Yoann Louis
email: louis@univ-tln.fr

Petra Cmuk
email: pcmuk@irb.hr

Dario Omanović
email: omanovic@irb.hr

Cédric Garnier
email: c.garnier@ism.u-bordeaux1.fr

Véronique Lenoble
email: lenoble@univ-tln.fr

Stéphane Mounier
email: mounier@univ-tln.fr

Ivanka Pi Žeta
email: pizeta@irb.hr

Speciation of trace metals in natural waters: The influence of an adsorbed layer of natural organic matter (NOM) on voltammetric behaviour of copper

Keywords:

The influence of an adsorbed layer of the natural organic matter (NOM) on voltammetric behaviour of copper on a mercury drop electrode in natural water samples was studied. The adsorption of NOM strongly affects the differential pulse anodic stripping voltammogram (DPASV) of copper, leading to its distortion. Phase sensitive ac voltammetry confirmed that desorption of adsorbed NOM occurs in general at accumulation potentials more negative than -1.4 V. Accordingly, an application of negative potential (-1.6 V) for a very short time at the end of the accumulation time (1% of total accumulation time) to remove the adsorbed NOM was introduced in the measuring procedure. Using this protocol, a well-resolved peak without interferences was obtained. It was shown that stripping chronopotentiogram of copper (SCP) in the depletive mode is influenced by the adsorbed layer in the same manner as DPASV. The influence of the adsorbed NOM on pseudopolarographic measurements of copper and on determination of copper complexing capacity (CuCC) was demonstrated.

A shift of the peak potential and the change of the half-peak width on the accumulation potential (for pseudopolarography) and on copper concentration in solution (for CuCC) were observed. By applying a desorption step these effects vanished, yielding different final results.

Introduction

The bioavailability and toxicity of metal ions to aquatic organisms is mainly related to concentrations of their free (hydrated) form [START_REF] Campbell | Metal Speciation and Bioavailability in Aquatic Systems[END_REF][START_REF] Buffle | Complexation Reactions in Aquatic Systems; An Analytical Approach[END_REF][START_REF] Batley | [END_REF]. The distribution of inorganic metal species in natural waters with known composition of major stripping voltammetry (ASV) is an attractive method which satisfies the requirements concerning sensitivity and selectivity, while offering the benefit of low contamination risk and alteration of original sample composition during analysis. Speciation of trace metals by ASV is usually performed by determination of metal complexing capacity (MCC) [START_REF] Batley | [END_REF][4][5].

As a final result, concentration of ligands forming stable inert complexes with a metal of interest and the corresponding conditional stability constants (K ) are determined. It was recently shown that voltammetric titration data (interactions of trace metals with active sites of NOM) are mainly fitted to model encompassing two discrete classes of ligands [4]. To ensure the reliability of the obtained information, application of a wider detection window (using logarithmic additions) is proposed [5].

According to some studies, the limitation of ASV for metal speciation is that it is a kinetically dependent technique [6], i.e. some inert complexes may dissociate during the accumulation period, which could lead to underestimation of complexing parameters. However, this could be a good point because such processes might occur on the biological cell membranes as well [6].

The well-known pseudopolarographic approach in trace metal speciation [7][8][9][10][11][12][13][14][15][16][17][18][19][20] has recently attracted new attention due to the possibility to automate the process of recording of pseudopolarograms. The benefit of using pseudopolarography and a recently developed analogous technique, scanned (deposition potential) stripping chronopotentiometry (SSCP), is that obtained information about metal complexation are similar to classical polarography, but achieved at significantly lower metal concentrations [11,[22][23][24]. As it is theoretically described and experimentally verified, the complexation of a metal by a ligand will cause a (slight) shift of reduction wave of labile complexes towards more negative potentials, while the inert complexes are separately reduced at more negative potentials comparing to labile complexes [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. The magnitude of these shifts (for both labile and inert complexes) is related to the stability constant of the formed complex and to the concentration of the free complexing ligand in the case of labile complexes.

The interferences in voltammetry [START_REF] Batley | [END_REF][23][24][25][26][27][28][29][30][31] are well known, and one important problem is adsorption of surface active substances (SAS) on the electrode surface. As a significant part of NOM usually exhibit the properties of SAS [START_REF] Hunter | Organic sea surface films[END_REF], this process is likely to occur when dealing with real sample in ASV. In the literature it has been reported how the adsorption of SAS influences the electrochemical reactions of copper [25][26][27] and also that the desorption of adsorbed SAS occurs at more negative potentials, producing well-resolved copper peak [25,31].

Each interference affecting measurement is in some extent converted into final speciation data and contributes to more or less erroneous data. Thus, the intention is to exclude interferences as much as possible during measurements.

The main objective of this paper is to explore the impact of an adsorbed layer of SAS on a mercury electrode in measurements of copper complexing properties. The benefit of removing the adsorbed layer by applying desorption step in the procedure of complexing capacity determination and pseudopolarographic measurement in seawater samples is demonstrated.

Experimental

All voltammetric measurements were carried out using an Autolab potentiostat (EcoChemie, Utrecht, The Netherlands) (Autolab or PGSTAT12) equipped with a Metrohm 663 VA stand (Metrohm, Herisau, Switzerland). The instrument was computer-controlled using GPES 4.9 control software. The working electrode was a static mercury drop electrode (size 1, 0.25 mm 2 ), the counter electrode was a platinum wire or a glassy carbon rod, and the reference electrode was Ag|AgCl (sat. NaCl). Stirring of the solution was performed by built-in Teflon stirrer rotating at 3000 rpm. All measurements were performed at room temperature (22 ± 1 • C). pH was controlled by a Metrohm pH meter (Model 713) with a glass pH electrode. Differential pulse anodic stripping voltammetry (DPASV), phase sensitive ac voltammetry and stripping chronopotentiometry (SCP) were used as measuring techniques under the following conditions (if not otherwise noted in figure captions): pulse amplitude, A = 20 mV; potential step increment, E st inc = 2 mV; time between pulses, t int = 0.1 s; pulse duration, t mod = 0.04 s; phase angle, Â = 90 • . Other procedure parameters are given in figure captions. For pseudopolarographic measurements, accumulation of metal ions was performed during the deposition time (t acc ) consecutively, at a selected range of deposition potentials (E acc ) in defined steps.

Pseudopolarographic measurements were carried out automatically using the measuring procedure integrated in the PROJECT mode offered by the GPES 4.9 software. A homewritten program capable to change either conditioning or deposition potential was integrated in the measurement protocol to simplify the measurement protocol/project. Analysis of voltammetric peaks and construction of pseudopolarograms was performed using home-written software [10] (the two home-written programs are available on request free of charge).

