1 Introduction

Environmental pollution by heavy metals underwent a marked increase in the last decades due to their continuous release and accumulation in the terrestrial and aquatic ecosystems. Such metals can accumulate in the plants growing on contaminated soils; they then enter the food chain through consumption of plant material and finally affect the health of animal and human systems. The heavy metals are toxic even at low doses because they are not biodegradable (Migid et al. 2007). Arsenic, cadmium, cobalt, chromium, copper, mercury, manganese, nickel, lead, tin, and thallium are considered by the European Union to be of the highest concern (Santos and Rodriguez-Gomez 2012).

Chromium, widely present in environment and in various products or compounds with which people come in contact (photographic films, leather in shoes, toner in photocopiers, cosmetics, vitamins), became a serious environmental pollutant. It is one of the most abundant elements in the Earth’s crust (100–300 μg g−1) (Cervantes et al. 2001; Castro et al. 2007), but the principal causes of the increasing chromium pollution are the specific activities developed in certain industrial and manufacturing sectors (metallurgy, electroplating, production of chromium-pigment-based paints and of fungicides, leather tanning, wood preservation, chemical industry, paper production) in which chromium compounds are used (Cervantes et al. 2001; Ali et al. 2004; Ozdener et al. 2011).

In normal soils, chromium ranges from 5 to 3,000 μg g−1 (Skeffington et al. 1976) or 4,000 μg g−1 (Dayan and Paine 2001), but extremely contaminated surface soils were found in proximity of chromium smelter heaps where the metal concentration exceeded 10,000 μg g−1 (Kabata-Pendias and Pendias 2001). Its presence in agricultural soils can be attributed to the use of organic wastes as fertilizers and of waste waters for irrigation or to the high chromium amount included in some phosphate fertilizers (Kabata-Pendias and Pendias 2001; Oliveira 2012). In Romania, the chromium amount in podzols and sandy soils is 12–86 μg g−1 dry weight, in loamy and clay soils is 19–73 μg g−1 dry weight, whereas kastanozems and brown soils contain 15–67 μg g−1 dry weight (Rauta et al. 1987). As concerns the atmospheric heavy metal deposition, Romania had the highest concentration of cadmium, chromium, and zinc among eastern European countries which participate in biomonitoring studies by moss surveys (Blum 2007). Despite the fact that the exploitation of the rich sources of heavy metals has been reduced or abandoned, the level of these elements in Romanian soils is still high and the risk for population health via consumption of the vegetables grown in old mining areas is proven by several studies (Harmanescu et al. 2011).

A number of international guidelines establish the maximum accepted levels for heavy metals in soils and agricultural purpose waters. For example, the regulatory limits on heavy metals applied to soils stipulate a maximum concentration of chromium in sludge of 3,000 mg kg−1 (USDA-NRCS 2000) but, despite the specific measures carried out in many countries, it seems impossible to completely stop the environmental chromium accumulation which in excess affects the living systems by inducing the chromosome damage in plants and increasing the carcinogenic risk in humans (Oliveira 2012).

Chromium is an essential element for humans and animals, but not for plants and microorganisms (Cervantes et al. 2001; Singh et al. 2013) although it was proved that at low concentrations (0.05–1.00 mg l−1), this heavy metal promoted plant growth (Zou et al. 2006; Oliveira 2012). Although chromium exists in several oxidation states, the most stable and common are the trivalent and hexavalent forms which have very different chemical and hence biological properties (Dayan and Paine 2001). The hexavalent chromium is several times more toxic than the less mobile trivalent form (Cervantes et al. 2001; Jamal et al. 2006; Pohren et al. 2013). The presence of hexavalent chromium over safe levels in edible plants represents a potential hazard to animals and humans (Oliveira 2012), existing sufficient evidence in humans which allowed its introduction in the group I of carcinogens (IARC 1990). Concentrations of hexavalent chromium above 30 mg kg−1 and of trivalent chromium above 10,000 mg kg−1 may cause adverse health effects in humans (USEPA 2006).

