Kirsten H Lund

Jens Højslev Petersen

Keywords: Monomer, Migration, Simulants Food Types: Acidic Food

Migration of formaldehyde and melamine monomers from kitchen-and tableware made of melamine plastic

and kitchenware such as bowls and ladles. In its native form the polymer is a polycondensation product of the monomers formaldehyde and melamine in a proportion of 2:1 on a molar basis, but amino plastics with a slightly different composition also tend to be sold under the common name "melamine".

Residual monomers can be left in the finished plastic item after manufacturing and their migration to the foodstuffs must be in compliance with the current legislation. Formaldehyde and melamine are both compounds on the positive list for monomers and other starting substances of the EU Plastics Directive having specific migration limits (SML's) of 15 and 30 mg/kg of food or food simulant, respectively. When the volume of food contained in the article is below 500 mL (e.g. a cup) or when the surface to volume ratio in actual use cannot be estimated (e.g. a ladle) the limits are expressed in mg/dm 2 . The corresponding limit for formaldehyde is 2.5 mg/dm 2 and for melamine 5 mg/dm 2 (EC, 2002).

Despite the widespread use of melamine articles only few researchers have published studies addressing results from migration testing of the monomers. However, a Japanese group observed a general tendency towards higher migration of the monomers when the food or food simulant is acidic and when the test temperature is high [START_REF] Ishiwata | Migration of melamine and formaldehyde from tableware made of melamine resin[END_REF][START_REF] Sugita | Release of formaldehyde and melamine from tableware made of melamin-formaldehyde resin[END_REF]. Moreover, some tarnishing of the surface was reported to take place after repeated testing with acidic food simulant [START_REF] Ishiwata | Migration of melamine and formaldehyde from tableware made of melamine resin[END_REF], a physical phenomenon often seen on melamine articles that have been used for some years. The deterioration of the surface has inspired monitoring the development of migration of the monomers from cups used in a cafeteria over several years [START_REF] Ishiwata | Effect of long-term use of melamine table ware on the release of monomers and some other changes[END_REF], and by up to 20 repeated tests of virgin tableware from the Philippines [START_REF] Martin | Release of formaldehyde and melamine from melamine tableware manufactured in Philippines[END_REF].

The test conditions with respect to time, temperature and food simulant were in all cases different from what is currently prescribed in the EU legislation.

In none of the above mentioned papers violations of the EU SML's were seen although the test conditions chosen in general were quite severe. Recently, a UK-study of 50 different melamine samples reported formaldehyde migration significantly above the SML in 5 of the samples (10%).

In that study, migration of formaldehyde was seen from all samples and migration of melamine monomer was seen from 43 samples (always below 36% of SML in the third exposure) [START_REF] Fsa | Chemicals used in plastic materials and articles in contact with food: compliance with statutory limits on composition and migration -year 2[END_REF][START_REF] Bradley | Survey of the migration of melamine and formaldehyde from melamine food contact articles available on the UK market[END_REF].

A common tendency in the published work concerned with migration testing of melamine articles seems to be a gradual shift in the molar proportion of formaldehyde (CH 2 0) to melamine (MEL) during continuous use towards lower CH 2 0/MEL-values. This indicates a relatively high amount of unreacted formaldehyde (or its precursors, e.g. hexamethylenetetramine) being present in the newly manufactured item. It seems also clear that the migration of the monomers continues during the lifetime of melamine articles, but ending up with a "stable" migration with a stable CH 2 0/MELratio originating from a gradual breakdown of the polymer. A similar report of migration of bisphenol A, the breakdown product of polycarbonate, from reusable polycarbonate articles during long-term use was published recently [START_REF] Brede | Increased migration levels of bisphenol A from polycarbonate baby bottles after dishwashing, boiling and brushing[END_REF]. The present study included testing of a selection of virgin melamine articles bought on the Danish market. Moreover, to acquire more knowledge of the effect of long-term use, migration tests were performed on cups, which had been used for several years in a day nursery. Furthermore, the migration pattern for medium term use was illustrated by 10 repeated tests applied to two types of virgin melamine tableware.

Materials and Methods

Sampling

Ten different brands of new melamine plastic kitchenware and tableware were bought in retail shops. When possible at least three test specimens from each brand was bought. No restrictions on use was prescribed by the manufacturers with the exception of that a few of the items, were marked as not recommended for use in the microwave oven. Additionally, a series of used cups (9 red and 2 white test specimens, all of the same brand) from a day nursery was analysed. An overview of all the analysed samples and number of test specimens (replicates) is presented in Table 1.

[Insert table 1 about here]

For each item, transmission infrared spectra were recorded (1725X FT-IR, Perkin Elmer, Beaconsfield, UK) of a tablet pressed from 1-4 mg pulverized sample and KBr. In this way, it was confirmed that all items in Table 1 were made of melamine plastic. Spectra of samples known to be of melamine plastic and the digitalised Hummel polymer library of IR-spectra were used for the identification. In addition to the samples mentioned in simulant up to 0.5 cm from the rim i.e. single sided testing [CEN, 2004]. The ladle (sample identity (i.d.) G) was placed in a glass and covered with simulant up to the beginning of the shaft i.e.

immersion testing. After each exposure period the simulant was removed and combined with additional two times 10 ml (used for washing the article and an eventual migration cell) before analysis of the formaldehyde and melamine concentration.

For each sample the actual surface area in contact with food simulant was measured and when appropriate the concentration of monomer in the volume of food simulant (mg/kg) was converted into migration per exposed surface unit (mg/dm²). Surface to volume ratios from 4,8 dm²/liter to 8,4 dm² /liter were obtained.

Spectrophotometric determination of formaldehyde

Two spectrophotometric CEN methods [CEN part23, 2005] were used for the determination of formaldehyde; called the main method and the verification method. Principle of the main method:

Formaldehyde in the food simulant reacts with chromotropic acid in the presence of sulphuric acid to give a violet reaction product, for which the absorbance is measured at a wavelength of 574 nm with spectrophotometer (Perkin Elmer UV/VIS Lambda 10, Beaconsfield, UK). Each sample and each standard were measured twice and the mean absorbance was used for quantification.

Principle of the verification method: Formaldehyde in the food simulant reacts with pentane-2,4dione to form 3,5-diacetyl-1,4-dihydrolutidine, which is measured at a wavelength of 410 nm with spectrophotometer.

For more details about the two methods consult the CEN standard [CEN part23, 2005]. Acetonitrile and 25% 5mM phosphate buffer, flow 1 mL/min. The absorption at 230 nm was used for quantification. Further all DAD-spectra were qualitatively checked against the spectrum of the melamine standard (max. absorption 205 nm). Retention time of melamine was 3.9 min.

Quality assurance

In each analytical series, 1-3 reagent blanks in glass bottles were included. The reagent blank from each series was used as zero reference for the spectrophotometer before measuring the formaldehyde content of the samples. Melamine was not observed in any reagent blank.

Quantification was carried out using linear calibration curves (R 2 > 0.996) ranging from 5 to 60 mg/L (melamine) and 1.5 to 30 mg/L (formaldehyde). It should be noted that for the main formaldehyde method deviations from linearity were seen several times. In those cases the highest point 30 mg/L was omitted from the calibration curve, the highest point then being 20 mg/L.

The limit of quantification (LOQ) in each series was determined from two independent calibration curves as the lowest concentration where the two curves differed less than 5% as required in the CEN standard. The LOQ for formaldehyde using the main method differed between the different series and was between 0.3 and 2.5 mg/kg. Expressed on the basis of exposed food contact area this corresponds to LOQs between 0.14 and 0.36 mg/dm 2 . The LOQ for melamine was 1.8 mg/kg corresponding to LOQs between 0.24 and 0.38 mg/dm 2 .

The inter-day repeatability of the formaldehyde determination with the main method was better than 2% as determined from the relative standard deviation (RSD) of 17 repeated measurements of a standard with a concentration of 13.1 mg/kg (close to the SML-value). The inter-day repeatability of the melamine determination was found in the same way to be 4% at a level of 5.6 mg/kg (n=8)

and 2% at a level of 22.5 mg/kg (n=11).

To demonstrate the trueness of the formaldehyde determination 6 test specimens were analysed using the verification method as well. A satisfactory agreement was seen between the results obtained with the two methods. The trueness of the melamine determination was demonstrated by participation in a FAPAS proficiency test [FAPAS, 2000]. The average concentration of melamine found in a melamine solution in 3% acetic acid by the 11 participating laboratories was 22.9 mg/kg.

We reported a concentration of 23.2 mg/kg, which resulted in a z-score of 0.1.

The FAPAS proficiency round included exposure of a melamine plastic plate (sample A) to 3% acetic acid for 2 hours at 70 o C followed by measurements of the two monomers in the food simulant. Unfortunately, due to inhomogeneity of the test material, it was not possible for FAPAS to calculate the participants z-scores for formaldehyde as well as for melamine. For both monomers, our results were in the same order of magnitude as the results from the main part of the other laboratories. The median value for formaldehyde of the 14 participating laboratories was 3.9 mg/kg (minimum and maximum-values: 0.1 and 314.7 mg/kg); our laboratory found 6.2 mg/kg (average test result of three plates, standard deviation 0.6 mg/kg). The median of melamine of the 11 participating laboratories was 2.2 mg/kg (minimum and maximum-values: <0.5 and 14.6 mg/kg); we found 2.6 mg/kg (average test result of three plates, standard deviation 0.5 mg/kg).

Overall, the two CEN methods performed well in our laboratory.

