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Article

Traffic-Related Heavy Metal Stress in the Medicinal Plant Plantago lanceolata L.

by
Agata Bartkowiak
1,* and
Joanna Lemanowicz
2
1
Department of Biogeochemistry, Soil Science and Irrigation and Drainage, Faculty of Agriculture and Biotechnology, Bydgoszcz University of Science and Technology, Bernardyńska 6 St., 85-029 Bydgoszcz, Poland
2
Division of Biochemistry, Faculty of Medicine, Bydgoszcz University of Science and Technology, Bernardyńska 6 St., 85-029 Bydgoszcz, Poland
*
Author to whom correspondence should be addressed.
Sustainability 2026, 18(9), 4561; https://doi.org/10.3390/su18094561
Submission received: 20 February 2026 / Revised: 27 April 2026 / Accepted: 30 April 2026 / Published: 5 May 2026
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Abstract

Ensuring the safety of sustainably managed medicinal plants is closely linked to the quality of plant raw materials, including the presence of heavy metals within safe limits. Sustainable management in the context of herbal raw materials therefore entails responsible management of herbal plant resources, integrating environmental protection with ensuring long-term economic profitability. The aim of this study was to analyze selected biochemical parameters and to determine metal concentrations in soils and leaves of Plantago lanceolata L. collected from natural habitats at increasing distances from traffic routes. The content of Zn, Cu, Ni, and Pb was determined in the soils and leaves of Plantago lanceolata L. Assessing the content of these elements in plant raw materials allows for: the prevention of harmful substances in final products, adaptation of raw materials to applicable safety standards (avoiding toxicity), and protection of consumer health. This promotes sustainable development by building a safe supply chain. The leaves of Plantago lanceolata L. were also tested for biochemical enzymatic (catalase (CAT) and superoxide dismutase (SOD)) and non-enzymatic (chlorophyll a and b (Chl a and b), carotenoids (Car), ascorbic acid (AAC)), and mechanisms regulating the activity of reactive oxygen species (ROS) were determined in the leaves of Plantago lanceolata L. Based on the results of leaf pH, relative water content (RWC), ascorbic acid content, and total chlorophyll content, the air pollution tolerance index (APTI) was calculated. The distance from the road has a significant impact on the concentration of the heavy metals analyzed. The soils were found to be free of Zn, Cu, Pb, and Ni contamination. However, analysis of Plantago lanceolata L. leaves revealed exceedances of acceptable lead limits for herbal plants. The content of pigments, the ratio of Chl a/b, and Chl (a + b)/Car in the leaves of Plantago lanceolata L. was significantly dependent on the distance from the road. The activity of CAT and SOD in the leaves of Plantago lanceolata L. growing closest to the road was significantly higher compared to the others. APTI values suggest that Plantago lanceolata L. exhibits sensitivity to pollution, independent of its distance from the emission source.

