Guillaume Lorette
email: lorette.guillaume@gmail.com

Nicolas Peyraube

Roland Lastennet

Alain Denis

Jonathan Sabidussi

Matthieu Fournier

David Viennet

Julie Gonand

Jessica D Villanueva

Tracing water perturbation using NO3-, doc, particles size determination, and bacteria: A method development for karst aquifer water quality hazard assessment

Keywords: karst aquifer, high resolution monitoring, nitrate, bacteria, particle, water quality hazard, water resource management

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. Introduction

Karst aquifers are major water resources, supplying approximately 25% of the world's population (Ford and Williams, 2007). Karst landscapes, associated with anthropogenic activities, are foremost concerns in determining karst water quality hazards specifically in terms of contamination. Since the beginning of the year 2000, researches on the issue of karst aquifers being vulnerable to water quality deterioration has increased. These are presented in the surface mapping studies [START_REF] Andreo | Source vulnerability mapping in carbonate (karst) aquifers by extension of the COP method: application to pilot sites[END_REF][START_REF] Kavouri | PaPRIKa : a method for estimating karst resource and source vulnerability-application to the Ouysse karst system (southwest France)[END_REF][START_REF] Kazakis | Groundwater vulnerability and pollution risk assessment with disparate models in karstic, porous, and fissured rock aquifers using remote sensing techniques and GIS in Anthemoutas basin, Greece[END_REF][START_REF] Marin | Vulnerability mapping and protection zoning of karst springs. Validation by multitracer tests[END_REF][START_REF] Kazakis | Management and research strategies of karst aquifers in Greece: Literature overview and exemplification based on hydrodynamic modelling and vulnerability assessment of a strategic karst aquifer[END_REF][START_REF] Ollivier | Challenges and limitations of karst aquifer vulnerability mapping based on the PaPRIKa method -Application to a large european karst aquifer (Fontaine de Vaucluse, France)[END_REF] and on the results of the analyses of the water temporal evolution at the outlets of karst systems [START_REF] Pronk | Dynamics and interaction of organic carbon, turbidity and bacteria in a karst aquifer system[END_REF][START_REF] Goldscheider | New insights into the transport of sediments and microorganisms in karst groundwater by continuous monitoring of particle-size distribution[END_REF][START_REF] Le Cann | Microbial contamination detection in water resources: interest of current optical methods, trends and needs in the context of climate change[END_REF][START_REF] Schiperski | Relationship between organic micropolluant and hydrosedimentary processes at a karst spring in south-west Germany[END_REF][START_REF] Doummar | Assessment of the origin and transport of four selected emerging micropoluttants sucralose, Acesulfame-K, gemfibrozil, and ioxehol in a karst spring during a multi-event spring response[END_REF][START_REF] Yang | Coupled hydroeochemical evaluation of a vulnerable karst aquifer impacted by septic effluent in a protected natural area[END_REF].

Exokarstic landforms such as sinkholes or sinking stream allow punctual infiltration of pollutants [START_REF] Fournier | Using turbidity dynamics and geochemical variability as a tool for understanding the behavior and vulnerability of karst aquifer[END_REF][START_REF] Kavouri | PaPRIKa : a method for estimating karst resource and source vulnerability-application to the Ouysse karst system (southwest France)[END_REF][START_REF] Huneau | Intrinsic vulnerability mapping for small moutainous karst aquifers, implementation of the new PapRIKa method to Western Pyrenees (France)[END_REF][START_REF] Sivelle | Short-term variations in tracer-test responses in a highly karstified watershed[END_REF]. Fast infiltration and rapid transport of water can occur through fissure and conduits. This is characteristic of karst aquifers, and contribute to having incidences of contamination [START_REF] Vesper | Spring and conduit sediments as storage reservoirs for heavy metals in karst aquifers[END_REF][START_REF] Geyer | Multitracer test approach to characterize reactive transport in karst aquifers[END_REF][START_REF] Hillebrand | Caffeine as and indicator for the quantification of untreated wastewater in karst system[END_REF][START_REF] He | The use of nitrate, bacteria and fluorescent tracers to characterize groundwater recharge and contamination in a karst catchment, Chongqing, China[END_REF][START_REF] Bauer | Well water in karst regions of Northeastern Wisconsin contains estrogenic factors, nitrate, and bacteria[END_REF].

Identifying water resource hazards implies understanding the movement of water from different origins which can be done through the analysis of spring's natural and artificial responses [START_REF] Massei | Transport of suspended solids from a karstic to an alluvial aquifer: the role of the karst/alluvium interface[END_REF][START_REF] Pronk | Dynamics and interaction of organic carbon, turbidity and bacteria in a karst aquifer system[END_REF][START_REF] Heinz | Water quality deterioration at a karst spring (Gallusquelle, Germany) due to combined sewer overflow: evidence of bacterial and micro-polluant contamination[END_REF][START_REF] Goldscheider | New insights into the transport of sediments and microorganisms in karst groundwater by continuous monitoring of particle-size distribution[END_REF][START_REF] Goeppert | Hydrogeological controls of variable microbial water quality in a complex subtropical karst system in Northern Vietnam[END_REF][START_REF] Sivelle | Short-term variations in tracer-test responses in a highly karstified watershed[END_REF][START_REF] Goeppert | Improved understanding of particle transport in karst groundwater using natural sediments as tracers[END_REF].

