M Peters

T T Nguyen-Dang

C Cornaggia

S Saugout

E Charron

A Keller

O Atabek

Ultrafast Molecular Imaging by Laser Induced Electron Diffraction

We address the feasibility of imaging geometric and orbital structure of a polyatomic molecule on an attosecond time-scale using the laser induced electron diffraction (LIED) technique. We present numerical results for the highest molecular orbitals of the CO2 molecule excited by a near infrared few-cycle laser pulse. The molecular geometry (bond-lengths) is determined within 3% of accuracy from a diffraction pattern which also reflects the nodal properties of the initial molecular orbital. Robustness of the structure determination is discussed with respect to vibrational and rotational motions with a complete interpretation of the laser-induced mechanisms.

With the development of attosecond laser sources [1], ultrafast molecular imaging has become a major research topic in modern physics. On one hand, attosecond laser pulses are directly used to image dynamical processes in schemes such as the attosecond pump-probe spectroscopic mapping of molecular motions [2] or the interferometric real-time observation of electronic motions [3]. On the other hand, a number of schemes have been proposed to image molecular structure, which are all based on the rescattering mechanism [4]. This phenomenon consists of the tunnel ionization of an electron followed by its acceleration and its return by the field, to end with its recollision with the molecular ionic core. The possible outcomes of this mechanism are all relevant to molecular imaging. The elastic scattering of the returning electron with the ion core defines the LIED [5,6], which can be compared with ultrafast electron diffraction using an external electron source [START_REF] Lobastov | Proc. Natl. Acad. Sci[END_REF]. Inelastic scattering of the returning electron may be accompanied by electronic excitation of the parent ion, its further ionization or the emission of high-energy radiation. This last process, high-harmonic generation (HHG), has been used directly as a probe or indirectly to unveil orbital and molecular structure, and even to image rotational and vibrational motions [8].

We wish to assess the feasibility of imaging polyatomic molecular structure, e.g., measuring its geometrical parameters, such as bond-lengths and/or bond-angles), on an attosecond time-scale using the LIED technique. We show how a diffraction pattern constructed from measurable photo-electron momentum distribution encodes informations on the electronic orbital nodal properties but also on the geometry of the nuclei. We assess the robustness of this two-fold structure determination procedure with respect to inevitable uncertainties about the alignment of the molecule relative to the field polarization direction, its bond lengths and angles. We consider a relatively simple model of the CO 2 molecule, the choice of which being motivated by different considerations: Being linear in its symmetric equilibrium geometry, it is simple enough, in particular with respect to its alignment properties, and yet with three nuclei, it has enough internal degrees of freedom to make its molecular structure determination challenging.

To concentrate on the problem of reading geometrical informations out of the photo-electron diffraction pattern in momentum space, we develop a theoretical model based on an effective single active electron (SAE) Hamiltonian. Nuclear dynamics and its coupling to the electron ionized out of a definite orbital (initial state) can be ignored at the time scale of a few-cycle pulse. Nuclear degrees of freedom are thus taken into account only through statistical distributions of CO bond-lengths R and polar angles θ with respect to the laboratory y-axis along which CO 2 is supposed initially aligned. The intense laser pulse, which monitors the electron dynamics, is linearly polarized along the laboratory x-axis and the essential of the electron diffraction process takes place in the (x, y)-plane. Electronic wave packets are generated by solving the time-dependent Schrödinger equation (atomic units are used):

i ∂ ∂t Φ( r, t) = Ĥ( r, t)Φ( r, t) (1)
with a SAE Hamiltonian using a soft-Coulomb potential and an electron-laser dipole coupling in the length gauge:

Ĥ( r, t) = p 2 2 + 3 α=1 -Z α ( r) | r -ρ α | 2 + a 2 α + r • E (t) , (2)
r and p being the electron position and momentum, and α labeling the three nuclei located at fixed position vectors ρ α . The laser field E (t) is characterized by the angular frequency ω L and the amplitude E 0 . The r-dependent

effective charge Z α ( r) = Z ∞ α + (Z 0 α -Z ∞ α ) exp(-| r - ρ α | 2 /σ 2 α
) involves a single adjustable parameter σ α . It is defined, together with the softening parameter a α , so as to give field-free eigenstates of the SAE Hamiltonian identifiable with the highest occupied molecular orbitals, HOMO-n (n = 0, 1, 2), in the sense of giving the same ionization energy and the same nodal properties as the actual molecular orbital of the 22-electron molecule. Numerical wave packet propagation uses a third-order split operator formula on a finite 2D grid continued analytically by projection onto Volkov states for | r | > 45 Å.

The four panels on the second row of Fig. 1 show the results of the time-propagation from an initial state Φ( r, t 0 ) taken as the HOMO, characterized by a perpendicular nodal plane which is preserved through time-evolution, when the laser polarization is orthogonal to the molecular axis. Each panel depicts the electronic density at a different time within a single optical cycle, as indicated by the corresponding letter on the waveform shown in the middle panel of the first row. The most important observations from these spatial wave functions are first the fringes of panel (c) obtained during the forward (x < 0) electron motion as a signature of the two oxygen atoms acting as electron ejection sources, and second the rich pattern of interference fringes of panel (d) in the backward (x > 0) motion resulting from the electron diffraction analogous to Young's slit experiment with photons. More relevant for structure determination is the diffraction pattern in reciprocal momentum space, accessible experimentally through time-of-flight electron velocity mapping, and determined numerically by the Fourier transform Φ(k x , k y ) of the asymptotic electron wave packet. To illustrate this, we show, in the rightmost panel on the first row of Fig. 1, | Φ(k x , k y )| 2 for the CO bond length R = 4.8 Å, corresponding to a symmetrically stretched molecule. The features of this momentum distribution, with a succession of vertical interference fringes, are in agreement with the interpretation of Ref. [6]. From this distribution, a k x -averaged diffraction pattern can be defined as

S(k y ) = Γ | Φ(k x , k y )| 2 dk x , (3)
where Γ denotes the domain of integration. In analogy with Young's double slit experiment, we expect a good resolution of the interference signal if the electron de Broglie wavelength λ DB = h/p is smaller than the slit separation d = 2R. This criterion corresponds to |k| > π/R and therefore to the high energy range of the photo-electron spectrum. To insure a resolution of the order of 0.5 Å, we define here the domain of integration Γ as the region where |k| > 3.15 a.u. For a single-cycle pulse, and a perfect alignment situation, the averaged distribution S(k y ) associated with the diffraction of an electron ejected from the HOMO is shown as a black solid line in the upper panel of Fig. 2. One can notice that S(k y ) exhibits clear interference fringes at large k y , characterized by a regular succession of peaks, from which geometrical parameters will be inferred. Indeed, that S(k y ) directly gives informations on the nuclear geometry can be seen by the following simple symmetry considerations: The CO 2 HOMO initial molecular orbital is anti-symmetric with respect to the perpendicular σ h mirror plane containing the carbon atom, and so is the electronic wave function at all time, assuming perfect alignment of the molecule orthogonal to the laser polarization. It can approximately be written as

Φ πg ( r, t) ∝ f (x, y -R, t) -f (x, y + R, t) , (4)
which, through Fourier transformation, gives Φπg ( k, t) ∝ e iRky -e -iRky f ( k, t) .

(

) 5
The squared modulus of this wave function behaves as sin 2 (R k y ) and thus exhibits zeros for k y = nπ/R. The positions of the dark fringes should thus be directly related to the internuclear distance R. These positions are indicated by dotted vertical gray lines in Fig. 2. The local minima in the calculated spectrum S(k y ) are seen to agree fairly well with these predicted positions. The complete quantitative analysis gives, within the model assumptions, R ≃ 4.92 Å, with a deviation of less than 3% from the initial input value R = 4.8 Å.

