Marianne Caldow
email: m.caldow@csl.gov.uk

Richard John Fussell

Frankie Smith

Matthew Sharman

Development and validation of a method for the analysis of total amitraz in fruit and honey with quantification by gas chromatography-mass spectrometry

Keywords: amitraz, 2, 4-dimethylaniline, gas chromatography-mass spectrometry (GC-MS), hydrolysis

Development and validation of a method for the analysis of total amitraz in fruit and honey with quantification by gas chromatography-mass spectrometry

Introduction

Amitraz is a non-systemic acaricide and insecticide, with contact and respiratory action. Prior to the partial withdrawal of amitraz-based products, on 12 August 2004 (Annex 1, Council Directive 91/141/EEC), its uses included control of all the growth stages of mites, insects, flies and aphids in a wide variety of crops including pome/citrus/stone/bush/soft fruit, curcubits, capsicums and tomatoes. It was also used as an animal ectoparasiticide to control ticks, mites and lice on domestic and farm animals (The Pesticides Manual, 2003). Following this date the use of amitraz is still permitted on pear trees, after harvest, in The Netherlands, UK and Portugal until 30 June 2007. In apiculture amitraz is used to control the honeybee parasite Varroa destructor [START_REF] Korta | Study of Acaracide Stability in Honey, Characterization of Amitraz Degradation Products in Honey and Beeswax[END_REF].

The MRL definition for amitraz is 'the sum of amitraz plus all its metabolites containing the 2,4 aniline moiety'. In plants amitraz is rapidly degraded to N-(2,4dimethylphenyl)-N'-methylformamidine and 2,4-dimethylformanilide (The Pesticides Manual 2003). Methods that are based solely on the analysis of parent amitraz could potentially underestimate the concentration of total amitraz in the sample. The hydrolysis pathway of amitraz (figure 1) [START_REF] Pierpoint | Kinetics and Mechanism of Amitraz Hydrolysis[END_REF] shows that amitraz is readily hydrolysed (under acidic conditions) to 2,4-dimethylphenyl formamide, which can be rapidly hydrolysed to 2,4-dimethylaniline under alkaline conditions. It is commonly accepted that 2,4-dimethylaniline is the stable end-point of amitraz degradation [START_REF] Taccheo | Determination of Total Amitraz Residue in Honey by Electron Capture Capillary Gas Chromatography -A Simplified Method[END_REF]. Methods of analysis should therefore include a hydrolysis step to meet the MRL definition.

[Insert Figure 1 about here] Existing methods for the analysis of amitraz are time-consuming and do not always include the analysis of the metabolites or provide satisfactory confirmation of the analyte. For example [START_REF] Muíño | Gas Chromatographic-Mass Spectrometric Method for the Simultaneous Determination of Amitraz, Bromopropylate, Coumaphos, Cymiazole and Fluvalinate Residues in Honey[END_REF][START_REF] Muíño | Gas Chromatographic-Mass Spectrometric Method for the Simultaneous Determination of Amitraz, Bromopropylate, Coumaphos, Cymiazole and Fluvalinate Residues in Honey[END_REF][START_REF] Maver | Coumaphos and amitraz residues in Slovenian honey[END_REF]Poklukar 2003 both developed methods for the analysis of the parent analyte only in honey using gas chromatography-mass spectrometry (GC-MS) detection. [START_REF] Hornish | Total Residue Analysis of Amitraz [1,5-Bis(2,4-dimethylphenyl)-3-methyl-1,3,5triazapenta-1,4-diene] Residues in Fruit and Soil Samples by Electron Capture Gas Chromatography[END_REF][START_REF] Taccheo | Determination of Total Amitraz Residue in Honey by Electron Capture Capillary Gas Chromatography -A Simplified Method[END_REF][START_REF] Jiménez | Factors affecting the extraction, hydrolysis and derivatization steps for the quantitation of total residues of amitraz in honey by gas chromatography with electron capture detection[END_REF] all developed methods involving steam distillation and/or hydrolysis with a derivitisation prior to detection using an electroncapture detector (ECD).

The aim of this work was to develop a rapid method which would allow the quantification of 'total' amitraz and provide confirmation of the identify of 2,4dimethylaniline in representative matrices including those for which there is still permitted use, to ensure compliance with recent legislation.

Material and methods

Chemicals

Hydrochloric acid (laboratory reagent grade), sodium hydroxide pellets (analytical reagent grade), sodium chloride (analytical reagent grade) and 2,2,4-trimethylpentane (iso -octane), methanol and acetonitrile (all HPLC grade) were obtained from Fischer Scientific (Loughborough, UK). A standard of amitraz (96.4 % purity) was obtained from Sigma-Aldrich (Dorset, UK). 2,4-Dimethylaniline (99 % purity) was obtained from Greyhound Chromatography and Allied Chemicals (Birkenhead, UK).

Materials

Representative matrices: strawberries, pears and oranges (labelled as organically produced) were purchased from local retailers and used in both the method development and validation studies. Honey (Yorkshire Blossom) was supplied by the National Bee Unit (Central Science Laboratory, UK).

Preparation of standard solutions

A stock solution of 2,4-dimethylaniline was prepared in methanol and stored at -20 °C. Intermediate dilutions were prepared in 2,2,4-trimethylpentane and/or methanol and stored at +4 °C.

A stock solution of amitraz was prepared in acetonitrile -it is unstable and degrades rapidly in methanol [START_REF] Pierpoint | Kinetics and Mechanism of Amitraz Hydrolysis[END_REF][START_REF] Bernal | Influence of solvent and storage conditions on the stability of acaricide standard stock solutions[END_REF]). The stock standard solution was stored at -20 °C. Intermediate dilutions were prepared in 2,2,4trimethylpentane and stored at +4 °C.

Sample Preparation

Samples of pears, oranges and strawberries were frozen and comminuted cryogenically in the presence of dry ice (frozen carbon dioxide); sub-samples were stored at -20 °C until required. The sample of honey was well mixed and stored at room temperature. Extraction Sub-samples (20 g) were weighed into Schott bottles (with a septum to seal) and heated at 80 °C for 1 hour in the presence of 100 ml of 0.5 M hydrochloric acid in a water bath, the contents were swirled intermittently. The extract was cooled using a cooling waterbath (10 °C), and the pH adjusted to 11-12 with 5 M sodium hydroxide using a pH meter. The alkaline extract was heated at 80 °C for 1 hour in a water bath; the contents were again swirled intermittently. The extract was cooled using a cooling waterbath (10 °C) and sodium chloride (50 g) was added. The mixture was shaken vigorously until saturation was achieved to allow optimal partitioning of the 2,4-dimethylaniline into the 2,2,4-trimethylpentane (20 ml, shake for 30 seconds, centrifuge at 2350 g for 5 minutes). The (upper) 2,2,4-trimethylpentane layer was removed and an aliquot retained for analysis by GC-MS. A second volume of 2,2,4trimethylpentane was added to the hydrolysed extract (20 ml) and vigorously shaken (30 seconds), followed by centrifugation (2350 g for 5 minutes). Again an aliquot of the supernatant was retained for analysis by GC-MS. The supernatants were analysed separately. [Note for screening purposes only one partition is required. To increase the percentage recovery a second partition can be undertaken and the extracts pooled.]

GC-MS analysis

Quantification was performed using an Agilent 6890 gas chromatograph connected to a 5973 Agilent Mass Selective Detector operated in selected ion monitoring mode (SIM). The injection (2 µl) was splitless at 250 °C and the detector temperature was set at 280 °C. A fused silica capillary column; Rtx5-MS (stationary phase 5% diphenyl/95% dimethyl polysiloxane stationary phase, 30 m x 0.25 mm i.d. x 0.25 µm film thickness) with the carrier gas (helium) set at 0.9 ml minute -1 (constant flow) was [Insert Figure 2 about here]

Calibration Curves

The calibration curves obtained for 2,4-dimethylaniline, using matrix-matched standards and non-weighted linear regression, were linear across the range 0.002 -0.5 µg ml -1 (figure 3) with correlation coefficients >0.990 for all commodities (table 1), with a signal to noise ratio at 0.002 µg/ml -1 (equivalent to 0.002 mg kg -1 2,4dimethylaniline with a final volume of 20 ml) of >10:1.

[insert Figure 3 about here] Calculations [START_REF] Taccheo | Determination of Total Amitraz Residue in Honey by Electron Capture Capillary Gas Chromatography -A Simplified Method[END_REF] To express the results as amitraz a conversion factor must be applied; the conversion factor between amitraz (molecular weight 293 Da) and 2,4-dimethylaniline (molecular 1 mg kg -1 amitraz = 0.826 mg kg -1 2,4-dimethylaniline. To comply with EU guidelines (SANCO/10232/2006), the validation of amitraz in each matrix incorporated the analysis of five replicate recoveries at two spiking levels (the reporting limit (RL) and five times the RL), and included duplicate analysis of blank material. The EU MRL's are variable (0.05 -0.5 mg kg -1 ) depending on the commodity. In-house validation experiments were undertaken at 0.02 mg kg -1 (equivalent to 0.0165 mg kg -1 2,4-dimethylaniline) and 0.1 mg kg -1 amitraz (equivalent to 0.0826 mg kg -1 2,4-dimethylaniline) for each commodity (pears, oranges, strawberries and honey). A summary of the validation results are shown in Table 1. Mean recoveries obtained using a single partition were in the range 55 -73 % with % CV's in the range 4.4 -10.9. When both partitions were taken into account the mean recoveries were in the range 66 -96 % with % CV's in the range 3.3 -9.4.

Results and Discussion

Jiménez

Indications were that a third partition would not significantly improve the recovery of the analyte obtained.

[insert Table 1 Mean recoveries of 55 -73 %, obtained using a single partition, are considered sufficient to permit cost effective screening of samples. The second partition provides an additional recovery of approximately 10 -20 %. Although in this case these data were measured at/or around the limit of detection and may have some error associated with it, during actual analysis this problem would not exist, as the two supernatants would be combined. Combination of the two supernatants would give a final volume of 40 ml and accordingly the limit of detection would be decreased two-fold. As the sensitivity of the method is excellent this would have no consequence on the detection of the lowest calibrated level used which was 0.002 µg ml -1 , which gives a signal to noise ratio > 10:1. No interferences were observed during the analysis of blank sample material (figure 2).

Current analysis methods, involving steam distillation and/or derivitization with heptafluorobutyric acid to enable detection by GC-ECD, are time-consuming. The hydrolysis method developed and validated here is much simpler than existing methods and is applicable to different matrices, as demonstrated. It is possible for an analyst to analyse 30 samples per day using the rapid screening method and 20 samples per day if a double liquid/liquid partition is used for confirmatory quantitative analysis.

  . The typical oven programme was as follows: initial temperature 50 °C, held for 1.5 minutes, programmed to 120 °C at 20 °C minute -1 , then programmed to 200 °C at 15 °C minute -1 , then finally programmed to 280 °C at 40 °C minute -1 (held for 5 minutes). The total run time was 17.33 minutes. The ions monitored for 2,4-dimethylaniline were m/z 106, 120, 121 (figure2) and for amitraz m/ z 121, 132, 162, 293. Quantification was undertaken using m/z 106. The identity of 2,4dimethylaniline was confirmed using the criteria specified in the Document N o SANCO/10232/2006 document 'Quality Control Procedures for Pesticide Residues Analysis': confirmation of identity for the principal ions are within 70 -130 % of those obtained for the standard and data is available for three ions with m/z >100.