Chromium was genotoxic in some prokaryotes (Petrilli and De Flora 1977), lower eukaryotes such as fission yeast (Papp et al. 2011), insects (Michailova et al. 2001), fish species (Yadav and Trivedi 2006; Normann et al. 2008; Velma and Tchounwou 2010), rodents (Seoane and Dulout 1999; Balansky et al. 2000), and humans, where especially hexavalent chromium compounds have mutagenic and carcinogenic properties (Benova et al. 2010; Maeng et al. 2004; Patlolla et al. 2009; Halasova et al. 2012). There are also many studies on negative effects determined by chromium in plant metabolism (Cervantes et al. 2001; Ali et al. 2004; Shanker et al. 2005; Jamal et al. 2006). The stress induced by hexavalent chromium in plants such as rice and maize is analyzed inclusively at transcriptomic, proteomic, and metabolomic levels (Dubey et al. 2010; Wang et al. 2013) but, unlike other heavy metals, relatively few information is available on chromium-induced effects on plant genetic material (Villalobos-Pietrini et al. 1986; Rodriguez-Gomez 2011; Srivastava and Jain 2011; Mishra et al. 2012). The reported range of phytotoxic concentrations is variable: 18–24 μg g−1 dry weight in tobacco, 4–8 μg g−1 dry weight in corn, 10 μg g−1 dry weight in barley, 10–100 μg g−1 dry weight in rice, and 1–2 μg g−1 dry weight in some sensitive plants (Kabata-Pendias and Pendias 2001). According to Akinci and Akinci (2010), the critical level of chromium in soil solutions ranges between 1 and 5 μg g−1 dry weight for many plants, whereas Sinha et al. (2005) noted a larger interval of chromium concentrations before appearance of toxicity symptoms in plant tissues, depending on species, from about 5 μg g−1 dry weight (barley, corn, oats, citrus) to 175 μg g−1 dry weight (tobacco).

Sometimes, even at values significantly lower than the maximum levels accepted by the legislation of some countries, chromium induced genotoxic effects in several plant and animal systems (Depault et al. 2006; Matsumoto et al. 2006; Yadav and Trivedi 2006; El-Yamani et al. 2011). Even trivalent chromium, although less toxic, can produce serious injuries to living tissues—in some cases it has more toxic effects than hexavalent chromium, but at higher concentrations than this (Cervantes et al. 2001; Gardea-Torresdey et al. 2005). It is constitutive part in some nutritional supplements for humans and animals, but several researchers have recently raised the question of the long-term use risk of these trivalent chromium-containing supplements because it was proved that they exert genotoxic action under certain conditions (Eastmond et al. 2008).

In view of these, it is necessary to extend the evaluation of chromium genotoxicity on a larger spectrum of plant species. In this study, barley (Hordeum vulgare L.) was selected as experimental biological material not only because of its great economical, edible, medicinal, and pharmacological importance, but also because it has a chromosome set constituted by a small number of large chromosomes (2n = 14) suitable for the analysis of the damage amplitude in the genetic material, expressed in chromosome aberrations and disturbances of the mitotic cycle.

The main objective of this study is to evaluate and to provide new data on the genotoxic potential of chromium, provided as K2Cr2O7 and CrCl3, by quantifying the rates of the ana-telophase chromosome aberrations and of metaphase disturbances induced in root tips of barley seedlings after short-term seed exposure to chromium and by analyzing the chromium-induced effects on mitotic index and on the frequency of division stages. A second objective of this paper was to analyze the influence of chromium on length growth in the early ontogeny of barley plantlets.

2 Materials and Methods

2.1 Plant Material and Treatment Conditions

Seeds of Hordeum vulgare L. cv. Madalin (Center for Agricultural Research and Development Secuieni—Neamt, Romania) were utilized. Chromium was provided as potassium dichromate [Cr(VI)], K2Cr2O7 (CAS registry number 7778-50-9, molecular weight 294.185 g/mol) and chromium chloride [Cr(III)], CrCl3.6H2O (Merck, Darmstadt; CAS registry number 10060-12-5, molecular weight 266.45 g/mol). Barley seeds were immersed for 3 h in each of the aqueous solutions of 10, 100, 250, and 500 μM K2Cr2O7, corresponding to a chromium concentration of 1.04, 10.39, 25.99, and 51.99 μg ml−1 and in each of the solutions of 10, 100, 250, and 500 μM CrCl3.6H2O, corresponding to a chromium concentration of 0.52, 5.19, 12.99, and 25.99 μg ml−1. Controls were prepared in distilled water. After chromium treatment and several rinses with running tap water, the seeds (50 per variant) were placed on moistened filter paper, in covered glass Petri dishes, and incubated in the dark, at 20 °C, until germination took place. The germinated seeds were then maintained at a photoperiod of 16 h/8 h (light/dark), at 23 ± 1 °C, in order to determine the growth in length of the barley plantlets in early ontogeny.

