sustainability

Article Characteristics of Cadmium and Accumulation and Transfer by Chenopodium Quinoa Will

Vesna Radovanovic 1, Ilija Djekic 2 and Branka Zarkovic 3,*

1 Environmental Consulting Agency, B.E.A Better Environmental Activity, 11185 Belgrade, Serbia; [email protected] 2 Department of Food Safety and Quality Management, Faculty of Agriculture, University of Belgrade, 11080 Belgrade, Serbia; [email protected] 3 Department of Agrochemistry and Plant Physiology, Faculty of Agriculture, University of Belgrade, 11080 Belgrade, Serbia * Correspondence: [email protected]

 Received: 5 April 2020; Accepted: 5 May 2020; Published: 7 May 2020 

Abstract: Potentially toxic elements are persistent in the environment and plants have the ability to absorb and transfer them from in edible parts. The objectives of this study were to characterize the distribution of Cd and Pb in quinoa tissues and to investigate their accumulation and transfer from irrigated water in edible parts of quinoa. For the purpose of this study experiment and simulated in the form of different concentration in water that was used for irrigation was designed. Distribution of in quinoa were determined and analyzed in seed formation and maturation stage. Bioaccumulation and translocation factors were calculated to characterize the efficiency of quinoa to absorb metals. The results of our study indicated that quinoa adopts potentially toxic metals from substrate but does not accumulate them. The potential of such a conclusion is useful for exploring the use of quinoa as lead and cadmium excluders.

Keywords: potentially toxic elements; water-to-plant transfer; bioaccumulation factor; translocation factor; metal excluders

1. Introduction In numerous publications in recent decades, the term “” is a term for metals that are contaminants. The of a substance can only be defined if the relationship between dose and effect is known. This relationship is a characteristic of each substance and it is necessary to know this relationship when determining whether a substance is toxic or not. The term “Potentially Toxic Element(s)” (PTEs) can include uniform and comparative use, and meanings on the basis of more reliable characterization [1]. Potentially toxic elements (PTEs) are not susceptible to biodegradation, they are persistent in the environment and plants can absorb them from soil and accumulate them in edible parts. Consequently, PTEs can enter into the food chain, creating a risk to human health [2,3]. Cadmium (Cd) is extremely toxic at very low concentration for both plants and humans [4]. Toxic effects of cadmium in plants is reflected through its affinity for the thiol group as Cd can functional groups of biomolecules, which results in the inhibition of enzyme activity of important plant metabolic processes, such as photosynthesis and respiration [5]. Since it belongs to the group of metals whose ions can rapidly be absorbed by plant roots, Cd is very mobile in soil and available to plants, and can easily be implemented into the food chain [6]. The toxic effect of lead (Pb) is more expressed at higher concentration and duration of exposure. These levels of Pb concentration in the plant affect germination, growth, photosynthesis, water regime,

Sustainability 2020, 12, 3789; doi:10.3390/su12093789 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 3789 2 of 11 nutrition and enzyme activity [7,8]. Accumulation of Pb by plants has been reported in roots, shoots, leaves and seeds. However, the majority of absorbed Pb by plants resides in the root, having only small amounts translocated to the aerial parts of plants [8]. Despite their relatively low concentration in edible parts of plant, both Cd and Pb are reported as food contaminants and food agencies highlight their threat to human health [9]. The uptake, accumulation and translocation of toxic metals by plants can differ widely even across cultivars within the same species [10]. Quinoa (Chenopodium quinoa Will) is a pseudocereal originated in the Andean region of South America. This cereal has received an increased interest across Europe lately due to its high protein content and compositions of protein [11]. Because of its nutritional value (rich in protein, fat, dietary fiber and ) quinoa is applied in the production of bread, pasta and baby food [12]. The Food and Agriculture Organization declared 2013 to be the “International of Quinoa” [13]. In 1995, 61.0 tons of quinoa was exported from Peru; in 2014, global demand for quinoa was 600 times higher, so they exported 36,000 tons of quinoa [14]. Estimated high nutritional value in alternative cereals, including quinoa, is associated with their qualitative and quantitative composition of protein [15,16]. The content of protein in quinoa ranges in value from 12.1% to 14.5% [17,18], with the maximum content being up to 22.08% [19,20]. The bioaccumulation and translocation potentials of PTEs by plants grown on contaminated soil were studied on Plantago major L. [21], Triticum aestivum [22,23], 30 varieties of Brassica pekinensis L. [24], miscanthus [25,26] and 32 hybrid varieties [27]. Transfer of PTEs from water-to-plant were studied on 14 sunflower hybrids [28], two eucalypt hybrid clones [29], wheat and different vegetables [30]. In spite of some research characterizing accumulation and transfer of PTEs in crops, the literature search revealed that quinoa has not been a focus of such research, and so this was identified as a research gap by the authors of this paper. The objectives of this study were to characterize the distribution of Cd and Pb in quinoa tissues and to investigate accumulation and transfer of Cd and Pb from irrigated water in edible parts of quinoa.

