Structure/Fertilizer Properties of Byproducts in EDDHA Synthesis
J. Agric. Food Chem., Vol. 54, No. 12, 2006 4357
(see the Supporting Information for complete detailed experimental
procedure). A modified Langmuir equation (amount of Fe in solution
vs time) was applied to the experimental data
o,o-EDDHA/Fe3+ and o,p-EDDHA/Fe3+). Since EDTA/Fe3+ does not
present a significant absorbance at 535 or 480 nm, the [Fe2+(BPDS)3]
concentration was directly calculated from the absorbency at 535 nm.
The slope of the plots of Fe2+ (µmol g-1 dry root) produced versus
time (h) was used as the Fe2+ reduction rate for each pair of plants.
Data were expressed as the mean reduction rate and the standard error,
corresponding to five plant replications for each treatment.
[Mmax]t
[M] )
t1/2 + t
where [M] is the amount of soluble metal per mass unit (µmol g-1),
t1/2 (halftime) is the time used to dissolve half of the maximum
concentration of the metal, and Mmax is the maximum amount of metal
dissolved.
Efficiency to Provide Fe to Soybean Plants. Soybean plants were
used in this experiment since they are considered susceptible to chlorosis
and are considered as a model for crops normally treated with chelates.
Soybean seeds (Glycine max L. cv. Oshumi) were germinated at 28
°C on paper moistened with 1 mM CaSO4 in the dark for 3 days.
Afterward, the seedlings were placed in 10 L containers (27 seedlings
per container) filled with a 1/5 diluted EDTA buffered nutrient solution
of the same composition as in the cucumber experiment and grown
for 7 days. On the eighth day, to induce iron chlorosis, seedlings were
transferred to 12 L polypropylene buckets containing an aerated full-
strength EDTA buffered nutrient solution but without an Fe source.
Plants were grown under these conditions until severe symptoms of
Fe deficiency were observed in the upper leaves (6 days), and then
plants were placed in 2 L pots (2 plants per pot) covered with black
plastic to avoid light exposure, and treatments (5 µM o,o-EDDHA/
Fe3+, o,p-EDDHA/Fe3+, sample A/Fe3+, or sample B/Fe3+) were
applied. The nutrient solution contained macronutrients and anionic
micronutrients as for the cucumber experiment, and cationic micronu-
trients were added at (µM) 1.0 MnSO4, 0.5 CuSO4, 0.5 ZnSO4, 0.1
NiCl2, and 0.1 CoSO4. Water was added every 2 days, and the solution
was renewed weekly. The treatments were repeated four times in a
completely randomized design. Plants were harvested after 14 days.
The growth chamber conditions were the same as those used in the
cucumber experiment.
Chelate Adsorption in Soils and Soil Materials. The sample
adsorption rate in soils and soil materials was determined by a known
procedure (15). The soil used was a clay loam with pH (water extract)
of 7.75, 15 g kg-1 organic matter, 430 and 140 g kg-1 total and active
lime, respectively, and 12.1 mg kg-1 DTPA (Soltanpour and Schawb
method) extractable Fe (Soltanpour and Schawb method) (10). The soil
materials used were (a) a standard of calcareous soil (16); (b) ferrihydrite
(5Fe2O3‚H2O) (17, 18); (c) acidic mountain Sphagnum peat, provided
by Tolsa S.A. (Buyos, Lugo, Spain) [its chemical characteristics were
pH (saturated paste), 4.0; dichromate oxidizable OM (%), 85.4; total
OM (%) (determined by loss of weight by ashing), 99.5; C in humic
acid (%), 30.2; C in fulvic acid (%), 18.3; Nkj (%), 1.4%; C/N, 35.4;
CEC (cmolc kg-1), 150; and DTPA-extractable Fe and Mn, 295 and
8.2 mg kg-1, respectively]; (d) calcium-montmorillonite (STX-1,
Gonzalez County, TX), obtained from the Clay Minerals Society Source
(Clay Minerals Repository, Department of Geology, University of
Missouri, Columbia, MO) [this reference material has been well-
characterized elsewhere (19), and so despite its differences, it has been
considered as an acceptable model for soil smectites]; and (e) CaCO3
analytical grade (Panreac).