All solutions were prepared in deionised water from a Millipore Milli-Q system (Bedford, USA). Stock metal solutions (Cu, Zn) were prepared by diluting a commercial standard solution (1.0 g L -1 , Fluka, Buchs) in MQ-water. Concentrated nitric acid was of supra pur grade (Merck, Darmstadt). Samples in the cell (except for a.c voltammetric measurements) were purged with oxygen-free nitrogen for at least 5 min prior the measurement, followed by maintaining a nitrogen blanket above the electrolyte surface during the measurement.

Natural water samples were taken in estuary of the Gapeau river (Hy ères, France) (sample 1, salinity = 35‰), in a small port on island Prvi ć ( Šepurine, Croatia) (sample 2, salinity = 38‰) and in estuary of the Krka river ( Šibenik, Croatia) (sample 3, salinity = 24‰). Surface water samples (0.2 m) were sampled and stored in pre-cleaned 1 L high-density polyethylene bottles (HDPE) or in 1 L FEP (fluorinated ethylene propylene) bottles (both from Nalgene Labware). Samples were filtered through 0.45 m cellulose-nitrate filters and stored at 4 • C until analysed.

Results and discussions

The influence of SAS on the voltammetric behaviour of copper was studied by anodic stripping voltammetry (ASV), phase sensitive ac voltammetry, stripping chronopotentiometry (SCP) and pseudopolarography.

Influence of SAS on ASV of copper

Voltammograms of copper (1.4 × 10 -7 mol L -1 , determined after acidification and UV digestion) in seawater using DPASV and different parameters during the accumulation period are shown in Fig. 1. An appropriate accumulation potential to reduce the labile part only was selected on the basis of corresponding pseudopolarograms [10]. Electrochemical reaction of copper in chloride media occurs via a monovalent copper (Cu(I)) intermediate [27,[START_REF] Nelson | [END_REF], which causes a shift in the copper peak position toward a more negative potential, a half-peak width which corresponds to a one-electron charge transfer reaction, and a peak height about half the value of that recorded in the absence of chloride (oxidation from monovalent copper to divalent copper also occurs but overlaps with the mercury oxidation and is therefore not observed). The anodic peak observed at -0.17 V in curve 1 (Fig. 1) was obtained with an accumulation potential of -0.45 V. The measured half-peak width is 115 mV, which is wider than expected.

The relatively high and slowly rising background current and the poorly defined peak made it difficult to determine exactly the copper peak height. Furthermore, an additional small peak was observed on the anodic side of the main peak, indicating occurrence of non-faradaic surface reactions from the adsorbed state [25,26]. Curve 2 (Fig. 1) was obtained using an accumulation potential of -1.6 V. Compared to curve 1, a more pronounced oxidation peak of copper was observed, together with a significant decrease in the background current. The peak current was increased by a factor of three, and the half-peak width was reduced to 85 mV (corresponding to a reversible one-electron charge transfer). Taking into account that more stable metal complexes also should be reduced at very negative potentials [12][13][14][15][16][17], the results obtained in curves 1 and 2 indicate that only approximately 30% of the total dissolved copper exists in labile metal complexes in the actual sample. However, on the basis of curve 3 (Fig. 1), a completely different conclusion is drawn. In this experiment, the same accumulation potential as in curve 1 (-0.45 V) was used for 297 s, and then for the last 3 s the applied potential was set to -1.6 V. The peak height decreased only by about 30% compared to curve 2, and the half-peak width remained surprisingly the same (87 mV). It is obvious that, by applying a very negative potential for only 1% of the total accumulation time, significant changes regarding the electrochemical processes at the mercury surface occur. This phenomenon is described in the literature for model solutions using extracted hydrophobic organic matter from seawater added to perchlorate supporting electrolyte [25]. It is supposed that the adsorbed SAS hinders the electrochemical reaction of copper during the stripping mode (curve 1) although, by applying a very negative potential, desorption of SAS takes place and produces a stripping peak without interferences (curve 3). The small difference between peak heights in curves 2 and 3 could be attributed to the actual difference between the fraction of labile and stronger metal complexes. If experiments are performed in such a way that initial potential is in the range of SAS adsorption (more positive that -1.1 V), some small portion of SAS will be adsorbed on the electrode surface during the equilibrium time and stripping time. However, as during this time the mass transfer is controlled mainly by the diffusion, the extent of adsorbed SAS could be considered as minor. To explore this phenomenon of SAS adsorption, experiments utilizing phase sensitive ac voltammetry were performed.

It is well known that adsorption of surface active substances (SAS) on a mercury electrode surface occurs usually in a potential range from 0.1 to -1.3 V [34][35][36]. Adsorption of SAS is usually the strongest in the range of an electrocapillary maximum of the mercury electrode (around -0.6 V) and depends both on the type of SAS and on the sample composition (major ions). The content of SAS in a natural sample could be easily quantified and characterized using phase sensitive ac voltammetry (phase angle = 90 • ) [34][35][36]. The procedure is based on the measurement of the capacitive current (or capacitance) at a potential of maximum adsorption (around -0.6 V) after applying an accumulation step (usually at accumulation potential of -0.6 V) during the chosen accumulation time. The resulting decrease in capacitive current corresponds to the concentration of SAS, while the capacitive current at zero accumulation time was taken as the reference value without adsorbed SAS. To explore these phenomena observed in Fig. 1, measurements of capacitive current at different accumulation potentials were performed (sample 1). Fig. 2 shows the differences in capacitive currents (related to the current at the most negative accumulation potential, -1.6 V) in relation to the accumulation potential. The procedure for curve adsorbed SAS is removed from the mercury surface. From the inset to Fig. 2, it can be evaluated that a degree of surface coverage attained during 30 s at -0.6 V without the desorption step (curve 1) corresponds to the degree attained during 600 s of accumulation time for curve with the potential jump (curve 2). However, compared to the dependence obtained for the accumulation at only the most negative potential (-1.6 V, curve 3), some SAS still remained on the mercury surface. For comparison, measurements in a UV irradiated sample without using the desorption step was performed (curve 3). As the NOM was destroyed by the prolonged exposure of the sample to UV light (14 h), the observed effect of adsorbed layer decreased to less than 10% of that obtained in the UV untreated sample (curve 1).

From the results given in Figs. 1 and2, it is obvious that the ASV of copper (curve 1, Fig. 1) is strongly influenced by the adsorbed SAS layer. The simple experiments presented above show that such undesired effects can easily be eliminated or reduced, giving more correct information about speciation.

