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Naturally-assisted metal phytoextraction by Brassica carinata: Role ofroot exudates
Environmental Pollution 157 (2009) 2697–2703
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Naturally-assisted metal phytoextraction by Brassica carinata: Role of root exudates Mike F. Quartacci a, *, Barbara Irtelli a, Cristina Gonnelli b, Roberto Gabbrielli b, Flavia Navari-Izzo a a b
` di Pisa, Via del Borghetto 80, 56124 Pisa, Italy Dipartimento di Chimica e Biotecnologie Agrarie, Universita ` di Firenze, Via Micheli 1, 50121 Firenze, Italy Dipartimento di Biologia Vegetale, Sezione di Ecologia e Fisiologia Vegetale, Universita
Phytoextraction of metals is enhanced in Brassica carinata grown in succession to metallicolous populations of spontaneous species.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2008 Received in revised form 28 April 2009 Accepted 29 April 2009
Due to relatively high chelant dosages and potential environmental risks it is necessary to explore different approaches in the remediation of metal-contaminated soils. The present study focussed on the removal of metals (As, Cd, Cu, Pb and Zn) from a multiple metal-contaminated soil by growing Brassica carinata plants in succession to spontaneous metallicolous populations of Pinus pinaster, Plantago lanceolata and Silene paradoxa. The results showed that the growth of the metallicolous populations increased the extractable metal levels in the soil, which resulted in a higher accumulation of metals in the above-ground parts of B. carinata. Root exudates of the three metallicolous species were analysed to elucidate their possible role in the enhanced metal availability. The presence of metals stimulated the exudation of organic and phenolic acids as well as flavonoids. It was suggested that root exudates played an important role in solubilising metals in soil and in favouring their uptake by roots. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Brassica carinata Metals Phytoextraction Plant succession Root exudates
1. Introduction The availability of metals for plant uptake is greatly restricted by their adsorption to solid soil fractions. Chelant-assisted phytoextraction has been used to improve the effectiveness of conventional phytoextraction of metal polluted soils by dissolving target metals from soil and making them more available for plant uptake and translocation to harvestable above-ground parts of high biomass crops (Meers et al., 2008). Recently, aminopolycarboxylic acids such as nitrilotriacetic acid (NTA) and (S,S)-ethylenediaminedisuccinic acid (EDDS) have been proposed as an alternative to EDTA and other persistent synthetic chelants (Evangelou et al., 2007; Luo et al., 2008a; Meers et al., 2008; Quartacci et al., 2006, 2007; Tandy et al., 2006). Even though the application of such biodegradable chelants could minimize the risks of potential off-site migration of metals – either in surface runoff or by leaching into groundwater (Nowack et al., 2006) – the accumulation of metals such as Cd, Cu and Zn increased only by a factor of 2–3, although their solubility in soil increased by a much higher factor (Grcˇman et al., 2003; Kayser et al., 2000; Kos and Lesˇtan, 2004; Kulli et al., 1999; Quartacci et al., 2007). Therefore, efforts have been focussed on screening plant species that are more
* Corresponding author. Tel.: þ39 50 2216633; fax: þ39 50 2216630. E-mail address: [email protected] (M.F. Quartacci). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.04.035
sensitive to chelant treatments and on developing new phytotechnologies that will help reduce and/or eliminate the amount of chelant applied to the field decreasing the environmental risk of mobilised metals (Luo et al., 2008a,b). Root exudates may influence the availability of nutrients – enhancing or reducing it – by a direct impact on the uptake of metals by acidification, chelation, precipitation and redox reactions, as well as by an indirect impact through their effects on microbial activity, the physical and chemical properties of the rhizosphere and root growth patterns (Tao et al., 2004; Uren and Reisenauer, 1988). Molecules naturally exuded by plants such as organic acids have been used in chemically-assisted phytoextraction programmes to enhance soil metal availability and root uptake by high biomass accumulating crop species (Evangelou et al., 2007). On the other hand, the exudation of organic molecules from the roots is also considered one of the most important strategies by which plants can exclude metals such as Al, Cd and Pb chelating them in the rhizosphere or in the apoplastic space and preventing their entry into the symplast (Hill et al., 2002; Watanabe and Osaki, 2002; Yang et al., 2000; Zheng et al., 1998). In this way, some metaltolerant species can restrict uptake and translocation of metals maintaining low shoot level over a wide range of external concentrations (Baker, 1981). It is still a matter of debate whether root exudates by different species vary in terms of quality and exudation rate. If so, such variations may significantly contribute to explain the different metal accumulation/tolerance capacity shown
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by plant species. Thus, the same chemical might play a role in limiting the uptake of metals in metal-tolerant populations or in enhancing metal mobility in chelant-assisted phytoextraction programmes carried out using accumulating crops. The fact that compounds commonly exuded from roots are useful in metal complexation have suggested the idea to grow a metal accumulating crop in succession to metallicolous populations in order to verify whether its phytoextraction efficiency may be improved. In this study three species from metalliferous soils were used as potential natural chelant producers for naturallyassisted phytoextraction of metals from a multiple contaminated soil by Brassica carinata, a species that has been seen to accumulate significant amounts of more metals at the same time (Quartacci et al., 2007). The results in terms of above-ground metal accumulation, soil metal dissolution as well as nature and amount of root exudates are discussed. 2. Materials and methods 2.1. Chemicals All chemicals used were analytical grade reagents and were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). 2.2. Soil characteristics and plant growth The metal-contaminated area is represented by a site used as a dump for pyrite ashes derived from the ore roasting processes for sulphur extraction. At present, the site is included within the perimeter of the polluted area named ‘Grado and Marano lagoon and neighbouring water-courses’ identified as a site of national interest by the Italian Cabinet Decree 468/2001 ‘National Programme for Environmental Restoration of Polluted Sites’. Preliminary tests showed that B. carinata performed poorly when sown in pots containing the contaminated soil. A milder substrate was then prepared by mixing the contaminated soil with peat in a 2:1 ratio by volume. After mixing, the soil mixture was allowed to equilibrate for a period of four weeks, undergoing three cycles of saturation with water and air-drying before being remixed and finally planted. Mixed soil characterisation was carried out according to Italian Standard Methods (Mi.P.A.F., 2000). Standards (National Institute of Standards and Technology) and reagent blanks (aqua regia) were run with all samples to ensure accuracy and precision in the analyses. The standard reference material used was SRM 2586. As for arsenic, preliminary tests showed that sample digestion by HNO3 resulted in a better recovery of the metal in comparison with aqua regia and HNO3/H2SO4/HClO4 (10:1:4, by vol.). The average recovery of As was 93% and 89% for SRM and soil samples, respectively. Physical and chemical characteristics of the mixed soil are reported in Table 1. The mixed soil was contaminated by As (total amount of 639 mg kg1 soil), Cd (61 mg kg1 soil), Cu (1846 mg kg1 soil), Pb (246 mg kg1 soil) and Zn (1143 mg kg1 soil). The polluted mixed soil was then used to fill 9 9 12 cm diameter plastic pots (700 g dry weight soil per pot) and moistened with water to approximately 80% field capacity. Basal fertilisers (150, 75 and 95 mg kg1 N, P and K, supplied as NH4NO3 and KH2PO4) were also applied. Pots were divided into five groups. One set of pots Table 1 Physical and chemical characteristics of the contaminated soil. Parameter Sand (%) Silt (%) Clay (%) pH (H2O) Organic matter (%) CEC (cmol(þ) kg1) Total S (%) Total N (g kg1) Exchangeable P (mg kg1) Exchangeable K (mg kg1) Available metals As (mg kg1) Cd (mg kg1) Cu (mg kg1) Pb (mg kg1) Zn (mg kg1)
79 14 7 7.3 6.8 4.4 0.01 0.7 11.7 170.8 0.8 0.6 29.4 2.7 3.4
Results are the means of five replicates. The standard deviation (SD) was always less than 10%.
