Ecological and health risk assessment of heavy metals bioaccumulation in Ganges �sh near Varanasi, India

Heavy metal contamination in river Ganga is one of the factor for deterioration in its water quality, also adds to human health risks. We designed our study to achieve a holistic approach by not only estimating the concentration of heavy metals (Lead, Manganese, Chromium and Cadmium) in the river water at different sites based on human anthropogenic activities but also in the �shes residing in the same sites that are collected for human consumption on daily basis. We found, Ganga river in Varanasi is highly loaded with metals (PLI = 6.698) Mean concentration in water was: Pb 1.29 mg/L, Mn 1.325 mg/L, Cr 0.169 mg/L and Cd 0.161mg/L, which were above the permissible limits stated by Environment Protection Agency (EPA) in drinking water. Fishes including exotic and invasive species were collected from the wild and were processed for the presence of these metals in their living tissues. Degree of heavy metal concentration followed liver > gills > muscles. Highest accumulation of Pb was observed in Carpio (Cyprinus carpio) liver (8.86 µg/g) and lowest in Baikari (Clupisoma garua) muscles (0.07 µg/g). Total Target Hazard Quotient (THQ) value i.e. hazard index (HI) showed values in following sequence: C.carpio > O. nilotus > C.punctatus > J.coitor > M.armatus > M.tengara > Baikari. Maximum HI was recorded in Carpio, which is highly consumed �sh by human, hence may be harmful to them.


