G Fayet

P Rotureau

L Joubert

C Adamo

Development of a new QSPR based tool to predict explosibility properties of chemical substances within the framework of REACH and GHS

Introduction

The new European régulation of Chemicals named REACH (for "Registration, Evaluation and Authorization of Chemicals") turned out in the practical registration phase in December 2008. It requires the new assessment of hazard properties for up to 140000 substances. However, the complète expérimental characterization of toxicological, ecotoxicological and physicochemical hazards is time-consuming, costly, sometimes not feasible at the R&D stage and potentially risky. In this context, the development of alternative prédictive methods for assessing hazardous properties of chemical substances is promoted in REACH and in the related new European classification System of substances CLP (Classification, Labelling and Packaging of chemical substances and mixtures). Upon the available alternative approaches, Quantitative Structure-Property Relationships (QSPR), extensively already used in biological and toxicological applications to reduce unnecessary animal testing, are now also developed to predict physico-chemical properties. This contribution focuses on models established to predict accurately two physico-chemical properties of potentially explosive nitroaromatic compounds: the heat of décomposition and the electric spark sensitivity. An original approach associating the QSPR method to quantum chemical calculations was developed. Once validated, models are expected to be integrated into a global tool for the estimation of explosibility hazards.

Présentation of the tool

Regulatory frameworks consider explosion hazards by characterizing différent aspects of the phenomena. Tests to déflagration and détonation estimate if the décomposition can initiate déflagration and, then, propagate to détonation. Explosive power represents the amount of energy released during the explosive décomposition. Sensitivities characterize the ability to react under external solicitation: mechanical (impact or friction sensitivity), thermal (heat sensitivity) or electrostatic (electric spark sensitivity). Our project aims to develop a tool that gathers and uses prédictive models to give a first évaluation of explosibility hazards to complément the expérimental assessment process. This tool, presented in figure 1, comprises 4 main modules. The input module collects molecular géométrie and electronic structures of the investigated substances and calculâtes molecular descriptors. In our combined DFT-QSPR approach, models are based on molecular descriptors extracted from density functional theory (DFT) calculated structures, optimized at PBE0/6-31+G(d,p) level in GaussianO3 package [1] and more than 300 molecular descriptors are computed using CodessaPro software [2].

QSPR models

The QSPR approach consists in correlating quantitatively the expérimental property with the previously calculated descriptors.

Property = f (Descriptors) (1 )
An expérimental data set gives the property measured values. Thèse values hâve to be obtained within a single protocol and with a high accuracy to ensure the best reliability. Différent kinds of descriptors characterize the molecular géométrie and electronic structure:

-Constitutional: number of spécifie atoms, functional groups, bonds; -Topological: atomic connectivity giving information about size, branching degree; -Géométrie: distances, angles, molecular volume; -Quantum chemical: atomic charges, molecular orbital énergies, reactivity indices. Then, the model can be set up using artificial neural networks or genetic algorithms. Hère, statistical multilinear régressions were computed in CodessaPro. The fitting of the models was characterized by the corrélation coefficient (R 2 ) and their robustness was estimated using the cross-validation method (R 2 CV ).

Décision making tool

Once QSPR models are validated, they can be used within a classification procédure. A décision making tool gathers QSPR calculated properties following the gênerai regulatory guidance. Adéquate models are selected upon the studied molécule and the properties to be determined. A key point concerns uncertainties to estimate, not only accuracy but also the need of further expérimental characterization.

Output

To the end, this global tool gives a first évaluation of the possible classification of substances based on calculated properties and indicates whether further expérimental characterization is needed or not. It could also help in substitution goals or R&D processes to estimate the explosive properties of new substances before synthesis.

Example of QSPR models

Heat of décomposition

Thermal stability is an important behavior of explosive substances since it gives information about the energy released during décomposition. If expérimental characterization is well defined, using calorimetric analyses [START_REF] Grewer | Thermal Hazards of Chemical Reactions[END_REF], only few prédictive models hâve been up to now developed. Grewer [START_REF] Grewer | [END_REF] revealed the influence of some chemical groups on décomposition températures. More recently, Saraf et al. [5] simply assumed that the heat of décomposition was proportional to the number of nitro groups in the molécule (n NO 2)-In our study [6], the heats of décomposition of 22 nitroaromatic compounds were extracted from literature [7]. The computed multi-linear model reveals highly correlated with experiments (R 2 =0.98), as shown on figure 2, and is based on a restricted number of parameters to ensure against any over-parameterization, which would reduce predictivity.

-AH (kJ/mol) = 401,6 n N + 2092,2 BO N , avg + 13287 E 0 ,max-3148,5

where n N is the number of nitrogen atoms, BO N , a vg the average bond order for a nitrogen atom and E o , m ax the maximum electrophilic reactivity index for an oxygen atom. Thèse descriptors characterize the number of nitro groups, via n N , and their ability to leave the molécule, via two local reactivity indices (BO N , avg and E 0 ,max)-This is consistent with chemical knowledge since heat of décomposition represents the amount of released energy by the loss of nitro groups. So, the model is not only correlated but also chemically consistent. Moreover, the obtained 3-parameter model gives good predictivity for the TNT molécule, out of the initial set of data. To strongly evaluate the prédictive power of the model, further external validation is needed, but the small size of our data set (22 molécules) was not sufficient to divide it into training and validation sets. Nevertheless, cross-validation method demonstrated good robustness for this model (R 2 cv=0.97).

Electric spark sensitivity

Concerning electric spark sensitivity (E E s), only few prédictive approaches hâve been proposed so far, due to the complexity of expérimental characterization, currently performed with various protocols. Moreover, microscale mechanisms are, up to now, not completely clarified, even if relations were exhibited with the molecular structure, thermal reactivity, mechanic sensitivity or détonation properties [START_REF] Zeman | A study ofchemical micro-mechanisms of initiation oforganic polynitro compounds[END_REF][START_REF] Hosoya | [END_REF]. To our knowledge, the only QSPR type approach was proposed by Keshavarz recently for nitroaromatic compounds using constitutional descriptors (R 2 =0.77) [10].

Our study was based on a more extended set of descriptors for 26 nitroaromatic compounds. A 4-parameter model was developed [START_REF] Fayet | Proceedings ofthe 43rd Loss Prévention Symposium and 5th Global Congress on Process Safety[END_REF] with high corrélation with experiments (R 2 =0.90).

E ES (J) = 29.6 n sing ie + 63.3 N c , ma x + 168.4 Qc, min -27.8 Vc, min + 99.4

where n S in g ie is the relative number of single bonds and N c ,max> Qc.min and V c ,min are the maximum nucleophilic reactivity index, the minimum charge and valence for a carbon atom, that can be related to the C-NO 2 bond. Beside, gênerai assumption considers the loss of nitro group as the rate limiting step of décomposition in nitro compounds. So, this new model is chemically consistent, llmprovement is expected by enlarging the data set, making external validation possible, but its current performances look yet better than Keshavarz one (R 2 =0.90 vs. 0.77), as shown in figure 3.

25 30

Figure 3: Plot of calculated electric spark sensitivity (in J) versus expérimental values according to équation 4 (in plain circles) and Keshavarz's model [10] (in wide circles).

Conclusion

Two physico-chemical properties related to the explosibility of nitroaromatic compounds hâve been investigated using a combined DFT-QSPR approach: the heat of décomposition and the electric spark sensitivity. The developed models need consolidations to validate their predictivity using extended data sets, but they already upgrade the existing models with corrélations up to R 2 =0.98 and 0.90. Moreover, they integrate chemically consistent descriptors related to the C-NO 2 bond, the critical reactive site for the décomposition of nitroaromatic compounds [START_REF] Fayet | [END_REF]13].

Once strongly validated models are obtained, they will be associated within a global tool to assess explosive hazards in the line of the search of new methods (required by REACH), not replacing but complementary to expérimental tests, to provide first évaluation of hazards and to guide to further expérimental investigation when necessary.

Figure 1 :

 1 Figure 1 : Global tool for the évaluation of explosibility hazards

Figure 2 :

 2 Figure 2: Plot of calculated décomposition enthalpies (in kJ/mol) versus expérimental values according to équation (2)


F Antoine
email: frederic.antoine@ineris.fr

Assessment of the evaporation corrélations performance 13 th International Symposium on Loss Prévention

Introduction

When performing a risk analysis on industrial facilities, various hazardous scénarios must be imagined. Thèse scénarios may lead to the formation of a toxic or flammable gas cloud and it is therefore important to détermine precisely the pool evaporation so as to estimate the size and the concentration of the cloud. Currently, différent mass transfer evaporation corrélations are used to quantify the evaporation rate. Some of them let easily calculate the evaporation rate only taking into account few parameters as the saturated vapour pressure, the liquid température, the substance molecular weight, the air wind velocity and the evaporation area. However, when thèse corrélations are compared each other on a specified case, the evaporation rate results obtained are not necessary similar. A significant gap may be even noted between them. This point lets think that the use of thèse tools should be done with caution. A survey, focusing on three corrélations which appear regularly in the safety surveys, has been set up in order to well understand why such a différence may appear between results. Then, from this first work, the fair conditions of use, regarding the three différent tools, hâve been identified. The three tools analyzed are adapted to the evaporation phenomena of pure evaporating liquids whom the saturated vapour pressure is weaker than the atmospheric pressure. Consequently, only the mass transfer evaporation phenomena may be assessed using thèse formulas.

The différent corrélations

The three corrélations examined in this document are the followings:

• The UIC corrélation: in the UIC «cahier de sécurité n°11 » [1], a mass transfer evaporation formula is proposed, based on the work of Clancey [2], • The "Mackay and Matsugu's" corrélation : based on their expérimental work but also on Sutton's work [3] [4], Mackay and Matsugu [5] established a mass transfer corrélation to calculate the pool evaporation rate, • The "TNO" corrélation: in the 3rd édition of the TNO « Yellow book » [6], an analytical corrélation is proposed to assess the pool evaporation rate by mass transfer.

