A Janes

A Janes -J

Chaineaux -D

A Carson -P

Le Lore

MIKE 3 versus HARTMANN apparatus: Comparison of measured minimum ignition energy (MIE)

Keywords:

In this study, MIE values measured with two different explosion tubes, HARTMANN and MIKE 3, are compared. Generally, MIKE 3 apparatus provides MIE results which are equal or lower to those measured with the HARTMANN apparatus; this is particularly true for the energy ranges between 1 and 10 mJ and higher than 100 mJ.

Differences observed can modify samples classification according to their sensitivity to electrostatic ignition sources.

Nevertheless, ignition of a dust cloud by an electrostatic discharge is complex, and implies a different mechanism from that occurring during MIE tests. Thus, it seems difficult to synchronise dust dispersion and spark triggering to obtain optimal concentration in the spark area. Moreover, spark characteristics such as duration or energy feeding rate of spark can not reproduce exactly industrialworld ones. On this point, it is not possibly to conclude if characteristics of MIKE 3 electric circuit, e.g. resistance and inductance, are more relevant than HARTMANN circuit ones.

INTRODUCTION

Prevention of dust explosion in industries manufacturing or handling combustible powder or dust is a major challenge [START_REF] Eckhoff | Dust Explosions in the Process Industries[END_REF] and [START_REF] Bartknecht | Explosionsschutz[END_REF]. Minimum ignition energy (MIE) determination of dust/air mixtures is useful for risk assessment, since MIE value is linked to the sensitivity of a sample to be ignited by an electrostatic source. But does experimental determination of MIE in explosion tubes such it is presently proposed in standards, using different apparatuses, conduce to the same results. In this paper, MIE values measured with two different explosion tubes, HARTMANN and MIKE 3, are compared and discussed.

PRINCIPLE OF MIE OF DUST/AIR MIXTURE DETERMINATION

MIE of a combustible dust cloud is the lowest energy that is sufficient to ignite a dust-air mixture, under specified test conditions.

The determination of the MIE requires pneumatically dispersing of a given amount of dust in a test chamber. An electric spark of a theoretical energy level is then triggered between two electrodes located inside the chamber, which is an open transparent tube. Then, the diagnosis of the ignition is visual: propagation or non-propagation of a flame inside the tube. Standards also allowed implementing pressure-based detection instead of this visual detection in a closed system.

The main influencing factors on MIE recorded values, for a given dust, are:  (1) delay between dust dispersion and sparkover,  (2) dispersion method, influenced e.g. by the nozzle size and shape,  (3) spark characteristics e.g. duration and energy, influenced by electric circuit design such as including capacity, inductance and resistance values,  (4) amount of dust and particle distribution placed in the bottom cup of the explosion chamber prior to the test.

The two first items influence turbulence, dust concentration and particle distribution in the gap between electrodes at the time of sparkover. The third point is linked to the level of energy needed to ignite combustion. Finally, the last item influences average dust concentration and particle distribution in the explosion tube.

The MIE is the energy initially stored in the electric circuit that is just sufficient to cause the ignition of the dust/air suspension, under the optimum conditions of delay between dust dispersion and sparkover and dust concentration.

STUDY CONTEXT AND OBJECTIVE

Before INERIS acquired in 2002 a MIKE 3 apparatus [START_REF] Cesana | MIKE 3. Minimum Ignition Energy, B021_033[END_REF], MIE tests were carried out with a HARTMANN tube built by CERCHAR in accordance with a standard published by the International Electrotechnical Commission (IEC) [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF].

After giving details about the two applicable standards and the two apparatuses operated, this paper comments results of comparative experiments on HARTMANN and MIKE 3 explosion tubes.

APPLICABLE STANDARDS

The two reference standards for MIE determination of dust/air mixtures applied in this study are IEC 61241-2-3 [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF], applied to HARTMANN apparatus, and EN 13821 [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF], applied to MIKE 3 explosion tube. Major differences between these two references are shortly discussed below.

Several convenient spark-generating systems are listed in both standards (Table 1).

Standard IEC 1241-2-3 [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF] does not specify any constraint related to the dust dispersion system. On the contrary, the standard EN 13821 [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF] specifies that dust dispersion must be triggered by an air blast powered at 7 bar. These differences in dust dispersion systems will necessary induce effects on turbulence in the tube and on dust concentration and particle distribution into spark area.

According to EN 13821 [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF], a non-ignition result must be recorded if the cloud does not ignite after only 10 successive attempts, whereas IEC 1241-2-3 [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF] requires 20 successive unsuccessful ignition attempts to record a non-ignition result. This may have an influence on the result of a MIE test, especially because of the two following aspects:

 multiplication of successive attempts without removing dust, cleaning the tube and recharging it with a new sample can modify particle distribution of friable dust or hygrometry of very hygroscopic samples, e.g. corn starch. Moreover, successive attempts will disperse a fraction of sample outside the test chamber,  ignition by an electrostatic discharge, such as occurring in explosion tube during MIE tests, is a stochastic phenomenon. So, the less attempts are done, the more uncertainties are associate to the test result.

USUAL INTERPRETATION OF MIE RESULTS

Usually, MIE results are referred to electrostatic ignition risk assessment. It is established that MIE is representative of the sensitivity of a sample to be ignited by an electrostatic source. In the same way, the comparison of MIE results indicates the relative sensitivity of samples.

