Kevin Mark Cooper

David Glenn Kennedy

Stability studies of the metabolites of nitrofuran antibiotics during storage and cooking

Keywords: nitrofurans, metabolites, stability, cooking, freezer storage, semicarbazide, FoodBRAND

published or not. The documents may come

Stability studies of the metabolites of nitrofuran antibiotics during storage and cooking

Introduction

Food safety and the exposure of the consumer to chemical residues in their food are of growing concern and the subject of extensive worldwide research. The year 2001 saw the beginning of what became known as the "nitrofuran crisis" in world food production.

The nitrofurans are a group of antibiotics prohibited within the European Union (EU) for use in food-producing animals (Commission Regulation 1995) because of their potentially carcinogenic and mutagenic effects on human health (Van Koten-Vermeulen et al. 1993). They have been used previously for prevention and treatment of various gastrointestinal infections and as growth promoters in livestock. Nitrofurans are metabolised rapidly by animals in vivo [START_REF] Mccracken | Determination of furazolidone in porcine tissue using thermospray liquid chromatography-mass spectrometry and a study of the pharmacokinetics and stability of its residues[END_REF] but persistent tissuebound metabolites are formed which may be released by mild acid hydrolysis and used as marker residues (Hoogenboom et al. 1991). For example, the stable tissue-bound metabolite 3-amino-2-oxazolidinone is monitored as a marker residue for the parent nitrofuran furazolidone.

The four main nitrofuran antibiotics are furazolidone, furaltadone, nitrofurantoin and nitrofurazone. Their marker residues are 3-amino-2oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) respectively.

The widespread use of nitrofuran drugs in food production, in particular the poultry and aquaculture industries, was uncovered by partner laboratories in the EU-funded R&D project FoodBRAND (www.afsni.ac.uk/foodbrand), primarily AFBI (formerly DARD)

Belfast and RIKILT, Wageningen, The Netherlands. The first major nitrofuran food scare concerned prawns and chicken from Vietnam, Thailand and Brazil. This led the reviewed [START_REF] Kennedy | Analysis of veterinary drug residues in food: the nitrofuran issue[END_REF]. Legislators and consumers may also wish to know if nitrofuran residues in food are destroyed when that food is cooked. Between 1995 and 1999, Rose and co-workers in the Central Science Laboratory, Norwich demonstrated that residues of a range of veterinary drugs have varying degrees of stability during cooking and, therefore, that cooking influences the level of risk posed by such residues (Rose et al. 1999). However, with the exception of AOZ, nitrofuran stability during cooking has not been assessed. [START_REF] Mccracken | The bioavailability of residues of the furazolidone metabolite 3-amino-2-oxazolidinone in porcine tissues and the effect of cooking upon residue concentrations[END_REF] demonstrated that AOZ residues were stable in meat, liver and kidney of furazolidone treated pigs following cooking by microwave, grilling and frying. However, no reports have been published

concerning other nitrofuran residues.

Furthermore, very little information is available concerning the stability of nitrofuran metabolite residues in food during long-term frozen storage. Residues inspection laboratories will wish to know if metabolite residues in incurred tissues degrade significantly during storage. In addition, the stability during refrigerated storage of standard solutions of the metabolites used for analytical monitoring has not been defined.

The current study aims to determine the stability of the four major nitrofuran metabolites during cooking and storage.

Materials and methods

Materials and instrumentation

Internal standards D 4 -3-amino-2-oxazolidinone, D 5 -3-amino-5-morpholinomethyl-2oxazolidinone, 13 C 3 -1-aminohydantoin and

Cooking stability study

Three muscle samples and 3 liver samples from one withdrawal time-point were selected from pigs fed each of the 4 medicated diets (6 weeks withdrawal for furazolidone, furaltadone and nitrofurazone, 3 weeks for nitrofurantoin). After thawing to room temperature, muscle samples were cut into 4 portions (40 to 60 g). Three of these portions were weighed immediately before and after cooking by grilling, microwaving or roasting. Samples were then re-frozen at -20°C along with the fourth uncooked muscle sample. Liver samples were cut into 3 portions (30 to 50 g). Two of these portions were weighed immediately before and after cooking by frying or roasting. Samples were then re-frozen at -20°C along with the third uncooked liver sample. Cooking conditions were as follows.

Grilling. Muscle was grilled for 8 min, turning once, under medium heat in a pre-heated domestic grill on a grill rack, which allowed meat juices to escape. bag on a rotating plate in an 800 Watt domestic microwave oven at 50% setting, which was taken from a recipe for the cooking of meat..

Roasting.

Muscle and liver were roasted for 20 min at 170°C in a pre-heated fan-assisted domestic oven, in ovenproof plastic cooking bags on an uncovered tray on the middle shelf of the oven.

Frying. Liver was fried for 2.5 to 3 min each side at medium heat setting on a domestic ceramic hob, in a minimal volume of sunflower oil to prevent burning.

In all cases, excess juices and cooking oil were removed from raw and cooked tissues using absorbent tissue paper before weighing the samples. The maximum internal temperature of each sample was measured on completion of cooking by inserting a digital penetration probe thermometer into the centre of the sample. All samples, raw and cooked, were analysed by LC-MS/MS for total nitrofuran metabolites as described below.

Tissue storage stability study

The stability of total nitrofuran metabolites in incurred porcine muscle and liver was assessed during 8 months storage at -20°C. Five muscle samples and 5 liver samples were selected from pigs fed each of the 4 medicated diets. Tissues were homogenised in domestic food processor and divided into 4 separate aliquots to avoid repeated freezethawing. Tissues were stored at -20°C. At 0, 2, 4 and 8 month intervals an aliquot of each sample was thawed to room temperature and analysed for total nitrofuran metabolites as described below. For each of these 4 analytical runs, completely new sets of calibration standard solutions were prepared freshly from stock nitrofuran metabolite standard powders as described below. The use of fresh standards would ensure that any observed reduction in nitrofuran concentration in tissue would not be masked by a commensurate degradation of metabolites in stored standard solutions.

Standard storage stability study

The stability of solutions of each of the four nitrofuran metabolites, in methanol, was assessed at two concentrations (1.0 mg ml -1 and 10 ng ml -1 ) during storage at 4°C for 0, 2, 6, 8 and 10 months. At each time point, the concentration in the stored standards was measured against analytical standards, freshly prepared from powdered stocks. These powders were stored desiccated at room temperature throughout this period. Separate 1 mg ml -1 stock solutions were prepared in methanol for each metabolite at each time-point.

Mixed standard solutions (10 Pg ml -1 , 1 Pg ml -1 , 100 ng ml -1 and 10 ng ml -1 ) were prepared by volumetric serial dilution of the freshly prepared 1 mg ml -1 stock solutions at each time-point. Using the LC-MS/MS nitrophenyl derivatisation and extraction method described below, a calibration standard curve (consisting of duplicate analyses of 6 standards in the range equivalent to 0 to 25 ng ml -1 ) was prepared using the freshly prepared 10 ng ml -1 mixed standard solution. Each of the stored 10 ng ml -1 solutions were analysed and quantified, as five replicates, against this calibration curve. Each of the stored 1 mg ml -1 solutions were serially diluted to 10 ng ml -1 immediately prior to analysis (to bring them within the range of the standard curve), and were analysed and quantified, as five replicates, against the same calibration curve. Results obtained following the analysis of the dilutions made from the stored 1.0 mg ml -1 solutions are presented after correction for the 100,000-fold dilution that was necessary to bring them within the range of the standard curve.

Sample derivatisation and LC-MS/MS analysis

This analytical method is based on the protocol for total nitrofuran metabolites (sum of Tissues were minced in a domestic food processor and samples (1.00 ± 0.02 g) weighed into 30 ml glass tubes. Mixed internal standard (100 Pl of a 1 Pg ml -1 solution) was added to all samples, controls and standards. To all tubes were added 0.1 M hydrochloric acid (9 ml) and 2-nitrobenzaldehyde (150 Pl, 100 mM in DMSO). Samples were homogenised gently for 1 min in a laboratory homogeniser. The homogeniser was rinsed with 3 x 1 ml 0.1 M hydrochloric acid, which was added to sample tubes. Calibration standards were prepared by addition of mixed standard solutions to 30ml glass tubes.

Control muscle and liver tissues from nitrofuran-free pigs were fortified at 50 Pg kg -1 with mixed standard (50 Pl of a 1 Pg ml -1 solution) to act as recovery control samples.

