Functional Traits of Plant Species Suitable for Revegetation of Landfill Waste from Nickel Smelter

applied sciences

Article Functional Traits of Plant Species Suitable for Revegetation of Landﬁll Waste from Nickel Smelter

Malvína Ciernikovˇ á 1,*, Ivana Vykouková 1 , Tomáš Orfánus 2 and Elena Masaroviˇcová 1

1 Department of Soil Science, Faculty of Natural Sciences, Comenius University, Mlynská dolina, Ilkoviˇcova6, 842 15 Bratislava, Slovakia; [email protected] (I.V.); [email protected] (E.M.) 2 Institute of Hydrology SAS, Dúbravská cesta 9, 841 04 Bratislava, Slovakia; [email protected] * Correspondence: [email protected]

Abstract: The landfill waste of leached ore residue represents a serious environmental risk and may also negatively affect the appearance, growth and development of vegetation. Here we focused on the evaluation of functional traits of selected plant species Populus alba, Calamagrostis epigejos, and Diplotaxis muralis growing in an unfavourable environment. We determined different adaptive strategies of selected species to extreme conditions. For Diplotaxis muralis the highest values of the leaf dry matter content (LDMC) and the lowest values of the specific leaf area (SLA) were determined, while for Calamagrostis epigejos these two traits correlated in opposite directions. Populus alba reached the lowest value of the water saturation deficit (WSD), suggesting that this species was most affected by soil water deficiency. The leaf water content (LWC) correlated negatively with the LDMC and positively with the SLA (narrow leaf blade). Although each plant species belongs to a different strategic group (therophyte, hemicryptophyte and phanerophyte in the juvenile stage), they are all very plastic and therefore suitable for remediation. Despite the unfavourable conditions, selected plant species were able to adapt to poor conditions and form more or less vital populations, which indicate the revegetation as a key measure for remediation of landfill waste from nickel smelter.

Citation: Cierniková,ˇ M.; Keywords: landfill waste; revegetation; functional traits; extreme conditions Vykouková, I.; Orfánus, T.; Masaroviˇcová,E. Functional Traits of Plant Species Suitable for Revegetation of Landfill Waste from 1. Introduction Nickel Smelter. Appl. Sci. 2021, 11, 658. https://doi.org/10.3390/ Industrial activities leave unwanted traces in the surrounding territory and have a app11020658 severe impact on the environment. As is well known, industrial and mining wastes with a high content of heavy metals pose a risk to the environment and have a demonstrable Received: 7 December 2020 toxic effect on vegetation and soil microorganisms and their life processes [1–3]. These Accepted: 8 January 2021 environmental burdens represent a very negative factor influencing the functional spatial Published: 12 January 2021 structure of the landscape. There is not only contaminated groundwater around the landfill but also a high level of air pollution with toxic dust. [4]. Recently, various methods have Publisher’s Note: MDPI stays neu- been used worldwide for the remediation of similarly contaminated sites: isolation and sta- tral with regard to jurisdictional clai- bilization of geochemically transformed elements [5–7], using natural alkaline material [8], ms in published maps and institutio- covering of mine tailings by using inert materials [9], soil washing [10], electrokinetic nal affiliations. remediation [11], microbiological methods [12] and many others. These technologies can be successful but generally require several specific processes, which take a long time. They are often expensive and can be practically used only in small areas [13]. Therefore, there is currently an increasing emphasis on the use of various phytotechnologies, including Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. phytoextraction, phytodegradation, rhizofiltration, phytostabilization, phytovolatilization This article is an open access article or phytocapping [1,13–15]. Revegetation is a low-cost measure based on the use of naturally distributed under the terms and con- occurring plants to cover mining sites to not allow a release of heavy metals from mining ditions of the Creative Commons At- waste into surrounding soil, groundwater and air by creating natural surface biomass layer tribution (CC BY) license (https:// as well as a branched root system. Thanks to this plant cover, water and wind erosion creativecommons.org/licenses/by/ are also prevented, rhizosphere properties can be improved and thus the landfill can be 4.0/). gradually revitalized [13].

Appl. Sci. 2021, 11, 658. https://doi.org/10.3390/app11020658 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 14

Appl. Sci. 2021, 11, 658 2 of 14 and wind erosion are also prevented, rhizosphere properties can be improved and thus the landfill can be gradually revitalized [13]. The investigated site was the landfill waste of black mud near the town of Sereď (SW Slovakia),The investigated which is a sitesource was of the heavy landfill metal waste contamination. of black mud The near landfill the town environment of Sered’ (SW is Slovakia),unfavourable which for plant is a source growth. of Landfill heavy metal material contamination. is not soil in Thethe real landfill sense; environment it is industrial is unfavourable for plant growth. Landfill material is not soil in the real sense; it is industrial waste that has poor physical properties without developed soil structure. Due to its black waste that has poor physical properties without developed soil structure. Due to its black colour, the landfill surface heats up and dries out very quickly. The material is strongly colour, the landfill surface heats up and dries out very quickly. The material is strongly alkaline (pH 8.5) and the organic content is very low 0.05%. The C:N ratio is 0.74:0.05. alkaline (pH 8.5) and the organic content is very low 0.05%. The C:N ratio is 0.74:0.05. Total content of Fe2O3 is 78% and Al2O3 3.27%. Content of chromium is the highest 24,300 Total content of Fe O is 78% and Al O 3.27%. Content of chromium is the highest mg/kg, nickel 2920 2mg/kg,3 zinc 300 mg/kg2 3 and copper 79 mg/kg. [16]. It does not contain 24,300 mg/kg, nickel 2920 mg/kg, zinc 300 mg/kg and copper 79 mg/kg. [16]. It does not enough biogenic elements for the proper plant growth and nutrition. Greening of the land- contain enough biogenic elements for the proper plant growth and nutrition. Greening of fill waste can be considered as one of the most natural, and in this case, the most appro- the landfill waste can be considered as one of the most natural, and in this case, the most priate remediation methods. appropriate remediation methods. This work was aimed to characterise native plant species that have been able to adapt This work was aimed to characterise native plant species that have been able to adapt inin anan extremeextreme environmentenvironment unfavourableunfavourable forfor plantplant growth.growth. TheThe functionalfunctional traitstraits ofof threethree plantplant species species PopulusPopulus alba alba,, CalamagrostisCalamagrostis epigejos and DiplotaxisDiplotaxis muralis were evaluated, evaluated, whichwhich reflectreflect thethe plant’splant’s responseresponse toto thethe habitathabitat andand aa wayway ofof adaptingadapting toto environmentalenvironmental conditions.

