Biosolids, mycorrhizal fungi and Eucalypts for phytostabilization

Transcription

1 Biosolids, myorrhizl fungi nd Eulypts for phytostiliztion purposes of sulphidi mine tilings E. Mdejón* 1, A. I. Doronil 2, P. Mdejón 1, A. J. M. Bker 2, I. E. Woodrow 2 1 Instituto de Reursos Nturles y Agroiologí de Sevill (IRNAS-CSIC). Av. Rein Meredes 10, Sevill, Spin. 2 Shool of Botny, The University of Melourne, Prkville, VIC 3010, Austrli *Corresponding uthor: emdejon@irnse.si.es. Tel , FAX

2 Astrt In mine tilings filities, phytostilistion is n ttrtive tehnology for long-term remedition. We tested the effet of the ddition of iosolids omined with ntive rusulr myorrhizl inoulum (AMF) on growth of eulypt (E. ldolyx), nd on tre element stiliztion of sulphidi gold mine tilings. A glsshouse tril ws estlished using four sustrtes: tilings (T); tilings with lyer of 5 m topsoil (TS); tilings mended with 100 dry t h -1 iosolids (LB), nd tilings mended with 500 dry t h -1 iosolids (HB). Hlf pots were inoulted with mixture of Glomus sp. (WUM ), Sutelspor uriglo (WUM51-53) nd Aulospor levis (WUM46) ulture mix, nd others were uninoulted ontrols. Two seeds per pot were sown in the pots nd fter 30 dys ll pots were uniformly thinned to 1 plnt/pot. Plnts were destrutively hrvested t 150 dys fter the sowing. Lef nd stem weights, lef re nd plnt height were mesured for eh plnt. Nutrients nd tre element onentrtions in leves nd stems were lso determined. Addition of iosolids signifintly inresed myorrhizl oloniztion (oth etomyorrhizl (ECM) nd rusulr myorrhizl) in roots ompred with the other tretments. Biosolids lerly improved the estlishment nd growth of the eulypts. At low iosolid doses, myorrhizl inoulum inresed plnt iomss prodution nd the effetiveness of nutrient uptke. Tre element onentrtions in trees of the iosolid-mended pots were in generl high. It is therefore importnt to redue the uptke of toxi elements y plnts, whih n e done suessfully y djusting mendment ddition nd the use of myorrhizl inoultion, nd then monitoring tre element ontents of different tx growing in the ffeted re. Keywords: Biomss prodution, Tre elements, Glomus, Sutelspor, Aulospor. 2

3 Introdution Mny urrent gold mining opertions exploit very deep ore odies whih invrily genertes very lrge volumes of wste tilings whih re often finely rushed sulphidi minerls. Due to the urrent high vlue of the gold even low grde deposits ontining < 5 mg/kg re very profitle to mine nd proess. Wstes re often sline nd ontin toxi guest elements suh s dmium, led, mngnese, nd prtiulrly rseni nd invrily ynide, whih is used to soluilize the fine deseminted gold prtiles. Mny Gold Mines in Vitori (Austrli) re urrently mining deep ores whih ontin rsenopyrite in whih As hs een deteted in onentrtions of up to 4000 mg kg -1, 130 times the mximum llowle onentrtion for len soil (ANZEC/NHMRC 1992). Currently, numer of pprohes, suh s phytostilistion, re eing investigted s remedition mehnisms for low vlue, highly ontminted lnd suh s mine tilings (Cunninghm et l. 1995). For phytostilistion purposes, plnts must demonstrte suitility for growth on tilings; this wste mteril hs poor soil struture, miniml nutrient ontent nd is extremely sline. Root development provides greter stility within the sustrte, while ove ground iomss helps to prevent eolin dispersion of ontminnts (Cunninghm et l. 1995). Root development lso filittes dehydrtion of tilings nd n immoilise ontminnts through sorption (Mendez nd Mier 2008). In this sense the use of orgni mendment might improve the qulity of the tilings fvouring the estlishment of plnts. The ddition of orgni mtter into the surfe lyers of the tilings improves soil physil properties suh s wter retention pity, ulk density nd ertion (Entry et l., 1997). Moreover, the pplition of orgni mendments inreses soil nutrient nd soil iohemil sttus (Pérez de Mor et l., 2005). 3

