Claire Weeks

Mark Osborne

Leisha Hewitt

Patrick Miller

Michelle Croasdale

Malcolm Baxter

Paul Robb

Paul Warriss

Toby Knowles

Multi-element survey of wild edible fungi and blackberries in the UK

teaching and research institutions in France or abroad, or from public or private research centers.

The survey aimed to provide an index of levels of environmental pollution, and to 3 provide a baseline against which to monitor future changes in pollution. Levels of 4 arsenic, cadmium, chromium, copper, lead, manganese, mercury, nickel, platinum, 5 tin, titanium and zinc were measured. It also assessed the levels in the most popular 6 types of wild food collected and eaten in the UK, but not seafoods or game, which are 7 now often farmed and are included in other MAFF surveys (Veterinary Residues 8 Committee, 2002). Therefore, the chosen wild foods were fungi and blackberries. 9

Edible wild fungi were selected because they can accumulate contaminants from the 10 soil (e.g. cadmium, mercury and lead). The accumulation of contaminants by fungi is 11 dependant both on environmental factors such as metal concentrations in the soil and 12 pH, and on factors such as fungal structure (Garcia et al. 1998). Several studies have 13 shown that the fruiting bodies (i.e. mushrooms) of many fungal species can 14 accumulate mercury, cadmium and lead (Kalac et al. 1991, Garcia et al. 1998, Kalac 15 et al. 1996, Falandysz et al. 2004), with some very high lead concentrations being 16 reported in mushrooms growing in the vicinity of highways or other sources of lead 17 (Kalac et al. 1996). 18 19 Wild blackberries were analysed as indicators of surface pollution (e.g. from traffic 20 exhausts). There have been recent concerns about the potential for increases in the 21 levels of platinum in the environment due to emissions from catalytic converters in 22 car exhausts (Anon, 1998, Farago et al., 1998, Hees et al., 1998, Ravindra et al, 23 2004). At the time of sampling, it is believed that approximately half the cars 24 registered in the UK had catalytic converters fitted, whereas virtually none was fitted 25 before 1993. Platinum was therefore included in this survey to compare its levels of 26 contamination in wild foods growing close to roads with those grown away from 27 traffic. 28 29 Manganese was also included in this survey in order to provide baseline data against 30 which any increase in contamination can be compared, should manganese-based fuel 31 additives be used in the future as replacements for leaded fuel. Titanium was 32 analysed as an indicator of soil contamination. Plants do not take up titanium 33 Samples of wild fungi and blackberries were taken from both rural and urban 4 locations, and from roadside sites and those distant from roads around the UK in order 5 to reflect potential localised sources of contamination. There are no data for wild 6 foods from previous UK surveys, however several surveys have measured elements in 7 commercially-grown mushrooms and blackberries (MAFF, 1982(MAFF, , 1983(MAFF, , 1985(MAFF, , 8 1987a(MAFF, ,b, 1997a)). Such commercial samples are likely to have been produced under 9 much more controlled conditions and, for fungi, comprise different species to some of 10 the wild fungi in this survey. 11 12 Some metals and other elements (e.g. zinc, selenium, cobalt, copper) can act as 13 nutrients and are essential for health, while others (e.g. mercury, cadmium, lead) have 14 no known beneficial health effects (MAFF, 1998 a,b). All may be harmful if 15 excessive amounts are consumed. Metals and other elements are present in foods 16 naturally; as a result of human activities (e.g. agricultural practices, industrial 17 emissions, car exhausts); from contamination during manufacture/processing and 18 storage; or may be added directly (MAFF, 1998 a,b, [START_REF] Peshin | Potential food contaminants and 23 associated health risks[END_REF]. 'rural' or 'urban' location. The volunteers were asked to obtain mushrooms, mainly 2 of 'Agaricus' species, which are the most commonly eaten wild fungi. Where this was 3 not possible, other common edible species, which they would also normally collect, 4 could be substituted. The Latin and common names of the fungi collected are given 5 in table 1. Once collected, the fresh samples were sealed in air and water tight plastic 6 bags. More samples of wild fungi than the planned 24 were collected, and so all 34 7 samples were analysed in order to give a better representation of results. A minimum 8 of 25g of fresh mushroom was required for analysis but collectors were requested to 9 provide at least 50g. 10 11 [table 1 about here] 12 13 Blackberry samples 14 15

The survey was designed to collect 48 samples of wild blackberries. Within the 16 three regions, four samples each came from 'rural' sites, 'urban' sites, 'beside a rural 17 road' and 'beside a main road'. Once collected, the samples were washed with 18 distilled water, dried using absorbent paper (Kimwipes Classic (Steel Blue), 19 Kimberly-Clark) and then sealed in plastic bags prior to analysis. A minimum of 200g 20 fresh weight were collected for each sample. 21

22

Soil samples 23 24

A sample of the soil substrate was collected from each site of fungi and blackberry 25 collection and this comprised random sub-samples taken from within the area of the 26 site to a depth of 15 cm, using a soil auger. The soil samples were stored in sealed 27 plastic sample bags. The soil samples were not analysed, but were stored against the 28 need for analysis to investigate any unexpected or unusual results. The wild fungi and blackberry samples were prepared and analysed for arsenic, 1 cadmium, chromium, copper, lead, manganese, mercury, nickel, platinum, tin, 2 titanium, and zinc. 3 4 All samples were washed thoroughly with distilled water and the edible portions 5 prepared. Aliquots of the homogenised edible portion of each sample (equivalent to 6 approximately 0.5 g dry weight) were digested with nitric acid by microwave heating 7 in a CEM MDS 2000 UDV system. All elements were measured by inductively 8 coupled plasma-mass spectrometry (ICP-MS) using a Perkin Elmer Elan 6000. All 9 measurements were covered by the United Kingdom Accreditation Service (UKAS) 10 accreditation, except titanium which was used as a screen only and thus outside the 11 scope of UKAS accreditation, and platinum, which was analysed by a different 12 method in order to obtain a lower limit of detection (LOD). For the platinum 13 samples, aliquots of each sample (equivalent to approximately 0.5 g dry weight) were 14 digested a form of aqua regia (4+1, concentrated nitric and hydrochloric acids (v/v)) 15 by microwave heating in a Perkin Elmer Multiwave microwave digestion system. 16

This method was chosen because platinum that is emitted from car exhausts fitted 17 with catalytic converters is likely to be in resilient forms that are difficult to digest 18 fully by nitric acid digestion alone. However, this method was outside the scope of 19

UKAS accreditation. 20 21

Quality Control 22 23

The approach for quality control in multi-element analyses by the analyst has been 24

Results and discussion 18 19

There were detectable levels of titanium on all the samples, indicating contamination 20 by soil. For blackberries, this was significantly greater for samples obtained next to 21 major roads, presumably as a result of splashing from vehicle wheels in wet weather. 22 Titanium concentrations in urban fungi samples (1.4 mg/kg) were found to be 23 significantly higher (P < 0.05) than in rural samples (0.35 mg/kg), however, it should 24 be noted that species of fungi were not matched between the two types of site. For 25 blackberries, the highest concentration of 1.8 mg/kg was found in an urban roadside 26 sample and, as with the fungi, titanium was significantly higher (p<0.01) in the urban 27 samples. 28

29

The results for titanium suggest that, not surprisingly, significant levels of soil and 30 dust may remain in the wild fungi and berry samples even after washing with clean 31 water. Any surface soil remaining in samples would have contributed to the titanium 32 concentrations reported. However, since none of the reported concentrations were 33 above guideline limits, this was not pursued. 34 In general, the concentrations of the chemical elements in the samples of blackberries 1 were low, with many falling below the limits of detection. Kruskal Wallis tests were 2 used on the non-parametric data to test for regional and site variation. There were few 3 regional differences but levels of arsenic and chromium were significantly lower (P < 4 0.001) in the north than in the midlands and south of the UK. Levels of copper were 5 also lower in the north but platinum was higher in the samples from this region (P < 6 0.01). Manganese concentrations were significantly higher (P < 0.001) in samples 7 from rural locations, with median levels of 11.7 mg/kg almost six times as high as 8 urban samples (Table 4). Manganese is commonly applied to agricultural crops, 9 which are often deficient in this trace element. Mean concentrations of lead were 10 almost nine times higher (P < 0.001) in samples by major roads and at urban sites than 11 rural sites. Significantly elevated levels of tin (P < 0.01), chromium (P < 0.05), 12 arsenic (P < 0.05) and titanium (P = 0.001) were also found in blackberries in those 13 locations. These results for lead and platinum are as expected because road traffic 14 represents a significant localised source of emissions of these elements to the 15 environment. 16 17

F
The levels of the four elements (arsenic, cadmium, mercury and lead) with the 18 greatest potential to be toxic to humans are illustrated for edible fungi in figure 1 Several samples of fungi showed high arsenic levels, the highest being in Agaricus 26 macrosporus (0.97 mg/kg), Lepiota procera (0.83 mg/kg) and Agaricus arvensis (0.73 27 mg/kg). These concentrations are, nonetheless, below the statutory limit of 1 mg/kg 28 for arsenic in commercial foods set by The Arsenic in Food Regulations 1959, as 29 amended, although those Regulations do not apply to wild foods collected for 30 personal consumption. There was no significant difference between arsenic 31 concentrations in urban and rural samples of fungi. 32 Levels of arsenic were very low in all the blackberry samples with the highest 1 concentration being 0.069 mg/kg. The results show significantly (P < 0.05) higher 2 concentrations in urban samples (mean 0.0102 mg/kg, range 0.0015 mg/kg to 0.0690 3 mg/kg) than rural samples (mean 0.0032 mg/kg, range 0.0011 mg/kg to 0.0079 4 mg/kg). However, there were no significant differences in arsenic levels between 5 roadside and non-roadside samples of blackberries. 6 7 Cadmium 8 9 Cadmium concentrations in wild fungi ranged from 0.02 mg/kg to 20.0 mg/kg with a 10 mean of 1.6 mg/kg. Relatively high concentrations of cadmium were found in several 11 species (Agaricus macrosporus, 20 mg/kg; Agaricus campestris, 8.7 mg/kg; and 12

