Evaluation of Toxic Element Contamination Levels in the Environment of the Republic of Croatia Under Different Anthropogenic Influences

Abstract

Human activities inevitably affect natural ecosystems, the impact of which most often refers to negative factors resulting in the accumulation of toxic elements in environmental components. This study quantified the presence of 12 toxic elements (Cd, Co, Cr, Cu, Hg, Fe, Mn, Ni, Pb, Zn, As, and Se) in water, soil, and six melliferous plant species across the Republic of Croatia. Sampling sites were classified into four groups according to the dominant anthropogenic impact: agricultural areas, urban and traffic-affected zones, industrial vicinities, and forested hill regions. The results demonstrate the transfer of toxic elements from abiotic matrices into plants, indicating their potential as bioaccumulators. Soil contamination with toxic metals was identified as a relevant ecological risk factor, while contamination of melliferous plants highlights potential implications for human health through the production of honeybee-derived products. Element concentrations in water and soil were determined using three atomic absorption spectrometry techniques (FAAS, GFAAS, and CVAAS), whereas concentrations in floral samples of melliferous plants were measured using inductively coupled plasma mass spectrometry (ICP MS). The obtained results were interpreted in relation to natural background levels and the current national legislation. Anthropogenic impacts were further evaluated using environmental quality indices and bioaccumulation factors, revealing site-specific contamination patterns of both natural and anthropogenic origin.

1. Introduction

Humans, together with other forms of life, live and work in environments that may contain various substances in the form of contaminants with concentrations higher than naturally occurring elements. Soil, an environmental component, is the loose surface layer of the Earth’s crust that originates from bedrock exposed to the factors involved in pedogenesis through the action of the pedogenesis process [1]. In ecosystems, the soil is a living, complex, dynamic, and seasonally variable component [2]. Water is essential for life and is at the heart of natural ecosystems and climate regulation, with the free flow of uncontaminated water being important for the maintenance of all ecosystems. It is not only a consumer product, but a precious natural resource which is necessary for both present and future generations [3]. Therefore, the quality of water is of particular importance, which is generally expressed in terms of the concentrations of inorganic and organic material in the water. Water quality monitoring is necessary for assessing the quality status in relation to existing standards, as well as the suitability of water for various applications [4]. Heavy metals and metalloids—defined as those present in nature with a density equal to or higher than 5 g/cm³ and an atomic number over 20—found in environmental components are often associated with environmental contamination. Their name and classification are associated with their toxicity; therefore, they also include the metalloid arsenic and the non-metal selenium [5,6,7]. An important classification is also derived from the biological perspective, dividing heavy metals into those needed for the growth and development of plants, those that are essential for only certain organisms, and those that are phytotoxic. It is important to note that the boundary between the same element acting as a plant nutrient and a phytotoxin is narrow [1,8,9,10,11]. To date, although soil and/or water analyses have been conducted at certain locations throughout the Republic of Croatia, they have not been directly linked to biological indicators associated with human activities in the form and extent explored in this study. To gain insight into the state of various environmental components—including water, soil, and melliferous plants—in terms of contamination by 12 analyzed heavy and toxic metals, an extensive scientific study was conducted throughout Croatia. The purpose of the research was to determine the concentrations of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn at the studied locations, with the aim of quantifying anthropogenic impacts on the environment depending on the sampling location, expressed through bioaccumulation factors and quality indices. For this purpose, the locations were categorized as agricultural areas, those near urban and traffic zones, those near industrial facilities, and predominantly natural, forest areas, as well as an island location.

2. Heavy and Toxic Metals and Metalloids

Heavy metals interact with natural compounds, both chemically and physically, and can be transformed through these interactions; for example, they interact with natural substances through binding or sorbing, which increases or decreases their mobility. Increased concentrations in soil pose a threat to the environment, especially to plants, and poses a significant environmental problem as heavy and toxic metals are not biodegradable; instead, they accumulate in different organisms and environmental components. Metals can reach water by leaching through the soil layers, with their transport determined by both their physical and chemical characteristics and those of the soil [12,13].

Heavy and toxic metals are widely used in the chemical industry, metallurgical industry, mining, refineries, agricultural food production, brackish water irrigation, the pharmaceutical and medicine sector, the technological sector, and thermal (coal) power plants, with their environmental distribution resulting from activities such as fuel combustion, household activities, wastewater, and landfills. They pose a direct health threat due to their widespread presence. Natural sources of heavy and toxic metals include rocks and sediments that contain them; over time these erode, are washed away, or come to the Earth’s surface through volcanic eruptions. Metals may also arise through evaporation from both water and soil. Additionally, human activity is a major factor contributing to environmental contamination with heavy metals, with Cd, Cr, Cu, Fe, Hg, Mn, Ni, and Pb being among the most significant contaminants in this respect [10,14].

Heavy and toxic metal concentrations are lower in arable soil compared to industrial sites but may still be high enough in the former to pose potential risks to environmental and human health. Szczygłowska et al. found that about 10,000 ha of the land used for agricultural purposes in Germany should be excluded from food production because of heavy metal contamination and reviewed the situation regarding 100,000 ha of heavy metal-contaminated land across Europe and the USA [15]. Heavy metals are constantly released into the environment from natural or anthropogenic sources and enter biological cycles, where they continue to bioaccumulate and circulate. Regarding the concentrations of metals in melliferous plant species, it is necessary to consider their botanical origin (plants with more open flowers are more exposed to contaminants), season (in spring, nectar secretion is more abundant than in summer and autumn), and weather conditions (wind and rain can transfer contamination from the atmosphere to other components of the environment and far away from the contamination source) [16].

3. Bioaccumulation of Metals in Plants

Certain wild plant species vary in their ability to remove or absorb heavy and toxic metals from the soil, which is expressed by their phytoaccumulation factor [17]. The toxicity of metals in plants depends on the plant species and the specific metal, its chemical form and concentration, and soil properties such as the soil pH [10]; for example, the metals Cd and Zn have high transfer coefficients and are easily taken up by plants, while others, such as Cu, Cr, Co, and Pb, have low transfer coefficients [18]. The mobility of heavy and toxic metals in the soil varies with the soil pH; at pH > 6.5, the concentration of easily soluble forms of metals in the soil is significantly reduced, limiting their uptake and accumulation in plants [15].

Hyperaccumulators are plant species that can tolerate high concentrations of toxic substances in their roots and above-ground parts and can quickly transfer elements from the root system to the above-ground parts. They can tolerate a minimum of 0.001% Hg; 0.01% Cd and Se; 0.1% As, Co, Cr, Cu, Ni, Pb, Sb, Se, and Tl; and 1% Mn and Zn in the dry matter of the above-ground part of the plant. Furthermore, increased heavy metal concentrations in the plant itself should not cause any changes in it [1]. Over 400 plant species have been identified as natural hyperaccumulators of metals, representing about 0.2% of all angiosperms [15]. These species include medicinal and melliferous plants, such as sage and lavender [19].

Phytoremediation involves the use of plants and their root microorganisms for the removal, degradation, or retention of harmful chemical substances in the soil, surface and underground water, and the atmosphere. The success of phytoremediation efforts depends on the branching of the root network and the size of the above-ground part of the plant; in particular, larger root mass and above-ground mass of the plant are preferable for phytoextraction [1,10].

