Impact of Biofouling on Diffusive Gradient in Thin ... - ACS Publications

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Impact of Biofouling on Diffusive Gradient in Thin Film Measurements in Water Emmanuelle Uher,*,†,‡ Hao Zhang,§ Sarah Santos,† Marie-Hélène Tusseau-Vuillemin,∥ and Catherine Gourlay-Francé†,‡ †

Irstea, UR HBAN Hydrosystèmes et Bioprocédés, 1 rue Pierre-Gilles de Gennes, CS 10030, 92761 Antony Cedex, France FIRE FR-3020, 4 place Jussieu, 75005, Paris, France § Lancaster Environment Centre, Lancaster University, Bailrigg, LA1 4YQ , U.K. ∥ Ifremer, 155 Rue Jean-Jacques Rousseau, 92138 Issy-Les-Moulineaux, France ‡

S Supporting Information *

ABSTRACT: The technique of diffusive gradient in thin film (DGT) is commonly used to assess metal contamination in natural waters. In this paper, we assess the effect of biofouling on DGT measured labile concentrations in water and investigate whether an additional nuclepore polycarbonate membrane on the surface of DGT devices can limit biofilm growth. Simultaneous field deployments of DGT equipped with and without the additional membrane in a canal receiving wastewater were compared. The effect of the biofilm was also assessed in controlled laboratory experiments, completed by the experimental determination of several metals diffusion coefficients in the hydrogel and membrane systems. The biofilms effect was problematic only from the 10th day of accumulation. Accumulation of some elements is highly biased by the presence of a thick biofilm (Zn, Ni, Cd). The polycarbonate membrane improved the quantification of Cd and Ni but adversely affects the quantification of Cr and Co. A kinetic model is proposed to explain the biofilm role on the DGT measurement. Depending on the metals of interest, it is possible to limit bias due to biofilms by using an additional polycarbonate membrane.

T

he technique of diffusive gradient in thin film (DGT) developed by Zhang and Davison1 is now commonly used to measure in situ labile metals, metalloids, and phosphates in water. It has proved to be useful on account of its simplicity and wide applicability. DGT is an in situ passive sampling technique. It offers a large number of advantages in comparison with grab sampling: continuous monitoring of water bodies which have fluctuating levels of contaminants, minimizing matrix effects, and reducing analysis costs. It is suitable for obtaining a long time overview of pollutants in an environmental compartment. In natural waters, formation of biofilms at the surface of the samplers is a consequence of the exposure. Biofilm is composed of bacteria, algae, fungi, and extracellular polymers resulting from cell metabolism.2 These various elements interact with trace metals through physical, chemical, and biological processes in the water.3,4 Biofilm growth at the surface of the samplers may affect the thickness of the diffusion layer and/or modify the diffusion coefficient. Biofilms are considered as a nuisance for a lot of applications (industrial processes,5 passive samplers,6−9), and many studies have investigated methods to eliminate them.5,9,10 The most common solution is to use biocides, among them metals or coatings with polymers to modify the surface.2,10−12 © 2012 American Chemical Society

Although biofilms may affect the estimation of labile concentrations by DGT, very few studies have investigated their effect. In most cases, biofilms are not taken into account or they are considered to be insignificant.13 Pichette et al. studied the effect of algal biofilm development on phosphate measurement by DGT in a freshwater aquaculture pond.7 They observed that after 3−4 days of deployment, the accumulation of phosphate in the DGT binding layer was affected by the algal growth. To prevent biofilm formation, they pretreated the protective membrane at the surface of the DGT with copper or silver iodide11 and also tested the use of a polycarbonate nucleopore track-etched membrane as an alternative outer layer. The alternative membrane was not as successful as biocides in preventing algal growth. With another type of DGT device using a poly(4-styrenesulfonate) as a binding phase, Li et al.14 were able to exchange the dialysis membrane diffusive layer to overcome biofouling problems. Furthermore, they noticed that the very hydrophilic properties of the membrane prevent from important biofouling. Received: October 28, 2011 Accepted: March 7, 2012 Published: March 7, 2012 3111

