Fly ash (FA), the main by-product of coal-fired thermal power plants is a complex, heterogeneous material whose highly variable physical and chemical properties and toxicity are determined by the coal’s geological origin, the combustion process, the disposal method, the time the ash has been exposed to atmospheric conditions (age of the ash), and vegetation development (Haynes, 2009; Izquierdo and Querol, 2012; Bhatt et al., 2019; Kostić et al., 2022a). The intensification of activities related to this type of electricity generation is one of the major environmental problems today because the disposal of FA in dry or wet lagoons near thermal power plants contributes to the leaching of potentially toxic elements (PTEs) into soil and groundwater, while windblown micrometre-sized and poorly aggregated particles of FA from the dry surfaces of landfills pollute agricultural land and endanger the flora, fauna, and health of residents of nearby settlements (Raja et al., 2015; Ćujić et al., 2016; Khan and Umar, 2019).The formation of a vegetation cover, i.e. the revitalisation of FA landfills, is a cost-effective and environmentally sound method and the best method when it comes to stabilising this mobile substrate physically and chemically (Haynes, 2009; Yan et al., 2020; Kostić et al., 2022b). However, the synergistic effect of multiple abiotic stress factors, such as drought, low organic matter content, lack of essential nutrients (N, P, Mn, etc.), alkaline pH, and phytotoxicity due to high PTE and soluble salt content, limits the number of species that can grow under such conditions (Kostic et al., 2018; Kalashnikova et al., 2021). Due to its dark grey colour, FA absorbs enormous amounts of heat from the sun, which increases surface temperatures at the landfill and reduces humidity. This is further decreased by the ash’s sandy texture, which reduces water and nutrient retention capacity (Carlson and Adriano, 1991; Pavlović et al., 2004). Increased salinity, which can be as high as 13 dS m^-1 in raw FA, can have a similar effect to drought on plants growing in landfills (Carlson and Adriano, 1993). When coal is burned, carbon (C) and nitrogen (N) are oxidised and transition to the gas phase, meaning FA is poor in both (Carlson and Adriano, 1993; Haynes, 2009). During and after combustion, the mineral fraction of coal undergoes various transformations that make the chemical elements bound in the original coal matrix susceptible to leaching during transport and disposal in landfills, especially when in contact with water (Izquierdo and Querol, 2012). In addition to the aluminosilicate matrix containing Si, Al, Fe, Ca, Mg, Na and K, the chemical composition of FA includes numerous microelements (As, B, Cu, Cr, Cd, Mn, Ni, Pb, Co, Mo, Zn, Se, etc.) whose concentrations in FA can be 4-10 times higher compared to the parent coal (Tian et al., 2018). Some of these elements are essential for plants in minimal amounts, while their presence in high concentrations can be potentially toxic. The most susceptible to leaching, and therefore most accessible to plants, are the partially volatile chemical elements (As, B, Cr, Se, and Zn) that condense in the surface layer of ash particles as the flue gases cool during the coal combustion process, while the less volatile elements such as Mn and Ni accumulate in the inner layer and are not directly exposed to leaching (Tian et al., 2018). The alkalinity of FA can cause the formation of insoluble forms of elements and thus a deficit of essential nutrients, most commonly phosphorus (P), manganese (Mn), and copper (Cu), but also increased solubility of arsenic (As), boron (B), and selenium (Se) and their accumulation in plants in toxic concentrations (Pandey et al., 2011; Gajić et al., 2020; Kostic et al., 2022c).

