Latest Trends in Engineering Mechanics, Structures, Engineering Geology

Using Artificial DNA as tracer in a bedrock river of the Middle Karst System (, Vallo Diano and Alburni European&Global Geopark, southern Italy)

VITTORIO BOVOLIN, ALBINA CUOMO, DOMENICO GUIDA Department of Civil Engineering University of Salerno Via Giovanni Paolo II ITALY [email protected]

JAN WILLEM FOPPEN Institute of Water Education Unesco-IHE Delft THE NETHERLAND

Abstract: The paper deals with strengths and shortcomings of the use of artificial DNA as tracer in environments as such the fast flowing water in bedrock and step-and-pool streams. The use of synthetic DNA as tracer has been tested in rivers and brooks, and in carbonate rocks, but no attempt has been made, so far, to use this technique in torrents and ground water. One of the main strength is the low environmental impact which makes its use very appealing in protected areas. Taking into account these considerations an artificial DNA test was carried out in a fast flowing reach of the Middle Bussento Karst System. The artificial DNA technique was performed together with a traditional salt injection test. Results obtained from the experiment confirm the overall effectiveness of the use of artificial DNA in fast flowing environment, particularly excellent is the DNA mass conservation compared to the salt test. The main shortcoming of the artificial DNA test resides in the elaborate sampling procedure which requires the approximate knowledge of the transit time of the water into the system.

Key-Words: Karst, artificial DNA, tracer, Bussento river, National Park of the Cilento, Vallo Diano and Alburni-European&Global Geopark, southern Italy.

1 Introduction The level of complexity of such systems is dictated Environment protection and proper water resource by the geological litho-structures and hydro- assessment and management require a good geomorphic processes which have shaped both the knowledge of catchment basin structure and surface and the underground portion of the karst functioning [9]. This knowledge have to be acquired landscape. Understanding relationships existing by interdisciplinary studies regarding hydro- between these two “worlds” is paramount. This is geological setting, hydrological recharge-discharge particularly true for Mediterranean protected areas, monitoring and hydraulic modelling [3]. This is as such the Cilento, Vallo Diano and Alburni particularly important in the case of complex National Park (in the following National Park), situations, such as aquifers-rivers interaction in located in the southern of Italy, where the water mature karst-influenced hydro-systems [3]. Water quality must be kept at the highest standard in order resources from karst systems not only are one of the to ensure healthy populations of fishes and aquatic primary sources of drinking water in many areas, fauna (native trout, otter, fresh water crayfish, and but there is an increasingly recognition of the others). Monitoring and modeling of the riverine importance of assuring their quality as they eco-system has been carried in the past by the represents the basic abiotic support for all riverine University of Salerno and CUGRI [11] in habitats and fluvial ecosystems. cooperation with the Basin Authority and the

ISBN: 978-960-474-376-6 105 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

National Park, whose are in charge of water The aim of the test was to explore potentials and planning and management in the protected area [9]. shortcomings of the use of artificial DNA tracer in environments as such fast flowing water in bedrock Recently, the inclusion of the National Park into the and step-and-pool streams. European and European & Global Geopark Network, under the auspices of the UNESCO, has fostered further research agreements focused on 2 Hydro-geomorphological setting water research with international organizations [9]. One of the aim of these collaborations is to develop The Bussento river basin, located in the Campania new monitoring techniques that may overcome Region, is one of the more complex drainage river limitations of traditional ones and allow monitoring systems in Italy (Fig. 1). activities in sensitive environments [5].

In the National Park, the Bussento river basin has been identified as an appropriate playground for testing new techniques. Monitoring activity in this area is a challenging task both for the complexity of the system and for the strict regulation which set tight constrains to the use of traditional monitoring techniques.

Tracer testing is an efficient and useful method to characterize hydrological processes both in surface and subsurface waters [10]. The use of synthetic DNA (deoxyribonucleic acid) as tracer is a relatively new technique [4], whose use can be performed with a, theoretically, unlimited number of tracers.

