Physiological aspects of vetiver grass for rehabilitation in abandoned metalliferous mine wastes

Chemosphere 52 (2003) 1559 1570 www.elsevier.com/locate/chemosphere Physiological aspects of vetiver grass for rehabilitation in abandoned metalliferous mine wastes J. Pang a, G.S.Y. Chan b, J. Zhang c, J. Liang a, *, M.H. Wong c a College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, PR China b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Hom, Hong Kong c Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong Abstract Physiological aspects of why vetiver grass (Vetiveria zizanioides L.) can be tolerant to heavy metals and be used as an alternative method for rehabilitation of abandoned metalliferous mine wastelands have been investigated. The results showed that high proportions of lead and zinc (Pb/Zn) tailing greatly inhibited the leaf growth, dry matter accumulation, and photosynthesis of leaves, but stimulated the accumulation of proline and abscisic acid (ABA), and enhanced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), implying that different mechanisms to detoxify active oxygen species (AOS) existed in different parts of plants. Physiological responses to heavy metal treatments differed greatly between roots and shoots. Nitrogen fertilizer application could greatly alleviate the adverse effects of high proportions of Pb/Zn tailing on vetiver grass growth. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Pb/Zn tailing; Physiological responses; Rehabilitation of abandoned metalliferous mine wastes; Vetiver grass 1. Introduction The heavy metals contamination of the environment by soil erosion in agricultural lands, urban wastes and by-products of rural, industrial and mining industries attracts world-wide concern, especially in developing countries (Tordoff et al., 2000; Mejare and B ulow, 2001). In China, there are many abandoned metalliferous mine wastelands and the areas become larger and larger (Young, 1988). Economically there is an urgency to decontaminate or re-vegetate the mine wastelands in order to improve environment. Although there are many methods used to treat them, most of them are either expensive or impossible to carry out, as the volume of contaminated material is very large, such as the coal mine tailings (Salomons et al., 1995). Therefore, a more * Corresponding author. Tel.: +86-514-797-9320, fax: +86-514-799-1747. E-mail address: jsliang@yzu.edu.cn (J. Liang). economical and practical approach is urgently needed at present, especially for the developing countries. Vegetative methods are thought to be the most practical and economical method for rehabilitation of the mine wastelands (Flathman and Lanza, 1998). However, re-vegetation of these sites is often difficult and slow due to the hostile growing conditions, which include toxic levels of heavy metals. Therefore, selection or screening of plant species which are tolerant to toxic levels of heavy metals has attracted much attention in the treatment of the abandoned mine wastelands (Chaney et al., 1997; Salt et al., 1998). There are a wealthy of evidence to show that vetiver grass is highly tolerant to the hostile soil conditions and widely used as a natural, effective, and low-cost alternative mean to vegetate the heavy metal-contaminated lands (Truomg, 1996). The aim of this paper is to investigate the physiological responses of vetiver grass to heavy metals. The experiments were carried out in greenhouse, where vetiver plants were grown in different proportions of lead/ zinc (Pb/Zn) tailings collected from the abandoned mine 0045-6535/03/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/s0045-6535(03)00496-x