Firstly, in infiltrations, identifying water origins are important. DOC and NO3 -can be used as infiltration markers. These tracers can be used in assessing the flow of water from soil or unsaturated zone [START_REF] Celle-Jeanton | Contribution of time tracers (Mg 2+ , TOC, δ 13 C-DIC, NO3 -) to understand the role of the unsaturated zone : A case study -Karst aquifer in the Doubs valley, eastern France[END_REF][START_REF] Mahler | Using nitrate to quantify quickflow in karst aquifer[END_REF]Mudarra and Andreo 2011;Mudarra et al., 2011). Specifically, DOC in groundwater is used to assess a water origin [START_REF] Batiot | Total Organic Carbon (TOC) and magnesium (Mg 2+ ): two complementary tracers of residence time in karstic systems[END_REF]Charlier et al., 2011) and as a complementary marker of turbidity in identifying fecal contamination [START_REF] Pronk | Particle-size distribution as indicator for fecal bacteria contamination of drinking water from karst springs[END_REF]. In a rural context, the input of nitrogen, organic or inorganic matter, can lead to an accumulation of NO3 -in soils and unsaturated zone [START_REF] Thomas | Impacts of management and climate change on nitrate leaching in a forested karst area[END_REF]. Leaching of these may result to a nitrate increase at the outlet of kart systems [START_REF] Rowden | Results from big spring basin water quality monitoring and demonstration projects, Iowa, USA[END_REF][START_REF] Stueber | Origin and transport of dissolved chemicals in a karst watershed, southwestern Illinois[END_REF]Pronk et al., 2008;[START_REF] Pu | High-resolution monitoring of nitrate variations in a typical subterranean karst stream, Chongqing, China[END_REF].

The common use of NO3 -and DOC at the outlet of a karst system with high resolution monitoring will not only help in identifying the water origins but also facilitates in evaluating the identified water quality hazards (punctual infiltration and diffuse infiltration). This brought to the second and third concerns: (2) which type of water perturbation needs water quality treatment and particularly, (3) which type hosts bacteria? In these lines, particles and bacteria were used as supplementary parameters.

As for the particles, these can be resuspended due to a pressure wave induced by percolating storm water reaching the phreatic water level (pulse-through turbidity). These can also be introduced into the aquifer from the Earth surface (flushthrough turbidity) as a result of either slow percolation or a more rapid point recharge such as from sinking streams. The nature of turbidity can be determined looking at the particles if these are of autochthonous (originating from the weathering of material) or allochthonous (originating from the Earth's surface) origin [START_REF] Massei | Transport of suspended solids from a karstic to an alluvial aquifer: the role of the karst/alluvium interface[END_REF][START_REF] Massei | Assessment of direct transfer and resuspension of particles during turbid floods at a karstic spring[END_REF][START_REF] Valdes | Analysis of karst hydrodynamics through comparison of dissolved and suspended solid's transport[END_REF][START_REF] Fournier | Using turbidity dynamics and geochemical variability as a tool for understanding the behavior and vulnerability of karst aquifer[END_REF][START_REF] Schiperski | Relationship between organic micropolluant and hydrosedimentary processes at a karst spring in south-west Germany[END_REF].

Particle is an important subject matter as it can induce environmental problems due to its ability to sorb bacteria. There are studies which have demonstrated the increased survival of microorganisms when they are associated with particles [START_REF] Pommepuy | Enteric bacteria survival factors[END_REF][START_REF] Palmateer | Suspended particulates/bacterial interaction in agricultural drains[END_REF]. Some research results suggest that turbidity indicates microbial pathogen presence such as E. coli, enterococcus or total coliforms [START_REF] Ryan | An examination of short-term variations in water quality at a karst spring in Kentucky[END_REF][START_REF] Nebbache | Turbidity and microorganisms in a karst spring[END_REF][START_REF] Pronk | Dynamics and interaction of organic carbon, turbidity and bacteria in a karst aquifer system[END_REF]Pronk et al., , 2009;;[START_REF] Goldscheider | New insights into the transport of sediments and microorganisms in karst groundwater by continuous monitoring of particle-size distribution[END_REF]. However, others did not observed the same findings stating that turbidity is not always accompanied by surface contaminants [START_REF] Dussart-Baptista | Transfer of bacteria-contaminated particles in a karst aquifer: evolution of contaminated materials from a sinkhole to a spring[END_REF][START_REF] Pronk | Particle-size distribution as indicator for fecal bacteria contamination of drinking water from karst springs[END_REF]Pronk et al., , 2009;;[START_REF] Heinz | Water quality deterioration at a karst spring (Gallusquelle, Germany) due to combined sewer overflow: evidence of bacterial and micro-polluant contamination[END_REF]. In these lines, this study proposed a tool that can determine the type of turbidity that is accompanied by contaminants (e.g. NO3 -and microbial pathogens).

The proposed method was applied to the Toulon springs in Périgueux, France. The Toulon springs, as several karst springs, are a water resource being influenced by diffuse pollution and punctual infiltration [START_REF] Lastennet | Geochemical characterization of complex multilayer karstic systems. Springs of Périgueux, France[END_REF][START_REF] Lastennet | Groundwater-flow characterization in a multilayered karst aquifer on the edge of a sedimentary basin in western France[END_REF]. The hydrogeological catchment is mainly rural (agriculture). There is no livestock and few residential areas (less than 3 000 houses). The Toulon springs are experiencing an increase of nitrate for over 60 years. This is a foremost concern as it serves as a major source of drinking water for approximately 55 000 people.

The main objective of this research is to develop a method using high-resolution monitoring that will facilitate in determining origins of water flowing at the outlet of karst system during floods like in the Toulon. These origins are associated with water quality hazards. Specifically, this study would like to: (i) identify the types water quality hazards that springs like in the Toulon are facing brought about by the movement of water from different origins (proposing a conceptual model of transfer of contaminants) and (ii) propose a conceptual model/framework explaining the relationships between NO3 -and DOC in this karst environment.

Site description

Local geology and hydrogeology

The Toulon springs supply drinking water to the city of Périgueux (France) since 1832.

These consist of the Abîme Spring and the Cluzeau Spring, originating from the same karst conduit. The average discharge is 450 L s -1 , making it as one of the most important water resources in the Dordogne County. The springs are vauclusian type, water reaches the surface through localized faults at the extrados of an anticline fold oriented at N145° [START_REF] Lastennet | Geochemical characterization of complex multilayer karstic systems. Springs of Périgueux, France[END_REF].

