Phytoextraction potential of two Rumex acetosa L. accessions collected from metalliferous and non-metalliferous sites: Effect of fertilization

Chemosphere 74 (2009) 259–264

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Chemosphere j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / ch e m o s p h e r e

Phytoextraction potential of two Rumex acetosa L. accessions collected from metalliferous and non-metalliferous sites: Effect of fertilization O. Barrutia a, L. Epelde b, J.I. García-Plazaola a, C. Garbisu b, J.M. Becerril a,* a b

Depart­ment of Plant Biol­ogy and Ecol­ogy, Uni­ver­sity of the Basque Coun­try/EHU, P.O. Box 644, E-48080 Bil­bao, Spain NE­I­KER-Tec­na­lia, Basque Insti­tute of Agri­cul­tural Research and Devel­op­ment, c/ Berre­aga 1, E-48160 De­rio, Spain

a r t i c l e

i n f o

Article history: Received 21 April 2008 Received in revised form 9 September 2008 Accepted 11 September 2008 Available online 31 October 2008  Key­words: Phy­to­sta­bi­li­za­tion Heavy met­als Pseu­dome­tal­lo­phyte Mine tail­ing Reveg­e­ta­tion

a b s t r a c t Metal tol­er­ance and phy­toex­trac­tion potential of two com­mon sor­rel (Ru­mex ace­to­sa L.) acces­sions, col­lected from a Pb/Zn con­tam­i­nated site (CS, Lanes­to­sa) and an uncon­tam­i­nated site (UCS, Larr­au­ri), were stud­ied in fer­til­ized and non-fer­til­ized pots prepared by com­bin­ing soil sam­ples from both sites in dif­fer­ent pro­por­tions (i.e., 0%, 33%, 66% and 100% of Lanes­to­sa con­tam­i­nated soil). The original met­al­lif­er­ous mine soil con­tained 20480, 4950 and 14 mg kg¡1 of Zn, Pb and Cd, respec­tively. The micro­cosm exper­i­ment was car­ried out for two months under green­house con­trolled con­di­tions. It was found that fer­til­iza­tion increased mean plant bio­mass of both acces­sions as well as their tol­er­ance. How­ever, only the CS acces­sion sur­vived all treat­ments even though its bio­mass decreased pro­por­tion­ally accord­ing to the per­cent­age of con­tam­in ­ ated mine soil pres­ent in the pots. This me­tal­lic­o­lous acces­sion would be use­ful for the reveg­e­ta­tion and phy­to­sta­bi­li­za­tion of mine soils. Due to its high con­cen­tra­tion and bio­avail­abil­ity in the con­tam­in ­ ated soil, the high­est val­ues of metal phy­toex­tract­ed cor­re­sponded to Zn. The CS acces­sion was capa­ble of effi­ciently phy­toex­tract­ing metal from the 100% mine soil, indeed reach­ing very prom­is­ing phy­toex­trac­tion rates in the fer­til­ized pots (6.8 mg plant¡1 month¡1), sim­i­lar to the ones obtained with hyper­ac­cu­mu­la­tor plants. It was con­cluded that fer­til­ iza­tion is cer­tainly worth being con­sid­ered for phy­toex­trac­tion and reveg­e­ta­tion with native plants from met­al­lif­er­ous soils. © 2008 Else­vier Ltd. All rights reserved.

1. Intro­duc­tion Phy­toex­trac­tion is a tech­nol­ogy that uses uptake by plants to remove met­als and other con­tam­i­nants from soil, sed­i­ments and water (Salt et al., 1995). The amount of metal extracted depends on its con­cen­tra­tion in the har­vest­able parts of plants as well as on plant bio­mass. Thus, selec­tion of ade­quate plant spe­cies which pres­ent these two char­ac­ter­is­tics is cru­cial for suc­cess­ful phy­toex­ trac­tion pro­cesses. In this respect, although hy­per­ac­cu­mu­lat­ing plants con­cen­trate large amounts of met­als in their above­ground tis­sues, they fre­quently reach low bio­mass val­ues and lack any estab­lished cul­ti­va­tion, pest man­age­ment or har­vest­ing prac­tices, as major draw­backs for their uti­li­za­tion for metal phy­toex­trac­tion (Wen­zel et al., 1999). On the other hand, com­pared to hy­per­ac­ cu­mu­la­tors, most high bio­mass crop plant spe­cies usu­ally have lim­ited metal tol­er­ance (Cha­ney et al., 1997). A prom­is­ing alter­ na­tive, worth of in-depth explo­ra­tion for metal phy­toex­trac­tion, is the uti­li­za­tion of plants spon­ta­ne­ously grow­ing in metal-enriched soils (met­al­lif­er­ous sites), such as those from min­ing areas (Fre­itas et al., 2004). The study and selec­tion of native pseu­dome­tal­lo­ phytes (i.e., com­mon plant spe­cies, with great eco­log­ic­ al ampli­tude, * Cor­re­spond­ing author. Tel.: +34 94 601 5328; fax: +34 94 601 3500. E-mail address: jo­sema­ria.be­cer­[email protected] (J.M. Becerril). 0045-6535/$ - see front matter © 2008 Else­vier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.09.036

