To limit human contact with contaminated waters, fecal indicator bacteria (FIB) are used to evaluate risk of illness from recreational bathing at beaches. Enterococci are one of the most commonly used bacteria for confirmation of sewage presence in recreational water bodies. Recent studies at a subtropical non-point source beaches have identified the intertidal zone as a potential alternative source of FIBs; thus encouraging further experiments to obtain a better understanding of the behavior of FIBs, particularly enterococci, within the intertidal zone. Such experiments can aid in improved regulations and monitoring practices. This study evaluated enterococci behavior in simulated intertidal sand and ditch environments. The focus of the study was to determine whether there is die-off, persistence, or increases of cultural enterococci under saturated and unsaturated conditions in the beach environment. By utilizing a combination of experiments, the relationship of enterococci survival was evaluated. Results exhibited a constant die off of enterococci in all simulated ditch environments. Sand experiments showed an increase in enterococci concentration in dry sand after wetting events, however these were not statistically significant due to the limited number of samples (p=0.33, p=0.20 and p=0.14 respectively). All sand experiments showed a statistically significant difference between enterococci concentration variance in dry sand after simulated wetting (p<0.0001, p<0.0001, p= respectively). No significant change in enterococci die-off rates were observed in sand from both intertidal and ditch zones. Further studies are necessary to establish correlations and statistical significance of the change in concentration of enterococci in dry sand after wetting events. Survival of Enterococci in Unseeded Runoff Ditch and Sand at a Subtropical Non-point Source Beach J. DeFrancesco 1,2, C. Ortega-Castineiras 1,2, R. Ramoutar 1,2, H. Solo-Gabriele 1,2 Jenna DeFrancesco Methods Results Discussion Explore prolonged periods of “drying” before a wetting event as well as after to follow extended trends. Maintain a control throughout every experiment and repeat in triplicates to establish the significance of the increase in the enterococci colony counts. Explore varying percentages of water content to determine how dry sand must be to experience pronounced regrowth. (Between 0.2% and 4% according to data) *Further compare sterile ocean and rainwater in their effects on regrowth. (Rainwater showed most regrowth at 3 hours whereas Ocean showed peak regrowth at 6 hours post wetting event) Determine impact of increase in enterococci CFU’s in sand on the bacterial level in the water column. Repeat experiments with the addition of PCR analysis of the samples in order to establish whether there is actual regrowth or just a strengthening of the bacteria after a wetting event. Acknowledgements: NSF-NIEHS Oceans and Human Health Program (P50 ES NSF REU Program (NSF OCE ), Abstract Study Site Objectives Hobie Beach Outfall W WTP Outfall Sewage We are here RSMAS Hobie Beach. Abstract # 1 University of Miami, NSF NIEHS Oceans and Human Health Center, Miami, FL Department of Civil, Arch., and Environmental Engineering, University of Miami, Coral Gables, FL Rain Water Sample Collection at various sites. Ocean Water was collected using sterile 5L containers. Filtered through 0.45 µm filter for use in microcosm experiments. Ocean and Rain Water Sample Collection Sampling of FIBs in runoff water FIBs in runoff water tested Regulatory Standards for recreational water: U.S. (EPA) enterococci < 33 or 35 / 100 ml Evaluate the behavior of enterococci in simulated sand and ditch environments that replicated those found at the study beach. Determine whether there is die-off, persistence, or increases of culturable enterococci under saturated and unsaturated conditions. This study utilized a unique combination of experiments in order to simulate the different environmental conditions and evaluate their relationship to enterococci survival. Overall, the study also identifies possible implications for beach regulation practice. Ditch Microcosms (Undisturbed) Ditch Microcosms (Mixed) Replicates of runoff ditch environment using:20 g sand, 10 g seaweed and 800 ml of synthetic rainwater Used to determine the relationship between CFU/100ml in various ditch microcosms over time in relation to turbidity Microcosms were kept in the dark at a constant temperature Samples were taken without mixing the microcosms during 3 consecutive days Ditch microcosm experiment was repeated Agitation used to more closely replicate the natural environment and obtain a more representative enterococci count Used to determine the survival rate of enterococci in various runoff ditch microcosms over time Sand Microcosms before and after a Wetting Event Sand samples were procured via sterile containers from each representative sampling environment Moisture content over time was recorded CFU/100ml was tracked over time to determine natural enterococci trends in each microcosm environment Sterilized rain and ocean water were used to simulate natural wetting events 3 repetitions of this sand experiment were performed CFU/g of sand was tracked before and after wetting to observe behavior in each microcosm Water sample collection showed that rainwater was virtually Enterococci free before filtering, thus rainwater wasn’t contributing significantly to FIBs found after wetting events. Sampling of FIBs in standing ditch water and runoff water showed a significant FIB load, particularly Enterococci was well above regulatory standards Enterococci concentrations show a steady decrease in regrowth experiments in all water microcosm environments which correlate to decreases in turbidity. No significant regrowth was observed. In a controlled environment, Enterococci concentrations diminished as the sand from each microcosm dried. Water Experiments Sand Experiments Wetting each microcosm resulted in pronounced regrowth in only the very dry above sand. This was true for both rainwater and ocean water. A series of experiments showed that regrowth was seen in the dry above sand after both rainwater and ocean water wetting events. There was no significant regrowth found in either Ditch or Intertidal sand which may be attributed to varying moisture content.