Keywords: chlorpyrifos-methyl, pirimiphos-methyl, post-harvest treatment, wheat grain, processing products, residues, baby food

Study on the effect of grain storage and processing on chlorpyrifos-methyl and pirimiphos-methyl residues in post-harvest treated wheat with regard to baby food safety requirements

Anna Balinova, Rosica Mladenova, Doncho Obretenchev

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Abstract

A study was undertaken to assess the effects of storage intervals and of milling procedures on dissipation of chlorpyrifos-methyl and pirimiphos-methyl residues in post-harvest treated wheat grain with the aim to obtain scientific data on compliance of the processed products with the safety requirements concerning baby foods. The insecticide formulations were applied on stored wheat at recommended rates (20 ml.t -1 ). The initial concentration levels in whole grain were determined in samples taken 1 h after treatment. The dissipation of residues and their distribution in different fractions of the milled grain were studied after various storage intervals, from 7 to 270 days after treatment. Samples of treated grain were milled in a fractionating laboratory mill and 8 fractions -bran, semolina, 3 types of groats and 3 types of flour were collected and analysed for pesticide residues. The residues were determined by an analytical method based on acetone extraction, graphitized carbon clean-up and GC-ECD, respectively GC-NPD determination of residues. The limits of determination of both pesticides were 0.005 mg. Portugal, 1999). Studies on the effect of storage on insecticide residues following post-harvest treatments have generally shown that residues decline rather slowly [START_REF] Holland | Effects of storage and processing on pesticide residues in plant products[END_REF]. Typically concentrations showed little decrease over 32 weeks at 20 0 C and 50-70% relative humidity. Chlorpyrifos-methyl and pirimiphos-methyl were reported more persistent than malathion [START_REF] Holland | Effects of storage and processing on pesticide residues in plant products[END_REF]. For refining of dietary intake estimates of pesticide residues a generation of food processing studies data has been required. Milling is one of the 10 processing methods identified as deserving main attention (World Health Organisation 1997).

Studies on the fate of pesticide residues on grain subjected to milling and processing showed that the bulk of the insecticides deposited on the grain remained in the epidermis and was removed from the bran during milling. Most residues were found in the outer portion of grains [START_REF] Hajslova | Pesticides[END_REF]. The residues in bran have been reported to be consistently higher than in whole grain. The concentration on the bran was from 2 to 3.2 times the concentration on the whole wheat (Desmarchelier 1979a,b). Bran/grain concentration factors in the range of 2 to 6 were reported in many studies (Joint Meeting on Pesticide Residues 1988Residues , 1989Residues , 1991Residues , 1992)).

Generally the amount of insecticide in flour was 10% or less of the concentration on the whole wheat. Yet the reduction of fenitrothion concentrations in flour was not found sufficient enough to comply with some MRLs particularly when wheat available had been treated within the previous two to three months at the recommended levels (Murray andSnelson 1978, Snelson 1979).

Chlorpyrifos-methyl and pirimiphos-methyl are registered as grain protectants and have been used as such for more than 30 years. The MRLs established for wheat grain intended for general nutritional uses are 3 mg.kg -1 and 5 mg.kg -1 respectively for both insecticides (European Commission 2002) The general MRLs are set on the basis of supervised trials in which Good Agricultural Practice is observed, on results, obtained under national and international monitoring programs etc. and must not pose an unacceptable risk to human health. These levels are subject to the requirements of international trade and as a rule are not violated.

The enforcement of the very stringent European Directive on residues in processed cereal-based baby food imposes the necessity of careful tuning of all technological parameters in order to select procedures that enable production of baby food in The established MRL for residues in baby food is tens to hundreds times below the MRLs for food of general consumption. This imposes the necessity considerable attention to be paid to the problems of assurance the high quality of analytical methods both in respect of quantitative parameters (sensitivity, trueness, precision, and range) and of qualitative assessment (identification and confirmation).

Implementation of analytical methods capable to determine the pesticides studied at or below 0.01 mg.kg -1 was mandatory in the study.

The objectives of this work were: to study the influence of the wheat grain storage intervals and of milling on the decline and distribution of residues of the selected post-harvest applied insecticides in the processed fractions; to establish the residue balance of selected insecticides in processed products of post harvest treated wheat grain; to provide scientific data on compliance of the processed post-harvest treated wheat grain products with the safety requirements concerning baby foods.

Materials and methods

All processes applied to the study for processing effects assessment were planned in a way to correspond as closely as possible to those that occur in actual practices.

Wheat treatment, sampling and processing.

High grain quality wheat (Triticum estivum) cultivar "Momchil" was used in the experiments. The initial grain moisture was 14.0±0.5%. The commercial insecticide formulations were Reldan 50 EC (chlorpyrifos-methyl 500 g.l -1 active ingredient [a.i])

and Actellic 50 EC (pirimiphos-methyl 500 g.l -1 a.i). They were applied at recommended rate of 20 ml. were submitted for analysis as well as whole grain.

Chemicals

Pesticide standards. Analytical standards of pirimiphos-methyl, purity 99.8% and chlorpyrifos-methyl, purity 99.9% were obtained from LGC Promochem (Wesel, Germany). Stock solutions (1.0 mg.ml -1 ) of each pesticide were prepared in ethyl acetate and stored in a freezer at -23 0 C. Working standard solutions at concentration rates of 10 ng.ml -1 -500 ng.ml -1 were prepared by diluting of stock solutions with ethyl acetate and stored at 4°C in dark.

Solvents and reagents.

All organic solvents were of gas chromatography grade and were obtained from Merck KGaA (Darmstadt, Germany). Double distilled water was used when needed in experiments. Solid phase extraction (SPE) sorbent: graphitized carbon ENVI-Carb 120/400 mesh, surface area 100 m 2 /g, was obtained from Supelco (Bellefonte, PA, USA).

Instrumentation.

Hewlett-Packard gas chromatograph Model 5890 Series II equipped with 63 Ni electron capture detector (ECD) and nitrogen-phosphorus detector (NPD) was used.

Split-splitless injector operated in the splitless mode. Capillary column AT™-5ms, (30 Belgium). Nitrogen of purity 99.9995 % was used for gas chromatography.

Vacuum manifold (Varian BV) was used for SPE.

Analytical method

Extraction. The weight of the analytical samples taken of different products varied depending on the expected levels of residue concentrations. The whole wheat grain laboratory samples were homogenised by mixing, analytical samples of 10 g were weight, 50 ml mixed solvent acetone-water (8:2) were added and samples were extracted by shaking for 30 min on a shaking machine and left over night. Analytical samples of 10 g were weight of bran and semolina and extracted with 100 ml of the solvent. 20 g analytical samples of flour and groats were extracted with 100 ml of the solvent. The row extracts were filtered through filter paper and 25 ml aliquot of each extract was taken for further SPE cleanup and pre-concentration.

Sample clean up.

25 ml of the acetone extracts were diluted with 200 ml bidistilled water and the diluted extracts were submitted to SPE clean-up/preconcentration. SPE was carried out by using polypropylene cartridges manually packed with 400 mg ENVI-Carb, conditioned with 10 ml ethyl acetate:methanol (8:2) followed by 5 ml methanol and washed with 10 ml water. The sorbent was never allowed to dry up during the conditioning and sample loading steps. The samples were forced to pass through the sorbent under vacuum at rate of 10 ml min -1 . After the passage of the sample, the cartridge was washed with 0.5 ml of methanol and dried under vacuum for 30 min. The pesticides were eluted with 6 ml ethyl acetate:methanol (8:2) at rate of 1 ml.min -1 and collected in 10 ml graduated tubes. The final volumes of the samples were adjusted by dilution or evaporation under gentle nitrogen flow in order to obtain analyte concentrations appropriate to quantitative determination.

Gas-Chromatographic determination.

GC-NPD operating conditions: carrier gas nitrogen, at flow rate 2.5 ml min -1 ; make up nitrogen 30 ml min -1 ; hydrogen -4 ml min -1 ; air -100 ml min -1 ; injector temperature 270 0 C; detector temperature 300°C. GC-ECD operating conditions: carrier gas nitrogen, at flow rate 2.5 ml min -1 ; make up nitrogen 30 ml min -1 ; injector temperature 270°C; detector temperature 300°C.

Oven temperature programme for both systems was: 100°C, hold 1 min; rate 20°Cmin -1 to 260°C final temperature, hold 3 min.

Injection volume was 1 µl for all samples. Quantification was performed by using external standards. Matrix-matched calibration standards were used for all quantifications.

The recovery studies were performed by using blank samples of untreated wheat grain. The weights of blanks were the same as the weights of the analytical samples of treated grains. The fortification was done by addition of calculated volumes of working standard solutions in order to obtain 3 concentration levels of the insecticides studied: at MRL (0.01 mg.kg -1 ), below MRL (0.005 mg.kg -1 ) and higher than MRL (0.02 mg.kg -1 ). Five parallel samples were prepared for each fortification level. After waiting time of 1 h for evaporation of the ethyl acetate from standard solutions the fortified samples were analysed according to the method described above.

Results and discussion

Single laboratory method validation:

The method was validated for the matrices studied.

The ECD response for chlorpyrifos-methyl was linear in the range of 0.005 ng -0.05 ng and the correlation coefficient r 2 was higher than 0.98 (five-point calibration curve).

The NPD response for pirimiphos-methyl was linear in the studied range of 0.01 ng -1.0 ng. The correlation coefficient r 2 for the five-point calibration curve was > 0.99.

Concentrations of the analytes in the samples were calculated by using single-level calibration method. This approach is recommended in case the detector response is variable with time (Document No SANCO/10476/2003). In our studies it was confirmed that single-level calibration gave more accurate results than multi-level calibration when the analytes concentrations in samples were adjusted in an interval ± 10% of the calibration level used. The mean recoveries and the repeatability expressed as standard deviation (SD), determined at three fortification levels (number of samples n=5) are presented in table 1.

The limits of quantification (LOQ) were adopted at 0.005 mg.kg -1 for both analytes.

