Sonia Marín

Hector Morales

Hassan Ahmed

Hassan A H Hasan

Antonio J Ramos

Vicent ; Sanchis

Antonio ; Ramos

A J Ramos

Patulin distribution in the tissue of Penicillium expansum-contaminated Fuji and Golden apples

Keywords: Chromatography -HPLC, Clean-up, Extraction, Mycology Additives/Contaminants: Mycotoxins -fungi, Mycotoxins -other Food Types: Fruit, Fruit juices patulin, apple, Penicillium expansum, trimming, storage 25

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

Patulin is a mycotoxin produced by a number of fungi common to fruit-and vegetable 28 products, most notably apples. Patulin production is usually, but not exclusively, 29 associated with apple soft rot and blue mould rot, most commonly caused by 30 Penicillium expansum (Brian et al. 1956). Within the food industry, apples and their 31 respective products are of greatest concern for patulin contamination. Numerous 32 studies around the world have examined the extent and degree to which apple 33 products have been contaminated by patulin, finding high percentages of apple juice 34 samples contaminated by patulin in levels exceeding the 50 ng g -1 limit imposed by the 35 EC (Comission Regulation 1425/2003) [START_REF] Moake | Comprehensive review of patulin 354 control methods in foods[END_REF].

37

The health risks posed by patulin necessitate its control and removal from apple 38 products, creating a demand for food-processing techniques capable of doing so, 39 preferably at low cost for industry [START_REF] Moake | Comprehensive review of patulin 354 control methods in foods[END_REF]. In many countries apple juice is 40 typically a by-product produced from culled apples unfit for other, higher quality and 41 higher profit, purposes. Bruises, skin breaks, and other physical damage within these 42 apples provide a perfect entry for P. expansum and other patulin-producing species 43 into the fruit [START_REF] Moake | Comprehensive review of patulin 354 control methods in foods[END_REF]. Low quality apples originating from storage rooms 44 (either controlled atmosphere (CA) or not) are also usually employed for apple juice 45 production. As a result, the use of low quality raw material leads to highly patulin 46 contaminated apple juices. In Spain, juice concentrates are mainly produced from 47 Golden apples, although Fuji apples are sometimes used when they are not suitable for 48 fresh consumption. The exposition of these P. expansum infected apples intended for 49 juice production to room temperature for short periods of time during postharvest 50 operations has been shown to be critical for the accumulation of patulin (Marín et al.

98

Apples were stored at 20°C. For each variety, half of the apples were removed from 99 incubation when their lesions were about 2-cm diameters (5 days), and the remaining 100 ones, when the lesions were 4-cm wide (7-8 days). Incubation at 20°C was carried out 101 to simulate the interval of time that apples may be at room temperature before with the aid of a die-cut. From each cylinder, 0.5 cm sections were cut across starting 107 from the surface of the apple (Figure 1); when no lesion was observed in one section, 108 one more section was taken in depth. From all the sections, those visible decayed were 109 divided in two samples: sound and rotten, while the sound sections were considered as 110 a single sample. The same procedure was applied to 4-cm lesions, although in this 111 case the cylinder had 5.5 cm of diameter. Eight ml distilled water were added to the sound/decayed samples. A "Polytron" unit of 117 Kinematica AG (Swizerland) was used to pulp them. Puree samples were 118 enzymatically treated to remove pectins and allowed to react for 1 h at 40°C. After that,

119

samples were centrifuged at 6000 rpm for 5 min and 5 ml of the supernatant were 120 analysed following AOAC method 995.10 ( [START_REF] Brause | Determination of patulin in 315 apple juice by liquid chromatography: Collaborative study[END_REF]

150

An initial analysis of the variance of the whole set of data revealed that the 151 concentration of patulin in both the decayed and sound portions significantly depended 152 on lesion diameter, apple variety, and on degree of ripeness for Fuji apples (Table II).

153

Patulin concentrations found in the sections were significantly higher for 2-cm lesions 154 than for 4-cm ones. Variety was the main factor affecting patulin content in the sections 155 analysed, due to the markedly higher concentration found in Golden than in Fuji For apples with a 2-cm lesion, Golden ones had mean concentrations of 9.67 and 0.28 164 µg g -1 , in the decayed portion and surrounding one, respectively. For Fuji apples these 165 concentrations were 3.31 and 0.13 µg g -1 , respectively (Figure 2). No significant 166 difference due to different degree of ripeness was observed as a single factor, 167 however, under ripe Fuji apples yielded significantly higher concentrations of patulin in 168 both sound and decayed sections. For both sound and decayed sections, most patulin 169 was found in the upper section, with the second section containing significantly less, and extent and levels of diffusion of patulin in both sound and decayed tissue 173 depended on the variety.

175

For apples with a 4-cm lesion, Golden ones had mean concentrations of 4.98 and 0.11 Total patulin contents found in each apple confirmed, besides the high variability of the 187 results, the higher accumulation of patulin in Golden apples, and the lower 188 accumulation in Fuji ones, mainly when they were riper (Table IV). Interestingly, total 189 levels were quite similar for 2 and 4 cm lesions, due to the lower concentrations found 190 in the sections in the 4 cm assay. Looking in detail at the results summarised in Fig. 2 191 and 3, it was observed a decrease in concentration in the first two sections in both 192 sound and rotten tissue, and an increase in the concentration in the subsequent 193 sections, plus the additional sections analysed and found to be positive in the 4-cm 194 assay.

196

Regarding diffusion, patulin found in healthy tissue accounted for 2-6% of the total, 197 more diffusion being observed for Fuji apples, and in under ripe apples for both 198 varieties. In general, patulin was always found in the rings surrounding the lesions, and 199 also in the 0.5-cm section next to the last section where rot symptoms were observed.

201

[insert table IV about here] 202 203 2-cm diameter lesions resulted in accumulated patulin to a depth of 1.5 cm, or even to 204 2 cm for under ripe Golden apples (Figure 4). For 4-cm diameter lesions, patulin was 205 found at 2 and 2.5 cm depth for ripe and under ripe Fuji apples, respectively, and to 2.5 206 and 3 cm for ripe and under ripe Golden apples, respectively. At a given lesion size in 207 the surface of the apples, the depth of the lesions reached in Golden apples was in 208 general higher than in Fuji ones. Patulin is a common contaminant of ripened apples used for the production of apple 214 juice concentrates [START_REF] Sydenham | The influence of deck storage and initial processing on patulin levels in apple 374 juice[END_REF]. No patulin has been detected in cider 215 pressed from tree-picked fruit (culled or not culled). In contrast, cider produced from 216 ground-harvested fruit contained up to 374 µg l -1 , depending on the varieties tested 217 [START_REF] Jackson | Apple quality, storage, and washing treatments affect patulin levels in apple 334 cider[END_REF]. No patulin was detected in Golden apples after storage periods 218 of 1-2.5 months of storage at 1°C (under CA or not) (Morales et al. 2006a[START_REF] Morales | Patulin accumulation in 362 apples during postharvest: Effect of controlled atmosphere storage and fungicide 363 treatments[END_REF].

219

Cold stored apples yielded free of patulin ciders if culled, but levels 0-64 µg l -1 if not 220 culled [START_REF] Jackson | Apple quality, storage, and washing treatments affect patulin levels in apple 334 cider[END_REF]. The removal of fungally decayed and the surrounding 221 tissue from apples, prior to further processing, has been reported to reduce significantly 222 patulin levels in juiced products [START_REF] Lovett | Trimming as a means of removing patulin 341 from fungus-rotted apples[END_REF]Sydenham et al. 1995).

224

In the present study, brown, watery rot increasing depth in rot as the lesion diameter 225 increased was observed at 20°C. In agreement with [START_REF] Laidou | Diffusion of 337 patulin in the flesh of pears inoculated with four post-harvest pathogens[END_REF], an increase in 226 lesion diameter corresponded to an increase in depth of penetration of the fungi.

227

Stand-by steps at room temperature (e.g. 20°C) prior juice concentrates manufacture

231

Patulin concentrations found in the sections were significantly higher for 2-cm lesions 232 than for 4-cm ones, although the overall amount of patulin was similar in both cases. In Although high patulin concentrations were found in apples after 5 days, a decrease of 240 concentration found in the rotten tissue was observed after 7-8 days, for both varieties.

241

In rotten Golden delicious apples, in the decayed tissue, after a 2-days induction 242 period, patulin increased and, after running through a maximum of 185000 ng g -1 after 243 5 days, decreased to 0.060 ng g -1 after 15 days [START_REF] Rychlik | Model studies on the diffusion behaviour of the 365 mycotoxin patulin in apples, tomatoes, and wheat bread[END_REF]. Patulin 244 metabolisation by fungal enzymes has been proposed [START_REF] Damoglou | Some factors governing the production 322 of patulin in apples[END_REF]. In their 245 study, however, although patulin was significantly lower in the rotten area after 5 days, 246 its content in juice made from the whole fruit remained on a high level of 1700 ng g -1

247

until the tenth day of incubation, before it finally decreased; this is in accordance with 248 the trend observed in this study, because total patulin content in apples was similar for 249 5 and 8 days of incubation. This phenomenon may be attributed to the exponential 250 increase of the rotten part by volume during fungal invasion and to patulin diffusion into 251 the sound tissue [START_REF] Rychlik | Model studies on the diffusion behaviour of the 365 mycotoxin patulin in apples, tomatoes, and wheat bread[END_REF].

253

Our results showed that between 2 and 6% of total patulin was found in the sound 254 tissues of apples. The total patulin detected in apparently sound tissues of pears 255 inoculated with P. expansum was nearly 25% of the total pear tissues (diseased and 256 healthy) [START_REF] Laidou | Diffusion of 337 patulin in the flesh of pears inoculated with four post-harvest pathogens[END_REF]. Less than 1% of the total patulin was found in sound 257 tissues of apples inoculated with P. expansum [START_REF] Harwing | Occurrence of patulin and 329 patulin-producing strains of Penicillium expansum in natural rots of apple in Canada[END_REF]. A similar study 258 was carried out in P. expansum inoculated pears kept at room temperature (18-20°C)

259

till lesions expanded to 10 mm in diameter; in this case 6 mm thick sections were 260 obtained. Patulin was detected in the first three sections at concentrations of 288000, 261 144000 and 144000 ng g -1 from outside to inside of fruits. Patulin could diffuse through 262 6 mm in sound tissues [START_REF] Laidou | Diffusion of 337 patulin in the flesh of pears inoculated with four post-harvest pathogens[END_REF]. Apples inoculated with P.expansum

296

As a conclusion, patulin accumulation can occur in a short period of time in P.

297

expansum infected apples left at room temperature and attain dramatically high levels.

298

The concentration of patulin seems to be dependent on apple variety and, in particular, 299 on acidity, more acidic conditions leading to a higher production of patulin. In addition, 300 trimming just the rotten tissue is not enough to exclude all patulin, but trimming apple 301 tissue around the rotten part may be a good preventative practice for industry using 302 apple products. Accordingly, [START_REF] Lovett | Trimming as a means of removing patulin 341 from fungus-rotted apples[END_REF] removed more than 93% of patulin by 303 trimming effective apple tissue from mould-infected rotten apples. One more conclusion 304 is that washing with a high-pressure water spray, is a good control method, but not 305 sufficient to totally eliminate the toxin as it may remove just the soft rotten tissue.

306

Consequently, either trimming or culling should be considered as a routine practice in 307 industries using apples as raw materials.

  apple inoculation and incubation 92 Petri dishes of PDA were point inoculated with the strain and incubated at 25°C until 93 sporulation. A conidial suspension was prepared in Tween 80 (0.005% (v/v)) in sterile 94 water and adjusted to 10 6 conidia ml -1 . Apples were wounded 2 mm deep with a 2 mm 95 diameter needle twice in two opposite sides. Both points were inoculated with 20 µl of 96 the conidial suspension.

