1. Introduction
Presence of enterococci in the pumping sites for drinking water supply of Bokanjac–Poličnik karst aquifer is an indicator of faecal pollution which is very dangerous for the environment and people living in that area. The Bokanjac–Poličnik aquifer is burdened by pollution caused by seepage of polluted water from the surface (septic tanks). The die-off coefficient of faecal indicator bacteria, such as enterococci (ENT), in typical conditions of groundwater flow through the karst aquifer (fracture porosity) is unknown. The objective of this study is to define appropriate methodology for determining the die-off coefficient of faecal indicator bacteria, ENT, when transported in a karst environment. Besides that, additional goals are to determine self-purification capacity (sorption and die-off expressed with associated coefficients) in case of incident events or continuous pollution processes and to determine the dominant process in removal of ENT in the observed karst environment. The proposed methodology strives to cope with limited amount of available hydrological, hydrogeological and biochemical data on the analysed karst aquifer.
Karst aquifers are formed in a soluble rock, usually limestone, and they are characterised by distinctive heterogeneity in a sense of structure and indentedness of rock mass, the so-called matrix, inside which one could find different karst phenomena such as karst sinkholes, springs, caverns, fractures and caves. Over the years, many researchers dealt with the conceptual solutions and the idea of structural schema of karst [
1,
2,
3,
4,
5]. In general, a karst system could be divided into four subsystems:
Soil: surface, infiltration part over which aquifer is water-supplied—water recharge can be autogenous, allogeneic, diffuse or concentrated, depending on the characteristics of the surface layer.
Epikarst: subsurface layer which stores part of the infiltrated water; it accepts the water which is drained through the vertical flow in a unsaturated zone.
Vadose zone: connects subsurface layers with a saturated zone;
Saturated zone: it consists of a low permeable matrix and a net of highly permeable karst channels and fractures.
In a karst environment, groundwater flow is mostly conducted through fractures and karst channels where quite high flow velocities can be achieved. Although the matrix can be very porous due to small primary permeability, it has no contribution to the flow to a large extent, which is why diffuse flow happening inside of it, is often neglected. On the other hand, the retention capacity of the matrix is quite big and inside its huge amounts of water can be stored; that water is gradually seeped towards springs, fractures and karst channels. Therefore, karst rock and aquifer are characterised by triple porosity (the matrix, fractures, karst channels), laminarly-diffuse flow inside the matrix and turbulent flow inside the karst channels and fractures.
Different types of mathematical and numerical models could be applied to deal with hydrological and ecological problems of karst aquifers with dual and/or triple porosity depending on available data and identified problems. Distributed parameter models are often applied to systems of karst aquifers [
6,
7,
8,
9]. Those types of models are usually applied to places where enough data about the features of aquifer is gathered. Those features include permeable and non-permeable boundaries, zones of inter-layer seepage, positions of faults, springs and sinks. Distributed parameter models require division of the aquifer into small representative three-dimensional volumes such as finite difference cells, or finite element cells. Basic hydraulic features of aquifer (hydraulic permeability, transmissivity, storage, effective and total porosity) are spaced and can be distinguished and changed for every model cell or element. Various numerical strategies have been used in karst aquifers analysis over the years. The simplest approach is the one which assumes that karst aquifer conducts oneself equivalently towards the medium of intergranular porosity in the conditions of flow and transport. The approach is called single continuum porous equivalent (SCPE) model. SCPE is based on the acquisition of the continuum scheme and flow simulation by the potential flow equation, and the model itself simulates laminar flow. Relevant research in [
10,
11,
12,
13,
14,
15,
16] are just a few examples of successful implementations of the SCPE approach when it comes to researches of water resource. The approach is based on the fact that higher values of hydraulic conductivity are given to the model cells for which it is known or assumed that go through the fractures compared to the surrounding cells which therefore represent a weakly permeable matrix. Computer codes mostly used are different variants of the MODFLOW software.
