UCLA UCLA Electronic Theses and Dissertations Title Evaluation and Performance of Rapid Methods for Identifying and Tracking Sources of Fecal Pollution in Coastal Watersheds Permalink https://escholarship.org/uc/item/5sr2k7mq Author Thulsiraj, Vanessa Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles Evaluation and Performance of Rapid Methods for Identifying and Tracking Sources of Fecal Pollution in Coastal Watersheds A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Civil Engineering by Vanessa Thulsiraj 2014 © Copyright by Vanessa Thulsiraj 2014 ABSTRACT OF THE DISSERTATION Application of Rapid Methods for Identifying and Tracking Sources of Fecal Pollution in Coastal Watersheds by Vanessa Thulsiraj Doctor of Philosophy in Civil Engineering University of California, Los Angeles, 2014 Professor Jennifer A. Jay, Chair Fecal contamination of coastal waters is known to degrade the environment and poses a health risk to recreational beach users. Fecal indicator bacteria (FIB) are used around the world to assess water quality and characterize fecal contamination. Elevated levels of FIB have been linked to health risks in epidemiological studies. However, some limitations exist with this indicator. FIB cannot be used to identify the specific sources as they originate from both human and animal sources. FIB may also persist and regrow in the environment. In order to effectively remediate the cause of pollution and characterize the hazards at chronically impaired beaches it is necessary to measure indicators that can provide information about the sources of the general fecal pollution. Tracking pollution sources at impaired beaches is critical to ensuring the health of coastal watersheds and reducing the incidence of swimming related illness. Molecular methods have gained popularity to identify and detect sources of fecal ii contamination using host-associated markers. The work presented here addresses areas warranting further research in the state of the science of water quality monitoring. In Chapter 2, we demonstrate that host-associated markers exhibit similar limits of detection in different water types and are robust in environmental field applications. Additionally, we provide a cost-benefit analysis and provide water quality managers with information supporting the inclusion of molecular methods in current monitoring practices. This body of work also presents novel methods for rapid and viability-based detection of recent fecal contamination with propidium monoazide (PMA-qPCR) and a field portable method covalently-linked IMS/ATP technique (Cov-IMS/ATP). In Chapters 3 and 4, we present results on optimization and specificity of the Cov-IMS/ATP. We evaluated the performance of Cov- IMS/ATP at three different watersheds for rapid quantification of enterococci, and show this method to be a robust tool in assessing water quality at complex sites. This work also addresses drawbacks of traditional qPCR to quantify viable fecal contamination. We validate the PMA- qPCR method and demonstrate its performance in detecting recent fecal contamination in environmental waters. Use of these methods demonstrates a new framework that can enhance current microbial source tracking studies and water quality monitoring. iii The dissertation of Vanessa Thulsiraj is approved. Richard F. Ambrose Michael K. Stenstrom Shaily Mahendra Jennifer A Jay, Committee Chair University of California, Los Angeles 2014 iv Dedication Page To my family, friends and better half for all the love and support throughout the years, I could never have achieved this goal without you all by my side. v Table of Contents Chapter 1: Introduction to the evaluation and performance of rapid methods for identifying and tracking sources of fecal pollution in coastal watersheds ........................................................................ 1 1. Introduction......................................................................................................................................... 1 2. References........................................................................................................................................... 5 Chapter 2: Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters ................................................ 8 Abstract: ................................................................................................................................................. 8 1. Introduction......................................................................................................................................... 9 2. Methodology ..................................................................................................................................... 11 3. Results .............................................................................................................................................. 18 4. Discussion ......................................................................................................................................... 24 5. Conclusion ........................................................................................................................................ 29 6. Appendix A ....................................................................................................................................... 36 7. References......................................................................................................................................... 48 Chapter 3. Optimization of the Cov-IMS/ATP method for rapid assessment of coastal water quality ...... 53 1. Introduction....................................................................................................................................... 54 2. Materials and Methods ...................................................................................................................... 56 3. Results .............................................................................................................................................. 63 4. Discussion ......................................................................................................................................... 70 5. Conclusions....................................................................................................................................... 74 6. Appendix B ....................................................................................................................................... 85 7. References......................................................................................................................................... 92 Chapter 4. Use of viability-based methods for improved detection of recent fecal contamination in a coastal watershed near Tijuana, Mexico .................................................................................. 96 1. Introduction....................................................................................................................................... 97 2. Methods .......................................................................................................................................... 100 3. Results ............................................................................................................................................ 106 4. Discussion ....................................................................................................................................... 111 5. Conclusion ...................................................................................................................................... 115 6. Appendix C ..................................................................................................................................... 129 7. References....................................................................................................................................... 131 Chapter 5. Molecular marker data suggests that FIB inputs to coastal waters at Topanga State Beach are from multiple animal sources each with different seasonal patterns. ..................................... 137 vi 1. Introduction..................................................................................................................................... 138 2. Materials and Methods .................................................................................................................... 139 3. Results ............................................................................................................................................ 146 4. Discussion ....................................................................................................................................... 156 5. Conclusions..................................................................................................................................... 159 6. Appendix D ..................................................................................................................................... 174 7. References....................................................................................................................................... 181 Chapter 6. Summary: Evaluation