Gianfranco Brambilla

Igor Fochi

Stefania Paola De Filippis

Nicola Iacovella

Alessandro Di Domenico

Alessandro Di

Domenico Pcp

Stefania P De Filippis

PCP , PCDD, AND PCDF DEPLETION OF CONGENERS IN EGGS FROM HENS EXPOSED TO CONTAMINATED WOOD SHAVINGS

Keywords: pentachlorophenol, Dioxins, Dioxins -TEQs, Preservatives eggs, hen, pentachlorophenol, dioxins, bedding material, depletion 5

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

Improved legislation on animal welfare has prompted farmers to move from caged rearing systems 9 toward free-range ones (EC Council Directive 1999/74). This change in animal farming is currently 10 highlighting the relevance of some non-feed contributions to unacceptable residue levels of 11 polychlorodibenzodioxins (PCDDs) and polychlorodibenzofurans (PCDFs) -both also known as 12 "dioxins" -in eggs (Schoeters and Hoogenboom, 2006;Van Overmeire et al., 2006). According to 13 the results of investigations carried out in different countries, eggs produced under free-range 14 conditions and/or relevant environmental exposures may show higher levels of PCDD and PCDF 15 contamination compared to those from caged systems (Figure 1). 16 17 Previous papers have focused their attention on the relationship between PCDD, PCDF and DL-PCBs 18 intakes and contamination levels in eggs, and in other edible tissues (Stephens et al., 1995;19 Hoogenboom et al., 2006;Pirard and De Pauw, 2006), proposing also a toxicokinetics/toxicodynamics 20 -based modelling approach to estimate the carry-over rate (van Eijkeren et al., 2007). Some of these 21 papers have already dealt with uptake exposures, where the main source of PCDDs and PCDFs in eggs 22 was the soil (Schuler et al., 1997;Le Bizec et al., 2005) and the materials originating from dust and 23 shavings recovered from timber treated with pentachlorophenol (PCP) (Lynn et al., 2000;Diletti et al., 24 It is well known that PCP, according to its grade, may contain some PCDD congeners, such as 1 the hepta-and octachloro-substituted congeners, as technical contaminants (US EPA 2006). Moreover, 2 these congeners may results from PCP degradation, i.e. if treated timbers were exposed to sunlight or 3 burnt (Draper et al. 1988). According to Fries and co-workers (2002), intermediate timber treatment 4 could result in a litter contamination of 880 µg/g product for PCP and of 53 pg WHO-TE/g product for 5

PCDDs and PCDFs. 6 7 At present, for materials in contact with food specific legislation has been set up (EC Regulation 8 1935/2004), whereas for exposure of food producing animals to PCP-contaminated materials, such as 9 barns, stables, and litters and the consequent possible bio-accumulation of such persistent organic 10 pollutants, only some general recommendations have been issued (EC Regulation 852/2004). 11 12 In this paper, we report the results of a depletion study carried out on eggs after a laying hen flock was 13 reared on a PCP-contaminated floor, with the aim to give useful information for appropriate 14 management actions based on a deeper knowledge of PCP, PCDD, and PCDF depletion in eggs, as 15 consequence of possible aggregate exposures. 16 17

MATERIALS AND METHODS 18

Animals. During monitoring activities, eggs with violative PCDD and PCDF levels were found in a 19 hen flock reared on wood shavings. Epidemiological investigation to trace back the contamination 20 revealed the presence of PCP at 5 mg/kg product and of PCDDs and PCDFs at 50 ng WHO-TE/g 21 product in the litter, with 1,2,3,6,7,8-H 6 CDD, H 7 CDD and O 8 CDD congeners at 70, 3,077 and 23,273 22 ng/kg product, respectively, whereas the contribution to contamination of commercial feed seemed to 23 be negligible (<0.15 pgWHO-TE/g dry matter, 12% moisture). According to farm records, such 24 purchased from local craftsman and originating from imported Ayous timber. Six Brown Swiss laying 2 hens of 1.5 kg body weight were randomly sampled from a 100-animal flock and placed on 3 uncontaminated bedding, under controlled feed conditions. The average egg production was 4 approximately 4.9 egg/head per week; each egg weighing approximately 55 g, with an average fat 5 content of 12 %. The depletion study lasted eight weeks; the first day of the experiment under 6 controlled conditions was considered Day 0. Observation points were planned every two weeks, by 7 drawing one egg from each animal at each sampling time. Pools of six eggs were gently whipped until 8 the mixture was homogeneous and then kept frozen at -20 °C until analyzed. 9 10 Standards and Reagents. Acetonitrile, methanol (VWR, Buffalo Grove, IL, USA), diethyl ether, n-11 hexane (Carlo Erba, Rodano, Italy), acetone, sulphuric acid (95-97 %), sodium oxalate, ammonium 12 acetate (Fluka, St. Louis, MO, USA), cyclohexane, dichloromethane, sodium sulphate (Riedel de Haen,13 Seelze, Germany), and Extrelut ™ (Merck, Darmstadt, Germany) were purchased of the best 14 commercially available quality. Gases (purity, 5.5) were purchased from Rivoira (Turin, Italy). Native 15 and fully 13 C labelled PCP were certified standards (purity, 98 %) provided by Aldrich (St. Louis, MO, 16 USA). Native and 13 C 12 labelled PCDDs, PCDFs were certified standards (purity, 99 %) provided by 17 CIL (Andover, MA, USA). 18 19 PCDD and PCDF analytical procedure. The analytical procedure was adapted from the US EPA 20

Method 1613 and validated in-house (US EPA, 1994). Briefly, after fortification with 13 C-labelled 21 congeners, each test portion (50 g from a six-egg homogenate) was allowed to rest for 24 hours at 4 °C 22 and then subjected to extraction after addition of 100 mL of distilled water and a thorough mixing. 23

After addition of 1.5 g sodium oxalate, the mixture was extracted three times with a liquid-liquid 24 5 extraction procedure involving methanol, diethyl ether, and n-hexane (50 mL of each solvent) (Fürst, 1 2006). The organic extract was purified by eluting through a column of Extrelut ™ impregnated with 2 96% sulfuric acid, followed by an automated cleanup with a Power-Prep ™ unit. In this unit, three 3 sequential chromatographic steps take place on columns packed with silica gel, alumina, and graphitic 4 carbon. PCDDs and PCDFs were quantified by high resolution gas chromatography on a 60m, I.D. 5 0.25 µm BPX-DXN column (SGE, Ringwood, Australia), coupled with high resolution mass 6 spectrometry (VG Autospec HRGC-HRMS) used in the selected ion monitoring mode (SIM). 7

Acceptable recovery rates for labelled internal standards were within the 60 -120 % range. Average 8 extended uncertainty was within 20% for each congener quantified. 9 10 PCP analytical procedure. Determination was carried out by HRGC-LRMS/MS, without the 11 derivatization of the analyte, according to a validated internal procedure. Briefly, a 300-mg test portion 12 of each homogenate sample was fortified with the internal standard 13 C-PCP at a level of 50 ng/g and 13 allowed to equilibrate, after thorough mixing, for 2 hours at room temperature. Then, 3-mL sodium 14 carbonate (0.1 M pH = 10.5) were added to the sample, that was defatted with two independent 15 liquid/liquid partitions with 3 mL of diethyl ether and 3 mL of n-hexane, respectively. After the 16 removal of the organic phase, the aqueous layer was acidified with the addition of 2-mL HCl 1.0 N and 17 the PCP back-extracted twice in organic phase, with the addition of 5-mL of t-butyl methyl ether, at 18 each extraction. Using tetradecane as keeper, the 10-mL extract was then carefully reduced to dryness 19 under a nitrogen stream and redissolved in 500 µL of iso-octane containing hexachlorobenzene (HCB) 20 (100 pg/µL) as injection standard for the GC-MS/MS analysis on a GCQ system 21 Milan, Italy). The chromatography was carried out on a 10-m 0.32-mm-i. product m/z 255. In both cases, the first transition was utilized for quantification. Recovery rates for 6 labelled standard were above 85 %; the calibration line had R = 0.9979. Results were expressed as the 7 mean of two independent extractions on the same sample with 25% of extended uncertainty. Definitive 8 confirmation of the PCP presence was also achieved with high resolution mass spectrometry on 9 Prospec instrument (Micromass, Manchester, UK), at a resolution of 5000 at 10% of peak height with 10 on-column injection and a chromatography setting similar to the GC-MS/MS analysis. 11 12 Data processing. The litter-to-eggs transfer ratio, (ratio between the contamination in eggs on lipid 13 base, and that in litter, on weight base) was classically considered, for possible aggregate exposures. 14 Congeners half-lives (HLs) were calculated from the linear regression of log-transformed concentration 15 data vs. time, according to the following canonical equation (Eqn. 1): ln(C) = ln(C 0 ) -kt , where the 16 C 0 represents the extrapolated contamination level, at time 0, according to the best fitting of the 17 regression curve, and k is its slope. The regression line fitness was tested with the Fisher test (P ≤ 18 0.01). The statistical work was performed by the Excel XP software. 19 20

RESULTS AND DISCUSSION 21

The depletion curves of PCP and total PCDDs and PCDFs (WHO-TEQs) are reported in Figure 2; 22 Figure 3 shows the depletion curves of selected PCDD and PCDF congeners. The pertinent statistical 23 coefficients are summarized in Table 1 . PCP seems to show a steady state up to the second week 24 after withdraw, with a sudden drop between the second and the fourth week; at this time-point, the PCP 1 in eggs was approximately 2.5 % of the highest contamination recorded. The observed trend does not 2 allow suitable modelling of depletion, likely due to PCP metabolism chemical features. PCP allows 3 two different mechanisms of interaction with macromolecules, according to the non-dissociated or the 4 anionic form. The latter can bind positive charged groups of proteins (Vidal-Madjar et al., 1992). The 5 rate of such protein interaction may be affected by a quite large variability, possibly involving a 6 distribution in compartments different from fat (around 2 % of body weight in hens), that could 7 influence the ways of excretion and the kinetics of depletion (Lin et al. 1996). 8 9 On the contrary, a log-linear trend was observed for PCDD and PCDF (TEQs) depletion with an 10 overall half life estimated as 3.8 weeks. The design of the study did not allow us to verify the possible 11 presence of the few-day α-phase of depletion, already described in other papers (Stephens et al., 1995;12 Le Bizec et al., 2005, van Eijkeren et al., 2006). However, the α-phase results are not able to influence 13 the overall half-life, as indicated by the good fitting of the linear regression of WHO-TEQ values vs 14 time (R > 0.998; F = 789; P F <0.01) (Figure 2), a finding in agreement with the conclusions reported by 15 Hoogenboom et al., (2006) and attributable to a reduced body burden contribution, a metabolism-16 driven scenario observable in high-production laying hen (Gregory and Robins, 1998). Previous papers 17 dealing with PCDD and PCDF depletion in eggs after feeding exposures reported total TEQ half-lives 18 of approximately 1.5 weeks after one week of exposure to contaminated feed (Traag et al., 2006), and, 19 after the steady state was reached, in the range of 4 -8 weeks, (Stephens et al., 1995;Pirard et al., 20 2006); a half-life of 7 weeks (referred to the second phase of depletion) has been reported in eggs from 21 flocks reared on naturally contaminated soil (Le Bizec et al., 2005). 22 minimizing possible body burden contributions (Pirard et al., 2006;Hoogenboom et al., 2006); (c) an 3 egg production rate approximately 25 % higher than that reported by Le Bizec and co-workers, along 4 with a more efficient depletion of such contaminants. 5 6 From the analytical profile derived from the C 0 values of individual congeners (Figure 3), the PCP 7 origin of PCDD and PCDF contamination in eggscharacterized by the predominant contribution of 8 the H 7 -and O 8 CDD congenersseems to be evident up to the second week from withdrawal. In fact, 9 the congener profile and the ratio between the aforesaid congeners in the eggs and in the wood 10 shavings not contaminated by animal dejects (roughly 1: 5) is in good agreement with that reported by 11 US EPA for PCP (US EPA, 2006). 12 13