Nevertheless, ignition of a dust cloud by an electrostatic phenomenon is complex and differs from the test mechanism. This is due to the difficulty to quantify and reproduce experimentally real-world spark characteristics and especially spark duration and synchronisation between dust dispersion and sparkover [START_REF] Boudalaa | Energie minimale d'inflammation des nuages de poussières, Méthode de mesure et application aux situations industriellescas des étincelles électrostatiques[END_REF] - [START_REF] Randeberg | Measurement of minimum ignition energies of dust clouds in the < 1 mJ region[END_REF] (see discussion in section 3.2). However, it is not possible to accurately qualify the ignition sensitivity of a sample according to the test result.

EXPERIMENTAL

HARTMANN APPARATUS

HARTMANN apparatus was designed and manufactured by CERCHAR in the seventies. This equipment was systematically employed for measuring the MIE of dust/air mixtures until its progressive take over by the MIKE 3 apparatus until the end of 2004.

Dust dispersion and ignition take place in 1.6 L Plexiglas tube open at the top.

The diameter of the explosion chamber is 71 mm and the height 420 mm. The dust placed in the bottom cup prior to the test is dispersed by two successive air blasts powered with a mushroom shaped nozzle at 450 mbars (pre-rising) and then 500 mbars (dispersing), in order to generate an homogeneous cloud. Fig. 1 shows the low bottom cup of the HARTMANN apparatus on the top, of which the Plexiglas tube is settled.

Our HARTMANN apparatus exhibits only one electric circuit. The spark is triggered by a transformer and this circuit operate a two-electrode system. More details are given in the A6 appendix of EN 13821 standard [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF] as well as in the A5 appendix of the IEC 1241-2-3 [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF] standard (Table 1). The gap between the tapered tips of the stainless steel electrodes (diameter: 2.4 mm) is 6 mm.

The available energy values (mJ) are chosen among: 1200 ; 810 ; 540 ; 360 ; 225 ; 158 ; 105 ; 77 ; 58 ; 45 ; 36 ; 27 ; 21 ; 16 ; 12 ; 9.4 ; 7.6 ; 5.9 and 4.5. Due to a breakdown that occurred during the course of the study, the 1200 mJ condenser was replaced. Delivered energy is now close to 1100 mJ.

The inductance of the circuit of our HARTMANN explosion tube is not adjustable: a value of 570 mH was measured.

The delay between the second air blast and the triggering of the spark can be selected as follow (in seconds): 0.3 ; 0.35 ; 0.4 ; 0.45 ; 0.5 ; 0.6 ; 0.7 ; 0.8 ; 0.9 and 1. Fig. 2 shows the control and operation interface of our HARTMANN apparatus.

MIKE 3 APPARATUS

The MIKE 3 apparatus was acquired from the KUHNER Company [START_REF] Cesana | MIKE 3. Minimum Ignition Energy, B021_033[END_REF].

The dispersion and ignition occur in a 1,2 L glass tube. A removable vent is located at the top of the tube. The diameter of the explosion chamber is 68.5 mm and the height 315 mm. The dust is dispersed by an air blast with a mushroom shaped nozzle, at the pressure of 7 bar. As for the HARTMANN explosion tube, the gap between the tapered tips of the stainless steel electrodes (diameter: 2.0 mm) is 6 mm. However, when using the method of triggering by electrode movement, the electrode gap cannot be known at the time of sparkover. MIKE 3 can operate with one of the following circuits (Table 1):  triggering by high-voltage relay, using a two-electrode system. This circuit is employed for low energies (1 and 3 mJ). It is described more precisely in the A2 appendix of the EN 13821 standard [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF],  triggering by electrode movement, using a two-electrode system. This circuit is operated for high energies (10, 30, 100, 300 and 1000 mJ). It is described in details in the A3 appendix of the EN 13821 standard [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF] and in the A3 annex of the IEC 1241-2-3 standard [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF].

The inductance of the electric circuit is adjustable to either 0 or 1 mH.

The usual delays between dust dispersion and sparkover vary from 60 ms to 180 ms, by 30 ms time steps.

PROCEDURE

TEST PROCEDURE

Each series of tests is carried out for a given concentration of dust in air and a given delay between dust dispersion and sparkover.

In order not to sidestep the issue of this study, for both HARTMANN (IEC 61241-2-3) and MIKE 3 (EN 13821) test procedures, 10 successive unsuccessful ignition attempts to record a non-ignition result were required.

Moreover, dust was removed after a maximum number of five non-ignition attempts and a new sample was then prepared for the following attempts.

The tests begin with the highest of ignition energy value (1000 mJ for MIKE 3 and 1200 mJ in the case of HARTMANN).

It is also necessary to set a definite value of average dust concentration. The tests begin with an average concentration close to 750 g/m 3 .

The delay between dust dispersion and sparkover set for the first series is 120 ms in the case of MIKE 3 and 300 ms for HARTMANN.

Lastly, circuit inductance is kept constant during the complete course of the procedure. Tests with MIKE 3 explosion tube were carried out with an inductance of 1 mH only. Some tests were carried out in order to see the influence of the selected inductance value (either 0 or 1 mH) on results obtained for one sample.

Several series of tests are then conducted in the range of optimal dust cloud concentrations at the available delays between dust dispersion and sparkover (MIKE 3: 60 ms and 180 ms ; HARTMANN: 700 ms).

