Virus occurrence in private and public wells in a fractured dolostone aquifer in Canada

The array of 22 sampling wells (Table 2) is located approximately 100 km west of Toronto, Ontario, Canada in various urban and rural communities of southern Wellington County (Fig. 1). The area surrounding the City of Guelph (population: 130,000) is dominated by agriculture and small towns. Of the 2,511 farms located in Wellington County in 2011, there were 363 dairy cattle and milk production farms, 423 beef cattle ranching and farming farms, 120 hog and pig farms, and 166 poultry and egg farms, making Wellington County one of the largest agricultural hubs in Ontario. These farms host a total of 142,197 cattle and calves, 236,144 pigs, 5,706,394 hens and chickens, and 248,811 turkeys. To the south of Guelph are two agricultural and residential areas, Arkell and Aberfoyle (combined population: 7,000) and to the northeast is Eden Mills (population: 350) built along the banks of the Eramosa River. Northwest of Guelph are Fergus (population 19,000) and Elora (population: 4,000), the two largest communities within Centre Wellington Township. As Guelph, Fergus, and Elora are larger communities, they have both public water distribution systems and sanitary sewers serving the majority of their populations with septic systems and private wells on the outskirts of the villages and rural areas. Despite the availability of municipal drinking water works in the older sections of town, many residents of Fergus and Elora still rely on private wells and septic systems even within highly populated centres of the communities. The populations of Arkell, Aberfoyle, and Eden Mills are entirely reliant upon private wells and septic systems.

Although all of the communities chosen for this investigation are entirely encompassed by the 6,800 km² of the Grand River watershed, the belt of Silurian dolostone that subcrops beneath the glacial sediments along the Niagara Escarpment and extends from Lake Huron/Georgian Bay to Niagara Falls, Ontario serves as a regionally important aquifer for nearly 800,000 people in southern Ontario including over 200,000 residents of Wellington County, and therefore, groundwater is the primary source of drinking water in the study area. All of the wells chosen for this investigation were completed in the Lower Silurian sequences as described by Brunton (2009) and consist of the Guelph, Eramosa, Goat Island, Gasport, Irondeqouit, Rockway and Merritton formations and regionally bounded by the underlying Cabot Head shale Formation. Both the Guelph and Gasport formations act as the predominant fractured bedrock aquifers in the region. In the study area, the Guelph Formation is characterized by crinoidal grainstones and wackestones, is generally 15–22 m thick, and exhibits a sharp basal contact with the shaley facies and crinoidal grainstones of the underlying Eramosa and Goat Island formations, respectively. Although the Eramosa and Goat Island are water-bearing formations, their relatively lower transmissivities cause them to be considered regional aquitards (Golder Associates 2006). When vertical fractures and preferential pathways in the form of cross-connecting boreholes are not present, the Eramosa and Goat Island formations can limit the transport of some surface-derived contaminants present in the unconfined Guelph aquifer to the underlying Gasport aquifer. The thickness and presence of the Eramosa and Goat Island formations, however, are highly variable in the study area and these formations are sometimes entirely absent (Belan 2010). The 25–70 m of the blue-grey crinoidal grainstones of the Gasport Formation can therefore be in direct contact with the overlying Guelph Formation, which in turn is overlain by various types of glacial deposits that potentially offer a source of attenuation of surface contaminants before they enter the rapid flow regime of the underlying fractured bedrock aquifers. The presence and thickness of both of the Port Stanley and Wentworth Tills is highly variable in the study area throughout southern Wellington County (Fig. 3b,c) and studies investigating the vulnerability of the underlying fractured bedrock aquifers have established that the varying thicknesses and degrees of permeability of these sediments result in the aquifers being highly susceptible to surface contamination (Lake Erie Region Source Protection Committee 2012).

The 22 wells sampled during this investigation (Table 2; Fig. 2) consisted of existing private (PW), municipal supply (MSW), and monitoring (MW) wells completed in the local fractured dolostone aquifers within a 20-km radius of the City of Guelph. Each of the wells was chosen in order to represent the range of well construction and hydrological conditions that might affect the vulnerability of typical bedrock wells to virus and bacterial contamination and the variety of well constructions and pumping volumes common to the study area. Surficial geology data were obtained from Ontario Ministry of Northern Development and Mines (2013). Final selections were based on seven criteria: (1) well type, i.e., private, municipal supply, or monitoring well; (2) well completion, including total depth, length of open interval, and bedrock aquifer segment; (3) the well’s proximity to potential sources such as sanitary sewers, septic systems, animal agriculture operations, or surface water bodies; (4) the spatial distribution across the study area and flow system; (5) surrounding land use, i.e., urban versus rural;( 6) overburden thickness (i.e., the thickness of unconsolidated materials overlying bedrock facies); and (7) accessibility/permission.