Given the role that lower-lying valence molecular orbitals play for CO 2 in intense laser fields [9], the averaged S(k y ) distribution for the HOMO-2 is shown as a green dotted line in the upper panel of Fig. 2. Though a bonding σ u orbital, the HOMO-2 presents the same symmetry property as the π g HOMO with respect to the σ h mirror plane containing the carbon atom. Eqs. ( 4) and ( 5) are thus valid for the HOMO-2 also, and the photo-electron signal exhibits the same interference pattern for large k y values. In contrast, the bonding π u HOMO-1, being symmetric with respect to the σ h mirror plane, gives rise to fringes out-of-phase with respect to those associated with the HOMO and HOMO-2. The nodal structure of this orbital is reflected through (6) which, through Fourier transformation, gives Φπu ( k, t) ∝ e iRky + 1 + e -iRky f ( k, t) .

Φ πu ( r, t) ∝ f (x, y -R, t) + f (x, y, t) + f (x, y + R, t) ,
The squared modulus of this wave function, which behaves as [1 + 2 cos(R k y )] 2 , is characterized by a regular succession of two peaks of different amplitudes. Their maxima, located at k y = nπ/R, and their relative amplitudes are reproduced in the full quantum calculation shown in the lower panel of Fig. 2. The total photoelectron signal, constituted by combined contributions from the HOMO, HOMO-1 and HOMO-2 which can be controlled by the field intensity, could be used to read accurately the CO bond length from the fringe pattern observed in the large k y -range. The structural reading made above of the diffraction pattern refers to a perfectly aligned molecule in a fixed nuclear geometry configuration. In reality, molecules are subject to rotations and vibrations, and these geometrical parameters are usually distributed over some range dictated by the rotational and vibrational state in which the parent molecule is initially prepared. A first aligning laser pulse can be used to create a rotational wave packet which periodically rephases, thus yielding, at the revival time, a strong molecular alignment along the polarization direction. Angular distributions confined in a cone angle of about 20 • have been reached experimentally with CO 2 [10]. The left column of Fig. 3 shows, in red dotted lines, the diffraction pattern incoherently averaged over a Gaussian distribution of alignment angles θ, with a characteristic width of 20 sensitivity to alignment defects. However, the fringes are still clearly observable at their original positions in the range |k y | > 0.7 a.u. The extraction of geometrical informations thus remains achievable even in case of imperfect initial alignment of the molecular target. To study the effect of variations of the bond length, we also considered a Gaussian distribution of internuclear distances with a characteristic width of 0.2 Å. Note that this width is larger than the typical size of the ground vibrational level associated with the stretching and bending modes of CO 2 . The results are shown in the middle column of Fig. 3. One can notice that the diffraction pattern S(k y ) is almost insensitive to the initial distribution of R. The zeroes in the averaged S(k y ) remain basically unshifted can therefore be used for geometrical information and imaging purposes. To conclude, we expect that a measurement on imperfectly aligned CO 2 molecules in their ground vibrational level would provide diffraction patterns that are no less analyzable than those shown in Fig. 2, and from which the CO equilibrium bond length could be extracted unambiguously. We also note in Fig. 3 that, as expected, the oscillation period of S(k y ) is 4 times smaller with R = 4.8 Å than with R = 1.2 Å.

Finally, the right column of Fig. 3 shows the results obtained with a 10 fs FWHM sine-square pulse of same carrier wavelength, 800 nm. In this case, the 2D photo-electron momentum spectrum (not shown) is different from the one obtained under the single-cycle pulse (Fig. 1). It is more symmetric with respect to k x , reflecting a different rescattering physics: Concentrating on what happens near the peak of the pulse envelope, one distinguishes two separate electron trajectories, which correspond to ionization events taking place at different times. The associated electron wave packets are then driven by the field in opposite directions. Contrary to what happens in the single-cycle case, these trajectories can both return to the ion and contribute to the diffraction event. The returning electron waves overlap and interfere with each other in the low-energy region of the 2D momentum distribution, corresponding to |k| ≤ 2 a.u., blurring out the diffraction pattern and prohibiting a simple reading of the fringes in this region. However, even with a long pulse duration, only a few trajectories contribute to the highest energy domain of the spectrum. An average of the 2D diffraction map over the domain Γ of Eq. ( 3) corresponding for instance to |k x | > 3.15 a.u. helps recovering clearly visible interference fringes. In the right column of Fig. 3, the black solid line shows the result obtained using this integration domain in the case of a single cycle pulse, while the red dotted line refers to a 10 fs FWHM sine-square pulse. It is seen that with this pulse, not only does the diffraction signal S(k y ) continue to reveal the geometrical and orbital structure of the parent molecule, but it also is amplified by about an order of magnitude as compared to the single-cycle signal.

To conclude, we have suggested a simple yet robust method to extract the molecular structure from the photo-electron spectra of a laser-driven linear, symmetric molecule. It is applicable to any sufficiently symmetric molecular system for which the HOMO, or any molecular orbital contributing to the ionization signal, has specific symmetry properties that would allow for a simple interpretation of the diffraction patterns. The detailed structure of the diffraction image reflects the symmetry of the molecular orbital from which the recolliding electron emanates since this symmetry is conserved during the timeevolution of the system under the combined effect of the Coulomb forces and the laser field. Such a symmetry conservation actually goes beyond the SAE approximation, and we expect the diffraction images calculated here to survive inclusion of many-electron effects, contrary to what was found relative to corrections to the strong field approximation [14], which is not assumed here.

As the acquisition process, relying on the rescattering mechanism, is faster than nuclear motions, it can be used to produce a sequence of stroboscopic pictures, making up, for example, a movie of vibrational dynamics. The imaging technique could be implemented using electron spectroscopy based on different schemes such as angleresolved time-of-flight detection or velocity map imaging [11]. These methods are currently used in the atomic, molecular and optical physics community, and offer an energy resolution ∆ε/ε of the order or better than 1% at 50 eV, which turns out to be largely sufficient to resolve the fringe separations suggested by the present simulations. While, in the present theoretical study, a typical 800 nm laser radiation was considered, preliminary numerical simulations also show that our proposal could be used efficiently with few-cycle pulses produced nowadays in the IR domain (1-3 µm) [12]. Practically, a single-cycle pulse has been produced at λ=1.8 µm [13]. Higher recollision energies are then obtained, yielding shorter electron de Broglie wavelengths. These laser systems should therefore provide a better resolution in terms of internuclear distances at fixed laser intensity.

  FIG. 1. (Color online) Ionized electron motion in laser-driven CO2 molecule. First row, from left to right: Field-free (πg) HOMO of CO2, external field's waveform, and calculated 2D asymptotic electron momentum distribution for the CO bond length 4.8 Å. Various snapshots of the calculated 2D electron density are shown in panels (a) to (d) in the second row, corresponding to different times within the optical cycle (ωL = 0.06 a.u., E0 = 0.15 a.u.) as marked on the field waveform above.

log 10 [

 10 FIG. 2. (Color online) Averaged normalized electron diffraction pattern S(ky) obtained with a single cycle pulse (see Fig. 1) for a perfectly aligned molecule, with R = 4.8 Å. In the upper panel, the black solid and green dashed lines correspond to the diffraction patterns obtained from the (πg) HOMO and (σu) HOMO-2 molecular orbitals of CO2. The red solid line in the lower panel is for the (πu) HOMO-1 initial state.

  • for R = 1.2 Å (first row) and R = 4.8 Å (second row). This Figure also shows, in black solid lines, the result obtained for a perfect alignment. The contrast loss seen in this Figure denotes a certain

FIG. 3 .

 3 FIG. 3. (Color online) Averaged normalized electron diffraction patterns S(ky) resulting from ionizing the HOMO of CO2 under the same field conditions as in Fig. 1 for R = 1.2 Å (first row) and R = 4.8 Å (second row). The solid black lines correspond to a perfect alignment at fixed internuclear distance for a single optical cycle pulse. The red dashed lines correspond to, column (a), an imperfect alignment, column (b), a distribution of R and, column (c), a 10 fs FWHM sine-square pulse.

