Enhancement of metal(loid)s phytoextraction by Cannabis sativa L.

Transcription

1 WFL Pulisher Science nd Technology Meri-Rstilntie 3 B, FI Helsinki, Finlnd e-mil: info@world-food.net Journl of Food, Agriculture & Environment Vol.10 (1): Enhncement of metl(loid)s phytoextrction y Cnnis stiv L. Šárk Petrová, Dgmr Benešová, Petr Soudek nd Tomáš Vnĕk * Lortory of Plnt Biotechnologies, Joint Lortory of Institute of Experimentl Botny AS CR nd Crop Reserch Institute, Rozvojová 263, Prgue 6, Czech Repulic. *e-mil: vnek@ue.cs.cz Received 6 Septemer 2011, ccepted 10 Jnury Astrct The key fctor for the phytoextrction efficiency is iovilility of extrcted element. Moility nd iovilility of metl(loid)s is ffected y numerous soil fctors. To improve the metl(loid)s ccumultion cpcities, the ddition of chelting gents hs een proposed. The ojectives of this reserch ws to investigte the ility of different chelting gents (EDTA, humic sustnces) nd glutthione to enhnce the metl(loid)s phytoextrction y four Cnnis stiv L. cultivrs. Our results showed tht metl(loid)s ccumultion in plnts incresed with incresing concentrtion of metl(loid)s in growing solution; lthough, the metl distriution in plnt prts ws vrious. Generlly, ll metl(loid)s were ccumulted minly in roots except rsenic tht ws detected primrily in shoots. However, our results showed tht metl(loid)s ccumultion depended on chosen cultivr nd there ws no existing strtegy for metl detoxifiction in C.stiv. Tested cheltes enhnced the trnsfer from roots to shoots. Assuming tht EDTA hd positive effect on the metl(loid)s moility, lrger mount of metl(loid)s is tken up nd trnslocted to the shoots, while n effect of humic sustnces wsn t sttisticlly proved. Nevertheless, glutthione ppliction incresed metl(loid)s ccumultion in roots. C.stiv plnts demonstrted to possess the ility to trnsfer rsenic, mium, copper nd zinc from root to shoot, one of the criteri tht must e met to consider plnt well suited for phytoextrction. Key words: Phytoextrction, chelte, glutthione, Cnnis stiv L., metl. Introduction Low contminted sites ner the industril plnts re not suitle for food crops cultivtion 1. Contminnts ccumulted in food crop my cuse dnger to humn helth. Though, low contminted sites seem to e pplicle for other useful plnts. Nowdys, there is huge energy demnd; therefore, the growth of energy crops on polluted sites cn e fesile. Industril hemp is crop with rod rnge of pplictions. Its est known products re sed on hemp fiers. Generlly, the fier content in plnt is out 25% 2, the rest of the wood mteril is pressed into riquettes or it is used s therml insulting mterils 3. Due to high iomss production, hemp cn e used s n energy crop nd t the sme time it cn clen up the environment. Its growth on contminted soil cn reduce the pollution through trnsfer of the contminnt from soil to hrvestle plnt prts 4. Green technologies s phytoremedition hve recently ttrcted gret del of ttention s n lterntive mens of soil nd wter pollution 5-8. For exmple, phytoextrction technique uses n ility of plnts to uptke of toxic metl(loid)s from soil A potentil of different plnt species for phytoextrction ws rodly studied 12, 13. Phytoextrction y hemp ws studied for mium, led, zinc, copper, chromium nd nickel Fewer studies focused on fire qulity. Linger et l. 6 hve recently shown no effect of hevy metl contmintion on fire fineness nd strength. According to other uthors, hemp is suitle for growing in industrilly polluted regions, nd it cn e used for reclmtion of hevy metl contminted soils with further utiliztion of iomss in the industry 4, 17. Hemp is lso fesile for neroic sewge sludge mngement. It cn remove hevy metls from sludge nd t the sme time the ddition of sludge increses hemp iomss production 18. Plnts ccumulte from soil metl(loid)s tht hve either essentil or nonessentil function. Ech plnt species requires different element composition nd concentrtion. Generlly, plnts tke up mcronutrients s nitrte nd phosphte, essentil elements such s chromium, copper, iron, mngnese nd zinc 19, nonessentil elements such s mium, colt, mercury, led nd vndium 9, 16 nd rdioctive elements such s cesium, strontium nd urnium 20, 21. Lck of nutrients cn cuse serious crop production prolems 22. Such conditions led to synthesis of specific sustnces tht cn mke the lcking elements ioville. For instnce, roots of higher plnts cultivted in solution without nutrients could secrete proteses nd increse the level of free mino cids in the soil solution s