1. Introduction

Globalization and the development of economic activity in countries determine the level of demand for transport services. Road transport is dominant in European countries, which is primarily determined by the availability of road transport infrastructure and relatively low costs [1]. The increase in the density of the transport network and the intensity of road traffic leads to increased pressure from road transport on the quality of the environment, the extent of which depends on the transport technologies used, the quality and capacity of transport routes, and the technical solutions applied in vehicles [2,3,4,5].
Heavy metals are pollutants characterized by a very high potential risk to the environment. In addition to industrial and agricultural activities, road transport is a major source of environmental pollution with heavy metals such as Zn, Cu, Pb, Ni and Cd [2,6,7]. Vehicle-derived pollutants are considered more hazardous than industrial emissions because they disperse at high concentrations, at low altitudes, and in close proximity to humans. The heavy metals come from exhaust gases, car oil leaks, the wearing of tires and brake discs, and corrosion of metal vehicle parts. Regardless of the emission source and type, heavy metals accumulate on the soil surface, leading to their build-up in the organogenic horizon and subsequent absorption by plant roots [8]. Therefore, the accumulation of metals within the soil–plant system should be thoroughly investigated. Among these elements, both those essential for living organisms—micronutrients (e.g., Cu, Zn)—and non-essential elements, whose physiological functions have not yet been fully elucidated (e.g., Pb, Cd), can be distinguished. Zinc and copper are key micronutrients in plants, playing important roles in photosynthesis, plant development, and protein synthesis, as well as enhancing resistance to diseases and protecting against oxidative stress. The roles of lead and nickel in plants differ: nickel is an essential micronutrient, whereas lead is a toxic element. A common feature of all heavy metals is their toxicity (at high concentrations) to the biotic components of the environment. The availability and toxicity of heavy metals to plants depend primarily on the chemical forms in which these metals occur and on the prevailing soil conditions. Soil contamination with heavy metals is rarely visible; however, it is associated with serious, delayed effects from an ecotoxicological perspective.
Medicinal plants are widely used as home remedies and as raw materials for the pharmaceutical industry. Over the past decade, the use of herbal medicine has increased significantly. According to a report by the World Health Organization (WHO), approximately 80% of people are prone to using herbs as a first line of treatment for their illnesses. Medicinal plants are particularly important in developing countries because, in addition to herbal medicine, they are also used as dietary ingredients [9]. Although herbal medicine is officially recognized as beneficial to human health, there are few guidelines and regulations for its safe use. Therefore, special attention should be paid to plant species that are sources of herbal raw materials for humans. They are obtained from both natural and anthropogenic habitats. Very often, herbaceous plants grow along roadsides and are collected because of their easy access and transport. However, medicinal plants often show the ability to selectively accumulate toxic elements, and the concentration of heavy metals in many herbaceous plant species may exceed acceptable, safe concentrations [10,11,12]. Analyzing the heavy metal content of these plants has a direct and indirect impact on sustainable development in the context of medicinal plant sourcing and production. It influences, among other things, consumer health and environmental protection, risk management, and raw material quality and sourcing. Sustainable management, in the context of herbs, means above all responsible management of herbal resources, taking into account environmental protection as well as economic profitability [13]. Sustainable harvesting of herbs from natural resources requires caution due to their potential contamination with heavy metals, which can affect the health and condition of humans and animals [14]. Compared to synthetic drugs manufactured under strictly controlled industrial conditions, where heavy metal contamination is minimized within closed technological processes, the analysis of herbal plants provides an additional environmental function. In addition to ensuring the quality of the final product, it serves as a tool for assessing the impact of anthropogenic activity on the natural environment, emphasizing the role of phytotherapy in the context of sustainable healthcare systems.
Plantago lanceolata L. is a common plant growing along roadsides, widely distributed throughout the world. It belongs to the Plantaginaceae family, which is very often found in degraded habitats such as forest edges, roadsides, railway tracks, river banks, as well as grassy and sandy areas [15]. The family is widely distributed worldwide and comprises approximately 90 genera and 1900 species, occurring in temperate regions [16]. Its habitat is mainly associated with the presence of calcium sources. Plantago lanceolata L. can be classified as a wild edible plant [17]. Young leaves of plantain can be added to salads, soups, or as a sandwich filling. Older leaves can be bitter and are better suited for cooking. Plantago lanceolata L. also has numerous medicinal properties. It has anti-inflammatory, expectorant, and astringent effects, and also promotes wound healing. It is helpful for coughs, digestive problems, and even for relieving the symptoms of colds and flu [18,19,20,21]. Furthermore, due to its ability to accumulate heavy metals, Plantago lanceolata L. is considered a suitable species for phytoremediation applications [22].
Leaves are a good indicator for assessing air pollution. Abiotic stress leads to the production of reactive oxygen species (ROS). Increased ROS production (superoxide radical (O2•−), singlet oxygen (1O2), hydroxyl radical (•OH), and hydrogen peroxide (H2O2)) is one of the main responses to stress [23]. The primary damage caused by ROS is related to the inactivation of proteins, nucleic acids, and lipids. Maintaining plant cell homeostasis is important, which is why the antioxidant system includes enzymes, mainly superoxide dismutase (SOD) and catalase (CAT) [24]. Under the influence of negative anthropogenic factors, plants modulate the process of photosynthesis, thereby minimizing ROS production [25,26]. They develop various physiological and biochemical enzymatic and non-enzymatic mechanisms that help them adapt to stress factors [27,28]. Enzymatic systems include catalase, superoxide dismutase, and ascorbate peroxidase, which directly remove ROS. The first line of defense against the toxic effects of O2 is superoxide dismutase (EC 1.15.1.1). Another important mechanism in the regulation of ROS activity is the level of catalase activity (EC 1.11.1.6). This is an enzyme that catalyzes the degradation of H2O2 produced in cells exposed to stressors as part of the oxidative burst phenomenon. The decomposition of hydrogen peroxide proceeds through a disproportionation reaction to water and oxygen [29]. Non-enzymatic systems include vitamins and pigments (e.g., chlorophylls, carotenoids, ascorbate, flavonoids, plastochinol, glutathione, as well as pH and RWC (Relative Water Content) [25,30]. Based on selected biochemical properties of leaves (ascorbic acid, total chlorophyll content, pH and relative water content), Singh and Rao [31] developed the air pollution tolerance index (APTI). According to Enitan et al. [32], this index is a natural characteristic of plants and facilitates the determination of plant tolerance to pollution.
The aim of this study was to conduct a comparative analysis of selected biochemical parameters and to determine the concentration of metals in soils and Plantago lanceolata L. specimens collected from sites located at varying distances from roads. Plantago lanceolata L. was selected as the plant model due to its wide application and geographical range, which is not limited to Europe. The data obtained will enable an assessment of the species’ potential as a source of herbal raw material derived from natural habitats exposed to different levels of anthropogenic pollution. The study also attempted to assess the air pollution tolerance index to determine the tolerance of Plantago lanceolata L. to road pollution.

2. Materials and Methods

2.1. Study Area

The study area is a rural area with a typical layout: roadside green belts, drainage ditches, crop fields separated by field margins, and field woodlots. The area exhibits characteristics of a temperate, transitional, warm climate typical of central Poland. The average air temperature for the multi-annual period 1991–2010 for this area is 8.6 °C, with the lowest monthly temperature in January at −1.1 °C and the highest monthly average in July at 19.3 °C. The average annual atmospheric precipitation in this area during this multi-annual period is 503.9 mm. The highest average is recorded in July (86.3 mm), and the lowest in February (22.2 mm). The samples for testing consisted of soil and plants collected from natural habitats located at various distances from national road No. 25 (DK 25) in Lipinki (Kujawsko-Pomorskie Province, central Poland). This road connects Central Pomerania with the Wrocław agglomeration. The section under study has GP class parameters (commonly referred to as an expressway). The average number of vehicles passing through a given section of the road in a single day, calculated on an annual basis, is 8563.
The sampling sites were located directly next to the road (approx. 1 m from the edge of the roadway) and at distances of 10, 50, and 150 m from the road. Samples were also taken from a control site located approx. 500 m from the tested DK 25 pollutant emitter. There were 25 sampling sites designated, five at each distance from the road. At each designated sampling site, measuring 1 m2, five plants and five soil samples were collected from the root system. Every five samples collected from a single sampling point (1 m2) were combined into one representative sample (total of 25 soil and 25 plant samples).

