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Sub-cellular localization and quantitative estimation of heavy metals in lemongrass plants grown in multi-metal contaminated tannery sludge
South African Journal of Botany 131 (2020) 74 83
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Sub-cellular localization and quantitative estimation of heavy metals in lemongrass plants grown in multi-metal contaminated tannery sludge Janhvi Pandeya,b, Sougata Sarkarc, Rajesh Kumar Vermaa,b, Saudan Singha,b,* a
Academy of Scientific and Innovative Research (AcSIR), India Division of Agronomy & Soil Science, CSIR-Central Institute of Medicinal and Aromatic Plants (Council of Scientific and Industrial Research) PO- CIMAP, Lucknow 220615 Uttar Pradesh, India c Genetic Resources and Agro-Technology Division, CSIR-Indian Institute of Integrative Medicine (IIIM), Jammu 180001, India b
A R T I C L E
I N F O
Article History: Received 12 July 2018 Revised 11 September 2019 Accepted 24 January 2020 Available online xxx Edited by M Vaculik Keywords: Heavy metals Histochemical Fluorescence Lemongrass Localization Phytoextractor
A B S T R A C T
Major objective of the study was to assess the localization pattern of heavy metals in different parts of lemongrass plant when grown in multi-metal contaminated sites. Microscopic and quantitative investigations (by ICP-OES) were carried out in root and leaves of plant to examine the compartmentalization of heavy metals. Along with it, the morphological variations in the size of trichomes in test plants were also perceived. Differential distribution of Cr, Pb, Ni and Cd was observed in root and leaf sections. Localization of these heavy metals in different parts of the plant cultivated in sole tannery sludge was compared with the plants grown in garden soil; which served as control. Histochemical methods for Pb, Cd and Ni detection revealed their significant accumulation in the root and leaf sections. Cr accumulation in roots of test plants was confirmed by fluorescence staining. Translocation factor <1 was recorded for Cr but greater than 1 for Ni and Pb. Moreover, Bioaccumulation factor >1 was observed for Ni and Pb. SEM studies revealed that the size of trichomes enlarged in plants grown in tannery sludge. Hence, our study provides an insight into the localization pattern of heavy metals in lemongrass plants and suggests that lemongrass can serve as a Ni and Pb phytoextractor when grown in multi- metal contaminated sites. In addition, enhancement in the size of trichomes was detected due to heavy metal stress which may prompt increment in essential oil yield thus; cultivation of lemongrass in heavy metal contaminated sites can also prove profitable economically. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction In the present era of industrialization, the tremendous amount of untreated waste from industries is posing a serious threat to the ecological wellbeing. The problem of pollution generated by heavy metals is among one of these risks hence, it needs to be addressed at a priority (Kumar et al., 2018). Leather industry is among one of the significant contributors of hazardous waste, particularly heavy metals (Kocurek et al., 2015). Sludge from tannery industry contains significant amounts of heavy metals especially Cr, Pb, Ni and Cd. At present, a proper sludge disposal system is lacking in many countries worldwide. A major threat for environment is the presence of several landfills containing heavy metal rich sludge from these industries (Ahmad and Misra, 2014). The impact of heavy metals on plants requires significant consideration in relation to expanding ecological Abbreviations: ICP-OES, Inductively Coupled Plasma-Optical Emission Analyzer; SEM, Scanning Electron Microscopy * Corresponding author at: Division of Agronomy & Soil Science, CSIR-Central Institute of Medicinal and Aromatic Plants (Council of Scientific and Industrial Research) PO- CIMAP, Lucknow 220615 Uttar Pradesh, India. E-mail address: [email protected] (S. Singh). https://doi.org/10.1016/j.sajb.2020.01.034 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
contamination problem. In this context, it is critical to examine the compartmentalization of retained heavy metals in different parts of the plant (Seregin and Kozhevnikova, 2011). Diverse plant species respond distinctively to toxicity caused by heavy metals likeaccumulation in various parts, prohibition, chelation or immobilization (Yousefi et al., 2018). For some plant species, the concentration of heavy metals in their aboveground parts are generally lower than the accumulation in roots, which is controlled by the hindrance capacity of the root framework (Carrier et al., 2003; Yuan et al., 2011). Histochemical methods may greatly help to investigate metal localization, distribution and accumulation in plant tissues. These methods provide information that supplements the results of quantitative analysis. It is important that the results of histochemical analysis should always be compared with the data obtained by other methods like SEM-EDAX, ICP, etc. Studies of metal distribution and accumulation in plants should rest on the combination of these methods in order to produce an integral pattern of translocation fluxes of the metal under investigation (Seregin and Kozhevnikova, 2011). Aromatic grasses can be grown in multi-metal contaminated areas as they serve the purpose of phytoremediation along with obtaining monetary benefits without the risk of food chain contamination (Lal
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et al., 2013). Lemongrass (Cymbopogon flexuosus (Nees ex Steud.) W. Watson) is an enduring perennial aromatic grass which produces a huge biomass. Essential oil is the main by-product of this crop for which it is widely cultivated all over the world (Zheljazkov et al., 2011). It is a metal-tolerant plant that withstands harsh environmental conditions (Das and Maiti, 2009). Lemongrass has been recommended for the phytoremediation of metal-polluted sites as it is a potential metal accumulator especially Cd, Ni and Pb (Israila et al., 2015). Though lemongrass has been recommended as a potential metal accumulator yet no significant work has been done to study the localization pattern of heavy metals specifically in its plant parts. Our present study has been performed to investigate the localization of heavy metals (Cr, Ni, Pb and Cd) in different parts of lemongrass plant when grown in multi-metal contaminated tannery sludge. For this, microscopy techniques (histochemical, SEM-EDAX and confocal) have been used and further confirmed by quantitative estimation (through ICP-OES). This study can provide an insight into the heavy metal compartmentalization pattern in lemongrass plant when grown in contaminated sites and the effect of heavy metal stress on