source of nitrogen 23. However, phytotoxic mounts of oth essentil nd nonessentil metl(loid)s in plnts cn led to n inhiition of plnt growth 24, 25. Metl(loid)s ffect enzyme mechnisms y inding to sulphydryl groups in proteins or y displcing of the essentil metls in metlloproteins or metlprotein complexes 26. Some metls ffect chloroplst pprtus. For instnce, mium ffects chlorophyll synthesis, wter splitting, Clvin cycle enzymes nd regultion of energy distriution of PS Indeed, plnts response to the stress with incresing production of rective oxygen species (ROS) 28, 29. Low moleculr weight thiols, s glutthione (GSH), ply importnt role in defense mechnisms ginst ROS. GSH detoxifies xenoiotics y prior ctivtion nd conjugtion with such compounds 30, 31. Further, GSH cts s precursor for the synthesis of phytocheltins 32. The effectivity of phytoextrction is strongly dependent on the iomss production, metl(loid)s concentrtions in plnt tissues nd iovilility of extrcted element 33. The key fctor is Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury

2 iovilility. Moility nd iovilility of metl(loid)s is ffected y numerous soil fctors, such s ction exchnge cpcity, ph nd orgnic mtter content 34, 35. To improve the metl(loid)s ccumultion cpcities, nd the enhncement of metl(loid)s vilility in soil, the ddition of chelting gents hs een proposed. Recently, the synthetic chelting gents such s ethylenediminetetrcetic cid (EDTA) hve een used 36. Nowdys, the iodegrdle chelting gents such s ethylenedimine disuccinte (EDDS) hve een tested 14, 33. As n lterntive to ove mentioned cheltes nturl sources such s humic sustnces (HS) cn e used 37. They re composed from three min frctions: humic cid, fulvic cid nd humin 38. Solule complexes of HS nd metl ions reduce metl sorption onto soil surfce while increse the metl uptke into plnts 39. Humic cids (HA) were found more effective in enhncing metls uptke thn EDTA in sunflower nd corn 40. Furthermore, it ws stted tht HAs re idel soil mendments for phytoextrction enhncement ut more studies re needed to prove the potentil of HS to ecome effective phytoextrction enhncers 41. The ojectives of this reserch were to investigte the ility of different chelting gents (EDTA, humic sustnces) nd glutthione to enhnce the metl(loid)s phytoextrction y four Cnnis stiv L. cultivrs. Mterils nd Methods Plnt mteril: Cnnis stiv L. cv. Beniko, Bilorzskie, Firol nd Monoic, fire hemp cultivrs (Agritec, Ltd.), were cultivted in cultivtion room under controlled conditions (23 C, humidity out 60%, dily light phse of 16 hours) with supplementry light (irrdince of 115 µmol/m 2 s) in Hoglnd s solution 42. Four weeks old plnts were replced into solution with toxic metl(loid)s. The experiments were performed in triplictes. Firstly, n ility of phytoextrction of hemp cultivrs ws tested. Plnts were cultivted in the solution with ions of rsenic (NAsO 2 ), mium (Cd(NO 3 ) 2 4H 2 O), copper (Cu(NO 3 ) 2 3H 2 O), led (P(NO 3 ) 2 ) or zinc (Zn(NO 3 ) 2 6H 2 O) t metl(loid)s concentrtions 50, 100, 200, 500, 1000, 2000 nd 5000 µmol/l. Secondly, mendment effects on metl(loid)s ccumultion were studied. Plnts grew in modified solution. The modifiction ws relized s n mendment of 100 µmol/l of EDTA slt (C 10 H 14 N 2 N 2 O 8.2H 2 O), GSH (Sigm-Aldrich), or 100 mg/l of mixture of humic nd fulvic cids (Lignohumte AM - AMARGO s.r.o., Czech Repulic) to Hoglnd s solution. The solution lso contined 100 µmol/l of metl(loid). After one week, the plnts were hrvested. Roots were wshed susequently in doule distilled wter, in solution of EDTA (0.1 mol/l) nd doule distilled wter. Plnt leves, stems nd roots were seprted, frozen t -70 C in liquid nitrogen nd freeze-dried. Metl determintion: The dried plnt tissues were ground to powder nd digested in 5 ml of cid mixture of 65% HNO 3 nd 60% HClO 4 (v/v rtio 85/15) 43. Contents of mium, copper, led nd zinc were mesured y tomic sorption spectroscopy (SensAA, GBS, Austrli). Determintion of rsenic ws relized y mesurement t Optim 2000 DV ICP spectrometer (Perkin Elmer, USA). Metl(loid)s concentrtions were clculted s proportion of the metl(loid) mount to dry weight (DW) of the plnt prt. Dt nlysis: The sttisticl tretment included clcultion of men concentrtions of elements nd nlysis of vrinces to estimte sttisticlly significnt differences etween groups of smples. The significnce of differences ws determined using Student s t-test for α The differences mong mendment tretments of prticulr cultivrs were