Phytoaccumulation of Heavy Metals in Flowers of Tilia cordata Mill. and Soil on Background Enzymatic Activity

Abstract

The phytoaccumulation of Fe, Mn, Cu, Zn, and Pb in Tilia cordata flowers and soils from six locations with varying degrees of anthropopressure in Bydgoszcz city and its surroundings (Poland) was assessed. Additionally, metal concentrations and soil enzymatic activity were analyzed. Enrichment Factor analysis revealed significant Zn enrichment at only one locality, supported by a geoaccumulation index value indicating moderate soil pollution. Total metal content in soils correlated with total organic carbon (TOC), while total iron content correlated with the clay fraction (<0.002 mm). Metal concentrations were comparable to the geochemical background levels for soils in Poland. Assessment of total metal contents in the topsoil surface layer from the six locations indicated that concentrations did not exceed permissible limits established by applicable legislation. The study showed that sampling locations influenced the activities of dehydrogenase (DHA), fluorescein diacetate hydrolysis (FDA), β-glucosidase (GL), and arylsulfatase (AR), and these activities correlated more strongly with pedogenic factors than with metal content. No elevated metal levels were detected in the dry mass of T. cordata flowers. Lead content did not exceed 10 mg·kg⁻¹ dry matter, in accordance with World Health Organization (WHO) recommendations. Continued monitoring of trace element levels in soils and T. cordata flowers, particularly in urban environments, is advisable.

1. Introduction

Expansive urban infrastructure development, urban activities, agricultural development, industrial processing, vehicular traffic, air deposition, and domestic and commercial wastes result in environmental pollution. Of all types of pollutants, heavy metals above acceptable limits are the major pollutants and cause for concern due to their persistent nature and health hazards. Such activities can cause water, air, and soil pollution with toxic heavy metals and pose a threat to the health and life of people, animals, as well as to biodiversity and the structure of plant communities [1,2]. The type of pollution depends mainly on the local infrastructure and traffic intensity [3]. The main pollutants degrading the environment include hydrocarbon compounds, pesticide residues, sulfur, and, above all, heavy metals [4]. The presence of metals such as nickel (Ni), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), and chromium (Cr) in the atmosphere, soil, or plant tissues has become a serious problem in most highly developed and industrialized countries [1]. Special attention and highest priority pollutants for control are given to metals such as Hg, Pb, Cd, and As [5]. According to the guidelines of the Agency for Toxic Substances and Disease Registry (ATSDR), these elements in amounts exceeding the established safe limits are harmful to both plants and humans [6].

Heavy metals in biological systems are classified as essential and non-essential. Essential heavy metals such as Mn, Fe, Cu, and Zn are important for living organisms and can be present in the body in low, safe concentrations and their deficiency can result in significant disturbances in plant development. Non-essential heavy metals such as Cd, Pb, and Hg can be found in plant organisms but have no known biological role in living organisms [7]. Environmental pollutants, including heavy metals that disrupt many biochemical and metabolic processes, can be transferred to leaf tissues—mainly through adsorption and internalization by the cuticle and penetration through stomata or taken up by roots [8,9,10]. Heavy metals may pollute the food chain, and in grains, vegetables, fruits, and herbs we can find elements which are extremely dangerous for the safety of human health and the quality of food [11]. Most of them are resistant to degradation but can be accumulated in living organisms [12].

Plants have the ability to phytoaccumulate some heavy metals. Long-term exposure to heavy metals causes numerous changes in cells such as lipid peroxidation, enzyme inactivation, DNA damage, inhibition of respiration, gas exchange, a limitation of photosynthesis, and disturbance of water balance. The effects of these disturbances are changes in plant morphology, wilting, aging, reduced biomass production, and plant death [13]. The main source of exposure to heavy metals for humans is their consumption through food and drinking water. Growing food ingredients in an environment contaminated with heavy metals leads to the bioaccumulation of these elements in human food chains, where they ultimately enter the body and may cause risk to human health [12].

With the increased intensity of various lifestyle diseases associated with stress and diet, there is a growing interest in natural methods of treating or preventing diseases and herbal medicine. Despite the constant expansion of the range of cultivated herbs and the growth of the area of field crops, collection from the natural state is still one of the most important sources of supply for the herbal industry in raw materials. In addition, the raw material of some species is obtained mainly from natural sites. The most important herbal raw materials obtained in Poland include chamomile flower, mint leaf, or linden flower [14].

Lime (Tilia L.) belongs to the Malvaceae family and is common in Europe, North America, and Asia. This plant was described by Carl Linnaeus in 1753 and has since gained popularity as an ornamental and medicinal tree. It is an example of a multifunctional species with high aesthetic value and wide possibilities of use. The flowers are a source of bees’ food and together with the leaves constitute a herbal raw material, the wood can be used to woodcarving. Lime seeds are also used, from which a chocolate substitute or oil can be produced. In addition, the roots of Tilia L. are a component of a mycorrhizal system with truffles, which are a valuable culinary ingredient achieving high value on the consumer market [15].

Lime trees grow up to 40 m tall, have a round, broad crown. The leaves are arranged alternately and have a heart shape. The flowers have an intense fragrance and their color ranges from green to yellow, depending on the species. The fruit of the Tilia is a round, woody nut [16,17]. In Europe and America, the genus Tilia L. is widely used as a street tree due to its ability to absorb pollutants and heavy metals, thus purifying the air and regulating the ecosystem [15].

Tilia cordata Mill., native to Europe [18], is a popular tree planted in Western, Nordic, and Central European cities and parks [19,20], which may be due to the fact that it is sensitive to drought but tolerant to very low temperatures and is regarded as tolerant of urban pollution [21].

In Poland, two species of the Tilia genus occur naturally in forests, as park plantings, in gardens, along streets and roads—the small-leaved lime (Tilia cordata Mill.), which is more popular—and the less common broad-leaved lime (Tilia plathyphyllos Scop.) [22]. The typical habitat of T. cordata is deciduous forests and city parks. It used to be one of the most important forest-forming species in Poland. Its ecological importance is mainly due to the beneficial effect it has on the habitat, enriching the soil with humus through abundant leaf fall, and it does well on poorer and acidic soils [23]. The popularity of this genus is due to the fact that it is a melliferous plant. Lime honey is known for its unique smell, taste, and medicinal properties [24]. Due to the content of biologically active compounds valuable herbal raw materials are mainly flowers and, to a lesser extent, leaves [15,17,22,25,26].

The valuable properties of T. cordata are used in folk medicine. The herbal raw material is mainly T. cordata flowers. Phytochemical studies have shown that the tissues contain carbohydrates, flavonoids quercetin, kaempferol, rhamnoside, tiliroside, phenolic compounds, tannins, coumarin, saponins, scopoletin, and essential oils, the main components of which are mono- and sesquiterpenes (linalool, germacrene, farnesene) [27,28,29,30,31,32,33].