dx.doi.org/10.1021/ac2028535 | Anal. Chem. 2012, 84, 3111−3118

Analytical Chemistry

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by inductively coupled plasma mass spectrometry (ICPMS; Thermo X7). The temperature, pH, and volume of liquid were monitored at the beginning and at the end of the experiment and have to be the same in both compartments. To spike the source compartment, a multielement solution in HNO3 5% (SCP-28-AES, SCP Science) was used. Diffusion coefficients of Ba, Cd, Cr, Co, Cu, Mn, Ni, Pb, Ti, V, and Zn were measured simultaneously. In this study, the water was adjusted at pH 2 with HNO3 in the receptor compartment to ensure that all metals are under ionic form. The diffusion coefficient measurements were run with four configurations of diffusion layer: open pores gel (OP), restricted pores gel (RG), open pores gel + PES membrane + PC membrane (OP + m), restricted pores gel + PES membrane + PC membrane (RG + m). The determination of the diffusion coefficient D is based on Fick’s first law. At steady state, a gradient of concentration ΔC is established through the diffusion layer (thickness Δx) between the two compartments. The flux F of metal through the diffusion layer is

The use of silver and copper biocides is prohibited for metal measurement by DGT. A physical method, which modifies the surface of the membrane, is then more appropriate to limit the biofilm formation in this case. It has been demonstrated that smooth surfaces (glass, polished surfaces) are less prone to biofilm deposition.2 Polyethersulfone (PES) membranes, usually used as the outer protective layer of the device, are rough. Consequently alternative smoother membranes, like polycarbonate nucleopore track-etched membranes, have been proposed. Although less effective than biocides for the limitation of algae-rich biofilm formation,11 these techniques were applied in wastewater,15,16 where biofilms are very likely composed of bacteria and quite different from the algal-dominated ones observed in aquaculture waters. Yet, the quantitative effect of biofilms with or without alternative membranes has not been investigated. The purpose of this study is to assess the effect of biofilms on the measurement of labile metals by DGTs under in situ deployment conditions and then to verify to what extent nuclepore membranes can limit biofouling. The effect of the additional membrane on the diffusion coefficients was investigated independently. We then discuss how the membrane modifies the diffusion coefficients, if the membrane is effective in limiting the biofilm effect, and the impact of the biofilm on DGT measurement.

F=D

ΔC Δx

(1)

When the concentration in the source compartment is sufficiently high, the mass transferred to the receptor compartment does not affect significantly the concentration of the source, and the flux is constant. The flux in the receptor compartment is calculated as follows:

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EXPERIMENTAL SECTION DGT, Gel, and Membranes Materials. Two types of gel supplied by DGT Research Ltd. (U.K) were investigated: classical diffusive open pores gels (Δg = 0.76 mm) and gels with restricted pores (Δg = 0.78 mm). Gels are made up of crosslinked polyacrylamide. The pore size and structure of the gels, which can be easily manipulated by varying the chemistry and the concentration of cross-linker, determine the rate of diffusion of the species in the gels. Gels with restricted pores severely retard the diffusion of metal-humic substances but allow free diffusion of simple metal ions, while open pores gels allow diffusion of organic species,17 but with a much lower diffusion coefficient than in water. Membranes of polyethersulfone (PES) (Pall, 0.45 μm pore diameter, 2.5 cm diameter, 140 μm thickness) were washed in suprapur 1 mol L−1 HNO3 (Merck) and stored in NaNO3 suprapur solution. Polycarbonate nuclepore membranes PC were purchased from Whatman (0.4 μm pore diameter, 2.5 cm diameter, 10 μm thickness). DGT holders, gels, and Chelex-resin were purchased from DGT Research Ltd. The DGT devices were assembled under a laminar flow hood at the laboratory before deployment. Acid and DGT assembly blanks were also prepared. Diffusion Coefficients Measurements. The diffusion coefficients were measured with a diaphragm cell, as described previously.18,19 The cell was comprised of two 70 mL perspex compartments, each with an interconnecting 1.5 cm diameter opening. A 2.5 cm diameter disk of gel, or gel plus membranes, was placed between the openings, and the whole assembly was clamped together. A volume of 50 mL of carrier solution made up of Milli-Q water and 10 mmol L−1 NaNO3 were introduced simultaneously in each compartment. The carrier solution of the source compartment was spiked with metallic ions of interest at 2 ppm, which diffused into the receptor compartment. Both compartments were stirred continuously using an overhead stirrer. Subsamples of 0.2 mL were taken from each compartment at constant intervals (10−15 min) and analyzed

