Accumulation of Heavy Metals in Blueberry (Vaccinium myrtillus L.) and Dominant Mosses (Pleurozium schreberi (Willd. ex Brid.) Mitt.) as Bioindicators of the Expressway Influence on Forest Ecosystems

Abstract

The intensive use, development, and expansion of the road network is expanding the zones of direct impact of road transport on forest ecosystems. Issues related to the mobility of trace elements in forest ecosystems along motorways are very important due to the numerous environmental risks associated with the excessive accumulation of metals, the ability to migrate and accumulate in plants and animals, and the risk of transferring these elements to higher trophic levels. The aim of this article was therefore to determine the impact of road traffic on the basis of contents of trace metals Cd, Cr, Cu, Ni, Pb, and Zn and to describe the relationship of these contents in moss gametophytes and blueberry leaves taken in the vicinity of an existing and variously expanded expressway (S7, Poland, Europe). Analyses of transport impacts included the effects of distance and time of pollutant deposition and road transport on habitat and stand conditions. The highest contents of Cd, Cr, Cu, Ni, Pb, and Zn in moss tissues were found in fir stands and the contents were, respectively, 0.36 mg·kg⁻¹, 5.91 mg·kg⁻¹, 12.5 mg·kg⁻¹, 3.26 mg·kg⁻¹, 8.82 mg·kg⁻¹, and 55.28 mg·kg⁻¹. Mosses showed the best bioindication capacity of all of the studied ecosystem elements. The Pb, Zn, Cr, Cu, and Ni contents were particularly markedly elevated in moss tissues relative to non-anthropopressured areas and dependent on distance from the emitter (road). Blueberry proved to be a less useful bioindicator, as the contents of Cd, Cr, Cu, Ni, Pb, and Zn found were similar to the data from non-anthropopressured areas and were, respectively, 0.09 mg·kg⁻¹, 0.98 mg·kg⁻¹, 7.12 mg·kg⁻¹, 2.49 mg·kg⁻¹, 1.18 mg·kg⁻¹, and 15.91 mg·kg⁻¹ in fir stands and 0.04 mg·kg⁻¹, 0.47 mg·kg⁻¹, 6.63 mg·kg⁻¹, 1.65 mg·kg⁻¹, 0.72 mg·kg⁻¹, and 17.44 mg·kg⁻¹ in pine stands.

1. Introduction

The intensive use, development, and expansion of the road network are often associated with agrotechnical measures, which result in expanding the zones of direct impact of road transport on forest ecosystems. Emission pollution related to road use causes a polluted environment, such as dust, trace metals, and polycyclic aromatic hydrocarbons [1,2,3,4,5,6]. Issues related to trace element mobility in forest ecosystems adjacent to highways are critical in terms of the numerous environmental risks associated with excessive heavy metal accumulation, the ability to migrate and accumulate in plants and animals, and the risk of transfer of these elements to higher levels trophic [7,8,9,10,11,12,13,14].

The pollution problem is growing almost exponentially due to the continuous expansion of road networks [15,16,17,18,19]. Pollutants originating from roadways continuously enter adjacent ecosystems in the form of dry and wet deposition [20,21,22,23]. High traffic volumes result in elevated contents in the environment of heavy metals by wear and tear of tires, brake linings, and various vehicle components [22,23,24,25,26]. Exhaust fumes also hurt the environment in addition to the lubricants used in various engine components and unburned fuel residues [27,28,29,30,31,32].

Road traffic emissions include many metals that come from asphalt abrasion (Cd, Cr, Cu, Ni, Pb, Zn, and V) [25], battery corrosion and leakage (Hg, Ni, and Pb), emissions from fuel combustion (As, Cd, Cr, Cu, Hg, Mn, Ni, Pb, and Zn) [33], brake wear (Cu, Pb, and Zn) [34], leakage engine oil (Cd, Cr, Cu, Ni, and Zn), corrosion of galvanized structures (Cd, Cu, and Zn) [24], and tire wear (Cd, Co, Cr, Cu, Ni, Pb, and Zn) [26,29,31].

The assessment of pollution status and the impact of road transport on roadside areas is most often conducted based on monitoring and analysis of trace metal concentrations [34,35,36,37,38] including Cd, Cr, Cu, Pb, Ni, and Zn, as well as others that show elevated contents, such as Hg, As, Co, Sb, Se, Sr, and V. Of the elements listed, the most hazardous are As, Pb, Cd, and Hg [18,39,40,41].

Biomonitoring-based methods have been used to assess environmental status for quite a long time [42]. Biomonitoring methods use bioindicator species [20,21,22], among which trees are surveyed, as well as undergrowth plants, including mosses [43,44,45,46], and vascular plants such as bilberry (Vaccinium myrtillus L.) [47,48,49,50].

