Biol Trace Elem Res (2013) 156:288–297 DOI 10.1007/s12011-013-9848-8

Toxic Element Profiles in Selected Medicinal Plants Growing on Serpentines in Bulgaria Dolja Pavlova & Irina Karadjova

Received: 28 July 2013 / Accepted: 15 October 2013 / Published online: 31 October 2013 # Springer Science+Business Media New York 2013

Abstract Populations of medicinal plants growing on serpentines and their respective soils were analyzed for Fe, Ni, Mn, Cr, Co, Cd, Cu, Zn, and Pb using inductively coupled plasma atomic emission spectrometry. Aqua regia extraction and 0.43 M acetic acid extraction were used for the quantification of pseudototal and bioavailable fractions, respectively, of elements in soil and nitric acid digestion for determination of total element content in plants. Screening was performed to (1) document levels of toxic metals in herbs extensively used in preparation of products and standardized extracts, (2) compare accumulation abilities of ferns and seed plants, and (3) estimate correlations between metal content in plants and their soils. The toxic element content of plants varied from site to site on a large scale. The concentrations of Fe and Ni were elevated while those of Cu, Zn, and Pb were close to average values usually found in plants. The highest concentrations for almost all elements were measured in both Teucrium species. Specific differences in metal accumulation between ferns and seed plants were not recorded. The investigated species are not hyperaccumulators but can accumulate toxic elements, in some cases exceeding permissible levels proposed by the World Health Organization and European Pharmacopoeia. The harvesting of medicinal plants from serpentines could be hazardous to humans.

Keywords Medicinal plants . Serpentine . Toxic metals . Bulgaria

D. Pavlova (*) Department of Botany, Faculty of Biology, University of Sofia, Blvd. Dragan Tzankov 8, 1164 Sofia, Bulgaria e-mail: [email protected] I. Karadjova Department of Analytical Chemistry, Faculty of Chemistry and Pharmacy, University of Sofia, Blvd. J. Bouchier 1, 1164 Sofia, Bulgaria e-mail: [email protected]

Introduction Ultramafic (serpentine) soils are considered adverse habitats for plants because of many factors: low nutrient levels, calcium deficiency, magnesium toxicity, and high concentrations of potentially toxic elements, such as chromium, nickel, cobalt, and soil xericity [1, 2]. The high concentrations of Mg in relation to Ca and elevated concentrations of Ni are considered to be important factors affecting plant growth and survival on serpentine soils [3]. Medicinal plants growing in nature can accumulate toxic elements depending on their individual properties; concentrations of metals in soil, air, and water [4]; climatic factors; plant species; and other environmental factors [5]. The most important natural source of toxic metals is soil parent minerals and chemical element bioavailability to plants [6]. All chemical elements, both essential and nonessential, can have toxic effects on plants and further to humans if found at elevated concentrations. Medicinal plants used in therapy after collection from a polluted environment and transformed into dosage form may have serious toxic effects to humans [7]. Recently, the use of herbal products has increased in developed countries, due in part to the widespread assumption that “natural” implies “harmless.” That is why, with their increased popularity and global market expansion, the safety of herbal products has become a major concern in public health [8]. The World Health Organization also emphasized the need to ensure the quality of medicinal plants and their products applying suitable quality standards which are controlled by using modern analytical techniques [9]. It is emphasized that it is necessary to improve quality standards for herbal medicines after revising the maximum allowable values for toxic element content in medicinal plants based on modern research studies [10]. Bulgaria and other southeastern and eastern European countries are rich sources of botanical drug species within Europe [11]. Every year more than 10,000 tons of botanical drugs is collected, purchased, and processed in Bulgaria.

