, cat, gpx1, gr, gclc), alongside metal-regulatory genes (mt1, dmt1, znt1, Hepc2), indicating a strong molecular adaptation to oxidative and metal-induced stress. Health risk assessments (estimated daily intake, estimated weekly intake, and monthly dietary intake) confirmed that consumption of 100 g/d or 500 g/d remains within international safety thresholds for all age groups, posing no non-carcinogenic risk. While these findings indicate safe consumption under current conditions, interpretation should consider the study’s limited sample size and the need for broader temporal monitoring. Under monitored ecological conditions, sustainable harvesting of O. niloticus from Lake Mariout can enhance human nutrition and regional food security, as the species remains a safe and nutrient-dense resource.