UK Monitoring Programme for Nitrate in Lettuce and Spinach 2001

• Nitrate is a natural component of vegetables although the levels present are affected by growing conditions, fertiliser use and plant species / variety.
• The UK has carried out annual monitoring for nitrate in lettuce and spinach since 1996. Every Member State is required to monitor and report levels of nitrate in lettuce and spinach as part of a European Commission Regulation.
• The European Commission Regulation sets maximum levels for nitrate in lettuce and spinach, however UK growers are currently exempted from meeting these limits for a transitional period.
• The levels of nitrate detected in lettuce and spinach do not pose a risk to consumer's health; all dietary exposure estimates were below the Acceptable Daily Intake (ADI) set by the European Commission's Scientific Committee for Food (SCF).

Summary

The results of the 1999 Monitoring Programme for nitrate in lettuce and spinach are reported. Samples of UK lettuces and spinach were obtained from six growing regions (South East, Humberside, East Anglia, Lancashire, South West and Northern Ireland). Trading Standards Officers co-ordinated the collection of samples and Public Analyst laboratories performed the analyses for nitrate. Samples of imported lettuce and spinach were also analysed in this survey.

One hundred and fourteen samples of lettuce were analysed with results in the range 244-3073 mg/kg for summer outdoor crops, 937-3740 mg/kg for summer indoor crops, 670-3000 mg/kg for winter outdoor crops and 1040-4425 mg/kg for winter indoor crops. Only 3.5 per cent of samples tested exceeded the legal limit.

Thirty-four samples of spinach were analysed with the results in the range 25-3910 mg/kg for summer crops and 2180-2560 mg/kg for winter crops. Fifteen percent of samples tested exceeded the legal limit. All concentrations relate to fresh weight. UK growers are currently exempted from the maximum levels for lettuce and spinach for a transitional period.

Background
Nitrate in food
Nitrate is a natural component of vegetables although the levels present are affected by growing conditions, fertiliser use and plant species / variety. Light is the main influence on nitrate concentrations in plants. Plants take up nitrogen in the form of nitrate and convert it into proteins via amino acids (photosynthesis). Poor light (such as in winter or during cloudy spells in summer) can result in a lower rate of photosynthesis, creating an accumulation of nitrate in the tissues.

Vegetables are the main source of nitrate in the diet and contribute to 70 to 90 per cent of the total nitrate intake. Green leafy vegetables, such as lettuce and spinach, naturally contain higher levels of nitrate than other foods.

Studies of nitrate in food have demonstrated that there can be both beneficial and potentially detrimental health effects. In 1995, the EC Scientific Committee on Food (SCF) agreed to retain its earlier 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).

European Commission legislation on nitrate in lettuce and spinach
European Commission Regulation (EC) No. 194/97 which sets maximum levels (limits) for nitrate in lettuce and spinach has applied in the UK and other Member States since 15 February 1997. In 1998, the EC reviewed these levels. However, there were insufficient data available and the review period was extended to the end of 2001 to allow more time for data to be collected (EC Regulation No. 864/99). This EC Regulation is implemented in the UK by a Statutory Instrument (The Contaminants in Food Regulations Act 1997 (S.I. [1997] No. 1499)) which came into force in February 1997. The limits set by this EC Regulation vary according to season with higher nitrate levels permitted in crops grown in winter compared with those grown in the summer and for lettuce different limits apply to glasshouse-grown and outdoor crops. These are summarised in Table 1.

Derogation
Most lettuce and spinach grown in the UK meets the EC levels for nitrates; but some summer spinach and protected lettuces fail to meet these levels when weather during the growth period, or more particularly at harvest, has included cloudy spells. Other Member States also experience these problems.

EC Regulation 194/97 (amended by EC Regulation No. 864/99) allows for an optional derogation (exemption) from the limits for nitrate in lettuce and spinach grown and sold in individual Member States for a transitional period. This is provided that nitrate levels in these foods are acceptable on public health grounds and that growers follow Good Agricultural Practice (GAP). The UK is currently applying this optional derogation along with Ireland for lettuce, and Ireland, Finland, Denmark and the Netherlands for spinach. The average nitrate levels for UK consumers of lettuce and spinach would not cause even high-level consumers to exceed the SCF ADI, justifying the UK's use of the derogation. The maximum levels do apply to lettuce and spinach imported into the UK from Member States and third countries.

UK Monitoring Programme for Nitrate in Lettuce and Spinach
It is a requirement of EC Regulation 194/97 (amended by EC Regulation No. 864/99) that all Member States carry out monitoring for nitrate in lettuce and spinach and report the results annually to the EC. Data on nitrate levels in lettuce and spinach obtained from Monitoring Programmes carried out in each Member State will be used as part of the review of the EC Regulation, which began in February 2001 and is due to be completed by 1 January 2002.

