Georges Boudebs
email: georges.boudebs@univ-angers.fr

Julien-Bilal Zinoune

Christophe Cassagne

Mihaela Chis

Thermal lens Z-scan measurements for the determination of fluorescence quantum yields: theoretical and experimental uncertainties for low and high quantum yields

The single-beam Z-scan thermal lens (TL) technique is conducted to evaluate the fluorescence quantum yield of various solutions in the case of high-moderate absorption. Considering both scenarios: solutions with substantial fluorescence and solutions with high thermal efficiency but low fluorescence. An analytical calculation is performed to determine the uncertainties associated to the random errors introduced by optical detectors. The results reveal that solutions with low fluorescence led to a significant error, whereas higher fluorescence can help decreasing the uncertainty. Additionally, the issue of random errors arising when multiple measurements are needed to accurately estimate the fluorescence of a solution will be discussed in different situations.

Introduction

Thermal lens measurement techniques encompass a broad spectrum of applications, with a notable focus on calorimetry and spectroscopy. These techniques have been extensively employed in a multitude of studies aimed at precisely characterizing the thermal [START_REF] Cabrera | Frequency-resolved photothermal lens: An alternative approach for thermal diffusivity measurements in weak absorbing thin samples[END_REF][START_REF] Safi Keykeleh | Investigation of the effect of temperature on opto-thermal parameters of glycerol and ethylene glycol using laser thermal lens spectroscopy[END_REF][START_REF] Badran | Thermal properties of a new dye compound measured by thermal lens effect and Z-scan technique[END_REF] and optical [START_REF] Estupiñán-López | Eclipsing thermal lens spectroscopy for fluorescence quantum yield measurement[END_REF][START_REF] Mohebbifar | Investigation of thermal lens performance of rhodamine 6G and rhodamine B at different concentration using pump/probe laser thermal lens spectroscopy[END_REF][START_REF] Cabrera | Trace detection and photothermal spectral characterization by a tuneable thermal lens spectrometer with white-light excitation[END_REF] properties of various compounds and materials. One of the first was the measurement of small absorption in low-loss liquids and solids as low as [START_REF] Leite | Low absorption measurements by means of the thermal lens effect using an He-Ne laser[END_REF] showing that it can serve as highly sensitive absorption spectrometers especially using pumbprobe mode mismatched measurements [START_REF] Marcano | Pump-probe mode-mismatched thermal-lens Z scan[END_REF][START_REF] Marcano | High-sensitivity absorption measurement in water and glass samples using a mode-mismatched pump-probe thermal lens method[END_REF]. On the other hand, the single-beam Z-scan method has often been used with pulsed lasers for third-order nonlinear susceptibility measurements (see for example [START_REF] Besse | Determination of the third-and fifth-order optical nonlinearities: the general case[END_REF][START_REF] Falcão-Filho | Nonlinear optical properties of tungstate fluorophosphate glasses[END_REF][START_REF] Vermeulen | Post-2000 nonlinear optical materials and measurements: Data tables and best practices[END_REF]) distinguishing between the real and imaginary parts of this physical quantity. Basically, these two methods are based on the far field diffraction phenomenon and are designed and known to measure relatively small, induced phase shifts with a sensitivity of a few tens of mrad at room temperature [START_REF] De Araújo | Techniques for nonlinear optical characterization of materials: a review[END_REF]. The combination of these two techniques should give even greater robustness to the so-called Zscan TL based measurements which will be considered here. In their significant contribution, Hu and Whinnery [START_REF] Hu | New thermooptical measurement method and a comparison with other methods[END_REF] highlighted an additional use of thermal lensing measurement, which can be combined with conventional transmission data to calculate the fluorescence quantum yield. Initially, the total power absorbed by the dye solution is determined through energy transmission measurement. Subsequently, the power converted into heat is measured using the TL method. The difference between these two measurements is assumed to represent the power that has been emitted through fluorescence. This constitutes an indirect yet absolute method for measuring fluorescence. Several techniques are available for fluorescence measurements [START_REF] Würth | Relative and absolute determination of fluorescence quantum yields of transparent samples[END_REF][START_REF] Rth | Comparison of methods and achievable uncertainties for the relative and absolute measurement of photoluminescence quantum yields[END_REF][START_REF] Würth | Determination of the absolute fluorescence quantum yield of rhodamine 6G with optical and photoacoustic methods-Providing the basis for fluorescence quantum yield standards[END_REF], with the most widely used one being the spectrofluorometer. Initially conceived by Vavilov [START_REF] Vavilov | The fluorescence efficiency of dye solutions[END_REF] and subsequently refined by Parker et al. [START_REF] Parker | Correction of fluorescence spectra and measurement of fluorescence quantum efficiency[END_REF], the spectrofluorometer performs direct measurements relative to a known standard sample. The diversity of techniques and protocols has, in turn, led to variations in outcomes when applied to the same species [START_REF] Rth | Comparison of methods and achievable uncertainties for the relative and absolute measurement of photoluminescence quantum yields[END_REF][START_REF] Rurack | Fluorescence quantum yields: methods of determination and standards (Standardization and quality assurance in fluorescence measurements I: techniques[END_REF][START_REF] Fery-Forgues | Are fluorescence quantum yields so tricky to measure? A demonstration using familiar stationery products[END_REF]. The determination of the absolute value of fluorescence quantum efficiency has been proven to be challenging, leading to conflicting results in the literature. While pure optical techniques can be employed to determine quantum efficiency, calibration is often problematic. The Z-scan technique relies on the absorption of light by the sample to generate a TL effect. If the sample has low extinction absorption, the sensitivity of the measurement decreases, making it challenging to accurately measure fluorescence signals [START_REF] Jacinto | Thermal lens and Z-scan measurements: Thermal and optical properties of laser glasses-A review[END_REF]. On the other hand, if absorption is increased, mathematical formulas using the TL technique assume a first-order expansion of Beer-Lambert-Bouguer's law, and results derived from this approximation become unreliable.

Recently, experimental determination of the absorption and scattering efficiencies of spherical gold nanoparticles (NPs) with different diameters was carried out using the TL effect at three distinct wavelengths: blue, green, and red. The measurement method's key features, including the experimental procedure for weak absorption using Gaussian beams and its extension to higher absorption with top-hat beams, are thoroughly explained to provide a comprehensive understanding. In order to validate our approach, the obtained experimental TL data was compared to the theoretical values derived from Mie theory. Remarkably, the comparison showed a high level of agreement, within the limitations of experimental errors [START_REF] Zinoune | Wavelength-dependence of the photothermal efficiency of gold nanoparticles in solution by Z-scan photothermal lens spectroscopy[END_REF].

It would nevertheless be useful to recall the advantages and disadvantages of the Z-scan TL method, which we have frequently used for third-order nonlinear optical measurements. It is indeed a valuable technique for fluorescence measurement in certain situations. This method offers high sensitivity, and it can detect very low levels of fluorescence signals. One of the key advantages is its ability to suppress background noise effectively. By detecting the signal in the far field ( away from the cell) with a small-aperture PD, there is very little risk of detecting signals from scattering and fluorescence diffused in all directions around the focal spot-line, while only thermal effect optical signals propagate axially through a very small solid angle to reach the detector. This enables precise measurement of the desired fluorescence signal without significant background contamination. The TL method is a gentle, generally non-destructive method. It does not alter or damage the sample during measurement using relatively moderate power, making it suitable for analyzing delicate or valuable samples in thin films [START_REF] De Araújo | Giant third-order nonlinearity of lead and germanium based films in the visible and in the infrared[END_REF] or those having two photon absorption [START_REF] Fedus | Nonlinear characterization of GeS2-Sb2S3-CsI glass system[END_REF]. Z-scan TL can provide high spatial resolution in fluorescence measurements. By focusing the laser beam to a small spot, it allows for localized analysis, enabling to investigate specific regions or features within the solution and obtain detailed information about their fluorescence properties. So, Z-scan TL qualitative imaging method can be applied to various types of samples as in thin films [START_REF] Ba | Phase shift imaging in thin films using CW Z-scan based technique[END_REF], inhomogeneous solid surfaces [START_REF] Falcão-Filho | Nonlinear optical properties of tungstate fluorophosphate glasses[END_REF][START_REF] Manzani | The role of Bi2O3 on the thermal, structural, and optical properties of tungsten-phosphate glasses[END_REF], and even biological tissues [START_REF] Angert | The Reliability of Fluorescence Z-Scan Analysis in the Complex Environment of the Living Cell[END_REF]. This versatility makes it a useful tool in many fields, such as environmental monitoring, pharmaceutical analysis, and bioimaging but one need to take more parameters into account than those considered so far. It could be combined with a phase object to differentiate the various phenomena that can occur in light-matter interaction as it has been done in [START_REF] Boudebs | Nonlinear refraction measurements in presence of nonlinear absorption using phase object in a 4f system[END_REF]. In many cases, the TL method requires minimal sample preparation. It can directly analyze samples without the need for complex labeling or immobilization procedures, saving time and resources. It can provide information about the fluorescence lifetime of a sample. By analyzing the TL decay curve, it is possible to extract valuable kinetic and dynamic information, which can be useful for studying molecular interactions, photochemical processes, and other time-dependent phenomena. Moreover, in terms of microscopic dimensional metrology, incorporating doping fluorescent nanoparticles could serve as a viable solution for specific measurement-problem types [START_REF] Boudebs | Contrast increasing by third-order nonlinear image processing: a numerical study for microscopic rectangular objects[END_REF].

However, Z-scan TL efficiency depends on various factors and limitations of the method in relation to the particular application at hand. Measuring the fluorescence of a solution has certain disadvantages. It relies on the absorption of light by the sample to generate a TL effect. If the sample has low extinction coefficient, the sensitivity of the measurement decreases, making it challenging to accurately measure fluorescence signals having even more lower absorption TL coefficients. It is not well-suited for measuring the fluorescence of solutions throughout their entire volume, thickness must be less than the Rayleigh range of the incident beam. So, the TL effect is limited in depth profiling capabilities. Interpreting the Z-scan TL data can be complex, especially when dealing with samples that have complex optical properties or heterogeneous compositions. The analysis often requires sophisticated mathematical models and assumptions, which can introduce uncertainties in the measurement. Moreover, this technique, sometimes, involves focusing a high-intensity laser beam onto the sample, which can lead to photothermal damage [START_REF] Boudebs | Characterization of light-induced modification of the nonlinear refractive index using a one-laser-shot nonlinear imaging technique[END_REF], particularly in sensitive samples or those prone to photochemical reactions. This can alter the fluorescence properties, affecting the accuracy and reliability of the measurements. Z-scan experiments typically involve multiple measurements. The Z-scanning process can be time-consuming, especially when trying to obtain a reliable and statistically significant dataset. In addition, the accuracy of Zscan TL measurements is highly dependent on the stability of the laser source which is another parameter which will not be considered here. Any fluctuations or instabilities in the laser power or beam profile can introduce errors or noise into the measurements, affecting the precision and reproducibility of the results.

