María-José ; Moreno

Emma Plana

Ángel Montoya

Pasquale Caputo

Application of a monoclonal-based immunoassay for the screening of imazalil in fruit juices

Keywords: Imazalil, Azole fungicide, Monoclonal antibodies, Immunoassay, ELISA, Fruit juices

Introduction

Imazalil [(RS)-1-β-allyloxy-2,4-dichlorophenylethyl)imidazole], is a systemic imidazole fungicide used to control a wide range of fungi on fruit, vegetables, and ornamental plants. It is also used for the treatment of barley and wheat seeds prior to planting, and to treat equipment and egg storage areas in chicken hatcheries. Imazalil was initially classified as likely to be carcinogenic in humans, but recent studies have provided sufficient evidence that imazalil is a threshold carcinogen. Exposure studies concluded that there is a reasonable certainty of no harm to any population subgroup from aggregate exposure to imazalil from dietary (food and water) exposure and all other non-occupational sources. On the basis of risk quotients, imazalil used at the recommended application rates should not result in an acute or chronic risk to mammal and avian species. Imazalil is moderately toxic to rats, freshwater fish and invertebrates following acute exposure. Results of imazalil ecological risk assessments show that none of the risk quotient values exceed the level of concerns for either terrestrial or aquatic non-target organisms. Fate studies showed that imazalil degrades slowly in soil and is not mobile, which mitigates the likelihood that this chemical will reach surface water and accumulate in fish (US Environmental Protection Agency 2006;[START_REF] Tomlin | The pesticide manual: A world Compendium[END_REF]). As mentioned above, imazalil is particularly widely used for post-harvest treatment of citrus, banana, and other fruit to control storage decay. Due to this application, imazalil residues can remain in fruit and, obviously, in their derivatives.

This fact poses the need to establish legal directives to control their levels through the Maximum Residue Levels (MRLs), which are established by each country. In this sense, in Europe, the current legislation allows imazalil residue levels of up to 5 mg kg -1 in fresh fruit (FAO/WHO 2006). Therefore, it is necessary to develop of appropriate analytical methods for the follow-up of imazalil residues in food. Imazalil has been analysed by various chromatographic methods. They involve different extraction procedures followed by gas chromatography [START_REF] Garrido | Chromatographic analysis of imazalil and carbendazim in fruits: Method validation and residue monitoring program 1995[END_REF][START_REF] Matsumoto | Simultaneous determination of imazalil and its major metabolite in citrus fruit by solid-phase extraction and capillary gas chromatography with electron capture detection[END_REF][START_REF] Yamazaki | Determination of imazalil residues in lemons by gas chromatography with nitrogen-phosphorus detection[END_REF], liquid chromatography [START_REF] Blasco | Evaluation of 10 pesticide residues in oranges and tangerines from Valencia (Spain)[END_REF][START_REF] Yoshioka | Rapid simultaneous determination of ophenylphenol, diphenyl, thiabendazole, imazalil and its major metabolite in citrus fruits by liquid chromatography-mass spectrometry using atmospheric pressure photoionization[END_REF][START_REF] Zamora | Determination of tridemorph and other fungicide residues in fruit samples by liquid chromatography-electrospray tandem mass spectrometry[END_REF] or gas-liquid chromatography [START_REF] López | Residue levels of ethoxyquin, imazalil, and iprodione in pears under cold-storage conditions[END_REF] analysis, coupled to different detection systems. The limits of detection of these techniques were in the range of 1-100 ng g -1 . Recently, a new method for rapid quantification of imazalil in citrus fruit has been developed, which uses flow-injection electrospray ionization tandem mass spectrometry [START_REF] Ito | Simple and rapid determination of thiabendazole, imazalil, and o-phenyl phenol in citrus fruit using flow-injection electrospray ionization tandem mass spectrometry[END_REF]. To date, these chromatographic methods are the most used, but they generally require a laborious sample pre-treatment and high-cost equipment. Immunochemical techniques, mainly enzyme-linked immunosorbent assays (ELISA), are alternative or complementary analytical tools to conventional methods. Immunochemical methods are based on the interaction of an analyte with an antibody that recognizes it with high affinity and specificity. They are simple, cost-effective, and field-portable, do not require sophisticated instrumentation, and are able to analyse a large number of samples. In practice, one of the most important advantages of immunoassays for pesticide analysis lies on their minimal requirement of sample cleanup. Over the last decade, several ELISAs based on polyclonal antibodies for residue analyses of azole fungicides have been developed [START_REF] Cairoli | Enzyme-linked immunosorbent assay for the quantitation of the fungicide tetraconazole in fruits and fruit juices[END_REF][START_REF] Chen | Development of an enzyme-linked immunosorbent assay for a broad spectrum triazole fungicide: hexaconazole[END_REF][START_REF] Forlani | Development of an enzyme-linked immunosorbent assay for triazole fungicides[END_REF]). Nevertheless, the amount of antiserum produced by even a group of animals is limited, and its characteristics vary from animal to animal and also between different breedings from the same animal. Monoclonal antibodies (MAb) can be produced in unlimited quantities and offer the possibility to standardize the assay method. Recently, the development of a sensitive ELISA for the detection of imazalil in citrus fruits samples based on monoclonal antibodies has been described [START_REF] Watanabe | Development of an enzyme-linked immunosorbent assay for the fungicide imazalil in citrus fruits[END_REF]. This ELISA required imazalil extraction from samples, although no further clean-up was needed other than appropriate dilution of filtrated methanolic extracts. The same authors used this MAb in an immunoaffinity column clean-up for imazalil determination in citrus fruits by HPLC [START_REF] Watanabe | Immunoaffinity column clean-up for the determination of imazalil in citrus fruits[END_REF].

The analysis of fungicide residue in juice matrices by immunoassays offers considerable advantages over chromatographic techniques, since analyte extraction is not required and samples could be directly analysed by diluting them adequately.

Therefore, the aim of this work was to obtain MAbs to imazalil for the development of enzyme-linked immunosorbent assays, whose analytical parameters could allow the simple detection of imazalil in fruit juice samples with a high sample throughput.

Materials and methods

Reagents and instrumentation

Analytical standard of imazalil and azole fungicides were from Riedel-de Haën (Seelze, Germany). Working stock solutions were prepared in N,N-dimethylformamide and stored at -20ºC. Analytical grade solvents and silica gel 60, 0.06-0.2 mm, for column chromatography, were from Scharlau (Barcelona, Spain). Imazalil precursor, 1-(2, 4dichlorophenyl)-2-(1-imidazolyl) ethanol, was from Acros Organic (Geel, Belgium).

Potassium hydride 30 % wt dispersion in mineral oil, ethyl 6-bromohexanoate, ovalbumin (OVA), o-phenylenediamine (OPD), Sephadex G-50, and Freund´s complete and incomplete adjuvants were obtained from Sigma-Aldrich Química (Madrid, Spain).

Bovine serum albumin fraction V (BSA), enzyme-immunoassay grade horseradish peroxidase (HRP), hybridoma fusion and cloning supplement (HFCS), and polyethylene glycol (PEG) 1500 were purchased from Roche Diagnostics (Barcelona, Spain).

Peroxidase-labelled rabbit anti-mouse immunoglobulins was obtained from Dako ELISA plates were washed with a 96 PW microplate washer from SLT Labinstruments GmbH (Salzburg, Austria), and absorbances were read in dualwavelength mode (490-650 nm) with a Emax microplate reader from Molecular Devices (Sunnyvale, USA). Ultraviolet-visible reported spectra were recorded on a UV-160A Shimadzu spectrophotometer (Kyoto, Japan).

Preparation of imazalil protein conjugates

The structure of the imazalil hapten IMH [1-(2, 4-dichlorophenyl)-2-(1-imidazolyl) hexanoic acid] used to prepare imazalil protein conjugates is depicted in Figure 1. IMH was prepared by introduction of an alkyl chain spacer, ending in a carboxylic acid, by O-alkylation of the hydroxyl group of an imazalil precursor [START_REF] Ito | Hapten compound and antibody of imazalil and measurement[END_REF]).

[Insert Figure 1 about here] Preparation of immunizing conjugates. IMH hapten was covalently attached to BSA using the modified active ester method [START_REF] Langone | Radioimmunoassay of nicotine, cotinine, and γ-(3piridyl)-γ-oxo-N-methylbutyramide[END_REF] and the mixedanhydride method [START_REF] Rajskowsky | The conjugation of testosterona with horseradish peroxidase and a sensitive enzyme assay for the conjugate[END_REF]. According to the first method, 15 µmol of the hapten was reacted with stoichiometric amounts of N-hydroxysuccinimide and dicyclohexylcarbodiimide in 0.1 ml of DMF overnight at room temperature. After centrifuging, 0.083 ml of the clear supernatant containing the active ester was dissolved in 0.317 ml of DMF and slowly added to 0.6 ml of a 25 mg ml -1 BSA solution in 0.2 M The mixture was allowed to react at room temperature for 4 h with stirring, and finally the conjugate was purified by gel filtration on Sephadex G-50 using 100 mM sodium phosphate buffer, pH 7.4, as eluant. The extent of coupling of the hapten to BSA was estimated by the determination of the number of protein free amino groups before and after conjugation, using 2,4,6-trinitrobenzenesulfonic acid (TNBS) as the titration reagent [START_REF] Habeeb | Determination of free amino groups in proteins by trinitrobenzenesulfonic acid[END_REF]. The hapten to protein molar ratio was evaluated as 20.

According to the mixed-anhydride method, 16.70 µmol of the hapten was treated with stoichiometric amounts of tri-butylamine and isobutyl chloroformate in 0.229 ml of DMF for 30 min at room temperature. To the reaction mixture was added DMF up to 0.4 ml, and was slowly added to a solution of 15 mg of BSA in 0.6 ml of 0.2 M borate buffer, pH 9. The initial hapten to protein molar ration in the mixture was 73:1. After stirring for 2 h at room temperature, the conjugate was purified as above. The hapten to protein molar ratio in the conjugate was evaluated as 10.

Preparation of the coating conjugate. IMH hapten was covalently attached to OVA using the mixed-anhydride method. The hapten (11.2 :mol) was treated with stoichiometric amounts of tri-n-butylamine and isobutyl chloroformate in 0.153 ml of DMF for 30 min at room temperature. To the reaction mixture was added DMF up to 0.5 ml, and then was slowly added to 1.5 ml of a 20 mg ml -1 OVA solution in 0.2 M borate buffer, pH 9. The initial hapten to protein molar ration in the mixture was 17:1.

After stirring for 2 h at room temperature, the conjugate was purified by gel filtration on Sephadex G-50 using 100 mM sodium phosphate buffer, pH 7.4 as eluant. The extent of coupling of the hapten to OVA was determined by the TNBS protocol above described.

The hapten to protein molar ratio in the conjugate was evaluated as 5. Preparation of the enzyme tracer. IMH hapten was covalently attached to HRP using the mixed-anhydride method. 11.2 :mol of the hapten was treated with stoichiometric amounts of tri-n-butylamine and isobutyl chloroformate in 0.153 ml of DMF for 30 min at room temperature. To 0.1 ml of the reaction mixture was added 0.9 ml of DMF, and 0.1 ml of this dilution (0.73 :mol) was slowly added to 1 ml of a 2.2 mg ml -1 HRP solution in 0.2 M borate buffer, pH 9. The initial hapten to protein molar ration in the mixture was 15:1. After stirring for 2 h at room temperature, the conjugate was purified by gel filtration on Sephadex G-50 using 100 mM sodium phosphate buffer, pH 7.4, as eluant.