Introduction:
The worldwide issue of heavy metal pollution poses a signi cant threat to both aquatic and terrestrial ecosystems.Several rivers are known to be affected by this pollution with particular concern for those that hold sacred signi cance, as they are often subjected to extensive human activities and interventions.Our very prestigious and holy river Ganga is the largest river (travels 2525 km and covers 8,61,404 km 2 of the drainage basin, travelling through Uttarakhand, Uttar Pradesh, Bihar, Jharkhand and West Bengal, nally discharges its water into the Bay of Bengal) of the Indian subcontinent, with spiritual signi cance in Indian culture.Unfortunately, in recent times, the Ganga River has encountered signi cant sequential and spatial challenges concerning its water quality and biodiversity, primarily due to poor sanitation, agricultural runoff, household waste, and rapid anthropogenic activities.Among these challenges, heavy metal pollutants stand out as exceptionally persistent and long-lasting, remaining in the ecosystem and impacting various trophic levels (Maurya et al., 2016).Aquatic organisms including sh, zooplankton, benthic algae etc. are frequently exposed to heavy metals and show signi cant variance in metal accumulation.Furthermore, it adversely impacts the diversity of ora and fauna, leading to alterations in the physical, chemical, and biological properties of the river (Usmani et al., 2017).Various site and season speci c studies have consistently found that carcinogenic metals, such as Arsenic (As), Cadmium (Cd), Chromium (Cr), Mercury (Hg), Nickel (Ni), and Lead (Pb), exceed the World Health Organization's (WHO) permissible limit for drinking water in the Ganga River (Pandey et al., 2010;Katiyar, 2011) and concluded that Ganga water is un t for human consumption even for household works.
As the River Ganga is home to about 140 species of shes including exotic varieties, numerous hazardous elements bio-accumulate and biomagni es in the shes of Ganga (Mitra et al., 2012;Sudhakar et al., 2014).These sh species play a crucial role as prominent bio-indicators, re ecting the extent of metal pollution present in the ecosystem (Sarkar et al., 2012).Gills and oral ingestion of contaminated food and sediment particles are the main sources of entry, while the liver, kidneys, gills, muscles, skin and brain are the main organs of their accumulation in shes (Vaseem et al., 2013).As a potential source of protein, essential minerals, vitamins, and unsaturated fatty acids in the human diet, about 20 kg year − 1 per capita, Indian consumers alone account for 50% of this global consumption, contributing around 8-9 kg per year for those who include sh in their diet.(Medeirost al., 2012;FAO, 2022).From the contaminated shes, these metals can enter the human system and contribute to a serious health risk to a person through contaminated environmental and food exposure.
In 2010, the World Health Organization (WHO) identi ed lead (Pb), cadmium (Cd), and mercury (Hg) as three of the top ten chemicals with signi cant public health implications.These metals can adversely affect critical organs such as the kidneys, liver, and brain, leading to nephrotoxicity, hepatotoxicity, and neurotoxicity, respectively.Once inside the body, some metals have the ability to bind to enzymes containing sulfur, disturbing their normal functioning.Additionally, certain metals can induce oxidative stress by generating free radicals (Jaishankar et al., 2014;Maurya et al., 2016).The amount of heavy metals present in the water bodies reaches to human beings via consumable shes present in them.Therefore, shes are one of the primary source of entry of these metals.