Abiotic stress factors lead to an overproduction of reactive oxygen species (ROS). These toxic products of incomplete oxygen reduction, which are common by-products of regular aerobic metabolic processes, are most commonly formed in chloroplasts, mitochondria, and cytoplasm, but also in membrane-bound exocellular enzymes involved in redox reactions, especially during photosynthetic electron transport and respiration (Sharma et al., 2020; Sachdev et al., 2021). Abiotic stresses such as high temperatures, drought, high salinity, and heavy metal pollution, as well as the combination of these stresses, disrupt the metabolic balance of cells, leading to increased production of ROS (Qadir et al., 2021; Mishra et al., 2023). In plants, this causes oxidative damage that disrupts many physiological processes, including photosynthesis as the main process of plant metabolism (Pavlović et al., 2007; Mitrović et al., 2012; Huang et al., 2019; Muhammad et al., 2021; Della Maggiora et al., 2023). Despite all the physical and chemical limitations and toxicity of FA, various plant species have been found growing on it (Kostić et al., 2012; Técher et al., 2012; Mukhopadhyay et al., 2017; Yadav et al., 2022). Thanks to the development of adaptation mechanisms based on accumulation and exclusion, tolerant species survive under conditions of PTE deficiency or toxicity in the substrate. By regulating the transfer between soil and roots and between roots and leaves, concentrations of these elements are kept within a normal range in their tissue (Pandey et al., 2012; Mukhopadhyay et al., 2017; Kostić et al., 2022b). Tolerant plant species also respond to abiotic stress by increasing their antioxidant capacity, which helps them to maintain a normal cellular balance between production and binding, degradation, and neutralisation of ROS by enzymatic or non-enzymatic antioxidants (carotenoids and anthocyanins) and secondary metabolites (phenols) (Cervilla et al., 2012; Gajić et al., 2020; Kebert et al., 2022; Vuksanović et al., 2022). Intolerant species exibit decreased antioxidant capacity and fewer mechanisms for detoxification in relation to ROS production, resulting in chain reactions in which free radicals damage important biomolecules, such as chloroplast pigments, lipids, and nucleic acids, oxidatively. Inactivation of enzymes and disruption of membrane structure follows, which leads to tissue damage and finally cell death (Moreno-Jiménez et al., 2009; Love et al., 2013; Stojnić et al., 2016; Gajić et al., 2020; Pilipović et al., 2020). In this sense, choosing the right plant species is an important factor in determining the efficiency of the FA landfill revitalisation process.

Previous studies have shown that autochthonous, long-living woody species characterised by high below- and above-ground biomass and rapid growth, as well as the ability to reproduce vegetatively and capacity to tolerate various stressors, are best suited to stabilising FA landfills and reducing health problems associated with FA while restoring ecosystem functions (Carlson and Adriano, 1991; Kostić et al., 2012; Mitrović et al., 2012; Pietrzykowski et al., 2018; Kostić et al., 2022b). Species from the Salicaceae family stand out when it comes to being used for the phytoremediation and revitalisation of polluted habitats, and in particular those from the Populus genus, which is characterised by high tolerance to a variety of environments (Zacchini et al., 2009; Guerra et al., 2011). However, species of Populus, as a thoroughly mapped genus, differ widely in their uptake patterns, as well as adaptation abilities under varying abiotic conditions (Dos Santos Utmazian et al., 2007; Zacchini et al., 2009; Di Lonardo et al., 2011; Tőzsér et al., 2023). To date, research has mostly focused on accumulation, phytoremediation potential, and tolerance of poplars to metal pollution (Madejón et al., 2004; Robinson et al., 2005; Borghi et al., 2008; Todeschini et al., 2011; Kostić et al., 2022b) or their physiological response to individual stress factors such as drought, salinity or high temperatures (Chen and Polle, 2010; Yoon et al., 2014; Zhang et al., 2019; Kim et al., 2022). However, studies on the ecophysiological response of Populus alba L. to the combined effects of multiple stress factors are rare, particularly at FA landfills. Therefore, the aim of this study is to contribute to the expansion of knowledge on the ecophysiological response of P. alba to single and combined multiple abiotic stresses at FA landfills and to test its suitability for the potential revitalisation of such habitats. We hypothesised that P. alba, which spontaneously colonised two FA disposal lagoons at the ‘Nikola Tesla A’ thermal power plant (TENT A), Obrenovac, Serbia, 3 years ago (L3) and 11 years ago (L11), has high restoration potential thanks to its stress tolerance. The first research objective was to evaluate some of the abiotic stress factors in FA lagoons at different stages of weathering by analysing (1) basic physical and chemical properties and (2) PTE (As, B, Cr, Cu, Mn, Ni, Se, and Zn) concentrations. The second objective was to assess the ecophysiological response of P. alba to abiotic stresses based on (1) determining its ability to regulate PTE transfer from soil to root and root to leaf and (2) its photosynthetic efficiency parameters (Fv/Fm), photosynthetic pigment content, non-enzymatic antioxidant defence parameters (carotenoids, anthocyanins, and phenols), oxidative stress parameters by measuring the concentration of malondialdehyde (MDA), and the total antioxidant capacity (IC50) for neutralizing the activity of DPPH free radicals. The results of this study, which would determine the temporal dynamics of the ecophysiological response of P. alba to single and combined abiotic stresses, could be of importance both for its application in the planned revitalisation of the TENT A landfill and for restoring similar landfills around the world.