The bioaccumulation of heavy metals and toxic elements is a very important indicator of both the state of the analyzed organism and its environment [1]. Heavy metals affect biochemical processes in plants and their physiological activities, leading to visible effects on plant growth and photosynthetic pigments. Plants respond to heavy metal accumulation in their tissues in two ways. First, heavy metals can simply have a toxic effect on the plant; second, the plant may possess certain resistance mechanisms against the toxic effects of heavy metals [1].

4. Materials and Methods

4.1. Sampling and Preparation

Water, soil, and melliferous plants were sampled at 20 locations across the territory of the Republic of Croatia, reflecting varying levels of anthropogenic influence, as shown in Figure 1. At the selected locations, water, soil, and specific melliferous plant species were sampled during the blossoming season where they represent abundant honey bee foraging (

Table 1. List of locations, sampling dates, and sampled materials.

LOCATION MARK	LOCATION	SAMPLING DATE	SAMPLED MATERIAL	MELLIFEROUS PLANT
L1	Donja Dubrava	26 April 2023	water, soil, melliferous plant	rapeseed
L2	Gornji Kučan	5 May 2023	water, soil, melliferous plant	rapeseed
L3	Leščinci-Đurđevac	9 May 2023	water, soil, melliferous plant	rapeseed
L4	Kutina	9 May 2023	water, soil, melliferous plant	rapeseed
L5	Preloščica-Sisak	9 May 2023	water, soil, melliferous plant	rapeseed
L6	Orlec-Cres	16 May 2023	water, soil, melliferous plant	black locust + sage
L7	Jalkovec-Varaždin	23 May 2023	water, soil, melliferous plant	black locust
L8	Kutina	29 May 2023	water, soil, melliferous plant	black locust
L9	Sisak	29 May 2023	water, soil, melliferous plant	black locust
L10	Bisag	29 May 2023	water, soil, melliferous plant	black locust
L11	Korenjak	2 June 2023	water, soil, melliferous plant	black locust
L12	Trakošćan	3 June 2023	water, soil, melliferous plant	black locust
L13	Varaždin	14 June 2023	water, soil, melliferous plant	linden
L14	Zagreb	22 June 2023	water, soil, melliferous plant	linden
L15	Drežnica	29 June 2023	water, soil, melliferous plant	alder buckthorn
L16	Zečev Varoš-Slunj	29 June 2023	water, soil, melliferous plant	chestnut
L17	Vukovar	29 June 2023	soil, melliferous plant	linden
L18	Trstenik-Vukovar	15 July 2023	water, soil, melliferous plant	sunflower
L19	Viškovci-Đakovo	17 July 2023	water, soil, melliferous plant	sunflower
L20	Varaždin	21 July 2023	water, soil, melliferous plant	sunflower

).

Water was sampled from surface streams or reservoirs, collected below the surface of the water body using 1 L plastic bottles. The samples in the bottles were marked with the name, location, and time of sampling. Upon arrival at the laboratory, the water was filtered through ReliaDisc sterile membrane filters with a pore size of 0.45 µm, manufactured by Ahlstrom, Bärenstein, Germany, into 250 mL plastic vials. The samples were then acidified by adding 1 mL of ultrapure nitric acid (67–69%, Honeywell Fluka, Seelze, Germany).

Soil was sampled from the surface layer to a depth of 30 cm from plots in a random dotted pattern, and one pooled sample was taken per location. The samples were collected from locations where a specific melliferous plant grows, which blooms during a certain period. Soil samples were placed in 2 kg plastic bags and marked with the name, location, and time of sampling. Upon arrival at the laboratory, they were prepared using the quartering method and placed in similarly marked plastic trays for air drying.

Melliferous plants were sampled at the time of soil and water sampling at sites where they provided the most abundant honey bee foraging. The plant samples were transported in paper bags labeled with the name, location, and time of sampling. Upon arrival at the laboratory, the flowers were placed on similarly labeled paper mats for air drying.

The dry samples were manually ground in an agate mortar. They were then sieved through a stainless steel sieve certified for pedogeochemical analysis with a 2 mm mesh size and stored in a plastic bag. The samples prepared in this way were digested using aqua regia prepared from ultrapure hydrochloric acid (37%, Merck, Darmstadt, Germany) and ultrapure nitric acid (67–69%, Honeywell Fluka, Seelze, Germany) at a ratio of 3:1. For a weight of ~2 g of soil in Teflon tubes, 15 mL of HCl was added to 5 mL of HNO₃, maintaining a soil-to-aqua regia ratio of 1:10. In the same way, but without a soil sample, a reagent blank was also prepared. Sample digestion was performed in a Speedwave microwave system manufactured by Berghof, Eningen, Germany.

4.2. Sample Analysis

Water and soil samples were analyzed using a Sension156 multimeter manufactured by HACH, Loveland, CO, USA, previously calibrated with pH standards of 4005 and 7000 pH units (Hach Lange GmbH, Düsseldorf, Germany), with IUPAC and CRM traceability. A calibration of −58 (±3) mV was acceptable. The pH value of the soil samples was determined in a 1 M solution of potassium chloride of pro analysis purity grade.

Soil humus was determined using the dichromate method, which involves wet burning of organic matter with a 0.33 M potassium dichromate solution and the addition of concentrated sulfuric acid (95%), both of pro analysis purity grade. The concentration of humus was measured on a Hach DR 5000 spectrophotometer, manufactured by Hach Lange GmbH, Düsseldorf, Germany, and expressed in %.

The heavy and toxic metals and metalloids analyzed in the samples included As, Cd, Co, Cr, Cu, Hg, Fe, Mn, Ni, Pb, Se, and Zn. These were determined using an Atomic Absorption Spectrometer PerkinElmer AAnalyst 800, Shelton, CT, USA. The following techniques were used. FAAS is a flame technique that measures element concentrations in ppm or mg/L (for Cr, Cu, Fe, Mn, and Zn). GFAAS is a technique using a graphite furnace that measures concentrations in ppb or µg/L (for As, Cd, Co, Ni, Pb, and Se). Using the FIAS 100 flow injection system by PerkinElmer, Shelton, CT, USA, with 0.2% sodium boron hydride solution and 3% hydrochloric acid for trace metal analysis, the CVAAS technique was used to determine the concentration of Hg, whereby the concentration was obtained in ppb or µg/L. For each individual element, the instrument was previously calibrated using certified reference standards from Inorganic Ventures, Christiansburg, VA, USA, with a minimum of three calibration points. An acceptable calibration required a correlation coefficient of 0.9999 and an RSD of up to 2% across three consecutive measurements. The obtained results represent the mean values of three measurements of each metal’s concentration, with an acceptable RSD of up to 2%. Additionally, intermediate standard solutions obtained from certified reference materials were used for verification.

Prior to analysis, the soil samples were prepared through decomposition using the Speedwave Xpert microwave digestion system manufactured by Berghof, Eningen, Germany. A precisely measured amount of pre-ground air-dried soil sample was placed into Teflon containers (~2 g) and treated with aqua regia (20 mL). The method reaction time (25 min) was followed by cooling and removal from the Teflon containers. The soil samples were filtered through blue ribbon Munktell filter paper, Bärenstein, Germany, into glass volumetric flasks that were filled with ultrapure water produced using a Millipore Direct-Q3 UV, Millipore, Molsheim, France, to a final volume of 100 mL.