Stripping chronopotentiometric (SCP) measurements

Usually, substances adsorbed on an electrode surface have more effect on voltammetric shape obtained using pulse techniques than using direct mode techniques. It was demonstrated in model electrolyte solutions that stripping chronopotentiometry (SCP) in depletive mode has an advantage over ASV in pulsed mode if adsorption processes take place at the electrode surface [21][22][23][24]. Because of NOM heterogeneity, such situations are likely to occur in analysis of natural water samples, and SCP should be taken into account in the measurement protocols as a comparative technique. In general, SCP offers better resolution/separation of adjacent peaks than ASV, but its detection limit is in most cases poorer than that of ASV. Fig. 3 shows the stripping chronopotentiograms of copper in sample 1. According to the literature, SCP in depletive mode is recommended because all the accumulated metal is stripped out from the mercury, while at the same time slow oxidation produces less interferences on the mercury surface [23]. Thus, the stripping current of 2 × 10 -9 A was applied in the stripping stage. Curve 1 is acquired by accumulation at -0.45 V during 300 s. Comparing to curve 1 in Fig. 1 a similar shape is obtained, but taking into account the background current, the copper peak is poorly defined, which supports the thesis of lower sensitivity of SCP compared to ASV in pulse mode. Small shoulder on the anodic side of the peak is also noticeable, indicating some surface reaction, probably nonfaradic. Similarly to ASV, keeping the electrode for only three (3) seconds at the end of the accumulation time at a very negative potential (-1.6 V), resulted in a more pronounced peak (curve 2, Fig. 3). This experiment shows that, although the SCP in some cases is superior to ASV, in our sample of seawater the improvement of the signal resolution/evolution was not registered, and all the subsequent experiments were carried out employing only ASV.

Pseudopolarographic measurements

The influence of SAS adsorption on a mercury electrode on the shape of copper pseudopolarograms in the seawater sample (sample 2) was investigated. Copper pseudopolarograms were recorded without added copper, because relatively high concentration of copper was found in the sample (1.8 × 10 -7 mol L -1 ). Fig. 4a shows two pseudopolarograms of copper obtained with different voltammetric parameters during the accumulation period. To exclude a possible change in the sample properties and experimental conditions during prolonged measurements (several hours), the pseudopolarograms were recorded in a way that both voltammetric procedures (with 3 s of desorption time at -1.6 V and without it) were applied for each accumulation potential one after the other (in a commutation mode). As in previous figures, curve 1 represents the procedure performed without applying the desorption step, while for curve 2, a potential jump to -1.6 V for 3 s at the end of the accumulation time was applied. The difference between the two pseudopolarograms is obvious. Using the desorption step (curve 2), two very well defined waves were obtained. The first wave at a potential of -0.2 V corresponds to the reduction of labile copper complexes. In the potential range between -0.4 and -1.15 V, a wide plateau was registered, which indicates that no reduction process of any additional copper species took place. At more negative potentials (<-1.15 V) the copper peak current increased, producing a wave that extended over the potential range of 300 mV. Several speculations could be drawn for this wave: (1) the reduction of a group of inert copper complexes with very high stability constants [9,[12][13][14][15], (2) an intermetallic compound of Cu and Zn, which is formed in this range of potentials, usually increases the copper peak height [20,23], and (3) a decrease in SAS adsorption on the mercury surface with an increase in the accumulation potential to more negative values (see Fig. 2). The detailed analysis of the pseudopolarograms and additional measurements performed on separate samples showed that the first (1) scenario is the most probable one, i.e. the reduction of inert metal complexes. Assumption 2 was discarded by additional insight in the zinc pseudopolarogram (Fig. 4a), which shows that the rising part of the copper peak height started at potentials where the main portion of the limiting current of zinc pseudopolarogram has already been reached. In the case of formation of an intermetallic compound between Cu and Zn, the starting point of the wave should be more positive than what we observed. Assumption 3 is less probable than assumption 1 since by comparing Fig. 4b andc, it can be noticed that both the peak potential and the half-peak width in this potential range do not change after using the desorption step. As the increase of this wave, compared to the preceding plateau, is about 20%, it is to expect a small change in values of these two parameters (peak potential and half-peak width). This assumption is drawn on the basis of the dependences of peak potential and half-peak width on the accumulation potential without the desorption step, where a considerable change of these two parameters occurred (curves 1).

The three plots in Fig. 4 clearly evidence that the adsorbed layer of SAS has a significant impact on all three parameters of the voltammetric peak (height, potential and half-width). The intensity of this influence is dependent on the accumulation potential, as well as on the concentration of copper inside the mercury drop. The obtained results are in a very good agreement with our results obtained with ac voltammetry. At potentials more negative than -1.4 V the effect is negligible. However, by increasing the accumulation potential, the influence of SAS also increases and remains constant around the potential of maximal adsorption (∼-0.6 V). At accumulation potentials more positive than -0.2 V, the peak height decreased (reduction wave of labile copper) causing a shift in peak potential and broadening of the peak.

A detailed examination of curve 1 in Fig. 4a reveals three separate waves. The height of the first wave at -0.2 V barely reaches 50% of the equivalent wave using the desorption step (curve 2), while the wave at the more negative potential (around -1.3 V) expands from -0.9 to -1.5 V. An additional (third) poorly resolved wave appears at a potential around -0.5 V. Compared to curve 2, a considerable detachment is evident by applying the desorption step. As pointed out in several papers, the position, numbers, and shape of the pseudopolarographic waves is dependent on the metal complexes existing in the sample [10,[12][13][14][15][16][17][18][19][20][21][22][23]37], and stability constants of labile metal complexes can easily be calculated [12][13][14][15]38]. The more negative pseudopolarographic wave that we obtained using the desorption step corresponds to a thermodynamic stability constant of the formed complex in the order of log K ∼39 (estimated according to "chelate scale" previously presented by group of Luther III [12][13][14][15]). Such a high stability constant of copper complexes has previously been reported by pseudopolarography and ascribed to the complex formed with a ligand produced by cyanobacteria [13]. However, a more detailed study should be performed to confirm such an assumption for our sample.

Similarly to the more negative wave, the small (third) wave around -0.5 V observed on curve 1 could be assigned to the reduction of a copper complex. However, as this wave is not registered using the desorption step (curve 2), it is fully clear that its formation is the result of surface reactions on the mercury electrode caused by the adsorbed organic matter, and that metal complex does not exist in the bulk of the sample in sufficient concentration to produce measurable wave.

The curves representing the dependence of the peak position and half-peak width on the accumulation potential (Fig. 4b andc) show strong interaction of copper with ligands from the adsorbed layer during oxidation process and indicate formation of kinetically labile complexes which affect the peak position and half-peak width [26,28]. Although studies of such interactions are important and useful for interpretation of voltammetric data [28,35], the main goal in speciation measurements is characterization of trace metal interactions with ligands within the bulk of the sample [START_REF] Buffle | Complexation Reactions in Aquatic Systems; An Analytical Approach[END_REF]. Thus, any interference in the voltammetric measurement occurring at the surface of the electrode is inauspicious and should be avoided. This evident example shows a delicate situation, which could initiate inaccurate conclusion about metal speciation in natural water sample.

