Bull Environ Contam Toxicol (2013) 91:469–474 DOI 10.1007/s00128-013-1074-8
Arabidopsis arenosa (L.) Law. On Metalliferous and Non-metalliferous Sites in Central Slovakia Ingrid Turisova´ • Toma´sˇ Sˇtrba • Sˇtefan Aschenbrenner Peter Andra´sˇ
•
Received: 7 March 2013 / Accepted: 26 July 2013 / Published online: 4 August 2013 Ó Springer Science+Business Media New York 2013
Abstract This study compared morphological characteristics and seed germinative capabilities of the metallophyte Arabidopsis arenosa grown at a copper mining heap with individuals of the same species grown at a reference site. We observed the height of the plant, the width of rosette leaves at ground level, the width and length of the lowest stem leaf, the number of seeds per silique, the belowground biomass weight and the above-ground biomass weight. We found that the pH and the Eh of soil taken from the root sphere of A. arenosa were similar on both sites, and the pH ranged from 5.87 on the heap to 7.03 on the reference site. The measured morphological attributes and the number of seeds produced were significantly reduced (p \ 0.01) in plants from the metalliferous site. The biggest difference was in leaf length, where plants from the heap were almost 2.5-times smaller. The mean germinative capacity of seeds ranged from 87 % to 93 %, and was not different between sites. The length of roots of germinated seeds from the heap (9.14 mm) was significantly longer than those from the reference site (8.27 mm). Results support the hypothesis of the influence of site conditions and heavy metals on the habitus of the plant and its development. Keywords Morphometry Germination Cold dormancy Heavy metals
In Slovakia, the mining and processing of ores have a long tradition, as evidenced by finds dating back to the I. Turisova´ (&) T. Sˇtrba Sˇ. Aschenbrenner P. Andra´sˇ Faculty of Natural Sciences, Matej Bel University Banska´ Bystrica, Tajovske´ho 40, 974 01 Banska´ Bystrica, Slovakia e-mail:
[email protected]
Early Bronze Age, and reaching a climax in the Middle Ages. In central Slovakia, in the surroundings of Banska´ Bystrica, this period is associated especially with the extraction of copper carried out by the world-famous Thurza-Fugger company that operated here in the fifteenth and the sixteenth century (Jurkovicˇ 2005). Even after the dissolution of the company, the extraction of copper continued until the beginning of the twentieth century. The intensive mining activity left its traces in the landscape, which are visible above all in the form of heaps. These heaps are sites having specific ecological conditions that distinguish them from the surrounding environment. They often contain increased to extreme amounts of heavy metals in comparison with their natural levels in soil not affected by human activity; there, only such plants can grow that are able to survive in extreme conditions thanks to their specific strategies and mechanisms (Bana´sova´ 1976). Already in the sixteenth century, Agricola (2011) took notice of this in observing only certain plant species along the ore vein. The intensity of research into the influence of heavy metals on vegetation, flora, tissues and physiological processes has considerably increased since then. The aim of the submitted contribution was the evaluation of morphometric analyses and seed germinative capacity in the case of Arabidopsis arenosa (L.) Law., as a typical representative of the heaps from copper mining and processing in the Stare´ Hory—Sˇpania Dolina ore district in central Slovakia. Individuals growing on a copper mining and processing heap were compared with individuals growing on a reference site. Results should point to specific site conditions associated with the increased content of heavy metals in the heaps from copper mining and processing.