Blank tissues were also included in every analytical run. Tubes were vortex mixed for 10 sec to disperse the froth produced during homogenisation, then were incubated overnight (approximately 16 hr) in a water bath held at 37°C. All tubes were then adjusted to pH 7.2 ± 0.2 with 0.3 M tri-sodium orthophosphate (2.2 ml) and 1 M sodium hydroxide (approximately 6 drops). Liquid-liquid extraction was carried out using ethyl acetate (2 x 8 ml, inversion for 1 min and centrifugation at 2000 rpm for 15 min at 4°C) and the organic phase evaporated to dryness at 50°C under nitrogen. Residues were re-dissolved in methanol : water (50 : 50 v/v; 2 ml) and an aliquot transferred to HPLC microvials (200 Pl) and stored at 4°C. Prior to LC-MS/MS analysis, microvials were centrifuged at 13 000 rpm for 15 min to remove precipitated material. LC-MS/MS was carried out using the conditions described previously [START_REF] Cooper | Depletion of four nitrofuran antibiotics and their tissuebound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV[END_REF]. Analyte concentrations in samples were calculated by comparing the ratio of an analyte base peak response to its appropriate internal standard response with the same ratio in calibration curve standards. Tissue sample chromatograms were clean, with no interferences arising from cooking processes. This method has been subject to extensive validation in a wide range of tissues (albeit uncooked). In all cases, CC and CC were less than the EU Minimum Required Performance Limit (MRPL, 1.0 Pg/kg) for all of the metabolites, indicating fitness-for-purpose and CVs obtained during validation, above and below the MRPL, were less than 15%. and grilling (74°C).

[Insert TABLE I about here]

Concentrations of all four nitrofuran metabolites dropped during cooking of incurred liver, losses ranging from 6 to 33%. Losses were consistently greater following roasting of liver than frying, possibly due to the higher internal temperatures achieved during roasting. Concentrations of AOZ, AMOZ and SEM also dropped during cooking of incurred muscle (losses ranging from 7 to 15%) but no loss of AHD was observed. This was surprising, as anecdotal evidence previously suggested that AHD was the least stable of the nitrofuran metabolites. There was no clear correlation between the drop in metabolite concentrations in muscle observed during different cooking methods and their respective internal cooking temperatures. These data clearly demonstrate that all four major nitrofuran metabolites are sufficiently stable to survive conventional cooking procedures in sufficient quantities to continue to pose a health risk to the consumer. This finding agrees with McCracken and [START_REF] Mccracken | The bioavailability of residues of the furazolidone metabolite 3-amino-2-oxazolidinone in porcine tissues and the effect of cooking upon residue concentrations[END_REF] who observed no significant reduction in total AOZ in porcine liver or muscle following frying, grilling or microwaving. For the consumer, the loss of (at best) one third of the chemical residues in their food by cooking is of little comfort. Consumers want their meat to be residue-free on the supermarket shelf, and on their plate.

It could be argued that the tissue slices used for comparative cooking procedures did not contain equal concentrations of residues prior to cooking. Various studies have demonstrated that concentrations of some veterinary drug residues such as tilmicosin [START_REF] Beechinor | Variability in residue concentrations of tilmicosin in cattle muscle[END_REF]Bloomfield 2001), clenbuterol (Rose et al. 1995) and chloramphenicol [START_REF] Cooper | Aspects of extraction, spiking and distribution in the determination of incurred residues of chloramphenicol in animal tissues[END_REF] are not evenly distributed throughout a tissue sample. Nonhomogenous residue distribution potentially raises problems for residues surveillance, in particular when intact A and B samples are sub-sampled from a tissue prior to tissue homogenisation. In cases of prosecution of a food producer when a second quantitative, confirmatory analysis is required, the second sample may give a different result to the first sample. This may lead to confusion in the case of legal medicines where a maximum residue limit (MRL) applies -sample A may be above the MRL while sample B may be below. However, in the case of banned substances such as the nitrofurans, such minor quantitative variations are not as problematic, at least in principle, since the confirmation of any concentration of the substance is a non-compliance. . Furthermore, inherent variation in tissue distribution can be minimised by sampling adjacent slices of a tissue sample. This was the practice in the current study where adjacent tissue slices were sub-sampled for each cooking procedure and the uncooked control sample. Furthermore, non-homogenous residue distribution within a tissue is not an issue in the tissue storage stability study described below. In this case tissue samples were thoroughly homogenised before dividing into aliquots for storage at -20°C.

The presence of metabolites in cooked meat indicates that nitrofuran antibiotics have been administered to the livestock or have contaminated the food product at a later stage.

The metabolic breakdown of the nitrofuran drugs is complex and the routes leading to toxicological effects in animals and humans are unclear. It is thought that the nitrofurans bind covalently to macromolecules via the opening of the furan ring structure (Silva et al. 1993[START_REF] Debnath | Mechanistic interpretation of the genotoxicity of nitrofurans (antibacterial agents) using quantitative structureactivity-relationships and comparative molecular-field analysis[END_REF]. A variety of metabolite structures have been postulated, some of which contain side chains including the marker residues measured in this study.

It is likely that the acidic conditions in the human stomach would liberate these residues, making them available for absorption. When rats were fed tissues from furazolidone treated pigs, AOZ was detected subsequently in the rat tissues [START_REF] Mccracken | The bioavailability of residues of the furazolidone metabolite 3-amino-2-oxazolidinone in porcine tissues and the effect of cooking upon residue concentrations[END_REF]. It is not clear to what extent the marker residues AOZ, AMOZ, AHD and SEM are themselves toxic. For example, it has been suggested that the in vivo inhibition of the enzyme monoamine oxidase (MAO) results from the formation of U-hydroxyethylhydrazine from the ring cleavage of AOZ (Hoogenboom et al. 1991).

However, the MAO inhibitory effect of AMOZ is lower than that of AOZ (Hoogenboom et al. 1994). The authors are unaware of any studies of the toxicity of AHD. Following a major food scare in 2004 involving the discovery of SEM in baby food (SEM was a byproduct of the manufacture of the plastic gaskets used in the jar lids) the European Food Safety Authority (www.efsa/eu/int) commissioned studies into the toxicity of SEM. The opinion of the EFSA Panel was that "the issue of carcinogenicity is not of concern for human health at the concentrations of SEM encountered in food" (EFSA 2005).

However, it must be emphasised that the marker metabolites measured in the present study are merely fragments of many unidentified and potentially toxic metabolites that will be present in tissue of an animal treated with a nitrofuran antibiotic.

Tissue storage stability study

Data relating to the stability of the nitrofuran metabolites during long-term storage at -20°C are shown in Table II. At each time point (0, 2, 4 and 8 months storage) tissues were analysed using standards prepared freshly from stock powders. Each variable (for example, AOZ concentration in muscle) was analysed statistically using the following technique. A separate linear regression was fitted against storage time for each sample, generating a regression coefficient (or slope). If the concentration within a variable falls significantly over time, then the slope of this regression should be negative.

Consequently, the standard one tailed t-test was applied to the five regression coefficients (for example the five AOZ muscle samples) to test the null hypothesis that the nitrofuran concentration does not fall over time, against the alternative hypothesis that the concentration does fall (a one-tailed test).

[Insert TABLE II about here]

There was no significant reduction (P>0.05) in the concentration of any of the four nitrofuran metabolites in either muscle or liver over the 8 months period of storage at -20°C. Residues inspection authorities, official control and research laboratories can be reassured that the total nitrofuran metabolite content of incurred tissue samples will not change significantly during extended storage at -20°C.

Standard storage stability study

Data relating to the stability of methanolic standard solutions of the nitrofuran metabolites during storage at 4°C are shown in Table III. Each variable (for example, AOZ concentration in 1 mg ml -1 stock standard solution) was analysed statistically using the same linear regression and one-tailed t-test technique as applied above to the tissue storage data, the only difference being that the regression was applied within a replicate analysis as opposed to within a repeated analysis of a sample. No significant reduction (P>0.05) was seen in the concentration of any of the four nitrofuran metabolites in either 1 mg ml -1 or 10 ng ml -1 methanolic solutions over the 10 month period of storage at 4°C

with the exception of SEM in 10 ng ml -1 solution (P = 0.015). The equation of the linear regression line for SEM in 10 ng ml -1 solution was y = -0.0127x + 1.0604. The slope of this regression indicates that the concentration of a 10 ng ml -1 SEM standard solution will fall by 5% in 3.9 months. The 1 mg ml -1 stock SEM solution, however, was stable for at least 10 months. The authors therefore suggest that stock solutions of nitrofuran metabolites in methanol may be retained for 10 months, whilst working standard dilutions prepared from these stocks should be used for no longer than 4 months. Some laboratories currently place expiry dates of 1-2 months on nitrofuran working standards.

