2

Nitrate and Edible Plants in the Mediterranean

Region of TTurk
urkey
ey:: An Over
urkey view
Overview
MÜNIR ÖZTÜRK1, SALIH GÜCEL2, SERDAL SAKÇALI3 AND SÜLEYMAN BASLAR4
1
Ege University, Botany Department, Bornova, Izmir, Turkey
Near East University, Environmental Sciences Institute, Nicosia, Cyprus
2

3
Fatih University, Biology Department, Istanbul,Turkey
4
Faculty of Education, Dokuzeylül University, Buca- Izmir, Turkey
e-mail: munirozturk@gmail.com

ABSTRACT

Turkey is divided in three main phytogeographical regions; one of these is the

Mediterranean. The total gene pool of wild species and land races of cultivated plants
has a great potential in the plant cover of this region. The studies undertaken in this
region revealed that large levels of fertilizers, in particular nitrates, are used in
thousands of greenhouses and on the farms, most of these being used unknowingly. A
large number of producers apply both inorganic as well as organic fertilizers. The
amount varies between 40–75 kg/da fertilizer, which is tenfold of the average
recommended for the area. The excessive use of these fertilizers under long periods
of drought to leads accumulation of nitrates in plants, which in turn poses a great
threat as a potential pollution source for the soils and waters of the region. This paper
presents an overview of the results published on the nitrate pollution and accumulation
in the plants of this region.

Keywords: Turkey, Mediterranean region, Nitrates, Fertilizers

Chapter-2..p65 17 6/6/2013, 5:21 PM

 18 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

INTRODUCTION
Nitrogen as an important part of plants is one of the most abundant elements on
the earth, but its deficiency is probably the most common nutritional problem
affecting plant growth globally. The nitrogen in soil that might eventually be used
by plants has two sources—nitrogen containing minerals and the vast storehouse
of nitrogen in the atmosphere. The nitrogen in soil minerals is released as the
mineral decomposes. This process is generally quite slow and contributes only
slightly to nitrogen nutrition on most soils. In the soil it exists in three general
forms—organic nitrogen compounds, ammonium (NH4+) ions, and nitrate (NO3–)
ions. At any given time, nearly 96 percent of the potentially available nitrogen in
the soil is in organic form either in plants or animal residues (manure, residues of
forage legumes, etc.), relatively stable soil organic matter, or living soil organisms,
mainly microbes such as bacteria. This nitrogen is not directly available to plants,
but some can be converted to available forms by microorganisms (Fig. 2.1) (Ozturk
and Secmen 1995). The concentration of this essential element in the plants can be
as high as 4–5 percent of the dry matter. It is impossible to generalise the
profitability of use of nitrogen fertilizers. These are used to help fill the feed gaps
on large number of farmlands. However, it is necessarry to establish if there is a
need for extra pasture to feed the animals before using nitrogen. Application of
nitrogen fertilizers to the pastures is an important management practice at many
places. The principles governing their use are not affected by differences between
the pastures, but differences in growth patterns can prove effective to increase the
production.
Fertile soils contain large amounts of nitrogen in plant and animal residues,
animal excreta and humified soil (Whitehead 1995). This nitrogen is not taken up
by the plants. But microbial breakdown of these leads to the plant available nitrogen
with its release in ammonium forms through mineralization, and a part is converted
to nitrate forms (nitrification). These processes are influenced by both soil water
availability and temperature. The amount of plant available N in soil at any one
time (the ammonium and nitrate pools) is usually relatively low under irrigated
conditions, at 10–15 kg N/ha in the 0–30 cm horizon. The majority of this is in the
ammonium form and the amount of nitrate can be as low as 1 kg N/ha. The rate
of mineralisation of organic-N in soils can limit the plant growth. When the available
N supply is less than that required for potential grass growth, the plant will respond
to N fertilizer.
The quantity of nitrogen released from any of these materials depends on the
composition of the material, particularly its ratio of carbon to nitrogen, and the
weather. When microorganisms decompose manure and alfalfa residue having
C/N ratios of 10:1 to 20:1, net mineralization occurs because the microbes are
releasing nitrogen from the residue faster than they can utilize it (Guleryuz et al.
2009). On soils containing large quantities of NH4-rich clays, however, nitrogen
supplied by the mineral fraction may be significant in some years.
A very small amount of organic nitrogen may exist in soluble organic
compounds such as urea that may be slightly available to plants. The majority of

Chapter-2..p65 18 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 19

plant-available nitrogen is in the inorganic NH4+ and NO3-forms. Ammonium ions

bind to the negatively charged cation exchange complex of the soil and behave
much like other cations in the soil. Nitrate ions do not bind to the soil solids
because they carry negative charges, but exist dissolved in the soil water, or
precipitated as soluble salts under dry conditions. Some NH4+ and NO3– may also
exist in the crystal structure of certain soil minerals, and may be quite available.
Immobilization and mineralization by microorganisms are most rapid when
soils are warm and moist, but not saturated with water. The quantity of inorganic
nitrogen available for crop use often depends on the amount of mineralization
occurring, and the balance between mineralization and immobilization (Guleryuz
et al. 2009). The nitrogen cycle contains several routes by which plant-available
nitrogen can be lost from the soil. Nitrate-nitrogen is usually more subject to loss
than ammonium-nitrogen. Significant loss mechanisms include leaching,
denitrification, volatilization, and crop Fig. 2.1. The nitrate form of nitrogen is so
soluble that it leaches easily when excess water percolates through the soil. This
can be a major loss mechanism in coarse-textured soils where water percolates
freely, but is less of a problem in finer-textured, more impermeable soils, where
percolation is very slow. Denitrification can cause major losses of nitrogen when
soils are warm and remain saturated for more than a few days. Crop removal
represents a loss because nitrogen in the harvested portions of the crop plant is
removed from the field completely. The nitrogen in crop residues is recycled back
into the system and is better thought of as immobilized rather than removed.

Fig. 2.1. A schematic representation of the nitrogen cycle (modified from Öztürk ve Seçmen, 1995).

Chapter-2..p65 19 6/6/2013, 5:21 PM

 20 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

Anthropogenic activities aimed at enhancing food production facilitate

accumulation of nitrates in plants and affect the quality of soil and water resources
adversely. The reason being when input of nitrogen exceeds the demand, plants
are no longer able to absorb it, and nitrogen builds up in the soil, mostly as
nitrates. This causes imbalance of nutrients in the soil and increases the nitrate
level in groundwater supplies, which influences the nitrate content of plants,
especially the leafy vegetables (Vieira et al. 1998; Dapoigny et al. 2000; Nosengo
2003; NAAS 2005; Anjana et al. 2007 a,b,c). At the same time, long periods of
drought also result in an accumulation of nitrates in plants. When more soil nitrogen
is present than that needed for maximum growth, some plants tend to accumulate
nitrate even without environmental stress. This response is particularly true with
hardy soil feeders like sorghum, noted for “luxury consumption” of certain
nutrients.
Farmers generally apply excessive amounts of nitrogenous fertilizers to their
crops as an insurance against yield losses. The situation in Turkey is not different
in particular in the mediterranean phytogeographical region, which is characterized
by dry hot summers and moderate winters, with low precipitation. The soils are a
mixture of old and newer soils, but distinctive terra rossa soil is widely distributed
in the region. The bare and steep slopes, broad floodplains and torrential forms
are the main kinds of landforms. It is an area of megadiversity of important food
crops, pasture and range land species. The total gene pool of wild species and
land races of cultivated plants has a great potential in its flora. Excessive levels of
nitrogen in the form of nitrate have been found in some groundwaters in the
region. Well waters, especially from shallow wells, are sometimes high in nitrates.
Similarly, runoff from feedlot grounds is reported to be high in nitrite (Atilgan et
al. 2007). The aim of this review is thus to present an overview of the current
situation on the nitrate accumulation in the plants in the Mediterranean part of
Turkey.

