Chiang Mai J. Sci. 2017; 44(4) 1367 Chiang Mai J. Sci. 2017; 44(4) : 1367-1378 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Tolerance Threshold and Phyto-assessment of Cadmium and Lead in Vetiver Grass, Vetiveria zizanioides (Linn.) Nash Chuck Chuan Ng* [a], Amru Nasrulhaq Boyce [a], Md Motior Rahman [a,b] and Mhd Radzi Abas [c] [a] Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. [b] Department of Plant Agriculture, Ontario Agricultural College, University of Guelph, Ontario, Canada. [c] Chemistry Department, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. *Author for correspondence; e-mail: chuckz89@gmail.com Received: 29 April 2016 Accepted: 8 July 2016 ABSTRACT Various types of plant species have been extensively used for heavy metals phyto-remediation without taking into consideration its tolerance threshold. In this study, Vetiver grass, Vetiveria zizanioides (Linn.) Nash was evaluated under five different sets of contaminated spiked cadmium (5Cd, 10Cd, 50Cd, 100Cd and 150Cd mg/kg) and lead (50Pb, 100Pb, 200Pb, 400Pb and 800Pb mg/kg) concentration levels in soil. The growth performance, metal tolerance and phyto-assessment of Cd and Pb in the roots and tillers were assessed using flame atomic absorption spectrometry (FAAS). Tolerance index (TI), translocation factor (TF), biological transfer factor (BTF), biological accumulation coefficient (BAC) and metal uptake efficacy were used to determine the Cd and Pb translocation capability in Vetiver grass. Significantly higher (p<0.05) accumulation of Cd and Pb was recorded in the roots of all spiked treatments. Furthermore, strong and significantly positive correlations were exhibited between the increased levels of spiked heavy metal concentrations with both Cd (r=0.975) and Pb (r=0.952) accumulations. The results of this study showed Vetiver grass as an effective phyto-stabilizer for both Cd and Pb. Nevertheless, the growth of Vetiver grass was restricted when the tolerance threshold of 100 mg/kg (dry weight basis) Cd was exceeded in the contaminated soil. Keywords: heavy metal, spiked metal, phyto-stablizer, Vetiver grass, threshold 1. INTRODUCTION Over the years, soil contamination has attracted much global attention as it instigates considerable risks to human health and the environment. Anthropogenic sources of soil contaminants such as heavy metals released by human activities via industrial and agricultural practices, urban activities and transportation have caused serious threats to the environment [1, 2]. The term heavy metal is widely used to describe a large group of 1368 Chiang Mai J. Sci. 2017; 44(4) elements in the periodic table with an atomic density greater than 6 g/cm3 but can also be defined in relation to its natural chemical properties [3]. Among various types of heavy metals, both cadmium (Cd) and lead (Pb) are regarded to be highly toxic pollutants even at low concentration levels. Although naturally occurring, both Cd and Pb are often imperceptible, non-biodegradable and are persistent in soils over a long duration [4, 5]. These metals are hazardous to human health as it is easily bio-accumulated via the food chain including direct inhalation, ingestion of soil and/or consumption of contaminated plants [6, 7]. The exposure of plants in heavy metal contaminated soil could possibly affect the inhibition of plant growth; reduced metabolism and lower biomass due to the toxicity effects of both Cd and Pb [8, 9]. As a result, the United States Priority Pollutant List [10] has recognised both Cd and Pb to be among the 126 