1

The Pilot Epidemiologic Assessment of Microbial Indicators 

for 

Monitoring Recreational Water Quality 

in 

Marine Sub/Tropical Environments 

Investigators: 

Lora E Fleming MD PhD MPH MSc, Principal Investigator (University of Miami) 

Dr Helena Solo Gabriele PhD, Co Principal Investigator (University of Miami), Jay M.Fleisher PhD, CoPrincipal Investigator (Nova Southeastern University), Dr Kelly Goodwin PhD, Collaborator (NOAA Southeast 

Miami Lab), Dr Lorrie Backer PhD, Collaborator (CDC), Dr Samir Elmir PE, Collaborator (Miami Dade County 

Public Health Department), Dr John Wang, Collaborator (University of Miami). 

FL DOH Proposed costs and time period: total $150,000 (direct + 8% indirect) from 9/1/07 to 8/31/08 

Note: 

The successful completion of this Pilot Epidemiology Proposal is contingent on starting this Pilot 

Epidemiology by September 2007 since it must be performed in conjunction with timeline of the 

ongoing NSF NIEHS OHH Research Project Environmental Monitoring Project. 
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Abstract

Microbial water quality indicators are found in high concentrations in sewage, and are thus used to determine 

whether or not water is safe for recreational purposes. Recently, concerns have been raised about the 

appropriate use of microbial indicators to regulate recreational uses of water bodies. This is particularly true 

for water bodies located in sub/tropical environments with no known point sources, due to the potential for 

microbial regrowth in sub/tropics, and the fact that studies used to set national guidelines were not conducted 

in sub/tropical environments. The primary goal of this proposed study is to evaluate the relationship between 

human health, and the physical and microbial characteristics of coastal marine waters and its shoreline. This 

goal will be addressed by measuring human health effects and microbial water quality at a recreational beach 

site within a sub/tropical climate in conjunction with ongoing development of a predictive, coupled 

hydrodynamic and microbial fate model that can be used to distinguish impacts from sewage sources versus 

regrowth of microbes, each of these sources possibly resulting in different health effects as observed at the 

beach site. 

Substantial environmental pilot data have been collected for the sub/tropical Study Site chosen for this 

research. These pilot data, as well as the literature, indicate that exposure to contaminated recreational marine 

waters may result in human health effects, and that the shoreline sediments is one likely source of indicator 

microbes. Human health will be evaluated in the proposed Pilot Project through an epidemiologic study which 

will randomly assign volunteer participants to bather and non-bather groups. This epidemiologic effort will be 

conducted in conjunction with an ongoing extensive sampling of indicator organisms and pathogens as well as 

hydrodynamic modeling. The environmental assessment will be conducted such that measures of indicator 

organism exposure will be assigned directly to each individual bather. Microbe measurements will include 

traditional (enterococci, fecal coliform) and non-traditional (C. perfringens, Bacteroides) microbial indicators, as 

well as direct measurement of microbial pathogens (S. aureus, enterovirus, C. parvum, and G. lamblia). The 

hydrodynamic modeling will refine an existing model of the Study Site, incorporating wind, tide, run off, 

microbial sampling, and ultimately the epidemiologic information. 

The current FL DOH External Funding Proposal below is for funding for a PILOT PROJECT of the 

epidemiologic portion of the “Recreational Microbes” Study; the extensive environmental monitoring 

and hydrodynamic modeling which will occur concomitantly with the proposed epidemiologic study 

are currently funded by an ongoing Research Study of the NSF NIEHS Oceans and Human Health 

Center of the University of Miami with collaborators from NOAA, the CDC, and the Miami Dade County 

Dept of Health. This current FL DOH proposal will allow for the collection of human epidemiologic data in 

conjunction with the ongoing already-funded environmental monitoring and hydrodynamic modeling, allowing 

for “leverage” of funding and resources. Ultimately, the human, environmental and oceanographic data will be 

used to develop a predictive model with broad applicability to beaches in the sub/tropics with appropriate 

modifications for local conditions. The final result will be the potential improvement of recreational water 

quality monitoring in the sub/tropical marine environment without a known point source. 

Statement of Problem 

Recently, concerns have been raised about the appropriate use of microbial indicators to regulate recreational 

uses of water bodies, particularly true for water bodies located in sub/tropical environments and without known 

point sources, due to the potential for microbial regrowth in sub/tropics, and the fact that studies used to set 

national guidelines were not conducted in sub/tropical environments and lacked individual exposure 

information. 

Specific Aims: The objective of this proposed Pilot Epidemiologic Study will be to begin to evaluate the 

relationship between microbe densities and reported symptoms in human subjects with random 

assignment to recreational marine water exposure and intensive individual microbial monitoring. 

Ultimately, the human, environmental and oceanographic data will be used to develop a predictive 

model with broad applicability to beaches in the sub/tropics with appropriate modifications for local 

conditions. 
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Background 

The primary significance of the proposed Pilot Study is that it begins to address an important data gap. The 

use of indicator microbes for water quality monitoring in sub/tropical (tropical and sub-tropical) environments 

has been questioned, in particular for sub/tropical coastal zones where there are no known point sources of 

pollution. The premise is that indicator microbes within these regions regrow due to climate conditions 

conducive for their regrowth, and are thus not representative of sewage sources of pathogens. The few 

epidemiologic studies conducted in sub/tropical environments (Fujioka 1994, Kueh 1995) showed no 

statistically significant relationship between human health and the current U.S. recommended indicator 

microbe for evaluating beach water quality in sub/tropical regions (particularly without point sources). Although 

these studies challenged the use of current methods for sub/tropical environments, they did not establish a 

substantial case for a new method or set of indicators. These prior studies did not include an oceanographic 

component aimed at simulating the distribution and number of microbes in water during different environmental 

conditions. The proposed epidemiologic study differs in that it will be conducted in conjunction with 

coordinated ongoing interdisciplinary efforts including intensive individual microbial exposure monitoring and a 

strong oceanographic component to evaluate the distribution and inter-relation of microbes, human illness and 

other environmental factors. The researchers proposing this Pilot Epidemiologic Project consider that this 

combination of interdisciplinary efforts will build a much unique stronger case concerning which microbe or 

monitoring methodology is the most effective for establishing human related illness in sub/tropical marine 

environments and non point sources. 

