*2.5. Plants Analysis*

Several analytical techniques were applied to determine the elemental composition of plants materials. Content of As, Br, K, La, Na, Mo, Sm, U, W, Ba, Ce, Co, Cr, Cs, Hf, Ni, Rb, Sb, Sc, Sr, Ta, Tb, Th, Yb, Zn, Al, Ca, Cl, I, Mn, and V was determined using instrumental neutron activation analysis (INAA) at the fast-pulsed reactor IBR-2 in FLNP JINR. For Al, Ca, Cl, I, Mn, and V determination, samples of about 0.3 g were irradiated for 3 min and measured for 15 min. To determine As, Br, K, La, Na, Mo, Sm, U, W, Ba, Ce, Co, Cr, Cs, Hf, Ni, Rb, Sb, Sc, Sr, Ta, Tb, Th, Yb, and Zn, samples were irradiated for 3 days and measured after 4 and 20 days of irradiation. Gamma spectra of induced activity were measured using three spectrometers based on HPGe detectors with an efficiency of 40–55% and resolution of 1.8–2.0 keV for total absorption peak 1332 keV of the isotope 60Co and Canberra spectrometric electronics. The analysis of the spectra was performed using the Genie2000 software from Canberra, while the calculation of concentration was carried out using software "Concentration" developed in FLNP. The quality control of measurement was assured by the use of the following reference materials: IAEA-336 (Lichen), NIST SRM 1572 (*Citrus* Leaves), NIST SRM 1575 (*Pine* Needles).

The content of Cu, Pb, and Cd in samples was determined by using an iCE 3400 Atomic Absorption Spectrometer (AAS) with electrothermal (graphite furnace) atomization (Thermo Fisher Scientific, Waltham, MA, USA). Details of samples preparation for analysis and measurement can be found in [40].

Along with INAA, elemental composition of plant samples grown on contaminated was determined using an Element 2 mass spectrometer (Thermo Fisher Scientific of GmbH, Dreieich, Germany) after adding indium as an internal standard. Before the measurement, the instrument was adjusted in such a way that the sensitivity was at least 1,000,000 cps when analyzing a solution of indium with a concentration of 1 μg/L. The instrument was calibrated using multielement standard solutions ICP-MS-68A Solution B, ICP-MS-E, and ICP-MS-B (High-Purity Standards, Charleston, SC, USA). The good correlation of the concentration of elements obtained by two technique was attained.

To determine Mg and Fe content, a KVANT-2 spectrometer (KORTEK, Moscow, Russia) was used. The measurement was carried out in an air-acetylene flame using absorption lines of 248.3 nm for Fe and 285.2 for Mg. The instrument was calibrated using a multielement standard solution ICP-MS-68A Solution A (High-Purity Standards, USA).

For ICP-MS and AAS analysis, plant samples were digested using a microwave system (MARS5, CEM Corporation, Charlotte, NC, USA) in XP-1500 Teflon liners. The liners were preliminarily kept with 10.0 mL of an aqueous solution of nitric acid (1:1) at 160 ◦C, then cooled and rinsed with deionized water (18.2 M Ω.cm, Milli-Q, ADVANTAGE A10, Millipore Corporation, Molsheim, France). A 0.5 g sample was placed in a liner and nitric acid was added, then the mixture was kept at room temperature for 48 h. Then, hydrogen peroxide was added, and after the end of the vigorous reaction, decomposition was carried out in a microwave system at 180 ◦C. The resulting solution was analyzed after dilution with deionized water. Each average plant sample was analyzed twice, the content of the element in the sample was calculated as the average of two independent values.

Simultaneously with the plants, the analysis of "blank" samples and standard samples of plants certified for microelement composition was carried out. Elodea canadensis EK-1, birch leaf LB-1, and grass mixture Tr-1 (Institute of Geochemistry SB RAS, Irkutsk, Russia) were used as standard samples. For every 10 routine samples, one blank sample and one standard sample were analyzed.

#### *2.6. Determination of Ascorbic Acid (AA) and Glutathione (GSH)*

Determination of ascorbic acid and glutathione was carried out by titrimetric method using fresh shoots of *Echinochloa frumentacea*. To determine the content of ascorbic acid, an aliquot of the centrifuged supernatant of plant shoots was titrated in a solution of metaphosphoric acid (5 mL) with 0.001 N solution of 2,6-dichlorophenolindophenol (2,6- DCPIP) to a slightly pink color.

