Aurelia Tubaro
email: tubaro@units.it

Elena Bandi

Silvio Sosa

Maria Rosa Soranzo

Anna Giangaspero

Valeria De Ninis

Takeshi Yasumoto

Paola Lorenzon

Food Additives

Keywords: yessotoxin, skeletal muscle cells, calcium, videomicroscopy, transmission electron microscopy

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.

 1) and analogues (YTXs) are ladder-shaped lipophilic polycyclic ether compounds of algal origin, initially included in the Diarrheic Shellfish Poisoning (DSP) toxins group, and often detected in mussels together with the primary DSP toxin okadaic acid (OA). YTXs are produced by Protoceratium reticulatum (= Gonyaulax grindley) (Satake et al. 1997a), Lingulodinium polyedrum (= Gonyaulax polyedra) [START_REF] Paz | Production and release of yessotoxin by the dinoflagellates Protoceratium reticulatum, and Lingulodinium polyedrum in culture[END_REF] or Gonyaulax spinifera [START_REF] Rhodes | Yessotoxin production by Gonyaulax spinifera[END_REF], organisms that are distributed. worldwide. So far, no human poisoning induced by YTXs has been reported [START_REF] Toyofuku | Joint FAO/WHO/IOC activities to provide scientific advice on marine biotoxins (research report)[END_REF]. Moreover, since YTXs do not provoke diarrhoea, they have been recently reclassified and separately regulated from DSP toxins [START_REF] Cee | Commission decision of 15[END_REF].

"[Insert Figure 1 about here]"

The oral toxicity of YTXs appears to be low. After acute oral administration of YTX (1-10 mg kg -1 ) in mice, only ultrastructural changes in cardiomyocytes were observed: the morphological alterations were detectable only in restricted areas of the heart close to the capillaries [START_REF] Terao | Histopathological studies on experimental marine toxin poisoning-5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative[END_REF][START_REF] Aune | Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice[END_REF][START_REF] Tubaro | Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice[END_REF]. In a more recent study consisting of 7 oral doses administered over a 3 weeks, a No Observable Adverse Effect Level (NOAEL) of 5 mg YTX kg -1 was established [START_REF] Espenes | Toxicity of yessotoxin in mice after repeated oral exposure. Galway (Ireland)[END_REF]). In the absence of human data and applying a safety factor of 100, a provisional acute reference dose of 50 µg kg -1 , corresponding to a Regulatory Limit (RL) of 12 mg YTXs kg -1 shellfish meat, was recently proposed [START_REF] Toyofuku | Joint FAO/WHO/IOC activities to provide scientific advice on marine biotoxins (research report)[END_REF]. However, repeated daily oral administration of YTX (1 or 2 mg kg -1 day -1 , for 7 days) in mice induced ultrastructural changes such as alterations of fibrils and packages of swollen 4 mitochondria in cardiomyocytes near capillaries, similar to those observed after acute oral intake [START_REF] Tubaro | Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice[END_REF][START_REF] Callegari | Oral administration of yessotoxin stabilizes E-cadherin in mouse colon[END_REF].

In vitro studies showed that YTX causes apoptosis in skeletal muscle cell lines via a mitochondrial pathway [START_REF] Korsnes | Induction of apoptosis by YTX in myoblast cell lines via mitochondrial signalling transduction pathway[END_REF], suggesting that also the skeletal muscle could represent a target tissue for the toxin. The aim of the present study was to further investigate the effects of YTX on the skeletal muscle both in vitro and in vivo. In vitro experiments were performed to investigate if YTX affected the intracellular calcium transients generated by the action potentials. In vivo experiments were performed to analyze the possible ultrastructural effects of YTX on skeletal muscle cells of mice because no effect was previously observed by light microscopic examinations after acute or repeated oral administration of YTX [START_REF] Tubaro | Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice[END_REF]2004).

Materials and methods

Materials

YTX was isolated from P. reticulatum collected in Mutsu Bay (Japan) [START_REF] Murata | Isolation and structure of yessotoxin, a novel polyether compound implicated in diarrhetic shellfish poisoning[END_REF]. The purity of the final preparation was confirmed by mass spectrometry and liquid chromatographic methods (Satake et al. 1997b;[START_REF] Yasumoto | Fluorometric measurement of yessotoxins in shellfish by high-pressure liquid chromatography[END_REF].

YTX was dissolved in 95 % aqueous ethanol and, for oral administration, aliquots of the stock solutions were properly diluted with saline solution (0.9 % NaCl), in order to administer the appropriate dose of the toxin and reduce the concentration of ethanol to 1.8 % (v/v). For in vitro studies, aliquots of the stock solution of YTX were properly diluted, in order to reach a concentration of ethanol in the cells medium lower than 1 % (v/v).

Cell cultures

Skeletal muscle cell cultures (i28) were established from mouse satellite cells [START_REF] Irintchev | Functional improvement of damaged adult mouse by implantation of primary myoblasts[END_REF]. Briefly, satellite cells were isolated from the hind leg muscles of 7-day-old male Balb/c mice killed by cervical dislocation. Muscle tissue was minced and then treated with collagenase and trypsin. Expansion and enrichment of myogenic cells were achieved by repeated plating and cultivation in DMEM with D-valine containing 20 % fetal bovine serum. Muscle cells could be maintained as exponentially growing myoblasts over 40 passages in the presence of a medium consisting of HAM'S F-10 containing 20 % fetal calf serum (FCS), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). To induce cell differentiation and myotube fusion the medium was replaced, 1 day after plating, with DMEM supplemented with 2 % horse serum and L-glutamine, penicillin, and streptomycin as above. Cells were maintained at 37 °C in CO 2 (5 %)-enriched air. Experiments were performed on myoblasts maintained in culture from the 5 th to the 40 th passage and cultured up to 10 days in differentiation medium. Experiments were carried out at 37 °C on Fura-2 loaded myotubes attached to a coverslip treated with matrigel. All the experiments were performed on myotubes bathed in an external solution containing 2 mM CaCl 2 . Briefly, the cells were alternatively excited at 340 and 380 nm and the fluorescence images were acquired with an intensified CCD camera [Hamamatsu Photonics, Hamamatsu, Japan]. The acquisition rate was 4 frames/s. The calculation of ratio images [START_REF] Grynkiewicz | A new generation of Ca 2+ indicators with greatly improved fluorescence properties[END_REF] and the temporal plots of the fluorescence signals were performed off-line.

Animals and experimental conditions

Experiments were carried out in conformity with the Italian D.L. n. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC), concerning animal welfare.

Female CD-1 mice (18-20 g) were purchased from Harlan Italy (S. Pietro al Natisone, Italy). Groups of three animals were acclimated for 1 week under the conditions of controlled room temperature (21 ± 1°C), humidity (60-70 %), and a fixed artificial light cycle (7.00 -19.00 h). Dust-free poplar chips were used as bedding (Harlan Italy; S.

Pietro al Natisone, Italy). Mice were fed a global diet for rodents containing 18.5 % protein (Harlan Italy; S. Pietro al Natisone, Italy), and had free access to food and water. YTX, dissolved in saline solution (0.9 % NaCl) containing 1.8 % ethanol (v/v), was administered to mice by gavage in a volume of 10 ml kg -1 body weight. The toxin was administered as a single dose (1 mg kg -1 ) or as repeated daily doses for 7 days (1 mg ). Control animals were given 10 ml kg -1 day -1 of the vehicle (saline solution containing 1.8 % ethanol v/v). The toxin or vehicle was administered in the morning (9.00-10.00 h). Twenty-four hours after the last administration, the mice were killed by ketamine hydrochloride (350 mg kg -1 , administered intraperitoneally). Skeletal muscle (right hind leg) was removed and fixed for examination by transmission electron microscopy (TEM).

Transmission electron microscopy analysis

Blocks of skeletal muscle smaller than 1 mm 3 were promptly fixed in a solution of 3 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 h at room temperature and 2 h at 4 °C, rinsed three times (10 min each wash) in the same buffer and post fixed in 1 % osmium tetroxide for 1 h at 4 °C. The samples were then dehydrated in graded ethanols and embedded in Dow Epoxy Resin 332 [START_REF] Lockwood | A reliable and easily sectioned epoxy embedding medium[END_REF]. Ultrathin sections were cut by an ultratome Leica Ultracut UCT8 (Leica, Mikrosysteme Aktiengesellschaft, Wirn, Austria), double stained with uranyl acetate and lead citrate [START_REF] Venable | A simplified lead citrate stain for use in electron microscopy[END_REF] and examined with a Philips EM 208 transmission electron microscope.

Statistical analysis

In vitro data are expressed as means ± standard error. The Student's t test was used to examine statistical significance, assumed for p values lower than 0.05.

Results and discussion

Mouse myotubes differentiated in vitro generate spontaneous action potentials, accompanied by [Ca 2+ ] i transients and contractions. The mean event frequency is 0.16 Incubation of myotubes with YTX (1.0 µM for 20 min) did not affect the frequency of [Ca 2+ ] i transients (Figure 2). However, after 1 hour, an almost complete inhibition of spontaneous activity was observed: the percentage of active cells was reduced by 96 % with respect to the control cells (Figure 3). No effect was observed at the lower concentrations used (0.1 and 0.01 µM).

"[Insert Figure 2 about here]"

When the incubation time was increased to 24 hours, 0.1 µM YTX significantly reduced the spontaneous activity (77 %). After 48 hours of incubation, the percentage of active cells was reduced by 97 % at all the YTX concentrations tested (Figure 3). It has to be pointed out that the inhibition of the spontaneous activity after 48 hours incubation was associated with a dramatic reduction in the number of viable cells. Thus, YTX affected both the excitability and the viability of skeletal myotubes in vitro and its effect was concentration-and time-dependent.

