Bioaccumulation and Health Risk Assessment of Heavy Metals in Labeo rohita and Mystus seenghala from Jhelum River, Punjab, Pakistan

Abstract

The bioaccumulation of trace elements and heavy metals in aquatic organisms is a critical environmental concern due to its potential impact on ecosystem health and human safety. This study investigated the level of trace elements and heavy metals bioaccumulation in Labeo rohita and Mystus seenghala from the River Jhelum in the district Khushab, Punjab, Pakistan. The concentration of calcium, magnesium, iron, nickel, copper, arsenic, cadmium, zinc, chromium, manganese, cobalt, and lead in the gills, liver, and muscle tissues of these fish was measured using inductively coupled plasma-mass spectrometry. Then, the extent of contamination and its possible health risks were assayed. Our findings indicate significant variations in the elemental and metal concentrations among different organs and between species, reflecting their diverse feeding habits and habitats. The health risk assessment based on the estimated daily intake, estimated weekly intake, maximum permissible intake, target hazard quotient, hazard index or total target hazard quotient, health risk index, and target cancer risk revealed potential risks to human consumers of these fish. This study emphasizes the need for continuous monitoring, as new data and insights are crucial for understanding and mitigating these risks. Strict regulatory measures are also necessary to safeguard public health and preserve the ecosystem of Jhelum River.

1. Introduction

Heavy metals have become an increasing global concern due to their persistence in the environment, impact on biogeochemical cycles, and associated ecological risks [1,2,3,4,5,6,7,8]. In aquatic ecosystems, heavy metals can originate from several sources including industrial discharges, mining activities, agricultural practices, urban runoff, inadequately treated sewage, atmospheric deposition, and the improper disposal of metal-containing products [9]. Once introduced into the aquatic environment, these metals can persist for long periods, leading to long-term ecological consequences [10,11,12,13,14,15]. These metals are typically classified into two categories: potentially toxic metals such as cadmium (Cd), lead (Pb), and nickel (Ni), and essential metals such as copper (Cu), zinc (Zn), iron (Fe), and manganese [16]. Potentially toxic metals pose significant health risks even at low concentrations when ingested over extended periods, potentially leading to serious health conditions including neurological disorders, kidney damage, and various forms of cancer. On the other hand, while essential metals are required for various biological functions, excessive intake can result in toxic effects, disrupting metabolic processes and leading to health issues such as gastrointestinal distress, liver damage, and other systemic effects [16,17,18].

Heavy metal pollution negatively impacts environmental health and biodiversity [13]. Moreover, heavy metals, such as mercury and lead, can accumulate in seafood, posing serious health risks like neurological disorders and cancer. Therefore, bioaccumulation and health risk assessments of heavy metals in finfish and shellfish are crucial to protect human health and environmental quality [12]. Prabakaran et al. [19] found that although zinc (Zn) was the most common metal in fish from the Gulf of Thailand, high levels of mercury (Hg) in some species posed potential health risks to consumers. In Ghana, Blankson et al. [20] observed higher metal accumulation in the fish liver and gills compared to the muscles; however, the health risk indices remained below critical thresholds, indicating no immediate health concerns. Ali et al. [21] reported that arsenic (As) was the most concentrated metal in fish from Bangladesh, with risk assessments indicating minimal health risks despite seasonal variations. Rani et al. [22] found that metal concentrations in brackish water fish from India were generally safe, but continuous monitoring was recommended due to potential long-term health impacts. Hashim et al. [23] showed that fish from the Zhob River had safe metal concentrations, although high levels in the water and soil pointed to potential risks. Köse [24] reported that while metal levels of fish in Türkiye’s Meriç Delta were within the permissible values, the high phosphorus content in water warranted further monitoring. Ray and Vashishth [25] emphasized the global concern of heavy metal bioaccumulation in fish and called for more research on its long-term health effects. Moreover, Habib et al. [26] found that wild fish in Pakistan had higher metal concentrations than farmed fish, although health risks were low, with target hazard quotient (THQ) values below 1. Therefore, assessing these metals helps ensure that seafood remains safe for consumption and informs regulations to maintain food safety standards. Furthermore, high levels of heavy metals in aquatic organisms can indicate broader environmental pollution, which affects entire ecosystems and wildlife [3,6,7,27,28,29].