128

  Regarding repeatability the S r of five determinations (25 ng g -1 ) carried out under 129 repeatability conditions was 0.005. Finally the limit of detection of the analysis was 130 10 ng g -1 (depending on the initial amount of sample) for the sound 131 samples and between 3 and 100 ng g -1 for the decayed ones.132 133Statistical analysis of the results134The whole experiment was designed as a full factorial one, with 4 factors to be tested 135 on the patulin accumulated by P. expansum: apple variety, degree of ripeness, size of 136 lesion, depth from the surface of the apple. The variables recorded were concentration 137 of patulin both in the decayed and the sound samples from each section. All treatments 138 were repeated three times. Patulin concentrations for each treatment were evaluated 139 by analysis of variance using the SAS version 8.2 (SAS Institute Inc., Cary, NC, USA). 140 Significant differences (P<0.05) in parameters were tested by a Duncan's Multiple 141 patulin (µg) in each section and for both healthy and decayed 144 fractions was calculated taking into account both patulin concentration and the weight 145 of each sample. Percentage of difused patulin was calculated by adding the patulin 146 contained in the healthy fractions of the sections and dividing by the total patulin found 147 in the decayed fractions of the sections. 148 149 Results

  156apples, regardless of the other factors tested. For the patulin content in the sound 157 ripeness had some influence on the results, depending on variety 158 and lesion size. Data were thus analysed separately for each lesion size tested (2 and 159 4 cm diameter) (

  228have been shown to be a crucial point in patulin accumulation prevention(Morales et 229 al. 2006a).

  263showed diffusion of patulin up to approximately 10 mm into the surrounding healthy 264 tissues; most of the patulin being present in the first 1 cm of the lesion (6.5 to >10 000 265 ground for a certain period of time, and to any other harvested apples which 293 undergo any standby period at room temperature before processing, as long as they 294 have been infected.

Figure. 1 . 4 Figure 2 .Figure 3 .Figure 4 .

 14234 FIGURE CAPTIONS

. Patulin was extracted 121 with ethyl acetate and then cleaned up by extraction with sodium carbonate solution. 122 Extracted samples were dried with anhydrous sodium sulphate. After evaporation of 123

	124	ethyl acetate, patulin was re-suspended in water pH 4 (adjusted with acetic acid) and
	125	determined by reversed-phase LC with UV detection. The mobile phase was
	126	water+tetrahydrofuran (100+0.8) at a flow rate of 1.2 ml min -1 . Patulin was detected at
	127	UV at 276 nm wavelength. The recovery rates obtained by spiking apple pieces with
		10, 40, and 100 ng patulin g -1 in duplicate were of 96.2, 88.5 and 69.1%, respectively.

  Table III).

	160
	161
	162	[insert table II and III about here]
	163

Table I .

 I Relevant quality parameters for the two different lots of Golden and Fuji apples. Values are the mean of three determinations made for 3 different apples. For each row, different letters mean significant differences among the mean values of the

	measured parameter.
	Quality parameters			Golden		Fuji
	Firmness (kg) Soluble solids F o r	Lot A 6.77b 14.8a	Lot B 4.74c 14.7a	Lot A 7.85a 14.5a	Lot B 7.55a 15.17a
	concentration (°Brix)	P
	Acidity (g malic acid l -1 )	10.68a e	9.03b	6.88c	5.36d
				e r
				R
				e
				v i e
					w
					O n l
						y

Table II .

 II Analysis of variance of patulin concentration found in decayed and sound sections of apples.

	Source of variation			Decayed samples	Sound samples
			DF	MS	F	MS	F
	Ripeness (R) Variety (V) Depth section (D) F o r	1 1 5	18.26 1731.39 1320.05	0.53 50.01** 38.13**	0.06 1.05 1.92	1.25 23.90** 43.50**
	Lesion size (L)	P	1	1045.36	30.20**	1.87	42.40**
	R x D	5 e	23.23	0.67	0.01	0.23
	R x V V x D	1 5 e r 169.44 360.11	4.89* 10.40**	0.36 0.45	8.10** 10.27**
	L x R		1	33.83 R	0.98	0.00	0.09
	L x V		1	99.07 e	2.86	0.23	5.33*
	L x D R x V x D		4 4	859.79 9.81 v 24.84** 0.28 i e	1.05 0.15	23.78** 3.39*
	L x R x D		3	6.20	0.18 w	0.00	0.01
	L x R x V L x V x D L x V x D x R		1 4 3	59.60 210.76 31.60	1.73 6.09** 0.91 O 0.40 0.18 0.17 n l	9.02** 4.09** 3.96**
	**P<0.01					y
	*P<0.05

Table III .

 III Analysis of variance of patulin concentration found in decayed and sound sections of apples; separate analysis are shown for each lesion diameter.

	Source of		Decayed samples		Sound samples
	variation
	2 cm MS 33.85 0.79 F r DF 1 F Ripeness (R) o	4 cm MS 1.70 0.12 0.02 0.27 2 cm F MS F	4 cm MS 0.05 8.59* F
	Variety (V)	1 1457.5 P	34.02	554.62 37.55	1.27 18.88* 0.16 26.82*
			3 e	*		*
	Depth section (D) 5 2346.3 5 e 54.77 * r	156.06 10.57 *	3.06 45.30* 0.15 25.65*
	R x D	5	24.97 0.58 R 5.67	0.38 0.01 0.11	0.01	1.05
	R x V	1 151.66 3.54* 12.58 0.85 0.51 7.50* 0.00 e	0.17
	V x D	5 559.37 13.06 *	57.96 3.92* 0.56 8.35* 0.05 9.02* v i e
	R x V x D	5	38.84 0.91	3.59	0.24 0.26 3.91* 0.01 w	1.29
	*P<0.05					O n l
							y

Table IV .

 IV Mean total patulin (µg) found for each treatment.

	Page 25 of 26			Food Additives and Contaminants
	Rotten	Apple	Ripeness Mean total patulin ± SD	% in decayed	% Diffused
	zone	variety				portion
	2 cm	Fuji	Lot A		33.44±10.53	94	6
		Lot B Golden Lot A Lot B F o r	15.46±5.07 65.10±24.49 93.71±61.32	96 96 97	4 4 3
	4 cm	Fuji	Lot A P	39.12±22.51	94	6
			Lot B	e	16.20±7.55	94	6
		Golden Lot A Lot B	115.89±84.47 91.75±63.90 e r	96 98	4 2
					R
					e
					v i e
					w
						O n l
						y

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titrated with a 0.1 mol l -1 NaOH solution. The acidity was expressed in grams of malic

and finally levels of patulin found in the remaining sections were not significantly

different. Due to the higher patulin levels produced in Golden apples, the distribution,

of more than 2 cm from the infected zone (83700 ng g -1 ) in Golden delicious apples

storage, the results may also be applied to ground-picked P.expansum infected apples,

Brian PW, Elson GW, Lowe D. 1956. Production of patulin in apple fruits by Penicillium

0.5 cm 0.5 cm

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


Marín, S Neus ; Bellí

Vicent ; Sanchis

Sanchis V Ramos

Dr Antonio J Ramos
email: ajramos@tecal.udl.es

Impact of fungicides on Aspergillus carbonarius growth and ochratoxin 1 A production on synthetic grape-like medium and on grapes 2 3

Keywords: fungicides, A. carbonarius, ochratoxin A, grapes. 40

evaluate the impact of the application of several fungicide 16 treatments used in Spanish vines, on Aspergillus carbonarius growth and ochratoxin

and 30 ºC; ii) find out the minimum inhibitory concentration (MIC) of each fungicide 20 for A. carbonarius growth on synthetic medium, and iii) investigate the effect of several 21 fungicides on A. carbonarius-inoculated grapes. In synthetic medium, nine fungicides 22 significantly reduced A. carbonarius growth rate, meanwhile 13 completely inhibited its 23 growth. In general, growth was faster at 30 ºC than at 20 ºC, contrary to OTA 24 production. Fungicides that stopped fungal growth also inhibited OTA production, but 25 not all the fungicides that reduced growth reduced the OTA synthesis. In general, 26 fungicides that contained copper or strobilurins reduced both growth and OTA 27 production, contrary to sulfur fungicides. At the optimum temperature for A. 28 carbonarius growth of 30 ºC, higher amounts of fungicide were needed to prevent 29 fungal growth than at 20 ºC. Among the fungicides that inhibited A. carbonarius growth 30 on SNM at the initial doses, cyprodinil seemed to be the active ingredient more 31 effective to stop fungal growth when testing reduced doses. Fungicide effect on grapes 32 was similar to that on synthetic medium. Both infection and OTA production were 33 reduced when using cyprodinil (37.5%) + fludioxonil (25%) and azoxystrobin (25%). 34 Penconazole (10%) also showed a clear reduction in OTA production at both 35 temperatures, although infection was only reduced at 20 ºC. OTA reduction was strain 36 and temperature-dependent. In general, fenhexamid (50%), mancozeb (80%) and copper 37 hydroxide (80%) + copper (50%) enhanced infection and OTA production. 38 Introduction 42

43

Ochratoxins are fungal secondary metabolites produced mainly by fungi from the 44 genera Aspergillus and Penicillium, which are present in a wide variety of foods. 45

Ochratoxin A is one of the more studied mycotoxins in wines nowadays, being recently 46 regulated in the European Union (Commission Regulation 123/2005), mainly due to its 47 high toxicity and its presence in wines over the world. Its production in grapes from 48

Mediterranean area is associated with different Aspergillus spp., mostly black aspergilli 49 and among them, A. carbonarius [START_REF] Cabañes | What is the source of ochratoxin A in wine?[END_REF], Battilani et al. 2003, Bellí et al. 50 2004a). 51 52 Prevention of growth of mycotoxin-producing fungi is the most effective strategy for 53 controlling the presence of mycotoxins in foods. This could be achieved by knowing the 54 critical limits of different eco-physiological factors affecting fungal infection and 55 mycotoxin synthesis, but in many cases, the use of fungicides is the only efficient, cost-56 effective, and often successful way to prevent the mould growth (Munimbazi et al. 57 1997). 58

59

The aim of this study was to evaluate the impact of the application of several fungicides 60 to grapes, on A. carbonarius growth and OTA production. The experimental design was 61 divided in three parts in order to: i) screen the main fungicides used in Spanish vines, to 62 test their efficiency against A. carbonarius growth and OTA production on synthetic 63 nutrient medium (SNM); ii) find out the minimum inhibitory concentration (MIC) of 64

Material and methods 68

Screening of the main fungicides used in vines on synthetic grape-like medium 69 Three ochratoxigenic A. carbonarius strains (3.161, 3.162 and 3.168, grape-isolated 70 from Italy, France and Spain, respectively), were used to centrally inoculate (10 6 spores 71 ml -1 ) Petri dishes containing 20 ml of synthetic nutrient medium (SNM), which had a 72 composition similar to grapes, and a water activity (a w ) level of 0.99 a w (Bellí et al. 73 2004b). Strains are held in the culture collection of the Food Technology Department, 74 University of Lleida, Spain. 75 76 Twenty-six fungicides commonly used on Spanish vines, were added to the medium at 77 the doses recommended by manufacturers (Table 1). Each fungicide was aseptically 78 added to the autoclaved medium before plating it. No fungicides were added to control 79 plates. Plates were incubated at 20 ºC and 30 ºC inside plastic bags. Colony diameters 80 were measured after 3, 5 and 7 days and OTA was extracted after 7 days following the 81 method of [START_REF] Bragulat | An easy screening 368 method for fungi producing ochratoxin A in pure culture[END_REF]. 82

