Raphaël Pesche

Georges Bossis

Alain Meunier

SIMULATION OF PARTICLE SEGREGATION IN A BIDISPERSE SUSPENSION

Introduction

It is now well known that a gradient in shear rate can induce particle migration in a concentrated suspension. [START_REF] Philips | A constitutive equation for concentrated suspensions that accounts for shear-induced particle migration[END_REF] have developed a constitutive equation for monodisperse suspensions based on the different shear induced diffusion mechanisms first described by Leighton and Acrivos (1987). This equation gives the equilibrium concentration profile from a balance between the primary flux, induced by shear rate gradients, and the secondary flux coming from concentration and viscosity gradients. Their model is in fair agreement with experimental results obtained by NMR imaging in a Couette geometry. In particular they recover, at steady state, a higher concentration in the lower shear rate region.

More recently, [START_REF] Chow | Shear-induced particle migration in Couette and parallel-plate viscometers : NMR imaging and stress measurements[END_REF] have also reported a migration towards higher shear rate region in a Couette flow but they did not observe such a phenomenon in parallel plate device contrary to the prediction of the model of Philips & al. The reason is that the constitutive equation of this model doesn't take into account the flux induced by the curvature of streamlines. Krishnan & al. (1996) have reported curvature contribution for bidisperse suspensions with one of the species being present only as tracer particles. A more general model for bidisperse suspensions was proposed recently by Bossis & al (1997) considering that the trajectory of the big particles were not perturbed by the small ones.

Basic models for suspensions in a Couette flow

We consider a suspension of spheres of radius a in a Newtonian fluid with viscosity η. We assume that the Reynolds number is sufficiently small for inertial forces to be negligible. On the other hand, we suppose that the spheres are large enough for Brownian motion to be negligible i.e. Peclet number (Pe = 6πηa 3 γ/ kT) tends to infinity. The local shear rate γ is due to the cylindrical Couette flow with the inner cylinder rotating with angular velocity Ω.

Monodisperse suspension

Ω

We have to consider two different kinds of flux that cause particles migration. First, the effect of a spatially varying collision frequency in the gap between the cylinders gives rise to a flux J c :

J K a d dr d dr c c = - + 2 2 ( ) Φ Φ Φ γ γ (1)
γ is the local shear rate. K c is a proportionality constant of order unity that is found from experimental data. We can see from (1) that even with no gradient in the particle volume fraction, migration will result because of the spatially varying shear rate. The minus sign indicates that the particles will migrate from high to low shear rate. The flux J c induces a non uniform particle volume fraction and therefore a spatially varying viscosity. This gives rise to another flux J η :

J K a d dy η η γ η η = - 2 2 Φ (2)
η is the local viscosity which is a function of the local volume fraction and K η is found from experimental data.

A conservation equation can be written in a Lagrangian frame as :

∂ ∂ Φ Φ t = -∇ + .( ) v J (3)
Where J = Jη+ J c . With the condition of incompressibility and taking into account that the radial particles flux is zero on the boundaries, we must have at equilibrium :

J = 0 (4)
The solution of eq. 4 with (1) and ( 2) gives the result of Philips & al.:

Φ Φ Φ Φ ( ) / . ( ) / . . ( / ) r r R r W W W K K C = - -       - 2 2 1 82 1 1 0 68 1 0 68 η (5)
In this equation, Φ W is the particles' concentration on one wall of the cylinders with radius R W . We choose the empirical law for the relative viscosity (Krieger 1972):

η r ( ) max . Φ Φ Φ = -       - 1 1 82 (6)
Φ max being the volume fraction at which h tends to infinity, about 0.68 for hard spheres.

In equation ( 5) the quantity K c /Kη is a parameter. It was found to be equal to 0.66 by Philips & al. Noting that 1.82 (1-K η /K c ) = -1 allows to find the analytical solution of (5).

In contrast with the previous theory, migration has not been observed in a parallel plate device. In fact, we have to take into account the flux J r due to the curvature of the streamlines (Krishnan & al., 1996) :

J K a r r r = γ 2 2 Φ (7)
In a parallel plate device, the particle volume fraction is initially constant (and therefore the viscosity), so J η = 0 ( cf eq. ( 2) ) and the total initial flux is :

J = J c + J r = A(r) (K r -K c ) (8)
As no migration has been observed experimentally (J=0), we shall take K r =K c . Finally, imposing that the total flux (Eqs (1), ( 2), ( 7)) is zero, we obtain an equation for Φ including the participation of all the fluxes :

Φ Φ Φ Φ ( ) / . ( ) / . . ( / ) r r R r W W W K K C = - -       - 3 3 1 82 1 1 0 68 1 0 68 η (9)

Bidisperse suspension

In a further work we shall describe in more details the extension of Philips & al. theory for bidisperse suspensions with the addition of streamlines curvature flux. The only hypothesis is that the big particles are not perturbed by the small ones. We then rewrite the fluxes associated with the different kinds of collision and end up with two coupled equations for the volume fractions of big and small particles. In the next section of this work, we shall compare the predictions of the bidisperse model with a first numerical result we have obtained for a given ratio of sizes : a 1 /a 2 =0.2.

Numerical results

Monodisperse system

A Stokesian dynamic numerical method is used to simulate the whole system [START_REF] Durlofsky | Dynamic simulation of hydrodynamically interacting particles[END_REF]. This method takes into account multi-body hydrodynamic interactions and the effects of lubrication layers between nearly touching particles. In order to save computing time we have simulated a monolayer of particles and therefore we only consider the two dimensional motion of the particles. The translational and rotational velocities of the spheres can be related to the hydrodynamic forces and torques (F H ) exerted on the suspended spheres through the relations :

F H =R(U ∞ -U) (10)
U contains the translational and rotational velocities of the N suspended particles ; U ∞ contains the translational and rotational components of the bulk velocity that would exist at the centre of the particles in the absence of the particles.

The coefficients in the matrix R can be found in Jeffrey & al. (1984[START_REF] Jeffrey | The calculation of the low-Reynolds-number resistance functions for two unequal spheres[END_REF]. Because of the presence of the cylinders walls, we have to introduce the particle-wall interactions as in [START_REF] Bossis | Stokesian dynamics simulations of particle trajectories near a plane[END_REF].

In addition to hydrodynamic interactions we have to take into account a repulsive potential which can be related to the rugosity or ionic repulsion between particles. An important point is that this potential introduces an asymmetrical trajectory between two spheres. We choose a pairwise repulsive DLVO-type colloidal force of the form (Brady and [START_REF] Bossis | Dynamic simulation of a bidisperse system[END_REF]:

F ab = τF 0 e e - - - τ ε τ ε 1 (11)
F ab is the force on particle a due to particle b, ε is the separation between the particle surfaces, τ is the Debye length which gives the range of the repulsive force. In this work, we have taken 1/ τ = 2.2 10 -3 radius.

The amplitude F 0 is chosen in order to obtain hydrodynamic forces between particles a and b equal to F ab when the surface separation is 0.01 radius. In Fig. 2 (a), our numerical simulation is compared with an analytic solution obtained by solving equation ( 9) taking K c /K η =0.66 as proposed by Philips. The quantity Φ w is obtained from the condition that the integral of the volume fraction gives the average volume fraction. The dotted line is the prediction of Eq. ( 5) whereas the solid line is the prediction of Eq. ( 9). It appears that the inclusion of the radial flux markedly improve the model.

Bidisperse system

We apply the same numerical method with two different sizes of spheres of radius ratio a 1 /a 2 =0.2. The functions that we need to build have been studied by Jeffrey for unequal rigid spheres.

We observe a strong segregation in sizes with larger particles migrating towards lower shear rate region (i.e. the outer cylinder). The concentration profiles are plotted (solid dots for large particles and solid squares for small particles in fig. 3a). The theoretical curves obtained from our model are respectively the solid line and the dotted line for the big and the small particles. The agreement is fair especially taking into account that the only parameter of the bidisperse model is the same as for the monodisperse one (K c /K η =0.66).

Phase separation under simple shear rate

We also performed simulation of a 2 dimensional bidisperse system undergoing simple shear flow in order to determine the importance of hydrodynamic interactions. Suspended particles interact each other with a Debye-Huckel (see [START_REF] Russel | Colloidal dispersions[END_REF] repulsive force (F rep ). Each particles undergoes a Stokes drag force F η as if it were alone in the fluid. At each time step, we have :

F F rep + = η 0 (neutrally buoyant particles)
The velocity of the particles is given by :

v F e 1 = + rep a y 6π η γ
With a velocity in the x-direction due to the shear given by γy ; a is the radius of a particle and η the viscosity of the fluid.

Figure 4 shows that the system becomes stripe structured. [START_REF] Santra | Fluid-induced particle-size segregation in sheared granular assemblies[END_REF] have obtained such a phase separation taking into account inertia of particles.

Effect of hydrodynamic interactions between particles

We performed numerical simulation of the same system including a Stokesian Dynamic method [START_REF] Durlofsky | Dynamic simulation of hydrodynamically interacting particles[END_REF]. Because of the computational time increasing, we used a small number of particles. Figure 5 allows to compare the effect of hydrodynamic interactions -that make little clusters of aggregating particles and prevents the stripes formation -with the case of a single repulsive force.

Conclusion

We have developed a numerical simulation including many body hydrodynamic interactions which allows to reproduce particle's migration in a monodisperse or a bidisperse suspension placed in a two dimensional Couette flow. The concentration profiles obtained by numerical simulation have been compared to the predictions of models based on the ideas of Leighton and Acrivos. For monodisperse suspensions it appears that taking into account the flux due to the curvature of streamlines well improve the agreement of the model with the numerical simulation. In the bidisperse case for a ratio of sizes a 1 /a 2 =0.2, we observe a strong segregation with the large particles going towards the lower shear rate gradients. Our model for bidisperse suspensions fairly well predicts this behaviour without the addition of others parameters. The comparison between numerical simulations and the bidisperse model will be worked out for different size ratios and volume fractions.