Elemental analysis of the plant material samples was carried out using inductively coupled plasma mass spectrometry (ICP MS 7900, manufactured by Agilent, Santa Clara, CA, USA), after sample preparation using microwave digestion. The samples were placed in a 0.1 g Teflon cuvette, then treated with 2.5 mL of concentrated nitric acid (min. 65%, manufactured by Scharlau, Barcelona, Spain) and 0.5 mL of hydrogen peroxide (30%, manufactured by Alkaloid, Skopje, North Macedonia) using a dispenser. After digestion and cooling, the cuvette was opened, and the clear solution was quantitatively transferred to a 10 mL glass volumetric flask using a glass funnel and rinsing with deionized water. The flask was filled to the mark with ultrapure water. Certified reference materials were used to calibrate each individual chemical element.

4.3. Bioaccumulation Factors

Bioaccumulation can be expressed using factors that represent the ratio of a metal’s concentration in a plant to its concentration in the soil [1]. The purpose of calculating bioaccumulation factors is to numerically express the impact of contamination on a living organism, reflecting it as an anthropogenic footprint. In this process, matrices that are considered to be in direct contact are compared; in this case, expressing the relationship between the melliferous plant and the soil. The influence of water was not considered for the plants, in addition to the influence of soil, as the sampled water does not necessarily relate to the growth of all analyzed melliferous plants. Therefore, the bioaccumulation factor (BAF) for melliferous plants was obtained and presented as the ratio of the concentration of a particular metal in the plant’s flowers to its concentration in the soil, as shown in Equation (1):

$${BAF}_{plant}=\sum {\displaystyle \frac{\left[{metal}_{plant}\right]}{\left[{metal}_{soil}\right]}}$$

(1)

4.4. Quantification of Anthropogenic Pressure—Quality Indices

Anthropogenic pressure has resulted in significant ecological degradation, with negative and permanent changes in the environment affecting large areas worldwide [21]. Using anthropogenic footprint models, land use can be planned and areas of special conservation value can be identified in local landscapes within a regional context. Such models can serve as a tool for studies investigating the responses of flora and fauna to gradients of intensity of human disturbance. The anthropogenic footprint can directly affect ecosystems through human activity, which can lead to land cover changes, or indirectly through degradation of individual ecosystem functions [22]. Chmielewski et al. stated that the above disasters are the consequences of recklessness, greed, and unlimited production [23], and consider examples of ecosystem degradation as evidence of low ecological awareness, which should serve as a warning when setting guidelines for the rational management and use of the environment.

The anthropogenic footprint is evident in environmental components such as water and soil, as well as in melliferous plants. To quantify anthropogenic pressure on environmental components, the water quality index (WQI) and the soil quality index (SQI) were calculated. The water and soil quality indices for each determined metal were obtained and presented as the ratio of the metal’s concentration to its maximum allowable concentration ordained by regulations, as shown in Equations (2) and (3):

$${WQI}_{metal}={\displaystyle \frac{\left[metal concentration\right]}{\left[{MAC}_{water}\right]}}$$

(2)

$${SQI}_{metal}={\displaystyle \frac{\left[metal concentration\right]}{\left[{MAC}_{soil}\right]}}$$

(3)

The total water quality index and soil quality index for a specific location were calculated as the mean value of the impact of each individual metal; that is, as the ratio of the sum of the indices for all metals to a value of 11 or 9, respectively, representing the number of metals prescribed by the Ordinance on Water for Human Consumption [24] and the Ordinance on the Protection of Agricultural Land from Contamination [25]. The associated formulas are shown in Equations (4) and (5):

$${WQI}_{L1-L20}={\displaystyle \frac{\sum {WQI}_{metal}}{11}}$$

(4)

$${SQI}_{L1-L20}={\displaystyle \frac{\sum {SQI}_{metal}}{9}}$$

(5)

where

QI(W,S) = 0–1 🡺 satisfactory;

QI(W,S) = >1 🡺 unsatisfactory.

5. Results

The results of the analyses of 12 heavy metals and toxic elements in the three different sampled materials are presented in

Table 2. Concentrations of heavy metals and toxic elements and pH values in water samples from the investigated locations.

SAMPLING LOCATION	pH	As µg/L	Cd µg/L	Co µg/L	Cr mg/L	Cu mg/L	Fe mg/L	Hg µg/L	Mn mg/L	Ni µg/L	Pb µg/L	Se µg/L	Zn mg/L
L1	8.06	<0.05	<0.002	0.103	<0.003	<0.0015	<0.005	0.022	<0.0015	0.428	<0.05	<0.05	0.031
L2	7.91	0.594	<0.002	0.179	<0.003	<0.0015	<0.005	0.015	<0.0015	0.804	<0.05	<0.05	0.023
L3	7.39	3.656	<0.002	0.275	<0.003	<0.0015	0.027	0.021	<0.0015	3.383	0.082	<0.05	0.019
L4	6.74	1.470	<0.002	0.195	<0.003	<0.0015	<0.005	0.035	<0.0015	3.918	<0.05	<0.05	0.007
L5	8.08	3.927	<0.002	0.344	<0.003	<0.0015	<0.005	0.012	0.018	2.453	<0.05	<0.05	0.034
L6	7.25	1.065	<0.002	0.529	<0.003	<0.0015	<0.005	0.024	<0.0015	0.303	<0.05	<0.05	0.012
L7	7.9	0.694	<0.002	<0.15	<0.003	<0.0015	<0.005	0.012	<0.0015	1.118	<0.05	<0.05	0.028
L8	7.56	1.072	<0.002	0.112	<0.003	<0.0015	<0.005	0.028	<0.0015	1.628	<0.05	<0.05	0.011
L9	7.87	0.271	<0.002	0.118	<0.003	<0.0015	<0.005	0.02	<0.0015	0.382	<0.05	<0.05	0.005
L10	8	0.756	<0.002	<0.15	<0.003	<0.0015	<0.005	0.041	<0.0015	0.78	<0.05	<0.05	<0.0015
L11	7.26	0.99	<0.002	<0.15	<0.003	<0.0015	<0.005	0.018	<0.0015	1.151	<0.05	<0.05	<0.0015
L12	8.11	0.288	<0.002	<0.15	<0.003	<0.0015	<0.005	0.003	<0.0015	0.342	<0.05	<0.05	0.015
L13	7.29	0.287	<0.002	1.672	<0.003	<0.0015	<0.005	0.015	<0.0015	0.372	20.91	<0.05	0.733
L14	5.72	<0.05	<0.002	0.262	<0.003	<0.0015	<0.005	0.017	<0.0015	0.407	0.205	<0.05	0.079
L15	7	<0.05	<0.002	0.032	<0.003	<0.0015	<0.005	0.011	<0.0015	<0.07	0.275	<0.05	0.026
L16	7.63	0.242	<0.002	0.172	<0.003	<0.0015	<0.005	0.009	<0.0015	0.623	0.05	<0.05	0.003
L17	-	-	-	-	-	-	-	-	-	-	-	-	-
L18	8.19	1.298	<0.002	<0.15	<0.003	<0.0015	<0.005	0.021	<0.0015	0.554	<0.05	<0.05	<0.0015
L19	8.92	12.28	<0.002	0.194	<0.003	<0.0015	<0.005	0.027	<0.0015	0.834	<0.05	<0.05	0.004
L20	6	0.108	<0.002	7.206	<0.003	<0.0015	<0.005	0.015	<0.0015	0.549	27.75	<0.05	0.135

,

Table 3. Concentrations of heavy metals and toxic elements, as well as pH values and humus content, in soil samples from the investigated locations.