Production of monoclonal antibodies to imazalil

Four BALB/c female mice (8-10 weeks old) were immunized with the BSA-IMH conjugates. First dose consisted of 100 :g of conjugate intraperitoneally injected as an emulsion of 250 :l of a 1:1 (v/v) mixture of PBS (10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and complete Freund's adjuvant. Three subsequent injections were given at three-week intervals using the immunogen emulsified in incomplete Freund's adjuvant. After a resting period of at least three weeks from the last injection in adjuvant, mice received a final soluble intraperitoneal injection of conjugate in PBS, four days prior to cell fusion. Cell fusion and hybridoma selection procedures were carried out essentially as previously described by [START_REF] Manclús | Development of a monoclonal immunoassay selective for chlorinated cyclodiene insecticides[END_REF].

Antibodies were directly purified from late stationary phase hybridoma culture supernatants by saline precipitation with saturated ammonium sulfate (1:1, v/v), followed by affinity chromatography on a HiTrap Protein G column (Amersham Biosciences, Uppsala, Sweden). ELISA plates were coated overnight with conjugate or antibody solutions in 50 mM carbonate buffer, pH 9.6. A volume of 100 :l per well was used throughout all assay steps, and all incubations were carried out at room temperature. After each incubation, plates were washed four times with washing solution (0.15 M NaCl containing 0.05% Tween 20). Two basic formats were used depending on the assay component immobilized into the ELISA plates. In the conjugate-coated format, an indirect ELISA was used for the screening of culture supernatants, and a competitive indirect ELISA was used for the study of antibody sensitivity and specificity to analytes. In the antibody-coated format, the specific antibody was coated directly or by using a capture auxiliary antibody, and competitive ELISAs were followed to evaluate the assay properties using the HRP enzyme tracer. For competition assays the concentrations of antibodies, hapten conjugates, or enzyme tracers were optimized by checkerboard titration.

ELISA procedures

Conjugate-coated format. A competitive ELISA, based on an homologous conjugatecoated format, was chosen. Immunoassays were performed as follows: ELISA plates were coated overnight with 0.75 :g ml -1 of OVA-IMH conjugate. Then, culture supernatant, or antibody dilutions in PBST (PBS containing 0.05% Tween 20) were added and incubated for 1 h. Next, plates were incubated for 1 h with peroxidaselabelled rabbit anti-mouse immunoglobulins diluted 1/2000 in PBST. Finally, peroxidase activity bound to the wells was determined by adding the substrate solution (2 mg ml -1 OPD and 0.012% H 2 O 2 in 25 mM citrate and 62 mM sodium phosphate, pH 5.35). After 10 min, the reaction was stopped with 2.5 M sulfuric acid, and the absorbance at 490 nm was read and recorded. For competitive assays, the procedure was the same except that after coating a competition step was introduced by adding 50 :l of Antibody-coated format. In this format, plates were coated overnight with monoclonal antibodies either directly at 1 :g ml -1 or indirectly with goat anti-mouse immunoglobulins at 2 :g ml -1 . Next, the competition was established for 1 h between imazalil standards and the selected dilutions of the enzyme tracer. Peroxidase activity was measured as above.

Preparation of imazalil standards and spiked juice samples

From a 1 mM stock solution of imazalil in DMF, serial dilutions in this solvent from 1 mM to 12.8 nM were prepared. From these concentrations, standards from 2 :M to 25.6 pM were prepared by 1/500 dilution in PBS. As the assay procedure involved the addition of the same volume of the appropriate immunoreagent concentration, imazalil standards in the final assay ranged from 1 :M to 12.8 pM (297.17 to 3.8 10 -3 ng ml -1 ).

Tomato, orange and apple juices were obtained from a local supermarket and were used without any sample pretreatment. For ELISA determination, spiked juice samples were daily prepared by dilution in PBS, and each dilution was analyzed in triplicate. The imazalil concentration in the samples was calculated by averaging values obtained at each dilution.

Data analysis

Competitive curves were obtained by plotting absorbance values against the logarithm of analyte concentration. Using a commercial software (Sigmaplot, Jandel Scientific), sigmoid curves were fitted to a four-parameter logistic equation: where A is the asymptotic maximum (maximum absorbance in absence of analyte, A max ), B is the curve slope at the inflection point, C is the x value at the inflection point (corresponding to the analyte concentration giving 50% inhibition of A max , I 50 ), and D is the asymptotic minimum. Imazalil concentration of fruit juice samples was determined by interpolation of the mean absorbance values on the standard curve run in the same plate (SOFTmax Pro Software, Molecular Devices).

y = {(A-D)/[1+(x/C)B]}+D

Results and discussion

Imazalil immunoassay development

Imazalil, like most pesticides, is a small organic molecule which is non-immunogenic by itself and lacks a functional group for coupling to proteins. Therefore, the synthesis of pesticide-like molecules containing a functional group suitable for protein conjugation is a necessary and critical step in the production of high-affinity antibodies.

The hapten IMH used to obtain monoclonal antibodies was synthesized by introduction of a spacer arm through the hydroxyl group of 1-(2, 4-dichlorophenyl)-2-(1-imidazolyl) ethanol, an imazalil synthesis precursor. Four cell fusion experiments were performed with each of the BSA conjugates of this hapten. In the screening of culture supernatants only wells showing a 50% signal inhibition in the presence of 1 µM imazalil, with respect to that obtained in the absence of analyte, were considered as containing highaffinity antibody-secreting clones. From all fusion experiments, seven hybridomas fulfilled the above-mentioned criterion. These hybridomas were subsequently cloned and expanded in culture medium.

For the evaluation of the conjugate-coated format, non competitive experiments were carried out to estimate MAb titer. Thereafter, inhibition curves using imazalil as competitor were performed with the coating conjugate. Under these conditions, I 50 I). With respect to the antibodycoated formats, noncompetitive experiments were carried out prior to inhibition assays.

Concentrations of HRP-IMH conjugate from 2 to 0.016 µg ml -1 were tested in plates previously coated with the MAbs directly immobilized to the plate. HRP-IMH was only recognized by two MAbs . Imazalil inhibition curves were performed for these two MAbs. I 50 values of 118 and 130 nM were obtained for IMH-41 and IMH-42, respectively (Table I). The effect of a pre-coating step with goat antimouse Igs (indirect antibody-coated format) was also evaluated. This approach improved the assay sensitivity, as evidenced by a two-fold decrease of I 50 values.

As shown in Table I, among the monoclonal antibodies and ELISA formats evaluated MAb IMH-51 used in the conjugate-coated format provided the highest affinity ELISA to imazalil and, therefore, this combination of antibody and ELISA format was selected to carry out this study. The influence of the nonionic surfactant Tween 20, BSA (commonly used in ELISA to reduce nonspecific interactions), ionic strength, and pH on ELISA characteristics was examined (data not shown). The addition of Tween 20 to the assay buffer of the competitive step slightly improved the assay sensitivity (five-fold decrease of the I 50 values). The highest assay sensitivity was achieved with a PBS buffer at neutral pH containing only a small amount of Tween 20 (0.0025%).

[Insert Table I about here]

Specificity of the imazalil immunoassay

The specificity of the IMH-51 monoclonal antibody was evaluated by performing competitive assays with other imidazole and triazole pesticides and determining their respective I 50 values. As shown in Table II, the assay was very specific for imazalil, since the assayed pesticides were not significantly recognized. In detail, the imidazole moiety has an important role in the analyte-antibody interaction since just the change to Furthermore, the imazalil metabolite, from which the IMH hapten was synthesized, was poorly recognized.

[Insert Table II about here] [Insert Figure 2 about here] Table III summarizes the variability of the mathematical parameters that define the sigmoid equation. Results are the average of 8 independent runs, each one fitted without normalization as used for direct interpolation of samples. Coefficients of variation (CV) were around 20% for A, B and C. The high CV of parameter D is due to the very low values obtained for asymptotic minimum absorbances, normally below the microplate reader precision. Referring to assay analytical parameters, the mean imazalil I 50 value was 1.6 nM (0.5 ng ml -1 ) and the detection limit, estimated as the imazalil concentration that reduced absorbance to 90% of the maximum, was 0.2 nM (0.06 ng ml -1 ). The working range was defined as the concentrations giving 20-80% inhibition, i.e., 0.4-8.0 nM (0.12 -2.4 ng ml -1 ). These parameters indicate that this ELISA can be considered sensitive enough for imazalil determination in fruit juices.

Analytical characteristics of the imazalil immunoassay

[Insert Table III about here]

Evaluation of matrix effects Food samples contain substances that may interfere in analyte determinations. These interferences are known as matrix effect. In immunoassays, these effects are manifested in different ways, the most common ones being a loss of assay sensitivity (increase of I 50 ) or a decrease of colour development (overestimate), though a combination of both effects is also possible. Thus, the influence of the selected juices on imazalil immunoassays should be determined prior to the application of the method to samples spiked with the analyte. With this idea in mind, two experiments were performed.

In a first experiment, the matrix influence on standard curve parameters was analysed. Imazalil standards were prepared in duplicate in PBS buffer containing a variable proportion of each juice. A decrease of the maximum absorbance and of the assay sensitivity were observed at matrix proportions higher than 1/25, 1/50 and 1/100 for tomato, orange, and apple juice, respectively (data not shown). These results suggested that imazalil could be analysed in juices simply by appropriately diluting samples in PBS, without the need of a cleanup step.

In a second experiment, juice samples were spiked with imazalil at several levels and they were properly diluted to give a concentration close to the midpoint of the working range. The objective of this experience was to determine the matrix dilution that provided accurate and precise determinations of imazalil. Table IV shows the statistical analysis obtained for each fortified juice sample determined four times at the indicated dilutions. Matrix effects were reduced to acceptable levels when tomato, orange and apple juice samples were diluted at least 1/25, 1/50 and 1/100, respectively, as proved by the recovery and the precision data obtained. Mean recoveries were 81.4 %, 100.8 %, and 97.7 % for tomato, orange and apple, respectively, and most interassay CV were below 20 %.

[Insert Table IV

Analysis of spiked juice samples

To evaluate the performance of the imazalil immunoassay, fruit juices were spiked with known concentrations of imazalil in the 10-500 ng ml -1 range, and directly analysed by ELISA without any pre-treatment other than a 1/25, 1/50 and 1/100 dilution for tomato, orange and apple juice, respectively. After dilution, these fortification levels entered directly into the assay working range of the imazalil ELISA, and they are well below the maximum residue limits for imazalil in these crops (2-5 mg kg -1 ). Spiked samples were analysed four times independently. In order to obtain the imazalil concentrations, absorbances from triplicate wells were interpolated in the standard curve performed in the same plate. Table V summarizes the results obtained for each spiked juice sample.

Coefficients of variation can be considered as acceptable, ranging from 8.3 to 30.8, while recoveries were fairly good (mean values of 88%, 106%, and 97% for tomato, orange and apple juices, respectively). Taking all together, linear regression analysis indicated a good correlation between the concentration of imazalil added to juice samples and the concentration determined by ELISA (y = 0.987x -2.042, r 2 = 0.993).

