COMPARISON OF EDTA AND EDDS ENHANCTED PHYTOEXTRACTION OF Cr AND Pb FROM CONTAMINATED SOIL BY ANANAS COMOSUS (L.) MERR

The effects of chelating agents on Chromium (Cr) an d lead (Pb) absorption were studied by planting pineapple, Ananas comosus (L.) Merr. in contaminated soil. All plant samples were grown in a nursery for 30 days and then separated into seven sets: Set (1) ha d nothing added (Blank); (2) had Pb added as Pb(II) Nitrate (Pb(NO3)2) at 500 mg kg −1 soil; (3) added Pb(II) nitrate and EDTA, a chelati ng agent; (4) contained both Pb(II) nitrate and EDDS, a second chelating agent; (5) onl y added Cr as potassium dichromate (K 2Cr2O7), at 400 mg kg soil; (6) was treated with Cr (potassium dichromat e) plus EDTA; and (7) contained both Cr (potassium dichromate) and EDDS. The chelating agent concentra tions were 2 millimoles per kilogram soil. Soil and plant contamination levels were measured by analyzing the Cr, Cr(VI) and total Pb after growing for 30, 60, 90 and 120 days. The analysis divided the plant samples in to two parts: Aboveground and underground. Plant gr owth was also analyzed by dry weight, root length and ex pression of toxicity through withered leaves and ye llow leaf symptoms. The results of this study indicate t hat after 60 days, the EDTA agent had the highest P b absorption efficiency, with the plant sample absorb ing 288.14 mg Pb per kg soil in the aboveground par t and 796.66 mg kg soil in the underground part. The EDTA agent had h igh Cr absorption efficiency, with the plant sample absorbing Cr at 545.72 mg kg −1 soil in the aboveground part and 2267.99 mg kg −1 soil in the underground part after 90 days. The EDTA and EDDS a gents did not affect pineapple growth and expressio n of toxicity symptoms were statistically significant (p≤0.05) compared with the control sets.


1.INTRODUCTION
Heavy metals affect human daily life because of their long-term environmental stability. The main sources of these hazardous substances are industrial and agricultural activities.Some hevy metals such as Chromium (Cr) and lead (Pb) are toxic to humans at low levels. The presence of heavy metal contamination in water, air and soil can disrupt human and animal life cycles and cause damage to the food chain through bio-accumulation. Soil remediation methods include physical, chemical and biological techniques. For example, one physical technique involves washing contaminated soil to achieve a steady state; a chemical technique uses sedimentation and chemical reduction and biological techniques make use of absorption by microorganism and using plants for containment, degradation or extraction of xenobiotics from water and soil substrates through a process known as phytoremediation (Sampanpanish et al., 2006;Abdu et al., 2011). This biological technique offers an economical and non-invasive alternative for treating polluted soils. Phytoremediation is also recognized as a green technology (Kumar et al., 1995;Huang et al., 1997;Salt et al., 1997;Blaylock et al., 1997;Vassil et al., 1998;Mohd et al., 2013)

and removes
Science Publications AJABS pollutants from contaminated soils by root absorption and translocation to harvestable plant parts. Rajoo et al. (2013).
This study examined enhanced phytoremediation of heavy metals using chelating agents to improve the solubility of heavy metals from soil. Two chelating agents, Ethylene Diaminete Traacetic Acid (EDTA) and Ethylene Diamine Disuccinate (EDDS), were selected for their efficiency as soil amendments. EDTA and EDDS differ significantly in their distinguishing characteristics. EDTA is resistant to biodegradation and has a high environmental persistence. EDTA therefore exhibits a prolonged presence in the soil and presents an increased risk of leaching. On the other hand, EDDS is produced naturally by a number of microorganisms and is readily biodegradable.
The plant species used for phytoremediation in this study was Ananas comosus (L.) Merr. (Pineapple), grown in an agricultural soil contaminated with Cr and Pb, resulting from disposal of solid hazardous waste from a nearby industrial zone in the eastern part of Thailand. Pineapple is the dominant crop in this area, favored due to its rapid growth and high yield. Therefore, it is pertinent to explore pineapple's ability to absorb and translocate heavy metals as a potential means of bioremediation. The objective of this study was to compare the ability of the two chelating agents, EDTA and EDDS, in increasing uptake of Cr and Pb.