Geology of the area is composed of carbonated rocks from upper Jurassic and upper Cretaceous [START_REF] Stempel | Etude des ressources en eau de la région de Périgueux (Dordogne)[END_REF][START_REF]Fonctionnement et vulnérabilité d'un système karstique multicouche à partir d'une approche multi-traceurs et d'un suivi haute-résolution[END_REF]. At the bottom, the upper Jurassic formations (Oxfordian and Kimmeridgian) consist of dolomitic limestone with a thickness varying from 100 m to 150 m. At the top, the upper Cretaceous formations (Turonian, Coniacian and Santonian) is made up of limestone with a thickness varying from 200 m to 250 m. At the top of the carbonate rocks is an epikarst, surmounted by a soil layer of few meters. The Toulon karst system also presents fold and faults with a principal direction NW-SE (Fig. 1).

Hydrogeological context, on the other hand, considers two main multilayered aquifers: Functioning of the Toulon springs was explained by [START_REF] Lastennet | Examining the functioning of a multilayer karst aquifer. The case of Toulon Springs[END_REF][START_REF] Lastennet | Groundwater-flow characterization in a multilayered karst aquifer on the edge of a sedimentary basin in western France[END_REF] and Lorette (2019). The system is an example of springs originating from confined Jurassic aquifer and unconfined Cretaceous aquifer according to the hydrogeological conditions.

During low-water period, there is a large water input coming from confined Jurassic aquifer and saturated part of the unconfined Cretaceous aquifer (Turonian). Thus, implying that the Jurassic aquifer has a reservoir function. In a high-water period, infiltration water activates the drainage network in the formation above the Turonian (i.e. Coniacian and Santonian).

Hence, this infers that the Cretaceous aquifer has a transmission function. The Cretaceous aquifer is responsible for the hydrodynamic and hydrochemical variations. This enables the rapid transmission of the elements from the surface and subsurface. In terms of surface infiltration (e.g. NO3 -, DOC, bacteria, and turbidity), the Cretaceous aquifer contributes to the Toulon springs' water quality hazards.

Local geomorphology

The surface area presents typical karstic landforms. Several exokarstic forms such as sinkholes and surface water (e.g. stream river) loss (Fig. 2) are noticed on the plateau above the Toulon springs. Most of the losses are temporary and activated only after heavy rainfall.

Sinkholes are mostly located on the plateau, in thin soil layer area. Tracer test is an efficient tool to show relationships between the surface water losses and springs. In the area, several dye tracers were performed. Some of the results showed a rapid transit, from 5 m.h -1 to 130 m.h -1 . This underlines the karstic behavior of the aquifer and the nature of the water quality hazard that can be brought to the system. Surface water losses are considered as major source of water quality hazard in terms of contamination due to the surface runoff [START_REF] Pronk | Dynamics and interaction of organic carbon, turbidity and bacteria in a karst aquifer system[END_REF][START_REF] Goldscheider | New insights into the transport of sediments and microorganisms in karst groundwater by continuous monitoring of particle-size distribution[END_REF].

The area, localized in the north of the city of Périgueux, is considered as a rural area.

Land use is mainly forest (50%) and agricultural (40%) (Fig. 2). Urban land use is only 10%.

Agriculture lands mostly consist of wheat, corn, sunflower, and colza. Some livestock farming is present, raising cows and ducks. The use of nitrogen synthetic fertilizer and manure increased for the last decades, leading to the augmentation of nitrate concentration in the groundwater (Lorette 2019). The local hydrochemical background was ~1 mg L -1 in 1960 at low-flow conditions and is now close to ~12 mg L -1 on the average with a maximum peak concentration of ~20 mg L -1 during floods. Data from 1960 to 2014 are provided by administration of Dordogne, in addition, we measure it since 2014 (Lorette 2019). For the year scale, about 40 TN/year are exported at the Toulon springs. Nitrate stored in the unsaturated zone of the aquifer may be transported to the saturated zone after some rainfall events.

Climate

Since January 2016, 3 rainfall stations and one meteorological station (recording temperature, humidity, wind, and the rate of sunshine) are installed at the plateau above the Toulon springs (Fig. 1). Périgueux climate is tempered, with an average of 13°C (since 2016). In summer, average temperature ranges from 20°C to 21°C. In winter, average temperature ranges from 5°C to 6°C. Annual average rainfall is close to 800 mm. Real evapotranspiration is calculated between 200 mm and 350 mm per year (using Penman-Monteith equation).

Material and methods

Hydrological Approach

The sampling strategy aims to identify the water quality hazards of the Toulon springs in relation to the movement of water from different origins. High resolution monitoring of hydrochemical parameters combined with particle sampling measurement and bacteriology sampling were performed during the first flood events of each cycles: (i) February 2017 for the 2016-2017 cycle and (ii) December 2017 for the 2017-2018 cycle. The first floods are identifiable as these were associated with important rainfall after a long low-water period.

Nitrate, turbidity, and DOC were monitored in the Toulon springs. These parameters show similar trends at each first flood of the cycle. The flood of February 2017 was chosen to establish the links between the bacterial contamination and other monitored parameters. Once relationships are established, an innovative method was developed to identify the origin of the water flowing in the karst outlet during flood events. Once the NO3 -and DOC trend is known, this can be applied for the first flood of every hydrological cycles (for example December 2017).

Continuous data monitoring and discrete sampling

Since March 2016, turbidity, NO3 -, and DOC are measured at the Toulon springs.

Turbidity is measured using an optical sensor (Solitax sc TLine, HACH) every 10 minutes, with a relative precision of 1%, and a range of measurement of 0.001 NTU to 4 000 NTU.

