Number 177 May 1999
MAFF UK - NITRATE IN LETTUCE AND SPINACH
Index to MAFF UK Food Surveillance Information
91: MAFF UK -
Nitrate in Vegetables (July 1996)
121: MAFF UK - 1996/97 UK Monitoring
Programme for Nitrate in Lettuce and Spinach (August 1997)
137: MAFF UK - 1994 Total Diet Study -
Nitrate and Nitrite (December 1997)
142: MAFF UK - Survey of Nitrite and
Nitrate in Bacon and Cured Meat Products (February 1998)
154: MAFF UK - 1997/98 UK Monitoring
Programme for Nitrate in Lettuce and Spinach (August 1998)
158: MAFF UK - Nitrate in Vegetables
163: MAFF UK -
1997 Total Diet Study: Nitrate and Nitrite. (October 1998)
165: MAFF UK - Duplicate Diet Study of
Vegetarians-Nitrate Analyses (November 1998)
- This survey was carried out to provide up-to-date information on nitrate
levels in lettuce and spinach on sale in the UK for negotiations on a review of
European Commission (EC) limits for nitrate, and to check that nitrate
concentrations in these vegetables are not a risk to consumers' health.
- Nitrate levels in lettuce and spinach on sale in the UK are generally
below EC limits but 13 per cent were above. There are no health concerns for
consumers from the nitrate levels found in this survey.
- Consumers do not need to make any changes to their diets from the results
of this survey.
A survey of lettuce and spinach on sale in the UK was carried out in 1998
and 1999 to provide up-to-date information on nitrate concentrations in these
vegetables for negotiations on a review of European Commission (EC)
Regulation No. 194/97 (which sets maximum levels for nitrate in lettuce and
spinach). The results were also used to estimate dietary exposures for
consumers of lettuce and spinach to assess the risks to health.
Most samples contained nitrate concentrations below the maximum levels
(limits) specified by EC Regulation No. 194/97. However, 6 per cent
of UK-grown lettuce samples were above the EC maximum levels as were 30 per cent
of UK-produced and 22 per cent of imported spinach samples. These results have
been submitted to the EC. Retailers of the imported spinach samples which
exceeded the maximum levels have been informed of their results by the Joint
Food Safety and Standards Group (JFSSG). These retailers have also been invited
to submit brief statements on these results for inclusion in this Food
Surveillance Information Sheet, and those that were received are reproduced at
Dietary exposures of both mean and upper range (97.5 percentile) consumers
of lettuce and spinach are below the Acceptable Daily Intake (ADI) for nitrate
set by the EC's Scientific Committee for Food (SCF). There are therefore no
health concerns for consumers.
Nitrate in food
Most foods contain nitrate. This is
present naturally, or may be present as a result of the use of fertilisers on
crops,1 or from its use as a preservative.2 The nitrate content of foods has been of
interest for many years because of its possible health effects.3
However, the chemistry of nitrate metabolism is complex and there are a number
of potentially detrimental and beneficial health effects. An example of a
possible detrimental effect is the metabolism in the gut of dietary nitrate to
potentially carcinogenic N-nitroso compounds.4
Conversely, the acidic conditions in the stomach cause the formation of nitric
oxide which has recently been investigated for its role in the body's defence
against pathogenic bacteria.5,6
The SCF recommended an Acceptable Daily Intake (ADI) for the nitrate ion of
3.65 mg/kg body weight (equivalent to 219 mg/day for a 60 kg person).1,7 The SCF ADI for nitrate has been endorsed by
the UK Committee on Toxicity of Chemicals in Food, Consumer Products and the
Environment (COT).8 More information on how
the ADI for nitrate was derived is given in the SCF reports, Food Surveillance
Paper Number 32 and Food Surveillance Information Sheet No. 163.1,7-9
JFSSG has carried out a number of surveys of nitrate in food.8-13
The results of these surveys have been used to assess the risks to health of UK
consumers from nitrate in food by estimating dietary exposures and comparing
them with the ADI. For example, dietary exposures to nitrate for adult
consumers, estimated from the results of the 1997 UK Total Diet Study, were 57
mg/day for mean (average) consumers and 105 mg/day for upper range (97.5
percentile) consumers.9 These exposure
estimates are all below the ADI.