High performance liquid chromatography (HPLC) with fluorescence detection (Waters 83 474, Milford, Massachusetts, U.S.A.) (λ exc 330 nm; λ em 460 nm) was used for OTA 84 analysis. The mobile phase was acetonitrile-water-acetic acid (57:41:2) (1.0 ml min -1 ) 85 and a C 18 column (Waters Spherisorb 5 µm, ODS2, 4.6x250 mm) was used. The 86 injection volume and the retention time were 25 µl and 7.1 min, respectively. The 87 detection limit of the analysis was 0.02 µg OTA g -1 of SNM, based on a signal-to-noise 88 All the fungicides that prevented A. carbonarius growth at the doses recommended by 97 the manufacturers in the previous experiment (n=13) were selected for this study in 98 order to find the minimum concentration that inhibited the growth of this mould (MIC). 99 Spore suspensions of 3.162 and 3.168 A. carbonarius strains were adjusted to contain 100 approximately 10 6 spores ml -1 for use as inoculum. SNM plates (0.99 a w ) with 101 decreasing concentrations of those fungicides (D, dose recommended by the 102 manufacturer; d1, 0.75xD; d2, 0.5xD; d3, 0.25xD; d4, 0.1xD; d5, 0.01xD and d6, 103 0.005xD) were single-point inoculated and incubated at 20 ºC and 30 ºC. Growth was 104 measured daily during a period of 30 days. Three repetitions were carried out. 105 106

F

Effect of fungicides on grapes 107

The effect of six fungicides applied directly to grapes was investigated. F3, F14 and F26 108 were used at the dose recommended by the manufacturer and F1, F9 and F23 were used 109 at d4 (0.1xD). 6 ml of water to keep a high relative humidity (90-100%) throughout the experiment. In 114 control treatments, grapes were dipped in water instead of fungicide. For culturing, 10 3 115 spore ml -1 suspensions of two A. carbonarius strains (3.162 and 3.168), were sprayed 116 onto the grapes. After 7 days of incubation at 20 ºC and 30 ºC, the percentage of grapes 117 infected by A. carbonarius was assessed. The whole set of grapes of each treatment 118 were crushed, and filtered through Whatman nº1 filter paper under vacuum. OTA was 119 extracted from this must following the method of [START_REF] Bezzo | A method for the 364 determination of specific mycotic contaminants random occurring in wines[END_REF]. Twenty-five µl of 120 each sample were injected into the HPLC system equipped with a fluorescence detector 121 (Waters 474, Milford, Massachusetts, U.S.A.) (λ exc 230 nm; λ em 458 nm) and a C 18 122 column (Waters Spherisorb 5 µm, ODS2, 4.6x250 mm). The analysis was performed 123 under isocratic conditions, with acetonitrile 48% -sodium acetate 4mM/acetic acid 124 (19/1)-52%, as the mobile phase, pumped at a flow rate of 1 ml min -1 . The injection 125 volume and the retention time were 25 µl and 12 min, respectively. The detection limit 126 of the analysis was 0.05 µg l -1 , based on a signal: noise ratio of 3:1. OTA was quantified 127 by the external standard method. The ochratoxin standard was from Aspergillus 128 ochraceus (Sigma-Aldrich, Steinheim, Germany). The standard solution was made in 129 methanol and confirmed by using an UV spectrophotometer. 130 131

Statistical treatment of the results 132

The regression lines of colony diameters against days after inoculation were calculated 133 for each fungicide and were used to obtain the growth rate under each treatment 134 conditions. Fungicide effect on mycelial growth on SNM and OTA production, both in 135 medium and in natural grapes, were statistically analysed with SAS Enterprise Guide 136 software (SAS Institute, version 2.0, Inc., Cary, N.C., U.S.A.) by analysis of variance 137 Screening of the main fungicides used in vines on synthetic grape-like medium 142 Significant differences were detected for the single factors temperature and fungicide 143 and their interaction, while all the strains showed statistically similar growth, regardless 144 of the assayed levels of the remaining factors. Growth was faster at 30 ºC than at 20 ºC, 145 except for F2 and F11. Nine fungicides significantly reduced A. carbonarius growth 146 rate at both temperatures (F2, F5, F7, F11, F13, F14, F20, F21 and F26) in comparison 147 with the control treatment, meanwhile thirteen fungicides completely inhibited fungal 148 growth at the dose assayed (F1, F4, F6, F9, F10, F12, F15, F16, F18, F19, F23, F24 and 149 F25) (Table 2). No significant effects were observed for the remaining four fungicides 150 on growth. 151 152

The fungicides had a significant effect on OTA production by A. carbonarius, because 153 obviously the 13 fungicides that prevented fungal growth also inhibited OTA 154 production. Among the remaining fungicides, analysis of variance showed that none of 155 them reduced significantly OTA production. No significant differences were found 156 among the isolates tested, either in their response to temperature and fungicide 157 treatments. The interaction fungicide x temperature was also significant. Contrary to the 158 growth pattern, OTA production was in general higher at 20 ºC than at 30 ºC. Although 159 not having a significant weight, general trends can be drawn from the results. Mean 160 levels of OTA production showed that most of the fungicides that reduced A. 161 carbonarius growth, also reduced OTA production, with the exception of F2, F5 and 162 the addition of F7 at 20 ºC and F14, F21 and F26 at 30 ºC. OTA was also higher than 164 the control under the effect of F8 at both temperatures although growth was only 165 stimulated at 30 ºC. Contrary, F17 reduced OTA production although growth was 166 favoured at 20 ºC. OTA was favoured at 20 ºC and 30 ºC under the effect of F22, the 167 unique fungicide that increased fungal growth at both temperatures. Figure 1 compares 168 the growth rate and the amount of OTA detected at both temperatures after the 169 application of each fungicide with the control treatment, which is represented at the 170 origin of coordinates. Control growth and OTA production detected after the application 171 of each fungicide has been subtracted from growth and OTA production by the control 172 treatment. Thus, fungicides in the third quadrant of the graphic resulted in a reduction of 173 both growth and OTA production. 174 175 [Insert table 3 and figure 1 about here] 176 177 Minima doses preventing A. carbonarius growth on synthetic grape-like medium 178 No significant differences were found between the two strains of A. carbonarius tested 179 (data not shown). At the optimum temperature for A. carbonarius growth, 30 ºC, higher 180 concentration of fungicide was needed to prevent fungal growth than at 20 ºC. Each 181 fungicide had a different effect on A. carbonarius growth, but most of them were 182 effective at doses around 1/4 th (d3) of the dose recommended by the manufacturer (D) 183 (Table 3). Only three fungicides did not prevent growth at this dose: F4 at D (d1) and 184 F1 and F16 at 0.50 x D (d2), in the assays at 30 ºC. 1/10 th of D (d4), was the MIC of 185 fungicides F4 and captan 50% (F18) at 20 ºC, and fludioxonil (F6) and tolyfluanid 186 (F15) at both temperatures. A mixture of cyprodinil 37.5% and fludioxonil 25% (F9) 187 Half of the fungicides (penconazole 10%, azoxystrobin 25% and cyprodinil production at both temperatures, in comparison with the control treatment (Figure 3). 217

For these fungicides, reduction was higher at 20 ºC than at 30 ºC, except for 218 penconazole 10% (F1) for 3.162 strain. Differences in the percentage of reduction were 219 also observed between both strains, especially at 30 ºC, where fungicides were more 220 effective against strain 3.168. At 20 ºC, fenhexamid 50% (F14), mancozeb 80% (F23) 221 and copper hydroxide 80%+copper 50% (F26) reduced the OTA production for strain 222 3.168 meanwhile they produced the opposite effect on strain 3.162, increasing OTA 223 production more than 200 % sometimes. At 30 ºC, these three fungicides increased up 224 to 20 % OTA production of both strains. the control of other food-spoilage fungi like Fusarium spp. [START_REF] Mathies | Investigations on the action of different 399 active ingredients on the biosynthesis of mycotoxins in Fusarium culmorum and F. 400 graminearum. Modern fungicides and antifungal compounds[END_REF]Buchenauer 231 1996, Moss and[START_REF] Moss | The influence of the fungicide tridemorph on T-2 409 toxin production by Fusarium sporotrichioides[END_REF], Aspergillus flavus and A. ochraceus (Munimbazi et al. 232 1997), Botrytis cinerea [START_REF] Slawecki | Novel fungitoxicity assays 423 for inhibition of germination -associated adhesion of Botrytis cinerea and Puccinia 424 recondite spores[END_REF], etc, but not any studies on grapes. 233

Existing earlier studies have shown that combinations of Euparen (a sulfamide type 234 fungicide) and Mycodifol [START_REF] Karadimcheva | Effect of some fungicides on the epiphytic microflora in 392 grapes[END_REF], or captan [START_REF] Tandon | Chemical control of 431 decay of fruit of Vitis vinifera caused by Aspergillus niger and Penicillium sp[END_REF] were 235 found to be effective against black aspergilli colonising grape berries. Data on 236 resistance of A. carbonarius to fungicide treatments is non-existent so far. Therefore, 237 To discuss the results obtained, the different fungicides may be grouped according to 243 their active ingredients. A. carbonarius growth and OTA production were minimised 244 when using fungicides with copper in their composition (F20, F21, F24, F25 and F26). 245 However, copper oxychloride was also present in F5, which although limiting A. 246 carbonarius growth, did not decrease OTA production. The same OTA-enhancing 247 effect was detected for fluazinam (F2) and procymidone (F11), which were classified as 248 dinitro aniline and dicarboximide fungicides, respectively. It is known that fluazinam 249 (F2), together with azoxystrobin (F3, F13 and F17), thifluzamide and carboxin can 250 interfere with respiration processes [START_REF] Corbett | The biochemical mode of 385 action of pesticides[END_REF], Guo et al. 1991, Sauter et al. 251 1995). Both A. carbonarius growth and OTA production were increased, although not 252 significantly, when adding inorganic fungicides containing sulfur to the medium (F8 253 and F22). Similar effects were detected in a study of the OTA content in red wines 254 produced from vineyards treated with different pesticides [START_REF] Lo Curto | 395 Ochratoxin A occurrence in experimental wines in relationship with different pesticide 396 treatments on grapes[END_REF]). The 255 level of OTA in wines from sulfur-treated grapes was higher than in the other samples. 256 Furthermore, those authors reported azoxystrobin as a fungicide able to reduce OTA 257 concentration in wine, with 96.5 % of reduction. Data on OTA concentration in wine 258 are not directly comparable to the screening in this study, because OTA concentration in 259 wine or grape accounts for total OTA. In our study, both OTA producing capacity must 260 be coupled to colony size data to have an idea of the total OTA accumulation. Thus, in 261 the present study, the total amount of OTA produced by A. carbonarius growing with 262 12 sulfur fungicides increased due to both the higher OTA-producing capacity detected and 263 the bigger diameters of the colonies. However, it is not clear whether the total amount 264 of OTA accumulated by A. carbonarius treated with fluazinam (F2) and procymidone 265 (F11) increased or not because OTA production stimulation occurred but, in contrast, 266 smaller colonies were observed. Some othe fungicides have been found to stimulate 267 OTA production in grapes (Battilani et al. 2003). 268 269Little is known about the mechanism of action of the active ingredients of the 270 fungicides assayed. For many compounds, spore germination is the growth stage that is 271 most sensitive to inhibition [START_REF] Slawecki | Novel fungitoxicity assays 423 for inhibition of germination -associated adhesion of Botrytis cinerea and Puccinia 424 recondite spores[END_REF]. In the present study, fungicides that 272 completely inhibited germination were enclosed in several groups according to their 273 active ingredients: amide (F12 -one component-, F15) and dicarboximide fungicides 274 (F12 -one component-, F18), triazol fungicides (F1 and F16), benzimidazole (F19) and 275 dithiocarbamate fungicides (F23, F24 and F25 -one component-), pyrimidine fungicides 276 (F9 -one component-and F10), phenylpyrrole fungicides (F6 and F9 -one component-), 277 etc. It would be interesting to study growth for longer periods in order to know if this 278 last group of fungicides at the doses assayed, totally inhibited growth or only prolonged 279 the lag phase of the mould. Doses of these fungicides were reduced in a subsequent 280 experiment in order to find the threshold dose preventing A. carbonarius growth. In 281 general, fungicides with the same active ingredients seemed to have similar effects 282 when reducing the doses. Triazol fungicides (F1 and F16) were the less effective 283 fungicides against A. carbonarius growth, as just reducing up to one quarter the initial 284 dose the inhibitory growth effect disappeared. Fungicides with the MIC at one quarter 285 of the initial one (F19, F23, F24 and F25), were classified as carbamate and Greek authors observed that pesticides such as Carbendazim and Chorus were 290 ineffective in controlling sour rot caused by aspergilli [START_REF] Papageorgiou | Aspergillus niger and A. carbonarius[END_REF]). However, 291 the application of Switch led to significant decrease in incidence of black aspergilli on 292 grapes. The fungicide Switch contains cyprodinil and fludioxonil which belong to the 293 pyrimidine and pyrrolnitrin classes of fungicides, respectively. Since the fungicide 294 Chorus contains cyprodinil and was ineffective against aspergilli, it was concluded that 295 fludioxonil was the active ingredient of Switch [START_REF] Papageorgiou | Aspergillus niger and A. carbonarius[END_REF]. 296 297 Sauter et al. (1995) noted that the group of fungicides containing the strobilurins, 298 blocked electron transport at the cytochrome bc 1 complex of the mitochondrial electron 299 transport chain, and therefore were extremely potent inhibitors of spore germination, but 300 much less active as inhibitors of mycelial growth. No germination-inhibitory effect of 301 fungicides grouped as strobilurin fungicides (F3, F13 and F17) was noticeable in this 302 study, as the three of them allowed A. carbonarius growth, although less than the 303 control treatment. Other reported fungicides that typically acted after germination in 304 filamentous fungi by strongly inhibiting mycelial growth, included antimicrotubule 305 agents (carbendazim and N-phenylcarbamates, which inhibited nuclear division (Suzuki 306 et al. 1984)), and inhibitors of ergosterol biosynthesis [START_REF] Buchenauer | Mechanism of action of triazolyl fungicides and related 372 compounds. Modern selective fungicides: properties, applications, mechanisms of 373 action[END_REF]. However, in 307 this study, benzimidazol (F19) and dithiocarbamate fungicides (F23, F24 and F25 -one 308 component-) showed a completely inhibition of germination as mentioned before. 309 310 On grapes, a mixture of cyprodinil (37.5 %) and fludioxonil (25 %) (F9) seemed the 311 best fungicide that controlled A. carbonarius growth and OTA production together with 312 and Mikal (containing fosetyl-Al and the dicarboximide folpel) were found to be the 319 most effective for lowering fungal colonization and OTA content of wines Fenhexamid 320 50% (F14) and copper 50% (F26) showed the same effects on synthetic nutrient 321 medium than on grapes, as they increased OTA production, especially at 30 ºC. 322

