Chemistry and Ecology Vol. 26, No. 4, August 2010, E.C.H.E.T. Lucassen a *, M.M.L. van Kempen b, J.G.M. Roelofs a,b and G.

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1 Chemistry n Eology Vol. 26, No. 4, August 21, Deline in metllophytes in tertiry pollute flooplin grsslns in the Netherlns: experimentl eviene for metl n nutritionl hnges in soil s river ftors E.C.H.E.T. Lussen *, M.M.L. vn Kempen, J.G.M. Roelofs, n G. vn er Vele, Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 B-WARE Reserh Centre, Rou University Nijmegen, Nijmegen, the Netherlns; Deprtment of Aquti Eology & Environmentl Biology, Rou University Nijmegen, Nijmegen, the Netherlns; Deprtment of Animl Eology n Eophysiology, Rou University Nijmegen, Nijmegen, the Netherlns; Netherlns Centre for Bioiversity Nturlis, Leien, the Netherlns (Reeive 24 Mrh 21; finl version reeive 3 April 21 ) Metllophyte vegettion long the River Geul hs een lmost ompletely reple y grsses uring the lst ees. Fiel investigtions inite tht this ws ompnie y higher lklinity n phosphte vilility in the soil, relte to the losure of the metl inustry n intensifition of griulturl prties. An experiment with full ftoril esign for phosphte n zin vilility inite tht the metllophytes Silene vulgris n Thlspi erulesens i not grow on zin-poor soils, irrespetive of phosphte vilility. The grss Holus lntus performe well on phosphte-rih soil, irrespetive of zin vilility. An experiment with zin-poor n zin-rih flooplin soils onfirme the high zin emn of the metllophytes T. erulesens n Armeri mritim n the zin inepenene of H. lntus.a thir experiment inite tht reue zin vilility ue to liming ffete only the metllophyte T. erulesens; it h no effet on the growth of the grss Festu rur. This mens tht inresing lklinity les to erese in zin vilility, limiting the growth of t lest some metllophyte speies. An inrese in phosphte vilility stimultes growth in more ompetitive fst-growing grsses uner zin-rih s well s zin-poor onitions. Keywors: lklinistion; flooplin grsslns; metllophytes; metl vilility; phosphte; restortion 1. Introution Soils enrihe or pollute with metls, n their hrteristi vegettion, n e foun in mny prts of the worl [1]. In generl, three types of metlliferous soils re istinguishe: (1) primry pollute soils, inluing nturlly enrihe soils in the viinity of minerl veins; (2) seonry pollute soils on metlliferous mine spoils; n (3) tertiry pollute soils iniretly pollute vi strems or the tmosphere, inluing flooplin grsslns [2]. Metllophytes re plnts tht hve evolve iologil mehnisms to survive on these metl-rih soils [3]. They re the result *Corresponing uthor. Emil: e.lussen@-wre.eu ISSN print/issn online 21 Tylor & Frnis DOI: 1.18/

2 274 E.C.H.E.T. Lussen et l. Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 of tens, hunres n often thousns yers of strong seletive pressure exerte y the soils [4 6]. The restrite geogrphil re of metllophytes is key ftor in their high rtes of popultion eline n extintion [7,8]. Metllophytes re inresingly uner thret n tions towrs their onservtion re therefore impertive [8,9]. The River Geul re is situte ner the orers etween Germny, Belgium n the Netherlns. In L Clmine n Plomières (Belgium) over 2.3 million tons of zin n le hve een proue sine pre-romn times. The most intense perio of proution ws uring In 188 n 192, metl extrtion stoppe ut proessing of ores ontinue until 195. Wste wter ws e iretly to the River Geul, using wiespre ispersl of pyrite- n metl-ontining mu in Belgin n Duth flooplin grsslns [1 12]. As result of these proesses, very speifi zin-tolernt vegettion type, syntxonomilly elonging to the Violetum lminrie Shwikenrth, evelope not only in the seonry pollute Belgin mine spoil res, ut lso in the tertiry pollute flooplin grsslns [13 15]. At the time tht the Belgin zin inustry eme intive, metllophyte vegettion in the reltively metl-rih seonry pollute mine spoil res in Belgium remine more or less stle, lthough it showe strong eline in the reltively metl-poor tertiry pollute flooplin grsslns of Belgium n the Netherlns [16]. More n more flooplin grsslns (reltively poor in metls ompre with mine spoil res) me into ultivtion n were use for griulturl purposes. Sine 1933, grsses initive of fertilise griulturl lns, like Holus lntus L., Agrostis pillris L. n Festu rur L., hve egun to evelop t the ost of the zin vegettion [17]. By 196, the overll re of zin vegettion in the Duth flooplin grsslns h grully erese y 8% [16,18,19]. The hitt of the metllophyte vegettion is now protete on oth ntionl n Europen sle euse the flooplin re forms prt of the Europen Eologil Network Ntur 2 [2]. Reserh rrie out in the Duth flooplin of the River Geul, previously n urrently vegette y metllophytes, inite tht metllophytes h een mintine only t reltively ii lotions (ph-h 2 O = ) with reltively high zin vilility (totl Zn > 4 μmol g 1 w; Zn/C >.8; Zn-H 2 O > 59 μmol kg 1 w) in omintion with low phosphte vilility (Olsen-P 125 μmol kg 1 w). Uner ll other soil onitions in this re, no metllophytes were present n the grss H. lntus h eome the ominnt plnt speies [21]. Totl zin onentrtions were high throughout the soil profile ( 5 m), wheres the top lyer ontine reltively high phosphte vilility n lium onentrtion. Removl of the lkline n phosphte-rih soil top lyer le to n inrese in the unne of metllophytes uring t lest the first four yers, while re-vegettion of H. lntus ws nil [21,22]. Fiel investigtions inite tht the eline in metllophytes might e relte to hnges in ln use (inrese use of lime-ontining fertiliser) n losure of the Belgin metl inustry, leing to phosphte enrihment n erese zin vilility. In this stuy, plnt growth experiments were performe uner lortory onitions to test the (omine) effets of zin n phosphte in the soil on the growth of severl grsses n lol metllophytes. It is hypothesise tht the growth of grsses is stimulte t the ost of the originl metllophyte vegettion when zin vilility ereses n/or phosphte vilility inreses. Growth of the grss H. lntus n of two lol metllophytes ws teste on rtifiilly rete soil types with full ftoril esign for phosphte n zin vilility. In ition, the effet of lklinistion n zin vilility on flooplin grsses (Festu rur) n metllophytes (Thlspi erulesens (J. & C. Presl) n Armeri mritim Will. susp. hlleri (Wllr.) Rothm.) ws teste. Plnt evelopment ws reore n the elementl omposition of the shoot mteril ws ompre with plnt mteril ollete uner fiel onitions.