Vegetables, particularly green vegetables, contain higher nitrate
concentrations than other types of food and make the main contribution to total
dietary exposure. In the 1997 Total Diet Study, green vegetables, potatoes and
other vegetables were found to contribute 21 per cent, 33 per cent and 15 per
cent respectively to total dietary exposure to nitrate.9
European Commission legislation on nitrate in lettuce and spinach
1997, Member States agreed an EC Regulation setting levels (limits) for nitrate
in lettuce and spinach (EC Regulation No. 194/97).14
The maximum levels set by this Regulation are summarised in
Table 1. This Regulation has applied in the UK and
other Member States since 15 February 1997. The Contaminants in Food
Regulations 1997 (S.I.  No. 1499), which came into force on 4 July
1997, allow for enforcement of the EC Regulation in the UK.15
The main purpose of this EC Regulation is to harmonise limits for nitrate in
these vegetables as the different national limits set by some Member States were
causing trade difficulties across the European Union. The EC Regulation is also
intended to protect public health in response to the SCF's considerations of
nitrate in food.1,7
European Commission Regulation No. 194/97 allows for an optional
derogation (exemption) from the maximum levels, for a transitional period, for
lettuce and spinach grown and sold for consumption in individual Member States.
This is provided that growers follow Codes of Good Agricultural Practice (GAP)
and that the nitrate concentrations in lettuce and spinach do not pose a risk to
public health.14 The UK and several other
Member States are currently operating this derogation. It should be noted that
the maximum levels do however apply to lettuce and spinach imported into the UK
from other Member States and third countries.
Review of EC Regulation
It is a requirement of EC
Regulation No. 194/97 that Member States monitor nitrate levels in lettuce
and spinach and report the results to the EC.14
For example, the UK has been carrying out a Monitoring Programme for Nitrate in
Lettuce and Spinach since June 1996.16,17 The intention of this monitoring is to
provide information for a review of the EC Regulation which started in September
1998. The results of monitoring by Member States showed that there are wide
variations in nitrate concentrations between Member States and over time.
However, the results were insufficient to determine if GAP is effective in
controlling nitrate concentrations in lettuce and spinach. It was therefore
decided to extend the review period for a further 3 years to obtain more data.
A Regulation amending
EC Regulation No. 194/97, to bring this extension into effect, was
agreed by Member States on 9 March 1999.18
This survey was carried out to obtain recent
information on nitrate concentrations in lettuce and spinach on sale in the UK
to support the UK position in negotiations on the extended review of EC
Regulation No. 194/97. Results were provided to the EC in October 1998
(for samples obtained in July and August) and February 1999 (for all samples).
The survey was also carried out to compare estimated dietary exposures from
nitrate in lettuce and spinach with the ADI.
In accordance with the MAFF policy for the
release of brand names when reporting the results of food chemical surveillance,19 details of the lettuce and spinach samples
obtained in this survey are given in full in Annex II.
It should be noted that the absence of any particular brand from this survey
means only that the brand has not been included in the survey. No further
meaning should be read into the absence of any brands from this Food
Surveillance Information Sheet.
A total of 105 samples of lettuce and spinach
were obtained from retail outlets (e.g. supermarkets, grocers, market stalls,
etc.). Most samples were obtained locally to ADAS Laboratories in the
Wolverhampton area. To determine if there were any seasonal differences in
nitrate concentrations in lettuce and spinach and to reflect the different
maximum levels specified by EC Regulation No. 194/97 for summer and
winter produce, samples were taken in two phases:
- summer: July and August 1998 (52 samples)
- winter: November 1998 (53 samples).
As one of the purposes of the survey was to provide data to support the UK
position in negotiations on a review of EC Regulation No. 194/97, the
sampling plan was biased to UK produce. Most of the samples taken were
therefore of UK origin. However, the original sampling plan had to be revised
to reflect the produce available during the two sampling periods. In
particular, it was found that little imported spinach was on sale in the summer
and that UK-grown spinach was not readily available in the winter. Priority was
therefore given to obtaining the number of samples required from a wide range of
origins even if this meant obtaining a greater number of samples from some
retailers than others. It is however reasonable to assume that imported lettuce
and spinach sold by the major retailers are grown under similar conditions and,
like UK produce, nitrate concentrations in these crops will be influenced by
weather conditions. The numbers of samples of UK and imported lettuce and
spinach taken are shown in Table 2 and full sample
details are given in Annex II.