In this paper, we will present the argument that the single beam Z-scan TL method is not just an alternative approach to measuring fluorescence quantum yields, but rather offers substantial advantages provided that the appropriate measurement errors are taken into account [START_REF] Cruz | Quantum yield excitation spectrum (UV-visible) of CdSe/ZnS core-shell quantum dots by thermal lens spectrometry[END_REF][START_REF] Cruz | Absolute photoluminescence quantum efficiency of P3HT/CHCl3 solution by Thermal Lens Spectrometry[END_REF][START_REF] Würth | Critical review of the determination of photoluminescence quantum yields of luminescent reporters[END_REF]. To achieve this, we will extend the application of this method to solutions with relatively higher absorbance. We will conduct measurements across various compounds, ranging from low to high fluorescence yields. Afterwards, this paper aims to examine the potential sources of errors that may arise and their impact on the measurement of fluorescence quantum yield. We are only interested in the error generated by the photodetectors, bearing in mind that there are other errors that can also influence the final result of the absolute measurement relative to the fluorescence quantum yield. Relative methods, however, yield the highest accuracy. To address this, it might be reasonable to create customized standards tailored to specific requirements. For instance, one could employ the TL method to measure very low fluorescent sample and promptly compare it to the current unknown of interest.

Our findings in this paper clearly demonstrate that the measurements uncertainties are not constant as is often considered in the literature but variable, increasing rapidly as quantum yield decreases. The number of measurements for low yields must therefore be increased to compensate for the higher relative error.

Theory

The theory relies on the concept of thermal lensing, which arises from localized variations in the optical index caused by the temperature distribution resulting from the absorption of light, specifically in our case, a single Gaussian laser beam. Let us start by distinguishing and clearly defining the two coefficients needed to measure the fluorescence efficiency (hereafter denoted as ). The absorption coefficient , expressed in units of inverse length (e.g., in

), is a measure of the material's ability to absorb photons of specific energy levels. It is defined according to the Beer-Lambert-Bouguer law relating the optical attenuation in a solution to the path length . The intensity change in the laser light as it passes the absorbing medium is expressed by: with being the radial intensity profile at the entry. The extinction coefficient is theoretically defined using the same law by measuring the transmitted power through two identical cells using a first cell filled with pure solvent, and a second one filled with the tested solution (see Appendix 1). It is related to absorption and other energy-loss processes that occur when light interacts with the travelled medium. It characterizes the total reduction in light power due to absorption, scattering, fluorescence luminescence… It is assumed here that the incident energy is transformed only into heat or fluorescence. Other losses are considered negligible.

The following symbols will be used: density in ; beam intensity in ; thickness of the medium (cell) in ; beam power in (taking into account the Fresnel reflection); specific heat in ; beam radius in ; temperature variation in ; thermal conductivity in ; wavelength in . The usual TL approximations are assumed following references [START_REF] Zinoune | Wavelength-dependence of the photothermal efficiency of gold nanoparticles in solution by Z-scan photothermal lens spectroscopy[END_REF][START_REF] Cassagne | Time-Resolved cw Thermal Z-scan for Nanoparticles Scattering Evaluation in Liquid Suspension[END_REF][START_REF] Sheldon | Laser-induced thermal lens effect: a new theoretical model[END_REF]: i) thin sample; ii) beam-waist considered small when compared to the dimensions of the cell. An expression of the temperature change as a function of radius and time can be obtained by solving the non-steady state heat equation with a Gaussian beam illuminating the medium [START_REF] Zinoune | Wavelength-dependence of the photothermal efficiency of gold nanoparticles in solution by Z-scan photothermal lens spectroscopy[END_REF][START_REF] Carter | Comparison of models describing the thermal lens effect[END_REF]:

(1)
where and is the characteristic buildup time constant.

Note that in the special case where we come back to the approximation used in [START_REF] Sheldon | Laser-induced thermal lens effect: a new theoretical model[END_REF] (

) but for the more general case with relatively larger absorbance (while remaining with should be considered. Here, could replace the absorption in the derived equations of Sheldon et al. [START_REF] Sheldon | Laser-induced thermal lens effect: a new theoretical model[END_REF] to define the phase lag characteristic quantity relative to :

, (2) with
denoting the algebraic value of the thermo-optical coefficient. The change in the linear index due to this temperature variation is:

(3) with where is the refractive index at the initial temperature. The phase shift is related to as usual, using . The single-beam configuration that we consider here states that the beam induces and probes it at the same time.

Principle of the optical method

The principle of the single beam method, which is now well known, has already been explained in detail in [START_REF] Zinoune | Wavelength-dependence of the photothermal efficiency of gold nanoparticles in solution by Z-scan photothermal lens spectroscopy[END_REF][START_REF] Cassagne | Time-Resolved cw Thermal Z-scan for Nanoparticles Scattering Evaluation in Liquid Suspension[END_REF]. For self-consistency, we summarize here the most significant features of the technique. The setup (Fig. 1) has the advantage of combining a Z-scan configuration when the sample moves along z (focal region of lens L 1 ) and a TL signal describing the time response profile when the chopper rotates for each position z. The signal is obtained on a photodiode (PD) that acquires the central variation of the diffracted far field beam intensity versus z, the position of the specimen. This is equivalent to performing what is commonly referred to as a "closed aperture Z-scan". The PD is connected to a digital storage oscilloscope where the signal is only revealed by a TL effect in the cell which allows the measurement of . is defined as the fractional change in intensity when the cell is located at after reaching the steady state regime as approaching infinity:

. ( 4
)
The analytical calculations presented in [START_REF] Cassagne | Time-Resolved cw Thermal Z-scan for Nanoparticles Scattering Evaluation in Liquid Suspension[END_REF] yield the far field intensity variation at :

(5

)
where with the Rayleigh distance and being the focused beamwaist at (the focus of lens ). is a calibration factor that needs to be adjusted for each experiment, as it may depend on the shape of the input beam, which is not perfectly Gaussian, and all the experimental imperfections that could be encountered. This factor could be used as well when considering a reference solution with a specific characteristic. Eq. ( 5) allows to obtain by fitting the experimental data. Following relationship (2), one can then find , i.e., the light truly absorbed in the medium:

. ( 6
)
In contrast, the specimen's average extinction coefficient value is determined by using an optical power meter (OPM) (Ophir, PD300UV, aperture [ 38 ]). This measurement is conducted by repeatedly varying the incident power and performing multiple measurements: with being the transmitted power by the pure solvent and the transmitted one by the solution that will be used in the experiment. The latter equation is employed to consider the Fresnel reflection that occurs between two identical cells.

Let

represent the ratio between the number of fluorescent photons to that of extinguished ones. It is assumed that the incident energy is transformed only into heat or fluorescence. Other losses are considered negligible: scattering, luminescence or other photoinduced light phenomena which spread out in the solution. This results in the determination of the light fluorescence efficiency Φ in relation to the overall light extinction, as calculated using the following equation (refer to the calculation details in Appendix A of reference [START_REF] Zinoune | Wavelength-dependence of the photothermal efficiency of gold nanoparticles in solution by Z-scan photothermal lens spectroscopy[END_REF]): , [START_REF] Leite | Low absorption measurements by means of the thermal lens effect using an He-Ne laser[END_REF] with and representing the respective frequencies of incident photons and the average frequency of the emitted fluorescence. Note two points: i) this relationship reduces to a simpler expression, used up to now, if we consider the limiting case with low absorbances:

; ii) represents the portion of light extinction that is absorbed by the solutions, resulting in photothermal heating and the detection of the TL signal.

Experimental results

The room temperature was consistently maintained at 21°C throughout all the measurements. The study has been performed using cw laser at with methanol-based solutions. The output laser beam was spatially filtered before collimation and truncated by a circular aperture resulting in a "top hat" beam of diameter ≈ . It was then focused into the quartz cell (Thorlabs, CV10Q35) containing the solutions to be tested. The rotation of the two-bladed chopper was set at a frequency of which gives of opening time during which the solution is irradiated and of obstruction allowing the liquid to cool down between two acquisitions. These exposure times provide a sufficient duration for the temporal evolution to reach the nearly stationary regime. Typically, with , the thermal response time of the methanol solvent is characterized by . This time can be regarded as very small when compared to the opening duration of the chopper. The scan on z is performed in the interval so that remains at most 10 times smaller than the opening time:

. As an illustration, an experimental result is provided here using the laser with a solution of Rhodamine B (RhB), purchased from [39], and taking into account . The blue stars experimental data in Fig. 2 ( are calculated using Eq. ( 4) and show a characteristic valley-peak profile. They are numerically processed to be symmetrical with respect to . Using Eq. ( 5), the fitting of allows to estimate the beam-waist of the focusing beam corresponding to a Rayleigh distance of using a focal lens . The thickness of the cell is therefore compatible to be considered as a thin sample. Note that the incident power (inside the liquid) in this example was resulting in a central peak intensity in the focus at , .

The solid red line in Fig. 2 shows the fitting of the experimental data (blue stars) giving with a solution having an overall extinction coefficient measured in a standard light transmission experiment via the OPM. Knowing , and for methanol, the value of the TL absorption can be obtained from Eq. ( 6) giving and therefore the value of . Finally, according to Eq. ( 7) and considering the average emission photon energy ( ) for RhB [START_REF] Velapoldi | Corrected emission spectra and quantum yields for a series of fluorescent compounds in the visible spectral region[END_REF], the measured fluorescence yield is . It is important to evaluate measurement error. Excessive measurement error compromises the validity and reliability of the results, necessitating careful assessment and mitigation strategies. Measurement error is categorized into two main types: random error and systematic error. Random error, generally defined as precision, focuses on the reproducibility and consistency of measurements; it occurs by chance and leads to slightly different results when measuring the same item multiple times, even if all steps are performed correctly. On the other hand, systematic error which is connected to accuracy focuses on how close those measurements are to the true or target value. It arises when the measurement system consistently makes the same type of mistake, often due to issues with the measuring tool.