Toxic Element Profiles in Selected Medicinal Plants

Around 60–70 % of the plant material is exported, while the other 30–40 % remain in the country for the production of phytopharmaceuticals, herbal teas, and spices. Around 20 % of wild botanical drug species collected in Bulgaria are weeds or occur in rural habitats and are not threatened by collection [12]. Although Bulgaria has a comprehensive policy on harvesting, conservation, and use of medicinal plants, still little is known about serpentine habitats and their distribution and effect on plants. People living in such regions, which are mainly mountains, considered them clean, being far from factories and roads, and these people collected plants for medicinal uses. Some of the species examined in this study have a history of traditional use in Bulgaria and in other Balkan countries for herbal teas and basic medicinal healing treatments [13–15]. The species Hypericum perforatum L., Teucrium chamaedrys L., and Teucrium polium L. are included in the list of medicinal plants permitted for export, and annually, hundreds of tons are exported from Bulgaria [16]. The species Rhodiola rosea L., which is protected by the Law of the Medicinal Plants in Bulgaria (2002) and used in pharmacy after cultivation, was also included and checked for metal accumulation. The boreal species Empetrum nigrum L. is traditionally used as a medicinal plant in Canada [17] and in other northern countries. This species is not used in Bulgaria for pharmaceutical purposes, but it was collected and analyzed for comparison with the medicinal plant R. rosea. Different plant parts (rhizome, stem, leaves, and fruits) from both species were analyzed for their metal concentrations. Screening of the selected medicinal plants and their populations growing on serpentine areas in Bulgaria was carried out to (1) document evidence of toxic metals in herbs, some of them extensively used in preparation of products and standardized extracts; (2) compare accumulation abilities of ferns and seed plants; and (3) estimate correlations between metal content in plants and their respective soils.

Material and Methods

289

was shaken with 40 ml 0.43 M acetic acid for 16 h. Suspensions were centrifuged at 14,000 rpm and elements measured in the supernatant by ICP-AES or electrothermal atomic absorption spectrometry (ETAAS) (PerkinElmer Zeeman 3030). Extractable fraction is calculated as a percent from the pseudo total (aqua regia) amount according to the formula: D% =A e/A tot ⋅100, where D % is the extractable fraction, A e, [mg kg−1] is the extractable amount in 0.43 M acetic acid, and A tot , [mg kg−1] is the pseudo total amount extractable aqua regia. Plant Sampling and Analysis The medicinal plants H. perforatum , T. chamaedrys , T. polium , Asplenium trichomanes L., Ceterach officinarum DC., E. nigrum, and R. rosea growing on serpentines in the Rhodope Mts. and the Rila Mt. were selected for this study (Table 1). The total amounts of essential (Fe, Mn, Cu, Zn) and nonessential elements (Ni, Cr, Co, Cd, Pb) were determined in plant samples after nitric acid digestion and ICP-AES/ETAAS measurement (Table 2). Plant parts analyzed varied for different species, coincide with those most frequently used in traditional medicine or for pharmaceutical applications, and could be summarized as follows: aerial plant parts as mixed sample from stems, leaves, and inflorescence for H. perforatum, T. chamaedrys, and T. polium; fronds for ferns; and separate analysis of different plant parts (rhizome, stems, leaves, and fruits) for E. nigrum and R. rosea. Plant parts most used in traditional medicine and pharmacy for E. nigrum are the fruits, and for R. rosea, the rhizomes, that is why they were analyzed separately and compared for their accumulation abilities with other plant tissues. Samples (e.g., required plant parts) were prepared from fresh plants and then air-dried at room temperature. A sample amount of around 0.3 g was treated with 10 ml conc. HNO3 overnight. Then, solutions were digested on a sand bath for about 3 h and transferred in a volumetric flask of 25 ml. ICP-AES and ETAAS under optimal instrumental parameters were used for the determination of the metal concentrations.

Soil Sampling and Characteristics Data Analysis Soil samples were collected from 1 to 10 cm depth near the roots of plants studied from different sites. They were airdried, sieved to pass a 2-mm nylon mesh, and ground. For the determination of pseudototal amount of elements, a sample amount of 0.5 g was digested for about 5 h with 15 ml aqua regia. The elements Fe, Ni, Mn, Cr, Co, Cd, Cu, Zn, and Pb were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), Jobin Yvon Ultima 2 under optimal instrumental parameters. For the determination of bioavailable fraction of elements in soils, single-step extraction was performed: 1 g soil sample

Cluster analysis using Euclidean distance and unweighted pair group mean average (UPGMA) was used as the computational criteria to express the similarities between studied species based on element concentrations measured in the aerial plant parts (Fig. 1). Pearson correlations between the measured elemental concentrations in plant populations and their rhizospheric soils were also performed. A bioaccumulation factor calculated as ratio of elemental concentrations in plants to that in soil was performed to evaluate the capacity of the analyzed species for accumulation

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Table 1 List of the localities of the studied species populations, Abbr.=abbreviations used for the populations №

