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1. Introduction
With the increasing industrial activity of the growing human population, the demand for metal materials currently exceeds the available supply. This has led to an increase in recycling technology, such as steel scrap recycling via electric arc furnaces. Steel recycling is a process in which iron is melted from waste iron in electric arc furnaces for steel production [1]. The problem of accidental melting of sealed radioactive sources from the steel-making process is notoriously like that of electric arc furnace dust contaminated with 137Cs (137Cs-EAFD) [2]. Therefore, 137Cs-EAFD is considered dangerous and causes significant health and safety risks to living organisms and their surroundings [3]. This radioactive waste contains high levels of 137Cs contamination, with a half-life of 30.17 years, and generates the ionizing radiation of gamma-ray (γ) emission (centroid peak energy at 661.7 keV), after which 137Cs decays into 137mBa through beta (β) emission [4]. Accordingly, 137Cs-EAFD is classified as radioactive waste, and it must be treated with appropriate management following its supporting regulations [5]. In this case, the leaching process is more favorable for reducing the proportion of 137Cs-EAFD to be responsible for the waste management strategy, which will dissolve 137Cs-EAFD and release 137Cs in a liquid that can then be remediated [6]. Moreover, our laboratory is now carrying out inspections of 137Cs-EAFD removal by washing treatment under laboratory-scale experiments [7,8].
137Cs is a non-degradable radionuclide notable for its moderately long half-life, and translocation in the ecosystem pose a great threat to public health and the environment. In this regard, with the objective to cope the defect of this radionuclides, remediation technologies of 137Cs contaminated areas have been pursued [9]. The conventional remediation of 137Cs uses mainly physical and chemical approaches. However, of these protocols need advanced machinery that is exorbitant cost, and the activities utilized in remediation operation have a possibility of by-product production causing further environmental damage [10]. In comparison, phytoremediation is an environment-friendly and sustainable approach that could be an effective alleviation method to remediate 137Cs-polluted water in a cost-effective way. Moreover, in term of waste management, plant residues after phytoremediation of 137Cs can be accomplish by pyrolysis and compressed that the best way to reduce the mass and volume of waste before it goes to a landfill [11]. Several plants have been identified for effective use in facilitating the phytoremediation of environmental pollutants, such as Vetiver grass was exhibited effectively remove 137Cs from low-level nuclear waste water [12]. In addition, phytoremediation is in line with worldwide efforts for sustainable development and environmental protection initiatives, making is the procedure of choice for remediation that led to increased public acceptance. Legumes are phytoremediators that have several advantages, such as nitrogen fixation, which compete with other plants for limited supplies of available nutrients at contaminated sites [13]. For this reason, legumes can act as pioneer plants in nonarable areas where other species are unable to grow. 137Cs tolerance is especially critical during germination because the higher activity concentrations of ionizing gamma radiation, such as from 137Cs, can affect plant growth and development [14]. Moreover, it is known that ionizing radiation does not only affect germination and initial seedling growth, but also has effects at physiological, biochemical and molecular levels [15,16,17]. Seed germination is the first stage of plant life, and this stage is more susceptible to highly contaminated environments such as radioactive nuclides. Thus, the germination assay and early seedling stage are essential because these initial phases are related to the environment [18].
This investigation aims to determine the effect of 137Cs stress from 137Cs-EAFD leaching effluent on legume species at the germination and initial seedling growth. This study compared the germination and initial seedling growth of five legume species at different activity concentrations of 137Cs. It is essential to select legume species that can be recommended as effective phytoremediators for 137Cs phytoremediation. The results of this research will probably establish a comprehensive of the performance of legume species, and they will serve future research in investigating their phytoremediation efficiency and potential to restore the 137Cs-EAFD leaching effluent.
2. Materials and Methods
2.1 Plant Materials and Experimental Design
The study was conducted at a radioactive waste management laboratory at the Thailand Institute of Nuclear Technology (Public Organization).
Five legume species from the Fabaceae plant family were selected for the experiment. These species were selected because they have various characteristics; some are perennial, others are annual, and they differ in their morphological characteristics of various external features, including roots, stems, leaves, flowers, fruits, and seeds. The species evaluated were Crotalaria juncea L., Mimosa pigra L., Neptunia plena L., Sesbania javanica Miq., and Vigna mungo L.