3 Chemistry n Eology Mterils n methos 2.1. Bkgroun informtion Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 Sees of Thlspi erulesens, Holus lntus n Festu rur were ollete in the zinflor nture reserve situte in the tertiry pollute flooplin grsslns in Epen long the River Geul (the Netherlns: N, E). Sees of Silene vulgris (Moenh) Grke vr. humilis n Armeri mritim were ollete upstrem of the River Geul t seonry pollute mine spoil in Plomières euse oth speies hve eome extint in Epen [16]. Sees were grully rie t room temperture for three months. Germintion took ple (fter ol perio of one week t 4 C) on wet filter pper. After germintion, plnts were grown up in mixture of nutrient-poor river sn n metlliferous flooplin soil (2:1) for one week prior to the experiments. Seelings h the following men length (±SEM) t the strt of the experiments: T. erulesens, 1. ±.1 m; F. rur, 1.5 ±.2 m; S. vulgris,.9 ±.1 m; A. mritim,.7 ±.1 m; H. lntus,2.5 ±.3 m. Before filling the pots, textile fire ws ple t the ottom to prevent the soil flling out. All pots were inoulte with soil suspension (25 ml) rete y mixing flooplin soil (originting from n re with metllophytes present) with eminerlise wter (1kg L 1 ) t 5 rpm for one y. This ws to exlue possile effet of the sene of re-speifi or metl-tolernt miroorgnisms in the ifferent soil types. Pots with the sme seiment type were put in the sme wtertight try to prevent wter shortge without using ontmintion etween the soil types. In the first week, rtifiil rinwter (5 mg se slt L 1, rn: Tropi Mrin) ws e on top of the pots. After this, seelings were settle n eminerlise wter mixe with tp wter (9:1) ws e to the ottom of the trys weekly ( 3L week 1 ). The trys were move eh week to minimise ny effet of ifferenes in mirolimte. The experiments were performe in limte-ontrolle room with temperture of 18 C, y/night regime of 15/9 h, reltive humiity of 6% n light intensity of 25 μmol m 2 s Experiment 1: omine effets of phosphte n zin vilility Growth of the metllophytes T. erulesens n S. vulgris n the grss H. lntus ws teste in lortory experiment with full ftoril esign for phosphte n zin vilility in the soil. Plnts n rete vrious grients in the vilility of phosphte n metls in soil s onsequene of rnge of proesses inue y the tivity of either plnt roots or the rhizosphere miroflor [23]. Therefore, we eie to rry out our experiments in soil inste of in hyroponis. We eie to rete sustrtes iffering in Zn n P vilility y mixing soil types. In ft, mixing of soils lso hppens uring flooing events n ploughing of the soil y frmers. The following three soil types were use to rete four sustrte types iffering in Zn n P vilility, s inite in Tle 1: river sn from nture reserve long the River Meuse (poor in phosphte n zin), griulturl soil from flooplin long the River Meuse (rih in phosphte n poor in zin), n metlliferous soil from Plomières (poor in phosphte n rih in zin). The soils were ollete with shovel t epth of 2 m. The homogenous river sn n griulturl soil were not sieve. The ry n heterogenous metlliferous soil ws sieve (<2 μm). For eh sustrte type, five iniviuls per plnt speies were plnte in lk plsti pot (length: 11 m, with: 11 m, height: 12 m). The numer of leves, the length of the longest lef n the with of the wiest lef were reore t t =, 2, 4 n 6 weeks. Estimte shoot size ws lulte y multiplition of the numer of leves, the mximum lef length n with (W mx ) [24]. Plnts were hrveste fter six weeks. Shoots n roots were seprte, rie (24ht7 C) n weighe.

