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Plant and Soil 258: 57–68, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Metal-humic complexes and plant micronutrient uptake: a study based ondifferent plant species cultivated in diverse soil types

J.M. Garcı́a-Mina1,2,4, M.C. Antolı́n3 & M. Sanchez-Diaz3

1R&D Department. Inabonos-Roullier Group; Poligono Arazuri-Orcoyen, C/ C no 32, 31160 Orcoyen (Navarra),Spain. 2Department of Chemistry and Soil Chemistry, Faculty of Sciences, University of Navarra, PO Box 273,31080 Pamplona (Navarra), Spain. 3Department of Plant Physiology, Faculty of Sciences, University of Navarra,PO Box 273, 31080 Pamplona (Navarra), Spain. 4Corresponding author∗

Received 17 June 2002. Accepted in revised form 18 August 2003.

Key words: humic substances, metal-humic complexes, micronutrient uptake, plant development, plant nutrition,soil-plant interactions

Abstract

There are several studies in the literature dealing with the effect of metal-humic complexes on plant metal uptake,but none of them correlate the physicochemical properties of the complexes with agronomic results. Our studycovers both aspects under various experimental conditions. A humic extract (SHE) obtained from a sapric peat wasselected for preparing the metal–humic complexes used in plant experiments. Fe–, Zn– and Cu–humic complexeswith a reaction stoichiometry of 2:0.25 (humic:metal, w/w) were chosen after studying their stability and solubilitywith respect to pH (6–9) and the humic:metal reaction stoichiometry. Wheat and alfalfa plants were greenhouse cul-tured in pots containing one of three model soils: an acid, sandy soil and two alkaline, calcareous soils. Treatmentswere: control (no additions), SHE (53 mg kg−1 of SHE), and metal (Cu, Zn and Fe)–SHE complexes (2.5 and 5 mgkg−1 of metal rate and a SHE concentration to make 53 mg kg −1). Cu- and Zn–humic complexes significantly(p≤0.05) increased the plant uptake and the DTPA-extractable soil fraction of complexed micronutrients in mostplant–soil systems. However, these effects were associated with significant increases (p≤0.05) of shoot and rootdry weight only in alfalfa plants. In wheat, significant increases of root and shoot dry matter were only observedin the Cu–humic treated plants growing in the acid soil, where Cu deficiency was more intense. The Fe–humiccomplex did not increase Fe plant assimilation in any plant–soil system, but SHE increased Fe-uptake and/orDTPA-extractable soil Fe in the wheat–calcareous soil systems. These results, taken together with those obtainedfrom the study of the pH- and SHE:metal ratio-dependent SHE complex solubility and stability, highlight theimportance of the humic:Fe complex stoichiometry on iron bioavailability as a result of its influence on complexsolubility.

Introduction

Excellent studies and reviews on the ability of humicsubstances (HS) to affect plant micronutrient uptakedue to their ability to complex metals under differentenvironmental conditions have been published (Chen,1996; Chen and Aviad, 1991). Likewise, a numberof studies carried out using nutrient solutions or in-ert substrates have shown plant capacity to assimilate

∗ FAX No: +34-948-324032. E-mail: [email protected]

micronutrients from metal–humic complexes (M-HC).In this way, Pinton et al. (1999) and Chen et al. (1999)have pointed out the participation of Fe-HC in Fe up-take mechanisms in cucumber, melon and bentgrass.Similar results had been obtained by Miravé et al.(1987) and Lobartini and Orioli (1988) using otherplant species, as well as by Mirave and Orioli (1989)and Barnard et al. (1992) in relation to Zn plant up-take from Zn–HC complexes, and by others in relationto Cu plant uptake from Cu–HC complexes (Ennis,1962; Ennis and Brogan, 1961; Gupta, 1986; Gupta

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and Häni, 1980). However, in the case of plants grownin soil, published studies are scant and results far fromclear. In fact, no clear improvement in metal plantuptake from M–HC has been observed in plant–soilsystems (Alva and Obreza, 1998; Burau et al., 1960;Kumar and Prasad, 1989; Pandeya et al., 1998). Thatis why some authors have expressed serious doubtsabout the real usefulness and efficiency of these or-ganic complexes as valuable sources of micronutrientsfor crops cultivated in soils that present micronutrientdeficiency (Burau et al., 1960).

In order to explain this apparent contradictionbetween results obtained from soil and from inert sub-strate or nutrient solution experiments several factorshave to be taken into account. For instance, the ca-pacity of M–HC to keep complexed micronutrients insoil solution with pH values or chemical compositionwhich favor metal precipitation. It is clear that thiscapacity will be related to the stability and solubil-ity of M–HC in soil solution. In fact, the importanceof the complexing capacity of soluble organic matterfor the availability of micronutrients has long beenacknowledged, especially under adverse soil condi-tions (Hodgson, 1969). Thus, Hodgson et al. (1965,1966) and Geering et al. (1969) found that most Cu,Zn, Fe and Mn in soil solution in limy, alkaline soilswas present in the form of organic matter complexes.Similarly, several studies have demonstrated the rela-tionship between on the one hand complexed metalsolubility and mobility in soil, and on the other hand,the solubility and stability of the metal–humic com-plexes in soil solution (Bar-Ness and Chen, 1991;Mirave and Orioli, 1987). What is more, the import-ance of metal chelate solubility and stability in metalbioavailability in soil systems has also become thecentral topic of many studies into Fe availability fromsynthetic chelates such as Fe-EDDHA, Fe-EDTA andFe-DTPA (O’Connor et al., 1971). From these, it hasbecome clear that Fe cannot be available unless tightlybound to soluble chelates able to withstand the unfa-vorable milieu of alkaline, calcareous soils (basic pHand competing cations principally). Nevertheless, che-late stability must not be so high that plants are unableto take up the chelated metal (Lucena et al., 1987).