The issue of heavy metals and trace elements in Pakistan’s water ecosystem is a significant environmental and public health concern. Previous studies have documented a significant deterioration in the quality of aquatic environments in Pakistan due to the direct discharge of toxic chemicals into rivers [30,31]. This pollution can turn these water bodies into death traps for aquatic organisms including fish. Fish are particularly vulnerable to exposure of various toxic elements through bioaccumulation processes within the food chain, potentially becoming hazardous to human health upon consumption [32,33].

The Jhelum River, flowing through Punjab, Pakistan, is a crucial waterway that supports a diverse aquatic ecosystem and is a significant resource for local communities. However, increasing pollution from various anthropogenic activities has compromised the health of this river [34]. It has been observed that the waste from the markets across the district of Khushab is dumped into the river. These markets are located in Jauharabad, Quaidabad, Khushab, and Noshehra. The most notable sources of pollution in the Jhelum river in district Khushab are wastes from markets, agricultural runoff, and domestic waste. Recent studies on the physicochemical characterization of the Jhelum River have indicated varying ranges of various physical and chemical parameters at different sites. However, most of these studies have focused on seasonal fluctuations and water suitability indices at different locations including Azad Jammu and Kashmir, the Kashmir Valley, Kashmir Himalayas, and northern Punjab [35,36,37]. Javed et al. [38,39,40] observed a high contamination degree and moderate risks of potentially toxic elements in the Jhelum River in the district of Jhang. However, studies concerning trace elements or heavy metals in the Jhelum River basin at Khushab are still scarce. The current study is the first of its nature on the Jhelum River in the district of Khushab.

In this study, we focused on two economically and ecologically significant fish species, Labeo rohita (rohu) and Mystus seenghala (singhari), which are commonly found in the Jhelum River and are vital to local fisheries. These species are also a key part of the diet for many local residents. By analyzing the concentrations of heavy elements in different organs of these fish species, specifically the liver, muscles, and gills, this research aims to provide a comprehensive assessment of the contamination levels and potential health risks.

Due to their physiological roles, the liver, muscles, and gills are important organs for metal bioaccumulation [3,6,41,42,43,44]. The liver often acts as a detoxification and storage site for metals [45], while the muscles and gills are involved in nutrient absorption and respiration, respectively [46]. Understanding the distribution of heavy metals in these organs can offer insights into the extent of contamination and the potential health risks posed to humans who consume these fish [47].

Studies concerning the bioaccumulation of heavy metals in fish tissues have been regularly conducted on different fish species across the globe as well as Pakistan. However, there is not a single study on the Jhelum River in the district of Khushab. Therefore, this study was aimed to determine the concentrations of trace elements and heavy metals (Ca, Mg, Fe, Ni, Cu, As, Cd, Zn, Cr, Mn, Co, and Pb) in the dominant fish species of the Jhelum River in the district of Khushab and assess the associated health risks. By comparing these concentrations with the established safety thresholds, the research will evaluate the potential health impacts on consumers and provide recommendations for mitigating pollution and ensuring safe fish consumption.

2. Materials and Methods

2.1. Study Area

The Jhelum River is a significant river within the Indus River system. Originating in the Indian-administered territory of Jammu and Kashmir, it flows through Pakistan’s Punjab province, passing through various ecological zones. The Jhelum River stretches approximately 725 km and has a catchment area that includes parts of both India and Pakistan. The region experiences a subtropical climate, with hot summers and mild winters. Monsoon rains from July to September significantly impact the river’s flow, leading to seasonal water levels and quality variations.

The Jhelum River basin is home to a rich biodiversity including numerous fish species, aquatic plants, and invertebrates. This diverse ecosystem highlights the river’s ecological importance and its role in supporting local livelihoods and environmental health.

