Environ. Sci. Technol. 2003, 37, 673-680
Tiered Approach for Identification of a Human Fecal Pollution Source at a Recreational Beach: Case Study at Avalon Bay, Catalina Island, California ALEXANDRIA B. BOEHM Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305-4020 JED A. FUHRMAN Department of Biology, University of Southern California, Los Angeles, California 90089-0371 R O B E R T D . M R Sˇ E † A N D STANLEY B. GRANT* Henry Samueli School of Engineering, University of California, Irvine, California 92697-2575
Recreational marine beaches in California are posted as unfit for swimming when the concentration of fecal indicator bacteria (FIB) exceeds any of seven concentration standards. Finding and mitigating sources of shoreline FIB is complicated by the many potential human and nonhuman sources of these organisms and the complex fate and transport processes that control their concentrations. In this study, a three-tiered approach is used to identify human and nonhuman sources of FIB in Avalon Bay, a popular resort community on Catalina Island in southern California. The first and second tiers utilize standard FIB tests to spatially isolate the FIB signal, to characterize the variability of FIB over a range of temporal scales, and to measure FIB concentrations in potential sources of these organisms. In the third tier, water samples from FIB “hot spots” and sources are tested for human-specific bacteria Bacteroides/ Prevotella and enterovirus to determine whether the FIB are from human sewage or from nonhuman sources such as bird feces. FIB in Avalon Bay appear to be from multiple, primarily land-based, sources including bird droppings, contaminated subsurface water, leaking drains, and runoff from street wash-down activities. Multiple shoreline samples and two subsurface water samples tested positive for human-specific bacteria and enterovirus, suggesting that at least a portion of the FIB contamination is from human sewage.
Introduction Coastal managers are responsible for protecting recreational beach users from exposure to water-borne pathogens. Sources of pathogens include treated and untreated sewage, nuisance and agricultural runoff, and bathers themselves. * Corresponding author e-mail:
[email protected]; phone: (949)824-7320; fax: (949)824-2541. † Present address: RBF Consulting, 14725 Alton Pkwy, Irvine, CA 92618-2027. 10.1021/es025934x CCC: $25.00 Published on Web 01/09/2003
2003 American Chemical Society
Detection of human pathogens in bathing waters, although theoretically the most direct method of assessing a human health risk, is problematic because assays are expensive and time-consuming and many pathogens are, as of yet, unidentified (1). Fecal indicator bacteria (FIB) are used as surrogates for pathogens in assessing water quality throughout the world because they are present in high concentrations in sewage and urban runoff (2), epidemiological studies have documented an increased risk of contracting gastrointestinal and respiratory illnesses after recreating in waters with elevated concentrations of FIB from sewage and urban runoff (3-7), and standardized inexpensive assays are available for their detection. The FIB groups most commonly used as indices of water quality are total coliform (TC), fecal coliform (FC), Escherichia coli (EC) (which is a fecal coliform), and enterococci (ENT) (2). Despite widespread implementation of FIB monitoring programs, recent studies have identified a number of problems associated with their use. Researchers have documented that FIB are present in the feces of a wide variety of warm-blooded animals (2), including sea gulls (8) and pigeons (9) that congregate near shorelines. In addition, the growth of FIB in soils in Hawaii (10), Guam (11), and Florida (12) and on marine vegetation in New Zealand (13) has been reported. The absence of human pathogens and zoonoses associated with FIB from these sources confound their use as indicators of human pathogens. Novel human-specific chemical and biological fecal indicators have been proposed including fecal sterols, caffeine, male-specific bacteriophage, enteric virus, and enteric bacteria such as Bacteroides/Prevotella (2). In particular, PCR-based nucleic acid detection techniques that take advantage of the unique genomic RNA or DNA sequences of human viruses or rDNA sequences of human-specific bacteria are becoming increasingly popular. Interpreting positive results from the gene-based tests is not straightforward because the presence of a specific nucleic acid sequence in a water sample does not definitively prove that the organism with which it is associated was present in the sample, viable, and (if pathogenic) infectious. In general, interpreting positive results from any of the novel indicators is difficult because there is no epidemiological evidence to support their use, and thus it is yet to be seen how well they relate by themselves to risk of infection. However, by analogy to FIB, we might consider these novel indicators as specific indicators of the recent or current presence of human fecal material and its associated microbes. The identification and mitigation of coastal FIB sources is complicated by the multiple spatially and temporally variable sources of FIB from both human and nonhuman sources, the highly dynamic currents that transport them, and the complex biological and physicochemical processes that influence the rate at which they are removed from the water column (14). In this study, we describe a three-tiered approach for determining sources of human and nonhuman FIB at a recreation beach that utilizes both standard assays for FIB and novel detection techniques for human-specific bacteria Bacteroides/Prevotella and enterovirus. The first tier documents the spatiotemporal variability of the pollution signal and takes into account the possible influence of sunlight and tides on FIB concentrations in coastal waters. The second tier consists of source studies. Studies in the first two tiers identify pollution sources and “hot spots” using only standard FIB tests. The third and final tier consists of selectively sampling FIB sources and hot spots for the enteric bacteria Bacteroides/Prevotella and enterovirus using nucleic VOL. 37, NO. 4, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
673
FIGURE 1. Avalon Bay is on the southeast side of Catalina Island, which is located off the coast of southern California. Sampling sites are shown in the bottom panels. An expanded view on the lower right shows the sites sampled twice daily for an entire month.