1.1. Site Characterisation InIn the the second second half half of of the the last last century, century, nickel nickel ore orewas was processed processed near nearthe town the townof Sereď of Sered’(SW Slovakia, (SW Slovakia, Figure Figure1), where1), where the landfill the landfill waste wasteof black of blackmud (waste mud (waste from the from colour the colourmetallurgy metallurgy of nickel of and nickel cobalt) and cobalt)is a direct is a evidence direct evidence of this activity. of this activity. The tailin Theg is tailing 45 m ishigh, 45 m800 high, m long 800 mand long 550 and m wide 550 m(Figure wide (Figure2). In the2). south, In the west south, and west east, and it east,is in itcontact is in contactwith agricultural with agricultural land and land represents and represents a significant a significant anthropogenic anthropogenic barrier [16]. barrier It is [ 16a ].dis- It istinct a distinct anthropogenic anthropogenic form of form relief of with relief a withtypical a typicalconvex convexunit (Fi unitgure (Figure 2). The2 ).site The is non- site iscombustible noncombustible and has and a hasdistinctive a distinctive shape shape of a tabular of a tabular terrace, terrace, which which was wasformed formed by the by thegradual gradual deposition deposition of waste of waste layers layers—black—black nickel nickel mud. mud. It has Itsteep has steepslopes slopes of up to of 45 up de- to 45grees degrees (anthropogenic (anthropogenic table table mountain). mountain). According According to a to field a field survey survey by by Michaeli Michaeli et et al. al. [[4],4], thethe landfilllandfill wastewaste hashas high permeability;permeability; therefore,therefore, rainwaterrainwater quicklyquickly penetratespenetrates thethe lowerlower layers,layers, andand thethe remainingremaining waterwater evaporatesevaporates quicklyquickly sincesince thethe blackblack nickelnickel mudmud stronglystrongly absorbs sunlight andand thus rapidlyrapidly overheats.overheats.

Figure 1. Map of Slovakia with the landfilllandfill wastewaste location.location. After the closure of the smelting factory in Sered’, the landfill waste was formed by After the closure of the smelting factory in Sereď, the landfill waste was formed by reclamation processes, geomorphological processes and processes from anthropogenic reclamation processes, geomorphological processes and processes from anthropogenic ac- activities (mining of black mud). The landfill soil can be classified as technosol developed tivities (mining of black mud). The landfill soil can be classified as technosol developed form a substrate of technogenic origin [16]. form a substrate of technogenic origin [16]. 1.2. Characteristics of Investigated Plant Species To determine the functional traits, three plant species were selected, which represent different life forms—therophyte, hemicryptophyte and phanerophyte (in the juvenile stage). Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 14

Appl. Sci. 20202121, 11, 658x FOR PEER REVIEW 3 of 14 Sampling site

Sampling site

Figure 2. Actual view of the landfill.

1.2. Characteristics of Investigated Plant Species To determine the functional traits, three plant species were selected, which represent different lifeFigure forms 2.—Actualtherophyte, view of hemicryptophyte the landfill. and phanerophyte (in the juvenile stage). Figure 2. Actual view of the landfill. PopulusPopulus albaalba (Figure(Figure3 ,3, white white poplar—phanerophyte poplar—phanerophyte [ [17]17] inin thethe juvenilejuvenile stage)stage) isis aa 1.2. Characteristics of Investigated Plant Species fast-growingfast-growing treetree withwith deciduousdeciduous leaves,leaves, whichwhich cancan growgrow toto aa heightheight ofof 2020 toto 3535 m.m. DarkDark green,green,To lobed lobeddetermine leaves,leaves, the thethe functional undersideunderside traits, isis slightlyslightly three plant felted,felted, species colouredcoloured were white. white.selected, ItIt is iswhich widespreadwidespread represent inin differentCentral,Central, SouthernSouthern life forms andand—therophyte, EasternEastern Europe, hemicryptophyte across Siberia and to the phanerophyte Yenisei River,River, (in in in the AsiaAsia juvenile toto thethe stage).westernwestern Himalayas.Himalayas. ItIt waswas introducedintroduced toto Micronesia,Micronesia, AustraliaAustralia andand NewNew ZealandZealand andand isis alsoalso spreadingPopulusspreading alba invasivelyinvasively (Figure 3, inin white thethe CanaryCanary poplar Islands.Islands.—phanerophyte ItIt isis aa treetree [17] ofof floodplainfloodplain in the juvenile forests;forests; stage) itit occursoccurs is a fastinin coastalcoastal-growing shrubsshrubs tree andwithand on ondeciduo riverriver alluvium.usalluvium. leaves, InwhichIn thethe softsoftcan meadow,growmeadow, to a ititheight formsforms of communitiescommunities 20 to 35 m. Dark withwith green,SalixSalix albaalba lobed (white(white leaves, willow)willow) the underside andand PopulusPopulus is slightly nigranigra(black (black felted, poplar).poplar). coloured WhiteWhite white. poplarpoplar It is is iswidespread aa light-lovinglight-loving in Central,tree,tree, onlyonly Southern younger and individuals individuals Eastern Europe, tolerate tolerate acrossweaker weaker Siberia shading. shading. to the It Ittolerates Yenisei tolerates River,changes changes in inAsia water in to water the re- westernregimegime very veryHimalayas well well and and. isIt alsowas is also resistant introduced resistant to long to to Micronesia,- long-termterm floods. floods.Australia It is secondary It and is secondary New to quarri Zealand toes quarries in and sand- is alsoinpits, sandpits, spreading brickyards, brickyards, invasively embankments, embankments, in the Canarylandfills, Islands. landfills, mining It miningheapsis a tree and heapsof similarfloodplain and places similar forests; with places it exposed occurs with inexposedsurfaces coastal surfaces[18]. shrubs In Slovakia, and [18]. on In river Slovakia, it is widespreadalluvium. it is widespread In in the the soft lowlands inmeadow, the lowlands [19]. it forms [19 ].communities with Salix alba (white willow) and Populus nigra (black poplar). White poplar is a light-loving tree, only younger individuals tolerate weaker shading. It tolerates changes in water regime very well and is also resistant to long-term floods. It is secondary to quarries in sandpits, brickyards, embankments, landfills, mining heaps and similar places with exposed surfaces [18]. In Slovakia, it is widespread in the lowlands [19].