At FA landfills, numerous kinds of abiotic stress simultaneously and continuously affect plants as a result of the unfavourable physical and chemical properties of the ash, the extreme microclimatic conditions, and the deficit or toxicity of essential and non-essential chemical elements. Such conditions hinder the survival and development of vegetation because they threaten the normal course of physiological processes in plants, including photosynthesis, which is extremely sensitive to heat, drought, salinity, heavy metal toxicity, and nutrient deficiency or toxicity (Muhammad et al., 2021). Research has shown that a combination of multiple stressors has a cumulative effect on almost all the physiological parameters that result in increased plant damage (Pandey et al., 2015; Zandalinas et al., 2021). Many studies have shown that plants vary in terms of their ability to adapt and survive under conditions of multiple and varying stresses at ash landfills and that their survival is most often based on a strong functional response and their antioxidant capacity (Gajić et al., 2020; Kalashnikova et al., 2021; Qadir et al., 2021; Qadir et al., 2022). In addition, successful plants are able to immobilise PTEs in the root zone rather than accumulate them in leaves (Mitrović et al., 2008; Kostić et al., 2022b). Therefore, by analysing the functional properties of plants that survive in conditions of multiple stresses, it is possible to select species that are suitable for the processes of the revitalisation of polluted habitats and thus contribute to reducing pollution in the vicinity of large industrial complexes.

Analysis of chlorophyll fluorescence is a useful tool for studying the impact of abiotic stress on plants (Guidi et al., 2019). It provides information on the state of PSII, i.e. to what extent it uses the energy absorbed by chlorophyll and to what extent these stress conditions lead to damage to the photosynthetic apparatus. In this sense, calculating the photosynthetic efficiency parameter (Fv/Fm) is crucial for detecting the photoinhibition of PSII under stress conditions. The results of our study showed that at the control site there was good water availability due to occasional flooding, an adequate water table, and well-structured silty clay loam. The soil was also slightly alkaline (7.4-7.8; Soil Survey Division Staff, 2017), there was an adequate mineral supply, and concentrations of the studied PTEs in the leaves of the control plants were in a range below the critical values for plants (Kabata-Pendias and Pendias, 2001). This provided optimal conditions for the growth of P. alba as shown by Fv/Fm values, which were in the optimal range for plants (0.750-0.850; Bjorkman and Demmig, 1987). However, Fv/Fm values for P. alba leaves at the FA lagoons were significantly lower at L3 (ranging between 0.650-0.687), indicating photoinhibition of PSII and a decrease in P. alba vitality with a tendency to further decrease at L11 (ranging between 0.510-0.585) due to prolonged stress and the accumulation of photosynthetic tissue damage, visible through pronounced morphological symptoms in the form of necrosis and leaf drying. A decrease in photosynthetic efficiency has been found in many plants at FA landfills such as Dactylis glomerata L., Ricinus communis L., Solanum lycopersicum L., Tamarix tetrandra Pall. ex M. Bieb., Pinus sylvestris L., Picea abies (L.) H. Karst., Fagus sylvatica L., and Alnus glutinosa (L.) Gaertn. (Gajić et al., 2020; Bęś et al., 2021; Kostic et al., 2022c; Qadir et al., 2022).