Nitrate and DOC are measured every 6 minutes using a UV-visible scanning spectrophotometer (Spectro::lyser, S::CAN), with a precision of 0.1 mg L -1 and 0.05 mg L -1 , respectively, and a range of measurement of 0 to 46 mg L -1 and 0 to 133 mg L -1 , respectively.

To avoid measurement errors due to matrix interferences when turbidity increases, a manual calibration of the spectro::lyser was made with laboratory measurements (Lorette, 2019).

Simultaneously, hydrodynamic data (rainfall and discharge) were also monitored.

Rainfall was measured at the Toulon springs hydrogeological catchment every 15 minutes.

Only accumulated rainfall over a day for the central station is presented in this article.

Finally, discharge was measured in a gauging station every 10 minutes at the Toulon springs, with an accuracy of 0.5%.

From 08/02/17 to 24/02/17, 12 sampling campaigns were performed to analyze the bacteriology water samples (Escherichia coli, total coliforms, and enterococcus) and particles (particle size distribution, observation, and chemistry) in the water. Bacteriology water samples were taken using HDPE 500 mL bottles with thiosulfate and kept at 4°C before the analysis on the same day of sampling. To sample particle, an HDPE bottle of 2 L is immerged and filled up in the Toulon spring. These bottles were kept in darkness until the analysis is performed.

Analytical methods

Bacteriological analyses were done at the Laboratory of Dordogne Departmental

Research (LDAR24). Measured parameters are: (i) Escherichia coli (E. coli), (ii) total coliforms (both using French norm NF EN ISO 9308-1), and (iii) enterococcus (using French norm NF EN ISO 7899-2).

Analyses on particles were done at the University of Rouen, in coastal and Continental Morpho-dynamic Laboratory. Particle counting for sizes from 0.04 µm to 2000 µm was done using a laser granulometer Beckman coulter LS 13 320. Observation of particles and particle chemistry characterization was done using a Scanning Electron Microscope Ziss EVO 40 Ep and a Brucker microprobe for each sample.

Normalized data analysis

Normalized data enables studying the relative variations of a parameter free from units.

Comparing normalized data of river flow and sediment concentration during a flood event [START_REF] Williams | Sediment concentration versus water discharge during single hydrologic event in rivers[END_REF] described several possibilities of solid and dissolved phases transport. [START_REF] Valdes | Analysis of karst hydrodynamics through comparison of dissolved and suspended solid's transport[END_REF], [START_REF] Fournier | Using turbidity dynamics and geochemical variability as a tool for understanding the behavior and vulnerability of karst aquifer[END_REF] and [START_REF] Schiperski | Relationship between organic micropolluant and hydrosedimentary processes at a karst spring in south-west Germany[END_REF] used normalized data on turbidity and electrical conductivity measurements to highlight the sedimentary process.

In this paper, application of the method using data normalization of nitrate and DOC data is proposed as water origin markers. Using these infiltration markers will help in describing the process occurring in an anthropogenically influenced alimentation area of the Toulon springs. DOC can be used to identify water originating from soil or punctual infiltration. NO3 -can be used as a tracer of diffuse infiltration through soil and unsaturated zone.

Normalization was made using the maximum and minimum values during the flood events (equation 1 and 2):

(1)

(2)

Results

Geochemical variability

During February 2017 and December 2017 flood event, high-resolution monitoring of hydrodynamic (discharge and rainfall) and hydrochemical parameters (turbidity, nitrate, and dissolved organic carbon) were obtained at the outlet of the Toulon karst system. Table 1 summarizes data of the Toulon springs. Fig. 3 illustrates the temporal evolution of discharge and hydrochemical parameters from the Toulon springs.

First cycle: February 2017

From February 1 st to February 8 th , a rainfall event of 51.4 mm led to an increase of water discharge of the Toulon springs (Fig. 3A). The previous water discharge was about 300 L s -1 which is characterized as the regular flow level the Toulon springs [START_REF] Lastennet | Groundwater-flow characterization in a multilayered karst aquifer on the edge of a sedimentary basin in western France[END_REF]. The weak rainfall intensity (5 mm.d -1 on the average) implied slow flow, taking 6 days to increase water discharge in the Toulon springs.

The maximum water discharge is recorded on 08/02/2017 having 511 L s -1 . This high water flow influenced the hydrochemical parameters as well as the particles. The turbidity presented an inconsequential variation with minimum and maximum values of 0.70 NTU and 3.95 NTU, respectively. Four increases are recorded during this water flow: three during high water flow and one when there was low water flow.

NO3 -and DOC increased during the high water flow (Fig. 3A). The average concentrations of NO3 -and DOC were 14.9 mg L -1 and 0.7 mg L -1 , respectively, with corresponding minimum values of 12.0 mg L -1 and 0.20 mg L -1 . The maximum values of NO3 -and DOC are 20.0 mg L -1 and 1.30 mg L -1 , respectively. These parameters did not show the same dynamic evolution during this flood event.

DOC appeared to have three stages: (i) a slow increase from 04/02/2017 to 10/02/2017 then (ii) rapid increase from 10/02/2017 to 14/02/2017, finally, (iii) another slow increase during 14/02/2017 until 20/02/2017.

Second cycle: December 2017

From December 7 to December 15, two rainfall events for 6 days brought to an increase of the discharge at the outlet of the Toulon karst system (Fig. 3B). The first peak accounted 50.4 mm of rainfall for 2 days. Water discharge increased from 315 L s -1 (10/12/2017) to 590 L s -1 (12/12/2017). The second increase in water discharge is, however, associated with less intensive rainfall, accumulating with a total of 30.2 mm in 4 days. This gave an augmentation of 90 L s -1 (14/12/2017 to 15/12/2017) reaching a maximum water flow of 615 L s -1 .