Human Health Data: Several epidemiologic studies have found that, compared with bathing in 

uncontaminated waters, bathing in temperate recreational waters contaminated with domestic sewage or storm 

drain runoff has been associated with an increased risk for transmission of infectious diseases, including 

gastroenteritis, febrile respiratory illness, eye and ear infections, and skin infections and rashes. However, 

there were weaknesses in the designs of many of these studies, including the absence of data capable of 

assigning individual estimates of exposure to each bather resulting in exposure misclassification, a failure to 

account for other risk factors for the illnesses of interest, the presence of a known point source (rather than the 

more common scenario of no known point source), and temperate not sub/tropical environment. 

Pruss (1998) reviewed all the significant existing epidemiologic studies on the health effects from exposure to 

recreational water. She found that most studies reported a dose-related increase in swimming-related 

illnesses associated with increases in recreational water indicator bacteria counts. The indicator organisms 

that reportedly correlated best with the health outcomes were enterococci/fecal streptococci for marine and 

freshwater, and E. coli for freshwater. Of note, the majority of these studies were conducted in the northern 

US and the UK, with few studies performed in sub/tropical marine recreational waters and/or without known 

point sources. 

In a series of studies of randomly assigned bathers vs non-bathers in the United Kingdom, Fleisher, et al. 

(1993, 1996, 1998) attempted to address some of the methodologic issues of prior studies. A summary of 

findings is presented in Table 1. These results, coupled with the evidence that infectious microorganisms can 

occur in sub/tropical waters in the absence of specific contamination events and the dirth of information 

describing the impact of these illnesses (i.e., the severity of the illness or need for medical care), indicate that 

the actual public health risk associated with bathing in sub/tropical waters containing microorganisms, including 

those from EPA’s list of microbial indicators, is unknown. 

Table 1: Summary of results from the work of Fleisher et al. (1991, 1993, 1996, 1998) 

Number of Study

Subjects 

Indicator 

(Concentration) 

Reporting Symptoms 

(Percentage) 

Statistical Test 

Significance 

Fleisher et al (1993) 

Fecal streptococcus* GI Chi Square(trend) 

278 Non-bathers 7-14 

78 <35 7-18 

128 >35 20-24 P< 0.01- 0.11 
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Fleisher et al (1996) 

Fecal streptococcus* 

(quartiles) 

Acute febrile Respiratory 

illness 

Chi Square (trend) 

665 Non-bathers 3 

124 0-14 4 

138 15-27 5.8 

125 28*50 1.6 

158 51-158 7.6 P<0.04 

Fecal Coliforms* Ear ailments 

636 Non-bathers 2.8 

120 0-40 6.7 

113 41-79 6.2 

152 80-133 5.9 

134 134-661 14.2 P< 0.001 

Fleisher et al (1998) 

Not specified GI illness Attributable Proportion

605 Non-bathers 9.7 

546 Bathers 14.8 34% 

Acute Febrile Resp. Illness 

665 Non-bathers 3.0 

546 Bathers 5.0 40% 

Ear Ailments 

636 Non-bathers 2.8 

525 Bathers 8.2 66% 

Eye Ailments 

658 Non-bathers 2.1 

538 Bathers 4.5 53% 

*Number of organisms per 100 ml 

 

Preliminary Data 

The Investigators from the NSF NIEHS Oceans and Human Health Center at the University of Miami Research 

Project, as well as NOAA, Miami Dade County Public Health Department and CDC possess a considerable 

amount of experience in the area of recreational microbial pollution and are considered experts in their fields. 

Drs Fleming and Solo Gabriele with Dr Elmir have performed pilot studies at the proposed Study Site (see 

below); Dr Jay M Fleisher is the author of numerous studies on the issue of the human health effects of 

microbial pollution in recreational marine waters in temperate waters (see Table 1); Drs Backer and Goodwin 

have extensive experience in epidemiologic and microbial pollutant studies. Letters of Support are included 

from the Collaborators. Furthermore, this proposed epidemiologic study will be integrated with the ongoing 

extensive environmental monitoring and hydrodynamic modeling of an ongoing research study of the NSF 

NIEHS Oceans and Human Health Center of the University of Miami with collaborators from NOAA, the CDC, 

and the Miami Dade County Dept of Health. 

Drs Solo-Gabriele, Fleming and Elmir (Fleming 2004, Shibata 2004) conducted a pilot epidemiological and 

water quality study at two sub/tropical public beaches, Hobie and Crandon, located in the southern part of 

Biscayne Bay, Miami (FL) next to both the University of Miami Rosenstiel School and the NOAA Southeast 

Center. This very small pilot study was funded by the NIEHS Marine and Fresh Water Biomedical Sciences 

Center and the Miami Dade Dept of Health. Hobie Beach (also the study site for the Proposed Study) is 

approximately one mile long, relatively shallow, and characterized by poor water circulation; its shoreline is 

covered with seaweed over a silty and muddy floor. The beach has a history of poor water quality: during 
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2000, the beach exceeded the EPA Poor Water Quality Guideline (PWQG) for enterococci 29.2%. Probably 

due to the very limited sample size, results showed that there was no significant relationship between density 

of microbes and health effects; the water quality at Crandon Beach was better than Hobie Beach regardless of 

the season (wet vs. dry); there was no fecal pollution point source identified in the sanitary survey; intensive 

spatial water quality monitoring indicated the southern tip of the shoreline at Hobie Beach appeared to be the 

source of microbes; and the concentrations of indicator microbes were considerably lower during low tide as 

compared to high tide. The detection of those indicators in the soil/vegetation of the shoreline without a known 

point source of fecal pollution again raised issues concerning the suitability of those indicators for measuring 

the sanitary water quality in sub/tropical climates. 