tested y one-wy ANOVA with Tukey s HSD multiple comprison test. Significnce level P ws 0.05 for oth nlyses. Ech tretment ws represented y three iologicl replictes. STATISTICA 8 (SttSoft, Tuls, OK, USA) softwre ws used for ll the computtions. Results nd Discussion Phytoextrction of metl(loid)s: Our results showed tht metl(loid)s ccumultion in plnts incresed with incresing concentrtion in the growing solution, lthough the metl(loid)s distriution in plnt prts ws vrious. Tested metls (mium, copper, nd zinc) were ccumulted primrily in roots while rsenic occurred mostly in shoots. Moreover, ccumultion trends of mium, copper, nd zinc were very similr to ech other. Higher concentrtions of metl(loid)s in solution incresed their trnsfer to the shoots. Metl sorption nd the restriction of trnsloction to the shoots my e the voidnce of toxic effect of the metl on the roots 44. Arsenic: When cultivted with the ddition of rsenite, plnts in 5000 µmol/l solution egn to wilt next dy in 2000 µmol/l, third dy in 1000 µmol/l, nd sixth dy in other concentrtions (Tle 1). Arsenite ions reduced reltive growth rte nd developed severe rowning which progressed to necrosis. At low concentrtions (up to 200 µmol/l) rsenic ws ccumulted mostly in stems of tested cultivrs, nd t higher concentrtions in leves (Tle 2). At 5000 µmol/l, the highest rsenic concentrtion ws detected in the leves of Beniko cultivr (18 mg/g DW) nd the lowest in the stems of Firol nd Monoic cultivrs (oth, 3 mg/g DW). In the cultivr Bilorzskie the stems nd the leves rsenic contents were comprle, nd moreover t the highest concentrtion the metlloid vlue in the stems exceeded its vlue in the leves (15 nd 13 mg/g DW, respectively). Conversely, it ws reported tht rsenic is stored primrily in roots 45, 46. However, different rsenic trnsloction in plnt prts depends on their sensitivity or resistnce to rsenic. The ccumultion of rsenic in hyperccumultor P. vittt ws much lower in the roots thn in the fronds, while the opposite ws true in non-hyperccumultor P. ensiformis 47. It ws demonstrted tht tomto plnts stored rsenic in roots, while rsenic in en plnts ws redily trnsported into the shoots nd ccumulted in high levels in the leves 48. Limiting of rsenic trnsfer to shoots helped tomto plnts ginst rsenic phytotoxicity, so they were more tolernt to rsenic thn en plnts. Phytotoxicity of rsenite is in greement with pulished dt 25, 49. It is known tht rsenite is moile nd it is tken up into the plnt y P-independent mechnism 50. Once inside the plnt, rsenite ound to thiol groups nd interferes with enzymes mechnisms 45. Its rection with sulphydryl groups of proteins cuses disruption of root functions, nd even cellulr deth 51. Trnsport to the shoots might e supported y complex of As-thiol. It ws demonstrted the formtion of As (III)-S complexes in the roots of Prosopis, which were freely trnsported into the shoots 52. Arsenic uptke nd distriution is lso strongly dependent on the type of rsenic. 632 Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury 2012

3 According to Cronell et l. 49, the ility to uptke of rsenic into the roots of Sprtin lterniflor grew in order: dimethylrsenic cid (DMAA) < monomethylrsenic cid (MMAA) <As (V) <As (III). Inorgnic rsenic nd MMAA were ccumulted minly in the root system, while DMAA ws redily trnslocted to the shoots. Cdmium: The ddition of mium led to grdul wilting of plnts (Tle 1). Third dy plnts strted to wilt in 5000 nd 2000 µmol/l mium solution, fourth dy in 1000 µmol/l nd sixth dy in 500 nd 200 µmol/l. Roots ecme rownish, less rnching nd reduced growth. Toxicity symptoms s stunting nd chlorosis lso ppered. Cdmium toxicity might cuse trnsloction to the shoots s the voidnce of toxic effect of the metl on the roots. At the highest concentrtion (5000 µmol/l), the mount of the metl in the stems gretly rised up to the root levels (Tle 3). Furthermore, mium content in the stem of Bilorzskie cultivr ws twice more higher thn in the root. The plnts from the solutions with other concentrtions ccumulted mium chiefly in roots. The mount in the roots vried etween 22 mg/g DW (Monoic) nd 30 mg/g DW (Beniko, Bilorzskie, Firol). It corresponds to the literture. It ws found tht cnnis plnts ccumulted 4 g/g in root nd 0.1 g/g in shoots t the mium concentrtion of 200 mg/kg in the cultivtion medium 53. Energy plnts ccumulted mium more in root, less in shoots. These results re consistent with the generl ssumption tht the metls deposited especilly in roots 46, which is