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Materials and Methods The field research into A. arenosa (Brassicaceae family) was done at a heap at Richta´rova´ near Banska´ Bystrica (central Slovakia) and a reference site in its vicinity in the course of the growing season in the years 2011–2012. The measurements of selected attributes were taken in May, when all above-ground organs of individuals were fully developed. In each of the two areas, 30 individuals were selected by stratified sampling, and the main stem height, length and width of blade of the lowest assimilatory leaf on the main stem, and leaf rosette diameter were measured. We also counted the number of seeds in the oldest silique on the main stem. We carried out the stratified sampling of 60 individuals (30 from each of the areas) with fully developed seeds in July–August 2011. At the same time, we took soil from the area of the root ball of each plant from the heap and reference site; to obtain two representative soil samples (one from the heap, one from reference site), we homogenized the soil and determined the pH and Eh of soil using distilled water and 1 M KCl solution (cf. Sobek et al. 1978). The content of heavy metals in soils was measured using an ICP-MS method by ACME Analytical Laboratories (Vancouver, BC, Canada). The concentrations of 16 metals were measured in the homogenized soil sample from each of the two sites. The resulting value is the average of two parallel control measurements. For the determination of dry aboveground biomass and below-ground (root) biomass, we collected and weighed 16 plants (the number of them was limited by the number of individuals on the reference site) from each of the areas. The germinative capacity of seeds was observed in three periods to determine the effect of cold dormancy at -8°C for 8, 10 and 12 weeks. A week before the experiment, the seeds were stratified (moistened with water and stored in wrapping paper at a temperature of 5°C). We carried out the observation of germinative capacity on filter paper in petri plates on 50 seeds randomly selected from the heap and the reference site at compensated ambient temperature of 20°C (±1.5) with a natural photoperiod. The germinative capacity was evaluated daily; we considered seeds with roots of 1 mm as a minimum to be germinated. From the daily germination percentages, cumulative germination percentages after 5, 10 and 15 days and the final germination percentage were calculated. The length of the root of each new germinating seed was measured as well (cf. ISTA 1985). The nomenclature of plant taxa followed Marhold and Hinda´k (1998). Student‘s t test was used to statistically evaluate differences between means. Floristic and ecological characteristics of the localities are as follows: (1) The heap from copper mining and
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processing at Richta´rova´, Stare´ Hory cadastral area, Starohorske´ vrchy Mts., central Slovakia is 653 m above sea level, with GPS coordinates of N48.493141, E19.075282. It has Agrostis capillaris forming an almost continuous cover in places with well-developed soil. Acetosella vulgaris, A. arenosa and S. dioica are other predominant species on the site. Of lichenized fungi, Cladonia mitis and another Cladonia species predominate (Bana´sova´ et al. 2006; Aschenbrenner et al. 2011). The pedogenically young soil types are formatted on natural cambisols. These are skeletal soils with a shortage of essential nutrients and water. (2) The Dolny´ Sˇtu´rec reference site, Stare´ Hory cadastral area, Starohorske´ vrchy Mts., central Slovakia is a clearing above the heap at 949 m above sea level, with GPS coordinates of N 48.485329, E 19.085240. On the site, there are substitute associations of class Epilobietea angustifolii Tu¨xen et Preising ex von Rochow 1951 with a predominance of Rubus idaeus and a relatively abundant population of A. arenosa. Soil type is represented by skeletal cambisol.
Results and Discussion For the assessment of homogeneity of and variability in the attributes measured on A. arenosa we calculated the indicators of variability and central values (Table 1), from which it follows that the average values of particular morphometric attributes measured in the heap were in all cases statistically significantly lower than the average values from measurement on the reference site. Based on the coefficient of variation (CV), we can confirm a relatively high variability in each attribute in the framework of individuals from both the heap and the reference site, which indicates that A. arenosa is a morphologically variable species regardless of the type of site. For the weight of above-ground parts of A. arenosa and the weight of roots (Table 2), we did not observe any significant difference in average value, but we observed high CV values in the case of individuals from the reference site. This indicated a higher morphological plasticity of the species in non-disturbed areas. In stress conditions induced by specific site conditions in the heap (mainly by microclimate and trophic conditions and by increased content of heavy metals in the substrate), plants produce, on an average, similar weights of above-ground and below-ground biomass. We recorded the statistically significant difference at a level of significance p \ 0.01 also in the number of seeds of the oldest silique, where the number of seeds in the plants from the heap was smaller by 6 on an average. The pH values on the reference site (Table 3) were neutral, while those in the heap were slightly acidic (however, at normal values of soil pH). In soils, Eh values
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Table 1 Central values, indicators of variability and results of Student’s t test for measurements of selected attributes on A. arenosa Central values
Leaf length
Leaf width
Plant height
Width of rosette of leaves
Number of seeds per one silique
Heap
Heap
Heap
Heap
Heap
RS
RS
