Olga ; Pardo
email: pardo_olg@gva.es

Vicent Yusà

Pilar Villalba

Jose Antonio Perez

Csisp

Olga Pardo-Marín

Vicent Yusà-Pelechà

Pilar Villalba-Martín

José Antonio Perez- Dasí

Monitoring programme on nitrate in vegetables and vegetable-based baby foods marketed in the Region of Valencia: levels and estimated daily intake

Keywords: Exposure, Chromatography -HPLC, Risk assessment, Quality assurance Additives/Contaminants: Nitrate Food Types: Baby food, Vegetables nitrate, vegetable, baby foods, dietary intake

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Vegetables play an important role in human nutrition since they are an outstanding source of vitamins, minerals and biologically active compounds [START_REF] Favell | A comparison of the vitamin C content of fresh and frozen vegetables[END_REF]. The health benefits of a diet rich in vegetables and fruits have been recognized for some time. The World Health Organization (WHO) recommends to consume at least 400 g or five portions of fruits and vegetables (excluding tubers) a day [START_REF] Who | Diet, nutrition and the prevention of chronic diseases[END_REF]. However, these plant foods can also be a source of unfavourable contaminants, e.g. nitrates, heavy metals and residues of pesticides.

Several sources are responsible for the dietary intake of nitrates, of which vegetables and drinking water are quantitatively the most important [START_REF] Martin | Determination of nitrates by novel ion chromatographic method: occurrence in leaky vegetables (organic and conventional) and exposure assessment for italian consumers[END_REF]. Nitrates and nitrites are also used as antimicrobial and flavouring/colouring agents in meat and fish products. About 90 % of the total nitrate concentration in an european normal diet is believed to be a direct result of vegetable intake [START_REF] Hambridge | Nitrate and nitrite: intake assessment[END_REF]. The presence of nitrates in vegetables is one of the consequences of the mechanism by wich plants absorb the nitrogen element, in the form of NO 3 -, from fertilisers or organic materials [START_REF] Schuddeboom | Nitrates and nitrites in foodstuffs[END_REF], which are essential to the process of protein synthesis. Their concentration depends on the biological properties of the plant culture, lightning conditions, 3 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]. It has been discovered that green leafy vegetables contain the highest levels of nitrates [START_REF] Wolff | Nitrates, nitrites and nitrosamines[END_REF]. Nitrate contamination in vegetables occurs when crops absorb more than they require for their sustainable growth. Spinach, lettuce, broccoli, cabbage, celery, radish, beetroot, etc. possess the tendency to accumulate nitrates. On the other hand, vegetables such as carrots, cauliflower, French beans, peas and potatoes seldom accumulate nitrates. Nitrate content of vegetables may range from 1 to 10,000 mg kg -1 [START_REF] Ximenes | Polarographic determination of nitrate in vegetables[END_REF]. The limit concentrations for nitrates have been established in EU for spinach, lettuce and baby foods (European Commission 2006). ML prescribed for lettuce (2000-4000 mg kg -1 of fresh sample) depend on the time of yield harvest and the type of growing (in the open or under protected conditions), and in spinach (2000-3000 mg kg -1 of fresh sample) on the time of yield harvest and the type of processing (canned or frozen spinach).

The nitrate ion has a low level of acute toxicity, but if transformed into nitrite, it may constitute a health problem. Some researches suggested that the vegetables with high nitrate in the diet could put a human into the risk of gastrointestinal cancer and methemoglobinaemia [START_REF] Bartsch | Inhibitors of endogenous nitrosation: Mechanisms and implications in human cancer prevention[END_REF]. It has been estimated that 5-8% 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]. The elevation of gastric pH higher than 5.5 leads to bacterial growth followed by rapid conversion of nitrate to nitrite. Nitrite is a precursor in the formation of nitrosamines [START_REF] Tannenbaum | Nitrate and gastric cancer risks[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]. Vegetables are an important part of [START_REF] Huarte-Mendicoa | Nitrate and nitrite levels in frozen broccoli. Effect of freezing and cooking[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] Sanchez-Echaniz | Methemoglobinemia and consumption of vegetables in infants[END_REF]. Processed infant foods may also contain increased levels of nitrites (Hill 1996).

Young babies with low stomach acidity may suffer from infantile methemoglobinemia due to excessive nitrates in their diet, where nitrite is substituted for oxygen in hemoglobin and death may occur [START_REF] Ezeagu | Nitrate and nitrite contents in ogi and the changes occurring during storage[END_REF][START_REF] Swann | The toxicology of nitrate, nitrite, and N-nitroso compounds[END_REF].

Therefore, there is great concern about the nitrate content in the daily diet, especially in vegetables. In recent decades, considerable researches have been done to minimize the nitrate accumulation in vegetables, mainly in plant nutrient management, horticultural technology, and breeding. Interest in the dietary intakes of nitrates and nitrites has arisen from the concern about their possible adverse effect on health. An acceptable daily intake (ADI) of 0-3.7 mg kg -1 , equivalent to 222 mg nitrate per day for a 60 mg kg adult was established by the former Scientific Committee for Food (EU SCF 1995)

Materials and methods

Samples and sampling

Samples of vegetables and vegetable-based baby foods were randomly acquired in local markets, large supermarkets and grocery stores of the Valencia region (Spain) and stored at 4 ºC until analysis. The vegetable species included in the study are lettuce (lactuca sativa), iceberg-type lettuce (lactuca sativa var. capitata), spinach (spinacia oleracea), potato (solanum tuberosum), carrot (daucus carota), chard (beta vulgaris var. cicla), artichoke (cynara scolymus), rucola (eruca sativa) and lamb's lettuce (vallerianella locusta). These species were chosen because of their high consumption in the Valencian diet.

Nitrate concentrations in vegetables vary according to season and condition of cultivation, as well as according to condition of storage. Therefore the following food commodities were collected: fresh spinach, preserved, deep-frozen or frozen spinach; fresh lettuce

Reagent and standard solutions

Nitrate calibration standard solutions were prepared from certified commercial standard solutions (Merck, Darmstadt, Germany) by dilution in water and stored in capped amber vials at 4 ºC. Concentrations of the calibration standards ranged between 0.5-100 µg ml -1 . Water was Milli-Q water from a Millipore purification system (Millipore, Bedford, MA, USA). Borax was supplied by Panreac (Barcelona, Spain). Carrez I and II were purchased from Scharlau (Barcelona, Spain). Methanol, monopotassium phosphate and disodium posphate were of high analytical grade (Merck, Darmstadt, Germany). Nitrate reference test materials were provided by CSL (Central Science Laboratory, York, UK).

Sample treatment

Before testing, any inedible parts were removed. The hole samples were quartered, grinded and homogenised (Ultra-Turrax TR-50, Germany). An analytical aliquot of approximately 5 g was weighed. The subsample was transfer into a 200 ml volumetric flask and about 150 ml of water and 5 ml of borax solution were added. The whole content was shaked and after 15 minuts in warm water, 2 ml of Carrez I and 2 ml of Carrez II were added. The mixture was then brought to volume and filtered into a 250 ml Erlenmeyer flask through a 0.45 µm pleated filter.

An SPE column (C18/1g, Sep-Pak ® , Waters Corporation, Milford, Massachusetts, USA) was activated with 5 ml methanol and 5 ml Milli Q water. A total of 4 ml of the filtered sample was loaded onto the column to saturate it and the eluate was discarded; another 4 ml of the sample were then passed through. Final extract was collected and filtered through 0.45 µm prior to the LC analysis.

Instrumental

The liquid chromatograph was an isocratic system equipped with an ultraviolet (UV) diode array detector. Chromatographic separations were performed at 30 ºC on a Waters IC-PAK HC anion exchanger column (4.6 x 150 mm) using a 4.6 x 50 guard column of the same phase (Waters Corporation, Milford, Massachusetts, USA). Mobile phase was phosphate 5

Method validation and analytical quality assurance (AQA)

The analytical method was validated. The main performance characteristics of the method were: limits of detection (LOD) and quantitation (LOQ) of 7 and 20 mg kg -1 respectively, recoveries from 83 to 106 % and precision between 5 and 12 %, estimated as relative standard deviation (less than the recommended values derived from Horwitz Equation). The performance criteria fullfill the specific criteria for methods of analysis used in the monitoring of nitrate levels established in the european legislation (European Commission 2006b). The linear model was statistically validated taking into account that residual values were randomly distributed about the regression line, the p-value of the F-test statistic was <0.05, and the coefficient of determination (R 2 ) was better than 0.999 [25,33] (Eurachem [START_REF] Guide | The fitnesspurpose of analytical methods. A laboratory guide to method validation and related topics[END_REF].

Dietary exposure estimates

The daily intake of nitrates through vegetables was calculated by multiplying the respective average concentration (median) by the weight of vegetables consumed by an average adult from Spain (17 years and over, 68.5 kg body weight) per day. Also exposures for children (7-12 years; average weight, 34.5 kg) and adult extreme fish consumers were estimated.

Daily intake (mg week -1 )= occurrence (mg kg -1 ) x consumption (kg person -1 day -1 ) / body weight (kg)

The daily intake of nitrates through vegetable-based baby foods was calculated by multiplying the respective average concentration (median) by the weight of vegetable-based baby foods consumed by an average baby from Spain (0-1 years, 7.4 kg body weight; 1-2 years, 11.7 kg body weight) per day.

In this work food consumption data from the Spanish Food Safety Authority (AESAN 2006) were used in the calculation of average intake for the whole population (children 7-12 years, adults and extreme consumers). Intake of nitrates by infants (0-1 and 1-2 years) were estimated using the consumption data from the Spanish Nuclear Safety Council (CSN 2002).

For calculations, when a nitrate concentration was under the limit of quantification (LOQ), the value was assumed to be the respective limit, which was 20 mg kg -1 .

Results and discussion

Concentration of nitrates in vegetables

Nitrate concentrations found, expressed in mg kg -1 of wet weight (w.w.) in the different vegetable species monitored are summarised in Table 1.

Cultivar and harvest date can affect the nitrate levels of selected vegetables (Arm & Hadidi 2001). For this reason, european legislation (European Commission 2006a) fix different maximum nitrate levels in certain vegetables such as lettuce and spinach depending on the cultivar and the season.