2. Materials and Methods

2.1. Experimental Design For the purpose of this experiment, plant growth chamber (FITOCLIMA 20000HP MI234INR00—Ingles, 20–25 ◦C, 12–15 h photoperiod, RH 60%) was used. Composition of Floradur B (Floragard, Germany) was: 70% of German white peat + 30% German black peat + lime + macro and micro elements (Mg, B, Cu, Mn, Mo, Fe and Zn) + wetting agent. A Superfine structure was used with a pH of 5.6 and a salt content of 0.8 g/l. Such an experiment simulated pollution in the form of different metal concentrations in water that was used for irrigation. The experiment had 12 treatments with 5 simultaneous repetitions, leading to a total of 60 variations. Solutions for plant watering were made by dissolving salts of lead (Pb (CH COO) 3H O) and cadmium (3CdSO 8H O) in tap water. 3 2 × 2 4 × 2 During the entire duration of the experiment, soil moisture was maintained at level of 60% of the water retention capacity. After 4 weeks, continuously to the end of the experiment, the following treatments were applied: I—tap water, II—5 mg/kg Pb, III—50 mg/kg Pb, IV—100 mg/kg Pb, V—5 mg/kg Cd, VI—50 mg/kg Cd, VII—100 mg/kg Cd, VIII—5 mg/kg Pb and Cd, IX—50 mg/kg Pb and Cd, X—100 mg/kg Pb and Cd, XI—100 mg/kg Pb and 5 mg/kg Cd, XII—5 mg/kg Pb and 100 mg/kg Cd (Table1). Grain formation was observed after 100 days and two plants from each treatment were harvested. After 20 days, maturation of grain was completed and the rest of the plants were harvested. Sustainability 2020, 12, 3789 3 of 11

Table 1. Experimental design, applied treatments.

Treatment Number Treatment Applied I tap water II 5 mg/kg Pb III 50 mg/kg Pb IV 100 mg/kg Pb V 5 mg/kg Cd VI 50 mg/kg Cd VII 100 mg/kg Cd VIII 5 mg/kg Pb and Cd IX 50 mg/kg Pb and Cd X 100 mg/kg Pb and Cd XI 100 mg/kg Pb and 5 mg/kg Cd XII 5 mg/kg Pb and 100 mg/kg Cd

2.2. Chemical Analysis The first set of harvested plants were separated into root, shoot, leaf and seed; while the second set of harvested plants were separated into root, shoot and seed. The plant samples were first air-drayed at room temperature and then in a drying chamber at 105 ◦C to constant weights. After drying, the samples were powered with IKA A11 mill for plant material. For analysis of total Cd and Pb concentration in substrate, 2.0000 g of sample was weighted and digested with 20 mL of concentrated HNO3 and slowly heated to boiling temperatures. The reaction mixture was heated until no brown vapors were generated. A mixture of 3 mL of concentrated H2O2 was added into the cooled reaction and the reaction mixture was heated slowly to boiling. After the termination of brown vapor generation, the reaction mixture was re-cooled and 3 mL of concentrated H2O2 was added. The reaction mixture was gradually heated to boiling and after no brown vapor was generated, the reaction mixture was cooled and quantitatively transferred to a volumetric flask of 100 mL [31]. A similar procedure was used for the plant samples digestion [32]. Preparation of the substrate samples for determination of the available forms of Pb and Cd was carried out by extraction with a DTPA (diethylenetriaminepentaacetic acid) buffer solution [33]. The concentration of cadmium and lead in the solution obtained by digestion were determined by the inductively coupled plasma-mass spectrometry, ICP-OES, Thermo ICAP 6300 duo [34].

2.3. Bioaccumulation Factor (BAF) and Translocation Factor (TF) Analysis The bioaccumulation factor (BAF) was defined as the ratio of Cd or Pb concentration in the roots and those in the substrate used for plant growth [35], and it was defined using Equation (1). The BAF was calculated as: BAF = Cplant/Csoil, (1) where Cplant and Csoil represent Pb or Cd concentration in plants and substrate on dry weight basis, respectively. The translocation factor is defined as the ratio of Cd or Pb concentration in the seed to those in the root [21], as given in Equation (2): TF = Cgrain/Croot, (2) where Cgrain and Croot represent Pb or Cd concentration in plants on dry weight basis.

