Iron addition as a shallow lake restoration measure: impacts on charophyte growth

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1 DOI /s SHALLOW LAKE ECOSYSTEMS Iron ddition s shllow lke restortion mesure: impcts on chrophyte growth Anne K. Immers Msh T. Vn der Snde Rene M. Vn der Znde Jeroen J. M. Geurts Ellen Vn Donk Elisbeth S. Bkker Received: 1 July 2011 / Accepted: 30 December 2011 Ó The Author(s) This rticle is published with open ccess t Springerlink.com Abstrct Eutrophiction hs cused decline of chrophyte species in mny shllow lkes in Europe. Even though externl inputs of phosphorus re declining, internl loding of P from the sediment seems to dely the recovery of these systems. Iron is useful chemicl binding gent to combt internl phosphorus loding. However, the effects of iron ddition on chrophytes re not yet known. In this study we experimentlly tested the potentil toxicity of iron(iii)chloride (FeCl 3 ) on two different chrophytes, Chr virgt Kützing nd Chr globulris Thuiller dded t the concentrtion of 20 g Fe m -2 nd 40 g Fe m -2 to the surfce wter. C. virgt growth ws not significntly ffected, wheres C. globulris growth significntly decresed with incresing iron concentrtions. Nonetheless, biomss of both species incresed in ll tretments reltive to strting conditions. The decrese of C. globulris biomss with high iron dditions my hve been cused by drop in ph nd lklinity in combintion with iron induced light limittion. Iron ddition over longer time scle, however, will not cuse this rpid drop in ph. Therefore, we conclude tht dding iron(iii)chloride in these mounts to the surfce wter of lke cn potentilly be useful restortion method. Keywords Chrophyte Mcrolge Iron Phosphte Shllow lke restortion Guest editors: Zhengwen Liu, Bo-Ping Hn & Rmesh D. Gulti / Conservtion, mngement nd restortion of shllow lke ecosystems fcing multiple stressors A. K. Immers (&) M. T. Vn der Snde R. M. Vn der Znde E. Vn Donk E. S. Bkker Deprtment of Aqutic Ecology, Netherlnds Institute of Ecology (NIOO-KNAW), Droevendlsesteeg 10, 6708 PB Wgeningen, The Netherlnds e-mil:.immers@nioo.knw.nl J. J. M. Geurts Institute for Wetlnd nd Wter Reserch, Heyendlseweg 135, 6525 AJ Nijmegen, The Netherlnds E. Vn Donk Institute of Environmentl Biology, Utrecht University, Pduln 8, 3584 CH Utrecht, The Netherlnds Introduction Submerged mcrophytes ply crucil role in the mintennce of wter trnsprency nd qutic biodiversity in shllow wter bodies (Timms & Moss, 1984; Scheffer et l., 1993). However, mcrophyte species seem to differ in the success t which they perform this role (Engelhrdt & Ritchie, 2001). Prticulrly the group of chrophytes (Chrcee) hs been documented to be more successful in mintining wter clrity thn for exmple Potmogeton species (Hrgeby et l., 2007, Ibelings et l., 2007, Bkker

2 et l., 2010). Chrophytes re green mcrolge, the closest ncestors of lnd plnts (Krol et l., 2001), which re known s species of high conservtion vlue (Lmers et l., 2006) nd re commonly found in cler, hrd, nd nutrient poor wter bodies of reltively high lklinity (Simons & Nt, 1996; Vn den Berg et l., 1998b; Kufel & Kufel, 2002). Under these conditions, chrophytes cn improve their own light climte by forming dense beds on the sediment surfce (Kufel & Kufel, 2002; Vn Donk & Vn de Bund, 2002), which hve high nutrient uptke, enhnce sedimenttion nd counterct fish or wind induced sediment resuspension (Scheffer et l., 1993; Vn den Berg et l., 1998; Vn den Berg et l., 1999; Kufel & Kufel, 2002). Chrophytes my lso directly reduce phytoplnkton nd periphyton growth by relesing llelopthic substnces (Mulderij et l., 2003). High nutrient loding nd subsequent increse in wter turbidity due to phytoplnkton surfce blooms hve led to decrese of chrophytes in mny shllow lkes in Europe (Vn den Berg et l., 1998, b; Klosowski et l., 2006; Lmbert & Dvy, 2010). Recent restortion mesures, where externl phosphorus (P) input nd wter turbidity were experimentlly reduced, hve led to the return of dense chrophyte beds (Vn den Berg et l., 1998; Meijer et l., 1999; Ibelings et l., 2007). These restortion mesures, however, were performed in sndy lkes, wheres