FigureFigure 3.3. PopulusPopulus albaalba growinggrowing inin thethe landﬁll.landfill.

CalamagrostisCalamagrostis epigejos epigejos(Figure (Figure4, bush 4, bush grass—hemicryptophyte grass—hemicryptophyte [ 17] [17]is a perennial is a perennial grey- greengrey-green bushy bushy grass that grass has that strong has strongcreeping creeping subsoils subsoils with long with shoots. long Itshoots. inhabits It inhabits mainly sparsemainly forests, sparse logforests, cabins, log quarries, cabins, quarries, neglected neglected pastures pastures and ruderal and areas. ruderal In pasturesareas. In andpas- Figuremeadows,tures and3. Populus meadows, this speciesalba growing this is invasivespecies in the landfill.is and invasi suppresses ve and suppresses various meadow various communities.meadow communi- Bush grass is an ecologically plastic and can colonize multiple substrates. It is widespread in Europe,Calamagrostis North Asia, epigejos and secondarily (Figure 4, in bush North gra Americass—hemicryptophyte and South Africa. [17] is It inhabits a perennial the greywhole-green territory bushy of Slovakia grass that [20 has]. strong creeping subsoils with long shoots. It inhabits mainly sparse forests, log cabins, quarries, neglected pastures and ruderal areas. In pastures and meadows, this species is invasive and suppresses various meadow communi- Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 14

ties. Bush grass is an ecologically plastic and can colonize multiple substrates. It is wide- Appl. Sci. 2021, 11, 658 ties. Bush grass is an ecologically plastic and can colonize multiple substrates. It is wide-4 of 14 spread in Europe, North Asia, and secondarily in North America and South Africa. It inhabits the whole territory of Slovakia [20].

FigureFigure 4.4. CalamagrostisCalamagrostis epigejosepigejos growinggrowing inin thethe landﬁll.landfill.

DiplotaxisDiplotaxis muralismuralis (Figure(Figure5 5,, annual annual wall-rocket—therophyte wall-rocket—therophyte [[17])17]) isis anan annual,annual, bien-bien- nial,nial, rarelyrarely perennialperennial herb,herb, 15–5015–50 cmcm tall.tall. ItIt inhabitsinhabits mainlymainly rubbles,rubbles, ﬁelds,fields, fallowsfallows andand vineyards;vineyards; itit alsoalso occurs occurs along along roads, roads, on on landﬁlls, landfills, ruderalized ruderalized lawns, lawns, composts composts and and various vari- anthropogenicous anthropogenic habitats. habitats. It occurs It occurs mostly mostly on on clayey clayey or or gravelly, gravelly, nutrient-rich nutrient-rich soilssoils [[21].21]. ItIt isis originallyoriginally widespreadwidespread throughoutthroughout CentralCentral andand SouthernSouthern EuropeEurope (up(up toto NorthwestNorthwest Africa),Africa), secondarilysecondarily it it occurs occurs in in Northern Northern Europe, Europe, in in parts parts of of Russia Russia and and northern northern China, China, in Southin South Africa, Africa, as wellas well as inas Australia in Australia and and Tasmania, Tasmania, New New Zealand, Zealand, New New Mexico, Mexico, in almost in al- themost entire the entire United United States States and southernand southern Canada, Canada, and and Central Central and and South South America America [22]. [22]. In Slovakia,In Slovakia, it is it a is common a common species, species, especially especially in warmerin warmer areas areas and and in habitatsin habitats with with mild mild to mediumto medium soil soil moisture. moisture.

FigureFigure 5.5. DiplotaxisDiplotaxis muralismuralis growinggrowing inin thethe landﬁll.landfill.