2.2 Critic Acid Leaching Process of 137Cs-EAFD
Before conducting the experiments, 137Cs-EAFD obtained from a steel production factory in Thailand (activity concentration 153.42 ± 3.20 Bq·g−1) was incubated in a hot air oven at 105°C for 24 h. For the batch of leaching experiments, the following conditions were used: solid-to-liquid (S:L) ratio was 1:2 at 1,000 g 137Cs-EAF; 0.1 M citric acid was added and mixed in appropriate proportions; then the mixture was subjected to stirring conditions of 137Cs-EAFD resuspension via jar tests (WiZard plus6S). The solid-liquid mixture was vortexed under the following constant conditions: leaching time, 120 min; stirring speed, 150 rpm; room temperature, ~25°C. After the treatment, the suspension was then filtrated through a polypropylene mesh filter bag (5 μm). After separating the obtained 137Cs-EAFD, leaching effluent was chemically analyzed for the content of heavy metals using ICP-MS. High Purity Germanium (HPGE) gamma spectrometer was used to determine the specific activity (A) of 137Cs in EAFD leaching effluent, which was calculated using the following equation [6]:
where A is the specific activity in units (Bq·L−1); cps is the gamma radiation readings in counts per second; V is the volume of the model; %Eff is the percentage of efficiency; %Iγ is the intensity of gamma rays.
2.3 Germination Experiment
In the germination experiment, surface sterilized seeds were conducted using 95% ethanol for 30 s and 2% sodium hypochlorite solution for 5 min and thoroughly rinsed with distilled water three times. Additionally, the seeds of Mimosa pigra L. and Neptunia plena L. have a thick seed coat, and hot water seed priming was performed to break the thick seed coat. The ½ MS medium was supplemented with various activity concentrations of 137Cs, pH = 5.7. Subsequently, the media were poured onto the bottom of a 10 cm diameter Petri plate. The experiment involved seven treatment levels, including a control treatment (distilled water) and six different activity concentrations of 137Cs: 5,000, 10,000, 20,000, 30,000, 40,000, and 50,000 Bq·L−1. Then 10 seeds of each legume species were placed on ½ MS medium in each Petri Dish in triplicate. Seeds were considered for initial germination whenever the radicle emerged. The experiment was conducted for 10 days. Legume seeds were allowed to germinate on tissue culture racks at a temperature of 22°C and 65% humidity. The number of germinated seeds was counted daily for 10 days. The following are the main parameters used to describe the germination of the tested plants: germination percentage, mean germination time, mean germination rate, coefficient of variation of germination time, coefficient of velocity of germination, germination index, and stress tolerance index; all parameters were calculated using Khalid’s formula [19]. Furthermore, the fresh weight of each legume species was measured 10 days later.
The percentage of seed germination data at various activity concentration of 137Cs was calculated by using a formula:
where N: Total number of seeds and Ni: germinated seeds at the end of counting days.
The Mean germination time (MGT), which is a measure of the speed of germination was estimated by using a formula:
where N: Total number of seeds Ni: germinated seeds per day and di: counting day.
The Mean germination rate (MGR), which is an measure of seedling emergence and is elucidate as convertible of the mean germination time (1/MGT):
where N: Total number of seeds Ni: germinated seeds per day and di: counting day.
The coefficient of velocity germination (CVG), gives an indication of the rapidity of germination. Its value increases when the number of germinated seeds increases and the time required for germination decreases.
where Ni = the number of germinated seeds and Ti = the last day of germination.
The coefficient of variation of the germination time (CVt), which is evaluate the germination uniformity or variability in relation to the mean germination time:
where St and t stand for the standard deviation of the germination time and mean germination time.
The germination index (GI), which is a measure of the percentage and rate of germination was calculated by using a formula:
The stress tolerance index (SI), was evaluated for plant weight by comparing the mean weight of treated plants to that of control plants under various 137Cs treatments for test species. This comparison was conducted using a formula:
2.4 Statistical Analysis
All the experiments were carried out with triplicates and all the data from legume seed germination experiments were subjected to one-factor analysis of variance (ANOVA) to test the significant effects of the measured variables of treatments, the means were separated using Tukey’s test at p ≤ 0.05. All the statistical analyses were performed using the statistical program SPSS (version 26).