2.4. Statistical Analysis Data were processed using one-way ANOVA and Tukey’s HSD post hoc test to distinguish statistical differences between the transfer of Cd and Pb from water—to—plant, bioaccumulation factors of Cd and Pb during formation and maturation of seed and transfer coefficients of Cd and Pb during formation and maturation of seed. An independent sample t-test was performed to analyze Sustainability 2020, 12, x FOR PEER REVIEW 4 of 11

2.4. Statistical Analysis Data were processed using one-way ANOVA and Tukey’s HSD post hoc test to distinguish statistical differences between the transfer of Cd and Pb from water – to – plant, bioaccumulation factors of Cd and Pb during formation and maturation of seed and transfer coefficients of Cd and Pb during formation and maturation of seed. An independent sample t-test was performed to analyze the bioaccumulation factor between Cd and Pb. In such a way, we analyzed the statistical difference between the two toxic metals. The level of statistical significance was set at 0.05. Statistical processing was performed using Microsoft Excel 2010 and SPSS Statistics 17.0. Two way ANOVA and Tukey’s post hoc test was also used to show interaction between treatments and PTEs. Sustainability 2020, 12, 3789 4 of 11 3. Results

3.1.the Transfer bioaccumulation of PTEs from factor water-to-plant between Cd and Pb. In such a way, we analyzed the statistical difference between the two toxic metals. The level of statistical significance was set at 0.05. Statistical processing Bioaccumulation and translocation of cadmium in quinoa during formation and maturation of was performed using Microsoft Excel 2010 and SPSS Statistics 17.0. Two way ANOVA and Tukey’s seed within the 12 applied treatments is presented in Figure 1. Values in all parts of the plant for the post hoc test was also used to show interaction between treatments and PTEs. treatments VII and IX and the difference between the content in the root and seed of the plant for the treatments3. Results IV, V, VI, VIII, X, XI and XII during formation of the seed were statistically significant (p < 0.05). At the stage of seed maturation, analysis showed a statistically significant difference (p < 0.05) between3.1. Transfer the root, of PTEs stem from and Water-to-Plant seed within treatments V, VI, VII, VIII, IX, X, XI and XII. Figure 2 depicts the distribution of Pb in quinoa during the two observed stages of seed and the Bioaccumulation and translocation of cadmium in quinoa during formation and maturation of 12 treatments applied. Statistically significant differences were not found only within the treatments seed within the 12 applied treatments is presented in Figure1. Values in all parts of the plant for the V, VI and VII (p < 0.05). For all other treatments, significant differences exist between the content in treatments VII and IX and the difference between the content in the root and seed of the plant for the roots and seeds. the treatments IV, V, VI, VIII, X, XI and XII during formation of the seed were statistically significant As for the seed maturation stage, the highest concentration of Pb was determined in the root for (p < 0.05). At the stage of seed maturation, analysis showed a statistically significant difference the treatment IV (21.82 mg/kg). (p < 0.05) between the root, stem and seed within treatments V, VI, VII, VIII, IX, X, XI and XII.

F.I 0.00 Root Stem M.I 0.00 Root Stem Fruit/Seed Leaf Fruit/Seed F.II 0.00 M.II 0.00

F.III 0.00 M.III 0.00

F.IV 0.00 M.IV 0.00 0.18a 1.82b 6.31c F.V 0.23a 2.55b M.V 1.05a 3.12b F.VI 0.73a 1.43a 4.36b 12.02c M.VI 2.24a 7.24b 24.66c 2.46b F.VII 1.06a 3.35c 21.39d M.VII 2.85a 10.93b 35.45c 0.18a 1.57b 3.75b a F.VIII 0.19a 2.31c M.VIII 1.28 6.81c

0.60a c d a b c F.IX 1.21b 4.06 10.03 M.IX 3.58 8.47 17.64

0.95a 2.88b F.X 3.42b 19.76c M.X 5.93a 13.96b 30.01c

0.14a 1.68b F.XI M.XI 2.32a 5.19b 15.34c a 0.18 1.63b F.XII 0.54a 2.05b 19.75c M.XII 2.78a 7.06b 23.35c 1.29a,b -5 0 5 10 15 20 25 -5 0 5 10 15 20 25 30 35 40

(a) (b)