1560 J. Pang et al. / Chemosphere 52 (2003) 1559 1570 near Guangdong province, China. The results showed that vetiver plants grown well in suitable proportions of tailings-contained soil medium, and there exited doseand time-effects of the responses of vetiver plants to Pb/ Zn-tailings, and when being grew in the high proportion of tailings-contained soil and/or for a extended period of treatment, the growth of vetiver plants was significantly influenced. The growth of vetiver plants when grew in high tailing-contained soil could be greatly improved if nitrogen fertilizer is applied. 2. Materials and methods 2.1. Plant materials and treatments Vetiver plants (Vetiveria zizanioides L.) were provided by Zhongshan University, Guangzhou and precultivated in the John Innes No. 2 soil compost for about 30 days when the vetiver grasses grown well. One hundred and fifty uniform-sized plants (30 plants per treatment) were selected and five treatments were designed (30 plants for each treatment). Plants were transplanted into the plastic pots (18 cm in diameter, 25 cm in height; one plant per pot) containing different proportions of Pb/Zn tailing (by weight): 100% of John Innes No. 2 soil compost (JI-2) (Liang et al., 1996) (treatment 1), 25% Pb/Zn + 75% JI-2 (treatment 2), 50% Pb/Zn tailing + 50% JI-2 (treatment 3), 100% Pb/Zn tailing (treatment 4), and 100% Pb/Zn tailing + 0.1 g nitrogen fertilizer ((NH 4 ) 2 SO 4 ) (treatment 5), respectively. Plants were grown in greenhouse at the temperature of 25 28 C, 16/8 h (D/N) photoperiod and a photosynthetic photo-flux density of 300 lmol m 2 s 1 and watered daily. The numbers of the tillers per plant were recorded. 2.2. Measurements of leaf growth and biomass of the plants The growing leaves with the same leaf-age were labeled from each plant and the leaf length was measured at an interval of 10 days with a ruler and the leaf growth rate was calculated accordingly. At day 30 and 50 after treatment, five plants were harvested and washed with tap water thoroughly. The shoots and roots were separated and oven-dried to a constant dry weight for the measurements of biomass and heavy metals. 2.3. Measurements of photosynthetic rate and chlorophyll fluorescence Twelve fully expanded leaves were selected from each treatment for the measurements of leaf photosynthetic rate, chlorophyll fluorescence, enzyme activities, etc. Photosynthetic rate (A) of the fully expanded leaves were measured with a gas exchange system (CIRAA-1, PP System, Hitchin, Herts, UK) at ambient CO 2 concentration and light intensity 400 lmol quanta m 2 s 1. Leaf fluorescence emission was measured on darkadapted leaves with a plant efficiency analyzer (PEA System Hansatech, Norfolk, UK). The results of the measurement were computed by the equipment as F v =F m, a ratio of maximal variable fluorescence out of fully light-saturated peak fluorescence. Variable fluorescence is subtracted from peak fluorescence with a constant fluorescence of dark-adapted leaves. Dark-adaptation was achieved with specially designed, light-proof clips attached on leaves for at least 30 min (Liang et al., 1997). 2.4. Measurements of water potential and electric conductivity of leaves Leaf water potential was measured with a pressure chamber (Model 3000, Soil Moisture Equipment Co., USA). Excised leaves were immediately placed into the chamber lined with a moisture filter paper around the inner wall to minimize evaporative water loss and pressurized to the balance pressure. For measurement of electric conductivity of leaves, leaves were excised from plants and cut into 0.5 cm segments and weighed. After being washed with distilled water for at least three times, the leaf segments were washed with deionized water for three times, and then transferred into 25 ml beakers containing 10 ml deionized water. The leaf segments were vacuum-treated for 15 min leaf segments to be immersed fully into water. The electric conductivity was measured with an electric conductivity meter after 4 h under room temperature (EC1). Thereafter, the beakers containing leaf segments were placed into a boiling water bath for 15 min and then cooled to room temperature, second measurement was made as above (EC2). The percentage of electrolyte leakage was calculated as (EC1/EC2) 100. 2.5. Enzyme extraction and assays Leaf samples were harvested at a given sampling time and plunged into liquid nitrogen and stored at )80 C pending enzyme assay. For enzyme extraction, frozen leaves (0.5 g) were grinded in liquid nitrogen in a precold mortar. Ten ml of 50 mm pre-cold PBS buffer (ph 7.2) containing 1 mm EDTA and 1% (w/v) PVP was added to the mortar and homogenized. The homogenate was then filtered through four layers of cheesecloth and centrifuged for 20 min at 15 000 g and 4 C. The supernatant was used for enzyme assays without further purification. Superoxide dismutase (SOD) (EC 1.15.1.1) activity was analyzed based on the procedure described by Beauchamp and Fridovich (1971) with minor modifica-