In silent myotubes incubated for 24 hours with 0.1 or 1.0 µM YTX, we tested the responsiveness to physiological stimuli, such as depolarization induced by perfusion The percentage of cells responsive to KCl and caffeine was similar to that observed under control conditions (Figure 4). Therefore, 24 hours incubation with YTX had no effects on the mechanism controlling Ca 2+ homeostasis in skeletal muscle cells. "[insert Figure 4 about here]"

The effects of YTX on skeletal muscle were also evaluated in vivo after single (1 and 2 mg kg -1 ) and repeated daily oral administration in mice (1 or 2 mg kg -1 day -1 , for 7 days). The mice were sacrificed 24 hours after the single or last dose and morphological changes in skeletal muscle were analyzed by TEM. No morphological differences in the skeletal muscle sections were noted between control and YTX-treated mice, neither after acute administration nor at the end of the repeated YTX treatment. Particular attention was given to the mitochondria and myofibrils. The myofibrils presented a normal fine structure similar to the controls and unaltered mitochondria were observed between the fibrils (Figure 5). In conclusion, we observed significant in vitro effects on cell excitability and viability when skeletal muscle cells were incubated with YTX. However the toxin did not cause tissue damage or ultrastructural changes in the skeletal muscle in vivo. These results support the hypothesis that YTX is poorly absorbed after per os intake and that its bioavailability to skeletal muscle is too low to provoke significant morphological changes. On the contrary, under the same experimental conditions, in vivo YTX provokes ultrastructural myocardium changes. Thus, cardiac tissue seems to be the specific target for YTX. Further studies are in progress to further clarify the differing responses of cardiac and skeletal muscle, an aspect that could be an important determinant in view of the proposed deregulation for YTX . The present EU Regulatory Limit for YTX is 1 mg of YTX equivalents per kg of shellfish meat [START_REF] Cee | Commission decision of 15[END_REF].

However, this tolerance level was established considering the estimated No Observable Adverse Effect Level of 1 mg YTX kg -1 body weight, based on the results obtained from acute oral exposure to mice, as well as assuming an average shellfish consumption of 100 g and applying a safety factor of 600 due to the lack of data and indications of effects on the cardiac muscle [START_REF] Espenes | Toxicity of yessotoxin in mice after repeated oral exposure. Galway (Ireland)[END_REF].

  modified Eagle's medium (DMEM), horse serum, penicillin, streptomycin and glutamine were purchased from ICN Biomedicals (Irvine, USA). Bovine fetal calf serum was purchased from PAA Laboratories (Pasching, Austria), 3 % glutaraldehyde from Serva (Heidelberg, Germany) and Dow Epoxy Resin (DER 332; Unione Chimica Europea; Milan, Italy). All other chemicals were Carlo Erba products of analytical grade (Milan, Italy).

  Video-imaging was used to measure the intracellular calcium concentration ([Ca 2+ ] i ).

  spontaneous action potentials, [Ca 2+ ] i transients and contractions resemble those triggered by the nerve in skeletal muscle fibers being inhibited by tetrodotoxin and verapamil[START_REF] Lorenzon | Properties of primary mouse myoblasts expanded in culture[END_REF]. The video-imaging technique was used to study the effect of YTX on the skeletal muscle cell excitability, measuring the spontaneous [Ca 2+ ] i transients.

  KCl, a classic membrane depolarizing agent, or 20 mM caffeine, a well known agonist of the intracellular Ca 2+ channels present on the surface of the sarcoplasmic reticulum. "[Insert Figure 3 about here]"

"

Figure 1 .

 1 Figure captions:

Figure 2 .

 2 Figure 2. Representative trace showing the [Ca 2+ ] i transients generated by myotubes

Figure 3 .

 3 Figure 3. Effect of YTX (0.01, 0.1 and 1.0 µM) on the number of myotubes exhibiting Ca 2+ spiking activity after 1, 24 or 48 hours incubation. For each experimental point, 10

Figure 4 .

 4 Figure 4. Responsiveness of myotubes to depolarisation by KCl (40 mM) or caffeine

Figure 5 .

 5 Figure 5. Electron micrographs of longitudinal sections of skeletal muscle from control (upper image) and YTX-treated mice (2 mg kg -1 day -1 , for 7 days; lower image).

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

Acknowledgements

This work was supported by a grant of the Italian Ministry of Instruction, University and Research. The authors are grateful to Prof. Roberto Della Loggia (University of Trieste, Italy) for the stimulating discussion, and to Mr. Claudio Gamboz, "Centro Coordinamento e Sviluppo Progetti e Apparecchiature -CSPA", University of Trieste, for his careful technical work.


Ron Laurentius Hoogenboom

Jan G Bokhorst

Martin D Northolt

Lucy Van De Vijver

Nico J G Broex

Dik J Mevius

Jac A C Meijs

Joop Van Der Roest

Ron ; Hoogenboom

L A P Hoogenboom

M.D Northolt

Contaminants and micro-organisms in Dutch organic food products; comparison with conventional products

Keywords: Screening -microbial screening Mycotoxins -fusarium, Nitrate, Pesticides, Veterinary drug residues Food Types: Cereals and grain, Meat, Organic foods, Vegetables Organic, lettuce, carrots, pigs, broilers, antibiotic resistance, Salmonella, Campylobacter

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.

Introduction

Organic production differs from conventional production in a number of ways. The use of synthetic pesticides is e.g. prohibited, the use of antibiotics is restricted and under strict control, only animal manure is used for fertilization and animals are allowed to forage outside. Theoretically this may result in a different composition of the products, both in terms of natural ingredients, but also with regard to contaminants and micro-organisms. It has e.g. been suggested that wheat may contain higher levels of mycotoxins, since no fungicides are used during production. On the other hand, the mechanical treatment of the soil against weeds, which is not so common in conventional practice, and the much poorer fertilization may create less advantageous conditions for survival of moulds during the winter. Foraging outside might expose the animals to higher levels of environmental contaminants, which end up in the edible products. This has e.g. been demonstrated in the case of organic eggs, which may contain elevated levels of dioxins and dioxin-like PCBs (Traag et al. 2005[START_REF] Schoeters | Dioxin contamination in laying hens and eggs[END_REF][START_REF] Van Overmeire | Chemical contamination in eggs of free-range hens in Belgium[END_REF], Kijlstra et al. 2007). The use of animal manure may also result in increased levels of heavy metals in organically produced vegetables and the presence of dangerous micro-organisms like Salmonella and E. coli O157. The restricted use of antibiotics might be expected to result in a decreased antibiotic resistance, especially against the newer generation of antibiotics.

A number of studies have focussed on potential differences between organic and conventional products, but these are still limited [START_REF] Woese | A comparison of organically and conventionally grown foods-results of a review of the relevant literature[END_REF], Soil Association 2001[START_REF] Magkos | Organic food: buying more safety or just peace of mind? A critical review of the literature[END_REF]. The aim of the present study was first to examine the food safety aspects of organic products and secondly to look for potential differences with conventional products, focussing on a limited number of contaminants and micro-organisms. A major issue in this type of studies is the sampling of the products, since various factors may influence the results.

It was decided to focus on Dutch products and to sample these as much as possible at the Certain conventional products were sampled at stores. In other cases data were compared with data from existing monitoring programmes. The study included the most important organic vegetable products in The Netherlands, being lettuce, wheat, carrots and potatoes. In addition, eggs, broilers, pigs and cows were studied. Investigated were factors like mycotoxins, nitrate, heavy metals, pesticides and E. coli O157 in vegetables, and Salmonella, E. coli O157, antibiotic resistant bacteria, heavy metals and veterinary drugs in animals or their products.

As such the study contributes to the essential and fortunately growing amount of information about the food safety of organically produced food products.

Materials and Methods

Sampling of products

Vegetables

Samples of 1 kg organic or conventional wheat were collected in August 2003 and August/September 2004 at processing plants. Due to limited availability, this comprised in 2003 nine samples conventional winter wheat and 9 samples organic summer wheat, and in 2004 fourteen samples conventional winter wheat, 17 samples conventional summer wheat and 22 samples organic summer wheat. Samples came from several provinces in the Netherlands, with the exception of the organic summer wheat in 2004 that came primarily from the centre of the Netherlands, Flevoland.

Organic lettuce was collected in 2003 and 2004 at 3 distribution centres and at small farms. Samples came from various locations in The Netherlands. Conventional lettuce was sampled at super markets. Each sample contained 3-8 pieces of lettuce. The lettuce produced in the field was sampled in 2003 in September/October, and in 2004 in June/July. Lettuce Organic carrots were sampled in 2003 at farms shortly after the harvest (October-November). In addition, organic and conventional carrots were sampled at farms (15 organic, 4 conventional), distribution centres (4 conventional) and shops (7 conventional) in March-April 2004. Although spread all over the Netherlands, most farms were located in Flevoland.

Each sample contained 40 carrots.

Organic potatoes were to a large extent sampled at the same farms as the carrots in October-November 2004 shortly after the harvest. Each sample contained 40 potatoes.

Animal products

In the case of pigs, 31 organic pig farms were visited, 10 in December 2003-January 2004 and 21 in April-May 2005. For analysis on Salmonella and E. coli O157, at each farm faeces sampled from 12 pigs, short before slaughter, were pooled into a combined sample. On farms with more stables, 2 or 3 pooled samples were taken. To study the incidence of antibioticresistant bacteria, faecal samples were taken from 5 pigs per farm. All samples were immediately sent to the laboratories. Pig kidneys and diaphragm from 20 animals of 20 different organic farms were sampled at the slaughterhouse.

Samples of bovine faeces were collected at 10 organic farms according to routine procedures of the Food and Consumer Products Safety Authority. The ten farms were visited in October-November 2003. One farm with a positive result on E. coli O157 was revisited in June 2004. Each pooled sample consisted of faeces from 12 cows. The number of samples per farm was 2 for less than 40 cows, 3 for 40-59 cows, 4 for 60-199 cows and 5 for more than 200 cows. Samples were put in polystyrene boxes and shipped immediately to the laboratory.