  ) must be taken into account. Since one mole of amitraz produces two moles of 2,4-dimethylaniline, then:

  et al 2002 recommended an extraction pH of 11 for the analysis of total amitraz in honey, to ensure complete extraction of 2,4-dimethylaniline. Initial experiments conducted in 0.5 M NaOH (aq) showed the complete conversion of amitraz to the 2,4-dimethylaniline under alkaline conditions. However in the presence of matrix it was necessary to also include an acidic hydrolysis stage to ensure complete conversion occurred. During validation, the absence of amitraz parent in the final extract was used as an indication that complete conversion to 2,4dimethylaniline had occurred during the two-stage hydrolysis. An internal standard was not included in the extraction as this would give no indication to the degree of conversion achieved. 4-chloro-2-methylaniline would be a suitable internal standard to correct for volumetric injection errors.Initial experiments conducted in 0.5 M NaOH (aq) showed that 2,4-dimethylaniline in hydrolysed alkaline extract could be successfully retained on a solid phase extraction (SPE) (Chromabond HR-P cartridges (3 ml, 200 mg, MN New Application Database), and then eluted with acetonitrile/methanol, 1/1, v/v prior to quantification by HPLC-MS. However due to the high viscosity of the acid-hydrolysed sample extracts the use of SPE cartridges was not practicable even with pre-filtration of the hydrolysate , celite or depth filters and the use of positive pressure the sample extracts could not be loaded onto the column within a reasonable time.A liquid/liquid partition of the 2,4-dimethylaniline into 2,2,4-trimethylpentane from the aqueous phase (saturated with sodium chloride) negated the necessity for the SPE clean-up. No concentration of the extract was required prior to quantification by GC-MS.

Figure 3 .

 3 Figure 1. Hydrolysis pathway of amitraz

F

Table 1 .

 1 Mean validation results (n=5) for amitraz measured as 2,4-dimethylaniline

	Matrix type	Correlation spiking level	Mean (%)/ % CV
		coefficient	mg kg -1	partition 1 partition 2	total
	pear	0.991	0.02 0.1	61 (8.2) 61 (6.2)	10 (22.4) 71 (6.1) 15 (11.8) 77 (7.4)
	strawberry	0.996	0.02 0.1	61 (4.4) 64 (5.7)	9 (14.8) 10 (13.3) 73 (5.2) 71 (3.3)
	orange	0.998	0.02 0.1	73 (4.5) 63 (10.9)	23 (8.4) 13 (8.3)	96 (4.7) 77 (9.4)
	honey	0.997	0.02 0.1	60 (5.3) 55 (7.3)	12 (0.0) 11 (11.1) 66 (7.4) 72 (3.7)
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http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.ukFood Additives and Contaminants

Acknowledgements

We gratefully acknowledge Defra (Department for Environmental Food and Rural Affairs) for funding and M. Hetmanski for helpful discussions.


Sonia Lucía Blanco

Cristina Sobrado

Corina Porro

Santiago Cabaleiro

José Carlos González

Juan Manuel Vieites

Corina Quintela

Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Spanish aquacultured turbot (Psetta maxima)

Abstract

The human population is exposed to dioxins (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) mainly through diet; bioaccumulation and biomagnification in aquatic environment results in fishery products and by-products being an important vector to humans. The determination of PCDD/Fs and dl-PCBs in fillets of young turbots (Psetta maxima) (0 -2 years) from aquaculture plant (Galicia, Spain) (N = 21) and in feedingstuffs were carried out, and dietary accumulation values and lipidnormalized biomagnification factors (BMF) relating concentration in fish and in feed were calculated. Levels found in feedingstuffs (0.5 pg TEQ-PCDD/F / g and 1.6 pg TEQ-dl-PCB / g), and turbots (0.13 -0.27 pg TEQ-PCDD/F / g fresh weight and 0.35 -1.2 pg TEQ-dl-PCB / g fresh weight) were below maximum permitted levels set by EC. Levels of toxic compounds in feeding stuff are reflected in fish fillets; predominant isomers are 2,3,7,8-TCDF, OCDD,1,2,3,7,2,3,4,7,2,3,4,6,7,and PCBs 118,105,156 and 167. Relevant compounds accounting for total toxicity are the same congeners in feeding stuff and turbots: 2, 3,4,7,2,3,7,2,3,7,2,3,7,and PCB 126. Higher accumulation efficiency values were obtained for dl-PCBs (30 -46%); tetra-and penta-chloro substituted PCDD/Fs showed the highest values (27 -34%) of the PCDD/F group. Biomagnification was shown for these compounds (BMF around 1.5).

Introduction

Turbot aquaculture is a very important activity in Spain, producing fish of remarkable quality due to optimal conditions of sea water of the northwest of the country.

According to the United Nations Food and Agriculture Organization (FAO), by the year 2030 more than half of marine products for human consumption will come from aquaculture. Aquaculture is the only way to keep sea products in the daily diet in view of the stagnation of extractive fishing. In this sector, Galicia, in the northwest of Spain, is a world leader thanks to its natural conditions, especially its "rias", and the commitment undertaken by Galicia's companies and Administration. In the year 2002, Galician aquaculture attained an estimated production of more than 267,000 tons, dominating the Spanish market as one of the main suppliers of European markets [START_REF] Anonymous | Fishing -Aquaculture: the future of the sector. Xunta de Galicia[END_REF]. Galician aquaculture experienced continuous growth, in both the volume of its production and first-sale value, between 1994 and 2002, increasing from a value of 93.65 million euros to a total of 193.79 million, turbot representing 14.8% of this economical value [START_REF] Anonymous | Fishing -Aquaculture: the future of the sector. Xunta de Galicia[END_REF]. Many technical advances have been made in breeding turbot in Galicia, the European region where turbot growing is completed in the least time, thanks to the richness and temperature of its waters; at present, there are 17 farms in Galicia, including the largest in the world, in Lira, Carnota (A Coruña).

Feeding stuffs used in cultured fish are made mainly from fish oil and fish meal, being fish fat one of the main components where persistent contaminants such as dioxins and PCBs are accumulated, due to bioaccumulation and biomagnification in the aquatic environment. Due to aerial deposition and diffuse contamination of soil, these contaminants are found in roughages and other types of animal feed of vegetable origin. Contaminated surface waters, sediments and prey lead to dioxins and dl-PCBs in fish, and thus to contamination of fish meal and fish oils used as animal feed. Fishery products and by-products might constitute an important vector of dioxins in humans.

Due to the high toxicity of these compounds, particularly chlorinated dioxins and furans Regulation No. 199/2006 (food).

In order to evaluate the safety of this aquaculture product and the relevance of the feeding stuffs in the final levels of contaminants found in fish, determinations of PCDD/Fs and dl-PCBs in turbots and in feeding stuffs have been carried out in this study.

Materials and methods

Sampling

Two different feedingstuffs were used to feed the turbots: 8 mm diameter (fish younger than 1 year), and 15 mm diameter (fish older than 1 year). Both feedingstuffs showed similar proximal composition: about 20% fat, 53% protein, and 8% moisture. Both feedingstuffs contained a proportion of marine fish oil (data not supplied). Fish of three different age/size groups (from 0 to 2 years old) of turbots (N=21) (Table 1) were pooled from aquaculture plant in March 2005. Fish were frozen until analysis.

Determinations of PCDD/Fs and dl-PCBs in fish individual samples (N=21) and in feedingstuffs used with them were carried out; analyses were performed in April 2005 -June 2005. Fat content was determined by Soxhlet extraction procedure (AOAC 1990).

Dietary accumulation efficiencies were calculated using the equation α = C fish / (F t C feed ), where C fish is concentration in fish, C feed is concentration in feed, F is feeding rate and t is time, for each toxic congener in the turbots of 1-2 years old under study.

Feed conversion ratio (Kg food consumed / kg weight gain) of 1.1 was applied to calculate the consumed quantity by each individual. This value is applied under conventional turbot aquaculture conditions in the region, and represents the total consumption by a total mass of fish.

Sample pre-treatment

Frozen fish were allowed to thaw (a few hours) and edible parts (fillets) were divided into small pieces and homogenized with a food processor. Fish samples were previously dried for 24 h using sodium sulphate. Feeding stuffs were grinded and directly processed. Sub-samples of 20 g were taken for analysis.

For sample preparation and purification, open adsorption chromatography columns and

Supelco Dioxin Prep System were used, including empty tubes for multi-layer silica gel columns, vacuum manifold, and accessory glassware and connectors. Preparation for PCDD/Fs analysis was carried out as described by Sobrado et al. (2004). Sub-samples obtained as described above were previously homogenized, next, they were spiked with known amounts of mixtures of 13 C 12 -PCDD/Fs and 13 C 12 -dioxin-like PCBs (EPA-1613LCS and WP-LCS WHO/EPA from Wellington Laboratories) and extracted using adsorption chromatography with n-hexane:acetone (1:1). The extracts were rotary evaporated and transferred to 5 mL n-hexane. In order to remove organic components, fat and other interfering substances, the n-hexane extracts were passed through chromatographic columns packed with silica and acid silica, modified with sulphuric acid, using n-hexane as eluent. After solvent evaporation to 5 mL, the extract is cleanedup in a multilayer column, packed with silica, acid silica modified with sulphuric acid and basic silica modified with potassium hydroxide, eluted with n-hexane, rotary evaporated to 1 mL, and solid phase-extracted using carbon cartridge Supelclean-Envicarb, from Supelco. Dioxins and furans are retained in carbon structure while interferences are eluted by a direct flow of n-hexane:toluene (99:1 and 75:25 alternatively) and then eluted by reversed flow of toluene. Extract is evaporated to dryness and re-dissolved in 5 µL of nonane and 5 µL of labelled internal standard solution EPA-1613ISS (Wellington Laboratories), prior to injection.

Preparation for dl-PCBs analysis was carried out using adsorption chromatography, following a similar procedure, but introducing the use of Supelco Prep System, by one step method as described by [START_REF] Maeoka | Quick Pretreatment Prep Study for Dioxin Analysis[END_REF]. This one-step method consists of a multi-layer silica gel column, as described above for dioxins and furans, directly coupled to a dual layer carbon column composed of two different carbon layers in series with distinct binding characteristics, Carboxen 1016 and Carboxen 1000, from Supelco, that retains dioxins and co-planar PCBs which are eluted by reversed flow of toluene.

Extract is evaporated to dryness and re-dissolved in 38 µL of nonane and 2 µL of labelled internal standard solution WP-ISS WHO/EPA (Wellington Laboratories), prior to injection. (hold 0 min), 20ºC min -1 , 300ºC (hold 9 min). Detection was performed with an ion trap mass spectrometer Varian 4000 GC/MS, based on the pattern of fragmentation of the congeners by MS/MS. Quantification was based on the isotope dilution method; calibration was previously performed using native and labelled dioxins/furans solutions from Wellington Laboratories (EPA-1613CVS, CS1-CS-5) and dl-PCBs (WP-CVS WHO/EPA, CS1-CS7) .

High resolution mass spectrometry (HRGC-HRMS) operating in electron ionization (EI) mode and at a resolving power of 10,000 was also applied as confirmation method for PCDD/Fs and dl-PCBs since the values for some congeners obtained in many of the samples were below our detection limits; monitored masses in SIR mode were M and M+2 or M+4.

Quality

HRGC-MS/MS

Identity was confirmed by comparing retention time between native and labelled analogue isomers and the ion ratio is fulfilled between M and M+2 considering an interval ±25% of the theoretical ion ratio. Mass spectra similarity of sample and standard peaks was checked.