Considering the accurate determination of the real power entering the sample is crucial as it directly impacts the measurement of , then . Ideally, both high precision and high accuracy are required in a measurement system. In our case, to determine , we must perform measurements on and . The assessment of exclusively relies on the OPM, which introduces a total error, as indicated by the OPM data sheet [38,41], with an accuracy of ±3% considering expanded error within 2.0 standard deviations with 95% confidence level. On the other hand, the measurement of entails the utilization of both the OPM and the photodetector PD, resulting in a measurement error that is predominantly random in nature. Consequently, our statistical evaluation yields a total error, which is a composite of systematic and random errors. This error is referred to as a relative error throughout the following text. The mathematical details of the absolute random error calculation are reported to the Appendix 1:

(8)
where, to lighten the notation, we designed by θ the characteristic phase parameter defined in Eq. ( 2). Based on this last equation, a simulation program has been developed to find the variation of as a function of taking into account the following experimental conditions: 1-Knowing , and for methanol with .

2-

Maintaining the linearity of with a good signal-to-noise ratio: [START_REF] Zinoune | Wavelength-dependence of the photothermal efficiency of gold nanoparticles in solution by Z-scan photothermal lens spectroscopy[END_REF]. So, having the highest signal at the PD corresponds to or .

3-

Knowing that (see Appendix 1) relative to our OPM, we need to decrease the relative error with (conditions for moderate absorptions) which requires .

4-

The simulation program therefore consists of varying for a given which allows to find through Eq. ( 7). allows to find with from Eq. ( 6). The obtained for the varying allows to calculate (Eq. 3) and therefore by taking the ratio. The results of the numerical calculation are shown in Fig. 3. The coordinates of the point that appear on this figure demonstrate that when the yield is less than 9%, the relative error on the measurement exceeds 100%. Moreover, Fig. 3 shows that in particular, the error will be less than 40% and therefore acceptable when Φ is greater than 20%. For lower , this indicates that the measurement ceases to provide accurate and consistent results, making it uncertain and untrustworthy. This result could have been obtained more quickly by noting that tends towards 0, forcing the ratio, of course, to tend towards infinity. But here significant values are obtained when is large. For example, the relative error tends to cancel out when tends towards 1, allowing to have lower relative errors on the quantum yield than those considered for the estimation of the incident power and the diffracted signal through .

It would be interesting to determine the most crucial parameter that requires meticulous measurement considering the experimental used value of which is the approximate value used for assessing various fluorescent compounds hereafter. In fine, there are two parameters that we quantify with a silicon detector, the 1st on the OPM to measure power and the 2nd on the PD to measure . Two simulations were carried out, the first dividing the relative error on by and the second dividing the error on by . The following Fig. 4 shows the two results in comparison with that given by the standard errors shown in blue solid line (3% with the OPM and 12% with the PD).

In Fig. 4, the simulation reveals that the primary source of critical error for low values of comes from the photodetector (PD). To minimize the overall measurement error, it becomes necessary to effectively decrease the PD error. Specifically, when measuring with a range of , the relative error exhibits 12%. Even with a PD that produces measurements with only a 1.2% error (as depicted by the green curve), it would be unrealistic to expect reliable measurement of a quantum yield below 10% with such high relative errors. Of course, by increasing the number of measurements, errors can be reduced. Given the trend in uncertainty shown in Figs. 3 and4, it seems obvious that the number of measurements needs to be increased more and more as yield decreases. On the other hand, one might think that it would be possible to calibrate the system with known solutions such as Rh6G with a high quantum yield ( , in order to make reliable measurements with products presenting lower yields. In reality, the sensitivity of the measurement is very low when the yield is high: the absolute value of the curve slopes given in Figs. 3&4 decreases exponentially with increasing . Then a dilemma presents itself: faced with the decision of either conducting measurements with heightened sensitivity but significant uncertainties or opting for lower uncertainty and sensitivity simultaneously. We opted for the 1st solution, increasing sensitivity but taking many measures to reduce uncertainty. Using hexaaquacobalt(II) nitrate [42], [Co(OH 2 ) 6 ][NO 3 ] 2 (CoN), as a reference with the lowest fluorescence efficiency, after a considerable number of measurements (68), a calibration coefficient of was derived adjusting the mean value of to be close to . Consequently, table 1 represents the average of the obtained quantum yield values of different solutions in this scenario while the detailed measurements according to Eq. ( 2) are given in the Appendix 2. Note that each value obtained from Z-scan profile represents a mean value based on fitting performed on approximately twenty positions of the sample along the z-axis (see Fig. 2). This should normally give a more reliable measurement than a single measurement taken with a given cell position proceeding with TL method alone by fitting the time evolution of the signal using Eq. ( 36) in [START_REF] Sheldon | Laser-induced thermal lens effect: a new theoretical model[END_REF]. In Table 1, we give at the first line the average value of the absorption coefficient due to the thermal effect of the pure solvent (methanol purchased from [43]) as an indication. Additionally, we verified that when the same power was applied, the empty quartz cell did not exhibit a Z-scan signature indicative of any absorption. To better visualize the distribution of the quantum-yield relative to the different solutions, we have reproduced the results of the measurements shown in table 1 on Fig. 5(a) as a function of input power. For the two products where is low (CoN and pararosaniline base [44]) measured values are much more dispersed than for the others, noting that in contrast, Rh6G values are well confined around 94% with very little variation.

In Fig. 5 (b), the dispersion of the relative error with the quantum yield can be seen more clearly. Indeed, a comparison of the experimental results is made with the theoretical one shown in Fig. 3. The colored rectangular dots are taken from the last column of table 1 by dividing by obtained following the method detailed in Appendix 2. Note that the agreement is almost perfect from a qualitative point of view, with a rapid decrease in relative error describing the uncertainties as a function of the yield. Obviously, the point representing the CoN with a zero Φ is not shown, as its value is indeterminate on y-axis since it tends towards infinity.

Comparison is done between our measurements and some measurements found in the literature. The measured value for cresyl violet perchlorate [45] ( see table 1) compared with that obtained by Isak and Eyring [START_REF] Isak | Fluorescence quantum yield of cresyl violet in methanol and water as a function of concentration[END_REF] or Magde et al. [START_REF] Magde | Absolute luminescence yield of cresyl violet. A standard for the red[END_REF] considering an average concentration of , gives us essentially the same value to within experimental errors, whether the solution is made in methanol or ethanol. Another solution where measurements converge towards a stable value reported by several authors [START_REF] Drexhage | Structure and properties of laser dyes[END_REF][START_REF] Magde | Fluorescence quantum yields and relation to lifetimes of rhodamine 6G and fluorescein in nine solvents: Improved absolute standards for quantum yields[END_REF][START_REF] Mertens | Ultra-precise photothermal measurements reveal near unity photoluminescence quantum yields of molecular emitters in solution[END_REF][START_REF] Kubin | Fluorescence quantum yields of some rhodamine dyes[END_REF] around a quantum yield of 0.95 is rhodamine 6G that was obtained from sigma [52]. Despite the relatively high concentrations we have considered (

), the mean value obtained using our technique corroborate exactly the results already established in these references considering the limit of low concentration. One point that should be stressed here is that the relative error on the measurements can be very low (as low as 1% when , see measurements done in [START_REF] Mertens | Ultra-precise photothermal measurements reveal near unity photoluminescence quantum yields of molecular emitters in solution[END_REF]), whereas silicon-based detectors have a much larger relative error on the measurement at room temperature. For moderate quantum yields, the value measured for rhodamine B here, , shows very good agreement with the mean values obtained for the base and acid forms in [START_REF] Snare | The photophysics of rhodamine B[END_REF] with . As already seen, when dealing with low quantum yields, TL techniques lose their effectiveness. In such cases, it is preferable to be careful with measurements, even at a value as low as [START_REF] Brannon | Absolute quantum yield determination by thermal blooming. Fluorescein[END_REF]. The result of our theoretical and experimental calculations in Fig. 5(b) shows why there are not many measured values in the literature for the last two products we tried to use as references. Besides, it should be noted that, according to our measurements, pararosaniline has a slightly higher fluorescence yield than CoN. This result has been confirmed qualitatively by the naked eye, where a faint yellowish fluorescent line delimits the axis of light propagation inside the cell filled with this solution. To conclude this paragraph, it would nevertheless be useful to recall that single-beam Zscan TL method offers high sensitivity, and it can detect very low levels of fluorescence signals but remains highly unreliable requiring a very large number of measurements for low values of the quantum yield. Measuring quantum yields with conventional OPM for values below 20% (if a threshold is to be set) is fraught with uncertainty. To mitigate this uncertainty, the fundamental approach would involve reducing , which represents the uncertainty in the Z-scan signal measurement, and to use a more sensitive photodetector with improved resolution. Achieving this can be accomplished through cooling, a technique already employed in infrared spectroscopy. One could also consider using a thermoelectrically cooled CCD camera, using a Peltier cooler to maintain a lower temperature of the sensor, resulting in improved signal quality and sensitivity. Of course, this would come at a significantly higher cost.

Conclusions

In this study, we have expanded the utilization of the single-beam thermal lens Z-scan technique to measure the fluorescence quantum yield of solutions with moderately high absorption. We have formulated analytical expressions to assess the measurement errors, revealing that uncertainty rapidly increases as the quantum yield decreases. When quantum efficiency is below approximately 20%, it is advisable to conduct the measurement a significant number of times. Our experiments covered a range of compounds, spanning from those with low to high fluorescence yields, validating the trends observed in our analytical models. The single-beam thermal lens Z-scan technique offers several advantages, make it a valuable tool for investigating the fluorescence properties of solutions, provided that the inherent measurement errors associated with this technique are accurately estimated.

Funding. Univ Angers. Laboratoire de Photonique d'Angers (LPHIA, EA 4464). SFR MATRIX.

The estimation of and are obtained using two Matlab functions: i) to evaluate the first-degree polynomial fit in at the points in ; ii) to specify the error estimation structure as the third input so that calculates an estimate of the standard error which is returned in . This provides the slope of the least-squares line and displays the absolute error of this slope with a 95% confidence interval considering (green lines in Fig. A-1). Then, Eq. A2-1 is derived to evaluate . In the example shown here for cresyl violet, we have obtained:

. Thus, knowing the extinction coefficient, , we can deduce the quantum yield according to Eq. [START_REF] Leite | Low absorption measurements by means of the thermal lens effect using an He-Ne laser[END_REF]. Finally, taking into account the calculation detailed in section 3 of Appendix 1, we obtain . Hence .

