Biofortification of green bean (Phaseolus vulgaris L.) and lettuce (Lactuca sativa L.) with iodine in a plant-calcareous sandy soil system irrigated with water containing KI

Journal of Food Composition and Analysis 88 (2020) 103434

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Original Research Article

Biofortification of green bean (Phaseolus vulgaris L.) and lettuce (Lactuca sativa L.) with iodine in a plant-calcareous sandy soil system irrigated with water containing KI

T

Péter Dobosya, Krisztina Kröpfla, Mihály Óvária, Sirat Sandilb, Kitti Németha, Attila Englonera, Tünde Takácsc, Gyula Záraya,b,* a b c

MTA Centre for Ecological Research, Danube Research Institute, Karolina út 29-31, H-1113 Budapest, Hungary Cooperative Research Centre of Environmental Sciences, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, Hungary MTA Centre for Agricultural Research, Institute for Soil Sciences and Agricultural Chemistry, Herman Ottó út 15, H-1022 Budapest, Hungary

A R T I C LE I N FO

A B S T R A C T

Keywords: green bean lettuce biofortification iodine food analyses food composition

Uptake and translocation of iodine by green bean (Phaseolus vulgaris L.) and lettuce (Lactuca sativa L.) were investigated in a calcareous sandy soil-plant system. These plants were cultivated in calcareous sandy soil in presence of irrigation water with the iodine concentrations of 0.10, 0.25 and 0.50 mg/L. The growth of these plants was stimulated at the two lower iodine concentrations of 0.10 and 0.25 mg/L, but hampered at 0.50 mg/L. In lettuce the highest iodine dosage resulted in smaller leaf surface and higher number of leaves, nearly with the same biomass production. In the edible parts of green bean and lettuce plants irrigated with 0.25 mg/L iodine containing water, the iodine concentration levels amounted to 0.6 and 5.2 mg/kg dry weight (DW), respectively. In lettuce, the uptake and translocation of selected essential elements (P, Mg, Mn, Fe, Cu, Zn, B) were also stimulated (20-260%) by iodine treatment; however, in fresh bean pods this phenomenon was negligible. Considering the iodine level and moisture content of the fresh lettuce leaves and fresh bean pods, the consumption of 100 g fresh vegetable covers about 66%, 3% of the recommended dietary allowance (150 μg).

1. Introduction Iodine is an essential component of the thyroid hormones, which regulate many metabolic processes and play a dominant role in the early growth and development of most organs, especially the brain (Fuge and Johnson, 2015). Consequently, if iodine deficiency is severe enough to affect thyroid hormone synthesis it can result in hypothyroidism and brain damage (Andersson et al. 2007; Denage et al., 2002). The WHO recommended daily iodine intake for the age groups: 0-5 years, 6-12 years, and 13 and above years, is 90 μg, 120 μg and 150 μg, respectively, while in case of pregnant and lactating women it is 250 μg (WHO 2004). In some countries iodine intake is insufficient, while in other countries like in the USA and Canada the intake generally exceeds the recommended value (Zimmermann & Boelaert 2015). Iodine is not an essential element for terrestrial plants, and its concentration in typical land plants and food crops amounts to only 0.07-10 mg/kg (Robertson et al. 2003). The main intervention strategy for iodine deficiency monitoring and prevention is “universal” salt iodization (Andersson et al. 2007). It implies that all salt products intended for

⁎

human consumption, including that used in processed foods, and for animal feeding, are iodized. Iodine can be added to table salt as potassium iodide, potassium iodate or sodium iodide. Potassium salts are the most frequently used compounds, and iodate is more preferred due to its higher stability and lower solubility than that of iodide. The iodized table salt as a simple prophylaxis tool has been successfully introduced in several countries in spite of possible iodine losses during transportation, storage or cooking itself (Kapusta-Duch et al. 2017; Rana & Raghuvanshi 2013) Another possibility to eliminate the iodine deficiency is the consumption of iodized oils, like the most commonly used Lipiodol, a poppy seed oil containing 40% iodine per weight (Azizi 2007; Wolff 2001). In addition to these relatively efficient and extensively applied methods, many different experiments have been carried out in various countries e.g., production of bread fortified with iodine, application of iodized drinking water or iodized sugar (Andersson et al. 2007). However, the efficiency and applicability of these experiments are not comparable with that of the iodized table salts. Indirect iodization of food materials has been receiving widespread attention due to adoption of new policies by many countries to

Corresponding author. E-mail address: [email protected] (G. Záray).

https://doi.org/10.1016/j.jfca.2020.103434 Received 7 May 2019; Received in revised form 23 January 2020; Accepted 26 January 2020 Available online 01 February 2020 0889-1575/ © 2020 Elsevier Inc. All rights reserved.

