Phytotoxicity of polymetallic mine wastes from southern Tuscany and Saxony

Ecotoxicology and Environmental Safety 162 (2018) 505–513

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Phytotoxicity of polymetallic mine wastes from southern Tuscany and Saxony☆

T

⁎

J. Franzaringa, , S. Ancorab, L. Paolic, A.H. Fongoha, P. Büttnere, A. Fangmeiera, S. Schlosserd, F. Monacic a

University of Hohenheim, Institute for Landscape and Plant Ecology (320), August-von-Hartmann-Str. 3, D-70599 Stuttgart, Germany University of Siena, Dept. of Physical Sciences, Earth and Environment, University of Siena, Via Mattioli 4, I-53100 Siena, Italy c University of Siena, Dept. of Life Sciences, University of Siena, Via Mattioli 4, I-53100 Siena, Italy d Core Facility Hohenheim (CFH), Emil Wolff Str. 12, D-70599 Stuttgart, Germany e Helmholtz Institute Freiberg for Resource Technology, Halsbrücker Str. 34, D-09599 Freiberg, Germany b

A R T I C LE I N FO

A B S T R A C T

Keywords: Mine wastes Phytoremediation Heavy metals Oilseed rape Buckwheat Soil amendment

Restoration potential of mine wastes or approaches to improve soil conditions and to ameliorate phytotoxicity on these sites may be simulated in standardized greenhouse experiments. Plants can be cultivated side by side on materials from different origins in dilution series with defined admixtures of certain aggregates. Mine wastes used in the present study originated from Fenice Capanne (FC, Tuscany, Italy) and Altenberg (ALT, Saxony, Germany). Tailings of the Italian site contain high concentrations of lead, zinc, arsenic and sulphur while tin, wolfram, molybdenum and lithium are highly elevated in the German mine waste. We tested growth responses of five crop species and analyzed concentrations of various metals and nutrients in the shoot to evaluate the toxicity of the FC mine waste and found oilseed rape being the most and corn the least resistant crop. Interestingly, oilseed rape accumulated seven times higher levels of lead than corn without showing adverse effects on productivity. In a subsequent comparison of FC and ALT mine waste, we cultivated different species of buckwheat (Fagopyrum spec.), a fast growing genus that evolved in mountain areas and that has been shown to be tolerant to low pH and high concentrations of metals. We found that the FC mine waste was more toxic than the ALT substrate in F. tataricum and F. esculentum. However, lower admixtures of FC material (10%) resulted in stronger growth reductions than higher proportions (25%) of the mine waste which was primarily related to the slightly lower pH and higher availability of essential metals due to the admixture of sand. These results confirm the importance of managing the soil chemical and physical characteristics of wastelands and call for the development of assisted reclamation to prepare sites for regular biomass production.

1. Introduction In many regions of Europe historical metal extraction and smelting activities left behind large deposits of mine wastes and slags, which over the time became re-vegetated by grassy and shrubby pioneer vegetation and secondary forests without human intervention. Today, only few mines and smelters remain active due to economic and environmental constraints and the operation of these facilities as well as the controlled flooding of underground mines are highly regulated using best available technologies. Adverse mining and smelting impacts in modern times are mostly locally restricted, while historical activities affected larger areas by the deposition of airborne particles and unmanaged discharges of metal sludge into the rivers (Ernst et al., 2009).

Furthermore, the long-term and partly continued use of metal slags as liming materials and fertilizers in agriculture and as road construction materials have added up to the geogenic metal levels Europe wide. Repeated fine-scale, pan-European geochemical mapping projects will enable us to identify pollution hotspots and to assess the enrichment of toxic metals in soils and sediments over time (e.g. Fauth, 1985; Salminen et al., 2005; Reimann et al., 2014; Tóth et al., 2016; Birke et al., 2017). Historical mining sites are an often neglected cultural and industrial heritage and knowledge about their geochemistry, geomorphology and ecology should not fall into oblivion. Since heavy metals will not degrade in the soils, ancient mining sites may be useful outdoor laboratories for the study of long-term effects of these pollutants on biota

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Corresponding author. E-mail address: [email protected] (J. Franzaring).

https://doi.org/10.1016/j.ecoenv.2018.07.034 Received 29 May 2018; Received in revised form 4 July 2018; Accepted 8 July 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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German polymetallic mining site. The objective of the investigation was to develop a standard phytotoxicity testing scheme for mine wastes using crop plants. Since these plants produce more biomass than the metallicolous species naturally invading former mine sites, a combined approach based on soil improvement (fertilization and substrate loosening) of the mine waste and a directed cultivation of crop and bioenergy plants may favor the stabilization and carbon accumulation of those substrates. Provided that heavy metals are retained in the root zone, shoot mass can be subjected to bioenergectic uses, while accumulation of heavy metals and certain strategic elements in the hyperaccumulating shoot would be a prerequisite in phytomining.