This study demonstrates that such standards may be retained for at least 4 months.

[Insert

  these countries(Commission Decision 2002a, b, c). These restrictions have now been lifted following proactive measures taken by the Governments concerned to reduce nitrofuran use in their food production industries and to implement effective residues monitoring programmes. However, nitrofuran use has been shown to be more widespread than initially thought. Furaltadone contamination of the Portuguese poultry industry led to the destruction of 1.5 million birds in 2002. Metabolites of furazolidone and furaltadone were discovered by the FoodBRAND project consortium (O'Keeffe et al. 2004) in pork meat purchased in Portugal, Greece and Italy. Furthermore, the European Commission issued notifications to Member States via its Rapid Alert System for Food and Feed concerning findings of nitrofurans in fish from Taiwan, crayfish and salted hog casings from China, prawns from Bangladesh, India and Indonesia, catfish from Thailand, egg powders from India, Brazil, Israel, France and Mexico, honey from Vietnam, Argentina, Turkey and various European countries, and poultry meat products from Argentina, Romania and Bulgaria. In light of these findings, the consumer will want to know what health risks are posed by nitrofuran residues in their food.AOZ in tissues of pigs fed furazolidone has been shown to be bioavailable to the rat[START_REF] Mccracken | The bioavailability of residues of the furazolidone metabolite 3-amino-2-oxazolidinone in porcine tissues and the effect of cooking upon residue concentrations[END_REF]. It is likely that the acidic conditions in the human stomach would also liberate these potentially carcinogenic metabolite side chains and pose a threat to human health. Consequently, there is a "zero tolerance" towards the of nitrofurans in food-producing animals within the EU. The development of methods and legislation with regard to the monitoring of nitrofuran residues has recently been

  extractable and tissue-bound metabolites) developed by RIKILT Wageningen and AFBI VSD Belfast as part of the FoodBRAND project and disseminated to the EU National and Community Reference Laboratory networks and 3 rd countries during 2001-2003.

  Data relating to the stability of the nitrofuran metabolites during cooking are shown in TableI. Each datum is a mean of three different tissue samples selected to contain similar concentrations of metabolites before cooking. All concentrations are based on the wet weight of tissue samples, following correction for loss of water during the cooking process, as judged from the figures contained in columns 4 and 5 of TableI. Internal cooking temperatures ranged from 68 to 98°C. Higher internal temperatures were achieved during roasting of liver (mean 92°C) than frying (84°C). Microwaving of muscle achieved the highest temperatures (mean 95°C) compared with roasting (86°C)

  13 C 15 N 2 -semicarbazide were supplied by

	furazolidone, furaltadone, nitrofurantoin and nitrofurazone at 400 mg per kg feed, the
	oxazolidinone (AOZ) and semicarbazide (SEM) were supplied by Sigma-Aldrich (Poole, recommended therapeutic dose for furazolidone preparations prior to it being banned in
	UK) and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) by Chemical Synthesis 1995 (National Office of Animal Health Limited 1992). Medicated feed was provided ad
	Services (Belfast, UK). 1-Aminohydantoin (AHD) was a gift from Proctor and Gamble libitum for 10 days after which it was withdrawn and replaced with conventional
	Pharmaceuticals USA. Unless stated, all other chemicals were obtained from Sigma-unmedicated feed for a 6 weeks drug withdrawal period. At weekly intervals following
	F o withdrawal of medicated feed, 3 pigs from each feed group were euthanized by captive Aldrich. F o bolt and samples of Longissimus dorsi muscle, liver and kidney removed and stored at
	r P e e r An Agilent 1100 Series HPLC system (Agilent Technologies, USA) coupled to a Quattro operating under MassLynx ® software, were used for sample analysis. The mass spectrometer operated in electrospray positive mode and data acquisition was in multiple r e e Ultima ® Platinum tandem mass detector (Micromass/Waters, Manchester, UK), both r P -20°C.
	R e v i e w reaction monitoring mode (MRM). The HPLC system was equipped with a Luna C18(2) 3 Pm, 2.0 x 150 mm column (Phenomenex, UK). A binary gradient mobile phase was used at a flow rate of 0.2 ml/min, solvent A being 0.5 mM ammonium acetate and methanol (80 : 20 v/v mix), solvent B being 100% methanol. A Testo 915-1 Universal Thermometer was obtained from, Testo AG, Lenzkirch, Germany. Tissues were minced R e v i e w
	O n in a Mini Prep Plus domestic food processor (Waring, Torrington, CT, USA) before homogenising in a SL2 laboratory homogeniser (Silverson Machines Ltd, Chesham, O n
	England).	l y l y
	Production of incurred pig tissues
	Weaned piglets, approximately 8 weeks of age, were divided into 4 groups and housed in
	concrete floored pens.	Four nitrofuran medicated feeds were prepared using
		Page 7 of 24

TABLE III

 III The various stability data presented here demonstrate that the metabolites AOZ, AMOZ, AHD and SEM, which are used as marker residues of their parent nitrofuran antibiotics, show remarkable chemical stability. They are resistant to conventional domestic cooking procedures and are not degraded in edible animal tissues during many months storage in the freezer or in pure standard solution in the refrigerator. Whilst this chemical stability is advantageous to the analyst monitoring the nitrofuran drugs in food production, it is to the detriment of the consumer of that food. This study highlights the need for continued vigilance in the global monitoring of nitrofuran abuse and possible routes of contamination. Keeffe, M., Conneely, A., Cooper, K.M.,Kennedy, D.G., Kovacsics, L., Fodor, A., Mulder, P.P.J., van Rhijn, J.A., Trigueros, G. 2004. Nitrofuran antibiotic residues in pork: the FoodBRAND retail survey. Analytica Chimica Acta 520: 125-131.

	O'Rose, M.D., Bygrave, J., Sharman M. 1999. Effect of cooking on veterinary drug residues
	in food part 9: Nitroimidazoles. Analyst 124: 289-294.
	F o F o F o F o Rose, M.D., Shearer, G., Farrington, W.H.H. 1995. The effect of cooking on veterinary drug residues in food: 1. clenbuterol. Food Additives and Contaminants 12: 67-76. F o
	r P r P r P r P r Silva, J.M., Khan, S., O'Brien, P.J. 1993. Molecular mechanisms of nitrofurantoin-induced hepatocyte toxicity in aerobic versus hypoxic conditions. Archives of P
	e e r e e r e e r e e r Biochemistry and Biophysics 305: 362-369. van Koten-Vermeulen, J.E.M., Wouters, M.F.A., van Leeuwen, F.X.R. 1993. Report of 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 the 40 th Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), World Health Organisation, Geneva, pp. 85-123. R e v i e w
	O n O n O n O n O n
	l y l y l y l y l y
	about here]
	Page 17 of 24 Page 20 of 24 Page 21 of 24

TABLE AND FIGURE CAPTIONS:

 AND

	Table I. Effect of various cooking methods on the concentration (Pg kg -1 ) of total
	nitrofuran metabolites in pig muscle and liver. Data are means ± standard errors of 3
	different tissue samples from a single withdrawal date.	Expected metabolite
	concentrations are corrected for weight loss during cooking.

Table II .

 II Effect of long term storage at -20°C on the concentration (Pg kg -1 ) of total nitrofuran metabolites in pig muscle and liver. The figures in bold are the mean ± SE of the normalised concentration (relative to the concentration at t = 0) measured in each tissue sample.

Table III .

 III Stability of nitrofuran metabolites (1.0 mg ml -1 and 10 ng ml -1 in methanol) during storage at 4°C.