J. Pang et al. / Chemosphere 52 (2003) 1559 1570 1561 tion. In brief, the reaction mixture contained 50 mm PBS (ph 7.8), 8.6 lmol/l methionine, 42 lmol/l NBT, 1.3 lmol/l riboflavin, and 0.1 mmol/l EDTA, 50 ll crude enzyme extract. The reaction was initiated by placing the test tubes containing reaction mixture under 100 lmol quanta m 2 s 1 of light intensity for 15 min later and the color formed was determined spectrophotometrically at 560 nm. One unit of SOD activity is defined as the amount of enzyme required to inhibit 50% of the color formation. Analyses of peroxidase (POD) (EC. 1.11.1.7) and catalase (CAT) (EC. 1.11.1.6) activities were performed using guaiacol and H 2 O 2 substrates, respectively, as described by Chance and Maehly (1995). 2.6. Quantification of leaf ABA The concentration of abscisic acid (ABA) in the leaves was determined by methods of the radio immunoassay (RIA) according to the procedure described by Liang et al. (1996). 2.7. Chlorophyll determination The chlorophyll content was determined in 80% acetone extract of 0.2 g of dry weight of leaves based on the procedure described by Arnon (1949). 3. Results Pb/Zn mine tailing contains high levels of heavy metals, especially Pb and Zn (Table 1), which are toxic to plants or inhibitory to plant growth. Experiment was conducted with different proportions of tailings to determine the tolerance of vetiver grass to high levels of heavy metals. Vetiver plants were transplanted into pots containing different proportions of tailings, and Table 1 Heavy metal contents of different proportions of mine tailings (lgg 1 dry weight) Heavy metals Treatment 1 (100% tailing) Treatment 2 (50% tailing + 50% soil) Treatment 3 (25% tailing + 75% soil) Treatment 4 (100% soil) Pb 1980 1110 530 100 Zn 1700 1000 550 150 Fe 36 870 28 280 17 450 13 550 Cu 30 20 10 10 Tailings collected from the abandoned mine near Guangdong province, China. Fig. 1. Effects of different proportions of Pb/Zn tailings on tillering capacity of vetiver grass. Filled circles indicated control treatment (100% soil), empty circles: 75% soil plus 25% Pb/Zn tailing, filled triangles: 50% soil plus 50% Pb/Zn tailing, empty triangles: 100% Pb/Zn tailing, and filled squares: 100% Pb/Zn tailing plus nitrogen fertilizer. Data shown are the means of five plants ± SD.

1562 J. Pang et al. / Chemosphere 52 (2003) 1559 1570 Fig. 2. Effects of different proportions of Pb/Zn tailings on leaf growth of vetiver grass. The treatments were similar to Fig. 1. Each point is mean of 12 measurements ± SD. physiological parameters were measured. Fig. 1 showed the effects of different proportions of tailings on the tillering capacity of vetiver grass. As compared with the control treatment (100% soil), there is no influence of 25% tailing treatment on the tillering capacity of vetiver grass, and the numbers of tillers is about 8 per plant after 50 days of treatment. However, the tillering capacity is significantly reduced as the tailing content increased and the tiller numbers decreased to only 60% of those of control treatment when vetiver was treated with 100% tailing for 48 days. However, if nitrogen fertilizer was applied to the 100% tailing treatment, the tillering capacity was greatly improved and the tiller numbers Fig. 3. Effects of different proportions of Pb/Zn tailings on dry matter accumulation in shoots and roots of vetiver grass. Each point is mean of five plants ± SD. Fig. 4. Effects of different proportions of Pb/Zn tailings on leaf water potential of vetiver grass. The symbols are the same to those in Fig. 1. Each data is mean of five measurements ± SD.

J. Pang et al. / Chemosphere 52 (2003) 1559 1570 1563 Fig. 5. Effects of different proportions of Pb/Zn tailings on chlorophyll content, photosynthetic rate and photochemical activity of vetiver leaves. The symbols are the same to those in Fig. 1. Each data is mean of 12 measurements ± SD.