  M.P. and T.T.N.D. acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial supports. The authors acknowledge supports from CFQCU (contract 2010-19), from ANR (contracts ImageFemto ANR-07-BLAN-0162 and Attowave ANR-09-BLAN-0031-01), from FCS Digiteo -Triangle de la Physique (project 2010-078T -High Rep Image), and from the EU (ITN-2010-264951 -CORINF).


Valerie Gaudin
email: v.gaudin@fougères.afssa.fr

Valérie Gaudin

Céline Hedou

Annie Rault

Eric Verdon

of antibiotic residues in muscle from different animal species

Keywords: Screening -microbial screening Additives/Contaminants: Residues, Veterinary drug residues -antibiotics, Veterinary drug residues -antimicrobials, Veterinary drugs Food Types: Animal products -meat, Meat Validation, decision 2002/657/EC, STAR protocol, screening, antimicrobial 48 residues, muscle, plate test

    Validation of a Five Plate Test, the STAR protocol, for the screening of antibiotic residues in muscle from different animal species according to the European decision 2002/657/EC

144 [START_REF] Anonymous | Document of 21 st of January 2010[END_REF]. This guideline deals with the initial validation and also a shortened or 145 'abridged' validation, which under certain conditions, allows for the transfer of methods 146 already validated in one laboratory to a second one. The guideline proposes some 147 recommendations to implement a validation protocol for screening methods. It explains the 148 performance characteristics to be determined (specificity, detection capability, robustness, 149 …) and how to determine them in practice. Moreover, the number of samples necessary to 150 validate a screening method is discussed. Finally, some new concepts have been introduced 151 in the guideline: the preparation of "simulated tissues" and a list of representative substances 152 to be validated. These two concepts will be detailed below in the validation protocol.

154

The validation conducted in this study is based on this validation guideline which is also the 155 internal guideline in our laboratory for the validation of screening methods. Two objectives 156 have been set: firstly to validate the STAR protocol for its application to the detection of 157 antibiotic residues in muscles from different animal species according to the decision 158 2002/657/EC [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council 887 Directive 96/23/EC concerning the performance of analytical methods and interpretation of 888 results[END_REF]; secondly to show that the European guideline for the validation 159 [START_REF] Anonymous | Document of 21 st of January 2010[END_REF]) and based on the decision 2002/657/EC [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council 887 Directive 96/23/EC concerning the performance of analytical methods and interpretation of 888 results[END_REF] was applicable to the 160 validation of a microbiological screening method. to "simulated tissues". Finally the plates are incubated: at 30°C for at least 18 hours for Bs8 184 and Bc6, at 37°C for at least 24 hours for Kv8, at 37°C for at least 18 hours for Bc6 and at depends on the results of the positive controls which have to be included in the following 200 intervals: 5.5 ± 1.5 mm, 6.5 ± 1.5 mm, 6.0 ± 1.5 mm, 7.0 ± 1.5 mm and 6.0 ± 1.5 mm for Bs8, 201 Kv8, Bc6, Ec8 and Bst respectively. discs. However, this way of working was not completely satisfying because the interference 207 of the muscle matrix was absent. In fact, Okerman et al (1998a) showed that the tissue 208 matrix has an effect on the sensitivity of the test plates. During that study, pieces of frozen 209 meat laid on paper discs impregnated with antibiotic standard solutions have been used for 210 the validation. Usually inhibition zones decreased when spiked meat samples have been

211

analysed, compared to antibiotic spiked discs without meat. The same conclusions were 212 reported by [START_REF] Pikkemaat | Improved microbial screening assay for the detection of quinolone residues in 932 poultry and eggs[END_REF]. Because of the difficulty and the high cost of

297

One wants to underline that the analysis of the 60 samples for each antimicrobial were 298 performed step by step. When one false negative result or more was obtained after the 299 analysis of 10 samples, either the validation was stopped at this concentration and started 300 again with an increased concentration if it was of interest (e.g. near the MRL) or the 301 validation went on with the same antibiotic and the same concentration because the 302 concentration was already much higher than the concerned MRL. The STAR protocol is implemented in parallel with the Four Plate Test for the analysis of 310 muscles from different animal species , during the routine analysis of field samples (cattle, 311 sheep, poultry, ...) in National Monitoring Plans since at least 5 years. Then, the positive 312 samples at the screening step are confirmed by a multi-residue LC-MS/MS method (data to 313 be published). Therefore, we have a lot of experience about the applicability of the method to 314 muscles from different animal species.

316

However, to complete this study, we decided to perform a study to determine the CCβ of 317 some antibiotics in cattle, sheep and poultry muscles, antibiotics for which the CCβ were 318 determined previously in porcine muscle. Therefore, the CCβ of 5 antimicrobials (penicillin G 319 at 25 µg kg -1 , doxycycline at 100 µg kg -1 , erythromycin at 400 µg kg -1 , gentamicin at 6000 µg 320 kg -1 , enrofloxacin at 200 µg kg -1 ) have been determined with 20 samples from each of the 3 321 species over 5 days and have been compared with the CCβ calculated for porcine muscle. In 322 the applicability study, only the specific plate (which has presented inhibition zones for pig 323 muscle) was tested with the corresponding antibiotic.

325

The applicability would be proved if the CCβ determined for these species are similar to the 326 CCβ determined for porcine muscle (the average inhibition zones obtained for each species 327 on the specific plate should be similar: accepted deviation ± 25%). A variability of 25 % is 328 usually accepted with microbiological methods (for positive controls or spiked muscles).

329

Moreover, this range of variability was observed during the validation in porcine muscle. The 330 specificity of the STAR protocol with other species has also been studied by the analysis of 331 different blank muscle samples.

333

Ruggedness study

334

The ruggedness of the method has been partly evaluated during the validation study, since 335 different batches of media, bacterial strains, antimicrobial standards have been used. In 336 addition, 2 technicians have been involved in the study, analysing each half of the samples 337 and the variability between technicians could be estimated.

339

In the decision 2002/657/EC [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council 887 Directive 96/23/EC concerning the performance of analytical methods and interpretation of 888 results[END_REF], the ruggedness is defined as " the susceptibility of 340 an analytical method to changes in experimental conditions […]

357

The ruggedness study has been focused on 6 different representative antimicrobials which 358 were specifically detected on each of the 5 plates on muscle: penicillin G at 25 µg kg -1 and 359 sulfadimethoxine at 300 µg kg -1 on plate Bst, doxycycline at 100 µg kg -1 on plate Bc6, 360 erythromycin at 400 µg kg -1 on plate Kv8, gentamicin at 6000 µg kg -1 on plate Bs8,

377

The specificity of the STAR protocol for the detection of antimicrobial residues in porcine 378 muscle was very satisfactory.

381

The results presented in table 3 are those obtained from 60 samples for each antibiotic at 382 least one tested concentration.

384

Insert Table 3 about here 385 386 For some antibiotics, after 15 or 30 analyzes by one or two technicians, either it turned out 387 that too many false negative results were obtained at the selected concentration or on the 388 opposite the first tested concentration gave higher inhibition zones than anticipated,

389

suggesting that the validation could be performed at a lower concentration. At these 390 concentrations, less than 60 samples have been analyzed, however it was helpful for the 391 determination of the detection capability. 419 neomycin 1500 µg l -1 (MRL = 1500 µg l -1 ), streptomycin 1000 µg l -1 (MRL = 200 µg l -1 ), 420 kanamycin 1000 µg l -1 (MRL = 150 µg l -1 ), spectinomycin 40000 µg l -1 (MRL = 300 µg l -1 ), 421 paromomycin 2000 µg l -1 (not authorised in milk), apramycin 4000 µg l -1 (not authorised in 422 milk). Therefore, the sensitivities for GTM and DHS were much better in milk than in muscle.