These results prove that this immunoassay is able to quantitatively detect concentrations of imazalil as low as 10-50 ng ml -1 in these juices.

[Insert Table V about here]

Conclusions

Following general guidelines for hapten design, IMH hapten was synthesized and used to produce monoclonal antibodies to imazalil. One of them, IMH-51, was selected for the development of a homologous indirect ELISA on the basis of its affinity and specificity to imazalil. The sensitivity of this immunoassay, expressed as the I 50 value for standards, was 1.6 nM imazalil (0.5 ng ml -1 ). Most important, the ELISA presented here is able to determine down to 10-50 ng ml -1 of imazalil in tomato, orange, and

N N CH O Cl C O C(CH3)3 < 0.1 Prochloraz N N C N CH 2 CH 2 O Cl Cl Cl CH2CH2CH3 O < 0.1 Hexaconazole N N N CH 2 C Cl OH CH 2 CH 2 CH 2 CH 3 Cl < 0.1 Myclobutanil N N N CH 2 C CH 2 CH 2 CH 2 CH 3 Cl C N < 0.1 Penconazole N N N CH 2 CH Cl Cl CH 2 CH 2 CH 3 0.12 Propiconazole N N N CH 2 O O Cl Cl CH 2 CH 2 CH 3 < 0.1 Tebuconazole N N N CH 2 C CH 2 CH 2 Cl OH C(CH 3 ) 3 < 0.1 Tetraconazole N N N CH 2 CH Cl Cl CH 2 OCF 2 CHF 2 < 0.1 Triadimefon N N N CH O Cl C O C(CH 3 ) 3 < 0.

  Denmark). Culture media (high-glucose Dulbecco´s Modified Eagle´s medium with GLUTAMAX I and sodium pyruvate, DMEM), fetal calf serum (Myoclone Super Plus), and supplements were from Gibco (Paisley, UK). Culture plasticware was from Corning (New York, USA). P3-X63-Ag8.653 mouse plasmacytoma line was from American Tissue Type Culture Collection (Rockville, USA). Polystyrene culture plates (High Binding Plates, cat. no. 3590) were from Costar (Cambridge, USA).

  pH 9. The initial hapten to protein molar ration in the mixture was 55:1.

  by 50 :l of the appropriate concentration of antibody (culture supernatant or purified MAb).

  nanomolar range were obtained (Table

  , while maintaining a similar exposed structure as represented by penconazole, drastically reduces the compound recognition (cross-reactivity < 0.12%).

Figure 2

 2 Figure2shows the standard curve obtained for the optimized imazalil competitive

Figure 1 .Figure 2 .

 12 Figure 1. Structures of imazalil and of the IMH hapten. 63x67mm (600 x 600 DPI)

  about here]

Table I .

 I apple juices with acceptable accuracy and precision for a residue analysis method.Furthermore, the simplicity of the immunoassay is clearly evidenced by its high sample throughput, since dozens of samples can be simultaneously analyzed simply by dilution. Sensitivity of antibodies estimated as I 50 values for imazalil (ng ml -1 ), obtained with different ELISA formats. 1

	MAb	Conjugate-coated format	Direct antibody-coated format	Indirect antibody-coated format
	IMH-41	61		35	20
	IMH-42	180		39	20
	IMH-51	2		-2	-
	IMH-52 IMH-53 IMH-54	F o r F o r F o r F o r 80 77 10 F o r	---	---
	IMH-55	6		-	-
	P P P P 1 Competitive ELISAs were performed in optimum conditions, i.e., limiting concentrations of immunoreagents giving maximum P
	absorbance around 1.0. 2 No recognition.	e e r e e r e e r e e r e e r
				R R R R R
				e e e e e
					v i e v i e v i e v i e v i e
					w w w w w
					O n l O n l O n l O n l O n l
					y y y y y

Table II .

 II Cross-reactivity 1 of several compounds with LIB-IMH51 monoclonal antibody.

	Compound	Chemical structure	Cross-reactivity (%)
		N	N CH 2 CH	OCH 2 CH CH 2
			Cl
	Imazalil		Cl	100
	Climbazole

Table III .

 III Variability of the mathematical parameters of the sigmoid standard curve. 1 Asymptotic maximum absorbance (A), slope at the inflexion point (B), concentration value in nM at the inflexion point (C), and asymptotic minimum absorbance (D) are the parameters which fit the sigmoid equation y = {(A-D)/[1+(x/C) B ]}+D. 2 n = 8.

	Page 19 of 24		Food Additives and Contaminants
		A	B	C (I 50 )	D
	Mean 2	0.91	1.02	1.61	0.024
	SD	0.2	0.21	0.4	0.012
	CV (%)	22	21	25	70
	Minimum	0.64	0.8	1.03	0.008
	Maximum	1.28	1.41	2.11	0.039
	F o r 1 F o r
		P P
		e e
			e r e r
				R R
				e e
				v i e v i e
					w w
					O n l O n l
					y y
					1
	1 Cross-reactivity was calculated as (I 50 imazalil/I 50 compound) x 100.

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Table IV .

 IV Influence of matrix dilution on the reliability of the imazalil ELISA. Sample dilution before adding to ELISA plate. 2 Data obtained from five independent determinations.

		Spiked levels	Sample dilution	Imazalil found 2
		(ng ml -1 )	factor 1	(ng ml -1 )	Recovery (%) CV (%)
	Tomato juice	10	5	5.1	51.0	33.4
		50	25	41.0	82.0	22.6
		100	50	75.9	75.9	20.4
		200	100	177.3	88.7	11.3
		500	250	394.7	78.9	10.5
	10 50 100 200 500 10 50 100 200 500 o r P 100 5 25 50 250 5 25 50 100 250 e e r 1 F Orange juice Apple juice R 102.8 26.2 39.7 213.0 465.7 3.3 35.8 76.5 183.7 516.9	262.0 79.4 102.8 106.5 93.1 33.0 71.6 76.5 91.9 103.4	21.7 20.0 14.5 24.2 11.2 73.6 14.3 29.6 9.8 19.8
				e
				v i e
				w
					O n l
						y

Table V .

 V ELISA reproducibility and accuracy in spiked juice samples. Data are the average of 4 independent determinations.

	Imazalil spiked level (ng ml -1 ) Orange juice 25 50 100 33.4 44.1 98.8 262.2 250 10.3 11.1 22.3 39.2 30.8 25.2 22.6 14.9 21.8 33.6 73.9 217.1 43.5 54.3 116.8 287.8 133.6 88.2 98.8 104.9 Food Additives and Contaminants 100 94.6 19.4 20.5 72.2 44.2 107.3 Tomato juice 25 50 22.8 35.0 5.4 8.5 23.6 24.3 17.7 26.8 27.8 91.2 70.0 94.6 F 10 9.7 1.6 16.5 8.7 11.6 97.0 o 1 Page 22 of 24 Apple juice 50 100 250 500 Mean value found 1 36.2 108.0 273.6 486.7 SD 3.5 28.2 44.1 40.2 CV (%) 9.7 26.1 16.1 8.3 Minimum 32.7 75.7 224.4 440.3 Maximum 39.7 127.8 309.6 510.3 Recovery (%) 72.4 108.0 109.4 97.3 r
	P
	e e r
	R e v i e w
	O n
	l y

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Acknowledgements


Simon Navarro
email: snavarro@um.es

Nuria Vela

Gabriel Pérez

AND Ginés Navarro

Simón Navarro

Decline of Pesticide Residues from Barley to Malt

Keywords: Barley, malt, malting, malt storage, pesticide residues

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

from 13 to 51% for fenitrothion and nuarimol, respectively. Steeping was 31 the most important stage in the removal of pesticide residues (52%) 32 followed by germination (25%), and kilning (drying and curing, 23%).

33

During malt storage (3 months) the fall in pesticide residues was not 34 significant. Applying the standard first-order kinetics equation (r>0.95), the 35 half-lives obtained for the pesticides during malt storage varied from 244 to 36 1,533 days for myclobutanil and nuarimol, respectively.

Brewing is the production of alcoholic beverages through fermentation. This 40 is the method used in beer production, although the term can be used for 41 other drinks. Four raw materials are required for beer production: barley, 42 hops, water and yeasts. The quality of these raw materials has a decisive 43 influence on the quality of the final product (Kunze, 2004).

44

Because of their high content of starch and storage proteins, barley According, the objective of this research was to know the fate of several 92 pesticides commonly used on barley from this cereal to malt for assist 93 maltsters in preventing the occurrence of pesticide levels higher than those 94 allowed.

95

Material and Methods

96

Pesticides and reagents. Pesticide standards with purity higher 97 than 98% were obtained from Dr. Ehrenstorfer GmbH (Augsburg, 98 Germany). Table 1 shows the pesticides used and their main 99 physicochemical characteristics (Tomlin, 2003). Stock standard solutions of (µg/L), 45.2 and pesticides below detection limit) was obtained from the 118 municipal network. After approximately 24 h, the first visible sign of 119 germination was the appearance of the root, as a white "chit". The grains 120 were then transferred to malting beds where germination was performed at 121 15±1ºC over a period of around four days. The speed of germination was 122 controlled by temperature and aeration of the malt bed, while moisture 123 content was maintained by spraying. The grain bed was regularly turned to 124 prevent grains matting together. Finally, after a period of germination, the 125 "green malt" was kiln dried from 50 to 85ºC over 24 h in a forced flow of 126 hot air to arrest germination and stabilize the malt by moisture level of 4-127 5% and then the malt was "cured" increasing temperature to 90-100ºC and 128 reducing the speed of air flow. At the end of kilning the malt was cooled to 129 arrest colour formation and to stabilize enzyme levels. Finally, the brittle 130 malt rootlets were separated and the rest milled. Figure 1 shows the 131 experimental procedure for malting process.

132

Samples and sampling methods. Different samples (barley grains, 133 steeped grains, steeping water, green malt, dried malt, and cured malt) 134 were taken during malting process (Figure 1). Bearing in mind the yields 135 obtained after the different elaboration steps, the total residual amount 136 present in each vessel was calculated, as shown in Table 2. The total 137 amount present in barley grains (100%) was considered to be the starting 138 point to study the dissipation of the pesticides. To known the dissipation of 139 pesticide residues from stored malt four samples (0, 30, 60, and 90 days) 140 were taken, starting the study when the cured malt was cooled and milled.

197

The slopes of the calibration graphs with the standards directly prepared in 198 2,2,4-trymethyl pentane/toluene (1/1, v/v) and the standard addition 199 calibration graphs obtained from the different samples were similar, 200 confirming the absence of any matrix effect.

201

The limit of quantitation (LOQ), defined as the lowest fortification 202 level attempted, was lower than 5 ng/g with the exception of malathion ( 14203 ng/g). These limits are, in all cases, below the maximum residue limits

254

Finally, curing removed fenitrothion and propiconazole residues in 255 proportions of 39% and 23%, respectively while the losses of the other 256 pesticides ranged from 2-16%. Figure 3 shows the total amounts of 257 pesticide residues removed during the different stages of malting. As can be 258 observed the most important stage in the removal of pesticide residues was 259 steeping (52%) followed by germination (25%), and kilning (drying and 260 curing, 23%)..