	Page 21 of 24	Food Additives and Contaminants
		F o r P e e r R e v i e w	O n l y
		Page 22 of 24

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk

Table I

 I

				Food Additives and Contaminants
	Tissue Nitrofuran	Treatment	Weight	Weight	Temp °C	$g kg	-1 before	$g kg	-1	$g kg	-1 found	Net change (%)
	metabolite AOZ Muscle AOZ Liver Liver AMOZ Muscle AMOZ Liver AHD Muscle AHD Liver SEM	before (g) 42.3 46.9 54.2 41.3 56.2 38.0 37.8 53.0 51.8 50.4 44.4 42.9 45.7 40.8 53.5 36.3 F o r P e e r R e v i e w after (g) cooking 29.6 83.9 Roast Fry 27.3 94.1 Uncooked 51.3 ± 4.1 Grill 37.2 75.3 Microwave 24.8 85.7 Roast 36.7 88.5 Uncooked 45.6 ± 2.8 Fry 27.9 83.5 Roast 23.8 89.5 Uncooked 32.8 ± 1.7 Grill 41.0 67.5 Microwave 32.2 98.1 Roast 32.5 85.1 Uncooked 72.3 ± 7.2 Fry 32.5 82.2 Roast 28.0 91.9 Uncooked 41.0 ± 1.2 Grill 32.8 76.8 Microwave 25.6 96.7 Roast 35.9 83.2 Uncooked 25.4 ± 1.2 Fry 26.0 84.4	expected after cooking 75.3 ± 5.6 88.8 ± 1.9 66.5 ± 5.3 75.9 ± 5.6 69.7 ± 5.2 44.6 ± 1.8 52.4 ± 4.1 93.6 ± 9.5 116.2 ± 9.8 112.5 ± 10.9 56.1 ± 2.1 62.6 ± 1.6 35.4 ± 1.7 41.1 ± 2.2 38.0 ± 2.5 57.8 ± 8.7 O n l y after cooking 59.4 ± 6.6 59.6 ± 2.7 59.7 ± 6.3 63.9 ± 1.9 62.0 ± 4.6 39.7 ± 2.2 38.9 ± 3.0 86.8 ± 7.3 99.5 ± 2.1 94.4 ± 7.6 46.5 ± 1.8 48.5 ± 0.6 36.6 ± 0.4 41.4 ± 1.5 37.9 ± 1.7 53.1 ± 5.4	during cooking -21 -33 -10 -15 -11 -11 -26 -7 -14 -15 -17 -22 +4 +1 0 -6
		Roast	39.6	23.6	93.4			68.2 ± 8.2	51.8 ± 6.4	-24
		Uncooked				40.2 ± 4.1
	Muscle SEM	Grill	54.8	38.4	77.0			343.3 ± 14.0	293.6 ± 10.4	-14
		Microwave	47.2	29.5	98.1			387.5 ± 19.2	338.2 ± 12.3	-13
		Roast	55.6	37.0	87.3			361.4 ± 15.8	320.3 ± 22.5	-11
		Uncooked				240.9 ± 10.0
					Page 23 of 24

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk

Table II

 II

	Page 23 of 24
	Nitrofuran	Muscle	Muscle	Muscle	Muscle	Liver	Liver	Liver	Liver
	metabolite	0 months	2 months	4 months	8 months	0 months	2 months	4 months	8 months
	AOZ	54.0	63.4	60.6	70.3	226.4	232.3	*	198.6
		65.3	72.7	76.1	86.3	219.8	208.4	253.4	210.6
		44.6	44.1	45.8	55.8	54.2	62.4	65.5	63.8
		35.6	36.7	40.6	43.6	39.7	40.5	47.0	44.4
		32.0	32.0	32.5	36.5	60.5	62.7	60.9	59.0
	Normalised Mean ± SE	100	106.1 ± 3.2	109.4 ± 2.7	124.8 ± 2.9	100	103.6 ± 2.9	113.8 ± 3.9	102.1 ± 4.9
	AMOZ	126.0	103.3	102.3	105.5	98.7	97.6	103.2	106.0
		86.6	79.8	86.2	81.5	93.7	80.7	90.0	104.1
		59.8	51.9	54.7	54.2	28.6	27.6	28.2	28.3
		53.3	45.6	51.7	52.3	27.5	24.4	23.6	25.2
		70.7	58.8	70.3	68.3	30.5	28.7	27.8	30.0
	Normalised Mean ± SE	100	85.9 ± 1.6	96.9 ± 3.5	92.6 ± 2.3	100	92.9 ± 2.1	92.9 ± 3.2	101.5 ± 3.1
	AHD	54.5	50.1	47.5	51.7	95.9	91.2	85.1	87.7
		50.7	55.5	53.5	53.8	116.7	101.3	113.7	109.6
		29.3	25.4	27.8	29.5	42.6	35.5	36.5	38.3
		27.9	27.8	28.5	27.8	41.9	41.8	38.2	41.0
		28.0	23.5	26.0	26.5	42.2	41.8	44.1	42.7
	Normalised Mean ± SE	100	94.3 ± 4.1	98.9 ± 3.3	99.2 ± 1.9	100	92.0 ± 3.2	94.7 ± 3.4	94.9 ± 1.9
	SEM	304.0	253.5	299.0	281.3	96.6	86.4	92.1	91.5
		260.6	274.7	251.4	252.9	74.9	72.4	72.8	79.1
		203.6	219.4	221.5	216.7	27.9	32.9	30.4	34.9
		210.5	202.9	188.0	185.4	39.7	41.2	46.2	47.1
		201.1	194.0	201.9	187.8	40.7	40.5	45.1	41.8
	Normalised Mean ± SE	100	97.9 ± 3.8	98.7 ± 3.1	95.5 ± 2.8	100	101.5 ± 4.2	108.3 ± 4.1	109.4 ± 4.9

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y Page 24 of 24

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.ukFood Additives and Contaminants

Acknowledgements

The authors acknowledge the financial support of the European Commission for the project QLK1-CT1999-00142 'FoodBRAND' which funded part of this work. Grateful thanks are expressed to the farm and post-mortem room staff of AFBI Veterinary Sciences Division, Belfast.


C Crews
email: c.crews@csl.gov.uk

S Hasnip

D R Roberts

L Castle

THE ANALYSIS OF FURAN IN HEATED FOODS

Keywords: Process contaminants, Volatiles Food Types: Canned foods, Coffee, Cooked foods, Processed foods furan, heated foods, heat-induced toxicant, analysis, determination

Various experimental factors that might affect the measurement of furan by automated headspace gas chromatography/mass spectrometry have been investigated. It was established that furan was not lost during sample heating through leakage or decomposition and that deuterium labelled furan used as an internal standard was stable with respect to incubation in the presence of food and stable in model food systems at raised temperature. Saturation of the aqueous phase of the sample with ammonium sulphate improved the partitioning of furan from samples into the headspace. There was possibly a very small decrease in the peak areas of both d 0 -furan and d 4 -furan when heated at acid pH. For food samples heated at normal headspace incubation temperature (50 °C), the level of furan measured was highest at the most acidic conditions used (pH 2.4) and did not differ significantly between pH 5 and 10. Under strong heating, production of furan decreased markedly at very high pH. Quantification based on standard additions or on external calibration gave comparable results for foods containing furan at relatively low, moderate and high levels.

INTRODUCTION Background

Furan is a possible human carcinogen found in a number of heated foods, particularly those heat-processed in cans and jars, and in strongly heated foods such as roasted coffees. On account of its high volatility furan is best measured by headspace sampling techniques including direct headspace sampling [Hoenicke et al., 2004] or solid-phase microextraction [START_REF] Goldmann | Rapid determination of furan in heated foodstuffs by isotope dilution solid phase micro-extraction-gas chromatography -mass spectrometry (SPME-GC-MS)[END_REF][START_REF] Bianchi | Development and validation of a solid phase micro-extraction-gas chromatography-mass spectrometry method for the determination of furan in baby-food[END_REF]. In both of these approaches the food sample is weighed into a sealed vial which is heated (incubated) to effect a favourable partition of furan between the sample and the headspace gas.

Quantification is usually achieved by means of the method of standard addition or by using external calibration standards, both using deuterated furan as an internal standard.

The first publication of a method suitable for the quantification of furan at the low levels found in foods was issued by the US FDA [FDA, 2004a], and was used to collect data from a number of foods likely to contain furan [FDA, 2004b]. Since the issue of this method a number of refinements have been made, most notably a reduction in the incubation temperature from 80° to 50ºC or lower, because it has been established that furan can be formed in some foods heated at temperatures above 50ºC [START_REF] Hasnip | Some factors affecting the formation of furan in heated foods[END_REF]Senyuva and Gokmen, 2005].

