REGULAR ARTICLE

Considerations on the shuttle mechanism of FeEDDHAchelates at the soil-root interface in case of Fe deficiency

Walter D. C. Schenkeveld & Arjen M. Reichwein &

Erwin J. M. Temminghoff & Willem H. van Riemsdijk

Received: 12 August 2013 /Accepted: 6 February 2014 /Published online: 9 March 2014# Springer International Publishing Switzerland 2014

AbstractAim A mechanism of action for the performance of Fechelates as soil-applied fertilizer has been hypothesizedby Lindsay and Schwab (J Plant Nutr 5:821–840, 1982),in which the ligand participates in a cyclic process ofdelivering Fe at the root surface and mobilizing Fe fromthe soil. This “shuttle mechanism” seems appealing inview of fertilizer efficiency, but little is known about itsperformance. The chelate FeEDDHA is a commonlyused Fe fertilizer on calcareous soils.Methods In this study, the performance of the shuttlemechanism has been examined for FeEDDHA chelatesin soil interaction and pot trial experiments.Results The specificity of EDDHA ligands for chelatingFe from soils of low Fe availability is limited.

Experimental support for a shuttle mechanism in soil-plant systems with FeEDDHAwas found: specific metalmobilization only occurred upon FeEDDHA-facilitatedFe uptake. The mobilized metals originated at least inpart from the root surface instead of the soil.Conclusion The results from this study support theexistence of a shuttle mechanism with FeEDDHA in soilapplication. If the efficiency of the shuttle mechanism ishowever largely controlled by metal availability in thebulk soil, it is heavily compromised by complexation ofcompeting cations: Al, Mn and particularly Cu.

Keywords CuEDDHA . EDDHA isomers .

FeEDDHA . Iron chelates . Iron chlorosis . Shuttlemechanism

Abbreviationso,o-FeEDDHA Iron (3+) ethylene diamine-N,N′-

bis(2-hydroxy phenyl acetic acid)complex

o,p-FeEDDHA Iron (3+) ethylene diamine-N-(2-hydroxy phenyl acetic acid)-N′-(4-hydroxy phenyl acetic acid) complex

DOC Dissolved organic carbonDTPA Diethylene triamine penta acetic acidEDTA Ethylene diamine tetra acetic acidICP-MS/AES Inductively coupled plasma mass

spectrometry/atomic emissionspectrometry

SOC Soil organic carbonSOM Soil organic matterHFO Hydrous ferric oxide

Plant Soil (2014) 379:373–387DOI 10.1007/s11104-014-2057-1

Responsible Editor: Jian Feng Ma.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-014-2057-1) contains supplementarymaterial, which is available to authorized users.

W. D. C. Schenkeveld : E. J. M. Temminghoff :W. H. van RiemsdijkDepartment of Soil Quality, Wageningen University,P.O. Box 47, 6700 AAWageningen, The Netherlands

A. M. ReichweinDepartment CFC-CMS, AkzoNobel Chemicals BV,P.O. Box 10, 7400 AA Deventer, The Netherlands

W. D. C. Schenkeveld (*)Department of Environmental Geosciences, Center for EarthScience, 1090 WienAlthanstraße 14 (UZA II), Austriae-mail: walter.schenkeveld@univie.ac.at

Introduction

Fe deficiency chlorosis is a common nutrient disor-der in plants grown on calcareous soils. It is char-acterized by a significant decrease in chlorophyllcontent in the leaves and impacts growers returnsby decreasing crop yield and quality (Chaney 1984;Mortvedt 1991).

The limited bioavailability of Fe in calcareous soilsarises from the poor solubility of Fe(hydr)oxides at highsoil pH (7–8.5) and the presence of a bicarbonate buffer(Boxma 1972; Lindsay 1979). The most common prac-tice to prevent and remedy Fe chlorosis is through theapplication of synthetic Fe chelates, which can greatlyincrease the Fe concentration in soil solution.FeEDDHA (iron (3+) ethylene diamine-N,N ′-bis(hydroxy phenyl acetic acid)) is among the mosteffective chelates in soil application (Reed et al. 1988;Lucena 2003).

Commercially available FeEDDHA products consistof a mixture of FeEDDHA components. The quantita-tively most important components are the isomers race-mic o,o-FeEDDHA, meso o,o-FeEDDHA ando,p-FeEDDHA. The physical and chemical propertiesof these isomers differ and, as a consequence, so doestheir effectiveness in delivering Fe to the plant.

Lindsay and Schwab (1982) proposed a mechanismof action for the performance of Fe chelates in soils, inwhich the chelating agent, i.c. EDDHA, ideally func-tions as a shuttle transporter for Fe between soil particlesand plant roots (Fig. 1). This mechanism was laterreferred to as “the shuttle effect” (Lucena 2003).Driving forces behind the mechanism are the concentra-tion gradients arising from chelate splitting, Fe uptakeand chelation of soil-Fe, which induce diffusive trans-port. The applicability of the shuttle mechanism conceptto the performance of FeEDDHA has been experimen-tally supported by the ability of EDDHA ligands tomobilize Fe from synthetic Fe oxides and soils (Pérez-Sanz and Lucena 1995; García-Marco et al. 2006), aswell as from an adsorbed Fe siderophore (ferrioxamineB) (Siebner-Freibach et al. 2003). In the latter study, Fewas actually subsequently transported to and taken upby plants grown in hydroponic culture.

Although the concept of recycling the chelating agentseems particularly appealing in view of fertilizer effi-ciency, hardly anything is known, about the actual per-formance of the shuttle mechanism in soil-plant sys-tems. In accordance with Lindsay and Schwab’s

concept, the effectiveness of the shuttle is determinedby the rates at which diffusive transport takes place,EDDHA ligands mobilize soil-Fe and plants take upFe from FeEDDHA chelates. The efficiency of theshuttle will however be compromised by processes oc-curring in the soil (Fig. 1). FeEDDHA components mayleach out of the root zone as a result of excessiveirrigation or atmospheric precipitation (Rombolà andTagliavini 2006), or be transported upwards due toevapotranspiration and precipitate on the soil surface(Schenkeveld et al. 2008). Reactive soil constituentsadsorb FeEDDHA components (Hernández-Apaolazaand Lucena 2001, 2011; García-Marco et al. 2006;Hernández-Apaolaza et al. 2006) and competing cationssuch as Cu can displace Fe from the FeEDDHA chelate(Schenkeveld et al. 2010c, 2012b). Biodegradation ofEDDHA ligands presents a potential sink, but needs tobe further examined; biodegradation did not affectFeEDDHA chelate concentrations in a soil incubationstudy (Schenkeveld et al. 2012a). Although Fe uptakefrom FeEDDHA is generally preceded by chelate split-ting, Bienfait et al. (2004) and Orera et al. (2010) foundFeEDDHA inside plants grown on substrate with nutri-ent solution, suggesting passive uptake of theFeEDDHA chelate as a whole. Finally, EDDHA ligandsdo not exclusively chelate Fe upon interaction with soil,but also competing cations such as Cu and Al (García-Marco et al. 2006; Schenkeveld et al. 2007).

