1. Determination of concentration levels of anticoccidials in eggs due to the presence of low levels of those compounds in feed for laying hens caused by carryover at the feeding mill E. Daeseleire, L. Mortier, Ph. Delahaut and G. Huyghebaert

2. Brussels, 03/05/2005 Anticoccidials compounds that are widely used as feed additives to prevent and treat avian coccidiosis avian coccidiosis: contagious amoebic disease affecting livestock carried by unicellular organisms belonging to the genus Eimeria causes poor weight gain, reduced egg production, intestinal lesions resulting in diarrhoea and related health problems, can cause death most important parasitic disease in poultry

3. Brussels, 03/05/2005 Legislation Regulation (EC) N° 1831/2003 on additives for use in animal nutrition although pure medicines, regarded as additives : not subject to MRL-legislation (MRL = maximum residue limit) phasing out by 31/12/2012; Commission shall submit a report on the use of these substances as feed additives and available alternatives before 1/1/2008 No MRLs available  “zero tolerance” principle : no residues allowed

4. Brussels, 03/05/2005 main metabolite Selected compounds dimetridazole halofuginone robenidine diclazuril nicarbazin an equimolar mixture of

5. Brussels, 03/05/2005 LC-MS/MS-method HPLC (high performance liquid chromatography) –instrument : 2695 Alliance LC system (Waters) –conditions : column Waters C 18, 5 µ m, 150 mm x 2.1 mm i.d. pre-column Alltima C 18, 5 µ m, 7.5 x 2.1 mm i.d. eluent A H 2 O/ACN : 95/5 + 0.1% HCOOH eluent BACN flow rate0.25ml/min column temperature 35°C injection volume 10 µ l gradi ë nt gradient elution Isocratic for narasin MS (mass spectrometry) –instrument : Quattro Ultima Pt (Waters Micromass) –MS/MS : principle of MRM : fragmentation of precursor ion into product ions

6. Brussels, 03/05/2005 Mass spectrometric conditions compound ionisation mode Precursor ion (m/z) cone (V) Product ions (m/z) *: most abundant collision energy (eV) dimetridazoleES +142.150 96.1 * 81.2 12 20 2-hydroxy dimetridazole ES +158.140 140.1* 55.2 9 14 halofuginoneES +416.050 100.2* 120.1 20 18 robenidineES +334.150 155.1* 138.1 18 24 nicarbazinES -301.135 137.1* 107.1 8 30 diclazurilES - 404.9 406.9 50 334.1* 336.1 16 narasinES +787.480 431.2* 531.2 50 43

7. Brussels, 03/05/2005 Animal experiment : –location : Department of animal nutrition and husbandry, Merelbeke –Laying hens : ISA-brown, selected on egg production and body weight, in week 30 of laying cycle at start of the experiment –Period of adjustment : 4 weeks before the experiment hens were placed in the cages –feed : mash of 11.7 MJ MEn/kg, containing 16 % crude protein –housing : 4 hens/cage, conventional conditions of ventilation, temperature (18-22°C) and light (16h light/day)

8. Brussels, 03/05/2005 GROUPS : 5 compounds diclazuril, halofuginone, robenidine, nicarbazin, dimetridazole x 2 concentrations/compound 100% (= max. conc. in feed), 5% (carry-over) + blank group compoundadditive/premixconc. 100% diclazurilClinacox1 mg/kg halofuginoneStenerol3 mg/kg robenidineCycostat36 mg/kg nicarbazinMaxiban40 mg/kg nica+40 mg/kg narasin dimetridazolestandaard Sigma200 mg/kg dimetridazole : only 1 group (100%) as this is a forbidden compound (Annex 4) TOTAL : 10 groups x 12 hens/group

9. Brussels, 03/05/2005 PREPARATION OF FEED : at DVV per group 40 kg feed was prepared first 5 %, than 100 % concentration between 5 % and 100 %, 3 rinses were scheduled between 2 different compounds, 5 rinses were done ANALYSIS FEED SAMPLES : feed samples were analysed with a validated LC-MS/MS method the rinses in between were negative : no carry-over took place

10. Brussels, 03/05/2005 compoundgrouptheoretical conc.measured conc. % (compared to theoretical value) diclazuril 100%1000 µg/kg926 µg/kg93 5%50 µg/kg47 µg/kg94 halofuginone 100%3000 µg/kg1475 µg/kg49 5%150 µg/kg162 µg/kg108 robenidine 100%36 mg/kg39 mg/kg108 5%1800 µg/kg1597 µg/kg89 nicarbazin+narasin 100% nicarbazine : 40 mg/kg41 mg/kg103 narasine : 40 mg/kg41 mg/kg103 5% nicarbazine : 2000 µg/kg2114 µg/kg106 narasine : 2000 µg/kg2144 µg/kg107 dimetridazole100%200 mg/kg101 mg/kg51 results analysis feed containing anticoccidials :

11. Brussels, 03/05/2005 a. halofuginone first pos. sampleplateau concentrationconcentration below CCα 5%day 3 (18 µg/kg)30 µg/kg8 days after withdrawal feed 50%day 2 (3 µg/kg)450 µg/kg19 days after withdrawal feed

12. Brussels, 03/05/2005 b. dimetridazole first pos. sampleplateau concentrationconcentration below CCα DMZday 2 (676 µg/kg)650 µg/kg9 days after withdrawal feed DMZ-OHday 2 (1513 µg/kg)1700 µg/kg 13 days after withdrawal feed

13. Brussels, 03/05/2005 c. robenidine first pos. sampleplateau concentrationconcentration below CCα 5%day 3 (6 µg/kg) 70 µg/kg 13 days after withdrawal feed 100%day 2 (90 µg/kg)1300 µg/kg26 days after withdrawal feed

14. Brussels, 03/05/2005 d. narasin first pos. sampleplateau concentrationconcentration below CCα 5%day 3 (1 µg/kg)6 µg/kg11 days after withdrawal feed 100%day 3 (40 µg/kg)90 µg/kg18 days after withdrawal feed

15. Brussels, 03/05/2005 e. nicarbazin (dinitrocarbanilide) first pos. sampleplateau concentrationconcentration below CCα 5%day 3 (10 µg/kg)320 µg/kg15 days after withdrawal feed 100%day 3 (200 µg/kg)7000 µg/kg> 23 days after withdrawal feed

16. Brussels, 03/05/2005 f. diclazuril first pos. sampleplateau concentrationconcentration below CCα 5%day 3 (1 µg/kg)5 µg/kg11 days after withdrawal feed 100%day 3 (18 µg/kg)110 µg/kg> 22 days after withdrawal feed

17. Brussels, 03/05/2005 Advice 24-2004 of the scientific committee of the Belgian Federal Food Agency: action limit of 10 µg/kg for diclazuril, narasin, nicarbazin and robenidine compoundgroupmax. concentration # days after withdrawal < 10 µg/kg diclazuril 100 %100 µg/kg11 5 %5 µg/kg- halofuginone 50%501 µg/kg10 5%36 µg4 robenidine 100%1535 µg/kg15 5%70 µg/kg8 narasin 100%97 µg/kg8 5%7 µg/kg- nicarbazin100 %6500 µg/kg19 5 %300 µg/kg11 dimetridazole50% DMZ711 µg/kg (dag 7)5 DMZ-OH1915 µg/kg (dag 3)6