Water, Air, and Soil Pollution

, Volume 188, Issue 1, pp 335–343

Humic Acid Addition Enhances B and Pb Phytoextraction by Vetiver Grass (Vetiveria zizanioides (L.) Nash)


  • Ilker Angin
    • Faculty of Agriculture, Department of Agricultural Structures and IrrigationAtatürk University
    • Faculty of Agriculture, Department of Soil ScienceAtatürk University
  • Quirine M. Ketterings
    • College of Agriculture and Life Sciences, Department of Crop and Soil SciencesCornell University
  • Avni Cakici
    • Faculty of Engineering, Department of Environmental EngineeringAtatürk University

DOI: 10.1007/s11270-007-9548-0

Cite this article as:
Angin, I., Turan, M., Ketterings, Q.M. et al. Water Air Soil Pollut (2008) 188: 335. doi:10.1007/s11270-007-9548-0


Phytoremediation is an attractive, economic alternative to soil removal and burial methods to remediate contaminated soil. However, it is also a slow process. The effect of humic acid in enhancing B and Pb phytoextraction from contaminated soils was studied (pot experiment) using transplanted vetiver grass (Vetiveria zizanioides (L.) Nash). Boron was applied at 0, 45, 90 and 180 kg B ha−1 soil (as H3BO3) in 16 replicates. Of the 64 pots, four pots each were treated with 0, 100, 200 and 400 kg ha−1 humic acid (HA) solution. In a separate experiment, Pb was applied (as Pb(NO3)2) at 0, 45, 90 and 180 kg Pb ha−1 prior to addition of HA solutions at levels identical to the B experiment. Experiments were conducted using a randomized complete block design with four replicates. Vetiver grass was harvested 90 days after planting. Lead addition beyond 45 kg Pb ha−1 decreased Pb uptake mostly due to a yield decline. Humic acid application increased Pb availability in soil and enhanced Pb uptake while maintaining or enhancing yield. An application of 200 kg HA ha−1 was optimal for maintaining yield at elevated Pb levels. Boron application did not impact yield but greatly increased B content of roots and shoot. Boron uptake was greatest upon addition of 400 kg HA ha−1. We conclude that HA addition to vetiver grass can be an effective way to enhance phytoremediation of B and Pb but optimum rates differ depending on soil B and Pb contamination levels.


BoronHumic acidLeadPhytoremediationVetiver grass

1 Introduction

Heavy metals have been and continue to be used for industrial, agricultural, and domestic purposes. For example, mining, smelting, electroplating, energy and fuel production, power transmission, intensive agriculture, sludge dumping and military operations have all used metals such as Cd, Cu, Pb and Zn (Herawati et al. 2000; Brun et al. 2001). Some heavy metals, e.g. Mn, Fe, Cu, Zn, Mo, and Ni, are essential or beneficial micronutrients for micro organisms, plants and animals, whereas others such as Cd, Pb, and Hg have no known biological or physiological function. At high concentrations, heavy metals can have strong toxic effects and be a threat to the environment (Chatterjee and Chatterjee 2000; Kim et al. 2001). Elevated soil metal concentrations can not only negatively impact plant growth, but also deteriorate human and animal health through ingestion of water, soil and food produced on contaminated soils (Angin and Turan 2004).

Boron toxicity is a common problem in semiarid regions like Eastern Anatolia in Turkey, where soil and irrigation water B levels are frequently high (Soylu et al. 2004). As with Se, a narrow range in B concentration can mean the difference between plant deficiency and plant toxicity (Choi et al. 2006).

Traditional ex-situ treatments for B and Pb contamination in soils such as high temperature exposure (produce a vitrified, granular, non-leachable material), addition of solidifying agents and soil washing processes are expensive and cost prohibitive when large areas are contaminated (USDA-NRCS 2000). Therefore, cost-effective in situ environmentally technologies for the remediation of heavy metal contaminated soils are needed.