Preparation of Soil, Plants and Reagents
Samples were first collected from uncontaminated soils in eastern of Thailand. Samples were randomly taken from depths of 0-30 centimeters. The samples were dried and analyzed for their general properties and total heavy metal content. This step involved determining aspects such as soil texture, moisture content, pH, the Cation Exchange Capacity (CEC), Conductivity (EC) and Organic Matter (OM), total nitrogen (N), Phosphorus (P) and potassium (K) as well as the presence of naturally occurring heavy metals (e.g., total Cr, Cr(VI) and total Pb).
Black plastic 8 inch diameter pots containing 5 kilograms of dry soil each were used in the experiment. A total of 84 pots covered with thick transparent plastic bags were prepared. The plants used were Ananas comosus (L.) Merr. Selected for uniform size and weight.
The Cr and Pb contaminants used (K 2 Cr 2 O 7 and Pb(NO 3 ) 2 ) were in the ratio of 400 mg kg −1 soil for Cr and 500 mg kg −1 of soil for Pb. Each soil sample weighed 5 kg.
The two chelating agents, EDTA and EDDS, were prepared at a concentration of 2 millimoles per kg soil. Na 2 EDTA and Na 3 EDDS salt were used to make a solution with a soil concentration ratio of 774.48 and 578.39 mg kg −1 for EDTA and EDDS, respectively.

The Experiment
Experimental plants were thinned to 1 per pot and allowed to grow for approximately 30 days. Soil moisture content was maintained by adding 100 mg of water per pot every day. After selecting plants with healthy root systems, the Cr and Pb contaminates were introduced using a solutions of K 2 Cr 2 O 7 and Pb(NO 3 ) 2 at concentrations of 400 and 500 mg kg −1 of soil, respectively. The pots were separated into seven sets: (1) had 12 pots without any treatments (blank); (2) had 12 pots to which Pb but no chelating agents was added; (3) had 12 pots to which both Pb and EDTA were added; (4) had 12 pots to which both Pb and EDDS were added; (5) had 12 pots to which Cr but no chelating agents were added; (6) had 12 pots to which both Cr and EDTA were added; and (7) had 12 pots to which both Cr and EDDS were added. The experimental design tested these seven sets in three replicates. Addition of 100 milliliters of water every two days in the morning was necessary to keep the soil moist. However, the absorption mechanism might be from the plastic bags that covered the pot when the plants needed more water in the drier part of the day. No fertilizers were used in the experiment.

Sampling and Analysis
Soil and plant samples were taken at intervals of 30, 60, 90 and 120 days after adding the Cr, Pb and chelating agents. Soil and plants were analyzed for total Cr, Cr(VI) and total Pb. The soil samples were dried at room temperature and separated into two parts. One part was ground and sieved by a No.2 sieve apparatus, kept in a storage bag and analyzed for background. The other part was dried in an oven at 105°C for 48 h, ground and sieved by a No.2 sieve apparatus, then analyzed for Cr and Pb concentrations. The plants were washed 2-3 times with tap water, using distilled water for the final rinse. Also, the plants were separated into two classes: Aboveground parts (stems and leaves); and underground parts (stem and roots). The fresh plants were weighed then oven-dried at 105°C for 48 h until a stable dry weight was reached and recorded. The dried plant samples were ground and sieved using a No.2 sieve apparatus and analyzed for total Cr, Cr(VI) and total Pb, in each part of the plant. Total Cr and Pb in the soil and Science Publications AJABS plant samples were determined using the USEPA 3052 method,acid digestion and by microwave digestion with the total amount calculated by Atomic Absorption Spectrometer; AAS. The Cr(VI) concentration in soil and plant was analyzed by using USEPA 3060 method and the total amount calculated by UV spectrophotometer.

Statistical Analysis
Descriptive statistics were performed using the Statistic Package for Social Science; SPSS. The variance absorption data and the accumulation of Cr and Pb within plants were analyzed using ANOVA and the results were compared to different data by Duncan's New Multiple Range Test; DMRT.

The Properties of Soil Samples
The uncontaminated soil used in the experiment was a sandy clay loam with sand: Silt: Clay ratio of 63.80: 5.40: 30.80. The other soil properties are shown in Table 1 and demonstrate the acidity of the soil.

Accumulation of Chromium and Lead in Soil Samples
The accumulation of Cr and Pb in the soil is shown in Table 2. Soil samples at 30, 60, 90 and 120 days showed that the total amounts of Cr and Pb in the soil decreased over time. The soil samples had low levels of organic matter and low pH, which increase solubility of Cr and Pb compounds, enhancing root uptake. High plant growth rate was associated with enhanced reduction in total soil Cr and Pb. The results for experimental sets 5, 6 and 7 showed that the total amount of Cr(VI) in the soil decreased as time increased from 30 to 120 days. By contrast, the total amount of Cr(III) increased as time increased from 30 to 120 days. Cr(VI) is reduced in the soil to Cr(III) (Grohse et al., 1988).