2.2. Sampling of Fish

For this study, fish were sampled from different sites in district Khushab along the Jhelum River in Punjab, Pakistan. This site was strategically chosen because it is situated downstream from several industrial zones and agricultural fields, making it an essential location for evaluating the impact of pollutants on aquatic life. The area is influenced by a combination of industrial effluent, agricultural runoff, and domestic waste, which contribute to various contaminants in the water. Two fish species, Labeo rohita (Rohu) and Mystus seenghala (Singhara), were selected for sampling due to their ecological significance and higher consumption rates among the local population. Fishes (nine specimens of each fish species were collected from three different sites such as upstream, mid-stream, and downstream and a total of 27 specimens were collected for each species; n = 27; N = 54) were collected between November 2022 and January 2023 with the help of local professional fishermen, who used cast nets for fish collection. After collection, the dead fish samples were placed in clean, ice-filled containers. The specimens were placed in ice from all sides to avoid contact of the fish specimens with the walls of the container. These specimens were then transported to the “Fisheries and Aquaculture Research Laboratory (Headed by Dr. Sana Ullah)” at the University of Education (Jauharabad Campus) for subsequent analysis.

2.3. Preparation of Samples

The collected fish specimens were rinsed with distilled water to remove surface contaminants. Scales were removed using a stainless steel knife, and tissues such as the muscles, gills, and liver were dissected out. These samples were then weighed, packed, and stored at −20 °C until further analysis. The aqua regia method was employed for digestion, which involves a mixture of concentrated nitric and hydrochloric acids handled with extreme care. The defrosted tissues were washed, blotted, and transferred to digestion flasks. About 2 g of each sample was weighed and digested in a 3:1 solution of HCl (Sigma-Aldrich) and HNO₃ (Sigma-Aldrich). The mixture was heated until reduced to 5 mL, cooled to room temperature, and filtered (through 0.45 µm membrane filters—nylon) to remove the undigested material. The filtrate was diluted to 50 mL with distilled water [48].

2.4. Heavy Metal Analysis

The samples were tested for essential elements [calcium (Ca), magnesium (Mg), iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn)], trace element [nickel (Ni)], and heavy metals [arsenic (As), cadmium (Cd), lead (Pb), and chromium (Cr)] at the Hi-Tech Laboratory of Government College University, Faisalabad, using inductively coupled plasma-mass spectrometry (ICP-MS). The regulatory guidelines of ASTM [49] were followed. To determine the accuracy of the heavy metal results, the following points were considered: (1) Quality control measures including calibration verification (analyzing standards before and after sample analysis), instrument performance check (monitoring sensitivity, resolution, and mass axis stability), and method validation (verification of method detection limits, precision, and accuracy); (2) Interference checks including spectral interferences (monitor potential overlaps) and matrix effects (evaluation of sample matrix impact on analyte signals); (3) Contamination control include blank samples (analysis of procedural blanks to detect contamination), duplicates (running duplicate samples for assessing precision), and spikes (spiked samples were analyzed to evaluate recovery); and (4) Data evaluation including signal-to-noise ratio (S/N), isotopic ration, and result validation. The common interference in ICP-MS used was spectral (argon-based polyatomic ions such as ArO⁺ or ArN⁺, and/or molecular ions such as NO⁺). For the mitigation strategy, matrix-matching standards (for improved accuracy, reduced matrix interference, and enhanced robustness of the method) were employed.

2.5. Human Health Risk Assessment

Evaluating the health risks associated with fish consumption was a vital component of this research. This assessment employed various indices such as the estimated daily intake (EDI) [1], estimated weekly intake [50], target hazard quotient (THQ), total target hazard quotient (TTHQ) or hazard index (HI) [32,41,45,46,47], health risk index (HRI) [48,50,51], target cancer risk (TR) [52,53,54], and maximum permissible intake (MPI) [39,55]. These indices provide a comprehensive understanding of the potential exposure to contaminants and were calculated using data derived from the muscle samples.

2.5.1. Consumption Data

Due to the lack of existing data on daily fish consumption in the study area, a survey was conducted to gather this information. The survey found that a total of 50,000 kg of fish was sold daily within the district (unpublished data). By dividing this quantity by the district’s total population, it was estimated that an average of 39.03 g of fish was consumed per person per day in the district Khushab.

