Analysis of Selected Elements in Rivers and Streams of Papuk Nature Park, Croatia

Abstract

This study is the first investigation into the content of heavy metals (ecotoxic) and metalloids in the available water resources of the Papuk Nature Park (Croatia), conducted after the war in Croatia. Analyses of 13 elements, As, B, Ba, Cd, Co, Cr, Cu, Fe, Ni, Pb, Sr, V and Zn, were carried out on 21 water samples from rivers and their tributaries using the ICP-MS method. The concentration of heavy metals and metalloids in the water of the rivers and their tributaries was low, revealing an intact water environment. Depending on the area of sampling, medians (in µgL⁻¹) were 0.50–4.22 (As), 1.90–141.78 (B), 0.31–60.01 (Ba), 0.16–0.57 (Cd), 0.095–0.62 (Co), 0.18–0.66 (Cr), 1.59–15.89 (Cu), 12.1–1692.2 (Fe), 0.26–8.21 (Ni), 0.017–4.45 (Pb), 0.039–126.09 (Sr), 0.48–2.53 (V), and 3.01–25.95 (Zn). Higher concentrations of Fe (median ranged from 155.42 to 968.20 µgL⁻¹) were found in the northern part of the Papuk Nature Park and are considered to be of natural origin; therefore, anthropogenic influences are excluded. The watercourses of the Papuk Nature Park are generally characterized as a clean ecosystem. This, in part, can be attributed to the human activism legislation that declared Papuk a Nature Park, and the self-healing potential of nature.

1. Introduction

The Papuk Mountain is a part of the Slavonian Mountains, known for its abundance of surface and underground water. This has led to the creation of numerous mountain streams that eventually join larger rivers in the lowlands, offering a habitat for various species.

The area is located in the vicinity of the Drava River and Hungary, and it represents the central part of Croatia’s eastern region of Slavonia. The area is known for natural beauties, enormous forest wealth, wildlife, and water streams including one of the few Croatian waterfalls—Jankovac.

The watercourses in the Papuk area are mountain streams and rivers, known for their fast currents, high levels of dissolved oxygen, and lower water temperatures. Based on the classification of the Croatian hydrographic network, all of Papuk’s watercourses can be categorized as small mountain and sub-mountain streams on a silicate substrate. When we talk about the watercourses on Papuk, in addition to the possible consequences of the war, it is necessary to refer to the dangers of flooding, pumping water from springs, the illegal removal of gravel, illegal fishing, stone quarrying, the arrangement of watercourses, and exploitation fields. Starting from the Roman era, Papuk has been a location of many quarries and inhabited by populations that lived on land and farming. Nowadays, Papuk, unlike most similar parks and reservations in the world, contains four active quarries, the largest of which is Veličanka, located on the outskirts of the national park. Quarries create dust and expose deeper layers of soil [1]. A study conducted in 2011 revealed the detrimental effects of quarries on the watercourses located in their vicinity, including the Radlovačka River, Vetovka, Veličanka, and Brzaja. The negative impact was attributed to fine particles from the quarries that enter the watercourses, causing their mudding [1].

Starting from the 19th century, on the southern slopes of Papuk, in the wider Požega area, significant food, textile, and chemical factories have developed. From this period, the railway was built, crossing the Papuk Mountain. This was followed by roads and the D1 state motorway and railway, passing through the entire northern slope of Papuk, connecting Osijek and Zagreb. In 1974, the largest Croatian cement factory was built in Našice, located on the northeastern edge of the Papuk Nature Park. Increased emissions of cement dust from the Našice Cement Factory can contribute to air pollution and thus affect the environment. Research on spruce needles grown near the Našice Cement Factory revealed changes in anatomy and histochemical alternations that indicate a stressful situation caused by the direct deposition of alkaline dust, which can spread over long areas [2]. Over centuries, timber from Papuk was exploited, and numerous furniture companies (Požega, Đurđenovac) were built. These employed thousands of workers in the region, with Papuk oak being a raw material for the furniture industry. This made the wider Papuk surrounding area one of the most industrially developed regions of Croatia and Yugoslavia before the Homeland war. The war in this part of Croatia had an impact on the destruction of industrial plants, civilian objects, infrastructure, and the displacement of population, leaving Papuk with one-third of the population and a significantly reduced industrial capacity. The war in Croatia resulted in a significant release of contaminants into the environment as a result of the use of combat assets [3]. Although the combat around Papuk was just one part of the war in Croatia, the region experienced significant consequences. The combat also took a toll on the environment of the Papuk Nature Park area. Forests were damaged due to bombing, artillery strikes, and numerous mine fields [4,5,6]. The studies conducted in Croatia suggested a correlation between heavy metal exposure and 1991–1995 war actions. Heavy metal monitoring in the Drava River showed that concentrations of Pb, Cd, and Hg were increased during the war period [7].

