Research Ideas and Outcomes 5: e48898

doi: 10.3897/rio.5.e48898

Grant Proposal

Monitoring and risk assessment for groundwater

sources in rural communities of Romania
(GROUNDWATERISK)
Oana Teodora Moldovan‡, Rannveig Øvrevik Skoglund§, Horia Leonard Banciu|, Alexandra Dinu
Cucoș|, Erika Andrea Levei¶, Aurel Perșoiu‡, Stein-Erik Lauritzen§
‡ Emil Racovitza Institute of Speleology, Cluj-Napoca, Romania
§ University of Bergen, Bergen, Norway
| Babes Bolyai University, Cluj Napoca, Romania
¶ INCDO INOE 2000 Research Institute for Analytical Instrumentation, Cluj-Napoca, Romania

Corresponding author: Oana Teodora Moldovan ([email protected]) Reviewable v1

Received: 28 Nov 2019 | Published: 05 Dec 2019

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

Abstract
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

© Moldovan O et al. 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.
2 Moldovan O et al

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.

Keywords
groundwater, springs, microbiology, chemistry, stable isotopes, radon, risk assessment,
ecosystem services, rural communities, Romania, Norway

Overall aim of the project

The research aims of the GROUNDWATERISK project (Fig. 1) are to improve the
methods of groundwater monitoring for a better quality of life in rural communities that use
local sources of water and to raise the awareness of groundwater users on potential health
risks and the need to protect vulnerable groundwater ecosystems in connection to the
surface land use.

Figure 1.
The project logo.
 Monitoring and risk assessment for groundwater sources in rural communities ... 3

The proposed OBJECTIVES are:

1. Interdisciplinary evaluation of groundwater ecosystems used as sources of drinking

water in rural communities
2. Finding the most feasible method for groundwater microbiological monitoring to
ensure water quality for human use and to better protect against possible outbreaks
of pathogenic bacteria caused by contaminated drinking water
3. Risk assessment for the groundwater sources used by rural communities
4. Assessment of the groundwater ecosystem services to enhance conservation
practices in rural communities.

The target group of our research are the rural communities that are not connected to a
public water supply. According to Eurostat 2019, in Romania in 2017, only 67.5% of the
population is connected to a public water supply, a number which is continuously
increasing. It means that the rest of more than 32.5% of the population, mostly rural
inhabitants, has to use local sources of water. These sources are represented by water
taken directly from caves, wells or springs. The quality of these water sources are not
monitored on a regular basis by the water agencies or by agencies for public health. To the
danger represented by the lack of water quality monitoring adds, in rural areas, the
agricultural practices and the lack of waste management. Agriculture and household
wastes accumulation on the surface are two main groundwater polluting factors. Moreover,
the waters taken directly from caves can be polluted by the inhabiting bats that adds to the
inherent lack of mechanical filtering of karst waters.

Key targets to be achieved in the project are related to the proposed objectives:

• An evaluation of the quality and sustainability of groundwater sources used by the

rural communities across Romania;
• Establishing the most feasible method for microbiological monitoring and radon
concentration measurements of drinking water to be applied by responsible
agencies;
• To assess the environmental and human-induced risks for groundwater sources in
different regions of Romania;
• To educate people for the protection of their local source of water and its
sustainable use.

State of the art

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 (Gibert et al.
1994). It is therefore a vital resource, which requires constant protection. The Groundwater
Directive 2006/118/EC, a derived directive of the WFD, is protecting groundwater in terms
of quality (chemical composition) and quantity, while attention is paid to groundwater as
4 Moldovan O et al

ecosystem only in association with surface systems through the Groundwater Directive
2014/80/EU and the Groundwater Directive 2006/118/EU.

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
(Bitton 2014, World Health Organization 2015) contribute to a high cost for prevention and
treatment. Bacterial indicators such as total coliforms, faecal coliforms, E. coli, faecal
streptococci, and many others are widely used for the assessment of water quality in
Europe, including Romania. They are used for detection of anthropogenic impacts such as
faecal pollution of water, mainly caused by raw and treated sewage and diffuse impacts
from farmlands and pastures.

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 (Ramírez-Castillo et al. 2015).