Corrélations analysis

The table below compares the expressions of each corrélation : Table 1 : The expression of the three différent corrélations: The parameters v, r, P, T and M, appearing in the three first corrélations associated with the same exponents values, may be clearly identifiée! in this expression. Just the opposite, the parameters Di, k, z-! do not appear explicitly in the three corrélations.

The accurate assessment of thèse last ones seems to be really more difficult to carry out than the one of the parameters v, r, P, T and M. Di is the coefficient diffusion, k is the Von Karman's constant whose value is estimated to 0.4 by Pasquill [7] and the parameter Zi, minimal height from which the maximal wind velocity is reached. So, it appears possible that the three corrélations don't use this kind of parameters due to the difficulty to détermine it. To replace it, some fitting parameters or coefficients seem to be présent in the corrélations expressions.

It implies that the expérimental conditions (device, substances, environmental conditions...) from which the corrélations are based on, may hâve a great influence on the détermination of the coefficients. And, consequently, the corrélations should be applied in similar conditions to be efficient. AH of this leads to clearly analyse the testing conditions.

The following examples let lighten the importance that may hâve some fitting parameters or coefficients on the final evaporation results:

-The diffusion coefficient: the Mackay and Matsugu corrélation takes directly into account the laminar diffusion coefficient Di through the laminar Schmidt number Se.

The expressions of the two other corrélations don't let clearly take into account the laminar diffusion. However, after analyse, the fitting coefficients supposes a value regarding this point. So, the UIC corrélation takes into account a diffusion coefficient about 7.5.10" 6 m 2 /s and the TNO corrélation uses a diffusion coefficient of 1.9.10" 5 m 2 /s. Supposing a large part of Chemicals is characterised by a laminar Schmidt number between 0.5 and 3, the evaporation rate, from the use of the Mackay and Matsugu corrélation, is three times higher in the first case than in the second one. -The height of référence Zi : Pasquill [7] considers the assessment of the appropriate values of z-i difficult. However, he explains that an error of 500 % regarding Zi has only an influence of 20 % on the final results. In any case, this parameter is one of the toughest to assess and can vary significantly depending on the experiments conditions. Finally, to increase the importance of this matter, it seems relevant to notice that ail the exponents values which appear in the corrélations are partly based on expérimental results. So, even if thèse last parameters don't appear clearly in the différent corrélations, some fitting coefficients in the corrélations determined experimentaly may indirectly take into account the parameters X, k and zi.

Expérimental conditions

UIC Corrélation (Pasquill experiments)

The experiments were carried out with four substances: water, aniline, methyl salicylate and bromobenzene. The saturated vapour pressure of the substances was always below 2000 Pa. The evaporation surface was about 200 and 450 cm 2 . The application of équation (1.4) on bromobenzene was quite interesting: the maximal différence between the calculations and the expérimental results was about 10 %. However, the use of the équation (1.4) to estimate evaporation rate of other substances, whom the characteristics were différent from the bromobenzene ones, was not so efficient. Pasquill [7] proposed to replace the kinematic viscosity k (m 2 /s) in équation (1.4) by the diffusion coefficient Di (m 2 /s) of the vapour in the air. The results were improved. In this case, taking into account the diffusion of the evaporating substance seems to be relevant. 4.2 Mackay and Matsugu, TNO corrélations (Mackay and Matsugu experiments) Cumene (450 Pa at 290 K), water (2300 Pa) and gasoline (between 50-100 kPa ) hâve been tested. Tests were achieved with evaporation surfaces between 700 cm 2 and 3.14 m 2 . From this work, Mackay and Matsugu set up a transfer coefficient to détermine an evaporation corrélation. Satisfying results were obtained for water and cumene, not for gasoline. Mackay and Matsugu suggested there might be a "liquid phase résistance" to evaporation in the gasoline due to the fact it is made up of différent Chemicals with différent volatilities. The most volatile Chemicals could create a fall of the liquid surface température hence reducing the saturated vapour pressure and in turn the evaporation rate. That could be an explanation of the overestimation of the Mackay and Matsugu's calculations regarding the gasoline evaporation.

Mackay and Matsugu, TNO corrélations (Woodward [8] experiments)

The author developed an evaporation model in which Mackay and Matsugu's [5] mass transfer coefficient is used. However, the author modified their original works regarding the mass transfer coefficient in order to get a better fit between expérimental results and calculations. According to Woodward [8], the observed discrepancy is due to the différence between the substances nature tested in this case and the Chemicals used by Mackay and Matsugu. So, using the original Mackay and Matsugu's [5] corrélation, the evaporation rate tended to be overestimated for the volatile ones and underestimated for the less volatile ones. This example distinctly lightens the influence of the expérimental results on the final assessment of the evaporation rate.

Corrélations field of use

For each of the reviewed corrélation, the table below lists the conditions which are more likely to ensure a good prédiction of the evaporation rate : Table 2: The TNO and Mackay and Matsugu's corrélations can be applied for the same evaporating substances in similar conditions. The same theory and the same expérimental results hâve been used to develop the corrélations. However, a major importance regarding the choice of the diffusivity value may hâve a significant influence on the final evaporation resuit. The Mackay and Matsugu's one takes into account the evaporating substance diffusivity through the Schmidt number whereas a fixed value of 0.8 is set for this parameter in the TNO équation. The range of application of the UIC corrélation is significantly différent from the two other ones especially in terms of evaporating substance volatility and diffusivity. The evaporation conditions are also quite différent especially concerning the environmental conditions and the evaporating area.

  and Matsugu : D = 1.67 * Se 067 *10" 3 * r 189 * v 078 * M * P/T (1.2) TNO : D = 1.95* 1Q-3 * r 189 * y 078 * M * P/T (1.3)The main comment arising from this comparison is the strong similarity between the three différent expressions. Five parameters appear in thèse formulas associated with the same exponent value: the wind velocity v, the pool radius r, the saturated vapour pressure P, the pool température T and the molecular weight M. The three corrélations are based on Sutton's work[3] [4] regarding the pool evaporation phenomena. This work is based on the advection-diffusion équation. From this équation, Sutton proposed the following simplified expression to estimate the evaporation rate: proposed to replace X appearing into équation (4) by the diffusion coefficient. D ^ 0.38 * Di 022 * k 133 *zr°1 1 * r 189 *v 078 *M*P/ T (1.5)

Conclusion

The "Mackay and Matsugu", the TNO", and the "UIC" corrélations dérive, partly, from the same fundamental works of Sutton [3] [4] based on the diffusion-advection équation solving. After several simplifications of thèse works, several evaporation corrélations hâve been set up as the three ones discussed hère. A part of the simplifications realised is based on the use of expérimental results from différent expérimental works. The comparison between the results from the corrélations and the expérimental results lightened some fitting parameters which hâve been required to improve the équations performance. Thèse fitting parameters are, however, strongly dépendent on the substances tested and the expérimental conditions. Consequently, it clearly appears that the three corrélations, focused on hère, are a mixing of a theoretical and expérimental works. The use of thèse last ones narrow the field of use of the différent corrélations. So, thèse observations tend to warn concerning the appropriate use of the various mass transfer evaporation tools. Thèse latters should be used having in mind the theory used but also the expérimental conditions which let the équation détermination. Thèse two determining parts hâve been identifiée! in this article in orderto let a performant use of the three corrélations studied. This watching also reveals that more experiental tests would let better distinguish the field of use of thèse corrélations and would let improve it. So, information regarding the assessment of the evaporation of large pools whom surface is about 100 m 2 or more, the evaporation of high volatile liquids (» 50 kPa) or the evaporation with a low wind velocity below 1 m/s seems to miss.

Références


S Di Tommaso

' P Rotureau

A Vignes

M Alzerreca

W Benaissa

C Adamo

Study of the peroxidation mechanism of diethyl ether (DEE) 13 th International Symposium on Loss Prévention

Introduction

A great number of organic compounds spontaneously form unstable peroxides by reaction of the carbon chain with molecular oxygen in a process of auto-oxidation, the so-called "peroxidation". This is a free-radical and self-propagating reaction that may generate a wide variety of peroxide molécules (above ail hydroperoxides and dialkyl peroxides) for each initiating event.

In figure 1 are presented some of the involved prototypical reactions)

Initiation Oxygen addition Hydroperoxide formation Dialkyl peroxide formation

R+ÔH-> R + 0 2 -> R00-+ H- ROO + R' RO + R' O R + H 2 0 ROO ->ROOH -> ROOR' -> ROOR' Fig 1.