FERTILIZERS AND NITRATES

The role of fertilizers came to forefront in the 19th century. After the discovery of
potassium and phosphorus as the key elements in the growth of plants, these
were easily added to the soil due to their presence in huge amounts on earth.
However, the same was not true for nitrogen. The programme for the synthetic
production of nitrogen began, and for the first time, Fritz Haber, a Chemist
produced ammonia from the atmospheric nitrogen and hydrogen. He was awarded
a Nobel Prize in 1918. The industrial production of nitrogen fertilizer was given a
start through the joint efforts of Karl Bosch and Haber, and in 1913, a factory
with the production capacity of 60,000 tons was established in Germany. This
method is still used in the production of nitrogenous fertilizers. Today, the amount
of nitrogenous fertilizers produced has reached a level of around 175 million tons.
The developments in this direction followed the trend in Turkey. The first fertilizer
production was started in 1939 at Karabuk. In this iron and steel factory, sulfuric
acid and ammonia gas were used to produce ammonium sulfate (21%N), followed
by the production of super phosphate. An impetus was given to the production

Chapter-2..p65 20 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 21

from 1954 onwards when nitrogen industry was developed with facilities in
Kutahya, Samsun, Gemlik, and Elazig. In 1984, the name of this industry was
changed to Turkey Fertilizer Industry Inc. (TÜGSAŞ) as an entire state-owned
institution. However, with the decision of the Supreme Council of Privatization,
TÜGSAŞ was privatized in 1998, but in the year 2000, Elazig Fertilizer Industries
Inc. was transferred to Firat University. Presently, the total production is nearly
six million tons. Out of this 35–40 percent is provided by TÜGSAŞ, 25 percent by
Toros Fertilizers, and 15 percent by Gübretaş. The import of fertilizers increased
after the year 2000 at a level of 110 percent as compared to 1996, but has decreased
to 3 percent after 2005. Toros Tarim Mersin Factory (Figs. 2.2, 2.3) is a fertilizer
production complex producing ammonium nitrate (26–33 percent nitrogen) and
diamonium phosphate (18 percent nitrogen and 46 percent P2O5). In the ammonium
nitrate unit, calcium ammonium nitrate (CAN) fertilizer with 26 percent nitrogen
is produced together with ammonium nitrate with 33 percent nitrogen. In the
main production units, 5,94,000 tons/year CAN and 1,50,000 tons/year DAP are
produced. It produces 38 percent of CAN, and 28 percent of DAP out of the
countries’ total production.

Fig. 2.2. A general view of Toros Tarim Mersin Factory alongside the Mediterranean coast in Turkey.

Chapter-2..p65 21 6/6/2013, 5:21 PM

 22 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

Fig. 2.3. Fertilizer use (tons) (Ministry of Agriculture and Rural Affairs—On the basis of Sales).

Fertilizers like urea, ammonium nitrate and diammonium phosphate (DAP)

have single chemical compound with more than one element, and mixed ones like
nitrogen, phosphorus and potassium blends, nitrogen and phosphorus blends and
nitrogen and nitrogen blends have more than one chemical compound. They have
different contents and ratios of nitrogen (Fig. 2.4).
Nitrogenous fertilizers are the most important input in agicultural production.
Several reports suggest that some forms of N are more effective for plant growth
than others. Annually, the N2O levels in the atmosphere increase by 0.2–0.3 percent
globally (Bacon 1995; Abdalla et al. 1998). Asian countries consume 48 percent of
the total fertilizer production in the world (Yilmaz 2004). When applied
insufficiently, losses in yield and quality occur, however, overusage causes washing
of nitrogen and phosphorous fertilizers away, leading to the pollution of surface
and groundwaters and air pollution with the emission of nitrogen oxides (Guler
2004; Atilgan et al. 2007). All nitrogen materials are usually manufactured from
ammonia. Such materials are less expensive, more concentrated, and just as plant-
available as the organics used in the past. The readily available soluble forms of
nitrogen are anhydrous ammonia and aqua ammonia, ammonium nitrate, urea,
non-pressure nitrogen solutions, ammonium sulfate, and ammonium phosphates.
The rate at which these should be applied depends on the weather and soil
conditions. When the soils are warm and moist, decomposition proceeds rapidly,
and nitrogen released may be significant, but when the soils are cold and wet, or
very dry, nitrogen release may be very much less than expected.
There are two major types of fertilizers consumed in the Mediterranean region
of Turkey. These are solid soil chemical fertilizers and liquid leaf fertilizers. The
former includes ammonium sulphate, ammonium nitrate, diammonium phosphate,

Chapter-2..p65 22 6/6/2013, 5:21 PM

 24 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

micro- and macronutrients. These provide nutrients and improve the soil structure
as well. Some synthetic organic fertilizers (Ormin K, Biyofarm, NOF, Organica,
Coplex, Orgevit, liquid and powder organic fertilizer, liquid and powdered chicken
manure) are also used. The maximum levels of nitrogen used in the region are 10
times more. The situation for phosphorus, potassium, magnesium, iron, zinc,
manganese, and copper is much worse (Kaplan et al. 2001). The danger increases if
we consider the waste water effluents from the factories established to meet the
increasing fertilizer demand. The wastewater effluents of fertilizer factories contain
ammonium and nitrate nitrogen levels well above the limits specified in the
regulations (Anonymous 2004).

NITRATES AND LIVING BEINGS

Ammonium nitrate fertilizers are more complex in their mode of action. The
ammonium portion is immediately available in the rumen and is passed across the
rumen wall leading to an increase in the blood ammonia levels very shortly after
consumption. The nitrate fraction, accumulates in the rumen and is converted to
nitrite before crossing the rumen wall, creating nitrate poisoning conditions. Toxicity
due to nitrates can be either acute or chronic (Hill 1999; Bjorne et al. 2004). Acute
toxicity is expressed when nitrite enters the bloodstream in large quantities and
oxidizes the ferrous iron of hemoglobin to the ferric form producing methemoglobin,
which cannot transport oxygen and results in methemoglobinemia (Szaleczky 2000;
Fewtrell 2004). It has been reported that unless immediate relief is provided, a
proportion of methemoglobin that reaches to approximately 70 percent or more of
the total hemoglobin may be fatal. The methemoglobinemia cases are generally
restricted to anemic adults who genetically have high methemoglobin levels in
their blood, and the situation is visually prominent in infants under 3 months of
age and is known as blue baby disease (Al-Redhaiman 2000). The reports published
by some researchers reveal that nitrate, nitrite and some other compounds can get
converted to nitrosamines in human and animal digestive systems, which could
have carcinogenic effects (Atilgan et al. 2007).
The reports show that over 80 percent of the total nitrate intake in humans
originates from vegetables and some part from the drinking water (Croen et al.
2001; Anjana et al. 2007 a,b,c). Although the risks have not been fully evaluated,
several developed countries have established maximum permissible levels for
nitrate concentration in vegetable food crops, and other countries are considering
the establishment of similar standards. As a result of increasing pollution of sources
adjacent to agricultural activities, the World Health Organization has set a limit of
10 mg of nitrate nitrogen per litre in domestic water supplies (Van Diest 1986;
Pionke et al. 1990; McPharlin and Aylam 1995). Research has shown that high
concentrations of nitrate in plant food can decrease the level of ascorbic acid, a
known inhibitor of the formation of carcinogenic compounds that contain nitrite.
There are acute and chronic effects of nitrate poisoning in animals (Cash et al.
2004). When acute poisoning occurs, most animals die before any signs of toxicity