Over the past 2.5 years of NSF NIEHS Oceans and Human Health Center and collaborative NOAA funding, 

the Recreational Microbes Research Group has made significant progress. The group developed and 

implemented 4 sediment and water sampling and testing efforts within the Hobie Beach study site. The overall 

results suggest that the inter-tidal zone (sand that is periodically wet and dry between low and high tide) is the 

source of indicator microbes at this beach. The release of indicators from this source is a function of tidal 

height. Runoff also contributes to the indicator microbe burden of the water column. The sampling efforts 

supporting these observations included: 1) intensive sediment sampling effort, 2) hourly sampling continuously 

for 48 hours, 3) sampling along transects at set tidal heights, 4) direct sampling of runoff, and 5) evaluation of 

the microbial contribution from runoff, human swimming and animal feces (Elmir in Press and PhD 

Dissertation). Finally, many important logistical, administrative and training activities accompanied those 

intensive sampling efforts, including: developing and implementing safety, sampling training and analyses 

protocols, meetings with the epidemiological, sediment and modeling groups, governmental agencies, and 

working with a variety of universities and public and private local laboratories. Those activities were essential 

for the success of the sampling efforts and the epidemiologic study proposed below. 

Goals and Hypotheses 

The primary goal of the proposed study is to conduct a prospective randomized epidemiologic study to assess 

the human health risks associated with swimming in sub/tropical seawater contaminated with infectious 

microorganisms while minimizing the significant bias seen in prior epidemiological studies, in collaboration with 

ongoing intensive environmental monitoring and hydrodynamic modeling. The proposed Pilot Epidemiological 

Study is designed to address 3 major methodological weaknesses inherent in previously published 

epidemiological studies assessing the health effects of bathing in waters contaminated with domestic sewage. 

These methodological weaknesses are: 1) Failure to control for the substantial amount of temporal and spatial 

variation in indicator organism density shown to occur within just a few hours at marine bathing locations; 2) 

Failure to relate indicator organism density directly to the individual bather; and 3) Failure to rigorously control for 

non-water-related risk factors for the outcome illness under study. The design of the proposed epidemiological 

study addresses these weaknesses by controlling bather exposure, by selected individual study participant 

monitoring of indicator organism densities, and by pre- and post-exposure interviews with each study participant. 

The hypotheses to be tested for the proposed epidemiologic study are: 

H1: People exposed to recreational waters contaminated with infectious microorganisms will be 

significantly more likely to report symptoms than unexposed similar people who go to the beach but 

do not go into the water. 

H2: The organism densities sampled at the time of exposure in the marine waters will correlate with 

reported human symptoms. 

The following related coordinated questions will be explored by the NSF NIEHS Oceans and Human Health 

Center Research Project, with the NOAA, CDC and Miami Dade County Public Health collaborators including: 

1) Coupled Hydrodynamic/Microbial Water Quality Model: This model will be used to assess environmental 

factors that influence indicator microbe levels. Such information will be useful in establishing the field sampling 

plan for the epidemiologic study; 2) Intensive Environmental Monitoring of Enterococci: Update sanitary survey 

for site. This will involve spatially and temporally intense sample collection in water and sand. Laboratory 

work will also be included to further establish source functions needed for the water quality model; 3) Sediment 

Study: Characterize sediment types, study of spatial variability in different types of sediment, and analyze 
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temporal changes in sediment distribution. Results will be used in support of developing source functions for 

suspended sediments within the hydrodynamic model; 4) Microbe Monitoring Using Traditional Methods: 

Monitor water during the epidemiologic study for the numbers of pathogens and microbiological indicators, 

using traditional analysis methods. Microbes measured will include the traditional indicators (enterococci, fecal 

coliform), non-traditional indicators (C. perfringens, and Bacteroides), and pathogens (S. aureus, enterovirus, 

Cryptosporidium parvum, and Giardia lamblia). 

Study Methods 

The design of the proposed epidemiologic study will be based on information collected by the Investigators 

during past studies at Hobie Beach described above (Shibata 2004; Fleming 2004) and on the work of Fleisher 

et al (1991, 1993, 1996, 1998) (see Table 1 above). The investigators will conduct a prospective randomized 

intervention study with study participant exposure randomly assigned to either potentially contaminated marine 

recreational water or beach-only exposure. 

Local adult residents (at least 18 years old) will be recruited to participate in the study. After recruitment, the 

participants will be screened for basic demographics, current illnesses and symptoms, alternative risk factors, 

and a brief health history. Study participants will then be randomized to “water exposure” or “no water 

exposure.” Study participants will then: 1) Spend 10 minutes in waist-deep water in a demarcated study 

environmental sampling area and dunk their heads completely under water at least 3 times (“water exposure”), 

or 2) Spend time on the beach at a demarcated study environmental sampling site away from the water (“no 

water exposure”). Seven (7) days after the beach visit, study participants will complete a short telephone 

follow-up interview to assess the occurrence of illnesses caused by the majority of known infectious agents 

(including questions about symptoms occurring since the beach visit, whether they sought medical care for 

their symptoms, and whether the symptoms caused them to miss work or school), as well as alternative risk 

factors for the study outcomes (including subsequent marine water exposures and food consumption) and 

subsequent beach visits. After completing all study activities, study participants will be given $25 to 

compensate them for any costs associated with participating in the study. 