Concerning the phase separation in bidisperse 2D suspension under simple shear rate, we perform now a theoretical interpretation including diffusion coefficients in order to predict the appearance of stripes depending on the radius ratio and concentration of the spheres.

Fig

  Fig.1 Couette flow geometry

Fig. 2 :

 2 Fig.2: Results obtained for a monodisperse suspension with mean volume fraction Φ = 0.5. (a) concentration profile compared with theoretical prediction ; (b) picture of half Couette system. The full spheres are those taken into account in the calculations. The others are obtained with angular periodicity. Units are in particle's radius.

  Results obtained for a bidisperse suspension with mean volume fraction Φ = 0.45 (large spheres) and 0.02 (small spheres). The radius ratio a 1 /a 2 is 0.2. (a) concentration profile compared with theoretical prediction ; (b) picture of half Couette system. Units are in large particle's radius (i.e. a 2 ).

Fig. 4 :

 4 Fig. 4 : 2000 particles, φ B =0.21, φ S =0.20 (surface concentration of big and small particles) at dimensionless time γt = 400.

  fig. 5 : Results obtained for a bidisperse suspension with mean volume fraction Φ = 0.42. (a) agregations caused by lubrication forces. (b) stripes when lubrication forces are replaced by short ranged repulsive forces.

Acknowledgement

We thank the French company SNPE for his financial support, and the IDRIS center in France who provided the computer time.


Sébastien Zamith

Pierre Labastie

Jean-Marc L'hermite

Fragmentation cross sections of protonated water clusters

We have measured fragmentation cross-sections of protonated water cluster cations

by collision with water molecules. The clusters have well defined sizes and internal energies. The collision energy has been varied from 0.5 to 300 eV. We also performed the same

colliding with deuterated water molecules. The main fragmentation channel is shown to be a sequential thermal evaporation of single molecules following an initial transfer of relative kinetic energy into internal energy of the cluster. Unexpectedly, that initial transfer is very low on average, of the order of 1 % of collision energy. We evaluate that for direct collisions (i.e. within the hard sphere radius), the probability for observing no fragmentation at all is more than 35 %, independently of cluster size and collision energy, over our range of study. Such an effect is well known at higher energies, where it is attributed to electronic effects, but has been reported only in a theoretical study of the collision of helium atoms with sodium clusters in that energy range, where only vibrational excitation occurs.

I.

Introduction

When a molecule, an atom or an ion collides with a solid target, it loses its energy in small steps through collisions with the constituents of the target. At energies above 1 keV or so, the energy loss is mainly through electronic excitation of the target. The energy loss per unit length (the "stopping power") increases as the velocity decreases and reaches a maximum, which leads to the well-known "Bragg peak" at some penetration depth. At this point, the particle loses so much energy that it may damage or otherwise modify the target. Afterwards, the projectile keeps flying, with much less energy, in the 10-100 eV range. Electronic excitation is then impossible, so that the energy loss is through "nuclear" (or "vibrational") excitation of the target. This type of excitation does not lead to mere structural modification of the target, only to some heating, which is almost not observable in bulk targets.

Collisions between clusters and molecules, atoms or ions lead basically to the same effects. High energy collisions, where electronic excitation occurs, have been widely studied, both experimentally and theoretically [1,2,3,4,5,6,7 and references therein]. In those studies, an effect specific to binary collisions, charge exchange, often occurs [1,8]. Collisions in the energy range where nuclear excitation occurs have been much less studied per se [3,9]. On the other hand, they are often used as a mean to determine the stability of complexes, as in Ref. 10. A variety of effects are expected. The impinging particle may:

(i) Kick out a constituent of the cluster: this is the mechanism of "direct" or "impulsive" fragmentation. A variant is a "direct exchange" between the projectile and one constituent of the cluster.

(ii) Transfer some energy to the cluster, which is therefore heated, thus leading to the subsequent evaporation of one or more particles: this is the mechanism of "thermal fragmentation". (iii) Attach to the cluster for some time. If this time is long enough to observe the resulting cluster, the collision is considered "sticking" or attaching. If the impinging molecule evaporates before the cluster is observed, some energy has nevertheless been transferred to the cluster and the subsequent behavior is the same as in case (ii). Another eventuality after attachment is that another constituent of the cluster is evaporated first, leading to "thermal exchange". (iv) Undergo an almost elastic collision with the cluster, which leads to unobservable consequences in our experiment (no change of the mass of the cluster).

Since in all cases except (iv), the initial process may be followed by other evaporations, the different cases can only be discriminated experimentally if one is able to detect all the collision products in coincidence. Some of the studies performed at higher energies use coincidence devices [6], but all the works we are aware of in the low energy range are based on the observation of fragmentation patterns, as in Ref. 9, 10 and 11.

In this paper, we present the measurement of absolute fragmentation cross sections of positively charged protonated water clusters induced by collisions with water molecules. The clusters have a controlled internal energy, a well-defined size, and the collision energy in the center-of-mass (CM) frame can be accurately varied from  0.1 eV to 300 eV. Fragmentation of water clusters induced by collisions with different targets has already been reported [7,12,13,14]. Dissociation cross-sections have been reported by Armentrout's group [15,16], for small (less than seven water molecules) protonated water clusters. Those experiments allow reaching very low collision energies, but lack control of the cluster temperature. In this size range, our cross section measurements are very close to theirs. We are able to reach much higher sizes, and to fix the clusters temperature, which allows a better control of the collision parameters.

The main result of our work is that in a wide collision energy range (roughly between 5 eV and a few hundreds of eV), and for roughly one third of the head-on (i.e. inside the geometrical cross section of the cluster) collisions, such a small amount of the collision energy is transferred into internal energy of the cluster, that the cluster does not fragment at all, i.e. case (iv) above is observed. The high rate of almost elastic collisions is very puzzling, since simple models of clustermolecule collisions lead to a significant energy transfer for head-on collisions. One of those models, the line-of-centers one [START_REF] Levine | Molecular reaction dynamics and chemical reactivity[END_REF] is used below to model the shape of the cross section as a function of energy.

An amazing "transparency effect" has been predicted in Ref. 3 for sodium cluster, where the projectile goes all the way through the target almost without loss of energy. While the electronic and spatial structures are very different in sodium and in water, it is possible that we observe a similar process. Actually, this effect has been reported in Ref. 6 in the case of fullerene-He + collisions at high energy, although the observation is based on the measurement of relative cross-sections, while absolute measurements would be more conclusive. In Ref. 7, the authors have studied the collision of protonated water clusters at high collision energy with He and Xe atoms and observed a transparency effect. Absolute cross sections have been measured in Ref. 9 for fullerene-rare gas collisions, and are again much lower than the geometric cross section. It should be mentioned that channeling effects [START_REF] Feldman | Material Analysis by Ion Channelling[END_REF], i.e. the almost lossless travel of an ion through a macroscopic crystal, have long been known. Those effects are very similar to Saalmann and Schmidt's [3] transparency effect.

II. Experimental setup

The experimental setup has already been detailed elsewhere [START_REF] Chirot | [END_REF]. Water clusters are produced in a gas aggregation source. Right after production, they enter a cell whose temperature can be varied between 25 and 400 K. In this cell, clusters undergo thousands of collisions with the carrier gas, which is enough to accurately fix their temperature. The clusters are then mass-selected and their velocity can be reduced, using a special electrostatic device [START_REF] Chirot | [END_REF], to reach kinetic energies as low as a few tenths of an eV in the CM frame. At this point, we get clusters with a known size, a known kinetic energy and a Boltzmann internal energy distribution at a known temperature. Those clusters then enter a 5 cm long collision cell that contains a controlled pressure of water vapor. Typical values for the water vapor pressure in the cell range from 1 to 3.10 -4 mbar in order to keep the average number of collisions below unity. In the case of water, evaporation occurs before clusters reach the collision cell when their temperature is above roughly 130K; to prevent this phenomenon, clusters' temperature has been kept below 130 K all along the experiments. These clusters undergo collisions with the water molecules of the vapor, and the products are mass analyzed in a high resolution time of flight mass spectrometer. From these mass spectra, absolute cross sections of the reaction that took place in the cell are deduced. Two kinds of reactions can be studied. First, owing to the very low collision energy, long lived products can be created, which allows measuring attachment cross sections [20,21,22]. Second, fragmentation cross sections can also be measured at higher collision energies, whose analysis is the aim of this paper.

III. Computation and calibration of fragmentation cross sections

Our raw experimental data are fragmentation patterns such as that of (H 2 O) 30 H + displayed in figure 1 for different CM collision energies. To deduce absolute cross-sections, we use a procedure close to that of Kinsey et al [23]. We recall it briefly: first, for a given incident cluster's velocity 0 v , an apparent fragmentation cross section is deduced from the mass spectra by the relation:

  l I I v app   0 0 ln ) (   (1)
where 0 I I is the ratio between the integrated intensity of parent I and the total integrated intensity 0 I , including the fragments and the parent,  is the density of the vapor in the collision cell, l is the length of this cell.

Relation (1) must then be corrected in order to account for two factors: a) the velocity distribution of molecular targets, and b) the velocity dependence of cross sections.

a) The velocity 0 v is associated to a mean relative velocity:

              a e a a a v v a  erf 2 1 erf 0 (2) with cell B lab T nk E a 
, where lab E is the kinetic energy of the cluster in the laboratory frame, n is the size of the cluster (rigorously, it should be the ratio of the mass of the cluster to the mass of the molecule), B k is the Boltzmann constant and cell T is the temperature of the vapor in the cell, which is roughly 300 K in our case. b) To account for the velocity dependence of cross-sections, the "Kinsey factor" ) , ( 0 cell T v f is introduced [23]. This factor, which depends on the shape of the function

) (v
 , is very close to 1, whenever 0 v is higher than the mean thermal velocity of the targets.