that can grow either in non-con­tam­i­nated or metal con­tam­i­nated soils) that pres­ent high metal tol­er­ance, mod­er­ate plant bio­mass, and ele­vated capac­ity to accu­mu­late met­als, is a most inter­est­ing approach in an attempt to search for new plant spe­cies of potential for metal phy­toex­trac­tion. Cer­tainly, indig­en ­ ous plant spe­cies are most likely to fit into a fully func­tional eco­sys­tem (Li, 2006). Reveg­e­ta­tion and imple­men­ta­tion of remediation ­phyto­technol­o­gies in metal pol­luted mine soils is not an easy, ­straight­for­ward task. Apart from the high lev­els of toxic heavy metal char­ac­ter­is­tics of mine soils, these par­tic­ul­ ar ­envi­ron­ments ­usu­ally pres­ent sev­eral other cru­cial con­straints for the ­estab­lish­ment of plant spe­cies (e.g., absence of top­soil, drought, sur­face ­mobil­ity, com­pac­tion, wide tem­per­a­ture fluc­tu­a­tion, and short­age of ­essen­tial nutri­ents) (Wong, 2003). Among the ­afore­men­tioned, nutri­ent defi­ ciency has been reported to be one of the most ­lim­it­ing fac­tors for the reveg­e­ta­tion of Pb/Zn mine tail­ings (Ye et al., 2002). Fer­til­iza­tion of mine-tail­ing soils could facil­i­tate plant sur­vival, alle­vi­ate growth inhi­bi­tion and pro­cure a bet­ter plant devel­op­ment. The peren­nial herb Ru­mex ace­to­sa L. (com­mon sor­rel) is a pseu­dome­tal­lo­phyte that has already been iden­ti­fied in sev­eral metal con­tam­i­nated sites (Wang et al., 2003; Ernst et al., 2004). We iden­ti­fied a R. ace­to­sa acces­sion grow­ing in a nat­u­rally rev­eg­ e­tat­ed Zn/Pb min­ing area of the Basque Coun­try (north­ern Spain) that was aban­doned 30 years ago. This spe­cies ful­fils many of the

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require­ments of a use­ful plant for metal phy­toex­trac­tion, i.e., it con­cen­trates high lev­els of met­als in its tis­sues (John­ston and Proc­ tor, 1977), and it can pro­duce a bio­mass of 7 t dry weight shoot ha¡1 yield¡1 (Tang et al., 1999). In this study, we inves­ti­gated the tol­er­ance and heavy metal phy­toex­trac­tion potential of two R. ace­to­sa acces­sions, one col­ lected from the afore­men­tioned aban­doned Zn/Pb mine area and the other from an agri­cul­tural site. To this aim, R. ace­to­sa plants were grown, under green­house con­trolled con­di­tions, for 2 months in fer­til­ized and non-fer­til­ized pots that were prepared by com­bin­ ing original soil sam­ples from both sites in dif­fer­ent pro­por­tions (i.e., 0%, 33%, 66% and 100% of Lanes­to­sa con­tam­i­nated soil). The objec­tives of this study were to deter­mine: (i) dif­fer­ences in heavy metal tol­er­ance, uptake and phy­toex­trac­tion rate between the two acces­sions and (ii) the effect of fer­til­iza­tion on metal tol­er­ance and phy­toex­trac­tion. The suit­abil­ity of using native plant acces­sions for the reveg­e­ta­tion and phy­toex­trac­tion of aban­doned mine soils is dis­cussed. 2. Mate­ri­als and meth­ods 2.1. Soil char­ac­ter­iza­tion Two types of soils col­lected (depth: 0–25 cm) in the Basque Coun­try (north­ern Spain) were used in this study: (i) one from a nat­ur­ ally rev­eg­e­tat­ed aban­doned mine that is cur­rently con­tam­i­ nated with Pb, Zn and Cd and located in Lanes­to­sa (lat­it­ ude 43°139; lon­gi­tude 3°269), (ii) the other from a non-con­tam­i­nated agri­cul­ tural site located in Larr­au­ri (lat­i­tude 43°229; lon­gi­tude 2°489). Table 1 shows the phys­i­co­chem­i­cal prop­er­ties of both soils. For chem­i­cal anal­y­sis, soils were air dried at 70 °C for 72 h and sieved to <2 mm. Soil pH was mea­sured in a 1:2.5 (w/v) sus­pen­sion of soil and water. Organic mat­ter (OM) con­tent, total N, elec­tri­cal con­duc­tiv­ity, par­ti­cle size dis­tri­bu­tion, Ol­sen P, and exchange­able K, Ca and Mg were deter­mined fol­low­ing stan­dard meth­ods (MAPA, 1994). Water hold­ing capac­ity (WHC) at 33 and 1500 kPa was mea­ sured in a Rich­ard’s mem­brane-plate extrac­tor (Klute, 1986). Total con­cen­tra­tions of heavy met­als in soils were deter­mined using ICPAES fol­low­ing aqua re­gia diges­tion (McG­rath and Cun­liffe, 1985).