The UK Monitoring Programme for Nitrate in Lettuce and Spinach has been carried out since May 1996. Results for previous surveys are reported in MAFF Food Surveillance Information Sheets 121 and 154.

Brand names
UK growers of lettuce and spinach are exempt from the brand naming policy as explained in the Agency's Guidelines for Planning and Reporting Surveys.

Methodology
Sampling The number of samples taken in the 1999 UK Monitoring Programme was in accordance with European Commission guidelines. These guidelines state that one sample is to be taken per 2000 tonnes of production or import with a minimum of 12 samples and a maximum of 120 samples a year for one product from each Member State.

Trading Standards officers collected samples from UK and imported produce. Samples were taken in accordance with Commission Directive 79/700/EEC. At all stages, samples were kept below 10^oC and reached the laboratory within 24 hours of harvest or purchase. They were then analysed within three working days of receipt.

Four types of UK produce were sampled: glasshouse-grown lettuce, outdoor lettuce and spinach (fresh and canned). Samples of glasshouse-grown lettuce were taken throughout the year, samples of outdoor lettuce from between April and November and samples of fresh spinach from April to November. Samples of canned spinach were collected in November and December.

Analyses
Samples of lettuces and spinach were prepared and analysed in accordance with European Commission Guideline Document VI/4800/96. Each sample comprised 10 heads of lettuce or packets/boxes of spinach, which were combined and homogenised to produce a representative test sample. The guidelines do not specify particular analytical methods, but set criteria for analytical performance (including recovery and precision) of the methods used.

All samples were analysed by Public Analysts' laboratories. Their methods meet the assessment criteria set out in Article 3 of Council Directive 93/99/EEC relating to official food laboratories. All laboratories participated, and achieved satisfactory performances in the nitrate rounds of the Food Analysis Performance Assessment Scheme (FAPAS).

Results
UK glasshouse-grown lettuce
A total of 37 samples of glasshouse-grown lettuce were taken from the South East, Humberside, East Anglia and Northern Ireland. The results of the analyses of these samples are summarised in Table 2. Nitrate concentrations were higher during the winter months (i.e. October to March) with a mean level of 3158 mg/kg than in the summer months (i.e. April to September) when the mean nitrate level was 2247 mg/kg. Only 1 sample of the 18 indoor lettuces obtained in the summer exceeded the maximum level specified in the Regulation and none of the winter lettuces.

The nitrate levels from the 1999 monitoring programme are similar to those which have been reported in previous years. The mean nitrate levels in the 1997/1998 programme were 3432 mg/kg during winter and 2545 mg/kg during summer.

UK outdoor lettuce
A total of 56 samples of outdoor lettuce were taken from the South East, East Anglia, Lancashire, South West and Northern Ireland. Only 2 of the 26 samples taken in the mid-summer period exceeded the EC maximum levels as shown in Table 3. No samples were over the maximum levels in the early- / late-summer periods or winter. The results of the 1999 Monitoring Programme are similar to those found in previous years. Mean nitrate levels for the period May to August in 1999 were 1045 mg/kg, and for the same period in 1996 and 1997 they were 1089 mg/kg and 937 mg/kg respectively. Between 1 September and 30 September 1999 the mean nitrate level was 1090 mg/kg. The nitrate level in 1997 for the same period was 850.

UK fresh spinach
Twenty-three samples of UK produced fresh spinach were obtained. Twenty-one samples were taken in summer and 2 in winter. Of the 21 samples taken in summer,4 exceeded the EC limit. The mean levels were 1487 mg/kg for the summer samples and 2370 mg/kg for the winter samples. In 1997, 30 per cent of samples in the summer exceeded the limit.

UK canned spinach
Six samples of canned spinach were obtained during November and December. The nitrate levels in these samples were all below the maximum of 2000mg/kg. The mean level was 545 mg/kg. Samples in 1997 were also below the maximum limit at a mean level of 787 mg/kg (Table 5).

Imported fresh spinach
Five samples of imported fresh spinach were obtained during November and December (Table 6). Only 1 sample (3100 mg/kg) exceeded the maximum limit of 3000 mg/kg shown in Table 6. This sample was imported from Italy. The mean nitrate level for the 5 samples was 2367mg/kg. In 1997 only 2 samples of imported fresh spinach were taken and both of these were below the EC maximum levels.