Chapter-2..p65 24 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 25

are observed. Death usually occurs within a few hours of feeding (Al-Redhaiman
2000). The clinical signs of acute poisoning are increased heart rate, blue-grey
mucous membranes, excess saliva and tear production, weakness, depression,
laboured or violent breathing, staggered gait, frequent urination, vomiting, muscle
tremors, low body temperature, disorientation and an inability to get up. Animals
are found in a lying position after a short struggle.
Chronic nitrate toxicity is a form of nitrate poisoning where the clinical signs
of the disease are not observed. It is more common to see a reduction in the rate
of weight gain, lower milk production, depressed appetite, and a greater
susceptibility to infections. These production-related problems or losses are not
often recognized and will occur when nitrate levels are at 0.5 to 1.0 per cent of the
feed consumed (on a dry weight basis). This type of poisoning can cause abortions
within the first 100 days of pregnancy by interfering with the implantation of the
egg in the uterus. Calves affected by nitrate poisoning in the last 3 months of
gestation are usually born 1 to 4 weeks premature, and most appear normal but
die within 18 to 24 hours of birth. Newborn calves that survive, but are affected
by nitrate poisoning, may have convulsions and seizures. Nitrates reduce thyroid
gland activity and therefore, the metabolic rate of the animal, causing a depression
in appetite and lower weight gains. Animals exhibiting signs of distress from chronic
toxicosis may appear uncoordinated, belligerent, or have uncoordinated hind quarter
movement, together with diarrhea, and eye lesions (Al-Redhaiman 2000; Gupta
2006).
In the Mediterranean region the greenhouse agriculture is intense, at many
places drinking water pipelines are lacking and the wells in the vicinity are used
by the labourers working in the area as well as by local inhabitants. A large number
of these greenhouses lack drainage systems. The excess irrigation water thus moves
into deeper parts of the soils and gets mixed up either with the groundwater or
reaches the wells lying in the vicinity of the greenhouses or farms, thus polluting
not only the waters but also producing adverse effects on soil flora. In some cases
the nitrate concentration is (45 ppm) above the levels recommended by WHO
(Kaplan et al. 1999). Nitrate contaminated well water, consumed along with certain
feeds that are high in nitrate, can pose health hazards to domestic animals. These
waters may cause livestock losses. Occasionally, when water contains insufficient
nitrate to be toxic by itself, it may add to the toxicity of nitrate-containing feeds.
Nitrite in water is 10 times more toxic to animals compared to nitrate in feeds.
The critical toxic level of nitrate varies according to the animal group (Smith
and Guthrie 2004). Molybdenum, copper, iron, magnesium and trace elements such
as manganese, are effective in the nitrate to ammonium conversion. Therefore, in
animals are feed with plant material lacking these elements, toxic effects of nitrite
occur in higher levels. The animals fed on a single type hay or silage with high
nitrate content are most likely to get poisoned. Depending on the nitrate content
of plants in the grasslands, poisoning can be seen in grazing animals. The symptoms
start within 1-2 hours after the consumption of high nitrate feed; negative effects

Chapter-2..p65 25 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 27

canola plants, kochia, nightshade, smartweed, and white ragweed (Gastal and
Lemaire 2002). Critical level of nitrate varies between 2000–2500 ppm in forage
crops, which is related to drinking water. Cereal crops are usually planted into
well fertilized, manured or recently plowed/broken grassland or pasture. These
crops are also harvested at an early stage of development (milk to dough stage)
when they contain the highest nitrate content. Some plants usually do not require
soil available nitrogen due to the presence of nodules on their roots, which absorb
gaseous nitrogen from the atmosphere and convert it into ammonia and
subsequently other forms of plant nitrogen with the help of Rhizobium bacteria
located in the nodules through nitrogen fixation contributes up to 85 percent of
the nitrogen content in plants.
Studies on the effect of nitrogen rate and its comparison with the production
of plants has revealed that after applying different amounts of nitrogenous
fertilizers to the plants in a farmland, an increase is observed in the plant growth
at 50 kg N/ha as compared to 10 and 25 kg, but overapplication of nitrogen, 75
and 100 kg adversely affects the plant growth, depression in growth being more
prominent at 100 as compared to 75 kg, through a decline in the final dry matter
production. The cumulative adverse effects become more significant following
applications for a few years. This stresses the fact that low to moderate rates of
nitrogen should be used to strategically increase plant production on the farmlands.
In addition, the component of the nitrogen-fixing species and seasonal effects of
the application on a farmland are important in the nitrogen nutrition of the plants
used for feeding animals (Fig. 2.5). Usually, economic responses to nitrogen
fertilizers can be obtained following application in late winter, provided there are

Fig. 2.5. Responses of plants after applying 50 kg N/ha to two farmlands in different seasons.

Chapter-2..p65 27 6/6/2013, 5:21 PM

 28 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

sufficient other companion species that can grow well at that time of year. Generally,
fertilizers change the plant composition of farmlands, and plant response to nitrogen
is best when it limits their potential to grow and the level of response depends
largely on its deficiency at that time. Sometimes responses which are not economic
can also occur when fertilizers are applied during autumn-early winter. Lower
nitrogen application rates reduce potential changes in the composition of farmlands
because some plants are stimulated less than with heavier applications.
The levels of nitrogen in the farmland species get affected by the rotation
length and rate of fertilizer applied. Rotational grazing is widely practised on
irrigated lands. The use of nitrogen is well suited to this form of farm management
because resting between grazings gives time to respond to the applied fertilizer
before it is grazed again. The length of a pasture rotation affects the quantity of
pasture grown within each rotation, but also can change the relative response to
nitrogen (Fig. 2.6). When rotation on the farmland is short, there is loss of response
to nitrogen fertilizer and in the case of marginally responding plants, this affects
the economic application of the fertilizer. Although N rate increased the N content
of the pasture, the effectiveness of the fertilizer in achieving this declined with
increased rotation length. Generally, if weather conditions are favorable for plant
growth, plants take nearly a month from the time of application to the next grazing,
to respond adequately. Under less suitable growth conditions, it will take longer
for most of the response to occur. Therefore, the rotation needs to be extended
by an appropriate amount. Consumption by stock of relatively small amounts of N
fertilizer granules can be toxic to the stock. Dissolving the nitrogen fertilizer
granules by irrigation or rainfall before allowing stock access to the pasture should

Fig. 2.6. Effect of nitrogen rate and rotation length on plant nitrogen content.

Chapter-2..p65 28 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 29

reduce any potential problems with stock consuming fertilizer on the pasture or
from spillage from a spreader.
Several studies have indicated that plant responses could be much greater when
nitrogen is applied within seven days after grazing (Fig. 2.7). The timing of the
application of nitrogen is probably more critical with lower rates of fertilizer applied.

Fig. 2.7. Cumulative response of plants after single application of nitrogen at different times.

Plant growth varies from day-to-day and week-to-week throughout the season.
The frequency of nitrogen application to plants greatly influences the production,
but not the response. Plant responses with less frequent applications will fluctuate
depending on nitrogen application. However, residual effects of the fertilizer on
plant growth are often small in comparison with the initial response obtained
after application.
The repeated use of nitrogen to increase the production within a short period
reduces legume growth, unless the amount of nitrogen applied during this period
is restricted. In the case of fields with a mixture of grass and legume, 50 kg N/ha
applied over a season is not effective in the production of legumes, but if 100 kg
N/ha is applied, total legume production is significantly reduced. The application
of 50 and 100 kg N/ha over a season to a cereal-legume field has no significant
effect on total legume production (Fig. 2.8). At the same time total dry matter
production in such fields (Fig. 2.9) shows that the effect of intensive nitrogen use
in the cereal-legume field is related to the quality and possibly milk production
due to relatively low digestible cereal domination in the field.
Nitrogen fertilizers are used widely in banana plantations in our Mediterranean
region. Its use over a number of times in a year to maintain a higher rate of plant
growth and with the expectation that it will fill the feed gaps may limit the feed

Chapter-2..p65 29 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 31

(a) Grass–Legume

(b) Cereal–Legume

Fig. 2.9. Production of two fields after applying 0 or 20 or 40 kg N/ha.

Chapter-2..p65 31 6/6/2013, 5:21 PM

 32 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

availability for prolonged periods. The consequences of repeated applications need

to be understood if its use is to remain economical.
An excess use of nitrogen fertilizers results in the accumulation of potentially
harmful nitrate concentrations especially in the leaves of edible plants like spinach,
lettuce, cabbage, etc. (Lorenz 1987; Alt and Fuller 1988; Luo et al. 2006; Anjana et
al. 2007 a,b,c; Anjana et al. 2009 a,b). In general, the harmful effects originating
from an overuse of nitrogenous fertilizers in the plants are bad quality of fruits
(thick peel, spongy, not resistant) (Fig. 2.10), decrease in the resistance to drought,
cold, diseases and pests, soft tissue and cell walls, fast growth followed by lying
of whole plant and thus lower grain yield, but increased shoot growth, and finally
monetary loss. The spinach and chenopodium leaves contain nitrate in concentrations
higher than the recommended daily intake limits (Anjana et al. 2007 a,b,c). (Roorda
van Eysinga 1984; Dich et al. 1996; Turkdogan et al. 2003; Anjana et al. 2007 a,b,c).
Generally, healthy plants often contain 3–4 percent nitrogen in the shoots, which
is a higher concentration than many other nutrients. The important winter crops
like celery are grown on the soils rich in nitrogen. A supplemental fertilization is
essential for optimum production. Nitrogen fertilizers are seldom applied because
soil oxidation generally supplies celery’s requirements. There are situations in which
nitrogen applications can be reduced or eliminated when a crop follows nitrogen
fixing plants. Application of nitrogenous fertilizers used in the dairy farms is very
important to increase the plant production and help reduce expected feed gaps.
Therefore, strategic nitrogen fertilizer use is defined as its addition to increase
plant growth. The benefits of applying these fertilisers are best with responsive
plants, but at present no soil test is available for predicting the response of such
fertilizers. However, indicators of responsive plants are obvious urine patches
after pasture topping, pale green colour in the plants (Fig. 2.11), and decrease in
the efficiency with increasing rates of fertilizer application.