Mosses, as bioindicative species, are used to monitor pollution levels, including those from traffic and those associated with wet and dry deposition. Analyzing the prevalence of mosses in Europe, they were found to account for 7.2% of the total number of known plants species, hence the widespread use of mosses in biomonitoring studies [51,52]. These plants have several different characteristics that influence their potential for use in numerous environmental studies. These include, inter alia, that they have a wide distribution range and that the absence of cuticle and epidermis allows free penetration of metal ions [52,53,54,55].

Blueberries (Vaccinium myrtillus L.) are widely used in biomonitoring studies in areas subjected to anthropogenic pressures, as indicated by many studies [56,57,58,59]. The blueberry, as one of the few species, can exist in areas heavily contaminated by trace elements [60,61,62]. Trace element uptake in plants and accumulation in tissues depends on the plant species, as there are different strategies for trace element contents in plants [63]. As indicated in the literature [59], trace element contents in different blueberry organs can vary. Zinc in blueberries accumulates in higher amounts in shoots than in leaves and copper in higher amounts in leaves than in roots.

Mosses are indicators in the biomonitoring of metals and markings in the environment not only in Poland but also in various applications around the world, e.g., in Spain [64], Italy [65], Croatia [66], China [67], Mexico [68], and Serbia [69]. Blueberry is also available in environmental biomonitoring in various locations, e.g., in Finland [70], Russia [57], Slovakia [71], and Serbia [72], given applications of the plants used in environmental biomonitoring.

Therefore, this article aimed to determine the impact of vehicular traffic based on contents of trace metals, viz: Cd, Cr, Cu, Ni, Pb, and Zn, and to describe the interrelationship of these contents in mosses and blueberry leaves sampled near the existing and variously extended expressway (S7, Poland, Europe). Analyses of the impact of transport included the influence of distance and time of deposition of pollution and road transport against habitat and stand conditions. The studies were conducted to assess biosorption risks and threats to forest ecosystems.

2. Materials and Methods

2.1. Area of Study

The study sites (Figure 1) were located along the S7 expressway in the Swietokrzyskie Mountains region, in central Poland, and included habitats dominated by Scots pine and silver fir. The study area is characterized by a typical upland climate. The average air temperature ranges from 7 °C to 8 °C. The average annual temperature amplitude is 21 °C. The length of the growing season is 190–210 days. Precipitation in the Swietokrzyskie Mountains region is approximately 800 mm, while in lower areas it does not exceed 600 mm. The prevailing winds are west [73].

The studies were carried out to take into account the period of pollutant deposition. For this purpose, two categories of plots were distinguished:

• Stands “newly opened” to the impact of deposition—stands around strips cut for the construction of the S7 route, in use since 2009–2011 (5 plots; plots: no. 1–5);
• Stands under the influence of deposition for a long time—stands around strips cut for the construction of the S7 route in the 1970s (3 transects; plots: no. 6–8).

The study also took into account distance from the road, selected stand characteristics, soil and habitat characteristics, and location of study plots in relation to the road (microtopography).

2.2. Sampling Scheme

Sampling points (Figure 2) were located in the form of a wide transect running perpendicular to the road up to 110 m into the forest stand. On each transect, there were four sampling zones: -Zone I at 0–10 m from the road, -Zone II at 20–30 m from the road, -Zone III at 45–55 m from the road, and -Zone IV at 100–110 m from the road. There were a total of 8 study plots. In plots 1, 4, 5, and 8 there was a fir stand and in plots 2, 3, 6, and 7 there was a pine stand. In each plot, a total of 20 moss (Pleurozium schreberi (Willd. ex Brid.) Mitt.) samples and 15 blueberry (Vaccinium myrtillus L.) samples (5 samples from each study zone) were collected from designated points. This gives us a total of 160 moss samples and 120 blueberry samples. Samples collected in 2019 were collected in the summer.

The sampling scheme is presented in Figure 2.

Blueberry leaves were used for analyses; moss samples were devoid of the grasping and ground parts. The samples were not washed. The samples were then air-dried at room temperature. The next step was to grind all of the samples and prepare them for further analysis. In the plant samples, the determination of elemental contents Cd, Cu, Cr, Ni, Pb, and Zn were determined on an Thermo Scientific ICP OES ICAP 6000 Series apparatus after mineralization in concentrated nitric acid (HNO₃).

The term reference areas was used in the paper; it refers to areas that are not under anthropogenic influence in Poland and are treated in the literature as areas where natural concentrations of the trace elements in question occur, namely Puszcza Borecka Forest, Puszcza Knyszyńska Forest, Puszcza Białowieska Forest [74], or Puszcza Biała [75,76].