2.2 Preparation and Analysis of Slides

For the cytogenetic examination, barley roots (15–20 mm in length) were fixed for 24 h in alcohol/acetic acid (3:1, v/v), at room temperature, then they were washed, transferred to 70 % ethyl alcohol, and stored at 4 °C. The plant material was hydrolyzed for 20 min in 37 % HCl/distilled water (1:1, v/v), and then stained in modified charbol-fuchsin solution (Gamborg and Wetter 1975). Three rootlets/germinated seed were mounted on a slide and squashed in a drop of 45 % acid acetic, under a coverslip. Five slides/variant and 10 fields/slide were analyzed using Novex light microscope to score the cell cycle phases (interphases, prophases, metaphases, anaphases, and telophases) and the chromosome aberrations. The best preparations were photographed using Nikon Eclipse 600 microscope, with Nikon Cool Pix 950 digital camera (1,600 × 1,200 dpi) at ×100 objective.

2.3 Cytogenetic Parameters

The mitotic index (MI%) and the frequencies of the division stages (prophase index (PI%), metaphase index (MeI%), anaphase index (AI%), telophase index (TI%)) were calculated according to Eqs. 1 and 2, respectively:

$$ \mathrm{MI}\left(\%\right)=\frac{\mathrm{total}\;\mathrm{dividing}\;\mathrm{cells}}{\mathrm{total}\;\mathrm{analyzed}\;\mathrm{cells}}\times 100 $$
(1)
$$ \mathrm{Phase}\;\mathrm{in}\mathrm{dex}\left(\%\right)=\frac{\mathrm{cells}\;\mathrm{in}\;\mathrm{respective}\;\mathrm{mitotic}\;\mathrm{phase}}{\mathrm{total}\;\mathrm{dividing}\;\mathrm{cells}}\times 100 $$
(2)

To calculate the frequency of the ana-telophase chromosomal aberrations (CAA-T%) and the rate of the metaphase disturbances (M abn%), the Eqs. 3 and 4 were used:

$$ {\mathrm{CA}}_{\mathrm{A}\hbox{-} \mathrm{T}}\left(\%\right)=\frac{\mathrm{chromosomal}\;\mathrm{aberrations}\;\mathrm{in}\;\mathrm{ana}\hbox{-} \mathrm{telophase}}{\mathrm{total}\;\mathrm{dividing}\;\mathrm{cells}}\times 100 $$
(3)
$$ {M}_{\mathrm{abn}}\left(\%\right)=\frac{\mathrm{total}\;\mathrm{abnormalities}\;\mathrm{in}\;\mathrm{metaphases}}{\mathrm{total}\;\mathrm{dividing}\;\mathrm{cells}}\times 100 $$
(4)

The quantitative results are based on at least 6,500 cells counted in each experimental variant, including controls.

2.4 Statistical Analysis

The results are expressed as the mean values and standard error of the means \( \left(\overline{x}\pm \mathrm{SE}\right) \) for all groups of investigated parameters. The estimation of the increase/decrease (±) rate was based on Eq. 5:

$$ \pm \mathrm{rate}\left(\%\right)=\left(1-\frac{x}{y}\right)\times 100, $$
(5)

where y is the average value detected in the control and x is one of each treated samples (Liu et al. 2005). Statistical analysis was carried out according to Student’s t test for each parameter in order to establish the differences between the treated variants and the controls. The differences were considered as significant at levels of *p < 0.05, **p < 0.01, and ***p < 0.001. The Microsoft Office Excel 2003 software of Windows XP operating system was used to calculate and to graphically represent the statistical parameters.