Infusions or teas prepared from T. cordata flowers have calming properties, facilitating sleep. They also have diuretic, diaphoretic, anti-inflammatory, and soothing properties in the treatment of respiratory diseases, fever, cough, and asthma [28,34,35]. Herbal substances from Tilia are also essential raw materials for the pharmaceutical and cosmetics industries. Cosmetics from small-leaved linden can take various forms, such as creams, tonics, oils, or micellar fluids. Herbal dermatological preparations from T. cordata are suitable for skin care. They can be used for the care of dry, sensitive, and irritated skin due to their soothing and anti-inflammatory properties [36,37]. T. cordata also has culinary uses. The leaves are edible and have a delicate honey flavor. They can be used in salads or as an ingredient in tea.

Poland has a long tradition of growing and obtaining herbal raw materials and is one of the largest exporters of herbal raw materials in Europe. Over the past 10 years, Poland has been among the top ten exporters of medicinal and aromatic plants in the world [38]. Poland’s share in herb production compared to other European countries is around 30%. Poland has the largest area of herb cultivation in Europe (30,000 ha) [39]. In Poland, herbs are also obtained in their natural state and the individual collection of herbs still occurs.

Since herbal medicinal products are obtained not only from controlled plantations but also from uncontrolled habitats; it is very important that the quality of herbal medicines is controlled in the same way as the quality of chemically synthesized drugs [40]. The WHO reported that approximately 80% of the population of developing countries rely on traditional herbal medicine [41]. In this regard, the quality and safety of herbal products are extremely important. Herbal raw materials originating from natural habitats require the use of special control measures; if the permissible limits for heavy metal content are exceeded, the collection of the plant must be limited [40]. By using advanced quality control techniques and appropriate standards, it is possible to ensure the quality of herbal medicines. Initial identification and the evaluation of physical, chemical, and biological properties contribute to maintaining the quality standard of herbs. Appropriate harvesting time, drying techniques, plant origin, the presence of heavy metals, and microorganism content are the main reasons for the change in the quality of herbal medicinal and prophylactic products [42].

The development of modern cities and rapid urbanization are the main causes of environmental pollution with heavy metals [43]. The dynamics of organic matter and enzymatic activity is an early diagnostic indicator of negative changes in urban pedogenesis. Enzymes in soil play crucial roles in nutrient cycling and energy transformation by catalyzing numerous chemical reactions [44]. Soil enzymes are proteins and therefore are likely to be sensitive to variations induced by natural and anthropogenic disturbances [45]. Various studies have presented that soil enzymes can be used as bioindicators of soil contamination and soil health [46].

The aim of this study is to quantify heavy metals in traditional herbal raw material (Tilia cordata flowers) used in the treatment of various diseases in Poland and other European countries. Due to the fact that T. cordata is one of the species included in the list of medicinal and aromatic plants and plant parts subject to commercial trade, collected from natural sites in Poland [38], it is necessary to indicate the safest possible collection sites for this raw material, taking into account the constantly growing threat from anthropological pollution and the fact that Tilia spp. leaves show significant seasonal accumulation of heavy metals [47]. In this study, habitats at different distances from communication routes, residential and agricultural habitats, fields, and forests were compared to compare the degree of heavy metal contamination and indicate the best sources for obtaining this herb. Phytoaccumulation of Fe, Mn, Cu, Zn, and Pb in T. cordata flowers and the soil of six locations with different degrees of anthropopressure in the Kuyavian–Pomeranian province (Bydgoszcz city and its surroundings) was assessed. We also assayed the content of metals and the enzymatic activity in the rhizosphere soil. The research hypothesis assumed that soils and plants collected in the vicinity of places with heavy traffic are contaminated with heavy metals (Fe, Mn, Cu, Zn, Pb) and show lower dehydrogenasefluorescein diacetate hydrolysis (FDA), β-glucosidase (GL), and arylsulfatase (AR) activities.

2. Materials and Methods

2.1. Study Areas

The content of Zn, Cu, Mn, Fe, and Pb was assessed in small-leaved lime (Tilia cordata Mill.) and soil collected from 6 locations in Poland in the Kuyavian–Pomeranian province (Bydgoszcz city and its surroundings). The places where flowers of T. cordata and soil samples were taken, their location, and the degree of traffic intensity in the examined place are presented in

Table 1. Places of collecting flowers of T. cordata and soil samples.

Sampling Location	The Place Where the Material Was Collected	The GPS Coordinates	Road Distance	The Degree of Traffic Intensity *	Surroundings
A	Bydgoszcz city	53°07′15.6″ N 18°00′23.1″ E	10 m	a	A tree in the old town center stands on a regularly trimmed lawn, surrounded by old, tall trees, separated from the road by a lawn and other lower plants.
B	Łochowo village	53°07′30.8″ N 17°49′30.3″ E	5 m	b	A tree growing near the road and urbanized area, separated from the road by an area of wild weeds and grass.
C	Łochowo village	53°08′01.2″ N 17°49′24.0″ E	200 m	c	A tree growing near the field, on the edges of mixed forests with a predominance of deciduous trees, away from roads and urban area.
D	Węgorzyn village	53°12′21.7″ N 18°47′18.2″ E	2 m	b	A tree growing near the field, near the rural road on rural area close to residential and farm buildings.
E	Pszczółczyn village	53°00′41.0″ N 17°53′45.0″ E	2 m	b	A tree growing in the area of the Aleja Lipowa Reserve protected by law, by the road. Nearby there are meadows and fields. In the area there are fragments of a deciduous forest with limes, alders, and ash trees.
F	Olimpin village	53°01′39.0″ N 17°57′50.3″ E	15 m	b	A tree growing near the field, near a rural road, far from residential and farm buildings.

* The degree of traffic intensity based on the estimated number of vehicles per day; a—places with heavy traffic; b—places with medium traffic intensity; c—places away from road traffic.

and Figure 1.

2.2. Sampling Procedure

The soil and plants of T. cordata were harvested in early July 2021 during the flowering phase. All trees from which the research material was taken were old and over 100 years old. Plant and soil material was dried on paper at room temperature to an air-dry state and flowers ground in a laboratory grinder. Before drying, the T. cordata flowers were thoroughly washed under top water. The plant material were collected from trees from 6 locations (Table 1). In this study, habitats at different distances from communication routes, residential and agricultural habitats, fields, and forests were selected. The soil was sampled from the plant root zone (0–25 cm). A total of 50 flowers were collected from different sides of each T. cordata tree examined. Soil samples were taken from 3 places at a distance of 20 cm from the trunk, and this distance was standardized for all trees. The soil was dried and sieved through a sieve with a mesh diameter of 2 mm. From the pooled flower and soil samples, analyses were performed in triplicate for each location.

2.3. Analytical Procedure

The homogenized material (300 mg) of soil samples and flowers of T. cordata was microwave-digested in the Speedwave Two mineraliser (Berghof Speedwave, Eningen, Germany) with the wet mineralization method (5 mL 65% HNO₃, 1 mL 30% H₂O₂). In mineralized plant samples and soil, the total concentrations of Zn, Cu, Mn, and Fe was determined by atomic absorption spectrometry (AAS) with the SOLAAR S4 spectrometer (ThermoElemental, Cambridge, UK). Analyzes were performed in three replications.