The extent to which EDDHA ligands are capable ofspecifically mobilizing Fe from target soils is presum-ably a key factor in determining the success of theshuttle mechanism, but has hardly been studied.Moreover, the validity of the mechanism as proposedby Lindsay and Schwab (1982) has not yet been expe-rimentally demonstrated for EDDHA ligands in soil-plant systems. The aim of the present study was twofold.First, the specificity of EDDHA ligands for chelating Fefrom soil was examined in a series of soil interactionexperiments involving 4 calcareous soils and 3 separat-ed EDDHA isomers (soil interaction experiments 1–3).Secondly, experimental support was sought for the shut-tle mechanism actually taking place in soil-plant sys-tems with FeEDDHA. This was done using data fromtwo previously reported pot trial studies (pot experi-ments 1–2), in which FeEDDHAwas shown to facilitateFe uptake by soybean plants grown on calcareous soil(Schenkeveld et al. 2008, 2010b). Emphasis has been onthe o,o-EDDHA ligands, because thus far only racemicand meso o,o-FeEDDHA have been reported to supply

374 Plant Soil (2014) 379:373–387

soil grown plants with Fe (Rojas et al. 2008;Schenkeveld et al. 2010a), which is a prerequisite forthe shuttle mechanism.

Material and methods

Soils

Calcareous soils (two clay soils, two sandy soils)were collected from sites, located in Spain(Santomera and Xeraco L) and Saudi Arabia(Nadec and Hofuf). At all sites the top layer(0–20 cm) was sampled, with exception of the Xeracosite (20–40 cm). The soils were selected for their low Feavailability (DTPA-extractable Fe < 11 mg kg−1). Fechlorosis was manifest in crops grown at all sites, andcould be reproduced in pot experiments with theSpanish soils (Schenkeveld et al. 2008, 2010b).Pretreatment consisted of air drying and sieving (meshsize: 1 cm (pot experiments); 0.2 cm (soil interactionexperiments)). Relevant soil characteristics are present-ed in Table 1.

Experimental solutions

Racemic and meso o,o-H4EDDHA were obtained byseparation of an o,o-H4EDDHA mixture (99 % pure;49 % racemic o,o-EDDHA, 51 % meso o,o-EDDHA)1,as described by Bannochie and Martell (1989) andBailey et al. (1981). EDDHA solutions were preparedby dissolving racemic o,o-H4EDDHA (purity: 100 %),meso o ,o -H4EDDHA (pur i ty : 99 .5 %) ando,p-H4EDDHA (purity: 90 %)1 in sufficient 1 MNaOH. pH was set to 7 (±0.5). 0.01 M CaCl2 was usedas background electrolyte.

Soil interaction experiments

Metal mobilization upon interaction of separatedEDDHA isomers with soil was examined as a functionof: interaction time (soil interaction experiment 1), con-centration of the chelating agent added (soil interactionexperiment 2), and interaction time with periodicalEDDHA addition (soil interaction experiment 3). Soil

1 o,o-H4EDDHA and o,p-H4EDDHA were kindly provided byAKZO-Nobel

FeEDDHA

EDDHA

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FeEDDHAleaching

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FeEDDHA uptake

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Fe uptake

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HA

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Fig. 1 Shuttle mechanism for EDDHA ligands in soil solution asproposed by Lindsay and Schwab 1982. FeEDDHA is transportedthrough the rhizosphere by diffusive transport; chelate splittingoccurs; Fe is taken up and further allocated inside the plant;EDDHA ligands are transported from the root surface along theconcentration gradient; EDDHA ligands chelate and solubilize Fefrom soil particles. Potential sinks for the EDDHA ligand are

indicated in italic. Chelate splitting can be preceded by Fe reduc-tion (Chaney et al. 1972) at ferric chelate reductase (FCR) sites(Robinson et al. 1999) – this step has not been included in thefigure. FeEDDHA facilitated Fe uptake, as illustrated with theshuttle mechanism, does not necessarily exclude “conventionalFe uptake” without synthetic chelates; conventional Fe uptakehas not been included in the figure

Plant Soil (2014) 379:373–387 375

interaction experiment 3 was done to mimic thegradual release of EDDHA in soil-plant systems,resulting from Fe uptake from FeEDDHA by plants.All soil interaction experiments were executed induplicates.

In soil interaction experiment 1, all four soils wereleft to interact with 89.5 μM solutions of all threeEDDHA isomers, for 1, 8, 24, 48, 96 and 168 h.

In soil interaction experiment 2, all four soils wereleft to interact for 24 h with solutions of all threeEDDHA isomers, at the following concentrations: 1.8,3.6, 7.2, 14.4, 28.6 and 57.3 μM.

In soil interaction experiment 3, Santomera soilinteracted with racemic and meso o,o-EDDHA. After2 days of equilibration with 0.01 M CaCl2 solution,EDDHA concentration was raised by 1.8 μM each time,three times per week for 5 weeks, through addition ofaliquots of EDDHA solution.Metal concentrations weremonitored throughout the experiment.