424

During the determination of detection capabilities in porcine muscle, it was demonstrated that 425 the STAR protocol could provide an help to confirm the identity of a family of molecules 426 present in the sample. Table 4 shows the plates on which each antibiotic reacted 427 preferentially. Some antibiotics (e.g. cefquinome, ceftiofur, doxycycline, enrofloxacin and The overall average inter-species was equal to 9.0 ± 1.3 mm, 5.2 ± 1.0 mm, 5.2 ± 1.4 mm, 465 6.1 ± 1.1 mm and 5.4 ± 0.9 mm for Bst, Bc6, Kv8, Ec8 and Bs8 respectively. Whichever was 466 the tested antibiotic and the plate, the discrimination between blank and spiked samples was 467 very clear and easy, for each species. Moreover, the average inhibition zone (IZ) for cattle,

468

sheep and poultry muscle was in the fixed interval of plus or minus 25% compared to the 469 average IZ for porcine muscle for plates Bst, Bc6, Ec8 and Bs8. Concerning plate Kv8, the 470 average inhibition zone (IZ) for cattle and sheep muscle was in the fixed interval of plus or 471 minus 25% compared to the average IZ for porcine muscle. The poultry muscle was out of 472 the interval for plate Kv8, but with an average inhibition zone of 33% higher than the porcine 473 muscle, so the sensitivity was better in the poultry muscle.

475

Two blank samples of cattle, 2 sheep and 2 poultry were analyzed for each plate and the 476 analyses were repeated for 5 days. Over 10 cattle tested, we got 2 false-positive results, 1 477 for sheep and 1 for poultry on Bst plate. The blank bovine and ovine samples gave IZ of 3.7 478 ± 0.7 and 3.3 ± 0.4 mm, higher than porcine (1.8 ± 1.4 mm) and poultry (2.0 ± 1.4 mm) blank 479 samples. All blank samples gave non specific inhibition on the plate Bst, higher than for the 480 other plates. That is why the positivity threshold of the plate Bst was set at 4 mm, while the 481 positivity threshold for the other plates was set at 2 mm (Fuselier at al. 2000). No false-482 positive results were obtained on the plate, whatever the species, on the 4 other plates.

483

In conclusion, the STAR protocol is applicable for the screening of antibiotic residues in the 484 muscle of the 4 major species: pig, cattle, sheep, poultry and by extension the muscle of 485 minor species, whatever is the plate.

492

Looking at the quantitative results (inhibition zones) obtained by the 2 technicians on muscle 493 samples, , the variability (standard deviations) was rather low, similar to or lower than the 494 variability that is fixed for the positive controls of the method. During this period, the positive 495 controls were included in the intervals set in the STAR protocol. This is the first track to 496 conclude that the STAR method is robust.

498

The average inhibition zones and the standard deviations obtained during the ruggedness 499 study and during the first part of the validation (determination of CCβ) have been compared 500 and are presented in Table 5.

502

Insert Table 5 about here

504

When the standard deviation in ruggedness conditions (SDi) is significantly larger than the 505 standard deviation of the method in terms of reproducibility, the conclusion is predictable that 506 all factors taken together have an impact on the results, even if no single factor has 507 significant influence. In this case, the method is not sufficiently robust regarding the range of 508 tested variations. From a quantitative point of view, we did not observe significant differences 509 between the SD in ruggedness conditions and the SD during the first part of the validation for 510 the 6 tested antibiotics. Therefore, the STAR protocol seemed to be robust for the 5 plates 511 regarding the range of tested variations.

513

If we look at the mean inhibition zones obtained at each day (run) during the ruggedness 514 study to the mean inhibition zones obtained during the determination of detection 515 capabilities, it seemed that the sensitivity is very near, even if the average inhibition zones 516 were often lower during the ruggedness study. In fact, the average inhibition zone of SDMX 517 on plate Bst was significantly lower during the ruggedness study (Table 5). Therefore some 2). The responses for each plate 529 for each run (day) were indicated (mean inhibition zone (IZ), the coefficient of variation (CV 530 %) on the inhibition zone and false positive and false negative rates when influence was 531 observed). From these results, the exploitation of the experimental design has been

532

performed. The results are presented in Table 6 for the 6 antibiotics and the 5 plates.

534

Insert Table 6 about here 535 536

In conclusion, whatever the plates, there was no significant effect of analytical factors tested 537 on the qualitative results of the STAR method (no influence on the false-positive rate and 538 false-negative rate), except for the plate Bst with SDMX (Table 6). When testing SDMX, on 539 day 4, the 4 tested samples gave false-negative results (Table 7). Therefore it has a strong 6). This is the reason why a longer incubation time (at least 24 hours) 564 compared to the other plates is recommended in the STAR protocol. However, even at the 565 lowest incubation time (21 hours), the results were satisfactory.

567

Finally, it has been demonstrated that a pre-incubation of 1 hour at room temperature could 568 have a positive effect on the sensitivity of the test (plates Ec8, Bst (SDMX), Kv8) or no effect 569 (plates Bc6, Bs8, Bst (penicillin G)), compared to no pre-incubation. However, pre-incubation 570 is not a critical parameter because if people does not apply a pre-incubation, the results

571

would be satisfactory and if pre-incubation is performed, the results would be equal or better.

637

Moreover, it was determined during the validation of the STAR protocol in milk 638 [START_REF] Gaudin | Validation of a 900 microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic 901 residues in milk[END_REF]) that the least detected cephalosporins were cefquinome (MRL = 639 50 µg l -1 ) and cefalexin (MRL = 200 µg l -1 ). On the opposite, the best detected 640 cephalosporins were cefazolin (Annex II for all tissues except milk) and cephapirin

641

(MRL =50 µg l -1 ). Finally, ceftiofur (MRL = 1000 µg l -1 ) was better detected than 642 cefquinome but less detected than cefazolin. Therefore, we have chosen to validate

682

When the STAR protocol was validated in milk, we have observed that enrofloxacin

683

(detection limit = 20 µg l -1 ), ciprofloxacin (10 µg l -1 ), marbofloxacin (30 µg l -1 ) and 684 danofloxacin (15 µg l -1 ) had the same activity profiles, with similar sensitivities (Gaudin et al.

694

gentamicin in milk was the best detected antibiotic (limit of detection 300 µg l -1 ) and 695 spectinomycin was the least detected aminoglycoside (20000 to 40000 µg l -1 ). The limit of 696 detection for neomycin, kanamycin, streptomycin and DHS was equal to 1000 µg l -1 .

697

Therefore DHS is representative of the 3 other aminoglycosides. Lincomycin was detected at 698 350 µg l -1 and pirlimycin at 100 µg l -1 . Therefore lincomycin was the least detected and is 699 representative of the detection of lincosamides.

701

The specificity of the STAR protocol for the detection of antimicrobial residues in porcine 702 muscle was very satisfactory. A similar result was obtained by Pikkemaat et al. (2009a) when The results were obviously similar to the global orientation given in the STAR protocol: beta-708 lactams and sulphonamides were detected preferentially on plate Bst, tetracyclines on plate 709 Bc6, quinolones on plate Ec8 and macrolides on plate Kv8. However, there were 2 710 exceptions. The aminoglycosides have been detected on plate Bst with the concentrations 711 tested for the determination of CCβ, whereas for higher concentrations than CCβ, they were 712 detected on plate Bs8 (= Bs7.2). However, when the validation of the STAR protocol has 713 been performed in milk, Bs8 was the specific plate for the detection of aminoglycosides.