SAMPLING LOCATION	pH	As mg/kg	Cd mg/kg	Co mg/kg	Cr mg/kg	Cu mg/kg	Fe mg/kg	Hg mg/kg	Mn mg/kg	Ni mg/kg	Pb mg/kg	Se mg/kg	Zn mg/kg	HUMUS %
L1	5.62	15.018	0.191	13.303	42.638	38.528	30,475.46	0.057	596.933	45.460	29.782	0.431	116.258	3.44
L2	4.25	9.058	0.133	7.964	31.262	23.107	19,093.85	0.043	604.207	25.110	17.236	0.238	80.906	2.44
L3	3.59	5.703	0.097	6.637	20.500	13.188	16,675.00	0.037	397.813	16.850	11.187	0.091	177.500	2.08
L4	4.24	6.323	0.134	9.537	25.182	14.620	19,099.01	0.035	530.693	23.832	13.069	0.085	133.663	1.97
L5	6.48	13.840	0.337	13.466	45.276	26.350	29,309.82	0.072	1.017.178	50.307	30.853	0.566	106.442	2.84
L6	6.9	19.238	0.609	12.513	67.523	51.870	28,644.86	0.215	743.925	64.019	82.747	0.768	248.131	14.1
L7	7.26	8.244	0.13	5.109	16.463	14.619	13,315.55	0.407	329.116	17.226	13.582	0.304	121.341	2.66
L8	4.87	8.291	0.133	10.373	32.517	186.258	19,079.47	0.044	514.901	24.533	17.447	0.11	126.821	5.36
L9	7.01	7.498	0.266	6.305	16.607	20.770	15,663.93	0.106	342.951	24.279	23.711	0.3	93.279	4.02
L10	6.82	12.117	0.138	10.061	40.860	26.834	28,896.10	0.086	470.130	35.032	21.312	0.371	147.240	4.73
L11	3.3	9.050	0.064	8.824	26.894	18.538	27,151.16	0.038	306.312	30.409	16.641	0.207	58.970	2.22
L12	4.67	9.945	0.167	11.263	45.793	23.657	32,427.18	0.095	331.715	49.968	23.472	0.455	107.767	5.2
L13	5.85	11.188	0.149	8.358	33.092	22.566	20,947.37	0.162	637.171	28.609	33.513	0.26	91.118	5.23
L14	7.04	10.609	0.302	7.844	25.801	37.837	20,392.63	0.205	426.282	32.660	50.157	0.438	124.679	3.97
L15	5.82	11.346	0.347	10.948	44.871	23.107	26,359.22	0.113	541.424	46.699	35.822	0.465	109.061	9.5
L16	5.71	17.168	0.22	22.167	49.917	22.110	30,722.59	0.077	493.355	50.033	36.101	0.492	90.864	4.65
L17	7.02	10.508	0.167	8.262	27.957	17.940	21,088.04	0.019	561.794	33.854	15.566	0.242	64.618	2.33
L18	8.19	9.631	0.338	7.714	25.482	58.073	20,265.78	0.022	426.910	36.130	14.242	0.31	54.817	2.23
L19	5.04	8.326	0.332	9.999	35.814	17.342	20,332.23	0.023	677.741	40.797	15.674	0.167	161.794	1.72
L20	6.32	7.508	0.166	6.216	28.370	23.887	15,573.67	0.571	618.182	27.235	39.655	0.158	109.718	2.61

and

Table 4. Concentrations of heavy metals and toxic elements in melliferous plant samples from the investigated locations.

SAMPLING LOCATION	As mg/kg	Cd mg/kg	Co mg/kg	Cr mg/kg	Cu mg/kg	Fe mg/kg	Hg mg/kg	Mn mg/kg	Ni mg/kg	Pb mg/kg	Se mg/kg	Zn mg/kg
L1	<0.03	0.09	0.19	0.25	6.37	87.91	<0.01	35.15	1.78	0.05	0.22	81.85
L2	<0.03	0.08	0.07	0.36	6.11	102.51	<0.01	31.34	1.16	0.17	1.82	82.07
L3	<0.03	0.09	0.42	0.27	9.13	147.16	<0.01	109.72	8.68	0.09	2.82	86.89
L4	0.03	0.02	0.1	0.31	19.83	139.19	<0.01	44.45	30.8	0.12	0.44	60.06
L5	<0.03	0.09	0.31	0.2	8.58	116.59	<0.01	47.44	1.16	0.08	0.27	84.93
L6	<0.03	<0.01	0.07	0.28	13.57	24.74	<0.01	30.62	0.57	0.08	<0.05	46.35
L7	<0.03	0.02	0.07	0.27	15.68	91.45	<0.01	22.52	5.32	0.11	0.6	49.64
L8	<0.03	0.15	0.87	0.21	8.84	105.02	<0.01	152.15	10.06	0.08	0.33	75.91
L9	0.03	0.03	0.08	0.59	13.57	152.81	0.01	32.23	12.03	0.22	0.39	51.97
L10	0.05	<0.01	0.14	0.45	15.68	160.66	0.01	22.86	1.81	0.17	0.16	43.96
L11	<0.03	<0.01	0.07	0.25	17.5	130.25	0.01	35.64	23.46	0.09	0.07	87.4
L12	<0.03	<0.01	0.06	0.29	13.49	103.86	<0.01	37.63	12.19	0.07	0.06	44.49
L13	<0.03	0.05	<0.05	0.17	7.59	64.19	0.01	28.36	0.43	0.07	<0.05	22.33
L14	0.05	0.02	0.07	0.33	7.21	143.51	<0.01	28.24	0.62	0.13	0.09	22.19
L15	<0.03	0.07	0.06	0.22	7.31	44.63	<0.01	46.26	1.18	0.07	0.07	30.77
L16	<0.03	0.13	0.25	0.4	11.23	57.13	<0.01	414.7	2.04	0.08	<0.05	20.63
L17	0.04	0.07	0.08	0.27	11.68	59.5	<0.01	95.16	1.26	0.07	<0.05	18.34
L18	0.12	0.05	0.19	0.93	22.75	183.33	0.01	34.23	1.49	0.39	0.11	36
L19	0.07	0.15	0.09	0.56	17.56	167.13	0.01	14.58	3.42	0.09	<0.05	44.01
L20	<0.03	0.04	0.08	0.36	15.08	76.62	0.01	16.07	0.43	0.1	<0.05	32.21

.

The bioaccumulation factors for the determined heavy and toxic metals in the flowers of melliferous plants are shown in

Table 5. Bioaccumulation factors for metals in melliferous plants.