2.2. Soil Analysis

The following parameters were determined in soil material dried and sieved through a 2 mm mesh sieve: granulometric composition using a Mastersizer MS 2000 particle analyzer (Malvern Instruments, Worcestershire, UK); pH in 1M KCl potentiometrically using a CPC-551 pH meter (Elmetron, Zabrze, Poland) [33]; organic carbon content (Corg) using the Tiurin method by wet oxidation at 180 °C with a mixture of potassium dichromate and sulfuric acid [34]; the total content of heavy metals (Zn, Cu, Ni, Pb) was determined by atomic absorption spectrometry (ASA) method using a Solaar S4 instrument (Thermo Elemental, Cambridge, UK) after mineralization in a mixture of HF + HClO4 acids according to the method of Crock and Severson [35] and bioavailable forms after extraction with 1M HCl (Rinkis method). Calibration was carried out using aqueous standard solutions, obtaining a linear relationship of the analytical signal over the tested concentration range. The sensitivity of the method was characterized by determining the limit of detection (LOD), which was 0.02 mg kg−1 for the tested metals. To verify the accuracy of the results, the analysis of the certified material Loam Soil No. ERM–CC141 as well as the so-called zero tests were made, which were exposed to the identical analytic procedure as the soil samples. Good compatibility between the certified and determined values was obtained.
Knowing the total heavy metal content and their bioavailable forms, the availability factor (AF) was calculated [36] according to the formula:
A F = C A C T × 100 %
where: CA—content of forms extracted with 1M HCl (mg kg−1); CT—content of total forms (mg kg−1).

2.3. Plant Analysis

2.3.1. Heavy Metal Content in Plantago lanceolata L. Leaves

The collected plants were washed with tap water and distilled water. The plants were dried, crushed using a grinder, and subjected to wet mineralization in a microwave oven in a mixture of HNO3 and H2O2. The concentrations of Zn, Cu, Pb, and Ni in the Plantago lanceolata L. leaves were determined using AAS with a Solaar S4 instrument. All metal concentrations in plant samples were expressed on a dry mass basis (d.m.).
Knowing the heavy metal content in the plant leaves and in the soil, the bioaccumulation factor (BCF) was calculated for each metal, which provides information on the transfer of the metal from the soil solution to the plant. The value of the factor was calculated according to the formula [37]:
B C F = C p l a n t C T
where: Cplant—heavy metal ion content in plant leaves (mg kg−1); CT—heavy metal ion concentration in soil (mg kg−1).

2.3.2. Biochemical Analysis of Plantago lanceolata L. Leaves

−
The content of chlorophylls a and b (Chl a and Chl b), carotenoids (Car) was determined according to Lichtenthaler [38] and Lichtenthaler and Buschmann [39]. Measurements were performed at wavelengths (λ max) of 645 nm for chlorophyll a content, 662 nm for chlorophyll b content, and 470 nm for carotenoid content. Based on the concentrations of chlorophyll a (Chl a) and chlorophyll b (Chl b), the Chl a/b ratio (an indicator of leaf physiological status) was calculated, along with the total chlorophyll content (Chl a + Chl b). Ascorbic acid (AAC) content was determined in accordance with PN A-04019:1998 [40] using a titration method in an acidic medium with a dye indicator, with the endpoint identified by the of a pink coloration. Antioxidant activity (AA) was assessed following the procedure described by Zeipina et al. [41].
−
Leaf pH was measured potentiometrically after homogenization of 5 g of fresh plant tissue in 10 mL of deionized water [42].
−
Relative water content (RWC in %) determined according to the method of Rai and Panda [42].
The Air Pollution Tolerance Index (APTI) was determined based on four biochemical parameters of Plantago lanceolata leaves, namely ascorbic acid content (AAC), total chlorophyll (Chl a + Chl b), leaf extract pH, and relative water content (RWC), following the method proposed by Prajapati and Tripathi [43]:
A P T I = A A C × [ ( C h l   a + C h l   b ) + P ] + R 10
where: AAC is the ascorbic acid content; (Chl a + Chl b) is the total chlorophyll content in leaves; P is the pH of the leaf extract; R is the relative water content.
Plants with APTI > 17 are resistant, 10 < APTI < 16 are moderately sensitive, and APTI < 10 are sensitive to air pollution [31].
−
Catalase (CAT) activity was determined following the method of Kar and Mishra [44], based on the quantification of purpurogallin formation measured spectrophotometrically at 420 nm.
−
Superoxide dismutase (SOD) activity was assayed according to Beauchamp and Fridovich [45]. This method is based on the enzyme’s ability to inhibit the photochemical reduction in nitro blue tetrazolium, with absorbance measured at 560 nm. One unit of SOD activity (U) is defined as the amount of enzyme required to cause 50% inhibition of the reduction reaction mediated by the superoxide anion (O2•−).
All chemical and biochemical determinations were performed in triplicate, and the results are expressed as arithmetic means.

