Wang Hong-qi, Lu Si-jin, Li hua, Yao Zhi-hua

College of Water Sciences, Beijing Normal University, Beijing ,China,100875

Abstract: Phytoremediation is a novel cleanup technology for the removal of heavy metals from contaminated soils.In phytoremediation, the plant uptake capability and the availability of the pollutant in the media are important. Bidens bipinnata has been found to be a new Pb hyperaccumulator,which not only has remarkable tolerance to Pb but also has extraordinary uptake and accumulation capacity for this element. The concentration of Pb in plants increased linearly with Pb concentration in pot experiment. The maximum Pb concentration was 1509.3 mg·kg^-1 in roots and 2164.7 mg/kgin shoots at an Pb concentration of 2000 mg/L in the culture solution. Bidens bipinnata has high effectiveness in translocating Pb from root to Shoot.Transfer factor (TF) was greater than 1 in all Pb concentration.The effects of ethylenediaminetetraacetic acid (EDTA) on Pb uptake by Bidens bipinnata was also studyed. Applied EDTA sig­nificantly both increased the mobility of soil Pb and the Pb concen­trations in roots and aboveground parts of Bidens bipinnata. And EDTA greatly promoted Pb to translocate from roots to shoots.Compared with CK,the Pb concen­trations in aboveground parts of Bidens bipinnata increased from 24.23mg/kg~680.56mg/kg to 29.07 mg/kg ~1905.57mg/kg by applying EDTA.These results confirm that Bidens bipinnata appeared to be a moderate Pb accumulator making it suitable for phytoremediation of Pb contaminated soil which grows rapidly, has substantial biomass, wide distribution and a broad ecological amplitude.

Key Words: Phytoremediation, Bidens bipinnata,Lead, Admend­ment, EDTA

Introduction

Lead (Pb) is a major anthropogenic pollutant and has accumulated in different terrestrial and aquatic ecosystems^[1]. Because of potential toxicity and high persistence, It presents potential high risks to humanhealth,the clean up of Pb contaminated soils is one of the most difficult tasks for environmental engineering.Some methods,such as immobilisation or extraction by physicochemical techniques, have an adverse effect on soil structure and require engineering costs^[2]. Phytoremediation has been put forward since the late 1980s,to remove heavy metals from contaminated soild by harvesting the plants without damaging the soil ^[2]. It has attracted much attention be­cause it is environmentally friendly and relatively cheap^[3,4].

Phytoremediation is defined as the use of plants to remove pollutants from the environment or to render them harmless^[3]. Much interest has focused on hyperaccumulator plants capable of accumulating potentiallyphytotoxic ele­ments to concentrations more than 100 times than those found in nonaccumulators^[2,3].These hyperaccumulators have strongly expressed metal sequestration mechanisms and metal accmulations in theirshoots.The discovery of hyperaccumulator plants prompted its use to remediate contaminated soils. The ideal hyperaccumulator plants employed for metal removal combine a high biomass plant, rapid growth, and highhyperaccumulation, such as oat (Avena sativa), barley (Hordeum vulgare), and Indian mustard (Brassica juncea) ^[5].

The use of hyperaccumulators is often coupled with manipu­lation of soil conditions either to increase the bioavailability of metals^[3]. Pb has limited solubility in soil and unavailability for plant uptake due tocomplexation with organic matter,sorption on oxides and clays or precipitation as carbonates, hydroxides and phosphates^[5].So,the two major limitations to Pb phytoremediation are: the low bio­availability in soil and poor translocation from roots to shoots. The poten­tial of adding chelates to Pb-contaminated soils to increase Pb bio­availability and accumulation in plants has also been investigated^[6~8].And the results show ethyl­enediaminetetraacetic acid (EDTA) was the most effective chelate to increase Pb desorption and increased the translocation of Pb from roots to shoots^[8].EDTA had been proved to be a strong and relatively biostablechelating agent that has poten­tial for soil remediation applications^[9~12].