pety lkes re suffering from high internl loding of P from the sediment nd re more prone to sediment resuspension (Cooke et l., 1993; Jeppesen et l., 1998; Søndergrd et l., 2003). Under nturl conditions, pety lkes in the Netherlnds would not suffer from internl P loding, s upwelling iron rich groundwter binds to phosphorus (in the form of phosphte, PO 4 ) in the sediment. This seepge, however, hs disppered over the yers due to high regionl nd locl use of groundwter (Smolders & Roelofs, 1996; Vn der Welle et l., 2007). Wter mngers hve tried to resolve this problem by dding iron (Fe), in the form of iron(iii)chloride, to the lke sediment s nturl P binding gent (Cooke et l., 1993; Boers et l., 1994; Burley et l., 2001). In this wy, the iron would not only precipitte with the vilble P in the sediment, but would lso form brrier on the top lyer of the sediment, preventing internl P loding of the lke in the future. However, lke restortion by dding iron in the lke sediment is costly nd time consuming process, therefore dding iron to the surfce wter my be more fesible in cse of restortion of whole lke. The effect of this iron ddition, nd the consequentil potentil drop in ph, on vrious orgnisms in the qutic food web is not yet well studied, wheres it is very importnt to know whether iron ddition my be hrmful for the trget species tht re imed to return to the restored lke. Chrophytes re desirble species for wter mngers to grow in lke s they re indictors of good wter qulity (Lmbert & Dvy, 2010) nd hve been shown to return in pet lkes fter restortion mesures hd been tken including externl nutrient reduction (Rip et l., 1992) nd biomnipultion (Ter Heerdt & Hootsmns, 2007). As chrophytes primrily utilize nutrients from the wter column insted of the sediment (Kufel & Kufel, 2002; Hidding et l., 2010), possible effects of iron on chrophytes would be more pronounced when dding iron in the wter column. The im of this study ws to test whether iron ffects the growth, biomss lloction nd nutrient concentrtion of two different chrophyte species. The experiment ws bsed upon the sitution of Lke Terr Nov, the Netherlnds, in which this method of FeCl 3 ddition to the surfce wter is now being pplied. Methods Experimentl set-up Mesocosm experiments were performed in My 2010 in 45 Perspex cylinders (d 9 h = cm) which were plced in temperture controlled culture room t the NIOO-KNAW in Nieuwersluis. Temperture ws kept constnt t 19 C nd light regime ws set t 12 h light nd 12 h drkness with light intensity t the wter surfce of 100 ± 5 lmol photons m -2 s -1. Ech cylinder ws filled up with 0.50 l pet sediment, collected on April 2010 in Lke Terr Nov ( N, E, The Netherlnds), nd subsequently very crefully 3.25 l of filtrted (0.2 lm, ME 24, Whtmn, Brentford, UK) Terr Nov wter ws poured on the sediment. To enble pore wter smpling, Rhizon soil moisture smplers (Eijkelkmp Agriserch Equipment, Giesbeek, The Netherlnds) ttched to 50 ml vcuum syringes were inserted into the upper lyer of the sediment.

3 During the experiment we mnipulted two fctors: nmely the iron ddition nd the plnts on which the effects of iron ddition were tested. The iron nd plnt tretments consisted ech of three levels. The effects of iron ddition were tested during 5 weeks, with three different levels of iron which would correspond to dditions in Lke Terr Nov of 20 g Fe m -2 (low) nd 40 g Fe m -2 (high) in the form of FeCl 3 nd control ddition (0 g Fe m -2 ) ws designed which received NCl in equl molr mounts of chloride in the high iron dditions. The plnt tretment levels consisted of cylinders filled with C. virgt Kützing, Chr globulris Thuiller nd empty cylinders. All nine combintions of levels were experimentlly tested with five replictes, which were rndomized in blocks. Chr virgt ws collected from experimentl ponds in Loenderveen ( N, E, The Netherlnds) on 29 April C. globulris ws prior to the experiment grown in quri from propgules in Terr Nov sediment. A bundle composed of 3 C. virgt shoots ws plnted in the sediment of 15 cylinders (totl FW per cylinder 0.16 ± 0.04 g), bundle of 3 C. globulris shoots in 15 other cylinders (totl FW per cylinder 