Temperature was a determinant factor and could influence in the effectiveness of the 323 fungicides. Results are in accordance with previous work, reporting optimum 324 temperatures for A. carbonarius growth and OTA production at 30 ºC and 20 ºC, 325 respectively [START_REF] Bellí | 360 Aspergillus carbonarius growth and ochratoxin A production on a synthetic grape 361 Page 15 of 28[END_REF]

  Impact of fungicides on Aspergillus carbonarius growth andochratoxin A production on synthetic grape-like medium and on grapes.

  A. carbonarius growth on SNM; iii) investigate the effect of several 65 fungicides on growth and OTA production of A. carbonarius inoculated on grapes. 66 67

  favoured toxin production at both temperatures. OTA was also favoured by 163

  (F10), were the most effective fungicides, as they hinder growth at 188 the minimum doses assayed (d5 and d6, respectively)completely inhibited A. carbonarius on SNM: penconazole 10% 194 (F1), cyprodinil 37.5% + fludioxonil 25% (F9) and mancozeb 80% (F23), and two 195 fungicides that reduced its growth at the initial doses assayed: fenhexamid 50% (F14) 196 and copper hydroxide 80%+copper 50% (F26), plus azoxystrobin 25% (F3) were 197 chosen for this study. The factors fungicide and temperature were significant in both 198 grape infection and OTA production experiments, and the factor strain only in the OTA 199 production trial (data not shown). The percentage of grapes infected by A. carbonarius 200 was calculated for each treatment. Infection was significantly higher at 30 ºC than at 20 201 ºC. A percentage of reduction of the percentage of infection for each treatment was 202 determined by comparison with the control. The average of the percentage of reduction 203 of both strains is shown in Figure2. Infection was reduced at both temperatures with 204 azoxystrobin 25% (F3) and cyprodinil 37.5%+fludioxonil 25% (F9), and with 205 penconazole 10% (F1) at 20 ºC. Maximum reduction (> 95 %) was achieved with 206 cyprodinil 37.5%+fludioxonil 25% (F9) at 20 ºC, followed by azoxystrobin 25% (F3) 207 (65 %) and penconazole 10% (F1) (59 %) at the same temperature. Around 10 % 208 reduction in the infection percentage was also detected with mancozeb 80% (F23) at 20 209 ºC. In general, fenhexamid 50% (F14), mancozeb 80% (F23) and copper hydroxide 210 80%+copper 50% (F26) enhanced A. carbonarius grape infection, especially at 30 ºC.

  has been concentrated on the development and use of fungicides for 230

  this mould of a range of fungicides designed to control other 238 species infecting vines, was tested in this study. Moreover, it has to be underlined, that 239 the doses proposed by the manufacturers and used in the present study, were also 240 designed for the control of other moulds. 241 242

  growth, as it was a component of the pyrimidine fungicides F9 (Switch) 288 and F10 (Chorus), which showed the minimum threshold concentrations. Oppositely, 289

  (F9), Scala (containing the pyrimidine fungicide pyrimethanil) 318

Figure 1 .Figure 2 .Figure 3 .

 123 Figure1. Growth rate (mm day -1 ) and OTA production (µg g -1 ) at 20 ºC and 30 ºC 457

Table grapes

 grapes

		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
	110	(Red Globe var.) were surface disinfected by dipping them
	111	in NaClO (0.1 % Cl) and ethanol (70 %) solutions for 30 seconds and excess of
	112	moisture was aseptically removed. Afterwards, 20 grapes were dipped in a fungicide
	113	solution for 30 seconds and were placed onto a grid inside plastic boxes containing 300

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	437	in Corinth raisin and wine-producing vineyards in Greece: population composition,
	438	ochratoxin A production and chemical control. Journal of Phytopathology, 152, 250-
	439	255.
		F o r F o r F o r F o r F o r F o r
		P P P P P P
		e e e e e e
		e r e r e r e r e r e r
		R R R R R R
	326	. Propitious levels of other environmental factors, such as e e e e e e
	327 328 329	humidity, could also interfere in the efficacy of the fungicides assayed, together with the reiterative application of the same fungicide, as it could modify the equilibrium in the v i e v i e v i e v i e v i e v i e ecosystem, enhancing other microorganisms development, as competing fungi are w w w w w w
	330 331 332	removed. Additional in vitro studies on grapes testing the whole range of fungicides are needed in O n l O n l O n l O n l O n l O n l y y y y y y
	333	order to find out the best active ingredients against A. carbonarius development and
	334	mycotoxin production. Afterwards, further studies of the in situ efficiency of pesticide
	335	treatments against A. carbonarius infection and OTA production in vines would be
	336	required.
		19 20

Table 1 .

 1 Fungicides used in the study, their composition, origin and doses recommended 440

	441	by the manufacturers.
	442
		Code Fungicide		Company	Composition	Dose
		F1	TOPAS		Syngenta	Penconazole 10% p/v	0.35 ml l -1
		F2	SHIRLAN 500SC	Syngenta	Fluazinam 50% p/v	2 ml l -1
		F3	QUADRIS		Syngenta	Azoxystrobin 25 % p/v	2.25 ml l -1
		F4	Experimental product Syngenta	CGA302130	2 ml l -1
		F5	CUPROCOL		Syngenta	Copper oxychloride 70% p/v 2 ml l -1
		F6	GEOXE		Syngenta	Fludioxonil 50%	0.5 g l -1
		F7	Experimental product Syngenta	CGA379438	1 g l -1
		F8	THIOVIT JET		Syngenta	Sulfur 80% WG	4 g l -1
		F9	SWITCH		Syngenta	Cyprodinil 37.5% +	1.8 g l -1
						Fludioxonil 25%
		F10	CHORUS 50 WG	Syngenta	Cyprodinil 50%	2 g l -1
		F11	SUMISCLEX 50WP Masso	Procymidone	1 g l -1
		F12	RIDOMIL	GOLD	Syngenta	Folpet 40% + Mefenoxam	2 g l -1
			COMBI			5% WP
		F13	QUADRIS DUO	Syngenta	Azoxystrobin 18.7% +	2.25 g l -1
						Cymoxanil 12% WG
		F14	TELDOR		Bayer	Fenhexamid 50% p/p	2 g l -1
		F15	EUPAREN M		Bayer	Tolyfluanid 50% p/p	1.75 g l -1
		F16	FOLICUR 25EW	Bayer	Tebuconazole 25% p/v	0.70 ml l -1
		F17	FLINT		Bayer	Trifloxystrobin 50% p/p	0.13 g l -1
		F18	CAPLUQ-50		Luqsa	Captan 50% p/p	3.5 g l -1

(OTA) production. Three trials were designed in order to: i) screen 26 fungicides at the

doses recommended by manufacturers on grape-like synthetic medium (SNM) at 20 ºC

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

37.5%+fludioxonil 25%; F1, F3 and F9) showed a clear reduction of the OTA

penconazole 10% (F1) and azoxystrobin 25% (F3). All three were also restrictive

fungicides in terms of growth and mycotoxin production when tested on SNM medium.

% the level of OTA in wines made from grapes treated with this fungicide (Lo Curto et

al. 2004). In another study carried out at the ITV France by[START_REF] Molot | Ochratoxine: prévention du risque. Etude au 404 vignoble de fongicides actifs contre Aspergillus carbonarius incidences sur la présence 405 aux vendanges. Les Entretiens Viti-Vinicoles Rhone-Mediterranée[END_REF],

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Generalitat de Catalunya), the Spanish Government (CICYT, Comisión Interministerial

de Ciencia y Tecnología, AGL2004-07549-C05-01, and Ramon y Cajal program) and

the EC, Quality of Life Programme (QoL), Key Action 1 (KA1) on Food, Nutrition and

medium in relation to environmental factors. Journal of Applied Microbiology, 98, 839-

Uncoupling activity of a newly developed fungicide, fluazinam[3-chloro-N-(3-chloro-

iprodione. Journal of Food Protection, 60, 849-852.

Table 2. Mean growth rates (mm day -1 ) and OTA production on Synthetic Nutrient

Medium (µg g -1 SNM) at two temperatures (20 ºC and 30 ºC) by three strains of A.

replicates of each ± standard deviation. Data in each column followed by different

Acknowledgments 338

The work was funded by the Catalonian Government (Direcció General de Recerca,

Health (QLRT-2000-01761). The authors are grateful to Syngenta-Agro S.A. (


Juli Broggi

Magdalene Langset

Bernt Rønning

Jorg Welcker

Claus Bech

P H J Becker

Broggi

Á B Rønning

Á C Bech

Estacio ´n Biolo ´gica Don ˜ana

Parent Kittiwakes experience a decrease in cell-mediated immunity as they breed

Keywords: Costs of reproduction, Immunocompetence, PHA, Phytohemagglutinin, High arctic, Rissa tridactyla

The maintenance of an effective immune system may entail physiological costs only affordable by individuals in good condition. Some species, like Blacklegged Kittiwakes (Rissa tridactyla) in the high arctic, experience important physiological changes during breeding in order to fulfil their reproductive goals. We studied the effects of breeding effort on the cell-mediated immunity (CMI) of adult breeding Kittiwakes. Parent Kittiwakes showed a significant decrease in CMI from hatching to 15 days into the chick-rearing period, when both parents are known to experience a drastic increase in energy expenditure together with important physiological adjustments. Despite being a long-lived species with biparental care, parent Kittiwakes at the northern border of their breeding range, seem to experience important reproductive immunosupression, even during favourable breeding years.