Soils were left to interact with EDDHA solutions in a1:1 (w/v) soil-solution ratio, in 50 ml polypropylene testtubes (Greiner bio-one, Cat No 210296) (soil interactionexperiment 1 and 2) and 0.5 l polyethylene shakingbottles (soil interaction experiment 3). Tubes and bottles

Table 1 Soil characteristics

Soil (origin) Country Region Soilclass.

pH-CaCl2

aSOCb

(g kg−1)Clayc

(g kg−1)CaCO3

d

(g kg−1)Oxalatee

Al(g kg−1)

Fe(g kg−1)

Santomera Spain Murcia Entisol 8.0 5.4 260 520 0.44 0.30Xeraco L Spain Valencia Entisol 7.7 13.7 360 150 1.74 0.90Nadec Saudi Arabia Near Persian Gulf Aridisol 8.0 8.7 70 140 0.18 0.13Hofuf Saudi Arabia Near Persian Gulf Aridisol 7.8 7.1 40 60 0.08 0.19

Soil (origin) 0.01 M CaCl2 DTPA-extractableg

DOCf

(mg kg−1)Fe(mg kg−1)

Cu(mg kg−1)

Mn(mg kg−1)

Ni(mg kg−1)

Zn(mg kg−1)

Santomera 55 3.5 4.1 4.6 0.20 0.90Xeraco L 108 10.5 3.0 5.3 0.20 6.7Nadec 101 2.1 0.13 5.7 0.13 0.47Hofuf 78 6.7 2.3 3.8 0.10 5.0

Soil (origin) 0.43 M HNO3-extractableh

Al(g kg−1)

Fe(g kg−1)

Co(mg kg−1)

Cu(mg kg−1)

Mn(mg kg−1)

Ni(mg kg−1)

Zn(mg kg−1)

Santomera 0.70 0.49 2.6 10.1 180 3.7 4.5

Xeraco L 2.1 0.46 2.8 12.6 240 3.5 24

Nadec 0.32 0.18 0.70 0.77 50 1.9 2.3

Hofuf 0.16 0.17 0.34 6.3 41 1.1 24

a ISO/DIS 10390 Soil Quality – Measurement of pHbWalinga et al. (1992)c Houba et al. (1997)d ISO 10693, Soil Quality – Determination of carbonate content, volumetric methode Schwertmann (1964)f Houba et al. (2000)g Lindsay and Norvell (1978) and Quevauviller et al. (1996)h Tipping et al. (2003) and Fest et al. (2005)

376 Plant Soil (2014) 379:373–387

were placed in an end-over-end shaker, rotating at18 rpm in absence of light. Room temperature was keptat 20 (± 1) °C. Blank treatments without EDDHAwereincluded in all soil interaction experiments. After inter-action, the samples from soil interaction experiment 1and 2 were centrifuged for 15 min at 3,000 rpm, and pHand EC of the supernatant were measured.

In soil interaction experiment 3, sampling directlypreceded each EDDHA addition. Sample and aliquotof EDDHA solution were equal in volume, to preserve aconstant total volume. Before sampling, pH and redoxpotential were measured in suspension; neither changedsubstantially during the experiment. The solid phasewas allowed to settle and a sample was drawn fromthe clear top layer of the supernatant accounting for1 % of the solution volume. Subsequently, the shakingbottle was stirred up and placed back in the end-over-end shaker. After termination of soil interaction experi-ment 3, the soil was centrifuged for 15 min at 3,000 rpmand extracted for 48 h with a mixture of 10 mMKH2PO4 and 10 mM K2HPO4 (1:1, 1:5, 1:25 and1:100 (w/v)), to gain insight in the amounts of metal-EDDHA complexes adsorbed. PO4 was used as extract-ant because it has a high affinity for Fe(hydr)oxidesurfaces and strongly competes with other adsorbedspecies for reactive surface sites. A mixture ofKH2PO4 en K2HPO4 was applied to avoid strong pHeffects from the extractant. Extractions were done as insoil interaction experiment 1 and 2. All samples andextracts were filtered over 0.45 μm cellulose acetatemicro pore filters (Schleicher & Schuell, ref no:10462650). The filtrates were further analyzed.

Pot experiments

Metal concentrations in soil solution from two previous-ly reported pot trial studies in which FeEDDHA facili-tated Fe uptake had been established, were examinedregarding the shuttle mechanism. Details regarding thepreparation of the pot trials, the experimental solutionsand plant care are presented in Schenkeveld et al.(2008).

In pot experiment 1, metal concentrations in the porewater of Santomera soil were monitored for 6 weeksusing rhizon pore water samplers (rhizons) (SMSMOM, Rhizosphere Research Products, Wageningen,The Netherlands). The pot trial included a blank andtwo FeEDDHA treatments, applied to pots both withand without soybean plants. FeEDDHA treatments were

equal in Fe dose (4 mg l−1 Fe (72 μM)), but differed inrelative o,o-FeEDDHA content (i.e. the fraction of theFe applied that was chelated by the sum of racemic andmeso o,o-FeEDDHA): 30 % and 100 %. In the 30%o,otreatment, Fe not chelated by o,o-EDDHAwas presentas o,p-FeEDDHA and rest-FeEDDHA. Rest-EDDHAmainly consisted of polycondensates (Schenkeveld et al.2010b). Rhizons consist of a cylindrical polyethersul-fone (PES) membrane (diameter: 2.5 mm; length:10 cm; pore size: <0.2 μm) connected to a PVC/PEtube. Before use, the rhizons were cleaned with0.14 M HNO3 and ultrapure water, rinsed with 1 mMNaNO3 and stored in a 1 mM NaNO3 solution. Therhizons were incorporated in the soil when the pots werefilled, with the membrane placed horizontally at a heightof approximately 10 cm above the bottom plate. The endof the PVC/PE tube was connected to the rim of the pot.Sampling was done twice per week by imposing avacuum on the inside of the rhizon with a 10 ml syringewith luer lock (SS*10LZ1, Terumo) for 16 h at most.The first ml sampled was used for rinsing and led backinto the pot. The sample size did not exceed 6 ml andexposure of the sample to light was prevented.Experiment 1 was executed in duplicates.

In pot experiment 2, soybean plants grown on cal-careous soil from Santomera were offered FeEDDHAtreatments, equal in Fe dose (7 mg l−1 Fe (0.13mM Fe))but differing in relative o,o-FeEDDHA content. Twocontrol treatments were included: 1) with plants, withoutFeEDDHA addition, 2) without plants with FeEDDHAaddition. Eight weeks after transfer of the seedlings tothe pots, the plants were harvested. Collection andanalysis of roots and pore water are described inSchenkeveld et al. (2008, 2010b). Additionally, aftercollection and rinsing with demineralized water, theroots were extracted with 150 ml 10 mM Na2H2EDTAsolution by manual shaking for one minute. The amountof metals extracted represents a measure for the amountof metals adsorbed onto the root surface (Bates et al.1982; Hassler et al. 2004; Kalis et al. 2006, 2007). Theextracts were filtered over a 0.45 μm cellulose acetatemicro pore filter (Schleicher & Schuell, ref no:10462650). The filtrate was further analyzed. Pot expe-riment 2 was conducted in triplicates.