  Ap

Fig. 1 :

 1 Fig. 1: Scheme showing the different position of the optical elements. The cell is scanned along the beam direction around the focal plane (z=0). The labels refer to: lens (L1), chopper (Ch), circular aperture (Ap), computer (Cp) and photodiode (PD).

Fig. 2 :

 2 Fig. 2: Fitting (red solid line) of the Z-scan profile (blue stars) versus the normalized position V allowing to obtain . The inset shows the temporal response at the PD.

Fig. 3 :

 3 Fig.3: Simulation of the relative error when measuring quantum yield as a function of under the best experimental conditions given by our measuring instruments.

Fig. 5 :

 5 Fig. 5: (a) Distribution of values for the different methanol-based solutions measured as a function of P. (b) Comparison of the experimental measurements (colored squares, results of table 1) with the theoretical calculations (solid line) already shown in Fig. 3.

Table 1 : The quantum yield measured by Z-scan TL method using CoN as a reference solution with .

 1

	Reference: CoN
	Solution	N files
	Methanol	4

Acknowledgments. The authors are thankful to Dr. Martinus H. V. Werts and Matthieu Loumaigne for helpful discussions and for donating the basic solutes used to prepare the solutions.

Data availability. Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.

Disclosures.

The authors declare no conflicts of interest.

Appendix 1

Here, we derive the analytical details of the mathematical calculation to evaluate the precision estimating the total error on the fluorescence quantum yield taking into account all measured parameters: and to find . Note that is defined by Eq. ( 2).

1)

This parameter is measured from 2 OPM measurements through two identical cells: , using a first cell filled with pure solvent, and , through a second cell filled with solution at concentration C.

If we consider the error on L to be negligible, we get:

Now, and which results in:

Considering 3% random error and thick cell:

2)

Neglecting the errors on and and following Eq. ( 6) [START_REF] Marcano | Pump-probe mode-mismatched thermal-lens Z scan[END_REF]:

It can be shown that: and resulting in:

To determine the absolute random error one can estimate the error range in which it is reasonable to think the true value lies. Experimentally, it appeared to be unable to resolve a signal having less than , it would be logical to think that and where is the value given by the fit of the acquired data. Then the probabilities show that with a 95% confidence interval.

3)

Leaving aside the corrective coefficient in Eq. [START_REF] Leite | Low absorption measurements by means of the thermal lens effect using an He-Ne laser[END_REF] , the yield is given by: Yet, and resulting in:

By replacing and found here in 1) and 2), one can finally obtain:

Appendix 2

Here we measure using least squares regression analysis. Using Eq. ( 5), defined in Eq. ( 2) is measured as a function of varying to obtain the slope of the linear regression line as shown in


Léa Bazus
email: lea.bazus@ensc-rennes.fr

Nicolas Cimetiere

Laurent Legentil

Note : les règles d'écriture des références bibliographiques peuvent varier selon les différents domaines du savoir.

RECHERCHE DE DÉRIVÉS CONJUGUÉS DE MÉDICAMENTS DANS L'EAU DE SURFACE ET EN COURS DE POTABILISATION

Search for conjugated drug metabolites in surface water and during drinking water treatment Léa BAZUS 1,2 *, Nicolas CIMETIERE 1,2 , Laurent LEGENTIL 1,2 , Guy RANDON 3 , Dominique WOLBERT

RÉSUMÉ

Les médicaments à usage humain ou vétérinaire se retrouvent, par différents moyens, dans les milieux aquatiques et notamment dans les filières de potabilisation des eaux. Les médicaments sont en partie métabolisés après ingestion (le pourcentage dépend du principe actif et est compris entre 1 et 99 %). Des médicaments se retrouvent alors dans les eaux destinées à la consommation humaine avec des concentrations allant du ng/L à la centaine de ng/L. Différents types de métabolites existent, et parmi eux les dérivés conjugués. Ils peuvent représenter 90 % des métabolites d'un principe actif. Le travail présenté ici décrit le développement d'une méthode analytique par « Solid Phase Extraction » (SPE) en lignechromatographie liquide haute performance -spectrométrie de masse en tandem (LC-MS/MS) pour la détermination et la quantification de principes actifs et leurs dérivés conjugués dans les eaux de surface et en cours de potabilisation. Ces dérivés conjugués sont susceptibles de subir une déconjugaison lors des traitements de potabilisation conduisant au relargage du principe actif. C'est pourquoi il est nécessaire de s'intéresser à ces molécules et en particulier les conjugués glucuronides. Comme tous les standards ne se sont pas disponibles dans le commerce, une nouvelle voie de synthèse en milieu aqueux a été explorée. Des analyses sont effectuées en entrée et sortie de filière de potabilisation d'eau afin de déterminer les concentrations présentes en principes actifs et leurs dérivés conjugués. Les limites de détection et de quantification obtenues sont de l'ordre du ng/L ou la dizaine de ng/L.

Mots-clés : Médicaments, dérivés conjugués, SPE-LC-MS/ MS, eau de surface, eau en cours de potabilisation

ABSTRACT

Pharmaceuticals for human or veterinary use are found, by various means, in aquatic environments and particularly in drinking water treatment plants. Pharmaceuticals are partially metabolized after ingestion (the percentage depends on the parent drug and falls between 1 and 99%), and consequently pharmaceuticals can be found in drinking water at concentrations ranging from the ng×L -1 to hundreds of ng×L -1 . Different types of metabolites exist, among them conjugates that can represent up to 90% of the released metabolites of a pharmaceutical. The present work describes the development of an analytical method using "on-line Solid Phase Extraction" (SPE) -high performance liquid chromatography -tandem mass spectrometry (LC-MS/MS) for the determination and quantification of the active substances and their conjugates in surface waters and during drinking water treatment. These conjugates may be subjected to a deconjugation during treatment, resulting in the release of the parent pharmaceutical. Hence, it is necessary to focus on these molecules and in particular on the glucuronide conjugates. As all standards are not commercially available, a new synthetic route in an aqueous medium has been explored. Analyses are performed on the input and output of drinking water treatment plants to determine the concentrations of parent drugs and their conjugates. The obtained limits of detection and quantification are in the order of 1 to 10 ng×L -1 .

Key words: Pharmaceuticals, conjugated metabolite, SPE-LC-MS/MS, surface water, drinking water treatment

INTRODUCTION

La forte consommation de médicaments à usage humain ou vétérinaire conduit inévitablement à leur présence dans les milieux aquatiques et par la suite dans les filières de production d'eau destinée à la consommation humaine [START_REF] Miege C | Fate of pharmaceuticals and personal care products in wastewater treatment plants -Conception of a database and first results[END_REF]. En effet, après l'ingestion par les patients, les médicaments sont excrétés soit en l'état, soit en partie métabolisés, puis se retrouvent dans le milieu environnemental via les réseaux d'assainissement. Le taux de métabolisation, la nature des métabolites et les performances d'élimination des stations d'épuration envers ces composés sont très variables et dépendent du principe actif (COLLODO et al., 2014).

Différents types de métabolites existent, et parmi eux les dérivés conjugués. Même si ces composés peuvent représenter 90 % des métabolites d'une molécule mère (Vidal 2013), la présence et le devenir de ces dérivés dans les eaux en cours de potabilisation ne sont que peu étudiés. À l'inverse, d'autres formes de métabolites résultant d'une modification irréversible de la molécule, les dérivés conjugués, peuvent subir une déconjugaison lors des traitements de potabilisation libérant à nouveau le principe actif. C'est pourquoi il est nécessaire de s'intéresser à ces molécules et en particulier les conjugués glucuronides. Des développements analytiques ont déjà été menés pour certaines de ces molécules dans le plasma ou les urines [START_REF] Tonoli | Quantification of acetaminophen and two of its metabolites in human plasma by ultra-high performance liquid chromatography-low and high resolution tandem mass spectrometry[END_REF][START_REF] Wang | Extending the detection window of diazepam by directly analyzing its glucuronide metabolites in human urine using liquid chromatography-tandem mass spectrometry[END_REF]. Pour notre étude, une liste de dérivés conjugués a été établie en fonction du pourcentage de métabolisation de la molécule mère et des probabilités d'occurrence dans les eaux. Cette liste est constituée des septs médicaments et leurs dérivés glucuronides associés (oxazepam, paracétamol, ibuprofène, kétoprofène, diclofenac, gemfibrozil, acide salicylique). Afin de permettre leurs quantifications, certains standards non disponibles commercialement ont dû être synthétisés. Une nouvelle voie de synthèse en milieu aqueux a été explorée. Cette communication ne présente les résultats que pour deux d'entre eux, l'oxazepam et le paracétamol.

Les méthodes analytiques utilisées pour la quantification des médicaments dans les eaux sont souvent réalisées par chromatographie liquide couplée à la spectrométrie de masse en tandem, LC-MS/MS (CASTIGLIONI et al., 2005 ;GROS et al., 2012). La technique de SPE « on-line » est de plus en plus utilisée pour ces études [START_REF] Idder | Quantitative on-line preconcentration-liquid chromatography coupled with tandem mass spectrometry method for the determination of pharmaceutical compounds in water[END_REF][START_REF] Lopez-Serna | Fully automated determination of 74 pharmaceuticals in environmental and waste waters by online solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry[END_REF].

Une méthode d'extraction et une technique analytique performante, « on line SPE »-LC-MS/MS, a été développée pour quantifier ces principes actifs et métabolites glucuronides dans les eaux de surface et en cours de potabilisation. Cette étude devra à terme pouvoir évaluer les performances des filières pour l'élimination des formes conjuguées et le risque de libération de principes actifs dans les eaux destinées à la consommation humaine.

MATÉRIELS ET MÉTHODES

Produits chimiques et solvants

Les principes actifs et les deux métabolites glucuronides disponibles ont été achetés chez Sigma-Aldrich (France) sous forme de poudre. Le tableau 1 montre leurs formules développées. Des solutions mères ont été réalisées à 500 mg/L pour chaque composé dans du méthanol et stockées à -20 °C.

