Human Viruses Found in
Groundwater Recharge Sites
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 environmentmaking
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
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
Elusive viral populations
In the past, viral waterborne outbreaks have been difficult to document
and researchers believe that weve 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, dont 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 waterto
determine the safety of groundwater recharge practices using the ICC/PCR
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
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. Whats
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.
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
were 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 arent
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
Just as weve 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 theres
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
productespecially in light of increased public awareness, growing
immunocom-promised populations and improvement in monitoring technologyin
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.
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
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,
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.
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