Murielle Rabiller-Baudry
email: murielle.rabiller-baudry@univ-rennes1.fr

Wemsy Ndeye

Dorian Diagne

Lebordais

Wemsy Ndéye

How the experimental knowledge of the irreversible fouling distribution can contribute to understand the fluid circulation in a spiral ultrafiltration membrane

Keywords: Ultrafiltration, spiral membrane, flat membrane, hydrodynamic, critical flux, limiting flux, fouling, proteins, membrane autopsy

HAL is

INTRODUCTION

Fouling systematically occurs during ultrafiltration (UF) of skim milk as for all dairy fluids filtered by polyethersulfone (PES) based membranes. Part of this fouling is irreversible (not removed by a simple water rinsing) and is known to be only made of proteins in the particular case of skim milk UF [START_REF] Rabiller-Baudry | A dual approach of membrane cleaning based on physico-chemistry and hydrodynamics: Application to PES membrane of dairy industry[END_REF][START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF]. This irreversible part represents the target of the cleaning operation that is a well-known bottleneck of membrane processes in food industry. The physico-chemical interactions between the filtered fluid and the membrane material have a crucial role on the membrane fouling [START_REF] Bouzid | Impact of zeta potential and size of caseins as precursors of fouling deposit on limiting and critical fluxes in spiral ultrafiltration of modified skim milks[END_REF], but the membrane geometry, impacting the overall hydrodynamic, is also an important criterion. Spiral membrane is a quite complicated geometry in which the fluid circulation, responsible of part of the fouling deposit origin, is still not well understood.

In literature several approaches are reported when studying fouling distribution in close correlation with hydrodynamics. One type of approach deals with simulations at local scale, mainly using computational fluid dynamics (CFD) to investigate flow patterns [START_REF] Santos | Investigation of flow patterns and mass transfer in membrane module channels filled with flow-aligned spacers using computational fluid dynamics (CFD)[END_REF][START_REF] Rahimi | CFD and experimental studies of fouling of a microfiltration membrane[END_REF][START_REF] Li | CFD simulation of fluid flow through spacer-filled membrane module: selecting suitable cell types for periodic boundary conditions[END_REF][START_REF] Schwing | A CFD study of unsteady flow in narrow spacerfilled channels for spiral-wound membrane modules[END_REF] and foulant deposition [START_REF] Li | CFD analysis of the initial stages of particle deposition in spiral-wound membrane modules[END_REF] around feed spacers. For instance, experimental and numerical (CFD) results, obtained for twelve different flow-aligned spacer structures under different hydrodynamic conditions are investigated in order to gain insight into the flow and mass transfer profiles inside the channels of membrane modules: mass transfer results demonstrated that the modified friction factor could be used for selecting the best spacer in terms of mass transfer efficiency [START_REF] Santos | Investigation of flow patterns and mass transfer in membrane module channels filled with flow-aligned spacers using computational fluid dynamics (CFD)[END_REF]. Rahimi et al. [START_REF] Rahimi | CFD and experimental studies of fouling of a microfiltration membrane[END_REF] report CFD and experimental studies on microfiltration fouling of a blue indigo solution. A hydrophilized PVDF membrane is used and experiments are carried out at cross-flow velocities ranging from 0.5 to 1.3 m.s -1 . The fouled membranes are analysed using Scanning Electron Microscope (SEM) pictures. The predicted shear stress distributions upon the membrane, determined from a 3-D CFD modelling carried out using FLUENT 6.2 code, are used to explain the observed fouling. CFD simulations of particle deposition in a spiral-wound membrane module show that there are inherent changes in the deposition profile in the spacer-filled channel due to variations in curvature on particle transport [START_REF] Li | CFD analysis of the initial stages of particle deposition in spiral-wound membrane modules[END_REF]. According to the authors, a microscopic understanding derived from the CFD analysis could improve module design and enhance membrane module performance. All these studies suffer more or less from a lack of experimental validation at a scale compatible with a whole spiral element.

Nevertheless, some recent studies attempt to overcome this conclusion coupling CFD calculations and in-situ analysis of fouling distribution in spiral membrane [START_REF] Graf Von Der Schulenburg | Nuclear magnetic resonance microscopy studies of membrane biofouling[END_REF][START_REF] Creber | Chemical cleaning of biofouling in reverse osmosis membranes evaluated using magnetic resonance imaging[END_REF][START_REF] Creber | Magnetic resonance imaging and 3D simulation studies of biofilm accumulation and cleaning on reverse osmosis membranes[END_REF][START_REF] Creber | Quantification of the velocity acceleration factor for colloidal transport in porous media using NMR[END_REF][START_REF] Vrouwenvelder | Biofouling in spiral wound membrane systems: Three-dimensional CFD model based evaluation of experimental data[END_REF][START_REF] Vrouwenvelder | Biofouling of spiral wound nanofiltration and reverse osmosis membranes: a feed spacer problem[END_REF]. Graf von der Schulenburg et al. [START_REF] Graf Von Der Schulenburg | Nuclear magnetic resonance microscopy studies of membrane biofouling[END_REF] and Creber et al. [START_REF] Creber | Chemical cleaning of biofouling in reverse osmosis membranes evaluated using magnetic resonance imaging[END_REF][START_REF] Creber | Magnetic resonance imaging and 3D simulation studies of biofilm accumulation and cleaning on reverse osmosis membranes[END_REF][START_REF] Creber | Quantification of the velocity acceleration factor for colloidal transport in porous media using NMR[END_REF] studied the membrane fouling directly in spiral wounded membranes using nuclear magnetic resonance (NMR) for magnetic resonance imaging (MRI). This non-invasive technique enables quite thick biofilm to be identified (few microns) directly in a whole RO spiral element used for water treatment. The effective membrane surface area is quantified from images and conclusions drawn from the reduction of the feed channel observed by MRI are in reasonable agreement with simulations proposed by the authors. Vrouwenvelder et al. [START_REF] Vrouwenvelder | Biofouling of spiral wound nanofiltration and reverse osmosis membranes: a feed spacer problem[END_REF] explain that in spiral wound membrane modules, two types of pressure drop can be discriminated: the trans-membrane pressure drop (TMP) and the feed spacer channel pressure drop (FCP), also named longitudinal pressure drop. They suggest that in extensively pre-treated water of NF and RO, biofouling (mainly corresponding to biofilm of micro-organisms) is a feed channel pressure drop problem. Moreover, in a another paper [START_REF] Vrouwenvelder | Biofouling in spiral wound membrane systems: Three-dimensional CFD model based evaluation of experimental data[END_REF] they propose a 3D mathematical model showing the same trends for (i) feed channel pressure drop, (ii) biomass accumulation, (iii) velocity distribution profile resulting in regions of low and high liquid flow velocity.

So it appears from literature that among the possible origins of variation in fouling deposit in the spiral membrane and at least in the efficiency of the subsequent step of cleaning, is the important pressure drop existing along the spiral element and leading to a transmembrane pressure (TMP) gradient. Moreover, if an average cross-flow velocity can be estimated from the retentate flow rate and the thickness of the liquid channel, assumed to be that of the retentate spacers inserted between two membrane sheets, the local velocity distribution due to the occurrence of retentate spacers is far from being well understood. Of course, it is quite well known that the insertion of spacers in the liquid channel is a real need to promote provoke turbulences and to lower the overall fouling [1, 2, 4-7].

In the particular case of fouling by dairy fluids, membrane irreversible fouling is assumed to depend on both the transmembrane pressure (TMP) used and on the cross-flow velocity (v) of the feed fluid, and also probably on a combination of these two parameters. For instance, at a given average apparent constant velocity (corresponding to the velocity calculated in the free channel) we have shown in a previous study that the irreversible fouling (target of the cleaning) obtained by UF of skim milk with a PES membrane at critical TMP is half that obtained at higher TMP (including TMP slightly higher than the limiting one) [START_REF] Diagne | On the actual cleanability of polyethersulfone membrane fouled by proteins at critical or limiting[END_REF].

-Remember that the concept of critical flux, firstly introduced by Field et al. [START_REF] Field | Critical flux concept for microfiltration fouling[END_REF] in 1995 and modified by the same main author in 2011 [START_REF] Field | Critical, sustainable and threshold fluxes for membrane filtration with water industry applications[END_REF], proposes a theoretical base for the fouling mastering during filtration by the minimization of its irreversible part according to the adequate choice of the TMP and its corresponding permeate flux (J) that must be lower than a critical value (J critical ). The critical flux concept was initially developed for a steady state system with a well-mixed feed compartment such as stirred cell and, of course, spiral membranes don't achieve this condition and consequently, significant variations are expected in both radial and axial dimensions. Nevertheless, even if this concept is, at first sight, of limited applicability to spiral elements because of great deviation from ideal conditions, it has been previously shown that it can be used [START_REF] Bouzid | Impact of zeta potential and size of caseins as precursors of fouling deposit on limiting and critical fluxes in spiral ultrafiltration of modified skim milks[END_REF] and applied in a close form, more recently called "threshold flux concept" [START_REF] Field | Critical, sustainable and threshold fluxes for membrane filtration with water industry applications[END_REF] which does not rely on the establishment of steady state conditions with a homogeneous concentration in the feed -Diagne et al. [START_REF] Diagne | On the actual cleanability of polyethersulfone membrane fouled by proteins at critical or limiting[END_REF] have observed the threshold type of the critical flux in the case of skim milk UF at 50°C with a 5-10 kg.mol -1 PES membrane. At an average estimated cross-flow velocity close to 0.3 m.s -1 either in spiral and flat configuration (both in presence of turbulences that were not quantified but due to similar 1 mm thickness spacers inserted in the feed liquid channel), the critical TMP was observed to be 1.5 bar, whereas the limiting one was shown to be 3.5 bar. This means that:

-for J < J critical , the flux lowering (when compared to the water flux) is due to the superimposition of the concentration polarisation (fully reversible) and of irreversible fouling due to adsorption (in the same amount as without any applied pressure).

