María Dolores

Pérez Bendito

Soledad Rubio Bravo

María Loreto

Lunar Reyes

Amalia García Prieto

Determination of Bisphenol A in canned fatty foods

Keywords: Chromatography -HPLC, Extraction Additives/Contaminants: Bis-phenol bisphenol A, canned fatty food, microextraction, coacervate, food analysis, liquid chromatography-florescence, reverse micelles

Introduction

Bisphenol A (4-4'-isopropylidenediphenol, CAS: 80-05-7; BPA) is a recognized environmental estrogen [START_REF] Brotons | Xenoestrogens released from lacquer coatings in food cans[END_REF][START_REF] Chen | Acute toxicity, mutagenicity, and estrogenicity of bisphenol A and other bisphenols[END_REF][START_REF] Saal | An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment[END_REF] widely used in the manufacturing of the most important internal polymeric coatings of food cans [START_REF] Goodson | Migration of bisphenol A from can coating-effects of damages, storage conditions and heating[END_REF]. Human exposure to BPA occurs from the migration of this contaminant into food (FSA 2001;[START_REF] Kang | Human exposure to bisphenol A[END_REF]. The tolerable daily intake (TDI) level established by the U.S. Environmental Protection Agency, EPA [START_REF] Iris | Bisphenol A[END_REF], as well as that recently recommended by the European Food Safety Authority, EFSA [START_REF] Iris | Bisphenol A[END_REF][START_REF] Saal | An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment[END_REF], is 50 ng g -1 body weight 3 day -1 . The amount of BPA legally permitted to migrate from packaging into food, known as the specific migration limit (SML), is based on the TDI and it was set at 600 ng g -1 by the EC in 2004 [START_REF] Ec | Commission Directive of 1 March 2004 relating to plastic materials and articles intended to come into contact with foodstuffs. (2004[END_REF].

Occurrence of BPA in canned foods has widely been reported (FSA 2001;[START_REF] Munguía-López | Migration of bisphenol A (BPA) from epoxy can coatings to Jalapeño and an acid food simulant[END_REF]Braunrath et al. 2005;[START_REF] Munguía-López | Migration of bisphenol A (BPA) from can coatings into a fatty-food simulant and tuna fish[END_REF][START_REF] Thomson | Bisphenol A in canned foods in New Zealand: an exposure assessment[END_REF][START_REF] Sun | Single laboratory validation of a method for the determination of bisphenol A, bisphenol A diglycidyl ether and its derivatives in canned foods by reversed-phase liquid chromatography[END_REF][START_REF] Podlipna | Determination of bisphenol A in canned fish by sol-gel immunoaffinity chromatography, HPLC and fluorescence detection[END_REF]. In 1995, Brotons and colleagues detected BPA in the liquid portion of several types of canned vegetables in the range 22-76 ng g -1 [START_REF] Brotons | Xenoestrogens released from lacquer coatings in food cans[END_REF]. Later, Goodson et al. conducted a wide study in the UK that involved the analysis of 62 samples of canned foods and drinks [START_REF] Goodson | Survey of bisphenol A and bisphenol F in canned foods[END_REF]. BPA was quantified in 37 samples of canned food at levels from 7 up to 70 ng g -1 , with one sample of meat containing a mean level of 380 ng g -1 . Recently, a number of studies have confirmed the presence of BPA in canned meat (7.6-140 ng g -1 ), fish (0.3-110 ng g -1 ), beverages (0.1-3.4 ng mL -1 ), vegetables (1-212 ng g -1 ), fruits (1.7-24 ng g -1 ), pet food (12-206 ng g -1 ) and so on [START_REF] Brotons | Xenoestrogens released from lacquer coatings in food cans[END_REF]FSA 2001;[START_REF] Imanaka | Determination of bisphenol A in foods using GC/MS[END_REF][START_REF] Yoshida | Determination of bisphenol A in canned vegetables and fruit by high performance liquid chromatography[END_REF][START_REF] Kang | Determination of bisphenol A in canned pet foods[END_REF]Braunrath et al. 2005;[START_REF] Munguía-López | Migration of bisphenol A (BPA) from can coatings into a fatty-food simulant and tuna fish[END_REF][START_REF] Thomson | Bisphenol A in canned foods in New Zealand: an exposure assessment[END_REF][START_REF] Sun | Single laboratory validation of a method for the determination of bisphenol A, bisphenol A diglycidyl ether and its derivatives in canned foods by reversed-phase liquid chromatography[END_REF]Podlipna andCichna-Markl 2007, García-Prieto et al. 2008). The widespread human exposure to BPA from food and beverage containers has opened a strong debate in the scientific community about the low-dose reproductive and developmental effects of this contaminant, the conclusions going from no risk to human health (FSA 2001) to the need for a new risk assessment [START_REF] Chen | Acute toxicity, mutagenicity, and estrogenicity of bisphenol A and other bisphenols[END_REF][START_REF] Saal | An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment[END_REF]. Determination of BPA in food is required for both the control of the compliance of current legislation and the assessment of human exposure. Most analytical methods proposed in recent years for the determination of BPA in canned foods invariably involve the extraction of samples with organic solvents followed by liquid BPA isolation procedure so far [START_REF] Goodson | Survey of bisphenol A and bisphenol F in canned foods[END_REF]. It involves the blending of twenty grams of fatty-sample with 20 mL of n-heptane for fat removal, duplicate extraction with 20+20 mL of acetonitrile, filtration, addition of anhydrous sodium sulphate and evaporation under nitrogen up to 5 mL. Other authors have proposed some minor modifications to this procedure, namely the treatment of ten grams of fatty sample, which reduces by half the solvent required [START_REF] Podlipna | Determination of bisphenol A in canned fish by sol-gel immunoaffinity chromatography, HPLC and fluorescence detection[END_REF], and the replacement of n-heptane by trimethylpentane, which is more effective for fat removal [START_REF] Thomson | Bisphenol A in canned foods in New Zealand: an exposure assessment[END_REF]. More organic solvent-consuming procedures (e.g 100 mL of methanol and 100 mL of hexane) have been proposed recently (Munguia-López et al. 2005). After isolation of BPA, the extracts are usually subjected to GC-MS, previous derivatisation of BPA, or they are analyzed by LC-FL, after a clean-up step.

For this purpose, high selectivity can be achieved using sol-gel immunoaffinity columns (Braunrath et al. 2005;Braunrath and Cichna 2005;[START_REF] Podlipna | Determination of bisphenol A in canned fish by sol-gel immunoaffinity chromatography, HPLC and fluorescence detection[END_REF].

The strong demand for simple, rapid, low-cost and environmentally friendly methods for food analysis has forced researchers to introduce new approaches and highly innovative technologies in food laboratories. In this context, the use of solvent-free (IUPAC 1972;[START_REF] Gander | Coacervation/phase separation[END_REF]. After separation, the coacervate, a low-volume phase, contains most of the colloid and it is in a dynamic equilibrium with the bulk solution. Coacervates are well-known in food industry where they are widely used for encapsulation of active ingredients [START_REF] Gander | Coacervation/phase separation[END_REF]. In extraction processes, applications have mainly focused on the use of surfactant aggregate-based coacervates for the extraction of pollutants from water [START_REF] Saitoh | Concentration of hydrophobic organic compounds and extraction of protein using alkylammoniosulfate zwitterionic surfactant mediated phase separations (cloud point extractions)[END_REF][START_REF] Jin | Surfactant-mediated extraction technique using alkyltrimethylammonium surfactants. Extraction of selected chlorophenols from river water[END_REF][START_REF] Casero | An acid-induced phase cloud point separation aproach using anionic surfactants for the extraction and preconcentration of organic compounds[END_REF][START_REF] Carabias-Martínez | Surfactant cloud point extraction and preconcentration of organic compounds prior to chromatography adn capillary electrophoresis[END_REF][START_REF] Rubio | Supramolecular assemblies for extracting organic compounds[END_REF][START_REF] Merino | Supramolecular systems-based extraction-separation techniques coupled to mass spectrometry[END_REF][START_REF] Ruíz | Tetrabutyl ammonium-induced coacervation in vesicular solutions of alkylcarboxylic acids for the extractions of organic compounds[END_REF][START_REF] Ballesteros-Gómez | Determination of bisphenols A and F and their diglycidyl ethers in wastewater and river water by coacervative extraction and liquid chromatography-fluorimetry[END_REF]), soil, sediment [START_REF] Merino | Acid-induced cloud point extraction and preconcentration of polycyclic aromatic hydrocarbons from environmental solid samples[END_REF] and sludge [START_REF] Merino | Mixed aggregate-based acid-induced cloud-point extraction and ion-trap liquid chromatography-mass spectrometry for the determination of cationic surfactants in sewage sludge[END_REF][START_REF] Ruíz | Potential of coacervation processes for the extraction of amphiphiles (linear alkyl benzenesulphonates) from sewage sludge samples prior to liquid chromatography[END_REF].

The most used surfactant aggregates have been aqueous non-ionic (Ishii et al. 1977;[START_REF] Hinze | Critical review of surfactant-mediated phase separation (cloud-point extraction): theory and applications[END_REF]Carabias-Martinez et al. 2000), amphoteric [START_REF] Saitoh | Concentration of hydrophobic organic compounds and extraction of protein using alkylammoniosulfate zwitterionic surfactant mediated phase separations (cloud point extractions)[END_REF], anionic [START_REF] Casero | An acid-induced phase cloud point separation aproach using anionic surfactants for the extraction and preconcentration of organic compounds[END_REF]) and cationic [START_REF] Jin | Surfactant-mediated extraction technique using alkyltrimethylammonium surfactants. Extraction of selected chlorophenols from river water[END_REF]) micelles.