In order to help restore Papuk to its previous condition, the Croatian Parliament declared the “Papuk Nature Park “area on 23 April 1999 based on the Act on the Proclamation of the Papuk Nature Park (“Official Gazette” 45/1999). With an area of 34.307 ha, it is the third largest of the 11 nature parks in the Republic of Croatia. Within the area of the “Papuk” Nature Park there are five more protected areas, declared based on the Nature Protection Act. The area of the “Streams around Papuk” Nature Park, in accordance with the Decree on the Ecological Network (“Official Gazette” 124/2013, 105/2015), is included in one of the three areas of the ecological network that also belong to the European Union’s Natura 2000 ecological network. The richness of hydrological and hydrogeological resources in the region is shaped by its geological structure, relief, and climate.

The term environmental contamination denotes the presence of undesirable materials in the air, water, and soil at concentrations exceeding permissible level. Furthermore, it is regarded as an undesirable change in the natural environment, leading to harmful effects on humans, as well as on both flora and fauna [8]. Clean drinking water is essential for life, making water quality monitoring a key aspect of drinking water management. Water pollution is a global issue, with the world’s population facing the consequences of contaminated water [9]. In addition to human activities, such as urban development replacing natural forests and further damaging the environment, agricultural, atmospheric, and mining sources also contribute to the accumulation of heavy metals in rivers and lakes. This buildup negatively affects both humans and animals. Heavy metals are among the most hazardous inorganic pollutants due to their non-biodegradability, ability to accumulate in living organisms, and toxic effects even at low concentrations. They can also enter the food chain. Furthermore, human activities, such as urban expansion and agriculture, increase environmental pressure, while agricultural, atmospheric, and mining sources may lead to the accumulation of heavy metals, adversely affecting plants, animals, and humans. The interaction of water with rocks can also introduce heavy metals and metalloids into water systems. These metals are naturally present in the earth, and they can enter water systems through various routes. Mining poses a substantial risk as it can displace and disperse heavy metals to surrounding regions during flood or windstorms [10]. The water of some watercourses in the area of the Papuk is still being used for human consumption, and for the purpose of protecting these watercourses and springs, sanitary protection zones have been declared that cover approximately 18% of the park. The water quality in the Papuk Nature Park has ecological significance and is one of the most important factors for nature lovers, hikers, plant diversity, and wildlife, as well as for the local population at the foothills of Papuk, who use some of the water sources for drinking. The Pannonian Basin is widely recognized for its groundwater resources with naturally high levels of arsenic, making it the largest region in Europe impacted by elevated concentrations of this element in drinking water. Around 1 million people are exposed to arsenic concentrations in their drinking water that exceed the EU’s maximum allowable limit of 10 μgL⁻¹ [11]. Also, groundwater in the lowland area of eastern Croatia contains high concentrations of Fe, Mn, ammonia, organic substances, and As [12,13,14]. In view of the mentioned problems, it is essential to explore alternative water sources in this region and take additional measures to protect sensitive and vulnerable areas, such as the Papuk Nature Park. Systematic investigations into water in this area have never been conducted. Similar studies were conducted only in the regional capital—Osijek and Baranya area—and were mainly carried out only on samples collected from well water and water from local water supply systems [12,13]. Previous research in the Papuk area has been mostly limited to uranium studies in metasediments [1]. A collection of uranium minerals, meta-torbernite, meta-uranospinite, and meta-zeunerite has been identified in metasediments of the Radlovac series in the Papuk area [15]. Although there are a large number of water sources in the Papuk Nature Park, they have never been systematically registered or analyzed. The aim of this study was to determine the metal and metalloid content in the available water sources in the wider Papuk area, since watercourses in the Papuk area are still being used for human and animal consumption. No comprehensive study has ever been conducted that takes into account the content of metals and metalloids in river water and assesses the potential environmental and human risks.