4 However, in As rih tilings there is lso the onern tht this metlloid whih is similr to phosphorus, my e tken up y plnt roots nd use toxiity. Phytostilistion using plnts tht n exlude As onentrtions uptke in stems, leves nd tips will gretly redue the risk of food-hin trnsfer (Mendez nd Mier 2008). Furthermore, s long-term ontminted lnd mngement solution, its viility relies on understnding of As-plnt intertions over n extended period of time (Tordoff et l. 2000). There re urrently very few studies on the use of woody speies for phytostilistion of As rih soils hene the knowledge of As tolerne nd uptke in woody speies is sre. Mny woody speies exhiit rpid growth of deep root systems, providing inresed stility, thus emphsising the importne of investigting their use in ontminted lnd mngement (Frenh et l. 2006). Idelly, phytostilistion should utilise speies endemi to the region where the ontminted site our. The hoie of these endemi speies reduing inherent risks ssoited with introdued speies. This my lso help to minimise long-term fter re requirements. In Austrli, the predominnt tree speies re from the genus Eulyptus whih ontins pproximtely 600 speies, diversified ross wide rnge of hitts. Thus, Eulyptus is very ttrtive s potentil resoure for rehilittion given the wide rnge of environments ontminted with As (Smith et l. 2003). Severl Eulyptus speies, inluding E. ldolyx, E. melliodor, E. polyrte nd E. viridis, exhiit tolerne of hrsh environmentl onditions nd suitility for estlishment on gold mine-wstes (Doronil 2006). Eulyptus is eologilly importnt euse it is one of the reltively few gener tht n form symioti ssoition with oth rusulr (AMF) nd etomyorrhizl (ECM) fungi (Lodge, 2000). Currently, the possile role of myorrhizl fungi in the 4

5 metl tolerne of higher plnts is poorly understood. However, reports on erieous myorrhizs (Brdley et l. 1982) nd etomyorrhizs (Denny nd Wilkins 1987) indited the importne of the myoionts in improving metl tolerne of their host plnts y primrily umulting metls in the wlls of extrmtril hyphe nd extrhyphl slime; thus pssge of metls to the shoots is restrited. The im of this work is to improve remedition strtegies for As rih tilings using eulypts mended with iosolid stilized mteril nd inoulted with mixture of ntive myorrhizl inoulum. A ftoril experimentl design ws utilized to determine ny possile intertion etween myorrhizl tretment nd iosolid pplition. We hypothesized tht the ddition of iosolid ould improve hemil nd physil properties of the tilings inresing seedling nd tree development. Myorrhyze ould enhne the effetiveness of the iosolid in remedition. To test this we studied root oloniztion, iomss prodution nd nutrients nd tre element onentrtions in shoots. Mterils nd Methods Design of the experiment Eulyptus ldolyx ws seleted for the experiments sed on results of previous studies of phytostiliztion using different Eulyptus speies in As ontminted tilings (King et l., 2008). A glsshouse tril ws estlished whih employed four sustrtes: tilings (T), tilings nd superfiil lyer of 5 m of top soil without tillge (TS), tilings mixed with 100 dry t h -1 of iosolids (LB), nd tiling mixed with 500 dry t h -1 of iosolids (HB). Tilings were olleted from the 15 yer-old stokpiles t Stwell Gold Mine, Vitori in Otoer The topsoil ws olleted from the surrounding res of the mine. 5

6 Biosolids were olleted from stokpile t Melourne Wter s Western Tretment Plnt t Werriee, Vitori, Austrli. Sulphidi tilings in the different tretments were mesured for severl physiohemil properties. These re summrized in Tle 1. The sustrtes were pled in drined pots of 1.2 L pity, with 12 pots per sustrte, giving totl of 48 experimentl units. Six replites per tretment (24 in totl) were inoulted with ulture of Glomus sp. (WUM ), Sutelspor uriglo (WUM51-53) nd Aulospor levis (WUM46) supplied y the Deprtment of Soil Siene, University of Western Austrli. The 3 AMF speies were isolted from lteriti ly topsoil whih hd een removed prior to uxite mining in the Drling esrpment ner Perth, Western Austrli. The ultures were mintined using Trifolium suterrneum host in psteurized 1:1:1 orse snd, fine snd nd rushed pet moss potting mix. Nutrients were supplied using low phosphorus, slow relese fertilizer. Inoulum ws prepred y hopping up the roots of the host plnts then thoroughly mixing these with proportionl mounts of the ulture potting mix. Approximtely 30 g ws dded on to the surfe of eh pot with the AMF tretment then grss seeds were sown nd overed with lyer of sterilized orse snd. Two seeds per pot of E. ldolyx were sown in the pots nd fter 30 dys ll pots were uniformly thinned to 1 plnt/pot. The pots were rrnged in rndomized pttern within glsshouse nd rndomly rerrnged every two weeks to minimize the influene of mirolimti vrition. All pots were initilly wtered to mximum pity nd then wtered y individul drippers every seond dy for 2 minutes t flow rte of 4L/hour. Nitrogen ws supplied dding 60 mg of N s NO 3 NH 4 per pot every 20 dys. Plnt smpling nd nlysis Plnts were destrutively hrvested t 150 dys fter the sowing. Lef re nd plnt 6

