Materials and methods

Study area

Fish samples were collected from local fish markets in Mansoura City, Egypt (Fig. 1). Mansoura lies on the Nile’s east bank, approximately 120 km northeast of Cairo28. The city serves as a major commercial hub with high fish consumption. Fish markets in Mansoura provide a wide variety of marine fish, either from the Egyptian Mediterranean coastal fisheries, mainly Kafr El Sheikh, El Bahira, Damietta, and Port Said governorates, or imported frozen fish. The mackerel samples (Scomber scombrus) were commercially available imported frozen fish, primarily sourced from Norway and the Netherlands, according to vendors’ information.

Fig. 1: Map of the sampling areas for the fish analyzed in the present study. Satellite basemap imagery was obtained from Google Maps (Google LLC; https://www.google.com/maps/). Labels, sampling-site coordinates, arrows, and other graphical elements were added by the authors using Microsoft PowerPoint (Version 2021; Microsoft Corporation).

Sample collection

A total of 250 fish samples, including 50 flathead grey mullet (Mugil cephalus) and 100 each of sardine (Sardinella aurita) and mackerel (Scomber scombrus) with different lengths and weights, were collected from six retail fish outlets in East Mansoura City, Egypt (Fig. 1), across ten sampling occasions between August 2023 and March 2024. During each sampling occasion, five outlets were randomly selected from which one mullet, two sardines, and two mackerel were purchased per outlet. Total body length ranged from 31.0 to 43.0 cm for flathead grey mullet, 10.7 to 14.0 cm for sardine, and 27.0 to 39.0 cm for mackerel. Body weight ranged from 332.0 to 485.0 g for flathead grey mullet, 19.0 to 43.0 g for sardine, and 394.0 to 517.0 g for mackerel. Each sample was individually placed in an impermeable polyethylene bag, placed in an icebox (4 °C), and immediately transported to the Department of Food Hygiene, Safety, and Technology, Faculty of Veterinary Medicine, Mansoura University, Egypt. At the laboratory, each sample was weighed by a digital balance, transferred to a clean polyethylene bag, then labeled with the collection date and identification number, and kept at -20 °C till HMs are analyzed. As the study involved only commercially available fish samples obtained from retail markets and did not involve human participants or live animal experimentation, ethical approval for human or animal subjects was not applicable. The study was conducted in accordance with the research integrity and ethical guidelines of Mansoura University. The graphical abstract summarizing the plan of the current study is presented in Fig. 2.

Fig. 2: A graphical abstract of the study design, workflow, and results.

Chemicals and laboratory wares preparation

All chemicals used were of ultrapure grade (Merck KGaA, Darmstadt, Germany), including perchloric acid (70%), nitric acid (65%), hydrochloric acid (37%), and hydrogen peroxide (30%). All laboratory wares used for fish sample digestion, handling, and storage were thoroughly cleaned by soaking in soap and water for at least 2 h, followed by several rinses with running tap water. The wares were then sequentially rinsed once with deionized water, once with Therands mixture [250 ml deionized water + 200 ml concentrated HCl (37%) + 80 ml H₂O₂ (30%)], once with washing acid [900 ml deionized water + 100 ml concentrated HCl (37%)], and finally with deionized water, following the procedure described by Sallam et al.29, after cleaning, the wares were dried in an incubator until use.

Sample digestion

Fish tissue samples were individually digested following the wet digestion technique previously outlined by Sallam et al.29. In brief, 2 g portions of dorsal muscle along with the overlying skin from each selected sample were excised aseptically with a sterile stainless-steel scalpel and forceps, macerated using a ceramic knife, and put into a screw-capped tube containing 8 ml of nitric acid (65%) and 4 ml of perchloric acid (70%). To ensure laboratory safety and allow gas release during acid reaction, the tubes were loosely covered and incubated in a water bath at 53 °C overnight. This procedure resulted in a clear, colorless digest with no visible residual organic matter. All tubes were then allowed to cool to room temperature. The cooled digest was then diluted with deionized water and filtered through a Whatman filter number 42 (Merck, Darmstadt, Germany) into clean glass beakers. The filtrate was subsequently diluted with deionized water to a final volume of 50 ml. The resultant filtrates were placed into clean, screw-capped bottles, labeled with the fish species and sample number, and stored at room temperature until HMs analysis. Blank solutions were prepared following the same wet digestion procedure, but without adding samples to detect any background contamination from reagents or equipment. To guarantee the accuracy of HMs determination, any detected residues were deducted from the final results.