-for J > J critical the irreversible part of the fouling severely increases. Nevertheless, at this average cross-flow velocity estimated to be 0.34-0.37 m.s -1 , using a 4333 spiral membrane (4.3" diameter and 33" length), when the membrane works at an average TMP of 2.6 bar, the TMP along the membrane varies in the 3.7-1.5 bar range because of the 2.2 bar pressure drop along the spiral element. Consequently, in this particular case, the main part of the membrane filters at flux higher than the limiting flux (associated to the limiting TMP, TMP limiting = 3.5 bar) and only a little area is submitted to conditions leading to filtration at critical flux (associated to the critical TMP, TMP critical = 1.5 bar).

Even if the impact of the pressure drop and of the turbulences due to spacers on the local fouling/cleaning is commonly suggested, experimental investigations remain particularly difficult to hold in order to confirm this assumption and mainly global observations are commonly available, except those from NMR studies dealing only with quite thick fouling whereas in the case of skim milk UF the irreversible fouling layer is equal or lower than one micrometre.

This paper aims at evidencing how the knowledge of the distribution of a very thin layer of irreversible fouling can help to go ahead in the understanding of the fluid circulation in a spiral membrane. The membrane destruction for analysis of fouling due to proteins quantitatively determined by mean of FTIR-ATR is an integrant part of the approach together with the characterisation of local hydrodynamic using UF experiments performed in small plate and frame module working in dedicated conditions.

EXPERIMENTAL

Solutions

The skim milk used is a commercial one (UHT, Lait de Montagne, Carrefour, France) containing an average of 32 g.L -1 proteins and 48 g.L -1 carbohydrates (mainly lactose) and only tracks of lipids (< 0.5 %).

Water used either for solution preparation and membrane rinsing is deionised and 1 µm filtered. Its conductivity is always lower than 1 µS.cm -1 .

Membrane and ultrafiltration loops

Membrane material and membrane preparation

A PES membrane (5-10 kg mol -1 , HFK-131, Koch, USA) is selected because it is the worldwide reference for the skim milk UF application at industrial scale. At laboratory scale the spiral module is chosen in order to have a filtering area of several square meters (6.5 m 2 , 4333 K131 VYV module). It can be noticed that according to their date of acquisition, the spiral membranes, are made of 3 double sheets of membranes (old version, bought in 2003, membrane reference KM8243083017V, corresponding to the spiral membrane used here for skim milk UF) then they are made of 4 double sheets of membranes (new version buy after 2006, corresponding to source of flat membranes used in this study, see below).

-Spiral membrane:

The spiral membrane (old version, called CIP-1, 2003) used for UF of skim milk is installed on the UF pilot of our laboratory since December 2003. It is regularly checked that the membrane permeability to water at 50°C remains constant at 50 ± 5 L.h -1 .m -2 .bar -1 and that the permeability in skim milk remains constant at 15 ± 1 L.h -1 .m -2 .bar -1 at 50°C. This membrane has been regularly cleaned, mainly with alkaline detergents at pH 11.5-12.0.

During the 9 years of its service life, the cleaning time by alkaline bleach at 200 ppm in total free chlorine has been negligible thus limiting the chemical ageing of the membrane material.

Between each UF, the membrane has been stocked in sodium metabisulfite (5 g.L -1 ) to avoid micro-organisms growth. Even if the permeability to water remains always correct, we have autopsied the membrane, to understand its fouling behaviour.

-Flat membranes:

Several flat membranes of 127 cm 2 filtering area and spacers (V type, 1 mm thickness) are sampled in a second virgin spiral membrane of the same reference as described above (new version commercialised after 2006). A new flat membrane is used for each experiment and is firstly rinsed with warm water to remove the preservative and then inserted in a plate and frame module. Secondly a procedure of compaction is applied to reach a stable water flux.

The compaction step consists in UF of deionised water during 6 h at 50°C applying an increasing TMP from 1 to 4 bar. The permeability to water is measured on all flat membranes.

We have observed that the water flux of the flat membrane depends on the location of the membrane coupon in the spiral membrane. Figure 1 highlights this phenomenon, evidencing that the flux is greater in the spiral sheet centre and can be very low on the sides (especially near the glue, our hypothesis is that this difference in the heterogeneousness of the membrane permeability could be partly attributed to the diffusion of the glue on the edges of the membrane together with heterogeneity of the membrane material itself due to its fabrication process, but experiments are needed to confirm this assumption). This allows establishing mapping of the permeability distribution in a virgin spiral membrane. As shown on Figure 1 the variation on the membrane permeability can be 10 % or 20 % depending on the sheet in the same spiral membrane.

A similar procedure was punctually applied to another virgin spiral membrane allowing confirming the general trends in the description of the permeability distribution and up to 50% variation can sometimes be observed.

UF with the spiral membrane

The spiral membrane is installed on a pilot provided by TIA (TIA 3093, Bollène, France)

shown on Figure 2. The two pumps impose a cross-flow velocity close to 0.37 m.s -1 (see Appendix 1) in the recirculation loop thanks to the 10.5 m 3 .h -1 flow rate.

In such configuration, the feed is introduced perpendicularly to the membrane axis and against a sharp right angle provoking turbulences in the carter. Nevertheless the membrane entrance is few centimetre after this angle and a stable regime is probably established before the membrane entrance (Figure 2, Figure 3).

The average TMP is classically calculated as the mean value when considering the membrane inlet TMP and the membrane outlet TMP. Nevertheless, it must be kept in mind that the pressure drop along the membrane element is 2.2 bar that is not a negligible value by comparison to the average TMP (see below).

Fouling of the PES membrane is achieved by UF of skim milk during 3 h at 50°C in batch mode, meaning here that both the retentate and the permeate are fully recycled in the feed tank. This corresponds to a volume reduction ratio (VRR) of 1. Because of the dead volume of the pilot, 24 L is the minimum volume to be processed to allow filtration without any vortex in the feed tank and some sampling. Several TMPs ranging from 1 to 4 bar can be applied (see results). The membrane flux (J UF ) is followed all over the skim milk UF. The irreversible part of the fouling is evaluated from the water flux measurement (J irrev,initial ) after a careful water rinsing.

UF with the flat membranes

The plate and frame module (Ray-Flow X100, Novasep-Process, France) allows using two membranes in series. Two new membranes (2 x 127 cm 2 ) are used for each experiment. After the compaction procedure, the water flux (J 0 ) is further used as reference for the pristine membrane.

Fouling of two PES membranes is simultaneously achieved by UF of skim milk during 3 h at 46°C ± 1°C in batch mode. Because of the dead volume of the pilot, the skim milk volume used is the minimum one, equal to 4 L. The average cross flow velocity is v= 0.34 m.s -1 and spacers are added in the liquid channel, in order to be, as close as possible, in the same hydrodynamic conditions as with the spiral membrane. Various TMPs ranging from 1 to 4 bar are applied (see results) and the membrane flux (J UF ) is followed all over the skim milk filtration.

Then membranes are carefully rinsed with deionised water and the final water flux (J irrev, initial ) is determined for both membranes, evidencing the good agreement between the two membrane behaviors.

Resistances due to membrane and fouling (both overall and irreversible one remaining after water rinsing) are determined from the well-known resistance in series model.

Determination of the limiting and critical/threshold fluxes

The protocol used has been already described in [START_REF] Bouzid | Impact of zeta potential and size of caseins as precursors of fouling deposit on limiting and critical fluxes in spiral ultrafiltration of modified skim milks[END_REF] for the spiral membrane and adapted for the flat membrane as described in [START_REF] Diagne | On the actual cleanability of polyethersulfone membrane fouled by proteins at critical or limiting[END_REF]. Some details are briefly repealed here for sake of clarity. Permeate fluxes are measured during skim milk UF by increasing step by step the TMP from 1 to 4 bar (Figure 4). The permeate flux at plateau value corresponds to the limiting flux (J limiting ) and in the following the (TMP limiting , J limiting ) point is determined as the first point for which the plateau value of flux is reached. The critical/threshold point is determined from the shape of the J vs TMP curve: the relationship remains linear below the (J critical/threshold , TMP critical/threshold ) point allowing its determination. It is noticeable that without retentate spacers in the liquid channel, the limiting flux is quite immediately reached highlighting the role of local turbulences due to the presence of spacers when the cross-flow velocity is so low that only a laminar regime is established from the fluid circulation.

Quantification of residual proteins on membranes

We have previously shown that the irreversible fouling is only made of proteins as we have not been able to show the presence of other compounds on the membrane [START_REF] Rabiller-Baudry | A dual approach of membrane cleaning based on physico-chemistry and hydrodynamics: Application to PES membrane of dairy industry[END_REF][START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF].

Consequently, the fouling seems to be valuably appreciated from the protein quantification that can be achieved by an infra-red technique according to a procedure established in our laboratory [START_REF] Rabiller-Baudry | Coupling of SEM_EDX and FTIR-ATR to (quantitatively) investigate organic on porous organic composite membranes[END_REF] and previously described in [START_REF] Rabiller-Baudry | A dual approach of membrane cleaning based on physico-chemistry and hydrodynamics: Application to PES membrane of dairy industry[END_REF][START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF]. The calibration range is 1 -350 µg.cm -2 .

Infra-red (FTIR) analyses are performed using the Attenuated Total Reflection mode (ATR) directly on fouled membranes. The FTIR-ATR spectra are recorded between 4000 and 600 cm -1 with a spectrometer provided by Perkin-Elmer (Paragon 1000, spectrum for windows software) equipped with a ZnSe crystal with an incidence angle of 45° allowing 12 reflections.

The background spectrum is recorded in the air. The conditions of acquisition are as follows: 20 scans, 2 cm -1 resolution. The membrane samples are carefully dried under dynamic vacuum before registration.

Two specific procedures are used: one for the flat fouled membranes and the other for the spiral fouled membrane.