RS
RS
RS
Measurements
30
30
30
30
30
30
30
30
30
30
Average
19.4
49.4
7.6
15.9
18.6
30.6
5.0
10.5
13.3
19.4
Median
20
48.5
6.5
16
19
29.3
4.6
10.3
12
19
Mode
20
38
5
17
20
27
3
9.5
13
21
Variance
56.8
127
10.0
15.7
17.3
21.2
3.9
5.7
7.9
5.4
SD
7.5
11.3
3.2
4.0
4.2
4.6
2.0
2.4
2.8
2.3
CV t test (sl)
38.9 22.8 0 (99 %)
39.4 0 (99 %)
22.9
21.3 0 (99 %)
12.0
41.6 24.9 0 (99 %)
22.4 15.1 0 (99 %)
SD standard deviation, CV coefficient of variation, sl significance level, RS reference site Table 2 Central values, indicators of variability and results of Student’s t test for weights of roots and above-ground parts of A. arenosa Central values
Weight of roots (g)
Weight of above-ground parts (g)
Heap
RS
Heap
RS
Measurements
16
16
16
16
Average
0.25
0.40
0.76
1.39
Median
0.22
0.4
0.65
1.18
Variance
0.01
0.06
0.26
0.51
Standard deviation
0.09
0.23
0.49
0.72
CV
36.7
50.03
65.02
51.68
t test (sl)
0.001 (99 %)
0.013 (95 %)
RS reference site, CV coefficient of variation, sl significance level
Table 3 Values of soil pH and Eh from the root system of plants Water
1 M KCL solution
pH
Eh
pH
Eh
Reference site
7.03
0
6.53
29.4
Heap
5.87
25
5.57
45
were measured that indicated unambiguously neither oxidation nor reduction processes; nevertheless, the value of the Eh readings were not stable. They fluctuated even with very small distances, and were dependent, upon the amount of precipitation. Results for the mean germinative capacity of seeds did not show a significant difference between seeds from the heap and those from the reference site at any of the experimental variants of cold dormancy time or days of germination (Fig. 1). This indicates a resistant genetic apparatus of the studied species for which propagation by seeds is the main type of reproduction. This hypothesis is also supported by the high average germinative capacity of
seeds that ranged from 87 % to 93 % with 10 or 15 days of germination time. The duration of cold dormancy, as well as the duration of germination, affected the germinative capacity. We recorded the highest number of germinating plants after 10 weeks of cold dormancy; already after five days, almost 70 % of seeds had germinated. The maximum daily germination percentage was achieved between the 4th and the 6th day, after which the germinative capacity decreased in all variants until the 11th to the 14th day; after that, we no longer recorded any new germinating seeds. In comparison with the germinative capacity of other species, this was however very high (87 %–93 %), which was confirmed in the case of genus Arabidopsis by Rivero-Lepinckas et al. (1998) as well. Cambpell (1985) stated in the case of Hypericum perforatum that the germinative capacity after cold dormancy was 68 %. Although stratification has been reported to favorably influence germinative capacity (cf. Cambpell and Delfosse 1984; Baskin and Baskin 2001), this was not confirmed in our study. The stress environment of the heap with a lack of nutrients and soil moisture probably manifests itself in the length of roots of germinating seeds that are statistically significantly longer than those of germinating seeds from the reference site (Table 4). Heaps represent a specific type of biotope in which plants are exposed to permanent stress. The specifics of this biotope are very sandy, skeletal and dry soils, relatively long direct sunshine duration, frequent wind, lack of basic soil nutrients, minimum of water, and increased concentrations of heavy metals. Metal concentrations are especially high in historically old heaps, when metal-extraction technologies were less than perfect (Bana´sova´ 1976). The taxa that are generally designated as heavy metaltolerant taxa are often characterised by the high viability that is a result of good adaptive mechanisms and an ability to eliminate nutrient-competitive species that are less adaptive (Lambinon and Auquier 1963). This may be a
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Fig. 1 Cumulative germination percentages of seeds of A. arenosa after 8, 10 and 12-week cold dormancy. RS reference site, SG 5 summary germination after 5 days, SG 10 summary germination after 10 days, TG (15) total germination after 15 days
Table 4 Central values, indicators of variability and result of Student’s t test for the length of the roots of germinating seeds
Table 5 Contents of heavy metals in soil from the root system of A. arenosa
Central values
Element
Heap
Reference site
-1
Mo
Cu
Pb
Zn
Ag
Ni
Co
Mn
50.3
70
2.5
17.7
18.2
770
Th
Sr
Cd
Sb
Bi
19.8
62
0.3
307.4
13.6
Measurements
30
30
Heap (mg kg )
0.7
938.5
Average
9.14
8.27
Element
As
U
Median
7.9
6.9
Mode (modus)
9
8.15
Heap (mg kg-1)
166
4.7 \0.1
Variance
0.95
1.32
Standard deviation
0.97
1.15
Coefficient of variation
10.66
13.88
t test (significance level)
p \ 0.01 (99 %)
p value
0.007
result of phylogenesis not only within the taxonomic group, but even among populations of the same species (Ernst 2006), or microevolutionary changes manifesting themselves in the development of tolerant ecotypes (Ernst et al. 1992; Ye et al. 1997; Wierzbicka and Rostan´ski 2002; Zhang et al. 2011). We assumed that the most significant influences on the whole ontogenesis of individuals and the development of populations in our study were those of an increased content of heavy metals in sediments and soil substrate (Table 5), availability of nutrients and microclimate characteristics. The concentrations of heavy metals in soil from the heap, especially Cu, As and Sb, were much higher in comparison to soil from the reference site, probably due to polymetallic mineralization. These values exceeded the limit set by Slovak legislative standards. These factors have been noted to influence species such as Acetosella vulgaris, Agrostis capillaris, Avenella flexuosa, Silene dioica, Thlaspi caerulescens (Ernst 1974; Bana´sova´ 1983; Baker et al. 2000; Bana´sova´ et al. 2006; Przedpelska and Wierzbicka 2007; Milner and Kochian 2008; Cˇiamporova´ et al. 2009).
