Abstract
We evaluated the application of DGT (diffusion gradients in thin films) as a tool to determine Cu, Zn, Ni, Cd, Pb and Mn concentrations and speciation in a hardwater eutrophic lake. This technique was used in situ during six sampling periods over one year in Lake Greifen. The DGT-labile species of Cu and Ni amounted to 15 -25 % of the total dissolved concentrations. Speciation by ligand-exchange/DPCSV indicated that Cu and Ni were predominantly organically complexed (> 99 %). Thus, the DGT-labile species for Cu and Ni were much more abundant than the free ionic and inorganic species determined by ligand-exchange/DPCSV. The results can be explained by incomplete metal exchange of very strong complexes with the chelating resin in the DGT devices, metal exchange of less abundant weaker complexes, and by slow diffusion of exchangeable organic Aquat. Sci. 64 (2002) 
Abstract
complexes. For Zn (36 to > 90 % DGT-labile) and Mn (50 to 100 % DGT-labile), the results indicated that these metals are less strongly organically complexed. A larger fraction of Zn occurred in DGT-labile species in the hypolimnion than it did in the surface water, probably due to a larger concentration of strong ligands in the productive surface water. DGT-labile Cd-(0.01 -0.02 nM) and Pbspecies (0.03 -0.06 nM) were detected at very low levels. The combination of measurements of dissolved and DGT-labile species showed a decrease of Cu, Zn, Cd and Mn vpeters@example.com m from June to August, which was probably linked to intensive sedimentation of organic matter during summer stagnation. Mixing and oxygenation of the lake in winter-spring led to an increase in dissolved and DGT-labile Ni, Zn and Cd, whereas Mn decreased in the hypolimnion.
Introduction
Trace metals such as Cu, Zn, Mn and Ni play an important role as essential elements in aquatic systems. Elevated concentrations of these elements and of non-essential elements such as Cd and Pb, however, may be toxic. Investigation of the speciation of trace metals is a prerequisite to evaluating their bioavailability and mobility. Biological responses of organisms often have been shown to be related to the free-ion activity of a metal ion or to the concentration of labile metal species in solution (Hudson and Morel, 1990; Morel and Hering, 1993) .
Introduction
Reliable measurements of trace metal speciation in the aquatic environment are essential for studies of trace metal cycling and metal bioavailability. Most of the techniques involved in such measurements (ASV, ligand-exchange/CSV, extraction on C-18 cartridges, etc.) require that analyses be completed on samples brought to the laboratory. The results obtained with these techniques will thus be affected by uncontrolled changes in temperature, pressure, pH, P CO2 and P H2S during sample collection, storage or treatment (Tercier and Buffle, 1993; van den Berg and Achterberg, 1994) . Also, many of these techniques require the addition of reagents to the sample that modify the metal speciation. To overcome such problems, the development of techniques capable of measuring metal speciation in situ is highly desirable. The technique of diffusive gradients in thin films (DGT), which provides an in situ means of quantitatively measuring labile trace metal species in aqueous systems (Davison and Zhang, 1994; Davison, 1995, 2000; Denney et al., 1999; Davison et al., 2000; Twiss and Moffett, 2002) , is very promising.
Introduction
The DGT technique is based on Fick's first law of diffusion. By ensuring that transport of metal ions to an exchange resin is solely by free diffusion through a gel layer of known thickness, the concentration in the bulk solution can be calculated from the metal mass measured in the resin. If a sufficiently thick diffusion layer is selected, the flux of metal to the resin is independent of the hydrodynamics in solution above a threshold level of convection (Zhang and Davison, 1995) .
Introduction
In principle, three factors determine which species are measured by DGT, the binding agent, the diffusion layer thickness and the pore size of the gel. Depending on these factors, various fractions of the dissolved metal species are determined by DGT, such as the inorganic complexes and some fraction of the organic complexes, depending on their stability, lability and molecular size (Zhang and Davison, 1999; Twiss and Moffett, 2002) . In other words, DGT will measure free aquo ions, inorganic complexes and some fraction of the organic metal complexes (depending on the diffusion coefficients and the stability of these organic metal complexes). Colloidal and particulate species are excluded by the pore size of the gel.