Chemical analyses

Cu, Al, Mn, Zn, Ni and Co concentrations were mea-sured by ICP-MS (Perkin Elmer, ELAN 6000). Samples

Plant Soil (2014) 379:373–387 377

were acidified with nitric acid before ICP-measurement,with exception of the EDTA extracts. FeEDDHA isomerconcentrations were determined after separation throughhigh-performance liquid chromatography (HPLC) asdescribed in Schenkeveld et al. (2007). The concentra-tions of metals other than Fe, chelated to EDDHAcomponents were calculated as the difference betweenthe metal concentration in the EDDHA treatment andthe blank.

Statistical analysis

Statistical analysis of the data was performed usingSPSS 12.0. Homogeneity of the data was tested withthe Levene’s test (α=0.05). Differences among treat-ments were determined by applying the multivariategeneral linear model procedure with a Tukey post-hoctest (α=0.05).

Results

Soil interaction experiment 1 Metal mobilization wasexamined as a function of time upon interaction ofEDDHA ligands with 4 calcareous soils. ForSantomera soil, soil solution concentrations of the quan-titatively most important cations (Al, Cu, Fe and Mn)are presented in Fig. 2; for the 3 other soils, the resultsare presented as Supporting Information (SI-Figure 1–3). The kinetics of metal mobilization from soil byEDDHA ligands are fast: after 1 h, mobilizedmetals accounted for 90–92 % of the racemico,o-EDDHA, 83–88 % of the meso o,o-EDDHA, and36–67 % of the o,p-EDDHA added (Fig. 2 and SI-Figures 1–3). This implies the average residence timeof EDDHA ligands in soil solution is short. Metal mo-bilization by racemic and meso o,o-EDDHA is fairlysimilar, while metal mobilization by o,p-EDDHA dif-fers strongly. In all soils o,p-EDDHA mobilized moreCu and less Fe than o,o-EDDHA ligands (Fig. 2 and SI-Figures 1–3). Furthermore, contrary to o,o-EDDHA,o,p-EDDHA hardly mobilized Al or Mn from any soil.To a substantial degree the different mobilization behav-iour results from the fact that phenolic hydroxyl groupsin para position are sterically inhibited from contributingto chelation. Therefore, the lack of a second phenolichydroxyl group binding the metal considerably affectsthe relative affinity of o,p-EDDHA for metals in com-parison to o,o-EDDHA.

The concentrations of Fe and Al mobilized byo,o-EDDHA remained relatively constant over time,while the concentrations of Cu and Mn decreased andeventually approached zero, except in the Hofuf soil(Fig. 2 and SI-Figures 1–3). The fact that Fe and Alconcentrations remained unaffected by the decrease inCu and Mn concentration suggests that o,o-CuEDDHAand o,o-MnEDDHA complexes were adsorbed and nosubstantial degree of complex dissociation or cationdisplacement reaction took place; the Fe availabilityparameter DTPA extractable Fe (Table 1) suggests that,at the point that Cu and Mn concentrations started todecline, Fe availability was not limiting furthermobilization of Fe in case the o,o-CuEDDHA ando,o-MnEDDHA complexes would have dissociated.The strong tendency of o,o-CuEDDHA to be adsorbedto soil has been previously reported by Schenkeveldet al. (2010c), and the relatively slow decline in Cu andMn concentration in the Hofuf soil (SI-Figure 3), whichhas the lowest content of reactive soil compounds, fur-ther supports the involvement of adsorption. Foro,o-MnEDDHA, also oxidation of the ligand by chelatedMn(III) may have contributed to the decrease in concen-tration (Patch et al. 1982).

o,o-CuEDDHA is removed from solution morequickly than o,p-CuEDDHA and thus displays a stron-ger tendency to be adsorbed, despite its higher complex-ation constant. Because adsorption processes generallystart without lag time, it is remarkable that the decreasein CuEDDHA concentration did not set in immediately,but, in the case of Santomera soil, only after 2 days.

Only in the Hofuf soil, EDDHA ligands also tempo-rarily mobilized Zn to a substantial degree (9–18% after1 h; data not shown); however, the ZnEDDHA concen-trations dropped rapidly and after 24 h, Zn mobilizationaccounted for less than 1 % of any EDDHA ligandadded to any of the soils included. For this reason Znwas not further considered in subsequent soil interactionexperiments.

Soil interaction experiment 2 Because the Fe uptakerate at plant roots is relatively low and complexationkinetics are fast, the EDDHA concentration near theroots resulting from Fe uptake from FeEDDHAwill beclose to zero. In soil interaction experiment 1 the con-centration administered was orders of magnitude higherthan the low levels expected, and mobilization of certainmetals can then become limited by decreasing metalavailability on the soil reactive surfaces. In soil

378 Plant Soil (2014) 379:373–387

interaction experiment 2, metal mobilization from soilwas examined as a function of the administeredEDDHA concentration, to address metal-EDDHA spe-ciation resulting from extremely low EDDHA doses, aswould be expected near the root surface as a result of Feuptake from FeEDDHA. Interaction time was set at24 h, because, on average, overall metal mobilizationwas highest at that moment (Fig. 2). For Santomera soil,mobilized Al, Cu, Fe and Mn concentrations are pre-sented in Fig. 3. For the other soils, the results arepresented as Supporting Information (SI-Figure 4–6).

In Santomera soil, metal concentrations increasedapproximately linearly with increasing EDDHA con-centration added (Fig. 3); for the other soils this linearrelationship was not always observed (SI-Figure 4–6).Deviation from the linear increase was most pronouncedfor racemic and meso o,o-EDDHA interacting withHofuf soil (SI-Figure 6): at low EDDHA concentrationsmainly Cu was mobilized, whereas at high EDDHAconcentrations, as a result of decreased Cu availabilitydue to extraction, mainly Fe was mobilized.

