Arsenic accumulation in Brassicaceae seedlings and its effects on growth and plant anatomy

Ecotoxicology and Environmental Safety 124 (2016) 1–9

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Arsenic accumulation in Brassicaceae seedlings and its effects on growth and plant anatomy Larisse de Freitas-Silva, Talita Oliveira de Araújo, Luzimar Campos da Silva n, Juraci Alves de Oliveira, João Marcos de Araujo Universidade Federal de Viçosa, 36570-900 Viçosa, Minas Gerais, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 19 March 2015 Received in revised form 18 September 2015 Accepted 19 September 2015

We wished to evaluate the effects of arsenic on the morphology and anatomy of Brassica oleracea, Raphanus sativus, Brassica juncea, Brassica oleracea var. capitata and Brassica oleracea var. italica. Seeds were subjected to concentrations 0 mM, 250 mM, 350 mM and 450 mM arsenic in the form of sodium arsenate (Na2HAsO4
7H2O) during 12 days. All species accumulated more arsenic in the roots than in the shoots, except for B. oleracea var. capitata. There was no difference of translocation factor between species and treatments. Growth decrease was observed in roots of B. oleracea and R. sativus, and in shoots of R. sativus and B. oleracea var. italica. All species presented anatomical alterations in the roots, such as: cell hypertrophy, protoplast retraction, cellular plasmolysis, and necrotic regions. B. juncea presented collapse and hypertrophy of cells from the leaf blade tissues. Quantitative anatomical analyses performed on the root and leaves of B. oleracea and B. juncea revealed that arsenic interfered on the root vascular cylinder diameter and on height of epidermal cells of the adaxial leaf surface of both species. We concluded that arsenic was absorbed from the culture medium and induced alterations both on root and shoot growth of the seedlings. Retention of arsenic within the root was responsible for major damage in this organ. & 2015 Elsevier Inc. All rights reserved.

Keywords: Brassica oleracea L. Raphanus sativus L. Brassica juncea (L.) Czern. Brassica oleracea L. var. capitata L. Brassica oleracea L. var. italica Plenck Light microscopy

1. Introduction Arsenic is a metalloid widely distributed across the Earth’s crust (Melo et al., 2012). Its availability in arable soils might get increased by the application of herbicides and pesticides in crops, by the disposal of industrial wastes in whose composition it is present, or by gold mining (Ali et al., 2009; Dho et al., 2010; Guala et al., 2010; Zhao et al., 2010). The main route of entry of this pollutant in the food chain is through ingestion of contaminated water, seafood, grains and vegetables, due either to cultivation in polluted soils or to irrigation with water containing excessive amounts of the element (Carbonell-Barrachina et al., 2009; Bhattacharya et al., 2010). Arsenic-contaminated foods contribute with 93% of the total arsenic consume by humans (Bhattacharya et al., 2010). Thus, a health risk issue is posed since arsenic is considered one of the most hazardous substances to humans by the Agency for Toxic Substances and Disease Registry (Liao et al., 2005; ATSDR, 2007; Khan et al., 2009). When exposed to arsenic, sensitive plant species usually present several symptoms, which may indicate the presence of the metalloid. These symptoms include decreased growth, biomass n

Corresponding author. E-mail address: [email protected] (L.C. da Silva).

http://dx.doi.org/10.1016/j.ecoenv.2015.09.028 0147-6513/& 2015 Elsevier Inc. All rights reserved.

and photosynthetic rate; appearance of necrotic regions; accumulation of anthocyanins; leaf senescence (Stoeva and Bineva, 2003; Gisbert et al., 2006; Pigna et al., 2009; Rahman et al., 2007; Panda et al., 2010); and alterations on the antioxidant mechanisms (Singh et al., 2007; Gomes et al., 2012; Ahmad and Gupta, 2013). Several structural alterations on plant cells and tissues have been reported as a consequence of arsenic toxicity, such as: chlorosis, necrosis, wilting, size increase in phenolic idioblasts, alterations on chloroplast structure, root length decrease, and alterations on root epidermal and cortical cells (Li et al., 2006; Singh et al., 2007; Shaibur and Kawai, 2009; Campos et al., 2014). Plant anatomy allied to morphological analyses is important in studies on environmental pollutants as it provides prognostic information on the deleterious effects that they may have on the studied plant species (Sant’Anna-Santos et al., 2006; Sant’Anna-Santos and Azevedo, 2007; Shaibur and Kawai, 2009; Santana et al., 2014). The low cost of these techniques, the reliability of the results and the rapidity with which these results are obtained make such techniques applicable in analyses of the quality of large-scale produced crop species (Shaibur and Kawai, 2009), such as several representatives of the Brassiaceae. Brassicaceae is one of the largest Angiosperm families, with c.a. 380 genera and 3700 species. The family includes species of great economic importance, e.g. in the Brassica genus. This genus covers

