Plant and Soil 258: 5768, 2004.

2004 Kluwer Academic Publishers. Printed in the Netherlands.

57

Metal-humic complexes and plant micronutrient uptake: a study based on

different plant species cultivated in diverse soil types
J.M. Garca-Mina1,2,4 , M.C. Antoln3 & M. Sanchez-Diaz3
1 R&D

Department. Inabonos-Roullier Group; Poligono Arazuri-Orcoyen, C/ C no 32, 31160 Orcoyen (Navarra),

Spain. 2 Department of Chemistry and Soil Chemistry, Faculty of Sciences, University of Navarra, PO Box 273,
31080 Pamplona (Navarra), Spain. 3 Department of Plant Physiology, Faculty of Sciences, University of Navarra,
PO Box 273, 31080 Pamplona (Navarra), Spain. 4 Corresponding 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 study
covers both aspects under various experimental conditions. A humic extract (SHE) obtained from a sapric peat was
selected for preparing the metalhumic complexes used in plant experiments. Fe, Zn and Cuhumic complexes
with a reaction stoichiometry of 2:0.25 (humic:metal, w/w) were chosen after studying their stability and solubility
with respect to pH (69) and the humic:metal reaction stoichiometry. Wheat and alfalfa plants were greenhouse cultured in pots containing one of three model soils: an acid, sandy soil and two alkaline, calcareous soils. Treatments
were: control (no additions), SHE (53 mg kg1 of SHE), and metal (Cu, Zn and Fe)SHE complexes (2.5 and 5 mg
kg1 of metal rate and a SHE concentration to make 53 mg kg 1 ). Cu- and Znhumic complexes significantly
(p0.05) increased the plant uptake and the DTPA-extractable soil fraction of complexed micronutrients in most
plantsoil systems. However, these effects were associated with significant increases (p0.05) of shoot and root
dry weight only in alfalfa plants. In wheat, significant increases of root and shoot dry matter were only observed
in the Cuhumic treated plants growing in the acid soil, where Cu deficiency was more intense. The Fehumic
complex did not increase Fe plant assimilation in any plantsoil system, but SHE increased Fe-uptake and/or
DTPA-extractable soil Fe in the wheatcalcareous soil systems. These results, taken together with those obtained
from the study of the pH- and SHE:metal ratio-dependent SHE complex solubility and stability, highlight the
importance of the humic:Fe complex stoichiometry on iron bioavailability as a result of its influence on complex
solubility.

Introduction
Excellent studies and reviews on the ability of humic
substances (HS) to affect plant micronutrient uptake
due to their ability to complex metals under different
environmental conditions have been published (Chen,
1996; Chen and Aviad, 1991). Likewise, a number
of studies carried out using nutrient solutions or inert substrates have shown plant capacity to assimilate
FAX No: +34-948-324032. E-mail: jgmina@inabonos.com

micronutrients from metalhumic 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 uptake mechanisms in cucumber, melon and bentgrass.
Similar results had been obtained by Mirav et al.
(1987) and Lobartini and Orioli (1988) using other
plant species, as well as by Mirave and Orioli (1989)
and Barnard et al. (1992) in relation to Zn plant uptake from ZnHC complexes, and by others in relation
to Cu plant uptake from CuHC complexes (Ennis,
1962; Ennis and Brogan, 1961; Gupta, 1986; Gupta

58
and Hni, 1980). However, in the case of plants grown
in soil, published studies are scant and results far from
clear. In fact, no clear improvement in metal plant
uptake from MHC has been observed in plantsoil
systems (Alva and Obreza, 1998; Burau et al., 1960;
Kumar and Prasad, 1989; Pandeya et al., 1998). That
is why some authors have expressed serious doubts
about the real usefulness and efficiency of these organic complexes as valuable sources of micronutrients
for crops cultivated in soils that present micronutrient
deficiency (Burau et al., 1960).
In order to explain this apparent contradiction
between results obtained from soil and from inert substrate or nutrient solution experiments several factors
have to be taken into account. For instance, the capacity of MHC to keep complexed micronutrients in
soil solution with pH values or chemical composition
which favor metal precipitation. It is clear that this
capacity will be related to the stability and solubility of MHC in soil solution. In fact, the importance
of the complexing capacity of soluble organic matter
for the availability of micronutrients has long been
acknowledged, especially under adverse soil conditions (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 soils
was present in the form of organic matter complexes.
Similarly, several studies have demonstrated the relationship between on the one hand complexed metal
solubility and mobility in soil, and on the other hand,
the solubility and stability of the metalhumic complexes in soil solution (Bar-Ness and Chen, 1991;
Mirave and Orioli, 1987). What is more, the importance of metal chelate solubility and stability in metal
bioavailability in soil systems has also become the
central topic of many studies into Fe availability from
synthetic chelates such as Fe-EDDHA, Fe-EDTA and
Fe-DTPA (OConnor et al., 1971). From these, it has
become clear that Fe cannot be available unless tightly
bound to soluble chelates able to withstand the unfavorable milieu of alkaline, calcareous soils (basic pH
and competing cations principally). Nevertheless, chelate stability must not be so high that plants are unable
to take up the chelated metal (Lucena et al., 1987).
The aforementioned data lead us to conclude that
the discrepancy between results from soil and from
nutrient solution studies must be directly related to
the physicochemical properties of MHC used in the
experiments, and essentially to their stability and solubility. Unfortunately, in the above-mentioned works
no study was made of the stability and solubility of

the metalhumic complexes employed in soilplant

experiments.
Thus, the aim of our research was to clarify previous studies using metalhumic complexes (MHC)
of known stability and solubility as starting point,
and testing their ability to supply the complexed micronutrients to different plant species cultivated in
several soil types. To this end, we studied: (i) the
stability and solubility of the humic complexes used
in plant experiments with defined conditions of pH
(6,7,8 and 9) and ionic strength (I = 3 mM) matching common soil values (Elprince, 1986; Garca-Mina,
1998); (ii) the preparation of metalhumic complexes
for plant experiments based on the results obtained in
the stabilitysolubility study, in order to ensure their
adequate stability and solubility and (iii) the effect of
the metalhumic complexes on micronutrient uptake
and development of two plant species (wheat and alfalfa) cultivated in different soil types (two alkaline
calcareous soils and an acid organic soil). This experimental design included graminaceous and dicotyledonous plants, with different Fe uptake mechanisms
(Schmidt, 1999), and soils favouring micronutrient
deficiencies (basic pH, high carbonates, low organic
matter content, and poorly humified acid soils) (Loue,
1993).

