Archives of Environmental Contamination and Toxicology

, Volume 60, Issue 2, pp 233–240

Assessment of Heavy Metal Pollution in Republic of Macedonia Using a Plant Assay


    • Faculty of Medical SciencesUniversity “Goce Delčev”
  • Tatjana Kadifkova-Panovska
    • Faculty of PharmacyUniversity “Ss. Cyril and Methodius”
  • Katerina Bačeva
    • Faculty of Natural Sciences and MathematicsUniversity “Ss. Cyril and Methodius”
  • Trajče Stafilov
    • Faculty of Natural Sciences and MathematicsUniversity “Ss. Cyril and Methodius”

DOI: 10.1007/s00244-010-9543-0

Cite this article as:
Gjorgieva, D., Kadifkova-Panovska, T., Bačeva, K. et al. Arch Environ Contam Toxicol (2011) 60: 233. doi:10.1007/s00244-010-9543-0


Different plant organs (leaves, flowers, stems, or roots) from four plant species—Urtica dioica L. (Urticaceae), Robinia pseudoacacia L. (Fabaceae), Taraxacum officinale (Asteraceae), and Matricaria recutita (Asteraceae)—were evaluated as possible bioindicators of heavy-metal pollution in Republic of Macedonia. Concentrations of Pb, Cu, Cd, Mn, Ni, and Zn were determined in unwashed plant parts collected from areas with different degrees of metal pollution by ICP-AES. All these elements were found to be at high levels in samples collected from an industrial area. Maximum Pb concentration was 174.52 ± 1.04 mg kg−1 in R. pseudoacacia flowers sampled from the Veles area, where lead and zinc metallurgical activities were present. In all control samples, the Cd concentrations were found to be under the limit of detection (LOD <0.1 mg kg−1) except for R. pseudoacacia flowers and T. officinale roots. The maximum Cd concentration was 7.97 ± 0.15 mg kg−1 in R. pseudoacacia flowers from the Veles area. Nickel concentrations were in the range from 1.90 ± 0.04 to 5.74 ± 0.03 mg kg−1. For U. dioica leaves and R. pseudoacacia flowers sampled near a lead-smelting plant, concentrations of 465.0 ± 0.55 and 403.56 ± 0.34 mg kg−1 Zn were detected, respectively. In all control samples, results for Zn were low, ranging from 10.2 ± 0.05 to 38.70 ± 0.18 mg kg−1. In this study, it was found that the flower of R. pseudoacacia was a better bioindicator of heavy-metal pollution than other plant parts. Summarizing the results, it can be concluded that T. officinale, U. dioica, and R. pseudoacacia were better metal accumulators and M. recutita was a metal avoider.

Pollution of the environment refers to pollution due to the release (into any environmental medium) from any process of substances which are capable of causing harm to humans or any other living organisms (UK Environmental Protection Act 1990). Monitoring the pollution status of the environment using plants is one of the main topics of environmental biogeochemistry (Diatta et al. 2003). The interest in phytoindicators arises from the fact that plants quickly react to chemical changes in the environment and are affected by a wide array of substances that contaminate air, water, and soil (Kabata-Pendias and Pendias 1992).

Pollution of the environment with toxic metals has increased dramatically since the onset of the Industrial Revolution. From an environmental point of view, all heavy metals are very important because they cannot be biodegraded in soils, so they tend to accumulate and persist in urban soils for a very long time (Kabata-Pendias and Dudka 1991). Lead, cadmium, copper, zinc, and nickel are metals frequently reported to have the highest impact on organisms.

The aim of the present study was to look at the pollution levels of Pb, Cu, Cd, Mn, Ni, and Zn, by using different plant organs from four plant species: stinging nettle, Urtica dioica L. (Urticaceae); black locust, Robinia pseudoacacia L. (Fabaceae); dandelion, Taraxacum officinale (Asteraceae); and chamomile, Matricaria recutita (Asteraceae). These plants were selected as possible biomonitors of heavy-metal pollution in Republic of Macedonia, because of the following characteristics: widespread occurrence from rural to urban areas; wide geographical rangel ecological distribution throughout the world; and ease of identification, sampling, growing, and cultivating. A comparison among leaves, roots, stems, and flowers of the four analyzed plant species was performed to evaluate their ability to reflect heavy-metal pollution in some regions in Republic of Macedonia.