Prototypical reactions of auto-oxidation process

Peroxidation is promoted by a prolonged exposure to air, heat and light during storage and the accumulation of produced peroxides can lead to unexpected explosions. Whereas reactive hazards of organic peroxides are often well-known (usually they are characterized by shock, heat or friction sensitivity), no data are available about the concentration at which thèse molécules or a mixing pose a potentially explosive hazard 1 ' 2 . The reaction of peroxidation is most likely to occur in compounds with activated hydrogen atom in which the intermediate free radical can be stabilized through electronic résonance structures. For instance activated hydrogen atoms occur on carbon linked by oxygen as ethers, on tertiary carbon atoms, on carbon atoms in vinyl structures and in allylic and benzylic structures. In figure 2 3 are presented peroxidizable moieties in order of decreasing hazard. Among the Chemicals widely used in a lot of laboratories and industries as solvent or reactive, ethers are, as shown in figure 2, the most notorious peroxide formers. However, few works propose mechanistic study of their oxidation reactions. The dimethyl ether (DME) is the most investigated ether both experimentally 4 " 6 and theoretically 7 " 9 , since it has a relevant rôle, due to its good ignition properties, in the domain of biofuel. In particular, Andersen and coworkers propose a mechanism for the reaction of low température oxidation of DME. They suggest that, analogously to the alkane Systems, the reaction occurs when reactive species (the hydroxyl radical for instance) abstract a hydrogen atom from the ether. This initiation step is followed by the reaction of the radical (R ) with molecularoxygen. The newadduct (ROO) can décompose, toform two formaldehyde molécules and a HO 1 radical. Alternative^ it can isomerize by intra-molecular hydrogen transfer and then décompose to form various neutral molécules and two HO 1 radicals instead of one: this leads to a branched chain reaction which causes an increase in the rate of DME oxidation. Expérimental study also exist on the oxidation of the other ethers often used as additives in fuel and biofuel like the diethyl ether (DEE) [START_REF] Naito | A comparative study on the autoxidation ofdimethyl ether (DME) comparison with diethyl ether (DEE) and diisopropyl ether (DIPE), International Conférence on Bhopal Gas Tragedy and Its Effecton Process Safety[END_REF] ' [START_REF] Orlando | [END_REF] , the diisopropyl ether (DIPE) 7 orthe methyl tert-butyl ether (MTBE) 12 , but, in gênerai, they do not suggest any mechanism. However, since DEE and DIPE are known to be responsible of a great numbers of laboratory accidents 13 " 16 for their ability to form explosive peroxides, mechanistic study on the ethers oxidation reaction is fundamental to better understand and to control their accidentai risks. The diethyl ether is particularly studied because it is largely used, above ail as a very common laboratory solvent. Due to its use as additive in biofuel 17 , it is also présent in gas phase in the terrestrial atmosphère and it is studied as a contributor to the formation of photochemical smog 8 . From the expérimental characterization of final products of décomposition pathways (ethyl formate, ethyl acétate, formaldehyde, methanol, formic acid), Orlando suggests a mechanism for the atmospheric oxidation of DEE. He proposes that the initiation process (via reaction with HO 1 or CI 1 radical) and the reaction of the radical [CH3CH2OCHCH3] 1 with molecular oxygen, are followed by self-reactions of the 1ethoxyethylperoxy radical [CH 3 CH 2 OCH(OO)CH 3 ] 1 . Thèse self-reactions are a fundamental step for the décomposition of the molécule. In this scientific context, the présent work, carried out in the domain of the INERIS research project named RIPER (for "study of Rlsk linked to the PERoxidation of chemical products"), proposes the study of the peroxidation of diethyl ether at both expérimental level, through calorimetric tests and analytical methods aiming to detect and measure the amount of produced peroxides, and at theoretical level to identify reaction mechanism, principal products and unstable species. This study will lead to develop a detailed chemical kinetic model of the oxidation process of DEE and, coupled with expérimental data, should allow a better understanding of its accidentai risks.

l.Ethers and acetals with a-hvdroaen

H -i-o-< I x

Materials and methods

2.1 Computational method AH calculations were performed using the Gaussian 03 set of programs [START_REF] Frisch | Gaussian 03[END_REF] . DFT calculations were performed with the hybrid B3LYP functional [START_REF] Becke | [END_REF] . The 6-31+G(d,p) basis set was used to optimize geometries and characterize stationary points in the potential energy surface as minima orsaddle point (transition state). The intrinsic reaction coordinate (IRC) calculations were also performed to verify that the products and the reactants were correctly connected 20 . Gibbs free énergies hâve been calculated at standard conditions (T=298.15K, P=1atm).

Expérimental method

The work presented in this paper deals with the évolution of the stability of diethyl ether exposed to air and light during 2 months. Samples were investigated by means of a Setaram DSC 131 at 5 K/min heating rate in order to détermine the reactivity of the System. Each sample were weighted to a mass of few milligrams and placed into a stainless-steel crucible.

Thermal analysis was performed between 20 and 500 °C. It should be noted that iron may slightly influence the décomposition process within the crucible. However, it is not taken into account at the moment. DEE (99.7%) was obtained from Sigma-AIdrich.

Results and discussion

3.1 Theoretical results: the case of one of the possible intramolecular isomerization reaction AH the chain reactions of DEE (1 ) décomposition that we hâve characterized start with the abstraction of a hydrogen atom from the neutral molécule by an initiator (the HO 3a radical can follow more than one décomposition pathway: it can react with other radicals (for instance with H to give a hydroperoxide ROOH) or, as proposed also for DME, it can isomerize by intramolecular hydrogen transfer in a more complex mechanism 5 .

In figure 3 is presented the potential energy surface of the isomerization mechanism. This mechanism starts with the transfer of a hydrogen atom from a carbon atom to the oxygen radical: this first step is endothermic with an energetic barrier of 18.6 kcal/mol.

OC"

The product of this reaction can follow, as showed in figures 3 and 5, two possible paths: it can directly décompose in two acetaldehyde molécules (12) and a HO 1 radical or react with another O 2 molécule to give 10a. This radical can then isomerize to form the neutral molécule hydroperoxyethyl acétate (11a) and, also in this case, a HO' radical. Thèse processes are both exothermic, but the energetic barriers are very différent: 6.1 kcal/mol for the décomposition and 36.5 for the isomerization path. It is verified, as also shown in table 1, that 11 a is the more stabilized product for about 25 kcal/mol. Hydroperaxyethyl acétate (11a) is not the final product of the reaction of DEE oxidation. We hâve found a lot of possible competing décomposition paths of the 11a neutral molécule, that are collected in figure 6, in order of increasing activation énergies (table 2). AH the stationary points and transition states involved in the identified paths are above the energetic zéro represented by the sum of Gibbs énergies of DEE and of the radical initiator. The reaction paths a, b and c are characterized by the lowest energetic barriers (i.e. kinetically favored). However in ail thèse three cases the final reaction products are less stable than 11a. The reaction of dehydration of hydroperoxyethyl acétate (path d) is the thermodynamically favored one. The energetic barrier (see table 2) for this path is not the smallest (43.4 kcal/mol for the rate determining step), but the stabilization of final products (acetic acid, water and CO-CH 2 formed by anhydride décomposition) is the greatest (i.e. thermodynamically controlled). The two last reaction channels (e, f) for the 11a décomposition are both unfavourable due to their prohibitive energetic barriers.

Expérimental results

Auto-oxidation process of DEE is mainly characterized by a long induction period (i.e. few months at 298 K and atmospheric pressure) 21 . Relatively slow until 155°C , peroxidation leads to the formation of hydroperoxides that décompose to organic compounds such as ethyl formate, ethyl acétate, formaldehyde, methanol, formic acid, acetic acid, ethanol 13 ' 22 as well as some gases like méthane or carbon dioxide 2324 . Thèse early results seem to be in accordance with the latest scientific work performed by Orlando 11 , who also proposed an atmospheric oxidation mechanism of DEE initiated by HO 1 or CI 1 radical. In fact, they showed by expérimental characterization (infrared spectra) that the final products of the décomposition pathways of hydroperoxydes were the same compounds as above mentioned. Hydroperoxides are relatively stable and may not trigger any explosion event. However, hydroperoxides tend sometimes to polymerize to form a highly reactive compound which is likely responsible of the accidentai events 1323 . Peroxidation process is underthe dependence of several parameters, like température, pressure, geometry of the storage, exposure to light, air heat, which détermine the long term stability of the product. In order to decrease the complexity of the study, it is necessary to consider spécifie tests allowing to détermine globally the évolution of the System. Thus it was decided to perform in a first step a preliminary study enabling to characterize in a simple way the évolution of the reactivity of the System along the time by using calorimetric tools. Hère are briefly presented the first results of that expérimental work which hâve just started and will last two years.

In the thermal analysis performed on the samples, a single exothermic peak (22 J/g) was observed around 410°C for each "old" samples (exposed to air and light during two months), but no peak was previously observed in the case of fresh DEE. It was first thought that it would witness peroxide compounds formation, but qualitative tests with Quantofix tests strips were négative, i.e that ether samples do not contain peracetic acid nor peroxides compounds. Further tests with 2,4 DNPH were then carried out, showing a slightyellow precipitate. It means that the exothermic peak may be due to the présence of aldéhydes or ketones compounds and not peroxide compounds. At the moment, the risk stability of our samples is still low but the évolution of this parameter needs to be studied in a long-term period to define more accurate and reliable conclusions which is the présent work performed atlNERIS.

Conclusion

The theoretical study of the oxidation reaction of diethyl ether molécule by DFT calculations confirms the great variety of the possible décomposition mechanisms supposed in literature. At présent, we hâve characterized (in structures and énergies) more than ten différent radical chains. The objective of the work is now to collect thermodynamical data of ail the available reaction paths. Furthermore this theoretical study has as fundamental aim also the development of a detailed chemical kinetic model of the auto-oxidation process of DEE. Thèse informations, coupled with the data obtained from the expérimental work (that is at présent just at the beginning), will lead to better understand the accidentai risks of diethyl ether.

Fig 3 .Fig 4 .Fig 5 .

 345 Fig 3. Potential energy surface of the isomerization reaction

22 Fig 6 .

 226 Fig 6. Competing paths of décomposition of the hydroperoxyethyl acétate in order of increasing activation énergies

  1 radical for instance) to form a radical at the carbon atom next to the oxygen (2a) or at the terminal carbon (2b):

	1	^^^o^\-+ H 2 O ^^O^^ + H 2°2 < 2b a
	It has been verified by DFT calculations that the 2a radical is more stable than the 2b one for
	8.2 kcal/mol. The 2a radical (or 2b) can then react with molecular oxygen to form the 3a
	peroxy radical:
		00"
		+o 2
	2a	3a

Table 1 .