The granulometric composition was determined using the Mastersizer 2000 analyzer (Malvern Instrument, Malvern, UK), the total organic carbon content (TOC) using the Vario Max CN analyzer from Elementar provided by Analysensysteme GmbH (Elementar, Analysensysteme GmbH, Hanau, Germany), and the pH using the potentiometric method on a pH meter after adding a 1 M KCl solution to the soil samples, at a soil/solution ratio of 1:2.5. The total metal contents were determined by applying digestion with HF and HClO₄ acids solutions according to the Crock and Severson [48] method. Certified reference material TILL-3 (Canadian Certified Reference Materials Project) was used to verify analytical accuracy. The recovery rates for the analyzed elements were as follows: Fe—97%, Mn—101%, Zn—102%, Cu—103%, and Pb—97%. All analyses were performed in triplicate. Total metal concentrations were determined using atomic absorption spectrometry (AAS) with a SOLAAR S4 spectrometer (Thermo Elemental, Cambridge, UK). Electrical conductivity (EC), which is defined as the ability of a given material to conduct electric current, was also determined in soil samples.

Dehydrogenase (DHA) activity was assayed by Thalmann [49] based on a colorimetric assay with a buffered pH 7.6 tetrazolium salts (TTC) and glucose were incubation at 30 °C for 24 h. DHA activity was expressed as µg TPF·g⁻¹·h⁻¹. The rate of fluorescein hydrolysis (FDA) was estimated according to the report Adam and Duncan [50]. Soil samples were incubated with buffer pH 7.6 and fluorescein by hour in 37 °C. The FDA were determined by released fluorescein concentration (µg F·g⁻¹·h⁻¹) at 490 nm. β-glucosidase (GL) activity was assayed by a colorimetric method, using 4-nitrophenyl-β-D-glucopyranoside as substrate, and buffer pH 6.0 soil samples were incubated at 37 °C for 60 min, expressed as µg pNP·g⁻¹·h⁻¹ [51]. Similarly, the activity of arylsulfatase (AR) used acetic buffer pH 5.8 and 4-nitrophenyl-sulfate potassium salt expressed as µg p-nitrophenol g^–1 soil h^–1 was determined according to Tabatabai and Bremner [52]. In each biochemical analysis, three performing samples and three controls were taken, and the results were calculated into appropriate units per one hour and gram of soil.

2.4. Mathematical Calculations

The bioconcentration factor (BCF) was calculated based on a ratio of the total content of the elements studied in flowers of T. cordata CS_f (mg·kg⁻¹ dry weight) to their content in the rhizosphere soil CS_s (mg·kg⁻¹ dry weight). The values of the BCF (relative content) of Fe, Mn, and Zn as well as Cu in the plant material were calculated following the formula:

BCF_f flowers in T. cordata = CS_f flowers/CS_s soil.

To evaluate the degree of accumulation of Zn, Cu, Mn, as well as Fe, a four-degree scale provided by Kabata-Pendias et al. [53] was used. The values of the BCF were interpreted as follows: 0.001–0.01—lack; 0.01–0.1—weak; 0.1–1.0—medium; 1.0–10.0 intensive of accumulation.

The coefficient of variation (CV) was also calculated in the study. The CV is a measure of relative variability, expressed as the ratio of the standard deviation to the mean, expressed as a percentage. The CV of the parameters mathematically can be expressed as

CV = (S/X) × 100%,

where CV is the coefficient of variation (%), S is the standard deviation, and X is the arithmetic mean. The values 0%–15%, 16%–35%, and >36% indicate low moderate or high variability, respectively [54].

The anthropogenic impact was assessed using the Enrichment Factor (EF). Iron (Fe) was used as the reference element for these calculations, applying the following formula:

EF = (Cn/CnFe)/(Bn/BnFe),

where Cn is the total metal concentration in the surface soil layer, CnFe is the total Fe concentration in the surface soil layer, Bn is the total metal concentration in the geochemical background, and BnFe is the total Fe concentration in the geochemical background. The average metal concentration in the parent material, constituting the geochemical background of the region’s soils [40], were as follows: Zn—31.50 mg·kg⁻¹, Cu—6.17 mg·kg⁻¹, Mn—450.7 mg·kg⁻¹, Fe—9.13 g·kg⁻¹, and Pb—15.90 mg·kg⁻¹. The calculated EF values are interpreted as follows: EF < 2: deficiency to minimal enrichment, EF = 2–5: moderate enrichment, EF = 5–20: considerable enrichment, EF = 20–40: very high enrichment, EF > 40: extremely high enrichment.

Soil pollution was also evaluated using the geoaccumulation index (I_geo), calculated as

I_geo = log₂ (Cn/(1.5 × Bn)),

where Cn is the measured metal concentration in the soil, and Bn is its geochemical background value. The degree of heavy metal pollution is categorized by Förstner et al. [55], and I_geo values are as follows: I_geo ≤ 0: unpolluted, 0 < I_geo < 1: unpolluted to moderately polluted, 1 < I_geo < 2: moderately polluted, 2 < I_geo < 3: moderately to heavily polluted, 3 < I_geo < 4: heavily polluted, 4 < I_geo < 5: strongly to extremely polluted, and I_geo > 5: extremely polluted.

2.5. Statistical Analysis

The obtained physicochemical and biochemical analysis data were first subjected to Pearson correlation to determine the adequacy of the data for multivariate statistics for p-value thresholds (α = 0.05). Statistical analyses were performed using the “Statistica 12.0 for Windows PL” package [56]. The results of heavy metal content in soil and plant parts were expressed as mean ± standard deviation (SD). The soil and metal content in T. cordata flowers, as well as the activity of all enzymes in the soil, were assessed using principal component analysis (PCA). The first two principal components (PC1 and PC2) were selected to determine which soil properties differentiated individual sampling sites. The dendrogram obtained by the Ward’s Euclidean linkage distance method was confirmed by PCA results.

3. Results and Discussion

3.1. Physical and Chemical Properties and Heavy Metal Content in Soil

Soil samples from locations A, B, D, E, and F exhibited a sandy loam texture, while the sample from location C was classified as loamy sand (

Table 2. Selected physical and chemical properties of soil samples (0–25 cm).

Sampling Location *	Grain Size Composition [%]			Textural Class USDA ***	pH **	TOCs	ECs
	Sand 2.0–0.05 mm	Silt 0.05–0.002 mm	Clay <0.002 mm		1 M KCl	g∙kg−1	µS∙cm−1
A	68.5	27.7	3.8	SL	6.74 ± 0.04	19.3 ± 0.26	185.0
B	64.6	31.2	4.2	SL	7.12 ± 0.05	13.1 ± 0.28	111.5
C	74.4	22.8	2.8	LS	6.96 ± 0.07	10.3 ± 0.23	89.7
D	59.9	33.7	6.4	SL	6.87 ± 0.05	14.1 ± 0.30	124.0
E	65.5	31.1	3.4	SL	5.97 ± 0.03	13.3 ± 0.28	89.9
F	68.7	28.1	3.2	SL	7.10 ± 0.05	9.9 ± 0.38	97.5
mean	66.9	29.10	3.97		6.79	13.3	116.3
SD	4.86	3.80	1.29		0.43	3.39	36.2
CV (%)	7.3	13.1	32.5		6.3	25.5	31.1

* Sampling location explanations as in Table 1; ** results are the mean of 3 replicates ± SD (standard deviation); CV—coefficient of variation; EC—electrical conductivity; TOC_s—total organic carbon in soil; SL—sandy loam; LS—loamy sand; *** USDA [58].