Les solvants utilisés pour la partie SPE et LC sont les suivants : l'eau Ultra-Pure (EUP) est produite par un système ElgaPureLab (18.2 MW.cm). L'acétonitrile (ACN), avec le cas échéant 0.1 % d'acide formique (AF), et du méthanol pour LC-MS de marque JT Baker. Le spectromètre de masse (Quattro Premier ; Micromass) est réglé avec les conditions suivantes : gaz de cône (N2, 50 L/h, 120 °C), gaz de désolvatation (N2, 750 L/h, 350 °C), gaz de collision (Ar, 0.1 mL/min), tension du capillaire (3 000 V). La source d'ionisation du spectromètre de masse est un Electrospray (ESI) qui est utilisé en mode positif ou négatif en fonction des composés pharmaceutiques. Pour nos analyses, le spectromètre de masse est utilisé en mode « multiple reaction monitoring » (MRM), l'ion parent à étudier est sélectionné dans le premier quadripôle puis fragmenté dans le second qui sert de cellule de collision. Le ou les ions fils sont ensuite isolés par le troisième quadripôle avant d'être détectés par un photomultiplicateur. Ce mode de fonctionnement présente une double sélectivité, au niveau des sélections de l'ion parent et de l'ion fils ainsi qu'une très grande sensibilité.

SPE "on-line"-LC-MS/MS

RÉSULTATS ET DISCUSSION

Synthèse des dérivés glucuronides

Pour les essais de synthèse, deux molécules mères ont été choisies : le paracétamol et l'ibuprofène. Pour la synthèse de leurs dérivés conjugués, deux voies d'accès majeures sont pour le moment développées afin d'accéder aux paracetamol-b-D-glucopyranosiduronate et ibuprofen-b-D-glucopyranosiduronates. La première repose sur la glucuronylation du paracétamol à partir du trichloroacetimidoyle de glucuronide catalysée par un acide de Lewis. L'autre réside dans l'attaque nucléophile de la fonction acide de l'ibuprofène sur le bromure de glucuronyle afin d'introduire le lien 1b-acyle glucuronide. Concernant cette dernière synthèse, une nouvelle stratégie en milieu aqueux sera explorée. L'objectif sera i) l'acylation de l'acide glucuronique

Analyses en SPE "on-line"-LC-MS/MS

Dans un premier temps, pour détecter et quantifier le paracétamol, l'oxazepam et leurs dérivés glucuronides, un gradient d'élution a été choisi partant à 90 % d'EUP et 10 % ACN pour arriver à 90 % ACN et 10 % EUP. Le temps d'analyse est de 8,5 minutes pour la partie SPE et de sept minutes pour la partie séparation chromatographique.

Pour la partie détection, les transitions ion parent-ion fils de chaque composé ont été déterminées en réalisant des infusions. Cela consiste à faire passer directement dans le spectromètre de masse une solution mère d'un composé cible à 5 mg/L. Cela permet de déterminer le mode d'ionisation (positif ou négatif ) et les transitions ion parent-ion fils pour la détection et la quantification. Pour la plupart des composés, deux transitions ont été choisies, une pour la quantification (la plus intense) et une pour la confirmation. La figure 1 montre les chromatogrammes correspondant au paracétamol, l'oxazepam et leurs conjugués respectifs. Pour ces deux molécules, nous avons pu déterminer les limites de détection et de quantification dans l'EUP, elles sont respectivement comprises entre 2 et 5 ng/L et entre 10 et 20 ng/L respectivement.

Des études préliminaires (CIMETIÈRE et al., 2013) ont montré que la prise en compte des effets de matrice est cruciale pour obtenir un résultat représentatif, ainsi la stratégie adoptée pour la quantification de nos molécules cibles est basée sur la méthode des ajouts dosés.

Analyses dans les eaux naturelles

Des prélèvements en entrée et sortie de six filières de production d'eau potable ont été réalisés en mai, juin et juillet 2014. De plus, le suivi de ces molécules a été réalisé après chaque étape de traitement d'une filière en juin 2014. Les moyennes des concentrations trouvées en entrée et sortie des six filières sont notées dans le tableau 2. Seul l'oxazepamglucuronide n'est pas détecté dans les eaux de surface ou destinées à la consommation humaine. Le paracétamolglucuronide et le paracétamol sont présents en entrée et sortie des six filières avec des moyennes de concentration élevées. On constate qu'en entrée de filières les concentrations en paracétamol glucuronide sont cinq fois plus élevées que celles du paracétamol : la moyenne des concentrations en entrée de filières est de l'ordre de 90 ng/L pour le paracétamol et de 530 ng/L pour son conjugué. En sortie de filières, le rapport entre les concentrations des deux molécules varie : les concentrations en paracétamol sont de l'ordre de 30 ng/L et de 100 ng/L pour son conjugué. Une des filières permet une élimination complète du paracétamol-glucuronide. Il faudra, dans la suite de l'étude, regarder plus précisément quelles sont les différentes étapes traitements utilisées dans chaque filière et leurs efficacités. Pour le moment seule une filière a été suivie. Celle-ci possède cinq étapes de traitement : Décantation, Réacteur CAP, Filtre à sable, Ultrafiltration et Chloration. Dans cette filière, une eau de surface subit une décantation avant d'être mélangée à de l'eau provenant de captages avec un taux de 50 %, pour ensuite poursuivre son parcours dans la filière. La Figure 2 montre l'évolution des concentrations de nos quatre molécules cibles dans l'eau en cours de potabilisation. Nous constatons la présence de trois molécules sur quatre en entrée de filière. La concentration en paracétamol-glucuronide est élevée : 605 ng/L. En sortie de filière, le paracétamol et son conjugué sont présents avec des concentrations respectivement de 50 ng/L et 190 ng/L. Le tableau 3 donne l'efficacité d'élimination des procédés présents dans cette filière. Le réacteur CAP est le procédé le plus efficace dans cette filière pour l'élimination de nos molécules avec des pourcentages d'élimination allant jusqu'à 95 % pour l'oxazepam.

CONCLUSION

Les premiers résultats montrent que la technique analytique SPE « on-line »-LC-MS/MS permet d'analyser et de quantifier aussi bien les principes actifs que leurs conjugués glucuronides. Les temps d'analyse sont de 8,5 min pour la partie SPE et de 7 min pour la partie chromatographique, soit 16 min. Les limites de détection et de quantification obtenues pour le paracétamol, l'oxazepam et leurs conjugués sont comprises entre 2 et 5 ng/L et entre 10 et 20 ng/L respectivement. Les premières analyses réalisées montrent la présence de molécules cibles dans les eaux de surface et les eaux destinées à la consommation humaine de six filières de potabilisation d'eau. Seul l'oxazepamglucuronide n'est pas retrouvé dans nos échantillons. Le suivi des concentrations en molécules cible dans l'eau en cours de potabilisation dans une des filières montre que, dans cette filière, le réacteur CAP est le procédé le plus efficace. Cependant, les rendements d'élimination ne sont que d'environ 35 % pour le paracétamol et son conjugué. Les campagnes de mesures en entrée et sortie de filières et les prélèvements de l'eau en cours de potabilisation sur chaque filière de notre étude vont se poursuivre pour obtenir plus de données pour déterminer les efficacités des procédés de traitement pour l'élimination de principes actifs et de ces métabolites. Cinq autres médicaments et leurs dérivés glucuronides vont être ajoutés au protocole pour élargir la gamme des principes actifs.

  Le système analytique consiste en un système SPE « on-line » développé par Waters et constitué d'un passeur d'échantillon 2777, d'une pompe quaternaire (Acquity BSM) et de deux colonnes Oasis HLB dont le fonctionnement en parallèle est assuré par deux vannes Everflow six positions (Waters) . La partie chromatographie liquide haute performance (Acquity, Waters) est équipée d'une pompe binaire, d'une colonne en phase inverse de chez Waters (Acquity C18 BEH, 100 mm x 2.1 mm ID, 17 μm) et thermostatée à 45 °C.

Tableau 2 .Figure 1 .Figure 2 .

 212 Figure 1. Chromatogrammes du paracétamol, de l'oxazepam et leurs conjugués glucuronides Chromatograms of acetaminophen, oxazepam and their conjugated metabolites

  1,23 1 Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, Avenue du Général Leclerc, CS 50837, 35708 Rennes Cedex 7, France

2 Université Européenne de Bretagne, 5, Boulevard Laënnec, 35000 Rennes, France. 3 VEOLIA EAU, Direction Technique Région Centre Ouest, 8, Allée Adolphe Bobierre, TSA 12007, 35020 Rennes, Cedex 9, France. Reçu le 25 novembre 2014, accepté le 16 janvier 2015 Revue des Sciences de l'Eau 28(1) (2015) 19-25 ISSN : 1718-8598

Tableau 1. Formules développées des molécules d'intérêt et leurs conjugués glucuronides Table 1. Structural formulas of the pharmaceuticals and their glucuronide conjugates

	à l'aide d'un bio-catalyseur (lipase) ii) le couplage en milieu
	aqueux du glucuronate sur l'ibuprofène. L'objectif commun de
	ces approches est l'économie d'atome et la minimisation des
	solvants organiques. Les formules développées des principes
	actifs et de leurs conjugués sont représentées.
	Classe de molécules	Molécules mères	Dérivés glucuronides
		Oxazepam	Oxazepam-glucuronide
	Neuroleptique
		Paracétamol	Paracetamol-glucuronide
	NSAID
		Ibuprofène	Ibuprofène-acyl-β-D-glucuronide

Table 3 . Characteristics of rainfall events occurring during the month of October 2012. RÉFÉRENCES BIBLIOGRAPHIQUES CASTIGLIONI

 3 , S., R. BAGNATI, D. CALAMARI, R. FANELLI et E.ZUCCATO (2005). A multiresidue analytical method using solid-phase extraction and highpressure liquid chromatography tandem mass spectrometry to measure pharmaceuticals of different therapeutic classes in urban wastewaters. J. Chromatogr. A 1092, 206-215. Standard addition method for the determination of pharmaceutical residues in drinking water by SPE-LC-MS/MS. Environ.Technol., 34,[3031][3032][3033][3034][3035][3036][3037][3038][3039][3040][3041] COLLADO, N., S. RODRIGUEZ-MOZAZ, M. GROS, A.RUBIROLA, J. COMAS, I.RODRIGUEZ-RODA et G. BUTTIGLIERI (2014). Pharmaceuticals occurrence in a WWTP with significant industrial contribution and its input into the river system. Environ.Pollut., 185,[202][203][204][205][206][207][208][209][210][211][212]

CIMETIERE, N., I. SOUTREL, M. LEMASLE, A. LAPLANCHE et A. CROCQ (2013). GROS, M., S. RODRIGUEZ-MOZAZ et D. BARCELO (2012). Fast and comprehensive multi-residue analysis of a


Davide Furceri
email: davide.furceri@oecd.org.