Results and discussion

Migration testing of all virgin materials was conducted under test conditions selected from the current EU legislation (EEC 1982, EEC 1985) to be the worst foreseeable use in normal Danish household or day nursery. The foreseeable uses of the melamine articles include contact to acidic foods like juice and stewed apples and the official food simulant 3% acetic acid was chosen.

Distilled water is the official food simulant for other aqueous foods like milk, tea and coffee but other studies have shown only a limited migration from melamine plastics to this simulant [START_REF] Ishiwata | Migration of melamine and formaldehyde from tableware made of melamine resin[END_REF][START_REF] Sugita | Release of formaldehyde and melamine from tableware made of melamin-formaldehyde resin[END_REF]. When the manufacturer does not deliver any restrictions in service temperature in a user instruction, the current EU-legislation, for enforcement purpose, prescribe testing in three successive four hours test with a new portion of simulant at 100°C with the aqueous food simulants. However, in this case it was decided for each item to simulate a situation considered closer to every-day use. In the majority of cases an exposure period of 2 hour at 70°C was selected: These (so called "hot-fill") test conditions are prescribed in the current legislation when an article is filled with a hot aqueous liquid and the temperature falls below 70°C within 15 min after filling.

An overview of the analysed samples is given in Table 1 as well as the number of repeated exposures and the test conditions applied. In general, no further testing was performed of samples where none of the three test specimens showed any measurable migration in the first test. Three out Table 2 contains the results from the six samples exposed to three successive tests at 70ºC in 2 hours. Only in one test specimen (E-1), the migration of 1.5 mg formaldehyde/dm² to the food simulant in the third test was close to the SML of 2.5 mg/dm 2 . An interesting observation is that the difference in migration behaviour between individual items of the same brand is quite significant:

the difference between test specimens with the highest and lowest migration of formaldehyde is about a factor of 8 (sample E-1 and E-3). This phenomenon, possibly connected to small differences in the manufacturing processes, was observed in other studies as well [START_REF] Fsa | Chemicals used in plastic materials and articles in contact with food: compliance with statutory limits on composition and migration -year 2[END_REF]FAPAS, 2000). The migration of melamine was limited and only in three test portions a migration above the LOQ was measured.

[Insert Table 2 about here]

All three test specimens of two of the samples in Table 2 (B, a plate and C, a bowl) were exposed in seven additional exposure periods with new portions of food simulant, which were all analysed for both monomers. For one of the samples (C) no migration above the LOQ was seen in these additional tests. For another sample (B), some migration of formaldehyde took place into about half of the test portions but the migration of melamine was, with a few exceptions, below the LOQ. The formaldehyde data are presented in In a previous study, a significant increase in the migration of monomers to the food simulant was found when the test temperature was increased from 60 through 80 to 95ºC [START_REF] Sugita | Release of formaldehyde and melamine from tableware made of melamin-formaldehyde resin[END_REF].

The test conditions giving rise to the highest migration in that study were more severe than the "hot fill"-conditions applied for most of the samples in this study. Therefore, it was decided to use the test conditions: 30 min at 95ºC for a virgin sample showing a measurable migration.

In Figure 1, the migration of formaldehyde during 10 successive exposures of sample M is illustrated. Migration measurements were applied for all six test specimens analysing simulant portions from exposures number 1, 2, 3, 5 and 10.

[Insert Figure 1 about here]

Although the migration results obtained from the individual test specimens of sample M are quite different, they follows the same pattern as sample B in table 3. Values for the migration of formaldehyde and melamine at exposures 1, 2, 3, 5 and 10 are seen in Table 5.

A general interpretation of the migration pattern could be the following: The main contribution to the migration in the first and second exposure periods is from residual monomers (migration speed could be dependent on the presence of a surface treatment or not). In the following tests, the monomer migration probably primarily comes from the degradation of the polymer. Others have continued testing for up to 20 exposure periods to investigate the degradation pattern [START_REF] Martin | Release of formaldehyde and melamine from melamine tableware manufactured in Philippines[END_REF]. However, in this study we decided to analyse samples, which had been used for several years in a day nursery. Such cups have been exposed to acidic foods like juice and also to high temperature and an acidic environment when frequently cleaned in the dishwasher. The cups were exposed to the acidic food simulant for only one exposure period at "hot-fill" conditions or one period at 2 days at 20ºC. The results of the testing are presented in Table 4.

[Insert Table 4 about here]

It was obvious from the rather rugged surface that the cups from the day nursery were well used. At the lower test temperature migration of the monomers was always below the LOQ. At hot-fill conditions, two of the six test specimens showed a measurable migration of formaldehyde and three of the test specimens showed a measurable migration of melamine. The levels of formaldehyde migration from these old cups are comparable to the level seen for other melamine articles after only 10 exposures (see Table 3).

As mentioned in the introduction, a certain shift in the molar proportion between formaldehyde and melamine could be expected when the samples are tested (or used) several times. Others have shown that the molar proportion of formaldehyde to melamine (the CH 2 0/MEL-ratio) in repeated testing of a plate exposed to 4% acetic acid at 95ºC in 30 min decreases from about 15 to about 1.8 after 12 exposures [START_REF] Sugita | Release of formaldehyde and melamine from tableware made of melamin-formaldehyde resin[END_REF]. In a recent UK survey, 50 samples were exposed for 3 successive test periods to 3% acetic acid at 2 hours at 70ºC. Calculation of the CH 2 0/MEL-ratio in the third test for 43 of the samples from this study gave a ratio of 14 (range 7-32) (Bradley et. al.2005). In Table 5, the change in CH 2 0/MEL-ratio obtained with plate M in the present study is presented.

[Insert The CH 2 0/MEL-ratio of 12 obtained in the 2nd exposure are reasonably in line with the values found by others and a decrease in the ratio can be seen towards the 10th exposure. The lower CH 2 0/MEL -value obtained in the first exposure period could be due to a surface treatment (see discussion above).

The CH 2 0/MEL-ratio found in the well-used cups from a day nursery was close to 3 for the only test specimen where a calculation was possible. This is in line with or slightly higher than other reported values from long term use studies [START_REF] Ishiwata | Migration of melamine and formaldehyde from tableware made of melamine resin[END_REF][START_REF] Sugita | Release of formaldehyde and melamine from tableware made of melamin-formaldehyde resin[END_REF][START_REF] Martin | Release of formaldehyde and melamine from melamine tableware manufactured in Philippines[END_REF] Until recently the EU Scientific Committee for Food (SCF) performed evaluations of the health aspect of chemicals migrating from food contact materials, a function which is now transferred to the European Food Safety Agency (EFSA). SCF did evaluate melamine in 1987 and the tolerable daily intake (TDI) for melamine was established at 0.5 mg/kg bodyweight/day [START_REF] Scf | Report of the Scientific Committee for Food concerning certain monomers and other starting substances to be used in the manufacture of plastic materials and articles intended to come into contact with foodstuffs, 17th Series[END_REF]. This value leads to the current SML-value of 30 mg/kg food using the conventional procedure of the EU-Commission taking in consideration a "standard EU-citizen" with a bodyweight of 60 kg consuming one kg of food per day. However, small infants and young children are a major group of users of melamine tableware and consume several times more food per kg bodyweight than a 60 kg person [START_REF] Petersen | Migration of 2-butoxyethyl acetate from polycarbonate infant feeding bottles[END_REF]. Anyway, due to the rather low migration of melamine found in this survey there cannot in all probability be an intake above the tolerable daily intake from the melamine plastics alone.

For formaldehyde, neither SCF nor EFSA did yet agree upon a TDI but the EU-Commission has established a SML of 15 mg/kg food. The SML could be compared to a reported naturally occurring formaldehyde concentration of 17 mg/kg in apples and of 60 mg/kg in pears (WHO, 1989). Again the migration values from melamine plastics like those found in this study could not alone result in children intake of excessive amounts of formaldehyde.

Conclusions

In general, at low test temperatures, the migration of formaldehyde and melamine was negligible while being measurable at higher test temperatures. Only in one sample a migration of 1.5 mg/dm² of the monomer formaldehyde came close to the specific EU migration limit of 2.5 mg/dm² when exposed to the food simulant 3% acetic acid.

The results of this study confirm what has been demonstrated in the few other studies published; a continuing migration of monomers especially to hot acidic foods takes place from melamine plastics during the whole lifetime of the product. This phenomenon must be attributed to a gradual breakdown of the polymer, which potentially could have adverse effects on the foreseeable users of such articles, namely children. Fortunately, the migration was low compared to the current restrictions for the articles included in this limited study.

It could be worth paying more attention to consumer exposure related to breakdown products from used table and kitchenware, an aspect normally remaining unnoticed in exposure assessments.

F

Figure 1 :

 1 Figure 1: Migration of formaldehyde from 6 individual test specimens of sample M (a plate) after

  method[CEN part27, 2005] was used for the determination of melamine, see this method for details. A Hewlett-Packard 1100 series HPLC equipped with an autosampler, diode array detector (DAD) and HP ChemStation (ver A.06.01) was used. Column: Lichrosorb NH2 (Merck) 4.6 x 200 mm, particle size 5 µm. Injection volume 20 µL, mobil phase isocratic 75%

  virgin samples (H, I and J) did not show any migration above the LOQ of either formaldehyde or melamine in the first test (LOQ in mg/dm²: 0.4 for formaldehyde and 0.3 for melamine).

Table 1 :

 1 Overview of analysed samples, the number of test specimens for each sample, number of repeated exposures and test conditions.