3 Results and Discussion

3.1 Chromium Influence on Growth of Young Barley Plantlets

Both K2Cr2O7 and CrCl3 generally promoted the growth of barley seedlings in the early ontogeny (Fig. 1). Statistical analysis revealed that the stimulative action of the two chromium-containing compounds on shoot length was significantly higher in younger plantlets (p < 0.001 for 100 and 500 μM K2Cr2O7; p < 0.01 in the variants treated with 10, 100, and 500 μM CrCl3). In older plantlets, only K2Cr2O7 at 500 μM concentration determined significant differences as compared to the untreated control (p < 0.05). In 9-day-old plantlets, chromium at 250 μM concentration inhibited the growth for both compounds, but not in a significant manner (inhibition rate was −3.79 %, for K2Cr2O7, and −2.18 %, for CrCl3, respectively).

Fig. 1
figure 1

Growth of the barley plantlets in early ontogeny, after seed exposure to different concentrations of K2Cr2O7 and CrCl3. Vertical bars represent the standard errors of the means. Asterisks indicate the levels of significance in the differences between the control and the treatments: *p < 0.05, **p < 0.01, and ***p < 0.001

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In literature, both inhibitory and stimulatory effects of chromium on plant growth have been reported, depending on experimental coordinates. Low chromium concentrations (0.05–1.00 mg L−1) promoted plant growth in bean (Bonet et al. 1991), maize (Mengel and Kirkby 2001), and green amaranth (Zou et al. 2006), whereas other works evidenced inhibitive effect of the heavy metal to wheat growth (Jamal et al 2006).

Jun et al. (2009) noted the differentiated behavior of the plant growth to chromium exposure depending on species. At levels significantly higher than those tested in our experiment (above 145 mg kg−1 Cr), chromium suppressed the growth of pea plantlets (Bishnoi et al. 1993), while Castro et al. (2007) observed the stimulation of Arabidopsis thaliana seedling growth at concentrations of chromium lower than 100 μM and the inhibition of growth at levels of hexavalent chromium higher than 200 μM correlated to reductions in mitotic index.

3.2 Chromium Influence on Division Indices

Chromium also exerted stimulative action on cell division. Both chromium-containing compounds induced increases in mitotic index (MI%) in all treated variants (Table 1, Fig. 2). The stimulatory rate ranged between 20.36 and 42.52 % in the variants exposed to K2Cr2O7 and from 33.24 to 36.85 % in the variants treated with CrCl3. There were significant differences between MI% of the control and treatment groups: p < 0.05, for 100 μM K2Cr2O7 and p < 0.01, for the other treatment groups, except for 500 μM.

Table 1 Behavior of the genetic indices in barley root meristems after seed exposure to different concentrations of K2Cr2O7 and CrCl3
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Fig. 2
figure 2

Evolution of MI% in barley root meristems after exposure to chromium. Vertical bars represent the standard errors of the means. Asterisks indicate the levels of significance in the differences between the control and the treatments: *p < 0.05 and **p < 0.01

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The highest tested concentration induced a nonsignificant increase in the frequency of dividing cells (4.67 ± 0.44 %, for K2Cr2O7, and 4.12 ± 0.20, for CrCl3, as compared to the control, 3.88 ± 0.18 %). The results did not show a linear dose–response relationship.

Chromium influence on the frequency of division stages mainly consisted of prophase and telophase accumulation and diminution of metaphase and anaphase proportion in almost all chromium-treated variants (Table 1). CrCl3 induced significant increase (p < 0.05) of prophases at concentration of 250 μm (39.51 ± 2.37 % as compared to 29.27 ± 2.28 %, for the control), while 10 μm K2Cr2O7 significantly lowered the anaphase frequency (p < 0.05), in this case, the decrease rate being −38.5 % as compared to the control.

A variable profile of MI% induced by chromium treatment was reported in literature, depending on tested compound, plant species, heavy metal concentration, oxidation state, and exposure duration. So, in green amaranth, at the maximum tested concentration (10−3 M), hexavalent chromium determined a marked decline of MI% as compared to the control (Zou et al. 2006), whereas in Vicia faba, increases were observed in MI% after the treatment with K2Cr2O7 (Qian 2004). In this species, other authors (Mishra et al. 2012) established that the exposure to chromium trioxide resulted in values significantly lower than the control in longer treatments (6, 12 h), but it was stimulative in short-term treatments (4 h).

