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TECHNICAL REPORT
Ground Water Quality

A Field Study of Virus Removal in Septic Tank Drainfields

^a Dep. of Marine Science, Univ. of South Florida, 140 7th Ave. S., St. Petersburg, FL 33701
^b Tampa Dep. of Health, 3952 W. Dr. Martin Luther King Jr. Blvd., Tampa, FL 33614
^c Dep. of Geology, Univ. of South Florida, Fowler Avenue, Tampa, FL 33602

* Corresponding author (lncartaya{at}earthlink.net)

Received for publication February 21, 2000.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Two field studies were conducted at a research station in Tampa, Florida to assess the removal of bacteriophage PRD1 from wastewater in septic tank drainfields. Infiltration cells were seeded with PRD1 and bromide and the effects of effluent hydraulic loading rate and rainfall on virus removal were monitored. Septic tank effluent samples were collected after passage through 0.6 m of unsaturated fine sand and PRD1 was detected over an average of 67 d. Bacteriophage PRD1 breakthrough was detected at approximately the same time as bromide in all three cells except for the low-load cell (Study 1), where bromide was never detected. Log₁₀ removals of PRD1 were 1.43 and 1.91 for the high-load cells (hydraulic loading rate = 0.063 m/d) and 2.21 for the low-load cell (hydraulic loading rate = 0.032 m/d). Virus attenuation is attributed to dispersion, dilution, and inactivation. Significant increases in PRD1 elution with rainfall were observed in the first 10 d of the study. Approximately 125 mm of rainfall caused a 1.2 log₁₀ increase of PRD1 detected at the 0.6-m depth. Current Florida on-site wastewater disposal standards, which specify a 0.6-m distance from the drainfield to the water table, may not provide sufficient removal of viruses, particularly during the wet season.

Abbreviations: pfu, plaque-forming units • PVC, polyvinyl chloride • TSB, tryptic soy broth

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
SEPTIC tanks are the most frequently reported source of ground water contamination in the USA (Yates, 1985). Occurrence data indicate that at least 60 to 70% of ground water sources in the USA have been contaminated with fecal viruses and/or bacteria (Macler, 1996). Thirty percent of Florida's population disposes of wastewater through on-site wastewater disposal systems or septic tanks, which translates into 1.6 million septic tanks and cesspools, disposing of 798 million liters per day (210 million gallons) (Marella, 1990). On-site wastewater disposal systems pose a public health threat to drinking water because approximately 90% of the people in Florida rely on ground water as a potable water supply. Septic tanks also pose an environmental threat in Florida due the connection between the surficial aquifers and coastal surface waters. Low levels of human enteric viruses have consistently been detected in Sarasota Bay, Florida; an area with variable densities of septic tanks (Lipp et al., 2001a).

The Florida Department of Health sets standards for on-site wastewater disposal systems. Septic tanks must be located and installed to function sanitarily with proper maintenance and to pose no hazard to the domestic water supply, ground water, or surface water (Florida Administrative Code, 1986). Soils at the installation site are rated slightly to severely limiting on their suitability to properly assimilate sewage effluent. The effective soil depth is defined as the depth of slightly or moderately limiting soil material at a drainfield site. Slightly limiting soils are not subject to further evaluation in site evaluation. A minimum effective soil depth of 0.6 m (2 ft) is required between the bottom of the drainfield and the water table during the wet season, and a depth of 1.1 m (3.5 ft) is required during the remainder of the year. Soil ratings also determine septic tank effluent hydraulic loading rates.

The effectiveness of these criteria on virus removal was assessed using the bacteriophage PRD1 as a human enteric virus surrogate. Bacteriophage PRD1 is a relatively conservative model virus, similar to the coliphage MS2, and attaches poorly under field conditions in sandy soils at pH 6 to 8 (Schijven and Hassanizadeh, 2000) due to its relatively low isoelectric point (Bales et al., 1991). Bacteriophage PRD1 is a more favorable surrogate for ground water studies due to its stability at higher temperatures. It has a low inactivation rate between 10 and 23°C (Yahya et al., 1993). Soil characteristics affect virus removal and in general, virus adsorption capacities increase as clay content, cation exchange capacity, and specific surface area increase, and as organic content decreases (Burge and Enkiri, 1978).

The purpose of this study was to evaluate the efficacy of Florida on-site wastewater disposal standards on the removal of viruses, using a conservative viral tracer. Bacteriophage PRD1 removal was measured after passage through a 0.6-m-thick unsaturated sandy drainfield or infiltration cell under septic tank effluent hydraulic loading rates above and below Florida regulations. The effect of rainfall on removal was also investigated.

