DETECTION AND QUANTIFICATION OF SOURCES OF HUMAN FECAL POLLUTION TO THE MARINE ENVIRONMENT OF THE SOUTHWESTERN U.S. BORDER BACKGROUND Imperial Beach, located within San Diego County, between Coronado and the U.S./Mexico Border, has a history of high levels of microbial pollution. According to data collected by the San Diego County Department of Environmental Health spanning the last 10 years (2006-2017), Imperial beach was closed 437 days due to elevated fecal indicator bacterial (FIB) levels. FIB can originate from both human and non-human sources, with the human sources representing the greatest health risk. At Imperial Beach, sources causing poor beach conditions are unknown and of concern to public health officials given the proximity of multiple potential inputs of human fecal contamination. These include 1) the outfall of the San Antonio de los Buenos (SAB) wastewater treatment plant (WTP) at Punta Bandera in Tijuana, Mexico, 2) the South Bay International Wastewater Treatment Plant (SBIWTP) which discharges to the South Bay ocean outfall, and 3) flows from the Tijuana River, which in large part are diverted to the SBIWTP during dry weather. While all potential human contamination sources will be characterized, a primary focus of this study is the SAB WTP, which serves communities just south of Tijuana. It has been hypothesized that flows from the SAB WTP outfall at Punta Bandera, Tijuana, Mexico, may contribute to elevated bacterial levels and introduce human fecal contamination into Imperial Beach waters during periods when ocean currents are flowing South to North. The plant uses primary treatment to treat wastewater, this effluent is then piped to a coastal stream which ultimately discharges to the ocean approximately three miles south of the treatment plant. Previous studies conducted in the same area have found widespread contamination associated with the SAB WTP plume in Tijuana, Mexico (Orozco-Borbon et al. 2008; Sassoubre et a. 2012; Thulsiraj et al. 2017). Multiple Mexican beaches frequented by swimmers and surfers dot the coast between the San Antonio de Los Buenos treatment plant and Imperial Beach, underscoring the value of investigating the plant’s role in potentially contributing to beach fecal contamination. In this study, a multi-pronged approach will be used to evaluate impacts of specific sources of human fecal contamination to Imperial Beach water quality. Microbial source tracking (MST) technology will be used to distinguish human fecal sources from non-human ones, and to differentiate contributions from specific locations where human contamination is likely originating, using a next generation sequencing (NGS) approach. Emerging NGS technologies have the potential to characterize and track bacterial contamination from multiple sources. This study will serve to answer questions regarding application and performance of these methods in coastal waters. MST results will also be compared to and used to ground-truth nearshore 3- dimensional transport and mixing models of specific sources, being developed at Scripps Institution of Oceanography (SIO). Questions ● Is there a link between water quality at Imperial Beach, CA and SAB WTP effluent discharged at Punta Bandera in Tijuana, MX? ● What is the effectiveness and feasibility of microbial community-based source tracking methods at tracking specific human sources in the coastal environment? ● Can MST results be used to help validate/inform nearshore plume transport models? APPROACH Water samples will be collected from approximately 16 sites, located between Punta Bandera in Tijuana, MX and Silver Strand State Beach, CA, and analyzed with molecular methods. Microbial Community-Based Source Tracking Next generation sequencing, performed on the Illumina MiSeq platform, will be used to characterize microbial community structure and diversity at coastal sites along the U.S/Mexico border region, and at sites potentially contributing nonindigenous bacteria to coastal waters. Environmental waters in the study area are likely to be a mixture of potential source waters including 1) effluent from the Pt Loma WWTP, 2) effluent from the SBIWTP, 3) SAB WTP effluent discharged at Punta Bandera, 4) Tijuana River water, and 5) marine water (Table 1). NGS DNA sequencing will be used to characterize these source waters along with environmental water (“sink”) samples. Sink and source microbial community profiles will be compared using SourceTracker, an algorithm that utilizes Bayesian statistics to estimate the source of microbial communities in a set of sink samples (Knights et al. 2013). Several studies have successfully applied a similar approach to characterize bacterial contamination sources in recreational fresh (Baral et al. 2018; Staley et al. 2018) and estuarine (McCarthy et al. 2017) waters. Success of these methods relies on microbial community profiles that are unique to each source. If the SAB WTP has a profile that is unique to the plant, it will be used to track extent and dispersal of the of the