The aforementioned data lead us to conclude thatthe discrepancy between results from soil and fromnutrient solution studies must be directly related tothe physicochemical properties of M–HC used in theexperiments, and essentially to their stability and sol-ubility. Unfortunately, in the above-mentioned worksno study was made of the stability and solubility of

the metal–humic complexes employed in soil–plantexperiments.

Thus, the aim of our research was to clarify pre-vious studies using metal–humic complexes (M–HC)of known stability and solubility as starting point,and testing their ability to supply the complexed mi-cronutrients to different plant species cultivated inseveral soil types. To this end, we studied: (i) thestability and solubility of the humic complexes usedin plant experiments with defined conditions of pH(6,7,8 and 9) and ionic strength (I = 3 mM) match-ing common soil values (Elprince, 1986; García-Mina,1998); (ii) the preparation of metal–humic complexesfor plant experiments based on the results obtained inthe stability–solubility study, in order to ensure theiradequate stability and solubility and (iii) the effect ofthe metal–humic complexes on micronutrient uptakeand development of two plant species (wheat and al-falfa) cultivated in different soil types (two alkalinecalcareous soils and an acid organic soil). This exper-imental design included graminaceous and dicotyle-donous plants, with different Fe uptake mechanisms(Schmidt, 1999), and soils favouring micronutrientdeficiencies (basic pH, high carbonates, low organicmatter content, and poorly humified acid soils) (Loue,1993).

Materials and methods

Extraction and physicochemical characterisation ofthe selected humic extract (SHE)

SHE was extracted from a sapric peat from Cantabria(Spain) using the methodology described by Steven-son (1994) with some operational modifications. Inshort, 10 g of non dried organic material was weighedin a 250-mL flask to which 0.1 M NaOH was addeduntil all the air had been displaced. After 48 h stirringat 25 ◦C in darkness, the supernatant containing theSHE was separated from the solid fraction by cent-rifugation at 7650 × g for 30 min. Subsequently thesupernatant was treated with the necessary amount ofcation exchange resin (Amberlite IRA –118H+) untilpH values were in the 3.5 – 4 range. The resin was sep-arated by filtration (Whatman no. 42 filter paper), andthe supernatant containing the whole humic extract(SHE) was freeze-dried.

The main chemical properties of SHE were:56.23% C, 4.87% H, 37.03% O and 1.87% N (multi-elemental analysis. Carlo Erba EA7), E4/E6: 5.52

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(Chen et al., 1977), 2.35 and 2.74 meq g−1 ofcarboxylic and phenolic groups, respectively (Poten-tiometric titration. Takamatsu and Yoshida, 1978)and 8.07% of degree of aromaticity (X-ray analysis.Schnitzer et al., 1991).

Study of the stability and solubility of the metal humiccomplexes (M–SHE)

In order to study the influence of the SHE:metal reac-tion stoichiometry on the stability and solubility of thecomplexes at different pH values, the reaction betweena constant concentration of SHE (90 mg L−1 ) anddifferent concentrations of each micronutrient (Cu(II),Zn(II), and Fe(II)) was carried out at the followingpH values: 6, 7, 8 and 9 (soil pH range). I (3 mM)was chosen according to its main value in soil solutionfrom soils under irrigation (Elprince, 1986). pH andI were kept constant in all the reactions. The SHE(mg):metal (mg) ratios in the reaction were: 2:0.10,2:0.25, 2:0.40, 2:0.55, 2:0.70, 2:0.85, 2:1.00, 2:1.50,and 2:2.00.

All the reactions were carried out in darkness,with constant stirring, at a temperature of 25 ◦C, fora period of 24 h. The air was displaced by the liquid.Metals (Cu(II), Fe(II) and Zn(II)) were added veryslowly as sulphate solutions, adjusting pH with 0.1 MNaOH.

In order to measure complexed metal in solution,free metal and SHE, the following methodology wasused:1. Once the reaction had finished, the precipitated

fraction containing metal hydroxides and insolublehumic complexes was separated by centrifugationat 10 000 × g for 30 min.

2. Ultrafiltration was used to separate complexed anduncomplexed metal in the solution obtained fromstep 1. A 1000 D ultrafiltration cell (Filtron, poly-ethersulfon type) was used and ultrafiltration plotswere first obtained with known concentrations ofeach metal in similar conditions of pH and I(García-Mina, 1998). In all cases, the retentioncoefficient of the ultrafiltration cell for M-SHE andSHE was 1 (full retention).From the supernatant, the concentration of SHE

in solution was determined using oxidation with 0.01N KMnO4 in an alkaline medium (Primo Yufera andCarrasco, 1987) (1 meq kmnO4 oxidises 9.4 mg SHE).Metal concentration in solution, before and after ul-trafiltration, was analysed using atomic absorptionspectrometry (AAS). Values of SHE in solution, total

complexed metal and total free metal (total metal –total complexed metal) selected for stability studieswere obtained from reactions in which there were noprecipitation of SHE.

