Fact sheet:

Nitrate and nitrite

Version: 09/01/2015

GMP+ International B.V.

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 Content

General Summary

Summary of GMP+ products standards for the animal feed sector

More Facts
1. Nature, history and prevalence of nitrate and nitrite
2. Transmission to the environment, plants, animals and humans
3. Diagnose of poisoning
4. Potential hazards and adverse effects
5. Severity of the hazard
6. Standards
7. Analysis methods
8. Control measures
9. References
10. Websites

APPENDIX / APPENDICES

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 General Summary

Name: Nitrate and nitrite

code: C31

Description: Nitrate (formula NO3) and nitrite (formula NO2) are nitrogen compounds which are
part of the nitrogen cycle. Through human activity, especially excess use of fertiliser,
large amounts of extra nitrogen are added to certain phases of the natural nitrogen
cycle. This leads to leaching and acidification. Nitrite is the conversion product of
nitrate.
Type: chemical

Severity: high

Control measures: - Analysis of spring/treated water quality

- Establish purchasing requirements
- Expert use of fertilisers
- Finished product analysis
- Monitor cultivation area (certified)
- Plot history
- Prevent overdosering of nitrate
- Received product/raw material analysis
- Soil history
- Water management

The control measures specified in this fact sheet are all control measures which can be used depending on the
product and/or process step.

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 SUMMARY OF GMP+ SPECIFIC FEED SAFETY LIMITS FOR THE ANIMAL FEED SECTOR
Contaminant Product Action limit(1) Rejection limit(1) Source Supplementary Analysis method7
requirements
Chemical: Other undesirable substances and products

C31 Nitrites Feed materials, with the exception of: - 15 mg/kg Commission OZM Part 2; OSP-7
(expressed in Regulation (EU) No
sodium nitrite) 1275/2013
amending Annex I
- Fish meal - 30 mg/kg to Directive
(expressed in 2002/32/EC
sodium nitrite)
- silage -

- Products and by-products from sugar beet and - -

sugarcane and from starch production and
alcoholic drink production.
Complete feed, with the exception of: - 15 mg/kg
(expressed in
sodium nitrite)
- complete feed for dogs and cats with a moisture - -
content exceeding 20 %.

[1 ] Action limit: A feasible limit agreed in consultation with the sector, supplier or customer. If this limit is exceeded then an investigation into the cause should be undertaken and corrective measures should be
taken to remove or control that cause. Maximum levels in mg/kg (ppm) of the feed materials or compound feeds, derived to a moisture content of 12% unless mentioned differently.
Rejection limit: A feasible limit agreed in consultation with the sector, supplier or customer. If this limit is exceeded then the product is not suitable for use as feed material or animal feed. Maximum levels in mg/kg
(ppm) of the feed materials or compound feeds, derived to a moisture content of 12% unless mentioned differently.
[7] The research methods (OZM) can be found via the PDV website (www.pdv.nl ; quality; research methods)

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 More facts

Chemical name
Nitrite (expressed as sodium nitrite)

CAS-number
Nitrite : n.a.
Sodium nitrite : 7632-00-0
Potassium nitrite : 7758-09-0

Synonyms
Nitrite : n.a.
Sodium nitrite : Nitrous acid sodium salt.
Potassium nitrite : Nitrous acid potassium salt.

1. Nature, history and prevalence of nitrite

Nitrite is the anion of inorganic nitrite salts, the most important of which are sodium nitrite and
potassium nitrite. It is formed naturally by the nitrogen cycle during the process of nitrogen
fixation. It is subsequently converted to nitrate, a major plant nutrient. In animal tissues,
nitrite is naturally present mainly as the result of endogenous nitrate conversion (EFSA1,
2009). The main anthropogenic sources result from the use of N-fertilizers but also from
nitrates present in animal, municipal, industrial and transport wastes (EC, 2003). Animal feed
represents a natural source of exogenous nitrite. Nitrate levels in forages are naturally high
and the inter-conversion of nitrate to nitrite is the largest contributor to nitrite exposure in
food-producing animals. Additionally, water can be an important dietary source of nitrite via
reduction of nitrate. Nitrite in drinking water is regulated in Europe (EFSA1, 2009), USA (US-
EPA1, 2009), Australia (AG, 2011) and other countries in the world. However well water
throughout the world especially contribute to nitrate-related toxicity problems (WHO2, 2003).

Toxicity is usually the result of the conversion of nitrate into the more toxic nitrite. There are
two major toxicological concerns regarding nitrite. First, nitrite may induce
methaemoglobinaemia, which can result in tissue hypoxia, and possibly death. Secondly,
nitrite may interact with secondary or N-alkyl-amides to form N-nitroso carcinogens
(Mensinga et al., 2003).

Natural occurrence of nitrite in the environment is a consequence of the nitrogen cycle.

Nitrite is formed in nature by the action of nitrifying bacteria as an intermediate stage in the
formation of nitrates (EFSA1, 2009). Nitrites in commercial use are all of synthetic origin
(WHO3, 1996). Synthetically, nitrites of the alkali earth metals can be produced by reacting a
mixture of nitrogen monoxide (NO) and nitrogen dioxide (NO2) with the corresponding metal
hydroxide solution, as well as through the thermal decomposition of the corresponding nitrate
(EFSA1, 2009).

The molecular formula of nitrite, sodium nitrite, potassium nitrite and nitrate are (NO2-),
NaNO2, KNO2 and (NO3-) respectively. The chemical structure of nitrite is shown in figure 1.

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 Figure 1. Chemical structure of nitrite

Sodium nitrite is a white to slightly yellowish crystalline powder. It is soluble in water and
slightly soluble in alcohol and is hygroscopic. It has a melting point of 270C and
decomposes above 320C. It slowly oxidizes in the air to sodium nitrate. Potassium nitrite,
with chemical formula KNO2, is a white to slightly yellow crystalline powder. The melting
point is at 441C but decomposition starts at 350C. It is very soluble in water and slightly in
alcohol (ONeil, 2006).

Sodium nitrite is used as an antimicrobial agent, a preservative and a colour fixative in meats
and fish. It is known as E250. As a food additive it stabilizes the colour of preserved fish and
meats (EFSA, 2009), especially cured meats (WHO1, 2007) and also it inhibits the growth of
Clostridium botulinum, the bacterium, which produces the botulinum toxin. Maximal residual
amounts have been set for various types of meat products. It is also used in the
manufacturing of dyes, nitroso compounds and other organic compounds. Sodium nitrite has
been used in human and veterinary medicine, as a vasodilator, a bronchodilator and as an
antidote for cyanide poisoning. In veterinary medicine the substance is intended for use
(together with other antimicrobial agents or biocides) as an antiseptic by topical application to
the teats of dairy cows after milking in order to prevent mastitis. Other applications are:
dyeing and printing of textile fabrics, bleaching of fibres, in photography, in metal coatings for
phosphatising and detinning and as an analytical reagent (EFSA1, 2009).
Potassium nitrite is used as a colour fixative in fish products and in pickling and curing meat,
sometimes in combination with sodium nitrite and with potassium and sodium nitrates. Like
sodium nitrite it inhibits the growth of the botulism-causing bacterium Clostridium botulinum.
As a permitted food additive, it is known in the EU as E249 (EFSA1, 2009).
Nitrite has been used in some countries for the preservation of fish meal submitted to heat
treatment, but is no longer permitted as it has been suspected to generate nitrosamines
when reacting with higher amines present in fish (EC, 2003). It is also known to be added as
a preservative in the production of silage (EFSA1, 2009). The WHO2 (2003) reports that other
applications of nitrite in the food industry are in cheese and cheese products, edible casings
(for e.g. sausages) and roe (eggs of marine animals). The US-EPA2 (2007) reports that
nitrates and nitrites are also used in rodenticides.