Locality

Rhodopes Mt. 1 Eastern Rhodope Mts.—southward from G. Kamenjane Village, scree slope 2 Eastern Rhodope Mts.—southward from Chernichevo Village 3 Eastern Rhodope Mts.—northwestward from Chichevo Village, open rocky places 4 Eastern Rhodope Mts.—southwestward from Fetler Village 5 Eastern Rhodope Mts.—westward from Dobromirtzi Village 6 Eastern Rhodope Mts.—near Kazak Village 7 Central Rhodope Mts.—near Parvenetz Village southeastward, open stony slope Rila Mt. 8 Seven Rila lakes—north-facing slope near Trilistnika Lake

of a specific metal [2, 18]. Correlation between bioaccumulation factor and chemical element bioavailability from soil (presented as fraction extracted from soil with 0.43 M acetic acid) was also discussed. All statistics were performed using the Statistica 7 program, StatSoft.

Results Soil Analysis The soils in the study sites are thin and characterized by elevated levels of the metals Fe, Ni, Cr, Co, and Mn, typical for ultramafic environments (Table 2). The concentrations of the investigated elements from serpentines from the Rhodope Mts. vary from site to site and confirm previous data reported for this area [19–21]. Relatively lower concentrations were found for the same elements (Ni, Mn, Cr, Co) in the serpentine soils from the Rila Mt. While Ni concentrations in the sites from the Rhodope Mts. were in a range considered as typical (500–5,000 mg kg−1) for serpentine and other Mg- and Fe-rich soils [18, 22, 23], those from the Rila Mt. were in the range from 98 to 161 mg kg−1. The Cu and Zn concentrations in all studied soils from the Rhodope Mts. were within the range considered as usual for soils [22], while those of Pb were above the proposed limits. This is obviously related to Pb/Zn mineralization in this region of the mountain. Unusually, high concentrations of 9.3 and 10.6 mg kg−1 for Cd in soils were measured in two sites in the Rhodope Mts. (Chichevo and G. Kamenjane) where the soils are rich in Zn ores. This result confirms Kabata-Pendias and Pendias [22] who reported very high concentration of Cd in the vicinities of Pb and Zn mines and, in particular, close to smelting operations. The concentrations of Zn in soils from the Rila Mt. (112– 286 mg kg−1) were elevated compared to those determined

Altitude Geographical coordinates

Abbreviations used for the populations

414 614 478 425 389 350 360

41° 24.068′ N, 25° 42.231′ E 41° 20′43.04″ N, 25° 45′36.83″E 41° 21′03.84″ N, 25° 17′23.18″ E 41° 21.800′ N, 25° 18.628′ E 41° 23.053′ N, 25° 12.096′ E 41° 24.619′ N, 25° 53.166′ E 42° 03.948′ N, 24° 39.472′ E

GK Che Ch F D Kaz Pz

2,100

42° 12′53.43″ N, 23° 19′ 09.14″E R

in soils from the Rhodope Mts. The amounts of Pb and Cu were relatively low, below 31 and 28 mg kg−1, respectively.

Plant Analysis The concentration of essential and nonessential elements in the selected medicinal plants growing on serpentine are presented in Table 2. Elevated concentrations of metals Fe, Ni, Cr, and Co, which are usually in high concentrations in serpentine soils, were also in elevated values in medicinal plants while Mn was below the critical level of 300– 500 mg kg − 1 dry weight [24]. The highest mean concentrations for Fe and Co were measured in T. chamaedrys; for Mn in E. nigrum; and for Ni, Cr, Cd, Cu, Zn, and Pb in T. polium. The measured Ni concentrations were the highest in both Teucrium species and the lowest in H. perforatum (1.2 mg kg−1). The total concentrations of Cr and Co in plant aerial biomass in all species from serpentines in the Rhodope Mts. were over the proposed limits [22] and obviously are in relation to the elevated values of these metals in the serpentine soils. The same elements were also elevated in plants from the Rila Mt., but they reached the highest level in R. rosea. The lowest concentrations of Co were found in H. perforatum . The fern species demonstrate lower concentrations for Cr in comparison with other plants. Both the elements Cd and Pb were in high concentrations for most of the populations of the studied species. Their amounts in plant material varied considerably, reaching maximal levels in T. polium. The contents of the essential trace elements Fe, Mn, Cu, and Zn in all plants studied also varied considerably between species and populations. The concentrations of Mn in plants were close to typical despite high Mn concentrations in serpentine soils. The studied plants were found growing together only in one of the sites in the Rhodope Mts. (G. Kamenjane). A