The general reduction in photosynthetic efficiency in the examined species at both FA lagoons is the result of the influence of multiple stressors such as drought, heat, and salt, but also inadequate mineral nutrition (PTE toxicity or deficit). In general, drought, salt, and heavy metal stress have been found to have almost similar effects on plant growth as they result in the production of ROS and oxidative stress (Zhang et al., 2019; Krzyżak et al., 2023). Specifically, the weaker binding properties of FA particles due to their texture, which corresponds to the texture of loamy sand soils, and low organic matter content resulted in the ash having a lower water-retention capacity, which exposed P. alba to drought stress at L3. Despite the fact that the hydraulic transport of raw FA during its disposal at L3 caused a significant reduction in the ash’s salinity due to drainage of the transport water and the drying out of the FA (Kostic et al., 2018), the salt content in the ash at that lagoon was still significantly higher (242.60µS cm^-1) than in the control soil. This has a drought-like effect on the plants and is reflected in the reduction in photosynthetic efficiency (Técher et al., 2012). It should be noted that at L11 both abiotic (weathering) and biotic (vegetation development) factors that inevitably accompany the FA disposal process at landfills contribute to the increase in the Silt+Clay fraction and the narrowing of the C/N ratio as the best indicator of organic matter accumulation. They also result in a reduction in pH and the content of soluble salts in FA due to their leaching, which should increase the capacity of the FA to retain water and nutrients (Uzarowicz et al., 2017; Konstantinov et al., 2020; Petrović et al., 2023). However, at the time of our research, maintenance of the vegetative cover at L11 was no longer carried out (i.e. it was not watered), unlike at L3, so there was a moisture deficit despite the changes in the physical and chemical characteristics. The moisture deficit at the TENT A landfills is further intensified by the excessive heating of the surface layers of FA due to its dark grey colour, which was confirmed by the extremely high temperatures that were measured on the surface of the FA lagoons in the summer months. Moreover, due to the pozzolanic properties of FA particles, their wetting in the presence of liming material (CaSO4) cements them together, which is why compacted layers, which reduce aeration, water infiltration, and root penetration, form in places (Carlson and Adriano, 1993). Although flexible plant roots can grow horizontally or develop through zones of least resistance such as root canals, root elongation and water infiltration is reduced by the hard, compact layers of FA (Haynes, 2009). Stomatal closure as a common response to drought reduces CO2 uptake and fixation, but also reduces transpiration, limiting the cooling of the leaf’s surface, resulting in thermal stress that has negative effects on metabolic processes, primarily photosynthesis. Thermal stress also causes changes in chlorophyll pigment and carotenoid content in leaves, thus affecting photoinhibition, which results in a reduced quantum yield of PSII (lower Fv/Fm), enhanced peroxidation in the leaf cell membrane (an increase in MDA), and reduced membrane thermostability (Yoon et al., 2014; Stojnić et al., 2016; Pilipović et al., 2020; Rosso et al., 2023). Increased salinity limits plant growth by inducing osmotic stress, impairing the uptake of ions leading to their imbalance or toxicity, inhibiting metabolic processes and enzyme activity, damaging thylakoid membranes, reducing photosynthetic pigments, and disrupting photosynthetic activity (Chen and Polle, 2010; Zhang et al., 2019).

Two of poplar’s main strategies to cope with drought stress are avoidance mechanisms that control transpiration through the regulation of stomatal conductance, the deposition of cuticular waxes to limit non-stomatal transpiration, increased root growth, and reduced leaf area (Attia et al., 2015) and a stress tolerance strategy based on mechanisms to maintain biological functions through the synthesis of protective molecules and compounds under stress conditions (Viger et al., 2016). Salt tolerant poplar species are characterised by restricted xylem translocation and salt accumulation in the roots (Chen and Polle, 2010). However, our research found a reduction in photosynthetic efficiency of P. alba at both FA lagoons, which confirms earlier findings concerning P. alba x P. glandulosa (Yoon et al., 2014) and P. alba × P. davidiana (Kim et al., 2022) on the sensitivity of poplar to drought and salt stress, which leads to changes in their metabolic activity.