Determination of metal complexing capacity (MCC)

Determination of metal complexing capacity (MCC) is very often used as a method to characterize particular natural water, either as a parameter in metal speciation or the characterization of the natural organic mater (NOM) [START_REF] Campbell | Metal Speciation and Bioavailability in Aquatic Systems[END_REF][START_REF] Buffle | Complexation Reactions in Aquatic Systems; An Analytical Approach[END_REF][START_REF] Batley | [END_REF][4][5]. Copper is usually used as the target metal, because it forms the most stable complexes among metals which are easily determined by ASV. However, the above-mentioned complications regarding the adsorption of NOM in ASV are strongly pronounced in a stripping process of copper [25].

Fig. 5 shows an example of determination of copper complexing capacity (CuCC) in a brackish water sample (sample 3) using the two different protocols mentioned in previous sections. Curve 1 on each plot corresponds to the protocol without using the desorption step, while all curves 2 were obtained by applying a potential jump to -1.65 V for 3 s at the end of the accumulation time (297 s). The initial (ambient) copper concentration in the sample was 1.05 × 10 -8 mol L -1 . As the reduction of only labile fraction of copper is a prerequisite in CuCC determination, a proper accumulation potential of -0.5 V is selected on the basis of the copper pseudopolarogram shown in Fig. 4a, curve 2. The difference between the complexing curves recorded using the two protocols is evident (Fig. 5a). Although the shape is not strongly influenced, the relatively large difference in peak currents is obvious. The ratio of peak currents (curve 3) decreases by addition of copper, which indicates a relatively diminishing influence of the adsorbed SAS on copper electrochemical reactions occurring during the stripping step. Complexing capacity parameters (ligand concentration and stability constant) were obtained using the Ru ži ć-van den Berg linearization method [39,40] assuming the 1:1 metal-ligand interaction (Table 1). Without the desorption step, almost a 2.5 times higher concentration of complexing ligand was obtained, while the apparent stability constant increased only slightly. It is obvious that the increased ligand concentration obtained without the desorption step is the result of the adsorbed SAS. Although the absolute difference in ligand concentration is not large, it is evident that without the desorption step, the ligand concentration is overestimated. As a consequence, different speciation results concerning copper distribution in a particular sample are inevitable. Very important parameter in the protocol of MCC determination is the sensitivity, which is used for transformation of peak heights into the concentration of metal found in the sample (this step is a prerequisite for all calculations in metal speciation studies using voltammetric methods) [5]. Usually, authors use the sensitivity obtained either in the range where, according to the titration curve, all sites are almost saturated (linear part of the plot) [39,41] or from the separately determined sensitivity in a UV digested sample (where all organic matter is destroyed) [31]. According to the theoretical calculations for metal complexes with known stability constants, the slope which corresponds to the titration curve without the ligands is never reached in real samples (only asymptotically approaching) [5]. The intensity of convergence depends on the stability constant of formed metal complexes. As the apparent stability constant is lower, higher concentrations of added metal are needed in order to be closer to the real slope, and the logarithmic model of metal addition is advisable [5]. If the slope/sensitivity obtained in the UV sample is used for data transformation, more erroneous results could be obtained, if the protocol without the desorption step is used (in the UV sample, SAS are destroyed and the effect of adsorbed SAS on the peak height vanishes, see curve 3 in Fig. 2). However, for the transformation of data obtained using the desorption step, the sensitivity obtained in the UV sample is recommended, but critical examination of both slopes should be done before the final data treatment/conversion.

Conclusion

As shown in many papers, the adsorption of natural organic matter (NOM) on the surface of working electrodes produces effects which interfere with electrochemical reactions of metal ions. Such interferences are amplified in ASV of copper in natural water samples. In this work, we used a protocol which utilizes desorption of the NOM to obtain reliable copper voltammograms, free of interferences. According to phase sensitive ac voltammetric measurements, most of the NOM is desorbed at potentials <-1.4 V. Application of a very negative potential in ASV at the end of the accumulation time for a short period of time (3 s) produced anodic peaks of copper free of artefacts. Although it is shown in the literature that stripping chronopotentiometry in the depletive mode is less sensitive to adsorption processes than ASV in the pulse mode, no benefit is obtained using this technique in measurements in natural samples without the desorption step. The application of desorption step could be very beneficial in ASV measurements using solid electrodes.

Recently, it was shown that pseudopolarography (and an analogue method, scanned stripping chronopotentiometry) is a powerful technique in the speciation of trace metals in natural waters. Various theories, describing probable conditions which could arise in such measurements, were developed and tested in model solutions. However, as the natural water is a mixture of many different compounds, it is very important to resolve/recognize processes which occur during the measurements. We showed that the adsorbed layer of NOM strongly influences a pseudopolarogram of copper, producing changes in its shape and intensity. It also influences the peak potential and half-peak width of single copper anodic voltammograms. Analysis of such pseudopolarograms according to theoretical postulates could lead to incorrect conclusions. Application of the desorption step produces very well resolved pseudopolarograms of copper, without any of the above mentioned drawbacks, which is a benefit in copper speciation consideration.

Similar benefits of the desorption step were registered in the determination of copper complexing capacity (CuCC). The adsorbed layer of NOM caused an apparent 2.5-fold increase in ligand concentration compared to the protocol which utilizes the desorption of NOM, while the conditional stability constant slightly increased. It is important to note that the amount of reduced copper from inert copper complexes during short desorption step at -1.6 V is negligible comparing to amount of labile copper reduced at more positive potentials (∼-0.5 V), which is prerequisite in determination of CuCC (desorption step usually contributes to only 1% of the total accumulation).

These experiments show the importance of distinguishing processes which occur as the consequence of metal complexation with ligands from the bulk of the solution, and processes arising as the product of a surface reaction with adsorbed ligands.

To eliminate the artefacts of the adsorption of NOM upon the copper speciation results using ASV, the application of described desorption step is recommended.

Fig. 1 -

 1 Fig. 1 -DPAS voltammograms of copper in seawater sample (sample 1) obtained using different experimental parameters: (1) t acc = 300 s at E acc = -0.45 V; (2) t acc = 300 s at E acc = -1.6 V; (3) t acc = 297 s at E acc = -0.45 V and t acc = 3 s at E acc = -1.6 V.

Fig. 2 -

 2 Fig. 2 -Difference of capacitive current (measured at -0.6 V) vs. accumulation potential obtained in seawater sample (sample 1) using accumulation time of 60 s and without (1) and with (2) 1 s at -1.6 V at the end of accumulation time (-1.6 V used as reference potential). Curve 3 corresponds to UV irradiated sample without desorption step. Inset: dependence of the difference of capacitive current on the accumulation time: (1) E acc = -0.6 V; (2) E acc = -0.6 V and E acc = -1.6 V for 1 s; (3) E acc = -1.6 V. Parameters: f = 75.12 Hz, t mod = 0.21 s, t int = 0.61 s, phase angle = 90 • .