proly prt of the defense mechnism of plnts ginst to toxic sustnces. It ws found tht roots of lettuce relesed much more of their sored mium for trnsloction to the roots thn ryegrss or orchrdgrss 54. The effect of the incresed mium ccumultion in shoot ws confirmed in Ipomoe stem 55. Cdmium trnsloction to the shoots my e the voidnce of toxic effect of the metl on the roots 44. Cdmium detrimentl effect on photosynthetic ctivity, chlorophyll content, nd plnt growth ws lso proved 27, 56. Metl induced oxidtive stress tht led to protein degrdtion through mino cid metolism resulting in decrese of plnt growth 16. Copper: Copper is n essentil element nd thus plnts were more tolernt to its presence in solution. Tested concentrtions of copper did not cuse evident toxic symptoms (Tle 1). It is known tht copper plys roles in photosynthesis, respirtion, ntioxidnt ctivity, cell wll metolism nd hormone perception 57. However, it ws reported tht copper concentrtion in noncontminted soils is out µg/g ut in contminted soils cn rech levels one hundred times higher 58. Our tested concentrtions were higher thn nturl ckground nd copper ws concentrted minly in the roots (Tle 4). On the other hnd, the mount of copper in the roots ws independent on the mount of copper in solution. It ws evident tht ll four cultivrs reched similr copper level in the roots (out 15 mg/g DW) nd even the higher concentrtion in solution did not increse it. Moreover, t the highest concentrtion (5000 µmol/l) the mount of the metl in the stems gretly rised up (from 15 mg/g DW for Bilorzskie to 30 mg/g DW for Firol nd Monoic) so copper content in the stems ws igger thn in the roots. It ws demonstrted tht toxicity symptoms induced y copper (1500 µmol/l) were linked to shrp rise of copper content in lef of Hordeum vulgre, ccompnied y oxidtive stress 59. However, ny dt regrding copper toxicity on C.stiv hve not een pulished. Hemp plnts seemed to e more resistnt compring to rley. Our results corresponded with the ccumultion nd distriution of copper in Elsholtzi splendens 60, which is known for its tolernce to high concentrtions of this metl. The mount of copper in plnt prts lso decresed in the line: root > stem > lef. At 500 µmol/l copper content in roots ws out 8 mg/g, 1 mg/g in stems nd 0.25 mg/g in leves. The mounts of copper in plnt prts of E. splendens greed with our results (9-12 mg/g DW in roots, mg/g DW in stems, nd mg/g DW in leves). Plnt species hve different tolernce strtegies tht protected themselves ginst copper toxicity. It ws descried y reserchers tht compred plnts nturlly growing on contminted site 61. In Mlv sylvestris, exclusion of copper from the roots or its stiliztion in the soil restricted its toxicity effects. Chenopodium mrosioides ccumulted copper in roots nd then in leves nd in Dtur strmonium most of copper ws ccumulted in leves. Moreover, D. strmonium nd C. mrosioides elevted their ntioxidtive enzyme ctivities in response to copper toxicity. The protection strtegy in C. stiv seems to e similr to C. mrosioides. Due to esily inding of copper to the sulfhydryl groups of memrne proteins, dmge of the proteins cn e provoked 62. A model for effect of copper on C. stiv roots ws lso proposed 63. Aldo/keto reductse is the first protein intercting with copper ions, it could reduce copper to Cu(I), so ions could e ville for interction with other prtner proteins, like phytocheltins, which usully ind Cu(I), nd from this loction copper cn e trnsported to the vcuole. Led: Toxic effect of led ppered third dy (Tle 1). Plnts wilted grdully in solution concentrtion of ove 1000 µmol/l. Roots ecme rownish nd reduced growth. Sixth dy, wilting of plnts ws visile lso t lower concentrtions (200 nd 500 µmol/ l). Wilting of plnts growing in solution with higher concentrtions of led ws cused y n increse of led mount in the stems (Tle 5). Led concentrtions in the leves vried from 7 to 24 mg/g DW, nd t lower concentrtions were comprle to the control plnts (from 3 to 14 mg/g DW). Generlly, led ws ccumulted primrily in the roots, nd then in the stems. At 5000 µmol/l, the highest led concentrtion ws detected in the roots of Firol nd Monoic cultivrs (oth 60 mg/g DW) nd the lowest one in Beniko cultivr (30 mg/g DW), Our results re in greement with literture 4. Linum usittissimum nd C.stiv plnts growing t n industrilly polluted region ccumulted led minly in roots nd less in stem nd in leves. Similrly, the ccumultion of led y Elsholtzi sp. lso decresed in line root> stem> lef 