Using Microbial Source Tracking to Identify Pollution Sources in Pathogen Impaired Embayments in Long Island, New York

Science Center Objects

Problem The presence of pathogens in Long Island marine embayments and the hazards they pose to marine resources and human health is of increasing concern. Many waterbodies on the New York State Section 303(d) List of Impaired Waters have pathogens listed as the primary pollutant that are suspected to originate from urban/storm runoff. There is neither a clear understanding of the relative mag...


The presence of pathogens in Long Island marine embayments and the hazards they pose to marine resources and human health is of increasing concern.  Many waterbodies on the New York State Section 303(d) List of Impaired Waters have pathogens listed as the primary pollutant that are suspected to originate from urban/storm runoff. There is neither a clear understanding of the relative magnitude and geographic origin of sources of loadings of pathogens (from urban/storm runoff, submarine groundwater discharge, etc) on Long Island, nor clear understanding about the host organisms from which they originate (such as human, mammals, or birds).

Pathogen loads to specific embayments are affected by watershed land-use, proximity of point sources such as wastewater treatment plants (WWTP) and municipal separate storm sewer systems (MS4s), tidal exchange/circulation, sediment re-suspension, and human recreation (including boating). Point source discharges and stormwater runoff are not the only contributors of pathogens to our coastal waters. It is suspected that a significant source of pathogens may come from shallow groundwater discharge, as much of Long Island still relies on onsite wastewater disposal systems (OWDS), such as cesspools and septic systems.  For example, groundwater samples collected downgradient of OWDS were found to contain wastewater associated compounds (pharmaceuticals, personal care and domestic use products, and endocrine active compounds) indicating a human source (Fisher and others, 2016). Human and animal sources of pathogens pose the greatest human health risk (Cabral, 2010).


It is a complex task to understand the sources of pathogen contamination and relative loads to Long Island’s marine embayments. Multiple nonpoint conveyances from the watershed coupled with dynamic, tidally-influenced waterbodies makes a multi-tiered and site-specific analytical approach necessary. Regulatory agencies can generate effective pathogen-control measures once the significant contributing hosts and transport mechanisms are identified. 



Standard measurements of pathogens using fecal-indicator bacteria (FIB) along with known discharges in MS4 areas allow for the identification and prioritization of embayments that are impaired for pathogens. The DEC routinely collects FIB data in embayments where shellfishing is listed as a designated use. The National Shellfish Sanitation Program (NSSP) standards and New York State’s water quality standards for certified shellfish lands can provide the basis for Total Maximum Daily Load (TMDL) development for pathogens. In order to effectively improve water quality and meet any TMDL goals, both the host source and geographical origin of pathogenic bacteria must be understood. Estimates of pathogen loading to the marine embayments surrounding Long Island have been modeled previously however these efforts have had limited success in adequately identifying the sources of the loads.


Microbial Source Tracking (MST) is a tool used to better classify and allocate the contributions of fecal contamination, particularly from nonpoint sources, once a problem is identified. MST protocols followed by the U.S. Geological Survey (USGS) typically include several microbiological targets or source identifiers, detection methods, and analytical approaches to link data from water samples to the fecal sources, depending on the objectives of the study (Stoeckel, D.M., 2005). Male-specific (F+) coliphages (FRNA) are an example of a source specific viral indicator organism that has been successfully used to identify the source of fecal contamination in surface waters (Cole and others, 2003; Griffin and others, 2000). Coliphage are viruses that infect the host bacteria E.coli, and their presence in the environment is directly related to the presence of the host. There are four genetic groupings of F+ specific RNA coliphages: Group I is indicative of an animal origin (such as cattle, sheep, pigs, and others); group II  is indicative of a swine or human source, group III is exclusively human, and group IV designates animal origin (predominantly birds) (Griffin and others, 2000; Osawa and others, 1981). FRNA source tracking would most likely detect contamination from recent fecal inputs though the persistence of coliphage that may vary significantly with temperature and host availability (Ravva, S.V., 2016). The U.S. Environmental Protection Agency (EPA) is currently developing water-quality criteria for coliphage as a formal fecal indicator organism for regulatory standards (EPA, 2016; EPA, 2015). Linking the host-source identifier for MST in this study to an impending regulatory indicator that will be routinely sampled will add value, in terms of potential sources and loads, to the results obtained by monitoring efforts in the future.


Complementary data that can help refine the geographical origins of pathogens and, potentially, the relative contributions of the host-source include stable isotope analyses of nitrogen and oxygen in the inorganic forms of nitrate and ammonium. Ratios of the stable isotopes of nitrogen, 14N and 15N, can be helpful in differentiating atmospheric, wastewater, fertilizer, and pet waste sources (Abbene and others, 2010). Oxygen isotope ratios (18O and 16O) in nitrate can be useful to distinguish the nutrient source and process where ratios of nitrogen overlap.  In this way, the relative contribution of commercial fertilizers, animal and septic waste, and organic nitrogen can be evaluated and used in conjunction with MST to provide stronger evidence of the “waste” component (human or animal) in complex mixtures of storm water and groundwater. 


