Accumulation, translocation, and toxicity of arsenic in barley grown in contaminated soil

Arsenic is a nonessential element for plants; however, high levels of As can inhibit plant growth. The toxicity of As is influenced mainly by its speciation in soil. The objectives of the present study were to determine the fractional composition of As in soil, its accumulation in plants, and its potentially toxic effects on the morphological, anatomical, and ultrastructural levels. In a model experiment, barley (Hordeum sativumL.) was planted in Haplic Chernozem spiked with three different concentrations of As (20, 50 and 100 mg/kg). The fraction composition of As in the experimental soil was analysed using a method of sequential fractionation. The phytotoxic effects of As were analysed microscopically at the tissue, cellular, and intracellular levels. Analysis of the fraction composition of As revealed a higher amount of mobile forms of As that contaminated the generative organs of plants. Oxides of Fe, Al, and Mn became the main soil components to retain As when contamination of As increased. Arsenic toxicity inhibited plant growth by affecting morphological parameters (shape, size, and colour). It caused impairment in the root cells and a reduction in the size of the chlorophyllic parenchyma in the leaves. The ultrastructural analysis found changes in the main cellular organelles (chloroplasts, mitochondria, and peroxisomes). The bioconcentration factor (BCF), bioaccumulation factor (BF-soluble), and translocation factor (TF) allowed evaluation of plant protection mechanisms and determination of hazardous concentrations of As in soil. Despite the high buffering capacity of the soil, high As concentration affected morphological and ultrastructural parameters of the H. sativum.

2020). The content of As in Chernozem soils in the Russian steppes ranges from 1.1 mg/kg to 150 mg/ kg (Motuzova et al. 2006) in association with its presence in soil-forming rocks. In the North Caucasus region, a high concentration of As is attributed to the presence of rocks rich in polymetallic ores in the region. Typical concentrations of As in uncontaminated soils range from 0.1 mg/kg to 40 mg/kg (Bowen 1979). The total amount of As in earth's upper crust is estimated to be 4.01•10 16 kg with an average content of 6 mg/kg (Taylor and McLennan 1985;Nriagu and Azcue 1994;Matschullat 2000). In addition to natural sources, As may enter into the soil as a byproduct of anthropogenic activities such as manufacturing and widespread application of insecticides, herbicides, defoliants, feed additives for livestock, wood preservatives, and the burning, mining, and smelting of coal and other nonferrous metals (Smith et al. 1998).
The transfer of As from contaminated soils to agricultural crops poses potential health risks to humans through exposure to contaminated soils and consumption of food crops grown on them (Otones et al. 2011;Antoniadis et al. 2017). Moreover, plant growth, metabolism, respiration, photosynthesis, and opening size of stomata can be adversely affected by elevated levels of As. The accumulation of As and other pollutants in plant tissue depends on the plant species, and their capacity to adsorb pollutants which can be assessed by measuring rates of uptake and other soil-to-plant transfer parameters (Adamo et al. 2014;Antoniadis et al. 2017). In food crops, As uptake is highly variable and appears to be higher in green vegetables and grain crops compared to other crops grown in contaminated soils (Kabata-Pendias and Mukherjee 2007;Rahman et al. 2007). Researchers (Williams et al. 2007) showed the transition of As from various types of soils (anaerobic paddy soil and aerobic wheat soils) to barley, wheat and rice. There is a report indicating that Barley uptakes less As than rice. Transfer of As from soil to grain was an order of magnitude greater in rice than for wheat and barley, despite lower rates of shoot-to-grain transfer. The bioavailability of As for plants is determined by soil properties, notably mineral composition, organic matter content, pH, and redox potential (Tu and Ma 2003;Warren et al. 2003;Antoniadis et al. 2017;Zhao and Wang 2020). The biogeochemistry of As is not well understood due to the complexity of the fractioning methods used to analyse the composition and diversity of various As compounds (Motuzova et al. 2006;Adamo et al. 2014;Ma et al. 2017;Mathee et al. 2018).
To date, there is no reliable data on soil components that fix As and contribute to its bioavailability in soils in natural landscapes. Exchangeable fractions and carbonate are considered to be bioavailable; oxide-and organic matter-bound fractions may be potentially bioavailable; while the mineral fraction is mainly not available to either plants or microorganisms (He et al. 2005;Larios et al. 2013;Pavlović et al. 2019;Srithongkul et al. 2020;Alan and Kara, 2019).
The biogeochemistry and bioavailability of As in Chernozem soils are still insufficiently studied; thus, analysis of As uptake in Chernozem soils is an important research objective because of the effect of As on the morphological, biometric, and ultrastructural parameters of plants grown in contaminated soil (Fitz and Wenzel 2002;Otones et al. 2011;Pandey and Bhatt 2016;Williams et al. 2007). Speciation of As in Haplic Chernozem was investigated for the first time using a sequential extraction procedure (Motuzova et al. 2006). This method has advantages in extracting As compounds and can isolate up to six fractions. A combination of sequential extraction and microscopic analysis allowed us to estimate the bioavailability, uptake mechanism, and potential risk posed by As for barley (Hordeum sativum L.), one of the most important food crops in the world (Arendt and Zannini 2013) and widely used as a bioindicator to assess the effects and accumulation of soil pollutants Rajput et al. 2020).
Thereby, the main objective of this study was to investigate As uptake by the H. sativum from Haplic Chernozem from North Caucasus and to identify the impact of As on the morphological, anatomical, and ultrastructural indices of the plants.