Table 1 shows that spinach has the highest nitrate content (median of 1410 mg kg -1 ) compared with the other vegetable species (lettuce and iceberg-type lettuce) which maximum limit are regulated. This finding correlated well with the levels found in other Mediterranean countries as France or Italy [START_REF] Malmauret | Contaminants in organic and conventional foodstuffs in France[END_REF][START_REF] Menard | Assessment of dietary exposure of nitrate and nitrite in France[END_REF], De Martin et al. 2003) and are lower than those reported by [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF] or [START_REF] Hsu | Nitrate and nitrite quantification from cured meat and vegetables and their estimated dietary intake in Australians[END_REF] from Estonia and Australia respectively. The levels found were, in general, below the maximum limits established by the European Commission (European Commission 2006a). However, thirteen fresh spinach samples exceeded the regulatory limits (see Table 2): ten were summer samples and three winter samples. Nitrate concentration in some 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. As can be seen in Table 1 spinachs harvested during autumn-winter (median of 1755 mg kg -1 ) have higher content of nitrate comparing with those harvested in spring-summer (median of 1269 mg kg -1 ). These differences can be (2004), processed spinachs (frozen, deep-frozen or preserved) contain the lowest levels of nitrate due to the reduction of the content during the blanching process.

For lettuce and iceberg-type lettuce, the nitrate contents found (median of 1156 and 798 mg kg -1 respectively) are similar to those reported by [START_REF] Malmauret | Contaminants in organic and conventional foodstuffs in France[END_REF] and [START_REF] Merino | Lvels of nitrate in Swedish lettuce and spinach over the past 10 years[END_REF] respectively in studies carried out in France. Iceberg-type lettuce levels are considerabily higher than thos reported by [START_REF] Hsu | Nitrate and nitrite quantification from cured meat and vegetables and their estimated dietary intake in Australians[END_REF] in Australia and lettuce levels are lower than those reported by [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF] in Estonia. As can be seen in Table 1, fresh lettuce grown under cover (potencially reduced light intensity) contain higher levels of nitrate comparing with those grown in the open air (both collected in winter and summer seasons). The nitrate values found in lettuce and iceberg-type lettuce were, in all cases, below the maximum limits established by the European Commission (European

Commission 2006a).

Nitrate content in other vegetables (potato, carrot, chard, artichoke, rucola and lab's lettuce) for which a maximum limit has not been fixed by the European Commission were investigated. The species with the highest content of nitrates were rucola (median of 4474 mg kg -1 ), lamb's lettuce (median of 2572 mg kg -1 ) and chard (median of 1597 mg kg -1 ).

Rucola levels are higher than those reported in Italia by De [START_REF] Martin | Determination of nitrates by novel ion chromatographic method: occurrence in leaky vegetables (organic and conventional) and exposure assessment for italian consumers[END_REF]. The use of rucola and lamb's lettuce seems to be increasing in several European diets, therefore it can mean a large contribution to the nitrate daily intake. Elevated consumption of rucola, more than 47 g, at the median nitrate concentration mean to exceed the ADI without taking into account any other source of nitrate exposure (EFSA 2008). The lowest median values of (median of 96 mg kg -1 ) is higher than the levels found in France [START_REF] Menard | Assessment of dietary exposure of nitrate and nitrite in France[END_REF]). The median nitrate content of potato (median of 196 mg kg -1 ) is also comparable to those obtained in France [START_REF] Menard | Assessment of dietary exposure of nitrate and nitrite in France[END_REF], China [START_REF] Zhong | Nitrate and nitrite in vegetables from north China: Content and intake[END_REF] and Slovenia [START_REF] Susin | A survey of nitrate and nitrite content of fruit and vegetables grown in Slovenia during[END_REF]) with median values of 191.8, 164 and 158 mg kg -1 respectively. [START_REF] Ierna | Influence of harvested date on nitrate contents of three potato varieties for of-season production[END_REF] has recently investigated the influence of variety and harvest time in potato nitrate content concluding that tuber nitrate contents vary among the potato variety and that nitrate contents decrease in winter-spring crops, whereas they increase in summer-autumn crops.

The nitrate level found in carrot was 203 mg kg -1 and is similar to that found in Slovenia [START_REF] Susin | A survey of nitrate and nitrite content of fruit and vegetables grown in Slovenia during[END_REF].

The nitrate levels in the different vegetals show similar profiles (see Figure1) to those reported by [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF], [START_REF] Menard | Assessment of dietary exposure of nitrate and nitrite in France[END_REF] or [START_REF] Susin | A survey of nitrate and nitrite content of fruit and vegetables grown in Slovenia during[END_REF] in Estonia, France and Slovenia, respectively. Leafy vegetables accumulate high amounts of nitrates, concentrations reaching up to 6000 mg kg -1 [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF]). On the other hand, roots and tubers contain generaly low nitrate levels.

Concentration of nitrates in vegetable-based baby foods

Sixty seven samples of vegetable-based baby foods were analysed and the levels found were, in all cases, lower the maximum level proposed by European legislation (European Commission 2006a). Nitrate content close to EU limit concentration of 200 mg kg -1 was detected in one sample.

The mean nitrate content in vegetable-based baby foods (taking into account the samples with concentrations above the minimum reporting levels) was 60.4 mg kg -1 with a standard deviation of 38.6 mg kg -1 . This level is lower than those in a Spain [START_REF] Hardisson | The evaluation of the content of nitrates and nitrites in food products for infants[END_REF] [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF], where mean levels of 92 and 88 mg kg -1 were reported. [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF] detected the highest levels in the baby foods containing carrot and pumpkin (62-148 mg kg -1 and 124-162 mg kg -1 , respectively). 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 concentration exceeding the regulatory limit were found in baby foods in wich the main ingredient was carrot.

Evaluation of dietary exposure

Human exposure to nitrate is mainly exogenous through the consumption of vegetables, and to a less extent through water and other foods (nitrate is an approved food additive). It is estimated that approximately 80-95% of the daily nitrate intake comes from plant foods, mostly vegetables and a few fruits [START_REF] Pennington | Dietary exposure models for nitrates and nitrites[END_REF]. Consequently, information about dietary intake is necessary in order to evaluate the potencial risk for humans. Apart from comparing the concentrations of nitrate with the maximum levels fixed by European legislation (European Commission 2006a), a more signifficant assessment of potencial health hazards for consumers can be obtained by calculating the nitrate daily intake deriving from the consumption of vegetables and comparing it with the acceptable daily intake (ADI) set by international organizations. It should be stated that the purpose here is not to evaluate the total diet exposure, but rather to study the intake of nitrate from vegetables. Vegetables with the highest consumption values for the total Spanish population (AESAN 2006) have been selected for the daily intake estimation.

An Acceptable Daily Intake (ADI) for nitrate of 3.7 mg kg b.w. -1 day -1 , equivalent to 222 mg nitrate per day for a 60 kg adult was established by the former Scientific Committe on Food (EU SCF 1995). Table 3 shows the nitrate daily intake through vegetables based on the concentrations found in this study, calculated by taking into account the consumption http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk 3.

The nitrate daily intake through vegetables was 69 mg person -1 day -1 , wich corresponds to 29% of the ADI. This intake is similar than those reported by other countries, as can be seen in Table 4. The main contribution to the daily intake comes from lettuce, with medium levels of nitrates, and is six times higher approximately than the spinach contribution. The use of rucola, lamb's lettuce seems to be increasing in the Spanish salads, so a high consumption of these vegetable spices can contribute to increase the nitrate daily intake. Elevated consumption of rucola , more than 47 g day -1 , at the median nitrate concentration mean to exceed the ADI without taking into account any other source of nitrate exposure (EFSA 2008). On the other hand, potatoes often containing low levels of nitrates, but eaten more frequently, have an important influence in the total dietary intake, similar for adults and children, and mean an important contribution for the children nitrate daily intake, since they are an important part of their diet (4.5% aproximately). Chard contribution to nitrate daily intake is slightly higher than that reported for spinach. The nitrate intakes owing to artichoke and carrot consumption (0.1% and 0.8% of the ADI respectively) are the lowest of the commodities studied.

For children the mean nitrate daily weekly from vegetables achieve a 15.1% of ADI. This value is directly related with the low consumption of green salad by children population.

The estimation of the dietary exposure for individuals who eat huge amounts of vegetables shows that these extreme vegetable consumers have a nitrate daily intake of 78.9% of ADI. This value is higher than that reported by [START_REF] Clarke | Levels of phytoestrgens, inorganic trace-elements, natural toxicants and nitrate in vegetarian duplicate diets[END_REF] in vegetarian diets.

The nitrate daily intake through vegetable-based baby foods based on the concentrations found in this study, calculated by taking into account the Spanish infants consumption data 5. The estimated nitrate daily intake through vegetablebased baby foods corresponds to 13% and 18% of the ADI for infants from 0 to 1 year and from 1 to 2 years, respectively. This intake is lower than those reported by [START_REF] Tamme | Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population[END_REF] and the estimated value is adequate in order to protect against infant methemoglobinemia.

Conclusiones

Taking into account the vegetable species considered, the data obtained in the study do not show evidence of risk for the average consumers. Nitrate values were, in general, lower than the maximum limits established by the EU legislation. Thirteen fresh spinach samples, ten summer samples and three winter samples, exceeded the regulatory limits. In case of vegetable-based baby foods the levels found were, in all cases, lower the maximum level proposed by EU legislation.

This study also reports the dietary intakes of nitrates through vegetables consumption in adults living in the Region of Valencia on the basis of food consumption data at the national level. It turns out that the contribution of vegetable to the intake of nitrate is extensively below the ADI values indicated by international regulation bodies. The estimated daily intake accounts for 29% of ADI, which is similar to those found in other countries. The estimated daily intake for extreme vegetable consumers is 78.9% ADI higher than that reported in studies of vegetarian diets. Aulthought the main human exposure to nitrate is through the consumption of vegetables, it should be highlight that for the total nitrate daily intake must be taken into account the water and meat products contribution.