). In location A, there is a Technosol soil with a transformed soil profile, but with a significant thickness of the humus horizon. The other soils belong to Luvsols, with a characteristic eluvial horizon below the humus horizon and parent material of glacial origin [57]. In the surface layer, soil samples contained between 59.9% and 74.4% of sand (2.0–0.05 mm), 22.8% to 33.7% of silt (0.05–0.002 mm), and 2.8% to 6.4% of clay (<0.002 mm). The pH values ranged from 5.97 to 7.12, and total organic carbon (TOC) content ranged from 9.9 to 19.3 g·kg⁻¹. The mean electrical conductivity was 116.3 µS∙cm⁻¹. ECs values indicate that the soil is not salinized and measuring of the water-soluble salt content in soil materials and serves as an important indicator of soil quality. EC values influence nutrient availability and the activity of soil microorganisms. Among the measured parameters, the clay content and electrical conductivity showed the highest variability, as indicated by their respective coefficients of variation (CV) of 32.5% and 31.1% (Table 2).

Data on the metal concentrations in the analyzed soil samples are presented in

Table 3. Total content of metals (Zn, Cu, Mn, Fe, Pb) in topsoil (0–25 cm).

Sampling Location *	Zn	Cu	Mn	Fe	Pb
	mg·kg−1			g·kg⁻1	mg·kg−1
A	164.7 ± 47.4	16.7 ± 6.11	265.5 ± 23.7	6.37 ± 0.78	14.0 ± 1.34
B	45.3 ± 3.12	6.9 ± 1.85	152.2 ± 15.8	3.43 ± 0.68	12.5 ± 2.26
C	16.6 ± 2.89	5.67 ± 0.98	75.5 ± 14.9	3.08 ± 0.28	11.2 ± 1.21
D	46.9 ± 5.88	8.67 ± 5.34	179.0 ± 34.8	8.83 ± 0.65	13.1 ± 1.42
E	48.9 ± 6.25	2.00 ± 0.87	141.6 ± 29.0	3.18 ± 0.39	10.9 ± 1.32
F	33.6 ± 5.65	4.73 ± 0.46	124.4 ± 36.8	3.03 ± 0.41	9.9 ± 1.25
Mean for location	59.33	7.45	156.37	3.82	11.93
SD	48.39	4.61	58.05	1.17	1.39
CV (%)	81.6	61.9	37.1	30.6	11.7

* Sampling location explanations as in Table 1; results are the mean of 3 replicates ± SD (standard deviation); CV—coefficient of variation.

. An assessment of the total metal content in the surface layer of soils from six sites indicates that the concentrations did not exceed the permissible limits established by applicable legislation [59]. The Fe content in the surface layer ranged from 3.03 g·kg⁻¹ (location F) to 8.83 g·kg⁻¹ (location D), with a mean value of 3.82 g∙kg⁻¹. The Zn concentration varied considerably, as indicated by a high coefficient of variation (CV). The total content of Zn in soil ranged from 16.6 mg·kg⁻¹ (location C) to 164.7 mg·kg⁻¹ (location A), with a mean of 59.33 mg·kg⁻¹. A similar pattern was observed for Cu, which ranged from 2.0 mg·kg⁻¹ (location E) to 16.7 mg·kg⁻¹ (location A), with a mean of 7.45 mg·kg⁻¹. According to Kabata-Pendias [53], the total Zn content in surface soil layers typically ranges from 50 to 100 mg·kg⁻¹. The mean total manganese content was 156.4 mg·kg⁻¹, with moderate variability (CV = 37.1%). In the soil samples analyzed, the total concentration of Pb did not exceed natural background levels, ranging from 9.9 mg∙kg⁻¹ (location F) to 14.0 mg·kg⁻¹ (location A), with a mean of 11.93 mg·kg⁻¹. Lead is a heavy metal and one of the most common soil contaminants [60]. In Poland, the permissible Pb content in surface soil horizons ranges from 30 to 70 mg·kg⁻¹ [61]. The concentrations of the studied metals were comparable to the geochemical background levels for soils in Poland [62].

Average heavy metal concentrations in some roadside soils in Poland have been found to be significantly higher than geochemical background levels, indicating moderate contamination. Studies of roadside soils in northeastern Poland have reported elevated levels of Zn, Cu, and Pb, suggesting the influence of land use and traffic emissions [63]. The total Zn content was found to reach up to 70 mg·kg⁻¹ at location A. This concentration is considered a critical threshold, above which plants, under favorable soil conditions, may accumulate excessive amounts of this metal [40]. Copper concentrations were found to vary depending on land use, with levels in some cases exceeding established toxicity thresholds. Some studies have documented Cu concentrations above recognized safety limits, underlining the need for ongoing monitoring and assessment of contamination risks [64]. Anthropogenic activities, particularly urbanization and vehicle emissions, have been linked to elevated Pb levels in soils, especially in urban and peri-urban environments. Pb is a frequent soil contaminant, and its accumulation presents serious ecological risks [65].

Overall, the elevated average concentrations of Zn, Cu, and Pb in Polish soils reflect a concerning trend of anthropogenic pollution, underscoring the substantial impact of transportation, urban development, and agricultural practices. To ensure environmental safety, continuous monitoring and, where necessary, remediation strategies are essential. The total contents of metals Zn, Cu, Mn, and Pb were correlated with total organic carbon (TOC). The analysis also showed significant positive relationships between clay fraction (<0.002) and total content of Fe (r = 0.869) (Table 10 and Table S1). The total content of Zn in the topsoil showed highly significant positive correlations with both the total contents of Cu and Mn in soil. Pb also exhibited a significant positive correlation with the total content of Cu. A strong positive correlation between TOC_s and the total content of Zn in soil (r = 0.937) and Zn available to plants has been previously reported in the literature [40,66]. This significant relationship suggests that Zn is one of the more mobile heavy metals and is strongly associated with organic matter. The correlation analysis also revealed a significant positive relationship between TOC_s and the total content of Cu in soil (r = 0.973), which is consistent with findings by Figas et al. [40] and Skwaryło-Bednarz et al. [66].

The highest contents of Zn, Cu, and Mn in forms available to plants were recorded at locality A, amounting to 38.3, 4.24, and 58.8 mg·kg⁻¹, respectively (

Table 4. Concentrations of metals available to plants after 1 M HCl extraction and their proportion of total metal content.

Sampling Location *	Zna	Cua	Mna	Pba	Fea	Zna/Znt	Cua/Cut	Mna/Mnt	Pba/Pbt	Fea/Fet
	mg·kg⁻1					%
A	38.3	4.24	58.8	1.20	232.1	23.2	25.4	22.1	8.6	3.64
B	5.21	0.91	21.1	0.89	78.4	11.5	13.2	13.9	7.1	2.29
C	2.32	1.32	8.32	0.67	87.5	14.0	23.3	11.0	6.0	2.84
D	5.47	1.81	32.2	0.90	83.9	11.7	20.9	18.0	6.9	0.95
E	7.20	0.32	20.5	0.98	98.7	14.7	16.0	14.5	9.0	3.10
F	8.80	1.43	31.4	1.30	245.6	26.2	30.2	25.2	13.1	8.11

* Sampling location explanations as in Table 1; _t—total contents of metals; _a—metals available to plants.