4 276 E.C.H.E.T. Lussen et l. Tle 1. Reltive ontriutions (volume %) of river sn, griulturl soil n metlliferous soil in orer to rete four soil types iffering in P n Zn vilility. Tretment River sn Agriulturl soil Metlliferous soil P Zn 1 P Zn P+Zn P+Zn Note: P =P-poor; P+ =P-rih; Zn =Zn-poor; Zn+ =Zn-rih. Tle 2. Prmeters of the four soil types (n = 4). P Zn P Zn+ P+Zn P+Zn+ Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 Orgni mtter (%).3 (.3) 4.4 (.4).7 (.4) 4.9 (.3) Moisture (%) 18.9 (.4) 17.2 (.2) 17.6 (.3) 17.6 (.3) ph-h 2 O 8.42 (.4) 7.76 (.4) 7.23 (.15) 7.7 (.7) Olsen-P (4.3) 42.6 (48.5) 1596 (82.7) 12.4 (61.1) Zn-totl.74 (.9) 21.5 (2.4).75 (.5) 32.2 (6.3) C-totl 47.5 (4.3) 57.1 (6.3) 48.8 (6.9) 46.9 (7.) Zn/C.2 (.).46 (.6).2 (.).71 (.15) Zn-DTPA 58.5 (1.9) (236.5) 42.6 (2.4) (118.5) NH + 4 NCl 8.9 (1.4) 21.1 (4.3) 17.3 (.5) 9.1 (1.1) Notes: P =P-poor; P+ =P-rih; Zn =Zn-poor; Zn+ =Zn-rih. Totl onentrtions re given in μ mol g 1 w, extrtle onentrtions re given in μ mol kg 1 w. Mens n (SEM) re given. Mens (within row) with sme letter o not signifintly iffer. Soil mixing my le to ltertions in NH + 4 versus NO 3 vilility, whih my in turn le to onsierle hnges in rhizosphere ph n thus Zn vilility [25]. However, this effet n e exlue in our experiment euse the NCl-extrtle NH + 4 onentrtion ws very low n ph ws high in ll soil types use (Tle 2). Previous ttempts to rete these sustrtes y ing fertiliser n metls file. As uner nturl onitions, quik ition of lrge mounts of fertiliser n metls les to shok effet resulting in ie k of plnts. Only fter geing will new soil equilirium estlish whih is suitle for plnt growth (Lussen, unpulishe results) Experiment 2: effet of lklinistion Growth of the metllophyte T. erulesens n the grss F. rur ws teste on flooplin soil ollete with shovel ( 4 m) from site previously vegette y metllophytes. The soil ws thoroughly mixe n istriute into 1 plsti pots (imeter: 18 m, height: 1 m). Hlf of the pots were spike with 2 g of CCO 3 whih ws mixe to erese the Zn/C rtio of the soil to elow the ritil vlue of.66 hrteristi of flooplin soils with zin-flor growth [21]. Five iniviuls per plnt speies were susequently plnte in eh pot (n = 5). Plnt mteril ws ollete fter three months Experiment 3: effet of zin vilility in flooplin soils with moerte phosphte vilility Growth of the metllophytes T. erulesens n A. mritim n the grss H. lntus ws teste on flooplin soil ollete t zin-rih lotion urrently vegette y metllophytes n t zin-poor lotion vegette y H. lntus. Previous reserh hs shown tht phosphte ws minly umulte in the first 2 m of the soil [21]. Soil ws ollete with shovel t epth of

5 Chemistry n Eology m to prevent n interting effet of ifferenes in phosphte vilility on plnt growth etween the two soil types. For eh sustrte type, 12 plsti pots (length: 2 m, with: 11 m, height 5 m) were fille with unsieve mteril. For eh plnt speies, four pots were plnte with 1 speimens per seiment type (n = 4). The five lrgest plnts were hrveste per pot fter three months Fiel mesurements In orer to etermine relisti rnge for the elementl omposition of metllophytes n grsses, shoot mteril of severl plnt speies ws ollete t metlliferous sites in Plomières (Belgium), L Clmine (Belgium) n Epen (the Netherlns) in August Proessing of soil n plnt mteril Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 An elementl nlysis ws rrie out to etermine the totl mount of elements (metls, totl-p n totl C) in the vrious soil types s well s in the plnt mteril. An mount of 2 mg of rie mteril (7 C for 24 h) ws igeste for 17 min with 4 ml of onentrte nitri i n 1 ml 3% hyrogen peroxie using Milestone type mls 12 Meg mirowve. An Olsen-P extrtion ws rrie out to etermine the onentrtion of plnt-ville phosphorus in the soil [26]. A H 2 O-extrtion (17.5 g of fresh soil n 5 ml of eminerlise wter) ws rrie out to etermine the ph of the soil. A iethylene trimine penteti i (DTPA)-extrtion ws rrie out to etermine the onentrtion of extrtle Zn in the soil [27]. A NCl-extrtion (17.5 g of fresh soil n 5 ml.1 M NCl t 1 rpm for 2 h) ws rrie out to etermine the mount of soil-extrtle ions in the soil. All extrts were smple with Teflon rhizon soil smplers (Eijkelkmp Agriserh, the Netherlns) onnete to vuum nitrogen pre-flushe serum ottles (3 ml). Orgni mtter ontent ws etermine y loss on ignition (55 C for 4 h) Chemil nlysis Totl onentrtions of elements (Zn, P, C, C, Mg, S, Mn, K, Fe, Al, P) in igests, Olsen-P n DTPA-extrts were nlyse using n inutively ouple plsm mss spetrophotometer (ICP-MS). Qulity ssurne mesures inlue lnks, replite nlyses n mtrix spikes (stnr ertifie referene hemils). Reoveries from mtrix spikes rnge from 95 to 17%. Repete nlyses i not revel ifferenes >5%. NH + 4 n NO 3 onentrtions in the NCl extrts were nlyse with TehnionAA II systems using sliylte [28]. ph-h 2 O ws mesure with omintion ph eletroe with n Ag/AgCl internl referene (Orion Reserh) Dt nlysis Overll effets on plnt ry weight n plnt omposition t the en of the experimentl perio were nlyse using generl liner moel (GLM) multivrite nlyses with esign (speies n soil type) s the fixe ftor. Differenes within soil type or tretments were nlyse using GLM univrite nlyses with soil type or speies s the fixe ftor. Dt on estimte shoot size were nlyse using GLM repete mesures. Sttistil ifferenes etween groups were nlyse using Tukey s HSD post-ho tests. Kolmogorov Smirnov tests were use in ll ses to investigte whether the responses met the prmetri ssumptions of normlity. Homogenity of vrine ws heke with Levene s test. Prmeters on plnt omposition of experiment 1 n on plnt mteril from the fiel were sujete to nturl log trnsformtion. SPSS 16. ws use s the sttistil pkge.