7 height ws reorded for eh plnt. Fresh weight of eh hrvest (lef nd stem) ws determined. Plnt smples were wshed for 15 s with 0.1N HCl solution then for 10 s with distilled wter. Wshed smples were oven- dried t 70 ºC nd then the dried plnt mteril ws ground nd pssed through 500 µm stinless steel sieve prior to nlysis. Shoots were digested y wet oxidtion with onentrted HNO 3 under pressure in mirowve digester. Anlysis of nutrients (P, K, C, Mg, K, S, nd N) nd tre elements (As, Cd, Cu, P nd Zn) in the extrts ws performed y ICP-OES. The N ontent ws determined y Kjeldhl digestion. Susmples of fresh roots were tken nd stored in vils. Root frgments were lered nd stined ording to the method of Brundrett (1996). Root smples were wshed nd then dispensed into MCrtney ottles hlf-filled (25 ml) with 10% KOH solution. These were utolved t C for 30 min. Smples were poured into fine mesh striner nd rinsed under tp wter. Clered roots were soked in the stined solution (0.05% trypn lue stin in 1:1 80% lti id, glyerol solution). Stined root frgments were pled on slide nd then oserved under ompound mirosope to determine the perentge of myorrhyzl oloniztion long the length of root. Thirty root frgments from eh smple were mesured ording to the tehnique of Giovnnetti nd Mose (1990) in whih the smple ws mesured longside grtiule nd the degree of infetion estimted long 1 m length. The ECM root tips were ounted under disseting mirosope, wheres the perentges of root length olonized y AM fungl strutures were mesured using ompound mirosope (Brundrett et l. 1996). Coloniztion of roots y AM fungi ws determined y the presene of hyphe, rusules, or vesiles t n intersetion. Sustrte nlysis 7

8 Sustrte smples from eh tretment were tken fter the seond grss hrvest. For hemil nlysis, soil smples were ir-dried rushed, sieved (< 2 mm) nd ground (< 60 µm). Soil smples for miroiologil nlysis were sieved (< 2 mm) nd stored t 4ºC prior to nlysis. Dehydrogense tivity ws determined in 1 M Tris-HCl uffer (ph 7.5) y the method of Trevors (1984), using INT (2 (p-iodophenyl)-3-(p-nytrophenyl) 5-phenyl tetrzolium hloride) s the eletron eptor. The iodonitrotetrzolium formzn (INTF) produed ws mesured spetrophotometrilly t 490 nm. The ph of the sustrtes nd the iosolids ws mesured in 1:2.5 smple:h 2 O extrts fter shking for 1 hour. Totl hevy metls (HM) nd As onentrtions in the tilings (< 60 µm) nd in the iosolids were determined y ICP-OES (IRIS ADVANTAGE, Thermo Jrrel Ash Corportion, MA, US) fter qu regi digestion in mirowve oven (Mirowve Lortory Sttion Mileston ETHOS 900, Milestone s.r.l., Sorisole, Itly). Sttistil nlysis Comprison of the different tretment effets y nlysis of vrine (ANOVA) ws performed using the sttistil softwre SPSS 15. Approprite trnsformtions on the dt were mde prior to nlysis to redue the heterogeneity of the vrine. A orreltion mtrix etween sustrte hemil nd plnt-relted prmeters ws lulted. The signifine levels reported (p <0.01 nd p <0.05) re sed on Person oeffiients. Results Biomss prodution Sttistil nlysis of the min effets showed tht ddition of iosolids nd AMF signifintly improved the growth of the eulypt in terms of plnt height, plnt weight 8

9 nd lef re (Figure 1 nd Tle 2). This signifint inrese ws mesured in LB+ nd HB+ tretments. Inoultion with myorhize lso signifintly improved the growth of the eulypt s it n e shown y the differenes etween eh sustrte with nd without inoultion. However, intertion etween the two min ftors myorrhize nd sustrte use ws not signifint for ny of the iomss prmeter studied (Tle 2). Root oloniztion Tle 3 shows the perentge of oloniztion oserved in the roots. There were signifint differenes etween mended tretments (TS, LB nd HB) nd unmended tretments (T) in men oloniztion rtes of inoulted tretments. Exmintion of myorrhizl oloniztion of roots showed tht oloniztion rte for eh tretment rnged from 16.8 to 32.8% for ECM nd from 10.3 to 18.7% for AM (Tle 3). A low residul oloniztion (<2.5%) ws lso deteted in the no inoulted tretments. Plnt nutrient uptke Mronutrient onentrtions in leves nd in stems t hrvest re presented here (Figure 2). In the se of the T tretment, iomss prodution in the tretment without ny mendment (T) ws very low nd the quntity of plnt mteril ws not suffiient to perform hemil nlysis. Vlues for these tretments (T nd T+) were therefore exluded from the sttistil nlysis. In generl, men onentrtions of mronutrients (N, S, C, Mg, K nd P) were higher in the leves of plnts mended with iosolids (LB nd HB) in inoulted nd uninoulted soils (Figure 2) thn in leves of plnts grown in topsoil (TS). In most ses, signifint differenes were found for N, C, Mg, K nd P nd S uptke. We found the sme tendeny in stems lthough nutrients vlues in woody tissues were slightly lower thn those found in leves. The AMF inoultion did not hve signifint influene in nutrient uptke. Intertion etween the two min 9