The LOQ of the analytical method is strongly dependent not only on the response of the detector to the analyte but also on the matrix co-extractives in the final extract. It is possible to obtain higher sensitivity of the method for analytes of lower detector response if the sample extract does not contain interfering co-extractives. In case of pirimiphos-methyl the lowest calibrated level (LCL) was 0.01 microgram.ml -1 . The clean extracts free of co-extractives made it possible to adjust the matrix concentration in the sample final solution to 2g.ml -1 so the LOQ was 0.005 mg.kg -1 . In case of chlorpyrifos-methyl the LCL was 0.005 microgram.ml -1 but the matrix concentration in the sample final solution was only 1g.ml -1 because some coextractives caused higher chromatographic noise. For that reason the LOQ for chlorpyrifos-methyl was also 0.005 mg.kg -1 , despite its higher absolute sensitivity compared to pirimiphos-methyl. The LOQ was specified for both pesticides at this concentration level because it ensured compliance with European Commission criteria for quantitative residue methods (Document No SANCO/10476/2003).

Processing.

The laboratory mill used for processing of the grain was a conventional device used for simulated fractionation that corresponds closely to the fractionation under the real industrial process. The results of residue analysis obtained from processing experiments carried out on stored wheat grain, treated post harvest with chlorpyrifosmethyl and pirimiphos-methyl are presented in tables 2 and 3.

Both insecticides were persistent under the conditions studied. After storage period of 270 days the average level of pirimiphos-methyl residues in the whole grain was 5.2 mg.kg -1 which was 53.6% of the concentration determined in the day of treatment.

Under similar conditions chlorpyrifos-methyl was less persistent. The average residue level in grain after storage period of 270 days was 3.3 mg.kg -1 -42.3% of the initial concentration determined in the day of treatment. These results show that the residues on grain following post-harvest treatments with insecticides decline rather slowly. This corresponds with the results of long residual effectiveness of postharvest applied insecticides on storage pests [START_REF] Snelson | Grain Protectants[END_REF][START_REF] Holland | Effects of storage and processing on pesticide residues in plant products[END_REF].

Pirimiphos-methyl was reported as very stable in stored wheat with half-life at 30 0 C storage temperature 70 weeks. Chlorpyrifos-methyl under similar conditions was found to be less stable with half-life 19 weeks [START_REF] Holland | Effects of storage and processing on pesticide residues in plant products[END_REF].

The distribution of chlorpyrifos-methyl and pirimiphos-methyl residues in fractions from processed wheat is presented in tables 2, 3. The bulk of the insecticides deposited on the grain remain in the bran. The concentrations of chlorpyrifos-methyl in the bran are from 3.6 to 4.5 times as high as those present on the whole wheat grain. The residue levels of pirimiphos-methyl in the bran show a concentrating effect from 1.6 to 4.7 in comparison with the whole grain. Considerable part of the insecticides is distributed in the semolina fractions. The residues of chlorpyrifosmethyl and pirimiphos-methyl determined in semolina are only slightly lower the residues in bran and are respectively 2.0 -3.6 and 1.3 -3.2 times as high as those on the whole grain. Generally the residue levels of chlorpyrifos-methyl in flour are 1.6 -5.6 times lower of the concentration on the whole grain. The concentration interval of pirimiphos-methyl residues in flour is 2.1 -3.4 times lower than the residues on the whole grain. The reduction of residues in flour and groats is mostly due to the removal of the insecticides with bran and partly with semolina fractions.

In order to determine the percentage loss of residues during processing the residual amounts in the separate fractions were calculated on basis of the weight of each fraction and the corresponding residue concentration. The balance of residues in all fractions compared to the amount on whole grain before milling, determined in samples taken after 270 days storage (Table 4), shows that the loss of chlorpyrifosmethyl during processing is less than 2% of the total amount on whole grain.

Pirimiphos-methyl undergoes comparatively more substantial loss during milling -the loss of residues is determined to be about 10% (Table 4). Taking into consideration that volatilisation is one of the basic processes acting on pesticide residues this could be explained by the higher vapour pressure of pirimiphos-methyl-V.p.(40 0 C)=22mPa [START_REF] Tomlin | The Pesticide Manual, Twelfth Edition, British Crop Protection Council World Health Organisation[END_REF] and the processes favourable to volatilisation -grinding, screening etc.

Conclusions

The analytical method used was sensitive enough for reliable determination of chlorpyrifos-methyl and pirimiphos-methyl in wheat at concentration 0.01 mg.kg -1 .

The storage time and milling of wheat grain, treated post-harvest against storedproduct pests with chlorpyrifos-methyl and pirimiphos-methyl at recommended application rates, are not effective enough to reduce the residues in grain and in milled products to levels at/below the general MRL of 0.01 mg.

  established MRL from potentially contaminated raw materials. A study was thus undertaken aimed at assessment of the risk of baby food MRL violation due to utilisation of contaminated wheat grain for processing and production of baby food. It was focused on the influence of storage intervals and milling processes on behaviour of chlorpyrifos-methyl and pirimiphos-methyl applied on stored wheat grain.

  t -1 , at working solution volume 1 l. 40 kg of wheat grain for each treatment were spread in about 1 cm thick layer and sprayed with 40 ml of working solution. The application was accomplished by means of hand-operated After treatment the wheat grain of each particular variant (and of untreated control) was mixed thoroughly and kept in open containers in a storehouse at conditions simulating the practical grain storage. The room temperature was 20±2 0 C and the air moisture -70±5%. Samples of 0.5 kg grain of each variant were taken 1 hr after treatment, when the grain became dry, for determination of the initial concentrations of pesticides in the whole grain. The following samplings were made after 7, 30, 90, 180 and 270 days storage periods. 3 kg of each treated grain variant and of untreated control were taken on particular sampling dates and submitted to milling in laboratory mill, simulating the industrial processing.Milling was carried out in a fractionating laboratory mill Buhler MLU 202. Eight fractions: bran, semolina, groats (3 fractions), flour (3 fractions) were obtained and

  25 mm × 0.25 µm) was obtained from Alltech Associates, Inc. (Lokeren,

  kg -1 established by EC for any individual pesticide in processed cereal-based baby foods. The bulk of the insecticides deposited on the grain is removed with the bran and partly with the semolina fractions during milling but yet the reduction of residue concentrations in the flour and groats fractions is not sufficient for compliance with the baby foods safety requirements. All EC Directives concerning baby food safety stipulate that the exposure of infants and children to food contaminants is of particular concern because of their possible increased susceptibility for adverse effects. Growing and developing organisms have particular sensitivity to toxic compounds[START_REF] Dencker | Susceptibility in utero and upon neonatal exposure[END_REF]. The EC Directives on infant formulas and baby foods state that these products shall not contain any substance in such quantity as to endanger the health of infants and young children. The results of the work presented here indicate that the processed foods based on post-harvest treated wheat grain are not eligible for nutrition of infants and little children.Taking into consideration the MRLs for foodstuffs of general consumption, the results of this study indicate that processed products (flour, groats) of wheat, treated postharvest with the insecticides studied, are in compliance with the safety requirements for general population.

Table 1 .

 1 Recoveries and standard deviations of fortified pesticides from wheat versus matrix-

	matched standard calibration (n=5)
	Pesticide	Fortification level, mg.kg -1 Mean recovery ±SD, %
	Pirimiphos-	0.005	106±4.2
	methyl	0.01	98.5±2.2
		0.02	102±4.0
	Chlorpyriphos-	0.005	122±12.5
	methyl	0.01	123±6.0
		0.02	120±9.4

Table 2 .

 2 Decline of chlorpyrifos-methyl residues in whole grain and processed products with

	time (n=3)
				Mean residue, mg kg -1 ± SD
	Matrix			Days after treatment
		0	7	30	90	180	270
	Whole grain	7.8±0.6	5.4±0.4	3.4±0.6	3.0±0.2	3.1±0.05	3.3±0.04
	Bran		19.4±0.0	10.1±0.03 13.4±0.3	13.6±0.0	11.6±0.2
	Semolina		19.6±0.7	8.0± 0.02 6.1±0.1	6.80±0.6	8.0±0.1
	Groats 1 st fraction		1.5±0.1	1.0±0.02	1.1±0.02	1.01±0.06 1.1±0.0
	Groats 2 d fraction		3.0±0.1	1.3±0.1	1.1±0.001	1.3±0.06	1.1±0.0
	Groats 3 d fraction		4.7±0.4	1.6±0.1	1.4±0.001	0.9±0.03	1.3±0.03
	Flour 1 st fraction		3.7±0.3	0.9±0.003 0.7±0.03	1.2±0.06	0.8±0.01
	Flour 2 d fraction		5.1±0.04	0.6±0.03	0.9±0.01	1.1±0.0	1.2±0.0
	Flour 3 d fraction		4.7±0.4	1.0±0.001 1.4±0.01	1.5±0.0	2.1±0.1

Table 3 .

 3 Decline of pirimiphos-methyl residues in whole grain and processed products with

	time (n=3)
				Mean residue, mg.kg -1 ± SD
	Matrix			Days after treatment
		0	7	30	90	180	270
	Whole grain	9.7±0.7	6.8±0.6	7.2±0.1	6.9±0.3	6.5±0.2	5.2±0.1
	Bran		31.9±0.4	24.4±3.3	29.0±0.5	10.4±0.1	15.4±0.2
	Semolina		21.7±1.0	18.8±1.1	21.6±0.4	8.3±0.4	13.9±0.5
	Groats 1 st fraction		1.6±0.02	2.5±0.2	3.3±0.1	2.8±0.1	1.9±0.02
	Groats 2 d fraction		2.8±0.1	3.1±0.2	3.7±0.1	3.5±0.2	1.8±0.01
	Groats 3 d fraction		4.8±0.2	4.2±1.0	3.7±0.1	3.5±0.0	2.2±0.04
	Flour 1 st fraction		3.7±0.1	1.9±0.1	2.8±0.1	2.8±0.03	0.6±0.01
	Flour 2 d fraction		4.1±0.1	2.6±0.1	3.8±0.2	1.5±0.3	1.9±0.05
	Flour 3 d fraction		5.9±0.2	3.6±0.2	3.9±0.1	1.3±0.2	3.7±0.1

Table 4 .

 4 Residue balance of insecticides in processed products after 270 days of storage

	Product	Chlorpyrifos-methyl	Pirimiphos-methyl
		Amount (mean),mg	%	Amount (mean),mg	%
	Bran	5.0	50.5	6.8	43.6
	Semolina	2.7	27.3	5.2	33.4
	Groats 1 st fraction	0.1	1.0	0.2	1.3
	Groats 2 d fraction	0.2	2.0	0.3	1.8
	Groats 3 d fraction	0.1	1.0	0.2	1.2
	Flour 1 st fraction	1.0	10.1	0.07	0.4
	Flour 2 d fraction	0.5	5.1	0.8	5.3
	Flour 3 d fraction	0.2	2.0	0.3	2.1
	Total	9.8	99.0	13.9	89.1
	Whole grain	9.9	100	15.6	100

Study on the effect of grain storage and processing on chlorpyrifos- methyl and pirimiphos-methyl residues in post-harvest treated wheat with regard to baby food safety requirements

	A. Balinova, R. Mladenova and D. Obretenchev
	Plant Protection Institute, Kostinbrod 2230, Bulgaria

E-mail: balinova@internet-bg.net

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

Acknowledgements

The authors acknowledge the financial support of the NATO Program "Science for Peace" for Project № SfP 977983.