The instrumental limit of detection established for each compound was estimated as the concentration providing a signal-to-noise ratio of 3:1. On a fresh-weight basis, the limits of detection were 0.05-0.2 pg/g for tetra-and penta-congeners PCDD/Fs, 0.2-1 pg/g for hexa-, hepta-and octa-congeners PCDD/Fs, and 0.3-1.5 pg/g for dl-PCBs. The limits of detection were 0.01-0.02 pg/g for tetra-, penta-, hexa-and heptachlorinated congeners PCDD/Fs, 0.10-0.15 pg/g for octa-chlorinated congeners PCDD/Fs.

HRGC-HRMS

Results and Discussion

PCDD/Fs and dl-PCBs in Feeding stuffs

Results for the two different feedingstuffs used to feed turbots, 8 mm diameter and 15 mm diameter, are shown figures 1 -2.

[ Insert Figure 1 about here ] [ Insert Figure 2 about here ] Although most abundant isomers in both feeds are 2,3,7,8-TCDF, followed by OCDD and 2,3,4,7,8-PCDF, and PCBs 118, 105, 156 and 167, most contributing to total TEQ are 2,3,4,7,8-PCDF, 2,3,7,8-TCDF, 2,3,7,8-TCDD and 1,2,3,7,8-PCDD, and PCBs 126, 118, 156 and 105. Relatively low levels of PCDD/Fs and dl-PCBs were found in the feeding stuffs (0.52 pg TEQ/g PCDD/Fs and 1.62 pg TEQ/g dl-PCBs), far below maximum levels set by the European Commission (2.25 pg/g TEQ-WHO PCDD/Fs, relative to a moisture content of 12%, and 4.75 pg/g dl-PCBs), and below the action limit (1.75 pg/g TEQ-WHO PCDD/Fs and 3.5 pg/g dl-PCBs) stated by Directive 2006/13/EC. The most abundant contaminants in the feedingstuff are the most abundant in the fish, these are: 2,3,7,8-TCDF, OCDD,1,2,3,7,2,3,4,7,2,3,4,6,7,and PCBs 118,105,156 and 167. The most relevant compounds accounting for the total toxicity in turbots are 2,3,4,7,8-PeCDF, 2,3,7,8-TCDF, 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD, and PCB 126 (figures 3 -4), the same congeners accounting for the total toxicity in feeding stuff. As reported by other authors in salmon [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF], the concentration of dioxins and dl-PCBs in turbots reflect the levels present in the feed.

PCDD/Fs and dl-PCBs in fish

TEQ PCDD/Fs and dl-PCBs in turbots analysed were below the maximum permitted levels (4 pg/g fresh weight TEQ-WHO PCDD/F and 4 pg/g fresh weight TEQ-WHO PCBs, Regulation EC No. 199/2006) and below the action limit (3 pg/g fresh weight TEQ-WHO PCDD/F and 3 pg/g fresh weight TEQ-WHO PCBs, Recommendation 2006/88/EC).

Other authors have reported similar results in Spanish farmed fish, showing low levels of PCDD/Fs in general, with a pattern characterized by the presence of toxic TCDF, TCDD, PeCDD and PeCDFs (Abad et al. 2003), in trout, sea bass, gilthead and turbot.

Trouts from French aquaculture showed very similar values (Marchand et al. 2004) for the same parameters, with mean values of 0.17 and 0.58 pg/g fresh weight for WHO-TEQ PCDD/Fs and dl-PCBs respectively. Other farmed fish, such as salmon, has been widely analyzed in many European countries for these contaminants, showing higher values of PCDD/Fs and dl-PCBs than wild salmon, associated with the use of fish oil [START_REF] Karl | Dioxins, dioxin-like PCBs and organochlorine pesticides in farmed salmon of various origins[END_REF]. Levels obtained in salmon are usually higher than values in turbots obtained in this paper, expressed on a wet weight basis, as reported by many authors [START_REF] Jacobs | Investigation of polychlorinated dibenzo-pdioxins, dibenzo-p-furans and selected coplanar biphenyls in Scottish farmed Atlantic salmon (Salmo salar)[END_REF][START_REF] Karl | Dioxins, dioxin-like PCBs and organochlorine pesticides in farmed salmon of various origins[END_REF]).

Dietary uptake

Correlation coefficients (Pearson) for TEQ levels and fat content were calculated (SPSS 12.0 for Windows, SPSS Inc., 1989-2003), and values obtained showed a significant correlation at the 0.05 level (2-tailed) for tetra-and penta-chlorinated PCDD/F congeners and hexa-and hepta-chlorinated dioxins 1,2,3,6,7,8-HxCDD and 1,2,3,4,6,7,8-HpCDD as well as for total TEQ (0.5 < R < 0.6). The 12 dl-PCBs show a significant correlation at the 0.05 level (2-tailed) between mentioned parameters (0.5 < 2. Accumulation efficiency values around 30-46% for the 12 dl-PCBs and 27-34% for PCDD/Fs tetra-and penta-chloro substituted eaten by turbots ended up in the fish fillets. Tetra-and penta-chlorinated PCDD/F congeners show a mean accumulation of 31%, while higher chlorinated PCDD/F congeners show lower efficiencies (< 22 %). Total WHO-TEQ accumulation efficiencies were higher for dl-PCBs than for TCDD/Fs and around 35% of the total ingested TEQ dl-PCBs remain in the organism, versus 30% of TEQ PCDD/Fs.

[ Insert Table 2 about here ] These data show that dioxin-like PCBs accumulate more efficiently than PCDD/Fs in the fillets of farmed turbots. Similarly, Isosaari et al. (2002) reported higher accumulation efficiency for dl-PCBs in rainbow trout (Oncorhynchus mykiss) and [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF] reported higher retention of dl-PCBs than for PCDD/Fs in cultured Atlantic salmon (Salmo salar). The high tendency of the toxic PCB congeners to be retained in fish tissues might explain why PCBs have a high contribution to the total WHO-TEQ in fish tissues. [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF] in salmon were higher both for PCDD/Fs and PCBs: 57-64% of the total TEQ PCDD/Fs and 86-94% TEQ dl-PCBs.

Accumulation values reported by

These values have been obtained from whole fish,adult salmon, while ours correspond to the edible portion of fish, the fillets, of young turbots up to 2 years old; we have to consider that maturation takes place at the age of 5 years (natural environment), but aquaculture industry usually produce turbots of around 2 Kg (2-3 years old). According to data from these authors, approximately 30% of both pollutants (WHO-TEQ) found in fish were located in skinned fillet [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF], which is concordant with accumulation efficiency values obtained for turbot in this study.

Log K ow are in the range 6.6 -8.0 for tetra-to octa-substituted PCDD/F congeners, and increase with increasing number of chlorine atoms (Rittler et al., 1996). Correlation between log K ow and bioaccumulation of several hydrophobic compounds shows that the highest bioconcentration factors of non-ionic compounds and the longest half-lives of several hydrophobic organochlorine compounds occur at log K ow values of approximately seven (Fisk et al., 1998;Meylan et al., 1999). This is concordant with the higher accumulation mean values in tetra-and penta-substituted congeners (27.6 -34.0%) obtained in present study.

Log K ow for PCBs indicate the potential for bioaccumulation varies between different congeners with the more chlorinated PCBs having a much higher potential for bioaccumulation than less-chlorinated congeners (5.6 -6.5 for tetra-, 6.2 -6.5 for penta-, 6.7 -7.3 for hexa-chlorobiphenyl isomers) (Mackey et al., 1992;Ritter et al., 1996), although our results do not support this observation, tetra-substituted congeners (77 and 81) showing similar accumulation values to higher chlorinated PCBs (see Table 2). [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF] have obtained preferential accumulation of tetrachlorinated biphenyls in salmon, finding no significant differences in the accumulation efficiencies among PCBs with five to eight chlorines.

Biomagnification factors

Lipid-normalized biomagnification factors (BMFs) were calculated and results are shown in Table 2. Congener-specific differences in biomagnification factor were similar to the trends observed in accumulation efficiencies, which is concordant because both parameters were based on the same fish fillets concentrations. Biomagnification (> 1) was shown for tetra-and pentachlorinated PCDD/Fs (mean BMF 1.5), but not for all the higher-chlorinated congeners. Biomagnification factors were similar for all dl-PCB congeners, with the same mean of 1.5 as for tetra-and pentachlorinated PCDD/Fs, and all the measured PCB congeners biomagnified, except PCB 169. Similar results have been obtained for whole salmon [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF]), although higher BMF for all dl-PCB congeners than for PCDD/Fs were obtained by these authors, who have described that the lipid-normalized concentration ratios of salmon fillet to whole fish showed an equal partitioning of these contaminants.

Dietary intakes of PCDD/Fs and dl-PCBs recently calculated in Spanish population (Fernández et al. 2004) show that fish and seafood accounted for 11% of the intake, being dl-PCBs an important component in the total WHO-TEQ, up to 80% of this value in fish food. Considering the more efficient accumulation of PCBs in fish, that might explain the high contribution to the total TEQ in fish tissues [START_REF] Lundebye | Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Atlantic salmon (Salmo salar)[END_REF], and the relatively important contribution of fish to dietary intake, it will be necessary to ensure the use of uncontaminated fish feed in aquaculture industry.

Conclusions

These preliminary results for young turbots may be helpful to approach the pattern of dioxins and related contaminants retention from the diet in aquaculture fish, showing that the levels of toxic compounds in feed are reflected in fish fillets and biomagnification for relevant compounds accounting for total toxicity takes place, the dl-PCBs being more efficiently accumulated. Considering the biomagnification of the most toxic PCDD/Fs and dl-PCBs and the more efficient accumulation of PCBs in fish, as well as the relatively important contribution of fish to dietary intake of total WHO-TEQ, it will be necessary to ensure the use of uncontaminated fish feed in aquaculture industry. The inclusion of dl-PCBs in this study provides valuable information for forthcoming risk assessments and contributes to establish a relationship between dioxins and similar contaminants in farmed fish meat and diet, to help assure consumer confidence on farmed fish. Official Journal of the European Communities L32, 4.2.2006, pp. 44-53. Anonymous, 2006, Comission L42, 14.2.2006, pp. 26-28. AOAC 1990. Helrich, K. (editor)

(

  PCDD/Fs) and dioxin-like PCBs (dl-PCBs), it has been necessary to establish the maximum permitted level (MPL): Directive 2002/32/EC establishes the maximum dioxins and furans content in feeding stuffs, and Regulation (EC) No. 2375/2001 in food. Recently established MPLs for the sum of dioxins/furans and dioxin-like PCBs in Directive 2006/13/EC (feeding stuffs) and Commission

  Solvent concentration was carried out in a Laborota 4000 Heidolph connected to a Büchi V-500 vacuum pump. Solvent evaporation was carried out under nitrogen in a Reacti-Vap evaporator 18780 coupled to a Reacti-Therm heating module 18790, Pierce. Organic solvents were for residue analysis; sodium sulphate granular was 99+% A.C.S. reagent (Aldrich); silica gel 60 (0.063-0.200 mm) was for column chromatography (Merck).Determination of PCDD/Fs and dl-PCBsPCDD/Fs and dl-PCBs were determined using high resolution gas chromatography coupled to an ion trap tandem mass spectrometer (HRGC-MS/MS). Gas chromatograph Varian CP-3800, equipped with an autosampler Varian CP-8400, a VF-5ms capillary column (Factor Four, 60m x 0.25 mm ID, DF = 0.25, Varian); for PCDD/Fs separation, 1 µL of sample is injected in splitless mode at 300º C, followed by column temperature program 90º C (hold 2 min), 20º C min -1 , 200º C (hold 1.3 min), 1º C min -1 , 230º C (7 min), 10º C min -1 , 300º C (hold 20 min); for PCBs separation, injection of 10 µL sample in LVI (large volume injection) mode at 95º C (hold 0.5 min), applying injector temperature program of 100º C min -1 , 300ºC (hold 12.55 min), and column temperature program of 60ºC (hold 3 min), 20ºC min -1 , 235ºC (hold 10 min), 10ºC min -1 , 260ºC

  All PCDD/F and dl-PCB data were assessed for compliance with the methods performance criteria guidelines laid down in EU Commission Directives 2002/69/EC and 2002/70/EC. Dioxin, furans and dioxin-like PCBs were quantified by isotopic dilution, based on the linearity of the detector signal of each isomer. The stability of the relative response factor has been checked during successive calibrations, supporting the stability and robustness of the methods. Recoveries for each labelled congener have been studied in diverse matrix samples. Recovery average values are 60-70%, ranging from 50 to 85%; it is quite constant among congeners and also among tested sample matrices. Repeatability and reproducibility were checked, using MS/MS method and HRMS as confirmation method. Certified Reference Materials have been used during the 2004-2006 period (Carp muscle CARP-2, National Research Council Canada NRC-CNRC, Cod liver oil FAPAS test material, Central Science Laboratory). Blanks were also checked, and routinely included in each set of analyses.

