Dorevitch et al. Critical Research Questions Four groups of critical questions should be answered regarding measures or models of water quality as a means of assessing recreational waterborne illness risk in IW (Appendix 1): 1. Microbial indicators as predictors of pathogen exposure and health risks. To advance our ability to model health risk, we must characterize the transport, survival, fate, and re-growth (for bacteria) of indica- tors and pathogens in flowing and nonflowing IW. Persistence of pathogenic bacteria needs to be better understood, as Campylobacter, Salmonella, Shigella, and shigatoxin-producing E. coli have been found on algae growing in surface waters (Ishii et al. 2006). Similarly E. coli O157:H7 can persist in sediments (Bruce et al. 2003). Although the dynam- ics of FIB have been studied in some coastal contexts (Boehm et al. 2002; Whitman and Nevers 2004), FIB spatiotemporal variability and its determinants need to be characterized in hydrologically diverse IW. 2. Fecal pollution sources as predictors of pathogen exposure and health risk. It is important to determine if the source of fecal pollution modifies the indicator-health asso- ciation. The assumption that human fecal pol- lution presents the greatest health risk needs further evaluation. Prior discussions, including those at the workshop, focused on rates of ill- ness seen among swimmers at beaches, gener- ally mild and self-limited. Future work should also consider illness severity, which may be substantial if agricultural animals, wildlife, or other bathers are sources of pathogens at waters where dilution is limited. The health risks to recreators at IW impacted by con- fined animal feeding operations have not been the subject of epidemiologic studies but are a potential concern. The possibility of zoonotic waterborne viral infections should be inves- tigated, as recent evidence supports possible zoonotic origins of some human rotaviruses (Banyai et al. 2009; Matthijnssens et al. 2009). 3. Molecular methods for water qual- ity testing. Sediments, which likely contain qPCR inhibitors, FIB (viable and nonviable), and cell-free FIB DNA present a challenge to IW monitoring. It is important to know how insolation, water chemistry, wastewater treatment, hydrologic parameters, season, and water matrix differentially affect these com- ponents of the qPCR signal. Optimizing the primers and probes, particularly those that differentiate human from other sources, and establishing procedures for minimizing natu- rally occurring PCR inhibitors are priorities. 4. Other approaches to predicting IW recreation health risks. Procedures for opti- mizing and validating predictive models, sanitary surveys, and QMRA approaches are important for both coastal and IW. The U.S. EPA Great Lakes survey tool (http://www. epa.gov/waterscience/beaches/sanitarysurvey/) should be modifiable for IW. For QMRA and mechanistic models, inputs such as indicator and pathogen concentrations, pathogen load- ing estimates, and health risks posed by vari- ous animal species are needed. An uncertainty in estimating the health risk associated with livestock is the high variability of protozoa excretion rates (Ferguson et al. 2009). An IW Research Agenda To provide answers to the questions identified in Appendix 1, short-term research (within 2 years) and longer-term projects (2–5 years) are needed. These questions can be addressed through targeted literature reviews, com- puter modeling, field sampling for environ- mental microbes, laboratory research on analytic methods, and human health stud- ies. Interdisciplinary studies would compile sediment, soil, hydrology, microbiology, and health data, all of which could be used to identify predictors of health risk. Other than epidemiologic and QMRA research, these studies would not directly inform the estab- lishment of AWQC but could advance water quality modeling and our understanding of sources of risk and uncertainty. Research agenda elements are listed in Table 1. Limited explanations of several items follow. Short-term research. Molecular tests for FIB. Rapid tests that are strongly correlated with pathogen densities are needed to sup- port the development of improved predictive models of health risk. Pretreatment with pro- pidium monoazide (PMA) may allow the dif- ferentiation of DNA from intact viable cells, as opposed to extracellular DNA or DNA in cells without a functioning membrane (Nocker et al. 2007). This PMA-qPCR approach has been used to demonstrate a faster decay of the qPCR signal of Bacteroidales compared with conven- tional qPCR (Bae and Wuertz 2009). PMA pretreatment or other approaches for quantify- ing elements of the overall qPCR signal should be evaluated for their ability to improve qPCR predictions of pathogen presence. Predictive modeling of health risk. Comparison of retrospective QMRA analyses with previously conducted epidemiologic stud- ies could lead to revisions in QMRA model assumptions and inputs to bring projected levels of risk in line with risk levels observed Table 1. Research to address critical IW criteria questions. Type of research required Library computer simulation Laboratory Critical questions Determinants of indicator- pathogen relationships in IW Sources of indicators, pathogens, and health risk Molecular methods in IW: interpretation, standardization X X X X X X X X X X X X X X Field X X X X X X X X X X Study overview L: Advance mechanistic modeling of FIB and pathogens, supported by sampling of water, sediment and soil in diverse IW. Repeated sampling to characterize the fate, transport, persistence, and re-growth S: Meta-analysis of epidemiologic studies to evaluate fecal pollutant source as a modifier of the indicator–health risk relationship S: Optimize and anchor QMRA models based on prior epidemiologic study results S: Develop a sanitary survey tool for use in future IW epidemiologic and QMRA studies S: Field sampling of feces from agricultural animals and wildlife to determine human pathogenic potential and dynamics L: Epidemiologic studies conducted at diverse IW sites, each with a different dominant source of fecal pollution (agricultural animals, wildlife, urban runoff, wastewater) S: Develop a database of relationships between rapid molecular-based and culture-based measures of indicator microbes described in the literature S: Characterize the persistence of specific molecular targets (human vs. other) in a variety of environmental settings and wastewater, looking at viable and nonintact cells and cell-free DNA S: Optimize, simplify, and standardize qPCR methods (particularly for source-specific markers) and other rapid methods; use these in epidemiologic studies L: Develop rapid methods for concentrating, identifying, and quantifying pathogens in recreational waters QMRA validation studies, S (retrospective) and L (prospective) S: Measure real-time physicochemical, hydrologic, meteorologic parameters, with microbes Modeling health risk and real-time water quality Abbreviations: S, short term (< 2 years); L, longer term (2–5 years). 874 ovlume 118 | number 6 | June 2010 • Environmental Health Perspectives