1.3.1.3. FunctionalFunctional TraitsTraits FunctionalFunctional traits are are defined defined as as morpho morpho-physio-phenological-physio-phenological traits, traits, which which impact impact the thefitness fitness of plant of plant indirectly indirectly via their via their effects effects on growth, on growth, reproduction reproduction and survival and survival [23]. Some [23]. Someplants plants have the have ability the ability to adapt to adapt to changes to changes in the in environment; the environment; this feature this feature is called is called phe- phenotypic plasticity. Plasticity may vary from species to species, some species are unable to notypic plasticity. Plasticity may vary from species to species, some species are unable to adapt to changes in the environment, but some have evolved, and they are able to respond adapt to changes in the environment, but some have evolved, and they are able to respond positively to changes, i.e., changes in soil properties due to contamination, changes in positively to changes, i.e., changes in soil properties due to contamination, changes in wa- water management, climatic changes, etc. [24]. ter management, climatic changes, etc. [24]. Functional traits can be divided into simple traits—soft traits, which can be quantified relatively quickly and easily, and hard traits, which are more difficult to quantify. Soft traits include the water and dry matter content of leaves, the specific leaf area, the number of seeds, the relative length of the roots, the bark thickness or the height of the plant. Appl. Sci. 2021, 11, 658 5 of 14

1.3.1. Specific Leaf Area (SLA) It represents the area of one side of a fresh leaf divided by its dry matter. It is expressed in cm2·g−1. For example, species with higher SLAs (less carbon invested per leaf area) achieve higher specific growth rates (RGRs), which is an essential criterion for assessing biomass production, to compare plant species performance as well or to observe different impacts on plants. Plants with higher SLA values have thinner leaves and higher chlorophyll concentration per unit weight [25,26]. Further studies also found a positive correlation between SLA and the rate of net photosynthesis. SLA has also proven to be a key parameter that allows a plant to expose a large area of leaves to CO2 and light, and thus use them efficiently. Species found in environments with enough nutrients, water, light, etc., generally have higher SLA values than species living in a resource-poor environment. Low values tend to correspond to long leaf life and relatively high contributions to the defence mechanisms of plant species [27].

1.3.2. Leaf Dry Matter Content (LDMC) It is expressed in units of g·g−1 and represents the dry weight of the leaves divided by its fresh weight. In this case, the fresh weight corresponds to 100%. The remainder is water in the leaves, and therefore the water content of the leaves (LWC) can be derived as 100%—LDMC or 1—LDMC [25,28]. LDMC is closely related to leaf tissue density. There is a negative correlation with the relative potential growth rate but a positive correlation with leaf life. However, the resulting beneﬁts are likely to be less than those of the SLA. Leaves with a high LDMC value are relatively stiff so that they may be more resistant to physical risks (hail, wind, herbivores). Leaves with low LDMC are associated with a considerably disturbed environment. The dry matter content of the leaves is more useful and can provide more information than the SLA in cases where it is difﬁcult to measure the leaf area [27].

1.3.3. Water Saturation Deficit (WSD) Water saturation deficit means the amount of water that is missing (or part of it) in the plant until it is fully saturated; in other words, the difference between the water content at maximum saturation and its content at a given moment. It is expressed as a percentage of the maximum water content. A water deficit occurs when the water balance is disrupted, and the plant releases more water than it receives [25]. Water balance describes the correlation between water loss and water uptake. When the plant is optimally saturated with water, an optimal water balance occurs. Temporary or permanent decrease of water saturation of the plant with negative influence and effects occurs after depletion of physiologically available water. Water saturation of the plant occurs at an active water balance, and thus a water deficit occurs at a negative water balance. A sublethal water deficit is typical water stress and thus a borderline case of the water deficit. It expresses the value of WSD when 5–10% of leaf area is already irreversibly damaged [29].

1.3.4. Leaf Water Content (LWC) In the physiological processes of plants, the water content of the leaves is an essential indicator of photosynthesis, transpiration or respiration [30]. The water regime of the plant is crucial for the proper functioning of the cycle of substances in plant tissues. The water content in the leaves is thus an essential prerequisite for plant growth, especially to prevent drought stress. The water content at leaf level is derived from the difference between the weight of freshly harvested leaves and their dry weight or the 1-LDMC relationship [31]. Appl. Sci. 2021, 11, 658 6 of 14

2. Materials and Methods 2.1. Field Work and Collection of Leaves Regarding the aims of the work, samples of plant material were randomly taken from an area of approximately 20 × 20 m2 (Figure2). Three dominant species were sampled: Populus alba, Calamagrostis epigejos and Diplotaxis muralis. Ten leaf samples, for Populus alba in the juvenile stage, were taken in July and September from ten individuals per genus that were not damaged by external negative inﬂuences (herbivores, pathogens). We removed the leaves, which were vital, healthy, developed, together with the stem, in the place of natural separation at the leaf fall.

2.2. Functional Traits The measurement of the functional traits of the plants was in accordance with the procedure described in Cornelissen et al. [27]. The leaf characteristics of the plants that have been determined include: • FM—fresh leaf weight [g] • SM—saturated leaf weight [g] • DM—dry weight of the leaf [g] • LA—leaf area [cm2] Using these values, the functional traits were calculated as • Speciﬁc leaf area: SLA = LA/DM [cm2·g−1] • Leaf dry matter content: LDMC = DM/FM [g·g−1] • Water saturation deﬁcit: WSD = (SM-FM)/SM [%] • Leaf water content: LWC = 1-LDMC [g·g−1]

2.3. Statistical Analysis Box plots in the graphs show the variability of individual functional leaf traits (SLA, LDMC, WSD, LWC) between 3 selected species. Boxplots are a standardized way of displaying the distribution of data based on a five-number summary: minimum, first quartile, median, third quartile and maximum. The edges of the box represent the upper and lower quartiles (values in the range of 25–75% of the total range of values). Pearson’s correlation coefficients (r) quantified the linear relationships between functional traits. Correlation relationships were classified as: small dependence for r ≤ 0.4, slight dependence for 0.4 < r ≤ 0.8 and strong dependence for r > 0.8.

3. Results 3.1. Specific Leaf Area The Diplotaxis muralis had the highest average values of specific leaf area (SLA) of 148.7 cm2·g−1, the Calamagrostis epigejos followed with an average value of 130.77 cm2·g−1 and the lowest values were found for the Populus alba 98.7 cm2·g−1 (Figure6). In terms of functional groups, according to the values shown in Figure6, we could include the species Calamagrostis epigejos and Diplotaxis muralis in one group and the Popu- lus alba in another group. This probably reflects different life strategies, various adaptations of species and acclimatization of their organs to given environmental conditions.