). The concentration of plant-available Pb ranged from 0.67 mg·kg⁻¹ in soil samples from locality C to 1.30 mg·kg⁻¹ at locality F. A similar concentration of the studied microelements in the soils of the analyzed region was described by Kobierski et al. [67] and Figas et al. [68]. The highest percentage of available forms in relation to the total metal content was observed for copper (up to 30.2% at locality F), whereas the lowest was recorded for iron (only 0.95% at locality D) (Table 4). Based on the Enrichment Factor (EF), a considerable enrichment of Zn was identified only at locality A (EF = 7.5), which is further supported by a geoaccumulation index (I_geo) value of 1.8, indicating a moderately polluted soil environment. The surface soil layer at locality A exhibited an I_geo value of 0.85 for Cu, suggesting an unpolluted to moderately polluted soil condition. For all other localities, the I_geo values for the analyzed metals were below zero (I_geo < 0), indicating unpolluted soils. Moderate enrichment of Zn total content was recorded at localities B (EF = 3.8), E (EF = 4.5), and F (EF = 3.2). Soil samples from localities A, B, C, and F also showed moderate accumulation of total Cu, with EF values of 3.9, 3.0, 2.7, and 2.3, respectively. Regarding the geochemical background of Pb, deficiency to minimal enrichment was noted at localities A (EF = 1.3), D (EF = 0.9), E (EF = 2.0), and F (EF = 1.9), while moderate accumulation was observed in the surface soils at localities B (EF = 2.1) and C (EF = 2.1) (

Table 5. Values of Enrichment Factor EF and geoaccumulation index I_geo in soil surface.

Sampling Location *	Zn (EF)	Zn (I_geo)	Cu (EF)	Cu (I_geo)	Mn (EF)	Mn (I_geo)	Pb (EF)	Pb (I_geo)	HC **
A	7.5	1.80	3.9	0.85	0.8	−1.35	1.3	−0.77	2
B	3.8	−0.06	3.0	−0.42	0.9	−2.15	2.1	−0.93	3
C	1.6	−1.51	2.7	−0.71	0.5	−3.16	2.1	−1.09	5
D	1.5	−0.01	1.5	−0.09	0.4	−1.92	0.9	−0.86	3
E	4.5	0.05	0.9	−2.21	0.9	−2.26	2.0	−1.13	4
F	3.2	−0.49	2.3	−0.97	0.8	−2.44	1.9	−1.27	4

* Sampling location explanations as in Table 1; EF—Enrichment Factor; I_geo—geoaccumulation index; ** HC—Hazard Classes: 1 (Extremely Hazard), 2 (Considerable Hazard), 3 (Moderate Hazard, 4) (Low Hazard), 5 (Non-hazardous).

). According to the Polish regulation of the Minister of the Environment, the total concentrations of the examined metals indicate that the areas remain unpolluted [69].

3.2. Heavy Metals Content in T. cordata Flowers

The studied flowers of T. cordata were characterized by different trace element contents depending on the sampling location. The concentrations in the plant samples were in the following decreasing order: Fe > Mn > Zn > Cu > Pb. In European Union (EU) countries and worldwide, herbal raw materials are standardized according to the requirements of the European Pharmacopoeia [70] and the World Health Organization [71]. These standards aim to define permissible limits for heavy metals such as cadmium, lead, and mercury. The content of trace elements Fe, Mn, Zn, and Cu in herbs is not regulated by law. According to the WHO, the permissible lead content in plants with medicinal properties is Pb 10 mg∙kg⁻¹ d.m. In Poland [72], the regulations are more restrictive and specify the limit values of Pb concentrations below 5.0 mg∙kg⁻¹ d.m. in herbs.

Iron (Fe) is an essential micronutrient for almost all living organisms because it plays critical role in metabolic processes. Iron occurs mainly in the form of Fe chelate⁺³ in soil. Fe deficiency results in low plant yields and reduced nutritional value. The physiological function of iron is participation in chlorophyll synthesis and maintenance of chloroplast structure and function. Plants growing at high soil pH conditions are not very efficient in developing and stabilizing chlorophyll, resulting in leaf chlorosis, poor growth, and reduced yields [73,74]. The exposure of plants to above-optimal levels of iron results in browning plants due to connective tissue necrosis, as well as blackening of roots [73,75]. In our study the contents of Fe in flowers of T. cordata ranged from 380 mg∙kg⁻¹ d.m. (location E) to 1560 mg∙kg⁻¹ d.m. (location D) with a mean of 760 mg∙kg⁻¹ (

Table 6. Content of metals (Zn, Cu, Mn, Fe, Pb) in dry weight of flowers of T. cordata.

Sampling Location *	Zn **	Cu	Mn	Fe	Pb
	mg·kg−1
A	23.0 ± 4.58	4.27 ± 2.25	15.5 ± 0.46	930.0 ± 60.1	4.0 ± 0.34
B	23.0 ± 1.28	9.73 ± 2.80	24.2 ± 0.1	530.0 ± 20.3	3.5 ± 0.26
C	31.7 ± 0.85	11.4 ± 1.07	38.1 ± 1.72	490.0 ± 50.2	2.2 ± 0.21
D	18.6 ± 0.98	10.7 ± 1.31	13.3 ± 1.44	1560.0 ± 150.2	3.1 ± 0.41
E	33.9 ± 0.98	10.9 ± 0.45	104.7 ± 1.72	380.0 ± 10.9	3.2 ± 0.32
F	26.4 ± 0.51	10.8 ± 0.61	57.5 ± 0.85	670.0 ± 10.7	2.9 ± 0.25
Mean for location	26.10	9.63	42.22	760.00	3.15
SD	5.29	2.45	31.70	397.32	0.55
CV (%)	20.3	25.4	75.1	52.3	17.5

* Sampling location explanations as in Table 1; ** results are the mean of 3 replicates ± SD (standard deviation); CV—coefficient of variation.

). Figas et al. [68] determined the content of Fe in flowers in black elderberry Sambucus nigra L. flowers in the range from 105.8 mg·kg⁻¹ to 281.7 mg·kg⁻¹. In turn, Tomaszewska-Sowa et al. [76] determined the content of this metal in sand everlasting Helichrysum arenarium (L.) Moench inflorescences in the range from 248.6 to 271.8 mg·kg⁻¹, yarrow Achillea millefolium L. from 458.7 to 496.5 mg·kg⁻¹, and stinging nettle Urtica dioica L. from 335.1 to 704.2 mg·kg⁻¹.