Figures 3 -

 3 Figures 3 -4 show individual WHO-TEQ for each fish sample.

  mentioned PCDD/Fs congeners and dl-PCBs. The other PCDD/Fs showed very low levels both in the feeding stuff and fish muscle tissue, under the limit of detection. Dietary accumulation efficiencies were calculated for each toxic congener in turbots of 1-2 years old under study. Feed conversion ratio (kg food consumed / kg weight gain) of 1.1 was applied to calculate the consumed quantity by each individual. Dietary accumulation values obtained are shown in Table

  in animal feed as regards dioxins and dioxin-like PCBs.

  , Oficial Methods of Analysis, Vol I and II. Association of Official Analytical Chemists, Arlington, VA. Fernández, M.A., Gomara, B., Bordajandi, L.R., Herrero, L., Abad, E. Abalos, M., Rivera, J., González, M.J., 2004, Dietary intakes of plychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls in Spain. Food Additives and Contaminants, 21, 983-991. Fisk, A.T., Norstrom, R.J., Cymbalisty, C.D., Muir, D.C.G., 1998, Dietary accumulation and depuration of hydrophobic organochlorines: Bioaccumulation parameters and their relationship with the octanol/water partition coefficient. Environ Toxicol Chem, 17, 951-961. Isosaari, P., Vartiainen, T., Hallikainen, A., Ruohonen, K., 2002, Feeding trial on rainbow trout: comparison of dry fish feed and Baltic herring as a source of PCDD/F and PCBs. Chemosphere, 48, 795-804.

  .,Matayron, G., Gade, C., LeBizec, B., Andre, F., 2004, PCDD/F, dioxinlike and markers PCBs in trouts from French aquaculture. OrganohalogenCompounds, 66, 1932-1939. Meylan, W.M., Howard, P.H., Boethling, R.S., Aronson, D., Printup, H., Gouchie, S., 1999, Improved method for estimating bioconcentration/bioaccumulation factor from octanol/water partition coefficient. Environ Toxicol Chem, 18, 664-672.Ritter, L., K.R. Solomon, J. Forget, M. Sternoff and C.O.Leary, 1996, Persistent Organic Pollutants. An Assessment Report on: DDT-Aldrin-Dieldrin-Endrin-Chlordane Heptachlor-Hexachlorobenzene Mirex-Toxaphene Polychlorinated Biphenyls Dioxins and Furans. Prepared for the International Programme on Chemical Safety. Sobrado, C., Quintela, C., Blanco, L., Cabaleiro, S., González, J.C., Vieites, J.M., 2004, Suitability of an ion trap GC/MS/MS method for routine analysis of PCDD/Fs in fishery products and by-products. Organohalogen Compounds, 66, 369-375.

  Figure 1

  Table 1 shows fat and total WHO-TEQ data in each age/size turbot group. [ Insert Table 1 about here ]

  Commission Recommendation of 6 February 2006 on the reduction of the presence of dioxins, furans and PCBs in feedingstuffs and foodstuffs. 2006 / 88 / EC. Official Journal of the European Communities

	Regulation (EC) Nº 199 / 2006 of 3 February 2006
	amending Regulation (EC) No 466 / 2001 setting maximum levels for certain
	contaminants in foodstuffs as regards dioxins and dioxin-like PCBs. Official Journal of
	the European Communities L32, 4.2.2006, pp. 34-38.
	Anonymous, 2006,

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

Acknowledgements

This work was funded with Grant CAL 01-031 from National Institute for Agriculture and Nutrition Research and Technology, Spanish Ministry of Science and Technology (INIA-MCYT). Authors are grateful to the contributions of Ana I. Bernardino and Gerardo Fernández.


Ulla Maria Karlström

Lasse Nuotio

Erja Lindfors

Riitta Maijala

Ulla Karlstrom

Ritta Maijala

Simulation of human exposure to benzylpenicillin residues via pork originating from pigs fed with a dairy by-product

Keywords: Exposure assessment, HPLC, Probabalistic modelling, Risk assessment Additives/Contaminants: Veterinary drug residues -antimicrobials By-product, feed, antimicrobial drug residues, benzylpenicillin, pork, simulation model, exposure assessment, risk assessment, Finland

come

Simulation of human exposure to benzylpenicillin residues via pork originating from pigs fed with a dairy by-product

Simulation of human exposure to benzylpenicillin residues via pork originating from pigs fed with a dairy by-product

Introduction

The food industry produces extensive amounts of various by-products, many of which serve as animal feed. For example, in 2004, the dairy industry in Finland dispensed over 140 000 tons of by-products for feeding, most of which, 82%, consisted of whey and whey concentrate (National Food Administration [NFA] 2005).The new EU regulation on animal by-products (1774/2002) limits the use of such biomaterial as an animal feed, and member states must either ascertain the safety of such products as animal feed, or come up with sustainable solutions on how to dispose of these products (European Union [START_REF] Eu | Laying down health rules concerning animal by-products not intended for human consumption[END_REF]. This precautionary approach is justified, e.g. BSE is generally believed to have spread by the recycling of the proteinaceous material from the carcases of infected cattle into meat and bone meal. This permitted the amplification of what was previously a very rare cattle disease [START_REF] Anderson | Transmission dynamics and epidemiology of BSE in British cattle[END_REF][START_REF] Wilesmith | Bovine spongiform encephalopathy: Epidemiological studies[END_REF]). However, many animal by-products have a high nutritional value and most likely do not pose any significant risks when used as feed. Only a few studies have evaluated the risks imposed by such products. Milk rejected for human consumption is used, after mixing with other ingredients, as feed for finisher pigs. This long-standing practise is at odds with the new EU by-products regulation. In theory, such a practice has the potential to lead to BP residues in pork and indirectly expose consumers to the residues.

A total exposure of as negligible as 3 µg of BP has been reported to elicit allergic reactions, such as rash or urticaria, in individuals previously sensitized to penicillin [START_REF] Borrie | Dermatitis caused by penicillin in bulked milk supplies[END_REF]Barret 1961, Dewdney et al. 1991).

The occurrence of test positive milk loads in Finland is a rare (ca 50 annually) and random event. Timely tracing of the farms receiving the rejected lots and representative sampling of the prepared feed, as well as reliable sampling and determination of the residue level in retail pork, were considered infeasible. The objective of this study was to construct a probabilistic dilution and exposure model for the residue, starting from the rejected milk and arriving to the concentration in pork. The aim was to assess the safety of the long-standing feeding practice to consumers by comparing the modelled probable distribution of BP residue concentration in pork with the appropriate Maximum Residue Limit (MRL) -value (European Community [START_REF] Ec | Council Regulation 2377/90 laying down a community procedure for the establishment of maximum residue limits of veterinary medical products in foodstuffs of animal origin[END_REF]EMEA 2005a). Data about the amounts of milk containing antimicrobial drug residues rejected at dairies and the amount of other dairy by-products mixed with the milk prior to the delivery to the pig farms were obtained from the National Food Agency. The data about the feed processing practices on the farms, consumption data and the duration of the time when milk was used on farms were obtained from the Plant Production Inspection Centre. In addition, laboratory analyses were carried out to obtain information on the BP concentrations in the rejected bulk milk lots. Since no data on the concentration of BP in the final feed mixture were available, a simulation model was developed to estimate the daily exposures of fattening pigs and thereafter the amount of BP residues in pork meat.

Materials and methods

F

Other parameters taken into consideration in the simulation model were the, oral bioavailability and metabolism of BP. Based on literature, benzylpenicillin is not chemically stable at the low pH present in the stomach. The gastric acid hydrolyses the beta-lactam ring and only a minor proportion, 15-30%, of the orally ingested BP dose is bioavailable [START_REF] Plumb | Veterinary drug handbook[END_REF]. Additionally, the half-life of BP in serum is comparatively short, 1.7 hours [START_REF] Luthman | Influence of the local tolerance on the pharmacokinetics of two penicillin G preparations in cattle and swine[END_REF]. These parameters that reduce the possible amount of residues in pork meat were included into the evaluation (see Figure 1).

Chemical analysis

Benzylpenicillin in milk samples was analysed using a multiresidue method detecting also phenoxymethylpenicillin, amoxicillin, ampicillin, oxacillin and cloxacillin. The method was based on the one used for meat products devised by [START_REF] Verdon | Multiresidue analytical method for the determination of eight penicillin antibiotics in muscle tissue by ion-pair reversedphase HPLC after precolumn derivatization[END_REF] and was applied for milk with some modifications.

Briefly, the milk samples were centrifuged at 10 °C for defatting. The sample tubes were kept at -18 °C for 10 min and the fat button was removed with a spatula. The defatted milk samples were transferred with sodium phosphate buffer (3x5 ml; 0.1M at pH 8) into ultracentrifuge tubes, ensuring that all of the precipitate on the bottom was transferred. The pH was adjusted to 8.0-8.1 and the capped tubes were shaken horizontally for 10 min. The samples were clarified by centrifugation (12 000 g for 15 min at 10 °C) and vacuum filtration using glass fibre filters (Millipore). The extracts were further purified with C18 solid phase extraction (SPE). The eluents were derivatized with benzoic acid anhydride and mercuric chloride, diluted with phosphate buffer (1:1; 0.1M pH 8) and analysed with high performance liquid chromatography (HPLC) using a reversed phase column (Hypersil BDS C18 5 µm 250x4 mm Agilent Technologies, Germany) and UV (diode array) detection. The SPE, derivatization and HPLC procedures followed in general the method of [START_REF] Verdon | Multiresidue analytical method for the determination of eight penicillin antibiotics in muscle tissue by ion-pair reversedphase HPLC after precolumn derivatization[END_REF]. Positive samples were confirmed with UV spectrum and penicillinase treatment. The limit of detection (LOD) was calculated on the basis of a 3:1 S/N ratio from blank milk and standard solution (10 µg l -1 ) taking into account correction for the recovery. The limit of quantification (LOQ) was calculated similarly based on a 6:1 S/N ratio. To determine the recovery, blank milk samples were spiked at a level of 10 µg l -1 with six replicates. The measurement uncertainty was 3%.

Construction of the simulation model

The model consisted of five consecutive steps. Three steps described the dilution processes, one step simulated the exposure of fattening pigs and the final step estimated the amount of residues in pork meat and its relation to the MRL-value. The calculations in the five steps were as follows:

Step 1. Residue concentration in dairy silo was calculated by multiplying the respective concentration in truck or trailer (a distribution) with the ratio of the quantity of the milk on truck or trailer (a distribution) to the quantity of milk in silo (a distribution).