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species that have been cultivated and consumed worldwide for centuries (Katz, 2003; Pedras and Yaya, 2010). Several Brassicaceae species are classified as tolerant to metals (Wu et al., 2004; Shiyab et al., 2009), including arsenic (Irtelli and Navari-Izzo, 2008; Karimi et al., 2009; Srivastava et al., 2009). However, plant sensitivity to pollutants is related not only to the applied dose and time of exposition, but also to the plant developmental stage. Organisms at an initial developmental stage are more susceptible to the toxic effects, as their defense mechanisms are not yet fully developed (Chaves et al., 2002; Liu et al., 2005). Arsenic can be associated to sulfur in minerals, such as Arsenopyrite (FeAsS) (Daus et al., 2005; Zhao et al., 2010), which is one of the main minerals associated to gold. This renders gold mining the main anthropic activity responsible for releasing arsenic in the environment (Borba et al., 2000). In Brazil, the Iron Quadrangle, located in Minas Gerais state, constitutes one of the natural sources of arsenic contamination, due to intense mining of gold and other metals (Borba et al., 2000). The cultivation of plant species in arsenic-contaminated soils can lead to the absorption and accumulation of this element in the edible plant parts, thus enabling its entrance in the food chain (Bhattacharya et al., 2010). In view of the wide human consume of representatives of the Brassicaceae and of the ease of their cultivation in domestic areas, we wished to test the following hypotheses: Brassicaceae seedlings, when cultivated in the presence of arsenic, even at initial developmental conditions, are capable of absorbing and accumulating it in their organs; and the metalloid is capable of provoking morphoanatomical alterations in the seedlings. Thus, this study aimed at evaluating arsenic absorption in Brassicaceae seedlings, by determining its content in roots and shoots, and assessing its phytotoxicity, through anatomical and visual analyses on leaves and roots.

2. Material and methods 2.1. Plant material and cultivation conditions Seeds of the species Brassica oleracea L. (kale), Raphanus sativus L. (radish), Brassica juncea (L.) Czern. (mustard), Brassica oleracea L. var. capitata L. (cabbage) and Brassica oleracea L. var. italica Plenck (broccoli), all from the Brassicaceae family, were obtained in local commerce, superficially disinfected with 5% sodium hypochlorite during 5 min and washed with distilled water. Three seeds were germinated in test-tubes with 30 cm length containing 28 mL of semisolid culture medium (Supplementary material 1), which was composed of 10 mL of macronutrient solution, 1 mL of micronutrient solution and 1 mL of ferric chloride (Heller, 1953), in the control treatment; arsenic in the form of sodium arsenate (Na2HAsO4
7H2O) was added to the medium at concentrations 250 mM, 350 mM and 450 mM. Culture medium pH was adjusted to 7.0 and the test-tubes were kept for 12 days in a growth room with controlled temperature (25 72 °C), luminosity (230 mE s  1 m  2) and photoperiod (16 h). This time period was enough for seed germination and seedling initial development to occur. 2.2. Determination of arsenic content and determination of translocation factor For quantification of arsenic content, roots and shoots were previously separated and dried in a forced ventilation oven at 65 °C until constant weight. Dry matter samples were predigested with a mixture of nitric acid (3 mL) and perchloric acid (1 mL) for 24 h at room temperature (Tedesco et al., 1995). Then, a heating program was performed, keeping temperature at 50 °C for 30 min,