In the past few years, several studies in various regions across the globe has highlighted, about the detrimental impacts of metals on both the water ecosystems and human health.Matta G et al, 2022 has studied the Himalayan region of Ganga river and its health risk for resident population, using Hazard Quotient (HQ) and Hazard index (HI).Han et al, 2021 investigated the levels of As, Cd, Cr, Hg and Pb in marine shes of Zhejiang province, which is a coastal area of East China Sea and evaluated the health risk based on daily consumption of these shes.The metals in question originate from both natural and anthropogenic sources.According to Hossain et al. 2023, Pb, As, Co, and Ni primarily stem from natural sources, while Cu, Mn, Zn, and Cr are mainly linked to anthropogenic activities, including industrial e uents, domestic wastewater, agricultural practices, vessel emissions, and river con uences.Nevertheless, some water bodies like Lake Hawassa, Ethiopia are free from metal pollution to the extent that consuming sh from them poses no health risk to humans (Melake et al, 2022).Thus regularly conducting studies on major river bodies is crucial to understand the potential harmful effects of heavy metals in river water and its associated health risks to human beings.The Minamata catastrophe in the 1950s in Japan, due to mercury poisoning, serves as a stark reminder of the devastating consequences of such contamination.Itai Itai disease is way similar which has also affected the mass population.
The current study was conducted in the Varanasi district of Uttar Pradesh, which is one of the busiest industrialized cities in India.The primary objective of the present study is to assess the presence of heavy metals especially Pb, Mn, Cr and Cd in river sediment, water and in tissues (gills, liver and muscle) of seven different consumable sh species, collected directly from river Ganga.This comprehensive study is the rst of its kind conducted in the Ganga River in Varanasi, providing insights into the extent of heavy metal pollution in the environment and its potential impact on human health.
Furthermore, the study aimed to determine the ecological risk using sediment quality indices and assess the potential health risk for individuals who consume these sh contaminated with metals.

Study area
Present study is done in Varanasi (25° 20'N and 83° 00'E) an industrially and spiritually important resident hub of north India.The district is registered with several small and large-scale industries including carpet, textiles, Diesel and locomotive workshops, paper, food, medical and rubber-plastic, and glass industries whose e uents are either partially or without treatment discharged into the river.Ten different study sites (Fig. 1) were selected based on severe anthropogenic activities like ritual bathing, religious offerings, cremation of dead bodies and the places where city's heavy discharge of sewage and waste water takes place.

Sediment
Sediment sample from the bank of river Ganga with the help of core sampler (0-10 cm depth) were collected from the mentioned 10 stations (Fig. 1) labelled and transported to the laboratory in airtight plastic bags at 4°C.

Water
Water samples were collected from approximately 50-100 cm from the bank of each collection sites (Fig. 1) in 1000 ml pre-sterilized bottles from 10 different locations.Sampling was done at the early morning because morning hours are with least human interventions, water ow is stable and its contents are uniformly distributed.The water was collected in plastic bottles and immediately kept in an ice box (4°C) and transported to the laboratory.