Finally, the cross-section at mean velocity v is deduced from the following relation [23]:

) ( ) , ( ) ( 0 0 0 v v T v f v v cell app    (3)
As soon as 20  a , which corresponds to an incident energy of 15 eV for a cluster of size 30, all the correction factors are very close to 1 and can be dismissed. This criterion is valid provided that the cross-section is measured for collision energies high enough above the threshold for dissociation, which is the case in our experiment.

To apply Eq. ( 1), an important parameter must be determined, namely the product of the density  and the length l. A careful determination of both parameters has been described in Ref. 20. We use the same calibration here. In particular we have checked that the quantity   0 ln I I  evolves linearly with l  for a given collision energy and deduced the cross-section from the slope. An example of such a curve is given in figure 2, for n=30 protonated clusters colliding with water molecules at a collision energy of 2.3 eV. From the slope of this curve the fragmentation crosssection is readily deduced as 125 Å 2 . Since the cross-section is deduced from the slope of such a curve, there is no need in knowing the absolute density in the cell but rather to have a correct measurement of its variation. The density  in the cell is deduced from the relation

cel B cel T k P /  
where P cel is the pressure measured in the collision cell, T cel its temperature and k B the Boltzmann constant. The pressure is simultaneously measured by an ionization gauge (Leybold Ionivac ITR 90) and a capacitive gauge (Leybold Ceravac CTR 91). The capacitive gauge provides accurate gas independent pressure variations. Of course the pressures given by these gauges might be off by an unknown amount, however, this would not affect the major conclusions of the present paper since fragmentation cross-sections can be directly compared with attachment cross-sections previously measured with exactly the same experimental setup and using the same data processing method. In particular the same gauges were used, so that any systematic error in pressure measurements would have the same effect in both experiments. Since all the attachment cross sections measured in Ref. 20 converge towards the same values at large sizes (see Fig. 9 of [20]), this common value is likely to be close to the geometrical cross section, which is calculated as follows: define an effective interaction radius

0 * R R R c  
, where 0 R is the molecular radius and

3 1 0 n R R c 
is the cluster radius , assuming that the clusters are spherical; the geometrical cross section is then:

2 * R geo    (4)
In bulk ice, the value of 0 R is 1.98 Å. In Ref. 20, a slightly higher value has been found, namely 2.17 Å for clusters in the 100  n size range. However, it is shown in Ref. 20 (see Fig. 11 of this reference) that some models could lead to a higher cross section than geometrical. We therefore adopt a conservative attitude in this paper and use a geometrical cross section given by Eq. ( 4) and a value 98 . 1 0  R Å. Doing this, the ratio of the measured cross section to the geometrical cross section might be overestimated by at most 17 %. This means that all the experimental ratios presented below are upper bounds for the actual ratios. Our point is that those experimental ratios are too low by about 35 %. Actual ratios might be even lower. Anyway, the absolute values deduced for the cross-sections compare well to values found in the literature (see section IV 1.).

One may be concerned by the influence of multiple collisions on the determination of the crosssections. Indeed, if for some collisions the energy transfer is rather inefficient, only a small portion of the collision energy is converted into internal energy of the clusters, which is just not enough to lead to fragmentation. But a second collision, still with a rather inefficient energy transfer, could induce fragmentation. Thus, multiple collisions might well artificially increase the measured cross-section. One way to ensure that one measures cross-sections in single collision conditions is to extrapolate the cross-sections measured at different pressures to zero pressure value. Equivalently, as already mentioned, we have checked that the quantity

  0 ln I I 
evolves linearly with the density in the collision cell (see Fig. 2). In any event, multiple collisions will induce a measured cross-section higher than it is actually.

Another issue with the way we compute 0 I is that we suppose that all the charged fragments are detected, so that the sum of all the detected intensities is the same as the intensity of the parent cluster entering the cell. This might not be so if either some fragment gets undetected because it has been deflected away, or if a small charged fragment is ejected, which cannot be detected. Another way to measure 0 I , which is more experimentally demanding, is to take the intensity of the parent cluster when there is no gas in the cell, and compare it with the intensity I at a given pressure in the cell. For this to be done, a reference mass spectrum at zero pressure must be recorded before and after each fragmentation spectrum. The results obtained are the same as previously, which indicates first that our measurements using Eq. ( 1) are valid, and second that the main evaporation channel is, unsurprisingly, the loss of small neutral monomers.

IV. Experimental Results

Fragmentation cross section

The fragmentation cross sections have been measured for both protonated water clusters (H 2 O) n H + impinging on H 2 O water molecules and deuterated heavy water clusters (D 2 O) n D + impinging on D 2 O heavy water molecules. There is no major qualitative difference between both cases. The ratios of the fragmentation cross sections to the geometric cross sections First of all let us compare our results with some available data from the literature. In ref. 15, Armentrout and coworkers report a fragmentation cross-section of 49 Å 2 for the collision of (H 2 O) 5 H + with Xenon atoms at 20 eV collision energy. In the present study, we find a cross-section of 55 Å 2 for (D 2 O) 5 D + clusters colliding with D 2 O molecules at 100 eV collision energy. In [10] the authors give a value of about 70 Å 2 for the collision of Cd 2+ (H 2 O) 10 in collisions with Xenon between 1 and 6 eV. This value compares rather well with the present results on (D 2 O) 10 D + since we measure a cross-section of 70 Å as well. Of course we are aware that the comparisons are not strictly correct (different targets, H/D difference). Nevertheless we believe that the agreement with previously measured crosssections tends to validate the present measurements.

The most noticeable feature in the evolution with collision energy of the fragmentation of water clusters is that, even at collision energies of hundreds of eV, the fragmentation cross section never reaches the geometrical cross section. Up to about 5 eV, the fragmentation unsurprisingly increases with collision energy, although the large size dependence of the curves is hard to interpret. For higher energies, the ratio At larger collision energies, the fragmentation cross section even decreases. This means that 40 % [respectively 35%] of D 2 O [respectively H 2 O] molecules that directly encounter D 2 O [respectively H 2 O] clusters (those inside the geometrical cross section) get out of the collision process without losing a noticeable amount of energy. Actually, the statistical model presented below shows that less than about 1 eV is deposited inside the cluster if no fragmentation occurs. This kind of fragmentation behavior, characterized by a maximum in the fragmentation probability plotted as a function of collision energy, has been also observed, in a comparable energy range, for the fragmentation of positively charged fullerenes induced by collisions with Ne and Ar [9]. However, the observations of ref. [9] are different from ours. First, in C 60 + +Ar collisions, the maximum fragmentation cross section reaches, within experimental accuracy, the geometrical cross section (in the case of Ne, the maximum is at 80 % of the geometrical cross section). It is clearly not the case for (H 2 O) n H + -H 2 O collisions, where absolute cross sections are much lower compared to geometrical cross sections. Furthermore our results are more reliable since they can be compared to the results obtained in attachment experiments. Second, the fall in the cross section at high energy in [9] is mainly attributed to the time available for thermal fragmentation, which decreases as the collision energy increases. As shown by our simulation of thermal evaporation presented in Sec. IV C, which takes into account the propagation time after the collision cell and its variation with size and collision energy, this effect cannot explain the fall of the cross section at high energy in our case.

Thermal effect

Total fragmentation cross sections are not enough to discriminate between the various collision processes described in the introduction. However our experiment gives more information. First, the initial temperature of the clusters can be varied: it is very likely that the temperature dependence of the cross section be different whether the fragmentation is direct or statistical. Second, the fragmentation patterns also contain relevant information: we postpone the results on fragmentation patterns to the next paragraph and concentrate for now on the temperature effects. We first rule out another temperature effect, namely fragmentation induced by absorption of black body background radiation of the experiment chamber at room temperature. This source of fragmentation can be excluded since the lifetime of protonated clusters exposed to a black body radiation spectrum at 300K, in the size range

50 5   n
, is larger than 10 -1 s [24], which exceeds by far the time of flight of the clusters in our experiment, which is in the 100 µs range.

The collision-induced fragmentation cross sections observed in the collision (H 2 O) 30 H + + H 2 O are shown in figure 4, for two different initial temperatures, namely 25K and 100 K. At those temperatures there is no spontaneous thermal evaporation before the clusters enter the collision cell [22]. If there is indeed a statistical redistribution of the collision energy among all internal degrees of freedom followed by thermal evaporation, the fragmentation rate should depend only on the total internal energy after the collision, which is the sum of cluster's initial thermal energy and collision energy. Then, in a thermal evaporation scheme, increasing the initial temperature of the cluster is expected to lower the collision energy that leads to a given fragmentation rate. It is exactly what is observed by comparing the fragmentation rates obtained at two different initial temperatures, which is a first indication that the main fragmentation mechanism is thermal.

Fragmentation patterns

Fragmentation patterns may be used in order to discriminate between thermal and direct fragmentation: in the case of thermal fragmentation, they can be reproduced with a model based on classical evaporation theories, while in the case of direct fragmentation, the intensities of the peaks, at least those close to the parent, are not expected to be reproduced by a statistical model. On the other hand, the interpretation is model dependent. The model itself is made of two pieces: first, a model for the collision process, which gives the distribution of energy deposited in the cluster; second, a model for the evaporation itself. The second one has been the most studied in cluster physics, and is now well established. Two main analytical approaches can be used, which give roughly the same results for our purpose: an Rice-Ramsperger-Kassel model or a Weisskopf model. They assume that the cluster evaporates monomers by sequential unimolecular decay. We used Engelking's derivation of the Weisskopf model [25,26] to describe this decay. The Weisskopf expression of the decay rate of a cluster made of n monomers and bearing the internal energy E is:

  2 5 ) 1 ( 4 ) 1 ( 7 3 0 1 ) 1 ( 8                      E E D n h W n n n i geo , ( 5
)
where  0 is the intermolecular vibrational frequency,  the reduced mass,  geo the geometrical cross section as given by Eq. ( 4), and α the number of degree of freedom per molecule. The sizedependent dissociation energies D are taken from the experimental work by Hansen and coworkers [27].