Having the increase of the water flow, the turbidity increased as well identifying 2 large peaks. The first peak is recorded during the augmentation of the water flow. Turbidity was accounted from 0.36 NTU (10/12/2017) to 3.88 NTU (11/12/2017). The second is recorded from 13/12/2017 to 16/12/2017 with values of 2.58 NTU and 8.37 NTU, respectively (Fig. 3B).

The average concentrations of NO3 -and DOC were 14.58 mg L -1 and 1.66 mg L -1 , respectively, with corresponding minimum values of 10.84 mg L -1 and 0.17 mg L -1 . The maximum value of NO3 -was 19.12 mg L -1 , while, DOC was 2.30 mg L -1 . The same as the water flow during February 2017, DOC had three distinct peaks (Fig. 3B

DOCnorm over NO3 -norm: Normalized data analysis

Normalized data were performed using the maximum and minimum values during the flood events to determine the relative variations of a parameter free from units. Normalized data analysis from DOCnorm vs NO3 -norm was performed on the two flood events previously presented.

February 2017 (Fig. 3A) and December 2017 (Fig. 3B) flood events presented the same trend as the DOCnorm vs NO3 -norm. In Fig. 4, the start of the high water flow is characterized by a slope inferior to 1 (α<1). This means that the increase of NO3 -is higher compared to the increase of DOC. It is followed by a change of slope going to the right, having α>1. This signifies that the increase of DOC is more pronounced at this time than NO3 -. Then, the slope went back to α<1.

Particles and bacteria

Particle size distribution

The particle with size more than 100 µm dominates the whole flooding event with 91% during the 14/02/2017 to 48% during the 23/02/2017). After the peaks, the particles of this size decreased in terms of percentage with respect to other smaller sizes.

In Fig. 5, the sample of February 14 th shows the transfer of a new water mass. This water mass is characterized by an increase of the small size particles (10-50 µm) and a decrease of the large size particles (100-2000 µm). The flowing of this new water mass is confirmed by a stagnation of nitrate and an increase of DOC (Fig. 3A).

Particle Characterization using Scanning Electron Microscope (SEM)

Characterization of the chemistry of the particle helps in distinguishing allochthonous and autochthonous particles. Microprobe and Scanning electron microscope were used.

Analysis of particle nature and size distribution during the flood of February 2017 showed variations of the particle type along the flood event.

During the flood happened last February 2017, particles flowing throughout the rising of the flood were bigger than 100 µm (we call it large particles given the range of particles size at the Toulon springs) and mainly composed by minerals like quartz (Fig. 6a) or calcite (Fig. 6b and Fig. 6c). This corresponds to the composition of the rock and implies autochthonous origin of the particles.

During the lowering of the flood, another nature of particle appeared, associated with an increase of the turbidity. These particles are smaller than 100 µm (we call it small particles) and mainly organics: organic-mineral flocs (Fig. 6d) and vegetal debris (Fig. 6f).

Bacterial colonies were adsorbed on vegetal debris (Fig. 6e). Also, there was an increase of fecal contaminants showing a surface origin of the water. This denotes allochthonous origin of these particles.

Fecal bacteria

The maximum value of E. coli detected is 300 CFU/100 mL with a minimum value of 10 CFU/100 mL and an average of 80 CFU/100 mL (Fig. 3A). This maximum value was observed during 13/02/2017 (high flow), 4 days after the maximum of discharge. The minimum value, however, was observed during 24/02/2017, at the end of the low-flow.

The number of total coliforms is higher than E. coli. It has a maximum value of 600 CFU/100 mL with a minimum value of 20 CFU/100 mL and an average of 146 CFU/100 mL (Fig. 3A). This maximum value was observed during 13/02/2017, high flow, 4 days after the maximum water discharge. The minimum value, however, was observed during 10/02/2017, also within the high flow event, 1 day after the maximum of water discharge.

Enterococcus has the least number of detected bacterial species compared to E. coli and total coliforms (Fig. 3A). The maximum value identified is 54 CFU/100 mL with a minimum value of 2 CFU/100 mL and an average of 21 CFU/100 mL. This maximum value was observed during 13/02/2017, high-flow, 4 days after the maximum water discharge. The minimum value, however, was observed during 09/02/2017, still high flow, at the date of the maximum water discharge.

Discussion

Bacterial contamination

The concentration of E. coli that can be found in some karst springs are as follows: 183 CFU/100 mL from Cossaux Spring (Pronk et al., 2009), 648 CFU /100 mL from Moulinet Spring (Pronk et al., 2009), 1088 from Moulinet Spring [START_REF] Pronk | Particle-size distribution as indicator for fecal bacteria contamination of drinking water from karst springs[END_REF]. The Toulon springs has non-negligible E. coli concentration having the maximum recorded during the sampling campaign as 300 CFU/100 mL. The highest concentration of E. coli recorded in the Toulon springs is 700 CFU/100 mL during 2017 (Lorette 2019).

For the total coliforms, high values were found in the karst springs: 794 CFU/100 mL from AB Spring (Bucci et al., 2009), 2420 CFU /100 mL from Lez Spring [START_REF] Bicalho | Geochemical evidence of water characterization and hydrodynamic responses in a karst aquifer[END_REF], 8680 CFU /100 mL from Moulinet Spring (Pronk et al., 2009). These values are higher than the maximum concentration found in the Toulon springs during the sampling campaign. However, a higher value was recorded during 2003 with a concentration of 2000 CFU/mL (Lorette 2019). Therefore, the concentrations measured in the Toulon springs are somehow in between the measured values of AB Spring and Lez Spring (Table 2).

The maximum concentration of enterecoccus measured during the sampling campaign is lesser than other karst springs. Cossaux Spring for example has a concentration of 123 CFU/mL (Pronk et al., 2009). Moulinet Spring and AB Spring recorded 430 CFU/mL and 1216 CFU/mL, respectively. Nonetheless, vigilance should be applied in terms of water monitoring as the concentration of enterecoccus in the Toulon springs can reach up to 250 CFU/mL as recorded last 2004 (Lorette 2019).

