4.2. Enrichment of Se in Plants
In this present study, the Se concentration in untreated control pea grain was 118 μg kg
−1 (DW), which was higher than in those grown under field conditions in Slovenia (11 μg Se kg
−1 DW) [
52] and in Spain (57 μg Se kg
−1 DW) [
17], and in greenhouses in Australia (38 μg Se kg
−1 DW) [
53]. In a comprehensive survey of 293 green pea samples in Canada, Gawalko et al. [
54] found that the average Se content was 331 μg kg
−1 (DW), while 56% of the samples tested had a Se content higher than 300 μg kg
−1 due to the naturally Se-rich soils. In this work, in carrot root, 78 μg kg
−1 (DW) Se was measured in the control soil. De Temmerman et al. [
49] examined 121 carrot samples grown in Belgium and recorded an average Se content of 43.4 μg kg
−1 (DW).
Some plant species, such as certain members of the Brassicaceae family, are capable of hyperaccumulation, i.e., they may have a very high Se content without any signs of toxicity [
12]. However, carrots and green peas may exhibit severe toxicity as a result of high soil Se doses and, in extreme cases, when the Se content in carrots and green peas exceeds 63 and 176 mg kg
−1 (DW), respectively, the crops may be completely eradicated [
55,
56,
57]. This is because Se shows structural similarity to sulphur, making it easy for plants to take up. Subsequently, sulphur is replaced by Se in certain proteins, which consequently lose their function. Toxicity may also be caused by oxidative stress due to Se [
58]. Several studies report that Se promotes plant development in low concentrations but inhibits it in high concentrations. Hegedűsová et al. [
59] found that higher Se treatments reduced germination and root and shoot formation in seedlings, while low-dose Se increased root and shoot length by about 25%. Landberg and Greger [
60] and Łukaszewicz et al. [
61,
62] reported a decrease in the root and shoot biomass in young pea plants grown in nutrient solution as a result of selenate treatments. Regarding the yield of mature green pea grain in the present experiment, no clear decreasing or increasing trend was observed in response to the Se doses, except for the increased fresh grain weight in silty sand (
Table 4). Thus, it should be noted that no yield depression was observed even when a relatively high average concentration of 30 mg Se kg
−1 developed in the pea grains in the Se-2 treatment (
Table 5). All the other experiments described in the literature used foliar spray to enrich ripe green peas with Se. Nevertheless, these results are partly consistent with the results of the present experiment. Poblaciones and Rengel [
63] found that foliar Se treatment increased the Se content of field-grown green pea grain to 1.415 mg Se kg
−1 at the highest dose of Na
2SeO
4 but had no effect on shoot biomass or grain yield, while the root weight and 100-grain weight increased. Hegedűsová et al. [
64] applied 50 g and 100 g Se ha
−1 sodium selenate during pea flowering by leaf spraying in a field experiment, which increased the Se content of the pea grain from 90–100 μg kg
−1 to 1.16–1.30 mg kg
−1 and 2.22–2.29 mg kg
−1, respectively, which is roughly half the Se content achieved in the Se-1 (100 µg L
−1) treatment used in the present experiment (
Table 5). Nearly half of the Se content obtained in the Se-2 (500 µg L
−1) treatment was achieved by Poblaciones et al. [
17], who increased the Se content of field-grown green pea grain to a maximum of 12.2 mg Se kg
−1 and found no effect on the grain yield.
The carrot root FW showed a mostly decreasing trend, whereas Se-2 treatment had a significant negative effect on root DW compared to the control when averaged over the three soils (
Table 4). This is partly in agreement with Smoleń et al. [
13], who applied 0.5 kg Se ha
−1 to the soil in the form of Na
2SeO
4 in a field experiment, and observed no changes in carrot root biomass yield, except for a minimal decrease when the Se content reached 25 mg kg
−1 (DW) in the root, which is approximately comparable to the effect of the Se-2 treatment used in the present experiment (
Table 5). Oliveira et al. [
14] also applied sodium selenate to the soil, which had a very slight non-significant negative effect on the root weight while increasing the Se content of carrot roots to nearly 10 mg kg
−1. Bañuelos et al. [
65,
66] investigated the effect of mixing Se-rich
Stanleya pinnata plant residues with the soil. The Se content of carrots reached a maximum concentration of 6.28 mg kg
−1 as a result of the treatments without any decrease in biomass or any stress symptoms.
