Research Ideas and Outcomes : Grant Proposal
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Corresponding author: Oana Teodora Moldovan (oanamol35@gmail.com)
Received: 28 Nov 2019 | Published: 05 Dec 2019
© 2019 Oana Teodora Moldovan, Rannveig Øvrevik Skoglund, Horia Leonard Banciu, Alexandra Dinu Cucoș, Erika Andrea Levei, Aurel Perșoiu, Stein-Erik Lauritzen
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Moldovan OT, Øvrevik Skoglund R, Banciu HL, Dinu Cucoș A, Levei EA, Perșoiu A, Lauritzen S-E (2019) Monitoring and risk assessment for groundwater sources in rural communities of Romania (GROUNDWATERISK). Research Ideas and Outcomes 5: e48898. https://doi.org/10.3897/rio.5.e48898
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In the past 100 years, a decreasing rainfall trend has been recorded on Romanian territory, a trend that continues today. Therefore, realistic estimation of the groundwater resources is crucial, especially for the rural communities lacking the economic power to use alternative sources of drinking water. The groundwater sources used by rural communities in Romania generally originate directly from caves, wells or springs with no proper evaluation of the water quality. Groundwater is exposed to different pollutants, as bats' guano in caves, fertilizers in agricultural areas or livestock (cattle, sheep, goats, etc.) farms on the surface. On the other hand, the water extracted directly from inside the caves is affecting groundwater ecosystems, highly vulnerable to any human impact and neglected by European legislation so far. The project aims to monitor, during two consecutive years, groundwater sources with different degrees of above- and underground pollution, from different regions of Romania. To achieve the goals of the project, a multidisciplinary monitoring strategy that will include measurements of hydrological, physico-chemical and biological (microbiology and aquatic invertebrates’ assessment) parameters alongside the quantification of radon and stable isotopes, rainfall or possible inflows of water. The specific outcomes of this project are: i) to test, develop and validate a new, more rapid and efficient method for monitoring and risk assessment of groundwater sources – and not only – by using molecular techniques, and propose this method to the water agencies in Romania; ii) to propose for Romanian authorities to implement a harmonized coherent methodology to measure radon concentration in water, as a consequence of EURATOM Directive; and iii) to educate local communities that are using groundwater as source for drinking water and raise young people’s awareness on the benefits of ecosystem services provided by the groundwater.
groundwater, springs, microbiology, chemistry, stable isotopes, radon, risk assessment, ecosystem services, rural communities, Romania, Norway
The research aims of the GROUNDWATERISK project (Fig.
The proposed OBJECTIVES are:
The target group of our research are the rural communities that are not connected to a public water supply. According to
Key targets to be achieved in the project are related to the proposed objectives:
Groundwater is defined in the Water Framework Directive (WFD), Directive 2000/20/EC, as “all water which is below the surface of the ground in the saturation zone and in direct contact with the ground or subsoil”. Groundwater is the largest supply of water for human consumption with 97% of all freshwater on the Globe being underground (
Groundwater harbors a unique and vulnerable ecosystem characterized by lack of light and primary producers, relatively stable physico-chemical conditions and poor nutrients content - unless human-induces changes are interfering. The poor food resources originate from the surface as particulate organic carbon (POC) or dissolved organic carbon (DOC) and microbial activity, which is low per volume of water. Groundwater animals in Romania are invertebrates, mostly Crustaceans, which have particular adaptations to life underground: lack or reduction of eyes, depigmentation, elongated appendages, fine body shape, slow metabolism and high vulnerability to high variation of their physico-chemical environment. They are used in ecological studies as an biondicators of water quality, their presence and diversity indicating the ecological state of the environment.
Waterborne diseases are a global burden which is estimated to cause more than 2.2 million deaths/year and an even higher number of recorded and unrecorded illnesses (
For the monitoring of the quality of water used for drinking, irrigation and bathing along the physico-chemical parameters, the examination of the microbiological standard parameters is mandatory: EU-Surface & Drinking Water Directive 75/440/EEC and EU-Bathing Water Directive 76/160/EEC. Nevertheless, the methods applied in the microbiological monitoring of waters are outdated and underestimate the level of microbial pathogens (e.g. the multiple-tubes method is used as a standard for Romanian and other European countries water estimation of pathogen bacteria such as E. coli) while groundwater microbiological monitoring is not performed at all. Although molecular techniques might improve the identification and abundance estimation of these pathogens, several disadvantages such as the lack of standardization of protocols and sample processing are still a challenge (
Radon (222Rn), found in soil, rocks and water all over the Earth, is listed by the World Health Organization as the second leading cause of lung cancer after cigarette smoking. Areal variations of radon levels in houses depend on numerous factors, such as geological features, environmental parameters or occupational patterns. Most of the cancer risks from radon in drinking water arise from the transfer of radon into indoor air, and the exposure through inhalation (
Stable isotopes used for environmental studies. Due to the direct relationship between air temperature and δ18O and δ2H in rainfall and spring water, we can establish the moment when karst aquifers recharge occurs and the delay between the moment of surface rainfall and runoff and underground recharge (e.g., by determining the time difference between the moment of winter precipitation with very low δ18O and δ2H and the moment these low values are registered in the underground streams). The hypothesis is that, for hydrokarstic systems with diffuse feeding, there is a several months interval between rainfall and the moment when water reaches the subsurface karst. For karst systems fed directly through ponors and caves, the rainfall (including the potential contaminants) reaches the subsurface within days.