In onion root meristems, chromium nitrate lowered MI% and modified the proportion of division phases (Liu et al. 1992; Glinska et al. 2007). Concerning the frequency of division stages consisting in prophase and telophase accumulation, our results are in agreement with those reported in blackgram (Chidambaram et al. 2009) and sugarcane (Mishra et al. 2012).

3.3 Chromium-Induced Disturbances in Mitotic Ana-Telophases and Metaphases

As Fig. 3 shows, the profile of the rates of total chromosome aberrations (CAA-T%) induced by chromium in ana-telophases of root meristems of barley seedlings is relatively similar for K2Cr2O7 and CrCl3, namely, CAA-T% registered an ascendant trend from 10 to 250 μM, followed by a decline at the maximum tested concentration, although the level still maintained over the average control values at 500 μM (Fig. 3). Significant differences in the percentage of chromosomal aberrations were observed in chromium-treated variants as compared to the control average value (6.83 ± 1.25 %).

Fig. 3
figure 3

Frequency of the ana-telophase chromosome aberrations and metaphase disturbances in barley root meristems after seed short-term exposure to different concentrations of K2Cr2O7 and CrCl3. Vertical bars represent the standard errors of the means. Asterisks indicate the levels of significance in the differences between the control and the treatments: *p < 0.05, **p < 0.01, and ***p < 0.001

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The exposure to K2Cr2O7 induced increments by 1.3–2.3 times in CAA-T%—the differences between treatment groups and control were found significant (p < 0.01, p < 0.05) at concentrations of 100, 250, and 500 μM. In the variants treated with CrCl3, CAA-T% average values surpassed 1.7–2.2 times the control. This increase was significant at 100 and 250 μM (p < 0.05).

The chromosome bridges involving one or more chromosomes (Fig. 4c, d) represented the prevalent category among ana-telophase aberrations, for both chromium-containing compounds. Their frequency in the treated groups was 1.4–2.1 times higher than control. The complex aberrations such as bridges + lagging chromosomes (Fig. 4g), multipolarity + bridges + vagrant chromosomes (Fig. 4h), etc. occupy the second place as frequency, followed by lagging chromosomes (Fig. 4e), registered in higher percentage at the concentrations of 100 and 250 μM, for both compounds. In addition to these aberrations, vagrant chromosomes and multipolar ana-telophases (Fig. 4f) were observed, but in smaller proportion.

Fig. 4
figure 4

Normal and aberrant ana-telophases in barley root meristems after chromium treatment. a Normal anaphase. b Normal telophase. c Anaphase with one chromatid bridge, 100 μM CrCl3. d Anaphase with multiple chromatid bridges, 10 μM CrCl3. e Anaphase with lagging chromosome, 250 μM K2Cr2O7. f Tripolar telophase, 250 μM CrCl3. g, h Complex aberrations (g telophase with chromatid bridge and lagging chromosomes, 250 μM K2Cr2O7; h tripolar anaphase with chromatid bridges and vagrants, 250 μM CrCl3). Scale bar = 10 μm

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As concerns the micronuclei (Fig. 5), they were sporadically observed in this study and not in all chromium-treated variants. Their presence was noted in 500 μM K2Cr2O7 (Fig. 5b) and in 250 μM CrCl3 (Fig. 5c).

Fig. 5
figure 5

Micronuclei in barley root meristems after chromium treatments. a Normal interphases. b Small micronucleus, 500 μM K2Cr2O7. c Large micronucleus, 250 μM CrCl3. Scale bar = 10 μm

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They can result from an aneugenic (mitotic spindle malfunctioning) or a clastogenic event (chromosome breakage) induced by the stressor (Medeiros et al. 2003; Attia 2011). Unlike our results, several authors reported substantially increased frequencies of micronuclei after chromium exposure in many plant species—Tradescantia (Villalobos-Pietrini et al. 1986), V. faba (Qian 2004; Zhang et al. 2009), and blackgram (Chidambaram et al. 2009) as well as in animals (Balansky et al. 2000). In onion root meristems, Eleftheriou et al. (2012) observed that micronuclei frequency was higher after long treatments with chromium (48, 72 h), not in short exposure as in our work.