3. Results and Discussion
The present study investigated the effect of 137Cs-EAFD leaching effluent on the germination and early seedling stage of legume species with the aim of establishing its phytoremediation potential. This result revealed that legume seeds of different species responded differently to the 137Cs gradient and that the increased activity concentration of the 137Cs-EAFD leaching effluent treatment generally decreased the seed germination parameters of all studied species.
3.1 Citric Acid Leaching of 137Cs-EAFD
137Cs and metallic elements were separated from 137Cs- EAFD by leaching or lixiviation using 0.1 M citric acid. The findings of the chemical characterization of 137Cs- EAFD leaching obtained using ICP-MS are presented in Table 1. Based on the results obtained by chemical analysis (Table 1), the analyzed 137Cs-EAFD leachate sample contained a large number of metallic elements. Zinc had the most significant amount in the 137Cs-EAFD leaching effluent (14,345 mg·kg−1), followed by magnesium and sodium, whereas other elements, including Al, As, Cd, Co, Cr, Cs, Cu, Fe, K, Mn, Mo, Ni, and Pb, were less abundant in the leachate. In general, a citric acid solution was applied to leach 137Cs from EAFD, which simultaneously acts as a chelating agent to increase plant growth and increase metal uptake by plants [20]. Therefore, assessing 137Cs tolerance during the germination and early seedling phases may provide insight into the tolerance of legume species throughout their life cycle [21,22].
Table 1
Chemical of metallic composition of a 137Cs-EAFD removal by leaching treatment using ICP-MS. The means ± standard deviation are represented averages of leaching treatment triplications. Different letters indicate in the column as significant difference according to Tukey’s test at p ≤ 0.05
| Element | Content, mg·kg−1 | Element | Content, mg·kg−1 |
|---|---|---|---|
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| Aluminum (Al) | 67.01 ± 4.27e | Potassium (K) | 1,776.67 ± 57.71c |
| Arsenic (As) | 0.14 ± 0.02e | Magnesium (Mg) | 2,323.18 ± 117.49b |
| Cadmium (Cd) | 64.05 ± 2.09e | Manganese (Mn) | 737.97 ± 13.00d |
| Cobalt (Co) | 0.06 ± 0.01e | Molybdenum (Mo) | 1.26 ± 0.03e |
| Chromium (Cr) | 61.8 ± 1.50e | Sodium (Na) | 2,390.95 ± 75.82b |
| Cesium (Cs) | 3.42 ± 0.10e | Nickle (Ni) | 0.3 ± 0.02e |
| Cu (Copper) | 146.90 ± 4.52e | Lead (Pb) | 0.5 ± 0.02e |
| Iron (Fe) | 1,382.87 ± 52.06c | Zinc (Zn) | 14,345 ± 742.93a |
3.2 Effect of 137Cs on Seed Germination Parameters
137Cs is one of the most essential artificial radionuclides that has been introduced into the environment by the discharge of nuclear waste and accidental release and longterm adverse impacts on living organisms. The effects of different levels of 137Cs activity concentration stress on seed germination and stress tolerance in five legume species are shown in Fig. 1. As the activity concentration of 137Cs increased, a gradually decreasing trend of final germination was detected for all species. Almost all the species did not germinate at higher 137Cs activity (40,000 and 50,000 Bq·L−1). Approximately 50% of the seeds of Crotalaria juncea L. germinated at a maximum of 50,000 Bq·L−1137Cs. However, germination decreased significantly to 0% at the higher activity concentration. Analysis testing of this study revealed that Neptunia plena L. was the most susceptible legume species.
Fig. 1
Cumulative percentage of seed germination of legumes species as dependent on 137Cs radioactivity. Values shown are means ± SD. Values in a figure with lower-case letters are significantly different for the same treatment of 137Cs activity concentration (Bq∙L−1) according to Tukey’s test at p ≤ 0.05. The absence of letters in a graph means no significance among treatments. Five legume species: CJ (Crotalaria juncea L.), MP (Mimosa pigra L.), NP (Neptunia plena L.), SJ (Sesbania javanica Miq.), and VM (Vigna mungo L.).