Introduction

As predicted by life history theory, individuals adjust their breeding effort in order to maximise their fitness reward [START_REF] Stearns | Life history evolution: successes, limitations, and prospects[END_REF]. Accordingly, long-lived bird species are often confronted with trade-offs between their own survival and reproduction in the allocation of resources. The maintenance of an effective immune system is critical for adult survival, but involves important physiological costs [START_REF] Harshman | The cost of reproduction: the devil in the details[END_REF][START_REF] Norris | Ecological immunology: life history trade-offs and immune defence in birds[END_REF][START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF]. Immunocompetence can be understood as a surrogate estimation of general parasite resistance of an organism, and the ability to minimise the fitness costs of an infection. The vertebrate immune system comprises diverse components that interact in a complex way, one of these components being acquired cell-mediated immunity (CMI) [START_REF] Kennedy | The perils and prospects of using phytohaemagglutinin in evolutionary ecology[END_REF][START_REF] Tella | The PHA test reflects acquired t-cell mediated immunocompetence in birds[END_REF].

Some studies suggest that immunocompetence is costly, and that stressful situations such as heavy workload can compromise the immune response [START_REF] Bonneaud | Assessing the cost of mounting an immune response[END_REF][START_REF] Hanssen | Cost of reproduction in a long-lived bird: incubation effort reduces immune function and future reproduction[END_REF][START_REF] Knowles | Elevated reproductive effort increases blood parasitemia and decreases immune function in birds: a meta-regression approach[END_REF]. For example, food manipulation experiments have shown that immunocompetence can be negatively affected by limited resources (Alonso-Alvarez and Tella 2001; [START_REF] Gasparini | In kittiwakes food availability partially explains the seasonal decline in humoral immunocompetence[END_REF]). On the other hand, some studies have demonstrated that the costs, in terms of nutrients or energy, of building and maintaining an immune defence are probably not large enough to induce a trade-off, suggesting that other factors may regulate immunocompetence (e.g. avoidance of immunopathology) [START_REF] Hanssen | Costs of immunity: immune responsiveness reduces survival in a vertebrate[END_REF][START_REF] Nilsson | Does the strength of an immune response reflect its energetic cost?[END_REF][START_REF] Ra ˚berg | On the adaptive significance of stress-induced immunosuppression[END_REF][START_REF] Speakman | Body size, energy metabolism and lifespan[END_REF]. However, despite the fact that the immune response may not involve severe direct energetic costs, it may still have non-trivial effects on individuals' life-history strategies [START_REF] Martin | Immune activity elevates energy expenditure of house sparrows: a link between direct and indirect costs?[END_REF][START_REF] Martin | Investment in immune defence is linked to pace of life in house sparrows[END_REF][START_REF] Ots | Immune challenge affects basal metabolic activity in wintering great tits[END_REF].

Many studies of ecological immunology on breeding individuals have focused on the immune response of offspring exposed to different levels of stress [START_REF] Gasparini | In kittiwakes food availability partially explains the seasonal decline in humoral immunocompetence[END_REF][START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF][START_REF] Soler | Environmental, genetic and maternal components of immunocompetence of nestling pied flycatchers from a cross-fostering study[END_REF]. However, parents are the ones paying most of the costs of unsuitable breeding conditions, especially in capital breeding and short-lived species, from which comes most evidence on wild populations [START_REF] Ardia | Individual quality mediates trade-offs between reproductive effort and immune function in tree swallows[END_REF][START_REF] Morales | Associations between immune parameters, parasitism, and stress in breeding pied flycatcher (Ficedula hypoleuca) females[END_REF][START_REF] Moreno | Reproductive effort and T-lymphocyte cell-mediated immunocompetence in female pied flycatchers Ficedula hypoleuca[END_REF][START_REF] Nordling | Reproductive effort reduces specific immune response and parasite resistance[END_REF]. Costs of reproduction on immunocompetence have been demonstrated in incubating females of long-lived species like the Common Eider (Somateria mollissima) during a period of fasting which is known to potentially have long-lasting costs [START_REF] Bourgeon | Phytohemagglutinin response and immunoglobulin index decrease during incubation fasting in female common eiders[END_REF][START_REF] Hanssen | Cost of reproduction in a long-lived bird: incubation effort reduces immune function and future reproduction[END_REF]. Studies on parent immunocompetence have often focused on breeding females, since in many cases they are solely responsible for incubation and brooding, and thus are expected to be more constrained than their partners in resources that could be potentially traded-off against immunocompetence [START_REF] Cichon | The effect of elevated reproductive effort on humoral immune function in collared flycatcher females[END_REF][START_REF] Ilmonen | Are incubation costs in female pied flycatchers expressed in humoral immune responsiveness or breeding success?[END_REF][START_REF] Merino | Effects of parental effort on blood stress protein HSP60 and immunoglobulins in female blue tits: a brood size manipulation experiment[END_REF][START_REF] Moreno | Reproductive effort and T-lymphocyte cell-mediated immunocompetence in female pied flycatchers Ficedula hypoleuca[END_REF][START_REF] Nordling | Reproductive effort reduces specific immune response and parasite resistance[END_REF]. However, information from long-lived species where the costs of reproduction are equally shared between parents is scant.

The Black-legged Kittiwake (Rissa tridactyla) is a longlived medium-sized seagull species having a northern hemisphere circumpolar distribution. It is a semi-precocial species where nestlings require a high degree of parental care [START_REF] Golet | Energy costs of chick rearing in black-legged kittiwakes (Rissa tridactyla)[END_REF]. Chicks are normally brooded until 15 days of age [START_REF] Moe | Changes in body condition in breeding kittiwakes Rissa tridactyla[END_REF], and are totally dependent on their parents during the first 6-8 days as they are unable to thermoregulate [START_REF] Barrett | Adult body temperatures and the development of endothermy in the kittiwake (Rissa tridactyla)[END_REF]. Chicks fledge at an age of 5-6 weeks and regularly return to the nest after that as they still depend on the parents for food [START_REF] Cam | Long-term fitness consequences of early conditions in the kittiwake[END_REF]. Such long-lasting parental dependence, as opposed to precocial species, e.g. eiders, imposes important physiological challenges to parent Kittiwakes. Furthermore, parental duties are shared between parent Kittiwakes [START_REF] Fyhn | Individual variation in field metabolic rate of kittiwakes (Rissa tridactyla) during the chick-rearing period[END_REF], suggesting that costs of reproduction may be traded differently compared to species with different degrees of investment between the sexes.

Previous studies have demonstrated that adult Kittiwakes suffer from a considerable reduction in body condition from hatching until the end of the chick-rearing period, when chicks are around 15 days of age. During this period, parental duties change from incubating to brooding and food-provisioning, and the parents total energy expenditure increases by 24% [START_REF] Moe | Changes in body condition in breeding kittiwakes Rissa tridactyla[END_REF]. Despite this considerable increase in energy expenditure, their body mass and basal metabolic rate decreases significantly, suggesting that birds depress certain physiological functions (possibly related to digestive efficiency) in order to fulfil their parental role [START_REF] Bech | The energy economy of the arctic-breeding kittiwake (Rissa tridactyla): a review[END_REF][START_REF] Langseth | Reduction in body mass and basal metabolic rate in breeding female black-legged kittiwakes Rissa tridactyla: an adaptation to reduce maintenance costs[END_REF][START_REF] Moe | Changes in body condition in breeding kittiwakes Rissa tridactyla[END_REF][START_REF] Rønning | Metabolic adjustments in breeding female kittiwakes (Rissa tridactyla) include changes in kidney metabolic intensity[END_REF]).

In the present study, we investigated if parent Kittiwakes do experience changes in their cell-mediated immunocompetece parallel to their temporal reallocation of energy from self-maintenance to chick rearing.

Methods

Study area

The Kittiwakes were studied during the breeding season of 2005 at two breeding sites in Kongsfjorden, on the west coast of Spitsbergen, Svalbard (78°54 0 N, 12°13 0 E). Basic breeding data (clutch size, hatching date and success, and fledging date and success) were collected by regular inspections of the nests throughout the breeding season, which lasts from mid-June to mid-August. Hatching date (day 0) of a nest was defined as the date on which the first egg hatched, and all subsequent events were related to this date.

Bird manipulation

A total of 36 adult Kittiwakes (16 males and 20 females) from 28 nests were used in the present study. The birds were caught at the nests by means of a telescopic fishing rod with a nylon snare fitted to its end. Birds were banded if caught for the first time, and body mass (to the nearest 5 g with a spring balance; Mess und Wiegetechnik, Wennigsen, Germany), and skull, tarsus and wing lengths were measured; see [START_REF] Moe | Changes in body condition in breeding kittiwakes Rissa tridactyla[END_REF] for details on the measurement and sexing procedures. Individuals were marked according to their sex with non-permanent ink that lasted for about 1 week, so individuals from each nest could be selectively recaptured.

Measurement of cell-mediated immunity (CMI)

The phytohemagglutinin (PHA)-injection assay was used to evaluate CMI in vivo following the simplified protocol of [START_REF] Smits | Simplifying the phytohemagglutinin skin-testing technique in studies of avian immunocompetence[END_REF]. PHA is a non-pathogenic mitogen of plant origin used to stimulate an unspecific immune response, which is commonly used as an index of cellmediated immunity [START_REF] Kennedy | The perils and prospects of using phytohaemagglutinin in evolutionary ecology[END_REF][START_REF] Tella | The PHA test reflects acquired t-cell mediated immunocompetence in birds[END_REF]. Each individual was challenged once by subcutaneous injecting 50 ll of 1 mg/ml PHA (Sigma, St. Louis, Missouri) in phosphate-buffered saline (PBS) in the wing web (patagia). The thickness of the wing web was measured (to the nearest 0.01 mm) with a digital micrometer (Mitutoyo, Tokyo, Japan) at the injection site just prior to and approximately 24 h after challenge. Because time between measurements varied slightly between individuals, due to the fact that any given individual could not be recaptured at an exact time, time from immune challenge to measurement was used as a covariate in all statistical analyses. Swelling of the wing web (i.e. CMI) was calculated as the difference in wing web thickness prior to and 24 h after injection. The repeatability [START_REF] Lessells | Unrepeatable repeatabilities: a common mistake[END_REF] of our measurements of patagia thickness was very high (R = 0.968, P \ 0.0001; based on 36 individuals measured 5 times) and therefore mean values were used in the analyses. Birds were separated in two groups according to the phase of their breeding cycle. The first group included individuals that were challenged around the day their chicks hatched (hereafter around hatching group, AH). The second group included individuals that were challenged around day 14, when brooding duties finalise (hereafter post-brooding group, PB). Details of breeding characteristics and parental profiles of both groups are shown in Table 1.

Statistical analyses

We studied which factors explained variation in CMI response by means of a general linear model, with breeding stage group and sex as categorical predictors, body mass at the time of immune challenge, and time between measurements as a continuous predictor. We succeeded in measuring CMI of both parents only in eight nests. To explore the association of CMI response between members of a same pair, we ran a separate GLM with all other predictors except sex, but including nest as a random effect. As nest did not explain any significant part of the variation among the subsample (F 6,6 = 0.692, P = 0.667), we did not include nest as a factor in our final analyses. Reproductive and morphological differences between breeding stage groups were assessed beforehand, and removed from later analyses in order to improve statistical power (Table 1). We conducted all the statistical analyses with Statistica 6.0 (Statsoft, Tulsa, OK, USA). All statistical tests are two-tailed.