Materials and methods

Extraction and physicochemical characterisation of
the selected humic extract (SHE)
SHE was extracted from a sapric peat from Cantabria
(Spain) using the methodology described by Stevenson (1994) with some operational modifications. In
short, 10 g of non dried organic material was weighed
in a 250-mL flask to which 0.1 M NaOH was added
until all the air had been displaced. After 48 h stirring
at 25 C in darkness, the supernatant containing the
SHE was separated from the solid fraction by centrifugation at 7650 g for 30 min. Subsequently the
supernatant was treated with the necessary amount of
cation exchange resin (Amberlite IRA 118H+) until
pH values were in the 3.5 4 range. The resin was separated by filtration (Whatman no. 42 filter paper), and
the 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 (multielemental analysis. Carlo Erba EA7), E4/E6: 5.52

59
(Chen et al., 1977), 2.35 and 2.74 meq g1 of
carboxylic and phenolic groups, respectively (Potentiometric 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 humic
complexes (MSHE)
In order to study the influence of the SHE:metal reaction stoichiometry on the stability and solubility of the
complexes at different pH values, the reaction between
a constant concentration of SHE (90 mg L1 ) and
different concentrations of each micronutrient (Cu(II),
Zn(II), and Fe(II)) was carried out at the following
pH values: 6, 7, 8 and 9 (soil pH range). I (3 mM)
was chosen according to its main value in soil solution
from soils under irrigation (Elprince, 1986). pH and
I 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, for
a period of 24 h. The air was displaced by the liquid.
Metals (Cu(II), Fe(II) and Zn(II)) were added very
slowly as sulphate solutions, adjusting pH with 0.1 M
NaOH.
In order to measure complexed metal in solution,
free metal and SHE, the following methodology was
used:
1. Once the reaction had finished, the precipitated
fraction containing metal hydroxides and insoluble
humic complexes was separated by centrifugation
at 10 000 g for 30 min.
2. Ultrafiltration was used to separate complexed and
uncomplexed metal in the solution obtained from
step 1. A 1000 D ultrafiltration cell (Filtron, polyethersulfon type) was used and ultrafiltration plots
were first obtained with known concentrations of
each metal in similar conditions of pH and I
(Garca-Mina, 1998). In all cases, the retention
coefficient of the ultrafiltration cell for M-SHE and
SHE was 1 (full retention).
From the supernatant, the concentration of SHE
in solution was determined using oxidation with 0.01
N KMnO4 in an alkaline medium (Primo Yufera and
Carrasco, 1987) (1 meq kmnO4 oxidises 9.4 mg SHE).
Metal concentration in solution, before and after ultrafiltration, was analysed using atomic absorption
spectrometry (AAS). Values of SHE in solution, total

complexed metal and total free metal (total metal

total complexed metal) selected for stability studies
were obtained from reactions in which there were no
precipitation of SHE.
The relative solubility of humic complexes with respect to pH and SHE:metal reaction stoichiometry was
determined by means of a solubility index (IS) defined
as the SHE:metal ratio in the reaction corresponding
to the precipitation of SHE over 50%.
PH-dependent stability of the humic complexes
in solution was estimated using an apparent stability constant (K0 ) calculated by the Scatchard method
(Stevenson, 1994):
/MF T = K0 - K0
MF T being the total free metal in the equilibrium; MCT the total complexed metal; and the
Sites bound/Maximum metal binding capacity (MBA)
(MCT / MBA) ratio, where K0 (K01 for low metal ion
saturation and K02 for high metal ion saturation) was
obtained from the plot /MF T vs. .
MBA was obtained from experimental data using the double-surface Langmuir equation (Fitch and
Stevenson, 1984):
MCT = [b1-MCT /K1 MF T ]+ [b2 -MCT /K2 MF T ].
MBA was estimated by extrapolating the straightline segment corresponding to binding at high metal
ion saturation in a MCT vs. MCT /MF T plot. Results
corresponding to SHE:metal reaction ratios without
precipitation of SHE were only considered in the
calculation of K0 .
Preparation of MSHE complexes used in soil-plant
experiments
MSHE complexes were prepared with 1 g of freezedried SHE. This was dissolved in 25 mL 0.1 M NaOH,
diluted with 250 mL distilled water and pH adjusted
to 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 stoichiometry 2:0.25 (SHE:metal, w/w) while maintaining
pH 8 by the addition of NaOH. Solutions were stirred
in darkness at 25 C for 24 h. After this time, Zn-SHE
solution was further alkalinised to pH 9.5 and stirred
for two additional hours to precipitate any remaining
uncomplexed Zn. It was assumed that some complexed Zn could also be lost. The complete extent of
free metal precipitation under the conditions described
above (pH 8 for Cu and Fe, and pH 9.5 for Zn) was
substantiated in previous studies (Garca-Mina, 1998).

60
The MSHE solutions thus prepared were centrifuged
again at 7650 g for 30 min and the supernatants
analysed to obtain final SHE:metal stoichiometries in
the complexes. SHE was analysed by oxidation with
0.01 N KMnO4 in an alkaline medium (Primo Yfera
and Carrasco, 1987) (1 meq KMnO4 oxidizes 9.4 mg
SHE). 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.

shown to be sufficient to compensate for variability

(Garca-Mina, 1998). The experiments were carried
out in May, June and July. Greenhouse photoperiod
was approximately 14/10 h day/night using additional
lighting when needed. The average temperature was
25/15 C day/night although variations of up to + 5 C
were occasionally observed on very sunny days. The
relative humidity was between 50 and 70% depending
on the time of day. Plants were watered daily to field
capacity to avoid any stress due to drought.