Sgoifo Rossi
email: fac@tandf.co.uk

Neoformation of boldenone and related steroids in faeces of veal calves

Keywords: boldenone, veal calves, faeces, urine

Conflicting findings regarding the boldenone content of bovine faeces suggests it may be synthesised de novo in emitted faeces. We tested this hypothesis by analysing uncontaminated urine, fresh and various forms of dried faeces from 10 calves (not given boldenone) by liquid chromatography/tandem mass spectrometry for 17α-and 17β-boldenone (α and β BOL); 1,4-androstadiene-3,17-dione (ADD); 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET). Urine contained no α BOL, β BOL or ADD. The analysed substances were variably present in the rectal faeces, and at generally higher levels in faeces scraped from skin or stall floor. In pooled rectal faeces naturally dried for 13 days, α BOL, ADD, AED and ET levels were extremely high (much higher than accounted for by increases due to drying), and β BOL and T were absent. It is concluded that de novo synthesis of α BOL and metabolites occurs naturally in bovine faeces and only uncontaminated urine should be analysed for illegal boldenone.

Introduction

The first report of the presence of boldenone in bovine faeces, published in 1998 (Van Puymbroeck et al.), proposed that faeces could be analysed as an alternative to urine to detect illegal dosing of cattle with this steroid. The authors attributed the high concentrations of 17α-boldenone (α BOL), 1,4-androstadiene-3,17-dione (ADD) and, in some samples, of 17β-boldenone (β BOL) present in slaughterhouse faeces, to oral administration of β BOL esters or precursors. More recently, [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF] found α BOL (520.0 ng/g) and β BOL (21.0 ng/g) in dried faeces from calves reported to be untreated; [START_REF] Sangiorgi | Boldenone and related hormones quantification by liquid chromatography-mass spectrometry in urine and faeces after calf administration of boldenone undecanoate[END_REF] detected these substances in rectal faeces from untreated calves, while [START_REF] Brabander | Presence and metabolism of anabolic steroid boldenone in various animal species: a review[END_REF] reported low concentrations of α BOL and (1->10 ng/g) and β BOL (0.1-2.0 ng/g) in faeces scraped off the skin of calves. It therefore seems that α and β BOL can be present in a wide range of concentrations in faecal materials sampled outside the animals.

Previous work from our group (Sgoifo [START_REF] Rossi | Evidence for false-positive results for boldenone testing of veal urine due to faecal cross-contamination during sampling[END_REF]) demonstrated that deliberate contamination of calves urine with faeces can render the urine positive for α BOL and occasionally β BOL. By contrast, [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF] found that boldenone epimers were absent from faeces collected from the rectum but present in The results of these analyses on various faecal samples (rectal faeces, dried faeces, faeces adhering to the animal's skin) from untreated animals, and faecescontaminated urine, cast doubt on the utility and reliability of measuring boldenone in faeces to provide and indication as to whether this substance is being administered illegally to veal calves. We hypothesised that the reason for these conflicting results might be that boldenone and related steroids may form de novo in calf faeces after emission. We therefore determined these steroids and the related substances ADD, 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET) in various faecal samples (fresh from individual animals, from animals' skins, and pooled naturally dried samples) as well as in urine, from animals not dosed with boldenone

Materials and methods

Animals

Ten Friesian veal calves, age 135±5 days, fed with milk replacer (2.8 kg /calf/day) and corn silage (1.3 kg/calf/day) were studied. The lipid content in the milk replacer was 20% (35% coconut and 65% tallow + lard). The animals were housed in individual stalls within a single cowshed and cared for in accordance with European

Materials

Analytical-grade solvents and reagents were purchased from Merck (Darmstadt, Germany). AED, ADD, β BOL, ET, T, d3 testosterone and β-glucuronidase from Helix pomatia (containing also sulfatase activity) were from Sigma-Aldrich (St Louis, MO, USA). α BOL was synthesized by modified Mitsunobo protocol [START_REF] Dodge | Alcohol inversion of 17-β-steroids[END_REF]. OASIS ® SPE columns (HLB 60 mg ) were from Waters (Millford, MA, USA). Bakerbond 500 mg SPE columns were from J.T Baker (Philipsburg, NJ, USA).

Urine collection and extraction

Urine (pH ranged from 6.8 to7.1) was collected into long-handled container after cleaning and trimming the sheath area and extracted according to the method described by [START_REF] Sangiorgi | Boldenone and related hormones quantification by liquid chromatography-mass spectrometry in urine and faeces after calf administration of boldenone undecanoate[END_REF] with slight modifications. Briefly urine samples (10 ml) from each of the ten animals were spiked with trideuterated (d3) testosterone (2 ng/ml) as internal standard, and brought to pH 4.5 with acetic acid; 100 µl of β-glucuronidase from Helix pomatia extract was added, and after overnight incubation at 37°C, the digest was solid-phase extracted (SPE) on a column Oasis® HLB (60 mg) previously activated with 3 ml methanol and 3 ml water. The column was washed with 2 ml methanol:NaOH 0.01 M (60:40) followed

Faeces collection and extraction

Faecal samples were collected as follows: (a) rectal faeces from each of the ten animals, (b) wet and dried faeces from the shed floor; (c) faeces scrapped from skin or fur of three animals; and (d) pooled rectal faeces samples from the 10 animals and allowed to dry for 13 days in the cowshed. Samples a-c were analysed immediately; pooled faeces were analysed at 4 and 13 days.

Faeces samples (2 g) were spiked (10 ng/g) with (d3) testosterone and dissolved in 6 ml acetate buffer (pH 4.8). After sonication and vortexing, 1 ml NaOH 1M plus 6 ml diethyl ether were added and the mixture head-over-head incubated for 20 min.

The sample was then centrifuged for 15 min at 2000 g and the organic layer collected. The ether extraction was repeated twice more. The organic phase was evaporated under nitrogen at 40°C and the residue dissolved in 800 µl ethanol:methanol (3:1). Isooctane (1.2 ml) and acetic acid 0.2 M (0.4 ml) were then added. The mixture was loaded onto a C18 SPE cartridge (Bakerbond SPE 500 mg) previously activated with methanol (5 ml) followed by water (5 ml). After washing with 3 ml H 2 O:methanol (95:5) and 3 ml n-hexane, and drying under vacuum the analytes were eluted with 2 x 2ml diethyl ether. The eluate was evaporated to held at 90% for 1 min, and then reduced to 65% in 1 min. An isocratic period of 5 min followed with 65% solvent B. Quantitative analyses were carried out comparing the peak areas of analytes with those of standard solutions.

The matrix calibration curves (four replications; six concentration points) were prepared by fortifying and then extracting blank samples with mixtures of the six analytes at 0.2-0.5-2.0-10-50-100 ng/ml (urine) or ng/g (faeces) levels and with I.S. at the concentration of 2.0 ng/ml in urine and 10 ng/g in faeces. For the full identification of the analytes the criteria established in the 2002/657/EC Commission Decision were followed.

Results and discussion

In urine, the calibration curve of AED was linear in the range 0.2 (LOQ) -100 ng/ml, while other analytes showed linearity from 0.5 ng/ml (LOQ). In faeces the curves were always linear in the range 2.0(LOQ)-100 ng/g. The correlation coefficients (r 2 ) ranged between 0.93 and 0.98.

Under our operating conditions the limits of detection (LODs), considered as the analyte amount giving a signal to noise ratio ≥ 3, resulted about 0.1 ng/g in urine and 0.5 ng/g in faeces. Recoveries determined on blank samples fortified with a mixture of the six steroids at 5, 10 and 50 ng/ml (urine) or ng/g (faeces) were α BOL, β BOL and ADD were never detected in urine samples, while AED, T and ET were always present (Table I).

[insert Table I about here]

Figures 1 and2 show the reconstructed LC/MS/MS chromatograms and the related MS/MS spectra of the analytes more frequently found in rectal faeces and in stall faeces, respectively, as below explained.

[insert Figs. 1 and 2 about here]

In rectal faeces (Table II), β BOL was present in all samples, α BOL was detected in four samples only, and at lower concentrations than β BOL. ADD and T were present in one sample each. ET was always present. AED was always absent.

[insert Table II about 2004) reported α BOL and β BOL at levels of about 10 ng/g, in rectal faeces from a six month-old calf not given boldenone or precursors, but not in another animal from the same shed and fed on the same diet.

We found that β BOL was always present in the rectal faeces from the animals studied. However additional rectal faecal sampling of these animals and of others housed in the same shed showed that β BOL was not always present, although it was present more often than α BOL (data not shown). Our findings therefore suggest that both α BOL and β BOL can be present in rectal faeces from untreated animals and that this presence is highly variable. This would explain contradictory literature findings, and furthermore indicates that analysis of rectal faecal samples is not a reliable method for detecting illegal dosing of boldenone since positive findings, particularly of β BOL, are common in the absence of boldenone administration. It is possible that boldenone was present in 'untreated' animals' faeces because it was given without the experimenters' knowledge. However, the results of the urine analyses exclude this in the present study (Table I): BOL 10 epimers and their metabolites were never detected. These substances always occur in urine following oral or parenteral boldenone administration [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF]Sangiorgi et al., 2004;[START_REF] Brabander | Presence and metabolism of anabolic steroid boldenone in various animal species: a review[END_REF].

The results of the analyses of faeces from the skin are shown in Table III.

[insert Table III about here]

β BOL was present in all faeces taken from the skins of the three animals sampled.

However, unlike rectal faeces, α BOL was present in most (7/9) of these samples, while ADD was present in three samples from skin, yet in only one rectal faeces sample. ET was always present in skin faeces, while T was present in one sample.

Thus α BOL, β BOL and ADD were more often present in crusted skin samples than in rectal faeces samples and generally at higher concentrations. Nevertheless concentrations of α BOL (0-988 ng/g) and β BOL (2.1-482 ng/g) were much more variable than concentrations in rectal faeces (2.6-89.0 ng/g both epimers).