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reduce the salt consumption to 5 g/day, so as to tackle hypertension and cardiovascular diseases. One way of achieving adequate iodization is through iodine fortification of animal fodder and food derived from animal sources. The alternative way is through iodine fortification of edible plants by application of irrigation water containing iodine. On basis of literature studies the agronomic biofortification of crops with iodine appears to be a promising way to increase the iodine intake of the population (Azizi 2007). Approximately 80% of the iodine in the human body and animals originates from plants, and nearly 99% of this iodine is bioavailable and can be easily assimilated (Gonzali et al. 2017). Therefore, plant-based foods with increased iodine content offer an attractive and cost-effective approach to reduce iodine deficiency. Recently, hydroponic (Kato et al. 2013; Landini et al. 2011; Li et al. 2016; Voogt et al. 2010; Weng et al., 2008a, 2008b, 2008c; Zhu et al. 2003; Zhu et al. 2004) pot (Blasco et al. 2008; Blasco et al. 2012; Caffagni et al. 2011; Dai et al. 2006; Hong et al. 2008; Hong et al., 2009a, 2009b; Voogt et al. 2014; Weng et al., 2008a, 2008b, 2008c) and field (Lawson et al. 2015; Smoleń et al. 2011) experiments have been carried out to produce iodine-enriched crops, by applying iodine containing nutrient solutions or irrigation water, as well as solid fertilizers (e.g. algal-based). In addition to the iodine dosage, the chemical forms of iodine (iodide or iodate) were also investigated. For these experiments, different vegetables were selected such as lettuce, Lactuca sativa L.(Blasco et al. 2008; Blasco et al. 2012; Hong et al. 2008; Lawson et al. 2015; Voogt et al. 2010); spinach, Spinacia oleracea L. (Dai et al. 2006; Weng et al., 2008a, 2008b, 2008c; Zhu et al. 2003; Zhu et al. 2004); pakchoi, Brassica chinensis L. (Hong et al., 2009a, 2009b); cabbage, B. oleracea var. capitata (Weng et al., 2008a, 2008b, 2008c); Chinese cabbage, B. chinesis L. (Hong et al. 2008); tomato, Solanum lycopersicum L. (Caffagni et al. 2011; Hong et al. 2008; Landini et al. 2011); strawberry, Fragaria ananassa (Li et al. 2016); pepper, Capsicum annuum L. (Hong et al., 2009a, 2009b); cucumber, Cucumis sativus L. (Voogt et al. 2014); carrot, Daucus carota L.var. sativus (Hong et al. 2008); celery, Apium graveolens L. var. dulce (Mill.) DC. (Hong et al., 2009a, 2009b); radish, Raphanus sativus L. (Hong et al., 2009a, 2009b; Lawson et al. 2015); potato, Solanum tuberosum L. (Caffagni et al. 2011); rice, Oryza sativa L. (Kato et al. 2013); barley, Hordeum vulgare L. wheat, Triticum aestivum L.; ryegrass, Lolium perenne L.; buckwheat, Fagopyrum esculentum; flax, Linum usitatissimum L.; tobacco, Nicotiana tabacum L. (Hong et al. 2007, Hong et al., 2009a, 2009b; Umaly & Poel 1971; Xie et al. 2007; Yu et al. 2011). All research groups determined the concentration of iodine in different plant parts, however, only a few of them studied the effect of iodine on the accumulation of macro- and micro elements (Smoleń et al. 2011; Smoleń and Sady, 2012; Smoleń et al. 2014; Krzepiłko et al. 2016). On basis of literature related to biofortification of different plants with iodine the following observations were made:

•

We studied the uptake and translocation of iodine in a leafy vegetable, lettuce and a fruit vegetable, green bean in a pot-soil system with calcareous sandy soil. Irrigation water containing KI at concentration of 0.10, 0.25 and 0.50 mg/L was added to the soil. The iodine concentration of different plant parts and the iodine distribution within the plants was investigated by inductively coupled plasma mass spectrometer (ICP-MS) following their microwave-assisted (MW) acid digestion. In addition to these measurements, the effect of iodine on plant growth, morphological and anatomical features of plants, as well as the uptake and translocation of selected essential elements (P, Mg, K, Fe, Mn, Cu, Zn, B) was also studied. 2. Materials and Methods 2.1. Chemicals All chemicals used during the experiments were of analytical grade. The ultra-pure water (resistivity: 18 MΩ cm-1) was taken from an ELGA Ultra Purelab unit (ELGA LabWater/VWS Ltd., High Wycombe, UK). For the quantitative determination of iodine a standard solution was prepared using solid KIO3 (Sigma Aldrich Ltd., Missouri, USA), and for the analyses of P, Mg, K, Fe, Mn, Cu, Zn and B, ICP-MS multi-element standard solution (Sigma Aldrich Ltd., Missouri, USA) was applied. In order to check the accuracy of the analytical method the certified reference material, NIST SRM 1573a, Tomato leaf (National Institute of Standards and Technology, Gaithersburg, USA) was used. 2.2. Characterization of soil The pH was measured according to the Hungarian Standard (MSZ08-0206/2:1978) in 1:2.5 soil:water suspension after mixing for 12 h. The CaCO3 content was determined using the Scheibler gas-volumetric method (MSZ-08-0206/2:1978)· The organic matter (OM) content was measured using the modified Walkley-Black method (MSZ-080452:1980). Plant-available P and K concentrations were determined after extraction with ammonium-acetate lactate (AL-P2O5 and AL-K2O) (Egnér et al. 1960). The total N content was measured by the Kjeldahl method (ISO 11261:1995). The NH4-N and NO3-N concentrations were determined from KCl (Sigma Aldrich Ltd., Missouri, USA) extracts according to the Hungarian Standard (MSZ 20135:1999MSZ 5:, 1999MSZ 20135:1999). The cation exchange capacity (CEC) values were measured by applying the modified method of Mehlich (MSZ-08-0215:1978). The iodine concentrations were determined by ICP-MS following microwave assisted aqua regia digestion (Table 1).