The internal energy of clusters as a function of their temperature ( E in relation ( 5)), is given by the Einstein model for the phonon statistics:

    1 exp 6 2 ) 6 ( 0 0 0      T k h h n h n E B      (6)
where B k is the Boltzmann constant. In the temperature range (25K-100K) covered in our fragmentation experiments, the internal energy calculated using Eq. ( 6) is close to that determined for water clusters by Hock and coworkers [28], who measured the heat capacity of negatively charged water clusters for 48  n and 118  n . The heat capacities per molecules of water clusters, which do not vary noticeably between the two sizes 48 and 118, are not expected to vary dramatically from negatively to positively charged clusters [30].

Note that the number of degrees of freedom per molecule  has not been fixed so far to a particular value in the above relations ( 5) and (6). As a matter of fact, this parameter cannot be determined unambiguously and must be considered in such a simplified model as an effective value. First, this quantity varies with temperature: For instance, the number of degrees of freedom per molecule deduced from the ice caloric curve varies between 0.5 and 4.5 when the temperature goes from 25 K to 270 K.. Furthermore some internal modes might not couple well with the dissociation coordinates [15]. Moreover, there are discrepancies in the literature concerning the effective number of degrees of freedom of water clusters: Hock et al find that their caloric curve is similar to the one of bulk ice at low temperature [28] whereas others deduce a value close to 6k B [29,30], much higher than the one deduced from bulk ice caloric curve. Anyway, changing the value of the number of degree of freedom in eq. ( 5) can be easily compensated by slightly changing the prefactor.

We performed several calculations using a number of degrees of freedom varying from 3 to 6 per molecule. The frequency 0  needed to be slightly adjusted, but remained always close (within  20%) to the water intermolecular stretch vibrational energy



. Any value between 3 and 6 gives a comparable agreement between experiment and theory.

Since the energy transfer during the collision depends in a complicated manner on the impact parameter and the initial orientation of the cluster, it is much harder to model the collision process. We used the well-known line of centers model [START_REF] Levine | Molecular reaction dynamics and chemical reactivity[END_REF], which assumes that the energy transferred t E is the collision energy diminished by the centrifugal energy at distance

0 * R R R c  
, as shown in Fig. 5:

  2 2 2 * 1 2 1 R b E v E c radial t     (7)
When the statistical distribution of impact parameters is taken into account, the distribution of deposited energy is uniform between 0 and the maximum available energy, that is, the collision energy.

Using a Monte-Carlo sampling of the energy transferred, fragmentation patterns and dissociation cross sections can both be simulated using the above models. The comparison with experimental results is shown as solid lines on Fig. 1 for fragmentation patterns and Fig. 4 for cross sections. The agreement is very satisfactory, which indicates that sequential unimolecular evaporation is the most probable fragmentation mechanism in our experiments. However, to reproduce the experimental results in both cases, we had to adjust the only free parameter of the model, namely the molecular radius 0 : we had to take a molecular radius 62 . 1 0  R Å, much the value deduced from either attachment cross sections or ice density (

98 . 1 0  R Å)
. This means that the cross sections are reduced by a factor of about 0.67 respective to the geometric ones. On the other hand, the fragmentation patterns do not depend on the choice of 0 R , since the evaporation rates computed with Eq. ( 5) are rather insensitive to the prefactors.

The main conclusion of this section on experimental results is that all the observed effects are compatible with a thermal mechanism, except that roughly 35 % of the head-on collision do not lead to fragmentation and thus are unobservable in our experiment.

V. Discussion

We first want to stress that the above value of 35% for non-fragmenting collision is huge: for instance, the experimental fragmentation cross section of    H O H 30 2 at a collision energy of 40 eV (with an initial cluster temperature of 25 K) is 140 Å 2 , whereas the geometrical cross section is 210 Å 2 : this means that the experimental fragmentation probability is 65%, and 35% of the collisions that occurs within the hard sphere cross section do not undergo fragmentation. On the other hand, the Weisskopf model (Eq. ( 5)) predicts that the fragmentation time becomes of the order of the observation time when the energy transferred is about 1.5 eV. If the hypothesis of uniform distribution of the transferred energy were true, this would mean that at most 4% of the collisions within the geometrical cross section should be non-fragmenting at 40 eV collision energy.

It is hard to interpret the above result without detailed simulations. In the following, we propose a few hypotheses, which might lead to the observed effect. A first possibility is that the incoming molecule undergoes an elastic collision with a unique molecule of the cluster. However, either the target molecule stays in place, and it receives less than about 1.5eV, which means that the impinging molecule undergoes a very small deviation, and has a high probability to interact with another molecule of the cluster, or the whole energy (minus maybe about 1.5 eV) is transferred to the target molecule, which is then ejected. We have ruled out this second process by performing collision between H 2 O clusters and D 2 O molecules, which show negligible exchange. And the first process is very likely to eventually deposit a significant amount of energy inside the cluster.

Another possibility is that the interaction between the molecule dipole and the cluster charge is repulsive, due to the orientation of the dipole, which for some reason remains frozen during the collision. This could explain why the impinging molecule would be substantially deviated without exciting the cluster, since the repulsion would be at long distance. We have no however of the probability of such an event. We can anticipate that it might be dependent on the collision time, since the dipole orientation and the cluster vibration have to be considered frozen.

Actually, a striking feature is displayed in Fig. 6, which might support the hypothesis of a temporal effect related to the vibrational excitation of the clusters. In this figure, the fragmentation cross sections are plotted as a function of the interaction time. The collision time as a function of size and kinetic energy in the lab frame is given by [22]:

  2 1 0 3 1 3 2 2   m T k nm E R n B k (8)
Here, n is the number of molecules in the cluster, m and 98 . 1 0  R Å are the mass and radius of a single water molecule, respectively, T is the temperature of the collision cell. Fig. 6 shows that, for collision times greater than the intermolecular vibrational period (deduced from the value of 0  used in expressions ( 5) and ( 6) ), the energy transfer, thus the fragmentation rate, increases as expected with increasing the collision energy (i.e. with reducing the interaction time). However, when the collision time becomes smaller than the intermolecular vibrational period, the fragmentation rate stops increasing with collision energy, and even decreases for D 2 O clusters. In this case, the energy transfer decreases as the interaction time decreases. In the frame of simple impulsive models where the impinging water molecules hits an individual water molecule of the cluster, this behavior cannot be supported by classical approaches such as the well known "spectator model" [START_REF] Levine | Molecular reaction dynamics and chemical reactivity[END_REF] in which the energy transfer is expected to be more efficient as the interaction time decreases.

A third hypothesis is that the impinging molecule goes through the cluster without transferring a noticeable energy, as observed in numerical simulations by U. Saalmann and R. Schmidt [3] on sodium clusters. Of course, the structures of sodium and water clusters are different. However, it is known that water can form clathrate structures, with a lot of empty space inside, and it has been shown that water clusters in our size range are amorphous [32]. Whether those structures might allow or hinder an energy-lossless traversal is of course unknown. As pointed out in [3], when the collision time becomes smaller than the typical vibrational period of the cluster, the energy transfer between the projectile and the target becomes less efficient. This is again in agreement with the behavior shown in Fig. 6.

VI. Conclusion

Measurements of collision-induced fragmentation cross sections in water cluster-water molecule collisions have shown that the part of collision energy transferred into cluster's internal energy is significantly smaller than expected in the collision energies window 5-100 eV, where 35 % of the collisions that occur within the geometric cross section do not lead to fragmentation although the energy transferred to the cluster is expected to be by far large enough to induce thermal evaporation.

The measured fragmentation cross sections are compatible with an interpretation in the frame of a thermal evaporation model. It has been shown that, according to the hypothesis of statistical thermal decay, the relevant parameter for calculating the evaporation rate is the total internal energy, that is the sum of the initial thermal energy plus the collision energy, and should consequently depend on both collision energy and initial cluster temperature: At constant collision energy, in this energy range, the measured fragmentation rate indeed varies with the temperature of the cluster, in good agreement with the predictions of the thermal model.

For collision energies ranging from about 5 eV up to tens of eV however, the fragmentation cross sections of water clusters colliding with single water molecules is roughly constant, and reach a value significantly smaller than the geometric collision cross section. This means that, in this energy range, water molecules hit water clusters at higher and higher velocity without increasing the cluster internal energy, as if water clusters were partially "transparent". At the energies considered, the collision energy is likely to be transferred directly to the cluster's vibrational modes without involving electronic excitation. This puzzling behavior is not yet really understood. However, it is worth noting that it seems to be similar, to some extent, to the one predicted for sodium-rare gas collisions by U. Saalmann and R. Schmidt [3], who ascribed tentatively this vibronic transparency effect to the relative velocity between projectile and target becoming too large to excite cluster's vibrational degrees of freedom.

Figure captions

  and deuterated water clusters are displayed in figure3.

  between roughly 10 and 50 eV. The values of the ratio at the plateau, about 0.6 for D 2 O and 0.65 for H 2 O, do not depend significantly on the size.

  . The results presented in figures 1 and 4 were calculated with =6, Hz

Figure 1 :

 1 Figure 1:Experimental relative intensities of fragments (H 2 O) n H + (black lines) from the collision of (H 2 0) 30 H + with H 2 O molecules at four center-of-mass collision energies E cm . Continuous and dashed red lines are obtained from Monte Carlo simulations (Continuous line: 3n-6 degrees of freedom, ν 0 =5.5 THz, Dashed line: 6n-6 degrees of freedom, ν 0 =6.5 THz, see text).

