Indian Journal of Geo Marine Sciences Vol. 47 (05), May 2018, pp. 1075-1080

Comparative study of Salmonella and Vibrio spp. distribution in the Karnafuly River estuary at with response to microbial pollution

Md. Wahidul Alam* & Mohammad Mizanur Rahman Institute of Marine Sciences and Fisheries, University of Chittagong, Chittagong-4331, Bangladesh

*[Email: [email protected]]

Received 03 August 2017; revised 22 November 2017

The present study carried out to examine the present status and contribution of Salmonella spp. and Vibrio spp. presence in soil and water along with water parameters at the Karnafuly River estuary during March-June 2015 from three stations to find out the microbial load. Water temperature ranges from 26.5 - 32ºC, salinity ranged between 0ppt to 5ppt; pH 6.5 – 8.5 and DO varies 1.20 – 2.91mg/L in water samples. Maximum Salmonella spp. were observed 5cells/gm. in soil at St-1 in May and maximum Vibrio spp. was 6 cells/ml in water sample of St-1 during May. Dissolve oxygen concentration found in most influential factor for occurrence of Salmonella where salinity found for Vibrio spp. at the estuary. Between the two species, Vibrio spp. contributed 67.23% where Salmonella contributed 32.77% response to microbial pollution.

[Keywords: Comparative study; Microbial pollution; Karnafuly River; Salmonella and Vibrio spp.]

Introduction water from the Bay of . Pollution of Aquatic environment contaminated by limited watercourses associated with industrial discharge and microbial population when little amount of organic refuse from human settlements is a global problem15 matters support but higher concentration of organic that everything carried by rivers ultimately goes into matter can support a higher population of bacterial oceans through estuaries. The pollutants are disperse growth.1 Salmonella spp. and Vibrio spp. in water and by turbulence, ocean currents, and tidal action firstly soil can lead to diseases i.e. Typhoid, dysentery and in the estuary then concentrated in the food chain cholera is transmitted by water. The sites of infections components through microbial action or deposited in in animal and human body are the alimentary canal, the bottom soils1. People in Chittagong city are ears, eyes, nasal cavity, skin and upper respiratory depending on Karnafuly River for drinking or tract. Consumption of contaminated shellfishes also another household purpose. Bangladesh developed the exposes humans to marine pathogens. In Bangladesh, first Water Quality Standards in 1976 based on pathogenic bacteria contamination can also lead to International Drinking Water Standards.16 Bangladesh infectious hepatitis and dysentery, which are more standard specification for drinking water (BDS 1240: common. From health hazard point of view, Wood2 1989) was prepared and published by the Bangladesh has identified many indicator microorganisms. Standard and Testing Institution (BSTI) for the Salmonella spp. and Vibrio spp. traditionally control of drinking water including bacterial load. monitored by control to help limit the According to World Health Organization17 and spread of environmental diseases3. Concentration Environment Quality standard for Bangladesh18, the levels of Salmonella and Vibrio spp. are significantly standard value of a total load of Salmonella and influenced by various nonpoint sources such as Vibrio spp. for drinking water is nil/ml of water. surface runoff, bank soils, recreational activity, and Different researchers identified different pathogenic animal excreta.4-8 Studies indicate that soils can bacteria in respect of contaminated fish of the harbor much higher populations of both fecal coliform Karnafuly River but it is rare to identify the presence and Vibrio spp. than the overlying water column9-14. of Salmonella spp. and Vibrio spp. in water and soil in Karnafuly river estuary is one of the most respect of this area. Consider the importance of water important estuaries in Bangladesh and hydro- and soil of the estuary in daily life, the present biologically, it is the meeting place in which fresh research work aimed to estimate the presence of water from upstream in continuously mixing with salt Salmonella and Vibrio spp. in water and soil of 1076 INDIAN J. MAR. SCI., VOL. 47, NO. 05, MAY 2018