COMPARISON CRITERION

According to the EN 13821 standard [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF], validation tests must be carried out on at least five different dust type for each three different energy ranges: 1 mJ-10 mJ, 10 mJ-100 mJ and 100 mJ-1 J.

The different dusts to be tested include at least two metal powders, two natural organic powders, two synthetic organic powders and two coal dust.

With the aim of comparing various equipment, this standard also specifies that only one value of MIE (Es) must be kept, instead of the range of measured energy for witch ignition took place. This Es value is calculated on the basis of a statistical formula given in the standard. Conformity between two apparatuses is proven when Es values for all the dust tested differ by a factor less than 3.

However, the comparison between HARTMANN and MIKE 3 results was carried out on the basis of the interval defined by minimum and maximum energies for which an ignition is observed. Thus, the value Es was not calculated.

We compared the results obtained with both apparatuses for samples belonging to the three energy ranges (1 mJ -10 mJ ; 10 mJ -100 mJ and 100 mJ -1 J).

As far as possible, we tried to comply with the minimum number of five samples by energy range as well as with the criterion relating to the type of samples.

SAMPLES TESTED

Selected samples are listed in table 2, where the classification in energy range is based on MIKE 3 results.

RESULTS AND DISCUSSION

COMPARISON OF MIE RESULTS

Results are presented in table 3. Fig. 4 shows MIE measured values for tested samples with both explosion tubes.

RANGE FROM 1 TO 10 MJ

Generally, MIKE 3 results are lower than those obtained with the HARTMANN apparatus.

In addition, the criterion of the maximum ratio of 3 between two valid results is only confirmed for the lower limit of the result interval of calcium stearate.

On the contrary, this criterion is valid in every case with regard to the upper limit, except for niacin (CaRo 05 1 ) [START_REF] Cesana | Calibration-Round-Robin CaRo[END_REF]. This had also been witnessed during round robin tests carried out in 2003 [START_REF] Cesana | Calibration-Round-Robin CaRo 05[END_REF].

RANGE FROM 10 TO 100 MJ

In general, the range of results obtained with the HARTMANN tube is within the range obtained with the MIKE 3. This can be explained by the available energy levels:  for the HARTMANN apparatus, seven levels are available between the two levels 12 and 105 mJ of the range considered,  for the MIKE 3 apparatus, there is only one level available (30 mJ) between the two levels 10 and 100 mJ of the range considered.

In the particular case of one of the agricultural products, the HARTMANN result is significantly higher than that with the MIKE 3.

In all other cases, the criterion of the maximum ratio of 3 between two results can be considered as met.

RANGE FROM 100 TO 1000 MJ

Only tests carried out with aluminium dust lead to coherent results between the two apparatuses. The HARTMANN result being however higher than that obtained with the MIKE 3.

In three cases, an ignition was observed with the MIKE 3 apparatus whereas there was none with the HARTMANN. Moreover, the criterion of the maximum ratio of 3 between two results was never met.

A doubt remains for two products for which no ignition was observed whatever the apparatus.

SYNTHESIS

Table 4 itemizes, for the various samples studied, the sensitivity to electrostatic ignition sources, which can be deducted from MIE determination results.

1 CaRo 05 (pyridine-3-carboxamide) is the reference sample provided for round robin tests the dust cloud is a sufficient low distance between particles. Thus, the particle concentration into the dust/air suspension has an influence on ignition probability. This is confirmed by the results of the tests carried out in this study. Fig. 5 shows the evolution of MIE with particle concentration for sulphur powder dust on MIKE 3 apparatus. Nevertheless, Fig. 6, which compares MIE evolution with particle concentration for the same sulphur powder on both MIKE 3 and HARTMANN explosion tubes, shows that the average particle concentration in the tube isn't the only factor to be considered. Indeed, the method of dust dispersion operated and the delay between dispersion and sparkover are influent on turbulence inside the tube, dust concentration and particle distribution in the spark area. Especially, nozzle size, shape and pressure are very different on MIKE 3 and HARTMANN apparatuses, as same as the range of delay available between dust dispersion and sparkover. Thus, such differences on this combined parameters lead certainly to the gap between optimal concentrations observed on Fig. 6. It is interesting to notice that this gap is not a constant for all the samples tested, as shows Table 5. It is supposed that the influence of these factors depends on the nature and maybe the particle size distribution of the dust, but any simple correlation can be found at this stage. Randberg and Eckhoff [START_REF] Randeberg | Measurement of minimum ignition energies of dust clouds in the < 1 mJ region[END_REF] pointed that MIE tests by using independent dust dispersion and spark triggering is not really representative of electrostatic discharges which actually occur inside a dust cloud in industrial situations, because of the difficulty to synchronise sparkover and optimal concentration. When using a method with a spark triggering by the dust cloud itself, very low MIE (< 1 mJ) were measured [START_REF] Randeberg | Measurement of minimum ignition energies of dust clouds in the < 1 mJ region[END_REF]. Thus, independent dust dispersion and spark triggering, such as carried out in this study, conduce to quite conservative results.