10 ftors myorrhize nd sustrte ws not signifint for ny plnt nutrient in leves (Tle 2). Tre element uptke Tre element onentrtions in leves nd in stems t hrvest re presented in Figure 3. Arseni onentrtions in leves were low ( mg kg -1 ) in plnts grown in ll tretments, nd no signifint differenes etween tretments were found (Figure 3). Arseni onentrtions in stems were even lower nd in most ses these vlues were elow the detetion limit (0.5 mg kg -1 ). Men onentrtions of Cd vried etween 0.42 nd 4.82 mg kg -1 (Figure 3). Cd onentrtions were signifintly higher in iosolid (LB nd HB) tretments. Cd onentrtions in stems were slightly lower thn in leves. Similrly, Zn onentrtions were signifintly higher in leves ( mg kg -1 ) nd in stems ( mg kg -1 ) in plnts in iosolid tretments (Figure 3). Men onentrtions of Cu vried only etween 8.53 nd 14.5 mg kg -1 in leves nd etween 19.1 nd 24.6 mg kg -1 in stems (Figure 3) nd were very similr in ll tretments. Men vlues of P were lso very low oth in stem nd lef tissues nd were similr for ll tretments. Men vlues of Ni in leves were slightly higher in iosolids tretments wheres no differenes etween tretments were oserved in stems. AMF inoultion did not ffet plnt tre element onentrtions nd the intertion etween myorrhize nd sustrtes ws only signifint for Cd (Tle 2). The orreltion oeffiients etween the onentrtion of Cd, nd Zn in leves nd their vlues in the soil of the tree rooting volume re shown in Figure 4. In generl, lef onentrtions of these tre elements were highly orrelted (higher orreltion oeffiients) with the metl onentrtions in the tilings. The strongest orreltion ws otined for Cd, (r=0.84, p<0.01, Fig. 6). Zn onentrtions in eulyptus leves were 10

11 lso signifintly orrelted with Zn ontent in tilings (r=0.68, p<0.001, Fig. 4). For the rest of the elements nlysed, signifint orreltion ws not deteted (dt no shown). Sustrte nlysis. Anlysis of the sustrtes t the end of the experiment showed tht the ddition of iosolids or top soil did not ffet the ph or the EC vlues of the tilings (Tle 4). Similrly, AMF inoultion did not ffet ph or EC vlues in the sustrtes. In ll tretments eulypt roots idify soil ph in 1 unit. Dehydrogense tivity ws higher in tilings mended with iosolids. In generl, dehydrogense tivity ws higher for inoulted tilings nd in some ses these differenes were signifint. In ft the two ftor nlysis showed tht the myorrhizl inoulums hd signifint effet on dehydrogense tivity (Tle 5). Tre element onentrtions were relted to the nture of the sustrte used. TS tretment present the lowest onentrtion for ll tre elements studied. AMF did not hve ny ler influene on tre element ontents (Tles 4 nd 5). Intertion etween the two min ftors, myorrhize nd sustrtes, ws not signifint for ny vrile (Tle 5). Disussion The results from this experiment show tht the ddition of iosolids enhned the growth of the eulypt in rseni-rih tilings. Enhned plnt iomss prodution in term of height, weight nd lef re ws mesured in the LB nd HB tretments (Figure 1). This ould e ttriuted to improvement of tilings onditions (espeilly physil, hemil, iohemil nd nutrient sttus). Inoulted plnts lso showed in generl etter growth response. For most woody plnt speies, seedling estlishment is the ritil phse in their yle life (Rey nd Alántr, 2000). In this experiment the 11