To determine the amount of glutathione, 2–3 drops of a 15% KI solution and 5 drops of a 1% starch solution were added to an aliquot of the supernatant (5 mL) and titrated with a 0.001 N KIO3 solution until a faint blue color was obtained.

The content of AA and GSH was calculated using the following formulas and expressed in mg/g of fresh weight

$$\mathbf{c}\left(\mathbf{A}\mathbf{A}\right) = \left[\left(\mathbf{a} \times \mathbf{K}\right) \times 0.88 \times \mathbf{M}\right] \div \left(\mathbf{m} \times \mathbf{n}\right)$$

$$\mathbf{c} \text{ (GSH)} = [(\mathbf{b} - \mathbf{a}) \times \mathbf{K} \times 0.307 \times \mathbf{M}] \div (\mathbf{m} \times \mathbf{n})$$

where c(AA), c(GSH) is content of ascorbic acid and glutathione in plant material (mg/g fresh weight), a is the volume of titrated 2,6-dichlorophenolindophenol (mL), b is the volume of titrated KIO3 (mL), K is the ratio of volumes of titrated KIO3 and 2,6-dichlorophenolindophenol (in the present study K = 1.5 ± 0.02), n is the sample weight (mg), M is the total volume of extract (mL), m is the volume of aliquot (mL), 0.88 is the volume of AA (mL), equivalent to 1 mL of 0.001 N solution of 2,6-dichlorophenolindophenol, and 0.307 is the volume of GSH (mL), (equivalent to 1 mL of 0.001 N solution of KIO3) [41].

#### *2.7. Microbiological Analysis of Rhizosphere Soil*

To analyze the rhizosphere soil, the plants were taken out from the pot, the bulk soil was shaken off from the plant roots, and the roots with the remaining adhering soil (thickness, no more than 2–3 mm) were used for analysis. The sampling was made in three replicates per pot. The sample of root with attached rhizosphere soil (1.5–2.0 g) was placed into 0.25-mL Erlenmeyer flask with 100 mL of sterile tap water and was shaken for 30 min. Then, the roots were taken out and the suspension was kept to let the soil particles settle out, after which a range of dilutions was prepared for isolation of rhizosphere microorganisms.

The total numbers of culturable heterotrophic bacteria, actinomycetes, and micromycetes after plant growth were estimated. For isolation and enumeration of the total number of culturable heterotrophs, we used the GRM-agar medium (State Research Center for Applied Biotechnology and Microbiology, Obolensk, Russia) of the following composition: fish meal pancreatic hydrolysate 12 g/L, enzymatic peptone 12 g/L, NaCl 6 g/L, and agaragar 10–12 g/L. The number of actinomycetes was determined using a starch–ammonium agar medium of the following composition: (NH4)2SO4 1.0 g/L, MgSO4·7H2O 1.0 g/L, NaCl 1.0 g/L, CaCO3 3.0 g/L, and agar-agar 20 g/L. The number of microscopic fungi was determined using Martin's medium of the following composition: glucose 10 g/L, KH2PO4 5 g/L, MgSO4·7H2O 0.5 g/L, peptone 5 g/L, agar-agar 20 g/L, and tap water 1 L (pH 5.5). The number of microorganisms resistant to Zn2+ and Pb2+ ions was determined on LB agar medium [42]. After sterilization, the water-soluble salts of heavy metals ZnSO4·7H2O, Pb(CH3COO)2, or CuSO4 were added to the culture medium to a final metal concentration of 0.5 mmol/L. The inoculated plates were incubated at 28–30 ◦C for 5–10 days, after which microbial colonies and colony-forming units (CFU) were counted and the morphological diversity of the microorganisms was measured.

The study of the taxonomic structure of rhizosphere microbial communities was carried out using metagenomic analysis of rhizosphere soil samples for the 16S rRNA gene. The purified DNA preparation was used as a template in the PCR reaction with universal primers (27f/533r) to the variable V4 region of the 16S rRNA gene using GS Junior Technology (Roche 454 Life Sciences, Branford, New Haven, CT, USA). Sequencing was performed on a MiSeq platform (Illumina, San Diego, CA, USA). The obtained data were analyzed using the QIIME v. 1.9.1.