Recently, coacervates made up of vesicles [START_REF] Ruíz | Tetrabutyl ammonium-induced coacervation in vesicular solutions of alkylcarboxylic acids for the extractions of organic compounds[END_REF]Ruíz et al. 2007a) and reversed micelles (Ruíz et al. 2007b;[START_REF] Ballesteros-Gómez | Determination of bisphenols A and F and their diglycidyl ethers in wastewater and river water by coacervative extraction and liquid chromatography-fluorimetry[END_REF]) of alkyl carboxylic acids have been reported, which permit the extraction of organic compounds in a wide polarity range. Because of their excellent solvation and stabilizing properties, which can be varied by changing the nature of the macromolecule or the coacervating agent, coacervates have the potential to simplify extraction procedures in food analysis. This paper focuses on the use of decanoic acid reverse micelle-based coacervates for the extraction of BPA from canned fish and meat using minute amounts of food sample and coacervate. The research includes the study of the parameters affecting the extraction efficiency of BPA, the study of the quantitative performance of the method using LC-FL and its application to the determination of BPA in several samples of canned fish and meat purchased in local supermarkets. Bisphenol A (BPA) and decanoic acid (capric) were obtained from Fluka (Buchs, Switzerland). HPLC-grade acetronitrile, hydrochloric acid 37% and tetrahydrofuran were purchased from Panreac (Barcelona, Spain). Stock solutions of BPA (0.5 g L -1 ) were prepared in acetonitrile and stored under dark conditions at 4ºC not more than three months. Ultra-high-quality water was obtained from a Milli-Q water purification system (Millipore, Madrid, Spain).

Materials and methods

Reagents

Apparatus

The liquid chromatrographic system used (Spectra System SCM1000, ThermoQuest, San Jose, CA, USA) consisted of a P4000 quaternary pump, a UV6000LP diode-array detector and a FL3000 fluorescence detector. In all experiments, a Rheodyne 7125 NS injection valve, with a 20 µl sample loop, was used. The stationary-phase column was a Hypersil ODS C 18 column (5µm, 4.6 x 150 mm) from Análisis Vinicos (Tomelloso, Spain). A magnetic stirrer Basicmagmix from Ovan (Barcelona, Spain) and a digitally regulated centrifuge Mixtasel from JP-Selecta (Abrera, Spain) were used for sample preparation. Volumes of coacervate were measured using a digital calliper from Medid Precision, S.A. (Barcelona). Centrifuge tubes with narrow necks (Figure 1 sterilized meatballs and lean pork cooked in its own juices) were bought in supermarkets in Córdoba (Spain). The unopened cans were stored at room temperature. For analysis, the overall content of each can was drained in a mesh strainer (holes size ~0.85 mm 2 ) and the liquid discarded. Then, the overall solid portion was blotted with a filter paper (base weight of 73 g/m 2 , Anoia S.A., Barcelona, Spain) and homogenized using a kitchen food mixer (~9000 rpm, Thermomix TM 21, Vorwerk, Spain). Aliquots of 200-400 mg were taken for analysis and recovery experiments, which were made in triplicate. Spiking of samples for recovery rates was carried out by adding minute volumes of a BPA standard solution in acetonitrile to the solid portion of the food before its homogenization. Samples not immediately analyzed were stored at -20 ºC until analysis. The type of the internal coatings of the cans was not investigated.

Coacervative microextraction of BPA. Decanoic acid (200 mg) was dissolved in THF (2 mL) in a glass centrifuge tube (Figure 1). Then, 8 mL of distilled water and 140 µl of HCl 0.5 M were added. Immediately, the coacervate phase (around 550 µl) separated from the bulk solution. Next, the food (200-400 mg) was added using a small spatula (100 mm long and 4 mm width, supplied by Selecta, Barcelona, Spain). The mixture was magnetically stirred at 1200 rpm for 15 min to favour BPA extraction and then centrifuged at 4000 rpm for 15 minutes to accelerate phase separation. Next, the volume of the coacervate, which was standing at the narrow neck of the glass tube, was measured with a digital calliper. Finally, an aliquot of 20 µl of the coacervate was withdrawn with a microsyringe and injected into the chromatograph. (A:B) in 13 minutes and then isocratic initial conditions (70:30) for 7 min to clean and stabilize the chromatographic system. The selected wavelengths were 276 nm (excitation) and 306 nm (emission). BPA eluted to a retention time of 8.8 min.

Calibration was run by injecting 20 µl of standard solutions in acetonitrile containing between 0.2 and 60 ng of BPA. Quantification was performed by measuring peak areas. Correlation coefficients between peak area and BPA were in the range 0.9995-0.99990.

Results and discussion

Formation and structure of decanoic acid reverse micelle-based coacervates

Decanoic acid is highly soluble in THF where it self-assembles as reverse micelles (size around 4-8 nm), according to a sequential-type self-association model (Ruiz et al. 2007b). The addition of water, in which decanoic acid is scarcely soluble, causes the partial desolvation of the reverse micelles and makes easier micelle-micelle interaction.

As a result, the reverse micelles self-assemble in larger aggregates with a wide size distribution in the nano and micro scale regimes and separate from the bulk solution as an immiscible liquid phase (the coacervate). At a microscopic level, the coacervate consists of spherical droplets made up of reverse micelles which are dispersed in a THF continuous phase containing a minimal amount of water that is essential to keep the coacervate structure. "[Insert Figure 2 about here]"

Figure 2 shows the relative concentration of decanoic acid:THF:water at which the coacervation occurs. Beyond the boundaries of this region, the decanoic acid precipitates or produces a homogeneous micellar solution where the size of the aggregates is considerable smaller than those in the coacervate. Figure 3 They were estimated from the volume of the coacervate (Vc), the recovery for BPA (R) and the instrumental quantification limit (IQL) for this contaminant, which was 0.2 ng (absolute amount injected in the chromatographic system). Conditions giving minimal method quantitation limits (MQL) were selected provided that the extraction for BPA was quantitative and the relative standard deviation of the method was below 10% [START_REF] Green | A practical guide to analytical method validation[END_REF].

Influence of the coacervate amount and composition. Coacervates are mainly made up of decanoic acid and THF, so the amount of these components in the samples determines the coacervate volume and greatly influences the BPA recovery. Figure 4 shows the dependence of Vc on both the amount of decanoic acid (a) and the percentage of THF (b). The volume of coacervate increased linearly as the amount of decanoic acid did independently of the percentage of THF investigated. This type of dependence indicated that the composition of the coacervate kept constant when the other variables remained unchanged. The corresponding equations for 5% and 20% of , where y was the volume of coacervate in µl and z the percentage of THF. The corresponding equations for 200 and 400 mg of decanoic acid were y = (223±11) e (0.045±0.003)z and y = (433±17) e (0.046±0.002)z respectively, with correlation coefficients of 0.997 and 0.998. This type of dependence indicated that progressively more THF was incorporated to the coacervate as the percentage of THF in the solution increased and consequently, the reverse micelles became more and more diluted. As these equations were also similar to those obtained in the absence of food sample, the general equation previously derived for the prediction of the volume of coacervate (y) obtained as a function of the amount of decanoic acid (x) and percentage of THF (z) was also applicable to tuna samples [START_REF] Ballesteros-Gómez | Determination of bisphenols A and F and their diglycidyl ethers in wastewater and river water by coacervative extraction and liquid chromatography-fluorimetry[END_REF]). So, it is possible to estimate the volume of coacervate and therefore the maximum concentration factor that can be obtained under given experimental conditions through the equation y = 1.06 x e 0.04731z .

"[Insert Table I about here]"

Table I shows the volumes of coacervate estimated and the recoveries obtained for BPA as a function of the amount of decanoic acid and the percentage of THF. The I. Quantitative recoveries were obtained for BPA at the selected THF (20%) and decanoic acid ( 200 mg) values and the corresponding MQL (29 ng g -1 ) was far below the specific migration limit (SML) set by the EC (600 ng g -1 , EC 2004).

Under these experimental conditions, it was found that the volume of coacervate measured with a digital calliper (550.0 ± 0.4µl) coincided with that calculated by the general equation (550 µl, Table I) thus confirming the high capability of prediction of the equation used.

Influence of operational parameters. The optimization of such parameters as extraction time and temperature and stirring rate was carried out in order to select those conditions under which the extraction became faster. None of these parameters modified the volume of coacervate yielded, so MQLs directly depended on the recoveries obtained for BPA.

The stirring rate influenced the kinetics of the extraction and consequently, more time was required to reach equilibrium conditions at the lowest stirring rates tested (600-1000 rpm). A value of 1200 rpm was selected as optimal since larger stirring rates did not reduce the extraction time. Equilibrium conditions and quantitative recoveries were obtained after 15 min of extraction and this time was selected as optimal for further studies. The temperature scarcely influenced the extraction kinetics for BPA in the range investigated (20-60 ºC), so the whole procedure was carried out at room temperature. Influence of matrix components. Table II shows the concentrations of the major components in the selected foods. The data were obtained from either the can label or significant databases. Preliminary experiments indicated that independently of the food analyzed, a whitish precipitate, which was standing as a very thin layer between the bottom of the coacervate and the THF:water solution, was extracted. This precipitate decreased as the amount of sample did, so it was matrix-dependent. It was found that the precipitate was caused by the proteins present in the samples, which were agglutinated by the reverse micelles and extracted by the coacervate, but they did not interfere in the recovery of BPA.

"[Insert Table II about here]"

As salt was a component of samples (see Table II Lipids in the samples are expected to be extracted by the coacervate by the formation of mixed micelles with decanoic acid. As incorporated at enough proportion, the lipids should produce an increase in the volume of coacervate obtained. The effect of lipids on the extraction process could not be directly investigated because of both the exact proportion of lipids in the solid portion of the food is unknown (Table II gives the amount of total fat) and it is very difficult to simulate the exact composition of the lipids (i.e. free, triglyceride, phospholipids, etc) and their exact interactions with the rest of matrix components. So, we decided to asses the overall effect of matrix components, which also include lipids, on the extraction of BPA through two experiments. In the first one, different amounts (150, 200 and 400 mg) of an unfortified tuna sample were analyzed by triplicate. The concentrations of BPA found (135±6, 129±6 and 130±5, respectively) were similar in the range investigated, so it was assumed that matrix components did not influence the extraction process. In the second one, different amounts (50-400 mg) of both tuna in olive oil and mackerel in vegetable oil were spiked with 50 ng of BPA and analyzed following the procedure recommended (see the section materials and methods). The results obtained are shown in Table III.

Recoveries were quantitative and independent of the amount of sample thus confirming that matrix components, including lipids, did not affect the extraction process. On the other hand, the 95% confidence interval for the volumes of coacervate obtained in both experiments was 545±19 µL, so the content of the lipids extracted from the minute amounts of sample analyzed was not enough to produce detectable changes in the volume of coacervate. III about here]"

Analytical performance

Calibration curves for BPA were run using standard solutions prepared in acetonitrile.