Soil characteristics
The upper horizon of uncontaminated soil (Haplic Chernozem according to the WRB 2015) was collected from the Persianovskaya Steppe Nature Reserve (Rostov region) and five initial well-nixed samples from surface horizons (0 to 20 cm) were sampled in polyethylene bags and immediately taken directly to the laboratory for analysis. The soil samples were mixed, air dried, sifted through a 2 mm sieve, and homogenized. Soil sampling was performed as per ISO 18,400-104 (2018) procedures and standards.
The main physical-chemical soil properties analysed are presented in Table 1. The hydrogen ion concentration (pH) was analysed by potentiometric titration in a soil paste saturated with water. The cation exchange capacity (CEC) was estimated using the ammonium acetate method (Tan 1996), and the exchangeable cations were analysed using 1 M NH 4 OAc (Vorob'eva 2006). Organic matter was evaluated by dichromate oxidation using the Tyurin method (Jackson 1960), and particle-size distribution was determined using the pipette method (pyrophosphate procedure for soil preparation (Shein 2009).
The composition of the exchangeable bases, a high amount of organic matter, the presence of highly dispersed micellar forms of carbonates, and loess-like parent rocks produced favourable physical properties in Haplic Chernozem (i.e., permeability to water and air, high porosity, and moisture capacity).

Plant growth conditions and sampling methods
The artificial application of different doses of As allowed us to trace the changes of the soil indices under varying anthropogenic loads to predict the strength of metalloid fixation by the soil components at different levels of contamination. A 3-cm layer of washed glass was placed on the bottom of 2-L plastic vessels to provide liter for drainage, and 2 kg soil was added. Next, different doses of arsenic oxide (As 2 O 3 ) were added as dry powder to each of the treatment vessels and thoroughly mixed into the soil. Approximate permissible concentrations (APC) for As in neutral and slightly alkaline soils is 10 mg/ kg (GN 2.1.7.2511/09). The doses of As spiked in this experimental soils were similar to levels of As in anthropogenically-polluted soils. The doses of As corresponded to low (2 APC), high (5 APC), and very high (10 APC) degrees of contamination in soil. The study included three treatment doses (20, 50, and 100 mg/kg of As) and a control (uncontaminated soil sample). Each treatment was prepared in triplicate.
Soil incubation took place at a temperature of 20-22 °C under natural light conditions. The optimal soil moisture was maintained in the vessels throughout the experiment. After two months of soil incubation, the H. sativum was sown. Previous research demonstrated that two months is sufficient time for the transformation process of pollutants in the Haplic Chernozem . Twelve plants were grown in each vessel (4 treatment variants with three replications each, for a total of 144 plants). No additional fertilisers or pesticides were applied during the experiment. Each treatment was prepared in triplicate, and the position of the vessels was changed randomly every week. After the harvesting plants, the soil samples were taken for determination of As.

