THE HYPERACCUMULATOR PLANT ALYSSUM MURALE AS A POTENTIAL AGENT FOR PHYTOMINING OF NICKEL IN AN ALBANIAN SITE Aida BANIa,b, Teuta TOPIc, Ardian MAÇIa, Guillaume ECHEVARRIAb, Alkeda KALAJNXHIUa, Sulejman SULÇEa, Jean Louis MORELb a Agricultural University of Tirana, ALBANIA Laboratoire Sols et Environnment, Nancy-Université, INRA, Vandœuvre lès Nancy, FRANCE c Ministry of Agriculture, Food and Consumer Protection, Tirana, ALBANIA E-mail: aida_alushi@hotmail.com b ABSTRACT Serpentine soils, developed upon ultrama•c rock, contain relatively high concentrations of heavy metals and are the preferred substrate for several adapted plants, especially those that accumulate Ni in their above-ground tissues. A serpentinized peridotite outcrop located in Pojske hosts a population of the Ni-hyperaccumulator species Alyssum murale. Recent work has been carried out on the site of Pojske to optimize agronomic practices which may affect the ef•ciency of Ni phytoextraction by native stands of A. murale on 18-m2 plots in natural conditions (three replicates for each treatment). In soil fertility management studies, we have used 120 kg ha-1 NPK. Only two different fertilization regimes (fertilized vs. non-fertilized treatments) were tested in 2005. In 2006, we studied the effect of targeted herbicide treatment to control the main competing weed (Chrysopogon gryllus). The two treatments were crossed (herbicide + fertilization). We found that NPK application signi•cantly increased shoot biomass yield, without reducing shoot Ni concentration. In weed control practices, the use of anti-monocots herbicide (FocusTM ultra) allowed for the full development of A. murale stands. In this two-year experiment, biomass yields in fertilized and herbicide treated plots have progressively improved: 2.6-3.7t ha-1. Aktet e Takimit Vjetor, Vëll. II, Nr. 2 The plant of Alyssum murale (Waldst. & Kit.) So have phytoextracted Ni quantities: 22.6-29.5 kg ha-1. Such crop management practice studies have improved phytoextraction ef•ciency and extensive phytomining could be promising in the Albanian context by domesticating already 21 A. BANI, T. TOPI, A. MAÇI, G. ECHEVARRIA, A. KALAJNXHIU, S. SULÇE, J.L. MOREL installed natural populations. Key-words: Ultrama•c soil, Nickel hyperaccumulator plant, Serpentine !ora, Bioavailability, Phytomining PËRMBLEDHJE Tokat serpentine të formuara nga alterimi i shkëmbinjve ultrama•ke, përmbajnë përqëndrime të larta të metaleve të rënda dhe janë zona të preferuara për disa bimë, veçanërisht për ato që akumulojnë nikel në indet e tyre. Zona e Pojskës (Pogradec) me toka me prejardhje nga peridotite të serpentinizuara është e populluar nga hiperakumulatorja e nikelit, Alyssum murale. Ky studim u krye në zonën e Pojskës me qëllim optimizimin e praktikave agronomike të cilat mund të ndikojnë efektivitetin e •toekstraktimit të nikelit nga popullimet e A. murale në parcela me sipërfaqe 18 m2 në kushte natyrore (3 përsëritje për secilin trajtim). Në kuadrin e menaxhimit të pjellorisë së tokës, ne kemi përdorur 120 kg ha-1 NPK (AzotFosfor-Potas). Në 2005 u testuan dy rregjime plehrimi (trajtimi i plehruar dhe ai i paplehruar). Në vitin 2006, u studiua efekti i trajtimit me herbicide për kontrollin e barërave të këqija më konkurente si Chrysopogon gryllus. U ndërthurën dy trajtime (herbicid + plehrim). Nga rezultatet gjetëm se aplikimi i NPK-së rrit prodhimin e biomasës së pjesës mbitokësore të bimës, pa reduktuar përqendrimin e nikelit (Ni) në të. Në praktikat për kontrollin e barërave të këqija përdorimi i herbicidit anti-monokotiledon (FocusTM ultra), lejon zhvillim të plotë të