Evaluating the dilution of wastewater treatment plant effluent and viral impacts on shellfish growing areas in Mobile Bay, Alabama.
Subject: Water treatment plants (Health aspects)
Sewage (Purification)
Sewage (Health aspects)
Authors: Goblick, Gregory N.
Anbarchian, Julie Mayer
Woods, Jacquelina
Burkhardt, William, III
Calci, Kevin
Pub Date: 12/01/2011
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2011 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2011 Source Volume: 30 Source Issue: 3
Organization: Government Agency: United States. Food and Drug Administration
Accession Number: 278595643
Full Text: ABSTRACT The U.S. Food and Drug Administration (FDA) provides guidance to state shellfish control authorities on establishing prohibitive closure zones in proximity to wastewater treatment plant (WWTP) discharges with the purpose of minimizing the exposure of molluscan shellfish to health hazards posed by bacterial and viral pathogens present in wastewater effluents. For more than 25 years, the FDA has recognized conditional area management as an option to minimize the size of a prohibitive closure zone, and to enlarge the size and productivity of shellfish growing areas. To use this option, the FDA has recommended achieving a 1,000:1 dilution of effluent within the perimeter of the prohibited closure zone. Using newly available analytical methods and hydrographic equipment, the FDA is undertaking studies to determine whether its 1,000:1 dilution recommendation is supported by the findings. From 2007 through 2009, the FDA conducted field investigations to assess the impacts of wastewater effluent from a large municipal WWTP that discharges into Alabama's Mobile Bay. The dilution of the effluent in the bay was ascertained by conducting a hydrographic dye study using rhodamine WT tracer dye. Submersible fluorometers fastened to oyster cages at sentinel stations were used to determine continuously the dilution of the dye-tagged effluent throughout a 4-day study period. In addition, dilution and dispersion of the dye-tagged effluent was tracked throughout Mobile Bay by fluorometric measurements made while conducting boat transects. The microbiological impacts of the wastewater on molluscan shellfish were assessed by testing oysters placed in cages at sentinel stations at various distances along the anticipated path of the effluent. Levels of fecal coliforms, Escherichia coil, male-specific coliphage, and norovirus genogroups I and II were determined. Norovirus genogroup II was detected in oysters that were located as far as 5.74 km from the discharge, an area in close proximity to the calculated 1,000:1 dilution line. Results also showed that the levels of indicator microorganisms and viral pathogens in the shellfish inversely correlated with increased dilutions of the wastewater effluent in Mobile Bay.

KEY WORDS: wastewater, shellfish, growing are& treatment plant


State shellfish control authorities are keenly aware of the increased public health threats posed by wastewater treatment plant (WWTP) failures, bypasses, and combined sewer overflows that release untreated or minimally treated human sewage directly into estuary receiving waters. However, it has been demonstrated that even under normal WWTP operation, human enteric viruses are not eliminated as effectively as bacterial pathogens and indicators during wastewater treatment (Baggi et al. 2001, Burkhardt et al. 2005).

Bivalve molluscan shellfish feed on algae, detritus, and other particulates from the surrounding water, and during this feeding process each animal can filter up to 3.9 L/h (Galtstoff 1964). Filter-feeding shellfish have been reported to concentrate coliphage S-13 at levels up to 1,000-fold greater than levels found in the overlying water (Canzonier 1971). Likewise, the typically reported bioaccumulation of enteric viruses (e.g., norovirus (NOV), poliovirus) in shellfish is up to 100-fold greater (Seraichekas et al. 1968, Maalouf et al. 2011). The ability to concentrate contaminants varies, depending on shellfish species, microorganism type, environmental conditions, and season (Bedford et al. 1978, Metcalf et al. 1979, Burkhardt et al. 1992, Enriquez et al. 1992, Burkhardt & Calci 2000). Of significance to public health officials, molluscan shellfish retain viruses to a greater extent and for longer periods than the bacteria relied on as indicators of sanitary quality (Sobsey et al. 1987, Dore & Lees 1995).

Recognizing that enteric viral pathogens, including NoV and hepatitis A viruses, can persist in estuarine and marine waters and within shellfish from a few days to several months (Bosch 1995, Callahan et al. 1995) and that these pathogens are bioaccumulated by shellfish, the occurrence of significant levels of enteric viruses being discharged in sewage effluent can pose a considerable health hazard to shellfish consumers.