0.89 ± 0.38 g), nd the lst 15 cylinders were not plnted with mcrolge s controls. To distinguish between the effects of iron toxicity nd P limittion we reduced P in control iron dditions t the onset of the experiment with low dose of 0.33 mg FeCl 3 per cylinder. During the experiment, iron ws dded two times every week on 8 ddition dys, which corresponds to the low nd high iron ddition of nd mg FeCl 3 per ddition dy, respectively. Smpling nd nlysis Once every week during the experiment, 35 ml smples of surfce wter were tken from ech cylinder for chemicl nlyses. A subsmple of 10 ml from ech cylinder ws filtrted over Whtmn GF/C (1.2 lm) filters nd subsequently stored t -20 C before nutrient nlysis. The remining 25 ml subsmple ws used to mesure ph nd lklinity with TIM840 titrtion mnger (Rdiometer Anlyticl, Copenhgen, Denmrk). Alklinity ws determined by titrting with 0.01 M HCl down to ph 4.2. The stored 10 ml subsmples were used to colorimetriclly determine PO 4,NH 4, nd NO 3 with QuAAtro CFA flow nlyzer (Sel Anlyticl, Norderstedt, Germny). During the lst smple dy, in ddition to prior nlyses, 50 ml of sediment pore wter smples were collected from ech cylinder using Rhizon soil moisture smplers. Smples were stored in 50 ml centrifuge tubes t -20 C directly fter the pore wter hd been collected. The sme volume of surfce wter ws, prior to storge in 50 ml centrifuge tubes t -20 C, filtrted over 0.45 lm membrne filter (ME 25, Whtmn, Brentford, UK). Membrne filters tht were used were fterwrd dried for 24 h t 60 C nd lter stored in 50 ml centrifuge tubes t -20 C. Anlyses of stored smples were performed using n inductively coupled plsm emission spectrophotometer (ICP; Liberty 2, Vrin, Bergen op Zoom, The Netherlnds) ccording to the Dutch NEN-EN-ISO to estimte dissolved Fe, Al, C, nd S in surfce nd pore wter. The sme method ws used to mesure precipitted Fe in the surfce wter, which ws prior to nlysis collected by filtrtion of surfce wter on 0.45 lm membrne filters (ME 25, Whtmn, Brentford, UK), tht were subsequently treted with 8 ml nitric cid (2 M). At the end of the experiment, ±3 cm of shoot mteril from ech cylinder ws plced in plstic cup with 20 ml of deminerlized wter for periphyton determintion following Zimb & Hopson (1997). Ech cup ws shken gently for 1 min nd subsequently shoot mteril ws tken out, dried for 24 h t 60 C nd weighed. Deminerlized wter with periphyton ws filtered over Whtmn GF/C (1.2 lm) filter, nd fterwrd filters were dried for 24 h t 60 C nd weighed. Subsequently ll chrophytes were hrvested nd seprted in bove- nd belowground mteril. All mteril ws dried for 24 h t 60 C, dried shoots from periphyton determintion were dded nd subsequently ll mteril ws weighed to determine the totl bove- nd belowground dry weight. Totl dry weight t the strt of the experiment ws clculted with conversion fctor, which ws cquired from the fresh nd dry weight of severl subsmples (for C. virgt dry weight = 30% of fresh weight, for C. globulris dry weight = 18% of fresh weight). A homogenized portion of dry chrophyte mteril ws used to determine both C nd N concentrtions with FLASH 2000 Orgnic Elementl Anlyzer (Interscience, Bred, The Netherlnds). Chrophyte P concentrtions were cquired by incinerting homogenized dry mteril for 30 min t

4 500 C, followed by digestion in H 2 O 2 (Murphy & Riley, 1962) before nlysis with QuAAtro CFA flow nlyzer. Sttisticl nlysis Sttisticl nlyses were crried out with SPSS 18.0 (SPSS, Chicgo, IL, USA). Differences between tretments for plnt biomss, shoot:rhizoid rtio nd plnt nutrient composition were tested with one-wy ANOVA s with iron tretment s fixed fctor followed by Tukey s post hoc test. Differences in chemicl vribles nd periphyton growth were tested with two-wy ANOVA s with iron tretment nd plnt tretment (consisting of the levels C. virgt, C. globulris or empty cylinders) s fixed fctors followed by Tukey s post hoc test. Prior to nlysis, ll dt were tested for normlity nd homogeneity of vrince, nd if necessry, dt were log 10 trnsformed. For dt tht hd no norml distribution, even fter trnsformtion, non-prmetric Kruskl Wllis test ws used with Sttistic 9.1 (SttSoft Inc., Tuls, OK, USA) to nlyze vrinces. Results were expressed s men ± stndrd error of men nd P B 0.05 ws ccepted for sttisticl significnce. Results Chrophyte response Both chrophyte species biomss incresed notbly over the 5 weeks tht the experiment rn. C. virgt experienced on verge fourfold increse, from 0.05 ± 0.00 to 0.20 ± 0.02 g dry weight, wheres C. globulris, which strted with higher men biomss of 0.15 ± 0.02 g dry weight, incresed on verge threefold to 0.51 ± 0.04 g dry weight. Iron dditions hd different effects on the two species (Fig. 1). C. virgt bove ground nd below ground biomss were not significntly ffected by iron dditions (Tble 1), lthough t the highest level of iron ddition C. virgt biomss tended to be somewht lower (Fig. 1). The growth of C. globulris, however, ws negtively ffected by iron dditions (Fig. 1). C. globulris below ground mteril, which only on verge mde up 6% of totl biomss, did not differ between iron dditions, but bove ground mteril ws considerbly lower in cylinders which Fig. 1 Biomss increse (verge ± SEM) in rection to iron ddition fter 5 weeks for Chr virgt nd Chr globulris. White, grey, nd blck brs represent, respectively, dditions of 0, 20, nd 40 g Fe m -2. Significnt differences between iron dditions re indicted for ech species seprtely by different letters (Anlysis of vrince, Tukey test, P B 0.05) received iron compred to cylinders in which no iron ws dded (Tble 1). Totl biomss, which ws on verge composed of 94% bove ground mteril thus decresed with incresing iron concentrtions (Tble 1). Biomss lloction of both C. virgt nd C. globulris ws not ffected by iron ddition, s chrophyte shoot:rhizoid rtio did not differ between iron dditions (Tble 1). Tissue nutrient concentrtions for C. virgt incresed significntly during the experiment for N nd P, respectively, from ± 0.35 to men end concentrtions of ± 1.14 mg N g dry weight -1 nd from 1.05 ± 0.01 to men end concentrtions of 1.76 ± 0.06 mg P g dry weight -1. Different iron dditions, however, did not induce ny differences in N or P concentrtions nd their reltive rtios in this chrophyte (Tble 1). This reltionship ws not seen in the tissue of C. globulris, where the control iron ddition (0 g Fe m -2 ) remined similr to the strt conditions (1.18 ± 0.01 mg P g dry weight -1 nd ± 0.52 mg N g dry weight -1 ) nd only the iron dditions of 20 nd 40 g Fe m -2 induced significnt increse in N nd P concentrtions nd their reltive rtios (Tble 1). The mount of periphyton, the reddish colored mteril growing on the chrophyte shoots (Fig. 2), ws clerly ffected by iron dditions. For cylinders contining C. virgt, the high iron ddition (40 g Fe m -2 ) yielded significntly more periphyton thn the low iron ddition (20 g Fe m -2 ). Cylinders

5 Tble 1 Men (± sem) end results of chrophyte biomss, growth, shoot:rhizoid rtio nd nutrient composition of C. virgt nd C. globulris t different iron dditions Men ± SEM Effect iron mount df = 2,14 0gFem g Fe m g Fe m -2 F P C. virgt Biomss below ground (g) 0.03 ± ± ± Biomss bove ground (g) 0.19 ± ± ± Totl biomss (g) 0.22 ± ± ± Totl biomss increse (g) 0.17 ± ± ± Shoot:rhizoid rtio (g g -1 ) 0.87 ± ± ± C (mg g dryweight -1 ) ± ± ± N (mg g dryweight -1 ) ± ± ± P (mg g dryweight -1 ) 1.81 ± ± ± C:N rtio (mol mol -1 ) ± ± ± N:P rtio (mol mol -1 ) ± ± ± Periphyton (g g dryweight -1 ) 0.38 ± 0.06 b 0.21 ± ± 0.07 b C. globulris Biomss below ground (g) 0.03 ± ± ± Biomss bove ground (g) 0.65 ± ± 0.02 b 0.34 ± 0.03 b <0.001 Totl biomss (g) 0.69 ± ± 0.02 b 0.39 ± 0.02 b <0.001 Totl biomss increse (g) 0.51 ± ± 0.01 b 0.23 ± 0.01 c <0.001 Shoot:rhizoid rtio (g g -1 ) 0.96 ± ± ± C (mg g dryweight -1 ) ± ± ± N (mg g dryweight -1 ) ± ± 1.20 b ± 1.70 b P (mg g dryweight -1 ) 1.10 ± ± 0.01 b 1.46 ± 0.03 c <0.001 C:N rtio (mol mol -1 ) ± ± 0.83 b ± 0.48 b N:P rtio (mol mol -1 ) ± ± 1.90 b ± 1.80 b Periphyton (g g dryweight -1 ) 0.17 ± ± 0.08 b 0.81 ± 0.18 b Dt were nlyzed with one-wy ANOVA with the levels of iron tretment (0, 20, or 40 g m -2 ) s fixed fctor, n = 5. Significnt differences between iron dditions re indicted for ech species seprtely by different letters (nlysis of vrince, Tukey test, P