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Influence of partial
replacement of nitrate by
amino acid nitrogen or urea
in the nutrient medium
on nitrate accumulation in
NFT grown winter lettuce
a b
Aydin Gunes , Wietse N. K. Post , Ernest A.
c a
Kirkby & Mehmet Aktas
a
Department of Soil Science, Faculty of
Agriculture , University of Ankara , Ankara,
06110, Turkey
b
Glasshouse Crops Research Station ,
Kruisbroekweg 5, Postbus 8, Naaldwijk, 2670
AA, The Netherlands
c
Department of Pure and Applied Biology ,
University of Leeds , Leeds, LS2 9JT, United
Kingdom
Published online: 21 Nov 2008.

To cite this article: Aydin Gunes , Wietse N. K. Post , Ernest A. Kirkby &
Mehmet Aktas (1994) Influence of partial replacement of nitrate by amino
acid nitrogen or urea in the nutrient medium on nitrate accumulation in NFT
grown winter lettuce, Journal of Plant Nutrition, 17:11, 1929-1938, DOI:
10.1080/01904169409364855

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 JOURNAL OF PLANT NUTRITION, 17(11), 1929-1938 (1994)

INFLUENCE OF PARTIAL REPLACEMENT OF NITRATE BY

AMINO ACID NITROGEN OR UREA IN THE NUTRIENT
MEDIUM ON NITRATE ACCUMULATION IN NFT GROWN
WINTER LETTUCE
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Aydin Gunes
Department of Soil Science, Faculty of Agriculture, University of Ankara, 06110
Ankara, Turkey
Wietse N. K. Post
Glasshouse Crops Research Station, Kruisbroekweg 5, Postbus 8, 2670 AA
Naaldwijk, The Netherlands

Ernest A. Kirkby
Department of Pure and Applied Biology, University of Leeds, Leeds, LS2 9JT,
United Kingdom

Mehmet Aktas
Department of Soil Science, Faculty of Agriculture, University of Ankara, 06110
Ankara, Turkey

ABSTRACT: Two cultivars of lettuce plants (Lactuca sativa L. cvs Berlo and
Kirsten) were grown for two months in late fall in NFT under three different
nitrogen (N)-regimes but with the same total N concentration (13.4 mM). A
reference treatment with a nutrient solution containing 94% nitrate (NO3) and 6%
ammonium (NH4) was compared with urea and proteinate treatments in which
20% of the NO 3 of the reference treatment was replaced by either of these two N
sources. Proteinate is a fertilizer produced in Turkey containing 8% amino acid N
and 8% NO 3 -N. For both cultivars the fresh weights of the harvested plants were
unaffected by the N source as was also the total N uptake. Nitrate content,
however, was considerably lower in the urea and proteinate plants, values for the
three treatments ranging from 3314 to 4579 mg NO3/kg fresh wt making up from
between 44.3% to 55.4% of the total plant N. Of the two cultivars, Berlo
accumulated greater concentrations of NO3 than did Kirsten. The accumulation of

1929

Copyright © 1994 by Marcel Dekker, Inc.

 1930 GUNES ET AL.

chloride (Cl) was in the reverse order of that of NO3 with greatest concentrations
occurring in the proteinate plants.

INTRODUCTION
Nitrate accumulation in crop plants is undesirable for a number of reasons. It
has been known for many years that cows may die from ingesting herbage rich in
NO3 as the NO3 may be reduced to nitrite (NO2) under the anaerobic conditions in
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the rumen where it can combine with haemoglobin to form methaemoglobin to