714

Secondly, tylosin was detected preferentially on Bst instead of Kv8 at this concentration, 715 which had already been observed during the validation of the STAR protocol in milk. So if a 716 positive result is reported on plate Bst, the confirmation should be directed onto beta-717 lactams, sulphonamides, but also tylosin and aminoglycosides. A comparative study of 3 718 microbial screening tests including the STAR protocol applied to routine monitoring samples 719 (Pikkemaat et al. 2009a) has similarly, shown that a macrolide (tulathromycin) was 720 preferentially detected on plate Bc6 and Bst instead of plate Kv8. Therefore if no 721 tetracyclines could be confirmed in a positive sample on plate Bc6, the confirmation could be 722 directed towards tulathromycin. During this comparative study, the STAR protocol was able 723 to detect the 4 MRL samples which contained antibiotic concentrations higher than their 724 respective MRLs. Three of them were tetracyclines, detected preferentially on plate Bc6 and 725 the remaining residue was sulfadiazine at 172 µg kg -1 . No information were available for 726 beta-lactams, macrolides and quinolones because none of these families were found in the 727 routine monitoring samples. The NAT-screening test was able to detect 4 samples containing 728 aminoglycosides, but not the STAR protocol because this method is not enough sensitive 729 towards the aminoglycoside family. Furthermore, the NAT-screening test is applied to kidney, 730 while the STAR protocol is recommended for muscle. Moreover, it is well known that 731 aminoglycosides concentrated in kidney, while very low concentrations could be found in 732 muscle. The muscle matrix is not a satisfactory matrix for the screening of aminoglycosides.

733

The results of this comparative study are in accordance with our validation data.

750

The validation of microbiological plate test is also a complex issue because of the solid 751 matrices like muscle and kidney. In fact, the ideal solution would be to produce incurred 752 samples from animal treatment for all antibiotics having MRL in the corresponding matrix and 753 to validate all these antibiotics. However this is extremely time consuming and expensive.

754

Furthermore, it is really difficult to obtain exactly a target concentration after the treatment of 755 animals. So there are different possibilities to implement the validation of a microbiological 756 plate test.

758

The first option is to use spiked paper discs to determine the sensitivity of the method from 759 antibiotic standard solutions (Koenen-Dierick et al. 1995;[START_REF] Calderon | Evaluation of a multiple bioassay 874 technique for determination of antibiotic residues in meat with standard solutions of 875 antimicrobials[END_REF], Currie et al. 760 1998;Ferrini et al. 2006). The main advantages are that this solution is less expensive and 761 quicker. The validation could be implemented for a wide list of antibiotic residues. However, 762 matrix components could affect the detection capabilities of a method. Okerman et al.

763

(1998a) showed that only tetracyclines and quinolones were similarly detected with or 764 without tissue. However, the detection of beta-lactams was better with antibiotic spiked discs 765 without tissue than with tissues. For some antibiotics, the difference was only observed at 766 low concentrations. Therefore, the use of antibiotic spiked discs is not totally satisfactory.

767

The second possibility is to use monitoring routine samples (Okerman et al. 1998b; 768 Pikkemaat et al. 2009a;[START_REF] Schneider | Comparison of 945 screening methods for antibiotics in beef kidney juice and serum[END_REF]. Routine samples from monitoring plans have

839

In the guideline for validation of screening methods [START_REF] Anonymous | Document of 21 st of January 2010[END_REF], one of the 840 recommendations is to reduce the work of validation when the method is transferred to 841 another laboratory which would want to implement it in routine conditions. In fact, the number 842 of samples to be analysed is reduced. The performance characteristics (e.g. detection The antibiotics in italics are the antibiotics which have been substituted to the antibiotics initially included in the list of representative antibiotics.

  on the DGSANCO website since the 21 st of January 2010

  was considered positive when the inhibition zone around meat sample was 188 equal or superior to 2 mm in width on plates Bs 8, Kv 8, Bc 6 and Ec 8 and / or the inhibition 189 zone equal or superior to 4 mm in width on plate Bst. 190 191 Since 2004, medium test agar pH 7.2 has been replaced by antibiotic medium II at pH 8.0 192 (Difco, reference 259310) because it was not commercialised anymore.. Moreover, the 193 positive control of plate Bst was modified for practical reasons: sulfamethazine at 1000 µg l -1 194 has been replaced by amoxicillin at 40 µg l -1 . Positive controls consist of 30 µl of antibiotic 195 solutions which are put on paper discs of 9 mm diameter (Durieux, France). There is a 196 specific positive control for each plate: streptomycin at 2000 µg l -1 on plate Bs8, tylosin at 197 1000 µg l -1 on plate Kv8, oxytetracycline at 800 µg l -1 on plate Bc6, enrofloxacin 800 µg l -1 on 198 amoxicillin at 40 µg l -1 on plate Bst. The validity of each day of analysis 199

  we have studied the sensitivity of 35 antibiotics by the way of antibiotic spiked 206

  213 production of incurred materials for validation of a microbiological plate test, it was decided 214 to work on what we called "simulated tissue. Furthermore, it was impossible to mix several 215 antibiotics in the same sample, since the method was not specific and did not identify the 216 molecule that produced an inhibitory effect. Moreover, one could observe a cumulative 217 effect of antibiotics in their inhibitory activity when several antibiotics are contained in one preliminary study was conducted to determine what was the best way to 221 prepare simulated tissues and what kind of preparation would give the closest result to the 222 actual samples. This study was based on the experience of 2 National Reference 223 Laboratories (Vicente Calderon, AESAN, Spain; Anna Liisa Myllyniemi, EVIRA, Finland) 224 that had already worked on such samples. During this preliminary study, the way of 225 preparation of simulated tissue was also tested for homogeneity of the material. Regarding 226 the results, the homogeneity was satisfactory. 227 228 Production of spiked materials ("Simulated tissue") 229 A preliminary study has been conducted to determine the concentrations of antibiotics to be 230 spiked in muscle samples for later validation. Sixteen different antibiotics have been tested at 231 various concentrations (the MRL and according to the assumed detection limit at half MRL, 2 232 times the MRL or more) in simulated tissues. The tested concentrations were based on the 233 results of the validation of the STAR protocol in milk (Gaudin et al. 2004). These tests were 234 repeated several times. The concentration which always gave positive results (inhibition 235 one antibiotic concentration, instead of 20 samples to reach the most 272 statically significant determination of the CCβ (EC 2002). the samples is detailed in figure 1. To introduce a maximum of variability 277 in the determination of the detection capabilities, 30 samples have been prepared and 278 analyzed by one technician and another 30 samples by other technician. In addition, the 30 279 samples have been divided into 2 sets of preparation. In each set, 15 samples for the same 280 antibiotic concentration have been prepared and analyzed by each technician. For each 281 antibiotic and each technician, the 2 sets have been spaced out, to introduce variability in the 282 period of analysis (ambient conditions) and therefore variability of batches (media, bacteria, 283 antibiotic standard...). Moreover, in each set of preparation of 4 antibiotics, 4 different 284 batches of porcine muscle have been used. Finally, on each day of preparation, 2 different 285 working solutions have been prepared from the stock solution of antibiotic and used to spike 286 the different batches of muscle. The coding of the samples has been performed by the head 287 of the study, to ensure that the analyses would be performed blindly, and then frozen atof specificity and detection capabilities, all samples have been analyzed 291 on the 5 plates of the STAR protocol to check the specificity of the plates for the different 292 families of antibiotics. Five days of analyses have been performed with the STAR protocol for 293 each set of preparation and each technician. Each day, 14 samples have been analysed 294 blindly (12 spiked samples and 2 blank samples). Therefore, all blank and spiked samples 295 have been analyzed twice or 3 times, on 2 or 3 different days.

  : After the analysis of the 60 spiked samples, the concentration level,305where only less than 5 % of false compliant results remains was the detection capability CCβ 306 of the method (3 false compliant results maximum out of 60 spiked samples).

  350 concentration (A), medium quantity (B) in the plate, incubation time (C) and pre-incubation 351 time at room temperature (D). These factors have been modified in an order of magnitude 352 corresponding to the usual differences: factor A concentration in bacteria: ± 30%; factor B 353 quantity of medium 5 ± 0.5 ml; factor C incubation time ± 10%; factor D pre-incubation time 354 (period at room temperature before incubation in the incubator: 1hour of pre-incubation or no 355 pre-incubation).

