Effect of Chromium VI on edible plants and their health risks: case of Radish (Raphanus sativus L.)

Radish (raphanus sativus L) is a vegetable very rich in vitamin C and fiber, this plant belonging to the family Brassicacae characterized by their great capacity to accumulate heavy metals such as Chromium. The aim of our work is the study of the effect of Chromium VI on the morpho-physiological parameters of radish and the assessment of health risk related to the bioaccumulation of Cr in the edible parts. The plantation of radish was made on a soil artificially contaminated by 4 concentrations of Cr(VI) (10, 20, 40 and 60ppm). After comparing the results obtained with the results of non-contaminated soil, it is observed that the Cr affects negatively the growth, yield and the content of chlorophyll, On the other hand, it is noticed that there is a slight increase of sugars, proteins and Proline content with the increase of CrVI concentration in the soil, we can explain this increase by the development of defense mechanisms by the radish plant against the stress caused by CrVI. Regarding the bioaccumulation of Cr we found that the concentration of Cr in different parts of radish is too high compared to the recommended daily dose (120μg), so it is not recommended to consume radish grown in areas contaminated by Cr.


Introduction
The human and industrial activities have led to contamination of soils by various pollutants such as heavy metals, contamination of agricultural soils leads to losses in agricultural production and yield, and an hazardous effects on humain and animals health. Large soils all over the world are contaminated with heavy metals [1], the strong global demand for agricultural production can lead to use the contaminated or marginal soils and increase the risk of food contamination, each metal act in a different way depending on their role in cell metabolism. Microelements, such as zinc, are essential and participate in many physiological processes [2]. However, at optimal doses they are highly toxic and inhibit plant growth. Heavy metals such as chromium are toxic even at low concentrations [3]. The main sources of chromium contamination in leather processing, landfill leachate, Anthropogenic activities, industrial effluents, automobile exhausts, waste incinerators, metal plating and finishing operations, pesticides and fertilizers manufacturing, cement factories, wood processing, metallization, oxidative dyeing, metal finishing, tobacco emissions, acid manufacture chromic, cement plants and paper production [1,4,5].
metal stress-induced reductions in plant growth parameters may be ascribed to low water potential, nutritional imbalance, and reactive oxygen species (ROS) mediated oxidative stress [6]. [7] argued that plants exposed to high concentrations of trace elements synthesized less chlorophylls, carotenoids, and other photosynthetic pigments. In addition, as a self-defense mechanism, plants have antioxidant systems to remove ROS. However, in the case of severe toxicity, this defense system may not be able to mitigate the toxic effects that have resulted in reduced plant functioning and normal growth [8]. Some edible plants, including family of Brassica plants, are characterized for their ability to accumulate large quantities of heavy metals such as radish. This has led to the search for species that can be used for phytoremediation of sole, with the following main features [9] (i) the ability to accumulate heavy metals in non-edible parts; (ii) tolerance to high doses of metals; (iii) rapid growth and high accumulation biomass; (iv) easy to grow as an agricultural crop and easily harvested.
In this study, we evaluated the effect of chromium metal concentrations (10 to 60 ppm) on growth parameters, chlorophyll content, TSS content, proteins content and proline content of Radish (Raphanus sativus L.)

Germination seeds
Dry seeds were washed with sodium hypochlorite 20% for 5 min, and then rinsed with distilled water. The seeds were placed on double layered the seeds were placed glass Petri dishes with double layer filter papers, soaked with 15 ml of H2O for the control and for the treatment soaked with 5ml of chromium solution [10].

Growth parameters
The Radish plants were harvested after 1 month. The plants were oven dried at 80°C for 24h to determine the dry weight of root, shoot and hypocotyl of radish. The content of chlorophyll was estimated using the method of [11]. Pigment concentrations are expressed as mg/g FW (Fresh Weight).

Chlorophyll content
Leaf pigment contents including chlorophyll a andb, were extracted as described by [11].160 mg leaf samples were broyed in 4 ml of acetone (80% ). The mixture was centrifuged for 10 min at 5000×g, and the absorbance ofthe supernatant was noted at 662 and 645 nm using a UV-Vis spectrophotometer (Lambda 25, PerkinElmer, Inc. USA).The pigment contents were calculated using theequations and coefficients given by [11]. The pigment fractions were calculated as mg g-1 fresh leaf weight.