Manganese (Mn) as an essential element for all living organisms can act as an enzyme cofactor or occur in the body as a metal with catalytic activity in biological clusters [77,78]. In humans, too much manganese can cause poisoning that can result in liver cirrhosis, polycythemia, and symptoms that resemble Parkinson’s disease [78,79]. For plants, although it is needed in small amounts, it is as critical for growth and development as other nutrients. In plants, Mn is an element of the metalloenzyme cluster of the oxygen-evolving complex (OEC) in photosystem II (PSII). Mn plays a role in physiological processes such as photosynthesis and respiration. It also participates in the scavenging of reactive oxygen species (ROS) as well as in pathogen defense and hormone signaling [78]. Toxic Mn concentrations are dependent on plant species [80,81]. Excess Mn can be stored in vacuoles and cell walls [82]. It can also be distributed to different leaf tissues [83]. For most plant species, the content of 10 to 25 mg·kg⁻¹ is sufficient [53]. In our study, the Mn concentration in flowers of T. cordata ranged from 13.3 mg·kg⁻¹ d.m. (location D) to 104.7 mg·kg⁻¹ d.m. (location E) with a mean of 42.22 mg∙kg⁻¹. The highest variability in plant materials collected from six locations was found in relation to Mn in flower content (CV = 75.1%) (Table 6). The Mn content in plants growing outside the direct influence of pollution is most often 340–1339 mg·kg⁻¹ [84].

Zinc (Zn) is an essential trace element for all living organisms, performing important functions in biological processes (growth, development, defense). Zn is the most common deficiency in crops, particularly in soils with high and low pH [85,86,87]. However, excessive amounts of this metal can be harmful to plants, although tolerance levels are usually high. Plant species and genotypes differ in their tolerance to high zinc concentrations [88,89]. This element is usually taken up by the roots of plants and transported to the aboveground part in a certain amount [86,87]. Zn activates or is a component of many enzymes and therefore influences many metabolic processes in the plant. Zn is the only metal represented in oxidoreductases, hydrolases, lyases, ligases, transferases, and isomerases [86]. In our study, the analyzed flower of T. cordata samples Zn concentration in ranges from 23.0 mg∙kg⁻¹ d.m. (location A and location B) to 33.9 mg∙kg⁻¹ d.m. (location E) with a mean of 26.10 mg∙kg⁻¹ (Table 6). According to the World Health Organization [90], the average concentration of Zn in plant crops ranges from 10 to 100 mg∙kg⁻¹ of dry matter. The range of 15–30 mg·kg⁻¹ of dry mass in the aboveground parts of plants is considered sufficient to meet the physiological needs of most plants [91]. Zn deficiency in plants is usually detected when they contain less than 20 mg∙kg⁻¹. On the other hand, toxic effects appear when the content exceeds 300–400 mg∙kg⁻¹ [7]. This means that most of the plants tested, except for locations C and F, were characterized by a Zn concentration in flowers that covered the average physiological needs of plants. Sampling from locations C and F were located close to the field. Zn is a ubiquitous metal particularly correlated with the agriculture (Zn containing fertilizer) and industry [92]. The results show that the T. cordata flowers collected from all locations met the conditions specified in the WHO standard for herbal raw materials used in herbal medicine. According to this standard, the Zn content cannot exceed 50 mg·kg⁻¹.

Copper (Cu) belongs to the group of essential elements that play a role in physiological processes and plant growth. Cu acts as a structural element in regulatory proteins and participates in photosynthetic electron transport, mitochondrial respiration, oxidative stress responses, cell wall metabolism, and hormonal signaling, acting as cofactors in many enzymes [93,94]. However, too high concentrations of this metal have a toxic effect, inhibiting plant growth and disrupting normal physiological [95,96,97]. Cu can cause chlorosis, necrosis, stunted growth, leaf discoloration, and inhibited root [93,98]. In our research, the Cu_f concentration in flowers ranged from 4.27 mg∙kg⁻¹ d.m. (location A) to 11.4 mg∙kg⁻¹ d.m. (location C) with a mean of 9.63 mg∙kg⁻¹ (Table 6). The Cu content in flowers of T. cordata showed a significant positive correlation (r = 0.909) with the content in soil Cu_t (Table 10 and Table S1). The highest values for our samples were recorded for an agricultural field (location C), which can be explained by the fact that Cu concentrations depend primarily on the parent soil material. Cu is not very mobile in soil and tends to accumulate in the topsoil due to specific adsorption on mineral and organic fractions. Cu accumulation in soil may be the result of mining, dust fall, urban pollution, organic residues, and the use of Cu-based fungicides in crops or the use of organic fertilization, e.g., pig slurry, are important cases of anthropogenic Cu contamination of soil [99]. The impact of environmental pollution is confirmed by Georgieva et al.’s [100] research, who found that the Cu content level in linden flowers collected from the urban area was 8.90 mg∙kg⁻¹ and from less polluted areas it was 7.09 mg∙kg⁻¹. In lime flowers collected from urban areas in Serbia, Mitrović et al. [101] found higher values ranging from 12.4 to 22.49 mg∙kg⁻¹. Similar amounts of Cu in medicinally used lime herbs were determined by Basgel and Erdemoglu [102]. Such high amounts of Cu in aboveground organs such as leaves or flowers may result from high concentrations of this metal in the soil. Cu is highly capable of translocating from its roots to the leaves and flowers [101]. For this reason the level of this metal should be monitored in these herbal medicines. Since the results obtained for the Cu concentration in T. cordata flowers from all the studied locations do not exceed the permissible standards for flowers (>20 mg∙kg⁻¹) [7], they can be recommended as a source of Cu supporting health.

Lead (Pb) accumulation is affected by anthropogenic activity. Pb has no biological use in plants. It can change the morphology of the plant and adversely affect morphological, physiological, and biochemical processes [60,103]. Pb is adsorbed by roots and accumulates there. Only a small amount Pb is transported to the aboveground parts of plants [103]. The lead content in different plant organs is as follows: roots > leaves > stem > inflorescence > seeds [104]. In our study the contents of Pb in flowers ranged from 2.2 (location C) to 4.0 mg·kg⁻¹ (location A). The lowest variability in flowers collected from six locations was found in relation to Pb in flower content (CV = 17.5%) (Table 6). The Pb content in flowers of T. cordata plants correlated significantly (r = 0.863) with TOC_s (Table 10 and Table S1). The content of Pb and other metals in medicinal plants was also studied by Hung et al. [105] in Stevia rebaudiana plants (herbal sweetener). The authors obtained Pb concentrations in flowers at the level of 1.1 mg·kg⁻¹ d.m. In turn, Tomaszewska-Sowa et al. [76] determined the content of this metal in plants with medicinal properties such as common yarrow Achillea millefolium L., sand everlasting Helichrysum arenarium L., and stinging nettle Urtica dioica L. in the range from 3.8 to 15.7 mg·kg⁻¹ d.m. The highest content was found in the inflorescences of the everlasting H. arenarium, which amounted to 15.7 mg Pb·kg⁻¹ d.m. The legislation regulating the content of heavy metals in plant materials worldwide is guided by the requirements of the World Health Organization [71]. The WHO proposes a limit of 10 mg Pb·kg⁻¹ for lead in dried herbs. In turn, the regulations of the Polish Pharmacopoeia are more restrictive and allow Pb concentrations even below 5.0 mg·kg⁻¹ d.m. in herbs [72]. The obtained results of our analyses indicate that the T. cordata flowers collected from all the tested sites are safe for use in phytotherapy because the Pb content is lower than 10 mg Pb·kg⁻¹ d.m.

Values of the bioconcentration factor (BCF_f) of Zn, Cu, Mn, and Fe in T. cordata flowers are presented in

Table 7. Values of bioconcentration factor (BCF_f) metals (Zn, Cu, Mn, Fe) for flowers T. cordata.