Another influent factor on MIE results is the design of the ignition energy power supply. Nifuku and Katoh [START_REF] Nifuku | Incendiary characteristics of electrostatic discharge for dust and gas explosion[END_REF] showed that the feeding time and the feeding rate of the ignition energy influence considerably the ignition of a dust cloud. This was confirmed either by Randberg et al. [START_REF] Randeberg | Initiation of dust explosions by electric spark discharges triggered by the explosion cloud itself[END_REF], or by Bennett et al. [START_REF] Bennett | A test for electrical ignition of flammable dust clouds[END_REF] who demonstrated through a literature review [START_REF] Parker | Electric spark ignition of gases and dusts[END_REF]- [START_REF] Norberg | Powder Ignition energy measured utilizing a new fluidized bed ignition chamber[END_REF] how adding either a larger inductance or a larger resistance can increase the spark duration and thus decrease the MIE of a given dust. This is confirmed by Fig. 7, on which can be compared MIE results for lycopodium on MIKE 3 apparatus, with and without inductance in electric power supply. Considering these results, it seems than adding an inductance increased the probability of ignition.

In [START_REF] Nifuku | A study on the static electrification of powders during pneumatic transportation and the ignition of dust cloud[END_REF], Nifuku and Katoh also pointed that the larger the feeding rate of spark energy, the higher the ignition probability.

CONCLUSION

The results of this study are concluded as follows.

1. Generally, MIKE 3 apparatus provides MIE results which are equal or lower to those measured with the HARTMANN apparatus ; this is particularly true for the energy ranges between 1 and 10 mJ and higher than 100 mJ. Differences observed can alter samples classification according to their sensitivity to electrostatic ignition sources.

2. According to the results obtained by testing CaRo05 (pyridine-3carboxamide) with both apparatuses, it is tempting to consider that using MIKE 3 explosion tube instead HARTMANN can discriminate more efficiently the ignition sensitivity of dust, and thus support the recommendation of more relevant ignition prevention measures, especially for dust presenting low MIE.

The literature reviewed shows that particle size distribution and dust concentration in the spark area are influent factors on MIE results, as well as method of dust dispersion in the explosion chamber and delay between dispersion and sparkover. These two last points may explain some differences observed between MIKE 3 and HARTMANN apparatuses, but it appears that this gap is not a constant for all samples tested.

Circuit design

Use in the study Reference in CEI 1241-2-3 [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF] Reference in EN 13821 [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF] Triggering by high-voltage relay, using a two-electrode system MIKE 3 apparatus for Table 1: Spark-generating systems referred by each explosion tube and standard used. Further details about precise design of these circuits are given in [START_REF] Iec | Electrical apparatus for use in the presence of combustible dust, Part 2: Test methods, Section 3: Method of determining minimum ignition energy of dust/air mixtures, IEC International Standard[END_REF] and [START_REF] Cen | Potentially explosive atmospheres, Explosion prevention and protection, Determination of minimum ignition energy of dust/air mixtures[END_REF].

  The recommendations for interpreting MIE results are based on energy levels available on MIKE 3 apparatus. According to the INERIS usual practice, MIE can be ranked as follow:  MIE > 1000 mJ: sample almost insensitive to electrostatic ignition,  300 mJ < MIE < 1000 mJ, 100 mJ < MIE < 300 mJ and 30 mJ < MIE < 100 mJ: sample sensitive to electrostatic ignition,  10 mJ < MIE < 30 mJ and 3 mJ < MIE < 10 mJ : sample very sensitive to electrostatic ignition,  1 mJ < MIE < 3 mJ and MIE < 1 mJ : sample extremely sensitive to electrostatic ignition.

Figure 1 :

 1 Figure 1: View of the low bottom part of the HARTMANN apparatus (electrodes and dispersion mushroom) on the top of which the Plexiglas tube is settled prior to a test.

Figure 4 :

 4 Figure 4: MIE measured results for tested substances with both explosion tubes.

Table 2 :

 2 List of substances tested in the study. Granulometry of aluminium powder is thinner than aluminium dust one. Classification in energy range is based on MIKE 3 results.

	Energy range	Substances	Type (according to EN 13821 [5])
		Sulphur powder	Natural mineral
		Toner	Synthetic organic
	1 mJ-10 mJ	Anthraquinone	Synthetic organic
		Niacin (pyridine-3-carboxamide) (sample provided for the round robin tests CaRo 05)	Synthetic organic
		Calcium stearate	Synthetic organic
		Lycopodium	Natural organic
		Starch	Natural organic
		Corn starch	Natural organic
	10 mJ-100 mJ	Wood dust	Natural organic
		Aluminum powder	Metal
		Agroalimentary product 1	Natural organic
		Agroalimentary product 2	Natural organic
		Aluminum dust	Metal
		Crushed pea fiber	Natural organic
		Cocoa	Natural organic
	100 mJ-1 J
		Atomised arabic gum	Natural organic
		Coal dust	Coal
		Pharmaceutical product	Synthetic organic

Table 4 :

 4 Comparison of the sensitivity of tested samples to electrostatic ignition sources based on MIE measurement results. Granulometry of aluminium powder is thinner than aluminium dust one. Classification in energy range is based on MIKE 3 results.

