Water Quality and Associated Human Health Risk Assessment Related to Some Ions and Trace Elements in a Series of Rural Roma Communities in Transylvania, Romania

This research aims to assess the content of some ions and trace elements in water sources in 24 rural Roma communities in Transylvania in order to assess the human health risk associated with exposure to such elements and ions. To this end, eight ions (F−, Cl−, Br−, NO2−, NO3−, SO42−, PO43−, NH4+) and ten trace elements (Cr, Ni, As, Pb, Cd, Mn, Cu, Zn, Fe, and Hg) were determined in 71 water samples by ion chromatography coupled with a conductivity detector for ions and atomic absorption spectrophotometry for all trace elements. General parameters were also determined. Non-conformity (as number of samples), according to the EU Drinking Water Directive, was observed as follows: pH (7), EC (7), hardness (1), oxidizability (15), Cl− (4), NO3− (30), SO42− (6), Fe (16), Mn (14), As (3), and Ni (1 sample). The incidence of ions was Cl− (71), SO42− (70), F− (67), NO3− (65), NH4+ (21), Br− (10), PO43−, and NO2− (1 sample) and for trace elements, Mn (59), Fe (50), As (38), Ni (32), Cu (29), Zn (28), Cd (12), Cr (11), and Pb (3 samples). Hg was not detected. Non-carcinogenic (HI) values exceeded one for As in 13 Roma communities, with higher values for children than for adults. For NO3−, the HI values were >1 in 12 for adults and 14 communities for children. The carcinogenic risk (CR) for As through ingestion ranged from 0.795 to 3.50 × 10−4 for adults and from 1.215 to 5.30 × 10−4 for children. CR by dermal contact was in the range of ×10−6 both for adults and children.


Introduction
Water is considered a primary need [1], which is both limited [2] and essential for sustaining life [3]; therefore, water quality is crucial for the health and well-being of a country's inhabitants.Since agriculture, urbanization, industrialization, and other human activities have increased, the general quality of both surface and underground water has suffered [4].
One of the most important and widespread contaminants in groundwater worldwide is nitrate (NO 3 − ), which has high solubility, mobility, and stability in water [6][7][8].Possible Foods 2024, 13, 496 2 of 13 sources of nitrate include animal waste, use of nitrogen-containing fertilizers (also a source of nitrites (NO 2 − )) [9], and irrigation using wastewater [7,8].The consumption of water with high concentrations of nitrates and nitrites, especially among infants, leads to methemoglobinemia ("blue baby syndrome") [8]; among adults, it can cause gastric cancer [9].In the digestive tract, nitrate is converted to nitrite [10].Within the European Union, the Drinking Water Directive (EU/2020/2184) [11] sets the standards for nitrate at 50 mg/L and for nitrite at 0.5 mg/L for water intended for human consumption.
Bromine is an element present in the Earth's crust and therefore automatically occurs in water.Following the water disinfection process, it can react with organic matter and form brominated disinfection by-products, which are carcinogenic [12].The threshold for bromate in the drinking water in the European Union is set at 10 µg/L [11].Although in low concentrations (<0.5 mg/L), fluoride (F − ) is necessary for bone mineralization and the formation of dental enamel, while in high concentrations (>1.5 mg/L) it can cause dental and skeletal fluorosis [5,8].For this reason, in the European Union, the concentration of fluoride in drinking water must not exceed the threshold of 1.5 mg/L [11].
Nitrate, chloride, and sulfate (SO 4 2− ) are considered indicators of anthropogenic contamination; sodium and chloride indicate salinization, bicarbonate (HCO 3 − ) shows acidification or water hardening, while fluoride indicates an intensive evaporation [13].In the European Union, the concentrations of chloride and sulfate in drinking water must not exceed the threshold of 250 mg/L [11].
Along with anions and cations, toxic metals are usually present in water, endangering the health of both humans and aquatic organisms.Toxic metals are often associated with anthropogenic activities (like agricultural practices, mining and smelting activities, domestic and industrial wastewaters, and even emissions due to traffic) and with natural sources (erosion and weathering of crustal materials) [14][15][16][17][18]. High temperatures and low pH values allow the toxic metals to be more easily released into the water [19].Because of these reasons, toxic metals are a significant global concern, being ubiquitous, persistent, nonbiodegradable, and bioaccumulative [18,20,21].Long-term exposure to water contaminated with toxic metals can drastically affect the normal functioning of the human body, since they interact with biological molecules containing nitrogen, oxygen, and sulfur, triggering structural changes and functional effects.This can eventually lead to central nervous system disorders, skin lesions and blood vessel damage, immune system dysfunction, gastrointestinal and kidney problems, and sometimes cancer [22].For example, the inorganic form of arsenic is much more toxic than the organic form [5]; exposure can lead to dermal lesions, cardiovascular disease, as well as skin, bladder, and lung cancer [23].Cadmium poisoning can produce kidney, lung, and liver dysfunction, bone fracture, and cancer [21].Lead intake can cause anemia, central nervous system dysfunctions, as well as kidney and fertility problems [1,21,23].Nickel can cause skin irritation, as well as kidney, liver, and brain dysfunctions, while copper ingestion causes dizziness, nausea, and respiratory and gastrointestinal problems.Mercury intake may lead to the development of neurological injuries, and chromium can generate skin irritation and kidney dysfunction [16,21].Ingesting zinc can lead to digestive disorders and even death [24].In the European Union, the thresholds of heavy metals as contaminants present in drinking water are As 10 µg/L; Cd 5 µg/L; Cr 50 µg/L; Cu 2 mg/L; Pb 10 µg/L; Hg 1 µg/L; and Ni 20 µg/L [11].
Evaluating health risk by investigating some of the anions and toxic metals in water is essential to determine the probable illnesses caused by drinking water and to protect public health as well; this risk assessment is being carried out all around the world [1,3,7,8,10,15,19].The most commonly applied methods are chronic daily intake, hazard quotient, and total carcinogenic risk [19,[25][26][27][28][29][30][31][32][33][34].Human health risk assessment is defined by the United States Environmental Protection Agency (US EPA) [25] as a systematic approach to assess the potential health effects of exposure to certain harmful substances that are present in polluted environmental media [25,26].Simply said, it is a systematic model for quantitative or semi-quantitative description of the negative effects of exposure to harmful substances [8].In the less developed areas of Romania, including those inhabited by Roma rural communities, wells are fed with underground water and provide drinking water to a large part of that community's population.Groundwater is used without any prior treatment or analysis, therefore posing a significant health risk of exposure to contaminants.According to the European Union Drinking Water Directive [11], Romania, as a member state, must take all the measures to ensure that the water for human consumption is free of chemicals, parasites, and microorganisms that could endanger human health; also, ensuring access to water must be, as a main sustainable development objective, a priority in the case of marginalized population categories such as the Roma.
The main objective of this study was to evaluate water contamination with certain ions and toxic metals in a series of rural Roma communities in Transylvania, Romania, and then to assess the human health risk due to water consumption.In addition, the findings of this study will help to design and implement a set of guide recommendations for improving rural drinking water quality for the residents of the mentioned communities.