6 278 E.C.H.E.T. Lussen et l. 3. Results 3.1. Experiment 1: omine effets of phosphte n zin vilility Results re shown in Tle 2 n Figures 1 3. The onentrtion of plnt-ville phosphte (Olsen-P) ws, s expete, signifintly higher in soil types enrihe with griulturl soil 1 Holus lntus Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 Estimte shoot re (m 2 ) Estimte shoot re (m 2 ) Silene vulgris Thlspi erulesens Estimte shoot re (m 2 ) Time (weeks) Figure 1. Men (+SE) estimte shoot re of the grss H. lntus n the metllophytes S. vulgris n T. erulesens growing on soil with full ftoril esign for zin n phosphte vilility for 1.5 months. ( ) Phosphte- n zin-poor (n = 4).( ) Phosphte-poor n zin-rih (n = 4).( ) Phosphte-rih n zin-poor (n = 4).( ) Phosphten zin-rih (n = 4). Mens with the sme letter o not iffer signifintly. 6

7 Chemistry n Eology 279 Shoot iomss (g w) H. lntus S. vulgris T. erulesens P Zn P Zn+ P+Zn P+Zn+ Soil type Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 Root iomss (g w) H. lntus S. vulgris T. erulesens P Zn P Zn+ P+Zn P+Zn+ Soil type Figure 2. Men (+SE) shoot n root iomss of the grss H. lntus n the metllophytes S. vulgris n T. erulesens fter 6 weeks of growth on soils with full ftoril esign for phosphte (P) n zin (Zn) vilility., Poor; +, rih. Mens with the sme letter o not iffer signifintly. (P+Zn n P+Zn+) thn in those without griulturl soil (P Zn n P Zn+). The onentrtions of totl (Zn-totl) n extrtle zin (Zn-DTPA) were, s expete, signifintly higher in soil types enrihe with metlliferous soil (P Zn+ n P+Zn+) thn in those without metlliferous soil (P Zn n P+Zn ) (Tle 2). Root n shoot iomss proution were signifintly ffete y soil type (p <.1), plnt speies (p <.1) n their intertion (p <.1). Shoot iomss of S. vulgris remine low if growing on zin-poor soil uner phosphte-poor s well s phosphte-rih onitions (P Zn, P+Zn ). Shoot iomss proution inrese signifintly on zin-rih soil uner phosphtepoor s well s phosphte-rih onitions (P Zn+, P+Zn+). In this se, the mximum length n with of the leves ws lower n the totl length of the plnts higher, explining the lower estimte shoot size of the plnts on zin-rih soil uner P-rih onitions ompre with P-poor onitions. Shoot iomss proution of T. erulesens inrese signifintly only if growing on soil rih in oth zin n phosphte (P+Zn+). Growth of H. lntus inrese signifintly if growing on the phosphte-rih soil type, espeilly in omintion with high zin vilility (P+Zn, P+Zn ). H. lntus growing on phosphte-rih soil ontine the highest estimte shoot sizes of the investigte plnt speies. Ptterns in shoot iomss evelopment etween the vrious soil types were relte to the totl onentrtion of metls (Zn, P n C) n phosphorus in the shoot mteril. T. erulesens

8 28 E.C.H.E.T. Lussen et l. 1 H. lntus S. vulgris T. erulesens f 1 H. lntus S. vulgris T. erulesens e Zn (µmol g w 1 ) 1 1 e 1 e e e e e P Zn P Zn+ P+Zn P+Zn+ P Zn P Zn+ P+Zn P+Zn+ P (µmol g w 1 ) 1. H. lntus S. vulgris T. erulesens 15 H. lntus S. vulgris T. erulesens g Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 C (µmol g w 1 ) e P Zn P Zn+ P+Zn P+Zn+ P (µmol g w 1 ) ef ef fg ef P Zn P Zn+ P+Zn P+Zn+ Figure 3. Men (+SE) onentrtions of Zn, P, C n P in the shoot of the grss H. lntus, n the metllophytes S. vulgris n T. erulesens growing on soil with full ftoril esign for phosphte (P) n zin (Zn) vilility for 1.5 months., Poor; +, rih. Mens with the sme letter o not iffer signifintly. grew est on zin-rih soil rih in phosphte (P+Zn+). Uner tht onition, its shoots ontine omine high onentrtions of zin n phosphorus. S. vulgris grew est on zin-rih soil inepenent of the phosphorus ontent. Uner oth onitions, its shoots ontine omine high onentrtions of zin n phosphorus. The grss H. lntus grew est on oth phosphte-rih soil types. Uner oth onitions its shoots ontine high onentrtions of phosphorus, wheres, y ontrst to the two metllophyte speies, zin onentrtions in the shoots were vrile. e 3.2. Experiment 2: effet of lklinistion The results of experiment 2 re shown in Tle 3 n Figure 4. The totl C onentrtion in the flooplin soil inrese signifintly from 42 to 245 μmol g 1 w ue to liming. As result, the Tle 3. Soil hrteristis of the flooplin soil (previously ominte y metllophytes) with n without CCO 3 ition. CCO 3 (n = 5) +CCO 3 (n = 5) Zn-totl 53.4 (.3) 52.1 (.5) C-totl 41.9 (.6) (9.9)** Zn/C 1.27 (.3).21 (.1)** ph-h 2 O 6.55 (.1) 7.29 (.17)* Olsen-P (15.2) 24.2 (1.2) Zn-DTPA 6111 (282) 422 (71)** Notes: Totl onentrtions re given in μmol g 1 w, extrtle onentrtions re given in μmol kg 1 w. Men n (SEM) re given. *p <.5; **p <.1.