Fig. 3 -

 3 Fig. 3 -Stripping chronopotentiograms of copper in seawater sample (sample 1) obtained using different experimental parameters: (1) t acc = 300 s at E acc = -0.45 V; (2) t acc = 297 s at E acc = -0.45 V and t acc = 3 s at E acc = -1.6 V. I stripp = 2 × 10 -9 A. For both curves, an equilibrium potential of -0.5 V for 10 s was used.

Fig. 4 -

 4 Fig. 4 -Pseudopolarograms of copper in seawater sample (sample 2) obtained without (curve 1) and with (curve 2) desorption step constructed using peak height (a), anodic peak potential (b) and half-peak width (c). Total accumulation time was t acc = 300 s. Solid line in (a) corresponds to pseudopolarogram of zinc performed separately in a new sample (peak currents of zinc are scaled by factor of 0.75 to correspond to peak currents of copper).

Fig. 5 -

 5 Fig. 5 -Copper ion titration curve for the determination of CuCC in brackish water sample (sample 3) without (curve 1) and with (curve 2) applied desorption step. Procedure parameters: total t acc = 300 s, E acc = -0.5 V, desorption potential, E acc = -1.65 V. Curve 3 corresponds to peak height ratio of curve 2 and 1.

Table 1 -Ligand concentration, [L] and apparent stability constant, K obtained in brackish water sample using two different protocols

 1

		[L] (mol L -1 )	K (mol -1 L)
	Without desorption step	3.40 × 10 -8	4.14 × 10 8 (log K = 8.62)
	With desorption step	1.41 × 10 -8	2.70 × 10 8 (log K = 8.43)

was the following: accumulation at desired potentials (during 60 s) followed by measurement of the capacitive current at -0.60 V, while for

curve 2 at the end of accumulation time the potential of -1.6 V was applied for 1 s. It is evident that by applying the negative potential, almost a complete layer of

Acknowledgments

This work was accomplished within the framework of French project (grant no. 03/1214910/T) called MONALISA (Mati ère Organique NAturelle en miLIeu SAl é). The grant of the University of the South -Toulon-Var for Ph.D. and researcher exchanges between France and Croatia, as well as financial supports of the Ministry of Science, Education and Sports of the Republic of Croatia (through grant no. 098-0982934-2720) are gratefully acknowledged. r e f e r e n c e s


Rudy Nicolau
email: rudy.nicolau@ensil.unilim.fr

Yoann Louis
email: louis@univ-tln.fr

Dario Omanovic
email: omanovic@irb.hr

Cédric Garnier
email: c.garnier@ism.u-bordeaux1.fr

Stéphane Mounier
email: mounier@univ-tln.fr

Ivanka Pizeta
email: pizeta@irb.hr

Dario Omanović

Ivanka Pi Žeta

Study of interactions of concentrated marine dissolved organic matter with copper and zinc by pseudopolarography

Keywords:

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Introduction

Dissolved organic matter (DOM) in natural aquatic systems, such as marine waters and estuaries, is well known to interact with metals and organic pollutants, thus modifying their speciation and subsequently their transport, fate, and bioavailability towards micro-and macro-organisms [START_REF] Buffle | Complexation Reactions in Aquatic Systems[END_REF][START_REF] Byrne | [END_REF]. Organic pollutants-DOM associations occur mostly due to the DOM hydrophobic nature, and are simply characterised by par-tition coefficients (K DOC ), often correlated to the octanol/water partition coefficient (K ow ) [3]. In contrast, metal ions-DOM associations, although often of great intensity (e.g. more than 99% of Fe III or Cu II are strongly bound by organic ligands in surface seawaters [4][5][6]), are metal-and DOM-specific complexes, dependent on the physico-chemical conditions: ionic strength, pH, presence of other major and trace cations. Characterisation of the DOM binding properties has been a subject of numerous studies, leading to the development of analyti-0003-2670/$ -see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.04.038 cal techniques and modelling tools (e.g. [7][8][9]). However, due to low organic carbon content (0.1-1 mg C L -1 ) and trace levels of metals (rarely higher than several nmol L -1 ), a study of DOM-metal binding properties in unpolluted marine waters still remains a challenge. In order to understand the role of the marine DOM in metals speciation, various techniques have been used ( [10], and references cited therein). Among them anodic stripping methods offer satisfactory sensitivity and selectivity and could provide useful information about the DOM-metal association (complexing parameters) required by the mathematical speciation programs [11][12][13]. Titration of the marine DOM with metals (preferably in a non-linear addition mode [14]) and/or methods based on ASV measurement at different deposition potentials (pseudopolarography, pseudovoltammetry or scanned (deposition potential) stripping technique) were widely used to accomplish this [15][16][17][18][19][20][21][22][23][24][25][26][27][28].

Pseudopolarography and analogues techniques have been used for speciation studies for the last 30 years [15][16][17][18][19][20][21][22][23][24][25][26][27][28]. But, as it is a time-consuming technique, it has seldom been used until the last decade, when new computercontrolled instruments offered the possibility to automate the whole process. The theory and experimental verification of pseudopolarography [15][16][17][18][19][20][21][22][23] and scanned stripping chrono-potentiometry [24,25] are well described. The main benefit of using pseudopolarography when compared to the classical polarography/voltammetry is the possibility to measure speciation at natural concentration level of trace metals (<10 -8 mol L -1 ). This technique allows discrimination/separation of different fractions of metals, from the electrochemically labile to the inert ones, regarding the analytical window of the method. It is successfully applied for the speciation of metals in model solutions and in natural waters, both for the labile and for the inert metal complexes. According to the theory and to experimental evidences [15][16][17]22], one or more reduction waves could be obtained (similarly like in classical polarography) which correspond to one labile and/or to one or more inert metal complexes. The position of those waves (half-wave potentials) depends on the corresponding stability constants of the formed metal complexes. Although the theory for inert metal complexes is valid only for the standard electrode potentials [16], experimentally obtained dependence of the reduction half-wave potential on the stability constants ("chelate scale") was successfully applied for the estimation of the stability constants of unknown inert metal complexes in natural waters [19][20][21][22]. Since the DOM is of complex and heterogeneous nature, complications such as its adsorption on electrode surface could occur and lead to erroneous results [29][30][31]. Thus, special attention should be given to the interpretation of obtained results.

In an unpolluted marine environment, average DOM concentration is about 1 mg L -1 . In such clean seawater systems the determination of complexing parameters by stripping voltammetry was often achieved near the detection limit or resolution capability of the method [14]. So, the opportunity has been taken to join the project which concentrated the DOM in a marine sample [START_REF] Huguet | Universit ät Karlsruhe, Proceedings of the 13th Meeting of the International Humic Substances Society[END_REF], and to analyse its interactions with metal by pseudopolarography, exploring and showing more clearly the possibilities of the method.

The objective of this work is to explore the pseudopolarographic behaviour of the copper and zinc complexes present in the seawater sample with high concentration of DOM. An attempt has been made to extract some quantitative data regarding the metal complexes in the sample and to point out certain features of the pseudopolarographic approach as a kind of "finger-print" method.