• Low

• •

•

(Dai et al. 2006; Weng et al., 2008a, 2008b, 2008c; Zhu et al. 2003; Zhu et al. 2004), and Chinese cabbage (Hong et al. 2008) have high translocation rates due to their larger leaf surface area and thus accumulate high amounts of iodine in the leaves. This makes them ideal candidates for biofortification with iodine. However, some fruits (strawberry, tomato) and tuber vegetables (potato) can also store high amount of iodine (Caffagni et al. 2011; Landini et al. 2011; Li et al. 2016). Iodine at a concentration range of 0.05-1.0 mg/L was found to have a minimal effect on the macro- and micro-elements concentration in spinach and lettuce (Smoleń and Sady, 2012; Smoleń et al. 2014; Krzepiłko et al. 2016).

concentration of iodine has been found to enhance plant growth in plants like barley, ryegrass, tomato, buckwheat, flax, strawberry, tobacco (Hong et al. 2007; Hong et al., 2009a, 2009b; Umaly & Poel 1971; Xie et al. 2007; Yu et al. 2011). However, over a certain threshold concentration (e.g. 25 mg/kg and 1 mg/L in case of fertilizer and hydroponic technology, respectively), iodine becomes toxic, resulting in negative biomass production (Hong et al., 2009a, 2009b; Weng et al., 2008a, 2008b, 2008c). Iodide is more detrimental to plant growth as compared to iodate due to higher uptake (Blasco et al. 2008). Iodine concentration decreased along the uptake pathway, with roots having the maximum concentration and fruits or leaves having the minimum concentration. Iodine transport occurs mainly through the xylem (Hong et al., 2009a, 2009b; Li et al. 2016; Weng et al., 2008a, 2008b, 2008c), but phloem transport was also observed in lettuce and tomato (Landini et al. 2011; Smoleń et al. 2014). Leafy vegetables like lettuce (Blasco et al. 2008; Blasco et al. 2012; Hong et al. 2008; Lawson et al. 2015; Voogt et al. 2010), spinach

2.3. Plant material and treatments Pot experiments were carried out in a climatic chamber at controlled temperature and light conditions (25-27 °C/17 °C for day/night and 16 h lighting at 500 μmol/m2/s photon flux density). Cylindrical, transparent rhizoboxes were filled with calcareous sandy soil and watered up to 60% of field capacity. The transparent walls of the pots 2

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Table 1 Major physical-chemical properties of the calcareous sandy soil. pH-H2O

7.71

OM (m/m%) CaCO3 (m/m%) Total-N (m/m%) NH4-N (mg/kg) NO3-N (mg/kg) AL - K2O (mg/kg) AL - P2O5 (mg/kg) CEC (meqNa/100 g) Total iodine (mg/kg)

0.502 16 0.067 3.2 3.2 48 129 4.8 0.2

Table 2 Operating conditions of the ICP-MS.

allowed the observation of the root growth of seedlings in the first few weeks. 2.4. Plant growing

Plasma power

1290 W

Outer gas (Ar) Intermediate gas (Ar) Aerosol carrier gas (Ar) Collision gas (He) Reaction gas (H2) Sample uptake Nebulizer Spray chamber Sampler cone Skimmer cone Analytical isotopes Internal standards Data acquisition Dwell time Replicates

7.5 L/min 1.5 L/min 1.0 L/min 90 m L/min 110 m L/min 0.30 mL/min Meinhard double pass Ni. 1.1 mm orifice Ni. 0.5 mm orifice 11 26 B; Mg; 31P;39K; 55Mn; 45 Sc; 89Y;115In;159Tb peak jumping 30 ms 5 × 20

56

Fe;

63

Cu;66Zn;

127

I

concentrations of iodine, and selected macro and micro- elements were determined by ICP-MS (Plasma Quant MS Elite, Analytik Jena, Germany). The operating conditions of the ICP-MS are listed in Table 2. The recovery values obtained for the investigated elements, by analyzing the CRM, NIST SRM 1573a, were between 90 and 111% (Table 3).

A pre-germinated bean (Phaseolus vulgaris L., variety: Golden Goal) seed was planted in each pot. Pots were weighed ( ± 1 g) and irrigated with standing tap water three times a week to maintain the water status of the soil (60% of field capacity). Irrigation was supplemented by modified Hoagland nutrient solution (Varga et al. 2000) and KI (Sigma Aldrich Ltd., Missouri, USA) solution at concentrations 0.10, 0.25 and 0.50 mg/L, from the third week of plantation. The same volume of nutrients and KI solution was added to each pot. The total volume of Hoagland solution in the third phenophase supplied to each plant was 760 ml, in the entire plant growth period (180 ml, 520 ml, and 760 ml solution was added at the first [Vn:2-3 trifoliolate leaves], second [R1:flowering], and third [R4:young pods 2-3 inches long] developmental stage, respectively). The volume of KI solution supplied was 0.18 L, 1.16 L, and 2.31 L until the end of first, second and third stage, respectively. A random experiment design was applied with 15 parallel plants in all treatments (5 pots or replicates were harvested at the end of every stage). Three pre-germinated lettuce (Lactuca sativa L., variety: “Május királya”) seeds were planted in each pot. Plants were thinned to one plant per pot after a week. Irrigation procedure was the same as in case of bean plants. The total volume of Hoagland solution supplied to each plant was 500 ml and 780 ml, until the end of the first [7-8 leaves] and second [formation of head] development stage, respectively. The volume of KI solution supplied was 0.46 L and 0.92 L at the first and second stage, respectively. A random experiment design was applied with 10 parallel plants in all treatments (5 pots or replicates were harvested at the end of every stage).

