Science of the Total Environment 775 (2021) 145738

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Microbial pathogens and contaminants of emerging concern in

groundwater at an urban subsurface stormwater infiltration site
Jane R. de Lambert a,⁎, James F. Walsh a, Deanna P. Scher a, Aaron D. Firnstahl b, Mark A. Borchardt c
a
Minnesota Department of Health, 625 Robert St. N, St. Paul, MN 55164, United States
b
U.S. Geological Survey Upper Midwest Water Science Center, 2615 Yellowstone Drive, Marshfield, WI 54449, United States
c
U.S. Department of Agriculture-Agricultural Research Service, Environmentally Integrated Dairy Management Research Unit, 2615 Yellowstone Drive, Marshfield, WI 54449, United States

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Infiltration to control stormwater runoff

is common but may degrade groundwa-
ter.
• Contaminants measured beneath storm
water infiltration sites at multiple depths.
• Rain a significant driver in microbial var-
iation across sampling events.
• Pathogens present at all depths but low-
est below fine-grained sediment.
• Infiltrating urban stormwater may
threaten geologically sensitive aquifers.

a r t i c l e i n f o a b s t r a c t

Article history: Urban stormwater may contain a variety of pollutants, including viruses and other pathogens, and contaminants
Received 28 September 2020 of emerging concern (pharmaceuticals, artificial sweeteners, and personal care products). In vulnerable geologic
Received in revised form 29 December 2020 settings, the potential exists for these contaminants to reach underlying aquifers and contaminate drinking water
Accepted 4 February 2021
wells. Viruses and other pathogens, as well as other contaminants of emerging concern, were measured in
Available online 10 February 2021
stormwater and groundwater at an urban site containing a stormwater cistern and related subsurface infiltration
Editor: Jay Gan gallery, three shallow lysimeter wells, and a monitoring well. Five of 12 microbial targets were detected more
than once across the eight rounds of sampling and at multiple sampling points, with human-specific Bacteroides
Keywords: detected most frequently. The microbial and chemical contaminants present in urban stormwater were much
Water microbiology lower in the water table monitoring well than the vadose zone lysimeters. There may be numerous causes for
Zoonotic pathogens these reductions, but they are most likely related to transit across fine-grained sediments that separate the
Contaminant water table from the vadose zone at this location. Precipitation amount prior to sample collection was signifi-
Hydrogeology cantly associated with microbial load. A significant relation between microbial load and chloride-bromide ratio
Water quality
was also observed. The reduction in number and concentrations of contaminants found in the monitoring well
Runoff infiltration
indicates that although geologically sensitive aquifers receiving urban stormwater effluent in the subsurface
may be prone to contamination, those with a protective cap of fine-grained sediments are less vulnerable.
These results can inform stormwater infiltration guidance relative to drinking water wells, with an emphasis
on restricting infiltration near water supply wells finished in geologically sensitive aquifers to reduce public
health risks.
© 2021 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Minnesota Department of Health, 625 Robert St. N, St. Paul, MN 55164, United States.
E-mail address: jane.de.lambert@state.mn.us (J.R. de Lambert).