361

  enrofloxacin at 200 µg kg -1 on plate Ec8. The chosen concentrations were equal to the 362 detection capabilities determined in the first part of the validation study for each of these 363 antibiotics, except for gentamicin. The samples were prepared from pig muscle only. All 364 b²lank and spiked minced muscle samples were prepared the day before starting the 365 ruggedness study and were frozen at -20°C. The study was performed blindly (codified 366 samples). Then each day of analysis (each run), 4 different blank materials and 4 different 367 spiked materials per antibiotic were analysed. capabilities were determined for pig matrix because it is the most 372 common species analyzed in the laboratory. 49 different batches of porcine muscle have 373 been tested. Few variations have been observed. Of 176 analyses in total, only 7 gave false 374 positive results (4%). The 7 false positive results were obtained with 5 different muscle 375 batches (2 false positive results for 2 batches and 1 false positive result for each of 3 376 batches). All false positive results have appeared on the plate Bst, none on the other plates.

Figure 2

 2 Figure 2 is a graphical representation of the relationship between detection capabilities

Figure 3

 3 Figure3represents the average inhibition zones on the 5 different plates(Bst, Bc6, Kv8, Ec8

  During the determination of specificity and detection capabilities of the 16 antimicrobials, lots 490 of different media (different preparation and validity date), different preparations of bacteria, 491 different batches of antibiotic standards have been used over a period of 9 months of testing.

518

  of the tested factors had an effect on the sensitivity. Sulphonamides tested concentrations 519 often gave Partial Inhibition Zones (PIZ), which are sometimes quite tricky to read for 520 someone not trained. On the opposite, beta-lactams usually gave clear inhibition zones. So, 521 slight variations like in the ruggedness study, even for a trained people, led to more effect on 522 these PIZ than on clear inhibition zones. Therefore, it is logical that the Bst plate is less 523 robust for the detection of SDMX than for penicillin G. the STAR method has been evaluated using an experimental design. The 525 influence of the 4 factors (concentration of bacteria, quantity of medium, incubation time and 526 pre-incubation time) on the mean inhibition zone (quantitative result), repeatability (coefficient 527 of variation CV%) and false positive and false negative rate (qualitative result), as well as the 528 interactions between factors, have been evaluated (Table

540

  impact of the combination of factors tested that day: A +, B +, C-, D-, i.e. increasing the 541 concentration of bacteria, increasing the amount of medium, reducing the incubation period 542 and without pre-incubation. In the optimization of microbiological methods, it is well known 543 that increasing the concentration of bacteria or increasing the amount of medium may 544 decrease the sensitivity of the plate. Moreover, as seen with other plates, a decrease of the 545 incubation period can indeed reduce the sensitivity. It is also logical that a lack of pre-546 incubation may decrease the sensitivity of the plate. It was observed in fact that an increase 547 of factors A and B may increase the false negative rate, while an increase of the incubation 548 time and pre-incubation of one hour may increase the sensitivity. It is noticeable that 1 hour 549 of pre-incubation at room temperature increased the mean inhibition zone of SDMX of 17%. variability of results of microbiological plate tests, none of the factors 554 or combination of factors had significant effect on the mean inhibition zone (quantitative 555 result) for any of the tested antibiotics on the 5 plates. Finally, no significant effect has been556observed on the repeatability of the method. Moreover, slights changes in the coefficient of 557 variation never had effect on the qualitative results of the test for the tested antibiotics.558Therefore, the STAR protocol is a robust method for the detection of antibiotic residues in 559 muscle. Some recommendations are given in the STAR protocol, concerning the different 560 incubation periods for the 5 plates, because incubation time was already known as a critical 561 the incubation time for plate Kv8 increased the sensitivity of the plate 563 of 13 % (Table

572A

  recommendation of pre-incubation 1 hour at room temperature, should be added in the 573 next version of the STAR protocol. Moreover, every day of analysis, specific positive control 574 antibiotic paper discs are put on each plate. The results of these positive controls should be 575 included in the range given in the STAR protocol. If it is the case, the results are valid. , very few laboratories tried to validate their screening methods (microbiological or 579 immunological methods) according to the decision 2002/657/EC (EC 2002) for 2 main 580 reasons. Firstly, the decision 2002/657/EC (EC 2002) gave very little information and no 581 technical recommendations for the implementation of the validation of a screening method. 582 Secondly, especially for the validation of microbiological plate tests, the validation requires a 583 long work, is time-consuming and quite expensive. In our laboratory, which is the Community 584 Reference Laboratory for antibiotic residues, the validation of screening methods has been a 585 subject of interest for many years. At this time, the validation of immunological tests and 586 microbiological tests (tube tests and plate test) have been performed in our laboratory 587 according to the European decision 2002/657/EC[START_REF] Gaudin | Validation of a 900 microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic 901 residues in milk[END_REF][START_REF] Gaudin | Validation of a Biacore Method for Screening Eight 903 Sulfonamides in Milk and Porcine Muscle Tissues According to European Decision 904 2002/657/EC[END_REF] 588Gaudin et al. 2009a; Gaudin et al. 2009b). The former "detection limit" has been replaced by 589 a new performance characteristic called "detection capability CCβ". To our knowledge, the 590 other validation studies of microbiological plate tests according to the European decision (EC 591 2002) were from a National Reference Laboratory in the Netherlands[START_REF] Pikkemaat | Improved microbial screening assay for the detection of quinolone residues in 932 poultry and eggs[END_REF] 592[START_REF] Pikkemaat | A new 934 microbial screening method for the detection of antimicrobial residues in slaughter 935 animals: The Nouws antibiotic test (NAT-screening)[END_REF] Pikkemaat et al. 2009b).

593 594-

 593 Regarding the time needed for this validation study, it would have been impossible to 595 validate the STAR protocol for all the antibiotics having a MRL in muscle matrix (more than 596 50 antibiotics). The work of validation would be too long, expensive and laborious. Therefore 597 Some antibiotics are only used as intra-mammary treatment (e.g. cefalonium has 635 been replaced by ceftiofur because ceftiofur is widely used in cattle and swine to treat 636 respiratory diseases while cefalonium is only administered intra-mammary),

643-

  for ceftiofur and cefquinome because one was the least detected antibiotic of the 644 family, with a low MRL (cefquinome) and the other (ceftiofur) has an intermediary 645 detection with a high MRL. Regarding the activity profiles on the STAR protocol, it 646 was assumed that if cefquinome is detected at 2000 µg kg -1 (CCbeta), cefazolin and 647 cephapirin would be easily detected.648 649The antibiotic should be largely used in the country of implementation of the method 650 (e.g. in France, sulfamethazine replaced sulfathiazole). Moreover, colistin which 651 belongs to the polymyxin family has been replaced by a second macrolide: tylosin 652 because macrolides are often used for animal treatment (cattle, swine, poultry).

--

  During the development of the STAR protocol, it was already shown that the method 655 had a very poor sensitivity for some antibiotics (e.g. flumequine, sulfaguanidine, 656 colistin and spectinomycin), largely above the respective MRLs. So 657 dihydrostreptomycin (DHS) replaced spectinomycin (level of detection in milk: 20000 658 to 40000 µg l -1 ). During the validation of the STAR protocol in milk (Gaudin et al. 659 2004), the level of detection of colistin was included between 200 and 2000 times its 660 MRL (MRL 50 µg l -1 in milk and 150 µg l -1 in muscle). Therefore the STAR protocol is 661 not fitted to the detection of colistin or of spectinomycin. 662 663 Some antibiotics are frequently detected in routine monitoring samples: the 664 tetracyclines, particularly oxytetracycline (OTC). Thus, tetracycline has been replaced 665 by oxytetracycline and doxycycline was added instead of flumequine. During the 666 validation of the STAR protocol in milk, the limits of detection for OTC and TTC were 667 both equal to 250 µg l -1[START_REF] Gaudin | Validation of a 900 microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic 901 residues in milk[END_REF]). The detection limits of doxycyline (50 µg 668 l -1 ) and CTC (75 µg l -1 ) were better. Therefore tetracycline family has been divided in 669 2 groups from which OTC (the least detected) and doxycycline (the best detected) 670 were chosen as representative compounds. of MRL (tylosin 100 µg kg -1 and erythromycin 200 µg kg -1 ). Concerning 680 quinolones, enrofloxacin was considered as representative of the quinolones family, except 681 flumequine. In fact, flumequine is usually badly detected by many microbiological methods.

