ARTICLE pubs.acs.org/est
High-Resolution, Two-Dimensional Measurement of Dissolved Reactive Phosphorus in Sediments Using the Diffusive Gradients in Thin Films Technique in Combination with a Routine Procedure Shiming Ding,*,† Fei Jia,‡ Di Xu,† Qin Sun,‡ Lei Zhang,† Chengxin Fan,† and Chaosheng Zhang§ †
State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China ‡ College of Environmental Science and Engineering, Hohai University, Nanjing, China § GIS Centre, Ryan Institute and School of Geography and Archaeology, National University of Ireland, Galway, Ireland
bS Supporting Information ABSTRACT: Dissolved reactive phosphorus (DRP) is the most available P form in sediments and often directly controls phytoplankton blooms in aquatic systems. In this study, a novel procedure was developed for two-dimensional (2D) measurement of DRP in sediments at a spatial resolution of 0.45 mm using the diffusive gradients in thin films (DGT) technique with a revised high-capacity binding phase (Zr oxide gel). This procedure involves DGT uptake of P in sediments, 2D slicing of the binding gel on a 0.45 0.45-mm grid system, elution of P from each gel square with 1 M NaOH, and microcolorimetric determination of DRP in each eluted solution using 384-microwell plates. Measurements of DRP via this procedure were tested in homogeneous solutions and sediments and produced an acceptable error (100 μg P cm2) for DGT measurements in various environments. The aim of the present study was to develop and test a novel procedure for the 2D measurement of DRP in sediments at submm resolution using the Zr-oxide DGT technique.
’ EXPERIMENTAL SECTION Preparation and Characterization of the Zr-Oxide Binding Gel. The procedure for the preparation of the Zr-oxide binding
gel was modified, based on a report by Ding et al.,26 in order to enable a high-resolution measurement. Half-dried amorphous zirconium hydroxide (2 g) was added to 4 mL of gel solution composed of 28.5% acrylamide (w/v) and 1.5% methacrylamide (w/v). The mixture was thoroughly ground in an agate mortar, followed by further dispersion in an ultrasonic disruptor. The mixture was left to stand for 5 min to remove settled particles, and then 3.0 μL tetramethylethylenediamine (TEMED) catalyst and 75 μL freshly prepared ammonium persulfate initiator (10%, w/v) were added. After mixing, the solution was immediately cast between glass plates separated by 0.4-mm plastic spacers. The glass plate assembly was placed in an incubator at 10 ( 1 °C for 0.5 h, to allow the zirconium hydroxide to settle by gravity to one side of the gel, and was then transferred to an oven at 45 ( 1 °C to allow the gel to polymerize for 1 h. The gel sheet removed from the glass plates was soaked in deionized water for at least 24 h (the water was replaced 23 times) and stored in deionized water prior to use. The distribution of zirconium hydroxide on the modified binding gel was checked by scanning electron microscopy (SEM) and compared to a binding gel prepared according to the original procedure.26 The gels were placed on a gel dryer for 4 h at 60 °C under vacuum. The dried gels were analyzed on a Cambridge Stereoscan 120 instrument at an accelerating voltage of 20 kV after gold-coating using a Balzers Union SCD 40 sputter-coater. The DGT performance of this binding gel was evaluated using the DGT uptake of P with the deployment time in a well-stirred solution containing 21.5 mg L1 P (KH2PO4) and 0.03 M
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NaNO3 at pH 7.0 and 25 °C, with the diffusive gel (0.80 mm thickness) prepared from 15% acrylamide and 0.3% agarosederived cross-linker following the published procedure.7 A 0.13 mm cellulose nitrate filter membrane (Whatman, 0.45 μm pore size) was used as an extension of the diffusion layer in contact with the solution. Phosphorus accumulated in the binding gels was eluted with 1 M NaOH 26 and measured using the molybdenum blue method.19 The elution factor was detected at 0.95 (Figure S1), which is the same as reported previously.26 This elution factor was adopted for the calculation of the accumulated mass of P. Operational Procedure for the High-Resolution, 2D Measurement of DRP. A procedure for the high-resolution, 2D DGT measurement of DRP was developed in this study. This involved DGT uptake in various P-containing environments, 2D slicing of the binding gel on a submm scale, elution of P from each gel square, and microcolorimetric analysis of DRP in each eluted solution (Figure 1). A special cutter was made to slice the binding gel to yield a 0.45-mm resolution after DGT uptake. The cutter was made by stacking a total of 80 commercial Teflon-coated razor blades (0.10 mm thickness, Gillette, China), with each pair of neighboring blades separated by a 0.35-mm plastic spacer (see “The details about the special cutter and 2D slicing” and Figure S2A-D in the Supporting Information). Prior to cutting, the gel was fixed on one side of a commercial double-faced adhesive tape, with the Zr-oxide side facing outward. The other side of the tape was adhered to a clean perspex plate. The fixed gel was cut in one direction by pressing the cutter directly into the gel, followed by a second cut perpendicular to the first one. The twodimensional cuts produced a square array with each gel square having a size of 0.45 0.45 mm (Figure S2E). The gel square array was covered with a wet filter paper (made by immerging Whatman No. 40 into deionized water) for 5 min to remove the adhesive of the tape from the gel squares. Each gel square was then sequentially picked using a needle and transported into a polystyrene microwell composed of 16-microwell strips in a 384microwell plate holder (Jingrui, Shanghai GenoMintel Bioscience & Technique Development Co., Ltd.). 