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Project Proposal Specific Aims
and Hypotheses
I. Monitor the microbiological water quality at the 2 study sites daily for 2 months using multiple bacteriological indicators (ie. fecal coliforms, enterococci, and C. perfringens). II.
Administer an existing epidemiological questionnaire to evaluate
swimming-associated reported symptoms and exposures. III.
Evaluate the relationship between organism density and reported
symptoms between beach sites. H1: The
organism density will be higher for the beach with historically elevated
concentrations of indicator microbes (Hobie Beach) compared to the beach with
historically lower concentrations of indicator microbes (Crandon Park Beach). H2: The organism density at each beach
will correlate with reported human symptoms. H3: There will be significantly higher reported human symptoms
for Hobie Beach compared to Crandon Park Beach. IV. Evaluate the relationship between seasonal conditions and organism density/reported symptoms. H1: The organism density will be higher during the wet season. H2: The reported human symptoms will be higher during the wet season. V. Evaluate the relationship between the 3 indicator microbes and reported symptoms. H1: The reported human symptoms will correlate highest with C.
perfringens compared to fecal coliforms or enterococci. VI. Once the data are evaluated, future research goals will be established to further support the preliminary conclusions obtained from this pilot study. A set of recommendations will be provided to improve recreational water quality monitoring and protect public health in tropical marine environments. Background and Significance
Traditionally, analogous to drinking water, monitoring of water quality for coastal waters used for recreational purposes has been regulated by measuring concentrations of indicator microbes. The microbes utilized are those typically found in human feces in high concentrations. An elevated concentration of these indicator microbes within a coastal water body would thus indicate that the water body has been contaminated by human waste and is unsafe for recreational use. Recently the use of indicator microbes to regulate the recreational use of coastal waters has come into question, particularly in the tropical and sub-tropical marine environment. The U.S. Environmental Protection Agency recommends (USEPA 1986) that States utilize the indicator microbes enterococci and/or Escherichia coli to determine whether health advisories or closures should be issued for recreational coastal waters. E. coli is recommended for freshwaters and enterococci is recommended for both fresh and marine waters. Many States, on the other hand, have NOT adopted USEPA's 1986 criteria, and utilize USEPA’s 1976 recommendation of fecal and/or total coliform as the indicator microbes of choice. Fecal coliform is a subset of total coliform, while E. coli is one of several fecal coliform bacteria. Studies have also shown, however, that total coliforms proliferate naturally in soil (See review article by Toranzos 1991). Both enterococci and E. coli have been found in the environment in areas where no sources of sewage contamination are apparent (Carillo et al. 1985; Rivera et al. 1988, Wright 1986). These indicator microbes have also been observed to multiply in warm tropical environments outside of the human intestinal tract, thereby calling into question the use of these indicator microbes as indicators of sewage (Hardina and Fujioka 1991; Roll and Fujioka 1997; Solo-Gabriele et al. 2000; Wright 1989). Limited studies found that C. perfringens, a spore-forming obligate anaerobic bacteria, is consistently present in wastewater at high concentrations, resistant to chlorination and environmental stresses. 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 and Shizumura 1985; Fujioka et al. 1997). Therefore, using C. perfringens as part of a monitoring program for the microbiological recreational water quality can be a valuable alternative, particularly in tropical and 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. To make matters even more complicated, there have
been documented cases where coastal waters monitored for both sets of
microbial indicators (enterococci and members of the coliform group) have
pass regulatory limits for enterococci and not for fecal coliform, and vice
versa (Elmir, Department of Health, personal communication). So a regulator is left with a perplexing
situation where it is not clear which indicator microbe(s) should be utilized,
and once the data are obtained, how these data should be interpreted? The U.S. EPA guidelines (using E. coli and enterococci) for coastal waters were established based on only 3 water quality and epidemiological studies performed in the 1980s. All of these studies were conducted in the northern part of the U.S. As discussed above, questions have been raised about whether or not the results of such studies are applicable to the tropical coastal ecosystems, given the differences in hydro-climatologic and microbiologic conditions. Human Health Data
Pruss (1998) reviewed all significant existing epidemiologic studies on the health effects from exposure to recreational water. She found that most studies reported a dose related increase of health risk in swimmers with an increase in the indicator bacteria count in recreational water. The relative risks for reported symptoms (either gastrointestinal symptoms or highly credible gastro-enteritis) ranged from 1<RR<3 in these studies. The indicator organisms that correlated best with the health outcomes were enterococci/fecal streptococci for marine and freshwater, and E. coli for freshwater. In both marine and freshwater, the increased risk of gastro-intestinal symptoms was associated with water quality values ranging from only a few indicator counts/100 ml to about 30 indicator counts/100 ml. These values are low compared to water qualities frequently encountered in coastal recreational waters (See Appendix Historical Study Site Data). Of note, the majority of these studies were conducted in the US and UK with few studies evaluated in tropical marine recreational waters. Haile et al. (1999) among others (Fleisher 1998, Pruss 1998) evaluated the risk of reported gastrointestinal symptoms, highly credible gastro-enteritis and other symptoms with respect to reported distance from storm drains with untreated run off in the County of Los Angeles. Over 22,000 persons were interviewed 9 days after their facial immersion exposure to recreational beach waters concerning their symptoms. An increased risk of adverse health outcomes associated with swimming in ocean water contaminated by untreated urban runoff was found with a significant dose response relationship. Miami-Dade County