FigureFigure 1. 1. TheThe transfer transfer of ofCadmium Cadmium from from water water—to—plant, – to – plant, Legend: (a (a) )Seed Seed formation, formation, F.I–F.XII—treatmentF.I–F.XII—treatment of plants during formationformation ofof seed;seed; (b(b)) Seed Seed maturation, maturation, M.I–MXII—treatment M.I–MXII—treatment of ofplants plants during during maturation maturation of seed. of seed. I—tap I—ta water;p water; II—5 mgII—5/kg mg/kg of Pb; III—50of Pb; mgIII—50/kg Pb;mg/kg IV—100 Pb; mgIV—100/kg Pb; mg/kgV—5 mg Pb;/kg V—5 Cd; mg/kg VI—50 Cd; mg /VI—50kg Cd; VII—100mg/kg Cd; pm VII—100 Cd; VIII—5 pm mgCd;/ kgVIII—5 Pb and mg/kg 5 mg /Pbkg and Cd; IX—505 mg/kg mg Cd;/kg IX—50Pb and mg/kg 50 mg /kgPb Cd;and X—100 50 mg/kg mg/ kgCd; Pb X—100 and 100 mg/kg mg/kg Pb Cd; and XI—Pbmax 100 mg/kg Cdmin; Cd; XII—PbXI—Pbmax min Cdmin; Cd max. XII—PbMean values min withinCd max. the Mean same rowvalues with within the di fftheerent same superscripts row with di theffer significantlydifferent superscripts (p < 0.05). differ significantly (p < 0.05). Figure2 depicts the distribution of Pb in quinoa during the two observed stages of seed and the 12 treatments applied. Statistically significant differences were not found only within the treatments V, VI and VII (p < 0.05). For all other treatments, significant differences exist between the content in the roots and seeds. As for the seed maturation stage, the highest concentration of Pb was determined in the root for the treatment IV (21.82 mg/kg). SustainabilitySustainability 20202020, 12, 12, x, 3789FOR PEER REVIEW 5 of5 of11 11

F.I 0.00 Root Stem M.I 0.00 0.09 Root Stem Fruit/Seed

a b 0.07 c Leaf Fruit/Seed a 1.37 F.II b 2.74 M.II 0.00 0.17a 0.72 7.00c F.III 0.08a 0.80c 4.27d M.III 0.00a 1.96b 0.22b F.IV 0.14a 1.06b 11.05c M.IV 0.00a 2.92b 21.82c 0.69a,b F.V 0.00 M.V 0.00

F.VI 0.00 M.VI 0.00

F.VII 0.00 M.VII 0.00 0.11a F.VIII 0.05a 0.62b 2.55c M.VIII 0.00a 1.97b

a 0.10 c F.IX 0.01a 1.62c M.IX 0.00a 1.63 0.89b 1.02b 13.76b F.X 0.58a 1.03a 3.48b 11.79c M.X 0.00a 0.72a F.XI 0.12a 1.46b 8.27c M.XI 0.00a 0.44a 31.79b

a a F.XII 0.07 1.05c M.XII 0.00 2.85b 0.09a 0.58b -5 0 5 10 15 -5 0 5 10 15 20 25 30 35

(a) (b)

FigureFigure 2. TheThe transfer transfer of Leadof fromLead water—to—plant.from water – Legend:to – plant. (a) Seed Legend: formation, (a) F.I–F.XII—treatmentsSeed formation, F.I–F.XII—treatmentsof plants during formation of plants of during seed; formation (b) Seed maturation,of seed; (b) Seed M.I–MXII—treatment maturation, M.I–MXII—treatment of plants during maturation of seed. I—potable water; II—5 mg/kg of Pb; III—50 mg/kg Pb; IV—100 mg/kg Pb; of plants during maturation of seed. I—potable water; II—5 mg/kg of Pb; III—50 mg/kg Pb; IV—100 V—5 mg/kg Cd; VI—50 mg/kg Cd; VII—100 pm Cd; VIII—5 mg/kg PB and 5 mg/kg Cd; IX—50 mg/kg mg/kg Pb; V—5 mg/kg Cd; VI—50 mg/kg Cd; VII—100 pm Cd; VIII—5 mg/kg PB and 5 mg/kg Cd; Pb and 50 mg/kg Cd; X—100 mg/kg Pb and 100 mg/kg Cd; XI—Pbmax Cdmin; XII—Pb min Cd max. IX—50 mg/kg Pb and 50 mg/kg Cd; X—100 mg/kg Pb and 100 mg/kg Cd; XI—Pbmax Cdmin; Mean values within the same row with the different superscripts differ significantly (p < 0.05). XII—Pb min Cd max. Mean values within the same row with the different superscripts differ 3.2.significantly Interaction between(p < 0.05). Treatments and PTEs