40 μL of 1 M NaOH was added to each microwell using a multichannel pipet, and the solutions were left standing for 16 h at room temperature to elute P from each gel square. The determination of DRP was based on the molybdenum blue method of Murphy and Riley.19 A volume of 24 μL elution solution was withdrawn from each microwell and transferred into a new microwell. A volume of 6 μLof 2 M H2SO4 was added, and the microwells were left to stand for 1 h to neutralize the alkali. Then a volume of 3 μL of reagent (see “The mixed reagent used for analysis of DRP” in the Supporting Information) was added, and the microwells were centrifuged at 2000 rpm for 5 min to remove any gas bubbles that were formed during the neutralization. The mixtures (33 μL in each microwell) were incubated at 35 °C for 1 h to allow colorimetric formation. The microtiter plates were finally read at 700 nm using an Epoch Microplate Spectrophotometer (BioTek, USA). Reproducibility of the DGT Measurement in Homogeneous Media. To test the reproducibility of the DGT measurement of DRP in the above procedure, DGT deployments were sequentially performed in P-containing solutions and aquatic microcosms with thoroughly homogeneous sediments. Standard piston-type DGT units (DGT Research Ltd.) containing the Zr-oxide gel were exposed to 4 L KH2PO4 solutions (pH 7.0) containing 0.03 M NaNO3 at 20 °C. The concentrations of 9681
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Figure 1. Operational procedure for high-resolution, 2D measurement of DRP (CDGT) in sediments.
KH2PO4 varied between 1 and 20 mg P L1. These solutions at pH 7 contain roughly equal concentrations of the hydrogen phosphate ion and the dihydrogen phosphate ion. Both would diffuse through the diffusive layers to adsorb on the Zr-oxide gel. Each DGT unit was retrieved 2 h after deployment. The accumulated masses of P in the binding gels were characterized by random sampling of ∼80 gel squares within each gel using the above procedure. A total of 10 different sediments were collected from several lakes in China. The fresh sediments were lyophilized at 80 °C and passed through a 0.15 mm-mesh sieve prior to a thorough combination and homogenization. A weight of 50 g of the dried sediment sample was transferred into a 100-mL beaker, followed by addition of 50 mL of deionized water. The mixture was shaken slightly by hand to produce a flat interface between the sediment and aqueous phase. The beakers were covered with nylon nets (0.25 mm mesh) or aluminum foils to create oxygenated or oxygen-limited conditions, respectively. All beakers were left standing at room temperature for 1 month to enable sufficient settlement of the sediment particles. Standard piston-type DGT units containing the Zr-oxide gel were then placed into the beakers (one DGT unit per beaker) by pressing them gently onto the sediment-water interface (SWI) to create good contact between the membranes of the DGT units and the SWI. The beakers were further left to stand for different periods ranging from 3 to 10 d. After retrieval, the accumulated masses of P in the binding gels were characterized by random sampling of ∼80 gel squares from each gel sheet using the above procedure.
DGT Measurements in Sediments. High-resolution, 2D measurements of DRP using the Zr-oxide DGT technique were performed to investigate the influence of tubificid worm bioturbation on DRP distributions in sediment profiles. An incubation microcosm was designed based on the report by Zhang et al.27 Two sediment cores as well as the overlying water from the estuary of the Dapu River, Lake Taihu (31°180 42.700 N, 119°560 52.200 E), were collected into plexiglass tubes (11 cm i.d., 50 cm long) using a gravity core sampler. The top 12-cm sediment sections were sliced at 2-cm intervals. Sediments of the corresponding intervals from the two cores were combined and sieved through a 0.6-mm mesh to remove macrofauna and large particles. Each sediment sample was then fully homogenized and put back into the two plexiglass tubes at 2-cm intervals according to the original sequence. Filtered overlying water was gently added to the top of the sediment using intravenous needles. The two tubes, each containing 12-cm sediment, were stored in a dark tank and further submerged in filtered lake water. The microcosm was preincubated at 15 ( 1 °C. Tubificid worms were collected from the same sampling area with a Peterson Grab. Active worms (3545 mm in body length) were selected, counted, and introduced into one tube on day 17 after the preincubation. The amount of worms added was on average 17.1 g ww/m2, which equaled the largest biomass found in the estuary of the Dapu River.27 Two Zr-oxide DGT probes assembled with standard DGT holders for sediment measurement (DGT Research Ltd.) were deoxygenated with nitrogen for 16 h and then 9682
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Figure 2. SEM images of the binding gels prepared according to Ding et al.26 (left) and this study (right).