The relative solubility of humic complexes with re-spect to pH and SHE:metal reaction stoichiometry wasdetermined by means of a solubility index (IS) definedas the SHE:metal ratio in the reaction correspondingto the precipitation of SHE over 50%.

PH-dependent stability of the humic complexesin solution was estimated using an apparent stabil-ity constant (K0) calculated by the Scatchard method(Stevenson, 1994):

θ /MFT = K0 - θ K0

MFT being the total free metal in the equilib-rium; MCT the total complexed metal; and θ theSites bound/Maximum metal binding capacity (MBA)(MCT / MBA) ratio, where K0 (K01 for low metal ionsaturation and K02 for high metal ion saturation) wasobtained from the plot θ /MFT vs. θ .

MBA was obtained from experimental data us-ing the double-surface Langmuir equation (Fitch andStevenson, 1984):

MCT = [b1-MCT /K1MFT ]+ [b2-MCT /K2MFT ].

MBA was estimated by extrapolating the straight-line segment corresponding to binding at high metalion saturation in a MCT vs. MCT /MFT plot. Resultscorresponding to SHE:metal reaction ratios withoutprecipitation of SHE were only considered in thecalculation of K0.

Preparation of M–SHE complexes used in soil-plantexperiments

M–SHE complexes were prepared with 1 g of freeze-dried SHE. This was dissolved in 25 mL 0.1 M NaOH,diluted with 250 mL distilled water and pH adjustedto 8 with 0.1 M NaOH or 0.1 M HCl. Fe(II), Cu(II)and Zn(II) were added as sulphates to attain a stoi-chiometry 2:0.25 (SHE:metal, w/w) while maintainingpH 8 by the addition of NaOH. Solutions were stirredin darkness at 25 ◦C for 24 h. After this time, Zn-SHEsolution was further alkalinised to pH 9.5 and stirredfor two additional hours to precipitate any remaininguncomplexed Zn. It was assumed that some com-plexed Zn could also be lost. The complete extent offree metal precipitation under the conditions describedabove (pH 8 for Cu and Fe, and pH 9.5 for Zn) wassubstantiated in previous studies (García-Mina, 1998).

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The M–SHE solutions thus prepared were centrifugedagain at 7650 × g for 30 min and the supernatantsanalysed to obtain final SHE:metal stoichiometries inthe complexes. SHE was analysed by oxidation with0.01 N KMnO4 in an alkaline medium (Primo Yúferaand Carrasco, 1987) (1 meq KMnO4 oxidizes 9.4 mgSHE). Metal content was analysed by AAS. Final SHE(mg):metal (mg) stoichiometries were Fe-SHE 2:0.22,Zn-SHE 2:0.165 and Cu-SHE 2:0.17.

Plant experiments

Pots were prepared using the following soils: twoalkaline, Typic Xerorthent calcareous, low organicmatter soils, one a clay soil very prone to compactiondue to its high clay content (Legarda soil); the other ahigh sulphate silt soil with little tendency to compac-tion (Mendigorría soil). The third soil type was an acidsandy Typic Dystrupdet soil rich in poorly humifiedorganic matter (Velate soil). The main soil propertieswere: for Legarda soil, 8.0 pH (water), 2% of totalorganic matter, 52.3% of total carbonates and 3.2 mg,9.08 and 0.48 mg kg−1 of DTPA-extractable Cu, Feand Zn, respectively (Lindsay and Norvell, 1978); forMendigorría soil, 8.2 pH (water), 1.4% of total organicmatter, 39.9% of total carbonates, 11.7% of calciumsulphate and 1.02, 4.86 and 0.48 mg kg−1 of DTPA-extractable Cu, Fe and Zn, respectively, and for Velatesoil, 5.5 pH (water), 9.8% of total organic matter and0.56, 196 and 1.2 mg kg−1 of DTPA-extractable Cu,Fe and Zn respectively (Norvell, 1984). These ana-lyses were carried out on air-dried soil samples atroom temperature after sieving using a plastic sieve(0–2.5 mm).

The procedure for obtaining and preparing the dif-ferent soils was as follows: having eliminated the first10 cm, samples corresponding to 40 cm of soil weretaken. Non-cultivated areas of each soil were selec-ted in order to eliminate the residual effects of bothordinary fertilisation and crop development on the mi-cronutrient available soil fraction. Soil samples wereair-dried at room temperature and soil aggregates werereduced using a Teflon hammer. Finally, soil sampleswere sieved using a plastic sieve (size 0–2.5 mm) toavoid possible metal contamination. Soil samples werealso well mixed before the experiments.

Wheat plants (Triticum aestivum L. cv. Marius)were grown in each soil; alfalfa (Medicago sativa L.cv. Aragon) only in Velate and Legarda soils. Pots con-tained 800 g soil, with six plants each, three pots forwheat and five for alfalfa. These numbers have been

shown to be sufficient to compensate for variability(García-Mina, 1998). The experiments were carriedout in May, June and July. Greenhouse photoperiodwas approximately 14/10 h day/night using additionallighting when needed. The average temperature was25/15 ◦C day/night although variations of up to + 5 ◦Cwere occasionally observed on very sunny days. Therelative humidity was between 50 and 70% dependingon the time of day. Plants were watered daily to fieldcapacity to avoid any stress due to drought.