Individual samples were selected, as far as possible, at random from the
produce on sale. Each sample comprised at least three heads of lettuce or packs
of spinach (or equivalent quantity if sold loose) from the same batch and source
according to the information given on the packaging and at the point of
After purchase, samples were
transported to the laboratory in cool boxes. On receipt at the laboratory,
samples were stored at 2-4oC. Inedible (heavily soiled or damaged)
leaves were removed and, if necessary, the remaining sample was rinsed using
deionised water. Samples were prepared by combining representative portions of
the edible portions of at least three individual lettuce heads or packs of
spinach. All samples were chopped, freeze-dried, ground and stored frozen until
All samples were analysed by a method based
on that of Hunt and Seymour (1985).20
Nitrate was extracted by the following method. A scoop of activated carbon (100
mg to 250 mg) was added to 0.1 g to 0.2 g (weighed accurately) of the
freeze-dried sample along with 50 ml of water. The sample was then shaken for
30 minutes. An aliquot of the sample was filtered through a 0.45 micrometer
membrane filter into an autosampler vial ready for analysis.
Nitrate determination: Nitrate ion was separated by High
Performance Liquid Chromatography (HPLC) using a Chrompack Ionosphere 5A column
eluted with a flow rate of 1.5 ml/minute with analytical grade potassium
dihydrogen orthophosphate (0.045M) adjusted to pH3 with orthophosphoric acid.
The nitrate ion was detected at 210 nm after approximately 6 minutes. The total
run time was 10 minutes.
ADAS Laboratories are UKAS (United
Kingdom Accreditation Service) accredited and take part in the nitrate rounds in
FAPAS (Food Analysis Performance Assessment Scheme).
A spinach sample of known nitrate content (i.e. a FAPAS sample with a
nitrate content of 1097 mg/kg) was analysed with each batch of 10 samples.
Batches were rejected if the concentration detected in this FAPAS sample was
outside the range of 974 mg/kg to 1220 mg/kg. The recovery of this method was
104 per cent.
All samples were analysed in duplicate and the final result for each sample
is reported as the mean of duplicate analyses. The weights of each sample after
preparation and freeze drying were recorded so that results could be expressed
on an 'as received' basis rather than on the concentration detected in the
freeze-dried sample. The limit of detection (LOD) of this method for
freeze-dried samples was 7.5 mg/kg.
Further information on the sampling plan, and preparation and analysis of
samples is given in the final report of this survey which is available for
public access in MAFF's library.21
The five samples of imported spinach which
were found to contain nitrate concentrations above the maximum level of 3000
mg/kg, were re-analysed by a second laboratory to confirm the results. These
five samples were analysed by West Yorkshire Analytical Services. This is a
Public Analyst Laboratory and takes part in the nitrate rounds in FAPAS. The
results of the re-analyses are given in Table 3.
Nitrate was extracted from freeze-dried samples using boiling water and
analysed by ion chromatography using suppressed conductivity detection. The
recovery of nitrate ion using this method was 100.5 per cent.
Dietary exposures were estimated for
mean and upper range (97.5 percentile) consumers of lettuce and spinach using
the mean concentrations detected and consumption data from the Dietary and
Nutritional Survey of British Adults.22 To
derive estimates of total dietary exposure of consumers of lettuce and spinach,
nitrate exposure from the rest of the diet was added using the exposure for a
mean adult consumer estimated from the results of the 1997 Total Diet Study
(i.e. 57 mg/day).9
Results of nitrate analyses of lettuce and spinach samples are summarised in
Table 2. The full results and sample details are
given in Annex II.
The nitrate concentrations in UK-produced lettuce and spinach found in this
survey are similar to those reported previously.17
The proportions of samples of UK-grown lettuce and spinach which had nitrate
concentrations above the maximum levels in the EC Regulation are also similar to
previous surveys. For example in this 1998 survey, 30 per cent of UK-grown
spinach and 6 per cent of lettuce samples had nitrate concentrations above the
maximum levels. In comparison, 35 per cent of UK-grown spinach and 7 per cent
of lettuce samples from the 1997/98 UK Monitoring Programme for Nitrate in
Lettuce and Spinach had nitrate concentrations above the maximum levels.17
The UK is currently operating the optional derogation provided for by EC
Regulation No. 194/97. This means that the maximum levels do not
apply to lettuce and spinach grown and sold for consumption in the UK. However,
the maximum levels do apply to any imported lettuce and
spinach. Five samples of imported winter spinach were found to have nitrate
concentrations above the maximum levels (Table 3).