Toxic Element Profiles in Selected Medicinal Plants

291

Table 2 Elemental concentrations of plant and soil samples. Recorded concentrations are in milligrams per kilogram dry material (mean ± standard deviation of three replicates). Abbreviations of the populations

are given in Table 1. Mean values of the investigated plant parts for E. nigrum and R. rosea were included

Study sites

Cr

Ceterach officinarum G. Kamenjane Village Plant (GK) Soil Chichevo Village (Ch) Plant Soil Parvenetz Village (Pz) Plant Soil Asplenium trichomanes G. Kamenjane Village Plant (GK) Soil Chichevo Village (Ch) Plant Soil Fetler Village (F) Plant Soil Chernichevo Village Plant (Che) Soil Teucrium chamaedrys

Fe

Ni

Co

Cd

Cu

Zn

Pb

2,233±183 70±6 84±6 1.2±0.2 3.1±0.2 78,389±5,487 927±65 796±40 1,713±103 82±3 1,088±98 22±2 42±3 1.2±0.7 1.7±0.1 88,699±6,209 1,340±94 849±42 1,229±79 112±4 861±69 27±2 44±3 1.4±0.2 1.5±0.1 82,380±894 1,156±116 1,113±134 1,422±156 619±43

2.69±0.22 8.7±0.7 1.4±0.2 34.5±1.4 0.48±0.04 4.8±0.4 9.3±0.6 22.5±7.4 0.8±0.1 6.0±0.5 2.3±0.2 34±2

22±2 70±5 40±3 73±5 57±4 102±10

0.9±0.1 40±3 1.2±0.2 48±3 1.4±0.2 51±4

242±24 21±2 23±2 1.4±0.2 1.1±0.1 84,903±86 1,018±102 722±61 1,936±213 108±9 269±22 24±2 18±1 1.3±0.1 1.3±0.1 88,699±6,209 1,340±94 849±42 1,229±79 112±4 338±31 46±3 21±1 1.6±0.2 3.8±0.3 87,048±621 1,217±122 1,757±193 1,157±104 180±13 156±12 22.4±1.8 14±1 0.9±0.2 1.1±0.1 27,511±2,201 631±50 487±39 556±44 36±3

0.61±0.04 2.9±0.2 1.3±0.1 63±4 0.48±0.03 4.0±0.3 9.3±0.6 22.5±7.4 0.4±0.03 4.1±0.3 2.3±0.2 27±2 0.45±0.04 6.5±0.5 6.2±0.5 11.3±0.9

21±2 71±7 37±3 73±5 26±2 99±7 24±2 51±4

0.6±0.1 15±1 1.1±0.2 48±3 0.9±0.8 58±5 0.67±0.05 25±2

1.43±0.1 7.6±0.3 7.9±0.5 23.2±0.9 1.16±0.07 7.1±0.3 9.3±0.6 22.5±7.4 0.62±0.04 8.9±0.7 2.3±0.2 27±2 0.2±0.02 5.7±0.5 2.9±0.2 41±2

24±2 47±3 31±2 73±5 45±3 99±7 31±2 70±7

1.4±0.1 12±1 4.5±0.3 48±3 11.3±0.9 58±5 3.9±0.3 43.9±3.5

G. Kamenjane Village Plant 2,740±192 109±7.6 (GK) Soil 75,926±5,315 1,493±105 Chichevo Village (Ch) Plant 1,385±97 49±3.4 Soil 88,699±6,209 1,340±94 Fetler Village (F) Plant 1,380±120 63±6 Soil 87,048±621 1,217±122 Dobromirtzi (D) Plant 569±50 31±3 Soil 95,238±1,180 1,162±116 Teucrium polium G. Kamenjane Village Plant 2,348±264 156±14 (GK) Soil 84,903±986 1,018±102 Parvenetz Village (Pz) Plant 412±33 16±1 Soil 82,380±894 1,156±116 Kazak Village (Kaz) Plant 1,241±99 21±1 Soil 110,831±1,239 1,491±149 Hypericum perforatum G. Kamenjane Village Plant 40±2 3.5±0.2 (GK) Soil 90,860±6,360 1,380±97 Chernichevo Village (Che)

Mn

Plant 99±5 Soil 102,343±948 Fetler Village (F) Plant 63±2 Soil 86,749±585 Dobromirtzi (D) Plant 57±2 Soil 91,773±568 Parvenetz Village (Pz) Plant 52±3 Soil 82,380±894 Rhodiola rosea Rila Mt. (R) Plant 638±35 Soil 14,326±453