2. Transmission and likelihood of occurrence

Environment
The occurrence of nitrite in the environment is a consequence of the nitrogen cycle as shown
in figure 2.

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 Figure 2. Nitrogen cycle (source: PhysicalGeography.net)

Nitrate and nitrite are naturally occurring ions that are part of the nitrogen cycle and are
ubiquitous in the environment. Both are products of the oxidation of nitrogen (which
comprises approximately 78% of the earths atmosphere) by microorganisms in plants, soil or
water (IARC, 2010).

The atmosphere receives nitrogen mainly as air emissions of NOx, NH3, and N2O from
aquatic and terrestrial ecosystems and of NOx from combustion of biomass or fossil fuels.
NOx and NHx (NH3 andNH4+) can accumulate in the troposphere on a regional scale. But
because NOx and NHx have a short residence time in the atmosphere and lack potential to
form N2 by denitrification almost all N remitted as NOx and NHx is transferred back to Earth's
surface within hours to days (Galloway et al., 2003). Nitrogen oxide and dioxide present in
the atmosphere from combustion processes are sources of nitrite and nitrate in soils and
surface waters, delivered via acid rain. Naturally occurring nitrogen oxide and dioxide in the
atmosphere are also possible sources of nitrite (OECD, 2005).
In soil inorganic and organic nitrogen are first decomposed to give ammonia, which is then
oxidized to nitrite and nitrate (WHO1, 2007). Nitrite is formed in nature by the action of
nitrifying bacteria. Conversely, microbiological conversion of nitrate to nitrite may also occur.
Nitrite is not normally present in soils to any significant extent (EFSA1, 2009), it oxidizes
easily to nitrate (ATSDR, 2011). As stated, environmentally, the most significant source of
nitrate and the cause for much of the higher levels noted groundwater is from the nitrogen in
animal manure and fertiliser. When animal manures and fertilisers are repeatedly applied to
limited areas of land, nitrates tend to build up in the soil (Robinson, 1999; EFSA2, 2008).
Nitrite occurs in water via reduction of nitrate and can be an important source of nitrite. Its
presence in water is usually due to the excessive nitrite inter-conversion from nitrate. This
can result from water polluted by animal waste, particularly in the presence of bacterial
contamination (EFSA1, 2009). Nitrates build up in the soil leach to groundwater (Robinson,
1999; EFSA2, 2008). Nitrate is extremely soluble in water and can move easily through soil
(EFSA1, 2009). Nitrate movement into surface and ground waters is of concern both for
health and environmental quality reasons (Galloway et al., 2003). In the Merck Veterinary
Manual (Kahn, 2005) is mentioned that nitrate concentrations may be hazardous in ponds
that receive manure or fertilizer runoff; these types of nitrate sources may also
contaminate shallow, poorly cased wells.

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 Plants
Nitrogen is essential to the nutrition and function of plants, so plants exert a close metabolic
control on the concentration of nitrate and other nitrogen compounds (EFSA2, 2008). Nitrite
is not normally present in soils to any significant extent, and as a result it is not normally
available for uptake by plants. However the majority of nitrite exposure in livestock results
from the endogenous inter-conversion of nitrate in feed materials into nitrite (EFSA1, 2009).
Nitrate moves from the bulk soil to the root surface mainly by convection rather than
diffusion, so shortage of water will restrict nitrate uptake. Excess soil water dilutes the nitrate
in the soil solution and can make the soil anoxic, thereby restricting crop growth and causing
loss of nitrate by denitrification. Both environmental and agricultural factors can influence the
nitrate concentrations in vegetables. The former include soil moisture, light intensity and
temperature and the latter fertilizers, variety and crop protection strategies (EFSA2, 2008).
Excess nitrate in plants is generally associated with damp weather conditions and cool
temperatures (13C), although high concentrations are also likely to develop when growth is
rapid during hot, humid weather. Drought conditions, particularly if occurring when plants are
immature, may leave the vegetation with high nitrate content. Decreased light, cloudy
weather; and shading associated with crowding conditions can also cause increased
concentrations of nitrate within plants. Well-aerated soil with a low pH, and low or deficient
amounts of molybdenum, sulphur, or phosphorus in soil tend to enhance nitrate uptake,
whereas soil deficiencies of copper, cobalt or manganese tend to have opposing effects.
Anything that inhibits growth increases nitrate accumulation in the lower part of the plant
(Kahn, 2005).

Nitrate, which does not selectively accumulate in fruits or grain, is found chiefly in the lower
stalk with lesser amounts in the upper stalk and leaves. Nitrate in plants can be converted to
nitrite under the proper conditions of moisture, heat and microbial activity after harvesting
(Kahn, 2005).
Leaf crops such as cabbage, lettuce and spinach have fairly large nitrate concentrations
whereas storage organs such as potato tubers, carrots, leeks, onions, seeds and pods of
pea and bean plants have relatively small concentrations (EFSA2, 2008). The IARC (2010)
also report that high nitrate levels (above 1000 mg/kg) are found in celery leaves and
beetroot leaves. Other vegetables reported by Santamaria (2006) containing high nitrate
levels (1000-2500 mg/kg product) are celeriac, endive, fennel, kohlrabi, leaf chicory, leek
and parsley and very high levels (above 2500 mg/kg product) chervil, cress, radish and
chard.
The WHO4 (2003) has reported nitrate and nitrite concentrations in food, as illustrated in
table 1.
4
Table 1. Nitrate and nitrite concentrations in food (WHO , 2003).
Food Nitrate (mg/kg) Nitrite (mg/kg)
Turnip 4800 Not reported
Chinese cabbage 3500 Not reported
Celery 2900 Not reported
Kohlrabi 2700 Not reported
Radish 2100 Not reported
Spinach 1900 Not reported
Endive 1900 Not reported
Lettuce 1700 Not reported
Rhubarb 1600 Not reported
Beetroot 1500 Not reported
Eggplant 440 Not reported
Broccoli 440 Not reported
Cabbage 340 Not reported
Carrot 220 Not reported
Potato 180 0.7
Turnip 120 Not reported
Sweet pepper 120 Not reported
Onion 110 Not reported
Cauliflower 85 Not reported

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 Food Nitrate (mg/kg) Nitrite (mg/kg)
Grape 70 Not reported
Shallot 65 Not reported
Brussels sprouts 35 Not reported
Garlic 35 Not reported
Apple 30 Not reported
Asparagus 30 Not reported
Pear 20 Not reported
Tomato 15 Not reported
Cereals 10 2.6

More than 80 plant species, which include a number of common agricultural crops that have
been associated with nitrite poisoning, can accumulate nitrate to high levels under extreme
conditions (EFSA1, 2009). These include grains (especially oats, millet, maize, sorghum
and rye) and sunflower. Some weeds can also contain high nitrate levels: pigweed, lambs
quarter, thistle, Datura stramonium, fireweed, smartweed, dock weed and Johnson
grass (Kahn, 2006). In the GMP+ Monitoring database nitrite data indicate that safflower
products (e.g. extracted safflower seeds) and grain distillers may contain high levels of
nitrite.
Nitrate levels in forages (e.g. hay) and silages are naturally high. Also sodium nitrite, often
in combination with other products, can be used in silages (maize, grasses, legumes,
wheat and alfalfa) as an antimicrobial for preservation, aiming at the suppression of C.
botulinum (EFSA1, 2009) and other micro flora present in the silage (Knicky and Sprndly,
2011).

