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Introduction

Chromium Contamination

The extensive use of chromium, an indispensable metal but a persistent contaminant has resulted in its accumulation in various environmental compartments. The major contributor of Cr pollution to the biosphere is the leather industry accounting for 40% of the total industrial use [1]. In India, about 2000–32,000 tons of elemental Cr annually escapes into the environment from tanning industries. In recent times, there is thrust to explore the mechanism of absorption, uptake, translocation, tolerance, bonding and especially the chemical form of chromium in plants to understand its phytoavailability from contaminated soils and waters, their further transfer through agricultural food chains and to evaluate the potential of plants to exploit them for phytoremediation to treat Cr contaminated soil [3,4].

Chemical Speciation of Chromium

Chemical speciation is significantly applicable in the case of chromium as its two important and stable states viz. hexavalent and trivalent species show contrasting behavior with respect to reactivity, solubility, mobility and toxicity. Cr (III) is considered an essential micronutrient needed by the body for the maintenance of normal glucose tolerance levels [5] whereas anionic Cr (VI) is toxic as an oxidizing agent and even carcinogenic [6].

Chromium Uptake and Speciation in Plants

The uptake of Cr by plants depends on Chemical Speciation changes in soil and water, in the plant root environment (rhizosphere), in the root, in the xylem sap and in the leaves and fruits [7]. There are several studies on the uptake, accumulation and phytotoxicity of the two Cr species in different plant parts in various cereal crops and vegetables [8–11]. The few studies on the chemical form of Cr in different plant components of different plant species indicate the conversion of toxic Cr (VI) to the harmless Cr (III) species in root and the presence of Cr as Cr (III)-organic acid complex in the roots and leaves [4, 12–15].

Chromium Speciation in Xylem Sap

There is a dearth of studies on the translocation and speciation of Cr in the xylem sap [16–18]. The xylem sap translocates water, nutrient and non-nutrient ions after absorption by roots, to the aerial parts i.e. shoot and fruits. This component of the plant can provide a unique insight into the basic mechanisms employed to transport nutrient as well as pollutant metals in plants. Besides inorganic ions, xylem sap has various organic ligands of which the carboxylic acids are the major complexants [19, 20]. Heavy metals that exist in many oxidation states and/or are prone to hydrolysis are translocated after interaction with the organic ligands of the xylem sap [7, 16–18, 21–22].

In a series of studies aimed on chemical speciation of Cr in plants, in the present study, the chemical form in which Cr(III) and Cr(VI) is translocated by the xylem sap in 90 days old maize plants (Zea mays. L; Ganga 5) representing the most important stage of the crop, the cob stage was determined. Simultaneous cation exchange (Dowex-50 X8) and anion exchange (Dowex-1 X8) column elution chromatography was used for Speciation analysis using radiotagged Cr. The latter was detected and quantified by Gamma Ray Spectrometer.

Experimental

Plant Material

Seeds of maize (Zea mays L.; seed type: Ganga 5; sowing season: October–November) procured from National Seed Corporation Ltd., Indian Agricultural Research Institute, New Delhi, India were sterilized and soaked for 24 hours. They were sown in polyethylene pots filled with washed dried quartz sand (pH, conductivity of washing: 6.5 – 7.0 and 1.4 – 1.6 mmho respectively) and the pots placed in a green house. Till the seeds germinated, water was sprinkled twice a day. There after, they were irrigated alternately by water and nutrient solution. The nutrient solution [23] contained the following nutrient millimolar concentrations: K, 5.0; N, 15.0; Ca 5.0; P, 1.33; Mg, 3.0; S, 1.5; Fe, 0.1; Mn, 0.01; Cu, 0.001; Zn, 0.002; B, 0.03; Mo, 0.0002; Co, 0.0001; Na, 0.1, Cl, 0.04. Citrate was replaced by EDTA to maintain the solubility of nutrient Fe.

Collection of Xylem Sap Samples

Plant stems were cut perpendicular to the stem axis with sterilized stainless-steel razor blades at approximately 12cm above the root. The first drop of exudate from the cut surface was discarded and the cut surface washed and wiped gently. The subsequent drops that emerged were sucked by a micropipette and collected as xylem sap samples in ice cooled sterilized borosil glass vials. Plant stem was severed in a basipetal fashion and fresh cuts were made after every 1.5 – 2.0 hours at a distance of 1 – 2cm from the previous cut till the junction of stem and root. The sampling was done for a period of 24 hours and the xylem sap of various plants was pooled. The sap was filtered with Millipore GV nylon filters (0.22mm), purged with pure nitrogen (N2) gas and stored at freezing temperatures.

Separation of Carboxylic Acid Fraction of Xylem Sap

Separation of carboxylic acids (major complexants) from the other constituents of xylem sap mainly, amino acids was carried out using cation exchange resin (Dowex-50 X8). pH of the sap was adjusted to 7.0, and loaded on a 1 X 20cm column with a bed height of 6cm. The carboxylic acids were eluted with deionized distilled water (pH 6.95) [24]. This separated fraction was utilized for in vitro studies as well as quantification of the major carboxylic acids by ion suppressed reversed phase HPLC.