-Specific procedure for the flat membranes

To take into account possible variations of the irreversible fouling amount due to small variations of the velocity profile in the plate and frame module [START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF] an average value is calculated from the quantification of the fouling deposit measured on nine equivalent pieces cut in the membrane. Generally the difference does not exceed 3 µg.cm -2 between the 9 samples of a 127 cm 2 membrane coupons. As two flat membranes are simultaneously fouled on the plate and frame module, it has been preliminary checked that the average amount of proteins are similar for the two membranes in series.

-Specific procedure for the spiral membrane

The spiral membrane after fouling by skim milk and rinsing by deionised water can also be analyzed. This procedure can only be one time applied at the end of the overall study. It can be underlined that the limitation is only due to economic considerations and time consumption and not because of technical aspects. The overall spiral membrane (6.5 m 2 ) is cut in 336 pieces of 127 cm 2 area. Only the center of each sample is analyzed by FTIR-ATR for quantification purpose (of course each sample could be divided in 9 smaller pieces as for the flat membranes, but it has been checked that the variations were not very different and a single result seems quite acceptable for the followed purpose). This autopsy allows establishing a mapping of the irreversible fouling that would be compared to the fouling amount obtained for the flat membranes at various TMPs (see results and discussion).

RESULTS AND DISCUSSION

Fouling

Hydraulic characterisation

The limiting and critical points are determined either with the flat and spiral membranes. It has been checked that in both cases the critical TMP is 1.5 bar whereas the limiting TMP is 3.5 bar.

Then UF has been performed at constant TMP values including TMP critical and TMP limiting , but also an intermediate value of 2.0 bar. A fourth value greater than TMP limiting is also tested, namely 4.0 bar. Table 1 summarizes the resistances due to the overall fouling (R f ) for the set of experiments. R f can be considered as roughly constant from 1.5 bar to 4.0 bar, regardless of the membrane geometry.

For a given TMP of fouling and after water rinsing, the remaining fouling resistance (R irrev,initial ) is roughly the same for both flat and spiral membranes, even if it seems to slightly increase with the TMP increase in this last case. More or less, 35 % of the fouling is irreversible in the 1.5-4.0 bar TMP range.

Finally, on a global hydraulic point of view, fouling obtained with the two membranes geometries can be considered as similar.

Physico-chemical characterisation

Table 2 shows the residual amount of proteins after fouling of flat membranes obtained at constant TMP ranging from 1.5 to 4.0 bar and after water rinsing. Contrary to hydraulic characterisation, this quantification highlights differences according to the TMP. The protein amount at 1.5 bar (critical TMP) is about half that obtained for higher TMPs.

Similar quantifications are performed on the spiral membrane fouled at an average TMP of 2.6 bar (3.7 bar inlet and 1.5 bar outlet). This spiral membrane is made of 3 sheets of membrane corresponding to 6 different flat membranes assembled by two, back to back (Figure 3, Figure 5).

Figure 6 shows the distribution of the residual protein amount according to the location on the six membranes issued from the autopsied fouled spiral membrane. At first sight, regardless of the membrane, the zone in close vicinity to the permeate collector axis is always the most fouled one. The other parts of the membranes are heterogeneously fouled.

In a first attempt of simplification, an average value is calculated representing 85 % of the membrane area (except values in close vicinity to the collector axis), that corresponds to 40 ± 20 µ g.cm -2 and (Figure 7).

The fouling can also be discussed with more accuracy. For instance, considering an average TMP of 2.6 bar, the expected amount of the residual fouling calculated from the flat membrane experiments would be close to 22 µg.cm -2 (Table 2) that is half of the average value calculated above. Figure 6 and Figure 7 show clearly that some zones of the spiral membrane are fouled with this protein amount but some others are much more fouled.

In order to propose a more accurate analysis of the irreversible fouling distribution in the spiral membrane, attempt of correlation with the local TMP would be interesting. As no local measurement can be available, an attempt of calculation is made. On an experimental point of view the pressure drop on the retentate side is measured at 2.2 bar. It can be reasonably assumed that the pressure drop is linear along the membrane length (that must be understood as parallel to the direction of the permeate axis, in other words the axial direction, Figure 5b).

As permeate side is opened to air (and then set at atmospheric pressure), the variation of the permeate pressure with the location on membrane can be is neglected at first sight.

Nevertheless, it is probable that some variations also occurred with respect to the membrane wounding and the presence of permeate spacers that are able to generate some turbulences and consequently a pressure drop. It can be guessed that near the permeate axis the pressure is that of atmosphere but at the end of each sheet the pressure could be greater (Figure 5c, Figure 5d). Consequently the TMP is probably a little overestimated in our approach for the zones far from the permeate axis. This hypothesis leads to the assumption that firstly TMP mainly depends on the position along the collector axis (feed flow direction, axial dimension) and then decreases from the inlet to the outlet. Secondly, it is considered that for a given distance from the membrane inlet (feed flow direction along the permeate axis) the TMP variation with the distance (d, Figure 7) from the permeate axis is negligible (in other words the radial dimension, Figure 5c and Figure 5d). It is then inferred that the spiral membrane can be divided in four main zones of TMP, defined by reference to the 127 cm 2 samples that will be cut in the membrane for autopsy purpose and corresponding to "local" TMP values (at sample centre) of 3.5, 3.0, 2.5 and 2.0 bar, respectively (Figure 6). An average of protein amount is then determined for each "local" TMP (Table 2). In the spiral module the same protein average amount is obtained for TMP in the range 2.0 to 3.5 bar that is in good agreement with experiments on flat membrane. But, it is also observed that the level of the irreversible fouling is significantly higher in the spiral membrane than with the flat one for the same TMP range.

Looking carefully at the quantification of the protein amount in the spiral membrane, a fouling distribution can be observed for a given average local TMP on a single membrane sheet (Figure 6). All membrane sheet demonstrate different fouling amount. The possible origins of this fouling distribution are discussed below.

Discussion on the possible origin of fouling distribution

Different hypotheses can be made to explain these differences either based on the initial membrane material heterogeneity and on the variable local hydrodynamic conditions.

. Heterogeneity of the virgin membrane permeability

The heteregoneity of the membrane material is observed from water permeability measurements performed on several membrane pieces that are sampled in a spiral virgin membrane (Figure 1). Similar trends are observed for all sheets of this spiral membrane and on all sheets of a second spiral membrane (not shown here). In our laboratory we performed such measurements systematically from more than six years now, and finally, the permeability is generally higher in the spiral membrane center, in other words far from the edges where is applied the glue between two membranes of a same double sheet.

It can be noticed that FTIR-ATR spectrum of membrane (active layer side) allows to demonstrate the presence of glue at membrane edges (superimposition of FTIR spectra of PES and additives containing C=O bound that could correspond to polyurethane glue type).

We guess that the membrane permeability is probably decreased by the diffusion of the glue from the edges toward the center, but we have failed to prove its presence on the active layer side when going away from the center. Another explanation could be the heterogeneity of the active layer thanks to the process achieved to obtain polymer large sheets.

Nevertheless, permeability in skim milk is always the same regardless of the initial water permeability of the membrane coupon. So in the following we suggest to ignore this assumption as a possible main origin of fouling variation.

. TMP gradient in the spiral membrane

The second hypothesis to explain the heterogeneity of the irreversible protein amount on the spiral membrane is linked to the TMP variation as discussed in the following.

Generalising results obtained with the flat membranes at various TMPs (Table 2), we can consider that all TMPs higher than the critical TMP might lead to the same fouling amount.

Because of the pressure drop, the center of all pieces sampled in the spiral membrane (location of the protein quantification) are in the 2.0 -3.5 bar range, corresponding to a TMP range higher than the critical TMP (1.5 bar). Nevertheless a small part of the membrane outlet is set at the critical TMP and is probably fouled in a lower extend but this phenomenon can be considered as negligible for this particular membrane.

Consequently the variation of the fouling amount from the inlet to the outlet at a given distance from the permeate collector axis (radial dimension, Figure 8) cannot be mainly attributed to the TMP variation and the main origin must be found elsewhere.

. Gradient of velocity/turbulences in the spiral membrane

The third hypothesis to explain the heterogeneity of the irreversible protein amount on the spiral membrane is linked to the fluid distribution and fluid velocity/turbulences in this quite complex geometry. Of course, as the filtered fluid is newtonien, it is not compressible. So in abscence of local turbulences, the fluid velocity must remain constant along the membrane length. But to avoid a very important fouling, lowering the process productivity, retentate spacers are added in the liquid channel to promote local turbulences. The result would be the creation of a local field of velocity/turbulences.

It can be underlined that up to now computational fluid dynamic (CFD) is not able to propose theoretical approach of the fluid velocity profile in a whole spiral membrane and calculations are mainly achieved in close vicinity of a single cross of the retentate spacers, as seen above.

Even though, the validation of theoretical proposal will always be a need. The mapping of fouling has been shown to be an experimental way to study the velocity field in the case of a flat membrane inserted in a plate and frame module, by comparing the fouling amount and the velocity gradient calculated from CFD [START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF]. This methodology has also been validated to discuss the shear stress in dynamic filtration with circular membrane inserted on a V-Sep vibrating module [START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF]. These experimental results are in good accordance with some NMR observations achieved on a whole spiral membrane fouled by micro-organisms leading to a biofilm (in this case the fouling layer was very thick contrary to the thickness of the irreversible deposit of proteins of the present study) [START_REF] Graf Von Der Schulenburg | Nuclear magnetic resonance microscopy studies of membrane biofouling[END_REF][START_REF] Creber | Chemical cleaning of biofouling in reverse osmosis membranes evaluated using magnetic resonance imaging[END_REF][START_REF] Creber | Magnetic resonance imaging and 3D simulation studies of biofilm accumulation and cleaning on reverse osmosis membranes[END_REF][START_REF] Creber | Quantification of the velocity acceleration factor for colloidal transport in porous media using NMR[END_REF].

The discussion proposes here is in the continuity of the experimental approch described in [START_REF] Delaunay | Mapping of protein fouling by FTIR-ATR as experimental tool to study membrane fouling and fluid velocity profile in various geometries and validation by CFD simulation[END_REF].