was planted with seeds of B. carinata cv. 079444 (10 seeds per pot), whereas other three groups were planted with seeds (10 seeds per pot) of Pinus pinaster, Plantago lanceolata and Silene paradoxa collected at the polymetallic sulphide deposit of Fenice Capanne (Mascaro et al., 2001). The last set of pots was not sowed with any species. Metallicolous species were also grown separately in a non-contaminated control soil (commercial mould) with the same experimental procedure above reported. All pots were placed outdoor on the soil surface. The climatic conditions (Pisa, year 2007) were as follow: temperature 11–22 C, humidity 59–84%, average daily eliophany 8 h (May), and temperature 14–26 C, humidity 62–86%, average daily eliophany 9 h (June). Following seedling emergence, pots planted with B. carinata were thinned to two plants per pot. When soil moisture content decreased to 75% field capacity, pots were watered with 150 ml distilled water. Due to the fact that metallicolous species grow at a much lower rate compared to agronomic crops, after five and eight weeks of growth (for B. carinata and metallicolous species, respectively) all plants were harvested by cutting stems 1 cm above the soil surface and the soils were analysed for DTPA-extractable metals and for pH (Mi.P.A.F., 2000). For pH determination soil samples were collected at different depths (4 and 8 cm) and at about 2 cm far from roots. Thereafter, the same pots used for B. carinata growth were planted with seeds of the three metallicolous species (each species separately), whereas those in which the spontaneous plants were cultivated were sown with B. carinata seeds at the same density previously reported. In addition, one set of B. carinata seeds was planted in pots containing the contaminated soil in which no other species was previously grown (control). Pots were placed outdoors on the soil surface, and plants were watered, fertilised and harvested as reported. The climatic conditions (Pisa, year 2007) were as follow: temperature 17–29 C, humidity 45–75%, average daily eliophany 10 h (July), and temperature 17–29 C, humidity 46–84%, average daily eliophany 9 h (August). The above-ground parts of all four species collected were washed carefully with distilled water to remove any soil splash and oven-dried at 110 C for 24 h. Dried material was then digested with concentrated HNO3 in a capped Teflon pressure digestion vessel and analysed for As, Cd, Cu, Pb and Zn using a Perkin–Elmer Optimal DV 2100 ICP OES. Standards (National Institute of Standards and Technology) and reagent blanks (HNO3 65%) were run with all samples to ensure accuracy and precision in the analyses. The standard reference material used was SRM 1570a. 2.3. Determination of extractable metals in the soil Soils were analysed for DTPA (diethylenetriaminepentaacetic acid)-extractable metals by adding 5 mM DTPA, 10 mM CaCl2 and 0.1 M triethanolamine (pH 7.3) to give a 1:2 (w/v) soil:solution ratio (Mi.P.A.F., 2000). After shaking for 120 min, tubes were centrifuged at 17 400 g for 10 min and the supernatants collected after filtering through a Whatman No. 41 filter paper (pore size 20–25 mm). Analysis of the filtrate for As, Cd, Cu, Pb and Zn was performed as reported. 2.4. Determination of flavonoids, organic and phenolic acids in root exudates Seeds of P. pinaster, P. lanceolata and S. paradoxa were sterilized with 70% ethanol for 20 s and with 2.5% NaClO for 20 min and washed three times with deionised water. Seeds were then germinated for 3 days in the dark on floating trays containing 400 ml of continuously aerated nutrient solution composed of 0.25 mM NH4H2PO4, 1 mM Ca(NO3)2, 0.5 mM MgSO4, 1.5 mM KNO3, 11.5 mM H3BO3, 5 mM ferric tartrate [Fe2(C4H4O6)3], 3.5 mM MnCl2, 0.03 mM MoO3, 0.3 mM ZnSO4 and 0.12 mM CuSO4 (pH 5.5). Plants were grown at 16-h photoperiod, 400 mmol m2 s1 photon flux density, 23 1 C temperature and 70–75% relative humidity. After 15 days, plants were removed from the floating trays and transferred to vials, one plant per vial. Vials contained 20 ml of nutrient solution added with Cd(NO3)2, Pb(NO3)2, Zn(NO3)2, Cu(NO3)2 and Na2HAsO4 in order to reach the concentrations of the available metals in the Brassica-planted soil (see values in Fig. 1) calculated taking into consideration plant available water content (PAWC). One set of plants, kept as control, was transferred to vials containing 20 ml of nutrient solution. After 24 h seedlings were removed from vials, and roots were collected and weighted for dry weight determination. The control and metal-added solutions containing dissolved root exudates were lyophilised and the dry residues stored at 80 C till analysed. Immediately before analysis, the dry lyophilised residue was dissolved in deionised water and root exudates were analysed for flavonoids, organic and phenolic acids with a Waters HPLC system consisting of two 515 pumps and a 2487 programmable UV detector. Organic acids were separated on a Waters Nova-Pack C18 reverse-phase column (150 3.9 mm, 4 mm) and detected at 210 nm wavelength. A 10 mM phosphate buffer (pH 2.3) at a flow rate of 0.4 ml min1 was used as mobile phase. Phenolic acids and flavonoids were separated on a Waters Simmetry C18 reversephase column (150 3.9 mm, 5 mm). Phenolic acids were detected at 254 nm wavelength. Solvent A was 2% acetic acid and 30% acetonitrile, whereas 2% acetic acid was used as solvent B. A linear gradient from 10 to 95% solvent B for 55 min was used at a flow rate of 1 ml min1. Non-conjugated flavonoids were detected at 365 nm (quercetin and kaempferol) and 290 nm (naringenin). The mobile phase was formed by 55% of methanol and 45% of 0.05% trifluoroacetic acid eluted at a flow rate of 1 ml min1. Identification of flavonoids, organic and phenolic acids was obtained by comparison of their retention times with those of authentic standards (Sigma
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Fig. 1. DTPA-extractable metals in the multiple metal-contaminated soil after Brassica carinata, Pinus pinaster, Plantago lanceolata and Silene paradoxa growth. Control represents the multiple metal-contaminated soil in which no species was grown. Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05). Chemical). Quantification of individual compounds was obtained by comparing calibration curves. Chromatogram analyses were performed by Millennium 32 software (Waters). Total concentrations were calculated as sum of individual amounts. 2.5. Statistical analysis All statistical analysis was carried out using Costat 6.4 (CoHort software). The error bars shown in the figures represent the standard deviation of the mean of three independent experiments (n ¼ 3) each analysed twice. The effects of experimental factors were evaluated by one-way ANOVA and comparisons between means were carried out using the Tukey’s HSD multiple range test at the significance level of P 0.05.
Fig. 2. Metal concentrations (a) and uptakes (b) in the above-ground parts of B. carinata grown in the multiple metal-contaminated soil in succession to P. pinaster, P. lanceolata or S. paradoxa. Control represents the multiple metal-contaminated soil in which no species was grown. Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05).