popullimit natyral të A. murale. Në këto dy vite eksperimentimi, prodhimi i biomasës së A. murale në parcelat e trajtuara u përmirësua progresivisht deri në 2.6-3.7t ha-1. Kështu, u •toekstraktuan sasitë e Ni: 22.6-29.5 kg ha-1. Studimi i praktikave të tilla menaxhuese përmirësoi e•kasitetin e •toekstraktimit dhe ky ekstraktim i nikelit me anë të Alyssum murale mund të jetë premtues në konteksin shqiptar duke përdorur popullime natyrale të kësaj bime. INTRODUCTION Ultrama•c terrains occupy 1 % of the earth’s land surface and are host to distinctive !ora [5]. Ultrama•c rocks and particularly serpentinites containing very high magnesium (18-24%) and high iron (6-9%) but very low Ca (1-4%) and aluminium (1-2%) [2]. The soils derived from ultrama•c rocks, such as peridotites, dunites and serpentinites – termed serpentine soils – are also strongly in!uenced by the geochemistry and mineralogy of the parent material [2]. These soils share a number of chemical properties, including a high content of speci•c heavy metals (nickel, chromium and cobalt), a low Ca:Mg concentration ratio and low concentrations of macronutrients [5]. In general, the vegetation of serpentine soils is well differentiated with respect to the surrounding areas. Among the metals widely presents in ultrama•c soils, Ni is probably the one that causes the most signi•cant toxicity to nonadapted plants. Ni hyperaccumulation has been de•ned as the accumulation of at least 1000 mg kg-1 Ni in the dry biomass of plants grown on a natural substrate [6, 14]. Hyperaccumulation has become recognized as an unusual response and speci•c ecophysiological adaptation to the elevated metal concentrations generally found in soils derived from ultrama•c areas. Magnesium-rich soils in Albania consist of about 10,000 ha out of 700,000 ha of total agricultural land [15]. One of many ophiolitic massifs called Shebeniku is located in the ophiolites of eastern Albania and it hosts several Ni hyperaccumulator species (e.g., Alyssum murale Waldst. & Kit.) [4]. A. murale can be used to pro•tably phytoextract Ni from ultrama•c soils as an alternative to the traditional cropping in metal-toxic soils. Phytoextraction of Ni in ultrama•c areas is called “phytomining” and has been successfully implemented in soils containing natural high concentrations of Ni, Co and Cr [12]. The objectives of this work were to assess: 1) the approximate yield of Ni; 2) the relation between Ni content in harvested plant parts and the status of Ni available in the soil; 3) the effect of fertilization and weed on biomass and Ni content of harvests; This is of particular interest nowadays since several potential applications of hyperaccumulators, such as phytoremediation and phytomining, are being tested. MATERIALS AND METHODS The experiment was carried out in Pojskë (700 m, Pogradec), with latitude of 40°59’55, 28” N and longitude of 20°38’03, 92” E, and with a Mediterranean climate characterized by annual rainfall averages of ~730 mm and a mean temperature of ~10°C. The experimental site 22 www.alb-shkenca.org THE HYPERACCUMULATOR PLANT ALYSSUM MURALE AS AN AGENT OF PHYTOMINING species). For each plot and species, plant samples were taken, rinsed with deionized water and dried at 80°C for 24 h. Trace metal contents in shoots were analyzed by plasma emission (ICP) spectrometry after the digestion of plant samples in microwave oven. A 0.25-g DM plant aliquot was digested by adding 8 mL of 69% HNO3, 2 mL H2O2. Solution were •ltered and adjusted to 25 mL with 0.1 M HNO3. was a colluvial downslope (10-15%) characterized by spontaneous native ultrama•c vegetation. The