TL21 - MICROBIAL SOURCE TRACKING AT AN URBAN ESTUARY (GOLDEN HORN, TURKEY) .... Challenges in implementing new technology for beach water.
TL21 - MICROBIAL SOURCE TRACKING AT AN URBAN ESTUARY (GOLDEN HORN, TURKEY) Zeki, S1; Aslan, A2; Rose, J3 1 Institute of Marine Sciences and Management, Istanbul University, Turkey; 2Jiann-Ping Hsu College of Public Health, Georgia Southern University, USA; 3Department of Fisheries and Wildlife, Michigan State University, USA
AIMS Fecal pollution is a worldwide environmental and human health problem that affects many coastal and inland waters. Using modern rapid technologies and adding the identification of the sources of pollution component can improve protection and restoration of water quality of these environments. In this study, we evaluated a human specific fecal pollution marker (Bacteroides thetaiotaomicron alpha-1-6, mannanase) in an urban estuary located at the Istanbul metropolitan area. We used quantitative polymerase chain reaction (q-PCR) method for detection. Particular advantages of this method over current recreational water testing methods is its capability to provide more timely results, specificity, sensitivity and quantification (Griffith and Weisberg, 2011, Aslan and Rose, 2012; Layton et al., 2013). The estuary receives freshwater from two creeks and marine water from the Strait of Istanbul. The study area had historically been polluted by urban pollution and non-point sources and has recently gone through a series of rehabilitation efforts. Today, the estuary is open for recreational activities but the microbiological analysis only depends on the national criteria (fecal coliforms) that are not rapid enough to detect the pollution on the same day of sample collection and cannot identify the pollution sources. The goal of this study was to test a rapid qPCR method and Bacteroides thetaiotaomicron alpha-1-6, mannanase as an alternative indicator and compare it with the local criteria and in-situ environmental parameters. METHODS A total of 108 samples were analyzed monthly from February 2011 to January 2012. Fecal coliforms were enumerated by using membrane filtration technique (APHA, 2005). Samples (100 ml) were filtered from sterile 0.45 µm membrane filters (Sartorius 13906-50-AJN) and were incubated on mFC medium (Sartorius 14068-50-N) at 44,5 ± 0,1°C for 24 hours. B. thetaiotaomicron alpha-1-6, mannanase genetic marker was quantified by using qPCR. DNA was extracted from samples (300 ml) by using QIAGEN DNA Mini kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). qPCR reaction mix contained 10 µl of Taqman Light Cycler Master Mix (Roche, Indianapolis, IN, USA), 0.4 µl each of forward and reverse primers, 0.2 µl of the probe, 1 µl of bovine serum albumin, 5 µl of template and nucleasefree water to a final volume of 20 µl. All qPCR analysis were performed by Roche Light-Cycler 480 Instrument (Roche Applied Sciences, Indianapolis, IN). The program was set to 15 min at 95°C, followed by 45 cycles at 94°C for 15s, annealing at 60°C for 60 s, extension at 72°C for 5 s, with final step for 30s at 40°C (Yampara et al, 2008; Aslan and Rose, 2012). In situ environmental parameters were also collected during the study. Winkler Method was used to detect the dissolved oxygen (APHA, 2005). In situ water temperature, salinity and pH measured by WTW Multi 340i probe system. The rainfall data were obtained from the Turkish State Meteorological Service.
RESULTS All 108 samples were positive for fecal coliforms. There was a gradual increase of water degradation towards the upper estuary. The concentrations were below the national criteria at the lower part whereas the fecal coliform compliance remained below 10% at the upper estuary (Table 1). Unlike fecal coliforms, B. thetaiotaomicron was positive in only 8% of the samples at the lower estuary. The concentrations and occurrence followed similar pattern to fecal coliforms and increased towards the upper part (Table 1). Moderate positive correlation was observed between B. thetaiotaomicron and fecal coliform (r = 0.66, p < 0,007). Relationships between B. thetaiotaomicron marker and salinity, temperature, dissolved oxygen, pH, and rainfall were examined. Among all environmental parameters, B. thetaiotaomicron concentrations were negatively correlated to salinity (r = -0.55) and temperature (r = -0.52), and positively correlated to 2 days rainfall (r = 0.79, p < 0.001). Strong seasonality (p