Considering a given distance from the permate axis (d, radial dimension), the fouling varies from the inlet to the outlet but it is not in a continuous trends (Figure 8a). Generally in a first section (further called zone A) the fouling at inlet is greater than the fouling at outlet, then the fouling is roughly the same at inlet and outlet of the membrane (Zone B), finally the fouling is systematically higher at outlet than at inlet (Zone C) (Figure 8b).

From one membrane to another, the size of each three zones varies as shown in Table 3.

Nevertheless as first attempt we can considered that zone A corresponds to the first 300 x 10 -3 m, zone B to the 300 x 10 -3 -1000 x 10 -3 m range and zone C to the last part of the unwounded membrane in the 1000 x 10 Considering that the lower the fouling is the higher velocity/turbulences are, it can be drawn that (i) in zone A, velocity/turbulences increase from inlet to outlet, (ii) in zone B, there is no significant variation of velocity/turbulences from inlet to outlet, (iii) in zone C, velocity/turbulences decrease from inlet to outlet. Nevertheless, if similar trends are shown, our results also highlight the difference in local fluid distribution from one membrane sheet to another, as fouling level is different from one membrane to another. Li et al. [START_REF] Li | Mitigating the Curvature Effect of the Spacer-Filled Channel in a Spiral-Wound Membrane Module[END_REF] have proposed an interesting discussion about the curvature effect of the spacer in a spiral membrane. They finally suggest to optimize the design of the spiral module by varying that of the spacers from one feed channel to another, aiming at the decrease of the imbalance shear stress between the inner and outer membrane walls. We agree with such proposals. If such module could be fabricated, the fouling mapping we proposed in this paper would be a way to experimentally establish the proof of concept.

CONCLUSION

The approach discussed in this paper aims at show that analysis of a thin layer of irreversible fouling in a spiral membrane, each zone of which filtering in conditions over the critical one, is a powerful approach to deal with the role of the velocity/turbulences independently of the transmembrane pressure gradient. Considering that the lower the fouling is the higher the velocity/turbulences are, it can be drawn that the 6.5 m 2 membrane studied here made of three double sheets of membrane can be roughly described from only one sheet. Each sheet can be divided in 3 zones having more or less the same size when comparing one sheet to another.

These zones are defined according to their distance toward the permeate collector axis (radial dimension) and not regarding the position according to inlet or outlet of the spiral element (axial dimension). Fouling amount suggests that the velocity/turbulences are constant in the main middle part of the membrane (zone B, 50% of the global area). But for the area in close vicinity to the permeate axis, and corresponding to 30 % of the whole membrane (zone A), the fouling decreases from inlet to outlet meaning that velocity/turbulences increase. On the contrary, for the area far from the permeate axis and corresponding to the last 20 % of the membrane (zone C) the fouling increases from inlet to outlet meaning that velocity/turbulences decrease. Differences between the behaviors of zone A and zone C could perhaps be related to an additional gradient of centrifuge forces. To go ahead, a similar study applied to several a spiral membranes of same area but made of four double sheets of membranes instead of three in the present case is in progress, also taking into account other TMP gradient ranges applied to the filtering area. Moreover, in order to have a better insight, we need now to establish a correlation between the fouling amount and controlled velocity/turbulences in order to appreciate the real gradient in a more accurate way. A first limitation is already observed depending on the membrane material itself, as irreversible fouling clearly increases with membrane ageing [START_REF] Rabiller-Baudry | On the use of microwaves to accelerate ageing of an ultrafiltration PES membrane by sodium hypochlorite to obtain similar ageing state to that obtained for membranes working at industrial scale[END_REF] and consequently the age of the membrane material must be carefully mastered in addition to the control of hydrodynamics. This work is also currently in progress. Then two assumptions are considered to model the membrane.

Hypothesis 1: In the spiral membrane the feed flow is equally distributed along the surface seen at entrance. In other terms the feed see the whole L length at membrane entrance (Figure A2)

Figure A2: Hypothesis of equal distribution of fluid at spiral membrane entrance Hypothesis 2: The thickness of the liquid channel is that of the retentate spacer as the spiral membrane is very compact. Consequently, the area in which the fluid is distributed is a small rectangle (Figure A3). Then Area = 1 mm x 7.74 m 7.74 x 10 -3 m 2 Consequently, the cross-flow velocity v = Qfeed / area = 2.92 x 10 -3 / 7.74 x10 -3 =0.37 m.s -1 * TMP in flat configuration is the average value calculated from TMP at inlet and outlet, the pressure drop in the plate and frame module is negligible (less than 100 mbar). each value is the average of 9 analyses. ** TMP in the spiral configuration is the local value calculated from TMP at inlet and outlet and assuming a linear decrease of the pressure drop of 2.2 bar along the spiral membrane element. In this case the average TMP is 2.6 bar and the TMP at inlet is 3.7 bar. Each value is the average of 84 analyses.

9 .

 9 -3 -1200 x 10 -3 m range. The 3 zones (A, B, C) are schematically shown Figure For the most inner membrane sheet (F1C1), zone A corresponds more or less to the two first turns of the membrane in the wound, zone B to the 3 and 4 turns plus part of turn number 5, whereas zone C corresponds to the last part of turn 5 and to turn 6. Similar drawn can be established for the other membrane sheets and clearly the three zones are not correlated to the upper or lower positions in the carter toward fluid entrance.

Appendix 1 -

 1 FigureA1describes the unwounded spiral membrane as an equivalent rectangle of the same area as the whole spiral membrane. This membrane is made of 3 double sheets of membrane • Length of permeate axis is 84 cm • Membrane area is 6.5 m 2• Membrane modelized as a rectangle of 6.5 m 2 (allowing the determination of L)• Area = 84x10 -2 x L = 6.5 L = 7.74 m

Figure A3 :Figure captions Figure 1 :

 A3captions1 Figure A3: Hypothesis on the thickness of the liquid channel in the spiral membrane

Figure 2 .

 2 Figure 2. Scheme of the UF pilot equipped with the 4333 spiral membrane allowing the feed entrance in the carter first perpendicularly to the membrane axis.

Figure 3 .

 3 Figure 3. Pictures of the spiral membrane of this study -(a) inserted in the stainless steel carter on the pilot -(b) evidencing the distribution system at outlet of the spiral membrane.

Figure 4 -

 4 Figure 4 -Typical UF of skim milk at 46°C, VRR= 1, 0.34 m.s -1 with step by step increase of TMP for a flat membrane inserted in the plate and frame module with (•) or without ( ) retentate spacers of 1 mm inserted in the liquid channel.

Figure 5 .

 5 Figure 5. Schemes of a spiral membrane (a) with 3 double sheets highlighting the different channels in which are inserted retentate or permeate spacer, respectively. Each membrane is labelled according to the same nomenclature as those used for the quantification of protein amount for the autopsied membrane. F1C1 is the innermost membrane whereas F3C2 is the outermost one -(b) single sheet made of two membranes glued back to back and direction of feed flow-(c) single sheet of membrane and direction of permeate when crossing the membrane -(d) single sheet of membrane and direction of permeate moving toward the permeate axis in the permeate channel shared by two membranes glued by to back.

Figure 6 .Figure 7 .

 67 Figure 6. Mapping of the protein irreversible deposit in the spiral membrane determined from FTIR-ATR quantification (protein amount in µg.cm -2 ) according to the location in the spiral membrane. The local TMP is calculated from the assumption of a linear pressure drop decrease. The membrane labels are defined on Figure 5. (nd = not determined)

Figure 8 :

 8 Figure 8 : Amount of protein irreversible deposit on the spiral membrane versus the distance from the permeate collector axis for inlet (♦, local TMP = 3.5 bar) and outlet (×, local TMP = 2.0 bar) samples . (a) All membranes -(b) F1C2 membrane (see Figure 5 for definition). The estimation of the local average TMP is calculated from TMP at inlet and outlet and assuming a linear decrease of the pressure drop along the spiral membrane element.

Figure 9 :

 9 Figure 9: The 3 zones A, B, C (defined Table3) on a membrane sheet and variation of fouling from which is deduced variation of velocity/turbulences

Figure 2 .Figure 4 -

 24 Figure 2. Scheme of the UF pilot equipped with the 4333 spiral membrane allowing the feed entrance in the carter first perpendicularly to the membrane axis.

Figure 5 .

 5 Figure 5. Schemes of a spiral membrane (a) with 3 double sheets highlighting the different channels in which are inserted retentate or permeate spacer, respectively. Each membrane is labelled according to the same nomenclature as those used for the quantification of protein amount for the autopsied membrane. F1C1 is the innermost membrane whereas F3C2 is the outermost one -(b) single sheet made of two membranes glued back to back and direction of feed flow-(c) single sheet of membrane and direction of permeate when crossing the membrane -(d) single sheet of membrane and direction of permeate moving toward the permeate axis in the permeate channel shared by two membranes glued by to back.

Figure 6 .

 6 Figure 6. Mapping of the protein irreversible deposit in the spiral membrane determined from FTIR-ATR quantification (protein amount in µg.cm -2 ) according to the location in the spiral membrane. The local TMP is calculated from the assumption of a linear pressure drop decrease. The membrane labels are defined on Figure 5. (nd = not determined)

Figure 7 .

 7 Figure 7. Irreversible amount of proteins on the spiral membrane versus the distance from the permeate collector axis (at a given distance each point correspond to a local TMP for the 6 membrane sheets). The estimation of the local average TMP is calculated from TMP at inletand outlet and assuming a linear decrease of the pressure drop of 2.2 bar along the spiral membrane element.