prevent the transfer of oxygen (Van Diest, 1986). In humans, a similar very rare
illness known as Blue Baby Syndrome has been found to be associated with high
NO3 levels in drinking water. Additionally it has been suggested that NO2
produced in the stomach following the reduction of ingested NO3 may react with
secondary amines to produce nitroso compounds which are said to induce gastric
cancer although whether or not there is a direct association between dietary NO3
level and the occurrence of gastric cancer is a matter of controversy (Greenwood,
1990).
Leafy vegetable crops, such as lettuce, contain particularly high amounts of
NO3-N and there have been moves to limit the NO3 levels in these crops when
used for human consumption. The acceptable limit set by the Dutch Government
for winter-grown lettuce is 4500 mg NO3 per kg fresh weight, while that for
summer harvested lettuce is 2500 mg NO3 per kg fresh weight (Anonymous,
1982, 1985). Recently the German Government has proposed a maximum level
for the winter-grown crop of 3500 mg NO3 per kg fresh weight (Anonymous,
1993). If this limit were to be accepted by the EEC, it may place the winter lettuce
growing industry in jeopardy unless current fertilizer practice were changed. There
is, therefore, a need to study means by which NO3 concentration can be depressed
in winter grown lettuce without affecting yield. Some success has been obtained in
this direction by replacing a small part of NO3 supply by NH4 (Ikeda and Osawa,
1984; Van der Boon and Steinhuizen, 1986; Van der Boon et al., 1990) or by
increasing the level of Cl nutrition (Van der Boon et al., 1990). Another approach
to lower the NO3 concentration has been to use Triamide Phosphoryl [PO(NH2)3]
as a N source. Lettuce plants growing in this form of N contained 10 times less
NO3 at maturity than plants grown with ammonium nitrate (Beresniewiecz et al.,
1988). In a study described by Aktas et al. (1992), it was found that maize plants
 PARTIAL REPLACEMENT OF NITRATE 1931

TABLE 1. Composition of the basal nutrient solution

(mmol/L).

Source Reference Urea Proteinate

N 13.40 13.40 13.40

H2PO4 1.35 1.60 1.60
SO4 0.76 1.00 1.00
Cl Trace 0.60 0.60
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K 7.40 6.70 6.70

Ca 3.03 2.74 2.74
Mg 0.67 0.62 0.62

grown in soil which had received the same amount of N but in different forms,
those plants supplied with proteinate (a fertilizer produced in Turkey containing
8% amino acid-N and 8% NO3-N) was lower in NO3 than the plants which had
received N from either ammonium nitrate, ammonium sulphate, or urea.
The object of the present work was to determine whether it was also possible
to decrease the NO3 content in two cultivars of butterhead lettuce growing in NFT
by replacing a small part of the NO3 supply (20%) by either urea or proteinate
fertilizer.

MATERIALS AND METHODS

Two cultivars of lettuce (Lactuca sativa L.), Berlo and Kirsten, were grown
using NFT in a recirculating solution. Three-week-old seedlings in peat blocks
were transplanted at the rate of four plants per meter in gutters of 10 meters in
length, spaced 0.2 meters apart, and held at a slope of 5% and with the nutrient
solution flowing at the rate of 20 mL/s. The solutions were held in 500-L
containers, each container supplying four gutters (i.e. one treatment). Three
treatments were compared in which the N source was varied but with the same
total N concentration (13.4 mM) in the nutrient solution. The composition of the
basal nutrient solution is given in Table 1. Iron (Fe), boron (B), manganese (Mn),
 1932 GUNES ET AL.

TABLE 2. Proportions of the N source in total-N (%).

Treatments
Source Reference Urea Proteinate

NO3 94 74 74*
NH4 6 6 6
Urea — 20 —
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Proteinate - - 20

* Since half the total N in the proteinate treatment was in the

form of N03-N the proportion of "actual" NO3 in this
treatment was 84%.

zinc (Zn), copper (Cu ), and molybdenum (Mo) were applied to all treatments at
the rates of 40, 30, 5, 4, 0.75 and 0.5 jimol/L, respectively.
As shown in Table 2, the reference treatment consisted of a mixture of 94%
NO3 and 6% NH4. This is used at Glasshouse Crops Research Station
(Naaldwijk, The Netherlands) and is known to produce lettuce crops of high yield
and quality. For the other two treatments, 20% NO3-N was replaced by either urea
or proteinate. During the experiment the pH of the nutrient solution was
maintained between 5.9 and 6.2 by automatic addition of either K2CO3 or H2SO4
(0.1 equivalent H + or OH"/L). The nutrient composition of the growing solution
was monitored by weekly analysis of the recirculating solution.
The plants were grown for two months in late Autumn, sowing,
transplanting, and harvesting dates being as follows: 11/9/1992, 29/9/1992, and
16/11/1992, respectively. Treatments were arranged in a randomized complete
block design with four replications. At the end of the experiment, five plants were
harvested and a quarter of each harvested plant was taken for NO3 determination
on the fresh material. The remainder was washed and dried 70°C in order to
determine dry matter and subsequently ground to provide dried plant material for
the determination of total N and Cl.
 PARTIAL REPLACEMENT OF NITRATE 1933

TABLE 3. Fresh weight yields (g/plant) and dry matter percentages of lettuce
plant in relation to the form of N.