Determination of arsenic in soil samples
The composition of As complexes in the experimental soil were analysed using a method of sequential fractionation of metalloids developed by Motuzova et al. (2006). In the present study, the most ecologically-important compounds were extracted. Metalloid compounds were divided into mobile forms (specifically and nonspecifically adsorbed arsenate ions), strongly bound forms (low-solubility arsenates and As in primary and secondary minerals), and As compounds associated with oxides of Fe, Al, and Mn and organic substances ( Table 2). The main advantage of this method is the use of 0.1 N NaOH instead of 30% H 2 O 2 followed by dissolution of the residue in 1 N CH 3 COONH 4 and is often used to extract trace elements from soil organic matter (Tessier et al. 1979;Ure et al. 1993;Matera et al. 2003). Nonspecifically adsorbed arsenate ions were extracted using 1% (NH 4 ) 2 SO 4 (pH 6.0-6.2) with addition of ammonium molybdate (1% (NH 4 ) 2 SO 4 + 0.25% (NH 4 ) 2 MoO 4 , pH 5.5-6.0) to prevent re-precipitation of As into the solution. Specifically, adsorbed arsenate ions were extracted with 1 M NH 4 H 2 PO 4 . The As compounds retained by oxides of Fe, Al, and Mn compounds were determined using the Mehra-Jackson extract (0.5 M Na 3 C 6 H 5 O 7 + 1 M NaHCO 3 + 0.13 M Na 2 S 2 O 4 •2H 2 O with pH 7.3, Table 2). The As-containing compounds bound with organic substances were extracted with 0.1 N NaOH and bound to carbonates; low solubility salts were isolated with 1 N HNO 3 . Arsenic in the structure of the minerals comprising the soil was represented by residual fractions remaining after extraction was extracted by melting with NaOH. Analyses of As content in the soil extracts were determined by atomic absorption spectrophotometry (AAS, KVANT 2-AT, Kortec Ltd., Russia).

Preparation of samples for cytological and ultrastructural observation
The samples of plants with the maximum doses of contamination (100 mg/kg) were studied by transmission electron microscopy (TEM) analyses, because the changes in the ultrastructure are more visible with the high level of contamination in soil and plants (Fedorenko et al. 2020a, b;Minkina et al. 2020). When the H. sativum reached the bootand-head emergence phase (day 45 after sowing, Pérez-Harguindeguy 2013), 24 fresh samples were selected for analysis using light optical and electron microscope (2 plants each were taken for 4 treatment variants with 3 replications each). Samples of plants were taken from the central part of the second or third leaf (2 × 2 mm) and from the root fibrils (2 mm). All stages of tissue preparation for cytological and ultrastructural observations were performed using common techniques and included contrasting, dehydration, fixing in a polymerising mixture, and preparation and staining of semi-thin sections for light optical analysis .
Semi-thin Sections 0.5-1 µm wide were analysed by light optical microscopy using a LOMO microscope (Russia) at magnifications of 100 × and 400x. Ultrathin sections were obtained using an ultramicrotome EM UC6 (Leica, Germany) and contrasted with lead citrate. Microscopic analysis was performed using TEM, Tecnai G2 (Phillips, Netherlands) at the Centre of collective usage of Modern Microscopy of the Southern Federal University (Rostovon-Don, Russia). Ultrastructural measurements were performed using 10 sections from each sample for light optical microscopy and 10 sections for electron microscopy with an automated system for image analysis (Olympus Soft Imaging Solution iTEM).