FertilisationEach pot received a single NPK fertiliser dose perkg of soil of 100 mg N as NO3NH4, 42.7 mg P asKH2PO4, 244.8 mg K as KH2PO4, KCl and K2SO4,each 30 days as a liquid solution along with the ir-rigation water. The concentration of the nutrients waschosen according to the results of the soil nutrientanalyses.

TreatmentsM–SHE pots (Fe-SHE, Cu-SHE and Zn-SHE) re-ceived 2.5 or 5.0 mg kg−1 of complexed metal in asingle application. At the same time, SHE pots re-ceived an SHE amount (53 mg kg−1) equivalent toSHE content added as a complex in M–SHE pots.Control pots received no treatment.

N-P-K fertilisation was applied 48 h after planting.The SHE complexes and the SHE were applied 48 hafter the N-P-K fertilizer application.

Plants were harvested at random 90 days aftertreatment. Air-dried soil samples at room temperaturefrom each pot were used to analyse soil micronutri-ent availability using the DTPA method as describedbelow. These soil samples corresponded to the rhizo-sphere area. Although the root material was carefullyseparated previously, some root material could haveremained in soil samples.

Plant analysisShoot and root dry weight was determined after dry-ing at 85 ◦C for 48 h. Shoot nutrient content (mgkg−1) was measured by AAS according to Gárate etal. (1984).

A post-hoc comparison of means was carried outwith the LSD test for paired treatments. Statistic ofinter-group significance was fixed at p≤0.05.

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Analysis of the DTPA-extractable soil fraction ofmicronutrients in plant experiments

Soil levels of potentially plant available Cu, Fe, and Znwere determined using the DTPA method, pH 7.3 foralkaline, calcareus soils (Lindsay and Norvell, 1978)and pH 5.3 for acid soil (Norvell, 1984). The valuesobtained were referred to as an index of plant mi-cronutrient availability. The analysis was carried outon dried soil samples from each pot after plant rootswere separated.

Results and discussion

Stability and solubility studies

It is not the purpose of this research to investigatethe stability of humic complexes from a general pointof view, but rather to better understand the effects ofthese complexes on plant nutrition and development insoil systems. Furthermore, there are a great number ofstudies in the literature on the stability of this kind oforganic compound and on the development of physi-cochemical models which allow adequate descriptionof the complexing process (Kinniburgh et al., 1996;Stevenson, 1994; Tipping et al., 1995). Therefore,we will only briefly describe our results in the lightof their possible relationships with plant developmentand plant micronutrient uptake.

As can be seen in Table 1, all M–SHE complexesshowed a considerable stability in the pH interval thatcorresponds to soil pH values. The stability constantvalues were also consistent with those reported inprevious work (Stevenson, 1994). Regarding Cu com-plexes, as reported by other authors (Cao et al., 1995),the value of the stability constant (log K01, log K02)decreased as the pH becomes more basic. This resultsuggests a specific role in the complexing process ofchemical groups whose chemical reactivity is modi-fied as the pH varies from acid to basic values. Someauthors have related these groups to amino groupslinked to aliphatic or aromatic substructures (Senesi,1992). In the case of Zn complexes, the greatest sta-bility was found for pH values of 7 and 8, which cor-respond to those of neutral and alkaline soils. This factsuggests the intervention of strongly acidic carboxylicand phenol groups in the complexing process at thesepH levels. At pH 6, stability was also important al-though lower than at alkaline pH levels. Stability atpH 9 decreased significantly, probably because of the

Table 1. Stability (Log K0) and solubility (IS) of differentM–SHE complexes

M–SHE Log K0 IS†

pH Log K01 Log K02 SHE(mg):metal (mg)

ratio

Cu–SHE

6 4.20 – 2:0.40

7 3.74 – 2:0.55

8 3.49 1.89 2:0.70

9 3.37 1.59 2:1.50

Zn–SHE

6 2.01 1.53 2:1.50

7 3.99 1.58 2:1.50

8 3.72 1.66 2:1.50

9 1.80 1.15 2:2.00

Fe–SHE

6 3.09 1.45 2:1.00

7 2.91 1.40 2:1.50

8 4.11 1.03 2:1.50

9 1.74 0.79 2:2.00

† IS is defined as the SHE:metal ratio in the reaction corres-ponding to the precipitation of SHE over 50%.

competition between the complexing process and theformation of the insoluble hydroxide (Zn(OH)2). Fur-thermore, in this case the importance of the ionisedamine groups which has been observed by other au-thors in other humic extracts (Senesi, 1992), is notas clear as in the case of Cu complexes, although thestability at pH 6 is suggestive of this fact.

In the case of Fe complexes, a clear maximumstability was observed at pH 8. The stability at pH 6and 7 was similar and also important. At pH value of9, a clear reduction in stability was observed. Such apronounced increase in stability at pH 8 could reflectthe role of phenolic groups – those most markedlyacid in nature – in the complexing process, in ad-dition to carboxylic groups. The fall in stability atpH 9 suggests that other factors exert an importantinfluence on the complexing process apart from theincrease in the ionisation of the phenolic groups. Suchfactors are probably directly associated with the con-formational changes accompanying the increase in pH(Swift, 1989), and with the formation of Fe(OH)−4that might cause a significant change in the model ofchemical interaction between Fe and the complexinggroups.

