2.1 Study AreaThe Rupsha river skilled Khulna metropolitan city was selected for this study. Three sampling points were selected relying on the proximity to the polluted source: S1: at main industrial discharge point near glass and plastic industries, paint and dye manufacturing plants, soaps and detergent factories, paper mills also as steel mills; S2: near Rupsha fish market and Rupshaghat where the effluents of fish processing plants, wastes of brickyards and thus the wastes from agriculture runoff are discharged; S3: at low industrial discharge area during which sewage and municipal wastes unwanted residues admixing with Fe2O3.2H2O resulted from the activities of Shipbreaking yard are directly discharged. 2.2 Sample CollectionIn this present investigation, a complete of 12 effluent samples, 36 water samples, 36 sediment samples, and 40 fish samples of 10 species from the three pointing zones also as nearby areas were collected within the pre-monsoon and post-monsoon, respectively with the help of the fishermen of that locality. All samples were collected in duplicate to research the heavy metals; this analysis was performed accordingly for checking repeatability. 2.4 Water and Sediment SamplesWater and sediment samples were collected from three sampling points in Rupsha River. Water samples were collected in acid-washed polyethylene bottles and Whatman no. 541 paper was used. The samples were then preserved with the addition of 2ml ultrapure HNO3 per litre of water sample to attenuate the absorption of metals into the wall of the containers. Sediment samples were collected with a grab sampler and were placed in pre-cleaned polyethylene bags. Then, the samples were transferred to the analytical chemistry laboratory of the Institute of Nuclear Science and Technology, BAEC to analyze these. Before analysis, the sediment samples were dried, ground, sieved with a 2mm sieve, and stored in airtight plastic vials inside desiccators. 2.5 Fish Samples: Forty fish samples of 10 species (i.e., Mastacembelusarmatus (common Bengali name Baim), Gudusiachapra (Chaplin), Puntiusticto (Puti), Notopterusnotopterus (Foli), Coricasoborna (Kachki), Setipinnaphasa (Fhassa), Amblypharyngodonmola (Mola), Mystusvittatus (Tangra), Heterponeustesfossilis (Singh), and Clupisomapseudeutropius (Batashi)) were collected from the three pointing zones also the closest areas at different times of the year with the help of local fishermen from the Rupsha River. For the preparation and preservation of the fish samples, an in-depth method is described. 2.6 Heavy Metal Analysis: Initially, for the analysis of the heavy metals, effluent and water samples were filtered through paper Whatman no. 541 (Whatman, Germany) into 100 mL of prewashed plastic bottles and thus the analytical grade HCl (Assay: 36.5%; Fisher Scientific, USA) was used to adjust water pH to 3.5. For the heavy metal analysis in sediment samples, the dried sediment samples were digested first according to the procedure of Thompson and Benerjee (1991), during which accurately weighed sediment samples (0.25 g) were treated with 1.0 ml 70% HClO4and 4 ml concentrated HNO3during a clean Pyrex tube (12 × 2 cm). The tube was placed during a laboratory-made heating block and heated gently for 2–3 hours at 150°Cin an oil bath (paraffin) until the copious fumes ceased to evolve. The temperature of the bath was raised to about 190°Cand heating was continued at that temperature until the residue became dry. The dried residue sample was cooled to ambient temperature and later, it's treated with 4 ml HCl (2M). The solution was diluted using double-distilled deionized water and filtered quantitatively (Whatman no. 541) into a 50 ml volumetric flask. Fish digestion was administered according to Hanson (1973) with some modifications. An entire of 0.5 g of fish powder was taken during a digestion apparatus and 2.5 mL of concentrated H2SO4 (Assay: 98%; Fisher Scientific, USA) and 4 mL of concentrated HNO3 (Fisher Scientific, USA) was added. When the initial vigorous reaction subsided, the mixture was heated slowly in an oil bath with the addition of 3/4 drops of H2O2 (Assay: 30%; Sigma Aldrich, USA). This step was repeated until the mixture became clear. The mixture was heated for an extra 20 min at about 150°Cand allowed to relax at a temperature (25 ± 1°C). Then, the mixture was diluted properly with the double-distilled water then filtered quantitatively into a 50 mL volumetric flask.The concentration of the subsequent heavy metals (Pb, Cd, Ni, Cr, Cu, Zn, Mn and As) in water, sediments and fish samples were determined by AAS (Model AA-6800, Shimadzu Corporation Japan) with digital read-out system. 2.7 Statistical Analysis All samples were collected and analyzed in a minimum of twice and thus the 2 tests were statistically similar in paired-samples t-test, at a 95% confidence level. The typical results were wont to represent the informative data during this study. Analysis of variance test was performed to figure out any significant difference in heavy metals concentration among effluents and water samples of the Rupsha River. The various difference of Rupsha River sedimentary metals content was shown employing a student’s t-test. The info of the concentration of the metals within the ten different fish species was also subjected to ANOVA test. To spot the connection among the metals within the studied samples and to infer the hypothetical sources of these metals (natural or anthropogenic), Person’s coefficient of correlation analysis, principal component analysis, and cluster analyses were administered. All calculations were performed using the statistical software SPSS 15 for Windows (SPSS Inc., Chicago, IL, USA). Only cluster analysis was performed using Minitab 16 (MINITAB Inc., State College, PA, USA).