the size of trichomes as well. 2. Materials and methods 2.1. Cultivation of experimental test plants For the present trial, slips of lemongrass (variety- Suwarna) cultivated in nurseries were utilized. The slips were transplanted in 140 kg capacity cemented barrels present in the experimental farm of CSIR-Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow, India for the duration of one year (2017 2018). The barrels were filled with garden soil and sole tannery sludge respectively. Cemented barrels were used to mimic field conditions without the risk of horizontal leaching of metal concentrations from tannery sludge during irrigations. Tannery sludge was collected from Common Effluent Treatment Plant (CETP) located nearby CSIR-Central Leather Research Institute (CLRI resource center), Kanpur (India). It was processed and filled in the barrels and then left for 30 days incubation period before the slips were planted. Plants grown in garden soil served as control and both control and test plants were replicated five times each. Data of four harvests were recorded during the duration of one year. 2.2. Microscopy procedures for determining heavy metal localization in plant parts
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(Seregin et al., 2003). Five replicates of each sample were taken for control and test plants. 1% solution of dimethylglyoxime in 1.5% solution of NaOH in 0.05 M borax (pH 9.8 10.4) was made. It forms nickel dimethylglyoximine, a reddish-brown complex with nickel (II). Three to four drops of dimethylglyoxime were added on root and leaf sections and observed under Leica DM 750 microscope equipped with a Leica ICC 50 HD camera and LAS EZ software. 2.2.3. Fluorescence staining of chromium Method given by Kovacik et al. (2014) was used for the fluorescence visualization of Cr in root sections. A working solution of 0.25% 1, 5-diphenylcarbazide was made in acetone diluted with distilled water which was then diluted to 0.05% with hydrochloric acid/potassium chloride buffer (0.1 M, pH 1.8). Transverse sections of roots were incubated in the working solution for 30 min at RT. After incubation, washing of sections with hydrochloric acid/potassium chloride buffer was done and observed using a Zeiss LSM880 confocal microscope equipped with a multiline Argon Laser and ZEN BLACK software. Images were collected at 500 to 560 nm (FITC) using excitation at a wavelength of 488 nm from an argon laser. 2.2.4. Scanning electron microscopy and energy dispersive X-ray analysis Morphological variations in trichome size in lemongrass leaves were observed by SEM analysis. Leaf and root sections were fixed with 2% glutaraldehyde maintained in phosphate buffer saline (0.1 M, pH 6.8) for 12 18 h at 40 °C; washed and fixed for 2 h in osmium tetra oxide (1%) in PBS at 40 °C. The specimen was washed with phosphate buffer; dehydrated in a series of ethanol water solutions (30%, 50%, 70% and 90% and 100% ethanol) and dried for 20 min. Mounting was performed on aluminum stubs, and the cells were coated with 90 nm thick palladium coating in polar sputter coater for 10 min. Coated sections were viewed at 15 kV under scanning electron microscope equipped with EDAX (JEOL JSM-6490LV). 2.3. Heavy metal analysis using ICP-OES Root and shoot samples of plants were washed with 0.1 N HCl, deionized water and then with distilled water. They were then oven dried at 70 °C. 0.25 g of powdered sample was acid digested (HNO3: HClO4 = 10:4) for heavy metal analysis via ICP-OES, Perkin-Elmer model 53,000 V (Piper, 1966, Page et al., 1992, Jones 1990). 2.4. Statistical analysis
To carry out microscopy procedures, leaves and root samples were collected prior to each harvest from control and test plants (from all five replicates). 2.2.1. Histochemical determination of lead and cadmium Seregin and Ivanov method (1997) was used to determine Cd and Pb speciation in the transverse sections of roots and vertical sections of leaves in test plants. Five replicates of each sample were observed for control and test plants. Hand cut root and leaf sections were stained by dithizone. Dithizone solution was made in acetone (0.5 mg/ml) which was then diluted with distilled water in 3:1 ratio and few drops of glacial acetic were added in the solution to prevent the leaching out of metals due to solvent medium. The sections were then placed on a glass slide and stained by adding a drop of dithizone solution. Dark red to blue-black coloured cadmium and lead dithizonate crystals were observed using a Leica DM 750 microscope equipped with a Leica ICC 50 HD camera and LAS EZ software. 2.2.2. Histochemical determination of nickel For determination of nickel localization in the root and leaf sections of test plants, modified dimethylglyoxime method was used
Normality distribution in data sets was evaluated using Shapiro Wilk test. Owing to the non-normal distribution of data sets, nonparametric Kruskal Wallis ANOVA was performed. Each data point in Figures represents an average value. Standard deviation obtained from five replicates for each sample is shown in the Figures as an error bar using the Origin version 8.0 software. 3. Results and discussions 3.1. Initial and post-harvest physico-chemical and heavy metal concentration status in tannery sludge and garden soil The initial status (prior to planting) of tannery sludge and garden soil comprising physico-chemical parameters and heavy metal concentration has been given in Table 1. A marked reduction in heavy metal concentration was recorded after each harvest. After final harvest, a percentage reduction of 46.57% in Cr concentration, 30.22% in Cd, 41.89% in Ni and 37.94% reduction in Pb concentration, in post-harvest sludge samples was observed. It can be attributed to the uptake of these metals by test
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3.2.3. Concentration of heavy metals in clumps at the time of harvest After final harvest, clumps of test plants were also analyzed for their heavy metal concentration. The data recorded revealed an interesting result that maximum concentration of Pb followed by Cr, Ni and Cd was found in clumps. Pb and Cr concentration was found in significant amounts in control plants too. It supports the fact that Pb was being translocated to upper parts of lemongrass plants which was justified by maximum concentration of Pb in leaves of test plants but there was less translocation of Cr in upper parts; it may be attributed to complex formation of Cr with sulfhydryl groups which inhibited their movement (Malec et al., 2010). As clump gives rise to shoots repeatedly, hence the translocation rate of metals could be greater in this portion of the plant. We tried to explore this hypothesis. Concentration of heavy metals in clumps followed the same pattern as leaves in tests plants which is shown in Fig. 1.