(Camizuli et al., 2018). While information on the geographical distribution of mines is available for sites that continued to produce until modern times, cadasters of long abandoned ancient mines will be necessary for environmental impact studies as well as for assessing their potential re-use or re-mining of strategic metals. Some of the places may be prone to ground collapses and the subsequent leaching of acid mine drainage (AMD) and toxic metals (e.g. Environment Agency, 2008). Erosion of the deposited material will be more pronounced when vegetation is removed or damaged e.g. by severe weather events and fires. Recent examples for ecological studies on ancient mine wastes in Wales and Spain are presented by Davies et al. (2016), Rossini-Oliva et al. (2016) and Fernández et al. (2017). The increased chemical weathering under climatic change (higher temperatures and precipitation) will probably lead to higher releases of AMD and metals in the future. On the other hand, former mining sites may offer chances as a readily available, pre-processed substrate for re-mining e.g. for strategic and rare earth metals. In nature conservation they may be used for the creation of wealthy secondary habitats, i.e. calaminarian and serpentine grasslands hosting highly adapted and endemic plants and insects (Baumbach et al., 2007). In the EU, these open semi-natural grasslands (habitat code 6130) have been protected under the habitats directive and the Natura2000 network. While the establishment of secondary nature should be restricted to outcrops with high metal pollution, flat basins of moderately polluted former mine sites may offer some potential for the cultivation of bioenergy crops and short rotation coppices. Since plants are the first component of the food chain and metals can be both essential and non-essential for plant growth, it is important to know how these metals will interact with the physiology and ecology of plants and why some of the plant taxa were able to develop metal tolerant lines (metallophytes). These ecotypes are able to persist on former mine sites and have developed mechanisms to detoxify or exclude metals. The presence of and the metal concentrations in certain plant species can be used for the environmental monitoring at abandoned mines, but the involved taxa can also be used in vegetation assisted clean-up and remediation approaches, often referred to as phytoremediation and phytostabilization (Mahar et al., 2016). However, such plants are rather unproductive and are adapted to the harsh conditions and nutrient poor conditions in metal enriched soils. In order to reduce the stress created from a low availability of nutrients and essential elements, fertilization of such sites and soil amendments can be an option to improve the growth conditions and productivity locally with changes in plant composition (Chiarucci et al., 1998). While metallophytes and the associated vegetation and mechanisms for metal accumulation on polymetallic sites were first addressed by Ernst (1974) and Baker (1981), many studies thereafter dealt with the effects of heavy metals on plants in laboratory and field studies. When addressing growth responses to heavy metals, native soils from metal sites are better suited than artificial substrates and the addition of rapidly soluble metal salts. Combining field and lab approaches gives a realistic impression of the toxicity and restoration potentials of postmining substrates (Tesnerová et al., 2017). Using solid substrates with near-natural soil properties and relevant plant species should be preferred over using hydroponic cultures and cell cultures of model plants, which will not be able to mimic the conditions that drive the phytoavailability of elements in the infertile polymetallic sites. However, labbased approaches and the use of defined metal solutions may help to investigate the involved biochemical and molecular biological mechanisms. Besides phytotoxicity studies, classical OECD acute toxicity tests can simulate whether soil biota would also be affected at such sites (Finngean et al., 2018). Before going into the field, the amendments with fertilizers, chelators, sorbents, organic matter as well as pH stabilizers and the improvement of the soil texture of mine wastes can all first be studied on the lab scale. In present study we performed pot experiments with dilution series using a standard growth medium and wastes from an Italian and a

2. Materials and methods 2.1. Mine waste materials used in the study The Fenice Capanne (FC) sulphide deposit belongs to the Tuscan Colline Metallifere and the extraction and smelting of metals (Zn, Cu, Pb, Fe, Ag) dates back to the Etruscan times. Activities at the site were given up in 1985 and the flotation tailings and roasting piles remained untreated ever since. Environmental pollution at the site has been studied extensively by Benvenuti et al. (1997) and Mascaro et al. (2001) and studies related to the heavy metal tolerance of a metallicolous ecotype of Silene paradoxa L. and other species present in FC, were performed by Gonnelli et al. (2001), Arnetoli et al. (2008), Marchand et al. (2014) and Colzi et al. (2014). While these investigations focused on sulphidic substrates and plants adapted to rather acidic soils, the Italian researchers Angelone et al. (1993), Bini et al. (2017) and Selvi et al. (2017) focused on serpentine soils close to the study site and often addressed the nickel accumulator Alyssum bertolonii L. A composite sample of 50 kg was obtained in April 2017 from tailing basin no. 1 (see photo in Mascaro et al., 2001) at the former mine at FC. The material had been deposited between 1957 and 1964 and contains high concentrations of heavy metals (Table 1). Soil cores of the yellowish to reddish sandy-silty material were taken at 20 vegetation-free positions from the upper layer (0–20 cm). A list of the few plant species present at the site is given in the results section. After the transfer of the FC material sample to Germany, it was homogenized with a concrete mixer, air dried for a few days and sieved to 2 mm. Then the material was blended with washed river sand and a standard fertilized growth substrate (LD80) at defined proportions to generate five treatments ranging from 0% of FC material (the control) to the highest treatment containing 25% of the FC material. Every treatment was made up with 50% of the LD80 material to guarantee a basic supply of nutrients. Although the percentage of sand varied between 25% and 50%, we assume that differences in soil texture did not largely affect the outcome of the experiment. Fruhstorfer LD80 (Gebr. Patzer GmbH & Co. KG, Sinntal Jossa) is a standard plant growth medium. It is composed of peat, volcanic clay, bark humus and is enriched with a slow-release fertilizer. Having a pH of 5.9, the substrate contains 35% organic matter and has a salt content of 1 g L-1 KCl. Plant available nutrient concentrations are 150 mg L-1 each for N and P and 250 mg L-1 of K. The high share of the standard earth in each of the treatments guaranteed an adequate and similar supply of nutrients during the experiments. While LD80 and sand are free from heavy metals, we assumed sand and the FC substrate had low nutrient concentrations. We addressed the chemical composition (heavy metals and nutrients) of the material used in present study and compared these results to published data (Table 1). However, we determined the HNO3 extractable pseudo-total concentrations and did not include other extraction methods. In the study of Pignattelli et al. (2012) only low amounts of these metals proved to be phytoavailable after an extraction with 0.01 M CaCl2 solution. When comparing the heavy metal loads of the diluted FC material to the precautionary values of the German soil protection and contaminated sites ordinance (BBSchV, 1999), we may 506