2.5.2. Estimated Daily Intake of Heavy Metals

The estimated daily intake (EDI) of trace elements and heavy metals through fish consumption was calculated using the metal concentrations in the edible part (muscle tissues—flesh—because of its consumption), daily consumption rates, and body weight. The following equation was used to calculate the EDI [51,54].

EDI = (FIR × C)/BW

where FIR is the food ingestion rate (39.03 g), C is the metal concentration in the samples (mg/g), and BW is the body weight of the consumer (70 kg) [52,53].

The estimated weekly intake was calculated using the following equation [50]

EWI = EDI × 7

The estimated daily intake (EDI) can be compared to the provisional tolerable daily Intake (PTDI). In contrast, the EWI can be compared to the provisional tolerable weekly intake (PTWI) to assess the potential human health risks.

2.5.3. Target Hazard Quotient (THQ)

The THQ can be calculated by using the following equation [50]

THQ = (C × IR × EF × ED)/(BW × RfD × AT)

where C represents the contaminant concentration in fish (mg/g), IR is the ingestion rate of fish (g/day), EF denotes the exposure frequency (365 days/year), ED is the exposure duration (67 years), BW refers to the body weight of the consumer (70 kg), RfD is the reference dose (mg/kg/day) [39,40], and AT is the averaging time (EF × ED) [55]. A THQ value less than 1 indicates a negligible risk, while a THQ value greater than 1 indicates a significant health risk [56,57].

2.5.4. Hazard Index (HI)

The hazard index (HI) was calculated by summing the target hazard quotient (THQ) values for each contaminant. The formula for HI is provided in the equation below [58].

Hazard Index HI = ∑THQ

If the HI is less than 1, the risk is considered low. If it exceeds 1, there may be a significant health risk [59].

2.5.5. Daily Metal Intake/Health Risk Index (HRI)

The health risk index (HRI) measures the potential health risk of consuming contaminants and is calculated using the daily metal intake (DIM) from food and the oral reference dose (RfD).

HRI = DIM/RfD

If the HRI is below 1, the risk is minimal. If the HRI is above 1, there is a significant health hazard [57,60].

2.5.6. Target Carcinogenic Risk (TR)

The target carcinogenic risk (TR) factor represents the increased likelihood of an individual developing cancer over their lifetime due to exposure to a potential carcinogen. The TR was calculated using the formula:

TR = (C × IR × CPSo × EF × ED)/(BW × AT) × 10⁻³

In this formula, C is the concentration of the carcinogenic contaminant in the fish (mg/g), IR is the ingestion rate of fish (g/day), EF is the exposure frequency (days/year), ED is the exposure duration (years), BW represents the body weight of the consumer (in kg), AT stands for the time period over which the average exposure is calculated (in days), and CPSo is the carcinogenic potency slope via oral exposure (mg/kg/BW/day) [61,62].

If the TCR value is below 10⁻⁶, the hazard is considered negligible. A TCR value between 10⁻⁶ and 10⁻⁴ indicates a low hazard, while a TCR value above 10⁻⁴ is classified as a high risk [63,64].

2.5.7. Metal Pollution Index (MPI)

The metal pollution index (MPI) is a measure used to estimate the overall bioaccumulation of trace elements and heavy metals in different fish tissues. It is calculated using the formula:

MPI = M1 × M2 × M3 … Mn^1/n

where M1, M2, M3, …, Mn represent the concentrations of various metals in the tissues, and (n) is the number of metals analyzed [65,66].

2.6. Statistical Analysis

Analyses were carried out to statistically compare the results observed to determine whether there were significant differences in the elemental and metal concentrations across the liver, gills, and muscles of the studied fish species. Analysis of variance (ANOVA) was performed followed by least significant difference (LSD) to test the homogeneity of variance (multiple variance analysis) [67] at a significance level (p value) of less than 0.05. Furthermore, the two-sample t-test was performed to compare the mean concentration of the studied elements and metals between Labeo rohita and Mystus seenghala at significance levels (p value) less than 0.001, 0.01, and 0.05. Pearson correlation was carried out to study the correlation of the studied heavy metals across the sampled tissues in both fish species. The statistical analysis was carried out using MS Excel (2016) and Statistix (V. 9).

