Heavy Metals Status in Some Commercially Important Fishes of Meghna River Adjacent to Narsingdi District, Bangladesh: Health Risk Assessment

The present study was carried out to determine the heavy metals (Cd, Pb, Zn, Al, Cu, Ni, Fe, Mn, Cr, Co) in the muscles of 32 fish species for 3 seasons in Meghna River. The heavy metals were analyzed by Atomic Absorption Spectrophotometer. The estimated concentrations of all metals in the present study were lower than the limits permitted by FAO, WHO, EU, United States Food and Drug Administration (USFDA), US Environment Protection Agency (US/EPA) and England Guidelines except the concentrations of Pb & Zn that were found above the allowable ranges in different fishes namely Amblypharyngnodon mola. Colisa lalia, Tetraodon cutcutia, Barbodes sarana, Labeo calbasu, Puntius sarena, Ompok pabda, Aila coila, Mastacembelus armatus, Glossogobius giuris, Nandus nandu, Tenualosa ilisha, Lepidocephalichthys guntea, Xenentodon cancila, Stinging catfish. Multivariate statistical analysis such as principal component analysis and correlation matrix showed significant anthropogenic intrusions of Zn, Al, Cd, Pb, Cu, Ni, Fe, Mn, Cr, Co in fishes. There was significant positive correlation between Cd vs Co (0.733), moderate positive correlation between Fe vs Al (0.568), Ni vs Co (0.482), Mn vs Co (0.395) which indicate that their common origin especially from industrial effluents, municipal wastes and agricultural inputs.


Introduction
Industrialization is a term associated with socio-economic activities (Richard 2005; Jaillon and Poon 2009, Thanoon et al. 2003) that alter the society infrastructure (Abdullah et al. 2009) through the huge production (Abdullah et al. 2009; Thanoon et al. 2003). Most of these industries discharge untreated wastes that contribute heavy metal hugely. Industrial and agricultural as well as natural activities are the leading responsible sources of metal contamination in aquatic environment ( Islam et al. 2015a,c). Heavy metal in aquatic ecosystems measured by monitoring concentrations in water, sediments and biota (Camusso et al. 1995) but considerable amount was found in fishes (Rashed 2001) and sediments (Namminga and Wilhm 1976). Several studies have been conducted in rivers and lakes giving special preference to environment during the last decade (

Sampling Sites
Fish samples were collected from two points (Effluent discharge area and far from the discharge area) of Meghna River near Narsingdi District (23º55'28.52"N and 90º45'12.06"E). Sampling procedures were performed in three phases: firstly, September, 2015 (Rainy season); secondly, January, 2016 (Winter season) and thirdly, March, 2016 (Pre-monsoon).

Sample Collection and Preservation
A total of 32 fish species were collected from fishermen for individual season and then identified according to  Quddus and Shafi (1983). After collection the fish were placed immediately in poly-ethylene bags and then kept into isolated container of polystyrene icebox. Finally, the samples were transferred to the Bangladesh Council of Scientific and Industrial Research (BCSIR) in ice box (Irwandi and Farida 2009;Ismail and Saleh 2012) where the fish were first washed with deionized water sealed in poly-ethylene bags subsequently kept in a freezer at -20ºC until analysis (Elnabris et al. 2012).

Heavy Metal Determination
The heavy metal contents were determined by AAS using standard analytical procedure. Sample collection is important stage for metal analysis. Samples were generally carefully handled to avoid contamination. Glassware was properly cleaned, and the reagents were of analytical grade. Distilled water was used throughout the study. Reagents blank determinations were used to correct the instrument readings. The techniques for samples preparation, standard Preparation, analysis for metal analyses have been briefly described below.

Sample Preparation (Dry Ashing Method)
This procedure was also used for destruction of organic Adjacent to Narsingdi District, Bangladesh: Health Risk Assessment matter. Precaution was to be taken to avoid losses by volatilization of elements. At first samples were homogenized. Then the samples were weighed accurately a suitable quantity (10 to 20 g) of the homogenized samples in a tared silica dish. After that the samples were dried at 100°C in a laboratory oven. These dishes were then placed in the muffle furnace at ambient temperature and slowly raised temperature to 450°C at a rate of no more than 50°C/h. The samples were ignited in a Muffle furnace at 450°C for at least 8 hrs. After ashing was completed and cool, then the dishes were removed from furnace. Then the ashes were dissolved in diluted nitric acid (Afthan et al. 2000). The solutions were returned to a hot plate and continued heating, adding additional acid as necessary until digestion was completed. Then the samples were filtrated into a 100 ml volumetric flask using Whatman No. 44 filter paper and washed the residue. Each sample solution was made up to the mark with distilled water.