plume at Punta Bandera and as it moves alongshore South to North, potentially impacting Imperial beach water quality. Human Source Identification Environmental water samples will be analyzed for human-associated fecal marker (HF183) and Enterococcus using digital polymerase chain reaction (PCR) methods previously developed (Cao et al. 2015). Human marker results will be used to gauge the presence and extent of human fecal material at each site and across the region. SIO Plume Model Professors Falk Feddersen and Sarah Giddings and colleagues are developing models capable of describing transport, mixing, and dilution of the SAB WTP plume. These models will help inform understanding of dispersal of the SAB WTP plume as it moves from Punta Bandera Northward, potentially impacting Imperial Beach water quality. Specific times water quality sampling will be conducted will be selected to match when there is the highest potential for transport of sewage from the SAB WTP to Imperial Beach. STUDY DESIGN Approximately 16 sites will be sampled during dry weather along the coastline from the outfall of the SAB WTP at Punta Bandera to Silver Strand State Beach, CA over a one-year period. Four dry weather, south swell events will be targeted for sampling. Three days will be sampled per south swell event (n= 12 sampling dates total). The Imperial Beach site will be sampled for an additional three days (six days total), in order to better understand persistence of water quality impacts related to transport of the SAB WTP plume. Proposed sampling locations are shown in Figure 1 and described in Table 1. Samples will be collected collaboratively with Mexico-based NGO’s already conducting citizen monitoring in this region. Nearshore grab samples for water will be collected from approximately 8 sites spanning the U.S./Mexico border region. In addition, paired surfzone samples (approximately 100 m offshore) will be collected from approximately three sites located North of the U.S/Mexico border, with the support of the City of Imperial Beach Ocean Safety Department. Samples from potential sources (n=5) will also be collected, for a total of 16 sites. All environmental sites will be sampled in the morning, to limit degradation of the bacterial signal. Water samples will be transported to the City of San Diego Water Quality Laboratory for FIB analysis and filtered for bacterial DNA. Processed samples will then be transported to SCCWRP’s laboratory in Costa Mesa, CA for analysis by sequencing and digital PCR. Additional Measurements Salinity and water temperature will be measured at all sites using a portable meter. TRAINING Training on sample collection and handling, and filtration, will be provided by SCCWRP prior to the beginning of sampling and will include distribution of appropriate SOPs. SCHEDULE Kick Off Meeting – June 2018 Sample collection begins – Fall 2018 Molecular analyses begin – Spring 2018 Draft Report- January 31, 2020 Final Report- by March 31, 2020 DATA REPORTING AND ANALYSIS SCCWRP will perform molecular analysis on environmental water samples, including nucleic acid extraction, performing digital PCR assays, and sequencing and bioinformatic analysis. SCCWRP will compile and conduct the analysis of the study data. SIO will develop and calibrate nearshore plume transport models and conduct analysis of model results. Figure 1. Map of potential sampling locations. Table 1. Description of potential sampling locations Site Latitude Longitude Description SAB South 32.43786 -117.10189 South of SAB WTP discharge point SAB 32.4466 -117.10788 Ocean outfall for SAB WTP Southern end of Playas de Tijuana- El Vigia 32.47061 -117.11980 sampled by Tijuana Waterkeeper Playas 32.50937 -117.12411 Northern end of Playas de Tijuana Beach Watch Station; Border Field State IB-020 32.5434 -117.125 Park Beach Watch Station; Tijuana Slough IB-040 32.561 -117.132 National Wildlife Refuge; 3/4 mi N of TJ River Beach Watch Station; Imperial Beach PL-010 32.5847 -117.133 municipal beach Beach Watch Station; N Silver Strand State IB-070 32.6296 -117.141 Beach Pt Loma WTP 32.66528 -117.32361 Wastewater effluent: Advanced Primary South Bay 32.44804 -117.10527 Wastewater effluent: Secondary International WTP Receives urban runoff from U.S. and Tijuana River 32.55299 -117.12776 Mexico SAB WTP 32.54083 -117.18333 Wastewater effluent: Primary Scripps Pier at La 32.867034 -117.25738 Marine water (background) Jolla Beach, CA REFERENCES Baral, D., Speicher, A., Dvorak, B., Admiraal, D., & Li, X. (2018). Quantifying the relative contributions of environmental sources to the microbial community in an urban stream under dry and wet weather conditions. Applied and Environmental Microbiology, 84(15), 1– 36. https://doi.org/10.1128/AEM.00896-18 Cao, Y., Raith, M. R., & Griffith, J. F. (2015). Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. Water Research, 70, 337–349. https://doi.org/10.1016/j.watres.2014.12.008 Orozco-Borbón, M. V., Rico-Mora, R., Weisberg, S. B., Noble, R. T., Dorsey, J. H., Leecaster, M.