Morphological characterisation
At the ripening stage (day 80), the remaining plants were harvested for morphological measurements (10 plants were taken from 4 treatment variants with 3 replications each for a total of 120 plants). The following parameters were measured: root length, plant height, stem length, leaf length, spike length, length of the spike with setas, 1000 grains weight, and fresh weight and dry weight of the plant. Next, the plants were divided into stem, roots, and grains for further analysis.
Determination of arsenic in H. sativum L. tissue and its accumulation in the soil-plant system The plant parts (stems, roots, grains) were dried at 105 °C for 30 min and then at 70 °C to a constant weight to determine dry biomass. The dry biomass was ground to a powder, then ashed in a muffle furnace at 450 °C for 6 h dissolved in 5 ml of 20% HCl, filtered through 0.45 µm Whatman filter paper, washed into a 50-mL flask, and brought to the required volume with deionised water. Mineralisation of the plant samples was carried out according to the Standard Russian method (GOST 26,929-94).
The toxicity level of As in the H. sativum tissues was determined by comparison with the Standard Russian maximum permissible concentration (MPC) of As in grain and grain products which is 0.2 mg/kg (Sanitary Regulations and Norms 2.3.2.1078-01). Depending on the As concentration, the standard sample of H. sativum obtained from the Federal Agency for Technical Regulation and Metrology of the Russian Federation was used for the digestion procedure (standard sample 10-234-2019).
To assess As accumulation in the soil-plant system, the following factors were calculated. The bioconcentration factor (BCF) was defined as the ratio of the content of the pollutant in the shoots to the total content in the soil (Edwards et al. 1998) and indicates the degree of 'biophilicity' of the element and any changes (Brooks 1983). If BCF < 1, the barrier functions of the root system manifest. Soil properties include the amount of organic matter, texture, CEC, and the effect of speciation of As on its bioavailability to plants (Huang et al. 2006;Zhao et al. 2009;Sharma et al. 2020).
To obtain the soluble As concentration in the soil (BF-soluble), the bioaccumulation factor (BF) was calculated as the ratio of the concentration of As in the plant biomass (roots and aerial parts) to the concentration of soluble As in the soil (Brooks 1998;Brunetti et al. 2012). The first two fractions (nonspecifically and specifically adsorbed As compounds) were used to calculate the BF-soluble factor.
The translocation factor (TF) was defined as the ratio between the pollutant content in the stems and that in the roots (S tem /R oot ), as well as between the content in grains and stems (G rain / S tem ) (Brunetti et al. 2012;Otones et al. 2011). According to Kachenko and Singh (2006), the TF can be used to quantify existing differences in the bioavailability of pollutants to plants, thus indicating their mobility in the soil.

Statistical analyses
All laboratory tests were performed in triplicate. The results obtained were processed mathematically using Microsoft Excel. The Mann-Whitney test (U-test) was used to assess the statistical significance of differences between the mean values. The influence of the metalloid content in the soil on changes in the morphological parameters of barley was estimated using the Spearman correlation coefficient. The calculations were performed at a given p-level < 0.05.

Arsenic fractional composition in Haplic Chernozem
Regardless of the dose of the metalloid in the unpolluted soil, a statistically significant increase in all mobile forms of As, as well as their total content relative to the soil of the control variant, was revealed ( Table 3). The main proportion of As compounds in unpolluted soil was found in the residual fraction (57%) characteristic of bonding with silicates ( Table 3). The content of the most mobile fractions of As in unpolluted Haplic Chernozem was less than 6% of the total specifically and nonspecifically adsorbed fractions. The specifically adsorbed fraction of As was higher than the As content in the nonspecifically adsorbed fraction. The amount of As in the fraction bound with organic substances was 7%. The As compounds bound with Fe, Al, and Mn oxides (29%) play an important role in the fixation of As.
When metalloid is introduced into uncontaminated soil, the average values of the sum of all the nonspecific and specifically-bound As compounds, as well as other fractions of As, significantly increase relative to the content of pollutants in the soil of the control variant (Table 3). The addition of As into soil in various doses increased the content of more mobile nonspecific and specifically-bound As from 3 (2 APC) to 43 folds (10 APC) compared to unpolluted soil. The fraction of As bound with organic substances increased from 7 to 11% at a very high degree of pollution (10 APC). The proportion of As in Fe, Al, Mn oxides during contamination is up to 36-43%. The fraction of low-solubility As compounds, represented mostly by Fe and Ca arsenates, increased from 1 to 8% with increasing of soil pollution (from 2 to 10 APC). The portion of As in stable compounds not transferred into extracts reduced the degree of pollution in the structure of the minerals comprising the soil and comprised 47-25% of the total fraction of the As.