Toxic and Priority Pollutants due to its lethal characteristics in nature. There are numerous remediation technologies including both physical (excavation, containment and fracturing) and chemical (soil washing, solidificationstabilization and chemical redox) assisted methods that have been tested to clean up contaminated heavy metals in soils [11, 12]. Nonetheless, phyto-remediation has evolved to be an alternative biological assisted method that is cost-effective, non-destructive and environmentally friendly solution for heavy metals soil contamination compared to other techniques [13-15]. Among various types of plants, Vetiver grass, Vetiveria zizanioides (Linn.) Nash has been reported to be one of the most promising species with great potential for heavy metals phyto-remediation due to its fast growth, extensive deep root system and high tolerance to a wide range of adverse soil conditions [16-19]. However, little information is available concerning the effect of different levels of spiked heavy metals in Vetiver grass. Recent studies [20-22] have solely reported on the phyto-extraction and phyto-stabilization effects of heavy metals uptake in Vetiver grass. Nevertheless, there are currently no robust studies that have been tested to evaluate the comparative and empirical phyto-assessment using Vetiver grass growing under different levels of spiked Cd and Pb conditions. Hence, the purpose of this study was to (i) evaluate the growth performance; (ii) assess the metal uptake ability and accumulation trends; and (iii) examine the phyto-tolerance and threshold limits of Vetiver grass growing under different levels of spiked Cd and Pb contaminated soil conditions. 2. MATERIALS AND METHODS 2.1 Samples Preparation and Experimental Design The study was conducted using pot experiments in the planthouse of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur with the average room temperature ranging between 27°C and 36°C throughout the day. Top soil (0-20cm) collected from uncontaminated field located in the University of Malaya, Kuala Lumpur (3° 7′ N latitude and 101° 39′ E longitude) was used as the tested soil in the experiment after undergoing preliminary physico-chemical soil assessment (Table 1). The dull reddish brown soil composed of 90.47% sand, 7.89% silt and 1.64% clay. All collected soil was air-dried for a week followed by < 4mm sieving using test sieve to remove gravels and large non-soil particles. The artificially spiked heavy metal treatments were prepared using cadmium nitrate tetrahydrate [Cd(NO3)2.4H2O] and lead (II) Chiang Mai J. Sci. 2017; 44(4) 1369 nitrate [Pb(NO 3 ) 2 ] salt compounds that exceed the median permissible in the natural occurring levels of the Malaysian [23], Canadian [24] and European Union [25] soil contamination guidelines. The amended soil was then continuously stirred and incubated for a week to ensure that the homogeneousity of the desired spiked heavy metal concentration is obtained. The control and five different levels of spiked Cd and Pb treatments (Table 2) were tested using Vetiver grass, Vetiveria zizanioides (Linn.) Nash in plastic pots (0.18m diameter × 0.16m depth) filled with 2kg of soil, respectively. Vetiver saplings were obtained from Humibox Malaysia whereby only fresh and healthy plant saplings with an initial average tiller number (10-15), plant height (30-35cm) and basal diameter (2.5-3.5cm) were selected for the experiment. All of the spiked heavy metal treatments were watered evenly with 50mL of tap water once a day and the plant growth parameters