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The effect of citric acid application on phytoextraction of as, Cr and Cu by Maize from CCA contaminated soil.

Introduction

The contamination of soils with metals is a major environment problem throughout the world. Although a small portion of heavy metals in soils is derived from natural processes, such as bed rock weathering, a much higher amount comes from anthropogenic sources such as the mining and smelting industry, use of mineral fertilizers and pesticides, sewage sludge and animal (poultry manure) waste application, etc (Adriano, 2001). More traditional soil remediation methods (in situ and ex situ) such as soil excavation and dumping, containment methods (e.g. vitrification, stabilization), soil washing/flushing, etc are generally costly and harmful to soil properties that are needed for a successful remediation of contaminated soil (Mulligan et al., 2001, Komarek et al., 2007). Phytoremediation, the use of green plants to remove pollutants from the environment or render them harmless (Raskin et al., 1997) with its low cost and environmental friendly nature, has received much attention in the last several years (Meers et al., 2005), Vyslouzilova et al., 2003, Garbisu and Alkorta, 2001). In most soils only a fraction of heavy metal is readily available for plant uptake, and the potential for metal hyperaccumulating plant species such as Thlaspi, Urtica, Chenopodium, Polygonum sachalate, Alyssum is limited by the fact that they are slow growing and have small biomass (Mulligan et al., 2001, do Nascimento et al., 2006). Synthetic chelaters are common chemical amendments used in chemical assisted phytoextraction of heavy metals from soils (Liu et al., 2008). Such substances are capable of forming chemical complexes with metals ions thereby modifying their bioavailability in soils (Quartalli, et al., 2006).

A number of soil amendments have been reported in literature which would render soil trace metals more phytoavailable, among which ethylene diammine tetra-acetate (EDTA) has taken a predominant place (Cooper et al., 1999, Shen et al., 2002). Whilst EDTA does enhance phytoextraction of metals from soil, it has disadvantages resulting from poor degradability. This, in combination with its high affinity for heavy metal complexation, results in an increase risk of leaching (Nowack, 2002). There has therefore been interest in degradable low molecular weight organic acids as potential alternatives to EDTA in enhancing phytoextraction of heavy metals from contaminated soils. In this study, the effect of citric acid application on the phytoextraction of As, Cr and Cu by maize (Zea mays L.) from chromated-copper-arsenate contaminated soil is examined. Previous studies established that the soil is moderate to highly contaminated by As, Cr and Cu (Uwumarongie, 2009) and recommended phytoremediation as a remediation technology for the soil (Okieimen and Uwumarongie-Ilori, 2010)

Materials and Methods

Soil samples were collected from five locations with the premises of an active wood treatment factory in Benin City, Nigeria. The samples were pooled, sieved through 2mm screen and air-dried. The physicochemical properties and As, Cr and Cu levels in the soil sample are given in Table 1.

The water soluble fractions of As, Cr and Cu in the soil sample were determined by agitating 5g portion in the 25mL of distilled water for 6h followed by centrifugation at 1500rpm. The amount of the metals in the water extract determined by AAS is reported as water soluble fractions. The bioavailable fractions of As, Cr and Cu in the soil was determined by extraction of an aliquot sample of the soil with 0.01M Ca[Cl.sub.2] solution following the method described by Oliver et al. (1999).

Air-dried soil samples (1kg) were placed in plastic pots and maintained at 60% filed water capacity by adding deionised water. Four grams were sown in each pot. Fifteen days after germination, subsets of pots were treated with 100mL of 0, 20, 40, 60 and 100mM citric acid solution. Soil treatment was performed by applying the solution of the amendment (citric acid) on the top of the pots. Post-germination treatment as opposed to previous treatment was adopted to avoid detrimental phytoxic growth depressions (Meers et al., 2004). The plants were harvested five days after application of citric acid solution by cutting the shoots 0.5cm above the surface of soil, and the roots were steeped in 0.01m Ca[Cl.sub.2] for 30min to extract the exogeneous metals and thereafter washed free of the salt solution. The roots and shoots were washed and rinsed with deionised water and thereafter dried at 70[degrees]C until constant weight. The dried plant parts were ground using agate mill.