Materials and Methods

Sampling Area

The city of Veles is located in the valley of the Vardar River, about 55 km south of the capital Skopje. The town is an industrial area in Republic of Macedonia, located at 160–200-m altitude, with a continental climate and a mean annual precipitation of about 469 mm. The lead and smelting plant in Veles was chosen as the study area because it is the largest complex and important source of lead and zinc pollution in Macedonia, with estimated lead emission of 83 tons per year according to the National Environmental Action Plan (NEAP 1996), and there were several investigations of soil, vegetables, and fruit produced in the region of Veles for heavy-metal content (Stafilov and Jordanovska 1997; Barandovski et al. 2008; Stafilov et al. 2008, 2010).

Lower or higher plants can act as bioindicators, biomonitors, and bioaccumulators (Markert et al. 1999; Chehregani et al. 2009). Botanical materials such as lichens (Garty et al. 1997), mosses (Poikolainen et al. 2004; Harmens et al. 2008), ferns, and tree bark have been used for biomonitoring of heavy metals since 1950s. These lower plants, due to their higher capacity for metal accumulation, are probably the organisms most frequently used for monitoring metal pollution in environments. But in the past few decades, there has been increasing use of higher plants as biomonitors of heavy-metal pollution (Pyatt 2001; Oliva and Rautio 2004; Çelik et al. 2005). Most studies have focused on perennial plants rather than annual or short-lived perennial plants, although the latter have a potential advantage for monitoring short-term or yearly changes in pollution. Therefore, a good biomonitor will indicate the presence of the pollutant and also attempt to provide additional information about the amount and intensity of the exposure (Wolterbeek 2002).

In this study, samples from an industrialized area (U. dioica, R. pseudoacacia, and M. recutita) were taken from different places between 10 and 100 m from the lead smelting plant “MHK Zletovo” in the Veles area (V; Fig. 1). Samples from the urban roadside (T. officinale) were collected about 2 m away from the main road, which is also about 500 m from the lead smelting plant (Vr), and from a road (R. pseudoacacia) about 15 km from the city of Veles (Ro). For uncontaminated controls, samples from all four investigated plant species were taken from Plackovica Mountain (P), about 60 km from the city of Veles (Fig. 1). The Mountain is located in the eastern part of Republic of Macedonia and its highest point is peak Lisec (1754 m). Plants were identified at the Department of Pharmacognosy, Faculty of Pharmacy, Skopje, where specimens of all the plants are deposited in the herbarium. Sample locations are given in Table 1.
Fig. 1

Map showing the city of Veles and Mount Plackovica, the area in Republic of Macedonia where plants were sampled in this study

Table 1

Plant, plant organ, and sample location

Plant, organ


Altitude above sea level (m)




Robinia pseudoacacia


Veles (V)





Road (Ro)





Plackovica Mountain (P)




Urtica dioica


Veles (V)





Plackovica Mountain (P)




Taraxacum officinale


Urban road (Ur)





Plackovica Mountain (P)




Matricaria recutita


Veles (V)





Plackovica Mountain (P)




Sample Collection and Preparation

All samples were collected during May 2009. Characteristics of studied plants are given in Table 2. About 200 g (fresh weight) of each plant part investigated was collected as follows: R. pseudoacacia—leaves and flowers from the middle section of the main leafy area of the plant from each direction, west, east, south, and north; T. officinale—similar–sized, well-developed rosette leaves and roots; U. dioica—similar-sized leaves and stems cut with a nonmetal knife; and M. recutita—stems with flowers and leaves.
Table 2

Characteristics of studied plants



Robinia pseudoacacia

Taxonomy: Fabaceae; genus Robinia, sp. pseudoacacia

Synonyms: False acacia, black locust

Morphology: Large deciduous flowering tree, growing to 25 m tall; dark-green epileptic leaflets; white flowers