such as height, tiller number, basal diameter and percentage plant survivorship were continuously monitored throughout the 60-day period of the experiment. The study was conducted under the completely randomized design (CRD) with three replications. Table 1. Physico-chemical parameters of growth media soil. Very coarse sand (%) Coarse sand (%) Medium coarse sand (%) Fine sand (%) Very fine sand (%) Cd Pb Dry Dull reddish brown Mean ± standard deviation Parameters (Unit) pH Soil texture Sand (%) Silt (%) Clay (%) Metal contents (mg/kg) Bulk density (g/cm3) Porosity (%) Field capacity (%) Saturation level (%) Colour (Munsell colour charts) Mean 6.62 ± 0.09 90.47 1.93 50.45 32.19 10.22 5.21 7.89 1.64 1.23 ± 0.46 9.25 ± 1.42 1.49 ± 0.56 43.77 ± 2.14 24.32 ± 2.16 15.83 2.5YR 5/4 1370 Chiang Mai J. Sci. 2017; 44(4) 2.2 Samples and Chemical Analyses At the end of the 60-day of the experimental period, all Vetiver treatments were uprooted and brought into the laboratory for chemical analysis. Freshly harvested Vetiver were washed in running water followed by deionized water to remove any adhering soil particles before it was sectioned into parts of roots and tillers (shoots). All soil and plant samples were oven-dried for 72 hours at 70°C to obtain a constant weight of dry matter content before it was homogenized in a mortar and pestle. Approximately, 0.5g of the homogenized samples underwent acid digestion with hydrochloric acid (HCl), nitric acid (HNO 3 ) and hydrogen peroxide (H 2 O 2 ) according to Method 3050B [26] followed by Method 7000B [27] for total recoverable elemental analysis of both Cd and Pb using the PerkinElmer AAnalyst 400 flame atomic absorption spectrometer (FAAS). The highly precise technique of determining the spiked heavy metal concentrations and chemical analysis was controlled using the BAM Germany (BRM#12-mixed sandy soil) certified reference material with an average rate of metal recovery for Cd (93.46%) and Pb (108.25%), respectively (Table 3). Table 2. Treatment variables. Table 3. Concentrations of initial and spiked heavy metal (mg/kg), certified reference material (CRM) and metal recovery (%) for Cd and Pb metals. *BAM Germany (BRM#12-mixed sandy soil) certified reference material Mean ± standard deviation; NA = Not applicable Treatment Control 5Cd 10Cd 50Cd 100Cd 150Cd 50Pb 100Pb 200Pb 400Pb 800Pb Detail of spiked heavy metal No heavy metal added 5 mg/kg of Cd 10 mg/kg of Cd 50 mg/kg of Cd 100 mg/kg of Cd 150 mg/kg of Cd 50 mg/kg of Pb 100 mg/kg of Pb 200 mg/kg of Pb 400 mg/kg of Pb 800 mg/kg of Pb Treatment Control Spiked Cd 5Cd 10Cd 50Cd 100Cd 150Cd Spiked Pb 50Pb 100Pb 200Pb 400Pb 800Pb Initial soil (mg/kg) Cd 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 1.23 ± 0.46 Pb 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 9.25 ± 1.42 Spiked metal (mg/kg) Cd NA 5.31 ± 0.94 13.83 ± 2.76 51.77 ± 5.32 107.68 ± 8.90 155.14 ± 4.61 NA NA NA NA NA Pb NA NA NA NA NA NA 52.18 ± 4.82 102.44 ± 8.31 207.63 ± 3.19 404.94 ± 8.09 811.76 ± 13.98 CRM* (mg/kg) NA 4.04 ± 0.22 NA NA NA NA NA 204.00 ± 6.00 NA NA NA NA NA Measured (mg/kg) NA 3.78 ± 1.42 NA NA NA NA NA 220.83 ± 34.92 NA NA NA NA NA Metal recovery (%) NA 93.46 NA NA NA NA NA 108.25 NA NA NA NA NA Chiang Mai J. Sci. 2017; 44(4) 1371 2.3 Statistical Analyses and Data Interpretation The plant growth performance were evaluated using the root-tiller (R/T) ratio and tolerance index (TI) whilst the ability and proficiency for heavy metal accumulation and translocation upwards (soil-to-root and root-to-tiller) in Vetiver grass were