Fish
Fishes were collected directly from the wild with the help of local shermen.Gill net was used for capturing the shes.Fishes were captured consecutively for 3 years from 2019 to 2021, except in rainy season.Seven species of shes were randomly selected from the wild, from site no.1 to 10 (Fig. 1) on the basis of their availability.Collected samples were bought to the laboratory in water lled container.Identi cation of shes were done by key identifying features, based on scales, ns, presence of barbles, body shape and size (Srivastava, 2019).
. Within 2 hours of collection, biometrics study was done and shes were dissected to collect their tissues (gills, liver and muscles).The tissues were stored at -20°C until further processing.

Atomic Absorption Spectrometer (AAS) technique
A Perkin Elmer, PinAAcle Atomic Absorption Spectrometer (AAS) with Zeeman background correction system equipped with a ame furnace was used to measure Mn, Pb, Cd and Cr in the samples using an external standard method.Detection limit of the instrument was 0.127 mg/L for Mn, 0.18 mg/L for Pb, 0.052 mg/L for Cd and 0.096 mg/L for Cr.
2.4.1.Quality assurance and quality control (QA/QC) of AAS The quality assurance and quality control (QA/QC) Analytical estimation was performed using NIST traceable Certi ed Reference Material of Multielement standard with purity of more than 99.99%.AAS, Perkin Elmer, PinAAcle 900F was calibrated with three-point calibration in triplicate.The sample were analyzed in triplicate with a mandatory blank sample for all the estimations during study.The QA/QC data is depicted in Table 2. no.of colonies×100 dilution ………….Eq. 2 Baseline concentration was taken as per reference no (Salomons et al., 1984).
The CF is divided into these categories as (Patel et al., 2018).

Enrichment Factor
Enrichment Factor (EF) denotes the anthropogenic and geogenic in ux of metals in the water (Salomons et al., 1984).Eq. 3 is used to calculate EF (Delgado et al., 2010).

………….Eq. 3
Here, Fe is used as conservative metal and its baseline was obtained from (Muller, 1979).The value of enrichment factor if estimated close to or < 1, re ects the primary source of trace elements from natural source like crustal or marine environments and if > 1 shows the source is from anthropogenic activities.
Enrichment factor is categorized as

Pollution Load Index
Pollution Load Index (PLI) gives the overall picture of pollution load at a particular site (Angulo, 1996) PLI was calculated as: …………Equation 4 PLI if obtained 1 indicates baseline levels and > 1 indicates progressive alteration of toxicity.

Geo-accumulation Index
Geo-accumulation Index (Igeo) suggested by Muller represents the degree of metal pollution in terms of metal accumulation and considers anthropogenic input in pollution load (Muller, 1979).It is given by following formula: …………Equation 5 Where, Cn = concentration of element n, in sediment in mg/kg Bn = background concentration 1.5 represents a constant, which compensates for weathering and lithogenic effects (Huang et al., 2020).
Table 3 shows the seven classi cation levels of heavy metal pollution in sediment, with standard values of Igeo as de ned by Muller, 1979.

Ecological risk level
The potential ecological risk index from heavy metals to the aquatic ecosystem is assessed by calculating ecological risk level.This index was developed to evaluate the potential risk of heavy metal contamination in sediments by (Hakanson, 1980).The nal value of RI is obtained by calculating using following formula: …………(Eq.6) …………(Eq.7) RI is the sum of potential risk of individual heavy metal.Er is the potential ecological risk of individual metal.Tr denotes toxicity response index.Tr values for Pb, Mn, Cr, and Cd are 5, 1, 2, and 30 respectively (Hakanson, 1980) and CF is the contamination factor of individual metal.