Ten bovine kidneys from 10 organic farms were sampled at the slaughterhouse. For examining Salmonella in faeces of laying hens and broilers, respectively 10 (April-May 2004) and 9 (April-June 2005) organic farms were visited and 5 pooled samples, each from 12 fresh droppings, were collected and sent immediately to the laboratory, according to the protocol of the Product Boards for Livestock, Meat and Eggs (PVE). For antibioticresistant bacteria in broilers, 5 samples of 1 fresh dropping per farm were collected and send to the laboratory, according to standard protocols. At 10 organic farms with eggs, 30 fresh eggs per farm were collected and mixed at the laboratory into one pooled sample per farm.

Analysis of samples

For chemical analysis, lettuces, carrots and potatoes were frozen in liquid nitrogen and homogenized. Wheat was milled with a sieve of 1 mm. For microbiological examination samples were not pre-treated.

Mycotoxins in wheat

The 71 wheat samples were analyzed for mycotoxins using an LC-MS/MS multi-method, including aflatoxin B 1 , fumonisins B 1 and B 2 , ochratoxin A, zearalenone, deoxynivalenol, T-2 and HT-2 toxins. Zearalenone was also examined with an HPLC method and fluorescence detection. In short, from each sample two portions of 10 g were taken. One portion was extracted with water and the other portion with a mixture of acetonitrile/water (84:16 v./v).

Both portions were extracted by shaking intensively for at least 3 hours. After filtering from each extract 250 µL was mixed and injected on a LC-MS-MS system in multiple reaction monitoring (MRM) mode. To diminish matrix influences, the analytical method was performed by a standard addition protocol. The analysis of mycotoxins was performed on a Waters Ultima LC-MS/MS system in the MRM-mode. A water (5 mM formic acid)/acetonitrile (5 mM formic acid) gradient was run at a flow of 0.20 ml/min on a Phenomenex Synergy HydroRP column (150 x 2 mm, 5 µm). All mycotoxins were analysed under ESI+ conditions with the exception of zearalenone which was analysed under ESIconditions. Reference substances (>95% purity) were obtained from Biopure Reference Substanzen GmbH (Tulln, Austria). Reference materials (Test materials) were obtained from FAPAS (Central Science Laboratory, York, Great-Britain). Detection limits were 0.1 mg/kg for deoxynivalenol and T-2 toxin, 0.1 mg/kg for HT-2 and fumonisins B 1 and B 2 , 5 µg/kg for aflatoxin B 1 , and 50 µg/kg for ochratoxin A and zearalenone.

Heavy metals and arsenic

Samples were treated with 70% HNO 3 in a microwave oven (MARS 5). An aliquot was subsequently analyzed in an electrothermical atomic absorption spectrophotometer (ETAAS, SIMAA 6100) for cadmium, led and arsenic by atomic adsorption at 228.8, 283.3 and 193.7 nm. Concentration measurements were determined from a working curve (linear) after calibrating the instrument with standards of known concentration.

Another aliquot was analyzed for mercury in a Flow Injection Mercury System, FIMS 100 (Perkin Elmer, Groningen, The Netherlands). In short, the mercury in the digested samples was reduced to the elemental state by tin (II) chloride solution and liberated from solution in a closed vessel system, cold vapor technique (CV AAS). The mercury vapor passed through a cell positioned in the light path of an atomic absorption spectrometer (FIMS). Its absorbance is measured at a wavelength of 253.7 nm. The absorbance signal is a function of the mercury concentration in the samples.

Nitrate

Samples of lettuce, carrots and potatoes were analyzed according to the SKALAR method. In short, any debris (e.g. soil) from the sample was removed (without water) and a representative 8 part of the sample was heated with water for 10-15 min on a boiling water bath. After cooling down, the volume was fixed to a known amount and the samples filtered. The method procedure for the determination of nitrate is based on the hydrazinium reduction method (Skalar Methods, In short, the nitrate in the sample was reduced to nitrite with hydrazinium sulphate and the nitrite was determined by diazotizing with sulphanilamide and coupling with naphthylethylenediamine dihydrochloride to form a highly coloured azo dye.

This dye was measured at 540 nm in a continuous flow detector (Skalar, Breda, The Netherlands).

Pesticides

Pesticides were analyzed by a multi-method based on [START_REF] Anastassiades | Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and "Dispersive Solid-Phase Extraction" for the Determination of Pesticide Residues in Produce[END_REF]. Samples were extracted by a modified Quechers method [START_REF] Díez | Comparison of an acetonitrile extraction/partitioning and "dispersive solid-phase extraction" method with classical multi-residue methods for the extraction of herbicide residues in barley samples[END_REF]). After addition of magnesium sulphate and salt, the mixture was centrifuged and an aliquot of the acetonitrile was purified with primary-secondary amine. After cleaning by dispersive solid-phase extraction the extracts were analysed on an Agilent Technologies 6890N GC (Waldbronn, Germany) equipped with an ATAS Optic 3 PTV injector (Veldhoven, The Netherlands) and a LECO Pegasus III TOF-MS (St. Joseph, MI, USA). A Restek RTX-CL Pesticides column (30m x 0.25 mm, 0.25 µm, Bellefonte, PA, USA) was employed with helium as carrier gas and a linear programmed temperature program from 40°C to 280°C with 10°C/min. All analytes were measured in EI+ mode. Reference substances were obtained from Dr.

Ehrenstorfer (Augsburg, Germany). The method quantifies a number of selected pesticides and detects a large number of others qualitatively. If detected, these pesticides are quantified by injection of standards. LOQs vary between 0.01 and 0.5 mg/kg. The method is able to detect over 300 pesticides, including the major organophosphates, organochlorines and pyrethroids. Eggs, kidneys and meat were investigated for the presence of antibiotics (macrolides, aminoglycosides, sulphonamides, tetracyclines, quinolones, colstin) using microbial methods [START_REF] Nouws | A microbiological assay system for assessment of raw milk exceeding EU maximum residue levels[END_REF], Pikkemaat et al. 2007a). Test systems consisted of a series of agar plates, each one specific for a group of residues and optimized for a particular matrix [START_REF] Pikkemaat | A new microbial screening method for the detection of antimicrobial residues in slaughter animals: The Nouws antibiotic test (NAT-screening). Food control In Press[END_REF]. Methods were validated conform 657/2002/EC. Detection limits (CCß) were below the EC maximum residue limits, except for valnemulin, cloxacillin and dicloxacillin in kidney and valnemulin, cefapirin, cefalexin, kanamycin, cloxacillin and dicloxacillin in meat ( 1 to 4 times the MRL).

Eggs were analysed for residues of toltrazuril and its metabolite ponazuril by LC-UV according to the method described by [START_REF] Mulder | Deposition and depletion of the coccidiostats toltrazuril and halofuginone in eggs[END_REF]. In brief, homogenized whole egg was extracted with ethyl acetate. The solvent was evaporated and the residue dissolved in 0.02 M trisodium phosphate buffer. Purification was carried out by solid phase extraction on an Oasis ® HLB cartridge. LC-UV analysis was performed on a Symmetry ® C18 150 x 3.0 mm column with a methanol/water/acetic acid gradient and UV detection at 245 nm. The detection limit was 25 µg/kg for toltrazuril and 10 µg/kg for ponazuril.

Livers from broilers were analysed for residues of nitrofuran metabolites by LC-MS/MS based on the method of [START_REF] Hoogenboom | The use of pig hepatocytes to study the nature of protein-bound metabolites of furazolidone; a new analytical method for their detection[END_REF] as modified by [START_REF] Cooper | Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV[END_REF] In brief, homogenized liver samples were treated overnight with 0.1 M hydrochloric acid in the presence of 2-nitrobenzaldehyde. The samples were extracted twice with ethyl acetate. After evaporation the residue was dissolved in mobile phase. LC-MS/MS analysis was performed on a Symmetry ® C18 150 x 3.0 mm column with a acetonitrile/water/acetic acid gradient and detection with positive ESI in multiple reaction monitoring mode. The detection limit for

Salmonella and E coli O157

Lettuce was examined for Salmonella using two methods. An aliquot of 25 gram of sample (outer leaves) was incubated in 225 mL buffered peptone water (BPW) for 18 h at 37ºC. This pre-enrichment broth was subcultured in 2 selective enrichment media: Rappaport Vassiliadis soya broth (RVS) and Müller-Kaufmann tetrathionate novobiocin broth (MKTTn). After 24 h incubation at respectively 41.5 and 37°C aliquots of the selective media were streaked at both XLD and BGA selective media. After 24 h incubation at 37°C the plates were evaluated for the presence of specific Salmonella colonies. Suspected colonies were confirmed using biochemical and serological methods. The VIDAS screenings method, based on an Enzyme Linked Fluorescent Assay, was started from the selective enrichment broths after 6-8 h. VIDAS-negative samples do not require confirmation and can be considered "Salmonella not detected". VIDAS-positive samples were further treated as described above.

Lettuce was examined for E. coli O157 as follows. An aliquot of 25 gram of sample (outer leaves) was incubated in 225 mL modified tryptone soy bouillon at 41.5°C. After both 6 h and 18-24 h an immunomagnetic separation was carried out with Dynabeads ® anti-E.coli O157.

Aliquots were plated on 2 selective plates (CT-SMAC and Chromagar O157™). After 18-24 h incubation at 37°C the plates were evaluated for the presence of specific colonies.

Suspected colonies were confirmed based on indole-production and agglutination with specific antiserum. Positive isolates were also confirmed by the RIVM (Bilthoven, The Netherlands).

Faeces samples were examined for Salmonella according to both the VIDAS method or the ISO 6579:2002 method, as described for lettuce. From February 2004 faeces samples, 11 after enrichment in BPW, were also inoculated in modified semi-solid Rappaport Vassiliadis medium (MSRV) with 0.01 g novobiocin/L. After incubation for 24 and 48 h at 41.5°C, plates were evaluated for specific growth areas, if necessary inoculated at selective growth media XLD and BGA, and further treated as described above. E. coli O157 in faeces was examined as described above for lettuce.