2.6. Morphological and anatomical measurements Morphological and anatomical investigations were performed on the plants grown under control conditions and on those treated with irrigation water containing iodine at concentration of 0.50 mg/L. For all plants, the total leaf biomass and total leaf number was determined. Leaf widths and lengths and the anatomical features of the mesophyll were studied in the oldest leaves of lettuce and in the terminal leaflets of the oldest leaves of green bean. Cross-sections were taken from the middle part of each leaf or leaflet. Plant materials were embedded with polar resin (Leica Biosystems, Wetzlar, Germany) and sections were prepared by Leica microtome RM2265 (Leica Biosystems, Wetzlar, Germany) equipped with a glass knife. After digitalization by means of Olympus BX43 light microscope and Canon EOS 1200D digital camera, the following mesophyll characteristics were measured: total thickness of mesophyll, total thickness at the midrib, thickness of parenchyma, spongy and palisade mesophyll layers, and area of the main vascular bundles and xylem (Rasband, 2012) (Fig. 1). 2.7. Statistical analysis Statistical differences between iodine concentrations of the control and treatment plants were determined by one-way analysis of variance (ANOVA) and Tukey’s test at a significance level of 0.05, using R 3.5.3 and RStudio 1.1.463 software (R Core Team 2019, R Studio Team

2.5. Sample preparation and elemental analysis of plants At the end of different growth stages, plants were harvested and cleaned with deionized water. The root, stem, leaf and fresh pods of the bean, and the root and leaves of lettuce were separated. Samples were dried in a hot air laboratory oven (UF 450 Plus Memmert, Labsystem Ltd., Hungary) at 40 °C for two days to achieve a constant weight, following which the dry mass of different plant parts was measured. The dried samples were homogenized using an agate mortar and pestle. Then the samples were mineralized by applying a microwave-assisted acid digestion system (TopWave, Analytik Jena, Germany). Twelve PTFE vessels were used (one for blank and eleven for samples). Blank analysis was carried out for every batch. 100-500 mg samples were digested in a mixture of 7 mL Suprapur® 65% nitric acid (VWR International, Pennsylvania, USA) and 3 mL Suprapur® 30% hydrogenperoxide (VWR International, Pennsylvania, USA) using a three-step heating program at temperature of 90, 160 and 200 °C for 10 min each. After digestion internal standards were added to the samples and the volume was made up to 15 mL with de-ionized water. The

Table 3 Certified and measured concentration values of the tomato leaf CRM and the recoveries obtained by ICP-MS.

I Mg P Mn Fe Cu Zn K B

Certified (mg/kg)

Measured (mg/kg)

Recovery (%)

(0.85)* (12000)* 2160 ± 40 246 ± 8 368 ± 7 4.70 ± 0.14 30.9 ± 0.7 27000 ± 500 33.3 ± 0.7

0.93 ± 0.07 10800 ± 200 2080 ± 60 230 ± 2 365 ± 14 4.57 ± 0.13 32.8 ± 0.3 25900 ± 200 36.9 ± 0.4

(110) (90) 96 94 99 97 106 97 111

* indicative values. 3

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Fig. 1. Investigated tissue features in a green bean leaflet (A) and a lettuce leaf (B, C). A5, C5: total mesophyll thickness; A6, C7: palisade mesophyll layer; A7, C6: spongy mesophyll layer; A8, B8: total thickness at the midrib; A9-10, B9, 11-12: parenchyma layer; A11: rib hight; B10: schizogenous intercellular space; A12, B13: total area of the main vascular bundle; A13, B14: area of the xylem. The numbering refers to variables in Fig. 3, 4.

2015). Morphological and anatomical data were analyzed by standardized Principal Components Analysis (PCA) using SYN-TAX 2000 computer program package (Podani 2001). 3. Results and Discussion 3.1. Effect of iodine on the growth of green bean and lettuce plants In the first and second phenophases of green bean plants the addition of iodine to the irrigation water at concentration of 0.10-0.50 mg/L had practically no influence on the dry biomass of leaves and stems. The dry biomass of aerial parts for both the control and the treated plants amounted to about 60-64% and 67-71% of the total mass in the first and second phenophase, respectively. It means that there were only moderate observable differences in the mass distributions. However, in the third phenophase the development of fresh pods resulted in considerable changes in the mass of bean plants as compared to the control plants (Fig. 2/a) and the inhibitory effect of iodine was well detectable. The mass ratio of aerial parts of green bean plants increased to 77% for the control and 73-75% for the plants irrigated with water containing iodine at concentration of 0.10 and 0.25 mg/L. At iodine concentration of 0.5 mg/L the mass of all plant parts decreased, and the mass ratios shifted to the roots and stems. Considering this reduced biomass production, it is recommended to irrigate bean plants cultivated on calcareous sandy soil with water containing iodine in concentration less than 0.50 mg/L to avoid the reduction of plant growth. In case of lettuce, the presence of iodine in the irrigation water resulted in a lower root and higher leaf biomass production (Table 4). However, it should be mentioned, that the increment in leaf biomass values at iodine concentration of 0.50 mg/L decreased considerably to the level of control plants (Fig. 2/b). When comparing the effect of iodine concentration on the growth of bean and lettuce plants a similar phenomenon can be observed. At concentration of 0.10 and 0.25 mg/L the iodine has a stimulating effect on growth, while at concentration of 0.50 mg/L an inhibitory effect of different degrees can be observed. A moderate stimulating effect of iodine on the growth of lettuce was also observed by Hong et al., 2008, but other authors did not observe any significant differences in biomass production on application of iodine containing fertilizers (Lawson et al. 2015; Smoleń et al. 2011). These varying observations can be attributed to the deviations in the experimental and environmental conditions.

Fig. 2. Effects of iodine concentrations of the irrigation water on the dry biomass of green bean (a) and lettuce (b) plant parts related to the control plants.