Preparation of Reagents

All solutions were prepared in deionized distilled water using ‚Analytical Grade Reagents‘. The radioactive 51Cr isotope was obtained as aqueous CrO4 2- (specific activity 50μci/mg) from Board of Radiation and Isotope Technology (BRIT), Mumbai, India. 200μM radiotagged Cr (III) stock solution was prepared. 1ml of 1000 μM Cr (VI) stock was mixed with active Cr (VI) (41.7μci) and reduced with Na2SO3 such that the pH of the resultant solution was 4.5. The prepared oxidation state of radiotagged Cr (III) was checked by batch cation and anion exchange experiments. 0.0192 gm citric acid was made up to 50ml at pH 5.0 to prepare 2000μM stock solutions.

0.01M, 0.1M, 1M and 2.0M NaNO3 at pH 4.5 were the eluents used in anion exchange elution chromatography and 0.01M, 0.1M and 1M HClO4 and 0.25M, 0.5M and 1M Ca (NO3)2 solutions prepared in 0.01M HClO4 solution were the eluents for cation exchange elution chromatography.

Cr (III) and Cr (VI) Complexation and in Vitro Studies

Cr (III) and citric acid was mixed in the ratio of 1:10 (100μM: 1000μM) at pH 4.5–5.0 at 25°C. The complexation analysis was carried out on the 4th, 7th, 10th and 30th day of mixing. Only the results of 10th and 30th day of complexation are shown.

For the in vitro studies, 0.5ml of Cr (III)/Cr (VI) solution containing 100μM of Cr (III)/Cr (VI) was mixed with 0.5ml of carboxylic acid fraction of sap or 0.125ml of pure sap corresponding to 1000μM of total carboxylic acid as determined by HPLC. The total volume was made up to 1ml. The pH of these solutions was in the range of 4.5 and 5.0 and they were kept at 25°C. For Cr (III), the analysis was carried out just as in synthetic Cr (III) complexation studies whereas for Cr (VI), analysis was carried out after a period of 20 days at 25°C.

Ion Exchange Elution Chromatography and Preparation of Elution Curves

Dowex-1 and Dowex-50 ion exchange columns with bed heights 3cm and 2cm respectively were prepared by pouring the slurry of the respective resin in the first eluent in columns of dimensions 1 X 20cm. The elution was carried out at a flow rate of 0.5ml/min. 5ml fractions of each eluent were collected from the column in scintillation vials. Each fraction was counted using Gamma Ray Spectrometer at 0.320 MeV corresponding to the photopeak of 51Cr. The Gamma ray Spectrometer model used for the present work was a NaI (Tl) Detector coupled to a 4K Multichannel Analyser (Canberra Accuspec Card PC386AT). The elution pattern was obtained by plotting the volume (ml) of eluent against the percentage of total counts obtained in each fraction using MS Office Excel Software.

The elution profiles of pure Cr (III), pure Cr (VI) and Cr (III)-citric acid complexation were compared with elution curves obtained for interaction of pure Cr (III) with the carboxylic acid fraction and the pure sap in the in vitro studies. The Cr (III) complexes which are chiefly anionic, were separated by anion exchange elution chromatography. The extent of reaction or the unreacted Cr (III) and formation of any positive complexes was ascertained by comparison with the elution curve of pure Cr (III) from the cation exchanger.

Results and Discussion

Cr (III) In Vitro Studies Results

Time based interaction of radiotagged Cr (III) with the carboxylic acid fraction of maize sap (WIIIx) indicates gradual formation of anionic complexes (Fig. 1a). The anionic complexes elute as peaks at 30 and 65ml, with the dominance of the 65ml peak on 30th day. The elution curve profiles for Cr (III) complexation with pure xylem sap (WIIIy) are comparable to the carboxylic acid fraction with similar dominant anionic complexes being eluted at 30ml and 65ml (Fig. 1b).

Fig. 1a:
figure 1a

Cr (III)-Carboxylic acid fraction (WIIIx) elution curve on Dowex-1

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Fig. 1b:
figure 1b

Cr(III)-Pure sap (WIIIy) elution curve on anionic Dowex-1

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The above elution curves obtained are similar to Cr (III) complexation studies carried out with citric acid (Fig. 2). Three major peaks elute from the anion exchange column at 30, 65 and 80ml. The 30ml peak is considerably reduced whereas the 65ml peak becomes dominant on the 30th day.

These peaks along with the other minor peaks represent various 1:1 citrate complexes.

The extent of complex formation is substantiated by the bar diagram illustrated for elution from the cation exchanger on the 10th day (Fig 3a,b) for Cr(III)-WIIIx and Cr(III)-WIIIy respectively as compared to pure Cr (III).