3. Results 3.1. Soil extractable metals following plant growth The cultivation of the three metallicolous species had a positive effect on metal dissolution in the contaminated soil (Fig. 1). In comparison with the control (contaminated soil in which no species was grown), the growth of the three metallicolous species caused an average enhancement of extractable metals by about 3fold, with the exception of Cd that showed on average an 11-fold increase mostly due to P. pinaster contribution. In particular, S. paradoxa was the most effective species in enhancing Cu and Zn extractable amounts in soil, whereas available Cd and Pb were increased more by the presence of P. pinaster. No differences were observed for extractable As amounts. P. lanceolata growth resulted in a lower extractable metal level in the contaminated soil compared to the other two species. Compared to the control, the presence of B. carinata in the metal-contaminated soil had no effect on soil available metal concentrations. The cultivation of the metallicolous plants caused a significant decrease in soil pH from 7.3 0.3 (control) to 5.4 0.2 for P. pinaster and S. paradoxa, and to 5.6 0.5 for P. lanceolata, whereas B. carinata growth did not change soil pH. 3.2. Metal uptake by B. carinata In comparison with the control (contaminated soil in which no species was previously grown), B. carinata cultivated in the soil previously planted with the three metallicolous species showed a higher metal concentration in the shoots (Fig. 2a). Cd, Cu and Pb accumulated in the above-ground parts of B. carinata especially following P. pinaster growth (4.4-, 3.0- and 2.5-fold increase,
respectively), whereas the highest Zn concentration in Brassica shoots (1.6-fold increase compared to control plants) was observed following S. paradoxa growth. No significant differences between control and planted soils were detected for As. Compared to control plants (0.16 g dry weight plant1), the dry matter of the aboveground parts of B. carinata grown in succession to the three spontaneous species suffered a slight not significant reduction, (0.14 g dry weight plant1) following Plantago and Silene growth and a significant 25% decrease (0.12 g dry weight plant1) following Pinus growth. The amount of metals expressed on a plant basis followed almost the same behaviour observed for their concentrations (Fig. 2b). Cd, Cu and Pb showed the highest content in Brassica above-ground parts following Pinus growth (9.6, 20.3 and 8.0 mg plant1, respectively), whereas Zn accumulated mostly following Silene cultivation (15.6 mg plant1). 3.3. Metal uptake by metallicolous species As regards metal concentrations in the above-ground part of metallicolous species, in general the growth of P. pinaster and S. paradoxa in the multiple metal-contaminated soil (MCS) resulted in slight differences compared to a non-contaminated control soil (Fig. 3a and c) composed of commercial mould. On the contrary, P. lanceolata showed a remarkable increase in Pb, Zn and, in particular, a 47-fold increase in Cu (Fig. 3b). Arsenic concentrations did not change following treatments. Metallicolous species grown in the metal-contaminated soil in succession to B. carinata (MCS þ Bc) showed a reduction of shoot metal concentrations in comparison with plants not preceded by B. carinata growth (MCS), and in most cases values returned to the control ones or were even lower (Fig. 3).
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Fig. 4. Dry weights of the above-ground parts of P. pinaster, P. lanceolata and S. paradoxa grown in non-contaminated control soil, in the multiple metal-contaminated soil (MCS) and in the multiple metal-contaminated soil in succession to B. carinata (MCS þ Bc). Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05).
Fig. 3. Metal concentrations in the above-ground parts of P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) grown in non-contaminated control soil, in the multiple metalcontaminated soil (MCS) and in the multiple metal-contaminated soil in succession to B. carinata (MCS þ Bc). Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05).
Compared to the non-contaminated control soil (Fig. 4), the metal-contaminated soil (MCS) caused a remarkable reduction of dry biomass in the above-ground part of the metallicolous species (60, 68 and 49% decrease for Pinus, Plantago and Silene, respectively). The previous growth of B. carinata in the multiple metal-contaminated soil (MCS þ Bc) had beneficial effects on biomass production limiting shoot dry mass loss in the metallicolous plants (Fig. 4). 3.4. Root exudates The main components of root exudates were organic acids followed by lower amounts of phenolic acids and flavonoids (Table 2). The highest amount of organic acids was observed in the presence of metals (about 28-fold increase on average compared to the control), whereas the concentrations of phenolic acids and flavonoids were enhanced by a factor of 1.5 and 3, respectively. It should be pointed out that, differently from the other two species, in S. paradoxa total phenolic acids were halved after metal treatment.
Six different organic acids were detected, being their typology and amount strictly species-dependent (Fig. 5). Oxalic and malic acids were detected in all the three metallicolous species, while tartaric and malonic were present in P. pinaster, citric and fumaric in P. lanceolata, and malonic and fumaric acids in S. paradoxa. Oxalic and malic were the main representative organic acids in the exudates of both control and treated roots. The kind of organic acid present in root exudates did not change following metal incubation, although the rate of increase was dependent on the species investigated. Under metal excess the highest increase in P. pinaster exudates was shown for oxalic acid (173-fold) followed by malic acid (31-fold) (Fig. 5a). Similar remarkable increases were detected in response to metal treatment also in S. paradoxa, whose oxalic and malic acid concentrations increased by about 143- and 78-fold, respectively (Fig. 5c). P. lanceolata was the species which showed the lowest rates of increase, fumaric and oxalic acids increasing by a factor of 10 (Fig. 5b). As for organic acids, the amount and type of phenolic acids detected in root exudates were also strictly dependent on the species analysed (Fig. 6). In control conditions the main phenolic acids exuded by P. pinaster were p-OH-benzoic (37% of total concentration), syringic (33%) and protocatechuic (30%). Ferulic (31% of total concentration), syringic (29%), p-cumaric (11%) and caffeic (11%) acids were the main constituents of phenolic exudates of P. lanceolata. The phenolic acids present in the exudates of S. paradoxa grown in control solution were mainly protocatechuic (36% of total concentration), syringic (31%), p-OH-benzoic (23%) and vanillic (10%). Differently from organic acids, the treatment of the metallicolous species with metals caused both an increase and a decrease in the amount of exuded phenolic compounds (Fig. 6) depending on the species and the individual compound investigated. The most remarkable enhancements concerned chlorogenic acid (70- and 13-fold increase compared to control in P. pinaster and S. paradoxa, respectively) and vanillic acid (8-fold increase in P. lanceolata), whereas syringic acid was the phenolic compound that suffered from the strongest reductions in all species (82, 42 and 98% decrease in comparison with non-treated plants for Pinus, Plantago and Silene, respectively). Kaempferol, naringenin and quercetin were the free flavonoids found in the exudates of control roots (Fig. 7). Naringenin was the flavonoid which production was the most stimulated by the metal treatment in all species. In comparison with control plants naringenin increased by about 20-, 7- and 3-fold in Pinus, Plantago and Silene exudates, respectively. Following metal treatment
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Table 2 Organic acid (mg g1 root dry weight), phenolic acid and flavonoid (mg g1 root dry weight) total contents in root exudates of plants incubated for 24 h in nutrient solution (control) and in nutrient solution added with metals. Organic acids
Pinus pinaster Plantago lanceolata Silene paradoxa
Phenolic acids
Flavonoids
Control
Treated
Control
Treated
Control
Treated
8.1 0.5 11.1 0.5 36.2 1.9
440.2 20.1* 99.1 3.9* 713.1 21.7*
14.0 0.9 23.6 1.2 79.3 2.8
22.8 0.8* 32.2 1.8* 39.9 1.9*
0.4 0.1 1.9 0.2 3.1 0.2
0.9 0.1* 9.6 0.3* 6.6 0.4*
Values are shown as means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05).
kaempferol and quercetin showed a lowest rate of increase or, as in the case of P. pinaster, remained almost unchanged. 4. Discussion Although EDDS is one of the most biodegradable among metal chelants, Meers et al. (2005) suggested that under less optimal conditions ligand persistence or ligand effect (i.e. the concentration of metal(s) mobilised into the soil solution following chelant
Fig. 5. Organic acids in root exudates of 15-days-old P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) incubated for 24 h in nutrient solutions added with As, Cd, Cu, Pb and Zn. Control represents nutrient solutions without metals. Values are means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05).
addition) can increase dramatically. The evidence that metaltolerant species exude organic compounds in order to complex and mobilize metals suggests the possibility to use those plants for producing chelants for naturally-assisted phytoextraction programmes. In general, if roots exudates remain stable in soils for sufficient time, they may improve the capacity of crops to uptake
Fig. 6. Phenolic acids in root exudates of 15-days-old P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) incubated for 24 h in nutrient solutions added with As, Cd, Cu, Pb and Zn. Control represents nutrient solutions without metals. Values are means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05). Ben, p-hydroxybenzoic acid; Caf, caffeic acid; Chl, chlorogenic acid; Cum, p-cumaric acid; Fer, ferulic acid; Gal, gallic acid; Pro, protocatechuic acid; Syr, syringic acid; Van, vanillic acid.