Because of this range of factors and particularly the impact of fertiliser application rates,
levels of nitrate in forage crops can vary widely, but normally remain within safe ranges. To
illustrate the impact for common feeds and forages of different fertiliser application rates and
harvesting regimens, resultant nitrate levels are shown in table 2.

Table 2. Nitrate concentration in vegetal feed materials after different fertiliser or harvesting regimens (adapted
from Cash et al., 2007).
Feed material Harvesting regime Fertiliser Nitrate (mg/kg dry
application rate matter)
Oat hay Grain at soft dough 67 kg/ha 2149
Oat hay Grain at soft dough 134 kg/ha 5613
Barley hay Grain at soft dough 67 kg/ha 868
Barley hay Grain at soft dough 134 kg/ha 2627
Alfalfa First crop, vegetative Not reported 1800
Alfalfa Second crop, vegetative Not reported 3200
Alfalfa Silage Not reported 880
Maize Fresh, chopped, before ensiling Not reported 4400
Maize Silage, after ensiling Not reported 2200
Soybean meal - Not reported 4

Levels of nitrate and nitrite in raw agricultural commodities can be influenced by a number of
factors such as storage time and conditions (i.e. ambient, refrigerated, frozen), and food
processing (i.e. washing, peeling, blanching, boiling (EFSA2, 2008) and ensiling).

In the GMP+ Monitoring database data can be found concerning nitrite in feed materials of
vegetal origin.

Overall nitrite data in food and feed materials are scarce.

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 Animals
Exposure:
Animal feed can contain nitrite from its natural plant constituents (generally very low levels),
from microbial action in moist feeds/stored plant materials e.g. hay and silage, or when nitrite
is added directly as a preservative. Nevertheless, the majority of nitrite exposure in livestock
results from the endogenous inter-conversion of nitrate in feed and water into nitrite (EFSA1,
2009).
Dietary intake of nitrite per se results from vegetable feeds and forages, fertilisers / manures
or when nitrite is used as an antimicrobial for preservation, for example in silage and in
former times fish meal. Additionally, water can be an important dietary source of nitrite via
reduction of nitrate. Forages are naturally high and the inter-conversion of nitrate to nitrite is
the largest contributor to nitrite exposure in food-producing animals (EFSA1, 2009).

The EFSA1 (2009) estimated the nitrite exposure of monogastric food producing animals
from feed and water given a diet containing the maximum permitted sodium nitrite
concentration and water at the EU maximum limit value, as shown in table 3.

Table 3. Estimated exposure of monogastric food producing animals to nitrite from feed and water given a diet
containing the maximum permitted sodium nitrite concentration (15 mg/kg) expressed as nitrite ion (10 mg/kg)
1
and water at the EU maximum limit value (EFSA , 2009).
Live Consumption Nitrite intake from
1 2
weight Complete feed Water Complete feed Water
Species kg kg/day l/day mg/kg b.w./day
Pigs 100 3.7 10 0.37 0.05
Sows 250 6.5 25 0.26 0.05
Poultry (boilers) 2.1 0.15 0.02 0.71 0.001
Poultry (laying hens) 1.9 0.115 0.02 0.61 0.001
1 2
Nitrite in total complete feed assumed equal to 10 mg/kg; Water contribution using the EU maximum limit value
of 0.5 mg/L is given for comparative purposes.

Table 4 illustrates the estimated exposure of ruminants to nitrite given compound feed
containing the maximum permitted nitrite concentration (10 mg/kg), forage at the maximum
value (26.2 mg/kg), as reported to the EFSA by Slovenia and water at the EU maximum
value for nitrite.

Table 4. Estimated exposure of ruminants to nitrite from feed and water where the diet contains the maximum
permitted sodium nitrite concentration (15 mg/kg) in compound feed and the maximum reported concentration in
forage, expressed as nitrite. These calculations assume typical livestock feed and represent a worst-case
1
scenario (EFSA , 2009).
Species Live Consumption Nitrite intake from
weight Forages Compound Water Forages Compound Water
feed feed
kg kg/day kg/day l/day mg/kg b.w./day
Dairy cow 625 14.0 10.0 120 0.59 0.18 0.1
Cattle 300 7.0 1.0 30 0.61 0.04 0.05
Lactating ewe 70 1.5 0.3 15 0.56 0.05 0.11
Growing lamb 20 0.45 0.15 5 0.59 0.09 0.13
Dairy goat 65 1.5 0.7 15 0.6 0.12 0.12

In table 5 the total estimated nitrite exposure from feed (incl. forages) of monogastric animals
and ruminants, (modified from EFSA1, 2009) is shown, using the data presented in tables 3
and 4.

Table 5. Total estimated nitrite exposure from feed (incl. forages) and water of monogastric animals and
1
ruminants, (modified from EFSA , 2009), using the data presented in tables 2 and 3.
Nitrite intake from Total
Forages Other feed Total feed Water exposure
Species mg/kg b.w./day
Pigs - 0.37 0.37 0.05 0.42
Sows - 0.26 0.26 0.05 0.31

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 Nitrite intake from Total
Forages Other feed Total feed Water exposure
Species mg/kg b.w./day
Poultry (boilers) - 0.71 0.71 0.001 0.71
Poultry (laying hens) - 0.61 0.61 0.001 0.61
Dairy cow 0.59 0.18 0.77 0.1 0.87
Cattle 0.61 0.04 0.65 0.05 0.7
Lactating ewe 0.56 0.05 0.61 0.11 0.72
Growing lamb 0.59 0.09 0.68 0.13 0.81
Dairy goat 0.6 0.12 0.72 0.12 0.84

Exposure to nitrite in grazing animals may also arise from soil ingestion (EFSA1, 2009).
However to what extent is not known.
Nitrite has been used as an additive for preservation of animal feed, in particular fish meal
and forages. In the EU in 1993/94 the use of sodium nitrite as a preservative was forbidden
in fish meal because it has been suspected to generate toxic nitrosamines when reacting
with the amines present in fish. Today, preservation of fish caught at sea is achieved by a
combination of the use of refrigerated seawater and acetic acid. The EFSA1 (2009) states
that in the absence of exogenously applied nitrite for preservation, the levels in fresh fish and
derived fish meals are likely to be low. In the EU still maximum nitrite levels are stated for fish
meal. Limited data are available on current levels (EFSA1, 2009). Recent fish meal data in
the GMP+ Monitoring database indicate nitrite levels below detection limits and below the in
the EU stated maximum. Based on the history of the use of sodium nitrite as a preservative,
its prohibition in fish meal in the EU and the current EU legislation fish meal is for the
moment indicated as a nitrite risk product.
Nitrate can be used as an additive in the manufacture of cheese to prevent fermentative
reactions. Cheese itself should not be a potential risk, since 67% of the nitrate and nitrite
added to milk are leached out to the whey. Spray-drying could lead to increased nitrate and
nitrite levels and also to the formation of nitrosamines (Oliveira et al., 1995). Consequently
whey-based products, especially dried whey-based products are of risk containing
hazardous levels of nitrate and nitrite.