F=

M At

(2)

where M is the mass transferred between both compartments, A is the exposed area, and t the time of deployment. Combining eqs 1 and 2 and assuming that ΔC is constant ≈ C (concentration in the source compartment), M is expressed using eq 3:

M=

ADC t Δx

(3)

The mass transferred is deduced from the concentration of the sub samples taken in the receptor compartment at a constant interval of time. M is plotted versus time, and D is calculated from the slope of the least-squares linear regression given by Excel. The standard deviation is calculated by propagating uncertainties in a first-order approximation to the model of the measurement system. 2 ⎛ sA ⎞2 ⎛ sC ⎞2 ⎛ sΔx ⎞2 ⎛ sslope ⎞ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ sD = D + + +⎜ ⎟ ⎝A⎠ ⎝C ⎠ ⎝ Δx ⎠ ⎝ slope ⎠

(4)

where sA, sC, sDx are the standard deviation associated with area, concentration, and thickness of the diffusion layer. sA and sDx are 5%, sC is calculated with the concentrations measured in the source compartment, and sslope is the standard deviation associated with the slope of the regression, performed by Excel. Deployment of DGT Devices in the Canal Experiment. DGT devices equipped with restricted gels and with or without PC alternative membranes were deployed to investigate the effect of PC membranes on metal accumulation and biofilm formation. DGT were exposed in a canal receiving raw settled wastewater. This effluent was rich in urban organic matter which allowed fast growth of a predominantly bacterial biofilm. The velocity of the water was controlled (0.07 cm s−1) and 3112



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regression was used to calculate the mean labile concentration over the total deployment time.

remained very low compared to water velocity in natural waters (3−30 cm s−1). A low water velocity is expected to favor biofilm development. Temperature was recorded by a HOBO temperature data logger (Prosensor, France) and rose steadily from 21 to 24 °C during the deployment. Two series of 15 DGT devices were deployed at t = 0, one with PES + PC membranes and one with PES membranes only. Three DGT of each series were taken at deployment times of 1, 3, 7, 10, and 14 days. At d = 10, 3 new DGT devices were added to each series. After their retrieval, DGTs were disassembled and metals were eluted from Chelex-resin by soaking in 1 mL of HNO3 (suprapur, Merck) 1.2 mol L−1 for a minimum time of 24 h. The resulting extracts were diluted with ultrapure water, spiked with internal standards, and analyzed by ICPMS (X series 2 Thermo Fisher Scientific). Deployment of Clean DGT Devices with Biofouled Membranes in Laboratory Experiment. PC membranes were removed from DGT devices deployed in treated wastewater for 14 days and used to assembly new, otherwise clean DGT devices. In parallel, clean DGT devices with clean membranes were also assembled. All DGTs were equipped with restricted gels. Triplicates of DGT (biofouled and clean) were deployed in two 5 L containers filled with inorganic media made up with ultrapure water and NaNO3 10 mmol.L−1. Monoelemental solutions (100 mg L−1) were prepared with the following salts: Ba(NO 3 ) 2 , CdNO 3 ·4H 2 O, CoCl 2 , Cr(NO3)3·9H2O, CuSO4·5H2O, MnCl2·4H2O, NiSO4·7H2O, Pb(NO3)2, and ZnSO4·7H2O. All salts were purchased from Acros Organics. One container was spiked with 500 μL of each solution to reach about 10 μg L−1 concentration for each metal. The concentration in the container was verified by analysis of samples of the exposure medium taken on three occasions during the deployment. DGTs were deployed for 7 h under controlled temperature and continuous magnetic stirring. pH and temperature were measured at the beginning and at the end of the experiment. After the deployment, DGT devices were treated as described in the previous section. DGTs and water samples were analyzed by ICPMS. DGT Labile Concentration Calculations. The calculation of the labile DGT concentration is based on recent papers,20,21 where the method to assess the aqueous diffusive boundary layer δ is described. In this layer, the species diffuse with the coefficient in water Dw. Two sampling areas are used: Ag = 3.14 cm2 in the water, and the effective sampling area As = 3.8 cm2. As, which is higher than the sampling area because of lateral diffusion, was measured by Warnken et al.20 The labile concentration in the bulk medium is given by eq 5: C=

