Elemental composition of carnivorous fish Wallago attu (Siluridae, Siluriformes) from River Chenab, Pakistan

Over the decades, riverine ecosystems is suffering from intense human intervention resulting in degradation and habitat loss as a consequence, many fish species become endangered, particularly in rivers where heavy demand is placed on freshwater. Wallago attu is fast growing catfish belongs to the family siluridae has good market demand having high protein content and nutritional value in it’s flesh. The data was obtained for metal concentration in wet and dry body weight in wild Wallago attu . All metal was found under permissible limit as recommended by different international organizations. Calcium was found highest, while cadmium in lowest concentration. Fe, Zn, Cu, Ni, Mn, K, Na, Ca and Mg showed highly significant (P<0.001) positive correlation with wet body weight except Co and Cd showed non-significant (P>0.05) correlation. Metals such as Fe, Cu, Zn, Cd, Pb, K, Ca, Mg and Co shown negative allometric pattern with increasing body weight concentration of Cu, Fe, Zn, Ni, Pb, Mn, Na, Ca and K showed positive relationship (P<0.001) with total length (cm), except for Co and Cd, which indicated non significant correlation (P>0.05). Present Study will helpful to assess toxicity due to presence of heavy metals for researchers and different organizations from River Chenab, Punjab, Pakistan.


Introduction
If the quantity of heavy metals in the environment is higher than the permissible limits, the aquatic environment and the organisms are seriously threatened by heavy metal pollution (Shahjahan et al., 2022).Erosion, volcanism, and weathering are examples of natural processes that produce heavy metals.Moreover, heavy metals are produced by human activities such as smelting, oil refinement, consistent in Incucell Oven (Memmert; Germany).After Specimen became dried, grind the samples and put it in labelled box for further analysis.1 g of grinded powder was taken and put into muffle furnace at 500 °C for 24h for ashing.Ash was mixed with 25 mL prepared solution of 1% Nitric acid.Samples were filtered (Whatman 41; Ashless circles 125mm; Cat No. 1441 125) and were stored in labelled polythene bottles for further analysis of different metals.Dilluted sample were analyzed for elemental composition analysis by Agilent atomic absorption (200 Series AA) in Lab.

Chemical reagents
For analysis of metals on Agilent atomic absorption different glasswares were used.Before usage of glasswares, all glassware were washed and autoclaved for approximately 6h, at 150 °C for processing.

Statistical analysis
Statistical data analysis was used dteremine the correlation of body size (Body weight and Total length) and condition factor with studied metals samples of Wallago attu by Regression analysis on MS Excel (Version 16).
Regression analyses were done by Equation 1: where: X = Total length (cm) / Body Weight (g)/Condition Factor; Y= Concentration of Metals (µg/g); a= Constant; b= Slope.By correlation cofficient analysis to access the significant values (P<0.001;P<0.01, and P<0.05).Data of log transformed was explain by isometric and allomeric pattern statistically with metal concentration by interpreting b value of data.
Multiple regression analyses was used for inter relationships of fish size (Total length (TL) and Wet body weight (WW)) with metal concentration as general Formula 2: where: 'a' is constant, 'b 1 ,b 2 ' are slopes, X 1 is WW and X 2 is TL/condition factor; while Y for concentration of metals.
The descending order of analyzed elements concentration is as follows:

Ca K Mg Na Fe Ni Zn Mn Pb Cu Co Cd
To study the analyzed elements the given orders showed that calcium was found highet in concentration, while cadmium was found in lowest concentration.Concentration because they do not disintegrate when they are heavily concentrated in a niche or medium like soil, water, or air.Because fish are a necessary component of the food chain in the environment and human health is affected by the accumulation of heavy metals in fish, living species, notably humans, get poisoned through connection with aquatic systems (Agbugui et al., 2019).There is growing concern about the value of foods in numerous parts of world.The evaluation of toxic elements in food has provoked investigations on toxicological consequence of these elements in food (Eletta et al., 2003).There are several aspects play important part in controlling the concentration and poisonousness of elements on aquatic animals and fish in specific.These features involved physicochemical parameters and biological parameters (Christophe et al., 2009).
Even though to its toxicity, long-term persistence, bio-magnifications and bioaccumulation in the food chain, water contamination has long been considered the main hazard to the aquatic ecosystem (Morillo et al., 2004).Any metallic element that is particularly dense, dangerous or deadly even at low concentrations is referred to as a "heavy metal."Both natural and human-made heavy metals are dispersed throughout animals' organs and tissues when they enter ecosystems.While some of these metals are required by living things in tiny amounts, excessive concentrations of them may make them extremely poisonous (Ogbuagu and Iwuchukwu, 2014).
Hundreds of living beings are present in the aquatic environment which can majorly benefit humans.Though, because of the continuous rise in the growth of industries, numerous of these water beings are killed because of pollution.This lead to a many investigators in analyzing toxicants in the marine habitat.Presently, the pollution of the aquatic environment and its other forms of life became a popular field of research (Ashraf, 2006).Seeing the above evidences, the current study was commenced to examine and assess the presence of heavy elements in fish species.The issue of heavy elements contamination in aquatic habitat has become progressively serious problem over the previous decades in China (Wang et al., 2008).Present Study is used to access toxicity due to presence of heavy metals for researchers and different organizations of Wallago attu from River Chenab, Punjab, Pakistan.