M. PLANT	BAF-As	BAF-Cd	BAF-Co	BAF-Cr	BAF-Cu	BAF-Fe	BAF-Hg	BAF-Mn	BAF-Ni	BAF-Pb	BAF-Se	BAF-Zn	BAFp loc
L1	0.000	0.471	0.014	0.000	0.059	0.039	0.000	0.059	0.039	0.002	0.510	0.704	0.164
L2	0.000	0.602	0.009	0.000	0.052	0.046	0.000	0.052	0.046	0.010	7.647	1.014	0.805
L3	0.000	0.928	0.063	0.000	0.276	0.515	0.000	0.276	0.515	0.008	30.989	0.490	2.832
L4	0.000	0.149	0.010	0.000	0.084	1.292	0.000	0.084	1.292	0.009	5.176	0.449	0.712
L5	0.000	0.267	0.023	0.000	0.047	0.023	0.000	0.047	0.023	0.003	0.477	0.798	0.164
L6	0.000	0.000	0.006	0.000	0.041	0.009	0.000	0.041	0.009	0.001	0.000	0.187	0.043
L7	0.000	0.154	0.014	0.000	0.068	0.309	0.000	0.068	0.309	0.008	2.034	0.409	0.341
L8	0.000	1.128	0.084	0.000	0.295	0.410	0.000	0.295	0.410	0.005	3.000	0.599	0.465
L9	0.004	0.113	0.013	0.094	0.094	0.495	0.094	0.094	0.495	0.009	1.300	0.557	0.282
L10	0.004	0.000	0.014	0.116	0.049	0.394	0.116	0.049	0.394	0.008	0.431	0.299	0.160
L11	0.000	0.000	0.008	0.263	0.116	0.771	0.263	0.116	0.771	0.005	0.338	1.482	0.329
L12	0.000	0.000	0.005	0.000	0.113	0.244	0.000	0.113	0.244	0.003	0.132	0.413	0.124
L13	0.000	0.336	0.000	0.062	0.045	0.015	0.062	0.045	0.015	0.002	0.000	0.245	0.087
L14	0.005	0.066	0.009	0.000	0.066	0.019	0.000	0.066	0.019	0.003	0.205	0.178	0.063
L15	0.000	0.202	0.005	0.000	0.085	0.025	0.000	0.085	0.025	0.002	0.151	0.282	0.090
L16	0.000	0.591	0.011	0.000	0.841	0.041	0.000	0.841	0.041	0.002	0.000	0.227	0.186
L17	0.004	0.419	0.010	0.000	0.169	0.037	0.000	0.169	0.037	0.004	0.000	0.284	0.133
L18	0.012	0.148	0.025	0.455	0.080	0.041	0.455	0.080	0.041	0.027	0.355	0.657	0.186
L19	0.008	0.452	0.009	0.435	0.022	0.084	0.435	0.022	0.084	0.006	0.000	0.272	0.194
L20	0.000	0.241	0.013	0.018	0.026	0.016	0.018	0.026	0.016	0.003	0.000	0.294	0.105

.

The calculation results for the water quality and soil quality indices are presented in

Table 6. Water quality index results.

WATER	WQI-As	WQI-Cd	WQI-Cr	WQI-Cu	WQI-Fe	WQI-Hg	WQI-Mn	WQI-Ni	WQI-Pb	WQI-Se	WQI-Zn	WQI loc
L1	0.000	0	0	0	0	0.022	0	0.021	0	0	0.016	0.005
L2	0.059	0	0	0	0	0.015	0	0.040	0	0	0.012	0.011
L3	0.366	0	0	0.001	0.135	0.021	0	0.169	0	0	0.000	0.063
L4	0.147	0	0	0	0	0.035	0	0.196	0	0	0.004	0.035
L5	0.393	0	0	0	0	0.012	0.360	0.123	0	0	0.017	0.082
L6	0.107	0	0	0	0	0.024	0	0.015	0	0	0.006	0.014
L7	0.069	0	0	0	0	0.012	0	0.056	0	0	0.014	0.014
L8	0.107	0	0	0	0	0.028	0	0.081	0	0	0.006	0.020
L9	0.027	0	0	0	0	0.020	0	0.019	0	0	0.003	0.006
L10	0.076	0	0	0	0	0.041	0	0.039	0	0	0.000	0.014
L11	0.099	0	0	0	0	0.018	0	0.058	0	0	0.000	0.016
L12	0.029	0	0	0	0	0.003	0	0.017	0	0	0.008	0.005
L13	0.029	0	0	0	0	0.015	0	0.019	4.128	0	0.367	0.419
L14	0.000	0	0	0	0	0.017	0	0.020	0.041	0	0.040	0.011
L15	0.000	0	0	0	0	0.011	0	0.000	0.055	0	0.013	0.007
L16	0.024	0	0	0	0	0.009	0	0.031	0.010	0	0.002	0.007
L17	-	-	-	-	-	-	-	-	-	-	-	-
L18	0.130	0	0	0	0	0.021	0	0.028	0	0	0.000	0.016
L19	1.228	0	0	0	0	0.027	0	0.042	0	0	0.002	0.118
L20	0.011	0	0	0	0	0.015	0	0.027	5.550	0	0.068	0.516

and

Table 7. Soil quality index results.

SOIL	pH	SQI-As	SQI-Cd	SQI-Co	SQI-Cr	SQI-Cu	SQI-Hg	SQI-Ni	SQI-Pb	SQI-Zn	SQI loc
L1	5.62	0.601	0.127	0.267	0.533	0.428	0.057	0.909	0.298	0.775	0.444
L2	4.25	0.604	0.133	0.266	0.782	0.385	0.086	0.837	0.345	1.348	0.532
L3	3.59	0.380	0.097	0.222	0.513	0.220	0.074	0.562	0.224	2.958	0.583
L4	4.24	0.422	0.134	0.319	0.630	0.244	0.070	0.794	0.261	2.228	0.567
L5	6.48	0.461	0.169	0.225	0.377	0.220	0.048	0.671	0.206	0.532	0.323
L6	6.9	0.641	0.305	0.209	0.563	0.432	0.143	0.854	0.552	1.241	0.549
L7	7.26	0.275	0.065	0.085	0.137	0.122	0.271	0.230	0.091	0.607	0.209
L8	4.87	0.553	0.133	0.347	0.813	3.104	0.088	0.818	0.349	2.114	0.924
L9	7.01	0.250	0.133	0.105	0.138	0.173	0.071	0.324	0.158	0.466	0.202
L10	6.82	0.404	0.069	0.168	0.341	0.224	0.057	0.467	0.142	0.736	0.290
L11	3.3	0.603	0.064	0.295	0.672	0.309	0.076	1.014	0.333	0.983	0.483
L12	4.67	0.663	0.167	0.376	1.195	0.394	0.190	1.666	0.469	1.796	0.768
L13	5.85	0.448	0.099	0.168	0.414	0.251	0.162	0.572	0.335	0.607	0.340
L14	7.04	0.354	0.151	0.131	0.215	0.315	0.137	0.435	0.334	0.623	0.300
L15	5.82	0.454	0.231	0.219	0.561	0.257	0.113	0.934	0.358	0.727	0.428
L16	5.71	0.687	0.147	0.444	0.624	0.246	0.077	1.001	0.361	0.606	0.466
L17	7.02	0.350	0.084	0.138	0.233	0.150	0.013	0.451	0.104	0.323	0.205
L18	8.19	0.321	0.169	0.129	0.212	0.484	0.015	0.482	0.095	0.274	0.242
L19	5.04	0.333	0.221	0.200	0.448	0.193	0.023	0.816	0.157	1.079	0.386
L20	6.32	0.250	0.083	0.104	0.236	0.199	0.381	0.363	0.264	0.549	0.270

, respectively.

6. Discussion

6.1. Metal Content in Water Samples

To compare the concentration values of the determined metals in water samples against established quality limits, the Ordinance on Compliance Parameters, Methods of Analysis, and Monitoring of Water Intended for Human Consumption were considered [24]. According to the ordinance, the concentration of hydrogen ions (i.e., the pH value) in water intended for human consumption should be between 6.5 and 9.5, indicating that the pH values of the water samples from almost all investigated locations meet the prescribed range. Exceptions include sample L14, with the lowest measured pH value of 5.72, followed by sample L20, with a pH value of 6.00. Both water samples—L14 from Zagreb near the Faculty of Veterinary Medicine and L20 from a public orchard in Varaždin—were precipitation or rainwater samples. At the time of sampling, no natural source of water was available from location L17.