M A sDδ + A g Dw Δg t A sA g Dw D

M=

■

A sA g Dw D A sDδ + A g Dw Δg

Ct (6)

RESULTS Diffusion Coefficients. The mass of metal transferred in the receptor compartment was linearly related to time (r2 > 0.99 in most cases; r2 > 0.95 for Zn). The diffusion coefficients are given in Table 1. When the PC membranes were added, no significant difference was observed for the RG. A single average coefficient was calculated for the two configurations. The coefficients of OP gels with PC membranes (OP + m) decrease compared to OP alone. All diffusion coefficients calculated for gels alone were close to the reference coefficients given by DGT Research Ltd.22 for OP and deduced from Scally et al.19 for RG. Canal Experiment. After retrieval from the canal, a thick brown biofilm was present on the surface of the DGT units. Nevertheless, all metals except Ti and Ba were accumulated in the resin. Figure 1 shows examples of the metal accumulation pattern: Zn (a and b), Pb (c and d), and Co (e and f). The 3 DGT added at d = 10 (“fresh” DGT) are plotted on the same graph. All accumulation patterns are displayed in the Supporting Information. In the case of Zn, the accumulation tends to be linear until the 10th day and then it reaches a plateau, whatever the membrane used. The same behavior is observed for Cd, Ni, and V, but unlike Zn, the accumulation of these elements remains linear for the 14 days measured in DGTs equipped with the PC membrane. Pb was linear too for DGTs equipped with the PC membrane but not linear from the 7th day of accumulation when the PC membrane was absent. The accumulation of Cr in DGTs without PC membranes was linear, whereas it curved in DGT equipped with PC membranes from d = 10. Co, Mn, and Cu accumulations in DGTs with and without PC membranes were linear all along the exposure and could not be distinguished apart when we look at the whole deployment. Nevertheless, the mean total metal accumulated in the resin of the last DGT was higher when DGTs were equipped with the PC membrane. For Cd, Ni, Pb, V, Zn and Mn, there was less metal accumulated after 14 days of deployment in the resin of DGTs without the PC membrane (25% on average for the first five elements, 10% for Mn), reflecting a consistently less efficient uptake for devices without PC membranes. Unlike the other metals, the masses of Cr and Cu were higher (40%) without PC membranes. Considering the whole data set of mass accumulated in the resin according to the time, the mean labile concentrations over the 2 weeks of deployment were calculated using eq 6 (Figure 2a). Labile concentrations of Mn, Zn, and Co were significantly higher when a PC membrane was used. On the contrary, the concentration of Cu measured with PC membranes is lower than the concentration measured without PC membranes. The labile concentrations for the last 4 days of deployment, plotted in Figure 2b, are calculated with “biofouled” and “fresh” DGT devices to assess how the biofilm affects the labile metal measurement. The concentration of the “biofouled” DGTs corresponds to the difference of the mass accumulated in DGTs retrieved at d = 14 and d = 10. The “fresh” DGT concentration

(5)

The diffusive boundary layer δ was measured according to Garmo et al.21 by using 9 other DGTs with varying hydrogel thicknesses (Uher et al., unpublished). Values of 0.5 mm for the deployment in the canal and 0.3 mm in the laboratory deployment were calculated. When several deployment times were available, M was plotted versus time and the slope of the corresponding linear 3113



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Table 1. Diffusion Coefficients (×10‑6 cm2 s−1) in the Gels and in the Gel + Membranes Layer at 25 °Ca OP Ba Cd Cr Co Cu Mn Ni Pb Ti V Zn