Avail­able mobile frac­tions of heavy met­als in soil were deter­mined using 0.01 M CaCl2 as extract­ant (Hou­ba et al., 2000). 2.2. Exper­i­men­tal design and plant cul­ti­va­tion A micro­cosm phy­toex­trac­tion exper­i­ment was car­ried out in fer­til­ized and non-fer­til­ized pots that were prepared by com­bin­ing (w/w) soil sam­ples from both sites (met­al­lif­er­ous Lanes­to­sa and non-met­al­lif­er­ous Larr­au­ri) in dif­fer­ent pro­por­tions (i.e., 0%, 33%, 66% and 100% of the Lanes­to­sa mine soil). Thus, four treat­ments were con­sid­ered in this study: (i) treat­ment 0: 0% mine soil, 100% agri­cul­tural soil; (ii) treat­ment 33: 33% mine soil, 67% agri­cul­tural soil; (iii) treat­ment 66: 66% mine soil, 34% agri­cul­tural soil; and (iv) treat­ment 100: 100% mine soil, 0% agri­cul­tural soil. Soils were then used to fill 12 cm-diam­e­ter and 11 cm-height plas­tic pots (700 g of soil pot¡1) and moist­ened with dis­tilled water to ­approx­i­mately 80% WHC. Two assays were car­ried out in par­al­lel: (i) one with nonfer­til­ized pots, and (ii) the other with pots ­fer­til­ized with a sin­gle dose of 1 g kg¡1 of a com­mer­cial fer­til­izer con­tain­ing 20/8/40+0.1 N/P/K+B (Massó Gar­den, Bar­ce­lona, Spain). Acces­sions of R. ace­to­sa were col­lected from the con­tam­i­nated mine area in Lanes­to­sa (CS) and the uncon­tam­i­nated site in Larr­ au­ri (UCS). Both acces­sions were grown under green­house con­ di­tions for one year prior to the exper­i­ment. Clonal plants from the two selected acces­sions were grown from root stock. Once plant shoot emerged these seed­lings were used for the exper­i­ ment. Three weeks old R. ace­to­sa seed­lings (approx­i­mately, 10 g fresh weight) were trans­planted into pots (one seedling pot¡1) and placed in a green­house under con­trolled con­di­tions of tem­per­a­ ture and humid­ity as fol­lows: tem­per­a­ture 25/18 °C day/night, rel­ a­tive humid­ity 60/80% day/night. Nat­u­ral light was sup­ple­mented with white cold lamps (Phil­lips SON-T AGRO 400, Bel­gium) to reach an illu­mi­na­tion of 400 lmol pho­ton m¡2 s¡1 pho­to­syn­thetic flux den­sity under a pho­to­pe­riod of 16 h d¡1. All treat­ments were con­ducted in qua­dru­pli­cate. Pots were watered from under­neath (sub-irri­ga­tion). After two months, plants were har­vested and sep­a­rated into roots and shoots. Shoots and roots were care­fully rinsed in tape water, soaked twice in deion­ized water and finally oven dried at 70 °C for 48 h. Dry weights were recorded and plant mate­rial was prepared for acid diges­tion. 2.3. Fluo­res­cence mea­sure­ments

Table 1 Phys­i­co­chem­i­cal prop­er­ties of the con­tam­i­nated mine CS soil and the uncon­tam­i­ nated agri­cul­tural UCS soil used in this study Param­e­ter

UCS soil

CS soil

Texture pH OM (%)a Coarse sand, 1–2 mm (%) Fine sand, 0.05–1 mm (%) Lime frac­tion, 2–50 lm (%) Clay frac­tion, <2 lm (%) Total N (%) WHC-33 kPab WHC-1500 kPac P (mg kg¡1) K (mg kg¡1) Ca (mg kg¡1) Mg (mg kg¡1) Total Cd (mg kg¡1) Total Pb (mg kg¡1) Total Zn (mg kg¡1) Extract­able Cd (mg kg¡1) Extract­able Pb (mg kg¡1) Extract­able Zn (mg kg¡1)

Clay loam 6.9 15.4 ± 0.2 16 ± 4 36 ± 9 24 ± 2 24 ± 3 0.64 ± 0.1 48 ± 3 33 ± 2 >120 536 ± 32 5260 ± 101 690 ± 54 1 ± 0 44 ± 1 173 ± 2 0.01 ± 0 0.16 ± 0.1 1.6 ± 0.4

Sandy loam 6.6–6.8 4.8 ± 0.1 23 ± 2 50 ± 4 14 ± 1 13 ± 3 0.2 ± 0 21 ± 0.8 12 ± 0.2 6 ± 1.0 49 ± 4.0 1110 ± 120 134 ± 15 14 ± 2.0 4950 ± 178 20480 ± 201 2.5 ± 0.01 13.9 ± 0.4 748.3 ± 10.1

UCS: uncon­tam­i­nated site; CS: con­tam­i­nated site. a Organic mat­ter. b Water hold­ing capac­ity at 33 kPa. c Water hold­ing capac­ity at 1500 kPa.

Prior to plant har­vest Chl a fluo­res­cence was mea­sured using a portable mod­u­lated fluo­rim­e­ter (OS 5-FL, Op­ti­science, Tyn­gs­boro, USA). Ini­tial (Fo), max­i­mal (Fm), and var­i­able (Fv) fluo­res­cence were mea­sured in dark adapted leaves with a sat­u­rat­ing light pulse of 0.8 s. The max­i­mal pho­to­chem­i­cal effi­ciency of pho­to­sys­tem II was esti­mated by the ratio Fv/Fm = (Fm ¡ Fo)/Fm (Gen­ty et al., 1989). 2.4. Heavy metal anal­y­ses in plant sam­ples Dried plant sam­ples were milled, homog­en ­ ised to obtain a r­ ep­re­sen­ta­tive sam­ple, and sub­se­quently digested with a mix­ture of HNO3/HClO4 (Zhao et al., 1994). Con­cen­tra­tions of Pb, Zn, and Cd in the digested plant sam­ples were deter­mined using an Atomic Absorp­tion Spec­trom­e­ter (Spec­tra AA-250 plus, Var­ian, Aus­tra­lia) equipped with an auto­matic sam­pler (Sps-5, Var­ian, Aus­tra­lia). Cal­i­bra­tion curves were made up from com­mer­cial stan­dard solu­ tions of 1 g L¡1 (Sigma). 2.5. Sta­tis­ti­cal anal­y­ses Sta­tis­ti­cal anal­y­ses were per­formed using SPSS/PC Sta­tis­ti­cal Anal­y­sis Soft­ware. One-way anal­y­sis of var­i­ance was used to com­ pare treat­ments within the same R. ace­to­sa acces­sion. Dun­can test