Agaricus arvensis, 5.3 mg/kg). This could reflect either a higher rate of cadmium 13 accumulation in these species, or it could be that these samples happened to be 14 growing in soil that had higher levels of cadmium. 15 16 Some results from this study are somewhat higher than those from a study of wild 17 fungi undertaken in Croatia [START_REF] Mandic | The natural levels of aluminium, 15 cadmium and lead in wild mushrooms in Eastern Croatia[END_REF]) (mean 1.2 mg/kg, range 0.3 mg/kg 18 to 4.87 mg/kg dry weight) although different species were analysed. The mean 19 concentration of 1.6 mg/kg from this study is greater than the mean concentration of 20 0.018 mg/kg found in samples of commercial fungi reported elsewhere (MAFF 1997) 21 and also in 4 samples of mushroom in an earlier study of background levels of 22 cadmium in foods (MAFF 1983) where the mean concentration was 0.06 mg/kg (0.02 23 mg/kg to 0.08 mg/kg). Directive 2001/22/EC sets a limit of 0.2 mg/kg wet weight for 24 Cd in all cultivated fungi. Fourteen out of 34 samples exceeded this limit,which is not 25 applicable to wild fungi. 26 27 Cadmium concentrations in the blackberry samples ranged from 0.0007 mg/kg to 28 0.094 mg/kg with a mean concentration of 0.011 mg/kg. There was no significant 29 difference between the urban and rural samples or between roadside and non-roadside 30 samples. A MAFF study analysing foods grown in the Shipham area (MAFF 1983) 31 where elevated levels of cadmium are found as a result of historic mining activity, 32 found a commercial sample of blackberry with a cadmium level of 0.08 mg/kg, which 33 is at the higher end of the range found in samples from this survey. Although not a 34 Lead was detected in all of the wild fungi samples (mean 0.7 mg/kg, range 0.012 7 mg/kg to 6 mg/kg). The majority of the samples had lead concentrations below the 8 EU statutory limit of 0.3 mg/kg for lead in commercial fungi (Directive 2001/22/EC), 9 although, as noted above for arsenic, these statutory limits do not apply to wild foods 10 collected for personal consumption. Five samples with higher lead concentrations 11 were found, with the greatest (6.0 mg/kg) being in a sample of 'Wrinkled Club' 12 (Clavaria species). Statistical analysis showed there to be no significant difference 13 between urban and rural samples. 14

15

The mean concentration from this survey (0.7 mg/kg) is greater than results for 16 commercial fungi tested in an earlier JFSSG study, (MAFF, 1997a) where a mean 17 concentration of 0.01 mg/kg was obtained. However, this result is expected as 18 commercial fungi are different species, grown in controlled conditions and are 19 unlikely to be exposed to the same types of contaminant as those growing in the wild. 20

This has been shown in a previous study (Borella et al., 1994). A study in Croatia 21 measured a range of lead concentrations in edible wild fungi up to a high of 7.72 22 mg/kg [START_REF] Mandic | The natural levels of aluminium, 15 cadmium and lead in wild mushrooms in Eastern Croatia[END_REF]. 23 24 None of the wild blackberry samples had lead concentrations above 1 mg/kg. There 25 was no significant difference between the samples taken from urban sites and main 26 road sites. However, the mean concentration of lead in urban samples (0.093 mg/kg) 27 was significantly greater (P < 0.01) than in the rural samples (0.012 mg/kg), which 28 suggests that localised environmental contamination in urban areas and near main 29 roads does contribute to higher lead levels in wild blackberries. The mean 30 concentration of lead found in blackberries in this survey (0.05 mg/kg) is less than 31 that found in commercial fresh blackberries (0.07 mg/kg) in a previous survey 32 (MAFF, 1987a). 33 The concentrations, on a fresh weight basis, in all of the wild fungi samples were 3 above the LOD of 0.0006 mg/kg (mean 0.64 mg/kg, range 0.006 mg/kg to 4.2 mg/kg) 4 and there was no significant difference in levels of mercury between urban and rural 5 samples of fungi. Mean levels measured were greater than those for commercial 6 fungi (mean 0.02 mg/kg) in a survey of individual foods (MAFF, 1987b). However, 7 as noted above, commercial fungi samples may be different species and are grown in 8 controlled conditions and so this difference is not surprising. Falandysz et al. ( 2004) 9

found that bioconcentration factors of different species of wild edible mushroom 10 sampled in Poland varied on average between none and 73 times the concentration of 11 the soil substrate. The range for the majority of species was between 4 and 23 in the 12 caps. Actual values (on a dry matter basis) were between 0.17 mg/kg in the stalks and 13 0.92 mg/kg in the caps. They concluded from their study that even at a high 14 consumption level of 23 g per day of the most contaminated species (Amanita 15 muscaria), there was no risk to human health. 16 17 In wild blackberries, the mercury concentrations were very low (mean 0.00088 18 mg/kg, range 0.0006 mg/kg to 0.0079 mg/kg) with only 8 samples having mercury 19 concentrations above the LOD of 0.0006 mg/kg. 20 21

Conclusions 22 23

The levels of potentially harmful elements in wild foods were in general these low, 24 and often below the limit of detection. Concentrations of tin and titanium were 25 significantly higher at urban sites than at rural sites in both wild fungi and 26 blackberries. Platinum concentrations in blackberries were significantly higher at 27 roadside locations. Levels of platinum are believed to reflect its use in catalytic 28 converters and could be used to monitor future pollution. Levels of lead, which were 29 significantly greater in blackberries sampled within urban areas and adjacent to main 30 roads, might be expected to decrease in future, as lead is no longer added to petrol in 31 the UK. 32

  so the presence of titanium in plant samples can be used to indicate the 1 presence of soil or dust on the outer surface of the plant sample. 2 3

  undertaken between June and November 1998. Samples (ideally at 24 least 200 g) of wild fungi and blackberries were collected from the 'North', the 25 'Midlands' and the 'South' of mainland Great Britain. by Members of the British Mycological Society, with half 31 from urban and half from rural locations. These volunteers were used 32 as types of edible fungi can be difficult to find and to identify. They were provided 33 with written instructions on sample collection. Records included the date of 34 ordnance survey grid reference and whether the sample was from a 1

••••3

  discussed elsewhere (Baxter et al. 1997). Every multi-element analysis in this study 25 included the following quality control checks: 26 27 Measurement of a calibration standard at the start and end of each ICP-MS run -28 values to be within ± 20% of each other; 29 Spiked reagent blank recoveries to be within ± 20% of the expected value (and all 30 results are corrected for spike recovery); 31 Replicate determinations to give a relative standard deviation of ≤ 20% or twice Certified Reference Material (CRM) results -values to be within the certified 1 range or ± 40% of the certified value, whichever was the greater. 2 Every microwave digestion batch included a National Institute of Standards 4 Technology (NIST) certified reference material (CRM) (BCR 60 -Aquatic Plant; 5 BCR 61 -Aquatic Moss; NIST 1547 -Peach leaves; NIST 1570 -Spinach; NIST 6 1573 -Tomato leaves) and a duplicate sample. Each batch also contained reagent 7 blanks, and a reagent blank spiked with a known amount of each element for recovery 8 estimate purposes. Rhodium and indium were added to the digests as internal 9 standards before element concentrations were measured by ICP-MS. 10 11 The limits of detection of the elements analysed in the fungi and blackberries 12 surveyed are shown in

  and 19 for blackberries in figure2, with further comments on these results given below.

  Comment [C1]: Toby -can we give the exact numbers or proportion of samples over 0.2 mg/kg? strawberries analysed in a Finnish study[START_REF] Tahvonen | Lead and Cadmium in some Berries and 29 Vegetables on the Finnish Market in 1991-1993[END_REF]) 1 had a mean cadmium concentration of 0.007 mg/kg, which is similar to concentrations

6 7

 6 , Determination of bioavailability: platinum particles emitted from 4 automobile exhaust catalysts. News report -Institut Toxikologie und 5 Aerosolforschung. The Arsenic in Food Regulations 1959 (S.I. [1959] No. 831), as amended by The 8 Arsenic in Food (Amendment) Regulations 1960 (S.I. [1960] No. 2261) and The 9 Arsenic in Food (Amendment) Regulations 1973 (S.I. [1973] No. 1052). .S.H., Kovnatsky, E.F., Smetannikova, M.S., 1998, Absorption of heavy 13 metals in wild berries and edible mushrooms in an area affected by smelter emissions. 14 Water, Air and Soil Pollution, 103, 173-195. 15 16 Baxter, M.J., Crews, H.M., Robb, P., Strutt, P., 1997, Quality control in the multi-17 element analysis of foods using ICP-MS. Plasma source mass spectrometry: 18 developments and applications, edited by G. Holland and S.D. Tanner. The Royal 19 Society of Chemistry, London. 20 21 Borella, P., Caselgrandi, E., Fabio, G., Gibertoni, C., 1994, Risk of intake for 22 cadmium and lead with consumption of fresh mushrooms on sale. L'Igiene Moderna, 23 101, 323-331. 24 25 Falandysz, J., Jedrusiak, A., Lipka, K., Kannan, K., Kanwo. M., Gucia, M., 26 Brzostowski, A. and Dadej, M. (2004) Mercury in wild mushrooms and underlying 27 soil substrate from Koszalin, North-central Poland. Chemosphere, 54, 461-466. 28 29 Farago, M.E, Kavanagh, P., Blanks, R., Kelly, J., Kazantis, G., Thornton, I., Simpson, 30 P.R., Cook, J.M., Delves, H.T., Hall, G.E.M., 1998, Platinum concentrations in urban 31 road dust and soil, and in blood and urine in the United Kingdom.

table 2

 2

	13	.
	14
	15	[table 2 about here]
	16
	17

  This is consistent with previous studies(Kalac et al., 1991, Kalac et al., 10 1996, Barcan et al., 1998, Garcia et al., 1998[START_REF] Michelot | Metal content profiles in 19 mushrooms collected in primary forests of Latin America[END_REF].Over half the samples collected were Agaricus species. No urban samples were 15 collected from the north but two extra samples were collected from each of urban 16 midlands and urban south. These four extra samples were used in an analysis of urban 17 -rural differences in order to maintain the urban/rural balance within the survey. 18Several samples of wild fungi did show the ability to accumulate some relatively high 19 concentrations of certain elements, especially cadmium, copper, arsenic, mercury, 20 zinc and lead. This is consistent with earlier studies, which show that certain fungi 21 can accumulate very high concentrations of metals(Anon, 1998, Barcan et al., 1998, 22 Farago et al., 1998, Garcia et al. 1998, Hees et al., 1998, Kalac et al. 1991, Kalac et 23 a.l 1996, Michelot et al., 1999, Falandysz et al. 2004).