acid detection techniques to determine if FIB are of human origin. This study illustrates how measurements made with traditional indicators, in conjunction with more novel indicators, can successfully lead to source identification and mitigation.
Field Site The impacted coastline is a 500-m stretch of sandy beach located in Avalon Bay on the southeast side of Catalina Island (area 200 km2), at approximately 33°20.9′ N, 118°19.5′ W (Figure 1). The beach is tide energy dominated with the ratio of tide range to wave height typically greater than or equal to 1. Two breakwaters surround the entrance to Avalon Bay; however, it has relatively good circulation based on the rapid (1 h) dissipation of dye patches introduced into its waters during the summer of 2001 (16). Avalon (area 6.9 km2) is the largest town on the island with 3500 year-round residents. The city’s primary source of revenue is tourism (17); on a typical summer day 17 500 tourists arrive via ferry, cruise ship, or personal vessel, and up to 400 vessels are anchored in the bay. Rainfall in this region occurs primarily from November through March (18), and consequently, during our summer-time study, there was no rainfall. As is the case for virtually any coastal community, there are many potential sources of fecal contamination in Avalon Bay. Sewer trunk lines run parallel to the beach, approximately 20 m from the shoreline. Nuisance runoff is directed into the sewer system by low-flow diverters; however, some of the runoff enters the ocean untreated through small drains that discharge to the sand, particularly during periods when streets are being washed down by City staff (19). Secondary treated sewage is released at a rate of approximately 2158 m3 d-1 southeast of the bay through an outfall that terminates 100 m from the coast at a depth of 65 m. A pier with restrooms, restaurants, and recreational 674
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 4, 2003
establishments extends from the shoreline near the southeast end of the beach. A wharf with restaurants overhangs a portion of the beach (Figure 1). In addition, pigeons and sea gulls congregate to feed and nest near the shoreline. Avalon Beach is tested weekly for FIB at six stations: Tuna, Busy Bee, Middle, Pier, South, and Channel (lower right panel of Figure 1, red asterisks). If FIB levels at these sites fail to meet any of the seven marine bathing water standards set by California Department of Health Services (CDHS), the beach must be posted with signs that warn beachgoers to stay out of the water, and the beach is closed to the public if the health officer has reason to believe that the water is contaminated with sewage. The CDHS standards for FIB in marine bathing waters are as follows. A single sample of water must not contain more than 10 000, 400, or 104 MPN/ 100 mL of TC, FC, and ENT, respectively, and the geometric mean of samples collected within the prior 30 days (the geometric mean standard) cannot be greater than 1000, 200, or 35 MPN/100 mL for TC, FC, and ENT, respectively. In addition, if the TC/FC ratio falls below 10, then TC levels may not exceed 1000 MPN/100 mL. During the summers of 2000 and 2001, water samples from Avalon Beach frequently exceeded the single sample standard for ENT, and thus, signs were posted at the beach warning swimmers not to enter the water. City officials were not able to readily identify and remedy the pollution source, and thus the present study was commissioned. At the outset of this study, it was not clear to what extent the following potential sources impacted water quality in Avalon Bay: effluent from the sewage treatment plant, nuisance runoff, feces of birds and other wild animals, contaminated subsurface water, and boat sewage collection tanks. The latter is not expected to contribute much to the pollution because the city has an aggressive dye program to reduce illicit discharges into the bay.