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24

was used to estab­lish the sig­nif­i­cance of the dif­fer­ences among means. Com­par­i­sons between the two acces­sions under the same treat­ment were per­formed using t-test with a con­fi­dence inter­val of 95%.

3.1. Phys­i­co­chem­i­cal char­ac­ter­iza­tion of the original soils Lead/zinc mine tail­ings have fre­quently a poor phys­i­cal ­struc­ture, high lev­els of avail­able met­als and low nutri­ent con­ tents (Ye et al., 2002). In con­se­quence, for the reveg­e­ta­tion and ­phy­to­reme­di­a­tion of mine soils, it is essen­tial to choose plant spe­ cies which are not only tol­er­ant to met­als but also to the other adverse envi­ron­men­tal fac­tors pre­vail­ing in these areas, so that a self-sus­tain­able ­veg­e­ta­tion is achieved (Wong et al., 2003). As observed in Table 1, as far as phys­i­co­chem­i­cal prop­er­ties are con­cerned, the main dif­fer­ences between the mine soil from Lanes­to­sa (CS soil) and the agri­cul­tural uncon­tam­i­nated soil from Larr­au­ri (UCS soil) related to phys­i­cal prop­er­ties and the con­tent of nutri­ents, OM and heavy met­als. The CS soil (sandy loam) had a lower WHC and also lower con­tents of OM, total N and other ma­cronu­tri­ents (i.e., P, K, Ca and Mg) than the UCS soil. In con­ trast, both soils pre­sented a sim­i­lar neu­tral pH. The con­tents of Pb, Zn and Cd in the CS mine soil were much higher (about 1000-, 100- and 14-fold higher for Pb, Zn and Cd, respec­tively) than those in the UCS agri­cul­tural soil. How­ever, total metal con­cen­tra­tion in soil does not give enough infor­ma­tion on metal mobil­ity, bio­ avail­abil­ity and phy­to­tox­ic­ity. Instead, assess­ment of soil con­ tam­i­na­tion can also be deter­mined by esti­mat­ing the bio­avail­able frac­tion of toxic met­als (Bo­u­lar­bah et al., 2006). In the CS met­al­ lif­er­ous soil the high­est val­ues of avail­able metal con­cen­tra­tion in the mobile frac­tion of the soil were found for Zn, i.e. 748 mg kg¡1 dry weight (cor­re­spond­ing val­ues for Cd and Pb were 2.5 and 14 mg kg¡1 dry weight soil, respec­tively). The avail­able metal pool is a most impor­tant param­e­ter for metal phy­toex­trac­tion, since it not only influ­ences phy­to­tox­ic­ity and envi­ron­men­tal risk but also ­deter­mines the effec­tive­ness of the phy­toex­trac­tion pro­cess itself ­(Her­nán­dez-Al­li­ca et al., 2006).

18

Dry weight shoot biomass plant -1(g)

3. Results and dis­cus­sion

261

b

b

a

UCS

b* c*

12

c*

6

d d

0 18

CS

A

A A

B*

12

BC*

B*

6

D

CD

0 0

33

66

100

Treatment Fig. 1. Dry weight plant¡1 (g) of aer­ial tis­sues of UCS and CS acces­sions of R. ace­to­sa plants after two months of growth in non-fer­til­ized (white bars) and fer­til­ized soils (black bars) con­tain­ing dif­fer­ent per­cent­ages of the mine tail­ing soil: 0% (treat­ment 0), 33% (treat­ment 33), 66% (treat­ment 66) and 100% (treat­ment 100). Data rep­re­ sent mean val­ues (n = 4). Bars rep­re­sent stan­dard devi­a­tion. Dif­fer­ent let­ters denote sta­tis­ti­cally sig­nif­i­cant dif­fer­ences at p < 0.05 among treat­ments for each metal and within each acces­sion (lower case let­ters for UCS acces­sion; upper case let­ters for CS acces­sion). Aster­isks stand for sig­nif­i­cant dif­fer­ences at p < 0.05 between acces­ sions under the same treat­ment and for the same metal.