1564 J. Pang et al. / Chemosphere 52 (2003) 1559 1570 Fig. 6. Effects of different proportions of Pb/Zn tailings on electric leakage of vetiver leaves and roots. The symbols are the same to those in Fig. 1. Each data is mean of 5 measurements ± SD. was about 3 more than that of 100% tailing treatment per plant (Fig. 1). The leaf growth rate and dry matter accumulation of both shoots and roots of vetiver grass were also inhibited when grown in the tailing-contained soil and the degrees of inhibition increased with the increment of tailing content and prolongation of treatment (Figs. 2 and 3). The effect was more pronounced on roots than on shoots, and also application of nitrogen fertilizer could significantly alleviate the inhibitory effects of heavy metals (Fig. 3). When plants were grown in a heavy metal-contaminated soil, the roots were the primary site of heavy metal accumulation. Many reports showed that root growth was severely inhibited by heavy metal treatment. The inhibition of roots may lead to the decrease in water and nutrient absorption. Fig. 4 showed the effects of different Pb/Zn tailing treatments on leaf water potential. The results indicated that the leaf water potential was almost not significantly influenced when plants were grown in the relatively low contents of tailing and short time of treatments. However, obvious decrease in leaf water potential was observed when plants were grown in 100% tailing medium and the treatment time was longer than 13 days when the roots displayed an obvious injury (data not shown).

J. Pang et al. / Chemosphere 52 (2003) 1559 1570 1565 Fig. 7. Effects of different proportions of Pb/Zn tailings on SOD activity of vetiver leaves and roots. The symbols are the same to those in Fig. 1. Each data is mean of 3 measurements ± SD. No significant inhibitory effects of tailing treatment on chlorophyll content, photosynthetic rate and photosynthetic photochemical activity of leaves were observed, except for 50% and 100% tailing treatments (Fig. 5). In fact, nitrogen fertilizer treatment can greatly improve the photosynthetic characteristic of leaves of vetiver plants when grown in the tailing-containing soil medium. One of the most important effects of heavy metals at the cellular level are the alteration of membrane integrity and the formation of active oxygen species (AOS) (Dietz et al., 1999). In order to determine whether high tolerance of vetiver grass to heavy metal is related to its effective protective mechanisms to eliminate or reduce AOS caused damages, the dose- and time-responses of electric conductivity and the enzymatic antioxidant system of leaves were investigated (Figs. 6 9). The results of Fig. 6 shown that electrolyte leakage of both roots and leaves increased with the increment of tailing contents and the progress of the treatment time, but a significant increase was observed only for the 50% and 100% tailing treatments and the increase in the relative electric conductivity was more pronounced in roots than in shoots, implying that the injury of roots was severer than that of shoots (Fig. 6). There were great differences in the activities of SOD, POD and CAT between roots

1566 J. Pang et al. / Chemosphere 52 (2003) 1559 1570 Fig. 8. Effects of different proportions of Pb/Zn tailings on POD activity of vetiver leaves and roots. The symbols are the same to those in Fig. 1. Each data is mean of 3 measurements ± SD. and shoots in response to tailing treatments. Increases in activities of all three enzymes were observed in roots and in shoots after treatment of tailings. However, the increases in the activities of SOD and CAT were much more distinct in shoots than in roots, whereas the activity of POD increased more greatly in shoots than that in roots (Figs. 7 9), suggesting that the mechanisms to scavenge reactive oxygen species used were different between various parts of plants. Abscisic acid (ABA), well known as a plant stress hormones, plays an important role in the improvement of plant tolerance to adverse environmental conditions. As shown in Fig. 10, ABA concentrations in leaves and roots increased with the increment of proportions of tailing and the progress of treatments, but significant increase in ABA concentration was observed only in leaves of 100% tailing treatment. Nitrogen fertilizer treatment had no significant influence on ABA concentrations in leaves and roots, as compared with 100% tailing treatment without nitrogen fertilization. Similar increases in proline concentrations were observed in both leaves and roots (Fig. 11). However, the proline concentration was much higher in roots than in leaves after 50 days of treatments, especially in 100% tailing-treated roots. Fertilizer treatment could stimulate the accumulation of proline in both leaves and roots (Fig. 11).