The research which dealt with controlled discharge of sewage water from septic tanks showed the importance of enough thickness of the unsaturated zone. It also showed that the concentration of the faecal indicator bacteria is largely decreased with the flow heading towards the depth [
17,
18]. However, the research also indicated that the transport of the bacteria through the soil intensified due to more pronounced precipitation, and the increased concentration of bacteria on the pumping sites coincided with the periods of intensive precipitation [
19,
20]. In case that bacteria reach the saturated zone relatively quickly and in high concentration, it will represent danger for the ecosystem in which they are found. Several studies [
21,
22,
23,
24,
25] have shown that the microorganisms and colloids can easily be transported by preferential flows. Firouzi et al. [
26] analysed transport and bacteria removal in a limestone soil under the saturated conditions. He noticed that the breach of bacteria mass is enlarged with the increase of velocity of groundwater flow. Potential presence of pathogens in groundwater could be discovered by monitoring the presence of faecal indicator bacteria such as ENT. A general model of bacteria transport consists of advection, dispersion and the process of attachment (sorption), detachment (desorption), filtration, blocking, die-off in contact with the porous medium and die-off in a liquid phase [
27]. A high rate of attachment and removal of bacteria on limestone grounds could be ascribed primarily to the fact that calcium carbonate is mostly positively charged when in neutral pH level surroundings which makes it convenient for the attachment of negatively charged bacteria. According to [
27], blockage and detachment do not have a significant effect on bacteria transport, and the most plausible reason is the die-off that is happening on the surface of particles, that again enables the particle surface to be free for attachment. The bacterial die-off rate in a liquid phase is under the influence of factors such as temperature, light, pH level, amount of poisoned substances, dissolved oxygen and protozoa. The die-off coefficients,
k, of the faecal indicator bacteria are defined mainly for the conditions of flow through the aquifer with the intergranular porosity, and with somewhat different values depending on all the above-mentioned factors [
28,
29,
30]. One of the most important factors that affect die-off of the bacteria is the temperature. John and Rose [
31] compared approximately ten studies which dealt with the investigation of the die-off of the Escherichia coli (EC) and ENT in a temperature range from 3 to 37 °C. In a total sample of 35 values, the die-off coefficient of the EC varies from 0.01 to 1.5 day
−1. Higher levels of die-off (above 0.5 day
−1) is noticed with temperatures above 20 °C. According to [
27], despite heterogeneous conditions during different experiments, from the gathered data, it can be concluded that the value of the die-off coefficient increases with the temperature rise. The average die-off coefficient value with the temperature of 10 °C is 0.15 day
−1 that is 0.5 day
−1 with the temperature of 20 °C. Impact of the soil to the die-off rate depends primarily on the local conditions on the catchment, and the effect can be positive or negative which depends on the presence of nutrients, toxic substance, oxygen, etc. Heavy metals have toxic impact on bacteria and with its presence in ground water and soil, die-off level is increased [
32,
33]. The level of die-off of the faecal indicator bacteria in oxygen-saturated water is significantly lower in relation to water with low oxygen saturation [
32].
Based on the above mentioned, one can conclude that the chances for the bacteria removal in karst channels are very small due to preferential flows and high velocities of the groundwater found inside of them. Mechanisms of bacteria removal from water are conducted by percolation through the surface layer, epikarst and unsaturated zone of karst rock where soil pores and percolation velocities are significantly lower in relation to karst channels. Furthermore, it is determined that intensive precipitation can trigger a major flow of water through the epikarst by virtue of binding smaller fractures and creating a continuous flow. In such conditions, hydraulic pressure can mobilise water stored inside an epikarst towards larger fractures, and eventually saturated zone. Such a flow concept is called piston-type flow [
34]. Schwarz et al. [
34] noticed that the largest number of bacteria reaches the saturated zone shortly after intensive precipitation. The phenomenon is also explained because of smaller fractures binding inside the epikarst and moving of the bacteria sedimented within it. Williams [
35] also recognised the piston-type flows when he noticed that the concentration of pollution passing through the epikarst zone exponentially decreases over time. Concentration of the bacteria can be increased with the emergence of “new” precipitation (so-called “pulse” effect). That is why each precipitation can be looked at as a separate event, i.e., as an impulse that triggers pollution towards the saturated zone.
2. Study Site
The Dinaric karst in Croatia, globally known as “locus typicus" of karst landscapes, is characterised by very irregular karstification which is caused by tectonics, compression, reverse faults and over thrusting structures. The research area is generally located between the Adriatic microplate to the southwest and the Dinaric regional structural unit to the northeast. Their contact is represented by reverse faults that strike northeast-southwest [
36]. Eocene and Cretaceous limestone is a main component of the terrain [
37,
38]. Carbonate rocks, clastic rocks and quaternary soil are basic rock masses that build the terrain. The most represented rock from those three is the most permeable one, the limestone. The whole hydrogeological system of Bokanjac–Poličnik covers an area of 235.7 km
2. The Bokanjac–Poličnik aquifer takes around 86% of the Bokanjac–Poličnik catchment, and it spreads around the carbonate rocks formation. The remaining part, around 33 km
2 belongs mainly to non-permeable flysch surfaces [
39].