 1 Gibbs énergies of stationary points and transition states of the two competing paths

		Isomerization		Décomposition
	species	ÛG 0lSOlï (kcal/mol)	Species	ÛG 0lSOlï (kcal/mol)
	1+HO	0
	2a + H 2 O	-23.7
	3a	-48.0
	TS5a	-29.4
	9a + O 2 10a	-33.7 -59.7	TS7a	-27.6
	TS6a	-23.2	2x12 +HO	-80.8
	11a + HO	-103.0

Table 2 .

 2 Gibbs énergies of stationary points and transition states of competing paths of 11 a décomposition

		Species	ÙS 0 ** 1 (kcal/mol)
		11a	0
	a	16a + HO	24.6
	b	TS9a	27.0
		17	15.2
		18 + 19	22.9
	c	TS10a	29.8
		18+ 20 +HO	8

.0 d e f species TS11a 21 + H 2 O TS12 18 + 22 TS13a 23 TS14a 24 + H 2 + 22

	AG 0lSOlï (kcal/mol)
	43.4
	-

73.1 -40.7 -60.5 61.0 69.3 74.2 16.5


A Janes

G Marlair
email: guy.marlair@ineris.fr

P Rotureau

Biodiesel: a case study of the impact of new rules regarding the classification and labelling of physical and chemical properties of chemicals

Introduction

Biodiesel and any co-products or intermediate chemicals produced and used from the biodiesel industry fall under the scope of the regulation related to chemical substances. The criteria and methods for classification of physical hazards of substances and preparations used so far in the European Union were defined since 1967 in 67/548/EEC Directive [START_REF]on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances[END_REF] so called "substances Directive" and in 1999/45/EC Directive [START_REF]EC of the European Parliament and of the Council of 31 May 1999 concerning the approximation of the laws, regulations and administrative provisions of the Member States relating to the classification, packaging and labelling of dangerous[END_REF], dealing with preparations. The European regulation CLP [START_REF]Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing[END_REF] -Classification, Packaging and Labelling -came into force on 20 January 2009 and implements the recommendations of Globally Harmonised Systemso called GHS -in the European Union. CLP Regulation is an essential tool for the implementation of REACh Regulation [START_REF]the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)[END_REF]. The GHS is developed at the international level and describes the classification criteria of the hazards of chemicals. It also provides new harmonized elements related to safety information on these hazards. These are intended to develop new labels and material safety data sheets. It is expected that the 67/548/EEC and 1999/45/EC Directives are repealed with effect from 1st June 2015. It corresponds to the end of the transitional period before full implementation of the CLP Regulation for mixtures. Full enforcement for substances will be effective from 1 December 2010. This paper provides a comparison between previous EU on the one hand and CLP on the other hand regulation criteria and methods, focusing on physical and chemical hazards. The impact of these new rules on the biodiesel industry is specifically investigated.

Update of new rules about physical and chemical hazards evaluation in EU

CLP Regulation modifies the rules on classification and labelling for substances and mixtures (hazard categorisation, labelling, MSDS ...). Enforcement of CLP involves the shift from 5 categories for physical hazards in the EU previous system to 16 classes in the new one. Some hazard categories are modified and new classes do appear. Therefore, significant differences appear as to the assessment of physical hazards between the EU classification and labelling system applied so far and in the CLP Regulation: reorganization of the hazards already defined, definition of new hazard classes, such as "Gases under pressure", "Self-reactive substances and mixtures" and "Corrosive to metals", modification of criteria and thresholds for the classification and/or assessment methods. This is true for many hazard classes and differences are particularly significant for "Oxidising solids" and 'Explosives" classes. Only two hazard classes are strictly identical in the two classification systems: "Pyrophoric solids" and "Pyrophoric liquids".

As an example, table 1 below provides the changes involved for the classification and the labelling concerning the "Flammable liquids" class. Although the evaluation method -which consists in flash point measurement -does not change, thresholds selected for classification are modified. This increases the severity of classification. So far, every industry that uses a family of chemicals, such as biodiesel, is -more or lessimpacted by the implementation of the CLP Regulation. It requires significant changes in current benchmarks related to classification and labelling of hazardous chemicals. This implies that each industry make an overview analysis concerning expected evolution.

How is the biodiesel industry impacted?

Biodiesel has been produced on an industrial scale in the European Union since 1992, largely in response to positive signals from the EU institutions. In the transport sector, it can be used both when blended with fossil diesel fuel and in pure form. Today, there are approximately 120 plants in the EU producing annually up to 6,100,000 tonnes of biodiesel. These plants are mainly located in Germany, Italy, Austria, France and Sweden [START_REF]European Biodiesel Board[END_REF].

Biodiesel is typically produced through the reaction of a vegetable oil or animal fat with methanol (which is preferred for cost reasons) or ethanol, in presence of a catalyst, to yield methyl or ethyl esters, i.e. mainly called FAME for fatty acid methyl ester and FAEE for fatty acid ethyl ester, and glycerine.

Biodiesel derived from rapeseed oil is the most common biodiesel available in Europe, while soybean biodiesel predominate in the United States [START_REF]Biodiesel -A realistic Fuel Alternative for diesel Engines[END_REF]. Most common catalysts are sodium or potassium hydroxide, sodium or potassium methoxide and boron tri-fluoride [START_REF]Biodiesel -A realistic Fuel Alternative for diesel Engines[END_REF] and [START_REF]Biodiesel -Basics and beyond -A comprehensive Guide to Production and Use for the Home and Farm[END_REF].

In fact, "biodiesel" covers a wide range of products: there are about 50 substances on EINECS that may be manufactured and use as "biodiesel" [START_REF]UK REACH Competent Authority Information Leaflet Number 15 -REACH & Biodiesel[END_REF]. The EU has published guidelines in compliance with CEN Standard -EN 14214 [START_REF]Automotive fuels -Fatty acid methyl esters (FAME) for diesel engines -Requirements and test methods[END_REF] -in order to insure consistent quality and performance. Despite these specifications, there is enough leeway as regards to the composition and physical and chemical properties of biodiesel.

Table 2 shows the changes involved for the physical and chemical hazards classification for the most common alcohols and catalysts used in the biodiesel industry. It indicates that among these examples, alcohols classification is impacted and two new classes appears concerning the catalysts.

For diesel fuel, FAME, FAEE, vegetable oils and glycerine, classification do not change, considering the "Flammable liquids" class. In both systems, all are excluded due to their high flash point value. In fact, it is known that biodiesel is better than diesel fuel in terms of sulphur content, flash point, aromatic content and biodegradability [START_REF]Biodiesel -A realistic Fuel Alternative for diesel Engines[END_REF].

Classification in other physical and chemical hazard classes should be considered on a case by case basis, considering the physical and chemical properties, which may be obtained from standard tests. Particularly, it is known that sensitivity of vegetable oils to self-heating is highly dependent on its composition. This may lead to classify some of them in the "selfheating substances and mixtures" class.

Conclusion

Biodiesel has been demonstrated to have some significant environmental benefits in terms of limited global warming impacts, reduced emissions and greater energy independence.

Recent developments of the European legislation, such as CLP Regulation implementation, require every industry to assess the changes involved in classification and labelling of chemical used.

In the case of the biodiesel industry, CLP Regulation induces a more detailed classification of chemical and physical hazards, particularly considering some catalysts. Safety information about these hazards, especially concerning pictograms and hazards statements, is also significantly modified. Although this is not constitutive of a real revolution, one has to get accustomed with these new practical references. It will take a while for people to shift from previous references to the new ones. This concerns the whole life cycle of chemicals and involves producers, users, authorities, emergency services... Finally, for the majority of process chemicals used in biodiesel industry, changes in health and environmental hazards classification and labelling must also be investigated.

Table 1 :

 1 Comparison between "Flammable liquids" classification in old and new systems, based on ebullition temperature and flash point value

	Flash point	< 0°C	< 21°C	< 23°C	≤ 55°C	≤ 60°C
	67/548/EC Directive	if T eb ≤ 35°C: Extremely flammable -R12 if T eb > 35°C:	Easily flammable -R11	No pictogram Flammable -R10	Not included in « Flammable liquids » class
		Flammable -R11
	CLP		if T
	Regulation

eb ≤ 35°C: Flammable liquid Cat. 1 -Danger -H224 if T eb > 35°C: Flammable liquid Cat. 2 -Danger -H225 Flammable liquid Cat. 3 -Warning -H226 T eb : ebullition temperature Hazard statements: R12: Extremely flammable H224: Very highly flammable liquid and vapour R11: Easily flammable H225: Highly flammable liquid and vapour R10: Flammable H226: Flammable liquid and vapour

Table 2 :

 2 Harmonised physical and chemical hazards classification of some process chemical used in biodiesel production in both 67/548/EEC Directive and CLP Regulation systems[START_REF]Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing[END_REF]

				Health and/or
	Products (CAS number)	67/548/EEC Directive	CLP Regulation	environmental hazards to be
				considered
	Methanol (67-56-1)	Easily flammable R11	Flammable liquid cat. 2 H225	Yes
	Ethanol (64-17-5)	Easily flammable R11	Flammable liquid cat. 2 H225	No
	Sodium hydroxide (1310-73-2)	-	-	Yes
	Potassium hydroxide (1310-58-3)	-	-	Yes
	Sodium methoxide (124-41-4)	Easily flammable R11 -R14	Self-heating cat. 1 H251 -EUH014	Yes
	Potassium methoxide (865-33-8)	Easily flammable R11 -R14	Self-heating cat. 1 H251 -EUH014	Yes
	Boron trifluoride (7637-07-02)	-	Gas under pressure EUH014	Yes
	Hazard statements:
	R14 and EUH014: Reacts violently with water	H251: Self-heating: may catch fire


T Pénelon

D Jamois

C Prousf
email: proust@ineris.fr

Liquefied Gas Over Water : An Expérimental Approach at Médium Scale to assess gas/water interactions and vapor dispersion 13 th International Symposium on Loss Prévention

Introduction

In case of a marine accident, gas or liquid substances could be released from the damaged ship and generate important risks for the crew, for the approaching emergency services and even for the population if the damage occurs close to the sea coast. In particular, some delayed hazardous phenomena may occur, like ignition of the spilled gas leading to a fire (jet fire, pool fire) or an explosion (overpressure added to flash fire effects), instantaneous vaporisation of liquefied gas still confined (BLEVE) or toxic effects due to evaporation of spreading pools over water. Even though improvements hâve been made in ship structures and safety equipments, the increasing volume and number of marine tankers in the récent years make this threat more acute.