Habitats: Humid climate, variety of soils, especially limestone

Zones 3–8; Europe, Asia, North America

Flowering time: May and early June

Taraxacum officinale

Taxonomy: Asteraceae; genus Taraxacum, sp. officinale

Synonyms: Dandelion, blowball

Morphology: Perennial herb; rapid growing; single crown with green leaves and yellow flowers

Habitats: Variety of soils (pH 4.8–7.5)

Europe, Asia, North America

Growth period: Spring and fall

Flowering time: Early spring

Urtica dioica

Taxonomy: Urticaceae; genus Urtica, sp. dioica

Synonym: Stinging nettle

Morphology: Herbaceous perennial plant, 1–2 m; soft green leaves; small greenish or brownish flowers; leaves and stems with stinging hairs (trichomes)

Habitats: Woodland garden; sunny edge; dappled shade

Europe, Asia, North Africa

Flowering time: Summer, June to September

Matricaria recutita

Taxonomy: Asteraceae; genus Matricaria, sp. recutita

Synonyms: German chamomile; chamomile

Morphology: Annual plant; growing 0.5–0.25 m; long narrow leaves and white florets with yellow discs and strong, aromatic smell

Habitats: Sandy or loamy arable soil; near roads

Europe, East to West Asia, North America, Australia

Flowering time: May, June–July

Heavy metals are mostly taken up into leaves by the roots via transpiration stream from soil water (root uptake). However, a certain portion may also pass through the stomata on the surface of the foliage or via the cuticle directly from wet and/or dry deposition, and in addition, some amounts are fixed to hairy or waxy cuticles. Unwashed plants were used to obtain the total content of heavy metals, as recommended by Oliva and Rautio (2004). All plant samples, unrinsed, except for dandelion roots (from soil), were air-dried, milled in a microhammer (without metal parts in it), and stored in clean paper bags.

Analytical Techniques

For standard and sample preparation demineralized water and high-purity reagents (Tracepur and p.a.) were used. Standards of selected metals were set by dilution of stock standards which were prepared using analytical grade salts of metals (Merck multielement standard, 1000 mg/L) with HNO3 and results were corrected for reagent blanks.

From fine crushed tissues of each item, approximately 0.5 g was weighed and placed in PTFE vessels, and a 5 ml HNO3 (69% Merck; Tracepur) and 2 ml H2O2 (30%, mass/vol; Merck) mixture was left at room temperature for 1 h, then placed in a rotary microwave oven (Mars CEM XP 1500) and mineralized using the two-step procedure reported in Table 3. Ventilation was performed for 20 min after the end of the second step. Finally, the vessels were cooled and carefully opened, and digests were filtered on filter paper (Munkteil), quantitatively transferred in 25-ml calibrated flasks, diluted with demineralized water, and analyzed by ICP-AES (Varian715-ES) for selected metals.
Table 3

Procedure used for digestion of plant samples


Temp. (°C)

Time (min)

Power (W)



10 (ramp time)




15 (hold time)


The instrumental parameters for determination of the investigated elements are reported in Table 4. All samples were analyzed three times. Each value represent the mean ± SD of three samples. All results were calculated on a dry weight basis (mg kg−1 dw).
Table 4

Instrumentation and operating conditions for the ICP-AES system (Varian 715ES)

Radiofrequency generator

 Operating frequency

40.68 MHz; free-running, air-cooled

 Power output

700–1700 W in 50 -W increments

 Power output stability


Introduction area

 Sample nebulizer


 Spray chamber

Double-pass cyclone

 Peristaltic pump

0–50 rpm

 Plasma configuration

Radially viewed


 Optical arrangement

Echelle optical design


400-mm focal length

 Echelle grating

94.74 lines/mm

 Polychromator purge

0.5 L min−1

 Megapixel CCD detector

1.12 million pixels

 Wavelength coverage

177–785 nm

Conditions for program

 RF generator power

1.0 Kw

 Plasma Ar flow rate

15 L min−1

 Auxiliary Ar flow

1.5 L min−1

 Nebulizer Ar flow rate

0.75 L min−1

 Pump speed

25 rpm

 Stabilization time

30 s

 Rinse time

30 s

 Sample delay

30 s

 Background correction


 No. of replicates




226.50 nm


324.754 nm


257.610 nm


231.604 nm


220.353 nm


213.857 nm


The results for the content of the investigated heavy metals in different plant organs of four plant species are given in Fig. 2. The concentrations of Pb (mg kg−1) in samples from the Veles area around the lead and zinc smelting plant range from 7.94 ± 0.08 (M. recutita flowers) to 174.52 ± 1.04 (R. pseudoacacia flowers), and those in samples from Plackovica Mountain range from 1.00 ± 0.03 to 3.86 ± 0.1 (U. dioica leaves).
Fig. 2