The discovery of metal hyper-accumulating properties in certain plants resulted in the study of phytoremediation (Ensley 2000). The design of a phytoremediation system varies among contaminants, the conditions at the site, the level of clean-up required, and the plants used (Schnoor 1997). However, the solubility of heavy metals in soil tends to be low due to complexation with organic matter, adsorption on clays and oxides, and precipitation as carbonates, hydroxides and phosphates (McBride 1994).

Addition of chelates such as ethylene-diamine-tetra-acetic acid (EDTA) and organic ligands to soil can enhance metal activity in the soil (Naidu and Harter 1998). Chelates were traditionally used in hydroponic solutions to buffer or decrease the availability of metals for uptake. In soils, EDTA has been used to increase the availability of heavy metals for plant uptake and to facilitate metal movement to plant roots (Norvell 1991). However, EDTA is not easily biodegradable and non-selective (Evangelou et al. 2007) and alternative methods are needed.

Humic acids (HA) contain acidic groups such as carboxyl and phenolic OH functional groups (Hofrichter and Steinbüchel 2004) and, therefore, provide organic macromolecules with an important role in the transport, bioavailability, and solubility of heavy metals (Lagier et al. 2000). Turan and Angin (2004) found HA effective in enhancing Cd, Mo, Pb and B desorption from soil and increasing their accumulation in maize (Zea mays L.) and sunflower (Helianthus annus L.). The lower stability constant of HA, compared to synthetic chelates for metals, makes HA an ideal soil amendment for phytoextraction and prevents the possible movement of heavy metal-humic acid complexes across the soil profile (Chen and Aviad 1990; Mackowiak et al. 2001; Zhang et al. 2003).

The ideal plant species for phytoremediation of contaminated soil is a plant species that has high yields and both tolerates and accumulates the targeted contaminants. Vetiver grass (Vetiveria zizanioides (L.) Nash) has been widely known for its effectiveness in erosion and sediment control. It has a strong root system which can reach up to 3 m depth, is fast growing, and can survive in harsh environments including sites with high metal levels (Truong et al. 1995; Chen et al. 2000). Pot experiments using heavy metal contaminated soil from suburban areas of Nanjing in China showed that the application of EDTA to the soils resulted in a surge of Pb concentrations in the shoots and roots of vetiver grass (Chen et al. 2004) and it is thus hypothesized that vetiver grass can be used to remediate sites contaminated with Pb and possibly B as well.

The objective of this study was to investigate the impact of HA addition on B and Pb phytoextraction by vetiver grass.

2 Materials and Methods

2.1 Pot Experiments

Two pot experiments were conducted using a complete randomized block design with two factors (Pb or B and HA addition) and four replicates. The studies were done with an Ustorthents (Soil Survey Staff 1992) sampled to a depth of 0–15 cm from agricultural fields in Erzurum province (39° 55′ N, 41° 61′ E) in Turkey. Soil was taken over an area of 10 ha using a grid sampling pattern.

The soil was air-dried, well-mixed and crumbled to pass 4 mm. For each experiment 1,000 g soil was transferred to 64 polyethylene pots (20 cm diameter and 15 cm depth). Boron and Pb were added as H3BO3 and Pb(NO3)2 solutions to obtain application rates of 0, 45, 90 and 180 kg ha−1 (0, 45, 90 and 180 mg kg−1) for both contaminants (assuming 1 Mg soil ha−1). Soils were saturated with dionized water and kept wet for 24 h prior to 24 h of drying at 35°C in a fan-forced oven and further mixing according to procedures outlined in Qadir et al. (2003). Ten such cycles of wetting, drying and mixing were conducted to ensure equilibrium 1 month after incubation. To support optimum vetiver grass growth, urea ((NH2)2CO) (120 kg N ha−1), tri-calcium phosphate (Ca(H2PO4)2) (100 kg P ha−1), and potassium sulphate (K2SO4) (50 kg K ha−1) were applied before planting. One month after the addition of Pb and B, vetiver grass was planted (1 plant pot−1) using 10 cm tall plants that had been grown from seedlings in a heated greenhouse under natural light (14 h day length), a minimum temperature of 10–11°C and maximum of 25–30°C, and a relative humidity of 30–40%. The pots were treated with 0, 100, 200 and 400 kg ha−1 HA (0, 100, 200 and 400 mg kg−1 HA) solutions applied to the soil equally split over three applications with 15 days interval after planting. Granular HA extracted from leonardite was purchased from Biyotar Organik Tarim A.S. (Ankara, Turkey). The water content of the soil was maintained at 70% of field capacity (375 g kg−1) throughout the 90 days experiments by daily additions of dionized water. Shoots and roots were harvested 90 days after planting and washed with dionized water to remove soil particles.