Study Populations: Local adult residents (not tourists) will be recruited to participate in the study due to 

logistics of the several week follow up. An individual will meet the study inclusion criteria if: 1) at least 18 

years old; 2) Do not have any chronic gastrointestinal (GI), respiratory disease, skin, or eye disease; 3) do not 

currently use prescription medications that may predispose to gastroenteritis; 4) was not exposed to bathing 

waters in the prior 1 week; 5) is willing to be exposed to bathing waters at the Study Site; 6) willing to be 

randomized; and 7) not ill on the study day. 

Disease Endpoint Criteria: The disease endpoints of interest are self-reported symptoms of gastrointestinal 

illness, acute febrile respiratory illness (as specified by ICD 9 461-466, 480), eye or ear infections, and skin 

infections/irritation within seven days of going to the beach. These health endpoints are based on information 

collected during past studies by the Investigators at Hobie beach and on the work of Fleisher et al. (1991, 

1993, 1996, 1998) (see Table 1). 

Recruitment Methods: Recruitment efforts will be initiated several weeks prior to the actual Study Site data 

collection days. Local health authorities and local civic organizations, as well as the local media, will used in the 

recruitment effort. Potential study subjects age 18 and older will be approached by staff members and asked if 

they would like to participate in the study. 

Study Site Data Collection: The proposed epidemiologic study data collection activities will be scheduled over 

a total data and sample collection period of 20 days in coordination with the environmental research group over 

a total period of several months. Each sampling day, approximately 65 participants will be randomly assigned 

to simultaneously go into the water or go to the site on the beach away from the water (i.e., approximately 32 

people/group). On the actual day of the study data collection prior to beginning the Study activities, an IRBapproved Consent Form will be explained and signed, and then an interview will be administered to each study 

participant. This interview will be administered by a trained interviewer, and focus on any non-water-related 
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behavior that could either cause or confound any association found between bathing and the outcome illnesses 

under study, any current illnesses, and any recent exposure to bathing waters. 

Study subjects will be randomly assigned to either a bather group or a non-bather group. The ratio of staff 

members to study subjects will be approximately 1:6. Individual bathers will then assigned to staff members who 

will supervise the exposure activity of the bather, including recording the time, location, activities, and duration 

each individual bather spent in the water. Bathers will be required to spend at least 10 minutes in the water at 

waist height, and to immerse their heads three times. Using ropes, a 60-meter stretch of beach will subdivided 

into 20-meter intervals forming three bathing exposure zones, each zone being separated by ropes. Exposure of 

any individual bather will therefore be restricted to a single 20-meter-wide swim zone. Each subject will be 

instructed on the technique to take their own water sample near the surface during their head immersions as well 

as provided with an appropriate water sampling container. Staff members will observe the subjects at all times to 

insure that the subjects perform the water sampling appropriately as well as follow the 3 head immersion protocol. 

When the subjects leave the water, they will give their individual water samples to the Study Staff for microbial 

analysis processing (as described below). 

No bather will be allowed to enter the water more than once. The non-bather group will be restricted to a roped-off 

area distant from the water. Staff members will be assigned to ensure that non-bathers do not enter the water. 

Each study participant will be given a box lunch to eat, and will be asked not to bring any additional food or 

beverage to the beach (to avoid possible illness due to contaminated food). 

Environmental Sampling: The Study Site will be Hobie Beach (described above in Preliminary Data) which 

has been and is being well characterized in terms of sediments, microbial content and sources for water and 

sediments, and hydrodynamics from the prior pilot study and the ongoing research project of the NSF NIEHS 

Oceans and Human Health Center at the University of Miami, as well as NOAA, Miami Dade County Public 

Health Department and CDC. Of note, the environmental monitoring and assessment are ongoing and funded 

by the NSF NIEHS Oceans and Human Health Center Recreational Microbes Research Project and 

collaborators; these funds do not cover the performance of the Proposed Epidemiologic Study 

described above. The environmental assessment described below will focus on the analysis of several 

microbes in water including pathogenic viruses, bacteria, and protozoa and the traditional indicator microbes. 

Funds have already been provided with the NSF NIEHS Oceans and Human Health Research Project 

and collaborators for microbial analyses described below. 

Water Quality Monitoring at Study Site: While each study participant is actually in the water, each subject will 

collect their own environmental water sample from the demarcated study sites as described above. All water 

samples will be iced and transported back to the Microbe Laboratory at the University of Miami Rosentiel School 

(<5 minutes) for processing, separation, storage and distribution. 

The environmental sample will be split 3 ways: one for enterococci analysis and one separate 

enterococci duplicate sample/each subject, and one for a daily pathogen/indicator sample composite 

corresponding to 35 study participants. This daily composite sample will be analyzed for a suite of 

additional indicators/pathogenic bacteria (fecal coliform, Staph aureus, C. perfringens and Bacteroides), 

viruses (enterovirus) and protozoans (Giardia and Cryptosporidium); the composite sample is necessary due to 

the high expense of the numerous pathogen analyses. Investigators will also survey the bather density and 

animal populations in the study area during participant exposure, as well as evaluate data from a 24 hour 365 

day per year video camera focused on the Study Site and installed by the NSF NIEHS OHH Center 

Recreational Microbes Research Project with permission of Miami Dade County. Of note, the issue of the 

variability between replicate samples for the indicator organisms and the pathogens is currently being 

investigated by the Investigators under the ongoing NSF NIEHS Oceans and Human Health Research Project. 

