Journal of Applied Botany and Food Quality 91, 8 - 13 (2018), DOI:10.5073/JABFQ.2018.091.002 1Forestry, Wildlife and Fisheries Department, Government of the Punjab, Lahore, Pakistan 2Institute of Geology, University of the Punjab, Lahore, Pakistan 3Department of Botany, University of the Punjab, Lahore, Pakistan 4Department of Zoology, University of Gujrat, Gujrat, Pakistan 5Department of Agricultural Sciences, University of Haripur, Haripur, Pakistan Phytoremediation of arsenic-contaminated soils by Eucalyptus camaldulensis, Terminalia arjuna and Salix tetrasperma Firoz ud Din Ahmad1, Nasir Ahmad2, Khan Rass Masood3, Mubashar Hussain4*, Muhammad Faheem Malik4, Abdul Qayyum5 (Submitted September 23, 2017, Accepted January 2, 2018) * Corresponding author Summary Rising levels of arsenic in ground water posing threats to the lives of millions of people residing in the Indus plains, whereas the magnitude of the risk is alarming that needs to control arsenic from leaching down into the ground water becomes essential. The study was designed to assess the potential of three tree seedlings to reclaim the arsenic-affected soils in Pakistan and determining the impact of arsenic on growth parameters and its accumulation in tree seedlings. The experiment conducted at the Botanical Garden, University of the Punjab, Lahore, revealed that Eucalyptus camaldulensis, Termina- lia arjuna and Salix tetrasperma showed varying adaptability to survive under the arsenic stress environment, suggesting them as strong candidates to be exploited for arsenic remediation. Arsenic- treated tree seedlings showed reduced growth in terms of stem height (21.76%), stem diameter (25%), number of branches (22.3%), number of leaves (21%), root length (24.8%), total plant length (25.6%), fresh biomass production (40%) and dry biomass production (49%) as compared to plants grown without arsenic treatment. Arsenic accumulated in all vegetative parts of the seedlings, however maximum arsenic accumulation was recorded in roots of E. camaldulensis (37.25 mg kg-1) followed by S. tetrasperma (35.76 mg kg-1) and T. arjuna (24.13 mg kg-1) when arsenic was applied at a concentration of 4.0 mg L-1. The study has shown that these tree species can be grown on arsenic-contaminated fields to reclaim the soil from arsenic content resulting in its substantial reduction in leaching to the groundwater. The results suggest that E. camaldulensis plants accumulated maximum arsenic in its vegetative parts and confirmed it as a tree species with a potential of arsenic phyto-extraction and a good candidate for phytoremediation. Key words: Arsenic accumulation, metalloid, phytoremediation, reclamation. Introduction Arsenic-contaminated soils and water cause environmental and human health problems and the situation is becoming grave with rapid industrialization and disturbance in natural geochemical cycle (Mieke and Rengel, 2005; MadhuMita et al., 2014; Paul et al., 2015; Singh et al., 2015). Many areas of the world are affected by arsenic contamination of the underground water (MukheRjee et al., 2006; Singh et al., 2015). Arsenic-contaminated soils and water have been attempted to remove arsenic using a wide range of remediation technologies (uddin et al., 2001; Manning et al., 2002; gavRileScu, 2006; tonni et al., 2006; Pavel and gavRileScu, 2008; Singh et al., 2015). Generally all these methods are costly, require high technology and by-products from these technologies can be harmful (ali et al., 2013). Phytoremediation of toxic substances has emerged as a promising, cost-effective, eco-friendly and sustainable approach by using green plants (trees, shrubs, grasses and aquatic plants) to remove and uptake, degrade or isolate toxic substances (heavy metals, metalloids, trace elements, organic and inorganic compounds, and radioactive compounds) from the soils and water (enSley, 2000; Mendez and MaieR, 2008; dickinSon et al., 2009; luqMan et al., 2013). In this study, tree seedlings of E. camaldulensis, T. arjuna and