The studies presented here were part of a wider investigation into the factors affecting the formation of furan in foods and were carried out as part of the EU 'Heatox' project on heat-generated food toxicants [START_REF] Hellenas | The HEATOX project[END_REF]. Our aim was to further characterize and improve the methodology for testing foods and model foods systems for furan.

To this effect, studies were made into several areas of the methodology. These were:

1. The potential for losses of furan through leakage through the headspace vial seals.

2. The potential for loss or decomposition of furan or deuterated furan on heated incubation for headspace sampling.

Materials and methods

Reagents and samples

Furan (99+%), d 4 -furan (>99 atom % D) and ammonium sulphate were purchased from Sigma-Aldrich (Poole, Dorset). Sodium hydroxide, hydrochloric acid, sodium dihydrogen orthophosphate and disodium hydrogen orthophosphate (Analar grades) were purchased from Fisher Scientific UK Ltd. (Loughborough, Leicestershire).

Vegetable samples -fresh green (French) beans and a dried bean-based vegetable soup mixture containing dried peas, lentils, beans, onion, pearl barley, and pasta, were obtained from a local retailer.

Apparatus

Furan was measured using gas chromatography-mass spectrometry (GC-MS) using a ThermoFinnigan Voyager benchtop system fitted with a HS2000 headspace autosampler. The temperature of the headspace incubation oven was 50°C and the syringe needle was held at 100°C. Samples were incubated for 30 minutes with shaking.

The injection volume was 1 ml and the injection speed was 30 ml/minute. Spit injections were made at a ratio of 2:1 on to a HP-Plot Q column of 15 m x 0.32 mm i.d. with a 20 µm phase thickness. Helium carrier gas was used at a constant flow of 1.7 mL/min. The GC oven was held at 50°C for 1 min, then heated at 10°C min -1 to 225°C and held for 12.5 minutes. The mass spectrometer was operated in electron ionization mode. Furan was detected by selected-ion monitoring of the molecular ion at m/z 68 and confirmed by monitoring the fragment ion, [C 3 H 3 ] + at m/z 39. The internal standard was detected by monitoring the molecular ion at m/z 72.

Each analytical batch included a reagent blank and a tomato sauce in-house reference material which had been characterised by repeated analysis as containing furan at 2 µg/kg ± 1.8 µg/kg.

Experimental approaches

Losses of furan through leakage or decomposition

There were concerns that furan might leak from the vial seals during, for example, hightemperature experiments of model systems, or that the deuterium labelling of the internal standard would be lost in similar circumstances, or through reaction with food matrices. Possible loss of furan was tested for by measuring the stability of furan and deuterated furan heated in sealed vials with and without a food matrix. Aqueous solutions (1 ml) containing 2.5 µg of unlabelled furan and deuterium labelled furan were prepared in 20 ml headspace vials which were sealed with PTFE faced silicone septa. One set was heated at 100°C for 1 hour and the other was left at room temperature for the same time. The samples were analysed by GC-MS and the responses measured for the molecular ion of d 4 -furan (m/z 72), and the ions corresponding to successive substitution of deuterium atoms by hydrogen, i.e. m/z 71, 70, 69 and also for undeuterated furan (m/z 68).

Potential reaction of the internal standard with food was studied by heating 0.5 g of a dried bean-based vegetable mixture in water (8 ml) with 2.5 µg d 4 -furan internal standard which was added before or after the heating period. This was intended to reveal any deuterium exchange with hydrogen from water or food components. water with the pH adjusted to acid (pH 4), neutral (pH 7) or basic (pH 11) conditions using 0.2M phosphate buffer adjusted with sodium hydroxide and hydrochloric acid. In these experiments the peak area response of the d 4 -furan was recorded.

Stability with respect to pH

Effect of pH on furan quantification

The effect of pH on furan determination was also tested on two sample matrices, runner beans and a dried bean-based vegetable mixture. The runner beans were purchased fresh and homogenised in a domestic food blender. The dry bean-based vegetable mixture was milled to a homogenous fine powder. Portions of about 5g fresh bean and about 0.5g dry bean-based vegetable mix were mixed with 8 ml of pH buffer solution in 20 ml headspace vials. Each matrix was prepared in duplicate at pH 2.4, 4.7, 7.1, 10.1 and 11.6.

The samples were warmed in the headspace autosampler incubation oven under the normal operating conditions of 50°C for 30 minutes. To amplify any effect of pH further samples were heated at 80°C or 120°C for 60 minutes. After cooling internal standard was added and furan was measured using a single point standard addition.

Addition of ammonium sulphate

Addition of inorganic salts is known to improve the partition of volatile organic compounds from aqueous solution into the headspace and ammonium sulphate is often used for this purpose. Two samples of tomato sauce, which contained about 15 µg/kg furan, were mixed in a headspace vial with 2 ml water and the aqueous phase saturated by the addition of 5 g ammonium sulphate. These samples were compared with two others without ammonium sulphate saturation. The samples were analysed under the standard conditions with incubation at 50°C and the peak area response of the furan was recorded. Sets of calibration standards containing 0.025, 0.050, 0.125, 0.25, 0.5, and 1.0 µg furan per vial were prepared in 2 ml water with and without ammonium sulphate addition. These were analysed as above, recording the peak area of the d 0 and d 4 -furan.

Comparison of quantification methods

Two of the different methods used to quantify furan were compared on the same samples. These were the standard additions procedure of the FDA method [FDA, 2004a] and external calibration used in this laboratory and others [Hoenicke et al., 2004;[START_REF] Hasnip | Some factors affecting the formation of furan in heated foods[END_REF]. The procedures were applied to samples having low (tomato sauce), medium (tomato soup) and high (coffee powder) levels of furan. Each of the samples was analysed on five separate days with furan quantified by standard additions at five levels (5, 10, 25, 50, 100 µg/kg) and by means of an external calibration graph.

RESULTS

Potential for loss of furan through leakage or decomposition

The effects of heating unlabelled and deuterium labelled furan in headspace vials at 100°C for 1 hour are shown in Table 1. The repeatability of the determination was good. The peak area for the unlabelled furan molecular ion (m/z 68) had a range of 598 million to 640 million units in the unheated sample, and 550 to 661 million units in the heated sample. Similar data were obtained for the d 4 -labelled furan. These sets of results could not be differentiated and thus it was clear that there were no physical losses from the headspace vial or decomposition of the furan or internal standard caused by heating.

Table 1 also shows the percentage of the response corresponding to the molecular ion of furan with full deuteration (m/z 72) and with successive substitutions of deuterium by hydrogen. The relative abundance of the molecular ion of the d 4 -furan remained at 95% throughout, demonstrating that there was no isotopic exchange with hydrogen atoms in the matrix.

The range and average response for d 4 -furan in five samples of vegetable matrix when the internal standard was added before or after heating for one hour at 100°C were very similar. When the internal standard was added before heating the range was 71400 to 96800 peak area units, mean 85600 peak area units, and when it was added after heating the range was 77000 to 88800, mean 82900 peak area units. The peak areas were also similar in the unheated sample (82500 to 100800, mean 93200 peak area units). This showed that there was no significant isotopic exchange of deuterium with the food sample, or other losses of d 4 -furan.

Stability with respect to pH

Because deuterium loss by deuteron-proton exchange could be catalysed by acid or base, the influence of pH was investigated. When d 0 -furan and d 4 -furan standards were heated together at 100°C for 1 hour in water at pH 4, 7 and 11 there was a possible small decrease (4%) in the d 0 -furan and d 4 -furan absolute peak areas in the acidic medium (pH 4), but no difference between the samples heated at neutral and alkaline pH. The peak area ratios remained constant across the samples. The slight decrease in the peak areas at low pH, but constant peak area ratio of d 0 /d 4 -furan, is interpreted as being due to a partial protonation of the ether oxygen atom of furan, which would reduce the concentration of free (neutral, uncharged) furan in solution and thus lead to a reduction in the partial pressure of furan in the headspace gas.

Effect of pH on furan quantification

Table 2 shows the furan levels measured with warming at the incubation temperature of 50°C for 30 minutes as part of the normal analytical procedure. For both green beans and the dried vegetable mixture the level of furan measured was highest at the most acid pH (2.4). Furan levels did not differ significantly between pH 4.7 and 10.1 for the dried vegetable mixture, but at pH 11.6 very little furan was detected in the green beans.