	1
	2	The results for each element are shown in tables 3 for wild fungi and 4 for wild
	3	blackberries. Further details can be found elsewhere (University of Bristol, 1999;
	4	MAFF 2000) . All results are reported on a fresh weight basis.
	5
	6		[tables 3 and 4 about here]
	7
	8	Several of the samples for both fungi and blackberries contained relatively high
	9	concentrations of some elements, and highly skewed distributions of values were
	obtained. 11 12 Fungi 13	o r F o r
	14		P P
			e e
			e r e r
			R R
			e e
	24 25		v i e v i e
	26 27	w Overall, there appeared to be few large or significant differences between samples from the rural and urban sites. Statistical analysis showed samples of fungi from w
	28 29 30 31 32	O n l y urban sites contained significantly higher concentrations of titanium, tin and platinum and lower levels of zinc (Mann Whitney, exact P values = 0.014, 0.019, 0.003, 0.028 respectively). O n l Blackberries y
	33
			8 9

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  Report on Surveillance for Veterinary Residues in Food in the UK, 2002. Veterinary 2

	1 2 3 4 5 6 7 8 9 10 11 12 13 1 3 4 5	F o r Garcia, M.A., Alonso, J., Fernandez, M.I., Melgar, M.J., 1998, Lead content in edible wild mushrooms in northwest Spain as indicator of environmental contamination. Archives of Environmental Contamination and Toxicology, 34, 330-335. Hees, T., Wenclawiak, B., Lustig, S., Schramel, P., Schwarzer, M., Schuster, M., Verstraete, D., Dams, D., Helmers, E., 1998, Distribution of platinum group elements (Pt, Pd, Rh) in environmental and clinical matrices: composition, analytical techniques and scientific outlook. Environmental Science and Pollution Research, 5, 105-111. Kalac, P., Burda, J., Staskova, I., 1991, Concentrations of lead, cadmium, mercury and copper in mushrooms in the vicinity of a lead smelter. Science of the Total Environment, 105, 109-119. F o r F o r Veterinary Residues Committee, 2002, The Veterinary Residues Committee Annual Residues Committee, Addlestone, Surrey. Page F o r
	14 15	P Kalac, P., Niznanska, M., Bevilaqua, D., Staskova, I., 1996, Concentrations of P P P
	16	e mercury, copper, cadmium and lead in fruiting bodies of edible mushrooms in the e e e
	17 18 19	e r vicinity of a mercury smelter and a copper smelter. The Science of the Total Environment, 177, 251-258. e r e r e r
	20 21 22 23 24 25	R e v i e MAFF, 1982, Survey of Lead in Food: Second Supplementary Report. Food R R R Surveillance paper No.10. HMSO, London. e e e MAFF, 1983, Survey of Cadmium in Foods: First Supplementary Report. Food Surveillance Paper No. 12. HMSO, London. v i e v i e v i e
	26 27	w MAFF, 1985, Survey of Aluminium, Antimony, Chromium, Cobalt, Indium, Nickel, w w w
	32 28 29 30 31 32	Analyst, 123, 451-O n l y Thallium and Tin in Food. Food Surveillance Paper No. 15. HMSO, London. MAFF, 1987a, Lead in Food: Progress Report. Food Surveillance Paper No.27. HMSO, London. O n l O n l O n l y y y
	33 33	454. MAFF, 1987b, Survey of Mercury in Food: Second Supplementary Report. Food
	34 34	Surveillance Paper No. 17. HMSO, London.
		14 16 17

Table 1 .

 1 Common names of fungi collected.

	Page
	1
	2
	LATIN NAME	COMMON NAME	Number sampled
	Corprinus atramentarius	Shaggy Ink Cap	1
	Corprinus comatus	Common Ink Cap	3
	Agaricus campestris	Common Field Mushroom	9
	Agaricus augustus	The Prince	1
	Agaricus arvensis	Horse Mushroom	5
	Agaricus silvaticus	Brown Wood Mushroom	2
	Agaricus langei	No widely used specific	1
		common name
	Agaricus macrosporus	No widely used specific	1
		common name
	Agaricus bisporus	Cultivated Mushroom	1
	Mycena pura	No widely used specific	1
		common name
	Lepista nuda	Wood Blewit	3
	Lepiota procera	Parasol Mushroom	2
	Hygrocybe pratensis	Meadow Wax Cap	2
	Langermannia gigantea	Giant Puff Ball	1
	Clavariaceae	Wrinkled Club	1
	3
	4

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				Page
	1
	2	Table 2. Limits of Detection (LOD)
	3
		ELEMENT SYMBOL	LOD (mg/kg)*
		Arsenic	As	0.0005
		Cadmium	Cd	0.0001
		Chromium	Cr	0.01
		Copper	Cu	0.002
		Lead	Pb	0.0006
		Manganese	Mn	0.01
		Mercury	Hg	0.0006
		Nickel	Ni	0.002
		Platinum	Pt	0.00001**
	4 5 6	Tin Titanium Zinc * fresh weight NB The LODs shown above are for a nominal sample weight of 4g, as taken in this survey Sn 0.001 Ti 0.001 F Zn 0.04 o r
	7 8 9	** For the wild blackberries, a more sensitive procedure using aqua regia digestion was used for which an LOD of 0.000006 mg/kg was obtained (see text) P
				e
				e r
				R
				e
				v i e
				w
				O n l
				y
				18

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	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	19

Table 3 .

 3 Summary statistics of the levels of the elements found within the mushroom

	1
	2	samples (mg/kg fresh weight).
	3
		N Median Range Minimum Maximum
		Ti 34 0.41	6.03	0.03	6.06
		Cr 34 0.04	0.96	0.01	0.97
		Mn 34 1.44	8.36	0.45	8.81
		Ni 34 0.044 0.348 0.009	0.357
		Cu 34 6.0	32.1	2.7	34.8
		Zn 34 11.4	31.0	3.6	34.6
		As 34 0.109 0.961 0.011	0.972
		Cd 34 0.17	19.58 0.02	19.60
		Sn 34 0.003 0.079 0.001	0.080
		Pt 34 0.00002 0.00019 0.00001 0.00020
		Hg 34 0.36	4.14	0.01	4.15
		Pb 34 0.22	5.98	0.01	5.99

Table 4 .

 4 Summary statistics of the levels of the elements found within the blackberry 2 samples (mg/kg (*Pt µg/kg) fresh weight).

	3
	4
	N Median Range	Minimum Maximum
	Ti 48 0.071	1.777	0.033	1.810
	Cr 48 0.02	1.23	0.01	1.24
	Mn 48 4.61	151.88	1.12	153.00
	Ni 48 0.087	0.670	0.025	0.695
	Cu 48 0.938	2.694	0.596	3.290
	Zn 48 2.55	4.03	1.52	5.55
	As 48 0.0035 0.0680	0.0011	0.0691
	Cd 48 0.0044 0.0936	0.0007	0.0943
	Sn 48 0.001	0.189	0.001	0.190
	Pt* 48 0.006	0.070	0.006	0.076
	Hg 48 0.0006 0.0073	0.0006	0.0079
	Pb 48 0.0128 0.5458	0.0032	0.5490
	5

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  Figure 1. Levels of arsenic, cadmium, mercury and lead in wild mushrooms. Two 2 extreme sample values for cadmium have not been plotted and were 8.67 and 19.6 3 mg/kg. Values are fresh weight basis with the centre line of the box representing the 4 median of the distribution, the box the interquartile range. The whiskers extend to 1.5 5 times the box length with outliers (circles) from 1.5 to 3.0 x box length and extreme 6 values (stars) above 3 x box length. Levels of arsenic, cadmium, mercury and lead in wild blackberries. See Fig 5 1 for explanation of box and whisker plots. Levels of lead (Pb) were variable and five 6 extreme values (0.127, 0.160, .0365, 0.382, 0.549) have not been plotted for clarity. 7 Note the scale is different from fig 1 and values are also on a fresh weight basis.

	Page 21 of 22			Food Additives and Contaminants	Page 22 of 22
	1 1 2 3 4
	7 8 9 Figure 2 8 9 10 11
		6.00 0.100
		5.00 0.080
	mg/kg mg/kg	1.00 0.020 2.00 3.00 4.00 0.040 0.060	F o r P e e r r e e P F o r
	10 11 12 13 14 15 16 17 19 20 21 22 25 26 27 28 29 30 31 32 34 35 36 40 39 38 37 33 24 23 18 12 13 14	0.000 0.00	As As	Cd Cd	R e v i e w O n l y Hg R Hg Pb Pb y l n O w e e i v
					21 22

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Monitoring of pesticide residues in apples, lettuce and potato of the Slovene origin in the years 2001 to 2004

Table 1. Sampling of agricultural products in the period 2001-2004 2001 2002 2003 2004 check the proper use of pesticides and be assured of the quality of fresh food produced by Slovene producers for the market.

.0 fludioxonil 0 1 0 2 0.0 3.3 0.0 7.1 iprodione 1 0 0 0 6.7 0.0 0.0 0.0 metalaxyl 0 2 0 0 0.0 6.7 0.0 0.0 pirimiphos-methyl 0 1 0 0 0.0 3.3 0.0 0.0 procymidone 1 1 0 1 6.7 3.3 0.0 3.6 tolylfluanid 0 0 0 1 0.0 0.0 0.0 3.6 vinclozolin 3 0 0 0 20.0 0.0 0.0 0.0
The purpose of the present paper is to present the results of pesticide residues in apples, lettuce and potatoes from 2001 to 2004 and compare with the results of monitoring in those three matrices in Belgium [START_REF] Dejonckheere | Monitoring of Pesticide Residues in Fresh Vegetables, Fruits, and Other Selected Food Items in Belgium, 1991-1993[END_REF] Egypt [START_REF] Dogheim | Monitoring of pesticide residues in Egyptian fruits and vegetables during 1997[END_REF] and Denmark [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market[END_REF].

Materials and methods

Sampling

In the period 2001 to 2004, 404 apple, lettuce and potato samples were collected. The sampling was random and it was carried out in eight agricultural production areas of Slovenia (Ljubljana, Kranj, Nova Gorica, Koper, Novo mesto, Murska Sobota, Maribor and Celje). Agricultural products were taken directly from the field or from storehouses after the pre-harvest interval for the plant protection products that had been used. The sampling was performed by agricultural inspectors and a sampler from the Agricultural Institute of Slovenia in collaboration with Agricultural Advisory Service during the period from May to November. The programme of sampling is shown in Table 1.

[Insert table 1 about here]

Analytical methods

Multi-residue method

The following pesticides were analysed using a multi-residue method in the years 2001 heptenophos, imazalil, iprodione, lindane, malathion, mecarbam, metalaxyl, methamidophos, methidathion, parathion, permethrin, phosalone, pirimiphos-methyl, procymidone, pyridaphenthion, quinalphos, thiabendazole, triazophos and vinclozolin. In 2003 the following pesticides were added to the programme: azoxystrobin, bromopropylate, chlorothalonil, cypermethrin, dichlofluanid, omethoate, oxydemetonmethyl, phorate, propyzamide and tolylfluanid.

In 2004 the following pesticides were added to the choice of those from 2003: cyprodinil, diphenylamine, kresoxim-methyl, myclobutanil, pyrimethanil and spiroxamine.

Extraction and clean-up

The sample was cut in four pieces; two opposite quarters were taken, mixed and stirred.

The mixed sample was extracted immediately or it was kept in plastic vessels at -20ºC.

To 20 g of mixed sample the following mixture of three solvents was added; acetone, petroleum ether and dichloromethane at a ratio of 1:2:2, homogenised with an Ultraturrax and filtered through filter paper (black strip) into a porcelain Büchner funnel apparatus. The lower water phase was separated and the upper organic one was filtered through filter paper (black strip) into a Soxhlet flask. The solvents were evaporated on a rotary-evaporator and blown to dryness under nitrogen. The extract was dissolved in a mixture of cyclohexane and ethyl acetate at a ratio of 1:1, filtered through filter, pore size 0,2 µm, and cleaned-up by gel permeation chromatography. The eluate was evaporated on a rotary-evaporator and blown down with nitrogen. The dry residue was dissolved in acetone (Baša [START_REF] Česnik | Multirezidualna metoda za določevanje ostankov pesticidov v sadju in zelenjavi, Zbornik Biotehniške fakultete univerze v Ljubljani, Kmetijstvo[END_REF][START_REF] Makovi | Pesticide Analytical Manual, Vol. 1, Extraction with acetone, liquid-liquid partitioning with petroleum ether/methylene chloride[END_REF]Thier and Zeumer, 1987;Thier and Zeumer, 1992).