Heavy metal concentration assessment in water and tissues
AAS readings obtained for the different heavy metals present in the water samples and the sh tissue analyzed was then used for further estimations.
The Heavy metals concentration in water samples from river Ganga was calculated by the formula given below: …………(Eq.8) The concentration of heavy metals in sh tissue was calculated as: …………(Eq.9)

Bioaccumulation factor (BCF)
BCF is the ratio of the contaminant in an organism to the concentration in the ambient environment in the steady state, where the organism can take in the contaminant through ingestion with its food as well as through direct contact.BCF was calculated using the formula suggested by

Human health risk assessment
In order to assess the health risk due to consumption of metal-loaded shes, we calculated EDI, THQ and HI which gives an account of potential risk on health due to heavy metals consumption through contaminated food.(Chary et al., 2008;Hough et al., 2004) and calculated as …………(Eq.12) Where EDI is calculated as per Eq. 5. RfD is the standard dose intake of a particular metal in a day (mg/kg body weight/day) that is under tolerable and healthy range (USEPA, 2011) given in table no 10.A THQ more than 1 indicates deleterious health effect due to contaminated food exposure in the population.

Statistical analysis:
The signi cant difference between heavy metal contaminations at different sampling sites were compared using one-way ANOVA.The signi cant difference between concentration of heavy metals and their bioaccumulation in various sh tissues were compared using Two-Way ANOVA test and correlation test was done by Pearson's correlation matrix to show the inter elemental relation.
3. Results and Discussion:

Analysis of physicochemical properties of water in Varanasi
The physicochemical qualities of river water samples collected from ten different sites of Varanasi are summarized in Table 4.The temperature of the river water in the studied period ranged between 18 and 30°C with an average temperature of 27°C, which was stable over the period.pH was within the permissible range, comparatively lower towards the drainage site (7.5)  *Other river sites are site no.3-9 as per mentioned in Fig. 1.
DO determines the purity of water and its tness for the survival of life within the water body.It is seen that the observed DO (Table 4) is below the permissible limit at the drainage site and within range at other river sites, indicating poor water quality at the drainage site, which might be caused due to heavy sewage discharge from the city.Aquatic aerobic bacteria consume oxygen from water for the decomposition of wastes, thus increasing BOD.
In the present study, BOD was found to be 53.77mg/L at the drainage site (Table 4).This could be due to untreated domestic sewage, agriculture runoff, and residual fertilizers.Other sites of the river also showed increase in the BOD as compared to the permissible limit stated by BIS.Notably, in Table 4, TDS, BOD and fecal coliform are increased with higher metal concentration whereas pH and DO is decreased with increased metal concentration at drainage site and vice versa for other river sites.These physicochemical parameter indicates the deterioration of the quality of river water at drainage site due to increasing metal pollution load.Besides these the addition of non-biodegradable pollutants keep on adding to the associated problems.Aquatic species thriving in such waters are subjected to poor quality of environment as well as bioaccumulation of several pollutants in their body tissues.(fecal coliforms in heavy metals)

Heavy metal assessment in sediment
Sediment sample collected from different sites mentioned in Fig. 1 were screened for the occurrence of heavy metals in it.Result obtained is summarised in Table 5. Mean metal concentration in sediment followed the trend as follows Mn > Pb > Cr > Cd.The mean concentration of Pb in sediment was 158.455 mg/kg, CF is 8.33 and EF is 40.21 indicating very high lead contamination in sediment.Geo accumulation factor (GAF) 1.66 indicates Class 2 category (Table 3) with moderately polluted intensity and ecological risk level of 41.695 (Table 5) reveals moderate ecological risk due to lead.In a similar study lead accumulation was found from moderate to strong in river Ganga in Varanasi (Pandey et al., 2015).The primary sources of Pb contamination in the river Ganga in Varanasi are metal processing and battery manufacturing industries, the use of lead-based paints, pesticides, and fertilizers in agriculture, and gold extraction processes involving lead in amalgamation techniques.These activities result in the leaching of Pb into the soil, which eventually gets transported into the river.However, the mean Mn concentration in sediment was found to be 355.622mg/kg, CF is 0.46 and EF is 2.25 which indicates low contamination, GAF is 0.092, depicts Class 1 category (Table 3), i.e, unpolluted to modrately polluted, Er: 0.461 indicate low ecological risk due to manganese.This indicates that river is safe with Mn concentration.Cadmium toxicity is highest in river sediment and has surpassed safe concentration limit.The elevated Cd level in the river Ganga is likely to stem from different tanning, electroplating and dye industries in Varanasi district and several domestic channels induced in the Ganga River may be the source of Cd contamination.Additionally, improper disposal and recycling of electronic wastes might also contribute to the presence of Cd in the river.
Overall pollution load index (PLI) of heavy metals was 6.698 which clearly indicates that river Ganga is polluted with heavy metals beyond the permissible limits in Varanasi district and people living in its vicinity are subjected to health risk.