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 (World Health Organization 2009). The latter resulting from tap water
utilized by households. Since radon is soluble in water, its degassing is added to the indoor
exposure (Kendall and Smith 2002). Special attention is needed when groundwater is used
for drinking water, as radon can become a risk factor for users if the radon concentration in
the aquifer is high (Committee on Risk Assessment of Exposure to Radon in Drinking
Water 1999). Generally, groundwater contains, potentially, much higher concentrations of
 Monitoring and risk assessment for groundwater sources in rural communities ... 5

radon than surface water (Cosma et al. 2008). The radon activity concentrations in surface
waters is low, usually below 1 Bq/L, while in groundwater vary from 1 to 50 Bq/L for rock
aquifers in sedimentary rocks, 10 to 300 Bq/L for wells dug in soil, and 100 to 50 000 Bq/L
in crystalline rocks. The highest concentrations are usually associated with high uranium
concentrations in the bedrock. A characteristic of radon concentrations in rock aquifers is
their variability; within a region with fairly uniform rock types, some wells having
concentrations far above the average for that region (European Commission 2001). On the
basis of Articles 35-36 of the Euratom Treaty, The European Commission’s Drinking Water
Directive (98/83/EC) established a maximum effective dose of 0.1 mSv/y for ingestion of
water supplied by the public network (Council Directive 98/83/EC 1998, Euratom 2010).
Radon and its decay products were excepted from the calculation of this maximum
effective dose. Commission Recommendation 2001/928/Euratom (European Commission
2001) comes to meet this omission by establishing maximum concentration values for
radon (100 Bq/L) and its long-lived decay products (210Po: 0.1 Bq/L and 210Pb: 0.2 Bq/L).
The latest European Union 2013/51/Euratom (Council Directive 2013/51/Euratom 2013)
states that Member States may set a level for radon which is judged inappropriate to be
exceeded and below which optimization of protection should be continued, without
compromising water supply on a national or regional scale. The level set by a Member
State may be higher than 100 Bq/L but lower than 1,000 Bq/L. In order to simplify national
legislation, Member States may choose to adjust the parametric value to this level.
Remedial action is deemed to be justified on radiological protection grounds, without
further consideration, where radon concentrations exceed 1,000 Bq/L. Recently the
Romanian government has adopted the Law no. 301/2015 (Official Journal, Monitorul
Oficial 2015) regarding the requirements for the protection of the health of the general
public with regard to radioactive substances in drinking water, which transposes Directive
2013/51/Euratom laying down requirements for the protection of public health in radioactive
substances in water intended for human consumption.

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 (Schwab et al. 2017, Smith et al. 2012, Korbel et al.
2017).
6 Moldovan O et al

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 (Korbel et al. 2017). Groundwater near animal farms may be a source of
pathogenic Campylobacter sp., E. coli, Yersinia sp. (Pitkänen et al. 2011). Several human
pathogens (E. coli, enterococci, Pseudomonas aeruginosa, Salmonella, Bacillus, Shigella
and Staphylococcus aureus) have been detected in groundwater (Grisey et al. 2010),
posing a serious risk in water resources, as these microbes drastically influence
groundwater purity and availability of drinking water.

Risk analysis requires a holistic approach to assess stress and vulnerability of

groundwater resources. Risk assessment of water supplies aims at:

1. identifying causes that may threaten the quality of the water supply;
2. assess whether the harm can be eliminated; and if not
3. suggest preventive or protective measures that can control and reduce the risk.

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 (Civita 2010). Once groundwater is contaminated, it is very
costly to clean up and takes a very long time to recover. In addition, groundwater
monitoring is time-consuming and too costly to adequately define the geographic extent of
contamination at a regional scale. Thus, the appropriate way to manage groundwater
contamination is to identify the spatial distribution of the areas with contamination risk or
vulnerable to contamination (Lahr and Kooistra 2010, Mimi and Assi 2009). Groundwater
risk is determined by combining the vulnerability and the hazard assessments. The highest
groundwater contamination risk appears when hazards occur in a high vulnerability zone
(Ravbar and Goldscheider 2007).

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 (Zwahlen 2003). The methodology
for assessing the intrinsic vulnerability of karst areas (Goldscheider et al. 2000, Vías et al.
2006), include evaluation of the overlying layer or protective cover, and the infiltration
conditions like concentration of flow and precipitation. The European Approach also add
the K-factor, the degree of karst and flow system development (Zwahlen 2003). A thick soil
 Monitoring and risk assessment for groundwater sources in rural communities ... 7

cover with low permeability where dispersion, dilution and degradation may occur, have a
protective function to the groundwater resource and may prevent contamination from the
terrain surface to enter the karst system. Assessment of the quality and presence of a
cover layer, and identification of swallow holes where the cover layer may be bypassed is
an essential step in the risk assessment. Field survey and local information combined with
remote sensing in GIS-analysis provide the basis for the intrinsic vulnerability maps for the
ground surface.