In the weeks following withdrawal, such ratio is no longer mantained, as a consequence of the different 14 toxicokinetics of the H 7 -and O 8 CDD congeners in eggs (Figure 2, Table 1). Nevertheless, those 15 PCDDs still represent up to the 70 % of the overall contamination on a gravimetric basis and these 16 data could still represent a marker for the source of exposure up to the fourth week from that 17 withdrawal. 18 19 For most congeners, the F-test related to regression analyses indicates that such model was suitable to 20 describe PCDD and PCDF depletion (P F ≤ 0.01). Only for 1,2,3,7,8-P 5 CDD, the P F value fell within 21 0.01 and 0.05 (Table 1). The total contribution of the aforesaid congeners to the cumulative TEQ levels resulted lower than 5 % 2 throughout the depletion process. 3 4

In the case of 2,3,7,8-T 4 CDF congener, the pertinent regression equation resulted in a negative value of 5 the rate constant k, thus highlighting a possible transfer mechanism not only based on the fugacity 6 (Sweetman et al., 1999), as already described in other depletion studies related to clams (Miniero et al., 7 2005), trout (Brambilla et al., 2007) and cows ( Brambilla et al., 2008). 8 9 According to a an audit at the farm, as the flock has been reared on contaminated litter more than two 10 months before the first violative findings, the transfer ratio referred to a steady state were estimated 0.9 11 for PCDD and PCDF WHO-TEQs and 0.03 for PCP. The 0.9 value for PCDDs and PCDFs is higher 12 than the 0.6 derived from Le Bizec study (2005) for a 11.4 pgWHO-TE/g top soil contamination, and 13 lower than the 1.2 described by Schuler et al., (1997) for a 13 pgI-TE/g soil contamination. Such 14 differences may account for the different contamination patterns of the soils and the litter and for the 15 diversity in hens husbandry and management. 16 17 In our study, when calculated on gravimetric basis, the transfer ratio for 1,2,3,6,7,8-Hexa-, Hepta-and 18 O 8 -CCD result in 1.5, 0.49, and 0.31, respectively, in line with the general rule of a lower bio-19 availability for higher chlorine-substituted congeners. Such transfer ratios are in good agreement with 20 those calculated by Schuler and co-workers (1997) for the same congeners (1.6; 0.4, and 0.1, 21 respectively). It is worth noting that corresponding congeners COR of 42, 21, and 8 % have been 22

reported for feed exposures, (Hoogenboom et al., 2006). A possible explanation could rely on the 23 different ways of exposure: in our case, the inhalation as a consequence of dustiness and contaminants 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk The results of our work highlight the relevance of assessing the environmental quality of materials in 9 contact with food-producing animals that could contribute to the aggregate (dermal, inhalatory, and 10 alimentary) exposure in non-caged flocks. Due to their peculiar behaviour, the aforesaid requirement 11 appears to be more stringent in laying hens reared on PCP-contaminated litter, where a PCDD and 12 PCDF transfer ratio both on WHO-TE and gravimetric basis (for those congeners most representative 13 of a PCP exposure) higher than the corresponding COR described for feed exposures were found. For 14 monitoring purpose, PCP should be investigated on timber-derived materials; however, it would not 15 represent a reliable marker for the presence of violative residues of PCDDs and PCDFs in eggs due to 16 the strong differences in compound toxicokinetics. On the contrary, analytical congener patterns where 17 H 7 -and O 8 CDD account for more than the 70 % of the total TEQ amount, may help to trace back the 18 origin of contamination. Last but not least, the present study highlights some interesting features in the 19 depletion of T 4 CDF, that may expand the withdrawal time requested to recover the alimentary 20 soundness of the produce, when the contribution of such congener to total TEQs were relevant (Table 21 1). It is our aim to deepen such aspect in the near future.

  in the contamination profile may help to trace back to the source of contamination. Among 1 congeners, the 2,3,7,8 T 4 CDF exhibited a different depletion pattern, indicating a possible mechanism 2 of active transport. 3INTRODUCTION 8

  more than two months earlier, along with the change of the animal bedding, 1

Food

  d. FFAP column(HP Italia, 22 Milan, Italy), with a PTV injector (0.2 min hold at 60 °C, then to 200 °C at 14.5 °C/ sec) in splitless 23 mode (1.5 min). Ramp started from 80 °C (1 min), then to 135 °C (30 °C/min), and to 165 °C (10 ° 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk ). After a 2 min hold, 250 °C were reached at 20 °C/min. Carrier gas (He) was set at 3mL/min. 1 Ionization was achieved with electronic impact, positive ions, at 70 eV. Transfer line and source were 2 set at 240 °C, and 200 °C, respectively. MS/MS acquisition was carried out in Multiple Reaction 3Monitoring mode with the following ion species: precursor m/z 266 for native PCP, products m/z 202 4 and 230; precursor m/z 272 for 13 C-PCP, products m/z 207 and 236; precursor m/z 290 for HCB, 5

  (a) an exposure to wood shavings of more than eight weeks, sufficient to reach a 1 steady state(Pirard and De Pauw, 2005); (b) an exposure occurred in already egg-producing hens, thus 2

Page

  3,7,8-T 4 CDD and 2,3,4,7,8-P 5 CDF showed a correlation coefficient less than 0.878 24 for P = 95 %), and for this reason their depletion patterns are not reported in Figure3. 1

Food3

  litter (Lorber et al., 2002), facilitated by the long time spent in close contact with 1 the litter of nesting and laying 41-°C warm-blooded animals. 2On the contrary the poor PCP transfer ratio can be due to the following factors: a) the ionic behaviour 4 of the contaminant at the alkaline pH 7.4 -8.0 of the litter, where the PCP log Kow is estimated below 5 3; b) the possibility that depletion mainly occurs through pathways different from eggs. 6 7 CONCLUSION 8

PagePagePageFigure 1 .Figure 2 .Figure 3 .Figure 1 .Figure 2 .Figure 3 .

 123123 FIGURES CAPTIONS 10

Table 2 .

 2 Mean C 0 , k, and half-life (HL) values and their confidence intervals (P = 95%) of PCDD and PCDF congeners in eggs and total TEQs estimated from the depletion study (n = 5). Values rounded off to two figures. Critical value for R respectively 0.878 (P R = 95%) and 0.959 (P R = 99%)

		C 0 (pg/g fat)	k (weeks -1 )	HLs (weeks)	R	F	P F
		Mean	CI [95%]	Mean	CI [95%]	Mean CI [95%]
	2,3,7,8-T 4 CDD	-	-	-	-	-	-	0.803	5.43	0.10
	1,2,3,7,8-P 5 CDD	4.5	3.4-6.0	0.079	0.023-0.14	8.7	5.1-31	0.932	19.9	<0.05
	1,2,3,4,7,8-H 6 CDD	17	13-22	0.15	0.10-0.21	4.5	3.4-6.7	0.984	93.6	<0.01
	1,2,3,6,7,8-H 6 CDD	110	86-150	0.14	0.078-0.20	5.1	3.5-8.9	0.974	54.6	<0.01
	1,2,3,7,8,9-H 6 CDD	27	22-32	0.21	0.17-0.25	3.3	2.8-4.1	0.995	296	<0.001
	1,2,3,4,6,7,8-H 7 CDD	1300	714-2500	0.35	0.22-0.48	2.0	1.4-3.1	0.981	75.0	<0.01
	O 8 CDD	5800 1100-30000	0.55	0.21-0.88	1.3	0.79-3.3	0.949	27.0	<0.05
	2,3,7,8-T 4 CDF	-	-	-	-	-	-	0.914	15.1	<0.05
	1,2,3,7,8-P 5 CDF	2.1	1.8-2.5	0.072	0.036-0.11	9.7	6.5-19	0.965	41.0	<0.01
	2,3,4,7,8-P 5 CDF	-	-	-	-	-	-	0.713	3.10	0.18
	1,2,3,4,7,8-H 6 CDF	50	45-55	0.17	0.15-0.19	4.0	3.6-4.6	0.998	662	<0.001
	1,2,3,6,7,8-H 6 CDF	14	12-17	0.18	0.14-0.22	3.9	3.2-4.9	0.994	241	<0.001
	1,2,3,7,8,9-H 6 CDF	0.83	0.72-0.96	0.14	0.11-0.17	5.0	4.1-6.4	0.993	227	<0.001
	2,3,4,6,7,8-H 6 CDF	14	11-17	0.25	0.21-0.29	2.8	2.4-3.3	0.996	348	<0.001
	1,2,3,4,6,7,8-H 7 CDF	180	85-400	0.39	0.24-0.55	1.8	1.3-2.9	0.977	63.3	<0.01
	1,2,3,4,7,8,9-H 7 CDF	36	21-62	0.33	0.22-0.44	2.1	1.6-3.2	0.984	91.6	<0.01
	O 8 CDF	310	83-1100	0.48	0.22-0.75	1.4	0.92-3.2	0.958	33.9	<0.05
	Total WHO-TEQs	46	41-50	0.18	0.16-0.21	3.8	3.4-4.2	0.998	789	<<0.001

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


Susana M Rodrigues

P Vale

M J Botelho

S S Gomes

Extraction recoveries and stability of DSP toxins in naturally contaminated samples

Keywords: Okadaic acid, dinophysistoxin-2, acyl esters, solvent extraction, shellfish

During the last few years the occurrence of a high percentage of esters of DSP toxins has been observed in shellfish from the Portuguese coast. Most of the commercial bivalves contain DSP toxins in ester forms, either acyl derivatives of okadaic acid (OA) or of dinophysistoxin-2 (DTX-2). The stability of these toxins in shellfish tissues and in raw methanol extracts was investigated in two different species naturally contaminated, mussel and carpet shell, over a four week period. The results for both species revealed that DSP toxins were more stable in tissue than in raw methanol extracts. Losses of DSP toxins were seen in the first two weeks and were more than 30%, but after that a period of stabilisation was observed. The decrease was due probably from losses of esters of OA and DTX-2, the free toxins were stable over the period studied. The extraction most commonly used for chemical and biochemical assays relied on methanolic extraction with aqueous 80% methanol. In this work we have tested the extraction solvent on the extractability of DSP toxins from several naturally contaminated species. A single dispersive extraction with methanol, with solvent ratios of 70%, 80%, 90% and 100% were tested. After alkaline hydrolysis of esterified toxins and cleanup with hexane and dichloromethane, the samples were analysed by LC-MS. The recovery of DSP toxins increased with increasing percentage of methanol up to 90%. A decrease in recovery with 100% methanol was observed probably due to problems during the liquid-liquid partitioning.

Introduction

Diarrheic shellfish poisoning (DSP) is the marine syndrome that affects most recurrently shellfish from the Portuguese coast. Most of the commercial bivalves contain DSP toxins in ester forms, either acyl derivatives of okadaic acid or acyl derivatives of dinophysistoxin-2 [START_REF] Quilliam | Direct detection of acyl esters of okadaic acid and dinophysistoxin-2 in Portuguese shellfish by LC-MS[END_REF], Vale, 2006a). In highly contaminated Portuguese shellfish, it was demonstrated that 98-99% of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) were found in the ester form (DTX-3), being detected only after hydrolysis of shellfish extracts. For mussels (Mytilus galloprovincialis) and donax clams (Donax spp.)