No differences in peak areas or retention times were observed for BPA injected in acetonitrile or coacervate. The retention time for BPA was 8.8 min. Correlation coefficient between peak areas and the amount of BPA injected every day (0.2, 1, 2, 10, 20, 30 and 60 ng) was in the range 0.9995-0.99990 indicating good fits. The slope of the calibration curve was (4.63± 0.04) * 10 3 fluorescence intensity units ng -1 . The instrumental detection limit (IDL) was calculated from blank determinations by using a signal-to-noise ratio of 3 and it was calculated to be ~ 0.06 ng. From this value and the average percentage of recovery of BPA in food samples (95±8%, data calculated from Table IV), an average volume of coacervate of around 550 µl and an amount of sample handled of 200 mg, the minimum detection limit that could be reached by the method was calculated to be ~ 9 ng g -1 . Lower detection limits can be achieved if necessary by increasing the amount of sample treated (e.g. 400 mg) or decreasing the amount of decanoic acid used for extraction (e.g. see in Table I, data for 20% THF and 100-200 mg of decanoic) since recoveries above 70% are allowed (AOAC/FAO/IAEA/IUPAC 1999, [START_REF] Green | A practical guide to analytical method validation[END_REF]. The relative standard deviation, calculated from the analysis of a tuna sample during five days, two complete analyses per day, was ±6%.

The accuracy of the quantitation of BPA in fatty foods using external calibration was assessed by comparison of the slopes of the calibration curves obtained from standards in acetonitrile with those obtained from 200 mg of lean pork, tuna and mackerel samples fortified with known amounts of BPA (10-300 ng) and run using the whole procedure. Six different concentrations were used for construction of each calibration curve. The slopes and correlations coefficients found for lean pork, tuna and and 92% for lean pork, tuna and mackerel, respectively), so calibration external is recommended for quantitation of BPA in fatty foods.

Analysis of canned fatty foods

The suitability of the proposed analytical method for the determination of BPA in canned fatty food was assessed by analyzing four samples of canned fish (tuna, mackerel, sardine and mussel) and two samples of canned meat (meatball and lean pork). Table IV shows some of the characteristics of the cans analyzed as well as the concentrations of BPA found and the recoveries obtained after spiking the samples with 50 ng of BPA. Values for concentrations and recoveries were expressed as the mean value of three independent determinations, besides their corresponding standard deviations. Recoveries of BPA were always above 90% and the standard deviations were between 2 and 7% thus indicating that the characteristic of the method were matrix-independent. BPA was present at quantifiable levels in all the canned foodstuffs analyzed except in the cooked meatballs. The quantification of BPA in the canned mackerel involved the analysis of 400 mg of sample owing to its low content. BPA in meatballs, if any, was below the detection limit of the method (i.e. 9 ng g -1 ).

Concentrations of BPA were always below the specific migration level of 600 ng g -1 set by the European Commission [START_REF] Ec | Commission Directive of 1 March 2004 relating to plastic materials and articles intended to come into contact with foodstuffs. (2004[END_REF].

"[Insert Table IV andFigure evaporation is necessary. As a result, the whole treatment procedure takes about 30 min and several samples can be simultaneously extracted, so sample throughput is considerable increased. There are additional assets associated with the proposed method; such as requires only minute amounts of sample (200-400 mg), features low cost (the consumption of organic solvent is greatly reduced and the use of SPE columns is avoided), no special equipment is required for sample treatment and it uses liquid chromatography-fluorescence for separation-detection, so the method can be applied in routine analysis in labs without extra investment. The quantitation limit of the method is about 29 ng g -1 , so it can be used for the routine control of BPA in canned fish and meat below the current specific migration limit (SML) of 600 ng g Analysis of * 200 and † 400 mg of fatty food. ‡ Standard deviation, n = 3. ¶ Food samples spiked with 50 ng of BPA. § : below the detection limit of the method.

1

  Determination of Bisphenol A in canned fatty foods by coacervative microextraction, liquid chromatography and fluorimetry

  (LC-FL) or gas chromatography-mass spectrometry (GC-MS). Sample treatment is by far the most laborious and critical step. Concerning the extraction of fatty foods, Goodson et al. have proposed the simplest

  techniques and other isolation emerging technologies is essential to increase sample throughput. The present work proposes the coacervative microextraction of BPA from fatty-foods as an innovative isolation technique to reduce time, cost and toxicity of current extraction methodologies.Coacervates are water immiscible liquids that separate from the bulk of colloidal solutions by the action of a dehydrating agent, namely temperature, pH, electrolyte or a

  ) were designed by authors in order to make easier the measurement and collection of the coacervate after extraction. Pobel S.A. (Madrid, Spain, web page: www.pobel.com) constructed them from commercial heavy-duty glass cylindrical centrifuge tubes with round-bottom (ref. 159050) by keeping their basic structure at the bottom (34 mm of outside diameter) but reducing the diameter from a specified height, which depended on the required tube capacity. "[Insert Figure 1 about here]" Determination of BPA in canned fatty foods Sample pretreatment. Spanish manufactured cans of fish (tuna in olive oil, mackerel in vegetable oil, sardines in olive oil and mussels in pickled sauce) and meat (cooked and

  Quantitation by liquid chromatography-fluorescence. Quantification of BPA and separation from the matrix components was carried out by liquid chromatographyfluorescence. The mobile phase consisted of water (A) and acetonitrile (B). The flowrate was 1 mL min -1 . The gradient elution program was: linear from 70:30 to 45:55

  shows a picture of the surfactant aggregates present in the different steps of the extraction process. The reversed micelles in the coacervate provide a 2-fold mechanism for BPA octanol-water partition coefficient, log K o/w , 2.91 and pK a 9.73), namely van der Waals interactions in the decanoic acid hydrocarbon chains and hydrogen bonds in the micellar core, so it can be extracted efficiently from food samples. Since reverse micelles only occur from protonated decanoic acid (pKa = 4.8±0.2), extractions must be carried out below pH 4. "[Insert Figure3about here]"Optimisation of the coacervative microextraction of BPAOptimisation was carried out by extracting both fortified (n= 3; 50 ng BPA) and non fortified (n=3) tuna in olive oil samples (50-400 mg) under a variety of experimental conditions (100-600 mg decanoic acid; 5-40 % THF; pH 1-4; extraction time 10-60 min; temperature 20-60 ºC, stirring rate 600-1500 rpm). Method limits of quantification (MQL) were used as a criterion for the selection of the optimal conditions for extraction.

  y = (29±28) + (1.27±0.08) x and y = (9±15) + (2.7±0.1) x, respectively, where y was the volume of coacervate in µl and x the amount of decanoic acid in mg. The correlation coefficients were 0.996 and 0.998. The slopes of the linear relationship were similar to that obtained in the absence of food sample (Ballesteros et al. 2007), so although matrix components could be incorporated to the coacervate they did not influence its volume. "[Insert Figure 4 about here]" The relationship between the volume of coacervate and the percentage of THF was exponential (Fig. 4 (b)) and fit to the equation y = b 0 e b 1 z

  became essential to get quantitative recoveries. A THF concentration of ca. 20% was found to be necessary for efficient coacervative extraction of BPA. The recovery decreased at THF percentages above 30%, which can be explained by the increase of BPA solubility in the THF:water solution in equilibrium with the coacervate, that resulting in decreased BPA partition coefficients. From these results, the corresponding MQL values were calculated and they are included included in the Table

  were not affected by the pH of the extraction solution in the range 1 to 4, which is logical considering the type of interactions expected to be the driving forces for the extraction. So, the pH of this solution was adjusted at 3-3.5 by the addition of hydrochloric acid.

  (4.6 ± 0.2) * 10 3 , 0.998; (4.4 ± 0.1) * 10 3 , 0.9990 and (4.26 ± 0.03) * 10 3 , 0.9998; respectively. The differences found between these calibration slopes and that obtained in acetonitrile (4.63 ± 0.04) * 10 3 were exclusively due to the percentages of recovery obtained for BPA (e.g. mean recovery values of around 99, 95

Figure 5

 5 Figure5shows the LC-fluorescence chromatograms obtained for the analysis of unfortified (a1-d1) and fortified with 50 ng de BPA (a2-d2) canned foods.Chromatograms were clear enough for the detection and quantitation of BPA in all the samples analyzed. Identification of the target analyte was based on the retention time and the UV spectrum, which was obtained from the diode array in line with the fluorescence detector. Analysis of the UV spectrum included both peak purity testing and spectrum matching. To calculate the purity a scan threshold of 1 mAU and peak coverage of 95% were considered. With regard to spectrum matching, the similarity threshold was set at 0.98.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Phase diagram of decanoic acid in binary mixtures of THF:water. Final volume of the solution (THF+ Water) = 40 mL. Experiments made at room temperature.

Figure 3 .

 3 Figure 3. Illustration of the different steps and surfactant aggregates involved in the coacervative extraction of BPA.

Figure 4 .

 4 Figure 4. Volume of coacervate as a function of: a) the amount of decanoic acid at two percentages of THF, and b) the concentration of tetrahydrofuran at two amounts of decanoic acid. Extraction of 200 mg of tuna with a water:THF solution (10 mL)

  NaCl g sample, so the volume of coacervate increased at a rate of 6.6 µl per 100 mg salt g -1 sample. Such low rate was undetectable for the low concentrations of salt present in the samples but this increase of volume should be taken into account if samples with a high salt content are analyzed.

	relationship was 0.066±0.003 µl mg -1
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	), its influence on the extraction
	process was investigated by adding NaCl to 200 mg of tuna at the following concentrations: 1, 5, 10, 15 and 25 mg per gram of sample. Neither the recoveries for O n l
	BPA nor the volume of coacervate were affected by NaCl in the range of y
	concentrations usually present in these types of samples. The effect of larger amounts
	of salt: 500, 1170, 1750, 2300 and 2500 mg NaCl g -1 tuna sample was also
	investigated in order to determine if they caused a salting out effect on the extraction of
	13

BPA. Salt did not influence on the efficiency of the extraction process, so its addition to samples is not recommended. It was clearly observed in this study that the volume of coacervate increased linearly with the NaCl concentration. The slope of this linear

  food and those previously obtained by the authors for the extraction of BPA from canned vegetable and fruits[START_REF] García-Prieto | Decanoic acid reverse micelle-based coacervates for the microextraction of bisphenol A from canned vegetables and fruits[END_REF]) are not essentially different, which proves the suitability of decanoic-based coacervates for the efficient and simple extraction of this contaminant from a variety of canned foods. Because of the absence of clean-up steps, chromatographic conditions are simpler for canned fruits than those for canned vegetable and fatty-foods in order to assure the exact quantitation of BPA.

