Toxicity and Translocation of Selenium in Phaseolus vulgaris L

Selenium (Se) is not considered an essential nutrient for plants, although trace amounts of this element can enhance the growth and yield of some plant species. The application of sodium selenate in staple foods has been proposed as an alternative to minimize Se deficiency in the human diet. However, the threshold between deficiency and toxicity for Se is very narrow. Different plant species vary considerably in the absorption and accumulation of Se in shoots and other edible parts, and also in the tolerance to high Se concentrations in the soil. Therefore, this study aimed to evaluate the Se toxicity in common bean plants grown under high doses of sodium selenate, and the Se translocation of contaminated bean seeds to next generation grains. The study was carried out on a field experiment with the application of four rates of sodium selenate (0, 50, 500 and 5000 g/ha) to the soil were common bean crop was grown. Following, greenhouse conditions were used to investigate the translocation of Se from enriched seeds to the grains. The common bean showed tolerance to sodium selenate rates up to 500 g/ha, with reduction of yield observed at rate of 5000 g/ha. Even with no symptoms of toxicity the application rates of 500 g/ha of sodium selenate to the soil produced grains with concentrations of Se that surpass the limit established by Brazilian food law. The seeds enriched with Se can translocate this nutrient to the next generation.


Introduction
The concentration of selenium (Se) is highly variable in the soils of the globe, ranging from 0.005 to 8000 mg/kg (Mora et al., 2015). Although not considered an essential nutrient for plants, trace amounts of Se can enhance the growth and yield of some plant species due to the antioxidant effect that protects plants from a variety of oxidative stresses caused by oxygen free radicals (Cartes et al., 2010;Pandey & Gupta, 2015;Kumar et al., 2012).
In soils with low levels of Se the application of sodium selenate in staple foods has been proposed as an alternative to minimize selenium deficiency in the human diet (Nestel et al., 2006;Mayer et al., 2008;Zhao & McGrath, 2009). However, the threshold between deficiency and toxicity is very narrow for Se and high levels of this element may cause toxicity symptoms such as reduced growth, leaf chlorosis, decreased protein synthesis and premature plant death. Anthropogenic activities like the disposal of coal generated fly ash, agricultural drainage water and use of fertilizers for crop production have led to Se toxicity (Lemly, 2017).
The mechanism of absorption, translocation, and distribution of Se by plants are related to Se concentration, to its chemical form found in soil, as well as the soil chemical and physical properties, besides the genetic variation of plant species (Lakin, 1972;Haudin, 2007;Renkema et al., 2012;Souza et al., 2013). Sodium selenate (Na 2 SeO 4 ) is more easily transported from soil to plants compared to sodium selenite (Na 2 SeO 3 ). For this reason, sodium selenate is mainly found in shoots while selenite is retained in roots (Ríos et al., 2008;Malagoli et al., 2015).
Different plant species vary considerably in the absorption and accumulation of Se in shoots and other edible parts, and also in the tolerance to high Se concentrations in the soil (Marschner, 1995). In addition, Se translocation from the contaminated seeds to the next generation is yet a knowledge gap.
Therefore, this study aimed to evaluate the Se fitotoxicity in common bean plants grown under high doses of sodium selenate, and the Se translocation of contaminated bean seeds to next generation grains.

Field Experiment
The experiment was carried out at the experimental station of the Agronomic Institute of Campinas, State of São Paulo, Brazil (22º89′S; 47º06′W; 674 m a.s.l.). The climate is as a humid tropical with a clearly defined rainy summer and dry winter, with an average rainfall of 1060 mm, as a clayey Distrophic Haplustox, according to the FAO Soil Classification System (FAO, 1988). The soil characteristics are shown in Table 1 and they were determined according to Raij et al. (2001). Note. a CEC: Cation Exhange Capacity.
The experiment consisted of a randomized complete block design with four Se rates and five replications, totalizing 20 experimental plots. Se was applied in the furrow as a sodium selenate solution (Na 2 SeO 42--Sigma-Aldrich, Dorset, UK) at rates of 0, 50, 500 and 5000 g/ha. Common bean (Phaseolus vulgaris L. cv. IAC carioca) was sown in 2 m × 7 m plots with four rows spaced 0.5 m apart at a seeding density of 70 seeds/row. Chemical fertilizers applied consisted of N (10 kg/ha as ammonium sulfate), P 2 O 5 (4 kg/ha as triple superphosphate) and K 2 O (25 kg/ha as KCl) at sowing and N by side dressing (40 kg/ha). Plants grown without Se application were used as a control. Shoots were harvested at the physiological grain maturity and separated into stems, leaves and seeds.