Table III also presents the results of the analyses wet faeces collected from the cowshed floor and trampled by the calves (samples A, B, C and D) and of old dried faeces taken from the side of the cowshed (samples E and F). Samples E and F were the oldest faecal material analysed, and the concentrations of most steroids analysed (the exception being β BOL) were much higher in these than in wet samples. To assess de novo boldenone formation in faeces after emission, fresh samples from each animal were pooled, naturally dried in the cowshed, and analysed after 4 and 13 days (Table IV).

[insert Table IV about here]

As expected the dry matter content increased during drying (50% at day 4 and 85% at day 13) but the water loss was not sufficient to explain the overall increases in steroid concentrations shown in Table IV. In particular α BOL increased by about 1600-fold over the 13 days. ADD, AED and ET also increased massively. These findings indicate de novo synthesis. Our data also indicate that α BOL and ADD could not have originated from the β BOL present in rectal faeces. Although the β BOL level at day 4 increased by an amount compatible with drying, at day 13 it was not detected. Yet levels of α BOL and ADD at day 13 were very much higher can We therefore conclude from our data that one or more precursors in rectal faeces can be biotransformed into ET, ADD, AED and α BOL under certain conditions. These same conditions do not permit the production of β BOL, which nevertheless is usually present in rectal faeces. We propose that the anaerobic condition within the gut allow the production of β BOL from the same precursors. [START_REF] Groh | Steroid metabolism with intestinal microorganisms[END_REF] widely described the metabolic activities of intestinal micro-organisms on substrates like steroids, that can be degraded via reduction of the A ring, the delta 5bond, the 17-keto or cleavage of the C17 side chain. In his review, for instance, a strain of Pseudomonas was reported to anaerobically transform cholesterol into ADD.

Nevertheless at present we cannot indicate which are the precursors of BOL and related steroids in faeces. To elucidate it, studies are in progress in our laboratory.

The results of this study on rectal faeces apparently do not agree with those obtained by [START_REF] Van Puymbroeck | Identification of some important[END_REF] who found that, in faeces positive on routine screening α BOL and ADD were ubiquitously present, but β BOL was present in only 17 samples. Furthermore ADD concentrations were higher (up to 632 ng/g) than α BOL (up to 240 ng/g) and β BOL (up to 24.0 ng/g) concentrations. 13 However these authors stated that preliminary screening was preformed to detect α BOL, and this suggests that the quantitative analysis was not performed immediately after faeces emission. If the faeces had been in contact with air for some time it is possible that the transformations we observed in the present study occurred in their study. The study of [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF] on untreated calves, reported in dried faeces similar concentrations and concentration ratios (ADD 790 ng/g, α BOL 520 ng/g, β BOL 21.0 ng/g) to ours.

In the light of these considerations, the proposal of [START_REF] Van Puymbroeck | Identification of some important[END_REF] that illegal oral treatment with boldenone precursors or esters is the only explanation for high concentrations of α BOL and ADD in cattle faeces, has to be revised.

The significance of α BOL and β BOL findings bovine urine must also be reassessed in the light of our findings on faeces. [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF] suggested that urine contamination by faecal matter must be unrealistically high in order to give a positive β BOL result in urine. However this conclusion was based on the finding of 21.0 ng/g β BOL in one faeces sample only. We found that faeces from skin could have a β BOL concentration of up to 482 ng/g, suggesting that contamination with a few milligrams of such material would be enough to give a positive urine result.

These considerations also apply to α BOL and ADD. We calculate that 4 mg of dried faeces containing 2680 ng/g of α BOL and 13550 ng/g of ADD (Table IV) 14 would produce detectable contamination of 10 ml of urine (1.0 ng/ml of α BOL and 5.4 ng/ml of ADD). Contamination at these levels is possible when urine is collected with a container without prior cleaning and trimming of the sheath area or when a collection apron (tied round the animal) is used.

However, simple transfer of steroids to urine is only one aspect of faecal contamination. The high de novo production of α BOL, ADD, AED, and ET occurring in faeces after deposition is almost certainly due to microbial activity and bacteria, as well as undigested food and catabolism products would be transferred to urine by faecal contamination. It is therefore possible that de novo synthesis of these  steroids also occurs in urine. This hypotesis is in agreement with the data of de la Torre et al. (2001), which demonstrated that a de novo synthesis of androgen steroids (e.g. AED from dehydroepiandrosterone sulfate) can occur in urine contaminated with bacteria, so causing alterations of the urine steroid profile.

Contamination of urine with faecal matter is therefore likely to result in highly variable presence of α BOL or β BOL since microbic conversion and/or de novo synthesis will be influenced by variable storage conditions prior to analysis.

The contamination with faeces can make urine positive (via a transfer or a neo formation) to α BOL and β BOL that will be detected in their free (unconjugated) form [START_REF] Rossi | Evidence for false-positive results for boldenone testing of veal urine due to faecal cross-contamination during sampling[END_REF][START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF]: the detection of these steroids in 15 their conjugated form could therefore be used to discriminate between the presence due to contamination and the presence due to treatment [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF][START_REF] Brabander | Presence and metabolism of anabolic steroid boldenone in various animal species: a review[END_REF]. At the moment, however, it is not yet possible to affirm that the presence in veal urine of conjugated β BOL surely indicates an illicit treatment.

It is moreover unclear whether boldenone and metabolites in rectal faeces derive from biliary excretion or anaerobic conversion of the same precursors that give rise to them outside the animal. The second possibility would explain the occasional finding of α BOL and β BOL in rectal faeces for untreated calves, in which testicular synthesis has been excluded [START_REF] Brabander | Presence and metabolism of anabolic steroid boldenone in various animal species: a review[END_REF]. In such cases the presence of conjugates of α BOL and β BOL in urine might be due to production in, and absorption from the enteric tract, but our results (presence of β BOL in faeces and absence in urine from the same animal) seem to contrast with this hypothesis. However factors influencing these processes are unknown and it is therefore not yet possible to correlate the faecal concentration of β BOL with the presence of α and β BOL in veal urine.

Moreover, conjugated α BOL (4ng/ml), was found in bovine colostrum samples by [START_REF] Brambilla | Trends in animal feed composition and the possible consequences on residue tests[END_REF] and α and β BOL (7 ng/g and 3 ng/g, respectively) were detected in a sample of milk replacer by [START_REF] Nielen | Confirmatory analysis of 17-β-boldenone, 17-α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF], so these steroids and/or their precursors could come from feed and be excreted in urine according to the mechanism reported by [START_REF] Rapp | Determination of hormone contaminants in milk replacers by High-Performance Liquid Chromatography and immunoassay[END_REF] with regard to 19-nortestosterone. In this contest the intake by the animals (licking activity) of dried and skin faeces containing the high concentrations of ADD, AED and BOL reported in this paper could represent a considerable source of these steroids. Further studies are necessary to understand the amount of boldeneone an/or precursor taken by this way.

Finally, this study showed that large amounts of α BOL and ADD are ejected with veal calves faeces and that these steroids must be added in the list of natural hormones (T, ET and AED) present in livestock excreta reported by [START_REF] Lange | Sex hormones originating from different livestock production system: fete and potential disrupting activity in the environment[END_REF]. An evaluation of their fate in the environment should be carried out.

F

  the low β BOL level of 21.0 ng/g, and concluded that only massive and unlikely contamination would result in urine positivity to β BOL.

  No. 86/609/EEC) approved by the Italian Ministry of Health (L.116/92).

  purified water and the analytes eluted with methanol (2 x 2 ml). The eluate was evaporated to dryness under nitrogen at 40°C and the residue dissolved in 200 µl MeOH:H 2 O (50:50). 10 µl samples analysed by LC/MS/MS.

  at 40 °C and the residue dissolved in 200 µl methanol:H 2 O (50:50). 10 µl samples were analysed by LC/MS/MS.The water content of faeces was determined after drying till no further weight loss in an oven at 100°C.LC/MS/MS analysisThe LC/MS system consisted of a ThermoFinnigan LC Surveyor (S. José CA, USA) equipped with a LCQ Deca XP Max ion trap mass spectrometer (ThermoFinnigan, S. José, CA, USA). The mass spectrometer was operated in positive atmospheric pressure chemical ionization (APCI) mode with capillary voltage of 31 V, capillary temperature of 220°C, discharge current of 4.5 µA, vaporizer temperature of 350°C and sheath and auxiliary gas (nitrogen) flow rates of 20 and 8 arbitrary units, respectively. Helium was used for collision induced dissociation (CID). The molecular ions (α BOL, β BOL and AED m/z 287; ADD m/z 285; and T and ET m/z 289) were precursors for CID. The most abundant product ions were identified: the associations m/z 287→173, 287→147 and 287→135 for α BOL and β BOL; 285→151, 285→147 and 285→121 for ADD; 287→97 and 287→109 for AED, and 289→253 and 289→271 for T and ET. The analytical column was a 150 mm x 2.1 mm i.d., 5 µm ODS Hypersil-Keystone (ThermoFinnigan, S. José, CA, USA) in a column oven at 30°C. The mobile phase was water with 0.1% acetic acid (A) and methanol (B), flow rate 0.3 ml/min. Solvent (B) was kept at 65% for 1 min, increased to 90% over 7 min,

  122%. No significant differences in recoveries were noted between 17 -keto and 17 hydroxy steroids.

  here] Data regarding the presence of boldenone in rectal faeces are contradictory. Studies by Rijkskwaliteitsinstituut voor Land en Tuinbouwproducten (RIKILT) and α BOL (42.4 ng/g) and β BOL traces (0.04 ng/g) in rectal faeces from an untreated calf, although neither substance was present in faeces from the same animal sampled five days later. Recently, Sangiorgi et al. (

  content of dried faeces (from skin and stall floor) cannot be explained by concentration of analytes due to the water loss. The dry matter of rectal faeces was 10-15% and complete water loss could result in an increase in analyte concentrations by a maximum of 10 times. Since the dry matter content of the samples shown in TableIIIranged from 40% (A, B, C and D) to 85% (E and F), only neoformation of α β BOL, ADD, AED and ET can explain the higher concentrations in dried material.

  conversion of the β BOL originally. It is also evident that ET and AED could not have been produced by transformation of T, since this was present initially -at very low levels -in only one sample of rectal faeces.

Figure 1 -

 1 Figure 1 -Reconstructed LC-MS/MS chromatograms of a sample of rectal

Figure 2 -

 2 Figure 2 -Reconstructed LC-MS/MS chromatograms of a sample of stall faeces

Table I .