100 °C for 30 min, and 150 °C until complete acid digestion of the organic matter. Arsenic content in the resulting solution was determined by atomic absorption spectrophotometry (model Spectr AA220, Varian Medical Systems, Inc., USA) and the results were expressed in mg g  1 dry matter. After quantification of arsenic accumulated in shoots and roots, translocation factor (TF) were calculated according to the methodology proposed by Bao et al. (2009). TF is defined as the ratio between arsenic concentration in shoots and arsenic concentration in roots. 2.3. Visual characterization and growth analysis For the evaluation of visual alterations, seedlings were observed on a daily basis. After the experimental period, they were removed from the culture medium and the roots were washed with 1% nitric acid, then with distilled water, and were ultimately photographed with a digital camera (model Cyber-Shot DSCW310, Sony Corporation, Japan). In order to assess the influence of arsenic on growth, at the end of the experimental period the length (cm) of the main root and of the shoot were measured in seedlings from each test tube. The shoot was regarded as the region between the stem base and the eldest leaf node. 2.4. Structural characterization in light microscopy Samples of leaf median and marginal regions and roots were collected from seedlings of all species, subjected to all treatments, and processed for anatomical analysis. The samples were fixed in a solution of 4% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer pH 7.0 (Karnovsky, 1965, modified), dehydrated in an ethyl series and embedded in methacrylate (Historesin, Leica Instruments Heidelberg, Germany). Cross sections 5 mm thick were obtained with a rotary automatic microtome (model RM2255, Leica Microsystems Inc., Deerfield, Illinois, USA) and stained with 0.05% toluidine blue ph 4.7 (O’Brian and McCully, 1981). Glass slides were mounted in Eukitt (Eukitt Mounting Medium, Sigma-Aldrich Corporation, USA) and photographed in a light microscope (model AX70RF, Olympus Optical, Tokyo, Japan) equipped with a U-Photo system and a coupled digital camera (model Spot Insightcolour 3.2.0, Diagnostic Instruments Inc., New York, USA), at the Laboratory of Plant Anatomy of the Department of Plant Biology, UFV. 2.5. Micromorphometrical analyses in light microscopy For the micromorphometrical analyses, root and leaf fragments of B. oleracea and B. juncea were collected, processed and photographed as described above. Previous studies showed that B. juncea individuals are capable of tolerating high arsenic contents in their tissues. We wished to assess whether the morphometrical results found in B. juncea samples would be similar to those in B. oleracea ones. The latter species has not yet been given any report on its tolerance or sensitivity to arsenic, yet it may be the most consumed species as human food among all species we studied. On the leaves, measurements were taken of the leaf blade and mesophyll thickness, and of the height of epidermal cells of the adaxial and abaxial leaf surfaces. On the root, measurements were taken of the vascular cylinder diameter, cortex and epidermis thickness, and the total diameter of the organ. The measuring process was carried out with image analysis software AnatiQuanti (Aguiar et al., 2007) and the results were expressed in micrometers (mm). For each evaluation, four repetitions per treatment were used and three glass slides were made out of each repetition. In each slide, three sections were randomly chosen and for each parameter three measurements were performed, totalizing 108

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measurements on each parameter per treatment. 2.6. Statistical analyses Statistical analyses were performed based on a completely randomized design in a 5  4 factorial scheme, with five species, four treatments and four repetitions. The experimental plot was represented by one test-tube containing three seedlings. A total of 240 seeds were germinated; 3 seeds per test tube. Each seedling constituted a subrepetition, and each tube constituted an experimental unit (repetition). The values presented are means of each repetition, which, in turn, are means of 3 subrepetitions. Data was submitted to analysis of variance (ANOVA) using software SAEG – Sistema de Análises Estatísticas e Genéticas (Statistical and Genetic Analysis System) of UFV (Euclydes, 2004) and treatment means were compared by Tukey’s test at 5% probability.

Table 1 Arsenic content (μg g  1) in root and shoot dry matter and translocation factor (TF) of Brassica oleracea, Raphanus sativus, Brassica juncea, Brassica oleraceae var. capitata and Brassica oleracea var. italica after 12 days of treatment. Species

B. oleracea

R. sativus

B. juncea

B. oleracea var. capitata

3. Results 3.1. Arsenic content in dry matter and determination of translocation factor The arsenic content in the seedlings showed significant difference on the element concentration as a function of the applied doses, the analyzed species and the interaction between these factors, both in the root and in the shoot. All studied species presented higher arsenic content in the roots, except for B. oleracea var. capitata at concentrations 250 mM and 450 mM arsenic (Table 1). Among treatments, there was a significant increase in the arsenic content from concentration 250 mM on when compared to the control treatment, in the roots of B. oleracea and B. oleracea var. italica and in the shoot of R. sativus. Significant increase was observed at concentration 450 mM in the root of R. sativus and in the shoot of B. oleracea and B. oleracea var. capitata. Among species, B. oleracea var. capitata presented lower concentrations of the metalloid in the root than B. juncea and B. oleracea at dose 350 mM, and lower values than the other studied species at concentration 450 mM. B. juncea and B. oleracea var. italica presented lower arsenic contents in the shoots than the other studied species at treatment 450 mM arsenic (Table 1). For the determination of translocation factor, there was no difference between species and treatments.