	F o r F o r F o r F o r F o r F o r F o r F o r
	P P P P P P P P
	e e e e e e e e
	e r e r e r e r e r e r e r e r
	R R R R R R R R
	e e e e e e e e
	v i e v i e v i e v i e v i e v i e v i e v i e
	w w w w w w w w
	O n l O n l O n l O n l O n l O n l O n l O n l
	y y y y y y y y
	24

-1 . The experimental conditions established for the coacervative extraction of BPA from fatty-

Table III .

 III Mean recoveries and method quantification limit as a function of the amount of sample analysed TableIV. Characteristics of the cans analysed, mean concentrations found for BPA and recoveries obtained after spiking of the samplesProductLacquer area (cm 2 ) Best before date Net weight (g) Drained weight (g) BPA * ±S ‡ (ng g -1 ) Recovery *, ¶ ± S ‡ (%)

	Food Additives and Contaminants
	Sample Tuna in olive oil Mackerel in vegetable oil Fish Tuna in olive oil Mackerel in vegetable oil Sample amount (mg) 50 150 200 400 50 75 120 200 400 110 120 Sardines in olive oil 120 Mussels in pickled sauce 110 Meat Meatballs 300 F o r P e e r R e v i e w Recovery * ± S † MQL ‡ (%) (ng g -1 ) 97 ±4 113 104 ± 5 35 95± 2 29 98 ± 7 14 104 ± 6 105 111 ± 3 66 108 ± 3 42 98 ± 4 28 97 ± 2 15 12/2011 80 52 12/2012 115 81 12/2011 125 87 12/2010 80 43 12/1012 420 390 Lean pork 243 08/2009 220 210	O n l y 129 ± 6 † 20 ± 5 119 ± 5 121 ± 2 <MDL § 37 ± 5	95 ± 2 † 92 ± 6 90 ± 5 95 ± 6 98 ± 2 99 ± 7

* Spiking level: 50 ng of BPA † Standard deviation, n = 3 ‡ Method quantification limit estimated from an average volume of coacervate of 550 µl Page 32 of 33 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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Acknowledgments

The authors gratefully acknowledge financial support from the Andalusian Government (Junta de Andalucía, Spain, Project FQM 166). A. García-Prieto acknowledges to the Spanish MEC the doctoral fellowship awarded (AP 2003-5344).


Mario ; Schirra
email: mario.schirra@ispa.cnr.it

' Aquino

Quirico Migheli

Filippo M Pirisi

Salvatore D'aquino

& Alberto

Angioni

Influence of postharvest treatments with fludioxonil and soy lecithin co-1 application in controlling blue and grey mold and fludioxonil residues in 2

Keywords:

    Influence of treatment time and soy lecithin co-application on residue levels and efficacy of fludioxonil in controlling Penicillium expansum and Botrytis cinerea of inoculated 'Coscia' pear [Pyrus communis L.] fruit

treatment with water and FLU mixtures was investigated on artificially inoculated pears for 5 the control of postharvest decay caused by blue (Penicillium expansum Link) and grey 6 (Botrytis cinerea Pers. ex Fr.) mold. Treatment with 300 mg l -1 FLU at 20°C increased 7 residues significantly when treatment time rose from 1 to 2 min; no further increase was 8 recorded when dip time raised from 2 to 4 min. FLU residue rates were unaffected by 9 treatment time when 300 mg l -1 a.i. was applied in combination with lecithin at 20°C. While 10 treatment with 100 mg l -1 a.i. at 50°C for 1 and 2 min resulted in similar residue levels, 11 significantly higher residues were detected when dip time increased from 1 to 4 min. Co-12 application of lecithin significantly decreased FLU residues with respect to fruit treated 13 with FLU alone. Treatments with FLU at 20 or 50°C effectively controlled decay over 10 14 days of incubation. While co-application of lecithin did not affect the efficacy of FLU at 15 300 mg l -1 and 20°C, treatment efficacy decreased when lecithin was applied in combination [START_REF] Errampalli | Control of blue 17 mold (Penicillium expansum) by fludioxonil in apples (cv Empire) under controlled 18 atmosphere and cold storage conditions[END_REF] with 100 mg l -1 FLU and 50°C for 4 min and to a greater extent when dip time was 1-2 min. 17 18 19 Keywords: Pyrus communis L, disease control, hot water dip, fludioxonil, residues, 20 lecithin. 21 22 Running title: Decay control on pears by fludioxonil and lecithin 23 The post-harvest life of summer pear cultivars is generally shorter than that of autumn and 3 winter pears [START_REF] Bell | Pears. In: Fruit Breeding Vol 1 2 Tree and Tropical Fruits[END_REF]. Under shelf-life conditions summer pears soften rapidly, develop 4 storage disorders that spoil their appearance and texture and become susceptible to decay 5 caused by various pathogens, especially blue (Penicillium expansum) and grey (Botrytis 6 cinerea) mold. Pre-and/or postharvest treatments and cold storage are therefore needed to 7 delay senescence and to control postharvest decay. 8

Certain edible coatings have been reported as effective in preventing deteriorative 9 changes and prolonging postharvest life when applied to fresh fruits and vegetables. 10 Lysophosphatidylethanolamine (LPE) phospholipid, for example, which is present in egg 11 yolk and soy lecithin, has beneficial effects on plants or parts thereof by protecting against 12 stress-related injuries, improving keeping quality during storage and shelf-life and delaying 13 senescence [START_REF] Farag | 24 management for stone fruit crops in California using the "reduced-risk" fungicides 2 fludioxonil and fenhexamid[END_REF][START_REF] Ozgen | Use of lysophosphatidylethanolamine (LPE), a natural lipid, to 15 accelerate ripening and enhance shelf life of cranberry fruit[END_REF]. Beneficial effects such as 14 reducing internal breakdown of 'Granny Smith' apples [START_REF] Watkins | Effects of lecithin, calcium, and antioxidant 3 formulations on superficial scald and internal breakdown of 'Granny Smith' apples[END_REF]), delaying or 15 preventing the formation of blemishes or micro-lesions brought about by physical and/or 16 chemical treatments [START_REF] Sardo | Method for processing fruits and vegetables on the base of lecithin[END_REF]), improving turgidity, colour and flavour of fruit and 17 vegetables and reducing fruit cracking [START_REF] Rowley | 4 Methods for treating plants and part plants[END_REF]) have been observed following 18 treatment with lecithins and/or their derivatives. However, as lecithin has no antifungal 19 activity, co-application of a fungicide is needed to control postharvest decay. 20

In the pear packing-house, immersion dumping in a water mixture containing a flotation 21 agent and disinfectants is common practice for removing fruit gently from field bins (Sugar 22 and Basile 2005), whilst current control of storage decay is based on postharvest drench or 23 line spray application of thiabendazole (TBZ) [START_REF] Kupferman | Practices to reduce postharvest pear diseases[END_REF] water, and lower rates of active ingredient were needed with respect to standard treatment 14 performed at ambient temperature, to achieve a comparable control of decay. Results 15 reported herein provide information on the influence of treatment time and soy lecithin co-16 application on residue levels and the effectiveness of fludioxonil in Coscia pears following 17 dip treatment in an aqueous mixture containing soy lecithin and FLU at ambient 18 temperature or reduced rates of FLU at 50°C. The effectiveness of heat treatments with 19 water, and fludioxonil applied separately or in combination with soy lecithin, was 20 investigated on artificially inoculated fruits for the control of postharvest blue and gray 21 mold. 22

Material and Methods 23

Fruit samples 24 Commercially mature 'Coscia' pear (Pyrus communis L.) fruit were hand-harvested from 1 trees which had received standard horticultural practices, from an orchard located in the 2 north-west Sardinia (Italy). Fruits were placed in plastic trays and delivered to the 3 laboratory immediately after harvest. Then, fruit were selected, returned to each box and 4 left overnight at 20°C. 5 6 Chemicals 7

The fungicide used was a commercial formulation of FLU (SAVIOR FLOWABLE 20, 8 Syngenta, USA) containing 20% active ingredient (a.i.). A commercial formulation 9 (Xedabio  ) containing soy lecithin (lecithin) was obtained from CEDAX s.r.l., Italy. 10 11

Fludioxonil residues in fruit 12

To determine the influence of treatment time and lecithin co-application on FLU residue 13 levels in fruit, pear samples were dipped for 1, 2 or 4 min in water mixtures containing 14 FLU at 300 mg l -1 and 20°C with or without 2% lecithin or FLU at 100 mg l -1 and 50°C 15 with or without 2% lecithin. Each treatment was replicated four times. After treatments, 16 fruits were allowed to dry and stored for 14 days at 17°C and 80% relative humidity 17 (simulated shelf-life conditions). Analyses were performed following treatment and after A total of twelve of the halves, each from different fruit from those in a replicate, were 5 stored in a freezer at -20°C until analysis. Then, frozen samples were chopped and 6 homogenized using a semi-industrial blender (Malavasi, Bo, Italy) and 5 g of the material 7 was placed in a 40-ml capacity flask with 10 ml of acetone/hexane (1/1) and 6 g of NaCl. 8

The flasks were shaken in a rotary shaker for 20 min. Subsequently, the phases were 9 allowed to separate and the organic layer was injected into a gas chromatograph for the 10 analysis, without any clean-up step. Residue analysis of FLU was performed by a TQ Trace 11 Gas Chromatographer, coupled with a NPD 80 detector, a split/splitless injector, and a 12 AS200 auto sampler (Termo Quest, Milan, Italy) as described in a previous paper (Schirra 13 et al. 2005). [START_REF] Ozgen | Use of lysophosphatidylethanolamine (LPE), a natural lipid, to 15 accelerate ripening and enhance shelf life of cranberry fruit[END_REF] 15