Varieties
Berlo Kirsten

Treatments Fresh Dry Fresh Dry

Weight Matter Weight Matter
g/plant % g/plant %
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Reference 237±6.1 2.89+0.07 232+0.5 2.93+0.04

Urea 250±9.3 2.79+0.08 247±0.8 2.82+0.05
Proteinate 249±4.8 2.82±0.03 245+7.1 2.77+0.05

Total-N was determined by the Kjeldahl method as described by Bremner

(1965). Nitrate-N and Cl were determined using an AutoAnalyser as described by
Bes (1986) and Van de Woestijne (1990). Yield and chemical analytical data were
analyzed statistically by ANOVA and treatment means were compared with LSD
(p=0.05).

RESULTS
Neither fresh weight nor percentage dry matter values was affected by the
three sources of N supply for either of the cultivars (Table 3). The concentration
of NO3 in the plants of both cultivars, however, was considerably affected by the
N source—being depressed in the urea and proteinate treatments. Values are given
in mg/kg fresh weight (Table 4) since this is the form of expression commonly
used in horticultural practice. In winter grown plants, the values are high
particularly in the reference treatment and at the limit of acceptability for human
consumption in the Netherlands (i.e. 4500 mg NO3/kg FW). Berlo accumulated
more NO3 than did Kirsten as is indicated in the experiment and is also clear from
our unpublished data. The NO3 levels are considerably greater than those obtained
 1934 GUNES ET AL,

TABLE 4. Nitrate contents of the plants (mg

NO3/kg FW) in relation to nitrogen
supply.

Varieties
Treatments
Berlo Kirsten

Reference 4579±244 4332±122

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Urea 3761±55 3672+134

Proteinate 3817±88 3314+122

LSD (p=0.05) 468 490

in a similar experiment carried out in late summer, for Berlo, values for the
reference, urea and proteinate, respectively, being 3920, 3861, and 3315 mg
NO3/kg fresh weight, again showing the depressing effect that proteinate had on
the NO3 concentration even during summer conditions.
Total N concentrations were unaffected by the treatments for either cultivar
(Table 5). The higher NO3 concentration in the reference treatment is, therefore,
reflected by a higher percentage of NO3 of total N in this treatment. Even in the
urea and proteinate treatments, NO3-N made up an amount approaching 50% of
the total N.
Chloride and NO3 values given in Table 6 are expressed in mmol/kg on a
fresh weight basis since both these ions function as osmotica in plant cells and
expression in this form is more appropriate in considering interrelationships
between these anions. The results presented here shows that the fall in NO3
concentration in the urea and proteinate-supplied plants is accompanied by an
increase in Cl, the total Cl and NO3 concentration being more or less constant at
around 70 mmol regardless of the form of N supply.

DISCUSSION
The main finding of this work is that replacement of 20% of the NO3 in the
nutrient medium by either urea or proteinate for lettuce plants grown in an NFT
 PARTIAL REPLACEMENT OF NITRATE 1935

TABLE 5. Total N, N03-N and % NO3-N of the total N of lettuce plant in relation to N
supply.

Berlo Kirsten
Treatments Total N NO3-N N03-N% Total N NO3-N NO3-N
%DW %DW % of Total N %DW %DW %of Total N

Reference 6.46±0.06 3.58+0.13 55.4 6.3510.14 3.33+0.12 52.4

Urea 6.2610.11 3.05+0.07 48.7 6.07+X).07 2.9410.10 48.4
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Proteinate 6.3010.05 3.05+0.04 48.5 6.0910.07 2.70+0.09 44.3

LSD (p=0.05) 0.33 0.39

TABLE 6. Chloride and NO3 contents of lettuce plants (mmol/kg FW).