https://doi.org/10.1016/j.scitotenv.2021.145738
0048-9697/© 2021 Elsevier B.V. All rights reserved.
J.R. de Lambert, J.F. Walsh, D.P. Scher et al.
1. Introduction
The high demand for water resources has led stormwater capture
and reuse to become a popular practice in many communities across
the United States, particularly those affected by droughts (USEPA,
2019). Population growth, changes in climate, and greater irrigation
needs can stress the demand for high quality water, even in water-
rich states such as Minnesota (Interagency Workgroup on Water
Reuse, 2018). Capturing stormwater for irrigation reduces the use of po-
table water sources and minimizes pollutant loads reaching surface wa-
ters, including vulnerable wetland systems.
Despite its utility in providing an alternative use to potable water
sources, urban stormwater has been widely found to carry human
enteric pathogens and other microbiological and chemical contami-
nants (Sauer et al., 2011; Sercu et al., 2009; Sidhu et al., 2012, 2013).
Little is known about the fate and transport of these constituents in
the subsurface during stormwater infiltration and the resulting ef-
fects on groundwater quality (NAS, 2016). From 2016 to 2017, the
Minnesota Department of Health (MDH) conducted a monitoring
study to characterize viruses, other pathogens, and chemical con-
taminants of emerging concern in stormwater at varying depths in
the vadose and phreatic zones beneath a subsurface stormwater in-
filtration gallery.
The main goals of this work were to increase understanding of the
transport and fate of stormwater contaminants in the subsurface and
improve assessment of groundwater contamination risk related to this
practice. We also sought to improve upon the study design of larger-
scale, future monitoring efforts through evaluation of this pilot study.
To target future sampling, we looked for the relation between microbial
concentration and general water quality parameters to determine
whether any of these routinely collected and lower-cost analytes may
Fig. 1. Upper Villa Park site location.
2
Science of the Total Environment 775 (2021) 145738
serve as indicators of elevated microbial load. We also assessed the rela-
tion between precipitation amount prior to sampling and microbial
concentration to determine if targeting sample collection after rainfall
events increases the likelihood of finding a detectible or above-normal
level of microbial contamination.
2. Methods
2.1. Site description
The study site is located at Upper Villa Park in Roseville, Minnesota,
USA, in Ramsey County. This park houses a baseball field and is
surrounded by developed land to the north and west and deciduous for-
est, wetlands, and a small, unnamed lake to the east (Fig. 1). The
stormwater infrastructure at the site consists of a concrete cistern (ap-
proximately 18 m × 14.5 m and 2.5 m high) and related subsurface in-
filtration gallery. The cistern was built to harvest and hold stormwater
entering from a municipal storm sewer, and then release to a 1700
cubic meter underground infiltration gallery located beneath an asphalt
parking lot and constructed of perforated, corrugated metal pipe. Runoff
to the storm sewer is primarily from roadways and residential proper-
ties. The infiltration system automatically releases water from the cis-
tern to the gallery after a rain event. A portion of the infiltrated water
is pumped out of the cistern and used to irrigate the baseball field in
the park; the remaining water in the cistern travels to the infiltration
gallery at a depth of approximately 10 to 15 ft below ground surface.
Three non-weighing lysimeters were installed at the site and finished
at depths of 3, 5, and 7 ft below the infiltration gallery. These will be re-
ferred to as Lysimeter 3, Lysimeter 5, and Lysimeter 7, respectively. Ad-
ditionally, a monitoring well was installed at the park and is located
between the infiltration system and the receiving wetland (Fig. 1). A
J.R. de Lambert, J.F. Walsh, D.P. Scher et al.
municipal sanitary sewer runs from west to east along the length of
the park.
2.2. Site geology and hydrogeology
The surficial geology of the study site consists of approximately
200 ft of unconsolidated Pleistocene sediments overlying bedrock of
the Ordovician-age St. Peter Sandstone (Meyer and Swanson, 1992).
The water table at the site is approximately 30 ft below the land surface,
and groundwater flow is southeasterly, towards McCarrons Lake, into
which the unnamed lake directly east of the site flows. The generalized
geologic soil profile inferred by the site boring logs indicates 2 to 6.5 ft of
fill, consisting mostly of lean clay, overlying native soils. These are gen-
erally composed of coarse alluvium to depths of approximately 23 ft and
represent glacial meltwater sediments of the Grantsburg sublobe
(Meyer and Swanson, 1992). Finer-grained sediments (silty till and
fine alluvium) are noted locally below 23 ft, especially near the monitor-
ing well, until coarse alluvium is again encountered from 31 to 43 ft.
Mixed silty till and fine alluvium are noted below that depth. The sedi-
ment surrounding the cistern, three lysimeter wells, and the infiltration
gallery is primarily native fill material that was replaced after excava-
tion and installation of the infiltration system. Given that the lysimeters
are completed at depths of 3, 5, and 7-ft below the base of the infiltra-
tion gallery, which is itself buried approximately 15 ft below grade,
they are completed in coarse alluvium with an estimated infiltration
rate of 6–7 in/h (American Engineering and Testing, Inc., 2013). The
monitoring well, screened from 33 to 43 ft below grade, is open to a
mix of coarse alluvium and mixed silty till and fine alluvium that is sep-
arated from the lysimeters by intervening silty or clayey sediments with
an estimated infiltration rate of less than or equal to 1.4 in/h (Fig. 2). The
lysimeters capture stormwater and groundwater percolating rapidly
Fig. 2. Upper Villa Park geologic cross-section.
3
Science of the Total Environment 775 (2021) 145738
through the vadose zone following rain events. It is estimated that the
flush of stormwater released to the infiltration gallery following a
storm event passes by the lysimeters within as little as 40 h (Capitol
Region Watershed District, 2017). In contrast, the water in the monitor-
ing well has an estimated recharge age of approximately 1 year based
on sulfur hexafluoride (SF6) analysis (Sharon Kroening, Minnesota Pol-
lution Control Agency, written communication, 2019). As such, the
lysimeters represent a geologically sensitive portion of the groundwater
system, whereas the monitoring well is drawing from a less sensitive
portion due to its greater depth and presence of relatively low-
permeability sediments above the well screen (Minnesota DNR, 1991).
2.3. Stormwater collection system
Stormwater flow through the cistern and infiltration gallery is
guided by the Opti-RTC system. Opti-RTC constantly checks forecasted
precipitation 48 h into the future. If a rain event that is greater than
0.05 in. and greater than 60% probability is forecasted, the Opti-RTC sys-
tem will open the valve between the cistern and the infiltration gallery
(generally 24 h prior to the forecasted rain event), emptying the stored
cistern water into the infiltration gallery. The stormwater from the fore-
casted event refills the cistern. Therefore, the water being collected in
the three lysimeters is water that was stored in the cistern from a previ-
ous event. In some circumstances, the cistern is evacuated, and then the
forecasted event does not occur. Alternately, the forecasted event could
be much larger than predicted causing the cistern to reach the overflow
level, whereby stormwater from the current event overflows into the
infiltration gallery. In this case, sampling efforts after a cistern release
and event overflow could represent multiple storm events (Sarah
Wein, Capitol Region Watershed District, written communication,
2020). Additionally, precipitation from the predicted rain event also
J.R. de Lambert, J.F. Walsh, D.P. Scher et al.