Recovery assays 16

Samples of untreated pears were fortified with appropriate volumes of stock standard 17 solutions to reach concentrations of 0.1, 1.5, and 2.0 mg kg -1 . The samples were allowed to 18 settle for 30 min prior to extraction and then processed according to the above procedure. [START_REF] Errampalli | Evaluation of fludioxonil for the control of 20 post-harvest gray mould and blue mold in pears in cold and controlled atmosphere 21 storages[END_REF] Average recovery from four replicates showed values of FLU ranging from 97 to 108% 20 with a maximum coefficient of variation of 11%. All analyses were performed on 12 fruits 21 per replicate. Residues were reported as mg kg -1 of the fresh weight of the fruit. 22

Fruit inoculation and treatments 23

Pears were disinfected by immersion for 1 min in diluted household bleach (1% sodium 24 hypochlorite), rinsed with tap water by 30 s dips followed by 30 s shower and allowed to 25 dry at room temperature. Fruit samples were subdivided into two groups and each fruit was 1 punctured twice equatorially at the opposite sites, and inoculated with a mixture of 1•10 4 2 conidia ml -1 P. expansum or 1•10 5 conidia ml -1 B. cinerea isolates, respectively. After 3 inoculation, fruit were incubated for 20 h at 20°C before treatment. The treatments were 4 carried out approximately 20 h after fruit inoculation. Pears were subjected to dip 5 treatments for 1, 2 or 4 min with: (1) water at 20°C (control fruit); [START_REF] Watkins | Effects of lecithin, calcium, and antioxidant 3 formulations on superficial scald and internal breakdown of 'Granny Smith' apples[END_REF] water plus lecithin at 6 20°C; (3); FLU at 300 mg l -1 and 20°C; (4); FLU at 300 mg -1 and 20°C plus lecithin; (5) 7 water at 50°C; (6) water plus lecithin at 50°C; (7) FLU at 100 mg l -1 and 50°C; (8) FLU at 8 100 mg l -1 and 50°C plus lecithin. Lecithin was always applied at 2% (wt vol -1 ).. Each 9 treatment was carried out in triplicate (three replicates of 30 fruit each) and the experiment 10 was repeated two times. Fruits were evaluated for incidence of decay caused by blue or 11 gray mold (percentage of fruit with any size of decay lesions of the total number of fruit) 12 after 7 and 10 days of incubation at 20°C and 95% relative humidity. 13

Statistical analysis 14

Statistical analysis was performed by Statgraphics software (Manugistics, version 5 15 Professional, 2000) statistical program. Analysis of variance (ANOVA) was carried out 16 using a unifactorial complete randomized block design. Percentages were not transformed 17 or transformed in arcsin√x or √x before the ANOVA, depending on the range of variation 18 of data [START_REF] Gomez | Statistical procedures for agricultural research[END_REF]. Mean comparisons of the effects of treatments were 19 calculated by the Fisher's least significant difference test at P ≤ 0.05. 20 21

Results and discussion 22

FLU residue level in pear fruit treated for 1 min with 300 mg l -1 at 20°C was 1.57 mg l - 23 1 . When treatment time was increased from 1 to 2 min, FLU residues significantly increased 24 (ca 71%); no significant changes were recorded when dip time went from 2 to 4 min. 25 Treatment with 100 mg l -1 a.i. at 50°C did not significantly change residue rates when dip 1 time was increased from 1 to 2 min but showed significantly higher values (> 98%) when 2 dip time increased from 1 to 4 min. While co-application of 300 mg l -1 a.i. and lecithin at 3 20°C did not affect fruit FLU residue rates as a function of treatment time, treatment with 4 100 mg l -1 FLU and lecithin at 50°C increased residue levels when dip time rose from 1 to 2 5 min; no further increase was found when dip time went from 2 to 4 min. 6

In the present study the highest levels of FLU residues (3.52 mg kg -1 ) were found in 7 fruit treated with 100 mg l -1 FLU at 50°C and were below the maximum residue limit set in 8 the USA (5.0 mg kg -1 ) for apples and pears. 9

Trials on oranges [START_REF] Schirra | Residue level, persistence and storage performance of citrus fruit treated with 9 fludioxonil[END_REF] have shown that 3-min dip treatment with 150 10 mg l -1 FLU at 50°C produced a residue level approximately 2.5-fold higher than treatment 11 at 20°C. In apricots, FLU residue levels at 48°C were ca. 0.5-2-fold higher than that of 12 treatments at 20°C, whereas in peaches and in nectarines, FLU residues after dipping at 13 48°C were 2.6-6.4-fold higher than those left at 20°C. The amount of fungicide deposition 14 as a function of treatment time and temperature is thus dependent on fruit species. [START_REF] Smilanick | The effectiveness of 16 pyrimethanil to inhibit germination of Penicillium digitatum and to control citrus green 17 mold after harvest[END_REF] Various factors are known to affect fungicide uptake in fruit, including the method of 16 application such as aqueous or wax-based mixture (see reviews by [START_REF] Dezman | Postharvest fungal decay control chemicals: 12 Treatments and residues in citrus fruits[END_REF]17 Papadopoulou-Mourkidou 1991). Our study shows that when the fungicide was applied in 18 combination with lecithin, residue levels in fruit were lower than residues left by FLU 19 treatment applied alone, with decreases averaging from 18 to 52% at 20°C and from 51 to 20 67% at 50°C. 21

All inoculated wounds of control fruit were infected by P. expansum after 7 days of 22 incubation (Table 2). B. cinerea infections were much less aggressive than P. expansum, 23 accounting for an incidence of grey mold in untreated fruit of 43.3%. Water dip at 50°C for 24 1-2 min resulted in 27-31% less blue mold decay, while the 4-min dip yielded 90% less 25 decay than in control fruit. While the incidence of lesions caused by grey mold was 1 unaffected by water dip at 50°C for 1 min, it was significantly reduced when dip time was 2 2 and 4 min. After 10 days of incubation the benefit of hot water dip for 1-2 min in 3 controlling blue mold was lost and notably reduced when dip time was 4 min. While water 4 dip for 1 min had a slight but significant effect against grey mold, it was more effective 5 when fruits were submerged for 2 and 4 min. Best results in controlling decay were 6 achieved with 300 mg l -1 FLU at 20°C, the effect being suppressive and long-lasting. 7

Treatment with 100 mg l -1 FLU at 50°C was equally effective in controlling blue and grey 8 mold decay, indicating the positive synergistic effects of heat treatment and FLU. These 9 results corroborate previous findings on various fruit species [START_REF] Schirra | Residue level, persistence and storage performance of citrus fruit treated with 9 fludioxonil[END_REF]; D' Aquino 10 et al. 2007;[START_REF] Palou | Combination of postharvest antifungal chemical 18 treatments and controlled atmosphere storage to control gray mold and improve 19 storability of 'Wonderful' pomegranates[END_REF]). 11

The sorption ability of cuticular wax is generally low for most agricultural chemicals 12 being affected by wax composition (Jenks and Ashworth 1999), permeability of cuticles 13 and active ingredient diffusion through the plant cuticle (Riederer and Schreiber 1995) and 14 increases with increasing temperature [START_REF] Schönherr | Effects of temperature, surfactants and other adjuvants on rates 13 of uptake of organic compounds. In: Plant cuticles -An integrated functional approach[END_REF]. For this reason, when 15 treatments are performed with heated fungicides lower concentrations are required to 16 supply equal residue levels and effectiveness with respect to treatments performed at 17 ambient temperature [START_REF] Schirra | Host pathogen interaction 11 modulated by heat treatment[END_REF]. Although reduced treatment time would be 18 desirable so as to increase packinghouse output and shorten delays in fruit marketing, 19 treatment duration should be long enough to produce the heat-induced beneficial effects in 20 terms of physical changes of epicuticular wax, host defensive responses and inhibition of 21 pathogen development [START_REF] Schirra | Host pathogen interaction 11 modulated by heat treatment[END_REF]. [START_REF] Farag | 24 management for stone fruit crops in California using the "reduced-risk" fungicides 2 fludioxonil and fenhexamid[END_REF] It is recognized that the co-aplication of coatings such as waxes reduces the 23 effectiveness of certain fungicides [START_REF] Dezman | Postharvest fungal decay control chemicals: 12 Treatments and residues in citrus fruits[END_REF][START_REF] Eckert | Postharvest Disorders and Diseases of Citrus Fruits[END_REF]. When 24 imazalil (IMZ) is applied in water, for example, it controls green mold significantly better 25 than when it is applied in wax mixture [START_REF] Brown | Residues from postharvest nonrecovery spray 5 application of imazalil to oranges and effects on green mold caused by Penicillium 6 digitatum[END_REF]. This because part of the a.i. is 1 not available, being immobilised in the wax, and because the wax, being more viscous than 2 water, penetrates less effectively into the wound-infection that are exploited by P. digitatum 3 on the rind of citrus fruit. Accordingly, pyrimethanil was found to be more effective in 4 aqueous suspensions than when suspended in fruit waxes [START_REF] Smilanick | The effectiveness of 16 pyrimethanil to inhibit germination of Penicillium digitatum and to control citrus green 17 mold after harvest[END_REF]. Similar 5 results are reported by [START_REF] Kanetis | Comparative efficacy of the new postharvest 10 fungicides azoxystrobin, fludioxonil and pyrimethanil for managing citrus green mold[END_REF], who applied the new fungicides, azoxystrobin, 6 fludioxonil and pyrimethanil as well as imazalil and thiabendazole. Thus, to achieve a 7 comparable effect to that in water mixture, the fungicide concentration should be increased 8 [START_REF] Eckert | Postharvest Disorders and Diseases of Citrus Fruits[END_REF]. Our results show that the efficacy of FLU was not compromised 9 with co-aplication of lecithin at 300 mg l -1 and 20°C but significantly decreased with 100 10 mg l -1 FLU at 50°C for 4 min and to a greater extent for 1-2 min, respectively, due to the 11 reduced levels of residues left by these treatments. Thus, to compensate for the reduced 12 residues caused by lecithin co-treatment, higher FLU rates should be applied to achieve 13 similar control of grey and blue mold decay. Each value is the mean of two experiments (three replicates of 30 fruit each). In each column grouping means, means followed by a common letter are not significant different by Fisher's least significant difference (LSD) procedure, P ≤ 0.05.

  50°C, respectively, with or without 2% soy lecithin. The efficacy of heat 4

  refers to active ingredient. y

Table 1 .