3.2. Leaf Dry Matter Content The differences in values of the dry matter content in the leaves (LDMC) between selected species are shown in Figure7. The LDMC values are the lowest for the Diplotaxis muralis and, conversely, the LDMC values are much higher for the other two species Popu- lus alba and Calamagrostis epigejos. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 14

250

Appl.Appl. Sci. Sci. 20212021, 11, ,11 x, FOR 658 PEER REVIEW 200 7 of7 of14 14

- 150 .g

2 250

cm Mean

SLA SLA 100 200

50 1

- 150

.g 2

cm 0 Mean

SLA SLA 100 PA DM CE

Figure 6. Specific leaf area (SLA) of investigated plant species (PA—Populus alba, DM—Diplotaxis muralis, CE50 —Calamagrostis epigejos).

3.2. Leaf Dry Matter Content 0 The differencesPA in values DMof the dry matterCE content in the leaves (LDMC) between selected species are shown in Figure 7. The LDMC values are the lowest for the Diplotaxis Figure 6. Speciﬁc leaf area (SLA) of investigated plant species (PA—Populus alba, DM—Diplotaxis Figuremuralis 6. and, Specific conversely, leaf area (SLA) the LDMC of investigated values are plant much species higher (PA —forPopulus the other alba, twoDM— speciesDiplotaxis Po p- muralis, CE—Calamagrostis epigejos). muralisulus alba, CE and—Calamagrostis Calamagrostis epigejos epigejos). .

3.2. Leaf Dry Matter Content 0.5 The differences in values of the dry matter content in the leaves (LDMC) between selected species are shown in Figure 7. The LDMC values are the lowest for the Diplotaxis muralis and, conversely, the LDMC values are much higher for the other two species Pop- 0.4

ulus alba and Calamagrostis epigejos.

)

1 -

0.5 g.g 0.3

Mean LDMC ( LDMC

0.4

0.2

)

- g.g 0.3 0.1 Mean

LDMC ( LDMC PA DM CE

Figure 7. Dry matter content in the leaves (LDMC) of investigated species (PA—Populus alba, DM— Figure 0.27. Dry matter content in the leaves (LDMC) of investigated species (PA—Populus alba, DMDiplotaxis—Diplotaxis muralis muralis, CE—, CECalamagrostis—Calamagrostis epigejos epigejos). ). The LDMC values are the lowest for the Diplotaxis muralis and, conversely, the LDMC The LDMC values are the lowest for the Diplotaxis muralis and, conversely, the LDMC values0.1 are much higher for the other two species Populus alba and Calamagrostis epigejos. values are much higher for the other two species Populus alba and Calamagrostis epigejos. Therefore, we couldPA include PopulusDM alba andCECalamagrostis epigejos in one functional group, Therefore, we could include Populus alba and Calamagrostis epigejos in one functional which indicate their similar mechanism of adaptation to extreme properties of soil on Figuregroup, 7. which Dry matter indicate content their in similar the leaves mechanism (LDMC) of of investigated adaptation species to extre (PAme— Populusproperties alba ,of soil the landfill. DMon the—Diplotaxis landfill. muralis , CE—Calamagrostis epigejos). We found the highest average values of the LDMC in the Populus alba (0.37 g·g−1) We found the highest average values of the LDMC in the Populus alba (0.37 g·g−1) and and Calamagrostis epigejos (0.38 g·g−1), and the lowest values for the Diplotaxis muralis CalamagrostisThe LDMC epigejos values (0.38 are the g·g lowest−1), and for the the lowest Diplotaxis values muralis for and, the Diplotaxis conversel y, muralis the LDMC (0.17 (0.17 g·g−1). valuesg·g−1). are much higher for the other two species Populus alba and Calamagrostis epigejos. Therefore,3.3. Water we Saturation could include Deficit Populus alba and Calamagrostis epigejos in one functional group, which indicate their similar mechanism of adaptation to extreme properties of soil Figure8 shows the differences between water saturation deficits in the selected species. on the landfill. The highest average value was determined in the Populus alba with the WSD of 33.2%; for We found the highest average values of the LDMC in the Populus alba (0.37 g·g−1) and the Diplotaxis muralis it was 20.8% and for the Calamagrostis epigejos the lowest average value Calamagrostis epigejos (0.38 g·g−1), and the lowest values for the Diplotaxis muralis (0.17 was 8.1%. The Calamagrostis epigejos probably has the best strategy for water management g·g−1). under stress.

Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 14 Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 14

3.3. Water Saturation Deficit 3.3. Water Saturation Deficit Figure 8 shows the differences between water saturation deficits in the selected species. TheFigure highest 8 shows average the differences value was between determined water in saturation the Populus deficits alba in with the selected the WSD spe- of 33.2cies.% The; for highestthe Diplotaxis average muralis value it waswas determined20.8% and for in the the Calamagrostis Populus alba epigejos with the the WSD lowest of Appl. Sci. 2021, 11, 658 average33.2%; for value the wasDiplotaxis 8.1%. Themuralis Calamagrostis it was 20.8 epigejos% and probablyfor the Calamagrostis has the best epigejosstrategy the for lowwater8 ofest 14 managementaverage value under was 8.1 stress.%. The Calamagrostis epigejos probably has the best strategy for water management under stress. 90 90 80 80 70 70 60 60 50 50 40 Mean