Miami-Dade County, Florida is the ideal site in which to study the issues of recreational water quality and possible water quality indicators in the tropical marine environment. There is approximately 25 miles of beach available for public recreational use, with significant amounts of historic water quality data collected regularly by the Miami-Dade County Health Dept as well as other regulatory agencies. These monitoring data have shown variable water quality in recreational areas throughout Miami Dade. In particular, there have been a range of indicator organisms recorded from Hobie Beach (located on Virginia Key and heavily used particularly by wind surfers) (See Appendix Historical Study Site Data). Hobie Beach is one of Miami-Dade County’s heavily visited beaches by the locals and tourists, and it is legally used by other animals. It is located in Biscayne Bay just West of Miami Seaquarium and approximately 3.5 miles South East of the mouth of Biscayne Bay and the highly contaminated Miami River. It is about 1 mile long surrounded by urban areas (high rise condominiums on Brickell Avenue, shopping centers, Port of Miami, restaurants, Miami Seaqarium and other commercial and industrial facilities). It is used all year round for various water recreational activities. Furthermore, with its prolonged shallow mud and sand bottom, Hobie Beach has the topographical and ecological qualities associated with apparently natural microbacterial regrowth observed to occur in tropical/sub-tropical marine environments. Based on this background, there is a need to evaluate the water quality at Hobie Beach and possible associated health risks using the best available scientific and investigative techniques available. Methods
The proposed pilot study will consist of 2 months of daily monitoring for recreational water quality using multiple bacteriologic indicators, with 2 cross sectional surveys of reported symptoms from exposed beach users at 2 beach locations performed in the middle of the month. Study Sites
The 2 beach locations, Hobie Beach (Virginia Key) and Crandon Park Beach (Key Biscayne) were selected on historic water quality monitoring data. These beaches are located within 1 mile of each other, yet historically have had dramatically different water qualities. Hobie Beach as noted above has had consistently high indicator organisms, while Crandon Park has had consistently low indicator organisms indicative of good water quality (See Appendix Historical Study Site Data). The 2 beaches will be monitored daily during 1 dry season month (November) and 1 wet season month (July or August) because there is some historic data to indicate that the wet season may be associated with higher levels of indicator organisms. One possible reason is the increase in temperature which is suspected to be conducive for the regrowth of some indicator microbes and the increased run off from contaminated land areas into shallow recreational coastal marine waters. In the case of Hobie Beach, there is also increased outflow during the rainy season from the highly contaminated Miami River. Both beaches are used heavily for all types of water sports throughout the year by Miami natives and tourists (the latter more in the winter months). Population
With approval of Dr. Robert Haile et al. (1999), the Investigators will use the adapted questionnaire and consent form developed for the 1995 Santa Monica Bay Study to recruit and survey beach goers to the 2 study sites in the middle of each of the 2 sampling months. The original questionnaire was validated and administered to over 22,000 recreational beach goers in Southern California in English and Spanish (See Appendix). After obtaining approval from the Human Subjects Committee (IRB) of the University of Miami (application submitted) and the Miami Dade County Health Dept IRB, the Investigators will enroll at least 120 beach goers who immerse their face in the beach water during the sampling periods. To decrease the effects of confounding, the number of enrollment days will be kept to 2 days in each month. On the days of enrollment, after reviewing and signing the Informed Consent, brief demographic and contact information will be collected. Then, as per the 1995 Santa Monica Bay Study (Haile et al. 1999) and per expected incubation periods for enteric diseases, the participants will be contacted by phone 9 days from exposure to ascertain symptoms, particularly gastrointestinal symptoms, since the visit to the beach as well as any additional beach exposures. Any person who immerses their face in the beach water on the date of enrollment and agrees to the telephone follow up in Spanish or English will be eligible to participate. Parents will be interviewed about symptoms for exposed children age 17 and younger. Sampling
The 2 beach sites will be monitored daily for each 1 month period. Monitoring will take place at 2 different sites for each beach for a total of 4 sampling stations. The location of each site will be fixed by GPS (Global Positioning System). The sampling sites will be selected to provide maximal geographic coverage of the study sites. The collection procedures are described in detail in an Appendix. Information concerning antecedent weather conditions will be collected for each site from available rain gauges operated by the South Florida Water Management District. Laboratory Methods
For this pilot study, microbiological recreational water quality will be assessed using multiple bacteriological indicators: fecal coliforms, enterococci, and C. perfringens. In addition to the microbial indicators below, the water will be analyzed for basic physico-chemical parameters including pH, salinity, temperature, and dissolved oxygen using a multi-parameter probe (Model 600R, YSI, Yellow Springs, Ohio) and turbidity using a standard nephelometer (Model 40, Turner Designs, Sunnyvale, California). Enterococci using defined substrate technique (IDEXX, Westbrook, Maine): EnterolertTM reagent is an easy to use method for the detection of enterococci in marine and fresh waters (Budnick et al. 1996). This reagent utilizes nutrient indicators that fluoresce when metabolized by enterococci. Enterococci can be detected in 24 hours after incubation at 41 +or- 0.5 degrees Celsius. Enterococci using the membrane filter(MF) method (USEPA 1997): The MF method provides a direct count of bacteria base on the development of colonies on the surface of the filter. The water sample is filtered through a sterile membrane filter. The membrane containing the bacterial cells are placed on a medium, mEI agar, and incubated for 24 hours at 41 degrees Celsius. After incubation colonies with a blue halo are recorded as enterococci. Fecal Coliforms using membrane filter method (APHA 1995): A water sample is filtered through a sterile membrane that retains the bacteria. The membrane is then placed on a modified mFC agar and incubated for 24 hr at 44.5+or- 0.2 degree Celsius. Blue colonies are recorded as fecal coliform. Clostridium perfringens using the membrane filter(MF) method (USEPA
1995): The MF method provides a
direct count of spores and vegetative cells based on the development of
colonies on the surface of the membrane. The MF is placed on mCP agar and
incubated anaerobically for 24 hr at 44.5 degrees Celsius. The plates are exposed to ammonium
hydroxide fumes and all dark pink colonies are counted as C. perfringens.