Materials and Methods Tier One: Pollution Patterns. (a) Bay Survey. To pinpoint the spatial extent of the pollution and determine if FIB sources were located inside or outside the bay, we conducted a water quality survey at a series of shoreline and offshore sites inside and outside of Avalon Bay (lower left panel of Figure 1, blue solid circles). The sampling period (1900 to 2100 on September 19, 2001) was chosen to coincide with the beginning of a flood tide so that the net movement of bay water would be toward the shoreline. In addition, sampling commenced after sunset so that effects of sunlight on FIB survival and/or detection would not affect their measured concentration (20). Offshore and shoreline sampling was initiated in the southern portion of the study area and progressed northward with the first samples located directly over and shoreward of the wastewater outfall. Ankle depth samples and surface samples were collected from all shoreline and offshore sites, respectively. In addition, a subset of shoreline and offshore sites were sampled respectively at waist and 1-m depth. All samples were collected in 500-mL Nalgene bottles, immediately placed on ice, and delivered to the laboratory within 6 h. Sample pH (Thermo Orion 720A+ pH probe, Beverly, MA), salinity (Thermo Orion 162A conductivity and temperature probes, Beverly, MA), and turbidity (HF Scientific DRT-15CE, Fort Meyers, FL) were recorded, and FIB analyses were performed using Colilert-18 (TC, EC) and Enterolert (ENT) defined substrate tests (IDEXX, Westbrook, MN, implemented in a 97-well Quanti-Tray). The Colilert and Enterolert systems were chosen because they are utilized for Avalon’s routine water quality monitoring program. Throughout the study, we compared the concentration of EC (from Colilert-18) with the CDHS standard for FC. Because the FC group contains strains of Klebsiella, Enterobacter, and Citrobacter in addition to EC and other strains of Escherichia (21, 22), the EC level in a water sample is always less than or equal to the corresponding FC level. However, EC is the only FC that is almost always of fecal origin (10, 23). (b) Cross-Shore Transect. To investigate whether sediment within the bay could be a source of FIB, we collected sediment samples and samples of the overlying water column at stations extending from ankle depth at the shoreline to waters approximately 10 m deep (lower left panel in Figure 1, green asterisks). Sampling commenced during a falling tide in the early afternoon on September 18, 2001. At each station a diver collected 1 kg of sediment from the sediment/ water interface and approximately 1 L of water at a series of depths in the overlaying water column, including the surface. Water and sediment samples were placed on ice and transported to the laboratory in under 6 h where they were analyzed for physical characteristics and FIB. Sediment samples were processed by suspending 50 g of sediment in 450 mL of phosphate-buffered saline (PBS) solution, shaking gently, allowing the suspension to settle for 10 min, and then drawing off 10 mL of the supernatant for FIB analysis. All measurements were conducted as described above. (c) Twenty-Four Hour Study. To characterize the diurnal variability in the concentration of FIB, water samples were collected from two shoreline sites (S1 and S11) and one offshore site (P) every other hour for 24 h (lower left panel of Figure 1, purple triangles). Stations S1 and S11 are located shoreward of the wastewater outfall and near the center of Avalon Beach, respectively. Station P is located 20 m away from the shore at the end of a floating dock, which is connected to the pier. At the shoreline sites, both ankle and waist depth samples were collected. At station P, surface and 1-m-deep samples were collected. The study commenced at 1800 h on September 20, 2001. The samples were immediately placed on ice and transported to the laboratory where they
were analyzed for physical characteristics and FIB following procedures outlined above. (d) Month-Long Twice Daily Shoreline Survey. Day-today changes in the FIB levels were characterized during a month-long intensive sampling study. Water samples were collected from the six shoreline sites routinely monitored by the City of Avalon (lower right panel of Figure 1; Channel, South, Pier, Middle, Busy Bee, and Tuna) twice daily from September 25 through October 20, 2001. Shoreline sites were sampled at ankle and waist depths in the morning at 900 h and only at ankle depth in the evening at 1800 h. After being collected, the samples were immediately placed on ice, transported to the laboratory, and analyzed for physical characteristics and FIB. Each morning and evening