Fecal contamination in Midwestern karst regions was evaluated by a combination of Bacteroidales-based microbial source tracking (MST), traditional bacterial indicators, and environmental variables. Water samples from springs and wells were collected from Illinois, Wisconsin, Kentucky, and Missouri. A quantitative polymerase chain reaction (PCR) with seven primer sets targeting different members of Bacteroidales was used to discriminate human and livestock fecal pollution. The results suggested that most samples were contaminated by a mixture of human and animal waste sources, with only a few samples showing pollution solely by humans or animals. Spring water tended to be more contaminated than well water, and fecal biomarkers were detected in higher numbers in urban spring systems than rural spring systems. Correlation between traditional bacterial indicators and fecal contamination determined by Bacteroidales-based MST was weak, with the exception of E. coli, chloride, and dissolved oxygen (DO), which correlated well with the level of Bacteroidales fecal biomarkers. The Bacteroidales-based MST method used in this study did not give definitive results, but nevertheless we believe it could be a powerful tool used in conjunction with other measures, including traditional bacterial indicators and a variety of environmental variables. It appears that there currently is no single “silver bullet” for MST in karst aquifers, and future studies should continue to combine chemical-MST and microbe-MST techniques.
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