Determination

The content of pesticide residues was determined by GC/MS (MSD). The compounds were separated on a HP-5 MS column (30 m x 0.25 mm x 0.25 µm). Selective ion monitoring was carried out for 2 -4 characteristic ions. In order to avoid the influence of matrix the calibration was carried out using matrix matched standards. Identification was based on a comparison of retention times and mass spectra of standards and samples. Quantitative was carried out on target ion (Baša [START_REF] Česnik | Multirezidualna metoda za določevanje ostankov pesticidov v sadju in zelenjavi, Zbornik Biotehniške fakultete univerze v Ljubljani, Kmetijstvo[END_REF][START_REF] Fillion | Multiresidue Method for the Determination of Residues of 251 Pesticides in Fruit and Vegetables by Gas Chromatography/Mass Spectrometry and Liquid Chromatography with Fluorescence Detection[END_REF].

The multi-residue method was validated for apples, lettuce and potatoes. The limit of detection, limit of quantification, repeatability, standard deviation and recoveries of this method are shown in Tables 2 to 7.

[Insert tables 2 to 7 about here]

Determination of dithiocarbamates

Extraction

The sample was cut in four pieces; two opposite quarters were taken and cut into smaller units. The sample was extracted immediately or it was kept until analysed in plastic vessels at -20ºC. To 50 g of unfrozen sample, 40 ml isooctane and 100 ml solution of tin (II) chloride (20 g tin (II) chloride + 500 ml HCl filled up to 1L with MilliQ water) was added. The samples were heated for at least 60 min at 80ºC. This yielded carbon disulphide (CS 2 ) which was dissolved in the organic phase (isooctane) (Restec Laboratories Limited, 1997).

Determination

The CS 2 in the isooctane phase was determined using GC/MS with selective ion monitoring. (Restec Laboratories Limited, 1997).

Determination of benomyl, carbendazim and thiabendazole

During the extraction benomyl is transformed into carbendazim.

Extraction and clean-up:

The sample received was cut in four pieces; two opposite quarters were taken, mixed and stirred. The mixed sample was extracted immediately or it was kept in plastic vessels at -20ºC. 8 g of unfrozen sample was weighed into centrifuge tube. To the sample 15 ml acetone was added and homogenised with an Ultra Turax blender for 30s.

15 mL dichloromethane and 15 mL petroleum ether were added and homogenised for 60 s. The homogenate was centrifuged for 5 min at 400 rpm. 30 mL of the upper, organic phase was pipetted into a Soxhlet flask and evaporated to dryness on a rotary evaporator. The residue was dissolved in 2 mL solution of internal standard benzimidazole (10 mg benzimidazole are diluted to 100 ml with methanol; from this solution 0.6 ml are pipetted and diluted to 100 ml with methanol). For the clean-up diol bonded silica extraction columns were used which were first conditioned with 2 mL methanol. Then 1 mL of extract was applied and washed with 1 mL methanol. The column was dry in approximately 1 min, then it was eluted with 2 mL solution of Health, Welfare and Sport, 1996).

Quality assurance

Each series of analyses included two spiked samples of each commodity analysed. For each batch recoveries had to be 70 -120 % to be acceptable. The recoveries were allowed to be higher for those active compounds which were not found in the samples. In potato samples, residues of only dithiocarbamates or no residues at all were found.

Active substances found during the four year monitoring in apple, lettuce and potatoes samples are shown in table 8. The frequency of pesticides found in apples, lettuce and potato for each year of sampling are shown in tables 9-11.

[Insert tables 8-11 about here]

A comparison of the results for apples in Slovenia with the results obtained for apples in Denmark has shown captan, folpet, dichlofluanid and dithiocarbamates to be found in both countries [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market[END_REF]. Bromopropylate and phosalone were found in apples both in Egypt and in Slovenia [START_REF] Dogheim | Monitoring of pesticide residues in Egyptian fruits and vegetables during 1997[END_REF] captan, diazinon and tolylfluanid were found in apples in Belgium and in Slovenia [START_REF] Dejonckheere | Monitoring of Pesticide Residues in Fresh Vegetables, Fruits, and Other Selected Food Items in Belgium, 1991-1993[END_REF].

[Insert table 12 about here]

A comparison of the results obtained for lettuce in Slovenia with those obtained in Denmark indicated comparable levels of dithiocarbamates and iprodione in both countries [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market[END_REF]. Dimethoate was found in lettuce both in Egypt and in Slovenia [START_REF] Dogheim | Monitoring of pesticide residues in Egyptian fruits and vegetables during 1997[END_REF]. Dithiocarbamates, iprodione, metalaxyl, procymidone, tolylfluanid and vinclozolin were found in lettuce in Belgium and in Slovenia [START_REF] Dejonckheere | Monitoring of Pesticide Residues in Fresh Vegetables, Fruits, and Other Selected Food Items in Belgium, 1991-1993[END_REF].

[Insert table 13 about here]

In the potatoes grown in Slovenia only dithiocarbamates were found but they were not found either in Denmark or in Belgium [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market[END_REF].

[Insert table 14 about here]

The active substances which exceeded the MRL in apples in Slovenia were phosalone in 1 sample (0.7%) and tolylfluanid in 2 samples (1.3%). The content of phosalone in the sample was 2.24 mg/kg, compared to the MRL of 2 mg/kg. The content of tolylfluanid was 0.33 mg/kg in one sample and 0.95 mg/kg in the other one (MRL for tolylfluanid in apples is 0.21 mg/kg). In lettuce from Slovenia, the MRL was exceeded by: dimethoate, dithiocarbamates and metalaxyl, each in 1 sample (1.0%). Dimethoate was present at 2.56 mg/kg (MRL of 1 mg/kg), dithiocarbamates at 6.36 mg/kg (MRL of 5 mg/kg) and metalaxyl was present at 0.14 mg/kg (MRL was 0.05 mg/kg).

In potatoes from Slovenia, the MRL of 0.05 mg/kg was exceeded by dithiocarbamates in 36 samples (23.1%) at levels ranging from 0.06 to 0.51 mg/kg. Considering these exceedances for dithiocarbamates in potatoes a question arises as to whether the treatment of this crop was really carried out in accordance with the registered use. No exceedances of MRLs were found in apples, lettuce or potatoes in Denmark [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market[END_REF]. In Egypt no exceedances of MRLs were found in apples whilst in lettuce dimethoate was exceeded. The maximum value of dimethoate was 2.90 mg/kg, with an MRL of 2 mg/kg [START_REF] Dogheim | Monitoring of pesticide residues in Egyptian fruits and vegetables during 1997[END_REF]. Vinclozolin was exceeded in apples in

Belgium at a maximum level 1.05 mg/kg (MRL of 1 mg/kg). In lettuce the following substances were exceeded: bromide at a maximum level of 351 mg/kg, (MRL of 50 mg/kg), dithiocarbamates at a maximum level of 33.18 mg/kg, (MRL of 5 mg/kg), iprodione at a maximum level of 61.5 mg/kg, (MRL of 10 mg/kg), metalaxyl at maximum level of 0.38 mg/kg, (MRL of 0.1 mg/kg), propamocarb at a maximum level of 10.61 mg/kg, (MRL of 5 mg/kg), propyzamide at a maximum level of 0.12 mg/kg, (MRL of 0.1 mg/kg) and vinclozolin at a maximum level of 13.73 mg/kg, (MRL of 5 mg/kg). In potato chlorpropham was found at a maximum level of 7.2 mg/kg, (MRL of 5 mg/kg) and thiabendazole was found at a maximum level of 5.97 mg/kg, (MRL of 3 mg/kg) [START_REF] Dejonckheere | Monitoring of Pesticide Residues in Fresh Vegetables, Fruits, and Other Selected Food Items in Belgium, 1991-1993[END_REF].

Conclusions

The contamination with pesticide residues of apples, lettuce and potato produced in Slovenia in the years 2001 to 2004 is not any cause for concern. Fifty-one percent of samples of apples, lettuce and potatoes did not contain detectable residues. In 10.4 % of apple, lettuce and potatoes the MRL was exceeded, but to a large extent (8.9 %, 36 samples) this was caused by dithiocarbamates in potatoes. Dithiocarbamates are the only active substance found in potato. The limit of quantification for dithiocarbamates is 0.05 mg/kg, which was the MRL for potatoes. In January 2005, according to the new

Table 4 :

 4 Repeatability standard deviation of multiresidual method for apple, lettuce and potato

						total
	apples	15	30	36	70	151
	lettuce	15	30	24	28	97
	potato	30	30	35	61	156
	total	60	90	95 159	404

Table 5 :

 5 Recoveries (mg/kg) of multiresidual method for apple

	Pesticide	spiking level (mg/kg)	recovery (%)	CV (%)
	acephate	0.2	103	8.7
	aldrin	0.2	88	3.2
	azinphos-methyl	0.2	89	14.5
	captan	0.2	105	27.5
	carbofuran	0.2	90	4.6
	chlorpyriphos	0.2	89	3.1
	chlorpyriphos-methyl	0.2	90	4.1
	cyhalotrin-λ	0.2	90	6.0
	DDD-o,p	0.2	86	2.5
	DDD-p,p in DDT-o,p	0.2	92	11.8
	DDE-p,p	0.2	89	2.6
	DDT-p,p	0.2	112	31.6
	deltamethrin	0.2	90	21.0
	diazinon	0.2	89	2.7
	dimethoate	0.2	91	5.6
	endosulfan-α	0.2	89	4.2
	endosulfan-β	0.2	91	6.5
	endrin	0.2	93	13.7
	fenitrothion	0.2	94	6.2
	fenthion	0.2	64	14.0
	fludioxonil	0.2	56	18.2
	folpet	0.2	119	34.6
	HCH-α	0.2	89	4.7
	heptachlor	0.2	89	12.0
	heptenophos	0.2	91	3.6
	iprodione	0.2	89	9.4
	lindane	0.2	93	10.0
	malathion	0.2	90	2.8
	mecarbam	0.2	89	3.3
	metalaxyl	0.2	89	2.7
	methamidophos	0.2	80	10.4
	methidathion	0.2	92	5.2
	parathion	0.2	90	5.7
	permethrin-cis	0.2	91	8.1
	permethrin-trans	0.2	91	9.3
	phosalone	0.2	92	9.0
	pirimiphos-methyl	0.2	89	3.3
	procymidone	0.2	89	2.5
	pyridaphenthion	0.2	89	6.9
	quinalphos	0.2	89	3.8
	thiabendazole	0.2	78	9.0
	triazophos	0.2	88	3.8
	vinclozolin	0.2	89	3.1

Table 6 :