assessed by determining the translocation factor (TF), biological transfer factor (BTF), biological accumulation coefficient (BAC) and percentage of metal uptake efficacy [3, 15, 28] as follows: R/T ratio = Dry matter content in root/Dry matter content in tiller TI = Total dry matter content in spiked heavy metal treatment/Total dry matter content in control TF = Concentration of heavy metal in tiller/ Concentration of heavy metal in root BTF = Concentration of heavy metal in root/ Concentration of heavy metal in soil BAC = Concentration of heavy metal in tiller/ Concentration of heavy metal in soil Metal uptake efficacy (%) = [Concentration of heavy metal in tiller/Total concentration of heavy metal accumulated in Vetiver] × 100 All experimental data were analysed by performing the one-way analysis of variance (ANOVA) and further statistical validity test for significant differences among treatment means was conducted by employing the Fisher's least significant difference (LSD) tests at the 95% level of confidence. 3. RESULTS AND DISCUSSION 3.1 Responses of Soil pH and Plant Growth Performance The initial soil pH varied from 4.12 to 5.57 where control soil recorded the highest pH of 5.57 while the lowest pH of 4.12 was observed in 800Pb treatment (Figure 1). Upon harvesting, all spiked Cd and Pb treatments, except for 800Pb treatment, showed a decline in pH ranging from 4.20 to 4.62, where the highest pH reduction (-1.03 pH units) was recorded in 50Pb treatment. No significant difference (F=1.45; d.f.=35; p>0.05) in soil pH was observed among all Cd spiked treatments. On the other hand; 100Pb, 200Pb, 400Pb and 800Pb treatments significantly (F=4.05; d.f.=35; p<0.05) affected the soil pH levels compared to the control. The changes in soil pH observed could be related to the application of high levels of spiked Pb as more positively charge (proton ions) are present in the soils. Similar studies [29, 30] have revealed that the bioavailability of metal uptake in plants is strongly associated with the soil pH conditions. Figure 1. Changes in soil pH in Vetiver as influenced by different levels of spiked Cd and Pb concentrations. Vertical bars represent standard deviation in treatment means and same letters are not significantly different at 0.05 levels of probability. 1372 Chiang Mai J. Sci. 2017; 44(4) Table 4 shows that a significantly lower (F=5.98, 8.42; d.f.=35; p<0.05) tiller number and plant height were obtained in 150 Cd treatment compared to other spiked Cd treatments and control. Both 100Cd (64.44%) and 150Cd (37.22%) treatments demonstrated significantly lower (F=13.09; d.f.=35; p<0.05) percentage of survival compared with the control and other spiked Cd treatments. Among spiked Cd treatments, 100Cd and 150Cd recorded significantly lower growth performance, as the plants started to wither during the second week of the experimental period due to the presence of high concentrations of spiked Cd in the soils. In contrast, there was no significant difference (F=0.80, 0.35, 0.02, 1.05; d.f.