Experimental

Sample preparation

The seawater sample was taken in the coastal area of southern France. The sampling site (bay of Balaguier: (500-1000 L), using two subsequent concentrations steps: nanofiltration followed by reverse-osmosis [START_REF] Huguet | Universit ät Karlsruhe, Proceedings of the 13th Meeting of the International Humic Substances Society[END_REF]. The concentration step is necessary to obtain a sufficient quantity of DOM for the subsequent characterisation of its properties by other MONALISA participants by using NMR 13 C, NMR 113 Cd, pyrolysis-GC-MS, HPSEC and acido-basic titrations. The method was conducted in two steps: [START_REF] Buffle | Complexation Reactions in Aquatic Systems[END_REF] with osmosis equipment (TIA) by nanofiltration (Osmonics DL2540) from 940 L of filtered sample of the salinity S = 37 and DOC = 1.1 mg L -1 , the sample reached 32 L with an addition of MilliQ water at the end to decrease the salinity to the value of S = 10.2 and DOC = 10.8 mg L -1 , and (2) with reverse-osmosis (Filmtec SW30-2540) the final 10.5 L of the sample with the salinity of S = 28 and DOC = 30.4 mg L -1 was obtained. The obtained DOM concentrated sample, stored in the dark at 4 • C after spiking with NaN 3 (100 L of 0.1 mol L -1 ) to prevent any biological activity, was subsequently analysed with voltammetric methods by the addition of copper(II) ions and by changing the pH of the sample.

Chemicals, instrumentation and measurement protocol

A copper stock solution was prepared using a salt of copper nitrate trihydrate of reagent grade (MERCK, Darmstadt). Samples were acidified using 69% HNO 3 (J.T. Baker). All measurements were carried out with voltammetric analyser PGSTAT 12 with GPES 4.9 software (EcoChemie, Utrecht, The Netherlands) coupled to a three-electrode system of a Metrohm 663 VA Stand. The working electrode was a static mercury drop electrode (SMDE) (drop area of 0.25 mm 2 ). All potentials were referred to Ag|AgCl|KCl 3 mol L -1 reference electrode. A carbon rod was used as a counter electrode. The electrolyte solution was stirred at 3000 rpm using a Teflon rotating stirrer. To minimise adsorption of metal ions a Teflon electrochemical cell was used. Before each measurement all vessels, the cell and electrodes were cleaned with 10% (analytical grade) nitric acid and rinsed with ultra-pure MilliQ-water. pH was controlled by a pH meter (Metrohm 713) with a combined pH-micro-electrode (Mettler, Inlab422, reference: Ag|AgCl|KCl 3.0 mol L -1 ).

For each experiment, 20 mL of sample was placed in the voltammetric cell. Dissolved oxygen was removed from the solution prior to the measurements by purging with water-saturated nitrogen for 15 min and a nitrogen blanket was maintained above the sample surface during the measurement. Measurement protocol: total metal concentrations were determined by standard additions of Cu and Zn ions by differential pulse anodic stripping voltammetry (DPASV) after UV-irradiation (Hanovia, 450 W medium Hg pressure, 12 h) at acidic pH. Voltammograms for pseudopolarography were recorded using DPASV with the following measurement parameters: deposition potential E dep = -1.6 to -0.020 V in steps of 0.020 V (altogether 80 scans), deposition time t dep = 360 s, pulse amplitude A = 25 mV, interval time t int = 0.15 s, modulation time t mod = 0.04 s, step increment E st = 0.002 V, initial potential E i = -1.2 V, final potential E f = 0.01 V. An equilibration at -1.2 V for 15 s was applied at the end of the accumulation period. During this period, desorption of adsorbed surface active substances on the mercury surface occurs, preventing negative effects on the copper anodic peak [31]. A home-written program incorporated in the Project mode of the GPES software was used to automate pseudopolarographic measurements. The voltammetric parameters (peak height, peak position and half-peak width) were automatically determined by the home-written software, as well.

Results and discussion

The purpose of this work was a pseudopolarographic study of copper complexes in concentrated seawater sample. As the procedure of voltammetric measurements included a zinc oxidation peak as well, it was found very interesting to present and to try to interpret behaviour of zinc pseudopolarograms in addition to the copper ones. The formation of CuZn intermetallic compound is not likely to occur, because of much lower concentrations of copper and zinc in our experiments when compared to those reported in literature, which should be in a milli-molar range [START_REF] Wise | [END_REF]34]. While in the original sample total copper(II) concentration was found to be (1.48 ± 0.12) × 10 -8 mol L -1 , the concentration was 100 times higher (1.43 ± 0.17) × 10 -6 mol L -1 in the concentrated sample. Total zinc concentration in the concentrated sample was (3.9 ± 0.3) × 10 -7 mol L -1 . Dissolved organic carbon (DOC) before and after pre-concentration was 1.1 and 30.4 mg L -1 , respectively. This showed that during the concentration process ratios of analytes were changed, but it is not the subject of this study.

A set of anodic stripping voltammograms obtained in a concentrated seawater sample without metal additions appears in Fig. 1. Two well separated and expressed peaks related to zinc (∼-1.0 V) and copper (∼-0.18 V) are evident, ensuring the accurate and reliable peak parameters read outs needed for the construction of corresponding pseudopolarograms. It is interesting to note that although total copper concentration was 3.5 times higher than that of zinc, the obtained anodic peak of copper (at E dep = -1.6 V) was about half of that of zinc. Taking into account that copper in chloride solutions undergoes one-electron charge transfer [35] and that the sensitivity is half of that required for two-electron charge transfer, still remains the factor of 3.5 to be explained. Such a smaller copper peak could be attributed to the presence of its organic complexes which are either (i) non-electroactive at this potential, (ii) have much lower diffusion coefficient from that of free/labile copper, or both of this. This example simply illustrates that experiments performed in natural samples should be very carefully analysed in respect to the expected theoretical behaviour.

Pseudopolarograms of copper

Experimental conditions and labelling of the results are summarised in Table 1 and are valid for Figs. 234. Fig. 2a shows untreated pseudopolarograms obtained without added copper and with additions of 4 × 10 -6 and 8 × 10 -6 mol L -1 of copper(II) ions at natural pH 8.3 and 4. In addition, two pseudopolarograms at pH 2 were also recorded for the two additions of copper, but they were practically the same as the pseudopolarograms obtained at pH 4 (data not shown).

The three doted lines correspond to the peak currents anticipated for reduction of only free/labile inorganic copper complexes (in DOM-free sample). These values were obtained according to the sensitivity obtained in UV-digested sample at natural pH. To get better overview and relations between pseudopolarograms, each of them was normalised to the above-mentioned projected currents in non-complexing media (i NC ) (Fig. 2b). 2 3c

Conditions and labelling of the curves.