OA and DTX-2 were found both esterified and in free form, with variable percentage of acyl esters [START_REF] Vale | Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS -a widespread phenomenon[END_REF][START_REF] Vale | Differential dynamics of dinophysistoxins and pectenotoxins between blue mussel and common cockle: a phenomenon originating from the complex toxin profile of Dinophysis acuta[END_REF]Vale, 2006b). Dinophysistoxin-1 (DTX-1) was not found until now in Portuguese shellfish [START_REF] Vale | Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS -a widespread phenomenon[END_REF].

Due to the absence of commercial standards and the large diversity of acyl esters that can be present in shellfish samples, they are indirectly analysed by an alkaline hydrolysis reaction that releases fatty acids from the parent toxins and gives an indirect estimation of their abundance. The hydrolysis procedure is a good option to quantify the amount of toxin esters belonging to OA-group and the effectiveness of hydrolysis depends on the binominous temperature/time of reaction and the base concentration used [START_REF] Rodrigues | Optimisation of the determination of DSP esters and their cytotoxicity evaluation[END_REF], Villar-González et al., 2008).

Few studies have been conducted about the behaviour of DSP toxins with storage at freezing temperatures immediately after harvesting the samples. The stability of DSP toxins in shellfish tissues and in raw methanol extracts was studied in two different naturally contaminated species, mussel and carpet shell, after harvesting and stored at -30ºC over a four week period.

The extraction most commonly used until recently for chemical and biochemical assays was based on the method of [START_REF] Lee | Fluorometric determination of diarrhetic shellfish toxins by high-performance liquid chromatography[END_REF] and relied on extraction with aqueous 80% methanol. New information was reported recently about the low extractability of DSP esters with the use of 80% methanol [START_REF] Holland | LC-MS methods for marine biotoxins and their introduction into the New Zealand shellfish regulatory programme[END_REF][START_REF] Mcnabb | Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study[END_REF]. Due to the high percentage of acyl esters commonly found in shellfish it is important to use efficient procedures that can extract DSP toxins, particularly ester forms, from shellfish tissues. In this work we have also tested the extraction solvent on the extractability of DSP toxins from several naturally contaminated shellfish species.

Material and methods

Shellfish samples used in this study were collected from the northwest coast of Portugal in the summer of 2005 and were obtained as part of the biotoxins monitoring programme. Samples were received in the laboratory within 24 h of being removed from the field and were analysed as soon as they arrived at the laboratory.

Stability of DSP toxins

The study on stability of DSP toxins was performed in two different species naturally contaminated with DSP toxins, mussel (Mytilus galloprovincialis) and carpet shell (Venerupis pullastra). Stability studies were carried out on shellfish tissues and on raw methanol extracts for different times of storage and were performed in triplicate (three aliquots were selected on each day).

Fresh edible tissues were removed from the shell, homogenised and 5g aliquots were weigh into 18 capped polypropylene tubes. All the aliquots were stored at -30ºC with the exception of the aliquots that were processed at the same day (day 0). Day 0: For each shellfish species 3 tubes with 5g homogenate were extracted with 20 ml of methanol:water (80:20, v/v) using dispersive extraction procedure and centrifuged. From each tube a 2 ml supernatant aliquot was taken for total DSP determination and a 2 ml supernatant aliquot for free DSP toxins determination. The remaining raw methanol extracts were stored at -30ºC. Hydrolysis was performed with 400 µl of 1M NaOH at 40ºC for 45 min. and neutralised with 425 µl of 1M HCl.

The cleanup of the hydrolysed and unhydrolysed extracts was performed through double washing with 2 ml hexane, addition of 0.5 ml of water and extracted twice with 2 ml of dichloromethane. The combined dicloromethane extracts were dried at 38ºC under reduced pressure on a Rapidvap and ressuspended in 500 µl of methanol:water (80:20, v/v). 2.5 µl aliquots were injected in triplicate on the LC-MS system according with the method described by [START_REF] Vale | Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS -a widespread phenomenon[END_REF].

Day 4: Stability on raw methanol extract: From the raw methanol extracts stored at day 0 a 2 ml supernatant aliquot was taken for total DSP determination and a 2 ml supernatant aliquot for free DSP toxins determination. The hydrolysis and cleanup procedure was the same described above.

Stability on shellfish tissues: From the shellfish tissues stored at day 0 a 5 g homogenate were taken in triplicate and extracted with 20 ml of methanol:water (80:20, v/v). After centrifugation a 2 ml supernatant aliquot was taken for total DSP determination and a 2 ml supernatant aliquot for free DSP toxins determination. The hydrolysis and cleanup procedure was the same described above. Day 7, 14, 21 and 28: The same procedure described for day 4.

Solvent Extraction

Seven shellfish samples were analysed and included mussel (Mytilus galoprovinciallis), cockle (Cerastoderma edule), grooved carpet shell (Ruditapes decussates), carpet shell (Venerupis pullastra), surf clam (Spisula solida), razor clam (Ensis spp.) and donax clam (Donax spp.). For each species the The extraction procedure was performed in triplicate with methanol:water ratios of 70%, 80%, 90% and 100% using a single dispersive extraction with 20 ml of the methanol solutions to be tested. A 2 ml aliquot of methanol extracts were hydrolysed with 400 µl of 1M NaOH at 50ºC for 1h and neutralised with 425 µl of 1M HCl. After alkaline hydrolysis of esterified toxins, the extracts were cleaned twice with 2 ml of hexane and dichloromethane extracted. For 70% and 80% methanolic extracts 0.5 ml of water was added before dichloromethane cleanup, while for 90% and 100% extracts 1 and 1.5 ml of water was added respectively. The combined dichloromethane layers were dried under reduced pressure and re-suspended in 500 µl of 80% aqueous methanol. 2.5 µl aliquots were injected in triplicate into the LC-MS.

Liquid chromatography-mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) was performed on a Hewlett-Packard (HP) Model 1100 coupled with an HP model 1946A Series single quadrupole mass spectrometer trough an atmospheric pressure electrospray ionization (ESI) interface operated in the negative ion mode according with the method described by [START_REF] Vale | Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS -a widespread phenomenon[END_REF]. Briefly a 2.5 µl aliquot was separated on a Merck Lichrospher 100 RP-18 column (5 µm, 125×2 mm) protected by a guard column (5 µm, 4×4 mm) with a mobile phase consisting of acetonitrile: 0.5% acetic acid (65:35, v/v) pumped at 200 µl/min. The column temperature was kept at 30ºC. After 2 min. of separation the effluent was directly coupled with the mass spectrometer and selected ion monitoring (SIM) was used to record the signals from the [M-H] -ions at m/z 803.5 for OA and DTX-2. Identities of OA homologues peaks were confirmed by matching of retention time with standards compounds. The linear calibration curve consisted of six standard solution in the range corresponding to 0-0.96 µg/ml of OA in methanol:water (80:20, v/v), prepared from the OA certified reference material (NRC CRM-OA-b) from Marine Biosciences Institute (NRC, Canada). For DTX-2 the response was assumed identical to OA on a peak area basis.

Results

Stability of DSP toxins

The stability of DSP toxins in shellfish tissues and in raw methanol extracts was evaluated during 28 days of storage. For both species, the concentration of total DSP toxins was determined by LC-MS in the hydrolysed extracts and represents the concentration of total OA plus total DTX-2 (free OA and free DTX-2 plus the OA and DTX-2 that resulted from the hydrolysis of acyl esters of OA and acyl esters of DTX-2). Free DSP toxins (free OA plus free DTX-2) were also quantified by LC-MS in the unhydrolysed extracts. DTX-1 was not found in these samples. The results for both species are presented in Figure 1. For carpet shell total DSP concentration decreased 20% in tissues homogenates and 35% in raw methanolic extract in the first two weeks, and no further decrease was observed in the two remaining weeks of storage at -30ºC (Figure 1a and Table 1). For carpet shell the concentrations of free DSP toxins were very low, near the quantification limit. Like in other reports for this species OA and DTX-2 were found 97% esterified [START_REF] Vale | Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS -a widespread phenomenon[END_REF][START_REF] Vale | Differential dynamics of dinophysistoxins and pectenotoxins between blue mussel and common cockle: a phenomenon originating from the complex toxin profile of Dinophysis acuta[END_REF]Vale, 2006b). For free DSP toxins the concentration in raw methanol extracts seems to have increased slightly, while in shellfish tissue a slight decrease was observed (Figure 1b). A decrease of concentration of DSP esters (acyl OA and acyl DTX-2) was observed in shellfish tissues and in methanolic extracts, with losses higher than 20% in shellfish tissue and higher than 35% in methanolic extracts after two weeks (Figure 1c). The stability of total OA was similar to the stability of total DTX-2 both in shellfish tissues and in raw extracts, as well as the stability of acyl esters of OA and DTX-2.

For mussels the same behaviour was observed for total DSP toxins in tissues homogenates and raw methanolic extracts with a decrease of 25% in total DSP concentration in the first two weeks of storage at -30ºC (Figure 1d and Table 2). The stability of OA and DTX-2 was similar in tissues and in raw extracts. For free DSP toxins no significant variations occurred in tissues homogenates and the slight increased observed in the methanolic extracts was not statistically significant (Figure 1e). The concentration of DSP esters decreased in methanolic extracts from the first week of storage and after two weeks losses of 40% were observed either in shellfish tissues or in methanolic extracts. In both cases comparing the stability OA acyl and DTX-2 acyl esters, the decrease of DTX-2 esters concentration was higher than the observed for OA esters. For acyl OA esters losses of 36% and 40% were found in tissues and in raw extracts, while for acyl DTX-2 esters the losses were higher than 65%.

Solvent Extraction

The extraction efficiency was tested for different shellfish matrices with different profiles of DSP contamination. For this study two shellfish species contaminated with only acyl esters of OA (carpet shell and donax clam) were chosen and five species contaminated with acyl esters of OA and DTX-2 (cockle, grooved carpet shell, mussel, razor clam and surf clam). The results obtained for the different methanol:water ratios tested with single dispersive extraction are presented in Table 3 andFigure 2. The highest DSP concentrations were obtained with 90% methanol for all the matrices analysed with the exception of carpet shell and grooved carpet shell. 90% methanol was able to extract 10 to 28% more DSP toxins from shellfish tissues than 80% methanol (Table 3 and4). Low recoveries were obtained for OA and DTX-2 with 70% methanol for all the species analysed, with this extraction solvent more than 40% of DSP toxins remained in shellfish tissues (Table 4). The DSP concentrations obtained with 100% methanol were lower than 90% methanol and high variability was observed between triplicates (Table 3).

Discussion

Some studies on the stability of DSP toxins in shellfish tissues have been conducted with the scope of preparing contaminated shellfish tissues to use as reference materials [START_REF] Hess | Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins[END_REF]McCarron et al., 2007). [START_REF] Mccarron | Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (Mytilus edulis) reference materials[END_REF] reported in a mussel tissue contaminated with OA and DTX-2 that these compounds were stable during the storage at -20ºC, but the mussel material used was frozen for several years prior to the study.

