Human Viruses Found in
Groundwater Recharge Sites
By Kelly A. Reynolds, Ph.D.

Recharge of groundwater with reclaimed water is an idea that has expanded in popularity as water demands continue to increase with population growth. Interest is particularly great in the United States’ arid Southwest, where rainfall is infrequent and recharge of the vast underground reservoirs, formed by aquifers, constitutes a valuable drinking water supply. Treatment technology is thought to be able to render wastewater drinkable by intentionally mixing treated, reclaimed water with groundwater intended for consumption.
However, a major concern with recharge of groundwater is the possible introduction of disease-causing organisms from inadequately treated wastewater. Although harmful bacteria, viruses and protozoa may be present in wastewater, viruses cause the greatest concern regarding groundwater contamination due to their small size and long-term survival capabilities in the environment—making them less likely to be removed by the process of ground filtration. Few scientific studies have been conducted using the latest virus detection methodologies; therefore, little information is available regarding the true public health impact of reclaimed wastewater on groundwater recharge basins.

Groundwater contamination
More than 140 different types of viruses are known to infect the human intestinal tract and are subsequently excreted in feces. Enteric viruses previously associated with waterborne outbreaks include the enterovirus group (poliovirus, coxsackievirus and echovirus), hepatitis A virus, rotavirus, adenovirus and Norwalk virus. These viruses are responsible for a wide range of illnesses including meningitis, paralysis, myocarditis, hepatitis, encephalitis, diabetes, respiratory illness and, perhaps the most commonly identified symptom, diarrhea. These human pathogens find their way into the environment via municipal waste disposal, septic tank seepage, stormwater runoff, wastewater reclamation practices and recreational bathing, just to name a few. Transmitted by the fecal-oral route through contaminated water, low numbers of these pathogens are able to initiate infection in humans. In fact, an infectious dose may be as low as one culturable organism. Therefore, effective methods of virus monitoring must be able to detect very low levels of viruses in very large water volumes.
Groundwater contamination may originate from a variety of sources including injection wells, land application of wastes, septic tanks, faulty sewer lines, defective well casings and underground channelization and fault lines. Characteristics of the subsurface environment, such as pH, permeability, ionic strength, particle size, texture, adsorptive nature and organic load, also play a role in the ability of the soil/aquifer to filter out possible groundwater contaminants. In addition, the depth to the groundwater table is an important factor in determining the success of soil/aquifer treatment applications.

Elusive viral populations
In the past, viral waterborne outbreaks have been difficult to document and researchers believe that we’ve only identified the tip of the iceberg concerning viral waterborne illness. Although no etiological agent has been identified in nearly half of all waterborne outbreaks, probably due to lack of efficient detection methods, viruses are known to be the causative agent in 15 percent of all documented incidents.
The standard method of enterovirus assay and detection is through use of animal cell culture. The presence of virus is indicated by the destruction of the cells referred to as the cytopathogenic effects (CPE). Virus detection requires up to 14 days for environmental strains, when tested in first passage cell culture. Some viruses, however, don’t produce CPE and therefore elude detection by conventional methods. A new method of virus detection utilizing cultural and molecular techniques, known as the integrated cell culture-polymerase chain reaction (ICC-PCR), provides a more rapid and sensitive means for isolating low levels of infective virus, including elusive strains that do not produce CPE.1,2, 3
ICC/PCR has recently been used to detect viruses after exposure to chlorine disinfectant.1 This method detected viruses after eight minutes of chlorine treatment, four times longer than the recommended exposure time of two minutes based on cell culture analysis alone.4 This has serious implications since the determination of inactivation rates of waterborne virus is crucial to the drinking water industry. This phenomenon could help to explain why infectious viruses have been detected in drinking water receiving what was believed to be adequate disinfecting.5,6
Currently, the University of Arizona and County Sanitation Districts of Los Angeles County are conducting a collaborative study to assess potential virus contamination in: 1) water reclamation plant disinfected tertiary effluents used for recharge, 2) groundwater monitoring wells, 3) sites impacted by reclaimed water, and 4) sites not exposed to reclaimed water—to determine the safety of groundwater recharge practices using the ICC/PCR methodology.7,8