(Fig. 3/a,b). Based on the dry mass and iodine concentration values of different plant parts, the distribution of iodine among the plant parts were calculated (Table 5). In case of green bean, about 83-87% of iodine accumulated in the roots and only 1.0% was translocated to the fresh pods. Lettuce presented a different picture with a lower amount (4256%) of iodine accumulating in the roots, as compared to the green beans, and an equivalent amount (44-58%) of iodine being translocated to the leaves. The highest iodine concentrations in the fresh bean pods (1.8 mg/kg DW) and in the lettuce leaves (5.6 mg/kg DW) were achieved at 0.50 mg/L iodine concentration, however, as mentioned in section 3.1, the biomass production was hampered at this concentration. Therefore,

3.2. Uptake and translocation of iodine The iodine concentration of the various plant parts (root, leaves, stem and fresh pods) was determined immediately after the harvest. The iodine content of both plants and all plant tissues increased upon application of higher iodine concentration in the irrigation water 4

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Table 4 Effects of iodine concentrations of the irrigation water on the distribution of biomass among the plant parts of green bean and lettuce plants in percent (n = 5). Iodine concentration (mg/L)

Control 0.10 0.25 0.50

Root

Leaves

Stem

Fresh pod

green bean

lettuce

green bean

lettuce

green bean

green bean

24 27 25 35

40 48 44 60

36 28 31 23

60 52 56 40

16 14 15 18

25 31 29 24

± ± ± ±

10 2 6 12

± ± ± ±

15 16 17 18

± ± ± ±

11 6 6 8

± ± ± ±

16 14 11 6

± ± ± ±

3 2 1 4

± ± ± ±

5 7 5 7

iodine in lettuce under different environmental conditions and fertilization technologies was widely studied. In lettuce leaves, the iodine concentrations changed in the range of 3-30, 5-40, 12-18 and 1254 mg/kg DW (Voogt et al. 2010; Hong et al. 2008; Smoleń et al. 2011) cultivated the plants in greenhouse or in field trials applying different fertilization technologies. It indicates that in lettuce leaves up to ten times higher iodine concentration can be achieved as compared to our results. However, it is necessary to find an equally favorable solution for all parameters involved: biomass production, iodine content, and chemical composition of soil and groundwater.

3.3. Effect of iodine on the selected essential element transport The relative concentration changes of some macro and micro nutrients in the edible parts of green bean and lettuce plants related to the control plants are listed in Table 6. It can be seen, that the concentration changes caused by iodine addition are much higher in case of lettuce leaves, than in the fresh bean pods. At iodine concentration of 0.10 and 0.25 mg/L where the lettuce growth was stimulated, the concentration of macro and micro nutrients increased nearly to the same extent. At iodine concentration of 0.50 mg/L where the lettuce growth was slightly inhibited the P and Zn concentration further increased, while the concentration of Mg, Mn, Fe decreased. It should be noted that the Cu concentration was not significantly influenced by the increasing iodine concentration. Our results do not harmonize with the literature data published by Smoleń et al. 2014 and Krzepiłko et al. 2016. These research groups cultivated the lettuce plants in hydroponic system containing iodine in concentration range of 0.05-1.0 mg/L. While Smoleń et al. 2014 did not find any sigificant changes in the concentration of macro- and micro elements, Krzepiłko et al. 2016 detected a moderate reduction (10-17%) of Na, Mn and Mg. These discrepant observations can be attributed to the different cultivation technologies. In case of fresh bean pods at iodine concentration of 0.10 mg/L a moderate reduction of Mg, Mn, Fe, P, and Zn concentrations was observed in spite of the fact that the plant growth was considerably stimulated. Increasing iodine concentration resulted in a continuous increment of elemental content for these macro- and micronutrients, however, the Fe translocation became extremely high at the iodine concentration of 0.50 mg/L, where the plant growth was significantly inhibited. Similarly, as in the lettuce leaves, the concentration of Cu in the fresh bean pods was not influenced by the iodine treatment.

Fig. 3. Iodine concentration in different parts of green bean (a) and lettuce (b) plants. “a, b, c, d” letters indicate significant difference (p < 0.05, Tukey's test).

0.25 mg/L iodine concentration can be recommended for biofortification, where the plant growth was moderately stimulated, and the iodine concentration in fresh bean pods and lettuce leaves amounted to 0.6 and 5.2 mg/kg DW, respectively. It should be mentioned, that in the literature there is no experimental data for green bean plants, but, the uptake and translocation of

Table 5 Effects of iodine concentrations of the irrigation water on the distribution of iodine among the plant parts of bean and lettuce plants in percent (n = 5). Iodine concentration (mg/L)

Control 0.10 0.25 0.50

Root

Leaves

Stem

Fresh pod

green bean

lettuce

green bean

lettuce

green bean

green bean

64 83 86 87

28 42 49 56

22 ± 3 7±3 6±2 5±1

72 58 51 44

9 8 7 7

5 1 1 1

± ± ± ±

9 16 14 13

± ± ± ±

4 12 2 6

5

± ± ± ±

18 15 11 10

± ± ± ±

4 2 4 3

± ± ± ±

1 0.2 0.2 0.2

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tissues, however, were highest in plants grown in control conditions, while the total leaf number was the highest in plants treated with irrigation water containing iodine at concentration of 0.50 mg/L. As a result of iodine treatment, the biomass, number, and size of the green bean leaves decreased. When lettuce plants were irrigated with water containing 0.50 mg/L iodine, the size of the leaves decreased but the number increased. As a result, the total leaf biomass of the control and treated lettuce plants remained practically the same (Fig. 2/b). According to our literature screening, similar observations have not been published.