Samples and Sampling Areas
A total of 71 water samples were collected in 24 rural Roma communities in Transylvania, Romania (Figure 1).
are present in polluted environmental media [25,26].Simply said, it is a systematic m for quantitative or semi-quantitative description of the negative effects of exposu harmful substances [8].
In the less developed areas of Romania, including those inhabited by Roma communities, wells are fed with underground water and provide drinking water to a part of that community's population.Groundwater is used without any prior treat or analysis, therefore posing a significant health risk of exposure to contaminants.Ac ing to the European Union Drinking Water Directive [11], Romania, as a member must take all the measures to ensure that the water for human consumption is fr chemicals, parasites, and microorganisms that could endanger human health; also, e ing access to water must be, as a main sustainable development objective, a priority i case of marginalized population categories such as the Roma.
The main objective of this study was to evaluate water contamination with ce ions and toxic metals in a series of rural Roma communities in Transylvania, Rom and then to assess the human health risk due to water consumption.In addition, the ings of this study will help to design and implement a set of guide recommendation improving rural drinking water quality for the residents of the mentioned communi
The water samples were collected in 1 L brown glass bottles between October and November 2021.After sampling, the samples were transported to the laboratory in refrigerated bags and analyzed within a maximum of 48 h after sampling.Until the analysis, the samples were kept at a temperature of 5 • C. Before analyses, the water samples were filtered through filters with a pore size of 0.45 µm.