9 Chemistry n Eology Unlime Lime 75 Unlime Lime Shoot iomss (mg) P (µmol g w -1 ) 5 25 T. erulesens F. rur T. erulesens F. rur 5 4 Unlime Lime 5 4 Unlime Lime Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 C (µmol g w -1 ) T. erulesens F. rur Zn (µmol g w -1 ) T. erulesens F. rur Figure 4. Men (+SE) shoot iomss evelopment n onentrtions of P, C n Zn in the shoot of the metllophyte T. erulesens n the grss F. rur growing on lime (Zn/C =.21, n = 5) n unlime (Zn/C = 1.27, n = 5) flooplin soil with low P vilility (Olsen-P = μmol kg 1 w) for 3 months. The soil (totl Zn = μmol g 1 w) originte from lotion previously ominte y metllophytes. p<.5. Zn/C rtio erese signifintly from 1.27 to.21 n the onentrtion of extrtle zin (Zn- DTPA) erese signifintly from 61 to 42 μmol kg 1 w. Shoot iomss evelopment ws signifintly ffete y soil type (p =.26) n plnt speies (p =.18), ut not y their intertive effet (p =.118). Shoot iomss for T. erulesens erese signifintly ue to liming, wheres shoot iomss for the grss F. rur i not. Derese growth in T. erulesens ws ompnie y signifint inrese in the onentrtion of C n signifint erese in the onentrtion of Zn in the shoot. There were no signifint ifferenes in the onentrtion of other elements mesure in the shoot inluing Fe (t not shown) Experiment 3: effet of Zn vilility in flooplin soils with moerte P vilility The results of experiment 3 re shown in Tle 4 n Figure 5. The Zn-poor flooplin soil showe signifintly lower Zn/C rtio lthough the totl onentrtion of C ws signifintly higher in this soil type. The vilility of nutrients i not iffer signifintly etween soil types. The mount of extrtle Zn (Zn-DTPA) ws, s expete, signifintly higher in the Zn-rih soil thn in the Zn-poor soil. Shoot iomss evelopment ws signifintly ffete y soil type (p =.4), plnt speies (p =.42) n their intertive effet (p =.32). On the Zn-poor soil, T. erulesens evelope signifintly lower shoot iomss ompre with the Zn-rih soil. The iomss evelopment of A. mritim showe similr tren ut the effet ws not signifint etween the two soil types (p =.167). Growth of the grss H. lntus ws not ffete y the Zn onentrtion of the soil (p =.672). The onentrtion of Zn, Mg, P n S in the shoots iffere signifintly etween the two soil types for most of the plnt speies. There were no suh signifint ifferenes in the onentrtions of other elements mesure. The onentrtion of Zn in shoots ws signifintly lower in A. mritim n H. lntus if growing on Zn-poor soil. T. erulesens i not evelop signifint ifferenes in Zn onentrtion in its shoots etween

10 282 E.C.H.E.T. Lussen et l. Tle 4. Soil hrteristis of two flooplin soil types with low P vilility (ollete t epth of 2 3 m) iffering in Zn onentrtion. High Zn (n = 12) LowZn(n = 12) Orgni mtter (%) 8.3 (.3) 8. (.2) Moisture (%) 21.1 (1.5) 2.3 (.5) Zn-totl 5. (.6) 2.3 (.5)** C-totl 44.4 (1.1) 89.8 (.5)* Zn/C 1.14 (.4).3 (.)** ph-h 2 O 5.95 (.1) 6.34 (.14) Olsen-P 41.2 (22.3) (12.6) Zn-DTPA 2567 (35) 11 (5)** Notes: Totl onentrtions re given in μmol g 1 w, extrtle onentrtions re given in μmol kg 1 w. Men n (SEM) re given. *p <.5; **p <.1. Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 Shoot iomss (g) P (µmol g -1 w) Zn-poor Zn-rih Zn (µmol g -1 w) T. erulesens A. mritim H. lntus T. erulesens A. mritim H. lntus T. erulesens A. mritim H. lntus S (µmol g -1 w) T. erulesens A. mritim H. lntus Figure 5. Men (+SE) shoot iomss evelopment n onentrtion of Zn, P n S in the shoot of the grss H. lntus (n = 4) n the metllophytes T. erulesens (n = 4) n A. mritim (n = 4) growing on Zn-poor (white rs: 2 μmol g 1 w) n Zn-rih (lk rs: 5 μmol g 1 w) flooplin soil with moertely high P vilility (Olsen-P = 4 42 μmol kg 1 w) for 3 months. Mens with sme letter o not signifintly iffer. oth soil types. For ll plnt speies, the onentrtion of Mg in their shoots ws higher if growing on the Zn-rih soil (results not shown). For ll plnt speies, the onentrtion of P n S ws signifintly higher if growing on the Zn-poor soil. Conentrtions of elements in the shoot mteril of the plnt speies uner fiel onitions re given in Figure 6. T. erulesens showe muh wier rnge in the onentrtion of metls (Zn, P n C) s well s in the onentrtions of C n S ompre with the other plnt speies. The men onentrtions of Zn, P n C in the shoots were signifintly higher in T. erulesens ompre with the other plnt speies. There were no signifint ifferenes in metl onentrtions in the shoots etween S. vulgris, A. mritim n H. lntus. The C onentrtions in the shoots were lower in H. lntus ompre with metllophyte plnt speies. There were no signifint ifferenes in the onentrtion of phosphorus in the shoots of the four plnt speies.