Table 1 Chemical characteristics, heavy metals and micronutrient concentration in tannery sludge and garden soil. Parameters
Tannery sludge
Garden soil
pH (1:2.5) Electrical conductivity (ds/m) Organic Carbon (%) Available Nitrogen (mg/kg) Available Phosphorous (mg/kg) Available Potassium (mg/kg) Iron (mg/kg) Copper (mg/kg) Manganese (mg/kg) Zinc (mg/kg) Lead (mg/kg) Cadmium (mg/kg) Chromium (mg/kg) Nickel (mg/kg)
7.9 § 0.09 1.4 § 0.9 10.4 § 1.3 875.34§2.1 65.67§1.2 291.83§2.7 2401.92§11.69 75.96§2.1 224.90§1.89 251.74§1.7 91.28§1.4 16.62§1.5 24,105§23.6 44.25§1.6
8.0 § 0.1 0.04§0.005 0.60§.009 125.01§1.36 11.9 § 1.1 65.60§1.95 1119.63§2.6 39.66§0.86 133.50§1.26 27.25§0.8 3.84§0.53 0.04§0.01 14.53§2.4 1.43§0.6
3.3. Translocation factor (TF), bio concentration factor (BCF) and bioaccumulation factor (BF)
*All the values are mean of triplicates § Standard deviation.
plant in roots or in above ground parts which led to its significant reduction in the post-harvest soil and sludge samples. 3.2. Quantitative estimation of heavy metal concentration in different parts of lemongrass by ICP To assess the uptake potential of lemongrass plant, a quantitative estimation was done to find out the concentration of heavy metals in its roots, leaves and clumps (post-harvest). For this plant samples (five replicates each) were analyzed through ICP-OES and following observations were made 3.2.1. Concentration of heavy metal in roots Roots were analyzed after each harvest to assess the heavy metal concentration. Maximum concentration of Cr was recorded in roots in each harvest followed by Pb, Ni and Cd respectively. Maximum Cr concentration was found in the third harvest which may be attributed to the unfavorable weather conditions in which metal uptake by roots was enhanced due to stress but it was not significantly translocated to the aerial parts as overall growth was low in winters. The heavy metal concentration in roots per harvest has been shown in Fig. 1 (a, b, c and d). 3.2.2. Concentration of heavy metals in shoots After each harvest, leaves of test plants were also analyzed to assess the heavy metal concentration. Pb concentration was found to be maximum in leaves of test plants followed by Cr, Ni and Cd. Concentration of metals in shoots in third harvest was found to be minimum; it may again be attributed to the unfavorable weather conditions which stunted the growth of plants and hence less uptake of metals was observed in shoot portion. The results obtained were in agreement to Israila et al. (2015) who reported lemongrass as a potential Pb accumulator especially in leaves. The heavy metal concentration in leaves per harvest has been shown in Fig. 1 (a, b, c and d).
The phytoremediation potential of plants can be assessed through indexes like translocation, bio-accumulation and bio concentration factor. Translocation factor signifies the ability of plants to translocate metals to aboveground parts from roots. In the present experiment, TF<1 was observed for Cr and Cd but TF>1 was observed for Pb and Ni. It indicated that significant amount of Ni and Pb was translocated from roots to shoots in test plants when grown in tannery sludge. Less translocation of some metals through roots occurs due to formation of complexes with sulfhydryl groups; the same may have been formed with Cr leading to the greater accumulation in roots and less translocation to shoots (Singh et al., 2004). In plants, due to the presence of cysteine sulfhydryl groups, complexes with metal ions are formed with various chelator groups which ultimately inhibit translocation of certain metals inside the cytosol (Malec et al., 2010). Data recorded revealed that Bioaccumulation factor (BF) was found to be greater than 1 for Pb and Ni when plants were grown in tannery sludge. It indicates that there is a significant accumulation of these metals in leaves as BF is the ratio of metal concentration in above ground compared to concentration in soil. It complemented with the results obtained from quantitative analysis by ICP. In addition to this, Bioconcentration factor (BCF) >1 was recorded for Cr. BCF is the ratio of metal concentration in the roots compared to concentration in soil. The results indicated that there is a significant accumulation of Cr in the roots of test plants. It was in agreement to the results obtained by ICP. Thus, our study confirms that lemongrass acts as a potential phytoextractor for Ni and Pb whereas it can be regarded as a phytostabilizer for Cr and Cd. Translocation factor; Bioaccumulation factor and Bioconcentration factor in different varieties of lemongrass grown in sole tannery sludge are shown in Table 2. 3.4. Histochemical localization assessment of Pb, Cd and Ni The quantitative estimation of heavy metal uptake pattern was further confirmed by histochemical detection of Pb, Cd and Ni in roots and leaves of test plants. In histochemical method for metal
Table 2 Translocation, Bioaccumulation and Bio concentration factors in four consecutive harvests of lemongrass grown in tannery sludge. Cr No. of harvests First Second Third Fourth
Cd
Pb
Ni
TF
BCF
BF
TF
BCF
BF
TF
BCF
BF
TF
BCF
BF
0.080 0.003 0.002 0.050
1.56 1.34 2.01 1.96
0.005 0.001 0.0001 0.001
0.14 0.39 0.07 0.72
0.57 0.74 0.87 0.75
0.08 0.29 0.06 0.54
0.98 1.03 1.00 0.99
0.550 0.470 0.696 0.640
1.36 1.62 1.67 1.09
0.38 0.39 1.01 0.94
0.19 0.57 0.05 0.34
0.51 1.46 1.54 1.01
*All the values are mean of triplicates.
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Fig. 1. Concentration of heavy metals in leaves and roots of lemongrass plant.