In terms of PTE pollution, among the PTEs determined in the FA at L3, As, B, and Cr were the main contaminants with concentrations exceeding critical levels for plants (As>20 mg kg^-1, B>25 mg kg^-1, Cr>75 mg kg^-1; Kloke et al., 1984; Kabata-Pendias and Pendias, 2001). Concentrations of Cu, Ni, and Se were higher than the average for sandy to silt-loam substrates and concentrations of Mn and Zn were lower than the range of average values (<270 mg kg^-1 and<45 mg kg^-1, respectively; Kabata-Pendias and Pendias, 2001). The surface association of As, B, Cr, and Zn on FA particles contributes to their greater solubility, making these elements extremely hazardous to plants at FA landfills if they are accumulated in high concentrations (Gajić et al., 2020; Kostić et al., 2022b; Kostic et al., 2022c). However, it also contributes to their significantly lower content in FA at L11, although the content of B at L11 was still in the critical range for plants. As essential elements, B and Zn are easily transported (BCF Leaf>1; TF>1) to leaves (Kabata-Pendias and Pendias, 2001; Robinson et al., 2005), while As and Cr as non-essential chemical elements mostly accumulate in roots (Wang et al., 2002; Singh et al., 2013). Under these conditions at the TENT A landfills, P. alba accumulated toxic concentrations of B and Zn (>50 mg kg^-1, >100mg kg^-1; Kabata-Pendias and Pendias, 2001) at L3 and L11 and also toxic concentrations of As (>5 mg kg^-1; Kabata-Pendias and Pendias, 2001) at L11, while Mn content was in the deficit range (<30 mg kg^-1; Kabata-Pendias and Pendias, 2001). The accumulation potential for B and Zn in P. alba leaves at L3 and L11 is also confirmed by earlier studies on poplars (Madejón et al., 2004; Robinson et al., 2005; Dos Santos Utmazian et al., 2007; Robinson et al., 2007; Di Lonardo et al., 2011; Rees et al., 2011; Huang et al., 2019; Pilipović et al., 2019; Kostić et al., 2022b; Tőzsér et al., 2023). The lower content of B in P. alba leaves at L11 compared to L3 was accompanied by changes in B content in FA because its solubility at pH>6 is conditioned only by its total concentration (Iwashita et al., 2005). The increase in Zn and As content in leaves at L11 compared to L3 is a result of their greater accumulation and translocation to leaves at this lagoon. This is due to the higher bioavailability of Zn and As, which is in line with the changed physical and chemical characteristics of FA at L11 compared to L3 – namely, a higher proportion of finer particles and organic matter, lower pH and salinity, and the absence of supplementation with phosphate fertilisers. This contributes to the greater solubility and mobility of the elements (Kostic et al., 2018; Balafrej et al., 2020; Gajić et al., 2020). Specifically, unlike at L3, where the application of phosphate fertilisers compensated for the phosphorus deficit in the FA, the cessation of phosphorus supplementation at a later stage of revitalisation and its leaching caused a deficit in the FA at L11, which can result in increased uptake of As by plants through the phosphate transport system (Wang et al., 2002). The poor solubility of Mn in FA is the cause of its characteristic deficit in plants growing at ash landfills (Adriano et al., 2002; Mitrović et al., 2008; Kostić et al., 2022b). Namely, earlier findings suggest that Mn content in P. alba leaves correlates to its availability in surface soil (Madejón et al., 2004) and that the accumulation potential of poplar for Mn is limited (Tőzsér et al., 2023). The higher uptake and transport of Zn through plant tissue to P. alba leaves at L11 may be caused by the lower Mn content in leaves resulting from its reduced bioavailability in FA at L11 due to the altered physical and chemical characteristics of FA when compared to L3 (Kabata-Pendias and Pendias, 2001; Aref, 2011). Cr content in P. alba leaves at L3 and L11 was higher than normal, but not toxic (>0.5 mg kg^-1; Kabata-Pendias and Pendias, 2001). In our study, much higher Cr concentrations were measured in P. alba roots than in leaves, confirming earlier findings for poplar trees (Pulford and Watson, 2003; Ancona et al., 2017; Tőzsér et al., 2023). BCF Root<1 and TF<1 make P. alba suitable for the phytostabilisation of this element.

The results of discriminatory analysis showed that of all the analysed PTEs in P. alba parts at the study sites, toxic concentrations of As, B, and Zn and a deficit of Mn contributed most to differences in the response of P. alba to the effects of stress. Of these differences, changes in the content of Chl a, Chl b, Tot Carot, MDA, and IC 50 were the most pronounced (Figure 5). Specifically, the toxic content of B in leaves may inhibit photosynthesis and significantly reduce Fv/Fm by damaging thylakoid membranes and reducing CO2 assimilation. This, in turn, causes disruption to photosynthetic electron transport, oxidative damage, and decreased photosynthetic enzyme activity, as well as reduced synthesis of δ-aminolevulinic acid and protochlorophyll as a precursor of chlorophyll biosynthesis as noted by a large number of researchers (Ardic et al., 2009; Han et al., 2009; Wang et al., 2011; Cervilla et al., 2012; Chen et al., 2012; Landi et al., 2012). At L3, typical visible symptoms of B toxicity were noted in the form of chlorosis and tip and marginal leaf necrosis (Rees et al., 2011). Nevertheless, the fact that there are differences in the tolerance levels of different poplar species is shown by the hybrid species Populus nigra × euramericana. Only 10% of its leaves