Introduction

Groundwater in arid regions is the most used for all activities (human consumption, agriculture, and industry), and therefore plays a very important socioeconomic role for many countries. Groundwater pollution is a serious problem for water managers. Taking measures in this regard is crucial for the proper care of these hydrological systems, which are fundamental for the societies that depend on them. In this sense, studies aimed at assessing the chemical and microbiological quality of water and its suitability for use (Ghalib 2017) are of extreme importance for regional development. Hydrological systems are vulnerable to pollution from point sources (feedlots, dairies, landfills, etc.) and diffuse sources such as agrochemicals and extensive livestock farming. Nitrates and micro-organisms are considered valuable indicators in different pollution scenarios. Nitrate is a valuable indicator in oxidising media and its natural background value (NBV) allows managers to better assess anthropogenic impacts. In the Pampean sedimentary plain of Córdoba, the NBV was estimated to be ~ 10 mg/L (Blarasin et al. 2014) for comparison with contaminated sites. The detection of faecal contamination is relevant when groundwater is used for human consumption, as it indicates the possible presence of pathogenic microorganisms and thus the potential risk to health (Ibekwe et al. 2011; Carlos et al. 2012). Although Escherichia coli (E. coli) is the best indicator of faecal contamination, its presence in water does not provide definitive information on its possible origin (Unno et al. 2010; Ibekwe et al. 2011). Phenotypic methods, such as antibiotic resistance profiles of E. coli, are a useful and cost-effective method to differentiate the source of faecal contamination in different aquatic environments (Meays et al. 2004; Gourmelon et al. 2007; Ksoll et al. 2007; Ibekwe et al. 2011). In rural areas, antibiotics are used in veterinary medicine either prophylactically (antiparasitic and antibiotic) or as growth promoters (antibiotics used at sub-therapeutic doses), creating a selective pressure on indicators of faecal contamination. This selective pressure may be a useful criterion for identifying sources of E. coli contamination in water by assessing antimicrobial susceptibility (Ibekwe et al. 2011). In addition, the use of these compounds in agro-ecosystems, coupled with the intensification of livestock production in small areas, increases surface and groundwater contamination with the above-mentioned substances (Kummerer 2003; Cirelli and Mortier 2005; Pruden 2009). Therefore, an integrated study that includes the analysis of antibiotic resistance profiles in relation to physico-chemical and hydrological characteristics and land use could provide a more complete result in aquifer contamination studies.

Objective

The objective of this study is to show the usefulness of chemical and microbiological indicators in the analysis of the impact generated by agricultural activities on groundwater in contrasting hydrogeological and land use situations.

Study area

Climatic, geologic, and geomorphologic characteristics

The research area (~ 500 km2) is located in the South of Córdoba Province (Argentina), about 80 km east to the Comechingones Mountains (Fig. 1). It is a vast rural area in the Pampean plain, apt for farming and cattle raising. There are also some small villages and towns, being San Basilio the most important with 7,987 inhabitants, according to NISP (National Institute of Statistics and Population) 2022 Survey. For all activities, the groundwater resource is used, being the unconfined aquifer the most consumed.

Fig. 1
figure 1

Location map of the study area

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The climate is subhumid-dry mesothermal, with an average rainfall of 801 mm (1945–2018), which is concentrated mainly in spring–summer (80%). The average potential evapotranspiration is 827 mm, which defines a water deficit in the order of 26 mm per year. However, a sequential monthly water balance shows notorious water excesses (< 198 mm), which mainly occur in the spring–summer seasons of the most humid years (Cabrera et al. 2017).

The Pampean plain of southern Cordoba is part of a sedimentary basin whose varied topography is related to the presence of differentially tilted and sunken structural blocks. This basin has suffered tectonic inversion from an extensional regimen at the end of the Mezosoic to a compressive one in the Paleogene (Chebli et al. 1999). The plain covers part of the sedimentary basin called Chacoparanaense basin and minor depocenters filled with continental, alluvial, and lacustrine sedimentary successions (Webster et al. 2004). In the litho-stratigraphical column up to 100 m depth there is a predominance of Quaternary aeolian sediments (very fine silty sands) with layers of cemented silts (calcretes), fine-medium sands, and clayish sands that are subordinated and associated to the fluvial belts.