S. tetrasperma were used to reclaim arsenic-contaminated soils. In this study, an attempt has been made to determine the potential of three tree species to uptake and accumulate arsenic from arsenic- contaminated soil in their vegetative parts and to demonstrate their tolerance against arsenic toxicity, so that these trees can be planted on arsenic-contaminated land to remediate the soil through phytoremediation. Materials and methods Raising of seedlings: Six month old seedlings of Eucalyptus camaldulensis dehnh., Myrtaceae, Terminalia arjuna (Roxb.) Wight & Arn., Combretaceae, and S. tetrasperma, Salicaceae, were obtained from a nursery of the Forest Department, Government of the Punjab, and were planted in plastic pots (40 cm height × 35 cm internal diameter) containing loamy soil at the Botanical Garden, University of the Punjab, Lahore. Application of arsenic: Plants were given different doses of arsenic (0.5, 1.0, 2.0, 4.0 mg L-1) for 18 months. These doses were given to plants once a week in the winter season (October - March) and on alternate days during summer (April - June) and plants were dosed twice a week during the monsoon season (July - September). Sodium arsenite salt (NaAsO2) was used by making stock solutions and given through irrigation water. The level of arsenic in the tap water given to control plants was 5.5 μg L-1. No fertilizer was used in this experiment. Determination of plant growth and biomass: The shoot length and diameter (of collar) of arsenic treated and untreated (control) plants were measured monthly during the experiment. At the end of experiment, seedlings were uprooted and thoroughly washed with tap water and rinsed with distilled water. The leaves and number of branches were counted and weighed separately. Dry weight of leaves, shoots and roots was determined after oven drying at 70 ºC for 48 hrs. Phytoremediation of arsenic-contaminated soils by different plant species 9 Arsenic analysis: Oven dried plant parts (roots, shoots and leaves) were ground separately using Willey mill (2000 SM, Retsch, Germany) and sieved with mesh (2 mm). One g of plant material was transferred into conical flasks and digested in a solution of HNO3 : HClO4 with 2:1 ratio (v/v) by placing it on a hot plate for about 2 hrs until the plant material became transparent. The concentrations of arsenic were determined by using hydride generation atomic absorption spectrophotometer (AA-7000, Shimadzu, Japan). Statistical analysis: The data recorded for each treatment was subjected to the Analysis of Variance (ANOVA) and means of various treatments were compared using Tukey’s HSD test at 5% probability level (Steel and toRRie, 1980). Results Effects of arsenic on plant growth Effects of various doses of arsenic on the growth of E. camaldulensis, S. tetrasperma and T. arjuna plants are shown in Tab. 1. Stem height (cm) Stem height varied significantly with increase in arsenic concentration. A reduced value of 272 cm in stem height of E. camaldulensis was recorded in T4 as compared with 331.3 cm in control (T0) while in the case of S. tetrasperma, it was reduced to 92 cm in T4 compared with 141.3 cm in T0. In the case of T. arjuna, however it was reduced to 166.7 cm in T4 from 221 cm in T0. A gradual smooth decreasing trend was observed in stem height (average reduction of 25.8% as compared to control) in all three plant species with the increase in the concentration of arsenic. Stem diameter (cm) Stem diameter significantly decreased with the increased con- centration of arsenic. A maximum reduction in stem diameter of E. camaldulensis was found to be 2.63 cm that was recorded in T4 compared with 3.8 cm recorded in control (T0) while in the case of S. tetrasperma, it was reduced to 1.4 cm in T4 down from 1.72 cm in T0. In case of T. arjuna plant, however it was recorded to be 2.65 cm at T4 down from 3.6 cm at T0. A gradual smooth decreasing trend in stem diameter was observed with an average reduction of 25% for all three plants species when the concentrations