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Table 1 Information on the chemistry of the original Fenice Capanne (FC) substrate from the literature and results of analyses performed on the mixed substrates of FC and Altenberg (ALT) used in present experiment. "LD80" refers to the fertilized standard earth that made up half of the substrates in each of the treatments and the controls. Precautionary values for three heavy metals addressed by the German legislation are included in the lower part of the table. pH

Pb ppm

Zn ppm

As ppm

Original material (Basin 1) according to literature Mascaro et al. (2001) Basin I 7.1 1310 5470 230 Pignattelli et al. (2012) Total 5599 5223 753 Plant available 25.1 32.8 3.35 (%) 0.4 0.6 0.4 Marchand et al. (2014) not specified 7.4 66 720 Chemistry of the mixed substrates Present study FC Sand LD80 (%) (%) (%) 25 25 50 6.1 1064 240 198 12.5 37.5 50 6.05 532 120 99 6.25 43.75 50 5.94 266 60 49.5 3.125 46.875 50 5.92 133 30 24.8 0 50 50 5.86 0 0 0 ALT Sand LD80 (%) (%) (%) 25 25 50 5.35 9.92 22.9 220 10 40 50 5.38 3.97 9.16 88 Precautionary values of the German Soil Protection and Contaminated Sites Ordinance (BBSchV, 1999) clayey soils (< 8% org. matter) 100 200 sandy soils (< 8% org. matter) 40 60

Cd ppm

K ppm

Mg ppm

P ppm

C %

N %

S %

7 2 0.19 9.5 1.03

0.09

0.01

0.6 0.3 0.15 0.07 0

1599

1290

425

7.76

0.27

0.77

0.11 0.04

3617

1243

363

7.52

0.26

0.09

1.5 0.4

week. 35 days after sowing (DAS) plants were harvested. Before destroying the plants, shoot length was determined and the leaf greenness i.e. the relative chlorophyll concentration of the first true leaf was measured with a SPAD-meter 502 Plus (Konica Minolta). The destructive harvest after five weeks served to address species-specific growth differences between the tested crop species subjected to different soil mixtures, i.e. treatments. After determining the leaf area (LiCor leaf area meter) and counting the leaves, fresh weights of leaves and stems were noted. Stems and leaves were put into labelled bags and dry weights of these fractions were determined after drying them at 80 °C until constant weight. After weighing, leaves and stems of each plant were combined and subjected to the determination of heavy metals in the shoots. For the comparison of phytotoxicity between the FC and ALT mine wastes (study II) we grew three species of buckwheat, i.e. the annual species common buckwheat (F. esculentum Moench) and tartary buckwheat (F. tataricum (L.) Gaertn.) and the perennial buckwheat (F. cymosum (Trev.) Meisn.). While F. tataricum (two cultivars) and F. esculentum (one cultivar) stemmed from commercial seed suppliers, germplasm of F. cycmosum (accession FAG 142) was supplied by IPK Gatersleben (Germany). In a previous experiment, common buckwheat had proved to be rather tolerant to the ALT mine waste (Franzaring et al., 2018), while tartary buckwheat died at a mine waste concentration of 25%. Five replicates per accession (four accessions of three species) and treatment (one control and two dilutions of two mine waste substrates) were cultivated in rectangular pots (1.73 L) that were filled with substrates made up with either 0 (control), 10 or 25% of the FC and ALT mine wastes. The experiments lasted for 90 days for a complete growth cycle and plants were kept outdoors under a roof. Watering was done manually. Plant assessments were based on weekly measurements of length increments and leaf greenness (SPAD) and the final dry shoot mass was determined at the end of the vegetation period.