Table 6 Relative concentration changes (%) of some macro and micro nutrients in the edible part of green bean (A) and lettuce (B) related to the control samples (RSD %). A

Relative concentration changes (%) 0.10 mg/L

0.25 mg/L

0.50 mg/L

Mg P Mn Fe Cu Zn K B

−3 (7) −27 (14) −26 (8) −4 (14) +8 (20) −14 (12) +2 (15) −24 (12)

+8 (7) −13 (14) −1 (29) +3 (28) +6 (22) −4 (13) +170 (20) −18 (16)

+27 (14) −11 (3) +8 (11) +210 (3) +7 (11) +24 (19) +128 (18) +4 (11)

B

Relative concentration changes (%)

Mg P Mn Fe Cu Zn K B

0.10 mg/L

0.25 mg/L

0.50 mg/L

+70 (32) +143 (14) +106 (21) +260 (24) +50 (37) +20 (18) +52 (21) +9 (22)

+72 (26) +147 (15) +108 (22) +215 (30) +53 (28) +26 (19) +58 (18) +12 (12)

+30 (7) +192 (26) +57 (6) +138 (33) +51 (33) +29 (37) + 68 (15) +5 (25)

3.5. Recommended consumption amount of biofortified crops "WHO/UNICEF/ICCIDD recommend that, in typical circumstances, where the iodine lost from salt is 20% from production site to household, another 20% is lost during cooking before consumption, and average salt intake is 10 g per person per day, iodine concentration in salt at the point of production should be within the range of 20–40 mg of iodine per kg of salt (i.e., 20–40 ppm of iodine) in order to provide 150 μg of iodine per person per day” (WHO, 1996). However, it must be taken into account, that high intake of table salt is associated with adverse health effects (e.g. hypertension and cardiovascular diseases). Therefore, the salt consumption should be reduced to < 5 g/day (WHO, 2014). The newly revised reference values for adequate intake of sodium and chlorine are 1.5 and 2.3 g/day, respectively (Strohm et al., 2018). This means that 3.8 g NaCl is the new recommended daily intake value. Since table salts often contain different additive materials - e.g. sodium carbonate or sodium bicarbonate to increase alkalinity, as well as sodium thiosulfate or dextrose to stabilize potassium iodide, - the daily intake of table salts can be changed in the range of 3.8-5.0 g depending on the producers. Considering the new regulations and recommendations there are two possibilities to achieve the recommended daily intake of iodine:

3.4. Evaluation of morphological and anatomical measurements The experimental data obtained by morphological and anatomical investigations of plant leaves were evaluated by applying Principal Components Analysis (Figs. 4,5). This method is useful to obtain a summary of the correlation structure of variables based on a pooled data set and it may be used to demonstrate which variables have high discriminatory power. In case of green bean (Fig. 4), clear separation was observed between the control and treated leaves on the first axis which itself shows account for 64% of the total variance. The vectors (representing variables) point towards the control leaves, demonstrating that all the investigated characteristics (total leaf biomass; the number, length and width of leaves; thickness and area of mesophyll tissues) were the highest in the control plants. This distinction was weaker in case of lettuce leaves (Fig. 5) and many vectors situate between the two groups of leaves. It means that the total leaf biomass, as well as the length and width of leaves did not differ significantly among the control and treated plants. The leaf thickness and bulk of mesophyll

1 the iodine concentration in table salts has to be increased to 39 mg/ kg (WHO, 2014), or 2 the consumption of iodine enriched vegetables should be encouraged. In the second case the consumption of fresh vegetables (e.g. lettuce, tomato, carrot) is recommended because cooking result in losses of iodine. On basis of the data published by the Statista Research Department in 2018, the mean yearly consumption of bean and lettuce in USA were 3.5 and 6.0 kg, respectively (Statista Research Department Fig. 4. PCA ordination of green bean leaves based on the morphological and anatomical measurements. Objects enclosed by polygons are green bean leaves grown in control conditions (b_c) or treated by irrigation water with the iodide concentration 0.50 mg/L (b_0.5). Variables are 1) total leaf biomass; 2) number of leaves; 3-5) length, width and thickness of terminal leaflet; 6-13) anatomical features of leaflet mesophyll (for details, see Fig. 1.A).

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Fig. 5. PCA ordination of lettuce leaves based on the morphological and anatomical measurements. Objects enclosed polygons are lettuce leaves grown in control conditions (l_c) or treated by irrigation water with the iodide concentration 0.50 mg/L (l_0.5). Variables are 1) total leaf biomass; 2) number of leaves; 3-5) length, width and thickness of leaf; 614) anatomical features of leaf mesophyll (for details, see Fig. 1.B-C).

References

in 2018). Considering our experimental results, the daily iodine intake by consumption of bean and lettuce amounts to 5 and 80 μg; therefore, only lettuce can be recommended for biofortification with iodine.