inserted across the SWI of each tube on day 30 after the preincubation. The DGT probes were deployed for 6 days, with the temperature maintained at 15 ( 1 °C. On retrieval, the SWI on the probe was immediately marked. Accumulated masses of P in the binding gel corresponding to the upper 3.0 cm of the sediment layers were measured using the high-resolution, 2D operation procedure described above. Concentrations of DRP (CDGT) were then calculated using the following equation7 CDGT
MΔg ¼ DAt
ð1Þ
where A is the surface area of the gel square (0.225 mm2), D is the diffusion coefficient of phosphate in the diffusive layer, t is the deployment time, and M is the corresponding accumulated mass of P over the deployment time.7 The 2D spatial distribution of DRP concentrations (with a total of ∼2222 data for one sediment profile) was plotted using the software Origin 8.0 (OriginLab Corporation, USA). Data Processing. As the sediments were fully homogenized prior to incubation, the 2D spatial distribution of DRP concentrations of the control could be regarded as the background without bioturbation. Taking the horizontal heterogeneity of DRP concentrations in sediments of the control into consideration, the influence of tubificid worm bioturbation on DRP concentration in a localized zone of sediments can be regarded as a probability event, which can be assessed by comparing its DRP concentration with those in all the localized zones of the control at the same horizontal level through meshing. Both 2D DRP distribution images obtained from DGT measurements (the control and the bioturbation treatment, with 64 rows and 32 columns and a square size of 0.45 0.45 mm) were first regridded at a 1.8 1.8-mm interval, yielding a total of 128 (16 columns 8 rows) subregions with each composed of 16 (4 4) DRP concentration data. The mean values of DRP in each subregion for the bioturbation treatment were compared to each of the 8 subregions of the control in the same horizontal layers using a nonparametric test (SPSS 10.5, USA). The number “0” or “1” was recorded, depending on whether the difference between the two mean concentrations was insignificant or significant (p < 0.05, 2-tailed). All numbers were summed for each subregion of the bioturbation treatment. The sums for the different subregions varied within a range of 0 to 8. A value of “0” means there was the least probability of an impact from tubificid worms, whereas “8” reflects the greatest probability of an impact.
Other sums indicate intermediate situations, with the probability increasing with an increase in the sum. The 2D spatial distribution of the sums for all subregions of the bioturbation treatment was plotted using Origin 8.0 (OriginLab Corporation, USA).
’ RESULTS AND DISCUSSION Performance of the Modified Binding Gel. To facilitate high-resolution and 2D DGT measurement, the particle size of the impregnated binding agent should be small enough to produce a homogeneous and compact distribution on the binding gel. For example, to measure trace metals with the DGT technique at high resolution, the suspended particulate reagent iminodiacetate is used instead of Chelex 100 as the binding agent for preparation of the binding gel, as it has a much smaller particle size (0.2 μm) than that of Chelex 100 (∼100 μm).28 The Zroxide binding gel that the authors initially developed was designed to measure DRP on a mm or lower resolution scale.26 Its SEM image shows obvious aggregation of the impregnated Zroxide to large particles (∼30 μm) with an uneven distribution (Figure 2), reflecting that this Zr-oxide binding gel is unsuitable for DGT measurements on a submm scale. Modification was performed to reduce the particle sizes of the Zr-oxide through grinding and ultrasonic disruption prior to gel casting. The SEM image of the modified binding gel showed no evident aggregation of the Zr-oxide particles, and their distribution was much more homogeneous and compact (Figure 2). This binding gel was thus considered suitable for high-resolution, 2D measurement of DRP with the DGT technique. The feasibility and capacity of the modified Zr-oxide binding gel for DGT measurements were examined by deployment in a solution containing a high concentration of phosphate (21.5 mg P L1). The measured mass of P accumulated in the binding gel increased linearly with an increase in deployment time over a 24-h experiment (r2 = 0.998) (Figure S3). The experimental data agreed well with the theoretical predictions calculated using eq 1, with measured-to-predicted ratios of 0.98 ( 0.05 (n = 21). The results validated the use of the Zr-oxide binding gel for DGT measurements. The capacity for DGT response was estimated at >100 μg P cm2 based on the response of the accumulation mass to the theoretical line, corresponding well to the authors’ previous report.26 This capacity is much greater than that of other binding gels used for DGT measurement of P, including conventional ferrihydrite gel (∼2 μg P cm2)23 and the recently 9683