2.2 Soil and Plant Analysis

Four subsamples of the untreated soil were sieved to pass 2 mm and analyzed for physical and chemical analysis. Particle size analysis was performed by the pipette method after pre-treatment with 35% H2O2 and 1.0 M HCl to remove organic matter and carbonates according to Gee and Bauder (1986). Cation exchange capacity (CEC) was determined using C2H3NaO2–C2H7NO2 buffered at pH 7 according to Sumner and Miller (1996). The Kjeldahl method (Bremner 1996) and a Vapodest 10 Rapid Kjeldahl Distillation Unit (Gerhardt, Konigswinter, Germany) were used to determine total N while plant-available P was determined using the NaHCO3 method of Olsen et al. (1954). Electrical conductivity (EC) was measured in saturation extracts according to Rhoades (1996). Soil pH determinations were done in 1:2.5 H2O extracts according to McLean (1982). Soil organic matter was determined using the Smith–Weldon method as described in Nelson and Sommers (1982). C2H7NO2 buffered at pH 7 (Rhoades 1982) was used to determine exchangeable cations. Total Pb content of soil was determined by digesting 2 g of soil in a mixture of concentrated HNO3/HCl (v:v, 1:3) (Burau 1982) and B contents of soil were determined by digesting 1 g of soil with Na2CO3, H2SO4 and HCl (Bingham 1982). Available Pb and B were determined by 1 M NH4NO3-extraction (DIN 1995) and azomethine-H extraction (Wolf 1972), respectively using a Perkin-Elmer 360 Atomic Absorption Spectrophotometer (Perkin-Elmer, Waltham, Massachusetts, USA) for Pb determination and a UV/VIS (Aqumat) spectrophotometer (Thermo Electron Spectroscopy LTD, Cambridge, UK) for B determination. The physical and chemical properties of the untreated soil are given in Table 1.
Table 1

Chemical and physical properties of the Ustorthents from Erzurum province, Turkey (0–15 cm depth, n = 4, mean ± standard deviation)


Particle size distribution




33.2 ± 2.1



36.8 ± 1.9



30.0 ± 2.0

Organic C

g kg−1

6.0 ± 0.2

pH (1:2.5 soil:water)


7.2 ± 0.3

Total N

g kg−1

1.12 ± 0.11

Olsen P

mg kg1

14.2 ± 1.1

Cation exchange capacity

cmol(+) kg−1

16.8 ± 1.4

Electrical conductivity

dS m−1

1.15 ± 0.18

Exchangeable cationsa



cmol(+) kg−1

3.20 ± 0.32


cmol(+) kg−1

11.75 ± 1.05


cmol(+) kg−1

2.42 ± 0.92


cmol(+) kg−1

0.25 ± 0.13

Azomethine-H extractable B

mg kg−1

0.85 ± 0.14

1 M NH4NO3 extractable Pb

mg kg−1

0.42 ± 0.11

HNO3/HCl (v:v, 1:3) extractable (total) B

mg kg−1

12.6 ± 1.3

Na2CO3, H2SO4 and HCl extractable (total) Pb

mg kg−1

8.20 ± 0.74

aDetermined using ammonium acetate buffered at pH 7 (Rhoades 1982)

Following harvest, plant shoots and roots were dried for 48 h at 68°C and ground to pass 1 mm. The Pb and B contents of shoots and roots were determined using HNO3–HClO4 acid mixture digestion (AOAC 1990). The Pb analysis of the extraction solution was done by atomic absorption spectrometry (Perkin-Elmer 360 Atomic Absorption Spectrophotometer, Waltham, MA, USA) while B analysis was done using the azomethine-H procedure (Zhao et al. 1994) and an Aqumat UV/VIS spectrophotometer (Thermo Electron Spectroscopy LTD, Cambridge, UK). After harvest, soils were analyzed for 1 M NH4NO3-extractable Pb and azomethine-H extractable B as described above.