7.86 6.46 5.16 6.40 7.16 6.44 6.19 8.38 6.53 6.48 6.26

(0.23) (1.14) (0.19) (0.20) (0.26) (0.20) (0.23) (0.23) (0.21) (0.21) (0.36)

OP + m 5.32 4.08 3.43 4.44 4.41 4.31 4.10 5.65 3.98 4.38 4.70

(0.08) (0.11) (0.08) (0.08) (0.22) (0.12) (0.19) (0.10) (0.09) (0.09) (0.40)

RG 4.66 3.58 2.70 3.64 3.72 3.64 3.75 5.28 3.24 3.45 4.59

(0.13) (0.09) (0.08) (0.11) (0.53) (0.10) (0.11) (0.14) (0.13) (0.10) (0.30)

RG + m 4.55 3.45 2.62 3.57 3.96 3.51 3.64 5.01 3.16 3.03 4.26

(0.09) (0.06) (0.06) (0.07) (0.13) (0.07) (0.09) (0.08) (0.08) (0.06) (0.38)

average RG/RG + m 4.60 3.52 2.66 3.61 3.84 3.57 3.69 5.14 3.20 3.24 4.43

(0.16) (0.11) (0.10) (0.13) (0.55) (0.12) (0.14) (0.16) (0.15) (0.12) (0.48)

a

OP, open pore gels; RG, restricted gels; m, PES + PC membranes. Standard deviations are given in parentheses. The averages for the RG and RG + m are also shown.

Figure 1. Mass of Pb, Co, and Zn accumulated in the chelex resin of DGTs equipped with (PES + PC) and without a PC membrane (PES) in relation to the time. The open symbols represent the DGT added at d = 10. The bold line represents the linear part of the metal accumulation in the DGT. The bold lines of parts a (extrapolated), c, and e are plotted as dotted lines in, respectively, parts b, d, and f.

biofilm grown at the surface of the devices.The “fresh” DGTs give the reference concentration. Some elements were strongly affected by the biofilm, and the estimated labile concentration was highly biased. The concentration could not be calculated when there was no difference

corresponds to the new DGTs added at d = 10. Because no difference between fresh DGT equipped with or without the PC membrane was noticed, the mean labile concentration was calculated from the whole set of fresh DGTs. The differences between “biofouled” and “fresh” DGTs can be attributed to the 3114



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Figure 3. Labile concentration measured in the control and in the spiked container, total dissolved concentration measured in the tub (black bar). Bars represent standard deviations.

difference between the labile concentration calculated with biofouled DGTs in the spiked medium and in the control is close to the total dissolved concentration for Zn and is equal to 40% of it for Mn.

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DISCUSSION

Diffusion Coefficients of Ba, Ti, V, and Cr. The diffusion coefficients of Ba and Ti had not been previously determined with the diaphragm cell method used in this study. Regarding V, this study is the second data about the diffusion coefficient and the first for the restricted gel. The diffusion coefficient of V in the OP gels is exactly the same as Luo et al.23 Garmo et al.24 derived diffusion coefficients values from DGT measurements equipped with OP gels in known solutions in a pH range of 4.7−5.9. Their values for these three elements (4.70, 1.30, and 4.16 for Ba, Ti, and V, respectively) and for Cr (3.00) are lower than the ones determined in this study, which was conducted at pH 2 to ensure that all metals are in an ionic form. They noticed that diffusion coefficients varied with pH. This can be attributed to changes in the selectivity of the Chelex resin and in the speciation of metals with pH. In the range of pH > 4.7, metals can have more complicated speciation than at pH 2 and could form nonlabile hydroxy/oxy species. The higher values observed in this study can be attributed both to the use of the diffusion cell, which eliminates any resin effects, and to the low pH that ensures there are no hydrolysis effects. The coefficients presented in this study represent diffusion coefficients of the ionic species only. Do Membranes Modify the Diffusion Coefficients? When the two membranes overlay the gel, no difference in diffusion coefficients of the metals was observed for the RG gel. Conversely, the diffusion coefficients for OP + m systems decreased comparing to OP only. Scally et al.19 observed how the overall diffusion coefficients varied with the presence or absence of the PES membrane. Using the OP gel, they were identical but they increased for the RG gel when PES was present. The authors deduced that the diffusion coefficient in the PES membrane was close to the one in the OP gel. In this study, the addition of the PC membrane led to the opposite observation. It appears that the apparent diffusion coefficient of metal ions in PC membranes is smaller than in PES membranes.