In terms of drinking water, the international regulation (WHO, 2006) requires 0 CFU/100 mL of E. coli, total coliforms, and enterococcus. Hence, even the minimum concentrations 10 CFU/100 mL, 20 CFU/100 mL, and 2 CFU/100 mL of the E. coli, total coliforms, and enterococcus, respectively, should be addressed. As a normal standard procedure, water is treated before distributing it to the community. What is important to retain is the probable event that the bacteria can enter, facilitating in anticipating the necessary actions. Previous analyses can describe several parameters considered as degrading water quality: (i) particles, (ii) dissolved organic carbon, (iii) microbial pathogens, and (iv) nitrate.

The flood event of February 2017 is used to evaluate the relationships between the particles and contaminant. A synopsis of this method is described in Fig. 7, leading to a conceptual model (Fig. 8). This identifies several types of water at the Toulon springs, pointing out several sources of water quality hazards:

(i)
The first one is the resuspension of particles deposed in the saturated zone of the karst system. This first water perturbation (Category I in Fig. 7) is associated with an increase of turbidity signal without hydrochemical evolution.

Particles measured are inorganic and considered as big (> 100 µm). These particles are associated with an autochthonous origin and are not correlated with microbial pathogens. Stability of NO3 -and DOC concentration during this perturbation confirms the saturated zone origin of this water type.

(ii)

The second (Category II in Fig. 7) is the punctual contamination of surficial water. It is characterized by a joint increase in DOC, turbidity, and bacteria.

Despite a decrease in energy within the karst system (recession), an increase in the turbidity signal was recorded showing the mass transfer. The analyzed particles correspond to organic particles (algae debris, organo-mineral flocs, and colonies of bacteria adsorbed on plant debris), and sizes are between 50 and 100 microns. This type of particles, of allochthonous origin, confirms the surface origin of the new water mass identified. In the diagram DOCnorm vs. NO3 -norm, the arrival of this mass of water is identified by a slope α> 1.

(iii)

The third (Category III in Fig. 7) is the diffuse infiltration of water through the soil and the unsaturated zone. It is characterized during the recession by a single increase of NO3 -, without increased DOC, bacteria, and turbidity. Particles associated with this water type are minerals and of size essentially between 50 µm and 100 µm. It is of autochthonous origin to the karstic system. In the DOCnorm vs. NO3 -norm diagram, the arrival of this new water type is identified by a slope α <1.

NO3 -and DOC relationship in karst environments and contribution in terms of resource management and protection

Analyses performed in this study showed complex evolution of NO3 -and DOC at the Toulon springs. Coming from a same theoretical origin (soil), the evolution of these parameters can be associated with anthropogenic nitrate input in the hydrogeological catchment.

Fig. 9 presents the implication of the result of the performed Normalized Data Analysis of NO3 -and DOC. This enables to identify the infiltration process (using the slope α from DOCnorm vs. NO3 -norm) and its respective progression (with particle suspension and bacterial input). Data presented in the DOCnorm vs. NO3 -norm diagram corresponds to the increase of NO3 -and DOC during the high flow event. Through time, NO3 -and DOC are increasing at a different phase. When NO3 -increases more than DOC, a linear regression will present a slope α lower than 1 (α<1). On the contrary, when NO3 -increases less than DOC, a linear regression will present a slope α higher than 1 (α>1). The common use of the DOCnorm-NO3 -norm diagram, and especially the analysis of the evolution of the slope with continuous data allowed distinguishing punctual infiltration from diffuse infiltration through time. This analysis was applied in both February 2017 flood and December 2017 flood events, leading to: (i) describing the precision of hydrogeological functioning of the karst system and (ii) identifying water quality hazards and the period associated with microbial pathogens.

According to this analysis of high-resolution monitoring of NO3 -and DOC, the two flood events studied have a similar functioning with:

(i) A flush of water close from the saturated zone, bringing particles deposed during low water period (Fig. 10). This water type is associated with the pressure transfer in the karst system and is not correlated with bacteriology.

This result confirms those of [START_REF] Dussart-Baptista | Transfer of bacteria-contaminated particles in a karst aquifer: evolution of contaminated materials from a sinkhole to a spring[END_REF] and [START_REF] Heinz | Water quality deterioration at a karst spring (Gallusquelle, Germany) due to combined sewer overflow: evidence of bacterial and micro-polluant contamination[END_REF];

(ii) There is also a contribution of water coming from the unsaturated zone (Fig. 10). This water type is associated to the mass transfer in the karst aquifer. It enables leaching of dissolved tracers. Inorganic nitrogen, as NO3 -is the main tracer this research study, it is also possible to use other tracers coming from the same origin, such as pesticides, to identify this water type;

(iii) A contribution of punctual infiltration coming from the surface run-off (Fig. 10) appears with hydrological condition enabling infiltration through swallow holes.

This supposition is correlated with allochthonous particles and is associated with sharp increase of DOC and microbial pathogens. This poses the highest risk to the human health. In the Toulon springs, bacteria are recorded 4 to 6 days after the beginning of the flood.