3.2. InteractionA two-way between ANOVA Treatments was conducted and PTEs that examined the transfer of Cd and Pb from water to plants during formation and maturation of seed. Analysis of the transfer of Cd and Pb from water to the roots of theA two-way plant during ANOVA formation was ofconducted the seed showedthat examined that there the was transfer no statistically of Cd and significant Pb from interactionwater to plantsbetween during Pb andformation Cd (F =and1.67; maturationp = 0.22). of However, seed. Anal theysis transfer of the transfer of Cd and of Cd Pb fromand Pb water from to water the seed to theshowed roots of a statisticallythe plant during significant formation interaction of the betweenseed showed the two that PTEs there (F was= 6.36; no statisticallyp = 0.028). significant interactionDuring between maturation Pb and of Cd the (F seed, = 1.67; analysis p = 0.22). of However, the transfer the of transfer Cd and of Pb Cd from and Pb water from to water the roots to theof seed the plant showed showed a statistically no statistically significant significant interaction interaction between between the two PbPTEs and (F Cd = 6.36; (F = p1.733; = 0.028).p = 0.21). TheDuring transfer maturation of Cd and Pbof the from seed, water analysis to the seedof the showed transfer a statisticallyof Cd and Pb significant from water interaction to the roots between of thetwo plant PTEs showed (F = 12.19; no statisticallyp = 0.005). significant Further analysis interaction confirmed between that Pb Cdand and Cd (F Pb = were 1.733; not p = significantly 0.21). The transferaffecting of treatmentsCd and Pb during from water formation to the and seed maturation showed a of statistically seed (p = 0.22; significantp = 0.49, interaction respectively). between two PTEs (F = 12.19; p = 0.005). Further analysis confirmed that Cd and Pb were not significantly affecting3.3. Bioaccumulation treatments during Factor formation and maturation of seed (p = 0.22; p = 0.49, respectively).

3.3. BioaccumulationBioaccumulation Factor factor (BAF), for values of total content of Pb and Cd in substrate, during seed formation are presented in Table2. These values indicate that the absorption was of medium intensity. TheBioaccumulation highest value of factor BAF for (BAF), Cd was for values within of the total treatment content IV of (1.27) Pb and and Cd for in Pbsubstrate, within theduring treatment seed formationX (0.35). are presented in Table 2. These values indicate that the absorption was of medium intensity.The The results highest show value that statistical of BAF for diff Cderences was werewithin observed the treatment for treatments IV (1.27) II, and VII, for IX andPb within X (p < 0.05the ). treatmentMedium X intensity (0.35). absorption was also observed, in the seed maturation stage (Table3) with the highest valueThe within results the show treatment that statistical XI for both differences Cd (0.78) were and Pb observed (0.88). Resultsfor treatments show statistical II, VII, IX di ffanderences X (p for< 0.05).treatments Medium III, intensity IV, V, VI, absorption VII, VIII and was IX also (p < 0.05).observed, in the seed maturation stage (Table 3) with the highestDTPA value extractable within BAFthe treatment of Cd and XI Pb for (Figure both Cd3) indicate(0.78) and that Pb bioaccumulation (0.88). Results show was statistical generally differencesmedium tofor high treatments intensity. III, ForIV, V, the VI, seed VII, formationVIII and IX the (p highest< 0.05). value of Cd was for the treatment VII (5.27), while treatment IV showed the highest values for Pb (2.58). The highest value of Cd for the seed maturationTable 2. Bioaccumulation was for the treatment factor of VICd (4.75),and Pb, and total for content Pb for in the substrate, treatment the seed XI (5.51). formation. Treatment Cd Pb I 0.2051 ± 0.1179a 0.1240 ± 0.0131a Sustainability 2020, 12, 3789 6 of 11

Table 2. Bioaccumulation factor of Cd and Pb, total content in substrate, the seed formation.

Treatment Cd Pb I 0.2051 0.1179 a 0.1240 0.0131 a ± ± II 1.0162 0.1198 a 0.2062 0.0389 b ± ± III 0.1133 0.0058 a 0.1600 0.0177 a ± ± IV 1.2690 1.0203 a 0.3325 0.0427 a ± ± V 0.1194 0.0206 a 0.0796 0.0021 a ± ± VI 0.2907 0.0141 a 0.1082 0.0041 b ± ± VII 0.3375 0.0099 a 0.0838 0.0258 b ± ± VIII 0.1746 0.0707 a 0.1889 0.0567 a ± ± IX 0.4009 0.0150 a 0.0857 0.0148 b ± ± X 0.3055 0.0105 a 0.3533 0.0523 a ± ± XI 0.1047 0.0013 a 0.2411 0.0229 a ± ± XII 0.3056 0.0311 a 0.0929 0.0121 b ± ± The statistical differences were expressed with different letters (a,b) (p < 0.05).