With regard to M–SHE solubility, the followinggeneral rules appeared to be present in these chemicalsystems (Table 1):

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- When a specific SHE:M ratio is considered, thereseems to be a directly proportional relationshipbetween the concentration of M–SHE in solutionand pH alkalinity. As the pH level increases sodoes the concentration of M–SHE remaining insolution.

- There seems to be a directly proportional rela-tionship between M–SHE concentration and theSHE:metal reaction stoichiometry, since a clearprecipitation of the complexes takes place as theSHE:metal ratio in the reaction decreases.These results are consistent with those obtained by

others studying the flocculation of humic extracts bymetallic cations (Spark et al., 1997; Stevenson, 1994).Furthermore, this variation in solubility is consistentwith the influence exerted on this property by theconformational changes which these molecules mayundergo in response to pH, I and the increase in thecomplexing process (Senesi, 1992; Swift, 1989). Sim-ilarly, these results show the considerable influence ofthe physicochemical characteristics of the M–HC ontheir solubility in soil solution, and therefore on theirpotential biological activity.

Effects on micronutrient uptake and development ofplants

It is interesting to note the significant increase ofDTPA-extractable soil fraction of micronutrients incontrol pots of every plant–soil system (Tables 2–6).This fact was especially clear for Fe and Zn in wheatplants, and for all studied micronutrients in alfalfaplants. These results show the significant efficiency ofthe specific mechanisms that plants have in order tomodify the dynamics of nutrients in the rhizosphereunder adverse soil conditions (Uren, 2001). Thus,under Fe deficiency, graminaceous species releaseto the rhizosphere non–proteinogenic amino acids(phytosiderophores) with the capacity to solubilizenon-available iron and other micronutrients (princip-ally Zn and Cu) by the formation of stable complexesthat are directly taken up by roots. The response ofdicots and non graminaceous monocots to Fe defi-ciency seems to be more complex including differentco-ordinated events such as an increase in root re-ductase activity, proton extrusion, the development oftransfer cells and the release of low molecular weightorganic compounds with chelation and/or reductionactivity (Schmidt, 1999). Likewise, different studieshave pointed out that these mechanisms could also beefficient to mobilise and increase the plant uptake of

other micronutrients such as Cu and Zn (Treeby etal., 1989). The fact of carrying out our experimentsin pots involves that the observed effects – either onplant micronutrient uptake or on the DTPA-extractablefraction – have to be referred to as rhizosphere effectswhich could have been quantitatively potentiated bythis closed experimental system. Finally, nor can theimpact of the microbial activity in the rhizophere areabe neglected.

In order to evaluate the degree of Fe-, Cu- andZn-deficiency in both plant species cultivated in thedifferent soils used, we have compared the levels ofthese micronutrients in the control plants with tabu-lated data from several sources (Loue, 1993). Thus,both wheat and alfalfa plants presented a clear defi-ciency state in Velate soil. Wheat plants also presentedlow shoot contents of Zn, Cu and Fe in Legardasoil and of Zn and Fe in Mendigorría soil while al-falfa plants only presented low shoot content of Fein Mendigorría soil. It is also interesting to note thevery high Fe-shoot content in alfalfa plants growingin Velate soil, which is close to toxicity levels. Ingeneral, these results were coherent with those of thepotential soil content of plant available micronutrientsexpressed by the DTPA-analysis.

Regarding SHE effects, no significant increaseswere found in shoot Cu or Zn content whatever theplant or soil type (Tables 2-6). Fe content significantlyincreased in wheat plants grown on the soil of lowestFe availability (Mendigorría soil, Table 4). This resultwas associated with an equally significant increase ofDTPA-extractable iron. In the wheat–Legarda soil sys-tem a significant increment of the DTPA-extractableiron was also observed but it was not associated with aconcomitant increase of shoot Fe content (Table 3). Anopposite effect was observed on alfalfa plants grown inhigh Fe availability soil (Velate soil, Table 5) wherea significant decrease of shoot Fe content was ob-served. These results, far from being contradictory, fitthe model proposed by several authors (Chen, 1996;Stevenson, 1994): SHE complexation of metals in-creases their solubility, mobility and availability underdeficiency conditions (Mendigorría or Legarda soilconditions). Conversely, complexation of highly avail-able metals results in a kind of sequestering effect(Velate soil conditions). Furthermore, there is closeagreement with the results obtained in relation to theinfluence of pH and SHE:metal stoichiometry on com-plex solubility: the greater the proportions of Fe withrespect to SHE, the lower the solubility of Fe–SHEat pH 6 (under the same conditions as in Velate soil:

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Table 2. Effect of metal-humic complexes (Cu–SHE, Zn–SHE and Fe–SHE) on the concentration of soil available Zn, Cuand Fe, and on shoot dry weight, root dry weight and shoot Zn-, Cu- and Fe-content of wheat plants cultivated in Velatesoil (humic rate in SHE and metal-SHE treatments was 53 mg kg−1)