Figure 2 :

 2 Figure 2:Experimental data points for -ln(I/Io) (see relation(1)) as a function of the product of water vapor density in the cell times the cell length. The continuous line is a linear fit through the data points. The fit yields a value of 125 Å 2 for the fragmentation cross-section. The data points are obtained for (H 2 O) 30 H + clusters at a collision energy of 2.3 eV.

Figure 3 :

 3 Figure 3: Fragmentation cross section, relative to the geometrical cross section, plotted for various cluster sizes and collision energies. a) Fragmentation of deuterated heavy water clusters induced by collisions with heavy water molecules. b) Fragmentation of protonated water clusters induced by collisions with water molecules.

Figure 4 :

 4 Figure 4:Cross section of the fragmentation of (H 2 O) 30 H + induced by collisions with H 2 0 molecules as a function of collision energy for two different initial temperatures of the cluster (25 K and 100 K). The continuous and dashed lines are obtained from a Monte Carlo simulation using the model described in the text. Continuous line: 3n-6 degrees of freedom, ν 0 =5.5 THz, Dashed line: 6n-6 degrees of freedom, ν 0 =6.5 THz. The straight line (red online) at 210 Å 2 is the geometrical cross section.

Figure 5 :

 5 Figure 5:The radial energy transfer: only the energy transferred along the radial component v radial is assumed to be transferred into internal energy.

Figure 6 :

 6 Figure 6: Fragmentation cross section, relative to the geometrical cross section, plotted as a function of the duration of the collisions for different cluster sizes for D 2 O (a) and H 2 O (b) clusters. The vertical lines indicate the vibrational period deduced here from ν 0 = 5.5 THz.

  Figure 5


Keywords: Aquaculture, Nitrogen, Phosphorus, Phytoplankton, Chlorophyll-a, Mediterranean Sea

While several studies point at off-shore aquaculture as a possible source of impacts on the local marine environment, very few have analysed its effects at large scales such as at the bay, gulf or basin levels. Similar analyses are hampered by the multiple sources of disturbance that may concomitantly affect a given area. The present paper addresses these issues taking the Gulf of Castellammare (Southern Tyrrhenian Sea) as an example.

Nitrogen (N) and phosphorous (P) loads were calculated for the period 1970-2007, and compared to chlorophyll-a concentration as measured inside and outside the Gulf over the same period. Results indicate that N and P catchment loading has constantly decreased because of improved environmental management. Nevertheless, nutrient concentration in the Gulf has steadily increased since the establishment of aquaculture facilities in 1999. Chlorophyll-a concentration followed this trend, showing a marked increase from 2001 onwards. In the same period, chlorophyll-a concentrations measured inside and outside the Gulf have significantly diverged. As all the other possible causes can be ruled out, aquaculture remains the sole explanation for the observed situation.

This paper demonstrates for the first time ever that off-shore aquaculture may affect the marine ecosystem well beyond the local scale and provides an additional element of concern to be kept into consideration when allocating oceans' space for new fish farming activities.

Introduction

Since the 1990s, coastal aquaculture has become a consolidated economic activity as a global alternative to land-based aquaculture [START_REF] Beveridge | Cage aquaculture, 2nd edn[END_REF], and has been the major cause of a rapid domestication of marine species [START_REF] Duarte | Rapid domestication of marine species[END_REF]. Over the last two decades, along with the almost exponential increase in economic gains provided worldwide, coastal aquaculture has attracted a large interest either from marine scientists and policy makers for the potential detrimental effects of this industry on the surrounding marine environment (e.g., [START_REF] Munday | Quantitative statistical analysis of the literature concerning the interaction of the environment and aquaculture -Identification of gap and lacks[END_REF][START_REF] Pillay | Aquaculture and the environment[END_REF][START_REF] Pusceddu | Effects of intensive mariculture on sediment biochemistry[END_REF]Holmer et al., 2008a-b;[START_REF] Borja | Assessing the suitability of a range M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT of benthic indices in the evaluation of environmental impact of fin and shellfish aquaculture located in sites across Europe[END_REF]. Most of the research carried out so far has, indeed, documented significant changes in the physical, chemical and biological attributes of sediments and the water column around off-shore aquaculture plants, and these include, among the others: i) increasing organic matter contents and compositional changes of the sediment below fish cages (e.g., [START_REF] Pusceddu | Effects of intensive mariculture on sediment biochemistry[END_REF]; ii) altered inorganic and organic chemistry of farm water and sediments (e.g., Sarà, 2007a-b-c;Sanguling et al., 2011); iii) altered abundance, biomass and biodiversity of micro-(La [START_REF] Rosa | Benthic microbial indicators of fish farm impact in a coastal area of the Tyrrhenian Sea[END_REF], meio- [START_REF] Mirto | Meiofauna response to fish farming in seagrass and softbottom sediments of the Mediterranean Sea[END_REF] and macro-benthic [START_REF] Kalantzi | Benthic impacts of fish farming: meta-analysis of community and geochemical data[END_REF] communities; iv) modified distributional patterns of phyto-and microplankton abundance and production (e.g., [START_REF] Pitta | Planktonic food web in marine mesocosms in the Eastern Mediterranean: Bottom-up or top-down regulation[END_REF]. The extension of the abovementioned changes either in water and benthic variables has been observed generally at spatial scales ranging from meters to a few hundred meters from the edge of the pens, generally not exceeding 1-2 kilometres from the rearing site (e.g., [START_REF] Gowen | The ecological impact of salmonid farming in coastal waters: A review[END_REF][START_REF] Modica | The effect of mariculture facilities on biochemical features of suspended organic matter (South Tyrrhenian, MED)[END_REF][START_REF] Pusceddu | Effects of intensive mariculture on sediment biochemistry[END_REF]Holmer et al., 2008a- et al., 2009). [START_REF] Machias | The response of demersal fish communities to the presence of fish farms[END_REF] showed that the presence of fish farms can increase the biomass of wild fish populations at a spatial scale larger than the immediate vicinity of fish cage, though with no clear impact on species diversity. However, to our best knowledge, information about aquaculture impacts on water and sediment variables at large spatial scale (e.g., at a basin scale) are practically not existent. Furthermore, three recent and independent meta-analyses of more than 1000 study cases have pinpointed the unevenly spatially resolved design of most of the reviewed experiments aimed at assessing the effects of aquaculture on a wide array of abiotic (i.e., temperature, salinity, pH, water turbidity, dissolved oxygen, inorganic nutrient) and biotic (e.g., chlorophylla, organic carbon and nitrogen, prokaryotic and phytoplankton abundance) variables in the water column (Sarà, 2007a-b-c).

The persistence of these gaps of knowledge is, on the one hand, partially due to the difficulty in disentangling, at large spatial scales, the ecological effects induced by coastal aquaculture from those induced by other natural (e.g., currents, circulation) and anthropogenic sources of environmental variability (e.g., urban and/or industrial agglomerates, terrestrial zootechny and agriculture). On the other hand, the sampling bias affecting many of the studies carried out on the impacts of aquaculture on the neighbouring environment (with a few exceptions, e.g., [START_REF] Pusceddu | Effects of intensive mariculture on sediment biochemistry[END_REF]Holmer et al., 2008a;Sanz-Lázaro et al., 2011) makes even more difficult the task of identifying large-scale ecological effects of aquaculture because of the presence of possibly confounding explanatory variables and uncontrolled sources of variability.

In spite of these premises, it could be hypothesised that waste nutrients released by fish farming activities might co-act with N and P released in near-shore waters through continental run-off, rivers and seasonal streams, altogether promoting

M A N U S C R I P T A C C E P T E D
ACCEPTED MANUSCRIPT eutrophication of coastal waters (i.e., either in terms of increased nutrient contents, enhanced primary production and accumulation of algal biomass; [START_REF] Cloern | Our evolving conceptual model of the coastal eutrophication problem[END_REF]. However, we still do not know yet whether additive loads released by aquaculture may trigger water eutrophication at a very large distance (>10 km) from the local area of waste release, or how the duration of exposure to aquaculture wastes may affect the ecological functioning of the receiving basin. As a consequence, large-scale mitigation of fish-farming impacts has been de facto neglected in the present management practices of coastal areas worldwide, thus posing a concern also in the light of the predicted tendency of fish farming industries to move in off-shore waters, as the space in coastal waters is increasingly becoming a limiting factor [START_REF] Holmer | Environmental issues of fish farming in offshore waters: perspectives, concerns and research needs[END_REF].

In this study, we aimed to evaluate, in an unprecedented manner: 1) whether and how N and P catchment loading may affect quasi-decadal variations in the pelagic phytoplankton biomass of a Mediterranean Sea Gulf (Gulf of Castellammare, Southern Tyrrhenian Sea), and 2) whether and how much additional N and P inputs released from off-shore fish farming in the same area have contributed to the observed variations. To achieve these objectives we: i) quantified the amount of N and P loads in the Gulf (using empirical and statistical data) coming from point (population, industry, terrestrial and marine farming) and non-point (uncultivated and cultivated soils) sources; ii) analysed the quasi-decadal fluctuations in phytoplankton biomass (expressed as chlorophyll-a concentrations in the seawater), and iii) analysed the difference in chlorophyll-a concentrations within the Gulf vs. those in open-sea waters, to understand if chlorophyll-a fluctuations occurred at the Gulf scale were due to local or basin-wide long-term changes of the whole Southern Tyrrhenian Sea.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Material and Methods

The study area

The Gulf of Castellammare (Tyrrhenian Sea, surface ~370 km 2 ) is the widest inlet along the northern coast of Sicily (Fig. 1a). Bordered by rocky cliffs, it opens into shallow sandy shores with patches of rocky bottoms and interspersed seagrass beds. The area is influenced by limited continental inputs (~88,500 x 10 3 m 3 y -1 ; about 600 times less than the Po River input in the northern Adriatic Sea and about 25 times less than the Simeto river inputs in the South Ionian Sea along the SE coasts of Sicily; Sarà et al., 1998;Sarà et al., 2004;[START_REF] Modica | The effect of mariculture facilities on biochemical features of suspended organic matter (South Tyrrhenian, MED)[END_REF]Sarà et al., 2007). Those inputs are discharged by two of the four seasonal streams located in the central sector of the Gulf, namely the Nocella (13 km long, with a surface of about 15,000 ha) and the S.