Metabarcoding of water samples and detection of pathogens. By next-generation sequencing methods, high diversity of prokaryotes pertaining to both Archaea and Bacteria has been detected in groundwater, including members of Crenarchaeota, Euryarchaeota, Proteobacteria, Planctomycetes, Actinobacteria, Chloroflexi, Chlorobi, Bacteroidetes, Firmicutes and Cyanobacteria phyla (
In areas with high population density and/or intensive land use, groundwater is vulnerable to contamination, as various pathogenic microorganisms may enter groundwater due to septic systems, livestock manure, contaminated wells or recharge waters, etc. Groundwater contaminants detected through DNA-based studies include members of Xanthomonadales (known crop pathogens), Pseudomonadales (components of biofertilizers), and Burkholderiales (Comamonadaceae) used as biocontrol agents in agriculture (
Risk analysis requires a holistic approach to assess stress and vulnerability of groundwater resources. Risk assessment of water supplies aims at:
In the case of lacking (economic) resources, identification of highest risks is essential so that these risks can be handled first.
The management of groundwater contamination is a very difficult task due to the spatial heterogeneity of the aquifers and the natural processes in the soil and the unsaturated and saturated zones of the karst (
Groundwater in karst terrain is vulnerable to contamination due to the concentrated channel flow with low transit time and little self-purification within the karstic system. The European Approach to karst vulnerability and risk mapping of karst aquifers, by the COST Action 620, define two central terms: the intrinsic vulnerability of groundwater to contaminant which considers the geological, hydrological and hydrogeological characteristics of the karst area, and the specific vulnerability that accounts for the properties of the contaminant or group of contaminants (
Karst aquifers are unique in the way that enlarged fissures, conduits and caves provide habitats for macro and microorganisms and may give humans direct access to the water resource inside the aquifer. Biological contamination inside the karstic system may be an important threat to the water quality and safety. An evaluation of the degree of karstification and the flow system development as well as human and biological activity in accessible caves is a second approach in the risk assessment. Risk and vulnerability maps are useful tools for limited monitoring resources and in such areas a major effort is required to avoid or mitigate the impact of human activities on the environment (
Development of a microbial contamination susceptibility model for private groundwater sources has been carried out by assessing the presence of thermotolerant coliform (TTC) in groundwater (
Ecosystem services. Ecosystem services are vital to human survival and wellbeing, and the judicious management of these systems being essential. Ecosystem service indicators are increasingly recognized as a key part of assessing whether ecosystem services are being managed appropriately and used sustainably (
The research we propose has several components that are new to science and others which were never applied in Romania, as follows:
The milestones and the expected results are as follows:
A. Sites selection. Sites will be selected in different regions of Romania, with different surface use, different origin of the water (surface – short flow underground, surface – long flow underground, aquifer), various hydrology, various human impacts on the surface, etc. Samples will be taken seasonally during a 2-years period.
B. Sampling. Water will be collected in special bottles for chemical analysis, microbiology, stable isotopes and radon:
C. Identification and molecular analysis of invertebrates. Samples will be sorted under the optical microscope and identified at least at fauna group level, except for the crustaceans that will be identified at species level. Specimens will be also sent to specialists for identification. Amphipods will be analyzed by molecular methods and a phylogeny with all the obtained sequences will be build-up.
D. Molecular identification of microorganisms and profiling of water-borne pathogens. We will attempt to explore the molecular diversity of putative pathogens in groundwater by sequentially and complementary using three different molecular biology approaches:
(a) Commercial films. The plates will be transported at constant temperature in a cooler bag, and placed in incubators. The plates will be analyzed at 24-hour intervals, the results being expressed in the total readings after five days (
(b) Metabarcoding. To rapidly screen for the putative diversity and abundances of bacteria (and Archaea altogether), the amplicon sequencing (or metabarcoding) technique targeting the highly conserved, taxonomic relevant 16S rRNA gene will be employed. Raw sequence data obtained by this approach will be analyzed. Several processing steps of joining pair-end reads, quality filtration, dereplication, singleton and chimera removal will provide good quality sequences for taxonomic assignment. Recently released DNA sequence databases are available and can be used for establishing taxonomic diversity (Silva 132, Greengenes ’13-8’, Ribosomal Database Project). The metabarcoding approach allows overpassing the limitations of culture-dependent techniques, being a cost-effective and fast assessment, providing data on the entire prokaryotic community including ‘unculturable’ or fastidious microbes. We expect that the metabarcoding approach will accurately resolve the microbial community composition down to family and genus level. Thus, the presence and abundances of bacterial families comprising pathogenic members (e.g., the Gram-negative Enterobaceriaceae, Campylobacteriaceae, Aeromonadaceae, the Gram-positive Streptococcaceae, Staphylococcaceae, etc.) will be quickly evidenced.