Results

Parental characteristics and breeding parameters from both breeding stage groups did not differ, except for body mass that declined significantly (Table 1). The CMI of parent Kittiwakes measured at 14 days of the chick-rearing period was significantly lower than the CMI measured at hatching (F 1,30 = 4.516, P = 0.041; Fig. 1). This decline occurred equally in both sexes (F 1,30 = 0.150, P = 0.701), and independently of body mass (F 1,30 = 0.056, P = 0.815). Furthemorer, time elapsed (\48 h) between PHA injection and measurements of wing web swelling did not affect CMI measurements (F 1,30 = 2.283, P = 0.141).

Discussion

Despite being a long-lived seabird, Kittiwakes appear to pay important physiological costs of reproduction. A previous study in the same population showed that body mass and body condition decreased during the chick-rearing period [START_REF] Moe | Changes in body condition in breeding kittiwakes Rissa tridactyla[END_REF]. We expand these results by showing that CMI also decreases from the incubation to the end of the chick-rearing period.

The high arctic environment imposes strong selective pressures on the reproductive strategies of seabirds. First, adequate environmental conditions to breed may last for only few weeks, with failure to synchronise the breeding attempt being fatal. Secondly, breeding success may vary drastically between years due to the fluctuating nature of the arctic sea environment and the related changes in prey abundance. In this scenario, and considering that 2005 was a good year in terms of breeding performance and environmental conditions [START_REF] Moe | Climate change and phenological responses of two seabird species breeding in the high-arctic[END_REF], the fact that both parent sexes incurred a reduction in CMI implies that such costs could be even higher in years with less favourable breeding conditions. Costs of reproduction in long-lived birds had previously been found in Common Eiders, where parental roles are quite different, as only females are in charge of the offspring, and chicks are almost self-sufficient upon hatching [START_REF] Hanssen | Cost of reproduction in a long-lived bird: incubation effort reduces immune function and future reproduction[END_REF]. Despite being a long-lived species, the fact that both parent Kittiwakes invest similarly in breeding and still experience important physiological costs suggests that the reproduction in this species in high arctic conditions is highly constrained.

Interestingly, it could be argued that adult birds could afford depressing immune functions during breeding without incurring real costs [START_REF] Piersma | Do global patterns of habitat use and migration strategies co-evolve with relative investments in immunocompetence due to spatial variation in parasite pressure?[END_REF]. High arctic populations suffer from low parasitic pressure, as insect vectors and ectoparasites are not common in these high latitudes [START_REF] Mccoy | Comparative host-parasite population structures: disentangling prospecting and dispersal in the black-legged kittiwake Rissa tridactyla[END_REF][START_REF] Staszewski | Interannual dynamics of antibody levels in naturally infected long-lived colonial birds[END_REF]). However, other factors than vector-borne diseases, such as intestinal parasites which can be acquired from food, are also known to have a strong impact on avian populations [START_REF] Clayton | Host-parasite evolution. General principles and avian models[END_REF]. Additionally, considering that Kittiwakes are a long-lived species (3-4 years to return to the breeding places and start breeding), and that the potential transmission with individuals from other colonies exposed to parasites is possible, it is not likely that birds suppress immune function adaptively as a consequence of low-parasite exposure. In any case, deciding whether parent Kittiwakes adjust their immune system adaptively, or suffer immunosuppression as a result of breeding costs, would require an experimental approach to disentangle both explanations for our results.

In the present study, we have reported a decline in CMI in breeding parent Kittiwakes, as found previously in parents from other long-lived species, but with asymmetric parental roles (e.g. [START_REF] Bourgeon | Phytohemagglutinin response and immunoglobulin index decrease during incubation fasting in female common eiders[END_REF]). These results highlight the levels of physiological stress that this species needs to endure in order to reproduce. Because the results were obtained during a ''good breeding'' year, the effects may presumably be even more important under less favourable breeding conditions. Due to the energetic constraints imposed on these border populations, susceptibility to changes in climate inducing variations in prey availability, breeding conditions and parasite burdens may be extremely high, and thus require careful and constant monitoring.

Zusammenfassung

  Dreizehenmo ¨weneltern erfahren wa ¨hrend des Bru ¨tens eine Abnahme der zellvermittelten Immunita ¨t Die Aufrechterhaltung eines effektiven Immunsystems ko ¨nnte physiologische Kosten mit sich bringen, die sich nur Individuen in guter Kondition leisten ko ¨nnen. Einige Arten, wie z.B. Dreizehenmo ¨wen (Rissa tridactyla) in der Hocharktis, erfahren wa ¨hrend des Bru ¨tens wichtige physiologische Vera ¨nderungen, um ihre Fortpflanzungsziele zu erfu ¨llen. Wir haben die Einflu ¨sse des Brutaufwandes auf die zellvermittelte Immunita ¨t (CMI) adulter bru ¨tender Dreizehenmo ¨wen untersucht. Dreizehenmo ¨weneltern zeigten eine signifikante Abnahme der CMI vom Schlupf bis 15 Tage in die Ku ¨kenaufzuchtsphase hinein, wenn beide Eltern einen drastischen Anstieg im Energieaufwand nebst wichtigen physiologischen Anpassungen erfahren. Obwohl diese Art langlebig ist und beide Eltern Brutpflege betreiben, scheinen Dreizehenmo ¨weneltern am no ¨rdlichen Rand ihres Brutgebietes wichtige reproduktive Immunsuppression zu erfahren, selbst in gu ¨nstigen Brutjahren.

Fig. 1

 1 Fig.1Cell-mediated immunocompetence measured as wing web swelling in two groups of adult Black-legged Kittiwakes (Rissa tridactyla) at different stages of the breeding cycle. Group AH includes individuals challenged around day of hatching. Group PB includes individuals challenged around day 14 after hatching, when the intensive brooding phase ends

Table 1

 1 Breeding information and parental characteristics of the two groups of Black-legged Kittiwakes (Rissa tridactyla) considered in the study. Average sampling day for each group is presented (hatching day = 0). Mean values and corresponding standard error (SE) are presented, with corresponding sample sizes (n), except when stated otherwise. Differences between groups are tested by means of t tests with the corresponding degrees of freedom in parentheses and P values

		Around hatching (AH)		Post-brooding (PB)		t (df)	P
		Day = -1 (±2 days)		Day = 14 (±4 days)
		Value	n	Value	n
	Clutch size	2.0 ± 0.0	14	1.9 ± 0.5	22	0.64 (34)	0.52
	Average hatching date (July)	9.9 ± 1.08	13	8.8 ± 0.55	22	1.01 (33)	0.32
	Body mass (g)	433.14 ± 10.09	14	382.27 ± 9.25	22	3.45 (34)	\0.01
	Wing length (mm)	319.07 ± 2.72	14	315.69 ± 1.54	22	1.15 (31)	0.26
	Tarsus (mm)	34.72 ± 0.48	14	34.15 ± 0.49	22	0.79 (34)	0.44
	Proportion of males in the sample (%)	50%	14	41%	22
	Time between measurements (h)	23.4 ± 0.40	14	23.7 ± 0.80	22	-0.39 (34)	0.70

Acknowledgments Permission to carry out the study in the Kittiwake colonies was provided by the Governor of Svalbard (Sysselmannen; ref.

2004/00884-4 a522-01), while permission to perform the PHA injections was given by the Norwegian National Animal Research Authority (Forsøksdyrutvalget; ref. 2005/20577). J.B. was funded by EC Marie Curie EVK1-CT-2002-57003 fellowship, while B.R., C.B. and M.L. were funded by Norwegian Polar Institute. Thanks are due to to the staff at Kings Bay and Norsk Polarinstitutt for logistic support. We also thank Geir Wing Gabrielsen and Olivier Chastel for help during the field season. We are indebted with Jordi Figuerola for his valuable comments on previous versions of the manuscript.


Earl Michael Thurman
email: mthurman@ual.es

Imma Ferrer

Amadeo Fernandez-Alba

Octavio Malato

Amadeo Rodriguez Fernández-Alba

Feasibility of LC/TOFMS and Elemental Database Searching As a Spectral Library for Pesticides in Food

Keywords: pesticide residues, LC/MS, TOF-MS, database, imazalil, iprodione, thiabendazole

Introduction

The identification and quantitation of unknown pesticides in fruit and vegetables are greatly important to individuals and health organizations around the world. In order to meet these health concerns, the European Union (EU) and the US have set new Directives for pesticides at low levels in vegetables. For example, new laws, such as the European Directive 91/414/EEC or the Food Quality Protection Act (FQPA) in the US, have changed the standards for human health, workers, and environmental protection, which require lower levels of pesticides in food [EU Food Directives, Food Quality Protection Act]. They also require re-registration for older pesticides. Furthermore, the review programs have withdrawn authorizations for many of the crop protection products currently on the market, 177 compounds in US and 320 in Europe. Moreover, it was announced in Europe that a total of 110 products would be withdrawn in the near future (EU Directive 91/414/EEC). Furthermore, the revision of the review program in Europe dictates that over 450 existing active substances will be taken off the market by 2008 with about 400 pesticides remaining in use. Next, the quality standards within the new regulations include the re-assessment of the maximum residue limits (MRLs) for vegetables. EU directives are setting different MRLs for each pesticide within each food group, and typically, the MRLs are lower than the previous ones. Furthermore, the new directives also lead to different MRLs for each EU country, which are still being decided. EU directives state that the individual country MRLs will be maintained in the new program. Finally, banned compounds have the lowest MRLs, which is set now at 0.01 mg/kg (ppm). With the planned program to remove so many compounds from the market, it is important, even necessary, that screening for unknown pesticides may be done by LC/MS on vegetable extracts. Because it is not always possible to know which banned substances may be used, it is of vital importance to environmental food monitoring that there be a system of unknown identification to give fast and accurate screening of unknown substances in food and food products.

Thus, there is an important need for research studies and methods development on the analysis and screening of pesticides in food by new LC/MS methods, such as accurate mass screening using LC/TOFMS and database analysis [Ferrer and Thurman, 2003a;Thurman et al. 2005a, Thurman et al. 2005b;Garcia-Reyes et al. 2005]. This combined with the use of large accurate mass databases of pesticides is an important and useful new screening procedure, especially when accurate mass is less than 3-ppm accuracy.

The combination of LC/MS TOF and database screening consists of three steps, which are outlined in detail below. They are: 1. Analyse the fruit or vegetable extract with LC/TOFMS in full scan looking for important unknown peaks using a mild in-source CID fragmentation.

2. Search the Access pesticide database using the accurate mass found in full scan for initial screening.

3. If samples screen positive for a pesticide, make identification using at least one fragment ion with its accurate mass combined with retention time.

This paper gives several detailed examples of this process using retail samples of apples and oranges, which show the screening by accurate mass and database entry, followed by identification of several pesticides that are major components of the samples.

Materials and Methods

Whole-vegetable extraction. Portions of apples (2 kg) were obtained from the market place and homogenized by chopping. Then 15 g portions of the homogenized chopped fruit were weighted into a 200 mL PTFE centrifuge tube. 45 mL ethyl acetate and 1 mL NaOH (6.5 M) were added and blended in a Polytron (high-speed blender) for 30 seconds at 21,000 rpm. The combined extracts were then filtered through a thin layer of 20 g of anhydrous Na 2 SO 4 . The solid was washed with 50 mL of ethyl acetate and the combined extracts were evaporated to dryness on a vacuum rotary evaporator using a water bath at 45 ± 5ºC. The dried residue was dissolved by sonication in 15 mL of methanol. The extracts, which contained 1 g of sample per mL, were filtered through 0.2 µm PTFE filters (Millex FG, Millipore) prior to LC/TOFMS analysis. (hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine) m/z, resolution: 9500+500 @ 922.0098 m/z, reference A sprayer 2 is constant flow rate (600 µL/hr) during the run.

Results and Discussion

Creation of Access Database. Table 1 shows the list of 350 compounds that were used in the database for the search of pesticides in fruit extracts. The list consists of the three major classes of pesticides that are commonly used in Europe for treating crops of fruits and vegetables, as well as many of the pesticide degradation products.