Aleksandra Zdzienicka

The Real Effect of Financial Crises in the European Transition Economies 1

Keywords: Output Growth, Financial Crisis, CEECs. JEL: G1, E6

The aim of this work is to assess the impact of financial crises on output for 11 European transition economies (CEECs). The results suggest that financial crises have a significant and permanent effect, lowering long-term output by about 17 percent. The effect is more important in smaller countries, with relative higher dependence on external financing, and in which the banking sector noticed more important financial disequilibria. We also found that fiscal policy measures have been the most efficient tools in dealing with the crises, while the role of monetary policy instruments has been rather blinded. Exchange rate resulted to be more a propagator than a crises absorber, while the IMF credit has been found to have positive (but not significant) impact on growth performance. Finally, the effect for the CEECs is much bigger than in the EU advanced economies, for which we found that financial crises lowers long-term output only by 2 percent.

Introduction

The current financial crisis that started to spread over the world in 2007 has led to a rapid and sharp deterioration of economic activity. The severity of this impact has brought once again the discussion about the real economic effects of financial crises. In the literature, only few studies report no or modest permanent impacts of financial crisis on economic output 2 [START_REF] Hutchison | How costly are IMF stabilization programs? FRBSF Economic Letter[END_REF][START_REF] Boyd | The Real Output Loss Associated with Modern Banking Crises[END_REF] and in general these findings concern developed economies under minor crises. Traditionally, financial crises are associated with severe economic downturns [START_REF] Reinhart | The Aftermath of Financial Crises NBER Working Papers 14656[END_REF].

The propagation channels of financial crises on the real economy pass from a sharp currency depreciation through wealth effects and aggregate demand, through increasing imported factors prices and production costs. Financial crises affect also private agents' behavior, increasing uncertainty about future gains and decreasing the level of investment and consumption. Other important channels of crises propagation work through the banking system by disturbing the process of credit intermediation, and in the payment system by decreasing collateral values. 3 These real effects seem to be more important and persistent in emerging countries. In fact, developing economies are more vulnerable to the factors that lead to crises, such as: banks and private agents exposure to currency and maturity mismatch, disruption in international capital markets, banks panic [START_REF] Chang | The Asian Liquidity Crisis[END_REF] and "sudden stops" in capital inflows [START_REF] Calvo | Monetary Policy Challenges in Emerging Markets: Sudden Stop, Liability Dollarization and Lender of Last Resort[END_REF]. The empirical findings in the literature corroborate this thesis   2 Some traditional views on the real effect of (currency) crises suggest that, under the existence of nominal rigidities, the real depreciation increases exportations and stimulates employment and output. For example, [START_REF] Demirgüç-Kunt | Inside the crisis: An empirical analysis of banking systems in distres[END_REF] find a positive impact in the case of 40 percent of the currency crises taken under consideration. 3 See for example the "Financial accelerator theory" in [START_REF] Bernanke | Agency Costs, Net Worth and Business Fluctuations[END_REF], [START_REF] Kiyotaki | Credit Cycles[END_REF].

 

and suggest that the effect of financial crises on economic activity in developing countries is bigger and more persistent than in developed economies. 4 In this context, the European transition economies (CEECs hereafter) constitute a very interesting group of countries. Indeed, despite increasing similarities to developed economies they still show important features of emerging markets. In addition, structural reforms, which could enhance the degree of resilience in these economies, are far to be completed making thus more likely that the effect of financial crises would be important and persistent.

The main objective of this work is to analyze the effect of financial crises on real output for 11 CEECs' economies. In doing so, we follow the methodology initiated by [START_REF] Romer | Does Monetary Policy Matter? A New Test in the Spirit of Friedman and Schwartz[END_REF] to assess the impact of monetary shock on output. More precisely we estimate an autoregressive with distributed lags (ADRL) equation of financial crises and output growth, and we derive the corresponding impulse response functions for real output. This method completes previous attempts to measure the output cost of the crises assessing their short-and long-term impact. In fact, traditional approaches (initiated by [START_REF] Kaminsky | The Twin Crises. The Causes of Banking and Balance-of-Payments Problems[END_REF][START_REF] Calvo | When Capital inflows come to a sudden stop: consequences and policy options[END_REF] consist of output regressions including several controls, contemporaneous and lagged variables (real growth, real GDP per capital) and financial crises in a panel of developed and emerging economies. 5 Cerra and Sexana (2008) were the first, to our knowledge, to add impulse response functions to distinguish between the short-term and the long-term impact of currency and banking crisis on real GDP. The same methodology was used by [START_REF] Furceri | The Effect of Financial Crises on Potential Output: New Empirical Evidence from OECD Countries OECD Economics Department Working Papers[END_REF] to assess the impact of financial    For example, [START_REF] Hutchison | How Bad Are Twins? Output Costs of Currency and Banking Crises Journal of Money[END_REF] analyzing the impact of currency and banking crises for a large set of countries find that while in a sample of 24 emerging economies real output contraction reaches 8 percent and last 2 years, when developed countries are included in the study the reduction in output is about 2 percent during only one year. Similarly, Dell 'Ariccia et al. (2008) find that the real effect of banking crises is about 1.5 percentage point bigger for developing countries. Moreover, the recent [START_REF] Reinhart | The Aftermath of Financial Crises NBER Working Papers 14656[END_REF] historical analysis underlines that a decline in real GDP is greater and more persistent for emerging than developed countries.  5 See, for example, [START_REF] Milesi-Ferretti | Current account Reversals and Currency Crises-Empirical Regularities[END_REF][START_REF] Barro | Economic Growth in East Asia Before and After the Financial Crisis NBER Working Papers 8330[END_REF][START_REF] Bordo | Is the Crisis Problem Growing More Severe?[END_REF][START_REF] Hutchison | How costly are IMF stabilization programs? FRBSF Economic Letter[END_REF]Huichison and Ilan, 2005;[START_REF] Demirgüç-Kunt | Inside the crisis: An empirical analysis of banking systems in distres[END_REF].

 

crises on potential output for a panel of 30 OECD economies, and more recently by the European Commission to assess the potential output and potential growth effect of financial crises (European Commission, 2009).

Our approach completes and improves exiting studies by: 1. Using two different methodologies to identify financial crises. First, we construct a market pressure index (MPI) that takes into account the pressure in the real exchange rate and in foreign exchange reserves, and which accounts for several financial (mostly currency) crises. Second, we use the recent IMF crises database [START_REF] Leaven | Systemic banking crises: a new database[END_REF] to construct a dummy in correspondence of financial (banking, debt and currency) crises.

Finally, given the importance of the ongoing financial crisis for the CEEC economies, we include it to both indexes as starting in 2007.

2. Comparing the real effects of financial crises between the CEEC economies and advanced European countries (EU-15 hereafter).

3. Controlling for structural heterogeneity and the role of macroeconomic policy response.

The rest of the chapter is organized as follows. Section 2 describes methodological and data issues. Section 3 presents the results and Section 4 concludes.

Methodology and Data

To assess the impact and persistence of financial crises occurrence on output we follow the methodology proposed by [START_REF] Romer | Does Monetary Policy Matter? A New Test in the Spirit of Friedman and Schwartz[END_REF]. In particular, the approach consists of estimating and ADRL (4, 4) equation and to derive the relative impulse response functions:

                          
where y is output, D is a dummy variable, which takes value equal to one in case of a financial crisis occurrence and zero otherwise,   are country fixed effects to control for unobservable countries specific characteristics and   is the error term. The numbers of lags has been tested, and the results suggest that the inclusion up to 4 lags produces the best specification.

We correct for heteroskedasticity, when this is the cases, using White robust standard errors, while the problem of autocorrelation related to our dependent variable is solved using their lags as explanatory variables.

The impulse response functions (IRFs) are obtained by simulating a one year crisis and by computing the response of output over time trough the estimated coefficients. In particular, the simultaneous response will be   , the one-ahead cumulative response will be            , and so on. Then, 95-percent level confidence bands are derived using Monte-Carlo simulations using one thousand of trials.

Since we are also interested in the role of structural and macroeconomic policy variables on growth performance during times of crises, we re-estimate equation (1) adding interactions terms between the above-mentioned variables, crises occurrence and lagged output growth. The expanded specification is then:

                                           
where   stands for structural or macroeconomic policy variables,   and   capture, respectively, the effects of   on output persistence and on the direct impact of financial crises on output. The parameter   captures the impact of control variables on output. In all, the simultaneous impact of the crisis on output as measured by the impulse response functions will be        , the one-ahead cumulative response will be                            and so on.

 

To deal with the endogeneity issue, since some control variables can be affected by the crisis occurrence, we often consider their lagged values. In addition, to capture the impact of some macroeconomic policy measures, such as changes in government spending or in interest rate, we focus on their value after the crises onset. Financial crises episodes are identified using two approaches. 7 The first is based on the work by Valencia and [START_REF] Leaven | Systemic banking crises: a new database[END_REF], which list financial crises (currency, debt and banking crises) for all the countries in our sample from 1970 to 2007. The current financial crisis is assumed to have taken place in 2007. The second approach is the market pressure index (MPI). Since currency crises are usually associated with severe pressure on the domestic currency, we follow the approach used by [START_REF] Kaminsky | Leading Indicators of Currency Crises[END_REF] and we adopt a criterion for the definition of a crisis that accounts for both pressures occurring in the real effective exchange rate market (REER) and diminishing official foreign exchange (FX) reserves. 8 In particular, the crisis occurs when the indicator is standard deviations above its mean. We

 

6 The EU-15 countries included in the analysis as comparison are: Austria, Belgium, Denmark, France, Finland, Germany, Greece, Italy, Ireland, Luxembourg, the Netherlands, Portugal, Spain, Sweden and the United Kingdom.  See Table1 for a detailed description of crises episodes.  The reason that we use the real exchange rate -rather than the nominal one -is that it gives a more realistic view of crises episodes in the CEECs economies since these countries have adopted many different exchange rate regimes since the beginning of their transition process. We use REER and official FX reserves quarterly data from the IMF database. Credit boom episodes are identified on the basis of excessive banking credit growth comparing to a country financial sector and economic development [START_REF] Zdzienicka | Financial Vulnerabilities in Central and Eastern Europe: Credit Growth[END_REF]. adopt a grid-search methodology which consists of seeking a value of  in the interval [0, 3]9 which best reproduces the historical crises of the countries under exam. Using this methodology, we find that the value of 1.5 correctly identifies major crises in CEECs.10

Empirical Analysis

Baseline

Basic Results

We start by estimating equation (1) using both the MPI index and the IMF financial crisis dummy. The results of these regressions are presented in Table 2. Looking at the table we can see that both financial crises dummies have a significant contemporaneous effect on growth. Moreover, while in the case of the MPI index the first and the third lags are statistically significant, in the case of the IMF dummy all lags are statistically significant.