of arsenic were increased. Number of branches Similarly, reductions in number of branches were observed with an increase in arsenic concentration. A maximum reduction to the level of 163 (Nos.) in T4 in number of branches of E. camaldulensis was recorded as compared to average 217.3 branches per plant in control (T0) while in the case of S. tetrasperma, it was recorded as 79.8 in T4 down from 103.7 in T0. In the case of T. arjuna plant, however, it was found to be reduced to 64.7 in T4 compared with 79.7 in control (T0). A gradual smooth decreasing trend was observed with an average reduction of 22.27% for all three plants species when the concentration of arsenic was increased. Number of leaves Number of leaves was also significantly varied with an increase in arsenic concentration in all species. A maximum reduction in total number of leaves of E. camaldulensis plant was recorded to be 449.7 in T4 as compared with 610.7 in control (T0) while in the case of S. tetrasperma, the number was reduced to 348 in T4 as compared with 430 in T0. In the case of T. arjuna, however it was reduced to 408.7 in T4 down from 510.7 in T0. A gradual smooth decreasing trend was observed with an average reduction of 21.76% for all three plants species as the doses of arsenic were increased. Tab. 1: Effects of arsenic application on plant growth parameters. Treatments Species Stem Height Stem Diameter No. of Branches No. of Leaves Root Length Total Length (cm) (cm) (cm) (cm) E. camaldulensis 331.3±3.41 a 3.8±0.09 a 217.3±2.22 a 610.7±3.5 a 165.3±1.45 a 496.7±4.77 a T0 S. tetrasperma 141.3±2.82 def 1.7±0.33 d 103.7±1.15 d 430.0±8.71 fgh 149.2±3.33 ab 290.5±3.21 def T. arjuna 221.0±4.50 bc 3.6±0.33 ab 79.7±0.88 ef 510.7±2.19 bc 134.0±2.50 ab 355.3±4.07 bcde E. camaldulensis 292.0±4.51 a 3.3±0.33 abc 193.7±2.70 b 564.0±15.10 ab 155.0±3.04 ab 447.0±2.52 ab T1 S. tetrasperma 130.3±2.71 def 1.6±0.08 d 90.0±1.48 de 400.3±1.04 ghij 132.3±1.86 ab 262.6±2.70 ef T. arjuna 189.5±2.88 cd 3.0±0.10 abc 78.0±1.51 ef 489.7±2.19 cde 117.0±1.76 ab 306.5±2.15 cdef E. camaldulensis 283.0±3.04 ab 3.0±0.16 abc 182.0 ±2.86 bc 502.0±7.51 cd 147.1±1.50 b 380.1±4.02 bcd T2 S. tetrasperma 120.2±1.00 ef 1.5±0.33 d 86.0±1.67 de 381.0±2.08 hij 121.0±1.15 ab 241.2±2.33 ff T. arjuna 178.0±1.53 cde 2.8±0.11 bc 72.3±0.23 ef 460.0±3.51 cdef 110.2±1.00 ab 288.2±1.88 def E. camaldulensis 276.0±1.86 ab 2.9±0.12 bc 171.0±1.15 c 485.0±10.54 cde 134.0±1.15 ab 410.0±2.85 abc T3 S. tetrasperma 95.3±1.86 f 1.5±0.10 bc 79.8±0.58 ef 362.0±0.44 ij 116.0±3.86 ab 211.3±2.69 f T. arjuna 170.0±2.61 cde 2.7±0.09 c 64.7±0.67 f 437.7±1.45 efg 102.0±0.00 ab 272.0±3.93 ef E. camaldulensis 272.0±3.30 ab 2.6±0.33 c 163.0±2.52 c 449.7±1.76 defg 130.3±0.88 ab 402.2±2.89 abc T4 S. tetrasperma 92.0±0.88 f 1.4±0.07 d 79.8±1.76 ef 348.0±0.20 j 111.1±1.00 ab 203.1±3.17 f T. arjuna 166.7±1.67 cde 2.7±0.10 c 64.7±0.58 f 408.7±1.76 fghi 97.0±0.58 b 263.7±2.37 f T0 = Tap water (control); T1 = 0.5 mg L-1 arsenic; T2 = 1.0 mg L-1 arsenic; T3 = 2.0 mg L-1 arsenic; T4 = 4.0 mg L-1 arsenic. Values with different letters are significantly different from each other at p ≤ 0.05. 10 F.u.D. Ahmad, N. Ahmad, K.R. Masood, M. Hussain, M.F. Malik, A. Qayyum Root length (cm) Variation in root length was observed with change in arsenic doses in all three species. A maximum reduction in root length of E. camaldulensis was recorded to be 130.3 cm in T4 as compared with 165.3 cm in T0, while in the case of S. tetrasperma it was reduced to 111.1 cm in T4 down from 149.2 cm in T0. In the case of T. arjuna, however, the root length was observed as reduced to 97 cm in T4 in comparison to 134 in control (T0). A gradual smooth decreasing trend was observed for all three plant species with increasing concentration of arsenic having an average reduction of 24.8%. Total plant length (cm) Total plant length varied significantly with an increase in arsenic doses. A maximum reduction in total plant length of E. camaldulensis was observed to be in T4 due to the highest dose