Heavy metal analysis in water
The heavy metals concentrations in Ganga water in Varanasi at ten selected sites are recorded and presented in Table 6.All the four metals surpassed the safe concentration limit set by EPA at all the ten studied sites.Concentration of Pb, Mn, Cd and Cr was recorded highest at Varuna Ganga con uence point, which were 1.29 mg/L, 1.32 mg/L, 0.169 mg/L and 0.161 mg/L respectively followed by Nagwa and Raj ghat.These points are noted to have highest sewage discharge from the city and are considered among drainage sites (Table 6).These metal contaminations were statistically signi cant at different sampling sites with p < 0.5.It was found that the concentration of heavy metals at all the sites studied were beyond the permissible limits of this heavy metals in water given by EPA posing the threat to the aquatic life.Similar study was conducted at Kanpur, Allahabad, Mirzapur and Varanasi districts of Uttar Pradesh and observed that water of river Ganga was loaded with Pb 0.24 mg/l, Cd 0.85 mg/l and Cr 0.45 mg/l concentrations in Varanasi in the year 2019 (Maurya et al., 2019).The presence of heavy metals in water, sediment and aquatic lives is responsible for their entry in the food chain ultimately reaching the human population (Sarkar et al., 2016).The results of the present study indicates that, industrial e uent discharge and agricultural runoff, released into the Ganga River in Varanasi is polluted with heavy metals and un t for human consumption.Both indigenous and exotic shes were investigated for the potential accumulation of heavy metals in their tissues.It was revealed that all the four heavy metals examined were accumulated in the gills, liver, and muscles of every sh species investigated (Table 7).
In all the seven sh species, the degree of heavy metal concentration followed liver > gills > muscles.Liver being an important organ for detoxi cation as well as for protein synthesis may be a possible reason for having the highest metal a nity (Fernandes et al., 2008).Gills have a large surface area, and are in continuous contact with the aquatic environment, therefore are the second most important site for metal concentration.Another reason may be due to the increased number of chloride cells that pick up metal ions from contaminated water (Mazon et al., 1999;Costa et al., 2002).Although sh muscles are consumed as protein source all over the globe, it is a metabolically less active tissue (Adhikari et al., 2009;Radhakrishnan, 2010) and thus, the reason for least accumulation of metals in muscles.Less extensive blood circulation in muscles in comparison to other vital organs like liver, kidney and gills is also a major factor.
In our eld study, heavy metal trend was Mn > Cr > Pb > Cd in almost all the species and tissues.Probable reason for more Mn concentration could be cumulative role of water contamination and essential elemental nature of Mn in enzymatic activity (Altaf et al., 2016).Cr enters the aquatic system via multiple industrial sources from where hexavalent form of chromium is reported to diffuses readily in the sh tissue and penetrates cell membrane (Ghosh, 2002;Bagchi et al., 2001) Report shows that Cd was accumulated highest in liver tissue of bottom feeder followed by column and surface feeder shes (Delgado et al., 2010).
In the present study, the highest concentration of Pb and Cd was observed in Carpio liver (8.86 µg/g and 3.27 µg/g respectively) while lowest Pb in Baikari muscles (0.07 µg/g) and Cd remains undetected in tengra, bam and pathari.The Food and Agricultural Organisation (FAO) proposed a limit of 0.5 µg/g for Pb in food (FAO, 1983) while Food and Environment Protection Act (FEPA,2003) set this value to 2.0 µg/g.Being an invasive and larger size species, Cyprinus carpio and exhibits aggressive behavior and is a bottom feeder, which increases their exposure to higher concentrations of contaminants, such as lead, found in the sediment.Likewise, Tilapia is an omnivorous and opportunistic feeder, which could be attributed to high Pb accumulation in its liver.However small size of pathari sh gains importance because smaller body size reduces the metal accumulation through surface action.In a similar previous study, in delta region of Nigeria, Pb was detected higher than acceptable limit of 0.5 µg/ in the shes collected from Finima creek.The reason was due to crude oil spills in that area (Abarshi et al, 2017).Other studies showed higher concentrations of Cd and Pb compared to Cr in sh tissues (Javed et al, 2013; Begum e al, 2013).These results imply that these external sources have a more signi cant in uence on the bioaccumulation process in sh.Mn was recorded highest in Pathari liver (53.19 µg/g) and lowest in Baikari muscles (1.10 µg/g).Cr was estimated highest in Pathari liver whereas lowest in Bam muscles.European Union Commission (EUC) suggested the daily tolerable chromium concentration to be 1 µg/g, while the FEPA,2003 suggested 0.15 µg/g and WHO suggested 0.15 µg/g..The concentration level of each metal in sh tissue was statistically signi cant at p < 0.05 In another study of pelagic and benthic shes of Ogbese River, Ondo State, South-Western Nigeria, Cd was detected as 0.001 ppm in the heart of only one benthic species C. gariepinus (Josephine Omowumi Olayinka-Olagunju et al,2021).However even a little Cd concentration is hazardous.Variation of metal concentration in sh tissues also depend on age because the time they spend in water decide the concentration of metals in their body since shes are captured at their different life period.