F

  and was reconfirmed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2002. The Food Safety Plan of Valencia Public Health Department contains a specific monitoring programme focused on nitrates in vegetables and vegetable-based baby foods. The aim of the present study is to present the results of the monitoring of nitrate content in vegetables and vegetable-based baby foods marketed in Comunitat Valenciana over the period 2000-2008, to compare them with the results of monitoring which are performed in the countries of European Union and in the world and estimate the toxicological risk associated with their intake.

(harvested between 1

 1 October and 31 March and harvested between 1 April and 30 September) or iceberg-type lettuce grown under cover and in the open air; and vegetablebased products for infants and young children. All the samples (1083 vegetables and 67 baby foods samples) were collected during 2000-2008 by food inspectors authorities within the frames of state official surveillance and monitoring programme and analysed in an accredited public laboratory: Public Health Laboratory of Valencia.

  6.5) at a flow rate of 1.2 ml min -1 . The injection volume was 20 µl and the total run time of the chromatograms was 20 min. The UV detector operated at 214 nm and a 200 to 400 nm spectrum was registered.

  The laboratory was accredited by the Spanish national body for accreditation (ENAC), and operates under the quality assurance system established by ISO/IEC 17025 (International Organization for Standardization/International Electrotechnical Commission 2005). All test batches were analysed under quality-assurance protocols, including duplicate samples, reagent blanks, spiked samples, and certified reference materials from FAPAS (Central Science Laboratory, Sand Hutton, York, United Kingdom). Six lettuce (FAPAS T1529, T1531, T1537, T1543, T1546 and T1555) and four spinach (FAPAS T1532, T1548, T1551 and T1557) reference materials were analysed during the period 2003-2008, and the results of analysis (z-scores values ranged from -1.1 to 1.5) were in good agreement with values. The laboratory participates regularly in laboratory proficiency testing provided by the Central Science Laboratory (York, UK), with satisfactory z-scores.

  higher irradiance in summer which tends to reduce nitrate, and also to higher growth rates which coincide with periods of high irradiance and warmer temperatures (Kannan & Economakis 1992). However, according with Walderman et al.

  in arichoke, potato and carrot. The content of nitrates in artichoke

Food

  and per child for the total Spanish population(AESAN 2006). The extreme exposure (high level of vegetables consumption) are also showed in Table

Figure 1 .

 1 Figure captions: Figure 1. Profile of vegetables nitrate contents (median values a ) in different studies a Median concentration listed in the present study were determined using all samples

  )

	and Estonia
	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	12

Table 1 .

 1 Nitrate contents of vegetables marketed in the region of Valencia during the period 2000-2008

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

a

The median concentrations listed were determined using all samples.

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Table 2 .

 2 Nitrate contents in fresh spinach samples (marketed in the region of Valencia) exceeding the regulatory

	limits during the period 2000-2008
	Harvesting date	Nitrate content mg kg -1	Regulatory limit a mg kg -1

Table 3 .

 3 Estimation of the nitrate dietary intake through vegetables consumption based on the median concentrations Notes: a Average adult (17 years and over) weight:68.5 kg; average children (7-12 years) weight: 34.5 kg.

			Food Additives and Contaminants
	found in the study
				Consumption	Daily intake
			(g day -1 per person a )	(mg kg b.w. -1 day -1 )		Per cent ADI
	Commodity	Occurrence (mg kg -1 )		Adults	Children	Adults		Children	Adults	Children
				High						High
		Median	Mean		Mean	Mean High level	Mean	Mean		Mean
				level b						level
	Lettuce	1156	35.5	76.6	13.9	0.6	1.3	0.2	17.1	36.9	6.7
	Potato	196	56.8	109.3	55.0	0.2	0.3	0.2	4.6	8.9	4.5
	Carrot	203	9.3	24.9	5.9	0.0	0.1	0.0	0.8	2.1	0.5
	Spinach	1410	4.9	24.2	2.7	0.1	0.5	0.1	2.9	14.2	1.6
	Chard	1597	5.2	24.2	2.6	0.1	0.6	0.1	3.5	16.1	1.7
	Artichoke	96	2.8	15.3	1.0	0.0	0.0	0.0	0.1	0.6	0.0
	Total					1.0	2.8	0.5	29.0	78.9	15.1

b Increasing one standard deviation the average consumption of each vegetable item. Page 22 of 26 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 4 .

 4 Estimated mean nitrate daily intake through vegetables and per cent ADI in different studiesCountryYear of study Estimated daily intake (mg kg b.w. -1 day -1 ) Per cent ADI

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

Table 5 .

 5 Estimation of the nitrate dietary intake through vegetable-based baby foods consumption based on the mean concentrations found in the study

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	Age		Average body	Consumption	Daily intake
		Occurrence (mg kg -1 )
			weight (kg)	(g day -1 per infant)	(mg kg b.w. -1 day -1 )	Per cent ADI
	0-1 year	60.4	7.4	35.5	0.5	13%
	1-2 years	60.4	11.7	56.8	0.7	18%

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uk Food Additives and Contaminants

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Acknowledgements 15

The authors are grateful to all inspectors of the Public Health Department of Valencia Regional Government who participated in the monitoring programme. They also thank the technicians of the Public Health laboratoriy of Valencia, Spain.


Chiara Cavalieri

Luciana Bolzoni

Mirella Bandini

Nicotine determination in mushrooms by LC-MS/MS and early studies on the impact of drying on nicotine formation

Keywords: nicotine, mushrooms, LC-MS-MS, method validation

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

Nicotine determination in mushrooms by LC-MS/MS and early studies on the impact of drying on nicotine formation

Introduction

Nicotine is the main alkaloid in tobacco (Nicotiana tabacum) and other tobacco species where it occurs at concentrations ranging from 2% to 8%; it is synthesised in the tobacco root from ornithine and/or arginine by way of putrescine [START_REF] Hibi | Gene expression in tobacco lownicotine mutants[END_REF]. Low concentrations were also measured in other plants belonging to the family of Solanaceae, such as tomatoes, aubergines, peppers and potatoes [START_REF] Andersson | Nicotine alkaloids in Solanaceous food plants[END_REF]. Mushrooms are usually not reported as a significant source of nicotine, although both amino acids, ornithine and arginine have been identified in them [START_REF] Ribeiro | Comparative study of free aminoacod composition of wild edible mushroom species[END_REF]. Nevertheless, recently significant amounts of nicotine were detected in Germany in dried wild mushrooms (ceps) from China [START_REF] Bfr | Nikotin in getrockneten Steinpilzen: Ursache der Belastung muss geklart warden[END_REF], raising questions about the origin of nicotine in these products and the possible concerns for human health. Recently, the European Commission has been informed by food business operators that dried wild mushrooms (mainly Boletus edulis, but also truffles and chanterelles) may contain levels of nicotine higher than 0.01 mg/kg on a fresh weight basis. This is the default maximum residue level (MRL) set by Article 18.1.b of Regulation (EC) No 396/2005. According to the information received, 99% of samples tested from 2008 production did not comply with the current MRL, irrespective of the wild mushrooms' origin, although most of them originated from China. No clear reason has thus far been established for this unexpected presence of nicotine that could be of various origin, but also results from a combination of different factors: 1) pesticide use; in organic farming nicotine is not allowed to be used as pesticide (Regulation EC No 889/2008) and in general in European countries the use of plant protection products containing nicotine will phase out by the latest in June 2010, but its use in Third Countries may continue and may lead to residues of nicotine in food; 2) inherent presence of nicotine in wild mushrooms and its eventually arise due to an increase of its biosynthesis (via Putrescine, [START_REF] Hibi | Gene expression in tobacco lownicotine mutants[END_REF] caused by some not properly controlled conditions of storage during the drying phase; 3) cross contamination due to bad practices in the storage/drying and packaging process (smoking people handling the mushrooms, storage in rooms that have been disinfected with nicotine, simultaneous drying of tobacco and mushrooms in the same room). As a consequence of this alert the European Food Safety Authority (EFSA) received an urgent request on 27 April 2009 from the European Commission for a scientific opinion in relation to the risks for human health due to the presence of nicotine in wild mushrooms at concentrations up to 0.5 mg kg -1 . In reply to this request, the EFSA issued on 7 May 2009 a statement, concluding that a level of 0.5 mg kg -1 is not safe, because it would lead an exceedence of the acute reference dose (ARfD). The EFSA has proposed alternative MRLs to be set on a temporary basis both for fresh (0.036 mg kg -1 ) and for dried (1.17 mg kg -1 ) wild mushrooms. Nevertheless, as regards dried ceps it would be more appropriate to restrict the evaluation of a safe level of nicotine to the consumption data available for the adult population because of the poor data as regards consumption by children. As a consequence, for dried ceps, an acceptable level of 2.3 mg kg -1 could be derived. The EFSA also highlighted that the evaluation performed was affected by a number of uncertainties and limitations on the data available (consumption of wild mushrooms in Europe and, in particular on dried ones, contamination levels, actual unit-to-unit variability in fresh mushrooms). Consequently, EFSA recommends that the proposed MRLs should be considered on a temporary basis only (Annex III of Regulation 396/2005). Finally, it is noted that a more robust basis for the exposure assessment and MRLs setting will derive from the data provided by the monitoring and testing programme recommended by the European Commission that will be launched by food business operators at the start of the forthcoming 2009 harvest season. A variety of methods for isolation, identification and quantification of nicotine and related compounds in plants and biological tissues have been published. These methods range from early paper chromatographic procedures through to GC/MS, thin-layer and HPLC procedures; also several radioimmunoassay have been developed for nicotine [START_REF] Castro | Dietary nicotine and its significance in studies on tobacco smoking[END_REF]. Sheen [START_REF] Sheen | Detection of nicotine in foods and plant materials[END_REF] investigated the nicotine content of Solanaceous food plants using a GC technique using alkali flame detector with a LOD of around 1 mg kg -1 in dry material. Davis et al. (Davis, 1991), Domino et al. (1993[START_REF] Siegmund | Determination of the nicotine content of various edible nightshades (Solanaceae) and their products and estimation of the associated dietary nicotine intake[END_REF] used GC/MS with LODs of around 1 µg/ kg -1 . Nevertheless, no methods are published yet concerning the determination of nicotine in wild mushrooms. The aim of the present study was to develop, validate and apply an analytical method for routine analysis of nicotine in fresh and dried mushrooms. The method should be quick and sufficiently sensitive for the detection of small concentrations of nicotine. An LC-MS-MS spectrometer was employed as detection system for the quantitative determination using a deuterated isotope of nicotine as internal standard .