Introduction
The DGT technique has several advantages over other techniques proposed for measuring trace metals in natural waters: a) it is easy to use, b) it concentrates metals in situ, c) many trace metals can be measured simultaneously, and d) it yields time-averaged concentrations over the length of the deployment period (Zhang and Davison, 1995; Davison et al., 2000) .
Introduction
The DGT technique has been used in situ for the measurements of trace metals in ocean seawater (Davison and Zhang, 1994; Zhang and Davison, 1995) , rivers (Denney et al., 1999; Zhang and Davison, 2000) , oligotrophic lakewater with low major cation concentrations (AlfaroDe la Torre et al., 2000) and coastal seawater (Twiss and Moffett, 2002) . Until now, there have been no reports of its use in highly eutrophic lakes. As part of extensive metal speciation studies in natural waters, we have deployed DGT assemblies 6 times over the period of one year in Lake Greifen, a eutrophic hardwater lake. Our main objective was to evaluate the application of DGT as a tool to determine trace metal concentrations and speciation in a eutrophic lake. The trace metals Cu, Zn, Ni, Cd, Pb and Mn were measured simultaneously by DGT. The results obtained by DGT are compared to measurements of dissolved metal concentrations by conventional sampling and to the results of competitive ligand-exchange/ voltammetric measurements, by which the free ionic species are determined using ligand equilibrium relationships.
Materials and methods

Investigation area
The in situ measurements were performed for two or three days, and samples were collected six times over the period of one year (June 1999 -June 2000 at depths of 2.5 and 28 meters near the deepest point (31 m) in Lake Greifen, Switzerland.
Investigation area
This lake is highly eutrophic with a seasonally anoxic hypolimnion (Fig. 1) . Its algal productivity is very high (500 g C m -2 y -1 ) (Xue and Sigg, 1998) . It has a surface area of 8.5 km 2 and a volume of 150 ¥10 6 m 3 . Its average depth is 17.7 m with a maximum of 31 m. The geology of the watershed is dominated by limestone. The major ion composition of Lake Greifen is characterised by Ca = 1.2-1.6 mM, Mg = 0.6 mM, and alkalinity = 3.0-4.2 mM (Xue and Sigg, 1993) . The pH varies with time and depth between 7.5 and 8.7. Dissolved organic C varies between 3 and 4 mg L -1 . Oxygen was always close to saturation with vpeters@example.com m depth and mostly below detection at 28 m, with exception of the sampling in March 2000 (Fig. 1) . Temperature varied over the year between 4 and 20°vpeters@example.com m and was always about 5°C at 28 m.
Sampling
Go-Flo sampling bottles (General Oceanics, 5 L) were used to collect water samples. To avoid oxygenation of anoxic water samples, samples were transferred to polyethylene bottles and were passed through 0.45-µm pore Millipore filters using N 2 gas flow. The samples were filtered in a laminar flow clean hood within 2 h of collection. Samples for total dissolved metal measurements were acidified immediately after filtration. The filtered samples were stored in the dark at 4°C for up to one week before the analysis. All sampling and filtration devices, bottles and membrane filters were washed with 0.01 M HNO 3 and rinsed with deionized water before use.
Diffusion Gradient in Thin-Film (DGT)
Diffusive and Resin gel preparation. Acrylamide, ultrapure (Boehringer), agarose cross-linker (2 %) (DGT Research Ltd, Lancaster, UK) and deionized water were used to prepare diffusive and resin hydrogel. Chemical polymerisation was initiated by daily prepared ammonium persulphate (10 %) and TEMED (N,N,N'N'-Tetramethylenediamine) (99 %) (Merck). To prepare the resin hydrogel, an ion-exchange resin (Chelex-100, 200-400 mesh, sodium form) (Bio-Rad) was used in combination with the chemicals mentioned above. Both diffusive gel (0.8 mm thickness) and resin gel (0.4 mm thickness) were prepared according to the slightly modified procedure recommended by . Before casting, the glass plates and spacers were acid washed and air-dried in a laminar flow hood. To avoid any possible contamination, the whole process of preparing the solutions and pipetting between the glass plates was performed in the clean hood. Also, to make pipetting of the gel solution easier and more uniform and to avoid the beginning of polymerisation before the pipetting was finished, the solutions and the glass plates were kept on ice. This procedure is very important, especially during resin solution pipetting. After pipetting the resin solution between two glass plates, the assembly was laid flat on ice for 30 min. After this time the resin (3.5 grams of resin per 10 mL of gel solution) was fully settled on one side of the gel with maximum density. Finally, the gels were maintained at 42°C for 60 min to accomplish the polymerisation.