 6 Recoveries (mg/kg) of multiresidual method for lettuce

	Pesticide	spiking level (mg/kg)	recovery (%)	CV (%)
	acephate	0.2	101	6.4
	aldrin	0.02	85	4.8
	azinphos-methyl	0.2	93	4.9
	captan	0.5	73	15.6
	carbofuran	0.05	100	4.5
	chlorpyriphos	0.05	105	4.4
	chlorpyriphos-methyl	0.05	102	4.1
	cyhalotrin-λ	0.05	106	8.3
	DDD-o,p	0.2	91	5.1
	DDD-p,p in DDT-o,p	0.1	101	7.5
	DDE-p,p	0.2	94	4.2
	DDT-p,p	0.2	93	5.0
	deltamethrin	0.2	98	5.4
	diazinon	0.02	85	5.0
	dimethoate	0.05	97	5.1
	endosulfan-α	0.13	89	6.6
	endosulfan-β	0.067	89	6.6
	endrin	0.05	106	7.2
	fenitrothion	0.05	93	5.9
	fenthion	0.05	98	5.1
	fludioxonil	0.05	103	8.7
	folpet	0.5	71	18.2
	HCH-α	0.02	87	4.9
	heptachlor	0.02	82	6.9
	heptenophos	0.2	96	3.7
	imazalil	0.05	93	8.3
	iprodione	0.5	94	7.7
	lindane	0.05	110	4.5
	malathion	0.2	92	3.9
	mecarbam	0.2	91	4.6
	metalaxyl	0.05	103	4.5
	methamidophos	0.05	99	17.3
	methidathion	0.2	89	6.5
	parathion	0.05	92	6.2
	permethrin-cis	0.073	94	4.7
	permethrin-trans	0.032	109	8.6
	phosalone	0.05	106	7.5
	pirimiphos-methyl	0.05	100	4.2
	procymidone	0.05	104	6.0
	pyridaphenthion	0.05	106	7.7
	quinalphos	0.05	101	6.7
	thiabendazole	0.05	103	10.1
	triazophos	0.2	93	5.0
	vinclozolin	0.05	106	4.6

Table 7 :

 7 Recoveries (mg/kg) of multiresidual method for potato

	Pesticide	spiking level (mg/kg)	recovery (%)	CV (%)
	acephate	0.2	100	19.6
	aldrin	0.05	95	3.7
	azinphos-methyl	0.05	102	6.9
	captan	0.2	97	4.9
	carbofuran	0.02	88	6.5
	chlorpyriphos	0.02	89	6.9
	chlorpyriphos-methyl	0.02	86	5.9
	cyhalotrin-λ	0.05	99	6.3
	DDD-o,p	0.02	87	6.6
	DDD-p,p in DDT-o,p	0.1	100	6.7
	DDE-p,p	0.02	88	5.8
	DDT-p,p	0.2	98	4.9
	deltamethrin	0.05	93	7.2
	diazinon	0.02	85	6.8
	dimethoate	0.02	86	6.7
	endosulfan-α	0.033	95	14.8
	endosulfan-β	0.067	96	4.0
	endrin	0.02	88	5.5
	fenitrothion	0.05	97	5.5
	fenthion	0.2	85	4.2
	fludioxonil	0.05	99	6.7
	folpet	0.2	101	8.7
	HCH-α	0.02	84	5.6
	heptachlor	0.02	83	6.1
	heptenophos	0.02	87	7.2
	imazalil	0.05	100	7.3
	iprodione	0.05	99	6.2
	lindane	0.2	94	4.6
	malathion	0.05	98	3.6
	mecarbam	0.02	87	6.8
	metalaxyl	0.02	85	5.8
	methamidophos	0.2	96	9.1
	methidathion	0.02	90	6.0
	parathion	0.05	100	5.7
	permethrin-cis	0.018	99	5.9
	permethrin-trans	0.032	99	6.3
	phosalone	0.05	99	6.5
	pirimiphos-methyl	0.02	86	5.9
	procymidone	0.02	86	6.0
	pyridaphenthion	0.05	102	5.3
	quinalphos	0.02	88	6.6
	thiabendazole	0.05	98	8.1
	triazophos	0.5	94	4.3
	vinclozolin	0.02	84	9.8

P e e r R e v i e w O n l y

Table 8 :

 8 Active substances found in apple, lettuce and potato samples

			apples			lettuce			potato
		no. of	portion	range	no. of	portion	range	no. of	portion	range
		samples	(%)	(mg/kg)	samples	(%)	(mg/kg)	samples	(%)	(mg/kg)
	bromopropylate	2	1.3	0.03 -0.03	0	0.0	-	0	0.0	-	... [1]
	captan chlorpyriphos-methyl cyhalotrin-λ	26 8 1	17.2 5.3 0.7	0.02 -0.97 0.01 -0.03 0.02	0 0 0	0.0 0.0 0.0	---	0 0 0	0.0 0.0 0.0	---	... [2] ... [3]
	cyprodinil	3	2.0	0.01 -0.02	2	2.1	0.02 -0.11	0	0.0	-	... [4]
	diazinon	46	30.5	0.01 -0.15	0	0.0	-	0	0.0	-	... [5]
	dichlofluanid	3	2.0	0.02 -0.51	0	0.0	-	0	0.0	-	... [6]
	dimethoate	0	0.0	-	5	5.2	0.03 -2.56	0	0.0	-	... [7]
	dithiocarbamates	66	43.7	0.05 -0.80	30	30.9	0.06 -6.36	38	24.4	0.05 -0.51	... [8]
	fludioxonil folpet	0 5	0.0 3.3	-0.05 -0.06	3 0	3.1 0.0	0.02 -0.12 -	0 0	0.0 0.0	--	... [9]
	iprodione	0	0.0	-	1	1.0	0.05	0	0.0	-	... [10]
	metalaxyl	0	0.0	-	2	2.1	0.03 -0.14	0	0.0	-	... [11]
	phosalone	56	37.1	0.01 -2.24	0	0.0	-	0	0.0	-	... [12]
	pirimiphos-methyl	0	0.0	-	1	1.0	0.03	0	0.0	-	... [13]
	procymidone	1	0.7	0.02	3	3.1	0.01 -1.28	0	0.0	-	... [14]
	pyrimethanil tolylfluanid vinclozolin	4 23 0	2.6 15.2 0.0	0.01 -0.03 0.01 -0.95 -	0 1 3	0.0 1.0 3.1	-0.02 0.02 -0.10	0 0 0	0.0 0.0 0.0	---	... [15] ... [16]
											... [17]
											... [18]
											... [19]

Table 9 :

 9 Frequency of pesticides found in apple samples

			number of samples			sample portion (%)
	active substance	2001	2002	2003	2004	2001	2002	2003	2004
	bromopropylate	0	0	2	0	0.0	0.0	5.6	0.0
	captan	4	1	0	21	26.7	3.3	0.0	30.0
	chlorpyriphos-methyl	0	0	2	6	0.0	0.0	5.6	8.6
	cyhalotrin-lambda	1	0	0	0	6.7	0.0	0.0	0.0
	cyprodinil	0	0	0	3	0.0	0.0	0.0	4.3
	diazinon	6	5	12	23	40.0	16.7	33.3	32.9
	dichlofluanid	0	0	2	1	0.0	0.0	5.6	1.4
	dithiocarbamates	5	13	23	25	33.3	43.3	63.9	35.7
	folpet	0	0	0	5	0.0	0.0	0.0	7.1
	phosalone	6	15	16	19	40.0	50.0	44.4	27.1
	procymidone	0	0	1	0	0.0	0.0	2.8	0.0
	pyrimethanil	0	0	0	4	0.0	0.0	0.0	5.7
	tolylfluanid	0	0	0	23	0.0	0.0	0.0	32.9

Table 10 :

 10 Frequency of pesticides found in lettuce samples

			number of samples			sample portion (%)
	active substance	2001	2002	2003	2004	2001	2002	2003	2004
	cyprodinil	0	0	0	2	0.0	0.0	0.0	7.1
	dimethoate	1	3	1	0	6.7	10.0	4.2	0.0
	dithiocarbamates	3	7	6	14	20.0	23.3	25.0	50

Table 11 :

 11 Frequency of dithiocarbamates found in potato samples

									Formatted: Centered
									Formatted Table
									Formatted: Centered
									Formatted Table
									Formatted: Centered
									Formatted: Centered
			number of samples			sample portion (%)	Formatted: Centered
	active substance	2001	2002	2003	2004	2001	2002	2003	2004
	dithiocarbamates	6	13	14	5	20.0	43.3	40.0	8.2
									Formatted
									Formatted: Centered

Table Deleted :

 Deleted Table 3: : Apple, lettuce and potato samples which exceeded maximum residue levels ¶ matrix

		... [20]
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	y

Table 12 :

 12 Active substances found in apple samples (%)

	Denmark		Egypt	Belgium	Slovenia	Formatted Table
	bifenthrin	n.d.	n.a.	0.9	n.a.
	bitertanol	1.2	n.a.	n.d.	n.a.
	bromopropylate	n.d.	6.8	51.3	1.3
	captan		n.a.	4.3	17.2	Formatted: Right
	captan + folpet	14.6	n.a.
	carbendazim	1.2	n.a.	9.6	n.d.
	carbosulfan	n.d.	1.4	n.d.	n.a.
	chlorpyrifos	n.d.	n.d.	0.9	n.d.
	chlorpyrifos-methyl	n.d.	n.d.	n.d.	5.3
	cyhalotrin-λ	n.a.	n.a.	n.d.	0.7	Formatted: Font: Symbol
	cypermethrin	n.d.	5.5	n.a.	n.d.	Formatted
	cyprodinil	n.a.	n.a.	n.a.	2.0
	diazinon	n.d.	n.d.	0.9	30.5
	dichlofluanid	1.2	n.d.	n.d.	2.0
	dicofol	n.d.	13.7	n.d.	n.a.
	dimethoate	n.d.	4.1	1.7	n.d.
	dithiocarbamates	15.9	n.d.	n.a.	43.7
	endosulfan I	n.d.	n.a.	0.9	n.d.
	endosulfan II	n.d.	n.a.	0.9	n.d.
	endosulfan SO 4	n.d.	n.a.	0.9	n.a.
	fenitrothion	n.d.	n.d.	0.9	n.d.
	fenpropathrin	1.2	n.a.	n.a.	n.a.
	fenvalerate	n.d.	1.4	n.a.	n.a.
	folpet		n.a.	n.d.	3.3
	malathion	n.d.	1.4	n.d.	n.d.
	metalaxyl	n.d.	n.d.	0.9	n.d.
	methoxychlor	n.d.	50.0	n.a.	n.a.
	phosalone	n.d.	5.5	n.a.	37.1
	pirimicarb	1.2	n.d.	1.7	n.a.
	procymidone	n.d.	n.d.	n.d.	0.7
	pyrimethanil	n.a.	n.a.	n.a.	2.6
	teradifon	n.a.	1.4	n.a.	n.a.
	thiabendazole	n.d.	n.a.	0.9	n.d.
	tolylfluanid	n.d.	n.a.	7.8	15.2
	triazophos	n.d.	1.4	n.a.	n.d.
	vinclozolin	n.d.	n.d.	14.8	n.d.

n.a. means not analysed n.d. means not detected

Table Formatted Table Formatted Table Formatted Table Deleted :

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Table 13 :