Imported lettuce
Twenty-one samples of lettuce imported from other Member States were included in the UK Monitoring Programme (Table 7). One sample, imported from the Netherlands, was above the limit

Dietary exposure estimates
Dietary exposures of nitrate have been estimated for adults who eat average amounts of lettuce, spinach and foods containing them (mean consumers) and for those who eat significantly more than average (high level consumers). These estimates can then be compared with the ADI for nitrate set by the SCF. The ADI can be used as a guideline to assess whether nitrate exposure through lettuce and spinach consumption is a risk to consumer health.

The Food Standard Agency's INTAKE computer programme was used to combine the distribution of food consumption by adult consumers (obtained from National Diet and Nutrition Survey of British Adults)13 with the level of nitrate in foods in the diet. A recipe database provided consumption data on processed foods containing lettuce or spinach as an ingredient. Data on nitrate levels in lettuce and spinach were obtained from the 1999 Monitoring Programme. The 1997 Total Diet Study (TDS) on Nitrate and Nitrite14 provided information on other sources of nitrate in foods such as potatoes and cured meat products. Estimates of the intake from water and beer were also included.

The results from the INTAKE program of UK consumers of nitrate from UK-grown glasshouse lettuce, outdoor lettuce and spinach are shown in Table 8. The mean intake for a consumer from lettuce was 17 mg/day of nitrate and for the high level consumer, 70 mg/day. For consumers of spinach, the mean intake of nitrate was 12 mg/day from spinach and for the high level consumer, 47 mg/day. The data indicate that individuals who consume large amounts of lettuce are unlikely also to consume large amount of spinach. Intake of nitrate from all sources in the diet totals 88 mg/day for the average consumer and 136 mg/day for the high level consumer. These calculations of nitrate intake were made using data from the Total Diet Study, which includes some contributions from green vegetables (including lettuce and spinach) and from canned vegetables (including spinach). Therefore, the estimates of the total nitrate intakes obtained by adding the values calculated from the results of this survey to those from the TDS will result in an overestimation of total exposure to nitrate.

Interpretation

All the dietary exposures for consumers of UK glasshouse lettuce, outdoor lettuce, fresh spinach and preserved spinach were below the SCF's ADI for nitrate, which is equivalent to 219mg/day for a 60kg adult (Table 8). No account has been taken for the reduction of nitrate concentrations during cooking. A previous study has shown that nitrate concentrations in spinach are reduced by approximately 75 per cent by cooking (providing the cooking water is discarded and not used to make gravy or sauces).15

The optional derogation from the Regulation, which the UK is applying, allows lettuce and spinach grown and sold in the UK to be exempt from the maximum levels, provided that their nitrate content is not so high as to present a risk to health. The mean nitrate levels found in the UK Monitoring Programme would not cause even high-level (97.5th percentile) consumers of lettuce or of spinach to exceed the SCF ADI.

The finding of one imported spinach sample and one imported lettuce sample at levels above the EU limit shows that levels of nitrate in these crops is not an issue for the UK alone.

Actions
The results of the UK Monitoring Programme have been submitted to the EC as have those from other Member States. These results will be used to inform the review of EC Regulation 194/97 (amended by EC Regulation No. 864/99) which is due to be completed by 1 January 2002.

Nitrate analyses of Total Diet Study samples obtained in 1999 are currently being carried out by Campden and Chorleywood Food Research Association in a Food Standards Agency study. The results of this survey will be reported for a Food Surveillance Information Sheet in autumn 2001.

The current monitoring programme for nitrate in lettuce and spinach is in progress.

Conclusion
The levels of nitrate in lettuce and spinach sampled in this survey generally meet maximum limits set by EC Regulation 194/97 (amended by EC Regulation No. 864/99) although some results exceed these limits (1 sample of the 37 indoor lettuces, 2 of the 56 outdoor lettuces, 4 out of 21 samples of UK fresh spinach, one sample of imported fresh spinach and one sample of imported lettuce). None of the samples pose a risk to consumer's health as dietary exposure is below the ADI set by the SCF.