Metal concentration data were fit and the fittingcurves extrapolated to [EDDHA]added = 0. The tangentof a fitting curve at [EDDHA]added = 0 corresponds tothe fraction of the EDDHA ligand, mobilizing a specificmetal upon addition of an infinitesimally low EDDHAconcentration. All tangents are presented in Table 2, aspercentages instead of fractions. Chelate adsorption bythe soil is the most plausible explanation for the fact thatthe sum of percentages (per EDDHA ligand, per soil)remained mostly below 100 %. Upon addition of(infinitesimally) low EDDHA ligand concentrations,only 21–48 % of racemic o,o-EDDHA, 21–52 % ofmeso o,o-EDDHA, and 0–15 % of o,p-EDDHAwouldmobilize Fe from soil (Table 2). This implies that theefficiency of a potential shuttle mechanism would beheavily compromised by the limited specificity ofEDDHA ligands for mobilizing Fe from soils with alow Fe availability. In general, Cu is the principal com-peting metal; 9–70% of racemic o,o-EDDHA, 15–69%of meso o,o-EDDHA and 14–90 % of o,p-EDDHAwould mobilize Cu (Table 2). Over 90 % of the

0%

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Fig. 2 Percentage of a racemico,o- EDDHA; b mesoo,o-EDDHA; and c o,p-EDDHAadded, chelated to Fe, Cu,Mn andAl in solution upon interactionwith Santomera soil as a functionof time. Error bars indicatestandard deviations

Plant Soil (2014) 379:373–387 379

mobilized metal by o,p-EDDHAwould be Cu, except inthe Nadec soil.

Although the relative affinity of racemic and mesoo,o-EDDHA for Fe and Cu differ (Schenkeveld et al.2010c), the extent to which they mobilize Fe and Cu aresimilar, except in the Nadec soil. Therefore metal mobi-lization must at least in part be governed by kineticrather than thermodynamic principles. The deviant met-al mobilization from Nadec soil is related to the fact thatracemic o,o-EDDHA mobilized more Mn than mesoo,o-EDDHA, which became particularly apparent dueto the very low Fe and Cu availability of Nadec soil(Tables 1 and 2).

Soil interaction experiment 3 The gradual release ofracemic and meso o,o-EDDHA at plant roots resultingfrom FeEDDHA facilitated Fe uptake was mimicked byperiodical addition of small amounts of o,o-EDDHA toSantomera soil. Over a period of 36 days, EDDHAwasadded in 15 aliquots to a total concentration of 27 μM.Less than 2 % of the EDDHA added was removed byperiodical sampling; this was calculated from the metal-EDDHA species concentrations in solution and the

sampled volumes. FeEDDHA and CuEDDHA concen-tration data are presented in Fig. 4.

The FeEDDHA concentration data presents 3 pointsof attention. First, the slope of the concentration curvenear t=0 indicates that both o,o-FeEDDHA isomersaccounted for only 10–12 % of the EDDHA added(Fig. 4a). This is a factor 2–2.5 lower than derived withthe tangent in soil interaction experiment 2 (Table 2).Contrary to soil interaction experiment 2, the soil hadbeen allowed to equilibrate 2 days with backgroundelectrolyte solution prior to the first EDDHA addition.Apparently, equilibration negatively affected the relativeavailability of Fe compared to other metals. Secondly,o,o-FeEDDHA concentrations did not increase linearlywith the amount of o,o-EDDHA added, as would beexpected from soil interaction experiment 2 (Fig. 3), butthe slope flattened (Fig. 4a). And thirdly, mesoo,o-FeEDDHA concentrations deviated more stronglyfrom the expected linear increase than racemico,o-FeEDDHA concentrations (Fig. 4a).

The o,o-CuEDDHA concentration curves are typicalfor repetitive dosing with elimination: initially the con-centration increased, but the slope flattened and a

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bFig. 3 Metal-EDDHAconcentrations in solution after24 h of interaction betweenEDDHA isomers and Santomerasoil as a function of theconcentration of a racemico,o-EDDHA;bmeso o,o-EDDHA;and c o,p-EDDHA added. Errorbars indicate standard deviations

380 Plant Soil (2014) 379:373–387

plateau was reached relatively quickly (Fig. 4b). A saw-tooth concentration pattern underlies the measured Cuconcentrations; upon o,o-EDDHA addition, Cu wasmobilized from the soil and o,o-CuEDDHA concentra-tion increased rapidly, after which it gradually decreasedagain due to adsorption, until new o,o-EDDHA wasadded again. The racemic and meso o,o-CuEDDHAconcentration curves are identical, because the corre-sponding o,o-EDDHA ligands mobilize Cu to the sameextent (Table 2), and adsorption kinetics of racemic andmeso o,o-CuEDDHA are similar (Fig. 2). The slopes ofthe concentration curves near t=0 indicate thato,o-CuEDDHA accounted for only 6 % of the

o,o-EDDHA added, i.e. much less than the 49–53 %derived with the tangent in soil interaction experiment 2(Table 2). In soil interaction experiment 1, a strongincrease in o,o-CuEDDHA adsorption was observedafter 2 days (Fig. 2a & b). The 2-day equilibration timein soil interaction experiment 3 probably resulted inmore favourable conditions for o,o-CuEDDHA adsorp-tion already upon the first addition of EDDHA ligand,resulting in less o,o-CuEDDHA mobilization (Fig. 4a).The overall EDDHA fraction mobilizing metals wasmuch lower than in soil interaction experiment 2. After2 days of interaction it equaled around 21 % for botho,o-EDDHA isomers, compared to 83–88 % in soilinteraction experiment 2. The fraction further decreasedthroughout the experiment.

Statistical analyses on metal concentrations in the1:1, 1:5 and 1:25 PO4-extracts (Table 3) indicate thatonly Cu concentrations were significantly elevated inthe racemic and meso o,o-EDDHA treatments in com-parison to the blank; approximately to the same extent,as would be expected from the CuEDDHA concentra-tions in soil solution. FeEDDHA concentrations in theextracts were below determination limit and could notbe measured. The increase in Al and Mn concentrationwith lower soil solution ratios is remarkable and proba-bly related to the formation of soluble Al- and Mn-phosphate complexes.

An additional 1:100 PO4-extraction was done, tocloser estimate the amount of o,o-CuEDDHA adsorbed.When averaging the results of the racemic and mesoo,o-EDDHA treatment, the extracted o,o-CuEDDHAaccounts for 5.2 μM of the 27 μM o,o-EDDHA added;pore water content of the extracted soil (16.8 %) and theCuEDDHA concentration therein (0.27 μM) werecorrected for. No mass balance for the o,o-EDDHAligands could be established and it is unclear whichfraction of the adsorbed o,o-CuEDDHAwas extracted.The results from the extractions do however clearlyindicate that o,o-CuEDDHA accounts for a substantialfraction of the o,o-EDDHA added (5.2 out of 27 μM),and that at least a factor 20 more o,o-CuEDDHA isadsorbed (corresponding to a soil solution concentrationof 5.2 μM), than that is present in soil solution(0.27 μM).