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Fig. 2. Histochemical determination of Pb accumulation in transverse sections of lemongrass roots. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
Fig. 3. Histochemical determination of Ni accumulation in transverse sections of lemongrass roots. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
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Fig. 4. Histochemical determination of Ni accumulation in vertical sections of lemongrass leaves. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
detection in plant tissues, the reagent used forms a complex with metals present in the sample and can be visualized under a microscope because the colored complexes formed reflect the distribution of the metal within the tissues (Seregin and Kozhevnikova, 2011). 3.4.1. Assessment of Pb and Cd in root and leaf Dithizone stain forms red to blue-black complexes with Pb and Cd ions present in the sample tissue (Seregin and Ivanov method, 1997). When transverse sections of roots of plants grown in tannery sludge were stained with dithizone, maximum staining was found in endodermis to pericycle cells and in xylem elements.
Dark coloured complexes were also observed at endodermis layer (Fig. 2d, e, f). Intensity of staining indicates the significant accumulation of Pb and Cd in plant tissues (Baranowska et al., 2004). Dark pigmentation was also found in epidermis layer of plants grown in TS when compared to control plants. Staining in xylem elements can be attributed to the fact that metals are being transported to above ground parts of the plant along with water and solutes. No such pigmentation and deposits were seen in roots of control plants which indicated that these were due to lead and cadmium deposition (Fig. 2a, b, c). Similar observations were made by Baranowska-Morek and Wierzbicka, 2004 in which they
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Fig. 5. Histochemical determination of Pb accumulation in vertical sections of lemongrass leaves. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
observed dark coloured lead deposits in the pericycle region of Dianthus carthusianorum root tissues. When vertical sections of leaves of test plants were stained with dithizone for Pb and Cd localization assessment, dark bluish black crystals were found near the stoma and scattered in mesophyll cells of plants grown in tannery sludge. These deposits were not seen in leaf sections of control plants which confirmed the presence of Pb and Cd in leaves of test plants (Fig. 5).
3.4.2. Assessment of Ni localization in root and leaf Staining of transverse root sections of test plants with dimethylglyoxime revealed the presence of nickel localization by formation of reddish brown nickel dimethylglyoximine crystals. In roots of plants grown in tannery sludge, such complexes were seen at the endodermis layer and in the cortical cells. Ni made its way through the endodermal barrier and accumulated in the endodermis and pericycle region. It was followed by staining in xylem and phloem elements. Dark staining
J. Pandey et al. / South African Journal of Botany 131 (2020) 74 83
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Fig. 6.1. Determination of Cr localization in T.S. of lemongrass roots by confocal microscopy. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Scale bar is equal to 100 mm.
in xylem elements justified the translocation of metal to above ground parts through water transport (Fig. 3). Such dark complexes were also found in the epidermis layer. Similar observations were made by Seregin et al., 2003 in maize roots. They proposed that Ni accumulation in the endodermis cells and often in the pericycle is the characteristic feature of tissue Ni distribution in different root regions. To ascertain Ni localization in leaves of test plants, vertical sections of leaves were stained with dimethylglyoxime. In leaf sections
of plants grown in tannery sludge, localization of Ni in the form dark red coloured complexes (nickel dimethylglyoximine) were found beside the stoma region. This was in agreement to the observation that nickel was being translocated to the leaves by roots. No such deposits were found in leaves of control plants which confirmed their presence as Ni deposits (Fig. 4). Thus, histochemical investigation confirmed the results obtained by quantitative analysis performed through ICP-OES.
Fig. 6.2. SEM-Energy dispersive X-ray analysis (EDAX) micrographs showing Cr accumulation in roots of lemongrass (a) grown in garden soil (b) grown in tannery sludge.
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more water and nutrients storage. Similar results were obtained in trichomes of H. annuus plants grown on 50% and 100% tannery sludge by Singh and Sinha, 2004. 4. Conclusions Our study provides an insight into the localization and uptake pattern of heavy metals in lemongrass plants when grown in heavy metal polluted sites. Results obtained were confirmed quantitatively as well as through various microscopic techniques. Morphological investigations made through SEM analysis revealed the increase in trichome size of test plants. It could be inferred from this observation that heavy metal stress causes enlargement of trichomes which could lead to increase in essential oil content of lemongrass plant when grown in heavy metal contaminated sites. It can also be concluded from the results that lemongrass plant serves as a potential phytoextractor for nickel and lead; and a phytostabilizer for chromium and cadmium. Hence, this plant can be successfully grown in heavy metal contaminated sites for serving two consecutive purposes at the same time i.e. phytoremediation with the least risk of food chain contamination along with increase in essential oil yield owing to monetary benefits. Declaration of Competing Interest No conflict of interest is declared by the authors. Acknowledgement
Fig. 7. Scanning electron micrographs showing variation in size of trichomes in lemongrass plants (a) grown in garden soil (b) grown in tannery sludge.