Thus, in this study, in the Papuk Nature Park, analyses of water from 21 rivers and streams were carried out. The concentrations of the following elements were determined using the ICP-MS method: As, B, Ba, Cd, Co, Cr, Cu, Fe, Ni, Pb, Sr, V, and Zn. The research covered the entire area of the Papuk Nature Park by taking water samples from the peripheral parts on the northern, southern, western, and eastern sides of the nature park, as well as from its central part. Principal Component Analysis (PCA) was selected as a powerful statistical tool for simplifying complex data matrices while retaining essential information, making it an invaluable method in exploratory data analysis. High-dimensional data sets are often difficult to analyze and visualize. PCA reduces the number of variables while preserving the most important patterns in the data. Since descriptive statistics provide only unidimensional insights into the data, two multivariate data analysis methods, cluster analysis and PCA, were applied to the complex data matrix.

2. Materials and Methods

2.1. Sampling and Analysis

The Papuk Nature Park is located in the continental part of the Republic of Croatia, where central and eastern Croatia meet (45°32′ N, 45°32′ E). According to the natural geographical regionalization, it belongs to the Pannonian megaregion, that is, to the area of the Slavonia Mountains. Administratively, the Papuk Nature Park is located in the territory of two Slavonian counties: Požega-Slavonska and Virovitica-Podravska. The sampling locations were placed in the north (N = 5), west (N = 4), southwest (N = 3), east (N = 3), south (N = 3), and central parts (N = 3) of Papuk (Figure 1).

The ICP-MS instrument was calibrated after every 12th sample using an external standard (“71-Element Group Multi Element Standard Solution”, Inorganic Ventures, Christiansburg, VA, USA), with the application of internal standards containing elements Y, In, Tb, and Bi (Inorganic Ventures, USA). Intercalibration (international laboratory verification) was conducted in collaboration with IFA Tulln (Department of the University of Natural Resources and Applied Life Sciences, Vienna, in cooperation with the Vienna University of Technology and the University of Veterinary Medicine). Analytical methods were validated using standard reference materials (ICP Multi Element Standard Solution X CertiPUR for Surface Water Testing, Merck, Darmstadt, Germany) and standard samples (“Trace Elements Urine Blank” and “Trace Elements Urine”, SERO AS, Hvalstad, Norway). Polyatomic interferences from elements such as Fe, As, Cr, etc. were eliminated in the Dynamic Reaction Cell (DRC) of the instrument with the help of the reactive gas methane (CH₄). Working conditions of the device and limits of detection and quantification were given in

Table 1. The working conditions of the device.

Parameters	Working Conditions
Spray gas flow rate, Argon > 99.99%	0.88 Lmin−1
Auxiliary gas flow rate	1.20 Lmin−1
Plasma flow rate	15.00 Lmin−1
Lens voltage	8.0 V
Power voltage	1050 W

and

Table 2. Limits of detection (LOD) and quantification (LQD) and recovery (%) determined by ICP-MS.

Element	LOD (µgL−1)	LQD (µgL−1)	Certified (µgL−1)	Found (µgL−1)	Recovery (%)
As	0.1	0.30	53	52.1	98
B	0.1	0.30	93	101	101.8
Ba	0.07	0.21	51	55	107.8
Cd	0.01	0.030	20	19.1	95.5
Co	0.01	0.030	25	24.8	99.2
Cr	0.01	0.03	21	20.5	97.6
Cu	0.1	0.30	21	22.3	106.1
Fe	3	9	102	110	107.8
Ni	0.04	0.12	50	48.8	97.6
Pb	0.005	0.015	27	25.9	95.2
Sr	0.01	0.03	97	101	104.1
V	0.04	0.12	51	50.1	98.2
Zn	0.5	1.5	52	55	105.7

.