Analysis Methods
After the samples arrived at the laboratory, a series of physicochemical parameters, including pH, electrical conductivity (EC), carbonate and non-carbonate hardness, and oxidizability indices, were measured.pH and EC were measured using a multiparameter (Hanna Instrument, Cluj-Napoca, Romania).Before each series of measurements, the pH electrode was calibrated using two buffer solutions of pH 4 and 9, respectively, while the conductometer was calibrated using standards of 450 µS/cm and 1000 µS/cm.Carbonate and non-carbonate hardness and oxidizability indices were determined by titrimetric methods, using standard solutions of 0.1 N HCl, 0.1 N EDTA, and 0.1 N KMnO 4 , respectively.Total hardness was expressed as the sum of carbonate and non-carbonate hardness and is given in German degrees (1 German degree = 10 mg CaO).
The determination of anions (F − , Cl − , Br − , NO 2 − , NO 3 − , SO 4 2− , PO 4 3− ) was carried out by ion exchange chromatography using a Shimadzu ion chromatograph equipped with a high-pressure pump, conductometric detector, thermostat for columns, autosampler, and suppressor.The separation was carried out on a Dionex Ion Pac AS22 column (4 × 250 mm), using a mobile phase containing 4.5 mM sodium carbonate, 1.4 mM sodium bicarbonate.The flow rate of the mobile phase was 1 mL/min, and the samples were injected with a Shimadzu SIL-10 ADVP autosampler.
Ammonium was also determined by ion chromatography, using a Dionex Ion Pac SCS1 column (4 × 250 mm), with a mobile phase of 1 mL/min of 3 mM methanesulfonic acid solution, without suppression.
For the determination of anions and ammonium, the water samples were filtered through filters with a pore size of 0.45 µm, and for the determination of metals, the water samples were acidified to pH < 2 with 65% nitric acid.

Health Risk Assessment
Human health risk assessment involves estimating and quantifying the probability of occurrence of an event and the magnitude of its effect on health with a given exposure over a defined period.To this end, the United States Environmental Protection Agency (US EPA) has proposed a human health risk assessment model [25] widely used worldwide [26][27][28][29][30][31] which is based on four steps as follows: hazard identification, exposure assessment, response to a certain dose, and risk characterization [26].
In this study, the health risk was assessed by non-carcinogenic and carcinogenic risk of pollutants according to the US EPA recommendation [25].
The ingestion route and dermal contact were considered for estimating the exposure of the local population.The average daily doses from ingestion (ADD ing ) and dermal absorption (ADD dermal ) of a pollutant via water as the exposure pathways are expressed in (µg/kg/day) and were estimated by the following equations [26,28,29,31,33]: where ADD ing and ADD dermal represent the average daily doses via ingestion and dermal adsorption (µg/kg/day); BW is the average body weight (kg); AT is the average time of exposure (days); C w is the average concentration of heavy metals in water (µg/L); IR is the ingestion rate (L/day); EF is the exposure frequency (days/year); ED is the exposure duration (year); SA is the exposed skin area (cm 2 ); ET is the exposure time (h/day); and K p is the dermal permeability coefficient in water (cm/h).All exposure parameters were referenced from EPA and the scientific literature [25,27,28].
The potential non-carcinogenic risk via ingestion and dermal routes was assessed by hazard quotient (HQ) and hazard index (HI) using the following equations [28,33,34]: H I = ∑ HQ ing + HQ dermal (5) where R f D oral and R f D dermal are the oral and dermal toxicity reference doses for a specific metal.
The carcinogenic risk (CR) was estimated only for As and was calculated using the following equations [28,33]: CR dermal = ADD dermal × SF dermal (7) where SF represents the carcinogenic slope factors via ingestion and dermal exposure.

General Characteristics of Water Quality
Minimum, maximum, and average values of physicochemical parameters, anions, ammonium, and trace element concentration of analyzed water samples as a mean for each selected rural Roma community, as well as the maximum allowable concentration (MAC) established by the EU Drinking Water Directive [11], are given in Table 2. NO 2 − and PO 4 3− were found in one sample at a concentration of 5.77 and 4.57 mg/L and do not exceed MAC."*" values taken from [11].
From the 71 water samples analyzed, non-conformities with the limits established by the EU Directive were detected as follows: pH-7, EC-7, hardness-1, oxidizability-15, Cl − -4, NO 3 − -30, SO 4 2− -6, Ni-1, As-3, Mn-14, and Fe-16 samples.For a better visualization of the amplitude of the concentrations found in each selected Roma community, the ions and metals are further treated separately.

Anions and Ammonium Content in Water Samples
The highest incidence of anions in the samples analyzed was found for chloride (71 samples), followed by sulfate (70 samples), fluoride (67 samples), nitrate (65 samples), bromide (10 samples), and phosphate and nitrite (1 sample).Ammonium was detected in 21 analyzed water samples (Supplementary Material Table S1).In order to estimate the population's exposure to anions and ammonium through water consumption, the mean value obtained for each locality was taken into account (Table 3)."*" values taken from [11], "-" not detected.
From Table 3, it can be concluded that Cl − exceeds the MAC in two communities, NO 3 − in fourteen communities, and SO 4 2− in three communities.F − and NH 4 + do not exceed MAC in any selected community.Considering that the nitrite anion NO 2 − was found in only one sample, we can assume that the nitrate (NO 3 − ) has a geological origin.