12 ddition of iosolids to tilings improved plnt seedling nd plnt development. Moreover, AMF inresed the totl weight of plnts. These effets re espeilly importnt in degrded or/nd ontminted soils. In this se, our findings re lso onsistent with the extensively doumented enhned growth of plnts whih hve the symioti ssoitions (Smith et l., 1997). Addition of iosolids inresed severl nutrients in the tilings nd in turn the nutrient ontent of the plnts in oth the LB nd HB tretments. Use of mendments nd introdution of metl-tolernt plnts hs een proposed s ost-effetive pproh to remedition of metl-ontminted soils tht most losely follow nturl proesses (Gunn et l., 1995). Applition of orgni mendments suh s sewge sludge hs permitted suessful oloniztion of the eulypts in soil (Meruri et l., 2005). Moreover myorrhizl ould inrese eulypt growth through proess of improved nutrient quisition, espeilly P nd N, lthough the effet vries drmtilly with the speies of eulypt studied nd the fungi inoulted (Adjoud et l., 1996; Lu et l., 1998). Furthermore, the importne of eh ssoition hnges during the life of the plnt. Myorrhizl oloniztion rtes in roots of E. ldolx grown in rsenous nd sulphidi tilings were higher to those reported for field oservtion in Chin for different speies of Eulyptus (0 to 15.2% short roots olonized y ECM nd from 0 to 16% root length for AM) (Chen et l., 2007) ut lower thn tht found in glsshouse ondition (Chen et l., 2000). Although tilings were inoulted with AMF, fter 150 dys the presene of ECM ws deteted in the roots. Aording to Chilvers, (2000), more thn 140 nmed ECM re known to e nturlly ssoited with eulypts round the world nd mong them 104 hve een reported from Austrli. Folir As onentrtions were generlly within norml levels for plnts ( mg kg -1 ) (Chney, 1989) nd similr to those reported y King et l., (2008) for E. ldolyx 12

13 This speies showed little As uptke when ompred to the totl As onentrtions in the tilings (Tle 1). Despite the high onentrtions of As in the sustrte, plnts did not show high levels of these elements in their tissues. This n e relted to rpid hnge of As from solule into preipitted forms nd to preferentil As umultion in roots or tuer tissues (Smith et l., 1998). Our results show tht E. ldolyx hs the potentil to e n idel ndidte for use for As phytostilistion. Cdmium onentrtions in iosolid treted eulypts exeeded the norml vlues in plnts (Chney, 1989) nd the limit for livestok (0.5 mg kg -1 ; Chney, 1989) ws exeeded in the HB nd LB tretments. In generl, Cd is effetively sored y plnt roots nd umulted in oveground orgns; hving thus omprtively high trnsfer soil plnt oeffiient (Kloke et l., 1984), espeilly in this se, in whih soil ph ws very low. The umultion of Cu, P nd Zn in leves nd stems ws similr in ll the tretments or slightly higher in iosolid tretments. In ny se, the mesured vlues were lwys within the norml rnge, nd elow the phytotoxi rnge for plnts (Chney, 1989). The men onentrtion of Zn in iosolid treted trees ws higher thn in TS treted ones, ut in ny se these vlues were elow the phytotoxi rnge ( mg kg 1 ; Chney, 1989). Like Cd, Zn hs reltively high trnsfer oeffiient from soil to plnt, nd most plnts n tolerte high Zn levels (Kloke et l., 1984). Other studies hve found high Zn onentrtions in the leves of Populus speies nd Slix pre up to 800 mg kg -1 (Lepp nd Mdejón 2007) nd up to 2000 mg kg 1 under extreme onditions of low ph nd orgni mtter ontent (Ernst, 1998). Tre element onentrtions in seedlings grown in the iosolid-mended tretments were in generl higher thn those of plnts grown in the tretment topsoil, nd the totl mount of tre elements extrted in the oth the low nd high iosolid mendment 13

14 ws muh higher. The level of risk to the food hin tht the mesured tre elements levels represent is diffiult to ssess. Folige of these E. ldolyx is rrely eten y mmmls sine they possess rnge of defense ompounds suh s terpenes (Brophy et l., 1991), phenolis nd ynogeni glyosides (Gledow nd Woodrow, 2002). A reent study of E. ldolyx showed tht over five yers of growth under field onditions, only limited inset dmge to leves ourred (King et l., 2008). Nevertheless it is importnt to redue the uptke of toxi elements y plnts, whih n e done suessfully y djusting mendment ddition nd the use of myorrhizl inoultion, nd then to monitor tre element ontents of different tx growing in the ffeted re. In ft, results of this experiment showed tht lower doses of iosolid with the presene of the inoulums produed the sme response s higher doses, nd the tre element uptke ws lower. Leves of trees hve een widely used for iomonitoring of tre element pollution. In prtiulr, leves of poplr (severl speies) hve een seleted in numer of studies (Mdejón et l., 2004; Roinson et l., 2000; Brggli, 1998). Brk of trees hs een lso used for pssive iomonitoring (Böhn et l., 1998). However, s rule, the xil trnsport of elements from roots (or other ssimiltion orgns) to the rk is ssumed to e negligile. In ft, leves re the min sink for mny pollutnts, nd therefore they re more sensitive to their effets thn flowers, fruits or other plnt orgns (Brggli, 1998). Little is know out the potentil use of Eulyptus speies s ioinditor of tre element ontminted soil. The results of this experiment showed n interesting pproh to onsider these trees s ioinditor lthough further reserh is needed to ssess this fesiility. The low vlues of dehydrogense tivity oserved in ll sustrtes indite the poor miroiologil sttus of the originl tilings. Nevertheless, the ddition of iosolids 14