Samples collection
Specimens of freshwater fish Wallago attu were collected from River Chenab, Multan, Punjab, Pakistan.Fish specimens were identified on the basis of external morphology.Collected fish samples were transported to the Fisheries lab, Institute of Zoology (IOZ), Bahauddin Zakariya University (BZU), Multan, Pakistan.Body size of specimens were accurately measured by using wooden measuring tray and electrical balance.

Samples preparation
Each specimen was put in pre weighed aluminum foil with label, sample were dried completely till weight became Brazilian Journal of Biology, 2024, vol.84, e270501 3/9 Elemental composition of carnivorous fish Wallago attu of these metals with their mean, standard deviation and ranges were mentioned in wet and dry weight of fish.Concentration of Iron in wet body weight was ranges from 0.2 to 36 with mean value of 15.71 µgg-1, while in dry body weight 0.93 to 168.13 with mean value of 74.37 µgg-1 as the concentration of copper in wet body weight were ranges from 0.54 to 2.65 with mean value of 1.81 µgg-1, while in dry body weight 2.75 to 12.75 with mean value of 8.51 µgg-1.Zinc concentration in wet body weight were ranges from 0.05 to 15.92 with mean value of 9.56 µgg-1, while in dry body weight 0.24-74.37 with mean value of 44.98 µgg-1.Nikal concentration in wet body weight were ranges from 0 to 18.36 with mean value of 11.94 µgg-1, while in dry body weight 0-83.28 with mean value of 56.29 µgg-1.Cadmium concentration in wet body weight were ranges from 0 to 0.01 with mean value of 0.0001 µgg-1, while in dry body weight 0-0.05 with mean value of 0 µgg-1.Manganese concentration in wet body weight were ranges from 2.10 to 5.35 with mean value of 3.58 µgg-1, while in dry body weight 10 to 25 with mean value of 16.86 µgg-1.Lead concentration in wet body weight were ranges from 0 to 3.83 with mean value of 2.47 µgg-1, while in dry body weight 0-17.5 with mean value of 11.62 µgg-1.Sodium concentration in wet body weight were ranges from 116.41 to 278.37 with mean value of 204.14 µgg-1, while in dry body weight 587.50 to 1300 with mean value of 962.81 µgg-1.Potassium concentration in wet body weight were ranges from 962.38 to 1524.46 with mean value of 1230 µgg-1, while in dry body weight 4767.50 to 7487.5 with mean value of 5794.47 µgg-1.Calcium concentration in wet body weight were ranges from 2644.26 to 4471.73 with mean value of 3635.02µgg-1, while in dry body weight 13345 to 20360 with mean value of 17109.87µgg-1.Magnessium concentration in wet body weight were ranges from 122.28 to 895.06 with mean value of 393.56 µgg-1, while in dry body weight 585.00 to 4137.50 with mean value of 1843.72 µgg-1.Cobalt concentration in wet body weight were ranges from 0.00 to 0.02 with mean value of 0.01 µgg-1, while in dry body weight 0.00 to 0.08 with mean value of 0.03 µgg-1 as presented in Table 1.Results of regression analyses represented that most of the studied metals which include Zn, Ni, Fe, Cu, K, Ca, Mn, Na and Mg showed highly significant positive correlation (P<0.001) with wet weight of wild captured W. attu, showing correlation coefficient (r) values ranging from 0.499 to 0.954.Highest correlation coefficient value was observed in a relation between Potassium burden element (µg) and wet body weight (g) in W. attu.Lead (Pb) represented significant positive correlation at P<0.01 (r= 0.459; b = 1.203).Only two studied metals, Cd (r= 0.137; b = 0.0004) and Co (r = 0.286; b = 0.004), remained non-significantly correlated with the body weight of W. attu.