The countries of origin of these samples were Cyprus (1 sample), Italy (2
samples) and Spain (2 samples).
A statistical analysis was carried out on the results to determine if there
were differences for UK-grown lettuce between crops grown in different seasons,
and those grown outdoors or under glass. No analysis of seasonal differences in
nitrate concentrations in spinach was made as most spinach on sale in the summer
was UK-grown compared with that on sale in winter which was imported.
Differences in country of origin are likely to obscure any seasonal differences.
The nitrate concentrations
detected in the lettuce and spinach samples in this survey are similar to those
found in previous JFSSG-funded surveys.10,11,16,17
Concentrations were higher in UK-grown lettuce samples obtained in the winter
compared with those from the summer (Table 2). This
difference was statistically significant at the 99.5 per cent level. Light
intensity has been shown to be the major factor controlling nitrate
concentrations in lettuce with lower concentrations present in crops grown
during the summer.23 This is reflected in the
maximum levels specified by EC Regulation No. 194/97 with higher
concentrations permitted in crops grown in the winter months (Table 1).14
Lettuces grown under glass had
higher nitrate concentrations than other UK-produced lettuce. This difference
was significant at the 95 per cent level. This can be partly explained by
differences in variety as lettuces grown under protected conditions tend to be
of the round/butterhead type which contains higher nitrate concentrations than
some other varieties.17 It may also be a
result of the lower light levels available to glasshouse crops compared with
those grown outdoors. This difference is also reflected in EC Regulation
No. 194/97 which sets a lower maximum level for summer-grown outdoor
Dietary exposure estimates for
consumers of lettuce and spinach based on the results of this survey are below
the ADI of 3.65 mg/kg bodyweight/day (equivalent to 219 mg/day for a 60 kg
adult) (Table 4).1,7
Total dietary exposure estimates for mean and upper range (97.5 percentile)
consumers of lettuce are 77 mg/day and 136 mg/day, respectively (Table 4). Total dietary exposure estimates for mean
and upper range (97.5 percentile) consumers of spinach are 71 mg/day and 121
mg/day, respectively (Table 4). Exposure estimates
for mean and upper range (97.5 percentile) consumers of both lettuce and spinach
are 78 mg/day and 142 mg/day, respectively (Table 4).
These values can be regarded as over-estimates as no correction has been made
for the lettuce and spinach included in the exposure from the rest of the diet
(in other words there is some small amount of double counting). In addition,
for dietary exposures of consumers of spinach, no account has been taken for the
reduction of nitrate concentrations during cooking. A previous JFSSG-funded
study has shown that nitrate concentrations in spinach are reduced by
approximately 75 per cent by cooking.10
To estimate total dietary exposure, it is important to take into account all
dietary sources of nitrate. Nitrate in water and beer can also contribute to
total dietary exposure. Nitrate exposure from drinking water varies across the
UK.24 However, if it is assumed that nitrate
exposure from water is 20 mg/day and that from beer is 11 mg/day,8
then total exposures can be derived by adding 33 mg/day to the dietary exposure
estimates given in Table 2.
The dietary exposures to nitrate estimated from the results of this survey
are below the ADI (which is equivalent to 219 mg/day for a 60 kg person) and are
similar to those estimated from the results of from previous JFSSG surveys of
nitrate in vegetables (Table 5) and those estimated
for consumers in other countries (Table 6).
The results of this survey were reported to
the EC in October 1998 (for samples obtained in July and August) and February
1999 (for all samples).