Effect of Chelating Agent on Cr and Pb Uptakein Plants
The study of Cr and Pb accumulation in plants was focused on two parts: Aboveground (stem and leaf) and underground (stem and roots). The results are shown in Fig. 1 and 2.

3.3.1.Cr Accumulation in Whole Part of Plants
The total amount of Cr absorbed is shown in Fig.  1a and b which depict absorption by chelating agents of Cr from soil into the plant. The most significant finding was the amount of Cr absorbed into whole part of plant. The Cr solution concentrations in soil of 400 mg kg −1 soil were applied to one kilogram of soil at various times: 30, 60, 90 and120 days. The result was that the underground parts (stem and roots) accumulated more Cr solution than the aboveground parts (stem and leaf). In particular, pineapple plants from experimental set 6 absorbed the most Cr after 90 days. The aboveground parts absorbed 545.72 milligrams per kilogram plant and underground parts absorbed 2,267.99 mg kg −1 of plant. A study by (Lombi et al., 2001) found that EDTA was highly efficient in transferring Cr from soil to root, but limited in its ability to transfer Cr from root to stem. A study by (Lemen et al., 2002) found that certain plants may limit Cr transfer from stem to leaf. Nevertheless, absorption of Cr(VI) was highest at 30 days in both parts of pineapple in this study, with 224.12 mg kg −1 plant in aboveground parts and 826.74 mg kg −1 plant for underground parts. The aboveground parts from experimental set 7 absorbed the TCr after 90 days, but the underground parts absorbed the TCr only after 120 days: 513.68 mg kg −1 plant and 2,124.48 mg kg −1 , respectively. However, the absorption of Cr(VI) by aboveground parts from experimental set 7 was highest at 60 days and absorption by underground parts was highest at 30 days: 198.88 mg kg −1 plant and 784.61 mg kg −1 plant, respectively. The experimental time periods of 30 and 60 days showed statistically significant relationships between experimental set 6 (which added both Cr and EDTA) and both experimental set 5 (added Cr but did not add chelating agents) and experimental set 7 (added both Cr and EDDS) at p≤0.05. 48.0 TCr (mg kg −1 ) ND* TPb (mg kg −1 ) ND* Note: * ND = Not Detectable (<0.5 ppm)    On the other hand, the Cr(VI) absorption mechanism of aboveground parts and underground parts decreased with increasing time from 30 days to 120 days in experimental set 5 (which added Cr but did not add chelating agents), experimental set 6 (which added both Cr and EDTA) and experimental set 7 (which added both Cr Science Publications AJABS and EDDS). That the reduction reaction is a significant plant mechanism that changes Cr(VI) to Cr(III) is noted in Shanker et al., 2005;Grohse et al., 1988).

Pb Accumulation in Whole Part of Plants
The total amount of Pb absorbed in plant is shown in Fig. 2a and b. These data describe the absorption effect of chelating agents on soil Pb, as well as the effect of the pineapple plant parts. The most significant finding is that the amount of Pb increased in all parts of the pineapple plant. Soil Pb concentrations of 500 milligrams were applied to one kilogram of soil at 30, 60, 90 and 120 days. Analysis indicated that the aboveground parts (stem and leaf) accumulated less Pb than the underground parts (stem and roots). The results showed that the pineapple from experimental set 4 (Pb+EDDS) had the highest ability to absorb Pb after 30 days when the aboveground parts held 195.12 mg kg −1 plant, while underground parts held 691.44 mg kg −1 plant. On the other hand, experimental set 3 (Pb+EDTA) had the highest ability to absorb Pb after 60 days, with aboveground parts holding 288.14 mg kg −1 plant and underground parts holding 796.66 mg kg −1 plant, respectively. The experimental time periods of 60, 90 and 120 days showed statistically significant relationships between experimental set 3 (Pb+EDTA) and both experimental set 2 (Pb only-no chelating agents added) andexperimental set 4 (Pb+EDDS) at p≤0.05. Therefore the Pb solution could be transferred more easily under acidic soil conditions and soil Pb was more available for root absorbtion (Al-Taisan, 2009). Findings from many research studies such as (Huang et al., 1997) who worked with pea (Pisumsativum L.cv. Sparkle) are consistent with the findings of the current study.
EDTA increased Pb absorption potential in soil and the chelating agent affected Pb accumulation in the aboveground plant parts. Significantly, this research confirmed the different characteristics of EDDS and EDTA as chelating agents. EDTA was found to act more slowly than EDDS and had ahigher absorption efficiency. The comparative analysis of the efficiency of EDTA and EDDS showed that heavy metal absorption in the soil increased at the same concentration and the EDTA agent had higher absorption efficiency than EDDS (Luo et al., 2005). Inanother study, BrassiarapaL. was used in order to study its absorption efficiency for Pb, Zn and Cd. In that experiment the effects of adding ETDA with EDDS were compared. This experiment also found higher efficiencies for EDTA, compared with EDDS (Grcman et al., 2003).