Andersson, M., Benoist, B., Darnton-Hill, I., Delange, F., 2007. Iodine deficiency in Europe - World Health Organization. WHO UNICEF report. Azizi, F., 2007. Iodized Oil: Its Role in the Management of Iodine Deficiency Disorders. International Journal Endocrinology and Metabolism 2, 91–98. Blasco, B., Rios, J.J., Cervilla, L.M., Sánchez-Rodrigez, E., Ruiz, J.M., Romero, L., 2008. Iodine biofortification and antioxidant capacity of lettuce: potential benefits for cultivation and human health. Annals of Applied Biology 152, 289–299. Blasco, B., Ríos, J.J., Sánchez-Rodríguez, E., Rubio-Wilhelmi, M.M., Leyva, R., Romero, L., Ruiz, M.J., 2012. Study of the interactions between iodine and mineral nutrients in lettuce plants. Journal of Plant Nutrition 35, 1958–1969. Caffagni, A., Arru, L., Meriggi, P., Milc, J., Perata, P., Pecchioni, N., 2011. Iodine Fortification Plant Screening Process and Accumulation in Tomato Fruits and Potato Tubers. Communications in Soil Science and Plant Analysis 42, 706–718. Dai, J.L., Zhu, Y.G., Huang, Y.Z., Zhang, M., Song, J.L., 2006. Availability of iodide and iodate to spinach (Spinacia oleracea L.) in relation to total iodine in soil solution. Plant and Soil 289, 301–308. Denage, F., Bürgi, H., Chen, Z.P., Dunn, J.T., 2002. World status of monitoring iodine deficiency disorders control programs. Thyroid 12, 915–924. Egnér, H., Riehm, H., Domingo, W.R., 1960. Investigations on chemical soil analysis as the basis for estimating soil fertility. II. Chemical extraction methods for phosphorus and potassium determination. Kungl Land. Annaler 26, 199–215. Fuge, R., Johnson, C.C., 2015. Iodine and human health, the role of environmental geochemistry and diet, a review. Applied Geochemistry 63, 282–302. Gonzali, S., Kiferle, C., Perata, P., 2017. Iodine biofortification of crops: agronomic biofortification, metabolic engineering and iodine bioavailability. Current Opinion in Biotechnology 44, 16–26. Hong, C.L., Weng, H.X., Yan, A.L., Xie, L.L., 2007. Characteristics of iodine uptake and accumulation by vegetables. Chinese Journal of Applied Ecology 18, 2313–2318. Hong, C., Weng, H., Yan, A., Islam, E., 2009a. The fate of exogenous iodine in pot soil cultivated with vegetables. Environmental Geochemistry and Health 31, 99–108. Hong, C.L., Weng, H.X., Yan, A.L., Xie, L.L., 2009b. Dynamic characterization of iodine uptake in vegetable plants. Acta Ecologica Sinica 29, 1438–1447. Hong, C.L., Weng, H.X., Qin, Y.C., Yan, A.L., Xie, L.L., 2008. Transfer of iodine from soil to vegetables by applying exogenous iodine. Agronomy for Sustainable Development 28, 575–583. ISO 11261:1995, 1995. Soil quality - Determination of total nitrogen - Modified Kjeldahl method. Kato, S., Wachi, T., Yoshihira, K., Nakagawa, T., Ishikawa, A., Takagi, D., Tezuka, A., Yoshida, H., Yoshida, S., Sekimoto, H., Takahashi, M., 2013. Rice (Oryza sativa L.) roots have iodate reduction activity in response to iodine. Frontiers in Plant Science 4, 1–11. Kapusta-Duch, J., Bieżanowska-Kopeć, R., Smoleń, S., Pysz1, M., Kopeć, A., Piątkowska, E., Rakoczy, R., Koronowicz, A., Skoczylas, L., Leszczyńska, T., 2017. The effect of preliminary processing and different methods of cooking on the iodine content and selected antioxidative properties of carrot (Daucus carota L.) biofortified with (potassium) iodine. Folia Horticulturae 29, 11–24. Krzepiłko, A., Zych-Wężyk, I., Święciło, A., Molas, J., Skwaryło-Bednarz, B., 2016. Effect of iodine biofortification of lettuce seedlings on their mineral composition and biological quality. Journal of Elementology 21, 1071–1080. Landini, M., Gonzali, S., Perata, P., 2011. Iodine biofortification in tomato. Journal of Plant Nutrition and Soil Science 174, 480–486. Lawson, P.G., Daum, D., Czauderna, R., Meuser, H., Härtling, J.W., 2015. Soil versus foliar iodine fertilization as a biofortification strategy for field-grown vegetables. Frontiers in Plant Science 6, 1–11.

4. Conclusion Addition of iodine in the concentration of 0.25 mg/L to the irrigation water resulted in a stimulated growth of green bean and lettuce plants cultivated in calcareous sandy soil. In case of lettuce, the uptake and translocation of micro and macro elements were also stimulated (e.g. Zn +26% and Fe +215%) at this iodine concentration, while in the fresh bean pods only moderate concentration changes (e.g. P -13% and Mg +8%) were measured. Considering the iodine concentration (5.2 mg/kg DW) and water content (81%) of the lettuce leaves, consuming 100 g of this vegetable (fresh weight) would cover about 66% of the recommended daily iodine intake. Due to the low iodine translocation from root to fresh bean pods (root/pod concentration ratio = 76) is not suitable for biofortification with iodine. The addition of iodine to the irrigation water appears to be a realistic way to increase the iodine intake for humans, however, it is necessary to study the long term effects of iodine on the biological organisms (nematodes, worms, bacteria, etc.) present in soils, and to determine any potential cross-effect of iodine on other food sources. Author Statement The following tasks were performed by the co-authors: Péter Dobosy: Original draft preparation, writing, analytical experimental work; Krisztina Kröpl: Sample-preparation; Mihály Óvári: Analytical experimental work; Sirat Sandil: Sample-preparation; Kitti Németh: Statistical data evaluation; Attila Engloner: Statistical data visualization; Tünde Takács: Plant growing; Gyula Záray: Conceptualization, editing, writing, reviewing. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgement This research was supported by the National Research Development and Innovation Office (Grant No. NVKP16-1-2016-0044). 7