Tracer testing with synthetic DNA experiments has been tested in rivers and brooks, [5] e [6], and in calcareous rock [2]. So far no attempt has been Fig. 1: Location of the Bussento river basin in the Cilento, Vallo made to use such technique in torrents and ground Diano and Alburni National Park – European and Global water included in protected areas. One of the main Geopark. interests in this technique, beside the reduced environmental impact [5], resides into the The complexity is due to the highly hydro- possibility of using, in the future, different multiple, geomorphological conditioning induced by karst synthetic DNA tracers at the same time [5]. This landforms and processes as such as: karst highlands would be particularly useful in cases, as such the with dolines and poljes, lowlands with blind valleys, Bussento karst-influenced river, where it is known streams disappearing into sinks, cave systems, karst- that many stream sinks converge in the same outlet induced groundwater aquifers with resurgences. [9] and bed stream seepage induces discharge losses The Bussento river originates from the upland along the river reach. In these cases, traditional springs of Mt. (1,888 m asl), one of the techniques do not allow to distinguish contribution highest mountain in the southern Apennines. coming from each stream sink as well as investigate the gaining-losing processes along the river reach. The Upper Bussento flows partly in wide alluvial The artificial DNA technique may, at least in valleys (i.e. Sanza valley) and partly along steep principle, mark each inlet or loss with a different gorges and rapids. Along this path, abundant artificial DNA signature and allow to identify its springs, emerging from karst aquifers, deliver fresh individual and distinctive contribution. Therefore, in water into the streambed, increasing progressively order to test the feasibility of this technique in a the river discharge. Near the main karst environment, an artificial DNA tracer test was river and some adjacent minor creeks flow into three carried out in the Middle Bussento Karst System active stream sink, named La Rupe, Orsivacca and (MBSKS). Bacuta-Caravo, respectively.

ISBN: 978-960-474-376-6 106 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

These geosites are recognized as “principal reach represent one of more relevant ecological geosites” in [1]. The cumulative river discharge is habitats in the National Park, hosting many conveyed into a converging hypo-karst cave system populations of fishes and aquatic fauna (native trout, and, after a path of few kilometers, re-emerges in otter, fresh water crayfish, river lamprey). For this the “Bussento Resurgence”, which is a “focal ecological relevance the site is currently a WWF geosite” in [1]. The underground karst system is Oasis. largely unknown since only few hundred meters downstream the sinkholes and upstream the resurgence (Fig. 2) have been explored. 3 Experiment layout and procedure

On April the 10th 2012, a coupled salt and artificial DNA tracer test was carried out in the MBSKS: the activities were carried out by the IHE-Unesco researchers (Delft, the Nederlands) in collaboration with researchers of CUGRI-Salerno University, supported financially and professionally by the National Park and the Southern Campania Regional Basin Authority.

The experiment was conceived according to a two- step procedure: in the first step a known, little amount of salt was injected at the upstream section of the test site (Fig. 3), then the hydro-chemograph behavior of the flowing water at the downstream section was monitored. This step was aimed to detect the salt transit times in to the system so to provide useful information for the implementation of the second step which involved the injection of the artificial DNA.

Fig. 2: Hydro-geomorphological map of the MBSKS. The location chosen for the test was the torrent Legend: gsl. gravelly sandy silty complex; dt. debris reach (Fig. 3) from the Old Mill spring outlet (Fig. complex; Ar: Sandstone Complex, corresponding to the 4) to a section located immediately upstream the Internal Units; Am: Marly-clayey complex, comprising junction between the Old Mill reach and the BfFrm and PgFrm; Cm: Marly limestone complex, Resurgence reach (Fig. 5). corresponding to above TFrm; C. Limestone complex, comprising CclL, RqL and RdL formations. Stars: Active sinkholes and stream sinks; Double circle: Arrows: Middle Pleistocene Bussento river path; Buried and fossil stream sink; Double dashed line: Middle Pleistocene Bussento river path ; White portal: Middle Pleistocene Bussento; Black circle: explored underground karst cave system; White dotted circle: un-explored underground karst cave system; Portal: Resourgence; Drop: karst spring; dot-dashed double line: “Le Valli” Sakung; blue arrows: more probable direction of groundwater flow.

Upstream and downstream the Bussento Resurgence, the Bussento river flows partly as bedrock and partly as a boulder step-and-pool stream type [11]. This river reach, which is known as the “ gorge”, is a typical epigenetic valley, where groundwater outflows from epikarst Fig. 3 Hydro-geomorfological sketch of the river reach DNA test site. For legend, see Fig. 2. springs, conduit springs and cave springs supply a perennial stream flow (Fig. 3). Finally, the river

ISBN: 978-960-474-376-6 107 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

lower Bussento Resurgence stretch through fissures and little conduits (Fig. 6) .