	Sample i.d..	No. of test specimens	Description of the items tested	No. of repeated exposures	Test conditions
		for each sample
					( 0 C)	(hours)
	A	3	Plate a	3	70	2
	B	3	Plate	10	70	2
	C	3	Bowl	10	70	2
	D	3	Plate	3 b	70	2
	E	3	Mug	3	70	2
	F	1	Jug	3	70	2
	G	3	Ladle	3	70	2
	H	1	Cup	1	70	2
	I	1	Cup	1	70	2
	J	1	Cup	1	70	2
	K	1	Used cup	1	70	2
	L	5	Used cup	1	70	2
	K	1	Used cup	1	20	48
	L	4	Used cup	1	20	48
	M	6	Plate	10	95	0.5

a Delivered by Food Analysis Performance Assessment Scheme

(FAPAS, 2000)

.

b One of the test specimens was only analysed once.

Table 2 :

 2 Migration of formaldehyde after repeated exposures to 3% acetic acid for 2 hours at 70 o C.Melamine migration was below the LOQ except for three exposures (marked test specimens).

	Sample id.	Migration of formaldehyde (mg/dm 2 ) a
		1. test	2. test	3.test
	B-1	0.98 b	< 0.21	0.50
	B-2	0.79	< 0.21	< 0.26
	B-3	0.68	< 0.21	0.35
	C-1	0.41	< 0.26	< 0.31
	C-2	0.22	< 0.26	< 0.31
	C-3	0.31	< 0.26	< 0.31
	D-1	0.39	< 0.20	< 0.25
	D-2	0.91 c	0.23	0.50
	D-3	0.70	n.a.	n.a.
	E-1	1.6	1.0	1.5 d
	E-2	0.41	0.32	0.35
	E-3	0.19	< 0.20	0.32
	F	1.2 mg/kg	1.5 mg/kg	1.9 mg/kg
	G-1	0.34	< 0.29	< 0.36
	G-2	0.23	< 0.29	< 0.36
	G-3	0.29	< 0.29	< 0.36
	H	< 0.36	n.a.	n.a.
	I	< 0.34	n.a.	n.a.
	J	< 0.36	n.a.	n.a.
	n.a.: not analysed
	a LOQ ranged from 0.14 to 0.36 mg/dm 2
	b melamine migration 0.41 mg/dm 2
	c melamine migration 0.45 mg/dm 2
	d melamine migration 0.28 mg/dm 2

Table 3 :

 3 Migration of formaldehyde in ten successive tests of three test specimens of sample number B into 3% acetic acid for 2 hours at 70 o C.

	Test number

Table 4 :

 4 Migration to 3% acetic acid, at two different sets of exposure conditions, of formaldehyde and melamine from melamine-cups K and L used for several years in a day nursery.

	Number of	Test conditions	Migration of formaldehyde	Migration of melamine
	test	(hours)	( o C)	(mg/dm 2 )	(mg/dm 2 )
	specimens
	6	2	70	0.52, 0.39 and 4 samples < 0.35	0.49, 0.39, 0.35 and 3 samples < 0.25
	5	48	20	All samples < 0.35	All samples < 0.25

Table 5 :

 5 Migration of formaldehyde and melamine and the molar ratio (CH 2 0/MEL ratio) for six test specimens of sample M (a plate) after migration into 3% acetic acid for 30 minutes at 95 o C. The

	number of test specimens (n) where a migration was measured above the LOQ's is given in each
	case. The number of test specimens (r) with a sufficient migration of both monomers to calculate a
	CH 2 0/MEL ratio is mentioned in each case. Migration measurements were performed after
	exposure no. 1, 2, 3, 5 and 10.

Table 1

 1 made of melamine plastic or not. From the IR-spectra, it was possible to distinguish between samples made of melamine plastics and samples made of other plastics.Exposure of sample to food simulantSamples were exposed to the food simulant 3% acetic acid as outlined in Table1; at 70 o C for 2 hours, at 95 o C for 30 minutes or at 20 o C for 2 days. Each article was filled with pre-warmed food

	Chemicals
	Glass distilled water was used for the food simulants. The food simulant 3% acetic acid in water
	(w/v) was made from acetic acid (JT Baker, 100%). The density of the simulants was F conventionally set to 1.0 g cm -3 . Formaldehyde solution (approx. 38%, exact concentration o r determined by titration [CEN part23, 2005]), pentan-2,4-dione (99%) and melamine (2,4,6-
	P
	e
	e r
	R
	e
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	w
	O n l
	y

, other samples were purchased. From the appearance and manufacturers information on the packaging it was not always clear if they were triamino-1,3,5-triazine, 99%) all from Acros. Chromotropic acid (min. 98.5%), starch solution (pro analysi) and iodine solution (0.05 mol/L titrisol) all from Merck. Acetonitrile (HPLC grade S) from Rathburn. Potassium bromide (spectroscopic grade powder) from Graceby Specac Ltd. Sodium hydroxide (99.0%), sulphoric acid (95-97%), sodium dihydrogen phosphate, H 2 O (99%), ammonium acetate (98%), sodium thiosulphate, 5H 2 O (99.5%) and hydrochloric acid (min 37%, pro analysi) were all from commercial suppliers. All chemicals were used as received.

Table 3 .

 3 The highest formaldehyde migration was seen in the first test. After a general trend towards lower migration during the following tests, apparently the migration becomes stable.

	[Insert Table 3 about here]
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 5

 5

	about here]

  come into contact with foodstuffs". Official Journal of the European Communities L 297: p 26 (23.10.1982). Amendments 93/8/EEC and 97/48/EC. EC 2002. Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intended to come into contact with foodstuffs, Official Journal of the European Communities. L 220, 2002. p 18. EEC 1985. Directive 85/572/EEC "Council directive of 19 December 1995 laying down the list ofsimulants to be used for testing migration of constituents of plastic materials and articles to come into contact with foodstuffs" Off. J.Eur. Comm. L. 372: p 14 (31.12. 1985)

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

Acknowledgement

Lisbeth Krüger Jensen, Jette Boyer, Annie Foverskov, Lisbeth Juul Hansen and Tamara Malinovskaia are thanked for their skilful analytical work.


Anne-Claire Martel
email: ac.martel@afssa.fr

Keywords: HPLC, Screening -ELISA Additives/Contaminants: Veterinary drug residues -tetracycline Honey, antibiotics, tetracycline, residues, stability

Tetracyclines are used to control bacterial diseases like European and American foulbrood which may cause severe losses in honey bee population and honey production. This study, using 24 hives randomly distributed into four groups of 6 hives, was performed to measure the occurrence of tetracycline (TC) residues in honey following two types of TC application. Two groups of colonies were treated 3 times with 0.5 g of TC in 1 litre of syrup (group S) or in 10 g of powdered sugar (group P). Six hives of a first control group (group C) fed with untreated syrup were installed at 20 and 45 metres from groups S and P respectively. A second control group (group DC) was set up 3 km away. Honey was sampled at different times from all hives and honey artificially contaminated with TC was stored in the laboratory at 4, 20 and 35°C, and all samples were analysed by ELISA and HPLC methods. One day after the last application, the mean TC concentration in brood chamber honey was ten times higher in group S (40.7 mg.kg -1 ) than in group P (4.34 mg.kg -1 ). After 8 days, TC residues were detected in all hives of group C. After 146 days, the mean TC concentration in harvested honey was 1.54, 0.35, and 0.15 mg.kg -1 for groups S, P and C respectively. The control group C had been contaminated with TC by drifting. In all hives of the group DC, no residues were detected at any time during the study. The honey collected at day 504 did not contained any detectable TC residues, except in one super from group C (0.026 mg.kg -1 ).

The half-life of TC in honey from supers was similar in groups C, S and P and equal to 65 days. This duration was twice lower than in honey stored in laboratory in similar conditions : at 35°C in the dark (t 1/2 = 121 days). In honey stored at 20°C, TC was quite stable and its half-life was 242 days. The data from these experiments indicate levels of TC residues in honey after a treatment in hives, their persistence and diffusion into the apiary. These results show that the TC must be used with precaution in honey production.

adding supers in all groups in brood chambers ND 0.03 (a) ND 0.01 (c)

D+504

in honey harvested in supers ND ND (b) ND ND T : day of the first application of TC D : day of the last application of TC ND : no residue detected (a) : no residues were detected except in one super with a value of 0.17 mg.kg -1 (b) : no residues were detected except in one super with a value of 0.026 mg.kg -1 (c) : no residues were detected except in one super with a value of 0.043 mg.kg

Introduction

Bacterial diseases of the honey bee Apis mellifera may severely decrease the honey bee population and honey production, causing significant damage to the beekeeping industry. Two of most important bacterial diseases that affect the larvae of the honey bee Apis mellifera are American foulbrood and European foulbrood caused by Paenibacillus larvae and Melissococcus pluton, respectively. These pathogens have been treated in bee colonies using antibiotics for many years.

Due to their low cost and large availability, tetracyclines are most commonly used in several countries even as a preventive treatment [START_REF] Evans | Diverse origins of tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae[END_REF].

This treatment risks tetracycline residues in honey as reported by many authors. Among them, [START_REF] Faucon | Sondage sur la qualité des miels de lavande-lavandin[END_REF] found tetracyclines residues in 34 out of 75 French lavender honeys, and 14 of them had residues above 0.020 mg.kg -1 . In 2002, residues of tetracyclines (ranging from 0.019 to 2.152 mg.kg -1 ) were detected in 19 of the 113 French honey samples collected according to the 96/23/EC Directive (National surveillance plan, note DGAL 2002). Only two producers had previously indicated they used tetracyclines [START_REF] Martel | Bilan du plan de contrôle 2002 de la contamination des miels[END_REF].