Step 2. The concentration in feed was calculated by dividing the concentration in silo (step 1) with the final feed dilution factor (a distribution).

Step 3. Daily exposure of pigs was calculated as the product of concentration in feed (step 2) and quantity of feed fed per day. The total exposure of pigs was calculated as a product of concentration in feed (step 2), quantity of feed fed per day and number of days fed.

Step 4. The maximum amount of residue in 1 kg of pork was calculated as a product of daily exposure of pigs (step 3) and bioavailability, divided by approximate weight of pig carcases. Step 5. The maximum amount of residue in 1 kg of pork (step 4) in relation to the appropriate MRL was calculated as the percentage of the latter.

The input for parameters of the model were given as probability distributions based on observed data points, or as fixed point estimates, i.e. means of the observed data and literature references (see Table 1). The goodness of fit of the observed data with the representative input distributions is given in Table 1.

The model employed Monte-Carlo sampling of the input distributions and the final concentration of the residues in feed was hence also obtained in the form of probability distributions.

[insert Table I about

Statistical procedures

The goodness of fit of the observed data with the employed probabilistic distributions to represent them was tested with Chi square test for the concentration in truck or trailer, and Kolmogorov-Smirnov test for the quantity of milk in truck or trailer, quantity of milk in dairy silo and final feed dilution factor.

The p-value of the latter three was obtained by Smirnov's chi square

Results

BP residues in rejected bulk milk

The LOD and LOQ for benzylpenicillin were determined to be 2 µg l -1 and 4 µg l -1 , respectively. The mean recovery of benzylpenicillin in the recovery study was 113 % and the relative standard deviation was 1.5 %.

The average concentration of BP in the analysed bulk milk samples was 16.9 µg l -1 (median 6.9 µg l -1 , 90% probability interval 2.3-37.9). The distribution of the observed values conformed to Lognormal distribution (@Risk module), with the parameters calculated from the observed data.

[insert Table II about here]

Simulated exposure of pigs to BP residues via feed

On average, each bulk milk lot rejected at the dairy, was diluted 13-fold (median 8.3, 90% probability interval 1.7-40.7) before it was fed to pigs. According to the simulation model, the average BP concentration in feed was 3.1 µg l -1 (median 1.2 µg l -1 , 90% probability interval 0.1-11.9 µg l -1 ). Approximately 80% of the results for the BP residue concentration in feed remained under the MRL of BP (4 µg l -1 ) set for milk for human consumption (EMEA 2005a) (see Figure 2).

[insert Figure 2 about here] According to the model, the average daily exposure of fattening pigs to BP residues was 31.9 µg day -1 (median 12.0 µg day -1 , 90% probability interval 1.2-119.2 µg day -1 ) during the time that they were fed with the feed containing rejected milk. In the current situation, exposure to BP residues at this level continued on average for 3.6 days (maximum of 7 days) resulting in a total exposure to BP residues during the pigs lifetime of an average of 111.0 µg (median 42.9 µg, 90% probability interval 4.5-411 µg).

Possible BP residues in pork

Only a fraction of the ingested BP in feed is absorbed and BP is quickly eliminated from the body, mainly via urine. Prior to its elimination, the average amount of the orally ingested BP absorbed was 9.3 µg day -1 . (median 3.6, 90% probability interval 0.4-34.4 µg day -1 ). As the daily feed proportion is broken down into 3-4 meals, the average amount of BP absorbed during each meal varies between 3.1-2.3 µg/meal. The figure 3 demonstrates a feeding schema where pigs are fed 3 times a day. In case of other feeding schemes, such as four meals per day, the exposure per meal is even lower as the proportions diminish when pigs are fed more often. In view of the rapid elimination, accumulation of BP at such low exposure levels is negligible (see Figure 3). To assess the potential health risk of BP residues in pork for humans a risk estimate was created by comparing the maximum concentration of BP in pork and the MRL of BP set for meat. Residue levels in meat below the MRL are considered to be safe for human consumption (EMEA 2005b).

According to the model, the BP concentration in pork would remain well below the MRL: after 3.5 days of feeding, the average total amount of BP in the pigs immediately after the last feeding would be 9.3 µg (median 0.4, 90% probability interval 3.6-35.8 µg), which would be distributed throughout the body, mainly to stomach and duodenum [START_REF] Mckellar | Penicillin therapy of spontaneous streptococcal meningitis in pigs[END_REF]. Pork was estimated to contain on average 0.2% of the MRL concentration (median 0.09%, 100% of the results less than 1%).

Discussion

The route of the exposure in this study has a novel perspective. If animal by-1 products are used as animal feed then they still have the possibility of gaining 2 access to the human food chain. Therefore, there is clearly an increased need 3 for realistic assessments of risks involved in the use of animal by-products. In 4 this research we have concentrated on benzylpenicillin as one of the potential 5 hazards involved in the process. Other measured antimicrobials are seldom 6 detected in milk. The half-life of these other measured betalactams is short and 7 they do not bioaccumulate more readily than BP. As other betalactam 8 antimicrobials are better tolerated by consumers than BP, they are not expected 9 to pose a greater risk and are not evaluated in this study. If BP was deliberately added to feed, a normal withdrawal period should be 1 applied prior to the slaughter. However, if feed contains rejected milk, there is 2 no withdrawal period. Exposed pigs are allowed to be slaughtered immediately 3 and residues of BP may thus occur in meat. Thereafter the exposure of pigs to 4 BP was only evaluated via this route of using dairy by-products as feed. 5

In order to assess the risk, reliable data on the level of hazard in commodities are needed. The benzylpenicillin analysis method, modified from a method used for muscle samples, worked well for milk. The sample preparation was rather straight-forward and the recovery and relative standard deviation were good in the recovery study. The method has been used in Finland for many years at the EU National Residue Control Program to detect and quantify BP in milk and has been shown to be a reliable method for this purpose, in several proficiency and intercalibration tests it has produced z-scores between + 2.

No MRL for BP residues in feed has been established. However, if the same concentration of BP residues was found in milk as estimated in the pig feed with current practice in Finland, this milk would be approved for human consumption.

This study employed probabilistic distributions instead of average point estimates used in many other exposure studies for concentrations and intake values (e.g. [START_REF] Park | Estimation of the daily exposure of Koreans to aflatoxin B 1 through food consumption[END_REF][START_REF] Caldas | Chronic dietary risk for pesticide residues in food in Brazil: an update[END_REF][START_REF] Rao | Exposure assessment to synthetic food colours of a selected population in Hyderabad, India[END_REF]). This approach permits a more realistic estimation of the variability in the exposure, and the ensuing residue levels. Risk assessment based on the described variation in the factors is well in line with the transparency objective (Codex Alimentarius Comission [CAC] 1999). Based on the model, due to the current practice pork was estimated never to contain over 1% of the MRL for BP, even if the animals were to be slaughtered immediately after feeding. This level of BP residues in pork is below the detection limits. In normal practice pigs are not fed for several hours before they are slaughtered and therefore due to the active elimination, the actual BP concentration in meat at slaughter would be even lower than estimated.

According to our data, pigs are exposed to this type of feed only once during their lifetime. Therefore this exposure can be considered as an acute instead of a continuous exposure.

Instead of using a model to assess the probable distribution of concentration of BP residues in pork, there is the theoretical possibility of obtaining actual samples of retail pork and performing the chemical analyses. However, the number of samples to confidently detect the residues, the concentration of which most likely would lie below the detection limit of the method, was considered prohibitive.

Usually the safety of the long-term exposure to different compounds found in human diets is evaluated by comparing the estimated intake with acceptable daily intake values (ADI) [START_REF] Reynolds | Occurrence and risks associated with chlormequat residues in a range of foodstuffs in the UK[END_REF][START_REF] Rao | Exposure assessment to synthetic food colours of a selected population in Hyderabad, India[END_REF][START_REF] Caldas | Chronic dietary risk for pesticide residues in food in Brazil: an update[END_REF], or with no observed adverse effect levels (NOAEL) [START_REF] Poole | Review of the toxicology, human exposure and safety assessment for bisphenol A diglycidylether (BADGE)[END_REF]). The consumption of pork in Finland was not estimated in this study and therefore the safety estimation was made by comparing the estimated BP residue concentrations in meat with MRL. The smallest amount of BP (3 µg) Animal by-products may pose a risk for consumer, if used for animal feeding.

Our study has shown that the risk can be vanishingly small and nutritionally valuable feed raw material should not be summarily rejected without a careful assessment of the situation in each case.

F

Finnish

  national legislation (Ministry of agriculture and forestry [MMM] 2001) demands that each raw milk lot arriving at dairy must be tested with a microbiological screening test for antimicrobial drug residues. National testing statistics indicate that antimicrobial drug residues are detected in 0.05% of milk lots (Finnish Association for Milk Hygiene [FAM] 2005). The most common residue is benzylpenicillin (BP), ensuing from treatment of cows for mastitis. Commercially available microbiological screening tests are very sensitive for and usually detect BP at its MRL (4 µg kg -1 ) (European Medicines Agency [EMEA] 2005a). The efficient testing practice in Finland ensures safe milk products for consumers and reduces problems in cheese production.

[

  insert Figure1about here]Sample collectionTwenty raw milk samples, drawn from bulk milk lots which were positive in the microbiological screening tests, were collected during 2001-2003 and their BP . The samples were stored at -70°C and analysed within two months.

  here] Each model run consisted of 10 000 simulation runs. The simulation model was implemented on commercial spreadsheet software (Excel 2002, Microsoft Corporation, USA), with a commercial Excel add-in module for risk analysis (@Risk, version 4.5.2, Palisade Corporation, USA).

  calculations were performed with Statistix for Windows software (Analytical Software, USA).

[

  observed to elicit allergic reactions in previously sensitized individuals would require, according to our model, a consumption of about 25 kg of pork before any adverse health effects would be expected even in the most sensitive individuals. The Finnish practice of disposing of this dairy by-product as feed can therefore be considered as safe from the residue point of view for pork consumers.