The U.S. Food and Drug Administration (FDA) has long recommended a minimum 1,000:1 dilution of estuarine water to treated wastewater effluent when authorities establish conditionally approved and restricted shellfish growing area classifications adjacent to WWTP discharges, and that waters with dilutions less than 1,000:1 be classified as prohibited to the harvest of shellfish. Recent advances in the detection and enumeration of enteric viral pathogens and the use of fluorescent dye tracing technologies have expanded and enhanced capabilities to assess comprehensively the potential impacts of viruses on shellfish in growing areas exposed to WWTP effluent. Using these advanced technologies, we conducted several studies to assess the efficacy of the minimum 1,000:1 dilution guidance to maintain the safety of molluscan shellfish. The 2007 to 2009 Mobile Bay, AL, study presented here is one such study to assess its efficacy. This investigation was initiated to assess the impact of sewage effluents from the Clifton C. Williams WWTP discharged in upper Mobile Bay, AL. This WWTP facility serves approximately 200,000 people in Mobile County, and discharges treated and disinfected effluent at an average rate of approximately 75 million L/day or 20 million gallons/day.


Preliminary Investigation

A preliminary investigation was conducted in March 2007 using drogues to estimate WWTP effluent dispersion and indicator bacteria to assess the efficiency of the WWTP. Winged drogues (constructed of PVC pipe and aluminum sheeting) and citrus fruits (oranges and grapefruits) were released at the WWTP sewage outfall to estimate the location and extent of WWTP effluent dispersion in Mobile Bay. These releases occurred at the beginning of an ebb tide, and dispersions of the drogues and fruits were monitored for 1 tidal day. Four sentinel shellfish and fluorometer station locations were established based on the observed drogue paths and plume dispersion models developed by FDA engineers. When determining station locations, consideration was also given to available structures (e.g., buoys) needed to deploy electronics and shellfish safely. Soon after establishing the shellfish sentinel stations, the FDA began monitoring oysters placed in the cages at these locations to assess the microbiological impacts from the Mobile WWTP effluent. Levels of indicator bacteria (fecal coliforms (FC) and Escherichia coli) and male-specific coliphage (MSC) were determined in the oysters and in WWTP influent and effluent samples to assess the efficiency of the WWTP in removing or inactivating these indicator bacteria and MSC.

Hydrographic Dye Study

In March 2008, a hydrographic dye dispersion and dilution study using rhodamine WT tracer dye was conducted. The dye was injected into the WWTP's final effluent as the effluent was being discharged into receiving waters. Dye measurements were then recorded to determine the buildup of pollution from the WWTP and the overall steady-state dilutions of the WWTP effluent in Mobile Bay.

The superposition method (Hubbard & Stamper 1972, Kilpatrick & Cobb 1985) was chosen to overcome the challenges of studying the steady-state impact from a WWTP with a large flow rate (75 114 million L/day). The steady-state condition represents the impact from the WWTP's normal operating status and is achieved when the rate of WWTP effluent entering the bay is equal to the rate at which the effluent is being flushed from the bay by tides. An empirical determination of the steady-state impact from the WWTP would have entailed a 3 to 4-day dye injection with more than 200 L of dye--a length of time and a volume of dye that were considered impractical. Shortening the duration of the dye injection from several days to 1 tidal day (24.8 h) using the superposition method made the dye amounts and the injection times acceptable. Use of this method allowed data collected on tidal days after termination of the injection to be superimposed with the data collected on the first tidal day, providing cumulative data. The cumulative measurements from each tidal day reflect the overall peak concentrations that likely would have been obtained in the estuary if the tracer dye were continuously injected until steady-state conditions were achieved in approximately 4 tidal days.

Dye injection commenced on March 1, 2008, at approximately 6:45 AM. This single tidal day (24.8 h) injection required 75 L rhodamine WT tracer dye to achieve a concentration in the WWTP effluent that would account for instrument sensitivity when measuring concentrations in the estuary. The 75-L volume of dye was evenly mixed with 75 L deionized water in a 378-L polyethylene container. A Masterflex high-pressure L/S peristaltic pump (Cole-Parmer, Vernon Hills, IL) was used to recirculate the dye/water mixture throughout the injection period, and a second peristaltic pump of the same type was used to inject the dye/water mixture into the waste stream at a constant rate of 153 mL/min, achieving an average dye concentration of approximately 174 ppb in the WWTP effluent during the 24.8-h injection period.

WET Laboratories FLRHB submersible fluorometers (WET Laboratories Inc., Philomath, OR) positioned at the 4 station locations recorded fluorescence once every 5 min, at 1-sec intervals, for a duration of 1 min. The fluorometers were calibrated for a range of 0-230 ppb and had a sensitivity of 0.01 ppb. The fluorometers began logging data approximately 24 h prior to the start of the dye injection to capture background readings. Data logging continued for an additional 4 tidal days until dye concentrations at all station locations approached background levels. Dye levels also were ascertained around the cages and in surrounding waters using a Turner Designs 10-AU field fluorometer (Turner Designs Inc., Sunnyvale, CA) aboard a boat used to track the dye-tagged WWTP effluent during daylight hours on the day of the injection and during the next 4 tidal days. The Turner Designs 10-AU and WET Laboratories instruments were calibrated using the same dye standards. The Turner fluorometer had a calibration range of (0-100 ppb with a sensitivity of 0.01 ppb.