Cd, Cu, Mn, Pb, Ni, and Zn content (mg kg−1) in plants and plant organs from different areas: V, Veles; P, Plackovica Mountain; Ro, urban road. aTaraxacum officinale; bRobinia pseudoacacia; cUrtica dioica; dMatricaria recutita

The content of Cu (mg kg−1) in samples from the Veles area ranges from 7.16 ± 0.15 (U. dioica stems) to 15.88 ± 0.19 (M. recutita leaves), and in samples from Plackovica Mountain it ranges from 5.57 ± 0.2 (T. officinale leaves) to 16.71 ± 0.12 (T. officinale roots).

The cadmium concentration in this study was found to be under the LOD (0.1 mg kg−1) in all control samples, except for R. pseudoacacia flowers (0.28 ± 0.03) and T. officinale roots (0.43 ± 0.02). The concentrations of Cd (mg kg−1) in samples from the Veles area around the lead and zinc smelting plant range from 1.57 ± 0.02 (M. recutita flowers) to 7.97 ± 0.15 (R. pseudoacacia flowers). A concentration of 7.67 ± 0.3 mg kg−1 Cd was observed in T. officinale roots in samples also taken from an area around the lead smelter plant. The concentration of Cd in leaves from the same plant was very similar (7.24 ± 0.19 mg kg−1).

Among all species analyzed, the highest Mn content was found in M. recutita leaves (90.01 ± 0.29) sampled from the Veles area. The concentration in flowers of the same plant was 42.96 ± 0.15 mg kg−1. Somewhat lower values were obtained for control plants: 75.92 ± 0.27 and 32.15 ± 1.25 mg kg−1, respectively. This relationship, a higher concentration in leaves than in other plant parts, is valid for all investigated plants, for example, 74.71 ± 0.13 mg kg−1 for U. dioica leaves and 24.67 ± 0.08 mg kg−1 for U. dioica stems sampled from the Veles area.

In this investigation, Ni was in the range <1 mg kg−1 (below the LOD) to 5.74 ± 0.03 mg kg−1 and was found to be lower than (for all control samples) or slightly above the phytotoxicity range (for R. pseudoacacia sampled from the Veles area) according to Gough et al. (1979). There was no statistically significant Ni distribution among different plant parts in any species.

Zinc was the element with the highest concentrations (min., 10.2 ± 0.05; max., 465.0 ± 0.55 mg kg−1) in all plant species in this study, especially in U. dioica leaves (465.0 ± 0.55) and R. pseudoacacia flowers (403.6 ± 0.34) sampled near the lead smelting plant. In U. dioica stems and R. pseudoacacia leaves from the same location, high Zn concentrations were also found: 230 ± 0.61 mg kg−1 and 129.23 ± 0.12 mg kg−1, respectively. Results for T. officinale sampled from the same location were in the critical range, too. In all control samples from Mount Plackovica, results for Zn were low, no higher than 38.70 ± 0.18 mg kg−1 (T. officinale roots).


The investigated elements are very important essential and toxic substances, and it is important to follow their presence in the environment. Thus, lead is available to plants from soil and aerosol sources. In the field most uptake has been demonstrated to be through the leaves. Normal concentrations of Pb in plants are 0.1–10 mg kg−1 dw, according to Kabata-Pendias and Pendias (1992), although Allen (1989) considers a value of 3 mg kg−1 a normal level. Generally, toxic concentrations of Pb are defined as 30–300 mg kg−1 (Kloke et al. 1984). Results for Pb in this study corresponded to those obtained in several moss samples collected from the same region (Barandovski et al. 2008). The heavy-metal concentration in the leaves is proportional to urbanization, industrial activity, and density of traffic.