Fig. 2.10. Nitrogen related developments in the orange symptoms in fruits (high nitrogen-left, optimum
nitrogen-right).

Chapter-2..p65 32 6/6/2013, 5:21 PM

 34 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

Nitrate content of plants is moderate in the leaves as compared to the stem

and roots, whereas in high-energy grains it is rather low. The form of nitrate
fertilizer applied is effective on the nitrate content of the plants. The results
published by Aktas et al. (1993) show that in corn accumulation of nitrate was
higher after an application of ammonium sulfate nitrate as compared to ammonium
nitrate. Generally, the nitrate content in plants decreases with maturity, however,
fodders containing high levels of nitrate do not show a decrease in the level of
nitrate (Seay 1996; Smith and Guthrie 2004). Ahmed et al. (2000) have shown a
reduction in nitrate content due to increasing phosphorus fertilization. The reason
being growth reduction associated with limiting phosphate, which results in an
increase in the root-shoot ratio, dry matter content, concentrations of sugars and
organic acids, and reduction in the concentration of nitrate in the shoots (Buwalda
and Warmenhoven 1999). Nitrate accumulation has also been studied in relation
to some other chemicals. Foliar application of salicylic acid, molybdenum fertilizers
and nitrification inhibitors and calcium can materially reduce the nitrate content
of plants (Tzung et al. 1995; Ahmed et al. 2000; Zhou et al. 2000; Xu et al. 2005). In
lettuce leaves, sugar levels and free amino acid concentrations increase under high
supply of calcium and replace nitrate in the vacuole of the lettuce cell (Ahmed,
1996). There exists a negative correlation between the nitrate content and sulfate
content in the lettuce plant (Blom- Zandstra and Lampe 1985) and, therefore, sulfur
deficiency might lead to an increase in nitrate content. The nitrate levels in most
crops (cabbage, carrot, beet, tomato, onion and potato) have been reported to
decrease significantly under the influence of humic acid as well as zeolite (Cardenas-
Navarro et al. 1998, 1999; Nazaryuk et al. 2002). Accumulation of salt in the soils
can alleviate nitrate accumulation in vegetable crops to high fertilization and poor
water supply, the water potential of the soil may become so negative that salination
takes place and plants take up and accumulate nitrate as an osmoticum to adapt to
water conditions. In such a situation, potassium compensates for the increased
nitrate in maintaining the electroneutrality of the plant (Ruiz and Romero 2002;
Chung et al. 2005; Gairola et al. 2009).
The amount of nitrate used by the plant also depends on its metabolic activity
level (Inal and Tarakcioglu 2001; Stitt et al. 2002). Nitrates combine with
carbohydrates during metabolic processes to form the amino acids glutamine and
asparagine. These two amino acids are the base units for the formation of all
other plant amino acids and subsequent protein. Nitrate that is brought into the
plant accumulates in the stems and leaves when the glutamine and asparagine
pathways are saturated. If the conditions are not optimal, the conversion rate of
nitrate into amino acids is impaired. The plant usually uses up all available plant
sugars, and this results in the plant accumulating soluble nitrogen as nitrate and
ammonia.
The nitrate accumulation by plants in toxic amounts on soil high in nitrates is
an exception rather than a rule. The factors responsible for nitrate accumulation in

Chapter-2..p65 34 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 35

plants are mainly nutritional, environmental and physiological. Fertilizing with

nitrogen or plowing under legumes can contribute to the nitrate content of the
soil. The nitrate-utilization pathway depends on an adequate water supply, energy
from sunlight and warm temperatures (above 13°C). Nitrogen fertilization and
light intensity, in particular the diurnal changes, have been identified as the major
factors together with many nutrients that influence the nitrate accumulation in
plants. It varies in various parts of a plant and their physiological age (Delhon
et al. 1995; Gaudreau et al. 1995; Santamaria et al. 1999; Anjana et al. 2007 a,b,c;
Santamaria 2006). Excess nitrate also accumulates in drought stressed plants. The
reason being that dry weather usually strikes at a time when nitrogen is being
rapidly absorbed by the plant, and drought thus causes a reduction in the
conversion of nitrates to protein due to water stress, therefore resulting in nitrate
accumulation. If any of these conditions appears in an agricultural area, the potential
for nitrate poisoning exists within a few days of harvest or grazing.
Generally, 85 percent of the nitrogen from urea is recovered from initially
moist and wet soils within 4 weeks after its application to the fields with dry soil
at the end of autumn season under different initial soil water contents and under
standard flood irrigation. The lowest recovery is around 70, which occurrs on the
initially dry soils; only 10 mm of rainfall is sufficient to improve the recovery. As
such, application of urea should be done just before the expected rainfall or at
places where irrigation facilities are avilable. The application of nitrogen fertilizers
to the dry soil without irrigation or follow-up rainfall will produce negative effects
on plant growth. Similarly, ammonium sulphate and DAP get easily lost from
alkaline or heavily limed acid soils. Ammonium nitrate and MAP are more suitable
alternatives for such soils due to less possibility to form the unstable ammonium
carbonate, from which ammonia is lost. The losses of nitrogen through ammonia
volatilization can also occur from DAP particularly on acid soils, but these losses
are around 35 percent. Application of urea in cooler months results in a loss of 25
percent of the nitrogen to the atmosphere by ammonia volatilization. Overnight
dews are possibly sufficient to dissolve urea, but are not sufficient to wash urea
into the soil.
The effect of climate on nitrate accumulation has been studied at length by
several workers, notable among these being Chadjaa et al. 2001 and Grzebelus
and Baranski 2001. Gruda (2005) published a detailed review on the effects of
different environmental factors on the greenhouse vegetables. Generally, low levels
of nitrate have been recorded during high rainfall, but an accumulation occurs in
warm and wet years, regardless of whether the nitrogen originates from organic
or mineral sources. Nitrate levels are influenced by weather conditions more
significantly than by the form and application rates of fertilizers (Custic et al.
2003). The accumulation varies with the season being higher in autumn-winter
than in spring (Vieira et al. 1998; Santamaria et al. 1997, 2001). The reason is that
in winter plants cannot use all the nitrogen available in the soil due to less
favourable light and temperature conditions.

Chapter-2..p65 35 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 37

that about 91 percent of the growers are aware of organic fertilizers, but only 35
pecent use organic fertilizers in greenhouses, 19 percent use them “for plant
nutrition, 15 percent because of recommendations from pesticide dealers, and 31
percent for the root development and land smoothing. Some use them simply to
add organic matter to the soil and for soil improvement. Nearly 54 percent of the
industries use organic manure for plant nutrition to increase the yield. Looking at
the quantities of farm fertilizers used, the results show that 38 percent of the
greenhouses use 2 tons, 6 percent 2–3 tons and 24 percent 3 tons or more. It was
observed that 32 percent of the industries did not use farm manure and were not
interested in explaining the amount used. Nearly 70 percent of the growers had
not analyzed the soils before applying the fertilizers, 55 percent had applied the
fertilizers according to the recommendations from dealers, 24 percent by past
experience, and only 21 percent were analyzing the soils depending on the plant
and air conditions. Out of these applications, 36 percent of the organic fertilizer
used was obtained from dealers, 45 percent from the market and 19 percent from
the livestock enterprises and villages. The producers were using 29 percent of the
organic fertilizer as farmyard manure, 18 percent from the one sold at the market
(Ormin K, Biyofarm, NOF, Organica, = oplex, Orgevit), 11 percent used liquid
chicken manure, 12 percent liquid manure and 30 percent other liquid and solid
commercial organic fertilizers. Nearly 87 percent of the total organic fertilizer used
has been applied to the tomato growing and 13 percent for other products.
Approximately 36 percent of the enterprises declared that they were using more
than 100 kg/da from the commercialised fertilizers.
The average nitrate content in fresh vegetables cultivated in the greenhouses
were 3.68, 3.40 and 2.86 g/kg for lettuce, parsley and radish respectively. In the
vegetables grown on the farm, the values for lettuce, cabbage, cauliflower, spinach,
parsley, carrot, radish were 1.56, 1.06, 0.42, 0.84, 0.84, 0.50, 1.53 g/kg respectively.
The fresh vegetables like lettuce, cabbage, cauliflower, spinach, radish and carrot
grown as organic farming had 1.19, 0.96, 0.35 , 0.97, 1.31, 0.20 g/kg of nitrate
(Alcicek and Baslar 1995) (Fig. 2.12a,b).