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Au
Element
Mo
Cu
Pb
Zn
Ag
Ni
Co
Mn
RS (mg kg-1)
5.4
196
39.6
95
0.9
27.5
11.7
558
Element
As
U
Au
Th
Sr
Cd
Sb
Bi
RS (mg kg-1)
68
3.5
\0.1
9.4
126
0.9
44.4
5.1
Marks an excess of heavy metal in comparison to published limits in ‘‘Decision of Ministry of Agriculture of the Slovak Republic on the maximum allowed values of pollutants in soil no. 531/1994–540’’ or with average and critical values by Bowen (1979) and Kabata-Pendias and Pendias (1992) RS reference site
The mentioned species are, in addition to A. arenosa, the main components of central Slovakian heaps from copper mining and processing, as well. Arabidopsis arenosa was also studied in the heap from lead–zinc mining and processing at Boleslaw in Poland (Przedpelska and Wierzbicka 2007). By comparing the results, we can state the same conclusions that confirm significant differences in morphometrics of observed attributes between the individuals in the heaps and those on the reference sites (Table 6). In the heap from copper mining and processing, the individuals were altogether more sturdy than those in the heap from lead–zinc mining and processing; in the same manner they differed on the
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Table 6 Comparison of selected morphometric characteristics of A. arenosa from the sites in Poland and Slovakia Measured attribute
Poland Heap Boleslaw
Slovakia Heap Richta´rova´
Plant height (mm)
125.5 ± 5.6
185.7 ± 41.5
Leaf width (mm)
5.0 ± 0.2
6.5 ± 3.17
Leaf length (mm)
15.8 ± 0.5
19.37 ± 7.54
Number of seed per one silique
11.9 ± 1.5
13.26 ± 2.83
Measured attribute
NP Kampinos (RS)
Plant height (mm)
244.9 ± 13.5
305.8 ± 1.98
Leaf width (mm)
11.7 ± 0.5
15.93 ± 3.97
Dolny´ Sˇtu´rec (RS)
Leaf length (mm)
35.8 ± 1.1
49.4 ± 11.29
Number of seed per one silique
22.5 ± 1.8
19.3 ± 2.33
Source: Przedpelska and Wierzbicka (2007), NP National park, RS reference site
reference sites as well (with the exception of the number of seeds per silique). Based on the results, we agree with the above-mentioned authors in the assumption that a decrease in the area on the plant for the evaporation of water is a typical adaptation to xerothermic site conditions. This is also confirmed by the finding that the roots of germinating seeds from the heap grow faster than those of germinating seeds from uncontaminated localities, which may be influenced by a low content of nutrients too. Longer roots are able to more readily and effectively accept scarce nutrients dissolved in a limited supply of water. Arabidopsis arenosa is one of the dominant species in the floristic composition of cuprous heaps and is often regarded as a metallophyte (Zarzycki et al. 2002). High germination percentages of seeds of individuals from the copper mining heaps together with their ability to survive the generally unfavorable conditions of the heaps may serve to make this species a viable candidate for the phytoremediation of metalliferous soils. This hypothesis is necessary to confirm in future research. Acknowledgments The work was financialy supported by grant scheme APVV-0663–10, VEGA 2/0065/11, VEGA 2/0099/13.
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