Table 3. Bioaccumulation factor of Cd and Pb, total content in substrate, the seed maturation.

Treatment Cd Pb I 0.1382 0.0424 a 0.0517 0.0896 a ± ± II 0.0870 0.0485 a 0.0899 0.0104 a ± ± III 0.0100 0.0072 a 0.2537 0.0227 b ± ± IV 0.1473 0.0294 a 0.5995 0.0612 b ± ± V 0.2965 0.0388 a 0.0045 0.0078 b ± ± VI 0.5905 0.0343 a 0.0179 0.0309 b ± ± VII 0.5124 0.0351 a 0.0085 0.0148 b ± ± VIII 0.4628 0.0952 a 0.1208 0.0039 b ± ± IX 0.6310 0.0160 a 0.0819 0.0030 b ± ± X 0.4245 0.0288 a 0.4006 0.0345 a ± ± XI 0.7787 0.1341 a 0.8867 0.0379 a ± ± XII 0.3671 0.0804 a 0.2317 0.0313 a Sustainability 2020, 12, x FOR PEER REVIEW ± ± 7 of 11 The statistical differences were expressed with different letters (a,b) (p < 0.05).

7.0 Formation of seed a 6.0 Maturation of seed a a b a 5.0 a a 4.0 a

3.0 a a a a b 2.0 a b b a a a b b 1.0 b b b 0.0 I II III IV V VI VII VIII IX X XI XII

(a) (b)

FigureFigure 3.3. DTPADTPA (diethylenetriaminepentaacetic (diethylenetriaminepentaacetic acid) acid) extractable extractable bioaccumulation bioaccumulation factors factors of ( ofa) (Cadmiuma) Cadmium and and (b) ( bLead) Lead during during formation formation and and maturation maturation of of seed. seed. The The diversities diversities were were expressed asas didifferentfferent lettersletters ((pp < < 0.05).0.05).

TheTranslocation seed forming factor stage (TF) has of strongercadmium absorption and lead ofduring Cd, except formation for the and treatment maturation VI (Figureof seed3 ).is Inpresented contrast in for the Pb, Figure the stronger 4. There absorption is no significant is generally difference observed between in the values seed maturationof TF in these stage. two stages for lead.Translocation However,factor for cadmium (TF) of cadmiumstatistically and signific leadant during difference formation exists and for maturationtreatments VIII, of seed IX, isX presentedand XII. TF’s in the values Figure of4. metals There isin no seed significant formation di ff stageerence are between lower valuesthan 1, of except TF in thesefor control two stages and treatment II for cadmium because of the low bioaccumulation in root for these two treatments.

7.0 a 1.20 0.00008 Formation of seed Formation of seed a Maturation of seed Maturation of seed 0.00007 6.0 1.00 a 0.00006 5.0 0.80 0.00005 4.0 0.60 0.00004 3.0 0.00003 0.40 Maturation of seed 2.0 0.00002 b a 0.20 1.0 a 0.00001 a a a a a a a a a b a b a b a a a b 0.0 0.00 0 I II III IV V VI VII VIII IX X XI XII I II III IV V VI VII VIII IX X XI XII

(a) (b)

Figure 4. Transfer coefficients of (a) Cadmium and (b) Lead during formation and maturation of seed. The diversities were expressed as different letters (p < 0.05). Where no letters are presented, there is no significant difference.

4. Discussion The dominant toxic forms, accessible fractions, of the PTEs included in this study are free ions. It has been proved that their bioavailability is decreasing when the soil pH and organic content is increasing [36]. Distribution of Cd in the seed formation stage shows that the highest content was in the root of quinoa, with the highest concentration Cd within the treatments VII, 100 mg/kg Cd, (21.39 mg/kg), X, 100 mg/kg Pb and Cd, (19.76 mg/kg) and XII, 5 mg/kg Pb and 100 mg/kg Cd, (19.75 mg/kg). These results are consistent with the fact that in the treatments VII, X and XII, the highest concentration of Cd were applied (100 mg/kg in water for irrigation). The lowest Cd content was determined in the seed. In the seed maturation stage, the highest content can be observed in root and the lowest content in the seed. The highest concentration of Cd in the root was again within the treatments VII (35.45 mg/kg). Similar studies have shown low levels of metals in the seed, as well as a low Sustainability 2020, 12, x FOR PEER REVIEW 7 of 11

7.0 Formation of seed a 6.0 Maturation of seed a a b a 5.0 a a 4.0 a

3.0 a a a a b 2.0 a b b a a a b b 1.0 b b b 0.0 I II III IV V VI VII VIII IX X XI XII

(a) (b)

Figure 3. DTPA (diethylenetriaminepentaacetic acid) extractable bioaccumulation factors of (a) Cadmium and (b) Lead during formation and maturation of seed. The diversities were expressed as different letters (p < 0.05).