Retailers of the imported winter spinach samples which were found to exceed
the maximum level were informed of their results by JFSSG and asked to submit
statements for inclusion in this Food Surveillance Information Sheet. The Local
Authorities Co-ordinating Body on Food and Trading Standards (LACOTS) were also
informed of these results. Statements received are reproduced at
JFSSG has notified the authorities in Spain and Italy that samples of
winter-grown spinach imported from these Member States exceeded the EC maximum
The UK Monitoring Programme for Nitrate in Lettuce and Spinach recommenced
in March 1999 for a period of 3 years. The results of the Monitoring Programme
will be reported to the EC annually to inform the extended review of EC
Regulation No. 194/97. Results will also be reported in the Food Safety
Information Bulletin and Food Surveillance Information Sheets.
The first versions of the Codes of GAP for the production of glasshouse
lettuce, outdoor lettuce and spinach were sent to the EC in 1997. These Codes
have recently been revised by growers and the National Farmers Union in light of
the results of a JFSSG-funded research project25
and discussions with JFSSG and MAFF officials. The revised versions of the
Codes of GAP will sent to the EC.
A further JFSSG-funded research project to test the effectiveness of the
Code of GAP for the production of spinach in controlling nitrate levels began in
March 1999 at Horticultural Research International. The results from this study
will be used to revise the Code of GAP.
The results of this and other surveys and Member States' Monitoring
Programmes were considered at EC Working Group meetings, where it was concluded
that further information was needed before final agreement of maximum levels set
by EC Regulation No. 194/97 and that the review period should therefore
A vote was taken at the EC's Standing Committee for Foodstuffs on 9 March
1999 to agree a Regulation amending Commission Regulation (EC) No. 194/97.18 This amending EC Regulation extends the
review period for three years and clarifies some of the existing provisions.
The Contaminants in Food Regulations 1997 are being changed accordingly.
Most samples of lettuce and spinach contained nitrate concentrations below
the maximum levels specified by EC Regulation No. 194/97. However, 30
per cent of UK-grown spinach, 6 per cent of UK-produced lettuce, and 22 per cent
of imported spinach had nitrate concentrations above the maximum levels.
Dietary exposures to nitrate estimated from the results of this survey are
similar to previous estimates and are below the ADI. The nitrate concentrations
found in lettuce and spinach in this survey are not considered to be of concern
Summary of units
||one thousand grams|
||one thousandth of a kilogram|
|| one thousandth of a gram|
||milligrams per kilogram (equivalent to parts per million)|
|| milligrams per day|
| millilitre (ml):
||one thousandth of a litre|
||millilitres per minute|
||one millionth of a metre|
||one mole of solute in 1 litre of solution|
|| degrees Celsius|
||one thousand millionth of a metre|
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1-33. Office for Official Publications of the European Communities, Luxembourg.
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(S.I.  No. 3187). The Stationery Office, London.
- Gangolli, S.D., van den Brandt, P.A., Feron, V.J.,
Janzowski, C., Koeman, J.H., Speijers, G.J.A., Spiegelhalder, B., Walker, R. and
Wishnok, J.S. (1994). Assessment: nitrate, nitrite and N-nitrosocompounds.
European Journal of Pharmacology, Environmental Toxicology and Pharmacology.
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production in the glandular stomach of the rat by N-methyl-N1-nitro-N-nitroguanidine.
Cancer Research 30, 455-465.
- Dykhuizen, R.S., Frazer, R., Duncan, C., Smith, C.C.,
Golden, M., Benjamin, N. and Leifert, C. (1996). Antimicrobial effect of
acidified nitrite on gut pathogens: importance of dietary nitrate in host
defence. Antimicrobial Agents and Chemotherapy 40, 1422-1425.
- McKnight, G.M., Smith, L.M., Drummond, R.S., Duncan, C.W.,
Golden, M. and Benjamin, N. (1997). Chemical synthesis of nitric oxide in the
stomach from dietary nitrate in humans. Gut 40, 211-214.
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October 1990 (26th series).
- Ministry of Agriculture, Fisheries and Food (1992).
Nitrate, Nitrite and
N-Nitroso Compounds in Food: Second Report. Food Surveillance Paper
Number 32. The Stationery Office, London.
- Ministry of
Agriculture, Fisheries and Food (1998). 1997 Total Diet Study - Nitrate and
Nitrite. Food Surveillance Information Sheet Number 163.
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Agriculture, Fisheries and Food (1998). Nitrate in Vegetables. Food
Surveillance Information Sheet Number 158.