0.87 a* 0.84

UCS a*

ab

a

0.81 3.2. Plant growth and pho­to­chem­ic­ al effi­ciency

b*

b*

Fv//Fm

0.78 As observed in Table 1, the UCS soil (treat­ment 0) was rich in nutri­ents and, con­se­quently, sup­ported the growth of both the me­tal­lic­o­lous and the non-me­tal­lic­o­lous acces­sions of R. ace­to­sa with­out any lim­i­ta­tion through­out the exper­i­men­tal period. Actu­ ally, in the UCS soil, fer­til­iza­tion treat­ment did not lead to sig­nif­i­ cantly higher val­ues of plant bio­mass for any of the two R. ace­to­sa acces­sions used in this study (Fig. 1). When increas­ing the pro­ por­tion of mine soil in the non-fer­til­ized exper­i­men­tal pots, the acces­sion col­lected from the non-met­al­lif­er­ous agri­cul­tural site (UCS) grew well under treat­ment 33, in fact reach­ing sim­i­lar val­ ues of plant bio­mass to those of con­trol plants (treat­ment 0) (Fig. 1). How­ever, this non-me­tal­lic­ol­ ous acces­sion showed some phy­ to­tox­ic­ity symp­toms under treat­ment 33, as reflected in its lower pho­to­chem­i­cal effi­ciency (Fv/Fm) mean val­ues com­pared to those dis­played by con­trol plants (treat­ment 0) (Fig. 2). On the con­trary, treat­ment 66 sig­nif­i­cantly reduced the growth of the non-me­tal­lic­ o­lous UCS acces­sion (a 30% inhi­bi­tion) as well as its pho­to­chem­i­cal effi­ciency, with plants show­ing clear visual symp­toms of phy­to­tox­ ic­ity (e.g., chlo­ro­sis and necrotic spots in leaves). Finally, treat­ment 100 inhib­ited almost com­pletely the growth of UCS plants (actu­ ally, UCS plants died after two weeks of expo­sure to the con­tam­i­ nated mine soil). Con­versely, the R. ace­to­sa acces­sion iso­lated from the con­tam­in ­ ated mine soil (CS acces­sion) sur­vived all treat­ments, although its growth decreased pro­por­tion­ally accord­ing to the

0.75 0.72 0.84

A* B*

A* CS

A B

B*

B B

0.81 0.78 0.75 0.72 0

33

66

100

Treatment Fig. 2. Pho­to­chem­ic­ al effi­ciency (Fv/Fm) of UCS and CS acces­sions of R. ace­to­sa after two months of growth in non-fer­til­ized (white bars) and fer­til­ized soils (black bars) con­tain­ing dif­fer­ent per­cent­ages of the mine tail­ing soil: 0% (treat­ment 0), 33% (treat­ment 33), 66% (treat­ment 66) and 100% (treat­ment 100). Data rep­re­sent mean val­ues (n = 4). Bars rep­re­sent stan­dard devi­a­tion. Dif­fer­ent let­ters denote sta­ tis­ti­cally sig­nif­i­cant dif­fer­ences at p < 0.05 among treat­ments for each metal and within each acces­sion (lower case let­ters for UCS acces­sion; upper case let­ters for CS acces­sion). Aster­isks stand for sig­nif­i­cant dif­fer­ences at p < 0.05 between acces­ sions under the same treat­ment and for the same metal.

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amount of mine soil pres­ent in the pots (the higher the pro­por­tion of mine soil, the lower the growth) (Fig. 1). These me­tal­lic­o­lous plants did not show any visual phy­to­tox­ic­ity symp­toms except for some necrotic spots and a red col­or­a­tion, prob­a­bly due to antho­cy­ a­nin accu­mu­la­tion, in fully expanded leaves under treat­ment 100. Fer­til­iza­tion greatly stim­u­lated growth of both acces­sions under treat­ment 33 (Fig. 1). Under treat­ments 66 and 100, instead of nutri­ents con­tent, metal tox­ic­ity seemed to be the most lim­ it­ing fac­tor con­trol­ling plant bio­mass pro­duc­tion. Indeed, under these 66 and 100 treat­ments, although fer­til­iza­tion did exert a slight positive effect on plant growth of both R. ace­to­sa acces­ sions, plants did not reach the bio­mass val­ues obtained in con­ trol pots (treat­ment 0). Growth inhi­bi­tion and bio­mass reduc­tion are gen­eral responses of vas­cu­lar plants to metal tox­ic­ity (Oua­ri­ti et al., 1997). Inhi­bi­tion of both cell elon­ga­tion and divi­sion by heavy met­als could explain, in part, the decline in bio­mass pro­duc­ tion (Ar­du­ini et al., 1994). In gen­eral, under treat­ments 33 and 66, fer­til­iza­tion exerted a slight positive effect on max­i­mal pho­to­chem­i­cal effi­ciency of both acces­sions (Fig. 2). This effect was even more pro­nounced in CS acces­sion plants, which main­tained the pho­to­chem­i­cal effi­ciency of plants grow­ing in fer­til­ized treat­ment 0 pots. This positive effect was also reported by Pang et al. (2003) in vet­i­ver grass (Ve­tive­ria ziz­ano­ides L.), find­ing out that fer­til­iza­tion alle­vi­ated the decrease in pho­to­chem­i­cal activ­ity (Fv/Fm) when plants were grown in a metal con­tam­i­nated tail­ing soil. How­ever, this positive effect of fer­til­iza­tion on pho­to­chem­i­cal effi­ciency was not observed in CS plants under treat­ment 100, sug­gest­ing that metal phy­to­tox­ic­ity was prob­a­bly too high to be par­tially alle­vi­ated by nutri­ent addi­ tion. Any­way, these plants showed less visual phy­to­tox­ic­ity symp­ toms than when grow­ing under non fer­til­ized treat­ment 100, i.e. less necrotic spots and red­dish tis­sues and a higher pro­duc­tion of sprouts. 3.3. Metal con­cen­tra­tion in plant tis­sues Metal con­cen­tra­tion in shoots of both acces­sions was sim­i­lar when plants were grown in low or mod­er­ately con­tam­i­nated soils (i.e., treat­ments 33 and 66), but not under treat­ment 100 (100% mine soil) (Table 2). This dif­fer­ence was prob­a­bly due to the fact that, in the heav­ily con­tam­i­nated mine soil, only the more tol­er­ant CS acces­sion was capa­ble of sur­viv­ing through­out the exper­i­men­ tal period, thus being able to accu­mu­late high lev­els of met­als in its tis­sues. Zinc was the metal that reached the high­est shoot con­ cen­tra­tion in both acces­sions, most likely due to the high avail­able lev­els of this metal pres­ent in soil (Table 1) as well as to its high mobil­ity. In fact, Zn was respon­si­ble for the main dif­fer­ences found between the two R. ace­to­sa acces­sions. In par­tic­u­lar, under the nonfer­til­ized treat­ment 100, the CS acces­sion pre­sented a far much higher con­cen­tra­tion of Zn (3079 mg kg¡1) than the UCS acces­sion (573 mg kg¡1) which did not sur­vive under this treat­ment 100. Its sur­vival in the original met­al­lif­er­ous soil through­out the twomonth exper­i­men­tal period, together with its capac­ity to con­cen­ trate almost 5-fold more Zn in its shoots than the UCS acces­sion dur­ing that time, high­lights the high metal tol­er­ance of the me­tal­ lic­o­lous CS acces­sion. The recorded zinc con­cen­tra­tion in shoots of CS acces­sion plants grow­ing in treat­ment 100 soil (original min­ing soil) is within the range of val­ues observed in native CS pop­u­la­ tion plants spon­ta­ne­ously grow­ing in the aban­doned Pb–Zn mine (between 199 and 7800 mg Zn kg¡1 shoot), where this pop­u­la­tion behaves like an indi­ca­tor plant reflect­ing soil Zn con­cen­tra­tion in shoot tis­sue. Accord­ing to Zhao et al. (2003), bio­ac­cu­mu­la­tion ­fac­tor (BF) (shoot/soil con­cen­tra­tion ratio) is more impor­tant than ­con­cen­tra­tion per se when one con­sid­ers the potential of ­phy­toex­trac­tion for a given spe­cies. In this study the BF of the CS