Copper is necessary for plant growth in low amounts and is a structural part of many enzymes. Although Cu is an essential micronutrient for plant growth, it can be more toxic than nonessential Pb to biota when extraneous Cu is present in soil environments (An 2006). Ouzounidou (1994) has suggested that photosynthetic function is highly sensitive to copper toxicity. The phytotoxic level for this element is 30 mg kg−1. Shaw et al. (2004) reported 4–15 mg kg−1 for normal copper concentrations in plants. Plant concentrations of Cu above 25 mg kg−1 are considered toxic to plants by Allen (1989). According to Allen’s criterion, investigated regions in Republic of Macedonia are not highly polluted by copper since its content in all plant species did not exceed the upper limit.

Cadmium is very toxic for any kind of organism, and as far as is known, Cd is not a constituent of any metabolically important compound. Cadmium is an especially mobile element in the soil, is taken up by plants primarily through the roots, and is easily transported within plants and distributed to all plant organs (Kabata-Pendias and Pendias 1992). Depending on their Cd content, plants are considered Cd accumulators or Cd avoiders. In soils, Cd rarely occurs as the only heavy-metal pollutant, as it is most frequently accompanied by Zn (Schulze et al. 2005). Generally, it is accepted that the normal Cd concentrations in plants are between 0.2 and 0.8 mg kg−1 and toxic concentrations of Cd are defined as 5–30 mg kg−1 (Kloke et al. 1984; Kabata-Pendias and Pendias 1992).

Manganese is an essential micronutrient for plants, but when in excess, it has some toxic effects on plant growth. Manganese in plants is present in the NAD-malic enzyme system found in the leaves. It is also a specific constituent of the photosynthetic oxygen-evolving system in chloroplasts (Kim and Jung 1993). The toxicity of Mn is commonly associated with acidic soils and warm climates and manganese toxicity is one of the important factors limiting plant growth on acidic soils, pH <5.5 (Foy et al. 1978). Markert (1994) has reported 200 mg kg−1 as a normal value for plants.

Nickel is an essential microelement for plants, animals, and humans. Exceeding the optimum values, it shows a toxic effect. With respect to the fact that Ni uptake depends on plant species, and that some of them show hyperaccumulation effects, Ni concentrations in normal plants range from 0.5 to 5 mg kg−1 dw, and values exceeding these limits are reported as poisonous (Allen 1989).

Zinc is an essential element in all organisms and plays an important role in the biosynthesis of enzymes, auxins, and proteins. Zn is not considered to be highly phytotoxic and the toxicity limit for Zn (300–400 mg kg−1) depends on the plant species as well as on the growth stage (Kabata-Pendias and Pendias 1992). But high concentrations of zinc in plants may cause the loss of leaf production, whereas low concentrations may cause deformation of leaves. A plant foliar concentration of 100 mg kg−1 Zn has been quoted by various authors (Allen 1989) as a critical indicator of whether the environment is polluted with Zn.

Accumulation of heavy metals in regions with similar plant species has been reported by a number of authors (Aksoy et al. 2000; Gaweda and Capecka 2001; Baranovska et al. 2002; Krolak 2003; Barandovski et al. 2008; Gűleryűz et al. 2008; Kováčik and Bačkor 2008; Samecka-Cymerman et al. 2009; Stafilov et al. 2010).

The results of the present study confirm previous investigations by various authors. Çelik et al. (2005) made a biomonitoring study using Robinia pseudoacacia as well. Their results for Pb are quite high (between 132.2 and 139 mg kg−1). Akgüç et al. (2008) carried out a similar study using Pyracantha coccinea (Rosaceae) as a biomonitor in Mugla, Turkey (max., 14.92 mg kg−1 dw). Calzoni et al. (2007), using Rosa rugosa, determined Pb between 1.0 and 9.0 mg kg−1 dw in unwashed leaves in Italy.