Analysis of HMs

Heavy metals (Hg, As, Pb, and Cd) were quantified using an Atomic Absorption Spectrophotometer (AAS) (Buck Scientific 210 VGP, Inc.) at the Central Laboratory, Faculty of Veterinary Medicine, Zagazig University, Egypt, following the method described by AOAC30. Hg and As were analysed by a flameless AAS equipped with a hydride generation system for As and a cold vapor system for Hg. Meanwhile, Pb and Cd were analysed by flame AAS with an oxidizing air–acetylene flame. The apparatus was set to detect Hg, As, Pb, and Cd at wavelengths of 253.7 nm, 193.7 nm, 283.3 nm, and 228.8 nm, respectively. The detection limits (µg/g) for these metals.

HM concentrations were expressed as µg/g wet weight (ppm). They were obtained from the digital readout of the AAS and calculated using Eq. (1):where C represents the heavy metal concentration (µg/g wet weight), R is the reading (ppm) from the digital scale of AAS, D is the dilution factor of the measured sample, and W is the sample weight (g).

1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C=~\frac{{R \times D}}{W}$$\end{document}

In addition, blank sample readings were measured and subtracted from all sample measurements to determine any potential contamination.

Validation of analytical methods for HM determination in fish samples

The validation of analytical parameters used for HM quantification in fish samples, including calibration curves’ linearity, limit of detection (LOD), and limit of quantification (LOQ), instrumental precision, spike recovery %, and precision of certified reference material (CRM), is presented in Table 1.

 | Precision criteria** | Heavy metals concentrations (µg/g)
Hg | As | Pb | Cd
Instrumental precision | LOD | 0.017 | 0.194 | 0.132 | 0.010
LOQ | 0.056 | 0.640 | 0.436 | 0.033
Precision (CV %) | 2.25 | 2.84 | 2.12 | 0.79
Mean of spiked amount * | 0.375 ± 0.03 | 1.50 ± 0.04 | 0.500 ± 0.02 | 0.25 ± 0.03
Mean of recovered amount * | 0.362 ± 0.02 | 1.52 ± 0.03 | 0.494 ± 0.02 | 0.243 ± 0.02
Mean of spike recovery % | 96.5% | 101.3% | 98.8% | 97.2%
Calibration curve | Standard solution concentrations (mg/L) for the calibration curve*** | 0.005, 0.01, 0.05, 0.1, 0.5, 2.0, 5.0 | 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 20.0 | 0.005, 0.01, 0.05, 0.25, 0.5, 5, 10 | 0.005, 0.01, 0.05, 0.25, 0.5, 2.0, 5
Correlation coefficient (R2) | 0.999 | 0.999 | 0.999 | 0.999
Precision (RSD %) of the digestion method for heavy metal determination in fish matrix (n = 5). | Concentration of metal in fish sample | 0.05 ± 0.01 | 2.69 ± 0.07 | 0.47 ± 0.03 | 0.04 ± 0.01
Amount of metal added | 0.15 | 5.0 | 2.0 | 0.15
Concentration of metal in the spiked sample | 0.196 ± 0.002 | 7.62 ± 0.09 | 2.38 ± 0.04 | 0.185 ± 0.004
Recovery (%) | 97.3 | 98.6 | 95.5 | 96.7
RSD (%) | 2.28 | 2.64 | 3.75 | 4.83
Certified reference material (CRM)**** validation | Certified value a | 0.44 ± 0.18 | 34.6 ± 2.4 | 0.162 ± 0.032 | 14.5 ± 0.6
Observed value b | 0.42 ± 0.03 | 34.3 ± 0.8 | 0.163 ± 0.01 | 14.1 ± 0.2
Recovery % | 95.5 | 99.1 | 100.6 | 97.2

To prepare the working standards, stock standard solutions (1000 mg/L) of Hg, As, Pb, and Cd were diluted with acidified ultrapure water (5% v/v HNO₃) according to AOAC Official Method31. Calibration curves were constructed using standard concentrations of 0.005, 0.01, 0.05, 0.1, 0.5, 2.0, and 5.0 mg/L for Hg; 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 20.0 mg/L for As; 0.005, 0.01, 0.05, 0.25, 0.5, 5, and 10 mg/L for Pb; and 0.005, 0.01, 0.05, 0.25, 0.5, 2.0, and 5.0 mg/L for Cd. The correlation coefficient (R²) for each calibration curve was 0.999, indicating excellent linearity.

For the instrumental precision, LOD and LOQ values were calculated using Eq. 2:where X represents either LOD or LOQ, F represents the factor of 3.3 for LOD and 10 for LOQ; SD: Standard deviation of the blank; b: Slope of the regression line.