 I Concentrations of α BOL, β BOL, ADD, AED, T and ET in urine (ng/ml)

	metabolites of boldenone in urine and faeces of cattle by gas chromatography-mass
	spectrometry. Analyst, 123: 2681-2686. Animal α α α α BOL β BOL ADD	AED	T	ET
	1	nd		nd		nd	0.25	1.45	3.40
	2	nd		nd		nd	0.60	1.30	3.40
	o r F o r F o r F o r 3 nd 4 nd F o r	nd nd		nd nd	0.59 0.45	2.15 1.40	6.40 9.65
	5	P P P P nd P	nd		nd	0.25	6.05	17.90
	6 7	nd nd	e e r e e r e e r e e r nd e nd e r	nd nd	0.86 1.12	8.15 5.45	22.80 31.4
	8	nd		nd	R R R R R	nd	0.57	1.23	8.00
	9	nd		nd	e e e e nd e	0.72	2.30	7.10
	10 nd: < LOD	nd		nd	v i e v i e v i e v i e nd 0.46 v i e	4.70	16.80
							w w w w w
							O n l O n l O n l O n l O n l
								y y y y y
									19 20 21

Table II .

 II Concentrations (ng/g) of α β BOL, β BOL, ADD, AED, T and ET in rectal

	faeces
	Animal	α BOL	β BOL	ADD	AED	T	ET
	1	nd	30.6		nd		nd	nd	47.9
	2 3 F o nd 5.9 r	54.6 29.8		nd nd		nd nd	nd 4,4	9.5 7.5
	4 5	nd nd P e 31.1 36.8		nd nd		nd nd	nd nd	6.5 2.4
	6 7	nd 5.1	55.2 54.9 e r	nd nd		nd nd	nd nd	17.4 26.4
	8	3.1	89.0 R	nd		nd	nd	44.1
	9 10 nd: < LOD	2.6 nd	35.3 27.6	nd e 20.9 v i e	nd nd	nd nd	11.1 2.4
						w
							O n l
								y
								22

Table III .

 III Concentrations (ng/g) of α BOL, β BOL, ADD, AED, T and ET in faeces

	from skin and stall
	Animal	Sample	α α α α BOL β BOL ADD	AED	T	ET
	1 1 1 F o skin skin skin r	14.5 nd 32.8		437 2.1 268	nd nd 4.3		nd nd nd	nd nd nd	17.0 16.1 120
	2	skin P e 988		318	76.7	nd	4,5	179
	2 2	skin skin	7.3 858 e r	3.9 194	nd 19.7	nd nd	nd nd	51.8 251
	3	skin	nd R 8.1	nd		nd	nd	16.8
	3 3 A* B*	skin skin stall stall	853 401 nd nd	482 e 308 nd v i nd nd 14.6 e nd 40.5 w	nd nd nd nd	nd nd nd nd	70.5 71.0 12.6 7.1
	C* D*	stall stall	15.3 6.4		18.6 28.5	nd nd	nd nd O n l nd nd	68.6 186
	E**	stall	3090		nd	1490	12070 y nd	11000
	F**	stall	459		nd	401	2900	nd	3770
	* wet samples collected from cowshed floor; ** old dry samples collected from
	corner of cowshed; nd: < LOD
									23

Table IV .

 IV Concentrations (ng/g) of α BOL, β BOL, ADD, AED, T and ET in Mean of the ten values reported in Table II; nd: < LOD

	naturally dried faeces
	Sample	α α α α BOL β BOL	ADD	AED	T	ET
	Pooled rectal faeces *	1.7	44.5	2.1	nd	0.5	17.5
	Pooled faeces at day 4	51.9	102.0	15.7	34.2	nd	195.0
	Poled faeces at day 13 2680	nd	13550	5790	nd	16580
	•

Rijksinstituut voor Volksgezonheid en Milieu (RIVM), as reported by De[START_REF] Brabander | Presence and metabolism of anabolic steroid boldenone in various animal species: a review[END_REF], found neither α BOL nor β in rectally sampled faeces from

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

Acknowledgements

We thank Dr. Silvana Casati of the Department of Preclinical Sciences LITA (Laboratorio Interdisciplinare Tecnologie Avanzate) Vialba (Milan, Italy) for the synthesis of α BOL.


Ron Laurentius Hoogenboom
email: ron.hoogenboom@wur.nl

Marco J Zeilmaker

Wim A Traag

Kees Kan

Ron ; Hoogenboom

L A P Hoogenboom

C A Kan

J Van Eijkeren

Carry-over of dioxins and PCBs from feed and soil to eggs at low 6 contamination levels -Influence of mycotoxin binders on the 7 carry-over from feed to eggs 8 9 10 11 12

Keywords: Dioxins, PCBs Food Types: Animal, Eggs, Animal feed dioxins, PCBs, carry-over, chickens, eggs, feed, soil 44

teaching and research institutions in France or abroad, or from public or private research centers.

Carry-over of dioxins and PCBs from feed and soil to eggs at low contamination levels; Influence of mycotoxin binders on the carry-over from feed to eggs

Incidents with residues of PCBs and dioxins have shown that these compounds pose a major 46 threat to the quality of edible products derived from food producing animals. As the exposure 47 of part of the populations in Western countries still exceeds the exposure limits set by e.g. the 48 Scientific Committee on Food [START_REF] Scf | Opinion of the Scientific Committee on Food on the risk assessment of dioxins 435 and dioxin-like PCBs in food[END_REF], the EU has developed a strategy for further 49 reducing the exposure to dioxins and dioxin-like PCBs. This includes the establishment of 50 residue limits for dioxins in food [START_REF] Ec | Council regulation (EC) No 2375/2001 of 29 November 2001 amending 393 Commission Regulation (EC) No 466/2001 setting maximum levels for certain 394 contaminants in foodstuffs[END_REF] and feed [START_REF] Ec | Council directive 2001/102/EC of 27 November 2001 amending Directive 397 a999/29/EC on the undesirable substances and products in animal nutrition[END_REF]. 51

Since the dioxin crisis in 1999 in Belgium, the Dutch government established a number 52 of monitoring programmes for dioxins aiming at the early detection of contaminated feed and 53 food and subsequently the revelation and elimination of novel sources of dioxins. In 2001, 54 over 300 samples of animal origen were screened in the DR CALUX-assay and if suspected 55 positive for dioxins, analysed by HRGC/HRMS. In one case, eggs obtained from a small farm 56 with free-range hens were shown to contain dioxin levels above the then existing Dutch limit 57 of 5 pg TEQ/g fat [START_REF] Traag | Residues of dioxins and coplanar PCBs in eggs of 455 free range chickens[END_REF]. This initial observation was followed by a focussed 58 action on these types of eggs and showed a clear elevation in dioxin levels in eggs from free-59 range hens, in particular those from farms producing eggs according to strict organic 60 standards. This was confirmed in a survey by The Dutch Food Inspection Service, showing 61 that eggs from six out of 68 farms would exceed the new EU dioxin limit for eggs of 3 pg 62 TEQ/g (as of July 2003), and three exceeded the then existing limit of 5 pg TEQ/g (De Vries, 63 2002). In addition some of the samples contained high levels of dioxin-like PCBs, with a 64 highest total TEQ level of 15 pg TEQ/g fat. Follow-up studies were performed during the 65 winter at the first farm, and the levels in eggs from hens that were kept inside, strongly 66 suggested that the source of the contamination was not the feed, but the outdoor environment 67 (unpublished). Similar has been observed in other studies, but in these cases the soil was 68 clearly contaminated with dioxins [START_REF] Stephens | 442 Bioaccumulation of dioxins in food animals II: Controlled exposure studies[END_REF], 1994[START_REF] Stephens | Biotransfer and bioaccumulation of dioxins 450 and furans from soil: Chickens as a model fro foraging animals[END_REF][START_REF] Schuler | The transfer of polychlorinated dibenzo-p-dioxins 439 and dibenzofurans from soil into eggs of foraging chicken[END_REF] [START_REF] Lovett | PCB and PCDD/DF concentrations in 418 egg and poultry meat samples from known urban and rural locations in Wales and 419 England[END_REF][START_REF] Harnly | Polychlorinated dibenzo-p-dioxin 401 and polychlorinated dibenzofuran contamination in soil and home-produced chicken eggs 402 near pentachlorophenol sources[END_REF], Petreas et al. 2001, Air et al. 2002, Pussemier et al. 70 2004). Another study was performed in the autumn of 2003, when a large number of farms 71 producing organic eggs were visited and investigated for possible sources of dioxins and 72 factors that may contribute to the exposure of hens to dioxins [START_REF] Brandsma | Onderzoek naar factoren die het 383 dioxinegehalte in biologische eieren kunnen beïnvloeden, Praktijkrapport 384 Pluimveehouderij nr. 1330386003[END_REF]). This 73 study again showed that most eggs would comply with the current dioxin limit of 3 pg TEQ/g 74 fat, but that 13% of the eggs, produced on 26% of the farms would not be in compliance with 75 this limit. 76

In July 2003, new EU limits for dioxins in food and feed became official, including limits 77 for eggs and feed, being respectively 3 pg TEQ/g fat and 0.75 ng TEQ/kg feed [START_REF] Ec | Council regulation (EC) No 2375/2001 of 29 November 2001 amending 393 Commission Regulation (EC) No 466/2001 setting maximum levels for certain 394 contaminants in foodstuffs[END_REF](EC , 78 2002)). Eggs from free-ranging hens were excluded from these limits until 1-1-2004, later 79 extended to 1-1-2005. At the same time, studies were started to investigate the carry-over of 80 dioxins and PCBs from feed and soil to eggs at relatively low levels, and to investigate 81 possible ways for reducing the exposure and carry-over. The current paper describes a 82 controlled study on the relationship between dioxin and PCB levels in eggs and feed (Study 83 I), as well as soil (Study II). Contrary to previous studies 6 different levels in feed were used 84 in a range around the current and proposed EU-limits for dioxins and dioxin-like PCBs. In 85 addition we studied the depletion of these contaminants, which is relevant for risk 86 management purposes in cases of crisis situations. As a potential tool to decrease the carry-87 over to eggs we also tested the effects of so-called mycotoxin binders on the carry-over of 88 dioxins and PCBs (Study III). The results of the studies were used to develop a mathematical 89 model based on the physiology of the laying hen, that can be used for predicting the levels in 90 eggs resulting from a certain feed contamination, and the effect of certain variables on this 91 relationship (Van Eijkeren et al. 2005). 92 Feed was prepared with soy oil spiked with the different dioxin and PCB congeners. The 98 composition of the feed is given in Table 1. A blanc oil, obtained from Nutreco (Boxmeer, 99