 1 Residues of Fludioxonil in 'Coscia' pears following 1, 2 or 4 min dip treatments with FLU-based commercially available fungicide mixture, without or with 2 % lecithin and after 14 days at 17°C.

	14
	15

x The fungicide concentration refers to active ingredient.

y

In each column grouping means, means followed by a common letter are not significant different by Fisher's least significant difference (LSD) procedure, P ≤ 0.05. Page 15 of 16 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 2 .

 2 Influence of postharvest dip treatments on the incidence of blue mold and gray mold caused by Penicillium expansum and Botrytis cinerea of inoculated 'Coscia' pears after selected incubation period.

	Treatments x	Dip time	P. expansum (%)	B. cinerea (%)
		(min)		Incubation period (days) y
			7	10	7	10
	Water, 20°C	1	100 a	100 a	43.3 a	100 a
		2	100 a	100 a	37.2 b	100 a
		4	100 a	100 a	37.2 b	100 a
	300 mg l -1 FLU, 20°C

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Dundee, Scotland, Vol. 6, p. 131-156.

Acknowledgements 16

The authors gratefully acknowledge the Syngenta Crop Protection, Italy and the CEDAX


Keywords: Inter-laboratory validation, Microbiology, Screening -microbial screening Additives/Contaminants: Veterinary drug residues -antibiotics

Collaborative study of a microbiological screening method (three-plates) for the banned antimicrobial growth promotors tylosin, virginiamycin, spiramycin, zinc bacitracin and avoparcin in animal feed

Irene Pol-Hofstad

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Abstract

A microbiological screening method (three-plate) for detection of the antimicrobial growth promoters tylosin, spiramycin, virginiamycin, zinc bacitracin and avoparcin in animal feed has been developed and validated successfully. A collaborative study involving 18 laboratories receiving 172 samples was carried out to verify the performance characteristics. The detection level for tylosin/virginiamycin/spiramycin, expressed in microbiological activity, was 1 mg kg -1 (false positives 2%, false negatives 3, 0, and 6% respectively). Avoparcin could be detected at 1 mg/kg in feed in general (false positives 2%, false negatives 0%). However, in calf feed, the sensitivity was lower. The percentage of false negatives was found to be 12%, 7%, 0% at 1, 3 and 5 mg kg -1 respectively (false positives 4%). The limit of detection for zinc bacitracin was 3 -5 mg kg -1 (false positives 5-10%, false negatives 77% at 1 mg kg -1 , 45% at 2 mg kg -1 , 12% at 3 mg kg -1 and 4% at 5 mg kg -1 . The method allowed for distinction between the groups of antibiotics: avoparcin/zinc bacitracin vs.

tylosin/virginiamycin/spiramycin. This definitely gives added value to the method in the framework of a follow-up of positive screening results by post-screening and confirmatory analysis. selection and amplification of vancomycin-resistant pathogens [START_REF] Acar | Avoparcin and virginiamycin as animal growth promotors: a plea for science in decision making[END_REF].

Because of possible transfer of resistance genes from animal enteric flora to human bacteria and pathogens and increased appearance of cross resistance, the medical effectiveness of antibiotics reserved for human treatment could be impaired. In accordance with consumers demand for more safe and healthy foods, the European Union has therefore withdrawn the authorisation of several antimicrobial growth promotors as feed additives. Avoparcin, zinc-bacitracin, spiramycin, tylosin and virginiamycin, were banned from use as animal feed additives in 1997 and 1998 (European Community, 1997[START_REF]Council Regulation (EC) No 2821/98 of 17 December 1998 amending, as regards withdrawal of the authorisation of certain antibiotics, Directive 70/524/EEC concerning additives in feedingstuffs[END_REF].

In order to effectively and efficiently control the possible illegal use of these substances multi-screening and confirmation methods with sufficiently low detection limits are required. The available methods of analysis (adopted in the EC) at that time were based on microbiological inhibition tests (European Community, 1972[START_REF]Ninth Commission Directive of 31 July 1981 establishing community methods of analysis for the official control of feedingstuffs (81/715/EEC)[END_REF], 1984a, 1984b). These specific methods mainly focussed on checking the content of one single compound at the registered additive level, and were therefore not suited as broad screening method for all substances [START_REF] Higgins | Evaluation of a single plate microbiology growth inhibition assay as a screening test for the presence of antimicrobial agents in compound animal feedingstuffs at therapeutic and contaminating concentrations[END_REF]). Gafner described a multianalyte method using TLC and bio-autography [START_REF] Gafner | Identification and semiquantitative estimation of antibiotics added to complete feeds, premixes and concentrates[END_REF] The training period.

The training period was designed as a specific exercise for the candidate laboratories in order to make them (more) familiar with the method protocol. In addition, their performance and their rigorous application of the protocol were evaluated. First, an audiovisual support (CD-ROM) containing the method description was sent to the participants, along with a specific training reporting sheet, a feedback questionnaire and some test samples and standards.

The first training period contained known and unknown samples of all target analytes. A second training period proved to be necessary for avoparcin and zinc bacitracin to be able to have sufficient experienced laboratories for the actual validation phase of the collaborative study.

Collaborative study.

In total eighteen laboratories participated in the two training periods. Laboratories that successfully implemented the method were invited to join the collaborative study. All eighteen labs were selected for the analysis of tylosin/spiramycin/virginiamycin and avoparcin in the collaborative study, two labs were disqualified for the analysis of zinc bacitracin. The participating laboratories received a detailed method protocol, samples to be analysed and electronic forms to report the results of the analysis. In total each participant received 172 samples to be analysed, including samples for an identification study and corresponding blanks. All participants were asked to evaluate the feed samples for the presence or absence of the banned antibiotics. This method is a qualitative method in nature, therefore only three responses were allowed: 1 positive (substance present), 2 negative (not present), 3 no results. Laboratories reported no results when they were not able to report (in time).

Additional collaborative study.

After analysing the results of the first collaborative study round, an additional collaborative study proved to be necessary for avoparcin, containing eight feed materials. Each participant received 20 extra samples including four blind replicates of each spiked feed material and one sample of each blank feed.

Statistics

All results for this method were of qualitative kind, therefore the usual procedures for quantitative results were not applicable. In order to be able to assess the method performance, the number of correct positives, correct negatives, false positives and false negatives were calculated in relation to the compound, its concentration and the different feed matrices [START_REF] Vincent | Validation of an analytical method for the determination of spiramycin, virginiamycin and tylosin in feeding-stuffs by thin-layer chromotography and bio-autography[END_REF].

To be able to describe the capability of the method, the accuracy, the sensitivity and the specificity were calculated according to following equations [START_REF] Vincent | Validation of an analytical method for the determination of spiramycin, virginiamycin and tylosin in feeding-stuffs by thin-layer chromotography and bio-autography[END_REF][START_REF] Langton | Analysing collaborative trails for qualitative microbiological methods: accordance and concordance[END_REF]: The sensitivity (SE) is the ability of the method to classify a positive sample as positive, whereas the specificity (SP) is the ability to classify a negative sample as negative.

False negative(s) are defined as samples that are truly non-compliant, even though a compliant measurement has been obtained. False positives(s) are defined as samples that are truly compliant, even though a non-compliant measurement has been obtained.

A 100% score for accuracy means that all positive samples were correctly classified as positive and all negative samples were correctly classified as negative. For all scores below 100%, indicating that not all samples were classified correctly, it was investigated whether this was caused by false negatives or false positive samples. The limit of detection for the different antibiotics was set at the lowest concentration at which 95% of the samples were still identified as positive. This means that the sensitivity (SE) has to be equal to or above 95% known amount of stock solution to each 10 g feed portion to obtain 1, 2, 3 and 5 mg kg - 1 (end concentration in feed). After addition of the antibiotic solution the feed was mixed and stored at -20°C.

Stability tests

Prior to the preparation of the collaborative study samples, a stability study was performed. In this study 3 mg kg -1 spikes of avoparcin, tylosin and spiramycin en 4 mg kg -1 spikes of virginiamycin and zinc bacitracin were prepared in 10 gram portions. The samples were stored at -20, 4, 20 and 30°C for one month. Sample transport was mimicked by storing part of the samples at 4, 20 or 30°C for two days, followed by storage at -20°C. The samples were considered as stable when the concentration in the stored samples was higher or equal to 95% compared to the concentration in freshly prepared samples (tested using the official EU microbiological quantification methods. including freshly prepared spikes and calibration curves (European Community, 1970[START_REF]Third Commission Directive of 27 April 1972 establishing community methods of analysis for the official control of feedingstuffs (72/199/EEC)[END_REF][START_REF]Ninth Commission Directive of 31 July 1981 establishing community methods of analysis for the official control of feedingstuffs (81/715/EEC)[END_REF], 1984a,b)). Sufficient stability was obtained for tylosin, avoparcin and virginiamycin in all tested conditions. Zinc bacitracin should be stored at 4°C and spiramycin at -20°C. As a result the participants were advised to keep the antibiotics frozen under all conditions. Combined with a limited (three-four weeks) time to perform the analysis, stability was guaranteed.

Method protocol

The overall purpose of the developed method was to screen animal feed for the presence of the five banned antibiotics. The detail protocol is described on the SIMBAG FEED website (van Egmond, 2005a) and will be published (in preparation). In short, the feed samples (10 g) are extracted with a mixture (475/25/500) of acetone, hydrochloric-acid (HCl) and water (20 ml) for 15 minutes followed by further extraction for another 15 minutes by addition of 10 ml phosphate buffer pH 6.5. The supernatant is adjusted to pH 6.0-6.5 and dispensed into wells in three different agar media. The agars differ in inoculated bacterial strains and pH (see table II for details). After incubation, presence of antibiotics is shown by the formation of zones of inhibition. An indication of the group of antibiotics present is possible by observing with which microorganisms zones of inhibition are produced.

Single laboratory validation of the method

Single laboratory validation of the method included sensitivity (SE), specificity (SP), interference and ruggedness. The detailed results of this in-house validation are described on the SIMBAG FEED website (de Jong et al, 2005) and will be published (in preparation). The method showed satisfactory performance for tylosin, avoparcin, spiramycin at 1 mg/kg (SE>95%) and for virginiamycin and zinc bacitracin at 2 mg/kg (SE >95%). Most antibiotics used as veterinary medicine (e.g. tetracyclines, quinolones, other macrolides, ß-lactams, aminoglycosides) showed interference, which can be recognized by the inclusion of three additional test plates. Avilamycin, flavomycin, ionophores, benzimidazolen, ivermectine, ronidazole, sulphonamides, maduramycin, colistin and, spectinomycin showed no interference.