The behaviour of spilled cryogénie hazardous gases like Liquefied Natural Gas -LNG -onto or under water has been studied in the late 70s (e.g. [START_REF] Références | LNG safety research in the U.S.A[END_REF][START_REF] Opschoor | The spreading and evaporation of LNG-and burning LNG-spills on water[END_REF] and some new studies hâve been recently published (e.g. [START_REF] Cormier | Application of computational fluid dynamics for LNG vapor dispersion modeling: A study of key parameters[END_REF][START_REF] Fay | Spread of large LNG pools on the sea[END_REF][START_REF] Luketa-Hanlin | A review of large-scale LNG spills: Experiments and modeling[END_REF] due to the revival of interest in natural gas in the last few years. However the interaction mechanisms between the water surface and a refrigerated or pressurised gas spilled at sea are quite complex and still need to be studied to improve their understanding.

This paper présents the main observations from expérimental tests carried out at INERIS (Verneuil-en-Halatte, France) to study the behaviour of refrigerated liquefied gas and liquids spilled over or below a water surface. The main topics addressed with this expérimental approach at médium scale are:

• the vaporisation of the liquefied gas,

• the spreading of the pool, with potential freezing effects at the surface for cryogen substances,

• the evaporation of the liquid pool. The tests with pentane are not discussed in this paper.

In Section 2, we présent the test device. Section 3 is dedicated to the observations and their analysis.

Test device and protocol

Test device and instrumentation

The test device consists of a 10 mx10 m-pool of water with 1 m depth, in which some liquid or liquefied gases hâve been spilled (see Figure 1). The release point is located at the centre of the basin. The spilled materials are refrigerated liquefied nitrogen (boiling température T eb =-196°C) as a safe substitute to LNG, pressurized liquefied propane (T e b=-42°C) and butane (T eb =-1°C) and finally liquid pentane (T e b=36°C) as a more persistent substance. The nozzle is connected to the storage tank by a 8-m flexible heat insulated hose of internai diameter 20 mm for liquefied gases and by a 8-m simple hose of internai diameter 4 mm for pentane. The nozzle diameter could be adjusted. In ail the trials except in trial 8, the gases were spilled vertically downward 20 cm onto water. In test 8, the gas was spilled 10 cm below the waterline in order to observe how the water and the liquefied gas mix together and how the gas then vaporizes to the atmosphère.

Instrumented masts are immersed into the basin, equipped with température sensors (thermocouples) at différent depths. A line of thermocouples floats at the water surface in order to detect a surface pool of spilled gas and its évolution (Figure 2). Three instrumented aboveground masts are placed downwind of the basin between 5 m and 11m from the release point on a quite flat and open area, for measuring some properties of the dispersing cloud: gas concentration, température, velocity and turbulence level from 20 cm up to 3 m above the ground (Figure 3). The concentration measurements are made by means of a O 2analyserwith regular sampling of the atmospheric cloud. Several caméras are immersed into the basin to visualize the interaction between spilled gas and water. AH trials hâve also been filmed with a normal caméra and an infrared caméra.

As wind direction is quite variable on the test site, two large fans hâve been placed upwind of the basin in order to channel the atmospheric flow towards the flat measurement area.

Release point

Release pipe

Storage tank The characteristics of ail trials are given in Table 1. The indicated release pressure values give an estimation of initial pressures in the storage tank. It appeared in the trials that the pressure dropped significantly during the release, especially for the highest spill rate, since the gas vaporization into the tank could not be fast enough to compensate the decreasing volume of gas in the tank. Thus the estimated release rate given in the table is a mean value.

The four trials (No. 17 to 20) with pentane are not discussed in this paper.

Observations and analysis

An first analysis of the test results is discussed in the next paragraphs, mostly on the basis of trial No.7 (LN 2 spilled at a rate of 0,9 kg/s).

Interaction liquefied gas-water

Water surface température data from trial No.7 show a significant température decrease at water surface close to the release point (approximately -8°C at TC 6, Figure not shown). The other thermocouples do not measure température variations higher than 1 °C. Thèse variations are negligible compared to the boiling température of liquid nitrogen close to -200°C. Thus it is probable that evaporation does not take place at water surface but into the water. By direct observation with traditional video caméra (Figure 4b), one sees a plume produced at the water surface inside a circle from approximately 1 m to 1,5 m of diameter.

The image extracted from the thermal imaging caméra (Figure 4a) confirms that the water surface température shows negligible variations out of this "circle of émission". One can also notice that the order of magnitude of the température of the emitted vapors roughiy deduced from this thermal video is -50°C and the order of magnitude of the température of the cloud when reaching the first mast is between 0 and 10°C. Thèse values are rather close to those extracted from the analysis of the measurements (not shown). Underwater caméras did not allow perceiving the présence of a pool of nitrogen at the water surface further than 1 m from the release point.

Figure 5 shows snapshots -in a vertical eut -of penetrating jets of liquefied gases into the water. In trial No.7, the penetrating jet of nitrogen produces a mixing cône with a depth of about 75 cm and a diameter of about 40 to 50 cm at water surface. The pénétration depth appears to be correlated to the release rate.

Nitrogen-20mm

Nitrogen-16mm C3H8 -9mm C4H10 -9mm H : 75cm H : 30cm H : 25cm H : 50cm Q : 0,9 kg/s Q : 0,5 kg/s Q : 0,5 kg/s Q : 0,7 kg/s Thèse pictures suggest that this intense underwater mixing leads to the important vaporization of liquefied gas with bubbles rising to the surface. We can believe that a part of the neighboring water cooled by the strong vaporization is also entrained by the bubbles rising to the surface. This current of cooler water at the surface could explain the slight decrease of température (less than 1°C) detected by surface thermocouples out of the "circle of émission" previously discussed. As the base of the atmospheric cloud observed above the basin has a size comparable to the base of the vaporization cône under the waterline, we can deduce that most of the heat transfer and the vaporization process take place in the mixing cône underwater. This strong underwater mixing let suppose that some water droplets would be entrained into the vapor cloud in the atmosphère.

Short-range atmospheric dispersion

We first focus on the température in the atmospheric plume. On the one hand we tried to evaluate the local density of the cloud to assess the incidence of the gravity forces in the dispersion process. On the other hand we examined the nature of the mixture (adiabatic or not) between the vapors from the basin and the atmosphère. In a previous study, the degree of adiabaticity of a plume which is formed above an evaporating pool was studied. It appears that the évolution of température in the plume is very mainly induced by the mass transfer between vapor and atmosphère rather than by heat transfer with the ground. Consequently it seems reasonable to consider that the mixture vapor/ambient air occurs in an adiabatic way, i.e. the enthalpy is preserved. With this assumption, one can obtain an approximate mathematical expression for the mass concentration C of spilled gas in the plume, depending on the measured température and the water content in the atmosphère. It reads:

C = [C p . air (T amb -T m ) + L H 2oxf H 2o]/[C p . g (T m -T ge )] (1 )
where C: mass concentration of spilled gas (N 2 ) Cp-air, C p . g : respectively, heat capacity of air and spilled gas T m . T ge , T amb : respectively measured température in the cloud, température of gas when emitted from the pool, ambient température L H2 o : latent heat for (atmospheric) water f H 2o: mass fraction of condensable water in the atmosphère.

The température of the vapor emitted from the pool, T ge , can be estimated from the measurement of gas concentrations in the cloud at the first mast. The estimated value of T ge is then applied to ail data at aerial masts. Figure 6 présents the comparison between the concentrations deduced from formulation (1) and measured by means of O 2 -analyser for trial No.7. They appear to be fairly consistent once the température T ge well estimated. The estimated values of T ge are given in Table 2. It appears that T ge can be very différent from the boiling température. This confirms that the boiling transition occurs essentially in the underwater mixing zone and not at the water surface, as explained before. In trial No.8, where the release happens below the waterline, the value of température T ge of the emitted gas at the water surface is significantly higher than températures obtained for trials No.6 and 7. This is consistent with what was expected since the underwater mixing with water is strongly increased in this case. The comparison of émission températures for trial No.6/7 and No.9 indicates that the deeper the jet pénétrâtes into the water, the lower the émission température is. We can also notice that the température of -45°C obtained for Trial No.7 are in good agreement with the thermal infrared image presented in Figure 4a.