Bidens bipinnata can uptake large amount of Pb ^[13].It appeares to be suitable for phytoremediation of Pb contaminated soils.This paper did some researches on Bidens bipinnata that can uptake lead by the methods of soil-planting. The primary objectives are:(1)To determine the ability of Bidens bipinnata to tolerate and accu­mulate Pb;(2) To find the distribution and accumulating rules of Pb in Bidens bipinnata;(3) To explore EDTA and other amend­ments for enhancing the mobility and plant uptake of Pb.

Materials and methods

Plant material and pot experiment

Bidens bipinnata seeds were col­lected from the channel beside Qinghe road of Beijing,China.Seeds were presoaked for five min in 100% ethanol and washed with sterilized water.Seeds were sprinkled into a moist soil in a seedbed covered with a plastic cling film to maintain moisture.when the spores germinated and grew into sporelings with leaves about 2cm in height. The plant was pre-cultured for one week in a modified Hoagland nutrient solution containing 2.0 mmol/L of Ca (NO₃)₂, 0.75 mmol/L of K₂SO₄, 0.1 mmol/L of KCl, 0.25 mmol/L of KH₂PO₄, 0.65 mmol/L of MgSO₄, 0.1 mmol/L of EDTA-Fe(Ⅱ), 0.01 mmol/L of H₃BO₃, 0.001 mmol/L of MnSO₄, 0.001 mmol/L of ZnSO₄, 0.000 1 mmol/L of CuSO₄ and 5.0´10^-6 mmol/L of (NH₄)Mo₇O₂₄. Then plants were transplanted into a plastic pot containing 0.85 kg quartz sand, which was pretreated with diluted HCl and washed with deionized water. The plant in individual treatment was cultured in the sandy media containing the nutrient solution with different treatments of Pb concentration(Table 1). The plant culture was conducted in a growth chamber with the following conditions: 12 h of light period with a light intensity of 4900µW·cm^-2, temperature of 26℃/18℃ (day/night), and average relative humidity of 60%. Each treatment was repeated triplicately.

Table1 The different treatments of Pb concentration (mg/L)

After days cultivating, the plant was harvested and washed with tap-water followed by three rinses with deionized water. Afterwards, plants were separated in roots,stems and leaves and dried and ground. The grounded sample (about 0.1 g) was weighted and digested with a mixture of HNO₃-HClO₄-H₂SO₄.The Pb content in roots,stems, and leaves was measured using an atomic fluorescence spectrometer (Z—6100).

The index of tolerance (IT) was determined according to the Wilkins’method^[14]:

IT=   average length of roots in tested solution ×100%

average length of roots in control

  Admend­ments experiment

The treatments comprised the following amend­ments: (1) control with no amendment (CK); (2) EDTA disodium salt (EDTA); (3) citric acid; (4) NaH₂PO₄. The soils used for these experiments are representative of a northern soil. Once collected, the soils were air dried at room temperature, followed by screening with a 2mm sieve. Soil texture, pH, Organic matter were determined according to Tan (1995). Total PbConcentration was measured using an atomic fluorescence spectrometer (Z—6100) after the soil sample (about 0.1 g) was weighted and digested with a mixture of HNO₃-HClO₄-H₂SO₄. The characteristics of the soils were listed in Table 2.Air-dried soil equivalent to 1.5 kg (oven dry basis) was placed in each pot. NH₄NO₃ and KH₂PO₄ were applied as basal fertilizers at the rates of 0.43 and 0.33g/kg, respectively. EDTA , citric acid and NaH₂PO₄ were added to the pots 50 days after sowing and plants were harvested after 62 days. In every case, EDTA, citric acid and NaH₂PO₄ concentrations were 3.148 mmol/kg, 3.148 mmol/kg and 4.006mmol/kg respectively.Fresh soil were also collected to determine 1 mol/L NH₄NO₃-extractable Pb. There were three repli­cates of each treatment.