3. Results

3.1. Heavy Metal Concentration

The average concentrations of the studied trace elements and heavy metals in the gills, liver, and muscles of Labeo rohita and Mystus seenghala are provided in Table S1. In the gill tissue of Mystus seenghala, the bioaccumulation of metals followed the order: Ca, Mg, Fe, Ni, Cu, Cr, Mn, Co, As, Cd, Zn, and Pb. In the liver tissue, the order was Mg, Ca, Fe, Cr, Cu, Ni, As, Co, Mn, Cd, Zn, and Pb. For the muscle tissue, the sequence was Mg, Ca, Ni, Cr, Cu, Fe, As, Mn, Co, Cd, Zn, and Pb, while for Labeo rohita, the sequence of metal bioaccumulation in the gill tissue was Mg, Ca, Fe, Mn, Ni, As, Cu, Cd, Cr, Co, Zn, and Pb. In the liver tissue, the order was Mg, Ca, Fe, Mn, Ni, Co, Cd, As, Cu, Zn, Cr, and Pb, and in the muscle tissue, the metals bioaccumulated in the following sequence: Mg, Ca, Fe, Mn, Ni, Cr, Cu, Co, As, Cd, Zn, and Pb.

The tissue-wise comparisons of the accumulation patterns of the studied element and heavy metals within the same species of L. rohita and M. seenghala are illustrated in Figure 1 and Figure 2, respectively.

Table 1. Correlation coefficient matrix of the studied trace elements and heavy metals in the muscle tissues of Labeo rohita.

Heavy Metals	Zn	Cd	Pb	Cr	Co	Ni	Mn	Cu	As	Fe	Mg	Ca
Zn	1
Cd	0.6732	1
Pb	−0.9983	−0.7145	1
Cr	−0.6776	−0.9998	0.7187	1
Co	0.9752	0.8201	−0.9863	−0.8235	1
Ni	0.7545	0.9932	−0.7910	−0.9939	0.8810	1
Mn	0.1470	−0.6325	−0.0899	0.6278	−0.0755	−0.5382	1
Cu	0.9991	0.6412	−0.9950	−0.6459	0.9650	0.7261	0.1888	1
As	−0.0232	−0.7549	0.0806	0.7509	−0.2437	−0.6736	0.9855	0.0192	1
Fe	0.1666	0.8413	−0.2230	−0.8380	0.3806	0.7728	−0.9508	0.1247	−0.9896	1
Mg	−0.1145	−0.8117	0.1714	0.8082	−0.3314	−0.7383	0.9658	−0.0723	0.9958	−0.9986	1
Ca	0.7385	−0.0014	−0.6986	−0.0046	0.5711	0.1148	0.7754	0.7664	0.6569	−0.5418	0.5852	1

Note: Bold r-values > 0.500 are significant at p < 0.05.

and

Table 2. Correlation coefficient matrix of the studied trace elements and heavy metals in the muscle tissues of M. seenghala.

Heavy Metals	Zn	Cd	Pb	Cr	Co	Ni	Mn	Cu	As	Fe	Mg	Ca
Zn	1
Cd	−0.3770	1
Pb	0.9450	−0.6592	1
Cr	0.9744	−0.1592	0.8473	1
Co	0.9280	−0.6950	0.9988	0.8205	1
Ni	−0.7950	0.8616	−0.9497	−0.6383	−0.9638	1
Mn	−0.1623	0.9751	−0.4762	0.0636	−0.5184	0.7276	1
Cu	−0.1996	0.9828	−0.5091	0.0258	−0.5504	0.7531	0.9993	1
As	−0.1968	0.9823	−0.5066	0.0286	−0.5480	0.7512	0.9994	0.999996	1
Fe	−0.6508	−0.4578	−0.3667	−0.8048	−0.3210	0.0568	−0.6435	−0.6141	−0.6163	1
Mg	−0.2786	0.9946	−0.5774	−0.0556	−0.6164	0.8041	0.9929	0.9967	0.9965	−0.5478	1
Ca	−0.8371	0.8223	−0.9700	−0.6927	−0.9807	0.9973	0.6757	0.7031	0.7011	0.1294	0.7586	1

Note: Bold r-values > 0.500 are significant at p < 0.05.

show the correlation coefficient matrix of the studied elements and heavy metals in Labeo rohita and Mystus seenghala, respectively. Tables S2 and S3 show the correlation coefficient matrix of the studied elements and heavy metals in liver and gills of Labeo rohita, respectively. Tables S4 and S5 show the correlation coefficient matrix of the studied elements and heavy metals in the liver and gills of Mystus seenghala, respectively.