(c) Quantitative PCR (qPCR). If possible, presence of pathogens is inferred by method (a), the more sensitive qPCR assay will be performed targeting selected marker genes (
All molecular methods described above will be applied on the same samples collected from the same sites following the sub-splitting of membrane filters. The environmental DNA will be extracted from biomass retained on hydrophilic filter membranes with 0.22 µm pore size and large diameter (90 mm) under negative pressure (i.e., generated by vacuum pump). The filtered groundwater volumes (up to 15 L expected) will depend on how quick the filter membranes will be clogged. Each membrane will be then separated into slices needed for DNA extraction for further molecular analysis. The unused extracted DNA will be stored under freezing conditions.
E. Radon measurements. The radon measurements in water will use the Luk-VR system, which involves connecting a VR-scrubber to a radon detector. This method requires mixing of the dissolved radon from the water sample with the air above the water in the volume of the glass vessel. Following this procedure, the sample of air is transferred to the Luk 3P, and measured by the Lucas cell method.
The water samples will be collected in glass bottle of 0.5 L, fully filled and tightly sealed and transported to the laboratory for measurement purposes. The time interval between sampling and measurement is recommended to be of maximum 48 hours, in which case the half time must be considered and corrections are made accordingly
F. Stable isotopes. Precipitation will be collected continuously using specially designed collectors, constructed according to IAEA specification. A 3-L HDPE plastic canister is fitted with a funnel, prolonged with a plastic tube, channeling water to the bottom of the container. Excess air escapes the canister through to a narrow, 3 m long plastic tube, to minimize air exchange between the container and the outside environment. The funnel will be “sealed” with table tennis balls, to restrain insect access, but allow water collection. At the end of each month, the amount of water in the canister will be measured (to be compared with data provided by the Romanian National Meteorological Administration), and a 25 mL aliquot of water will be sampled for stable isotope analyses. The aliquot will be stored in HDPE scintillation vials at 4oC. In winter, snow samples will be collected after each event, allowed to melt at room temperature in closed vessels, and stored in similar manner to liquid samples. River water will be collected at the end of each month, from boats or bridges, from ca. 15 cm below water table, in 25 mL HDPE vials and refrigerated until analysis. Groundwater will be sampled from dug wells and deep wells (where available), as well as from springs.
Climate and hydrologic data will be provided by the respective national authorities. Where such data will not be available, we will install temperature data loggers, measuring air temperature with hourly resolution. An ongoing study has shown that there are no systematic differences between data from the national meteorological service and data provided by the loggers.
Stable isotope analyses will be performed in the laboratory. Prior to analysis, samples are filtered using 0.45 nm nylon microfilters. The results are calibrated against two internal standards (Greenland and Hawaii waters) and checked against a third one (Romanian water). Per laboratory internal regulations, an aliquot from each sample will be stored in 3 mL paraffin-sealed, screw-cap, glass vials.
G. Chemical analysis. Chemical parameters will be determined by standardized or alternative methods. Trace metals (Cd, Cu, Cr, Pb, Ni) will be determined by ICP-MS, mercury by atomic fluorescence spectrometry, major cations and P by ICP-OES and anions (nitrates, nitrites, phosphate, sulphates) by ion chromatography. Organic carbon ant total nitrogen will be determined by a combustion analyzer with NDIR and respectively chemiluminescence detector. Chemical oxygen demand (COD) and alkalinity will be determined by volumetric, total dissolved solids by gravimetric, while phenol index, ammonium and cyanides by spectrophotometric methods. Whenever applicable, standardized methods will be applied. For organics, water will be sampled in glass bottles, while for metals and anions in polyethylene bottles. Samples will be stored and conserved using standardized protocols (metals by acidulating with concentrated nitric acid, phenols by addition of phosphoric acid, organics by refrigeration). Where recommended, analysis (COD, DOC, alkalinity) will be carried out in the day of sampling. Quality control will be made by reference materials analysis and inter-laboratory trials.