When the samples were heated at 80°C, furan levels produced in the beans were little changed between pH 2.4 and 10.1 (18 to 29, average 22 µg/kg), but decreased to less than 2 µg/kg at the high pH of 11.6. Furan levels in the dried bean based vegetable mixture appeared to decrease in a less linear fashion with increasing pH, from 50 to 7 µg/kg. These effects were more evident when the beans were heated at 120°C for 60 minutes. Figure 1 shows a linear decrease in the furan with rising pH measured in duplicate samples of heated green beans. Figure 2 shows that for a bean based vegetable mixture heated under the same conditions there is a sharp decrease in the level of furan formed between pH 2 and 4 from about 500 to 200 µg/kg followed by a steadier decline to about 20 µg/kg at the highest pH. The fact that the response profiles for different pH values are so different, suggests that different precursors and/or different formation mechanisms operate in the two food types when heated. There appears to be three or more important routes of furan formation in heated foods, depending on the food composition and therefore the precursors available in that type of food [Locas and Yaylayan, 2004;Mark et al., 2006;[START_REF] Crews | A review of the occurrence, formation and analysis of furan in heat-processed foods[END_REF]. With this diversity of formation routes in mind, the temperature used for headspace incubation has to be considered carefully. As a pragmatic measure, it can be assumed that in the absence of any specific information to the contrary, that an incubation temperature of 50ºC or lower is acceptable for all food types.

Addition of ammonium sulphate

The results of the determination of furan in tomato sauce made with or without saturation of the aqueous phase with ammonium sulphate are shown in Table 3. The addition of the salt caused a 2.5 fold increase in peak area for both d 0 -furan and d 4 -furan and the level of furan measured was the same within experimental error. Calibration lines for standards prepared with and without ammonium sulphate addition were both linear. Thus, as expected, the addition of ammonium sulphate improved the partitioning of furan from samples into the headspace. Such salting-out might be of some use should an increase in analytical sensitivity be required but for most food testing purposes the sensitivity is sufficient without this enhancement.

Comparison of quantification methods

The results of the experiments comparing the quantification method are summarised in These results are essentially identical and this provides further evidence that both methods of quantification appear to be equally suited. It means that, as anticipated, the deuterated furan behaves well as an internal standard and the relative partitioning of d 0 /d 4 furan is not affected significantly by the nature of the food matrix. The external calibration procedure is especially useful where a large number of food samples are analysed as it reduces the need for duplication of sample preparation.

CONCLUSIONS

Studies into the methodology of furan determination have been applied to identify and improve those methods currently available. This work has shown that there is no major difference in furan levels measured in standards over a range of pH values, or any loss of furan by leakage from automated headspace sampler vials, or any loss of deuterium from the labelled internal standard.

The labelled internal standard was shown to retain its deuterium when heated with both water and a vegetable material, confirming that analytical data were not affected by the normal analytical conditions or experimental heating of food samples or model systems.

Extreme acidic conditions increased the level of furan measured in food samples during normal incubation compared with neutral conditions, whereas very basic conditions reduced the amount furan measured. In food samples heated experimentally, the pH had a pronounced effect on the level of furan measured, indicating that control of the pH is required in quantitative experimental work.

Methods of quantification based on standard additions or external calibration have been reported as applied to surveys of furan in foods. The methods were compared to establish that results obtained by both procedures were equivalent. For foods containing furan at relatively low, moderate and high levels the two approaches were shown to be equally effective. Saturation of the samples with ammonium sulphate enhances the sensitivity of the analytical method but, in practice, is not normally required because direct headspace methods have sufficient sensitivity anyway. Hoenicke, K., Fritz, H., Gatermann, R. and Weidemann, S. (2004). Analysis of furan in different foodstuffs using gas chromatography mass spectrometry. Czech Journal of Food Science, 22, 357-358. Locas, C. P. and Yaylayan, V. A. (2004). Origin and mechanistic pathways of formation of the parent furan -A food toxicant. Journal of Agricultural and Food Chemistry, 52, 6830-6836. Mark, J., Pollien, P., Lindinger, C., Blank, I. and Mark, T. (2006). Quantitation of furan and methylfuran formed in different precursor systems by proton transfer reaction mass spectrometry. Journal of Agricultural and Food Chemistry, 54, 2786-2793. Senyuva, H. Z. and Gokmen, V. (2005). Analysis of furan in foods. Is headspace sampling a fit-for-purpose technique? Food Additives andContaminants, 22, 1198-1202. Figure 1 The effect of pH on furan formation in green beans heated at 120°C for 60 minutes (n=2).

  potential for loss of internal standard through deuterium exchange with water or by reaction with food.4. The effect of pH on the stability of furan and deuterated furan during the headspace incubation.5. The addition of salt (ammonium sulphate) to increase headspace partitioning and thus analytical sensitivity.6. A comparison of the use of external calibration with the standard additions method.

  Stability in different pH regimes was studied by heating d 4 -furan at 100°C for 1 hour in

Table 4 .

 4 The coffee powder sample contained 59 µg/kg furan (range 46-72 µg/kg) when measured by external calibration and 62 µg/kg furan (range 56-67 µg/kg) by standard additions. The tomato soup sample contained 24 µg/kg furan (range 20-29 µg/kg) by external calibration and 28 µg/kg furan (range 22-33 µg/kg) by standard additions. The tomato sauce sample contained 3 µg/kg furan (range 2-3 µg/kg) by external calibration and 2 µg/kg furan (range 2-3 µg/kg) by standard additions.

Figure 2

 2 Figure2The effect of pH on furan formation in a dried bean based mixture heated at 120°C for 60 minutes (n=2).

Table 1

 1 Effect of heating on furan and deuterated furan in solution

				Peak Area (million peak area units) at given m/z		Percent abundance at given m/z
	sample		68	69	70	71	72	Total area	68	69	70	71	72
	d0-furan	min	598	28.0	2.6	0.8	0.7	630	95% 4.4% 0.4% 0.1% 0.1%
	unheated max	640	30.0	2.7	0.8	0.8	675	95% 4.5% 0.4% 0.1% 0.1%
	n=5	average	621	29.1	2.7	0.8	0.8	654	95% 4.4% 0.4% 0.1% 0.1%
	d0-furan	min	550	25.9	2.3	0.7	0.7	580	95% 4.5% 0.4% 0.1% 0.1%
	at 100°C	max	661	31.1	2.8	0.9	0.8	697	95% 4.5% 0.4% 0.1% 0.1%
	n=5	average	622	29.2	2.7	0.8	0.8	656	95% 4.5% 0.4% 0.1% 0.1%
	d 4 -furan	min	1.6	0.2	0.8	5.7	171	179	0.9% 0.1% 0.5% 3.2% 95%
	unheated max	1.7	0.2	0.9	6.2	185	194	0.9% 0.1% 0.5% 3.2% 95%
	n=5	average	1.7	0.2	0.9	6.0	178	187	0.9% 0.1% 0.5% 3.2% 95%
	d 4 -furan	min	1.7	0.2	0.8	5.6	167	175	0.9% 0.1% 0.5% 3.2% 95%
	at 100°C	max	1.9	0.2	0.9	6.0	179	187	1.0% 0.1% 0.5% 3.2% 95%
	n=5	average	1.8	0.2	0.8	5.8	173	182	1.0% 0.1% 0.5% 3.2% 95%

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 4

 4 Furan levels (range and average, n=5) in coffee, tomato soup and tomato sauce, quantified by external calibration and standard additions methods

	Food Additives and Contaminants
	Sample	Furan µg/kg
		external	standard
		calibration	additions
	Ground coffee	46 to 72 (59)	56 to 67 (62)
	Tomato soup	20 to 29 (24)	22 to 33 (28)
	Tomato sauce	2 to 3 (3)	2 to 3 (2)

Page 16 of 18 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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

ACKNOWLEDGEMENTS

We gratefully acknowledge financial support for this work from the UK Food Standards Agency and from the EU 6 th Framework Programme within the HEATOX project. The statements and conclusions in this paper are the responsibility of the authors alone and they should not be taken to represent the opinion of the sponsors.


Maria Maddalena Storelli

Grazia Barone

Gaetano ; Piscitelli

Maddalena

G O Marcotrigiano
email: g.o.marcotrigiano@veterinaria.uniba.it

Mercury in fish: concentration versus fish-size and estimates of mercury intake

Keywords: Metals -uptake, Risk assessment, Total diet, Toxicology Additives/Contaminants: Heavy metals -mercury Food Types: Fish total mercury, fish, risk assessment, PTWI

Mercury in fish: concentration versus fish-size and estimates intake dose.