Correlation analysis of heavy metal in sh tissue
Inter elemental relation is shown by Pearson's correlation matrix (Table 8).The correlation coe cient ranges between − 1 to + 1.A positive correlation between two metals indicate that for every positive increase in one, there is a positive increase of a xed proportion in the other, whereas a negative correlation indicates that for every positive increase in one metal, there is a negative decrease of a xed proportion in the other.In our study, we found notable correlation between Pb and Cd (r = 0.72, p < 0.05).The probable reason is due to the high concentration of these two elements in Carpio and Tengra in all the selected sh organs.Cd and Pb is reported to occur from the common sources like leaded petrol, coal combustion, smelting and old pre industrial lead.Similar result was observed in one study, where positive correlation between Cd and Pb was observed by C. catla and C. mrigala (Dhanakumar et al., 2015).The negative correlation was seen in case of Cd and Mn, while in case of Pb to Mn, Pb to Cr, Mn to Cr, and Cr to Cd, there were non-signi cant positive correlations (p < 0.05 and r is < 0.5).

Determination of Bio-accumulation factor
Metal bioaccumulation in shes depend upon a nity of metals to sh tissues, variations in uptake, deposition, and excretion processes (Jezierska, B. and M. Witeska, 2001).Higher metal concentrations in the environment tend to lead to increased uptake and accumulation in sh and may differ in shes living in the same water stream.This relationship has earlier been observed in both eld and laboratory studies (Hongjun W. et al, 2013).As shown in Table 9, at statistical signi cant p < 0.05, BAF in each sh species followed the same trend in organs i.e., liver > gills > muscles and all metals had tissue concentrations higher than their corresponding concentrations in water.Figure 3 shows that Bioaccumulation Factor (BAF) for all four metals is consistently higher in metabolically active tissues such as the liver and gills as compared to less active tissues like the muscles in both indigenous and invasive sh species.This indicates that the uptake and accumulation of these metals are more pronounced in the liver and gills due to their physiological roles and higher metabolic activity, while the muscles tend to exhibit lower levels of metal bioaccumulation.
The carp species showed the highest concentration of all metals in its liver, may be due to its opportunistic feeding behavior and potentially higher metabolic rates.This suggests that carp may have a greater e ciency in taking up heavy metals from the environment.Indigenous species such as Tengra, Bam, and Pathri also exhibited relatively higher metal concentrations compared to other sh species.These heavy metals has the capacity to form harmful soluble compounds (Amin et al, 2021) and higher metal retention could be due to the formation of metal-protein complexes, which might impede the excretion process within the sh body.The retention of these metals within sh can lead to substantial changes, causing them to endure stressful conditions and potentially disrupting their hematological and biochemical indices (M Salaah al, 2020; Elhaddad et al., 2022).Health risk assessment.
The ndings of our study indicate a substantial decline in the sediment and water quality of the Ganga river, primarily attributed to the elevated concentrations of heavy metals.This contamination poses a potential risk to human health to the local residents, especially concerning the direct consumption of contaminated shes caught from the river.Figure 4 represents species wise EDI of metal concentration (mg per kg body weight) for consumers.The EDI for Pb was measured higher than the recommended daily allowance in Sauri, Carpio, Bam and Pathari as mentioned in Table .10.
EDI for Mn was estimated highest in Sauri followed by Pathari.For Cr, it was higher than the recommended daily allowance in all the species, highest in Carpio followed by Baikari and Sauri, while Cd was highest in Common carp, followed by Sauri and Tilapia.The study clearly demonstrates that among the sh species examined, Cyprinus carpio displayed the highest concentrations of Cr and Cd, whereas Sauri showed the highest levels of Pb and Mn in terms of EDI.Being at higher trophic level sh, common carp may accumulate Cd and Cr through biomagni cation (Riede, 2004).This observation indicates that individuals who regularly consume sh may experience the greatest intake of these metals by consuming common Carp and Sauri sh species.The THQ estimated for individual heavy metals through consumption of different sh species are presented in In our study, the total THQ value i.e.HI of metals was recorded in following sequence: common Carp > Tilapia > Sauri > Pathari > Bam > Tengra > Baikari.
The study found that the average HI (hazard index) value for common Carp and Tilapia exceeded 1, indicating a potential health hazard for humans who consume these contaminated sh.Although, Tilapia consumption has been found to pose a non-carcinogenic risk to human health (Hong-Giang Hoang, 2021).Maximum HI was recorded in common Carp, implying that its consumption may pose a health risk to humans.Common carp and Tilapia, being invasive species with opportunistic feeding behavior, can inadvertently consume various ora and fauna, including microplastics.In urban areas, the disposal of colored microplastics into water bodies can attract these voracious eaters, causing them to ingest the microplastics and potentially be exposed to heavy metals (Adji BK et al., 2022) To safeguard against excessive metal concentration in humans through the food chain, regular monitoring of heavy metals in shes is crucial.

Conclusion:
In this research, the presence of heavy metals was observed in the river water, sediment, and edible shes of the Ganga river in Varanasi district.Concentrations of studied heavy metals, namely Pb, Mn, Cr, and Cd, were found to exceed the permissible limits set by international standards (BIS and WHO) for drinking water.The sediment was particularly polluted with Cd, posing a potential threat to bottom-dwelling species in the river.Although the levels of heavy metals in the sh muscles were below the hazard quotient, it is evident that prolonged consumption of such contaminated shes could lead to bioaccumulation in the food chain.This, in turn, may result in the accumulation of heavy metals in humans, surpassing the hazard index and causing health risks to the local population.
This study is of great importance because regular assessments aids in identifying early signs of contamination, implementing necessary measures, and raising awareness about the dangers posed by toxic substances in water sources.
The Ganga River Route (Blue) in India.An enlarged image of the Ganga sampling stations in the Varanasi district of North India.