These results, together with the significant persistence of output growth, imply that the occurrence of a financial crisis may have long lasting effects on output. This is confirmed by the impulse response functions derived by the estimated coefficients presented in the table. In particular, when our MPI is applied (Figure 1a), a financial crisis lowers output by 14 percent in the long-run. The impact is even greater with the IMF crisis indicator, for which the cumulative output loss is close to 18 percent (Figure 1b). This is consistent with the finding that the impact of banking and twin (currency and banking) crises on output is bigger than the one of currency crises [START_REF] Kaminsky | The Twin Crises. The Causes of Banking and Balance-of-Payments Problems[END_REF][START_REF] Goldstein | Assessing Financial Vulnerability: An Early Warning System for Emerging Markets[END_REF][START_REF] Cerra | Growth Dynamics: The Myth of Economic Recovery[END_REF].

Robustness check and endogeneity test

To check for the robustness of our results, we replicate this empirical exercise including other time shocks (time dummies) and oil shocks (oil price). The results even in this     case are statistically significant and suggest that the financial crises have permanent effects on output. In detail, we find that, even if the impact of other shocks has a significant effect on annual growth, financial crises continue to have a negative short and long-term impact on output. The cumulative lost is however lower and accounts for 8 percent in the case of the MPI (Figure 2a) and 15 percent in the case of the IMF indicator (Figure 2b). Subsequently, we control for the endogeneity of financial crises dummies. In fact, it is possible that economic slowdowns lead to crises onset, and in that case our OLS estimation will be biased and inconsistent. However, the GMM estimations using the MPI (Figure 3a) and IMF index (Figure 3b) report an almost identical impact than that obtained with the OLS method. As additional test, the Probit estimates of the probability of a crisis as a function of past growth performance and previous financial crises corroborates the absence of endogeneity bias (Table 3).11

Are financial crises more pernicious in the EU transition economies?

We test whether CEEC countries are more resilient to financial crises than other EU countries. To this purpose we estimate equation (1) for the EU-15 for the period 1980-2008.

When we compare the results between CEEC and EU-15 countries, we can see that impulse functions show a bigger and more persistent effect in the case of the CEECs' economies.

More precisely, with the MPI (Figure 4a) the short-term effect of financial crisis on the EU-15 output is negligible while in the CEECs output decreases by 2 percent. The long-term crisis impact in the EU-15 is greater than the one in the short-run (almost 3 percent of cumulative loss), but still much less important than in the CEECs -12 percent. The estimates based on the IMF indicator (Figure 4b) confirm the significance of both short and long-term effects of financial crisis. However, while the short-term effect is less important in the CEECs, the     crises impact on output is more visible in the EU-15 (1 percent loss). In the long -term, output in the EU-15 countries recovers and the cumulative loss stabilizes at 2 percent. In all, the output loss associated to financial crises in the EU-15 countries is about 1-2 percent in the short-term. In the long-term, the crisis impact on the CEECs is much more important and persistent than in the EU-15 countries. These results are in line with previous findings. For example, according to European Commission, the European countries growth rate will be reduced by a half in the short-term. In the long-term, the impact on potential growth is supposed to be more persistent in the case of CEECs. In fact, given the crisis implications on the growth components (investment, structural unemployment, factors productivity), the long-term reduction in growth rate is expected to reach 3.8 percent in the EMU countries and almost 7 percent in CEECs.

Macroeconomic Features and Policy Variables

So far, we have studied how financial crises affect the output performance in the CEECs' economies. Now, we attempt to determine whether countries' structural heterogeneity and macroeconomic policy variables play a role in shaping the impact of financial crises. More precisely, we explore the role of country size, openness, dependence on external financing, current account disequilibria, FDI inflows, financial development, credit boom, and a set of macroeconomic policy variables (exchange rate regime, government spending, monetary policy rates, and IMF aid). 12

Size and Openness

First, we examine whether the size of the economy and its openness affect the impact and persistence of financial crises on output growth. It is reasonable to think that smaller economies are more exposed to external shocks [START_REF] Aghion | Capital Markets and the Instability of Open Economies CEPR Discussion Papers[END_REF], since they are less     diversified and face resources constraints. It is also reasonable to assume that in economies with a higher degree of trade openness, that are per se more exposed to external shocks (Rodrick, 1998), the impact of financial crises may be more important. However, while our results indicated that the impact of financial crises decrease with the size of the economy (Table 5), trade openness (measured as the GDP share of total exports and imports) is found to be not statically significant (Table 5). This "puzzling" findings for trade openness is also reported by other studies [START_REF] Cerra | International Evidence on Recovery from Recessions[END_REF], and may be partially explained by the reduced effect of fiscal stimulus in open economies.

When we analyze the effect of the crises controlling for the size of the economy the impact is lower for the countries in which the size exceeds the sample median. More precisely, with the MPI index (Figure 5a) the short-term and long-term effects of financial crises on output are respectively about 1 and 9.5 percent for the bigger countries, while for the smaller ones these losses are respectively about 2 and 10.5 percent. The estimates based on the IMF dummy (Figure 5b) produce similar results in the short-run, but the long-term impact is even more important (14 percent) for smaller countries.

External Financing

Subsequently, we explore the CEECs' heterogeneity in terms of dependence on external financing. From a theoretical point of view, we should expect that the more the country depends on external sources in financing its activities the larger will be the effect of financial crises on its growth performance. Indeed, countries relying hardly on external financing have private sector balance sheets more exposed and are more vulnerable to sudden reversals in foreign capital flows. Our results (Table 5) confirm this theory and corroborate previous findings (Braun and Larrain, 2003;Dell'Ariccia et al., 2004;[START_REF] Tong | Real Effects of the 2007-08 Financial Crisis around the World[END_REF].

In details, in countries with a higher external financing (the 4 th quartile of the foreign banks   liabilities' dependent distribution), the impact of financial crises on output is at least twice more important than for the countries with average dependence on external financing. In all, a cumulative output loss varies between over 20 (the IMF dummy; Figure 6b) and 90 (The MPI; Figure 6a) percent for the most exposed CEECs.

Current Account Disequilibria and FDI

Dependence on external financing is closely associated with current account disequilibria. In fact, we notice that an increase in the current account deficit in the pre-crisis period amplifies the crises impact. More precisely, the crisis occurrence decreases output about 1 percent in the short-term and 9 percent in the long-run (the MPI; Figure 7). However, albeit our results stay in line with those reported by the literature [START_REF] Bordo | Is the Crisis Problem Growing More Severe?[END_REF], the impact of growing current account deficit on growth performance seems to be less important and uncertain. Indeed, the difference in output contraction between the countries that noted one-percent point increase and those that noted similar decrease in current account deficit before the crisis onset is less than 0.5 percent. Moreover, with the IMF dummy, the impact of current account disequilibria is not significant (Table 6). This is partially due to the fact that the CEECs' current account deficit has been largely financed by foreign direct investments that are considered as more stable especially in times of crises [START_REF] Lipsey | Foreign Direct Investors in Three Financial Crises NBER Working Papers[END_REF]World Bank 2009). Actually, when we look at the crises impact on growth performance controlling for the change in FDI inflows before the crisis occurrence, we notice that the short-term effect of the crises is less important in the countries that observed an increase of FDI inflows above the sample median.13 However, when the FDI inflows decrease the impact of financial crises on output is about 1.5 percent when the MPI is considered (Figure 8a). The opposite can be seen when we include the IMF dummy (Figure 8b).

   

Financial Development and Credit Boom

We also examine how financial development affects the impact of the crises on the real economy. A priori, the influence of financial development on growth performance during the times of crises is uncertain. On the one hand, financial development has a positive impact on economic growth through saving and investment channels [START_REF] Levine | Financial Development and Economic Growth: Causality and Causes[END_REF]. On the other hand, financial development can increase financial instability leading to unsustainable credit expansion, deterioration of borrower capacity and balance sheets disequilibria, and thus deteriorates economic growth [START_REF] Bordo | Is the Crisis Problem Growing More Severe?[END_REF]. In this context, previous works found a short-term detrimental effect and a long-term positive impact of financial development on growth performance (Loayza and Rancière, 2005). When we look at financial development in the EU transition economies, we remark that the real impact of financial crises is much more important for the countries with financial development above the sample median. 14 More precisely, with the MPI (Figure 9a) and the IMF dummy (Figure 9b) the short-term negative effect of financial crises on output is about 2.5 percent for whole sample while the long-term impact for the countries with the highest financial depth (the 4 th quintile of the banking creditto-GDP distribution) is respectively 10 and 15 percent more important. These findings can be partially explained by the fact that financial development in some CEECs' transition economies can be "excessive" and "unsustainable" comparing to their economic development.

As matter of fact, when we test for credit boom, we can observe that the countries that have been characterized by "excessive" banking credit development are more heavily affected by financial crises (Table 6). In detail, for the countries with sustainable credit growth, the shortterm impact of financial crises is marginal (the MPI; Figure 10a) or lower than one percent (the IMF; Figure 10b), while the long-term output cumulative loss is respectively 11 and 12.5 percent. For the countries that experienced credit boom in the times of crises, the short-term   14 See also Table 10.

 

effect is respectively 4 and 10 percent, and in the long-run output performance deteriorates about 18 and 28 percent.