of arsenic whereas it was recorded to be 402.2 cm as compared with 496.7 cm at control (T0) while in the case of S. tetrasperma it reduced to 203.1 cm in T4 as compared with 290.5 cm in T0. In the case of T. arjuna, however, it was reduced to 263.7 cm down from 355.3 cm in control (T0). A gradual smooth decreasing trend was observed for all three plants species with increasing concentration of arsenic having an average reduction of 25.6%. Effect of arsenic doses on biomass production of plants The results of the study regarding effects of varying concentrations of arsenic application on biomass production of E. camaldulensis, T. arjuna and S. tetrasperma are shown in Tab. 2. Leaf fresh biomass (g) Increase in arsenic contents resulted in reduction of leaf fresh biomass. With an increase in arsenic doses, a reduced value of 201 g in leaf fresh biomass of E. camaldulensis was recorded in T4 as compared with 300.8 g in control (T0) while in the case of S. tetrasperma, it reduced to 28.3 g in T4 compared with 49.5 g in T0. In the case of T. arjuna, however, it was reduced to 61 g in T4 from 125.3 g at T0. Stem fresh biomass (g) Increase in arsenic contents resulted in reduction of stem fresh biomass. A 955.7 g in stem fresh biomass of E. camaldulensis was recorded in T4 as compared with 1221.7 g in control (T0) while in the case of S. tetrasperma, it reduced to 55 g in T4 compared with 195 g in T0. In the case of T. arjuna, however, it was reduced to 225 g in T4 from 372 g at T0. Root fresh biomass (g) Similarly, root fresh biomass also varied with arsenic increase. In response to arsenic doses, a reduced value of 457.8 g in root fresh biomass of E. camaldulensis was recorded in T4 as compared with 555.6 g in control (T0) while in the case of S. tetrasperma, it reduced to 65 g in T4 compared with 164.2 g in T0. In the case of T. arjuna, however, it was reduced to 146 g in T4 from 256.6 g at T0. Leaf dry biomass (g) Increase in arsenic contents resulted in reduction of leaf dry biomass. In response to arsenic doses, a reduced value of 105 g in leaf dry biomass of E. camaldulensis was recorded in T4 as compared with 160.5 g in control (T0) while in the case of S. tetrasperma, it reduced to 14 g at T4 compared with 29 g in T0. In the case of T. arjuna, however, it was reduced to 27 g in T4 from 62.5 g in T0. Stem dry biomass (g) Increase in arsenic contents resulted in reduction of stem dry biomass. A reduced value of 506.2 g in stem dry biomass of E. camaldulensis Tab. 2: Effects of arsenic doses on biomass production of tree seedlings of E. camaldulensis, S. tetrasperma and T. arjuna. Treatments Species Fresh Weight (g) Oven Dry Weight (g) Leaf Stem Root Leaf Stem Root E. camaldulensis 300.8 ± 3.23 a 1221.7 ± 9.99 ab 555.6 ± 2.67 b 160.5 ± 1.49 b 705.2 ± 8.45 a 216.0 ± 2.66 d T0 S. tetrasperma 49.5 ± 1.04 fgh 195.0 ± 2.00 b 164.2 ± 2.40 b 29.0 ± 1.76 b 92.3 ± 1.67 fg 80.0 ± 1.36 a T. arjuna 125.3 ± 2.14 d 372.0 ± 3.00 b 256.6 ± 3.12 b 62.5 ± 1.51 b 182.5 ± 3.49 c 141.7 ± 1.68 d E. camaldulensis 255.8 ± 2.73 b 1096.0 ± 8.53 ab 495.8 ± 1.53 b 128.9 ± 5.85 a 604.2 ± 2.19 b 192.5 ± 2.81 b T1 S. tetrasperma 41.0 ± 1.53 gh 125.3 ± 1.27 b 104.0 ± 3.22 b 22.0 ± 2.08 b 68.0 ± 1.16 efg 55.0 ± 1.16 ght T. arjuna 100.8 ± 1.82 de 297.0 ± 2.06 b 199.0 ± 3.53 b 47.5 ± 2.90 b 151.0 ± 2.08 cd 95.3 ± 1.76 e E. camaldulensis 235.8 ± 1.15 bc 1076.7 ± 5.33 ab 475.8 ± 2.00 b 119.5 ± 1.20 b 584.2 ± 4.70 b 170.8 ± 2.22 c T2 S. tetrasperma 35.0 ± 1.33 gh 101.7 ± 1.76 a 99.0 ± 1.56 b 20.8 ± 0.67 b 55.0 ± 1.53 fg 51.3 ± 1.33 hi T. arjuna 81.0 ± 0.22 ef 278.0 ± 3.35 b 177.7 ± 2.40 b 40.0 ± 2.52 b 137.0 ± 2.40 cd 87.0 ± 1.33 ef E. camaldulensis 222.8± 1.73 bc 1037.7 ± 6.67 a 466.0 ± 3.93 a 112.0 ± 2.00 b 561.5 ± 2.60 b 151.2 ± 2.69 cd T3 S. tetrasperma 27.0 ± 0.67 h 75.0 ± 3.51 b 88.0 ± 1.11 b 16.0 ± 1.67 b 45.0 ± 1.53 fg 46.0 ± 0.16 i T. arjuna 70.0 ± 1.16 efg 250.0 ± 2.67 b 161.0 ± 2.76 b 35.0 ± 1.76 b 126.0 ± 2.85 cde 75.0 ± 1.40 efgh E. camaldulensis 201.0 ± 1.00 c 955.7 ± 5.33 ab 457.8 ± 1.16 b 105.0 ± 1.00 b 506.2 ± 1.16 b 132.8 ± 1.20 cd T4 S. tetrasperma 28.3 ± 1.76 h 55.0 ± 3.06 b 65.0 ± 2.19 b 14.0 ± 2.03 b 40.0 ± 1.53 g 30.0 ± 0.73 i T. arjuna 61.0 ± 1.76 fgh 255.0 ± 3.06 b 146.0 ± 2.0 b 27.0 ± 2.00 b 115.0 ± 2.52 def 63.0 ± 1.73 efg T0 = Tap water (control); T1 = 0.5 mg L-1 arsenic; T2 = 1.0 mg L-1 arsenic; T3 = 2.0 mg L-1 arsenic; T4 = 4.0 mg L-1 arsenic. Values with different letters are significantly different from each other at p ≤ 0.05. Phytoremediation of arsenic-contaminated soils by different plant species 11 was recorded in T4 as compared with 705.2 g in control (T0) while in the case of S. tetrasperma, it reduced to 40 g in T4 compared with 92.3 g in T0. In the case of T. arjuna, however, it was reduced to 115 g in T4 from 182.5 g at T0. Root dry biomass (g) Increase in arsenic contents resulted in reduction of root dry biomass. In response to arsenic doses, a reduced value of 132.8 g in root dry biomass of E. camaldulensis was recorded in T4 as compared with 216 g in control (T0) while in the case of S. tetrasperma, it reduced to 30 g in T4 compared with 80 g in T0. In the case of T. arjuna, however, it was reduced to 63 g in T4 from 141.7 g in T0. Accumulation of arsenic by tree seedlings Effects of arsenic doses on the uptake and accumulation of arsenic in roots, shoots, leaves is shown in Tab. 3. Accumulation of arsenic in roots Arsenic accumulation tends to increase in roots with the higher dose of arsenic in all tested plant species. An increased value of 37.25 mg Kg-1 of As in roots of E. camaldulensis was recorded in T4 as compared with 1.83 mg Kg-1 in control (T0) while in the case of S. tetrasperma, it increased to 35.76 mg Kg-1 in T4 compared with 1.70 mg Kg-1 in T0. In the case of T. arjuna, however, it was increased to 24.13 mg Kg-1 in T4 from 1.98 mg Kg-1 at T0. Accumulation of arsenic in shoots Arsenic accumulation tends to increase in shoots with the higher dose of arsenic in all tested plant species. In response to arsenic doses, an increased value of 25.69 mg Kg-1 of arsenic in shoots of E. camaldulensis was recorded in T4 as compared with 1.08 mg Kg-1 in control (T0) while in the case of S. tetrasperma, it increased to 13.76 mg Kg-1 in T4 compared with 1.03 g in T0. In the case of T. arjuna, however, it was increased to 12.52 mg Kg-1 in T4 from 0.94 mg Kg-1 in T0. Accumulation of arsenic in leaves Arsenic accumulation tends to increase in leaves with the higher dose of arsenic in all tested plant species. In response to arsenic doses, an increased value of 6.55 mg Kg-1 of arsenic in leaves of E. camaldulensis was recorded in T4 as compared with 0.57 mg Kg-1 in control (T0) while in the case of S. tetrasperma, it increased to 6.03 mg Kg-1 in T4 compared with 0.57 g in T0. In the case of T. arjuna, however, it was increased to 4.22 mg Kg-1 in T4 from 0.67 mg Kg-1 in T0. Discussion An inhibition of plant growth is directly related to the enhanced uptake and accumulation of heavy metals in plants because metal elements adversely affect the plant growth through the generation of reactive oxygen species (ROS) causing constant damage to cel- lular structures, interfere metabolic process in plants (MaRin et al., 1993; Mallick et al., 2011). Upon translocation to the shoot, arse- nic severely inhibits plant growth by slowing biomass accumulation (Finnegan and chen, 2012). Results of this study shows that stem height of all three plants species gradually reduced with increas- ing doses of arsenic and averagely it reduced to 25.8% at 4 mg L-1 arsenic dose. Reduction in shoot growth could be attributed to the reduction in chlorophyll contents and activity of photosystem I (PSI) induced by heavy metal stress (SkoRzynSka-Polit and BaSzynSki, 1997). Moreover, accumulation of metal elements to above ground plant part can reduce plant height because of disturbance in cellular Tab. 3: Arsenic accumulation in leaves, shoots and roots of tree seedlings of E. camaldulensis, S. tetrasperma and T. arjuna. Treatments Species