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Fig. 7. Flavonoids in root exudates of 15-days-old P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) incubated for 24 h in nutrient solutions added with As, Cd, Cu, Pb and Zn. Control represents nutrient solutions without metals. Values are means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05).
metals. To date, this is the first report investigating the use of plant succession to improve metal phytoextraction from multiple contaminated soils. In agreement with the increase in available metal amounts in soil (Fig. 1), the growth of the three metallicolous species enhanced B. carinata capacity to accumulate and translocate Cd, Cu, Pb and Zn (Fig. 2). This result may be explained both by the acidification of the multiple contaminated soil and the formation of metal complexes that enhanced metal bioavailability for root uptake by B. carinata. In comparison with NTA- and EDDS-amended plants (Quartacci et al., 2007), plant succession determined on average a higher accumulation of Cd (3-fold) and similar amounts of As, Cu and Zn, whereas only Pb suffered a 1.8-fold reduction. The significant decrease of B. carinata above-ground biomass when grown in succession to P. pinaster was a consequence of the high concentrations of metals in the contaminated soil solution (Fig. 1) and in the above-ground tissues (Fig. 2a). The improved phytoextraction efficiency of
B. carinata determined by plant succession was relatively low (about 3- or 2-fold on average when expressed on a dry matter or plant basis, respectively) in comparison with the experiment in which no plants were grown before B. carinata (Fig. 2). Nonetheless, it indicates a potential approach to the remediation of metalcontaminated soils in the absence of exogenous compounds added to the soil and potential related risks for the environment. In spite of the limited metal accumulation due at least in part to a low dry biomass production (maximum of 50.4 mg metals plant1 following Pinus growth), B. carinata demonstrated the ability to survive and tolerate the toxic effects of more metals together, and in the presence of multiple contamination a compromise that makes possible the simultaneous uptake of more metals may not be discarded. Metallicolous species, and especially P. lanceolata, accumulated considerable amounts of metals after the period of growth in the multiple contaminated soil (Fig. 3), even though their use in phytoextraction programmes cannot be considered realistic due to their very low biomass and growth rate. The amounts of DTPA-extractable metals detected immediately after the harvest of the three metallicolous species showed that the growth of plants had a positive effect on metal mobilisation (Fig. 1) with values approaching or exceeding, as in the cases of Cd and Cu, those obtained after EDDS application (Quartacci et al., 2007). Similarly to the EDDS-treated soil (Quartacci et al., 2007), Cu was the metal extracted in the highest amount by Brassica, probably due to relatively high stability constants of its chelates with the various exudates (Martell and Smith, 1989). In comparison with nonplanted contaminated soil, B. carinata growth did not enhance metal availability for plant uptake (Fig. 1) indicating that this crop did not play a role in acidifying the rhizosphere and/or in dissolving metals, probably due to a qualitatively not adequate and/or quantitatively insufficient production of exudates. Mobilisation of metals in the rhizosphere has been related to its acidification and to complexation with organic acids present in root exudates. Organic acids typically flow across the lipid bilayer at a slow rate in response to the electrochemical gradient. However, efflux may be greatly increased in stress conditions such as metal ion toxicity due to the expression (encoding) of anion channels embedded in the plasma membrane or to their up-regulation (Rengel, 2002). The activation of Hþ-ATPases located at the plasma membrane level and the consequent release of organic exudates from roots could explain the decrease in soil pH observed following the growth of the metallicolous species. However, the fact that the decrease in the pH was similar for the three species whereas the capacity of mobilising metals was different among them (Fig. 1) suggests that the decrease of pH accounted only in part for the interspecific differences in metal mobilisation. The exudation of specific organic ligands from roots or the exudation rate could account for the rest of the difference as well as enhanced microbial activity linked to a higher availability of carbon sources deriving from root exudates (Tao et al., 2004). Indeed, it has been shown that the acidification of the soil solution was not the only reason for the increase in metal availability (Wenzel et al., 2003). Several organic acids, mainly oxalic and malic acids, have been found to be exuded in increased amounts by roots exposed to heavy metals (Mariano and Keltjens, 2003; Nguyen et al., 2003; Rengel, 2002). Also in the present study oxalic and malic acids were found as the most exuded organic acids in response to multiple metal stress (Fig. 5). In addition, other low molecular mass weight organic acids were detected, but these compounds did not show a constant presence being species-specific, and their concentrations, although in many cases significantly different between control and treatment, were much lower than those detected for oxalic and malic acids. Although citric acid is generally considered to play an important role in metal tolerance (Rengel, 2002), in this study it
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was released only by P. lanceolata and in a very small amount (Fig. 5). Metals also stimulated the excretion of fumaric and tartaric acids from roots of P. pinaster and P. lanceolata, respectively, suggesting that their release might take part to the tolerance strategies of the metallicolous populations of these two species. Only limited information exists about availability and uptake of metal–carboxylate complexes by roots. Hydroponic studies revealed that plant uptake is correlated mainly with the activity of free metal ions (Cd, Cu, and Mn) in solution which implies their dissociation from the complex likely mediated by rhizosphere acidification and reduction of metals (Neumann and Ro¨mheld, 2001). Although organic acids are the main components of Pinus, Plantago and Silene exudates, other organic compounds were exuded. Several phenolic acids and three flavonoids were detected both under control and metal excess conditions, although their release was one order of magnitude lower than organic acids (Table 2). While phenolic compounds showed a variable pattern in terms of composition and amount (Fig. 6), the three flavonoids were found to be exuded by all the metallicolous species in increased amounts following exposure to metals (Fig. 7). The low concentrations of phenolic acids and flavonoids, also reported for Norway spruce by Heim et al. (2001), do not suggest a relevant role in forming metal complexes in the contaminated soil where metallicolous species were grown, as only a minor part of the metals can be bound by these compounds, although both classes of compounds can form stable bonds with metals (Martell and Smith, 1989). Therefore, the exudation of phenolics – and flavonoids in particular – in plant adaptation to altered environmental conditions might be better explained if discussed in relation to their possible function as specific chemoattractant signal for soil microbes to the rhizosphere and/or growth promoters of rhizosphere bacteria as source of carbon substrate (Dakora and Phillips, 2002). In the case of our multiple contaminated soil a role of exudates as chelators of poorly soluble mineral nutrients cannot be excluded. It was observed that phenolics make Fe and P available by forming relatively stable chelates with Fe in insoluble Fe-phosphates thereby increasing the solubility of both Fe and P for plant uptake (Dakora and Phillips, 2002). 5. Conclusions The results of the present study show that growing B. carinata in succession to spontaneous species from metalliferous soils led to the accumulation of higher amounts of metals in the above-ground parts in comparison with the same plants grown in soil not previously planted. The fact that accumulation of metals is similar to that shown by B. carinata following amendments with biodegradable chelants such as NTA and EDDS indicates a possible approach to naturally enhanced phytoremediation of multiple metal-contaminated soils. Plant succession could improve phytoextraction efficiency of metals from soil without the application of any potentially dangerous chemical and with the advantage that chelants in this way are naturally produced by plants themselves. Further studies aimed to investigate the metal binding properties of each compounds released by roots and the possible involvement of other exudates are necessary in order to assess their role in the metal accumulating capacity of B. carinata. Acknowledgements Seeds of Brassica carinata cv. 079444 were kindly provided by Prof. M. Mazzoncini (Dipartimento di Agronomia e Gestione dell’Agrosistema, University of Pisa). This study was funded by the MIUR (Cofinanziamento 2005) and the University of Pisa (Fondi di Ateneo 2006).