The comparison of the sensitivity to electrostatic ignition sources deducted from the MIE results with both MIKE 3 and HARTMANN apparatuses, highlights the following points:  in the range of 1 to 10 mJ, operating with MIKE 3 can discriminate "extremely sensitive" from very "sensitive" samples. Indeed, in four out of five cases, samples seen as "extremely sensitive" based on MIKE 3 results were only seen as "very sensitive" according to the HARTMANN results. In only one case out of the five tests carried out with both apparatuses results converged in identifying the sample as "very sensitive",  in the range of 10 to 100 mJ, ignition sensitivities do not depend on the apparatus employed ("very sensitive" in one case and "sensitive" in five cases). However, one sample which is rated "very sensitive" with the HARTMANN apparatus is only seen as "sensitive" according to the MIKE 3 results,  in the range of 100 mJ to 1 J, both HARTMANN and MIKE 3 apparatuses discriminates three different ignition sensitivities. In three out of six cases, an agreement is reached: samples are rated "sensitive" (1 case) or almost "not sensitive" (2 cases), with both apparatuses. However, in three other cases, samples are rated "sensitive" by MIKE 3 results, whereas they are rated almost "not sensitive" by HARTMANN tests.

Round robin tests with CaRo05 (pyridine-3-carboxamide, year 2005), and previously with CaRo03 (pyridine-3-carboxamide, year 2003), showed that MIKE 3 apparatus and the test procedure implemented gave equivalent results than other laboratories. [START_REF] Nifuku | Incendiary characteristics of electrostatic discharge for dust and gas explosion[END_REF] studied the influence of particle size distribution on MIE.

INFLUENT FACTORS ON MIE RESULTS

Nifuku and Katoh

Their measurements showed that the smaller the particle size, the smaller the ignition energy, because of the larger specific surface area.

The same authors also reported the influence of dust concentration in the sparkover area [START_REF] Nifuku | A study on the static electrification of powders during pneumatic transportation and the ignition of dust cloud[END_REF]. They showed that a condition to forward the ignition inside

AKNOWLEDGMENTS

This work was supported financially by the French Ministry of Environment and Durable Development.


Wilfried Sanchez
email: wilfried.sanchez@ineris.fr

Benjamin Piccini

Jean-Marc Porcher

Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.)

Keywords: biomarkers, biotransformation enzymes, liver, oxidative stress, prochloraz, threespined stickleback

The aim of this study was to characterise biomarker responses in three-spined sticklebacks exposed to prochloraz (Pcz). For this purpose, adult sticklebacks were exposed for 2 weeks to prochloraz at 0, 10, 50, 100 and 500 µg/L prior to one week of depuration in clean water. At days 7, 14 and 21, several hepatic biomarkers were measured including 7-ethoxyresorufin-Odeethylase (EROD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), total glutathione (GSH) content and thiobarbituric acid reactive substances (TBARS).

Pcz induced a transient increase of antioxidant enzymes and a depletion of glutathione content during the first 7 days of exposure. This study showed that EROD activity and antioxidants were disrupted in a transient manner. GST was rapidly induced in a dose-dependent manner and this induction was persitent and observed also after depuration. GST appeared as a valuable biomarker to assess the exposure to Pcz.

INTRODUCTION

Azole fungicides play a key role as antifungals and are widely used in agriculture around the world. Among these compounds, the imidazole Pcz (N-propyl-N-(2-(2,4,6-trichlorophenoxy)ethyl)imidazole-1-carboxamide) is a broad-spectrum contact fungicide employed in Europe, South America, Asia and Australia, against several diseases in wheat, barley and oleaginous plants but also for the treatment of flower production and of turf on golf courses.

As with other imidazole compounds, the action of Pcz is based on the unspecific strong interaction with the iron atom of cytochrome P450. This binding inhibits the cytochrome P450-dependent 14α-demethylase activity required in the conversion of lanosterol to ergosterol, an essential component of fungal biological membranes, and disturbs the cell membrane assembly. [START_REF] Zarn | Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 -demethylase and aromatase[END_REF] Agricultural application of Pcz contributes to the diffuse freshwater pollution in several European countries including France and the United-Kingdom. [START_REF]DIREN Ile de France[END_REF][START_REF]The Government's Strategic Review of diffuse water pollution from agriculture in England -Paper 1: Agriculture and water: a diffuse pollution review[END_REF] In several rivers of Northern France, Pcz appears as one of the most quantified pesticides and its measured concentrations in surface waters are 5-fold higher than the official standard value of 0.5 µg of pesticides/L. [START_REF]DIREN Ile de France[END_REF] In freshwater ecosystems, Pcz can generate adverse effects to aquatic organisms. Pcz LC50 values for rainbow trout, harlequin fish and bluegill sunfish are in the range 1.5-2.90 mg / L. [START_REF] Larsen | The effect of esfenvalerate and prochloraz on amphibians with special reference to Xenopus laevis and Bombina bombina[END_REF] As in fungi, Pcz modulates fish cytochrome P450 enzyme activities. Pcz is able to modulate the rainbow trout CYP1A system involved in organic pollutant detoxification process, inducing both inhibitory and induction effects on CYP1A expression and EROD activity. [START_REF] Bach | Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout : in vivo induction[END_REF][START_REF] Sturm | Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes[END_REF] A recent study reported inhibition of CYP19 aromatase activity, an enzyme involved in steroid metabolism, in the brain of male and female fathead minnow. [START_REF] Ankley | Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas)[END_REF] In rainbow trout, Pcz can also modulate the phase II biotransformation enzymatic activities such as GST [START_REF] Bach | Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout : in vivo induction[END_REF] , a well known enzyme involved in protection against oxidative stress. [START_REF] Berhane | Detoxication of base propenals and other -unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases[END_REF] Pcz is a fungicide widely encountered in European freshwater ecosystem and can generate adverse effects in fish. However, to our knowledge, no study has investigated the early effect of Pcz on the biomarker responses in stickleback. This study was conducted to assess the biochemical responses in Pcz-exposed three-spined stickleback (Gasterosteus aculeatus L.) using biotransformation enzymes (EROD and GST) and oxidative stress parameters such as GPx, CAT and GSH content, as well as TBARS content (for review, see Van der Oost et al. [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF] ). Stickleback has been used as a model organism to study biochemical responses to various xenobiotics such as metals [START_REF] Sanchez | Copperinduced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF] , pesticides [START_REF] Sturm | Different sensitivity to organophosphates of acetylcholinesterase and butyrylcholinesterase from three-spined stickleback (Gasterosteus aculeatus): application in biomonitoring[END_REF][START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF] , halogenated organic compounds [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the three-spined stickleback, Gasterosteus aculeatus[END_REF] or environmental estrogens and androgens. [START_REF] Katsiadaki | Detection of environmental androgens: a novel method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterosteus aculeatus) glue protein[END_REF][START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF] More recently, stickleback has been described as a valuable sentinel fish species to assess sublethal stress in multipollution context. [START_REF] Sanchez | Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams[END_REF] Among the investigated parameters, EROD is a well documented biomarker in fish but its sensitivity and inducibility in stickleback are not described. Hence, the kinetic of EROD activity was first performed by using -naphtoflavone (BNF) as a reference inducing compound for this enzyme.