The results of this experiment confirmed that only a moderate non-significant decrease in yield if any can be expected for green peas and carrots with the high or higher Se contents achieved with the biofortification methods used so far. It should be noted that in a previous experiment with similar treatments, the fresh yield of green beans, tomato fruit, potato tubers and cabbage heads decreased slightly as a result of the Se treatment, but this effect was not significant. The Se content (DW) of green pea grain was quite similar to that of the green beans and tomato fruit, the lower Se content of carrot root was similar to or slightly higher than that of potato tuber, while the Se content of cabbage was much higher than that of the other vegetables studied [
33].
The effect of the soil type on the Se enrichment of plants was controversial in this experiment. In the case of green peas, the Se content in the treated plants tended to be much higher for sand soil. This may be explained by the fact that sand is looser with lower CEC, so it binds less Se, and a higher proportion remains easily available for green peas. The same was observed in a previous similar experiment with green beans, cabbage and potato plants [
33]. At the same time, in the case of carrots, the opposite trend was seen, though to a lesser extent, i.e., the highest Se contents in carrot roots were measured in silt soil. A possible explanation for this phenomenon is that pea roots are much denser, with more branching and a larger surface area than the taproot of carrot, enabling them to absorb the Se content of irrigation water much faster, while carrots might be more able to take up the Se content bound in the soil over a longer period of time. A similar conclusion was drawn by Bañuelos et al. [
66], who compared the uptake of Se from the soil with broccoli with that of carrots. De Temmerman et al. [
49] also found that on soils with similar Se concentrations, quite different Se concentrations may develop in different vegetable crops because the mode of uptake and accumulation is plant-specific.
4.3. Contribution of Se-Enriched Products to Human Se Intake
The daily RDA of Se may vary by country or region, but usually ranges from 25 to 60 μg day
−1 for adult women and from 30 to 75 μg day
−1 for adult men. A value of 55 μg day
−1 is mostly recommended in the EU, USA and Canada. However, the daily UL of Se lies between 350 and 450 μg day
−1 in different parts of the world, so the gap between deficient and toxic levels is relatively small [
48]. In Eastern European countries, the estimated daily intake is usually between 30 and 40 μg Se day
−1, which is below the RDA [
67].
Smoleń et al. [
68] applied 0.25 kg Se ha
−1 to the soil, which increased the Se content of carrot root from 2.21 to 10.97 mg kg
−1 (DW). The consumption of 100 g of untreated carrot root would cover only 46% of the RDA while eating treated carrots would exceed RDA by 2.4 times. Poblaciones and Rengel [
53] treated green peas with 0.03% or 0.06% (
w/
v) of SeNaO
4 leaf spray leading to Se contents of 67 and 95 μg Se, respectively, in 100 g of fresh grain weight, while Poblaciones et al. [
17] treated green peas with 10 g Se ha
−1 in the form of foliar application, resulting in 179 μg Se in 100 g of fresh grain weight, thus containing more than three times the RDA.