The determination of steady-state dilution was considered in three ways. The first approach considered the entire data set, for which the average dye concentrations per tidal day were determined. The second approach considered the highest instantaneous peak value measured at each station. For stations where pollutant buildup was not viewed as significant, this approach was considered to be the most conservative assessment with respect to a final dilution estimate. The third approach considered the peak l-h average concentrations on each tidal day, representing an hour when, at steady state, the expected exposure level will be greatest. Regression analyses of dilution versus distance from the WWTP outfall were performed considering the tidal day average, the highest instantaneous peak values, and the peak 1-h average concentrations for the shellfish stations to ascertain the approximate distance from the outfall at which a 1,000:1 dilution occurs. ArcGIS version 9.3 with Spatial Analyst (ESRI, Inc., Redlands, CA) was used to produce a contour map of the dye-tagged effluent to visualize the path of the dye and the relationship of dye concentration to distance from the outfall.

Indicator Microorganisms and Norovirus Analysis

In 2007, the year prior to the hydrographic dye study, caged oysters were deployed at four sentinel station locations as part of a preliminary study. After 6 weeks, the oysters were collected and analyzed for FC, E. coli, and MSC, as previously reported by Daskin et al. (2008). Additional testing for MSC was conducted on caged oysters from these locations throughout the course of the hydrographic dye study in March 2008 and again from September 2008 to March 2009. FC and E. coli levels in the oysters were determined using a 5-tube, 3-dilution MPN series as described by the American Public Health Association (1970, Rippey et al. 1987). MSC levels were determined using a double-agar overlay protocol using E. coli Famp as the host strain (Cabelli 1988).

NoV genogroup I (GI) and genogroup II (G I l) were detected and quantified in the shellfish using previously described extraction and detection methodologies (Burkhardt et al. 2004, Depaola et al. 2010, Woods 2010).


Hydrographic Dye Study

Table 1 shows peak and steady-state dye concentrations and dilution values at stations 1 to 4. These values were determined using data collected by the submersible fluorometers at the sentinel stations. Kilpatrick (1993) noted that decay rates for rhodamine WT are up to 5% per day in rivers and up to 3% per day in estuaries. Others have shown that rhodamine WT has a range of photochemical decay of approximately 2-4% in both field and laboratory tests (Hetling & O'Connell 1966, Tai & Rathbun 1988). To account for potential loss of dye from these factors, a value of 3% loss per tidal day was factored into dilution calculations, based on findings from field and laboratory studies (Hefting & O'Connell 1966, Tai & Rathbun 1988, Kilpatrick 1993). Because the largest dye readings occurred on the first tidal day, when little decay took place, the overall superposition values when accounting for dye loss were minimally affected. For example, accounting for the dye loss would result in an overall tidal day average steady-state dilution at station 4 of 556:1 (with dye decay), compared with 570:1 (without dye decay). The dilution values presented in Table I and the FDA's 1,000:1 dilution line estimates account for dye loss.

Dye concentration and superposition curves for each sentinel station's data set are provided in Figures 1-4. Figure 1 illustrates that station 1 reached a maximum concentration of 128.31 ppb, achieved shortly after dye injection commenced. However, dye concentrations were quickly reduced to trace amounts shortly after the dye injection concluded on the second tidal day, indicating that the dye-tagged effluent was quickly flushed from this area of Mobile Bay. This rapid change is attributable to the strong and continuous push of the freshwater inflow from the Mobile River. Therefore, little dye buildup occurred at station 1, as indicated by the relatively flat superposition lines on Figure 1. The overall steady state dilution was 5.44:1 based on the tidal day average or 1.49:1 based on the peak l-h average concentrations. The lowest dilution value for the first tidal day was determined to be 1.36:1.

The maximum concentration of rhodamine WT dye at station 2 was 63.58 ppb (Table 1). This was achieved 73 rain after the peak dye concentration was obtained at station 1. Knowing that stations 1 and 2 were 0.5 km apart from each other, the authors calculated that the dye was traveling at a rate of 0.4 km/h. On the tidal days after the day of the injection, higher amounts of dye were measured at station 2 than station 1. However, a minimal buildup of dye occurred at station 2, which is attributed to the freshwater input from the Mobile River and tributaries. The overall steady-state dilution of the dye based on the tidal day average for station 2 was 8.85:1, the peak 1-h average concentrations corresponded to a 2.76:1 dilution, and the lowest dilution value for the first tidal day was 2.74:1.