Antibiotic resistant bacteria

The isolation of E. coli, Enterococcus faecium, and Campylobacter spp. was started directly upon arrival of the samples at CIDC-Lelystad by making a 1:10 w/v suspension of the faeces in buffered peptone water with 20% glycerol, from which on the same day selective media were inoculated. Subsequently the suspensions were stored at -20°C.. E. coli and E. faecium were isolated on respectively MacConkey agar and Slanetz and Bartley agar by inoculating 50 µL of a serial dilution series of the 1:10 diluted sample with a spiral plater (E. faecium) or by direct inoculation of the plates with sterile swabs (E. coli). The MacConkey plates were incubated overnight aerobically at 37°C and the Slanetz Bartley plates during two days aerobically at 42°C. From each sample, an E. coli colony with the typical morphology was subcultured on Heart Infusion agar with 5% sheep blood and a colony typical of E. faecium on Columbia agar with 5% sheep blood. Both were incubated overnight at 37°C. Subsequently the strains were suspended in buffered peptone water with 20% glycerol and stored at -80°C.

All E. coli isolates were identified biochemically by the Indole reaction. The species identification of E. faecium was performed with a Polymerase Chain Reaction (PCR) described by Dutka [START_REF] Dutka-Malen | Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR[END_REF]. For the isolation of Campylobacter spp. CCDA-agar was used with 32 µg/mL cefoperazone and 10 µg/mL amphotericine B to suppress the growth of Gramnegative bacteria and moulds. Plates were inoculated directly upon arrival of the samples with a swab. The CCDA-plates were incubated for 48 h in microaerophilic conditions

Results and Discussion

The present study focussed on a limited number of contaminants and micro-organisms in organic products and organic production systems. Selection was based on a report of the Expertise Centre for Agriculture in the Netherlands, and information on other projects already running in this area. Contaminants already studied in ongoing studies, like dioxins in eggs (Kijlstra et al. 2007), were not included in this study. Results were subsequently compared with those from conventional agriculture, partly coming from this project and partly from other monitoring programmes in the Netherlands. In addition a comparison was made with results from other studies.

A very important issue is the sampling strategy and in particular the site of sampling; the farm or the shop/supermarket [START_REF] Magkos | Organic food: buying more safety or just peace of mind? A critical review of the literature[END_REF]. Samples taken at a shop, give information on the products actually bought by the consumer. However, in this study the choice was made to collect data as much as possible at the farm, thus allowing the control on the origin of the product and a survey on the actual farming conditions. This would also allow an investigation on potential causes for undesirable contaminants or micro-organisms. It should be stressed that organic farming in the Netherlands is a growing market and that most of the organic farms switched to organic practices only recently. In cases where sampling at the farms was not possible, due to logistic or hygienic reasons, samples were taken at distribution centres and slaughterhouses. At the time of this study there were about 500 farms involved in the organic production of vegetables, potatoes and grains. In the present study 24, 35 and 21 farms were sampled for lettuce, carrots and potatoes respectively. Results will be described and discussed for each type of product investigated. Wheat was collected in 2003 and 2004. Twelve of the 20 organic samples came from farms that switched to organic less than 5 years ago. The organic wheat covered races like Melon, Lavett and Pasteur, the conventional wheat was not inventoried. Yield of organic wheat varied between 4300 and 7900 kg/hectare. Eighteen pooled samples of organic wheat were investigated for non-polar pesticides but none of the more than 300 different compounds could be detected.

The levels of the 7 examined mycotoxins in wheat were below detection limits in the conventional and organic wheat samples, with the exception of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) (Table 1). In most of the samples collected in 2003, levels of DON were below the action limit of 0.1 mg/kg, with the exception of 6 conventional products showing levels between 0.1 and 1 mg/kg. Of the samples collected in 2004 before the end of August, nine out of 14 samples (64%) of conventional winter wheat and 8 out of 11 samples (73%) of conventional summer wheat, contained DON levels above the action limit (range 0.1-1.5 mg/kg), as compared to five out of seven (71%) samples organic summer wheat (range 0.1-1.8 mg/kg). However, after a period with heavy rain fall by the end of August 2004, DON was detected in all samples at levels up to 11 mg/kg. In these samples also zearalenone was increased with levels up to 5.7 mg/kg. Overall there was no significant difference between organic and conventional wheat. This is contradictory to studies by [START_REF] Malmauret | Contaminants in organic and conventional foodstuffs in France[END_REF] who did find higher levels of the Fusarium toxins DON, nivalenol and zearalenone in organic wheat and barley. However, [START_REF] Döll | Fusarium mycotoxins in conventionally and organically grown grain from Thuringia/Germany[END_REF] 15 high levels in some conventional wheat. In general, there is a tendency for organic cereals to contain lower mycotoxins levels but differences appear to be rather small [START_REF] Magkos | Organic food: buying more safety or just peace of mind? A critical review of the literature[END_REF]) and factors like climate may play a much more crucial role than the type of production.

Lead, mercury and arsenic levels in most samples were below the detection limits of respectively 0.1, 0.01 and 0.1 mg/kg. In 4 organic wheat samples mercury levels just above the LOD were detected, i.e. levels of 0.013, 0.011, 0.016 and 0.016 mg/kg. Cadmium could be detected in most samples but the highest level of 0.077 mg/kg was well below the EU-limit of 0.2 mg/kg. There was no difference in cadmium levels between organic and conventional wheat (data not shown). This is contrary to [START_REF] Rossi | organically or conventionally produced wheat on health, performance and mycotoxin residues in tissues and bile of growing pigs[END_REF], who found slightly lower cadmium but higher led levels in organic wheat, although not at levels exceeding the EUlimits.

Lettuce (micro-organisms, pesticides, nitrate, heavy metals)

Organic lettuce was sampled at 3 distribution centres, responsible for 70% of the organic lettuce. Half of the farms produced organically for two decades or more, the other half for less than ten years. Conventional lettuce was obtained from local stores. None of the samples tested positive for Salmonella or E. coli O157, two types of micro-organisms that might be introduced through the use of animal dung. This shows that this is not a common problem but cannot exclude the presence of these pathogenic micro-organisms in a small fraction of the products. To minimize the potential risk of contamination, several countries like the US and Canada do not allow the use of non-composted manure.

Ten pooled samples of organic and conventional lettuce were examined for non-polar pesticides but none of the organic products tested positive. Two pooled samples of conventional lettuce contained residues of the permitted pesticide vinclozolin and one of these in addition malathion and piperonylbutoxide. Levels were below the residue limits. In both organic and conventional iceberg lettuce produced outside, nitrate levels were below the limits, with a range of 370 to 1759 mg/kg for organic and 652 to 1367 mg/kg for conventional products (Table 2). In the case of organic head lettuce produced outside (19 samples) and the greenhouse (10 samples), one sample of each exceeded the limit. In the case of conventional head lettuce, this frequency was much higher. Levels above the limit were detected in 18 out of 19 samples grown outside and 4 out of 14 samples from the greenhouse.

In comparison with conventional head lettuce, nitrate levels were much lower for organic products. This confirms results from previous studies [START_REF] Consumentenbond | De schaduwkant van groente[END_REF][START_REF] Woese | A comparison of organically and conventionally grown foods-results of a review of the relevant literature[END_REF], Soil Association 2001). In the case of iceberg lettuce, no differences were observed.

Levels of arsenic and the heavy metals led, cadmium and mercury were below the limits.

Cadmium levels showed a large variation, as shown in Table 2. However, no differences were observed between organic and conventional products.

Carrots (pesticides, nitrate, heavy metals)

Organic carrots were sampled at farms with varying experience, ranging from 2-18 years.

Most of them switched to organic less than 5 years ago. More than 11 different varieties were used but Nerac was most popular. Yields varied between 35 and 75 tons/hectare. Four pooled samples of organic carrots were investigated for non-polar pesticides but none of the more than 300 different compounds could be detected.

Nitrate levels in organic carrots showed a large variation, with a range in 2004 of 11 to 864 mg/kg and a median of 244 mg/kg (Table 3). This was 3 times higher than in conventional carrots harvested in the same year, showing a median of 58 mg/kg and a range from 16 to 180 mg/kg. Previous studies showed lower levels in organic carrots [START_REF] Consumentenbond | De schaduwkant van groente[END_REF] but the present data confirm those from other studies showing an increase in nitrate levels in organic carrots (Bokhorst, personal communication). Malmauret et 2002) also observed higher levels in organic carrots, i.e. 394 versus 113 mg/kg product in conventional carrots. Studies at the farms showed that a relatively high use of dung and plant waste materials, as compared to previous years, is probably the cause of the rise in nitrate levels. This is partly related to farms that switched only recently to organic practices.

Heavy metals and arsenic were only analyzed in the organic carrots samples in 2003.

None of the levels exceeded the limits, although some cadmium levels were close to the limit of 0.1 mg/kg.

Potatoes (pesticides, nitrate, heavy metals)

Organic potatoes were sampled at the farms, conventional potatoes were not investigated.

Most of the farms were also sampled for carrots, again most of them with less than 5 years of experience. Agria and Sante were the most popular races with yields between 23 and 47 tons/hectare. Four pooled samples of organic potatoes were investigated for non-polar pesticides but none of the more than 300 different compounds could be detected.

The nitrate level in organic potatoes was low with an average of 87 mg/kg but the variation was quite large (range 8-390 mg/kg). There were no Dutch data on conventional potatoes for comparing the data. However, levels were lower than those reported by [START_REF] Hajšlová | Quality of organically and conventionally grown potatoes: four-year study of micronutrients, metals, secondary metabolites, enzymic browning and organoleptic properties[END_REF] for organic potatoes. In that study levels in conventional potatoes were higher than in organic ones.

None of the samples levels exceeded the limits for heavy metals, although some cadmium levels were close to the limit of 0.1 mg/kg. In total 31 organic farms with pigs were visited, representing 53% of the organic pig farms in the Netherlands. Fourteen farms were more experienced (6-14 years), the other 17 switched to organic only recently (1-4 years). The number of pigs varied between 20 and 1100 (Table 4).