Water samples from the watercourse for determining heavy metals and metalloids were collected in a clean 250 mL plastic bottle, which was first rinsed with the water sample and then preserved with 1.25 mL of concentrated nitric acid (HNO₃). After collecting, the samples were stored in a portable cooling box at a temperature of 6 (±2) °C and delivered to the laboratory for analysis. During transportation, additional care was taken to prevent any tilting or spillage of the samples from the containers. The samples were kept refrigerated at all times, from the moment of collection until delivery to the laboratory for analysis. In the laboratory, the samples were filtered through a filter paper membrane with a pore size of 0.45 µm.

2.2. Data Analysis

The power of chemometrics was used to identify the main relationships between variables (metal and metalloid concentrations) and objects (rivers). The (dis)similarities between objects were examined by cluster analysis method. The Euclidean distance and the Single Linkage Method were used. Also, the Principal Component Analysis (PCA) method was used.

Statistical software Statistica Tibco (Version 14.0.0.15) was used to analyze data.

3. Results and Discussion

This study assessed the water quality regarding 13 elements (heavy metals and metalloids) and the possible effect of anthropogenic activities on water chemistry in the rivers of the Papuk Nature Park area. The metal and metalloid concentrations were analyzed at 21 locations of the river waters in the Papuk Nature Park. The concentration (median values, 25th and 75th percentile values) of 13 elements in the samples of water are shown in

Table 3. The results of basic statistics.

Element (µgL−1)	MAC (µgL−1)	North Median (25–75%)	Central Part Median (25–75%)	West Median (25–75%)	Southwest Median (25–75%)	South Median (25–75%)	East Median (25–75%)
As	10	4.22 (4.01–4.93)	0.50 (0.433–1.63)	1.62 (0.83–2.57)	0.69 (0.21–1.18)	1.04 (0.60–1.88)	0.82 (0.66–1.48)
B	1500	65.78 (60.23–66.36)	40.39 (19.68–59.15)	141.78 (85.11–188.58)	140.52 (95.05–149.92)	96.78 (54.90–138.78)	1.90 (0.64–4.76)
Ba	700	32.3 (31.2–44.4)	60.01 (9.12–191.14)	18.89 (13.15–23.28)	25.62 (21.77–25.83)	35.02 (26.58–37.50)	0.31 (0.30–59.20)
Cd	5	0.37 (0.32–0.66)	0.29 (0.27–0.46)	0.21 (0.14–0.26)	0.19 (0.17–0.20)	0.16 (0.099–0.20)	0.57 (0.095–0.60)
Co	NR	0.62 (0.50–0.85)	0.18 (0.08–0.31)	0.15 (0.13–0.30)	0.15 (0.14–0.16)	0.095 (0.090–0.097)	0.13 (0.062–0.26)
Cr	25	6.66 (5.20–6.94)	6.27 (3.67–9.23)	6.26 (5.75–20.44)	2.07 (0.34–2.16)	1.52 (0.62–2.32)	0.18 (0.17–0.50)
Cu	2000	15.89 (13.34–16.42)	8.77 (8.10–9.57)	12.94 (11.40–14.39)	11.09 (3.35–12.67)	11.74 (7.57–16.15)	1.59 (1.56–1.99)
Fe	200	968.20 (557.10–1692.20)	189.42 (116.20–434.30)	171.68 (168.10–198.80)	250.89 (12.10–401.20)	155.42 (106.30–183.60)	190.12 (78.80–199.50)
Ni	20	8.21 (7.54–8.71)	6.04 (2.84–11.90)	4.24 (4.03–13.37)	1.85 (0.95–2.22)	1.60 (0.86–2.23)	0.26 (0.19–0.40)
Pb	5	4.78 (4.44–4.86)	2.93 (1.38–3.44)	2.60 (2.47–2.80)	1.73 (0.14–2.52)	1.38 (1.17–1.87)	0.0170 (0.015–0.85)
Sr	NR HBSL (4000 μgL−1)	62.4 (61.24–65.22)	86.35 (38.94–95.64)	71.69 (66.96–78.88)	65.22 (59.43–67.56)	126.09 (105.41–130.69)	0.039 (0.038–38.06)
V	5 μgL−1	2.53 (1.89–3.33)	0.53 (0.50–0.59)	1.08 (0.87–1.17)	0.73 (0.14–0.98)	0.48 (0.33–0.51)	0.57 (0.44–1.04)
Zn	3000 μgL−1	25.95 (24.99–109.86)	17.99 (15.06–19.24)	16.84 (11.30–27.64)	17.63 (10.11–18.81)	13.91 (12.85–18.16)	3.01 (2.49–3.86)

Notes: MAC—maximum permissible level of contaminant in drinking water established by Croatian Directives; HBSL—Health-Based Screening Level; NR—non-regulated; north—Rupnica and tributaries; central part—Papučica, Kovačica, and tributaries; west—Jovanovica and tributaries; southwest—Brzaja and tributaries; south—Dubočanka and tributaries; east—Babina, Pištanska, and Radlovačka Rivers.