(a) Lettuce (b) Rice

Fig. 2.12. Nitrogen fertilizers applied to lettuce (a) and rice (b).

Chapter-2..p65 37 6/6/2013, 5:21 PM

 38 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

Nitrogen fertilizers are very important for rice production (Fig. 6.12b). The
studies carried out show that in rice best dose is 15 kg/da nitrogen in the pure
form (Anonim 1990., Sürek ve Beşer 1997b). The nitrogen fertilizer should be applied
at least twice in batches—50 percent before sowing and at the time of seedling
appearance, the other 50 percent 55 days after sowing. If three applications are
given, these should follow as 1/3 before sowing, 1/3 at the start of vegetative
multiplication, and 1/3 definitely at the start of formation of panicles (Anonim
1990). Overdose application results in an increase in vegetative multiplication, as a
result differently ripened panicles are harvested, resulting in an increase in the
panicle sterility. Sometimes rice grains are not full, and with the decrease in the
temperatures, there is no production, increase in the laying down, disease
appearance increases, in particular risk of burning increases and production
decreases. A decrease in the production, grain diameter and protein content has
been recorded in case of low application of nitrogen fertilizers. If optimum dose is
not applied, production and quality decrease at the same time (Anonim 1991; Beşer
1997b; Sürek ve Beşer 1997b).
Among the fodder plants corn, British lawn grass, Italyan lawn grass, clover,
rape, sugarbeet leaf and mixed meadow grass contained 8.1, 8.2, 10.3, 6.4, 14.9,
14.1, and 5.4. g/kg dry weight of nitrate (Alcicek and Baslar 1995). Green fodder
plants containing 4400 mg/kg nitrate can be fed without problem, those having
4400–8800 mg/kg are tolerable but water should have no nitrate, however, in
those containing 8800–15000 mg/kg, the amount in the balanced diet should not
be beyond 50 percent (Santamaria et al. 1997, 2001). The fodder plants containing
nitrate 15,000 mg/kg and above are highly toxic (Alcicek and Baslar 1995). The
permissible limits of nitrate levels in water samples are 44 (safe for humans and
animals), 44–88 (normal for animals), 88–176 (not good for animals), 176–440 (can
be used in the absence of nitrates in the feedlot), and 440–880 (no toxical effect,
but fodder uptake effected unfavorably) mg NO/I (Alcicek and Baslar 1995).
Cereals are very responsive to nitrogen, but overapplication can reduce the
yields and quality. Their response depends on the soil type and the soil nitrogen
supply status. The yields are normal when nitrogen fertilizer is applied at lower
levels between 7–9 kg/ha or 7–8 kg/ha for wheat and barley respectively, but the
yields could be increased by applying 35 kg/ha in wheat and 25 kg/ha in feeding
barley. Depending on the soil type, weather conditions or management, the amount
applied can be lowered to 20 kg/ha. For spring-sown malting barley, total nitrogen
is applied to the seedbed. If it is grown after beet tops or turnips or a legume, the
amount is reduced to 25 kg/ha. However, if there is heavy rainfall during the
early growth period, additional 25 kg N/ha is applied (McNaeidhe 1985). In the
case of winter wheat, the nitrogen requirement is higher than other cereals, but
factors like sowing season as well as soil type affecting the yield potential of the
crop also affect its nitrogen requirement (McNaeidhe 1985). In general, 75 kg N/
ha should be the maximum level applied to avoid poor establishment in spring
cereals.

Chapter-2..p65 38 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 39

The response of sugarbeet to nitrogen fertilizers varies between 10–20 percent;

rest of the nitrogen required by the crop is provided by nitrogen reserves in soil
which are said to lie around 15,000 kg/ha N in the root profile (60 cm depth). In
the surface soils the amount available is around 15 percent, which is made available
by biological processes to different extents over different time periods. However,
the amount varies from field to field and is dependent on rainfall criteria and soil
type (Herlihy and Hegarty 2001; Coulter 2001).

MOLECULAR ASPECTS OF NITRATE ACCUMULATION

The nitrate content varies markedly with the plant species, cultivars of the same
species, the genotypes with different ploidy, and even in different parts of the
same plant (Ourry et al. 1997; Grzebelus and Baranski 2001; Harada et al. 2003;
Anjana et al. 2007 a,b,c). After seeding, much of the nitrate taken up by the plant
is used for root and shoot development. During the initial growth, the major part
is retained in the root, but as the growth proceeds the plant is able to convert
more nitrate to amino acids. This change in shoot to root ratio improves the nitrogen
balance. Nitrate accumulates in the plant as long as the roots supply more nitrate
than the plant can use. The highest nitrate levels are found in the lower one-third
of the stalk or stem. Concentrations in the leaves and flowers are lower and the
seeds or grain kernels are usually nitrate free.
The shoot nitrate content is genetically determined and likely to be controlled
by several genes (QTLs) (Harrison et al. 2004). The causal factors might include
genetic differences among genotypes in the enzymes of nitrogen metabolic pathway,
the uptake rate of nitrate as well as other elements needed for enzyme activity, or
differences in generation of electron donors required in the assimilative pathway.
The differing capacities of nitrate accumulation can also be correlated with differing
locations of nitrate reductase activity (Andrews 1986), differences in photosynthetic
capacity (Behr and Wiebe 1992), ability to generate and translocate respiratory
substrate and reducing equivalents, or differences in capacity to translocate the
absorbed nitrate to reduction sites. Nitrate accumulation decreases with increasing
carbohydrate concentration in the vacuoles, being negatively correlated to sugar
concentrations (Blom- Zandstra and Lampe 1985) and dry matter content (Reinink
et al. 1994). In general, the genotypes with high dry matter content could have a
high carbohydrate content in their vacuoles and thus need little nitrate to maintain
their osmotic value (Reinink et al. 1994). The studies using mutants and transgenic
plants have revealed a number of genes that can affect the concentration of nitrate
in a plant, e.g., genes encoding nitrate reductase, a putative anion channel, CLC-a,
glutamine synthetase, and ferredoxin-dependent glutamate synthase (Scheible et
al. 1997a,b; Geelen et al. 2000; Harada et al. 2004). Loudet et al. (2003) identified
eight quantitative trait loci (QTLs) for nitrate content on a dry matter basis, whereas
Hirel et al. (2001) identified five quantitative trait loci for nitrate content in dry
matter of maize, one of which included a gene encoding glutamine synthetase.
Nitrate storage in the vacuoles is affected by multiple processes. Therefore,

Chapter-2..p65 39 6/6/2013, 5:21 PM

 40 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

numerous gene products can potentially influence the naturally occurring variation
in free nitrate levels at the whole tissue level (Harada et al. 2004). Harrison et al.
(2004) have studied genotypic variability in shoot nitrate content and found that it
was mainly due to an increase in the ion uptake regardless of biomass production.
The positive correlation between the shoot nitrate content and the steady state
level of mRNA encoding high-affinity nitrate transporters suggests that the higher
nitrate flux is due to enhanced expression of transporters, indicating thus that
nitrate transport is one of the main checkpoints controlling shoot nitrate
accumulation. The nitrate content can therefore be reduced through breeding
strategies without affecting the biomass production. It is necessary to take into
account that the control of nitrate uptake and its accumulation in the plant may be
subjected to genetic variability regardless of the plant’s demand. This variability
allows for an adaptive regulatory control mechanism depending on soil nitrate
availability. Thus, the selection of genotypes that accumulate less nitrate may
contribute significantly to reduction in the nitrate consumption by humans through
vegetables and the subsequent risk of nitrate poisoning (Tohgi et al. 1998;
Ustyugova et al. 2002). Another method of circumventing the problems associated
with nitrate accumulation may be to breed cultivars that do not accumulate nitrate
even under heavy nitrogen fertilization.