Figure 3, Figure 4 and Figure 5 illustrate the tissue-wise comparison of the bioaccumulation patterns of the studied trace elements and heavy metals across species of L. rohita and M. seenghala.

3.2. Comparison of the Health Risks Associated with the Consumption of Labeo rohita and Mystus seenghala

The EDI and EWI values for Labeo rohita and Mystus seenghala were determined and contrasted with the PTDI and PTWI values, as shown in

Table 3. EDI and EWI values of L. rohita and M. seenghala.

Trace Elements and Heavy Metals	PTDI (mg/day/kg)	PTWI (mg/day/kg)	L. rohita		M. seenghala
			EDI	EWI	EDI	EWI
Zn	1	7	0.4503	3.1522	0.1234	0.8641
Cd	0.001	0.007	0.6320	4.4237	0.1814	1.2701
Pb	0.004	0.025	0.3064	2.1445	0.1118	0.7824
Cr	0.003	0.021	1.1111	7.7774	2.9396	20.5771
Co	0.06	0.42	0.8114	5.6801	0.8037	5.6261
Ni	0.005	0.035	1.2724	8.9068	3.3562	23.4933
Mn	0.14	0.98	1.2747	8.9228	0.8682	6.0775
Cu	0.5	3.5	0.8930	6.2507	2.4472	17.1307
As	0.002	0.015	0.7815	5.4705	2.0483	14.3383
Fe	0.8	5.6	1.8229	12.7605	2.3732	16.6123
Mg	5.833	40.83	13.8522	96.9651	50.2078	351.4548
Ca	14.19	99.33	12.3932	86.7523	29.8192	208.7343

.

Table 4. Comparison of the TR values of L. rohita and M. seenghala.

Trace Elements and Heavy Metals	CPSo	L. rohita	M. seenghala
Cd	6.3	3.981323 × 10−3	1.143088 × 10−3
Pb	0.0085	2.60409 × 10−6	9.50021 × 10−7
Ni	1.7	2.163089 × 10−3	5.705512 × 10−3
As	1.5	1.172257 × 10−3	3.072494 × 10−3

provides a comparison of the TR values for L. rohita and M. seenghala. Additionally, the THQ, TTHQ, HRI, and MPI values for both species are compared in

Table 5. Comparison of the THQ, TTHQ, HRI and MPI values of L. rohita and M. seenghala.

Trace Elements and Heavy Metals	L. rohita		M. seenghala
	THQ	HRI	THQ	HRI
Zn	0.0015	1.5011	0.0004	0.4115
Cd	0.6320	631.9560	0.1814	181.4425
Pb	0.0766	76.5910	0.0279	27.9418
Cr	0.3704	370.3526	0.9799	979.8626
Co	0.0135	13.5240	0.0134	13.3956
Ni	0.0636	63.6203	0.1678	167.8092
Mn	0.0091	9.1049	0.0062	6.2016
Cu	0.0223	22.3238	0.0612	61.1810
As	2.6050	2605.0166	6.8278	6827.7645
Fe	0.0026	2.6042	0.0034	3.3903
Mg	0.0024	2.3760	0.0086	8.6120
Ca	0.0009	0.9297	0.0022	2.2370
TTHQ (HI)	3.800		8.28
MPI	1796.59		8856.41

.

4. Discussion

Trace and heavy metals in aquatic environments pose significant health risks to both aquatic organisms and humans [2]. These metals can accumulate in fish tissues through various pathways such as water, sediment, and diet [68]. This bioaccumulation is influenced by factors like water quality, seasonal changes, fish species, and their life stages [69]. Trace elements and heavy metals are persistent in the environment and can magnify through the food chain, leading to elevated concentrations in top predators and consumers including humans [1].