Subsamples of the ground shoot (200mg) and root (100mg) were digested in a mixture of concentrated HNO3 and HclO4 (4:1 by volume) and the As, Cr and Cu in the digested solutions were determined by AAS. Reagent blank analytical duplicates were used to ensure accuracy and precision of analysis. The data reported in this paper were the mean values of three replicates.

Sequential extractions proposed by the European Communities Bureau Reference (BCR) were performed on 1g portion of the oven-dried (at 105[degrees]C for 2h) of the soils to assess the distribution of As, Cr and Cu among the operationally defined pools; extractable ([B.sub.1]); reducible ([B.sub.2]), organic matter bound ([B.sub.3]) and residual (R) before and after treatment (plant growth, citric acid application and harvesting). The extractions were performed in a mechanical shaker according to the procedures described by Ure et al. (1993), Tokalioglu et al. (2006) and Golia et al. (2007) and are summarised in Table 2.

Results and Discussion

Physico-chemical properties and contamination status of the soil:

Textural analysis showed a preponderance of sand fraction (73.10%) followed by clay (25.80%) then silt (2.1%) thus classifying the soil as sandy clay. Although sandy soils are known to have a poor retention capacity for water and metals, the relatively large proportion of clay (24.80%) in the contaminated soil sample suggests that the soil will not drain easily with implications for potential deleterious impact of retained pollutants on environmental receptors. The acidic pH 5.92 recorded for the soil is within the range for soil in the region. Soil pH play a major function in the sorption of heavy metals as it controls the solubility and hydrolysis of metal hydroxides, carbonates and phosphates. It also influences ion-pair formation solubility of organic matter, as well as surface charge of Fe, Mn and Al-oxidies, organic matter and clay edges (Tokalioglu et al, 2006). The soil showed moderate organic matter content (2.15%) and relatively high cation exchange capacity (CEC) (47.84meq/100g). The CEC measures the ability of soils to allow for easy exchange of cations between its surface and solutions. The relatively high level of clay and CEC indicate low permeability and leachability of metals in the soil and suggest that it might not be amenable to remediation by soil washing (Atafar, 2010).

Contamination status of soil sample

The soluble and plant available fraction and pseudototal levels of the As, Cr and Cu in the contaminated soil are given in Table 3. The results show that large proportions of the metals are potentially available; 50.5% As, 43.3% Cr and 42.3% Cu, suggesting significant risk to environmental receptors. The contamination status measured in terms of contamination factor, [C.sub.f], a ratio of the metal concentration in the contaminated soil to that in uncontaminated soil and degree of contamination, [C.sub.D], of the soil is given in Table 2. The values of contamination indices classify the soil as moderately contaminated to highly contaminated (Hakason, 1980).

Metal accumulation in maize plant

The analysis of the results of metal accumulation in maize plant grown on CCA contaminated soil is given in Table 4.

The results show that metal uptake was in the order Cu > Cr > As and that citric acid application markedly enhanced metal uptake levels by maize (Figs 1 and 2). For instance at 10 mmol citric acid/kg soil application, total levels of metal uptake by plant increased by 102.3% for As, 607% for Cr and 99% for Cu. Generally, the concentration of metals in shoots is lower than in roots (Gupta and Sinha, 2006). In this study, the levels of metals in maize shoots are about 5-fold lower than the levels in the roots. The transport of metals from the roots to shoots involves long distance translocation in the xylem and storage in the vacuole of the leaf cells and these processes are affected by many factors such as the concentration of available metals in soils, solubility sequences and nature of the plant species (Yang et al., 1997). The observed trend in total metal uptake by maize (Cu > Cr > As) did not quite correlate with the potentially available (soluble + 0.01M Ca[Cl.sub.2] extractable) fractions of As (16.0mg [kg.sub.-1]) < Cu (64.7mg [kg.sup.-1]) < Cr (104.5mg [kg.sup.-1]) (Table 3). This observation is consistent with the reports of Khan (2001) for Dalbergia sisso, Acacia arabica and Populus euroamericana grown on tannery effluent contaminated site and of Armienta et al. (2001) that Cr is mostly retained in the roots of plants and found no correlation between total Cr content in soils and plant shoots.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Soil-to-plant transfer ratio (coefficient) is one of the key components of phytoextraction. Figs 3 a and 3b show the values of the transfer coefficients for As, Cr and Cu to maize plant in CCA contaminated soil with respect to the pseudototal and plant available fraction of the metals respectively.