Recharge monitoring
Test sites for groundwater recharge monitoring in Arizona included a total of eight infiltration basins loaded with water from secondary effluent from a local wastewater treatment plant, filtered effluent from a water reclamation plant and wetlands effluent originating from each of the previously listed sites. This particular monitoring site recharges approximately 10,000 acre feet of treated wastewater per year. Treatment of the wastewater consists of primary sedimentation, biological treatment, clarification, chlorination and dechlorination prior to discharge to a surface water river basin.
Surprisingly, viruses were found in groundwater monitoring wells and in heavily disinfected effluents. In fact, of the 274 samples analyzed, none were positive by conventional cell culture methodologies, even after four passes (a total of 45 days of assay time) in cell culture, while at least five were positive by molecular-based methodologies (ICC/PCR). Specific sites positive for viruses included heavily disinfected tertiary effluents, shallow (30 feet deep) monitoring wells and other reclaimed water sites. Therefore, the data suggest that treated effluents and wells with short travel times are potentially vulnerable to virus contamination. What’s more, the data determined that conventional methods of virus detection failed at identifying a significant number of virus-positive samples. Genetic sequencing analysis of the viruses identified them as vaccine strain poliovirus, a common inhabitant of sewage due to the widespread practice of vaccinating humans. While not a pathogen itself, the vaccine strain poliovirus serves as a marker for sewage contamination and the potential for survival of other human viruses in the sample.

Disinfection concerns
Alarmingly, conventional methods of virus growth and isolation were not effective in detecting these certain virus populations. Instead, ICC/PCR was needed to determine the presence of these infectious agents. The fact that these viruses survive extensive disinfecting practices causes concern over whether these organisms are more resistant to disinfectants or perhaps altered by the disinfecting process. Certainly, evidence suggests that we’re overestimating the effectiveness of our disinfecting procedures while underestimating the survival and transport capabilities of these elusive virus populations.
Although the recharged groundwater wells monitored in this study aren’t currently used as potable water sources, the objective was to determine the safety of such practices in the future and the effectiveness of soil/aquifer filtration of potentially contaminated water, while using the best monitoring methods available.

Conclusion
Just as we’ve seen with chemical contaminants, methodology improvements for virus detection are revealing the presence of potentially harmful levels of pathogens in water environments previously thought to be safe for consumption. This is disconcerting, as it seems that there’s no end to the risk of exposure to contaminated water. The drinking water industry must continue to assume the challenge of producing a better quality product—especially in light of increased public awareness, growing immunocom-promised populations and improvement in monitoring technology—in order to promote the continued advancement of treatment technologies. More information is likely to become available on elusive and treatment resistant virus populations in the near future, including data on their occurrence, disinfectant resistance (ozone, chlorine, ultraviolet light, etc.), survival characteristics, genetic structure and infectious nature. q

References
1. Reynolds, K.A., M. Abbaszadegan, C.P. Gerba and I.L. Pepper, “Rapid ICC/PCR Detection of Enteroviruses and Hepatitis A Viruses in Water,” Proceedings of the Water Quality and Technology Conference (WQTC) 1997 (on CD-ROM), American Water Works Association (AWWA), Denver, November 1997.
2. Reynolds, K.A., “New methods for human virus detection: A new approach for ‘real time’ monitoring,” WC&P, Vol. 39, No. 6, 1997.
3. Reynolds, K.A., C.P. Gerba and I.L. Pepper, “Detection of infectious enteroviruses by an integrated cell culture-PCR procedure,” Applied Environmental Microbiology, 62:1424-1427, 1996.
4. Bitton, G., Wastewater Microbiology, Wiley-Liss Inc., New York, 1999.
5. Payment, P., M. Tremblay and M. Trudel, “Relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water,” Applied Environmental Microbiology, 49:981-983, 1985.
6. Rose, J.B., C.P. Gerba, S.N. Singh, G.H. Toranzos and B. Keswick, “Isolating viruses from finished water,” Journal AWWA, 78:56-61, 1986.
7. Seidel, G., C.P. Gerba and W. Yanko, “Application of Molecular Methods for the Detection of Enteroviruses at Recharge Facilities,” Artificial Recharge and Integrated Water Management (pp. 381-391), Proceedings from the 9th Biennial Symposium of the Artificial Recharge of Groundwater, Tempe, Ariz., June 10-12, 1999.
8. Seidel, G., C.P. Gerba and W. Yanko, “Application of Molecular Methods for the Detection of Non-CPE Enteroviruses at Recharge Facilities,” Proceedings of the 12th Annual Symposium of the Arizona Hydrological Society, White Mountains, Ariz., Sept. 8-11, 1999.
9. Reynolds, K.A., C.P. Gerba and I.L. Pepper, “Significance of noncytopathogenic viruses surviving chlorine disinfection,” AWWA, WQTC, San Diego, Nov. 1-4, 1998.
10. Seidel, G., C.P. Gerba and W. Yanko, “Application of Molecular Methods for the Detection of Enteroviruses at Recharge Facilities,” Proceedings of the 72nd Annual Conference of the Arizona Water and Pollution Control Association, May 3-5, 1999. Tucson, Ariz.

About the author Dr. Kelly A. Reynolds is a research scientist at the University of Arizona with a focus on the development of rapid methods for detecting human pathogenic viruses in drinking water. She is also a member of the WC&P Technical Review Committee.