Introduction

Fish provide a healthy source of dietary protein, and are relatively low in cholesterol and high in omega-3 (n-3) fatty acids (National Research Council 2000). Several studies have shown that fish consumption reduces the risk of coronary heart disease, decreases mild hypertension, prevents certain cardiac arrhythmias, lowers the incidence of diabetes and appears to alleviate the symptoms of rheumatoid arthritis [START_REF] Deckere | Health aspects of fish and n-3 polyunsaturated fatty acids from plant and marine origin[END_REF][START_REF] Billman | Prevention of sudden cardiac death by dietary pure omega-3 polyunsaturated fatty acids in dogs[END_REF], Rosenberg 2002). However, people who eat fish must balance the relative benefits from a low-fat source of protein against potential exposure to contaminants, above all mercury. Seafood consumption is, in fact, the main source of this toxin which accumulates in the human body and causes damage in many of its basic systems, particularly to the nervous system [START_REF] Dey | Developmental methylmercury administration alters cerebellar PSA-NCAM expression and Golgi sialyltransferase activity[END_REF].

Literature data indicate that predator fish occupying high trophic positions and therefore have generally higher amount of mercury because this metal is particularly liable to biomagnify along marine food chains [START_REF] Burger | Exposure assessment for heavy metal ingestion from a sport fish in Puerto Rico: estimating risk for local fisherman[END_REF][START_REF] Storelli | Total mercury in muscle of benthic and pelagic fish from the South Adriatic Sea (Italy)[END_REF][START_REF] Brabo | Mercury contamination of fish and exposures of an indigenous community in Parà State, Brazil[END_REF]. Added to this other studies show differences in mercury concentrations between pelagic and benthic species (Romeo et al. 1999[START_REF] Bustamante | Distribution of trace elements in tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF][START_REF] Henry | Heavy metals in four fish species from the French coast of the Eastern English Channel and Southern Bight of the North Sea[END_REF]. Animals living in close association with sediments in which they bury and from where they mainly feed, are more exposed to eventually sediment-associated contamination than other fish. These remarks can have implications for human health.

Earlier studies have, in fact, shown a high human dietary exposure associated with the consumption of predatory fish and bottom-dwelling fish [START_REF] Storelli | Total and methylmercury residues in tuna-fish from the Mediterranean Sea[END_REF] Of critical concern is also the fish-size because generally mercury levels in fish increase with body size. This issue has been discussed with supporting data in the environmental health sciences literature [START_REF] Dixon | Mercury concentrations in stomach contents and muscle[END_REF][START_REF] Storelli | Total mercury in muscle of benthic and pelagic fish from the South Adriatic Sea (Italy)[END_REF][START_REF] Joiris | Total and methylmercury in sardines Sardinella aurita and Sardina pilchardus from Tunisia[END_REF][START_REF] Storelli | Fish for human consumption: Risk of contamination by mercury[END_REF][START_REF] Storelli | Total mercury levels in muscle tissue of swordfish (Xiphias gladius) and bluefin tuna (Thunnus thynnus) from the Mediterranean Sea (Italy)[END_REF]) is a crucial point concerning consumer exposure. The positive relationship between fish-size and mercury levels suggests that consumers that eat larger fish might have higher exposure to mercury than those that eat smaller fish. In relation to this to address the people who eat smaller size fish might arise an effective risk reduction. In this contest we are particularly interested in examining variations in consumer exposure to mercury as a function of consumption of different size spectra of fish. Such information will be of value to those involved in risk communication. An understanding by consumers of all factors leading to an increase of exposure to mercury is the first step to enable them to make decisions about eating fish. each species, from the total number of specimens were formed pools within which individual fish were collected as a function of their similar size length to investigate the influence of size on mercury bioaccumulation.

Materials and methods

In

From the organisms of each pool muscle tissue was removed and preserved at -25 °C until analysis. The tissues were dissected with plastic materials that were washed with HNO 3 and rinsed with distilled and deionized water, in order to avoid metal contamination. For analyses of total Hg, homogenized samples of the tissue (2 g wet weight) were digested to a transparent solution with 10 mL of the mixture H 2 SO 4 -HNO 3

(1:1) under reflux. The resultant solutions were then diluted to a known volume with deionized water (Official Journal of the European Communities 1990) and the total Hg concentrations were measured by atomic absorption spectrophotometry (Analyst 800 Perkin Elmer) by the cold vapour technique, after reduction by SnCl 2 (FIMS-100, Perkin Elmer). Acid washed glassware, analytical grade reagents and double distilled deionized water were used in the tissue analysis.

In order to check on the purity of the chemical used, a number of chemicals blanks were run; there was no evidence of any contamination in these blanks. Analytical quality control was achieved using TORT-1 Lobster Hepatopancreas (National Research Council of Canada). Replicate analyses (n=3) (Hg total 0.32±0.02 µg g -1 dry weight) were in the range of the certified material (Hg total 0.33±0.06 µg g -1 dry weight). All data were computed on a µg g -1 wet weight basis. Mann-Whitney U test was used to test significance of differences between data sets. The level of significance was set at P <

Results and discussion

Among the different fish species examined the highest mean levels of total mercury were detected in conger eel (0.80 µg g -1 ) and in starry ray (0.75 µg g -1 ). Forkbeard (0.67 µg g -1 ), frostfish (0.59 µg g -1 ) and striped mullet (0.55 µg g -1 ) showed levels slightly lower, while red gurnard (0.33 µg g -1 ) and yellow gurnard (0.22 µg g -1 ) exhibited the lowest concentrations (Table 1). Despite of mercury level variability, statistical comparisons did not reveal significant differences among various fish species, except among Triglidae and the other species (P < 0.04).

It is known that differences in feeding habits generally assign the species to a trophic level and species belonging to higher trophic levels are considered to contain higher mercury concentrations [START_REF] Wiener | Environmental Contaminants in Wildlife: Interpreting Tissues Concentrations[END_REF][START_REF] Watras | Bioaccumulation of mercury in pelagic freshwater food webs[END_REF][START_REF] Snodgrass | Effects of trophic status, and wetland morphology, hydroperiod and water chemistry on mercury concentrations in fish[END_REF]. In our case the findings of statistical analysis led to suppose that all species analysed belonged to similar trophic levels. Published estimates of Mediterranean fish trophic levels confirmed this hypothesis being the species in question included in the same functional trophic group with the highest values corresponding to conger eel [START_REF] Stergiou | Feeding habits and trophic levels of Mediterranean fish[END_REF]. In this picture Triglidae were an exception in that their trophic levels was the lowest. This latter information corroborated further the thesis that mercury levels in fish reflect its trophic level. Significant differences in mercury concentrations were, in fact, detected solely between Triglidae with the lowest trophic levels and the other species assigned to the highest. Of primary importance in explaining mercury body burden of fish is also the habitat where they live. It is well known that benthic fish are species that tend to concentrate mercury to a higher degree than other organisms [START_REF] Campbell | Concentrations of heavy metals associated with urban runoff in fish living in stormwater treatment Ponds[END_REF][START_REF] Storelli | Merluccius merluccius) and striped mullet (Mullus barbatus) from the Mediterranean Sea: estimated weekly intake[END_REF], confirming the significant process of sedimentation and persistence of this metal in sea depths. For this reason the tested species were all benthic feeding fish but however, exhibiting different dietary preferences. It is clear from the above discussion that food habits as well as feeding location of fish, are factors of primary importance influencing mercury body load. However, independently from the complexity of interactions leading to different accumulation profiles among fish, a common point to all species in question was that metal concentration seemed to increase with size/age of fish suggesting that larger, older fish had higher mercury levels than smaller, younger fish.

The linear regression analyses confirmed this trend being mercury concentrations positively and significantly correlated with fish length (striped mullet: R = 0.94, P < 0.001; red gurnard: R = 0.92, P < 0.001; yellow gurnard: R = 0.96, P < 0.001; starry ray: R = 0.88, P < 0.001; conger eel: R = 0.89, P < 0.001; forkbeard: R = 0.93, P < 0.001; frostfish: R = 0.82, P < 0.001).

Generally, the evaluation of the toxicological risk of the metal for humans is carried out through comparison of measured concentrations of a certain element in food with the levels imposed by law or following guidelines proposed by different international organizations. In the case of mercury, European legislation (Official Journal of the European Communities 2001) sets the maximum limit in edible parts of fish-products at 0.5 µg g -1 wet wt, except for some species for which it is raised to 1.0 µg g -1 wet wt.