Table 2 Physical and chemical characteristics of the soil

Statistical analysis

The statistical analysis of the results was done with the use of the Statistica software, SPSS 10.0,counting arithmetic means and mean errors.

Results and discussion

The tolerance of Bidens bipinnata to different Pb levels

Firstly there was less symptom of Pb toxicity was found to appear in fronds of Bidens bipinnata even treated with high Pb treatment concentration.But after 40 d of the culture, symptom was observed mainly in height of frond. When Pb concentration was 200mg/L ,Bidens bipinnata growed more quickly and 3cm tower above those growed at other Pb concentration.After 60 d of the culture, the symptom of dark brown coloration at the tips and the margins of pinnae was observed mainly in the bottom pinnae of senescent fronds,especially when Pb concentration was above 1000 mg/L. This toxicity was not so serious that the plants could survive throughout the whole culture period.

Plant biomass can be used as an indicator for the overall health of Bidens bipinnata growing in the presence of Pb.The influence of different Pb concentration on the biomass of Bidens bipinnata was showed in Table3. Bidens bipinnata had biggest biomass including fresh weight and dry weight at low Pb concentration(200 mg/L).At first the biomass of Bidens bipinnata increased with Pb concentrationincreasement.Then the biomass of Bidens bipinnata decrease with Pb concentration increasement.At biggest Pb concentration (2000mg/L) the biomass of Bidens bipinnata reached its lowest value.The similar trend was also found in the change of height of fronds (Table 3).It shows Bidens bipinnata growth could be stimulated by low concentration Pb. The discrepancy of biomass of Bidens bipinnata at Pbsupplies(1000mg/L) compared with control was remarkably(p<0.05) by Statistical analyse.It means Pb inhibiting concentration might be 1000mg·L^-1.

  In general, The Bidens bipinnata has a strong tolerance to Pb and the growth of frond can be stimulated by low Pb supplies.But its Pb tolerance was not infinite, high Pb concentration would harm frond’s growth.

Table 3 Influence of different treatments of Pb on the growth of Bidens bipinnata (meansstandard deviation)

Growth rate of root length is also an important indicator of plant resistance to heavy metals described by Wilkins^[14]. This method consists in comparing the roots growing with the addition of the stress factor with the roots of control plants. The obtained results is described as the Index of tolerance (IT). Table 4 shows IT values was above 1 as the Pb concentration is under 800mg/L.It is indicated that at this concentration rangePb did not affect Bidens bipinnata growth,even stimulate its growth. While Pb concentration is above 1000mg/L, the IT values,decreased with Pb concentration increasement, was lower than 1.It is implyed that frond growth was baffled by high concentration Pb.This results was concide with those above.

Table 4 Root lengh and root Tolerance Index

Pb accumulation and distribution in Bidens bipinnata

  Table 5 shows the concentrations of Pb in leaves, stems and roots of Bidens bipinnat. In general concentrations of Pb in tissues (leaves, stems, roots) increased progressively with the increase of Pb supply.Whenthe Pb concentration was 400mg/L and 800mg/L, compared with control,the Pb concentration in the frond increased dramatically, increase extent reached 90.97%and 79.68%,respectively.The Pb concentration in the frond increased slowly when Pb concentration was above 1000 mg/L.From 1000 mg/L to 2000 mg/L, increase extent was only 2.92%.It indicates that the adsorption of Bidens bipinnata to Pb was limited and it may reach maximum.The reason may be Pb toxicity increased progressively to Bidens bipinnata with increase in Pb levels,which weaken frond’s adsorption ability of Pb. The maximal Pb accumulation concentration reached 1509.3mg/kg in roots and 2164.7 mg/kg in overground part when disposal concentration was 2000mg/L.