Three elements related to the structure of the forest edge zone, the age of the stand, and the position of the study plots in relation to the level of the road axis were taken into account in this study. According to Szymanski [77], he defines “a forest edge as comprising a 5–10 m wide forest fringe on both sides of an outermost row of trees and may be open or closed. A closed forest edge with a developed canopy and a forest edge part filled with tree crowns and undergrowth, reaching from the wall into the stand, at least for a distance equal to half the height of the old trees (10 m and more), protects the stand to a large extent from wind damage and contributes to the formation of a segregated atmosphere inside the stand”. In this study, this element was taken into account and, when analyzing trace element concentrations, it was checked whether the presence of a forest edge influenced the magnitude of element concentrations in the zones and plant components studied. Therefore, in the summaries prepared, ‘Yes’ was recorded in plots where the forest edge was present and ‘No’ in its absence. Another element investigated was the effect of plot position relative to the road axis level on the magnitude of trace element concentrations. Three designations were used for this purpose: “even”—surfaces located at the level of the road axis, “above”—surfaces located above the road axis, and “below”—surfaces located below the road axis. Another criterion analyzed was the age of the stand; three categories were each distinguished for pine stands (i.e., 53/57 years, 66 years, and 88 years) and fir stands (i.e., 65 years, 93/94 years, and 113 years).

2.3. Quality Control of the Obtained Measurement Results

Determinations of trace metals in each of the analyzed samples were carried out in three replications. Accuracy of the analytical methods was verified by determining the samples of certified reference materials and standard solutions: CRM023–050—Trace Metals—Sandy Loam 7 (RT Corporation Surrey, UK). The recovery within 80–120% of certified values was considered as acceptable.

2.4. Statistical Processing of Results

The significance of the differences between the mean values of the analyzed variables for the given deposition periods, the given distance zones from the road, and the selected characteristics of the stands and their locations (i.e., contents of trace elements accumulated in mosses and blueberry leaves) was checked using Tukey’s HSD test. The conformity of the distribution to the normal distribution was checked using the Shappiro–Wilk test. Statistica 10 software (StatSoft, Inc. 1984–2011 Tulsa, OK, USA) was used for the analyses.

2.5. Geoaccumulation Index—Igeo

The values of the Igeo index allowed the degree of heavy metal contamination of soils to be assessed on the basis of the ratio between trace metals (in the topsoil) and the heavy metal content of the parent rock or comparison with a specific geochemical background [78]. In the present study, the heavy metal content of the parent rock was considered as the background; data were obtained from the Atlas of Forest Soils of Poland [79].

$$I_{geo}={log}_2\left[{\displaystyle \frac{C}{1.5\times B_n}}\right]$$

(1)

where

• C—current content of heavy metals in the top soil layer
• Bn—heavy metal content of the parent rock or geochemical background
• 1.5—represents the background scattering due to lithogenic variations

The classification uses the Igeo classes introduced by [78], as follows:

• ≤0: Not to weakly polluted;
• 0–1: Weakly to moderately polluted;
• 1–2: Moderately polluted;
• 2–3: Moderately to strongly polluted;
• 3–4: Strongly polluted;
• 4–5: Strongly to excessively polluted;
• ≥5: Excessively polluted.

3. Results

It was observed that mosses collected in the undergrowth of pine and fir stands accumulated more trace metals, i.e., Cd, Cr, Cu, Ni, Pb, and Zn, than in reference areas not affected by anthropogenic factors (Figure 3 and Figure 4). Contents of trace elements in moss tissues were characterized by spatial variability related to distance from the road. It was found that the highest Cu, Pb, and Zn contents in mosses under fir stands were in the zone 0–10 m from the road. Elevated contents of Cd, Cu, and Zn were found throughout the study zone up to 110 m from the road in both fir and pine stands. Elevated Cr concentrations were found in mosses in the 0–10 m zone under pine stands and in the zone up to 55 m under fir stands. For Ni, higher concentrations are seen in the zone up to 30 m from the road under fir stands. However, in the case of Pb, exceedances were recorded in the zone up to 55 m under both fir and pine stands (Figure 3 and Figure 4). The time of deposition of pollutants counted from the time the road was made available did not affect the magnitude of trace element contents in mosses, with the only exception being Ni contents in mosses sampled in the undergrowth of fir stands (

Table 1. Content of trace elements and selected micronutrients in mosses in fir and pine stands as a function of pollutant deposition time (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Species
	Under Fir Stands		Under Pine Stands
	Pollution Deposition Period [Number of Years]
	9	36	9	36
Cd	0.36 ± 0.09 a	0.35 ± 0.22 a	0.28 ± 0.00 a	0.25 ± 0.04 a
Cr	7.21 ± 6.38 a	4.68 ± 2.99 a	3.69 ± 1.42 a	3.61 ± 1.00 a
Cu	11.25 ± 2.35 a	13.69 ± 4.45 a	10.27 ± 2.27 a	11.25 ± 6.37 a
Ni	3.93 ± 2.62 a	2.63 ± 1.26 b	1.84 ± 0.48 a	1.67 ± 0.37 a
Pb	10.06 ± 4.76 a	7.65 ± 3.66 a	7.61 ± 3.58 a	7.05 ± 2.16 a
Zn	52.85 ± 12.11 a	57.59 ± 16.84 a	58.58 ± 7.35 a	56.46 ± 6.15 a

^a,b—mean values with the same letter are not significantly different at p = 0.05.