2.3 Statistical Analysis

Soil, shoot and root Pb and B content, yield and uptake data were analyzed using PROC MIXED of SAS Institute Inc. (1999) with block effects as random effects and Pb or B and HA application level and their interactions as fixed effects. Because initial analyses showed significant interactions between contaminant application and HA treatment for almost all response parameters, the effect of HA addition on soil and plant Pb and B were analyzed for each contaminant addition level individually using HA application as fixed effect and block as random effect. Similarly, the effect of contaminant addition on soil and plant response were evaluated for each HA application level using contaminant addition as main effect and block as random effect. Mean differences were considered significant if P ≤ 0.05.

3 Results and Discussion

3.1 Vetiver Yield

The addition of Pb did not impact dry matter (DM) yield where no HA was added (Fig. 1a). These results are in contrast with studies by Chantachon et al. (2004) who noted a decrease in biomass of two vetiver species with Pb contamination level, possibly suggesting that the Pb additions in our study might not have been high enough to create a level of toxicity sufficient to induce yield declines similar to those seen by Chantachon et al. (2004).
Fig. 1

Effects of humic acid (HA) application on plant available lead (Pb) in the soil (1 M NH4NO3-extraction), Pb content of shoots and roots, and dry matter yield of vetiver grass grown on a Ustorthents from Turkey at four levels of Pb addition (mean ± standard deviation). Different letters within a Pb application indicate means are significantly different at P ≤ 0.05

The addition of 200 kg HA ha−1 did increase yields at the highest Pb application and in the control. A similar trend was seen where 90 kg Pb ha−1 was added but means were not significantly different, while HA addition did not impact yields of vetiver grass grown in soil to which 45 kg Pb ha−1 had been applied (Fig. 1a). The results at the higher Pb application levels are consistent with Atiyeh et al. (2002) who found that plant growth increased progressively with addition of HA in the range of 100–1000 kg ha−1. The lack of a response at the 45 kg Pb ha−1 level is consistent with findings in a tobacco (Nicotiana tabacum L.) study with Cd contaminated soil where application of HA did not impact DM production (Evangelou et al. 2004) reflecting tobacco’s ability to tolerate and accumulate high amounts of Cd (Kayser et al. 2000; Wenger et al. 2002). Similarly, our results at 45 kg Pb ha−1 addition suggest that vetiver has the ability to accumulate Pb without yield decline (Fig. 1a) at relatively low levels of Pb contamination but Pb becomes toxic, impacting DM production, at higher Pb levels. It is at these higher Pb contamination levels that HA addition appears most effective for Pb phytoremediation with vetiver.

Boron addition did not impact yield where no HA had been added (Fig. 2a). However, HA addition increased yield for all B treatments except the highest B application where no significant differences among HA treatment means were observed (Fig. 2a). These results are consistent with Bama and Selvakumari (2005) who recorded higher grain and straw yield in rice at 20 kg ha−1 HA application.
Fig. 2

Effects of humic acid (HA) application on plant available boron (B) in the soil (azomethine-H extraction), B content of shoots and roots, and dry matter yield of vetiver grass grown on a Ustorthents from Turkey at four levels of B addition (mean ± standard deviation). Different letters within a B application indicate means are significantly different at P ≤ 0.05

3.2 Soil B and Pb Availability

As expected, Pb and B addition greatly enhanced plant available Pb and B beyond levels in the control soil (Figs. 1b and 2b). Soil 1 M NH4NO3 extractable Pb increased from 0.42 to 61.26 mg kg−1 upon Pb addition, suggesting toxic Pb levels were obtained (Li et al. 2005). Soil azomethine-H extractable B levels increased from 0.85 to 44.75 mg kg−1 upon B addition, also suggesting toxic B levels were obtained (Choi et al. 2006).