261

Loss of pesticide residues from stored malt. Several models 262 [START_REF] Timme | Statistical interpretation and graphic 347 representation of the degradational behaviour of pesticide residues. 348 II[END_REF]Morton et al. 2001) are used to describe the decay of 263 pesticides in different matrices. Probably, the most commonly used model 264 to describe loss of grain protectants is the following equation, where R t is 265 the residue at time t, R 0 is the residue at time zero and K is the rate 266 constant [START_REF] Desmarchelier | A model of the breakdown of dichlorvos on grain[END_REF][START_REF] Desmarchelier | Loss of fenitrothion on grain in storage[END_REF]:

267 ln R t = ln R 0 -Kt

268

To know the rate of loss of residues in the stored malt, the 269 experimental data (Table 3) were fitted to the above model. Table 4 shows 270 the values derived from the fit. In all cases, there was a good linear

Propiconazole F (±)-1-([2-(2,4- Dichlorophenyl)-4- propyl-1,3-dioxolan- 2-yl]methyl)-1H- 1,2,4-triazole O O Cl Cl Pr H CH 2 N N N C 15 H 17 Cl 2 N 3 O 2 342.2 3.72 100 Myclobutanil F 2-(4-chloro-phenyl)- 2-(1H-1,2,4-triazol- 1-ylmethyl)-

  100 100 µg/ml were prepared by exact weighing and dissolving in acetone and 101 finally stored in the dark at 4 ºC. Working standard solutions were prepared 102 freshly by dilution in the same solvent. Solvents for pesticide residue 103 analysis (n-hexane, dichloromethane, and acetone) were supplied by 104 Scharlau Chemie S.A. (Barcelona, Spain). Helium (99.99% purity) was 105 purchased from Abelló Linde S.A. (Barcelona, Spain). 106 Raw material and malting process. Barley (11% moisture), two-107 row spring malting barley (Hordeum distichum) was obtained from the 108 Brewers Estrella de Levante Fábrica de Cerveza S.A. (Murcia, Spain). Barley 109 grains (100 g, n=3), once spiked with the pesticides (0.2-1 mg/kg) were 110 subjected to the process according to the common malting operations 111 (steeping; germination; and kilning) after evaporation of the spiking solvent112(3 h). The process commences with the steeping of barley by water 113 immersion (300 ml at 15ºC) followed by aeration until the water content of 114 the grain reaches to achieve a sufficient moisture level (42-44%). The 115 water used for the steeping process (EC -dS m -1 at 25°C-, 0.93; pH, 8.22;

141

  Extraction procedures. Water samples (20 ml) were placed in a 142 100 ml glass flask with hermetic closing, with 40 ml of n-, v/v) and 2 g of anhydrous NaCl. The flasks 144 were introduced into an ultrasonic bath (Ultrasons 613, Selecta) with 145 distilled water for 10 min and the liquid was passed through Phase 146 Separator Paper (Albet 302, 15 cm Ø). Finally, the organic phase was 147 concentrated by rotary vacuum evaporation (35 ºC) to dryness and 148 redissolved in 10 ml of acetone and injected into the GC/MS system in the 149 conditions specified previously. For barley and malt, samples (5 g) were 150 homogenized at 8,000 rpm for 5 min in a high-speed electric mixer 151 (Polytron-Aggregate, Kinematica) with 10 ml of distilled water and 20 ml of 152 n-hexane. After this time, 20 ml of dichloromethane were added and the 153 mixture was centrifuged for 10 min at 3,000 rpm. The liquid phase was 154 quantitatively transferred to a glass funnel provided with a phase separator 155 paper. After filtration, the organic phase was concentrated through rotary 156 vacuum evaporation to dryness and redissolved in 10 ml of acetone 157 Pesticide analysis. A Finnigan Trace Ultra GC (Thermo Electron 158 Corporation, USA) with split-splitless injector, AS2000 Autosampler and 159 directly coupled by a transfer line to a Finnigan Polaris Q Ion Trap MS 160 (Thermo Electron Corporation) was used. Updated Dell ChemStation 161 application with Xcalibur™ software was used to control and automate many 162 features of the GC/ITMS system. The Rtx®-5MS (Restek, USA) fused silica 163 capillary column (30 m x 0.25 mm x 0.25 µm film thickness) contained 5 % 164 diphenyl, 95 % dimethyl polysiloxane as liquid-phase. The oven 165 temperatures were programmed as follows: column oven: initial 70 ºC, hold 166 1 min, programming rate 10 ºC/min (70 to 160 ºC), hold 2 min; 5 ºC/min 167 (160 to 250 ºC), hold 0 min; 30 ºC/min (250 to 280 ºC) and hold 4 min at 168 280 ºC for a total run time of 35 min. The injector and MS transfer line were 169 operated at 230 and 275 ºC, respectively. The IT operation conditions were: , AGC 50. The carrier gas was He at 1.2 ml min -1 . The 173 sample (2 µl) was injected in the splitless mode (60 s). Identification of the 174 pesticides was based on the retention time of the target ion and on the 175 three qualifier-to-target ion ratios. The qualifier-to-target ion percentage 176 was then determined by dividing the abundance of the selected qualifier ion

  177(Q) by the target ion (T) and multiplying by 100. Quantitation was achieved from 3.6 to 8.7% and a mean recovery in excess of 89% in all cases.

204(

  MRLs) established in barley by the Spanish legislation for the studied 205 compounds(BOE, 2004).206Removal and transfer of pesticide residues from barley to 207 malt. Table2shows the amount of pesticide residues recovered from the 208 different samples. In all cases, RSDs were lower than 11%. The 209 percentages removed after each stage of the process were calculated 210 against the residue level on grains at the end of the previous stage. As can 211 be seen in Table2, steeping reduced residue levels significantly; the lowest 212 percentages removed at the end of the steeping process correspond to 213 dinitroaniline herbicides, pendimethalin (10%) and trifluralin (15%). Both 214 are hydrophobic pesticides because their high log P ow (>5) and low solubility 215 (0.2-0.3 mg/l). In consequence, a low proportion of their residues were 216 removed with the steeping water. Regarding to the organophosphorus 217 pesticides, malathion has the lower log P ow (2.75) and the higher water 218 solubility (145 mg/l) of the all studied pesticides. For this, 55% of its 219 residues were removed from the barley grains after steeping while 48% of 220 fenitrothion residues (log P ow = 3.5) were removed in this stage. Others 221 great extent. Only fenitrothion residues significantly declined 249 (32%) while the removal of the other compounds varied from 6-17%. Some 250 authors(Miyake et al. 2002) show that most of the residues on germinated 251 grain were lower than those on kilned malt which is attributed to the 252 inhibition of the extraction by the moisture present in germinated grains. In 253 our case, germinated grains were air-dried (30 ºC) to constant weigh.

Page

  271 correlation between ln R t and time (r≥0.95). Additionally, a perfect 272 correlation (r>0.99) between the analytical and theoretical concentration 273 calculated (R 0 ) at 0 days was observed which indicates that the model is 274 to the calculated values for the constant rate (K) and half-275 lives time, the following dissipation rate was observed: this research it can be concluded that residues of water 280 soluble pesticides on barley tend to be eliminated in the greatest amount 281 after steeping. Conversely, hydrophobic residues remain in steeped grain 282 which it is supported by the good correlation that exists between the 283 partition coefficients of the octanol-water values of the pesticides and the 284 decline in concentration. Germination and kilning processes also contribute 285 to reduce the residue content of malt although to a lesser extent. Finally, 286 malt storage does not significantly reduce pesticide residues. Therefore, the 287 maltsters should pay special attention to pesticide residues on barley, 288 especially for triazole fungicides as propiconazole, which has been 289 demonstrated to be a strong sterol biosynthesis-inhibiting compound 290 (Navarro et al., 2007).

Figure 1 :

 1 Figure 1: Scheme showing four primary steps of malting process and sample points (*) for pesticide analysis.

Figure 2 :

 2 Figure 2: Correlation between percentages of pesticides removed (n=3) after steeping and their log P OW values (solid line is 95% confidence interval and dash line 95% prediction interval).

Figure 3 :

 3 Figure 3: Mean amounts removed (n=3) of total pesticide residues into the different stages of malting (error bars are 95% confidence intervals).

  Figure1

  hexanetrile

			CN
	Cl		CBu
			CH 2		C 15 H 17 ClN 4	288.8	2.94	142
			N
			N
			N
	Nuarimol F
	(±)-(2-	Cl	OH
	Chlorophenyl)(4-fluorophenyl)-		C	F	C 17 H 12 ClFN 2 O	314.7	3.18	26.0
	pyrimidin-5-	N	N
	yl)methanol

*: H: Herbicide; I: Insecticide; and F: Fungicide.

Table 2

 2 Mean amount (n=3) of pesticide residues recovered in the whole weight or volume from different samples and percentage remaining during malting.

	Mean (µg, as dry) ± RSD and % remaining

(r>0.92) was observed between percentages removed after steeping and 29 the P OW values of pesticides. The amount remaining after malting ranged

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acquisition mode Sequential FS (50-400 m/z) & SIM range mode (exact

mass ± 0.4 amu), multiplier 1788, ion source temperature 230 ºC, emission

by linear regression against calibration standards (2-3,000 ng/mL) and

terbuthylazine, and their major metabolites in the brewing process.

Journal of Agricultural and Food Chemistry 45: 1375-1380.

Kunze W. 2004. Technology of Brewing and Malting (3 rd International

English Edition). The Research and Teaching Institute for Brewing in

Berlin (VLB). VLB's Publishing Department. Berlin.

Miyake Y, Koji K, Matsuki H, Tajima R, Ono M, Mine T. 1999. Fate of

agrochemical residues, associated with malt and hops, during

brewing. Journal of the American Society of Brewing Chemists 57:

46-54.

Miyake, Y.; Hashimoto, K.; Matsuki, H.; Ono, M.; Tajima, R. 2002. Fate of

insecticida and fungicide residues on barley during storage and

malting. Journal of the American Society of Brewing Chemists 69:

110-115

Morton R, Bryan JG, Desmarchelier JM, Dilli S, Haddad PR, Sharp GJ. 2001.

peas. Journal of Stored Products Research 37: 277-285.

Navarro S, Pérez G, Vela N, Mena L, Navarro G. 2005. Behaviour of

myclobutanil, propiconazole, and nuarimol residues during lager beer

brewing. Journal of Agricultural and Food Chemistry 53: 8572-8579.

Navarro S, Pérez G, Navarro G, Mena L, Vela N. 2007. Influence of fungicide

Principal physical-chemical characteristics of the studied compounds.

Acknowledgements

292

We thank the Ministerio de Educación y Ciencia of Spain (Project AGL2002-293 03560) for financial assistance and the Brewers, Estrella de Levante,

294

Fábrica de Cerveza, for the technical help with this research.