Standard Preparation
Every metal standard solution was prepared for calibration the instrument for each element being determined on the same day as the analyses were performed due to possible deterioration of standard with time. All samples were prepared from chemicals of analytical grade with distilled water. 1gm of metal Cadmium, Copper, Lead, Nickel were dissolved in HNO 3 solution; 1 g of Cobalt, Iron, Manganese, Zinc, Aluminum were dissolved in HCl solution; 2.8289 g K 2 Cr 2 O 7 (=1g Chromium) was dissolved in water and made up to 1 liter in volumetric flask with distilled water, thus stock solution of 1000 mg/l of Cd, Cu, Pb, Ni, Co, Fe, Mn, Zn, Al and Cr were prepared. (Cantle, J. E. 1982). Then 100 ml of 0.1, 0.25, 0.5, 0.75, 1.0 and 2.0 mg/l of working standards of each metal except iron were prepared from these stock using micropipettes in 5ml of 2N nitric acid. 100 ml of 2.0, 2.5, 5.0, 10.0 and 20.0 mg/l of working standards of iron metal were prepared from iron stock solution. Reagent blank was prepared in the same manners of sample preparation without sample to avoid reagents contamination.

Analysis of Sample
Finally, the atomic absorption instrument was set up carefully. At the meantime, flame condition and absorbance were optimized for the analyte. Then blanks (deionized water), standards, sample blank and samples were aspirated into the flame in AAS (Model-iCE 3300, Thermo Scientific, Designed in UK, Made by China)). The calibration curves were found for concentration vs. absorbance. Data were statistically analyzed using fitting of straight line by least square method. For more accuracy, a blank reading was also taken and necessary corrections were made during the calculation of various elements concentration.

Statistical Analysis
One Way Analysis of Variance (ANOVA) was done to show the variations in concentration of heavy metal in terms of seasons and fish. GGraph was used for graphical presentation of heavy metal against seasons. According to Dreher (2003), Principal Component Analysis (PCA) was performed on the original data set (without any weighting or standardization). Pearson's product moment correlation matrix was done to identify the relation among metals to make the result validate obtained from multivariate analysis.
During winter season, the highest value of Zn was recorded in Amblypharyngnodon mola (44.48mg/kg) and the lowest value was found in Johnius coitor (9.6mg/kg) ( Table 2). The highest concentration of Al was detected in Labeo rohita (106.70mg/kg) and the lowest value was documented in Nandus nandus (3.24mg/kg). The maximum value of Cu was measured in Glossogobius giuris (8.19mg/kg) and the minimum value was recorded in Macrognathus aculeatus (0.20mg/kg) ( Table 2). The maximum amount of Fe was detected in Mystus bleekeri (93.16mg/kg) and the lowest amount was found in Ctenopharyngodon idella (9.86mg/kg). The highest concentration of Mn was found in Colisa lalia (19.87mg/kg) and the lowest concentration was measured in Mastacembelus armatus (1.65mg/kg). Moreover, the maximum amount was recorded for Cd in Ompok pabda (0.21mg/kg); Pb in Colisa lalia (6.75mg/kg); Ni in Amblypharyngnodon mola (0.986mg/kg); Cr in Stinging catfish (3.01mg/kg) and Co in Labeo rohita (0.70mg/kg) while the lowest concentration of these metals were recorded below detection limit ( Table 2).
In Pre-monsoon, the highest value of Zn was recorded in Amblypharyngnodon mola (43.67mg/kg) and the lowest value was documented in Johnius coitor (8.65mg/kg) ( Table  3). The greatest amount of Al was measured in Labeo rohita (104.87mg/kg) and the lowest amount was found in Ctenopharyngodon idella (1.78mg/kg). The supreme concentration of Cu was detected in Barbodes sarana (26.67mg/kg) and the lowest concentration was measured in Macrognathus aculeatus (0.21mg/kg). The maximum value of Fe was recorded in Gudusiachapra (77.66mg/kg) and the minimum value was recorded in Xenentodon cancila (7.85mg/kg). The greatest amount of Mn was measured in Tetraodon cutcutia (20.01mg/kg) and the lowest value was found in Ctenopharyngodon idella (1.01mg/kg). The highest concentration was recorded for Cd in Ompok pabda (0.23mg/kg); Pb in Colisa lalia (6.85mg/kg); Ni in Amblypharyngnodon mola (0.98mg/kg); Cr in Aila coila (8.18mg/kg) and Co in Channa punctatus (0.51mg/kg) whereas the lowest concentration for these metals was documented below detection limit (Table 3).

Analysis of Variance (ANOVA) in Fish Species
There were significant variations in the concentrations of Zinc, aluminium, lead, copper, nickel, iron, cobalt, manganese found across the various fish species as the significance level (p<0.05). However, there were no significant variations (p>0.05) in cadmium and Chromium levels across the fish species. Moreover there was no prevalent variation (p>0.05) in the metal concentration in terms of seasons except for lead and manganese (p<0.05).