Translocation factor (TF) of cadmium and lead during formation and maturation of seed is Sustainability 2020, 12, 3789 7 of 11 presented in the Figure 4. There is no significant difference between values of TF in these two stages for lead. However, for cadmium statistically significant difference exists for treatments VIII, IX, X andfor lead.XII. TF’s However, values for of cadmiummetals in statisticallyseed formation significant stage diareff erencelower existsthan 1, for except treatments for control VIII, IX,and X treatmentand XII. TF’s II for values cadmium of metals because in seed of formationthe low bioa stageccumulation are lower thanin root 1, exceptfor these for two control treatments. and treatment II for cadmium because of the low bioaccumulation in root for these two treatments.

7.0 a 1.20 0.00008 Formation of seed Formation of seed a Maturation of seed Maturation of seed 0.00007 6.0 1.00 a 0.00006 5.0 0.80 0.00005 4.0 0.60 0.00004 3.0 0.00003 0.40 Maturation of seed 2.0 0.00002 b a 0.20 1.0 a 0.00001 a a a a a a a a a b a b a b a a a b 0.0 0.00 0 I II III IV V VI VII VIII IX X XI XII I II III IV V VI VII VIII IX X XI XII

(a) (b)

Figure 4. TransferTransfer coecoefficientsfficients of of (a ()a Cadmium) Cadmium and and (b )( Leadb) Lead during during formation formation and and maturation maturation of seed. of seed.The diversities The diversities were expressed were expressed as different as different letters (p le

4. Discussion Discussion The dominant toxic toxic forms, forms, accessible accessible fractions, fractions, of of the the PTEs PTEs included included in in this this study study are are free free ions. ions. It has been proved that their bioavailability is is decreasing when when the the soil soil pH and organic content is increasingincreasing [36]. [36]. Distribution of of Cd Cd in in the the seed seed formation formation stage stage sh showsows that that the the highest highest content content was was in inthe the root root of quinoa,of quinoa, with with the the highest highest concentration concentration Cd Cd within within the the treatments treatments VII, VII, 100 100 mg/kg mg/kg Cd, Cd, (21.39 (21.39 mg/kg), mg/kg), X, 100 100 mg/kg mg/kg Pb Pb and and Cd, Cd, (19.76 (19.76 mg/kg) mg/kg) and and XII, XII,5 mg/kg 5 mg Pb/kg and Pb 100 and mg/kg 100 mg Cd,/kg (19.75 Cd, mg/kg). (19.75 mg These/kg). resultsThese results are consistent are consistent with the with fact the that fact in that the in treatments the treatments VII, VII,X and X andXII, XII,the thehighest highest concentration concentration of Cdof Cdwere were applied applied (100 (100 mg/kg mg/ kgin water in water for forirrigation). irrigation). The Thelowest lowest Cd Cdcontent content was was determined determined in the in seed.the seed. In the In theseed seed maturation maturation stage, stage, the the highest highest content content can can be be observed observed in in root root and and the the lowest content in in the the seed. seed. The The highest highest concentration concentration of ofCd Cd in inthe the root root was was again again within within the thetreatments treatments VII (35.45VII (35.45 mg/kg). mg/kg). Similar Similar studies studies have have shown shown low low levels levels of ofmetals metals in inthe the seed, seed, as as well well as as a a low translocation factor [22]. Studies of Holm Oak (Quercus ilex L.) exposed to the urban environment showed a significant concentration of Pb and Cd in leaves [37]. The maximum content of Pb, observing the overall distribution in quinoa during the seed formation and maturation, was determined in the root and the lowest was found in the seed. Within the seed formation stage, the highest concentration of Pb in the roots was determined for the treatments IV at 100 mg/kg Pb (Cd: 11.05 mg/kg), and X at 100 mg/kg Pb, (Cd: 11.79 mg/kg). The highest concentration in the seed was determined for the treatment X (0.57 mg/kg) with the highest concentration of Pb applied. Investigations of lead content in 30 varieties of Chinese cabbage (Brassica pekinensis L.), in the experiment, have confirmed that the amount of lead in the soil had a significant effect on the lead concentration in plants [38]. As for seed maturation stage, the highest concentration of Pb was determined in the root for the treatment IV (21.82 mg/kg). Result of this study are consistent with the study of wheat (Triticum aestivum) that were grown on the contaminated soil in the vicinity of the mine, which contain high concentrations of Pb (17.23 mg/kg) and Cd (42 mg/kg), showing high accumulation of these elements in the root of the plants [23]. Similar distributions of both Cd and Pb, were observed also in the wheat with the highest content in the root [30]. According to some authors, the bioaccumulation factor is a compelling indicator of metal accumulation capacity [39]. Transfer of PTEs from soil into the plants is considered to be one of the main indicators of human exposure to contamination [24,40]. Sustainability 2020, 12, 3789 8 of 11