=35; p>0.05) in terms of tiller number, plant height, basal diameter and percentage survivorship recorded in all spiked Pb treatments compared with the control. The findings demonstrated adverse growth reduction in terms of tiller number (27.6-71.0%), plant height (26.8-77.8%) and plant survivorship (35.6-62.8%) when high amounts of spiked 100Cd and 150Cd treatments were applied to Vetiver grass, respectively. With regard to Pb, similar past studies [17, 31] had reported that Vetiver grass has progressive outgrowth under all different levels of spiked Pb concentrations. Table 4. The tiller number, plant height, basal diameter and plant survivorship of Vetiver as influenced by different levels of spiked heavy metal concentrations. Mean followed by the same letters are not significantly different for each treatment means at 0.05 levels of probability. Treatment Control 5Cd 10Cd 50Cd 100Cd 150Cd F-value LSD 95% Control 50Pb 100Pb 200Pb 400Pb 800Pb F-value LSD 95% Number of tillers 27.9 a 26.9 a 32.6 a 21.2 ab 20.2 b 8.1 c 5.984 12.075 27.9 a 21.0 a 19.0 a 21.4 a 21.1 a 23.0 a 0.802 11.868 Plant height (cm) 61.49 ab 73.26 a 75.58 a 61.93 ab 45.02 b 13.64 c 8.423 27.486 61.49 a 74.07 a 79.92 a 75.89 a 75.77 a 80.56 a 0.351 40.371 Basal diameter (cm) 7.73 ab 8.71 ab 9.64 a 7.95 ab 6.82 ab 3.38 b 1.472 6.203 7.73 a 8.28 a 8.37 a 8.37 a 8.44 a 8.13 a 0.019 6.557 Plant survivorship (%) 100.00 a 98.89 a 98.33 a 97.78 a 64.44 b 37.22 c 13.087 25.072 100.00 a 99.44 a 98.89 a 98.89 a 98.33 a 98.33 a 1.051 2.192 Chiang Mai J. Sci. 2017; 44(4) 1373 The roots, tillers and total dry matter contents in all spiked Pb treatments were not significantly affected (F=0.92, 0.14, 0.32; d.f.=17; p>0.05) with the application of different levels of Pb concentrations (Table 5). Only the 150Cd treatments recorded a significantly lower (F=4.66, 3.41; d.f.=35; p<0.05) tiller number and total dry matter content. The 150Cd (9.87 ± 0.12 g/m2) treatment displayed the lowest total dry matter content with an average of 31.3% reduction as compared to the control. The application of both root-tiller (R/T) ratios and tolerance index (TI), employed to assess the tolerance ability of Vetiver grass to grow under different levels of spiked heavy metal concentrations, showed that there was no significant difference observed in the R/T ratios (F=0.58, 1.07; d.f.=17; p>0.05) among all spiked Cd and Pb treatments. Furthermore, although no significant difference in TI (F=1.49, 0.23; d.f .=14; p>0.05) was observed in both spiked Cd and Pb treatments, all spiked Pb treatments recorded remarkably high TI values > 1 (1.021-1.099). The results indicated that the different levels of spiked Pb concentrations had no direct influence on the overall growth performance and dry matter contents in Vetiver grass. As the TI (1.001-1.099) was relatively > 1, Vetiver grass can be regarded to have good adaptability and high tolerance proficiency in Pb concentrated soils. Conversely, it can be concluded that Vetiver grass is not suitable to be used as a phyto-remediator for soils contaminated with Cd exceeding the threshold level of 100 mg/kg (dry weight basis). This is in agreement with the findings of past studies [17, 32-33]. Table 5. Dry matter content, root-tiller ratio and tolerance index of Vetiver as influenced by different levels of spiked heavy metal concentrations. Mean ± standard deviation followed by the same letters are not significantly different for each treatment means at 0.05 levels of probability. Treatment Control 5Cd 10Cd 50Cd 100Cd 150Cd F-value LSD 95% Control 50Pb 100Pb 200Pb 400Pb 800Pb F-value LSD 95% Dry matter content (g/m2) Root 8.20 ± 0.51 a 8.10 ± 1.15 a 8.04 ± 1.68 a 7.98 ± 1.56 a 7.56 ± 1.33 a 5.98 ± 0.40 a 1.467 2.411 8.20 ± 0.51 a 9.00 ± 0.81 a 9.73 ± 1.62 a 9.78 ± 1.70 a 9.54 ± 0.50 a 9.51 ± 0.68 a 0.919 2.178 Shoot 6.17 ± 0.93 a 6.11 ± 0.74 a 6.06 ± 0.79 a 5.83 ± 0.33 a 5.03 ± 0.87 ab 3.89 ± 0.48 b 4.662 1.445 6.17 ± 0.93 a 5.66 ± 0.80 a 5.86 ± 0.41 a 5.81 ± 0.81 a 6.01 ± 1.39 a 6.14 ± 0.87 a 0.141 1.829 Total 14.37 ± 1.43 a 14.21 ± 1.14 a 14.10 ± 2.32 a 13.81 ± 1.53 a 12.60 ± 2.19 ab 9.87 ± 0.12 b 3.408 3.251 14.37 ± 1.43 a 14.66 ± 1.50 a 15.60 ± 2.03 a 15.59 ± 2.47 a 15.56 ± 1.46 a 15.65 ± 1.27 a 0.320 3.484 R/T ratio 1.343 a 1.341 a 1.322 a 1.375 a 1.502 a 1.565 a 0.578 0.458 1.343 a 1.599 a 1.652 a 1.680 a 1.640 a 1.564 a 1.067 0.411 TI 1.001 a 0.994 a 0.969 a 0.890 a 0.692 a 1.488 0.385 1.021 a 1.084 a 1.087 a 1.086 a 1.099 a 0.229 0.236 1374 Chiang Mai J. Sci. 2017; 44(4) 3.2 Distribution of Metals in Plant The accumulation of both Cd and Pb in the roots and tillers of Vetiver grass was varied (Table 6). The roots (F=38.38; d.f.=17; p<0.05) and total accumulation (F=124.89; d.f.=17; p<0.05) of Cd in 50Cd, 100Cd and 150Cd treatments were found to be significantly higher than the control, 5Cd and 10Cd treatments. There was no Cd accumulation recorded in the tillers for both control and 5Cd treatments as its detection limits were lower than 0.01 mg/kg (dry weight basis), whilst the tillers of 150Cd treatment (66.85 ± 9.73 mg/kg) showed the highest accumulation Cd. Between roots and tillers, Cd accumulations were reasonably greater in the roots (1.28-112.67 mg/kg) than the tillers (4.16-66.85 mg/kg) in all treatments. The accumulation trend of Cd in Vetiver grass was in the following order: 150Cd > 100Cd > 50Cd > 10Cd > control. Similarly, with regard to Pb accumulation, the roots (F=160.28; d.f.=17; p<0.05) and total accumulation (F=228.32; d.f.=17; p<0.05) of Pb in 100Pb, 200Pb, 400Pb and 800Pb treatments were found to be significantly greater than the control and 50Pb treatments. A significantly higher accumulation of Pb was observed in the tillers (F=42.74; d.f.=17; p<0.05) of both 400Pb and 800Pb treatments compared to the control and other spiked Pb treatments. Between roots and tillers, Pb accumulated substantially higher in the roots (4.27-396.60 mg/kg) compared to the tillers (1.79-122.94 mg/kg) in all treatments. The trend for Pb accumulation in Vetiver grass was in the following order of 800Pb > 400Pb > 200Pb > 100Pb > 50Pb > control. In both spiked heavy metal treatments, Vetiver grass accumulated the highest total amount of Cd (179.52 ± 16.74 mg/kg) and Pb (519.54 ± 24.93 mg/kg) in the spiked 150Cd and 800Pb treatments, respectively. The results demonstrated that the accumulation of both Cd and Pb in Vetiver grass increases with the application of higher levels of spiked heavy metals concentrations. Both 100Cd and 150Cd treatments recorded fairly higher accumulation of Cd than the other spiked Cd treatments and the control whereby this was reflected in the noticeable reduction in plant growth. As a result, these findings indicated that the declining plant growth trends do not affect the overall bioavailability uptake of spiked heavy metals in Vetiver grass. On the other hand, the findings are supported by the past similar studies [18, 34] which revealed that the phyto-tolerance and growth threshold of Vetiver grass for Cd and Pb accumulations were 20-60 mg/kg Cd and > 1500 mg/kg Pb in soil, respectively. The obtained results are aligned yet it has been further extemporized to show that the Vetiver grass have limited phyto-tolerance and threshold levels when the accumulation of heavy metal is higher than 100 mg/kg of Cd in soil whilst no specific threshold level of Pb is recorded till present. However, the use of direct pot experiments in this study for the spiked heavy metals instead of in-situ (field) experiments may have possibly attributed to the high heavy metals accumulation in both the roots and tillers of Vetiver grass. 