Both K2Cr2O7 and CrCl3 increased significantly the frequency of metaphase disturbances (M abn%) in barley root meristems at all tested concentrations (Fig. 3). Their percentage was by ~5.0–7.5 times higher than in control. So, the difference was p < 0.05 in 500 μM K2Cr2O7, but p < 0.001 in the other treatments groups, as compared to the control (2.00 ± 0.35 %). The main categories of abnormal metaphases were c-like metaphases (Fig. 6b) which surpassed the control value in all chromium-treated variants. The most numerous were present in 100 and 250 μM CrCl3 (8.63 and 6.84 %, respectively) and in 250 μM K2Cr2O7 (5.64 %), as compared to the control (0.70 %). The occurrence of this type of configurations indicates an effect similar to that colchicine-induced, which consists in thick and shorter chromosomes blocked at metaphase and scattered throughout the cell as result of the damaged mitotic spindle. This action, proved for other heavy metals such as lead or cadmium (Wierzbicka 1999; Liu et al. 2003), was also previously confirmed for chromium in other works (Liu et al. 1992; Glinska et al. 2007).

Fig. 6
figure 6

Metaphases in barley root meristems after chromium treatment. a Normal metaphases. b C-metaphase, 100 μM CrCl3. c Sticky metaphase, 100 μM K2Cr2O7. d Metaphase with chromosome expulsed from equatorial plate, 100 μM K2Cr2O7. Scale bar = 10 μm

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A substantial increase in the number of metaphases with sticky chromosomes (Fig. 6c) was observed in all chromium-treated variants. Stickiness reflects the high toxicity of the tested compound on genetic material and can be attributed to the action of the heavy metal on chromosome DNA with occurrence of depolymerizations, condensation, or entanglement of interchromosomal chromatin fibers and subsequent formation of subchromatid connections between chromosomes (Yildiz et al. 2009). Metaphases with chromosomes expulsed from equatorial plate (Fig. 6d) were present in lesser extent than c-metaphases and stickiness.

Concerning the dose–effect relationship, the results herein did not show a linear relation between chromium concentration and the aberration rates. The data reported in literature on the amplitude of cytogenetic damage induced in plants depending on chromium concentration are variable. In some short-term treatments (1–3 h), no relation was found between the chromium trioxide concentration and proportion of the chromosome aberrations recorded in V. faba root meristems (Gomez-Arroyo et al. 1987), whereas in blackgram and sugarcane, the percentage of chromosome abnormalities increased with the increase of chromium concentration (Chidambaram et al. 2009; Srivastava and Jain 2011). In other research using Vicia bioassay, the occurrence of different types of chromosomal abnormalities observed after chromium trioxide treatment did not show any consistent pattern (Mishra et al. 2012). In literature, numerous situations of nonmonotonic and nonlinear relationships between dose and the analyzed endpoints have been in fact reported, especially at lower concentrations of a chemical stressor, and this fact must not be ignored (Vandenberg et al. 2012), although it is still looking for plausible explanations.

The high number of the chromosomal aberrations identified in chromium-treated variants could be the consequence of the heavy metal immobilization and accumulation in barley roots (Ali et al. 2004), as it was observed in many other plant species (Cervantes et al. 2001; Jamal et al. 2006; Scoccianti et al. 2006; Santos and Rodriguez-Gomez 2012). According to Zayed and Terry (2003), >99 % of the absorbed chromium is retained in the roots, a low translocation from root to plant aerial parts being evidenced (Singh et al. 2013). Rodriguez-Gomez (2011) considers that the damage induced by chromium on genetic material in root cells is larger than that observed in leaf cells because of the direct contact between the root, which acts as a barrier against chromium translocation, and the heavy metal. In our study, the genotoxic action of chromium is supported by the high levels of CAA-T% and M abn%. The potential of chromium, either trivalent or hexavalent, to produce lesions in genetic material was evidenced in many plant species, but the results existing in literature are sometimes conflicting, depending on plant species, tested chromium compound, concentration range, duration of exposure, etc. (Gomez-Arroyo et al. 1987; Liu et al. 1992; Matsumoto et al. 2006; Zou et al. 2006; Glinska et al. 2007; Chidambaram et al. 2009; Srivastava and Jain 2011; Mishra et al. 2012).