Table 2: Properties of gas cloud and atmosphère for trials No. 6, 7, 8, 12, 13, 14, 15, and 16. T ge : température of the gas emitted from the pool, deduced from comparison between thermocouple measurements and concentration measurements, T bO iiin g : boiling température of considered material. *release below the waterline. Thermocouples measure at a very higher frequency than the O 2 -analyser. Consequently the analysis of the behavior of the cloud is based on the data from thermocouples converted to nitrogen concentrations -based on the estimation of T ge and formulation (1). From trial No.7, we can observe from thèse mean concentrations (Figure 7) that at mast M1 the cloud of nitrogen is highly concentrated in the first meter above the ground and then concentration decreases rapidly with height, whereas further at masts 2 and 3 the concentration decreases more regularly with height. This highlights the decrease of the density in the cloud with the distance to the source: the cloud is dense close to the source due to its low températureand also water condensation -and then is gradually heated by the ambient air entrained. From wind speed and turbulence data measured in the cloud (not shown), it arises that no réduction of ambient vertical turbulent mixing or wind speed modification due to the présence of the dense cloud can be seen at this scale. Thèse effects should be visible with larger clouds as it had been clearly observed in Burro and Falcon experiments [START_REF] Koopman | Lessons learned from LNG safety research[END_REF]. We also suspect that thèse effects could hâve been masked by the quite high level of ambient turbulence induced by fans used to guide the wind flow.

Finally, Figure 8 shows the visible plumes from nitrogen, propane and butane spills. Thèse images and the measured concentrations (not shown) confirm that it cannot be assumed that the flammable région of a cloud should necessarily be included in the visible part of the cloud. Naturally, the visible part of a cloud dépends on the cloud température and the dew point of atmospheric water, which is related to ambient température and relative humidity. For example, the butane spill in trial No. 14 (Figure 8c) leads to no visible plume whereas measured concentrations at mast M1 are higher than the lower flammable limit of butane (1,3 %v/v). In this case, it appears that emitted vapours are close to 0°C whereas the dew température is around -0.5°C (cf. Table 2): atmospheric humidity cannot condense when mixed in the cloud, so the flammable cloud is not visible.

Conclusions

This medium-scale experiment allowed validating a measuring setup that permits to assess the behaviour of spilled hazardous liquid/liquefied gases over water. Only the spills of liquefied gas hâve been discussed. It appears that most of the gas vaporization occurs below the water line in the mixing cône produced when the jet pénétrâtes vertically into the water. The température of the emitted vapors at the water surface could be estimated and is shown to be significantly différent from the boiling point in some cases. Thus the dispersing cloud may be less cold and less dense than it would be expected for a cloud vaporizing from a still pool spreading at water surface. The physical process observed in our experiment is not taken into account by common conséquence models, which usually considerthat liquefied gas form a boiling pool at water surface. We thus expect that thèse conséquence models used for safety studies would give conservative results when mixing under water is important, since it can be expected that a lighter cloud would lead to shorter safety distances. Anyway, it is clear from the atmospheric measurements that the cloud is very dense close to the source, even though the ambient flow (turbulence and mean wind velocity) has not shown to be disturbed by the présence of the cold cloud of nitrogen -used as a substitute to LNG in the experiments -at this scale. Otherwise it was verified that the visible part of a cloud and its flammable part do not usually match, as the size of the visible cloud strongly dépends on cloud and air températures and relative humidity.

Globally, thèse experiments suggest that release conditions -over water, under water, impinging water surface,...-strongly détermine the actual hazardous areas.

Thèse trials can be seen as a first expérimental step towards large-scale experiments of liquefied gas releases at sea needed to assess the behaviour of large hazardous dense clouds over water and to validate accidentai dispersion models.

5.

FigureFigure 3 :

 3 Figure 1 : Expérimental device

Figure 4 :

 4 Figure 4: Images extracted from (a) thermal and (b) traditional videos of trial No.7

Figure 5 :

 5 Figure 5: Underwater mixing cônes for différent test cases. H: cône depth -Images extracted from trials No.7, 9, 13 and 16 respectively.

Figure 6 :

 6 Figure 6: Comparison of the averages of nitrogen concentrations measured and deduced from formulation (1) for trial No.7

Figure 7 :

 7 Figure 7: Calculated mean N 2 concentrations at the three atmospheric masts for trial No.7.

Figure 8 :

 8 Figure 8: Comparison of the atmospheric plume sizes for three test cases. Images extracted from trials No.7 nitrogen (a), 13 propane (b) and 14 butane (c).

Table 1 :

 1 List of trials and release conditions, where T wat e

r : ambient water température, T air : ambient air température, RH: ambient relative humidity, T dew : dew point température, P in it: estimated initial release pressure, M spi |i ed : mass of material spilled, d nozz i e : nozzle diameter, At: spill duration, Q me an: mean release rate. *release below the waterline.

Acknowledgements

The research presented hère was performed under the French research project GALERNE, which was partly financed by the French Agence Nationale de la Recherche.


D Calogine
email: didier.calogine@ineris.fr

S Duplantier
email: stéphane.duplantier@ineris.fr

Estimation of pollution by fire extinguishing water

Introduction

Industrial fires may be violent and significant and involve the combustion of large quantities of substances which could be hazardous. Therefore the environmental impact could be significant. Water is mainly used to extinguish industrial fires. Water is deployed to capture the energy released by combustion and consequently the water will be loaded with pollutants such as HCI and HCN. The project O2FEU is interested in analysing the life cycle of water in industrial fire fighting. This water présents a source of pollution for soil and subsoil. The project consists in studying the fate of run-off waters in environment using mathematical models. The aim of this work is the development of a mathematical model. This model will be used to détermine the infiltration of the contaminated water in soil.

Statement of the problem

Industrial fire fighting is usually based on the use of water. This water, in contact with smokes and toxic products of combustion, is loaded with toxic substances before seeping through the soil.

The knowledge of impact of extinguishing waters on environment is necessary considering the important number of industrial fires. In this work the infiltration of run-off waters in soil is considered. So mathematical modelling of infiltration in porous média is introduced. The soil is represented by the unsaturated zone and the saturated zone. Experiments were conducted to détermine the concentration of pollutants in the extinguishing waters. This data is necessary for the numerical simulation. Various charge and discharge curves of pollutant concentration are obtained from the collect of extinguishing water. Thèse initial concentrations will be used as référence source terms for the input of infiltration model. The experiments consist in producing wood fire containing two types of pollutants: PVC in powder (

Expérimental setup (left) and Variation ofchloride concentration in water versus time (right)

The gaseous effluents are analyzed and the extinguishing waters are collected to détermine their composition. The concentrations of chloride and sulphur were around 250 mg/l and 200 mg/l in extinguishing waters, respectively. Due to the significant concentration of pollutants the development of the mathematical model proves to be necessary.

Infiltration problem

The model is based on the transport équations in porous média. The model takes into account the rétention and biodégradation of the pollutants in soil. The model uses spécifie numerical resolution adapted for the transport équations. A simplified model of soil is used representing the unsaturated and saturated zone of the soil.

•

Computation domain

The équations used to simulate the infiltration of extinguishing waters in soil are the following:

• Unsaturated Zone: The Van-Genutchten model was used as it takes into account the variation of water content in soil [START_REF] Van Genutchen | A closed-form équation for predicting the hydraulic conductivity of unsaturated soils[END_REF]. According to the Van-Genutchen model, water content 6(h) is given by:

r (2.1)

Where 0 R = the residual moisture content û s = the moisture content at saturation a, n = empirical parameters

The transport équations are summarized by the following System [START_REF] Batu | Applied flow & soluté transport modelling in aquifers: Fundamental principles, analytical and numerical modelling[END_REF][START_REF] Richards | Capillary conduction of liquids through porous médiums[END_REF]:

dH dz (2.2) (2.3) (2.4)
This model is composed by the soluté transport équation (2.2), the Darcy équation (2.3) and the Richards équation (2.4).

In équations (2.3) and (2.4), the Mualem-Van Genutchen model used for the hydraulic conductivity K(O) is given by the following relation:

K(0) = K s Se(ôf [l -(1 -Se Vm ) m f (2.5)
Where K s Se the conductivity at saturation the effective saturation m a rétention parameter / = a coefficient of pore connectivity In équation (2.2) the addition of the coefficient fîallows to take into considération the delay due to the absorption phenomena in soil. So this équation computes the concentration of pollutant and takes into account the dispersion, convection and biodégradation of the pollutant in porous média.

• Saturated Zone:

The soluté transport équation is given by:

dt~ L dx 2 T dz 2 O s dx 9 S (2.6)
where D L and D T are the longitudinal and transversal diffusion coefficients.

Regarding boundary conditions, the relation C(x;z = 0;t) = C 0 (x;t) is adopted at z = 0.

Considering the sprinkling of fire zone by firefighters, boundary conditions at the surface of the soil is modelled by echelon-type solicitations. Three stages are considered. The first corresponds to situation where extinguishing waters contained pollutants. The second corresponds to situation where fire watering continues, but water does not contain pollutant anymore and the third corresponds to situation where sprinkling is ended.

The numerical method used a non-oscillatory scheme for the discretization of équations [START_REF] Harten | Uniformly high order essentially non-oscillatory schemes[END_REF].

Results

The software introduces user's interfaces for the model parameters. Various outputs are proposed: location of the peak of concentration and contours of concentration in 2D.

Local variation of concentration versus time (left) and Variation of (C/Co) max versus parameters Ks and 0 s (right)

Performed simulation allows good estimation of flow and pollutant transport in soil. First sensibility analysis has been performed for the model regarding conductivity and moisture content at saturation.

Perspectives

The developed model represents a practical, simple and quick tool for the estimation of pollution generated by tire extinguishing water. In case this tool predicts a high pollutant concentration, the use of 3D model proves to be necessary to refine calculation but it requires more calculation time. Improvements are necessary to reduce calculation time; the implementation of implicit numerical scheme is under way of implementation. The modelling of transport of pollutants in the soil is an over-defined problem. Moreover, some parameters are hardly measurable. To compensate for this problem, modelling with uncertain parameters could be a solution. Based on Mikhailov works [START_REF] Moreno | On the numerical simulation of uncertain parameters in a radionuclide transport model[END_REF], the introduction of parameters known a priori in form of distribution could be used to détermine probable concentration as a resuit of the model.