Table 5 The concentration of Pb in Bidens bipinnata / mg·kg-1(meansstandard deviation)

Previous experiments showed the concentrations of Pb in overground parts of Aneurolepidium chinense, Gnaphalium polycaulon and L.Medicogo sativa was 73.15mg/kg, 82.61 mg/kg and 389.61 mg/kg ,respectively when Pb disposal concentration is 400mg/L^[13]. But at the same Pb concentration Bidens bipinnata was 617.87 mg/kg,which was greater than those above.It is suggested Bidens bipinnata had a strong uptake to Pb and appeared to be a suitable Pb accumulator.

Transfer factor (TF), defined as the Pb concentration ratio between shoot and root biomass, is used to measure the effectiveness of a plant in translocating Pb from root to Shoot(Table 5). The results show TF was greater than 1 in all Pb concentration,and TF reach maximum(1.62) when Pb disposal concentration was 400mg/L. There was always a distinct difference in Pb distribution between overgroud parts and roots. Partitioning of Mn between overgroud parts and roots showed that about 80% of Pb was transported into overgroud tissue in all treatments, demonstrating the great capacity of transportation from roots to shoots in thisspecies.And the Pb concentration distribution order in the Bidens bipinnata was: leaf>stem>root.

Fig.1The concentration of Pb in different parts of Bidens bipinnata

These may indicate that Bidens bipinnata had special Pb transfer mechanism which can transfer mostly absorbed Pb to frond’s overground part. Maybe Bidens bipinnata had special Pb transport proteins which can easlily translocate Pb from roots to overgroud parts.

Bidens bipinnata Pb enrichment ability at the different growth period

In order to study Bidens bipinnata Pb enrichment ability at the different growth period and get its best Pb absorption period, the whole growth of Bidens bipinnata is divided into the four period:period1（0～20d）, period 2（20～40 d）, period 3（40～60 d）and period 4（60～80 d）in this pot experiment according to growth time.

Fig.2 Bidens bipinnata uptaking Pb speed at the difference growth periods

The result shows Bidens bipinnata’s adsorbtion speed of Pb was varied at different growth period(Fig.2). Bidens bipinnata get its maximal Pb uptake speed at period 3（40～60 d）, the whole absorbtion quantity of Pb was 15.81mg/kg·d in roots and 19.83mg/kg·d in shoots ,which were bigger than those at the period 1（0～20d）and  period 2（20～40 d）,but the margin was not big. The Pb absorption speed then descended quickly at period 4（60～80 d）.Therefore, at the spot repair, young sprout of Bidens bipinnata can be transplanted directly, and harvest the frond after 60 d, at that time the absorbtion quantity of Pb reached maximum.

Effect of admend­ments on Pb uptake by Bidens bipinnata

NH₄NO₃ is a weak extractant that extracts mainly the water-soluble and part of the exchangeable frac­tion of metals. Pb is readily adsorbed by soil colloids, hence its mobility is always very low and this is considered to be the main factor restrict­ing the phytoremediation of Pb-contaminated soils. Thus, although the soil was newly spiked with Pb in this experiment, the NH₄NO₃ extractable Pb in non admend­ments amended soil after plant growth was only17.29mg/kg when Pb disposal concentration was 800mg/L. But when 3.15 mmol/kg^-of EDTA was added, EDTA addition significantly increased soil NH₄NO₃-extractable Pb to 91.56mg/kg, almost a six-fold increase compared with the unamended control. However, soil NH₄NO₃-extractable Pb did not in­crease remarkablely when citric acid was applied at the same level (Fig. 3). On the contrary ,NaH₂PO₄ decreased the concentrations of Pb in the roots and overground parts of Bidens bipinnata. So applied EDTA sig­nificantly increased the mobility of soil Pb,its ability stronger than citric acid and NaH₂PO₄.The order is EDTA> citric acid> NaH₂PO₄.