Efficiency of Chelating Agents on Absorptionof Cr and Pb by Plant
Data on the efficiency of two chelating agents, EDTA and EDDS, are presented in Fig. 3a and b. The EDTA agent had Pb absorption efficiencies of 0.31, 0.46, 0.61 and 0.69% when time increased from 30 to 120 days, respectively. Moreover, the EDTA agent had higher Pb absorption efficiency than EDDS at 60, 90 and 120 days when statistically significant relationships were considered. On the other hand, the EDTA and EDDS agent had similar Cr absorption efficiencies at 30 and 60 days, but EDTA had higher efficiency after 90 days where EDTA was 5.3% and EDDS was 4.5%. Many other published research studies support these results. For example, a study of Cu, Pb, Zn and Cd absorption efficiency in Zea mays L. and Chrysanthemum coronariumshowed that the EDTA agent had higher Pb absorption efficiency than the EDDS agent (Luo et al., 2006). The EDTA agent had higher Cr absorption efficiency than oxalic acid (Hsiao et al., 2007) and the EDTA agent had high heavy metal absorption efficiency from soil, but had limited ability to transfer this solution from root to leaf (Madrid et al., 2003).

Effect of Chelating Agent on Growth of Plants
The study of pineapple growth considered plant dry weight and observed expressions of toxicity including withered or yellow leaves and truncated roots. The results are shown in Fig. 4.
Plant dry weight of aboveground and underground parts in samples with only Cr, Cr+EDTA and Cr+EDDS as shown in Fig. 4a and b, decreased with time from 60 to 120 days. Plant samples without added Cr or with Cr+chelating agent showed statistically significant relationships. These results indicate limited plant growth when Cr was absorbed, as seen in other studies. For example, dry weight of cabbage samples decreased from 88.4 to 28.4 grams when Cr was added at a level of 10 mg kg −1 soil (Hara and Sonoda, 1979). Table 3 shows root length data from all seven experimental sets: Blank, Cr or Pb only, Cr or Pb+EDTA and Cr or Pb+EDDS. Root length of the untreated control sample was longer than all other treatments, consistent with other studies of the effect of Cr on root elongation (Panda and Patra, 2000). Root length actually decreased when 1 micromole of Cr was added. Moreover, Caesalpiniapulcherrimaroot and dry weight were limited by Cr at a concentration of 100 mg kg −1 (Prasad et al., 2001).  Moreover, dry weight of aboveground and underground parts in samples that had only Pb, Pb+EDTA and Pb+EDDS (shown in Fig. 4c and d) was closer over time to samples that did not have Pb or both Pb and chelating agents. These were statistically significant relationships. From the Table 3 shows that the root length of the untreated control sample were similar to the samples with only Pb and Pb+chelating agents. Again, these were statistically significant relationships. The chelating agents did not affect dry weight of the plants and these results agree with findings from other research. For example, EDDS increased efficiency of heavy metal absorption but did not affect plant growth when compared to blank samples (Punshon, 1996). EDTA did not affect cabbage dry weight at a concentration of 3 millimoles per kilogram soil (Wu et al., 2004). EDTA and EDDS did not affect Helianthus annuus dry weight or growth at a concentration of 1.6 millimole per kilogram soil (Meer et al., 2005).

CONCLUSION
The conclusion from this study is that the EDTA agent promotes higher Cr and Pb uptake from soil than the EDDS agent. Moreover, two chelating agentsdid cause negative effects on growth rate of pineapple. In phytotoxicity studies of Cr and Pb no negative effects were observed on the pineapplein all of the experiment. Thus, the results of our experiment can be applied to help manage this problem. EDTA is suitable and should be promoted for use in Cr and Pb removal from soil using pineapple.

ACKNOWLEDGEMENT
We thank the Interdisciplinary Program of Environmental Science, Graduate School, Chulalongkorn University, the Environmental Research Institute (ERIC) and the Center of Excellence on Hazardous Substance Management (HSM), Chulalongkorn University for all the support, practical information, helpful instruments and excellent suggestions. Moreover, we are pleased to express our thanks to every person who gave us so much help.