F

uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y

	Page 17 of 22				Page 20 of 22
	Sample BP (µg l	-1 )
	1	29
	2	65
	Milk collection from farms Step in the model 3 4 4 10 5 6	Inputs	Symbol in Figure 1	Description	unit
	Testing of a STEP 1: Rejected milk lots 6 6 7 9 8 24 bulk milk lot 9 14 10 17	Quantity in transport truck BP concentrations	QT CT	Quantity of milk in transport truck rejected due to a test positive result Samples were drawn from the test positive	litres µg litre -1
	o r P e e r R e v i e w O n l F o r P e e r R e v i e w O n l F F o No residues detected Residues detected Milk rejected for human consumtion Dilution at dairy with other dairy by-products in transport truck milk lots and BP concentrations were analysed Dilution at farm with other feed components STEP 2: Dilution at dairy Quantity in dairy silo QS Quantity of rejected milk and other dairy by-products mixed together in dairy silo 11 6 12 8 13 3 F 14 6 o 15 92 o r CT: concentration of BP in milk QS: quantity of diluted milk in silo DF: dilution factor 16 22 r 17 0 r P e R e v i e l n l y y l O O n Human exposure n O w R e v i e w Residues in pork meat OB: bioavailability T 1/2 : metabolism WP: meat obtained from pigs Dairy processing Milk products Meat products STEP 5: Residues in meat Oral bioavailability OB Bioavailability of BP when ingested orally Elimination t 1/2 T 1/2 Half life of BP Weight of the pig carcases WP The average amount of meat which is obtained from each pig at slaughter MRL for BP in meat The maximum residue limits in meat set by the European Medicines Agency 1 p-value: probability of the data given the parametrized distribution w e i v e R r e P e e r QT: quantity of milk rejected Usage as feed/exposure QF: volume fed to pigs T: number of days used STEP 3: Dilution at farm Dilution factor at farms DF A factor indicating the dilution of rejected milk at farms due to the mixing with other feed components STEP 4: Exposure Quantity of feed eaten daily QF The total amount of feed fed to the pigs daily Number of days fed T Duration of time when feed contains rejected milk 18 5 19 6 P 20 5 Average 16.9 e r e Stdev 22.8	litres % h kg µg kg -1 litres day -1 days
					y y y

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Dr Ute Tiemann
email: tiemann@fbn-dummerstorf.de

S Dänicke

In vivo and in vitro effects of the mycotoxins zearalenone and 1 deoxynivalenol on different non-reproductive and reproductive organs in 2

Keywords: Fusarium toxins, zearalenone, deoxynivalenol, reproductive and non-reproductive 6 organs, female pig 7 8

In vivo and in vitro effects of the mycotoxins zearalenone and deoxynivalenol on different non-reproductive and reproductive organs in female pigs

reproductive and non-reproductive organs in female pigs. In vivo 22 mg ZON kg -1 in the diet 13 cause alterations in the reproductive tract of swine such as in the uterus, on the follicular and 14 embryo development. ZON and its metabolites have been shown to competitively bind to 15 estrogen receptors in an in vitro system. The feeding of pigs with 9 mg DON kg -1 diet 16 contaminated diet can act on protein synthesis, humoral and cellular immune response 17 depending on dose, exposure and timing of functional immune assay, and on liver and spleen 18 cell structures. Beside these effects, reproductive alterations were observed in pigs, too. Both 19 in vivo and in vitro exposure to DON decreased the oocyte and embryo development. In vitro 20 application of DON to uterine cells inhibits their proliferation rate and modulates the process 21 of translation at a different molecular level when compared with the in vivo application. The 22 histopathological results provide evidence of spleen and liver dysfunction in the absence of 23 clinical signs, especially in pigs fed higher concentrations of Fusarium toxin-contaminated 24 wheat. Prepuberal gilts react more sensitively to DON>ZON feeding compared to pregnant 25 sows. In the liver, histopathological changes such as glycogen decrease and interlobular 26 collagen uptake were observed only observed in prepuberal gilts, whereas enhancement of 27 haemosiderin was found in both perpuberal gilts and pregnant sows. This review presents 28 some of the current knowledge on the biological activities of ZON and DON in pig.

Introduction 1

Mycotoxins are biologically active secondary fungal metabolites found as contaminants of 2 feedstuffs and exert toxic effects in animals and human beings (Fink-Gremmels, 1999). 3 Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZON), contaminate wheat, 4 maize, and barley worldwide [START_REF] Abouzied | Immunochemical assessment of 7 mycotoxins in 1989 grain foods: Evidence for deoxynivalenol vomitoxin contamination[END_REF][START_REF] Chelkowski | Distribution of Fusarium species and their mycotoxins in cereal grains[END_REF]EFSA, 2004a,b). 5 These mycotoxins are produced mainly by F. graminearum, F. culmorum, and F. roseum 6 under predisposing environmental conditions during cereal flowering and include humidity 7 and fungus specific optimum temperature range [START_REF] Oldenburg | Risikofaktoren für die Fusariumtoxinbildung und 7 Vermeidungsstrategien bei der Futtermittelerzeugung und Fütterung[END_REF]. Fusarium species 8 are probably the most prevalent toxin-producing fungi of the northern temperate regions and 9 are commonly found in cereals grown in America, Europe and Asia. They cause a variety of 10 toxic effects in experimental animals and livestock. Different effects were described for 11

Fusarium toxins ZON (Kuiper-Goodman et al. 1987; [START_REF] Wood | Mycotoxins in food and feeds in the United States[END_REF] and DON (reviewed by 12 Pestka and Casale, 1990; [START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF]. 13 14 Zearalenone is biologically potent, but hardly toxic; rather, it sufficiently resembles 17ß-15 estradiol, the principal hormone produced by the human ovary, to allow it to bind to estrogen 16 receptors in mammalian target cells [START_REF] Katzenellenbogen | Zearalenones: 22 characterization of the oestrogenic potency and receptor interactions of a series of fungal 23 beta-resorcylic acid lactones[END_REF]. ZON is better classified as 17 a non-steroidal estrogen or mycoestrogen. Metabolism of ZON in monogastric animals takes 18 place in the liver and intestinal mucosa where it is reduced to αand β-zearalenol (α-ZOL and 19 β-ZOL) [START_REF] Olsen | Metabolism of 15 zearalenone by sow intestinal mucosa in vitro[END_REF][START_REF] Olsen | Metabolism of zearalenone in farm animals[END_REF], a reaction that competes with glucuronidation of the 20 parent molecule. Examination of excretory products indicated that in pig the α-epimer 21 predominates (Farnwoth and Trendholm, 1983;Olsen et al., 1985a,b;[START_REF] Biehl | Biliary 22 excretion and enterohepatic cycling of zearalenone in immature pigs[END_REF]22 Zöllner et al., 2002;[START_REF] Döll | Effects of graded levels of Fusarium toxin contaminated maize in 14 diets for female weaned piglets[END_REF]Dänicke et al., 2005b). Biotransformation studies with 23 pig liver sub-cellular fractions indicated that α-ZOL is the main hepatic metabolic of ZON in 24 pigs [START_REF] Malekinejad | Bioactivation of zearalenone by 19 porcine hepatic biotransformation[END_REF]. Additionally, the authors could detect that another extrahepatic 25 biotransformation of ZON into α-ZOL exists in porcine granulosa cells (Malekinejad et al. 1 2006). Alpha-ZOL binds more effectively to the receptor than the parent toxin (ZON) and 2 might therefore explain the sensitivity of pigs to this mycotoxin. 3 4 The effect of ZON and its metabolites depends upon the reproductive status (pre-puberal, 5 cycling or pregnant) of the affected animal [START_REF] Lopez | 13 Hyperestrogenism in swine due to natural poisoning with zearalenone[END_REF][START_REF] Diekman | Mycotoxins and reproduction in domestic livestock[END_REF]. 6

Pigs, especially gilts before the first oestrus, are the most susceptible to intoxication with 7 higher ZON doses (50-100 mg kg -1 ) and they can develop symptoms such as vulvae oedema 8 and erythrema, impairment of ovulation, conception, implantation, fetus development, and 9 newborn's viability (Mirocha et al. 1977;[START_REF] Weaver | 8 Mycotoxin-induced abortions in swine[END_REF][START_REF] Chang | Effects of the mycotoxin zearalenone on swine 4 reproduction[END_REF]Long and 10 Diekman, 1986;[START_REF] Price | Naturally occurring toxins in feedstuffs[END_REF][START_REF] Dacasto | Zearalenone mycotoxicosis 9 in piglets suckling sows fed contaminated grain[END_REF]Yang et al. 1995;[START_REF] Gajecki | Zearalenone -undesirable substances in feed[END_REF]. The 11 amount of 22.09 mg kg -1 of ZON in the ration of breeding gilts had an obvious harmful effect 12 on their reproductive performance: a decreased number of corpora lutea, a decreased weight 13 of ovaries, a decreased number of live embryos, an increased number of dead-born piglets, 14 and the occurrence of aborts. These effects were less pronounced in gilts fed mashes 15 containing 2.2 mg kg -1 ZON (Kordic et al. 1992). New guidance values recommended not to 16 exceed 0.1 mg ZON kg -1 for piglets and gilts, 0.25 mg ZON kg -1 for sows and fatting pigs, 17 and 0.9 mg ZON kg -1 diet for pigs in order to avoid adverse effects on reproductive traits (EU, 18 2006). EFSA (2004b) concluded from a recent literature evaluation that safe levels of ZON in 19 feed for pigs can not reliably established at present. 20 21 Deoxynivalenol, a Fusarium toxin belonging to the trichothecene group, has been reported to 22 produce a variety of adverse health effects in farm animals, such as inhibition of protein 23 synthesis, reduction of feed intake, and alteration of the immune system. The upper critical 24 concentrations in Germany was set at 0.9 mg DON kg -1 diet for all pigs, as a guideline level in 25 The presence of DON in pig diets decreases feed intake, causes feed refusal, and induces 8 occasional vomiting [START_REF] Diekman | Mycotoxins and reproduction in domestic livestock[END_REF]. Beside these effects, reproductive 9 alterations were observed in humans and pigs [START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans 17 and animals[END_REF][START_REF] Alm | 13 Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of 14 gilt oocytes[END_REF]. 10 11 Effects of a Fusarium toxin contaminated diet containing DON and/or ZON on the 12 fertility/reproduction of female pigs need to be viewed in the general context of the toxin 13 effects on porcine health and performance. Before the toxins are able to modify metabolic 14 processes they need to be consumed voluntarily by the pig. Data reported in the literature 15 indicate that the adverse effects of DON contaminated diets on growth performance of 16 growing pigs is primarily caused by depressing the voluntary feed intake [START_REF] Rotter | Influence of low-16 level exposure to Fusarium mycotoxins on selected immunological and hematological 17 parameters in young swine[END_REF]17 Swamy et al., 2002;Goyarts et al., 2005). The mycotoxins obviously act in a concerted 18 manner with regard to metabolic processes and consequently influence the health and 19 fertility/reproduction (Figure 1). 20 21 This review aims to give an overview about the effects of feeding with DON>ZON 22 contaminated diet on non-reproductive and reproductive organs which can influence 23 metabolism, health, and fertility/reproduction of female pigs. Figure 1 demonstrates a scheme 24 of the investigations on specific organs and parameters dealt with in the current study. 25 Deoxynivalenol is known to be a potent protein synthesis inhibitor according to research in 5 different in vitro and in vivo systems [START_REF] Feinberg | Biochemical mechanism of action of trichothecene 18 mycotoxins[END_REF]. However, the 6 significance of this toxic feature was not yet evaluated for the pig. Therefore, an experiment 7 was carried out by [START_REF] Dänicke | Effects of the 16 Fusarium toxin deoxynivalenol on tissue protein synthesis in pigs[END_REF] to measure porcine tissue protein synthesis 8 employing the so-called flooding dose technique using [ 2 H 5 ]-phenylalanine as a tracer in 9 dependence on different exposition of DON to pigs exposed to a DON-contaminated diet for 10 ~ 4 wks (chronic) or just uniquely (acute oral) to reflect different feeding scenarios. Moreover, 11 an additional group of pigs was injected with a single intravenous DON-dose (acute 12 intravenous) representing a complete systemic DON-availability. Protein synthesis expressed 13 as fractional synthesis rate (FSR) was significantly reduced in kidneys, spleen and ileum of 14 DON-exposed pigs. FSR of liver, skeletal and heart muscle, mesenteric lymph nodes, 15 duodenum, jejunum, jejunal mucosa cells, pancreas and lung were not affected by DON. [START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans 17 and animals[END_REF]

Goyarts et al. (2006a) have shown that DON decreased the FSR of albumin as the main 17

protein exported from the liver and as the predominant plasma protein. Pigs exposed to DON 18 5.7 mg kg -1 diet showed an increased protein synthesis capacity (RNA to protein ratio) caused 19 by an elevated liver RNA-concentration [START_REF] Dänicke | Effects of the 16 Fusarium toxin deoxynivalenol on tissue protein synthesis in pigs[END_REF] . 20 21