acces­sion con­sid­er­ing the avail­able Zn con­tent in soil (shoot Zn con­tent/soil avail­able Zn con­tent) under fer­til­ized and non-fer­til­ ized treat­ment 100 was around 4.7 and 4.1, respec­tively. The avail­ able Zn frac­tion of soils was used since there is a clear ten­dency for the BF to decrease with increas­ing soil total Zn (Zhao et al., 2003) and this mod­i­fied BF has also been used with other Ru­mex spe­cies when soil total metal con­tent is very high (Mo­ren­o-Jimé­nez et al., 2006). Besides BF, the trans­lo­ca­tion fac­tor (TF), or the rela­tion­ship between shoot and root metal con­cen­tra­tion is an impor­tant phy­ toex­trac­tion param­e­ter that can be used to eval­u­ate the capac­ity of each acces­sion to trans­lo­cate met­als from roots to shoot. Under our exper­i­men­tal con­di­tions, TF found on con­tam­i­nated soils were not higher than 1 for the three met­als (Table 3). How­ever, TF for Zn was much higher than those for the other met­als, in accor­dance to the high avail­abil­ity of this metal. The TF of this metal increased with mine soil pro­por­tion in pots for all sur­viv­ing plants. Fer­til­iza­tion did not affect TF. High­est TF val­ues were recorded under treat­ment 66 for the UCS acces­sion (2.5 to 3-fold higher than CS acces­sion). How­ever, final metal con­tent in shoots of both acces­sions did not dif­fer sig­nif­i­cantly. 3.4. Metal phy­toex­trac­tion Due to the high avail­able Zn con­cen­tra­tion in the original mine soil, this was the metal reach­ing high­est accu­mu­la­tion lev­els in shoots of both acces­sions (Table 4) and, in gen­eral, the amount of phy­toex­tract­ed Zn increased as a result of fer­til­iza­tion. The high tol­er­ance of the CS acces­sion of R. ace­to­sa to grow in pots con­tain­ ing real metal con­tam­i­nated mine soil, together with its capa­bil­ity to grow under the envi­ron­men­tal harsh con­di­tions in the aban­ doned mine, sup­ports the suit­abil­ity of this me­tal­lic­o­lous acces­ sion for the reveg­e­ta­tion and phy­to­sta­bi­li­za­tion of met­al­lif­er­ous mine soils. In terms of phy­toex­trac­tion, the CS acces­sion would be very effi­cient when grow­ing in highly pol­luted mine soils, since sur­viv­ ing in the original met­al­lif­er­ous soil con­tain­ing high avail­able Zn lev­els resulted in ele­vated Zn phy­toex­trac­tion rates by this acces­ sion when pots were fer­til­ized: 6.8 mg plant¡1 month¡1. This value is higher than that reported by San­tos et al. (2006), in a sim­i­lar micro­cosm study, with the high bio­mass plant Bra­chi­a­ria de­cum­ bens (0.46 mg Zn plant¡1 month¡1) and also than that obtained by Her­nán­dez-Al­li­ca et al. (2006) with Thl­as­pi cae­rules­cens var. Lanes­ to­sa grow­ing in the very same mine soil (i.e., approx­i­mately, 2.3 mg Zn plant¡1 month¡1). Tak­ing into account the Zn phy­toex­trac­tion rates observed for the CS acces­sion, together with its bio­mass pro­duc­tion when grown in the mine soil under green­house con­di­tions, a potential phy­toex­trac­tion rate of 7.6 kg Zn ha¡1 month¡1 could be expected under field con­di­tions. This value would be much higher than those found for com­mon crop cul­ti­vars such as Zea mays (2.8 kg Zn ha¡1 month¡1) or Nico­ti­ana ta­ba­cum (2.3 kg Zn ha¡1 month¡1) (Weng­er et al., 2002) and also for that reported by Zhu­ang et al. (2007) for other R. ace­to­sa acces­sions (0.531 kg Zn ha¡1 month¡1). In fact, this phy­toex­trac­tion rate would be com­pa­ra­ble to the one obtained by these authors (Zhu­ang et al., 2007) for the high bio­ mass spe­cies Ru­mex cris­pus (8.04 kg ha¡1 month¡1), and sim­i­lar to the ones observed with the hy­per­ac­cu­mu­la­tors Viola bao­shan­en­sis and Sedum al­fre­dii (9.81 kg Zn ha¡1 month¡1). Any­way, future field stud­ies are nec­es­sary to eval­u­ate real phy­toex­trac­tion rates under nat­u­ral con­di­tions. The uti­li­za­tion of the me­tal­lic­o­lous CS acces­sion of R. ace­to­sa here stud­ied seems highly fea­si­ble for the reveg­e­ta­tion and phy­ to­sta­bi­li­za­tion of met­al­lif­er­ous mine soils, not only due to its tol­ er­ance to the pres­ence of met­als, but also to nutri­ent defi­ciency and the other adverse envi­ron­men­tal fac­tors pre­vail­ing in mine dumps (Tord­off et al., 2000). In addi­tion, cul­ti­va­tion, pest con­trol