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Site Description
Studies were conducted at a research station located at the University of South Florida (Tampa, FL). The station was designed and constructed to study the behavior of septic tank effluent in a controlled septic tank drainfield. Soil analyses were performed prior to operation of the station. The subsurface at the station is typical of west-central Florida and is composed of a well-sorted fine- to very fine-grained sand. This soil has a high permeability and a low water capacity. Soil present at this study site is rated slightly limiting. A summary of the soil characteristics is listed in Table 1. The location at USF was chosen because the soil conditions typify those in which a significant number of on-site wastewater disposal systems are installed in Florida (D.L. Anderson et al., unpublished data, 1993).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Cross-sectional view of the research station (modified from D.L. Anderson et al., unpublished data, 1993). Bacteriophage PRD1 and bromide were added to cells via the observation port. Effluent was applied to each cell, six times daily. Two effluent loading rates were evaluated (0.032 and 0.063 m/d).

Infiltration cells were constructed by excavating soil, leaving 0.6 m of undisturbed soil above the artificial water table. Each cell was 0.6 m wide and 1.8 m long and was separated from other cells by a 0.3-m-thick divider wall to prevent the migration of wastewater from cell to cell. Polyvinyl chloride observation ports were installed on the infiltrative surface of each cell and extended above the grade to monitor for wastewater ponding and also served as a point of entry for tracer materials. Aggregate was placed in the cell to a depth of 22.5 cm to allow effluent migration to the infiltrative surface of the cell. A 1.8-m-long PVC pipe containing 0.9-cm holes spaced every 0.3 m was installed at the center of each cell to serve as effluent distribution line. An additional 7.5 cm of aggregate was installed and the cell areas were backfilled and graded.

Wastewater Distribution
Septic tank effluent was intercepted from a small dormitory, which houses three or four students and contains a kitchen and a laundry facility. A pump tank supplied septic tank effluent to a 4000-L storage tank. The storage tank contained a 0.25-kilowatt pump, which delivered septic tank effluent to dosing pots located in the roof of the station. The soil at the station site has a maximum septic tank effluent loading rate of 0.049 m/d (1.2 gpd/ft²). Cells received either high (0.063 m/d) or low (0.032 m/d) loading of septic tank effluent. Effluent was distributed to each cell, six times daily, at 0600, 0700, 0800, 1200, 1800, and 1900 h. Electrical valves regulated dosing times and upon activation, effluent flowed by gravity into the infiltration cells.

Bacteriophage PRD1 Propagation and Assay
Bacteriophage PRD1 was propagated to high titer and assayed in the host bacterium, Salmonella typhimurium in tryptic soy broth (TSB). Bacteriophage PRD1 was obtained from the University of Arizona (Dr. Charles P. Gerba) and is 62 nm in size (Olsen et al., 1974), has an isoelectric pH < 4, and contains an inner hydrophobic lipid layer (Bales et al., 1991). Distinguishable clearings in the bacterial host monolayer (logarithmic growth phase) were counted and enumerated as plaque-forming units (pfu) using the double agar plaque assay as described by Adams (1959).

Field Studies
Two field studies were conducted during April and May 1997. Each study included an evaluation of high and low dosing of septic tank effluent. A total of four cells, two per each study, were seeded with PRD1 and bromide. Tracers were delivered onto the surface of the infiltration cells via the observation ports originally installed to observe the drainfield (see Fig. 1) using a PVC pipe. Tracers were added immediately one after the other in 100-mL volumes. Bacteriophage PRD1 was added at an influent concentration of 6.0 x 10¹⁰ pfu/mL and 1.6 x 10¹¹ pfu/mL for the first and second study, respectively. Bromide concentrations for both studies were 1.3 x 10² mg/mL.

Effluent Collection
Effluent samples were collected at a depth of 0.6 m. Sampling commenced 16 h after tracers were introduced into the infiltration cells. An intensive sampling was conducted 1 d after cells were seeded. In the first study this was every 4 h, after which the samples were collected twice daily for 8 d and daily thereafter for 54 d. During the second study, samples were collected twice daily for 8 d, once daily for 39 d, and then five samples were taken over the next 4-wk period for a total sampling of 74 d after cells were seeded with tracers.

Large stainless steel pans (1.1 x 0.6 x 0.05 m) intercepted the percolating septic tank effluent. These pans had been pushed laterally into the subsurface 0.6 m below the drainfield from inside the observation gallery. A tube extended from the pan on the gallery side to serve as a conduit for effluent collection. Samples were collected using a vacuum pump into sterile 1-L polypropylene bottles capped with vacuum screw-caps. Approximately 1 L of effluent was collected per sampling time. Phage samples were kept on ice and the assay performed. If samples were not assayed within 10 h of collection, samples were stabilized by adding 1 mL of TSB (final concentration = 1.5 x 10^-3 g/mL) and stored at 4°C. All samples were assayed within 24 h. Ten replicates of each sample were assayed (1 mL/replicate) and the pfu/mL was calculated. Additionally, septic tank effluent was assayed for background Salmonella phage weekly but none were detected. Positive controls were conducted using PRD1 stock retained from the original lysate used to dose infiltration cells. Negative controls consisted of plating 1.5 mL of Salmonella host.