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and Food (1996). Nitrate in Vegetables. Food Surveillance Information Sheet
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and Food (1997). 1994 Total Diet Study - Nitrate and Nitrite. Food
Surveillance Information Sheet Number 137.
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N-Nitroso Compounds in Food. Food Surveillance Paper Number 20.
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No. 194/97 of 31 January 1997. Official Journal of the European Communities
- The Contaminants in Food Regulations 1997 (S.I.
 No. 1499). The Stationery Office, London.
- Ministry of
Agriculture, Fisheries and Food (1997). 1996/97 UK Monitoring Programme for
Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet Number 121.
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Agriculture, Fisheries and Food (1998). 1997/98 UK Monitoring Programme for
Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet Number 154.
- European Commission (1999). Draft Commission Regulation
amending Commission Regulation (EC) No. 194/97 setting maximum levels for
certain contaminants in foodstuffs. Document VI/8735/98 rev.2.
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Agriculture, Fisheries and Food/ Department of Health (1997). Food Safety
Information Bulletin Number 88.
- Hunt, J. and Seymour, D.J. (1985). Method for measuring
nitrate-nitrogen in vegetables using anion-exchange HPLC (high performance
liquid chromatography). Analyst 110, 131-133.
- ADAS (1999). Nitrate in Spinach and Lettuce. Report on
a survey undertaken on behalf of MAFF by ADAS (1998/99).
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(1990). The Dietary and Nutritional Survey of British Adults. The Stationery
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(1995). Regional variation in potable water nitrate concentration and its
effect on total dietary nitrate. Aqua 44, 224-229.
- Horticulture Research International (1998).
Effectiveness of the UK Codes of Good Agricultural Practice for the production
of lettuce and spinach in minimising nitrate residues. Final report of MAFF
project number CSA 4418.
- Dich, J., Järvinen, R., Knekt, P. and Penttilä, P. (1996).
Dietary intakes of nitrate, nitrite and NDMA in the Finnish Mobile Clinic Health
Examination Survey. Food Additives and Contaminants 13,
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(1990). Dietary intakes of some essential and non-essential trace elements,
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24-hour duplicate portion study. Food Additives and Contaminants 7,
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Kort, W.L.A.M. and Löwik, M.R.H. (1996). Dietary intake of food
contaminants in The Netherlands (Dutch Nutrition Surveillance System). Food
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Vasco (1997). Food Chemical Surveillance in the Basque Country: 1990-1995.
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Braeckman, H. (1994). Nitrate in food commodities of vegetable origin and the
total diet in Belgium (1992-1993). Microbiologie - Aliments - Nutrition
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chemical residues in an Egyptian total mixed diet. Food Chemistry 63,
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Naturally in Foods (2nd edition). National Academy of Sciences, Washington
- Pennington, J.A.T. (1998). Dietary Exposure Models for Nitrates and
Nitrites. Food Control 9, 385-395.
- Borawska, M., Markiewica, R., and Wikowska, A. (1998). Nitrate and
nitrite content in daily hospital diets during the winter season - comparison of
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- Gundimeda, U., Naidu, A.N. and Krishnaswamy, K. (1993). Dietary intake of
nitrate in India. Journal of Food Composition and Analysis 6,
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3.2.3. Assessment of dietary intake of nitrates by the population in the
European Union, as a consequence of the consumption of vegetables. Office for
Official Publications of the European Communities, Luxembourg.
Further information on this survey can be obtained from:
MAFF, Joint Food Safety and Standards Group, Food Contaminants
Room 238, Ergon House, c/o Nobel House
17 Smith Square
Tel: +44 (0)171 238 6756
Fax: +44 (0)171 238 5331
A copy of the full report of this survey has been placed in the MAFF
Library, Nobel House, London, SW1P 3JR, UK; Tel. No. +44 (0)171 238 6575. If
you wish to consult a copy please contact the library giving at least 24 hours
notice or alternatively copies can be obtained from the library: a charge will
be made to cover photocopying and postage.
Annex II: Nitrate Concentrations in Vegetables -
Full Sample Results
Click here to view the Excel 5.0 version of Annex II
Click here to view the .csv version of sheet 1 of Annex II
(if you have any other spreadsheet package)
here to view the .csv version of sheet 2 of Annex II (if you have any other
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