B 0.05). Bold vlues indicte P B 0.05 contining C. globulris, on the other hnd, showed no difference in periphyton biomss between the iron dditions, but the high iron ddition hd considerbly more periphyton biomss thn the control iron ddition (0 g Fe m -2 ; Tble 1). Moreover, during the experiment lrge number of chrophyte propgules sprouted from the sediment, which did not seem to be ffected by the different iron dditions. Chnges in wter properties Surfce wter ph decresed significntly due to iron dditions nd t the end of the experiment surfce wter ph reched men vlues of 6.95 ± 0.17 in the high iron dditions, 7.81 ± 0.13 in the low iron dditions nd men vlues of 8.35 ± 0.22 in the control dditions (Tble 2; Fig. 3). Alklinity showed the sme reltionship with low men vlues of 0.62 ± 0.04 meq l -1 in the high iron dditions, 0.95 ± 0.08 meq l -1 for the low iron dditions nd the highest men vlues of 1.55 ± 0.20 meq l -1 in the control dditions. Moreover, lklinity lso differed between the chrophyte species, with significnt lower lklinity of 0.62 ± 0.03 meq l -1 in the C. globulris cylinders compred to the empty cylinders or cylinders with C. virgt (1.25 ± 0.16 nd 1.24 ± 0.18 meq l -1 ; Tble 2; Fig. 3b).

6 () b Fig. 2 Periphyton mteril on shoots in g g dry weight -1 (verge ± SEM) in rection to different iron dditions. Periphyton my include other mteril such s precipitted iron. White, grey, nd blck brs represent, respectively, dditions of 0, 20, nd 40 g Fe m -2. Significnt differences between iron dditions re indicted for ech species seprtely by different letters (nlysis of vrince, Tukey test, P B 0.05). Pictures tken t the end of the experiment of Chr globulris receiving, b 0 g Fe m -2, nd c 40 g Fe m -2 (b) (c) Iron nd luminum concentrtions in the surfce wter decresed with higher iron dditions, however, concentrtions in the surfce wter were very low with men iron concentrtions rnging between 0.37 ± 0.05 nd 0.14 ± 0.04 lmol Fe l -1 nd men luminum concentrtions rnging between 1.93 ± 0.15 nd 0.21 ± 0.05 lmol Al l -1. This difference ws possibly due to the precipittion of iron with phosphte, however, phosphte concentrtions did not differ between iron nd control dditions, s P ws reduced in the control dditions (0 g Fe m -2 ) t the onset of the experiment. Iron nd phosphte concentrtions in the pore wter showed the sme rtio with the different iron dditions. As result Fe:PO 4 rtios in sediment, which re often used s tool to determine internl phosphorus loding, reched men vlues of ± 4.21 mol mol -1, but did not differ significntly between the iron dditions. Phosphte lso seemed to be lower in the surfce wter of the cylinders contining C. globulris where P decresed to men vlues of 0.05 ± 0.00 lmol l -1 compred to cylinders with C. virgt (0.08 ± 0.01 lmol l -1 ) nd empty cylinders (0.08 ± 0.01 lmol l -1 ), however, this difference ws not significnt (Tble 2; Fig. 3c). Precipitted iron, which ws mesured in the surfce wter, reched highest vlues in the cylinders which contined no chrophytes (Fig. 3d). No difference ws found for precipitted iron between iron dditions. Nitrogen, in the form of NO 3 nd NH 4, decresed significntly during the experiment in the surfce wter of ll cylinders. Nitrte showed cler significnt reltionship for the type of chrophyte presence in cylinders, with constntly lower vlues (pproching 0) in cylinders with C. globulris compred to higher vlues in empty cylinders nd cylinders with C. virgt (Tble 2; Fig. 3e). Ammonium reched highest men concentrtions in cylinders contining