[FIGURE 3 OMITTED]

The application of citric acid appeared to have significantly increased the amounts of the heavy metals in the shoots; 6-fold for As, 12-fold for Cr and 6-fold for Cu in comparison with the corresponding amounts in the unamended soil. On the basis of the metal fraction of environmental concern (the available fraction) the enhancement in phytoextraction following citric acid application for the contaminated soil was more marked with up to 30% of the available As and about 20% of available Cr and Cu transfered into the biomass of maize within the short duration (20 days) of this study.

It has been stressed that in evaluating a chelator; effectiveness in mobilizing metals to the root zone, the extent of translocation to the shoots should be determined (Lombi et al., 2001; Heiss et al., 2003). If for instance the metal concentration surrounding the roots rose too quickly the plant could exhibit toxic symptoms of damaged roots and growth inhibition. The translocation factor, TF, defined as the ratio of metal (loid) concentration in shoots to that in the roots, may be used to evaluate the capacity of a plant to transfer metals from roots to shoots (TF is usually > 1 or [greater than or equal to] 1) in (hyper) accumulators and < 1 in excluders (McGrath and Zhao, 2003). The TFs for As, Cr and Cu and the applied chelating agent citric acid are given in Table 5.

The results in Table 5 clearly showed marked improvements in the translocation of As, Cr and Cu from CCA contaminated soil by the application of citric acid by as much as 4100% for As, 186% for Cr and 342% for Cu. The substantial increase in the translocation factors for As, Cr and Cu following citric acid application to the contaminated soil is similar to the results of Luo et al. (2005) in the EDTA and EDSS enhanced phytoextraction of heavy metals from contaminated soils.

Heavy metal distribution in contaminated soil

The distribution patterns of As, Cr and Cu in the untreated contaminated soil was determined by the BCR sequential extraction procedure are given in Table 6 and Fig. 4. Mean concentrations in the exchangeable fraction 6.80mg [kg.sup.-1] As, 43.20mg [kg.sup.-1] Cr and 29.30 mg [kg.sup.-1] Cu corresponding to extraction yields of 23.12%, 18.98% and 20.36% for As, Cr and Cu respectively. Metal fractions in the reducible forms are 7.10 mg [kg.sup.-1] As, 49.70 mg [kg.sup.-1] Cr and 36.60 mg [kg.sup.-1] Cu. Approximately 45% As, 40% Cr and 41% Cu in this contaminated soil may be amenable (removable) to soil washing since they constitute the most loosely bound fractions to the soil matrix (Peters, 1999; Wuana et al., 2010). The organic matter bound and residual fractions of the metals varied in the order Cr > Cu > As.

[FIGURE 4 OMITTED]

Post-harvest distribution of As, Cr and Cu in contaminated soil

The post harvest distribution of As, Cr and Cu in the soil samples given in Figs 5a-c was carried out. to assess the amounts of the metals that were mobilized following citric acid application that were taken up by the plant. It has bee reported that during the application of chelated-assisted technology, plant only absorbed a limited fraction of mobilized metals in soil, hence post-harvest effects of chelators must be studied in view of the potential environmental risks (Quartacci et al, 2006, Meers et al., 2005, Lai and Chen, 2005). Prolonged mobilization of heavy metals after harvest is undesirable as the absence of an actively transpiring plant may result in percolation of mobilized metals. To examine these effects BCR sequential procedure was applied to the soil samples 3days after harvesting the plants. The results show that in comparison with the unamended soil, citric acid application markedly reduced the total levels of metal burden in the contaminated soil; 32% of Cu, 12% Cr and 14% As. The post-harvest distribution patterns of the metals show that citric acid amendment at 10mmol/kg soil application rate increased the leachable amounts of the metals by 95% for As, 72% for Cr and 27% for Cu in comparison with the unamended soil. These results are consistent with previous reports (Lai and Chen, 2005; Schmidt, 2003). However, the potential for these leachable fractions to be of environmental concern is precluded/reduced v\by the biodegradability and short lifespan of citric acid in soil environment. Facile degradation of leachable metal-citrate complexes will yield the metal ions adsorbed onto the soil matrix in unavailable forms.