Although various authors (Aksoy et al. 2000; Samecka-Cymerman et al. 2009) have reported that leaves of R. pseudoacacia are good bioindicators, our results for Pb concentrations in leaves and flowers of R. pseudoacacia from all locations (Fig. 2), did suggest that flowers could be used as better indicators of Pb pollution than leaves.

The results obtained, in agreement with results from the literature, suggest that leaves from plants in the polluted area investigated in this study are better indicators of Pb pollution than other plant organs. The exception was R. pseudoacacia’s flowers, as mentioned before.

There was no statistically significant difference in Cu levels obtained from different organs of the same plant, except for T. officinale leaves and roots. This corresponds to the fact that copper, as a redox active metal, is preferentially retained in the roots to protect shoots against oxidative damage (Kováčik et al. 2008). Metal concentrations in investigated plants were also in correlation with the results presented in maps 25a and 25b in the Geochemical Atlas of Veles and the Environs (Stafilov et al. 2008), where the highest content of copper is present in topsoils from the areas of the lead and zinc smelting plant and in the area of the city which is close to the smelter plant.

Aksoy et al. (2000) found a Cd concentration of between 0.47 and 3.39 mg kg−1 in unwashed leaves in their study realized in Kayseri (Turkey) using R. pseudoacacia as a biomonitor. A similar study was performed by other authors (Aksoy and Sahin 1999), where Elaeagnus angustifolia was analyzed. The results for Cd in their study were in the range of 0.50–3.45 mg kg−1.

All results for Cd in samples from the Veles area, where a factory produces metallic Cd, Pb, and Zn causing pollution, are high and in the toxic range. An exception was M. recutita, also sampled from the same area, but showing a lower Cd content than other investigated plants. This fact has been previously recorded in chamomile plants (Kováčik et al. 2006).

It can also be noted that Cd values obtained for different organs from one plant species are very similar. This may support the known fact that Cd root-to-shoot transport is most likely driven by a transpiration stream (Salt et al. 1995). We also noted that, in plants where high Cd levels were found, high Zn and Pb levels are always recorded too. To summarize, T. officinale, U. dioica, and R. pseudoacacia can be considered a Cd accumulator and M. recutita s a Cd avoider.

For manganese, in all cases the mean Mn concentration in other plant organs was lower than the mean content in leaves. These results are in agreement with previous investigations. The fact that Mn accumulates in plants leaves reflects its function in plant metabolism, particularly its important role in the Hill reaction, where H2O splits and O2 is released (Hagemeyer 2004).

Yusuf et al. (2003) have determined the Ni concentration in edible vegetables in the range from 0.48 to 1.31 mg kg−1. Palmieri et al. (2005), using Pittosporum tobira in Sicily, Italy, obtained a value of 129 mg kg−1 Zn in unwashed leaves and 128 mg kg−1 dw in washed leaves. Madejon et al. (2004)did a study using Populus alba (542.1 mg kg−1 dw Zn in unwashed leaves) in New Zealand.

Values obtained for Zn, indicated that all investigated species in this study had Zn accumulation capacity in their organs. Even M. recutita, the plant showing the lowest heavy-metal content in this study, had a high Zn concentration. Zn accumulation capacities of U. dioica (especially leaves) and R. pseudoacacia (especially flowers) indicated that U. dioica and R. pseudoacacia, can be used as possible bioindicators of Zn pollution. The high Zn content in all plant species from the Veles area corresponds to the fact that in the main polluted area, topsoils around the smelter plant were highly polluted with zinc, with an average content of 1100 mg kg−1 as presented in maps 50a and 50b in the Geochemical Atlas of Veles and the Environs (Stafilov et al. 2008).


The data obtained indicate that the distribution of heavy metals in the organs of plants is not homogeneous; it probably depends on the nature of plant species and the physicochemical characteristic of the element. The results suggest that R. pseudoacacia, U. dioica, and T. officinale may be considered bioaccumulator species for Pb, Cu, Cd, and Zn and can be used as bioindicators of pollution with these metals. Therefore, it seems that they can play a substantial role in remediation of polluted sites.

It was found that Matricaria recutita can be classified as a metal excluder. The analysis of heavy-metal concentration in plants is necessary also in the case of plants used for phytotherapeutical purposes.

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