conclude that the very high Pb levels would be of great concern even in the lowest dilution level. In contrast, levels of Cd and Zn would exceed the BBSchV-levels for clayey soils only in the highest treatment level (FC25%). As indicated by the pH values of the different treatments, mixing of the basic mine waste with sand and the slightly acidic LD80 material decreased the pH from the higher to the lower dilutions which may have increased the plant availability of metals in these treatments. The Altenberg (ALT) cassiterite (SnO2) occurrence stems from late variscan formations in the Saxonian Ore Mountains. Mining started in the Middle Ages and was ceased in 1991. Afterwards, most of the waste and flotation tailings were covered with a water proof layer and construction rubble. The involved mine waste contains high levels of Sn, As, W, Mo and Li (Büttner et al., 2018). For a detailed description of the metal concentrations, the sampling of the material and its phytotoxicity in selected crop species, refer to Franzaring et al. (2018). In the near future, mining activities will be starting again in Altenberg due to the anticipated Zinnwaldit (lithium) boom. In the present experiment (study II) we used the ALT and FC mine wastes to test different buckwheat species and to compare the toxicity of different mine wastes. Besides a control that was made up of 50% of LD80 growth substrate and 50% of sand, we prepared treatments that contained 10% and 25% of either the FC or the ALT mine waste. The chemical analysis of the ALT25% shows that it is slightly more acidic than the FC25% material and that it contains only a hundredth of Pb, a tenth of Zn, a fifth of the Cd, but similar concentrations of As as compared to the Italian mine waste. 2.2. Plant cultivation In order to test the phytotoxicity of the FC mine waste (study I), plants were cultivated in plastic pots (344 mL) and five replicates were provided per treatment and species. Originally we used seven crops, but quinoa (Chenopodium quinoa L.) and common buckwheat (Fagopyrum esculentum Moench) seeds did not germinate well so that replicate number was too low. In the five crops, Brassica napus L. (oilseed rape), Cucumis sativus L. (cucumber), Glycine max (L.) Merr. (soybean), Phaseolus vulgaris L. (garden bean) and Zea mays L. (corn) all replicates were available to study species specific growth responses and heavy metal uptake. Pots were equipped with saucers underneath, were watered on demand and were randomized on the growth benches once a

2.3. Chemical analyses Soil pH was determined with a WTW pH meter (Weilheim, Germany) in solutions resulting from mixing 20 g of dried and sieved (2 mm) FC or ALT substrates of the 10 or 25% dilutions in 50 mL deionized water. Plant and soil material was milled with a ball mill (MM2000, Retsch GmbH Haan, Germany) and digested with nitric acid 507

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Fig. 1. Photos showing growth responses of five crop species grown in different dilutions of FC mine waste (metal levels and soil pH increasing from left to right). Different letters denote significant differences in shoot mass accumulation as tested by post-hoc multiple comparisons (Tukey tests). Top right: combined doseresponse functions for growth reductions and underneath, lead accumulation in the five crops species based on the mean Pb levels (dots) determined in plants that were grown in substrates with defined levels of FC mine waste (x-axis, % of total substrate).

buckwheat experiment (study II), in which the toxicity of mine wastes from FC and ALT was compared, we used the ´multicomp´ package and Tukey tests to address significant differences between the different waste materials and concentrations. Pearson product moment correlation analyses were performed to address associations between metals, nutrients and plant biological parameters.

in an UltraCLAVE III microwave heated digestion unit (MLS GmbH, Leutkirch, Germany). Cd, Pb, Zn, Cr, Cu, Ni, Sb, V, Ba and As were determined using a NexION 300× inductively coupled plasma mass spectrometer (ICP-MS) by PerkinElmer (Rodgau, Germany). The nutrients K, Mg and P and Al and Fe were determined in the same solutions with an ICP-OES 5110 SVDV simultaneous inductively coupled plasma optical emission spectrometer (Agilent, Santa Clara, USA). For the determination of CNS, 15–30 mg of the milled samples were weighed into tin cups and analyzed using a vario EL CUBE element analyzer (Elementar Analysensysteme GmbH, Hanau) coupled to an automatic sampler.

3. Results and discussion 3.1. Phytotoxicity of mine waste from Fenice Capanne (FC) 3.1.1. Description of the plant species present on the tailing basin Comparing aerial views from the late 1980's (Mascaro et al., 2001) with recent photos shows that during the last 30 years the vegetation cover had not much spread towards the center of the basin owing to the compact structure, anoxic, dry and nutrient poor conditions and most likely, high heavy metal contents of the substrate. Typical pioneers on the site are the Caryophyllaceae Silene otites (L.) Wibel and Cerastium spec., while we did not observe the metallicolous species Silene paradoxa L., which has been observed elsewhere in the Fenice Capanne mine and which has been investigated extensively by Gonnelli et al. (2001), Arnetoli et al. (2008) and Colzi et al. (2014). We also found the legumes Spartium junceum L., Medicago minima L. and Dorycnium

2.4. Statistical analyses Graphical representations and statistical analyses were performed with Microsoft Excel® and R version 3.1.1, the language and environment for statistical computing and graphics (R Development Core Team, 2008). After the calculation of treatment means and standard deviations, dose-response curves were created using log-logistic functions from the ´drc´ package. For each of the tested plant species in the experiments with mine waste from FC (study I), effective doses (ED50) were calculated, i.e. the concentrations of mine waste that reduced growth by 50%. In both the FC phytotoxicity (study I) and the 508

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Table 2 Summary of mean concentrations of selected elements, nutrients and the biological parameters mean leaf area (MLA), specific leaf area (SLA), leaf area ratio (LAR) and water contents of the five tested species after treatment levels. Color coding refers to low (green), medium (yellow) and high (red) levels classified for each of the parameters (columns) across the five species. Values of the “reference plant” in the last line stem from Markert (1992) and can be used to evaluate the analytical results.