Bartolomeo (50 km long, 40,000 ha) streams (Fig. 1a). These two small rivers show only narrow fluctuations of discharge rates (on average 0.32 m 3 sec -1 and 0.24 m 3 sec -1 for the Nocella and S. Bartolomeo river, respectively; PTA, 2007) generally interrupted by seasonal rainfall episodes which cause short (i.e., a few days) flushing events (never exceeding 1.2 m 3 sec -1 ; PTA, 2007). The whole drainage basin of the Castellammare Gulf includes 16 small municipalities (a total of about 175,000 inhabitants), of which just six are located on the coast. The local population density is about 100 people per Km 2 (ISTAT, 2001).

The economy of the Gulf catchment is based on the tertiary sector, including tourism and small industries (fishery processing), for about 60-70% of the local gross domestic product. Agriculture, pastoral, fish farming and fisheries account altogether for about 30-40% of the local gross domestic product (ISTAT, 2005;PTA, 2007). In the Castellammare Gulf, fisheries have played a major role in the local economy for many

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
decades but only till 1990 [START_REF] Badalamenti | Artificial reefs in the Gulf of Castellammare (North West Sicily): a case study[END_REF], when bottom trawling was banned from the area [START_REF] Pipitone | Fish biomass increase after a four-year trawl ban in the Gulf of Castellammare (NW Sicily, Mediterranean Sea)[END_REF]. One of the biggest Mediterranean aquaculture companies has been operating in the eastern area of the Gulf since 1999, producing about 500 t y -1 of sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) ever since [START_REF] Mazzola | Cultivation of the Mediterranean amberjack, Seriola dumerili (Risso, 1810), in submerged cages in western Mediterranean Sea (Italy)[END_REF]Sarà et al., 2007). Since 2000, a bluefin tuna farming industry (only fattening) located in the central sector of the Gulf (off the small harbour in Castellammare del Golfo; Fig. 1a; [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | Qualità determination of Thunnus thynnus reared in sea cages in Western Sicily (Italy)[END_REF] produces about 1,000-1,100 tons of farmed tuna per year (from July to December).

Long-term variations in chlorophyll-a, total N and P concentrations in the Castellammare Gulf and the adjacent open waters

We inventoried records of chlorophyll-a (µg L -1 ), total nitrogen (N, µM) and phosphorus (P, µM) concentrations in the upper (within 1 m depth) surface layer of the Gulf of Castellammare waters in the period 1970-2007. Data were obtained from research and oceanographic cruises carried out by several Italian Academic Institutions and Governmental Agencies as well as from our own data (Fig. 1a). Those data, consistently obtained using spectrophotometric techniques (chlorophyll-a: [START_REF] Lorenzen | A note on the chlorophylla-a and pheophytin of the chlorophyll maximum[END_REF]total N and P: Valderrama, 1981), were averaged to obtain mean annual values and, though unevenly dispersed in space and time, used to assess inter-annual variations in the Gulf of Castellammare. Moreover, within the period 1998-2007, we inventoried also data of chlorophyll-a concentrations from five areas spread in the Southern Tyrrhenian Sea along the western coast of Sicily (Fig. 1b). These data, extracted from the Environmental Marine Information System (EMIS) published at http://emis.jrc.ec.europa.eu/4_1_gismap.php and maintained by the Joint Research

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Centre of the European Commission (2008), were used to compare inter-annual trends (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007) in phytoplankton standing stocks inside and far from the Gulf of Castellammare (Fig. 1a-b).

Estimates of N and P loads entering the Gulf

Wasted loads (tons per year) of total N and P coming from the most important point (i.e., identifiable localized sources, such as resident and touristic cities/villages; industries; zootechny plants; fish farms; cattle, swine, sheep and goat farms) and nonpoint (i.e., diffuse sources, such as agriculture and uncultivated soils) sources were estimated according to the transformation coefficients recently proposed by [START_REF] Pagnotta | Stima dei carichi inquinanti nell'ambiente marinocostiero[END_REF] Table 1) and adopted by the Italian Ministry of Public Health to assess the N and P loads along the Italian coasts. Since these coefficients were assumed to be constant across the year and since data about inputs coming from tourism are not available for the region, we are aware that our estimates of the N and P wastes entering annually the Castellammare Gulf from inland are affected by some uncertainty.

Nevertheless, statistical data released by the Italian Statistical Institute (ISTAT), similarly to those released by Statistical Institutes of most Western European countries, are only available as annual means, so that we were not able to carry out our estimates on shorter (e.g., monthly or seasonal) temporal scales.

Data on resident population, industry, terrestrial farming and cultivated and noncultivated soils in the study area were extracted from censuses carried out by the Italian Statistical Institute (ISTAT) in 1962(ISTAT) in , 1972(ISTAT) in , 1982(ISTAT) in , 1990(ISTAT) in , 2000(ISTAT) in and 2007 (only population) (only population).

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Estimates of nitrogen and phosphorus wastes released from seabream, seabass and bluefin tuna farms and entering the Castellammmare Gulf were based on individual gross metabolic waste outputs and coefficients of particulate and dissolved N and P released on a daily and biomass basis, as modelled in [START_REF] Piedecausa | Simulating the temporal pattern of waste M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT production in farmed gilthead seabream (Sparus aurata), European seabass Dicentrarchus labrax) and Atlantic bluefin tuna (Thunnus thynnus)[END_REF] and [START_REF] Aguado-Giménez | Gross metabolic waste output estimates using a nutritional approach in Atlantic bluefin tuna (Thunnus thynnus) under intensive fattening conditions in western Mediterranean Sea[END_REF]. These models allow simulations of real rearing and husbandry practices. Gross metabolic waste output estimates for three cultivated species (i.e., seabream, seabass and tuna) in the Gulf were made on a nutritional basis, using the following bioenergetic model [START_REF] Piedecausa | Simulating the temporal pattern of waste M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT production in farmed gilthead seabream (Sparus aurata), European seabass Dicentrarchus labrax) and Atlantic bluefin tuna (Thunnus thynnus)[END_REF]:

C =G+ E + F
where C is the nutrient (nitrogen, N and phosphorus, P) content in the food intake, G represents the retained nutrient as growth, E and F represent the excretory (dissolved) and faecal (particulate) losses, respectively. To calculate waste output coefficients of particulate and dissolved nitrogen and phosphorus on a daily and per biomass basis (mg N or mg P kg -1 d -1 ), the nitrogen and phosphorus content in the diet and faeces, the feeding conversion rate and nitrogen and phosphorus digestibility were considered [START_REF] Piedecausa | Simulating the temporal pattern of waste M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT production in farmed gilthead seabream (Sparus aurata), European seabass Dicentrarchus labrax) and Atlantic bluefin tuna (Thunnus thynnus)[END_REF].

Statistical analyses

Since we had gathered data from very different sources, we were forced to analyse our hypotheses using a combination of different statistical techniques. Therefore, we used the t-Student (Sokal and Rolhf, 1981) to test differences in the measured variables among cruises and years, while the Permutational Multivariate Analysis of Variance (PERMANOVA; [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF] was used to test the (null) hypothesis of no significant differences in chlorophyll-a concentrations between data collected during the

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
cruises (spectrophometrically determined) and the data extracted from the EMIS GIS database (obtained by means of satellite sensors). The latter comparison was done separately for each year as well as for the data recorded before and after the launch of aquaculture activities in the Gulf. We applied the linear correlation approach to relate chlorophyll-a and nutrients concentrations (Sokal and Rolhf, 1981) Over the study period , the main sources of nitrogen and phosphorus were from cultivation (on average 1,058 tons of N and 39.6 tons of P per year, representing ~67.2% and 36.7% of total N and P inputs, respectively) and human population (378.5 tons of N and 56.9 tons of P per year, representing 24.4% and 52.4% of total N and P inputs, respectively).

Results

Long-term changes in N and P loading from different sources

In 1999, a fish farm producing about 500 tons per year of sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) was installed in the Gulf. While no data are available for the 1999, the total amount of N and P discharged every year from 2000 and 2007 in the Gulf by sea bass, sea bream and tuna fish farming activities accounted up to 274.9 tons, representing about 17.4% and 34.2.5% of the relative N and P total inputs per year. These, in turn, represent about 51.5% and 122.0% of the N and P inputs derived from coastal discharge, respectively (Table 2-3).

Temporal changes in chlorophyll-a in the Gulf

Dissolved nutrient concentrations in the Gulf, as extrapolated from field campaigns data, showed a steady increase in mean annual values from 1980 to date (Fig. 3). On the contrary, in the upper 10-m layer of the water column annual mean values of chlorophyll-a concentration, used here as a proxy of phytoplankton biomass, decreased between 1980 and 1990 (by 30%), and another significant (t-Student p<0.05) drop was observed between 1991 (0.04 µg L -1 ) and 1995(0.01 µg L -1 ). Chlorophyll 4). Accordingly, since 2000, chlorophyll-a concentrations followed the increase in N and P loading (Table 2-3), and were significantly correlated to phosphate concentrations (Fig. 5).

Discussion

The most recent literature has provided consistent and repeated evidence that aquaculture can have remarkable effects on the surrounding environment with clearly detectable effects on the ecology of the benthos (Holmer et al., 2008a-b), but idiosyncratic effects on the ecology of the water column (Sarà, 2007a,b,c). Many of those investigations have proven that effects are detectable at a relatively short distance (i.e. hundreds of meters) from the farms, mainly because of the dilution effect promoted by the local currents [START_REF] Modica | The effect of mariculture facilities on biochemical features of suspended organic matter (South Tyrrhenian, MED)[END_REF][START_REF] Pusceddu | Effects of intensive mariculture on sediment biochemistry[END_REF][START_REF] Mirto | Meiofauna response to fish farming in seagrass and softbottom sediments of the Mediterranean Sea[END_REF].