Plant experiments
Pots were prepared using the following soils: two
alkaline, Typic Xerorthent calcareous, low organic
matter soils, one a clay soil very prone to compaction
due to its high clay content (Legarda soil); the other a
high sulphate silt soil with little tendency to compaction (Mendigorra soil). The third soil type was an acid
sandy Typic Dystrupdet soil rich in poorly humified
organic matter (Velate soil). The main soil properties
were: for Legarda soil, 8.0 pH (water), 2% of total
organic matter, 52.3% of total carbonates and 3.2 mg,
9.08 and 0.48 mg kg1 of DTPA-extractable Cu, Fe
and Zn, respectively (Lindsay and Norvell, 1978); for
Mendigorra soil, 8.2 pH (water), 1.4% of total organic
matter, 39.9% of total carbonates, 11.7% of calcium
sulphate and 1.02, 4.86 and 0.48 mg kg1 of DTPAextractable Cu, Fe and Zn, respectively, and for Velate
soil, 5.5 pH (water), 9.8% of total organic matter and
0.56, 196 and 1.2 mg kg1 of DTPA-extractable Cu,
Fe and Zn respectively (Norvell, 1984). These analyses were carried out on air-dried soil samples at
room temperature after sieving using a plastic sieve
(02.5 mm).
The procedure for obtaining and preparing the different soils was as follows: having eliminated the first
10 cm, samples corresponding to 40 cm of soil were
taken. Non-cultivated areas of each soil were selected in order to eliminate the residual effects of both
ordinary fertilisation and crop development on the micronutrient available soil fraction. Soil samples were
air-dried at room temperature and soil aggregates were
reduced using a Teflon hammer. Finally, soil samples
were sieved using a plastic sieve (size 02.5 mm) to
avoid possible metal contamination. Soil samples were
also 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 contained 800 g soil, with six plants each, three pots for
wheat and five for alfalfa. These numbers have been

Fertilisation
Each pot received a single NPK fertiliser dose per
kg of soil of 100 mg N as NO3 NH4 , 42.7 mg P as
KH2 PO4 , 244.8 mg K as KH2 PO4 , KCl and K2 SO4 ,
each 30 days as a liquid solution along with the irrigation water. The concentration of the nutrients was
chosen according to the results of the soil nutrient
analyses.
Treatments
MSHE pots (Fe-SHE, Cu-SHE and Zn-SHE) received 2.5 or 5.0 mg kg1 of complexed metal in a
single application. At the same time, SHE pots received an SHE amount (53 mg kg1 ) equivalent to
SHE content added as a complex in MSHE 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 h
after the N-P-K fertilizer application.
Plants were harvested at random 90 days after
treatment. Air-dried soil samples at room temperature
from each pot were used to analyse soil micronutrient availability using the DTPA method as described
below. These soil samples corresponded to the rhizosphere area. Although the root material was carefully
separated previously, some root material could have
remained in soil samples.
Plant analysis
Shoot and root dry weight was determined after drying at 85 C for 48 h. Shoot nutrient content (mg
kg1 ) was measured by AAS according to Grate et
al. (1984).
A post-hoc comparison of means was carried out
with the LSD test for paired treatments. Statistic of
inter-group significance was fixed at p0.05.

61
Analysis of the DTPA-extractable soil fraction of
micronutrients in plant experiments
Soil levels of potentially plant available Cu, Fe, and Zn
were determined using the DTPA method, pH 7.3 for
alkaline, calcareus soils (Lindsay and Norvell, 1978)
and pH 5.3 for acid soil (Norvell, 1984). The values
obtained were referred to as an index of plant micronutrient availability. The analysis was carried out
on dried soil samples from each pot after plant roots
were separated.

Results and discussion

Stability and solubility studies
It is not the purpose of this research to investigate
the stability of humic complexes from a general point
of view, but rather to better understand the effects of
these complexes on plant nutrition and development in
soil systems. Furthermore, there are a great number of
studies in the literature on the stability of this kind of
organic compound and on the development of physicochemical models which allow adequate description
of the complexing process (Kinniburgh et al., 1996;
Stevenson, 1994; Tipping et al., 1995). Therefore,
we will only briefly describe our results in the light
of their possible relationships with plant development
and plant micronutrient uptake.
As can be seen in Table 1, all MSHE complexes
showed a considerable stability in the pH interval that
corresponds to soil pH values. The stability constant
values were also consistent with those reported in
previous work (Stevenson, 1994). Regarding Cu complexes, 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 result
suggests a specific role in the complexing process of
chemical groups whose chemical reactivity is modified as the pH varies from acid to basic values. Some
authors have related these groups to amino groups
linked to aliphatic or aromatic substructures (Senesi,
1992). In the case of Zn complexes, the greatest stability was found for pH values of 7 and 8, which correspond to those of neutral and alkaline soils. This fact
suggests the intervention of strongly acidic carboxylic
and phenol groups in the complexing process at these
pH levels. At pH 6, stability was also important although lower than at alkaline pH levels. Stability at
pH 9 decreased significantly, probably because of the

Table 1. Stability (Log K0 ) and solubility (IS) of different

MSHE complexes
MSHE
pH

CuSHE
6
7
8
9
ZnSHE
6
7
8
9
FeSHE
6
7
8
9

Log K0
Log K01
Log K02

IS
SHE(mg):metal (mg)
ratio

4.20
3.74
3.49
3.37

1.89
1.59

2:0.40
2:0.55
2:0.70
2:1.50

2.01
3.99
3.72
1.80

1.53
1.58
1.66
1.15

2:1.50
2:1.50
2:1.50
2:2.00

3.09
2.91
4.11
1.74

1.45
1.40
1.03
0.79

2:1.00
2:1.50
2:1.50
2:2.00

IS is defined as the SHE:metal ratio in the reaction corresponding to the precipitation of SHE over 50%.