Metals such as Fe, Cu, Zn, Cd, Pb, K, Ca, Mg and Co shown negative allometric correlation with increasing body weight, representing significant decrease in concentration of these metals with increase in body weight of Wallago attu.Slope (b value) greater than 1 indicated positive allometry for nickel concentration representing significant increase in metal concentration with increase in body weight.While, Na and Mn is increases with increase in the body weight showing an isometric trend, when slope value 'b' is either not significantly differ from '1' or equal to '1' (Table 2).Regression analysis of total length (TL, cm) with log body burden element (µg/g) for wild captured Wallago attu.Concentrations (µg) of all of the studied elements including Fe (r = 0.693), Cu (r = 0.528), Zn (r = 0.584), Ni (r = 0.745), Mn (r = 0.846), Pb (r = 0.518), Na (r = 0.846), K (r = 0.850) and Ca (r = 0.845) represented strong positive relationship (P<0.001;r 0.518 to 0.850) with the fish total length (cm), except for Mg which was significant (P<0.01;r = 0.389), Co found least significant (P<0.05;r = 0.318) and Cd which indicated insignificant correlation (P>0.05;r=0.089) with TL of Wallago attu.
Regression analyses of log transformed data of total length (TL) with body burden element (µgg-1) indicated highly significant correlation (P<0.001) of Na, Cu, Mn, Ca and K with correlation coefficient (r) value 0.560, 0.818, 0.853, 0.863 and 0.879, respectively in wild W. attu, as represented in Table 3.On the other hand, log Fe and log Ni represented positive significance in correlation (P<0.01) with log TL containing r-value being 0.465 and 0.446, respectively for Wallago attu.Log Zn and log Mg in concentration (µg) in body burden of W. attu remained least significant (P<0.05) with log TL holding correlation coefficient value being 0.333 and 0.374, respectively.Whereas, concentrations of log Cd (r = 0.081), log Pb (r = 0.139) and log Co (r = 0.212) showed no relationships (P>0.05) with log TL of Wallago attu, as denoted in Table 3.
Table 4 also represent allometric approach for metal concentrations in the fish boy with increasing total length of the fish.For this purpose slope (b) value was compared with value being 3.00, as b=3 represents isometry in an    5. Multiple regression analysis of wet weight and condition factor with metal concentration (µg/g) for Wallago attu.Concentrations of Cu and Na were observed strongly correlated (P<0.001);Pb, K and Ca showed significant correaltion (P<0.01);while, Zn and Mg indicated least significant correlation (P<0.05) with condition factor and wet body weight.Though concentrations of Fe, Ni, Cd, Mn and Co were non significantly correlated (P>0.05) with condition factor and wet body weight in Wallago attu (Table 6).