2 )Figure 9 :

 29 Figure 9: The 3 zones A, B, C (defined Table3) on a membrane sheet and variation of fouling from which is deduced variation of velocity/turbulences

Table 3 )

 3 on a membrane sheet and variation of fouling from which is deduced variation of velocity/turbulences

	Figures

P Inlet P Outlet Zoom 1 sheet = 2 membranes Jpermeate membrane surface permeate axis Qpermeate // membrane surface permeate axis P end sheet P axis Zoom 1 sheet = 2 membranes

	Retentate spacer Retentate spacer Retentate spacer		membrane membrane membrane
		F2C2 F2C2
			F2C1 F2C1
	F3C1 F3C1
			membrane membrane membrane
		Permeate Permeate Permeate
	F3C2 F3C2	collector axis collector axis collector axis	feed / retentate feed / retentate
	(outermost) (outermost)
			F1C2 F1C2
		F1C1 F1C1
		(innermost) (innermost)
	Winding direction Winding direction		Permeate spacer Permeate spacer Permeate spacer

Table 3

 3 ) on a membrane sheet and variation of fouling from which is deduced variation of velocity/turbulences

	Inlet			Outlet
	TMP(bar)
	3.5	3.0	2.5	2.0	F1C1
	d (mm)	permeate axis			turn
	45				1
	135	zone A
	225	fouling decrease			2
	315
	405				3
	495	zone B
	585				4
	675	constant fouling
	765
	855				5
	945
	1035	zone C
	1125	fouling increase			6
	1215

Table 1 .

 1 Resistances of overall (R f ) and irreversible (R irrev,initial ) fouling due to skim milk UF at 46 -50 °C at several constant TMP(*) for the spiral and flat membranes * TMP in spiral configuration is the average value calculated from TMP at inlet and outlet in presence of a 2.2 bar pressure drop. The pressure drop in the plate and frame module is negligible (less than 100 mbar). ± 2 41 ± 1 45 ± 2 47 ± 5 49 ± 1 46 ± 3 50 ± 1 R irrev., initial 11 ± 2 15 ± 3 13 ± 3 17 ± 2 17 ± 3 19 ± 3 16 ± 3 20 ± 3

	TMP (bar) 1.5	2.0	3.5	4.0
	Resitance	Spiral Flat	Spiral Flat	Spiral Flat	Spiral Flat
	(10 12 m -1 )
	R f	40 ± 5 44

Table 2 .

 2 Protein amount (from FTIR-ATR) in the initial irreversible fouling due to skim milk UF at different TMP for the flat membranes (*) and the spiral one (**).

	TMP (bar)	1.5	2.0	2.5	3.0	3.5	4.0
	[Protein] (µg.cm -2 )	13 ± 2	22 ± 2	-	-	23 ± 2	26 ± 3
	flat membrane
	[Protein] (µg.cm -2 )		38 ± 4	35 ± 5 37 ± 5	37 ± 5
	spiral membrane

Table 3 :

 3 Size (according to the distance toward the permeate collector axis, d in 10 -3 m) of zones A, B, C (defined on Figure 8b) for each membranes. (nd = not determined) ŽĨ ŝƌƌĞ|ĞƌƐŝďůĞ ĨŽƵůŝŶŐ ĚƵĞ ƚŽ ƉƌŽƚĞŝŶƐ ŝƐ ƐŚŽǁŶ ŝŶ Ă ƐƉŝƌĂů ŵĞŵďƌĂŶĞ Ͳ Ͳ ĨŽƵůŝŶŐ ĚŝƐƚƌŝďƵƚŝŽŶ ŝƐ ŶŽƚ ƌĞůĂƚĞĚ ƚŽ ƉƌĞƐƐƵƌĞ ŝŶ ĐŚŽƐĞŶ ĨŝůƚƌĂƚŝŽŶ ĐŽŶĚŝƚŝŽŶƐ Ͳ Ͳ ĨŽƵůŝŶŐ ĚŝƐƚƌŝďƵƚŝŽŶ ŝƐ ŵĂŝŶůLJ ƌĞůĂƚĞĚ ƚŽ |ĂƌŝĂƚŝŽŶƐ ŽĨ |ĞůŽĐŝƚLJͬƚƵƌďƵůĞŶĐĞƐ Ͳ Ͳ ĨƌŽŵ ĨŽƵůŝŶŐ ĚŝƐƚƌŝďƵƚŝŽŶ ƚŚĞ ŚĞƚĞƌŽŐĞŶĞŝƚLJ ŽĨ ĨůƵŝĚ ĚŝƐƚƌŝďƵƚŝŽŶ ŝƐ ĚŝƐĐƵƐƐĞĚ Ͳ Ͳ Ͳ |ĂƌŝĂƚŝŽŶ ŽĨ |ĞůŽĐŝƚLJͬƚƵƌďƵůĞŶĐĞƐ ŝƐ ƉƌŽƉŽƐĞĚ ŽŶ Ă ƋƵĂůŝƚĂƚŝ|Ğ ƉŽŝŶƚ ŽĨ |ŝĞǁ Ͳ

	membrane Zone A	Zone B	Zone C
	F1C1	0-200	200-1200 nd
	F1C2	0-350	400-900 950-1200
	F2C1	0-250	300-1050 1100-1200
	F2C2	nd	200-900	950-1200
	F3C1	0-350	400-1100 1100-1200
	F3C2	0-350	400-900	950-1200

Acknowledgements

This study was financially supported by the "Region Bretagne" (Brittany) program DURAMEM for the PhD grant of NWD, France.


C Escolano
email: cescolano@gmail.com

M Navarro-Gil

J Garcia-Campayo

M Congedo

J Minguez

The Effects of Individual Upper Alpha Neurofeedback in ADHD: An Open-Label Pilot Study

Keywords: ADHD, neurofeedback, individual upper alpha, cognitive performance, EEG

Standardized neurofeedback (NF) protocols have been extensively evaluated in ADHD. However, such protocols do not account for the large EEG heterogeneity in ADHD. Thus, individualized approaches have been suggested to improve the clinical outcome. In this direction, an open-label pilot study was designed to evaluate a NF protocol of relative upper alpha power enhancement in fronto-central sites. Upper alpha band was individually determined using the alpha peak frequency as an anchor point. 20 ADHD children underwent 18 training sessions. Clinical and neurophysiological variables were measured pre-and post-training. EEG was recorded pre-and post-training, and preand post-the training trials within each session, in both eyes closed resting state and eyes open task-related activity. A power EEG analysis assessed long-term and within-session effects, in the trained parameter and in all the sensors in the (1-30) Hz spectral range. Learning curves over sessions were assessed as well. Parents rated a clinical improvement in children regarding inattention and hyperactivity/impulsivity. Neurophysiological tests showed an improvement in working memory, concentration and impulsivity (decreased number of commis-

Introduction

Attention-deficit/hyperactivity disorder (ADHD) is a behavioral disorder characterized by symptoms of inattention, impulsivity and hyperactivity according to DSM-IV (American Psychiatric Association 1994). This disorder is one of the most common psychiatric disorders of childhood, affecting up to 5% of children worldwide [START_REF] Polanczyk | The worldwide prevalence of ADHD: a systematic review and metaregression analysis[END_REF], presenting about 40-60% persistence in adolescence and adulthood [START_REF] Faraone | The age-dependent decline of attention deficit hyperactivity disorder: a metaanalysis of follow-up studies[END_REF]. Deficits in executive functioning, working memory and response inhibition have been repeatedly reported [START_REF] Barkley | Behavioral inhibition, sustained attention, and executive functions: constructing a unifying theory of ADHD[END_REF][START_REF] Martinussen | A meta-analysis of working memory impairments in children with attention-deficit/hyperactivity disorder[END_REF][START_REF] Castellanos | Neuroscience of attentiondeficit/hyperactivity disorder: the search for endophenotypes[END_REF].

The most accepted treatments for ADHD are stimulant medication and behavior therapy [START_REF] Barkley | Behavioral inhibition, sustained attention, and executive functions: constructing a unifying theory of ADHD[END_REF]. Stimulant medication has emerged as the primary treatment for the core symptoms of ADHD, however some children do not respond to medication or suffer from side effects such as headache, dizziness, insomnia, anxiety and gastroenterological problems [START_REF] Graham | European guidelines on managing adverse effects of medication for ADHD[END_REF]. In addition to that, the long-lasting effects of both stimulant medication and behavior therapy are uncertain, with some studies reporting limited effects [START_REF] Wang | Long-term stimulant treatment affects brain dopamine transporter level in patients with attention deficit hyperactive disorder[END_REF][START_REF] Molina | The MTA at 8 years: prospective follow-up of children treated for combined-type ADHD in a multisite study[END_REF]. Neurofeedback (NF) is a promising alternative treatment for ADHD [START_REF] Arns | Evaluation of neurofeedback in ADHD: The long and winding road[END_REF][START_REF] Loo | Clinical utility of EEG in attentiondeficit/hyperactivity disorder: a research update[END_REF].