competition between the complexing process and the

formation of the insoluble hydroxide (Zn(OH)2). Furthermore, in this case the importance of the ionised
amine groups which has been observed by other authors in other humic extracts (Senesi, 1992), is not
as clear as in the case of Cu complexes, although the
stability at pH 6 is suggestive of this fact.
In the case of Fe complexes, a clear maximum
stability was observed at pH 8. The stability at pH 6
and 7 was similar and also important. At pH value of
9, a clear reduction in stability was observed. Such a
pronounced increase in stability at pH 8 could reflect
the role of phenolic groups those most markedly
acid in nature in the complexing process, in addition to carboxylic groups. The fall in stability at
pH 9 suggests that other factors exert an important
influence on the complexing process apart from the
increase in the ionisation of the phenolic groups. Such
factors are probably directly associated with the conformational changes accompanying the increase in pH
(Swift, 1989), and with the formation of Fe(OH) 4
that might cause a significant change in the model of
chemical interaction between Fe and the complexing
groups.
With regard to MSHE solubility, the following
general rules appeared to be present in these chemical
systems (Table 1):

62
- When a specific SHE:M ratio is considered, there
seems to be a directly proportional relationship
between the concentration of MSHE in solution
and pH alkalinity. As the pH level increases so
does the concentration of MSHE remaining in
solution.
- There seems to be a directly proportional relationship between MSHE concentration and the
SHE:metal reaction stoichiometry, since a clear
precipitation of the complexes takes place as the
SHE:metal ratio in the reaction decreases.
These results are consistent with those obtained by
others studying the flocculation of humic extracts by
metallic cations (Spark et al., 1997; Stevenson, 1994).
Furthermore, this variation in solubility is consistent
with the influence exerted on this property by the
conformational changes which these molecules may
undergo in response to pH, I and the increase in the
complexing process (Senesi, 1992; Swift, 1989). Similarly, these results show the considerable influence of
the physicochemical characteristics of the MHC on
their solubility in soil solution, and therefore on their
potential biological activity.
Effects on micronutrient uptake and development of
plants
It is interesting to note the significant increase of
DTPA-extractable soil fraction of micronutrients in
control pots of every plantsoil system (Tables 26).
This fact was especially clear for Fe and Zn in wheat
plants, and for all studied micronutrients in alfalfa
plants. These results show the significant efficiency of
the specific mechanisms that plants have in order to
modify the dynamics of nutrients in the rhizosphere
under adverse soil conditions (Uren, 2001). Thus,
under Fe deficiency, graminaceous species release
to the rhizosphere nonproteinogenic amino acids
(phytosiderophores) with the capacity to solubilize
non-available iron and other micronutrients (principally Zn and Cu) by the formation of stable complexes
that are directly taken up by roots. The response of
dicots and non graminaceous monocots to Fe deficiency seems to be more complex including different
co-ordinated events such as an increase in root reductase activity, proton extrusion, the development of
transfer cells and the release of low molecular weight
organic compounds with chelation and/or reduction
activity (Schmidt, 1999). Likewise, different studies
have pointed out that these mechanisms could also be
efficient to mobilise and increase the plant uptake of

other micronutrients such as Cu and Zn (Treeby et

al., 1989). The fact of carrying out our experiments
in pots involves that the observed effects either on
plant micronutrient uptake or on the DTPA-extractable
fraction have to be referred to as rhizosphere effects
which could have been quantitatively potentiated by
this closed experimental system. Finally, nor can the
impact of the microbial activity in the rhizophere area
be neglected.
In order to evaluate the degree of Fe-, Cu- and
Zn-deficiency in both plant species cultivated in the
different soils used, we have compared the levels of
these micronutrients in the control plants with tabulated data from several sources (Loue, 1993). Thus,
both wheat and alfalfa plants presented a clear deficiency state in Velate soil. Wheat plants also presented
low shoot contents of Zn, Cu and Fe in Legarda
soil and of Zn and Fe in Mendigorra soil while alfalfa plants only presented low shoot content of Fe
in Mendigorra soil. It is also interesting to note the
very high Fe-shoot content in alfalfa plants growing
in Velate soil, which is close to toxicity levels. In
general, these results were coherent with those of the
potential soil content of plant available micronutrients
expressed by the DTPA-analysis.
Regarding SHE effects, no significant increases
were found in shoot Cu or Zn content whatever the
plant or soil type (Tables 2-6). Fe content significantly
increased in wheat plants grown on the soil of lowest
Fe availability (Mendigorra soil, Table 4). This result
was associated with an equally significant increase of
DTPA-extractable iron. In the wheatLegarda soil system a significant increment of the DTPA-extractable
iron was also observed but it was not associated with a
concomitant increase of shoot Fe content (Table 3). An
opposite effect was observed on alfalfa plants grown in
high Fe availability soil (Velate soil, Table 5) where
a significant decrease of shoot Fe content was observed. These results, far from being contradictory, fit
the model proposed by several authors (Chen, 1996;
Stevenson, 1994): SHE complexation of metals increases their solubility, mobility and availability under
deficiency conditions (Mendigorra or Legarda soil
conditions). Conversely, complexation of highly available metals results in a kind of sequestering effect
(Velate soil conditions). Furthermore, there is close
agreement with the results obtained in relation to the
influence of pH and SHE:metal stoichiometry on complex solubility: the greater the proportions of Fe with
respect to SHE, the lower the solubility of FeSHE
at pH 6 (under the same conditions as in Velate soil:

63
Table 2. Effect of metal-humic complexes (CuSHE, ZnSHE and FeSHE) on the concentration of soil available Zn, Cu
and Fe, and on shoot dry weight, root dry weight and shoot Zn-, Cu- and Fe-content of wheat plants cultivated in Velate
soil (humic rate in SHE and metal-SHE treatments was 53 mg kg1 )
Velate soil

Shoot dry
weight
(g pot1 )

Root dry
weight
(g pot1 )

Zn content

Cu content

Fe content

(mg kg1
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

2.31a
1.87a

1.41ab
1.48ab

21.89a
25.76a

2.22a
2.46a

2.87a
2.83a

0.28a
0.72a

66.35bc
61.87b

268a
269a

3.90c
3.23b

2.62c
2.67c

23.14a
28.57a

2.46a
2.73a

4.52b
6.76c

1.99b
3.19c

41.42a
82.02c

297a
284a

Zn SHE (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

1.80a
2.40a

0.89a
0.98a

34.58b
39.29b

3.55b
4.21b

2.61a
2.37a

0.46a
0.36a

55.53ab
84.37c

271a
267a

SHE Fe (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

2.31a
2.46a

1.56ab
1.70b

25.17a
23.17a

2.65a
2.61a

3.88ab
2.76a

0.48a
0.45a

82.79c
69.61bc

279a
285a

Treatments
Control
SHE
Cu SHE (metal rate)
Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

Table 3. Effect of metal-humic complexes (CuSHE, ZnSHE and FeSHE) 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 wheat plants cultivated in Legarda soil.
(humic rate in SHE and metalSHE treatments was 53 mg kg1 )
Legarda Soil

Shoot dry
weight
(g pot1 )

Root dry
weight
(g pot1 )

Zn content

Cu content

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

4.03a
4.44a

2.87b
2.20ab

6.10ab
5.51a

1.10a
1.33a

4.72a
4.72a

3.68a
4.25a

65.40bc
73.42c

21.52a
30.01b

4.49a
4.73a

2.07a
2.82b

5.79ab
5.60ab

0.97a
0.99a

6.18b
6.35b

4.42a
8.77b

49.18ab
61.40bc

21.39a
22.03a

Zn SHE (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

4.62a
4.78a

2.20ab
1.97a

10.23d
9.15cd

2.79b
3.13b

5.32a
4.83a

4.02a
3.62a

65.75bc
46.57a

24.35a
21.74a

SHE Fe (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

4.57a
4.50a

2.16ab
2.69ab

7.24b
8.09bc

0.95a
1.02a

4.98a
5.68ab

3.32a
3.51a

46.03a
55.52ab

25.27ab
26.23ab

Treatments
Control
SHE
Cu SHE (metal rate)
Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

Fe content

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 lower

the proportion of Fe with respect to SHE, the higher
the solubility of Fe-SHE at pH 8 (under the same conditions as in Mendigorra soil: alkaline pH, poor in
available Fe).

SHE did not significantly affect shoot or root dry

weight (Tables 26). Many studies have demonstrated
the ability of HS to increase plant growth in various soil types (Nardi et al., 1996). However, these
experiments were made at higher HS concentrations,

64
Table 4. Effect of metal-humic complexes (CuSHE, ZnSHE and FeSHE) 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 wheat plants cultivated in Mendigorra
soil (humic rate in SHE and metalSHE treatments was 53 mg kg1 )
Mendigorra Soil

Shoot dry
weight
(g pot 1 )

Root dry
weight
(g pot 1 )

Zn content

Cu content

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

4.48a
5.11abc

2.26a
2.69a

8.44a
7.19a

0.99a
1.15a

6.70ab
6.06a

2.38a
3.38a

50.47ab
80.62c

13.92a
18.15b

5.86c
5.15abc

2.54a
3.19ab

7.74a
7.70a

0.88a
1.37a

7.41bc
8.55c

6.07b
5.59b

52.07ab
63.69b

14.87a
13.65a

Zn SHE (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

5.29ab
4.96ab

3.16ab
3.07ab

11.50b
15.84c

2.43b
3.35b

5.68a
6.91ab

3.05a
3.97a

50.23ab
59.87b

16.43ab
14.09a

SHE Fe (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

5.66bc
5.04abc

2.63a
3.70b

7.13a
8.20a

0.81a
0.85a

5.97a
6.87ab

3.28a
2.82a

45.91a
44.07a

12.93a
16.09ab

Treatments
Control
SHE
Cu SHE (metal rate)
Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

Fe content

Different letters indicate significant (p 0.05) differences between treatments.

# DTPA, pH 7.3 (Lindsay and Norvell, 1978).

Table 5. Effect of metal-humic complexes (CuSHE, ZnSHE and FeSHE) 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. (humic
rate in SHE and metalSHE treatments was 53 mg kg1 )
Velate soil

Shoot dry
weight
(g pot 1 )

Root dry
weight
(g pot 1 )

1.71a
1.39a

0.93a
0.89a

57.45ab
99.20b

2.15ab
2.52bc

5.46a
4.08a

7.45a
7.49a

132.94b
89.39a

290ab
281ab

3.90cd
3.78cd

2.60bc
2.45bc

67.39ab
42.31a

2.64bc
2.32bc

8.70b
14.66c

7.51a
7.86b

77.06a
110.95ab

283ab
311b

Zn SHE (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

4.18d
2.31ab

2.05b
0.92a

2.74c
2.01ab

5.16a
3.03a

7.47a
7.25a

109.29ab
98.43ab

313b
265a

SHE Fe (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

3.11bc
4.05d

2.02b
3.15c

1.74a
1.99ab

4.14a
5.31a

7.32a
7.45a

94.39ab
89.98a

293ab
295ab

Treatments
Control
SHE
Cu SHE (metal rate)
Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

Zn content

Cu content

Fe content

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

152.37c
205.74d

75.54ab
33.13a

Different letters indicate significant (p 0.05) differences between treatments.