Figure 2. Labile concentration measured with DGTs with and without a PC membrane (a) over 14 days deployment (b) at the end of the deployment. Bars represent standard deviations. Concentrations marked with ∗ are significantly different according to the Student test.

between the accumulated mass at d = 10 and d = 14. This applied to Zn, Ni, and Cd (without a PC membrane) and Cr (with a PC membrane). The “biofouled” DGTs without a PC membrane underestimated the measurement for Mn, Pb, and V (40−75%), whereas Cu was overestimated (70%). The labile concentrations of Cr and Co were not significantly different from the reference concentration of fresh DGTs. The addition of the membrane led to an overestimation for Cd, Ni, and Pb of 70% on average. Co was underestimated by 85%. The labile concentrations of Cu, Mn, and V with a PC membrane were not significantly different from the reference concentration of fresh DGTs. Laboratory Experiment. The labile concentrations calculated from DGT deployments in the laboratory with biofouled or clean PC membranes are shown in Figure 3. No metal was added in the control medium. Nevertheless, some of them were measured in the resin of the biofouled DGTs. There was slight contamination of Co, Ni, and Pb, moderate for Cu and substantial contamination for Mn and Zn (equivalent to about 40 μg/L in the medium). Mn and Zn can be attributed to the release of metals from the biofouled membrane. In the spiked medium, the labile concentration estimated with DGTs was similar for both membranes used for all metals, except Mn and Zn. Moreover, labile concentrations were close to the measured total dissolved concentrations. For Mn and Zn, the labile concentration was overestimated by a factor of 3. The 3115



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and affinity, depending on the metal of interest. The actual accumulation of the metals within DGT depends on the relative strengths of the two sinks cited above. The composition, structure, and the thickness of the biofilm modulate its binding capacity. Moreover, its relative importance compared to the resin pumping depends also on the metal concentration in the water, its diffusion kinetics, and the possible saturation of the resin. A tentative representation of the “active” effect of the biofilm at the surface of the DGT device is represented in Figure 4.

Assuming that the diffusion in the membranes follows Fick’s first law, the diffusion coefficients should be related according to eq 7: Δg + m D

=

Δg Dg

+

Δm Dm

(7)

with Δg = thickness of the gel, Δm = thickness of the membranes (PES + PC), Δg+m = thickness of the diffusion layer (gel + PES + PC), Dg = diffusion coefficient in the gel, Dm = diffusion coefficient in the membranes (PES + PC), D = diffusion coefficient in the overall diffusion layer (gel + PES + PC). Using eq 7 and data given in Table 1, the diffusion coefficients in the membranes Dm were estimated. Dm values were calculated with the diffusion coefficients measured for both the OP and OP + m system and for the RP and RP + m system. Dm values calculated from RG experiments were on average 2 times higher than the ones estimated from OP experiments. Let us notice that the values of D and Dg for RG are very close and probably involve substantial error in the calculation of Dm. The values of Dm calculated with the OP experiment are equal to 20% of the diffusion coefficient in the water. The membranes slow the transport of the species through the diffusion layer. Because this was not observed by Scally et al.19 with the PES membrane alone, the polycarbonate membrane is supposed to be responsible for this phenomenon. Since the porosity of the PC membrane is much larger than the porosity of the gel and is close to the porosity of the PES membranes, the resistance is not likely due to steric hindrance. The most probable hypothesis is that, due to the functional groups present at the surface of the membranes, electrostatic interactions occur, as observed for Nafion membranes.10,25 In the case of Nafion, electrostatic cation-sulfonate interactions led to a lower diffusion coefficient. PC nuclepore membranes contain carboxylate groups which can produce similar interactions. In the following, the overall coefficients D given in Table 1 (RG/RG + m) were used in the calculations of labile metal concentrations in the canal and laboratory experiments. Biofilm Effect on DGT Measurement. Biofilm developing at the surface of passive samplers is usually expected to behave as an additional inert diffusion layer, which may reduce the uptake of analyzed species.8,9,11 Nevertheless, the results of this study suggest that biofilms at the surface of DGTs and metallic species interact, as when biofilm grows on any surface.4,26,27 To explain our results, we will consider two possible effects of the biofilm on the transport of the metallic species: clogging of the hydrogel pores by biofilm and interaction of metal with biofilms. If pores were clogged, diffusion of the metals in the gel would be stopped for all metals. Indeed, we observe such plateaus, but never for all metals at the same time, which leads us to reject this first hypothesis. Regarding the second effect, we propose to compare the relative importance of two sinks for the metals: the chelex resin that drives the diffusion through the DGT gel, and the embedding or binding within or onto the external biofilm. Biofilm overlaying the DGT devices interacts with metals in solution by various processes of varying importance and reversibility: biosorption, complexation, precipitation of insoluble salts, adsorption on iron and manganese oxides, and reduction.4,28,29 Microorganisms composing biofilms have a biological activity responsible for specific interactions as bio precipitations29−31 (Zn, Cu, Ni, Cd, Mn, Cr) or precipitations following enzymatic reductions (Cr).31,32 Biofilms exhibit metal-binding properties with varying degrees of specificity