11 Chemistry n Eology Zn (µmol g -1 w) H. lntus S. vulgris T. erulesens A. mritim H. lntus S. vulgris T. erulesens A. mritim P (µmol g -1 w) 3. 5 Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 C (µmol g -1 w) P (µmol g -1 w) H. lntus S. vulgris T. erulesens A. mritim C (µmol g -1 w) H. lntus S. vulgris T. erulesens A. mritim H. lntus S. vulgris T. erulesens A. mritim H. lntus S. vulgris T. erulesens A. mritim Figure 6. Shoot elementl omposition of the grss H. lntus (n = 6) n the metllophytes S. vulgris (n = 6), T. erulesens (n = 6) n A. mritim (n = 8) uner fiel onitions. Totl rnge n mein level re given. Mens with the sme symol o not iffer signifintly. S (µmol g -1 w) Disussion The lortory experiments suggest tht growth of metllophytes in tertiry pollute flooplin grsslns of the River Geul my e ffete y the vilility of zin n phosphte in the soil. At reltively low zin vilility (ue to erese zin pplition or lklinistion) iomss evelopment in metllophytes erese strongly. At reltively high phosphte vilility, growth of the more ompetitive grss H. lntus ws strongly enhne, irrespetive of the zin onentrtion in the soil. The experiment with full ftoril esign for phosphte n zin vilility in the soil inite tht metllophytes, suh s S. vulgris n T. erulesens whih re ominnt t metl-rih hitts, only performe well if growing on metl-rih soils, s inite y severl eologil fiel stuies [29 31]. The etter performne of T. erulesens, originlly from metl-rih hitt, with high Zn supply hs lso een shown in hyroponis [32]. However, nutritionl requirements of metllophytes n iffer n thus responses to soil fertilistion ue to griulturl prties my iffer. We showe tht S. vulgris only inrese in shoot iomss if growing on zin-rih soil, irrespetive of phosphte vilility (Figures 1 n 2). Uner these

12 284 E.C.H.E.T. Lussen et l. Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 onitions, shoots of S. vulgris ontine high onentrtions of metls (Zn, P n C) omine with high onentrtions of phosphorus (Figure 3). T. erulesens only showe n inrese in iomss on zin-rih soil if omine with high phosphte vilility (Figure 1). Uner this onition, shoots of T. erulesens lso ontine high onentrtions of metls (Zn, P n C) omine with high onentrtions of phosphorus (Figure 3). However, the grss H. lntus i not grow on soils poor in phosphte n strongly inrese in iomss in phosphte-rih soil, irrespetive of zin vilility (Figures 1 n 2). The hemil omposition of the shoots onfirme tht, in ontrst to the investigte metllophytes, growth of H. lntus i not epen upon the omintion of high phosphorus n high zin vilility. H. lntus plnts growing on soil rih in phosphte ut poor in zin performe well n evelope high onentrtions of phosphorus in omintion with low onentrtions of metls (Zn, P n C) in its shoots. These results inite tht H. lntus will esily outompete the slow-growing metllophyte vegettion when phosphte vilility inreses. It is well known tht metllophytes of Violetum lminrie Shwikenrth hve high zin emn n n suffer Zn-efiieny where Zn vilility ereses ue to the rpi physiologil intivtion of zin [29,33]. Eotypes of H. lntus tolernt to zin toxiity lso tolerte zin efiieny [34]. This is in greement with our finings. Eotypes of H. lntus re Zn tolernt euse of strong umultion of Zn in the roots. By mens of this tolerne mehnism, Zn-tolernt eotypes of H. lntus n even perform etter on soils with low Zn vilility ompre with Zn-sensitive eotypes [34]. The reltively high zin emn of metllophytes ws lso shown y the signifintly lower iomss evelopment of T. erulesens growing on flooplin soil poor in Zn (2 μmol g 1 w) ompre with growing on flooplin soil rih in Zn (5 μmol g 1 w). The iomss of A. mritim showe this tren lthough it ws not signifintly ifferent. By ontrst, iomss evelopment in H. lntus ws oviously unffete y the Zn ontent of the soil (Figure 5). In A. mritim n H. lntus, the Zn onentrtion in the shoot ws signifintly lower if grown on the Zn-poor soil. This ws not evient for T. erulesens whih is n hyperumultor [35]. In ll investigte plnt speies, the very low Zn vilility in the soil ws ompnie y the evelopment of extremely high onentrtions of P n S in the shoots (Figure 5). The vilility of P (Olsen-P) n the totl S onentrtion in the two soil types were, however, equl (Tle 4). The P onentrtion in the shoots of plnt speies growing on Zn-rih flooplin soil were omprly high ompre with onentrtions in plnts growing uner fiel onitions (Figure 6). However, if growing on the Zn-poor soil, P onentrtions in the shoots of T. erulesens n A. mritim were 5 1 times higher thn in plnts growing uner fiel onitions. For H. lntus growing on Znpoor soil, the P onentrtion in the shoots ws only two times higher thn for plnts growing uner fiel onitions. From solution ulture experiments rrie out with high phosphorus ut low Zn supply, it ppere tht zin efiieny is often ssoite with n exeptionlly high phosphorus ontent n even symptoms of phosphorus toxiity in mture leves [36 4]. The muh higher phosphorus ontent in shoot ry mtter of zin-efiient plnts supplie with high phosphorus onentrtions n, to some extent, e ttriute to onentrtion effet, prtiulrly in oler leves. Comprle effets hve een foun in experiments rrie out with rley (Horeum vulgre L. v Weeh) in soil n it ws onlue tht Zn plys speifi role in the signl-trnsution pthwy responsile for the regultion of genes enoing high-ffinity trnsporters in plnt roots [41]. The effet of zin efiieny on sulfur uptke is proly omprle with phosphorus. Phosphorus toxiity levels might vry etween plnt speies. P toxiity levels of 1% of its ry weight for tomto (Solnum lyopersium L.) plnts were foun [42]. It ws stte tht P ontents >2% of its ry weight n e generlly onsiere toxi to plnts [43]. The P onentrtions were 1.55 n 1.11% for A. mritim n T. erulesens, respetively, wheres the onentrtion ws.62% in H. lntus. This might inite tht the metllophytes hve suffere P toxiity uner lortory onitions. Uner nturl onitions, plnts will very