1.8±0.2 1,437±123 8.8±0.3 1,129±113 11.7±0.2 1,284±128 1.2±0.2 1,156±116 14.3±1 161±6

35±2 51±3 1,057±53 1,152±69 42±2 20±1 849±42 1,229±79 48±3 18.8±1.1 1,757±193 1,157±104 30±2 8±0.5 1,188±143 669±73.6

9.4±0.4 115±5 4.3±0.2 112±4 5.1±0.5 180±13 2.6±0.2 179±15

119±7 65±4.8 6.9±0.4 722±61 1,936±213 108±9 29±2 4.3±0.3 1.0±0.1 1,113±134 1,422±156 619±43 56±3 17±1 1.1±0.08 1,980±238 2,098±231 302±21 15±2 846±42

47±3 6.4±0.4 71±7 15±12 74±5 54.8±4.4 102±10 51±4.1 31±2 3.1±0.3 80±8 29±2.3

1.7±0.1 118±5

0.61±0.04 6.5±0.3 10.6±0.6 24.8±1

23±2 80±6

1.1±0.1 51±4

21±2 3.5±0.2 1.5±0.1 1,479±143 1,858±178 236±14 12±2 3.5±0.2 1.8±0.1 1,317±145 923±83 182±13 32±4 5.9±0.3 1.4±0.1 1,214±135 963±87 199±13 18±2 4.2±0.2 1.6±0.1 1,113±134 1,422±156 619±43

0.59±0.04 6.4±0.3 2.1±0.1 26±2 0.35±0.04 5.6±0.3 2.3±0.2 29.6±2 0.92±0.07 6.8±0.3 2.2±0.2 36±2 0.79±0.05 9.1±0.4 2.3±0.2 34±2

23±2 75±6 46±3 96.3±7 34±2 68±7 37±2 102±10

1.5±0.1 27±2 1.9±0.1 58±5 0.9±0.1 39±3.0 0.8±0.1 51±4

14±1 113±7

6.1±0.2 2,002±120

1.0±0.1 11.4±0.8 1.3±0.1 63±4 3.0±0.2 10±0.8 2.3±0.2 34±2 0.4±0.03 9.2±0.7 2.1±0.1 26±2

10±3 181±12

3.0±0.2 18±2

0.81±0.12 2.96±0.2 16.13±0.9 1.97±0.1 4±1 28±2 286±12 23±2

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Table 2 (continued) Study sites Empetrum nigrum Rila Mt. (R)

Fe

Plant Soil

165±14 25,542±112

Ni

Mn

16±2 98±4

90±5 123±7

comparison between their metal contents shows concentrations of Mn higher than Ni for C. officinarum , A. trichomanes , and H. perforatum while Ni concentrations prevail in both Teucrium species. The analysis of the rhizome, stems, leaves, and fruits of E. nigrum and R. rosea demonstrated a different distribution of trace elements in plant parts (Table 3). The highest concentrations of Fe, Mn, Ni, Co, and Pb in E. nigrum were found in leaves, while the highest concentrations for Cr, Cd, Cu, and Zn were in fruits. For most elements in R. rosea, the highest amounts were measured in the rhizome. The concentration of Ni varies from 8 mg kg−1 in fruits of E. nigrum to 30 mg kg−1 in the rhizome of R. rosea and Cr from 2 mg kg−1 in leaves in E. nigrum to 18 mg kg−1 in the rhizome of the R. rosea. The results obtained in this study showed concentrations of Ni and Cr over the limits of 10 and 1 mg kg−1 [22] considered usual for serpentine plants. The metals Cu, Zn, and Pb have low concentrations in both species (E. nigrum and R. rosea ) while the toxic metal Cd has concentrations over the limits of 0.5 mg kg−1 proposed by the European Pharmacopoeia [25]. Cluster Analysis The cluster analysis performed with mean values of the metal concentrations for each species is shown to express similarities between plants (Fig. 1). Two clusters (A and B) are formed at a linkage distance of around 400. The species R. Fig. 1 Cluster diagram showing the groups of similarity among the populations investigated

Cr

2.7±0.2 185±11

Co

Cd

2.6±0.3 18±2

0.32±0.03 3±1

Cu

7.3±0.3 11±3

Zn

25.8±1.7 112±10

Pb

1.9±0.1 31±2

rosea altogether with E. nigrum , H. perforatum , and A. trichomanes are separated from the other studied species due to the high concentrations of Fe. Lower concentrations of Mn, Zn, and Cu measured in samples of R. rosea are the reason for their separation from E. nigrum . Also, H. perforatum is in cluster A but separated because of a very low content of Ni and Fe and the highest Zn levels. Cluster B is formed from C. officinarum, T. chamaedrys, and T. polium characterized by higher concentrations for all elements.