 13 Active substances found in lettuce samples (%)

	Denmark		Egypt	Belgium	Slovenia
	bifenthrin	n.d.	n.a.	5.4	n.a.
	bromide	n.a.	n.a.	64.3	n.a.
	chlorpirifos	n.d.	n.d.	1.8	n.d.
	cypermethrin	2.3	n.d.	n.a.	n.d.
	cyprodinil	n.a.	n.a.	n.a.	2.1
	diclofluanid	n.d.	n.d.	0.9	n.d.
	dicloran	n.d.	n.a.	0.9	n.a.
	dimethoate	n.d.	3.6	n.d.	5.2
	dithiocarbamates	9.1	n.d.	14.3	30.9
	endosulfan	2.3	n.a.	n.d.	n.d.
	fludioxonil	n.a.	n.a.	n.a.	3.1
	folpet	n.d.	n.a.	0.9	n.d.
	iprodione	2.3	n.d.	26.8	1.0
	malathion	n.d.	3.6	n.d.	n.d.
	metalaxyl	n.d.	n.d.	0.9	2.1
	methomyl	n.a.	n.a.	6.3	n.a.
	parathion	n.d.	n.a.	0.9	n.d.
	permethrin	n.d.	n.d.	3.6	n.d.
	pirimicarb	n.d.	n.d.	2.7	n.a.
	pirimiphos-methyl	n.d.	n.d.	n.d.	1.0
	procymidone	n.d.	n.d.	0.9	3.1
	propamocarb	n.a.	n.a.	38.4	n.a.
	propyzamide	n.d.	n.a.	0.9	n.d.
	tolclofos-methyl	n.d.	n.d.	11.7	n.a.
	tolylfluanid	n.d.	n.a.	2.7	1.0
	vinclozolin	n.d.	n.d.	47.3	3.1
	n.a. means not analysed
	n.d. means not detected

Table 14 :

 14 Active substances found in potato samples (%)

	Denmark	Belgium	Slovenia
	carbendazim	n.d.	1.9	n.d.
	chlorpropham	n.d.	33.3	n.a.
	dieldrin	1.2	n.a.	n.a.
	dithiocarbamates	n.d.	n.a.	24.4
	propham	n.d.	10.2	n.a.
	thiabendazole	n.d.	13.9	n.d.
	tolclofos-methyl	0.6	n.a.	n.a.
	n.a. means not analysed
	n.d. means not detected

Table Formatted Table Formatted Table Formatted Table Formatted Table Formatted Table F

 FormattedFormattedFormattedFormattedFormattedF

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	r r r r r r
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	e e r e e r e e r e e r e e r e e r
		R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
		O n O n O n O n O n O n
			l y l y l y l y l y l y

Table 3 :

 3 : Apple, lettuce and potato samples which exceeded maximum residue levels

			exceeded result	MRL
	matrix active substance	(mg/kg)	(mg/kg)
		phosalone	2.24	2
	apples	tolylfluanid	0.33	0.21
		tolylfluanid	0.95	0.21
		dimethoate	2.56	1
	lettuce	dithiocarbamates	6.36	5
		metalaxyl	0.14	0.05
	potato	dithiocarbamates	0.06	0.05
		dithiocarbamates	0.06	0.05
		dithiocarbamates	0.06	0.05
		dithiocarbamates	0.06	0.05
		dithiocarbamates	0.06	0.05
		dithiocarbamates	0.08	0.05
		dithiocarbamates	0.08	0.05
		dithiocarbamates	0.09	0.05

Results of BIPEA proficiency tests for apple in the period from 2002 to 2004 BIPEA, apple

			Food Additives and Contaminants	Page 20 of 36
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Results of BIPEA proficiency tests for lettuce in the period from 2002 to 2004 BIPEA, lettuce

	Page 21 of 36		Food Additives and Contaminants
		3,00
	z -score	1,00 2,00 0,00 -1,00	F o r F o r 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 P P e e
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Monitoring of pesticide residues in apples, lettuce and potatoes from Slovenia in the years 2001 to 2004 Abstract Apples

  , lettuce and potatoes (404 samples) produced in the Republic of Slovenia were analysed for pesticide residues at the Agricultural Institute of Slovenia in the years 2001 to 2004. Sampling from eight agricultural production areas was performed at market producers at the harvesting of products or in storehouses, after the pre-harvest interval of plant protection products used. Samples exceeding the maximum residue levels were

	Introduction
	Following authorisation from by the Ministry of Agriculture, Forestry and Food the
	Agricultural Institute of Slovenia introduced permanent and systematic surveillance
	programme for pesticide residues in agricultural products in 1987. Until 1999 the
	monitoring of pesticide residues was carried out within framework of expert projects.
	F o r Since 1999 the Program of National Monitoring of Pesticide Residue in Foodstuffs and Agricultural Products has been performed on the basis of a Decree on Monitoring of F o r
	P P
	e e the following: 3 apple samples (2.0%) containing 0.33 -2.24 mg/kg (phosalone and tolylfluanid), 3 lettuce samples (3.1%) containing 0.14 -6.36 mg/kg (dimethoate, e r e r
	dithiocarbamates and metalaxyl and 36 potato samples (23.1%) containing 0.06 -0.51 R R
	mg/kg (dithiocarbamates). Residues of two or more active substances were found in 73 e e
	apple samples (48.3%), of which MRL was exceeded by 3 samples (2.0%) with the values ranging from 0.33 -2.24 mg/kg and 9 lettuce samples (9.3%) for which the v i e v i e
	MRL was exceeded by 3 samples (3.1%) with the values from 0.14 -6.36 mg/kg. In w w
	potato samples only residues of dithiocarbamates or no residues at all were found. Residues of the dithiocarbamate group were the most frequently found, i.e. in 66 apple O n l (Commission recommendation 99/333/EC, Commission recommendation 02/663/EC O n l samples (43.7%), 30 lettuce samples (30.9%) and 38 potato samples (24.4%). y and Commission recommendation 04/74/EC). The monitoring results are intended to y
	Keywords: monitoring, pesticides, plant protection products check compliance with maximum residue limits (MRL), to determine the origin or
	explanation for any contaminated agricultural products and to determine whether
	producers have followed good agricultural practice. By determining pesticide residues
	in agricultural products after picking, digging or harvesting (until marketing) we could

Pesticide in Foodstuffs and Agricultural Products (Official Gazette of the Republic of Slovenia No. 13/99). It was harmonised with the monitoring programme within the Ministry of Agriculture, Forestry and Food (Agricultural Institute of Slovenia) and Ministry of Public Health of the Republic of Slovenia (Institute of Public Health of RS and Institute of Public Health Maribor). The activity was based on guidelines and goals of the Law on Plant Protection Products (Of.G. RS No. 11/01) and of Regulations on Pesticide Residues in/on Foodstuffs and Agricultural Products (Of.G. RS No. 73/03).

Due to the characteristic type of foods consumed by Slovenes (Slovene Food Basket has not been made yet) pesticide residues were monitored each year in samples of potatoes, lettuce and apples while the choice of other agricultural products and active substances analysed was adjusted to the guidelines indicated in the EU recommendations

  Proficiency Test 06 and in 2005 in the European Proficiency Test 07. In 2005 our work was verified and accredited by French (COFRAC) accreditation body.

	substances were not found in any of the apple samples. The highest number of active
	substances found in one apple sample (0.7%) was eight.
	[Insert figures 1 and 2 about here] Nine out of 97 lettuce samples (9.3%) contained residues of two or more active
	substances. Residues of two active substances were found in 4 lettuce samples (4.1%) of
	F o r Results and discussion Out of the total of 151 apple samples, 3 samples (2.0%) exceeded the MRLs for which MRL for dimethoate was exceeded by one sample (1.0%) containing 2.56 mg/kg, F and residues of three active substances were found in 5 lettuce samples (5.2%) of which o r MRL was exceeded by 2 samples (2.1%) containing 0.14 -6.36 mg/kg (metalaxyl and
	P phosalone and tolylfluanid, 117 samples (77.5%) contained residues lower than MRLs, P dithiocarbamates). Residues of four or more active substances were not found in any
	e e r and no residues were found in 31 apple samples (20.5%). Out of the total of 97 lettuce samples, 3 samples (3.1%) exceeded MRLs for dimethoate, dithiocarbamates and e r e lettuce samples.
	R e metalaxyl), 37 samples (38.1%) contained residues lower than MRLs, and no residies R were detected in 57 lettuce samples (58.8%). Out of the total of 156 potato samples, 36 e
	v i e samples (23.1%) exceeded the MRL for dithiocarbamates. In 2 potato samples (1.3%) the residues were equal to the MRL. In 118 potato samples (75.6%) no residues were v i e
	w detected. 73 (48.3%) out of the total of 151 apple samples analysed contained residues w
	O n l of two or more active substances. Residues of two active substances were found in 39 apple samples (25.8%) of which MRL was exceeded by 2 samples (1.3%) with the levels from 0.95 -2.24 mg/kg (tolylfluanid and phosalone), residues of three active O n l
	The accuracy of methods has been verified since 2002 by participating in the French y substances were found in 25 apple samples (16.6%) for which MRL was exceeded by y
	interlaboratory proficiency scheme BIPEA (Bureau Interprofessionnel d′Etudes one sample (0.7%) at a level of 0.33 mg/kg (tolylfluanid), and residues of four or more
	Analytiques). Z-scores for apples and lettuce from 2002 to 2004 were mainly below ±2 active substances were found in 9 apple samples (6.0%). Residues were found of four
	and are presented in figures 1 and 2. In 2004 we also participated in the European active substances in 5 apple samples (3.3%), of five active substances in 2 apple
	samples (1.3%), and of six active substances in 1 apple sample (0.7%). Seven active

  Commission Directive 2004/115/EC, the MRL was raised to 0.1 mg/kg. If the new MRL is used, the number of samples exceeding the MRL would be reduced to 27 samples reducing the overall exceedances for apple, lettuce and potato samples to 8.2%. Official Gazette of the Republic of Slovenia No. 11/01. Official Gazette of the Republic of Slovenia No. 73/03. Restec Laboratories Limited, (1997) Determination of Dithiocarbamates and Thiuram Disulphide, Pesticide residues in fruit and vegetables. Thier, H.P., Zeumer, H., DFG (1987) Manual of Pesticide Residue Analysis, Vol. 1, Organochlorine, Organophosphorus, Nitrogen-Containing and Other Pesticides. Weinheim, VCH, Verlagsgesellschaft, 383 -400. Thier, H.P., Zeumer, H., DFG (1992) Manual of Pesticide Residue Analysis, Vol. 2, Cleanup Method 6. Weinheim, VCH, Verlagsgesellschaft, 31 -36.

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Food Additives and Contaminants

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

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Terje Tamme
email: fac@tandf.co.uk

Nitrates and nitrites in

Keywords: nitrate, nitrite, vegetables, intake http://mc.manuscriptcentral.com/

published or not. The documents may come

Introduction

Nitrate and nitrite can be found in food as naturally occurring compounds, drinking water and vegetables being substantial sources of nitrate intake. Interest in the dietary intakes of nitrates and nitrites has arisen from the concern about their possible adverse effect on health (Knekt et al.1999;[START_REF] Pegg | Nitrite curing of meat. The nitrosamine problem and nitrite alternatives[END_REF]. An acceptable daily intake (ADI) of 0 to 3.7 mg kg -1 body weight for nitrate and of 0 to 0.06 mg kg -1 body weight for nitrite has been established by EU Scientific Committee for Food ((EU Scientific Committee 1995). It has been estimated that vegetables constitute a major source of human exposure to nitrates contributing approximately 80 to 92% of the average daily intake ( [START_REF] Dich | Dietary intakes of nitrate, nitrite and NDMA in the Finnish Mobile Clinic Health Examination Survey[END_REF].