Treatment %

Oilseed rape 0 3.125 6.25 12.5 25 Bush bean 0 3.125 6.25 12.5 25 Corn 0 3.125 6.25 12.5 25 Soybean 0 3.125 6.25 12.5 25 Cucumber 0 3.125 6.25 12.5 25 Reference (Markert, 1992)

Al

As

Cd

Cu

Fe

Pb

Zn

Ni

Sb

C

N

S

K

P

Mg

C:N MLA 2

SLA 2

H2O

LAR -1

2

-1

cm g cm g

% DM

38 38 38 40 44

344 336 356 324 324

284 268 286 257 257

91 91 91 91 91

8.7 9.6 9.5 9.9 8.4

111 131 122 105 71

375 365 362 341 358

274 264 259 236 258

86 86 85 84 85

2955 3022 2454 2081 2233

11 9.9 11 12 12

98 86 93 83 54

415 430 409 425 457

210 231 211 221 234

90 90 90 89 89

5203 4006 3848 2525 1257

4622 4566 4466 4751 4342

9.5 9.6 9.3 9.6 11

20 24 20 20 18

369 414 378 364 349

257 290 258 254 235

82 83 83 82 79

5146 4133 3979 3212 1966

7411 7797 7638 8013 9229

7 6.5 5.9 6 7.7

123 124 118 113 67

357 356 352 376 291

259 259 255 272 219

91 91 92 91 88

0.30 19000 2000

2000

cm

ppm ppm ppm ppm ppm ppm ppm ppm ppm %

%

%

ppm

ppm

ppm

80 84 93 106 113

0.28 0.51 0.68 0.99 1.53

0.61 0.77 0.91 1.45 1.67

3.7 6.8 8.3 12.3 13.8

70 78 97 125 162

0.35 1.25 2.50 4.33 7.04

62 99 117 166 163

1.64 1.45 1.20 1.22 0.88

0.05 0.05 0.07 0.12 0.17

34.4 34.4 33.8 34.0 33.7

5.9 5.5 5.8 5.6 5.7

1.08 1.14 1.20 1.22 1.21

51961 48236 46089 48566 50065

4828 4275 4065 3873 2795

4996 4806 4478 4879 4583

5.8 6.2 5.8 6 5.9

48 51 51 54 79

0.35 0.43 0.43 0.40 0.93

0.13 0.14 0.13 0.19 0.29

3.2 5.2 5.1 4.9 7.6

57 66 62 60 106

0.41 0.56 0.64 1.39 4.26

37 41 40 45 62

2.05 1.81 1.96 2.16 2.73

0.05 0.06 0.07 0.05 0.10

37.4 38.5 38.4 38.5 35.9

4.3 4.0 4.1 3.9 4.3

0.28 0.28 0.31 0.30 0.53

26856 26059 27791 25172 19591

3310 2686 2889 1773 1105

3806 3953 4103 3736 4468

20 21 24 23 29

0.33 0.47 0.45 0.40 0.52

0.23 0.37 0.55 0.51 0.66

5.9 8.6 8.2 7.2 10.1

50 58 55 51 54

0.18 0.22 0.30 0.42 1.05

35 52 53 69 101

0.67 0.77 0.70 0.58 0.74

0.05 0.05 0.05 0.05 0.05

41.1 40.5 40.6 40.1 38.7

3.7 4.1 3.7 3.3 3.3

0.25 0.27 0.24 0.21 0.20

31960 36585 34548 37483 41966

3140 2947 2296 1650 1076

45 41 42 42 39

0.44 0.55 0.46 0.34 0.28

0.18 0.30 0.30 0.38 0.43

1.9 3.4 3.7 4.9 6.1

43 45 46 53 59

0.39 0.73 0.66 1.06 1.62

41 58 61 70 79

2.21 1.96 1.61 1.62 1.15

0.05 0.05 0.05 0.05 0.05

38.5 38.7 38.8 39.0 39.9

4.0 4.0 4.2 4.1 3.7

0.26 0.31 0.31 0.30 0.24

28867 29873 29645 28886 24954

48 50 54 54 59

0.55 0.79 0.78 0.71 0.80

0.33 0.29 0.31 0.41 0.65

4.6 9.4 9.4 9.4 7.4

57 74 64 72 98

0.49 0.83 0.87 1.48 2.61

50 50 52 54 64

1.64 1.40 1.55 1.55 1.65

0.05 0.05 0.05 0.05 0.05

35.1 34.5 33.1 32.8 33.4

5.0 5.3 5.6 5.5 4.4

0.55 0.71 0.76 0.86 0.97

39339 38607 44210 45338 31354

80

1

0.05 10

150

1

50

1.5

0.1

44.5 2.5

Fig. 1). Leaves showed intercostal reddening but no necrosis. Leaf greenness (SPAD values) did not significantly differ between the treatments, while plant length decreased significantly in three species (data not shown). A significant reduction in dry shoot mass with increasing concentrations of the FC mine waste was observed in the fast growing species corn, cucumber and bush bean. EC50 values were 21%, 25% and 27% of FC mine waste, respectively. Only soybean and oilseed rape were unaffected by the FC mine waste and latter species even showed a slight, though insignificant, growth stimulation at a concentration of 25%. Also Zhang et al. (2009) found a growth stimulation of oilseed rape growth at low Pb concentrations. Concentrations of the metals As, Cd, Pb, Zn, Al, Fe, Ba, Cr, Cu, Ni, Sb, V, the macronutrients N, P, K, S, Mg as well as the C contents were determined to study their accumulation and association in the different species. Oilseed rape, the most tolerant out of the five tested species, accumulated highest concentrations of As, Pb and Cd (see example of Pb, Fig. 1). Based on the fact that 0.4% and 9.5% of the Pb and the Cd would be plant available (Pignattelli et al., 2012), concentrations of plants grown on the 25% substrate should be 4.2 ppm for Pb and 0.056 ppm for Cd. The actual concentrations, i.e. 7 ppm Pb and 1.66 ppm Cd in the FC25% treatments, proved to be much higher in oilseed rape, suggesting that