2.4. Statistical Analysis

Statistical analyses were performed using Statistica 13 software (StatSoft Polska, Krakow, Poland). The effect of traffic-related pollution on selected biochemical and chemical parameters of soil and Plantago lanceolata L. was evaluated using one-way analysis of variance (ANOVA) under a completely randomized design. Results are presented as mean values with corresponding standard deviations (±SD). The normality of data distribution was assessed using the Shapiro–Wilk test. Post hoc comparisons between means were conducted using Duncan’s multiple range test. Pearson’s correlation coefficients among the analyzed variables were calculated with the PAST 4.13 software [46], and only statistically significant relationships were visualized in the form of a correlogram. Additionally, hierarchical cluster analysis (CA) was applied to classify sampling sites (control and locations at varying distances from the road). The Ward method [47] was used as the clustering algorithm to determine distances between groups.

3. Results and Discussion

3.1. Soil Parameters

The soils tested were characterized by a uniform granulometric composition and were all classified into one granulometric group—sandy loam [48]. The pH of the analyzed soils was slightly acidic in most samples and ranged from 6.18 to 6.40 (Table 1). This pH range is conducive to the growth of most plants, providing them with optimal nutrient availability. Only at one test point, 1 m from the road, was the pH alkaline (pH—7.30). The use of salt for de-icing roads in winter can lead to its accumulation in the soil, which affects the pH of the soil. The same point also had the lowest organic carbon content, at 7.45 g kg−1. At the other research points, the content ranged from 11.35 to 13.20 g kg−1.
The distance from the roadside had a significant impact on the content of the analyzed heavy metals. The highest total zinc (105.8 mg kg−1), copper (40.23 mg kg−1), nickel (6.92 mg kg−1), and lead (28.12 mg kg−1) contents were recorded closest to the roadside (Table 2). These concentrations were comparable to those obtained by Korzeniowska [49] in the soil along road No. 7 in the vicinity of Chyżne (southern Poland) and higher than those obtained by Kováčik et al. [50] for the vicinity of roads in Košice (Slovakia). The content of the analyzed heavy metals decreased with increasing distance from the road. The lowest amounts of the metals studied were recorded in the control sample and amounted to 29.66 mg kg−1 for Zn, 11.92 mg kg−1 for Cu, 3.69 mg kg−1 for Ni, and 6.25 mg kg−1 for Pb (Table 2). When assessing the metal content in soils based on the limit values for Polish soils specified in the Regulation of the Minister of Climate and Environment on the method of assessing land contamination of 13 November 2024 [51], it was found that they met the standards for soil groups I and II. The literature states that excessive accumulation of trace elements is limited to approximately 150 m on both sides of the roadway, and beyond this distance, it assumes values for uncontaminated areas [49,52,53,54]. Many environmental factors influence the ability of soil to adsorb heavy metals, but it is the colloidal components of the soil that mainly determine the amount of metals absorbed. In addition, factors such as soil pH, organic matter, ionic strength, and the amount of metals present in the soil also influence the extent of soil adsorption capacity [55,56,57,58,59]. Figure 1 shows the relationships between total heavy metal content and the soil properties studied.
The transport of chemical elements between soil and plants is part of the natural cycle of elements. However, the total concentration of elements in the soil cannot be considered an indicator of bioavailability. Determining the number of bioavailable metals allows for the assessment of a plant’s potential for bioaccumulation of these metals from the soil. In Poland, the assessment of plant-available forms of trace elements in agricultural soils is commonly based on a single extraction with 1 M HCl (the Rinkis method), which has been routinely used in agrochemical laboratories since the 1980s [60]. However, it should be noted that the 1 M HCl extraction represents a potentially mobile fraction, not the forms that are strictly available to plants. The content of forms extracted with the 1M HCl ranged from 3.36 to 10.20 mg kg−1 for Zn, from 1.02 to 4.13 mg kg−1 for Cu, from 0.72 to 2.37 mg kg−1 for Pb, and from 0.08 to 0.34 mg kg−1 for Ni. Significantly, the highest concentrations of the analyzed metals were recorded in samples taken 1 m from the road and the lowest in control samples (Table 3).
The mobility of nutrients depends on the granulometric composition, organic matter content, pH, and microbial activity [61,62]. Plants primarily absorb bioavailable active forms of elements [63]. Heavy metals accumulated in surface soil layers exhibit high chemical affinity for organic matter, which slows down their decomposition and reduces the bioavailability of these metals [59]. Correlation analysis showed significantly high negative correlations between the bioavailable forms of the analyzed metals and organic carbon (Figure 1). The calculated available factor (AF) for all heavy metals tested did not exceed 20% regardless of the sampling location (Figure 2) and did not pose a risk of metal accumulation in plants. Consequently, the use of 1 M HCl extraction in AF and soil–plant transfer analysis requires cautious interpretation. These indicators should be considered indicative of the potential mobility of metals rather than their actual bioavailability, thereby limiting direct inference regarding element uptake by plants. In the studies conducted, regardless of the soil sampling location, the highest AF values were found for zinc. This element belongs to the group of the most mobile heavy metals, and its mobility in the soil is closely related to its pH. In acidic soils, zinc is more soluble and available to plants, while in alkaline soils its mobility and availability decrease [62,64]. The results obtained confirm the literature data, according to which the solubility of zinc in soil increases with decreasing pH. This was proven by a correlation analysis, which showed a significant relationship between the bioavailable forms of zinc (r = −0.543, p < 0.05) and the availability factor (r = −0.510, p < 0.05) and soil pH (Figure 1).