NF provides the subjects with real-time feedback covarying with their own brain activity, thus promoting the self-regulation of brain activity by means of an operant conditioning paradigm. The rationale behind NF training in ADHD is the electrophysiological evidence collected over the last decades of abnormal brain oscillations in comparison to normal controls (see [START_REF] Barry | A review of electrophysiology in attention-deficit/hyperactivity disorder: I. Qualitative and quantitative electroencephalography[END_REF]; [START_REF] Loo | Clinical utility of EEG in attentiondeficit/hyperactivity disorder: a research update[END_REF] for reviews). The most reliable EEG pattern in ADHD to date is an excess of theta activity (4-8 Hz) in fronto-central sites, measured in resting state [START_REF] Barry | A review of electrophysiology in attention-deficit/hyperactivity disorder: I. Qualitative and quantitative electroencephalography[END_REF][START_REF] Snyder | A meta-analysis of quantitative EEG power associated with attention-deficit hyperactivity disorder[END_REF][START_REF] Clarke | EEG-defined subtypes of children with attentiondeficit/hyperactivity disorder[END_REF]. Reduced alpha (8-13 Hz) and beta activity (13-30 Hz) have been commonly reported as well, thus theta/beta and theta/alpha ratios have been traditionally considered reliable measures to discriminate between ADHD and normal individuals [START_REF] Barry | A review of electrophysiology in attention-deficit/hyperactivity disorder: I. Qualitative and quantitative electroencephalography[END_REF][START_REF] Snyder | A meta-analysis of quantitative EEG power associated with attention-deficit hyperactivity disorder[END_REF]. In this direction, NF studies have mostly used standardized protocols to "correct" the aforementioned abnormal EEG patterns. The most used protocol is theta suppression/beta enhancement, usually enhancing the sensorimotor rhythm (SMR) simultaneously [START_REF] Loo | Clinical utility of EEG in attentiondeficit/hyperactivity disorder: a research update[END_REF][START_REF] Monastra | Electroencephalographic biofeedback in the treatment of attention-deficit/hyperactivity disorder[END_REF][START_REF] Arns | Efficacy of neurofeedback treatment in ADHD: the effects on inattention, impulsivity and hyperactivity: a metaanalysis[END_REF]. The SMR term was coined to describe an EEG pattern measured over the somatosensory cortex in alert but motionless cats, in the (11-15) Hz range [START_REF] Sterman | Basic concepts and clinical findings in the treatment of seizure disorders with EEG operant conditioning[END_REF]. The SMR enhancement throughout NF is suggested to improve hyperactive symptoms in ADHD since the pioneer work of [START_REF] Lubar | EEG and behavioral changes in a hyperkinetic child concurrent with training of the sensorimotor rhythm (SMR)[END_REF].

An extensive evaluation of standardized NF protocols has been performed during the last 40 years in ADHD children, with recent reviews pointing out their effectiveness [START_REF] Arns | Evaluation of neurofeedback in ADHD: The long and winding road[END_REF][START_REF] Loo | Clinical utility of EEG in attentiondeficit/hyperactivity disorder: a research update[END_REF][START_REF] Heinrich | Annotation: Neurofeedback-train your brain to train behaviour[END_REF]. Despite the positive results, these protocols may not be able to account for the large EEG heterogeneity in ADHD [START_REF] Loo | Clinical utility of EEG in attentiondeficit/hyperactivity disorder: a research update[END_REF][START_REF] Arns | EEG phenotypes predict treatment outcome to stimulants in children with ADHD[END_REF]. In addition, recent findings challenge the theta/beta ratio as a marker for ADHD, which was found increased in only 20-30% of ADHD individuals [START_REF] Arns | A decade of EEG theta/beta ratio research in ADHD: A meta-analysis[END_REF][START_REF] Arns | The effects of QEEG-informed neurofeedback in ADHD: an open-label pilot study[END_REF]. This may be partially due to a subgroup of 10-15% ADHD individuals showing increased (rather than decreased) beta activity [START_REF] Clarke | Excess beta activity in the EEG of children with attention-deficit/hyperactivity disorder: A disorder of arousal?[END_REF][START_REF] Clarke | EEG-defined subtypes of children with attentiondeficit/hyperactivity disorder[END_REF]. Thus, individualized approaches may better cope with the EEG heterogeneity and improve the clinical outcome [START_REF] Arns | Evaluation of neurofeedback in ADHD: The long and winding road[END_REF]. Some recent NF studies are following this direction [START_REF] Arns | The effects of QEEG-informed neurofeedback in ADHD: an open-label pilot study[END_REF]Lansbergen et al 2011b;[START_REF] Logemann | The effectiveness of EEG-feedback on attention, impulsivity and EEG: a sham feedback controlled study[END_REF].

The current study evaluates an individualized NF protocol for ADHD children. This NF protocol aims at enhancing the relative upper alpha power in frontocentral sites, individually determined for each child using the individual alpha frequency (IAF, Klimesch 1999) as an anchor point. On one hand, this protocol has the potential to decrease the excess of absolute theta power (most reliable EEG pattern in ADHD to date) and the excess of slow frequency oscillations in general. On the other hand, this protocol builds upon the positive results of alpha-based protocols in cognitive enhancement, mainly evaluated in healthy users [START_REF] Gruzelier | EEG-neurofeedback for optimising performance. I: A review of cognitive and affective outcome in healthy participants[END_REF]. Positive results were obtained in working memory [START_REF] Escolano | EEG-based upper alpha neurofeedback training improves working memory performance[END_REF][START_REF] Nan | Individual alpha neurofeedback training effect on short term memory[END_REF], visuospatial rotation [START_REF] Zoefel | Neurofeedback training of the upper alpha frequency band in EEG improves cognitive performance[END_REF][START_REF] Hanslmayr | Increasing individual upper alpha power by neurofeedback improves cognitive performance in human subjects[END_REF] and procedural learning [START_REF] Ros | Neurofeedback facilitation of implicit motor learning[END_REF]. Thus, this NF protocol is meant to target the cognitive deficits of ADHD individuals. This paper reports an open-label pilot study with 20 ADHD children who underwent 18 NF sessions. Clinical and neurophysiological variables were measured pre-and post-training. EEG was recorded pre-and post-training, and pre-and postthe training trials within each session, in both eyes closed resting state and eyes open task-related activity. A power EEG analysis assessed long-term and withinsession effects, in the trained parameter and in all the sensors in the (1-30) Hz spectral range. Learning curves over sessions were assessed as well.

Methods

Participants

20 children with ADHD participated in the study. All children fulfilled DSM-IV1 criteria for ADHD (American Psychiatric Association 1994). Diagnoses were based on a semi-structured interview with the parents using the Structured Developmental History of the BASC [START_REF] Reynolds | Behavior assessment system for children[END_REF]. WISC-IV [START_REF] Wechsler | Wechsler intelligence scale for children-Fourth Edition (WISC-IV). The Psychological Corporation[END_REF] was administered to estimate IQ. All children were drug-free and without concurring psychotherapy for at least one month before starting the NF training. Children with comorbid neurological or psychiatric disorders, or IQ < 80 were excluded from the study. Three children did not complete the study, thus the final sample consisted of 17 children (mean ± SD age: 11.8 ± 2.2 years, one girl). Seven children were diagnosed with inattentive type, ten with combined type. Families were informed about the study from local professionals in the city of Zaragoza (Spain).

The experimental design was approved by the Ethical Review Board of the regional health authority and followed the Declaration of Helsinki. Parents and children signed informed consent.

Experimental design

An open-label pilot study was designed (Figure 1). After an intake evaluation, an initial and final behavioral assessment and EEG screening were carried out. The NF training was composed of 18 sessions, executed for two months (two or three sessions per week). Each session was composed of five trials of four min each for a total of 20 min of training, and a pre-and post-EEG screening. For each EEG screening we recorded threemin of eyes closed resting state activity and three-min of eyes open task-related activity. In the latter, children faced a computer screen showing a square that changed saturation color randomly from gray to red or blue (gradually). Children were instructed to count the number of saturation changes from gray to red as a cognitive challenge [START_REF] Zoefel | Neurofeedback training of the upper alpha frequency band in EEG improves cognitive performance[END_REF].

Behavioral assessments

Parents rated the clinical conditions of the children pre-and post-the training using the following scales: (i) Parent Rating Scales of the BASC (BASC-PRS, ) is a computerized assessment of attention-related problems. The CPT displayed letters on a computer screen, and children had to press the space bar except when the letter "X" was displayed.

The test scores were the number of omission and commission errors. Paired samples t-tests were performed for pre vs post comparisons.

EEG recording and neurofeedback procedure

EEG data was recorded from 16 electrodes placed at FP1, FP2, AFz, F3, Fz, F4, FCz, C3, Cz, C4, P3, Pz, P4, O1, Oz and O2 (subset of the 10/10 system), with the ground and reference electrodes on FPz and on the left earlobe, respectively. EEG was amplified and digitized using a g.tec amplifier (Guger Technologies, Graz, Austria) at a sampling rate of 256 Hz, power-line notchfiltered at 50 Hz and (0.5-60) Hz band-pass filtered. EEG recording and the NF procedure were developed using software of Bit&Brain Technologies, SL.

The NF training focused on the increase of the relative upper alpha power, averaged over fronto-central sites (AFz, F3, Fz, F4, FCz and Cz, referred to as feedback electrodes). EEG power was calculated through a short-term FFT with 1 s hamming window, 30 ms of overlapping, and zero-padded to 1024 points (0.25 Hz resolution). Relative power was computed in the (1-30) Hz range. For each session, the pre-NF EEG screening was recorded and then used to calibrate the training for each participant and session. In this calibration step, we automatically filtered out the blinking component from the task-related activity by Independent Component Analysis (ICA) using the FastICA algo-rithm [START_REF] Hyvarinen | Fast and robust fixed-point algorithms for independent component analysis[END_REF]. Furthermore, we removed the epochs with amplitude larger than 200µV at any electrode. The IAF was computed for each electrode on the power spectra of the filtered EEG data as the frequency bin with the maximum power value in the (7-13) Hz alpha range [START_REF] Klimesch | EEG alpha and theta oscillations reflect cognitive and memory performance: a review and analysis[END_REF]. Note that when no clear alpha peak was found, the IAF was computed on resting state EEG instead. The upper alpha band was then defined as (IAF, IAF+2) Hz [START_REF] Klimesch | EEG alpha and theta oscillations reflect cognitive and memory performance: a review and analysis[END_REF]. The baseline was computed as the mean upper alpha power averaged across the feedback electrodes, and (5th -95th) percentiles established the lower and upper limits, respectively. After the calibration, the participants performed the training trials. During online training, EEG data was online filtered from blinking artifacts (through the aforementioned ICA filter) and a visual feedback was then displayed every 30 ms on a computer screen in the form of a square with changing saturation colors.