after sample collection, we dropped an orange from the pier to obtain a crude measure of littoral drift. A velocity was calculated by estimating the distance the orange traveled in 30 min. We know from a previous circulation study (16) that water in Avalon Bay undergoes a net clockwise or counter-clockwise rotation. We estimated the direction of rotation using the orange as follows. If oranges drifted in the northeast to northwest direction (315-45°), then surface waters in the bay were designated as rotating in a clockwise direction; otherwise, circulation was designated as counter-clockwise. Tier Two: Source Investigations. Grab samples of nuisance runoff, sea gull feces, subsurface water, cooling water discharge from vessels, and discharge from leaking pipes under the pier and wharf were collected at various times and locations. Samples were stored on ice and transported to the lab where they were analyzed for pH, turbidity, salinity, and FIB utilizing the same methods as described for the tier one investigations. Bird feces were processed by suspending feces in 500 mL of PBS and then following the same procedure used for sediment processing described in the cross-shelf study. To test subsurface water, five 2-m-deep trenches were dug approximately 20 m landward of the shoreline (lower right panel of Figure 1, open triangles). The 500-mL samples were collected from the five trenches daily during the monthlong sampling effort and from trench G2 every other hour during the 24-h study. The pH, turbidity, salinity, and FIB levels were measured in each sample as outlined previously. Tier Three: Selective Human Bacteria and Virus Analysis. The goal of this tier was to determine if some portion of FIB in the bay is associated with human fecal pollution using nucleic acid based assays. Accordingly, we analyzed water samples from locations that were determined to be FIB hot spots or sources on the basis of results from the tier one and two investigations for human-specific bacteria Bacteroides/ Prevotella and enterovirus as described below. (a) Human-Specific Bacteria. Bacteria from water samples were collected by filtration of 1-4 L (depending on amount that could be filtered without clogging; for subsurface water, the sample volume was typically 40-150 mL) through 47 mm diameter, 0.22 µm pore size Durapore filters or 20 L through 142 mm Durapore filters (Millipore Corporation, Bedford, MA). No prefilters were used. Different types of samples required different DNA extraction procedures in order to minimize inhibition by humic acid-like substances. DNA from seawater was extracted by hot 1% SDS lysis and purified by phenol extraction (24), while effluent, subsurface water, and fecal samples were extracted by commercially available kits [Qiagen stool kit (Valencia, CA) or Bio 101 soil kit (Qbiogene, Carlsbad, CA)] following manufacturer’s instructions. DNA concentrations were measured by Pico Green fluorescence with a Bio-Rad fluorometer (Hercules, CA). Amplification of the human-specific Bacteroides/Prevotella marker followed the procedure of Bernhard and Field (25) VOL. 37, NO. 4, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
675
FIGURE 2. Results from the bay survey. Marker size is proportional to the log concentration of FIB with units MPN/100 mL in surface (offshore sites) and ankle-deep (shoreline sites) waters. Red, green, and black circles represent TC, EC, and ENT, respectively. Red arrow highlights a hot spot where FIB levels were most elevated. The inset shows locations of sites where samples were also analyzed for the human-specific bacteria and/or enterovirus. using the PCR primers that amplify partial rDNA that encodes 16S rRNA from the human fecal specific group. Most amplifications were from 1 and 10 ng of extracted DNA, equivalent to about 5-50 mL of seawater, chosen to provide an optimal compromise between sensitivity and avoidance of inhibition of the assay. All sets of assays included negative controls (no DNA added) and positive controls in which a small amount (1-100 pg) of human fecal DNA extract was added to replicates of the field samples to see if reactions were inhibited by substances from the sample. Only one analytical run had negative controls showing a positive result, and when the run was repeated, the negative control was negative. Any negative result from the positive controls rendered the overall result for that sample inconclusive. Inconclusive samples were re-run with less DNA amplified in an attempt to reduce inhibition. The PCR method utilized here successfully amplified the human marker from 24 192 24 192 >24 192 19 863 >2 419 200 17 300
EC
ENT
>24 192 >24 192 >24 192
>24 192 19 864 >24 192 >24 192