64. For exmple, t 200 µmol/l concentrtion plnts reched led concentrtion of 20 mg/g in roots, 2 mg/g in stems nd 0.15 mg/ g in leves. In our experiment we detected in roots 12 30, in stems nd in leves mg/g DW of led. It is evident tht C. stiv plnts trnsported less metl in the shoots, while plnts of Elsholtzi sp. trnsported more led in the shoots which corresponded with their known high tolernce to hevy metls. It ws reported tht once sored y the roots, led is rther immoile, showing very limited trnsloction into shoots 65, 66. According to literture, led retention in roots is sed on the inding of led to ion exchnge sites on the root cell wlls nd extrcellulr precipittion, minly in the form of P crontes 67. Led tretment of 300 µmol/l reduced root elongtion of Triticum Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury

4 Tle 1. Wilting of plnts during 7 dys exposure to rsenic, mium, copper, led, nd zinc t 0, 50, 100, 200, 500, 1000, 2000, or 5000 [µmol/l] concentrtion (+ mens no visile toxic symptoms, - mens visile toxic symptoms). Solution [µmol/l] As Cd Cu P Zn dy Tle 2. Arsenic concentrtion [µg/g] in root, shoot, nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 50, 100, 200, 500, 1000, 2000, or 5000 [µmol/l] rsenic concentrtion; control men Hoglnd solution (rsenic concentrtion under limit of quntifiction); DW men dry weight; stndrd devition is represented s ± S.D. (n=3). Beniko Bilorzskie Firol Monoic Solution Root Stem Lef Root Stem Lef Root Stem Lef Root Stem Lef [µmol/l] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] control 5.22 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1410 Tle 3. Cdmium concentrtion [µg/g] in root, shoot, nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 50, 100, 200, 500, 1000, 2000, or 5000 [µmol/l] concentrtion; control men Hoglnd solution (mium concentrtion under limit of quntifiction); DW men dry weight; stndrd devition is represented s ± S.D. (n = 3). Beniko Bilorzskie Firol Monoic Solution Root Stem Lef Root Stem Lef Root Stem Lef Root Stem Lef [µmol/l] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] control 4.00 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury 2012

5 estivum L. more thn three times nd significntly incresed ntioxidtive enzymes ctivities 68. It ws shown tht 500 µmol/l led concentrtion is modertely toxic nd 1000 µmol/l highly toxic to Oryz stiv L. plnts 69. On the other hnd, C.stiv plnts seemed to e more tolernt since the plnt survived even t 5000 µmol/l concentrtion. Zinc: Compring to mium nd led, zinc did not led to perishing of C. stiv plnts (Tle 1). Zinc content in plnt prts incresed with the increse of zinc exposure (Tle 6). The ccumultion trend in plnt prts decresed in the line: root > stem > lef, nd the trend ws equl to mium, copper nd led ccumultion. Plnts of Beniko nd Bilorzskie cultivted in 500 nd 1000 µmol/l solution showed shrp increse of zinc mount in the roots. At the highest zinc concentrtion (5000 µmol/l) the mount of zinc in the roots vried from 25 mg/g DW (Monoic) to 45 mg/g DW (Firol). Surprisingly, higher concentrtions of zinc (500, 1000 µmol/l) did not led to rise of zinc ccumultion in the roots ut led to steep increse of zinc mount in the stems. Zinc content in the stems gretly rose up to the root levels in Beniko nd Bilorzskie cultivrs nd zinc content in the stem t 5000 µmol/l ws 37nd 27 mg/g DW, respectively, nd in the roots 30 mg/g DW, ech. It ws proved tht zinc trnsloction grew with its mount in the soil, nd t higher concentrtion of zinc greter mount ws ccumulted in C.stiv plnts 18. Zinc content in plnt prts of C. stiv declined in the following sequence: root > lef > stem. Hydroponiclly growing plnts of Brssic npus nd Trifolium repens trnslocted most of the zinc in the leves nd less into the stems, wheres Agrostis stolonifer distriuted the metl eqully 70. All results confirmed tht excess of zinc ws trnsferred from the root into the shoot while zinc distriution in the shoot depended on plnt species. Zinc s n essentil element plys n importnt role in plnt metolism y n involvement in the ctivtion of mny enzymes nd supply of link etween enzyme nd sustrte 71. However, excess of zinc rises specific physiologicl nd morphologicl chnges such s n inhiition of photosynthesis, root system reduction, eril prt dwrfism, chlorosis formtion or disruption of mitochondril structures 72. Resistnt plnts such s Dtur innoxi didn t show ny visile injury t 5000 µmol/l ut high mount of zinc decresed the