3.2. Heavy Metals in Plants

The average concentrations of heavy metals in Plantago lanceolata L. leaves were as follows: Zn > Pb > Cu > Ni. The location where the plants were collected had a significant impact on their heavy metal content. The highest concentrations of Zn (86.23 mg kg−1), Pb (21.19 mg kg−1), Cu (15.98 mg kg−1), and Ni (4.41 mg kg−1) were recorded in plants growing in the immediate vicinity of the road (Table 4). This content decreased with increasing distance from the road, and the lowest contents of the tested metals were recorded at the control point.
Similar results for heavy metal content in Plantago lanceolata L. plant material were reported by Mazur et al. [54] and Drava et al. [65]. For most elements, there is insufficient data to determine the acceptable upper intake level, i.e., the maximum level of total chronic intake that will not pose a risk of adverse health effects in humans. The Scientific Committee on Food Scientific Panel on Dietetic Products, Nutrition and Allergies has established such a level only for Cu (from 1 mg/day for children to 5 mg/day for adults) and for Zn (from 7 to 25 mg/day) [66]. The WHO also does not provide a single set of heavy metal content limits for all herbs. According to WHO guidelines, the assessment of heavy metals in herbal materials typically includes elements such as Pb, Cd, As and Hg. Frequently cited permissible limits for herbal raw materials include 10 mg kg−1 for Pb and 0.3 mg kg−1 for Cd, although WHO emphasizes that specific limits should be adapted to national regulations and product types [67]. Therefore, considering the amount of Plantago lanceolata L. leaves used in food or herbal preparations, it can be concluded that none of the analyzed samples showed concentrations Zn, Cu and Ni that could pose a health risk. Conversely, the lead concentration of 21.19 mg kg−1 d. m. found in plants growing 1 m from the road and 15.19 mg kg−1 d.m. growing 10 m from the road exceeded the commonly accepted limits for herbal raw materials (10 mg kg−1 according to WHO guidelines). This indicates significant contamination and suggests an anthropogenic source of lead. Such levels of the lead in question may pose a threat to human health and disqualify the plant material from medicinal use. The literature indicates that lead can be taken up by plants both via the root pathway from the soil solution and via the foliar pathway through aboveground organs. Studies by Schreck et al. [68] demonstrated that fine Pb-containing particles can not only deposit on the leaf surface but also penetrate leaf tissues through the cuticle and stomata. The chemical composition of plants is dynamic and depends on many factors, primarily the plant species and the initial nutrient content of the soil and environmental conditions [63,69]. Correlation analysis confirmed the relationship between the content of the analyzed metals in the plant and soil (Figure 1).
One way to demonstrate a plant’s ability to accumulate heavy metals is the ratio of concentration in the plant and soil, also known as the bioaccumulation or bioconcentration factor (BCF). Our own research found that the bioaccumulation factor for Zn alone was greater than one (Figure 3). The value of the index increased with distance from the road (intensive accumulation) and reached its highest value of 1.27 at the control point. The BCF values obtained for the other metals showed moderate accumulation (0.1 < BCF < 1) [70]. For Pb, the highest value of the index was 0.75, for Ni 0.64, and for Cu 0.36. Drava et al. [65] also obtained maximum BCF values for Pb and Zn in their studies of Plantago lanceolata L. Kabata-Pendias [62] also reports similar bioaccumulation index values for herbaceous plants for Zn of about 1, and for Cu and Pb of 0.7.