FUTURE PROSPECTS
The fertiliser applications are becoming a great threat by becoming major polluters
as they enter our environment. People have started thinking about the future of
nitrogenous fertilizers due to their negative effects on human and environmental
health. The threat is extending from the local level to global scale, and from deep
groundwaters to the stratosphere. Its presence in our waters in high amounts
causes life-threatening “blue baby disease” in our children. Overapplication of
nitrogenous fertilizers around the agricultural fields is a continuous danger for us
through the wells and underground waters, which may have dangerous amounts
of nitrate accumulation. Excess nitrogen reaches the lakes and the sea leading to
eutrophication. The nitrogen fertilizers also increase the soil acidity by increasing
the degree of dissolution of trace elements and can result in the accumulation of
more trace elements in our drinking waters. It also increases the level of nitrogen
oxides in the atmosphere, thus contributing in the destruction of ozone layer, as
well as an increase in the greenhouse effect in the troposphere. Nitrogen oxides
stay more than 100 years in the atmosphere, and each molecule of nitrogen oxide
absorbs 200 times more radiation than carbon dioxide. This will also play an
important role in global climate change. As a result, it could be concluded that the
world has been caught to the desirability of nitrogen fertilizers very quickly, and
faced with its bad consequences before thinking and doing research on the effects
on environmental health and the possible influence on climate. As a remedy to
this dangerous trend, two focal points should be considered: first, we should
encourage organic farming, and reduce the use of artificial fertilizers and pesticides.

Chapter-2..p65 40 6/6/2013, 5:21 PM

 42 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

producers in order to increase their environmental awareness. The producers mainly

use the fertilizers according to the amount of production; they do not think of any
criteria apart from the yield and no attention is paid to the possible environmental
problems. The importance of environmental education comes to the forefront here.
With an increase in the level of education of the users, the importance given to the
analysis increases. Such people avoid unnecessary and excessive fertilizer usage
due to environmental awareness.
Major part of the soil nitrogen reserves occurs in complex organic forms, which
become available as the organic matter content of the soil is broken down to
release nitrate and ammonium forms of mineral nitrogen. The soil nitrogen supply
status can be deduced from the previous cropping, and manurial history of the
soil, and should be categorised into an index system.
For the usage of nitrate containing fodder, following recommendations can be
made: feed containing 1000 ppm nitrate could be used securely; feeds with nitrates
between 1000–2000 ppm are slightly risky; forages containing nitrate between 2000–
3400 ppm should not be given to animals, if given the ratio limit should not exceed
50 percent; forages containing nitrate between 3400–4000 ppm should be kept away
from the pregnant animals. If given the ratio limit should not exceed 25 percent;
feeds containing more than 4000 ppm nitrate forage are poisonous and should not
be given to the animals; high nitrate fodders must be given at 2–3 weeks intervals.
The nitrate levels in the forage crops growing on dry and wet soils could be
reduced by late harvesting. Animals should not consume high nitrate feed when
they are hungry. Intravenous injection of methylene blue in saline solution (4%)
for horses must be in the 1–2 mg/kg range, while cattle and sheep require 20 mg/
kg to obtain satisfactory results. By using sudans or sudan-sudan hybrids instead
of sorghum-sudan or sorgo-sudan hybrids, nitrate accumulation potential can be
reduced to a great extent. Although impractical, in most situations, contaminated
water supplies can be made safe by boiling.
Right amounts should be applied at the right time to prevent yield losses due
to wrong application of fertilizers and methods. Qualified researchers should guide
the producers about the use of fertilizers in vegetable farming in greenhouses.
The producers require a serious training from government or private sector (Kaplan
et al. 2001; Tuzel et al. 2005; Atilgan et al. 2007). With an increase in the level of
training, the number of producers using organic fertilizers as well as number of
producers approaching institutions for soil analysis will increase. Soil testing is
being suggested more often as an alternative for nitrogen fertiliser applications.
Testing soils for nitrogen has been a useful practice in the drier regions for many
years, and in such regions fertilizer rates can be adjusted to account for NO3–
found in the soil prior to planting. The producers should arrange the analysis of
soil and plant samples in the field and greenhouses for a sustainable production
and prevention of soil pollution through irrigation (Guo et al. 2006).
Easily accessible laboratories should be established in the region, which will
analyze the samples at a reasonable price. Restrictions should be imposed on the
producers in the use of different types of fertilizers in the irrigation water through

Chapter-2..p65 42 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 43

drip irrigation in order to prevent the wrong fertilization. While preparing a

fertilization program, help should be extended to the producers to have conscious
use of fertilizers, which is economically sustainable and enviroment-friendly. The
ammonium nitrogen and nitrate nitrogen amounts specified in the regulations are
very high in the wastewaters of fertilizer producing factories, which should be
controlled.
Effective means of minimizing the accumulation of nitrates in vegetables include
identification of low nitrate accumulating genotypes, manipulation of fertilizer
regimes, exploiting natural diurnal fluctuations in nitrate concentration, and use
of nitrification inhibitors. Rates needed to achieve different yields with different
crops vary by region, and such decisions are usually based on local
recommendations and experience. Nitrate consumption through vegetables can be
kept low by harvesting them at the proper time. Some crops should be harvested
in the afternoon of a sunny day when nitrate concentration in the leaves is low. It
has been found that the leaf nitrate concentration is at its lowest at 1:00 p.m.,
when the sunlight is maximum.
Nitrogen should be applied at a place where all plants have direct access to it.
Moist soil conditions are necessary for nutrient uptake. Placement below the soil
surface can increase nitrogen availability under dry conditions because roots are
more likely to find nitrogen in moist soil with such placement. Anhydrous ammonia
must be placed and sealed below the surface to eliminate direct volatilization losses
of the gaseous ammonia.
The major mechanisms for nitrogen fertilizer loss are denitrification, leaching,
and volatilization. Denitrification and leaching occur under very wet soil conditions,
while volatilization is most common when soils are only moist and drying (Benckiser
et al. 1995). Therefore wet and dry conditions shold be controlled before fertilizer
application as well as before grazing.
Although an inadequate use of nitrogen fertilizers reduces the benefits to the
farmers (Mengel et al. 2006), it provides huge benefits to the manufacturers when
sold at large amounts, who never warn the users about the negative environmental
impacts due to excessive usage. The most important problem which manufacturers
are not interested in solving is the optimal use of fertilizers.

CONCLUSION
Nitrogen losses from soil to water and air have become a concern at global level.
Attempts are being made to put forth some of the best management practices to
be used for controlling nitrogen loss from the fields or glasshouses (Magette and
Carton, 1997). The implementation of various EU Directives, particularly the Action
Programme (AP) required by the Nitrate Directive focuses an attention on tillage
farming as a source of nutrient emission or loss to the environment. It is important
that tillage farmers become more aware of the potential of their systems to cause
nutrient emissions that may result in damage to the environment. A major element
of this is the application of fertilizers to crops at optimum rates. One of the most

Chapter-2..p65 43 6/6/2013, 5:21 PM

 44 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

important EU Directive related to the water quality objectives is Nitrate Directive-

91/676/EEC which is important and challenging.
The Nitrate Directive introduced in 1991 focuses on nitrate losses from agriculture
to water. Its objective is “to reduce water pollution caused or induced by nitrates
from agricultural sources and prevent further such pollution”. The issue of nitrate in
water is of concern because elevated levels of nitrate in drinking water are considered
a danger to human health. In addition, nitrate in water, particularly estuarine waters,
results in the growth of algae and plants that cause undesirable shifts in the
ecosystem. The Directive requires designation of nitrate vulnerable zones and to
develop a list of measures to be implemented by the growers that will achieve the
Directive’s objective. There will be limits on the organic nitrogen loading from animals,
and nitrogen fertilizer and manure applications will be limited. It is important that
tillage farmers consider the potential pathways of nutrient loss from their systems
and the strategies available to control them.
The emphasis on nutrient emissions from agriculture has up to now been
primarily on water. There are three gases of concern—ammonia, methane and
nitrous oxide. Only nitrous oxide is of concern from the perspective of agroindustry.
This gas may account for up to 15 percent of the total global warming potential.
Its global warming potential is estimated to be more than 250 times that of carbon
dioxide. Moreover, its lifetime residency in the atmosphere is estimated to be
about 130 years. Nitrous oxide also leads to ozone depletion. Globally,
anthropogenic activity accounts for 64 percent of the total nitrous oxide emissions
of which agriculture accounts for 92 percent. Farmers will be required to
proportionally reduce the emission of greenhouse gases in the coming years.
Emissions of ammonia and methane relate primarily to animal production
systems. However, ammonia emission from the land spreading of manure is an
issue where a farmer uses animal manure as a nutrient source for crop production.
Agriculture accounts for a major part of ammonia emissions. The sources are
housing, manure storage, manure application to land, and urine and dung
deposition in the fields. Once in the atmosphere, gaseous ammonia reacts
progressively with acidic compounds forming ammonium salts, e.g., ammonium
sulfate and ammonium nitrate. It is this addition of nitrogen to natural ecosystems,
which is of environmental concern both in terms of its implications for biodiversity
and the acidification of soil and aquatic systems.
Ammonia is one of the three most important compounds that are responsible
for acid rain, the others being oxides of sulfur and nitrogen. Acid precursors in
the atmosphere can cross national borders, and emissions from one country can
adversely affect another country’s environment. Therefore, the responsibility of
achieving emission reduction targets rests primarily with the agricultural sector.
For this purpose, correct nutrient application is very important. This can be
achieved by avoiding excessive nitrogen application. The timing of nutrient
application is necessary and nutrients should be applied keeping with the Code of
Good Agriculture Practice. In the nutrient application only calibrated machinery