Pot experiment 1 Although quantitatively of little im-portance, Co mobilization by EDDHA is a suitableindicator for the shuttle mechanism in soil-plant sys-tems. Unlike Cu and Mn, Co largely remains in

Table 2 Metal mobilization by infinitesimally small amounts ofEDDHA ligands interacting with 4 calcareous soils

Santomera Xeraco L Nadec Hofuf

Racemic o,o-EDDHA

Al 5.5 % n.d. 9.3 % 0.4 %

Co 0.1 % 0.1 % 2.3 % 0.4 %

Cu 49.4 % 24.9 % 9.2 % 70.1 %

Ni 0.4 % 0.2 % 0.7 % 0.1 %

Mn 5.4 % 1.7 % 60.7 % 6.6 %

Fe 26.8 % 47.8 % 20.8 % 25.0 %

Total 87.6 % 74.8 % 102.9 % 102.5 %

Meso o,o-EDDHA

Al 5.6 % n.d. 24.4 % 2.9 %

Co 0.2 % 0.2 % 5.0 % 0.5 %

Cu 52.5 % 23.3 % 15.4 % 69.4 %

Ni 0.1 % 0.0 % 1.4 % 0.1 %

Mn 0.1 % 0.0 % 12.5 % 0.8 %

Fe 24.8 % 52.0 % 44.8 % 20.5 %

Total 83.3 % 75.6 % 103.5 % 94.1 %

o,p-EDDHA

Al n.d. n.d. n.d. n.d.

Co 0.1 % 0.1 % 4.2 % 0.3 %

Cu 79.0 % 58.3 % 14.3 % 89.5 %

Ni 0.0 % 0.2 % 3.3 % 0.5 %

Mn 0.7 % 0.1 % 6.1 % 1.2 %

Fe 0.0 % 2.1 % 14.6 % 3.1 %

Total 79.7 % 60.9 % 42.4 % 94.5 %

Metal mobilization is expressed as a percentage of the amount ofEDDHA ligand added, chelating a specific metal in soil solution.Percentages were determined from the tangents of concentrationcurves at [EDDHA]added = 0 (see Fig. 3)

n.d. not determined – concentration data could not be properly fitfor deriving the tangent

Plant Soil (2014) 379:373–387 381

solution,2 the variance in concentration among repli-cates and over time tends to be small, and backgroundCo concentrations in soil solution are generally very lowcompared to other metals. Therefore Co concentrationsin the pore water of Santomera soil were examined as afunction of time (Fig. 5).

Both plant and FeEDDHA treatment were required toobtain increased Co concentrations. Elevation in Coconcentration became substantial in the 3rd week(Fig. 5), when SPAD-indices of the youngest leaves inthe blank treatment became significantly lower thanthose in the FeEDDHA treatments (Schenkeveld et al.2010b). This implies that, from the 3rd week onwards,plants receiving FeEDDHA treatments were using theFe offered as FeEDDHA. So, utilization of Fe fromFeEDDHA and elevation in Co concentration coincided.The extent to which Co concentrations were increasedwas determined by the relative o,o-FeEDDHA content ofthe treatment. Fe uptake positively correlates to theamount of o,o-FeEDDHA applied (Schenkeveld et al.2008), so that a larger Fe uptake was related to more Comobilization. These findings supports the hypothesis thatupon FeEDDHA facilitated Fe uptake, EDDHA ligandsare released and mobilize metals including Co.

Pot experiment 2 Because the kinetics involved withchelating and mobilizing metals are fast (see soil

interaction experiment 1), EDDHA ligands should beexpected to mobilize metals from the direct vicinity ofFe uptake sites on the plasma membrane. Cell walls ofroot cells, both at the root surface and within the cortex,constitute a reactive surface onto which metals adsorband metal hydroxides precipitate (Bienfait et al. 1985;Kalis et al. 2007). These metals are in close proximity toFe uptake sites, and may comprise an important pool ofmetals for chelation by EDDHA ligands after Fe transfer.The effect of FeEDDHA application on metal availabil-ity at the root surface of soybean plants grown onSantomera soil was examined in pot experiment 2(Schenkeveld et al. 2008) by means of an EDTA extrac-tion of the roots. The amounts of Al, Co and Ni extractedfrom the root surface per gram dry weight root decreasedlinearly with increasing relative o,o-FeEDDHA contentof the treatment (Fig. 6). Blanks (without FeEDDHAtreatment) have been excluded from the regression, be-cause stress response mechanisms to Fe deficiency (rhi-zosphere acidification, excretion of chelating agents, etc.(Marschner et al. 1986)), cause metal mobilization fromthe root surface, unrelated to FeEDDHA application.

Consistently, the concentrations of Al, Co and Ni insoil solution increased linearly with increasing relativeo,o-FeEDDHA content of the treatment (Fig. 7). Inagreement with pot experiment 1, the data indicate thatincreased metal concentrations are a combined effect ofthe plant and the FeEDDHA application: both the treat-ment with plants without FeEDDHA, and withFeEDDHAwithout plants result in lower metal concen-trations than the treatments with both plants and

2 This is illustrated by the Co mobilization data from soil interac-tion experiment 1, presented as Supporting Information (SI-Figure 7).

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40time (days)

Cu

ED

DH

A (

aq)

(µm

ol l

-1)

racemic o,o-EDDHA

meso o,o-EDDHA

b

0

0.5

1

1.5

2

2.5

0 10 20 30 40time (days)

FeE

DD

HA

(aq

) (µ

mo

l l -1

)

racemic o,o-EDDHA

meso o,o-EDDHA

a

Fig. 4 a o,o-FeEDDHA, and b o,o-CuEDDHA concentration as afunction of time. After each sampling session EDDHAwas added,raising the concentration by 1.8 μmol l−1. The straight line in 4arepresents the o,o-FeEDDHAconcentration as a function of time ifthe increase had continued at the rate of t≈0. Because Fe

availability does not become limiting for Fe mobilization asFeEDDHA, deviation from the straight line must be caused by asecond process decreasing the FeEDDHA concentration. There-fore, the straight line represents the cumulative gross Femobilization

382 Plant Soil (2014) 379:373–387

FeEDDHA. For Fe and Cu, no significant decrease inEDTA-extractable amounts were observed as a functionof the relative o,o-FeEDDHA content of the treatments.