B. oleracea var. italica

As content (μg g  1dry matter)

As (μM) TF

0 250 350 450 0 250 350 450 0 250 350 450 0 250 350 450 0 250 350 450

Analysis of variance Arsenic (As) Species (Sp) As X Sp

Root

Shoot

0.54 7 0.11 0.217 0.03 0.28 7 0.10 0.29 7 0.01 0.40 7 0.06 0.767 0.19 0.577 0.04 0.32 7 0.06 0.117 0.03 0.22 7 0.02 0.157 0.07 0.117 0.01 0.647 0.20 1.167 0.05 1.20 7 0.49 1.26 7 0.56 0.717 0.06 0.187 0.02 0.197 0.02 0.147 0.01

6.43 7 1.82Bab 17.667 1.30Aab 16.40 7 2.09Aab 24.867 0.91Aab 3.85 7 0.56Bb 4.687 0.14Bb 6.737 0.81ABbc 15.28 7 3.49Ab 7.45 7 3.15Bab 12.96 7 1.67Bab 20.53 7 7.45ABab 25.357 2.20ABab 4.60 7 0.80Ab 3.357 0.53Ab 4.357 1.84Ac 5.677 2.84Ac 5.167 0.17Bb 20.36 7 1.20Aab 14.60 7 0.23Aabc 17.08 7 1.58Aab

3.05 70.11Bab 3.64 70.37Ba 4.2471.01Bab 7.20 70.12Aab 1.49 70.12Bb 3.51 70.79Aa 3.85 70.56Aab 4.45 70.10Ab 1.58 70.29Ab 2.75 70.05Aa 1.82 70.25Ab 2.69 70.54Ac 2.59 70.27Bab 3.86 70.49Ba 3.45 70.49Bab 5.92 70.34Aab 3.65 70.25Aab 3.64 70.46Aa 2.71 70.35Aab 2.34 70.24 Ac

n.s n.s n.s

* * *

* * *

Means followed by the same letter do not differ by Tukey’s test at 5% probability. Upper case letters compare arsenic treatments in the species, when there is interaction between these two factors; lower case letters compare species in the arsenic treatments. S.E. (n¼ 4) indicate the standard error. (*) significant differences at 5% probability. n.s. nonsignificant.

Table 2 Length of the shoot and main root (cm) in seedlings of species Brassica oleracea, Raphanus sativus, Brassica juncea, Brassica oleracea var. capitata and Brassica oleracea var. italica after 12 days of treatment. Species

As (μM)

Root (cm)

Shoot (cm)

B. oleracea

0 250 350 450 0 250 350 450 0 250 350 450 0 250 350 450 0 250 350 450

7.55 7 0.87Aabc 4.717 0.81Bab 4.53 7 1.33Babc 2.90 7 0.49Ba 9.687 0.28Aab 6.89 7 0.30BCab 5.707 1.04BCabc 3.87 7 0.46Ca 2.977 0.73Ad 3.247 0.07Ab 2.90 7 0.49Abc 2.707 0.06Aa 4.167 1.01Acd 2.977 0.29Ab 3.677 0.43Aabc 1.80 7 0.32Aa 6.02 7 0.51ABbcd 3.217 0.34ABb 1.08 7 0.27Bc 3.197 0.70Ba

2.40 7 0.30Ab 1.62 7 0.24Ab 1.767 0.33Aab 1.577 0.27Abc 5.62 7 0.46ABa 4.497 0.79ABa 3.137 0.25Bab 4.43 7 0.14ABab 1.62 7 0.38Ab 1.117 0.15Ab 2.29 7 0.33Aab 1.22 7 0.25Ac 2.52 7 0.15Ab 1.90 7 0.311Ab 2.677 0.14Aab 2.83 7 0.11Aabc 2.88 7 0.37ABb 1.62 7 0.22ABb 1.007 0.08Bb 1.58 7 0.11ABbc

* * *

* * *

R. sativus

3.2. Visual characterization and growth analysis Leaves of the arsenic-exposed seedlings were fully expanded presenting typical green color; no chlorosis or necrosis was visually observed. Roots visually presented an increased number of lateral ramifications and were intact, with a rigid texture and light color, as represented by B. oleracea (Supplementary material 2). Seedling growth showed a significant difference considering arsenic concentration as a function of the applied doses, the analyzed species and the interaction between these factors, in both root and shoot. From all analyzed species, B. oleracea and R. sativus presented root growth decrease from the control treatment on. The lowest values were found at concentration 450 mM, representing a reduction of 40% and 39% in relation to the control treatment, respectively. The highest root growth decrease in relation to the control treatment was observed in B. oleracea at the highest arsenic concentration. In the shoot, R. sativus and B. oleracea var. italica presented growth decrease also from the control treatment on. The lowest values were found at concentration 350 mM, with a reduction of 55% and 37% in relation to the control

3

B. juncea

B. oleracea var. capitata

B. oleracea var. italica

Analysis of variance Arsenic (As) Species (Sp) As X Sp

Means followed by the same letter do not differ by Tukey’s test at 5% probability. Upper case letters compare arsenic treatments in the species, when there is interaction between these two factors; lower case letters compare species in the arsenic treatments. S.E. (n¼ 4) indicate the standard error. (*) significant differences at 5% probability.