may reach the lysimeters through rapid recharge from the ground
surface.
2.4. Sample collection
MDH collected eight rounds of samples between November 2016
and November 2017 at the cistern, three lysimeters, and monitoring
well. Samples were collected on the following dates: November 9,
2016; and May 24, June 16, July 14, July 28, August 22, September 12,
and November 21 in 2017. Sampling events occurred at or greater
than 48 h following a release of the cistern (whether manual or auto-
matic) to ensure that the stored storm water had been emptied into
the infiltration gallery by the time of sample collection. The cistern re-
leases were generally timed to occur 24 h prior to forecasted precipita-
tion events; therefore, samples contained stormwater from a mix of
recent rain events, infiltration gallery recharge, infiltrating recharge
from the ground surface not associated with the infiltration gallery,
and in the case of the monitoring well, groundwater from outside the
infiltration gallery area. The sanitary sewer present onsite was also sam-
pled one time by MDH staff in November 2016.
Samples were collected at the cistern and monitoring well using a
Geotech submersible pump. Mean and range sample volumes for the cis-
tern and monitoring well were 339 L (range 57–693 L) and 91 L (range
1.9–144 L), respectively. The three lysimeter wells were pre-equipped
with individual Proactive Mini-Typhoon Purging pumps which were
used for sample collection. All tubing was replaced at the beginning of
each sampling event. At the cistern and monitoring well, microbial sam-
ples were collected using dead-end ultrafiltration using Hemodialyzer
Rexeed-25 s filters (Asahi Kasei Medical MT Corp., Oita, Japan). Due to
the presence of sediment and organic material, pre-filters were used
during five of eight sampling events at the cistern beginning in July
2017 to reduce ultrafilter fouling and risk of filter breakage.
At the three lysimeter wells, 1-l samples for microbial analyses were
collected in high-density polyethylene (HDPE) bottles. Microbial sam-
ples were shipped on ice and analyzed at the Laboratory for Infectious
Disease and the Environment in Marshfield, Wisconsin, by qPCR (quan-
titative polymerase chain reaction).
2.5. qPCR analyses
The 12 microbial targets analyzed are as follows: adenovirus groups
A-F and C,D,F, enteroviruses, hepatitis A virus, human polyomavirus,
norovirus genogroups I and II, pepper mild mottle virus (PMMV), rotavi-
rus group A (NSP3) gene, human-specific HF183 Bacteroides (hereafter
HF183 Bacteroides) and Bacteroidales-like Hum M2, and Cryptosporidium
spp. Upon arrival to the laboratory, ultrafilters were back flushed follow-
ing Smith and Hill (2009) and 1% of desiccated beef extract (mass/
volume) was added to backflush eluate. Pre-filters were transferred to
plastic bags and soaked in 200 mL of 3% beef extract solution containing
0.05 M glycine at a pH of 9.5 for 15 min. Liquid was then squeezed from
pre-filters and transferred to a sterile 500-mL bottle and pH adjusted to
7.0. Ultrafilter and pre-filter eluates were further concentrated by poly-
ethylene glycol precipitation (Lambertini et al., 2008; Stokdyk et al.,
2016). One-liter samples were supplemented with desiccated beef ex-
tract (1% mass/volume) and then concentrated using the polyethylene
glycol precipitation method. All final concentrated sample volumes
(FCSV) from ultrafilter and 1-l samples were stored at −80 °C until anal-
ysis. Nucleic acid extraction was performed with QIAcube® and QIAamp
DNA blood mini kits with buffer AVL (Qiagen, Valencia, California). To
obtain sufficient template, two 280 μL aliquots of FCSV were extracted,
each with a final elution volume of 120 μL. RNA gene targets were
reverse-transcribed using random hexamers (ProMega, Madison,
Wisconsin) and SuperScript® III reverse transcriptase (Invitrogen Life
Technologies, Rockville, Maryland) following procedures described in
Stokdyk et al. (2016). qPCR was performed using a LightCycler® 480
instrument (Roche Diagnostics, Mannheim, Germany) following
4
Science of the Total Environment 775 (2021) 145738
procedures described in Stokdyk et al. (2016). Hydrolysis probes
were used for quantification, and standard curves were created
from gBlocks® and Ultramer® oligos (Integrated DNA Technologies,
Coralville, Iowa). Forward and reverse primer sequences, probe se-
quences, and standard curve metrics for all qPCR assays are reported
in Supplemental Material (SM). Following Gibson et al. (2012),
lambda phage DNA and hepatitis G virus armored RNA were used to
evaluate all samples for inhibition of qPCR and reverse transcription-
qPCR, respectively. Negative controls were included at all processing
steps (secondary concentration, nucleic acid extraction, reverse tran-
scription, and qPCR) and must exhibit no fluorescence above the base-
line for data to be acceptable. Modified live virus vaccines (Zoetis Inc.,
Kalamazoo, Michigan) were used for DNA (bovine herpes virus) and
RNA (bovine respiratory syncytial virus) extraction positive controls,
with the latter serving also as the reverse transcription positive control.
2.6. Chemical parameter sample collection and analysis
Samples were collected once during November 2016 from the
sewer, cistern, Lysimeter 5, and the monitoring well. MDH shipped sam-
ples on ice to the Water and Environmental Analysis Laboratory (WEAL)
at the University of Wisconsin, Stevens Point. WEAL analyzed the sam-
ples for a suite of pharmaceuticals, artificial sweeteners, and personal
care products that are considered indicative of wastewater (Table II)
using high performance liquid chromatography and mass spectrometry
(ESI-LC/MS/MS). Prior to solid phase extraction, Benzoylecgonine-D3, a
surrogate standard, was added to all samples, blanks, and lab spikes. Du-
plicate, blank, and lab-spiked samples were prepared and analyzed for
approximately 10% of samples. A seven-point calibration was used for
each analyte. Deuterated analogs of target analytes were used as inter-
nal standards.
2.7. Water quality parameters
After microbial samples were collected, MDH collected additional
samples to measure general water quality parameters (Table III), unless
the available volume of water was insufficient for collection. Samples
were brought to the MDH Public Health Laboratory (PHL) in St. Paul,
Minnesota for analysis.
2.8. Statistical methods
Descriptive statistics on the individual microbial, chemical, and
water quality parameter results are reported by sampling location. We
also created an indicator of total microbial load by summing all micro-
bial targets with results available for the eight sampling events (herein
referred to as “microbial sum”). Because several microbial targets were
not detected in any of the five sampling locations, and Cryptosporidium
and Bacteroidales-like Hum M2 were not analyzed during the first round
of sample collection, microbial sum ultimately encompassed HF183
Bacteroides, rotavirus group A, and pepper mild mottle virus. The rela-
tion between the amount of precipitation prior to the sampling dates
(National Weather Service Station ID: GHCND:USC00218450, approxi-
mately 5.2 km from the studied site) and total microbial sum were eval-
uated using the total precipitation amount in the 7 days prior to the
sample collection date. Microbial sum was also assessed in relation to
the concentration of the following water quality parameter and bacte-
rial indicators (selected due to previously reported association with
sewage, wastewater, and/or stormwater (USEPA, 1986, Tiefenthaler
et al., 2008, NAS, 2016): boron, sulfate, potassium, chloride, the mass
ratio of chloride to bromide (Cl\\Br ratio), nitrate, Escherichia coli
(E. coli), and total coliform. The relation between microbial sum and
precipitation, and microbial sum and water quality parameters overall
and within each sampling location, were assessed visually. Due to the
non-normality of the data, non-parametric tests were used to assess
the strength and direction of correlation between total microbial sum
J.R. de Lambert, J.F. Walsh, D.P. Scher et al. Science of the Total Environment 775 (2021) 145738