Fig. 2:
figure 2

Cr (III)-citric acid complexation elution profile

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Cr(III) is present as different hydrolytic species of which [Cr(H2O)6]3+ is the most dominant and gets eluted at 65ml in the D fraction. Most species in the xylem sap samples are anionic as they elute in the first fraction from the cation exchanger. The D fraction is also reduced considerably. This signifies that Cr (III) forms anionic complexes or species with the pure sap (WIIIy) as well as the carboxylic acid fraction (WIIIx), though the extent of complexation is more with the former.

Fig. 3a:
figure 3

Comparison of elution of pure Cr(III) and Cr(III)-WIIIx from cation exchanger Dowex-50; Fig. 3b Comparison of elution of pure Cr(III) and Cr(III)-WIIIy from cation exchanger Dowex-50

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Cr (VI) In Vitro Studies Results

The elution profile of Cr (VI)-WIIIy (pure sap) interaction from the anion exchanger Dowex-1 shows shallow peaks at 65ml and 80ml (Fig. 4a) as compared to a single sharp peak for pure Cr (VI) at 65ml (Fig. 5a). Thus this profile is similar to Cr(III)-xylem sap curve (Fig. 1b) whereas the elution profile of this sample on cation exchanger Dowex-50 (Fig. 4b) is akin to pure Cr(III) curve (Fig. 5b). Thus there is a definite speciation change of Cr (VI) i.e. reduction as well as complexation by the organic ligands of the sap.

Fig. 4a:
figure 4

Cr (VI) – (WIIIy) pure sap elution curve on anionic Dowex-1. Fig. 4b: Cr (VI) – (WIIIy) pure sap elution curve on cationic Dowex-50

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Interestingly, the interaction of Cr (VI) with the carboxylic acid fraction does not reveal a significant reduction of Cr (VI) to form Cr (III) or Cr (III) complexes (Fig. 6a, b). The same was observed for Cr (VI)-citric acid complexation. The reduction of Cr (VI) to Cr (III) is not feasible. Reduction of Cr(VI) by oxygen-containing functional groups at pH values 4–8 is a slow process, though it occurs at significant rates in presence of catalysts specially Fe(II) [23]. Iron is absent in the carboxylic acid fraction, being held up in the cation exchange resin while separating the carboxylic acids in the separation procedure. Fe is a major ion present in the xylem sap and is present primarily as an organic acid complex [7, 21]. Fe (II) and Fe-(II) carboxylates are known to enhance Cr (VI) reduction and lead to the formation of complexed soluble Cr (III) [24]. This aspect needs to be further explored in the plant system. The reduction may also be attributed to other organic constituents (e.g. amino acids, sugars) [7, 13] that are present at a higher concentration in this mature stage [20].

Fig. 5a:
figure 5

Pure Cr (VI) elution profile on Dowex-1. Fig. 5b: Pure Cr (III) elution profile on Dowex-50

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Evaluation of Plant Role in Alleviation of Toxic Cr (VI)

The in vitro results are corroborated by similar studies carried out using electrophoresis technique [18] as well Cr (VI) in vivo analysis. Thus Cr(III) is present as an anionic carboxylate complex probably Cr(III)-citrate and Cr(VI) is also likely to be carried in the same form after reduction to Cr(III) in the xylem sap of mature maize plants.

The above findings correspond to other speciation studies on Cr in plants. Cr (VI) speciation analysis [16] in cabbage xylem sap, indicated that 95% of Cr (VI) was present in the trivalent form and it has been shown that once in the vascular system, low concentration of Cr (VI) was transported as a Cr (III)-organic complex in subterranean clover [14]. The Cr ion is found in an octahedral conformation bound to six oxygen atoms, with an absence of Cr (VI) in the shoots and leaves due to reduction to Cr (III) in the roots [11–18].

Fig. 6a:
figure 6

Cr (VI)-WIIIx elution curve on anionic Dowex-1. Fig. 6b: Cr(VI)- WIIIx elution curve on cationic Dowex-50

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This study also highlights the role of organic acids in the detoxification mechanism inside the plant. The detoxification starts in the roots, where the toxic Cr (VI) gets reduced within 4 h in the fine lateral roots [13] and is chelated and compartmentalized in the vacuole by low-molecular-weight organic acids (LMWOA) [9]. On translocation to the aerial parts, Cr(VI) gets reduced and bound by chelates of xylem sap [16–18] and further gets stored as less toxic Cr(III) complexes in the aerial parts [4,13–15]. The detoxification mechanism thus works at every step. The food chain also seems to be well protected from excess and toxic Cr by the Soil–plant Barrier [25] unless the conditions are drastic. In the in vivo study, no toxic symptoms were visible in the plants at a Cr (VI) concentration of 5.2 ppm in hydroponics. In field conditions, Cr (VI) is readily immobilized in soils by adsorption, reduction, and precipitation processes.

Thus the detoxification of Cr (VI) at each step enables that the phytotoxicity of chromium in edible plant tissues occurs at concentrations below that are injurious to animals or humans. In fact, this biotransformation of Cr(VI) to the less toxic and mobile Cr(III) complexes presents a significant approach for the Phytodetoxification or the in situ detoxification of Cr(VI) through plant-based reduction and chelation mechanisms for bioremediation of chromium contaminated wastelands.