Table 2
QA/QC results of analytical methods for heavy metal concentrations.
*Relative standard deviation (RSD) 2.5 Heavy metal pollution assessment in sediment 2.5.1 Contamination factor Contamination factor (CF) represents the concentration of selected metal higher than the baseline concentration of same metal in an uncontaminated site.CF in this study is calculated by following equation (Fukue et al., 2006).

Table 3
Pollution level classi cation of heavy metals in sediment.
Song et al., 2009.he major source of food for 50% of the human population.Therefore, sh muscles are used for calculating the human health risk through an estimated daily intake (EDI) of metals.EDI is calculated by the following equation as perSong et al., 2009.
(Jain et al., 1995)erageweight of sh consumed per day is 25 g as suggested by a North India survey study by Kumar et al, 2020 when average body weight of consumer is 52 (for Indian men)(Jain et al., 1995).
than other river siteGupta et al., 2013)ity of water observed in other river sites was due to dissolved carbonates, bicarbonates and hydroxides ions most probably due to increases in pollution load in the river from upstream to downstream, say from Kanpur to Varanasi(Maurya et al., 2019;Gupta et al., 2013).However, according to European Union, for sheries and aquatic life, pH of 6.0 to 9.0 is permissible.(USEPA, 1986; USEPA.1999a).Although Ganga water holds buffering capacity, nevertheless this pH is unsuitable for human consumption.
*Drainage sites are site no.1, 2 and 10 as per mentioned in Fig.1.

Table 5
Assessment of heavy metal pollution indices in sediment.
Mean chromium concentration in sediment was 154.328 mg/kg, CF is 2.14, EF is 10.45, which indicates moderate contamination of chromium in sediment and GAF is 0.428, depicts class 1 category (Table3), i.e. unpolluted to moderately polluted pollution intensity, Er is 4.286 showing low ecological risk.Mean cadmium concentration in sediment was found to be 46.717mg/kg, with CF is 274.96,EF is 1285.67 depicting very high contamination in the river sediment.GAF is 55.177 indicates alarming pollution intensity (Table3) whereas Er is 8248.8shows very high ecological risk due to cadmium.

Table 6
Heavy metal contamination in mg/L at different sampling sites.Statistical signi cance of heavy metal contamination at different sampling sites was done using One Way Anova and the differences were signi cant at p < 0.5, f = 1.6309.

Table 7
Concentration of heavy metals in different sh tissue (µg/g) and their permissible range by FAO.Statistical signi cance of heavy metal concentration in various sh tissues was done using Two Way ANOVA test and the differences were signi cant at p < 0.05, F tissue = 10.144,F metals 14.339 and F tissue × metals = 2.312.
*FAO: Food and Agriculture Organisation

Table 9
Bio-accumulation factor in different sh tissues.Statistical signi cance of heavy metal accumulation in various sh tissues was done using Two Way ANOVA test and the differences were signi cant at p < 0.05, F tissue = 9.052, F metals = 10.82 and F tissue × Metals = 2.2664.
(Ali MM et al., 2020)Ahmed et al., 2022;n our study, we found that every metal was below the hazard quotient except Cd in common Carp.This nding highlights the potential health risk associated with Cd exposure through the consumption of common Carp.Numerous scientists across the globe have experienced similar results.In an Indian study at Damodar River basin, Burnpur, West Bengal, children were found to be at higher risk of Cd toxicity when consuming three sh species, including Clupisoma garua, along with Puntius ticto and Labeo bata (Mohantaa et al., 2018).However, other studies have shown that the muscles of sh from various regions were within permissible limits for human consumption, with THQ values less than 1(Maurya et al., 2019;Ahmed et al., 2022; Ali et al., 2018).On the contrary, Yi et al. 2011 reported higher THQ values (THQ > 1) in the lower reaches of the Yangtze River basin in China.In the Karnaphuli River, Bangladesh, the THQ value for H. nehereus exceeded the reference value (higher than 1)(Ali MM et al., 2020).