soil pro•le was described and samples were taken from the horizons identi•ed in the •eld (0-30 cm, 30-50 cm, 50-70 cm, 70-120 cm). Physical and chemical characteristics of the soil samples were determined by the Soil Analyses Laboratory of INRA Arras in France [1]. X-Ray diffraction (XRD) was run on the 50 µm fraction to determine mineralogy. Ni-bearing phases were performed with transmission electron microscopy and coupled with X-ray spectroscopy (TEM-EDX) techniques to identify the minerals and their elemental composition. Ni chemical availability in soil samples of surfaces was characterized by DTPA-TEA [11]. Concentration of Ni in soil extracts were determined by plasma emission spectrometry (ICP-OES). A •eld experiment was conducted in 20052006. The experimental site was already covered by spontaneous native ultrama•c vegetation in March 2005 (an abandoned cropped •eld). The experimental area in 2005 was divided into six 36m2 plots, three of which were fertilized in April with 120 kg ha-1 N, P, K (NH4NO3, K2SO4 and Ca(H2PO4)2). In 2006, each experimental plot was divided into twelve 18-m2 plots, 3 of which were only fertilized (the same regime of fertilization, FNH), 3 were fertilized and treated with antimonocots herbicide (FocusTM ultra 33 mL in 3L water for 108 m2) (FH), 3 were only treated with herbicide (NFH) and 3 were not treated (NFNH). Plants were harvested each year on June 28th. The !ora of each plot was fully described in June, prior to harvest (with the help of European and Albanian Flora). The plant species were sampled at site ( s 3 & 4). The shoots of each species were individually collected and biomass yields were recorded for each plot. The rest of the biomass was pooled together (other non-frequent Particle size distribution Clay Silt pH % Horizon CO MO Sand H 2O 0 -30 cm 51.6 27.7 20.7 30-50 cm 52.2 14.8 50-70 cm 37.4 11.9 70-120 cm 57.5 23.3 19.2 N P total Olsen C/N CEC RESULTS AND DISCUSSION SOIL CHARACTERISTICS All soil characteristics con•rmed its ultrama•c nature: low concentration of Ca, K and P and elevated Ni, Cr, Co, Mn, Fe (Table 1). This soil showed high total Fe contents with values of 10 %. It had 6% Mg and a strong Ca de•ciency (0.3 %). The Mg:Ca ratio was high (20 as total concentration and 7.4 as exchangeable cations), a range that is commonly reported in serpentine soil material (13). Potassium total contents in soils were low (0.4%). According to FAO WRBSR (1998) the soil at the experimental site was classi•ed as Magnesic (Hypermagnesic) Hypereutric Vertisol. XRD analyses of the three horizons revealed that smectite and serpentine (undetermined type) were the two predominant minerals in the soil. TEM and EDX observations and analyses showed that both were in the Ni-bearing phases (Table 2). Ni availability assessed by DTPA was high, reaching 130 mg kg-1. Although soil pH was quite high (neutral to slightly alkaline), Ni availability in this soil was very high because it was associated with high charge phyllosilicates smectites (high charge phyllosilicates) and amorphous Fe oxides [10]. The soils were suitable for phytomining. EFFECT OF FERTILIZATION ON SPECIES COMPOSITION AND BIOMASS PRODUCTION A. murale, C. gryllus and T. nigriscens were the Exchangeable cations Ca2+ Mg 2+ % cmol+ kg 7.45 2.74 4.7 0.23 0.01 12 33 7.75 1.36 2.4 0.10 0.01 50.7 7.62 0.38 0.7 0.05 0.01 7.87 0.34 0.6 0.04 0.01 8.3 K2+ Na 2+ Total major elements Ca Mg -1 Al K Na Total trace elements Fe Co Cr % Mn mg kg Cu Zn Ni -1 38.9 5.42 40.5 0.37 0.08 0.30 6.01 2.7 0.5 0.2 10 207 1600 2060 23 130 3150 13 43.8 3.41 49.4 0.37 0.09 0.18 5.56 2.6 0.4 0.2 11 