Fig. 3 Soil NH4NO3-extractable metals

There were no obvious symptoms of Pb tox­icity in Bidens bipinnata during germination and growth. after the EDTA, citric acids and NAH₂PO₃ addition into the soil.But from Table 6,compared with CK, with the increase of Mn level, the biomass of Bidens bipinnata increased, and then decreased.

Applied admend­ments increased active Pb concentrations in soil. It shows again Bidens bipinnata growth could be stimulated by low Pb concentration.But when Pb concentrations was too high ,it also baffledBidens bipinnata growth.In this pot experiment the phytotoxicity of EDTA metals was under plant’s tolerance.There was less symptom of Pb toxicity was found to appear in fronds of Bidens bipinnata.

Table 6 Influence of admend­ments on the dry weight of Bidens bipinnata (g/frond )

Fig.4 shows the Pb concen­trations in roots and aboveground parts of Bidens bipinnata. Applied EDTA sig­nificantly increased the concentrations of Pb in the roots of Bidens bipinnata, whereas the citric acid also show some effect,but it is more less. Pb concentrations in overground parts was also increased by EDTA,especially at Pb concentrations(1000 mg/L).Although overground parts Pb uptake was also enhanced by addition by citric acids, the increases were smaller than those produced by EDTA.And NaH₂PO₄ decreased the concentrations of Pb in the roots and overground parts of Bidens bipinnata.

Fig.4 Pb concen­trations in roots and aboveground parts of Bidens bipinnata

It is seemed that applied EDTA sig­nificantly both increased the mobility of soil Pb and the Pb concen­trations in roots and aboveground parts of Bidens bipinnata.And the most important was EDTA could greatly promote Pb to translocate from roots to shoots.Compared with CK,the Pb concen­trations in aboveground parts of Bidens bipinnata increased from 24.23mg/kg~680.56mg/kgto 29.07 mg/kg~1905.57mg/kg by applying EDTA at Pb concentration(200~1000 mg/L). And it would be foreseed that the Pb concen­trations in aboveground parts of Bidens bipinnata would increas continually with Pb concentration increasement byEDTA.But this increasement was finite because Bidens bipinnata Pb tolerance was not infinite.

It is well known that EDTA acts as a chelating agent that has proven to be helpful in Pb movement from roots to leaves.In this study, the addition of EDTA greatly increased Pb translocation from roots tooverground parts. It is commonly assumed that type P ATP-ases are responsible for the translocation of both necessary (e.g. Cu²⁺, Zn²⁺,Mn²⁺) and nonessential metals (Cd²⁺, Pb²⁺, Hg²⁺)through the biological membranes ^[9,15,16].EDTA may induce the activation of the ATPases in the plasma membrane producing changes on transport of ions through the membrane.Additionally,possibly EDTA regulates a protein membrane which is related to Pb transport function. So Pb can easily be translocated from roots to overground parts through prevention of cell wall reten­tion.

It is seemed that applying EDTA could be considered as a feasible technique for the improvement of Pb accumulation in the aboveground parts of hyperaccumulator species.

Conclusions

  Overall, The Bidens bipinnata had a strong tolerance and big adsorption to Pb. Mostly absorbed Pb could be transfered from roots to overground part of fronds.Applying EDTA increased both the mobility of soilPb and the Pb concen­trations in Bidens bipinnata.But the most important was EDTA could greatly promote Pb to translocate from roots to overgroud parts.

The Bidens bipinnata has a broad ecological amplitude. It can grow in the majority of areas of China, and also in other country. Moreover, it has the characteristics of a fast growth rate, large biomass, abundant seed production, robust roots, which make it is easy to be harvested mechanically.Therefore, there is great potential for using Bidens bipinnata in the remediation of Pb-contaminated soils and it also provides a new resource for exploring the biochemical mechanisms of Pb hyperaccumulation and detoxification.And applying EDTA may be a feasible technique for the improvement of Pb accumulation in overground parts of fronds.

Acknowledgements

The study was supported by Natural Science Foundation of China (NSFC) (Grant No.: 40472129).

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