13 Chemistry n Eology 285 Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 likely not survive on these soil types s they re slow growing, smll-size plnts n thus wek ompetitors. Liming n erese the plnt vilility of Zn muh more thn of ny other minerl nutrient, inluing P, n my enhne the risk of Zn efiieny in plnts [44,45]. The results of the liming experiment inite tht liming le to erese Zn vilility, s reflete y the lower DTPA-extrtle Zn frtion, lthough P-vilility (Olsen-P) in the soil remine unffete (Tle 3). The ition of lime results in reue DTPA-extrtle Zn frtion y inresing the Zn frtion oun to iron hyroxies t the ost of the exhngele-zn frtion n the Zn frtion oun to orgni mtter [46]. At high ph, Zn vilility is limite y the low soluility of rontes n hyroxies [47]. The flooplins long the River Geul re very rih in iron (4 μmol g 1 w) n it is therefore likely tht sorption to iron hyroxies, next to the formtion of Zn rontes, plys n importnt role in the soluility of Zn in the soil [21].At low ph (5.) no Zn is oun to hyrous ferri iron, wheres t high ph (7.) ll Zn is oun n insolule [48]. In ition, it ws shown tht C inhiits Zn uptke in metllophytes y ompetition for the uptke of these elements t the roots [49]. Severl uthors onlue tht the Zn/C rtio in soil is etter mesure for expressing Zn vilility thn the totl Zn onentrtion in the soil [5 52]. Overll, liming of flooplin soil resulte in the evelopment of signifintly higher C n signifintly lower Zn onentrtions in the shoots of T. erulesens, ompnie y lower iomss evelopment. By ontrst, growth of the grss F. rur remine unffete. This is in greement with the finings of the first experiment showing the zin emn of metllophytes n zin inepenene of grsses. Liming n lso reue the vilility of iron. In our experiment, the onentrtion of Fe 2+ in the pore wter erese lthough not signifintly (from 225 ± 35 to 127 ± 19 μmol L 1 ; p =.85). We lso i not fin signifint erese in the onentrtion of iron in the shoot mteril for ny of the three investigte plnt speies following liming (results not shown). The lortory experiments suggest tht the eline in metllophyte vegettion in tertiry pollute flooplin grsslns of the River Geul my hve een use y the eresing zin vilility n/or inresing P vilility in the grsslns ourring sine the losure of the metl inustry n intensifition of griulturl tivities in the re in the previous entury. Pyrite-ering mine wstes t neutrl or slightly lkline ph n oxiise within months or few yers to proue extreme iity [53] n in the pst will hve le to iifition of the grsslns fter flooing events. Beuse of the losure of the mining inustry, potentilly iifying pyrite prtiles were no longer present in the river wter or h eome intive fter some yers [54]. A higher lklinity in the soil, leing to lower Zn vilility, will hve een itionlly stimulte y the inrese use n pplition of rtifiil lime-ontining fertiliser in griulture [19]. As result of these proesses, growth of metl-emning metllophyte vegettion my hve een hmpere. Owing to the inrese pplition of fertiliser leing to inrese P vilility, more ompetitive grsses inluing H. lntus n F. rur oul hve eome ominnt irrespetive of the zin onentrtion in the soil. A stuy rrie out t 23 sites in the Hrz Mountins in Germny inite tht the speifi metllophyte vegettion of reltively metl-rih seonry pollute mine spoils ws not only ontrolle y metl vilility ut lso y low soil fertility. It ws shown tht metllophyte vegettion types with some H. lntus over were positively orrelte with nitrogen vilility in the soil [3]. This inites tht inresing nutrient inputs might e thret to metllophyte vegettion in generl. It is likely tht metllophytes my e more expose to thret in low-metl pollute soils like tertiry pollute flooplin grsslns thn t high metl-pollute soils (like seonry pollute mine spoil res) s the first my e more esily reuse for griulturl purposes. Erlier fiel investigtions n the urrent lortory investigtions inite tht removl of the P- n C-enrihe top soil lyer might e n option to restore metllophyte vegettion in flooplin grsslns with high Zn ontent in the soil profile (>4 μmol g 1 w n Zn/C >.8).