3.3 Association of Metals Uptake in Plant The plant-soil association of both Cd and Pb accumulated from the spiked heavy metal soils into the roots and the tillers of Vetiver grass are shown in terms of translocation factor (TF), biological transfer factor (BTF), biological accumulation coefficient (BAC) and percentage of metal uptake efficacy in Table 6. Considering the tolerably lower accumulation of spiked heavy metals in the tillers than the roots in Vetiver grass, TF and BAC are employed to evaluate the capability of Vetiver to translocate Chiang Mai J. Sci. 2017; 44(4) 1375 heavy metals from soils and roots into the tillers. As reported in Table 5, all of the recorded TF and BAC values in both Cd and Pb accumulations were < 1. The 100Cd (0.446) and 150Cd (0.615) treatments recorded significantly higher (F=10.06; d.f.=17; p<0.05) TF values whilst the 50Pb (0.145), 100Pb (0.104) and 200Pb (0.145) treatments showed a significant decrease (F=3.82; d.f.=17; p<0.05) in TF values compared to the control for both Cd and Pb accumulations, respectively. No significant difference (F=1.56; d.f.=17; p>0.05) were found in BAC in all Pb spiked treatments. Meanwhile 10Cd, 50Cd, 100Cd and 150Cd treatments exhibited significantly higher (F=14.47; d.f.=17; p<0.05) BAC values than the control. Table 6. Metal accumulation of cadmium (Cd) and lead (Pb) with its translocation factor (TF), biological transfer factor (BTF), biological accumulation coefficient (BAC) and metal uptake efficacy (%) of Vetiver as influenced by different levels of spiked heavy metal concentrations. Mean ± standard deviation followed by the same letters are not significantly different for each treatment means at 0.05 levels of probability. ND = Not detected Nevertheless, in terms of BTF, no significant difference (F=1.90; d.f.=17; p>0.05) was observed in all spiked Cd treatments. However, 50Pb (0.739), 100Pb (1.207) and 200Pb (0.948) treatments documented significantly higher (F=13.82; d.f.=17; p<0.05) values compared to the control. Metal uptake efficacy (%) was used Treatment Cd accumulation Control 5Cd 10Cd 50Cd 100Cd 150Cd F-value LSD 95% Pb accumulation Control 50Pb 100Pb 200Pb 400Pb 800Pb F-value LSD 95% Metal concentration (mg/kg) Root 1.28 ± 0.72 c 6.71 ± 2.76 c 17.10 ± 6.05 c 59.12 ± 10.59 b 88.57 ± 18.89 a 112.67 ± 22.34 a 38.383 25.952 4.27 ± 0.43 e 36.94 ± 13.44 e 120.71 ± 16.02 d 189.58 ± 11.08 c 235.25 ± 28.49 b 396.60 ± 30.82 a 160.283 39.301 Shoot ND (< 0.01) ND (< 0.01) 4.16 ± 0.97 c 13.13 ± 7.79 c 37.32 ± 10.99 b 66.85 ± 9.73 a 46.660 13.579 1.79 ± 1.16 c 5.08 ± 1.61 c 12.52 ± 1.43 c 27.86 ± 9.22 c 86.57 ± 11.03 b 122.94 ± 29.11 a 42.741 26.554 Total 1.28 ± 0.72 d 6.71 ± 2.76 d 21.26 ± 6.10 d 72.24 ± 13.77 c 125.88 ± 15.50 b 179.52 ± 16.74 a 124.885 22.416 6.06 ± 1.13 e 42.02 ± 14.65 e 133.23 ± 16.86 d 217.44 ± 20.20 c 321.82 ± 37.04 b 519.54 ± 24.93 a 228.324 43.935 TF 0.010 c 0.002 c 0.262 bc 0.224 bc 0.446 ab 0.615 a 10.064 0.263 0.426 a 0.145 bc 0.104 c 0.145 bc 0.369 ab 0.314 abc 3.826 0.241 BTF 1.041 ab 1.341 ab 1.710 a 1.182 ab 0.886 ab 0.751 b 1.905 0.863 0.462 d 0.739 bc 1.207 a 0.948 ab 0.588 cd 0.496 cd 13.824 0.270 BAC 0.008 c 0.002 c 0.416 ab 0.263 b 0.373 ab 0.446 a 14.472 0.183 0.193 a 0.102 a 0.125 a 0.139 a 0.216 a 0.154 a 1.563 0.119 Efficacy (%) 0.98 c 0.17 c 20.49 b 17.65 b 29.89 ab 37.57 a 16.165 12.986 27.97 a 12.54 ab 9.45 b 12.63 ab 26.91 a 23.64 a 8.710 11.135 1376 Chiang Mai J. Sci. 2017; 44(4) to assess the total efficiency and potential of Cd and Pb uptake in Vetiver grass from the soil to its tillers. A significantly greater (F=16.17; d.f.