15 nd the presene of myorrhize inresed the iologil tivity of the sustrtes. The influene of orgni mendments on enzyme tivities in ontminted soils hs een the sujet of severl reent studies (Pérez de Mor et l., 2005). In the present study, we did not find ny ler reltionship etween tre element onentrtions nd dehydrogense tivity. AMF inresed this tivity of the enzyme inditing n improvement of the orgni mtter turnover. An inrese in enzyme tivities in the rhizosphere of inoulted plnts hs een oserved previously (Vivs et l., 2005). These uthors ttriuted this inrese to the effet of nutrient lekge from roots. Suh stimultory effets re inditive of etter iologil funtioning of the inoulted soils. Conlusions Although further field experiments re neessry, the omintion of rusulr myorrhizl fungi (AMF) nd iosolids ws shown to e promising tehnique for restortion of rsenil mine tilings. Addition of iosolids inresed the growth development of E. ldolyx in tre element-polluted tilings y improving nutrient uptke. The improvement of the physil, hemil nd iologil onditions of the polluted tilings used y the iosolids ddition ould lso influene this inrese in tree growth. Moreover, inoultion with AMF n lerly enhne the effetiveness of iosolid pplition in phytostiliztion of tilings. In ft the inoultion with myorrhizs permits the optimiztion of iosolid doses euse lower mounts of iosolid with the presene of the inoulums produed the sme response s higher mounts. Although further reserh is needed eulypts my prove useful ioinditor of tre element ontmintion Aknowledgements 15

16 Dr. E. Mdejón reeived finnil support for her work t The University of Melourne (Austrli) from Progrm Slvdor de Mdrig of the Spnish MEC. P Mdejón thnks her Rmon y Cjl Contrt finned y the Spnish Ministry of Innovtion nd Siene.This support is knowledged with thnks. Referenes Adjoud, D., Plenhette, C., Hlli-Hrgs, R., Lpeyrie, F Response of 11 eulyptus speies to inoultion with three rusulr myorrhizl fungi. Myorrhiz 6, Austrlin nd New Zelnd Environment Counil nd Ntionl Helth nd Medil Reserh Counil (ANZEC/NHMRC) Guidelines for the ssessment nd mngement of ontminted lnd. Cnerr, Austrli. Brggli, R Tre Elements in Terrestril Plnts: An Eophysiologil Approh to Biomonitoring nd Bioreovery. Springer-Verlg, Berlin, pp. Böhm, P., Woltereek, H., Verurg, T., Musílek, L The use of tree rk for environmentl pollution monitoring in the Czeh Repuli. Environ. Poll. 102, Brdley, R., Burt, A.J., Red, D.J The iology of myrorrhiz in the Eriee. VIII. The role of myorrhizl infetion in hevy metl resistne. New Phytol. 91, Brophy, J., House, A., Bolnd, D., Lssk, E Digests of the essentil oils of 111 speies from northern nd estern Austrli. In: Bolnd, D., Brophy, J., House, A., (Eds), Eulyptus lef oils, use, hemistry, distilltion nd mrketing. Inkrt Press, Melourne, pp. 16

17 Brundrett, M., Austrlin Centre for Interntionl Agriulturl Reserh Working with Myorrhizs in Forestry nd Agriulture. Austrlin Centre for Interntionl Agriulturl Reserh, Cnerr, A.C.T. Chney, R.L Toxi element umultion in soils nd rops: proteting soil fertility nd griulturl food-hins. In: Br-Yosef B.,. Brrow, N.J, Goldshmid, J. (Eds), Inorgni Contminnts in the Vdose Zone. Springer-Verlg, Berlin, pp Chen Y.L, Gong, M.Q, Xu, D.P, Zhong, C.L, Wng, F,Z., Chen, Y Sreening nd inoulnt effiy of Austrlin etomyorrhizl fungi on Eulyptus urophyll in field. For. Res. 13, Chen, Y.L., Liu, S., Dell B Myorrhizl sttus of Eulyptus plnttions in south Chin nd implitions for mngement. Myorrhiz 17, Chilvers, G.A Myorrhizs of eulypts. In: Kene, P.J, Kile, G.A, Podger, F.D, Brown, B.N. (Eds), Diseses nd pthogens of eulypts. CSIRO, Melourne, pp. Cunninghm, S.D., Berti, W.R., Hung, J.W Phytoremedition of ontminted soils. Trends Biotehnol. 13, Denny, H.J, Wilkins, D.A Zin tolerne in Betul spp. IV. The mehnism of etomyorrhizl meliortion of zin toxiity. New Phytol. 106, Doronil A Phytostilistion of rseni-rih sulphidi gold mine tilings in the Vitorin goldfields, Austrli. PhD thesis, Shool of Botny, University of Melourne. Ernst, W.H.O The origin nd eology of ontminted, stilized nd nonpristine soils Metl-Contminted Soils: In: Vngronsveld, J.,Cunninghm S.D. (Eds), Situ Intivtion nd Phytorestortion, Springer-Verlg, Georgetown, pp Frenh C.J., Dikinson N.M., Putwin P.D Woody iomss phytoremedition of ontminted rownfield lnd. Environ. Poll. 141,