Statistical analysis of decay of organophosphorus and pyrethroid

335

insecticides and carbaryl on paddy rice, maize, sunflowers and field


Vangelis Economou

Chrissanthy Moira Brett

Vassilios Papadopoulou

Stathis Frillingos

Tom Nichols

Vagelis Economou¹

Moira M Brett²

Chrissanthy Papadopoulou

Changes in histamine and microbiological analyses in

Keywords: Health significance, Microbiology, Quality assurance Additives/Contaminants: Animal products, Natural toxicants, Process contaminants Histamine, tuna, storage, temperature abuse, bacteria. 19

Histamine concentrations and bacterial counts in tuna loins stored at 3-4 o C and abused at 20 o C (histamine (ppm) in average and standard error in brackets, bacterial counts (CFU gr -1 ) in average of log 10 and standard error in brackets).

Days

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

Table 3 Histamine concentrations and microbial counts in fresh and frozen tuna loins stored at 0-2 o C and abused at 30 o C(histamine (ppm) in average and standard error in brackets, bacterial counts (CFU gr -1 ) in average of log 10 and standard error in brackets). Temperature abuse of tuna (Thunnus alalunga) was carried out in order to assess 3 the histamine build-up in fish processing facilities where fish can be exposed to high 4 temperatures for short periods of time. Histamine production was studied in tuna 5 loins under different storage and abuse conditions. Tuna was stored at 0-2 o C, 3-4 o C 6 and 6-7 o C and abused for 2 h daily at 20 o C and 30 o C for 7-12 days. Loins abused at 7 30 o C for 2 h daily, contained potentially toxic histamine concentrations (67-382 mg 8 kg -1 ) when stored at low refrigeration temperature (0-2 o C), whereas when stored at 6-9

Days

7 o C the loins contained highly toxic histamine concentrations (544.5-4156.6 mg kg -1 ). 10

Lower histamine concentrations (23 to 48 mg kg -1 in loins stored at 0-2 o C and 124.7-11 2435.8 mg kg -1 in loins stored at 6-7 o C) were observed in temperature abused loins 12 that were initially frozen. An increase over time was observed in most microbial 13 counts tested. Bacteria isolated from the temperature abused loins showed varied 14 ability of histamine production with Morganella morganii, Klebsiella oxytoca, 15

Staphylococcus hominis and Enterococcus hirae being the most active histamine 16 producing bacteria. 17 Proteus spp., Serratia macrescens) have been reported to be the most prolific 14 histamine producers [START_REF] Taylor | Histamine 20 production by food -borne bacterial species[END_REF] Scombroid or Histamine fish poisoning (SFP, HFP) is one of the most frequently 13 reported intoxications associated with the consumption of fish worldwide. The SFP 14 intoxication is generally related with high levels of histamine in spoiled fish. 15

Histamine, a biogenic amine involved in allergic reactions, is the main toxin involved 16 in SFP and its toxicity is enhanced by the presence of other biogenic amines in foods 17 [START_REF] Lehane | Histamine fish poisoning revisited[END_REF]. Histamine is formed by decarboxylation of histidine, which 18 is found in high levels in the muscle of fish belonging to the Scomberidae and 19

Scomberesocidae families such as tuna, albacore, mackerel and bonito. Still non-20 scombroid fish such as mahi-mahi, sardines, pilchards, anchovies, herring and marlin 21 have frequently been implicated in SFP (Taylor 1985;Clifford and Walker 1992). 22

Symptoms of SFP are cardiovascular, gastrointestinal and neurological, 23

including skin rashes, urticaria, oedema, localized inflammation, nausea, vomiting, 24 diarrhea, cramping, hypotension, headaches, palpitations and an oral burning and 25 Histamine is not inactivated by the usual thermal processes of the contaminated food 9 (freezing, chilling, cooking, canning etc) and the sole prevention is food storage at 10 temperatures below 4 o C and avoidance of prolonged handling at ambient 11 temperatures. The amount of histamine produced is a function of the fish species, the 12 individual fish and the fish part which is sampled, the time, the temperature and the 13 type and number of bacteria species present. The toxic dose is variable. Recent 14 reviews of the oral toxicity of histamine to humans in different foods concluded that 15

SFP is slight at 8-40 mg total dose, moderate at >40 mg and severe at >100 mg 16 [START_REF] Shalaby | Significance of biogenic amines to food safety and human health[END_REF][START_REF] Lehane | Histamine fish poisoning revisited[END_REF]. Scoging (1998a) analysed 405 incidents of 17 SFP in the UK and suggested the following classification: <100 mg kg -1 , safe for 18 consumption; 100-500 mg kg-1, mishandled and potentially toxic; 500-1000 mg kg -1 , 19 unsatisfactory and probably toxic; >1000 mg kg-1, toxic and unsafe for consumption. 20 where high concentrations of histamine are recorded in fish stored at appropriate 8 temperatures but thawed at the usually high room temperatures of the kitchen. Little 9

information is available about the effects of short -term temperature abuse and 10 bacterial flora on histamine production in tuna fish at the end-point (kitchen). The 11 objective of our study was to assess the effect of different temperature regimens, 12 simulating refrigerated storage and short -term temperature abuse, on histamine 13 formation together with the development of histamine forming microflora in tuna 14 steaks and loins, regardless of the temperature treatment they are subjected before 15 they reach a restaurant kitchen. This simulation of malpractices during the 16 preparation of tuna meals at a restaurant kitchen could be helpful to assess the risks 17 for public health and to implement proper control measures. Also an evaluation of the 18 histamine production capability of the different bacteria comprising the microflora of 19 spoiled fish was performed. incubator for 12 consequent days. The subgroup of 3 frozen loins was defrosted at 1 refrigeration temperature in a temperature monitored refrigerator. Histamine 2 concentration was tested prior to abuse and on the 4 th , 8 th and 12 th day of 3 temperature abuse, while microbiological analysis was performed prior to abuse and 4 on the 8 th and 12 th day of temperature abuse. The temperature abuse conditions are 5 concisely presented in Table 1. 6 7

Sampling for microbiological and histamine analysis 8

Sampling for microbiological and histamine analysis was carried out from the surface 9 of the middle part of the loin, from ~1cm depth, by cutting aseptically a vertical strip 10 weighing at least 30 g. The sample was homogenized in a laboratory blender (MSE 11 Atomix). 10 g were used for the microbiological analyses and 10 g for the histamine 12 analysis. Samples during the temperature abuse trial were taken from almost 13 adjacent areas so as to ensure uniformity in initial histamine content and microbial 14 populations. 15 16

Microbiological analysis 17

Bacterial enumeration 18

Bacterial counts were restricted to routinely detected food contaminants, which are 19 also supposed to be histamine producers (Enterobacteriaceae, Pseudomonas spp., 20 Staphylococcus spp., sulfite reducing bacteria, lactic acid bacteria, Photobacterium 21 spp. and Vibrio spp.) and was carried out in standard selective media. Ten grams of 22 homogenized muscle was combined with 90 ml of maximum recovery diluent and 23 ten-fold serial dilutions were performed. For surface plating 0.1 ml of each dilution 24 was used and for pour plating 1 ml of each dilution was used. 25

Isolation of bacteria 7

Characteristic colonies were picked from the selective media used and were 8 characterized to the genus level. All isolates were pure cultured and characterized 9

Gram stain was performed so as to test culture purity and the morphology of the 10 The bacterial isolates were stored until the examination of their histamine production 5 ability in beads of the Microbank system (ProLab Diagnostics, Ontario, Canada) 6 7

Histamine production by bacterial isolates 8

The ability of the isolated bacteria to produce histamine was measured in the culture 9 supernatant of Møller decarboxylase broth (MD broth) (Atlas, 1995). Prior to the 10 inoculation in MD broth the isolates were inoculated to Trypticase Soy broth 11 containing histidine (TSBH). 12 13 TSBH was prepared by addition of 30 g of Tryptone soy broth (Tryptone Soya Broth 14 (U.S.P.) CM0876, Oxoid Ltd, Wade Road, Basingstoke, UK), 1 g of histidine (L-15

Histidine dihydrochloride, Sigma -Aldrich) in 1 l of deionized water. The pH of TSBH 16 was adjusted to pH 6.8 before autoclaving for 10 min at 121°C. 17

The composition of MD broth was tryptone 0,5% (EZMix Tryptone, T-2559, 18

Sigma Chemical Co, St. Louis, USA), beef extract 0,5% (B-4888, Sigma Chemical 19 Co, St. Louis, USA), glucose 0,05% (α-D-Glucose analytical grade, 17023051, 20 SERVA Electrophoresis GmbH, Heidelberg, Germany), bromocresol purple 0,001% 21

(1.03025, Merck KgaA, Darmstadt, Germany), cresol red 0,0005% (11,447,2, Aldrich 22 Chemical Co, Milwaukee, USA), pyridoxal phosphate 0,0005% (pyridoxal 5'-23 phosphate, P-9255, Sigma) and histidine 1%. The pH of MD broth was adjusted to 24 pH 6.0 before autoclaving for 15 min at 121°C. 25

Histamine analysis 10

For the histamine analysis of tuna a fluorometric method was performed as described 11 by Taylor (1978). 10 g of fish flesh were mixed with 50 ml methanol (822283, Merck 12 KgaA, Darmstadt, Germany)and were incubated at 60°C in a water bath (type W760, 13

Memmert GmbH, Schwabach, Germany) for 15 min. 6 ml of water saturated butanol 14 (101990, Merck KgaA, Darmstadt, Germany), 2 g of sodium carbonate decahydrate 15 (106391, Merck KgaA, Darmstadt, Germany) and 1 ml of 5M NaOH (Sodium 16 hydroxide granulated, 106469, Merck KgaA, Darmstadt, Germany) was added to the 17 properly diluted methanol extract of the fish flesh. The tubes were shaken and were 18 allowed to settle. 3 ml of the organic phase was mixed with 3 ml of 0.1 M HCI 19 Hamburg GmbH, Hamburg, Germany). Histamine analysis was performed according 7

to the manufacturer's instructions. 8 9

Prior to histamine analysis of culture supernatants, a validation of the kit was 10 performed. 5 sterile spiked samples of the broth containing different histamine 11 concentrations (0, 5, 20, 50 and 100 mg kg-1) were analysed five times. The results 12 were sufficient for the use of this kit in the measurement of histamine in culture 13 supernatants. A plot of the measured histamine concentration against the actual 14 histamine concentration was drawn. The regression factor of the measured histamine 15 was r 2 = 0,982 and the difference between the measured and the actual 16 concentration of histamine did not exceed 2 standard deviations. These results were 17 judged as adequate for the employment of this method in the measurement of 18 histamine in MD broth supernatants. 19 20

Statistical analysis 21

The two-sided student t-test was used. Descriptive statistics of means, standard 22 deviation and standard error were applied in analyzing the results. A significance 23 level of 5% was used. The regression analysis between histamine concentration and 24

Temperature abuse 5

The histamine concentrations of the tuna samples upon delivery were ranging from 1 6 mg kg-1 to 47.1 mg kg-1. The histamine concentrations of the tuna during the time-7 temperature abuse procedure are presented in Tables 234. 8 9

In defrosted loins stored at 3 -4 o C, histamine concentrations were low (<60mg kg-1) 10 both in controls and loins abused daily at 20 o C for 2 h (Table 2), with only one 11 abused frozen loin exceeding this level (127.4 mg kg-1). There were only minor 12 differences between the different defrosting regimes, and all loins were characterized 13 according to their histamine content as safe for consumption at the end of the abuse 14 procedure. 15 16 In loins stored at 0-2 ο C the histamine concentration was low (<70mg kg-1) both in 17 controls and defrosted loins abused daily at 30 o C for 2 h. Higher concentrations (67-18 382 mg kg-1) were measured in fresh loins abused daily at 30 o C for 2h (Table 3). 19