7 Tble 2 Results of nlysis of the effects of iron ddition on surfce nd pore wter nutrient composition Effect Iron mount Mcrophyte species Iron x Mcrophyte df = 2,36 df = 2,36 df = 4,36 F/H P F/H P F/H P Surfce wter ph < Alklinity < < <0.001 Fe < Fe (precipitted) Al < <0.001 PO NO < <0.001 NH < <0.001 C S Pore wter Fe Al < PO 4 Fe:PO 4 NO 3 NH < C S Dt were nlyzed with two-wy ANOVA (F) or non-prmetric Kruskl Wllis (H) with the levels of iron tretment (0, 20, or 40 g m -2 ) nd the levels of plnt tretment (Chr virgt, Chr globulris or empty cylinders) s fixed fctors, n = 5. Bold vlues indicte P B 0.05 Non-prmetric Kruskl Wllis test (H) performed insted of ANOVA (F) C. virgt ( ± 0.42 lmol l -1 ), which differed significntly from cylinders contining C. globulris ( ± 0.20 lmol l -1 ) nd empty cylinders ( ± 0.19 lmol l -1 ; Fig. 3f). No significnt differences were found between tretments for clcium nd sulfur nd concentrtions remined constnt t ± nd ± lmol l -1 for C nd S, respectively. Over ll, pore wter nutrient concentrtions seemed to be less ffected by the presence/ bsence of chrophyte species (Tble 2). Discussion The decrese of C. globulris biomss with incresing iron concentrtions might be relted to iron toxicity. Negtive effects of iron ddition on the growth of mcrophytes re usully distinguished in two different kinds, nmely direct nd indirect (Wheeler et l., 1985). According to Vn der Welle et l. (2007), direct effects of iron toxicity cn be seen in the physicl structure of plnts. It cn ct on the leves by reducing the size or by the formtion of blck necrotic spots or complete discolortion of leves nd even die-bck of old leves, or in roots which cn blcken, stop growing or lck brnching (Vn der Welle et l., 2006). Other described unfvorble effects were the formtion of iron plques on roots, which could prevent plnt nutrient uptke (Vn der Welle et l., 2007). These physicl symptoms, indicting direct iron toxicity could not be detected in our experiment with C. virgt nd C. globulris. Chrophytes differ gretly from vsculr mcrophytes in hving only rhizoid system, on which they do not rely on for

8 () (b) (c) (d) (e) (f) Fig. 3 Surfce wter (), ph (b), lklinity (c), PO 4 (d), precipitted Fe (e), NO 3, nd (f)nh 4 concentrtions in meq l -1 nd lmol l -1 (verge ± SEM) fter 5 weeks for the different plnt tretment levels under different iron dditions. White, nutrient uptke (Kufel & Kufel, 2002). These processes of direct iron toxicity s found in vsculr mcrophytes therefore my not pply for chrophytes. For most higher plnt species, iron cn hve n indirect negtive effect on growth by minly limiting the mcronutrient P due to the precipittion of phosphte with iron (Wheeler et l., 1985). According to grey, nd blck brs represent, respectively, cylinders receiving iron dditions of 0, 20, nd 40 g F m -2. Significnt differences between iron dditions re indicted for ech species seprtely by different letters (Kruskl Wllis, P B 0.05) Koerselmn & Meulemn (1996), mcrophytes re P limited t N:P rtios mesured in plnt biomss bove 16 nd N limited t N:P rtios below 14. Chrophytes, however, re usully only found in lkes with low inorgnic P concentrtions (Bloemendl & Roelofs, 1988; Simons & Nt, 1996), nd re known to give wy to higher plnts with incresing phosphorus

9 concentrtions (Kufel & Kufel, 2002; Lmbert & Dvy, 2010). Moreover, for chrophyte species, the mesured concentrtions of the mcronutrients N nd P in plnt mteril not only vries gretly between species, it lso differs within species, nd usully only gives n indiction of the environment in which the chrophytes re growing (Kufel & Kufel, 2002). In our experiment N nd P concentrtions in C. globulris incresed with incresing iron ddition wheres this did not hppen in C. virgt, t lest not significntly. For both species, the N:P rtio ws bove 16, suggesting P limittion if this threshold cn be used for Chrcee. However, if considering ctul concentrtions for both N nd P, both species were lwys bove limiting levels of 13 nd 1.3 mg g dryweight -1 (Gerloff & Krombholz, 1966) for N nd P respectively, indicting tht these plnts were not limited by these nutrients. Mesurements of wter nutrients did not show evidence of incresing P limittion s well, but indicted strong reduction of nitrte by C. globulris reltive to C. virgt nd the cylinders with no plnts, wheres there were no differences in phosphte. Most reserch on chrophyte growth limittion hs focused on the effects of P, but recently Lmbert & Dvy (2010) showed tht N, prticulrly nitrte, my strongly ffect growth nd bundnce of Chrcee. The ccumultion of N nd P in the tissue of C. globulris in our study my be explined by the reduced growth of this species t higher iron ddition, which would simultneously explin the lck of significnt