hirsutum (L.) Ser., the gentian Blackstonia perfoliata (L.) Huds. and the conifer Pinus pinaster Ait.. Latter species indicates the onset of a primary succession in the southern part of the basin. In metalliferous (however, serpentine) soils nearby, Selvi et al. (2017) were able to show that the invasion of pines and the accumulation of needle litter leads to lower soil pH so that more metals will become phytoavailable over time. The same could be true for the mine waste at Fenice Capanne and could lead to long-term but subtle changes in biodiversity over time. 3.1.2. Chemistry of the substrate We analyzed pH, CNS and metal concentrations in the FC25% material and calculated the metal concentrations for the lower treatments (Table 1). Multiplying the metal levels by four should give comparable values like those reported in Mascaro et al. (2001) and Pignattelli et al. (2012). However, our composite sample had much lower zinc concentrations, while Pb and As concentrations agreed well with the results from Pignattelli et al. (2012). Obviously, metal concentrations vary across the flotation basins, but it cannot be excluded that zinc concentrations have indeed decreased over time. 3.1.3. Plant responses Foliar symptoms were observed only in corn in the highest treatment, i.e. the mixture that contained 25% of the FC material (see 509

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Table 3 Correlation matrixes (Pearson Product Moments) for the associations between heavy metal and nutrient concentrations and the biological parameters shoot mass, mean leaf area (MLA), water contents and specific leaf area (SLA). Pairs of values relate to the measurements taken on 25 plants per species (5 replicates x 5 treatments).