photosynthesis nd stomtl conductnce 73. Other plnts were more sensitive. Growth inhiition nd decrese of chlorophyll content in leves of S. lycopersicum ws oserved t concentrtion of 150 µmol/l 74. Moreover, scorte oxidtion occurred in the leves of Phseolus vulgris L. treted y 50 µmol/l 75. Plnts of C.stiv seemed to elong to resistnt plnts since the highest tested concentrtion (5000 µmol/l) did not show ny visile toxic symptoms. Amendments effect: Amendments of EDTA, GSH or humic cids mixture in solutions hd no significnt effect on plnts growth. In the presence of rsenic, mium nd led ions plnts wilted s ws mentioned previously. On the other hnd, our results demonstrted tht the solution enhncement hd n influence on metl(loid)s ccumultion nd distriution in plnts. EDTA incresed the trnsfer from roots to shoots, while GSH incresed metl(loid)s ccumultion in roots. Cheltes: The presence of EDTA in the solution hd no effect on rsenic uptke (Fig.1) ut it hd n effect on uptke of other metls (Figs 2-5). It significntly reduced the mount of ccumulted mium, copper nd zinc in the roots nd the stems, nd t the sme time it incresed metls trnsport into leves (Figs 2, 3 nd 5). Indeed, the metl contents in the roots were pproximtely twice (mium), three times (copper) or four times lower (zinc), wheres the metl contents in the leves were pproximtely twice (zinc) or six times (copper) higher. The increse of mium content in the leves ws strongly dependent on the cultivr nd vried from 0.2 to 0.7 mg/g DW, wheres its content in plnts grown in solution without EDTA vried from to 0.17 mg/g DW. Moreover, significnt increse of led ws oserved in the leves (Fig.4), the content ws forty times higher compring to the plnts treted y solution without EDTA. On the other hnd, humic sustnces (HS) hd only merely effect on metl(loid)s ccumultion. The most pronounced effect ws oserved in roots nd stems (Fig.1). For exmple, significnt increse (twice more) ws oserved in the ccumultion of led in the roots nd stems of Bilorzskie cultivr. Moreover, in the roots of the cultivr n increse of rsenic mount ws detected. In contrst, decrese in rsenic nd zinc ccumultion ws mesured in the stems of Bilorzskie nd Monoic cultivrs. It ws stted tht EDTA not only incresed the soluility nd hence iologicl vilility of metls in the soil 76, 77, ut it lso prticipted in the trnsport of metls in plnt prts 35, 76, 78. The effect of EDTA on led ccumultion in the shoots of Helinthus nnuus ws demonstrted. EDTA tretment incresed led content twelve times, nd significntly igger mount of led ws llocted in the shoots 79. The effect on other metls distriution ws lso reported. The ddition of EDTA incresed shoot concentrtion of mium, led nd zinc in Helinthus nnuus, Cnnis stiv nd Brssic rp 12. It ws reported tht led in the roots of Ze mys with EDTA ddition ws mostly distriuted in the poplst, while zinc ws mostly locted in the symplst; therefore, the cpcity of EDTA to enhnce the nonselective poplstic trnsport of metl my e most importnt for cheltes enhnced phytoextrction 80. According to literture, effectiveness of EDTA depends on its rte, contmintion level of led s well s complementry metls present in soils nd method of its ppliction 81. Although EDTA hs een shown to e effective, its toxic effect on soil microorgnisms ws proved 36. Humic sustnces (HS) were used s n lterntive to EDTA, HS hd more dvntges thn disdvntges. It ws reported tht HS mitigted dmging effects of rdition, pesticides nd excess of minerl fertilizers 82. Severl studies showed tht HS pplied into the soil incresed metl trnsfer into the shoot. Direct HS ddition significntly enhnced mium uptke y Nicotin tcum 37. The ddition of HS significntly incresed the copper content in roots, nd shoots of Elode nuttllii, wheres the presence of HS in the soil hd exctly opposite effect on mium content in the roots 83. Moreover, it ws proved tht irrigtion with wter contining HS incresed metls vilility tht led to incresed led nd mium ccumultion in Triticum estivum 39. Those studies indicted tht the enhncement effect is strongly dependent not only on plnt species, ut lso on the concentrtion of HS. However, grdul ppliction of smll doses of cheltes cn considerly reduce the toxicity nd environmentl prolems ssocited with its utiliztion 84. Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury

6 Tle 4. Copper concentrtion[µg/g] in root, shoot, nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 50, 100, 200, 500, 1000, 2000, or 5000 [µmol/l] concentrtion; control men Hoglnd solution (copper concentrtion 3.3 µmol/l); DW men dry weight; stndrd devition is represented s ± S.D. (n = 3). Beniko Bilorzskie Firol Monoic Solution Root Stem Lef Root Stem Lef Root Stem Lef Root Stem Lef [µmol/l] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] control 534 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 66.1 Tle 5. Led concentrtion[µg/g] in root, shoot, nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 50, 100, 200, 500, 1000, 2000, or 5000 [µmol/l] concentrtion; control men Hoglnd solution (led concentrtion under limit of quntifiction); DW men dry weight; stndrd devition is represented s ± S.D. (n = 3). Beniko Bilorzskie Firol Monoic Solution Root Stem Lef Root Stem Lef Root Stem Lef Root Stem Lef [µmol/l] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] control 74.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 8.02 Tle 6. Zinc concentrtion[µg/g] in root, shoot, nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 50, 100, 200, 500, 1000, 2000, or 5000 [µmol/l] concentrtion; control men Hoglnd solution (zinc concentrtion 2.3 µmol/l); DW men dry weight; stndrd devition is represented s ± S.D. (n = 3). Beniko Bilorzskie Firol Monoic Solution Root Stem Lef Root Stem Lef Root Stem Lef Root Stem Lef [µmol/l] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] [µg/g DW] control 435 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury 2012

7 As concentrtion [µg/g] c d c c c c EDTA +As GSH + As HS + As control As 0 root stem lef root stem lef root stem lef root stem lef Beniko Bilorzskie Firol Monoic Figure 1. Arsenic concentrtion [µg/g] in stem nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 100 [µmol/l] of metl(loid) concentrtion with different dditions (EDTA, GSH, or HS). Control mens solution without metl(loid) nd without mendments; stndrd devition is represented s ± S.D. (n = 3). The differences mong mendments tretments of the cultivrs were tested y one-wy ANOVA with Tukey s HSD multiple comprison test (significnce level P=0.05). Cd concentrtion [µg/g] e c c c c de e e e c e e d c EDTA +Cd GSH + Cd HS + Cd control Cd root stem lef root stem lef root stem lef root stem lef Beniko Bilorzskie Firol Monoic Figure 2. Cdmium concentrtion [µg/g] in stem nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 100 [µmol/l] of metl(loid) concentrtion with different dditions (EDTA, GSH, or HS). Control mens solution without metl(loid) nd without mendments; stndrd devition is represented s ± S.D. (n = 3). The differences mong mendments tretments of the cultivrs were tested y one-wy ANOVA with Tukey s HSD multiple comprison test (significnce level P = 0.05). Cu concentrtion [µg/g] c ef ef c def ef ef d c ef ef ef d e ef f ef d c c c EDTA + Cu GSH + Cu HS + Cu control Cu root stem lef root stem lef root stem lef root stem lef Beniko Bilorzskie Firol Monoic Figure 3. Copper concentrtion [µg/g] in stem nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 100 [µmol/l] of metl(loid) concentrtion with different dditions (EDTA, GSH, or HS). Control mens solution without metl(loid) nd without mendments; stndrd devition is represented s ± S.D. (n = 3). The differences mong mendments tretments of the cultivrs were tested y one-wy ANOVA with Tukey s HSD multiple comprison test (significnce level P = 0.05). Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury

8 P concentrtion [µg/g] Zn concentrtion [µg/g] ef ef c g efg e e c c e c e ef de efg ef g efg e ef c f c efg ef defg c c e c e e efg ef def e ef def fg defg efg e g ef fg root stem lef root stem lef root stem lef root stem lef Beniko Bilorzskie Firol Monoic EDTA +Zn GSH + Zn HS + Zn control Zn Figure 5. Zinc concentrtion [µg/g] in stem nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 100 [µmol/l] of metl(loid) concentrtion with different dditions (EDTA, GSH, or HS). Control mens solution without metl(loid) nd without mendments; stndrd devition is represented s ± S.D. (n = 3). The differences mong mendments tretments of the cultivrs were tested y one-wy ANOVA with Tukey s HSD multiple comprison test (significnce level P = 0.05). efg def e c e c f EDTA +P GSH + P HS + P control P root stem lef root stem lef root stem lef root stem lef Beniko Bilorzskie Firol Monoic Figure 4. Led concentrtion [µg/g] in stem nd lef of four Cnnis stiv L. cultivrs fter 7 dys of cultivtion in solution with 100 [µmol/l] of metl(loid) concentrtion with different dditions (EDTA, GSH, or HS). Control