Microbe Monitoring Using Traditional Methods: Analysis will include: pathogenic viruses (enterovirus), 

pathogenic protozoa (Cryptosporidium parvum and Giardia lamblia), pathogenic bacteria (S. aureus), as well 

as indicators including: enterococci, fecal coliform, C. perfringens, and Bacteroides. Water concentration for 

viruses and protozoa (described below) will be conducted at the University of Miami. Once concentrated onto 
8

filters, these samples will then be sent to a commercial or government laboratory for elution and analysis. Preprocessing of water samples will require filtration of a large volume of water (greater than 20 liters) using two 

sets of filters, one for protozoans which filter based upon size exclusion and another for viruses which filter 

based upon the sorption of the viruses. Furthermore, physico-chemical parameters (including pH, salinity, 

temperature, dissolved oxygen, and water turbidity) will be measured when sampling for the target microbes. 

Enterococci: Enterococci, the indicator currently recommended by the U.S. EPA for marine waters, will be 

enumerated using a traditional membrane filter method as recommended by the U.S. EPA (USEPA 1997, 

2000) and a chromogenic substrate method as proposed by IDEXX (Westbrook, Maine) (APHA 1995). The 

membrane filter method involves placing the membrane filter containing the bacteria on a selective medium 

(mEI agar) and incubating the sample at 41°C for 24 hours. Colonies with a blue halo are then counted as 

enterococci. The chromogenic substrate method in simple term utilizes enzymes that are specific to particular 

microbial groups. These enzymes are attached to dyes which are then released when the target microbe is 

present in the sample. Enumeration of the microbe population is based upon the use of a tray (QuantiTray/2000) which separates the sample into a set of test wells. The number of test wells that show the 

characteristic color are then counted and used in conjunction with a standardized table to provide the 

concentration in terms of the most probable number (MPN). 

S. aureus: S. aureus has been commonly associated with skin infections in marine waters (Charoenca 1993, 

Charoenca 1995). A membrane filtration method using Chromeagar medium will be utilized. Mauver colored 

colonies are counted as positive (Fowler et al. 2004). 

C. perfringens: There is scientific evidence that indicates that C. perfringens behave similar to enteric viruses 

in marine and fresh waters and does not multiply outside the human gastro-intestinal tract under aerobic 

conditions (Fujioka 1985; Fujioka 1997). Therefore, using C. perfringens as part of a monitoring program for 

the microbiological recreational water quality can be a valuable alternative, particularly in sub/tropical marine 

environments, where the regrowth of the other indicator microbes is suspected to occur. The state of Hawaii 

has elected to use C. perfringens in their water quality monitoring, and the USEPA has recently approved a 

membrane filtration method to detect and enumerate C. perfringens spores in water and sediments. C. 

perfringens will be analysed using the membrane filter method (USEPA 1995) requiring the use of mCP agar. 

Samples are incubated anaerobically at 44.5± 0.2°C for 24 hours. Plates are then exposed to ammonium 

hydroxide fumes after the incubation and dark pink to magenta colonies were counted as C. perfringens. 

Pathogenic Viruses: The presence of enteric viruses in coastal waters pose a public health risk since these 

viruses are typically transmitted via the fecal-oral route, and because for many viruses, only a small number of 

virus particles can initiate an infection. Because both the Florida Keys and the Tampa Bay region have been 

investigated for the presence of viruses in coastal waters and the presence of enteric viruses has been 

confirmed, it is likely that investigating the Miami region of Florida will also reveal the presence of viral 

pathogens in the coastal environment. After concentration using Filterite filters (with pH and salt adjustment), 

the samples will be sent to a commercial or government laboratory for elution and analysis using traditional 

virus culture methods. 

Pathogenic Protozoa: Cryptosporidium and Giardia will be analyzed using USEPA method 1623 (EPA 2001). 

This method requires the filtration of a large volume of water through size exclusion filters. The filters are then 

eluted, the pellet is concentrated, and the Cryptosporidium and Giardia are purified using immuno-magnetic 

separation. The purified pellet is stained with a fluorescent antibody and the number of oocysts/cysts are 

counted using a microscope. 

Coupled Hydrodynamic/Water Quality Model and Microbial Indicator Fate Model: The presence and 

temporal variation of microbial indicators in the sub/tropical coastal environment is profoundly influenced by 

water currents and mixing. In the presence of point and distributed sources of pathogens and indicator 

microbes, the interpretation of indicator levels is dependent on quantifying the pathways and fate leading to 

observed variations in spatial and temporal distribution. A model having two integrated components will be 

developed for assessing mechanisms that control microbe concentrations in beach areas. The two 
9

components are a hydrodynamic transport and mixing model and a microbe fate model that predicts the 

transformation kinetics of growth and mortality. 

To determine the current transport, an existing hydrodynamic model for Biscayne Bay will be used (Wang 

1986, Wang 1988, Ault 1999, Wang 2003). The CAFE3D model is based on a numerical solution of the full 

hydrodynamic equations of motion using the finite element method (Wang 1978, Wang 1994), and is similar in 

formulation to widely used finite difference models, such as the Princeton Ocean Model (Blumberg 1987). The 

general purpose CAFE3D model code was selected for its superior ability to handle complex topography and 

adapted to Biscayne Bay with support from NOAA’s Coastal Ocean Program as a tool for assessing the effect 

of natural and anthropogenic stressors on the Southeast Florida coastal environment. 