  685 2004). The quinolones which was badly detected was flumequine (> 600 µg l -1 ). During the 686 validation in milk, sulfanilamide, sulfapyridine, sulfadoxine, sulfacetamide, sulfaquanidine 687 were the least sensitive sulphonamides (3.5 to 20 times the MRL (MRL = 100 µg/l -1 ) and the 688 most sensitive sulphonamides were sulfaphenazole, sulfathiazole, sulfachloropyridazine 689 (MRL). The limits of detection of sulfadimethoxine (SDMX) (1.75 times the MRL) and 690 sulfamethazine (2.5 times the MRL) were in between. The choice of sulphonamide was not 691 based on the least detected compounds because it was too far from the MRL but was 692 focused on 2 antibiotics which had intermediary sensitivities. Moreover these 2 693 sulphonamides are commonly used for animal treatment. Concerning the aminoglycosides,

703

  implementing the STAR protocol on routine monitoring muscle samples in a comparative 704 study. Only 1 % of false positive results were observed (6/591), on plate Bst, after the 705 analyses of 591 routine monitoring samples, when the cut-off was set at 4 mm, which is the 706 recommended cut-off in the STAR protocol for the plate Bst.

  have been determined for 16 antibiotics from different families in 736 relation to their respective Maximum Residue Limit (MRL). The levels of detection of beta-737 lactams (penicillins and cephalosporins ≤ MRL) were very satisfactory because even the 738 least detected compound has been detected at or below MRL. Concerning tetracyclines, 739 OTC (the least detected) could be detected at levels below 2.5 times the MRL and 740 doxycycline (the best detected) at the MRL. The levels of detection of macrolides (2 MRL), 741 quinolones (≤ 2 MRL) and trimethoprim (2 MRL) were also satisfactory even for the least 742 detected compound. The levels of detection of one representative sulphonamide SDMX (≤ 3 743 MRL) were slightly higher than in milk, Finally, the sensitivity of the STAR protocol towards 744 was not satisfactory (>>MRL). Therefore the interest of the 745 plate Bs8 in the STAR protocol could be discussed. This plate should be improved for its 746 sensitivity or should be replaced by another plate. Other possibility is to use a 747 complementary method focused on aminoglycosides which could replace the use of this fifth 748 plate.

  769been analyzed with different methods including the method to be validated (including one or 770 several physico-chemical confirmatory methods). The first disadvantage is that the number of 771 samples containing residues is unknown. Moreover, very powerful confirmatory methods are 772 needed to confirm all the samples, preferably even negative results at the screening step to 773 check the false negative rate of the screening test. Therefore it is also costly and time 774 consuming. Finally only a narrow range of antibiotics (those which are most frequently used 775 in the country) is encountered. Schneider et al. (2009) confirmed the presence of antibiotics 776 in 29 samples, from which 23 belonged to the tetracycline family. This kind of validation led 777 very interesting information on the method, its practicability in routine conditions and some " (spiked tissues) when it concerns solid matrices and when it is impossible 828 to obtain incurred tissues. Moreover, the validation could be restricted to a list of 829 representative antibiotics. The validation which have been performed in the past on field 830 samples led very interesting information concerning the method. However this kind of 831 validation does not fit to the decision EC/2002/657 and to the recommendations of the 832 validation guideline (Anonymous 2010). In fact the number of antibiotics tested is always very 833 restricted. Moreover the number of samples to be analysed to determine the detection 834 capabilities of antibiotics is never respected. 835 836 As a conclusion, there is no ideal way of validating microbiological plate tests which would 837 not be time and money consuming. Obviously, validation of these kind of methods needs a 838 lot of efforts from the lab which would validate the protocol for the first time (initial validation).

  843 capabilities) determined in the transfer lab have to be compared to those determined during 844 the initial validation for a selected range of antibiotics. Then the participation to proficiency 845 testing studies could complete the validation dossier. of this validation study have been fulfilled. Firstly, the STAR protocol has 849 been validated for muscle by determining performance characteristics (specificity, detection 850 capabilities CCβ for 16 representative antimicrobials, applicability, ruggedness), according to 851 the European decision 2002/657/EC (EC 2002). The STAR protocol is applicable to the 852 broad spectrum detection of antibiotic residues in muscles of different animal species (pig, been determined for 16 antibiotics from different families in 855 relation to their respective Maximum Residue Limit (MRL): beta-lactams (penicillins and 856 cephalosporins ≤ MRL), tetracyclines (≤MRL and ≤ 2.5 MRL), macrolides (2 MRL), 857 quinolones (≤ 2 MRL), some sulphonamides (≤ 3 MRL), trimethoprim (2 MRL). However, the 858 sensitivity of the STAR protocol towards aminoglycosides and florfenicol was not satisfactory 859 (>>MRL). Finally, this study has shown that the STAR method is a robust screening method, 860 insensitive to reasonable variations analytical parameters such as concentration of bacteria, 861 amount of medium, incubation period and pre-incubation or not. 862 863 Secondly, the applicability of the European guideline (Anonymous 2010) to the validation of a 864 microbiological screening method and for muscle has been demonstrated. The use of 865 "simulated tissue" appeared to be a very good compromise between antibiotic spiked discs 866 and incurred samples. Moreover, the choice of a list of representative antimicrobials was also 867 very interesting to reduce the scope of validation. Explorer test, PremiTest 2 microbiological tube tests and a multi-907 sulphonamides ELISA kit, for the detection of antimicrobial and sulphonamide residues in 908 eggs. Food Additives & Contaminants: Part A. 26 (4):427-440. 909 Gaudin V, Hedou C, Verdon E. 2009b. Validation of a wide-spectrum microbiological tube 910 test, the EXPLORER® test, for the detection of antimicrobials in muscle from different 911 animal species. Food Additives & Contaminants: Part A. 26 (8) 1162-1171. 912 Koenen-Dierick K, Okerman L, De Zutter L, Degroodt JM, Van Hoof J, Srebnik S. 1995. A 913 one-plate microbiological screening test for antibiotic residue testing in kidney tissue and914meat : an alternative to the EEC four-plate method?. Food Additives and Contaminants.

  .-L., Rintala, R.,Backman, C., and Niemi, A., 1999, Microbiological and 917 chemical identification of antimicrobial drugs in kidney and muscle samples of bovine 918 cattle and pigs. Food

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Preparation of the simulated tissues.

  Figure 1.

  deviation; IZ: Inhibition zone (mm); +: positive result; -: negative result; D: doubtful result (0<ZI<2 mm).

346 347 Insert Table 2 about here 348 349

	345	chosen for the analysis of the effects and interactions of 4 independent factors (Renard et al.
		1992). The design matrix is presented in Table 2.
		Factors which may influence the measurement results have been selected: bacteria
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
	341	under which the method can
	342	be applied as presented or with specified minor modifications". A ruggedness study based on
	343	an experimental design was carried out and thus allowed testing analytical parameters which
		were different from those tested during the first step of validation. A full factorial design was
		11

Table 1 . Chosen antibiotics and corresponding concentrations for the validation study.

 1

	2a.

Table 4 .