Humoral and cellular immune response 22

DON affects the humoral immunity [START_REF] Bondy | Immunomodulation by fungal toxins[END_REF]. An overview on the 23 mechanisms of IgA production induced by DON is given by Pestka (2003). The effects of 24 DON on immunoglobulin levels of serum in swine are rather inconsistent. After feeding of 25 pure DON (600 µg kg -1 ) under highly standardized conditions. In contrast, some investigators 6

were not able to show interaction between DON and IgA serum concentrations (Bergsjø et al. 7 1992(Bergsjø et al. 7 , 1993 to DON>ZON feeding. In the liver, histopathological changes such as glycogen decrease and 1 interlobular collagen uptake were observed only in prepuberal gilts. In both animals an 2 enhancement of haemosiderin in liver and spleen was found. These results were supported by 3 ultrastructural investigations and can be due to a dysfunction in both spleen and liver cell 4 circulation. Because there was a statistically significant, but only a modest elevation of the 5 liver enzyme activities for aspartate aminotransferase (AST), alanine aminotransferase (ALT), 6 the authors suggest that the doses of mycotoxins used did not cause hepatic fibrosis. The 7 observed histological alterations in liver and spleen, however, were not clinically manifested. 8

There were no adverse effects on liver and spleen of piglets when their mothers consumed 9 diets containing up to 9,570 and 358 µg DON/ZON kg -1 diet (unpublished data). 10 11

Follicle development, oocyte maturation, and embryo development 12

Edwards et al. (1987) reported that pre-pubertal gilts fed zearalenone displayed first estrus 13 later than controls; however, in other investigations, pre-pubertal gilts consuming a milo diet 14 contaminated with zearalenone exhibited puberty at a younger age without an alteration in 15 conception rates, ovulation rates, or embryonic survival [START_REF] Rainey | Prepubertal exposure to dietary 11 zearalenone alters hypothalamo-hypophysial function but does not impair post-pubertal 12 reproductive function of gilts[END_REF]. 16 breeding were fragmentous, while those from control gilts were filamentous (Diekman and 17 [START_REF] Diekman | Blastocysts development on days 10 or 14 after consumption 10 of zearalenone by sows on days 7 to 10 after breeding[END_REF]. Degeneration in blastocysts, affecting both embryonic membranes and the 18 embryonic disk occurring in ZON-treated sows were observed by [START_REF] Long | Effect of zearaleone on days 7 to 10 10 post-mating on blastocyst development and endometrial morphology in sows[END_REF]. In 19 contrast, the authors observed no deleterious effects of DON on fetal development when feed 20 containing 8 mg kg -1 of purified DON was consumed (summarized Diekman and Green, 21

1992). 22 23

In vitro, the maturation of porcine oocytes was investigated after addition of αand β-ZOL 24 and DON. All three substances negatively affected oocyte maturation and degeneration rates 25 impairment of several enzymes on endometrial cell metabolism were not observed 6 (Wollenhaupt et al. 2006a). The data was supported by the fact that the quantity of 7 progesterone and estradiol receptors in the uterus was not affected by DON>ZON feeding. In 8 agreement to these results, Döll et al. ( 2004) reported that after 5 wk feeding of prepuberal 9 piglets with the concentrations of ZON up to 0.42 mg and DON up to 3.9 mg kg -1 originating 10 from Fusarium toxin contaminated maize, the mean weight of the uteri of animals receiving 11 the most highly contaminated diet was significantly increased at the time of slaughtering. 12 Although an uptake in uterus weight occurred the surface epithelium of the uterus, the uterine 13 glands and the vaginal epithelium were not altered by the treatment [START_REF] Döll | The effect of increasing concentrations of 16 Fusarium toxins in piglet diets on histological parameters of the uterus and vagina[END_REF]). 14 Enhancement of ZON (56 mg kg -1 ) and DON (4.99 mg kg -1 ) in the diet of pigs caused 15 histopatholoical changes of uterine tissue such as hyperplasia, hypertrophy, and metaplasia of 16 the myometrium [START_REF] Lopez | 13 Hyperestrogenism in swine due to natural poisoning with zearalenone[END_REF]). Application of ZON in crystalline form in the dose of 17 200-400 µg kg -1 body weight day -1 for a period of 7 days to immature gilts caused an 18 intensified cell proliferation which was expressed with the increase of PCNA (Prolifering Cell 19 Nuclear Antigen) index. This can be due to estrogenic effects and was observed in the 20 oviduct and uterus [START_REF] Obremski | Influence of zearalenone on reproductive system 5 cell proliferation in gilts[END_REF]. Higher concentrations of ZON, 2.2 mg kg -1 and 21 22.09 mg kg -1 in the ration of breeding gilts had an obvious harmful effect on their 22 reproductive performance, and the uterotropic effect of ZON was obvious in both groups 23 (Kordic et al. 1992). 24 In vitro, the affinity of ZON to uterine and oviduct estrogen receptors is greatest in pigs as 1 compared to rats and chickens (Fitzpatrick et al. 1989). The relative binding affinities to the 2 pig uterine cytoplasmic receptor for ZOL show that α-ZOL is more potent than ß- ZOL 3 (Tiemann et al. 2003b). Responsible for the higher binding affinity of α-ZOL to estrogen 4 receptor is the reduction of the 6-keto and also vinyl groups in its molecule, while the ß-5 epimer is much less active in this regard (Kuiper-Goodman et al. 1987). The mechanism by 6 which α-ZOL inhibits the formation of a hormone-receptor complex can be explained by the 7 fact that α-ZOL exerts its effect by competing with estrogen for cytosolic receptor on cells in 8 target tissue and this fact might upset the delicate balance necessary for normal reproductive 9 activity [START_REF] Jr | Ecoestrogens and embryos -Is there a 13 scientific basis for concern?[END_REF]. In contrast to the natural hormones, α-ZOL does not bind the 10 carrier protein [START_REF] Shrimanker | Binding of steroid hormones and anabolic 19 agents to bovine sex-hormone binding globulin[END_REF], and consequently may exhibit elevated unbound 11 serum concentrations, thereby increasing its ability to activate the estrogenic pathway (Martin 12 et al. 1978). Compared to α-ZOL, a strong anti-proliferative effect of β-ZOL and DON in 13 Previously, in vivo investigation were performed to determine different non-reproductive and 3 reproductive parameters in female pigs after feeding with DON and/or ZON contaminated 4 diets to evaluate the risks of their metabolism, health and fertility/reproduction. From all 5 described parameters, it is notable that the in vivo effects of DON on humoral immunity are 6 rather inconsistent. ZON has estrogenic activity and this is reflected by alterations of fertility 7 and reproduction in pigs. This review shows that besides the known effects of ZON on 8 reproductive organs, ingestion of DON also causes reproductive failure showing an 9 impairment of porcine oocyte and embryo development which may probably be related 10 directly to the toxic effect of DON. 1112 Table 1 indicates that in vivo effects of DON/ZON were more investigated than in vitro 13 effects. The in vivo investigations provide information on net effects in whole animals, 14 whereas cell specific answers result from in vitro investigations. The summarized results of 15 in vitro studies with porcine cell cultures indicate that there are only partial agreements with 16 that of in vivo experiments. An explanation could be the fact that sometimes the cells in vitro 17 react more sensitively than those in vivo, because a direct interaction of chemical substances 18 with the plasma membrane exists which may alter the membrane structure and function. 19 Therefore, in vitro experiments may contribute to risk assessments of toxins. 20

Pathophysiological studies are attributable on DON/ZON feeding in pigs and can be important 21 in understanding whether human exposure to DON/ZON may have unfavourable effects, 22 because swine are physiologically similar to humans [START_REF] Tumbleson | Advances in swine in biochemical research[END_REF] and are 23 widely used as models for human disease. The effects of a Fusarium toxin contaminated diet containing predominantly deoxynivalenol (DON) and zearalenone (ZON) on the fertility of female pigs need to be viewed in the general context of the toxin effects on animal health and performance. Before the toxins are able to modify metabolic processes they need to be consumed voluntarily by the pig. The effects on feed intake, which are mediated by DON, not only determine the amount of toxins entering the organism but also the metabolically available nutrients which might also markedly modulate processes involved in fertility. Although the primary molecular targets of DON (inhibition of protein synthesis) and ZON (interference with the oestrogen receptor) are different, they obviously act in a concerted manner with regard to health and fertility (further details in text).

3 4

 3 strategies(EU, 2006). EFSA (2004a) recommended the collection 1 of more data for refining or establishment of safe dietary DON levels, including the effects of 2 DON on the immune system. Macrophages, T cells, and B cells of the immune system are central targets of DON that can 5 be immunostimulatory or immunosuppressive depending on dose, exposure and timing of 6 functional immune assay[START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF][START_REF] Bondy | Immunomodulation by fungal toxins[END_REF][START_REF] Pestka | Cellular and molecular mechanisms for 6 immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox[END_REF]). 7

ZON

  Furthermore, we have focused on the comparison of in vivo and in vitro effects of DON and 1

  with DON>ZON for 5 wk the serum IgA concentration of 1 prepuberal pig increased significantly, while serum IgM and IgG concentrations were not 2 altered (Goyarts et al. 2006b; Tiemann et al. 2006a). Similarly, Swamy et al. (2002) observed 3 such effects in pigs fed with DON contaminated corn and wheat. Drocher et al. (2004) 4 demonstrated an IgA elevation in pigs fed a semisynthetic diet without grain components with 5

  ; Döll et al. 2003; Swamy et al. 2003; Dänicke et al. 2004a,b). No differences were 8observed in serum level of IgA between the pregnant sows fed 9.6 mg DON/0.358 mgZON 9 kg -1 diet and the controls for 35 days (Tiemann unpublished data). Table1summarized the 10 data and it can be noteable that the in vivo effects of DON on humoral immunity are rather 11 inconsistent. The reason may be methological differences in the experiments carried out by 12 several investigators. In vitro, the IgA concentration in the supernatant of ConA stimulated 13 porcine peripheral lymphocytes was dose-dependently inhibited with increasing DON 14 concentrations(Goyarts et al. 2006b).15 16 Mitogen-induced proliferation is a common technique to assess immunotoxicity. The 17 immunosuppressive effect of DON is primarily associated with cellular immune response. 18 Øvernes et al. (1997) observed a significant higher response to phytohemagglutinin in the high 19 DON group compared to the low DON group in growing pigs. In contrast, no effect of DON-20 contaminated feed on peripheral lymphocyte mitogen response was reported by Rotter et al. 21 (1994); Tiemann et al. (2006a, unpublished data). Compared to bloods lymphocytes, 22 splenocytes were more sensitive to ConA-induced response in perpuberal pigs fed the DON 23 contaminated diet compared to pregnant sows. The DON>ZON feeding of pregnant sows 24 the proliferative response in splenocytes in approximately the same manner, as 1 observed in prepuberal gilts (Tiemann et al. 2006a, unpublished data). 2 3 Mitogen-induced proliferation was impaired at low concentrations after in vitro exposure to 4 porcine lymphocytes or splenocytes (Goyarts et al. 2006b; Tiemann et al. 2006a). Both 5 authors found a reduced proliferative response which may be due to the capacity of DON to 6 inhibit protein synthesis (Ueno, 1983; Thompson and Wannemacher, 1986; Ehrlich and 7 Daigle, 1987). The latter assumption is supported by the findings of Goyarts et al. (2006a) 8 who measured the overall protein synthesis of porcine peripheral blood lymphocytes in vivo 9 by using a stable isotope labelled amino acid. Other toxic mechanisms that have been 10 associated with trichothecenes include impaired membrane function (Bunner and Morris, 11 1988), altered cellular communication (Jone et al. 1987), deregulation of calcium homeostasis 12 (Yoshino et al. 1996), and such as described above. 13 14 The capacity of DON to affect porcine neutrophils, critical mediators between innate and 15 acquired immunity (Ellis and Beaman, 2004), at pharmacological concentrations was 16 investigated by Takayama et al. (2005).