mens solution without metl(loid) nd without mendments; stndrd devition is represented s ± S.D. (n = 3). The differences mong mendments tretments of the cultivrs were tested y one-wy ANOVA with Tukey s HSD multiple comprison test (significnce level P = 0.05). Glutthione: The ddition of GSH hd n impct on uptke of ll metl(loid)s (Figs 1-5). It incresed mium nd zinc mounts in the roots of ll cultivrs (Figs 2 nd 5). Besides, n increse of led ccumultion in the roots of three cultivrs (Beniko, Bilorzskie nd Monoic) ws detected (Fig. 4). Moreover, enhnced rsenic uptke ws oserved in the roots of cultivrs Beniko, Bilorzskie nd Firol (Fig. 1). The trnsfer of metl(loid)s to the shoots ws not so ovious. An increse of rsenic in the leves (Beniko, Bilorzskie) nd copper in the stems (Bilorzskie) ws identified (Figs 1 nd 3). Incresed rsenic trnsfer corresponded with literture. It ws demonstrted tht low level of GSH (0.4 mmol/l) incresed rsenic uptke y Pteris vittt plnts, while higher concentrtion (0.8 mmol/l) hd no effect on the uptke 85. Nevertheless, the ddition of GSH helped rsenic trnsport from the root to the shoot of P. vittt (trnsfer fctor rose twice). It hs een presented tht glutthione is involved in the rections forming phytocheltins 32. Phytocheltins induced on exposure to rsenic formed complex with rsenite ions 45, 86. High mium levels in Brssic junce were lso ssocited with rpid ccumultion of phytocheltins in roots 87. The increse in glutthione synthetse ctivity ws dependent on the initil mium concentrtion. It ws found tht the ccumultion dynmic of mium or led ound with GSH ws chnged in Ze mys nd Brssic npus roots 88. Oserved inhiition of led uptke in the presence of incresing GSH concentrtion nd the pprent up-regultion of led uptke following pre-exposure to GSH were consistent with trnsport vi peptide trnsporter, tht did not differentited etween GSH nd the metl-gsh complex. Therefore, higher metl(loid)s contents in C.stiv might e cused y GSH involvement in production of phytocheltins. Conclusions Our results showed tht metl(loid)s ccumultion significntly vried with chosen cultivr. Generlly, ll metl(loid)s were ccumulted minly in roots except rsenic. Arsenic ccumultion trend ws similr to rsenic hyperccumultor P. vittt. Other metls showed different tendency. Cdmium content in the shoots incresed with incresing concentrtion of mium in solution. Copper ws ccumulted differently. It reched similr level in the roots of cultivrs nd even the higher concentrtion in solution 638 Journl of Food, Agriculture & Environment, Vol.10 (1), Jnury 2012

9 did not increse it. Moreover, t the highest concentrtion the mount of the metl in the stems gretly rised up to the roots levels. Zinc ccumultion trend ws comprle to copper. Higher concentrtions of zinc did not led to rise of zinc ccumultion in the roots ut led to steep increse of zinc mount in the stems. Its trnsloction to the shoots might e the voidnce of toxic effect of the metl on the roots. In contrst, led ws rther immoile, showing very limited trnsloction into the shoots. From the distriution of metls we cn suggest tht proly there is no existing strtegy for metl detoxifiction in C.stiv. Nevertheless, hemp demonstrted to possess the ility to trnsfer rsenic, mium, copper nd zinc from root to shoot, one of the criteri tht must e met to consider plnt well suited for phytoextrction. However, studies t rel contminted sites could give more informtion out the phytoextrction process. The effect of the mendments on the ccumultion of metl(loid)s vried depending on the cultivr nd the element. The cheltes enhnced the nonselective poplstic trnsport of metl(loid)s. Our results showed tht EDTA hd positive effect on the metls moility, lrger mount of metls ws tken up nd trnslocted into the shoots, while n effect of humic sustnces wsn t sttisticlly significnt. On the other hnd, the ddition of GSH incresed metl(loid)s mounts in the roots of plnts. Higher metl(loid)s contents in C.stiv my e cused y GSH involvement in production of phytocheltins, ut further studies re needed to explin the mechnisms. Acknowledgements This work ws supported y project NPVII 2B References 1 McLughlin, M. J., Prker, D. R. nd Clrke, J. M Metls nd micronutrients food sfety issues. Field Crops Res. 60(1-2): Snkri, H. S Comprison of st fire yield nd mechnicl fire properties of hemp (Cnnis stiv L.) cultivrs. Ind. Crop Prod. 11(1): Kymäläinen, H. R. nd Sjöerg, A. 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