Pigs (veterinary drugs, heavy metals, micro-organisms, antibiotic resistance)

No residues of antibiotics were detected with a bacterial test in samples of kidneys and meat of 20 organic pigs, sampled at the slaughterhouse. Only one kidney sample showed a slight inhibition of bacterial growth on the macrolides plate but below the action level. Levels of arsenic in meat were below the detection limit of 0.1 mg/kg. Similar was true for levels of led and mercury in the kidneys of these animals, being below respectively 0.05 and 0.005 mg/kg with one exception, being a mercury level of 0.008 mg/kg. Cadmium levels varied between less than 0.005 and 0.38 mg/kg (median 0.11 mg/kg), all of them being below the EU-limit of 1 mg/kg. Initially 10 farms were visited in the winter of 2003 and all the pooled faeces samples of the 12 animals per farm scored negative for Salmonella. Another set of 20 farms was sampled in the spring of 2005 and this time 8 farms were positive for Salmonella. Interestingly, this included 7 farms that only recently switched to organic (3 years or less). The other farm turned to organic in 1996 and on this farm 3 barns were sampled. One of these samples, derived from a stable with young pigs that were purchased elsewhere, was positive. Overall the incidence of Salmonella was 27% and comparable to data known from conventional farms in the Netherlands. However, the fact that Salmonella could not be isolated from dung collected at the more experienced organic pig farms, is of potential interest. Possible causes could be the use of more rough fibre rich and more diverse feeds at organic farms compared to conventional pig farms, which may influence the colonisation of Salmonella in the GI-tract [START_REF] Knudsen | Influence of feed and feed structure on disease and welfare of pigs (4 th NAHWOA Workshop[END_REF].

Campylobacter was one of the bacteria generally used for the assessment of antibiotic resistance and could be isolated from 56% of the faeces samples, being similar to or more of the 5 sampled pigs at 81% of the farms. E. coli O157 was not detected in any of the samples, but this was only analysed in samples from the first 10 farms. This was decided because the data were in line with conventional farms where the prevalence of E. coli O157 is 0-2%.

The use of antibiotics at organic farms is restricted although 19 of the 31 farms mentioned the occasional use of antibiotics. The resistance level to a number of antibiotics was investigated in E. coli, E. faecium and Campylobacter spp., isolated from faeces of the 5 animals per farm (1 isolate per sample). E. coli could be isolated from all 155 animals and resistance was only observed for older drugs like amoxicillin, neomycin, tetracycline, sulfamethoxazole, trimethoprim and chloramphenicol. No resistance was observed in E. coli from organic farms against newer drugs like cefotaxim, gentamicin, the quinolones ciprofloxacin and nalidixinic acid, and florfenicol. Figure 1A shows that in general the resistance in organic pigs is lower than observed in conventional pigs, based on data from the Dutch regular monitoring program (MARAN, 2004). In this program one animal per slaughter batch is sampled, according to guidelines of the European Food Safety Authority. Regarding the limited number of farms, it was decided to use five animals per farm in the present study, which could have some influence on the results. E. Faecium was isolated from 80% of the samples or on 97% of the farms. Significant resistance was only observed against doxycyclin, quinupristin/dalfopristin (Synercid ® ) and bacitracin, and to flavomycin but this Entorococcus species is intrinsically resistant to this drug. Figure 1B shows that also for this species in general the resistance is lower than in conventional pigs, in particular true for erythromycin and salinomycin. Bacitracin has been used as a growth promoter until 1999 and resistance is rather widespread in conventional pigs, and apparently also in organic pigs. Synercid is related to virginiamycin, also used as a growth promoter until 1999. As mentioned above, Campylobacter (jejuni and coli) could be isolated from 80% of the samples, or at 77% of the farms. In comparison to E. coli and E. faecium, Campylobacter showed resistance to many of the tested drugs (9 out of 12). Remarkable was the resistance against the quinolones ciprofloxacin and nalidixinic acid, since this type of drugs is only used to treat individual pigs. In general, resistance in organic pigs was lower than in conventional pigs (Figure 1C). When looking at the fraction of bacteria resistant to 1, 2, 3, 4 or more than 4 antibiotics, 32% of the E. coli isolates from organic pigs were multiple drug resistant (resistant to 2 or more independent classes of antibiotics), as compared to 55% in conventional pigs (Table 5). For E. faecium figures were similar for organic and conventional, being respectively 75% and 74%, but for Campylobacter spp. lower for organic, i.e. 45% versus 80%. Although the lack of difference for E. faecium seems remarkable, differences became more clear when focussing on multiple resistance against 3 or more antibiotic classes, showing 33% resistance for organic versus 46% for conventional. Furthermore, low level resistance (<10%) was observed in Enterococci isolated from organic farms for erythromycin and salinomycin compared to the resistance levels found in conventional animals.

Lactating cows (veterinary drugs, micro-organisms)

Ten kidney samples of cows, reared in organic farms, were collected at the slaughterhouse and tested for bacterial growth inhibition. Three samples showed a slight inhibition at the aminoglycosides plate but below the action limit.

Ten farms with lactating cows were visited in September-November 2003 and samples of dung were collected. Farm size varied between 35-74 cows and one bigger farm with 250 animals. E. coli O157 was detected in all three samples of one farm and in one of four samples of another farm, the latter collected at a pasture with non-lactating cows and young

Laying hens (veterinary drugs, micro-organisms, heavy metals)

Ten farms out of a total of 100 were visited and both eggs and dung collected. Number of hens per farm varied between 170 and 10900, and farms switched to organic between 1994 and 2003. Dung was tested for the presence of Salmonella using different methods, including the standard MSRV method. Most samples scored negative, except for one farm where one of the two barns tested positive. Except for one, hens were vaccinated against Salmonella, but antibiotics were reported not to be applied. All eggs tested negative for toltrazuril, and for the presence of aminoglycosides, sulfonamides, beta-lactam, tetracyclins, quinolones and colistin. Levels of cadmium, led, arsenic and mercury were below the detection limits of respectively 0.005, 0.05, 0.1 and 0.005 mg/kg. This is in contrast to the rather high levels of heavy metals observed by [START_REF] Van Overmeire | Chemical contamination in eggs of free-range hens in Belgium[END_REF] in eggs from private owners in Belgium, indicating that heavy metals may be transferred to eggs, as previously shown for dioxins.

Broilers (veterinary drugs, micro-organisms, heavy metals, antibiotic resistance)

The number of organic farms with broilers is rather limited in the Netherlands and these were all visited. Most farms turned organic since 1998 and the number of animals varied between 380 and 8000. Animals belonged to slow-growing varieties, as reflected by the age at slaughter of 70-81 days (for conventional broilers this is around 40 days). Antibiotics were reported not to be used. Other veterinary drugs and anthelmintics were used on 2 and 1 farms, respectively. Livers were checked for nitrofuran drugs and all tested negative. Led, arsenic and mercury in livers was below the detection limits of respectively 0.05, 0.1 and 0.01 mg/kg. Cadmium levels varied between <0.005 and 0.029 mg/kg, well below the EU-limit of 0.5 mg/kg.

On none of the 9 organic farms, animals were carriers of Salmonella as determined with the MSRV-method. Of course, the number of farms is very small but the data are in line with those observed by the VWA in 2003 and 2004 in meat, showing an incidence of 2 and 3% respectively, as compared to 11 and 7% in meat from conventional broilers (VWA 2004b[START_REF] Vwa | Survey pathogenen en bacteriële resistentie in kipproducten uit biologische teelt 2004[END_REF]. Campylobacter was detected in faeces samples at all farms, and overall could be isolated from 32 of the 45 samples. In conventional broilers the incidence is much lower, in 2000, 2001and 2002respectively 24, 16 and 27% (VWA 2004a)). The VWA detected Campylobacter in 36 and 44% of the meat products of organic broilers, as compared to 26 and 29% of the conventional products (VWA 2004b[START_REF] Vwa | Survey pathogenen en bacteriële resistentie in kipproducten uit biologische teelt 2004[END_REF]. [START_REF] Heuer | Prevalence and antimicrobial susceptibility of thermophilic Campylobacter in organic and conventional broiler flocks[END_REF] examined organic and conventional farms in Denmark and detected Campylobacter in respectively 49 and 100% of the faeces samples collected during slaughter. Remarkable was the high incidence of 91% for Campylobacter jejuni and only 9% C. coli, whereas [START_REF] Rodenburg | Campylobacter and Salmonella infections on organic broiler farms[END_REF], who also observed a high incidence of Campylobacter in organic broilers, reported incidences of 30% for C. jejuni and 70% for C. coli. This is relevant since in particular C. jejuni is responsible for infections in humans. Our data are however in line with those of [START_REF] Heuer | Prevalence and antimicrobial susceptibility of thermophilic Campylobacter in organic and conventional broiler flocks[END_REF], also reporting primarily C. jejuni, independent of the type of production.

In Campylobacter highest resistance against antibiotics was found against amoxicillin, doxycyclin, metronidazole and the quinolones nalidixinic acid and ciprofloxacin (Figure 2C). There were no clear differences in resistance proportions with data from 57 samples from conventional broilers. This was also the case for multi drug resistance (resistant against 2 or more independent classes of antibiotics) showing figures of 72 and 63% in organic versus conventional broilers (Table 6). Considerable resistance was observed in E. coli against amoxicillin, tetracycline, sulfamethoxazole, trimethoprim and to a lesser extent nalidixinic acid, but not against the newer antibiotics. Figure 2A shows a comparison with data from conventional broilers, demonstrating that in conventional broilers resistance occurred much more frequently than in organic animals. E. faecium showed considerable resistance against doxycyclin, bacitracin, synercid, and to a lesser extent to erythromycin, salinomycin and streptomycin but again much lower than in conventional broilers. This organism is known to be resistant against flavomycin as shown in Figure 2B. Overall, 27% of the E. coli's were multiple resistant (2 or more independent classes of antibiotics) as compared to 81% of the conventional broilers. For E. faecium figures are respectively 69 and 94% (Table 6). The observed reduced resistance against antibiotics confirms previous studies by the Dutch Food and Consumer Products Safety Authority in the Netherlands [START_REF] Vwa | Survey pathogenen en bacteriële resistentie in kipproducten uit biologische teelt 2004[END_REF].