Usleber et al. (1996) showed that seven days after the last treatment of bee colonies with tetracycline (TC) or oxytetracycline (OTC) in sucrose (10 mg.g -1 ), honey contained more than 0.4 mg of TC/kg and 1.5 to 3.5 mg of OTC/kg. [START_REF] Martel | Bilan du plan de contrôle 2002 de la contamination des miels[END_REF] showed that only two days after OTC administration, this antibiotic was already distributed in the whole hive (OTC concentration ranged between 15 and 91 mg.kg -1 depending on the proximity to the feeder) and was equalized within one week. Both authors observed that the degradation of OTC in hive was not complete after 6 weeks. However, currently, no maximum residue limits (MRL) have been defined for tetracyclines and their metabolites in honey contrary to other matrices such as milk, meat, liver and kidney whose MRL range between 0.1 and 0.6 mg.kg -1 . Switzerland and Belgium have established a tolerance limit of 0.02 mg of tetracyclines and their metabolites/kg of honey. Above this limit, honey must be removed from the market. Consequently, the present study was designed to contribute to a better knowledge of the consequences of antibiotic treatments on their residue levels in honey. Two of the current protocols used by beekeepers were tested : antibiotics in syrup or in powdered sugar given to colonies 4 weeks before the honey flow. The decline of TC in hive has been followed in brood chamber and super honey for 504 days following antibiotic treatment and compared to that in honey samples stored at different temperatures.

Experimental

Materials and reagents

The homogenisation equipment for TC extraction from honey samples consisted of a Vortex-mixer with variable speed from Fisher Bioblock Scientific (Illkirch, France) and an agitation system for centrifuge tubes from New Brunswick Scientific Co. (N.J., USA). The centrifuge GR 4.11 model from Jouan (Saint-Herblain, France) was used to eliminate pollen and other impurities in honey samples.

The TC analysis was performed by HPLC on an Agilent 1100 from Agilent (Karlsruhe, Germany) consisting of a quaternary pump, an autosampler with a 20 µl loop and a photodiode array detector (DAD). A second analytical method was based on the ELISA using a Ridascreen® Tetracyclines kit from R-Biopharm (Darmstadt, Germany) that contains all the reagents required for the assay. Test kits were stored at 4°C. Tetracycline hydrochloride (TC) was purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). TC standards solutions were prepared in methanol (100 mg.l -1 ) and kept at -18°C for one month in dark glass bottles. Just before use, these solutions were diluted to the required concentrations with 0.05 M ortho-phosphoric acid or PBS for HPLC or ELISA respectively.

Tetracycline analysis

Two analytical methods were employed to detect TC in honey samples : HPLC and ELISA. At the beginning of the experiment, TC analyses were undertaken by HPLC because residues levels were high. Under the level of 500 µg.kg -1 , the quantitative analysis was made by ELISA. According to the manufacturer description, the ELISA test kit recognizes several compounds of the chemical class of tetracyclines, including TC to which this test is reported to display a cross-reactivity of 100%.

HPLC

The procedure for extraction and analysis of TC was based on the Jürgens' (1981) method but was modified as follows. Honey (1 g) was mixed in a beaker with 3 ml of 0.05 M ortho-phosphoric acid and transferred to a 10 ml flask. The beaker was then rinsed with 5 ml of 0.05 M ortho-phosphoric

ELISA

Honey (1 g) was agitated using Vortex-mixer with 50 ml of PBS. This extract was subsequently centrifuged at 3290 g during 30 min at room temperature to eliminate pollen and other impurities.

According to the procedure described for the kit, 50 µl of TC standards prepared in PBS and the extracts of honey were added to the bottom of each well in duplicate. At the last step, in presence of TC, the addition of the stop reagent (which contains 1 M sulphuric acid) leads to a colour change from blue to yellow. The measurement is made by photometry at 450 nm. The absorption is inversely proportional to the TC concentration in the sample. Limits of detection and quantification were 10 and 15 µg.kg -1 respectively.

Protocols

Stability of tetracycline in honey stored under laboratory conditions

The honey used in this study had been harvested in the laboratory apiary at Sophia Antipolis at the end of summer 2002 from hives that had never been treated with TC. These hives were kept in a Three aliquots of 200 g of honey were spiked each with 4 ml of 100 mg.l -1 of TC solution prepared in methanol and manually homogenised using a spatula during 10 minutes. These samples with an initial level of TC equal to 2 mg.kg -1 were then kept in darkness at three different temperatures (4, 20 and 35°C). A fourth honey sample was added with 4 ml of methanol and used as control sample during analyses. TC analyses were performed on 3 aliquots of each of these samples at day 0, 2, 37, 90, 210 and 330 following this preparation.

Tetracycline found in honey following treatments of colonies

Honey bee colonies

Experiments were conducted on 24 10-frame Langstroth new hives purchased at Ickowicz (Bollène, France) equipped with crown board feeders. Colonies belonged to the small black local race of Apis mellifera mellifera of our laboratory's experimental apiary. They were transferred to new foundation combs on April 2002. Hives were randomly distributed into four groups of 6 hives. Two treated groups called 'the syrup' (S) and 'the powdered sugar' (P) and one untreated group called 'the control' (C) were installed at the same site in the premises of the laboratory at Sophia Antipolis, Alpes-Maritimes, France. For limiting drifting as much as possible, the distance between hives of the treated groups and the distance between treated and control hives (group C) were 10 metres and 20 metres respectively. The fourth group of 6 hives was disposed 3 km apart from the others and will be called 'the distant control' (DC) in the following lines. TC powdered sugar was processed by mixing manually 3.5 g of TC and 70 g of powdered sugar.

F

Each day of treatment, each colony of group P received 10 g of TC powdered sugar equally dropped between combs. An aliquot of each mix (saccharose syrup, TC saccharose syrup and TC powdered sugar) was kept at 4°C immediately after preparation for TC titration.

Honey sampling in colonies

In every colony, all frames of each brood chamber and super were identified with a number. On day 1,8,15,22,33,48,92,146,202,358,390 and 504 post treatment, individual samples of 10 g of honey were taken repetitively from the same five frames in the brood chamber and in the super from uncapped or sealed honey cells. Honey samples were filtered through a nylon sieve into a clean polyethylene jar appropriately labelled (identification of the colony, brood chamber or super, frame, treatment mode, date of sampling). All samples were stored at 4°C before analysis.

Until 22 days after treatments, each sample was analysed individually. After this date, the five samples from the same colony were pooled before analysis.

Supers

Data analysis

Degradation of tetracyline in spiked honey stored in laboratory conditions at various temperatures

We assessed that the degradation speed followed a first-order kinetic model according to the equation :

k = -ln(C/C 0 )/t (1)
where k, the degradation rate is expressed in day -1 , C and C 0 , the mean TC concentrations (mg.kg -1 ) of the three samples taken at day t and day 0 respectively. k was estimated by linear regression analysis. The persistence of the TC was estimated by the half-life time (t 1/2 ) using equation 2 :

t 1/2 = ln2/k (2)

Comparison between the mean tetracycline titre of the five honey samples of the same hives and the tetracycline titre measured on the pooled samples

At day 1, 8, 15 and 22, TC titre was measured independently on each honey sample (one sample per frame) and on the pool of the five samples taken from the same brood chamber. Means of the titres of these five samples and titres of the corresponding pools were compared using the mean square correlation method.

Results and discussion

Stability of tetracycline in honey stored under laboratory conditions

All linear adjustments according to the degradation speed model (1) were highly significant which ever was the storage temperature (Prob (theoretical F for 1 and 16 degrees of freedom > F obs ) < 0.0001). k was evaluated to 1.334 10 -3 , 2.862 10 -3 and 5.737 10 -3 day -1 at 4, 20 and 35°C respectively. Logically, higher storage temperatures resulted in a more rapid degradation of TC :

half-life time was evaluated to 520, 242 and 121 days at 4, 20 and 35°C respectively (figure 1). In other words, at 35°C, which is the internal hive temperature, the degradation of TC is twice faster than at ambient temperature (20°C). In our study, the honey had a pH equal to 4.5. As in general, the pH of honey ranges between 3.2 and 4.5, the observed stability of TC is consistent with the known stability of tetracyclines in acidic medium [START_REF] Chambonnaud | Contribution à la recherche des antibiotiques dans le miel[END_REF], Puyt 1997).

[Insert figure 1 about here]

Tetracycline found in honey following treatments of colonies

No residues of TC were detected in any samples 16 days before treatments (T-16). Treatments with TC were realised at T, T+8 days and T+14 days. Analyses of TC in each aliquot of preparation were realised by HPLC. Values measured for TC in syrup preparations used were 0.51, 0.44 and 0.35 g of TC/l of syrup at day 0, 8 and 14 respectively. The TC values measured in the powdered sugar preparations were 0.38, 0.5 and 0.49 g of TC/g of powdered sugar respectively. Most of these values are very close to the expected titre of 0.5 g of TC/l of syrup and 0.5 g of TC/g of powdered sugar.