18 Giovnnetti, M., Mosse, B An evlution of tehniques for mesuring vesiulr rusulr myorrhizl infetion in roots. New Phytol. 84, Gledow R.M, Woodrow I.E Defense hemistry of ynogeni Eulyptus ldolyx seedlings is ffeted y wter supply. Tree Physiol. 22, Gunn, J., Keller, W., Negusnti, J., Potvin, R., Bekett, P., Winterhlder. K Eosystem reovery fter emission redutions: Sudury, Cnd. Wter Air Soil Poll. 85, King, D.J., Doronil, A.I., Feenstr, C., Bker A.J.M., Woodrow, I.E Phytostilistion of rsenil gold mine tilings using four Eulyptus speies: Growth, rseni uptke nd vilility fter five yers. Si. Totl Environ. 406, Kloke, A., Suerek, D.R., Vetter, H The ontmintion of plnts nd soils with hevy metls nd the trnsport of metls in terrestril food hins. In: Nrigu J.O. (Ed), Chnging Metl Cyles nd Humn Helth. Springer-Verlg, Berlin, pp Lodge, D.J Eto-or rusulr myorrhizs, whih re est?. New Phytol. 146, Lepp, N.W., Mdejón, P Cdmium nd Zin in vegettion nd litter of voluntry woodlnd tht hs developed on ontminted sediment-derived soil. J Environ Qul. 36, Lu, X., Mljzuk, N., Dell, B Myorrhiz formtion nd growth of E. gloulus seedlings inoulted with spores of vrious etomyorrhizl fungi. Myorrhiz 8, Mdejón, P., Mrñón, T., Murillo, J.M., Roinson, B White poplr (Populus l) s iomonitor of tre elements in ontminted riprin forests Environ. Poll. 132,

19 Mendez, M.O., Mier, R.M Phytostiliztion of mine tilings in rid nd semirid environments n emerging remedition tehnology. Environ. Helth Perspet. 116, Pérez de Mor, A., Orteg-Clvo, J.J, Crer, F., Mdejón E Chnges in enzyme tivities nd miroil iomss fter in situ remedition of hevy metlontminted soil. Appl. Soil Eol. 28, Rey, P.J., Alántr, J.M Reruitment dynmis of fleshy-fruit plnt (Ole europe): onneting ptterns of seed dispersl to seedling estlishment. J. Eol. 88, Roinson, B.H., Mills, T.M., Petit, D., Fung, L.E., Green, S.R., Clothier, B.E Nturl nd indued dmium-umultion in poplr nd willow: Implitions for phytoremedition. Plnt Soil 227, Smith, E., Smith, J., Smith, L., Bisws, T., Correll, R., Nidu, R Arseni in Austrlin environment: n overview. J. Environ. Si. Helth Prt A-Toxi/Hzrd. Sust Environ Eng. 8, Smith, S.E., Red, D.J., Hrley. J.L Myorrhizl symiosis. Ademi Press, London. Tordoff, G.M., Bker, A.J.M. Willis. A.J Current pprohes to the revegettion nd relmtion of metlliferous mine wstes. Chemosphere 41, Trevors, J.T Dehydrogense tivity in soil: omprison etween the INT nd the TTC ssy. Soil Biol. Biohem. 1, Vivs, A., Bre, J.M., Azón, R Breviillus revis isolted from dmium- or zin-ontminted soils improves in vitro spore germintion nd growth of Glomus mossee under high Cd or Zn onentrtions. Miro. Eol. 3,

20 Tle 1. Men vlues (n=3) nd stndrd devition (in prenthesis) of the most relevnt hrteristis of the mterils nd the prepred sustrtes. T TS B LB HB ph 4.2 (0.25) 4.12 (0.32) (0.07) 4.7 (0.12) TOC g kg (0.02) 0.7 (0.05) (0.32) 3.1 (0.25) EC ms m Totl-C mg kg (2000) 219 (8.5) 8357 (121) (566) (1202) Totl-Mg mg kg (59) 379 (6.3) 2296 (110) (99) (28.3) Totl-K mg kg (155) 465 (47) 4736 (236) 4040 (35.3) 3830 (35.0) Totl-N mg kg (12) 137 (14) 179 (6.34) 340 (23) (9.2) Totl-S mg kg (700) 420 (2.8) (423) (353) (50.0) Totl-P mg kg (7.0) 89 (7.6) 8167 (321) 1932 (50) 2532 (49.0) 20