Diffusion Gradient in Thin-Film (DGT)
After hydration, the gel sheets were cut into 25 mm discs and assembled into the plastic gel holders (made by EAWAG Workshop) according to the procedure recommended by Zhang (1997 b) . To avoid adhesion of the fine particles from the lake water, a 100 mm thick, 0.45 mm pore size Millipore cellulose nitrate membrane was placed on top of the diffusive gel. DGT performance was tested in the laboratory using eight different metal concentrations (1-120 nmol L -1 ) in 0.01 M NaNO 3 . Six replicate DGT devices were deployed in each of the solutions (stirred and kept at 25°C) for eight hours. Correlation between DGT-measured and original concentrations was good, with regression coefficients between 0.9 and 1.0 for all metals tested.
Diffusion Gradient in Thin-Film (DGT)
DGT deployment and retrieval. In Lake Greifen, DGT units were vpeters@example.com and 28 meters six times for two or three days, over the period of one year (June 1999 -June 2000 . For each deployment, the same polystyrene buoy, 10 kg weight and 4 mm polyester rope (vpeters@example.com and 28 m depths) were used (Fig. 2) . At each depth, 4 DGT units were attached to the rope using an acid-washed reusable plastic strip (PE) made at the EAWAG Workshop (Fig. 2) . The strip had 4 holes with diameters slightly smaller than the outer sleeve of the DGT holder. Such a design enabled easy insertion and retrieval of the holders. On recovery, DGT holders were rinsed thoroughly with deionized water and immediately stored in clean zip-lock plastic bags.
Diffusion Gradient in Thin-Film (DGT)
On each deployment the background concentrations of trace metals were checked by analysing the resin gel layers from 3 or 4 DGT units prepared the same way but not deployed in the water column (blank samples). These concentrations were used to correct those of the units deployed and also to calculate the detection limits of the technique. The mean background trace metal concentrations in the resin gel eluent of the blank samples throughout the experimental period and the calculated detection limits are given in Table 1 .
Diffusion Gradient in Thin-Film (DGT)
Sample preparation and analysis. Upon return to the laboratory, the resin gel layers were carefully removed from the DGT holders and transferred to 15-mL pre-cleaned PP-Test tubes (Greiner labortechnik, Cellstar), each containing 2 mL of 2 M HNO 3 (Merck, Suprapur). This procedure was performed under a clean hood using plastic tools. To elute metals, the resin gel was placed in the acid for 24 h. After at least 8-fold dilution of 1 mL of extractant, the concentrations of Cu, Cd, Pb, Zn, Mn and Ni were analysed by inductively coupled plasma -mass spectrometry (ICP-MS, Perkin-Elmer ELAN 5000). When the concentrations were close or below the detection limit for ICP-MS, 1 mL of extractant (not diluted) was analysed by Zeeman furnace atomic absorption spectroscopy (ZFAAS, Perkin-Elmer Zeeman 5100 ZL). Equations used to calculate the DGT measured concentrations are explained elsewhere (Zhang and Davison, 1995) . The diffusion coefficients used for calculation of the DGT-labile concentrations are those for free aquo ions, which were measured by Hao Zhang, DGT Research Ltd., Lancaster, UK (personal communication).
Total dissolved concentrations
Total metal concentrations in filtered (0.45 mm) and acidified samples were analysed by ICP-MS (Perkin-Elmer ELAN 5000) using "Baker Instra-Analyzed" standard metal solutions. The methods for Cu, Cd, Zn, Ni, Mn and Pb determinations were checked using the Certified Reference Material SLRS-4 (River Water reference Material for Trace Metals) from the National Research Council of Canada. All results were within the standard deviations of the certified values for all metals investigated.