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Fig. 1. Leaf cross sections of Brassica juncea observed under light microscopy. (A) Control. (B–E) 450 μM arsenic. Decrease in leaf blade thickness (B). Hypertrophy of epidermal cells of the midrib region (C). Mesophyll cell collapse (D). Hypertrophy of epidermal cells at leaflet margin (E). S: stomata, Ead: epidermis of the leaf adaxial surface, Eab: epidermis of the leaf abaxial surface, Me: mesophyll, Vb: Vascular bundle, Thick arrow: decrease in leaf blade thickness, Asterisk: hypertrophied epidermal cells, Thin arrow: mesophyll cell collapse.

treatment, respectively. The highest shoot growth decrease in relation to the control treatment was observed in R. sativus at concentration 350 mM (Table 2). Among species, R. sativus presented the highest growth values in the shoot at the control treatment and at concentration 250 mM, when compared with the other species; and at concentration 450 mM, when compared with B. juncea (Table 2). 3.3. Structural characterization in light microscopy The studied plant species present similar anatomical features. The leaves possess a uniseriate epidermis and are amphistomatic. Mesophyll is homogeneous with eight to ten cell layers (Fig. 1a). Roots present a typical eudicotyledonous anatomy, with well-defined epidermis, cortex and vascular cylinder (Fig. 2a). In our work, B. juncea was the only species that presented anatomical damage on the leaves, and only when exposed to 450 mM arsenic. We observed regions with mesophyll cell collapse and the consequent reduction of leaf blade thickness (Fig. 1b). Some epidermal cells were hypertrophied (Fig. 1c–e). Roots of all species presented anatomical alterations resulting from the arsenic exposition. Damage was similar and recurrent among species and also among treatments, so that at the highest applied arsenic dose injuries were more evident: hypertrophy of cortical cells in B. oleracea (Fig. 2b) and of epidermal cells in B. oleracea var. capitata (Fig. 2c); protoplast retraction in epidermal and cortical cells of B. juncea (Fig. 2d) and in cortical cells of B. oleracea (Fig. 2e) and B. oleracea var. italica (Fig. 2f); and collapse of epidermal and cortical cells in R. sativus (Fig. 2g) and of endodermal cells in B. juncea (Fig. 2h). 3.4. Micromorphometrical analyses in light microscopy Significant difference between B. oleracea and B. juncea was observed for all measurements performed on the leaves. B. juncea presented higher height values for the epidermal cells of both leaf surfaces, while B. oleracea presented higher height values for mesophyll cells, which contributed to an increased leaf blade

thickness in this species. Significant difference was found in the interaction between arsenic doses and species for the measurements performed on the epidermis of the leaf adaxial surface at treatments 0 mM, 250 mM and 450 mM (Table 3). In the roots, significant difference between B. oleracea and B. juncea was observed for the measurements performed on the epidermis, the former species presenting higher cell height values. There was difference among treatments in all evaluated parameters. In the interaction between treatments and species, significant difference was observed only for the measurements performed in the vascular cylinder. In B. oleracea, there was a significant decrease in the vascular cylinder diameter between the control and the other treatments, while in B. juncea there was an increase in the vascular cylinder diameter at the highest arsenic dose (Table 4).

4. Discussion Plants are able to respond to environmental stresses, and pollution is a major variable that can affect plant development. Damage occurring in plants can be used to monitor the level of pollutants in the environment and the inflicted stress may influence the considered species, which can respond to it with changes in biomass, growth pattern, morphology, anatomy and physiology (Li et al., 2006; Balestri et al., 2014). Higher arsenic contents were found at the highest doses, except in the shoots of B. oleracea var. italica (broccoli) and B. juncea (mustard) and roots of B. oleracea var. capitata (cabbage) and B. juncea. Higher arsenic content with increasing amounts of the metalloid in solution has already been reported to B. juncea (Srivastava et al., 2009). Roots of the studied species, with the exception of B. oleracea var. capitata, accumulated higher arsenic amounts when compared to the shoots. Lower arsenic amounts in the shoots suggest that little translocation of the metalloid to the shoots occurred, which justifies the absence of morphological and anatomical damage on the leaves of the studied species, except for B. juncea. The absence