Although the coliphage method is highly sensitive, it is limited to four groups of animals that are only typically associated with types of mammals (Groups I-III) and birds (Groups I and IV). However, reference standards from samples collected along the shore will assist in matching the wildlife to the proper grouping. Additionally, host-associated genetic marker (Bacteroides) analyses will also be conducted on about 1/3 of samples. Further, the multiple-lines-of-evidence gained from the chemical and isotope analyses will aid in determining relative host contributions. When analyzed together; MST, stable isotope, and FIB data, along with ancillary data such as land use, locations of point sources, wildlife populations, and tidal exchange/circulation can indicate the sources of pathogens and allow us to make the best estimate of pathogen loading from each source.


Identify the source(s) and relative host contribution of bacterial contamination using microbial-source tracking (FRNA) in embayments identified by the DEC as pathogen impaired.


2.      Utilize stable isotopes to characterize nitrogen sources and transport mechanisms for each of the identified embayments.

3.      Estimate pathogen loads to each embayment in summer and winter seasons, during both wet and dry conditions, and during periods of concentrated bird populations.


4.      Sample TMDL waterbodies that do not have baseline FIB data to expand the ambient water-quality dataset.


 A three-tiered approach is needed to adequately determine the sources of pathogens and estimate relative loading to the 5 DEC funded study areas (9 priority waterbody segments) plus the 2 additional study areas supported by external partners (fig. 1), and is be outlined in the Quality Assurance Project Plans (QAPPs) that has been submitted to the DEC.


1.      Data and model review, site surveys, and sample point selection

a.       Working with local agencies, FIB data from existing monitoring networks (for example, the Suffolk County Department of Health Services (SCDHS) beach monitoring program and surface-water monitoring programs and the DEC shellfish monitoring program) will be evaluated for pathogen occurrence and spatial distribution. This includes taking into account the locations of storm drains (MS4s) and evaluating surrounding land use (for example, from 2012 Suffolk County Department of Planning digital land use maps).

b.      Design a sampling protocol based on storm drain/runoff points located around each bay/harbor/waterway, and other reliable data to identify points at which samples will be collected during each event. Points at which samples will be collected may include: storm drain outfalls (for water and sludge); surface waters (stations co-located with DEC shellfish monitoring sample sites, and SCDHS ambient water-quality monitoring sites); shallow groundwater at shores downgradient of onsite wastewater disposal systems; and bed sediment in areas adjacent to outfalls or in areas of dense boat anchorages.


2.      Collect samples and field data at each of the study areas during dry and wet weather conditions during both summer and winter seasons, and during periods of concentrated bird populations (amounting to five to six sample events per site per year), using analytical methods for the following factors to develop a better understanding of the sources of pathogens and the relative proportions of host organisms.

a.       Fecal indicator bacteria: Enterococci and coliforms (total, fecal, and E. coli).

b.      F+ specific coliphage for both qualification (host specificity) and semi-quantification for relative loading calculations. These data will provide information on animal type based on four groupings and host-specific genetic markers (Griffin and others, 2000).

c.       Host-specific genetic marker analysis and archives: 1/3 of water samples will be analyzed. Remaining water samples will be filtered and the filters will be frozen and stored for additional MST analysis using alternate source identifiers if necessary. Genetic markers would include those for cattle, human, dog, and waterfowl.


d.      Nutrients (USGS schedule LS1865/2702): inorganic forms of nitrogen and phosphorous for correlating with pathogen loading and necessary for nitrogen isotope analyses.

e.       Physical parameters (for water): temperature, specific conductance/salinity, pH, dissolved oxygen, turbidity, and fluorescent dissolved organic matter (fDOM).

f.      Weather and tide conditions from available National Weather Service (NWS), National Ocean Service (NOS), and USGS stations.

g.       Stable isotope analysis.

                                                              i.      Nitrogen and oxygen isotopes from nitrate will provide additional source characterization for waters associated with nutrient and pathogen load: nitrogen isotope ratios provide insight into the process (nitrification) and sources (human, pet, fertilizer, atmospheric); oxygen isotope ratios provide insight into processes (nitrification, deposition, biological versus physical reduction/oxidation).

                                                            ii.      Nitrogen from ammonium will provide insight into the nitrogen source, particularly in groundwater with low dissolved oxygen that is impacted by wastewater (fertilizer, septic effluent)


 The sampling effort will be divided into two parts in order to assess the receptor (the 16 receiving waters) and the transport mechanisms (stormwater runoff, groundwater, sediment, direct deposition).

           Receptor tracking: Correlating fecal indicator bacteria monitoring with hosts—screening at current network locations.