The data to be used for model forcing consist of wind, tides, and runoff. Historic and real-time hourly wind data 

are available from recording stations at the Rosenstiel School of Marine and Atmospheric Sciences (RSMAS) 

(1 miles from Hobie Beach), at the Fowey Rocks and Sombrero Key offshore Coastal Marine Automated 

Network stations operated by NOAA/NOS, and from Miami Airport. The sensors operated by the Remote 

Sensing Core of the NSF NIEHS OHH Center at RSMAS will include wind speed and direction (at two levels), 

air temperature and humidity, surface atmospheric pressure, rainfall rate and accumulated rainfall, broad-band 

insolation (0.3 to 3 μm wavelength) and broad-band infrared incident radiation (5-50 μm wavelength). The 

rainfall rate will be used to calibrate the radar reflectivity measured by the Miami NWS NEXRAD radar that will 

provide spatial distributions of rainfall for runoff estimation at Hobie Beach (see 

http://www.rsmas.miami.edu/environment/nexrad/). The NEXRAD data are available with a 6-minute time 

resolution. Tidal water levels are recorded at 6-min intervals right at Virginia Key, and both historic and realtime data are available to Dr Wang’s laboratory. In addition, Dr Wang has derived tidal harmonic components 

for several stations around the Bay including Rickenbacker Causeway in past efforts. These components 

provide sufficient information for including tides in the model. Hourly canal and Miami River discharges are 

monitored by the South Florida Water Management District. The Investigators have previously obtained the 

required data from their DBHYDRO data base server in suitable digital format. Finally, the Remote Sensing 

Core will provide satellite images from optical sensors with 10 m resolution to help quantify suspended matter 

and sediment transport in the study area. 

Microbial Indicator Fate Model: The hydrodynamic model provides the basic framework for transport and 

mixing of microbes. To develop a complete model of the fate of microbes, processes such as regrowth, dieoff, 

deposition and entrainment must be added to this framework. Previous work (Solo-Gabriele 2002, Shibata 

2004) has shown that microbes may be multiplying in the sediment on the beach and under water. The rates 

and effects of controlling factors are poorly understood, but will be better quantified through the field work and 

analysis of this project. As a first approximation, the investigators will model the dieoff and regrowth in water, 

sediment, and beach sand as separate 1st order processes with rates determined from the Phase I 

environmental assessment. The rates are expected to depend on temperature, salinity, solar radiation, 

sediment type and time. Further refinements will be made as more data become available. In addition source 

and sink functions due to deposition in and erosion from bottom sediment will be formulated as a function of 

water levels, wind speed and direction, rainfall, beach use, and sediment characteristics using the data from 

the literature (e.g. Burkhardt 2000) and from the first phase of environmental assessments. This innovative 

modeling approach will show whether indicator microbes correlate with pathogens and health effects, or it will 

provide an alternate means for determining the presence of pathogens from point sources if correlation with 

indicators is poor. 

Epidemiologic Study Sample Size Calculations: The sample size for the proposed epidemiologic study was 

calculated based on information collected during past studies by the Investigators at Hobie beach and on the 

work of Fleisher et al., (1993, 1996, 1998) (see Table I). Using GI symptoms as reported by Fleisher et al 

(1993) and again assuming 95% confidence and 80% power, approximately 1300 study participants (650 

bathers and 650 non-bathers) are needed to show an increase in reported GI symptoms of 5% in bathers 

exposed to fecal streptococcus at levels below the US EPA maximum level of 35 organisms/100 ml of water, 

with either bathing in waters below this level or not bathing. 
10

Epidemiologic Study Database & Statistical Analysis: The Study Database will be created and analyzed 

using SAS for the purposes of the epidemiologic analyses. These data will also be shared with the 

oceanographic risk modeling group as an Excel database. All univariate analyses, with the exception of any 

stratified analyses, will be carried out using the SAS package of statistical programs. Stratified analyses will be 

conducted using EPINFO, while all logistic modeling will be computed using SAS package of statistical programs. 

Initial analyses will evaluate the effects of randomization for all demographic variables to insure equal 

distribution of demographic risk factors among exposed (ie. bathers) and unexposed (ie. non bathers) 

populations. The basic analyses will be geared to answer two questions: (1) "Is there an excess of the illnesses 

under study among bathers vs. non-bathers?"; and (2) "Is there a dose-response relationship between indicator 

organism density and the incidence of the outcome illnesses studied among bathers?" 

1) Bather vs. Non-Bather Comparisons: Success of randomization will be assessed by comparing the distribution 

of non-water-related risk factors for the illness under study among bathers vs. non-bathers using univariate chisquare analysis. Assuming randomization was successful, the crude rates of illness for bathers and non-bathers 

will have been adjusted for any of these extraneous risk factors. If randomization is not successful for any nonwater-related risk factor, the effect of this failure will be assessed for any association found between illness among 

non-bathers vs. bathers at increasing indicator organism exposure using multiple logistic regression analysis. 

A second chi-square analysis will be conducted for each of the illnesses under study to assess at what indicator 

organism density rates of illness among bathers are significantly greater than rates of illness among non-bathers. 

In the context of public health planning, specifically the setting of recreational water quality criteria, this analysis 

could provide insight into possible thresholds of effect (i.e., an indicator organism density below which bathers 

experience no excess illness relative to non-bathers). It must be emphasized that this analysis is not meant to 

definitively define such a threshold, but rather to generate hypotheses regarding the possible existence of such 

thresholds to be tested by future epidemiological/statistical research, particularly for sub/tropical locations without 

known point sources. 

2) Analyses Restricted To Bathers Only: In order to assess the relationship between indicator organism density 

and each illness under study, while identifying and controlling for non-water-related risk factors for such illness, 

analyses will be restricted to bathers only. Exposure (i.e., indicator organism density) will be related to each 

bather using the time an individual exited the water, the swim zone used, and the indicator organism density 

recorded within that zone taken nearest to the time the individual exited the water. 

Chi-square analysis will then used to identify any statistically significant trends among the observed incidence of a 

particular outcome illness among non-bathers vs. bathers at increasing levels of indicator organism exposure. 