Dye readings measured at station 3 (Fig. 3) were significantly lower than had been anticipated, indicating that this station had been poorly positioned with respect to the concentrated center of the dye plume's path. This station's positioning was based in large part on proximity to a nearby mooring site, because it was important that caged oysters and a fluorometer be easily moored and protected from boat and barge traffic. However, in this investigation, the plume of dye traveled in a more westerly direction than anticipated. Subsequently, the waters around this site, east of the navigation channel, only briefly received minute traces of dye (<0.4 ppb) during the first tidal day. One and a half tidal days after the dye injection concluded, though, dye was detected continuously at this site, with a peak concentration of 0.32 ppb (Fig. 3). These data suggest that station 3 was influenced primarily by water associated with a flood tide on tidal day 3, a portion of which was dye-tagged wastewater effluent that was pushed back up into Mobile Bay. As shown in Table 1, the overall steady-state dilution for station 3 based on the tidal day average was 5,387:1, and based on the peak l-h average concentrations was 820:1. The lowest dye dilution value for the third tidal day was 501:1.

The leading edge of dye-tagged sewage effluent reached station 4, about 5.6 km from the discharge, approximately 11 h after the dye injection commenced. A peak dye concentration of 1.00 ppb was achieved around 11 PM at that station on the first tidal day (Fig. 4). In comparison with stations 1 and 2, significantly larger amounts of dye were measured at station 4 on tidal days 2 through 4, indicating that station 4's location was not as heavily influenced by water originating from the Mobile River. The average dye concentration on the first tidal day was 0.18 ppb and the peak 1-h average concentration was 0.36 ppb. Because more dye reached station 4 on tidal days 2-4 than on the first tidal day of the study, the overall steady-state average concentration at station 4 (0.31 ppb) was 66% higher than the first tidal day average. Similarly, the overall steady-state concentration for the peak 1-h averages was 1.07 ppb, a value 195% higher than that observed on the first tidal day.


The larger buildup of dye at station 4, evidenced by continuous dye readings at that station (Fig. 4), is attributed to a significant mass of dye remaining in the vicinity of station 4 for more than 3 tidal days after the termination of the dye injection. The overall steady-state tidal day average dilution of dye at station 4 was 556:1, whereas the steady-state peak l-h average dilution was 158:1 (Table 1). The lowest dilution at station 4, based on the peak concentration value measured on the first tidal day, was 173:1. Thus, of all the stations, only station 4 exhibited a lower steady-state peak l-h average dilution than the lowest dilution found at that station on the day of the dye injection. This indicates that dye-tagged sewage effluent remained in Mobile Bay for several days, and that steady-state concentrations in other parts of the bay may be significantly greater than even the highest initial concentrations measured in this study. Overall, these findings demonstrate that for estuaries, the buildup of pollutants is an important factor to consider in shellfish growing area management, particularly because some estuaries are even more susceptible to buildup than Mobile Bay.



A linear regression analysis was performed to establish the relationship between tidal day average dye concentrations (stations 1, 2, and 4) and distance from discharge (Fig. 5). Dye concentrations determined at station 3 were not used in this analysis because of its poor location in relation to the center of the dye plume, which was confirmed by data collected by a towed plume-tracking fluorometer. As shown in the ArcGIS-generated map in Figure 6, which represents all the plume tracking data collected on tidal days 1-3 during daylight hours, it is evident that station 3 is not well aligned with respect to the higher dye concentrations that passed station 2 and then tracked to the west of station 3. When the results from station 3 are excluded, the data indicate that the steady-state 1,000:l dilution would be achieved at 6.32 km from the WWTP discharge based on the tidal day average concentrations and at 7.71 km based on the 1-h average of tidal day peak concentrations (Table 1). The 1,000:1 dilution of estuarine water to dye based on the highest values measured at stations 1, 2, and 4 occurred at a distance of 7.89 km from the WWTP discharge (Fig. 6).




Illustration of the 1,000:1 dilution line based on the highest value measured by boat-towed plume-tracking fluorometers is provided in Figure 6, and is the farthest one from the WWTP's outfall. Nevertheless, it is reasonably close to the 1,000:1 dilution line determined based on the highest values measured by submersible fluorometers at the fixed station locations. The small discrepancy in the results between the boat-towed tracking fluorometers and submersible fluorometers is attributed to the fact that boat-towed tracking enables researchers to search for and target the greatest concentration of dye, whereas submersible fluorometers are stationary. The boat-towed fluorometers also provided a broader understanding of the dispersion and shifting location of the dye-tagged wastewater effluent plume, served as a backup source of dilution data, and verified that sentinel station locations could be better positioned in future studies, as in the case of station 3. However, boat tracking was only conducted during daylight hours, and the continuous data obtained from the submersible fluorometers provided a more complete set of dye readings over time to determine dilutions of wastewater in the vicinity of the sentinel shellfish stations and to calculate steady-state dilutions.