The maximum allowable concentration (MAC) for As is 10 µg/L [24]. The As concentrations in the water samples were lower than the specified value, except for that at L19, with the highest concentration of 12.28 µg/L. The Geochemical Atlas of the Republic of Croatia [26] reports a mean value of As in freshwater of 0.002 mg/L, which is generally consistent with the rest of the obtained results. Sample L19 likely had an elevated As concentration due to both its natural origin and the influence of surrounding intensive agriculture, which uses plant protection products and mineral fertilizers containing As [27].

The MAC for Cd is 5 µg/L [24]. All water samples showed Cd concentrations below the detection limit of the instrument and method; that is, less than 0.002 µg/L. The Geochemical Atlas [26] does not provide a mean value for the concentration of this element in freshwater.

The MAC for Co is not indicated [24]. The mean value of Co in freshwater is 0.0001 mg/L, or 0.1 µg/L [26], which generally corresponds to the measured values.

The MAC for Cr is 25 µg/L [24]. Analyses of water samples from all investigated locations showed Cr concentrations below the detection limit of the instrument and method; that is, less than 0.003 mg/L. The average concentration of Cr in freshwater is 0.001 mg/L [26], and there was no deviation from this data in the measured samples.

The MAC for Cu is 2 mg/L [24]. Analyses of water samples from all investigated locations showed Cu concentrations below the detection limit of the instrument and method; that is, less than 0.0015 mg/L. According to the data, the mean concentration of Cu in freshwater is 0.003 mg/L [26]; the obtained data show lower concentrations of this metal in the water samples.

The MAC for Fe is 200 µg/L [24]. All water samples, except L3, showed an Fe concentration lower than the detection limit of the instrument and method; that is, lower than 0.005 mg/L. The concentration of Fe in water sample L3 was below the prescribed limit at 0.027 mg/L. The average value of Fe in freshwater is 0.1 mg/L [26], with the obtained results showing slightly lower values.

The MAC for Hg is 1.0 µg/L [24]. All samples contained a certain concentration of Hg, but still below the maximum allowed concentration in each sample. The Geochemical Atlas does not provide a mean value for the Hg concentration in freshwater [26].

The MAC for Mn is 50 µg/L [24]. In all water samples, the concentration of Mn was below the detection limit of the instrument and method (i.e., below 0.0015 mg/L), except for sample L5 (with 0.018 mg/L). The mean value of Mn in freshwater is 0.015 mg/L [26]; the measured value in L5 in this study coincides with the given data, while those in the other samples are lower than the average value.

The MAC for Ni is 20 µg/L [24]. The Ni concentrations in all water samples were lower than the given value. In freshwater, the average concentration is 0.0015 mg/L [26], and the obtained results are close to this value.

The MAC for Pb is 5 µg/L [24]. The highest measured concentration was in L20, where it amounted to 27.75 µg/L and exceeded the MAC by five times. This can be explained by the fact that the rainwater, with an acidic pH, was sampled at L20 from a depression in a plastic slide, as Pb is used in pigments and as a stabilizer in plastic [28]. Given the acidic pH value of the water and that the plastic mass had been exposed to constant daily heating and expansion, nightly cooling and contraction, and freezing during the colder part of the year for a long time (years), it is possible to expect higher concentrations of Pb derived from the colored plastic material. The concentration of Pb in freshwater is 0.003 mg/L [26], with the measured values falling below the limit (except in sample L20, as explained above).

The MAC for Se is 20 µg/L [24]. Samples from all locations showed Se concentrations lower than the detection limit of the instrument and method; that is, lower than 0.05 µg/L. In the Geochemical Atlas, there is no data for Se in soil or water [26].

The MAC for Zn is 3 mg/L [24]. In all samples, the concentration of Zn was below this limit. The concentration of this element in freshwater is 0.02 mg/L [26]. This is in accordance with the water sample analysis results; with the exception of sample L13, where the Zn concentration was several times higher than the natural average, but still well below the MAC.

6.2. Metal Content in Soil Samples

Soil acidity (expressed as pH) at the studied locations varied from 3.30, which represents highly acidic soil, to 8.19, indicating alkaline soil [29]. The humus concentration in the studied soils ranged from 1.72 to 14.10, classifying the studied soils as poorly supplied with humus to those very richly supplied according to the categorization [29].

Arsenic (As) in the analyzed soil samples, according to the Ordinance on the Protection of Agricultural Land from Contamination [25], must not exceed 15, 25, or 30 mg/kg of dry matter depending on the pH value of the soil. All samples contained a certain concentration of As, but it was below the MAC in each sample. Therefore, it can be considered that the analyzed soils were not contaminated with arsenic. The Geochemical Atlas of the Republic of Croatia [26] states that the average concentration of As in soils is about 6 mg/kg. Samples such as L1 near Donja Dubrava, with 15.018 mg/kg, and L5 Prelošćica near Sisak, with 13.840 mg/kg, showed twice the average, which can be attributed to anthropogenic impacts from agricultural production due to the use of protective agents and certain mineral fertilizers containing As [27].

Cadmium (Cd) in soil, according to the ordinance [25], must not exceed 1, 1.5, or 2 mg/kg of dry matter. All samples contained a certain concentration of Cd, but at levels below the MAC; therefore, the analyzed soils can be considered uncontaminated by cadmium. According to data from the Geochemical Atlas [26], the average value of Cd concentration in soils is 0.5 mg/kg. The values in the samples were lower than the average for soils in Croatia, except in sample L6, where the naturally increased concentration may be due to the composition of the soil of coastal Croatia.

Cobalt (Co) in soil must not exceed 30, 50, or 60 mg/kg of dry matter [25]. All samples contained a certain concentration of Co, but below the MAC; therefore, the analyzed soils can be considered uncontaminated by cobalt. The Atlas [26] reports Co concentrations in soils ranging from 1 to 40 mg/kg, with a mean value of 13 mg/kg for Croatia. All samples fell within this range.

Chromium (Cr) in soil must not exceed 40, 80, or 120 mg/kg of dry matter [25]. All samples contained a certain concentration of Cr, but below the MAC; except in sample L12, where it amounted to 45.793 mg/kg. The rest of the analyzed soils were categorized as not contaminated with chromium. The Atlas [26] reports Cr concentrations in soils ranging from 5 to 1000 mg/kg or even more than 1%. All analyzed samples fell within this range.

Copper (Cu) in soil samples must not exceed 60, 90, or 120 mg/kg of dry matter [25]. All samples contained a certain concentration of Cu, but below the MAC; except for sample L8. While the other soils can be considered uncontaminated by copper, the highest concentration was in sample L8 from the Kutina area, which amounted to 186.258 mg/kg, exceeding the MAC by three times. According to data from the Geochemical Atlas [26], the Cu concentration in soils ranges from 2 to 250 mg/kg. All analyzed samples fell within this range. The significantly elevated concentration of copper in sample L8 can attributed to its proximity to industrial facilities and their long-term activity, as well as the use of copper-based protective agents, such as copper sulfate, for plant protection in surrounding orchards and vineyards [30].

Iron (Fe) in soil, according to the Geochemical Atlas [26], has an average value of 2.1%, which corresponds to the average concentration of this metal in the analyzed samples. The ordinance [26] does not set a MAC for iron.