Statistical Methods
The
data will be entered into a database, and initially analyzed for frequencies
and distributions. The geometric
means of the water quality indicators will be used in those analyses and
symptoms will be analyzed as dichotomous variables. Sample size Hypothesis 3H3 will be defined as a dichotomous variable (Gastro Intestinal symptoms yes/no). Using Epi Info 6 (CDC) Statistical Software, with an alpha level of 0.10 and power of 0.80, 1:1 exposed (Hobie) versus unexposed (Crandon) in a cross section study design seeking a relative risk of 3.0 for Hypothesis 3H3, then the sample size needed is approximately 60 exposed (Hobie) and 60 unexposed subjects. Due to limited resources, this sample size will be reached by pooling the 2 sampling periods. Sanitary Survey
Discussions have been initiated with the Miami-Dade Department of Health to fund a sanitary survey of the study sites as matching funds toward this proposed NIEHS pilot project. The purpose of the sanitary survey is to identify potential sources of fecal contamination to the beaches through additional field inspections and source-specific water quality measurements. Such a survey is of value given that results can be used to support conclusions and recommendations developed from this proposed NIEHS pilot project. Relevance to NIEHS Center Activities and Environmental
Health Sciences The NIEHS Marine and Freshwater Center is located on Virginia Key, less than 1 mile from the proposed contaminated site of Hobie Beach. As noted above, Hobie Beach is a favorite recreational area for Miami residents and tourists throughout the year, yet it has a long history of poor water quality. Most recently during the winter and spring of 1999-2000, there were numerous reports of inadequate water quality and increased human health complaints to the Miami Dade County Health Dept and the NIEHS Center. The NIEHS Center, as part of Center Outreach and Education (COEP) activities responded to these reports with information and the use of laboratory facilities for storing water samples taken by frequent beach users, particularly wind surfers. Therefore in part this proposed pilot study is in response to COEP activities of the NIEHS Center at the University of Miami. In addition, as described above, this proposed pilot study seeks to gather preliminary data for a larger study proposal to NIEHS and EPA to evaluate the growing problem of recreational water quality monitoring and possible human health effects in the tropical marine environment. Dr. Helena Solo-Gabriele, the Principal Investigator, is an environmental engineer with over 10 years of professional experience in water quality issues particularly in tropical environments, and she directs a water quality laboratory at the College of Engineering of the University of Miami. Mr. Samir Elmir is the Director of the Division of Environmental Health of the Miami Dade County Health Dept, responsible for supervising much of the ongoing recreational water quality monitoring in Miami Dade County. In addition, this pilot study will form the basis of Mr. Samir’s Ph.D. dissertation in civil and environmental engineering, under the supervision of Dr. Solo-Gabriele (dissertation advisor) and Dr. Fleming (dissertation committee member). Dr. Fleming is an epidemiologist and environmental and occupational health expert with 10 years experience in the human health effects of various aspects of the tropical marine environment. Dr. Fleming is also the Director of COEP and Associate Director of the NIEHS Center at the University of Miami. Furthermore, two students from Miami-Dade
Community College (MDCC), Margia Aguello and Wendy Quirino, have shown an
interest in participating on the project.
Both Ms. Aguello and Quirino are NIH-NIGMS (National Institute of
General Medical Sciences) Scholars.
Students enrolled in the NIH-NIGMS program receive an hourly salary
through the NIH-NIGMS program for their research activities. Both Ms. Aguello and Quirino are currently
seeking research projects. It is
envisioned that both of these students could potentially help to administer
the questionnaire and work with the field and laboratory assistant in
collecting and analyzing water samples.
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