3.3. Biochemical Parameters of Plantago lanceolata L. Leaves

Table 5 presents the results of photosynthetic pigment content. The lowest contents of (Chl a) and (Chl b) in Plantago lanceolata L. leaf samples were recorded at a site location 1 m from the road. These values were statistically significantly lower than those observed in the control site (Chl a 0.524 mg g˗1 FW and Chl b 0.185 mg g˗1 FW). It was 55% and 50% lower, respectively. No significant differences were found between chlorophylls in leaf samples from the 50 m and 150 m points. Chlorophylls are pigments that play a key role in photosynthetic activity. Variations in chlorophyll content are commonly associated with abiotic stress in plants, and their synthesis decreases under elevated stress conditions [71].
According to Dorgan [72], chlorophyll content decreases under the influence of air pollution. Research by Shrestha et al. [73] has shown that pollutants, once they enter the stomata, can cause damage to chloroplasts, reducing chlorophyll content.
Based on the contents of Chl a and Chl b, their total (Chl a + b) was calculated (Figure 4a). It was found that as the distance from the road increased, the value of Ch a + b increased significantly. No statistical difference in Ch a + b was found in leaves from locations 50 m and 150 m away. At a point 1 m away, the Ch a + b value was the lowest (0.328). This was 54% less than at the control point. The sum of chlorophyll a and b is an indicator of the total chlorophyll content in a plant. It provides information about the photosynthetic potential of the plant. Research by Lemanowicz and Bartkowiak [26] showed that Chl a + b content decreased significantly in the leaves of common nettle at lower N doses. The Chl a/b ratio in Plantago lanceolata L. leaf samples, depending on the distance from the road, is presented in Figure 4b. At a distance of 1 m, the Chl a/b value was significantly the lowest (2.57). No significant differences in Chl a/b were found in leaf samples taken from a distance of 10 m, 50 m, and the control site (C). It was found that the Chl (a + b)/Car ratio was significantly highest in Plantago lanceolata L. leaf samples collected at a distance of 50 m from the road, which is associated with a higher proportion of Chl (a + b) relative to Car (Figure 4c) [74].
The pH of extracts from Plantago lanceolata L. leaves ranged from 4.25 to 6.50 and was statistically significantly dependent on the road (Table 5). No significant differences were found between the pH of plants collected from locations 10 m, 50 m, and 150 m from the road. However, the lowest pH was obtained in Plantago lanceolata L. leaves from 1 m (4.25). Plants collected within the range of road impact showed an acidic reaction, while those at the control site were slightly acidic. According to Rai and Panda [42], pH serves as an indicator of plant’s sensitivity to air pollution. This effect is often associated with acidic pollutants, such as SO2 and NOX, which lower pH levels. In sensitive plants, the decrease is greater compared to tolerant plants. High pH helps convert six-carbon sugars into ascorbic acid, which participates in the removal of ROS [15].
The RWC of Plantago lanceolata L. collected at distances of 1 m and 10 m from the road was significantly lower than at the control location (C) and 150 m (Table 5). It was 40%, 29%, 17%, and 11% lower, respectively. A high RWC value promotes plant resistance to stress. The impact of pollution on the rate of transpiration in plant leaves can reduce the relative water content (RWC) in the leaves. Road dust settling on plants can absorb water from the surface of leaves or stems, which can lead to increased evaporation from the plant. The result is a decrease in RWC [75].
The highest AAC was found in the leaves of Plantago lanceolata L. at the control site, amounting to 0.621 mg g−1 FW, while the lowest content was found 1 m from the road, amounting to 0.418 mg g−1 FW (Table 6). A similar relationship was reported by Rai and Panda [42]. No statistical difference in AAC content was found in samples taken 1 m and 10 m apart, or between 50 m and 150 m. AAC is an antioxidant and plays a key role in plant tolerance to biotic and abiotic stress [76]. It scavenges O2•−, *OH, and 1O2, and can reduce H2O2 to H2O through the ascorbate peroxidase reaction [77].
According to Asif and Ma [75], higher ACC content indicates tolerance to pollution (e.g., SO2 and NO2). A significant effect of distance from the road on AA was observed (Table 6). Statistically, the lowest AA (36.17%) was recorded in Plantago lanceolata L. leaf samples collected 1 m from the road. This was 48% less than in the control samples (C) (69.12%). No statistically significant difference was observed between AA in leaves collected at 150 m and C, or between 10 m and 50 m. According to Lemanowicz and Bartkowiak [26], antioxidant (anti-radical) activity is the ability of plant substances to neutralize reactive molecules that damage cells.
Plants have developed an enzymatic antioxidant system to protect themselves from ROS. The enzyme that constitutes the first line of defense is SOD. It works by neutralizing ROS, especially superoxide anion (O2−), converting it into less harmful substances such as hydrogen peroxide (H2O2). CAT, on the other hand, removes H2O2 produced by superoxide dismutation rapidly and without consuming cellular energy [25]. Analysis of variance showed that the tested factor had a significant effect on SOD and CAT activity (Table 6). Statistical analysis indicated that the highest activities of both SOD (82.65 U g−1 FW) and CAT (5.32 mg H2O2 kg−1 h−1) were recorded in Plantago lanceolata L. leaves collected 1 m from the road, representing increases of 84% and 94%, respectively, compared to the control site (C). In the case of CAT, no significant differences were found between the samples from 10 m and 50 m and between 150 m and the control (C). The innate response of plants to counteract oxidative stress is the regulation of antioxidant enzyme activity [78].
The correlation analysis showed a positive and significant correlation between the total and available content of Zn, Cu, Ni, and Pb in the soil and the content of the tested plant pigments (Figure 1). Cu and Zn in low concentrations are essential for plants because they participate in a number of biochemical reactions [79]. Studies by Houri et al. [80] showed that excessive accumulation of heavy metals leads to damage to the photosynthesis system, resulting in an increase in chlorophyll content as a means of adaptation to stress. Ahmad et al. [81] showed that bioaccumulation, translocation, and biological concentration factors reduce the content of Chl a and b, and Car as a defense or survival mechanism against pollution. These results were confirmed by our research. According to Nigam et al. [82], the content of chlorophyll, which is an indicator of the photosynthesis process, can provide information about soil quality.
The activity of antioxidant enzymes CAT and SOD was positively correlated with both total and available heavy metal content in the soil, with correlation coefficients ranging from r = 0.734 to r = 0.915 for CAT and r = 0.775 to r = 0.974 for SOD (Figure 1). Nigam et al. [82] reported that stress caused by high Pb and Cd content caused a significant increase in SOD and POD activity. However, activity increased at low Pb and Cd concentrations.
The BCF Cu, BCF Ni, and BCF Pb indices reduced the accumulation of plant pigments in the leaves of Plantago lanceolata L., as evidenced by negative correlation values (Figure 1).
The APTI values calculated for Plantago lanceolata L. at various distances from the road are shown in Figure 5. The APTI range was between 2.09 (10 m) and 5.14 (C). The order of APTI values was determined as: C > 150 m > 50 m > 10 m > 1 m, although no statistically significant difference was found between 50 m and 150 m. ATPI values indicate the sensitivity of Plantago lanceolata L. to pollution regardless of the distance from the emitter. According to Yadav et al. [83], the APTI value indicates the susceptibility of plants to vehicle emissions, which are the main factor contributing to air pollution. Similar research results were obtained by Lemanowicz and Jaskulska [84]. Correlation analysis showed a positive relationship between the ATPI value and the content of Chl a (r = 0.946) and Car (r = 0.958). An increase in chlorophyll content in air-polluted areas is a way to combat oxidative stress generated by pollution. Variations in chlorophyll content can be used as indicators of atmospheric pollution Houri et al. [80].
Using Ward’s clustering method [47], based on soil properties and Plantago lanceolata L. parameters, similarities among all sampling sites were identified (Figure 6). CA generated a dendrogram grouping all sampling sites into two statistically significant clusters. Within cluster 1, three sampling sites located away from the road, which was the source of pollution, were identified: control, 100 m, and 50 m, while within cluster 2, two sites located closest to the road were identified: 1 m and 10 m.