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 (Almasri 2008, Thapinta and
Hudak 2003). Risk estimate of groundwater contamination at a basin/sub-basin scale will
help management plans and strategies to reduce the risk of surface and groundwater
contamination and provide communities a better understanding of potential impacts to local
water resources. A drinking water system is often described as an integrated chain of
supply from source to consumer (World Health Organization 2008). When groundwater is
the source for public water supply, understanding the impacts of land use and aquifer
vulnerability are fundamental to groundwater protection (Somaratne et al. 2013). One of
the important risks to groundwater is the adverse land use and therefore, it is important to
identify which aquifer systems are at high risk in order to adopt appropriate risk
management options. Several process-based approaches exist for assessing whether a
contaminated site or surface applied chemicals constitutes a risk to groundwater.

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 (Hylands et al. 2012). Risk analysis in this study shows that source type,
groundwater vulnerability, subsoil type, and set back distance from septic tanks are all
important factors for the presence of TTC. However, risk assessment tools and risk
management actions must be proactive rather than being a reactive response to the
detection of coliform bacteria.

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 (Brown et al. 2004). Several authors
(Haines-Young and Potschin 2010, Rounsevell et al. 2010) have defined and assessed
ecosystem services and highlighted key strengths and weaknesses within these
frameworks (Reyers et al. 2014). It further resulted in the development of new frameworks
which attempt to make the links between ecosystem structures, processes and human
well-being much more explicit (e.g. Haines-Young and Potschin 2010, Potschin and
Haines-Young 2011).
8 Moldovan O et al

The important outcomes of the project

Project contribution beyond the state-of-the-art

The research we propose has several components that are new to science and others
which were never applied in Romania, as follows:

• first overview of the quality of groundwater sources used as drinking water in

Romania by using multidisciplinary indicators (physical, chemical, biological and
microbiological);
• identifying a filter for microbiological sampling that will be effective not only in
detecting the presence/absence of microbial pathogens but also in identifying the
presence of pathogens at low concentrations;
• new protocol for groundwater microbiological monitoring and accurate identification
based on molecular methods;
• first radon analysis on water and first derived map on radon risks in Romania;
• identification of groundwater invertebrates that can be used as indicators of water
quality;
• identification of microorganisms possibly involved in water purification;
• first hydrological studies at a country level that will use stable isotopes as indicators
of water origin and water residence time underground;
• first risk maps for groundwater sources in Romania and the first maps that will
include microbial pathogens and radon in the analysis;
• first maps of groundwater ecosystem services in Romania.

Technical milestones and expected results

The milestones and the expected results are as follows:

• Compiling a database with all the results (physico-chemical, radon, stable isotopes,
biological, microbiological) after the monitoring period. This database will also
contain the identified invertebrate species, inferred pathogenic and nonpathogenic
microorganisms in each of the studied sites and will provide the basis for the risk
analysis and the ecosystem services approaches;
• Choosing both the optimal filter and filtration methods for retaining of pathogen's
biomass and the optimal test for pathogens in groundwater in terms of costs,
accuracy, efficiency and flexibility. The filter and the test will be used to validate a
protocol for best practices in microbiological monitoring for groundwater sources of
drinking water. The protocol will be published and distributed to water agencies;
• Risk analysis for all the studied sites. It will highlight the vulnerabilities of local
groundwater ecosystems and the possible risks for the human populations;
• A GIS model with ecosystem services in Romania. A simplified model will be
distributed in brochures and leaflets addressed to rural communities and local
schools and for the training of the personnel of water and health agencies;
• Conferences held in local communities and schools on the need to protect
groundwater sources and the importance of groundwater ecosystems;
 Monitoring and risk assessment for groundwater sources in rural communities ... 9

• A guideline on measurement procedures for the determination of radon

concentration in water;
• Publication of results in high impact factor journals and presentation of results at
national and international conferences;
• Implication of students and young researchers in new fields of research and in
interdisciplinary activities.

Methodologies to be used
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:

• Physico-chemical measurements. Electrical conductivity, temperature, pH, oxygen

and flow speed will be measured in situ. For the other chemical parameters 0.5-1 L
water sample will be collected in PTFE bottles. Samples will be stored and
conserved using standardized protocols (metals by acidulating with concentrated
nitric acid, organics by refrigeration).
• 25 ml aliquot of water will be sampled for stable isotope analyses. Every three
months, samples will be collected from the stations and transported to the
laboratory for analyses.
• Aquatic invertebrates will be sampled by using a 60 µm planktonic net directly from
springs for a standardized quantity of water. All samples will be preserved in 95%
ethanol.
• Microorganisms will be sampled on commercial films with growth media. At each
sampling site, 1 mL of water will be directly applied, with a sterile plastic pipette,
onto the growth medium surface, and then transported, on ice, to the laboratory for
incubation.

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
10 Moldovan O et al

being expressed in the total readings after five days (Bercea et al. 2018). The visible
colonies will be transferred from plates to agar for growth. After extraction, PCR products
will be purified. DNA will be sequenced. The sequences obtained will be deposited in an
online database.