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barley and pea plants in nutrient solution culture. Annals of Botany 35, 127–131. Varga, A., Záray, Gy., Ferenc, F., 2000. Investigation of element distributions between the symplasm and apoplasm of cucumber plants by TXRF spectrometry. Microchemical Journal 67, 257–264. Voogt, W., Holwerda, H.T., Khodabaks, R., 2010. Biofortification of lettuce (Lactuca sativa L.) with iodine: the effect of iodine form and concentration in the nutrient solution on growth, development and iodine uptake of lettuce grown in water culture. Journal of the Science of Food and Agriculture 90, 906–913. Voogt, W., Steenhuizen, J., Eveleens, B., 2014. Uptake and distribution of iodine in cucumber, sweet pepper, round, and cherry tomato. Wageningen UR Greenhouse Horticulture, Reports Wageningen UR Greenhouse Horiculture 1329, 1–72. Weng, H.X., Yan, A.L., Hong, C.L., Xie, L.L., Qin, Y.C., Cheng, C.Q., 2008a. Uptake of Different Species of Iodine by Water Spinach and Its Effect to Growth. Biological Trace Element Research 125, 184–194. Weng, H., Weng, J., Yan, A., Hong, C., Yong, W., Qin, Y., 2008b. Increment of iodine content in vegetable plants by applying iodized fertilizer and the residual characteristics of iodine in soil. Biological Trace Element Research 123, 218–228. Weng, H.X., Hong, C.L., Yan, A.L., Pan, L.H., Qin, Y.C., Bao, L.T., Xie, L.L., 2008c. Mechanism of Iodine Uptake by Cabbage: Effects of Iodine Species and Where It is Stored. Biological Trace Element Research 125, 59–71. WHO, 1996. Recommended iodine levels in salt and guidelines for monitoring their adequacy and effectiveness. WHO/FAO, 2004. Vitamin and mineral requirements in human nutrition, 2nd ed. joint FAO/WHO Expert Consultation in Human Vitamin and Mineral Requirements, Genova. WHO, 2014. Guideline: fortification of food-grade salt with iodine for the prevention and control of iodine deficiency disorders. Wolff, J., 2001. Physiology and pharmacology of iodized oil in goiter prophylaxis. Medicine 80, 20–36. Xie, L.L., Weng, H.X., Hong, C.L., Yan, A.L., 2007. Iodine uptake by bok-choy and Ipomoea aquatica Forsk. Plant Nutrition and Fertilizer Science 13, 123–128. Yu, W.J., Yao, Y., Wei, H.M., Long, M., Hand Tang, X.F., 2011. Absorption of exogenous iodine in rhizosphere and its effects on physiological parameters of cherry tomato plants. Guihaia 31, 513–519. Zhu, Y.G., Huang, Y.Z., Hu, Y., Liu, Y.X., 2003. Iodine uptake by spinach (Spinacia oleracea L.) plants grown in solution culture: effects of iodine species and solution concentrations. Environment International 29, 33–37. Zhu, Y.G., Huang, Y., Hu, Y., Liu, Y., Christie, P., 2004. Interactions between selenium and iodine uptake by spinach (Spinacia oleracea L.) in solution culture. Plant and Soil 261, 99–105. Zimmermann, B.M., Boelaert, K., 2015. Iodine deficiency and thyroid disorders. The Lancet Diabetes & Endocrinology 3, 286–295.

Li, R., Liu, H.P., Hong, C.L., Dai, Z.X., Liu, J.W., Zhou, J., Hua, C.Q., Wenga, H.X., 2016. Iodide and iodate effects on the growth and fruit quality of strawberry. Journal of the Science of Food and Agriculture 1, 231–235. MSZ-08-0206/2:1978, 1978. Evaluation of some chemical properties of the soil. Laboratory tests (pH value, phenolphtalein alkalinity expressed in soda, total water soluble salt content, hydrolytic (y1 value) and exchangeable acidity (y2 value). Hungarian Standard Association Budapest, Hungary. MSZ-08-0452:1980, 1980. Use of high-capacity analyser systems for soils analyses. Quantitative determination of the organic carbon content of the soil on Contiflo analyzer system. Hungarian Standard Association Budapest, Hungary. MSZ 20135:1999, 1999. Determination of the soluble nutrient element content of the soil. Hungarian Standard Association Budapest, Hungary. MSZ-08-0215:1978, 1978. Determination of the Cation Adsorption Capacity of the Soil. Modified Mechlich Technique. Hungarian Standard Association Budapest, Hungary. Podani, J., 2001. SYN-TAX 2000 Computer Programs for Data Analysis in Ecology and Systematics. User’s Manual. Scientia, Budapest. Rana, R., Raghuvanshi, R.S., 2013. Effect of different cooking methods on iodine losses. Journal of Food Science and Technology 50, 1212–1216. Rasband, W.S., 2012. Image J, U.S. National Institutes of Health, Bethesda, Maryland, USA. imagej.nih.gov/ij/, 1997—2012. . Robertson, D.E., Cataldo, D.A., Napier, B.A., Krupka, K.M., Sasser, L.B., 2003. Literature Review and Assessment of Plant andAnimal Transfer Factors Used in Performance Assessment Modeling. U.S. Nuclear Regulatory Commission Office of Nuclear Regulatory Research, Washington, DC 20555-0001. R Core Team, 2019. R. A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. RStudio Team, 2015. RStudio. Integrated Development for R. RStudio, Inc., Boston, MA. Smoleń, S., Ledwożyw-Smoleń, I., Rożek, S., Strzetelski, P., 2011. Preliminary evaluation of the influence of soil fertilization and foliar nutrition with iodine on the efficiency of iodine biofortification and chemical composition of lettuce. Journal of Elementology 16, 275–285. Smoleń, S., Sady, W., 2012. Influence of iodine form and application method on the effectiveness of iodine biofortification, nitrogen metabolism as well as the content of mineral nutrients and heavy metals in spinach plants (Spinacia oleracea L.). Scientia Horticulturae 143, 176–183. Smoleń, S., Kowalska, I., Sady, W., 2014. Assessment of biofortification with iodine and selenium of lettu ce cultivated in the NFT hydroponic system. Scientia Horticulturae 166, 9–16. Statista Research Department, 2018. United States of America. Strohm, D., Bechthold, A., Ellinger, S., Leschik-Bonnet, E., Stehle, P., Heseker, H., 2018. Revised Reference Values for the Intake of Sodium and Chloride. Annals of Nutrition and Metabolism 72, 12–17. Umaly, R.C., Poel, L.W., 1971. Effects of iodine in various formulations on the growth of

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