# DTPA, pH 5.3 (Norvell, 1984).

2004000 mg kg1 , than those used in our experiments. The low dose we used (53 mg kg1 ) was
chosen to allow a clearer discrimination between the
effect of MSHE and SHE alone.

As for CuSHE effects, this complex significantly

increased shoot Cu content in every test (Tables 2
6) except those made in the wheat-Mendigorria soil
system (low Cu dose) and the alfalfaLegarda soil sys-

65
Table 6. Effect of metalhumic complexes (CuSHE, ZnSHE and FeSHE) 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 Legarda soil.
(humic rate in SHE and metalSHE treatments was 53 mg kg1 )
Legarda soil

Shoot dry
weight
(g pot 1 )

Root dry
weight
(g pot 1 )

Zn content

Cu content

Fe content

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

(mg kg1 )
Shoot
Soil#

5.34a
5.87ab

2.34a
2.10a

17.02b
16.28ab

1.50a
1.43a

13.61b
12.68ab

10.71a
10.75a

81.92bc
80.30bc

25.28a
24.31a

7.16bc
9.28c

2.61a
4.71d

17.57b
15.26ab

1.43a
1.54a

14.07b
13.76b

12.71b
14.29c

65.82ab
59.67a

25.00a
26.16a

Zn SHE (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

9.35c
9.24c

3.16b
3.84c

26.49c
25.40c

2.54b
2.86b

10.44a
10.65ab

10.71a
10.05a

57.87a
64.23ab

24.15a
22.61a

SHE Fe (metal rate)

Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

6.48ab
7.50bc

2.10a
3.49bc

13.33a
16.53ab

1.29a
1.64a

10.39a
11.45ab

9.07a
10.79a

95.65c
80.33bc

23.19a
26.40a

Treatments
Control
SHE
Cu SHE (metal rate)
Dose 1 (2.50 mg kg1 )
Dose 2 (5.00 mg kg1 )

Different letters indicate significant (p 0.05) differences between treatments.

# DTPA, pH 7.3 (Lindsay and Norvell, 1978).

tem (both doses) possibly due to a dilution effect as

a result of higher shoot dry weight (Tables 4 and 6).
Differences were significant both in relation to control
and SHE treatment. These results were well correlated
to those of the Cu-DTPA soil fraction and they were
also consistent with those obtained for the solubility
and stability of CuSHE complexes at both acid and
basic pH (Table 1). In this sense the result obtained in
relation to plant Cu uptake in velate soil indicates that
this stability was sufficient to protect complexed Cu
against secondary reactions with non-humified organic
residues which would cause Cu unavailability.
Cu-SHE treatment significantly increased shoot
dry weight in relation to control and to SHE treatment
in every test, except that made in the wheatLegarda
soil system where the increases were not significant
(Tables 26). This was more apparent in plants grown
in Velate soil, very deficient in Cu, where root development also increased significantly (Tables 2 and
5).
No references have been found in the literature regarding CuHC effects on soil grown plants, but only
on inert substrates, mainly silica sand (Ennis, 1962;
Ennis and Brogan, 1961; Gupta, 1986; Gupta and
Hni, 1980).
With regard to Zn-SHE, this complex significantly
increased shoot Zn content as well as soil Zn-DTPA

in every test (Tables 26). Differences were significant in relation to both control and SHE. These
effects were proportional to dose for the wheatVelate
soil, wheatMendigorra soil and alfalfa-velate soil
(Tables 2, 4 and 5). The effects of ZnSHE on shoot
and root dry weight varied according to plant species
(Tables 26). Wheat plants did not increase shoot dry
weight, whereas alfalfa plants did (the reduction in
shoot dry weight at highest ZnSHE dose in alfalfavelate soil system may reflect a Zn toxicity effect).
These results reflect the different sensitivity of both
plant 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 plant
growth even under very severe micronutrient deficient
conditions except in the case of Cu deficiency.
Although several studies into the effects of Zn
HC complexes on Zn plant uptake have been carried
out on inert substrates (Barnard et al., 1992) or hydroponics (Mirav and Orioli, 1989) we have found
only one study specifically based on soil. Kumar and
Prasad (1989) studied the effect Znfulvic complexes
on maize grown in an alkaline, calcareous soil. They
found a significant shoot Zn uptake but not a shoot
Zn content increase. Control plants were only marginally Zn deficient, and Zn supplied was exiguously
absorbed (less than 7% of the largest dose of 5 mg

66
kg1 ). Therefore, it is tempting to attribute shoot Zn
uptake to growth stimulation caused by the fulvic acid
fraction of the complexes, and account for the poor
performance of Znfulvic complexes by reference to
low complex stability or solubility.
Unlike CuSHE and ZnSHE, FeSHE did not
increase Fe DTPA-extractable soil fraction, shoot Fe
content or shoot and root dry weight in the different
plantsoil system excepting the alfalfavelate system
where dose 2 of the complex caused a significant decrease of shoot Fe (Tables 26). This phenomenon
is probably related to a decrease in the iron content
in soil solution caused by the formation of FeSHE
complexes with a very small SHE:Fe ratio and low
solubility at acid pH (Table 1).
On the other hand, as has been previously discussed, Fe content significantly increased in wheat
plants treated with SHE alone grown on the soil of
lowest Fe availability (Mendigorra soil, Table 4), thus
showing the capacity of certain FeSHE complexes
to improve Fe plant uptake under adverse conditions.
In the light of these results, we have to conclude that
the FeSHE prepared for plant experiments lacked the
necessary stability, solubility or both to adequately
supply Fe to plants under our experimental conditions. Other contributing factors, along with SHE:Fe
complex stoichiometry, such as adsorption phenomena or interaction with Ca, mg or trace metals, may be
relevant to Fe-SHE stability and solubility.
The above results are in accordance with results
obtained by other authors. Thus, Burau et al. (1960)
studied the performance of FeHC as sources of Fe
in beans grown in alkaline, calcareous soil. These authors obtained no significant increases in Fe uptake
when results were compared with those of a control of acidified FeCl3 . In these experiments, Fe:HC
stoichiometry was much lower, and their complex
predictably less soluble than ours. Alva and Obreza
(1998) studied the effect of a Fe-rich organic product
on Fe uptake in several varieties of orange trees and
grapefruit grown in alkaline, calcareous soils. Leaf Fe
increase was significant in some varieties, but final leaf
Fe values were rather low, sometimes close to deficiency (Legaz et al., 1995). This Fe-rich product was
obtained as a by-product of water purification with
iron sulphate, with quite a high (18%) Fe content.
No further description of physicochemical properties
was provided, but taking into account the preparation
method and the low stoichiometry, complexes would
have been sparingly soluble in water.