21 Tle 2.Effet of the two min ftors (inoultion with AMF nd sustrte) nd intertion etween the two ftors presented s F- rtios for development prmeters, plnt nutrient nd tre element ontents in leves. Inoultion Sustrte Inoultion x Sustrte Height * Totl weight 4.29* 18.8* Totl lef re * C * 1.07 Mg * 0.32 K * 0.73 P * 0.01 S * 0.29 N * 0.52 As Cd * 7.26* Cu * 2.29 Ni * 0.57 P * 2.90 Zn * 2.79 *Indites sttistil signifine 21

22 Tle 3. Men vlues of perentge of myorrhyzl oloniztion (MC) in roots Tretment (-MC) (+MC) % Infetion % ECM % AM Infetion Infetion T ,3 TS LB HB ECM, etomyorrhiz, AM, rusulr myorrhiz. Vlues followed y the sme letter in the sme olumn, do not differ signifintly (p<0.05). 22

23 Tle 4. Men vlues of the most relevnt properties of the sustrtes t the end of the experiment. +/- refers to myorrhizl inoultion Tretment ph EC Dehydrogense As Cd Cu Ni P Zn (ms m -1 ) (mg kg -1 INTF h -1 ) T T TS TS LB LB HB d 60.0d HB d Vlues followed y the sme letter in the sme olumn, do not differ signifintly (p<0.05). 23

24 Tle 5. Effet of the two min ftors (inoultion with AMF nd sustrte) nd intertion etween the two ftors presented s F-rtios for some prmeters of the sustrtes. Inoultion Sustrte Inoultion ph * 0.26 EC * 0.81 Dehydrogense 10.4* 42.5* 059 As * 0.89 Cd * 0.86 Cu * 0.75 Ni * 0.74 P * 0.60 Zn * 0.38 *Indites sttistil signifine x Sustrte 24

25 Figure 1. Biomss development: ) Height (m); ) Weight (g/pot) nd ) Lef re (m 2 ) of the trees. Vlues followed y the sme letter ove the sme r, do not differ signifintly (p < 0.05). TS; topsoil. LB; sustrte low iosolids doses nd HB; sustrte high iosolids doses Figure 2. Nutrient onentrtions in leves (dry mtter). Vlues followed y the sme letter ove the sme r do not differ signifintly (p < 0.05). TS; topsoil. LB; sustrte low iosolids doses nd HB; sustrte high iosolids doses. Figure 3. Nutrient onentrtions in stems (dry mtter). Vlues followed y the sme letter ove the sme r do not differ signifintly (p < 0.05). TS; topsoil. LB; sustrte low iosolids doses nd HB; sustrte high iosolids doses. Figure 4. Tre element onentrtions in leves (dry mtter). Vlues followed y the sme letter ove the sme r do not differ signifintly (p < 0.05). TS; topsoil. LB; sustrte low iosolids doses nd HB; sustrte high iosolids doses Figure 5. Tre element onentrtions in stems (dry mtter). Vlues followed y the sme letter ove the sme r do not differ signifintly (p < 0.05). TS; topsoil. LB; sustrte low iosolids doses nd HB; sustrte high iosolids doses 25

26 70 60 ) Height m T T+ TS TS+ LB LB+ HB HB+ Tretment ) weight d d d g per pot d 5 0 T T+ TS TS+ LB LB+ HB HB+ Tretment ) LA m T T+ TS TS+ LB LB+ HB HB+ Figure 1 Tretment 26

27 g kg N stems N leves g kg P stems P leves TS TS+ LB LB+ HB HB+ Tretment 0.0 TS TS+ LB LB+ HB HB+ Tretment g kg C stems C leves g kg K stems K leves TS TS+ LB LB+ HB HB+ 0 TS TS+ LB LB+ HB HB+ Tretment Tretment 10 8 S stems S leves Mg stems Mg leves g kg g kg d Figure 2. 0 TS TS+ LB LB+ HB HB+ Tretment 0.0 TS TS+ LB LB+ HB HB+ Tretment 27

28 mg kg As leves mg kg Cd stems Cd leves d d d d TS TS+ LB LB+ HB HB+ Tretment 0 TS TS+ LB LB+ HB HB+ Tretment Cu stems Cu leves Ni stems Ni leves mg kg mg kg TS TS+ LB LB+ HB HB+ Tretment 0 TS TS+ LB LB+ HB HB+ Tretment mg kg P leves mg kg Zn stems Zn leves d d 0.0 TS TS+ LB LB+ HB HB+ Tretment Figure 3. 0 TS TS+ LB LB+ HB HB+ Tretment 28

29 7 6 5 r2=0.86 Experimentl point r2=0.67 Experimentl point Cd lef Zn lef Cd soil Zn soil Figure 4 29