The Netherlands) was analysed by both CALUX and by GC/MS and shown to be low in 100 dioxin-like compounds. Subsequently, a stock solution of contaminated oil was prepared with 101 about 7500 pg TEQ/g of dioxins and dioxin-like PCBs. An aliquot of 200-gram oil was mixed 102 with the following PCB stock solutions: 7.5 ml PCB 105 at 100 ng/ml, 15 ml PCB 118 at 100 103 ng/ml, 1.2 ml PCB 156 at 100 ng/ml, 7.5 ml PCB 28 at 100 ng/ml, 15 ml PCB 52 at 100 104 ng/ml, 30 ml PCB 101 at 100 ng/ml, 30 ml PCB 138 at 100 ng/ml, 30 ml PCB 153 at 100 105 ng/ml and 15 ml PCB 180 at 100 ng/ml. The organic solvent (nonane) was removed under 106 vacuum. Subsequently the following standard mixtures were added: 44 µl CIL EDF-4096 107 dioxins (tetra's 2.5 ng/µl, penta's, hexa's and hepta's 6.25 ng/µl and octa's 12.5 ng/µl), 408 µl 108 CIL EC-4986 non-ortho PCBs (10 µg/ml) and 785 µl CIL EC-4987 mono-ortho PCBs (10 109 µg/ml). The organic solvent again was removed under vacuum (1 hr, 63ºC). 110

The Hens (ISA Brown Warren, age about 25 weeks at the start) were housed in a three-tier battery 156 with 2-3 hens per cage. The seven groups of 5 animals followed an adaptation period of one 157 week on clean feed, and then were fed with the different feeds for 32 days. During the whole 158 32-day period eggs were collected and pooled per day per feeding group. On day 32 the hens 159 from each group were slaughtered and abdominal fat, livers and the ovaries collected. 160 161

Analysis of dioxins and dioxin-like PCBs 162

Levels of dioxins, non-ortho and mono-ortho PCBs were determined by high resolution 163 GC/MS, basically as described by [START_REF] Lgmth | The Dutch PCB/dioxin 458 study: development of a method for the determination of dioxins, planar and other PCBs in 459 human milk[END_REF]. Eggs and ovaries were freeze-dried 164 and subsequently fat was extracted using Accelerated Solvent Extraction (ASE). Abdominal fat 165 was melted in an oven at 70°C. After extraction or sample pre-treatment 13 C labeled dioxins and 166 dioxin-like PCBs were added to the fat phase of the samples. Feed samples were extracted with 167 ethyl acetate or, when minerals or soil was added to the feed (study II and III), with toluene using 168 ASE. Prior to extraction 13 C labeled dioxins and dioxin-like PCBs were added to the feed 169 samples. Separation between dioxins and fat was carried out using gel permeation 170 chromatography. The system consisted of an HPLC pump (Gilson,model 305,an autosampler 171 (Gilson,model 231) equipped to inject 12,5 ml of sample solution, and a fraction collector 172 (Gilson, model 202) adapted to collect 300 ml fractions using 500 ml glass collection flasks. The 173 glass GPC column (Spectrum) (60 x 2.5 cm) was packed with Biobeads SX 3. After an 174 additional clean up with activated Al 2 O 3 , separation between planar compounds (dioxins and 175 non-ortho PCBs) and non-planar compounds like other PCBs was carried out with porous 176 graphitised carbon. The alumina (basic) clean-up was performed with an automatic sample 177 preparation system using solid phase extraction columns (ASPEC, Gilson). The columns were 178 packed with 1 gram deactivated alumina (7% water) shortly before use. Porous graphitised 179 carbon clean up was performed using an HPLC system consisting of an HPLC pump (Gilson 180 model 205), a column switching device (Gilson, valvemate), a solvent switching device (Gilson,181 valvemate), an autosampler (Gilson model 231), equipped with a 5 ml loop and a fraction 182 collector (Gilson model 202) adapted to collect 100 ml fractions. The column used was 183 Hypercarb (100 X 4.6 mm) (Shandon). The final extract with planar compounds was concen-184 trated to 10 µl and the extract containing the non-planar PCBs to 200 µl. Both extracts were 185 analysed with gas chromato-graphy-high-resolution-mass spectrometry (HRGC-HRMS) 186 (Autospec, Micromass). The mass spectrometric method to determine the tetra through octa 187 dioxins is based on United States Environmental Protection Agency protocols. Included in the 188 analysis is a standard QA programme e.g. determination of recovery of internal standards, 189 accuracy of spiked samples and blanks. Non-detectable congeners were quantified at the limit of 190 detection (upperbound). Absolute levels were transfered to TEQ levels using the TEF values 191 described by [START_REF] Van Den Berg | Toxic Equivalency Factors (TEFs) for PCBs, 465 PCDDs, PCDFs for humans and wildlife[END_REF].

Analysis of indicator PCBs 193

The seven indicator PCBs (28, 52, 101, 118, 138, 153 and 180) were analysed by GC/MS 194 following on-line clean-up over a silica HPLC column using a large volume injector (LVI). In 195 short, 200 mg of the homogenised fat sample is mixed with 800 µl of the internal standard 196 solution of 13 C PCB 118 and PCB 198 (6.25 ng/ml). Subsequently, 50 µl is injected onto the 197 silica column and after elution of the fat with hexane, the direction of the flow is switched to 198 back-flush and the column is cleaned with 3 ml dichloromethane. The PCB containing 199 fraction (approximately 1 ml) is directly transferred to the gas chromatograph, which is 200 provided with an LVI. In the LVI the large amount of solvent was separated from the 201 compounds of interest by an uncoated retention gap of 10 meters, which ends up in a retaining 202 column of 1-meter coated with a non-polar phase. The temperature of the LVI, located in the 203 oven of the GC, is slightly (80°C) above the boiling point of the mobile phase. Hexane is 204 evaporated slowly and blown off via the Solvent Vapour Exit (SVE) while the PCB's are 205 retained on the 1-meter retaining column. After a specific time the majority of the solvent was 206 evaporated, the SVE closed and the temperature of the oven was raised in order to start 207 chromatography. Detection was done by mass-spectrometry with a bench top system in 208

Selected Ion Mode (SIM). 209 210

DR CALUX ® analysis 211

The DR CALUX ® test was carried out as described earlier by [START_REF] Bovee | Validation and use of the CALUX-bioassay for the detection of dioxins and coplanar 379 PCBs in bovine milk[END_REF]. An aliquot of 212 0.5 g fat was mixed with hexane and extracted on acid silica as described previously. In each 213 test series a blanc butter fat sample and butter fat samples containing 1, 2, 3 and 6 pg TEQ/g 214 fat were included. The response obtained with the butter fat sample of 3 pg TEQ/g, containing 215

1.5 pg TEQ/g dioxins and 1.5 pg TEQ/g non-ortho PCBs, was used as the reference signal. 216 PCBs to the total TEQ level was as intended, being respectively 50, 30 and 20%. Table 3 232 shows the levels of the various congeners in the two highest contaminated feeds. The overall 233 levels were higher than intended, which cannot be explained by the background levels in the 234 feed ingredients, as shown by the level in the blank feed (A). However, since a rather broad 235 range of different concentrations in feed was used, this deviation had no consequences for the 236 aim of the study. 237 238

Levels in eggs 239

Table 4 shows the average feed consumption and the productivity of the hens, both during and 240 after the exposure period. There was no effect of the treatment on these parameters. The 241 exposure did not affect the average egg weight, but in all groups a slight decrease was 242 observed after 3.5 weeks on clean feed, resulting in weights around 48-51 grams per egg 243 (without shell). There is no explanation for this decrease, or possible consequences for the 244 residue levels in the eggs. 245 that even after 56 days on contaminated feed, levels in eggs continued to increase. In a similar 248 study, [START_REF] Pirard | Uptake of polychlorodibenzo-p-dioxins, polychlorodibenzofurans 429 and coplanar polychlorobiphenyls in chickens[END_REF] exposed laying hens to a dioxin level ten-fold higher than 249 our highest concentration. They observed a continuous increase in the levels for 4 weeks, at 250 which time point the animals stopped laying eggs. As shown in Figure 1, after cessation of the 251 exposure, there was initially a small increase in the residues, than a rapid decline, and 252 subsequently a much slower decrease. A similar profile was observed previously in a study 253 with much higher dioxin and PCB levels [START_REF] Hoogenboom | Residues of dioxins and PCBs in eggs following short-term exposure of laying hens 406 to feed from the Belgian crisis[END_REF]). The explanation lies in 254 the fact that the production of a full-grown yolk requires about ten days. Thus yolks of eggs 255 laid during the first days on clean feed were largely formed and contaminated during the 256 exposure period. Redistribution of dioxins from abdominal fat to the general circulation and 257 thus to eggs, may explain the slow decrease in residues during the latter part of the study. 258

Figure 1A also shows that the current limit for eggs of 3 pg TEQ/g fat was rapidly exceeded, 259 when exposed to feed containing dioxins just above the current limit for feed of 0.75 ng 260 TEQ/kg. 261

Figure 2 shows the levels of the 7 indicator PCBs in egg fat during the exposure and 262 depletion period. In general the pattern was very similar to that of the dioxins and dioxin-like 263 PCBs. Again, data indicate that egg levels continued to increase even after 56 days. At similar 264 dose levels (1.8 or 6 µg/kg feed), De [START_REF] Vos | Digestibility, retention and incorporation of 390 low-level dietary PCB content in laying hens[END_REF] observed steady-state levels after 265 respectively 40 or 70 days. The highest level obtained at 56 days of exposure via the most 266 contaminated feed was around 325 ng/g fat. Based on 4% fat in the feed, the feeds E and F 267 would contain respectively 350 and 800 ng/g fat, thereby exceeding e.g. the Belgium limit for 268 feed of 200 ng/g. Only eggs from hens fed the highest contaminated feed F would actually 269 exceed the Belgium limit for indicator PCBs of 200 ng/g fat, although it cannot be excluded 270 that after longer exposure also eggs from hens fed with feed E might have approached the 271 limit. 272 The relationship between the dioxin levels in feed and eggs after different periods of 273 exposure is graphically presented in Figure 3. There is a clear linear relationship between the 274 feed levels and the levels in eggs at the different exposure times. This figure also clearly 275 demonstrates that even after a short period on feed contaminated at the current EU-limit of 276 0.75 ng TEQ/kg, the limit for eggs was exceeded. 277 278