exhibited damage at concentrations of up to 900 mg B kg⁻¹, while chlorosis occurred with concentrations of 1000-2000 mg B kg⁻¹ and tissue necrosis only at concentrations greater than >2000 mg B kg⁻¹ (Rees et al., 2011). Photosynthesis is the first metabolic process that will be disrupted by toxicity of Zn, which leads to a loss of plasma membrane integrity and a reduction in its permeability, damage to chloroplast functioning, and a reduction in photosynthetic electron transport (Huang et al., 2019; Balafrej et al., 2020). It has been found that high concentrations of Zn in poplar induce chlorotic and necrotic changes, alter leaf morphology and ultrastructure, leading to significant thickening of the leaf lamina and spongy tissue, and reduce above-ground biomass (Todeschini et al., 2011). It is possible that the extensive symptoms of damage in the form of chlorosis, necrosis, and leaf drying and also the visible smaller leaves with thicker lamina at L11 are the result of long-term toxic concentrations of Zn in poplar leaves. However, the toxic As levels measured in P. alba leaves at L11 can also adversely affect photosynthetic efficiency as it leads to disruption of the electronic transport chain, which conditions changes in the formation of NADPH and ATP and results in increased chlorophyll fluorescence (Stoeva and Bineva, 2003; Rahman et al., 2007; Gusman et al., 2013). This can be seen in our research through the decrease in Fv/Fm values, but also the visible morphological changes in the form of red-brown necrosis and the drying of older leaves (Kabata-Pendias and Pendias, 2001). Previous research has shown that P. alba accumulates higher amounts of As in leaves at contaminated sites than at uncontaminated ones (Madejón et al., 2004) and that the impact of As toxicity on chlorophyll synthesis is particularly pronounced (Moreno-Jiménez et al., 2009), which may be associated with the decrease in chlorophyll content at L11. Manganese is a chemical element that is directly involved in electron transport reactions and is essential for the synthesis of chlorophyll. Therefore, its deficit leads to the destruction of chloroplast thylakoid membrane structure, a reduction in the number of PSII reaction centres, and the degradation of complexes for the photolysis of water, in which four Mn atoms provide the energy necessary for the oxidation of two water molecules to oxygen (Alejandro et al., 2020).

In terms of photosynthetic pigments in leaves, measuring chlorophyll concentrations is an effective way to determine plants’ tolerance to stress. At L3, P. alba was found to have lower vitality, but significant differences in the content of Chl a, Chl b, Tot Carot, Anthoc, Ph Free, Ph Bound, and MDA compared to control individuals were not found. This may be the result firstly of NPK fertilisation, as N application improves the drought tolerance of poplars (Song et al., 2019), secondly of the higher Mn content in leaves compared to the other two sites as one of the mechanisms for overcoming stress (Della Maggiora et al., 2023), and thirdly the higher total antioxidant capacity of P. alba at this lagoon (<IC 50) as an indicator of the activation of enzymatic and non-enzymatic antioxidant systems (Zhang et al., 2019; Rosso et al., 2023). Also, the localisation of high concentrations of accumulated B in chlorotic and necrotic tissues in the tips and marginal parts of leaves is another adaptive mechanism of plants, which thus ensures a significant proportion of photosynthetic tissue remains functional (Rees et al., 2011). Unlike P. alba at L3 and the control site, a lower content of Chl a, Chl b, and Tot Carot and a higher content of MDA in the leaves along with a higher IC 50 was found at L11, indicating its less effective antioxidant defence system, which is consistent with the very low Fv/Fm values measured at that site. The significant correlation between As, Mn, and Zn in leaves and the analysed ecophysiological parameters (Table 4) indicates that the increase in As and Zn concentrations, as well as the decrease in Mn concentrations in leaves at L11 induces its weaker adaptive response at the older lagoon. The accumulation of both physiological and morphological damage indicates the importance of the long-term impact of stress on the process of photosynthesis in P. alba (Muhammad et al., 2021). Phenols as secondary metabolites contribute to physiological processes that are associated with protection against abiotic stress (Bencherif et al., 2020). As such, the higher Ph Free content in leaves at L11 when compared to the control site can be seen as the antioxidant response of P. alba to the environmental stresses at that particular site. However, it was not sufficient to quench lipid peroxidation and scavenge ROS under drought and salt stress conditions, as well as elevated PTE content. In particular, the toxic As and Zn concentrations contribute to this, as indicated by the positive correlation between Ph Free and the content of these two elements in leaves (Table 4). Also, the cytotoxic and prooxidative nature of phenolic compounds can be expressed in plants growing