. The metal concentrations in each sample, the sampling locations, and data deviations are given in Supplementary Tables S1 and S2.

Naturally occurring sources of boron include borosilicate minerals, volcanic eruptions, geothermal and groundwater streams, and seawater. Anthropogenic sources of boron released into the environment include irrigation wastewater, the use of boron-based fertilizers, and waste from mining and processing industries [16]. In Europe, boron concentrations in fresh surface water range from <1.0 to 2000 µgL⁻¹, with average values generally below 600 µgL⁻¹ [17]. The concentration of boron in fresh, uncontaminated surface waters range from <20 and 50 µgL⁻¹ (Austria, Turkey) to 870 µgL⁻¹ (England, Thames River) [16,18]. Boron concentrations in tap water samples collected from 75 cities in Turkey ranged between 1 and 5500 µgL⁻¹. The boron concentrations in the Papuk area ranged from 1.90 to 141.78 µgL⁻¹, which is about ten times lower than the maximum concentration (15,000 µgL⁻¹) prescribed by the regulations regarding water quality standards [19].

Barium is an alkaline earth metal naturally found in soils, as well as in igneous, metamorphic, and sedimentary rocks. The soluble form of barium sulfide (BaS) is highly toxic to humans, animals, and plants. Excessive intake of Ba(II) through food and drinking water can lead to symptoms such as diarrhea, nausea, hypertension, and breathing difficulties [20]. There are a large number of recent studies on barium levels in surface waters. In the Czech Republic, concentrations from 11.0 to 2561 µgL⁻¹ were recorded; in Germany, from 1 to 101 µgL⁻¹; Poland, from 4.3 to 430 µgL⁻¹; Portugal, from 4.09 to 10.49 µgL⁻¹; Serbia, 30 µgL⁻¹; and Turkey, from 85.1 to 86.1 µgL⁻¹ [21,22,23,24,25]. In our study, the highest barium concentrations ranged from 0.31 µgL⁻¹ (eastern part) to 32.05 µgL⁻¹ (southern part), with maximum concentrations of 191.14 µgL⁻¹ in the central part of Papuk. Median concentrations of barium in the river water of all investigated rivers were well bellow the maximum allowed concentration for barium of 700 µgL⁻¹ according to Croatian regulations for the safety of drinking water or the WHO guideline value for barium.

As the fifteenth most abundant element in the earth’s crust, strontium may be widely distributed in groundwater and some surface water near coastal and mountainous regions [21]. Although Sr is predominantly acquired from weathering processes, in some settings, concentrations are influenced by anthropogenic sources and activities (e.g., agricultural fertilizer application, evaporation of irrigation recharge) [22]. Since strontium is not included in the standards for drinking water quality in Croatia, its measurement is often neglected in most water supply systems. Therefore, it is crucial to investigate strontium concentrations in geologically diverse areas such as nature parks. Strontium intake in humans primarily comes from drinking water and food, including green leaf vegetables, grains, and dairy products. Due to its similarity to calcium, strontium acts as a bone-seeking trace metal (Agency for Toxic Substances and Disease Registry, 2004). Excessive strontium intake can result in abnormal bone development and osteomalacia (impaired bone mineralization), especially in young children whose skeletons are still developing [23]. As there is currently (2024) no regulated standard in Croatia, strontium in drinking water has a Health-Based Screening Level (HBSL) of 4000 μgL⁻¹. The Health-Based Screening Level (HBSL) is a non-enforceable water quality benchmark designed to complement the regulatory standards set by the US Environmental Protection Agency (USEPA). It is used to assess potential human health risks posed by contaminants in drinking water [24]. In our study, exposure to strontium in drinking water varied from very low 0.039 μgL⁻¹ in the eastern part to 126.09 μgL⁻¹ in the southern part of the Papuk Mountain, in which the highest concentration of strontium (130.69 μgL⁻¹) was recorded. For comparison, strontium concentrations in river water samples collected from the unpolluted Yamuna River and its tributaries draining down the southern slopes of the Himalayas and from the tributaries of the Chambal River ranged from 10.6 to 78 μgL⁻¹ [25]. The analysis of strontium in rivers and lakes from Ivvavik National Park (Canada) showed somewhat higher concentrations (from 141 μgL⁻¹ to 1148 μgL⁻¹) [26]. A previous study conducted in eastern Croatia also showed significantly higher B (max. = 338.30 µgL⁻¹), Ba (max. = 311.30 µgL⁻¹), and Sr (max. = 839.20 µgL⁻¹) concentrations in tap water of three settlements and two towns in eastern Croatia [13].