4. Conclusions

Ensuring food safety through the use of plant raw materials related to their quality, including the presence of heavy metals within safe limits, is one of the elements of sustainable management in herb harvesting.
The study showed that soils exposed to traffic pollution were not contaminated with heavy metals. However, Pb levels in the leaves of Plantago lanceolata L. exceeded permissible limits. Lead can enter plants not only through the root system but also through extra-root pathways—through leaves, where it settles along with air pollutants. Therefore, medicinal herbs should be grown and harvested outside the reach of pollutant emitters and only in controlled (unpolluted) areas.
It has been shown that traffic pollution affected changes in the content of Chl a, Chl b Car and the ratio of Chl a/b, Chl (a + b)/Car in the leaves of Plantago lanceolata L. growing at different distances from the road. In the leaves of Plantago lanceolata L. growing closest to the road, the activity of catalase and superoxide dismutase, antioxidant enzymes, was higher compared to the others. This indicates increased oxidative stress in plants growing in direct exposure to traffic. Plantago lanceolata L. demonstrates high sensitivity to traffic-related air pollution, regardless of the distance from the emission source.

Author Contributions

Conceptualization, A.B. and J.L.; methodology, AB. and J.L.; software, A.B. and J.L.; validation, A.B. and J.L.; formal analysis, A.B. and J.L.; investigation, A.B. and J.L.; resources, A.B. and J.L.; data curation, A.B. and J.L.; writing—original draft preparation, A.B. and J.L.; writing—review and editing, A.B. and J.L.; visualization, A.B. and J.L.; supervision, A.B. and J.L.; project administration, A.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Bydgoszcz University of Science and Technology under Grant BN-WRiB 2/2022.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Correlogram of the soil and Plantago lanceolata L. leaves parameters.
Figure 1. Correlogram of the soil and Plantago lanceolata L. leaves parameters.
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Figure 2. Available Factor.
Figure 2. Available Factor.
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Figure 3. Bioaccumulation Factor.
Figure 3. Bioaccumulation Factor.
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Figure 4. Variation in total chlorophyll content (a + b) (a), chlorophyll a/b ratio (b), and chlorophyll (a + b)/carotenoids ratio (c) in relation to distance from road traffic. Different lowercase letters indicate significant differences between distances from traffic at p < 0.05. a, b, c —different small letters indicate a comparison between distance from the road.
Figure 4. Variation in total chlorophyll content (a + b) (a), chlorophyll a/b ratio (b), and chlorophyll (a + b)/carotenoids ratio (c) in relation to distance from road traffic. Different lowercase letters indicate significant differences between distances from traffic at p < 0.05. a, b, c —different small letters indicate a comparison between distance from the road.
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Figure 5. APTI (air pollution tolerance index) value in Plantago lanceolata L. leaves depending on distance from the road. a, b, c —different small letters indicate a comparison between distance from the road.
Figure 5. APTI (air pollution tolerance index) value in Plantago lanceolata L. leaves depending on distance from the road. a, b, c —different small letters indicate a comparison between distance from the road.
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Figure 6. Cluster analysis.
Figure 6. Cluster analysis.
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Table 1. Soil characteristics in the studied area.
Table 1. Soil characteristics in the studied area.
SitesSandSiltClaypH 1M KClCorg
%g kg−1
C *55.40 ± 1.4439.87 ± 1.354.73 ± 0.216.37 b * ± 0.2413.20 a ± 0.08
1 m64.93 ± 2.3530.16 ± 1.124.91 ± 0.337.30 a ± 0.507.45 c ± 0.23
10 m58.48 ± 1.2636.14 ± 0.795.39 ± 1.126.18 b ± 0.1211.35 b ± 0.26
50 m56.42 ±1.3338.51 ± 0.665.07 ± 1.106.40 b ± 0.1611.92 ab ± 0.32
150 m56.72 ± 1.0238.68 ± 0.604.60 ± 0.606.31 b ± 0.1012.40 a ± 0.31