chnges in N nd P concentrtion in C. virgt tissue s this species did not experience significnt growth reduction with iron ddition. As less biomss is formed, nutrients my ccumulte in plnt tissue. Reduced growth cn in this cse be the result of toxic effects of iron, or the fct tht other fctors hve become limiting. In ddition to nutrients, light cn be limiting fctor of plnt growth. Another indirect negtive effect of iron ddition could be the formtion of iron precipittes nd their shding effect on shoots. No differences were found between iron dditions for the presence of precipitted iron; however, precipitted iron ws only mesured in surfce wter nd not on chrophyte shoots, cylinders or on the sediment surfce. Most of the iron could hve ccumulted on these surfces s ironphosphtes or iron oxides. The mount of mesured periphyton mteril on shoots did show reltion with iron concentrtions, s highest periphyton biomss for both species in the high iron dditions. Wheres the method of shking plnt shoots is commonly pplied to quntify periphyton biomss on the plnts, other mteril on the leves, such s the iron precipittes is included in this mesurement. When looking t the color of the periphyton nd the difference between periphyton in the high iron nd in the control dditions, the reddish colored periphyton in iron dditions does most probbly contin iron precipittes. For chrophytes, light is crucil fctor for growth (Kufel & Kufel, 2002; Rip et l., 2007). Consequently, dense growth of periphyton nd iron precipittion could hve limited chrophyte growth in high iron dditions. The ddition of iron lso resulted in decrese in ph nd lklinity in the cylinders receiving high iron dditions. Even though the ph styed well within the optiml rnge of 5 7 for mximl iron phosphte binding cpcity (Cooke et l., 1993), the lower ph nd lklinity were suboptiml for the chrophytes, s they require high ph nd high lklinity of the surfce wter (Vn den Berg et l., 1998b; Klosowski et l., 2006; Lmbert & Dvy, 2010). Not only ws there significnt difference in lklinity between the different iron dditions, there ws lso difference between chrophyte species. Cylinders contining C. globulris proved to hve lesser buffer cpcity thn empty cylinders nd cylinders contining C. virgt. This difference might well explin the difference in iron sensitivity, where C. globulris ws considerbly more ffected by iron dditions thn C. virgt. According to Vn den Berg et l. (2002), growth of chrophytes is strongly correlted to the bicrbonte (HCO 3 - ) concentrtions in the wter. The inbility of C. globulris to mintin the buffer cpcity in combintion with light limittion could therefore hve resulted in decresing photosynthesis rtes nd stedy drop in ph in cylinders of the iron dditions due to the quick ddition of iron. Iron s mesure to control eutrophiction The gol of dding Fe to the surfce wter of lkes is to lower surfce wter P nd to control internl P relese. The binding cpcity of Fe, however, is regulted by the redox stte of the gent (Burley et l., 2001). Under oxic conditions, oxidized ferric iron (Fe 3? ) cn freely precipitte with PO 4, but under noxic conditions, reduced ferrous iron (Fe 2? ) is formed nd Fe loses this binding cpcity nd consequently PO 4 will be relesed (Cooke et l., 1993). Chrophytes re ble

10 to oxidize the sediment, thereby preventing this redoxrection to occur (Kufel & Kufel, 2002). Moreover, the possibility for chrophytes to use bicrbonte s crbon source for photosynthesis leds to the formtion of crbonte, which in turn cn precipitte with clcium to form clcite (Otsuki & Wetzel, 1972). Clcite cn subsequently co-precipitte with phosphte, which is redox-insensitive rection (Otsuki & Wetzel, 1972). Chrophytes cn thus enhnce the binding cpcity of iron. The negtive effects of the ddition of 40 g Fe m -2 on C. globulris biomss my hve prtly been due to the fct tht iron ws dded over short period of 5 weeks. When using iron ddition s lke restortion mesure, the choice cn be mde for ddition distributed over longer time period. Moreover, drop in ph nd lklinity s observed in this experiment will probbly not occur in lke such s Terr Nov with the sme mount