Fish is a major source of easily digestible protein, supplying essential amino acids, fats, macro and trace elements, fat-soluble vitamins, and long-chain polyunsaturated omega-3 fatty acids. Omega-3 fatty acids like eicosapentaenoic acid (EPA, C20:5 n-3), docosahexaenoic acid (DHA, C22:6 n-3), and docosapentaenoic acid (DPA, C22:5 n-3) are crucial for reducing the risk of cancer, cardiovascular diseases, and neurological disorders [70]. With growing awareness of its nutritional and therapeutic benefits, global fish consumption has increased rapidly in recent years [16]. The American Heart Association recommends eating fish at least twice a week to meet the daily intake of omega-3 fatty acids [71]. However, the presence of toxic levels of heavy elements in fish can diminish these beneficial effects [33].

Studies have shown that the levels of heavy metals in fish can vary significantly based on geographical location, industrial activities, and agricultural practices [72]. In this study, we examined the concentrations of trace elements and heavy metals in the gills, liver, and muscle tissues of L. rohita and M. seenghala. One-way ANOVA tests revealed significant differences in the elemental and metal concentrations across the different tissues of the fish species, with a significance level of p < 0.05. A two-sample t-test demonstrated that the concentrations of Cr and Mg in the liver, Co, Cu, Ni, As, and Fe in the muscles, and Cu in the gills varied significantly across the species at a significance level of p < 0.05. Additionally, the concentration of Ca in the liver showed a significant difference at a significance level of p < 0.01, assuming equal variance. The levels of all the elements and metals analyzed in L. rohita and M. seenghala exceeded the international limits listed in

Table 6. Concentrations of trace elements and heavy metals in Labeo rohita and Mystus seenghala compared with international standards.

Trace Elements and Heavy Metals	Labeo rohita	Mystus seenghala	MPL (mg/g)	Reference
Zn	0.808	0.221	0.03	[73]
Cd	1.133	0.325	0.0005	[73]
Pb	0.549	0.200	0.0005	[73]
Cr	1.993	5.272	0.05	[73]
Co	1.455	1.441	0.0005	[73]
Ni	2.282	6.019	0.0005–0.0006	[74]
Mn	2.286	1.557	0.001	[75,76,77]
Cu	1.602	4.389	0.03	[75,76,77]
As	1.402	3.674	0.001	[75,76,77]
Fe	3.269	4.256	0.1	[75,76,77]

.

In this study, we found that the liver tissues had a high bioaccumulation of trace elements and heavy metals, consistent with findings from previous research [12,78]. Among all of the studied elements, Ca, Mg, and Fe were found to be highly bioaccumulated in both fish species because of their crucial roles in bone formation, muscle function, and oxygen transport [79]. These essential biological processes require increased uptake and storage of these metals, and this necessity leads to their significant presence in the tissues. Zn, Cd, and Pb were found to be less bioaccumulated in both fish species compared to other metals. In the liver, their concentrations were 1.220, 1.854, and 0.687 mg/g, respectively, in L. rohita, and 0.361, 0.530, and 0.312 mg/g, respectively, in M. seenghala. In the muscle tissues, L. rohita had concentrations of 0.808, 1.133, and 0.549 mg/g, while M. seenghala had lower levels of 0.221, 0.325, and 0.200 mg/g. In the gills, L. rohita showed concentrations of 0.332, 0.857, and 0.271 mg/g, whereas M. seenghala had even lower levels of 0.124, 0.156, and 0.116 mg/g. The results showed that Zn, Cd, and Pb were found to be higher in L. rohita compared to M. seenghala in all of the studied tissues. This difference might be due to variations in their metabolic rates and physiological mechanisms, leading L. rohita to have a greater capacity for the uptake and retention of these metals [65,66,80,81,82]. In M. seenghala, Ni, Cr, and Cu were found to be highly accumulated in the muscle tissues, with concentrations of 6.019 mg/g, 5.272 mg/g, and 4.389 mg/g, respectively. In the liver, their concentrations were 3.924 mg/g, 5.568 mg/g, and 5.118 mg/g, while in the gills, these were 3.184 mg/g, 1.855 mg/g, and 3.086 mg/g, respectively. These metals are essential for various biological functions in animals and humans, while their excessive intake can lead to adverse health effects. High levels of these metals can cause toxicity and disrupt physiological processes, potentially leading to health issues such as organ damage and metabolic disturbances [52]. The bioaccumulation of heavy elements in L. rohita investigated in the present study was found to be higher than that reported by scientists [83,84,85,86], while the concentration of heavy metals (Fe, Cu, Zn, Mn, Cd, Co, Ni, Pb, and Cr) in M. seenghala was found to be higher than that reported by Sofia and Teresa [87]. The levels of Cu, Zn, Cd, and Pb found in M. seenghala in this study were higher than those reported by Kumar et al. [84,88]. This study indicates that M. seenghala has higher concentrations of trace elements compared to L. rohita. The primary reasons for this difference are the distinct dietary habits and ecological roles of the two species. M. seenghala is a carnivorous fish, consuming other aquatic organisms that may already contain heavy elements, leading to bioaccumulation [89]. Moreover, as a bottom feeder, M. seenghala ingests sediments from the river or lakebed, where heavy metals often settle [90]. Therefore, M. seenghala may be more vulnerable to heavy metal bioaccumulation because of its slow metabolism and growth rate [91].