224 1610 2920 13 95 3050 8.3 42.4 1.47 48.6 0.32 0.09 0.11 7.00 1.9 0.2 0.1 12 160 1490 1460 11 91 3300 38.9 1.14 50.5 0.42 0.11 0.15 4.61 4.0 0.6 0.3 10 134 1170 1320 11 91 2070 Table 1. Physical-chemical characteristics of the four horizons of the soil at the experimental site of Pojskë (Albania) Aktet e Takimit Vjetor, Vëll. II, Nr. 2 23 A. BANI, T. TOPI, A. MAÇI, G. ECHEVARRIA, A. KALAJNXHIU, S. SULÇE, J.L. MOREL Bedrock 0-30 cm 30-50 cm 50-70 cm Ni-bearing phase Serpentine [8] Serpentine [8] Al-rich smectites [3] Mg = Al smectites[3] Mg-rich, Al poor smectites [1] Serpentine [3] Al rich smectites [6] Mg = Al smectites [3] Mg-rich, Al poor smectites [2] Serpentine [4] Al-rich smectites [1] Mg = Al smectites [4] Mg-rich, Al poor smectites [3] Ni concentration (%) 0.30 ± 0.17 0.69 ± 0.28 0.44 ± 0.22 1.00 ± 0.24 1.87 0.60 ± 0.31 0.57 ± 0.17 0.72 ± 0.28 3.12 ± 2.50 0.66 ± 0.12 0.66 0.99 ± 1.00 1.55 ± 0.54 Species Alyssum murale Waldst. et Kit Chrysopogon gryllus L. Trin Trifolium nigriscens Viv. Lolium perenne L. Aegilops geniculata Roth. Dasypyrum villosum L. P. Cond. Poa trivialis L. Centaura solstitialis L. Minuartia hybridaL. Lotus corniculatus L. Consolida regalis S.F.Gray Plantago lanceolata L. Petrorhagia prolifera L. Tragopogon pratesis L. Bromus racemosus L. Vicia villosa Roth. [n]: number of particles analysed (EDX) Table 2. Identi•cation of Ni-bearing phases and associated Ni content (%Wt) in the bedrock and horizons of the soil determined by TEM-EDX most frequent species in this site but other species were reported on the plots as well, although their contribution to biomass production was negligible (Table 3). According to what was expected on such soils with low K and P availability the overall vegetation responded dramatically to fertilization by doubling the biomass yield. However, the contribution of each species varied according to fertilization. In unfertilized plots, C. gryllus accounted for most of the biomass, whereas in fertilized plots, A. murale was the main contributor. A. murale biomass abundance increased dramatically from 6.1% to 40.7% whereas C. gryllus biomass abundance decreased from 77.5% to 54.5%. T. nigriscens decreased in fertilized plots (16.34.8%). The Ni phytoextraction yield was 22.6 kg Ni ha-1 in the fertilized plots compared to 1.67 kg Ni ha-1 in unfertilized plots. This difference was Year 2005 2006 Species A. murale Ch. gryllus T. nigriscens Total A. murale Ch. gryllus T. nigriscens Total A. murale T. nigriscens Other Total Plots F A. murale Ch.. gryllus T. nigriscens Total A. murale T. nigriscens Other Total A. murale Ch.. gryllus T. nigriscens Total FNH NF FH NFH NFNH Biomass (t ha-1) 2.56 ± 0.70 a 3.43 ± 0.35 0.30 ± 0.20 6.30 0.20 ± 0.44 b 2.53 ± 0.32 0.53 ± 0.12 3.27 3.70 ± 1.06 a 0.075 ± 0.04 0.38 ± 0.16 4.15 Ni yield (kg ha-1) 22.6 ± 4.4 a 2.17 ± 0.15 0.17 ± 0.12 24.93 1.67 ± 0.12 b 0.83 ± 0.49 0.60 ± 0.30 3.10 29.5 ± 8.6 a 0.06 ± 0.04 0.31 ± 0.13 29.87 2.16 ± 1.42 a 0.97 ± 0.45 0.025 ± 0.014 3.15 1.1 ± 0.3b 0.039 ± 0.024 0.19 ± 0.06 1.33 0.25 ± 0.05 c 0.47 ± 0.16 0.031 ± 0.026 0.75 18.4 ± 11.4 a 0.81 ± 0.43 0.0059 ± 0.004 19.1 8.9 ± 4.5b 0.013 ± 0003 0.08 ± 0.014 8.99 2.00 ± 0.34 c 0.17 ± 0.011 0.0021 ± 0.0007 2.17 Table 3. Biomass production and phytoextraction yield of the main species grown for each treatment. Values for each species within a plot are given as mean values ± standard deviation. For the biomass and phytoextraction yield of A murale, different letters indicate statistical difference. Family Brassicaceae Poaceae Fabacaeae Poaceae Poaceae Poaceae Poaceae Asteraceae Caryophyllaceae Fabacaeae Ranunculaceae