=17; p<0.05) percentage of Cd efficacy was recorded in the 10Cd, 50Cd, 100Cd and 150Cd treatments, whilst only 100Pb (9.45%) treatment showed a significantly lower (F=8.71; d.f.=17; p<0.05) percentage of Pb efficacy compared to the control. The overall findings of TF and BAC < 1, whilst BTF > 1, demonstrate that the translocation of both Cd and Pb were more favourably accumulated in the roots than the tillers in Vetiver grass. Besides, the noticeably higher Pb accumulation in the roots than the tillers of Vetiver grass could have caused the tolerably lower percentage of Pb efficacy among all spiked Pb treatments. Vetiver grass can be regarded as a suitable phyto-stabilizer for both Cd and Pb, owing to the BTF >1 value and its considerable ability to immobilize heavy metals in the soil [14, 35-36]. Vetiver grass showed a strong and significantly positive relationship with the levels of spiked concentrations for both Cd (r=0.975) and Pb (r=0.952) accumulations (Table 7). The slopes indicated that with each application of 1.0 mg/kg (dry weight basis) level of spiked Cd and Pb concentrations in soil, an approximately 1.181 mg/kg (Cd) and 0.617mg/kg (Pb) will be accumulated in Vetiver grass, respectively. However, significantly negative correlation relationships were exhibited between the dry matter content with Cd accumulations (r=0.491) and the levels of spiked Cd concentrations (r=0.508) in Vetiver grass. Dry matter content was negatively correlated due to the increased level of spiked Cd concentrations in the soil which affected higher accumulation of Cd in Vetiver grass. Table 7. Regression equation, coefficients of determination (R2) and F values of different parameters in Vetiver grass. X 1 = Level of spiked concentrations; X 2 = Dry matter contents * Significant at 0.01 level of probability ** Significant at < 0.001 level of probability Regression equation Relationship between level of spiked concentrations and Cd accumulation Y Cd = 6.059 + 1.181X 1 Relationship between level of spiked concentrations and Pb accumulation Y Pb = 49.690 + 0.617X 1 Relationship between dry matter contents and Cd accumulation Y Cd = 14.509 0.026X 2 Relationship between dry matter contents and Pb accumulation Y Pb = 380.678 -10.368X 2 Relationship between level of spiked concentrations and dry matter contents Y Cd = 14.509 0.026X 1 Y Pb = 15.690 0.0002X 1 R2 0.97507 0.95158 0.49136 0.00858 0.50833 0.00113 R 0.98746 0.97549 0.70097 0.09262 0.71297 0.09262 F value 586.795** 294.792** 14.490* 0.130 15.508* 0.130 Chiang Mai J. Sci. 2017; 44(4) 1377 4. CONCLUSIONS The inclination trend of heavy metals accumulation for both Cd and Pb in Vetiver grass were in the order of 150Cd > 100Cd > 50Cd > 10Cd > control and 800Pb > 400Pb > 200Pb > 100Pb > 50Pb > control, respectively. The accumulation of both Cd and Pb in the roots and tillers of Vetiver grass increased when higher levels of spiked heavy metals concentrations were applied into the soil. Vetiver grass can thus be suggested to be an effective Cd and Pb phyto-stabilizer, owing to the considerably high heavy metals accumulation in its roots. However, it is not suitable for phytoremediation if the level of contamination exceeded the threshold amount of 100 mg/ kg Cd (dry weight basis) as it would inhibit overall plant growth. Nonetheless, in terms of Pb accumulation, Vetiver grass could be used as a suitable phyto-remediator as its phyto-tolerance and threshold is expected to be higher than 800 mg/kg (dry weight basis). 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