Mercury (Hg) in soil must not exceed 0.5, 1.0, or 1.5 mg/kg of dry matter [25]. All samples contained a certain concentration of Hg, but below the MAC; therefore, all analyzed soils can be considered uncontaminated by mercury. The average value for mercury in soils is 0.05 mg/kg [26], which corresponds to the concentration in most of the analyzed samples. The concentration is higher in mountainous and coastal Croatia than in northern Croatia, as illustrated by the measured concentration of 0.215 mg/kg at L6 near Orlec on the island of Cres.

Manganese (Mn) in soil, according to the Atlas [26], has an average value of 1000 mg/kg and ranges from 20 to 10,000 mg/kg. This corresponds to the average concentration of this metal in the analyzed samples. The ordinance does not set a MAC for manganese [25].

Nickel (Ni) in soil must not exceed 30, 50, or 75 mg/kg of dry matter [25]. All samples contained a certain concentration of Ni. Concentrations were below the MAC in all samples except those from locations L11 (with 30.409 mg/kg), L12 (with 49.968 mg/kg), and L16 (with 50.033 mg/kg), indicating that these areas are the most contaminated with nickel. Nickel is reported to be below 100 mg/kg in most soils, and its concentration is between 20 and 30 mg/kg in temperate soils [1,26]. All analyzed samples fell within the specified average range.

Lead (Pb) in soil must not exceed 50, 100, or 150 mg/kg of dry matter [25]. All samples contained a certain concentration of Pb, but below the MAC; therefore, all analyzed soils can be considered uncontaminated by lead. Lead is reported to range from 2.6 to 83 mg/kg in soil, and its average concentration is around 14 mg/kg [1,26]. All samples fell within the specified range.

Selenium (Se) ranges from 0.1 to 1.0 mg/kg in soil [1]. All analyzed samples fell within this range. The Atlas [26] and the ordinance [25] do not state an average or MAC for selenium.

Zinc (Zn) in soil must not exceed 60, 150, or 200 mg/kg of dry matter [25]. According to the data [26], the average value for zinc in soils is between 10 and 300 mg/kg, which corresponds to the concentrations in all analyzed samples. Comparing the obtained values in individual samples with the ordinance [25] and the MAC, the following samples were considered to be contaminated: L2 with a value of 80.906 mg/kg, L3 with 177.500 mg/kg, L4 with 133.663 mg/kg, L6 with 248.131 mg/kg, L8 with 126.821 mg/kg, L12 with 107.767 mg/kg, and L19 with 161.794 mg/kg. The soil from location L3 had a concentration almost three times higher than that specified. This location is related to intensive agricultural production, and the contamination can be associated with the use of mineral phosphate fertilizers [31,32].

6.3. Metal Content in Melliferous Plant Samples

The maximum allowable concentrations of heavy and toxic metals in honey plants are not regulated by any ordinance. The analysis results for metals in the flowers of melliferous plants are presented in order from the lowest determined concentration to the highest concentration of each element. The samples of melliferous plant flowers contained the lowest concentration of Hg, with its concentration remaining below the detection limit of the instrument and method (i.e., <0.01 mg/kg) in 13 samples. The As concentration was <0.03 mg/kg in most of the soil samples, with the highest measured concentration occurring in sample L18 (0.12 mg/kg). The Cd concentration was <0.01 mg/kg in samples L6 and L10 to L12, while the highest cadmium concentrations were measured in samples L8 and L19 (0.15 mg/kg). Co was below the detection limit (i.e., <0.05 mg/kg) in sample L13, while the highest concentration of this metal was measured in sample L8 (with 0.87 mg/kg). Pb was present in samples from all locations and was highest in sample L18 (at 0.39 mg/kg). Cr was present in all samples, and its highest concentration was also in sample L18 (at 0.93 mg/kg). Se was not present in all plant samples and was below 0.05 mg/kg in samples L6, L13, L16, L17, L19, and L20; meanwhile, it was most abundant in sample L3 (at 2.82 mg/kg). Cu was also found in the plant materials from all locations, with the highest concentration observed in sample L18 (at 22.75 mg/kg). All samples also contained Ni, with the highest concentration in sample L4 (at 30.80 mg/kg). Mn was present in all 20 analyzed samples, with the highest concentration observed in sample L16 (at 414.70 mg/kg). All samples contained Zn, with the highest concentration in sample L11 (at 87.40 mg/kg). None of the plant flower samples contained Zn in a concentration considered toxic to plants, i.e., within the range from 150 to 200 mg/kg. Fe was present in the highest concentrations in the analyzed samples—especially in L18, where it amounted to 183.33 mg/kg.

Kumar et al. have provided a detailed table showing heavy metal concentrations in different plant species, depending on the part of the plant analyzed [18]. For brown mustard (Brassica juncea) from the area of Victoria in Australia and Amritsar, concentrations of 1.30 mg/kg Cu and 62 mg/kg Ni were determined in young leaves. In mature seeds of B. napus (Konya, Turkey), Cu was found at a concentration of 2.17 mg/kg and Mn was found at a concentration of 22.8 mg/kg. In the leaves of B. oleracea (Amritsar), the measured concentrations were as follows: Co (8.10 mg/kg), Cr (5.0 mg/kg), Cu (3.00 mg/kg), Mn (2.50 mg/kg), Ni (6.10 mg/kg) and Zn (33.6 mg/kg). The leaves of B. nigra (Amritsar) contained Co (2.80 mg/kg), Cr (5.10 mg/kg), Cu (7.50 mg/kg), Mn (2.70 mg/kg), Ni (4.60 mg/kg), and Zn (39.0 mg/kg). The results obtained from rapeseed flower samples from locations in the Republic of Croatia can be compared with those for mature rapeseed from Turkey as seeds are formed from fertilized flowers, thus providing a basis for comparison. There was a difference in the concentration of Cu, with that in samples L1 to L5 being up to five times lower than the quoted values. The case of Mn is the opposite, as its concentration in the mentioned samples was up to five times higher compared to those from Turkey. Furthermore, the pollen of broad-leaved linden (Tilia platyphyllos) from the area in Romania contained Cd (0.07 mg/kg), Cr (1.50 mg/kg), Cu (9.22 mg/kg), Mn (71.9 mg/kg), Ni (0.63 mg/kg), Pb (0.44 mg/kg), and Zn (18.8 mg/kg) [18]. In common linden (T. vulgaris) from the area of Katowice in Poland, the measured concentrations in the leaves were Cu (1.58 mg/kg), Mn (6.36 mg/kg), and Zn (55.1 mg/kg) [18]. Pollen analyses are similar to analyses of linden flowers, so it is interesting to compare the obtained data. The concentration of Cd in flowers compared to the concentration in pollen was almost identical in samples L13 and L17, while it was somewhat lower in sample L14. The Cr concentration was up to seven times lower than that in linden flowers. The concentration of Cu was lower in L13 and L14, and higher in L17, and the same pattern was observed for the concentration of Mn. The value for Ni was almost identical in sample L14, slightly lower in L13, and higher in L17. Pb showed significantly lower concentrations in flower samples than in pollen. Zn was present in the same concentration in pollen samples and L17, while it was somewhat increased in L13 and L14. The largest deviations in concentrations were observed for the metals Cr and Pb, which were many times lower in linden flowers. In samples of the whole sweet chestnut plant (C. sativa) from the area of Bozdag Izmir in Turkey, the following concentrations were measured: Mn (1.12 mg/kg), Ni (0.05 mg/kg), Pb (0.38 mg/kg), and Zn (0.40 mg/kg). Sunflower (H. annuus) from the area of Konya in Turkey was reported to contain Cu (18.1 mg/kg) and Mn (6.95 mg/kg) in mature seeds [18]. Comparing the concentrations of Mn, Ni, Pb, and Zn in the chestnut samples from Turkey with those obtained in this research, it is evident that Mn was about 370 times higher in the L16 chestnut flower sample from Slunj, Ni was 40 times higher, Pb was almost 5 times lower, and Zn was over 50 times higher than in the chestnut plant from Izmir. Regarding the contents of Cu and Mn in mature sunflower seeds from Turkey—which were compared with the contents of the same metals in sunflower flowers from locations L18, L19, and L20—it can be seen that the Cu concentration was almost the same as in the cited data; meanwhile, the Mn concentration was three to six times higher, depending on the individual sample.