on FA due to the high content of Fe and Al oxides with the formation of phenoxy radicals, the life of which is significantly extended in the presence of Zn (Sakihama and Yamasaki, 2002). Through the synthesis of carotenoids and anthocyanins, plants also actively increase tolerance to different types of stress (Cervilla et al., 2012). However, unlike some species of plant in which a high As content in leaves induces an increased accumulation of anthocyanins (Leão et al., 2014), in woody species, such as P. alba in this case, and in earlier studies on Tamarix tetrandra at FA landfills, their antioxidant role is absent (Kostic et al., 2022c). The degradation in carotenoids for P. alba observed at L11 results in a reduction in the plant’s resistance to ROS, which favours a rise in lipid peroxidation rates and MDA concentrations, as was also found in T. tetrandra, Cassia occidentalis, and Dactylis glomerata at FA landfills (Love et al., 2013; Gajić et al., 2020; Kostić et al., 2022b). The exposure of P. alba to oxidative stress at L11 is also indicated by the decrease in the Chl a + b/Tot Carot ratio compared to L3, which occurs because of the accumulation of leaf tissue damage and progressive tissue aging due to its prolonged exposure to environmental stresses (Stoeva and Bineva, 2003). This is unlike the Chl a/b ratio as an early warning indicator of the toxic effects of metal accumulation in plants (D’Addazio et al., 2023), which remains unchanged compared to L3. These results are in line with earlier research that also showed a decrease in chlorophyll and carotenoid content, as well as an increase in MDA in poplars in response to drought (Kim et al., 2022) and heavy metal stress (Chandra and Kang, 2016). A decrease in chlorophylls and carotenoids has also been found in many plants at FA landfills, such as T. tetrandra, Robinia pseudoacacia L., Amorpha fruticosa L., and Withania somnifera (Pavlović et al., 2004; Qadir et al., 2021; Kostic et al., 2022c).

This study was conducted with the aim of evaluating the ecophysiological response of Populus alba L., which spontaneously colonised two FA disposal lagoons at the ‘Nikola Tesla A’ thermal power plant (Obrenovac, Serbia) 3 years (L3) and 11 years (L11) before the beginning of the research, to simultaneous and continuous exposure to abiotic stressors. It was found that at both lagoons P. alba was exposed to multiple stress factors such as drought and salt stress, but also inadequate PTE concentrations. At L3 there were critical concentrations of As, B, and Cr in FA and at L11 critical concentrations of B, while a deficit of Mn is characteristic for all plants growing on such substrates. The P. alba individuals at both lagoons showed lower vitality than the individuals in their natural habitat. In addition, the effects of single and combined stressors on P. alba trees resulted in visible leaf damage symptoms in the form of chlorosis, necrosis, and drying of leaves. Some of these symptoms are characteristic of drought, increased salinity, toxic concentrations of B and Zn, and deficient Mn in P. alba leaves and were more pronounced due to the increase in toxic concentrations of As at L11. Nevertheless, care measures (e.g. watering, fertilisation) and the increased biosynthesis of enzymatic and non-enzymatic antioxidant compounds (<IC 50) at the beginning of the revitalisation process contributed to the better response of P. alba to stress at L3, as indicated by the absence of differences in levels of Chl a, Chl b, Tot Carot, Anthoc, Ph Free, Ph Bound, and MDA compared to the control individuals. In contrast to L3, the increased toxic concentrations of As and Zn in leaves at L11 resulted in oxidative stress in P. alba, as evidenced by lower vitality, reduced synthesis of chlorophyll, carotenoids and total antioxidant activity, and higher MDA content. This suggests that despite its rapid colonisation of FA lagoons, the stress tolerance of P. alba decreases with increasing intensity and duration of exposure to adverse abiotic factors. Furthermore, its weaker stabilisation potential for substrates with high As and B content makes P. alba highly unsuitable for the permanent revitalisation of such habitats. Although white poplar is a fast-growing species with a good ability to accumulate metals and a high stress tolerance, the above characteristics could give it an advantage, but only if it were used during the temporary, short-term restoration process in the cyclical disposal of FA in the passive lagoons at the landfill and with the application of care measures such as ash moistening and NPK supplementation.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

OK: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. SJ: Formal Analysis, Investigation, Validation, Writing – review & editing. DP: Formal Analysis, Validation, Writing – review & editing. MMa: Formal Analysis, Investigation, Writing – review & editing. NR: Formal Analysis, Investigation, Writing – original draft. MMi: Conceptualization, Methodology, Supervision, Writing – review & editing. PP: Funding acquisition, Supervision, Writing – review & editing.