Absorption:
The rate of absorption of nitrite and nitrate varies between species being relatively high in
humans and rats but it is lower in ruminants and both are quantitatively well absorbed from
the stomach and upper intestine/rumen. After transport to the stomach, the acidic conditions
will rapidly transform nitrite to nitrous acid, which in turn spontaneously decomposes to
nitrogen oxides including nitric oxide (EFSA1, 2009).
In monogastric animals, most nitrite is formed and absorbed in the upper digestive tract
(EFSA1, 2009). They convert nitrate to nitrite in the intestine closer to the end of their
digestive tracts with a minor absorption of nitrite. In contrast in ruminants nitrate is converted
to nitrite by bacteria in the rumen (Yaremcio, 1991). The nitrate pathway in ruminants is
shown in figure 3.

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 BLOOD
RUMEN STREAM

SALIVA Nitrate Nitrate

Feed nitrate
Nitrite Hemoglobin

Ammonia Methemoglobin

Ammonia

KIDNEY

Urea Urine

Figure 3. Nitrate pathway in ruminants (source: Yaremico, 1991)

In ruminants about 20% of nitrite and nitrate present in the rumen pass into the blood stream
as nitrite, while the rest of the nitrite and nitrate is metabolized by the microorganisms to
ammonia. The ammonia is utilized by rumen micro flora or eliminated with other gases during
eructation and the remainder is absorbed in considerable quantities directly into the blood
(Lewis, 1951; Wang et al., 1961). Absorbed nitrate can also be recycled into the rumen via
saliva (Yaremcio, 1991). Salivary, biliary, pancreatic or direct intestinal secretions of nitrate
may also contribute to the pool of nitrate present in the small and large intestines as was
observed in rats (Witter et al., 1979).
When high levels of nitrate are consumed (e.g. from contaminated water), normal microbial
conversion of nitrate to nitrite and assimilation post-absorption can be overwhelmed resulting
in excessive nitrite absorption and resultant methaemoglobin (MetHb) formation (EFSA1,
2009).

Distribution:
Once absorbed, nitrite is rapidly distributed in the plasma with rapid binding to the
erythrocytes (Jensen, 2005), which results in MetHb formation (Clark and Litchfield, 1973).
Due to the rapid metabolic turnover and excretion of nitrite and nitrate, the potential for
accumulation in animal tissues is low (EFSA1, 2009).
Concerning transfer to foetuses the OECD (2005) states that there is evidence for transfer of
sodium nitrite to foetuses in rats and mice. Concerning eggs, Lee et al. (1975) found that
increasing levels of sodium nitrate in drinking water resulted in increasing levels of nitrate in
the egg yolk and albumen of laying hens.

Metabolism:
Nitrate undergoes a number of metabolic inter-conversions, and is recycled between the
saliva and the gut and the bile and the gut (EFSA2, 2008). After binding to plasma and
erythrocytes, nitrite, when present at normal levels is metabolised to nitrate, which is then
metabolised to nitric oxide (EFSA1, 2009). In figure 3 the nitrate pathway in ruminants is
shown. If nitrite production is so high that rumen microorganisms, breaking it down to
ammonia, are overwhelmed, it will accumulate in the rumen, pass through the rumen wall to
the blood where it combines with haemoglobin (Hgb), by substituting for oxygen, to create
MetHb (Robinson, 1999). The formation of MetHb has also been observed in pig
erythrocytes (Jensen, 2005).
Micro-organisms of the alimentary tract play a significant role in the metabolism of nitrate as
has been demonstrated in experiments with germ-free (monogastric) rats which excreted in

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 urine approximately 20 % more nitrate than conventional rats (Schultz et al., 1985).

Excretion:
Nitrite is rapidly and extensively excreted in urine. However, nitrate has a slower rate of
excretion and the major part of the primary urinary nitrate (about 80 %) is pumped back to
the blood by an active transport mechanism (EFSA1, 2009). In ruminants nitrate is converted
to nitrite and ammonia. Excess ammonia is excreted as urea via urine (Yaremico, 1991).
Grner et al. (1973) found that suckling rats whose dams received nitrites in their drinking
water showed no rise in MetHb levels. By contrast, the dams showed high MetHb levels,
indicating that nitrites are not transferred in appreciable amounts to suckling rats via the milk.

Humans via animal products

The SCAN (Scientific Committee on Animal Nutrition) reports that no data were available
concerning either the endogenous nitrite contents of animal products or the conversion and
transfer rate of nitrates and nitrites through the animal food chain. However, the fast
excretion in urine of the absorbed nitrate and nitrite ions and their non-cumulative character
allows the conclusion that no bioaccumulation occurs in animal tissues and products (EC,
2003). In 2009 the EFSA1 also reported that although overall data on the carry-over and
residues of nitrite in animal tissues and animal-based products are very scarce. However,
because of the rapid excretion of nitrite and nitrate, the likelihood of accumulation in animal
tissues and animal-based products is considered to be low.

Cockburn et al. (2013) concluded that the potential health risk to humans from eating fresh
animal products containing nitrite is very low.

Since in several references is mentioned that the likelihood of accumulation of nitrite

and nitrate in animal tissues and animal-based products is considered to be low or is
concluded that no bioaccumulation occurs in animal tissues and products the
paragraphs humans will not be addressed to in the chapters below.
Mind that nitrite can be present in products of animal origin as a consequence of the use as
additive (e.g. in cured meat), however this route of exposure in not addressed to in this fact
sheet.

In the GMP+ Monitoring database can be found concerning nitrite in feed materials of animal
origin.

Humans
Exposure:
Not addressed to, see chapter 2, paragraph humans via animal products.

Absorption:
Not addressed to, see chapter 2, paragraph humans via animal products.

Distribution:
Not addressed to, see chapter 2, paragraph humans via animal products.

Metabolism:
Not addressed to, see chapter 2, paragraph humans via animal products.

Excretion:
Not addressed to, see chapter 2, paragraph humans via animal products.