Fig. 4 The Old Mill geosite

The injection section was located just downstream Fig. 6 Scheme of seepage flow from the Old Mill toward the Old Mill spring, the measuring section was the Bussento Resugence canal. located about 300 m downstream of the point of injection, just upstream the confluence (Fig. 5) with As far as the DNA experiment is concerned, prior to waters coming from the Bussento Resurgence. the injection a sample of 250 ml of stream water was taken in a PE bottle for background measurements of locations and it was used to determine a DNA standard curve [4] (Figure 7).

8 7 6 y = -0.3094x + 10.582 R² = 0.9964 5 4 3 Log (part/well) Log 2 1 0 10 15 20 25 30 35 Threshold cycle

Fig. 5 The Bussento Resurgence-Old Mil reach junction Fig. 7, Standard curve od DNA tracer T23 for Old Mill spring discharge water Before the confluence the stretch coming from the Old Mill runs at a level higher than the Bussento Then the entire contents of 1 vial of DNA tracer T23 Resurgence stretch (Fig. 6). purchased from Biolegio (The Netherlands) was put in a polypropylene co-polymer bottle filled with 100 The river reach where the experiment was carried ml membrane filtered, UV and diethyl out may be considered as a typical mountain stream pyrocarbonate treated water free from measurable having an alternating sequence of step-and-pools DNAs. After gently swirling, in order to and bedrock reaches. homogenize the solution in the bottle, 3 initial samples were taken in order to determine the The discharge along the Old Mill stretch of river injected DNA concentration and mass. was measured during the experiment. The discharge was not constant, it varied from 190 l/s upstream to In order to detect the salt transit time the hydro- 160 l/s at the measuring section, with a reduction of chemical monitoring at the downstream sections about 16%. Discharge reduction is caused by was carried through the use of an integrated water seepage from the higher Old Mill stretch toward the level and electrical conductivity sensor, type DLN70 produced by STS. Water Electro

ISBN: 978-960-474-376-6 108 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

Conductivity (EC) was monitored by display visual whereby the slope, given in Fig. 4, was 104%. We observation and recorded every five minutes. The considered this efficiency value to be excellent. In Electrical Conductivity parameter was preliminary addition, there was no inhibition of the PCR calibrated adding, to a sample of 200 ml of running reaction detected, and therefore, sample waters water, a total of 5 ml of salt solution in steps of 1 could be used undiluted. ml. However, the negative controls started to produce At 15:24, a total amount of 8 kg of salt was injected random amplifications at a threshold cycle of 30. in the inlet section. Monitoring of the EC at the This indicated that the lower limits of detection, or, downstream section provided a transit time of about the lowest quantity of DNA tracer that could be 12-13 minutes. This information was used in the distinguished as a positive, was at 27 cycles, or at DNA test so that the sampling for DNA started 10 850 particles/µl of sample (=8.5x108 particles/L). minutes after injection. Compared to other experiments carried out in the Netherlands (unpublished results) lower detection At 15:39 (15 minutes after the salt injection) the limit < 100 particles/µl of sample; threshold cycle = water with the DNA tracer was put in the river. At 33-36 , was considered to be high. the measuring section, samples of 0.5 ml of running water were taken at a 1 minute interval. The samples We hypothesize that the Old Mill spring water were collected with a sterile pipet tip with cotton contained relatively large Mg concentrations. plug in order to avoid cross-contamination, and Magnesium is required to carry out the PCR transferred into extra low-binding PE-C 1.5 ml reaction. However, too much magnesium might also Eppendorf vials. The Eppendorf vials were kept cause random amplification. From earlier work we cool (4 °C) immediately in the field. As such a total found that the artificial DNA tracers appeared to be of 75 samples were taken for determining artificial relatively sensitive to magnesium. However, since DNA concentrations in the laboratory. we added an 'overdose' of artificial DNA (minimum threshold cycle measured was 20.6), measuring the DNA breakthrough was not confounded by these 4 Experiment results relatively high detection limits. For mass recovery calculations, we assumed a discharge of 175 l/s DNA tracer samples were analyzed using a 25 (average between 190 l/s and 160 l/s). The time of nucleotide Taqman probe with a 6-carboxy- first arrival of the salt peak was measured 12-13 fluorescein fluorophore on one end and a black hole minutes after injection (Fig. 8a), while the peak quencher on the other end. concentration was measured 19 minutes after injection. For artificial DNA, the time of first arrival In order to determine the efficiency of the qPCR was measured at 12 minutes, while the peak was reaction with Old Mill discharge water, we prepared measured 17 minutes after injection. a standard curve comprising of 10-fold dilutions of known amounts of DNA tracer material (in Both salt peak and artificial DNA peak duplicate) to which Old Mill discharge water was demonstrated a modest tailing effect, giving the added. This enabled us also to assess whether breakthrough curves a slightly asymmetric inhibition was taking place, and to finally determine appearance. When expressed in mass (Figure 8b), it the time dependent DNA tracer mass balance during was clear that the relative salt mass was less than the the injection experiment. The PCR efficiency, relative artificial DNA mass. determined as Also, the total mass “recovered” from the salt 100%*(10 -1/slope-1))〗^( (1) injection experiment was only 2.67 kilo, 33.6% of the injected mass, while for the artificial DNA the “recovered” mass was 6.6x1015 particles, or 87.0% of the injected mass (Table 1).