3.5. Determination of Cr accumulation in lemongrass roots through confocal microscopy and SEM-EDAX For determination of Cr accumulation in roots of test plants, transverse sections of roots of test and control plants were stained by 1, 5- diphenylcarbazide work solution and observed under confocal microscope. In roots of plants grown in tannery sludge, maximum staining was found in epidermal layer region (rhizodermal region) and in the cortical cells as compared to control plants (Fig. 6.1.). It confirmed that staining was due to Cr accumulation in test plants. Similar observations were made by Kovacik et al., 2014 in chamomile roots. SEM- Energy dispersive X-ray analysis (EDAX) was also done to assess the elemental composition of root samples of control and test plants. It gives elemental information by analysis of X-ray emissions caused by a high-energy electron beam. Signal against Cr was observed in roots of plants grown in tannery sludge whereas no signal against Cr was observed in the control sample indicating the absence of Cr ion binding (Fig. 6.2.). This analysis further confirmed the observations in which Cr was found to be mainly accumulated in roots of test plants in comparison to other heavy metals. 3.6. Scanning electron microscopy to determine morphological changes Certain morphological changes were observed when leaf sections were observed under scanning electron microscopy, between control and test plants. Trichomes in leaves of plants grown in tannery sludge were found to be swollen at base and larger in size when compared to those of control plants (Fig. 7). The expansion in the size and the swelling of the base of trichomes may be ascribed to a strategy for
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Sub-cellular localization and quantitative estimation of heavy metals in lemongrass plants grown in multi-metal contaminated tannery sludge Janhvi Pandeya,b, Sougata Sarkarc, Rajesh Kumar Vermaa,b, Saudan Singha,b,* a
Academy of Scientific and Innovative Research (AcSIR), India Division of Agronomy & Soil Science, CSIR-Central Institute of Medicinal and Aromatic Plants (Council of Scientific and Industrial Research) PO- CIMAP, Lucknow 220615 Uttar Pradesh, India c Genetic Resources and Agro-Technology Division, CSIR-Indian Institute of Integrative Medicine (IIIM), Jammu 180001, India b
A R T I C L E
I N F O
Article History: Received 12 July 2018 Revised 11 September 2019 Accepted 24 January 2020 Available online xxx Edited by M Vaculik Keywords: Heavy metals Histochemical Fluorescence Lemongrass Localization Phytoextractor
A B S T R A C T
Major objective of the study was to assess the localization pattern of heavy metals in different parts of lemongrass plant when grown in multi-metal contaminated sites. Microscopic and quantitative investigations (by ICP-OES) were carried out in root and leaves of plant to examine the compartmentalization of heavy metals. Along with it, the morphological variations in the size of trichomes in test plants were also perceived. Differential distribution of Cr, Pb, Ni and Cd was observed in root and leaf sections. Localization of these heavy metals in different parts of the plant cultivated in sole tannery sludge was compared with the plants grown in garden soil; which served as control. Histochemical methods for Pb, Cd and Ni detection revealed their significant accumulation in the root and leaf sections. Cr accumulation in roots of test plants was confirmed by fluorescence staining. Translocation factor <1 was recorded for Cr but greater than 1 for Ni and Pb. Moreover, Bioaccumulation factor >1 was observed for Ni and Pb. SEM studies revealed that the size of trichomes enlarged in plants grown in tannery sludge. Hence, our study provides an insight into the localization pattern of heavy metals in lemongrass plants and suggests that lemongrass can serve as a Ni and Pb phytoextractor when grown in multi- metal contaminated sites. In addition, enhancement in the size of trichomes was detected due to heavy metal stress which may prompt increment in essential oil yield thus; cultivation of lemongrass in heavy metal contaminated sites can also prove profitable economically. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction In the present era of industrialization, the tremendous amount of untreated waste from industries is posing a serious threat to the ecological wellbeing. The problem of pollution generated by heavy metals is among one of these risks hence, it needs to be addressed at a priority (Kumar et al., 2018). Leather industry is among one of the significant contributors of hazardous waste, particularly heavy metals (Kocurek et al., 2015). Sludge from tannery industry contains significant amounts of heavy metals especially Cr, Pb, Ni and Cd. At present, a proper sludge disposal system is lacking in many countries worldwide. A major threat for environment is the presence of several landfills containing heavy metal rich sludge from these industries (Ahmad and Misra, 2014). The impact of heavy metals on plants requires significant consideration in relation to expanding ecological Abbreviations: ICP-OES, Inductively Coupled Plasma-Optical Emission Analyzer; SEM, Scanning Electron Microscopy * Corresponding author at: Division of Agronomy & Soil Science, CSIR-Central Institute of Medicinal and Aromatic Plants (Council of Scientific and Industrial Research) PO- CIMAP, Lucknow 220615 Uttar Pradesh, India. E-mail address: [email protected] (S. Singh). https://doi.org/10.1016/j.sajb.2020.01.034 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
contamination problem. In this context, it is critical to examine the compartmentalization of retained heavy metals in different parts of the plant (Seregin and Kozhevnikova, 2011). Diverse plant species respond distinctively to toxicity caused by heavy metals likeaccumulation in various parts, prohibition, chelation or immobilization (Yousefi et al., 2018). For some plant species, the concentration of heavy metals in their aboveground parts are generally lower than the accumulation in roots, which is controlled by the hindrance capacity of the root framework (Carrier et al., 2003; Yuan et al., 2011). Histochemical methods may greatly help to investigate metal localization, distribution and accumulation in plant tissues. These methods provide information that supplements the results of quantitative analysis. It is important that the results of histochemical analysis should always be compared with the data obtained by other methods like SEM-EDAX, ICP, etc. Studies of metal distribution and accumulation in plants should rest on the combination of these methods in order to produce an integral pattern of translocation fluxes of the metal under investigation (Seregin and Kozhevnikova, 2011). Aromatic grasses can be grown in multi-metal contaminated areas as they serve the purpose of phytoremediation along with obtaining monetary benefits without the risk of food chain contamination (Lal