As

Al

Cd

Cr

Cu

Fe

Sb

V

Pb

Zn

Shoot mass Oilseed rape Bush bean Corn Cucumber Soy bean

0.41 -0.7 -0.5 -0.4 -0.1

0.15 -0.8 -0.5 -0.6 -0.4

0.16 -0.7 -0.5 -0.9 -0.2

-0.2 -0.6 -0.2 -0.8 -0.2

0.25 -0.5 -0.7 -0.1 -0.4

0.35 -0.7 -0.1 -0.6 -0.6

0.39 -0.4 -0 -0 -0.3

0.18 -0.8 -0 -0.4 -0.1

0.42 -0.8 -0.8 -0.8 -0.5

MLA Oilseed rape Bush bean Corn Cucumber Soy bean

0.3 -0.7 -0.6 -0.4 0.14

0.11 -0.8 -0.5 -0.5 -0.3

0.11 -0.7 -0.5 -0.8 -0.1

-0.3 -0.7 -0.3 -0.6 0.07

0.19 -0.5 -0.7 0.06 -0.3

0.25 -0.7 0.02 -0.5 -0.5

0.33 -0.4 0 0 -0.2

0.13 -0.8 0 -0.3 -0.1

Water % Oilseed rape Bush bean Corn Cucumber Soy bean

-0.2 -0.3 -0 -0.3 0.23

-0.1 -0.3 -0.1 -0.5 0.12

-0.3 -0.4 -0.2 -0.8 -0.4

0 -0.3 0.19 -0.6 -0

-0.4 -0.3 -0 0.23 -0.4

-0.2 -0.3 0.56 -0.6 -0.6

-0.2 -0.2 0 -0 -0.1

SLA Oilseed rape Bush bean Corn Cucumber Soy bean

-0.1 -0.1 0.23 -0.3 0.34

0.07 -0.2 0.68 -0.3 -0

-0.2 -0.3 0.52 -0.6 -0.1

0.25 -0.1 0.3 -0.4 0.04

-0.2 -0.1 0.44 0.2 -0.2

-0.1 -0 0.42 -0.5 -0.2

-0.1 -0.2 -0 -0 0.05

Ni

Ba

Mg

C

N

P

K

S

0.1 -0.1 -0.6 -0.6 -0.8 -0.1 -0.8 -0.2 -0.2 0.18

-0.4 0.2 0.36 0.76 0.17

-0.4 -0.1 0.35 -0.6 0.11

-0.4 0.58 0.81 0.53 -0.2

-0.3 -0.4 0.31 0.41 0.6

-0.7 0.53 0.61 0.77 0.24

-0.2 0.6 -0.6 0.29 0.36

0.04 -0.7 0.5 -0.7 0.29

0.32 -0.8 -0.8 -0.7 -0.3

0.08 -0.6 -0.8 -0.7 -0.1

-0.2 -0.6 -0.1 -0.3 0.32

-0.3 0.38 0.29 0.58 0.18

-0.4 -0.3 -0.2 0.51 0.3 0.85 -0.6 0.36 0.1 -0.1

-0.1 -0.1 0.37 0.59 0.52

-0.6 0.71 0.64 0.68 0.22

-0.3 0.65 -0.7 0.43 0.21

0.22 -0.7 0.57 -0.6 0.36

-0.1 -0.4 0 -0.4 0.06

-0.2 -0.4 -0.4 -0.8 -0.5

-0.4 -0.2 -0.4 -0.6 -0.4

-0.2 -0.2 0.4 -0.1 0.29

0.49 0.45 0.31 0.58 0.32

0.16 0.05 0.68 -0.6 0.13

-0.2 -0 0.43 -0 -0.4

0.66 0.59 0.91 0.86 0.43

0.42 0.67 0.76 0.75 0.46

0.2 0.43 -0 0.78 0.53

0.04 -0.2 0.92 -0.5 0.34

0.05 -0.1 -0 -0.4 0.01

-0.2 -0.2 0.52 -0.6 -0.1

-0.2 -0 0.59 -0.5 -0.1

-0.1 -0 0.27 -0.2 0.3

0.38 0.41 -0.2 0.4 0.2

0.3 -0.2 -0.1 -0.5 -0.1

-0.1 0.06 -0.5 -0.1 -0.1

0.47 0.5 -0 0.73 0.3

0.52 0.27 -0.3 0.55 0.23

0.16 0.07 0.34 0.7 0.13

0.15 -0.1 -0.1 -0.4 0.25

of the tested species, cucumber and oilseed rape had the highest nutrient levels. Oilseed rape featured highest concentrations of NPK and S and cucumber highest levels of Mg. Interestingly, the legumes had a comparatively lower water content than the other species, which could relate to ecophysiological adaptations (e.g. reduced transpiration rates) and to high element concentrations in their dry mass. Although nutrient and metal concentrations were not elevated, the legumes had higher carbon acquisition than oilseed rape and cucumber. Overall, carbon levels were highest in the C4 species corn, while metal and nutrient concentrations were generally lowest in the grass species. Carbon concentrations decreased with increasing concentrations of FC mine waste in all species except in soybean, which may point to interactions of heavy metals with internal carbon cycling, allocation patterns as well as histological and morphological changes of plant tissues and organs. A negative effect of heavy metals on carbon contents was also observed in a mangrove species (Yadav et al., 2015), but it remains unclear whether this stems from a reduced carbon assimilation or from an increased respiration as a stress response. In order to address the differences in plant specific responses and to describe the strength of associations between heavy metal concentrations and biological responses, correlation analyses were performed for the five species and the endpoints shoot mass, leaf size, specific leaf area and shoot water contents (Table 3). Correlation matrixes for the complete dataset showing the associations between individual heavy metals and nutrients are presented in Supplement A1. Pb shoot levels

the species operates mechanisms, by which insoluble metals can be taken up from the soil. However, the plant availability of Pb decreased from 9.4% in the FC 3.125–6.6% in the FC25% treatment. The reason for the lower mobility of heavy metals in the latter treatment may be the slightly higher pH value of the substrate, which may have prevented most of the Pb and Cd from being released into the root zone. Oilseed rape, other Brassica species and slow-growing wild cruciferous species (e.g. Noccea caerulescens, Biscutella laevigata and Alyssum spec.) have often been shown to take up high concentrations of metals without showing toxicity symptoms (Turan and Esringu, 2007; Mourato et al., 2015; Pošćić et al., 2015; Pan et al., 2017; Drozdova et al., 2017). Also Ferreyroa et al. (2017) did not find any foliar symptoms and growth reductions of oilseed rape grown on Pb polluted soil and Pb concentrations in the shoot were comparable to present study. Brassica species are known for their high concentrations of sulphur-containing phytochemicals and Mendoza-Cózatl et al. (2008) showed that glutathione-derived peptides are involved in the long-distance transport and detoxification of heavy metals. A concise summary of mean element levels in the five species and in the different treatments is shown in Table 2, including the mean levels that have been established for a “reference plant” by Markert (1992) and a color coding to demonstrate differences between species and treatments. While oilseed rape had highest levels of iron group heavy metals, the legumes bush and soybean accumulated higher levels of nickel. Out 510

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Fig. 2. Photos showing differences in plant length 35 days after the onset of the experiment (left) and box-and-whisker plots (right) depicting the differences in final shoot masses determined at the end of the experiment (90 days). Numbers in the photos represent mean plant length (cm) and the color codes the ranking of effects with red indicating strongest adverse effects. Different letters in the box-plots denote significant differences in shoot mass accumulation between the FC and ALT dilutions as tested by posthoc multiple comparisons (Tukey).

metal levels and shoot mass in oilseed rape demonstrate that the species can deal with metals without having to invest much energy for detoxification. It may well be that the higher metal accumulation and tolerance in this species is related to its higher contents of sulphur-containing peptides (see above). Also Zhang et al. (2009) found that growth of oilseed rape was unaffected by soil Pb concentrations of up to 2000 ppm, which was double as high as in the FC25% treatment in present study. A continuing production of biomass at high Pb levels has previously been shown in oilseed rape by Oreščanin et al. (2012), but differences were found between different cultivars. Interestingly, Wu et al. (2011) found that oilseed rape cultivation and the accumulation of readily mobile metals can lower the Cd and Pb uptake in

were negatively correlated to shoot mass and MLA in bush bean, corn, cucumber and soybean, indicating that growth was reduced at higher internal levels of the metal. It must be noted however, that growth reductions and morphological changes are not depending on one single element alone, but are the result of various metals that cause similar toxic effects and interact with the nutrient acquisition. The presented results do neither point to toxic effects of heavy metals, nor do they clarify the involved physiological mechanisms that drive the transition from essentiality to toxicity. In cucumber, high internal Cd levels seemed to play a larger role on growth than Pb, probably due to the higher mobility of the metal and its interactions with Ca and Mg. Nevertheless, the slightly positive associations between internal 511