The fact that in organic broilers in the commensal gut flora (E. coli and E. faecium) less antimicrobial resistance occurs than in conventional broilers can mainly be explained by the absence of selection pressure in organic animals. The fact that in Campylobacter spp. this difference was not observed is likely to be caused by the fact that in broilers Campylobacter colonisation occurs after environmental contamination with this organism. In a densely populated country like the Netherlands it can be speculated that the Campylobacter spp. isolated from the organic broilers originate from conventionally reared poultry and were already resistant at the moment of colonisation.

Conclusions

Although some of the results suggest a difference between organic and conventional, it should be stressed that the present study was only a survey focussing on one set of samples and one or two production years. However, in a number of cases the results are similar to those from other studies, like the lower nitrate levels in head lettuce, the lack of increased mycotoxin levels in organically produced grains, less Salmonella but more Campylobacter in broilers, and less antibiotic-resistant bacteria in pigs and broilers. In the case of nitrate in carrots there are indications for increasing levels in organic cultivation and further studies are required to confirm this. Also the lower incidence of antibiotic resistant bacteria in pigs and the lower incidence of Salmonella in more experienced pig farms deserve more attention.

  allowing a check on the source. Furthermore it allowed an analysis of factors that might contribute to the occurrence or levels of certain contaminants or micro-organisms.

  was sampled in January till March in 2004. In each case similar numbers of samples were taken from both types of production.

  metabolites was 0.5 µg/kg and for nitrofurazone and nitrofurantoin 1.0 µg/kg. Quantification was performed by isotope dilution.

  Typical colonies were streaked on HIS-plates and again incubated for 48 h microaerophilically at 37°C. The isolates were all identified as Campylobacter coli by PCR.The susceptibility of the isolates was determined quantitatively using a broth micro dilution method according to CLSI guidelines (M31-A2 and M100-S16), with cation-adjusted Mueller Hinton broth. Microtiter plates with dehydrated dilution series of a panel of antibiotics (see Figures1 and 2) were provided by Trek Diagnostic Systems (Basingstoke, UK). For Campylobacter spp. the plates, after inoculation with 50 µL per well of a 200-fold diluted 0.5 McFarland suspension in NaCl 0.85% solution, were incubated microaerophilic in a shaking incubator at 37°C for 24 h. ATCC strains E. coli 25922, E. faecalis 29212 and C. jejuni ATCC 33560 were tested daily to control the quality of the results.The minimum inhibitory concentrations (MICs) were defined as the lowest antibiotic concentration at with visually bacterial growth was observed. Strains with MICs higher than the MIC breakpoints defined by CLSI were classified as resistant. For those antibiotics (e.g. neomycin and florfenicol for E. coli, the former growth promoters for the enterococci) and bacterial species (Campylobacter ssp.), for which CLSI has not defined clinical MICs, breakpoints used in the Dutch Resistance Monitoring Programme were used (Maran 2004).Subsequently resistance proportions were calculated for each individual antibiotic tested.Moreover, proportions of fully susceptible isolates and resistant to 1, 2, 3, 4 and more than 4 different antibiotic classes were calculated.

  , mycotoxins, heavy metals)

  [START_REF] Bernhoft | Less fusarium infection and mycotoxins in organically than in conventionally grown grain[END_REF],Schneweis et al. (2005),[START_REF] Rossi | organically or conventionally produced wheat on health, performance and mycotoxin residues in tissues and bile of growing pigs[END_REF] and[START_REF] Pussemier | Development and application of analytical methods for the determination of mycotoxins in organic and conventional wheat[END_REF] also observed lower average levels in organic cereals.[START_REF] Czerwiecki | On ochratoxin A and fungal flora in Polish cereals from conventional and ecological farms. Part 2: Occurrence of ochratoxin A and fungi in cereals in 1998[END_REF], analyzing various cereals in Poland in two subsequent years for ochratoxin A, observed higher frequencies of contamination of organic cereals in one year, but the opposite in the next year with some very

  When focussing on the farms, Campylobacter could be isolated from one

  first farm was revisited in September 2004 and found negative than, as was the case for the second farm revisited in June 2004.

  could be isolated from all 45 faecal samples, E. faecium from 89% of the samples..

Figure 1 .

 1 Figure 1. Incidence of antibiotic resistant E. coli's (A), E. faecium (B) and Campylobacter (C)

Figure 2 .

 2 Figure 2. Incidence of antibiotic resistant E. coli's (A), E. faecium (B) and Campylobacter (C)

F

  Figure1

Table 1 .

 1 Levels of DON and ZON (median and range) in organic and conventional wheat sampled in2003 and 2004.

	Sample date	Production type		DON	ZON	Number
				(mg/kg)	(mg/kg)	of samples
	2003	Organic	Summer	<0.1	<0.05	9
				(<0.1-0.2)
		Conventional	Winter	0.3	<0.05	9
				(<0.1-1.0)
	2004, until 24 th August o Organic r Conventional	Summer Summer	0.2 (<0.1-1.8) (<0.1-1.5) 0.2	<0.05 (<0.05-0.12) (<0.05-0.13) <0.05	7 11
	2004, after 24 th August	Conventional P Organic e r Winter Summer e Conventional Summer	<0.1 (<0.1-0.4) 1.7 (<0.1-11.0) (0.5-6.3) 2.0	<0.05 2.6 (<0.05-5.7) (<0.05-5.2) 0.7	14 15 6
	R No significant differences between conventional and organic (Student t-test, two-sided)
			e
			v i e
				w
				O n l
					y
						1

Table 2 .

 2 Nitrate and cadmium levels in organic (O) and conventional (C) iceberg and head lettuce produced outside and in the greenhouse. * EU-limits for head lettuce from the field are 2500 mg/kg, harvested between 1 st April and 30 th September, and 4000 mg/kg when harvested between 1 st October and 31 st March; for head lettuce from the greenhouse 3500 mg/kg, when harvested between 1 st April and 30 th

	Type	production			Nitrate**	n	Cadmium	n
				(mg NO 3 /kg)		(mg/kg)
				median	range		range
	Iceberg	field	O	939	370-1759	13	<0.02-0.022	5
	Iceberg	field	C	966	652-1367	13	<0.02-0.038	4
	Head lettuce	field	O	1275*	139-3212	19	<0.02-0.042	6
	Head lettuce	field	C	3280*	1818-4357	19	<0.02-0.052	8
	Head lettuce greenhouse O	3223	946-4129	10	<0.02-0.043	9
	Head lettuce greenhouse C	3515	2439-5197	14	<0.02-0.066	8
	* Differences between nitrate in conventional and organic head lettuce from the field were
	statistically significant (Student t-test, p<0.001)
	September, and 4500 mg/kg when harvested between 1 st October and 31 st March; for iceberg
	lettuce from the field 2000 mg/kg (EC 2001, 2002).

*

Table 4 .

 4 Description of the farms with organic pigs, including the results from the studies on Salmonella and Campylobacter.

	Farm	Organic	Animals	Salmonella Campylobacter* Rough fiber-
	number	since				rich feed
	1	1997	340	negative	5/5	+
	2	1990	75	negative	5/5	+++
	3	1998	500	negative	4/5	+
	4	1998	122	negative	3/5	++
	5	1998	200	negative	2/5	+
	6	1995	140	negative	4/5	+
	7	1999	120	negative	5/5	+
	8	1999	525	negative	5/5	+
	9	1997	850	negative	5/5	+
	10	1990	450	negative	0/5	+
	11	2003	500	positive	3/5	+
	12	2004	700	negative	4/5	±
	13	1996	460	positive	3/5	-
	14	2003	400	positive	2/5	+
	15	2002	325	negative	5/5	+
	16	2002	257	positive	3/5	+
	17	2002	950	negative	5/5	-
	18	2004	400	positive	3/5	-
	19	2003	106	negative	5/5	±
	20	2002	620	negative	1/5	+
	21	2001	400	negative	2/5	-
	22	2002	120	positive**	2/5	+
	23	1998	400	negative	5/5	-
	24	2003	1100	positive**	0/5	-
	25	2003	450	positive	0/5	+
	26	2002	80	negative	4/5	+
	27	2002	440	negative	1/5	+
	28	1998	700	negative	0/5	-
	29	1998	300	negative	0/5	+
	30	2002	450	negative	1/5	+
	31	1999	20	ni***	0/5	-
	* number of faeces samples from which Campylobacter could be isolated for antibiotic
	resistance
	** negative with the MSRV method
	*** ni: not investigated due to loss of sample

Table 5 .

 5 Resistance of bacteria from pig faeces against different classes of antibiotics. Data expressed as the percentage of bacteria resistant against 0, 1, 2, 3, 4 or more than 4 classes of antibiotics. Conventional data from the MARAN study(MARAN, 2004).

			Food Additives and Contaminants		Page 34 of 37
	Resistant	E. coli			E. faecium	Campylobacter
	to
	classes	Organic Conventional Organic Conventional Organic Conventional
	0	43	27	3	5	22	5
	1	25	19	22	21	33	15
	2	15	14	43	28	22	26
	3	7	16	22	17	14	25
	4	6	17	9	17	4	16
	>4	4	8	2	12	5	13

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 6 .

 6 Resistance of bacteria from broiler faeces against different classes of antibiotics.Data expressed as the percentage of bacteria resistant against 0, 1, 2, 3, 4 or more than 4 classes of antibiotics. Conventional data from the MARAN study(MARAN, 2004).