Materials and methods

Chemicals and samples

Nicotine and (±)-Nicotine-D 4 standards were purchased from Ehrenstorfer-Schafers ( Augsburg, Germany) and from Cerilliant (Round Rock, TX), respectively. Nicotine and (±)-Nicotine-D 4 standard stock solutions at 49192 mg kg -1 and 10 mg kg -1 , respectively, and nicotine working solutions in the range 2 -500 µg kg -1 were made with acetonitrile and stored at -25°C. For extraction and clean-up, bidistilled water, pesticide-quality acetonitrile (from VWR) and ammonia solution 30% (from Carlo Erba Reagenti) were used without further purification. QuEChERS kit were purchased from Waters (U.S.A.). Dried and frozen wild mushrooms were obtained from local producers.

Sample preparation

Extraction and clean-up of samples were done using QuEChERS kit. 0.5 g of dried or 5 g of fresh/frozen homogenized mushrooms were added to a 50 ml DisQuE extraction tube containing 1.5 g of sodium acetate and 6 g of magnesium sulphate. 14.5 g (for dried sample) or 10 g (for fresh/frozen sample) of NH 4 OH 0.1 M and the internal standard (75 µl of 10 mg/Kg (±)-Nicotine-D 4 solution in acetonitrile) were added. The sample was vigorously hand shaken and put into an ultrasonic bath for 30 min, than was left for other 30 min at room temperature without any shaking. 15 ml of acetonitrile was added and the sample was vigorously shaken by hand for 1-2 min, then was centrifuged at about 2000 rpm for 2-4 min. 1 ml of the acetonitrile extract was transferred into a 2 ml DisQuE extraction tube containing 50 mg of PSA and 150 mg of magnesium sulphate. The tube was shaken for 1-2 min using a vortex agitator than was centrifuged at about 2000 rpm for 2-4 min. The supernatant organic phase was injected to an LC-MS-MS spectrometer.

Drying of fresh/frozen mushrooms 10 g. of fresh or frozen homogenized mushrooms were weighted in a porcelain capsule and dried in an oven at 70°C under vacuum for about 2 hours, then sample was weighted again and the concentration ratio due to loss of water was calculated. A same experiment was done laying the sample exposed at the direct sun light until loss of water corresponding to 8-10, as normally done by dried mushrooms producers. Nicotine in the resultant final dried material was extracted following the procedure described in the previous paragraph.

LC-MS-MS analysis

The HPLC analyses were performed on an Agilent 1100 Series liquid chromatograph equipped with an Applied Biosystems API 2000 triple quadrupole mass detector. The column employed has been a Sinergy Polar-RP 80A 4 µm, 150x2 mm (Phenomenex USA). The operating conditions have been as follows: injection volume 2 µl; mobile phase of 30% component A (0.2% formic acid aqueous solution) and 70% component B (acetonitrile); total flow rate of 0.250 ml/min; total running time for each analysis of 7 min. The following characteristic transitions were monitored: m/z 163>80, 163>84, 163>106, 163>130, 163>132 for the nicotine and 167>84, 167>110, 167>121, 167>134 and 167>136 for the internal standard (±)-Nicotine-D 4 . For the quantitative determination of nicotine the following transitions were used: m/z 163>130,163>132, 167>134 and 167>136 (where the last two are referred to the internal standard). The calibration curve was prepared using acetonitrile solutions containing variable amounts of nicotine (2,5,20,100,200, 500 µg kg -1 ) and a constant concentration of the internal standard (50 µg kg -1 ); a linear regression was applied. Calibration curve was prepared before each set of sample measurements, using fresh, or properly stored (-18°C), standard solutions.

Method validation

The performance characteristics of the method were established by single-laboratory validation employing analyses with naturally nicotine-contaminated as well as spiked frozen and dried wild mushrooms. Repeatability, within and between days, as well as test of recoveries were evaluated.

For within-day and between-day studies one naturally nicotine-contaminated sample was analysed ten times on the same day (two samples at a time, five times, within five hours) and on ten different days, respectively, both for frozen and dried mushrooms. For the determination of recoveries, nicotine-spiked mushrooms samples were analysed. This last study was performed at two different levels for the dried matrix ( 0.2 and 2 mg kg -1 ) and at one level for the frozen material ( 50 µg kg -1 ).

Results and discussion

Sample preparation

Because of nicotine is a tertiary base with a pK a1 =6.16 and pK a2 =10.96 [START_REF] Siegmund | Determination of the nicotine content of various edible nightshades (Solanaceae) and their products and estimation of the associated dietary nicotine intake[END_REF] it is bound to the acid matrixes, as vegetables, as a salt. For this reason a particular attention must be done to the pH conditions of extraction solutions. Different aqueous solutions were tested: bidistelled water, NaOH 0.05 M, NaOH 0.1, NH 4 OH 0.1, 0.2 and 0.5 M. Extraction times for each of these solutions were tested: 10-30-45-60 minutes (half time in ultrasonic bath). Also two different organic phases were tested for the extraction of the nicotine from the aqueous solution: acetonitrile and acetonitrile containing 0.1% of acetic acid. From the results of these experiments it seems clear that a strong basic environment is absolutely necessary to completely extract nicotine from the matrix and the best results were obtained using NH 4 OH 0.1 M for a total of 60 minutes and acetonitrile as final organic solvent.

Development and validation of the method

The use of HPLC coupled with tandem mass-spectrometry provides a very selective and sensitive method for the nicotine analysis. The use of (±)-Nicotine-D 4 as internal standard has been suitable to compensate both for lack of fully quantitative extraction of the analyte and for the LC-MS-MS response suppression caused by the presence of sample matrix. In the described experimental conditions the retention time of the investigated compound and the deuterated internal standard is around 2.2 minutes. In these conditions to obtain repeatable retention times and good peak shapes for the analyte and the internal standard too a small injection volume has been required (2 µl) and special attention was also given to the equilibration of the column. All the characteristic transitions of the nicotine and the (±)-Nicotine-D 4 were monitored during all the instrumental analyses. For the quantification the ratio between the sums of the two most abundant transitions of the analyte (m/z 163>130,163>132) and the deuterated internal standard (m/z 167>134 and 167>136) was considered (Fig. 1). Standard solutions of nicotine in acetonitrile were analysed by HPLC with a triple quadrupole mass detector establishing a minimum detection limit of 1 µg kg -1 (S/N = 3) and a limit of quantification of 2 µg kg -1 (S/N =10); the concentration of the internal standard, (±)-Nicotine-D 4 , was 50 µg kg -1 for each solution (as in the final sample acetonitrile extract). The standard curve for nicotine was linear from 2 to 500 µg kg -1 (R 2 =0.999), equivalent to 0.06-15 mg kg -1 and 6-1500 µg kg -1 in dried and fresh/frozen samples, respectively. To test the repeatability of the method, naturally nicotine-contaminated samples of dried and frozen mushrooms were analysed ten times during the same days (within-day repeatability) and ten times in ten different days (between-day repeatability). Table 1 shows the calculated average data and the relative standard deviations (RSDs). For the recovery test, nicotine-free of dried and nicotine-low contaminated (10-15 mg kg -1 ) of frozen mushrooms samples were spiked with standard nicotine solutions at different levels. The tested levels were been 0.2 and 2 mg kg -1 for dried samples and 50 mg kg -1 for the frozen material; test was performed 8 times for the two different matrixes. As shown in Table 1 average recoveries of nicotine was ranged from 107 to 122% and RSDs was ranged from 2.9 to 10.1%. The combined uncertainty of the quantitative result of a sample has been calculated by using the bottom-up approach, taking account of the following contributes: uncertainty of repeatability; uncertainty of recovery; uncertainty of calibration curve; uncertainty of standard solutions; uncertainty of volumes and uncertainty of weight. Two different naturally nicotine-contaminated samples were analysed ten times each one: a dried mushrooms containing nicotine at level of 2 mg kg -1 and a frozen matrix with a contamination level of 0.015 mg kg -1 . The average results and the relative calculated uncertainties were been: 2.0 ± 0.2 mg kg -1 for the dried sample and 0.015 ± 0.002 mg kg -1 for the frozen one, both of them considering a coverage factor of 2, which gives a level of confidence of approximately 95%.

Naturally nicotine-contaminated mushrooms

After method development some samples of dried, frozen and also fresh mushrooms were analysed.

Results of the analyses of different kind (whole, sliced and diced) of frozen mushrooms purchased on the market show a range of nicotine from 0.01 to 0.03 mg kg -1 . This natural occurrence of nicotine is confirmed from the result of the analysis on a fresh matrix obtained from a local mushroom harvester that shows a nicotine-contamination at level of 0.04 mg kg -1 . Dried mushrooms from different origin (Europe, Asia and South Africa) were also analysed and in these samples nicotine is present in the range of concentration from < 0.1 (< L.Q.) to 4.5 mg kg -1 . From these data it seems that, in general, dried matrixes could contain an higher level of nicotine respect those that should be on the base of the results obtained on the fresh (or frozen) samples and considering a concentration factor of about 8 from fresh to dried material. To understand this fact, samples of fresh and frozen mushrooms were dried in laboratory and their nicotine content were measured before and after the drying treatment. In this experiment 10 g. of fresh/frozen homogenized mushrooms were dried in two different way: in an oven at 70°C after vacuum for about 2 hours and by exposition to the direct sun light for some days. For each experiment samples were weighted before and after the drying treatment and concentration factors due to loss of water were calculated. As it is evident from data show in table 2 the nicotine content in final dried matrixes has been not higher than those that were expected. These results, even still partially, don't get any confirm to the hypothesis that nicotine could be biosynthetized in mushrooms at higher level during drying phase in not properly conditions.