Chapter-2..p65 44 6/6/2013, 5:21 PM

 46 NITRATE IN LEAFY VEGETABLES: TOXICITY AND SAFETY MEASURES

Anonymous, (2004). Türkiye Cevre Atlasi. Cevresel Etki Degerlendirmesi ve Planlama Genel
Mudurlugu Cevre Envanteri Dairesi Baskanligi, Ankara.
Atilgan, A., Coskan, A., Saltuk, B. and Erkan, M. (2007). The level of chemical and organic
fertilizer usage and potential environmental impacts in greenhouses in Antalya
region. Ekoloji 15: 37-47.
Bacon, P.E. (1995). Nitrogen fertilization in the environment. Marcel Dekker, New York.
Balabanli, C., Albayrak, S., Turk, M. and Yuksel, O. (2006). Some toxic plants growing in
rangelands of Turkey and their effects on animals. Suleyman Demirel University,
Fores Faculty J. A2: 89-96.
Behr, U. and Wiebe, H.J. (1992). Relation between photosynthesis and nitrate content of
lettuce cultivars. Sci. Hortic. 49: 175-179.
Benckiser, G., Lorch, H.J. and Ottow, J.C.G. (1995). Quantification of total denitrification
losses from undisturbed field soils by the acetylene inhibition technique. In: Alef, K.
and Nannipieri, P. (Eds.). Methods in Applied Soil Microbiology and Biochemistry,
Academic Press Ltd., London, 473-478.
Bjorne, H., Petersson, J., Phillipson, M., Weitzberg, E., Holm, L. and Lundberg, J.O. (2004).
Nitrate in saliva increases gastric mucosal blood flow and mucus thickness. J. Clin.
Invest. 113: 106-114.
Blom-Zandstra, M. and Lampe, J.E.M. (1985). The role of nitrate in the osmoregulation of
lettuce (lactuca sativa l.) grown at different light intensities. J. Exp. Bot. 36: 2443-2452.
Blom-Zandstra, M. (1989). Nitrate accumulation in vegetables and its relationship to quality.
Ann. Appl. Biol. 115: 553-561.
Brown, J.R., Christy, M. and Smith, G.S. (1993). Nitrate in soils and plants. In: Agricultural
publication G9804, Missouri-Columbia.
Bruning-Fann, C.S. and Kaneene, J.B. (1993). The effects of nitrate, nitrite, and N-nitroso
compounds on animal health. Vet. Hum. Toxicol. 35: 237-253.
Buwalda, F. and Warmenhoven, M. (1999). Growth-limiting phosphate nutrition suppresses
nitrate accumulation in greenhouse lettuce. J. Exp. Bot. 50: 813-821.
Cardenas-Navarro, R., Adamowicz, S. and Robin, P. (1998). Diurnal nitrate uptake in
young tomato (Lycopersicon esculentum Mill.) plants: Test of a feedback based model.
J. Exp. Bot. 49: 721-730.
Cardenas-Navarro, R., Adamowicz, S. and Robin, P. (1999). Nitrate accumulation in plants:
a role for water. J. Exp. Bot. 50: 613-624.
Cash, D., Funston, R., King, M. and Wichman, D. (2004). Nitrate Toxicity of Montana
Forages www.animalrangeextension.montana.edu/articles/forage/general/nitrate-
tox.htm Moccasin.
Chadjaa, H., Vezina, L-P., Dorais, M. and Gosselin. A. (2001). Effects of lighting on the
growth, quality and primary nitrogen assimilation of greenhouse lettuce (Lactuca
sativa L.). Acta Hortic. 559: 325-331.
Chen, B-M., Wang, Z-H., Li, S-X., Wang, G-X., Song, H-X. and Wang, X-N. (2004). Effects of
nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration
and nitrate reductase activity in three leafy vegetables. Plant Sci. 167: 635-643.
Coulter, B.S. (2001). Nutrient and trace element advice for grassland and tillage crops.
Teagasc, Dublin.
Chung, J-B., Jin, S-J. and Cho, H-J. (2005). Low water potential in saline soils enhances
nitrate accumulation of lettuce. Commun. Soil Sci. Plant 36: 1773-1785.
Croen, L.A., Todoroff, K. and Shaw, G.M. (2001). Maternal exposure to nitrate from drinking
water and diet and risk of neural tube defects. Am. J. Epidemiol. 153: 325-331.

Chapter-2..p65 46 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 47

Custic, M., Poljak, M., Coga, L., Cosic, T., Toth, N. and Pecina, M. (2003). The influence of
organic and mineral fertilization on nutrient status, nitrate accumulation, and yield
of head chicory. Plant Soil Environ. 49: 218-222.
Dapoigny, L., Tourdonnet, S.D., Roger-Estrade, J., Jeuffroy, M-H. and Fluery, A. (2000).
Effect of nitrogen nutrition on growth and nitrate accumulation in lettuce (Lactuca
sativa L.) under various conditions of radiation and temperature. Agronomie 20:
843-855.
Delhon, P., Gojon, A., Tillard, P. and Passama, L. (1995). Diurnal regulation of nitrate
uptake in soybean plants. I. Changes in NO3 influx, efflux, and N utilization in the
plant during the day/night cycle. J. Exp. Bot. 46: 1585-1594.
Dich, J., Jivinen, R., Knekt, P. and Pentill, P.L. (1996). Dietary intakes of nitrate, nitrite and
NDMA in the Finish Mobile Clinic Health Examination Survey. Food Addit. Contam.
13: 541-552.
Durner, J. and Klessig, D.F. (1999). Nitric oxide as a signal in plants. Curr. Opin. Plant Biol.
2: 369-374.
Ebert, K., Koriath, H. and Rinno, G. (1997). The intensification of grass production through
nitrogen fertilization and the nitrate problem. XIII International Grassland Congress 2:
987-989.
EC (European Commission), Commission Regulation (EC) No. 1822/2005 of 8 November
2005 amending Regulation (EC) No. 466/2001 as regards nitrate in certain
vegetables. Official J. Eur. Union L293: 11-13.
Eichholzer, M. and Gutzwiller, F. (1998). Dietary nitrates, nitrites, and AA Nitroso
compounds and cancer risk: a review of the epidemiologic evidence. Nutr. Rev. 56:
95-105.
Fewtrell, L. (2004). Drinking-water nitrate, methemoglobinemia, and global burden of
disease: a discussion. Environ. Health Persp. 112: 1371-1374.
Gairola, S., Umar, S. and Suryapani, S. (2009). Nitrate accumulation, growth and leaf
quality of spinach beet (Beta vulgaris Linn.) as affected by NPK fertilization with
special reference to potassium. Indian J. Sci. Technol. 2: 35-40.
Gastal, F. and Lemaire, G. (2002). N uptake and distribution in crops: an agronomical and
ecophysiological perspective. Physiol. Plant 53: 789-799.
Gaudreau, L., Charbonneau, J., Venzina, L.P. and Gosselin, A. (1995). Effect of photoperiod
and photosynthetic photon flux on nitrate in greenhouse-grown lettuce. J. Plant
Nutr. 18: 437-453.
Geelen, D., Lurin, C., Bouchez, D., Frachisse, J.M., Lelievre, F., Courtil, B., Barbier-Brygoo,
H. and Maurel, C. (2000). Disruption of putative anion channel gene AtCLC-a in
Arabidopsis suggests a role in the regulation of nitrate content. Plant J. 21: 259-267.
Gok, M. and Ottow, J.C.G. (1998). Effect of cellulose and straw incorporation in soil on
total denitrification and nitrogen immobilization at initially aerobic and permanent
anaerobic conditions. Biol. Fert. Soils 5: 317-322.
Gruda, N. (2005). Impact of environmental factors on the product quality of greenhouse
vegetables for fresh consumption. Crit. Rev. Plant Sci. 24: 227-247.
Grzebelus, D. and Baranski, R. (2001). Identification of accessions showing low nitrate
accumulation in a germplasm collection of garden beet. Acta Hortic. 563: 253-255.
Guler, S. (2004). Dunya’da ve Turkiye’de Gubre Tuketiminde Yasanan Gelismeler. In:
Turkiye 3. Ulusal Gubre Kongresi, Karaman and Brohi (eds.), Tarim-Sanayi-Cevre,
11-13 Ekim 2004, Tokat, pp. 47-54.