MSC Accumulation Study Results

MSC has been used effectively as surrogates of sewage-borne enteric viruses and NoV (Dore & Lees 1995, Dore et al. 2000). In these studies, MSC served as a suitable surrogate for viral contamination from human sewage effluent in the wastewater and shellfish.

Overall, the dye tracking study results compared well with the 2007 FDA analysis of MSC in sentinel oysters (Daskin et al. 2008). Levels of FCs, E. coil. and MSC in oysters at each of the 4 stations are presented in Figure 7. MSC densities at stations 1,2, 3, and 4 were 2,296 pfu/100 g, 1,643 pfu/100 g, 197 pfu/100 g, and 171 pfu/100 g, respectively. In contrast, FC levels at stations 1,2, 3, and 4 were 540/100 g, 330/100 g, 130100 g, and 330; 100 g, respectively. Similar levels of these organisms in oysters relocated near stations 1 and 2 were previously reported in an earlier study (Shieh et al. 2003). This study demonstrates a significant inverse relationship between decreasing MSC levels in the shellfish and increasing wastewater dilution (Fig. 8), which is in turn strongly associated with increasing distance from the WWTP discharge (Fig. 5). These data demonstrate that continuous exposure of oysters to wastewater greatly increases the likelihood of their accumulation of enteric viruses, as demonstrated by the bioaccumulation of MSC, the enteric virus surrogate, in shellfish in close proximity to WWTP discharges. The results of this study also suggest that measuring FCs and E. coil in oysters provides insufficient insight into the potential risk of enteric virus accumulation in shellfish.


As previously noted, Figure 8 shows the relationship between MSC levels in the shellfish and wastewater dilution. The results from 4 separate study periods are presented in the figure July 2007, March 2008 (the time of the hydrographic dye study), December 2008, and February 2009. The linear regression analysis in Figure 8 shows the MSC levels that are projected to occur in shellfish placed at a 1,000:1 dilution line located past station 4 (i.e., the estimated 1,000:1 steady-state dilution lines in Fig. 6). Based on the mean value of the highest MSC results for each of the 4 study periods, MSC levels in oysters at a projected 1,000:1 line would be approximately 46 pfu/100 g. A linear regression analysis for the results from the February 2009 study period is also plotted in Figure 8 because that period had the highest MSC levels of any period. Based on the highest levels found in oysters sampled in February 2009, MSC levels at a projected 1,000:1 line would be approximately 132 pfu/100 g.

In 2009, the Interstate Shellfish Sanitation Conference (ISSC) officially recognized the utility of MS(7 as an indicator of viral contamination from sewage and established a critical limit of 50 pfu/100 g of MSC, The MSC results in Figure 8 demonstrate that even though the mean MSC level of 46 pfu/ 100 g at the projected 1,000:1 dilution line is below the ISSC critical limit for MSC, there are times of the year when the critical limit is exceeded at the projected 1,000:1 line.

Viral Accumulation Study Results

A summation of the occurrence and levels of NoV GI and GII, and MSC in oysters during March 2008. the timeframe of the comprehensive hydrographic dye study, and additional months of investigation at each of the 4 stations is provided in Figure 9 (Woods 2010).

Overall, the dye dilution analysis demonstrated comparable rest, Its with viral accumulation in shellfish analyzed during the same period (March 2008). A comparison of Figures 1,2, and 9 shows that stations 1 and 2 exhibited the highest dye concentrations and also had the highest levels of NoV detected in oysters at these stations (1,340 and 3,700 RT-PCR, respectively). Station 4 received a lower level of dye and had a lower level of NoV (28 RT-PCR units/100 g during the March 2008 investigation (Fig. 9)). While oysters from station 1 accumulated viruses to a lesser extent than those at station 2, oysters at both stations exhibited a substantial bioaccumulation of viruses discharged in the WWTP effluent. Although station 1 was located only about 70 m from the outfall discharge, the lesser extent of virus accumulation suggests that the caged oysters at this site may have been under greater physiological stress from continuous exposure to the predominantly freshwater associated with the effluent and Mobile River, and perhaps residual chlorine residue present in the effluent. As described earlier, a limited amount of the dye-tagged wastewater was detected at station 3. This finding is consistent with the analytical results that determined that NoV could not be detected in oysters at this location.