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 3. Diagnose of poisoning

Animals
Excess nitrate exposure can be assessed by laboratory analysis for nitrate in both pre- and
post-mortem specimens. Plasma is the preferred pre-mortem specimen, because some
plasma-protein-bound nitrate could be lost in the clot if serum was collected. Nitrite present in
whole blood also continues to react with haemoglobin in vitro, so these specimens must be
centrifuged immediately and plasma. Additional post-mortem specimens from either
toxicoses or abortions include ocular fluids, foetal pleural or thoracic fluids, foetal stomach
contents, and maternal uterine fluid. All specimens should be frozen in clean plastic or glass
containers before submission, except when whole blood is collected. Because the amount of
nitrate in rumen contents is not representative of concentrations in the diet, evaluation of
rumen contents is not indicated (Kahn, 2005).
Nitrite reacts with haemoglobin. In acute toxicosis MetHb analyses can be used. For non-
acute cases this alone is not a reliable indicator of excess nitrate or nitrite exposure because
50% of MetHb present will be converted back to haemoglobin in about 2 hours (Kahn, 2005).
Nitrate and nitrite concentrations >20 g NO3 /mL and >0.5 g NO2 /mL, respectively, in
maternal and perinatal serum, plasma, ocular fluid, and other similar biologic fluids are
usually indicative of excessive nitrate or nitrite exposure. Nitrate concentrations of up to 40
g NO3/mL have been present in the plasma of healthy calves at birth, but are reduced
rapidly as normal neonatal renal function eliminates nitrate in the urine. Normally expected
nitrate and nitrite concentrations in similar diagnostic specimens are usually <10 g NO 3/mL
and <0.2 g NO2/mL, respectively. Nitrate and nitrite concentrations >10 but <20 g NO3/mL
and >0.2 but <0.5 g NO2/mL, respectively, are suspect and indicate nitrate or nitrite
exposure of unknown duration, extent, or origin. The possible contribution of endogenous
nitrate or nitrite synthesis must also be considered (Kahn, 2005).

Also history of exposure and analysis of feed can be included.

Humans
Not addressed to, see chapter 2, paragraph humans via animal products.

4. Potential adverse effects

Environment
No data were studied.

Animals
Pigs and ruminants are particularly susceptible to nitrite toxicity, based on their respective
physiology (EFSA, 2009; Cockburn et al., 2013).
With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence a
reduced potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb
back to haemoglobin due to intrinsically low levels of MetHb reductase. In cattle the
sensitivity is due to the potential for high dietary intake and high levels of rumen conversion
of nitrate to nitrite, and an adaptable gut flora, which at normal loadings shunts nitrite to
ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite
builds up and can enter the blood stream resulting in methemoglobinemia (Cockburn et al.,
2013). Nitrate reduction (and nitrite production) occurs in the cecum of equids but not to the
same extent as in ruminants (Kahn, 2005).
Also free-range animals exposed to high levels of nitrate and/or nitrite via the ingesting
contaminated soil or via contaminated drinking water (ponds, groundwater, surface water)
might be susceptible for nitrite toxicosis.

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 Toxicity is usually the result of the conversion of nitrate into the more toxic nitrite (Mensinga
et al., 2003) with nitrite being about 10 times more toxic than nitrate (Kahn, 2005). There are
two major toxicological concerns regarding nitrite. First, nitrite may induce
methemoglobinaemia, which can result in tissue hypoxia (lack of oxygen in tissue), and
possibly death. Secondly, nitrite may interact with secondary or N-alkyl-amides to form N-
nitroso carcinogens (Mensinga et al., 2003). The severity of the intoxication from nitrite
exposure also depend on other factors such as dietary status, sudden changes in diet
composition (i.e. from grains to leafy forages) or the presence of metal ions and sulphur
compounds in the diet that may enhance/inhibit the reduction of nitrate to nitrite (EFSA1,
2009).

Although usually acute, the effects of nitrite or nitrate toxicity may be sub-acute or chronic
and are reported to include retarded growth, lowered milk production, vitamin A deficiency,
abortions (Kahn, 2005) (correlated with foetal hypoxia due to methemoglobinemia (EFSA1,
2009)), fetotoxicity, and increased susceptibility to infection (Kahn, 2005). The EFSA1 (2009)
also reports vitamin E reductions and reduced fertility.

Two year carcinogenicity studies (rat and mice) for sodium nitrite were conducted under the
National Toxicology Programme (NTP, 2001). In females, the authors concluded that there
was equivocal evidence for carcinogenic activity in the forestomach. The term equivocal
evidence of carcinogenic activity is defined in NTP as studies that are interpreted as showing
a marginal increase of neoplasms that may be chemical related.
A causative connection between nitrate/nitrite and cancer through the formation of N-nitroso
compounds is suspected (WHO3, 1996). The link between cancer risk and endogenous
nitrosation as a result of high intake of nitrate and/or nitrite and nitrosatable compounds is
discussed in other publications (WHO2,5, 2003, EFSA2, 2008).

In Appendix I the potential adverse effects of nitrite in animals are shown. For toxicity data,
see Appendix II.

Humans
Not addressed to, see chapter 2, paragraph humans via animal products.

5. Severity of the potential adverse effects

The severity of contamination of food and / or feed materials with nitrite is based on the worst
case scenario, as shown in table 6, and is based upon the potential adverse effects stated in
chapter 4 and Appendix I.

The severity of nitrite toxicosis in animals is classified as high because:

Nitrite may induce methemoglobinaemia, which can result in death (Mesinga et al.,
1983);
Reproductive effects were observed: abortions (Kahn, 2005), reduced fertility (EFSA1,
2009);
Nitrite may interact with secondary or N-alkyl-amides to form N-nitroso carcinogens
(Mesinga et al., 1983; WHO2,5,2003; EFSA2, 2008).

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 The severity of nitrite toxicosis in humans is not classified, see chapter 2: paragraph
humans via animal products.

Table 6. Severity of nitrite toxicosis

Severity
Low Medium High
Animals x
Humans Not classified

6. Legislation and standards

No data were studied.

7. Methods of analysis
Sampling: In GMP+ standards BA4 and BA13 requirements concerning sampling are stated
(GMP+1,2, 2013).

Analysis: No official analytical method for the determination of nitrite in animal feed is
prescribed in Commission Regulation (EC) No. 152/2009, laying down the topical methods of
sampling and analysis for he official control of feed in the EU. The European Committee of
Standardization (CEN) has elaborated a number of standards for the determination of nitrate
and/or nitrite content of vegetables, vegetable products, including vegetable containing food
for babies and infants as well as in meat and meat products but no specific methods for the
determination of nitrite in feed were established by CEN (EFSA1, 2009).
Chou et al. (2003) report that a variety of analytical methods for the determination of nitrate
and nitrite have been developed and applied to the analysis of food, water, plants and other
matrices. Besides spectrophotometry, High Performance Liquid Chromatography (HPLC),
Ion Chromatography (IC), Gas Chromatography (GC), polarographic methods and Capillary
Electrophoresis (CE).
The official method of analysis of the Association of Official Analytical Chemists (AOAC) for
the determination of nitrate and nitrite in animal feed is a colorimetric method (EFSA1, 2009).
The Product Board Animal Feed in the Netherlands describes the spectrophotometric
method. Two samples are prepared. One in which nitrate is reduced to nitrite followed by the
quantification of the nitrite content. The other sample, without reduction, is used to quantify
the nitrite content originally present. The nitrate content is calculated by the difference
between the sample with and without reduction (PDV, 1997). Moorcroft et al. (2001)
concluded that spectrophotometric methods lack a high sensitivity for the detection of trace
levels of the analytes could cause results to be unreliable due to sample matrix interferences.
A number of IC and HPLC methods have been developed, which are generally characterized
by faster, more accurate and higher sensitivity than the spectrophotometric methods. The
overall conclusion by Moorcroft et al. (2001) was that the use of spectroscopic analysis
appears to be the method of choice due to the simplicity of the protocols and the wide
availability of the instrumentation involved. The increasing demand for rapid on-site analysis
will ensure the continued development of both spectroscopic and electrochemical methods,
which are more applicable to miniaturisation and remote operation.

