BETTER WATER QUALITY INDICATORS FOR UNDERSTANDING MICROBIAL HEALTH RISKS A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Science in the University of Canterbury by P. Megan L. Devane Waterways Centre for Freshwater Management University of Canterbury 2016 Table of contents Table of contents ............................................................................................................................... i List of Tables .................................................................................................................................. iv List of Figures .................................................................................................................................. v Acknowledgements .......................................................................................................................... 1 Abstract ............................................................................................................................................ 2 Abbreviations ................................................................................................................................... 4 1 Chapter One: Introduction ....................................................................................................... 6 1.1 Faecal pollution and its effects .......................................................................................... 6 1.1.1 What is the nature of faecal pollution? ...................................................................... 6 1.1.2 Faecal pollution in water and its effects on human health and ecosystems ............... 6 1.2 Microbial faecal indicators .............................................................................................. 12 1.2.1 Indicator use in drinking and recreational water assessment ................................... 12 1.2.2 Limitations of current faecal indicator bacteria ....................................................... 13 1.3 Identifying Faecal sources: Faecal source tracking (FST) .............................................. 20 1.3.1 Chemical FST markers: Faecal Steroids .................................................................. 21 1.3.2 Chemical FST markers: Fluorescent Whitening Agents ......................................... 26 1.3.3 Microbial source tracking and PCR markers ........................................................... 27 1.4 Factors affecting FST marker persistence in the environment over time ....................... 31 1.4.1 The persistence of faecal steroids in the environment ............................................. 31 1.4.2 The persistence of Fluorescent Whitening agents in the environment .................... 33 1.4.3 PCR marker persistence in the environment ............................................................ 34 1.5 Limitations of current methods of faecal identification .................................................. 36 1.5.1 Correlations between FIB, FST markers and pathogens ......................................... 36 1.5.2 Faecal ageing ........................................................................................................... 37 1.6 Research aims .................................................................................................................. 40 1.7 Overview of the thesis structure ...................................................................................... 41 2 Chapter Two: Analytical Methods ......................................................................................... 43 2.1 Microbial analysis ........................................................................................................... 44 2.1.1 E. coli ....................................................................................................................... 44 2.1.2 F-RNA phage ........................................................................................................... 44 2.1.3 Clostridium perfringens ........................................................................................... 45 2.1.4 Campylobacter spp................................................................................................... 45 2.1.5 Analysis of Protozoa in water .................................................................................. 45 2.1.6 Faecal ageing ratio: AC/TC ..................................................................................... 47 2.2 Dry weight analysis ......................................................................................................... 47 2.3 PCR markers ................................................................................................................... 48 2.3.1 DNA extraction methods ......................................................................................... 48 2.3.2 PCR amplification conditions .................................................................................. 49 2.4 Metagenomic studies on irrigated cowpat faecal DNA extracts ..................................... 53 2.4.1 Amplicon preparation and sequencing..................................................................... 53 2.4.2 Data analysis of cowpat faecal DNA sequences ...................................................... 54 2.4.3 Microbial community diversity................................................................................ 55 2.5 Steroid analysis of water, sediment and cowpat runoff samples..................................... 56 2.5.1 Extraction of faecal steroids from environmental matrices ..................................... 56 2.5.2 GCMS protocol for analysis of steroids................................................................... 56 2.6 Fluorescent whitening agents (FWA) ............................................................................. 57 3 Chapter Three: Indicators and pathogens in urban river water and sediments after significant discharges of human raw sewage ................................................................................................... 60 i 3.1 Introduction ..................................................................................................................... 60 3.2 Methods ........................................................................................................................... 64 3.2.1 Site Location ............................................................................................................ 64 3.2.2 Collection of river water and sediment .................................................................... 65 3.2.3 Analysis Methods..................................................................................................... 66 3.2.4 Physical and chemical water parameters ................................................................. 66 3.2.5 Statistical analysis .................................................................................................... 66 3.3 Results ............................................................................................................................. 70 3.3.1 Water: Determining the source of faecal contamination ......................................... 71 3.3.2 Water: microbial indicators and potential pathogens ............................................... 73 3.3.3 Water: comparisons between discharge and post-discharge concentrations ........... 82 3.3.4 Water: relationships between microbial indicators and pathogens .......................... 85 3.3.5 Water: relationships between FST markers and microbes ....................................... 88 3.3.6 Water: the potential faecal ageing ratio of AC/TC .................................................. 95 3.3.7 Water: a steroid ratio indicative of untreated human faecal inputs ......................... 95 3.3.8 Sediments: Chemical FST markers .......................................................................... 96 3.3.9 Sediments: Microorganisms................................................................................... 101 3.3.10 Sediments: relationships between indicators and pathogens ................................. 106 3.3.11 Sediments: potential faecal ageing ratios ............................................................... 108 3.4 Discussion ..................................................................................................................... 110 3.4.1 Microbial indicators for assessing pathogen presence ........................................... 111 3.4.2 Pathogen concentrations from direct sewage discharge ........................................ 115 3.4.3 Wildfowl and canine markers ................................................................................ 116 3.4.4 Relationships between FST markers and microbes ............................................... 118 3.4.5 Sediments as a reservoir of microorganisms ......................................................... 119 3.4.6 Sediments as a reservoir of chemical FST markers ............................................... 121 3.4.7 Potential faecal ageing ratios ................................................................................. 123 3.4.8 Conclusions ............................................................................................................ 126 4 Chapter Four: Impacts on FST markers as cowpats decompose under field conditions 128 4.1 Introduction ..................................................................................................................