Discussion
Other than pollutants of environmental concern, contamination of fish food with various chemicals has turned into a global concern.While fish is regarded as a rich nutritional source, due to various anthropogenic activities, the wild fish population may have a greater concentration of heavy metals.Thus, it serves as an excellent signal to check for contamination (Bat et al., 2012).There is increases in accululation level of these metals day by day and can't easily digested in human body (Castro-Gonzalez and Mendez-Armenta, 2008).As accumulation of these metals in fish body to risky levels has turn issues and increases health concern.Accumulation in fish was as: Ca>K>Mg>Na>Fe>Ni>Zn>Mn>Pb>Cu>Co>Cd.Similar trend was reported in Oncorhynchus mykiss except Mg and Pb by Naeem et al. (2010) and in Ctenopharyngodon idella except Cu by Khalid et al. (2019).Highest concentration of these elements was observed in Calcium (3635.02± 471.40) while lowest in Cadmium (0.0001 ± 0.0003).Similarity the calcium concentration found highest and Cadium with lowest concentration as studied Oncorhynchus mykiss by Naeem et al. (2010); Aristichthys nobilis by Naeem et al. (2011) and in Ctenopharyngodon idella by Khalid et al. (2019).
Iron plays physiological role in living being, if concentration is in acces than it causes harmful effects (Misra and Mani, 1992).Access concentration of iron in fish did is harmful for fish acclimation and feed conversion efficieny of fish as it influences the growth (Javed and Saeed, 2010).Results in present study showed that Iron (Fe) mean concentrations in wet body weight of fish ranged as 15.71 ± 7.08 µgg-1, while it was 74.37 ± 33.91 µgg-1 in dry body weight (Table 1).
Concentration of iron (Fe) was found under permissible limit as reported by WHO and FAO, as cited in study by Kundu et al. (2017) as 80 µgg-1.Naeem et al. (2011) studies an dreported that the concentration of iron 61.64 ± 6.21 in Aristichthys nobilis and 38.57± 1.76 in Oncorhynchus mykiss (Naeem et al., 2010) as found close to the present study.Minimum concentration of Fe was was 2.805 µgg-1 in previous study in Heteropneustes fossilis while maximum 669 µg/g for Catla catla (Jaffar et al., 1988;Salam et al., 1998).As our study concentration is found in between these values.Fe in wet and dry body weight in Wallago attu was 21.672 ± 7.13 and 94.017 ± 42.68 reported by Yousaf et al. (2012) very similar to the present sudy.Maximum permissible limit for Fe as FAO ( 1989) is 100 µg/g as indicates that in present study Fe is under maximum permissible limit .
Zinc is trace element that is essential for the transfer of electron in different reactions.While high intake causes Elemental composition of carnivorous fish Wallago attu body organs bioaccumulation (Nussey et al., 2000) that caused toxicity (Kim et al., 2007).Zn is important for gene expression, cell based growth and for different metallo-enzymes (Mogobe et al., 2015).It is observed that the concentration of Zinc (Zn) in present study found in normal range as recommended by FAO (1989).Result of study indicated that the mean value is 9.56 and 44.98 µg/g in wet and dry wt.respectively.Result found closer with the study by Naeem et al. (2010) with mean value of 15.49 ± 1.41 in farmed Aristichthys nobilis; 16.96 µg/g in Mystus bleekeri by Naeem et al. (2011).Study by Ling et al. (2013) who reported the concentration 39.7 µgg-1 in Oreochromis niloticus.As this is also very close to our study.The mean value of concentration of Nikal in wet and dry wt. of fish 11.94 and 56.29 µg/g respectively as similar reported by Emeka (2014).Similarily, the studied concentration of Ni in Ctenopharyngodon idella wet wt. as 0.56 ± 0.04 in study was found higher than the evaluation of some essential and toxic metal reported values of 1.35 µg/g in Citharinus citharus and 2.96 µg/g in Eutropius niloticus by Ere et al. (2014).
The concentration of Cadmium is found lowest as compare to the other elements.The mean value Cadmium in 0.0001 and 0 in wet and dry wt. of fish.The concentration of Cd found below the limit in present study.Cadmium was not quantifiable in Oreochromis niloticus (Salam et al., 1996) and wild Oreochromis niloticus (Naeem et al., 2011).Observations found in general agreement with the results of present study as within permissible limit.
The mean concentrations of Mangnese (Mn) 16.86 (dry weight) and 3.58 µgg -1 (wet weight).The concentration of Mn 2.66 and 12.32 µgg -1 in wet and dry wt. in Onchorynchus mykiss by Naeem et al. (2010) as 2.81 and 14.01 µgg -1 in wet and dry wt. in Aristichthys nobilis by Naeem et al. (2011).These concentrations found very close to the our study as high values might be due to behaviour and feeding habits differences (Olatunde, 1978).
Lead (Pb) could be harmful for living organisms even if in low concentration.It causes Scoliosis, anaemia and caudal fin degeneration.In humans it causes nervous and the urinary tract problems (Burden et al., 1998).Lead in wet body weight 2.47 and 11.62 µg/g in dry body weight.The mean value of 1.63 and 2.25 for Onchorynchus mykiss and Aristicthys nobilis in wet weight as reported by Naeem et al. (2010Naeem et al. ( , 2011)).While, Tariq et al. (1993) reported 2.973 µg/g in wild Catla catla as close in present study.Study reported the presence of higher concentration of lead in present study than proposed by various international organization as 0.1 µg/g by EU (2001); 0.5 µg/g by FAO (1989) and 2.0 µg/g by Jones and Franklin (2000) and SASO (1997).Naeem et al. (2010Naeem et al. ( , 2011) ) and Yousaf et al. (2012) reported higher concentration in Wallago attu.As Chatta and Khan (2017) reported same concentration in Labeo rohita, Ctenopharyngodon idella, and Cirrhinus mrigala in wet wt. as studied in present study.