).

The contents of all studied trace elements in blueberry leaves were within the range characteristics of non-anthropopressured areas, while no spatial variability of element concentrations was found. Higher contents of Cd, Cr, Ni, and Pb were found in blueberry leaves sampled in the undergrowth of fir stands (Figure 5, Figure 6 and Figure 7).

The time of pollutant deposition counted from the time the road became available and had no effect on trace element contents in mosses, the only exception being increased Ni contents in mosses sampled in the undergrowth of fir stands under nine years of pollutant deposition (Table 1).

However, it was found that the highest contents of Cr, Ni, and Pb in blueberry leaves occurred in fir plots under a shorter period of road pollution deposition (9 years) and were 1.1 mg·kg⁻¹, 2.9 mg·kg⁻¹, and 1.3 mg·kg⁻¹, respectively (

Table 2. Trace element contents in blueberry leaves in fir and pine stands as a function of pollutant deposition time (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Species
	Under Fir Stands		Under Pine Stands
	Pollution Deposition Period [Number of Years]
	9	36	9	36
Cd	0.10 ± 0.04 a	0.10 ± 0.04 a	0.04 ± 0.02 a	0.10 ± 0.02 b
Cr	1.10 ± 0.30 a	0.80 ± 0.14 b	0.40 ± 0.23 a	0.60 ± 0.23 a
Cu	7.30 ± 1.30 a	6.70 ± 0.86 a	6.40 ± 1.21 a	7.20 ± 2.51 a
Ni	2.90 ± 0.53 a	1.70 ± 0.41 b	1.60 ± 0.59 a	1.70 ± 0.58 a
Pb	1.30 ± 0.38 a	0.90 ± 0.16 b	0.60 ± 0.26 a	1.00 ± 0.51 b
Zn	16.10 ± 1.77 a	15.60 ± 1.83 a	16.30 ± 2.48 a	20.80 ± 3.59 b

^a,b—mean values with the same letter are not significantly different at p = 0.05.

). In contrast, in blueberry leaves collected under pine stands, the concentrations of Cd, Pb, and Zn were highest in plots under long traffic influence and were 0.1 mg·kg⁻¹, 1.0 mg·kg⁻¹, and 20.8 mg·kg⁻¹, respectively (Table 2).

The presence of an established forest edge had little effect on the magnitude of trace element contents in mosses, which was found for both mosses collected in the undergrowth of pine and fir stands. The exception concerned the content of Zn in mosses collected in the undergrowth of pine stands in plots with a developed forest edge, where the contents of this element were the highest (

Table 3. Contents [mg·kg⁻¹] of selected trace elements in mosses in relation to the existence of an educated stand edge (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Scots Pine		Silver Fir
	Presence of Forest Edge
	No	Yes	No	Yes
	n = 58	n = 20	n = 20	n = 58
Cd	0.28 ± 0.07 a	0.25 ± 0.04 a	0.41 ± 0.09 a	0.34 ± 0.19 a
Cr	3.69 ± 1.42 a	3.61 ± 10 a	5.86 ± 2.31 a	5.93 ± 5.74 a
Cu	10.27 ± 2.27 a	11.25 ± 6.37 a	12.14 ± 1.72 a	12.63 ± 4.26 a
Ni	1.84 ± 0.48 a	1.67 ± 0.37 a	3.50 ± 1.32 a	3.18 ± 2.35 a
Pb	7.61 ± 3.58 a	7.05 ± 2.16 a	11.74 ± 5.45 a	7.82 ± 3.45 b
Zn	51.58 ± 7.35 a	56.46 ± 6.15 b	56.03 ± 12.53 a	55.02 ± 15.63 a

^a,b—mean values with the same letter are not significantly different at p = 0.05.

). A similar relationship was also found for Pb contents in mosses, where the highest values were found on plots without a forest edge zone in the undergrowth of fir stands (Table 3).

Different results were obtained for trace element contents in blueberry leaves, where the forest edge clearly influences the magnitude of trace element contents. It was found that the highest levels of Cd, Pb, and Zn were in blueberry leaves with a developed forest edge in the undergrowth of pine stands. The situation was different for the content of Cd, Cr, Ni, and Pb in blueberry leaves taken from the undergrowth of fir stands, where the highest values were found in plots without a developed forest edge (