Discussion

The substantially larger Mn mobilization by racemico,o-EDDHA than by meso o,o-EDDHA, especially

from the Nadec soil (Table 2), is possibly (co-)relatedto redox kinetics. Upon chelation by o,o-EDDHA underaerobic conditions, Mn(II) (from the soil) is oxidized toMn(III) (Patch et al. 1982). In plain solution, it wasobserved that Mn oxidation, which is accompanied bya change in colour of the complex from pale pinkto brown, is considerably faster for racemico,o-MnEDDHA. For ligands like EDDHA, primarilyconsisting of hard Lewis bases, complexation constantsare generally higher for the trivalent than for the divalentspecies of a metal. The faster formation of a more stableo,o-Mn(III)EDDHA chelate is advantageous in compe-tition with other metals. The redox behaviour and sta-bility of MnEDDHA complexes need to be furtherexamined, but were outside the scope of this study.

The observed differences in sorption behavior ofmetal-EDDHA complexes (Fig. 2) are presumably re-lated to differences in the geometry of the metal-EDDHA coordination complexes: FeEDDHA has amildly distorted octahedral structure, resulting fromheterogeneity in coordinating groups (Bailey et al.1981); AlEDDHA has an octahedral structure (Rajanet al. 1981), presumably also mildly distorted;

Table 3 a) Metal concentrations extracted from Santomera soil with 20 mM PO4-extractant in various soil-solution ratios. Standarddeviations are indicated between parentheses; b) P-values of the ANOVA; and c) P-values of the post-hoc test on treatment

(a) Extraction Treatment Al (μmol l−1) Co (nmol l−1) Cu (nmol l−1) Mn (nmol l−1) Ni (nmol l−1)

1:1 Blank 4.8 (0.54) 5.8 (1.4) 393 (9) 23.7 (3.1) 60.8 (0.7)

Racemic o,o-EDDHA 5.2 (0.12) 4.9 (0.2) 520 (52) 22.6 (1.5) 50.9 (9.9)

Meso o,o-EDDHA 5.9 (1.2) 5.6 (0.7) 520 (46) 23.5 (2.3) 46.3 (0.5)

1:5 Blank 16.5 (0.8) 3.4 (0) 345 (4) 59.9 (5.9) 40.9 (3.4)

Racemic o,o-EDDHA 23.7 (4.0) 3.4 (0.5) 402 (19) 87.0 (22.7) 57.7 (11.3)

Meso o,o-EDDHA 17.8 (−) 3.4 (−) 392 (−) 76.1 (−) 51.4 (−)1:25 Blank 16.9 (1.6) 1.4 (0.5) 161 (6) 91.0 (7.7) 29.6 (2.4)

Racemic o,o-EDDHA 19.7 (3.6) 1.4 (0) 186 (18) 92.1 (4.1) 29.5 (2.6)

Meso o,o-EDDHA 16.4 (2.2) 1.2 (0.2) 205 (12) 97.6 (6.2) 23.5 (1.0)

1:100 Blank 85 (4)

Racemic o,o-EDDHA 122 (3)

Meso o,o-EDDHA 136 (17)

(b) Effect of treatment (c) Cu

Al 0.059 Blank - racemic 0.005 (0.001)*

Co 0.761 Blank – meso 0.007 (0.002)

Cu 0.003 (0.000)* Racemic - meso 1.000 (0.909)

Mn 0.222

Ni 0.345

*In between parentheses: p-values if 1:100 extraction is included

0.0

0.1

0.2

0.3

0 2 4 6

time (weeks)

[Co

] (µ

mo

l l -1

)

100%o,o with plant

30%o,o with plant

blank with plant

100%o,o without plant

30%o,o without plant

blank without plant

Fig. 5 Co concentrations in the pore water of Santomera soil as afunction of time for treatments with and without FeEDDHAapplication, and with and without plants. Error bars indicatestandard deviations

Plant Soil (2014) 379:373–387 383

Mn(III)EDDHA has a more severely distorted octahe-dral structure with elongated metal-ligand bonds alongthe z-axis (a Jahn-Teller effect) (Bihari et al. 2002); andCuEDDHA has the most severe (Jahn-Teller) octahedraldistortion (Riley et al. 1983), in solution presumablyresulting in a square planar structure, in which thecarboxylic groups are detached at high pH and the(protonated) phenolate groups at low pH (Frost et al.1958). Functional groups which are detached or parti-cipate in relatively weak elongated bonds may bind tosoil reactive surface groups more easily, and positionson octahedral coordination complexes not occupied by,or bound relatively weakly to EDDHA ligands mayfacilitate ligand exchange reactions, e.g. with soil organ-ic matter. The adsorptionmechanisms of metal-EDDHAcomplexes need to be further examined in order toelucidate the differences in adsorption behavior.

In soil interaction experiment 3 it was observed that,upon repetitive EDDHA addition, the deviation from alinear increase in Fe concentration was stronger formeso o,o-EDDHA than for racemic o,o-EDDHA(Fig. 4a). This difference is unlikely to be caused byan ongoing decrease in relative Fe availability, affecting

Fe mobilization by meso o,o-EDDHA more stronglythan by racemic o,o-EDDHA. Instead, the involvementof a second process, causing a decrease ino,o-FeEDDHA concentration, simultaneous to the in-crease due to addition of o,o-EDDHA, is more likely.Assuming the relative availability of metals remainedconstant after equilibration and an equal amount ofFe was mobilized with each o,o-EDDHA addition,the rate of decrease was proportional to theo,o-FeEDDHA concentration, which suggests the sec-ond process caused an exponential decline. An exponen-tial decline in concentration of o,o-FeEDDHAinteracting with Santomera soil has been previously re-ported (Schenkeveld et al. 2010b, 2012a). Recent studiessuggest this decline is caused by displacement of Fe fromo,o-FeEDDHA by Cu (Schenkeveld et al. 2010c,2012b). Results from the present study support this,demonstrating the high relative affinity of o,o-EDDHAfor Cu, and the tendency of o,o-CuEDDHA to be re-moved from soil solution, inducing an ongoingreinstallment of the solution equilibria involvingEDDHA species and causing the o,o-FeEDDHA con-centrations to decline. Meso o,o-FeEDDHA is more

