Research Areas > Beach Water Quality > Science to Support the Commission’s Rapid Methods Task Force

Project: Science to Support the Commission’s Rapid Methods Task Force

Background and Objectives

In 2009, the SCCWRP Commission created a Rapid Methods Task Force to advance the use of rapid microbiological methods for beach monitoring and decision-making. The Task Force was formed of experts representing the wastewater treatment, regulatory, public health, local government, scientific, and environmental communities. They were tasked with reviewing the best available science regarding application of rapid methods, and making recommendations as to how to implement them for beach monitoring in Southern California. As part of their review, they discussed important logistics issues with regards to sampling, laboratory processing, data transfer, and public communication of beach warnings or closures. In addition, the Task Force requested that SCCWRP perform several additional technical studies to fill important data gaps. This project details the research that SCCWRP conducted in partnership with the Rapid Methods Task Force to gather data about the applicability of rapid methods. The goal of this project was to develop and refine a rapid microbiological method that could augment or replace the existing culture methods for one or more types of indicator bacteria. The objectives were to:

• Develop a method that will detect and quantify indicator organisms (or a molecular substructure of the organism) in less than two hours;
• Demonstrate equivalency to existing methods and a relationship to health risk in beachgoers;
• Evaluate the ability of the method to be transferred to end users at local laboratories; and
• Understand in situ factors that affect the results of the rapid analysis.

Status

This study was initiated in 2009 and completed in 2010.

Methods

Scientific findings in three topic areas were compiled, and two additional technical studies were conducted to support the Task Force.

• Side-by-side comparisons with traditional methods - Data was collated from trials conducted with developers (alpha testing) and end users (beta testing). In addition, data comparing rapid and traditional methods performed during epidemiology studies was examined.

• Epidemiology studies - Results from SCCWRP’s Southern California epidemiology studies at three beachs (Surfrider, Doheny, and Avalon) were examined to determine whether rapid methods were indicative of health risk. In these studies, swimmers were recruited at the beach and interviewed by phone 10-14 days later to identify whether they had experienced any health symptoms after swimming. Water quality measurements were taken at the time of exposure, and then compared with swimmer illness rates to produce an odds ratio that predicted the likelihood of illness and the relationship between illness and indicator organisms.

• Evaluation of method alternatives - This effort examined the effect of using different permutations of the qPCR method, including variation in the instrument, the chemistry, the primer-probe set, the internal control, and the calibration approach. It compared analyses from eight different laboratories analyzing identical sets of samples, but using three different brands of reagents and probes, four qPCR platforms, three calculation algorithms, and three calibration methods.

• Inhibition - This study assessed the effects of chemicals present in water samples that inhibit the polymerase chain reaction. Inhibition may lead to underestimation of bacteria levels and false negative test results, which could cause contaminated beaches to remain open. The study quantified the percentage of samples that were inhibited, the susceptibility of three different methods to inhibition, the effectiveness of four different internal controls, and the effects of adding in a dilution or DNA purification step.

• DNA degradation - This study examined differential degradation of the DNA signal detected by qPCR for waters influenced by disinfected effluent, in order to understand the chances of false positive test results from non-viable DNA. Factors assessed in the laboratory study included the disinfection procedure (chlorination vs. UV light), the discharge location (marine vs. freshwater), ambient water temperature, and exposure to light.

Findings

Findings from the studies to support the Task Force are summarized below:

• Side-by-side beta testing showed that introducing the rapid qPCR method along with the two existing traditional methods (EPA 1600 and Enterolert) adds slightly more variability, but nonetheless allows the user to get an answer a day faster. Side-by-side method comparisons in SCCWRP’s beach epidemiology studies also showed slightly more disagreement than that found for traditional methods. At Malibu, the relationships were poorer than the other sites, possibly due to disinfection issues.

• Epidemiology study results showed some significant health risk relationships for both traditional and rapid qPCR methods. Importantly, though, the relationships for the traditional methods were not significant when results were lagged by one day, reflecting the time it typically takes to process the samples.

• The evaluation of method alternatives indicated very little difference in results among laboratories, qPCR platforms, reagent and probe chemistry, calculation algorithms, and calibration methods.

• The inhibition study found the highest prevalence of substances inhibiting the qPCR reaction in storm drain samples, though some incidence of inhibition was detected across the board. In terms of internal controls for inhibition, the Scorpion assay with a salmon sperm control performed the best, producing about 75% agreement with the gold standard. A dramatic reduction in inhibition was seen using both the control and a 1:5 dilution of the sample. The other option, a 1-hour DNA purification, was extremely effective but at the expense of time.

• For the DNA degradation study, the difference in signal degradation time among QPCR and two traditional methods was evident, but was not large enough to concern the Task Force.

Based on the available information, the Task Force recommended moving forward with a pilot implementation project in summer 2010.

Partners This research was conducted in collaboration with the Rapid Methods Task Force, Orange County Sanitation District, Orange County Public Health Department, State Water Resources Control Board, LA Regional Water Quality Control Board, EPA Office of Science and Technology, South Orange County Wastewater Authority, University of North Carolina, Heal the Bay, and the City of Newport Beach. Members of the Rapid Methods Task Force: • Dr. Mas Dojiri - City of Los Angeles • Larry Honeyborne - Orange County Public Health Department • Charles McGee - Orange County Sanitation District • Darrin Polhemus - State Water Resources Control Board • Michael Lyons - Los Angeles Regional Water Quality Control Board • Dr. Mark Gold - Heal the Bay • Dave Kiff - City of Newport Beach • Shari Barash - US Environmental Protection Agency

Rapid Microbiological Monitoring Methods Fact Sheet