However, given the well documented consistency of nutrient release from fish farms into the marine environment (Piedicausa et al., 2010) and the crucial importance of water currents in the dispersion of wastes [START_REF] Pusceddu | Effects of intensive mariculture on sediment biochemistry[END_REF]Sanz-Lázaro et Contextually, agricultural run-off has significantly declined, reducing the discharge of N and P by at least 35% from the seventies to date (Tables 23). This has been probably due to the progressive shift of the local economy from agriculture to more profitable activities like tourism (Tyrakowski, 2004). Similar trends have been quite common in all North Mediterranean countries [START_REF] Milesi | Assessing the impact of urban land development on net primary productivity in the southeastern United States[END_REF], though in the North African countries the tendency appears to be inverse (e.g., agriculture, population and tourism are expected to increase till 2025, with effects still to be determined; Turley, 1999).

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Data for the Gulf of Castellammare do not strongly deviate from the common European scheme of the last decade. Indeed, suspended chlorophyll-a concentration was rather high in the 1980s, probably due to the almost total deregulation of N and P catchment loading, to the absence of wastewater treatment and to the intense bottom trawling activities carried out in the study area, which could have contributed to an enhanced mobilisation of benthic nutrients resuspended by fishing gears [START_REF] Pusceddu | Effects of natural and anthropogenic sediment resuspension on particulate organic matter dynamics in shallow coastal environments[END_REF].

At the end of the eighties, important steps towards the correct management of natural resources in the catchment area included the regulation and depuration of urban wastes released at sea and the ban on bottom trawling (since 1989;[START_REF] Pipitone | Fish biomass increase after a four-year trawl ban in the Gulf of Castellammare (NW Sicily, Mediterranean Sea)[END_REF]. As a plausible immediate consequence, the Gulf waters dropped to an ultra-oligotrophic status (i.e., <0.1 µg chlorophyll-a L -1 , according to the OECD scheme based on concentrations of chlorophyll-a; Zurlini, 1996;Sarà and Mazzola, 1997;Sarà et al., 1998), which lasted till 1999, when chlorophyll-a concentration in the surface waters of the Gulf steadily and significantly increased compared to open-sea baseline data sets (i.e., EMIS data). Between 2001 and 2007, the concentrations of chlorophyll-a in the surface waters of the Gulf were from 3 (0.15 µg L -1 , in 2001) to over 10 times (ca. 0.50 µg L -1 , as averaged for [2006][2007] higher than in the reference open-sea values. In the period 2004-2005 the concentration of chlorophyll-a in the eastern part of the Gulf was on average about 1.1 µg L -1 , but exceeded 1.5 µg L -1 (with frequent peaks over 3 µg L -1 ) close to the aquaculture facilities (Sarà et al., 2009).

The divergent temporal patterns in chlorophyll-a concentrations in the two investigated systems might be attributed to a wide and complex array of explanatory variables and processes. Many reasons, indeed, could be invoked for explaining the rise in chlorophyll-a concentration in the surface waters of the Gulf which, among the

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
others, include: i) increased terrestrial runoff due to rainfall; ii) increased release of nutrients from the sea bottom as affected by sediment resuspension induced by bottom trawling, iii) variations in global climate factors (e.g., temperature, storminess, etc.) and iv) increased nutrient loadings from local point and/or non-point sources. Over the last decades rainfall in the Gulf had a swinging trend (Fig. 6; data from SEDEMED II, 2009) and was accompanied by a general decrease of the number of wet days in Southern Italy [START_REF] Brunetti | Changes in daily precipitation frequency and distribution in Italy over the last 120 years[END_REF]. Nor rainfall or water temperature and salinity did explain significant proportions of chlorophyll-a variations in the Gulf (Table 4).

Therefore, we can conclude that variations in the thermohaline conditions of the water column and in rainfall did not have crucial roles in modifying the trophic status of the Gulf waters. Bottom trawling was the main economic activity in the Castellammare Gulf until the ban in 1989 [START_REF] Badalamenti | Artificial reefs in the Gulf of Castellammare (North West Sicily): a case study[END_REF][START_REF] Pipitone | Fish biomass increase after a four-year trawl ban in the Gulf of Castellammare (NW Sicily, Mediterranean Sea)[END_REF]. Disturbance of the seabed resulting from bottom trawling affects different ecosystem processes such as the rate and magnitude of nutrient regeneration [START_REF] Pusceddu | Effects of natural and anthropogenic sediment resuspension on particulate organic matter dynamics in shallow coastal environments[END_REF][START_REF] Dounas | Large-scale impacts of bottom trawling on shelf primary productivity[END_REF]. By cascade, plankton communities are expected to be influenced, which adds a pulsing component to local primary production [START_REF] Dounas | Large-scale impacts of bottom trawling on shelf primary productivity[END_REF]. The ban on bottom trawling in the Gulf (1989) could have led to a reduction of sedimentary nutrient contribution to the water column, so that we can exclude that the rise in nutrient and chlorophyll-a concentrations observed in the Gulf since 1999 was stimulated by bottom fisheries.

Along with a general temperature increase of sea surface waters, other climate related factors like ocean acidification could invoked as additive macro-causes of the deviation of ecosystem functions (including primary production) from their common natural patterns (IPCC, 2007). Although it has been shown that the Mediterranean Sea

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

has experienced a significant increase in CO 2 water concentrations in the last 25 years (Touratier and Goyet, 2011), it must be noticed that, whatever the (physical or biological) mechanism involved, it can be reliably hypothesised that climate change in the study area couldn't have determined a general increase in primary production. On the one hand, this hypothesis is supported by the fact that an increase in sea surface temperature, as the one observed in the whole study area, could have led -under constant CO 2 concentration in the atmosphere, that is not the case (Touratier and Goyet, 2011) -to a decreasing CO 2 availability for primary producers [START_REF] Harrison | Effects of a diel oxygen cycle on nitrogen transformations and greenhouse gas emissions in a eutrophied subtropical stream[END_REF].

On the other hand, phytoplankton possess C-concentrating mechanisms (counterbalancing CO 2 deficiency under warm temperature conditions; [START_REF] Giordano | CO 2 concentrating mechanisms in algae: mechanism, environmental modulation, and evolution[END_REF], and the eventual increase in CO 2 contents in the seawater may have had a mixture of relevant impacts on the availability of nutrients [START_REF] Raven | Ocean[END_REF].

Moreover, the temperature increase occurred in the whole Mediterranean Sea basin has considerably altered the water column stability and likely modified even the ecological response of deep fauna [START_REF] Coma | Global warming-enhanced stratification and mass mortality events in the Mediterranean[END_REF]. Altogether these data suggest that if a change in primary production would have occurred in the study area this have led most likely to a decrease or invariance rather than to an increase in chlorophyll-a concentrations in the Gulf. It must be noticed also that the expected diminished availability of nutrients under warmer conditions [START_REF] Coma | Global warming-enhanced stratification and mass mortality events in the Mediterranean[END_REF] might have favoured smaller phytoplankton taxa, with a consequently lower total primary production and C export to the sea bottom [START_REF] Danovaro | Biochemical composition of pico-, nano-and microparticulate organic matter and bacterioplankton biomass in the oligotrophic Cretan Sea (NE Mediterranean)[END_REF][START_REF] Danovaro | Deepsea ecosystem response to climate changes: The eastern Mediterranean case study[END_REF]. Therefore, we conclude that changes in the nutrient loads from inland, rainfall, Our conclusions, on the one hand, give us the opportunity to stress the need of: i) improved bio-monitoring protocols to detect possible impacts of aquaculture at spatial scales larger than usual (i.e., > 1-10 km from the farming structures) and ii) more appropriate temporal scales of investigation. On the other hand, our results provide an additional element of concern to be kept into consideration when, likely in the very next future, fish farming activities will expand in off-shore waters, with a potentially expectable impact on the trophic status of vaster regions of the coastal oceans [START_REF] Holmer | Environmental issues of fish farming in offshore waters: perspectives, concerns and research needs[END_REF]. and Environment (1984and Environment ( -1985)); black square = CNR Castellammare (1990); grey triangle = Sarà G. PhD Thesis (1991)(1992)(1993); black triangle = Dept. of Animal Biology and Marine Ecology, University of Palermo (1994Palermo ( -1995)); grey reversed triangle = Mediterranean Oceanological Centre (2000)(2001); black reversed triangle = Sarà G., unpublished data (2003)(2004)(2005); grey hexagon = Sarà G., unpublished data (2006)(2007); black hexagon = Environmental Marine Information System (EMIS;[START_REF] Pitta | Planktonic food web in marine mesocosms in the Eastern Mediterranean: Bottom-up or top-down regulation[END_REF]-2007).

In the small rectangle reported below on the left, the geographic area is showed with the black arrow indicating the location of the Gulf of Castellammare in the northern Sicily and five black circles indicating open-sea areas from which EMIS chlorophyll-a data were retrieved. , µM Figure 3 y -8 2 y -8 4 y -8 5 y -9 0 y -9 1 y -9 2 y -9 3 y -9 4 y -9 5 y -9 8 y -9 9 y -0 0 y -0 1 y -0 2 y -0 3 y -0 4 y -0 5 y -0 6 y -0 7 y -1 9 8 5 y -1 9 9 0 y -1 9 9 1 y -1 9 9 2 y -1 9 9 3 y -1 9 9 4 y -1 9 9 5 y -1 9 9 8 y -1 9 9 9 y -2 0 0 0 y -2 0 0 1 y -2 0 0 2 y -2 0 0 3 y -2 0 0 4 y -2 0 0 5 y -2 0 0 6

  The role of rainfall, water temperature and salinity on long-term (from 1980 to 2006) variations in chlorophyll-a annual mean annual concentrations was assessed by means of a distancebased multivariate multiple regression analysis with forward selection of explanatory variables, using the DISTLM Fortran-written routine (McArdle and Anderson, 2001).