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Fig. 2. Anatomical alterations on radicles of Brassica oleracea (A, B and E), Brassica oleracea var. capitata (C), Brassica juncea (D and H), Brassica oleracea var. italica (F) and Raphanus sativus (G). Cross sections observed under light microscopy. Control (A) and subjected to 450 mM arsenic (B–H). Hypertrophy of cortical cells (B). Hypertrophy of epidermal cell (C). Protoplast retraction on epidermal and cortical cells (D). Protoplast retraction on cortical cells (E and F). Collapse of epidermal and cortical cells (G). Collapse of endodermal cells (H). Ep: epidermis, Cx: cortex, En: endodermis, Pr: pericycle, Xy: xylem, Ph: phloem, Rh: root hair, Asterisk: cell hypertrophy, Arrow: protoplast retraction, Square: cell collapse.

of anatomical damage in Vicia faba exposed to metal toxicity on mine tailing substrate, can be justified by the retention of metal particles on the surface of the cell walls, and that chelation a defense strategy to minimize the input of the pollutant in the organ (Probst et al., 2009). In non-accumulator species arsenic is mainly accumulated in the roots (Stoeva and Bineva, 2003; Shaibur et al., 2006), and thus affects the anatomy and physiology of vegetative organs in different ways (Singh et al., 2007; Rahman et al., 2007). Metal accumulation in the roots has also been found by other authors (Wei et al., 2008; Gupta and Chakrabarti, 2013). Greater arsenic retention in the roots is one of the defense mechanisms of plants (Meharg and Macnair, 1991). This fact might be related to the

development of strategies capable of complexing arsenic in the root as molecules rich in –SH groups, which thus prevents displacement of the metalloid to the shoots (Sing and Agrawal, 2008), since its excess is sequestered in the intracellular medium and compartmentalized in regions such as vacuoles and the cell wall (Memon et al., 2001). Higher arsenic amounts in the shoots than in the roots of B. oleracea var. capitata might be justified by a less efficient chelating system in the roots, which did not happen with the other species. It is possible that the antioxidant system of B. oleracea var. capitata neutralized the damage by the metal in the roots in a less efficient way than the one of the other species. That which was neither neutralized nor compartmentalized might have been translocated

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Table 3 Leaf micromorphometrical analysis (mm) of Brassica oleracea and Brassica juncea after 12 days of treatment. Species

As (μM)

Epidermis of the adaxial surface

Epidermis of the abaxial surface

Mesophyll

Leaf blade

B. oleracea

0 250 350 450 0 250 350 450

28.577 1.38ABb 22.137 1.16Bb 28.52 7 1.76ABa 23.667 1.25ABb 33.967 2.0Aa 34.96 7 0.73Aa 31.90 7 0.8Aa 34.88 7 1.55Aa

36.08 7 1.87 26.517 1.30 31.53 7 1.24 27.197 0.98 58.42 7 6.5 65.92 7 17.63 40.777 2.96 42.317 1.58

318.277 17.73 295.357 13.16 362.217 30.28 343.187 24.58 282.017 13.6 251.90 7 15.1 267.65 7 13.36 228.23 7 24.0

366.98 7 17.74 335.95 7 12.39 411.977 33.81 387.46 7 26.42 350.227 17.34 315.30 7 16.13 327.617 15.59 293.617 24.43

n.s * *

n.s * n.s

n.s * n.s

n.s * n.s

B. juncea

Analysis of variance Arsenic (As) Species (Sp) As x Sp

Means followed by the same letter do not differ by Tukey’s test at 5% probability. Upper case letters compare arsenic treatments in the species, when there is interaction between these two factors; lower case letters compare species in the arsenic treatments. S.E. (n¼4) indicate the standard error. (*) significant differences at 5% probability. n. s. nonsignificant.

to the shoots. Interestingly, the arsenic contents found in the shoots of B. oleracea var. capitata were not sufficient to cause anatomical alterations in the leaves, which suggests that the detoxification mechanisms developed by the shoots of this species were efficient, having prevented the biochemical disorders produced to be reflected on its leaf anatomy. Arsenic contents were found in root dry matter of the control treatment of all species. We know that some reagents possess arsenic as a trace element in their constitution. In this experiment, the agarose used for mounting the culture medium has arsenic in the composition. In addition, many pesticides that preserve the quality of seeds for sale have arsenic. Probably, the arsenic values found in our controls result from this factor. The leaves of B. juncea presented hypertrophy of epidermal cells and collapse of mesophyll cells at concentration 450 mM arsenic. Adult plants of B. juncea are classified as hyperaccumulators (Gupta et al., 2009). However, organisms at an initial developmental stage are more susceptible to the toxic effects of pollutants (Liu et al., 2005). In our work, we found that in the early stages of development, B. juncea presents biochemical and physiological disorders that ultimately reflected on the species leaf anatomy, even with low amounts of the pollutant translocate in the shoots. Our study demonstrated that increasing arsenic doses promoted root growth decrease in B. oleracea (kale) and R. sativus (radish). Furthermore, a visual increase in the number of lateral ramifications in the roots of all species was also observed, which suggests that arsenic is capable of provoking mitotic alterations in the roots. Arsenic stress promotes inhibition of cell elongation and alterations in the division patterns of differentiating cells (Shaibur