Comparison between the mean tetracycline titre of the five honey samples of the same hives and the tetracycline titre measured on the pool of these samples

For groups C, S and P, the correlation between the mean of the individual measures performed on the honey taken from 5 frames and the measure on the pool of these samples is very highly significant with r=1.00, 0.99 and 0.94 respectively (P<0.0001). The slopes of the three correlation lines are not significantly different from 1 and the intercepts (equal to 0.06, 0.16 and 0.13 respectively) are not significantly different from 0. These slope and intercept values reveal that the measure on the pooled samples is a very precise and a non biased estimation of the mean of the measures of the individual samples. Consequently, after day 22, all titrations have been processed

on pooled samples only.

Tetracycline residues in hives

TC residue levels in the honey samples collected from brood chamber and super are described in figure 2 and table 1.

[Insert figure 2 about here]

[Insert table 1 about here] Honey samples from brood chambers One day after the last application, TC residues were detected in honey from brood chamber of all hives of groups S and P. Values ranged from 22.7 to 71.0 mg.kg -1 (mean = 40.7 mg.kg -1 ) and from 2.48 to 5.32 mg.kg -1 (mean = 4.34 mg.kg -1 ) for groups S and P respectively. Out of the 6 hives of the control group C, 3 had TC residues with values ranging from 0.06 to 0.72 mg.kg -1 (mean = 0.16 mg.kg -1 for the whole group C). The mean residues titre of group S is significantly higher than the means of groups P and C with no significant difference between these latest groups (Tukey-Kramer test with α equal to 5%). No TC residues were detected in the group DC.

Several conclusions may be drawn from the TC measurements in honey at day one : (i) the incorporation of TC in honey is very rapid; (ii) the treatment with syrup entailed a ten times higher concentration of TC in honey than treatment with powdered sugar. Both results are coherent with our observation of the rapid removal of syrup by bees and its storage in cells. As some traces of powdered sugar remained on the top of frames, TC was not so much incorporated in honey than when TC was given in syrup; (iii) non-treated hives may be contaminated with TC through drifting from neighbouring TC treated hives. This contamination is related with distance between hives :

whereas no TC residues were detected in the group DC, two of the three contaminated hives of group C were at the edge of this group i.e. at 20 metres from the first hive of group S. The farthest hive of this group was at 35 metres from the first hive of group S.

The rapid dissemination of syrup into the whole colony is a well known phenomenon : [START_REF] Courtois | Sur un procédé de marquage des abeilles butineuses au moyen d'un radioisotope[END_REF] demonstrated that 24 hours after application of syrup marked with 198 Au, all bees of the hive were radioactive with all the radioactivity detected in the digestive tract. In a similar experiment, [START_REF] Nixon | Food transmission within the honey bee community[END_REF] observed that following application of radioactive syrup to a colony, 43 to 60% of all bees of the colony were radioactive within 27 hours. Whereas, foragers became radioactive faster and at a higher rate (76%) than the rest of the colony, this enhanced their role of TC carrier to other colonies by drifting. All the large larvae in unsealed cells became radioactive within 48 hours. [START_REF] Mckee | Oxytetracycline hydrochloride activity in honey bee larvae (Apis mellifera) following medication with various doses[END_REF] confirmed that OTC treatment reaches larvae rapidly : on the day following a treatment with 0.3 to 1 g of OTC, they measured in larvae an OTC level ranging between 13.3 and 172.2 mg.kg -1 .

After 8 days, TC residues were detected in all hives of group C. The TC level decreased significantly in all groups whereas the differences previously observed between groups were maintained. The mean TC level of group S remained significantly higher than those of group P and C until day 358 post treatment. After approximately one year (358 days), mean TC residue levels were 2.57, 0.13 and 0.06 mg.kg -1 for groups S, P and C respectively. At day 390, mean values of TC residues were 1.50, 0.10 and 0.08 mg.kg -1 in groups S, P and C respectively. No residues were detected after 504 days in 3 hives on 4 for group S, in 2 hives on 3 for group P and in 4 hives on 5 for group C. At this date (D+504), residue levels detected became weak. It is worthwhile to note that at this time, the highest value (0.17 mg.kg -1 ) is measured in group C.

Honey samples from supers

At day 48 (15 days after adding supers), TC residues were detected in harvested honey in all hives of groups S and P (except for 2 hives of group S which did not produce any honey). Values ranged between 7.94 and 10.68 mg.kg -1 (mean = 9.19 mg.kg -1 ) and between 0.61 and 2.58 mg.kg -1 (mean = 1.35 mg.kg -1 ) for groups S and P respectively. TC residues measured in the 3 hives of group C which produced honey ranged between 0.41 and 1.31 mg.kg -1 (mean of the 5 hives of group C = 0.45 mg.kg -1 ).

At day 92 and 146, the mean TC level of group S was significantly higher than the means of groups P and C which were not significantly different (Tukey-Kramer test with α equal to 5%). In the honey harvested at day 146 (113 days after adding supers), TC concentrations had dramatically decreased. All samples from the treated groups (S and P) contained TC residues with values ranging from 0.87 to 1.94 mg.kg -1 (mean = 1.54 mg.kg -1 ) and 0.07 to 0.80 mg.kg -1 (mean = 0.35 mg.kg -1 ) in groups S and P respectively.

In group C, the mean residue level in honey increased as a result of the contamination by drifting of 4 of the 6 hives of the group with concentration ranging from 0.05 and 0.66 mg.kg -1 (mean for the 6 hives = 0.15 mg.kg -1 ). It is worthwhile to note that in 2 hives of group C, the contamination was as high as the level measured in several hives of group P whereas it did not reached values observed in group S.

In all hives of the group DC, no residues were detected at any time of the study. As expected, no drifting occurred from the treated colonies which were 3 km apart. Each super individually Data on the correlation between the amount of antibiotics in honey and the doses given as treatment are scarce. Such a correlation has been extrapolated by [START_REF] Douault | Etude du stockage des réserves par les abeilles au moyen d'un sirop de sucre marqué au 141 Ce[END_REF] following the feeding of colonies with 4 litres of saccharose syrup marked with 141 Ce : 46 days following the last administration of 1 litre of syrup and 40 days after adding supers, they demonstrated by autoradiography of a frame the flow of marked syrup to supers. The radioactivity in supers ranged between 2.4 and 6.3% of the theoretical residual radioactivity. According to these authors, this indicated that bees had moved 2.0 to 8.4 g of saccharose into every kg of honey gathered in supers.

Hypothesising that antibiotics distributed in syrup would follow the same flow as sugar, they concluded that a mean of 0.21% of an hypothetical antibiotic dose should finally be found in honey.

This evaluation is seven times lower than our experimental mean value of 1.5% of TC dose found in group S.

Comparison between tetracycline concentration in brood chambers and in supers

In every group, the TC concentration in honey from supers was significantly correlated to the TC concentration in honey from brood chambers measured the same date (table 2).

[Insert table 2 about here]

The correlation coefficient always lower than 1 and the constant term always not significantly different of 0 indicate that the TC concentration in honey from supers was in average always lower than the TC concentration in honey from brood chambers. The correlation coefficient decrease between day 48 and day 146 shows that the TC concentration in honey decreased more rapidly in supers than in brood chambers. This diminution in TC concentration was not due to a simple dilution of TC in the increased quantity of honey as no correlation could be demonstrated between the mass of honey harvested in the hive and TC concentration of this honey, or between the mass of honey harvested in the hive and the variation of TC concentration in brood chamber from day 22 to day 146. The amount of TC harvested with honey can be estimated to a range of 1.1 to 11.3 mg and 13.0 to 29.8 mg for groups P and S respectively which represented 0.07 to 0.75% and 0.9 to 2% of the TC dose applied in powdered sugar or syrup respectively.

These observations are in accordance with the results of [START_REF] Martel | Bilan du plan de contrôle 2002 de la contamination des miels[END_REF] who described a transitory increase in OTC concentration in honey from central combs until 5 to 7 days after treatment. They shown that this increase was not a result of water condensation in stored honey as there was no significant increase in sugar concentration but should be due to a new distribution of the medicated store by bees. After a week or more, they observed no apparent difference between the central combs and those next to the feeder comb.

Half-life time of TC in honey in brood chambers and supers

Considering the samples from brood chambers, all linear adjustments according to the degradation speed model (1) were highly significant (Prob (theoretical F for 1 and 9 degrees of freedom > F obs ) < 0.0001). Determination coefficient (r²) were 0.89, 0.78 and 0.92 for group C, S and P respectively. The degradation rate k and the t 1/2 of TC have been evaluated for the time interval day 1 to day 504, except for group C, for which the time interval was day 22 to day 504, as in this group, TC level increased until day 22. k was evaluated to 8.0 10 -3 , 12.6 10 -3 and 12.1 10 -3 day -1 for group C, S and P respectively.

We observe that the evaluations of the half-life time of TC in hives varied from 55 to 87 days, and were lower than the 121 days evaluated for honey stored at 35°C under laboratory conditions.

Considering honey from supers, due to the limited number of measures and to the limited variation of TC concentration during the period, the calculated half-life time values (77, 60 and 57 days for obtained for honey in brood chambers of the same groups.

In summary, TC decline in honey was more rapid in hive than when stored under laboratory conditions at 35°C. Whereas we did not measured the actual temperature in the hive in this experiment, hive temperature is considered to be equal to 35°C and fairly constant due to regulation by bees even if the external temperature is above 38°C (which was the case during summer 2003) [START_REF] Morse | The ABC&XYZ of Bee Culture[END_REF]). This raises the question of an active decline of TC by bees.