H. Risk analysis. We will use an integrated method for assessing groundwater contamination risk, based on the interaction between natural conditions and human activities, and by using analytical and numerical tools within a GIS framework. Different factors along the contaminant pathway from source to groundwater will be incorporated in the GIS database and analytical and numerical tools in GIS software will be used. The spatial groundwater contamination will be classified into categories based on the degree of risk (very high, high, moderate, low and very low). This classification is performed by considering the factors that influence groundwater contamination and assigning relative weights to them. This process is performed in a GIS environment in which thematic maps are produced for every factor. The linear combination of the thematic maps and the selection of the weights yield the final map of groundwater contamination risk.
In addition to the 30 Romanian sites, the project will also benefit from monitoring and investigation of two sites in Northern Norway where karst springs serve as water supplies for small communities. These two sites will be used during the project as models for the Romanian sites and as school-sites for the Romanian students. At both sites, cave systems upstream have been surveyed and investigated.
I. Ecosystem services’ evaluation. Identifying indicators takes a combination of scientific rigor and creative thinking. Creative thinking may be a surprising skill in this context, but the indicators with the greatest impact are often produced by combining different kinds of data. Scientific rigor is necessary to identify indicators that are conceptually valid and defensible for their purpose. In our case, the microbiological aspect will be important, because not only pathogens will be identified, but also microorganisms that could be of importance for water purification.
Data relevant for developing ecosystem service indicators will be available from our database. A wide range of models that exist for monitoring ecosystem services will be tested, as for example:
Co$ting Nature that calculates the spatial distribution of ecosystem services for water, carbon, hazard mitigation and tourism and combines these with maps of conservation priority, threatened biodiversity and endemism to understand the spatial distribution of critical ecosystems (
J. Communication of the obtained results to local communities and water and health agencies is an important component of our project. The obtained results will be published and also presented to the general public on the project site. The editing of a brochure and a leaflet for the local communities and the children in the respective communities will be presented in a friendly manner. We will present not only the obtained results but also impact messages regarding the conservation of groundwater sources. Public conferences and training of people from water and health agencies are also part of our strategy for the improvement of groundwater monitoring and protection.
During our field work we will also try to establish contacts in the local communities and involve the children and young people in our monitoring activities.
The project is structured in 4 Work Packages (WPs) distributed along the 48 months of the project (June 2019 – May 2023). The project is split in seasons as we will do the sampling seasonally and all the work will be organized, at least for the first 2 years, according to the sampling campaigns (see also Fig.
Objectives. Monitoring in different sites across Romania and Norway to ensure a multidisciplinary view on the groundwater quality used by rural communities as drinking water (wells, caves and springs).
Activities:
Deliverables. Press conference, Stations established, Kick-off meeting, Best filter for water pathogens identified, Common field-work in Romania, Devices installed, Common field-work in Norway, Project site opened for the public access, First data introduced in the database, Workshop on methods in groundwater monitoring, Database completed, Results dissemination, Phase report.
Objectives. Testing and validating a method for groundwater microbiological monitoring for water sources.
Activities:
Deliverables. Protocol for microbiological monitoring, Patent documentation, Results dissemination, Phase report.
Objectives. Survey at the surface of areas and water basins where the monitored sites are located and production of GIS maps where the risks for each of the studied site will be highlighted.
Activities:
Deliverables. Common field-trip in Romania, GIS vulnerability maps, Risk assessment reports, Report on radon, Results dissemination, Phase report.
Objectives. Raise the interest of the rural communities for the ecosystem services provided by groundwater, including the important drinking water source service, and develop indicators and maps for these services by using the obtained results.
Activities:
Deliverables. Ecosystem services indicators, Inter-comparison report for radon, GIS model of ecosystem services, Tool for good practices for radon, End of project workshop, Training for the representatives of the water and health Romanian agencies, Leaflets/brochures edited and distributed in local communities, Conferences for local communities, Results dissemination, Final report.
This proposal was reviewed by a team of international reviewers as a submitted research proposal before being awarded funding.
The EEA Grants CALL FOR PROPOSALS 2018 – Collaborative Research Projects
Monitoring and risk assessment for groundwater sources in rural communities of Romania (GROUNDWATERISK)
The Romanian Academy - Cluj Branch, Cluj Department of the Emil Racovitza Institute of Speleology, Romania
University of Bergen, Norway
Babeș-Bolyai University of Cluj, Romania
National Institute of Research and Development for Optoelectronics INOE 2000, Research Institute for Analytical Instrumentation Subsidiary, Cluj-Napoca, Romania
OTM wrote the proposal, ROS contributed to the risk assessment part, HLB contributed to the molecular biology methods, ADC contributed to the radon part, EAL contributed to the chemical methods, AP contributed to the stable izotopes method, SEL contributed to the Norwegian selection of sites. All authors corrected and approved the final manuscript.
There is no conflict of interests.