The mechanisms of the genotoxicity and mutagenicity induced by chromium are not yet entirely known (Matsumoto et al. 2006; Rodriguez-Gomez 2011; Santos and Rodriguez-Gomez 2012), but most of the scientists sustain that the formation of reactive intermediates and generation of oxidative stress is the main mechanism involved in DNA damage (Medeiros et al. 2003; Patlolla et al. 2009; Labra et al. 2010; Wang et al. 2013) and in occurrence of chromosomal aberrations (Shanker et al. 2005; Velma and Tchounwou 2010; Halasova et al. 2012). Changes in the activity of enzymes for antioxidative protection (superoxide dismutase, catalase, and peroxidase) under chromium stress have also been observed in barley seedlings in relation to heavy metal concentration and plantlet age (Olteanu et al. 2012). Therefore, the chromosome aberrations can be the result of either clastogenic or aneugenic action of chromium (Pohren et al. 2013). The clastogenic action implies chromosome structural damage during division, while the aneugenic effects are due to the noxious action on the fibers of cell division spindle, with repercussions in its assembly and functioning, or on the centromere structure, so disturbing the normal migration of the chromosomes to the cell poles (Parry et al. 2002; Attia 2011). Recently, Eleftheriou et al. (2012) established that the chromium-induced chromosome aberrations in Allium cepa L. were in relation with the changes in the organization of cytoskeleton microtubules (considered as main subcellular target for chromium) during mitotic division.

Some recent works, focused on chromium-induced DNA damage, provided supplementary information, but without elucidate the intimate mechanisms of this process. Two synthetic studies (Nickens et al. 2010; Tabrez et al. 2014) conclude that DNA adducts, DNA strand breaks, DNA-protein crosslinks, oxidized bases, abasic sites, and DNA inter and intrastrand crosslinks are the most abundant genetic lesions produced by Cr(VI), these modifications being considered to be primary causes of genotoxicity and mutagenicity of hexavalent chromium. Also, Khorsandi and Rabbani-Chadegani (2013) state that chromium oxide genotoxicity arises from DNA structural and conformational changes caused by chromium binding to both phosphate groups and base pairs of nuclear DNA. These modifications have negative repercussions on DNA function, replication, translation, and transfer of genetic information. Chromium can also lead to aberrant cell cycle checkpoints or to disturbances in regulation of DNA repair mechanisms so promoting genomic instability and favouring carcinogenesis (Tabrez et al. 2014). It is also important to recognize the role of nonmutagenic, but heritable, epigenetic alterations in chromium-induced genotoxicity and mutagenicity (Nickens et al. 2010).

Barley Madalin cultivar is recommended for malt and beer production, also for animal fodder; other cultivars are used to produce certain distilled beverages or as components of various health foods. Therefore, this major cereal—the fourth most important crop in the world—constitutes an important link in food chain and the results obtained in this study must be taken into consideration. The continuous increase of the pollution by heavy metals, including chromium, is a serious concern, and every positive result on their genotoxic, mutagenic, and carcinogenic potential must be considered as a warning signal about the risks of the uncontrolled release of the heavy metals in environment because they finally affect the plant, animal, and human systems.

4 Conclusions

This study provides useful information about genotoxic potential of chromium, a heavy metal which became a serious threat to environmental safety. Both potassium dichromate and chromium chloride generally stimulated the growth of barley seedlings in the early ontogeny. The mitotic index was significantly increased after short-term exposure to potassium dichromate and chromium chloride. Potassium dichromate and chromium chloride increased frequency of chromosomal aberrations and mitotic abnormalities, thus showing both clastogenic and aneugenic activity. In view of its overall sensitivity, the Barley test can be recommended as a useful cytogenetic assay to study the cytotoxic and genotoxic effects of heavy metals. We consider that further research should be carried out to clarify the intimate mechanisms of chromium action in living organisms and to estimate not only the extent of the injury induced on genetic material but also the repercussions of this damage on phenotype traits.