Finally, Pandeya et al. (1998) studied the effect of

Fefulvic complexes on the Fe uptake of rice plants
grown in an incubation medium containing a fraction of an alkaline, calcareous soil. Although these
authors made no reference to Fe:fulvic acid complex
stoichiometry, their results showed a clear correlation
between Fe uptake and complex diffusion in the soil.
In conclusion, our results prove the capability of
Cu and Zn SHE complexes to supply plant available
Cu and Zn under soil conditions favoring the deficiency of both micronutrients. In the case of FeSHE
complex, the obtained results emphasize the great influence of Fe:SHE stoichiometry on Fe bioavailability
due to its impact on complex solubility. Also, under the experimental conditions used in this study,
where increases of plant growth associated with M
SHE treatment were observed, they were caused by
an improvement in plant micronutrient uptake, since
the humic system alone did not show any significant
effect. These results are in agreement with the theory
proposed by some authors that the effect of humic
substances on plant development is only due to the
improved intake of some micronutrients (Chen et al.,
1999). However, our results do not allow us to rule
out the possibility of direct, pseudohormonal effects
of SHE since the SHE dose was much lower than the
stimulating doses proposed by most authors (Nardi et
al., 1996).

Acknowledgements
This research was funded by the CYCYT (SPAIN), the
Government of Navarra (Spain) and Roullier Group.
The authors thank Prof. Yona Chen for his valuable comments and suggestions; Rodrigo G. Cantera
and Paul Miller and David Rhymes for their help in
the preparation of the manuscript and Professor Jordi
Garrigo for his help in soil classification.

References
Abo F and Pinta, M 1982 Influence des oligoelements sur la production de matiere seche, laspect morphologique et le dveloppement du ble. CR Acad. Agric. 68, 756765.
Alva A K and Obreza T A 1998 By-product iron-humate increases tree growth and fruit production of orange and grapefruit.
Hortscience. 33, 7174.
Barnard R O, Watt H v H van der, Dekker J, Cronje I, Mentz W H,
Cillie G E B and Laker M C 1992 Application of Fe and Zn to
lime-rich soils in the form of formulated coal products. Sci. Total
Environ. 117/118, 569574.

67
Bar-Ness E and Chen Y 1991 Manure and peat based iron-organo
complexes. II. Transport in soils. Plant Soil. 130, 4550.
Burau R G, White R G and MacGregor J M 1960 Uptake of applied
iron by soybeans from calcareous soil treated with peat-based
humates and synthetic chelates. Proceedings 7th International
Congress of Soil Science, Madison, WI, vol 3, pp. 544553.
Cao Y, Conklin M and Betterton E 1995 Competitive complexation of trace metals with dissolved humic acid. Environ. Health
Perspect. 103, 2932.
Chen Y and Aviad T 1990 Effects of humic substances on plant
growth. In Humic Substances in Soil and Crop Sciences: Selected Readings. Ed. P MacCarthy, C E Clapp, R L Malcolm and
P R Bloom. pp. 161186. Soil Science Society of America, Inc,
Madison, WI.
Chen Y 1996 Organic Matter Reactions Involving Micronutrients
in Soils and their Effect on Plants. In Humic Substances in
terrestrial ecosystems. Ed. A. Piccolo. pp. 507530. Elsevier,
Amsterdam.
Chen Y., Senesi N and Schnitzer M 1977 Information provided on
humic substances by E4 /E6 ratios. Soil. Sci. Soc. Am. J. 41,
352358.
Chen Y, Clapp C E, Magen H and Cline V W 1999 Stimulation of
Plant Growth by Humic Substances: Effects of Iron Availability.
In Understanding Humic Substances. Advances Methods, Properties and Applications. Eds. E A Ghabbour and G Davies. pp.
255263. Royal Society of Chemistry, Cambridge.
Elprince A M 1986 Chemistry of Soil Solutions. Van Nostrand
Reinhold Company, New York, USA. 411 pp.
Ennis M T 1962 Some copper-complexing properties of peat. Irish
J Agric. Res. 1, 139147.
Ennis M T and Brogan J C 1961 The availability of copper from
copper-humic acid complexes. Irish J. Agric. Res. 1, 3542.
Fitch A and Stevenson F J 1984 Comparison of models for determining stability constants of metal complexes with humic
substances. Soil Sci. Soc. Am. J. 48, 10441050.
Grate A., Carpena O. and Ramon A M 1984 Determinacin de
boro en jugos de tejidos conductores. Anal. Edaf. Agrobiol. 43,
11231129.
Garca-Mina JM 1998 Metal-humic Complexes and Plant development. PhD. thesis. University of Navarra, Pamplona, Spain.
Geering H R, Hodgson J F and Sdano C 1969 Micronutrient cation
complexes in soil solution: IV. The chemical state of manganese
in soil solution. Soil Sci. Soc. Am. Proc. 33, 8185.
Gupta S K 1986 Bioverfuegbarkeit von Kupfer in einem
Huminsaeure-und Klaerschlamm-komplex. Mitteilgn. Dtsch.
Bodenkundl. Gesellsch. 45, 6166.
Gupta S K and Hni H 1980 Effect of copper supplied in the form
of different Cu-saturated sludge samples and copper salts on the
Cu-concentration and dry matter yield of corn grown in sand. In
Symposium on Copper in Animal Wastes and Sewage Sludge.
Eds. P. LHermite and J. Dehand-schutter. pp. 6769. Reidel
Publishing Co., Dordrecht.
Hodgson J F 1969 Contribution of metal-organic complexing agents
to the transport of metals to roots. Soil Sci. Soc. Am. Proc. 33,
6875.
Hodgson J F, Geering H R and Norwell W A 1965 Micronutrient
cation complexes in soil solution: I. Partition between complexed
and uncomplexed forms by solvent extraction. Soil Sci. Soc. Am.
Proc. 29, 665669.
Hodgson J F, Lindsay W L and Trierweiler J F 1966 Micronutrient
cation complexing in soil solution. II. Complexing of zinc and
cooper in displaced solution from calcareous soils. Soil Sci. Soc.
Am. Proc. 30, 723726.