Sample collection will be coordinated with the DEC Marine Resources, Shellfisheries Growing Area Classification Section and SCDHS to visit their monitoring networks’ locations twice in the summer months and twice in the winter months (one dry weather day and one wet weather day for each). Up to four surface water stations will be sampled per daily trip from each waterbody at points that have historically shown the greatest concentrations of FIB. If existing FIB data are unreliable or unavailable then other factors, such as, proximity to potential sources will be considered.

-          Surface waters will be screened for F+ coliphages, nutrients, and physical parameters; concurrently collected FIB data from the SCDHS and DEC will be used in lieu of USGS samples for FIB


Source tracking: Assessing pathogen introduction through various transport mechanisms.

The sample collection strategy will vary among the sites depending on historical data, land use, and other factors. For example, waterways with no contributing stormwater conveyance systems/outfalls discharging will instead have samples focused on groundwater, sediment, and direct overland runoff.

-          Stormwater samples will be composited by an automated sampler situated in select storm drains expected to contribute some of the highest pathogen concentrations to the receiving waters during an event. Grab samples will also be collected at all locations and compared to the composite samples and analyzed along with the chemical and physical data. Samples will be screened for F+ coliphages, FIB, nutrients, and physical parameters.

-          Groundwater samples will be collected by drive-point piezometers along the shore at shallow depths (just below to 5 feet below the water table) and assessed for F+ coliphages, FIB, nutrients, and nitrogen isotopes. These data will provide insight into the sources of nutrients and related bacteria and virus loading.

-          Bed sediment samples will collected by Ponar sampling (of the top 2 cm) and assessed for FIB and F+ coliphages. These samples will characterize the possible resuspension of pathogens, particularly during dry weather events.

-          Surface water samples will be collected throughout the waterbody.  The presence, abundance, and location of birds will be noted to better understand the importance of resident and migratory birds on pathogen loading. These coliphage data will be compared to samples collected during times when birds are less abundant and to the other types of source samples. The bird coliphage data will also be evaluated from a regional (estuary) and island-wide perspective.


2.      Data compiled and collected will be evaluated and modeled using a loading simulator determined following the complete evaluation of data to estimate concentrations of pathogens, their host sources, and their transport mechanisms. The ongoing USGS project to delineate groundwatersheds for all of Long Island’s surface waters will also be utilized for its high resolution groundwater contributing areas and outflows to this study’s embayments. Data will be compiled into national databases and, using the DEC’s preliminary Bacteria Trackdown document, into a geographic information system (GIS) product.


3.       Waterbodies with previous TMDLs that have no or few (<20) samples collected within the last 5 years will be eligible for the ambient water-quality network monitoring.  Samples will be analyzed for FIB with best efforts to retain one dataset each in wet and dry conditions.  Wet conditions will be defined as ½ inch of rainfall or more in 48 hours or 1 inch of rainfall or more in 72 hours. Sites under consideration are in Figure 2.

Abbene, I.J., 2010, Shallow groundwater quality in the Village of Patchogue, Suffolk County, New York: U.S. Geological Survey Scientific Investigations Report 2010–5132, 19 p., at
Cabral, J.P.S., 2010, Water Microbiology. Bacterial Pathogens and Water: International Journal of Environmental Research and Public Health, vol. 7, issue 10, p. 3657–3703.


Cole, D., Long, S.C., and Sobsey, M.D., 2003, Evaluation of F+RNA and DNA coliphages as source-specific indicators of fecal contamination in surface waters: Applied and Environmental Microbiology, v. 69, no. 11, p. 6507-6514.

Environmental Protection Agency, 2016, 2016 Coliphage Experts Workshop: Discussion Topics and Findings, Accessed January 17, 2017, at
Environmental Protection Agency, 2015, Review of Coliphages as Possible Indicators of Fecal Contamination for Ambient Water Quality, Accessed January 17, 2017, at
Griffin, D.W., Stokes, R., Rose, J.B,, and Paul, J.H., III, 2000, Bacterial indicator occurrence and the use of an F+ specific RNA coliphage assay to identify fecal sources in Homosassa Springs, Florida: Microbial Ecology, v. 39, no. 1, p. 56-64.
Fisher, I.J., et al., The impact of onsite wastewater disposal systems on groundwater in areas inundated by Hurricane Sandy in New York and New Jersey, Marine Pollution Bulletin (2016),
Osawa, S., Furuse, K., Watanabe, I., 1981, Distribution of ribonucleic acid coliphages in animals. Applied and Environmental Microbiology, v. 41, no. 1, p. 164-168.
Ravva, S.V., Sarreal, C.Z., 2016, Persistence of F-Specific RNA Coliphages in Surface Waters from a Produce Production Region along the Central Coast of California: PLoS ONE 11 (1): e0146623. doi:10. 1371/journal.pone.0146623
Stoeckel, D.M., 2005, Selection and application of microbial source tracking tools for water-quality investigations: U.S. Geological Survey Techniques and Methods Book 2, Chapter A3, 43 p.