Figure 4. Schematic representation of the role of the biofilm in the accumulation of metal by DGT.

Whenever metals interact with or within the biofilm, they are temporarily fixed by the biofilm. Metals reversibly retained by the biofilm eventually diffuse through the hydrogel toward the chelex resin. This step is possible if the metal-biofilm complexes dissociate so that the metal gets into the hydrogel. In this case, the diffusion gradient then has to be strong enough to disrupt metal-biofilm association in order to involve transport of metallic species toward the resin. If the dissociation of the metal from the biofilm is the limiting step, metal diffusion in the hydrogel might be severely retarded. If the complexes dissociate easily, accumulation of metal in the chelex-resin might happen without any significant effect. Let us turn now to the experimental results with this interpretation in mind. The biofilm effect on DGT measurement is assessed with the results of the DGTs with standard equipment in the canal experiment: we first examined how metals accumulate in resins of DGT equipped with PES membranes. Some elements (Zn, Cd, Ni, V, Pb) were not accumulated any more by the resin after the 10th day, whereas some other elements (Cr, Co, Cu, Mn) kept on linearly accumulating in the resin. These different behaviors among the metals in the presence of biofilm corroborate the hypothesis of chemical interactions between metals and biofilm and invalidate any physical obstruction to the accumulation, i.e., clogging of the pores. If the accumulation is retarded, metals might be then retained by the biofilm. If elements seem to be unaffected by the biofilm, the concentration gradient in the hydrogel due to the Chelex resin might be strong enough to counterbalance metal binding in the biofilm. The case of Cu, which has a notable behavior, suggests also chemical interactions: more labile Cu is measured with biofouled samples than with the fresh ones. Cu, which has a strong affinity for biofilms, is expected to accumulate in the biofilm layer. We suggest that some Cu-organic matter complexes that 3116



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CONCLUSION A considerable impact of the biofilm on the quantification of metals was observed. Indeed, extreme conditions were chosen to simulate a worst case of biofilm formation. The effect of the biofilm on the accuracy of labile concentrations measurement is actually very difficult to generalize. Since it is clearly different among the tested metals and the type of biofilm formed at the surface, it is also difficult to predict. Our interpretation enlights the role of physicochemical interactions between metals and the biofilm, which should be further studied, through a precise monitoring of the biofilm’s growing and binding capacity. Next, a simple but quantitative model, featuring biofilm interactions with metals in solution and in the hydrogel, would probably allow one to quantitatively verify the validity of the description of the phenomenon proposed in this study. The nucleopore track-etched polycarbonate membranes, used as an alternative to classical polyethersulfone, were expected to limit biofilm development by modifying the nature of the biofilm at the surface of the samplers. They improved the quantification for Cd, Cu, Mn, Pb, and V but worsened it for Cr and Co. For long deployment situations where conditions favor biofilm development, PC membranes could be an alternative to improve measurement of metals, depending on the metal of interest. Otherwise, in conditions which are in favor of biofilm growth like in this study, deployment times lower than 7 days are suitable to avoid a biofilm effect on DGT measurement. Because the biofouling effect is likely to change with biofilm type, we recommend being cautious with long time deployment. Practically, using several time deployments to calculate a mean labile concentration on the whole time of deployment seems to be a good way to minimize strong errors possibly made at the end of the deployment.