 4 Specificity of the plates.

	Antibiotic family	Antibiotic	Specific plate
	PENICILLINS	Penicillin G Cloxacilline	Bst Bst
	CEPHALOSPORINES	Cefquinome Ceftiofur	Kv8 (Bst and Ec8) Bst (and Ec8)
	TETRACYCLINES	Oxytetracycline (OTC) Doxycycline	Bc6 Bc6 (and Bs8)
	MACROLIDES	Erythromycin Tylosin	Kv8 Bst
	QUINOLONES	Enrofloxacin	Ec8 (and Bst)
	SULFONAMIDES	Sulfadimethoxine (SDMX) Sulfamethazine (SMZ)	Bst Bst
	AMINOGLYCOSIDES	Dihydrostreptomycin (DHS) Gentamicin (GTM)	Bst Bst
	LINCOSAMIDES	Lincomycin	Bst
	MISCELLANEOUS	Trimethoprim (TMP) Florfenicol	Bst Bs8 (Bc6 and Bst)

Table 5 . Comparison of the mean Inhibition zones (IZ) and the standard deviations (SD) calculated during the determination of the CCβ and during the ruggedness study.

 5

		Food Additives and Contaminants		Page 36 of 38
			Tested		Mean IZ * (mm) ±	Mean IZ * (mm) ±
	Antibiotic family	Antibiotic	concentratio n (µg kg -1 )	Plate	SD CCβ	SD Ruggedness
	PENICILLINS	Penicillin G	25	Bst	9.7 + /-0.2	8.5+/-1.2
	SULFONAMIDES	Sulfadimethoxine (SDMX)	300	Bst	8.8 + /-1.3	5.3+/-1.6
	TETRACYCLINES	Doxycycline	100	Bc6	5.4 +/-0.5	4.3+/-0.6
	MACROLIDES	Erythromycin	400	Kv8	5.1 +/-1.1	4.0+/-1.0
	QUINOLONES	Enrofloxacin	200	Ec8	6.2 +/-1.0	6.7+/-1.2
	AMINOGLYCOSIDES	GTM	6000	Bs8	6,0+/-0,7	6.2+/-0.5
	*IZ = Inhibition zone of the specific plate, mean of the values of the 4 sets (2 technicians)
	SD = Standard Deviation

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Table 7 . Ruggedness study for plate Bst: Design matrix and experimental design calculation.

 7 A strong impact of the combination of factors tested on day 4 was observed on false -rate: A +, B +, C-, D-, i.e. increasing the concentration of bacteria, increasing the amount of medium, reducing the incubation period and without pre-incubation.

	Run	Mean IZ SDMX	CV	False +	False -	Mean IZ Penicillin	CV	False +	False -
	1	4.8	17.5	0	0	9.1	10.9	0	0
	2	8.0	18.0	0	0	9.3	10.3	0	0
	3	4.7	13.9	0	0	7.5	22.5	0	0
	4	3.0	15.4	0	100*	10.0	4.6	0	0
	5	5.3	10.2	0	0	9.1	2.0	0	0
	6	4.6	7.6	0	0	7.5	7.1	0	0
	7	5.1	13.4	0	0	7.5	7.1	0	0
	8	6.9	11.8	0	0	8.4	8.9	0	0
	IZ: Inhibition zone (mm); False + = False positive rate; False -: false negative rate; CV: Coefficient of
	variation (%) = (mean IZ)/(SD IZ)*100; SD: standard deviation.
	Run 1 = Day 1

*

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Bst (figure3a), doxycycline at 100 µg kg -1 on Bc6 (figure3b), erythromycin at 400 µg kg -1 on

Kv8 (figure3c), enrofloxacin of 200 µg kg -1 on Ec8 (figure3d) and gentamicin at 6000 µg kg -1

for the penicillin family because penicillin G (LOD 5 µg l -1 ) was the best detected penicillin

during the validation of the STAR protocol in milk and cloxacillin (LOD 60 µg l -1 ) was the least

detected penicillin[START_REF] Gaudin | Validation of a 900 microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic 901 residues in milk[END_REF]). Moreover they belong to 2 groups of MRL (penicillin

G 50 µg kg -1 in muscle and cloxacillin 300 µg kg -1 ). Similarly, erythromycin (LOD 30 µg l -1 )

and tilmicosin (LOD 50 µg l -1 ) were the best detected macrolides in milk. On the contrary,

tylosin (LOD 200 µg l -1 ) was the least detected and was representative of the group of

spiramycin (LOD 300 µg l -1 ) and neospiramycin (LOD 200 µg l -1 ). Moreover they also belongs

In the decisionEC/2002/657 (EC 2002), there is no recommendation concerning the kind of

materials to be analysed (spiked or incurred matrix) during the validation. Therefore all of the

cattle, sheep, poultry.). The method has good specificity (false positive rate 4%). The

procedures for the establishment of residue limits of pharmacologically active substances

in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90. Official

Journal of the European Community. L152:11-22.

Fuselier R, Cadieu N, Maris P, in: L.A. van Ginkel, A. Ruiter (Eds.), proceedings of the

Euroresidue IV Conference, National Institute of Public Health (RVM),Bilthoven, 2000, p.

Gaudin V, Hedou C, Rault A, Sanders P, Verdon E. 2009a. Comparative study of 3

Myllyniemi AL, Nuotio L, Lindfors E, Rannikko R, Niemi A, Backmann C. 2001. A

resulting from animal treatment (Myllyniemi et al. 1999;Myllyniemi et al. 2001). In this case, 782 the authors used the activity patterns of antibiotic standard solutions to identify antibiotic 783 residues in incurred materials. Furthermore, the STAR protocol has been validated in our lab 784 using spiked paper discs, plus analysing field routine samples. The interest of spiked discs is 785 that many antibiotics at many concentrations could be tested but limitation is that no matrix 786 effect could be observed. It is interesting to obtain preliminary data on the evaluation of a 787 new method and activity profiles of many antibiotics. But we know that matrix will affect the 788 detection capabilities of the method. Therefore, field samples are very interesting because 789 they are incurred materials and matrix effect could be demonstrated. However the limitation 790 is that it is not known in advance how many positive samples are in the panel of field 791 samples, which antibiotics would be detected and at which concentrations. Moreover another 792 limitation is that a little variety of antibiotic residues or families are usually found, depending 793 on the matrix. [START_REF] Okerman | Evaluation 928 and establishing the performance of different screening tests for tetracycline residues in 929 animal tissues[END_REF] used artificially contaminated (spiked tissue fluid) as 794 well as incurred samples for the comparative study of 4 screening methods for the detection 795 of tetracyclines in muscle matrix. This approach is also interesting because matrix effect 796 could be observed both with spiked tissue fluid and incurred tissue.

798

The third possibility is the use of "simulated tissues" as it is proposed in this paper. It seemed 799 to be a good compromise between spiked discs and incurred tissues and the nearest 800 preparation from intact muscles because matrix effect could be evaluated. For the validation 801 of a microbial screening assay, [START_REF] Pikkemaat | Improved microbial screening assay for the detection of quinolone residues in 932 poultry and eggs[END_REF] used 2 different approaches to 802 produce fortified tissues. Firstly spiked minced was heated and centrifuged to extract meat 803 juice (supernatant) which was applied directly onto the plate. Secondly incurred tissues were 804 transformed into powder (using liquid nitrogen and blending the meat) and meat juice was 805 extracted. Therefore, it was possible to obtain accurate concentrations in the samples by 806 mixing incurred and blank materials. One conclusion was that "the sensitivity of the assay for 807 quinolones decreased 2 fold when matrix samples are analysed" by comparison with 808 antibiotic standard solutions. In these conditions, matrix effect was taken into account.

809

Moreover, these 2 approaches are fitted for this screening test because in routine, meat juice 810 samples would be analysed also. However, that sample preparation differed a lot from the 811 routine application of the STAR protocol which is based on pieces of raw muscles laid 812 directly on the plates. Therefore, in our case, our "simulated tissues" appeared to be as near