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et al., 2013). Lemongrass (Cymbopogon flexuosus (Nees ex Steud.) W. Watson) is an enduring perennial aromatic grass which produces a huge biomass. Essential oil is the main by-product of this crop for which it is widely cultivated all over the world (Zheljazkov et al., 2011). It is a metal-tolerant plant that withstands harsh environmental conditions (Das and Maiti, 2009). Lemongrass has been recommended for the phytoremediation of metal-polluted sites as it is a potential metal accumulator especially Cd, Ni and Pb (Israila et al., 2015). Though lemongrass has been recommended as a potential metal accumulator yet no significant work has been done to study the localization pattern of heavy metals specifically in its plant parts. Our present study has been performed to investigate the localization of heavy metals (Cr, Ni, Pb and Cd) in different parts of lemongrass plant when grown in multi-metal contaminated tannery sludge. For this, microscopy techniques (histochemical, SEM-EDAX and confocal) have been used and further confirmed by quantitative estimation (through ICP-OES). This study can provide an insight into the heavy metal compartmentalization pattern in lemongrass plant when grown in contaminated sites and the effect of heavy metal stress on the size of trichomes as well. 2. Materials and methods 2.1. Cultivation of experimental test plants For the present trial, slips of lemongrass (variety- Suwarna) cultivated in nurseries were utilized. The slips were transplanted in 140 kg capacity cemented barrels present in the experimental farm of CSIR-Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow, India for the duration of one year (2017 2018). The barrels were filled with garden soil and sole tannery sludge respectively. Cemented barrels were used to mimic field conditions without the risk of horizontal leaching of metal concentrations from tannery sludge during irrigations. Tannery sludge was collected from Common Effluent Treatment Plant (CETP) located nearby CSIR-Central Leather Research Institute (CLRI resource center), Kanpur (India). It was processed and filled in the barrels and then left for 30 days incubation period before the slips were planted. Plants grown in garden soil served as control and both control and test plants were replicated five times each. Data of four harvests were recorded during the duration of one year. 2.2. Microscopy procedures for determining heavy metal localization in plant parts
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(Seregin et al., 2003). Five replicates of each sample were taken for control and test plants. 1% solution of dimethylglyoxime in 1.5% solution of NaOH in 0.05 M borax (pH 9.8 10.4) was made. It forms nickel dimethylglyoximine, a reddish-brown complex with nickel (II). Three to four drops of dimethylglyoxime were added on root and leaf sections and observed under Leica DM 750 microscope equipped with a Leica ICC 50 HD camera and LAS EZ software. 2.2.3. Fluorescence staining of chromium Method given by Kovacik et al. (2014) was used for the fluorescence visualization of Cr in root sections. A working solution of 0.25% 1, 5-diphenylcarbazide was made in acetone diluted with distilled water which was then diluted to 0.05% with hydrochloric acid/potassium chloride buffer (0.1 M, pH 1.8). Transverse sections of roots were incubated in the working solution for 30 min at RT. After incubation, washing of sections with hydrochloric acid/potassium chloride buffer was done and observed using a Zeiss LSM880 confocal microscope equipped with a multiline Argon Laser and ZEN BLACK software. Images were collected at 500 to 560 nm (FITC) using excitation at a wavelength of 488 nm from an argon laser. 2.2.4. Scanning electron microscopy and energy dispersive X-ray analysis Morphological variations in trichome size in lemongrass leaves were observed by SEM analysis. Leaf and root sections were fixed with 2% glutaraldehyde maintained in phosphate buffer saline (0.1 M, pH 6.8) for 12 18 h at 40 °C; washed and fixed for 2 h in osmium tetra oxide (1%) in PBS at 40 °C. The specimen was washed with phosphate buffer; dehydrated in a series of ethanol water solutions (30%, 50%, 70% and 90% and 100% ethanol) and dried for 20 min. Mounting was performed on aluminum stubs, and the cells were coated with 90 nm thick palladium coating in polar sputter coater for 10 min. Coated sections were viewed at 15 kV under scanning electron microscope equipped with EDAX (JEOL JSM-6490LV). 2.3. Heavy metal analysis using ICP-OES Root and shoot samples of plants were washed with 0.1 N HCl, deionized water and then with distilled water. They were then oven dried at 70 °C. 0.25 g of powdered sample was acid digested (HNO3: HClO4 = 10:4) for heavy metal analysis via ICP-OES, Perkin-Elmer model 53,000 V (Piper, 1966, Page et al., 1992, Jones 1990). 2.4. Statistical analysis
To carry out microscopy procedures, leaves and root samples were collected prior to each harvest from control and test plants (from all five replicates). 2.2.1. Histochemical determination of lead and cadmium Seregin and Ivanov method (1997) was used to determine Cd and Pb speciation in the transverse sections of roots and vertical sections of leaves in test plants. Five replicates of each sample were observed for control and test plants. Hand cut root and leaf sections were stained by dithizone. Dithizone solution was made in acetone (0.5 mg/ml) which was then diluted with distilled water in 3:1 ratio and few drops of glacial acetic were added in the solution to prevent the leaching out of metals due to solvent medium. The sections were then placed on a glass slide and stained by adding a drop of dithizone solution. Dark red to blue-black coloured cadmium and lead dithizonate crystals were observed using a Leica DM 750 microscope equipped with a Leica ICC 50 HD camera and LAS EZ software. 2.2.2. Histochemical determination of nickel For determination of nickel localization in the root and leaf sections of test plants, modified dimethylglyoxime method was used
Normality distribution in data sets was evaluated using Shapiro Wilk test. Owing to the non-normal distribution of data sets, nonparametric Kruskal Wallis ANOVA was performed. Each data point in Figures represents an average value. Standard deviation obtained from five replicates for each sample is shown in the Figures as an error bar using the Origin version 8.0 software. 3. Results and discussions 3.1. Initial and post-harvest physico-chemical and heavy metal concentration status in tannery sludge and garden soil The initial status (prior to planting) of tannery sludge and garden soil comprising physico-chemical parameters and heavy metal concentration has been given in Table 1. A marked reduction in heavy metal concentration was recorded after each harvest. After final harvest, a percentage reduction of 46.57% in Cr concentration, 30.22% in Cd, 41.89% in Ni and 37.94% reduction in Pb concentration, in post-harvest sludge samples was observed. It can be attributed to the uptake of these metals by test
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3.2.3. Concentration of heavy metals in clumps at the time of harvest After final harvest, clumps of test plants were also analyzed for their heavy metal concentration. The data recorded revealed an interesting result that maximum concentration of Pb followed by Cr, Ni and Cd was found in clumps. Pb and Cr concentration was found in significant amounts in control plants too. It supports the fact that Pb was being translocated to upper parts of lemongrass plants which was justified by maximum concentration of Pb in leaves of test plants but there was less translocation of Cr in upper parts; it may be attributed to complex formation of Cr with sulfhydryl groups which inhibited their movement (Malec et al., 2010). As clump gives rise to shoots repeatedly, hence the translocation rate of metals could be greater in this portion of the plant. We tried to explore this hypothesis. Concentration of heavy metals in clumps followed the same pattern as leaves in tests plants which is shown in Fig. 1.