Karnafuly river estuary, which lead the microbial (SPC) techniques.20 As the Salmonella and Vibrio pollution. spp. were very rare in water and soils, so the author did the work without serial dilution in respect of Materials and Methods identifying the growth of colonies in selective The Karnafuly is the principal river of Chittagong medium. For this reason, SPC was conducted by district of Bangladesh. It originates in the Lushai Hills making without serial dilution, plating and identify of Mizoram (India), flows through Rangamati and the colony by presence or absence. Presence or absence port city of Chittagong and discharges into the Bay of of Salmonella spp. and Vibrio spp. estimated by using Bengal near Patenga. The river is flashy and its length pour plate techniques. 1 ml. of water (1ml: 9 ml of is about 131 km. Geographically this estuary located Distilled Water) or diluted soil samples (1gm: 9 ml of between latitude 22053/ N and longitude 92027/ E Distilled Water) were transferred by sterile pipettes and enters the districts of Chittagong from the into Petridis and the plates were poured with Brilliant northeastern side. Geologically, the entire catchment Green Agar (BGA) media for Salmonella spp. and consists of a substratum of tertiary rocks covered with Thio-sulphate Citrate Bile Salt (TCBS) Agar media alluvial deposits. The overlying deposits show that it for Vibrio spp. and incubated at room temperature for consists of successive layers of mud and sand.19 48 hours. The growth of Colonies having a pink Hydrological parameters i.e. temperature, salinity, opaque color surrounded by a bright red color pH, D.O fluctuate seasonally. Three sampling stations confirmed as Salmonella spp. present and colonies (Fig. 1) were select for the present investigation based having a greenish center confirmed as Vibrio spp. on different types of marine pollution. present. Present research work carried from March to June 2015. Water and soils collected from three stations Results and Discussion (St-1, St-2, and St-3) of the Karnafuly river estuary Results from the water samples showed distinct during this study period. In situ measurement of water hydrographic conditions (Fig. 2). Water temperature 0 0 temperature measured by centigrade thermometer varies between 26.5 C to 32.0 C. Maximum water 0 where water pH and salinity measured by pen temperature was recorded 32.0 C in May at St-1and pH meter and refract meter respectively. DO St-3 whereas minimum temperature recorded in concentration was measured in the laboratory by March at St-1. The monthly maximum average 0 following standard method.20 Collected water and soil temperature was found 31.67 C during May where 0 sample was preserved and microbial analysis was minimum 26.83 C during March. On the other hand conducted at the laboratory to identify the presence of average maximum water temperature was recorded 0 0 Salmonella and Vibrio in water and soil of the 29.25 C at St-2 and 3 and minimum 29.13 C at Karnafuly estuary. Salmonella spp. and Vibrio spp. of St-1. No significant difference observed in water water and soil identified by standard plate count temperature among the stations but significant

Fig. 1 — Map of the study area with sampling stations ALAM & RAHMAN: COMPARATIVE STUDY OF SALMONELLA AND VIBRIO SPP. DISTRIBUTION 1077

Fig. 2 — Physiochemical parameters of Karnafuly River estuary at different stations difference was found among the months (F=23, stations though significant difference was observed P<0.01). Salinity of the present study found between among months (F=25, P<0.01). 01ppt to 05ppt. Maximum recorded (05ppt) in May at Status of Salmonella spp. and Vibrio spp. colony in St-3 and minimum 0.3ppt at St-1, 2 of all months. water and soil sample shown in Fig. 3. No Salmonella Monthly maximum average salinity found 1.67ppt spp. was present in water sample except St-1. Only during May whereas minimum salinity was recorded 1cell/ml and 3cells/ml Salmonella spp. found in water 0.33ppt during March and April. Average maximum sample of St-1 during April and May respectively. salinity observed 2.3ppt at St-3 and average no Vibrio spp. concentration found nil during March and salinity found at St-1 and St-2. No significant June in water sample at St-1 but 4cells/ml and difference observed among the stations but have 6cells/ml concentrations observed during April and significant difference among months (F=26, P<0.001) May. On the other hand this species was absent in for salinity value. Water pH varies from 6.5 to 8.5 and St-2 during May and June but very few concentrations maximum pH value was recorded 8.5 at St-3 during (2cells/ml and 3cells/ml) was found during March and May and minimum 6.5 during June at St-2. Monthly April. No Vibrio spp. found throughout the study maximum mean pH was recorded 8.13 during May period in water sample at St-3. and minimum 7.2 during June. Average pH at In case of soil sample, 2, 5 and 3 cells/ml of maximum level occurred at St-1(7.7) where minimum Salmonella spp. found at St-1 during March, May and found at St-2 (7.6). No significant difference was June respectively whereas it was absent in April. found in pH values among the stations but significant During March and April Salmonella spp. was absent difference was found among the months (F=31, in the soil sample at St-2 but only few colony P<0.01). Dissolved Oxygen (DO) concentration in (2cells/ml and 1cell/ml) observed during May and water sample varies between 1.20mg/L to 2.91mg/L. June. No concentration of Salmonella spp. found in The maximum D.O. contents recorded in 2.91 in June soil sample at St-3. At St-1, Vibrio spp. found at a at St-3 and minimum recorded in May at St-2. concentration 3, 5, 7 and 4cells/ml in March, April, Monthly maximum mean DO found 2.71mg/L during May and June respectively. During March and June June and minimum 2.02mg/L during May. On the occurrence of this species was nil but 3cells/ml and other hand maximum mean DO 2.63mg/L was 4cells/ml concentration found during April and May. recorded at St-1 where minimum 2.25mg/L at St-2. Only 1cell/ml Vibrio spp. found in soil sample at St-3 No significant difference was found in DO among the during May and was absent in other months. 1078 INDIAN J. MAR. SCI., VOL. 47, NO. 05, MAY 2018

Fig. 3 — Salmonella and Vibrio spp. concentration in water and soil sample at three stations