Using these criteria alongside the Ontario Ministry of the Environment’s Well Record Database, 17 wells in the towns of Fergus, Elora, Arkell, Eden Mills, and Aberfoyle were chosen to be sampled. In addition to the aforementioned criteria and the Well Record Database, sewer invert elevations in Guelph were plotted relative to the top of bedrock (Fig. 3) and used to select five wells in areas where sewers were constructed into or near the top of bedrock. In the end, a total of 22 wells including 11 private wells, 8 municipal supply wells, and 3 monitoring wells were chosen to be sampled. The characteristics of each well are summarized in Table 2 and so only a brief summary of the wells will be presented here.

In Centre Wellington, six municipal supply wells were sampled, including three wells in Elora and three wells in Fergus. Each well was cased through the overburden sediments into the upper meter or so of the Guelph Formation. The wells were therefore open from the top of the Guelph Formation to varying depths within the Gasport Formation. In contrast to the wells sampled in Centre Wellington, those sampled within the City of Guelph did not consist of any actual municipal supply wells but two large capacity water supply wells operated by the University of Guelph and three monitoring wells. The three 2-inch (5 cm) diameter monitoring wells with varying completion depths are located along the Speed River at a site of known industry-derived contamination with various volatile organic compounds and within 10 m of the main trunk line of Guelph’s municipal sanitary sewer system that was constructed below the local water table and up to 4 m below the top of rock. The two University of Guelph water supply wells located within the centre of the City of Guelph are both 10-inch (25 cm) diameter wells and are pumped at an approximate rate of 280 L min^–1. For the purpose of this study, these wells are considered comparable to municipal supply wells and are labeled as such.

Each of the 11 private wells was located in a small residential or agricultural community outside the City of Guelph and had a unique well completion. In the small Puslinch communities of Arkell and Aberfoyle, four and five private wells of varying completion depths were sampled, respectively. The remaining wells added to the study were two private wells of different total depths and completion years located along the Eramosa River in Eden Mills.

Sampling began in the second week of June 2012 and extended until January 2013. Each of the wells was sampled once for general chemistry and several times for enteric pathogens within the 8-month sampling period. Most wells were sampled monthly for 6 months, while other wells were sampled less frequently due to limited accessibility. Temporal sampling for viruses is required as previous studies have shown virus concentrations in groundwater to be extremely variable (Bradbury et al. 2013). During virus sampling events in July and November 2012, samples for bacterial fecal indicators, E. coli and total coliforms, were also collected.

For the current study, all virus samples were collected by following a standard operating procedure (SOP) developed in accordance with the procedures outlined in Millen et al. (2012). Prior to sampling, all equipment set to come in contact with raw well water was soaked in a 0.52% NaClO solution for 30 min prior to being rinsed with 0.05 M sodium thiosulfate for 5 min. All hoses and tubes were then flushed with three volumes of DI water and covered with parafilm for transport to the sampling site. Samples from municipal supply wells were taken directly from raw water taps within each of the well houses, whereas private wells were sampled from external faucets bypassing water treatment such as filtration units or water softening systems. Monitoring wells were sampled using a Grundfos Redi-Flo2 electrical submersible pump. For all three well types, sampling hoses were connected to a flow-through cell with a YSI 556 multiprobe system to monitor field parameters including pH, temperature, and conductivity during purging. Once these parameters were constant, glass wool filters (Lambertini et al. 2008) were attached to well taps and faucets by sterile half-inch tubing. At one site known to have VOC contamination, filtered water was collected into 55-gal drums and treated at an onsite treatment centre. To maximize virus adsorption to glass wool well water pH levels ≥ 7.5 were adjusted to between 6.5 and 7.0 by injecting 0.5 M HCl before the glass wool filter using a Masterflex precision peristaltic pump. Four of the 22 study wells required pH adjustment during sampling. Mean sample volume was 877 L ± 210 l (±1 SD), range 483 –9,16 L, n = 118.

Glass wool filters were packed on ice and shipped overnight to the USDA Agriculture Research Service in Marshfield, WI for analysis using the procedures outlined by Borchardt et al. (2012). Immediately upon arrival glass wool filters were eluted using 3% beef extract containing 0.05 M glycine (pH 9.5) in the direction opposite of sample flow to maximize collection of bacterial and viral targets (Abd-Elmaksoud et al. 2014). Eluate pH was adjusted to 7.0–7.5 using 1 M HCl and flocculated with polyethylene glycol 8000 (8% wt/vol) and NaCl (final concentration, 0.2 M). This mixture was stirred for 1 h, incubated overnight at 4 °C, and centrifuged at 4200 × g for 45 min. The pellet was resuspended in 2 ml of sterile 0.15 M N₂HPO₄ and the final concentrated sample volume stored at −80 °C.