Arsenic accumulation and its distribution in H. sativum
Compared to barley grown on unpolluted soil, the metalloid content in all parts of the plant increased significantly with the introduction of As into the soil. The exception was the values obtained for barley grain grown in soil with 20 mg/kg As, where the average values of the metalloid content did not differ statistically compared to the grain of the control plant (Table 4). The results showed that the As content in different parts of plants grown in unpolluted soil was 0.01-0.2 mg/kg (Table 4). A 20 mg/kg dose of As caused 4, 3, and 2 times higher As content in the roots, stems, and grains, respectively, than in the control plants. The accumulation of metalloid in the parts of the plants increased as the applied dose increased ( Table 4).
The grains of H. sativum were contaminated at the highest added dose of As of 100 mg/kg (MPC for grain 0.2 mg/kg). Also, the addition of 100 mg/kg As to the soil caused the toxicity of the plants to exceed the MPC in plants (0.5 mg/kg) by 5 times.

Assessment of the effect of arsenic on morphological parameters in H. sativum
The total height of H. sativum (including the height of the stem from the tillering node and the height of the spike with awns) in the control plants was 85.9 cm (Table 5). A low dose of As (20 mg/kg) did not affect the biomass and plant height, roots, stems, or leaf length. Statistically significant changes in morphological parameters relative to the control variant were observed in plants grown in the soil with an As doses of 50 and 100 mg/kg: the length of the roots decreased by 24 and 58%, the height of the plants by 20 and 42%, the length of the stems by 21 and 40%, and spike length with awns by 18 and 48%, respectively (Table 5). The most sensitive morphological parameters, the CV of which is more than 30%, are the root length, fresh weight of root, dry weight of root and dry weight of stem with setas. At 100 mg/ kg, the length of the leaf decreased by 22% and spike length without awns decreased by 47%. Length of the leaf The length of the leaves is the most stable characteristic of the plant, since a significant change in the average values compared to the control is observed only when 100 mg/kg of As is added to the soil, the CV for this indicator is 11.1. These changes resulted in the curtailed fresh weight of roots and stems and reduced up to 24% dry weight of root and 17% dry weight of stem of H. sativum at the highest dose of As. The mass of 1000 grains decreased from 21 to 58% when the dose of As increased from 50 mg/kg to 100 mg/kg, respectively. A negative relationship was established between the content of nonspecifically absorbed, as well as specifically absorbed fraction As in the soil and morphological indicators of barley such as: "length of the leaf" and "1000 grains weight", "dry weight of root", "dry weight of stem with setas". For other studied characteristics of barley, with an increase in the content of the most mobile forms of As in the soil, a noticeable trend towards a decrease in indicators was observed (Fig. 1).

Light-optical microscopy of H. sativum tissues affected by arsenic
Light-optical microscopic observations of H. sativum tissues showed sparse and shorten root hairs, a smaller central cylinder than the in control (Fig. 2,  Table 6). A statistically significant decrease in "Cross-sectional area of the central cylinder", "The average number of cells per 1 mm 2 of the chlorenchyma", "Average cell size", "Number of plastids per cell" compared with control plants were shown only with a dose of 100 mg/kg As (Table 6). A change in the structure of the conductive tissue in the central cylinder was observed. The central cylinder had one large vessel in the center and many small radiallylocated vessels, and separated from the cortex by the endoderm which consisted of a single layer of cells and included the xylem vessels and phloem sieve tubes that form the conductive tissues. (Fig. 2). Most of the root cross-section (up to 70%) was primary bark (Table 6). Exposure to As at concentrations of 20, 50, and 100 mg/kg inhibited development of the epiblema, and the bark layer was thin, comprising less than 50% of the cross-section area of the root.
A layer of large cells of water-storage tissue was located under the epidermis of leaf cells. Mesophyll with a system of intercellular spaces and conductive bundles (the number that determines the rate of water uptake by the leaf plate) was located between the lower and upper epidermis. The spongy parenchyma was characterised by a large number of intercellular spaces that facilitate gas exchange. Whereas the leaves of the control plants were covered with a single epidermal layer of closely connected cells, the outer walls of which were covered with cuticles (Fig. 3). The pollutant violated the integrity of the epidermis, especially in the lower layer and significantly reduced the average number of cells per 1 mm 2 of the chlorenchyma and the number of plastids per cell when 100 mg/kg of Ac was introduced into the soil (Table 6).