ISBN: 978-960-474-376-6 109 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

0.12 100 9.0E+10 Added salt Added salt 90 0.1 DNA DNA 80 7.2E+10 DNA concentration DNA 0.08 70

) 60 5.4E+10 - ( 0.06 50 inject

40 3.6E+10 (part/L) 0.04 M/M 30

0.02 Concentration added of salt (mg/L) 20 1.8E+10

10 0 0 20 40 60 0 0.0E+00 0 10 20 30 40 50 60 Time since injection (min) Time since injection (min) Fig. 8: a) Breakthrough curve of salt and artificial DNA tracer T23 some 300 m downstream of the injection point; b) Breakthrough curve of salt and artificial DNA tracer T23 some 300 m downstream of the point of injection expressed as the ratio of measured mass passing the measuring location per minute and total injected mass.

Table 1: Overview of injected and recovered salt and DNA mass in the Old Mill Reach experiment Injected mass Recovered mass % Recovered mass SALT 8 kg 2.67 kg 33.5% DNA 7.6x1015 particles 6.6x1015 particles 87.0%

5 Discussion the pool the residence time of the tracer is lengthen, further more the higher density water resides in the The results of the experiment carried out in in the area where the seepage flow toward the Bussento MBSKS river reach river highlighted the following Resurgence is stronger. Pools in the underground issues. The Old Mill discharge water has no path may play a similar role with an even larger inhibitory effect on the qPCR reaction. However, effect due to the imperviousness of poll walls. likely due to relatively high magnesium concentrations, random amplification started to Longer term measurements of discharge and EC at occur at around 30 threshold cycles, thereby the Bussento Resurgence (Fig. 9) seems to support reducing the lower limit of detection to 850 the scheme described above. Figure 9 shows particles/µl of sample. Breakthrough of salt and discharge and EC measured at the Bussento artificial DNA at the measuring point in the Old resurgence in the days following the experiment. During the monitoring period the discharge Mill spring discharge some 300 m downstream of the point of injection almost coincided. The “lost” remained constant while the EC showed a hump of DNA is comparable the discharge reduction. with a moderate increase a return to the previous The total salt mass recovered was only 33.5% while value. the total mass of artificial DNA recovered was 0.50 0.50 0.45 0.45 87.0%. The amount of lost DNA is comparable with 0.40 0.40 the discharge reduction due to the seepage flow 0.35 0.35 0.30 0.30 from the Old Mill toward the Bussento Resurgence. Q0.25 EC0.25 The low recovery rate of the salt injection 0.20 0.20 0.15 Discharge 0.15 experiment requires a further explanation. The salt, 0.10 0.10 EC for obvious reason, was dissolved in a tank of 0.05 0.05 0.00 0.00 limited size, therefore the injected water had a 10/04/2012 11/04/2012 12/04/2012 13/04/2012 higher density compared to the stream water. The higher density may have caused in the upper pool of Fig. 9 Long term Discharge (m3/s) and EC (µS/cm) the river a density stratification which concentrated measurements at the Bussento Resurgence the higher density water toward the bottom of the pool. Once they end in the dead or less active part of

ISBN: 978-960-474-376-6 110 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