Conclusions

This work describes the development and the application of an analytical method to determine nicotine content in fresh and dried mushroom samples. The procedure for extraction and clean-up of nicotine from fresh, frozen and dried mushrooms by using QuEChERS kit is easy and fast. The final acetonitrile extract was analysed by LC-MS-MS and nicotine was determined with a limit of quantification limit (LOQ) of 2 µg kg -1 corresponding to 6 and 60 µg kg -1 in fresh/frozen and dried matrix, respectively. The method was validated by single-laboratory tests of repeatability (within and between days) and tests of recoveries. The combined uncertainty calculated at low level for frozen (0.015 mg kg -1 ) and high level for dried (2 mg kg -1 ) material was 13% and 10%, respectively (both of them considering a coverage factor of 2). Application of this method to analyse some real samples of fresh, frozen and dried mushrooms shows as results ranges of nicotine content of 0.01-0.04 mg kg -1 and 0.1-4.5 mg kg -1 in fresh/frozen and dried matrixes, respectively. Results of some experiments performed in laboratory to dry fresh and frozen mushrooms show that conditions of drying process seem not influence the nicotine content in final dried samples.

FigureFig. 1 .

 1 Figure

Table 1 .

 1 Average data and relative standard deviations of nicotine determination in recoveries, within-day and between-day repeatability tests

	A) m/z 163>130 + 163>132	B) m/z 167>134 + 167>136
	Rt= 2.19	Rt= 2.19

Table 2 .

 2 Data of the drying experiments on some fresh and frozen mushroom samples

	Experiment	Sample *	Drying conditions	Dried weight (Dw)	Fw/Dw	Nicotine in fresh/frozen (µ µ µ µg kg -1 ) sample (Nf)	Nicotine in dried (µ µ µ µg kg -1 ) (Nd) sample	Nd/Nf
	1	fresh	70°C,vacuum,2 h	1.27	8	45.4	319	7
	2	fresh	70°C,vacuum,2 h	1.28	8	45.4	321	7
	3	frozen	70°C,vacuum,2 h	0.92	11	20.1	309	15
	4	frozen	70°C,vacuum,2 h	0.91	11	20.1	252	12
	5	frozen	70°C,vacuum,2 h	0.88	11	20.2	239	12
	6	frozen	70°C,vacuum,2 h	0.88	11	20.2	213	11
	7	fresh	sun light	1.3	8	45.4	271	6
	8	fresh	sun light	1.3	8	45.4	283	6
	9	frozen	sun light	0.91	11	20.2	151	7
	10	frozen	sun light	0.90	11	20.2	162	8

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


Don Brian Clarke
email: don.clarke@fera.gsi.gov.uk

Antony S Lloyd

Simon Hird

David N Mortimer

Martin Gem

Victoria Adele Bailey

A Routledge

Dietary intake estimate for perfluorooctanesulphonic acid (PFOS) and other perfluorocompounds (PFCs) in English retail foods following

Keywords: perfluorooctanesulphonic acid, PFOS, perfluorocompounds, PFCs, food, fish, liver, TDI, dietary intake

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

PFOS is now the generally accepted term referring to the individual chemical perfluorooctanesulphonic acid and any closely related compounds that contain the PFOS moiety (C 8 F 17 SO 2 ), including those that may potentially degrade to PFOS in the environment [START_REF] Brooke | Environmental risk evaluation report: Perfluoroctanesulphonate (PFOS) Draft report prepared for the UK Environment Agency[END_REF]). This term is often used interchangeably with PerFluoro Compounds (PFCs), or PerFluoroAlkylated Substances (PFAS). The chemicals under consideration are best described as an homologous series of fullyfluorinated sulphonic acids, and these homologues can also be present as sulphonamide or carboxylic acid derivatives. The high-energy carbon-fluorine covalent bond makes the core perfluoroalkyl group resistant to hydrolysis, photolysis and microbial or mammalian metabolism. Standard metabolic processes can remove other constituents such as hydrocarbon groups. There are believed to be important introversions of larger to smaller molecular weight PFCs occurring in the environment. PFOS is therefore not readily degradable and can persist and bioaccumulate in the environment, probably by means of covalent protein binding.

The US Environmental Protection Agency (EPA) reported in May 2000 that PFOS combines persistence, bioaccumulation and toxicological properties to an extraordinary degree. PFOS exhibits hepatotoxicity, neurotoxicity, carcinogenicity and reproductive toxicity. One of the most disturbing findings was its unusual ability to influence second-generation rodent reproduction at 0.4 mg/kg body weight per day (bw/day) (repeated daily dosing). PFCs are also known to be endocrine disruptors [START_REF] Jensen | Emerging endocrine disruptors[END_REF]. patterns similar to other persistent organic pollutants (POPs). A recent review provides an up-to-date reference for sources, fate and transport of perfluorocarboxylates [START_REF] Prevedouros | Sources, fate and transport of perfluorocarboxylates[END_REF]. The increases in global exposure levels, have led to elevated circulatory concentrations of PFOS in mammalian plasma.

Retrospectively, this upward trend has been apparent for some decades. Aside from industrial exposure, it is not yet clear as to the exact mechanisms of exposure to specific fluorochemicals and their precursors, or their relative significance, e.g. via fire fighting foam, household and consumer products vs food and water, vs air and dust.

Historically the uses of PFOS have been widespread and include uses as; a metal surface treatment, as a fabric protector (stain repellents -carpets), impregnated in paper (grease proofing -pizza boxes), and leather, in waxes, polishes, paints, varnishes and cleaning products. In Europe the suggested scale of use is 80 tons of PFOS used between 1980 and 2005 by the North Sea oil industry (fire fighting) and an equivalent usage/emission as total European terrestrial pollution. 3M, the major PFC producer, had reduced PFOS production by 98% by the end of 2000 (equivalent to an 88% reduction in world-wide production), with total cessation in 2002 [START_REF] Olsen | Decline in Perfluorooctanesulfonate and Other Polyfluoroalkyl Chemicals in American Red Cross Adult Blood Donors, 2000-2006[END_REF][START_REF] Spliethoff | Use of Newborn Screening Program Blood Spots for Exposure Assessment: Declining Levels of Perfluorinated Compounds in New York State Infants[END_REF]. The global regulation of PFOS/PFOA, together with the voluntary change to alternative products that is already well underway in Westernised countries, should result in a decline in future environmental contamination and hence human exposure. The first declines in human exposure in localised areas have already been reported. The high human exposure to PFCs in North America led to this population and the blood samples collected by the American Red Cross, being the most highly contaminated in the world. However, their PFOS levels appear to have peaked, and are now declining in line with the previously reported half-life of 4-5 years in adult humans [START_REF] Olsen | Decline in Perfluorooctanesulfonate and Other Polyfluoroalkyl Chemicals in American Red Cross Adult Blood Donors, 2000-2006[END_REF]. A similar decline has very recently been observed in infants [START_REF] Spliethoff | Use of Newborn Screening Program Blood Spots for Exposure Assessment: Declining Levels of Perfluorinated Compounds in New York State Infants[END_REF]. While declines in US exposure to PFCs appear to have followed the fall in US production, this is not the case globally. PFOS levels appear to be continuing to increase in blood samples taken from Chinese citizens [START_REF] Jin | Historical Trends in Human Serum Levels of Perfluorooctanoate and Perfluorooctane Sulfonate in Shenyang, China[END_REF]. As the estimated half-life of PFOS in the environment is 41 years, the legacy of environmental contamination cannot be expected to be solved rapidly. One of the clearest examples of a temporal trend in the food chain was seen in guillemot eggs obtained from a Baltic island, where the guillemots feed on fish from the Baltic Sea, thus reflecting the general level of contamination in the region. The concentration in eggs rose at an almost linear rate of 18 µg/kg/per year between 1968-2003, increasing from 17 µg/kg in 1968 to 623 µg/kg in 2003 [START_REF] Holmstrom | Temporal trends of PFOS and PFOA in guillemot eggs from the Baltic sea, 1968-2003[END_REF]. As these eggs are a human food source, the trend seen in these eggs can be directly correlated with the human food chain.

Various regulatory limits for acceptable levels of PFOS in the environment are being discussed. The European Food Safety Authority (EFSA) identified 0.03 mg/kg bw per day of PFOS as the lowest no-observed-adverse-effect level (NOAEL, liver pathology) for monkeys and considered this suitable for deriving a human Tolerable Daily Intake (TDI) for PFOS of 0.15 µg/kg bw. Similarly a ten-fold higher human TDI for PFOA of 1.5 µg/kg bw has also been agreed [START_REF] Efsa | Opinion of the Scientific Panel on Contaminants in the Food chain on Perfluorooctane sulphonate (PFOS), perfluorooctanoic acid (PFOA) and their salts[END_REF].

To date there have been very few studies that have determined PFCs in the human diet. The prevalence of PFOS and its impact on the wider human food chain only became apparent when the 3M Multi-City Study reported that four of the twelve positive food concentrations were from 'control' cities, i.e., those without a fluorochemical industry (3M [START_REF] Company | Analysis of PFOS, FOSA and PFOA From Various Food Matrices Using HPLC Electrospray Mass Spectrometry[END_REF].

The emerging data on the concentrations of PFCs in food throughout the world are forming a picture of a generally low-level contamination in the wider diet, but with individual "hot spots" occurring within the fish and offal food groups. A survey of Swedish Total Diet Study (TDS) food composites [START_REF] Berger | Perfluorinated alkyl substances in market basket food samples and fish from Lake Vättern and the Baltic Sea[END_REF] focused on the four most likely food groups to be contaminated; meat products, dairy products, eggs and seafood. This survey did not positively identify any PFCs and reported less than the LOD (2.2, 3.2 µg/kg PFOS and PFOA, respectively) for all food composites surveyed. However, individual fish samples were found to contain up to 23 µg/kg PFOS [START_REF] Berger | Perfluorinated alkyl substances in market basket food samples and fish from Lake Vättern and the Baltic Sea[END_REF]. A Spanish survey of composite foodstuffs found numerous trace levels of PFOS with a maximum of 0.8 µg/kg [START_REF] Ericson | Human exposure to perfluorinated chemicals through the diet; intake of perfluorinated compounds in foods from the Catalan (Spain) market[END_REF] [START_REF] Fromme | Exposure of an Adult Population to Perfluorinated Substances Using Duplicate Diet Portions and Biomonitoring Data[END_REF]. For adults consuming 2.5 kg of food (solid and liquid) this represented mean daily dietary intakes of 123 and 270 ng of PFOS and PFOA, respectively, equivalent to 1.8 and 3.9

ng/kg bw/day. The duplicate diet collection model was therefore not dissimilar in outcome to the TDS model with regard to the intake estimates. Of interest in this study was the detailed analysis of each day's food in which just two PFOA values were unusually high. The higher of these was a concentration of 118 µg/kg in the daily diet. This one-off high value was undoubtedly from smaller portions of a single discrete foodstuff containing at least an order of magnitude higher concentration. This again is consistent with the emerging knowledge on upper-end mg/kg contamination levels in some European freshwater fish from heavily polluted industrialized areas.