Arsenic content Arsenic content Arsenic content accumulated in Leaves accumulated in Shoot accumulated in Root (mg kg-1) (mg kg-1) (mg kg-1) E. camaldulensis 0.57 ± 0.33 i 1.08 ± 0.06 j 1.83 ± 0.07 i T0 S. tetrasperma 0.57 ± 0.17 i 1.03 ± 0.04 j 1.70 ± 0.09 i T. arjuna 0.67 ± 0.06 i 0.94 ± 0.03 j 1.98 ± 0.11 i E. camaldulensis 2.73 ± 0.33 d 9.65 ± 0.38 f 14.84 ± 0.39 f T1 S. tetrasperma 1.87 ± 0.08 h 5.41 ± 0.33 hi 12.82 ± 0.46 g T. arjuna 1.73 ± 0.33 h 4.50 ± 0.33 i 10.45 ± 0.33 h E. camaldulensis 3.31 ± 0.33 c 15.61 ± 0.45 c 20.56 ± 0.67 e T2 S. tetrasperma 2.85 ± 0.39 fg 9.78 ± 0.30 g 19.98 ± 0.43 e T. arjuna 2.22 ± 0.17 gh 6.85 ± 0.58 h 13.01 ± 0.31 g E. camaldulensis 5.27 ± 0.33 b 20.94 ± 0.50 b 29.16 ± 0.58 b T3 S. tetrasperma 4.50 ± 0.37 e 10.91 ± 0.31 e 27.01± 0.58 c T. arjuna 3.22 ± 0.31 f 9.26 ± 0.33 g 19.92 ± 0.46 e E. camaldulensis 6.55 ± 0.67 a 25.69 ± 0.88 a 37.25 ± 0.61 a T4 S. tetrasperma 6.03 ± 0.33 d 13.76 ± 0.30 d 35.76 ± 1.44 a T. arjuna 4.22 ± 0.18 e 12.52 ± 0.33 de 24.13 ± 0.33 d T0 = Tap water (control); T1 = 0.5 mg L-1 arsenic; T2 = 1.0 mg L-1 arsenic; T3 = 2.0 mg L-1 arsenic; T4 = 4.0 mg L-1 arsenic. Values with different letters are significantly different from each other at p ≤ 0.05. 12 F.u.D. Ahmad, N. Ahmad, K.R. Masood, M. Hussain, M.F. Malik, A. Qayyum metabolism of shoots (ShankeR et al., 2005). A significant reduction in stem growth of pea (Pisum sativum) seedlings was also recorded when arsenic was applied at the rate of 5 mg L-1 (caStillo-Michel et al., 2007) which is almost similar to this study. Similarly, stem diameter gradually reduced with increasing doses of arsenic and on average it reduced to 25% at 4 mg L-1 arsenic dose in case of all three plants species. Leaf growth and total number of leaves are considered a suitable indicator of heavy metal toxicity. Reza et al. (2009) found a decrease in number of leaves per plant with gra- dual increase of As application to Amaranthus retroflexus plants. Similarly, a decrease in leaf number was observed in this study, with an average reduction of 22.27% for all three plants species as the doses of arsenic were increased. Root growth is also an important indicator related to the enhanced uptake and accumulation of heavy metals in plants. High concentration of heavy metals retard root res- piration leading to reduction in root growth (Menon et al., 2005). Moreover, due to high solute potential in the rhizosphere, nutrient uptake by plants is disturbed causing a decrease in mitotic division of meristematic cells (odjegBa and FaSidi, 2004). Root growth of all three plants species decreased with increasing doses of arsenic with an average reduction of 24.8% at 4 mg L-1 dose. ShRi et al. (2009) also observed a decrease in root growth of rice seedlings. The reduction in biomass at high doses of As might be due to the high lipid peroxidation which might have damaged chloroplast, and inhibited chlorophyll and protein contents (Singh et al., 2006; gaRg and Singla, 2011). Upon translocation to the shoot, arsenic severely inhibits plant growth by slowing biomass accumulation (Finnegan and chen, 2012) resulting in biomass reduction. aydin and akSoy (2009) applied arsenic on chickpea (Cicer arietinum L.) plants and found reduction in fresh and dry biomass production of chickpea plants with increasing arsenic levels. Arsenic is non-essential for plant growth; therefore, plants have no specific mechanism for its uptake and translocation in plant system. It enters by means of the same uptake transporter as essential nutri- ents. Higher levels of arsenic in soil interfere with nutrient uptake by plants and their distribution leading to a deficiency of essential nutrients in plants (MahiMaiRaja et al., 1994). Since roots are the first plant tissue to come in contact with soil and therefore, metal accumulation was more pronounced in roots compared to shoots and leaves (kuzovkina et al., 2004). Higher accumulation of heavy metals in roots might be due to poor translocation to above ground parts and