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only low contents in Pb so that other elements must have been involved in the observed phytotoxicity especially in the perennial F. cymosum (ALT25%). In a previous paper we suspected that lithium and element imbalances are responsible for the adverse effects on plant growth on ALT mine waste (Franzaring et al., 2018). The element Li is rather mobile and would therefore accumulate in higher quantities than heavy metals. Interestingly, we found large differences in responses between closely related species, which could be the product of different life histories and the ecological adaptations to different soils. Such adaptations often involve mycorrhiza and plant growth promoting rhizobacteria (PGPR) and the release of exudates in the presence of heavy metals above critical thresholds. In fact, buckwheat produced mycorrhiza and secondary roots, but this was not investigated systematically. Furthermore, the expression of detoxifying genes in buckwheat is depending on the concentration of heavy metals and the pH of the growth substrate (Nikolić et al., 2010; Yokosho et al., 2016), but these mechanisms are not yet fully understood.

subsequently grown rice. It thus appears that oilseed rape may be cultivated as a phytoremediator to reduce the plant available soil concentrations. Unfortunately, we were unable to address the soil metal concentrations and plant available concentrations that were left in the FC substrate after the experiments. The strong negative association between Cd and Pb and the water content observed in the fleshy cucumber leaves points to toxic effects of heavy metals on the level of hydration and osmotic regulation in some species. Such effects were less pronounced in corn, while the association between nutrient levels (NPK) and water contents was positive in all the species. Heavy metal effects on plant water relationships have been described in detail by Rucińska-Sobkowiak (2016) and relate to complex responses, e.g. decreased root length, xylem blockage and stomatal malfunctioning. Finally, the slight associations between internal heavy metal concentrations and SLA may relate to changes in cell volume and leaf thickness. While the negative association between metals and SLA indicated that leaves became thicker at elevated levels of heavy metals especially in cucumber, the opposite effect was observed in corn (see also Table 2).

4. Conclusions 3.2. Buckwheat performance on mine wastes from Fenice Capanne (FC) and Altenberg (ALT)

Comparing the phytotoxicity of different mine wastes in standardized pot experiments and determining shoot metal and nutrient concentrations and extraction rates are wealthy tools to identify tolerant species and cultivars that can be grown successfully on a variety of metal polluted sites. From our trials we concluded that high productivity and the uptake of high quantities of heavy metals determine the suitability of plants to act as phytoextractors. We suggest to install research platforms on different European mine waste tips and to use standardised cultivation approaches with productive annual (e.g. oilseed rape, buckwheat or equally productive metal tolerant herbs) and perennial bioenergy crops (e.g willow, miscanthus and virginia fanpetals) under a defined soil amendment scheme.

In the second part of the present study we compared the performance of different buckwheat species and accessions on the FC and ALT mine wastes. Common buckwheat has been shown to accumulate high concentrations of Al (Ma et al., 1998; Shen et al., 2006; Horbowicz et al., 2011), Cu and Cd (Nikolić et al., 2010) and Pb (Tamura et al., 2005; Honda et al., 2007; Katoh et al., 2015). We thus supposed that the species could be grown in soils polluted with different heavy metals, but wanted to test whether a high tolerance can be observed in other buckwheat species as well. While plants of all species had longer shoots in the controls during the first weeks, plant length differed between species, mine wastes and treatments (dilutions) 35 days after sowing (Fig. 2). In the early growth stage, plants of F. tataricum and F. cymosum were shortest in the ALT25% treatment, but F. esculentum had developed larger plants than the other species with shorter plants in the ALT and FC10% treatments. Likewise, both accessions of F. tataricum were shorter in the FC10 as compared to the FC25% five weeks after the onset of the experiment. As time progressed, the FC mine waste appeared to be more toxic than the ALT substrate, except in the slow-growing perennial F. cymosum (Fig. 2, righthand-side). Unexpectedly, the FC10% treatment resulted in stronger growth reductions than the FC25% material, so that in the end of the experiment, shoot mass was significantly reduced in the FC10% treatment in F. tataricum (accession PdD) and F. esculentum. In contrast, higher concentrations of ALT mine waste in the substrate created stronger growth reductions in all species, except in F. esculentum, which produced a significantly greater biomass in the ALT25% than in the control. These results are in line with Franzaring et al. (2018) who showed a higher tolerance to ALT mine waste in common buckwheat as compared to tatary buckwheat and other species. Since the pH values decreased slightly from 6.1 to 5.86 from the FC25% to the FC0% treatment (see Table 1), the higher toxicity observed in the FC10 as compared to the FC25% material may have been somewhat related to a higher availability of toxic metals under more acidic conditions. In addition, the higher proportion of sand in the FC10% substrate may have had subtle effects on cation exchange capacity. Since these differences were only small, we can not rule out hormetic effects which could have enabled plants to be more effective in stress avoidance in the FC25% treatments. However, we can not determine which of the involved heavy metals and combinations thereof were responsible for the phytotoxicity in present study. Based on final shoot mass across the tested species, the FC substrate appeared to be more toxic than the ALT mine waste most probable due to the very high lead and arsenic contents. The ALT substrate has similar As but

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