Plantaginaceae Caryophyllaceae Asteraceae Poaceae Fabacaeae Biological form H H Th H Th Th H H Th H Th H Th H H Th Lifespan perennial Perennial Annual Perennial Annual Biannual Perennial Biannual Perennial Perennial Annual Perennial Perennial Annual Annual Biennial H: Hemicryptophyte TH: Therophyte Table 4. List of plant species collected on the experimental plots of Pojskë (Albania) highly signi!cant (P < 0.01). A. murale was the main contributor in total phytoextraction yield (Table 4). In 2006, when the herbicide treatments were included to allow for the full development of A. murale we obtained a biomass of A murale of 3.7 t ha-1 (dry weight) in FH plots while the biomass in NFNH plots was only of 0.25 t ha-1. The herbicide treatment seemed to ef!ciently control the population of C. gryllus. The biomass production of A murale, C. gryllus and T. nigriscens in fertilized plots increased respectively 14.8-, 2.0- and 2.4-fold in comparison to the unfertilized plots. The biomass production of C. gryllus and T. nigriscens decreased in 2006 since we harvested before maturity of T. nigriscens (Therophyte) in untreated plots (2005) and the harvested buds of C. gryllus (Hemicryptophyte) on the experimental site were exposed to low temperatures in winter. The yield of Ni phytoextraction in 2006 was 29.5 kg Ni ha-1 in the fertilized and herbicide treated plot compared to 2 kg Ni ha-1 in the unfertilized with no herbicide plots. These differences were highly signi!cant (P < 0.01). The relative and net increase in biomass production of A. murale were the main reason for the increase of phytoextraction yield since the Ni concentration in shoots was not signi!cantly affected by the fertilization and herbicide treatments. In two years of experiment, species showed an increasing pattern in total biomass production in response to fertilization with signi!cant differences (p < 0.05) for A. murale only. A. murale was also the life-form showing the highest increase after fertilization (p < 0.05), and therefore a more competi24 www.alb-shkenca.org THE HYPERACCUMULATOR PLANT ALYSSUM MURALE AS AN AGENT OF PHYTOMINING and 0.8% in 2006. The Ni concentrations in shoots of A. murale were more than two times higher than in the soil. This ratio indicated for optimal hyperaccumulation conditions although the bioavailability of Ni is high. No particular trend regarding Fe and Mn accumulation was observed despite reports of Mn accumulation in Alyssum leaves in other studies. Cobalt concentration in shoots tissues varied from 0.29 to 4.9 mg kg-1. Some species in our site showed that they were able to colonize those soils in the presence of other tolerant populations. Ni content in these other species varied from 187 to 670 mg kg-1, whilst plants from non-serpentinitic substrata usually contain very low Ni concentrations (0.5 ± 10 mg kg-1) [14]. Centaurea solsticialis was accumulating at very high levels of Ni for non-accumulating species. tive specie in terms of ecological adaptation. This experiment showed that nutritional stress represents an important limiting factor for the existence of many species and plant productivity which is in accordance with the !ndings of Chiarucci (7) in serpentine vegetation of Tuscany. PLANT RESPONSES TO SERPENTINE The chemical analyses performed on plant bulk shoots showed different plant responses to the presence of trace metals and nutrients in soil of the experimental site (Table 5 & 6). Mg mean concentration for A murale was lower than Ca concentration while the inverse occurred in others species. The Mg: Ca ratio in A. murale was therefore lower than 