of iron, s the wter column bove the sediment is much lrger, nd therefore negtive consequences of iron ddition such s drop in ph nd lklinity would be much less drmtic (Boers et l., 1994). From the fct tht both species rected differently on iron ddition it might follow tht fter iron ddition, lkes would become dominted by more iron tolernt species, which could possibly cuse shift in community composition. However, the fct tht the ddition of iron to fresh wter ecosystem will reduce the phosphte concentrtion in the wter nd sediment by forming Fe-trpping brrier on the sediment wter interfce will be fvorble to push the equilibrium towrds cler, chrophyte-dominted ecosystem. And s chrophyte estblishment ws not hmpered by the iron lyer on the sediment, dense chrophyte beds cn provide positive feedbck loop resulting in resilient, cler wter stte. Acknowledgments The uthors would like to thnk Thijs de Boer, Koos Swrt, nd Mrtijn Dorenbosch for their prcticl ssistnce in the field nd Nico Helmsing nd Hrry Korthls for performing multiple chemicl nlyses in the lb. This study ws funded by the Wter Frmework Directive Innovtion Fund from Agentschp NL from the Dutch Ministry of Economic Affirs, Agriculture nd Innovtion. Open Access This rticle is distributed under the terms of the Cretive Commons Attribution Noncommercil License which permits ny noncommercil use, distribution, nd reproduction in ny medium, provided the originl uthor(s) nd source re credited. References Bkker, E. S., E. Vn Donk, S. A. J. Declerck, N. R. Helmsing, B. Hidding & B. A. Nolet, Effect of mcrophyte community composition nd nutrient enrichment on plnt biomss nd lgl blooms. Bsic nd Applied Ecology 11: Bloemendl, F. H. J. L. & J. G. M. Roelofs, Wterplnten en wterkwliteit. Koninklijke Nederlndse NtuurhistorischeVereniging, Utrecht, The Netherlnds (in Dutch). Boers, P., J. Vn der Does, M. Quk & J. Vn der Vlucht, Phosphorus fixtion with iron(iii)chloride: new method to combt phosphorus loding in shllow lkes? Archiv für Hydrobiologie 129: Burley, K. L., E. E. Preps & P. A. Chmbers, Phosphorus relese from sediments in hrdwter eutrophic lkes: the effects of redox-sensitive nd insensitive chemicl tretments. Freshwter Biology 46: Cooke, G. D., E. B. Welch, A. B. Mrtin, D. G. Fulmer, J. B. Hyde & G. D. Schrieve, Effectiveness of Al, C, nd Fe slts for control of internl phosphorus loding in shllow deep lkes. Hydrobiologi 253: Engelhrdt, K. A. M. & M. E. Ritchie, Effects of mcrophyte species richness on wetlnd ecosystem functioning nd services. Nture 411: Gerloff, G. C. & P. H. Krombholz, Tissue nlysis s mesure of nutrient vilbility for the growth of ngiosperm qutic plnts. Limnology nd Ocenogrphy 11: Hrgeby, A., I. Blindow & G. Andersson, Long-term ptterns of shifts between cler nd turbid sttes in Lke Krnkesjön nd Lke Tåkern. Ecosystems 10: Hidding, B., R. J. Brederveld & B. A. Nolet, How bottom-dweller bets the cnopy: inhibition of n qutic weed (Potmogeton pectintus) by mcrolge (Chr spp.). Freshwt. Biol. 55: Ibelings, B. W., R. Portielje, E. H. R. R. Lmmens, R. Noordhuis, M. S. Vn den Berg, W. Joosse & M.-L. Meijer, Resilience of lterntive stble sttes during recovery of shllow lkes from eutrophiction: Lke Veluwe s cse study. Ecosystems 10: Jeppesen, E., T. L. Luridsen, T. Kireslo & M. R. Perrow, 1998.Impct of submerged mcrophytes on fish-zooplnkton interctions in lkes. In Jeppesen, E., M. Søndergrd, M. Søndergrd & K. Christoffersen (eds), The Structuring Role of Submerged Mcrophytes in Lkes. Ecologicl Studies. Springer Verlg, New York 131: Krol, K. G., R. M. McCourt, M. T. Cimino & C. F. Delwiche, The closest living reltives of lnd plnts. Science 294: Klosowski, S., G. H. Tomszewicz & H. Tomszewicz, The expnsion nd decline of chrophyte communities in lkes within the Sejny Lke District (north-estern Polnd) nd chnges in wter chemistry. Limnologic 36: Koerselmn, W. & A. F. M. Meulemn, The vegettion N:P rtio: new tool to detect the nture of nutrient limittion. Journl of Applied Ecology 33: Kufel, L. & I. Kufel, Chr beds cting s nutrient sinks in shllow lkes review. Aqutic Botny 72:

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