Table I
Microbial results.a

Microbial target Concentrations in genomic copies per liter (gc/L)

Cistern Lysimeter 3 Lysimeter 5 Lysimeter 7 Monitoring well

HF183 Bacteroides # Positive: 5/8 # Positive: 6/8 # Positive: 7/8 # Positive: 7/8 # Positive: 7/8
p50: 8 p50: 1008 p50: 1920 p50: 1456 p50: 38
p75: 121 p75: 1350 p75: 7406 p75: 3425 p75: 141
Max: 96,429 Max: 2132 Max: 10,314 Max: 46,496 Max: 367
Rotavirus group A (NSP3 gene) # Positive: 2/8 # Positive: 0/8 # Positive: 1/8 # Positive: 1/8 # Positive: 0/8
p50: ND p50: ND p50: ND
p75: 5 p75: ND p75: ND
Max: 11,213 Max: 14,428 Max: 5141
Pepper mild mottle virus (PMMV) # Positive: 5/8 # Positive: 2/8 # Positive: 1/8 # Positive: 1/8 # Positive: 0/8
p50: 17 p50: ND p50: ND p50: ND
p75: 212 p75: 10,507 p75: ND p75: ND
Max: 21,013 Max: 43,027 Max: 218,783 Max: 9635
Cryptosporidium spp. # Positive: 2/7 # Positive: 4/7 # Positive: 2/7 # Positive: 0/7 # Positive: 0/7
p50: ND p50: 887 p50: ND
p75: 0.8 p75: 1330 p75: 1677
Max: 6 Max: 15,260 Max: 5930
Bacteroidales like Hum M2 # Positive: 1/7 # Positive: 0/7 # Positive: 1/7 # Positive: 1/7 # Positive: 1/7
p50: ND p50: ND p50: ND p50: ND
p75: ND p75: ND p75: ND p75: ND
Max: 55 Max: 513 Max: 9805 Max: 6
a
ND = no detection. p50 = 50th percentile, p75 = 75th percentile. Table excludes adenoviruses, enterovirus, hepatitis A, human polyomavirus, and norovirus genogroups I and II, as all
non-sewer results were negative. Cryptosporidium and Bacteroidales like Hum M2 were not analyzed during the first round of sample collection in November 2016 (n = 7).