MATERIALS AND METHODS

Fish Maintenance and Exposure

Male and female adult sticklebacks were collected in uncontaminated outdoor artificial streams (INERIS, France) and maintained in experimental water (alkalinity, 367 mg.L -1 as CaCO 3 ; pH, 8.35 ± 0.05) for 2 weeks prior to experimentation. The water temperature was 13.5 °C ± 1°C and the photoperiod was a light/dark cycle of 8/16 hours. Fish were fed every other day with frozen red mosquito larvae.

Fish were randomly distributed in 15 litres tanks and waterborne exposed to Pcz under semistatic conditions with complete water renewal and food supply every other day.

-naphtoflavone exposure BNF was dissolved in 5 mL of dimethyl sulphoxide (DMSO) and added to the experimental water with a maximal final concentration below 0.0001 %. Fish (n = 240) were exposed to 0.3, 0.9 and 2.7 µM for 12, 24, 48, 72 and 96 hours. In this experiment, one fish group was sampled before contamination as an initial control (0 h). Moreover, a control group was performed for each exposure time with and without solvent. At each sampling time, 10 fish per tested concentration were randomly collected for biochemical analysis.

Prochloraz exposure

Pcz was previously dissolved in 1 mL of absolute ethanol and added to the experimental tank water with a maximal solvant final concentration of 0.0001%. Fish (n = 160) were exposed to 10, 50, 100 and 500 µg/L (i.e. 0.03, 0.13, 0.3 and 1.3 µM) for 7 and 14 days. After 14 days, 10 fish per concentration were submitted to a depuration experiment and were maintained in uncontaminated water for 7 days with the same experimental conditions. In this experiment, an initial control (0 h) and controls for each exposure length (7, 14 days and 7 days of depuration) were performed. At each sampling time, 10 fish per tested concentration were randomly collected for biochemical analysis.

Biochemical Analysis

Fish were sacrificed, measured and weighed. Liver was dissected, weighed and homogenized in ice-cold phosphate buffer (100 mM, pH 7.8) supplemented with 20% v/v glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a protease inhibitor. The homogenate was centrifuged at 10,000 x g, 4°C, for 15 min and the supernatant was used for biochemical assays after measurement of total protein concentration using the method developed by Bradford et al. [START_REF] Bradford | A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] .

All biochemical assays (EROD, GST, GPx, CAT, GSH and TBARS) were carried out as previously described by Sanchez et al. [START_REF] Sanchez | Copperinduced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF][START_REF] Sanchez | Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams[END_REF]

Statistical Analysis

All data are reported as mean ± standard deviation obtained from the data of individual fish randomly sampled. SPSS 13.0 software was used for statistical analysis. Normal distribution and homoscedasticity of data were verified using respectively Kolmogorov-Smirnov and Levene tests. When data sets didn't have a normal distribution and/or homogeneity of variance, the biomarker data were log-transformed, using F(x) = log(1+x), prior to parametric analysis. Differences between biomarker responses in control of male and female fish were searched using a t-test. If a significant difference ( =0.05) was observed, male and female data were treated separately. A two-way analysis of variance, using time and concentration of exposure, was performed to assess the difference of evaluated parameters for all experimental conditions. When the null hypothesis was rejected ( =0.05), the position of significant difference with the control group were tested using Sidak post hoc test ( =0.05).