Macroeconomic Policy Variables

So far, we have focused on the CEECs' structural heterogeneity in explaining the impact of financial crises on output, but macroeconomic policy variables can influence the impact and persistence of financial crises. Here, we examine the effect of the following variables: exchange rate regime, changes in government spending and in central bank policy rates, and foreign aid approximated by the International Monetary Fund credits and loans. Firstly, we analyze the impact of exchange rate regime on growth performance after the crisis onset. Traditionally, the flexible exchange rate regimes are reported to perform better during financial crises [START_REF] Kaminsky | The Twin Crises. The Causes of Banking and Balance-of-Payments Problems[END_REF][START_REF] Goldstein | Assessing Financial Vulnerability: An Early Warning System for Emerging Markets[END_REF][START_REF] Cerra | International Evidence on Recovery from Recessions[END_REF]. Indeed, it is reasonable to think that under flexible exchange rate regime the monetary authorities are less financially constrained and dispose of at least one more policy instrument to deal with the crisis [START_REF] Tong | Real Effects of the 2007-08 Financial Crisis around the World[END_REF]. Our results reported in Table 7 partially confirm these findings. However, while flexible exchange rate attenuates the impact of crises as measured by the MPI, fixed exchange rate regimes perform better under financial crises detected by the IMF dummy. More precisely, with the MPI (Figure 11a) flexible exchange rate decreases the impact of the crises on output about 1.5 percent in the short-term and over 2 percents in the long-run. The estimates based on the IMF dummy (Figure 11b) report a marginal impact of financial crises on output in the short-term, but thecumulative output loss is over 4-percents less important when a country operates under fixed exchange rate. We can find two possible explanations for this finding. On the one side, a fixed exchange rate regime was adopted successively in CEECs at the beginning of their transition period as a part of stabilization programs and with the aim to gain the foreign investors trust. On the other   side, fixed exchange regime, by eliminating currency risk, attenuates the financial vulnerabilities linked to the CEECs' dependence on external financing.

When we look at the fiscal policy variable in transition economies, we notice a positive and significant effect of government spending increase on growth performance (Table7). 15 More precisely, with the IMF dummy (Figure 12b), the short-term effect of financial crises is marginal for the countries where the change in government spending is above the sample median and the long-term impact is 2-percents less important than for the countries with no change in government spending. When we use the MPI methodology (Figure 12a), the effect of fiscal stimulus is more delayed in the time, but the long-term impact of government spending is as much as important.

In the case of monetary policy, the effect on growth performance during periods of crises is less obvious, especially for developing economies. On the one hand, there is not unique recommendation concerning the monetary authorities' actions. Some economists advocate in favor of the interest rate increase to defend the currency and attract foreign capitals back into the country, while others encourage the opposite action to stimulate the economy (Krugman, 1999;[START_REF] Christiano | Monetary Policy in a Financial Crisis NBER Working Papers[END_REF]. On the other hand, the impact of monetary stimulus on growth performance also remains uncertain. More precisely, while a positive impact is reported for industrialized economies, for developing countries the monetary policy have only weak influence on economic growth [START_REF] Romer | Does Monetary Policy Matter? A New Test in the Spirit of Friedman and Schwartz[END_REF][START_REF] Cerra | International Evidence on Recovery from Recessions[END_REF].

For the CEEC economies we report a positive impact of changes in central bank interest rate on economic growth after the crisis onset (Table7). In details, with the MPI (Figure 13a) and IMF dummy (Figure13b), a decrease in interest rate starts to influence output only after the 5     periods and the impact of one percentage point decrease in interest rate accounts for less than 0.5-percent increase in output.

Finally, the last variable that we analyze is the IMF credit and loans. There is a large body of the literature debating about the impact of foreign aid on economic activity in times of crises. Some authors underline their positive impact on growth recovery [START_REF] Eichengreen | Does It Pay to Defend Against a Speculative Attack?[END_REF][START_REF] Park | Recovery and Sustainability in East Asia NBER Chapters[END_REF], while others find no significant effect (Honoham and Klingebiel, 2003;[START_REF] Cerra | International Evidence on Recovery from Recessions[END_REF]). In the case of transition economies, the impact of foreign liquidity support is not significant (Table7). This can be partially due to the reticence of national authorities to ask about the foreign financial aid just after the onset of the crisis. The ongoing financial crisis seems to be the exception, but it is too early to assess effects of the recent IMF aid on the CEECs' growth performance.

Conclusions

The aim of this research is to evaluate the short-and long-term impact of financial crises on output in the CEEC economies by estimating an autoregressive with distributed lags (ADRL) equation of financial crises and output growth and deriving the relative impulse response functions for real output.

The main results of the paper can be summarized as follows:

1) Financial crises have a significant impact on output both in the short and in the long-run. In particular, financial crises are found to lower output by 1 percent after one year, and by 17 percent after 5 years. The impact of currency crises is relatively less important (12 percent).

2) Comparing the effect of financial crises between the CEEC and the EU-15 economies, our results suggest that the effect is more important for the CEECs.

 

3) Controlling for structural heterogeneity among CEECs, the impact of financial crises is more important for smaller countries, with greater dependence on external financing, where the banking sector shows greater disequilibria (dominance of foreign sources of financing, excessive credit growth), and whose current account deficit is lower and less financed by the FDI inflows.

4) The impact of financial crises on growth performance is mostly influenced by fiscal policy (in terms of increase in government spending), while the effect of monetary policy is rather limited. Exchange rate can amplify the real effect of financial crises on the CEECs' economies. Moreover, the impact of foreign financial aid, albeit positive, has been found to be not significant.

In lights of these findings, the claim that these countries could be better off in joining the EMU in the very short-term may be only partially true. In fact, on the one hand the monetary integration process could favor other reforms that would increase the resilience of these economies to external shocks [START_REF] Duval | Is there a role for macroeconomic policy in fostering structural reforms? Panel evidence from OECD countries over the past two decades[END_REF]. On the other hand, even if the principal role in crises absorption belongs to fiscal policy, the large worsening of public finances would make domestic monetary and exchange rate policy the only (independent) tool to face the current and future significant output loss.

  All data are taken from the IMF International Statistics and World Economic Outlook database, except for government spending and central banks interest (REFI) rates that come from OECD Economic Outlook 85 (2009) and central banks statistics. The dataset consist of an unbalanced panel of annual observations from 1989 to 2008 for 11 CEECs' economies (Bulgaria, Croatia, the Czech Republic, Estonia, Hungary, Latvia, Lithuania, Poland Romania, Slovakia and Slovenia). 6

Figure 1 .Figure 4 .Figure 6 .Figure 8 .Figure 10 .Figure 12 .

 14681012 Figure 1.The impact of financial crises on the CEECs' output A. The MPI Index B. The IMF crisis dummy

Table 1 .

 1 Financial crises episodes.

	MPI	IMF

Table 2 .

 2 The effect of financial crises on output-OLS

	MPI	IMF

Table 3 .

 3 The effect of past growth on the probability of financial crisis (Probit)

		MPI	IMF
	Crisis (-1)	-6.484	-6.632
		(-0.00)	(-0.00)
	Crisis (-2)	-0.318	-0.170
		(-0.58)	(-0.27)
	Crisis (-3)	-0.445	-6.510
		(-0.00)	(-0.00)
	Crisis (-4)	-0.039	-0.270
		(-0.09)	(-0.44)
	Growth (-1)	-0.002	-0.076
		(-0.05)	(-1.20)
	Growth (-2)	-0.006	0.135
		(-0.09)	(1.72)
	Growth (-3)	0.003	-0.056
		(0.06)	(-1.05)
	Growth (-4)	0.020	0.022
		(0.58)	(0.58)
	N	156	156
	Note: z-statistics in parenthesis.

 

Table 4 .

 4 The sample means of structural and macroeconomic policy variables Decrease means increase in Current Account Deficit ** Decrease means decrease. Source: IMF, OECD, Central Banks Statics, Authors's Computations

		Size (log GDP)	Openness (%)	Dependence on External Financing (% change)	Change in Current Account Deficit*	Ratio of FDI over GDP (%)	Financial Development (% of GDP)	Number Year with Credit Boom Episodes	IMF Credit and Loans (millions USD)	Change in CB interest Rate (p.p)**	Change in Government Spending (%)**
	Bulgaria	0.78	112.30	67.63	215.26	34.44	34.91	4	-4.04	-2.72	-1.67
	Croatia	4.90	87.42	15.05	1.72	30.71	38.53	3	1.76	-1.50	1.50
	Czech Rep	7.71	125.01	10.25	-70.09	33.71	46.91	2	-69.98	-0.82	0.03
	Estonia	4.62	154.74	57.42	-32.67	56.70	48.78	3	0.25	0.07	2.25
	Hungary	9.50	110.98	19.61	-74.41	66.52	38.21	2	278.07	-1.25	0.59
	Latvia	1.64	98.62	34.77	-169.33	27.30	38.91	2	48.20	-2.36	2.74
	Lithuania	3.92	111.56	45.35	-0.93	21.17	26.69	2	-0.54	-4.68	1.51
	Poland	6.52	58.55	35.00	-87.85	19.96	27.63	0	-7.05	-2.60	1.24
	Romania	4.83	63.86	66.08	5.81	14.05	13.88	1	-10.95	-2.98	0.15
	Slovakia	6.87	141.48	14.81	-51.77	29.77	42.25	2	-34.44	-0.55	-0.43
	Slovenia	2.93	118.43	32.17	31.37	16.00	41.00	1	-0.06	-3.96	3.48
	*

Table 5 .

 5 The Impact of Financial Crises on Output controlling for: Size, Openness and External Financing

	Size		Openness		External Financing
	MPI	IMF	MPI	IMF	MPI	IMF

Note: t-statistics in parenthesis. ***,**,* denote significance at 1%,5% and 10%, respectively.

 

Table 6 .

 6 The Impact of Financial Crises on Output controlling for: Change in Current Account Deficit, FDI-to-GDP-ratio, Financial development and credit booms -statistics in parenthesis. ***,**,* denote significance at 1%,5% and 10%, respectively.

	Current account	FDI/GDP	Financial	Credit booms
	deficit				development
	MPI	IMF	MPI	IMF	MPI	IMF	MPI	IMF

The conventional choice of  is between 2.5 and 3(Edison, 2003).

Using the same method, we set the value  at 2 for the EU-15 countries.

The endogeneity and omitted variables test carried out for the actual EMU members confirms the predominance impact of financial crisis on growth rate and the validity of the exogeneity assumption. Results are available from the authors upon request.

See also Table9.

Previous findings corroborate a positive impact of fiscal stimulus on growth recovery in developing and developed countries[START_REF] Claessens | Resolving systemic financial crisis: policies and institutions Policy Research Working Paper Series 3377[END_REF][START_REF] Cerra | International Evidence on Recovery from Recessions[END_REF].

 

 

APPENDIX -DATA AND SOURCES