and water quality parameters (Kendall's tau (τ)) as well as differences
in total microbial sum between ordinal categories of precipitation and
water quality parameters (Kruskal-Wallis test). Two-sided p-values
<0.05 were considered significant. Due to a skewed and wide distribu-
tion, microbial sum was log transformed to aid visual interpretation; a
small value (0.01 gc/L) was assigned to microbial sum non-detections
in visual plots. All analyses were conducted using SAS 9.4 (SAS Institute
Inc. Cary, North Carolina).
3. Results
3.1. Microbial results
Enterovirus, human polyomavirus, and norovirus genogroup II were
detected at high concentrations in the sewer (1.3 × 105 gene copies L-1,
6.8 × 105 gene copies L-1, and 1.6 × 107 gene copies L-1, respectively),
but were not found in any other sampling location throughout the re-
mainder of the study. The microbial results from the sanitary sewer
are included in the SM. At the other sampling locations (cistern, lysim-
eters, monitoring well), five of 12 microbial targets were detected more
than once across the study period and at multiple sampling points
(Table I). HF183 Bacteroides was the most frequently detected microbial
target, with 80% positive samples overall. Percent detection was lowest
in the cistern (63%). The highest median concentrations of HF183
Bacteroides were found in the lysimeters. Bacteroidales-like Hum M2
was detected only once at all sampling sites except Lysimeter 3; the
highest concentration was found in Lysimeter 7. Cryptosporidium spp.
was detected most frequently and at the highest concentration in Ly-
simeter 3 (57% positive), with 29% of results positive from the cistern
and Lysimeter 5. It was not detected in Lysimeter 7 or the monitor-
ing well. Rotavirus group A (NSP3 gene) was detected in 25% of
samples from the cistern and in 13% of samples from both Lysime-
ters 5 and 7. It was not detected in Lysimeter 3, the monitoring
well, or the city sewer. Pepper mild mottle virus (PMMV) was de-
tected in varying concentrations at all sampling sites except for
the monitoring well. The highest frequency of detection was in
the cistern.
3.2. Chemical parameters
All chemicals except two pharmaceuticals were detected in the san-
itary sewer (Table II). Only cotinine and caffeine were detected in the
cistern sample, with the former also being detected in the sample col-
lected from Lysimeter 5. The monitoring well had no detections of phar-
maceuticals, artificial sweeteners, wastewater indicators, or personal
care products.

Table II
Chemical description and results.a

Chemical Description Group MRL (ng/L) Concentration (ng/L)

Sewer Cistern Lysimeter 5

Acesulfame Artificial sweetener Artificial sweeteners 5 31,735 <MRL <MRL

Sucralose 25 46,642 <MRL <MRL
Saccharin 25.0E 15,480 <MRL <MRL
Cotinine Nicotine metabolite Wastewater indicators 3 1911 5.2 5.1
Caffeine Stimulant 12 118,388 18.0 <MRL
Paraxanthine Caffeine metabolite 5 31,127 <MRL <MRL
Carbamazepine Antiepileptic Pharmaceuticals 2 1213 <MRL <MRL
Acetaminophen Analgesic 35.0E 204,774 <MRL <MRL
Trimethoprim Human antibiotic 5.0E 10.9 <MRL <MRL
Sulfamethazine Bovine antibiotic 1 <MRL <MRL <MRL
Sulfamethoxazole Human antibiotic 5.0E <MRL <MRL <MRL
Venlafaxine Antidepressant 5.0E 257 <MRL <MRL
Triclosan Antimicrobial Consumer products 75 411 <MRL <MRL
a
<MRL = less than method reporting limit. The monitoring well was also sampled – all results were < MRL. One sample per location.

5
J.R. de Lambert, J.F. Walsh, D.P. Scher et al. Science of the Total Environment 775 (2021) 145738

Table III
Water quality parameter results.a

Water quality parameter MRL Cistern Lysimeter 3 Lysimeter 5 Lysimeter 7 Monitoring well