17

 17

18

  Histopathology of spleen and liver 19After 5 weeks of feeding, the histopathological effects on spleens and livers in gilts(Tiemann 20 et al. 2006a,b) and pregnant sows (unpublished data) can be mostly observed at a dietary DON 21 concentration of 9.57 mg kg -1 , originating from naturally contaminated wheat containing only 22 minor traces of ZON (0.358 mg kg -1 diet), but in absence of clinical signs(Dänicke et al. 23 2005a, unpublished data). The authors observed the appearance of a dysfunction in porcine 24 liver and spleen which can be seen as hemosiderosis. Pre-puberal gilts react more sensitively 25

  Zwierzchowski et al.(2005) observed that the amount of ZON to a dose of 200 µg kg -1 body 17 weight in immature gilts leads to distribution in the development and maturation of the largest 18 follicles through the activation of an apototic-like process in the granulosa layer of single 19 mature follicles. In contrast,[START_REF] Alm | 13 Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of 14 gilt oocytes[END_REF] found that the size distribution of follicles was 20 not affected by feeding of gilts with different Fusarium-toxin contaminated diets. In these 21 animals the cumulus cell morphology was not changed by increasing dietary concentrations of 22 both toxins up to 9,570 and 0,358 mg kg -1 for DON and ZON, respectively. The authors 23 recovered oocytes from pig ovaries by follicle aspiration after ovario-hysterectomy. At the 24 concentration named above, at the time of recovery, oocytes with compact cumuli showed a 25 with immature chromatin. Here, the proportion of oocytes 1 having degenerated meiotic chromatin was significantly higher compared to lower 2 concentrations. The oocyte quality was significantly reduced by feeding of a DON>ZON 3 contaminated diet to gilts. The competence of oocytes in compact cumulus oocyte complexes 4 for in vitro maturation was likewise significantly reduced by high concentrations of Fusarium 5 toxin in feed; this is probably directly related to the reduced proportion of normal germinal 6 vesicle-stage oocytes, i.e., oocytes capable of maturation, in the high toxin groups.These 7 results are in agreement with data reported byKordic et al. (1992) who observed a doses-8 dependent effect of ZON on reproductive performance in breeding gilts, with decreased 9 numbers of live embryos as well as other reproductive problems including increased number 10 of dead-born piglets. Little information is available about effects of indirect (i.e., from mother 11 to young) exposure to DON/ZON on the health of piglets. Placenta transfer of mycotoxins can 12 indicate a potential risk for direct effects in the fetus.[START_REF] Long | Effect of purified zearalenone on early gestation in gilts[END_REF] reported that 13 gilts fed 5 to 30 mg kg -1 ZON from d 2 to 15 postmating had normal embryonic development, 14 but no fetuses were present in gilts fed 60 or 90 mg kg -1 ZON when killed d 40 to 43 post-15 mating. Embryos recovered on d 14 from gilts fed 60 mg kg -1 of ZON from d 7 to 10 post 16

3 4steroid synthesis 5

 35 -dependent manner, but to different extents (DON>α-ZOL>β-ZOL). In addition, α-1 ZOL directly affected the early embryonic development in vitro of in vivo-derived porcine 2 embryos[START_REF] Alm | The influence of the 10 mycotoxins deoxynivalenol and zearalenol on the in vitro maturation of pig oocytes and 11 in vitro culture of pig zygotes[END_REF].IntrafollicularThe feeding of wheat contaminated naturally with DON and ZON to gilts at increasing 6 proportions did not influenced the activity of enzymes involved in progesterone synthesis. In 7 vivo-derived porcine granulosa cells were analyzed for the mRNA expression of the 8 mitochondrial enzyme cytochrome P450, cholesterol side-chain cleavage (P450scc) and 9 microsomal enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) by RT-PCR, and 10 additionally for P450scc protein by Western blotting. Neither the expression of the P450scc 11 nor of the 3β-HSD mRNA, nor the abundance of the P450scc protein was significantly 12 influenced by the mycotoxin feeding[START_REF] Alm | 13 Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of 14 gilt oocytes[END_REF].[START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF] 14 Cultured primary porcine granulosa cells have been used to test the toxic potential of 15 xenobiotics on reproduction[START_REF] Haney | Screening of potential reproductive toxicants by use of 15 porcine granulosa cell cultures[END_REF]. In vitro addition of αand β-ZOL at 16 concentrations of 15 and 30 µmol l -1 inhibited the FSH-stimulated progesterone synthesis in 17 porcine granulosa cells. The inhibitory effect at a concentration of 30 µmol l -1 of both 18 mycotoxins could mainly be due to cell death. The antisteriodogenic effect of 15 µmol l -1 α-19 ZOL or β-ZOL was not due to cytotoxic effects. The enzyme activity of 3β-HSD and the 20 abundance of P450scc protein were reduced by both mycotoxins as well (Tiemann et al. feeding of pre-puberal pigs with a diet containing a concentration of DON which 3 was more than nine times, and of ZON only 0.43 times, higher than the upper critical 4 concentrations, hyperestrogenism, uterotrophic effects (Tiemann et al. 2006b) or an 5

Food

  porcine endometrial cells was observed by Tiemann et al. (2003b). This finding corresponds 14 to changes in the substructure and to a reduction in viability caused by apoptosis at β-ZOL 15 (Wollenhaupt et al. 2004) or necrosis at DON (Tiemann et al. 2003b). It can be assumed that 16 β-ZOL and DON modulate the process of translation at different molecular level. A molecular 17 target of DON in proliferating eukaryotic cells is the 60 S ribosomal subunit (Middlebrook 18 and Leatherman, 1989; Witt and Pestka, 1990). Following binding of DON to the ribosomal 19 RNA, a rapid activation of mitogen-activated protein kinases (MAPKs) could be observed in a 20 process that has been termed the "ribotoxic stress response" (Zhou et al. 2003; Pestka et al. 21 2004). The results of Wollenhaupt et al. (2004, 2006a, summerized 2006b) indicated that β-22 ZOL and DON showed a pronounced impact on MAPKs, which are in mRNA translation, and 23 different signalling pathways can be modulated resulting in the inhibition of cell proliferation 24 after β-ZOL and DON exposure. 25 Page 13 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

3 spleen

 3 Altogether, ZON affects reproduction of pigs most seriously because it possesses estrogenic 1 activity. Whereas DON affects reproduction in pigs via indirect effects such as reduced feed 2 intake, resulting in reduced growth or impairment of function in vital organs like liver and Edwards S, Cantley TC, Day BN. 1987. The effect of zearalenone on reproduction in pigs. II. 1 The effect of puberty attainment and postweaning rebreeding performance. 2 Theriogenology 28:51-58. 3 EFSA, 2004a. Opinion of the Scientific Panel on Contaminants in the Food Chain on a 4 request from the Commission related to Deoxynivalenol (DON) as undesirable substance in 5 animal feed. The EFSA Journal 73, 1-41 (available at: http://www.efsa.eu.int/). 6 EFSA, 2004b. Opinion of the Scientific Panel on Contaminants in the Food Chain on a 7 request from the Commission related to Zearalenone as undesirable substance in animal feed. 8The EFSA Journal 89, 1-35 (available at: http://www.efsa.eu.int/).

7 a

 7 Smith TK, MacDonald EJ, Karrow NA, Woodward B, Boermans HJ. 2003. 3 Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on 4 growth and immunological measurements of starter pigs, and the efficacy of a polymeric 5 glucomannan mycotoxin adsorbent. Journal of Animal Science 81:2792-2803. 6 Takayama H, Shimada N, Mikami O, Murata H. 2005. Suppressive effect of deoxynivalenol, Fusarium mycotoxin, on bovine and porcine neutrophil chemiluminescence: an in vitro 8 study. Journal of Veterinary Medicine A 67:531-3. 9 Thompson WL, Wannemacher RW Jr. 1986. Structure-function relationship of 12,13-10 epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. 11 Toxicon 24:985-994. 12 Tiemann U, Brüssow K-P, Jonas L, Pöhland R, Schneider F, Dänicke S. 2006a. Effects of 13 diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected 14 immunological and histological parameters of spleen in gilts. Journal of Animal Science 15 84:236-245. 16Tiemann U, Brüssow K-P, Küchenmeister U, Jonas L, Kohlschein P, Pöhland R, Dänicke S. 17

7 ( 21 Figure 1 .

 7211 Figure 1. The effects of a Fusarium toxin contaminated diet containing predominantly 22 deoxynivalenol (DON) and zearalenone (ZON) on the fertility of female pigs need to be 23 viewed in the general context of the toxin effects on animal health and performance. Before 24the toxins are able to modify metabolic processes they need to be consumed voluntarily by the 25 pig. The effects on feed intake, which are mediated by DON, not only determine the amount 26

  Figure1. The effects of a Fusarium toxin contaminated diet containing predominantly deoxynivalenol (DON) and zearalenone (ZON) on the fertility of female pigs need to be viewed in the general context of the toxin effects on animal health and performance. Before the toxins are able to modify metabolic processes they need to be consumed voluntarily by the pig. The effects on feed intake, which are mediated by DON, not only determine the amount of toxins entering the organism but also the metabolically available nutrients which might also markedly modulate processes involved in fertility. Although the primary molecular targets of DON (inhibition of protein synthesis) and ZON (interference with the oestrogen receptor) are different, they obviously act in a concerted manner with regard to health and fertility (further details in text).

Table 1 .

 1 Effects of deoxynivalenol (DON) and zearalenone (ZON) on selected reproductive and non-reproductive parameters in pigs

	Parameter	in vivo	in vitro	References
		Fusarium	DON	ZON
		toxin
	Protein synthesis + (DON)			Dänicke et al., 2006
		+ (DON)			Goyarts et al., 2006a
	Immune response -(DON)			Bergsjø et al., 1992, 1993
		-(DON)			Dänicke et al., 2004a,b
		-(DON)			Döll et al., 2003
		+ (DON)			Drochner et al., 2004
		+ (DON)	+		Goyarts et al., 2006b
		+ (DON)			Øvernes et al., 1997
		-(DON)			Rotter et al., 1994
		+ (DON)			Swamy et al., 2002
		-(DON)			Swamy et al., 2003
			+		Takyayama et al., 2005
		+ (DON>ZON)	+		Tiemann et al., 2006a
	Histopathology	+ (DON>ZON)			Tiemann et al., 2006a,b
	liver and spleen
	Follicular	-(DON>ZON)			Alm et al., 2006
	development	+ (ZON)			Zwierzchowski et al., 2005
	Oocyte		+	+	Alm et al., 2002
	maturation	+ (DON>ZON)			Alm et al., 2006
	Embryo		+	+	Alm et al., 2002
	development	+ (DON>ZON)			Alm et al., 2006
		+ (ZON)			Diekman and Long, 1989
		+ (ZON)			Kordic et al., 1992
		+ (ZON)			Long et al., 1992
	Steroid synthesis -(DON>ZON)			Alm et al., 2006
				+	Tiemann et al., 2003a
	Uterine effects	-(DON>ZON)			Döll et al., 2004
				+	Fitzpatrick et al., 1989
		+ (ZON)			Kordic et al., 1992
		+ (ZON>DON)			Lopez et al., 1988
		+ (ZON)			Obremski et al., 2003
			+	+	Tiemann et al., 2003b
		-(DON>ZON)			Tiemann et al., 2006b
				+	Wollenhaupt et al., 2004
		-(DON>ZON)	+		Wollenhaupt et al., 2006

'-' marked no effect; '+' marked significant effect

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