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Environmental Science & Technology developed precipitated ferrihydrite gel (7 μg P cm2)24 and Metsorb gel (∼12 μg P cm2).29 Analytical Errors from High-Resolution, 2D DGT Measurements. Prior to application, the analytical errors inherent in the high-resolution, 2D DGT measurement were estimated. Errors may result from all steps of the measurement, including DGT uptake, 2D slicing, P elution, and microcolorimetric determination (Figure 1). The error from DGT uptake was considered negligible with the use of the homogeneous Zr-oxide binding gel. For the slicing step, the distance between each pair of adjacent cutting edges in the cutter was examined using a microscope (Olympus BX51) and it varied within 2% (0.45 ( 0.01 mm) (Figure S2D). The resulting area of each gel square was further examined by randomly measuring ∼100 squares using the microscope, and it varied within 5% (0.2025 ( 0.001 mm2) (Figure S2E). The error from elution of P was also considered negligible since the eluting solution (1 M NaOH) had been mixed thoroughly prior to use. The error caused by
Figure 3. Analytical errors for high-resolution, 2D DGT measurement of DRP (CDGT) in solutions (0) and homogeneous sediments (9) at different accumulated masses of P in the binding gel.
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microcolorimetric determination of P was investigated by detecting different concentrations of phosphate (0.010.6 mg L1) spiked in 1 M NaOH solutions, with 16 duplicates for each concentration. The results showed that the relative standard deviation (RSD) was 0.03 mg L1, corresponding to P accumulation masses of >0.6 μg cm2 in the binding gel. The total errors during the entire procedure were investigated through DGT deployments in phosphate-containing solutions of different concentrations as well as in microcosms with homogeneously mixed sediments. The results showed that the RSD values were very similar for the two types of deployments, both exhibiting a sharp decrease and then remaining steady as the accumulated masses of P in the binding gels increased. The analytical errors were estimated to be within 20% and 10% once the mass of P was >1.2 μg cm2 and >4.0 μg cm2, respectively (Figure 3). These errors should have predominantly resulted from the 2D slicing and microcolorimetric determination processes as explained earlier. High-Resolution, 2D DGT Measurements in Sediments. As a pilot study, the newly designed high-resolution, 2D DGT operation procedure was used to investigate the impact of tubificid worms on DRP distribution in sediments sampled from a eutrophic lake. Tubificid worms represent an important fraction of the benthic community in eutrophic lake sediments.30 A similar study has been carried out by Zhang et al.27 via 1D measurements of DRP concentrations at 1-cm resolution. The field experimental design in this study was similar to that of Zhang et al.,27 but the measurements for DRP were performed at a finer resolution and at the 2D level. Two 2D DRP distribution profiles (for the control and bioturbation treatments) with a resolution of 0.45 0.45 mm were obtained in the upper 3.0 cm of the sediment layers (Figure 4), where the majority of tubificid worms were found (>60%).27 The analytical errors were controlled within 20% throughout the 6-d deployments, with most of the accumulated masses of DRP in the gel squares greater than 1.2 μg cm2.
Figure 4. 2D distribution images of DRP concentrations (CDGT) at a spatial resolution of 0.45 mm in sediments without (left) and with (right) tubificid worm bioturbation. The location of the sediment-water interface is represented by zero. 9684
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Figure 5. The effects of tubificid worm bioturbation on DRP concentrations (CDGT) in sediments based on 1D (left) and 2D (right) analyses. The 1D data were the average concentrations of DRP at a given depth, with a 0.45 mm interval for the depths. “CK” and “Bio” point to the control and tubificid worm treatments, respectively, with the asterisks indicating their difference at significance levels of p < 0.05 (*) and p < 0.01 (**). The 2D data were obtained through comparisons of DRP concentration in one localized zone (1.8 1.8 mm) of the tubificid worm treatment with those in all the localized zones of the control at the same horizontal level. The numbers of 0 to 8 indicate an increase in the probability of an impact of tubificid worms. The location of the sediment-water interface is represented by zero.
For the control, the 2D distribution of DRP showed systematic changes in the vertical direction, with low concentrations (