Conclusion

Tetracycline application in hives following protocols usually practiced in France with syrup or powdered sugar demonstrated that the antibiotic is very rapidly incorporated into honey. One day after the last TC application, treatment with syrup resulted in a concentration of TC ten times higher than in honey treated with powdered sugar : mean values were 4.34 and 40.7 mg.kg -1 for groups P and S respectively. After approximately one year (358 days), mean TC residue levels in brood chambers were 0.13 and 2.57 mg.kg -1 respectively and after 504 days, residue levels became weak.

Honey collected on day 146 had mean TC levels equal to 0.35 and 1.54 mg.kg -1 in these groups but no detectable residues in honey harvested on day 504. Storage temperature and probably activity of bees influence the half-life time of TC in honey : (i) the degradation of TC in honey kept in jars is twice faster at 35°C compared to 20°C and (ii) the estimated TC half-life time in honey is twice lower in hive (t 1/2 = 65 days) than when stored under laboratory conditions at 35°C (t 1/2 = 121 days).

Consequently, the relative stability of TC in honey requires appropriate regulatory measures for asserting honey quality. Either, no honey should be allowed for human consumption following a treatment of the colony with antibiotic during the year, or MRL or tolerance values for antibiotics should be defined. Our data could contribute to this definition. An important observation is that the honey of non-treated hives may be contaminated with TC through drifting from TC treated neighbouring hives. The contamination occurred from the 1 st day until three weeks after treatment and entailed TC concentrations in honey of the same order of magnitude as those measured in some treated hives (mean equal to 0.15 mg.kg -1 in honey harvested on day 146). Moreover, one hive of this control group still contained detectable residues at day 504 (0.026 mg.kg -1 ). This contamination is related with distance between hives : all contaminated hives were located within 20 to 35 metres from the nearest treated hives, whereas no residues were detected in control hives located 3 km apart.

F

  volume of the flask was completed to 10 ml. The extract obtained was ready for HPLC analysis after filtration on 0.45 µm filter.The liquid chromatographic column was a Hypersil BDS C18, 5 µm, 250 x 4.0 mm I.D. from Merck Eurolab-Prolabo. The mobile phase was 0.05 M ortho-phosphoric acid/acetonitrile (80:20, v/v) at a flow-rate of 1 ml.min -1 . Detection of TC was made at the 360 nm wavelength. The injection volume was 20 µl and an external calibration was employed. The chromatographic runs were performed at 15°C. Limits of detection and quantification were 200 and 500 µg.kg -1 respectively. Limits of detection (LOD) and quantification (LOQ) were calculated by a signal to noise ratio of 3:1 and of 10:1 (Directive 91/414/EEC modified by Directive 96/46/CE and Explantory Note EEC/844/87, European Union, Brussels).

  typical Mediterranean forest (Quercus suber and Q. ilex) and meadows.Therefore, additionally to Mediterranean oaks, sources of pollen, nectar and honey-dew were from a large variety of wild plants such as Dorycnium pentaphyllum, sages (Salvia sp.), thyme (Thymus sp.), rosemary (Rosmarinus officinalis), heather (Erica arborea and E. sp.), arbutus (Arbutus unedo), Inula viscosa, Solidago sp. This honey reflected this polyfloral origin.

  were added 33 days(14 April 2003) after the last treatment. On the 5 August 2003 (i.e. 113 days later), honey of each super was harvested separately for group DC, group C, group P and group S successively. The extractor was cleaned between each extraction. Honey harvests were weighed using an electronic balance (accuracy = +/-0.05 kg) and were ranged from 15.5 to 18 kg (mean = 16.5 kg), from 1 to 20 kg (mean = 12.7 kg), from 7.1 to 17 kg (mean = 12.6 kg) and from 11.6 to 16.8 kg (mean = 14.4 kg) for groups DC, C, P and S respectively.

  at day146 (5 August 2003) then replaced on their respective hive at day390 (5 April 2004). The honey gathered at day 504 (28 July 2004) did not contained any detectable TC residue, except in one super from group C with a value of 0.026 mg.kg -1 .

  S and P respectively) are non significant. Whereas, these values are very close to the t 1/2

Figure 1 .Page

 1 Figure 1. Degradation of tetracycline in honey stored in laboratory at 4, 20 and 35°C

Table 1 .

 1 Tetracycline residues in hives

	Mean residues (mg.kg -1 )

Table 2 .

 2 Comparison between tetracycline concentration in brood chambers and in supers

	-1

Tetracycline residues in honey after hive treatment

  Martel 1 Anne-Claire, Zeggane Sarah, Drajnudel Patrick, Faucon Jean-Paul and Aubert Michel

	Agence Française de Sécurité Sanitaire des Aliments (AFSSA) Site de Sophia Antipolis,
	Laboratoire d'Études et de Recherches sur les Petits Ruminants et les Abeilles (LERPRA),
	Unité de Pathologie de l'Abeille, 105 route des Chappes, F-06902 Sophia Antipolis Cedex, France F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

  Varroa destructor was preventively controlled using Apivar® : two stripes were inserted in the hives during ten weeks starting inSeptember 2002, in August 2003 and in September 2004.

	Additionally, a five-week treatment was initiated in May 2004 following the observation of
	parasites. All hives were artificially fed with commercial syrup (2.5 litres/hive of Apiinvert supplied
	from Nevières -Valensole, France) in September 2003 and April 2004 and with candy (2.5 kg/hive
	of Apifonda from the same supplier) in November 2003 and January 2004.
	o r Colony mortality was observed at day 146 (1 colony of group S), day 202 (2 colonies of group P), day 358 (1 colony of group P and 1 of group C) and day 504 (1 colony of group S). Similar mortality rates, colony weakness and a development of wax moth especially in supers were also F o r
	P observed by other beekeepers in the area and were attributed to the very high temperatures during P
	summer 2003.	e e
		e r e r
		R R
		e e
		v i e v i e
		w w
	O n l Syrup (saccharose/water 50:50 (w/v)) was prepared by mixing 5 kg of saccharose and 5 litres of O n l
	y water using a magnetic agitator during one night. TC syrup was processed extemporarily by mixing y
	during 4 hours 3.785 g of TC in 7.5 litres of saccharose syrup. Each day of treatment, each colony
	of groups C and S received 1 litre of syrup or TC syrup respectively, precisely measured with a
	graduated cylinder.

Treatment

Before the start of the experiment, analyses of TC were realised on honey collected in every combs of all hives 16 days before the first TC application. TC was applied to hives at the end of winter according to two modes of application : syrup or powdered sugar. Groups S and P received 3 applications of TC (0.5 g each per colony) 7 days apart (on the

26 February, 6 and 12 March 2003)

.

Group C received saccharose syrup only at the same dates.

  Statistical analyses were realised with the appropriate module of JMP® software (JMP® Design of Experiments, Version 5, 2002, SAS Institute Inc., Cary, NC, USA 27513) and multiple comparisons of means were performed on all pairs of means using Tukey-Kramer honestly significant difference test included in the same software.

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

  Puyt, J.D., 1997, Antibiotiques -Antibiomimétiques. Cours de chimiothérapie anti-infectieuse et antiparasitaire, Département de Biologie et Pharmacologie, Ecole Nationale Vétérinaire de Nantes, pp.

	77-90.
	Usleber, E., Dietrich, R., Märtlbauer, E., Unglaub, W., 1996, Antimicrobial residues in honey,
	Conference of residues of veterinary drugs in food. Euroresidue, Veldhoven, The Netherlands, 948-951.
	o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w
	O n l O n l
	y y

Table 1 .

 1 Tetracycline residues in hives : no residues were detected except in one super with a value of 0.17 mg.kg -1 (b) : no residues were detected except in one super with a value of 0.026 mg.kg -1 (c) : no residues were detected except in one super with a value of 0.043 mg.kg -1

	Mean residues (mg.kg -1 )

Table 2 .

 2 Comparison between tetracycline concentration in brood chambers and in supers

	Days after	Determination	Correlation	Significance of r	Constant	Significance of the
	treatment	coefficient (r 2 )	coefficient (r)		term	constant term
	48	0.92	0.68	P<0.0001 -S	0.36	P=0.32 -NS
	92	0.86	0.43	P<0.0001 -S	-0.13	P=0.44 -NS
	146	0.74	0.40	P<0.0001 -S	0.12	P=0.30 -NS
	504	0.93	0.14	P<0.0001 -S	-0.001	P=0.35 -NS
	NS : not significant
	S : significant

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

Acknowledgements

We thank Mr B. Delepine and Mr M. Laurentie from AFSSA Fougères for their advice.