(iv) Another contribution of water coming from the unsaturated zone (Fig. 10) is recorded again when hydrological conditions are not enough to generate infiltration through swallow holes. As a consequence, nitrate contamination increases. This study showed that turbidity alone is not always a reliable indicator of the presence of bacteria in the Toulon springs. Previous studies from [START_REF] Dussart-Baptista | Transfer of bacteria-contaminated particles in a karst aquifer: evolution of contaminated materials from a sinkhole to a spring[END_REF][START_REF] Pronk | Dynamics and interaction of organic carbon, turbidity and bacteria in a karst aquifer system[END_REF], [START_REF] Heinz | Water quality deterioration at a karst spring (Gallusquelle, Germany) due to combined sewer overflow: evidence of bacterial and micro-polluant contamination[END_REF][START_REF] Goeppert | Hydrogeological controls of variable microbial water quality in a complex subtropical karst system in Northern Vietnam[END_REF] also noticed this result in different region such as France, Switzerland, Germany, and Vietnam, respectively. In this study also, it is showed that the increase of DOC concentration does not mean that there will be increase on bacterial concentration. The contribution of high resolution monitoring of NO3 - concentration highlights the relationships of these parameters and their association with water origins. As for the NO3 -, concentrations increased during the first flood of the hydrological cycle. It can be associated to the mobilization of nitrate over the soil and unsaturated zone [START_REF] Rowden | Results from big spring basin water quality monitoring and demonstration projects, Iowa, USA[END_REF][START_REF] Stueber | Origin and transport of dissolved chemicals in a karst watershed, southwestern Illinois[END_REF]Pronk et al., 2008;[START_REF] Pu | High-resolution monitoring of nitrate variations in a typical subterranean karst stream, Chongqing, China[END_REF]. The high level of NO3 -in the Toulon springs is non-natural. This directly links to the agricultural land use over the hydrogeological catchment (Lorette, 2019). NO3 -can be used as a tracer of a specific water oriin such as water from unsaturated zone. Hence, this study presented that the comparison of relative evolution of DOC and NO3 -can help in distinguishing sources of water either from unsaturated zone or water from surface runoff. Table 3 is provided to give an overview of the application of the method used to water resource management and protection. In order to assure good spring water quality, recommendations on hydrogeological catchment management can be provided. The recommendations are as follows: (i) improve the protection zone in the surface catchment of swallow holes; (ii) install a monitoring device on the main swallow hole as an alert system of potential contamination; and (iii) restrict the agriculture land use to avoid further increase of NO3 -concentration that can reach the groundwater.

Conclusion

In water resource management, it is important to know the water quality hazards. In this study, a tool that can be used in tracing water perturbation in a karst aquifer is proposed.

First, method involves high-resolution monitoring of NO3 -and DOC. Second relies on the normalization of the data with NO3 -_norm=f(DOC_norm) reference frame. From these two steps, the infiltration process can be assessed based from the obtained slope. Applied on the data acquired during a flood event, the slope (α) showed that (i) if α>1, DOC increase is more pronounced while (ii) if α<1, contaminant of anthropogenic origin, e.g. NO3 -, is more distinct.

Third and fourth steps, NO3 -and DOC parameters were complemented by (a) determining and characterizing particle size and chemistry and (b) detecting bacterial contamination (e.g. E. coli, total coliforms, and enterococcus). Employing the particle size and determining the presence of the bacteria, kinds of infiltration can be further examined.

When α>1, it is observed that organic particles and bacteria are present. Whereas, when α<1, it has larger range of particle size without the bacterial incidence. The slope α>1 involves bacterial growth that can pose problem in terms of water quality. While, with the slope α<1, NO3 -is a contamination concern. This tool was applied on an important karst system the Toulon springs. Results showed that autochthonous and allochthonous type of turbidity can be identified at the Toulon springs. Authochthonous type of turbidity mostly consists of mineral particles. Allochthonous type, however, means that important contribution of recently infiltrated water occurs. Furthermore, three sources of water quality hazard were classified: (i) water from saturated zone, (ii) water from unsaturated zone, and (iii) water from surface runoff. Flushing of saturated water results to autochthonous type of turbidity. The punctual infiltration from surface runoff generates allochthonous type of turbidity with associated organic particles and bacteria. DOC is also a source of distress in this type. The infiltration from unsaturated zone engenders mineral type of particles associated with NO3 -increase.

As the only drinking water for more than 50 000 people, management, monitoring, and protection of the Toulon springs are keys for a sustainable water resource. Hence, the identification of the sources of the water quality hazards is an important step in managing the hydrogeological catchment better. In this study, the use of high-resolution monitoring of hydrochemical parameters (turbidity, DOC, and NO3 -) helped in determining the water quality hazards in the Toulon springs. Even if the trend of NO3 -concentration is of anthropogenic source, it is still below the French potability threshold (50 mg L -1 ). The main concern, then, for the water quality of the Toulon springs is the presence of bacteria coming from the surface runoff during flood. The presence of exokarstic forms such as swallow holes over the hydrogeological catchment is probably responsible of this contribution. The type of water perturbation associated with bacterial contamination is now identified. This is important to know as water with bacterial contamination needs treatment. This latter can help the water managers and providers in preparing necessary actions or water quality treatment before water will be supplied to the households. This proposed method can be applied on any karst system hosting features like sinkholes and swallow hole in their catchment area. This is as these system are vulnerable to infiltration of runoff water. The monitoring mostly relies on high resolution monitoring for NO3 -and DOC. Particle size and chemistry characterization and bacterial detection can be done just on few events, as they are mainly used to interpret the variations of NO3 -and DOC better. Employing this proposed method is rather simple. It can make used of in situ tools already existing in the markets. Furthermore, sampling of water does not need any new specific method. Personnel of the water providers and managers can be trained using this method straightforwardly. The future step is to characterize the NO3 -hazard by differentiating the fertilizer and organic inputs of NO3 -better. This would help in managing the land use of catchment area. The information gathered using the proposed method, will control the impact of allochtonous water during flood events

  (i) unconfined karstic upper Cretaceous aquifer: Turonian, Coniacian and Santonian aquifers and (ii) confined karstic upper Jurassic aquifer: Oxfordian and Kimmeridgian aquifers. The two main aquifers are separated by Cenomanian marls, considered impermeable. Spatial thickness (4-20 meters) and facies variations (marls-sand) over the study area induced an additional difficulty in understanding the relationship of the two main aquifers.

Fig. 1 :

 1 Fig. 1: Hydrogeological map of the Toulon karst system (Adapted from Lorette et al., 2018). AB: simplified SW-NE geological cross-section. The Cenomanian is considered as not an aquifer.