Ultrastructural changes in the cells of roots and leaves of H. sativum
The results of TEM studies revealed changes in the ultrastructure of roots and leaves of H. sativum. Dense flattened cytoplasm adjacent to the cell wall was filled with free ribosomes (Figs. 4a, c, e). Vacuole occupies the most area within the cell. Endoplasmic reticulum (EPR) and Golgi apparatus were not sufficiently developed. The EPR was represented by short cisterns, randomly scattered along the cell section, some of which were associated with ribosomes.
Round mitochondria with a small number of slightly extended flattened cristae contained a relatively electron-light matrix. Dictyosomes had small stacks of cisternae without expressed polarisation. The round or slightly lobed nucleus occupied a large volume of the cell.
In the roots of plants grown in the highly-high polluted soil, cytoplasm with a low ribosome count was highly vacuolated (Figs. 4b, d, f). Swollen dropshaped cristae were found in mitochondria on a background matrix with high electron density (Fig. 4f). Single cytoplasmic fragments were observed in the central vacuole due to a violation of the integrity of the cell membrane (Fig. 4b). Ultrastructural changes in single cells were destructive (Fig. 4d).
Most of the chlorenchyma leaf cells of the control plants were elliptical or round. The cytoplasm contained the nucleus, plastids, mitochondria, ribosomes, and other organelles in the form of a narrow strip adjoining the cell wall (Figs. 5a,c,e). The middle part of the cell section area was occupied by a large vacuole containing groups of small particles. Chloroplasts were elliptically shaped with a dense matrix. The system of internal membranes (lamellae) in the organelles was represented by single stromal thylakoids and densely-grouped grana of thylakoids. The number of lamellae in a granum varied from 2-3 to 20-30 units (Fig. 5c, insertion) and the majority of these were in plastid and large. Free single ribosomes were scattered in a narrow strip of cytoplasm tightly adjacent to the plastid, and a few dense 80 nm plastoglobules formed small groups of 2-3 units. Oval mitochondria (about 0.6 µm in diameter) contained a moderately-dense matrix with evenly distributed slightly swollen cristae (Fig. 5e). Peroxisomes contained a fine-grained homogeneous matrix with diameters of about 1 µm (Fig. 5e). A decrease in the electron density of the stroma was observed in the plastids of plants grown in polluted soil. Small grains with 2 to 5 lamellae increased in length and the inner thylakoid space expanded (Fig. 5d). The number of plastoglobules also increased. Large, elongated mitochondria, up to 2 µm in size, contained a light matrix and numerous swollen cristae appeared. In some cells, significant vacuolisation of the cytoplasm was observed as well as numerous agglomerations of condensed high-density nuclear chromatin evenly distributed over the entire surface of the organelle (Fig. 5f, g).