The shapes of the copper pseudopolarogram without addition and for the first addition of copper (curves 1a and 2a) clearly show that the wave which corresponds to the reduction of the free/labile copper species has not appeared. Only continuous increase of the peak current starting from E acc = -0.25 V was evolved. However, for second addition of copper (curve 3a) an evolution of the wave at E acc = -0.2 V occurred. This indicates a marked increase in concentration of free copper ion and its labile complexes comparing to copper-organic complexes. Several interpretations could be given for the continuous increase of the peak current at the accumulation potentials more negative than -0.3 V (for all three pseudopolarograms at pH 8.3). Many authors reported that an increase of the peak current more negative than Table 1. 1.

Fig. 3 -(a) Pseudopolarograms from Fig. 2 with the current normalised to the first point; (b) peak position vs. deposition potential curves of copper; (c) half-peak width vs. deposition potential curves of copper. Conditions and labelling of the curves are given in Table

reduction potential of free/labile copper corresponds to the reduction of various copper organic complexes [16][17][18][19][20][21][22]. Two or more well separated waves could be obtained depending on metal complexes having different stability constants [16,[19][20][21][22]36]. Several papers by the group of Luther III showed that thermodynamic stability constants of inert metal complexes could be estimated according to the formed "chelate scale" [19][20][21][22].

However, there are two completely distinct approaches concerning both, determination of complexing capacity and Table 1. pseudopolarography. The first approach considers the difference in diffusion coefficients of labile complexes as minor, while the direct reduction of inert metal complexes takes place at potentials more negative than labile metal complexes [16,[19][20][21][22]36]. In this case two or more reduction pseudopolarographic waves could be obtained. This approach is most widely used in the literature. The second approach assumes that all complexes are labile, and that there is only one pseudopolarographic wave having a flat limiting current controlled by the mean diffusion coefficient of both weak and strong metal complexes [17,28,37]. However, many authors recorded pseudopolarograms in a relatively narrow range of deposition potentials and there was no opportunity to see possible evaluation of pseudopolarographic waves of other directly reduced inert metal complexes [17,28].

Both approaches are very strict and often do not consider other possible options. Both approaches should be respected in the interpretation of the voltammetric data obtained in natural water samples. Taking everything mentioned above into consideration, there are no severe anchorages to apply one of those two approaches to our results to be able to extract some quantitative information at natural pH. The main unknown parameter is the diffusion coefficient which cannot be determined from this set of data. However, despite the lack of quantitative data, some relations could be assigned.

The shapes of all three pseudopolarograms at pH 8.3 showed increasing peak current trend towards negative potentials, without reaching a plateau in the analytical window of the method. It is reasonable to assume that this trend would continue for more negative potential which were not scanned in our experiments.

The parameters of the plateau could only be explained by speculations, because there are two unknown terms which were discussed above (diffusion coefficients and electroactivity and (non)lability of copper complexes). Gradual increase of peak current at potentials more negative than -0.3 V reflects the loss of lability of copper heterogeneous complexes [17] and could be assigned to the gradual reduction of numerous copper (inert) organic complexes having increasing stability constants [20]. In such a system the calculation of any binding parameter is precluded even for known sample composition [17]. However, as the diffusion coefficients and the electro(in)activity of all strong inert copper complexes in the sample are unknown it is not possible to declare the quantity of copper species. The absence of the wave of free/labile copper on the pseudopolarogram obtained without the copper addition implies that copper in this concentrated sample exists mainly in a form of relatively strong inert complexes. By gradual addition of copper, the distribution of copper species was changed thus forming more labile complexes, finally for the second addition of copper producing an evident wave. The position of this wave (Fig. 2, curve 3a) matched those obtained in the acidified sample (Fig. 2, curves 1b, 2b and 3b). Normalised pseudopolarograms at pH 8.3 show this evident increase of labile copper complexes with increasing concentrations of copper (Figs. 2b and3a).

The acidification of the sample leads to the redistribution of copper complexes. The redistribution of the organic fraction of copper complexes to inorganic forms during the acidification is a consequence of competition between Cu 2+ and H + for DOM binding sites that regards a part of carboxylic-like sites. The part of phenolic-like DOM sites with strong affinities towards proton was not importantly influenced as shown by Lu and Allen on concentrated freshwater DOM [8] (presuming the general distribution of DOM binding sites onto carboxylic-like sites (average pK a of 4.5) and the phenolic-like ones (average pK a of 10) [START_REF] Perdue | Acidic functional groups of humic substances[END_REF]). Three pseudopolarograms measured at pH 4 show that a large part of copper was released from its inert complexes. Limiting currents of pseudopolarograms for two copper additions approached the projected level for non-complexed copper. The difference between expected and obtained limiting current for all three pseudopolarograms remained the same, implying that not all of strong copper inert complexes (reduced within the scanned deposition potentials) were destroyed at pH 4. The portion of these complexes became smaller with a higher copper concentration which is illustrated in the normalised plot (Fig. 2b). These relations indicate that at such relatively weak acidic conditions (pH 4) the proton is a strong competitor to copper for these active sites of DOM, implying existence of weaker copper complexes in the sample solution. Additional experiments in the same concentrated sample involving competition with proton and calcium were already done and the paper is under submission.

It is evident from this entire consideration that numerous different experiments should be performed in the same sample to be able to correctly interpret obtained data in a manner of trace metal speciation of a particular natural water sample. Such an analysis can serve operationally to characterise a sample and to compare the sample with another one. In this way, pseudopolarograms of trace metals could be considered as a "fingerprint" of the sample.

In Fig. 3a pseudopolarograms are normalised to the most negative point. This transformation graphically illustrates the ability of the sample to complex copper(II) ions by either, the addition of copper or by changing of pH. The relationships of the peak potential and the half-peak width vs. deposition potential curves could provide additional information about electrochemical reactions (Fig. 3b andc). There is a clear distinction of the peak potentials of the copper anodic peak potentials obtained in samples with and without copper additions. This might indicate the existence of a fraction of labile copper(II) complexes which were concealed with higher copper(II) additions, this resulted in a peak shift towards more positive potentials. Shift towards more negative potentials when the deposition potential approaches peak potential has already been noticed [17,26,[START_REF] Buffle | [END_REF][40][41][42] and was ascribed to the existence of strong complexing ligands concentrated at the electrode surface. Half-peak width bears also some information. Its accurate determination is subjected to disturbances like humps affecting a baseline subtraction procedure and should be taken with attention. However, it should be considered of great interest as it could indicate, for example, the existence of two closely overlapped peaks that might come from adsorption processes, and could be a motivation for further investigation. The half-peak width of copper oxidation is about 90 mV that corresponds to one-electron charge transfer, as expected in a seawater sample [35].