Recent studies reported complexation of Cu, Zn, B, Cd, Mo and Pb by different organic and inorganic chelates could play an important role in controlling heavy metal solubility and concentration in soil (Naidu and Harter 1998; Turan and Angin 2004; Turan and Esringu 2007), rendering metals available for plant uptake, possibly due to a decrease in pH (Evangelou et al. 2004). In our study, although trends toward an increase in plant available Pb and B upon HA addition were seen with Pb and B additions of 45 and 90 kg ha−1, means were not significantly different (Figs. 1 and 2). These results might have been influenced by the fact that soil Pb and B levels were only determined after the 90 days plant uptake period, and relatively low HA application levels were used compared to studies by Evangelou et al. (2004) and Turan and Angin (2004) where 4,000 and 1,250 kg ha−1 HA were added, respectively.

3.3 Shoot and Root Pb and B Content

The addition of Pb and B increased Pb and B concentrations in both shoot and roots (Figs. 1c, d and 2c, d) with greater Pb and B contents in roots than in shoots. Application of HA did not impact the Pb content of shoots at any of the Pb application levels (Fig. 1c) but enhanced root B contents where 45 or 90 kg B ha−1 had been applied. This may be due to increased permeability of root cell membranes upon HA addition, allowing for easier transfer of metals (Valdrighi et al. 1996). Root Pb content increased with HA addition where 45 kg Pb ha−1 had been applied (Fig. 1d). Root B content increased linearly with HA addition for B applications of 45 and 90 kg B ha−1 but did not impact B concentrations for the control and the higher B application level (Fig. 2d). These results are consistent with work by Evangelou et al. (2004) who reported that HA increased Cd concentration in tobacco shoots and Turan and Angin (2004) who reported that HA addition enhanced root B, Cd, Mo and Pb uptake in sunflower and maize. A decrease in pH could explain the enhanced uptake (Evangelou et al. 2004) but HA application levels in the studies by Evangelou et al. (2004) and Turan and Angin (2004) were much higher (4,000 and 1,250 kg ha−1, respectively) and additional work is needed to determine processes involved in the enhance uptake of Pb and B upon HA addition.

3.4 Crop Removal of Pb and B

The addition of HA could not enhance Pb and B removal in above ground biomass (shoots) from the control soils, most likely due low Pb and B levels and a lack of a response in plant available Pb and B upon HA addition (Fig. 3). Humic acid addition was effective in enhancing Pb removal via vetiver grass above ground biomass only where 90 kg Pb ha−1 had been added to the soil. The amendment was effective in enhancing B uptake both at 90 and 180 kg B ha−1 additions and ineffective at lower B application rates (Fig. 3). Similar increases in crop removal were reported by Chen et al. (2004), who found that the translocation ratio of Pb from vetiver grass roots to shoots grew as larger amounts of EDTA were applied. However, in our experiments HA addition to Pb or B contaminated soil did not consistently impact the translocation ratio for either B or Pb, equally impacting root and shoot B and Pb uptake.
Fig. 3

Effect of humic acid (HA) application on vetiver uptake of lead (Pb) and boron (B) in above ground (harvestable) biomass at four levels of Pb and B contamination of a Ustorthents from Turkey (mean ± standard deviation). Different letters within a Pb or B application level indicate means are significantly different at P ≤ 0.05

4 Conclusions

This study showed that vetiver grass is tolerant of high Pb and B concentrations in soils and that HA addition can increase Pb and B removal but that its effectiveness in enhancing Pb and B removal depends on the initial level of Pb and B contamination and the amount of HA applied. Field studies are needed to further test and quantify the effectiveness of HA addition in enhancing phytoremediation of Pb and B contaminated soils.


We thank Muhammet Kilci for supplying the vetiver seedlings for this study.

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© Springer Science+Business Media B.V. 2007