and Kawai, 2009). An increased number of lateral ramifications has also been observed in studies on cadmium effects (Balestri et al., 2014). The shoots of R. sativus and B. oleracea var. italica presented growth decrease with increasing arsenic doses. Growth decrease in response to arsenic has already been observed in several plant species (Hartley-Whitaker et al., 2001; Meharg and Hartley-Whitaker, 2002; Kile et al., 2007; Shaibur and Kawai, 2009; Shri et al., 2009), and in some cases even led to plant death. When it is present in the substrate, arsenate, the main form of available arsenic, can replace phosphate in energy-generating metabolic processes such as respiration (Panda et al., 2010; Smith et al., 2008). Thus, the growth reduction observed might be related to decreased photosynthetic and respiratory rates (Mascher et al., 2002), which may have interfered in biomass production and consequently in plant growth. Probst et al. (2009) also reported the decrease of growth in edible species grown on soil contaminated by metals such as a defense strategy against the absorption of pollutants studied. Each species presented differential growth at the same pollutant dose. Some of them showed no arsenic influence on growth at all, like in the root system of B. juncea and B. oleracea var. capitata and in the shoots of B. oleracea, B. juncea and B. oleracea var. capitata. Arsenic tolerance is also related to genetic factors, through which each species has developed preferential adaptive strategies in order to tolerate the metalloid in its cells (Meharg and HartleyWhitaker, 2002; Zhao et al., 2010). While in initial developmental stages, B. oleracea, R. sativus, B. oleracea var. capitata, B. oleracea var. italica and B. juncea can be

Table 4 Root micromorphometrical analysis (mm) of Brassica oleracea and Brassica juncea after cultivation in medium with different arsenic concentrations. Species

As (μM)

Epidermis

Cortex

Vascular cylinder

Total diameter

B. oleracea

0 250 350 450 0 250 350 450

23.497 0.79 22.32 7 1.11 23.147 0.98 26.177 0.83 19.60 7 1.03 18.38 7 0.82 19.56 7 0.51 19.647 0.57

135.007 11.11 102.08 7 3.77 92.84 7 2.49 117.517 0.71 110.69 7 8.86 98.417 5.55 104.887 5.6 114.62 7 9.53

93.23 7 6.19Aa 66.627 5.25Ba 58.357 2.75Ba 69.677 1.13Bb 68.66 7 4.13Bb 60.29 7 3.55Ba 64.64 7 5.44Ba 85.727 1.56Aa

317.14 723.90 271.07711.76 244.95 77.42 292.66 75.92 269.09 72.17 244.36 713.01 253.80 716.40 287.61 713.87

* * n.s.

* n.s n.s.

* n.s. *

* n.s. n.s.

B. juncea

Analysis of variance Arsenic (As) Species (Sp) As x Sp

Means followed by the same letter do not differ by Tukey’s test at 5% probability. Upper case letters compare arsenic treatments in the species, when there is interaction between these two factors; lower case letters compare species in the arsenic treatments. S.E. (n¼4) indicate the standard error. (*) significant differences at 5% probability. n. s. nonsignificant.