J. Pang et al. / Chemosphere 52 (2003) 1559 1570 1567 Fig. 9. Effects of different proportions of Pb/Zn tailings on CAT activity of vetiver leaves and roots. The symbols are the same to those in Fig. 1. Each data is mean of 3 measurements ± SD. 4. Discussion The areas of heavy metal-polluted soils increased significantly throughout the world during past several decades, as the results of industry development, mining activity, irrigation of waste water, etc. (Smith et al., 1996; Herawati et al., 2000; Tordoff et al., 2000), which has become a global problem because of its deterious influences not only on plant growth (yield and quality) and environmental quality, but also on the health of human beings. Therefore, much effort has been made to decontaminate the polluted soil by using either chemical, physical or biological methods (Salt et al., 1998; Valls et al., 2000). For metalliferous mine wastelands, both the physi-chemical methods and biological methods are impossible to be used to decontaminate the heavy metal polluted wasteland as a consequence of the large amount of waste products of mining and ore-processing operations. Use of a vegetation cover gives a cost-effective and environmentally sustainable method of stabilizing and reclaiming wastes such as mine-spoils and tailings (Tordoff et al., 2000). Thus, screening of plant species which are high tolerance to high-level heavy metals is urgently needed in this aspect. Much progress has been made at the levels of physiology, biochemistry and molecular

1568 J. Pang et al. / Chemosphere 52 (2003) 1559 1570 Fig. 10. Effects of different proportions of Pb/Zn tailings on ABA contents of vetiver leaves and roots. The symbols are the same to those in Fig. 1. Each data is mean of 3 measurements ± SD. biology of plant tolerance to heavy metals in past decades (Chaney et al., 1997). Vetiver grass (V. zizanioides), due to its unique morphological and physiological characteristics, has been widely known for its effectiveness in erosion and sediment control, and has also been found to be highly tolerant to extreme soil conditions including heavy metal contamination (Truong and Baker, 1998). Nowadays vetiver grass has been widely used as an alternative method for rehabilitation of mine tailings in several countries, including in China. This paper highlights the physiological aspects of vetiver grass in responses to heavy metal treatment. The results showed that great physiolgical changes have occurred when vetiver grass grown in heavy metal-containing soil medium. The adverse effects were more pronounced in roots than in shoots (Figs. 3 and 6), which might be related to high heavy metal accumulation within the root cells as compared with that in the shoots. As a visible symptom in the leaves, significant decrease in chlorophyll content was observed, especially for the high proportions of tailing treatments (Fig. 5). The decrease in chlorophyll content may be due to the inhibition of chlorophyll biosynthesis (Stobart et al., 1985; Stiborova et al., 1986) or accelerated degradation of chlorophyll (Luna et al., 1994). The decrease in chlorophyll content leads to the decrease of photosyn-

J. Pang et al. / Chemosphere 52 (2003) 1559 1570 1569 Fig. 11. Effects of different proportions of Pb/Zn tailings on proline contents of vetiver leaves and roots. The symbols are the same to those in Fig. 1. Each data is mean of 3 measurements ± SD. thesis of leaves, and thus growth of plants (Figs. 2, 3 and 5). Of course, the inhibition of photosynthesis may also be due to inhibition of other photosynthesis-related factors, such as Rubisco and photochemical activity. However, the photochemical activity, indicated as F v =F m, decreased only when plants exposed to high level of heavy metals (Fig. 5). Application of nitrogen fertilizer combined with tailing treatment could greatly alleviate the decreases in chlorophyll content and photochemical activity, thus no influence on photosynthetic rate of leaves was observed, as compared with the corresponding treatments (Fig. 5). It is well known that exposure of plants to heavy metals induces the generation of AOS, which is harmful to plants (Zenk, 1996). The injury of plant cells caused by heavy metals is, to a great extent, related to the destruction of the balance between the generation and detoxification of AOS. Plants possess the protective mechanisms to scavenge the toxic AOS, but the ability to the balance between the generation and detoxification of AOS varies greatly among different plant species. The results shown in this paper suggest that tailing treatments could enhance the activities of POD, SOD and CAT, which are the major enzymes involving in scavenge of AOS (Figs. 7 9). However, great differences were observed in the changes of activities of these enzymes between shoots and roots, implying that different mechanisms were used to scavenge AOS in different parts of vetiver plants, which is wealthy of further study. Similar results were observed for protective substances, such ABA and proline (Figs. 10 and 11). In conclusion, the results shown in this paper can provide the guidelines in screening of plants high tolerant to heavy metals. Some physiological parameters

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