Continuous dye measurements obtained during March 2008 show that the WWTP effluent was generally more diluted at station 3 than at station 4, indicating that the oysters at station 4 were more consistently under the influence and in higher concentrations of the WWTP effluent than oysters at station 3 (Figs. 3 and 4). As previously described, the concentrated center of the dye traveled to the west of station 3 on the day of the injection and did not influence that station as much as the other stations. Because the evidence shows that the WWTP effluent was generally more diluted at station 3, even though closer to the discharge location, emphasis should be placed on the results for station 4 when estimating dilutions that achieve microbial reductions.

Figure 9 shows 2 survey periods where NoV GII was detected in oysters at station 4 (March 2008 and December 2008). The levels of NoV for March and December 2008 were determined as most probable number (MPN) values to be 28 RT-PCR units/100 g (CI, 3.9 200) and 65 (CI, 8.4 490) RT-PCR units/100 g, respectively. Even instances when the results of shellfish analyses for NoV indicate that less than 10 RT-PCR units,100 g are present, thorough consideration of all available information from other procedures, or other indicator levels, and from the various conditions and circumstances occurring just prior to sampling still is needed to assess reasonably the potential threat from NoV. There have been reported NoV outbreaks for which less than 10 RT-PCR units/ 100 g were found in samples of the implicated shellfish using extrapolation off the standard curve. Although comparing standard curve values and MPN values is not standard practice, side-by-side comparison of standard curve values and MPN values showed 94% agreement (Woods 2010). In this study, the repeatable positive NoV findings and those for viable MSC in oysters at station 4 were considered to signify a significant threat. The dilution at which no RT-PCR units for NoV in shellfish are detectable would need to be greater than the range of 158:1-556:1 measured for station 4.

Overall, the monthly survey results for MSC compare well with NoV GII (Fig. 9). Two of the 3 times MSC was found at station 4, NoV GII was also found, and the relative abundance of MSC and NoV GII was also comparable. Furthermore, the MSC results for samples collected in December 2008 and February 2009 demonstrated that MSC findings in oysters at station 4 exceeded the ISSC critical limit of 50 pfu/100 g. The consistency between the NoV GII results and the MSC results, and the repeatable findings of NoV GII indicate that the findings of NoV GII in the oyster sentinels located at station 4, although at levels less than 10 RT-PCR units/100 g, should be considered significant in a conservative analysis.


The NSSP relies on the establishment of prohibited areas around sewage discharges, such as WWTP effluents, to protect consumers of shellfish against exposures to enteric pathogens. For areas classified as conditionally approved under the NSSP, the FDA's 1,000:1 dilution recommendation represents the minimum amount of dilution needed when the WWTP is operating under normal conditions with disinfection.

This study investigated the accumulation of microorganisms by oysters and the hydrographic dilution and dispersion of wastewater effluent in tandem in Mobile Bay, AL. This was the first time that the FDA conducted a study comparing microbial indicator and NoV results in shellfish with the results of a hydrographic dye study. This research also used modern fluorometric equipment and advanced analytical techniques that the FDA never used in previous hydrographic studies. Using these advanced technologies, the efficacy of the minimum 1,000:1 dilution guidance to maintain the safety of molluscan shellfish was examined.

For station 4 in this study, the farthest station from the WWTP outfall, the dilution range of concern was from the period of low dilution (158:1) to a period of an overall average dilution (556:1). It is within these periods, or dilution ranges, that the highest virus levels in oysters are expected to occur. The range of 158:1 556:1 is less than the FDA's current dilution recommendation of 1,000:1. MSC findings above the ISSC critical limit of 50 pfu/100 g and positive NoV findings at station 4 indicate that the 158:1 556:1 range of dilution achieved at that station may be insufficient to guard against viruses.

Although there were no oyster sentinel stations beyond station 4, the FDA was able to project the possible location of the 1,000:1 dilution line using three approaches for assessing the data at each station and using linear regression analyses. The FDA was also able to project the anticipated MSC levels at a 1,000:1 dilution line using separate linear regression analyses for MSC versus dilution. The findings indicate that MSC levels at the projected 1.000:1 dilution line would be higher than the ISSC critical limit in some cases and lower than the limit in other cases. Furthermore, the FDA was unable to estimate with statistical significance what NoV GI or GII levels at the projected 1,000:1 dilution line would be. Therefore, no determination can be made regarding whether the 1,000:1 dilution policy is sufficiently protective in this case. However, based on the data gathered at station 4, it appears that a dilution level of 556:1 or less may be insufficient to protect the public health. Further research studies like the one conducted in Mobile Bay are needed to determine what dilution level above 556:1 is sufficiently protective against viruses and whether the FDA's 1,000:l dilution policy is adequate in this regard.