References

• Commission of the European Communities Scientific Committee for Food. Opinion on Nitrate and Nitrite, expressed on 22 September 1995. Annex 4 to Document III/5611/95, 1995.
• 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 Wishnock, J.S. Assessment: nitrate, nitrite and N-nitrosocompounds. European Journal of Pharmacology, Environmental Toxicity and Pharmacology. 1994, Section 292, 1-38.
• Sugimura, T., Fujimura, S. and Baba, T. Tumour production in the gladular stomach of the rat by N -methyl-N¹-nitro-N-nitroguanidine. Cancer Research. 1970, 30, 455-465.
• Duncan, C., Dougall, H., Johnston, P., Green, S., Brogan, R., Leifert, C., Smith, L., Golden, M. and Benjamin, N. Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate. Nature Medicine. 1995, 1, 546-551.
• Dykhuizen, R.S., Frazer, R., Duncan, C., Smith, C.C., Golden, M., Benjamin, N. and Leifert, C. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defence. Antimicrobial Agents and Chemotherapy. 1996, 40, 1422-1425.
• McKnight, G.M., Smith, L.M., Drummond, R.S., Duncan, C.W., Golden, M. and Benjamin, N. Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans. Gut. 1997, 40, 211-214.
• European Commission. Commission Regulation (EC) No 194/97 of 31 January 1997. Official Journal of the European Communities No. L31/48,1997.
• Ministry of Agriculture, Fisheries and Food. 1996/97 UK Monitoring Programme for Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet No. 121, 1997.
• Ministry of Agriculture, Fisheries and Food. 1997/1998 UK Monitoring Programme for Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet No. 154, 1998.
• European Commission. Monitoring Programme Nitrates: Number of Samples. Document VI/1655/97, 1997.
• European Commission. Guidelines for Laboratories Carrying Out The Determination of Nitrate in Lettuce and Spinach: EC Monitoring Programme. Document VI/4800/96, 1997.
• Council of the European Communities. Council Directive 93/99/EEC of 29 October 1993 on the subject of additional measures concerning the official control of foodstuffs. Official Journal of the European Communities No. L 290/14, 1993.
• Gregory, J., Foster, K., Tyler, H. and Wiseman, M. The Dietary and Nutritional Survey of British Adults. London: The Stationery Office, 1990.
• Ministry of Agriculture, Fisheries and Food. 1997 Total Diet Study- Nitrate and Nitrite. Food Surveillance Information Sheet No. 163, 1997.
• Ministry of Agriculture, Fisheries and Food. Nitrate in vegetables. Food Surveillance Information Sheet No. 158, 1998.

Product	Harvest period	Max. nitrate levels (mg/kg fresh product)
Spinach (fresh)	1 November to 31 March	3000
	1 April to 31 October	2500
Lettuce (protected and open-grown lettuce)	1 October to 31 March	4500
	1 April to 30 September	3500
	with the exception of open grown lettuce harvested from 1 May to 31 August	2500
Preserved, deep-frozen or frozen spinach		2000

Season	Sampling dates	EC Max. level (mg/kg)	No. samples	Nitrate levels (mg/kg fresh weight)		Number exceeding max. level
				range	mean
Summer	1 Apr 1999 - 30 Sept 1999	3500	18	937-3740	2247	1
Winter	1 Oct 1999 - March 2000	4500	19	1040-4425	3158	0

Season	Sampling dates	EC Max. level (mg/kg)	No. samples	Nitrate levels (mg/kg fresh weight)		Number exceeding max. level
				range	mean
Summer	1 April 1999- 30 April 1999	3500	2	775-1461	1118	0
	1 May 1999- 31 Aug 1999	2500	26	244-3073	1045	2
	1 Sept 1999- 30 Sept 1999	3500	17	308-2119	1090	0
Winter	1 Oct 1999- 31 Dec 1999	4500	11	670-3000	1348	0

Season	Sampling dates	EC Max. level (mg/kg)	No. samples	Nitrate levels (mg/kg fresh weight)		Number exceeding max. level
				range	mean
Summer	1 Apr 1999 - 31 October 1999	2500	21	25-3910	1487	4
Winter	1 Nov 1999 - 31 Dec 1999	3000	2	2180-2560	2370	0

Season	Sampling dates	EC Max. level (mg/kg)	No. samples	Nitrate levels (mg/kg fresh weight)		Number exceeding max. level
				range	mean
Winter	1 Nov - 31 Dec 1999	3000	5	1520-3100	2367	1

Season	Sampling dates	EC Max. level (mg/kg)	No. samples	Nitrate levels (mg/kg fresh weight)		Number exceeding max. level
				range	mean
Winter	1 Nov - 31 Dec 1999	3000	5	1520-3100	2367	1

Season	Sampling dates	EC Max. level (mg/kg)	No. samples	Nitrate levels (mg/kg fresh weight)		Number exceeding max. level
				range	mean
Summer	1 May - 31 August 1999	2500	8	340-2920	874	1
	1 Sept - 30 Sept 1999	3500	4	500-3100	2000	0
Winter	1 Oct - 31 Mar 1999	4500	9	780-1240	1037	0

	Dietary Intake (mg/day)
	lettuce		spinach
	mean	97.5%ile	mean	97.5%ile
From 1999 crop	17	70	12	47