Results and discussion

Training phase

The overall purpose of the study was to assess the fitness-for-purpose of the developed microbiological screening method for the screening of avoparcin, zinc bacitracin, spiramycin, tylosin and virginiamycin in feedingstuffs at the target level of 1 mg kg -1 . A training period was organised to get the participating laboratories acquainted with the developed method. In total 20 laboratories volunteered to participate in the training period of which 18 laboratories reported results (see table III). At the end of the training period all eighteen labs were selected for the analysis of tylosin/spiramycin/virginiamycin and avoparcin. Two labs were disqualified for the analysis of zinc bacitracin, due to lack of sensitivity of the bio-assay. Some of the remaining laboratories did not implement the method fully satisfactory or did not reported results of the training period (intermediate classification). The "intermediate" laboratories were invited for participation in the collaborative study with the restriction that results should be in accordance with those of the other laboratories, else their results would not be used in the calculations.

Collaborative study

In total 17 out of 18 laboratories reported results of the collaborative study (see table III). Two of these 17 laboratories (lab's 2 and 16), did not report results for tylosin/spiramycin/virginiamycin due to growth problems of the M.luteus ATCC 9341. , 7, 8) were not able to show improvements in their results and were therefore excluded from the study. These three laboratories had problems with positive blanks and unstable bio-assay systems.

For avoparcin, two laboratories (lab's 1 and 17) incubated part of the plates at wrong temperature and these data were excluded from the results. Four laboratories showed intermediate results for avoparcin after the training period. One of these four (lab. 2)

was not able to improve their results and was excluded from the collaborative study.

This laboratory had problems with positive blanks and an unstable bio-assay system.

In more detail: for the tylosin/ spiramycin/ virginiamycin group, 288 samples for each antibiotic were taken into account in the statistical evaluation originating from 12 laboratories. For zinc bacitracin, 288 samples were evaluated from 12 laboratories and for avoparcin 537 results were evaluated from 15 laboratories, including the samples from the extra collaborative study.

All data obtained from laboratories which successfully applied the method in the training period were used, except the avoparcin data originating from the incorrect incubation temperature. It is noticeable that one laboratory (lab. 2) was not able to produce acceptable results for all three groups of antibiotics For zinc bacitracin the number of false positives was rather high (11%). Most false positives originated from one laboratory (lab. 1). Excluding those results reduced the number of false positives to 5%. The target level of 1 mg kg -1 could not be reached in any type of feed. The limit of detection lies between 3 and 5 mg kg -1 , as the required 95% positive samples is only achieved at 5 mg kg -1 level. After analyzing the results of the collaborative study an additional collaborative study for avoparcin proved to be necessary. This was due to the fact that it was not possible to calculate the limit of detection of the method for the different types of feed. Most laboratories (79%) (unexpectedly) found a false negative result for 1 mg kg -1 in calf feed, whereas in cattle feed this concentration was positive for all participants. This result for calf feed (milk replacer) was not in accordance with the results obtained in the single laboratory validation and transferability studies where 1 mg/kg was good detectable.

It was decided to organise an additional collaborative study with three different levels of avoparcin in calf feed (1, 3, 5 mg kg -1 ) and a pig feed spiked with 1 mg kg -1 avoparcin.

With the results of this additional study the limit of detection in calf feed could be determined and it was proved that 1 mg kg -1 avoparcin was detectable in other feeds (SE = 96%). The number of false positives was only 2%. For calf feed, (milk replacer), the number of false positives was 4%.

The limit of detection for calf feed (milk replacer) is somewhat higher, viz. between 3 and 5 mg kg -1 . The number of false negatives (in the second study) is 12% (1 mg kg -1 ), 7% (3 mg kg -1 ) and 0% (5 mg kg -1 ). This is at the 1 mg kg -1 avoparcin level significantly lower than the observed 79% in the first study Most possibly the storage conditions of the calf feed has influenced the results negatively. The calf feed from the first collaborative study was stored in 10 gram portions at -18°C for almost a year, while the calf feed for the second collaborative study was stored in bulk quantities at -18°C and distributed shortly before use in 10 gram portions. The cause of this interference is not yet known but might be due to some physical or chemical changes in the feed during storage in contact with air. In practice these storage conditions are not regular (feed is normally stored for a maximum of three months) and therefore it was decided to only use the results of the second collaborative study for calf feed.

Discrimination study.

A study was set up to assess the discrimination properties of the method by the different laboratories. Therefore 20 unknown samples were sent to the participants to be analysed for antibiotics. In total 14 laboratories participated in this study of which 2 laboratories had difficulties in differentiating between tylosin/virginiamycin/spiramycin and zinc bacitracin/avoparcin. Most laboratories were able to detect the presence of tylosin/ virginiamycin/spiramycin (inhibition of M.luteus ATCC 9341) (see table VIII).

Distinction between zinc bacitracin and avoparcin turned out to be difficult. Samples containing 2-3 mg kg -1 zinc bacitracin were often classified as blanks. This is in accordance with the fact that the sensitivity of the method for zinc bacitracin lies around 3-5 mg kg -1 . All together, a first distinction between groups of blanks, tylosin/virginiamycin/spiramycin and zinc bacitracin/avoparcin proved to be possible.

Conclusions

In summary, this method showed good performance in detecting tylosin/virginiamycin/spiramycin at the target concentration of 1 mg kg -1 . Results were similar or even better in the case of spiramycin compared to the TLC chromatography and bio-autography described by [START_REF] Vincent | Validation of an analytical method for the determination of spiramycin, virginiamycin and tylosin in feeding-stuffs by thin-layer chromotography and bio-autography[END_REF]. For avoparcin, the level of sensitivity depended on the type of feed. In general, the target concentration of 1 mg kg -1 was reached for feed. When looking at milk replacers in particular, the level of detection lies between 3 and 5 mg kg -1 . Detection of zinc bacitracin proved to be more difficult. The target level of sensitivity could not be reached, but lies around 3-5 mg kg -1 .

Overall, the performance characteristics of this method prove that the method is

Key words :

 words Microbiological screening method, validation, animal feed, antibiotics, collaborative study, spiramycin, virginiamycin, tylosin, zinc bacitracin, avoparcin have been related to increased development of antibiotic resistance in bacterial strains. For instance use of avoparcin, which is structurally related to the human medicines vancomycine and teicoplanin, leads to

  agreements (correctly identified positives) NA = negative agreements (correctly identified negatives) ND = negative deviations (false negatives) PD = positive deviations (false positives) Sensitivity SE = (PA/ (PA+ND))*100% Specificity SP = (NA/(PD+NA))*100%

  the method was confirmed by two other laboratories, being able to produce comparable sensitivity results. The detailed results of transferability tests will be published (in preparation).

Table III :

 III Classification of the laboratories in the training period (T) and Collaborative study (C) in satisfactory performance (+),intermediate performance (±) or unsatisfactory performance (-)

											Formatted: Font: 8 pt
											Formatted
	Labo-	Tylosin/virginamycin/spiramycin			Avoparcin			Zinc bacitracin	Results
	ratory	T C Specification	T C Specification	T C	Specification	excluded
	1	+	+		+	+	C2: Incorrect inc. temperature	+	+		Avo (C2)
	2	+	-	C: No results, growth problems	±	±	T&C: blanks positive	±	±	T&C: blanks positive	Tyl/Vir/Spir/Bac/Avo
	3	+	+		+	+		+	+		-
	4	+	+		±	+	T: blanks positive	+	+		-
	5	+	+		+	+		±	+	T: blanks positive	-
	6	±	+	T: Sample exchange	+	+		+	+		-
	7	+	+		+	+		±	±	T&C: blanks positive	Bac
	8	+	+		+	+		±	±	T&C: blanks positive	Bac
	9	+	+		+	+		+	+		-
	10	+	+		+	+		+	+		-
	11	+	+		+	+		+	+		-
	12	±	±	T&C: Sensitivity bio-assay	±	+	T: Sensitivity bio-assay	-	n.a.	T: Sensitivity bio-assay	Tyl/Vir/Spir/Bac
	13	+	+		+	+		+	+		-
	14	±	±	T&C: Sensitivity bio-assay	+	+		+	+		Tyl/Vir/Spir
	15	+	+		±	+	T: Sensitivity bio-assay	+	+		-
	16	±	-	T&C:No results, growth problems	+	+		-	n.a.	T: Sensitivity bio-assay	Tyl/Vir/Spir/Bac
	17	±	±	T: No results, C: Sens. bio-assay	+	+	C1: Incorrect inc. temperature	+	+		Tyl/Vir/Spir/Avo (C1)
	18	+	-	C: withdrawn	+	-	C: withdrawn	+	-	C: withdrawn	n.a.
	C1 = regular avoparcin study, C2 = additional avoparcin study			n.a. = not applicable

Table Deleted :

 Deleted Section Break (Next Page)

Table IV :

 IV Final validation results of the materials spiked with tylosin, spiramycin and virginiamycin (results from 12 laboratories).

	Level	Total amount	Tylosin	positive results		Virginiamycin	positive results		Spiramycin	positive results		Blank	positive results
	(mg kg -1 )	of samples	Feed	n	%	mean%*	Feed	n	%	mean%*	Feed	n	%	mean%*	Feed	total	n
	1	48	Calf	45	94		Poultry	48	100		Cattle	42	88		Calf	24	0
		48	Pig	48	100	97	Pig	48	100	100	Poultry	48	100	94	Pig	24	0
	2	48	Calf	48	98		Calf	48	100		Pig	48	100		Poultry	24	0
		48	Cattle	48	100	99	Pig	48	100	100	Poultry	48	100	100	Cattle	24	2
	5	48	Poultry	48	100		Calf	48	100		Calf	48	100
		48	Cattle	48	100	100	Cattle	48	100	100	pig	48	100	100
	*mean% at each level

Table V :

 V Final validation results of the materials spiked with zinc bacitracin (n= 12 laboratories).