However, the increase was not statistically significant (p>0.10) and at the end of the 20 abuse procedure the controls and defrosted loins were characterized safe for 21 consumption whereas fresh loins were potentially toxic. 22

In loins stored at 6-7 o C there was a significant increase (p<0.05) in histamine levels 24 in the controls, resulting in final concentrations of 228.3-584.2 mg kg-1 (Table 4). 25 The final concentration of histamine in defrosted loins abused daily at 30 o C for 2 h 1 (124.7-2435.8 mg kg-1) was higher than the histamine concentration of the controls. 2

In fresh temperature-abused loins, histamine increased to final concentrations 544.5-3 4156.6 mg kg-1. At the end of the abuse procedure, both the controls and the 4 temperature abused loins were characterized as unsuitable for consumption, while 5 temperature -abused fresh loins being highly toxic. 6 7 No Vibrio spp. and Photobacterium spp. counts were recorded and no strains of 8 these species were isolated, although cultures of these species were performed 9 throughout the experimentations. In temperature abused loins and controls stored at 10 3 -4 o C (Table 2) the counts of Enterobacteriaceae and coliforms increased by 2.5-11 3.2 log 10 and 2.1-2.8 log 10 respectively whereas the ACC, LAB and Pseudomonas 12 spp. counts increased by approximately 3 log 10 (2.7 -4.5 log 10 , 3.1-5 log 10 and 1.5-13 5.7 log 10 respectively) after seven days of storage. However, minimal changes were 14 observed n the counts f SRB in this group of tuna loins. 15 16 In temperature abused loins and controls stored at 0-2 o C (Table 3), the counts of 17 In temperature abused loins and controls stored at 6-7 o C (Table 4), the 1 Enterobacteriaceae and coliforms counts increased by the end of the procedure by 2 approximately 3 log 10 (3.2 log 10 and 3.5 log 10 respectively) in the loins and by 1 log 10 3 in the controls. Also an increase was observed in ACC and Staphylococcus spp. Regression analysis of histamine concentrations and bacterial counts of loins stored 7 at 6 -7 o C revealed correlation between histamine concentrations and aerobic colony 8 counts (r 2 =0.84), Enterobacteriaceae counts (r 2 =0.86) and coliform counts (r 2 =0.87) 9

(Figure 1). A correlation between histamine concentrations and aerobic colony 10 counts (r 2 =0.81), Enterobacteriaceae counts (r 2 =0.67) and coliform counts (r 2 =0.62) 11 was also observed in loins stored at 3 -4 O C (Figure 1), whereas in loins stored at 0 -12 2 O C no correlation was observed between histamine concentrations and bacterial 13 counts (Figure 1). [START_REF] Holt | Bergey's manual of 15 determinative bacteriology[END_REF] 15

Bacterial isolates and their potential for histamine production 16

77 isolates were isolated from temperature abused loins with high histamine 17 concentration. The isolates are shown in Table 5. Forty of them belong to 18

Enterobacteriaceae (53% of the total isolated bacteria). These bacteria belong to the 19 respectively after 12 to 14 days of storage, which are lower than our measurements 17 obtained under similar conditions; however, the initial histamine concentration 18 measured in the loins by Frank and Yoshinaga was only 1.1 mg kg-1, while in our 19 loins ranged from 1 -47.1 mg kg-1. In our study histamine concentrations in control 20 loins stored at 0 -2 o C were similar to those reported in mackerel stored in ice 21 [START_REF] Bennour | Chemical 22 and microbiological assessments of mackerel (Scomber scombrus) stored in 23 ice[END_REF]. In Spanish mackerel stored at 0 o C, histamine concentrations 22 were lower (mean<6 mg kg-1) [START_REF] Middlebrook | Effects of 10 storage time and temperature on the microflora and amine development in 11 Spanish mackerel (Scomberomorus maculatus)[END_REF]. 23 Taylor and Speckhard (1983) reported that histamine-producing bacteria could not be 13 isolated from frozen tuna without using a 24 h recovery period. When Staruszkiewicz 14 et al. (2004) studied the changes in selected biogenic amines in mahi-mahi and tuna 15 held in seawater at 25 to 35 o C for up to 18 h, they observed that histidine 16 decarboxylase activity was retained in some frozen fish samples and could result in 17 further increases in histamine on thawing. 18 19 According to our results (Tables 2,3 2 5) varied in their capacity of producing histamine; isolate M4 was the most prolific 3 histamine producer (24 499 mg kg-1), while isolate M1 was a limited histamine 4 producer (20 mg kg-1). Other researchers reported M. morganii as the most active 5 histamine producer [START_REF] Frank | 17 Identification and decarboxylase activities of bacteria isolated from decomposed 18 mahimahi (Coryphaena hippurus) after incubation at 0 and 32 o C[END_REF] No correlation was found in loins stored at 0 -2 o C, with none of the tested bacterial 15 counts revealing any correlation with histamine production and probably, in this group 16 of loins, another bacterial group was responsible for the production of histamine. 17 epidermidis and S. capitis isolated from salted fish. [START_REF] Tham | Histamine formation by enterococci isolated from home -made goat 5 cheese[END_REF] reported production 8 of small amounts of histamine from E. faecalis and Ε. faecium isolated from cheese. 9

The measured histamine concentration from the EN2 isolate (2404 mg kg-1) is far 10 higher from the histamine concentration reported by Tham (4 mg kg-1). The 11 presence of Enterococcus spp. in fish is relatively rare and in most cases is attributed 12 to post -catch contamination (Gram και Huss 1996). We observed that the prolific 13 histamine producers presented only 10% of the isolates, while 39% were slow 14 producers and 51% were not histamine producing bacteria. The total histamine 15 produced by the prolific producers (47.4 mg) was almost 100 times more than the 16 histamine produced by the slow histamine producers (0.43 mg), indicating that the 17 contribution of slow histamine producing species to histamine build -up in fish flesh 18 is limited. ambient temperatures even for short periods should be avoided. In facilities where 2 temperature control is impossible or inadequate, tuna should be stocked up for 3 shorter periods (less than 12 days). Also, the practice of freezing the tuna prior to 4 storage at fridge temperature can restrain histamine production. 5

The authors wish to thank The British Council and the General Secretariat for 9

Research and Technology of the Greek Ministry of Development for financial support 10 of the present study. Also the authors wish to express special thanks to Dr. Diane 11

Roberts for her valuable advice throughout this study, to Mr. Jim Clements and Miss 12

Carla Milman for collection of samples and to all personnel of the Toxins Reference 13 Laboratory at Food Safety Microbiology Laboratory, CPHLS for their assistance.

FF

  Figure 1. Regression analysis of histamine and Aerobic Colony Counts, Enterobacteriaceae and coliforms 1

2 8 (

 8 Histamine formation in fish is related to the free histidine content of the fish muscle, 3 the presence of bacterial histidine decarboxylases and certain environmental 4 conditions. The production of histamine is largely induced by post-harvest exposure 5 to high temperature of the fish. Temperature enhances the multiplication of bacteria 6 and the bacterial production of histidine decarboxylases. In addition, histidine 7 decarboxylase activity is favoured by the exposure to temperatures higher than 15 o C Lehane and Olley 2000: Kanki et al. 2007). In general, histamine production is 9 favored by temperatures higher than 5 o C with an optimum of 20 -30 o C (Kim et al. 10 2000; Lehane and Olley 2000), and by the presence of bacterial species that are 11 prolific histamine producers. Enterobacteriaceae (Morganella morganii, Enterobacter 12 cloacae, Citrobacter freundii, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, 13

  [START_REF] Lehane | Histamine fish poisoning revisited[END_REF] Gillespie et al. 2001). The SFP 1 intoxication has to be differentiated from other food allergies. Manifestation and 2 severity of symptoms may vary according to individual susceptibility, dietary 3 components and medications, such as isoniazid and antimalarials, which may 4 increase susceptibility. The incubation period is short (less than 2 h from 5 consumption of food) and symptoms usually resolve within a few hours(Lerke et al. 6 1978; Taylor 1986; Lehane and Olley 2000; McLauchlin et al. 2005). 7 8

1 5 (

 5 According to the European legislation(Council Directive, 1991) among nine samples 21 from each batch the mean concentration of histamine must not exceed 100 mg kg -1 ; 22 two samples may have a concentration of histamine of more than 100 mg kg-1 but 23 less than 200 mg kg -1 and no sample may have a concentration of histamine 24 exceeding 200 mg kg -1 . 25Tuna fish is considered to be susceptible to histamine formation. Fresh tuna has a 2 shelf life of about 11 days from when they are caught when stored at temperature 3 >4 o C (Anonymous 1997). In recent years there are an augmenting number of 4 incidents of SFP attributed to consumption of fish (mainly tuna) in restaurants Anonymous 1986; Scoging 1998a; 1998b; Predy 2003; Wu and Chen 2003; Hall 6 2003; Feldman et al. 2005; McLauchlin et al. 2005; Sigmundsdóttir et al. 2005), 7

20

 20

3 4 21 22 19 20

 32119 The fish samples used for the trials were 30 tuna loins (Thunnus alalunga) collected 5 by Sampling Officers from consignments landed at Heathrow Airport. Six of the tuna 6 loins were imported from Brazil, 6 from Sri-Lanka, 6 from Maldives, 8 from Indonesia 7 and 4 from Yemen. The specimens were delivered to the Food Safety Microbiology 8 Laboratory (FSML), Central Public Health Laboratory Service (CPHLS) within 6 h 9 after collection. The average weight of the loins was 2.64 kg, ranging from 2.14 kg to 10 3.21 kg. The fresh loins were transported to the laboratory in insulated boxes and 11 reached the laboratory 1 to 2 days post catching. Every effort was made to keep the 12 temperature of storage of fresh loins at 3-4 o C from the point of collection to the use of 13 the specimens for the temperature abuse trial. 18 of the loins used (loins from Brazil, 14Sri -Lanka and Maldives) were frozen at the place of origin. They were transported 15 at the laboratory frozen in well insulated boxes and stored until defrosting in a 16temperature monitored freezer at -35 o C. All tuna fish samples were skinned and 17 placed into sterile plastic bags prior to any processing. For all samples the histamine 18 and microbiological analysis was performed the day of arrival at the laboratory to 19 ensure good quality of the loins. The temperature abuse trial has taken place one 20 day after the arrival of specimens to the laboratory.Temperature abuse process 23The tuna loins were divided into 3 groups. One group was stored at 3-4 o C and 24 consisted of 12 frozen (-35 o C) loins, divided into 4 subgroups (3 trial subgroups 25 each and one control subgroup containing 3 loins). The control 1 subgroup was defrosted at 3-4 o C and received no further treatment. The rest 3 2 subgroups were defrosted using 3 different regimes: at 3-4 o C in a temperature 3 monitored refrigerator, at 20 o C (room temperature) on the lab bench and in a 4 microwave oven (Philips Cooktronic M710, Philips Electronics UK Ltd, UK) according 5 to the manufacturer's instructions. The 3 trial subgroups were temperature abused 6 for 2 h daily at 20 o C for 7 consequent days. Histamine concentration and 7 microbiological tests were performed prior to temperature abuse and on the 5 th and 8 7 th day of temperature abuse. 9 10 The second group of loins was stored at 0-2 o C in ice inside a temperature monitored 11 refrigerator and consisted of 9 loins (6 fresh reserved at 3-4 o C, 3 frozen reserved at -12 35 o C). The 3 out of the 6 fresh loins served as controls and received no further 13 treatment, while the rest 3 fresh loins were temperature abused for 2 h daily at 30 o C 14 in a temperature monitored incubator for 12 consequent days. The 3 frozen loins 15 were defrosted at refrigeration temperature (3-4 o C) in a temperature monitored 16 refrigerator. All loins were tested for histamine prior to temperature abuse and on the 17 4 th , 8 th and 12 th day of temperature abuse and were microbiologically analyzed prior 18 to temperature abuse and on the 8 th and 12 th day of temperature abuse. The third group was stored at 6-7 o C using a cooled incubator (LMS Cooled incubator, 21 model No 303, LMS Ltd, UK) and consisted of 9 loins (6 fresh reserved at 3-4 o C, 3 22 frozen reserved at -35 o C) divided into 3 subgroups. One subgroup of 3 fresh loins 23 served as control and received no further treatment, while the other subgroup of 3 24 fresh loins was temperature abused for 2 h daily at 30 o C in a temperature monitored 25