Table 1 Chemical characteristics, heavy metals and micronutrient concentration in tannery sludge and garden soil. Parameters
Tannery sludge
Garden soil
pH (1:2.5) Electrical conductivity (ds/m) Organic Carbon (%) Available Nitrogen (mg/kg) Available Phosphorous (mg/kg) Available Potassium (mg/kg) Iron (mg/kg) Copper (mg/kg) Manganese (mg/kg) Zinc (mg/kg) Lead (mg/kg) Cadmium (mg/kg) Chromium (mg/kg) Nickel (mg/kg)
7.9 § 0.09 1.4 § 0.9 10.4 § 1.3 875.34§2.1 65.67§1.2 291.83§2.7 2401.92§11.69 75.96§2.1 224.90§1.89 251.74§1.7 91.28§1.4 16.62§1.5 24,105§23.6 44.25§1.6
8.0 § 0.1 0.04§0.005 0.60§.009 125.01§1.36 11.9 § 1.1 65.60§1.95 1119.63§2.6 39.66§0.86 133.50§1.26 27.25§0.8 3.84§0.53 0.04§0.01 14.53§2.4 1.43§0.6
3.3. Translocation factor (TF), bio concentration factor (BCF) and bioaccumulation factor (BF)
*All the values are mean of triplicates § Standard deviation.
plant in roots or in above ground parts which led to its significant reduction in the post-harvest soil and sludge samples. 3.2. Quantitative estimation of heavy metal concentration in different parts of lemongrass by ICP To assess the uptake potential of lemongrass plant, a quantitative estimation was done to find out the concentration of heavy metals in its roots, leaves and clumps (post-harvest). For this plant samples (five replicates each) were analyzed through ICP-OES and following observations were made 3.2.1. Concentration of heavy metal in roots Roots were analyzed after each harvest to assess the heavy metal concentration. Maximum concentration of Cr was recorded in roots in each harvest followed by Pb, Ni and Cd respectively. Maximum Cr concentration was found in the third harvest which may be attributed to the unfavorable weather conditions in which metal uptake by roots was enhanced due to stress but it was not significantly translocated to the aerial parts as overall growth was low in winters. The heavy metal concentration in roots per harvest has been shown in Fig. 1 (a, b, c and d). 3.2.2. Concentration of heavy metals in shoots After each harvest, leaves of test plants were also analyzed to assess the heavy metal concentration. Pb concentration was found to be maximum in leaves of test plants followed by Cr, Ni and Cd. Concentration of metals in shoots in third harvest was found to be minimum; it may again be attributed to the unfavorable weather conditions which stunted the growth of plants and hence less uptake of metals was observed in shoot portion. The results obtained were in agreement to Israila et al. (2015) who reported lemongrass as a potential Pb accumulator especially in leaves. The heavy metal concentration in leaves per harvest has been shown in Fig. 1 (a, b, c and d).
The phytoremediation potential of plants can be assessed through indexes like translocation, bio-accumulation and bio concentration factor. Translocation factor signifies the ability of plants to translocate metals to aboveground parts from roots. In the present experiment, TF<1 was observed for Cr and Cd but TF>1 was observed for Pb and Ni. It indicated that significant amount of Ni and Pb was translocated from roots to shoots in test plants when grown in tannery sludge. Less translocation of some metals through roots occurs due to formation of complexes with sulfhydryl groups; the same may have been formed with Cr leading to the greater accumulation in roots and less translocation to shoots (Singh et al., 2004). In plants, due to the presence of cysteine sulfhydryl groups, complexes with metal ions are formed with various chelator groups which ultimately inhibit translocation of certain metals inside the cytosol (Malec et al., 2010). Data recorded revealed that Bioaccumulation factor (BF) was found to be greater than 1 for Pb and Ni when plants were grown in tannery sludge. It indicates that there is a significant accumulation of these metals in leaves as BF is the ratio of metal concentration in above ground compared to concentration in soil. It complemented with the results obtained from quantitative analysis by ICP. In addition to this, Bioconcentration factor (BCF) >1 was recorded for Cr. BCF is the ratio of metal concentration in the roots compared to concentration in soil. The results indicated that there is a significant accumulation of Cr in the roots of test plants. It was in agreement to the results obtained by ICP. Thus, our study confirms that lemongrass acts as a potential phytoextractor for Ni and Pb whereas it can be regarded as a phytostabilizer for Cr and Cd. Translocation factor; Bioaccumulation factor and Bioconcentration factor in different varieties of lemongrass grown in sole tannery sludge are shown in Table 2. 3.4. Histochemical localization assessment of Pb, Cd and Ni The quantitative estimation of heavy metal uptake pattern was further confirmed by histochemical detection of Pb, Cd and Ni in roots and leaves of test plants. In histochemical method for metal
Table 2 Translocation, Bioaccumulation and Bio concentration factors in four consecutive harvests of lemongrass grown in tannery sludge. Cr No. of harvests First Second Third Fourth
Cd
Pb
Ni
TF
BCF
BF
TF
BCF
BF
TF
BCF
BF
TF
BCF
BF
0.080 0.003 0.002 0.050
1.56 1.34 2.01 1.96
0.005 0.001 0.0001 0.001
0.14 0.39 0.07 0.72
0.57 0.74 0.87 0.75
0.08 0.29 0.06 0.54
0.98 1.03 1.00 0.99
0.550 0.470 0.696 0.640
1.36 1.62 1.67 1.09
0.38 0.39 1.01 0.94
0.19 0.57 0.05 0.34
0.51 1.46 1.54 1.01
*All the values are mean of triplicates.
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Fig. 1. Concentration of heavy metals in leaves and roots of lemongrass plant.