	Page 35 of 37
	Resistant	E. coli			E. faecium	Campylobacter
	to
	classes	Organic Conventional Organic Conventional Organic Conventional
	0	53	10	3	1	28	19
	1	20	9	30	6	0	18
	2	16	8	38	10	31	23
	3	2	13	20	16	22	26
	4	9	22	8	22	13	7
	>4	0	38	3	46	6	7

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.

uk Food Additives and Contaminants

Schneweis I, Meyer K, Ritzmann M, Hoffmann P, Dempfle L, Bauer J. 2005. Influence of

Acknowledgements

This project was sponsored by the Dutch Ministry of Agriculture, Nature and Food Quality and the Food and Consumer Products Safety Authority. The authors also would like to thank Kees Veldman, Marga Japing, Jeannette Wup (Central Veterinary Institute), Riekje Bruinenberg (LBI), Grada Hazeleger-Druten, Ciska Jansen-Schalk, Wilma Jacobs-Reitsma, Wendy van Overbeek, Mariel Pikkemaat, Harry van Egmond, Cissy Warmerdam, Henk Bannink, Theo de Rijk and Paul Zomer (RIKILT) and Hilko van der Voet (Biometris) for their valuable contribution


P.-C Harizanis

E Alissandrakis

P.-A Tarantilis

& M Polissiou

Solid-phase microextraction /gas-chromatographic /massspectrometric analysis of p-dichlorobenzene and naphthalene in honey

Keywords: Pesticide residues, Pesticides -organochlorine honey, p-dichlorobenzene, naphthalene, solid-phase microextraction, gas chromarography-mass spectrometry

Protection of honey combs from wax moth Galleria mellonella involves the use of physical, biological or chemical control methods. Chemical control may result in residues in the extracted honey. The presence of residues of p-dichlorobenzene and naphthalene in honey were investigated by means of solid-phase microextraction (SPME) coupled to GC/MS. The method was linear between 5 µg/kg and 200 µg/kg of honey for p-dichlorobenzene and 1 µg/kg and 200 µg/kg of honey for naphthalene.

Limits of detection were 1 µg/kg and 0.1 µg/kg respectively. The relative standard deviations were 2.6 % and 7.9 % for p-dichlorobenzene and naphthalene respectively.

Application of the method to 90 unifloral Greek honeys revealed that in 25.6% of the samples the concentration of either one of the pesticides exceeded the Maximum Residue Level (MRL). Maximum concentrations were 163.03 µg/kg of honey for pdichlorobenzene and 193.74 µg/kg of honey for naphthalene. Naphthalene was found in traceable amounts in 78.9% of the samples, but only 5.6% of them contained concentrations above the MRL. This indicates the use of pre-contaminated honey comb foundations or built combs. Nevertheless, because naphthalene is naturally present in some plant species growing in Greece, the contribution of nectar from such a floral source should not be overlooked.

Introduction

Apiculture faces a great deal of pests, including mites, insects and a number of important diseases. Two insects problems that are fed on the comb wax are the greater wax moth, Galleria mellonella L., and the lesser wax moth Achroia grisella Fabricius.

Infestation of brood combs, extracting combs, comb honey and other wax materials that have been stored at temperatures above 15 o C especially by the greater wax moth G. mellonella, results in considerable economic lose. Beekeepers can protect their honey combs by storing them and applying physical, biological or chemical control methods [START_REF] Cantwell | Control of the greater wax moth, Galleria mellonella, in honeycomb and comb honey[END_REF][START_REF] Burges | Control of wax moths: physical, chemical and biological methods[END_REF].

Chemical control is effective and easy to use and usually is applied as fumigants such as p-dichlorobenzene and naphthalene. Wax moth action is prevented when the unused combs are stocked in the hive boxes and the fumigant is placed on top of the stock because the fumes are heavier than air [START_REF] Morse | The Illustrated Encyclopedia of Beekeeping[END_REF]. There is a risk of harmful residues in the extracted honey, regardless of the type of fumigant used [START_REF] Burges | Control of wax moths: physical, chemical and biological methods[END_REF]. In Greece, p-dichlorobenzene and naphthalene had been used for almost 60 years [START_REF] Nikolaidis | Bee Enemies[END_REF][START_REF] Dermatopoulos | Practical Beekeeping[END_REF], until the situation changed when European Regulation (EC) No 396/2005 set a limit of 10 µg/kg honey for those substances where no Maximum Residue Level (MRL) for honey had been established, including the two aforementioned insecticides.

Due to the lipophilic character of these two compounds, they are readily adsorbed by the wax and thereafter end up in honey stored by the bees. Their high volatility makes headspace techniques suitable for isolation and quantification of these analytes [START_REF] Bogdanov | Residues of p-dichlorobenzene in honey and beeswax[END_REF]Takanaki et al., 2005). In addition, many publications have shown that solid-phase microextraction is an effective procedure to isolate a variety of [START_REF] Jiménez | Solid-phase microextraction applied to the analysis of pesticide residues in honey using gas chromatography with electron-capture detection[END_REF][START_REF] Fernádez | Determination of organophosphorus pesticides in honeybees after solid-phase microextraction[END_REF][START_REF] Campillo | Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey[END_REF][START_REF] Rial-Otero | Gas chromatography mass spectrometry determination of acaricides from honey after a new fast ultrasonicbased solid phase micro-extraction sample treatment[END_REF]). This paper presents the development of an isolation and quantification method for p-dichlorobenzene and naphthalene residues in honey by means of solid-phase microextraction coupled toGC/MS. The results of the application of this method to 90 unifloral Greek honey samples are presented.

Experimental

Reagents

The reagents used for this work were of analytical grade (>99%). p-dichlorobenzene was purchased from Riedel de Haën and naphthalene from Fluka Chemika.

Benzophenone used as an internal standard was also purchased from Fluka Chemika.

Stock solutions of these analytes were prepared in methanol of analytical grade.

Honey samples

A total of 90 honey samples from various botanical sources were employed. These samples were collected during the years 1999-2003.

Extraction

Extraction was performed by means of a Solid-Phase Microextraction (SPME) device (Supelco, Bellefonte, USA), using a DVB/carboxen/PDMS fibre to isolate headspace volatiles. The samples (solution of 3g of honey for every mL of water) were placed in 15 ml screw-top vials with PTFE/silicone septa. Benzophenone was used as internal standard and a portion of 20 µL (10 µg/mL in methanol) was added prior to extraction.

The vials were maintained in a water bath at during equilibration and extraction and were partially submerged so that the liquid phase of the sample was in the water (Miller

Gas chromatography-Mass Spectrometry

The analysis of the extracts was performed using a Hewlett Packard 5890 II GC, equipped with a Hewlett Packard 5972 MS detector. The column used was an HP-5MS (Crosslinked 5% PH ME Siloxane) capillary column (30 m X 0.25 mm i.d., 0.25 µm film thickness) and the gas carrier was Helium, at 1 mL/min rate. The injector and MStransfer line temperatures were maintained at 220 o C and 290 o C respectively. Oven temperature was held at 40 o C for 3 min, raised to 160 o C at 3 o C/min and then to 200 o C at 10 o C/min. Electron impact mass spectra recorded at 40-500 mass range. An electron ionization system was used with ionization energy of 70 eV.

Quantification

The calibration curves were established by adding known concentrations of the analytes to a residue-free honey. Twelve concentrations were employed, ranging from 1 to 10 000 µg/kg of honey. For each concentration and analyte, two repetitions were made. For quantitative determination of p-dichlorobenzene and naphthalene in honey samples, three replicates were obtained.

Results and discussion

Method parameters

Limits of detection and quantification. The limit of detection (LOD) was considered as the amount of the analyte required to give a signal-to-noise ration of 3 and was found to be 1 µg/kg for p-dichlorobenzene and 0.1 µg/kg for naphthalene. The limit of 6 quantification (LOQ) requires a signal-to-noise ration more than 10 and it was found to be 5 µg/kg for p-dichlorobenzene and 1 µg/kg for naphthalene.

Linearity. Preliminary tests showed that the method was linear for concentrations up to 200 µg/kg for both pesticides, and, thus, calibration curves were prepared for concentrations up to this value. Specifically, the following concentrations were used; 1, 5, 10, 20, 50, 100, and 200 µg/kg of honey. Calibration curves (see Figures 1 and2) were prepared using the ratio of the peak area of the analyte against the peak area of the internal standard (R D/Β and R N/B for p-dichlorobenzene and naphthalene respectively). The correlation coefficients (R 2 ) were 0.9948 and 0.9902 respectively.

[Insert Figure 1 about here] [Insert Figure 2 about here] Repeatability. The repeatability of the procedure was estimated on a residue-free sample spiked with 10 µg/kg of honey for both analytes, which is the MRL value according to the European Regulation (EC) No 396/2005. Figure 3 shows a TIC chromatogram of this sample. The relative standard deviation (RSD) values were 2.6% and 7.9% for pdichlorobenzene and naphthalene respectively (n=5).

[Insert Figure 3 I. Honeys were placee in two groups, according to the season of harvest.

Group A includes summer (thyme, cotton) or early autumn (pine) harvests, while Group B comprises honeys harvested either in spring (citrus, fir chestnut, eucalyptus) or late autumn (heather). The maximum concentrations were 163 µg/kg of honey for pdichlorobenzene and 194 µg/kg of honey for naphthalene. Both samples possessing these concentrations belong to Group A, as do most of the samples in which high concentrations of the pesticides were estimated. In Figure 4, the chromatogram of a real honey sample (sample No 19) containing both p-dichlorobenzene and naphthalene at concentrations above the MRL values is shown.

[Insert Table I about here] [Insert Figure 4 about here] Table II summarizes the results of Table II, regardless of the botanical origin. The percentage of samples p-dichlorobenzene was detected was 46.7%, while for naphthalene it raised up to 78.9%. In 18.9% of the samples for p-dichlorobenzene and 5.6% for naphthalene, the concentration exceeded the MRL value of 10 µg/kg of honey.

Finally, 84.4% of the samples contained either one of the analytes, and 25.6% of them were improper for human consumption. It must be emphasized that the samples used were harvested prior to 2002, well before the limit of 10 µg/kg honey was established.