10 1

 10 Aerobic colony counts (ACC) were determined on Plate Count Agar using surface 2 plating after incubation for 2 days at 30 o C. Enterobacteriaceae, were determined on 3 MacConkey agar No.3 using surface plating after incubation for 1 day at 37 o C. 4MacConkey agar No.3 permits the growth of other bacteria, so the estimated counts 5 should be considered as presumptive Enterobacteriaceae. Staphylococcus spp. were 6 determined on Baird-Parker agar with egg yolk-tellurite supplement by surface plating 7 after incubation at 37 o C for 2 days. Characteristic colonies were tested for catalase 8 and coagulase production (Staphytect plus, Oxoid Ltd, Hampshire, England). 9Pseudomonas spp. were determined using Pseudomonas selective agar 10 supplemented with Cetrimide (10 mg l -1 ) -Fucidin (10 mg l -1 ) -Cephaloridine (50 11 mg l -1 ) by surface plating after incubation for 2 days at 30 o C. Characteristic colonies 12 were confirmed as Pseudomonas spp. by testing for oxidase production with the use 13 of oxidase identification sticks (Oxoid Ltd, Hampshire, England). Sulfite reducing 14 bacteria (SRB) were determined on Tryptose Sulfite Cycloserine agar supplemented 15 with Perfringens (TSC) selective supplement (D-cycloserine 400 mg l -1 ; Oxoid Ltd, 16Hampshire, England) using a pour plate with an overlay after incubation for 2 days at 17 37 o C in an anaerobic cabinet (Modular Atmosphere Controlled System, Don Whitley 18 Scientific Ltd, West Yorkshire, UK). Lactic acid bacteria (LAB) were determined on 19 DeMan-Rogosa-Sharpe agar by surface plating after incubation for 3 days at 37 o C in 20 an anaerobic cabinet. For the determination of Vibrio spp. and Photobacterium spp. 21ten fold serial dilutions were prepared in Alkaline peptone water (APW). 0,1 ml of 22 each dilution was plated on Thiosulphate Citrate Bile -salt Sucrose (TCBS) agar and 23 incubated at 37 o C for 24 h for the detection of Vibrio spp. and 20 o C for 48 h for the 24 detection of Photobacterium spp.(Holt et al, 1994). In addition, Gram staining 25 selected colonies was performed. The methodology followed 1 is described by[START_REF] Roberts | Practical Food Microbiology[END_REF]. All media and broths were supplied by the 2 PHLS Media Department (CPHLS Media Department, Colindale, London, UK) (ISO 3 9002 standard) and were quality controlled by the Media Department Quality Control 4 to UKAS standards. 5 6

isolates. 11 12

 11 The identification of the bacteria isolated from MacConkey agar was carried out with 13 the use of the API 20 E system (20 100, BioMérieux, Marcy-l'Etoile, France). The 14 tests used for the identification of Staphylococcus spp. were the catalase test, the 15 coagulase test (Staphytect plus, DR850M, Oxoid Ltd, Hampshire, England), the 16 DNAse production test (DNAase agar, Scharlau Chemie S.A., Sentmenat, Barcelona, 17 Spain), the ability of the isolates to grow in Nutrient agar (Scharlau Chemie S.A., 18 Sentmenat, Barcelona, Spain) supplemented with 7,5% NaCl and 10% NaCl and the 19 API 20 STAPH system (20 500, BioMérieux, Marcy-l'Etoile, France). Pseudomonas 20 spp. were identified according to their ability to produce oxidase (oxidase 21 identification sticks, BR64A, Oxoid Ltd, Hampshire, England) and with the use of the 22 API 20 NE system (20 050, BioMérieux, Marcy-l'Etoile, France). The identification of 23 Vibrio spp. and Photobacterium spp. was carried out with the use of the oxidase test, 24 the catalase test (Hydrogen peroxide 30%, analysis grade, 107209 Merck KgaA, 25 µg, DD15, Oxoid Ltd, Hampshire, England and the use of the API 2 20NE system (20 050, BioMérieux, Marcy-l'Etoile, France). 3 4

1

  The isolates were inoculated in TSBH and incubated at 37 o C for 24 h. 100 µl of the 2 culture was inoculated in MD broth containing 1% histidine and incubated at 37 o C for 3 48 h. After 48 h the culture supernatant was collected and centrifuged 2 times at 4 3500 mg kg-1 in a desktop centrifuge (Sorvall GLC-4, Sorvall Instruments, 5Wilmington, DE, USA). The supernatant was sterilized with the use of Acrodisc 6 syringe filters 0.45 µm (Pall, New York, USA). The filtered culture supernatants were 7 stored at -85 o C until histamine analysis. 8 9

( 320331 ,Food 2 3

 3203312 ACS reagent 37%, Aldrich Chemical Co, Milwaukee, USA). The organic 20 phase was discarded. 0.4 ml of 1M NaOH and 0.1 ml of 1% o-phthalaldehyde (P 21 0657, Sigma Chemical Co, St. Louis, USA) were added and fluorescent output was 22 measured by a fluorimeter (Kontron M25, Kontron Analytical, Kontron Instruments 23 Ltd, Herts, UK) at 360 nm excitation and 450 nm emission wavelengths. Histamine 24 Page 21 of 39 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk measured prior to any procedure and after time-1 temperature abuse of the samples.For the histamine analysis of culture supernatants a commercial ELISA kit for the in-4 vitro diagnostic quantitative determination of histamine in human plasma and urine 5 and in cell culture supernatants was used (Histamine ELISA, RE 592 21, IBL-6

Food 3 4

 34 bacterial counts was contacted by plotting scattered graphs and calculating the r 2 25 Page 22 of 39 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk 2 factors higher than 0.5 were accepted to indicate a correlation between the 1 histamine concentration and the bacterial counts measured. 2Results

Enterobacteriaceae

  and coliforms increased by approximately 2 log 10 (1.9-2.7 log 10 18 and 1.1-2.4 log 10 respectively). The ACC, Staphylococcus spp., LAB and 19 Pseudomonas spp. counts increased by 1.4-2.6 log 10 , 2-4.4 log 10 and 2.2-2.3 log 10 20respectively. In this group of abused tuna loins an increase of almost 1 log 10 was 21 observed in the counts of SRB. There was no difference in counts between controls 22 and temperature-abused loins.

4 counts ( 3

 43 -4.1 log 10 ) LAB (2.6-3.4 log 10 ) and Pseudomonas spp. (2.5-5.5 log 10 ). 5 6

species

  Citrobacter spp. (22% of Enterobacteriaceae), Enterobacter spp. (20%), 20 Klebsiella spp. (15%), Morganella spp. (10%) and Escherichia spp. (10%). The 21 Staphylococci that were isolated represent 21% of the total bacteria isolated and 22 consisted of Staphylococcus xylosus (24% of Staphylococci), S. epidermidis (19%), 23 S. hominis (19%), S. aureus (5%), S. auricularis (5%) και S. simulans (5%). The 24 Gram negative rods represented 21% of the total bacteria isolated and consisted of 25

18 3 4 14 F

 18314 Pseudomonas fluorescens (29% of Gram negative rods), Pseudomonas putida 1 (24%) Burkholderia cepacia (5%), Flavimonas oryzihabitans (5%) and Pseudomonas 2 aeruginosa (5%).Morganella morganii was the most active histamine producing species among the 5 bacteria isolated. In the cultures of the three out of four M. morganii the 6 concentrations of histamine were 24 499, 6875 and 6296 mg kg-1, with the 7 concentration of 24 499 mg kg-1 being the highest measured among the tested 8 bacterial cultures. High concentrations of histamine were also measured in the 9 cultures of one Staphylococcus hominis isolate (3050 mg kg-1), one Enterococcus 10 hirae isolate (2404 mg kg-1) and two Klebsiella oxytoca isolates (2156 and 2149 mg 11 kg-1 respectively). It should be noted that the four out of five S. hominis isolates 12 tested did not exhibit histamine production ability. The histamine concentration of the 13 culture supernatants of the bacterial isolates tested is shown in Table5.According to our results the temperature of abuse is the most important factor in 3 histamine formation (Tables2, 3, 4 and Figure 1). The loins abused at 20 o C (storage 4 at 3-4 o C) showed no increase in histamine, while the loins abused at 30 o C (storage 5 at 0-2 o C) presented toxic histamine levels. Histamine concentrations similar to those 6 observed in the present study have been reported by Guizani et al. (2005) in 7 yellowfin tuna. The histamine concentration in yellowfin tuna stored at 8 o C was 150 8 mg kg-1 after 8 days of storage and in our study it was 147.3 mg kg-1 after 8 days of 9 storage. Our results coincide with those of Kim and Price (1999), who detected low 10 concentrations of histamine (80 mg kg-1) in albacore loins stored at 0 o C for 18 days. 11 Lòpez-Sabater et al. (1996) detected histamine in large tuna fish pieces from the 4 th 12 day of storage at 8 o C, measuring concentrations >2500 mg kg-1 after 6 days of 13 storage at 8 o C, but no histamine was detected in large tuna fish pieces after 12 days 14 at 0 o C. Frank and Yoshinaga (1987) reported histamine concentrations 3.8 mg kg-1, 15 5.6 mg kg-1 and 169 mg kg-1 in skipjack tuna loins stored at -1 o C, 4.4 o C and 10 ο C 16

  was not statistically significant (p>0.05). The freeze -2 defrosting process appears to reduce the bacterial cell numbers or causes sub-lethal 3 injuries to bacterial cells causing poor histamine production.Baranowski et al. (1990) 4 observed that the duration of freezing of fish is an important factor for histamine 5 formation during storage of mahi-mahi fish at 32 o C for 1 day. Ben-Gigirey et al. 6(1998) reported that the survival of bacteria involved in biogenic amine formation is 7 poor at freezing temperatures. Recently[START_REF] Dalgaard | Biogenic amine formation 5 and microbial spoilage in chilled garfish (Belone belone belone) -effect of 6 modified atmosphere packaging and previous frozen storage[END_REF] evaluated the 8 biogenic amine formation and microbial spoilage in fresh and thawed chilled garfish, 9 concluding that histamine formation depends strongly on previous freezing of the 10 fish, which probably kills freeze -sensitive bacteria like Photobacterium spp. or 11Vibrio spp. In our study no Photobacterium spp. or Vibrio spp bacteria were isolated. 12