Table 4. Contents of selected trace elements in blueberry leaves in relation to existence of a developed stand edge (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Scots Pine		Silver Fir
	Presence of Forest Edge
	No	Yes	No	Yes
	n = 58	n = 20	n = 20	n = 58
Cd	0.04 ± 0.00 a	0.06 ± 0.02 b	0.12 ± 0.03 a	0.08 ± 0.04 b
Cr	0.44 ± 0.20 a	0.56 ± 0.23 a	1.11 ± 0.23 a	0.87 ± 0.24 b
Cu	6.44 ± 1.20 a	6.58 ± 0.87 a	7.56 ± 1.65 a	6.87 ± 0.76 a
Ni	1.61 ± 0.50 a	1.74 ± 0.58 a	2.94 ± 0.50 a	2.24 ± 0.75 b
Pb	0.60 ± 0.23 a	0.90 ± 0.20 b	1.46 ± 0.42 a	1.02 ± 0.22 b
Zn	16.30 ± 2.48 a	20.84 ± 3.59 b	16.29 ± 2.14 a	15.70 ± 1.57 a

^a,b—mean values with the same letter are not significantly different at p = 0.05.

).

It was found that the topography of the study plots influenced the content of elements in mosses as well as in blueberry leaves. It was shown that the highest contents of Cr, Pb, and Zn in mosses sampled in the undergrowth of pine stands were on plots located below the road axis. The exception was Cd, the highest contents of which were found in plots located above and below the road axis. The effect of plot position relative to the road axis on trace element content was less pronounced for mosses sampled from the undergrowth of fir stands. It was found that the highest contents of Cd and Pb were in plots above the road axis level and for Cu below the road axis level (

Table 5. Contents of selected trace elements in mosses in relation to microtopography (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Scots Pine			Silver Fir
	Position of Surface in Relation to the Road
	Equally	Above	Under	Equally	Above	Under
	n = 20	n = 18	n = 40		n = 18	n = 40
Cd	0.22 ± 0.06 a	0.27 ± 0.04 b	0.29 ± 0.06 b	Bd 1	0.40 ± 0.17 a	0.24 ± 0.09 b
Cr	2.97 ± 1.45 a	3.84 ± 1.19 ab	3.94 ± 1.20 b	Bd	6.26 ± 5.52 a	4.91 ± 3.38 a
Cu	11.06 ± 3.05 a	9.38 ± 0.87 a	10.76 ± 4.69 a	Bd	11.74 ± 3.45 a	14.70 ± 3.87 b
Ni	1.85 ± 0.53 a	1.83 ± 0.44 a	1.76 ± 0.43	Bd	3.40 ± 2.30 a	2.86 ± 1.49 a
Pb	5.53 ± 1.56 a	6.63 ± 1.63 a	8.81 ± 3.83 b	Bd	9.45 ± 4.76 a	7.01 ± 2.21 b
Zn	48.80 ± 7.99 a	51.42 ± 6.62 ab	55.48 ± 6.31 b	Bd	55.30 ± 15.91 a	55.22 ± 11.47 a

^a,b—mean values with the same letter are not significantly different at p = 0.05, ¹—no data.

and

Table 6. Contents of selected trace elements in blueberry leaves in relation to microtopography (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Scots Pine			Silver Fir
	Position of Surface in Relation to the Road
	Equally	Above	Under	Equally	Above	Under
	n = 20	n = 18	n = 40		n = 18	n = 40
Cd	0.04 ± 0.03 a	0.04 ± 0.02 a	0.05 ± 0.02 a	bd 1	0.09 ± 0.04	bd
Cr	0.18 ± 0.12 a	0.54 ± 0.18 b	0.58 ± 0.18 b	bd	0.95 ± 0.26	bd
Cu	7.57 ± 0.77 a	6.40 ± 1.01 b	5.94 ± 0.96 b	bd	7.12 ± 1.19	bd
Ni	2.13 ± 0.56 a	1.38 ± 0.49 b	1.54 ± 0.51 b	bd	2.49 ± 0.74	bd
Pb	0.38 ± 0.16 a	0.58 ± 0.16 b	0.86 ± 0.18 c	bd	1.18 ± 0.37	bd
Zn	14.63 ± 1.70 a	16.31 ± 2.18 a	19.40 ± 3.34 b	bd	15.91 ± 1.79	bd

^a,b,c—mean values with the same letter are not significantly different at p = 0.05, ¹—no data.

). Blueberry leaves occurring in the undergrowth of pine stands in plots below road level accumulated more Pb and Zn. In contrast, blueberries occurring in plots located at the road level accumulated more Cu and Ni. The highest Cr contents were found in plots above and below the road axis level (Table 6).

