Sample collection: The three commonly consumed fish species such as Rui (Labeo rohita), Gulsha (Mystus cavasius) and Shing (Heteropnuestes fossilis) of both wild and cultured type were studied to determine the heavy metal concentrations. Wild fish samples were collected directly from the fisherman of the Meghna River at Boidyer Bazar, Narayanganj District and cultured fish samples were collected from a culture pond of Rajoir fish farm, Rajoir upazila, Madaripur district, in November 2019. In case of Rui fish, ten fish samples were collected and incase of Gulsha and Shing, around three Kilogram fishes (55 Gulsha individuals and 45 Shing individuals) were collected. The samples were transported immediately to the fisheries laboratory of the Department of Zoology, Jagannath University into a chill-box. Collected samples were identified by using the morphological characteristics, following Shafi and Quddus (1982), Rahman (2005) and Fish base (2014), and then stored in freezer at -20 °C in jip-locked plastic bags with proper labeling.
Sample preparation: The samples were taken out from the freezer and thawed at room temperature. The muscles of fishes were taken with the help of steam cleaned stainless steel knife and washed with distilled water. The cleaned fish muscles were homogenized by a blender. Three replicates of each grinded fish sample were analyzed. The determination of moisture and ash contents and digestion were carried out in the Basic Facilities and Sample Processing Laboratory of Centre for Advanced Research in Science (CARS), University of Dhaka, Bangladesh. The analysis of eight different heavy metals namely chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) was carried out in BCSIR (Bangladesh Council of Scientific and Industrial Research), Dhaka.
Determination of moisture content: The weight of moisture free dried empty crucibles was taken, then 10 g blended fish sample were added in each crucible. The crucibles with samples were placed in an oven at 105 °C for 6 hours and then transferred into desiccators. The repeated weight was taken till constant weight was obtained and recorded. Determination of ash content: The crucible containing samples were placed in muffle furnace covering with a watch glass remaining a slight gap at 150 °C for 1 h and then the temperature of the furnace was raised to 200 °C, 300 °C and 400 °C gradually to avoid the loss of sample. The temperature was raised to 600 °C, keeping for 8 hours to obtain ash that is white and free from carbon. Afterwards crucibles were transferred to the desiccators, weighting were repeated till constant weight was obtained and recorded.
Digestion of fish samples: For digestion, 0.2 g dried sample was taken in a beaker and 5 mL of conc. HNO3 was added to the beaker. Then the samples were put on hot plate at 60 °C for digestion under fume hood. When the concentration drops, add 2 mL H2O2 to each sample. Finally, place the mixture on a hot plate at 100°C under a fume hood covering with a watch glass until it become transparent. The digests were allowed to cool and filtered with filter paper, then transferred to a different 50 mL volumetric flask and filled to the 25 mL mark with deionized water to obtain the final volume as a stock solution and kept ready for heavy metal analysis through Atomic Absorption Spectrometry (AAS).
Standards: Standard solutions of selected eight heavy metals such as Cr, Mn, Co, Ni, Cu, Zn, Cd, and Pb were provided by Fluka Analytical, SigmaAldrich (Germany). A calibration curve was prepared for all elements by running different concentrations of standard solutions prepared from certified reference materials (CRM) obtained from Fluka Analytical, Sigma-Aldrich. The standards were prepared from the individual 1000 mg/L standards (Merck) supplied in 0.1N HNO3. A series of working standards were prepared from these standard stock solutions.
Analysis of heavy metal by AAS: Eight heavy metal i.e. Cr, Mn, Co, Ni, Cu, Zn, Cd, Pb were determined by AAS using Zeeman Atomic Absorption Spectrophotometer (GTA 120-AA240Z with PSD 120 auto sampler, Varian, Australia). Measurements were carried out using standard hollow cathode lamps for Cr, Mn, Co, Ni, Cu, Zn, Cd, and Pb. The standard operating conditions for the analysis of heavy metals using AAS used in the experiments. The results were shown as mg/kg or ppm of dry weight. The glassware and containers used in the study were cleaned thoroughly then rinsed with double distilled water for 3-4 times and dried in air prior to use.
Health Risk assessment: The values of heavy metal concentration were used to calculate the estimated target hazard quotients (THQ), hazard index (HI), and target cancer risk (TR) of individuals.
Target Hazard Quotient: The Target Hazard Quotient (THQ) is a measure of the level of non-cancer risk associated with exposure to contaminants. THQ is evaluated using the US Environmental Protection Agency Risk-Based concentration Table-III to estimate the risk to human health from the consumption of metal-contaminated fish (US EPA 2011).