In order to put European and UK data into context, recent work in Japan [START_REF] Guruge | Species-specific concentration of perfluoroalkyl contaminants in farm and pet animals in Japan[END_REF]) reported cattle, pig and chicken livers to contain mean PFOS concentrations of 34, 54 and 67 µg/kg, respectively, with the highest individual PFOS value at 92 µg/kg in a chicken liver. Chinese chicken eggs were all found to contain PFOS in the range of 45-87 µg/kg [START_REF] Wang | Perfluorooctane sulfonate (PFOS) and related fluorochemicals in chicken egg in China[END_REF]. Oysters from Tokyo bay contained 3 µg/kg of PFOS [START_REF] So | Alkaline digestion and solid phase extraction methods for perfluorinated compounds in mussels and oysters from South China and Japan[END_REF].

The aim of the survey was to develop more robust analytical methods, in particular to lower the limit of detection, to enable improved estimates of dietary intakes of the chemicals and to provide data to inform negotiations regarding possible future limits for the chemicals in foods.

Materials and methods

Collection and preparation of food samples

Priority was given to foods in which the chemicals had been detected in the previous survey (potatoes and vegetables (FSA 2006), and foods in which it was anticipated that the chemicals would be present on the basis of literature reports (fish and offal). Chemical purities were all >90%, so no corrections were considered to be necessary.

F

Sample extractions

Quadruple 10 g portions of each homogenised food were weighed out into Falcon tubes (50 ml). The appropriate volumes of internal standard (IS) and standard addition Where clear solutions with no floating matter were obtained, these were simply added in repeated ca. 5 ml portions to the cartridges as the eluent went through (to ca. 1 ml remaining). The cartridges were loaded at a constant drip rate, by increasing from gravity feed to full vacuum as required. After loading, the cartridges were washed with ammonium acetate (2 x 6 ml, 25 mM, pH 4.5) and eluted with basic methanol (4 ml, 0.1% ammonia). The eluates were reduced under a stream of nitrogen gas (60 o C), just to dryness and the residues taken up in methanol (400 µl, sonication 10 min).

Extracts were transferred into silanised glass microvials (300 µl) for LC-MS/MS determination.

The analysis of cheeses was particularly problematic, due to the excessive amount of material (ca. 50 mg) left after the standard clean-up. The clean-up method was therefore extended. The residue was halved by elution of neutral amides off the SPE in methanol (4 ml, neutral pH), and then treating this fraction as a separate extract.

Acids, both sulphonic and carboxylic were then eluted as before, using basic methanol (4 ml, 0.1% ammonia). Each fraction still contained ca.

Chromatography and quantification

Analysis was undertaken by LC-MS/MS. A CTC Pal autosampler (Presearch, UK)

and an HP1100 HPLC system with column oven (Agilent, UK) were coupled to an API4000 triple quadrupole mass spectrometer (MDS Sciex Instruments, UK). The guard cartridge was C 8 . The HPLC column (5 µm, 60 Å, 2.1 x 150 mm) was

Fluorosep RP Octyl phase, thermostatically held at 30 o C in the column oven. The injection volume was generally 10 µl. The gradient programme (methanol: aqueous ammonium formate, 5 mM, pH 4) was: 10% methanol increasing to 30% at 0.1 min (linear gradient), to 75% at 7 min and 100% methanol at 10 min, this was held for 5 min (column washing), then decreased to 10% methanol at 15.1 min, this was held 4.9 min at 10% methanol (column re-conditioning). The eluate was diverted to the mass spectrometer between 7 and 19.5 min, whereas from 0-7 and 19.5-20 min it was discarded by valve switching to waste, in order to protect the ion source. A summary of MRM transitions, retention times and solvent-based LODs of the PFC analytical standards are given in Table 2 1, with representative chromatograms demonstrating the response of the less sensitive but highly selective secondary qualifying ion for PFOS in Figure 2.

Analytical quality assurance

Stock standard solutions were prepared at 1 mg/ml in methanol and stored at 4 o C.

Dilutions of these were prepared bi-weekly to minimise surface binding or evaporation losses. Dispensing of stock solutions was monitored for any possible losses, due to evaporation, by weighing. Working solutions of mixed standards were prepared at 5 and 0.5 µg/ml in methanol for spiking. Foods (10 g) were overspiked at 1 and 10 µg/kg by addition of 20 µl @ 0.5 or 5 µg/ml of a mixed standards solution.

Tetrahydro-PFOS (TH-PFOS), 13 C 4 -PFOA, 13 C 4 -PFOS, 13 C 2 -PFDeA, D 9 -n-Et-FOSE and D 3 -n-Me-FOSEA were used as internal standards (IS) by addition of 200 µl @ 0.5 µg/ml of a mixed solution, giving a 10 µg/kg IS addition level. Each food sample was analysed in duplicate throughout the entire extraction method to ensure that advantageous point contamination was not mistaken for the presence of any native PFC. Each sample was also overspiked at a minimum of two concentrations (1 & 10 µg/kg). For a specific analyte to be considered present in a sample extract the following criteria had to be met: i) the relative retention times of the analyte had to be comparable to those of a retention time marker, an internal standard, and to authentic analytical standards of each analyte; ii) the peak had to have the correct mass transition, maximising at the correct retention time; iii) the signal to noise ratio of any peak had to be greater than 3:1. In order to prove the absence of a given PFC, the internal standard had to be present in all extracts, the blank extract had to show no signal at the retention time of the target PFC, whilst the overspiked extracts had to show a peak for the target PFC at the required retention time. As many of the foods behave differently with respect to extraction, recovery of analytes through the SPE stages and matrix induced suppression of ionisation, a single LOD (reporting level) for all foods or all target PFCs was not possible. In the absence of any suitable certified reference material (CRM), a number of samples from previous surveys that had been found to contain measurable levels of specific PFCs were re-analysed to demonstrate inter-batch reproducibility (Table 3). required, the system identifies recipes containing these foods and the percentage within the recipe. For intake assessments, users apply levels of chemicals to the foods selected. Intake2 essentially identifies which consumers have eaten the foods of interest, the amount eaten, and the level of or contaminants within the foods, and provides summary distributional analyses across consumers or the survey population.

Dietary intakes

All calculations are consumer-based, i.e. where only those who eat a particular food item are included in the calculations, rather than the data being averaged over the whole population. Average and individual age group estimates are calculated based on the most recent UK consumption data for the elderly (65 years and over) [START_REF] Finch | National Diet and Nutrition Survey: People aged 65 years and over[END_REF] adults (19-64 years) (Henderson 2002), school children (4-18 years) [START_REF] Gregory | National Diet and Nutrition Survey: young people aged 4-18 years[END_REF] and toddlers (1½ -4½ years)(Gregory 1995)) surveys. (Table 7). High level consumer based estimates are taken as the 97.5% percentile (Table 8). Other calculations are illustrative and based on multiple consumption of single analytes contained in individual samples from Table 4 (Table 9).

Results

In almost three quarters of the samples, including all those of meat other than offal, none of the chemicals was observed above the limit of detection. PFOS was the chemical detected most often, especially in fish, shellfish, liver and kidney, and usually at the highest concentrations. 75 of the food samples contained trace levels of PFOS and other PFCs just above the reporting level of 1 µg/kg. All of the listed eleven perfluoro analytes were found to be present across these food samples. All of the foods found to contain concentrations exceeding the limit of detection have been reported individually, by analyte in descending order of the summed concentration of the individual PFCs (ΣPFCs) of 1-63 µg/kg (Tables 456). between the various mammalian, fish and avian sources. As the di-hydroxycholate epimers in foods are secondary metabolites, there is as yet no clear relationship between the primary bile salts in a species and the observed interferences.

The highest positive results were all in the fish and offal food groups, as anticipated.

Other primary produce foods, such as shellfish, meat, milk, butter, cheese, cereals and vegetables, were essentially free from contamination (Table 1). There was no observable trend in farming and husbandry practices; free range, organic, farmed and wild foods were all indistinguishable by PFC content.

Discussion

Analytical methods

The analytical method has been validated previously [START_REF] Lloyd | Mass spectral studies towards more reliable measurement of perfluorooctanesulfonic acid and other perfluorinated chemicals (PFCs) in food matrices using liquid chromatography/tandem mass spectrometry[END_REF]) and performed satisfactorily, with all samples being analysed to meet the required reporting level (RL) (Tables 12). The method proved reproducible and accurate over the course of this survey as determined by repeat analysis of in-house reference food samples (Table 3).

Intake estimates

Making the assumptions that these retail food samples reflect the general diet in the UK, it can be assumed that the latter is low in PFC. Dietary intakes of PFOS, PFOA and PFOSA were estimated from the results of this survey using food consumption data from various dietary surveys, in which the food eaten by each of up to 1,724 individual consumers was recorded over a 4-or 7-day period [START_REF] Gregory | National Diet and Nutrition Survey: children aged 1½ to 4½ years. Volume 1: report of the diet and nutrition survey[END_REF][START_REF] Gregory | National Diet and Nutrition Survey: young people aged 4-18 years[END_REF][START_REF] Finch | National Diet and Nutrition Survey: People aged 65 years and over[END_REF], Henderson 2002). Average concentrations of the chemicals found in the oily fish, white fish, shellfish, liver, kidney, meat, milk and milk products, cereals, popcorn, jams, potatoes and vegetables samples were applied to all samples of the relevant food type. These dietary intakes do not take account of food types that were not included in the survey, nor of carp for which there were no recorded consumers in any of the dietary surveys. Adults were assumed to have a body weight of 60 kg. For the other age groups the individual bodyweights of each recorded consumer were used.