It was found that mosses taken from older pine stands showed the highest contents of Cd, Cr, Pb, and Zn. A similar trend was also found for Cd, Cr, Ni, and Pb contents in mosses sampled from older fir stands, where these values were highest (

Table 7. Contents of selected trace elements in mosses in relation to stand age (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Scots Pine			Silver Fir
	Stand Age
	53 + 57	66	88	65	93 + 94	113
	n = 40	n = 20	n = 20	n = 20	n = 40	n = 18
Cd	0.26 ± 0.04 a	0.22 ± 0.06 b	0.33 ± 0.06 c	0.24 ± 0.09 a	0.43 ± 0.17 b	0.31 ± 0.07 a
Cr	3.72 ± 1.09 ab	2.97 ± 1.45 a	4.27 ± 1.31 b	4.91 ± 3.38 a	5.16 ± 2.46 a	8.71 ± 8.84 b
Cu	10.31 ± 3.62 a	11.06 ± 3.05 a	10.28 ± 2.02 a	14.70 ± 3.87 a	12.42 ± 3.29 b	10.25 ± 2.59 b
Ni	1.75 ± 0.40 a	1.85 ± 0.53 a	1.85 ± 0.49	2.86 ± 1.49 a	2.95 ± 1.15 a	4.41 ± 3.54 b
Pb	6.84 ± 1.89 a	5.53 ± 1.56 a	10.57 ± 4.36 b	7.01 ± 2.21 a	10.01 ± 5.06 b	8.20 ± 3.01 ab
Zn	53.94 ± 6.38 a	48.80 ± 7.99 b	54.5 ± 6.48 a	55.22 ± 11.47 a	57.99 ± 16.74 a	49.31 ± 10.90 a

^a,b,c—mean values with the same letter are not significantly different at p = 0.05.

). In the case of the influence of stand age on the magnitude of trace metal contents in blueberry leaves, the trends occurring cannot be clearly identified, despite the statistical differences found. Only in the case of Cu and Ni concentrations were higher contents found in blueberry leaves growing in older pine stands (

Table 8. Contents of selected trace elements in blueberry leaves in relation to stand age (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Scots Pine			Silver Fir
	Stand Age
	53 + 57	66	88	65	93 + 94	113
	n = 40	n = 20	n = 20	n = 20	n = 40	n = 18
Cd	0.05 ± 0.02 a	0.04 ± 0.03 a	0.05 ± 0.01 a	bd	0.11 ± 0.04 a	0.07 ± 0.02 b
Cr	0.55 ± 0.20 a	0.18 ± 0.12 b	0.61 ± 0.11 a	bd	0.93 ± 0.19 a	1.03 ± 0.25 a
Cu	6.49 ± 0.94 a	7.57 ± 0.77 b	5.35 ± 0.60 c	bd	7.14 ± 1.26 a	7.06 ± 0.61 a
Ni	1.56 ± 0.53 a	2.13 ± 0.56 b	1.33 ± 0.34 a	bd	2.34 ± 0.45 a	2.86 ± 0.58 a
Pb	0.74 ± 0.18 a	0.38 ± 0.16 b	0.82 ± 0.16 a	bd	1.20 ± 0.29 a	1.13 ± 0.23 a
Zn	18.58 ± 2.88 a	14.63 ± 1.70 b	17.96 ± 2.39 a	bd	15.92 ± 1.98 a	15.89 ± 1.20 a

^a,b,c—mean values with the same letter are not significantly different at p = 0.05.

).

The highest contents of selected trace elements in moss tissues were found in fir stands. The contents of Cd, Cr, Cu, Ni, Pb, and Zn were, respectively, 0.36 mg·kg⁻¹, 5.91 mg·kg⁻¹, 12.5 mg·kg⁻¹, 3.26 mg·kg⁻¹, 8.82 mg·kg⁻¹, and 55.28 mg·kg⁻¹. The highest contents of Cd, Cr, Ni, Pb, and Zn in blueberry leaves were also found in samples collected in the undergrowth of fir stands (

Table 9. Content of trace elements and selected micronutrients in bilberry leaves and moss tissues in fir and pine stands (ANOVA, Tukey’s post hoc test, p = 0.05).

Element [mg·kg−1]	Mosses		Blueberry
	Fir Stands n = 78	Pine Stands n = 78	Fir Stands n = 42	Pine Stands n = 60
Cd	0.36 ± 0.17 a	0.27 ± 0.07 b	0.09 ± 0.04 a	0.04 ± 0.02 b
Cr	5.91 ± 5.07 a	3.67 ± 1.32 b	0.98 ± 0.31 a	0.47 ± 0.23 b
Cu	12.5 ± 3.77 a	10.52 ± 3.74 b	7.12 ± 1.19 a	6.63 ± 1.64 a
Ni	3.26 ± 2.13 a	1.80 ± 0.46 b	2.49 ± 0.74 a	1.65 ± 0.59 b
Pb	8.82 ± 4.37 a	7.47 ± 3.27 b	1.18 ± 0.37 a	0.72 ± 0.38 b
Zn	55.28 ±14.83 a	52.83 ± 7.34 b	15.91 ± 1.79 a	17.44 ± 3.40 b

^a,b—mean values with the same letter are not significantly different at p = 0.05.

).

On the basis of the results obtained, it can be assumed that the deposition of trace elements in moss tissues was characterized by the following regularity under both fir and pine stands: Zn > Cu > Pb > Cr > Ni > Cd. However, in the case of blueberry leaves, deposition showed the following regularity regardless of stand species composition: Zn > Cu > Ni > Pb > Cr > Cd (Table 9).