WSD WSD (%) 40 Mean

WSD WSD (%) 30 30 20 20 10 10 0 0 PA DM CE PA DM CE

Figure 8. Water saturation deficit (WSD) in the leaves of investigated species (PA—Populus alba, Figure 8. Water saturation deﬁcit (WSD) in the leaves of investigated species (PA—Populus alba, DMFigure—Diplotaxis 8. Water muralissaturation, CE deficit—Calamagrostis (WSD) in epigejos the leaves). of investigated species (PA—Populus alba, DMDM——DiplotaxisDiplotaxis muralis muralis, CE, CE——CalamagrostisCalamagrostis epigejos epigejos).). 3.4.3.4. Leaf Leaf Water Water Content Content 3.4. Leaf Water Content TheThe measured measured values values of ofthe the LWC LWC (Figure (Figure 9)9 )showed showed that that the the DiplotaxisDiplotaxis muralis muralis is,is, despitedespiteThe the themeasured unfavourable unfavourable values conditions conditions of the LWC in the(Figure landﬁll,landfill, 9) ableshowedable to to work workthat withthe with Diplotaxis water water most most muralis effectively effec- is, tivelydespiteand keepand the keep it unfavourable intissues. it in tissues. The conditions highest The high measured inest the measured landfill, value for ablevalue the to Diplotaxisfor work the withDiplotaxis muralis waterwas muralis most 0.86 effec- gwas·g− 1. 0.86tivelyThe g· differencesgand−1. The keep differences it between in tissues. between the The studied high the specieseststudied measured were species not value were too high,for not the too and Diplotaxis high, the measured and muralis the meas- values was −1 ured0.86indicate gvalues·g . thatThe indicate thedifferencesPopulus that the between alba Populusand theCalamagrostis alba studied and Calamagrostis species epigejos wereare epigejos not also too able are high, toalso make and able the fullto meas-make use of fulluredtheir use values water of their indicate potential. water thatpot The ential.the highest Populus The measured highest alba and measured valueCalamagrostis for value the Populus epigejosfor the alba Populusare wasalso 0.68albaable was g to·g −make 0.681 and gfull·forg−1 useand the of Calamagrostisfor their the waterCalamagrostis epigejospotential. 0.67epigejos The g· ghighest −0.671. g· gmeasured−1. value for the Populus alba was 0.68 g·g−1 and for the Calamagrostis epigejos 0.67 g·g−1. 0.9 0.9

0.8

)



1 -

g 0.7  Mean 0.7

LWC LWC (g Mean LWC LWC (g

0.6 0.6

0.5 0.5 PA DM CE PA DM CE

FigureFigure 9. 9.LeafLeaf water water content content (LWC) (LWC) of of investigated investigated species species (PA (PA——Populus albaalba,, DM— DM—DiplotaxisDiplotaxis muralis , muralisFigureCE—,Calamagrostis 9.CE Leaf—Calamagrostis water epigejoscontent epigejos). (LWC)). of investigated species (PA—Populus alba, DM—Diplotaxis muralis, CE—Calamagrostis epigejos). 3.5. Correlation between SLA and LWC Based on the values of individual functional traits, we decided to determine whether the water content in the leaves (LWC) affects the speciﬁc leaf area (SLA). In the correlation diagram (Figure 10), the relationship between the SLA and LWC is plotted, which is signiﬁcant for the Populus alba with r = 0.716. Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 14

3.5. Correlation between SLA and LWC Based on the values of individual functional traits, we decided to determine whether the water content in the leaves (LWC) affects the specific leaf area (SLA). In the correlation diagram (Figure 10), the relationship between the SLA and LWC is plotted, which is significant for the Populus alba with r = 0.716. For the other two species, the dependence was not statistically significant. Low correlation between SLA and LWC for the Diplotaxis muralis (r = 0.386) is most likely caused by its adaptation to the lack of water in the habitat. The plant has scleromorphic leaves and can create an extensive root system [32]. The correlation coefficients in Figure 10 may indicate either different plant forms: the Populus alba is juvenile, the Calamagrostis epigejos is grass and Diplotaxis muralis is a herb; or it can mean a favourable leaf morphology as in the case of Populus alba, which is char- Appl. Sci. 2021, 11, 658 9 of 14 acterised with a larger leaf area and with the presence of hairs on the leaf surface, and thus it can better manage water.

0.9 r = 0.386

0.8

)

g  0.7 PA r = 0.716

DM LWC LWC (g CE 0.6 r = 0.500

0.5 50 100 150 200 250 SLA cm2g-1

Figure 10. Correlations between a speciﬁc leaf area (SLA) and leaf water content (LWC) of the Figure 10. Correlations between a specific leaf area (SLA) and leaf water content (LWC) of the selected species (PA— selected species (PA—Populus alba, DM—Diplotaxis muralis, CE—Calamagrostis epigejos) (values for Populus alba, DM—Diplotaxis muralis, CE—Calamagrostis epigejos) (values for SLA are given in cm2·g−1 and for LWC in g·g−1). SLA are given in cm2·g−1 and for LWC in g·g−1).