The results further demonstrated that measuring the physical dilution of human sewage discharges in receiving waters is a direct and effective way to assess the viral risks posed by these discharges. The greater the discharge volumes, the more water necessary to dilute the effluent to an acceptable level for growing and harvesting molluscan shellfish. In Mobile Bay, the bioaccumulation of viruses and microbial indicators at each of the stations was directly related to the dye concentrations detected at those stations. Reductions in viral indicators, such as MSC, and in human enteric viruses, such as NoV GII, occurred at substantial distances from the source and at relatively high dilutions. However, as shown by a comparison of the results at stations 3 and 4, the distance of shellfish from the wastewater discharge is not the lone factor in determining the potential viral impact. Shellfish that are relatively close to the discharge but are outside the concentrated path of the effluent plume may not bioaccumulate viruses to the degree that shellfish that are farther away but within the effluent plume's path do. Therefore, conducting a hydrographic dye study of this nature and mapping the size, shape, and path of the wastewater effluent plume is extremely useful in designating an area prohibited to the harvest of shellfish around a WWTP.


We acknowledge the State of Alabama Dauphin Island Sea Laboratory and Brandeis University for their technical contributions during the 2007 oyster relay study. We also acknowledge colleagues in the FDA Center for Food Safety and Applied Nutrition: Peter Pirillo and Paul DiStefano for field support, Guilan Huang for GIS-based analysis of the data, and William D. Watkins and Sebastian Cianci for their contributions to this paper.


American Public Health Association. 1970. Recommended procedures for the examination of seawater and shellfish, 4th ed. Washington, DC: American Public Health Association. 105 pp.

Baggi, F., A. Demarta & R. Peduzzi. 2001. Persistence of viral pathogens and bacteriophages during sewage treatment: lack of correlation with indicator bacteria. Res. Microbiol. 152:743-751.

Bedford, A. J., G. Williams & A. R. Bellamy. 1978. Virus accumulation by the rock oyster Crassostrea glomerata. Appl. Environ. Microbiol. 35:1012-1018.

Bosch, A. 1995. The survival of enteric viruses in the water environment. Microbiol. SEM 11:393-396.

Burkhardt, W., III & K. R. Calci. 2000. Selective accumulation may account for shellfish-associated viral illness. Appl. Environ. Microbiol. 66:1375-1378.

Burkhardt, W., III, W. D. Watkins & S. R. Rippey. 1992. Seasonal effects on accumulation of microbial indicator organisms by Mercenaria mercenaria. Appl. Environ. Microbiol. 58:826-831.

Burkhardt, W., III, J. W. Woods & K. R. Calci. 2005. Evaluation of wastewater treatment plant efficiency to reduce bacterial and viral loading using real-time RT-PCR. A poster presentation at the American Society of Microbiology, annual educational conference, Atlanta, GA.

Burkhardt, W., Ill, J. W. Woods & M. C. L. Vickery. 2004. Development of a quantitative multiplex RT-PCR assay for the detection of noro- and enteroviruses. In: Abstract presented at 104th General Meeting American Society for Microbiology, Washington, DC. p. Q-308.

Cabelli, V. J. 1990. Microbial indicator levels in shellfish, water, and sediments from upper Narragansett Bay conditional shellfish-growing area. Report to the Narragansett Bay Project (NBP-90-32), Providence, RI. Narragansett Bay Estuary Program. 67 pp.

Callahan, K. M., D. J. Taylor & M. Sobsey. 1995. Comparative survival of hepatitis A virus, poliovirus and indicator viruses in geographically diverse seawaters. Water Sci. Technol. 3l:189-193.

Canzonier, W. J. 1971. Accumulation and elimination of coliphage s-13 by the hard shelled clam, Mercenaria mercenaria. Appl. Microbiol. 21:1024-1031.

Carmichael, R. H., A. Brendan & I. Valiela. 2004. Nitrogen loading to Pleasant Bay, Cape Cod: application of models and stable isotopes to detect incipient nutrient enrichment of estuaries. Mar. Pollut. Bull. 48:137-143.

DePaola, A., J. L. Jones, J. Woods, W. Burkhardt, III, K. R. Calci, J. A. Krantz, J. C. Bowers, K. Kasturi, R. H. Byars, E. Jacobs, D. Williams-Hill & K. Nabe. 2010. Bacterial and viral pathogens in live oysters: 2007 United States market survey. Appl. Environ. Microbiol. 76:2754-2768.

Dore, W. J., K. Henshilwood & D. N. Lees. 2000. Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. Appl. Environ. Microbiol. 66:1280-1285.

Dore, W. J. & D. N. Lees. 1995. Behavior of Escherichia coli and male-specific bacteriophage in environmentally contaminated bivalve molluscs before and after depuration. Appl. Environ. Microbiol. 61:2830-2834.