Table 2 displays that the other attribute parameters related to seed germination measurements, including the mean germination time (MGT), mean germination rate (MGR), coefficient of variation of germination time (CVt), coefficient of the velocity of germination (CVG), and germination index (GI), differed significantly between the control and 137Cs-treated seeds. Accordingly, the stress of 137Cs at high activity concentration was increased on MGT and CVt values. In contrast, MGR, CVR, and GI were dramatically lower in the 137Cs-treated group than those in the control. In this study, increasing the 137Cs activity concentrations significantly delayed the germination process and decreased the germination parameters. The most tolerant species is Crotalaria juncea L., which can germinate at activity concentrations up to 50,000 Bq·L−1137Cs. These results are consistent with those in previously published reports that 137Cs has a negative effect on seed germination in various species [23,24].
Table 2
Cumulative parameters of seed germination of legume species as dependent on different activity concentration 137Cs; Mean germination time (MGT), Mean germination rate (MGR), Coefficient of variation of germination time (CVt), Coefficient of velocity of germination (CVG), Germination index (GI)
| 137Cs activity (Bq·L−1) | MGT (Day) | MGR (Day−1) | CVt (%) | CVG (%) | GI (Day) |
|---|---|---|---|---|---|
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| Crotalaria juncea L. | |||||
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| Control | 1.00 ± 0.0a | 1 ± 0.0d | 0 ± 0.00a | 100 ± 0.0d | 10 ± 0.0c |
| 5,000 | 1.03 ± 0.06a | 0.97 ± 0.05d | 9.583 ± 16.59b | 96.7 ± 5.24cd | 9.83 ± 0.28c |
| 10,000 | 1.03 ± 0.06a | 0.97 ± 0.05d | 9.583 ± 16.59b | 96.7 ± 5.24cd | 9.83 ± 0.28c |
| 20,000 | 1.03 ± 0.06a | 0.97 ± 0.05d | 9.583 ± 16.59b | 96.7 ± 5.24cd | 9.83 ± 0.28c |
| 30,000 | 1.77 ± 0.25a | 0.574 ± 0.08b | 41.432 ± 7.20d | 57.40 ± 8.48b | 6.72 ± 1.08b |
| 40,000 | 1.17 ± 0.06a | 0.850 ± 0.04c | 33.007 ± 3.68c | 85.85 ± 4.37c | 9.16 ± 0.28c |
| 50,000 | 3.58 ± 0.07b | 0.280 ± 0.005a | 49.775 ± 5.29d | 27.97 ± 0.52a | 1.43 ± 0.83a |
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| Mimosa pigra L. | |||||
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| Control | 1.00 ± 0.0a | 1.00 ± 0.0a | 0.0a | 100 ± 0.0b | 10.0 ± 0.0b |
| 5,000 | 1.00 ± 0.0a | 1.00 ± 0.0a | 0.0a | 100 ± 0.0b | 10.0 ± 0.0b |
| 10,000 | 1.00 ± 0.0a | 1.00 ± 0.0a | 0.0a | 100 ± 0.0b | 10.0 ± 0.0b |
| 20,000 | 2.57 ± 0.25b | 0.39 ± 0.04b | 44.14 ± 6.81b | 39.21 ± 3.93a | 5.19 ± 0.71a |
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| Neptunia plena L. | |||||
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| Control | 1.67 ± 0.31a | 0.61 ± 0.11b | 45.77 ± 9.61a | 61.42 ± 11.36a | 6.06 ± 0.51c |
| 5,000 | 1.39 ± 0.05b | 0.72 ± 0.03a | 54.09 ± 10.33b | 72.14 ± 2.57c | 4.22 ± 1.67b |
| 10,000 | 1.00 ± 0.87a | 0.44 ± 0.38c | 47.14 ± 0.0b | 66.67 ± 0.0b | 1.00 ± 0.87a |
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| Sesbania javanica Miq. | |||||
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| Control | 1.24 ± 0.13a | 0.811 ± 0.008d | 35.04 ± 3.23b | 81.14 ± 8.10d | 7.33 ± 0.76e |
| 5,000 | 1.52 ± 0.13a | 0.66 ± 0.05b | 43.37 ± 4.98c | 66.18 ± 5.83c | 6.38 ± 0.51d |