4.For Discussion the other two species, the dependence was not statistically significant. Low correlation4.1. Functional between Traits SLA and and Ecological LWC for thePlasticityDiplotaxis muralis (r = 0.386) is most likely caused by its adaptationLow SLA tovalues the lack are oftypical water for in thespecies habitat. inhabiting The plant environments has scleromorphic with low leaves nutrient andcontent, can create lack an of extensive water and root light system [27]. [ 32Our]. results confirmed low SLA values, mainly for theThe type correlation of Populus coefficients alba. However, in Figure it should 10 may be noted indicate that either all selected different species plant occurre forms:d in thethePopulus area outside alba is juvenile,the shade the withCalamagrostis enough light, epigejos whichis is grass an essential and Diplotaxis factor for muralis SLA, becauseis a herb;its orvalues it can are mean mainly a favourable related to leafthe photosynthetic morphology as activity in the case of the of leaves.Populus Despite alba, which the lowest is characterisedachieved SLA with values, a larger we leaf can area agree and with with the the findings presence of of Rafati hairs onet theal. [33] leaf that surface, the Populus and thusalba it canspecies better is manageable to colonize water. contaminated substrates and grow even in extreme conditions. In terms of its ecological plasticity, Populus alba showed rapid growth, high biomass 4. Discussion production, easy seed dispersal, fast attachment and root system development. 4.1. Functional Traits and Ecological Plasticity We can classify Calamagrostis epigejos among C-strategists at our site. In addition, ac- cordingLow SLA to Mitrovica values are et typical al. [32], for Calamagrostis species inhabiting epigejos environmentshas a high growth with potential, low nutrient which content,couldlack be used of water especially and light in disturbed [27]. Our habitats results confirmedfor shortening low succession SLA values, series mainly and forfaster thereclamation. type of Populus alba. However, it should be noted that all selected species occurred in the areaThe outsideresulting the SLA shade values with for enoughDiplotaxis light, muralis which indicate is an that essential this species factor is for the SLA, best of because its values are mainly related to the photosynthetic activity of the leaves. Despite the selected species adapted to the conditions of the habitat. This is probably due to the the lowest achieved SLA values, we can agree with the findings of Rafati et al. [33] that the fact that this species can be included among the R-strategists which enter the habitats in Populus alba species is able to colonize contaminated substrates and grow even in extreme conditions. In terms of its ecological plasticity, Populus alba showed rapid growth, high biomass production, easy seed dispersal, fast attachment and root system development. We can classify Calamagrostis epigejos among C-strategists at our site. In addition, according to Mitrovica et al. [32], Calamagrostis epigejos has a high growth potential, which could be used especially in disturbed habitats for shortening succession series and faster reclamation. The resulting SLA values for Diplotaxis muralis indicate that this species is the best of the selected species adapted to the conditions of the habitat. This is probably due to the fact that this species can be included among the R-strategists which enter the habitats in the early stages of succession, have the high reproductive capacity and rapid germination as well as rapid population growth with short life cycle and short vegetation phase. Higher water content in the leaves and a narrower leaf blade contribute to a higher SLA [34,35]. Plants with higher SLA flexibly respond to changes in resource availability in the soil [36], are more productive [37] but more prone to damage herbivores [38]. Cin- Appl. Sci. 2021, 11, 658 10 of 14

golani et al. [39] showed that productivity-related traits (SLA, LDMC) strongly reflect the soil moisture gradient. The LDMC reflects the availability of nutrients and water in the soil. Based on the obtained results, we can state that the Diplotaxis muralis species is well adapted to adverse conditions, as selected species were investigated in habitats with low soil moisture and nutrient content but with a high content of heavy metals. Its leaves are stiff, which is also a manifestation of its resistance to adverse external influences and its ability to use nutrients efficiently. Thus, it can survive long periods without water supply. For Populus alba and Calamagrostis epigejos species, regardless of their higher LDMC values, we assume that they are also resistant to adverse habitat conditions. In the case of the Populus alba, even though it requires higher humidity, it can also occur in water-deficient habitats. Calamagrostis epigejos is an ecologically plastic species which can occur in adverse habitats. We believe that this species was able to adapt well to extreme conditions in the habitat. The LDMC and SLA are highly correlated characteristics that reflect different plant adaptations. Each of these properties may indicate different functional strategies, where the SLA is mainly related to the light availability and photosynthetic activity, and the LDMC indicates the water and nutrient availability in soil. In terms of different adaptations and life strategies, we can divide selected species into two functional groups. Diplotaxis muralis belongs to the first functional group with lower LDMC values. We assume that it is well adapted to the given habitat and can effectively use available nutrients and water. Based on the higher values (dry matter content in the leaves) of the Populus alba and Calamagrostis epigejos species, we believe that they are more stressed by the adverse effects of the habitat and thus handle the obtained nutrients and water more gently. Therefore, in terms of the life strategy and the use of resources, we classify Populus alba and Calamagrostis epigejos into the second functional group of the examined habitat. The highest values of WSD were found in Populus alba. Its highest value reached up to 83.7%, which is the limit value of the water deficit [40]. The water deficit threshold, or sublethal water deficit, is a condition in which irreversible leaf damage occurs. Sublethal water deficit is achieved at values in the range of 70–80% WSD. In our case, one sample of Populus alba reached the value of the sublethal water deficit, the individual from which we took the leaf for WSD determination was exposed to considerable stress, and the leaf was damaged, even though we tried to take leaves healthy, vital and undamaged. The remaining WSD values for Populus alba were lower but indicate an adverse effect of the habitat on this species. The WSD values for Diplotaxis muralis species are lower than for Populus alba but higher than for Calamagrostis epigejos. This fact is related to the fact that the Calamagrostis epigejos species is best adapted to the lack of water in the habitat, has scleromorphic leaves and can form an effective root system [32]. Tissue hydration is a necessary condition for the physiological activity of the leaves. The current water deficit depends on the balance between water intake and expenditure by the plant, so it is conditioned by many environmental and biological factors [25]. Samples of selected species were taken in July under relatively unfavourable humidity conditions in the range of 18.1–22.9%. However, at the last field sampling in September, the relative air humidity ranged from 41.1 to 60.8%. Based on previous research, it has been found that higher water content in the leaves and narrower leaf blades contribute to a higher SLA [29]. On the other hand, leaf water content (LWC) correlates negatively with LDMC and positively with SLA [27].