Selenium Translocation Study
For Se translocation study, enriched seeds harvested at treatment with application of 5000 g/ha of sodium selenite were used. Plants grown without Se application were used as a control. Each treatment was conducted with five replications, totalizing 10 experimental units. After the physiological maturity of grain, seeds were analyzed in the laboratory and part of them was used in the greenhouse experiment. Ten seeds of enriched and non-enriched seeds were grown in 10 kg of soil pots. After 15 days of shoots emergence, plants were thinned to five seedlings per pot. At flowering, leaves were collected for nutritional diagnosis and after physiological maturity of the grains, the seeds and stems were collected.

Analytical Determinations
Leaves, stems and seeds were washed with tap water, rinsed with deionized water and oven-dried at 60 °C to constant weight. Dry matter weights of shoots and seeds were recorded and stored for Se analysis. The extraction procedure for Se content determination in the plant material and in the soil was in accordance with the EPA 3051a method (USEPA, 1995). The extracts were subjected to volume reduction on a hotplate at 100 °C to a volume of 5 ml. After cooling, each sample received 5 ml of concentrated HCl, followed by filtering with Whatman paper in filter 42, with the volume made up to 50 ml. Then, aliquots of the solution were taken for Se determination by ICP-OES with HGAAS as described by Welsch et al. (1990). Spinach leaves SRM 1570a (Se content 0.117±0.009 mg/kg) was obtained from NIST-National Institute of Standards and Technology and included in each analytical run as a quality assurance of the results.
Selenium absorption efficiency (%) was calculated for each treatment using the Equation (1): where, Se T Se in the c

Data A
The data w Brazil). Th

Seleniu
There wer the field. common b causing a ( Figure 2).    (Slekovec & Goessler, 2015). However, the rates of 500 and 5000 g/ha increased Se concentration in grain to 0.69 mg/kg and 1.94 mg/kg, respectively, which surpass the limit established by Brazilian law (Figure 3).
Se absorption was less than 0.3%, suggesting that Se remaining in the soil can affect to some extent the following culture (Table 2). These results are similar to those observed by Fernandes et al. (2014), who noted low Se absorption efficiency for rice and radish crops, where more than 98% of Se applied as sodium selenite has remained in the soil. Se mobility in soils increases with soil pH increase due to the predominance of selenate instead of selenite and decreases with high organic matter and the clay fraction content due to the retention of Se (Gissel-Nielsen 2002, Cartes et al., 2005Hlušek et al., 2005;Eich-Greatorex et al., 2007).

Translocation of Selenium from Enriched Seeds
Enriched seeds replanted in the greenhouse had a significant increase of Se content in the stem, leaves and grains compared to the non-enriched seeds (Table 3). However, we found the highest concentrations of Se in stem and leaves. Se content in the grains was below the limit established by the Ministry of Health of Brazil (2006) (0.3 mg/kg) and did not offer a risk for human consumption.
These results open the possibility of using areas that, for anthropological or natural reasons, present high soil concentrations of Se to produce seeds enriched with this nutrient, under controlled conditions, to address nutritional population deficiencies elsewhere, in regions with natural low levels of this nutrient in the soil, promoting concurrently some remediation of contaminated areas.
According to Reilly (1996) toxic levels of Se in plant tissues are generally above 5 mg/kg. Thus, despite the increase of Se in plant tissues of enriched seeds, the concentration was insufficient to cause toxicity. This can be observed by the no significant difference in the crop yields between the two plant groups (Table 3). The low Se concentrations found in this study show that even when the seeds were harvested in a contaminated area, only a small part was transported to the next generation.  Note. a CV: Coefficient of Variation.

Conclusion
The application of 50 g/ha of sodium selenate increase the common bean grain yield and produced grains with Se concentrations below the maximum levels allowed for consumption. Common bean showed tolerance to sodium selenate at the rate of 500 g/ha but the Se concentration in grains surpassed the limit of Brazilian food law limits. There was a reduction of grains yield, germination and initial plant growth at the rate of 5000 g/ha of sodium selenate in the soil. The seeds enriched with Se can translocate this nutrient to the next generation.