Voltammetric measurements
On two occasions (July 1999 and November 1999) ligand-exchange with catechol/DPCSV (Differential Pulse Cathodic Stripping Voltammetry) (Xue and Sigg, 1993) was used to measure free aquo Cu 2+ concentrations in the samples vpeters@example.com and 28 m depth. At the same time, free aquo Zn 2+ was measured using ligand-exchange with EDTA/DPASV (Differential Pulse Anodic Stripping Voltammetry) (Xue and Sigg, 1994) . Ligandexchange with ethylenediamine/DPASV (Xue and Sigg, 1998 ) was used to measure free aquo Cd 2+ in July 1999. Ligand-exchange with dimethylglyoxime/DPCSV was Zhang and Davison, 1995. used to measure free aquo Ni 2+ in July 1999 (Xue et al., 2001) . These methods yield free aquo metal ion concentrations, which are obtained by equilibrium calculations.
Results

Copper
The total dissolved Cu (ICP-MS) and DGT-labile Cu concentrations are shown in Figure 3 . Average values are given in Table 2 , which also shows the ligand-exchange/ DPCSV results. The total dissolved and DGT-labile concentrations were always somewhat vpeters@example.com m than at 28 m, except for the sampling in March 2000 when the lake was well-mixed. The DGT-labile Cu was on average about 22% of total dissolved vpeters@example.com m depth and 15% of total Cu at 28 m depth. The free aquo Cu 2+ concentrations obtained from the ligand-exchange/DPCSV method were several orders of magnitude lower than these concentrations, in agreement with previously obtained results (Xue and Sigg, 1993) .
Nickel
The total dissolved Ni concentrations were around 10 -20 nmol L -1 at both depths, except for a much higher value in March 2000 (Fig. 3) . The DGT-labile Ni concentrations were around 25 % of the total dissolved Ni concentration. The free aquo ion Ni 2+ concentration, which was measured once by ligand-exchange/DPCSV (July 1999), was several orders of magnitude lower than the DGT-labile concentrations.
Zinc
Total dissolved Zn concentration (ICP-MS measurements) and DGT-labile Zn in Lake Greifen differed significantly from one month to another (Fig. 3) . Total and labile Zn in the surface layer (2.5 m) decreased during the summer months. The Zn vpeters@example.com m were generally lower than those at 28 m, for both the total dissolved and the DGT-labile concentrations. The DGT-labile Zn was around 40 % of total Zn (ICP-MS measurements) at 2.5 m depth, and 94 % on average at 28 m depth. The free aquo Zn 2+ obtained from ligand-exchange/ DPASV was about 8-fold vpeters@example.com m and 2.5-fold lower at 28 m than the DGT-labile species.
Cadmium
Total dissolved Cd concentrations (ICP-MS measurements) in Lake Greifen were below the detection limit (< 0.09 nmol L -1 ) during most of the study (Fig. 3) . In June and July 1999, Cd concentrations were around the detection limit, and in March 2000 they were signifi-cantly above the detection limit. In contrast, DGT-labile Cd was measurable for each sampling because of the preconcentration in situ, giving DGT-labile Cd of about 0.01 nmol L -vpeters@example.com m and 0.02 nmol L -1 at 28 m. Ionic Cd (DPASV) was measured only vpeters@example.com and 28 m depth (July 1999) and gave values about 2 to 5 times lower than the DGT-labile species at the same time. Although total Cd concentrations measured by ICP-MS were vpeters@example.com m and 28 m depth, the DGT-labile and free ionic Cd concentrations (DPASV measurements) were higher at 28 m (Table 2) . By considering the values from July 1999 and March 2000, the DGT-labile Cd-species were about 14 % of total vpeters@example.com m and 21% at 28 m. The difference between the two depths comes mostly from July 1999, when anoxic conditions existed at 28 m.