In addition to being a potentially hazardous contaminant in the mercury, lead, and cadmium classes, vanadium is a trace element that has certain physiological functions [27]. Vanadium is essential for normal cell growth in trace amounts, but at higher concentrations it can become toxic. Since one of the pathways for vanadium to enter the human body is through drinking water, its detection in such samples is highly important. Depending on the amount consumed, vanadium can either be toxic or beneficial to health. Therefore, the determination and monitoring of vanadium levels is a crucial issue in environmental studies. The dissolved concentration in river water is about 0.7 μgL⁻¹, less than half of the concentration of ∼1.8 μgL⁻¹ in seawater [28]. The concentration of vanadium in ground water varies between 1 and 138 μgL⁻¹. In volcanic regions of Italy, vanadium levels can increase up to 100-fold compared to river water [29]. In mineral waters, vanadium concentrations range from 1 to 93 μgL⁻¹. This systematic review and meta-analysis was conducted for the first time to determine the vanadium concentration based on defined subgroups, analyzing data from 28 studies including 24 countries. The results of this study demonstrated that the MAC of vanadium in surface water varied from 0.010 μgL⁻¹ (USA) to 68 μgL⁻¹ (China), with an overall mean of 6.21 ± 13.3 μgL⁻¹ [30].

The current Croatian maximum allows for a concentration of vanadium of 5.0 µgL⁻¹. Vanadium was found in all analyzed water samples collected in the area of the Papuk Nature Park. The minimum and maximum vanadium concentrations found were 0.33 µgL⁻¹ and 3.33 µgL⁻¹, respectively. The median values varied from 0.48 µgL⁻¹ (south part) to 2.53 µgL⁻¹ (north part). It is obvious that the median values for vanadium in river water in the Papuk Nature Park were below the maximum allowed concentration for vanadium of 5 µgL⁻¹ according to the current Croatian regulatory water quality standard.

Inorganic arsenic has been confirmed as a human carcinogen, capable of inducing skin, lung, liver, prostate, and bladder cancer. Recent studies have also suggested a potential link between arsenic exposure and diabetes, neurological and cardiac disorders, as well as effects on reproductive organs. However, further research is needed to confirm these associations [31]. Increased levels of arsenic in potable water in eastern Croatia have been a subject of scientific interest for the past two decades due to various health effects, including carcinogenic risk [14,32,33]. The presence of elevated arsenic concentrations in local water supply systems across the lowland areas of eastern Croatia is not an isolated occurrence. Rather, it is a systemic hydrogeochemical phenomenon characteristic of the broader region surrounding the Danube and Drava Rivers. The spatial distribution of the total groundwater arsenic concentration is not homogenous over the area of eastern Croatia. Even in the same district, arsenic levels may vary significantly. Higher concentrations in tap water and well water (max. = 250 µgL⁻¹) were found in Osijek and its surroundings than in Baranja (max. = 93.8 µgL⁻¹), the western part of Osijek Baranja County [12].

There were no significant differences in the concentrations of arsenic in river water between different locations in the Papuk Natural Park area. Median values for arsenic in river water in the north (4.22 µgL⁻¹), west (1.62 µgL⁻¹), south/southwest (1.046 µgL⁻¹, 0.69 µgL⁻¹), east (0.82 µgL⁻¹), and central parts (0.50 µgL⁻¹) were of the same order of magnitude and well below the maximum allowed concentration for arsenic of 10 μgL⁻¹, according to Croatian regulations for the safety of drinking water, the WHO guideline values for arsenic, and the USEPA guidelines [19,34,35].