Field tests for nitrate are presumptive and should be confirmed by standard analytical
methods at a qualified laboratory. The diphenylamine blue test is more suitable to determine
the presence or absence of nitrate in suspected forages. Nitrate test strips (dipsticks) are
effective in determining nitrate values in water supplies and can be used to evaluate nitrate
and nitrite content in serum, plasma, ocular fluid, and urine (Kahn, 2005).

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 8. Possible control measures
The HACCP system should be used to assess the risk of nitrite/nitrate in feed and
consequently the control measures needed to control this risk. All parts in the feed and food
chain should be included, starting from cultivation or husbandry. These specific control
measures are included in the appendices:
Cultivation, harvesting, processing, transport and storage on farm, see Appendix III;
Husbandry, see Appendix IV;
Transport, see Appendix V;
Storage, see Appendix VI;
Processing of feed, see Appendix VII.

The possible control measures should focus on products with a higher risk to contain
nitrite/nitrate as summarised in table 7. It should be kept in mind that also products derived
from the risk products mentioned are also of risk. Examples of derived products are given in
table 7, however these examples are not exhaustive. Other derived products can also be of
risk.

Table 7. Products with a higher risk to contain nitrite/nitrate.

Nitrate and/or nitrite contaminated drinking water (incl. ground water, surface water, ponds
Products in

that, shallow and poorly cased wells) that receive manure or fertilizer runoff;
general

Soil contaminated with nitrate and/or nitrite via manure or fertilizer;

Products where during processing nitrate and/or nitrite containing additives are used or
are added directly to the product;

Forages (e.g. hay) and silages;

Grains e.g. oats, millet, sorghum, maize and rye);
Specific products of

Sunflower seeds;
vegetal origin

Safflower seeds (e.g. extracted safflower seed);

Weeds (e.g. pigweed, lambs quarter, thistle, Datura stramonium, fireweed, smartweed,
dock weed and Johnson grass);
Products where during processing nitrate and/or nitrite containing additives are used are added
directly to the product, e.g. silages (maize, grasses, legumes, wheat and alfalfa);
Leaf crops such as cabbage, lettuce, spinach, celery leaves, beetroot leaves, celeriac,
endive, fennel, kohlrabi, leaf chicory, leek, parsley, chervil, cress, radish and chard

Products where during processing nitrate and/or nitrite containing additives are used are added
of animal
products
Specific

directly to the product, e.g. whey-based products (especially dried whey products);
origin

Fish meal.

Pigs and ruminants are particularly susceptible to nitrite toxicity. Also free-range animals
exposed to high levels of nitrate and/or nitrite via the ingesting contaminated soil or via
contaminated drinking water (ponds, groundwater, surface water) might be susceptible for
nitrite toxicosis.

9. References
1. AG (Australian Government), Australian Drinking Water Guidelines 6, National Water
Quality Management Strategy, 2011
2. ATSDR (Agency for Toxic Substances and Disease Registry), Nitrates and Nitrites,
ToxFAQsTM, 2011
3. Cash et al., Nitrate Toxicity of Montana Forages, MontGuides 200205, 2006

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Version: 09/01/2015 GMP+ International B.V.
 4. Chou et al., A High Performance Liquid Chromatography Method for Determining Nitrate
and Nitrite Levels in Vegetables, Journal of Food and Drug Analysis, Volume 11(3),
2003, pages 233-238
5. Clark and Litchfield, Role of inorganic nitrite in methaemoglobin formation after nitrate
ester administration to the rat, British Journal of Pharmacology, Volume 48, 1971, pages
162-168
6. Cockburn et al., Nitrite in feed: From animal health to human health, Toxicology and
Applied Pharmacology, Volume 270(3), 2013, pages 209-217
7. EC (European Commission), Opinion of the Scientific Committee on Animal Nutrition on
Undesirable Substances in Feed (SCAN), adopted on 20 February 2003, updated on 25
April 2003, 2003
8. EFSA1, Nitrite as undesirable substances in animal feed, Scientific Opinion of the Panel
on Contaminants in the Food Chain, The EFSA Journal 1017, 2009
9. EFSA2, Nitrate in vegetables - Scientific Opinion of the Panel on Contaminants in the
Food Chain, The EFSA Journal 689, 2008
10. Galloway et al., The Nitrogen Cascade, BioScience, Volume 53(4), 2003, pages 341-356
11. GMP+1 International, Minimum requirements for sampling and analysis, GMP+ BA4,
2013
12. GMP+2 International, Minimum requirements for sampling, GMP+ BA13, 2013
13. GMP+3 International, GMP+ B6, Feed materials cultivation, GMP+ Feed Safety
Assurance scheme, 2013
14. GMP+4 International, GMP+ B1: Production, Trade and Services, GMP+ Feed Safety
Assurance scheme, 2013
15. GMP+5 International, GMP+ B4, Transport, GMP+ Feed Safety Assurance scheme, 2013
16. GMP+6 International, GMP+ B4.1, Road transport, GMP+ Feed Safety Assurance
scheme, 2013
17. GMP+7 International, GMP+ B4.2, Affreightment of short sea shipping and inland
waterway transport, GMP+ Feed Safety Assurance scheme, 2013
18. GMP+8 International, GMP+ B4.3, Short sea shipping and inland waterways transport,
GMP+ Feed Safety Assurance scheme, 2013
19. GMP+9 International, GMP+ B4.4, Sea transport and affreightment, GMP+ Feed Safety
Assurance scheme, 2013
20. GMP+10 International, GMP+ B4.5, Rail transport and affreightment, GMP+ Feed Safety
Assurance scheme, 2013
21. GMP+11 International, GMP+ B3, Trade, Collection and Storage & transhipment, GMP+
Feed Safety Assurance scheme, 2013
22. GMP+12 International, GMP+ B2, Quality control of feed materials, GMP+ Feed Safety
Assurance scheme, 2013
23. Grner et al., Methemoglobinemia induced by transplacental passage of nitrites in rats,
Bulletin of Environmental Contaminants & Toxicology, Volume 9(1), 1973, pages 44-48
24. IARC (International Agency for Research on Cancer), Ingested Nitrate and Nitrite and
Cyanobacterial Peptide Toxins, IARC Monographs on the Evaluation of Carcinogenic
Risks to Humans, Volume 94, 2010, pages 45-109
25. Jensen, Nitrite transport into pig erythrocytes and its potential biological role, Acta
Physiologica Scandinavia, Volume 184(3), 2005, pages 243-251
26. Kahn (ed.), The Merck Veterinary Manual, Ninth edition, 2005
27. Knicky and Sprndly, The ensiling capability of a mixture of sodium benzoate, potassium
sorbate, and sodium nitrite, Journal of Dairy Science, Volume 94(2), 2011, pages 824-
831
28. Lee et al., The influence of dietary nitrate on nitrate concentration in egg yolk and
albumen, Poultry Science, Volume 54(2), 1975, pages 475-478
29. Lewis, The metabolism of nitrate and nitrite in the sheep, The reduction of nitrate in the
rumen of the sheep, Biochemical Journal, Volume 48(2), 1951, pages 175-180
30. Mensinga et al., Health implications of exposure to environmental nitrogenous
compounds, Toxicological Reviews, Volume 22(1), 2003, pages 41-51