The dietary intakes so estimated are presented in 8).

An alternative method of estimating the dietary intakes of consumers of oily fish is to add the dietary intakes from typical portions of this food to that for the rest of the diet.

This method allows for more robust intake estimates from oily fish species for which there were very few or no recorded consumers, e.g. eel, whitebait and carp. The

Agency advises that all consumers should eat two portions of fish, one of which should be oily, as part of a balanced diet; that girls and women who may have children should not eat more than two portions of oily fish per week; and that boys, 9).

The concentrations of PFOS and PFOA in fish were consistent with those reported by other countries [START_REF] Senthilkumar | Perfluorinated compounds in river water, river sediment, market fish, and wildlife samples from Japan[END_REF][START_REF] De Voogt | Determination of perfluorinated organic compounds in food and dust[END_REF][START_REF] Nania | Monitoring of perfluorinated compounds in edible fish from the Mediterranean sea[END_REF]. The predominance of PFOS accords with evidence that pefluoroalkyl sulphonates such as PFOS are more bioaccumulative than perfluoroalkanoic acids, such as PFOA, with the same fluorinated carbon chain length (Condor 2008). The concentrations found suggest that, if regulatory limits were to be considered, these might be appropriate only for PFOS in fish and animal liver, Limits are not likely to be needed in other foods, or at all for PFOA. However, the results of this survey do not indicate a need for increased consumer protection. Accurate quantification of PFCs at concentrations below 1 µg/kg in food, while feasible, is complex and costly [START_REF] Lloyd | Mass spectral studies towards more reliable measurement of perfluorooctanesulfonic acid and other perfluorinated chemicals (PFCs) in food matrices using liquid chromatography/tandem mass spectrometry[END_REF]. It is clear from the results and the intake estimates, that it was possible to demonstrate that exposure to PFOS and PFOA would be considerably below the respective TDIs even for the highest consumers working to an LOD of 1 µg/kg. On this basis, the analytical method described and used in this paper to generate the intake estimates was fit-for-purpose. The lower bound estimate for PFOS dietary intake in the UK of 1 ng/kg/day as estimated from the results of this study, was calculated as a consumer rather than population based estimate, which gave a higher result, but this is still comparable to estimates calculated for Spain 1 ng/kg/day [START_REF] Ericson | Human exposure to perfluorinated chemicals through the diet; intake of perfluorinated compounds in foods from the Catalan (Spain) market[END_REF], Ireland 1 ng/kg/day (Clarke in press), Germany 2 ng/kg bw/day (Fromme), and Canada (4 ng/kg bw/day Tittlemier 2007).

Conclusions

The results from this study indicate an extremely low level of contamination in the English food chain, and they are directly comparable to the findings in other countries that have conducted similar studies. It would therefore be unlikely for any consumer, even an extreme consumer of the more contaminated foods, to exceed the TDI for PFOS or PFOA. b Concentrations expressed as µg/kg food equivalent, matrix matched. 10 g food matrix concentrated in the extract 25-fold. 3. Interbatch reproducibility data for repeat analysis of food sample contaminated with PFCs.

F

D 9 -n-Et-FOSE D 3 -n-Me-FOSA 2-(N-D 5 -ethylperfluoro-1-octanesulfonamido)-D 4 -ethanol) N-D 3 -methylperfluoro-1-octanesulfonamide S F F F F F F F F F F F F F F F F F O O N C 2 D 5 O H S F F F F F F F F F F F F F F
Measured PFC concentration µg/kg Sample PFBS PFHxS PFOS PFHxA PFHpA PFOA PFNA PFDeA PFUnA PFDoA PFOSA Mackerel 1[2] 1[1] Dressed crab 6[4] 9[8] 1[2] 1[1] Whitebait 11[15] 10[27] Whitebait 12[9] 6[4] Whitebait 48[40] 4[5] 1[2] Cromer crab 1[1] 14[12] 4[4] 2[1] 1[1] 1[2] Spider crab 2[1] 13[9] 5[
1 Smoked Eel < 1 <1 < 1 < 1 2 2 < 1 < 1 < 1 59 < 1 63 2 Whitebait <1 <1 5 <1 <1 2 <1 <1 1 40 14 62 3 Whitebait 7 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 15 27 49 4 Cromer crab (dressed) <1 <1 4 1 <1 1 <1 <1 2 12 <1 20 5 Spider crab (whole) <1 <1 6 1 <1 <1 <1 <1 <1 13 <1 20 6 Whitebait <1 <1 <1 <1 <1 <1 <1 <1 <1 9 7 16 7 Whitebait < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 9 6 15 8 Dressed Crab < 1 <1 8 2 < 1 <1 < 1 < 1 < 1 4 1 15 9 Brown crab (whole) <1 <1 6 3 <1 <1 <1 <1 <1 3
< 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 1 4 5 26 Cornish Sardines < 1 <1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 2 3 5 27 Ox Liver < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 5 < 1 5 28 Carp <1 <1 <1 <1 <1 <1 <1 <1 <1 4 <1 4 29 Bideford cod <1 <1 <1 <1 <1 <1 <1 <1 <1 2 2 4 30 Sardines <1 <1 <1 <1 <1 <1 <1 <1 <1 3 1 4 31 Pork Liver < 1 <1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 4 < 1 4 32 Lambs Liver < 1 <1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 4 < 1 4 33 Sardines <1 <1 <1 <1 <1 <1 <1 <1 <1 2

  biospheric distribution, bioaccumulation and biomagnification

(

  perfluoroundecanoic acid), PFDoA (perfluorododecanoic acid) and PFBSH (perfluorobutanesulphonic acid) and 3α,12α-dihydroxy-5β-cholic acid-N-(2sulfoethyl)-amide (taurodeoxycholic acid, TDCA) were supplied by Sigma (Poole, UK). PFOSA (perfluorooctanesulphonamide) and TH-PFOS (tetrahydro-PFOS) were supplied by ABCR GmbH (Karlsruhe, Germany), 13 C 4 -PFOA, 13 C 2 -PFDeA, D 9 -n-Et-FOSE (2-(N-D 5 -ethylperfluoro-1-octanesulfonamido)-D 4 -ethanol) and D 3 -n-Me-FOSA (N-D 3 -methylperfluoro-1-octanesulphonamide) were supplied by Greyhound Chromatography (Birkenhead, UK). Stock standards of those not already in solution were prepared at 1 mg/ml in methanol and stored at +4 o C. Stock solution volumes were monitored by recording the weight before and after each aliquot was removed, to check for possible losses of solvent due to evaporation. Working solutions and mixtures were prepared fortnightly and also stored at +4 o C. All fluorinated chemicals and reagents were assessed for purity and possible PFC cross contamination. For PFC analytical standards, the area responses of all multiple reaction monitoring (MRM) transitions in each separate 0.5 µg/ml solution in methanol were measured and assessed as a percentage of the total ion response [assuming equal ionization].

  to prepare two unspiked portions; one overspiked at the reporting level (1 µg/kg) and one at 10-times the reporting level (10 µg/kg). The food portions were homogenised for 1-3 mins as required in 20 ml of methanol with an Ultra Turrax (T25 basic with S25N blade). Once homogenised, more methanol was added (ca. 40 ml in total) and mixed, while withdrawing the Ultra Turrax blade.Samples were shaken vigorously overnight (16 h), then centrifuged (15 min, 5000 rpm)[START_REF] Tittlemier | Dietary exposure of Canadians to perfluorinated carboxylates and perfluorooctane sulfonate via consumption of meat, fish, fast foods, and food items prepared in their packaging[END_REF]. Eggs and milk samples required heating at 70 o C for 2 hr to induce protein precipitation. The supernatant methanol extracts were evaporated under a nitrogen stream (80 o C, in silanised glass vials) just to dryness, and the residues were re-dissolved in aqueous KOH (25 ml, 0.01 M, sonication 10 min). The aqueous solutions were then re-centrifuged (15 min, 5000 rpm). When required, the supernatants were poured in one continuous gentle movement, without breaking up the floating materials [fat] or disturbing the sediment, into a funnel connected onto the top of a preconditioned SPE cartridge (weak anion exchange)[START_REF] Taniyasu | Analysis of fluorotelomer alcohols, fluorotelomer acids, and short-and long-chain perfluorinated acids in water and biota[END_REF].

  25 mg of coextractives. A fluorous solvent mixture was then used to partition fluorinated from non-fluorinated compounds by liquid-liquid extraction (F-LLE). SPE eluates (neutral or basic) were dried and then redissolved in dichloromethane (10 ml, DCM saturated with water) by sonicating for 10 min. Trifluoroethanol (7.5 ml) and perfluorohexane (FC-72, 2.5 ml) added, the mixture was shaken (1 min), and then sonicated for 10 min. When the residues were in solution, the resulting triphasic system was heated to 80 o C for one hour to achieve a monophasic mixture. The mixtures were then cooled to -70 o C for one hour and the upper 10 ml of DCM was pipetted out and discarded as the nonfluorinated fraction. The lower two fluorous layers were pipetted out together and dried under a stream of nitrogen gas (30 o C) until just dry, and the residues taken up in methanol (400 µl, sonicated for 10 min). Extracts were transferred into silanised glass microvials (300 µl) for determination by LC-MS/MS.

  Calculations were performed by the UK Food Standards Agency using the Intake2 programme. Intake2 is the FSA in-house system supplied by Dietica for assessing exposure to chemicals in the food we eat and conducting nutrition and consumption assessments using NDNS (National Diet and Nutrition Survey) data. Intake2 is comprised of a bespoke VB front end and a Sagent back end database containing the NDNS consumption surveys, Nutrient information on all foods, and recipe composition data. Users set up a food group containing foods of interest and, if

  men and women who will not have children should not eat more than four portions of oily fish per week (Scientific Advisory Committee on Nutrition and Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment 2004, Food Standards Agency 2004). A man who ate four 70 g portions of consistently the most contaminated oily fish in the survey (i.e. smoked eel, 59 µg/kg) per week combined with average consumption of the rest of the diet (i.e. excluding the normal contribution from fish) would have a PFOS intake of 0.050 µg/kg bodyweight/day, which is again within the TDI (Table

  transitions, retention times and solvent based Limits of Determination LODs of PFC analytical standards. a Methanol based LODs (3x S/N) were all <10 ng/kg (0.25 ng/ml).