Correlation Matrix
In aquatic environment, the inter relationship among metals in fishes provide significant information sources and pathways of variables. The result of correlations between heavy metals acquiesced with the results obtained by PCA and CA that confirm some new associations between parameters. There was significant positive correlation between Cd vs Co (0.733), moderate positive correlation between Fe vs Al (0.568), Ni vs Co (0.482), Mn vs Co (0.395) and weak correlation exist between Cr vs Co (0.351), Pb vs Cr (0.283). The strong and moderate correlation indicates their sources of origin are similar especially from industrial effluents, municipal wastes and agricultural inputs. On the other hand, weak negative correlations were found between Cd vs. Zn (-0.241), Pb vs. Ni (-.155), Cd vs. Fe (-0.147) in river fish.

Principal Component Analysis
The extraction method was used to find out the principal components in PCA analysis that was Eigen values. The components were taken as principal components whose Eigen values was greater than 0.6 were taken into account. 6 PCs were extracted by using correlation matrix which reflects the processes influencing the heavy metals composition having 88.16 % of total sample variance ( Table 4). The total variance of the PCs were 29.0%, 18.81% and 14.48%, 10.91%, 8.74%, 6.22% for PC 1, PC 2, PC 3, PC 4, PC 5, PC 6 respectively. PC 1 is strongly correlated with Cd, Co, Ni, Mn and PC 2 with Al, Fe, Zn. Moreover, there was strong correlation of PC 3 with Pb, PC 5 with Cu and there was no strong correlation for PC 4 and PC 6. The source of PC 1 and PC 2 can be considered as mixed source from anthropogenic inputs particularly from industrial effluents and agricultural activities in the study area. Whereas PC 3, PC 4, PC 5 and PC 6 can be considered as different source from both lithogenic and anthropogenic inputs.

Discussion
The concentration of toxic metals in 32 fish species collected from Meghna River were found to be below the WHO permissible concentrations given for seafood (WHO 1972(WHO , 1987 Ni was found in very low concentration (BDL-0.986) mg/kg in fish samples. The maximum concentration was set by (USFDA 1993) for Ni is 70-80 mg/kg. The present concentration were far below the stipulated limit. Concentrations crossing the set limit may cause cancer of the lung and nasal cavity (USFDA 1993).
Cr is important element that helps the body use sugar, protein, and fat but excess amount have adverse effects on fish and wildlife (Akan et al. 2009) and sometimes Cr is carcinogenic (Institute of Medicine 2002). The concentration of Cr is below the limit (12-13) mg/kg set by the United States Food and Drug Administration (USFDA 1993). Moreover, the deficiency of Cr can affect the glucose, lipid and protein metabolism and impaired growth (Akoto et al. 2014).
Fe concentration varied between (7.85-147.77) mg/kg and highest concentration 147.77 mg/kg in Channa punctatus. Fe is essential element necessary for the production of hemoglobin, myoglobin and certain enzymes (Akoto et al. 2014). Fe deficiency causes anemia in human (Anderson and Fitzgerald 2010), weakness, inability to concentrate and susceptibility to infection (Akoto et al. 2014).
Mn in small amount needed for growth and prevention of cardiac arrest, heart attack, and stroke (Akoto et al. 2014;Ikem and Egiebor 2005). Acute toxicity causes psychologic and neurologic disorder (Saha and Zaman 2012). In present study the concentration of Mn in the fish muscles varied from 0.96-20.01 mg/kg. Similar result found by (Akoto et al. 2014) and this result was lower than Begum et al. (2005) in Dhamondi Lake, Dhaka, Bangladesh (8.8-23.5 µg/g) and 0.59-11.74 µg/g in Lake Tanganyika, Tanzania (Chale 2002).
Co concentrations was measured in all fish samples ranged from BDL-0.7 mg/kg wet weight. Co is beneficial to health but high level of Co may cause lung and heart effects and dermatitis (Agency for Toxic Substances and Disease Registry 2004).
The concentrations of Al ranged from 1.78-120.4 mg/kg in the fish muscles lower than the criteria for the maximum concentrations and criterion continuous concentration values of the US EPA (EPA 2002) water quality criteria. Aluminium frequently occur in the biosphere (Poleo 1995; Gromysz-Kalkowska and Szubartowska 1999; Weng et al. 2002) and the in concentration of Al in rivers and lakes may cause fish death (Reitz et al. 1996; Alloway and Ayres 1997). Ikem and Egiebor (2005) stated that some factors (the duration of exposure of fish to contaminants in water, feeding habit of fish, concentrations of contaminants in water column, water chemistry, contamination of fish during handling and processing) influenced the contamination of fish.

Conclusion
The findings of the present study indicate the fish community that are consumed regularly by the local people is being contaminated by different heavy metals discharged from the various industrial, urban as well as the agricultural activities occurred in the vicinity of the Meghna River. The elevated level of heavy metals found in most edible fishes ultimately will harm the human health. Therefore, the present research recommends that the point sources of heavy metals in the vicinity of the Meghna River should be strictly monitored for protecting the health of riverine ecosystem along with fish community.