RESULTS AND DISCUSSION

Sensitivity of EROD Induction in Stickleback

BNF exposure allows determination of a dose-response curve and completes the previous works showing the inductibility of stickleback EROD activty by chemicals in laboratory [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the three-spined stickleback, Gasterosteus aculeatus[END_REF][START_REF] Holm | Influences of halogenated organic substances on ovarian development and hepatic EROD activity in the thrre-spined stickleback, Gasterosteus aculeatus, and rainbow trout, Oncorhynchus mykiss[END_REF] and field experiments. [START_REF] Sanchez | Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams[END_REF] During this exposure no significant difference between male and female stickleback EROD activities was noticed. After 12 hours of BNF exposure, an increase of EROD activity was measured for each tested concentration (Fig. 1). This induction was also observed for all exposure times and appeared as dose-dependent after 48, 72 and 96 hours. Maximal inductions of 7, 15 and 18 fold were observed after 72 hours (Fig. 1).. The induction factors measured in the BNF exposure were similar to factors reported in fish species widely used in European field studies such as chub and gudgeon [START_REF] Flammarion | Cytochrome P450 1A induction in gudgeon Gobio gobio : laboratory and field studies[END_REF] but stickleback EROD appears less inducible than in rainbow trout, a fish species more widely used in laboratory for ecotoxicological studies. [START_REF] Haasch | CYP1A1 protein and mRNA in teleosts as an environmental bioindicator: laboratory and environmental studies[END_REF]

Effect of Pcz on Sticleback Biomarker Responses

Results obtained for EROD activity are presented in Figure 2A. A transient 3-and 7-fold EROD induction was measured after 7 days of exposure to 50 and 100 µg/L respectively. A significant EROD induction was also reported after prochloraz exposure to 500 µg/L for 7 and 14 days with an induction factor of 7 and 5 fold respectively. After 7 days of depuration, no significant difference was observed between exposed and unexposed groups (Fig. 2A). The present study shows that Pcz fungicide is able to induce EROD activity in three-spined stickleback. This induction appears as dose-dependent and moderate compared to the results obtained using a typical EROD inducer such as BNF. Scientific literature includes some examples of similar EROD induction generated by Pcz. [START_REF] Bach | Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout : in vivo induction[END_REF][START_REF] Sturm | Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes[END_REF] Previous studies reported a transient 3-fold increase of EROD activity in rainbow trout after 7 days of 100 µg/L waterborne exposure [START_REF] Sturm | Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes[END_REF] and intraperitoneal injection of Pcz. [START_REF] Bach | Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout : in vivo induction[END_REF] To our knowledge, the mechanism of EROD induction by Pcz is unknown but several studies have investigated the structural and molecular basis of EROD induction by imidazole compounds such as clotrimazole, 1-benzylimidazole and Pcz. [START_REF] Navas | Induction of CYP1A by the Nimidazole derivative, 1-benzylimidazole[END_REF][START_REF] Navas | Induction of cytochrome P4501A (CYP1A) by clotrimazole, a non-planar aromatic compound. Computational studies on structural features of clotrimazole and related imidazole derivatives[END_REF][START_REF] Babin | Cytochrome P4501A induction caused by the imidazole derivative prochloraz in a rainbow trout cell line[END_REF] These molecules present a non-planar conformation inconsistent with the fixation to the aryl hydrocarbon receptor (AhR) and an EROD induction. A possible explanation could be that the imidazoles are converted intracellularly into planar metabolites which then bind as ligands to the AhR. [START_REF] Babin | Cytochrome P4501A induction caused by the imidazole derivative prochloraz in a rainbow trout cell line[END_REF] This hypothesis is in accordance with the rapid and extensive metabolisation of the highly lipophilic Pcz fungicide (log K ow = 4.12) to more polar metabolites as suggested by Cravedi et al. [START_REF] Cravedi | Metabolic fate of 2,4dichloroaniline, prochloraz and nonylphenol diethoxylate in rainbow trout: a comparative in vivo/in vitro approach[END_REF] . In this context, the easier excretion of polar Pcz metabolites could explain the transient response observed for EROD activity.