Sampling Location *	BCFf
	Zn	Cu	Mn	Fe	Pb
A	0.14	0.26	0.06	0.15	0.28
	medium	medium	weak	medium	medium
B	0.51	1.41	0.16	0.15	0.28
	medium	intensive	medium	medium	medium
C	1.90	2.01	0.21	0.16	0.20
	intensive	intensive	medium	medium	medium
D	0.40	1.23	0.74	1.18	0.23
	medium	intensive	medium	intensive	medium
E	0.70	5.45	0.74	0.12	0.29
	medium	intensive	medium	medium	medium
F	0.79	2.28	0.46	0.22	0.29
	medium	intensive	medium	medium	medium

* Sampling location explanations as in Table 1; BCF_f—values of bioconcentration factor Zn, Cu, Mn, Fe, and Pb for flowers T. cordata.

. The BCF_f indicate that T. cordata flowers accumulated Cu and Zn to the greatest extent. Flowers were characterized by BCF_f value for Cu and Zn ranging from 0.26 (location A) to 5.45 (location E) and from 0.14 (location A) to 1.90 (location C), respectively. The BCF_f values for Fe indicate medium and intensive bioconcentration values ranging from 0.12 (location E) to 1.18 (location D). The bioconcentration of Pb in the studied sites was considered medium ranging from 0.20 (location C) to 0.29 (location E and F). The high coefficients for Zn and Cu are due to the fact that they are considered the most accessible trace elements [106,107]. High values of the bioconcentration factor of Zn in the inflorescences (BCF_f) of common yarrow Achillea millefolium L. and sand everlasting Helichrysum arenarium L. were obtained by Tomaszewska-Sowa et al. [76]. The authors noted average values of the BCF_f of Pb in the inflorescences of H. arenarium.

3.3. Enzyme Activity

The results of the activity of the enzymes studied are presented in

Table 8. Enzyme activity in topsoil (0–25 cm).

Sampling Location *	DHA **	FDA	GL	AR
	µg TPF·g−1·h−1	µg F·g−1·h−1	µg pNP·g−1·h−1	µg pNP·g−1·h−1
A	47.86 ± 7.26	35.78 ± 0.69	2.466 ± 0.03	0.030 ± 0.030
B	84.71 ± 17.64	59.76 ± 0.77	3.166 ± 0.12	0.062 ± 0.004
C	47.03 ± 4.20	38.07 ± 0.26	2.816 ± 0.27	0.035 ± 0.016
D	239.6 ± 17.94	42.10 ± 0.15	4.143 ± 0.24	0.051 ± 0.007
E	133.4 ± 16.53	43.70 ± 0.65	2.307 ± 0.02	0.018 ± 0.014
F	10.56 ± 2.70	48.30 ± 0.04	2.194 ± 0.07	0.124 ± 0.024
Mean for location	82.61	8.62	0.727	0.038
SD	93.85	44.62	2.849	0.054
CV (%)	88.02	19.31	25.53	70.91

* Sampling location explanations as in Table 1; ** results are the mean of 3 replicates ± SD (standard deviation); DHA—dehydrogenase activity; FDA—fluorescein diacetate hydrolysis activity; GL—β-glucosydase activity; AR—arylsulphatase activity.

. The greatest activity diversification among the enzymes analyzed was shown by dehydrogenases (the highest value of SD and CV). They are considered an indicator of the general microbiological activity of the soil, because they occur intracellularly in all living cells of microorganisms and do not accumulate outside the cell in the soil [108]. Its highest activity was determined at 239.6 µg TPF·g⁻¹·h⁻¹ in soil samples collected from Węgorzyn by the road (location D). The activity of this enzyme in these samples was about 96 times higher than in the soil collected from Olimpin (location F). The high activity of this enzyme indicates the high activity of microbial biomass in the soil. The positive correlation (r = 0.859) of dehydrogenase activity with the clay fraction of the soil (<0.002 mm), which affects many soil properties, including its structure and water capacity, may confirm a strong dependence on soil moisture. Dehydrogenase activity depends on soil moisture, which results from the fact that the metabolism and survival of soil microorganisms are also strongly dependent on water availability [109]. Similarly high activity and dependence on the clay fraction content of the soil were found for β-glucosidase activity. The activity of this hydrolase ranged from 2.194 µg pNP·g⁻¹·h⁻¹ (location F) to 4.143 µg pNP·g⁻¹·h⁻¹ (location D). This hydrolase is involved in the degradation of soil organic matter by participating in the enzymatic degradation of cellulose, the main component of plant polysaccharides. It is considered to be mostly extracellular. The strong association with the clay fraction of the soil may indicate that the activity of extracellular immobilized enzymes is of little importance. This is in agreement with the results of Datt and Singh [110], who stated that cellobiose degradation in soil was due to enzymes released from proliferating organisms and not to the accumulated enzyme fraction. Other studies [111] explain the relationship between β-glucosidase activity and clay content by the potential of enzyme immobilization in the soil and therefore the possibility of extracellular immobilized enzyme activity. In the case soil studied, similar activity of intracellular enzymes such as dehydrogenases, however, supports the first theory. High activity of dehydrogenases and β-glucosidase by proliferating microorganisms is induced by the existence of anaerobic conditions [112] and the availability of cellulose degradation products [113]. The highest FDA activity of 59.76 µg F·g⁻¹·h⁻¹ was determined in Łochowo by the road (location B). At this point, a fairly high activity of β-glucosidase (3.166 µg pNP·g⁻¹·h⁻¹) and arylsulfatase (0.062 µg pNP·g⁻¹·h⁻¹) was also determined. Enzymes responsible for the hydrolysis of FDA occur in large quantities in the soil environment. It is not a single enzyme but a series of nonspecific esterases, proteases, and lipases that hydrolyze FDA and participate in the decomposition of many types of tissues. The ability to hydrolyze FDA thus seems widespread, especially among the major decomposers, bacteria and fungi [114]. The high activity of these enzymes also suggests the existence of favorable conditions for the growth of microorganisms and the availability of substrates containing carbon and sulfur. However, the influence of organic and inorganic contaminants prevents the derivation of unequivocal relationships.

The Ward’s method was used in the cluster analysis. The data were grouped using Euclidean distance. In samples from locations C, E, and F, a similar content of metal and enzymes in soils and the concentration of metals in the dry matter of flowers was found (Figure 2) compared to the remaining samples. The samples from location D were different than the other by these properties. In order to identify environmental variables that affect the content of heavy metals in flowers, PCA was used. It is helpful in identifying variables that can be discarded to remove duplicate and difficult-to-measure information.

Principal component analysis (PCA) was performed to determine the mutual relationships between the studied parameters in different places of collecting flowers of T. cordata and soil samples. It showed general patterns in the relationships between variables, allowing the description and classification of the studied objects. Scatter plots of loadings for PC1 and PC2 illustrate which variables are important in flower and soil features (Figure 3A) and a plot of the distribution of the studied plant and soil sampling points in the arrangement of the first two components (Figure 3B).