Vincenzo ; Garau

Susana De

Melo Abreu

Pierluigi Caboni

Paolo Cabras

Arminda Alves

Filippo Pirisi

Residues of the fungicide famoxadone in grapes and its fate during wine production

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

Famoxadone is a recently applied fungicide to vines, belongings the oxazolidinedione family. In 29 this work, the fate of famoxadone was studied considering the decay ratio of this fungicide during 30 grapes maturation and wine production. The main factors affecting the presence of fungicide 31 residues such as fruit growth, photodegradation, evaporation, thermodegradation, and co-32 distillation were studied with model systems. 33

An experimental field was treated with a commercial product containing famoxadone at the 34 recommended dose. After this application, residues of famoxadone were found in grapes at the 35 concentration of 0.27 ± 0.06 mg kg -1 . In this field experiment, the half-life time (t ½ ) of 36 famoxadone, described as pseudo-first-order kinetics (R 2 = 0.74), was 18 ± 6 days, resulting from 37 the photodegradation. The famoxadone residue levels in grapes were below the established 38 Maximum Residues Level (MRL) for Europe (2 mg kg -1 ) whilst levels in wine, carried out with 39 and without maceration, were below the calculated limit of detection (LOD) of the method. 40 Protection, is applied to bunch and leaves to control downy mildew, which is an important 57 grapevine disease induced by the Plasmopora viticola [START_REF] Joshi | DPXP-JE874: A broad-spectrum fungicide with a new mode of 298 action[END_REF]. The chemistry 58 and biochemical mode of action of famoxadone is close to the known class of strobilurins (Bartlett 59 et al. 2002). The fungicidal activity of famoxadone consists in the inhibition of the mitochondrial 60 respiration by binding at the so-called Q 0 site of cytochrome b, located in the inner mitochondrial 61 membrane of the fungi (Jordan et al. 1999). 62 63 Famoxadone active ingredient can be used on grapes in Europe at a maximum application rate of 64 0.05 kg ha -1 (France and Greece), 0.09 kg ha -1 (Italy and Spain) or 0.14 kg ha -1 (Germany). Spain 65 and Greece allow a maximum of 6 applications per season and the other countries a maximum of 3 66 applications, except Germany (8). In Italy the maximum residue level (MRL) for famoxadone is 2 67 mg kg -1 with a safety interval (PHI) of 28 days 68 (http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/006/Y5221E/y5221e0e.htm). 69

The first analytical method reported in the literature to determine famoxadone in grapes and wine 70 used liquid-liquid extraction (LLE) and gel permeation chromatography (GPC) before a gas 71 chromatographic separation with electron capture detection (GC-ECD) [START_REF] Jernberg | Famoxadone. In Handbook of residue analytical methods for agrochemicals. 292[END_REF]. Our group 72 validated one method by GC/MS, using a simple and fast LLE procedure (de Melo Abreu et al. 73 2005). This method was used in the present work. To our knowledge no studies reported the fate of 74 (Cabras et al. 1997(Cabras et al. , 1998[START_REF] Cabras | Fenhexamid 279 residues in grapes and wine[END_REF][START_REF] Garau | Disappearance of 286 azoxystrobin, pyrimethanil, cyprodinil and fludioxonil on tomatoes in a greenhouse[END_REF] deal with the persistence and 78 the study of the mechanism of disappearance of pesticides with model systems in grapes and wine 79 production. Following treatment, pesticides spread into the epicuticular waxes present on fruits 80 surface and could affect the degradative process of the active ingredient (Cabras et al. 1997). This 81 paper aims to contribute to the knowledge of the persistence of famoxadone in grapes and its 82 behaviour during wine production. 83 84 Experimental 85

Fungicide treatment 86

The trial was carried out in a farm located in Santadi (Cagliari, Italy), in 240 vines of the vitis 87 variety Carignano, with a plant spacing of 1.9 m × 0.8 m. The treatment with the commercial 88 formulation Equation Pro, containing 22.5% of famoxadone, was done September 2, 2004. A 89 random block scheme with 4 replications for each test was used. Each block contained 60 vines. 90

The fungicide application was carried out with the dose recommended by the manufacturer (480 g 91 ha -1 ), using a Carpi portable motor sprayer (Modena, Italy). 92 93The weather conditions were continuously recorded with a Silimet AD-2 automatic station 94 (Modena, Italy), with an average total rainfall of 0.7 mm, a minimum temperature of 17.1 ºC, a 95 maximum temperature of 30.2 ºC, and an average solar radiation of 167 watt m 2 . 96 97 Grapes material and wine production 98 The amount of the famoxadone residues during the grapes maturation and wine-making was 107 determined. Crushed grape samples were directly analysed as described below. An aliquot of 100 g 108 of must was centrifuged at 4000 rpm for 5 min, the amount of cake and the concentration of 109 famoxadone residues in the cake and in the clear must were measured. Fermentation had been a 110 regular course in all samples and, after 15 days, the obtained wines were centrifuged in the same 111 manner as must and analysed. At the end of the vinification process without maceration, lees 112 amount and famoxadone residues were measured. In vinification process with maceration, after the 113 wine had been made, the amount of cake with lees, and famoxadone residues were measured. 114 115 Chemicals 116

The commercial formulation Equation Pro (22.5% of famoxadone and 30.0% of cimoxanil) and 117 the analytical standard of famoxadone (99.4%) were obtained from Dupont Crop Protection 118 (Newark, USA). Acetonitrile LiChrosolv isocratic grade for GC (≥99.8%) and chloroform 119 Normapur for GC (≥99.0%) were purchased from Merck (Darmstadt, Germany). Hexane 120 (≥95.0%), ethyl acetate (≥99.8%) and acetone (≥99.8%) were Pestanal Grade for residues analysis 121 from Riedel-de Haën (Seelze, Germany). The water was bidistillated and filtered with a Millipore 122 Milli-Q apparatus (Milan, Italy). 123

Standards preparation 125

A fungicide stock solution (1 g l -1 ), and a working solution (100 mg l -1 ) were prepared in 126 acetonitrile and stored in dark glass vials, and kept respectively at -20 ºC and 4 ºC. Appropriate 127 standards solutions for control quality assessment were made daily as described elsewhere (de 128

Melo [START_REF] De Melo Abreu | GC-MS determination of 282 famoxadone in grapes and wines[END_REF]. 129 130

Sample extraction 131

For extraction, crushed grapes (5 g), must and wine (5 ml) samples were weighed in 40 ml screw-132 capped tubes and 10 ml of ethyl acetate/hexane (50:50 v/v) was added. Tubes containing the 133 mixture were agitated for 15 min in a rotatory shaker, the organic phase was removed and injected 134 in the chromatographic system. The same procedure was used to analyse cake and lees. 135 136 Apparatus and chromatographic conditions 137 GC/MS analysis of famoxadone was carried out on a Hewlett-Packard (Palo Alto, USA) HP 5890 138

Series II gas chromatograph system, equipped with a HP 7673 GC/SFC autosampler, a 19251A 139 capillary split-splitless injector and a GC-MS HP 5971 Series detector. The analytical column used 140 was a J&W Scientific DB-17MS fused silica capillary column (Folsom, USA) with 15 m of length, 141 0.25 mm of internal diameter and 0.15 µm of film thickness. The injector temperature was set at 142 300 °C. The oven temperature program was as follows: initial temperature was kept at 200 o C for 1 143 min, increased to 300 o C at 10 o C min -1 and held at this temperature for 2 min. Total run time was 144 13.0 min. The volume of sample was 2 µl, injected in splitless mode with splitless time of 1 min. 145 Helium (99.9999% purity) was used as carrier gas at constant flow rate of 0.8 ml min -1 . The mass 146 spectrometer was operated in electron impact (65 eV of ion energy), with 10 minute solvent delay, 147 the interface temperature was kept at 300 ºC, and the ion source temperature was kept at 180 ºC. 148 The extraction of the grapes waxes was carried out as previously [START_REF] Mcdonald | Epicuticular wax morphology and composition 301 are related to grapefruit chilling injury[END_REF]. Grape 153 wax extraction was performed by the immersion of a known weight of untreated grapes in a known 154 volume of chloroform, for 1 min. The amount of waxes of the grape surface, expressed in µg cm -2 , 155 was determined by evaporating an aliquot of 10 ml of the chloroform extract to dryness and 156 calculating the surface area of the fruit. 157 158

Model systems 159

The model systems for photodegradation, codistillation, evaporation and thermodegradation were 160 studied according to Garau et al. 2002. Briefly, three tests, presenting below, were done: 161 Test A -Evaporation and Thermodegradation. In a membrane of regenerated cellulose there was 162 placed a solution of famoxadone with known concentration. After solvent evaporation, the 163 membrane was placed in a 10 ml dark screw-capped vial. This vial was putted in a thermostatic 164 stove at 50 ºC for 24 h. Then, the vial was removed and placed in freezer for 5h at -20 ºC to allow 165 the condensation of famoxadone. After removing the vial from the freezer, the membrane was 166 transferred to another vial with 5 ml of ethyl acetate/hexane (50:50 v/v) for the extraction of the 167 remained residues of famoxadone. The first vial was also washed with the same amount of the 168 solvent solution, allowing to measure the residues of famoxadone evaporated from the membrane. 169 At the same time, a control sample stored in the dark at room temperature was analysed with the 170 same procedure. After the GC/MS analysis of these extracted samples, the difference between the 171 amount of famoxadone of the control sample and the sum of the amounts of the famoxadone found 172 containing 5 ml of ultra-pure water previously weighted. This vial was placed in a thermostatic 177 stove in the same conditions of Test A, allowing the evaporation of the water and the co-178 distillation of the famoxadone. After 24 h, the vials were weighted to determine the amount of 179 water evaporated. The control sample was stored in the dark at room temperature. The loss of 180 famoxadone by codistillation was calculated by the difference between the amount of famoxadone 181 in the control sample and the amount of the famoxadone found in the membrane of the heated 182 sample estimated by GC-MS. For a suitable evaluation of codistillation, the possible losses of 183 famoxadone due to evaporation and thermo-degradation, determined with the Test A, must be 184

considered. 185

Test C -Sunlight Photodegradation. An aliquot of famoxadone solution at known concentration 186 was poured into Petri dishes with 5 cm of diameter and the solvent was allowed to evaporate, at 187 room temperature. The dishes were exposed to direct sunlight, removed at prefixed intervals and, 188 after re-dissolving the remained residues with 5 ml of ethyl acetate/hexane (50:50 v/v) injected for 189 GC-MS analysis. The control sample was stored in the dark at room temperature. provide insight into co-distillation, and these are presented in the Tables 3 and4 for famoxadone 229 and for the commercial product, respectively. Concerning the Test A for famoxadone and for the 230 commercial product, no evaporation phenomena occurred in either case, with and without waxes. 231

In fact, at 50 ºC, which is on average the higher temperature found in the field during grape 232 maturation, all the residues remained in the membrane (m). Considering the LOD of the method, 233 residues of famoxadone were not found in the vial (v) which contained the membrane. There were 234 no observed differences between the concentration of famoxadone in the control sample (c) and 235 the sum of the concentration in the vial and in the membrane indicating that there were no losses 236 by thermodegradation. In the Test B for the active ingredient and for the commercial product, it 237 was shown that co-distillation did not occur, either in the presence or absence of waxes. As shown 238 in Tables 3 and4 This field degradation trial showed that famoxadone residues at the pre-harvest interval and during 252 vinification were below the MRL (2 mg kg -1 ). During the wine-making process the fungicide is 253 completely adsorbed in the cake and lees. Data from the models system experiments suggest that 254 in real conditions evaporation, thermodegradation and co-distillation processes do not affect 255 residue levels of famoxadone and the mechanism responsible for the disappearance of famoxadone 256 was essentially that of photodegradation.