Stratigraphically, the upper zone of the terrain is shallow and highly karstified, while the deeper zone is characterised as a low permeability rock mass, with a network of karst fractures [
40]. Typical karst shapes in the analysed area are not particularly developed and the most important objects in the hydrogeological sense are active sinks. The most important sink is in Biljane Donje where tracer tests were carried out and where underground connection with the spring Golubinka was proven. It was there that the highest groundwater velocity was measured with apparent values of 8.1 cm/s [
41]. The flow direction goes from the mid and eastern parts of the catchment towards the shore. The flow begins on the higher parts of the relief, parts of Biljane Donje from where water flows towards northwest, and in that way, it forms the Oko spring. In cases of high waters, the water rises from the Oko spring and forms surface flow called Miljašić Jaruga. The flow then continues towards the Boljkovac and Golubinka springs, and due to “hanging” and complete barriers, part of the water is filtrated south towards Bokanjačko blato and Jezerce well. The tracer tests in the area conducted in 2007 [
39] suggests that apparent flow velocities, in the periods of hydrologically low groundwater levels (dry season), are very low compared to other karst terrains. In the periods of low groundwater level, in that morphologically low terrain, gradients are extremely small and water flows extremely slowly. That is why in such dry periods, the groundwater level in wells is rather low, but the inflows are still continuous, and with specific capacity decrease, it can always pump ten to twenty litres per second of the groundwater. Today, for the purpose of water supply, six wells are used. According to the information of municipal corporation in Zadar not a single settlement in the hinterland of Zadar has public sewage system and waste water from the households is treated in septic tanks. Such a way of dealing with waste water presents danger from polluting hydrogeological system Bokanjac–Poličnik and the springs within it.
The data on groundwater levels and concentrations of ENT on the wells in Jezerce and B4 were gathered for the period of 1 January 2012–19 October 2016. Based on [
42], an insight was given into the values of the hydraulic conductivity
K at the depths from 20 to 50 meters on several locations on the catchment (
Figure 1, purple dots). The groundwater level data for that period (1 November 1966–31 June 1967) were also gathered (
Figure 1, red dots).
5. Discussion and Conclusions
This paper presents a methodology for determining the die-off coefficient of faecal indicator bacteria, ENT, in terms of transport through the karst environment. The methodology assumes the successive application of statistical, numerical and analytical modelling in order to define the self-purification capacity of the analysed aquifer (the effects of sorption and die-off expressed by the corresponding coefficients).
With the transfer function, used as a method of time series analysis, a specific answer to the question of a dominant trigger of the enlarged ENT concentrations in the wells was obtained. In addition, time series analysis gave an insight into the average groundwater velocities later used to determine the potential source of pollution. In the applied analytical model, the calculation of ENT concentration removal was divided into two parts, the unsaturated and saturated transport phase. Removal of ENT concentrations in the unsaturated zone, due to the low percolation velocity and small size of the fractures, binds to two processes: natural die-off and sorption. By transporting through a saturated environment where much higher seepage velocities occur, the impact of sorption is negligible and the dominant process of ENT removal is a natural die-off.
The influence of the individual parameters used in the analytical model was analysed by sensitivity analysis. The diagram in
Figure 11 shows the influence of the turbulent filtration coefficient value at variable
. It is evident that
is smaller in the case of a turbulent filtration coefficient of
m/s, which is also confirmed by the lower values of the statistical criterion RMSE. Also, the calculated average seepage velocity (Equation (
9)) for the conductivity coefficient
m/s for the time period 1 January 2012—19 October 2016 is 1.26 cm/s and maximum 2.3 cm/s. Such values are consistent with the apparent velocity values in the area obtained from tracer tests [
40]. Therefore, in further analysis, the value of the turbulent filtration coefficient
m/s was used. The sensitivity analysis for the variable
was conducted for the range of
to
m/s. From
Figure 12 the exponential increase of the RMSE is clearly visible from the value
m/s upwards. The difference of the statistical criterion RMSE between
m/s and
m/s is approximately seven orders of magnitude (RMSE
for
m/s, RMSE ≈ 20 for
m/s). The analysis showed that the percolation velocity of
m/s results in the complete removal of faecal indicator bacteria to the contact with the saturated zone due to the extremely slow percolation which leaves enough time for ENT removal. With such velocity values, ENT to the saturated zone can only break through if ignoring the sorption effect, i.e., if
(Equation (
6)). On the other hand, due to the velocity of
m/s, the percolation is rapid and ENT encounter the saturated zone in virtually unchanged concentration. Therefore, value of the percolation velocity of
m/s was adopted, which, according to [
35], corresponds to the mean velocity in three recognised phases of percolation.
The mean value of the total die-off coefficient by transport through the unsaturated zone in the analysed case is . Within the saturated zone the total die-off coefficient is within the limits of 0.1 and 0.5. From the results of the analysis it can be concluded that the unsaturated zone represents a suitable environment for ENT removal (reduction of six orders of magnitude in relation to the initial concentration) where sorption is the dominant process of natural ENT removal.
Further research is planned to focus on the unsaturated zone and the processes within it, especially from the aspect of removing pollution from water. The significance of the unsaturated zone in pollution removal from the water has been proved by the implementation of this methodology.