6.4. Cluster Analysis

Hierarchical cluster analyses are increasingly used in environmental studies, including those involving heavy metal determination and monitoring [2,11,33]. The results of this research are presented through cluster analysis performed using the DATAtab program [34]. Cluster analysis was performed to identify clusters of sampling locations based on similar contamination characteristics, in order to reveal the similarity in contamination between each group at certain sampling locations. The cluster analysis results are presented with regard to the measured values of the soil quality indicators (Figure 2) because, in other matrices (water and melliferous plants), the concentrations of determined metals and metalloids were very low and uneven and were not present in all analyzed samples.

Hierarchical cluster analysis resulted in the grouping of indicators by location into six classes (distinguished by color in Figure 2). The distance between classes (or clusters) is inversely proportional to the similarities between them, which means that a greater distance indicates lower mutual similarity. In general, the division into two basic groups, recognizable by the level of anthropogenic impact, is evident. In the top group (dark green, purple, and red), most locations are related to urban areas (such as L7, L20, L9, L14, L17, L13, L8, L4, and L2) and some are related to intensive agricultural production (namely, L7, L3, L19, and L18). This cluster is characterized by anthropogenic influence in urban areas and intensive agriculture. The lower group (dark green, light green, and yellow) includes locations that are also characterized by agricultural production, possibly of lower intensity (such as L5 and L1), as well as an agricultural wooded area with a transit road (location L10). This group also includes locations from areas of presumed lower impact (such as L12, L15, L11, and L16), as well as the island location (L6). A more detailed analysis indicates that anthropogenic influence is not the only condition determining similarity, with the type of melliferous plant and natural differences in soil composition also affecting the groupings.

6.5. Correlation Analysis

Soils contaminated with heavy metals can pose a potential ecological risk and, consequently, plants contaminated with heavy metals can pose a risk to human health. To investigate these risks, research and correlation analyses have been conducted [4,35]. Thus, the results of this research are also presented from a correlational perspective to provide insight into the possible connections between the examined indicators. The results of the correlation analysis are shown in Figure 3, which were obtained using Excel.

There is no complete correlation (|r| = 1) between the determined heavy and toxic metals in the soil. The strongest positive correlation is between Cr and Ni, which amounts to 0.91. The analysis also determined strong correlations (0.8 ≤ |r| < 1) between As and Se (0.87), Ni and Se, As and Cr, and As and Ni (0.84). There is also a strong correlation between Fe and Ni (0.82), as well as between Cr and Fe (0.81). Pb shows a borderline strong correlation (0.79) with the humus in the soil, falling close to the line between a medium (0.5 ≤ |r| < 0.8) and strong correlation. Most of the remaining correlations between the metals, humus, and pH values are weak (0.2 ≤ |r| < 0.5) or insignificant (0 < |r| < 0.2), with 13 values indicating a complete absence of correlation between metals (|r| = 0).

6.6. Bioaccumulation Factors

The bioaccumulation factor (BAF) for melliferous plants represents the sum of the ratio of a metal’s concentration in the flower of the plant to its concentration in the soil. As shown in Table 5, the highest BAFs for melliferous plants across all heavy and toxic metals at were observed at L3 (at 2.832), followed by L2 (0.805) and L4 (0.712). These are locations on agricultural land where rapeseed was growing, indicating the highest bioaccumulation potential among the analyzed melliferous plants.

6.7. Water and Soil Quality Index

The results for each heavy metal and toxic element show that the water quality index was greater than 1 for As at location L19 (at 1.228). Elevated water quality index values were also observed for Pb at location L13 (4.182) and at location L20 (where it amounted to 5.55). However, the overall water quality index for all metals at each location did not exceed 1, indicating satisfactory water quality.

Based on the obtained results for each heavy metal and toxic element, the soil quality index was greater than 1 for Cr at location L12 (at 1.195). Elevated soil quality index values were also observed for Cu at location L8 (where it amounted to 3.104) and for Ni at locations L11 (1.014), L12 (1.666), and L16 (1.001). The index value was also higher for Zn at locations L2 (where it amounted to 1.348), L3 (2.958), L4 (2.228), L6 (1.241), L8 (2.114), L12 (1.796), and L19 (1.079). Despite these values, the overall soil quality index for all metals collectively at each location did not exceed a value of 1, indicating satisfactory soil quality.

7. Conclusions

This scientific research aimed to determine the presence of the elements As, Cd, Co, Cr, Cu, Hg, Fe, Mn, Ni, Pb, Se, and Zn in water, soil, and melliferous plants at locations differently impacted by anthropogenic activity, in order to provide insight into the conditions at the selected locations depending on the level of proposed anthropogenic pressure. The results of the research, involving 20 locations across the territory of the Republic of Croatia, demonstrate that water samples are mostly free from contamination regarding the specified values for the 12 heavy metals and metalloids analyzed, as the water quality index (WQI) for all metals at each location did not exceed a value of 1. Exceptions include the low pH values of rainwater samples from locations L14 and L20, as well as the concentrations of As in sample L19 and Pb in sample L20, which exceeded the MAC given by the Ordinance on Compliance Parameters, Methods of Analysis and Monitoring of Water Intended for Human Consumption. Analyses of soil samples revealed less satisfactory conditions regarding contamination. Cr in sample L12 stands out, with its concentration higher than the MAC given by the Ordinance on the Protection of Agricultural Land from Contamination. Cu exceeded the limit in sample L8, Ni in samples L11 and L12, and Zn showed the highest contamination in samples L2, L3, L4, L6, L8, L12, and L19. The analysis results for non-biogenic heavy and particularly toxic metals (As, Cd, Hg, and Pb) demonstrated that the analyzed soils in urban, traffic-affected, agricultural, and mainly forested areas in the Republic of Croatia were not contaminated by these metals. The soil quality index (SQI) for all metals at each location did not exceed a value of 1. There are no regulations for the concentrations of metals and metalloids in the analyzed melliferous plants, and the concentrations of non-biogenic heavy and toxic metals (Hg, As, Cd, and Pb) in their flowers were among the five lowest concentrations. Cluster analysis revealed one group comprising almost all locations related to anthropogenic impact, including urban areas and locations related to intensive agricultural production. Meanwhile, the other group also contained locations that are characterized by agricultural production, possibly of lower intensity. Based on the obtained results, the strongest positive correlation was observed between Cr and Ni. The bioaccumulation factor (BAF) for the flowers of the melliferous plants were the highest for rapeseed at three locations; namely, L3, L2, and L4. Considering that people consciously contaminate their environment, they are called upon to take responsibility for protecting it and keep it free from contaminants. Humans have to decide how to preserve the health of all people, which is inextricably linked to the health of the environment and its individual components. This research aims to provide a concrete foundation for future analyses, comparisons, and decision-making regarding the investigated locations and toxic elements.