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	Level	Total amount Zinc bacitracin positive results	Blank	Positive results		Formatted: Font: 9 pt
	(mg kg -1 ) of samples	Feed	n	%	mean%*	Feed	Total amount samples	n	Formatted Table
	1	48 48	Poultry Cattle	15 7	31 15	23	Calf Pig	23 22	6 -	Formatted: Font: 9 pt Formatted: Font: 9 pt
	2	48 48	Calf Pig	29 24	60 50	55	Poultry Cattle	24 24	2 2	Formatted: Font: 9 pt Formatted: Font: 9 pt
	3	48 48	Poultry Pig	48 36	100 75	88				Formatted: Font: 9 pt Formatted: Font: 9 pt
	5 *mean% at each level 48 48	Calf Cattle	47 45	98 94	96				Formatted: Font: 9 pt Formatted: Font: 9 pt
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Table VI :

 VI Final validation results of the materials spiked with avoparcin, (n = 15 laboratories). The feed samples marked with * are those from the additional avoparcin study

											Formatted: Left: 68.05 pt
	Level	Total amount	Avoparcin	positive results	Blank	Positive results		Formatted: Font: 9 pt
	(mg kg -1 )	of samples	Feed	n	%	mean%**	Feed	Total amount samples	n	Formatted Table
	1	68		Calf	14	21	#	Pig*	45	2	Formatted: Font: 9 pt
		60		Calf*	53	88	96	Poultry	30	-	Formatted: Font: 9 pt
		60		Pig*	60	100		Cattle	30	-	Formatted: Font: 9 pt
		59		Cattle	59	100		Calf*	45	2	Deleted: 96
	2	59		Poultry	59	100	100				Formatted: Font: 9 pt
		60		Cattle	60	100					Formatted: Font: 9 pt
	3 5	59 60 60		Calf* Calf* Poultry	55 60 60	93 100 100	93 100				Formatted: Font: 9 pt Formatted: Font: 9 pt
	60 **mean% at each level	Pig # data not used 60 F o r	100					Formatted: Font: 9 pt Formatted: Font: 9 pt Formatted: Font: 9 pt Formatted: Font: 9 pt Formatted Table Formatted: Font: 9 pt
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Table VII :

 VII Accuracy (AC), Sensitivity (SE) and Specificity (SP) for tylosin, virginiamycin, spiramycin, avoparcin and zinc bacitracin at different concentrations.

	Table VIII: Results of discrimination study
	compound	Level	Total amount	Classification results (amount of samples)
	Level (mg kg -1) compound (mg kg -1 )	of samples	AC	SE Tyl/virg/spi All	SP	Avo/bac	Blank	No result
	Tylosin	2		24			24	-			-	-	-
	1	5	Tylosin	12		97.4	96.9 11	1	97.9	-	-	-
	Virginiamycin	2	Virginiamycine 24		98.9	100 23	1	97.9	-	-	-
		5		12			11	1			-	-	-
	Spiramycin	2	Spiramycine 24		95.8	93.7 23	1	97.9	-	-	-
		5	Zinc Bacitracin 12		55.5	22.9 12	-	89.2	-	-	-
	Zinc Bacitracin	2	Avoparcin	12		96.7	96.0 -	-	97.4	5	6	1
		3		12			-	-			7	4	1
	2	5	Tylosin	24		98.4	98.9 -	-	97.9	21	-	3
	Avoparcin Blanks	2 5 -	Virginiamycin Spiramycin Zinc Bacitracin Avoparcin F o r 98.9 .98.9 71.9 98.5 24 12 48 F o r	100 100 55 100 ---	---	97.9 97.9 89.2 97.4	22 11 6	--37	2 1 5
	3 5		Zinc Bacitracin Avoparcin Tylosin Virginiamycin Spiramycin	87.5 96.2 P P 98.9 e 87.5 93.2 100 e 98.9 100 98.9 100 e r 89.2 97.4 97.9 97.9 97.9 e r
			Zinc Bacitracin Avoparcin		92.5 98.8	95.8 100	89.2 R R 97.4 e e
	Accuracy AC = PA+NA/(PA+ND+PD+NA) PA = positive agreements (correctly identified positives) NA = negative agreements (correctly identified negatives) ND = negative deviations (false negatives) PD = positive deviations (false positives)			v i e v i e w w	Deleted: Accurracy
	Sensitivity SE = PA/ (PA+ND) Specificity SP = NA/(PD+NA)								O n l O n l	Formatted: Dutch (Netherlands) Formatted: English (U.S.)
												y y
	collaborative study Irene Hofstad, 11-9-2007

1 Collaborative

study of a microbiological screening method (three-plate) for the banned antimicrobial growth promotors tylosin, virginiamycin, spiramycin, zinc bacitracin and avoparcin in animal feed

	F o r
	P Irene Pol-Hofstad, Wilma Driessen -van Lankveld, Marinka Tomassen, Jacob de Jong e and Harry van Egmond e r
	R RIKILT Institute of Food Safety, Bornsesteeg 54, PO Box 230, NL-6708 PD e
	Wageningen, The Netherlands	v i e
		w
		O n l
		y
	collaborative study Irene Hofstad, 11-9-2007	2

  The collaborative study was jointly conducted by RIKILT Institute for Food Safety in corporation with the European Commission's Joint Research Centre, Institute for Reference Materials and Measurements (JRC-IRMM) for overall coordination. JRC-IRMM also prepared the blank feed samples. The study was conducted with a step-bystep approach, where the laboratories were first involved in a training period consisting of specific training on the method through audiovisual material and known and unknown test samples. Laboratories showing good results were asked to participate in the final inter-comparison trail to validate the method.

	Banned Antibiotics and Growth promoters in FEEDingstuffs (SIMBAG FEED) various Method and materials
	analytical methodologies were developed and validated, including methods for Design of the study
	screening and confirmatory purposes. The developed methods should preferably have
	a multi-analyte approach and should meet the required specificity and sensitivity; the
	target limit for the antimicrobial growth promoters was 1 mg kg -1 (de Jong et al, 2006).
	After development, the analytical methods were subjected to a single-laboratory
	F o r validation. Due to lack of appropriate validation guidelines for the feed area, the validation of this method was carried out according to guidelines for determination of 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
	, however that method y y
	was not able to detect antibiotics at low levels, viz. five times lower than the contents
	formerly described in Council Directive 70/524/EEC (European Community, 1970).
	Within the European project: Screening and Identification Methods for official control of

residues in food matrices

(European community 2002)

, afterwards the transferability of the method to other laboratories was evaluated. Only methods successfully passing both these tests, where allowed to enter the validation phase consisting of an interlaboratory study. One of the major objectives of this collaborative study was to verify the performance characteristics of the developed microbiological screening method when used for identical samples by different laboratories.

This paper describes the interlaboratory validation of a microbiological screening method

(van Egmond et al. 2005)

. The method is based on plate inhibition tests, aiming at the detection of avoparcin, zinc bacitracin, tylosin, spiramycin and virginiamycin at detection limit of 1 mg kg -1 , expressed in microbiological activity

[START_REF] Van Egmond | Method protocol for screening on (banned) antibiotics in complete feedingstuffs and milk replacers by the microbiological plate test[END_REF]

The purpose of the method is to screen samples for the presence of one or more of the target analytes in feed without giving a quantitative statement on their concentration.

  Four different animal feed samples were used for sample preparation in this study: pig, poultry, cattle and calf feed. The different blank feeds were produced in bulk quantities for the SIMBAG FEED project and stored at -20°C until use. They were defrosted shortly before use and distributed in bottles in 10 g portions by IRMM (Belgium). Spiked samples were prepared by RIKILT. Pure antibiotic standards were used for the preparation of the spiked feed samples. Stock solutions were prepared according to TableI, and made freshly on the day of use. Samples were prepared by adding a

	Test materials
	F o r
	P
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	y
	(European Community, 2002; Vincent
	et al, 2007;).

Table IV ,

 IV V and VI show detailed information on the performance of the method for each single compound and the influence of the matrix on the detection of tylosin/virginiamycin/spiramycin, zinc bacitracin and avoparcin. The statistical calculations are reported in TableVII. For tylosin/virginiamycin/spiramycin the number of false positives was rather low with only 2%. At the target limit of detection of 1 mg kg - 1 the number of false negatives was respectively 3%, 0%, 6% (out of 96 results). The sensitivity (SE) shows acceptable values for detecting 1 mg kg -1 with respect to tylosin and virginiamycin. For spiramycin the SE values are just slightly below the required 95%, this is mainly caused by the results of cattle feed at this concentration.

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

Acknowledgments

The authors would like to acknowledge the financial support of the European Commission, DG RTD, Fifth Framework Programme, Competitive and Sustainable Growth (GROWTH) Programme, Generic Activity three: Measurements and Testing, Contract no. G6RD-CT-2000-00413 (SIMBAG-FEED). Entitled: "Screening and identification methods for official control of banned antibiotics and growth promoters in feedingstuffs". We would like to thank Gerard Kramer and his colleagues from JRC-IRMM for the preparation of the blank materials and Christoph von Holst, Gisele Gizzi and Ursula Vincent from JRC-IRMM for their advice about the design and interpretation of the results of the collaborative study. Furthermore we would like to thank the following collaborating laboratories for their cooperation and participation in this study: AGES, Austria; ALP, Switzerland, CCL, The Netherlands, Central Control and Test. Inst. Of Agric, Slovakia; Danish Plant Directory, Denmark, DGCCRF-L35, France; Fed. Voedingslab., Belgium; Istit. Zoo.Sper. Lobardia E. Romagna r.Bromat. Feeds Lab, Italy; Lab. Del Farmaco e Analysi Residui -CRNZB-Inst. Zoo. Sper. Sardegna, Italy; Forschunganstalt LUFA Rostock der LMS, Germany; LAREAL, France; LUFA Augustenberg, Germany; LUFA Dep. Microbiology, Germany; LUFA Leipzig, Germany; LUFA-ITL, Germany; Staatl. Tierarstl. Untersuchungsamt Aulendorf-Diagnostikzentrum, Germany; State Vet. Med. Diagnostic Centre, Latvia

suitable for inclusion in the control strategy as a screening method for the presence of the three groups of compounds, Moreover, the method allows to discriminate between the presence of tylosin/virginiamycin/spiramycin on the one hand and zinc bacitracin/avoparcin on the other hand providing valuable information for confirmatory analysis. This definitely has added value in decreasing the number of samples for confirmatory analysis resulting in lower costs.