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Water, Air, Soil Pollut.. doi:10.1007/s11270-008-9678-z. Mariano, E.D., Keltjens, W.G., 2003. Evaluating the role of root citrate exudation as a mechanism of aluminium resistance in maize genotypes. Plant Soil 256, 469–479. Martell, A.E., Smith, R.M., 1989. Critical Stability Constants, vol. 6. Plenum Press, New York. second supplement. Mascaro, I., Benvenuti, B., Corsini, F., Costagliola, P., Lattanzi, P., Parrini, P., Tanelli, G., 2001. Mine wastes at the polymetallic deposit of Fenice Capanne (southern Tuscany, Italy). Mineralogy, geochemistry, and environmental impact. Environ. Geol. 41, 417–429. Meers, E., Ruttens, A., Hopgood, M., Lesage, E., Tack, F.M.G., 2005. Potential of Brassica rapa, Cannabis sativa, Helianthus annuus and Zea mays for phytoextraction of heavy metals from calcareous dredged sediment derived soils. Chemosphere 61, 561–572. Meers, E., Tack, F.M.G., Verloo, M.G., 2008. 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Phytoextraction of metals from a multiple contaminated soil by Indian mustard. Chemosphere 63, 918–925. Quartacci, M.F., Irtelli, B., Baker, A.J.M., Navari-Izzo, F., 2007. The use of NTA and EDDS for enhanced phytoextraction of metals from a multiply contaminated soil by Brassica carinata. Chemosphere 68, 1920–1928. Rengel, Z., 2002. Genetic control of root exudation. Plant Soil 245, 59–70. Tandy, S., Schulin, R., Nowack, B., 2006. Uptake of metals during chelant-assisted phytoextraction with EDDS related to the solubilized metal concentration. Environ. Sci. Technol. 40, 2753–2758. Tao, S., Liu, W.X., Chen, Y.J., Xu, F.L., Dawson, R.W., Li, B.G., Cao, J., Wang, X.J., Hu, J.Y., Fang, J.Y., 2004. Evaluation of factors influencing root-induced changes of copper fractionation in rhizosphere of a calcareous soil. Environ. Pollut. 129, 5–12. Uren, N.C., Reisenauer, H.M., 1988. The role of root exudates in nutrient acquisition. In: Tinker, P.B., La¨uchli, A. 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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Naturally-assisted metal phytoextraction by Brassica carinata: Role of root exudates Mike F. Quartacci a, *, Barbara Irtelli a, Cristina Gonnelli b, Roberto Gabbrielli b, Flavia Navari-Izzo a a b
` di Pisa, Via del Borghetto 80, 56124 Pisa, Italy Dipartimento di Chimica e Biotecnologie Agrarie, Universita ` di Firenze, Via Micheli 1, 50121 Firenze, Italy Dipartimento di Biologia Vegetale, Sezione di Ecologia e Fisiologia Vegetale, Universita
Phytoextraction of metals is enhanced in Brassica carinata grown in succession to metallicolous populations of spontaneous species.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2008 Received in revised form 28 April 2009 Accepted 29 April 2009
Due to relatively high chelant dosages and potential environmental risks it is necessary to explore different approaches in the remediation of metal-contaminated soils. The present study focussed on the removal of metals (As, Cd, Cu, Pb and Zn) from a multiple metal-contaminated soil by growing Brassica carinata plants in succession to spontaneous metallicolous populations of Pinus pinaster, Plantago lanceolata and Silene paradoxa. The results showed that the growth of the metallicolous populations increased the extractable metal levels in the soil, which resulted in a higher accumulation of metals in the above-ground parts of B. carinata. Root exudates of the three metallicolous species were analysed to elucidate their possible role in the enhanced metal availability. The presence of metals stimulated the exudation of organic and phenolic acids as well as flavonoids. It was suggested that root exudates played an important role in solubilising metals in soil and in favouring their uptake by roots. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Brassica carinata Metals Phytoextraction Plant succession Root exudates
1. Introduction The availability of metals for plant uptake is greatly restricted by their adsorption to solid soil fractions. Chelant-assisted phytoextraction has been used to improve the effectiveness of conventional phytoextraction of metal polluted soils by dissolving target metals from soil and making them more available for plant uptake and translocation to harvestable above-ground parts of high biomass crops (Meers et al., 2008). Recently, aminopolycarboxylic acids such as nitrilotriacetic acid (NTA) and (S,S)-ethylenediaminedisuccinic acid (EDDS) have been proposed as an alternative to EDTA and other persistent synthetic chelants (Evangelou et al., 2007; Luo et al., 2008a; Meers et al., 2008; Quartacci et al., 2006, 2007; Tandy et al., 2006). Even though the application of such biodegradable chelants could minimize the risks of potential off-site migration of metals – either in surface runoff or by leaching into groundwater (Nowack et al., 2006) – the accumulation of metals such as Cd, Cu and Zn increased only by a factor of 2–3, although their solubility in soil increased by a much higher factor (Grcˇman et al., 2003; Kayser et al., 2000; Kos and Lesˇtan, 2004; Kulli et al., 1999; Quartacci et al., 2007). Therefore, efforts have been focussed on screening plant species that are more
* Corresponding author. Tel.: þ39 50 2216633; fax: þ39 50 2216630. E-mail address: [email protected] (M.F. Quartacci). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.04.035
sensitive to chelant treatments and on developing new phytotechnologies that will help reduce and/or eliminate the amount of chelant applied to the field decreasing the environmental risk of mobilised metals (Luo et al., 2008a,b). Root exudates may influence the availability of nutrients – enhancing or reducing it – by a direct impact on the uptake of metals by acidification, chelation, precipitation and redox reactions, as well as by an indirect impact through their effects on microbial activity, the physical and chemical properties of the rhizosphere and root growth patterns (Tao et al., 2004; Uren and Reisenauer, 1988). Molecules naturally exuded by plants such as organic acids have been used in chemically-assisted phytoextraction programmes to enhance soil metal availability and root uptake by high biomass accumulating crop species (Evangelou et al., 2007). On the other hand, the exudation of organic molecules from the roots is also considered one of the most important strategies by which plants can exclude metals such as Al, Cd and Pb chelating them in the rhizosphere or in the apoplastic space and preventing their entry into the symplast (Hill et al., 2002; Watanabe and Osaki, 2002; Yang et al., 2000; Zheng et al., 1998). In this way, some metaltolerant species can restrict uptake and translocation of metals maintaining low shoot level over a wide range of external concentrations (Baker, 1981). It is still a matter of debate whether root exudates by different species vary in terms of quality and exudation rate. If so, such variations may significantly contribute to explain the different metal accumulation/tolerance capacity shown
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by plant species. Thus, the same chemical might play a role in limiting the uptake of metals in metal-tolerant populations or in enhancing metal mobility in chelant-assisted phytoextraction programmes carried out using accumulating crops. The fact that compounds commonly exuded from roots are useful in metal complexation have suggested the idea to grow a metal accumulating crop in succession to metallicolous populations in order to verify whether its phytoextraction efficiency may be improved. In this study three species from metalliferous soils were used as potential natural chelant producers for naturallyassisted phytoextraction of metals from a multiple contaminated soil by Brassica carinata, a species that has been seen to accumulate significant amounts of more metals at the same time (Quartacci et al., 2007). The results in terms of above-ground metal accumulation, soil metal dissolution as well as nature and amount of root exudates are discussed. 2. Materials and methods 2.1. Chemicals All chemicals used were analytical grade reagents and were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). 2.2. Soil characteristics and plant growth The metal-contaminated area is represented by a site used as a dump for pyrite ashes derived from the ore roasting processes for sulphur extraction. At present, the site is included within the perimeter of the polluted area named ‘Grado and Marano lagoon and neighbouring water-courses’ identified as a site of national interest by the Italian Cabinet Decree 468/2001 ‘National Programme for Environmental Restoration of Polluted Sites’. Preliminary tests showed that B. carinata performed poorly when sown in pots containing the contaminated soil. A milder substrate was then prepared by mixing the contaminated soil with peat in a 2:1 ratio by volume. After mixing, the soil mixture was allowed to equilibrate for a period of four weeks, undergoing three cycles of saturation with water and air-drying before being remixed and finally planted. Mixed soil characterisation was carried out according to Italian Standard Methods (Mi.P.A.F., 2000). Standards (National Institute of Standards and Technology) and reagent blanks (aqua regia) were run with all samples to ensure accuracy and precision in the analyses. The standard reference material used was SRM 2586. As for arsenic, preliminary tests showed that sample digestion by HNO3 resulted in a better recovery of the metal in comparison with aqua regia and HNO3/H2SO4/HClO4 (10:1:4, by vol.). The average recovery of As was 93% and 89% for SRM and soil samples, respectively. Physical and chemical characteristics of the mixed soil are reported in Table 1. The mixed soil was contaminated by As (total amount of 639 mg kg1 soil), Cd (61 mg kg1 soil), Cu (1846 mg kg1 soil), Pb (246 mg kg1 soil) and Zn (1143 mg kg1 soil). The polluted mixed soil was then used to fill 9 9 12 cm diameter plastic pots (700 g dry weight soil per pot) and moistened with water to approximately 80% field capacity. Basal fertilisers (150, 75 and 95 mg kg1 N, P and K, supplied as NH4NO3 and KH2PO4) were also applied. Pots were divided into five groups. One set of pots Table 1 Physical and chemical characteristics of the contaminated soil. Parameter Sand (%) Silt (%) Clay (%) pH (H2O) Organic matter (%) CEC (cmol(þ) kg1) Total S (%) Total N (g kg1) Exchangeable P (mg kg1) Exchangeable K (mg kg1) Available metals As (mg kg1) Cd (mg kg1) Cu (mg kg1) Pb (mg kg1) Zn (mg kg1)
79 14 7 7.3 6.8 4.4 0.01 0.7 11.7 170.8 0.8 0.6 29.4 2.7 3.4
Results are the means of five replicates. The standard deviation (SD) was always less than 10%.