There are insecticides, herbicides, and fungicides. An Excel spreadsheet was used to calculate the accurate masses of each of the 350 pesticides. The compounds in the database are known to ionise in positive ion electrospray and were collated from a series of publications, as well as from approximately 100 standards analysed in our laboratory. Figure 1 shows the appearance of the Access database, called Pesticidas.

The front page of the database has 4 basic key functions. The first is called "Find Accurate Mass" and consists of the accurate masses of all 350 pesticides to the fourth decimal place. The program searches these and finds all compounds that have the correct mass to the second decimal place. This allows for compounds that have either similar or identical accurate masses. It turns out that although accurate mass is a very The second key of the database is "Find Pesticide". This key will locate any compound in the database and display its formula, structure, and accurate mass to 4 decimal points. The third key is "New Pesticide" key, which is used to add new compounds to the database. The fourth key is "Edit List", which allows one to change parameters of the compounds within the database. The edit key is disabled when the program is used in the laboratory in order to protect the safety of the program from error entries. The fifth and last key is the "Exit" key, which exits the database program. The logo of the University of Almeria (Almeria, Spain) is also shown on the front page of the database.

Advantages of using Access for this pesticide database is that this program is available as part of the software of Office (Microsoft) and thus is available to use on any computers that run XP Windows. The database is approximately 18 megabytes in size; thus, it is easily transported and available for use by other scientists that are working on pesticide analysis. It can be readily sent by email or by transfer by memory stick, CD etc. The database is used in manual mode by entering the accurate mass, this is a limitation at this time, which will be explained in the next section. for the extraction of a store purchased apple. There are approximately 20 major peaks in the chromatogram at retention times from 2 to 30 min. However, the matrix is not simple and over 1000 peaks are present in sample with a signal to noise of 10:1. However, in this manual application we are screening only the major peaks. Thus, two major peaks at 18.0 and at 25.3 min were screened as possible pesticides by the manual database search. Figure 2 shows an example positive pesticide screen. We start with an accurate mass, in this case, the m/z 297.0574 ion at 18.0 min. This ion was chosen because it is the protonated molecule of the peak at 18.0 min. The database gave a positive screen for imazalil (Figure 1). Here we differentiate between a screen and an identification of a pesticide.

Screening is a test that shows that a possible pesticide is present based on an analysis that is inadequate for confirmation, but that is rapid and inexpensive (i.e. the database search of the molecular ion with a retention time). The identification is based on further analysis, which includes the accurate mass of the protonated molecule and a one fragment ion with the correct retention-time match. Thus, continuing with the example of a positive screen, the database works by first entering the accurate mass of the protonated molecule. The mass is checked versus the database to two decimal places (note the value of 297.06 showing in Figure 1).

Next the compound name is entered in the database and the structure, formula, and accurate mass appear for imazalil. The accuracy in this case was a difference of 0.0018 u or ~6 ppm. The error in accuracy was caused by the very high concentration of imazalil in the sample, which saturated the detector. Typically, the extracted ion 8 should be less than 10 6 counts (10 5 is ideal), rather than the 10 7 value, which is two orders of magnitude above the ideal accurate mass window [Ferrer and Thurman, 2005]. Ideally the sample should be diluted 10 times and re-analyzed. Another quicker way is to take the accurate mass on the tail of the peak, which lowers the counts by an order of magnitude and gives an accurate mass of 297.0557, which is within 0.1 mD of the correct value (see Figure 1)! Note the lower intensity at 3 x 10 5 counts. This procedure completes the screen for imazalil, but the compound is not yet identified. Identification requires more information including fragment ion and retention time matching, which will be addressed in the following section. The sample can either be diluted and re-injected or, more simply, a smaller volume, such as 5 µL rather than the initial 50 µL can be injected. The concentration of imazalil is estimated at approximately 0.5 ppm in this apple extract.

Another screening example showing the power of the accurate mass and the database is the second positive screen at 25.3 min, which had an accurate mass of 330.0409.

Entering this value into the database gave the screening identification of iprodione (Figure 3). Note here that the intensity was 10 4 , which is in the "sweet zone" accurate mass range of the instrument [Ferrer and Thurman, 2005]. The accuracy is 0.2 mD for an accuracy of less than 1 ppm, which is typical of the instrument when carefully calibrated and run in its most accurate mode [Ferrer and Thurman, 2005]. The "sweet zone" is between 10 4 and 10 5 counts and reflects the instrument dynamics and tuning.

Using this approach it has been possible to measure the mass of an electron with reproducible results [Ferrer and Thurman, 2005]. Thus, this sample gave two positive screens for pesticides, imazalil and iprodione. The database also works well on compounds that are not chlorinated. For example, Figure 4 shows the TIC for an orange extract and the exact mass for a large peak at 7.6 minutes with a m/z of 202.0437. Entering the accurate mass in the database gives the formula and structure for only one compound, thiabendazole. Note that the mass spectrum also shows a sulphur isotope at m/z 204.0407, which is consistent with the structure of thiabendazole. Approximately 75% of the compounds in the pesticide database of 350 compounds contain Cl, Br, or S. These A+2 isotopes are useful in both the screening and the identification of the compounds. But they are not necessary as the next section will show.

Identification of Pesticides.

After the initial screening of the pesticides in the apple or orange extract, the next step is to identify the compounds. The usual approach is to acquire the standard and to measure both retention time and at least one fragment ion for identification [Stolker et al., 2003]. The accurate mass fragment ions for imazalil, iprodione, and thiabendazole are shown in Figure 5, along with the structure of the proposed fragment ion. According to the identification point system [Stolker et al., 2003, Ferrer andThurman, 2003b], the high resolution accurate mass of the protonated molecule and the fragment ion give a total of 4 IPs, which is used for identification along with the ion ratio. The IP system also requires a resolving power of 10,000 (defined as 10% valley), which is not possible with benchtop TOF instrumentation. Thus, unfortunately these data do not meet the IP standard. We consider this a shortcoming of the IP system, which was designed with GC/MS in mind rather than benchtop LC/TOFMS. The quality of the measurements meet the requirement of identification but not of the IP system. Nonetheless, further information is also obtained from the characteristic chlorine isotope cluster of both the protonated molecules and the fragment ions in these two cases. Note that the two chlorine isotope signature is obtained for the protonated molecule of imazalil m/z 297.0574 and 299.0038 (Figure 2) and for its fragment ion at m/z 255.0086 and 257.0045 in Figure 5.

Iprodione also showed a two chlorine isotopic signature at m/z 330.0409 and 332.0387 (Figure 4) for the protonated molecule and m/z 244.9881 and 246.9853 for the fragment ion (Figure 5). These chlorine signatures show the presence of two chlorine atoms in both molecules and give valuable identification information.

Likewise, thiabendazole has an accurate mass fragment ion at m/z 175.0340, which also has the S isotope signature at A+2 mass units for futher confidence in the identification.

Note that this two-step procedure requires first the screening of the pesticide followed by an identification step. The goal of our project, ultimately, is to include this information in the database, both fragment ion and retention time, and to automate the process. However, one must note that when this occurs the database becomes more of a user database because the retention time conditions may be variable from instrument to instrument. This problem of variability has been solved in GC/MS by the use of retention time locking and standardized electron impact spectra. Eventually this problem may be solved in LC/MS by similar approaches. Variation in fragmentation intensity is not critical with the use of accurate mass since the accurate mass of the fragment ion gives its molecular formula. Thus, at this time the database approach

Conclusions

The use of accurate mass LC/TOFMS combined with database searching is a powerful example of a new wave of technology for screening of pesticides in food.

The use of classical fragmentation libraries (i.e. comparison of spectra containing multiple ions) with LC/TOFMS is not needed, rather the use of molecular formula (accurate mass of the protonated molecule) and the accurate mass of fragment ion, when combined with retention time gives the identity of compounds without the worry of the intensity of the fragment ions and how this may vary from instrument to instrument and matrix to matrix. Thus, it is the view of the authors that LC/TOFMS will become a more important tool in the screening and identification of pesticides in the near future. However, there are problems to address before database screening is routine. First is the automation of the database. Second is to address the issue of false positives and false negatives. Of these, the false positives are easier to deal with because they involve the identification process. The false negative is more serious, since a pesticide is present in the food but is not detected. Also the topic of resolving power and identification points needs to be re-addressed in our opinion. These topics will be the subject of our future work on databases and LC/TOFMS.

  Methods. LC Pumps were HP 1100, injection volume 50 µL, column: ZORBAX Eclipse® XDB 4.6x150 mm C-8, 5-micron, mobile phase A=ACN and B=0.1% formic acid in water, gradient was 15%A to 100%A over 30 minutes at a flow rate of 0.6 mL/min, model LC/MSD TOF (Agilent Corp, Santa Clara, CA, USA) with electrospray source positive ESI+, capillary 4000V, nebulizer 40 psig, drying gas 9 L/min, gas temp 300°C, fragmentor 190V, skimmer 60V, Oct DC1 37.5 V, OCT RF V 250V, reference masses (Agilent Solution): 121.0509 (purine) and 922.0098

  it is possible for pesticides to have the same empirical formula, and thus the same accurate mass! In fact in this database of 350 compounds, 8 compounds have the same formula. This includes several degradate products of the triazines. These compounds also require retention time and fragment ions for a final confirmation.

  Database Search of Apple Extract. Figure2shows the total ion chromatogram (TIC)

  tool, but powerful and fast because only the molecular formula is needed.

  .M.; Ferrer, Imma, Fernandez-Alba, A.R., 2005a, Matching unknown empirical formulas to chemical structure using LC/MS TOF accurate mass and database searching: example of unknown pesticides on tomato skin. J. Chromatography A, 1067, 127-134. Thurman, E.M.; Ferrer, Imma; Zweigenbaum, J.A.; Garcia-Reyes, J.F.; Woodman, Michael, Fernandez-Alba, A.R., 2005b, Discovering metabolites of post harvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry and ion trap tandem mass spectrometry. J. Chromatography A, 1082, 71-80.

F

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 1 .

 12341 Figure 1. Access database search for accurate mass of the m/z 297.0574 ion.

FFigure 2 .FoodFigure 3 .FoodFigure 4 .FoodFigure 5 .

 2345 Figure 2. Total ion chromatogram of apple extract using LC/TOFMS. http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 1 .