Velate soil Shoot dry Root dry Zn content Cu content Fe content

weight weight

(g pot−1) (g pot−1) (mg kg−1 (mg kg−1) (mg kg−1)

Treatments Shoot Soil# Shoot Soil# Shoot Soil#

Control 2.31a† 1.41ab 21.89a 2.22a 2.87a 0.28a 66.35bc 268a

SHE 1.87a 1.48ab 25.76a 2.46a 2.83a 0.72a 61.87b 269a

Cu – SHE (metal rate)

Dose 1 (2.50 mg kg−1) 3.90c 2.62c 23.14a 2.46a 4.52b 1.99b 41.42a 297a

Dose 2 (5.00 mg kg−1) 3.23b 2.67c 28.57a 2.73a 6.76c 3.19c 82.02c 284a

Zn – SHE (metal rate)

Dose 1 (2.50 mg kg−1) 1.80a 0.89a 34.58b 3.55b 2.61a 0.46a 55.53ab 271a

Dose 2 (5.00 mg kg−1) 2.40a 0.98a 39.29b 4.21b 2.37a 0.36a 84.37c 267a

SHE – Fe (metal rate)

Dose 1 (2.50 mg kg−1) 2.31a 1.56ab 25.17a 2.65a 3.88ab 0.48a 82.79c 279a

Dose 2 (5.00 mg kg−1) 2.46a 1.70b 23.17a 2.61a 2.76a 0.45a 69.61bc 285a

Table 3. Effect of metal-humic complexes (Cu–SHE, Zn–SHE and Fe–SHE) on the concentration of soil available Zn, Cu andFe, and on the shoot dry weight, root dry weight and shoot Zn-, Cu- and Fe-content of wheat plants cultivated in Legarda soil.(humic rate in SHE and metal–SHE treatments was 53 mg kg−1)

Legarda Soil Shoot dry Root dry Zn content Cu content Fe content

weight weight

(g pot−1) (g pot−1) (mg kg−1) (mg kg−1) (mg kg−1)


Control 4.03a† 2.87b 6.10ab 1.10a 4.72a 3.68a 65.40bc 21.52a

SHE 4.44a 2.20ab 5.51a 1.33a 4.72a 4.25a 73.42c 30.01b


Dose 1 (2.50 mg kg−1) 4.49a 2.07a 5.79ab 0.97a 6.18b 4.42a 49.18ab 21.39a

Dose 2 (5.00 mg kg−1) 4.73a 2.82b 5.60ab 0.99a 6.35b 8.77b 61.40bc 22.03a


Dose 1 (2.50 mg kg−1) 4.62a 2.20ab 10.23d 2.79b 5.32a 4.02a 65.75bc 24.35a

Dose 2 (5.00 mg kg−1) 4.78a 1.97a 9.15cd 3.13b 4.83a 3.62a 46.57a 21.74a


Dose 1 (2.50 mg kg−1) 4.57a 2.16ab 7.24b 0.95a 4.98a 3.32a 46.03a 25.27ab

Dose 2 (5.00 mg kg−1) 4.50a 2.69ab 8.09bc 1.02a 5.68ab 3.51a 55.52ab 26.23ab

† Different letters indicate significant (p ≤ 0.05) differences between treatments.# DTPA, pH 7.3 (Lindsay and Norvell, 1978).

acid pH, very high in available Fe). Also, the lowerthe proportion of Fe with respect to SHE, the higherthe solubility of Fe-SHE at pH 8 (under the same con-ditions as in Mendigorría soil: alkaline pH, poor inavailable Fe).

SHE did not significantly affect shoot or root dryweight (Tables 2–6). Many studies have demonstratedthe ability of HS to increase plant growth in vari-ous soil types (Nardi et al., 1996). However, theseexperiments were made at higher HS concentrations,

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Table 4. Effect of metal-humic complexes (Cu–SHE, Zn–SHE and Fe–SHE) on the concentration of soil available Zn, Cu andFe, and on the shoot dry weight, root dry weight and shoot Zn-, Cu- and Fe-content of wheat plants cultivated in Mendigorrı́asoil (humic rate in SHE and metal–SHE treatments was 53 mg kg−1)

Mendigorrı́a Soil Shoot dry Root dry Zn content Cu content Fe content

weight weight

(g pot −1) (g pot −1) (mg kg−1) (mg kg−1) (mg kg−1)


Control 4.48a† 2.26a 8.44a 0.99a 6.70ab 2.38a 50.47ab 13.92a

SHE 5.11abc 2.69a 7.19a 1.15a 6.06a 3.38a 80.62c 18.15b


Dose 1 (2.50 mg kg−1) 5.86c 2.54a 7.74a 0.88a 7.41bc 6.07b 52.07ab 14.87a

Dose 2 (5.00 mg kg−1) 5.15abc 3.19ab 7.70a 1.37a 8.55c 5.59b 63.69b 13.65a


Dose 1 (2.50 mg kg−1) 5.29ab 3.16ab 11.50b 2.43b 5.68a 3.05a 50.23ab 16.43ab

Dose 2 (5.00 mg kg−1) 4.96ab 3.07ab 15.84c 3.35b 6.91ab 3.97a 59.87b 14.09a


Dose 1 (2.50 mg kg−1) 5.66bc 2.63a 7.13a 0.81a 5.97a 3.28a 45.91a 12.93a

Dose 2 (5.00 mg kg−1) 5.04abc 3.70b 8.20a 0.85a 6.87ab 2.82a 44.07a 16.09ab


Table 5. Effect of metal-humic complexes (Cu–SHE, Zn–SHE and Fe–SHE) on the concentration of soil available Zn, Cu and Fe,and on the shoot dry weight, root dry weight and shoot Zn-, Cu- and Fe- content of alfalfa plants cultivated in Velate soil. (humicrate in SHE and metal–SHE treatments was 53 mg kg−1)

