Environmental Pollution 214 (2016) 510e516 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol Pb uptake and toxicity to Iris halophila tested on Pb mine tailing materials* Yulin Han a, Lili Zhang a, Yongheng Yang b, Haiyan Yuan b, Jiuzhou Zhao a, Jiguang Gu c, * Suzhen Huang b, a Experimental Teaching Center of Ecological Environment of Jiangxi Province, Department of Landscape Architecture, Jiangxi University of Finance and Economics, Nanchang, 330032, China b Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing Sun Yat-Sen Botanical Garden, Nanjing, 210014, China c College of Life Science and Technology, Jinan University, Guangzhou, 510632, China article info abstract Article history: Pb tolerant mechanisms, plant physiological response and Pb sub-cellular localization in the root cells of Received 25 December 2015 Iris halophila were studied in sand culture and the Pb mine tailings. Results showed that the activities of Received in revised form superoxide dismutase (SOD) and peroxidase (POD) in the underground parts and the activity of catalase 8 April 2016 (CAT) in the aboveground and underground parts increased as Pb level was enhanced. Glutathione (GSH) Accepted 14 April 2016 and ascorbic acid (AsA) contents increased by Pb treatments. Pb deposits were found in the middle cell walls or along the inner side of epibiotic protoplasm of some cells which accumulated a large quantity of Pb and died. The dry weights (DWs) of aboveground parts under all Pb tailings treatments decreased Keywords: fi Iris halophila insigni cantly, while the DW of the underground parts growing in the pure Pb tailings decreased fi fi Compartmentalization signi cantly. Pb, Cu, Cd, and Zn contents increased signi cantly as the levels of Pb tailings were enhanced Antioxidant system and Pb contents in the aboveground and underground parts reached 64.75 and 751.75 mg/g DW, Lead (Pb) tailings respectively, at pure Pb tailings treatment. The results indicated that I. halophila is a promising plant in Phytoremediation the phytoremediation of Pb contaminated environment. Some antioxidant enzymes, antioxidants and compartmentalization of Pb were played major roles in Pb tolerance of I. halophila. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction antioxidants enable the cell to quench the oxidative stress in some species (Reddy et al., 2005; Zhou et al., 2010). Compartmentaliza- Heavy metals produced by the modern industries in the past tion of Pb is another mechanism for detoxification in plants (Han have reached the toxic levels in the land and water of many parts of et al., 2007). The determination of sub-cellular localization of Pb the world (Ha et al., 2011; Jiang et al., 2012). Lead (Pb) is one of the is useful to identify the pathways of Pb transport and localization in most toxic heavy metals causing the environmental contamination. the sub-cellular structure of the plant (Samardakiewicz and Wozny, Excessive Pb not only restrains the normal growth of the plant, but 2000). disrupts the balance of the ecosystem and has an extremely The wastes generated from mining processes may be the negative impact on the ecological environment and human health pollution sources to the environment, which includes a large (Mishra et al., 2006; Zeng et al., 2006). Pb may induce oxidative amount of waste rock, tailings and acidic water leaching from stress that damages plant cell membrane by free radicals and hy- tailings. Pb tailings contain higher concentrations of Pb, Cd, Zn, Cu droperoxides (Drazkiewicz et al., 2004) and affects normal physi- and other heavy metals and contaminate surroundings (Han et al., ological activity of plant. Chloroplast membrane is also damaged by 2013a). It was reported that the natural vegetation recovery of Pb and chlorophyll contents in the leaves indirectly affected tailings would take hundreds of years or even longer time (Burzyski and Kobus, 2004). Many antioxidant enzymes and (Bradshaw, 2000). Therefore, the governance of Pb tailings has been bound to arouse public attention. In recent years, people have been working on phytoremediation technology to control the contami- * This paper has been recommended for acceptance by Baoshan Xing. nated environment by heavy metals. For this reason, the values of * Corresponding author. heavy metal resistant and accumulated plants for environmental E-mail address: [email protected] (S. Huang). http://dx.doi.org/10.1016/j.envpol.2016.04.048 0269-7491/© 2016 Elsevier Ltd. All rights reserved. Y. Han et al. / Environmental Pollution 214 (2016) 510e516 511 remediation have been fully realized (Ke et al., 2007). However, the supplemented into each pot as the sand was relatively dry. The utilization of such plants could not fully meet the needs of phy- experiment was conducted