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Version: 09/01/2015 GMP+ International B.V.
 31. Moorecroft et al., Detection and determination of nitrate and nitrite: a review, Talanta,
Volume 54, 2001, pages 785-803
32. NTP (National Toxicology Programme), Toxicology and carcinogenesis studies of sodium
nitrite (CAS NO. 7632-00-0) in F344/N rats and B6C3F1 mice (drinking water studies).
NTP TR 495, 2001
33. OECD (Organisation for Economic Co-operation and Development), Sodium nitrite, SIDS
Initial Assessment Report for SIAM 20, Chemical Screening Information Dataset (SIDS),
United Nations Environment Programme (UNEP), 2005
34. Oliveira et al., Nitrate, Nitrite, and Volatile Nitrosamines in Whey-Containing Food
Products, Journal of Agricultural and Food Chemistry, Volume 43(4), 1995, pages 967-
969
35. ONeil (ed.), The Merck Index, Fourteenth edition, 2006
36. Oude Elferink et al., Silage fermentation processes and their manipulation, In: Silage
making in the tropics with particular emphasis on smallholders, FAO Plant Production
and Protection Paper 161, Paper 2.0, 2000
37. PDV (Productschap Diervoeder), Bepaling van nitriet- en nitraatgehalte, Bundel
Onderzoeksmethoden Diervoeder Deel II, OSP-7-1, 1997
38. Robinson, Nitrates and dairy cattle: cause for concern? Cooperative extension University
of California, Davis, 1999
39. Santamaria, Review: Nitrate in vegetables: toxicity, content, intake and EC regulation,
Journal of the Science of Food and Agriculture, volume 86, 2006, pages 10-17
40. Schultz et al., Pharmacokinetics of nitrate in humans: role of gastrointestinal absorption
and metabolism, Carcinogenesis, Volume 6(6), 1985, pages 847-852
41. Seerley et al., Effect of nitrate or nitrite administered continuously in drinking water for
swine and sheep, Journal of Animals Science, Volume 24(4), 1965, pages 1014-1019
42. US-EPA1, National Primary Drinking Water Regulations, 2009
43. US-EPA2, Nitrates and nitrites, TEACH Chemical Summary, 2007
44. Wang et al., Metabolism of nitrate by cattle, Biochemical Journal, Volume 81(2), 1961,
pages 237-242
45. WHO1, Nitrate and nitrite in drinking-water, Background document for development of
WHO Guidelines for Drinking-water Quality, 2007
46. WHO2, Nitrite (and potential endogenous formation of N-nitroso compounds), WHO Food
Additives Series, No. 50, 2003
47. WHO3, Nitrates and nitrites, Poisons Information Monograph (PIM), No. G016, 1996
48. WHO4, Nitrate and nitrite: intake assessment, WHO Food Additives Series, No. 50, 2003
49. WHO5, Nitrite, Summary of evaluations performed by the Joint FAO/WHO Expert
Committee on Food Additives (JECFA), 2003
50. WHO6, Sodium nitrite, Summary of evaluations performed by the Joint FAO/WHO Expert
Committee on Food Additives (JECFA), 2005
51. WHO7, Potassium nitrite, Summary of evaluations performed by the Joint FAO/WHO
Expert Committee on Food Additives (JECFA), 2005
52. Witter et al., Distribution of nitrogen-13 from labelled nitrate and nitrite in germfree and
conventional-flora rats, Applied Environmental Microbiology, Volume 38(5), 1979, pages
870-878
53. Yaremico, Nitrate poisoning and feeding nitrate feeds to livestock, Agdex 400/60-1, 1991

10. Websites
1. http://www.efsa.europa.eu/en/efsajournal/doc/1017.pdf
2. http://www.efsa.europa.eu/en/efsajournal/doc/689.pdf
3. http://www.efsa.europa.eu/en/efsajournal/doc/1538.pdf
4. http://www.inchem.org/documents/jecfa/jecmono/v50je07.htm
5. http://www.inchem.org/documents/jecfa/jecmono/v50je06.htm
6. http://www.inchem.org/documents/jecfa/jecmono/v50je05.htm
7. http://www.inchem.org/documents/pims/chemical/pimg016.htm

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 8. http://monographs.iarc.fr/ENG/Monographs/vol94/mono94-6A.pdf

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 APPENDIX I Potential adverse effects of nitrite

1,2 1,2 1,2 1,2 2 2 2

Death Carcinogen Mutagen Reproductive Internal injury Neurological Immunological Organs
(physical
1,2
contamination)
Animals
x x x x
Humans Not addressed to, see chapter 2, paragraph humans via animal products.

2 2 2 2
Dermal and Respiratory Musculo- Cardiovascular Gastrointestinal Hematological Endocrine Bodyweight
2 2
ocular skeletal
Animals
x x
Humans Not addressed to, see chapter 2, paragraph humans via animal products.

1
This potential adverse effect is classified as high severity for animals
2
This potential adverse effect is classified as high severity for humans

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 APPENDIX II Toxicity data of nitrite and nitrate

NOAEL (mg/kg BW) ADI (mg/kg BW) LD50 (mg/kg BW)

2 2 3
Nitrite 6.7 (rat) (EFSA , 2008; 0-0.07: expressed as 57-157 (rodents) (WHO ,
2 A 5
WHO , 2003) nitrite ion (WHO , 2003) 1996)
4
3.3 (swine) (derived from
Seerley et al., 1965 by
1
EFSA , 2009)
1
Sodium nitrite 190 (mice) (NTP, 2001) 0-0.06: expressed as
A 6
nitrite ion (WHO , 2005)
1
55 (rat) (NTP, 2001)
2 2
10 (rat) (EFSA , 2008;
2
WHO , 2003)
Potassium nitrite 0-0.06: expressed as
A 7
nitrite ion (WHO , 2005)
B 3
Nitrate 160 (transposed ADI) 1.2-6.6 g/kg BW (WHO ,
1
(dog) (EFSA , 2009) 1996)
3
Nitrate estimated to 3.3 (cattle, horse)
1 1
nitrite (EFSA , 2009) (EFSA , 2009)
A
ADI applies to all sources of intake but not to infants below the age of 3 months.
B 2
Transposed ADI = ADI based on Lehman (1958) study (cited in EFSA , 2008), reflecting the conversion of
nitrate to nitrite in the saliva. Transposed ADI = ADI applied by an uncertainty factor of 50 for normal converters
1
(EFSA , 2009).
1
during 14 weeks. NOAEL for reproductive effects.
2
during 2 years, NOAEL for heart and lung toxicity.
3
Estimated from feed exposure to nitrate using a 10:1 ratio for nitrite:nitrate ratio.
4
during 105 days, NOAEL for haematological effects (MetHb). A safety factor of 3 is applied to the LOAEL to
1
derive the NOAEL value (EFSA , 2009).