Velate soil Shoot dry Root dry Zn content Cu content Fe content

weight weight



Control 1.71a† 0.93a 57.45ab 2.15ab 5.46a 7.45a 132.94b 290ab

SHE 1.39a 0.89a 99.20b 2.52bc 4.08a 7.49a 89.39a 281ab


Dose 1 (2.50 mg kg−1) 3.90cd 2.60bc 67.39ab 2.64bc 8.70b 7.51a 77.06a 283ab

Dose 2 (5.00 mg kg−1) 3.78cd 2.45bc 42.31a 2.32bc 14.66c 7.86b 110.95ab 311b


Dose 1 (2.50 mg kg−1) 4.18d 2.05b 152.37c 2.74c 5.16a 7.47a 109.29ab 313b

Dose 2 (5.00 mg kg−1) 2.31ab 0.92a 205.74d 2.01ab 3.03a 7.25a 98.43ab 265a


Dose 1 (2.50 mg kg−1) 3.11bc 2.02b 75.54ab 1.74a 4.14a 7.32a 94.39ab 293ab

Dose 2 (5.00 mg kg−1) 4.05d 3.15c 33.13a 1.99ab 5.31a 7.45a 89.98a 295ab

† Different letters indicate significant (p ≤ 0.05) differences between treatments.# DTPA, pH 5.3 (Norvell, 1984).

200–4000 mg kg−1, than those used in our exper-iments. The low dose we used (53 mg kg−1) waschosen to allow a clearer discrimination between theeffect of M–SHE and SHE alone.

As for Cu–SHE effects, this complex significantlyincreased shoot Cu content in every test (Tables 2–6) except those made in the wheat-Mendigorria soilsystem (low Cu dose) and the alfalfa–Legarda soil sys-

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Table 6. Effect of metal–humic complexes (Cu–SHE, Zn–SHE and Fe–SHE) on the concentration of soil available Zn, Cu andFe, and on the shoot dry weight, root dry weight and shoot Zn-, Cu- and Fe- content of alfalfa plants cultivated in Legarda soil.(humic rate in SHE and metal–SHE treatments was 53 mg kg−1)

Legarda soil Shoot dry Root dry Zn content Cu content Fe content

weight weight



Control 5.34a† 2.34a 17.02b 1.50a 13.61b 10.71a 81.92bc 25.28a

SHE 5.87ab 2.10a 16.28ab 1.43a 12.68ab 10.75a 80.30bc 24.31a


Dose 1 (2.50 mg kg−1) 7.16bc 2.61a 17.57b 1.43a 14.07b 12.71b 65.82ab 25.00a

Dose 2 (5.00 mg kg−1) 9.28c 4.71d 15.26ab 1.54a 13.76b 14.29c 59.67a 26.16a


Dose 1 (2.50 mg kg−1) 9.35c 3.16b 26.49c 2.54b 10.44a 10.71a 57.87a 24.15a

Dose 2 (5.00 mg kg−1) 9.24c 3.84c 25.40c 2.86b 10.65ab 10.05a 64.23ab 22.61a


Dose 1 (2.50 mg kg−1) 6.48ab 2.10a 13.33a 1.29a 10.39a 9.07a 95.65c 23.19a

Dose 2 (5.00 mg kg−1) 7.50bc 3.49bc 16.53ab 1.64a 11.45ab 10.79a 80.33bc 26.40a


tem (both doses) possibly due to a dilution effect asa result of higher shoot dry weight (Tables 4 and 6).Differences were significant both in relation to controland SHE treatment. These results were well correlatedto those of the Cu-DTPA soil fraction and they werealso consistent with those obtained for the solubilityand stability of Cu–SHE complexes at both acid andbasic pH (Table 1). In this sense the result obtained inrelation to plant Cu uptake in velate soil indicates thatthis stability was sufficient to protect complexed Cuagainst secondary reactions with non-humified organicresidues which would cause Cu unavailability.

Cu-SHE treatment significantly increased shootdry weight in relation to control and to SHE treatmentin every test, except that made in the wheat–Legardasoil system where the increases were not significant(Tables 2–6). This was more apparent in plants grownin Velate soil, very deficient in Cu, where root de-velopment also increased significantly (Tables 2 and5).

No references have been found in the literature re-garding Cu–HC effects on soil grown plants, but onlyon inert substrates, mainly silica sand (Ennis, 1962;Ennis and Brogan, 1961; Gupta, 1986; Gupta andHäni, 1980).

