Fecal pollution contributes to global degradation of water quality and requires identification of the source(s) for predicting human health risk, tracking disease, and developing management strategies. While fecal indicator bacteria are commonly used to detect fecal pollution, they cannot identify sources. Novel approaches, such as microbial source tracking (MST), can be applied to evaluate the origin of fecal pollution. This study examined fecal pollution in the coral reef lagoons of Norfolk Island, Australia where reef health decline has been related to nutrient input. The primary objective of this study was to evaluate the host sensitivity and specificity of two human wastewater-associated marker genes (Bacteroides HF183 (HF183) and cross-assembly phage (crAssphage)) and four animal feces associated marker genes targeting avian, ruminant, dog, and pig (Helicobacter-associated GFD (GFD), Bacteroides BacR (BacR), Bacteroides DogBact (DogBact), and Bacteroides Pig-2-Bac (Pig-2-Bac)) in wastewater and animal fecal samples collected from Norfolk Island. The prevalence and concentrations of these marker genes along with enterococci genetic marker (ENT 23S rRNA) of general fecal pollution and human adenovirus (HAdV), which is considered predominantly a pathogen but also a human-wastewater associated marker gene, were determined in surface, ground, and marine water resources. A secondary objective of this study was to assess the sources and pathways of fecal pollution to a sensitive marine environment under rainfall events. HF183, crAssphage, HAdV, and BacR demonstrated absolute host sensitivity values of 1.00, while GFD and Pig-2-Bac had host sensitivity values of 0.60, and 0.20, respectively. Host specificity values were > 0.94 for all marker genes. Human and animal (avian, ruminant, dog) fecal sources were present in the coral reef lagoons and surface water whereas groundwater was polluted by human wastewater markers. This study provides understanding of fecal pollution in water resources on Norfolk Island, Australia after precipitation events. The results may aid in effective water quality management, mitigating potential adverse effects on both human and environmental health.

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