Kinniburg D, Milne CH, Benedetti M, Pinheiro J, Filius J, Koopal L

and Van Riemsdijk E H 1996 Metal ion binding by humic acid:
Application of the NICA-Donnan model. Environ. Sci. Technol.
30, 16871698.
Kumar M and Prasad B 1989 The relative contribution of natural
zinc complexing agents and ZnSO4 to growth and zinc nutrition
of maize. J. Nucl. Agric. Biol. 18, 2935.
Legaz F, Serna M D, Ferrer P, Cebolla V and Primo-Millo E 1995
Nutritional Diagnosis in Citrus plants. Generalitat Valenciana,
Valencia, Spain. 27 pp.
Lindsay W and Norvell W A 1978 Development of a DTPA soil test
for zinc, iron, manganese and copper. Soil Sci. Soc. Am. J. 42,
421428.
Lo S Y and Reisenauer M 1968 Zinc nutrition of alfalfa. Agron. J.
60, 464466.
Lobartini J C and Orioli G A 1988 Absorption of iron Fe-humate in
nutrient solutions by plants. Plant Soil 106, 153157.
Lou A 1993 Oligoelements en Agriculture. Editions Natham, Paris.
577 p.
Lucena J J, Grate A and Carpena O 1987 Iron-chelates evaluation
in a calcareous soil. Plant Soil 103, 134138.
Mirav J P and Orioli G A 1987 Edaphic mobility of complete
humic acid and fractions of high and medium molecular weight.
Plant Soil 104, 169174.
Mirav J P and Orioli G A 1989 Zinc absorption and transport
from complete humate and high and medium molecular weight
fractions. Sci. Total Environ. 81/82, 679682.
Mirav J P, Lobartini J C and Orioli G A 1987 Absorcin y transporte de hierro a partir de fuentes orgnicas de distinto peso
molecular. Ci. Suelo 5, 3135.
Nardi S, Concheri G and DellAgnola G 1996 Biological activity of
humus. In Humic Substances in Terrestrial Ecosystems. Ed. A.
Piccolo. pp. 361406. Elsevier, Amsterdam.
Norvell W A 1984 Comparison of chelating agents as extractants for
metals in diverse soil materials. Soil Sci. Soc. Am. J. 48, 1285
1292.
OConnor G.A, Lindsay W L and Olsen S R 1971 Diffusion of iron
chelates in soil. Soil Sci. Soc. Am. Proc. 35, 407410.
Pandeya S B, Singh A K and Dhar P 1998 Influence of fulvic acid
on transport of iron in soils and uptake by paddy seedlings. Plant
and Soil. 198, 117125.
Pinton R, Cesco S, Santi S, Agnolon F and Varanini Z 1999 Waterextractable humic substances enhance iron deficiency responses
by Fe-deficient cucumber plants. Plant Soil 210, 145157.
Primo Yfera E and Carrasco J M 1987 Qumica Agrcola. I. Suelos
y Fertilizantes. Alhambra, Madrid. 472 pp.
Schmidt W 1999 Mechanisms and regulation of reduction-based
iron uptake in plants. New Phytol. 141, 126.
Schnitzer M, Kodama H and Ripmeester J A 1991 Determination of the aromaticity of humic substances by X-ray diffraction
analysis. Soil Sci. Soc. Am. J. 55, 745750.
Senesi N 1992 Metal-humic substance complexes in the environment. Molecular and mechanistic aspects by multiple spectroscopic approach. In Biogeochemistry of Trace Metals. Ed. C M
Adriano. pp. 429496. Lewis Publishers, New York.
Spark K M, Wells J D and Johnson B B 1997 The interaction of a
humic acid with heavy metals. Ausy. J. Soil Res. 35, 89101.
Stevenson, F.J. 1994. Humus Chemistry. Genesis, Composition,
Reactions. John Wiley and Sons, Inc., New York. 496 pp.
Swift R S 1989 Molecular weight, size, shape, and charge characteristics of humic substances:some basic considerations. In Humic
Substances II. In Search of Structure. Eds. M H B Hayes, P MacCarthy, R L Malcolm and R S Swift. pp. 449466. J. Wiley, New
York.

68
Takamatsu T and Yoshida T 1978 Determination of stability constants of metalhumic acid complexes by potentiometric titration
and ion-selective electrodes. Soil Sci. 125, 377386.
Tipping E, Fitch A and Stevenson F J 1995 Proton and copper
binding by humic acid: application of a discrete-site/electrostatic
ion-binding model. Eur. J. Soil Sci. 46, 95101.
Treeby M, Marschner H and Rmheld V 1989 Mobilization of iron
and other micronutrient cations from a calcareous soil by plant-

borne, microbial and sinthetic metal chelators. Plant and Soil

114, 217226.
Uren N C 2001 Types, amounts, and possible functions of compounds released into the rhizosphere by soil-grown plants. In
The Rhizosphere. Eds. R Pinton, Z Varanini and P Nannipieri.
pp. 1940. Marcel Dekker, Inc, New York.
Section editor: Z. Rengel