The most outstanding geological-geomorphological characteristic of the research area is linked to the Tigre Muerto regional fault (N–S direction) that dips to the South/South East. This regional fault has originated a notorious incidence in the topography, which in turn has conditioned the pre-quaternary and quaternary sedimentary processes and the regional hydrological behavior (Cabrera et al. 2010; Blarasin et al. 2014). To the West, this fault defines the Lower Block Geomorphological Unit (LB), an area characterized by a very gently undulating relief that tilts to the South. In this area, the main deposits are made up of aeolian sediments (very fine silty sands) and fine-medium sands of the fluvial belts. To the East, the Upper Block Unit (UB) looks like a large asymmetrical hill with a flat top, which generates surface water and groundwater divides. The predominant materials are also Quaternary aeolian sediments (loessical type deposits) (Fig. 2).

Fig. 2
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Geological-geomorphological map (Cabrera et al. 2010)

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Hydrolithology and hydrodynamics

The unconfined aquifer, whose thickness is 75–100 m, is composed of fine aeolian Quaternary sediments characterized by a hydraulic conductivity (K) and an effective porosity (ρ) in the order of 1–5 m/day and 5–10%, respectively. Both surface and groundwater flow directions are coincident. In the LB Unit, the water flows to the South (Fig. 3), highlighting groundwater outcrops in local discharge sectors (lagoons and flooded areas). The streams are also preferential discharge zones of the aquifer. On the other hand, the UB Unit constitutes a preferential recharge zone for the unconfined aquifer. In this area water flows diverge from the top of the block towards the base (SW and SE). The hydraulic gradients are in the order of 0.2–0.3% and the groundwater flow velocity range is between 0.04 and 0.2 m/day.

Fig. 3
figure 3

Equipotential lines and water table depth lines for the unconfined aquifer of the study area (Cabrera et al. 2010)

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Materials and methods

Human activities and land uses were surveyed for the analysis of the pollution threat. Those located near the sampling sites (300 m in the surrounding area) were especially take into account. The vulnerability of the unconfined aquifer before the imposition of pollutant loads derived from the agro-ecosystem was analyzed based on the lithology of the unsaturated zone (UZ) and depth of the water table (WT).

Hydrological parameters were measured in 34 wells of the unconfined aquifer system. The multiparameter probe (Hanna HI 9828) was employed to record temperature, pH, electrical conductivity (EC), and dissolved oxygen (DO). The depth of the water table was measured with a piezometric probe (Solinst).

Water sample collection and chemical and microbiological variables analyzed

Based on standards guidelines of hydrogeological surveys, each well was properly purged prior groundwater sampling. Also, samples collection bottles were treated, cleaned and labeled before sampling. The chemical characteristics of groundwater were measured in the Geochemistry Laboratory of the Geology Department (National University of Rio Cuarto) following Standard Methods for Examination of Water and Wastewater (APHA/AWWA/WEF 2017). The analyzed chemical variables were: CO32−–HCO3−, Cl−, Ca2+, and Mg2+ through titration; SO42− by spectrometry; Na+ and K+ by flame photometry, and NO3− using an ion selective electrode method.

The water samples (500 mL) were taken for bacteriological indicator analysis according to the Argentine Food Code (AFC 2012). All samples were collected aseptically in sterile bottles, stored at 4 C and analyzed within 24 h of collection in the Microbiology Laboratory of the National University of Rio Cuarto. The determination of heterotrophic plate counts (HPC) was carried out in plate count agar (24 h incubation at 35°C). The total coliforms (TC) and thermotolerant or faecal coliforms (TTC) were determined by the multiple-tube fermentation (MTF) technique. The number of coliform organisms in 100 ml of water and the most probable number (MPN) were determined by Probably tables (McCrady tables). The TC were incubated in MacConkey broth (Britania, Argentina) at 35 °C for a period of 24–48 h and TTC in BRILA broth (brilliant green 2% bile lactose broth) (Britania, Argentina) at 44.5 °C for 24–48 h. The presence of E. coli was determined in 100 mL of the sample in MacConkey broth incubated at 35 °C for 24–48 h. Then, an aliquot was spread onto EMB agar (eosin methylene blue) (Britania, Argentina) plates and incubated at 35 °C for 24 h. Isolates were confirmed as E. coli by using a series of biochemical tests, including indole, methyl red, Voges-Proskauer tests, and the inability to grow on citrate agar (IMViC test) (Britania, Argentina). The presence of Pseudomonas aeruginosa (Pa) was determined on a volume of 100 mL of sample in asparagine broth incubated at 35 °C for a period of 24–48 h. The isolation was carried out in cetrimide agar (Britannia, Argentina) plates and colonies were confirmed by the following biochemical tests: oxidase, growth at 42 °C, and pigment production in pseudomonas agar P and F (Britannia, Argentina). The methodology was carried out according to APHA/AWWA/WEF (2017) and AFC (2012).