Bathers will be divided into quartiles of indicator organism exposure, and trend will then be assessed for each 

indicator organism assayed. These series of analyses will include the rates of illness observed among nonbathers, but a trend will be considered significant only if the chi-square for trend statistic remained statistically 

significant with and without the inclusion of the non-bather (reference) group. This insures that differences 

between the reference group and the lowest exposure grouping do not unduly influence the chi-square for trend 

statistic. When a particular outcome illness does not show a statistically significant trend for any of the indicator 

organisms assayed, rates of illness among bathers in the highest quartile of indicator organism exposure will be 

compared to rates of illness among bathers in the lowest quartile using chi-square analysis. 

Multiple logistic regression modeling will be used to evaluate relationships between indicator organism density and 

illness among bathers while controlling for extraneous non-water-related risk factors identified via a prior univariate 

chi square analysis of postulated non-water-related risk factors or confounders among ill vs. non-ill bathers. Initial 

criteria for entering non-water-related risk factors or possible confounders into the logistic regression model will be 

at p < 0.15, although retention in the model will be at p<0.05. In addition, a term will be included in the logistic 

model to indicate whether a bather reports the swallowing of any water. A term consisting of the period of time 

each bather spent in the water will also be included in each logistic regression model in order to assess whether 

duration of exposure affect risk of illness. If this term significantly adds to the fit of the model, the appropriate 

interaction term will be added to the model to test for possible interaction between the time a bather spent in the 
11

water and the indicator organism density the bather was exposed to. In this manner, the effect of duration of 

exposure on indicator organism exposure could be assessed. A backward step-wise elimination strategy will be 

employed in logistic regression modeling used to control for non-water-related risk factors or confounders for the 

illness under analysis. For convenience, based on the workings of the statistical package used for logistic 

regression modeling, a forward step-wise procedure will be used to assess the magnitude of any confounding of 

any observed indicator organism exposure-disease outcome relationships by non-water-related risk factors. 

Relation to Epidemiology and Public Health and Public Health Decision Making

The proposed epidemiologic study will provide individual exposure and health data which will allow an initial 

analysis of the risk of exposure to non point source microbial pollution in sub/tropical recreational marine 

waters. The data will also become part of a hydrodynamic and exposure model which will be used to predict 

the inherent factors in beach closure. Finally, the environmental collaborators will define the sources of 

microbes as well as develop more rapid inexpensive microbial exposure monitoring methods. 

Potential Challenges: By using only residents, not tourists, and in particular residents that are willing to go 

bathing at the Study Site, there is some indication from the Hawaii studies (Fujioka 1994) that we may be 

selecting for persons less susceptible to illnesses caused by exposure to microbial pollution in recreational 

marine waters due to self selection and the exclusion of persons with obvious immunologic susceptibility, 

although Fleisher et al found increased report of illness among his resident participants in the United Kingdom. 

In addition, if the microbial exposure levels are lower than predicted as might be expected with a non-point 

source site such as the selected Study SIte, it is possible that the sample size may be too small to detect a 

statistical difference. However, their calculations and study design are based on Dr Fleisher (Co PI) successful 

implementation with similar environmental levels of indicator organism in temperate waters (Fleisher et al 

1991, 1993, 1996, 1998) (see Table 1 above). Furthermore, with the limited monies available, this is the 

sample size that the investigators can attempt to obtain and process logistically. 

Time Line of Work 

The first 3 months of the proposed Pilot Project will consist of: hiring and training the epidemiologic staff, 

finalizing the study forms (including questionnaires, Human Subjects consent forms, subject information and 

recruitment materials), obtaining the Human Subjects Approvals, establishing the study database, setting up 

the study logistics for the beach screening days with the environmental research group, and recruiting the 1300 

study subjects. For the next 6 months, the epidemiologic research group will work closely with the 

environmental research group to coordinate 20 total days of randomization and individual environmental 

assessment at the Study Site for the 1300 study subjects; the epidemiologic research group will then perform 

the individual subject follow up at 1 week. Finally during the last 3 months of the Study, the epidemiologic data 

will be analyzed in conjunction with the environmental research group with publications submitted for peer 

review. Presentations will be made by the investigators at scientific meetings and community meetings as part 

of study outreach and education. 

Of note, for the successful completion of this Pilot Epidemiology Proposal, the proposed Pilot Study 

must begin by early Fall 2007 to be performed in conjunction with timeline of the ongoing NSF NIEHS 

OHH Research Project Environmental Monitoring Project. 
12

Reference 

American Public Health Association (APHA). 1995. Standard Methods for the Examination of Water and 

Wastewater, 19th ed. American Public Health Association, Inc., Washington D.C. 

Atmar RL, F. H. Neill, J. L. Romalde, et al. 1995. Detection of Norwalk virus and hepatitis A virus in shellfish 

tissues with the PCR. Appl. Environ. Microbiol. 61:3014-3018 

Ault, J.S., Luo, J., Smith, S.G., et al. 1999. A Spatial Dynamic Multistock Production Model. Can J. Fish. 

Aquat. Sci, 56(Suppl. 1), 4-25 

Blumberg, A.F., Mellor, G.L. 1987. Description of a Three-Dimensional Coastal Ocean Circulation Model. 

Three-dimensional Coastal Ocean Circulation Models, Coastal and Estuarine Sciences, 4, American 

Geophysical Union, Washington, DC. 

Burkhardt III, W., Calci, K.R., Watkins, W.D., et al. 2000. Inactivation of Indicator Microorganisms in Estuarine 

Waters. Water Research, 34(8), 2207-2214 

Charoenca N, Fujioka RS. 1993. Assessment of Staphylococcus Bacteria in Hawaii's Marine Recreational 

Waters. Water Science and Technology, 27(3/4): 283-289. 