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 APPENDIX III Nitrite/nitrate control measures: cultivation, harvesting, processing,
transport and storage of feed on farm.
Specific requirements and control measures related to feed ingredient cultivation are stated
in the GMP+ B6 standard (GMP+3, 2013). This standard should be complied with. Additional
recommendations for general control measures are stated below in this appendix.

Cultivation, harvesting, processing, transport and storage on farm

Use the HACCP system to assess the risk of nitrite/nitrate in feed. Besides others, the
following items should be addressed:
Minimise nitrogen fertilising. Good Agricultural Practices (GAP) schemes with regard to
nitrate were developed to help farmers to the European nitrate regulations. A range of
different GAP schemes operate within the EU Member States, each of which takes into
account the particular climatic conditions in that Member State. All the schemes focus on
abiotic factors shown to have a significant effect on nitrate concentrations in plants. First
among these in almost all GAP schemes is adequate light intensity, particularly for
vegetables grown under glass or plastic sheeting. GAP for nitrogen nutrition also aims to
minimize 'untimely nitrate', that is, nitrate that is in the soil when it is not needed by the
crop (EFSA2, 2008);
Raising cutter heads of machinery during harvesting operation selectively leaves the
more hazardous stalk bases in the field (Kahn, 2006);
Make sure that food producing animals do not have access to nitrate and/or nitrite
contaminated drinking water (e.g. ponds, surface water);
Do not transport water in improperly cleaned liquid fertilizer tanks as they may be
extremely high in nitrate (Kahn, 2006);
Apply ensiling methods that induce a rapid and sufficient drop in silage pH will therefore
help to decrease enterobacterial growth. A special characteristic of enterobacteria is their
capability to reduce nitrate to nitrite under silage conditions (Oude Elferink et al., 2000);
When using sodium nitrite in silages, keep in mind that nitrate and/or nitrite levels can be
high. Before feeding new silage nitrate and nitrite concentration should be determined;
Use animal feed low in nitrate and nitrite, see table 7 for feed of risk;
Feeding high-nitrate hay, straw or other forage that has been damp or wet for several
days, or stockpiled green-chopped forage should be avoided. Large round bales with
excess nitrate are especially dangerous if stored uncovered outside; rain or snow can
leach and subsequently concentrate most of the total nitrate present into the lower third
of these bales (Kahn, 2006).

Nitrite/nitrate monitoring programs:

Cultivation, harvesting, processing, transport and storage on farm

Implement nitrite/nitrate monitoring programs, as stated hereafter:

Concerning nitrite/nitrate monitoring programs for the transport and storage of feed is
referred to appendices V and VI respectively;
Implement a nitrite/nitrate monitoring program on the following products or critical control
points, which were determined of risk by the HACCP study: e.g. sowing seeds, soil,
fertilizer, pesticides, manure, compost materials, residues and spills in specific areas,
silage, other roughage and harvested crop, drinking water;
In the event of nitrite/nitrate contamination corrective measures will be taken immediately
(e.g. detect source, determine if other feed is contaminated).

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 APPENDIX IV Nitrite/nitrate control measures: husbandry.

Husbandry

Use the HACCP system to assess the risk of nitrite/nitrate in feed during husbandry. Besides
others, the following items should be addressed:
Do not use improperly cleaned liquid fertilizer tanks as drinking water unit as they may be
extremely high in nitrate (Kahn, 2006).

Nitrite/nitrate monitoring programs: husbandry

In case of home mixing of feed or drying of feed, the monitoring program on feed in Appendix
VII is referred to. Concerning nitrite/nitrate monitoring programs for the transport and storage
of feed is referred to appendices V and VI respectively. For specific information concerning
nitrite monitoring in husbandry, see below.
If applicable: implement a nitrite/nitrate monitoring program on the following products or
critical control points, which were determined of risk by the HACCP study: e.g. feed
purchased at third parties and drinking water;
In the event of nitrite/nitrate contamination corrective measures will be taken immediately
(e.g. detect source, determine if other feed is contaminated).

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 APPENDIX V Nitrite/nitrate control measures: transport of feed.
In GMP+ B1 standard general control measures and requirements are stated which should
be complied with (GMP+4, 2013). Specific requirements and control measures related to
transport are stated in various GMP+ standards (GMP+5,6,7,8,9,10, 2013). The applicable GMP+
standard depends on the means of transport. The applicable standard should be complied
with. There are no additional recommendations for general control measures.

Nitrite/nitrate monitoring programs: transport

Implement a nitrite/nitrate monitoring program, as stated hereafter.

If applicable: implement a nitrite/nitrate monitoring program on products or at critical
points in transporting feed, as is determined using the HACCP system, e.g. sample of
transported lot after transporting a nitrate/nitrite containing product (e.g. fertilizer) in order
to determine effectiveness of cleaning procedure.
In the event of nitrite/nitrate contamination corrective measures will be taken immediately
(e.g. detect source, determine if other feed is contaminated).

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 APPENDIX VI Nitrite/nitrate control measures: storage of feed.
In GMP+ B1 standard general control measures and requirements are stated which should
be complied with (GMP+4, 2013). Specific requirements and control measures related to
transport are stated in the GMP+ B3 standard (GMP+11, 2013). There are no additional
recommendations for general control measures.

Nitrite/nitrate monitoring programs: storage

If applicable: implement a nitrite/nitrate monitoring program on products or at critical

points in storing feed, as is determined using the HACCP system, e.g. sample of stored
lot after storing a nitrate/nitrite containing product (e.g. fertilizer) in order to determine
effectiveness of cleaning procedure.
In the event of nitrite/nitrate contamination corrective measures will be taken immediately
(e.g. detect source, determine if other feed is contaminated).

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 APPENDIX VII Nitrite/nitrate control measures: processing of feed.
In GMP+ B1 and B2 standard general control measures and requirements are stated which
should be complied with (GMP+4,12, 2013). Additional recommendations for general control
measures are stated below in this appendix.

Processing of feed or food

Verify if the supplier / producer of feed or food (also include premixes, additives etc.) has
assessed the hazard and risk of nitrite/nitrate;
Selecting of the suppliers based on their implementation of programs for the control of
nitrite/nitrate;
If water is used during processing, assure that nitrite/nitrate levels are safe;
If nitrite/nitrate containing additives are used, assure that residual nitrite/nitrate levels in
feed and food are safe. Apply statutory legislation and supplier instructions concerning
use.

Nitrite/nitrate monitoring programs: processing

Implement nitrite/nitrate monitoring programs, as stated hereafter:

Concerning nitrite/nitrate monitoring programs for the transport and storage of feed is
referred to appendices V and VI respectively;
If applicable: implement a nitrite/nitrate monitoring program on the following products or
critical control points, which were determined of risk by the HACCP study: e.g. premixes,
additives, processing aids, water, feed (incl. raw materials, compound feed and feed
directly delivered to farmers (e.g. cereals);
In the event of nitrite/nitrate contamination corrective measures will be taken immediately
(e.g. detect source, determine if other feed is contaminated).

Fact sheet: Nitrate and nitrite 27 / 27

Version: 09/01/2015 GMP+ International B.V.