Offline EEG pre-processing

EEG data from the EEG screenings and training trials was filtered from artifacts using a semi-automatic method based on Riemannian geometry [START_REF] Barachant | Multiclass brain-computer interface classification by Riemannian geometry[END_REF][START_REF] Barachant | The Riemannian Potato: an automatic and adaptive artifact detection method for online experiments using Riemannian geometry[END_REF]. This method was separately applied to each recording session, applied on one hand to the resting state activity and on the other hand to the taskrelated activity and training trials. We first selected 15-20 artifact-free 1-s epochs by visual inspection. Covariances matrices were computed in each artifact-free epoch, and the geometric mean was computed. The remaining EEG data was then parsed into 1-s epochs using a sliding window algorithm with 30 ms overlapping. The distribution of the Riemannian distances between the geometric mean and the covariance matrix of each epoch was computed. Epochs with an absolute z-score higher than 2.5 were removed. A slight variation of this method was applied to the task-related activity and training trials to be more sensitive to non-blinking artifacts such as eyes and body movements. Initially, the extended infomax ICA [START_REF] Lee | Independent component analysis using an extended infomax algorithm for mixed subgaussian and supergaussian sources[END_REF] was applied to remove the eye blinking component and artifact-free epochs were selected by visual inspection in the sensor space. The semi-automatic method was then applied on the source space (n -1 components) and clean EEG data was projected to the sensor space.

EEG analysis

Long-term effects assessed the power changes after the study, measured as the power comparison in the initial vs final EEG screening in both resting state and task-related activity. We performed a direct comparison in the trained parameter and an exploratory absolute/relative power spectral analysis in all the sensors in the (≈ 1-30) Hz range (section 2.7).

Learning curves over sessions assessed the power changes as a function of the number of sessions, measured as the Spearman correlation between the power computed in the pre-NF EEG screening of each session (recorded before the training trials) vs the session number. We assessed the effects in resting state and task-related activity. We performed the analysis in the trained parameter and an exploratory analysis in absolute/relative power in the feedback sites and parietooccipital sites (P3, Pz, P4, O1, Oz and O2) in the following bands: delta = (1, 3.5), theta = (IAF-6, IAF-4), lower alpha = (IAF-4, IAF), upper alpha = (IAF, IAF+2), beta1 = (IAF+2, IAF+8), beta2 = (IAF+8, IAF+14) and beta3 = (IAF+14, 30). A non-parametric randomization method using the r-max statistic was used to correct for the number of bands, i.e., to control the familywise type I error rate (FWER, [START_REF] Holmes | Nonparametric analysis of statistic images from functional mapping experiments[END_REF]. Following this method, the null distribution of the maximum absolute r-value across all bands was estimated by 5000 random permutations. Then the absolute observed r-value for each band was tested against the (1 -α)th percentile of the null distribution. Bonferroni correction was further applied to control for the comparisons in absolute/relative power and the number of sensor clusters. The FWER was set at α = .05.

Within-session effects assessed the power changes immediately after the training trials (in both resting state and task-related activity) and during training. First, the power values computed in the pre-and post-NF EEG screenings of each session were averaged across sessions. The power in the training trials were averaged across sessions as well, and further averaged across the five trials (averaged training power). Within-session effects in resting state and task-related activity were measured as the averaged pre-vs post-NF power comparison. We measured the effects during training as the averaged pre-NF power value in task-related activity (baseline) vs the averaged training power. We performed a direct comparison in the trained parameter and an exploratory absolute/relative power spectral analysis (section 2.7).

Cluster-based method for power EEG analysis

A cluster-based non-parametric randomization method [START_REF] Nichols | Nonparametric permutation tests for functional neuroimaging: a primer with examples[END_REF][START_REF] Maris | Nonparametric statistical testing of EEG-and MEG-data[END_REF] was used to assess pre vs post power changes in all the sensors in the (≈ 1-30) Hz range. This method is implemented in the Fieldtrip toolbox (FC Donders Cen- tre for Cognitive Neuroimaging, Nijmegen, The Netherlands; see http://www.ru.nl/fcdonders/fieldtrip). First, the power spectra of each subject was centered to the IAF and the (IAF-8, IAF+18) Hz range was considered. Since mean ± SD IAF was 9.25 ± 1.22 Hz, the (1.25-27.25) Hz range was covered on average. The clustering method computed the pre vs post difference by performing paired samples t-tests in the (sensor, frequency)-pairs. Those pairs exceeding a threshold (q = .05) were clustered on the basis of spatial and spectral adjacency, and cluster-level statistics were calculated as the sum of the t-values within every cluster. Finally, the significance probability at the clusterlevel was estimated by a permutation method [START_REF] Pesarin | Multivariate permutation tests: with applications in biostatistics[END_REF]. The null distribution of the cluster values was constructed by 5000 random permutations. The observed values were then tested against the (1 -α)th percentile of the null distribution. This method controls for the type I error rate and corrects for multiple comparisons across sensors and frequencies. The type I error at cluster-level was set to α = .05.

Results

Behavioral assessments

The scores of the clinical and neuropsychological variables are summarized in power in fronto-central sites) showed a long-term increase in task-related activity (paired samples t-test: t 16 = -2.44, p = .026), with an average increase of 13.4%. Figure 2A displays the results of the exploratory analysis. Significant clusters were only found in taskrelated activity, both in relative and absolute power. A relative power increase appeared in (IAF+1, IAF+3) Hz (p = .039), partially covering upper alpha and beta1. An absolute power increase was marginally sig-nificant in the same frequency range, apparent in central and parieto-occipital sites (p = .07). No significant effects were found in resting state.

Learning curves over sessions

Children with less than 30 s of artifact-free data in a given EEG screening of a session (in either resting state or task-related activity) were excluded from the analysis of that session. The mean ± SD number of children per session was 13.8 ± 1.3. Trained parameter (relative upper alpha power in fronto-central sites) showed a positive learning curve over sessions in task-related activity (r 17 = 0.62, p = .008), see Figure 3. The exploratory analysis in relative power revealed a marginally significant negative learning curve in parieto-occipital sites for delta power, measured in task-related activity (r = -0.65, p = .083). Absolute power analysis revealed a positive learning curve in parieto-occipital sites for upper alpha power, measured in task-related activity (r = 0.75, p = .01). No significant learning curves were found in resting state. Note that we are strictly controlling the FWER in the exploratory analysis by a randomization procedure plus Bonferroni correction.

Within-session effects

Trained parameter (relative upper alpha power in fronto-central sites) showed a within-session decrease in task-related activity (paired samples t-test: t 16 = 2.66, p = .017), with an average decrease of 4.4%. No significant effects in the trained parameter appeared either in resting state or during training. Figure 2B displays the results of the exploratory analysis. Regarding the resting state, a relative power decrease was found in lower alpha, (IAF-2, IAF) Hz, apparent in frontocentral and parietal sites (p = .063), and a power increase in beta1, (IAF+2, IAF+4) Hz (p = .083). An absolute power decrease was found in slow frequencies (delta and theta) and lower alpha, (IAF-8, IAF) Hz (p < .001), and a power decrease in beta1, (IAF+4, IAF+9) Hz, in central and parieto-occipital sites (p = .003). Regarding the task-related activity, a power decrease was found in upper alpha measured in relative (p = .005) and absolute power (p = .001). A relative power increase was found in beta3, (IAF+12, IAF+18) Hz, apparent in parieto-occipital sites (p = .007), and an absolute power decrease in theta and lower alpha, (IAF-6, IAF-2) Hz (p = .005). During training, slow frequencies and lower alpha, (IAF-8, IAF-2) Hz, showed a power decrease measured in relative (p = .008) and absolute power (p < .001). A power increase appeared in beta2 and beta3, (IAF+8, IAF+18) Hz, in relative (p < .001) and absolute power (p = .01).

Discussion

The current study evaluated an individualized NF protocol in children diagnosed with ADHD. Individualized approaches may better cope with the large EEG heterogeneity in ADHD and improve the clinical outcome [START_REF] Arns | Evaluation of neurofeedback in ADHD: The long and winding road[END_REF]. Recent NF studies have followed this direction [START_REF] Arns | The effects of QEEG-informed neurofeedback in ADHD: an open-label pilot study[END_REF]Lansbergen et al 2011b;[START_REF] Logemann | The effectiveness of EEG-feedback on attention, impulsivity and EEG: a sham feedback controlled study[END_REF]. Please note that in our study, "individualized NF approaches" refers to studies determining the EEG trained parameter according to the EEG activity of the individual rather than using a fixed EEG parameter for all the participants of the study. For instance, [START_REF] Arns | The effects of QEEG-informed neurofeedback in ADHD: an open-label pilot study[END_REF] classified each individual into a set of EEG clusters by a comparison to a normative database, and performed a different protocol according to the cluster (e.g., theta/beta, alpha or beta suppression, SMR enhancement). Lansbergen et al (2011b) and [START_REF] Logemann | The effectiveness of EEG-feedback on attention, impulsivity and EEG: a sham feedback controlled study[END_REF] performed a theta/beta protocol combined with SMR enhancement, in which the feedback sensors and range of frequency bands were determined by a comparison to a normative database.

The NF protocol herein proposed aimed at enhancing the relative upper alpha power in frontro-central sites, individually determined using the individual alpha frequency (IAF) as an anchor point. To the best of the authors knowledge, this is the first NF study evaluating such a protocol in ADHD individuals. In comparison to the aforementioned approaches, this NF proto-col does not rely on a normative database comparison and it can address recent concerns of children with slow IAF. For example, Lansbergen et al (2011a) found that children showing slow IAF may be clustered as an excess of theta activity. In addition to that, the use of an unique NF protocol makes possible to perform an homogenous group-level EEG analysis. On one hand, this protocol has the potential to deal with the excess of absolute theta power, which is the most reliable EEG pattern in ADHD to date [START_REF] Barry | A review of electrophysiology in attention-deficit/hyperactivity disorder: I. Qualitative and quantitative electroencephalography[END_REF][START_REF] Snyder | A meta-analysis of quantitative EEG power associated with attention-deficit hyperactivity disorder[END_REF]. Due to the 1/f distribution of EEG power spectra, we hypothetized stronger effects in slow frequencies (power decrease) and upper alpha (power increase). On the other hand, this protocol builds upon the positive results of alpha-based protocols in cognitive performance, mainly evaluated in healthy users (see [START_REF] Gruzelier | EEG-neurofeedback for optimising performance. I: A review of cognitive and affective outcome in healthy participants[END_REF] for a review on NF studies on cognitive enhancement). Thus, this NF protocol has the potential to alleviate the cognitive deficits of ADHD individuals. Note that deficits in executive functioning, including working memory, and response inhibition have been repeatedly reported [START_REF] Barkley | Behavioral inhibition, sustained attention, and executive functions: constructing a unifying theory of ADHD[END_REF][START_REF] Martinussen | A meta-analysis of working memory impairments in children with attention-deficit/hyperactivity disorder[END_REF][START_REF] Castellanos | Neuroscience of attentiondeficit/hyperactivity disorder: the search for endophenotypes[END_REF].