Levels of nitrates in vegetables differ to a large extent. According to nitrates content the vegetables can be divided into 3 groups:

1. Plants with high nitrate content (> 1000 mg kg -1 ) -beetroot, lettuce, spinach, other green leaf vegetables and herbs .; 2. Plants with average nitrate content (50 to 1000 mg kg -1 ) -potatoes and other vegetables;

3. Plants with low nitrate content (0.5 to 50 mg kg -1 ) -berries, fruits, cereals, pod vegetables.

Nitrate concentration in vegetables depends on the biological properties of the plant culture, lightning conditions, type of soil, temperature, humidity, frequency of plants in the field, plant maturity, vegetation period, harvesting time, size of vegetable unit, storage time and source of nitrogen [START_REF] Muramoto | Comparison of Nitrate Content in Leafy Vegetables from Organic and Conventional Farms in California[END_REF][START_REF] Fytianos | Nitrate and Nitrite Accumulation in Fresh Vegetables from Greece[END_REF].

Even among different samples of the same vegetable varieties, the range of concentrations can be manifold. The limit concentrations for nitrates have been established in EU for spinach, lettuce and baby foods (European Commission 2002;European Commission 2004). The nitrate ion has a low level of acute toxicity, but if transformed into nitrite, it may constitute a health problem. Reduction to nitrite may take place in the presence of bacteria or enzyme nitrate reductase, and in contact with metals. Nitrite is unstable at acidic pH values at which it can disproportionate to yield nitrate and nitrogen oxide and/or react with food components including amines, phenols and thiols [START_REF] Hill | Nitrate. Toxicological Evaluation of Certain Food Additives and Contaminants in Food[END_REF]. It has been estimated that 5 to 8% of the nitrate from the diet may be reduced to nitrite by the microflora in the oral cavity [START_REF] Mensinga | Health implications of exposure to environmental nitrogenous compounds[END_REF]. Although nitrates and nitrites have been used for centuries, it has only recently been discovered that nitrate is manufactured in mammals by the oxidation of nitric oxide and that the nitrate formed has the potential for disinfecting the food we eat [START_REF] Benjamin | Nitrates in the human diet -good or bad?[END_REF][START_REF] Archer | Evidence that ingested nitrate and nitrite are beneficial to health[END_REF].

Nitrite in food is primarily considered to be a health problem because its presence both in food and body may lead to the formation of carcinogenic nitrosoamines (JECFA 1996;Vermeer et al. 1998) and the clinical symptom of methemoglobinemia [START_REF] Who | Evaluation of certain food additives and contaminants[END_REF]. Although carcinogenicity of N-nitrosoamines in humans cannot be tested, epidemiological studies have suggested a possible link to the incidence of various cancers in humans [START_REF] Pegg | Nitrite curing of meat. The nitrosamine problem and nitrite alternatives[END_REF]. It is possible that other factors such as intake of vegetables, fruit and nitrosation inhibitors, or some component of cured meat and salted fish other than N-nitroso compounds could partly be responsible for the observed associations [START_REF] Eichholzer | Dietary nitrates, nitrites, and N-nitroso compounds and cancer risk with special emphasis on the epidemiological evidence[END_REF]. Consumption of vegetables containing high level of nitrates and incorrect storage of home-made vegetable purees has been found to be potential causes of infant methemoglobinemia [START_REF] Reinik | Estonian National Monitoring programme of Food Additives 2000[END_REF]. Processed infant foods may also contain increased levels of nitrites [START_REF] Hill | Nitrate. Toxicological Evaluation of Certain Food Additives and Contaminants in Food[END_REF]. Estonian food safety monitoring programme was initiated in 1998 [START_REF] Reinik | Estonian National Monitoring programme of Food Additives 2000[END_REF].

Concentrations of food additives and contaminants in main food groups are determined annually within the frames of the programme. Obtained data were used for assessment of dietary risks connected with consumption of food additives and contaminants.

Materials and methods

Samples

Altogether, 1349 samples of vegetables and ready-made food were analyzed in 2003 -2004. Samples were collected mainly at the retail level by food inspection authorities within the frames of state official surveillance and monitoring programme, by researchers within activities of the Estonian Science Foundation grant no. 5416, or were brought to the laboratory by the farmers with the aim of self-control.

Approximately 80% of the analyzed vegetables had been grown in Estonia. The minimal amount of vegetable sample was 1 kg. Sample collection covered all seasons.

In addition to raw vegetables, vegetable-based baby foods and ready-made soups were analyzed.

Methods of analysis

Nitrate concentrations in vegetables were determined by potentiometric method based on Russian standard GOST 4228-86 (Gosudarstvennõi agropromõšlennõi komitet 1986). Prior to analysis non-edible parts of the sample were removed. In case of small vegetables the whole sample was chopped. Large sample units (e.g. head cabbage) were cut vertically into four pieces, one quarter from each unit was taken for the analysis. Vegetable juice was prepared using a juice press. Leafy and low juice-containing vegetables were finely chopped. Nitrates were extracted by KAl(SO 4 ) 2 solution and determined potentiometrically by ion-selective electrode. The method has been accredited by Estonian Accreditation Body. Limit of determination was 30 mg kg -1 and measurement uncertainty up to 15 % (k=2, norm.) depending on the sample matrix.

The concentration of nitrates and nitrites in infant purees and ready-made food were determined by an HPLC method, based on NMKL (Nordic Committee on Food Analysis) method no. 165, 2000. Nitrates and nitrites were extracted from the samples by hot water. Acetonitrile was added for removal of interfering substances. The filtered solution was injected into a Shimadzu LC10 chromatograph, nitrate and nitrite were separated by Alltech C 18 column and detected by UV-detector at the wavelength of 205 nm. The limit of quantification for both ions was 5 mg kg -1 , measurement uncertainty (U) at the concentration of 100 mg kg -1 was 12 mg kg -1 (k=2, norm.). The method has been accredited by Estonian Accreditation Body. The Tartu Laboratory of Estonian Health Protection Inspectorate has successfully participated in collaborative study of the method [START_REF] Merino | Liquid Chromatographic Determination of Residual Nitrite/Nitrate in Foods: NMKL Collaborative Study[END_REF] and in intercomparison tests.

Method of intake calculation

Different methods can be used for the estimation of additive and contaminant intake from food: food supply data, published tables of the mean consumption of dietary items, duplicate portion analysis, dietary survey among the individuals, probabilistic and worse case screening models. Depending on the method used, intake estimates can differ to a large extent [START_REF] Kroes | Assessment of intake from the diet[END_REF].

Results and discussion

Concentration of nitrates in vegetables

Limit values for nitrate concentration in several vegetables including potato, cabbage, carrot, beetroot, onion, cucumber etc. had been valid in Estonia before joining the EU in 2004. Due to the existing legal limits, a lot of samples were analyzed annually within the framework of official control. Maximum permitted concentrations were exceeded in 17% of the samples, most frequently for cucumber in winter period, spring onion, cabbage, turnip and beetroot. Exceedance of the EU permitted concentrations were not detected, although the nitrate concentration was close to the MRL in one spinach sample.

The results for nitrate analyses in all vegetables are shown in Table I. As seen from the table, the highest mean values of nitrates were detected in dill, spinach, lettuce and beetroot. The mean concentrations were 2936, 2508, 2167 and 1446 mg kg -1 , respectively. The lowest mean values of nitrates were detected in tomato, onion and potato, the contents being 41, 55 and 94 mg kg -1 , respectively. In vegetables of Estonian origin, the content of nitrites was lower than 5 mg kg -1 -the detection limit of the method. As in Estonia potatoes are consumed in large amounts, the number of the studied samples is the largest. Statistical difference between nitrate content of early, late and stored potatoes was not observed. Average nitrate concentration in imported potatoes was 200 mg kg -1 , significantly higher than that of domestic tubers. Similar nitrate concentrations have also been detected in other countries (Table II), with the exception of nitrates content in spinach and potato, according to the data of [START_REF] Chung | Survey of nitrate and nitrite contents of vegetables grown in Korea[END_REF]. The contents of nitrate in spinach grown in Korea and Japan were 1.5 to 2.0 fold higher. The nitrate content in potatoes of Estonian and Finnish origin was 94 and 82 mg kg -1 , in those of Korean and Japanese origin it was 452 mg kg -1 and 713 mg kg -1 , respectively [START_REF] Penttilä | Estimation of Food Additive and Pesticide Intakes by Means of A Stepwise Method[END_REF][START_REF] Chung | Survey of nitrate and nitrite contents of vegetables grown in Korea[END_REF]).

[Insert Table I and II about here]

Leafy vegetables accumulate high amounts of nitrates, concentrations reaching up to 6000 mg kg -1 . Nitrate concentration in some salad crops of different varieties during summer and winter seasons were screened by [START_REF] Escobar-Gutierrez | Screening lettuce cultivars for low nitrate content during summer and winter production[END_REF]: nitrate concentration showed great variability between cultivars and also varieties within one cultivar, exceeding of limit concentrations was more frequent in summer season than in winter. In the present study the nitrate content of lettuce was lower in summer (average 1952 mg kg -1 ) than in winter (average 3024 mg kg -1 ), exceeding of limit concentrations was not detected. [START_REF] Worthington | Nutritional quality of organic versus conventional fruits, vegetables and grains[END_REF] states that organic crops contained significantly less nitrates than conventional crops. The review of literature conducted by [START_REF] Heaton | Organic farming, food quality and human health[END_REF] found 14 studies showing lower nitrate content (50%) in organically grown crops and two studies showing insignificant differences.

Concentration of nitrates and nitrites in vegetable-based products for infants

Thirty one samples of canned infant food and 10 carrot juice samples were analyzed for obtaining data for intake calculations. Nitrate contents close to EU limit concentration of 200 mg kg -1 were detected in two samples. Nitrate concentration was the highest in the purees containing carrot and pumpkin: 62 to 148 mg kg -1 and 124 to 162 mg kg -1 , respectively. The highest nitrate content, 251 mg kg -1 , was detected in fresh carrot juice, which is frequently fed to babies (Table III). Similar results have been reported in a Spanish study [START_REF] Hardisson | The Evaluation of the Content of Nitrates and Nitrites in Food Products for Infants[END_REF], where nitrate concentrations exceeding the level of 250 mg kg -1 were found in baby foods in which the main ingredient was carrot (Table III). The mean contents of nitrates in vegetable-based infant foods of Estonian and Spanish origin were 88 and 92 mg kg -1 , respectively. Nitrites were not detected in samples of infant and ready-made food of Estonian origin.

[Insert Table III about here]

Intake calculation

Intake of nitrates has been evaluated, using the data from three different consumption surveys in Estonia (see Tables IV, V and VI). Data were collected from the household The data for consumption levels in Table V were corrected for cooking and peeling loss for mean nitrate content in mg kg -1 . The consumption data in Table VI were calculated on the basis of the infant menu recommended by Estonian paediatricians. The recommended infant menu of the age group of 6 to 12 months should contain 50% of vegetable based foods and 50% of fruit based foods in addition to breast-feeding. The average content of nitrates in each vegetable commodity or vegetable based food has been used in all calculations of the intake. The average body weight used for comparison of the results with ADI (Acceptable Daily Intake) was 60 kg. Estonian and foreign products were not differentiated in the survey from the National Board of Statistics of Estonia, both white and red cabbages were included in the consumed amounts of cabbage (Table IV).