41

  Keywords: famoxadone residues; grapes; wine production; model systems. anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione) is a recently 54 developed highly active and broad spectrum fungicide used to control different crop diseases. In 55 viniculture, the commercial formulation, named Equation Pro and manufactured by Dupont Crop 56

  to wine. The application of the active ingredient close to the grapes harvest 75 could lead to the presence of famoxadone residues in wine. 76 77 Different papers

  added with 200 mg of sodium metabisulphite. One portion was fermented with 102 skin, for the vinification with maceration process, and the other without skin, being called 103 vinification without maceration process. To improve the fermentation process, 20 mg kg -1 of active 104 dry yeasts was added. 105 106

  for the ion monitoring was 100 ms per ion. Selected monitoring ion (SIM) mode 149 was used. The ions utilized for quantification of famoxadone were 196, 224, 330 and 374 m/z. 150 151 Extraction procedure for grape waxes 152

  and in the vial of the heated sample was calculated. This value indicates the 173 amount of thermo-degradation of famoxadone.

  , B and C were carried out with and without the presence of the epicuticular waxes 192 extracted from grapes, at the concentration of 0.37 µg cm -2 . Each experiment was replicated mg kg -1 ) at the end of the fermentation process. In both cases, all the residues of famoxadone 221 were adsorbed to the solid parts. 222223Model system studies (Tests A, B and C) were carried out with and without grape epicuticular 224 waxes (0.37 µg cm -2 ) because the latter could have an influence on the fate of pesticides on fruits 225(Cabras et al. 1997) enhancing or reducing t 1/2 of the active ingredient. Each test was carried out 226 with the active ingredient and with the commercial formulation. The results obtained for the Test 227 A, provide an insight into the mechanisms of evaporation and thermodegradation, and Test B, 228

  photodegradation of the commercial product with waxes was slightly lower than without waxes. 247

Table 1 .

 1 Concentration of famoxadone during grapes maturation, expressed by means of a relative 1

	2	standard deviation (SD).
	3
		Days after treatment	Concentration (mg kg -1 ) ± SD	t 1/2 (days)
		0		0.27 ± 0.06
		3		0.40 ± 0.11
		F r 7 14 o	0.30 ± 0.12 0.22 ± 0.11	18
	5 4	21 28	0.14 ± 0.10 r e P e 0.16 ± 0.07
	6			R
	7			e
	8 9			v i e
	10			w
	11 12 13			O n l
	14			y
	15
	16
	17
	18
	19
				1

Table 2 .

 2 Fate of famoxadone during wine production.

	20
	21

Table 3 .

 3 Results of Tests A (evaporation and thermodegradation) and B (codistillation) for the 36 famoxadone active ingredient, expressed in µg cm -2 ± SD.

	37
	38
			Test A		Test B
			without waxes	with waxes	without waxes	with waxes
		Control sample (c)	2.00 ± 0.06	1.40 ± 0.34	2.10 ± 0.20	1.70 ± 0.15
		Vial (v)	<LOD	<LOD	-	-
		Membrane (m)	2.00 ± 0.26	1.40 ± 0.29	1.90 ± 0.33	1.80 ± 0.10
		c -(v + m)	n.s.	n.s.	n.s.	n.s.
	39	n.s. -not significant
	40
	41
	42
	43
	44
	45
	46
	47
	48
	49
	50
	51
	52
	53
	54
	55

Table 4 .

 4 Results of Tests A (evaporation and thermodegradation) and B (codistillation) for the 56 commercial product Equation Pro, expressed in µg cm -2 ± SD.

	57
	58
			Test A		Test B
			without waxes	with waxes	without waxes	with waxes
		Control sample (c)	1.34 ± 0.01	1.69 ± 0.07	1.43 ± 0.05	1.63 ± 0.06
		Vial (v)	<LOD	<LOD	-	-
		Membrane (m)	1.34 ± 0.02	1.61 ± 0.15	1.44 ± 0.05	1.61 ± 0.05
		c -(v + m)	n.s.	n.s.	n.s.	n.s.
	59	n.s. -not significant
	60
	61
	62
	63
	64
	65
	66
	67
	68
	69
	70
	71
	72
	73
	74
	75

Table 5 .

 5 Results of Tests C (sunlight photodegradation) for the famoxadone active ingredient, 76

	77	expressed in µg cm -2 ± SD.
	78
			Test C
			without waxes	with waxes
		t 1/2 (days)	4.41	4.26
		R 2	0.77	0.99
	79
	80
	81
	82
	83
	84
	85
	86
	87
	88
	89
	90
	91
	92
	93
	94
	95
	96

Table 6 .

 6 Results of Tests C (sunlight photodegradation) for the commercial product Equation Pro, 97

	98	expressed in µg cm -2 ± SD.
	99
			Test C
			without waxes	with waxes
		t 1/2 (days)	3.75	4.61
		R 2	0.91	0.97
	100
	101

1 Residues

of the fungicide famoxadone in grapes and its fate during wine

	1 26
	2 27	production
	3 28	Abstract
	4	Susana de Melo Abreu † , Pierluigi Caboni ‡ , Filippo Maria Pirisi ‡ , Paolo Cabras ‡ ,
	5	Arminda Alves † , & Vincenzo Luigi Garau * ‡
	6 7 8	† LEPAE-DEQ, Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. F o r
	9	‡ Dipartimento di Tossicologia, Università di Cagliari, Via Ospedale, 72, 09124 Cagliari, Italy. P
	10	e
	11	* Correspondence: Prof Vincenzo Luigi Garau. E-mail: vlgarau@unica.it e r
	12 13 14 15 16 17	R e v i e w
	18 19	O n l
	20	y
	21
	22
	23
	24
	25
		2

Table 1 .

 1 Concentration of famoxadone during grapes maturation and half-life time..

	257
	258

Table 2 .

 2 Fate of famoxadone during wine production.

	322
	323

Table 3 .

 3 Results of Tests A (evaporation and thermodegradation) and B (codistillation) for the 338 famoxadone active ingredient, expressed in mg kg -1 ± SD.

	339
	340
			Test A		Test B
			without waxes	with waxes	without waxes	with waxes
		Control sample (c)	2.00 ± 0.06	1.40 ± 0.34	2.10 ± 0.20	1.70 ± 0.15
		Vial (v)	<0.02 (LOD)	<0.02 (LOD)	-	-
		Membrane (m)	2.00 ± 0.26	1.40 ± 0.29	1.90 ± 0.33	1.80 ± 0.10
		c -(v + m)	n.s.	n.s.	n.s.	n.s.
	341	n.s. -not significant
	342
	343
	344
	345
	346
	347
	348
	349
	350
	351

Table 4 .

 4 Results of Tests A (evaporation and thermodegradation) and B (codistillation) for the 358 commercial product, expressed in mg kg -1 ± SD.

	359
	360
			Test A		Test B
			without waxes	with waxes	without waxes	with waxes
		Control sample (c)	1.34 ± 0.01	1.69 ± 0.07	1.43 ± 0.05	1.63 ± 0.06
		Vial (v)	<0.02 (LOD)	<0.02 (LOD)	-	-
		Membrane (m)	1.34 ± 0.02	1.61 ± 0.15	1.44 ± 0.05	1.61 ± 0.05
		c -(v + m)	n.s.	n.s.	n.s.	n.s.
	361	n.s. -not significant
	362
	363
	364
	365
	366
	367
	368
	369
	370
	371

Table 5 .

 5 Results of Tests C (sunlight photodegradation) for the famoxadone and commercial 378 product, expressed in days ± standard error (SE).

	379
	380
	381
	382
	383
	384
	385
	386
	387
	388
	389
	390
	391

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Food Additives and Contaminants

September 2, before and after fungicide treatment. Then, the grapes were collected after 3, 7, 14,

and 28 days. In the wine production, each grape sample was divided in two portions, both of

Test B -Co-distillation. A regenerated cellulose membrane prepared in the same way of the Test A

was placed on the top of a 10 ml dark vial, with a screw-closed cap with a hole in the middle,

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

Strnberg JA, Tang XS. 1999. Mode of action of famoxadone. Pesticide Science 55: 105-118.

Acknowledgements 259

The authors wish to thank the FCT -Fundação para a Ciência e Tecnologia, in Portugal, for 260 financial support (SFRH/BD/4648/2001). 261

Sample

Concentration (mg kg -1 or mg l