| 10,000 | 2.14 ± 0.17b | 0.47 ± 0.03c | 32.15 ± 10.18b | 46.82 ± 3.63b | 5.08 ± 0.94c |
| 20,000 | 4.72 ± 0.25c | 0.25 ± 0.12a | 50.68 ± 36.35d | 25.27 ± 12.08a | 1.21 ± 0.82b |
| 30,000 | 4.33 ± 2.82c | 0.6 ± 0.056b | 0a | 40 ± 12.91b | 0.40 ± 0.52a |
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| Vigna mungo L. | |||||
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| Control | 1.0 ± 0.0a | 1.0 ± 0.0d | 0.0 ± 0.0a | 100 ± 0.0d | 10.0 ± 0.0e |
| 5,000 | 1.17 ± 0.15b | 0.87 ± 0.12c | 52.31 ± 0.55d | 86.75 ± 11.91c | 9.39 ± 0.59d |
| 10,000 | 1.81 ± 0.17d | 0.56 ± 0.05b | 48.04 ± 11.60c | 55.71 ± 5.15b | 2.94 ± 1.0b |
| 20,000 | 1.0 ± 0.0a | 1.0 ± 0.0d | 0.0 ± 0.0a | 100 ± 0.0d | 6.0 ± 1.0c |
| 30,000 | 1.67 ± 1.45c | 0.27 ± 0.23a | 23.19 ± 2.18b | 40.17 ± 3.79a | 0.83 ± 0.73a |
*Values shown are means ± SD. Values in a table with lower-case letters are significantly different for the same legume species according to Tukey’s test at p ≤ 0.05.
3.3 Effect of 137Cs on Seedling Growth
In addition to germination, the most crucial recognizable consequence of 137Cs toxicity in plants is root length suppression and shoot height inhibition [25]. Moreover, 10 days after the initiation of the investigation, a more pronounced effect of 137Cs stress on root length and shoot height was observed because the inhibition of these parameters was more significant in the 137Cs treatment than in the control test (Table 3). Remarkably, further increasing the activity concentration of 137Cs significantly reduced shoot height. Root length followed the same pattern as shoot height against 137Cs stress (Supplementary Fig. 1).
Table 3
Effects of different activity concentration 137Cs on in vitro root length and shoot height of legume species
| 137Cs activity (Bq·L−1) | Root length (cm) | ||||
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| CJ | MP | NP | SJ | VM | |
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| Control | 8.24 ± 1.93e | 2.215 ± 0.24c | 7.33 ± 1.87c | 4.19 ± 0.99d | 2.23 ± 0.73c |
| 5,000 | 7.44 ± 1.39d | 1.45 ± 0.12b | 2.07 ± 0.74b | 4.14 ± 1.43d | 1.73 ± 0.37b |
| 10,000 | 5.77 ± 2.94c | 1.59 ± 0.33b | 1.68 ± 0.29a | 3.51 ± 1.38c | 1.31 ± 0.22ab |
| 20,000 | 5.11 ± 2.84c | 1.09 ± 0.38a | - | 1.69 ± 1.44b | 1.80 ± 0.41b |
| 30,000 | 4.34 ± 2.19b | - | - | 1.05 ± 0.07a | 0.68 ± 0.21a |
| 40,000 | 4.56 ± 1.98b | - | - | - | - |
| 50,000 | 0.81 ± 0.98a | - | - | - | - |
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| 137Cs activity (Bq·L−1) | Shoot height (cm) | ||||
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| CJ | MP | NP | SJ | VM | |
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| Control | 5.83 ± 0.63d | 2.25 ± 0.53b | 4.62 ± 0.89c | 5.63 ± 1.01c | 6.30 ± 1.01c |
| 5,000 | 6.88 ± 1.07e | 2.57 ± 0.28b | 1.75 ± 0.49b | 5.15 ± 0.31c | 3.29 ± 0.72ab |
| 10,000 | 5.78 ± 1.71d | 1.95 ± 0.29ab | 1.22 ± 0.09a | 4.59 ± 1.37b | 3.60 ± 0.50b |
| 20,000 | 4.80 ± 1.67c | 1.61 ± 0.19a | - | 1.55 ± 0.86a | 3.90 ± 0.26b |
| 30,000 | 3.25 ± 1.26b | - | - | 1.80 ± 0.28a | 0.56 ± 0.65a |
| 40,000 | 3.97 ± 0.63b | - | - | - | - |
| 50,000 | 2.05 ± 0.94a | - | - | - | - |
* Values shown are means ± SD. Values in a table with lower-case letters are significantly different for the same legume species according to Tukey’s test at p ≤ 0.05; -: no growth.