was planted with seeds of B. carinata cv. 079444 (10 seeds per pot), whereas other three groups were planted with seeds (10 seeds per pot) of Pinus pinaster, Plantago lanceolata and Silene paradoxa collected at the polymetallic sulphide deposit of Fenice Capanne (Mascaro et al., 2001). The last set of pots was not sowed with any species. Metallicolous species were also grown separately in a non-contaminated control soil (commercial mould) with the same experimental procedure above reported. All pots were placed outdoor on the soil surface. The climatic conditions (Pisa, year 2007) were as follow: temperature 11–22 C, humidity 59–84%, average daily eliophany 8 h (May), and temperature 14–26 C, humidity 62–86%, average daily eliophany 9 h (June). Following seedling emergence, pots planted with B. carinata were thinned to two plants per pot. When soil moisture content decreased to 75% field capacity, pots were watered with 150 ml distilled water. Due to the fact that metallicolous species grow at a much lower rate compared to agronomic crops, after five and eight weeks of growth (for B. carinata and metallicolous species, respectively) all plants were harvested by cutting stems 1 cm above the soil surface and the soils were analysed for DTPA-extractable metals and for pH (Mi.P.A.F., 2000). For pH determination soil samples were collected at different depths (4 and 8 cm) and at about 2 cm far from roots. Thereafter, the same pots used for B. carinata growth were planted with seeds of the three metallicolous species (each species separately), whereas those in which the spontaneous plants were cultivated were sown with B. carinata seeds at the same density previously reported. In addition, one set of B. carinata seeds was planted in pots containing the contaminated soil in which no other species was previously grown (control). Pots were placed outdoors on the soil surface, and plants were watered, fertilised and harvested as reported. The climatic conditions (Pisa, year 2007) were as follow: temperature 17–29 C, humidity 45–75%, average daily eliophany 10 h (July), and temperature 17–29 C, humidity 46–84%, average daily eliophany 9 h (August). The above-ground parts of all four species collected were washed carefully with distilled water to remove any soil splash and oven-dried at 110 C for 24 h. Dried material was then digested with concentrated HNO3 in a capped Teflon pressure digestion vessel and analysed for As, Cd, Cu, Pb and Zn using a Perkin–Elmer Optimal DV 2100 ICP OES. Standards (National Institute of Standards and Technology) and reagent blanks (HNO3 65%) were run with all samples to ensure accuracy and precision in the analyses. The standard reference material used was SRM 1570a. 2.3. Determination of extractable metals in the soil Soils were analysed for DTPA (diethylenetriaminepentaacetic acid)-extractable metals by adding 5 mM DTPA, 10 mM CaCl2 and 0.1 M triethanolamine (pH 7.3) to give a 1:2 (w/v) soil:solution ratio (Mi.P.A.F., 2000). After shaking for 120 min, tubes were centrifuged at 17 400 g for 10 min and the supernatants collected after filtering through a Whatman No. 41 filter paper (pore size 20–25 mm). Analysis of the filtrate for As, Cd, Cu, Pb and Zn was performed as reported. 2.4. Determination of flavonoids, organic and phenolic acids in root exudates Seeds of P. pinaster, P. lanceolata and S. paradoxa were sterilized with 70% ethanol for 20 s and with 2.5% NaClO for 20 min and washed three times with deionised water. Seeds were then germinated for 3 days in the dark on floating trays containing 400 ml of continuously aerated nutrient solution composed of 0.25 mM NH4H2PO4, 1 mM Ca(NO3)2, 0.5 mM MgSO4, 1.5 mM KNO3, 11.5 mM H3BO3, 5 mM ferric tartrate [Fe2(C4H4O6)3], 3.5 mM MnCl2, 0.03 mM MoO3, 0.3 mM ZnSO4 and 0.12 mM CuSO4 (pH 5.5). Plants were grown at 16-h photoperiod, 400 mmol m2 s1 photon flux density, 23 1 C temperature and 70–75% relative humidity. After 15 days, plants were removed from the floating trays and transferred to vials, one plant per vial. Vials contained 20 ml of nutrient solution added with Cd(NO3)2, Pb(NO3)2, Zn(NO3)2, Cu(NO3)2 and Na2HAsO4 in order to reach the concentrations of the available metals in the Brassica-planted soil (see values in Fig. 1) calculated taking into consideration plant available water content (PAWC). One set of plants, kept as control, was transferred to vials containing 20 ml of nutrient solution. After 24 h seedlings were removed from vials, and roots were collected and weighted for dry weight determination. The control and metal-added solutions containing dissolved root exudates were lyophilised and the dry residues stored at 80 C till analysed. Immediately before analysis, the dry lyophilised residue was dissolved in deionised water and root exudates were analysed for flavonoids, organic and phenolic acids with a Waters HPLC system consisting of two 515 pumps and a 2487 programmable UV detector. Organic acids were separated on a Waters Nova-Pack C18 reverse-phase column (150 3.9 mm, 4 mm) and detected at 210 nm wavelength. A 10 mM phosphate buffer (pH 2.3) at a flow rate of 0.4 ml min1 was used as mobile phase. Phenolic acids and flavonoids were separated on a Waters Simmetry C18 reversephase column (150 3.9 mm, 5 mm). Phenolic acids were detected at 254 nm wavelength. Solvent A was 2% acetic acid and 30% acetonitrile, whereas 2% acetic acid was used as solvent B. A linear gradient from 10 to 95% solvent B for 55 min was used at a flow rate of 1 ml min1. Non-conjugated flavonoids were detected at 365 nm (quercetin and kaempferol) and 290 nm (naringenin). The mobile phase was formed by 55% of methanol and 45% of 0.05% trifluoroacetic acid eluted at a flow rate of 1 ml min1. Identification of flavonoids, organic and phenolic acids was obtained by comparison of their retention times with those of authentic standards (Sigma
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Fig. 1. DTPA-extractable metals in the multiple metal-contaminated soil after Brassica carinata, Pinus pinaster, Plantago lanceolata and Silene paradoxa growth. Control represents the multiple metal-contaminated soil in which no species was grown. Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05). Chemical). Quantification of individual compounds was obtained by comparing calibration curves. Chromatogram analyses were performed by Millennium 32 software (Waters). Total concentrations were calculated as sum of individual amounts. 2.5. Statistical analysis All statistical analysis was carried out using Costat 6.4 (CoHort software). The error bars shown in the figures represent the standard deviation of the mean of three independent experiments (n ¼ 3) each analysed twice. The effects of experimental factors were evaluated by one-way ANOVA and comparisons between means were carried out using the Tukey’s HSD multiple range test at the significance level of P 0.05.
Fig. 2. Metal concentrations (a) and uptakes (b) in the above-ground parts of B. carinata grown in the multiple metal-contaminated soil in succession to P. pinaster, P. lanceolata or S. paradoxa. Control represents the multiple metal-contaminated soil in which no species was grown. Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05).