L. de Freitas-Silva et al. / Ecotoxicology and Environmental Safety 124 (2016) 1–9

classified as arsenic-sensitive species. According to Sridhar et al. (2011) and De Temmerman et al. (2004), species that are sensitive to pollutants translocate low amounts of them to the shoots when compared to hyperaccumulator ones. In addition to the low arsenic amounts that were detected in the shoots, the studied species presented anatomical alterations and growth decrease. After a short exposition period to arsenic B. oleracea, R. sativus, B. oleracea var. capitata, B. oleracea var. italica and B. juncea presented toxicity symptoms such as growth decrease, increase in number of root lateral ramifications and anatomical alterations both in leaves and roots. Arsenic-sensitive plant species often undergo considerable stress when exposed to the metalloid, presenting symptoms like root growth decrease, appearance of necrotic regions, biomass depletion and leaf senescence. When manifested expressively, these symptoms might lead to inhibition of plant development and, in some cases, can even cause plant death (Gisbert et al., 2006; Gupta et al., 2009; Pigna et al., 2009; Panda et al., 2010). An apparent increase in the number of root lateral ramifications was the only visible morphological alteration, which reinforces the importance of anatomical studies for the precocious diagnose of the damage caused by arsenic or other pollutants (Sant’Anna-Santos et al., 2007; Tuffi-Santos et al., 2009; Silva et al., 2005; Siqueira-Silva et al., 2012; Santana et al., 2014). No anatomical damage was found in the vascular cylinder of any of the studied species, although damage could be observed in tissues from other root regions, including the endodermis of B. juncea. Roots possess barriers, such as the endodermis, that may limit pollutant absorption, thus minimizing the damage it may cause. Casparian strips may function as a barrier against metals (Lux et al., 2011; Gupta and Chakrabarti, 2013). Inan et al. (2004) reported the development of an extra endodermis in roots of Thellungiella halophila under salt stress. The endodermis might have been a functional barrier that prevented the pollutant from reaching the vascular cylinder, therefore protecting it from the deleterious effects of arsenic. The decreased root growth in the studied species might have promoted a lower water and nutrient uptake by the plants. Moreover, in situations of low energy production, water uptake through the root may be compromised (Alberts et al., 2008). Phosphate is easily substituted by arsenate in several biochemical processes, which can compromise innumerous cellular functions, including the generation of energy by the ATP complex, which becomes unstable in the form ADP-arsenic (Panda et al., 2010). This fact might have caused drought symptoms, which justifies the plasmolyzed aspect of the endodermal cells of B. oleracea and B. juncea, the cortical cells of B. oleracea, B. oleracea var. italica and R. sativus, and of the epidermal cells of R sativus. Anatomical alterations like protoplast retraction found in roots of B. juncea, B. oleracea and B. oleracea var. italica may be related to an increased lipid peroxidation (Mascher et al., 2002; Campos et al., 2014). The first plant response to arsenic exposition is the increased production of reactive oxygen species, which, when in excess, cause oxidative stress. This might alter the structure and permeability of cell membranes and consequently cause breakdown and electrolyte leakage (Khan et al., 2009; Singh et al., 2009; Yadav, 2010; Rai et al., 2011). Electrolyte leakage causes a decrease in cytoplasmic content, which can anatomically be identified as protoplast retraction. Future studies assessing the integrity of cell membranes of these species when exposed to arsenic should be performed in order to confirm this hypothesis. Other alterations such as mesophyll derangement, and cell hypertrophy and collapse, all of which were found in this work, were caused by the homeostatic disbalance provoked by arsenicinduced cellular stress and have also been reported by other authors in works not only with this metalloid but also with metals

7

(Rodríguez-Serrano et al., 2006; Vitória et al., 2006; Singh et al., 2007). Anatomical alterations caused tissue derangement, which was responsible for the alterations in the micromorphometrical parameters found in the epidermis of the leaf adaxial surface and in the root vascular cylinder of the two analyzed species. Micromorphometrical alterations can be justified not only by anatomical changes, but they can also be caused directly by arsenic accumulation within the measured tissues, as already described by other authors in studies on heavy metal compartmentalization (Cosio et al., 2005; McNear et al., 2005). The preferential sites of arsenic accumulations in these species need to be elucidated in further studies. Structural alterations on the leaves and roots of the arsenictreated plantlets indicate that the metalloid at the tested doses may also be capable of causing alterations on biochemical and physiological parameters of the studied species, at an initial developmental stage. This reinforces the need for further studies that might also elucidate these parameters.

5. Conclusions Arsenic induced alterations on root and shoot growth of the seedlings. The presence of mild visual symptoms such as growth decrease in organs of some species and the apparent increase in number of lateral ramifications on the root, and the anatomical alterations visualized endorse the need for prognostic studies with environmental pollutants. Arsenic was responsible for major damage in the roots, which suggests that these species, at an initial developmental stage, are sensitive to this metalloid. The hypothesis that arsenic can be absorbed by these species was confirmed in our study. The hypothesis that the metalloid is capable of causing morphoanatomical alterations was partially confirmed, since B. juncea was the only species that presented anatomical damage on the leaf.

Acknowledgments The authors thank: Minas Gerais State Foundation for Research Support (FAPEMIG), Brazil for financial support; Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, for the approval of a PNADB Project (AUX-PE-PNADB 1000/2010) (National Program for Botany Support and Development); National Council for Scientific and Technological Development (CNPq), Brazil , for the Research Productivity Scholarship granted to L.C. Silva (309170/2012-5); and Minas Gerais State Secretariat for Science, Technology and Higher Education (SECTES), Brazil and Project Floresta Escola, for funding the study.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.09. 028.

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