  .0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 .0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 .0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5

  [START_REF] Tittlemier | Dietary exposure of Canadians to perfluorinated carboxylates and perfluorooctane sulfonate via consumption of meat, fish, fast foods, and food items prepared in their packaging[END_REF]. A large 7-day duplicate diet study in Germany reported average values from 214 separate daily diets collected from 31 adults as 0.06 and 0.69 µg/kg for PFOS and PFOA, respectively

	freshwater fish (
	F o r
	P
	e
	e r
	R
	e
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	5

. A larger Canadian TDS study reported a maximum level of 5 µg/kg perfluorononanoic acid (PFNA) in beefsteak. PFOS was measured at 2.7 µg/kg in beefsteak, 2.6 µg/kg in marine fish, 2.1 µg/kg in ground beef and 2.0 µg/kg in

  . Analyst 1.4.2 software was used for instrument control, file acquisition and peak integration. The MS detector in MRM mode with a

	in Figure
	Turbo Ion Spray source was used for quantitative analysis. Data acquisition was conducted in one simultaneous acquisition schedule. Instrumental parameters were optimised by infusion of standard solutions directly into the MS detector (1 µg/ml in 1:1 methanol: aqueous ammonium formate (5 mM, pH 4). The Turbo Ion Spray (TIS) conditions were; turbo-gas 50 psi, curtain-gas 12 psi, nebuliser-gas 50 psi, desolvation o r P e e r R e v i e w O n l temperature 450 F y
	10

o C. An Excel spreadsheet was used to calculate PFC concentrations from the standard additions. The standard addition calculation approach is illustrated

  were found in samples of smoked eel and whitebait. PFOSA was the next most frequently detected chemical, but only in certain fish species and crab. PFOA was detected at low concentrations in some samples of whitebait, crab and liver. The other chemicals analysed were detected only occasionally and at low concentrations, but no chemical was absent from and totally undetected in all of the samples. A summary table of the results for all 252 samples, arranged by sample type, illustrates the frequency, range and mean levels of contamination (Table1). The highest individual concentrations were of 59 µg/kg of perfluorooctanesulphonic acid (PFOS) and 63 µg/kg total PFCs (ΣPFCs) in an eel sample, and 40 µg/kg PFOS (62 ΣPFCs) in a whitebait sample. There were 6 samples with ΣPFCs >15 µg/kg (fish &

	F o r
	P
	e
	e r
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	v i e
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	y
	The highest concentrations

crustaceans), a further 7 samples with ΣPFCs in the range 11-15 µg/kg (including a liver), nine with ΣPFCs in the range 6-10 µg/kg (fish and livers), 31 with ΣPFCs in the range 2-5 µg/kg (including kidneys, popcorn and processed peas) and 22 with

ΣPFCs at the reporting level of 1 µg/kg (including eggs and potatoes).

A set of representative chromatograms for one of the PFOS determinations is given (Figure 2). It was noted in separate LC-TOF experiments that PFOS [M-H] -m/z 498.927 can be readily distinguished from the isobaric interferences. These have a profile m/z 498.289, 499.289 and 500.289 which, within the constraint of containing a sulphonate grouping (MRM 499>80), gave a single fit [M-H] -formulae of C 26 H 44 NO 6 S, taurodeoxycholic acid, with the first natural abundance isotope peak [M+1,-H] -m/z 499.289 being the actual interfering ion. As there is a set of largely indistinguishable di-hydroxycholate epimers about the C 3 , C 6 , C 7 , C 12 positions, the actual interferences in the various foods differ between samples as do bile salts

Table 7

 7 included, the overall intakes shown in Table7also apply to the whole population.The estimated upper bound average adult dietary intakes of PFOS and PFOA 0.01 and 0.01 µg/kg bodyweight/day respectively (Table7) are within the Tolerable Daily

	for consumers in different

combined with the relatively high concentrations in these food types, inflate the consumer intakes from those food types. However, because foods bread, milk and potatoes, all of which are consumed by nearly all the UK population were also

Table 1 .

 1 Summary of the levels of PFCs by food type. ΣPFCs = the lowest and highest values of the combined concentration of all 11 Individual fluorinated analytes. Mean = lower bound, <LOD =0 N = The number of samples analysed in each group. N≥LOQ = The number of samples where residues were measured.Meat = pork, lamb, beef, venison, chicken, turkey.

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

uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 2Table 4 .

 4 Individual concentrations of PFCs in each food sample.

	1
			Measured PFC concentration µg/kg
	Sample	PFHxA PFHpA PFOA PFNA PFDeA PFUnA PFDoA	PFBS	PFHxS PFOS PFOSA	ΣPFCs
			6]
	Wild roe deer liver	10[11]	3[6]	1[2]
	Carp roe	765[757]
	Interbatch reproducibility data for repeat analysis of naturally incurred PFC containing food sample.
	Results for the second determination are presented in parenthesis.

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Table 5 .

 5 Concentrations (µg/kg fresh weight) of PFOS, PFOA, PFAS and PFOSA in fish and shellfishNote: * Means and ranges affected by one or more higher than usual LODs. PFAS = ΣPFCs.

	1	3

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co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 2Table 7 .

 7 Estimated average UK dietary intakes (µg/kg bodyweight/day) of fluorinated chemicals by consumers of different age ranges

					Food Additives and Contaminants		Page 28 of 32
	1
	2	from the diet in 2007-08
	3
		Food group		Estimated average dietary exposure (µg/kg bodyweight/day)
			PFOS		PFOA	PFOSA	PFAS
			Upper	Lower	Upper	Lower	Upper	Lower	Upper	Lower
		Senior citizens
	5 6 7	-living at home -in old peoples' homes Adults Schoolchildren -age 4-6 years -age 7-10 years -age 11-14 years -age 15-18 years Toddlers -age 1.5-2.5 years -age 2.5-3.5 years -age 3.5-4.5 years	0.009 0.008 0.01 0.02 0.02 0.01 0.01 0.04 0.03 0.03 F o r P e e r R e v i e w 0.0009 0.008 <0.00005 0.008 0.0004 0.007 <0.00005 0.007 0.0001 0.01 <0.00005 0.01 0.001 0.02 <0.00005 0.02 0.0008 0.02 <0.00005 0.02 0.0005 0.01 <0.00005 0.01 0.0005 0.009 <0.00005 0.009 0.0008 0.04 <0.00005 0.04 0.001 0.03 <0.00005 0.03 0.0009 0.03 <0.00005 0.03	0.0003 0.0001 0.0004 0.0003 0.0003 0.0002 0.0002 0.0002 0.0003 0.0003 O n l y 0.09 0.08 0.1 0.3 0.2 0.1 0.1 0.4 0.4 0.3	0.001 0.0007 0.002 0.002 0.001 0.001 0.001 0.001 0.002 0.002
						1

PFAS = ΣPFCs

The estimates for PFAS are both upper and lower bound. Intake estimates are consumer rather than population based, 4 where the non-consumers of a given food type are excluded, resulting in higher values.

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Table 8 .

 8 Estimated high-level 97.5 percentile UK dietary intakes (µg/kg bodyweight/day) of fluorinated chemicals by consumers of different age ranges from the diet in 2007-08

	Page 29 of 32			Food Additives and Contaminants
	Food group		Estimated average dietary exposure (µg/kg bodyweight/day)
		PFOS		PFOA	PFOSA		PFAS
		Upper	Lower	Upper	Lower	Upper	Lower	Upper	Lower
	Senior citizens
	-living at home	0.02	0.004	0.02	0.0002	0.02	0.002	0.2	0.007
	-in old peoples' homes	0.02	0.003	0.02	0.0001	0.02	0.001	0.2	0.004
	Adults	0.02	0.006	0.02	0.0002	0.02	0.002	0.2	0.009
	Schoolchildren
	-age 4-6 years	0.05	0.006	0.04	0.0002	0.05	0.002	0.5	0.01
	-age 7-10 years	0.05	0.004	0.03	0.0001	0.03	0.002	0.3	0.006
	-age 11-14 years	0.02	0.003	0.02	0.0001	0.02	0.001	0.2	0.004
	-age 15-18 years	0.02	0.003	0.02	0.0001	0.02	0.001	0.2	0.006
	Toddlers
	-age 1.5-2.5 years	0.08	0.005	0.08	0.0001	0.08	0.001	0.9	0.007
	-age 2.5-3.5 years	0.08	0.007	0.08	0.0002	0.08	0.002	0.8	0.01
	-age 3.5-4.5 years	0.06	0.005	0.06	0.0002	0.06	0.002	0.6	0.008
	PFAS = ΣPFCs

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Table 9 .

 9 Illustrative intakes (µg/kg bodyweight/day) of PFOS and PFOSA from selected fish compared to remainder of diet. The result is calculated where the line crosses the x-axis. Data are rounded to 1 significant figure, this sample was reported as PFOS contamination of 2 µg/g (primary 1.75, qualifier 1.81). The qualifying ion has a constant 24% of the response of the primary ion response (0.0897/0.3857). The average ion ratio for native and overspiked PFOS is equivalent to the precision ratio, 1.81/1.75 4%. Figure 2. A) Chromatogram of the primary ion 499.1>80.1[M>SO 3 ] -for PFOS in a sample of whitebait (sample 3 in Table1, containing 15 µg/kg naturally incurred PFOS) using; A) unspiked sample, B) overspiked at 1 µg/kg, C) overspiked at 5 µg/kg, D) overspiked at 10 µg/kg, E) overspiked at 20 µg/kg and F) overspiked at 50 µg/kg. Other peaks in this MRM are most likely due to epimers of taurodeoxycholic acid, specifically the 13 C 1 native abundance M+1 isotope .

	PFOS	PFOSA

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Acknowledgments

The authors would like to thank Ventress Technical Ltd for their assistance in preparing the sampling plan and for obtaining the samples. This work was funded by the UK Food Standards Agency.