In the present work, the study was conducted in fresh shellfish tissues after harvesting and the stability of DSP toxins was evaluated during the period of storage. For both species losses of DSP toxins in shellfish tissues up to 20-25% occurred in the first two weeks, after that period a stabilisation was observed. The decrease was due mainly from losses of esters of OA and DTX-2. The free toxins were stable over the period studied, however the concentration in raw methanol extracts seems to have increased slightly probably due to some enzymatic activity in the extract that originate some hydrolysis of esters forms with the release of the parent compounds. A possible route for DSP acyl esters degradation is the oxidation of the carbon chain of acyl groups bounded to OA and DTX-2. The fatty acids attached to OA and DTX-2 found in Portuguese shellfish ranged from a carbon chain of C14 to C22 with saturated and unsaturated carbon bonds [START_REF] Quilliam | Direct detection of acyl esters of okadaic acid and dinophysistoxin-2 in Portuguese shellfish by LC-MS[END_REF], Vale, 2006a, Vale, 2006b). Oxidative mechanisms of fatty acids can occur in the double bond of monoenoics acids, polyenoic acids and in conjugated double-bond systems. The last one oxidizes with considerable ease by oxidation of the terminal carbons in the conjugated system [START_REF] Streitweiser | Introduction to Organic Chemistry[END_REF].

The results for both species revealed that DSP toxins were more stable in tissue than in raw methanol extracts, losses of OA and DTX-2 were higher than the observed for tissues. These results are important for monitoring proposes when some times it is necessary to repeat the analyses. The study of the stability on raw methanol extract was performed in a way to evaluate the possibility of using methanol extracts. These extracts are routinely kept as safeguard measure in case of laboratory accidents during sample preparation, additionally tissue homogenates are also kept for monitoring and research purposes. To obtain reproducibility and reliable results it is recommended that DSP determination should be performed during the first week of storage and starting from the shellfish tissue rather than from methanolic extract

The study on the extraction efficiency with different methanol ratios revealed a low extractability of DSP toxins with 70% and 80% methanol. Aqueous 90% methanol was more efficient in recovering DSP toxins than 80% methanol as observed by [START_REF] Holland | LC-MS methods for marine biotoxins and their introduction into the New Zealand shellfish regulatory programme[END_REF] and [START_REF] Mcnabb | Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study[END_REF].

According to these authors it's due to an improved extraction of esters forms of OA and DTX-2 from shellfish tissues. The DSP concentrations obtained with pure methanol were lower than with 90% methanol. Poor recoveries were obtained with this solvent probably due to problems of phase 7 separation during liquid-liquid partitioning. The phase separation of 100% methanol and hexane is not complete, part of the hexane will stay in the methanol layer and the residual hexane can interfere when after addition of water a dichloromethane clean-up is applied resulting in problems of phase separation. The creation of an emulsion during dichloromethane extraction may have occurred. To use hexane to wash an extract to remove fat a maximum percentage of 80% methanol is required. If water was added before hexane washing in order to obtain an extract containing 80% methanol there will be probably less problems in phase separation. Some authors have reported that pure methanol could improve efficiency of extraction but the coeluting matrix compounds can affect the ionization efficiency and cause poor reproducibility and accuracy [START_REF] Goto | Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry[END_REF][START_REF] Ito | Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins[END_REF][START_REF] Mcnabb | Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study[END_REF]. [START_REF] Mcnabb | Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study[END_REF] recommended the use of 90% methanol because it provides high recoveries and keep the MS signal enhancement/suppression effects at acceptable levels. Table 1. DSP concentrations obtained in carpet shell tissues and raw methanol extracts during the 28 days of storage at -30ºC (mean ± SD).

Table 2. DSP concentrations obtained in mussel tissues and raw methanol extracts during the 28 days of storage at -30ºC (mean ± SD). Table 3. Total OA+DTX-2 concentration obtained in different matrices using different methanol ratios in the extraction procedure (mean ± SD). Table 4. Percentage recoveries of total DSP (OA+DTX-2) obtained in different matrices using different methanol ratios relatively to 90% methanol.

  were homogenised and 5 g aliquots were taken into 12 polypropylene tubes.

Figure 1 .

 1 Figure 1. Stability of DSP toxins in shellfish tissues and raw methanol extracts of carpet shell a), b), c) and mussel d), e), f) during 28 days of storage at -30ºC (each value is the mean ± SD). and d) total OA+DTX-2 , b) and e) free OA+DTX-2, c) and f) total acyl OA+acyl DTX-2.

Figure 2 .

 2 Figure 2. Concentration of a) total OA, b) total DTX-2 and c) total DSP determined by LC-MS for different shellfish species using different methanol ratios in the extraction procedure (each bar represents mean ± SD). For donax clam and grooved carpet shell the extraction was not performed with 70% methanol. DTX-2 was not present in carpet shell and donax clam samples.

Figure 1 .Figure 2 .

 12 Figure 1. Stability of DSP toxins in shellfish tissues and raw methanol extracts of carpet shell a), b), c) and mussel d), e), f) during 28 days of storage at -30ºC (each value is the mean ± SD). a) and d) total OA+DTX-2 , b) and e) free OA+DTX-2, c) and f) total acyl OA+acyl DTX-2.

Table 1 .

 1 DSP concentrations obtained in carpet shell tissues and raw methanol extracts during the 28 days of storage at -30ºC (mean ± SD). The acyl OA and acyl DTX-2 concentrations were obtained by the difference between total OA equivalents (with hydrolysis) and free OA and free DTX-2 (without hydrolysis), respectively.

		400				Total DSP
	µ µ µ µg OA equiv. /100 g edible parts	0 50 100 150 200 250 300 350											MeOH 70% MeOH 80% MeOH 90% MeOH 100%
		Carpet	shell	Mussel	Cockle	Surf clam	Razor	clam	Grooved	carpet	shell	Donax	clam

Table 2 .

 2 DSP concentrations obtained in mussel tissues and raw methanol extracts during the 28 days of storage at -30ºC (mean ± SD). The acyl OA and acyl DTX-2 concentrations were obtained by the difference between total OA equivalents (with hydrolysis) and free OA and free DTX-2 (without hydrolysis), respectively.

	Day	Free OA	Free DTX-2	Tissue (µ µ µ µg OA equiv./100g) Acyl OA 1 Acyl DTX-2 1	Total OA	Total DTX-2
	0	16.4 + 2.1	55.8 + 5.6	63.6 + 2.6	31.1 + 4.6	80.0 + 2.5	86.9 + 4.6
	4	15.6 + 0.8	53.9 + 2.9	69.0 + 1.1	32.4 + 2.3	84.6 + 3.9	86.3 + 4.5
	7	17.3 + 1.1	57.7 + 3.5	61.2 + 4.1	23.3 + 5.0	78.4 + 3.5	81.0 + 2.4
	14	14.4 + 1.4	54.2 + 4.2	42.9 + 3.9	17.3 + 1.8	57.3+ 4.1	71.5 + 8.9
	21	14.5 + 1.3	52.7 + 4.7	54.4 + 4.9	22.4 + 7.1	68.9 + 4.4	75.1 + 5.8
	28	13.6 + 0.8	59.6 + 3.0	40.9 + 3.3	6.4 + 4.3	54.5 + 2.8	66.0 + 4.4
	Day	Free OA	Methanol extract (µ µ µ µg OA equiv./100g) Free DTX-2 Acyl OA 1 Acyl DTX-2 1 Total OA	Total DTX-2
	0	16.4 + 2.1	55.8 + 5.6	63.6 + 2.6	31.1 + 4.6	80.0 + 2.5	86.9 + 4.6
	4	16.6 + 1.7	51.1 + 4.2	52.9 + 1.1	21.9 + 4.4	69.5 + 1.7	73.1 + 3.2
	7	18.5 + 1.7	53.2 + 2.7	53.8 + 4.3	21.9 + 2.7	72.3 + 2.9	75.2 + 2.1
	14	22.4 + 1.4	55.1 + 3.6	39.0 + 6.7	9.4 + 3.8	61.4+ 7.0	64.6 + 4.4
	21	19.7 + 1.3	60.2 + 2.8	43.5 + 4.3	16.1 + 2.6	63.2 + 4.2	72.6 + 6.5
	28	21.4 + 0.8	55.0 + 2.8	37.8 + 1.7	9.8 + 3.5	59.3 + 2.7	64.8 + 5.8
	1

Table 3 .

 3 Total OA+DTX-2 concentration obtained in different matrices using different methanol ratios in the extraction procedure (mean ± SD). The extraction with 70% methanol was not performed for grooved carpet shell and donax clam.

	Species	Total OA + DTX-2 (µ µ µ µg OA equiv./100g) 70% MeOH 80% MeOH 90% MeOH 100% MeOH
	Mussel	83.2 + 8.7	160.0 + 28	183.1 + 60.8	64.2 + 32.3
	Cockle	55.2 + 2.7	167.6 + 8.0	181.9 + 23.3	153.4 + 17.7
	Grooved carpet shell	---2	118.0 + 9.3	100.6 + 17.2	82.9 + 11.0
	Razor clam	106.5 + 25.3	170.4 + 14.1	197.8 + 31.7	111.3 + 65.6
	Surf clam	18.6 + 10.7	46.3 + 0.9	56.9 + 1.9	40.9 + 40.0
	Carpet shell	40.3 + 2.5	103.7 + 2.9	98.7 + 5.5	54.1 + 42.2
	Donax clam	----2	232.0 + 9.3	320.4 + 51.1	135.9 + 152.1
	2

Table 4 .

 4 Percentage recoveries of total DSP (OA+DTX-2) obtained in different matrices using different methanol ratios relatively to 90% methanol. The extraction with 70% methanol was not performed for grooved carpet shell and donax clam.

	Recovery (%) 70% MeOH 80% MeOH 100% MeOH 45.4 87.3 35.0 30.3 92.2 84.3 ---2 117.2 82.4 53.9 86.2 56.3 32.7 81.3 71.8 40.8 105.1 54.8 ----2 72.4 42.4 2 Page Species Mussel Cockle Grooved carpet shell Razor clam Surf clam Carpet shell Donax clam

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Acknowledgements

Programme "Safety, Surveillance and Quality of Bivalve Molluscs" (QCAIII/med.4/MARE Programme) supported this work.


De La Iglesia
email: fac@tandf.co.uk

Keywords: Phycotoxins, Phycotoxins -other, Phycotoxins - Capillary electrophoresis, mass spectrometry, sample stacking, yessotoxins, 21 pectenotoxins. 22 23 Yessotoxin 25 45-OHYTX: 45-hydroxyyessotoxin 26 PTX-6: pectenotoxin-6 27 CE-ESI-MS: Capillary electrophoresis-electrospray ionization-mass spectrometry 28

Conditions for the determination of lipophilic marine toxins, such as yessotoxins and 2 pectenotoxins-6 were investigated with capillary electrophoresis coupled to mass 3 spectrometry with an electrospray ionization source. After optimization, a simple and 4 MS compatible alkaline volatile buffer solution of ammonium acetate was selected as 5 background electrolyte, with isopropanol/water (80/20, v/v) sheath liquid modified with 6 ammonium acetate used at the ESI source. Previously to CE separations, the application 7 of an on-line sample pre-concentration approach based on field-amplified sample 8 stacking was accomplished to increase sensitivity. As a result, the limits of detection 9 provided by CE-ESI-MS were 0.02 µg mL -1 (0.01 µg g -1 ) which corresponded to 1.25 10 pg for yessotoxin and 0.25 µg mL -1 (0.13 µg g -1 and 13.25 pg on capillary) for Accuracy tests showed 97.7% recovery from spiked blank mussel samples which 12 showed no significant matrix influence running under optimal conditions. Intermediate 13 precision was close to 4% RSD for the migration time, and an RSD of 7.5% for peak 14 areas. The method was successfully applied to naturally contaminated seafood samples, 15 in which yessotoxins and pectenotoxins-6 were clearly determined. This work 16 demonstrated the potential of CE-ESI-MS to be applied for a sensitive determination of 17 lipophilic toxins from the marine environment as alternative to LC-ESI-MS for this 18 purpose.