Conclusion

To estimate the impact of fire extinguishing waters a model was developed. It is composed of two sub models. The first one, based in expérimental results, is dedicated to the calculation of pollutant concentration in run-off water. The second is dedicated to the modelling of water and pollutant infiltration in the ground. The infiltration model is based on a combination of classical équations for infiltration and transport in the unsaturated and saturated médium. It was implemented using a non oscillatory scheme. The first results are in good agreement with the literature data. The global model should be used for conséquence assessment in risk estimation of a variety of industrial sites as well as in emergency situations. Therefore, it requires to be usable without thorough data search. The next step is the development of a database of model parameters to be used in most typical situations. Future developments also concern the management of uncertainly with the capacity to estimate probabilistic distributions of concentration at various stages of the simulation.

T+at

  Plan of numerical resolution (left) and Side view of concentration in the soil (right)


Nicolas Creusot
email: nicolas.creusot@ineris.fr

Nathalie Tapie

Karyn Lemach

Patrick Balaguer

Emmanuelle Maillot-Maréchal

Jean-Marc Porcher

Hélène Budzinki

Selim Aït-Aïssa

Combined use of passive sampling and in vitro bioassays for the detection of emerging pollutants in surface water

Introduction

River systems are contaminated by various chemicals, including classical ones such as PCBs, PAHs, pesticides or alkylphenols as well as emerging pollutants such as pharmaceuticals compounds, personal care products (PCPs), steroid hormones and their metabolites. A number of these substances are described as endocrine disrupting compounds (EDCs), through their ability to modulate the synthesis, the secretion, the transport, the excretion or the binding of endogenous hormones [START_REF] Kavlock | Research needs for the risk assessment of health and environmental effects of endocrine disruptors: A report of the US EPA-sponsored workshop[END_REF]. In vitro bioassays based on mechanisms of action at receptor level are commonly used as rapid, sensitive and quantitative biological methods to assess the occurrence of EDCs in aquatic systems. While estrogen (ER) and aryl hydrocarbon receptors (AhR) activities are well documented [START_REF] Kinani | Bioanalytical characterisation of multiple endocrineand dioxin-like activities in sediments from reference and impacted small rivers[END_REF], there is still a lack of information on other nuclear receptor-related activities in the environment. Nonetheless, xenobiotic-sensing receptors [e.g. pregnane X receptor, PXR] or steroid receptors [e.g. androgen (AR), glucocorticoid (GR), progestagen (PR) receptors…] have been shown to be activated by environmental ligands like pesticides and pharmaceuticals. Hence, monitoring EDCs by using a pane l of complementary bioassays should increase the detection capacity of EDCs and allow a better hazard assessment by establishing toxicity profiles.

A number of emerging compounds are polar and occur mainly in surface water at very low concentration. Their contamination levels are function of the season and can fluctuate with episodic pollution. Until recently, the monitoring of environmental contaminants in water use discrete sampling but it gives an incomplete picture of the contamination level. Thus passive samplers giving an integrate picture in the time were developed (review in [START_REF] Vrana | Passive sampling techniques for monitoring pollutants in water[END_REF]). Among them the Polar Organic Compounds Integrative Sampler (POCIS) allow the sampling of pesticides, steroids and pharmaceuticals.

In this study, we report a multi-receptor approach based on a panel of different reporter cell lines (Table 1) to establish toxicity profiles in environmental samples and its combined use with passive sampling (POCIS) for the detection of polar emerging compounds in freshwater systems. By using this approach, we newly report the occurrence of steroid-like activities in the water phase but not in sediments at a river site that is subjected to pharmaceutical industry effluent and where development abnormalities in fish were noted. Mineralocorticoid (MR) HG5LN-hMR (GAL4RE-Luc/GAL4-hMR) Aldosterone (10 nM) Steroids Pharmaceuticals? [START_REF] Molina-Molina | Steroid receptor profiling of vinclozolin and its primary metabolites[END_REF] Progesterone (PR) HG5LN-hPR (GAL4RE-Luc/GAL4-hPR) R5020 (100 nM) Steroids Pharmaceuticals? [START_REF] Molina-Molina | Steroid receptor profiling of vinclozolin and its primary metabolites[END_REF] Table

Materials and methods

In vitro

1: In vitro bioassays based on reporter cell lines used in this study

POCIS property and calibration:

In order to calculate time weighted average (TWA) aquatic concentrations of studied compounds, Rs were calculated for several pollutants. To this end, POCIS (pharmaceutical configuration) were exposed to standard chemicals under controlled conditions in laboratory [START_REF] Togola | Development of polar organic integrative samplers for analysis of pharmaceuticals in aquatic systems[END_REF]. In each experiment, the water from the calibration system and the sorbent from the POCIS samplers were regularly analyzed for target analyte concentration in order to evaluate the sampling kinetics of the passive sampler.

Study sites and sample preparation:

The studied river is under mixed anthropogenic pressures (i.e. industrial and urban). Three different stations were sampled: the stations A, B and C were located upstream (A), 0.2 km downstream (B) and 1 km downstream (C) from a pharmaceutical industry effluent. Moreover, a municipal sewage treatment plant (1,000 eq-inhabitants) is located between stations B and C. In July 2009, sediments were sampled, sieved, freeze-dried and then 5 g were extracted (ASE) by heptane:acetone mixture (50:50). Extracts were evaporated to dryness and r edissolved in 1 mL of MeOH. POCIS were deployed from June to November 2009 (6 sampling campaigns of one month each). After exposure, each POCIS was rinsed with ultrapure water. The sorbent was transferred into glass SPE tube by rinsing with ultrapure water and then dried for 1h. Extraction of organic compounds was done with sequential elution with 10 mL of dichloromethane, 10 mL dichloromethane:methanol (50:50) and 10 mL of methanol. POCIS extracts were evaporated to dryness and redissolved in 200 µL of methanol. Methanol organic extracts were then used for bioassays.

Results and discussion

In sediments, weak estrogenic activity (E2-EQ = 0.05-0.3 ng/g), weak persistent dioxin-like activity (TCDD-EQ = 1-7 ng/g) and moderate non-persistent dioxin-like activity (BaP-EQ = 2-10 µg/g) were found at the three sites whereas no AR, (anti)-AR, GR, and PXR activities were found.

In POCIS, summer samples presented weak PAH-like activity (0.58-0.86 µg/g) and s trong PXR and E R activities were found at the tree stations, with the highest activities at the upstream station A. Interestingly, very strong GR activities were recorded at site B (Dex-EQ = 61 µg/g) and C (Dex-EQ = 69 µg/g) but none at site A located upstream from the pharmaceutical industry effluent. In addition, anti-MR, anti-PR and AR activity were also found downstream from the effluent hence suggesting a release of polar steroid compounds in the water phase during summer. Samples from the other campaigns are under analyses and results will be presented.

Conclusions

The combined use of POCIS and in vitro bioassays allowed detecting GR, AR, anti-MR and anti-PR activities downstream from a pharmaceutical industry. Such response profile strongly resembles that of certain classes of steroid drugs. Further in vitro bioassays are under progress to assess the seasonal fluctuation of these activities. Moreover, chemical analyses on POCIS extracts are under investigation in order to identify the substances responsible for activities.

Bioassays:

	Receptors	Cell lines (principle)	Reference ligands (EC50)	Examples of environmental ligands	Ref.
	Estrogen (ER)	MELN (MCF-7, ERE-LUC)	17β-E2 (0.01 nM)	(Xeno)Estrogens, Pharmaceuticals, PCPs	[4]
	Dioxin (AhR)	PLHC-1 (EROD induction)	TCDD (0.1/0.07 nM)	PAHs, PCBs, PCDD/Fs...	[2]
	Pregnane (PXR)	HG5LN-hPXR (GAL4RE-Luc/GAL4-hPXR)	SR12813 (70 nM)	Pesticides, Pharmaceuticals, Steroids, Plasticizers	[5]
	Androgen (AR)	MDA-kb2 (MDA-MD-453,MMTV-Luc)	DHT (0.1 nM)	Steroids, Pharmaceuticals	[6]
	Glucocorticoid (GR)	MDA-kb2 (MDA-MD-453,MMTV-Luc)	Dexamethasone (100 nM)	Steroids, Pharmaceuticals Vinclozolin	[6]


Julia Mouton

Karine Tack
email: karine.tack@ineris.fr

Sébastien Denys

Antimony-polluted soils risks assessment with special concern around speciation and bioaccessibility aspects

Introduction

Antimony is potentially toxic at very low concentrations and has no biological functions. Toxicity of Sb is strongly linked to its speciation. For instance Sb (III) was shown to be ten times more toxic than the form (V). For Sb contaminated soils, the exposure assessment of Humans is a crucial step. The exposure will depend on both the media (soil, water or air) from which the Humans are exposed and the route of exposure (inhalation, ingestion, dermal contact). In the case of polluted soils, direct ingestion is considered as the major exposure route. In this case, the total concentration is commonly characterized. However it does not reflect the actual concentration that is absorbed through the gastro-intestinal (GI) tract, i.e. the bioavailable concentration. The bioaccessibility research group of Europe (BARGE group) set an in vitro model (UBM test) to determine the fraction of pollutant potentially extractable during digestive processes, i.e. the bioaccessible fraction. Aims of this study were to combine bioaccessibility and speciation measurements to improve the human health risk assessment of Sb contaminated soils. If the concern of speciation as another tool for polluted matrixes characterization is growing (1), only few literature exists around pollutants speciation during the digestive processes [START_REF] Denys | Bioaccessibility of lead in high carbonate soils[END_REF][START_REF] Oomen | Lead speciation in artificial human digestive fluid[END_REF]. This research is devoted to the study of antimony species evolution during the UBM test, and aim to integrate this approach to polluted soil risks assessment.