Samples were analyzed by qPCR on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) for six human viruses (adenovirus, enterovirus, norovirus genogroups I and II, hepatitis A virus, and polyomavirus) bovine polyomavirus, influenza A virus, and C. jejuni following the reverse transcription and PCR procedures described in Borchardt et al. 2012. Primers (IDT, Coralville, IA) and hydrolysis probes (TIB Molbio, Berlin, Germany) are reported in Table 3. PCR inhibition was measured for every sample following the methods of Gibson et al. (2012). Among 118 samples, six were PCR-inhibited; inhibition was mitigated by 1:5 dilution with nuclease-free water. No-template controls were included for every batch of analysis steps: nucleic acid extraction, reverse transcription, and PCR. All no-template controls were negative—i.e., no cycle quantification (Cq) value. Performance parameters of the standard curves are reported in Table 4. Standards consisted of g-blocks (targets > 125 base pairs) and ultramers (targets ≤ 125 base pairs) both supplied by IDT. To facilitate identifying amplicon contamination from the standards, the sequence between primer and probe was scrambled while maintaining the wildtype base composition. One standard of each target with a Cq value near 30 was used as the positive control for every PCR analysis batch.

Glass wool filter performance was evaluated twice with samples collected from sites MW1 and MW3. Poliovirus Sabin type 3 was seeded into the samples and recovery measured following the methods described in Lambertini et al. (2008). Percent recovery for sites MW1 and MW3 were 53 and 39%, respectively, both which fall within the recovery range for enteroviruses concentrated by glass wool filtration (Lambertini et al. 2008).

Collection of groundwater samples for E. coli and total coliforms closely followed the guidelines set forth by US EPA Method 1604 (2002). As groundwater concentrations of E. coli and total coliforms were expected to be low, sample volumes were increased from the suggested 100 to 1,000 ml. Groundwater samples for bacterial analyses were collected in triplicate into autoclaved 1-L HDPE bottles and transported on ice to the University of Guelph.

Samples for E. coli and total coliform analyses were analyzed within 24 h of their collection using vacuum filtration following EPA Method 1604 (2002). Each groundwater sample was filtered onto pre-sterilized 47-mm diameter, grid-marked, cellulose ester membranes and placed on Oxoid CM 1038 Differential Coliform (DC) agar. After 24 h of incubation at 35 °C, the plates were removed and checked for bacterial growth. Pink colonies were counted as total coliforms and blue colonies were counted as E. coli. The plates were incubated for an additional 24 h followed by another round of colony counting. Blank samples were run between each filtration for quality control.

The association between well characteristics and virus levels was initially evaluated by Fisher’s exact test on a 4 × 2 table where the rows were categories of overburden type (gravel, diamicton, sand, none) and the columns represented the dichotomous response (virus detected versus virus not detected). The same analytic procedure was then undertaken separately for each of three continuous-scaled characteristic variables (total depth, overburden thickness, open interval length) to examine their relationship with virus levels. First, the response was treated as dichotomous and a parametric log-binomial regression model was fit to estimate the probability of virus detection across the observed range of values for the well characteristic variable (McNutt et al. 2003). To determine the appropriate representation (linear, quadratic, etc.) of the well characteristic in the log-binomial model, a preliminary step was undertaken where a graphic derived from a nonparametric generalized additive model (GAM) was examined (Hastie and Tibshirani 1990). In the second analysis for each continuous-scaled well characteristic, the data were restricted to wells where virus was detected and robust regression techniques were used to evaluate the association between the characteristic and continuous-scaled virus concentration levels (Huber 1973).

Precipitation data were acquired from the Guelph Turfgrass Institute and the Elora Research Station, University of Guelph. For each date on which sampling took place, precipitation levels were tabulated at lags of 0–7, 8–14 and 15–21 days prior to the sampling date; analyses were carried out separately for the three lag categories. The unit of analysis was a sampling date, and the same analytic approach as described above (GAM, log-binomial regression, robust regression) was used to examine the relationship between precipitation level and virus detection/concentration, except that a different robust regression technique was employed to accommodate the specific characteristics of the data (Yohai 1987). All analyses were performed in SAS 9.4 (SAS Institute Inc., Cary, NC).