14 286 E.C.H.E.T. Lussen et l. In ft, smll-sle restortion experiments rrie out in flooplins of the River Geul hve shown tht removl of the lkline n phosphte-rih soil top lyer enles reolonistion of introue metllophytes n suppression of grsses t lest uring the first four yers fter soil removl [21,22]. Aknowlegements The uthors wnt to thnk Mr Jelle Eygensteyn for his help with the hemil nlyses. Referenes Downloe By: [Vn er Vele, Gerr] At: 12:24 26 July 21 [1] W. Ernst, Shwermetllvegettion er Ere, Geootni selet, n V, Gustv Fisher Verlg, Stuttgrt, [2] A. Vn er Ent, Knsen voor herstel vn zinkflor in het oven-geull, De Levene Ntuur 18 (27), pp [3] W.H.O. Ernst, J.A.C. Verkleij, n H. Sht, Metl tolerne in plnts, At Bot. Neerl. 41 (1992), pp [4] J. Antonovis, A.D. Brshw, n R.G. Turner, Hevy metl tolerne in plnts, Av. Eol. Res. 7 (1971), pp [5] H. Wil n A.D. Brshw, The evolutionry effets of metlliferous n other nomlous soils in S. Centrl Afri, Evolution 31 (1977), pp [6] A.J. Shw, Hevy Metl Tolerne in Plnts: Evolutionry Aspets, CRC Press, Bo Rton, FL, 199. [7] S.N. Whiting, R.D. Reeves, n A.J.M. Bker, Conserving ioiversity: mining, metllophytes n ln relmtion, Min. Environ. Mnge. 1 (22), pp [8] S.N. Whiting, R.D. Reeves, D. Rihrs, M.S. Johnson, J.A. Cooke, F. Mlisse, A. Pton, J.A.C. Smith, J.S. Angle, R.L Chney, R. Ginohio, T. Jffré, R. Johns, T. MIntyre, O.W. Purvis, D.E. Slt, H. Sht, F.J. Zho, n A.J.M. Bker, Reserh priorities for onservtion of metllophyte ioiversity n their potentil for restortion n site remeition, Restor. Eol. 12 (24), pp [9] R. Ginohio n A.J.M. Bker, Metllophytes in Ltin Ameri: remrkle iologil n geneti resoure srely known n stuie in the region, Rev. Chil. Hist. Nt. 77 (24), pp [1] H. Leeners, The trnsport of hevy metls uring floo events in the pollute river Geul (The Netherlns), Hyrol. Pro. 3 (1989), pp [11] M.H. Stm, The ting of flooplin eposits with hevy metls 21 P n 137 Cs in the Geul thment (The Netherlns), Phys. Chem. Erth 24 (1999), pp [12] R. Swennen, I. Vn Keer, n W. De Vos, Hevy metl ontmintion in overnk seiments of the Geul river (Est Belgium): its reltion to former P Zn mining tivities, Environ. Geol. 24 (1994), pp [13] E. Heimns, Uit Ons Krijtln, W. Versluys, Amsterm, [14] F. Kurris n J. Pgnier, Botnish hemishe wrnemingen over e zinkvegettie in Epen, Ntuurhistorish Mnl 14 (1925), pp [15] J.P. Bizoux, F. Brevers, P. Meerts, E. Gritson, n G. Mhy, Eology n onservtion of Belgin popultions of Viol lminri, metllophyte with restrite geogrphi istriution, Belg. J. Bot. 137 (24), pp [16] B.P. Vn e Riet, E.C.H.E.T. Lussen, R. Boink, J.H. Willems, n J.G.M. Roelofs, OBN Previes Zinkflor, Report EC-LNV nr 25-Dk7-O, Expertiseentrum LNV, Duth Ministry of Agriulture, Nture Mngement n Fisheries, Ee Wgeningen, 25. [17] Th. Weevers, Iets over e vegettie vn Epen vn een soiologish eologish stnpunt, Ntur 1 (1933), pp [18] E. Heimns, Txonomi, phytogeogrphil n eologil prolems roun Viol lminri, the zin violet, Ntuurhistorish Genootshp Limurg 12 (1961), pp [19] D.J.W. Pool, De zinkvegettie vn het Geull, in Ons Krijtln Zui Limurg IV, KNNV Wetenshppelijke Meeelingen 76 (1968), pp [2] J.A.M. Jnssen n J.H.J. Shminée, Europen Nture in the Netherlns (in Duth), KNNV, Utreht, 23, pp [21] E.C.H.E.T. Lussen, J. Eygensteyn, R. Boink, A.J.P. Smolers, B.P. Vn e Riet, D.J.C. Kuijpers, n J.G.M. Roelofs, The eline of metllophyte vegettion in flooplin grsslns: implitions for onservtion n restortion, Appl. Veg. Si. 12 (29), pp [22] E.C.H.E.T. Lussen, J.G.M. Roelofs, n R. Boink, Herstel en herontwikkeling vn zinkvegettie, De Levene Ntuur 11 (29), pp [23] P. Hinsinger, Biovilility of soil inorgni P in the rhizosphere s ffete y root-inue hemil hnges: review, Plnt Soil 237 (21), pp [24] A.J.M. Vn er Smn, O.F.R. Vn Tongeren, n C.W.P.M. Blom, Growth n reproution of Rumex mritimus n Chenopoium rurum uner ifferent wterlogging regimes, At Bot. Neerl. 37 (1988), pp [25] M. Loosemore, A. Strzek, P. Hinsinger, n B. Jillr, Zin moilistion from ontminte soil y three genotypes of too s ffete y soil n rhizosphere ph, Plnt Soil 26 (24), pp [26] S.R. Olsen, C.V. Cole, F.S. Wtnee, n L.A. Den, Estimtion of ville phosphorus in soils y extrtion with soium ironte, USDA Cir 939, US Government Print Offie, Wshington, DC, 1954.

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