Analysis of the elemental geoaccumulation factor showed that cadmium contents in the topsoil layers (0–3 cm) ranged from moderately contaminated to highly contaminated. Higher values of the Igeo index were found in the zone 100–110 m from the road. For chromium, the highest index values were in the zone 100–110 m from the road and at the maximum ranges, which indicated severe contamination. Igeo index values for Cu ranged from uncontaminated to moderately contaminated soils. For Na in the zone 0–10 m from the road, the influence of road salinity in winter is evident and the values ranged from uncontaminated to heavily contaminated soils for the maximum values with a marked decrease in the index values at 45–55 m from the road. The value of the Igeo index for Pb indicates a clear influence of the surveyed road taking into account historical contamination; the values obtained are within the strong contamination range, while for the maximum ranges the values given indicate very strong contamination of the soils. The indicator values for zinc range from moderately contaminated to heavily contaminated. In the 0–10 m, 20–30 m, and 45–55 m zones, a decrease in the value of this indicator with distance from the road is evident (Figure 8).

The obtained Igeo index values for the 0–3 cm layer of soils indicate an anthropogenic origin of pollution, which is related to the existing S7 expressway.

4. Discussion

Analysis of the available literature allowed us to conclude that the obtained trace element contents in mosses also indicate pollution of forest ecosystems, by the impact of traffic and other associated emissions. This was found for all trace elements analyzed. Based on the available studies, the reference values of trace elements in mosses for Cd, Cr, Cu, Ni, Pb, and Zn were adopted and are, respectively, 0.24 mg·kg⁻¹, 3.90 mg·kg⁻¹, 5.80 mg·kg⁻¹, 3.50 mg·kg⁻¹, 6.50 mg·kg⁻¹, and 33.00 mg·kg⁻¹ (Puszcza Borecka Forest, Puszcza Knyszyńska Forest, Puszcza Białowieska Forest [74], or Puszcza Biała [75,76]). The mean contents of Cd, Cr, Cu, Ni, Pb, and Zn in mosses collected under fir stands near the S7 road under study were 0.36 mg·kg⁻¹, 5.91 mg·kg⁻¹, 12.50 mg·kg⁻¹, 3.26 mg·kg⁻¹, 8.82 mg·kg⁻¹, and 55.28 mg·kg⁻¹ and under pine stands they were, respectively, 0.27 mg·kg⁻¹, 3.67 mg·kg⁻¹, 10.52 mg·kg⁻¹, 1.80 mg·kg⁻¹, 7.47 mg·kg⁻¹ and 52.83 mg·kg⁻¹. Comparison with numerous works indicates the pollution of forest ecosystems, which is confirmed by numerous publications, viz: [53,54,74,75,80,81].

The analyses carried out showed that mosses did not reveal changes in the contents of the trace elements in question, depending on the time of exposure to pollutant deposition, related to the time of the start of road construction and the exposure of the interior of the stands, both in the case of mosses collected in the undergrowth of pine and fir stands. The exception was Ni contents in mosses collected in the undergrowth of fir stands, where higher contents were found in plots under shorter deposition (9 years). Depositions of other origins, e.g., those from low emissions from households (although it was not possible to isolate the influence of this parameter), could also have influenced the lack of visible differences. As reported by many authors, mosses have the ability to concentrate trace elements in their tissues for long periods of time [81,82,83]. Furthermore, these organisms do not have root systems but only grackles, which have a stabilizing function. Mosses are organisms that are evergreen and relatively long-lived, which affects their ability to accumulate elements over longer periods of time [44]. It should also be remembered that mosses are characterized by variability in element accumulation depending on the season, weather conditions, pH, or temperature [84,85]. The above variables and factors influenced the fact that the mosses did not indicate differences in trace element concentrations in the areas associated with the S7 expressway extension, in the sections in use since 1984, as well as the new section constructed in 2011.