Bioaccumulation Factor Bioaccumulation factor (BAF) is calculated as a ratio between the concentrations of elements in plants and in the respective soils. This factor has values Cd>Cu>Mn>Ni>Pb>Fe>Co>Cr A. trichomanes Zn>Cu>Cd>Ni>Pb>Mn>Co>Cr>Fe T. chamaedrys Zn>Cu>Cd>Pb>Ni>Co>Mn>Cr>Fe T. polium Cd>Pb>Zn>Cu>Mn>Ni>Cr>Fe>Co H. perforatum Zn>Cu>Cd>Pb>Mn>Co>Ni>Cr>Fe R. rosea Cd>Co>Mn>Cu>Ni>Pb>Zn>Cr>Fe E. nigrum Mn>Cu>Zn>Ni>Co>Cd>Pb>Cr>Fe It is worth mentioning that the bioaccumulation trend follows the bioavailability of trace elements as determined through extractable fraction in 0.43 M acetic acid (Fig. 2).

Toxic Element Profiles in Selected Medicinal Plants

293

Table 3 Metal concentrations in different plant parts of Empetrum nigrum and Rhodiola rosea. Recorded concentrations are in milligrams per kilogram dry material (mean ± standard deviation of three replicates) Plant organs

Fe

Ni

Mn

Cr

Co

Cd

Cu

Zn

Pb

Empetrum nigrum Stems 150±6 Leaves 211±15 Fruits 134±11

14±1 26±2 8.0±1

113±5 116±8 40±3

2.3±0.1 2.0±0.1 3.8±0.3

1.4±0.1 1.3±0.1 5.0±0.4

0.32±0.02 0.21±0.02 0.42±0.06

7.2±0.3 6.7±0.5 8.4±0.7

21.6±0.9 18.4±1.3 37.5±3

1.3±0.1 3.0±0.3 1.3±0.1

Rhodiola rosea Stems Leaves Rhizome

22±1 18±1 30±1

8±0 10±1 24±1

5±1 7±2 18±3

3.3±0.2 2.7±0.2 3.0±0.1

0.44±0.07 0.9±0.1 1.1±0.2

1.8±0.1 2.7±0.2 4.4±0.2

10.8±0.5 10.0±0.7 27.6±1.4

2.1±0.1 2.5±0.2 1.3±0.1

404±20 476±33 1,033±52

Higher BAFs could be expected for elements like Zn, Cu, Ni, and Pb. Extractable fraction for these elements is between 4 and 6 %, and there are much lower BAFs for elements like Fe and Cr with extractable fraction below 0.05 %. Although the species R. rosea and E. nigrum grow together on the same soil, they demonstrate different trends in accumulation of the metals which evidently shows that plant-specific physiology should also be taken into account.

Metal Correlations, Plant–Soil The correlations between measured elemental concentrations in plant populations and their rhizospheric soils are species specific. C. officinarum demonstrates highly significant correlations at level p C. officinarum > R. rosea > A. trichomanes > E. nigrum > H. perforatum . Impressively low are Fe concentrations measured in H. perforatum comparable to other studied species which obviously are related to its low Fe absorption abilities. This result is comparable to data of other authors for the same species [14, 41] and also to our unpublished data for other Hypericum species growing on serpentines in the Rhodope Mts. (Hypericum montbretii Spach, Hypericum olympicum L.) which are not medicinal plants. Except for H. perforatum and E. nigrum, all other studied species demonstrate abnormal levels of Fe (>200 mg kg−1) in their tissues according to the Element Concentration Cadastre in Ecosystems presented at the 25th General Assembly of the International Union of Biological Sciences [30]. Such behavior of some serpentine plants might