[FIGURE 5 OMITTED]

Conclusion

This study examined the effect of citric acid application on the phytoextraction of As, Cr and Cu from CCA contaminated soil in pots experiments. Most of the previous reports on chelant-enhanced phytoextraction focused on soil spiked artificially with high concentrations of a single metal. The use of soils directly from polluted site corresponds well to natural conditions in the area and therefore leads to mere representative results. The addition of citric acid led to significant increase in the values of transfer coefficient and translocation factor. Although the post-harvest leachable fractions of As, Cr and Cu were relatively high in the soil, the biodegradability of citric acid, and therefore its relatively short lifespan in soil environment would preclude the environmental risks presented by other less degradable chelating agents. There are no previous reports to the knowledge of the authors on citric acid-assisted phytoextraction of metals from CCA contaminated soil.

References

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Felix E. Okieimen (1), Gift O. Tsetimi (1) and Theresa O. Egbuchunam (2) *

(1) University of Benin, Department of Chemistry, GeoEnvironmental Research Laboratory, Benin City, Nigeria

(2) Department of Chemistry, Federal University of Petroleum Resources, Effurun, Delta State, Nigeria
Table 1: Physic-chemical properties of
CCA contaminated soil.

Properties

Physico-chemical properties:

pH 5.92 [+ or -] 0.10
Clay (%) 24. 80 [+ or -] 0.00
Silt (%) 2.10 [+ or -] 0.00
Sand (%) 73.10 [+ or -] 0.00
Nitrogen (%) 0.34 [+ or -] 0.08
Carbon (%) 1.22 [+ or -] 0.30
Organic matter (%) 2.15 [+ or -] 0. 40
Phosphorus (mg/kg) 44.74 [+ or -] 3.73
Calcium (meq/100g) 5.68 [+ or -] 0.40
Magnesium (meq/100g) 1.96 [+ or -] 0.30
Sodium (meq/100g) 0.19 [+ or -] 0.10
Potassium (meq/100g) 0.57 [+ or -] 0. 10
CEC (meq/100g) 48.74 [+ or -] 0.10

Table 2: BCR sequential extraction procedure
for heavy metal speciation.

Step Metal pools Extractant Agitation time

[B.sub.1] Extractable 40 mL of 0.11M 16 h at room
 C[H.sub.3]COOH temperature

[B.sub.2] Reducible 40 mL of 0.5M 16 h at room
 NH2OH.HCl (pH=2) temperature

[B.sub.3] Organic-bound 10 mL of 8.8M I h at room
 [H.sub.2][O.sub.2] temperature
 Then 1h at
 85[degrees]C

 Cool + 50 mL of 16 h at room
 IM C[H.sub.3] temperature
 COON[H.sub.4]
 (pH 2)

R Residual Aqua regia 16 h at
 digestion (21 mL 180[degrees]C
 concentration
 HCl + 7 mL
 concentration
 HN[O.sub.3])

Table 3: Contamination status of soil sample.

Metal Soluble Available
 fraction fraction
 (mg [kg.sup.-1]) (mg [kg.sup.-1])

As 6.20 [+ or -] 1.20 9.80 [+ or -] 3.90

Cr 34.70 [+ or -] 11.00 69.80 [+ or -] 14.40

Cu 18.40 [+ or -] 7.00 46.30 [+ or -] 11.50

CD = degree of
contamination
[SIGMA][C.sub.f]

Metal Psuedototal [C.sub.f]
 (mg [kg.sup.-1])

As 31.70 [+ or -] 2.90 90.57 (a); 21.13 (b)

Cr 241.40 [+ or -] 12.80 689.71 (a); 2.41 (b)

Cu 152.90 [+ or -] 18.20 16.09 (a); 3.06 (b)