2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X=~F~ \times \frac{{SD}}{b}$$\end{document}

To assess the digestion precision, fish samples were spiked with known concentrations of each metal (0.15 µg/g for Hg and Cd, 5 µg/g for As, and 2 µg/g for Pb) and analyzed following the same digestion and analytical procedures. The recovery rates for spiked samples were 97.3%, 98.6%, 95.5%, and 96.7% for Hg, As, Pb, and Cd, respectively (Table 1).

The accuracy of all analytical procedures was verified using DOLT-5, Dogfish liver CRM from the National Research Council of Canada (NRC-CNRC). The CRM recovery percentages ranged from 95.5% to 100.6% (Table 1).

Health risk assessment

Estimated daily intake (EDI)

The Estimated Daily Intake (EDI) of the analyzed HMs was calculated for both the general population and high fish consumers following Eq. (3), outlined by USEPA32.

3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$EDI=\frac{{{{\mathrm{C}}_{\mathrm{m}}} \times {\mathrm{IR}}}}{{BW}}$$\end{document}

EDI is the estimated daily intake (µg/g bw/day). Cm is the HM concentration in the sample and is expressed as µg/g wet weight. IR is the average fish consumption per day, which is 57.09 g/d for the general population2 and 200 g/d for high-fish consumers33. BW is the average body weight of Egyptian fish consumers (16–70 years), which equals 70 kg.

The EDIs were calculated and compared with their provisional tolerable daily intakes (PTDI) or benchmark dose levels (BMDL). The JECFA set the PTDI for MeHg and the BMDL for As at 2.30E−04 µg/g bw/day and 3.00E−03 µg/g bw/day, respectively34, while EFSA recommended the BMDL for Pb (6.30E−04 µg/g bw/day)35, and JECFA set the PTDI for Cd (8.30E−04 µg/g bw/day)36.

Assessment of Non-carcinogenic health risks using THQ and TTHQ

THQ and TTHQ are parameters used to assess the potential non-carcinogenic health risk linked to lifetime exposure to toxic metals from consuming fish. The THQ was determined using Eq. (4), previously described by USEPA32.

4THQ=EDI/RfD

where EDI refers to the estimated daily intake of HMs (µg/g/day), RfD (the oral reference dose) represents the estimated daily amount of contaminant that a person can ingest over a lifetime without causing any non-carcinogenic health risks. The RfD for Hg and that for As are 0.0001 and 0.0003, respectively26, while the RfD for Pb is 0.00437,38 and that for Cd is 0.00139.

Hazard Index (HI) or TTHQ was estimated using Eq. (5), endorsed by USEPA32.

5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{TTHQ}}={{{\Sigma}}}TH{Q_{Hg}}+TH{Q_{As}}+TH{Q_{Pb}}+TH{Q_{Cd}}$$\end{document}

THQ or TTHQ values > 1 indicate the potential for non-carcinogenic health risks associated with HM intake, with the risk increasing with higher values, whereas values below 1 reveal negligible risk to human health26.

Assessment of cancer risk (CR) in the general population and high-fish consumers

The probability of cancer occurrence over a lifetime because of the consumption of fish contaminated with HMs was calculated using Eq. (6), as described by the USEPA26.

6CR=CSF × EDI

where CR represents the cancer risk, CSF refers to the ingestion cancer slope factor (µg/g /day), and EDI is the estimated daily intake of HMs (µg/g/day). No oral CSF has been established for Hg in fish. The CSF values are 1.5 µg/g /day for inorganic As26, whereas CSF values of 0.0085 and 0.38 µg/g/day were adopted from published literature for Pb and Cd, respectively8,20,40.

The CR values between 1E−4 and 1E−6 are acceptable, whereas values exceeding 1E−4 indicate a potential carcinogenicity26.

Statistical analysis

All measurements were conducted in triplicate (analytical replicates), and the concentrations of HMs were expressed as means ± standard error (SE). Data were analyzed by one-way analysis of variance (ANOVA) to evaluate the differences in concentrations of HMs across all fish species examined. Mean differences were identified using Tukey’s honestly significant difference (HSD) test. Furthermore, Spearman’s rank correlation analysis was performed to evaluate the relationships between fish size parameters (total length and body weight) and heavy metal concentrations. Statistical significance was considered at P < 0.05 or P < 0.01. Data were analyzed by SPSS Statistics version 27.0 (IBM Corp., Armonk, NY, US).