Levels in abdominal and ovary fat 279

The time-related levels of the sum of dioxins and dioxin-like PCBs (A), as well as the 7 280

indicator PCBs (B) in egg, abdominal and ovary fat sampled from the chickens fed with feed 281 E are shown in Figure 4. During the 56-days exposure period, levels in eggs and ovary fat 282 were comparable and generally higher than in the abdominal fat. This was particularly true for 283 the indicator PCBs. This difference was also clear for either dioxins or dl-PCBs (data not 284 shown). After another 56 days on clean feed, levels of dioxin-like compounds were very 285 similar in ovary and egg fat and slightly lower than in the abdominal fat. However, levels of 286 the indicator PCBs were clearly elevated in the abdominal fat. The combined results indicate 287 that the decrease of dioxins and PCBs is much slower in the body fat than in the eggs and 288 related ovaries. From the model for carry-over (Van Eijkeren et al.) it appears that the levels 289 in eggs possess a clearly bi-phasic nature, an initial phase with fast rising or, after cessation of 290 contamination, fast declining levels, followed by a terminal phase with slowly increasing or 291 decreasing levels. On the other hand, in abdominal fat, which acts as a great capacitor for 292 these highly lipophilic compounds, the relative contribution of the fast rising phase is very 293

Carry-over rates of individual congeners from feed to egg 297

To compare the carry-over of individual congeners from feed to eggs, it is possible to 298 calculate the so-called Carry-Over Rate (COR), defined as the fraction of a certain congener 299 excreted in the eggs, in relation to the amount ingested from the feed. Ideally, this should be 300 done under steady-state conditions, but this was not achieved within the current study period. 301

Therefore, the COR values should be regarded as minimum values. Based on the daily egg fat 302 production of 5.1 g and daily feed intake of 116 g, CORs were calculated for feeds E and F in 303 I (Table 3). These data suggest a COR for the more important lower chlorinated congeners 304 around 40% and a lower COR for the higher chlorinated hepta and octa congeners. This is in 305 line with the observation by [START_REF] Pirard | Uptake of polychlorodibenzo-p-dioxins, polychlorodibenzofurans 429 and coplanar polychlorobiphenyls in chickens[END_REF] who also showed a much higher 306 excretion of the higher chlorinated PCDD/Fs into the feces. The non-ortho and mono-ortho 307 PCBs showed similar CORs as the lower chlorinated dioxins although some mono-ortho PCB 308 congeners appeared to have even higher CORs. These were present at relatively low 309 concentrations and in practice do not contribute significantly to the TEQ levels. In the group 310 of the indicator PCBs, the lower chlorinated PCBs 53 and 101 showed very low carry-over 311 rates, contrary to PCB 28. Similar has been observed previously in laying hens (De Vos et al. 312 2005) and broilers (Hoogenboom et al. 2004b[START_REF] Maervoet | 421 Accumulation and tissue distribution of selected polychlorinated biphenyl congeners in 422 chickens[END_REF]). Based on excretion in 313 the feces, De [START_REF] Vos | Digestibility, retention and incorporation of 390 low-level dietary PCB content in laying hens[END_REF] showed that active metabolism of these congeners must 314 underly this observation. 315

Before steady state conditions are reached, part of the absorbed compounds are still 316 accumulated in the body fat. Based on the mathematical modelling it is expected that after 317 reaching steady-state, COR values will be doubled, implying that the amounts excreted in the 318 eggs will be close to the amounts ingested (Van Eijkeren et al. 2005). As for total TEQ levels, 319 the absorbed fraction ranges between 90% and 100%, the COR for total TEQs at steady state 320 is expected to range between the same limits. 321

Carry-over from soil to eggs (study II) 323

Table 5 shows the levels of dioxins and PCBs in feed E, feed E mixed with 10% sand and the 324 two blank feeds mixed with 10% of the soils sampled at two chicken farms. The two soils 325 were reanalysed and shown to contain respectively 4.0 and 1.7 ng TEQ dioxins per kg and 326 only minor amounts of dioxin-like PCBs. The level in the feed containing soil A is in 327 agreement with the dilution, the level in the feed with soil B is relatively high which can best 328 be explained by the application of the upperbound principle. Lower bound levels (i.e. levels 329 of non-detects set at 0) in these two feeds were respectively 0.40 and 0.19 ng TEQ/kg, 330 reflecting the differences observed in the soil. 331

Figure 5 shows the dioxin and dioxin-like PCB levels in the eggs from the hens 332 obtaining these feeds. Time-related levels in the eggs from the hens fed with feed E, either 333 without or with 10% sand were very similar to the results obtained in study I. The dilution of 334 contaminated feed with 10% sand did not clearly influence the levels observed in the eggs and 335 abdominal fat. The results obtained with the blank feed mixed with 10% soil A or soil B 336 reflected the differences in the soil and feed levels. A plateau level appeared to be reached in 337 this study within a short period of exposure. Maximum dioxin and total TEQ levels measured 338 in eggs from hens exposed to soil A were 2.4 and 2.9 pg TEQ/g fat. In the case of soil B, 339 highest levels of 1.2 and 1.6 pg TEQ/g were observed. Again, levels in abdominal fat were 340 lower than those for egg fat (data not shown). For comparison, levels determined in pooled 341 egg samples from the farms where the soil was sampled were 7.3 and 1.0 pg TEQ/g fat. 342

343

Effect of binders (study III) 344

Table 5 includes the levels of dioxins and PCBs in the four feeds supplemented with the 345 different binders. Levels in egg and abdominal fat of the hens fed with these feeds showed 346 that the binders, at least at the levels used in this study, had little or no effect on the residue 347 levels of dioxins and PCBs in the eggs (Table 6), although in the case of MycoAd AZ and 348 especially Klinofeed, both aluminium silicates, there appeared to be a slight decrease in the 349 levels of the different contaminants in both the eggs and the abdominal fat. It seems 350 worthwhile to investigate if higher levels may have a more profound effect, and whether this 351 also works with the absorption of dioxins and PCBs from soil.. 352 353 354 Conclusions 355

• Laying hens are sensitive indicators for dioxin exposure, meaning that relatively low 356 intake levels result in high levels in the egg yolk. This is not only true for 357 contaminated feed but also for contaminated soil. 358

• The current EU limit for feed of 0.75 ng TEQ/kg, established in 2002, is insufficient to 359 guarantee that levels in eggs will not exceed the limit of 3 ng TEQ/kg fat. A further 360 decrease in the feed limit by a factor 4 is required to achieve this goal. The alternative 361 is to increase the limit in eggs, but this is not in line with the further reduction in the 362 exposure aimed at by the EU authorities. 363

• There are currently no Dutch or European limits for indicator PCBs in feed or eggs. 364 However, the Belgium limit of 200 ng/g fat for feed does ensure that the limit for eggs 365 of 200 ng/g fat is not exceeded. 366

• Addition of binders at the tested levels has minimal effects on the resulting residues of 367 dioxins and PCBs in hens. 368

  Carry-over of dioxins and PCBs from feed and soil to eggs at low contamination levels; Influence of mycotoxin binders on the carry-over from feed to eggs Introduction 45

  for Study I: relation between levels in feed and eggs 97

  200 gram oil was first diluted to 1500 gram, and subsequently diluted for 111 incorporation into the different feeds, as follows: 30 gram + 1470 gram blank oil (b used for 112 feed B), 60 gram + 1440 gram blank oil (c used for feed C), 265 gram + 3237 gram blank oil 113 (d used for feed D), 685 gram + 3882 gram blank oil (e used for feed E), and 420 gram + 980 114 gram blank oil (f used for feed F). 115Analysis of oil b by GC/MS showed a total TEQ content of 20 pg TEQ/g, with a 116 contribution of 11 pg TEQ/g for dioxins, 5.3 pg TEQ/g for non-ortho PCBs and 3.7 pg TEQ/g 117 ortho PCBs. The blank oil contained a total TEQ content of 0.4 pg TEQ/g fat 118 (upperbound). Based on these results the Nutreco feed mill in Heyen, The Netherlands, 119 prepared the feeds with 0.95% of oils (b) to (f), and the blank oil a in the following quantities: 120 290 and 650 kg blank feed (prepared in 2 batches), 110 kg of feeds B, C, D and F, and 400 kg 121 of feed E.

  192

  lower response were declared negative, samples showing a higher 217 response suspected. 218 Aliquots of 5 gram feed or binder were mixed with 15 ml methanol/water 85/15 (v/v) and 219 extracted twice with 20 ml hexane/diethyl ether 97/3 (v/v). The extract was reduced to 5 ml 220 and purified over acid silica columns as described above. Feed samples were included as from feed to eggs (study I) 225 226Dioxin and PCB levels in the feeds 227Table2shows the total levels of dioxins, dioxin-like PCBs and indicator PCBs in the various 228 feeds. Each feed was analysed 3 to 5 times, corresponding to the number of samples taken 229 from the different bags. The variation coefficients were in general below 5%, demonstrating 230 the good homogeneity of the feeds. The relative contribution of dioxins, non-ortho and mono-231

Figure 1

 1 Figure1presents the time-related levels of dioxins (A), non-ortho PCBs (B), mono-ortho 246

  low and the kinetics of contamination is almost mono-exponential with the rate of the slow

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. Levels of dioxins (A), non-ortho PCBs (B), mono-ortho PCBs (C) and total TEQ 474

FFigure 1 .

 1 Figure 1. Levels of dioxins (A), non-ortho PCBs (B), mono-ortho PCBs (C) and total TEQ (D) in egg fat from hens fed with feeds containing 0.04 (A, +), 0.34 (B, ∆, open triangle), 0.58 (C, ▲, closed triangle), 0.76 (D, ○, open circle), 1.85 (E, ◊, open diamond) and 3.95 (F, ▼, reversed triangles) ng TEQ/kg of dioxins and PCBs. Results from single analysis.

Figure 2 .Figure 3 .

 23 Figure 2. Levels of indicator PCBs in egg fat from hens fed with feeds containing 0.2 (A, +), 2.3 (B, ∆, open triangle), 4.3 (C, ▲, closed triangle), 6.0 (D, ○, open circle), 14.2 (E, ◊, open diamond) and 31.7 (F, ▼, reversed triangles) µg/kg feed of indicator PCBs. Results from single analysis.