The development of improved analytical methods for efficient and reliable 4 determination of marine toxins present in seafood is still challenging. Several methods 5 have been developed mostly focused in the improved sensitivity and selectivity of 6 which HPLC coupled with different detection modes has been the most widely applied. 7

Capillary Electrophoresis (CE) emerges as a very promising technique to be applied in 8 this field, and is considered one of the most powerful separation technique which has 9 rapidly grown and matured since its introduction in the early 80s by Jorgenson and 10 Lukacs [START_REF] Jorgenson | Zone electrophoresis in open-tubular glass capillaries[END_REF][START_REF] Jorgenson | Capillary zone electrophoresis[END_REF]. Indeed, CE has become 11 a well-established alternative to liquid chromatography (LC) since it is characterized by 12 some important features such as different separation mechanism, high efficiency, 13 simplicity, low-cost operation and usually short analysis time [START_REF] Altria | Validating CE Methods for Pharmaceutical 16 Analysis[END_REF]. 14 However, the lack of sensitivity has been found as the major limitation for the 15 application of CE to several contaminants such as marine biotoxins, since the limits of 16 detection provided by this technique in terms of concentration are often higher that 17 those achieved in LC. Consequently, only a few number of applications have been 18 reported in this particular field (Youyi Wu, et al. 2006 20 Shea 1997;Bouaïcha, et al. 1997a;Bouaïcha, et al. 1997b). This problem derives from 21 the low amount of sample that can be injected onto capillary without overloading effects 22 and moreover, when UV detection is used, the short optical path length. Nevertheless, 23 significant efforts have been focused in the last years to overcome this drawback, 24 resulting in several on-line pre-concentration modes that can be applied to improve the 25 sensitivity [START_REF] Osbourn | On-line preconcentration methods for 19 capillary electrophoresis[END_REF][START_REF] Chien | Sample stacking revisited: A personal perspective[END_REF]). Among them, sample stacking is one of 26 the most frequently applied with this aim since it: (i) is simple and can be performed in 27 a single buffer system, (ii) can be universally applied for every charged analytes, (iii) 28 does not require any additional instrumentation or device. In particular, previous work 29 carried out by this research group has focused on the development of strategies for on-30 line toxin enrichment based on sample stacking with successful results (Gago- Martínez, 31 et al. 2006). On the other hand, the coupling of CE with mass spectrometry (MS) 32 detection has been gaining attention since its introduction in 1987 by Olivares et al. 33 [START_REF] Olivares | On-line mass spectrometric 16 detection for capillary zone electrophoresis[END_REF] previously mentioned for CE with the ability of the mass spectrometer to provide highly 1 sensitive and selective detection. Furthermore, additional structural information can be 2 very useful to carry out unequivocal confirmation of the analytes, and easy separation of 3 co-eluting peaks can be performed by using the mass analyzers as second separation 4 dimension. Recently, relevant applications of CE-MS have been reviewed, among 5 others, in the fields of environmental and food analysis [START_REF] Klampfl | Recent advances in the application of capillary electrophoresis with 33 mass spectrometric detection. Electrophoresis 27 (1)[END_REF]Simó, et al. 6 2005). 7 8

The present work reports on the development of a CE-MS method involving a sample 9 stacking step to be applied to the determination of lipophilic toxins, mainly yessotoxins 10 (Figure 1A) which are polycyclic ether toxins produced by some dinoflagellates species 11 from the marine environment such as Protoceratium reticulatum [START_REF] Satake | Identification of Protoceratium 26 reticulatum as the biogenetic origin of yessotoxin[END_REF], [START_REF] Murata | Isolation and structure of 13 yessotoxin, a novel polyether compound implicated in diarrhetic shellfish 14 poisoning[END_REF] Lingulodinium polyedrum (=Gonyaulax polyedra) [START_REF] Draisci | High levels of yessotoxin in mussels and presence of 1[END_REF]) and Gonyaulax 13 spinifera [START_REF] Rhodes | 24 Yessotoxin production by Gonyaulax spinifera[END_REF]), but also pectenotoxin 6 (Figure 1B), which is a 14 macrolactone that contain multiple polyether ring units produced by Dynophysis spp 15 [START_REF] Mackenzie | 4 Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and 5 Dinophysis acuminata from New Zealand[END_REF][START_REF] Puente | Studies 11 of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland 12 using multiple tandem mass spectrometry[END_REF][START_REF] Mackenzie | 4 Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and 5 Dinophysis acuminata from New Zealand[END_REF]) . These 16 harmful algae species occur worldwide and seafood contamination as a consequence of 17 algae blooms has been reported worldwide. LC-MS methods have been often applied 18 for the determination of such toxins [START_REF] Draisci | High levels of yessotoxin in mussels and presence of 1[END_REF] were purchased at the market in Vigo (Spain) harvested from the Rías Bajas (NW 3 Spain) shellfish rafts. Stock standard solutions of YTXs and PTX-6 were prepared in 4 methanol, and stored at -18 ºC until analysis. HPLC-grade methanol, acetonitrile and 2-5 propanol were purchased from Merck (Darmstadt, Germany) and Scharlab (Sentmenat, 6 Spain), respectively. Ultrapure water (18.2 MΩ cm at 25 °C and total organic carbon 7 below 10 µg L -1 ) was produced with a Milli-Q water purification system (Millipore 8 Corporation, Bedford, MA, USA). Sodium hydroxide pellets and ammonium acetate 9

were both for analysis (puriss, ≥98%) Sigma Aldrich, St Louis, MO). NH 3 25% was 10 purchased from Panreac (Barcelona, Spain). 11 12Sample pre-treatment. [START_REF] Gago-Martínez | 14 Mycotoxins and Phycotoxins[END_REF] Shellfish samples (ca. 50 shellfish samples) were selectively extracted and further 14 purified with solid-phase extraction following the specific protocols previously 15 carried out with MS using YTX and PTX-6 standard solution (12 and 20 µg mL -1 16 respectively) at 400 µL min -1 of flow rate to characterize the MS spectra of both toxins. 17 ion was obtained for YTX compared to that achieved in FIA experiments, and the 1 pseudo-molecular ion at 1141.5 m/z showed in this case the highest relative abundance. 2

Hence, ions at 1141.5 and 1157.5 m/z were chosen for monitoring YTX and 45-3 OHYTX respectively, in selected ion monitoring mode. No changes were observed for 4 the PTX-6 molecular ion. The effects on signal were examined for coupling parameters 5 such as nebulizer pressure, drying gas flow, drying gas temperature, sheath liquid 6 composition and flow rate. The stability of the ESI source was effectively monitored by 7 plotting the current of the MS capillary which provided information about the flow of 8 ions reaching the MS entrance. 9 10The nebulizer pressure directly affects the ESI cone shape and droplet size so it must be 11 carefully optimized, and depends on the flow delivered to the interface. The optimal 12 value was obtained at 17 psi of pressure. Lower applied pressure led to lower ionization 13 yields and higher values showed decreased migration times as a consequence of the 14 Venturi effect at the end of the spray needle which soak the buffer up through the 15 capillary. This deleterious effect must be avoided since it generates a hydrodynamic 16 flow with a laminar flow profile into the capillary and consequently directly decrease 17 the CE performance. with this aim and their efficiency was tested also for this application. Methanol and 28 acetonitrile showed the best responses close to a 40% higher than that achieved with 2-29 propanol. However, 2-propanol provided the best ESI stability and therefore a sheath 30 liquid consisted in 2-propanol/water (80/20, v/v) was eventually selected as optimal 31 because it showed the best compromise between sensitivity and stability. The addition 32 of volatile organic modifiers such as formic or acetic acid or their ammonium salts are 33 commonly used in MS analysis specially to promote the analyte ionization at ESI 34 and to the high organic percentage used in the sheath liquid that promotes the 8 ionization. Higher concentration of ammonium acetate rapidly decreased the ionization 9 efficiency. The influence of the sheath liquid flow rate delivered to the interface was 10 examined between 2-15 µL min -1 (which corresponded to 0.2-1.5 mL min -1 pumped by 11 the auxiliary pump). Flow rates lower than 6 µL min -1 and higher than 12 µL min -1 did 12 not provide stable signal at the ESI source. Between 6-12 µL min -1 , the signal decreased 13 when the flow rate increased as a consequence of the dilution effect. Hence, 7 µL min -1 14 of flow rate was selected as optimum. 15 16

Electrophoretic conditions 17

Mass spectrometry compatible ammonium acetate buffer was selected as BGE. [START_REF] Osbourn | On-line preconcentration methods for 19 capillary electrophoresis[END_REF] Electrophoretic parameters were optimized in terms of buffer concentration, pH, 19 addition of organic modifiers and running voltage. The best efficiency was achieved 20 with 10 mM ammonium acetate adjusted at pH 8.5 with ammonium hydroxide. Higher 21 buffer concentration gave less signal intensity probably as a consequence of the 22 competitive effect of the buffer ions with the analyte in the ESI process. Resolution (R s ) 23 between two peaks was calculated through the expression R s = 2(t m2 -t m1 ) (w 2 + w 1 ) -1 , 24 where t m are the migration times and w the peak widths of the analytes 2 and 1 25 respectively. Efficiency in terms of theoretical number plates was experimentally 26 measured from the electropherograms through the expression N = 5.54 (tm/w ½ ) 2 , where 27 t m is the migration time of the analyte and w ½ is the peak width at half of the peak 28 height. Electrophoretic mobility (µ e ) was calculated by means of the apparent mobility 29 (µ a ) and the electroosmotic flow mobility (µ eof ) with the expression: µ a = µ e + µ eof . 30 Apparent mobility was directly calculated from the electropherograms through the 31 expresion µ a = l L t -1 V -1 , where L and l are the total and effective capillary length 32 respectively, V is the applied voltage and t the migration time. Results for YTXs and PTX-6 are summarized in Table 1. YTX and the analogue 45-1 OHYTX were not resolved by CE because their similarities in their q/r ratio eluting at 2 7.0 min, and no improvements in resolution were observed by addition of organic 3 modifiers (methanol, acetonitrile and isopropanol) to BGE. Nevertheless, the required 4 selectivity was obtained by means of the mass analyzer, extracting the signal 5 corresponding to each ion from the total ion electropherograms. PTX-6 eluted at 5.5 6 min, and an adequate Resolution (R s ) of 1.35 was found between PTX-6 and YTXs. 7 8 Theoretical number plates showed the high resolution provided by CE, higher than that 9 than usually achieved in chromatography. Electrophoretic mobilities are characteristic 10 for each analyte and only slight differences are observed even when changes on the 11 electrophoretic system or BGE are performed. Hence, this is valuable information if a 12 change in the buffer system is required in order to increase the applicability of CE to 13 other toxins not tested yet. 14 15 An on-line pre-concentration procedure based on field-amplified sample stacking 16 (FASS) was also applied in CE-ESI-MS. This procedure consists basically on 17 dissolving the sample in a less conductivity buffer than BGE used for separation. The 18 electric field strength will be amplified over the low conductivity region at the head of 19 the capillary where the charged analytes will migrate rapidly, slowing down when they 20 reach the buffer boundaries. Consequently, FASS mechanism allows stacking the 21 analytes from a large plug of sample into a narrow band enhancing the sensitivity. For 22 CE-MS, the optimal sample buffer consisted in a MeOH/BGE (70/30, v/v) solution 23 which was hydrodynamically loaded onto capillary by application of 50 mbar of 24 pressure for 20 s (corresponding to 53 nL of sample), and then stacking and separation 25 were performed by application of a running voltage of 20 kV with 0.30 min potential 26 ramp. Figures 2 and3 show examples of the analysis of YTXs and PTX-6 standard 27 solutions, where the high efficiency provided by CE for this application can be 28 observed. [START_REF] De La Iglesia | Advanced studies for the application of high-30 performance capillary electrophoresis for the analysis of yessotoxin and 45-31 hydroxiyessotoxin[END_REF] 30