4.2. Plant Utilisation in Landfill Remediation Strategies From the obtained data we could state that all plant species were able to be inhabited and grow in the landfill waste, while each species had a different life strategy and way to adapt to this habitat with extreme conditions. Calamagrostis epigejos proved to be a plastic species, and in the selected habitat it was probably the best of all species for water shortages. Diplotaxis muralis showed the best adaptation, mainly in terms of the specific Appl. Sci. 2021, 11, 658 11 of 14

leaf area, the dry matter content in the leaves but also values of the water saturation deficit. Populus alba, although in the juvenile stage and affected by the lack of substrate moisture, was also able to adapt, colonize and create populations in habitats with adverse conditions. All three species belong to the same ecological group in terms of salinity [41]. They are tolerant to the concentration of readily soluble salts, especially sulphates, chlorides and carbonates of sodium, potassium, calcium and magnesium. Despite the unfavourable conditions persisting in the landfill waste, all three species were able to create viable populations. Many plant species can accumulate heavy metals in their bodies and are therefore often sought after as indicators of contaminated areas or are used directly to revitalize and phytoextract heavy metals from soils [15]. Within the plant kingdom, about 500 species of higher plants are currently known to have hyperaccumu- lating properties [42]. Most of them can accumulate a specific element and can be used in phytoremediation methods. These species occur in 34 families, most of which belong to Brassicaceae family [42,43]. Diplotaxis muralis belongs to the family Brassicaceae, and even though it is not referred to as a hyperaccumulator, this species proved its ability to adapt to the high content of heavy metals in the substrate. In addition, the plasticity of species Populus alba and Calamagrostis epigejos allows them to grow in contaminated sites. These species are able to tolerate high levels of contaminants in the soil and partially accumulate them in their tissues [33,44]. Poplar is a sought-after species for the remediation of polluted sites due to its rapid growth, deep root system, large biomass production [45,46]. This species is suitable for soil rhizodegradation and phytoextraction of Cd, Ni, Fe, Zn and Pb [33,44,47,48]. Randelovic et al. [49] have been working directly with Calamagrostis epigejos. They con- cluded that its properties such as adaptation mechanism, phenotypic plasticity, genotypic variability, low nutrient requirements support its spread in this type of site and predestines it to indicate soil pollution and possible phytoremediation of contaminated areas. Its ability to colonize landfills after mining activities [50] plays an essential role in not spreading contaminants into the environment and crops. The species is suitable for remediation of soils polluted with heavy metals [48]. Phytocapping, as one of the phytoremediation technologies, is a new ecofriendly and cost-effective method working with overlapping landfill wastes with green naturally occurring plants. The main advantage of phytocapping is the reduction or complete stopping of heavy metal leaching into soil and groundwater. Plant biomass also slows down the spread of contaminants through the air and helps prevent erosion. Plants growing on landfill wastes are adapted to these conditions, and the concentration of heavy elements gradually decreases. Thanks to surface evaporation and plant transpiration, contaminated water is prevented from flowing down into the subsoil. Soil absorbs water and plants behave as “pumps” that can remove stored water [51–53]. The proposed phytocapping procedures recommend the use of hyperaccumulators and especially as the first step it is recommended to create a layer on contaminated soil, which will create a suitable environment for the plant roots [13]. For phytocapping, however, we can also use plants that themselves have the ability to adapt and grow on contaminated soils. If the landfill wastes are left without remediation methods for economic reasons, and natural vegetation is gradually created on it, we can talk about natural phytocapping.

5. Conclusions The landfill waste (SW Slovakia) and its surroundings represent, from the environmental point of view, a territory that is severely disturbed, ecologically unresolved and left to the self-cleaning ability of the natural landscape. The waste containing heavy metals has contaminated a wide area, and the landfill waste greening is considered the most suitable method of reclamation. For this reason, the site was selected for field research and more detailed study, which focused on its natural vegetation. The functional traits of three plant species that occur naturally at the site were evaluated, and the degree of adaptation of the plants to soil contaminated with heavy metals was Appl. Sci. 2021, 11, 658 12 of 14

determined. We focused on the species Populus alba, Diplotaxis muralis and Calamagrostis epigejos, which formed more numerous populations in this habitat. We evaluated leaf characteristics on selected individuals (specific leaf area, dry matter content in leaves, water saturation deficit and leaf water content). The highest values of the specific leaf area were determined for Diplotaxis muralis and the lowest in the case of Populus alba. On the contrary, Diplotaxis muralis showed the lowest values of leaf dry matter content, while the other two species, Populus alba and Calamagrostis epigejos, showed considerably higher values. The highest values of water saturation deficit were observed for Populus alba, Diplotaxis muralis had slightly lower values, and Calamagrostis epigejos showed the lowest values. Results of leaf water content revealed the best water management ability in the case of Diplotaxis muralis. This was confirmed by the significant correlation between specific leaf area and leaf water content of Diplotaxis muralis. We assume that this is related to the excellent plasticity of the species and its ability to adapt to adverse conditions. Based on the investigated functional traits, which are closely related to the ability of plants to adapt to the contaminated environment, we can conclude that if it is not possible to dispose of the landfill wastes conventionally, then natural greening is a suitable solution. The natural plant cover can be formed by plants that are able to plastically respond to the high contents of heavy metals in the soil and can create strong and viable populations. These plant species reduce water percolation due to interception and evapotranspiration, prevent water and wind erosion of the soil and therefore reduce the spread of contamination to the environment.

Author Contributions: Conceptualization, I.V., M.C.ˇ and T.O., methodology, I.V. and E.M.; formal analysis, T.O. and M.C.;ˇ investigation, I.V., M.C.;ˇ resources, E.M. and T.O.; data curation, M.C.;ˇ writing—original draft preparation, M.C.;ˇ writing—review and editing, M.C.;ˇ visualization, T.O.; supervision, I.V. and E.M.; project administration, M.C.,ˇ I.V. and T.O.; funding acquisition, I.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Slovak Scientific Grant Agency VEGA projects 1/0712/20 and 2/0096/19. Data Availability Statement: Data is contained within the article. Conflicts of Interest: The authors declare no conflict of interest.

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