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263

Table 2 Con­cen­tra­tions of Cd, Pb and Zn (mg metal kg¡1 DW shoot) of UCS and CS acces­sions of R. ace­to­sa after two months of growth in fer­til­ized (+F) and non-fer­til­ized soils con­ tain­ing dif­fer­ent per­cent­ages of the mine tail­ing soil: 0% (treat­ment 0), 33% (treat­ment 33), 66% (treat­ment 66) and 100% (treat­ment 100) Treat­ment

UCS

CS

Cd

Pb

Zn

Cd

Pb

Zn

0 0+F

0.0 ± 0.0 a 0.0 ± 0.0 a

0 ± 0 a 0 ± 0 a

38 ± 0 a 41 ± 7 a

0.0 ± 0.0 a 0.0 ± 0.0 a

0 ± 0 a 0 ± 0 a

48 ± 25 a 41 ± 16 a

33 33+F

0.0 ± 0.0 a 0.1 ± 0.1 a

17 ± 0 cd 5 ± 1 ab

189 ± 14 a* 170 ± 20 a

0.0 ± 0.0 a 0.0 ± 0.0 a

17 ± 1 c 7 ± 1 ab

134 ± 29 a* 157 ± 8 a

66 66+F

1.0 ± 0.6 b* 1.3 ± 0.3 b

7 ± 1 ab* 3 ± 0 ab*

512 ± 211 a 544 ± 67 a

0.2 ± 0.2 a* 0.9 ± 0.1 b

13 ± 1 bc* 6 ± 1 ab*

481 ± 133 a 384 ± 85 a

100 100+F

0.2 ± 0.2 a* 2.1 ± 0.5 c

29 ± 9 d 12 ± 3 bc*

573 ± 125 a* 1794 ± 601 b

1.7 ± 0.5 c* 1.8 ± 0.2 c

29 ± 8 d 28 ± 1 d*

3079 ± 537 b* 3490 ± 190 b

Dif­fer­ent let­ters denote sta­tis­ti­cally sig­nif­i­cant dif­fer­ences at p < 0.05 among treat­ments for each metal and within each acces­sion. Aster­isks stand for sig­nif­i­cant dif­fer­ences at p < 0.05 between acces­sions under the same treat­ment and for the same metal. Table 3 Trans­lo­ca­tion fac­tor (TF) (shoot/root metal con­cen­tra­tion) for Cd, Pb and Zn in UCS and CS acces­sions of R. ace­to­sa after two months of growth in fer­til­ized (+F) and non-fer­ til­ized soils con­tain­ing dif­fer­ent per­cent­ages of the mine tail­ing soil: 0% (treat­ment 0), 33% (treat­ment 33), 66% (treat­ment 66) and 100% (treat­ment 100) Treat­ment