3.4 Effect of 137Cs on the Stress Tolerance Index (SI)
To better screen legumes for 137Cs tolerant species during the germination process, Fig. 2 demonstrates a decreasing trend in the SI for all species with increasing 137Cs activity. In contrast, seeds exposed to 137Cs applied at 5,000 and 10,000 Bq·L−1 had greater SI than the control seeds for Mimosa pigra L. and Sesbania javanica Miq., respectively. Additionally, at activity concentrations of 20,000 Bq·L−1137Cs and higher, the latter exhibited a reduction of more than 50% in the SI compared to the control. Exposure to high activity concentrations of 137Cs not only reduced the total germination percentage but also decreased the SI (Fig. 2). Crotalaria juncea L. is widely used as a phytoremediator that can be established for remediation technology and can serve in ecological restoration for 137Cs contamination [26]. Among the five legume species, Crotalaria juncea L. was the most tolerant at germination, with the highest germination percentage and germination ability. Our study revealed that Crotalaria juncea L. tended to grow better under 137Cs treatment. Our findings were confirmed by Uchida and Tagami [27], who reported that leguminous species would have great 137Cs phytoaccumulation ability. Indeed, little information related to the specific difference in 137Cs tolerance among legume species is available. However, several studies have demonstrated that legumes are potentially able to perform phytoremediation in term 137Cs accumulation in leguminous crops. Findings reported by Kubo et al. [28] indicated that the transfer of 137Cs from contaminated site to grains is higher in soybean (Glycine max L.) than in rice (Oryza sativa L.). Moreover, findings reported by Uchida and tagami [27] who noted that leguminous species would have higher 137Cs uptake ability than Poaceae family plants. These evidences emphasize the phytoaccumulation of 137Cs by legume plants that can be used to enhance 137Cs remediation by legumes phytoremediation.
Fig. 2
Cumulative percentage of 137Cs stress tolerance index (SI) of legumes species as dependent on 137Cs radioactivity. Values shown are means ± SD. Values in a figure with lower-case letters are significantly different for the same treatment of 137Cs activity concentration (Bq·L−1) according to Tukey’s test at p ≤ 0.05. The absence of letters in a graph means no significance among treatments. Five legume species: CJ (Crotalaria juncea L.), MP (Mimosa pigra L.), NP (Neptunia plena L.), SJ (Sesbania javanica Miq.), and VM (Vigna mungo L.).
4. Conclusions
It is necessary to establish an effective screening method that allows accurate identification of 137Cs tolerant species useful as phytoremediators. Legume seeds were germinated under the tested activity concentrations of 137Cs (between 5,000 and 50,000 Bq·L−1). The outcomes of this study provide precedent information for future research on the phytoremediation of 137Cs. Legume seeds had different responses to 137Cs stress at the seed germination stage. Except for Crotalaria juncea L., all five species failed to germinate at an activity concentration of more than 30,000 Bq·L−1137Cs. Furthermore, experiments on seedling growth in the early days of development revealed a certain tolerance of legume seedlings growing in MS medium supplemented with 137Cs. Adverse effects caused by the studied 137Cs led to visible signs of inhibiting root length, shoot height, and SI. Thus, the legumes assessed in this study could constitute a promising approach for restoring contaminated 137Cs. The most significant aspect of phytoremediation of 137Cs- EAFD leaching effluent is the selection of the right plant. Diverse factors, such as the concentration of a 137Cs in the radioactive waste, composition heavy metals in the 137Cs- EAFD leaching effluent, the biomass of the plant, the plant species and the response effect of 137Cs in the plant, should be thoroughly investigated. Moreover, gaining insight into the biological processes underlying phytoremediation can lead to specific improvements. Further studies are needed to study the 137Cs uptake mechanisms and capacity by selected legume species. Moreover, specific contribution toward improving key of the plants-microbes interaction under stress conditions is much needed.