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Fig. 2. Histochemical determination of Pb accumulation in transverse sections of lemongrass roots. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
Fig. 3. Histochemical determination of Ni accumulation in transverse sections of lemongrass roots. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
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Fig. 4. Histochemical determination of Ni accumulation in vertical sections of lemongrass leaves. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
detection in plant tissues, the reagent used forms a complex with metals present in the sample and can be visualized under a microscope because the colored complexes formed reflect the distribution of the metal within the tissues (Seregin and Kozhevnikova, 2011). 3.4.1. Assessment of Pb and Cd in root and leaf Dithizone stain forms red to blue-black complexes with Pb and Cd ions present in the sample tissue (Seregin and Ivanov method, 1997). When transverse sections of roots of plants grown in tannery sludge were stained with dithizone, maximum staining was found in endodermis to pericycle cells and in xylem elements.
Dark coloured complexes were also observed at endodermis layer (Fig. 2d, e, f). Intensity of staining indicates the significant accumulation of Pb and Cd in plant tissues (Baranowska et al., 2004). Dark pigmentation was also found in epidermis layer of plants grown in TS when compared to control plants. Staining in xylem elements can be attributed to the fact that metals are being transported to above ground parts of the plant along with water and solutes. No such pigmentation and deposits were seen in roots of control plants which indicated that these were due to lead and cadmium deposition (Fig. 2a, b, c). Similar observations were made by Baranowska-Morek and Wierzbicka, 2004 in which they
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Fig. 5. Histochemical determination of Pb accumulation in vertical sections of lemongrass leaves. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Arrows indicate localization of metal. Scale bar is equal to 50 mm.
observed dark coloured lead deposits in the pericycle region of Dianthus carthusianorum root tissues. When vertical sections of leaves of test plants were stained with dithizone for Pb and Cd localization assessment, dark bluish black crystals were found near the stoma and scattered in mesophyll cells of plants grown in tannery sludge. These deposits were not seen in leaf sections of control plants which confirmed the presence of Pb and Cd in leaves of test plants (Fig. 5).
3.4.2. Assessment of Ni localization in root and leaf Staining of transverse root sections of test plants with dimethylglyoxime revealed the presence of nickel localization by formation of reddish brown nickel dimethylglyoximine crystals. In roots of plants grown in tannery sludge, such complexes were seen at the endodermis layer and in the cortical cells. Ni made its way through the endodermal barrier and accumulated in the endodermis and pericycle region. It was followed by staining in xylem and phloem elements. Dark staining
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Fig. 6.1. Determination of Cr localization in T.S. of lemongrass roots by confocal microscopy. Hand-cut transverse section of roots was done (five replicates for each sample) and representative photos are shown. Scale bar is equal to 100 mm.
in xylem elements justified the translocation of metal to above ground parts through water transport (Fig. 3). Such dark complexes were also found in the epidermis layer. Similar observations were made by Seregin et al., 2003 in maize roots. They proposed that Ni accumulation in the endodermis cells and often in the pericycle is the characteristic feature of tissue Ni distribution in different root regions. To ascertain Ni localization in leaves of test plants, vertical sections of leaves were stained with dimethylglyoxime. In leaf sections
of plants grown in tannery sludge, localization of Ni in the form dark red coloured complexes (nickel dimethylglyoximine) were found beside the stoma region. This was in agreement to the observation that nickel was being translocated to the leaves by roots. No such deposits were found in leaves of control plants which confirmed their presence as Ni deposits (Fig. 4). Thus, histochemical investigation confirmed the results obtained by quantitative analysis performed through ICP-OES.
Fig. 6.2. SEM-Energy dispersive X-ray analysis (EDAX) micrographs showing Cr accumulation in roots of lemongrass (a) grown in garden soil (b) grown in tannery sludge.
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more water and nutrients storage. Similar results were obtained in trichomes of H. annuus plants grown on 50% and 100% tannery sludge by Singh and Sinha, 2004. 4. Conclusions Our study provides an insight into the localization and uptake pattern of heavy metals in lemongrass plants when grown in heavy metal polluted sites. Results obtained were confirmed quantitatively as well as through various microscopic techniques. Morphological investigations made through SEM analysis revealed the increase in trichome size of test plants. It could be inferred from this observation that heavy metal stress causes enlargement of trichomes which could lead to increase in essential oil content of lemongrass plant when grown in heavy metal contaminated sites. It can also be concluded from the results that lemongrass plant serves as a potential phytoextractor for nickel and lead; and a phytostabilizer for chromium and cadmium. Hence, this plant can be successfully grown in heavy metal contaminated sites for serving two consecutive purposes at the same time i.e. phytoremediation with the least risk of food chain contamination along with increase in essential oil yield owing to monetary benefits. Declaration of Competing Interest No conflict of interest is declared by the authors. Acknowledgement
Fig. 7. Scanning electron micrographs showing variation in size of trichomes in lemongrass plants (a) grown in garden soil (b) grown in tannery sludge.
3.5. Determination of Cr accumulation in lemongrass roots through confocal microscopy and SEM-EDAX For determination of Cr accumulation in roots of test plants, transverse sections of roots of test and control plants were stained by 1, 5- diphenylcarbazide work solution and observed under confocal microscope. In roots of plants grown in tannery sludge, maximum staining was found in epidermal layer region (rhizodermal region) and in the cortical cells as compared to control plants (Fig. 6.1.). It confirmed that staining was due to Cr accumulation in test plants. Similar observations were made by Kovacik et al., 2014 in chamomile roots. SEM- Energy dispersive X-ray analysis (EDAX) was also done to assess the elemental composition of root samples of control and test plants. It gives elemental information by analysis of X-ray emissions caused by a high-energy electron beam. Signal against Cr was observed in roots of plants grown in tannery sludge whereas no signal against Cr was observed in the control sample indicating the absence of Cr ion binding (Fig. 6.2.). This analysis further confirmed the observations in which Cr was found to be mainly accumulated in roots of test plants in comparison to other heavy metals. 3.6. Scanning electron microscopy to determine morphological changes Certain morphological changes were observed when leaf sections were observed under scanning electron microscopy, between control and test plants. Trichomes in leaves of plants grown in tannery sludge were found to be swollen at base and larger in size when compared to those of control plants (Fig. 7). The expansion in the size and the swelling of the base of trichomes may be ascribed to a strategy for
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