The analysis showed that the two main hypothesized causes of variability (PC1, PC2) together accounted for 71.69% of this variation. The first principal component provides 46.74% of the information about the flower and soil properties contained in the input variables. Most of the variances included in the first component (PC1) correlated negatively with soil properties with TOC (−0.919), EC (−0.976), Zn_t (−0.900), Cu_t (−0.929), Mn_t (−0.968), Fe_t (−0.740), Pb_t (−0.865), Zn_a (−0.846), Cu_a (−0.882), Mn_a (−0.924), and metal content in flowers: Pb_f (−0.800) and positively with Cu_f (0.880). The second principal component (PC2) accounted for 24.46% of the data variance and correlated negatively with the sand fraction of soil (2–0.05 mm) (−0.777), silt fraction of soil (0.05–0.002) (−0.700), clay fraction of soil (<0.002) (−0.868), DHA (−0.899), and GL (−0.921) (

Table 9. Values of the two extracted factor loadings.

Component	PC1	PC2
sand fraction	0.34392	0.776781
silt fraction	−0.28147	−0.700461
clay fraction	−0.46887	−0.867700
TOC *	−0.91872 **	0.071594
EC	−0.97647	0.180229
Znt	−0.90019	0.377934
Cut	−0.92945	0.172712
Mnt	−0.96752	0.059829
Fet	−0.74030	−0.570136
Pbt	−0.86458	−0.269903
Cuf	0.88000	−0.428250
Pbf	−0.80075	0.086439
DHA	−0.17054	−0.899274
GL	−0.21352	−0.920609
Zna	−0.84578	0.521446
Cua	−0.88175	0.338314
Mna	−0.92369	0.254936

* TOC—total organic carbon in soil; EC—electrical conductivity in soil; Zn_t—total Zn in soil; Cu_t—total Cu in soil; Mn_t—total Mn in soil; Fe_t—total Fe in soil, Pb_t—total Pb in soil; Cu_f—total Cu in flowers of T. cordata; Pb_f—total Pb in flowers of T. cordata; DHA—dehydrogenase activity; GL—β-glucosydase activity; Zn_a—available Zn in soil; Cu_a—available Cu in soil; Mn_a—available Mn in soil; ** The bold highlights which components belong to a given principal component.

, Figure 3). Asitok et al. [115], in their research investigating the impact of cement dust pollution on the fertility parameters of an agricultural soil, evaluated the fertility parameters of soils, in particular, soil organic carbon (SOC), total nitrogen, pH, and enzyme activity: dehydrogenase, acid phosphatase, alkalinephosphatase, and β-glucosidase activity. The results have indicated the role of soil enzymes as the parameters of fertility.

Soil dehydrogenases were characterized by the greatest diversity of activity. The highest activity of enzymes in the soil was demonstrated in Węgorzyn by the road places with medium traffic intensity where the highest activity of dehydrogenases and β-glucosidase was determined. High activity was also determined in the location by the road in Łochowo where high activity of FDA and GL and AR was determined.

Table 10. Correlation coefficient (R) for selected properties of soils and T. cordata flowers (p ≤ 0.05).

Dependent (y)	Independent (x)	R
EC *	TOC	0.918
Znt	TOC	0.937
Cut	TOC	0.973
Mnt	TOC	0.953
Pbt	TOC	0.864
Pbf	TOC	0.863
Pbt	Cut	0.846
Znt	Cut	0.873
Znt	Mnt	0.931
Cut	Cuf	0.909
Zna	TOC	0.852
Zna	Znt	0.980
Cua	Cut	0.963
clay fraction	Fet	0.869
clay fraction	Fef	0.880
clay fraction	DHA	0.858
clay fraction	GL	0.884

* EC—electrical conductivity in soil; Zn_t—total Zn in soil; Cu_t—total Cu in soil; Mn_t—total Mn in soil; Fe_t—total Fe in soil; Pb_t—total Pb in soil; Pb_f—total Pb in flowers of T. cordata; Cu_f—total Cu in flowers of T. cordata; Fe_f—total Fe in flowers of T. cordata; Zn_a—available Zn in soil; Cu_a—available Cu in soil; DHA—dehydrogenase activity; GL—β-glucosydase activity; TOC—total organic carbon in soil.

Table 10. Correlation coefficient (R) for selected properties of soils and T. cordata flowers (p ≤ 0.05).

Dependent (y)	Independent (x)	R
EC *	TOC	0.918
Znt	TOC	0.937
Cut	TOC	0.973
Mnt	TOC	0.953
Pbt	TOC	0.864
Pbf	TOC	0.863
Pbt	Cut	0.846
Znt	Cut	0.873
Znt	Mnt	0.931
Cut	Cuf	0.909
Zna	TOC	0.852
Zna	Znt	0.980
Cua	Cut	0.963
clay fraction	Fet	0.869
clay fraction	Fef	0.880
clay fraction	DHA	0.858
clay fraction	GL	0.884

* EC—electrical conductivity in soil; Zn_t—total Zn in soil; Cu_t—total Cu in soil; Mn_t—total Mn in soil; Fe_t—total Fe in soil; Pb_t—total Pb in soil; Pb_f—total Pb in flowers of T. cordata; Cu_f—total Cu in flowers of T. cordata; Fe_f—total Fe in flowers of T. cordata; Zn_a—available Zn in soil; Cu_a—available Cu in soil; DHA—dehydrogenase activity; GL—β-glucosydase activity; TOC—total organic carbon in soil.

4. Conclusions

Small-leaved lime (T. cordata) is suitable for afforestation in rural and urban areas and for reclaiming former agricultural land. Its flowers are used in herbal medicine, making purity essential. The hypothesis that soils and flowers near heavy traffic areas are contaminated with heavy metals and exhibit reduced enzymatic activity was not confirmed. Metal concentrations (Zn, Cu, Mn, Fe, Pb) in soils from six sites were comparable to Poland’s geochemical background and did not exceed legal limits. Slightly elevated metals at some sites were linked to pedogenic factors and correlated with total organic carbon and clay content. Minor increases in metals may result from anthropogenic sources like dust and fumes. Sampling location affected soil enzyme activities including dehydrogenase (DHA), fluorescein diacetate hydrolysis (FDA), β-glucosidase (GL), and arylsulfatase (AR), which correlated more with pedogenic factors (TOC, clay fraction) than metal content. No elevated metal levels were found in T. cordata flowers; lead content complied with WHO guidelines, indicating the flowers are safe for phytotherapy. Enrichment Factor analysis showed significant Zn enrichment only at locality A (EF = 7.5), supported by a geoaccumulation index (I_geo = 1.8) indicating moderate soil pollution. Zn and Cu accumulation was noted only in the city center. Other sites had I_geo values below zero, indicating unpolluted soils. According to Polish environmental regulations, metal concentrations confirm that the studied areas remain unpolluted. It is recommended to continue monitoring trace element concentrations in soils and in T. cordata flowers, especially in urban areas. Long-term data will enable the detection of potential changes caused by industrial development and urbanization. The absence of elevated trace element concentrations in T. cordata flowers, along with compliance with WHO recommendations, confirms the safety of these flowers for use in phytotherapy. However, further research is warranted, particularly in new locations or environments with higher pollution levels. Despite the positive findings regarding enzymatic activity and soil purity, T. cordata remains a valuable species for land reclamation and afforestation.