Enriquez, R., G. G. Frosner, V. Hochstein-Mintzel, S. Riedermann & G. Reinhardt. 1992. Accumulation and persistence of hepatitis A virus in mussels. J. Med. Virol. 37:174-179.

Galtstoff, P. S. 1964. The American oyster, Crassostrea virginica, Gmelin. Fishery, Bulletin 64(1); 1-480.

Hetling, L. J. & R. L. O'Connell. 1966. A study of tidal dispersion in the Potomac River. Wrier Resour. Res. 2:825-841.

Hubbard, E. F. & W. G. Stamper. 1972. Movement and dispersion of soluble pollutants in the northeast Cape Fear estuary, North Carolina, Water-supply paper 1873-E. U.S. Geological Survey, United States Department of the Interior. Washington, DC: US Government Printing Office. 31 pp.

Kilpatrick, F. A. 1993. Techniques of water-resources investigations of the United States Geological Survey: simulation of soluble waste transport and buildup in surface waters using tracers. In: Application of Hydraulics, book 3, report no. TWRI3A20. Washington, DC: U.S. Geological Survey. Washington, DC: US Government Printing Office. pp. 2-14.

Kilpatrick, F. A. & E. D. Cobb. 1985. Techniques of water-resources investigations of the United States Geological Survey: measurement of discharge using tracers. In: Application of Hydraulics, book 3, report no. TWRI3A16. Washington, DC: U.S. Geological Survey. Washington, DC: US Government Printing Office. pp. 1-52.

Maalouf, H., J. Schaeffer, S. Parnaudeau, J. Le Pendu, R. L. Atmar, S. E. Crawford & F. S. Le Guyader. 2011. Strain-dependent norovirus bioaccumulation in oysters. Appl. Environ. Microbiol. 77:3189-3196.

Metcalf, T. G., B. Mullin, D. Eckerson, E. Moulton & E. P. Larkin. 1979. Accumulation and depuration of enteroviruses by the soft-shelled clam, Mya arenaria. Appl. Environ. Microbiol. 38:275-282.

Rippey, S. R., L. A. Chandler & W. D. Watkins. 1987. Fluorometric method for enumeration of Escherichia coli in molluscan shellfish. J. Food Prot. 50:685-690.

Seraichekas, H. R., D. A. Brashear, J. A. Barnick, P. F. Carey & O. C. Liu. 1968. Viral deputation by assaying individual shellfish. Appl. Microbiol. 16:1865-1871.

Shieh, C. Y., R. S. Baric, J. W. Woods & K. R. Calci. 2003. Molecular surveillance of enterovirus and Norwalk-like virus in oysters relocated to a municipal-sewage-impacted Gulf estuary. Appl. Environ. Microbiol. 69:7130-7136.

Sobsey, M. D., A. L. Davis & V. A. Rulhnan. 1987. Persistence of hepatitis A virus and other viruses in depurated eastern oysters, In: IEEE, editor. Proceedings: Oceans '87: Halifax, Nova Scotia. Piscataway, NJ: IEEE. pp. 1740-1745.

Tai, D. Y. & R. E. Rathbun. 1988. Photoanalysis of rhodamine-WT dye. Chemosphere 17:559-573.

Woods, J. S. 2010. Determining the relationship of human enteric viruses in clinical, wastewater, and environmental samples utilizing molecular and cell culture techniques. PhD diss., University of Souther Mississippi. 145 pp.


(1) Office of Food Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, HFS-325, College Park, MD 20740; and (2) U.S. Food and Drug Administration, Gulf Coast Seafood Laboratory One Iberville Drive, PO Box 158, HFS-400, Dauphin Island, AL 36528

* Corresponding author. E-mail: gregory.goblick(a!fda.hhs.gov

DOI: 10.2983/035.030.0341
Peak and steady-state rhodamine WT dye concentration and dilution
values at stations 1-4 in Mobile Bay.

          Approach 1

            Tidal Day       Tidal Day
             Average         Average
Station   Concentration     Dilution
No.           (ppb)

1                 31.61         5.44:1
2                 19.39         8.85:1
3                  0.03        5,387:1
4                  0.31          556:1

          Approach 2
                             at Peak
Station       Peak        (ppb) (Lowest
No.       Concentration     Dilution)

1                128.31         1.36:1
2                 63.58         2.74:1
3                  0.33          501:1
4                  1.00          173:1

          Approach 3

            Peak 1-h
             Average        Peak 1-h
          Concentration      Average
Station       (ppb)         Dilution

1                 85.05         1.49:1
2                 51.88         2.76:1
3                  0.17          820:1
4                  0.69          158:1
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