 1 List of 350 Pesticides in "Pesticidas" Database.

	Pesticide	Formula	MH+	Pesticide	Formula	MH+	Pesticide	Formula	MH+	Pesticide	Formula	MH+
	METHAMIDOPHOS	C2H8NO2PS	142.0086	ALDICARB SULFOXIDE C7H14N2O3S	207.0798	Trietazine	C9H16ClN5	230.1167	Diallate	C10H17Cl2NOS	270.0481
	8-Hydroxyquinoline	C9H7NO	146.0600	Isoproturon	C12H18N2O	207.1492	TERBUTHYLAZINE	C9H16ClN5	230.1167	Alachlor	C14H20ClNO2	270.1255
	Phenoxyacetic acid	C8H8O3	153.0546	Quinoclamine	C10H6ClNO2	208.0160	Methfuroxam	C14H15NO2	230.1176	5-Hydroxy-imidacloprid C9H10ClN5O3	272.0545
	Daminozide Dazomet METHOMYL Nicotine	C6H12N2O3 C5H10N2S2 C5H10N2O2S C10H14N2	161.0921 163.0358 163.0536 163.1230	PROMECARB Aminocarb PROPOXUR Acibenzolar-S-methyl	C12H17NO2 C11H16N2O2 C11H15NO3 C8H6N2OS2	208.1332 209.1285 210.1125 210.9994	DEMETON-S-METHYL Deethylcyanazine amide Deethylcyanazine acid DIURON Fluometuron	C6H15O3PS2 C7H11ClN6O C7H10ClN5O2 C9H10Cl2N2O C10H11F3N2O	231.0273 231.0756 232.0596 233.0243 233.0896	Napropamide BROMOXYNIL Fenpropidin Anilazine Difsulfoton	C17H21NO2 C7H3Br2NO C19H31N C9H5Cl3N4 C8H19O2PS3	272.1645 273.8508 274.2529 274.9653 275.0358
	Fenuron	C9H12N2O	165.1022	Propachlor	C11H14ClNO	212.0837	Dichlorprop	C9H8Cl2O3	234.9923	Neburon	C12H16Cl2N2O	275.0712
	2-Phenoxypropionic acid C9H10O3	167.0703	Deethylcyanazine	C7H9ClN6	213.0650	2,4-D Methyl ester	C9H8Cl2O3	234.9923	Paraoxon	C10H14NO6P	276.0632
	CYROMAZINE	C6H10N6	167.1040	Chlorotoluron	C10H13ClN2O	213.0789	Thiofanox sulfoxide	C9H18N2O3S	235.1111	imazameth	C14H17N3O3	276.1343
	Diphenylamine	C12H11N	170.0964	OMETHOATE	C5H12NO4PS	214.0297	Carboxin	C12H13NO2S	236.0740	Phorate sulfone	C7H17O5PS2	277.0328
	Deethylhydroxyatrazine	C6H11N5O	170.1036	Chlorpropham	C10H12ClNO2	214.0629	Fenpiclonil	C11H6Cl2N2	236.9981	2,4-D Butyl ester	C12H14Cl2O3	277.0393
	Deisopropylatrazine	C5H9N5Cl	174.0541	Simetryn	C8H15N5S	214.1121	Buturon	C12H13ClN2O	237.0789	Metazachlor	C14H16ClN3O	278.1055
	Propham	C10H13NO2	180.1019	Deethylterbutryn	C8H15N5S	214.1121	Carbetamide	C12H16N2O3	237.1234	Fenthion	C10H15O3PS2	279.0273
	ACEPHATE	C4H10NO3PS	184.0192	MCPA methylester	C10H11ClO3	215.0469	3-Hydroxy Carbofuran	C12H15NO4	238.1074	Ofurace	C14H16ClNO3	282.0891
	Demethyl monuron	C8H9ClN2O	185.0476	Monolinuron	C9H11ClN2O2	215.0582	PIRIMICARB	C11H18N4O2	239.1503	Propetamphos	C10H20NO4PS	282.0923
	Fuberidazole Deethylsimetryne 1-Napthalene acetamide	C11H8N2O C6H11N5S C12H11NO	185.0709 186.0808 186.0913	Metribuzin ATRAZINE Cycloate	C8H14N4OS C8H14ClN5 C11H21NOS	215.0961 216.1010 216.1417	Clomazone Thiram Cyanazine Prometryne	C12H14ClNO2 C6H12N2S4 C9H13ClN6 C10H19N5S	240.0786 240.9956 241.0963 242.1434	Pendimethalin Dodemorph Procymidone FOSTHIAZATE	C13H19N3O4 C18H35NO C13H11Cl2NO2 C9H18NO3PS2	282.1448 282.2791 284.0240 284.0538
	Chlorophenoxyacetic acid C8H7ClO3	187.0156	Terbacil	C9H13ClN2O2	217.0738	Terbutryn	C10H19N5S	242.1434	Penconazole	C13H15Cl2N3	284.0716
	1-Naphthaleneacetic acid C12H10O2	187.0754	THIABENDAZOLE	C10H7N3OS	218.0383	Dixanthogen	C6H10O2S4	242.9636	METOLACHLOR	C15H22ClNO2	284.1412
	Deethylatrazine	C6H11N5Cl	188.0697	PYMETROZINE	C10H11N5O	218.1036	Ethoprophos	C8H19O2PS2	243.0637	VINCLOZOLIN	C12H9Cl2NO3	286.0032
	Molinate	C9H17NOS	188.1104	2,4-D	C8H6Cl2O3	218.9621	METALAXYL	C15H21NO4	244.1543	Ethofumesate	C13H18O5S	287.0948
	PROPAMOCARB	C9H20N2O2	189.1598	Demethylfluometuron	C9H9F3N2O	219.0740	Phorate oxygen analogue	C7H17O3PS2	245.0429	Difenoxuron	C16H18N2O3	287.1390
	Tricyclazole	C9H7N3S	190.0433	THIOFANOX	C9H18N2O2S	219.1162	OXYDEMETON-METHYL	C6H15O4PS2	247.0222	VAMIDOTHION	C8H18NO4PS2	288.0488
	EPTC	C9H19NOS	190.1260	OXAMYL	C7H13N3O3S	220.0750	FLUDIOXONIL	C12H6F2N2O2	247.0324	Cyanofenphos oxygen C15H14NO3P	288.0784
	BUTOCARBOXIM	C7H14N2O2S	191.0849	Dichlorvos	C4H7Cl2O4P	220.9532	Paraoxon-methyl	C8H10NO6P	248.0319	Terbufos	C9H21O2PS3	289.0514
	ALDICARB Clopyralid CARBENDAZIM Demethylisoproturon 3,4,5-TRIMETHACARB Hydroxyatrazine Deethylterbumeton MCPA Monuron Cymoxanil Deisopropylprometryne	C7H14N2O2S C6H3Cl2NO2 C9H9N3O2 C11H16N2O C11H15NO2 C8H15N5O C8H15N5O C9H9ClO3 C9H11ClN2O C7H10N4O3 C7H13N5S	191.0849 191.9614 192.0768 193.1335 194.1176 198.1349 198.1349 199.0167 199.0633 199.0826 200.0964	QUINMERAC Chloridazon Methabenzthiazuron CARBOFURAN ACETAMIPRID ALDICARB SULPHONE C7H14N2O4S C11H8ClNO2 C10H8ClN3O C10H11N3OS C12H15NO3 C10H11N4Cl BUTOXYCARBOXIM C7H14N2O4S ALDOXYCARB C7H14N2O4S Sulfallate C8H14ClNS2 Monocrotophos C7H14NO5P BENDIOCARB C11H13NO4	222.0316 222.0429 222.0696 222.1125 223.0745 223.0747 223.0747 223.0747 224.0329 224.0682 224.0917	LINURON Heptenophos Thiofanox sulfone THIACLOPRID Hexazinone 2,4,5-T Fluroxypyr Propyzamide IMIDACLOPRID N-m-Tolylphthalamic acid Dimethachlor Trichlorfon	C9H10Cl2N2O2 C9H12ClO4P C9H18N2O4S C10H9ClN4S C12H20N4O2 C8H5Cl3O3 C7H5Cl2FN2O3 C12H11Cl2NO C9H10ClN5O2 C15H13NO3 C13H18ClNO2 C4H8Cl3O4P	249.0192 251.0235 251.1060 253.0309 253.1659 254.9377 254.9734 256.0290 256.0596 256.0968 256.1099 256.9299	Myclobutanil Chloroxuron Thiamethoxam Parathion Chlorbromuron Etrimfos Triadimefon Paclobutrazol Folpet Triadimenol 2,4,5-T Isopropyl ester IMAZALIL	C15H17ClN4 C15H15ClN2O2 C8H10ClN5O3S C10H14NO5PS C9H10BrClN2O2 C10H17N2O4PS C14H16ClN3O2 C15H20ClN3O C9H4Cl3NO2S C14H18ClN3O2 C11H11Cl3O3 C14H14Cl2N2O	289.1214 291.0895 292.0266 292.0403 292.9687 293.0719 294.1004 294.1368 295.9101 296.1160 296.9847 297.0556
	Deethylametryn	C7H13N5S	200.0964	Mepanipyrim	C14H13N3	224.1182	Metobromuron	C9H11BrN2O2	259.0077	SPIROXAMINE	C18H35NO2	298.2741
	PYRIMETHANIL	C12H13N3	200.1182	Desmethylpirimicarb	C10H16N4O2	225.1346	Cyanazine amide	C9H15ClN6O	259.1069	Phoxim	C12H15N2O3PS	299.0614
	MCPA	C9H9ClO3	201.0313	METHIOCARB	C11H15NO2S	226.0896	Cyanazine acid	C9H14ClN5O2	260.0909	Quinalphos	C12H15N2O3PS	299.0614
	acetate	C13H12O2	201.0910	ETHIOFENCARB	C11H15NO2S	226.0896	Phorate	C7H17O2PS3	261.0201	Merphos	C12H27PS3	299.1085
	THIABENDAZOLE	C10H7N3S	202.0433	CYPRODINIL	C14H15N3	226.1339	Bromacil	C9H13BrN2O2	261.0233	Captan	C9H8Cl3NO2S	299.9414
	SIMAZINE	C7H12ClN5	202.0854	Terbumeton	C10H19N5O	226.1662	phorate sulfoxide	C7H17O4PS2	261.0379	Phosphamidon	C10H19ClNO5P	300.0762
	CARBARYL	C12H11NO2	202.0863	Dichlone	C10H4Cl2O2	226.9661	2,4-D Isopropyl ester	C11H12Cl2O3	263.0236	Tolclofos methyl	C9H11Cl2O3PS	300.9616
	Fenfuram	C12H11NO2	202.0863	Ametryn	C9H17N5S	228.1277	Parathion-methyl	C8H10NO5PS	264.0090	DESMEDIPHAM	C16H16N2O4	301.1183
	2-Naphthoxyacetic acid	C12H10O3	203.0703	Metoxuron	C10H13ClN2O2	229.0738	aclonifen	C12H9ClN2O3	265.0374	PHENMEDIPHAM	C16H16N2O4	301.1183
	METAMITRON	C10H10N4O	203.0927	Tebuthiuron	C9H16N4OS	229.1118	Oxycarboxin	C12H13NO4S	268.0638	FENHEXAMID	C14H17Cl2NO2	302.0709
	Hormodin Pebulate	C12H13NO2 C10H21NOS	204.1019 204.1417	DIMETHOATE Dicryl	C5H12NO3PS2 C10H9Cl2NO	230.0069 230.0134	Diethofencarb Silvex	C14H21NO4 C9H7Cl3O3	268.1543 268.9534	Flutriafol FENOXYCARB	C16H13F2N3O C17H19NO4	302.1099 302.1387

Table 1 .

 1 Continued, "Pesticidas" Access Database

	Page 22 of 26

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Acknowledgments

This work was supported by the Ministerio de Ciencia y Tecnología (Spain, Contract: AGL2004-04838/ALI). I. Ferrer acknowledges her contract from the "Junta de

Andalucía". The first author acknowledges a grant from the University of Almeria for support of the database work.

EU Food Directives, 2002, 91/414/EEC Ferrer Imma and E.M. Thurman, 2003a, Liquid Chromatography/Mass Spectrometry/Mass Spectrometry and Time-of-Flight Mass Spectrometry for the analysis of emerging contaminants, American Chemical Society Symposium Volume 850. Ferrer, Imma; Thurman, E.M., 2003b, Analysis of Emerging Contaminants by LC-MS TOF. TrAC, 35, 304-312. Ferrer, Imma; Thurman, E.M., 2005, Measuring the mass of an electron by LC/TOF-MS. Analytical Chemistry, 77, 3394-3400. Garcia Reyes, J.F., Ferrer, Imma, Thurman, E.M., Molina-Diaz, A.; Fernandez-Alba, A.R.; 2005, Searching for non-target chlorinated pesticides in food by liquid chromatography/time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry, 19, 2780-2788. Food Quality Protection Act, 1998 (FQPA) Stolker, A.A.M.; Dijkman, E.; Niesing, W.; Hogendoorn, E.A., 2003, Identification of residues by LC/MS/MS according to the new European Union Guidelines: Application to the trace analysis of veterinary drugs and contaminants in biological and environmental matrices, Chapter 3, pp. 32-51, in Liquid Chromatography/Mass Spectrometry/Mass Spectrometry and Time-of-Flight Mass Spectrometry for the analysis of emerging contaminants, American Chemical Society Symposium Volume 850.