N

  wastes produced separately by all sources, with exception of agriculture, increased from 1970 to 1990 (by on average 15% for population, industry and zootechny), than all consistently decreased till 2007 (by about 30% on average; Table2). P wastes derived from all sources increased in the period 1970-1990 (by on average 16% for population, industry and zootechny); P released by resident population and tourists decreased in 2000 (by 4% when compared with the preceding ten years average) while increased again in 2007. P released by industry, terrestrial farming and non-cultivated landscapes decreased from 1990 to 2007 (~32%; Table3). N and P wastes derived from agriculture decreased almost constantly in the investigated period, with values in 2007 about 15% lower than in 1970. Generally, N and P wastes derived from diffuse sources (e.g., agriculture and non cultivated landscapes) decreased almost continuously from 1970 to 2007 (by about 34% for both N and P), whereas wastes derived from point sources M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT showed a biphasic temporal trend, increasing from 1970 to 1990 than decreasing again till 2007 (Fig. 2a-b).

  -a concentrations have significantly risen again in the Gulf after 1998 (t-Student test between before and after 1998 values: p<0.05), setting on constant values until 2001, positive shift led to the maximum value over the entire study period (0.36 µg L -1 in 2006-2007) (Fig. 4). Such increase in phytoplankton biomass observed in the Gulf of Castellammare was not observed in any of the five areas outside the Gulf spread in the Southern Tyrrhenian Sea. Indeed, while in 1998 and 1999 chlorophyll-a concentrations increased consistently either in the Gulf and the open sea area, showing not significant differences (PERMANOVA p>0.01; Fig. 4), from 2000 (about two years after the beginning of aquaculture operations), chlorophyll-a concentrations in the open-sea and the Gulf started to be significantly different (PERMANOVA, p<0.05; Fig.

  effects at large spatial scales may be reasonably expected in protected gulfs, bays and inlets, such as the area under scrutiny in this study.We show here that the surface waters of the Castellammare Gulf have experienced large-scale and long-term variations in the nutrient and chlorophyll-a concentrations, mostly as the result of the chronic release of nutrient waste produced by local aquaculture. Between the 1970s and the 1990s, N and P loads from the Gulf of Castellammare catchment have constantly increased, though diminishing around 2000 (Tab. 2-3). This is consistent with the socio-economic evolution of several Western Mediterranean and European countries over the last decades (Gurluk, 2009). The economic development of many Mediterranean coastal areas, led by tourism and resulting (among other effects) in the increased discharge of N and P into near shore waters (Garau-Taberner and Manera, 2006), was likely counteracted around 2000 by the effects of the European Union's (EU) Urban Wastewater Treatment Directive (91/271/EEC -21 May 1991). The implementation and enforcement of the EU Directive has resulted in a significant amelioration of wastewater treatment before discharge into European rivers, and in measurable improvements in the water quality. This has in turn led to a decrease in point sources of N and P reaching near-shore waters in 2000.

  bottom trawling activities and other climate-related factors have had a minor role in explaining the increase in chlorophyll-a concentrations in the sea surface waters of theM A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPTGulf, as observed from 1999 onwards. Moreover, we would stress here that the huge and continuous in situ nutrient enrichment due to aquaculture in the Gulf could have reasonably been a major factor determining the observed divergence from the open sea area. However, we also acknowledge that other local forcing factors (including circulation and altered food web characteristics), acting individually or synergistically, might have contributed to promote and maintain the observed shift in the productivity of Castellammare Gulf when compared with the adjacent open Tyrrhenian Sea. We also foster further research addressing these causes in future investigations. Nevertheless, the results of our study provide the first evidence that a long-term exposure of a certain coastal embayment to aquaculture wastes might result in a general modification of its trophic status (i.e. nutrient concentrations and phytoplankton biomass) at a spatial scale never considered before.

Figures CaptionsFigure 1 .

 1 Figures Captions

Figure 1 .

 1 Figure1

Figure 2 .

 2 Figure 2. Nitrogen (a) and phosphorus (b) loads released annually from point (resident and touristic population, industry and terrestrial farming) and non-point (agriculture and non-cultivated landscapes) sources in the Gulf of Castellammare.

Figure 3 .

 3 Figure 3. Annual means dissolved nitrogen (N tot, µM) and phosphorus (P-PO 4 , µM) concentrations in the surface waters of the Gulf of Castellammare from 1982 to 2007.

Figure 4 .

 4 Figure 4. Chlorophyll-a (µg L -1 ) inside and outside the Gulf of Castellammare between 1982 and 2007. Data have been obtained from cruises (squares) and satellite data (filled circles, inside the Gulf; triangles, outside the Gulf). Open circles represent the average values between cruise and satellite data inside the Gulf. No chlorophyll-a from satellite data before 1998 are available.

Figure 5 .

 5 Figure 5. Relationship between chlorophyll-a and phosphates in the Gulf of Castellammare between 1980 and 2007.

Figure 6 .

 6 Figure6. Temporal trend of rainfall in the Castellammare Gulf(PTA, 2007)

  Figure4

Acknowledgements

This study has been carried out within the projects INTERMED, under the CIRCLE Med Programme and ERA Net (European Commission 6th Framework Programme).

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

We are indebted with F. Aguado-Giménez (Murcia, Spain) for suggestions in calculating the aquaculture waste gross output of the Gulf. We thank G. Maio (Messina, Italy) and A. Tomasello (Palermo, Italy) for the help in gathering data from the field and the literature, M. Milanese (Genoa, Italy) and F. Marincioni (Ancona, Italy) for the fine tuning of English, M. Giordano (Ancona, Italy) and two anonymous referees for useful suggestions that helped to improve significantly an early version of the manuscript.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

acidification due to increasing atmospheric carbon dioxide. The Royal Society, The Clyvedon Press Ltd,Cardiff, UK, 56 pp. Šangulin, J., Babin, A., Škrgatić, Z., 2011. Physical, chemical and biological properties of water column and sediment at the fish farms in the middle adriatic, Zadar County. Fresenius Environmental Bulletin 19, 1869-1877Sanz-Lázaro, C., Belando, M.D., Marín-Guirao, L., Navarrete-Mier, F., Marín, A., 2011. Relationship between sedimentation rates and benthic impact on Maërl beds derived from fish farming in the Mediterranean. Marine Environmental Research 71, 22-30. Sarà, G., 2007a. A meta-analysis on the ecological effects of aquaculture on the water column: dissolved nutrients. Marine Environmental Research 63, 390-408. Sarà, G., 2007b. Ecological effects of aquaculture on living and non-living suspended fractions of the water column: a meta-analysis. Water Research 41, 3187-3200. Sarà, G., 2007c. Aquaculture effects on some physical and chemical properties of the water column: a meta-analysis. Chemistry and Ecology 23, 251-262. Sarà, G., Lo Martire, M., Buffa, G., Mannino, A.M., Badalamenti, F., 2007. The fouling community as an indicator of fish farming impact in Mediterranean. Aquaculture Research 38, 66-75. Sarà, G., Manganaro, A., Cortese, G., Pusceddu, A., Mazzola, A., 1998. The relationship between food availability and growth of Mytilus galloprovincialis (LMK, 1819) in the open-sea (South Mediterranean). Aquaculture 167, 1-15. Sarà, G., Mazzola, A., 1997. Effects of trophic and environmental conditions on the growth of Crassostrea gigas in culture. Aquaculture 153, 81-91.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Sarà, G., Scilipoti, D., Milazzo, M., [START_REF] Modica | The effect of mariculture facilities on biochemical features of suspended organic matter (South Tyrrhenian, MED)[END_REF]. Use of stable isotopes to investigate the dispersion of fish farming waste as a function of hydrodynamics. Series 313,261262263264265266267268269270Sarà, G., Scilipoti, D., Modica, A., Mazzola, A., 2004. Effect of fish farming wastes on sedimentary and particulate organic matter origin in a Southern Mediterranean area (Gulf of Castellammare, Sicily): a multiple stable isotope study (δ 13 C and δ 15 N). Aquaculture 234, 199-213. Sarà, G., Zenone, A., Tomasello, A., 2009

Marine Ecology Progress

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

The manuscript provides much-needed empirical data addressing the contentious but current question of whether aquaculture has an effect on local marine environment at a spatial scales larger than 10-km. We addressed this issues using long series data of the Gulf of Castellammare (Southern Tyrrhenian Sea) as an example. Nitrogen (N) and phosphorous (P) loads were calculated for the period 1970-2007, and compared to chlorophyll-a concentration as measured inside and outside the Gulf over the same period. Our study provides results about that although N and P catchment loading constantly decreased because of improved environmental management, the nutrient concentration in the Gulf increased since the establishment of aquaculture facilities in 1999. In addition, we showed that chlorophyll-a concentration in the Gulf markedly increased from 2001 onwards, while outside the Gulf was significantly lower. As all the other possible causes can be ruled out, aquaculture remains the sole explanation for the observed situation.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

1 Table 1. Transformation coefficients used to calculate gross wastes from point and nonpoint sources into dissolved N and P wastes entering annually the Gulf. Modified from [START_REF] Pagnotta | Stima dei carichi inquinanti nell'ambiente marinocostiero[END_REF] and [START_REF] Piedecausa | Simulating the temporal pattern of waste M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT production in farmed gilthead seabream (Sparus aurata), European seabass Dicentrarchus labrax) and Atlantic bluefin tuna (Thunnus thynnus)[END_REF]