3. Results 3.1. Soil extractable metals following plant growth The cultivation of the three metallicolous species had a positive effect on metal dissolution in the contaminated soil (Fig. 1). In comparison with the control (contaminated soil in which no species was grown), the growth of the three metallicolous species caused an average enhancement of extractable metals by about 3fold, with the exception of Cd that showed on average an 11-fold increase mostly due to P. pinaster contribution. In particular, S. paradoxa was the most effective species in enhancing Cu and Zn extractable amounts in soil, whereas available Cd and Pb were increased more by the presence of P. pinaster. No differences were observed for extractable As amounts. P. lanceolata growth resulted in a lower extractable metal level in the contaminated soil compared to the other two species. Compared to the control, the presence of B. carinata in the metal-contaminated soil had no effect on soil available metal concentrations. The cultivation of the metallicolous plants caused a significant decrease in soil pH from 7.3 0.3 (control) to 5.4 0.2 for P. pinaster and S. paradoxa, and to 5.6 0.5 for P. lanceolata, whereas B. carinata growth did not change soil pH. 3.2. Metal uptake by B. carinata In comparison with the control (contaminated soil in which no species was previously grown), B. carinata cultivated in the soil previously planted with the three metallicolous species showed a higher metal concentration in the shoots (Fig. 2a). Cd, Cu and Pb accumulated in the above-ground parts of B. carinata especially following P. pinaster growth (4.4-, 3.0- and 2.5-fold increase,
respectively), whereas the highest Zn concentration in Brassica shoots (1.6-fold increase compared to control plants) was observed following S. paradoxa growth. No significant differences between control and planted soils were detected for As. Compared to control plants (0.16 g dry weight plant1), the dry matter of the aboveground parts of B. carinata grown in succession to the three spontaneous species suffered a slight not significant reduction, (0.14 g dry weight plant1) following Plantago and Silene growth and a significant 25% decrease (0.12 g dry weight plant1) following Pinus growth. The amount of metals expressed on a plant basis followed almost the same behaviour observed for their concentrations (Fig. 2b). Cd, Cu and Pb showed the highest content in Brassica above-ground parts following Pinus growth (9.6, 20.3 and 8.0 mg plant1, respectively), whereas Zn accumulated mostly following Silene cultivation (15.6 mg plant1). 3.3. Metal uptake by metallicolous species As regards metal concentrations in the above-ground part of metallicolous species, in general the growth of P. pinaster and S. paradoxa in the multiple metal-contaminated soil (MCS) resulted in slight differences compared to a non-contaminated control soil (Fig. 3a and c) composed of commercial mould. On the contrary, P. lanceolata showed a remarkable increase in Pb, Zn and, in particular, a 47-fold increase in Cu (Fig. 3b). Arsenic concentrations did not change following treatments. Metallicolous species grown in the metal-contaminated soil in succession to B. carinata (MCS þ Bc) showed a reduction of shoot metal concentrations in comparison with plants not preceded by B. carinata growth (MCS), and in most cases values returned to the control ones or were even lower (Fig. 3).
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Fig. 4. Dry weights of the above-ground parts of P. pinaster, P. lanceolata and S. paradoxa grown in non-contaminated control soil, in the multiple metal-contaminated soil (MCS) and in the multiple metal-contaminated soil in succession to B. carinata (MCS þ Bc). Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05).
Fig. 3. Metal concentrations in the above-ground parts of P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) grown in non-contaminated control soil, in the multiple metalcontaminated soil (MCS) and in the multiple metal-contaminated soil in succession to B. carinata (MCS þ Bc). Values are means SD (n ¼ 3). Means followed by different letters are significantly different by ANOVA (P 0.05).
Compared to the non-contaminated control soil (Fig. 4), the metal-contaminated soil (MCS) caused a remarkable reduction of dry biomass in the above-ground part of the metallicolous species (60, 68 and 49% decrease for Pinus, Plantago and Silene, respectively). The previous growth of B. carinata in the multiple metal-contaminated soil (MCS þ Bc) had beneficial effects on biomass production limiting shoot dry mass loss in the metallicolous plants (Fig. 4). 3.4. Root exudates The main components of root exudates were organic acids followed by lower amounts of phenolic acids and flavonoids (Table 2). The highest amount of organic acids was observed in the presence of metals (about 28-fold increase on average compared to the control), whereas the concentrations of phenolic acids and flavonoids were enhanced by a factor of 1.5 and 3, respectively. It should be pointed out that, differently from the other two species, in S. paradoxa total phenolic acids were halved after metal treatment.
Six different organic acids were detected, being their typology and amount strictly species-dependent (Fig. 5). Oxalic and malic acids were detected in all the three metallicolous species, while tartaric and malonic were present in P. pinaster, citric and fumaric in P. lanceolata, and malonic and fumaric acids in S. paradoxa. Oxalic and malic were the main representative organic acids in the exudates of both control and treated roots. The kind of organic acid present in root exudates did not change following metal incubation, although the rate of increase was dependent on the species investigated. Under metal excess the highest increase in P. pinaster exudates was shown for oxalic acid (173-fold) followed by malic acid (31-fold) (Fig. 5a). Similar remarkable increases were detected in response to metal treatment also in S. paradoxa, whose oxalic and malic acid concentrations increased by about 143- and 78-fold, respectively (Fig. 5c). P. lanceolata was the species which showed the lowest rates of increase, fumaric and oxalic acids increasing by a factor of 10 (Fig. 5b). As for organic acids, the amount and type of phenolic acids detected in root exudates were also strictly dependent on the species analysed (Fig. 6). In control conditions the main phenolic acids exuded by P. pinaster were p-OH-benzoic (37% of total concentration), syringic (33%) and protocatechuic (30%). Ferulic (31% of total concentration), syringic (29%), p-cumaric (11%) and caffeic (11%) acids were the main constituents of phenolic exudates of P. lanceolata. The phenolic acids present in the exudates of S. paradoxa grown in control solution were mainly protocatechuic (36% of total concentration), syringic (31%), p-OH-benzoic (23%) and vanillic (10%). Differently from organic acids, the treatment of the metallicolous species with metals caused both an increase and a decrease in the amount of exuded phenolic compounds (Fig. 6) depending on the species and the individual compound investigated. The most remarkable enhancements concerned chlorogenic acid (70- and 13-fold increase compared to control in P. pinaster and S. paradoxa, respectively) and vanillic acid (8-fold increase in P. lanceolata), whereas syringic acid was the phenolic compound that suffered from the strongest reductions in all species (82, 42 and 98% decrease in comparison with non-treated plants for Pinus, Plantago and Silene, respectively). Kaempferol, naringenin and quercetin were the free flavonoids found in the exudates of control roots (Fig. 7). Naringenin was the flavonoid which production was the most stimulated by the metal treatment in all species. In comparison with control plants naringenin increased by about 20-, 7- and 3-fold in Pinus, Plantago and Silene exudates, respectively. Following metal treatment
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Table 2 Organic acid (mg g1 root dry weight), phenolic acid and flavonoid (mg g1 root dry weight) total contents in root exudates of plants incubated for 24 h in nutrient solution (control) and in nutrient solution added with metals. Organic acids
Pinus pinaster Plantago lanceolata Silene paradoxa
Phenolic acids
Flavonoids
Control
Treated
Control
Treated
Control
Treated
8.1 0.5 11.1 0.5 36.2 1.9
440.2 20.1* 99.1 3.9* 713.1 21.7*
14.0 0.9 23.6 1.2 79.3 2.8
22.8 0.8* 32.2 1.8* 39.9 1.9*
0.4 0.1 1.9 0.2 3.1 0.2
0.9 0.1* 9.6 0.3* 6.6 0.4*
Values are shown as means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05).
kaempferol and quercetin showed a lowest rate of increase or, as in the case of P. pinaster, remained almost unchanged. 4. Discussion Although EDDS is one of the most biodegradable among metal chelants, Meers et al. (2005) suggested that under less optimal conditions ligand persistence or ligand effect (i.e. the concentration of metal(s) mobilised into the soil solution following chelant
Fig. 5. Organic acids in root exudates of 15-days-old P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) incubated for 24 h in nutrient solutions added with As, Cd, Cu, Pb and Zn. Control represents nutrient solutions without metals. Values are means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05).
addition) can increase dramatically. The evidence that metaltolerant species exude organic compounds in order to complex and mobilize metals suggests the possibility to use those plants for producing chelants for naturally-assisted phytoextraction programmes. In general, if roots exudates remain stable in soils for sufficient time, they may improve the capacity of crops to uptake
Fig. 6. Phenolic acids in root exudates of 15-days-old P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) incubated for 24 h in nutrient solutions added with As, Cd, Cu, Pb and Zn. Control represents nutrient solutions without metals. Values are means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05). Ben, p-hydroxybenzoic acid; Caf, caffeic acid; Chl, chlorogenic acid; Cum, p-cumaric acid; Fer, ferulic acid; Gal, gallic acid; Pro, protocatechuic acid; Syr, syringic acid; Van, vanillic acid.