Figure 4 .Figure 5 .

 45 Figure 4. Levels of dioxins and dioxin-like PCBs (A), and indicator PCBs (B) in egg (▲, closed triangle), abdominal (▼, reversed triangles) and ovary fat (•, closed circle) from chickens fed with feed E. Results from single analysis.

Table 2

 2

	122	shows the analysed dioxin content of the different feeds
	123
	124	Preparation of feed for Study II: relation between levels in soil and eggs
	125	Aim of this study was to assess the bioavailability of dioxins from contaminated soil.
	126	Therefore, feeds were prepared by mixing the blank feed with 10% of soil collected from two
	127	chicken farms with elevated dioxin and dioxin-like PCB levels in eggs. The soils had been
	128	analysed previously showing dioxin levels of 5.88 ng TEQ/kg (soil A) and 1.94 ng TEQ/kg
	129	(soil B). Pooled egg samples collected at the same time at these farms were shown to contain
	130	dioxin levels of 1.0 and 7.3 pg TEQ/g fat. An additional feed was prepared as a control, by
	131	mixing feed E (study I) with 10% of clean sand.
	132
	133	Preparation of feed for Study III: effect of binders on carry-over from feed to eggs
		Aim of this

study was to assess whether or not binders would influence the bioavailability of 134 dioxins and PCBs in feed. A number of different binders were obtained for this study from the

	143	Animal studies
	144
	145	Study I
	146	Hens (Bovans Gold line, age about 45 weeks at the start) were housed in a three-tier battery
	147	with 3-4 hens per cage. The different groups contained 12, 12, 12, 13, 26 and 13 animals
	148	respectively. After an adaptation period on blank feed for 1 week, hens were fed with the
	149 150	F different feeds A to F for 56 days, followed by another 56 days on blank feed A. During the o r whole 112-day period eggs were collected and pooled per day per feeding group. On days 56
	151 152	and 112, at least five hens from each group were slaughtered and abdominal fat, livers and the P e ovaries collected. The group fed with feed E was larger allowing the collection of tissues of
	153 154	another five animals on days 10 and 28. e r
	155	Study II and III	R
			e
			v i e
			w
	135 136 137 138	producers. These materials were claimed to prevent the absorption of mycotoxins and as such might be potential candidates for reducing the absorption of dioxins as well. The materials O n l were first screened by DR CALUX ® and analysed by GC/MS, and shown not to contain y
	139	elevated dioxins and PCBs. The following feeds were prepared: feed E mixed with 0.5% Exal
	140	H (Tessenderlo, Madrid, Spain), feed E mixed with 0.5% MycoAd A-Z (Jadis, Schiedam, The
	141	Netherlands), and feed E mixed with 0.5% Klinofeed (Unipoint, Truttikon, Switzerland). The
	142	level of the binders was based on the recommendations of the providers.
			7

Table 1

 1 shows the ingredients of the feed, Topline extra.

	495
	496
	Ingredient

Table 2 .

 2 Levels of dioxins, non-ortho, mono-ortho and indicator PCBs in the different feeds used in 498 study I, as determined by GC/MS. Results are expressed as upperbound* levels and are the mean of 3-499 5 analyses. The % CV of the TEQ levels is given in brackets.

	500
	501
	Feed	Dioxins	no-PCBs	mo-PCBs	total TEQ	Indicator
						PCBs
		(ng TEQ/kg) (ng TEQ/kg) (ng TEQ/kg) (ng TEQ/kg)	(µg/kg)
	A	0.04 (10)	0.00 (16)	0.00 (3)	0.04 (10)	0.2 (14)
	B	0.20 (18)	0.09 (4)	0.06 (4)	0.34 (12)	2.3 (1)
	C	0.30 (2)	0.17 (3)	0.11 (1)	0.58 (2)	4.3 (7)
	D	0.40 (2)	0.22 (2)	0.14 (1)	0.76 (1)	6.0 (4)
	E	0.97 (1)	0.55 (2)	0.33 (1)	1.85 (1)	14.2 (2)
	F	2.04 (2)	1.19 (1)	0.72 (2)	3.95 (1)	31.7 (3)

Table 3 .

 3 Levels of the different dioxin and PCB congeners in feeds E and F and the eggs from 505 chickens fed with these feeds and sampled at day 56. Feed was analyzed in five-fold, eggs once. Based 506 on feed intake and egg production, carry-over rates for feed to eggs were determined for day 56.

	507
	508
		feed E	eggs E	carry-over	feed F	eggs F	carry-over
			day 56	(%)		day 56	(%)
	dioxins	ng/kg	pg/g fat		ng/kg	pg/g fat
	2,3,7,8-TCDF	0.12	1.10	40	0.25	2.33	40
	1,2,3,7,8-PeCDF	0.33	2.86	39	0.66	7.15	48
	2,3,4,7,8-PeCDF	0.33	2.83	38	0.67	6.76	44
	1,2,3,4,7,8-HxCDF	0.34	3.33	43	0.70	7.00	44
	1,2,3,6,7,8-HxCDF	0.33	2.98	40	0.68	6.44	42
	2,3,4,6,7,8-HxCDF	0.43	3.03	31	0.84	6.80	35
	1,2,3,7,8,9-HxCDF	0.32	2.85	39	0.67	6.28	41
	1,2,3,4,6,7,8-HpCDF	0.40	1.60	18	0.82	3.26	18
	1,2,3,4,7,8,9-HpCDF	0.32	1.44	20	0.66	3.06	21
	OCDF	0.63	0.98	7	1.39	1.55	5
	2,3,7,8-TCDD	0.14	1.31	41	0.30	3.26	47
	1,2,3,7,8-PeCDD	0.37	3.39	41	0.79	8.04	45
	1,2,3,4,7,8-HxCDD	0.40	3.80	42	0.84	8.38	44
	1,2,3,6,7,8-HxCDD	0.35	3.45	44	0.74	7.49	44
	1,2,3,7,8,9-HxCDD	0.40	2.87	32	0.84	6.49	34
	1,2,3,4,6,7,8-HpCDD	0.42	2.15	22	0.84	3.97	21
	OCDD	1.34	3.82	13	2.57	4.78	8
	non-ortho-PCBs	ng/kg	pg/g fat		ng/kg	pg/g fat
	PCB 81	5.12	46.70	40	10.86	102.0	41
	PCB 77	10.85	112.00	45	22.06	252.0	50
	PCB 126	4.98	54.10	48	10.86	123.0	50
	PCB 169	4.89	55.40	50	10.55	124.0	52
	mono-ortho PCBs	ng/kg	ng/g fat		ng/kg	ng/g fat
	PCB 123	5	0.10	78	14	0.22	70
	PCB 118	1576	17.10	48	3454	40.60	52
	PCB 114	6	0.07	52	14	0.16	51
	PCB 105	894	9.36	46	1940	23.00	52
	PCB 167	10	0.20	84	24	0.41	76
	PCB 156	139	1.75	55	307	4.03	58
	PCB 157	13	0.15	51	26	0.39	65
	PCB 189	10	0.14	61	21	0.29	61
	indicator-PCBs	µg/kg	ng/g fat		µg/kg	ng/g fat
	PCB 28	0.98	8.4	38	2.13	20.7	43
	PCB 52	1.30	1.6	5	2.91	2.9	4
	PCB 101	2.88	3.5	5	6.33	7.5	5
	PCB 138	2.99	34.6	51	6.72	83.1	54
	PCB 153	2.79	36.1	57	6.27	86.2	61
	PCB 180	1.89	25.6	59	4.27	58.5	41
	509

Table 4 .

 4 Feed consumption and productivity of the hens during and after the exposure period. Data are 510 based on the overall feed consumption and egg production over the whole exposure or withdrawal

	511
	512	period.
	513
				Exposure period		Withdrawal period
		Feed	Feed	Egg	Egg weight*	Feed	Egg	Egg weight*
			consumption	production	(g)	consumption	production	(g)
			(g/day)	(%)		(g/day)	(%)
		A	116	88	54	112	85	50
		B	108	82	56	111	86	51
		C	121	90	56	121	84	53
		D	113	80	55	110	86	52
		E	124	90	54	121	84	51
		F	108	90	54	111	79	51
	514	* average weight per egg without eggshell

Table 5 .

 5 Levels of dioxins, non-ortho and mono-ortho PCBs in the different feeds used in studies II 516 and III, as determined by GC/HRMS (mean of n=5, upperbound levels, lowerbound between brackets

	517
	518	where different from upperbound).
	519
		Feed	Dioxins	no-PCBs	mo-PCBs	total TEQ
			(ng TEQ/kg) (ng TEQ/kg) (ng TEQ/kg) (ng TEQ/kg)
		E	0.73	0.43	0.27	1.41
		E + 10% sand	0.65	0.39	0.25	1.28
		A + 10% soil A	0.44 (0.40)	0.10	0.05 (0.04)	0.64 (0.53)
		A + 10% soil B	0.35 (0.19)	0.11	0.08 (0.07)	0.54 (0.37)
		E + Exal H	0.87	0.53	0.33	1.73
		E + MycoAd Z	0.93	0.55	0.34	1.81
		E + Klinofeed	0.84	0.51	0.34	1.70
	520

Table 6 .

 6 Levels of dioxins, non-ortho, mono-ortho and indicator PCBs in the eggs and abdominal fat 521 of chickens exposed in the absence or presence of mycotoxin binders. Samples collected at day 32 522 were pooled from 5 hens or eggs and analyzed in single.

						Page 26 of 32
	523
	524
	Feed	Dioxins	no-PCBs	mo-PCBs	total TEQ	Indicator
						PCBs
		(pg TEQ/g)	(pg TEQ/g)	(pg TEQ/g)	(pg TEQ/g)	ng/g fat
	Eggs
	Feed E	7.4	5.3	3.1	15.8	117
	E + Exal H	7.4	4.7	2.9	15.0	109
	E + MycoAd Z	6.2	4.3	2.6	13.1	97
	E + Klinofeed	6.8	4.3	2.6	13.7	100
	Abdominal fat
	Feed E	6.2	4.5	3.0	13.7	84
	E + Exal H	5.7	4.3	2.9	12.9	89
	E + MycoAd Z	5.4	4.0	2.6	11.9	81
	E + Klinofeed	4.8	3.4	2.3	10.4	71
	525
	526
	527

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Acknowledgements 370

The study was sponsored by the Dutch Ministry of Agriculture, Nature and Food Quality. 371