in a greenhouse at ambient tempera- toremediation due to the limitation of the habitat condition, ture (15e25 C) under the natural light. biomass and propagation of these plants (Han et al., 2013b). Therefore, it is most important to effectively use these plants in the 2.2.2. Determination of physiological and biochemical parameters remediation of contaminated environment and to search more Leaves and roots were taken from same position of the seedlings plants which could accumulate heavy metals in the aboveground at 16 days Pb treatments and used for determinations of physio- parts and adapt different environments (Jiang et al., 2012; Han logical and biochemical parameters. The activities of peroxidase et al., 2013a,b). (POD) were assayed according to the method of Fang and Kao Iris halophila Pall. is a widely adapted perennial species of Iri- (2000). A unit of POD activity was expressed as the change in daceae, native to Xinjiang, Gansu of China and Roumania, Ukraine absorbance per minute and specific activity as enzyme units per g and Russia (Mathew, 1981). It is cold and drought tolerant species fresh weight (FW). The activities of superoxide dismutase (SOD) with relatively high biomass, nice leaves and flowers and often and catalase (CAT) were assayed according to Li et al. (2000). The grows in the meadow steppe, gravel slope and saline-alkali land enzyme activity of CAT was expressed in mgH2O2 destroyed À À (Huang et al., 2003). Moreover, I. halophila is a salt and Pb tolerant min 1 g 1 FW. One unit of SOD was defined as the enzyme amount species (Han, 2008). The objectives of this research were: (1) the causing 50% inhibition reduction of NBT, and the enzyme activity physiological and biochemical mechanism of Pb tolerance in was expressed in units per mg of protein. Proline was extracted and I. halophila; (2) sub-cellular localization of Pb in the root tip cells of its contents were determined by the method of Bates et al. (1973). I. halophila; (3) effects of different portions of Pb tailings on the Glutathione (GSH) and ascorbic acid (AsA) in the roots and leaves growth and tolerant index; (4) heavy metal absorption and trans- were extracted and the contents determined according to the location in the seedlings growing in the different portions of Pb method of Li et al. (2000). tailings. The results of the current work will help evaluate the ability of Pb tolerance and phytoremediation of I. halophila. 2.2.3. Sub-cellular localization of Pb in the root tip cells The sub-cellular localization of Pb in root tip cells was evaluated 2. Materials and methods according to Sahi et al. (2002) with slight modifications. Approxi- mately 5 mm long root tip segments were collected after 16 days 2.1. Materials exposure of Pb and fixed in 2% glutaradehyde in 50 mmol/L PIPES (pH 6.8). After 3 h incubation, the root tip segments were washed in 2.1.1. The physicochemical properties of Pb tailings and soil 50 mmol/L PIPES buffer and post fixed in 2% OsO4 for 2 h and then Pb mine tailings were collected from tailing sites of Dexing Pb dehydrated and embedded in Spurs epoxy resin. Ultrathin sections 0 0 Mine (29 43 N and117 02 E), Dexing, Jiangxi Province, China. were obtained by ultramicrotome (POWTIME-XL). The ultrathin Sample collection and treatment were based on the method of Jiang sections observed with H-7650 Transmission Electron Microscope et al. (2012). Garden soils were collected from the field in the west at 80 kV. side of Mailu Campus of Jiangxi University of Finance and Eco- nomics, Nanchang. Samples were then manually mixed. The 2.3. The experiment of potential phytoremediation of Pb tailings properties of Pb tailings were determined: pH (CaCl2) 6.23, organic matter 17.52%, total N 0.52 g/kg, total P 0.82 g/kg, available P 2.3.1. Experimental design 8.46 mg/kg, available K 23.27 mg/kg, Pb 706.93 mg/kg. The prop- The similar-sized seedlings of I. halophila were transplanted into erties of garden soil were determined: pH (CaCl2) 4.75, organic the pots with pure garden soil (CK), 3/4 garden soilþ1/4 Pb tailings matter 5.13%, total N 0.48 g/kg, total P 0.14 g/kg, available P 5.42 mg/ (1/4 Pb), 1/2 garden soilþ1/2 Pb tailings (1/2 Pb), 1/4 garden soilþ3/ kg, available K 11.56 mg/kg, Pb 8.64 mg/kg. 4 Pb tailings (3/4 Pb) and pure Pb tailings (Pb), respectively, with 6 seedlings per pot. Each treatment was in triplicates. Then an equal 2.1.2. Seedling cultivation quantity of tap water was supplemented into each pot when the The seeds of I. halophila were collected from the vegetative culture substrates were relatively dry until the seedlings were propagated plants in the Iris Resource Collection Garden, Jiangsu harvested. The experiment was conducted at ambient temperature Province and Chinese Academy of Science, Nanjing.