would not have diffused through a nonbiofouled gel, because of the diameter of the pores, might however be bound with or within the biofilm, thanks to the absence of size limitation before the biofilm layer. Only a strong concentration gradient in the hydrogel due to the high affinity of Cu for the Chelex resin might explain that we observe more accumulated Cu in the resin of the biofouled samples than in the fresh ones. It also corroborates the hypothesis of competition between metal binding in the biofilm and the concentration gradient due to the Chelex resin. Is the PC Membrane Effective in Limiting Biofilm Effects? Biofouling occurred on both types of membranes, but the biofilm did not affect the same elements in the presence or absence of the PC membrane. Indeed, biofilms developed on PES and on PC membrane are not similar, since they were grown on different substrates. It is already known that the composition and the origin of biofilms may change their interactions with metals.4 In our study, with a biofilm developing in raw wastewater, the PC membrane favors the quantification of Cd, Ni, and Pb, whereas Co and Cr are better assessed without PC membranes. Rather than limiting the biofilm growth, PC membranes may be selective for the nature of the biofilm and consequently modify the metal-biofilm interactions compared to those pertaining for a biofilm formed on a PES membrane. What Effect Do Biofilms Grown on PC Membranes Have on the DGT Measurement? First, release of Mn and Zn from biofilm at the surface of the PC membrane was observed during the laboratory experiment. Both elements were accumulated during the 15 days of deployment in the biofilm layer, showing the metal immobilization capacity of the biofilm grown on the DGT devices. Then, overlaying clean DGTs with the biofouled membranes disrupts the steady-state established between the solution, the biofilm layer, and the resin. The concentration gradient is established between the clean resin and the biofilm layer. The labile metal concentration calculated in the bulk medium, which is partly due to the additional release of Mn and Zn from the biofilm layer, is equivalent to 40 μg L−1. Regarding the other metals, no effect on labile metal measurement of the biofilm was noticed during the laboratory experiment. Unlike it was observed during the canal experiment with the PC membranes, the presence of biofilm does not modify the Cr, Co, and Zn labile concentration estimation. Several hypothesis can be put forward. First, the biofilm in both experiments is likely very different: the effluent in which DGTs were deployed prior to the laboratory experiments (treated wastewater) was different from the medium of the canal (raw wastewater). The biofilm formed is then different, and the interactions of the metals with the biofilm cannot be expected to be the same. Moreover, biofilms have been shown to be sloughed off when environmental parameters are drastically modified.4 Second, the metal exposure concentrations are different: the biofilm grew with concentrations in Cr and Co between 0.1 and 0.3 μg L−1 (data not shown), whereas metals concentrations in the lab were measured around 10 μg L−1. The concentration gradient induced in the DGT hydrogel is much stronger. The dissociation of metal-biofilm complexes, which was probably the limiting step in the canal, could have been forced by this stronger gradient. Moreover, binding sites in the biofilm could be saturated because of the much higher concentrations. In that case the effect on flux might be small. Finally, deployment times are different. Kinetic aspects could play a key role in metal biofilms interactions.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study is part of the EMESTox project, supported by the French National Research Agency. The Syndicat Interdépartemental de Assainissement de l’Agglomération Parisienne (SIAAP) is acknowledged for providing access to an experimental channel within the treatment plant. We thank Audrey Mayenaquiby, Hao Cheng, and Aurélie Germain for their help in experimental work and Chantal Compère for her reading and kind suggestions.

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