Plants’ ability to uptake and accumulate metals can vary greatly among different species or even within cultivars of the same species. An example of such a study is the research of 32 hybrid rice varieties grown on the contaminated soil. Results of this study indicated that there were significant differences in the accumulation and tolerance for lead and cadmium between all tested hybrids [27]. Also, 14 sunflower hybrids have shown significant differences in the accumulation of Cd and Zn [28]. The study of concentration levels of Cr, Cd and As in a soil-wheat system showed that bioaccumulation of Cd was much higher than other elements [41]. The experiment of the accumulation Pb by mandarin orange trees, irrigated with water containing different concentrations of Pb, showed that accumulation remained highest in plants that received the highest concentration of Pb via irrigation [42]. Investigation of bioaccumulation of PTEs by sorghum and sunflower on contaminated agricultural soil showed that the highest concentration of investigated elements (Cd, Cr, Cu, Ni, Pb) was in roots, while BF roots was the lowest [43]. Different plant species may accumulate lead with the highest concentration in the root [44] and, for this reason, an important step towards the control of health risks is obtaining an understanding of Pb accumulation mechanism, as well as its translocation to the aerial parts of plant. TF’s values of metals in seed formation stage are lower than 1, except for control and treatment II, 5 mg/kg Pb, for cadmium because of the low bioaccumulation in root for these two treatments. These values, TF < 1, indicate a low level of translocation from the roots to the seed. Also, TF’s values of metals in seed maturation stage are lower than 1, showing a deficient transport of metals, from the root to the seed, especially for lead. In this way, the edible parts of the plant are protected from potentially toxic elements contamination. These results indicate that quinoa only uptakes, but does not accumulate cadmium and lead, while the root is a barrier that prevents further translocation of metals to seed. Also, low metal accumulation indicates lower risk for primary consumers [45]. The high values of Cd and Pb in the root of pea plant with very low translocation to the edible parts of the plant were reported [45]. Similar studies of metal accumulation and translocation by miscanthus also reported limited translocation to aerial parts of plant [25,26]. Our results are not consistent with the study [39] that reported high TF values for Cd in wheat. The higher TF were found for Cd and Pb in Plantago major L. [21]. The highest concentration of Pb in beans was determined in shoot with very limited translocation to edible parts of the plant [46]. Metal excluders are defined as crops that can accumulate metals in small quantities in the edible parts, so their products are safe for consumption even when they are grown on the moderately contaminated [47]. This concept is based on the fact that several studies confirmed a significant difference in accumulation and distribution of metals between different varieties of the same culture. On the other hand, the plants that are suitable for phytostabilization accumulate trace elements in the roots and translocation of these elements are limited, which prevents their transfer into the food chain [48].

5. Conclusions The highest concentrations of metals were determined in the roots, for both studied growth stages of quinoa. BAF of Cd and Pb indicate that bioaccumulation was generally medium to high intensity. In addition, TF values were lower than 1, showing very limited translocation from root to seed and making quinoa suitable for Cd and Pb phytostabilization. The results of our study indicated that quinoa adopts potentially toxic metals from substrate with scarce translocation to the plant parts, in particular to the seed. This conclusion paves the way for quinoa as a potential lead and cadmium excluder. The limitation of this study is that they speciation/fractionation of Cd and Pb was not fully determined. Future research should analyzes the safety of quinoa grown in contaminated environments for human consumption. Also, it is suggested to compare the bioaccumulation efficacy of other PTEs from water to quinoa under irrigation with wastewater. Sustainability 2020, 12, 3789 9 of 11

Author Contributions: The manuscript was read, discussed, and approved by all authors. B.Z. and V.R. designed and performed the experiments; I.D. and V.R. analyzed the data and contributed to the writing of the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This research was supported by the Project: “Investigation of the possibility to use contaminated water in growing alternative, healthy cereals” (31006) financed by the Ministry of Education, Science and Technological Development of the Republic of Serbia for the 2011–2017. Conflicts of Interest: The authors declare no conflict of interest.

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