Sampling Area
The sampling sites were selected right from the upstream to downstream of the river limited to the area dominated by pollution status (Fig. 1). The upstream locations are significant and abound in sewage activities from the drainage system of Chittagong city. The six selected sampling stations are known as Chaktai Khal Matha, Firingee Bazar Ghat, Avaymitraghat (which is close to the most polluted water channel in Chittagong city known as Rajakhal), Sadarghat (known as the fishing hub of Chittagong), Bangla Bazar ghat and Saltgola respectively showed in Fig. 1. There are about 300 factories and industrial establishments along the 100-kilometer area of the Karnaphuli extending from Chandraghona to the mouth of the river at Patenga, including cement factories, fertilizer manufacturers, paper mills, rayon mills, tanneries, oil refineries, power plants, dyeing and washing plants (Azad, 2022). Among 36 canals that are connected to the Karnaphuli river, some significant canals known as Mohesh Khal, Chaktai Khal, Murari Khal, Khondokia Khal, Boalkhali canal, BoRubi Gate Khal, Maizpara Khal, and Rajakhali Khal, dump raw and partially decomposed sewages, solid wastes, domestic organic wastes and untreated industrial discharge into the Karnaphuli river (Azad, 2022); (Hossain et al., 2005).
Chaktai Khal, also known as the grief of Chittagong City, plays the most dominant role in the environment of the Karnafully river by dumping a large amount of solid and liquid wastes from the industrial, commercial, and residential areas adjacent to it. Again, the Saltgola canal contains a high amount of chemical waste from CEPZ and port-based industries which contain a large number of heavy mineral particles. Fish wastage, human wastage, oil spills of fishing vessels, market waste, etc. also caused pollution at Firingee Bazar Ghat and Sadarghat area of Karnaphuli River. Besides, there are 400 slaughterhouses in the Firingee Bazar and Dewanhat areas alone. The blood from the slaughtered animals directly finds its way into the river. Moreover, about 50,000 sanitary and 24,000 unhygienic traditional latrines of the port city are directly linked to the study area of this river (Hussain, 2019).
Sample Collection And Analysis With Preservation:
Present work was carried out in September; 2022 just after heavy rain. Water and soil samples were collected from six different stations of the Karnaphuli river estuary at low tide during this study period. In situ measurement water temperature was measured by a centigrade thermometer where water pH and salinity were measured by a pen pH meter and refractometer respectively. Dissolve Oxygen (DO) concentration was measured in the laboratory by the standard method (APHA, 1976). Water samples from three different stations were collected from 500 ml. bottles from the Karnaphuli River estuary with screw-capped and sealed with aluminum foil paper to control the bacterial growth before proceeding in an ice box at about 40C temperature until brought to the laboratory of the Department of Oceanography, CU. Soil samples were also collected from six same stations with grab samplers and stored in 500g jars. During the sampling, at first, the grab samplers were throughout to the bottom which carries a lot of soil filled with jars and then capped under an icebox and brought to the specified laboratory.
Bacterial isolation and Enumeration
The diversity of pathogenic bacteria (Salmonella spp.) in water and soil was identified by standard plate count (SPC) techniques (APHA, 1976). The above methods for enumeration of pathogenic bacteria were based on the fact that each of them gives characteristics type of colony confirmed by further gram straining, biochemical, and fermentation tests for accuracy.
As we are searching for Salmonella spp., we used specific agar media Bismuth sulfite agar (BSA) particularly useful for the isolation of lactose-fermenting Salmonellae. S. Typhi, S. Enteritidis, and S. Typhimurium typically grow as black colonies with a surrounding metallic sheen resulting from hydrogen sulfide production and reduction of sulfite to black ferric sulfide.
For isolation from the water sample, aseptically 1ml suspension from diluted suspensions of 10− 1 and 10− 2 was poured into BSA agar plates for isolation of Salmonella spp. and gently rotated for spreading the suspension. Agar plates were incubated for 48 hours at 37°C. In the case of sediment, 1 gm sediment from each sample was suspended into 9ml sterile distilled water and serially diluted to suspensions of 10− 2, and 1ml suspension from 10− 1 and 10− 2 dilution was poured into BSA agar plates for isolation and incubated for 48 hours in 37°C. After 48 hours All the black colonies with a surrounding metallic sheen are counted. The number of colonies is multiplied by the dilution factor and the mean result from dilution 10− 1 and 10− 2 plates is recorded. The result and the spatial distribution of Salmonella spp. are visualized with Arc Gis (10.8). The cultures were identified according to the bacteriological analytical manual (BAM) of the United States Food and Drug Administration (USFDA). 2 colonies from Petri dishes are further tested (Gram Staining, Indole Test, Methyl Red Test, Urease Test Voges Proskauer Test, Starch Hydrolysis Test, Catalase Activity Test, Fermentation Test) for confirmation of Salmonella spp. The relation between the physical parameters and Salmonella spp. from water and sediment are analyzed with R programming and visualized with the correlogram and similarity and dissimilarity among the parameters along with Salmonella spp. The count is shown in heatmap.