* C—control site; a, b, c—different small letters indicate a comparison between distance from the road.
Table 2. Total content forms of Zn, Cu, Ni and Pb in soil.
Table 2. Total content forms of Zn, Cu, Ni and Pb in soil.
SiteZnCuNiPb
mg kg−1
C *29.66 c ± 1.1811.92 d ± 0.143.69 c ± 0.446.25 d ± 1.01
1 m105.83 a ± 5.4640.23 a ± 3.246.92 a ± 2.1028.12 a ± 3.10
10 m65.56 b ± 3.3131.72 b ± 1.335.29 b ± 1.9521.40 b ± 1.20
50 m41.72 c ± 2.9219.33 c ± 2.163.88 c ± 0.2110.66 c ± 1.65
150 m30.99 c ± 5.1612.31 d ± 0.124.01 c ± 0.359.11 c ± 0.70
* C—control site; a, b, c, d—different small letters indicate a comparison between distance from the road.
Table 3. HCl-extractable forms of heavy metals in the soil samples.
Table 3. HCl-extractable forms of heavy metals in the soil samples.
SiteZnCuNiPb
mg kg−1
C *3.36 c * ± 1.261.02 d ± 0.300.08 d ± 0.050.66 c ± 0.40
1 m10.20 a ± 3.504.13 a ± 0.650.34 a ± 0.212.37 a ± 0.91
10 m7.98 b ± 1.303.91 ab ± 0.450.29 b ± 0.111.64 a ± 0.50
50 m4.10 c ± 1.053.02 b ± 0.500.16 c ± 0.091.11 b ± 0.33
150 m3.92 c ± 1.201.52 c ± 0.220.12 d ± 0.050.72 c ± 0.31
* C—control site; a, b, c, d—different small letters indicate a comparison between distance from the road.
Table 4. The content of heavy metals in plants.
Table 4. The content of heavy metals in plants.
SiteZnCuNiPb
mg kg−1 d.m.
C *37.70 d * ± 1.623.10 d ± 0.611.13 d ± 0.412.38 d ± 0.17
1 m86.23 a ± 4.3715.98 a ± 2.854.41 a ± 1.4521.19 a ± 1.55
10 m66.22 b ± 2.1111.05 b ± 1.662.73 b ± 1.4015.19 b ± 0.55
50 m50.88 c ± 1.716.23 c ± 1.312.16 bc ± 0.956.99 c ± 0.50
150 m38.69 d ± 1.443.12 d ± 3.711.19 d ± 0.652.33 d ± 0.20
* C—control site; a, b, c, d—different small letters indicate a comparison between distance from the road. d.m.—dry mass.
Table 5. Content of chlorophyll a, b, carotenoids, pH and RWC in Plantago lanceolata L. leaves.
Table 5. Content of chlorophyll a, b, carotenoids, pH and RWC in Plantago lanceolata L. leaves.
SitesChl aChl bCarpHpRWC
mg g˗1 FW%
C *0.524 a * ± 0.0110.185 a ± 0.0080.596 a ± 0.0126.50 a ± 0.02575 a ± 0.85
1 m0.236 d ± 0.0030.092 d ± 0.0060.268 c ± 0.0084.25 c ± 0.02145 c ± 0.56
10 m0.357 c ± 0.0040.125 c ± 0.0020.386 bc ± 0.0064.65 bc ± 0.01853 b ± 0.71
50 m0.406 b ± 0.0090.141 b ± 0.0030.422 b ± 0.0074.95 b ± 0.01662 ab ± 0.44
150 m0.411 b ± 0.0090.149 b ± 0.0020.438 b ± 0.0075.50 b ± 0.02267 a ± 0.63
* C—control site; a, b, c, d—different small letters indicate a comparison between distance from the road; Chl a—chlorophyll a; Chl b—chlorophyll b; Car—carotenoids; RWC—relative water content.
Table 6. The content of ascorbic acid and antioxidant, catalase and superoxide dismutase activity in Plantago lanceolata L. leaves.
Table 6. The content of ascorbic acid and antioxidant, catalase and superoxide dismutase activity in Plantago lanceolata L. leaves.
SitesAACAACATSOD
mg g˗1 FW%mg H2O2 kg−1 h−1U g−1 FW
C *0.621 a * ± 0.07169.12 a ± 7.122.89 c ± 0.12542.68 d ± 3.12
1 m0.418 c ± 0.05236.17 c ± 4.235.32 a ± 0.52382.65 a ± 4.58
10 m0.429 c ± 0.04243.55 b ± 4.654.23 b ± 0.48972.66 b ± 5.61
50 m0.502 b ± 0.06652.31 b ± 6.114.59 b ± 0.50169.23 b ± 5.07
150 m0.511 b ± 0.06164.56 a ± 7.522.54 c ± 0.13853.62 c ± 4.35
* C—control site; a, b, c, d—different small letters indicate a comparison between distance from the road; AAC—ascorbic acid content; AA—antioxidant activity; CAT—catalase; SOD—superoxide dismutase.
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Bartkowiak, A.; Lemanowicz, J. Traffic-Related Heavy Metal Stress in the Medicinal Plant Plantago lanceolata L. Sustainability 2026, 18, 4561. https://doi.org/10.3390/su18094561

AMA Style

Bartkowiak A, Lemanowicz J. Traffic-Related Heavy Metal Stress in the Medicinal Plant Plantago lanceolata L. Sustainability. 2026; 18(9):4561. https://doi.org/10.3390/su18094561

Chicago/Turabian Style

Bartkowiak, Agata, and Joanna Lemanowicz. 2026. "Traffic-Related Heavy Metal Stress in the Medicinal Plant Plantago lanceolata L." Sustainability 18, no. 9: 4561. https://doi.org/10.3390/su18094561

APA Style

Bartkowiak, A., & Lemanowicz, J. (2026). Traffic-Related Heavy Metal Stress in the Medicinal Plant Plantago lanceolata L. Sustainability, 18(9), 4561. https://doi.org/10.3390/su18094561

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