(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
(Szekeres et al. 2018). The high sensitivity and detection speed of this method is facilitated
by the simultaneous amplification and visualization of the newly formed DNA amplicons.
With the appropriate primers and optimized qPCR conditions, the amplified DNA sequence
can be unique to the level of genus, species, or even strain of microorganism (Schwab et
al. 2017, Rochelle and Schwab 2011). Thus, qPCR allows a rapid screen and
quantification of an array of pathogenic bacteria, which can be employed as tracers to
understand groundwater vulnerability to microbial contamination. To appropriately target
the microorganisms of interest in the qPCR assays, we will select specific primers
designed for functional genes distinctive of the pathogens detected by method (a). For fast
simultaneous microbial identification and profiling (i.e. detection of transcripts), dedicated
kits can be used. However, as the drawbacks of the qPCR include false-positive results we
will further proceed for the last, refining step that will clearly and most accurately evidence
the cells and vectors (plasmids, phages) bearing pathogenic traits.

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.
 Monitoring and risk assessment for groundwater sources in rural communities ... 11

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,
12 Moldovan O et al

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 (Mulligan 2015, Mulligan et al. 2010); ARtificial Intelligence for
Ecosystem Services (ARIES) that maps and values ecosystem services and assesses the
impacts of land use on them (Villa et al. 2014); Lund-Potsdam-Jena Managed Land model
(LPJmL) is designed to simulate vegetation composition and distribution as well as stocks
 Monitoring and risk assessment for groundwater sources in rural communities ... 13

and land-atmosphere exchange flows of carbon and water, for both natural and agricultural
ecosystems (Schaphoff et al. 2018b, Schaphoff et al. 2018a).

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.

Project structure
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. 2 and Fig. 3).

Figure 2.
Gantt Chart of the proposed activities during the project (June 2019 – May 2023). PR =
Phase Report, FR = Final Report; *only 1 month (1) or 2 months (17).
14 Moldovan O et al

Figure 3.
The flow of the WPs and their relationship.

WP 1. Evaluating the groundwater ecosystems used as sources of drinking

water

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:

1. Selection of sites to be studied and preliminary sampling;

2. Testing of an efficient water filtering method for pathogens;
3. Continuous sampling of precipitation for isotopic analysis;
4. Seasonal sampling of microorganisms and invertebrate fauna in water for two
successive years and on-site measurements;
5. Laboratory analysis;
6. Molecular identification of microorganisms and fauna identification in the laboratory
by use of conventional morphological methods;
7. Project management.

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.
 Monitoring and risk assessment for groundwater sources in rural communities ... 15

WP 2. Improving the methods for groundwater microbiological monitoring to

better protect against possible outbreaks of pathogen bacteria in the drinking
water

Objectives. Testing and validating a method for groundwater microbiological monitoring for
water sources.

Activities:

1. Laboratory testing of different methods for monitoring pathogenic bacteria;

2. Comparing the results of the three methods from all points of view;
3. Validating monitoring protocols on groundwaters of different qualities;
4. Project management.

Deliverables. Protocol for microbiological monitoring, Patent documentation, Results

dissemination, Phase report.

WP 3. Risk assessment for the improvised groundwater sources used by rural

communities

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:

1. Assessment of the quality and presence of a cover layer;

2. Identification of swallow holes and inlets on the surface;
3. Assessment of the human and agricultural activities on the surface;
4. Evaluation of the radiological risk for groundwater;
5. Production of GIS-based vulnerability maps;
6. Risk reports for the study cases;
7. Project management.

Deliverables. Common field-trip in Romania, GIS vulnerability maps, Risk assessment

reports, Report on radon, Results dissemination, Phase report.

WP 4. Assessment of the groundwater services in romania for enhancing

environmental awareness and education

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.
16 Moldovan O et al

Activities:

1. Developing groundwater ecosystem services indicators;

2. Mapping the ecosystem services in the studied areas;
3. Developing a tool for good practices on measurement procedures for the
determination of radon concentration in water;
4. Printing brochures and leaflets for local communities (for adults and for children);
5. Training for responsible water and health agencies for the best monitoring method
and protection of groundwater sources;
6. Educating the local communities on the potential health impact associated with the
pollution/radon from groundwater sources;
7. Project management.

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.

Acknowledgements
This proposal was reviewed by a team of international reviewers as a submitted research
proposal before being awarded funding.

Funding program
The EEA Grants CALL FOR PROPOSALS 2018 – Collaborative Research Projects

Grant title
Monitoring and risk assessment for groundwater sources in rural communities of
Romania (GROUNDWATERISK)

Hosting institution
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
 Monitoring and risk assessment for groundwater sources in rural communities ... 17

Author contributions
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.

Conflicts of interest
There is no conflict of interests.

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