Studies with FCR in Green Stressed Cucumber Plants. In this
study, cucumber plants were used since they are efficient and induce
the Fe chelate reductase when iron is limited. Cucumber seeds (Cucumis
satiVus L. cv. Ashley) were germinated on standard seed germination
papers moistened with a macronutrient solution in diffuse light in a
growth chamber for 7 days. Uniform seedlings were selected, and stems
of two individual plants were wrapped together with polyurethane foam
and placed in a 12 L polypropylene bucket (12 pairs of plants per
bucket) containing a continuously aerated EDTA buffered nutrient
solution with the following composition: macronutrients (mM) 1.0 Ca-
(NO3)2, 0.9 KNO3, 0.3 MgSO4, and 0.1 KH2PO4; cationic micronutrients
(µM) 5.0 EDTA/Fe3+, 2.5 MnSO4, 1.0 CuSO4, 10 ZnSO4, 1.0 CoSO4,
1.0 NiCl2, and 115.5 EDTANa2; anionic micronutrients (µM) 35 NaCl,
10 H3BO3, and 0.05 Na2MoO4; 0.1 mM HEPES; and 1 g L-1 CaCO3
to buffer pH at 7.5 to simulate conditions in a calcareous soil. Plants
were grown for 14 days in this nutrient solution in a Dycometal type
CCK growth chamber provided with fluorescent and sodium vapor
lamps with a 16 h/30 °C and 50% humidity day and 8 h/25 °C and
70% humidity night regime. Water was added every 2 days, and the
nutrient solution was renewed every 7 days. The amount of iron added
(5 µM) was found as the most adequate to produce green cucumber
plants but with a high FCR activity (stressed plants) in an assay with
similar experimental conditions (20).
During the experiment, SPAD readings with a chlorophyll meter
(Minolta SPAD-502) were taken for all the leaf stages (average of three
readings per leaf) at several times, although only values measured for
the second leaf stage (the youngest fully open leaf at the start of the
treatment period, t ) 0) have been presented in the results since they
were the most representative of the whole plant. Changes in the SPAD
index (∆SPAD) between the readings at the end of the experiment and
at the beginning of the treatments (t ) 0) have been used instead of
the actual SPAD readings due to the initial variability of developing
leaves. ∆SPAD gives a quantitative measurement of the recovery of
the plants from chlorosis and the relative effectiveness of Fe fertilization
treatment (21, 22). Negative values of ∆SPAD imply a lack of recovery
from the chlorosis, while positive values for a treatment mean that the
treatment is effective.
Root and shoot were separated and washed following the reported
procedure (23). Fresh and dry weights were determined, and after their
digestion, Fe, Mn, Cu, and Zn were assessed by atomic absorption
spectrometry (Perkin-Elmer Analyst 800).
Data were processed using the Statistical Package Social Science
PC 12.0. Duncan’s multiple range test (R ) 0.05) was used to test for
differences among means.
RESULTS AND DISCUSSION
For the measurement of FCR activity, 300 mL beakers, wrapped up
with tin foil to avoid light exposure, were placed in the growth chamber.
Each beaker contained 200 mL of reduction assay solution consisting
of macronutrient solution as in the growth period, 100 µM Fe chelate
Synthesis and Structure. Samples A and B were prepared
from phenol, ethylenediamine, glyoxylic acid, and NaOH,
following the known Mannich-like industrial procedure (4).
Sample A is representative of the mixture of compounds present
in a commercial EDDHA. In the preparation of sample A, a
great excess of o,o-EDDHA (the main product in the crude
reaction) was removed by successive precipitations at pH 6.87
and 6.63, respectively. Small amounts of o,p-EDDHA and p,p-
EDDHA were also removed by further precipitation at pH 5.70.
By following this procedure, sample A is enriched in the rest
of byproducts formed during the reaction. On the other hand,
sample B has been directly obtained by a modification of the
industrial method, carrying out the reaction in stoichiometric
amounts of phenol, ethylenediamine, and glyoxylic acid. Under
(o,o-EDDHA/Fe3+, o,p-EDDHA/Fe3+, sample A/Fe3+, sample B/Fe3+
,
and EDTA/Fe3+), 2 mM MES to buffer the pH at 6, and 300 µM Na2-
BPDS as the Fe2+ trapping and colorimetric reagent. Each solution was
continuously aerated. The roots of 21 day old plants were washed three
times in a macronutrient solution containing 37.5 µM Na2BPDS before
each individual pair of plants was transferred to one beaker. Aliquots
of 3 mL were withdrawn at 0, 10, 20, and 60 min after transfer for
absorbance measurements. Five replicates were prepared for each
treatment, and also five replicate blanks per chelate, consisting of
solutions without plants, were used.
The Fe2+(BPDS)3 concentration was calculated (20) after the
determination of the absorbencies at 535 nm (maximum absorbance
of the Fe2+(BPDS)3) and at 480 nm (near the maximum absorbance