Materials and methods

Six soils were used in this study: two certified reference materials (IAEA-356 obtained from the international atomic energy agency), PACS-2 obtained from national research council of Canada), two reference materials (IPL1 and MIX1 obtained from the Pasteur Institute of Lille in France), and t wo industrial soils named Soil-A and Soil-B sampled from a mining site (Glendinning, Scotland). Bioaccessibility measurements were performed using the Unified Barge Method (UBM) [START_REF] Denys | Bioaccessibility of lead in high carbonate soils[END_REF][START_REF] Denys | Bioaccessibility, solid phase distribution, and speciation of Sb in soils and digestive fluids[END_REF]. This procedure simulates the chemical digestive process taking place in mouth (five minutes), stomach (1 hour) and s mall intestine (4 hours) compartments within synthetic digestive fluids. The temperature in maintained at 37°C and the UBM test is applied to 0.6 g of soil. After the complete digestion procedure, samples were centrifuged at 3 000 g for 5 minutes. The supernatant was then sampled and analyzed for total Sb and Sb species. Sb species analyses were carried out within 24 hours following the test by HPLC-ICP-MS according to Zheng et al [START_REF] Zheng | Antimony speciation in environmental samples by using High Performance Liquid Chromatography coupled to Inductively Coupled Plasma Mass Spectrometry[END_REF], while total Sb analyses were performed by ICP-MS within the week following the test after having conditioned samples with 2% HNO 3

Results and discussion

according to AFNOR protocol (6). The UBM test was engaged on soil samples and on Sbspecies spiked solutions at different concentrations. All experiments were conducted in triplicates.

Bioaccessible antimony species from polluted soils

When applied on polluted soil samples, the amount of bioaccessible antimony was not shown to be correlated to the initial concentration of total Sb in the soil. Even if low bioaccessibilities were measured in real polluted soil samples, this information should be strongly considered. Indeed, for the soil A, for example, with the lowest bioaccessible percentage of Sb, the concentration of SbV reached 166±1 µg/L in the gastric juice and 4681±22 µg/L in the gastro-intestinal juice, while it was around 19.3±0.4 µg/L and 53.9±7.8 µg/L for SbIII respectively for the same fluids. These values were translated as the accessible dose for organism absorption via the soil ingestion (Table 1). The concentration of bioaccessible antimony measured with the UBM test can allow calculating the average daily accessible dose (ADAD) for absorption. 10 kg was used as the reference to the official minimal weight of a 1 year child [START_REF] Mathews | Healthy children : a guide for child care Marrickville[END_REF], that represents the worst scenario in case of soil ingestion by a 1 year old child. In this context, the toxicological reference values for total antimony can be used as reference values to appreciate the risk associated to children exposure to such kind of polluted soils due to the fact that no toxicological reference value exits for Sb(III) nor Sb(V).

Soil

Stability of Sb species in simulated digestive media for bioaccessibility measurements

) (RfD = reference dose and ADI = acceptable daily intake)

SbV specie was measured in the UBM fluids, while some SbIII was transformed in SbV when the UBM test was applied on S bIII-spiked solutions. This transformation seemed to take place during the entire gastrointestinal phase while no changes were observed during the single gastric phase. The part of SbIII transformed to SbV appeared to be dependant of the initial SbIII concentration in digestive fluids and could vary from 93% to 3% respectively for initial SbIII at 85 and 42 000 µg/L (Figure 1). A maximal concentration of SbIII transformed in SbV appeared around 550 µg/L in the gastro-intestinal fluid. The kinetic was fast (30 minutes) in the gastro-intestinal juice.

Conclusions

The UBM test is a quite fast and very simple in vitro test to express metallic pollutant bioaccessibility in case of soil ingestion. This test seems well appropriate for polluted soil characterization in terms of risks assessment. When coupled to speciation analysis, it offers a deaper understanding of real pollutant species that infants can be exposed to.

Figure 1 :

 1 Figure 1: SbV concentrations measured in the gastro-intestinal fluid after the digestion of SbIII-spiked solutions

Table 1 : Comparison of bioaccessible antimony concentration expressed as the interne dose accessible to a 10 kg child in case of soil ingestion to toxicological reference values (µg.kg -1

 1

		SoilA	SoilB	SoilA	SoilB
	Digestive phase		Gastric	Gastro-intestinal
	RfD (µg/kg/d) (USEPA)		0.4
	ADI (µg/kg/d) (WHO)		6
	SbV (µg/kg)	0.37	10.5	6.54	69.0
	SbIII (µg/kg)	0.04	0.03	0.32	1.34
	Total Sb (µg/kg)	0.56	9.97	8.46	74.0


Alain Devaux
email: alain.devaux@entpe.fr

Christian Gillet

Luc Fiat

Raphaël Santos

Wilfried Sanchez

Sylvie Bony

Functional significance of genotoxicity in fish germ cells

Introduction

Aquatic ecosystems are commonly considered as the ultimate receptacle for anthropogenic compounds. Recent works show that about one third of them could have genotoxic properties. If some great progress have been made in understanding the implications of genotoxin exposure to human health through water use, a huge gap remains in our understanding of the impacts on aquatic species. Indeed, DNA damage has been used since quite a l ong time as an ex posure biomarker in aquatic ecotoxicology, but the functional significance of this damage is still poorly understood, in particular in fish.

Genotoxins are prone to affect offspring directly or indirectly through the reproductive process. All this could influence the recruitment rate and hence the population dynamics [1]. However, assessment of the ecological risks associated with environmental genotoxic exposure is usually based on individual responses. Thus, there is a n eed for a bet ter understanding of the long term and pop ulation level implications of genotoxic insults in fish.

While low levels of DNA damage in somatic cells and oocytes can be efficiently repaired, mature sperm cells, i.e. spermatozoa, are susceptible to accumulate damage due to their lack of repair capacity. The present work aims to track the transfer of toxic effects across generations by studying the link between the level of DNA damage in fish sperm after parental exposure to the genotoxic compound methyl methane sulfonate (MMS) and the rate of development abnormalities measured in the offspring.

Three different fish species were chosen based either on their ecological importance or on their reproduction behavior, respectively brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus) and threespine stickleback (Gasterosteus aculeatus).

Materials and methods

A first experiment was carried out on 2 years old male brown trout and 3 years old male Arctic charr reared at the INRA hatchery (Thonon les Bains, France) in outdoor tanks continuously supplied with 36m deep water from Lake Geneva. Animals were fed daily ad libitum with dry pellets. Different modes of exposure to MMS (through water, food or intraperitoneal injection) were tested in preliminary experiments, and finally intraperitoneal injection was found to be the most effective (data not shown). Thus, male fish (n=31 trouts, n= 28 chars) were injected intraperitoneally with MMS previously dissolved in coconut oil. Exposed fish received 50 mg MMS / kg fresh body weight, control receiving vehicle alone (trout n=16, charr n=18). After 3 weeks of exposure DNA damage was measured in mature spermatozoa through the comet assay. Aliquotes from one egg pool obtained from nonexposed females were fertilized by each sperm of both control and exposed males. The rate of abnormalities development was then measured in the resulting offspring at various embryonic stages (neural plate, eyed stage) and larval stages (hatching, yolk resorption, swimming stage), as well as the survival during the first month post yolk resorption.

A second experiment following the same protocol is currently in progress in stickleback and will be presented.

Main results

Sperm DNA damage

After 3 w eeks of MMS exposure, a significant increase in sperm DNA damage was measured using the Comet assay reaching in average a 30% tail DNA value in both species (10 fold increase in trout and 20 fold increase in charr compared to the control, respectively). However fertilization rate was not affected by MMS treatment of male fish since it ranged in average 95% in trout and 84% in charr, whatever the treatment.

Embryo and larval development

Embryo abnormality rate clearly increased in offspring stemming from MMS exposed male fish but remains at these development stages to a low value (for example, 0.6% in treated trout versus 0.2 % in control). Male exposure to MMS led also to a large array of abnormalities when measured at later stages i.e. after hatching and in particular during yolk resorption (Figure 1). Such an effect was significant only in trout and reached 2% of abnormal larvae.

Figure 1. Different types of larvae abnormalities. A and E: yolk oedema in trout and charr respectively; B and C: trout spine deformation; D: charr jaw deformation; F: charr Siamese larvae

Skeleton development and larvae survival

If we look more accurately at the effects, bony staining reveals very frequent spine and cephalic deformation in swimming larvae stemming from DNA damaged sperm (Figure 2). It has to be stressed that those abnormalities reached around a 20 % value what is much higher than those observed at earlier stages in embryos. So it was observed that consequences of original sperm DNA damage appear to increase along with the progeny development. Trout larvae survival monitored one more month after the end of yolk resorption was 5 fold lower in larvae from MMS treated group compared to the control one. The increase in abnormality rate along the offspring development time underlines the interest to further study more integrated responses such as fish growth, survival and F1 reproductive success. Moreover, it would be interesting to evaluate both the oocyte capacity to repair spermatozoa DNA damage, and the contribution of a potential oocyte DNA damage to the reproduction impairment.

This work is a first step towards the understanding of the functional significance of fish germ cell primary genotoxic damage, revealed as a reproductive impairment.

Figure 2 .

 2 Figure 2. Spine deformation frequently observed in fish larvae

[ 1 ]

 1 . Newman M.C., Clements W.H. 2008. Ecotoxicology: a comprehensive treatment, CRC Press, 252 p. [2]. Lewis C., Galloway T. 2009. R eproductive consequences of paternal genotoxin exposure in marine invertebrates. Environ. Sci. Technol. 43: 928