With regard to Zn-SHE, this complex significantlyincreased shoot Zn content as well as soil Zn-DTPA

in every test (Tables 2–6). Differences were signi-ficant in relation to both control and SHE. Theseeffects were proportional to dose for the wheat–Velatesoil, wheat–Mendigorría soil and alfalfa-velate soil(Tables 2, 4 and 5). The effects of Zn–SHE on shootand root dry weight varied according to plant species(Tables 2–6). Wheat plants did not increase shoot dryweight, whereas alfalfa plants did (the reduction inshoot dry weight at highest Zn–SHE dose in alfalfa-velate soil system may reflect a Zn toxicity effect).These results reflect the different sensitivity of bothplant species to Zn deficiency (Abo and Pinta, 1982;Lo and Reisenauer, 1968). Thus, Abo and Pinta (1982)did not observe very significant changes in wheat plantgrowth even under very severe micronutrient deficientconditions except in the case of Cu deficiency.

Although several studies into the effects of Zn–HC complexes on Zn plant uptake have been carriedout on inert substrates (Barnard et al., 1992) or hy-droponics (Miravé and Orioli, 1989) we have foundonly one study specifically based on soil. Kumar andPrasad (1989) studied the effect Zn–fulvic complexeson maize grown in an alkaline, calcareous soil. Theyfound a significant shoot Zn uptake but not a shootZn content increase. Control plants were only mar-ginally Zn deficient, and Zn supplied was exiguouslyabsorbed (less than 7% of the largest dose of 5 mg

66

kg−1). Therefore, it is tempting to attribute shoot Znuptake to growth stimulation caused by the fulvic acidfraction of the complexes, and account for the poorperformance of Zn–fulvic complexes by reference tolow complex stability or solubility.

Unlike Cu–SHE and Zn–SHE, Fe–SHE did notincrease Fe DTPA-extractable soil fraction, shoot Fecontent or shoot and root dry weight in the differentplant–soil system excepting the alfalfa–velate systemwhere dose 2 of the complex caused a significant de-crease of shoot Fe (Tables 2–6). This phenomenonis probably related to a decrease in the iron contentin soil solution caused by the formation of Fe–SHEcomplexes with a very small SHE:Fe ratio and lowsolubility at acid pH (Table 1).

On the other hand, as has been previously dis-cussed, Fe content significantly increased in wheatplants treated with SHE alone grown on the soil oflowest Fe availability (Mendigorría soil, Table 4), thusshowing the capacity of certain Fe–SHE complexesto improve Fe plant uptake under adverse conditions.In the light of these results, we have to conclude thatthe Fe–SHE prepared for plant experiments lacked thenecessary stability, solubility or both to adequatelysupply Fe to plants under our experimental condi-tions. Other contributing factors, along with SHE:Fecomplex stoichiometry, such as adsorption phenom-ena or interaction with Ca, mg or trace metals, may berelevant to Fe-SHE stability and solubility.

The above results are in accordance with resultsobtained by other authors. Thus, Burau et al. (1960)studied the performance of Fe–HC as sources of Fein beans grown in alkaline, calcareous soil. These au-thors obtained no significant increases in Fe uptakewhen results were compared with those of a con-trol of acidified FeCl3. In these experiments, Fe:HCstoichiometry was much lower, and their complexpredictably less soluble than ours. Alva and Obreza(1998) studied the effect of a Fe-rich organic producton Fe uptake in several varieties of orange trees andgrapefruit grown in alkaline, calcareous soils. Leaf Feincrease was significant in some varieties, but final leafFe values were rather low, sometimes close to defi-ciency (Legaz et al., 1995). This Fe-rich product wasobtained as a by-product of water purification withiron sulphate, with quite a high (18%) Fe content.No further description of physicochemical propertieswas provided, but taking into account the preparationmethod and the low stoichiometry, complexes wouldhave been sparingly soluble in water.

Finally, Pandeya et al. (1998) studied the effect ofFe–fulvic complexes on the Fe uptake of rice plantsgrown in an incubation medium containing a frac-tion of an alkaline, calcareous soil. Although theseauthors made no reference to Fe:fulvic acid complexstoichiometry, their results showed a clear correlationbetween Fe uptake and complex diffusion in the soil.

In conclusion, our results prove the capability ofCu– and Zn– SHE complexes to supply plant availableCu and Zn under soil conditions favoring the defi-ciency of both micronutrients. In the case of Fe–SHEcomplex, the obtained results emphasize the great in-fluence of Fe:SHE stoichiometry on Fe bioavailabilitydue to its impact on complex solubility. Also, un-der the experimental conditions used in this study,where increases of plant growth associated with M–SHE treatment were observed, they were caused byan improvement in plant micronutrient uptake, sincethe humic system alone did not show any significanteffect. These results are in agreement with the theoryproposed by some authors that the effect of humicsubstances on plant development is only due to theimproved intake of some micronutrients (Chen et al.,1999). However, our results do not allow us to ruleout the possibility of direct, pseudohormonal effectsof SHE since the SHE dose was much lower than thestimulating doses proposed by most authors (Nardi etal., 1996).

Acknowledgements

This research was funded by the CYCYT (SPAIN), theGovernment of Navarra (Spain) and Roullier Group.The authors thank Prof. Yona Chen for his valu-able comments and suggestions; Rodrigo G. Canteraand Paul Miller and David Rhymes for their help inthe preparation of the manuscript and Professor JordiGarrigo for his help in soil classification.

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