In the present experiment, the Se content of 100 g of fresh green peas grown in the Se-1 treatment exceeded the RDA value of 55 μg Se day
−1 four times, but the average content of Se in carrots was 50.7 μg Se, so it was close to the RDA value (
Table 6 and
Table 7). Irrigation water containing 100 μg Se L
−1 can thus be considered a suitable method for the production of functional food from carrots in order to compensate for the low Se intake, but in the case of green peas, this concentration is too high. In a previous experiment, irrigation water containing 100 μg Se L
−1 resulted in a Se content close to the RDA value in green beans and potatoes, while in tomatoes the content was only two-thirds of RDA, and in cabbage, it was four times higher. Thus, the degree of Se enrichment in green peas is similar to that of cabbage, which absorbs Se highly efficiently compared to the other tested vegetables. Irrigation water containing 100 μg Se L
−1, therefore, resulted in excessive Se levels, equal to half of the UL for both species. The effect of the Se-2 treatment was also similar for cabbage and green peas in terms of the UL (
Table 8), because the Se contents were close to three-fold in both plants, while the enrichment of carrot was less than that of green bean and greater than that of potato, and about twice as much as that of tomato fruit [
33]. The use of 100 μg Se L
−1 concentration has already caused excessive Se enrichment in plants, so it is not recommended for biofortification.
The element content of raw vegetable products may change during the preparation of ready-to-eat food. For example, heat treatment reduces the Se content [
69]. The bioavailable organic Se fractions may decrease due to protein denaturalization as a result of boiling, baking, microwaving or frying [
70]. Cooking reduced the Se content of biofortified green peas by 7.4% [
53] and in another study, by 12% [
63].
4.4. Changes in Element Composition
Biofortification is a method to produce plant products rich in certain elements that are otherwise deficient, but the concentration of other nutrients important for human consumption should not be adversely affected. The antagonistic effect of Se on elements such as sulphur, mercury and molybdenum has been shown in previous studies [
71,
72,
73]. However, for most elements, the results have been conflicting, often depending on whether Se was applied to soil or hydroponic growing medium (nutrient solution culture) [
74] or as a leaf spray [
75]. For example, the results of an in vitro experiment proved the synergistic effect of Se on sulphur in the case of wheat and rape seedlings [
76]. These results underline the importance of investigating the effect of Se in irrigation water, as this application method differs from the methods commonly used. In a previous experiment, the concentrations of other elements (P, K, Fe, Mg, Zn, Cu) varied depending on the plant species or soil type or showed no substantial change (As, B, I, Mn), but no clear, consistent positive or negative effect of Se on the elements was observed [
33].
In the present experiment, the increase in P, K and Fe and the decrease in Zn in green peas partly confirmed and partly contradicted previous results (
Figure 2). Łukaszewicz et al. [
62] found that Se applied in the form of selenate in hydroponic cultivation decreased P and Mg while increasing K content in green pea shoots when the first pair of leaves appeared. Reynolds-Marzal et al. [
77] treated forage pea (
Pisum sativum L.) with a dose of 10 g Se ha
−1 in the form of Na
2SeO
4 in a field experiment, which increased the accumulation of Mg from 2.16 to 2.35 g kg
−1 and that of Ca from 8.74 to 9.55 g kg
−1 in relation to the control, but had no effect on Fe. According to Poblaciones and Rengel [
53], Se treatment caused a non-significant increase in the Zn concentration of peas from 33 to 38 mg kg
−1, while it had no great effect on the Ca, Fe or Mg contents. However, in another similar study, it was found that the Mg concentration of green peas decreased from 1260 to 946 mg kg
−1 in proportion to the increasing Se dose, while the Zn concentration increased from 43 to 47 mg kg
−1, and both changes were significant [
63].
The concentration-dependent effect found for Se on most elements in carrots agrees with the results of Filek et al. [
76], who recorded an increase in K, Mg, Mn, Zn and Fe compared to the control in a low Se treatment, but a decrease or a smaller increase due to a higher Se dose in rape and wheat seedlings. Oliveira et al. [
14] applied 1.0 mg Se dm
−3 to the soil in the form of Na
2SeO
4, which significantly reduced the K content of carrot shoots by 26% and non-significantly increased the Fe content by approx. 10%. Interestingly, both the Fe and Mn contents in the root were significantly reduced by the treatment.