1. This con!rms the ability of this plant to accumulate Ca and its positive response to Ca fertilization [9]. Among the species in the experimental site Ni hyperaccumulation was observed only in A murale. The average Ni content in shoots was about 0.9% at harvest time in 2005 in the fertilized plots Species Alyssum murale Trifolium nigriscens Alyssum murale Chrysopogon gryllus Trifolium nigriscens Alyssum murale Trifolium nigriscens Alyssum murale Chrysopogon gryllus Trifolium nigriscens Plots FH FNH NFH NFNH Ni (mg kg-1) 7887 ± 446 558 ± 528 8680 ± 617 812 ± 251 257 ± 196 7826 ± 1347 468 ± 278 7210 ± 1004 402 ± 141 90 ± 50 CONCLUSION The soil on the site of Pojska was clearly suitable for phytoextraction. Ni-bearing phases in the Mn(mg kg-1) 9.3 ± 1.8 18 ± 2.7 12 ± 3.7 28 ± 15 15 ± 4.4 9.9 ± 6.2 26 ± 17 7.9 ± 0.1 28 ± 3.3 16 ± 1.5 Co(mg kg-1) 2.1 ± 0.52 0.9 ± 0.34 2.6 ± 0.21 2.1 ± 2.2 0.8 ± 0.01 2.2 ± 1.14 2.1 ± 2.2 1.9 ± 0.01 1.2 ± 0.22 0.7 ± 0.1 Fe (%) 74 ± 30 327 ± 218 220 ± 180 513 ± 313 182 ± 141 263 ± 202 363 ± 62 145 ± 12 803 ± 287 252 ± 1.4 Ca (%) 0.49 ± 0.03 0.91 ± 0.07 0.64 ± 0.11 0.3 ± 0.03 0.8 ± 0.14 0.54 ± 0.1 0.88 ± 0.04 0.55 ± 0.02 0.23 ± 0.032 0.77 ± 0.19 Mg (%) 0.3 ± 0.01 0.9 ± 0.07 0.3 ± 0.05 0.38 ± 0.06 1.1 ± 0.34 0.36 ± 0.09 1.16 ± 0.18 0.36 ± 0.06 0.32 ± 0.06 0.97 ± 0.06 Table 5. Mean concentrations ± standard deviations of Ni, Mn, Co, Fe, Ca and Mg in three dominant species collected on the experimental site of Pojskë in 2006 Species Ni Co Fe Ca Mg Mn Mg/Ca mg kg-1 Lolium perenne 255 0.54 515 2314 4409 20 1.9 Aegilops geniculata Dasypyrum villosum Poa trivialis Centaurea solstitialis Minuartia hybrida Lotus corniculatus Consolida regalis Plantago lanceolata Petrorhagia prolifera Tropogon pratesis Bromus racemosus Vicia villosa 6.2 15 229 643 670 404 31.8 69.4 208 25.6 60.4 140 0.09 0.28 0.31 1.43 1.57 0.70 0.49 0.35 1.60 4.30 5.80 0.96 225 172 1105 1590 543 1075 142 408 1314 525 271 534 1200 1115 2268 5505 5298 4667 4627 3457 4895 2755 2148 5850 2212 2640 2872 6724 8703 7780 10969 6973 8584 7636 2794 7565 16 20 19 22 29 25 15 8 17 7 20 12 1.8 2.6 1.3 1.2 1.6 1.6 2.4 2.0 1.7 2.7 1.3 1.3 Table 6. Concentrations of Ni, Co, Fe, Mn, Ca and Mg in harvested plant parts of species collected on experimental site of Pojskë Aktet e Takimit Vjetor, Vëll. II, Nr. 2 25 A. BANI, T. TOPI, A. MAÇI, G. ECHEVARRIA, A. KALAJNXHIU, S. SULÇE, J.L. MOREL soil were mostly sources of highly available Ni. The experiment in serpentine vegetation showed that fertilization could enhance total plant cover and thus community production with slight changes in species richness and composition. Although these communities are mostly composed of different species, only hyperaccumulator plants are able to maintain a higher abundance under fertilization, whilst other species show a decrease of abundance. Fertilization, harvest and herbicide treatments affect plant community structure and productivity. Alyssum murale has a great phytoextraction potential in situations where native vegetation stand is enhanced by simple low-cost agronomic interventions. Fertilization treatment not only increased by more than 10 fold the biomass of A. murale production, but it also slightly increased the concentration of Ni in the harvested plant parts. 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(1997) Benchmark Soils of Albania: Resource Assessment for Sustainable Land Use. PhD thesis. Published by the USDA Natural Resources Conservation Service (NRCS), Washington DC and the International Fertilizer Development Center (IFDC), Muscle Shoals, Alabama. 2 Volumes. 293 pp. 26 www.alb-shkenca.org