Sodium Bromide Analysis
Bromide tracer was added simultaneously with the PRD1 tracer and effluent samples were split and analyzed for both tracers. The Florida Department of Health, Bureau of Laboratories, Tampa analyzed all samples for bromide. Sodium bromide measurements were made using a Corning Model 35 pH/ion meter, Corning double junction bromide reference (Catalog no. 476067) and Corning Bromide electrode (Catalog no. 80853) (Corning International, Corning, NY). Standards consisted of a 1:1 solution of 2 mol/L sodium bromide and 0.2 mol/L potassium nitrate and a curve developed over seven standard concentrations. Samples measuring at or below 0.799 mg/L (5 x 10^-6 mol/L) were recorded as below the detection limit.

Soil Temperature and Rainfall Measurements
Soil temperatures were recorded at each collection time with a stainless steel bi-metal thermometer (Reotemp [San Diego, CA] Model A) inserted into the soil at the 0.6-m depth in a nontest reference cell. Rainfall was measured using a Davis (Hayward, CA) Weather Wizard 3.0 located on-site at the research station.

Data Analysis
Log Removal
Log removal was computed as the PRD1 influent concentration (log₁₀ pfu/mL) minus the PRD1 effluent concentration (log₁₀ pfu/mL). Influent values equaled the total PRD1 particles seeded in the infiltration trenches (pfu). Effluent values were calculated by taking the geometric mean of the PRD1 concentrations (pfu/mL) detected at 0.6 m over the entire sampling time. The average log₁₀ pfu were multiplied by the total number of days in the study (pfu days/mL) and then multiplied by the effluent hydraulic loading rate [(pfu days/mL) (mL/day)] to give the total pfu exiting the cell throughout the study. The mL/day hydraulic loading rate was calculated based on the liters of effluent distributed daily.

Association with Rainfall
Relative changes in PRD1 concentrations with rainfall were examined to investigate the significance of rainfall on virus transport. Ratios of pfu/mL on Day 2 to Day 1, Day 3 to Day 2, etc. were calculated and correlated with rainfall. Breakthrough and peak concentrations in the first 4 to 6 d detected at the 0.6-m pan were not included in order to eliminate the gradual increase of phage concentrations due to dosing. Linear regressions were performed by plotting the 24-h concentration change against rainfall, in mm per day using Excel 7.0 (Microsoft, 1996). Regression output included correlation coefficients and r² values.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Bacteriophage PRD1 and Bromide Detection and Breakthrough Curves
Effluent collection for both studies commenced 16 h after the cells were seeded and continued for 61 d for Study 1 and 74 d for Study 2. Breakthrough curves for PRD1 and bromide are shown in Fig. 2 . Tracer breakthrough and peak concentrations and times are shown in Table 2. Breakthrough curves for either tracer were not established for the low-load cell in Study 1. Bromide was never detected in this cell and PRD1 was detected only briefly, relative to the high-load cell. Bacteriophage PRD1 was detected at a breakthrough concentration of 28.1 pfu/mL at 2 d and peaked at 3.7 x 10² pfu/mL, 4 d after the cell was seeded. Zero values were measured thereafter, with the exception of 2.1 pfu/mL detected at 11.3 d. Sampling continued five more days without detection of PRD1. Because bromide was never detected, the tracers may have been transported by preferential flow around the collection pan. It is not clear why a portion of the PRD1 lysate was detected. As a point of comparison, breakthrough and peak travel times are the same as the low-load cell, in the second study.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Bacteriophage PRD1 and bromide breakthrough curves. (a) High-load cell, Study 1; (b) High-load cell, Study 2; (c) Low-load cell, Study 2. C/C0 = log10 (effluent at 0.6 m)/log10 (influent). Bromide was not detected in the Study 1 low-load cell.

Bacteriophage PRD1 breakthrough was detected simultaneously with bromide (Fig. 2a,c) or shortly afterwards (Fig. 2b). This behavior has been observed in a number of field studies (Schijven et al., 1999; Pieper et al., 1997; Deborde et al., 1998). Contrasting the high-load cells, the PRD1 peak concentration was higher in Study 1, even though the influent concentration was almost a log lower for this study. This may also be attributed to greater saturation levels or failure to detect the true peak concentration. An intensive sampling schedule was conducted in Study 1 (every 4 h) but not in Study 2, during the time of peak detection of PRD1.