Isolation of microorganisms for resistance to antibiotics

The isolated and identified strains of E. coli were evaluated for resistance to antibiotics by the plaque diffusion method using ten antibiotic discs (Bauer et al. 1966; Gambero et al. 2016). They correspond to the drugs most commonly used in the treatment of infections caused by gram-negative bacilli in both humans and animals and on their use as a food additive and as growth promoters in animals according to Laplumé et al. (2011) (Table 1). An E. coli inoculum was prepared in tripticasa soya broth (Britania, Argentina) of approximately 2 × 108 cfu/mL, whose turbidity corresponded to tube number 0.5 on the McFarland scale. Therefore, 200 µL of the cultivation was placed in 5 mL of sterile physiological solution and the optical density (600 nm) of each mixture was adjusted to about 0.08. The bacterial suspension was inoculated onto plates with 150 mm of Mueller Hinton agar (Britannia, Argentina) and then the commercial antibiotic (Britannia, Argentina) discs were put in place. The plates were incubated at 35 °C for 18–20 h. Diameters (in mm) of the clear areas of growth inhibition around each antibiotic disk were measured with a precision caliper. The criterion of sensitivity or resistance to each antimicrobial was determined as established by CLSI (Clinical and Laboratory Standard Institute 2017). E. coli strain ATCC 25922 was used for routine quality control assay.

Table 1 Tested antibiotic
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Statistical analysis

For statistical analysis of bacterial contents and physicochemical variables values, the SSPS v.11.5 package was used. The multivariate analysis was performed using the factorial method by principal components (PC) to determine possible relationships between bacterial contents (TC– TTC– E. coli – Pa) and physicochemical variables (EC, T, DO, pH, HCO3−, SO42−, Cl−, Na+, K+, Ca2+, Mg2+ and NO3−). The variable TDS (total dissolved salts) was not considered in the multivariate analysis, because it is represented by the EC.

Results and discussions

Analysis of pressure factor on groundwater: land use

The most important activity is no-till direct seeding, so the threat of contaminant load to the unconfined aquifer is given by the excessive use of agrochemicals. Livestock is mainly of intensive type (dairy and feedlot cattle). In this case, potential contaminants derive from manure and effluents, which generates a high concentration of organic matter (OM). Also, leachate from fertilizers/pesticides could reach the aquifer (Gambero et al. 2018; Lutri et al. 2020).

Intrinsic vulnerability to contamination

In the LB Unit, the unconfined aquifer is more susceptible to contamination due to the shallow WT (0–6 m) and the presence of coarse-grained sediments in the UZ associated with the fluvial environments (Fig. 3).

By contrast, in the UB Unit the vulnerability of the aquifer system to be contaminated decreases due to a deeper WT (> 30 m at the top) and the dominance of fine-grained materials in the UZ. In this context, hydrochemical reactions (degradation, adsorption, etc.) are favored, which reduces the possibility of the eventual arrival of contaminants to groundwater (Figs. 2, 3).

Chemical indicators

In UB Unit, considered a local recharge zone, the groundwater has low salinity (EC: 1045–2050 µS/cm) and is sodium bicarbonate geochemical type. Instead, in LB Unit, groundwater is saltier (2190–6180 µS/cm) and sodium sulfate geochemical type, showing more geochemically evolved groundwater (~ 150 km).

In LB Unit, with the UZ of ~ 3 m thick and groundwater in oxidizing conditions (DO: 1.2–5.1 mg/L), NO3− exceeds the NBV in 75% of samples being sample D8 the one with the highest concentration (260 mg/L). In UB Unit (OD: 2.6–7.0 mg/L), it could be stated that: (a) WT sites < 10 m, show NO3− values that exceed NBV (25–60 mg/L) and, (b) WT sites > 10 m, show very low values (0-6 mg/L) (Fig. 4).