Discussion
The distribution of As was determined for the types of compounds found in the unpolluted Haplic Chernozem (Table 3) as follows (in descending order): As compounds within the structure of the minerals comprising the soil > compounds bound with Fe, Al, and Mn oxides > bound with organic substances > specifically adsorbed > nonspecifically adsorbed > bound with carbonates and low-solubility salts.
In the present findings, the main proportion of As compounds in unpolluted soil was found in the residual fraction characteristic of bonding with silicates. The presence of As in various minerals determines its content in various rocks (Price and Pichler 2006;Kabata-Pendias and Mukherjee 2007;Tabelin et al. 2018;Mensah et al. 2020). The source of the As and other chemical elements in the unpolluted soil were pedogenic minerals. Compounds of As, presumably bound with carbonates and low-solubility arsenates, were practically absent in Haplic Chernozem.
Despite the high humus content in the soil studied, As mostly formed compounds with Fe, Al, and Mn oxides. The absorption of As by soils depends on the content of amorphous Fe oxides. In contrast to crystalline structures, As is adsorbed on to the outer surface of crystals or enters the loose and highly hydrated structure (Smith et al. 1999;Mensah et al. 2020).
Moreover, the studied Haplic Chernozem was characterised by weak alkaline reactions in a medium that caused co-precipitation of arsenate ions by Fe and Al oxides. It increased the As concentration in the soil. The correlation coefficients between the sorption parameters of As (V) in some soils and the content of weakly-crystallised non-silicate Fe compounds (R = 0.80) reveals their interdependence (Smith et al. 1999). The arsenate ions were able to establish covalent bonds with the cations to form readily-soluble salts. The trend of decreasing of As content compounds in the structure of soil minerals indicate that increased mobility of As compounds in soil is related to the increase in the amount of specifically adsorbed and nonspecifically adsorbed forms of the element.
Distribution of As in soil fractions under contamination (concentrations of 20 and 50 mg/kg) was similar to its distribution in unpolluted soil; however, soil polluted with As in concentrations of 100 mg/kg redistributed the fractional composition characterised by a predominance of compounds bound with Fe, A1, and Mn oxides as follows (in descending order): As bound with Fe, Al, and Mn oxides > As compounds in the structure of soil minerals > As bound with organic substances > specifically adsorbed As > nonspecifically adsorbed As > As bound with carbonates and low-solubility salts. Changes in the fractional composition of As in polluted soil were reflected in its accumulation in plants. Excess of the permissible level of As contamination was detected at a contamination dose of 100 mg/kg.
The intensity of As accumulation by H. sativum grown in unpolluted soil (control) was very low (BCF = 0.01). With an increase in As contamination, the BCF factor did not change, indicating the barrier functions of the root system. BCF factor was getting higher only at the highest level of pollution (up to 0.02). Low values of shoot/soil ratio for barley were also obtained by the researchers (Williams et al. 2007). BF-soluble factor increased with the degree of soil contamination, indicating that unhindered flow of metalloid from the soil into plants. However, the The H. sativum grown in unpolluted soil (control) and in the variant with 20 mg/kg of As were characterised by high S tem /R oot TF values (< 0.55 and < 0.46, respectively). There is a decrease in the S tem /R oot TF factor with an increase in the level of pollution; the value of TF was 0.22 under the highest treatment dose. This regularity shows the role of the root/stem barrier function in case of contamination. The uptake of As from the stem in the grain is characterized by a lower intensity than from the roots in the stem. There is an increase in the G rain /S tem TF coefficient with an increase in the level of pollution. A down regulation of stem-to-grain export may occur in barley (Williams et al. 2007). The value of G rain /S tem TF and S tem /R oot TF was the same at the highest dose of treatment.