CD = degree of 796.39 (a); 26.60 (b)
contamination
[SIGMA][C.sub.f]

[C.sub.f] = [M.sub.contam]/Mref., a is with refernce to control soil
sample (0.35mg [kg.sup.-1] As, 0.35mg [kg.sup.-1] Cr and 9.50mg
[kg.sup.-1] Cu) and b is with reference to uncontaminated soil (1.50mg
[kg.sup.-1] As, 100.0mg [kg.sup.-1] Cr and 50.0mg [kg.sup.-1] Cu after
Sparks 2000)

Table 4: Effect of citric acid application on As, Cr and Cu uptake by
maize from CCA contaminated soil. Values are means [+ or -] SD
(n = 3).

Concentration Metal uptake by maize plant (mg [kg.sup.-1] dw)
of citric acid
applied Roots
(mmol/kg)
 As Cr Cu

0 5.90 [+ or -] 4.40 [+ or -] 36.10[+ or -]11.10
 1.00 0.80

2 6.40 [+ or -] 9.20 [+ or -] 40.20 [+ or -] 4.90
 1.30 1.20

4 6.90 [+ or -] 13.20 [+ or -] 46.40 [+ or -] 7.00
 1.90 2.10

6 7.50 [+ or -] 17.10 [+ or -] 51.20 [+ or -] 4.90
 1.90 2.00

8 8.60 [+ or -] 22.30 [+ or -] 55.30 [+ or -] 10.0
 2.00 2.40

10 9.70 [+ or -] 26.20 [+ or -] 59.20[+ or -]18.32
 1.40 3.90

Concentration Metal uptake by maize plant (mg [kg.sup.-1] dw)
of citric acid
applied Shoots
(mmol/kg)
 As Cr Cu

0 0.90 [+ or -] 1.30 [+ or -] 2.70 [+ or -]
 0.00 0.40 0.30

2 1.20 [+ or -] 2.40 [+ or -] 3.90 [+ or -]
 0.90 0.80 0.70

4 1.90 [+ or -] 3.00 [+ or -] 5.10 [+ or -]
 0.90 0.80 0.28

6 2.40 [+ or -] 6.30 [+ or -] 8.10 [+ or -]
 1.00 1.50 0.53

8 3.20 [+ or -] 9.66 [+ or -] 12.50 [+ or -]
 1.00 0.96 1.00

10 4.10 [+ or -] 14.10 [+ or -] 18.20 [+ or -]
 1.40 0.95 2.10

Table 5: Effect of citric acid application on translocation factors
of As, Cr and Cu from roots to shoots of maize 5 days after
treatment.

Concentration of citric TF
acid (mmol/kg soil)
 As Cr Cu

0 0.01 0.29 0.07
2 0.19 0.26 0.10
4 0.27 0.23 0.11
6 0.32 0.37 0.16
8 0.37 0.43 0.23
10 0.42 0.54 0.31

Table 6: Pseudototal and BCR extracted metal concentrations
(mg [kg.sup.-1]) and standard deviations (n = 3) of unamended
CCA contaminated soil.

Fraction As Cr

 (mg [kg.sup.-1])

[B.sub.1] 6.80 [+ or -] 2.70 43.20 [+ or -] 16.30
[B.sub.2] 7.10 [+ or -] 3.00 49.70 [+ or -] 24.70
[B.sub.3] 7.40 [+ or -] 4.20 57.90 [+ or -] 21.40
R 8.10 [+ or -] 4.00 76.80 [+ or -] 34.90
[SIGMA] 29.4 227.60
Mobility factor (%0 23.13 18.98
Recovery factor (%) 92.74 94.28

Fraction Cu

[B.sub.1] 29.30 [+ or -] 0.80
[B.sub.2] 33.60 [+ or -] 1.80
[B.sub.3] 37.10 [+ or -] 1.20
R 43.90 [+ or -] 3.10
[SIGMA] 143.90
Mobility factor (%0 20.36
Recovery factor (%) 94.11
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Author:Okieimen, Felix E.; Tsetimi, Gift O.; Egbuchunam, Theresa O.
Publication:International Journal of Applied Environmental Sciences
Date:Dec 1, 2010
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