Fig. 4
figure 4

Nitrates concentration and distribution in the unconfined aquifer of the study area

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Microbiological indicators

In the LB Unit (WT < 10 m) the greatest bacterial development was determined, while in the UB Unit, bacteria were only detected in sample D13 (Table 2). Among the TTC bacteria, although the predominant genus is Escherichia coli, there are also Citrobacter, Klebsiella, and Enterobacter, although the last three types have been isolated by several authors (Toranzos et al. 2007) from environmental samples without traces of faecal contamination. These results show that TTC is considered an acceptable faecal contamination index, but less reliable than E. coli.

Table 2 Microbial development
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Antibiotic resistance of E. coli strains

The antimicrobial resistance test of E. coli strains isolated from the unconfined aquifer showed resistance to ampicillin (AMP) and to tetracycline (TET), both antimicrobials commonly used in livestock, and to Cephalotine (CEF), an antibiotic mostly used in humans (Fig. 5). These results would indicate that the main source of faecal contamination would result from livestock activity and, subordinately, from human activity.

Fig. 5
figure 5

Antimicrobial resistance of different strains of E. coli. Example: strain D4 (2) = resistant to CEF and TET (LB)

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Multivariate analysis of hydrochemical and microbiological indicators

The factorial analysis carried out by PC method between chemical and microbiological variables shows PC4 that explains 76.6% of the total variance (Fig. 5). PC1 (44.8%) is determined by EC, SO4−2, Cl−, Na+, K+, Ca2+, Mg2+, and HPC and is inversely linked to the OD. PC1 represents the natural salinization of the groundwater, and explains those samples located in the LB Unit that are saltier, where more bacteria arrive and where the lower groundwater flow velocities control the low DO values. PC2 (11.7%) associates TTC, E. coli, and Pa. It explains a local aspect, such as the presence of these microorganisms in specific contamination scenarios. PC3 (10.7%) collect HCO3− with TC, suggesting bicarbonate as an indicator of the incorporation of CO2 by biological activity that can be associated with the arrival of faecal matter and bacteria to the aquifer. Finally, PC4 (9.4%) is associated with pH and HPC and inversely with NO3−, which would indicate another local situation given by an increase in NO3− and a decrease in pH due to the arrival of organic material (OM), whose association with bacteria also suggests local contamination sites by livestock waste (Fig. 6, Table 3).

Fig. 6
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Factorial analysis by PC of chemical and microbiological variables

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Table 3 Rotated component matrix
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The results show that such studies are very useful for assessing the state of groundwater. However, it can be considered that an important limitation for a better interpretation is the sampling density according to the scale of the survey and the need for monitoring in different seasons.

Conclusions

The main geological fault in the area, which generates a lower and an upper regional structural block, determines the hydrodynamics of the unconfined aquifer, giving rise to regional and local flow lines. This geological situation in turn controls the natural geochemistry of the groundwater. This is evidenced by highly evolved groundwater flows (saltwater) located in the discharge zones (LB Unit), and less evolved flows (freshwater) in the local recharge zone (UB unit). In addition, the structural control is also reflected in the vulnerability of the system to be contaminated by the agricultural activities that are carried out in the study area. Accordingly, more polluted waters were detected in the Lower Block and less polluted waters in the Upper Block. In this context of different contamination scenarios, the multivariate analysis allowed to simplify the analysis of the natural and anthropic processes that affect the unconfined aquifer. Comparing different pollution situations and land uses, it was observed that the values of NO3− and the counts of TC and TTC in wells adjacent to sites of punctual contamination (e.g.: feedlots), industrial and urban (septic wells, dumps, sewage effluents, cemetery) are significantly higher than those presented in diffuse pollution environments (agricultural). It should be noted that there are sites in the agricultural area belonging to the LB that presented an intermediate count of TC and TTC, but did not show the development of E. coli. TTC was considered an acceptable faecal contamination index, but less reliable than E. coli. The antimicrobial resistance test of E. coli strains isolated from unconfined aquifers showed resistance to antibiotics commonly used in livestock and, subordinately, from human activity.