Lead
The Pb concentrations were extremely low and often below the detection limit for ICP-MS (0.1 nmol L -1 ) and sometimes below the detection limit for DGT (0.02-0.03 nmol L -1 for a three day deployment) (Fig. 3) . Total dissolved Pb concentrations vpeters@example.com m by ICP-MS were slightly higher than the concentrations at 28 m (Table 2) . By contrast, labile Pb measured by DGT was two times higher at 28 m vpeters@example.com m.
Lead
In a limited number of samples, in which both techniques gave results above the detection limit, the DGTlabile species were around 10 % of total dissolved Pb vpeters@example.com m and around 90 % at 28 m. 
Manganese
Total dissolved Mn concentrations were on average two orders of magnitude higher at 28 m vpeters@example.com m because the hypolimnion of Lake Greifen is anoxic during most of the year (Figs. 1 and 3 , Table 2 ). Only in March 2000, when oxic conditions existed throughout the water column, were the vpeters@example.com m similar to those at 28 m depth. The DGT-labile species were ~50 % of the total dissolved Mn under oxic vpeters@example.com m. This ratio was very similar throughout the year. In contrast, the DGT-labile Mn was often higher than the measured total dissolved Mn in anoxic water at 28 m depth.
Discussion
To compare the three different techniques used in this study, it is important to note that sampling and sample preparation (storage, filtration, etc.) may have influenced the final results of ICP-MS and DPCSV or DPASV (DGT was the only in situ technique). The total dissolved (< 0.45 µm) metal concentration measured by ICP-MS may include some colloidal species. The ligand-exchange methods with DPCSV or DPASV give the free aquo ion concentrations (Me 2+ ) by equilibrium calculation. DGT measures the labile species under the given conditions (diffusion layer, ion-exchanger) considered to include the inorganic species and the weaker organic complexes smaller than a certain size, depending on the gel properties . An unconstrained gel has been used in the present study with pore size larger than 5 nm , so that most organic complexes of molecular weight up to a few thousand Daltons may diffuse through the gel. The contribution of various complex species to the DGT-labile concentration depends on the diffusion rate of the complexes. Humic acid complexes have diffusion coefficients that are substantially lower than those of free aquo ions . Since the calculations use the diffusion coefficient of the free aquo ions, the occurrence of organic complexes with smaller diffusion coefficients would result in underestimating the DGT-labile species. In addition, strong organic complexes may exchange incompletely with the ion-exchanger.
Discussion
Several points need to be considered in the interpretation of the DGT data. It has recently been observed that DGT devices with the Na form of Chelex resin as the binding agent cannot provide quantitative measurements of the mass transport of metals in waters of very low cation concentrations (S [cations] £ 0.2 mmol L -1 ) (AlfaroDe la Torre et al., 2000) . This problem is not likely to occur in Lake Greifen with Ca concentrations around 1.5 mmol L -1 and Mg 0.6 mmol L -1 . Cu and Ni show a similar behaviour regarding comparisons between the measured DGT-labile species and the ligand-exchange/DPCSV results. Both Cu and Ni appear by ligand-exchange/DPCSV to be very strongly complexed. The limited data obtained in the present study are supported by previous measurements of the Cu complexation in Lake Greifen (Xue and Sigg, 1993 ) and of Table 2 . Trace metal concentrations (June 1999 -June 2000 in Lake Greifen (Mean ± SD; n = 5-6).
Metal
Depth the Ni complexation in other systems (Xue et al., 2001) , all showing very strong complexation of Cu and Ni. The DGT-labile species of Cu and Ni are about 15 -25 % of the total dissolved concentrations, many orders of magnitude higher than concentrations of the free ionic and inorganic species, measured by the ligand-exchange/ DPCSV methods. They appear thus to include organic complexes that are sufficiently labile to undergo metal exchange with the Chelex resin. The difference between DGT-labile species and total dissolved concentration may be caused by incomplete exchange of very strong complexes, which bind a large fraction of Cu and Ni, and/or by low diffusion coefficients of metal complexes. Obviously, accurate DGT measurements of the distribution of species in a complex solution, such as the water of a eutrophic lake, is very difficult without knowing the diffusion coefficients of the metal complexes present in the water. According to Twiss and Moffett (2002) , it is even possible to calculate the effective diffusion coefficients of those complexes through the diffusive gel, if the mass of organically complexed DGT-labile Cu is accurately known. Furthermore, in the case of Ni, ligand-exchange reactions are very slow (Xue et al., 2001 ). However, the DGT measurements do confirm the significance of organic complexation for Cu and Ni in lake water.