The analysis of trace element contents in mosses, in comparison with other ecosystem elements, showed a clear effect of road traffic on content levels in zones in the vicinity of the S7 road. It was shown that Cr and Ni reached the highest contents in mosses in the zone up to 20 m from the road and in the case of Cu, Pb, and Zn, in the zone up to 10 m from the road in the undergrowth of fir stands. A similar relationship was found for mosses sampled in the undergrowth of pine stands, where the highest contents of Cr, Cu, Ni, Pb, and Zn were shown in the zone up to 10 m from the road. Cadmium, on the other hand, showed similar contents in all zones. An analysis of the impact of pollution using mosses was shown by [86], where Ni and Cu showed the highest contents in the zone of direct impact of the Hajravalta smelter in Finlia. Similarly, Zechmeister [87] used four moss species in his study to assess environmental pollution due to traffic. He found that the elements viz: Cr, Zn, Cu, and Ni had the highest contents in zones close to the road and were closely related to traffic. Cd contents in mosses, as in the 0–3 cm layer of soils, showed no spatial variation in content in the designated zones of distance from the road. This could be due to the high mobility of Cd in the environment, as mentioned earlier. In their study, Harmens [88] reported that the magnitude of Cd contents could be related to the impact of road salt, coming from the winter period, and that Cd and Pb deposition in mosses is related to atmospheric deposition. There are studies in which Cd contents in mosses may indicate a lack of correlation between traffic pollution and the origin of other sources. Hence, a lack of correlation with distance from the road has perhaps been shown [89]. Zinikovscaia [90] used mosses to assess the extent of air pollution in Chisinau, Moldova, where special bags of Sphagnum girgensohnii moss were used and exposed. Many studies have shown that mosses are used to map pollution and the direction of metal deposition [87,88,91]. Boquete [91] states that the results obtained should be considered as quantitative or semi-quantitative rather than absolute. This statement has to be agreed with, as many publications have shown a high variability of trace element concentrations depending on the sampling period, pH, humidity, weather conditions, acid rainfall, temperature influences, or moss morphology [26,84,92].

Aboal [84] indicated that mosses are not a good long-term indicator of environmental pollution, which they explain by the complex process of elemental uptake and physicochemical as well as biological processes that are closely linked to changing environmental conditions. Sorbed metals are strongly bound in moss tissues, making them resistant to desorption under outdoor conditions [93]. Biomonitoring of environmental contamination using mosses is intended to indicate the current state of the environment as a result of the impact of traffic or other emissions, so the comments given earlier do not disqualify mosses from the bioindicator group. The above results indicate that mosses can be considered as the best bioindicators for assessing the state of environmental pollution.

Analysis of the chemical composition of blueberry leaves indicated clear differences in element contents depending on the deposition time. It was shown that the highest contents of Cr, Ni, and Pb were in blueberry leaves collected in plots under shorter deposition under the canopy of fir stands and Cd, Pb, and Zn in plots collected in the undergrowth of pine stands influenced by the S7 road for several decades. Tahkokorpi [70] showed that Ni does not penetrate into plants through the root system and that this may be related to an ‘exclusion’ strategy for this element. The results obtained may indicate an anthropogenic origin of Ni in blueberry leaves. The expansion of the road contributed to the disturbance of the forest edge by cutting large belts of stands, which facilitated the migration of pollutants into the stand; this in turn increased the content of the elements in question in blueberry leaves under fir stands. For blueberry leaf samples collected under pine stands, the opposite relationship was found to that under fir stands. It was shown that the presence of the forest edge influenced the increased content of the elements in question. It was also found that the location of the plot influenced higher Pb and Zn contents. Some elements are inert to the forest ecosystem, some may have a stimulating effect through fertilization, and others may have a toxic effect. However, these results confirm that natural obstacles in the form of a dense forest edge can influence pollution levels in forest ecosystems. Hence, it is a valuable remark for road engineers, when designing new traffic routes, to analyze, in collaboration with foresters, the protection and restoration of natural barriers that protect forest stands from excessive pollution penetration. The ability of plants to retain pollutants has been confirmed in many studies. Popek [94] showed that trees and shrubs are very effective at trapping pollution generated by traffic. Similarly, the results have also been confirmed in other works [95,96,97,98]. Understanding the mechanism of contaminant spread in blueberries is very important because of the biogeochemical cycle of elements. In environments contaminated with trace metals, blueberries provide a link between the pool of elements accumulated in the soil and the primary consumers and higher trophic levels. A good example of this relationship is the Lepidoptera caterpillars that feed on blueberry leaves and further up the trophic chain, the caterpillars are food for various bird species through which metals move up the trophic chain [59]. The presented results regarding the use of mosses in environmental biomonitoring confirm their high usefulness in the analysis of trace elements.

5. Conclusions

Biomonitoring of environmental contamination using mosses indicates the current state of the environment as a result of traffic impacts or other emissions.

The survey carried out showed that the mosses collected in the vicinity of the S7 expressway were characterized by values indicative of pollution caused by traffic. Cr and Ni reached the highest concentrations in mosses in the zone up to 20 m from the road and in the case of Cu, Pb, and Zn, in the zone up to 10 m from the road in the undergrowth of fir stands. In contrast, the highest concentrations of Cr, Cu, Ni, Pb, and Zn were in mosses sampled in the undergrowth of pine stands and were shown in the zone up to 10 m from the road.

The mosses did not indicate differences in trace element concentrations in the areas associated with the S7 expressway extension, in the sections in use since 1984, as well as the new section constructed in 2011.

Natural obstacles in the form of a dense forest edge can influence and significantly reduce pollution levels in forest ecosystems. It is a valuable remark for road engineers when designing new traffic routes to analyze, in collaboration with foresters, the protection and restoration of natural barriers that protect forest stands from excessive pollution penetration.