Toxic Element Profiles in Selected Medicinal Plants

be explained from one side with their ability to acidify the rhizosphere and to increase the solubility of ferric ions and their reduction to even more soluble ferrous ion. However, from the other side, contamination problems also have to be taken into account. Some measured values in this study are extremely high, especially the values of over 2,000 mg kg−1 in T. chamaedrys, T. polium, and C. officinarum . Evidently, the usual cleaning procedures are not effective enough for such plants that are densely covered by hairs or scales. Higher values often indicate contamination of samples by serpentine soil or dust not always easily removed by simple washing procedures [42]. The sаme high concentrations have been also reported for different species growing both on serpentines and metalliferous soils [6, 34, 43–46]. High Fe concentrations found in C. officinarum samples and reasonable values for the other studied elements (Pb, Cr, Co, etc.) similar to these for other analyzed plants might be indicative and specific for this species. Probably, high Fe content in plants is not only a unique characteristic for the serpentine flora in Bosnia [47] and Greece [21] but typical for the serpentines of the Balkans as it was mentioned before [21]. High Fe content for C. officinarum in this study is almost identical to that reported by the abovementioned authors for species from different families. These cases are indicative of obligatory careful washing of the raw material before its use for medical treatments. The contents of Cu and Zn in all studied species are typical for plants. Their accumulation and concentration range in plants studied depends from one side on the species physiology and from the other side on their soil bioavailability. Zinc is the metal with the highest biouptake abilities among the studied species. Its toxicity is lower than copper's toxicity. The toxic concentration of zinc 200 mg/kg for dry plant matter [6] is quite above the concentrations found in studied plant samples. Even though E. nigrum and R. rosea are excluders growing on serpentine soils, the distribution of metals in plant organs is specific and certainly depends on biotic (species, genotype, development stage, etc.) and abiotic factors (bioavailability of chemical species of toxic elements in soils, their chelation properties, temperature, soil solution pH, chemical interactions, etc.) [48]. The concentrations of the most of studied metals (Fe, Ni, Cr, Co, Cd, and Pb) in stems of R. rosea were higher in comparison with those in E. nigrum . On the opposite, leaves of the last species demonstrate the highest concentrations of the elements Ni, Mn, Cu, Zn, and Pb. It is known that leaves usually have higher metal concentrations compared to the stems [39, 40, 48] which was confirmed with some exceptions (Zn, Co) in this study. While the highest concentrations for Cu were found in stems of E. nigrum, those for Ni and Pb were found in leaves. Increased accumulation due to increased tissue age reported for these species [49] was not confirmed. E. nigrum

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absorbed high concentrations of Cu and Ni [50], a fact also confirmed by higher BAFs received for these metals. Relatively high contents for most of the studied metals were found in fruits of E. nigrum and in rhizomes of R. rosea, and collecting them for medicinal purposes could be risky.

Conclusions The toxic element contents in plants vary from site to site on a large scale. The concentrations for Fe and Ni in plant samples from the Rhodope Mts. were elevated (except for H. perforatum) while those for Cu, Zn, and Pb were usual for most of the cases. The highest concentrations for all elements were measured in both Teucrium species. The ferns showed the lowest Cr concentrations. Toxic Cd was above 0.3 mg kg−1, a limit proposed for herbal drug [26] in all studied species. Specific differences in the accumulation of metals between ferns and seed plants were not recorded. The metal accumulation in plant parts of the studied species from serpentine soils is different and species specific; it correlates with the bioavailable fraction of trace elements in soils. The investigated species are not hyperaccumulators, but they can accumulate toxic elements from serpentine soils, thus exceeding permissible levels proposed by WHO [26] and European Pharmacopoeia [25]. The control of the quality of medicinal plants and herbal materials used in traditional medicine and pharmacy is an important step for consumer protection from contamination and health risks. Special care must be taken during the administration of routinely used medicinal plants. It is not enough to avoid collection of herbs from areas considered polluted, around factories, roads, etc. There are places like serpentines where natural contaminants such as toxic metals may cause harm to the consumers of herbal products and teas. It is necessary to introduce strict control on the origin of the harvest and harmonize permissible limits for toxic metals and standards at different administrative levels. Acknowledgments The research was realized within Project TK-02/ 39/2009 supported by the National Research Fund at the Ministry of Education Youth and Science in Sofia, Bulgaria. Both anonymous reviewers provided useful suggestions and comments to improve the manuscript.

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Toxic element profiles in selected medicinal plants growing on serpentines in Bulgaria.

Populations of medicinal plants growing on serpentines and their respective soils were analyzed for Fe, Ni, Mn, Cr, Co, Cd, Cu, Zn, and Pb using induc...
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