Table1 — SIMPER analysis of Vibrio and Salmonella concentration in water and soil sample Groups Average Contribution Dissimilarity (%) Vibrio-soil 24.88 40.46 Vibrio-water 16.46 26.77 Salmonella -soil 15.23 24.76 Salmonella -water 4.927 8.01 Total Vibrio 41.34 67.23 Total Salmonella 20.157 32.77 significantly correlated with dissolve oxygen concentration. SIMPER analysis (Table 1) showed that Vibrio spp. in soil contributed 40.46% whereas it contributed 26.77% for water sample. Salmonella spp. contributed 24.76% and 8.01% for soil and water

Fig. 4 — Canonical correspondence analysis (CCA) ordination of sample respectively. Between two species, Vibrio spp. species concentration and water parameters contributed 67.23% whereas Salmonella spp. Canonical correspondence analysis (CCA) contributed 32.77%, which indicate the abundance of ordination of species concentration and water total Vibrio spp. load at Karnafuly river estuary. parameters shown in Fig. 4. In CCA, species From the present physiochemical study of the concentration plotted closer to the vector, have water quality of the Karnafuly, it can be concluded stronger relationships with them. Species concentration that the condition of the Karnafuly river is critical. located near the origin either do not show a strong Decreasing trend of DO of the Karnafuly river water 21 relationship to any of the variables or are found at observed from the present and previous study. average values of water parameters. CCA Eigen Minimum DO value found for the Karnafuly as low as values of the first and second axis found 0.02 and 1.20mg/L. This indicates the critical condition of this 0.008 respectively. Species concentration and water River. DO values in the range (2.74-5.12 mg/L) parameters Pearson correlation coefficients for the and (3.95-5.97 mg/L) for river water samples and first and second axis found 0.62 and 0.41 respectively. (3.73-5.01 mg/L) and (5.04-5.49 mg/L) for lake water 22 The first and second axis modeled 72.01 and 27.94% samples in the dry and rainy seasons, respectively. data respectively. Water pH found significantly Mean pH value of the water was found in the correlated with Vibrio concentration in water sample alkaline range. Similar pH values 7.5-8.5 were reported where significant relationship found for St-2 and total by Chowdhury23 at the Naf river which almost similar Vibrio concentration with water temperature. to the present findings. McLusky24 stated that salinity Salinity showed significant correlation with St-1 and of an estuary ranged between 0.50ppt to 35ppt and St-3 and Vibrio concentration in soil. Occurrences of Ahmmad25 showed the salinity ranged between Salmonella concentration in soil sample found 14.43ppt to 25.92ppt salinity of the present work found ALAM & RAHMAN: COMPARATIVE STUDY OF SALMONELLA AND VIBRIO SPP. DISTRIBUTION 1079

at similar value. Kamal (1992)26 also reported variation number of chemical and fertilizer industries have been of water temperature for seasonal change. Kamal established on both the banks of the Karnafuly River. (1997)27 and Khan (2001)28 observed seasonal variation Effluents from these industries are directly and of water temperature ranged between 7.54ºC and 10ºC. indirectly discharge into this river. Huge amount of During the present study, monthly variation of water solid wastes and effluents are discharged through temperature also observed. different canals into the Karnafuly River, indicated In the Karnafuly estuarine area, many people used the increasing trends of river pollution day to day. estuarine water and soils for drinking, bathing and Leaking and leaching of oil from ships and boats are various houses hold purposes. Some pathogenic also polluting the water of the Karnafuly River bacteria contaminate estuarine resources and fish several times. Polluted water and soil creates a species because water and soils are the potential favourable condition for Salmonella and Vibrio spp. career of such types of bacteria. Health hazard multiplication. indicating bacteria are those, which are pathogenic to In the present work with compare between the organisms including fish species, human etc. or create water and soil, it found that soil sample carries more health hazard disease including diarrhea, dysentery, presence of bacterial colony in different stations than cholera, typhoid etc. So estuarine water and soils were water sample. In Karnafuly river area, people should be free from such types of microorganisms. suffering by various health hazard diseases due to During the study period, it was observe that uses of water for drinking, bathing, washing and soil Salmonella spp. and Vibrio spp. were very rare and for various houses hold purposes. Salmonella and sometimes absent in different station. The presence or Vibrio spp. contaminate water and soil resources and absence of Salmonella spp. and Vibrio spp. were also fish species of Karnafuly estuary. It concluded that observe in all the sampling stations during the Salmonella and Vibrio spp. in the Karnafuly estuary study period. The presences of these health exceed the standard level both in water and in soil hazard organisms indicate truly polluted aquatic and proved that, the abundance of Vibrio spp. environments because of producing many serious (67.23%) is higher than Salmonella spp. Therefore, it diseases in animal and human body. In the present is recommend that, the water of Karnafuly River is investigation, Vibrio spp. found more than Salmonella polluted by different microbial activity and is spp. whereas Salmonella spp. not found in both water unhygienic for drinking, even washing without and soils at St-3. 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