6 Conclusions [3] Bovolin V., Cuomo A. and Guida D., Monitoring activity at the Middle Bussento The analysis of the Old Mill DNA experiment Karst System (Cilento Geopark, southern Italy). allows the following conclusions. Engineering Geology for Society and Territory – Volume 3 River Basins, Reservoir The use of salt as a tracer seems to be limited by Sedimentation and Water Resources (IAEG XII processes as such as dilution and settling which Congress Turin Italy, September 15-19, 2014 weaken and may even obliterate the downstream [4] Foppen, J.W., Orup, C., Adell R., Poulalion, signal. The use of salt in an underground karst V., and Uhlenbrook, S., Using multiple system may face similar problems due to dilution in artificial DNA tracers in hydrology. the aquifer and/or precipitation at the bottom of the Hydrological Processes 25, 2011 pp. 3101– epi-phraetic pools/potholes. 3106 [5] Foppen J.W., Seopa J., Bakobie N., Bogaard The use of the artificial DNA tracers does not seem T., Development of a methodology for the to be affected by such processes, but its application of synthetic DNA in stream tracer implementation relies on the knowledge of the injection experiments. Water Resources travel times in systems. From this, we concluded Research, 49 (9) 2013 pp. 5369-5380. that artificial DNA is a suitable tracer technique, [6] Foppen J.W., and Bogaard T., Using synthetic which can be used in a wide variety of (multi-)tracer DNA tracers in environmental waters: effect of experiments both in the karst systems of the filtering. Rendiconti online della Società Bussento and in others karst river drainage system Geologica Italiana, Vol. 28,. 12th European of the Geopark, taking into account the low Geoparks Conference, 4-7 September 2013. pp. recovery rate of the pre-experiment salt injection. 70-73 The main shortcoming, resides in the manually [7] Guida D., Cuomo A., Longobardi A., Villani operated collection of samples, this requires to know P., Guida M., Guadagnuolo (2012), Integrated somehow in advance the time transit from the input Hydro-geomorphological Monitoring System to the outlet sections. of the Upper Bussento river basin (Cilento and Vallo Diano Geopark, S_Italy). Geophysical Research Abstracts, EGU General Assembly, Acknowledgments Vol. 14, EGU2012-14340 Vienna 2012 The authors tanks Paolo Villani and Giuseppe [8] Guida D., Longobardi A., & Villani P., Benevento, Director and Project Manager of the Hydrological modelling for river basin CUGRI-Salerno University, for scientific support; management in an highly hydro-geological Angelo De Vita and Aniello Aloia, Director and conditioned environment. Geo-Environment & Geopark Manager of the Cilento National Park- Landscape Evolution II. Eds. J. F. Martin- European Geopark, for institutional support; Stefano Duque, C. A. Brebbia, 3. D. Emmanouloudis, Sorvino and Raffaele Doto, Campania Sud Regional & U. Mander,, WIT Press. SOUTHAMPTON Basin Authority, for financial support. Pasqualino ISBN 1-84564-168-X .2005. pp. 283-292. Lovisi and Arnaldo Iudici, for field collaboration. [9] Guida, M., Guida, D., Guadagnuolo, D., Cuomo, A., & Siervo, V., Using Radon-222 as

a Naturally Occurring Tracer to investigate the

streamflow-groundwater interactions in typical References: Mediterranean fluvial-karst landscapes:

[1] Aloia A., Calcaterra D., Guida D., Valloni R., interdisciplinary studies in the Campania region Field Trip Guidebook – Four itineraries (southern Italy). Quinta convención cubana de through the geological and cultural heritage of ciencias de la tierra, geociencias 2013. 1-5 the Cilento, Vallo Diano and Alburni Geopark April Cuba 2013 2013 ISBN 978-88-907281-2-9. [10] Leibundgut C., Malozewski P., K¨ulls C., [2] Aquilanti L., Clementi F., Landolfo S., Nanni Tracers in Hydrology, John Wiley & Sons T., Palpacelli S., Tazioli, A., A DNA tracer Chichester UK 2009 used in column tests for hydrogeology [11] Longobardi A., Cuomo, A., Guida, D., & Villani P., Water Resources Assessment for applications. Environmental Earth Sciences, 70 (7), 2013 pp. 3143-3154. Karst Aquifer Conditioned River Basins: Conceptual Balance Model Results and Comparison with Experimental Environmental

ISBN: 978-960-474-376-6 111 Latest Trends in Engineering Mechanics, Structures, Engineering Geology

Tracers Evidences. in, Earth and Environmental Sciences eds. I. A. Dar, & M. A. Dar. InTech, ISBN 978-953-307-468-9. 2011 pp. 276-298 [12] Montgomery D. R. and Buffington J. M., Channel processes, classification, and response. In: River Ecology and Management (Naiman R.J. and Bilby R.E., Eds.), 1998, Springer Verlag.

ISBN: 978-960-474-376-6 112