R2 = 0.88

0

5

10

15

20

0 50 100o,o-FeEDDHA content (%)

Ni e

xtra

cted

(n

mo

l g(d

w)-1

)

blank with plant

FeEDDHA with plant

c

R2 = 1.00

0

200

400

600

0 50 100o,o-FeEDDHA content (%)

Al e

xtra

cted

(n

mo

l g(d

w) -1

)

blank with plant

FeEDDHA with plant

a

R2 = 0.98

0

5

10

15

20

25

0 50 100o,o-FeEDDHA content (%)

Co

ext

ract

ed (

nm

ol g

(dw

)-1)

blank with plant

FeEDDHA with plant

bFig. 6 EDTA-extractable a Al;b Co; and c Ni from roots ofsoybean plants grown onSantomera soil as a function ofthe o,o-FeEDDHA content of thetreatment. The slopes of theregression lines for Al (p=0.004),Co (p=0.000) and Ni (p=0.001)are significantly different from 0.Error bars indicate standarddeviations

384 Plant Soil (2014) 379:373–387

susceptible to Fe displacement by Cu than racemico,o-FeEDDHA (Schenkeveld et al. 2010c, 2012b),which corresponds with the stronger deviation fromlinear increase in meso o,o-FeEDDHA concentration(Fig. 4a). No substantial decrease in o,o-FeEDDHAconcentrations was however observed in soil interactionexperiment 1. This probably results from a combinationof factors: the shorter interaction time (without previousequilibration), a (partial) depletion of available Cu due tothe much higher o,o-EDDHA dose which caused a de-crease in Cu activity, and the lower relative rate ofdecline at (considerably) higher initial FeEDDHA con-centrations (Schenkeveld et al. 2010b).

The results from the pot experiments demonstratethat when Fe is taken up from o,o-FeEDDHA, theremaining o,o-EDDHA ligands mobilize metals fromthe root surface. The results from the soil interactionexperiments show that Fe and Cu are readily mobilizedby o,o-EDDHA ligands. Nevertheless, no decrease inEDTA-extractable Fe or Cu from the root surface wasobserved. The lack of a decrease in EDTA-extractableFe is possibly related to the fact that the method applied

does not deliver an adequate indication for Fe availabil-ity at the root surface due to the short extraction time andthe relatively slow kinetics involved with EDTA-enhanced dissolution of Fe(hydr)oxides (Nowack andSigg 1997). The lack of a decrease in EDTA-extractableCu might have been caused by the high affinity ofo,o-CuEDDHA for reactive surfaces; upon chelationof “root surface-Cu” by o,o-EDDHA, the resulting com-plex might have been sorbed to the root surface imme-diately. If so, the amount of root surface-Cu remainsunaffected by the FeEDDHA treatment; possibly, part ofthe Cu in the EDTA extract was or had been chelated too,o-EDDHA.

With the experimental setup of the pot experiments, itcould not be determined whether the Fe chelated too,o-EDDHA in soil solution was applied with the treat-ment or if it originated from the soil. Although, for thisreason, the evidence of the shuttle mechanism remainscircumstantial, the combined results of soil interaction andpot experiments are convincing: 1) Upon interaction ofEDDHA ligands with soil in soil interaction experiments,both Fe and competing metals were mobilized. 2) In pot

R2 = 0,94

0

0.5

1

1.5

2

2.5

0 50 100o,o-FeEDDHA content (%)

[Al]

(µ

mo

l l -1

)

blank with plantFeEDDHA with plantFeEDDHA without plant

a

R2 = 0,95

0

0.1

0.2

0.3

0.4

0 50 100o,o-FeEDDHA content (%)

[Co

] (µ

mo

l l -1

)

blank with plantFeEDDHA with plantFeEDDHA without plant

b

R2 = 0.97

0

0.2

0.4

0.6

0 50 100o,o-FeEDDHA content (%)

[Ni]

(µ

mo

l l -1

)

blank with plantFeEDDHA with plantFeEDDHA without plant

c

Fig. 7 Al (a), Co (b) and Ni (c)concentration in the pore water ofSantomera soil as a function ofthe relative o,o-FeEDDHAcontent of the treatment. Slopes ofthe regression lines for Al(p=0.000), Co (p=0.000) and Ni(p=0.001) deviate significantlyfrom 0. Error bars indicatestandard deviations

Plant Soil (2014) 379:373–387 385

experiments, the soil solution concentration of competingmetals that formEDDHAchelates with limited affinity forthe solid phase were only increased in treatments withplants receiving FeEDDHA; and elevation only startedwhen plants had started to utilize Fe from FeEDDHA.This suggests that, after Fe transfer to the plant, EDDHAligands hadmobilized competingmetals. Given the resultsfrom the soil interaction experiments, it is only likely thatEDDHA ligands will also have mobilized Fe.

If the efficiency of the Fe shuttle is largely controlledby metal availability in the bulk soil, it is heavily com-promised by competition from other cations. For thecalcareous soils included in the soil interaction experi-ments (SSR = 1), the Fe mobilizing efficiency was 21–52 % for direct o,o-EDDHA application (Table 2); equi-libration with electrolyte solution prior to o,o-EDDHAapplication to Santomera soil decreased the efficiencyfrom 25–27 % to 10–12 % (Table 2 and Fig. 4a).However, root extraction data from pot experiments sug-gest that at least part of the metals mobilized by EDDHAwere not adsorbed to soil, as suggested in Lindsay andSchwab’s conceptual model, but to the root surface.Metal availability in the rhizosphere and the potentialrole of the apoplastic Fe pool in the roots (Bienfait et al.1985; Strasser et al. 1999) need to be further examined inrelation to metal chelation by EDDHA ligands. Tracerstudies with isotopically labeled FeEDDHA (Orera et al.2010) could aid this and help to provide a better under-standing of the actual effectiveness of the shuttlemechanism in soil-plant systems.

Acknowledgments The authors wish to express their sincereappreciation and gratitude to the following: AkzoNobel for finan-cing this project which was initiated by P. Weijters and M. Bugter,Rob Dijcker and Marina Hernandez-Lopez for conducting a partof the experimental work and P. Nobels for his help with the ICP-measurements.

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