Evaluation of the CE-ESI-MS conditions. 31

Limits of detection (LOD) were determined from spiked blank mussel samples on the 32 basis of a signal-to-noise ratio of 3 were 0.02 µg mL -1 (0.01 µg g -1 ), corresponding to 33 1.25 pg of YTX, and 0.25 µg mL -1 (0.13 µg g -1 ), corresponding to 13.25 pg of PTX-6. 34 Furthermore, the application of FASS as on-line pre-concentration approach allowed 1 decreasing these limits in a simple and low-cost way that does not require additional 2 instrumentation. Thus, the LOD offered by CE-MS are similar to that provided by LC-3 MS although much lower mass of toxin is required for CE operation and fulfills the 4 requirements of Regulation (EC) No 853/2004 for these toxins (1µg g-1 for YTXs and 5 0.16 µg g-1 for PTXs). This fact means a great advantage over other liquid separation 6 techniques since the quantities available of standards of these toxins are often very low 7 or expensive. Furthermore, the low limits achieved makes feasible the application of CE 8 not only with food safety purposes but also to eco-physiological or toxicological studies 9

where the amount of sample can be much lower. Calibration for YTX was performed by 10 running triplicates at five levels over the range 0.25-6.0 µg mL -1 . The regression 11 equation obtained (y = 1194658 x + 52461) showed good regression coefficient, r = 12 0.9985, over the calibration range studied. Quantification of 45-OHYTX was performed 13 with calibration curve for YTX assuming the same response than that obtained for YTX 14 since the availability of standards for this analogue was very limited. Repeatability was 15 calculated by %RSD of seven replicates within a day while intermediate precision was 16 calculated by means of %RSD of nine replicates spread over 3 days. The results 17 revealed good precision for migration time with a RSD close to 4% in all cases. 18 However, peak area showed an intermediate precision of 7.5% RSD which should be 19 bore in mind for quantitative purposes. Accuracy study was considered because ion 20 suppression effects at ESI source has been described for many applications when 21 dealing with very complex matrices such as food samples, and occurs due to the 22 competitive effect at ESI source of some interference compounds which have not 23 previously resolved. In particular, this effect has already been described for YTXs 24 determination by LC-ESI-MS [START_REF] Ito | Matrix effect and correction by standard addition in 26 quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic 27 shellfish poisoning toxins[END_REF]. Matrix effects were evaluated for 25 CE-ESI-MS analysis by running triplicates of standards solution of YTX dissolved in 26 pure matrix and afterwards triplicates of shellfish matrix blank samples spiked with the 27 same amount of YTX standard. Recovery was 97.7% which means that for this 28 particular application, interferences co-existing with YTXs in naturally contaminated 29 samples are efficiently resolved by the separation mechanism provided by CE. For 30 PTX-6, it was not possible to carry out a complete evaluation of the CE-ESI-MS system 31 due to the low amount of this toxin available for this study, although preliminary 32 precision data obtained showed %RSD lower than 5% both for migration time and peak 33 area. 34 The applicability of CE-ESI-MS has been tested for food samples naturally 3 contaminated. Figure 4 shows the electropherograms obtained for the application of the 4 optimized conditions to the analysis of shellfish samples from Japan which was 5 naturally contaminated not only with YTXs but also with PTX-6. Hence, it was 6 demonstrated the potential of CE for this application, which was clearly enhanced with 7 the ability of MS for confirmation purposes. Moreover, the high efficiency provided by 8 CE as well as the improved sensitivity achieved by the application of a sample stacking 9 procedure allowed to determine these toxic compounds at levels as low as pg. Neither 10 YTXs nor PTX-6 were found in mussel samples from Galician Rías (Mytilus 11 galloprovincialis) harvested in the Galician rafts. 12 13

Conclusions 14

Capillary electrophoresis-electrospray ionization-mass spectrometry was developed and 15 applied to the determination of YTX, 45-OHYTX and PTX-6. Once the MS, 16 electrospray and electrophoretic parameters were optimized, this technique resulted in 17 an efficient alternative for the determination of these toxic compounds present in 18 seafood samples. Sensitivity was improved by the application of a field-amplified 19 sample stacking procedure and was shown to be comparable in terms of concentration 20 to that provided by LC-MS, although significantly lower mass analyte consumption is 21 required. In fact, the minimal sample loading requirements for full CE operation might 22 be very attractive in the marine toxins field, where the limited amount of standards is 23 one of the most important obstacles that have been found in research and routine 24 analysis. Besides, good reproducibility was achieved for migration times close to 4% 25 RSD whereas a slightly greater variation was obtained for peak areas. However, 26 accuracy test showed a recovery of 97.7% for YTX from real samples showing no 27 significant influence from the sample matrix. The ability to provide confirmation by MS 28 ensured highly specific identification of these toxins by means of the mass filters 29 enhancing the potential of CE for this particular application. Nevertheless, further work 30 should be performed to improve resolution on the capillary between YTXs as well as to 31 perform method validation for the studied compounds and other lipophilic toxins once 32 Certificate Standard Solutions and Reference Materials are available. 33

  Determination of yessotoxins and pectenotoxins in shellfish by1 capillary electrophoresis-electrospray ionization-mass spectrometry.

6 a 10 11and discussion 12 13

 61012 described in (De la Iglesia & Gago-Matínez 2007).16 17 Instrumentation and operation procedures.[START_REF] Osbourn | On-line preconcentration methods for 19 capillary electrophoresis[END_REF] Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) 19 analysis was performed using the 3D CE system (Agilent Technologies) with CE-MS 20 capillary cassette coupled to the Agilent 1100 Series single quadrupole mass 21spectrometer. An Agilent CE-ESI-MS sprayer kit (G1607A) and an Agilent CE ESI-MS 22 adapter kit (G1603A) were used to connect the CE system to MS. A binary pump 23 Agilent 1100 series (G1312A) equipped with a 1/100 splitter was employed to deliver 24 the sheath flow to ESI source. Nitrogen were used both for drying and nebulizer gases.25 Chemstation software also from Agilent was used for the entire instrument control, data 26 acquisition and data analysis.[START_REF] Shea | Analysis of brevetoxins by micellar electrokinetic capillary 28 chromatography and laser-induced fluorescence detection[END_REF] 28 All separations were performed on uncoated bare fused-silica capillaries of 80.0 cm of 29 total and 21.6 cm of effective length and 50 µm i.d. from Composite Metal Services 30 (The Chase, Hallow, UK). Capillaries were conditioned before its first use at 60 ºC with 31 the following solutions: 1 M NaOH for 15 min, then water for 15 min and eventually 32 background electrolyte (BGE) for 15 min. At the beginning of each day, the capillary 33 was rinsed with NH 4 OH 1.0 M for 15 min, then water for 15 min and finally BGE for 34 Between runs, the capillary was just flushed with BGE for 5 min. All CE 1 solutions were filtered through Millex®-HV 0.45 µm cut-off polyvinylidene-fluoride 2 (PVDF) membranes (Millipore, Bedford, MA, USA). BGE consisted in 10 mM 3 ammonium acetate solution adjusted at pH 8.5 and was prepared fresh daily by diluting 4 a 0.2 M ammonium acetate stock solution with Milli-Q water, and degassed for 15 min 5 in an ultrasonic bath to prevent bubble formation. The capillary was thermostated using Peltier system at 25 ºC. Sample buffer consisted in a MeOH/BGE (70/30, v/v). 7Hydrodynamic injection was performed at 50 mbar of pressure for 20 s, and afterwards 8 sample stacking and separation was carried out by application of 20 kV of running 9 voltage with 0.30 min potential ramp.ResultsPreliminary studies. 14Previous to CE-ESI-MS coupling, some flow injection analysis (FIA) experiments were 15

Full-scan mass

  spectra acquired in negative mode from 50 to 1200 m/z for YTX 18 standard gave majority signals at m/z 1163.5, 1141.5 and 1061.5, which corresponded to 19 [M-Na] -, [M-2Na+H] -and [M-2Na-SO 3 +H] -respectively[START_REF] Murata | Isolation and structure of 13 yessotoxin, a novel polyether compound implicated in diarrhetic shellfish 14 poisoning[END_REF] Draisci, 20 et al. 1998). The ion at m/z 1061.5 showed the highest relative abundance for suchFIA 21 analysis, while the ion [M-2Na] -2 at m/z 570.5 was not observed for the experimental 22 conditions applied. Signal of PTX-6 was obtained at 887.5 m/z which corresponded to 23 the molecular ion [M-H] -. MS was tuned and the optimized instrument parameters were 24 as follows: capillary voltage 4500V, fragmentor 275V, gain 3.0, peak width 0.30, high-25 resolution mode. 26 27Sheath-flow ESI source conditions 28A coaxial sheath-flow interface originally developed by Smith et al.(Smith, et al. 1988) 29 was used to couple CE with MS. This approach is still the most frequently applied for 30 CE-MS hyphenation[START_REF] Klampfl | Recent advances in the application of capillary electrophoresis with 33 mass spectrometric detection. Electrophoresis 27 (1)[END_REF]) and is based on a sheath liquid which flows around 31 the outer surface of the CE capillary, and mixes with the CE eluent at the end of the 32 capillary. The sheath flow enhances the CE flow to a level adequate for electrospray 33 operation and closes the electrical circuit. After CE-MS coupling, different predominant 34

18 19

 18 Drying gas flow and temperature were also optimized between 3-11 L min -1 and 250-20 350 ºC respectively, and the best response was achieved at 4 L min -1 and 350 ºC. 21 Furthermore, drying gas flows above 9 L min -1 led to unstable ESI operation. 22 23 As previously mentioned, the sheath liquid closes the CE electrical circuit and provides 24 an adequate flow rate for ESI operation. Since the sheath liquid is the dominant flow at 25 the ESI sprayer, a high content of an organic solvent improves the ionic evaporation 26 efficiency. Methanol, acetonitrile and 2-propanol are the most common solvents used 27

  acetate was selected as additive for sheath liquid in CE-ESI-MS 1 coupling bearing in mind that this modifier is also the most frequently added to the 2 mobile phase in LC-ESI-MS with this aim(Fernandez Amandi, et al. 2002; Ciminiello, 3 et al. 2002;[START_REF] Mackenzie | 7 Complex toxin profiles in phytoplankton and Greenshell mussels (Perna 8 canaliculus), revealed by LC-MS/MS analysis[END_REF] McNabb, et al. 2005). The highest ionization yield 4 was achieved by adding 0.1 mM ammonium acetate to the sheath liquid. This value was 5 lower than those found in the literature for LC-MS analysis of yessotoxins, although can 6 be explained due to the low flow delivered to the ESI source in CE-ESI-MS coupling, 7

Figure 1 9 Figure 2 .