Total soluble sugars (TSS) content
Total soluble sugars (TSS) were estimation by phenol sulfuric acid method [12]. The samples were measured spectrophotometrically at 485 nm by spectrophotometer (Palo Alto, California, USA). we used D-glucose as a standard.

Proteins content
Protein content in plant (50 mg) was determined by using BSA as standard protein following the method of [13].

Proline content
Proline content in Radish plant (250 mg FW) was measured by spectrophotometer (Palo Alto, California, USA) according to the method of [14]. A standard curve was obtained from known concentrations of proline.

Chromium accumulation
The standardized color method [15] was used to determine the concentration of Cr (VI) which forms a red-violet complex measured by spectrophotometry at 540 nm using a JENWAY 6300 spectrophotometer with 1.5-diphnylcarbazide (DPC).

Statistical analysis
All the measurements were triplicates. The analysis of variance (ANOVA) was carried out using a version 24 of SPSS and by Tukey's post-hoc test between treatment means [16].

Germination seeds
The chromium effect on radish seeds germination was shown in Fig. 1. Our results showed a decrease in germination rate by 1.65%, 3.3%, 13.3% and 38.3% for 10ppm, 20ppm, 40ppm, and 60ppm of Chromium respectively, as compared to control plant.

Growth parameters and Chlorophyll content
The results showed that chromium have a harmful effect on growth parameters, chlorophyll content, TSS content, proteins rate and proline content Table 1. In general, chlorophyll content decrease by 23%, 41%, 67%, and 75% for 10ppm, 20ppm, 40ppm, and 60ppm of chromium respectively, as compared to control plant.
The dry weight of shoot biomass of Raphanus sativus was influenced by the chromium, the metal caused a reduction of about 3%, 6%, 25%, 66.5% and in presence of 10ppm, 20ppm, 40ppm and 60ppm respectively, as compared to control plant. The dry weight of root of Raphanus sativus was affected by the metal.
The dry weight of hypocotyl was affected by the chromium, the metal caused a reduction of about 2%, 5.3%, 55.7%, 79% for 10ppm, 20ppm, 40ppm and 600ppm of metal respectively, as compared to control plant. In addition, However, we observed that TTS, total proteins and proline in Radish an increased slightly with the elevation of Cr (VI) concentration.

Total sugars, Proteins and Proline content
The results in Table 1 showed that exposure of radish plants to chromium slightly increases the production of soluble sugars, protein and proline. this increase is proportional to the increase in chromium concentration in the culture medium. it can be noted that the maximum production is recorded at the high chromium dose (60ppm) with an increase of 24%, 22%, 33% for SST, protein and proline respectively compared to the control plant.

Chromium accumulation
The variability of metal concentrations affected the chromium accumulation, Table 2 Showed that the accumulation in tissues increases with the increase of chromium concentrations in soil. The germination step is the first exchange interface with the surrounding medium and accordingly it is relatively sensitive to changing environmental conditions [17]. The present study showed that the Chromium metal reduced the germination rate, chlorophyll content, root and shoot dry weight of Radish (Raphanus sativus) as compared to control plant. These results correspond with that of [18] who observed that Chromium decreased the rate germination of Alfalfa seeds (Medicago sativa L.) by more than 50% and decrease the root and shoot weight Alfalfa plant. In addition [19] reported that the heavy metals reduced the root elongation of lettuce, broccoli, tomato and radish plant. However [20] noted that the germination of Coronilla varia was decreased by Ni, Cu, and Cd. and the inhibitory effect on the belowground growth was greater than on the aboveground growth. [

Conclusion
The present study revealed that growth parameters and chlorophyll contents were reduced under the influence of chromium treatments, while other parameters such as total soluble sugar, proteins content and proline content were slightly increased under similar conditions. Data indicated that Radis is moderately tolerant to chromium. it's could possibly be used with success in polluted soils and the extraction of heavy metals could be maintained at satisfying levels.