UCS

CS

Cd

Pb

Zn

Cd

Pb

Zn

0 0+F

0.00 ± 0.00 a 0.00 ± 0.00 a

0.00 ± 0.00 a 0.00 ± 0.00

1.1 ± 0.3 a 1.0 ± 0.1 a

0.0 ± 0.0 a 0.0 ± 0.0 a

0.00 ± 0.00 a 0.00 ± 0.00 a

0.8 ± 0.1 cd 0.9 ± 0.2 d

33 33+F

0.00 ± 0.00 a 0.15 ± 0.10 a

0.09 ± 0.03 b 0.02 ± 0.01 a*

0.4 ± 0.2 a 0.3 ± 0.1 a

0.0 ± 0.0 a 0.0 ± 0.0 a

0.08 ± 0.00 d 0.05 ± 0.06 c*

0.22 ± 0.02 a 0.3 ± 0.1 ab

66 66+F

0.12 ± 0.90 0.18 ± 0.10 a

0.02 ± 0.01 a 0.01 ± 0.01 a

0.9 ± 0.8 a 1.0 ± 0.7 a

0.01 ± 0.01 a 0.07 ± 0.02 c

0.02 ± 0.01 b 0.02 ± 0.01 b

0.3 ± 0.1 ab 0.4 ± 0.08 ab

100 100+F

0.01 ± 0.01 a 0.09 ± 0.05 a*

0.01 ± 0.01 a 0.01 ± 0.00 a*

0.1 ± 0.0 a 0.2 ± 0.03 a*

0.04 ± 0.02 b 0.02 ± 0.04 ab*

0.01 ± 0.01 ab 0.08 ± 0.00 ab*

0.64 ± 0.32 bcd 0.47 ± 0.06 abc*

Dif­fer­ent let­ters denote sta­tis­ti­cally sig­nif­i­cant dif­fer­ences at p < 0.05 among treat­ments for each metal and within each acces­sion. Aster­isks stand for sig­nif­i­cant dif­fer­ences at p < 0.05 between acces­sions under the same treat­ment and for the same metal. Table 4 Cd, Pb and Zn phy­toex­trac­tion rate of UCS and CS acces­sions of R. ace­to­sa (lg metal plant¡1 month¡1) grow­ing for two months in fer­til­ized (+F) and non-fer­til­ized soils con­ tain­ing dif­fer­ent per­cent­ages of the mine tail­ing soil: 0% (treat­ment 0), 33% (treat­ment 33), 66% (treat­ment 66) and 100% (treat­ment 100) Treat­ment

UCS

CS

Cd

Pb

Zn

Cd

Pb

Zn

0 0+F

0.0 ± 0.0 a 0.0 ± 0.0 a

0 ± 0 a 0 ± 0 a

319 ± 152 a 360 ± 106 a

0.0 ± 0.0 a 0.0 ± 0.0 a

0 ± 0 a 0 ± 0 a

340 ± 145 ab 344 ± 156 a

33 33+F

0.0 ± 0.0 a 1.3 ± 1.2 a

152 ± 2 f* 51 ± 9 e

1666 ± 128 b* 1753 ± 270 b

0.0 ± 0.0 a 0.0 ± 0.0 a

91 ± 5 e* 54 ± 12 d

714 ± 180 bc* 1266 ± 382 cd

66 66+F

5.5 ± 3.3 b 9.0 ± 2.9 c

40 ± 2 d 23 ± 5 c

2756 ± 999 c 3744 ± 706 d*

0.8 ± 1.3 ab 3.7 ± 0.5 c

46 ± 13 b 26 ± 3 b

1788 ± 880 d 1642 ± 382 d*

100 100+F

0.2 ± 0.1 a* 1.8 ± 0.8 a

15 ± 9 bc 11 ± 5 ab*

439 ± 154 a* 1500 ± 566 b*

1.8 ± 0.7 b* 3.5 ± 0.5 c

32 ± 15 bc 54 ± 5 d*

3174 ± 514 e* 6760 ± 297 f*

Dif­fer­ent let­ters denote sta­tis­ti­cally sig­nif­i­cant dif­fer­ences at p < 0.05 among treat­ments for each metal and within each acces­sion. Aster­isks stand for sig­nif­i­cant dif­fer­ences at p < 0.05 between acces­sions under the same treat­ment and for the same metal.

and har­vest­ing prac­tices for this plant spe­cies are well-known and, although it can be eaten by a few Lep­i­dop­tera (Karl and Fischer, 2008), its con­sump­tion by her­bi­vores is lim­ited (Schei­del and ­Brue­lhe­ide, 1999), which can be due to its high oxa­late con­tent ­(Noo­nan and Savage, 1999). Most inter­est­ingly, high avail­able Zn lev­els in the mine soil turn out in ele­vated Zn shoot lev­els in this tol­er­ant acces­sion, a remark­able phe­nom­e­non that can be exploited for phy­toex­trac­tion pur­poses. 4. Con­clu­sions Ru­mex ace­to­sa has a great potential for Zn phy­toex­trac­tion and reveg­e­ta­tion of metal con­tam­i­nated soils. For highly con­tam­i­nated soils, such as those char­ac­ter­is­tics of mine dumps, the R. ace­to­sa

acces­sion from the con­tam­i­nated met­al­lif­er­ous site (CS acces­sion) is the right choice, since the UCS acces­sion from the agri­cul­tural non-met­al­lif­er­ous site did not sur­vive under the harsh con­di­tions of the Lanes­to­sa mine soil. This CS acces­sion can be use­ful for the rec­la­ma­tion of Pb/Zn mine areas since it is adapted not only to high lev­els of bio­avail­able met­als in soil but also to nutri­ent defi­ ciency and to the other adverse envi­ron­men­tal fac­tors pre­vail­ing in mine areas. Phy­toex­trac­tion of met­als (Zn, Pb and Cd) in the CS acces­sion can be highly stim­u­lated via fer­til­iza­tion of the heav­ily con­tam­i­nated soil, reach­ing sim­i­lar Zn phy­toex­trac­tion rates than those of hyper­ac­cu­mu­la­tor plants. Efforts must be directed at cat­ a­logu­ing and pre­serv­ing mine adapted plant bio­di­ver­sity because of its potential use for phy­to­reme­di­a­tion and reveg­e­ta­tion of metal enriched envi­ron­ments. Indeed, it is of cru­cial ­impor­tance

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