sequestration in the vacuoles of root cells (ShankeR et al., 2005). Metal accumulation in plant tissues was in order of roots>shoot>leaves and the increase was proportional to the external application of metal elements. The variability of metals in various plant tissues may be due to compartmentalization and translocation in the vascular system (kiM et al., 2003). Arsenic in high concentra- tion may hamper with metabolism of plants and adversely affected the uptake of nutrients by competing with plant nutrients for trans- porters (MehaRg and MacnaiR, 1990; Mokgalaka-Matlala et al., 2008). Cell membranes are vulnerable targets of arsenic stress and damage of membrane is used as an indicator for tolerance to arsenic stress. Arsenic adversely affects the membrane leading to un- balanced nutrient uptake and water content in plant cells (gadallah, 1999; gaRg and Singla, 2011). Conclusion In this study the growth of tree seedlings was determined and shown to be affected by the uptake of arsenic as it accumulated in the vegetative parts of the plants grown under arsenic treated media. E. camaldulensis, T. arjuna and S. tetrasperma have demonstrated adaptability to recover the soil from arsenic with varying magnitude. Although arsenic-treated plants were affected and growth para- meters demonstrated reduced number of branches, leaves, reduced root length and low biomass production as compared to plants grown without arsenic treatment. They significantly reduced arsenic from the media. Among the three tree species investigated, E. camaldu- lensis showed the highest tolerance against arsenic toxicity and maxi- mum arsenic accumulation in its vegetative parts. Thus, the study has manifested success in the reclamation of affected soils with contri- buting environment cleaning and landscaping. References ali, h., khan, e., Sajad, M.a., 2013: Phytoremediation of heavy metals − concepts and applications. Chemosphere 91, 869-881. aydin, y.a., akSoy, n.d., 2009: Adsorption of chromium on chitosan: Optimization, kinetics and thermodynamics. Adv. Biochem. Bio- technol. 151, 188-194. caStillo-Michel, h., PaRSonS, j.g., PeRalta-videa, j.R., MaRtínez, a., dokken, k.M., gaRdea-toRReSdey, j.l., 2007: Plant Physiol. Biochem. 45, 457-463. dickinSon, n.M., BakeR, a.j.M., doRonila, a., laidlaw, S., ReeveS, R.d., 2009: Phytoremediation of inorganics, realism and synergies. Int. J. Phytoremediation 11, 97-114. enSley, B.d., 2000: Rationale for use of phytoremediation. Phytoremediation of toxic metals: using plants to clean-up the environment. New York, John Wiley & Sons, Inc, 3-12. Finnegan, P.M., chen, w., 2012: Arsenic toxicity: the effects on plant me- tabolism. Front Physiol. 3. DOI: 10.3389fphys.2012.00182 gadallah, M.a.a., 1999: Effects of proline and glycine betaine on Vicia faba responses to salt stress. Biol. Plant. 42, 249-257. gaRg, n., Singla, P., 2011: Arsenic toxicity in crop plants: physiological ef- fects and tolerance mechanisms. Environ. Chem. Lett. 9, 303-321. gavRileScu, M., 2006: Overview of in situ remediation technologies for sites and groundwater. Environ. Eng. Manag. J. 5, 79-114. kiM, S., kang, k.h., joSePh-gReen, P., lee, e.j., 2003: Investigation of heavy metal accumulation in Polugonum thunbergii for phytoextration. Environ. Pollut. 2, 235-243. kuzovkina, y.a., knee, M., quigley, M.F., 2004: Cadmium and Copper uptake and translocation in five willow (Salix L.) species. Int. J. Phytoremediation 6, 269-287. luqMan, M., Butt, t.M., tanviR, a., atiq, M., huSSan, M.z.y., yaSeen, M., 2013: Phytoremediation of polluted water by tress: A review. Afr. J. Agric. Res. 8, 1591-1595. MadhuMita, R., MukheRjee, a., MukheRjee, S., BiSwaS, j., 2014: Arsenic: an alarming global concern. Int. J. Curr. Microbiol. App. Sci. 3, 34-47. MahiMaiRaja, S., Bolan, n.S., hedley, M.j., MacgRegoR, a.n., 1994: Losses and transformation of nitrogen during composting of poultry ma- nure with different amendments: an incubation experiment. Bioresour. Technol. 