Chromium is a potentially toxic metal present in water and groundwater due to both natural and anthropogenic sources. Microbial interactions with mafic and ultramafic rocks, along with geogenic processes, can release hexavalent chromium (Cr VI) into the environment through the oxidation of chromite [36]. Chromium (Cr), mainly hexavalent chromium (Cr (VI)), is a chemical associated with cancer when found in drinking water, making it a major public health issue [37]. The WHO recommends a guideline value of 50 µgL⁻¹ for total chromium in drinking water, while the European Union (EU) has set a limit value of 100 µgL⁻¹ for total chromium in drinking water as part of its Drinking Water Directive. Chromium concentrations in Antarctic lakes increase with depth from <0.6 to 30 µgL⁻¹. Most surface waters contain chromium concentrations ranging between 1 and 10 µgL⁻¹ [38]. Chromium concentrations in the north, south, west, east, central, and southwestern parts were 6.66, 1.52, 6.26, 0.18, 6.27, and 2.07 µgL⁻¹, respectively. The median values of chromium concentrations of all water samples collected from the Papuk Nature Park area were much lower than the Croatian limit (25 µgL⁻¹).

The prolonged persistence of toxic cadmium (Cd) and lead (Pb) in the aquatic environment is due to their non-biodegradable nature. Cd is highly toxic even at low concentrations, leaching into the soil through water and accumulating in organisms and ecosystems. Moreover, cadmium has a long biological half-life in animals and the human body, ranging from 10 to 33 years [39]. In addition to anthropogenic sources, it is naturally incorporated into sulfides, carbonates, and phosphorites, leading to elevated Cd concentrations in these rock types. Weathering processes can result in Cd concentrations of up to 5 μgL⁻¹ in soil water and up to 1 μgL⁻¹ in groundwater [40]. The highest median Cd concentration in water was found in the eastern part of Papuk (0. 57 µgL⁻¹), and it was significantly below the Croatian maximum admissible level of 5 µgL⁻¹. In all other parts of Papuk, Cd concentrations in water were much lower, in the range from 0.16 to 0.37 µgL⁻¹.

Although Pb concentrations in water were close to the maximum allowed concentrations, they did not exceed this value (5 µgL⁻¹) and ranged, depending on the sampling area, from very low concentrations (0.017 µgL⁻¹) to slightly higher (4.78 µgL⁻¹) in the northern part of the Papuk Mountain.

Heavy metals copper, nickel, and zinc can be released into the environment from both natural and anthropogenic sources, but anthropogenic sources are much greater and more important [41]. The general concentrations found in the Papuk Nature Park are for Cu (1.56–16.42 µgL⁻¹), Ni (0.11–13.37 µgL⁻¹), and Zn (2.49–19.86 µgL⁻¹), and all are far below the permitted values for drinking water.

From the results of the analyses, it can be concluded that there is no major evidence of a significant anthropogenic effect on the concentrations of these metals in the Papuk rivers. Also, the results show low concentrations of cobalt in water samples analyses (0.061–0.85 µgL⁻¹). It has been found that in most drinking water samples in areas not exposed to anthropogenic influences, Co levels were less than 1–2 µgL⁻¹ [38]. Although no MAC has been established for Co, it is evident that cobalt concentrations in the rivers and tributaries of the Papuk Nature Park are lower than the usually measured concentrations in drinking water.

Iron concentrations were also elevated above the limit of the permissible value (MAC = 200 µgL⁻¹) in the northern part of the nature park (median = 986.2 µgL⁻¹), with the difference that elevated iron concentrations were also found in other parts, i.e., the southwestern part (median = 250.89 µgL⁻¹) of the Papuk Nature Park. The Sekuličanka Mountains are located within the Papuk Nature Park, on its northern and northwestern side—a special forest vegetation reservation protected since 1966. For this location, influences of anthropogenic factors that would contribute to higher concentrations of Fe can be excluded. The terrain is very steep and difficult to access. The soil within the special reserve is a district brown soil (district cambisol, pH < 5.5), which is characteristic of areas at higher altitudes. Subsequent rainfalls can flush accumulated acid from the soil into adjacent drains and waterways, reducing pH, mobilizing iron, and reducing dissolved oxygen. For example, chronic acid drains in the Tuckean Swamp and Rocky Mouth Creek exhibited high concentrations of dissolved metals (5 mgL⁻¹ Fe), a value of a similar order of magnitude to ours (max = 1.692 mgL⁻¹ Fe) [42].