The highest intakes of nitrates by Estonian population were obtained from consuming of cabbage and potato: 14 and 12 mg day -1 , respectively (Table IV). The following highest intakes were obtained by consuming of cucumber and beetroot: 8.2 and 7.3 mg day -1 , respectively. The mean total intake of nitrate per person in Europe ranges between 50 and 140 mg per day and in the USA about 40 to 100 mg per day [START_REF] Mensinga | Health implications of exposure to environmental nitrogenous compounds[END_REF][START_REF] Ysart | Dietary exposures to nitrate in UK[END_REF]. Mean daily intake of nitrates by Estonian 1-to 6-year-old children was calculated on the basis of the consumption survey from the menus and amounts of supplied vegetables of two kindergartens. Table V indicates the daily intakes of nitrates and the intake percentage from ADI value by children in the age group of 4 to 6 years, consuming vegetable-based foods. The intake of nitrates by 4-to 6-years-old children was 30 mg (40% of ADI), the average body weight of 20 kg was used in the calculations. The obtained results indicated that although the mean nitrate intakes did not exceed ADI, the maximum intakes were several times higher than mean values. The menu of the age group of 1-to 3-years-old was similar to 4-to 6-years-old children group with the difference of 15% less consumed food amount. According to the calculations, the intake of nitrates by this age group was 26 mg day -1 (52% of ADI). For calculation of intakes, the average body weight of 15 kg was used. The daily nitrate 11 intake by Finnish adolescents and children was 47.9 mg day -1 [START_REF] Penttilä | Estimation of Food Additive and Pesticide Intakes by Means of A Stepwise Method[END_REF]. In a Polish study [START_REF] Wawrzyniak | The evaluation of nitrate, nitrite and antioxidant vitamin intake in daily food ration of children aged 1-6 year of age[END_REF], nitrates intake for 1-to 6-years-old children exceeded ADI twice and the main food source of nitrates were vegetables (90%).

The mean daily intake of nitrate among Estonian infants aged 6 to 12 months was calculated on the basis of the recommended infant menu. Table VI indicates the recommended daily consumption amounts of vegetable-based purees and juices for infants. The mean contents of nitrates in infant foods are presented in Table III with the average content of 88 mg kg -1 . Table VI confirms that older infants get higher amounts of nitrates compared to younger ones. For instance, a 6 months old infant gets 4.4 mg and a 12 months old infant 11 mg nitrates per day. An infant's average daily intake of nitrates from consumption of vegetable-based infant foods was 7.8 mg (22% of ADI).

Conclusions

In the present investigation, the mean values and contents of nitrates and nitrites in vegetables and vegetable products were determined. In Estonia for the first time the daily intake of nitrates, obtained from vegetables by children and the whole population, was estimated. According to the EU legislation on food contaminants, exceedance of the permitted limits for nitrates in vegetables and infant food were not detected. The possible reason for low levels of nitrates in vegetables and infant foods was probably related with relatively low use of fertilizers in Estonia. The results showed that cabbage and potato were the main sources of nitrate intake. The mean intake of nitrates from vegetables by Estonian population was 58 mg per day, calculated on the basis of body weight of 60 kg, which made up 26% of ADI value. Nitrate intake by 1-to 6-years-old children was found to be 28 mg per day (46 % of ADI). The mean nitrates intake from infant food by children aged 6 to 12 months was 7.8 mg per day, calculated on the basis of body weight of 15 kg (22% of ADI). Considering the toxicity of nitrates and the possibility of their transformation to nitrite and carcinogenic N-nitrosoamines, the importance of information on daily intake by children is obvious. [START_REF] Penttilä | Estimation of Food Additive and Pesticide Intakes by Means of A Stepwise Method[END_REF][START_REF] Dejonckheere | Nitrate in Food Commodities of Vegetable Origin and The Total Diet in Belgium, 1992-1993[END_REF]Petersen & Stoltze, 1999;[START_REF] Belitz | Food Chemistry[END_REF][START_REF] Chung | Survey of nitrate and nitrite contents of vegetables grown in Korea[END_REF].

F

  food consumption data from the Board of Statistics were used in the calculation of average intake for the whole population. Intake of nitrates by children was estimated using the consumption data from kindergarten menus and nutrition recommendations for infants. Consumed amounts of vegetables were reduced, taking into account the effect of peeling, cleaning and removing of non-edible parts. Nitrate concentrations of the commodities were corrected for cooking loss.

  content was found in Italian organically grown green salad and rocket than in those conventionally produced[START_REF] Martin | Determination of nitrates by a novel ion chromatographic method: occurence in leafy vegetables (organic and conventional) and exposure assessment for Italian consumers[END_REF]. On the contrary, US investigation

  by the National Board of Statistics in Estonia. Data include the number of population and monthly average amounts of consumed vegetables by Estonian population in 2000-2002. Calculations of the mean daily intake of vegetables were made according to the data of the whole population. In Table IV the amounts of vegetables consumed daily are presented. Consumed amounts of vegetables were reduced, taking into account the effect of peeling, cleaning and removing of non-edible parts. Nitrate concentrations of the commodities were corrected for cooking loss. The consumption data in the tables were obtained in 2005 from the menu of two kindergartens. During the year, the consumption data according to the menu and amount of supplied vegetables were taken into account. The mean consumed amounts of vegetable-based foods by children in the age group of 1 to 6 years were calculated taking into consideration the average number of children (n=335).

[

  Table II Comparison of mean nitrate contents of vegetables (mg kg -1 ) in different countries

	Table I Nitrate contents of Estonian vegetables in 2003-2005
	Commodity		No. of samples	Minimum nitrate content, mg kg -1	Maximum nitrate content, mg kg -1	Average nitrate content, mg kg -1
	Potato		449		<30	360	94
	Carrot		202		<30	525	148
	Cabbage		168		74	1138	437
	Beetroot		130		214	3556	1446
	Cucumber		130		<30	1236	160
	o r F o r F o r F o r F o r F o r Chinese cabbage Turnip Tomato Onion Spring onion F o r	58 36 25 21 15		64 232 <30 30 160	1062 2236 100 92 920	307 1243 41 55 477
	Lettuce Pumpkin Radish Dill Rhubarb Celery Parsley	P e e r P e e r P e e r P e e r P e e r P e e r 14 8 6 P 5 e 4 4 4 e r	397 <30 670 2236 55 256 674	3230 445 1500 3267 376 830 1588	2167 174 1309 2936 201 565 966
	Strawberry Cauliflower Zucchini Watermelon Spinach Carrot juice Infant purees Ready-made salads Ready-made vegetable soups	3 3 2 1 1 10 31 14 5	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w <30 104 330 R 95 e 2508 76 26 80 v i e 588 w	111 404 511 95 2508 251 208 474 740	55 287 421 95 2508 136 83 259 639
						O n l O n l O n l O n l O n l O n l O n l
						y y y y y y y
						1 2

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  Table IV Mean daily nitrate intake in Estonia, calculated on the basis of the consumption survey from the National Board of Statistics

	Table III Comparison of the concentrations of nitrates in vegetable-based infant food
	Vegetable commodity Potato Carrots Lettuce Chinese cabbage Cabbage Turnip Tomato Description of food Finland Great Britain 82 150 264 274 1835 2330 1057 607 712 908 170 F o r Vegetable purees Commodity Consumption, Belgium Denmark United States 154 182 278 2782 1489 1859 333 451 36 Nitrate, mg kg -1 Korea Japan Germany 452 713 93 316 193 232 2430 1489 1740 1040 725 451 27 Estonia (this study) Spain (Hardisson et al.1996) Germany (Belitz and Grosch 1999) g per day Mean nitrate content, Mean nitrate mg kg -1 (cooking and intake, mg per % of ADI peeling losses taken day into account) 19-208 Vegetable and meat 32-148 Potato 206 57 12 5.3 20-204 Cabbage 36 382 14 6.2 purees Vegetable and cereal purees 24-162 Carrot 24 129 3.1 1.4 382 Carrots, carrot juice 76-251 104 average 88 (n = 41) 92 81 F o r Beetroot 6.7 1085 7.3 3.3 Turnip 4 269 1.1 0.5 Tomato 21 41 0.9 0.4 Cucumber 51 160 8.2 3.7 Onion 14 55 0.8 0.4 F o r
	Sweet pepper Cucumber Onion Beetroot Green beans Radish Spinach Leek Spring onion Garlic Green Lettuce Pumpkin and 140 240 140 1800 455 Zucchini Radish Ready-made salads Ready-made soups Frozen vegetables Herbs (celery, parsley, dill) Total ADI -Acceptable Daily Intake 151 235 2600 2470 P e 344 93 59 585 2136 2297 841 e r R 1505 1783 284 e v i e 965 P e e r R e v i e 2.8 2167 2.6 260 P 0.4 1300 e 2.9 259 e r 1.8 639 1.6 148 R 0.9 1489 e 376 v i e pepper w w w	212 23 1878 4259 56 436 124 76 6.1 0.7 0.5 0.8 1.2 0.2 1.3 58	384 1060 3560 145 455 99 2.7 0.3 0.2 0.3 0.5 0.1 0.6 26	1630 2030 965
	O n l O n l O n l
		y y y
				3 4 5

Table V

 V Mean daily nitrate intake of Estonian children, calculated on the basis of the consumption survey from the menu of kindergartens Table VI Mean daily nitrate intake of infants, calculated on the basis of the recommended infant menu

			Food Additives and Contaminants			Page 22 of 24
	Commodity Age 6 months 7 months	Consumption, g per day Body weight, kg Recommended Mean nitrate content, mg kg -1 (cooking and peeling losses taken into account) consumption of Mean nitrate Mean nitrate intake, intake, mg per day vegetable purees, g 7.8 50 4.4 mg per day 8.5 70 6.2	% of ADI % of ADI 15 20
	Potato 8 months	9	150	80	57	7.0	8.6	11 21
	Cabbage 9 months	9.4	17	90	382	7.9	6.5	8.6 23
	Carrot Beetroot Turnip Tomato Cucumber 10 months F 11 months 10.3 9.8 12 months 10.6 o Average 9.3 r ADI -Acceptable Daily Intake 21 7 5.7 4.3 8.5	100 110 120 89	129 1085 269 41 160	8.8 9.7 11 7.8	2.7 7.6 1.5 0.2 1.4	3.6 10 2.0 0.2 1.8 24 25 27 22
	Onion Spring onion Frozen vegetables Herbs Total, 4-to 6-years-old children Total, 1-to 3-years-old children ADI -Acceptable Daily Intake 10 0.5 1.6 P 0.7 e 226 e r 198 R	55 477 148 1489		0.6 0.2 0.2 1.0 30 26	0.7 0.3 0.3 1.4 40 52
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Acknowledgements

The study was supported by the Estonian Science Foundation, Grant no. 5416.