 192 Figure leyends: 5 6 Figure 1. (A) Chemical structures of yessotoxin (R=H) and 45-hydroxyyessotoxin 7 (R=OH); (B) Chemical structure of pectenotoxin 6 (R=COOH). 8 9 Figure 2. CE-ESI-MS analysis of YTXs standard solutions. A) Standard of 6 µg mL -1 10 YTX and B) Standard of 3 µg mL -1 45-OHYTX. Experimental conditions: bare fused-11 silica capillary of 80.0 cm 50 µm i.d.; BGE: 10 mM ammonium acetate, pH 8.5; 12 sample buffer: MeOH/BGE (70/30); injection FASS: 50 mbar for 20 s. Run: 20 kV, 1325ºC. MS conditions: acquisition in negative polarity mode; sheath-liquid: 14 isopropanol/water (80/20, v/v), 0.1 mM ammonium acetate; drying gas: nitrogen, 4 L 15 min -1 , 350 ºC; nebulizer pressure 17 psi; capillary voltage 275 V, SIM mode of 1141.5 16 and 1157.5 m/z for YTX and 45-OHYTX respectively. 17

18 Figure 3 . 21 Figure 4 .

 183214 Figure 3. CE-ESI-MS analysis of PTX-6 standard solution of 20 µg mL -1 . Extracted ion 19 electropherogram at 887.5 m/z. Experimental conditions as in Figure 2. 20 21 Figure 4. CE-ESI-MS analysis of a shellfish sample from Japan naturally contaminated 22 with 22.63 ± 1.36 µg g -1 of YTX, 12.34 ± 0.74 µg g -1 de 45-OHYTX and PTX-6 (not 23 quantified). (A) total ion electropherogram; (B) extracted ion electropherogram at 887.5 24 m/z; (C) extracted ion electropherogram at 1141.5 m/z; (D) extracted ion 25 electropherogram at 1157.5 m/z. Experimental conditions as in Figure 2. 26 27 28

  Figure 1B

  ; Kvasnicka, et al. 2006; Gago-19 Martínez, et al. 2006; Gago-Martínez, et al. 2003; Aguete, et al. 2003; Tan & Lau 2000;

  , as this hyphenated technique combines all the advantages 34

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while the application of CE-MS is still very limited. Preliminary 22 work has been carried out by this research group on the application of this technique to 23 yessotoxins (De la Iglesia & Gago-Matínez 2007) while the results of its application to 24

	19	; MacKenzie, et al. 2005;
	20	Fernández Puente, et al. 2004; Suzuki, et al. 2005; Fernandez Amandi, et al. 2002;
	21	Goto, et al. 2001; Ito & Tsukada 2002; Ciminiello, et al. 2002; MacKenzie, et al. 2002;
	25	Aasen, et al. 2005), pectenotoxins group are primarily described here. The paper provides comprehensive
	26	information about the development and evaluation of the optimal conditions for the
	27	simultaneous analysis of yessotoxins and pectenotoxins, as alternative to LC-MS for the
	28	analysis of such lipophilic toxins present in seafood samples.
	29

Materials and methods 30 Chemicals and materials 31

	1	extracts from Japan were kindly provided by Dr. Takeshi Yasumoto (Japan Food
	2	Research Laboratories, Tokyo, Japan). Blue mussel samples (Mytilus galloprovincialis)
	32 33	Research grade analytical standard of yessotoxin (YTX) was provided by Dr. Paul McNabb from Cawthron Institute (Nelson, New Zealand). Research-grade analytical o r P e e r R e v i e w O n l standard of 45-hydroxyyessotoxin (45-OHYTX), pectenotoxin 6 (PTX-6) and shellfish F y

manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

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Table 1 :

 1 Retention time, Theoretical number plates, Electrophoretic mobilities, limits of 16 detection (LODs) and resolution CE-ESI-MS applied to YTXs and PTX-6 17 determination. LODs were determined from spiked blank mussel samples on the basis of

	18
	19	a signal-to-noise ratio of 3
	20
	21
			PTX-6	YTX/ 45-OHYTX
	Retention time (min)		5.5	7.0
	Theoretical number plates (N)		34,560	26,463
	Electrophoretic mobility (cm 2 V -1 s -1 ).	-2.25 10 -5	-1.2 10 -4
	LOD (pg)		13.25	1.25
	LOD (µg mL -1 )		0.25	0.02
	LOD (µg g -1 )		0.13	0.01
	Resolution (PTX-6 -YTX)			1.35
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Acknowledgements


M Reverberi

S Zjalic

F Punelli

A Ricelli°

A A Fabbri

C Fanelli

Apyap1 affects aflatoxin biosynthesis during Aspergillus parasiticus growth in maize seeds

Keywords: Apyap1, oxidative stress, Aspergillus parasiticus, maize seeds, aflatoxin biosynthesis

In fungal cells grown in synthetic media we have demonstrated that the Apyap1 gene is implicated in the modulation of aflatoxin biosynthesis following the perturbation of the redox balance. In this study, we suggest that an association between oxidative stress and aflatoxin biosynthesis occurs also in maize seeds. We used ∆Apyap1, a strain in which the gene Apyap1 was disrupted, to verify whether this oxidative stress related transcription factor, by affecting cell redox balance, can have a role in the modulation of aflatoxin synthesis. The amount of hydroperoxides (ROOH) produced by wild type (WT) and ∆Apyap1, both grown in potato dextrose broth, was assayed in the filtrate. In maize seeds (30 g), which were inoculated with WT and ∆Apyap1conidia, and incubated at 30°C for 15 days, we analysed lipoxygenase activity (LOX), lipoperoxides (LOOH) production, fungal growth and aflatoxin biosynthesis. It was observed that ∆Apyap1 releases more hydroperoxides in the culture media and more aflatoxins in seeds, likely through a stronger stimulation of LOX, which, in turn led to a greater LOOH production in the seeds. On the basis of our results, we formulated hypothesis regarding strategies to control aflatoxin synthesis.

Introduction

A more detailed knowledge of the organization, regulation, and expression of genes linked to aflatoxin biosynthesis is important to detect early these mycotoxins and the mycotoxigenic fungi.

Additionally, it allows finding rapid prevention strategies to control their presence in different food sources. However, the molecular approach must advantage of information concerning those events that influence the fungal physiology in relation to aflatoxin production.

In the eighties we have demonstrated how lipoperoxidation can intervene in modulating the aflatoxin biosynthesis. This evidence was brought up both in vitro studies and in different seeds [START_REF] Fabbri | Lipoperoxidation and aflatoxin biosynthesis by Aspergillus parasiticus and A. flavus[END_REF][START_REF] Fanelli | Relationship between lipids and aflatoxin biosynthesis[END_REF]. In those systems, various antioxidants and free radicals scavengers affected the aflatoxin production, in different ways [START_REF] Fanelli | Effect of different antioxidants and free radical scavengers on aflatoxin production[END_REF]. Jayashreee and Subramanyam, (2000) further brought up oxidative stress, in particular the increase of lipid peroxidation and free radical generation, as a prerequisite for aflatoxin biosynthesis by Aspergillus parasiticus. Other researches have suggested the relationship between aflatoxin production and sporulation process [START_REF] De Pena | Relationship between aflatoxin biosynthesis and sporulation in Aspergillus parasiticus[END_REF]. The formation of conidia is an important factor for the infection onset and for the fungal colony formation on different synthetic and natural media. [START_REF] Calvo | Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp[END_REF][START_REF] Calvo | Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans[END_REF] pointed out the sporogenic effect of polyunsaturated fatty acid and their lipoperoxides (LOOH), during the development of Aspergillus spp. Hydroxylated derivatives of oleic, linoleic and linonenic acids, whose formation is directed by putative dioxygenase, modulate sporulation [START_REF] Tsitsigiannis | Endogenous lipogenic regulators of spore balance in Aspergillus nidulans[END_REF]. Such factors seemed to regulate the ratio of asexual to sexual development in A. nidulans [START_REF] Champe | Isolation of a sexual sporulation hormone from Aspergillus nidulans[END_REF]. 9-S and 13-S hydroperoxy-linoleic acids (9-S and 13-S HPODE), which are by-products of plant lipoxygenase, affect spore production in A. nidulans, A. parasiticus and A. flavus [START_REF] Calvo | Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp[END_REF]. In particular 9-S-HPODE enhanced spore production and aflatoxin biosynthesis. On the contrary, 13-S-HPODE inhibited both sexual spores and aflatoxin biosynthesis. Furthermore, these 3 compounds were able to play also a role in the formation of cleistothecia (A. nidulans) and sclerotia (A. flavus) [START_REF] Burow | Seed lipoxygenase products modulate Aspergillus mycotoxin biosynthesis[END_REF]. The biosynthesis of aflatoxins, like other compounds in the cell, is controlled by a signal transduction cascade that involves a G protein (FadA), a protein kinase (PKA) and other factors regulating the expression of AflR, the aflatoxin cluster regulator.

Compounds derived from ligninolitic fungi (Lentinula edodes and Trametes versicolor) are useful to inhibit aflatoxin production as reported by [START_REF] Fanelli | Inhibiting effect of medicinal mushroom Lentinus edodes (Berk.) Sing (Agaricomycetideae) on aflatoxinproduction by Aspergillus parasiticus Speare[END_REF] and [START_REF] Zjalic | Trametes versicolor: a possible tool for aflatoxin control[END_REF]. These findings land basis for innovating biological approaches to control aflatoxin biosynthesis and fungal growth. Antimicrobials and antioxidants compounds such as thioproline, mannitol, αand βglucans, caffeic acid and derivatives, have been detected in these basidiomycetes and plants and might be of significance in modulating oxidative stress and aflatoxin formation (Kurashima et al., 1990;[START_REF] Kim | Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link[END_REF]. In A. parasiticus we have observed [START_REF] Reverberi | Oxidant/antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis[END_REF] that the culture filtrates derived from some basidiomycetes affect transcription factors (such as Apyap1) regulating the expression of genes involved in the removal of reactive oxygen species (ROS), such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) enzymatic activities (Harschmann et al., 1988). In budding yeasts, Yap1, a transcription factor homologue of AP-1, is involved in the defence response against oxidative stress [START_REF] Estruch | Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast[END_REF]. Some other regulatory proteins such as Skn7 and Hsf 1-2 are also involved [START_REF] He | Elevated expression of heat shock factor (HSF) 2A stimulates HSF1-induced transcription during stress[END_REF][START_REF] Moye-Rowley | Regulation of transcriptional response top oxidative stress in fungi: similarities and differences[END_REF]. In a culture media conducive for aflatoxin biosynthesis, an increase of lipoperoxides appears in the trophophase followed by an enhancement of Apyap1 mRNA expression. Coincidentally, a rapid increase of SOD, CAT and GPX enzymatic activities accompanied by a decrease of aflatoxin biosynthesis, occurred. In the following phase, there is a decline of antioxidant enzymatic activities, while biosynthesis of lipoperoxides and the aflatoxin occurs [START_REF] Reverberi | Oxidant/antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis[END_REF]. From these results it emerges that the transcription factor Apyap1 regulates the natural antioxidant defence mechanisms of the aflatoxigenic fungi modulating the toxin biosynthesis.

In this paper the behaviour of two Aspergillus strains inoculated in maize seeds has been showed.