47, 265-273. Mallick, S., SinaM, g., Sinha, S., 2011: Study on arsenate tolerant and sensitive cultivars of Zea mays L.: Differential detoxification mechanism and effect on nutrients status. Ecotoxicol. Environ. Saf. 74, 1316-1324. Manning, B.a., FendoRF, S.e., BoStick, B., SuaRez, d.l., 2002: Arsenic (III) oxidation and Arsenic (V) adsorption reactions on synthetic Birnessite. Environ. Sci. Technol. 36, 976-98. MaRin, a.R., PezeSkhi, S.R., MaSScheleyn, P.h., choi, h.S., 1993: Effect of dimethyl arsenic acid (DMAA) on growth, tissue arsenic, and photo- synthesis of rice plants. J. Plant Nutr. 16, 865-880. MehaRg, a.a., MacnaiR, M.R., 1990: An altered phosphate uptake system in arsenate-tolerant Holcus lanatus L. New Phytol. 116, 29-35. Mendez, M.o., MaieR, R.M., 2008: Phytoremediation of mine tailings in temperate and arid environments. Rev. Environ. Sci. Bio. 7, 47-59. Menon, M., heRMle, S., aBBaSPouR, k.c., günthaRdt-goeRg, M.S., oSwald, S.e., Schulin, R., 2005: Water regime of metal-contaminated soil under juvenile forest vegetation. Plant Soil. 271, 227-241. Mieke, q., Rengel, z., 2005: Arsenic speciation governs arsenic uptake and transport in terrestrial plants. Microchim. Acta. 151, 141-152. Phytoremediation of arsenic-contaminated soils by different plant species 13 Mokgalaka-Matlala, n.S., FloReS-tavizon, e., caStillo-Michel, h., PeRalta-videa, j.R., gaRdea-toRReSdey, j.l., 2008: Toxicity of arse- nic (III) and (V) on plant growth, element uptake, and total amylolytic activity of mesquite (Prosopis spp.). Int. J. Phytoremediation. 10, 47-60. MukheRjee, a., SenguPta, M.k., hoSSain, M.a., 2006: Arsenic contami- nation in groundwater. A global perspective with emphasis on the Asian scenario. J. Health. Popul. Nutr. 24, 142-163. odjegBa, v.j., FaSidi, i.o., 2004: Accumulation of trace elements by Pistia stratiotes: implications for phytoremediation. Ecotoxicol. 13, 637-646. Paul, S., chakRaBoRty, S., ali, n. Md., Ray. d.P., 2015: Arsenic distri- bution in environment and its bioremediation, A review. Int. J. Agric. Environ. Biotechnol. 8, 189-204. Pavel, l.v., gavRileScu, M., 2008: Overview of ex-situ de-contamination techniques for soil cleanup. Environ. Eng. Manag. J. 7, 815-834. Reza, M.d., qaMRan, c., Shaikh, t.i., RaquiBul, a., Ranjit, S., haSan, g.M.j., chowdhuRy, M.d.i., 2009: Effect of Arsenic contaminated irrigation water on the cultivation of Red Amaranth. Am.-Eurasian J. Sustain. Agric. 4, 14-19. ShankeR, a.k., ceRvanteS, c., loza-taveRa, h., avudainayagaM, S., 2005: Chromium toxicity in plants. Environ. Int. 31, 739-753. ShRi, M., kuMaR, S., chakRaBaRty, d., tRivedi, P.k., Mallick, S., MiSRa, P., Shukla, d., MiShRa, S., SRivaStava, S., tRiPathi, R., tuli, R., 2009: Effect of arsenic on growth, oxidative stress, and antioxidant system in rice seedlings. Ecotoxicol. Environ. Saf. 72, 1102-1110. Singh, n., Ma, l.q., SRivaStava, M., RathinaSaBaPathi, B., 2006: © The Author(s) 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/). Metabolic adaptations to arsenic induced oxidative stress in Pteris vit- tata L. and Pteris ensiformis L. Plant Sci. 170, 274-282. Singh, R., Singh, S., PaRihaR, P., Singh, v.P., PRaSad, S.M., 2015: Arsenic contamination, consequences and remediation techniques: a review. Ecotoxicol. Environ. Saf. 112, 247-270. SkoRzynSka-Polit, e., BaSzynSki, t., 1997: Difference in sensitivity of the photosynthetic apparatus in Cd-stressed runner bean plants in rela- tion to their age. Plant Sci. 128, 11-21. Steel, R.g.d., toRRRie, h.M., 1980: Principles and procedures of statistics: A biometric approach of Statistics. 2nd ed. New York, USA: McGraw Hill press. tonni, a.k., chana, g.y.S., lo, w., Sandhya, B., 2006: Physico-chemical treatment techniques for wastewater laden with heavy metals. Adv. Biochem. Eng. 118, 83-98. uddin, M.t., MajuMdeR, M.S.i., Figoli, a., iSlaM, M.a., dRioli, e., 2001: Arsenic removal by conventional and membrane technology: An over- view. Indian J. Chem. Technol. 14, 441-45. Address of the corresponding author: E-mail: dr.mubashar@uog.edu.pk