Search

Estuarine water quality is declining worldwide due to increased tourism, coastal development, and a changing climate. Although well-established methods are in place to monitor water quality, municipalities struggle to use the data to prioritize infrastructure for monitoring and repair and to determine sources of contamination when they occur. The objective of this study was to assess water quality and prioritize sources of contamination within Town Creek Estuary (TCE), Beaufort, North Carolina, by combining culture, molecular, and geographic information systems (GIS) data into a novel contamination source ranking system. Water samples were collected from TCE at ten locations on eight sampling dates in Fall 2021 (n = 80). Microbiological water quality was assessed using US Environmental Protection Agency (U.S. EPA) approved culture-based methods for fecal indicator bacteria (FIB), including analysis of total coliforms (TC), Escherichia coli (EC), and Enterococcus spp. (ENT). The quantitative microbial source tracking (qMST) human-associated fecal marker, HF183, was quantified using droplet digital PCR (ddPCR). This information was combined with environmental data and GIS information detailing proximal sewer, septic, and stormwater infrastructure to determine potential sources of fecal contamination in the estuary. Results indicated FIB concentrations were significantly and positively correlated with precipitation and increased throughout the estuary following rainfall events (p < 0.01). Sampling sites with FIB concentrations above the U.S. EPA threshold also had the highest percentages of aged, less durable piping materials. Using a novel ranking system combining concentrations of FIB, HF183, and sewer infrastructure data at each site, we found that the two sites nearest the most aged sewage infrastructure and stormwater outflows were found to have the highest levels of measurable fecal contamination. This case study supports the inclusion of both traditional water quality measurements and local infrastructure data to support the current need for municipalities to identify, prioritize, and remediate failing infrastructure., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hynes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Wastewater based epidemiology (WBE) is useful for tracking and monitoring the level of disease prevalence in a community and has been used extensively to complement clinical testing during the current COVID-19 pandemic. Despite the numerous benefits, sources of variability in sample storage, handling, and processing methods can make WBE data difficult to generalize. We performed an experiment to determine sources of variability in WBE data including the impact of storage time, handling, and processing techniques on the concentration of SARS-CoV-2 in wastewater influent from three wastewater treatment plants (WWTP) in North Carolina over 19 days. The SARS-CoV-2 concentration in influent samples held at 4°C did not degrade significantly over the 19-day experiment. Heat pasteurization did not significantly impact the concentration of SARS-CoV-2 at two of the three WWTP but did reduce viral recovery at the WWTP with the smallest population size served. On each processing date, one filter from each sample was processed immediately while a replicate filter was frozen at -80°C. Once processed, filters previously frozen were found to contain slightly higher concentrations (<0.2 log copies/L) than their immediately processed counterparts, indicating freezing filters is a viable method for delayed quantification and may even improve recovery at WWTP with low viral concentrations. Investigation of factors contributing to variability during sample processing indicated that analyst experience level contributed significantly (p<0.001) to accepted droplet generation while extraction efficiency and reverse transcription efficiency contributed significantly (p<0.05) to day-to-day SARS-CoV-2 variability. This study provides valuable practical information for minimizing decay and/or loss of SARS CoV-2 in wastewater influent while adhering to safety procedures, promoting efficient laboratory workflows, and accounting for sources of variability., Competing Interests: The authors have declared that no competing interests exist.

Rapid molecular testing methods are poised to replace many of the conventional, culture-based tests currently used in fields such as water quality and food science. Rapid qPCR methods have the benefit of being faster than conventional methods and provide a means to more accurately protect public health. However, many scientists and technicians in water and food quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners can use and implement qPCR techniques successfully, we developed a week long workshop to provide hands-on training and exposure to rapid molecular methods for water quality management. This workshop trained academic professors, government employees, private industry representatives, and graduate students in rapid qPCR methods for monitoring recreational water quality. Attendees were immersed in these new methods with hands-on laboratory sessions, lectures, and one-on-one training. Upon completion, the attendees gained sufficient knowledge and practice to teach and share these new molecular techniques with colleagues at their respective laboratories. Key findings from this workshop demonstrated: 1) participants with no prior experience could be effectively trained to conduct highly repeatable qPCR analysis in one week; 2) participants with different desirable outcomes required exposure to a range of different platforms and sample processing approaches; and 3) the collaborative interaction amongst newly trained practitioners, workshop leaders, and members of the water quality community helped foster a cohesive cohort of individuals which can advocate powerful cohort for proper implementation of molecular methods.