Trace Element Content in Water Samples
The highest incidence of trace elements in the analyzed samples was found for Mn (59 samples), followed by Fe (50 samples), As (38 samples), Ni (32 samples), Cu (29 samples), Zn (28 samples), Cd (12 samples), Cr (11 samples), and Pb (3 samples) (Table S1).Hg was not detected in any analyzed sample.For a better estimate of the population's exposure to metals through water consumption, the mean value obtained for each locality was considered (Table 4)."*" values taken from [11], "-" not detected.
The results of the analyses carried out show that for Cr, Ni, As, Pb, Cd, and Cu, there are no MAC exceedances in any selected community.There are exceedances of the MAC for Mn in six communities and for Fe in seven communities.No MAC is established for Zn.Considering that in those investigated areas there were/are no anthropic activities that may generate heavy metals, we can assume that all investigated trace elements had a geological source.
Since the water sources are used in the studied communities both as a source of drinking water and as sources of domestic water, a simple comparison of the values found with the MAC is not sufficient.In order to study the effect on human health due to a longterm exposure to the studied elements, an evaluation of non-carcinogenic and carcinogenic risk is necessary.

Non-Carcinogenic Risk Assessment
According to the model recommended by the US EPA [25], the non-carcinogenic risk of trace elements and NO 3 − in water through ingestion and dermal routes was calculated and used as a health risk assessment tool (Tables S2-S4).The minimum, maximum, and average results of the HQ ing , HQ dermal , and HI results calculated using the average values of the elements measured in each Roma community are summarized in Table 5.
Apart from As and NO 3 − , the HI values were within the safety limits (<1), which indicates that the water from investigated rural Roma communities does not present a health risk [26] from the point of view of the investigated metals (Supplementary Materials Table S4).For As, in 13 of the 24 investigated Roma communities, the HI value is >1, which means that a significant health hazard occurs in these communities (Table S4) [26].The risk is higher for children than for adults, with the calculated HI values being higher in all 13 cases.In the case of NO 3 − , the HI values for adults are >1 in 12 Roma communities, compared to 14 of the 24 Roma communities in the case of children.
HQ values of metals and NO 3 − through dermal contact for adults and children were below 1 × 10 −3 (adults: 0.02 to 98.45 × 10 −3 ; children: 0.01 to 57.83 × 10 −3 ) (Table S3), indicating that the investigated elements presented a low hazard through dermal absorption [25,26].Except for As and NO 3 − , the HQ values of the investigated metals through the ingestion route ranged from 0.002 to 0.774 for adults and 0.002 to 1.129 (for Mn) (Table S2), indicating that ingestion exposure has a potential non-carcinogenic risk [26], being the main contributor to HI.For As, HQ ing values range from 1.76 to 7.76 for adults and from 2.70 to 11.76 for children.For NO 3 − , HQ ing values range from 0.003 to 6.13 for adults and from 0.004 to 9.32 for children.Values exceeding 1 indicate that the water sources present a significant non-carcinogenic risk for consumers [25,26].

Carcinogenic Risk Assessment
Among the elements studied, only As presents a carcinogenic risk, which is why the CR was calculated only for this element.CR was calculated for both ingestion and dermal contact routes (Table S5).
The obtained results show that in the 13 Roma communities in which As was identified, CR by ingestion for adults varied between 0.795 and 3.50 × 10 −4 and, for children, 1.215 and 5.30 × 10 −4 (Table 6).CR by dermal contact ranged from 1.01 to 4.43 × 10 −6 for adults and from 0.61 to 2.59 × 10 −6 for children (Table 6).

Figure 1 .
Figure 1.Map indication of all 24 water sampling points in the investigated area.

Figure 1 .
Figure 1.Map indication of all 24 water sampling points in the investigated area.

Table 1 .
The values of the variables used for estimation of ADD, HI, and CR.

Table 2 .
[11]es of physicochemical parameters, anions, ammonium, and trace element concentration of water samples in the study area and drinking water standard specifications given by EU Directive[11].

Table 3 .
Mean values of the ions in the analyzed water samples for each selected community.

Table 4 .
Mean values of the metals in the analyzed water samples for each selected community.

Table 5 .
The minimum, maximum, and average results of the HQ ing , HQ dermal , and HI of the investigated metals and NO 3 − in analyzed water samples.