For the health risk assessment, we used various health risk indices to estimate the level of risk posed to human health by consuming these fish species. We compared the estimated daily intake (EDI) with the provisional tolerable daily intake (PTDI) values and the estimated weekly intake [50] with the provisional tolerable weekly intake (PTWI) values for both fish species (Table 3). Based on the comparisons, it was evident that both fish species, L. rohita and M. seenghala, contained heavy metals in concentrations that generally exceeded the provisional tolerable intake limits for most of the analyzed metals [46,47,48,50,51,52,53,54,66]. In general, M. seenghala had higher EDI and EWI values for Cr, Co, Ni, Fe, Cu, Mg, and Ca compared to L. rohita, while some heavy elements like Zn in both fish species and Ca in L. rohita were within the safe limits.

Table 4 shows the TR value, which revealed that cadmium, nickel, and arsenic were associated with high carcinogenic risks in both fish species, with values significantly above 10⁻⁴, while lead showed a low hazard.

Table 5 shows the THQ, TTHQ, HRI, and MPI values of both fish species. The total target hazard quotient, indicating a considerable cumulative health risk, was significantly greater than 1 [46,47], with values 3.800 for L. rohita and 8.28 for M. seenghala. L. rohita showed an MPI value of 1796.59, reflecting a high level of metal pollution, whereas M. seenghala presented a value of 8856.41, indicating an even higher level of metal pollution. Both fish species had HRI values for several metals (Cd, Pb, Cr, Co, Ni, Cu, and As) that far exceeded the standard value of 1 [51,66], which shows a significant risk. Arsenic was the only heavy metal with a THQ value above 1, which was 2.6500 and 0.6828 in L. rohita and M. seenghala, respectively. M. seenghala generally exhibited higher HI, THQ, HRI, and MPI values compared to L. rohita, indicating a greater health risk from its consumption.

5. Conclusions

The investigation into the bioaccumulation of trace elements and heavy metals in Labeo rohita and Mystus seenghala from the Jhelum River has revealed critical insights into the extent of contamination and the associated health risks. The study identified significant trace elements and heavy metal bioaccumulation in the gills, liver, and muscle tissues of both fish species. The concentrations of all of the studied elements and metals were found to be higher than the international standards. The health risk assessment demonstrates potential hazards for humans consuming these fish, highlighting the need for comprehensive and continuous environmental monitoring, effective pollution control strategies, and increased public awareness. Collaborative efforts between governmental bodies, environmental organizations, and local communities are essential to address and mitigate the impacts of heavy metal pollution. Future research should aim at developing sustainable solutions and innovative approaches to ensure the long-term health and safety of both aquatic life and human populations relying on these ecosystems.