Charoenca N, Fujioka RS. 1995. Association of staphylococcal skin infections and swimming. Water Science 

and Technology, 31(5-6): 11-17.Current, W.L. and L.S. Garcia. Cryptosporidiosis. Clin. Lab. Med. 11 (1992), 

pp. 873–897. 

Elmir SM, Wright ME, Solo-Gabriele HM, Abdelzaher A, Fleming LE, Miller G, Rybolowik M, Shih M-T P, Pillai 

SP, Cooper JA, Quaye EA. In Press. Quantitative Evaluation of Bacteria Released by Bathers in a Marine 

Water. Water Research. 

Fleisher, J.M., Jones, F., Stanwell-Smith, R., et al. 1993. Water and non-water risk factors for gastroenteritis 

among bathers exposed to sewage-contaminated marine waters. International Journal of Epidemiology Vol. 

22, pp. 698-708. 

Fleisher JM. 1991. A Re-Analysis of the Data Supporting U.S. Federal Bacteriological Water Quality Criteria 

Governing Marine Recreational Waters. Water Pollution Control Federation Journal 63:259-265. 

Fleisher, J.M., Kay, D., Wyer, M.D., et al. 1998. Estimates of the severity of illnesses associated with bathing 

in marine recreational waters contaminated with domestic sewage. International Journal of Epidemiology Vol. 

27, pp. 722-726. 

Fleisher JM, Kay D, Jones F, et al. 1996. Marine Waters Contaminated with Domestic Sewage: Nonenteric Illness 

Associated with Bather Exposure in the United United Kingdom. American Journal of Public Health 86(9):1228-

1234. 

Fleming LE, Solo Gabriele H, Elmir S, Shibata T, et al. March 2004. A Pilot Study of Microbial Contamination of 

Subtropical Recreational Waters. Fl J Env Health 29-33. 

Fowler TL, Fujioka RS, Tice AD, Ishikawa AK, Yoneyanna BS. 2004. Modification of CHROMagar staph. 

aureus to enumerate staphylococcus aureus from marine recreational waters in Hawaii. ASM 104th General 

Meeting; May, 2004. 

Fujioka,R.S., Roll,K., and Morens,D. 1994. A Pilot Epidemiological Study of Health Risks Associated with 

Swimming at Kuhio Beach. Water Resources Research Center. 
13

Fujioka, R.S., Roll, B., Byappanahilli, M. 1997. Appropriate recreational water quality standards for Hawaii and 

other tropical regions based on concentrations of Clostridium perfringens. Proceedings of the Water 

Environment Federation, 70th Annual Conference and Exposition, 4: 406-411. 

Fujioka, R.S., Shizumura, L.K. 1985. Clostridium perfringens, a reliable indicator of stream water quality. 

Journal Water Pollution Control Federation, 57(10): 986-992. 

Kueh, C.S. Tam, T.Y. Lee,T. et al. 1995 Epidemiological study of swimming associated illnesses relating to 

bathing beach water quality.Wat.Sci.Tech,31(5):1-4. 

Pruss, A. 1998. Review of epidemiological studies on health effects from exposure to recreational water. 

International Journal of Epidemiology, 27(1):1-9. 

Shibata T, Solo Gabriele HM, Fleming LE, Elmir S. 2004. Monitoring Marine Recreational Water Quality Using 

Multiple Microbial Indicators in an Urban Tropical Environment. Water Research 38:3119-3131. 

Solo-Gabriele HM, Fleming L, and Elmir S. 2002. A Pilot Study Evaluation and Sanitary Survey of Microbial 

Recreational Water Quality Indicators in the Subtropical Marine Environment. Prepared for the National 

Institute of Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center, 

Rosenteil School of Marine and Atmospheric Sciences and the Miami-Dade County Health Department. 

U.S. Environmental Protection Agency. 1995. Method for Detection and Enumeration of Clostridium 

perfringens in Water and Sediments by Membrane Filtration, EPA-600-R-95-030. U.S. Environmental 

Protection Agency, Edison Laboratory, Environmental Services Division, Region 2 and Environmental 

Monitoring Systems Laboratory - Cincinnati, OH. 

U.S. Environmental Protection Agency. 1997. Membrane Filter Test Method for Enterococci in Water, EPA821-R-97-004. U.S. Environmental Protection Agency, Office of Water, Washington D.C. 

U.S. Environmental Protection Agency. 2000. Improved Enumeration Methods for the Recreational Water 

Quality Indicators: Enterococci and Escherichia coli. U.S. Environmental Protection Agency, Office of Science 

and Technology, Washington D.C. 

US EPA Method 1623, Cryptosporidium and Giardia in water by filtration/IMS/FA, EPA-821-R-01-025, April 

2001, Office of Water, Washington D.C. 

Wang, J.D. 1978. Real Time Flow in Unstratified Shallow Water. J. Waterway Port Coastal and Ocean Div., 

ASCE, 104(WW1), 53-68. 

Wang, J.D., J. van de Kreeke, N. Krishnan, D. Smith. 1994. Wind and Tide Response in Florida Bay. Bull. 

Marine Science, 54(3), 579-601. 

Wang, J.D., van de Kreeke J. 1986. Tidal Circulation in North Biscayne Bay. J. Waterway Port Coastal and 

Ocean Engineering, 112(6), 615-631. 

Wang, J.D., Cofer-Shabica S.V., J. Chin Fatt. 1988. Finite Element Characteristic Advection Model, J. Hyd. 

Eng., 114(9), 1098-1114. 

Wang, J.D., Luo, J., Ault, J.S. 2003. Flows, Salinity and Some Implications on Larval Transport in South 

Biscayne Bay, Florida. Bull. Marine Science.