Generally, these latter species are either high trophic level predators, that with their considerable mercury load demonstrate the importance of biomagnification process through food chain [START_REF] Monteiro | Mercury content of swordfish, Xiphias gladius, in relation to length weight, age, and sex[END_REF]Lopes 1990, Storelli and[START_REF] Storelli | Total mercury levels in muscle tissue of swordfish (Xiphias gladius) and bluefin tuna (Thunnus thynnus) from the Mediterranean Sea (Italy)[END_REF], or benthic organisms that spending a considerable time searching for food on the bottom of the sediment are particularly prone to accumulate higher amounts of mercury (Romeo et al. 1999[START_REF] Kljakovic | Cadmium and lead in selected tissues of two commercially important fish species from the Adriatic Sea[END_REF][START_REF] Storelli | Merluccius merluccius) and striped mullet (Mullus barbatus) from the Mediterranean Sea: estimated weekly intake[END_REF]. For fish analyzed in the present study total mercury concentrations should not exceed 0.5 µg g -1 wet wt, except for conger eel, starry ray and frostfish for which the established value is 1.0 µg g -1 . It is, in fact, not by chance that these latter species had a more consistent mercury load in their flesh respect to others. However, comparison of mercury mean concentrations detected in the present study with the levels imposed by law revealed that almost all fish in question (see conger eel, red and yellow gurnard, frostfish and starry ray) were suitable for human consumption with the metal concentrations under the prescribed legal limits.

Only two species, striped mullet and forkbeard, had mercury mean concentrations (striped mullet: 0.55 µg g -1 ; forkbeard: 0.67 µg g -1 ) slightly exceeding the standard of 0.5 µg g -1 wet wt established by European legislation. With these results, it is likely to conclude that the consumption of these fish by humans should be safe. However, this assertion was not corroborated by the concentrations relative to each fish-size classes.

Within each species, the larger size specimens showed, in fact, concentrations exceeding, to a more or less great extent, the maximum regulatory limits. What was observed might be particularly relevant with respect to potential risk on consumers' health. To test this hypothesis the metal intakes via dietary consumption of these fish were calculated and compared with the Provisional Tolerable Weekly Intake (PTWI) of 5 µg kg -1 body weight, set by the Joint Expert Committee on Food Additives (WHO 2003). The average weekly dietary intakes (0.52-1.90 µg kg -1 bw) calculated by taking into account mean mercury concentrations in each species and a mean weekly consumption of demersal fish of 142 g (FAO 2002), were below the fixed safe level (Table 1).

However, the purpose here was not to evaluate the exposure to mercury due to sea product consumption but rather to examine how varied the exposure according to consumption of different sized fish. In this respect, as shown in table 1, weekly mercury intakes were comprised in a wide range from 0.02 to 3.34 µg kg -1 body weight. As the figure indicated, mercury intake increased with consumption of larger size specimens for all species. However, the consumption of certain species, even of large size, such as yellow gurnard and red gurnard gave levels relatively small in comparison to PTWI value (25.0-27.4% of PTWI), whereas high exposure values, constituting from 49.2% to 66.8% of PTWI, were associated with consumption of largest specimens of the remained species. In particular, the highest mercury intakes resulted from the consumption of specimens of weight above 400 g of forkbeard (55.4-66.8% of PTWI), conger eel (51.0-62.4% of PTWI) and starry ray (50.2-56.8% of PTWI). These data clearly demonstrate that people that eat larger fish have higher exposure to mercury than those that eat smaller fish. This could be of concern for consumers, particularly if they repeatedly ate the largest individuals of some species. It would be, in fact, sufficient a weekly consumption of 250 g of largest specimens of these species to have a mercury intake close or that surpass the reference limit.

Potential public health risks from dietary exposure to mercury continue to be the subject of much research, regulation and debate. State and federal agencies can respond to potential health risks from mercury in fish by issuing consumption advice. For example, recently the US Food and Drug Administration (FDA 2002) has issued a series of consumption advice notes based on mercury that suggested that pregnant women should limit their fish consumption and avoid eating large predatory fish. However, the question of risk from eating fish is complicated by the positive health benefits of consuming fish. Because of this it becomes extremely important to reduce human exposure to mercury from fish consumption. The data here obtained clearly show that mercury levels vary in fish as a function of their trophic level and size. Consequently this means that the consumers could substantially reduce their exposure to mercury by eating selected species and fish of smaller size. However, such information is not helpful if the general public is unaware of this possibility. Understanding by consumers about the relationship between contaminant, fish-size, trophic level and mercury intake is crucial to enable them to be able to make better decisions about eating fish. On this basis educational programs to foster such an understanding and, thus, changes in the fish species and size consumed would constitute an useful tool to reduce human exposure to this neurotoxin.

  June-August 2005 during several trawl surveys, 100 conger eel (Conger conger, length range: 32.0-85.0 cm; median: 59.3 cm), 253 red gurnard (Aspitrigla cuculus, length range: 10.2-24.7 cm; median: 18.5 cm), 263 yellow gurnard (Trigla lucerna, length range: 20.4-55.5 cm; median: 35.0 cm), 464 striped mullet (Mullus barbatus, length range: 15.8-31.0 cm; median: 21.0 cm), 224 forkbeard (Phycis blennoides, length range: 12.4-49.2 cm; median: 26.5 cm), 879 frostfish (Lepidopus caudatus, length range: 110.0-144.0 cm; median: 128.0 cm) and 100 starry ray (Raja asterias, length range: 23.0-58.8 cm; median: 48.5 cm) specimens were caught in the Adriatic Sea. For

Table 1 .

 1 Total mercury concentrations (µg g -1 wet weight) and estimated weekly intake (EWI) of total mercury (µg kg -1 body weight) in dependence on consumption of differed sized fish.Figure1. Estimated weekly mercury intake (µg kg -1 body wt) versus fish length (cm).

	Pools	Striped mullet [Hg] EWI	Red gurnard [Hg] EWI	Yellow gurnard [Hg] EWI	Starry ray [Hg] EWI	Conger eel [Hg] EWI	Forkbeard [Hg] EWI	Frostfish [Hg] EWI
	1	0.16	0.38	0.07	0.17	0.01	0.02	0.15 0.35	0.26	0.61	0.30	0.71	0.16	0.38
	2	0.18	0.43	0.12	0.28	0.09	0.21	0.65 1.54	0.29	0.69	0.30	0.71	0.47	1.11
	3	0.39	0.92	0.40	0.95	0.08	0.19	0.30 0.71	0.55	1.30	0.42	0.99	0.39	0.92
	4	0.58	1.37	0.33	0.78	0.10	0.24	0.81 1.92	0.42	0.99	0.49	1.16	0.32	0.76
	5	0.63	1.49	0.42	0.99	0.08	0.19	0.60 1.42	0.43	1.02	0.60	1.42	0.48	1.14
	6	1.04	2.46	0.39	0.92	0.25	0.59	0.78 2.55	1.08	2.55	0.48	1.14	0.27	0.64
	7	0.88	2.08	0.58	1.37	0.15	0.35	1.08 2.51	1.32	3.12	0.64	1.51	0.68	1.61
	8					0.37	0.88	1.20 2.84	1.30	3.07	0.73	1.73	1.15	2.72
	9					0.35	0.83	1.19 2.81	1.08	2.55	1.17	2.77	0.80	1.89
	10					0.41	0.97		1.29	3.05	0.87	2.06	0.99	2.34
	11					0.53	1.25				1.41	3.34	0.82	1.94
	Min	0.16	0.38	0.07	0.17	0.01	0.02	0.15 0.35	0.26	0.61	0.30	0.71	0.16	0.38
	Max	1.04	2.46	0.58	1.37	0.53	1.25	1.20 2.84	1.32	3.12	1.41	3.34	1.15	2.72
	Mean	0.55	1.30	0.33	0.78	0.22	0.52	0.75 1.85	0.80	1.90	0.67	1.59	0.59	1.40
	Median	0.58	1.37	0.39	0.92	0.15	0.35	0.78 1.92	0.82	1.93	0.60	1.42	0.48	1.14
	St. Dev.	0.33	0.79	0.18	0.42	0.17	0.40	0.37 0.91	0.45	1.06	0.35	0.84	0.32	0.75