Metal
For Zn, a higher fraction of DGT-labile species was observed than for Cu and Ni (36 -> 90 % of total dissolved). Based on ligand-exchange/DPASV measurements, Zn is found to be much less strongly complexed than Cu and Ni. The DPASV measurements show that some zinc is strongly bound in non-labile complexes, but a substantial fraction occurs in weaker complexes and as free Zn 2+ (Xue and Sigg, 1994; Xue et al., 1995) . The results obtained by DGT are thus consistent with these observations. The weaker organic complexes of Zn are likely to contribute to the DGT-labile species, whereas some of the stronger complexes may exchange incompletely. It is noteworthy that a larger fraction of Zn occurs in DGT-labile species in the hypolimnion than it does in the surface water. These observations suggest a larger concentration of non-exchangeable or slowly diffusing strong ligands in the productive surface water.
Metal
Because of limitation by the detection limits, the results on Cd and Pb are not conclusive with regard to the fraction of DGT-labile species.
Metal
Under oxic conditions, a large fraction of Mn is measured as DGT-labile species. These observations are in agreement with the weak tendency of Mn 2+ to form organic complexes. Part of the Mn measured as "dissolved" (< 0.45 µm) may be bound in colloidal particles under oxic conditions, as also observed in other systems (DeVitre et al., 1988) . The anomalous results in the anoxic hypolimnion may result from experimental artifacts, such as differences in the depth sampled in a steep Mn(II) concentration gradient (at depths around 28 m) (Kuhn et al., 1994) , partial oxidation of Mn(II) during conventional sampling and filtration, or sediment resuspension. Although the sampling and filtration was performed using N 2 gas flow and the samples were immediately acidified after filtration, it is possible that some oxygen leaked into the system which caused partial oxidation of Mn(II). Also, DGT units were not deoxygenated before deployment. Possible precipitation of Mn(II) in the diffusive gel layer could have increased the Mn concentration measured by this technique. In this way, a higher dissolved Mn concentration may have been sensed by DGT than the concentration measured by conventional sampling.
Metal
The procedure recently proposed by Zhang and Davison (2000) , where the diffusive gel with the pore size > 5 nm (like the one used in this study) was used in combination with a more constrained gel (pore size < 1 nm) to quantify labile inorganic and organic species in natural water, would be of little help for metals that are nearly completely (99 %) organically complexed, as it appears to be for Cu and Ni. Even with a smaller pore size, some natural organic complexes will pass through the diffusive gel layer, and undergo ligand exchange with the Chelex resin. These complexes, thus, will contribute to the DGT labile species. For the metals like Zn, Cd, Pb and Mn, which are to a smaller extent bound to organic complexes, more precise quantification would be possible if multiple DGT devices with different gel compositions were used.
Metal
Some seasonal changes in metal concentrations in the lake were apparent from the combination of the measurements of dissolved and DGT-labile species. A decrease in dissolved and DGT-labile species of Cu, Zn, Cd and vpeters@example.com m was observed from June to August. This decrease was probably linked to the intensive sedimentation of organic matter during the summer stratification . Mixing and oxygenation of the lake in winterspring appeaed to lead to an increase in dissolved and DGT-labile Ni, Zn and Cd (in March 1999), whereas Mn clearly decreased in the hypolimnion. Oxidation of sulphides at the sediment-water interface may result in release of those elements from the sediments.
Metal
The DGT technique, thus, provides a convenient way of measuring in situ average concentrations of labile species over several days in the water column of a lake. The interpretation of these measurements with regard to speciation is, however, not straightforward due to the intricate behaviour of organic complexes during metal collection onto DGT-devices.

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