Pseudopolarograms of zinc

Eight pseudopolarograms of zinc, analogous to those of copper, were drawn by tracing the peak of oxidation of zinc in the same experiments as those described for copper. Similarly shaped pseudopolarograms were obtained in natural, non-concentrated sea water sample taken in the same bay of Toulon, France (data not shown). It is a good argument for accepting the proposed concentration procedure for the DOM investigation. In Fig. 4a the zinc peak current pseudopolarograms are shown in absolute values. Fig. 4b andc show the peak potential and the half-peak width vs. deposition potential curves of zinc. There is a strong dependence on pH that affects peak height, peak position and half-peak width, and groups the signals accordingly. Pseudopolarograms of the samples acidified to pH 2 have a particular shape. Their amplitude is lower (indicating possible blocking of the electrode by adsorption of protonated DOM) and they shifted negatively (variation about 200 mV), with a remarkable change of slope (indicating a quasi-reversible or irreversible cathodic reaction [23,36]). At the same time, peak potentials are about 60-70 mV more positive than for other conditions (pH 8.3 and 4), which might indicate the missing of some labile complexes that would shift the peaks negatively ( [START_REF] Crow | Polarography of Metal Complexes[END_REF], p. 49). The half-peak widths of the single curves indicate a simple reversible two-electron reaction ( [START_REF] Heyrovsk Ý | Principles of Polarography[END_REF], p. 129).

Regarding the complexation with DOM "the tails" of the pseudopolarograms representing the peaks of oxidation of zinc appeared to be of interest, while the deposition potential was more positive than the reduction potential, shown in detail in the inset of Fig. 4a. The maximum value of the tail moves from about -0.7 V for the natural pH to about -0.2 V for the pH 4. The tail does not exist at pH 2. At that pH most of the DOM is protonated and is not capable of complexing with zinc. Either adsorbed at the electrode or not, it does not help the accumulation of zinc at the electrode either during the deposition time or during the scanning time. As the tail exists at pH 4, one might conclude that the competition effect between Zn 2+ and H + towards the DOM binding sites in favour of H + prevalently occurs at the pH below 4, in contrast to Cu (see Section 3.1). It points out the important quantity of complexing sites of carboxylic type [8,[START_REF] Masini | [END_REF][46][47]. It also reveals that Zn-DOM binding sites and Cu-DOM ones are not identical. As the strength of Cu-DOM interactions is generally larger from that of Zn-DOM ones [6] and the total Cu concentration is increased, it is probable that Zn is not bound by the Cu-specific DOM sites, which are assumed to be of phenolic-like type (see Section 3.1), but by less strong sites with acidic properties supposed to be of carboxylic-like type that cover the pK a range of 1-7 [8,[START_REF] Perdue | Acidic functional groups of humic substances[END_REF]. This hypothesis has already been expressed to explain the weak competition effect between Cu and major divalent cations towards DOM binding sites [8].

A closer insight in the peak position and half-peak width vs. deposition potential curves (Fig. 4b andc) shows the redistribution of labile zinc species which was reflected in the peak position shift correlated with changing of pH, particularly complex shifting and width changing of the peak at pH 4. As mentioned previously, the presence of labile complexes is manifested by the shifting of the peaks in negative direction. However, the possible causes of the shifting of peaks vs. deposition potential are manifold. Taking into account the entire effects one might speculate about the adsorption of neutral organic matter: it is favoured at the potential of zero charge of the mercury drop, or the adsorption of charged species would influence the signal at the more extreme negative or positive potentials [48]. However, these effects appear to be interesting for further qualification of organic matter and for differentiation of the various dissociation constants of the numerous groups of organic ligands that could be traced by the combination of pH titration and pseudopolarography.

Conclusions

The ability of DOM to complex copper has been tested on a seawater sample concentrated by nanofiltration and reverse osmosis by titration with up to 8 × 10 -6 mol L -1 copper(II) ions applying pseudopolarography. The results obtained for the analysed sample clearly evidenced a distribution of copper in three categories: (i) free or labile (deposition potential at -0.3 V), (ii) complexes that are reduced and released within the analytical window of the method (deposition potential from -0.3 to -1.6 V), and (iii) electroinactive complexes. Their ratios change with the addition of copper(II) ions and with changing pH. Such an analysis can serve operationally to characterise a sample and to compare the sample to other ones. Zinc pseudopolarograms showed significant dependence on pH value. A large difference in pseudopolarograms of zinc between pH 4 and 2 reveals the existence of organic matter with protonation constant pK a between 2 and 4. Those analyses also indicate the differences of DOM sites for copper and zinc complexation regarding their stability and acidity constants. Additional experiments are necessary for more detailed DOM characterisation regarding zinc and possible zinc-copper competition. Experiments with the addition of other cations are planned for they might cause different reactions and characterisation of DOM, which would be reflected in the shapes of pseudopolarograms.

The complex structure of the obtained pseudopolarograms reflects and indicates composite structure of the ligands making complexes with metals. This raises a challenge to construct the models with such behaviour, and to match the curves with the theory of pseudopolarography for retrieving quantitative information out of the signals regarding the speciation in the bulk of the solution and regarding the electrode processes.

Fig. 1 -

 1 Fig. 1 -Set of voltammograms of pure concentrated sample of pH 8.3. Measurement parameters: DPASV method; E dep interval: from -1.6 to -0.02 V in steps of 20 mV, t dep = 360 s, A = 25 mV, t int = 0.15 s, t mod = 0.04 s, E st = 0.002 V, E i = -1.2 V, E f = 0.01 V.

Fig. 2 -

 2 Fig. 2 -Peak current pseudopolarograms of copper (a) row data and (b) data normalised to current expected for non-complexed copper (i NC ). Conditions and labelling of the curves are given inTable 1.

Fig. 4 -

 4 Fig. 4 -(a) Peak current pseudopolarograms of zinc. Inset: detail of Fig. 4a; (b) peak position vs. deposition potential curves of zinc; (c) half-peak width vs. deposition potential curves of zinc. Conditions and labelling of the curves are given in Table1.

Table 1 -Additions of copper to the sample containing (1.43

 1

		pH	Symbols	Curve labelling
	-	8.3		1a
	-	4	᭹	1b
	4 × 10 -6	8.3		2a
	4 × 10 -6	4		2b
	4 × 10 -6	2		2c
	8 × 10 -6	8.3	♦	3a
	8 × 10 -6	4		3b
	8 × 10 -6

± 0.17) × 10 -6 mol L -1 copper (II) ions Cu(II) added (mol L -1 )

Acknowledgements

This study was supported by the French MONALISA research project coordinated by the IFREMER (Grant No. 03/1214910/T). The grant of the University of Sud Toulon and Var for Ph.D. and researcher exchanges between France and Croatia, as well as financial support of the Ministry of Science, Education and Sports of the Republic of Croatia (through Grant No. 098-0982934-2720) are gratefully acknowledged. We thank Marta Plavši ć and Damir Krznari ć for their precious discussions. We also thank to the anonymous reviewers, especially to one of them.

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