Results obtained for GST are presented in Figure 2B. After 7 and 14 days of Pcz exposure, GST was increased for the two highest concentrations (i.e. 100 and 500 µg/L). GST increase was also observed after 14 days of exposure to the lower concentration (i.e. 10 µg/L). After 7 days of depuration in clean water, a significant induction of GST was noticed for all tested Pcz concentrations (Fig. 2B). Phase II biotransformation enzymes such as GST could play an important role in rapid and extensive metabolisation of Pcz. GST is implied in conjugation of electrophilic compounds with endogenous GSH. This reaction increases hydrophilicity of xenobiotics and thereby enhance excretion and consequently gives a detoxification effect. This hypothesis is consistent with the observed dose-dependent and persistent induction. Moreover, it is supported by previous studies investigating the effects of herbicides on stickleback hepatic enzymes that reported GST induction by various pesticides such as diquat [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF] and fomesafen. [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant and adjuvant-herbicide mistures on three-spined stickleback (Gasterosteus aculeatus L.)[END_REF] However, the response of GST could also be linked to Pcz-induced oxidative stress, since this enzyme is involved in the protection against reactive oxygen species. [START_REF] Halliwell | Free radicals in biology and medicine[END_REF] To our knowledge, no study has investigated the induction of oxidative stress by Pcz and the present results are the first evidence of Pcz-promoted oxidative stress in fish. Results obtained for oxidative stress parameters measured after 7 days of exposure are presented in Table 1. As previously reported, GPx is a gender dependent parameter with a higher activity in female than in male stickleback [START_REF] Sanchez | Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams[END_REF] so, data are presented separately. Three exposed groups (i.e. 50, 100 and 500 µg/L) contained only one male fish. Hence, results are not presented and only female data is interpreted. After 7 days of exposure to 100 and 500 µg/L, female fish exhibited an inhibition of GPx. This phenomenon could be explained by the suicidal substrate role played by Pcz. A similar mechanism has been reported for other imidazole compounds such as clotrimazole, which is oxidised to a highly reactive free radical before inactivating peroxidase. [START_REF] Trivedi | Clotrimazole inhibits hemoperoxidase of Plasmodium falciparum and induces oxidative stress: proposed antimalarial mechanism of clotrimazole[END_REF] This inhibition leads to an accumulation of H 2 O 2 and causes a decrease of GSH content and lipoperoxidation. [START_REF] Trivedi | Clotrimazole inhibits hemoperoxidase of Plasmodium falciparum and induces oxidative stress: proposed antimalarial mechanism of clotrimazole[END_REF] GPx inhibition was correlated with the decrease of GSH and the increase of CAT in fish exposed to 500 µg/L (Table 1) suggesting that high Pcz concentrations promote oxidative stress. The responses of enzymatic and non-enzymatic antioxidants appeared as transient while no significant variation was observed after 14 days of exposure and 7 days of depuration (data not shown). Moreover, no significant variation of TBARS content was noticed (Table 1) showing that no oxidative damages occurred. This decrease of GSH could be linked to the inhibition of GPx, but also to the increase of GST activity that used reduced GSH. Besides, an inhibitory effect on GSH regenerating systems (e.g. GSH reductase and/or GSH synthetase), which was not assessed in our study, might contribute also to the observed depletion of total GSH. Indeed, in a recent study, Knorpp et al. [START_REF] Knorpp | Glutathione peroxidase-1 contributes to the protection of glutamine synthetase in astrocytes during oxidative stress[END_REF] showed that catalytic activity of GSH synthetase is diminished by H 2 O 2 and pointed out the sensitivity of this enzyme to oxidative stress and the protective role of peroxidase. Hence, in the present work, Pcz could inhibit GSH synthetase activity following H 2 O 2 production and accumulation. The fact that we did not observe lipid peroxidation generated by Pcz indicates that antioxidant systems were able to protect the cells against the injuries of reactive oxygen species. This hypothesis was supported by the slow induction of CAT activity measured in stickleback after 7 days of exposure to 500 µg Pcz/L. CAT is involved in H 2 O 2 elimination [START_REF] Knorpp | Glutathione peroxidase-1 contributes to the protection of glutamine synthetase in astrocytes during oxidative stress[END_REF] and could eliminate the excess of this very toxic reactive oxygen species generated by GPx inhibition.

Interestingly, responses of enzymatic and non-enzymatic antioxidants were rapid and transient, suggesting the implication of Pcz metabolisation and elimination mechanisms involving phase II biotransformation enzymes such as GST, induced after the first week of exposure.

Implication for Environmental Biomonitoring

This study provides several elements to assess exposure to Pcz and other imidazole fungicides using biochemical biomarkers in three-spined stickleback. EROD activity was characterized by a fast dose-dependent induction followed by an adaptation phenomenon. This biomarker could provide a valuable information on Pcz exposure immediately after the contamination but, if the time between contamination and sampling is increased, the Pcz exposure cannot be detected. [START_REF] Matés | Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology[END_REF] EROD activity is a well documented biomarker of exposure to dioxin-like compounds but, in the light of these results, it could not be considered as a valuable biomarker to assess the exposure to Pcz fungicide. Enzymatic and non-enzymatic antioxidant parameters exhibited fast and transient responses. Moreover, antioxidant responses were only observed for the high tested concentrations. Consequently, biomarkers linked to oxidative stress could not giv valuable parameters to assess the imidazole exposures. In this work, GST appears as a more promising biochemical marker of exposure to Pcz. Our results showed that this phase II biotransformation enzyme is characterized by a fast induction followed by a slow recovery. Also, GST could provide a quantitative and time-integrated estimate of levels of pollution during environmental monitoring. [START_REF] Matés | Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology[END_REF] This conclusion confirms the results of previous study indicating that GST is a valuable biomarker of exposure to several phytopharmaceutical compounds in fish and particularly in three-spined stickleback. [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF]

CONCLUSION

This study showed that waterborne exposure to environmental Pcz concentrations induced Phase I and Phase II biotransformation enzymes in stickleback. EROD activity was moderately induced in a transient manner suggesting an extensive metabolisation. GST exhibited a dose-dependent induction that was also observed after depuration in clean water and could be considered as a valuable biomarker to assess the exposure to this imidazole compound while this enzyme is able to reflect present and past exposure to Pcz. For the high concentrations tested, Pcz promoted an oxidative stress characterised by depletion of GSH and perturbation of antioxidant enzyme activities. The mechanism by which Pcz promoted oxidative stress in fish is not fully understood but our results indicate that it is probably similar to the mecanism of other imidazole fungicides.

Figure 1 .

 1 Figure 1. Effect of 96 hours -naphtoflavone treatment on hepatic EROD activity. Data

Figure 2 .

 2 Figure 2. Effect of prochloraz 21 days treatment on biotransformation enzyme activities :

  Fig.1

Table 1 .

 1 Effect of 7 days prochloraz treatment on hepatic antioxidant parameters and TBARS content. Data represents mean ± SD of the measurements on ten fish per group except for GPx where males and females have been separated. The number of fish is indicated between brackets. (*) statistically different from control (P<0.05).

	Prochloraz (µg/L)

ACKNOWLEDGMENTS

We thank J. Sabarly for reviewing this manuscript. This work was supported by the French Ministry of Ecology and Sustainable Development (Budget Civil de la Recherche et du Développement AP-2004).