21 pneumoniae

 21 , 4 and Figure 1) the daily abuse of tuna 20 triggered the growth of bacteria. The correlation observed between histamine 21 concentration, ACC, Enterobacteriacae and coliform counts indicate that bacteria 22 belonging to these groups are responsible for histamine production in tuna loins 23 stored at temperatures >3 o C. Our results coincide with the statement by Stratton and 24 Taylor (1991) that enteric bacteria, specifically Morganella morganii, Klebsiella 25 and Hafnia alvei are the most prolific histamine producers in fish when 1 they are maintained at temperatures ≥4 o C. The Morganella morganii isolates (Table

2 some 3 (

 23 [START_REF] Okuzumi | Isolation of psychrophilic and halophilic 14 histamine-forming bacteria from Scomber japonicus[END_REF] have reported that Psychrophiles and especially halophilic N-18 group bacteria growing at 5 o C can produce histamine. By the end of temperature 19 abuse trials, the dominant microbial populations were LAB followed by the 20 Enterobacteriaceae. Similar results have been reported by Yoshinaga and Frank 21 (1982) and López-Sabater et al. (1996) when large fresh tuna fillets were examined. 22 Kim and Price (1999) reported lower levels (<10 4 CFU gr -1 ) of ACC in albacore stored 23 in ice, but the temperature of storage was 15 o C. Koutsoumanis et al. (1999) also 24 reported predominance of LAB and Enterobacteriaceae in steaks of Sparus aurata 25 ο C and 8 o C. In our study the SRB counts remained low during freezing and 1 storage at low temperatures. This observation may be attributed to the inability of Clostridia species (e.g. C. perfringens) to grow at temperatures below 15 o C McClane 1997). Ben-Gigigrey et al. (1998) reported absence of SRB in fresh 4 albacore too. From the Gram positive isolates, only one strain of Staphylococcus 5 hominis (S4) and one strain of Enterococcus hirae (EN2) were active histamine 6 producers. Hernández -Herrero et al. (1999) reported production of histamine by S. 7

19 20

 19 According to our results the histamine concentration was at toxic levels in tuna 21 samples which were exposed at temperatures ≥30 o C for short periods every day and 22 for longer than 3 days. Considering the public health protection, appropriate storage 23 at <4 o C and proper handling of tuna fish is essential for delaying histamine formation 24 and preventing SFP outbreaks. For food safety reasons the environmental 25 processing facilities should be kept cool (<20 o C) and exposure to 1

14

 14

3 Table 2 . 7 Table 3 . 11 Table 4 . 15 Table 5 .

 3273114155 Histamine concentrations and bacterial counts in tuna loins stored at 3-4 o C 4 and abused at 20 o C [histamine (mg kg-1) in average and standard error in 5 brackets, bacterial counts (CFU gr -1 ) in average of log 10 and standard error 6 in brackets]. Histamine concentrations and microbial counts in fresh and frozen tuna loins 8 stored at 0-2 o C and abused at 30 o C [histamine (mg kg-1) in average and 9 standard error in brackets, bacterial counts (CFU gr -1 ) in average of log 10 10 and standard error in brackets]. Histamine concentrations and microbial counts in fresh and frozen tuna loins 12 stored at 6-7 o C and abused at 30 o C [histamine (mg kg-1) in average and 13 standard error in brackets, bacterial counts (CFU gr -1 ) in average of log 10 14 and standard error in brackets]. Bacterial Isolates and histamine concentration in culture supernatants after 16 incubation at 37 o C for 48 h. 17

Fig 1 .

 1 Fig 1.Regression analysis of histamine and Aerobic Colony Counts, 18

  Aerobic colony count (30 o C), c Enterobacteriaceae, d Coliforms, e Staphylococcus spp., f Sulfite reducing bacteria, g Lactic acid bacteria, h Pseudomonas spp.

				H a	ACC b	E c	C d	ST e	SRB f	LAB g	PS h
		Controls	0 5 7	11.4 (3.3) 31 (2.5) 30 (6.1)	3.1 (0.1) 5.4 (1.7) 6.3 (1.1)	2.9 (0.2) 3.5 (1.1) 4 (1.2)	2.3 (0.1) 1.7 (1.2) 2.7 (1.9)	<2 3.4 (2.4) 4.9 (1.6)	<1 1.15 (0.8) <1	<2 3.4 (2.4) 3.6 (2.6)	2 5.5 (0.9) 5.9 (1.5)
						Defrosting at fridge temperature
	Storage at 3 -4 o C	2 h of temperature abuse at 20 o C	0 5 7 0 5 7 0 5	17.1 (0.5) 30.3 (0.6) 29.1 (1.7) 20.4 (0.6) 32 (8.5) 36 (3.5) 16.6 (3.7) 28.2 (6.5)	3.8 (0.4) 6.2 (0.5) 6.9 (0.5) 4.1 (0.8) 6.4 (0.4) 8.1 (0.1) 4 (0.8) 5.4 (1)	3.4 (0.2) 3.2 (0.4) 5.1 (0.6) Defrosting at room temperature 3.1 (0.3) 2.2 (0.9) 3.2 (0.5) 6 (0.3) 4.4 (0.7) 6.8 (0.2) 2.8 (0.4) 2.5 (0.2) 3.3 (0.6) 5.1 (0.6) 3.3 (1.3) 5.9 (0.7) 6.6 (0.4) 3.9 (0.8) 6.6 (0.6) Defrosting using a microwave oven 3.5 (0.4) 2.8 (0.2) 1.1 (0.9) 4.6 (0.7) 1.7 (1.4) 4.7 (1.1)	0.6 (0.5) 0.8 (0.6) 0.3 (0.3) <1 1.4 (0.7) <1 <1 1.6 (0.7)	1.3 (1) 5.2 (0.6) 5.8 (0.6) 2.2 (1.1) 5.4 (0.4) 6.5 (0.5) 1.6 (0.6) 3.4 (1.4)	2.2 (1.1) 6.5 (0.2) 7.1 (0.5) 3.3 (1.6) 6.4 (0.4) 8 (0.3) 3.1 (1.5) 6.6 (0.3)
			7	63 (26.4)	7 (0.8)	5.1 (1.4)	3.6 (1.4)	4.9 (2)	0.5 (0.4)	4.3 (1.9)	6.8 (0.7)

a Histamine, b Deleted: ., i Vibrio spp., j Photobacterium spp http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

Table 4

 4 Histamine concentrations and microbial counts in fresh and frozen tuna loins stored at 6-7 o C and abused at 30 o C (histamine (ppm) in average and standard error in brackets, bacterial counts (CFU gr -1 ) in average of log 10 and standard error in brackets).

			Days	H a	ACC b	E c	C d	ST e	SRB f	LAB g	PS h
			0	14.3 (2.6)	4.8 (0.3)	3.5 (0.2)	2.9 (0.2)	1.9 (0.8)	0.3 (0.3)	4.7 (0.2)	3 (0.2)
		Controls	4 8 12	48.2 (19.7) 147.3 (46.6) 350 (95.6)	7 (0.3) 7.2 (0.1)	4.6 (0.2) 4.8 (0.1)	3.6 (0.2) 3.8 (0.5)	5.7 (0.4) 5.9 (0.2)	1.7 (0.7) 1.6 (0.6)	7.2 (0.3) 7.6 (0.2)	5.2 (0.9) 7.2 (0.3)
	Storage at 6-7 o C	C 2 h of temperature abuse at 30 o	0 4 8 12 0 4 8 12	26.7 (7.72) 429.6 (316.8) 769 (398) 1962 (908.6) 35.1 (10.6) 921.6 (720.3) 318.5 (214.4) 1375.6 (550.3)	4.6 7.1 (0.3) 7.9 (0.2) 4.6 (0.5) 7.4 (0.1) 7.6 (0.1)	2.8 (0.4) 4.9 (0.5) 5.8 (0.4) 3.5 (0.1) 5.8 (0.3) 6.42 (0.3)	Fresh tuna 1.5 (0.6) 4.4 (0.7) 5.1 (0.8) Frozen tuna 0.9 (0.7) 4.1 (0.8) 4.9 (0.5)	1.2 (1) 5.5 (0.2) 6.2 (0.4) 3.2 (0.3) 6.4 (0.2) 6.9 (0.2)	<1 0.3 (0.3) 1.6 (0.7) 0.3 (0.3) 0.3 (0.3) 3 (0.3)	5.1 (0.5) 6.3 (0.2) 7.8 (0.5) 3.8 (0.2) 6.6 (0.2) 7.2 (0.3)	1.4 (0.6) 5.5 (0.2) 6.9 (0.1) 2.2 (0.2) 6.1 (0.3) 6.7 (0.3)

a Histamine, b Aerobic colony count (30 o C), c Enterobacteriaceae, d Coliforms, e Staphylococcus spp., f Sulfite reducing bacteria, g Lactic acid bacteria, h Pseudomonas spp. Deleted: , i Vibrio spp., j Photobacterium spp.

Table 5

 5 Bacterial isolates and histamine concentration in culture supernatants after incubation at 37 o C for 48 h.

			Number of	isolates	Strain	[Histamine	(ppm)			Number of	isolates	Strain	[Histamine	(ppm)
	Enterobacteriaceae						Gram positive cocci
	Citrobacter	braaki	2		C1	13	Staphylococcus aureus	1	S3	0
					C5	4		auricularis	1	S6	0
		freundii	7		C2	42		epidermidis	4	S1	0
					C3	19				S2	0
					C4	8				S5	0
					C6	6				S11	0
					C7	5		hominis	4	S4	305 0
					C8	2				S7	1
					C9	2				S9	0
	Enterobacter amnigenus	2		E3	0				S10	0
					E6	0		simulans	1	S15	1
		aerogenes	1		E4	60		xylosus	5	S8	1
		cloacae	7		E1	2				S12	0
					E2	0				S13	0
					E5	0				S14	0
					E6	0				S15	0
					E7	0	Enterococcus	faecalis	1	EN1	15
	Escherichia	coli	4		ES1	20		hirae	1	EN2	2404
					ES2	18

  ; Ababouch et al. 1991; López -Sabater et al. 6 1994; Dalgaard et al. 2006). Histamine -producing K. oxytoca strains have been 7 reported by López -Sabater et al. (1994), Rodriguez -Herez et al. (1994) and 8 Hernández -Herrero et al. (1999). Our K. pneumoniae strains produced no 9 detectable amounts of histamine. According to Taylor et al. (1979) Klebsiella 10 pneumoniae is capable of able histamine production, but Kanki et al. (2002) assume 11 that Taylor (1979) has mistakenly attributed histamine production to Klebsiella 12 pneumoniae, misidentifying strains of Raoultella planticola.

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Acknowledgements 7