Removal of Bacteriophage PRD1 in Infiltration Cells
Log₁₀ removals of PRD1 by the sandy soil shown in Table 2 were 1.43, 1.91, and 2.21 for the high-load cell (Study 1), high-load cell (Study 2), and low-load cell (Study 2), respectively. Removal was not calculated for the low-load cell in the first study. Minimal removal of PRD1 may be attributed to a high organic matter content in the soil. The station had been in operation for more than 4 yr when these studies were conducted. Studies have shown that organic matter interferes with virus adsorption by competing for adsorption sites on mineral surfaces (Rao and Melnick, 1986). High organic matter content was the primary factor promoting PRD1 transport in a contaminated sandy aquifer (Pieper et al., 1997). Removal in Study 2 was 0.3 log₁₀ greater in the low-load versus the high-load cell. Therefore, decreasing the loading rate did not appreciably enhance removal.

Soil temperatures ranged between 24 and 28°C throughout both studies, and averaged 24.6°C. An inactivation rate for PRD1 in ground water has not been calculated, however it has been shown to be stable between 18 and 23°C (Yahya et al., 1993). We can conclude that inactivation may be one of the major factors in PRD1 removal. Using the data from these studies and assuming that inactivation was the only factor contributing to PRD1 removal, the inactivation rate would equal 0.03 log₁₀/day. This is comparable to Poliovirus 1, which has an inactivation rate of 0.046 log₁₀/day in Florida ground water (Gerba and Bitton, 1984).

Comparison of Rainfall and Bacteriophage PRD1 Transport
Rainfall data were collected with each sampling and were plotted in Fig. 3 (Study 1) and Fig. 4 (Study 2) along with PRD1 concentration.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Bacteriophage PRD1 transport and rainfall received at the research station: Study 1. Total rainfall received = 240 mm.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Bacteriophage PRD1 transport and rainfall received at the research station: Study 2. Total rainfall received = 250 mm.

Study 2
A total of 250 mm of rainfall were received, however only 15 mm of rain were received during the first 10 d of sampling. Therefore, when considerable rainfall was received at the station, a greater portion of the lysate had already traveled through the cell and was beginning to decrease in concentration. This may explain why no substantial increases were observed during this study.

Prior to the first study, 58 mm of rain was received at the station for a total of approximately 300 mm for the month of April. Available data collected prior to the study indicated that soil moisture was highest throughout the cells at the start of the first study compared with measurements made during the previous 8 mo. Significant amounts of rainfall were received prior to and during the seeding, and most likely maintained elevated soil moisture. Fine sands have relatively high residual water contents, therefore increasing the possibility that the soil moisture was potentially high throughout the first study. In an unsaturated soil there is a critical point where the hydraulic conductivity changes rapidly with soil moisture content (Feter, 1988). Therefore, although a soil matrix is classified as being unsaturated, the water conductivity may be close to that for a saturated environment. The lack of consistently significant correlation between rainfall and PRD1 transport can also be explained by this phenomenon. The only significant correlation coefficient was observed for the Study 1 high-load cell, where the soil may have been near or at saturation due to rainfall received before and throughout the study. Studies in Charlotte Harbor, Florida found a statistically significant correlation between human enteroviruses and rainfall as well as coliphage and rainfall (Lipp et al., 2001b). Septic tanks were the major source of viruses.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
This study, although limited, provides specific fate and transport patterns for PRD1 in septic tank drainfields composed of fine sand under wet-season conditions. No clear recommendation can be made regarding the appropriate loading rate but a 50% decrease in hydraulic loading rate did not significantly increase virus removal. Bacteriophage PRD1 attachment was reversible and the phage particles slowly desorbed over time. Rainfall effects were observed to aid in desorption within the first 10 d of the study. Inactivation and dilution and/or dispersion were the main mechanisms contributing to virus attenuation.

Results from this study indicate the need to reassess the minimum (wet season) 0.6-m unsaturated zone. Florida onsite wastewater disposal regulations, specifically soil ratings and unsaturated depths, need to focus on pathogen removal along with effluent assimilation.

	ACKNOWLEDGMENTS

 
L.A. Nicosia would like to acknowledge Dr. Lillian Stark and the Florida Department of Health for the opportunity to participate in this research project, and her students: L. Heberlein-Larsen, C. Webb, and D. Scheer, without whom this project would not be possible. Also, thanks to Dr. Paul Sherbloom for rainfall and soil moisture data.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Nicosia, L.A. Articles by Stewart, M.T. Search for Related Content PubMed PubMed Citation Articles by Nicosia, L.A. Articles by Stewart, M.T. GeoRef GeoRef Citation Agricola Articles by Nicosia, L.A. Articles by Stewart, M.T. Related Collections Water Quality Water Pollution Other Waste Management Ground Water Quality