Bromide (mg/L) 0.005 # detects: 7/7 # detects: 7/7 # detects: 8/8 # detects: 8/8 # detects: 8/8
Min: 0.02 Min: 0.01 Min: 0.02 Min: 0.02 Min: 0.05
p50: 0.03 p50: 0.03 p50: 0.03 p50: 0.02 p50: 0.16
p75: 0.06 p75: 0.04 p75: 0.04 p75: 0.04 p75: 0.25
Max: 0.06 Max: 0.04 Max: 0.05 Max: 0.04 Max: 0.28
Chloride (mg/L) 0.500 # detects: 8/8 # detects = 7/7 # detects: 8/8 # detects: 8/8 # detects: 8/8
Min: 49.5 Min: 32.9 Min: 55.0 Min: 57.0 Min: 65.9
p50: 94.9 p50: 56.3 p50: 69.7 p50: 73.2 p50: 77.6
p75: 117.5 p75: 75.9 p75: 92.2 p75: 87.3 p75: 96.4
Max: 124 Max: 92.1 Max: 129.0 Max: 116.0 Max: 130.0
Chloride-bromide ratio N/A Min: 1452 Min: 1097 Min: 1536 Min: 1462 Min: 234
p50: 2368 p50: 2317 p50: 2829 p50: 2954 p50: 638
p75: 2987 p75: 2583 p75: 3294 p75: 3242 p75: 1690
Max: 3691 Max: 4572 Max: 4553 Max: 4413 Max: 2476
Nitrate + Nitrate Nitrogen (mg/L) 0.05 # detects: 7/7 # detects: 3/4 # detects: 4/4 # detects: 4/4 # detects: 6/7
Min: 0.3 Min: <MRL Min: 0.35 Min: 1.1 Min: <MRL
p50: 0.62 p50: 0.11 p50: 2.2 p50: 1.8 p50: 0.63
p75: 0.89 p75: 0.44 p75: 4.4 p75: 4.3 p75: 0.93
Max: 1.2 Max: 0.74 Max: 6.3 Max: 6.2 Max: 1.6
Escherichia coli (MPN/100 mL) 1 # detects: 6/6 # detects: 6/6 # detects: 5/6 # detects: 5/6 # detects: 0/6
Min: 2 Min: 52 Min: <MRL Min: <MRL
p50: 20 p50: 845 p50: 4.0 p50: 60.0
p75: 41 p75:4400 p75: 8.0 p75: 1200
Max: 190 Max: >MRL (>24,000) Max: 41.0 Max: 1700
Total coliform (MPN/100 mL) 1 # detects: 5/5 # detects: 5/5 # detects: 5/5 # detects: 5/5 # detects: 4/5
Min: 610 Min: >MRL (>24,000) Min: 180 Min: 2500 Min: <MRL
p50: 13,000 p50: >MRL (>24,000) p50: 990 p50: >MRL (>24,000) p50: 160
p75: 24,000 p75: >MRL (>24,000) p75: 8200 p75: >MRL (>24,000) p75: 640
Max: >MRL (>24,000) Max: >MRL (>24,000) Max: >MRL (>24,000) Max: >MRL (>24,000) Max: 2400
Potassium (mg/L) 0.50 # detects: 8/8 # detects: 8/8 # detects: 8/8 # detects: 8/8 # detects: 8/8
Min: 2.2 Min: 3.9 Min: 3.2 Min: 3.0 Min: 1.2
p50: 4.0 p50: 5.4 p50: 3.5 p50:4.6 p50: 2.7
p75: 4.4 p75: 7.5 p75: 4.7 p75: 5.7 p75: 3.4
Max: 5.5.5 Max: 8.52 Max: 5.4 Max: 8.5 Max: 15.0
Sulfate (mg/L) 0.50 # detects: 8/8 # detects: 5/5 # detects: 5/5 # detects: 5/5 # detects: 7/7
Min: 3.83 Min: 1.6 Min: 4.4 Min: 17.1
Min: 5.1
p50: 9.0 p50: 3.2 p50: 5.9 p50: 6.4 p50: 38.2
p75: 12.7 p75: 6.3 p75: 8.3 p75: 8.9 p75: 113.0
Max: 13.5 Max: 7.5 Max: 16.3 Max: 15.3 Max: 114.0
Boron (mg/L) 20 # detects: 5/8 # detects: 7/7 # detects: 5/8 # detects: 5/8 # detects: 7/7
Min: <MRL Min: 21.9 Min: <MRL Min: <MRL Min: 33.1
p50: 24.9 p50: 28.3 p50: 21.4 p50: 23.4 p50: 123.0
p75: 29.6 p75: 45.1 p75: 25.0 p75: 32.7 p75: 155.0
Max: 30.8 Max: 51.4 Max: 35.8 Max: 81.9 Max: 194.0
a
<MRL = less than method reporting limit. p50 = 50th percentile, p75 = 75th percentile.

3.3. Precipitation 3.4. Water quality parameters

Overall, a positive relation between total microbial sum and precip- Boron, chloride, Cl\\Br ratio, nitrate, potassium, sulfate, E. coli, and
itation was both visually apparent and statistically significant (Fig. 3a). total coliform were assessed in relation to total microbial sum. When
Within sampling locations, higher microbial sum was generally found combining results from all sampling locations, significant correlation
at higher precipitation amounts for the lysimeters only, but relations was found between microbial sum and Cl\\Br ratio (highest correlation
are more difficult to discern due to small sample size (Fig. 3b). coefficient of about 0.42), E. coli (0.26, borderline significant), and sul-
fate (−0.31, negative correlation) (Table IV). Microbial sum by tertiles
of these three general chemistry parameters are shown in Fig. 4; differ-
ences between the tertiles are the most obvious for Cl\\Br ratio. When
Table IV stratified by sample location, no strong or consistent monotonic or lin-
Kendall's Tau correlation coefficients for microbial sum vs. water quality parameters (all
ear relations between total microbial sum and assessed water quality
sampling locations combined).a
parameters were visually apparent in scatterplots (Fig. S1). However,
Parameter # Correlation coefficient (p-value) small sample sizes restricted the ability to visualize location-specific
Boron 38 −0.2093 (p = 0.0717) relations.
Chloride 39 −0.1075 (p = 0.3386)
Chloride-Bromide ratio 38 0.4175 (p = 0.0002)
4. Discussion
Nitrate 26 0.0373 (p = 0.7910)
Potassium 39 0.2000 (p = 0.0750)
Sulfate 30 −0.3120 (p = 0.0165) Stormwater infiltration practices have the potential to promote
Escherichia coli 30 0.2582 (p = 0.0513) recharge to groundwater on the urban landscape, reduce runoff, and
Total Coliform 25 0.2398 (p = 0.1100) limit the travel of pollutants to surface waters and wetland systems
a
Dependent variable = log microbial sum. Sampling location entered as random effect. (Weiss et al., 2008). However, our understanding of the effects of