EEG analysis

An extensive power EEG analysis was conducted. We assessed long-term and within-session effects in all sensors in the (≈ 1-30) Hz frequency range by a clusterbased randomization method, obtaining sensor x frequency maps of the power changes. Furthermore, the learning curve over sessions was assessed in frontocentral and parieto-occipital sites for a set of frequency bands covering the (1-30) Hz range. We believe that the present analyses can offer a clearer insight of the electrophysiological effects rather than traditional analyses only in the trained parameter.

Children showed long-term effects in the trained parameter: relative upper alpha power in fronto-central sites was significantly enhanced after the NF training, measured in task-related activity. An average increase of 13% was found, as well a significant positive learning curve over sessions. In line with these results, Nan et al (2012) performed a similar NF protocol of relative upper alpha enhancement in healthy users, obtaining a positive learning curve over sessions. We also found a significant absolute upper alpha power enhancement in parieto-occipital sites, and a learning curve over sessions. The increase of absolute upper alpha power in NF literature has been related to improvements (in healthy users) in working memory [START_REF] Escolano | EEG-based upper alpha neurofeedback training improves working memory performance[END_REF] and visuospatial rotation [START_REF] Zoefel | Neurofeedback training of the upper alpha frequency band in EEG improves cognitive performance[END_REF][START_REF] Hanslmayr | Increasing individual upper alpha power by neurofeedback improves cognitive performance in human subjects[END_REF]. The long-term effects in task-related activity were mainly restricted to the upper alpha band, with no significant effects in resting state. The stronger effects in task-related activity illustrate the importance of recording EEG in several conditions to provide additional information of the underlying brain processes. This is in contrast to the common practice to study only the resting state, either eyes closed or eyes open. Note that a correlation analysis was conducted between the behavioral and EEG variables, both in the initial and change scores, but no significant results were found.

The within-session effects measured the immediate effects after training in resting state and task-related activity, and the effects during training. To do so, EEG data was collected over sessions, and we compared the EEG screenings recorded immediately before vs after the training trials, and the EEG screenings recorded before vs the EEG during the training trials. A significant decrease in absolute and relative upper alpha power appeared in task-related activity, instead of the expected increase. This may be explained by an alpha "rebound" effect. While that kind of effect had been previously reported in EEG literature mainly related to motor acts in alpha or beta activity (Pfurtscheller and Lopes da Silva 1999), a recent alpha-based NF study with post-traumatic stress disorder (PTSD) patients reported that effect in alpha activity immediately after a single session of training, pointing to homeostatic/compensatory brain mechanisms [START_REF] Kluetsch | Plastic modulation of PTSD resting-state networks and subjective wellbeing by EEG neurofeedback[END_REF]. Regarding the resting state, an absolute power decrease was found in slow-frequency oscillations (delta and theta) and lower alpha, as well as a power decrease in lower part of beta. No significant effects in the trained parameter were found during training, however an absolute and relative power decrease appeared in slowfrequency oscillations and lower alpha, as well as an increase in upper part of beta. Thus, although the children were not able to increase the relative upper alpha during training, they showed a strong effect in slow frequencies (as hypothetized) and in upper part of beta to a lower extent. The latter effect was unexpected and should be further explored in future studies.

Behavioral assessments

Parents reported a significant reduction in the clinical symptoms of the children after the NF training. The externalizing and internalizing problems scores in the BASC test showed a significant improvement, as well as the inattention and hyperactivity/impulsivity scores in the CPRS test. The effect sizes in inattention and hyperactivity/impulsivity were 1.16 and 1.15, respectively. Regarding the neurophysiological tests, children showed a significant improvement in working memory as measured by the digit span and letter-number sequencing tests of the WISC-IV. We found a significant improvement in concentration as assessed by the D2 test. The number of commission errors in the CPT test was significantly decreased, thus suggesting an improvement in impulsivity. No significant change in the number of omission errors was found.

Interesting, slightly superior effect sizes in hyperactivity/impulsivity (d = 1.15) were found in comparison to literature (see [START_REF] Arns | Efficacy of neurofeedback treatment in ADHD: the effects on inattention, impulsivity and hyperactivity: a metaanalysis[END_REF] meta-analysis). In this direction, a large body of research has hypothetized that the neuronal substrates of inhibitory mechanisms are related to alpha oscillations [START_REF] Sauseng | Brain oscillatory substrates of visual shortterm memory capacity[END_REF][START_REF] Freunberger | Brain oscillatory correlates of working memory constraints[END_REF][START_REF] Klimesch | EEG alpha oscillations: the inhibition-timing hypothesis[END_REF]. Although it should be interpreted with caution, the upper alpha power enhancement herein reported may target mechanisms of behavioral inhibition, thus leading to higher outcomes in hyperactivity/impulsivity symptoms. SMR enhancement also has been traditionally hypothetized to alleviate hyperactivity. Due to the similitudes between these two protocols, the aforementioned relation may account for the clinical improvements and cognitive enhancement in ADHD. It was already pointed out by [START_REF] Hanslmayr | Increasing individual upper alpha power by neurofeedback improves cognitive performance in human subjects[END_REF] that the results in cognitive enhancement obtained after SMR-based NF (in healthy users) might be in part influenced by upper alpha activity. However, certainly more research is needed to elucidate the mechanisms of action underlying this protocol.

Limitations

Due to the novelty of the NF protocol in ADHD individuals an open-label pilot study was designed. The number of NF sessions was small in comparison with ADHD literature (30 to 40 sessions are usually executed). Furthermore, non-specific effects of the treatment cannot be ruled out due to the lack of a control group. The positive results of this NF protocol suggest that it should be further explored in a randomized controlled trial with a higher number of sessions and a larger sample size. Note that diagnosis was based on DSM-IV (American Psychiatric Association 1994) since it was the more recent edition at the beginning of our study, however it was recently updated to the fifth edition (DSM-5, American Psychiatric Association 2013).

Fig. 1

 1 Fig. 1 Experimental design of the study. After an intake evaluation, the children carried out an initial and final behavioral assessment (clinical and neuropsychological tests) and EEG screening within a two-months time interval. The NF training consisted of 18 sessions, which were composed of five training trials (four min each) and a pre-and post-EEG screening. The EEG screenings included eyes closed resting state activity (three min) and eyes open task-related activity (three min).

Fig. 2

 2 Fig. 2 Sensor x frequency maps displaying the (A) long-term effects and (B) within-session effects. Significant clusters (pre vs post power changes) are displayed. Left figures display the effects on relative power and right figures the effects on absolute power. Power spectra was centered per subject to the IAF. X axis shows the frequency bins in the (IAF-8, IAF+18) Hz range, whereas Y axis shows the sensor locations. Color scale represent t-values, with positive and negative values indicating a power increase or decrease, respectively.

Fig. 3

 3 Fig.3Relative upper alpha power in fronto-central sites (trained parameter) over sessions, measured in task-related activity. Dots depict the mean ± SEM power value in each session, computed in the EEG screenings recorded immediately before the training trials. Data was normalized per subject to the power in the initial EEG screening.

Table 1

 1 Results of the clinical and neuropsychological tests pre-and post-training. BASC Parent Rating Scales (BASC-PRS) with the composite scales. Conners' Parent Rating Scales (CPRS-R) with global index and DSM-IV items. Two tests of the WISC-IV evaluating working memory: digit span and letter-number sequencing, with the number of correct responses. D2 test with concentration index. Conners' Continuous Performance Test (CPT) with the number of omission and commission errors. t-and p-values for the paired samples t-tests are provided, as well as Cohen's d effect size (ES). Significant values are marked bold (p < .05).

		pre training post training	t-stat	p-value	ES
	Clinical scales
	BASC-PRS (T-scores)
	externalizing problems	61.44(2.94)	55.94(2.01)	t 16 = 3.52	.003	0.85
	internalizing problems	57.50(2.92)	50.41(2.23)	t 16 = 4.12	<.001	1.00
	adaptive skills	41.12(2.14)	41.53(1.92)	t 16 = -0.21	.833	0.05
	CPRS-R (T-scores)
	global index	68.38(2.65)	60.62(1.97)	t 16 = 4.86	<.001	1.18
	inattention (DSM-IV)	71.12(1.99)	62.65(1.82)	t 16 = 4.78	<.001	1.16
	hyperactivity/impulsivity (DSM-IV)	73.88(2.43)	64.32(1.71)	t 16 = 4.74	<.001	1.15
	total score (DSM-IV)	74.38(1.91)	64.50(1.57)	t 16 = 6.30	<.001	1.53
	Neuropsychological tests
	Digit span (WISC-IV)
	# correct responses	13.53(0.65)	15.76(0.85)	t 16 = -5.16	<.001	1.25
	Letter-number sequencing (WISC-IV)
	# correct responses	16.00(0.65)	17.65(0.66)	t 16 = -2.26	.038	0.55
	D2
	concentration index	48.76(6.44)	62.06(5.66)	t 16 = -3.29	.005	0.80
	CPT
	# omission errors	4.42(0.94)	4.79(0.98)	t 16 = -0.44	.664	0.11
	# commission errors	58.57(5.65)	45.10(5.51)	t 16 = 2.68	.016	0.65

DSM was recently updated to the fifth edition (DSM-5).