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Fig. 7. Flavonoids in root exudates of 15-days-old P. pinaster (a), P. lanceolata (b) and S. paradoxa (c) incubated for 24 h in nutrient solutions added with As, Cd, Cu, Pb and Zn. Control represents nutrient solutions without metals. Values are means SD (n ¼ 3). Means followed by asterisks are significantly different by ANOVA (P 0.05).
metals. To date, this is the first report investigating the use of plant succession to improve metal phytoextraction from multiple contaminated soils. In agreement with the increase in available metal amounts in soil (Fig. 1), the growth of the three metallicolous species enhanced B. carinata capacity to accumulate and translocate Cd, Cu, Pb and Zn (Fig. 2). This result may be explained both by the acidification of the multiple contaminated soil and the formation of metal complexes that enhanced metal bioavailability for root uptake by B. carinata. In comparison with NTA- and EDDS-amended plants (Quartacci et al., 2007), plant succession determined on average a higher accumulation of Cd (3-fold) and similar amounts of As, Cu and Zn, whereas only Pb suffered a 1.8-fold reduction. The significant decrease of B. carinata above-ground biomass when grown in succession to P. pinaster was a consequence of the high concentrations of metals in the contaminated soil solution (Fig. 1) and in the above-ground tissues (Fig. 2a). The improved phytoextraction efficiency of
B. carinata determined by plant succession was relatively low (about 3- or 2-fold on average when expressed on a dry matter or plant basis, respectively) in comparison with the experiment in which no plants were grown before B. carinata (Fig. 2). Nonetheless, it indicates a potential approach to the remediation of metalcontaminated soils in the absence of exogenous compounds added to the soil and potential related risks for the environment. In spite of the limited metal accumulation due at least in part to a low dry biomass production (maximum of 50.4 mg metals plant1 following Pinus growth), B. carinata demonstrated the ability to survive and tolerate the toxic effects of more metals together, and in the presence of multiple contamination a compromise that makes possible the simultaneous uptake of more metals may not be discarded. Metallicolous species, and especially P. lanceolata, accumulated considerable amounts of metals after the period of growth in the multiple contaminated soil (Fig. 3), even though their use in phytoextraction programmes cannot be considered realistic due to their very low biomass and growth rate. The amounts of DTPA-extractable metals detected immediately after the harvest of the three metallicolous species showed that the growth of plants had a positive effect on metal mobilisation (Fig. 1) with values approaching or exceeding, as in the cases of Cd and Cu, those obtained after EDDS application (Quartacci et al., 2007). Similarly to the EDDS-treated soil (Quartacci et al., 2007), Cu was the metal extracted in the highest amount by Brassica, probably due to relatively high stability constants of its chelates with the various exudates (Martell and Smith, 1989). In comparison with nonplanted contaminated soil, B. carinata growth did not enhance metal availability for plant uptake (Fig. 1) indicating that this crop did not play a role in acidifying the rhizosphere and/or in dissolving metals, probably due to a qualitatively not adequate and/or quantitatively insufficient production of exudates. Mobilisation of metals in the rhizosphere has been related to its acidification and to complexation with organic acids present in root exudates. Organic acids typically flow across the lipid bilayer at a slow rate in response to the electrochemical gradient. However, efflux may be greatly increased in stress conditions such as metal ion toxicity due to the expression (encoding) of anion channels embedded in the plasma membrane or to their up-regulation (Rengel, 2002). The activation of Hþ-ATPases located at the plasma membrane level and the consequent release of organic exudates from roots could explain the decrease in soil pH observed following the growth of the metallicolous species. However, the fact that the decrease in the pH was similar for the three species whereas the capacity of mobilising metals was different among them (Fig. 1) suggests that the decrease of pH accounted only in part for the interspecific differences in metal mobilisation. The exudation of specific organic ligands from roots or the exudation rate could account for the rest of the difference as well as enhanced microbial activity linked to a higher availability of carbon sources deriving from root exudates (Tao et al., 2004). Indeed, it has been shown that the acidification of the soil solution was not the only reason for the increase in metal availability (Wenzel et al., 2003). Several organic acids, mainly oxalic and malic acids, have been found to be exuded in increased amounts by roots exposed to heavy metals (Mariano and Keltjens, 2003; Nguyen et al., 2003; Rengel, 2002). Also in the present study oxalic and malic acids were found as the most exuded organic acids in response to multiple metal stress (Fig. 5). In addition, other low molecular mass weight organic acids were detected, but these compounds did not show a constant presence being species-specific, and their concentrations, although in many cases significantly different between control and treatment, were much lower than those detected for oxalic and malic acids. Although citric acid is generally considered to play an important role in metal tolerance (Rengel, 2002), in this study it
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was released only by P. lanceolata and in a very small amount (Fig. 5). Metals also stimulated the excretion of fumaric and tartaric acids from roots of P. pinaster and P. lanceolata, respectively, suggesting that their release might take part to the tolerance strategies of the metallicolous populations of these two species. Only limited information exists about availability and uptake of metal–carboxylate complexes by roots. Hydroponic studies revealed that plant uptake is correlated mainly with the activity of free metal ions (Cd, Cu, and Mn) in solution which implies their dissociation from the complex likely mediated by rhizosphere acidification and reduction of metals (Neumann and Ro¨mheld, 2001). Although organic acids are the main components of Pinus, Plantago and Silene exudates, other organic compounds were exuded. Several phenolic acids and three flavonoids were detected both under control and metal excess conditions, although their release was one order of magnitude lower than organic acids (Table 2). While phenolic compounds showed a variable pattern in terms of composition and amount (Fig. 6), the three flavonoids were found to be exuded by all the metallicolous species in increased amounts following exposure to metals (Fig. 7). The low concentrations of phenolic acids and flavonoids, also reported for Norway spruce by Heim et al. (2001), do not suggest a relevant role in forming metal complexes in the contaminated soil where metallicolous species were grown, as only a minor part of the metals can be bound by these compounds, although both classes of compounds can form stable bonds with metals (Martell and Smith, 1989). Therefore, the exudation of phenolics – and flavonoids in particular – in plant adaptation to altered environmental conditions might be better explained if discussed in relation to their possible function as specific chemoattractant signal for soil microbes to the rhizosphere and/or growth promoters of rhizosphere bacteria as source of carbon substrate (Dakora and Phillips, 2002). In the case of our multiple contaminated soil a role of exudates as chelators of poorly soluble mineral nutrients cannot be excluded. It was observed that phenolics make Fe and P available by forming relatively stable chelates with Fe in insoluble Fe-phosphates thereby increasing the solubility of both Fe and P for plant uptake (Dakora and Phillips, 2002). 5. Conclusions The results of the present study show that growing B. carinata in succession to spontaneous species from metalliferous soils led to the accumulation of higher amounts of metals in the above-ground parts in comparison with the same plants grown in soil not previously planted. The fact that accumulation of metals is similar to that shown by B. carinata following amendments with biodegradable chelants such as NTA and EDDS indicates a possible approach to naturally enhanced phytoremediation of multiple metal-contaminated soils. Plant succession could improve phytoextraction efficiency of metals from soil without the application of any potentially dangerous chemical and with the advantage that chelants in this way are naturally produced by plants themselves. Further studies aimed to investigate the metal binding properties of each compounds released by roots and the possible involvement of other exudates are necessary in order to assess their role in the metal accumulating capacity of B. carinata. Acknowledgements Seeds of Brassica carinata cv. 079444 were kindly provided by Prof. M. Mazzoncini (Dipartimento di Agronomia e Gestione dell’Agrosistema, University of Pisa). This study was funded by the MIUR (Cofinanziamento 2005) and the University of Pisa (Fondi di Ateneo 2006).
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