6
J.R. de Lambert, J.F. Walsh, D.P. Scher et al.
Fig. 3. Scatterplots of log microbial sum results by precipitation category: a) overall (n = 40) and b) by sampling location (n = 8)a. aDiamond represents mean value. Transparency is set so
that darker data points represent multiple results. K-W test = Kruskal-Wallis test.
infiltration on underlying aquifers is limited. Several factors affect the
potential for groundwater pollution, including land use practices, infra-
structure design, and the hydrogeologic setting (Edwards et al., 2016;
Nieber et al., 2014). Previous studies have found contamination from
stormwater infiltration in groundwater and drinking water sources
when infrastructure is poorly managed (Edwards et al., 2016). Other
studies have observed chemical changes in groundwater below infiltra-
tion basins (Datry et al., 2004). The results of this monitoring study may
help address data gaps in the understanding of stormwater infiltration
and groundwater interaction, particularly for microbial contaminants.
Aside from the sanitary sewer, the highest frequencies of detection
and highest concentrations of microbial targets were observed in the
cistern and three lysimeters, with lower frequencies and concentrations
of compounds observed in the monitoring well (Table I). Rotavirus,
Fig. 4. Vertical boxplots of log microbial sum by chloride-bromide ratio, Escherichia coli (E.coli), and sulfate tertilesa. aK-W test = Kruskal-Wallis test. Diamond = mean, horizontal line =
median.
7
Science of the Total Environment 775 (2021) 145738
PMMV, and Cryptosporidium spp. were not detected in the monitoring
well at any sampling event. HF183 Bacteroides was detected in the mon-
itoring well at multiple sampling events, but in concentrations much
lower than those measured at the lysimeters or cistern. The decreases
or absences of microbes in the monitoring well relative to the lysimeters
overall may be attributable to reductions via transit across the low per-
meability sediment that lies atop the sand in which the monitoring well
is finished, rather than reductions related to percolation through the
sandy vadose zone. However, the consistent decrease in Cryptosporid-
ium spp. detections with lysimeter depth, combined with the fact that
protozoa are one and two orders of magnitude larger in size than bacte-
ria and viruses, respectively (Blaschke et al., 2016), indicate that the re-
duction may be attributed to movement through the coarse sand of the
vadose zone. In addition, the groundwater well has a screen that is open
J.R. de Lambert, J.F. Walsh, D.P. Scher et al.
to a 10-ft profile of the aquifer approximately 3-ft below the water
table; samples from the groundwater well may have water recharged
from the infiltrating stormwater but also may have recharge from
non-infiltration gallery areas. Thus, lower microbial concentrations are
also likely related to dilution of vertical recharge from the infiltration
gallery with water recharged from non-infiltration areas.
Similar to the microbial results, chemical parameters reflective of
wastewater were observed in the single samples from the cistern and
Lysimeter 5, but none were detected in the sample from the monitoring
well (Table II). It is likely that as infiltrating stormwater moves down-
ward through the vadose zone, eventually reaching the water table, mi-
crobiological contaminants are lagged by relatively slow transit through
the clay-rich sediments that occur between the base of the lysimeters
and the monitoring well screen. Even then, some presence of the dis-
tinctive stormwater chemical parameters would have been expected.
However, when the well is sampled stormwater infiltration is expected
to mix with groundwater recharged from non-infiltration gallery areas.
In the process, particulate and dissolved contaminants, including mi-
crobes and chemical compounds, are reduced due to dilution, as well as
sorption, predation and dispersion that occurred during travel through
the vadose zone.
Microbial concentrations varied across sampling events and differ-
ences in concentration by precipitation amount indicate that rainfall
events are one driver in this variability. It has been observed that low
ionic strength water, typical of rainfall, may enhance virus transport
(Hunt and Johnson, 2016). Larger studies examining the role of precipita-
tion and recharge events on microbial contamination of both stormwater
and groundwater could be conducted to further assess this connection. In
addition, a more in-depth analysis of the travel of contaminants through
the vadose zone will be necessary in understanding the complete picture
of fate and transport in these systems. Because extensive microbial sam-
pling is time consuming and expensive to implement, we also examined
whether water quality parameters that are frequently part of routine
analysis may serve as predictors of microbial contamination. Although
sparse data created limitations, we found a significant, positive relation
between high Cl\\Br ratio and microbial load (Table IV). This may reflect
on the relative contribution of different sources of salinity within the
storm watershed. Ratios in excess of 250–300 are reflective of human-
caused sources, such as human or animal waste, water softener effluent
and road salt, with higher ratios like those observed at the cistern and ly-
simeters likely related to increasing amounts from the latter two sources
(Mullaney et al., 2009). This relation and other potential relations be-
tween microbial concentrations and standard water quality parameters
could be evaluated further in future studies with larger sample sizes
and higher sampling frequencies to determine whether any may serve
as a reasonable indicator of microbial contamination risk from urban
stormwater, although Hunt et al. (2010) showed that the extreme sensi-
tivity of the pathogen analytical methods generally makes them superior
tracers.
There are important study limitations. The sanitary sewer line
