## 2 Materials and Methods
### Analytic Methods  
The frogbit plants were taken from the containers and rinsed with distilled water. The number of rosettes (one developed leaf and one not developed leaf) were counted. The plant material was then dried in a dryer to a constant weight at 105 \({}^{\circ}\)C and weighed on a Radwag analytical balance with an accuracy of 0.001 g d.w. The dried plant material was ground and weighed, and its level of mineralization was determined by wet combustion. Briefly, powdered plant samples weighing 0.250-1.000 g  
Figure 1: Scheme of the experiment with a European frogbit (**a**) a 3-week exposure of the plants to the Cd solution (I term); (**b**) a 6-week exposure of the plants to the Cd solution (II term); four replications in each from three growing seasons, E12 replications. were placed in 100 cm\({}^{3}\) Kiejdahl flasks and flooded with a mixture of 69-70% nitric (V) and 65% chloric (VII) acids in a ratio of 3:1 by volume. Cold mineralization was carried out for about 12 h, and then at 200-220 \({}^{\circ}\)C until a colorless clear solution was obtained. The mineralized samples were diluted with distilled water (purified on an ion exchange bed and by reverse osmosis) and transferred to 50 cm\({}^{3}\) volumetric flasks, with a blank sample prepared for each measurement series [55]. The cadmium content was determined by flame atomic absorption spectrometry (FAAS). The analyses were carried out using a Solaar AA Series Spectrometer (Thermo Element, Thermo Scientific, Waltham, MA, USA). The accuracy was verified by comparison with certified reference material (BCR-670). The metal content was expressed as \(\mu\)g\(\cdot\)g\({}^{-1}\) plant dry weight (d.w). Before the tests, the soil was subjected to the following tests: granulometric composition [56], soil reaction (pH in 1 M KCl) [57], available phosphorus content [58], available potassium content [59], available magnesium content [60], organic carbon content [61], organic matter content [62], total nitrogen content [63], and salinity [64]; in addition, the total metal content (lead, copper, zinc, manganese, and iron) was determined after mineralization in a mixture of 69-70% nitric (V) and 65% chloric (VII) acids by FAAS [65]. After the tests were completed, samples of soil material taken from the containers were dried at a temperature of approx. 60 \({}^{\circ}\)C to a constant weight, ground in a mortar, and passed through a sieve with a mesh diameter of 1 mm. The samples were mineralized by wet combustion (0.250-1.000 g d.w.) using the 69-70% nitric (V) and 65% chloric (VII) acid mix. A blank sample was prepared for each measurement series. The metal content was determined by FAAS [55]. The determinations were validated by comparison with a standard sample of known chemical composition. The content is expressed as \(\mu\)g g\({}^{-1}\) d.w. plants. After the tests, water samples were taken from the containers. Cadmium concentration was determined after mineralization by FAAS; dissolved oxygen, reaction (pH), and electrical conductivity (EC) were determined in by using standard method for examination of water and wastewater [66]. The detection limit of Cd is 2.0 \(\mu\)g\(\cdot\)dm\({}^{-3}\)[67]. The degree of cadmium contamination of the aquatic ecosystem was determined based on the cadmium sensitivity index of frogbit (the degree of cadmium contamination \(-\) C\({}_{\rm P}\) c\({}_{\rm Cd}\); tolerance index \(-\) T\({}_{\rm i}\)), the water pollution index (C\({}_{\rm w\ Cd}\)), and the soil material of the substrate (C\({}_{\rm sm\ Cd}\)), as well as the index of cadmium uptake efficiency by the plant (E\({}_{\rm p}\)).