For proper functioning of all body parts and for maintainace of osmotic pressure is because of sodium and potassium in body for enzyme activation (Rajanna et al., 1981).As potassium and sodium is important for function of kidney, Muscle functioning and acid base balance (Woodling, 1999).Mean sodium concentration of Sodium 204.14 in wet body weight while 962.81 in dry body weight.Study by Tariq et al. (1993) reported 829 and 537 µg/g Catla catla by Tariq et al. (1993) and Salam et al. (1998); 770.48 µg/g in Aristichthys nobilis by Naeem et al. (2011) which is far from our study.A very close concentration of 339.42 µg/g in Oncorhynchus mykiss by Naeem et al. (2010).Mean value of Potassium (K) is 1230 in wet body weight while 5794.47 in dry body weight.The mean concentration of 1470.07 µg/g in Oncorhynchus mykiss by Naeem et al. (2010) which is very close to the present value.
Calcium and Magnesium in Wallago attu found 3635.02 and 393.56 µg/g in wet body weight while 17109.87 and 1843.72 µg/g in dry body weight.The concentration of Cobalt found to be 0.01 and 0.03 µg/g in wet and dry weight respectively.These reported concentrations found under permissible limit as 0.1 µgg -1 (Wyse et al., 2003).The mean concentration of Co 0.67 µg/g in Aristichthys nobilis (Naeem et al., 2011); 0.24 ± 0.01 µg/g in Wallago attu in wet and dry body weight (Yousaf et al., 2012).These are found to be very close with present study.
Metals such as Fe, Cu, Zn, Cd, Pb, K, Ca, Mg and Co shown negative allometric correlation with increasing total length and body weight, representing significant proportional decrease in metal concentration with increase in total length and body weight of Wallago attu.Slope (b) value was compared with value being 3.00, as b=3 represents isometry in an increase; while b value lower or higher than 3.00 shows allometry in metal concentrations with an increase in TL of the fish while slope (b value) greater than 1 indicated positive allometry for nickel concentration representing significant increase in concentrations with increase in body size.While, Mn and Na was increases in direct proportion with increases in the body weight showing isometric pattern, when slope value 'b' is either not significantly differ from '1' or equal to '1' or.As the study in Oncorhynchus mykiss by Naeem et al. (2010); Aristichthys nobilis by Naeem et al. (2011) and in Ctenopharyngodon idella by Khalid et al. (2019) provided reliable estimateby using predictive equations for regression cofficient analysis.Naeem et al. (2010) reported negative attometric relation in K and Na with body weight and total length of Oncorhynchus mykiss.
Regression parameters of metal concentration with condition factor in wet weight for Wallago attu showed that concentrations of Fe, Zn, Ni, Pb and Na represented significant positive correlation.However, other metals including Cu, Cd, Mn, K, Ca, Mg and Co remained constant showing no relationships with condition factor in Wallago attu.Iron showed significance with condition factor.Naeem et al. (2011) andFatima andNaeem (2016) have found insignificant correlation of Cu, Cd, Mn, K, Ca, Mg and Co concentrations in fish with condition factor.For multicollinearity, Variance inflation factor predictive equation showed less multicollinearity (VIF <10) with size (Body wight and total length).

Conclusion
It is concluded that studied metal concentrations founded lower than the permissible limits set by different international organization most specifically FAO/WHO.In respect to essential minerals, Wallago attu, collected from wild, has a good nutrition quality.Moreover, the fish size showed a definite effect on the metal concentrations in Wallago attu.However, Pd, Cd and Co did not significantly affected by the fish size.

Table 3 .
Regression analysis of total length (TL, cm) with log body burden element (µg/g) for Wallago attu (n=39).Elemental composition of carnivorous fish Wallago attu increase; while b value lower or higher than 3.00 shows allometry in metal concentrations with an increase in fish total length.Results of regression log transformed data of total length (TL) with body burden element (µg) indicated negative allometry in all the studied metals, as b-value remained significantly lower (b = 0.105 to b= 2.612) than 3 (an isometric slope) representing significant proportional decrease in metal concentration with increase in TL of Wallago attu, except for Ni concentration which represented significant proportional increase (b value = 5.351) in nickel concentration with increase in total length of Wallago attu.

Table 4 .
Regression parameters of metal concentration with condition factor (µg/g) in wet body weight for Wallago attu (n = 39).

Table 6 .
Multiple regression analysis of wet weight and condition factor with metal concentration (µg/g) for Wallago attu (n = 39).