When the concentrations of metals and metalloids in the rivers in the Papuk Nature Park area are compared with other rivers in Croatia located in national parks and nature parks, it is evident that the average values in the rivers of the Papuk Nature Park are higher than the concentrations of the same elements measured in the water of the Plitvice Lakes, water from karst springs of Mount Biokovo, and water from the Cetina River. The differences most likely occur due to the lithology of the rocks and geochemistry of the soil [43,44,45].

The results of a cluster analysis (dendrogram) indicate the presence of two main clusters (Figure 2). The first, a two-member cluster, consists of samples collected from the northern side of the Papuk Nature Park, while the second, a more complex cluster, contains the remaining samples. In order to identify the pattern of the data, we performed [43] Principal Component Analysis (PCA), a dimensionality reduction method commonly used in environmental data analysis. PCA can help reduce complexity and reveal underlying patterns in the complex data matrix.

Since cluster analysis does not provide relationships between variables (elements) and objects (samples) but rather identifies possible differences/similarities among the samples, the Principal Component method was applied (Figure 3). In addition, PCA was performed to help locate the main sources of heavy metals and metalloids.

The PCA results confirmed the findings obtained through cluster analysis, i.e., that samples collected from the northern side of Papuk differ from those collected in other parts of Papuk. The positive correlations of samples 1–5 with most of the studied elements indicate higher concentrations of As, Cd, Co, Cr, Cu, Fe, Ni, Pb, V, and Zn. Somewhat higher concentrations of B, Ba, and Sr are associated with samples from the western, south, southwestern, and central parts of the nature park. Generally, the concentrations of 13 analyzed elements were rather low. For most of the elements, median concentrations were at least 2 times lower than the maximum prescribed concentrations of heavy metals and metalloids in drinking water. None of the elements that could be related to anthropogenic influences (Pb, Ni, Cu, Cr, and Cd) were elevated. Spatial distributions were not particularly different. Sampling locations were grouped into two types of locations, the first consisting of samples from the northern part of the Papuk Nature Park, and the second consisting of the remaining locations, which is probably due to differences in the geological background. Multielemental analysis of heavy metals and metalloids in combination with multivariate analysis methods (cluster analysis and PCA) were shown to be useful methods for the recognition of sources of differences in complex data sets. Such an integrated approach is especially important in cases where a large number of elements is analyzed at several different locations, since the results of basic statistics are not sufficient to explain the spatial distribution of the data.

This research also has limitations regarding the lack of temporal data and the potential effects on seasonal variations on metal concentrations.

4. Conclusions

Water conservation is crucial for preserving ecosystems and biodiversity, helping to maintain healthy ecosystems and protect wildlife. Moreover, the unpolluted biodiversity of rivers is crucial not only for aquatic species, but also for humans, as it provides essential services, including a source of drinking water.

This study is the first to systematically investigate the concentrations of 13 elements in rivers and tributaries in the area of the Papuk Nature Park. Except for the concentrations of Fe, which were elevated in the northern part of Papuk as a consequence of the geochemical composition of the soil and the location, the concentrations of the remaining 12 elements were below the maximum permissible values for drinking water throughout the entire studied area. Despite significant sources of anthropogenic pollution, including severe war destruction, water sources of the Papuk area have remained of high quality. It should be noted that the political decision-making regarding the Papuk Nature Park in 1999 is an example of the positive human impact on nature. It is reasonable to presume that these favorable results of water sampling (after 25 years) are also a result of anthropogenic activity—a positive one helping nature in preventing further contamination and remediating the consequences of war. In the future, further extended research should be conducted to assess ecological impacts, seasonal fluctuations in metal levels, and the role of anthropogenic activities in metal distribution. These studies should involve wider research on soil, plants, animals, and humans residing in the park and its vicinity.