Chapter-2..p65 47 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 49

Magette, W.L. and Carton, O.T. (1997). Agricultural pollution control. In: Environmental
Engineering (Ed. Gerard Kiely) McGraw-Hill, 781-798.
Majumdar, D. and Gupta, N. (2000). Nitrate pollution of groundwater and associated
human health disorders. Indian J. Environ. Health 42: 28-39.
McNaeidhe, F.S. (1985). The effect of applying nitrogen at different times and rates on the
yield of winter cereals on peatland. Irish. J. Agric. Res. 24: 63-77.
McPharlin, I.R. and Aylam, P.M. (1995). Nitrogen requirements of lettuce under sprinkler
irrigation and fertilization on spearwood sand. J. Plant Nutr. 18: 219-241.
Mengel, K., Hutsch, B. and Kane, Y. (2006). Nitrogen fertilizer application rates on cereal
crops according to available mineral and organic soil nitrogen. Eur. J. Agronom. 24:
343-348.
Mensinga, T.T., Speijers, G.J.A. and Meulenbelt, J. (2003). Health implications of exposure
to environmental nitrogenous compounds. Toxicol. Rev. 22: 41-51.
Michal, F.D. (1998). A population based case control study on the association between
nitrate in drinking water and non-Hodgkin’s lymphoma. The Johns Hopkins
University Press, USA, p. 285.
NAAS, (2005). Policy options for efficient nitrogen use. Policy paper No. 33, National
Academy of Agricultural Sciences, New Delhi, pp. 1-4.
Nazaryuk, V.M., Klenova, M.L. and Kalimullina, F.R. (2002). Ecoagrochemical approaches
to the problem of nitrate pollution in agroecosystems. Russ. J. Ecol. 33: 392-397.
Nosengo, N. (2003). Fertilized to death. Nature 425: 894-895.
Oruc, H.H. and Ceylan, S. (2001a). Bursa Yoresinde Sigirlarin Yemlerinde, Icme Sularinda
ve Rumen Iceriginde Nitrat, Nitrit ve Kanda Methemoglobin Duzeylerinin
Arastirilmasi. J. Fac. Vet. Med. 20: 25-32.
Oruc, H.H. and Ceylan, S. (2001b). Bursa’da tuketilen bazi sebzelerde nitrat ve nitrit.
Uludagi University, J. Fac. Vet. Med. 20: 17-21.
Ourry, A., Gordon, A.J. and Macduff, J.H. (1997). Nitrogen uptake and assimilation in
roots and root nodules. In: Foyer, C.H. and Quick, W.P. (Eds.). A molecular approach
to primary metabolism in higher plants. Taylor and Francis, London, pp. 237-253.
Ozturk, M. and Secmen, O. (1995). Plant Ecology. Ege University Press, pp. 350.
Pionke, H.B., Sharma, M.L. and Hirschberg, K.J. (1990). Impact of irrigated horticulture on
nitrate concentration in ground water. Agric. Ecosys. Env. 32: 119-132.
Reinink, K. (1991). Genotype x environment interaction for nitrate concentration in lettuce.
Plant Breed 107: 39-49.
Reinink, K., Vannes, M. and Groenwold, R. (1994). Genetic variation for nitrate content
between cultivars of endive (Cichorium endiviae L.). Euphytica 75: 41-48.
Roorda van Eysinga, J.P.N.L. (1984). Nitrate and glasshouse vegetables. Fert. Res. 5: 149-
156.
Rubio, H.O., Wood, M.K., Gomez, A. and Reyes, G. (1996). Native forage quality, quantity,
and profitability as affected by fertilization in Northern Mexico. J. Range. Manag. 49:
315-319.
Ruiz, J.M. and Romero, L. (2002). Relationship between potassium fertilization and nitrate
assimilation in leaves and fruits of cucumber (Cucumis sativus) plants. Ann. Appl.
Biol. 140: 241-245.
Santamaria, P., Elia, A. and Gonnella, M. (1997). Changes in nitrate accumulation and
growth of endive plants during the light period as affected by nitrogen level and
form. J. Plant Nutr. 20: 1255-1266.

Chapter-2..p65 49 6/6/2013, 5:21 PM

 NITRATE AND EDIBLE PLANTS IN THE MEDITERRANEAN REGION... 51

Tzung, W-J., Po, W-Y. and Wu, J.T. et al. (1995). Effects of some environmental factors on
nitrate content of Chinese cabbage (Brassica chinensis L.). J. Chin. Agric. Chem. Soc. 33:
125-133.
Ustyugova, I.V., Zeman, C., Dhanwada, K. and Beltz, L.A. (2002). Nitrates/nitrite alter
human lymphocyte proliferation and cytokine production. Arch Environ. Contam.
Toxicol. 43: 270-276.
Van Diest, A. (1986). Means of preventing nitrate accumulation in vegetables and pasture
plants. In: Fundamentals, Ecological and Agricultural Aspects of Nitrogen
Metabolism in Higher Plants. Neeteson, L.H. and Stalen, J.J. (Eds.). Durdrecht: Martins
Nijhoft.
Vieira, I.S., Vasconselos, E.P. and Monteiro, A.A. (1998). Nitrate accumulation, yield and
leaf quality of turnip greens in response to nitrogen fertilisation. Nutr. Cycl.
Agroecosys. 51: 249-258.
Weiss, W.P. (2004). Nitrate toxicity in drought-stressed forages.
www.wvu.edu/agexten/forglvst/nitrate.htm Extension Service West Virginia University.
Whitehead, D.C. (1995). Grassland nitrogen. CAB International, Wallingford, Oxon, UK.
Xu, C., Wu, L.H. and Ju, X.T. et al. (2005). Role of nitrification inhibitor DMPP (3,4-
dimethylpyrazole phosphate) in nitrate accumulation in greengrocery (Brassica
campestris L. ssp. chinensis) and vegetable soil. J. Environ. Sci. (China) 17: 81-83.
Yilmaz, I., Ozkan, B., Akkaya, F., Yilmaz, S. and Kutlar, I. (2000). Antalya Ili Sera
Sebzeciliginde Ilac ve Gubre Kullaniminin Analizi. In: Turkiye 4. Ulusal Tarim
Ekonomisi Kongresi, 6-8 Eylul, Tekirdagi, pp. 1-10.
Yilmaz, H. (2004). Turkiye’de Kimyasal Gubre Uretim, Tuketim ve Dis Ticaretindeki
Gelismeler. In: Turkiye 3. Ulusal Gubre Kongresi, Tarim-Sanayi-Cevre, 11-13 Ekim,
Tokat, pp. 35-46.
Yilmaz, H. (2005) Kimyasal Gubre Kullaniminin Cevresel Etkilerinin Teknik ve Ekonomik
Acidan Degerlendirilmesi. In: I. Cevre ve Ormancilik Surasi, Tebligler, 21-24 Mart,
Antalya, pp. 118-128.
Zhou, Z.Y., Wang, W.J. and Wang, J.S. (2000). Nitrate and nitrite contamination in vegetables
in China. Food Rev. Int. 16: 61-76.
Zialdi, N., Simard, R.R., Allard, G. and Parent, G. (2000). Yield response of forage grasses
to N fertilizer as related to spring soil nitrate sorbed on anionic exchange
membranes. Canad. J. Soil Sci. 80: 203-212.

Chapter-2..p65 51 6/6/2013, 5:21 PM