The first strain has a functioning Apyap1 gene, whereas the second one presents a gene inactivated

Materials and methods

Plant and fungal material and culture conditions

The fungal strains used were Aspergillus parasiticus (NRRL 2999) wild type aflatoxins (B 1 , B 2 , G 1 , G 2 ) producer and a ∆Apyap1 mutant obtained from the same strain. The isolate was kept on PDA (Potato Dextrose Agar, Difco) for 7 days at incubation temperature of 30°C before the use. The inoculum of A. parasiticus was performed with conidia suspension (5x10 6 /ml in sterile distilled water) in PDB medium The same number of conidia of the two strains was inoculated in 30 g of maize seeds, cv Cecilia, moistened at a w 0.95. To avoid interferences due to the growth of other fungi, seeds were previously sterilized with γ-rays (8 Kgy, irradiation ratio 2602 Gy•h -1 . This procedure allowed obtaining micro flora sterilization and maintaining seed germination (about 97 %; data not shown). The inoculated seeds were kept in 100 ml Erlenmeyer flasks for 15 days at 30°C in the dark.

FOX-1 assay for the determination of hydroperoxides present in the culture filtrate

The total hydroperoxides (ROOH) produced by A. parasiticus grown in PDB were analyzed by spectrophotometric assay by monitoring the oxidation of xylenol orange at 560 nm (Ferrous ion Oxidation Xylenol orange: FOX-1). The sensitivity of the method was increased according to recent modification by the use of triphenylphosphine and stabilizing the reagent at pH 1.7-1.8 as reported by [START_REF] Banerjee | Measurement of plasma hydroperoxide concentration by FOX-1 assay in conjunction with triphenylphosphine[END_REF].

Assay of LOX activity in maize seeds

The activity of lipoxygenase (LOX, EC 1.13.11.12) was tested at different time points (0-15 days), in contaminated and not contaminated maize seeds. The assay was performed monitoring the diene conjugates formation at 234 nm as previously reported by Reverberi et al., (2005b).

Analysis of 9-and 13-HODE present on maize seeds surface

The seeds infected and not infected with WT and ∆Apyap1 were washed in distilled sterile water, stirring the specimens, to remove mycelia and conidia. Hydroperoxyoctadecadienoic acids (HPODEs) present on the surface of maize seeds were analysed after extraction of the samples with chloroform:methanol 2:1 v/v (20 ml x 3) in presence of 100 µg of butylated hydroxytoluene (BHT) as an antioxidant. The two hydroxyoctadienoic acids (9-HODE and 13-HODE) obtained by reduction of the respective HPODEs, were analyzed by HPLC-APCI-MS as reported by [START_REF] Reverberi | Oxidant/antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis[END_REF]. Data were collected and analyzed using the Chemstation LC/MSD revision A.09.01 (Agilent Technologies). Authentic 9-HODE and 13-HODE were purchased from Cayman (Ann Arbor, Michigan, USA).

Analysis of aflatoxins

The aflatoxins (B 1 + B 2 + G 1 + G 2 ) analyses were performed as previously reported [START_REF] Fanelli | Inhibiting effect of medicinal mushroom Lentinus edodes (Berk.) Sing (Agaricomycetideae) on aflatoxinproduction by Aspergillus parasiticus Speare[END_REF] by extracting inoculated maize seeds after homogenisation in chloroform:methanol (2:1 v/v) (60 ml x 3). The extracts were collected after filtration on anhydrous Na 2 SO 4 , concentrated under a N 2 stream and quantified by HPLC-UV-DAD.

Ergosterol analysis for fungal growth determination

Contaminated and not contaminated maize seeds were extracted with 250 ml chloroform:methanol (2:1 v/v) for 1 h in the dark, in the presence of 100 µg of buthylated hydroxytoluene as an antioxidant. After extraction, the solvents were filtered on anhydrous Na 2 SO 4 , collected and concentrated under a N 2 stream. Ergosterol was analysed by HPLC as reported by [START_REF] Fabbri | Effect of different antifungals on the control of paper biodeterioration caused by fungi[END_REF], using a Supelco LC-18, 5µm (25 cm x 4,6mm) column, methanol:water (98:2 v/v) as the mobile phase, and detecting at 282 nm.

Results

ROOH production, measured by FOX-1 assay, of the two Aspergillus strains (WT and ∆Apyap1) grown in PDB for 168 h at 30°C is reported in Figure 1. ∆Apyap1 presents a higher ROOH production compared with WT between 36 and 70 h with an evident peak at 42 h. After 60 h both strains show a similar trend of ROOH production. Figures 2 and3 report LOX activity (U/mg protein) and LOOH formation (ng/g seeds) of maize seeds inoculated or not (MS) with the WT and ∆Apyap1 strains and incubated for 15 days at 30°C. ∆Apyap1 promotes a stimulation of LOX activity in maize seeds up to 11 days (stronger at 5 and 11 days) in comparison with WT. Also LOOH were formed at greater extent until the first 7 days of incubation. LOOH and LOX activity of not inoculated seeds followed a trend similar to inoculated ones.

Production of 9-and 13-HODE during the growth of both strains on maize seeds is reported in table 1. In maize seeds, not inoculated (MS) and inoculated with the two strains, a slight prevalence of 13-HODE amount compared to 9-HODE occurred. These differences in the ratio between the two regioisomers in the contaminated and not contaminated maize seeds are not always significant.

The aflatoxin biosynthesis detected in maize seeds inoculated with WT and ∆Apyap1 for 15 days at 30°C is reported in Figure 4. It was evident that aflatoxins were produced before by the ∆Apyap1 strain (1.1 ± 0.1 µg/g maize seeds at the 2 nd day of incubation) compared with the WT (1.5 ± 0.2 µg/g maize seeds at the 5 th day of incubation). In addition the ∆Apyap1 produces significantly (p<0.001) more toxins in the intervals 3-6 days and 8-12 days. No significant differences in fungal growth occurred as evident from ergosterol results (Figure 5).

Discussion

Recently we have studied in vitro the regulation effect of some transcription factors, in particular Apyap1, hsf2-like and skn7-like, on the intracellular antioxidant defence mechanisms of some aflatoxigenic strains of A. parasiticus [START_REF] Reverberi | Oxidant/antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis[END_REF]. Notably, there is a direct correlation between levels of oxidative stressors in the cell, such as lipoperoxides, the activity of antioxidant enzymes (i.e. SOD, CAT and GPX), and aflatoxin biosynthesis. In maize seeds inoculated with ∆Apyap1, the activity of LOX and the LOOH amount were higher compared with the WT strain. It can be argued that when the seed is infected with ∆Apyap1 it produces more LOOH probably due to the ROOH produced at high concentration by this strain as suggested by FOX-1 assay. The oxidative stressors present on the surface of the seeds can trigger the activation of LOX and, in turn, lead to the release of LOOH. Thus, the fungus, unable to control the increase of oxidative stressors, such as LOOH, which are an important factor in the enhancement of aflatoxin biosynthesis on the surface of maize seeds, produces a higher amount of toxins and more rapidly. These results suggest that also in A. parasiticus -maize seeds interaction, oxidative stress and aflatoxin biosynthesis are correlated. It appears that the antioxidant defences that control the redox balance of fungal cell, also plays a role in the modulation of toxin formation on maize seeds contaminated by ∆Apyap1. This defence mechanism is a natural phenomenon that can be improved and controlled with different methods. For example the production of toxins can be modulated, as previously described, by the addition of antioxidants, which, in some cases, show also an intrinsic toxicity against pathogenic fungi [START_REF] Fanelli | Effect of different antioxidants and free radical scavengers on aflatoxin production[END_REF](Fanelli et al., , 1986[START_REF] Fanelli | Inhibiting effect of medicinal mushroom Lentinus edodes (Berk.) Sing (Agaricomycetideae) on aflatoxinproduction by Aspergillus parasiticus Speare[END_REF][START_REF] Kim | Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link[END_REF]. As previously reported [START_REF] Zjalic | Trametes versicolor: a possible tool for aflatoxin control[END_REF], comparable effects can be accomplished with compounds that protect the plant against oxidative stress. The ability of organisms to oppose oxidative stress has been exploited also as a biocontrol mechanism. Some yeasts, used as biocontrol agents [START_REF] Castoria | Resistance of post-harvest biocontrol yeasts to oxidative stress: a possible new mechanism of action[END_REF], successfully compete for space and nutrients over many pathogenic fungi [START_REF] Droby | Mode of action of biocontrol agents of postharvest diseases[END_REF]. These The results obtained in this paper suggest that Apyap1 plays a role in the regulation of antioxidant/oxidant balance of the cell in A. parasiticus also during maize seeds colonisation. Thus, it can be hypothesised that aflatoxin biosynthesis occurring during the interaction seed/pathogen is affected by their redox status. Some strategies can be advanced on the basis of our results, such as the use of antioxidants to minimise the formation of oxidants both in the pathogenic fungus and in the plant. WT or ∆Apyap1 incubated at 30°C for different periods (10, 12, 18, 21, 24, 30, 36, 42, 48, 60, 72, 96 and 168 h). The results represent the mean ± S.E.M. of three experiments. Table 1. 9 and 13 regioisomers of hydroxylated linoleic acid (ng/g maize seeds) produced on the surface of maize seeds at different times (0-15 days) not inoculated (MS) or inoculated with A.

parasiticus WT and ∆Apyap1. The results represent the mean ± S.E.M. of three separate experiments.

Time (days)

WT ∆Apyap1 MS 13-HODE 9-HODE 13-HODE 9-HODE 13-HODE 9-HODE 0 0.3 ± 0.1 0.2 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.1 ± 0.05 0.1 ± 0.05 1 1.1 ± 0.1 1.0 ± 0.1 1.5 ± 0.2 1.4 ± 0.2 0.6 ± 0.1 0.5 ± 0.1 2 2.9 ± 0.3 3.2 ± 0.2 3.5 ± 0.3 3.7 ± 0.5 0.9 ± 0.1 0.7 ± 0.1 3 1.8 ± 0.2 1.9 ± 0.2 3.2 ± 0.4 2.9 ± 0.4 1.0 ± 0.1 1.2 ± 0.2 5 0.6 ± 0.1 0.3 ± 0.1 2.0 ± 0.5 2.0 ± 0.3 0.3 ± 0.1 0.2 ± 0.1 7 0.9 ± 0.2 0.5 ± 0.1 1.2 ± 0.2 0.6 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 11 2.8 ± 0.3 1.9 ± 0.3 3.0 ± 0.4 1.9 ± 0.5 0.8 ± 0.1 1.0 ± 0.2

  . The results obtained suggested that the lipoperoxides produced by maize seeds can affect aflatoxin biosynthesis in both Aspergillus strain likely by Apyap1 pathway.

  were performed in triplicate and values, referred to minimum three replicates per sample, were averaged and standard deviations were determined. In all experiments, mean values were compared using Student's t-test.

  resisting to oxidative stress conditions, colonize wounds and protect the plant from the attack of different pathogens.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Lipoxygenase activity of maize seeds not inoculated (MS) or inoculated with A.

Figure 3 .

 3 Figure 3. Total lipoperoxides (LOOH) originated from linoleic acid, extracted from maize seeds

Figure 4 .

 4 Figure 4. Aflatoxin production in maize seeds inoculated with A. parasiticus WT or ∆Apyap1 and

Figure 5 .

 5 Figure 5. Fungal growth reported as ergosterol content (ng/g seeds) of A. parasiticus WT or

Figure 2 .

 2 Figure 1. Reverberi et al.

Figure 5 .

 5 Figure 3. Reverberi et al.

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