The analysis of samples collected after the European regulation 396/2005 EC shows a dramatic decrease in numbers of contaminated samples [START_REF] Tananaki | Contamination of honey by chemicals applied to protect honeybee combs from wax-moth (Galleria mellonela L.)[END_REF] due to the use of alternative ways for protecting honeybee wax combs. An important aspect of the results in Table II is the relatively few samples containing naphthalene at high concentrations, with respect to the number of samples in which this analyte was detected. Only 7% exceeded the MRL value, which suggests the use of wax foundation pre-contaminated with naphthalene. This is the case for pdichlorobenzene and it has been concluded that lipophilic substances accumulate in the wax and residues can be found in recycled wax used for the foundation [START_REF] Bogdanov | Residues of p-dichlorobenzene in honey and beeswax[END_REF]. In a likewise manner, detectable amounts can end up in honey by the use of built combs that had been treated with naphthalene in the past. For p-dichlorobenzene, the respective percentage was 40%.

Table III shows the results with respect to the botanical origin of the samples. The problem is crucial for group A, as 77.5% of the samples were found to contain pdichlorobenzene and 90% naphthalene, while those exceeding the MRL value were 30% and 10% respectively. During this period, the environmental conditions are favorable for the wax moth, and the use of pesticides seems indispensable for the beekeepers.

[Insert Table III about here]

In contrast, group B includes 22% of samples contaminated with pdichlorobenzene, 10% of which above the MRL. Naphthalene was detected in 70% of the samples, but only one (2%) was above the MRL. In particular, in 30 out of 33 citrus honeys naphthalene was detected, but in only one the concentration exceeded the MRL.

In fact, 29 out of 30 samples had concentrations below the LOQ (see Table I). Citrus honey is harvested in early to mid spring, a period with relatively low temperatures that are not suitable for the development of the wax moths, thus the application of pesticides is not essential. The occurrence of naphthalene in almost 91% of citrus honeys reinforces the hypothesis for pre-contaminated wax foundations or built combs.

Less likely, but still important to be mentioned, is the possibility that low proportions of naphthalene are of natural origin. Analysis of the essential oil of Valeriana tuberosa L., a common plant in southern Greece, revealed the presence of naphthalene at the steams, leaves and inflorescence [START_REF] Fokialakis | Essential oil constituents of Valeriana italica and Valeriana tuberosa. Stereochemical and conformational study of 15acetoxyvaleranone[END_REF].

Naphthalene has also been traced in the essential oil of Ononis viscosa subsp. breviflora [START_REF] Erdemgil | Composition of the essential oil of Ononis viscosa subsp. breviflora[END_REF].

Conclusions

The pesticides p-dichlorobenzene and naphthalene have been used as an effective tool to control the wax moths in stored honey combs. These substances are adsorbed in the comb and end up in honey, due to their lipophilic character. According to the European Regulation (EC) No 396/2005, the limit for these two substances is set at 10 µg/kg honey. Solid-phase microextraction proved efficient for isolating and quantifying these two analytes. LOD values were 1 µg/kg and 0.1 µg/kg and LOQ values 5 µg/kg and 1 µg/kg for p-dichlorobenzene and naphthalene respectively. The RSD values were 2.59% and 7.92%.

The method was applied in 90 samples of unifloral honeys from different botanical origin and 46.7% of them contained p-dichlorobenzene in traceable amounts, 78.9% naphthalene and 84.4% either of the two pesticides. In total, 25.6% of the samples were improper for human consumption, as either one of the pesticides exceeded the MRL value. Even though naphthalene was traced in 78.9% of the samples, only in

  ). Optimal conditions for the extraction were as follows: 30 min equilibration time, 60 min sampling time, 6 mL sample volume, and 60 o C water bath temperature.

  The regression equations are: for p-dichlorobenzene: R D/B = (0.097±0.004) D -(0.793±0.331), and for naphthalene: R N/B = (0.121±0.005) N -(0.710±0.471).

  applied to 90 samples of different botanical origin and the results are shown in Table

Figure 1 .Figure 3 .Figure 4 .

 134 Figure 1. Calibration curve of p-dichlorobenzene.

Table I .

 I Concentration of p-dichlorobenzene and naphthalene in analyzed Greek honeys.

	30 cotton		ND		<LOQ		70 citrus		<LOQ		<LOQ
	31 cotton		<LOQ		<LOQ		71 citrus		ND		<LOQ
	32 cotton		<LOQ		<LOQ		72 citrus		ND		<LOQ
	33 cotton		164.03	1.51	<LOQ		73 citrus		ND		<LOQ
	34 cotton		10.64	5.80	<LOQ		74 fir		11.60	2.76	ND
	35 cotton		Group A 19.07 7.88	9.51	2.37	B 75 fir		Group B ND	<LOQ
				p-dichlorobenzene	A naphthalene				p-dichlorobenzene	naphthalene
	36 pine		ND		<LOQ		76 fir		ND		ND
	No	Honey Type	µg/kg	SD (%)	µg/kg	SD (%)	No	Honey Type	µg/kg	SD (%)	µg/kg	SD (%)
	37 pine		<LOQ		ND		77 fir		ND		ND
	1 thyme		ND		<LOQ		41 citrus		ND		ND
	2 thyme 3 thyme 4 thyme 38 pine 39 pine 40 pine	F o r 12.01 3.10 ND ND F o r 11.44 6.74 <LOQ 9.78 3.60 F o r	<LOQ <LOQ <LOQ ND <LOQ ND		42 citrus 43 citrus 44 citrus 78 fir 79 fir 80 fir 81 heather	ND ND ND ND ND ND ND		ND ND <LOQ ND ND ND ND
	5 thyme		ND		<LOQ		45 citrus		14.65	2.71	<LOQ
	P e <LOQ 25.59 ND 6.57 <LOQ P e P e containing both p-dichlorobenzene (A) and naphthalene (B) at concentrations above the 6 thyme 46 citrus 10.86 4.74 <LOQ 7 thyme 47 citrus ND <LOQ 82 heather ND ND 83 heather ND <LOQ 84 heather <LOQ 8.41 3.98 MRL. e r 8 thyme 11.37 6.97 <LOQ 48 citrus ND <LOQ 9 thyme <LOQ 7.00 3.44 49 citrus 9.87 2.80 <LOQ e r 85 chestnut ND ND 86 chestnut 12.87 1.48 <LOQ e r
	10 thyme		ND		193.74	0.66	50 citrus		12.41	2.40	<LOQ
	11 thyme		12.18	12.39	R <LOQ R R	51 citrus 87 chestnut 88 eucalyptus	ND ND ND		<LOQ ND ND
	12 thyme 13 thyme 14 thyme 15 thyme ND: not detected. LOQ: limit of guantification. <LOQ <LOQ 9.69 1.63 <LOQ	21.08 7.14 9.16 <LOQ	e v i e 0.58 52 citrus 2.24 53 citrus e 89 eucalyptus e 1.48 54 citrus 55 citrus v i e 90 eucalyptus v i e	ND ND <LOQ ND ND ND		<LOQ <LOQ <LOQ <LOQ <LOQ ND
	16 thyme 17 thyme		9.38 <LOQ	6.12	<LOQ <LOQ		w 56 citrus 57 citrus w w	ND ND		<LOQ <LOQ
	18 thyme 19 thyme 20 thyme 21 thyme		<LOQ 31.19 <LOQ 10.84	3.34 5.87	<LOQ 18.19 82.58 7.06	1.36 1.14 2.68	58 citrus 59 citrus 60 citrus 61 citrus	O n l ND ND 38.26 1.06 ND O n l O n l	<LOQ <LOQ 22.03 <LOQ	1.52
	22 thyme		9.61	4.08	9.27	3.29	62 citrus		y ND y y	<LOQ
	23 thyme		<LOQ		<LOQ		63 citrus		ND		<LOQ
	24 thyme		ND		<LOQ		64 citrus		9.72	4.11	<LOQ
	25 thyme		23.63	2.17	<LOQ		65 citrus		ND		<LOQ
	26 thyme		15.98	3.61	8.02	2.07	66 citrus		ND		<LOQ
	27 thyme		13.25	3.14	9.64	1.67	67 citrus		ND		<LOQ
	28 thyme		<LOQ		ND		68 citrus		ND		<LOQ
	29 cotton		8.94	6.88	8.46	1.54	69 citrus		ND		<LOQ
							1 2 3

Table II .

 II Summarized results for p-dichlorobenzene and naphthalene in the 90 samples analysed.

	Residues	Detection number	Number of samples with concentration >
			(percentage)	10 µg/kg honey (percentage of samples)
	p-dichlorobenzene		42 (46.7%)	17 (18.9%)
	naphthalene p-dichlorobenzene or F o r	71 (78.9%) 76 (84.4%)	5 (5.6%) 23 (25.6%)
	naphthalene	P
		e
			e r
			R
			e
			v i e
				w
				O n l
				y
			4

Table III .

 III Detection results of p-dichlorobenzene and naphthalene in honey samples from various botanical origins.

		p-dichlorobenzene	naphthalene
			Number of		Number of
			samples with		samples with
	Botanical origin	Detection	concentration	Detection	concentration
	(number of samples)	number	> 10 µg/kg	number	> 10 µg/kg
		(percentage)	honey	(percentage)	honey
			(percentage of		(percentage of
			samples)		samples)
	Group A
	thyme (28)	21 (75.0%)	9 (32.1%)	27 (96.4%)	4 (14.3%)
	cotton (7)	6 (85.7%)	2 (28.6%)	7 (100.0%)	0 (0.0%)
	pine (5)	4 (80.0%)	1 (20.0%)	2 (40.0%)	0 (0.0%)

Sum (40) 31 (77.5%) 12 (30%) 36 (90.0%) 4 (10%)

					Page 20 of 20
	Group B
	citrus (33)	7 (21.2%)	3 (9.1%)	30 (90.9%)	1 (0.3%)
	fir (7)	1 (14.3%)	1 (14.3%)	1 (14.3%)	0 (0.0%)
	heather (4)	1 (25.0%)	0 (0.0%)	2 (50.0%)	0 (0.0%)
	chestnut (3)	1 (33.3%)	1 (33.3%)	1 (33.3%)	0 (0.0%)
	eucalyptus (3)	1 (33.3%)	0 (0.0%)	1 (33.3%)	0 (0.0%)
	Sum (50)	11 (22.0%)	5 (10.0%)	35 (70.0%)	1 (2.0%)

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