Berlo Kirsten
Treatments Cl NO3 CI NO3

Reference 3.3+0.2 73.9±3.9 3.6+0.6 69.9±1.9

Urea 8.8+0.4 60.7+0.9 8.9±0.5 59.2±2.2
Proteinate 13.4±0.3 61.6±1.4 14.4±0.6 53.5±2.0

LSD (p=0.05) 1.1 7.5 1.0 7.9

syste had no effect on fresh or dry weight yields or N uptake of either of the two
cultivars but substantially depressed the NO3 concentrations in the plants. Similar
findings have been reported by Van der Boon et al. (1990) in an experiment in
which 20% of NO3 was replaced by NH4.
Four forms of N, NO3, NH4, urea, and amino acid-N, were supplied in this
experiment, all of which can be taken up and utilized by the plants (Mengel and
Kirkby, 1987). Despite the relatively high NO3 supply in all four treatments, the
presence of small amounts of the other N containing constituents considerably
decreased the NO3 concentrations in the plants with lowest concentrations
 1936 GUNES ET AL.

occurring with the proteinate supply. The reason for this effect of proteinate
probably relates to the slow but continuous release of NH4-N from this N source.
Ammonium-N release also occurs on hydrolysis of urea when supplied in the
nutrient culture but urea is known to have other adverse effects, such as
depressing solution pH, decreasing nutrient uptake, and possibly inducing toxicity
per se(Luoetal, 1993).
It is well established that increasing concentration of Cl in the nutrient medium
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can depress NCo uptake by competition between these two ions (Kirkby and
Knight, 1977; Stienstrat, 1986; Van der Boon et al., 1990; Veen and
Kleinendorst, 1986; Wehrmann and Hahndel, 1984). The slightly higher Cl con-
centration in the urea and proteinate nutrient solution as compared with the
reference treatment (0.6 mM and trace), however, is unlikely to be the main factor
in depressing NO3 tissue concentration in these treatments (Table 6). This can be
deduced by comparing the proteinate and urea treatments both with the same Cl
concentration in the nutrient medium but with a much higher Cl concentration and
lower NO3 accumulation by the proteinate plants even though the NO3 concen-
tration was higher in the nutrient medium of the proteinate treatment (Table 2).
This finding is also confirmed by data for lettuce from another unpublished
experiment again comparing a 20% urea or proteinate substitution of NO3 and
NH4-N. With the same level and amounts of NO3 and NH4 in the nutrient
medium but a higher (3 mM) Cl concentration, NO3 accumulation in the lettuce
plant was much lower in the proteinate plants.
To account for the highest concentration of Cl and the lowest accumulation of
NO3 in the proteinate treatment, we suppose that the effect is one of ionic balance
in relation to uptake of NH4-N available on the breakdown of proteinate N.
Ammonium-fed plants in comparison with those supplied with NO3 are lower in
inorganic cations (Ca, Mg, and K) and organic anions (malate, citrate, etc), and a
higher proportion of the cation charge is satisfied by inorganic anions other than
NO3, e.g. Cl (Kirkby, 1968; Van Beusichem et al. 1988). Since Cl is readily
taken up, as is apparent from the data given in Table 6, it can replace organic
anions and may also replace NCo—presumably taking over its osmotic role. This
effect of NH4-nutrition in favouring Cl uptake has been reported recently by Cao
and Tibbits (1993) for potato. That less Cl is taken up by the plants supplied with
 PARTIAL REPLACEMENT OF NITRATE 1937

urea than with proteinate N may possibly relate to the higher stability of urea than
proteinate in the nutrient medium, and thus a lower rate of release of NH4.
The use of a proteinate N fertilizer as a means of depressing the NO3 concen-
trations in leafy vegetables is worthy of further consideration even though the
price of such fertilizer is about three times more than that of equivalent inorganic
N-sources. More information is needed concerning the stability of these amino N
containing fertilizers and the forms in which N is taken up from these compounds
when supplied in an NFT growing system under greenhouse conditions.
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