A low dose of As (20 mg/kg) did not affect plants, and higher doses (50 and 100 mg/kg of As) caused a high decrease in morphological parameters. Structural and ultrastructural examination of H. sativum tissues using light and TEM showed effects on  , d, f, g). CW = cell wall, Cl = chloroplast, P = plastoglobuli, GT = thylakoids gran, ST = thylakoids stroma, N = nucleus, M = mitochondria, Pr = peroxisomes, V = vacuole, EPR = endoplasmic reticulum. The scale bar (μm) is: a) 2, b) 2, c) 1 and for insert: 0.2, d) 1, e) 1, f) 0.5, g) 0.5 changes in cellular organelles. However, there were no qualitative changes in the ultrastructure of plant cells at doses of 20 and 50 mg/kg of As, and differences in the morphometric parameters of intermediate doses were not all significant. The toxic effects of metals on ultrastructural indices were also noted in recent findings on H. sativum anatomy; however, it was less expressed Minkina et al. 2020). The toxic effects of Cr, Ni, and Zn on Typha domingensis macrophytes increased the root crosssectional area of the stele (Hadad et al. 2010). Thus, it appears that structural changes in the root determine transport characteristics (capabilities) allow the plant to adapt to unfavourable environmental conditions in its habitat. The vascular bundle structure changed, and the division of the mesophyll into spongy and columnar parenchyma was poorly traced. The average size of chlorenchyma cells increased from 145 µm 2 in the control plants to 167 µm 2 in plants contaminated by 100 mg/kg of As. The number of plastids per cell decreased from 7.4 to 6.1. Plant samples grown in soil contaminated with 20 and 50 mg/kg of As had morphometric parameters similar to those observed in the control plants.
Data from ultrastructural analysis of plant cells grown at a 100 mg/kg contamination dose were the most informative and significant. The micrographs show that the ultrastructure of the root cells in the control plants is similar to data obtained in previous studies (Fedorenko et al. 2020a, b). Similar changes in the ultrastructure of root cells were observed in Typha angustifolia under the toxic influence of Pb(NO 3 ) 2 (20,000 mg/L) with partial degradation of the cell wall of the root parenchyma (Panich-pat et al. 2005). After treatment of T. angustifolia seedlings with Cr, Cd, and Pb, destructive changes in cytoplasmic membranes, mitochondria, and cell vacuoles were observed in the roots (Mohamed and Huaxin 2015). In addition, disorganisation of the thylakoid system and vacuolisation of chloroplasts were observed in the leaves of these plants. Based on these results, it can be concluded that these types of structural changes are adaptive and that homeostatic mechanisms enable the plant to resist (tolerate) pollution.
Photosynthesis is the fundamental function of plants, and the structures in the leaves are determined by photosynthetic activity. A decrease in the number of lamellae in the grana and a more homogenous distribution over the entire area of the chloroplast section optimises conditions for the contact of the thylakoid surface with the environment, providing enhanced 'pumping' of protons from the stroma (pH 8) into the inner thylakoid space (pH ≈ 5). This may create an additional proton-driving force against the thylakoid membrane, and as a result, the membrane syntax of ATP will synthesise the amount of ATP (Hinkle and McCarty 1978) required to supply cells with energy to function under extreme factors. It was evident that the change in donor-acceptor relations resulting from transformation of the thylakoid system of plastids may control the mechanism of plant adaptation to adverse environmental conditions (Klimov et al. 1990). The spatial rearrangement of the internal plastid membranes probably indicates functional activation of a modified thylakoid system. At the same time, an increase in the number of plastoglobules probably occurred due to changes in the membrane structure of the plastids because the constituent components of the photosynthetic membranes such as lipids, proteins, and pigments released during the rearrangement of grana accumulate directly in plastoglobules (Guiamet et al. 1999;Titov et al. 2007).

Conclusions
The pattern of formation of the fractional composition of As at different concentrations in Haplic Chernozem was studied using three different concentrations. The largest amount of As was concentrated in the residual fraction. Oxides of Fe, Al, and Mn played a key role in the behaviour in the soil samples. The fractional composition of As was significant changed in polluted soil compared to the control soil. The main change occurred in the content of nonspecifically and specifically adsorbed arsenate ions, the proportion of which increased noticeably. BF-soluble factor increased with the degree of soil contamination. This trend shows the great importance of mobile forms of As uptake of H. sativum.
Despite soil high buffering capacity, the application of high doses of As led to its accumulation in the tissues of the H. sativum and adversely affected the morphological and ultrastructural parameters of the plants. The introduction of As at a concentration of 100 mg/kg had a strong toxic effect associated with high accumulation of the metal in the roots and aboveground parts of the plants. Microscopic analysis of the plants revealed destructive changes in the cells of the bark layer of the root and a reduction in the size of the chlorophyllic parenchyma in the leaves. Ultrastructural analysis revealed changes in the main cellular organelles (chloroplasts, mitochondria, and peroxisomes). In conclusion, the behaviour of As in a soil-plant system is important to understand to evaluate its entry into the food chain and potential risk to human health.