Fig. 2 :

 2 Fig. 2: Geomorphological situation and land use of the Toulon karst springs.

Fig. 3 :

 3 Fig. 3: Temporal evolution of the water discharge and hydrochemical parameters from the Toulon springs with respect to the two flood events. (A) February 2017, (B) December 2017. Green items refers to manual sampling during February 2017 flood event.

Fig. 4 :

 4 Fig. 4: Normalized NO3 --DOC during two flood events from the Toulon springs. (A) February 2017, (B) December 2017. The blue line illustrates the slope α=1.

Fig. 5 :

 5 Fig. 5: Temporal evolution of the water dicharge, turbidity and particles during the February 2017 flood event.

Fig. 6 :

 6 Fig. 6: Nature of the particles found in the Toulon springs during the 2017 flood event observed by scanning electron microscope. a) quartz. b and c) calcite. d) organic-mineral flocs. e) bacterial colonies adsorbed on vegetal debris. f) vegetals debris.

Fig. 7 :Fig. 8 :

 78 Fig. 7: Synoptic for tracing water origins associated with hydrochemical parameters, particles and bacteria.

Fig. 9 :

 9 Fig. 9: Conceptual use of the NO3 -norm -DOCnorm diagram

Fig. 10 :

 10 Fig. 10: Continuous application of NO3 -norm -DOCnorm to differenciate concentrated recharge to diffuse recharge. A) February 2017. B) December 2017.

Table 1 : Statistical parameters of the hydrodynamic and hydrochemical parameters, and bacteria during February 2017 and December 2017 flood events. (n) number of samples, (σ) standard deviation, (CV) coefficient of variation.

 1

							Total
		Discharge	Turbidity	NO3 -	COD	E.coli	coliforms	Enterococcus
	Flood event
						(CFU / 100	(CFU / 100	(CFU / 100
		(L.s -1 )	(NTU)	(mg.L -1 )	(mg.L -1 )	mL)	mL)	mL)
	n	3600	2592	4388	4388	12	12	12
	Mean	386	1.89	14.93	0.70	80	147	21
	Min	277	0.70	12.06	0.18	10	20	2
	February 2017
	Max	511	3.95	20.03	1.30	300	600	54
	σ	51	0.51	2.88	0.39	88	155	16
	CV (%)	13	27	19	55	110	105	73
	n	2592	2592	4320	4320	-	-	-
	Mean	496	5.15	14.58	1.66	-	-	-
	Min	317	0.36	10.84	0.17	-	-	-
	December 2017
	Max	617	8.37	19.12	2.30	-	-	-
	σ	73	1.78	2.19	0.74	-	-	-
	CV (%)	15	35	15	45	-	-	-

Table 2 : Mimimum and maximum values of E.coli, total coliforms and enterococcus from several karst springs

 2

		E. Coli (CFU / 100	Total coliforms	Enterococcus
	Source		mL)	(CFU / 100 mL)	(CFU / 100 mL)	Reference
		Min	Max	Min	Max	Min	Max
	AB spring	-	-	0	794	0	1 216	Bucci et al., (2015)
	PB spring	-	-	0	895	0	740	Bucci et al., (2015)
	FC spring	-	-	0	280	0	1 456	Bucci et al., (2015)
	Moulinet spring	0	1 088	14	8 680	0	480	Pronk et al., (2007, 2009)
	Cossaux spring	0	183	2	2 150	0	123	Pronk et al., (2009)
	Lez spring	-	-	26.2	2 420	-	-	Bicalho et al., (2012)
	Lirou spring	-	-	13.5	2 420	-	-	Bicalho et al., (2012)
	Fleurette spring	-	-	60.2	2 420	-	-	Bicalho et al., (2012)
	Gallusquelle spring	0	7 000	0	35 000	-	-	Heinz et al., (2006)
	CSP1 spring	0	30	-	-	-	-	Ender et al., (2018)
	KS2 spring	250	> 2 420	-	-	-	-	Ender et al., (2018)
	The Toulon Springs	0	930	0	2 000	0	250	Lorette (2019)
	The Toulon Springs
	-February 2017	10	300	20	600	2	54
	5.2

Conceptual model exhibited by the Toulon springs during flood events (using the parameters: DOC, NO3 -, particles, and bacterial transfer)

Table 3 .

 3 Water quality hazards, indicators, causes, and suggested action for water resource management and protection

	Water	Indicator	Cause	Action for water
	perturbation/			resource management
	Water Origin			and protection
	Water Flush/	Turbidity	Pressure transfer	Check if the indicator is
	Saturated Zone	(autochthonous		more than the treatment
		particles)		possibility threshold:
				-If more than the
				threshold, then stop
				pumping for a while.
				-If less than the
				threshold, monitor and
				use classical treatment.
	Change in	Anthropogenic	Mass transfer	Check if the indicator is
	chemistry/ Unsaturated Zone	contaminant, in this study NO3 -	Leaching	more than the required water quality threshold
				-If more than the
				threshold, then
				recommendations in
				managing and
				controlling this specific
				contaminant are
				necessary
				-If less than the
				threshold, then
				monitoring and
				recommendations to
				control the concentration
				are necessary
	Punctual	COD	Infiltration through	Check the presence of
	infiltration/	Turbidity	swallow holes	bacteria, if there is, water
		(allochthones		treatment	such	as

Acknowledgements

This work was supported by the Région Nouvelle Aquitaine, the city of Périgueux, Suez, the Conseil Départemental de la Dordogne, and the Adour-Garonne Water Agency. The authors would like to thank Jean-Christophe Studer, Jerome Donnette, Julien Frant, Pierre Pinet, Emilien Castaing and Philippe Savy for their technical and field assistance contributed. This work benefited from fruitful discussion within the KARST national observatory network (SNO KARST) initiative from the INSU/CNRS.