present through the site presents an obstacle to definitive conclu-
sions about the source of microbiological contamination. Many of
the contaminants found in urban stormwater are identical to those
found in human sewage (Fairbairn et al., 2018). Without further in-
vestigation, it cannot be determined with certainty whether the san-
itary sewer is leaking and infiltrating contaminants to nearby
groundwater, although a recent video inspection of the line showed
no obvious flaws or leaks (City of Roseville, written communication,
2016). Moreover, the fact that three microbial contaminants (en-
terovirus, human polyomavirus, and norovirus genogroup II) were
detected in high concentrations in the sanitary sewer but not any
other sampling points indicates the sewer was not leaking. The
Cl\\Br ratios observed in samples from the monitoring well largely
fell in the range of waters affected by human or animal waste
(Mullaney et al., 2009). This indicates that leaking sewers may
have generally affected groundwater in the vicinity at one time,
8
Science of the Total Environment 775 (2021) 145738
though it is unclear if such leakage occurred in the past or from a dif-
ferent source, or if such leakage may have infiltrated the storm water
piping at some point in space or time. In addition, extrapolation of
study results to settings containing active pumping wells would
need to be cautioned, as this study design could not account for influ-
ences caused by well pumping, such as downward vertical hydraulic
gradients within a pumping well's zone of influence and mixing of
waters from within its zone of contribution. A further discussion of
the study limitations is included in the SM.
5. Conclusions
Stormwater infiltration projects have several environmental bene-
fits, including the capturing of total phosphorus, total suspended solids,
heavy metals, petroleum products, and other contaminants before they
enter nearby lakes and wetland systems. They can also conserve mil-
lions of gallons of potable water each year by reusing stormwater for ir-
rigation and aquifer recharge. However, the compounds present in
stormwater have the potential to affect surrounding areas when infil-
trated. Several microbial contaminants were detected throughout the
study period at multiple sampling points, with the lowest number of de-
tections and microbial concentrations observed in the monitoring well.
Wastewater indicators were detected in samples from the cistern and
from Lysimeter 5. A positive relation between microbial sum and pre-
cipitation was found to be statistically significant (Fig. 3a). Additionally,
a significant correlation was observed between microbial sum and
Cl\\Br ratio. The results of this study indicate that subsurface in compa-
rably sensitive geologic settings may be susceptible to contamination by
microbes and other wastewater-related contaminants when in proxim-
ity to subsurface stormwater infiltration and especially during periods
of substantial recharge.
In contrast, aquifers overlain by fine-grained sediments offering
moderately protective geology are expected to be less susceptible
to these contaminants. However, contaminants still have the po-
tential to reach these aquifers, perhaps at a longer time of travel
and in reduced concentrations. For example, John and Rose
(2005) described inactivation rates for a variety of microbes in
the 0.02–0.1 log 10day−1 range, indicating months may be required
to inactivate these constituents in some groundwater settings. As
geologic protection increases, the time it takes water to travel
from the stormwater discharge point to the aquifer is expected to
increase, therefore reducing the risk of contamination when com-
pared to shallower, more vulnerable aquifers.
In sum, when in proximity to subsurface stormwater infiltra-
tion, geologically sensitive aquifers may be susceptible to contam-
ination by the same microbes and wastewater compounds found in
storm water, particularly following rain events or an increase in re-
charge. Further, Cl\\Br ratio may be a useful indicator of microbial
load, in the absence of more advanced analytical technologies. The
presence of low permeability sediments above an aquifer can be an
important barrier for reducing contaminant loads. These results
may help inform siting decisions relative to urban storm water in-
frastructure and nearby drinking water supply sources and their
protection areas, although the water quality effects that might be
altered by the pumping effects of water supply wells could not be
evaluated in this study design.
CRediT authorship contribution statement
Jane R. de Lambert: Conceptualization, Methodology, Investiga-
tion, Writing – original draft. James F. Walsh: Conceptualization,
Supervision, Writing – original draft. Deanna P. Scher: Formal anal-
ysis, Visualization, Writing – original draft. Aaron D. Firnstahl:
Methodology, Investigation, Writing – review & editing. Mark A.
Borchardt: Conceptualization, Validation, Resources, Writing –
review & editing.
J.R. de Lambert, J.F. Walsh, D.P. Scher et al.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the Clean Water Fund of the Minnesota
Clean Water, Land, and Legacy Amendment. The authors thank Capitol
Region Watershed District, especially Britta Belden, Forrest Kelley, and
Sarah Wein, and the city of Roseville for their cooperation and partner-
ship on this project, and Walter Eshenauer of SRF Consulting Group, Inc.
for bringing this project to our attention and facilitating its creation. We
also wish to thank the Minnesota Pollution Control Agency for agreeing
to install the monitoring well at this site and for assisting with sample
collection during the study and sharing of analytical data. Finally, thanks
to Luke Pickman for his help with figure creation. Any use of trade, firm,
or product names is for descriptive purposes only and does not imply
endorsement by the U.S. Government.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.145738.
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