Gel-Based Coloration Technique for the Submillimeter-Scale Imaging

Jun 14, 2013 - School of Civil Engineering, Southeast University, Nanjing 210096, China ... Environmental Science & Technology 2017 51 (24), 14155-141...
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Gel-Based Coloration Technique for the Submillimeter-Scale Imaging of Labile Phosphorus in Sediments and Soils with Diffusive Gradients in Thin Films Shiming Ding,†,* Yan Wang,† Di Xu,† Chungang Zhu,‡ and Chaosheng Zhang§ †

State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, People’s Republic of China ‡ School of Civil Engineering, Southeast University, Nanjing 210096, China § GIS Centre, Ryan Institute and School of Geography and Archaeology, National University of Ireland, Galway, Ireland S Supporting Information *

ABSTRACT: We report a highly promising technique for the high-resolution imaging of labile phosphorus (P) in sediments and soils in combination with the diffusive gradients in thin films (DGT). This technique was based on the surface coloration of the Zr-oxide binding gel using the conventional molybdenum blue method following the DGT uptake of P to this gel. The accumulated mass of the P in the gel was then measured according to the grayscale intensity on the gel surface using computerimaging densitometry. A pretreatment of the gel in hot water (85 °C) for 5 d was required to immobilize the phosphate and the formed blue complex in the gel during the color development. The optimal time required for a complete color development was determined to be 45 min. The appropriate volume of the coloring reagent added was 200 times of that of the gel. A calibration equation was established under the optimized conditions, based on which a quantitative measurement of P was obtained when the concentration of P in solutions ranged from 0.04 mg L−1 to 4.1 mg L−1 for a 24 h deployment of typical DGT devices at 25 °C. The suitability of the coloration technique was well demonstrated by the observation of small, discrete spots with elevated P concentrations in a sediment profile.

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INTRODUCTION Phosphorus (P) is not only one of the most important nutrients for biological growth, but it is also the nutrient that most frequently limits biological productivity in terrestrial and aquatic environments.1,2 As most of the P in soils and sediments is bound to minerals or organic matter, only a small portion of P, which is present in the dissolved form, can be directly taken up by plant roots. As a consequence, the uptake of P by a plant is generally limited by the resupply of solid P to pore water P and the diffusion of pore water P to the plant roots, resulting in concentration gradients of P in the vicinity of plant roots, as recently observed.3 Such concentration gradients of P have also been widely observed in the vicinity of the sediment-water interface (SWI), resulting from a rate-determining resupply of solid P to pore water P and the diffusion of pore water P to the water column.4−6 According to © XXXX American Chemical Society

these source-sink interaction concepts of labile P, an accurate assessment of the P status of a soil or sediment should focus on the diffusive process of solution P to the sinks (e.g., roots or water column) coupled with its kinetic resupply from the solid phase. Additionally, capturing the heterogeneous feature of labile P in the root and SWI microenvironments necessitate their measurements at the two-dimensional (2D) level and at a high resolution. Traditional methods, mainly chemical extraction, can provide a simple differentiation of the labile and inert forms of P for soils and sediments, but they are developed based on an Received: January 14, 2013 Revised: June 4, 2013 Accepted: June 14, 2013

A

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Tu20 based on Murphy and Riley.21 It was prepared by dissolving 20 g ammonium molybdate tetrahydrate and 0.5 g potassium antimonyl tartrate each in 100 mL deionized water. The two solutions were slowly mixed with 194.6 mL concentrated sulfuric acid. After cooling to room temperature, the mixed solution was diluted to 1000 mL using deionized water. This stock solution can be stored in a brown bottle under room temperature for 1 month. Prior to colorimetric analysis, 1.5 g ascorbic acid was added to 100 mL of mixed solution, after which the solution was diluted to 1000 mL with deionized water preheated to 35 °C (the temperature required for color development). The final mixed reagents used for the colorimetric determination were prepared freshly and used within 2 h. This solution contained 0.113 M MoO42− and 8.6 mM Vc−, and had a pH value of 0.48. Preparation of the DGT Units and Special Assemblies. The diffusive gel was prepared with 15% acrylamide and a 0.3% agarose-derived cross-linker following published procedures.22 The revised Zr-oxide gel capable of a high-resolution measurement of P was prepared according to Ding et al.18 Briefly, 2 g of the half-dried Zr-oxide was added to 4 mL of the gel solution composed of 28.5% acrylamide (w/v) and 1.5% N,Ń -methylene bisacrylamide (w/v). The mixture was thoroughly ground in an agate mortar and followed by further dispersion in an ultrasonic disruptor. After standing for 5 min to remove settled particles, 3.0 μL tetramethylethylenediamine (TEMED) catalyst and 75 μL freshly prepared ammonium persulfate initiator (10%, w/v) were added to the mixture. The solution was 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 30 min to allow the Zr-oxide to settle to one side of the gel and was then transferred to an oven at 45 ± 1 °C to polymerize for 30 min. The gel sheet removed from the glass plates was soaked in deionized water for at least 24 h (the water was replaced 2−3 times) and stored in deionized water prior to use. The thickness of the gel remained at 0.4 mm after hydration, and the water content in the gel was approximately 70%. The gel sheet was cut into discs with a diameter of 2.5 cm or rectangles with a size of 3.0 ×15 cm. The discs and rectangles were used for the solution test and field deployment with the DGT, respectively. The piston-type DGT holder with a 2-cm diameter exposure window was obtained from DGT Research Limited (Lancaster, U.K.) and was used for the solution test. A flat probe with a 3.0 ×15 cm (width × length) exposure window was produced under the instruction of the authors and used for deployment in sediments. In both types of DGT assemblies, the Zr-oxide gel (with the Zr-oxide settled surface facing up) was covered sequentially by a diffusion gel and a 0.13-mm cellulose nitrate filter membrane (Whatman, 0.45-μm pore size). The thickness of the diffusive gel in the piston-type DGT unit was 0.80 mm, while a 0.40-mm-thick diffusive gel was used in the sediment probes. General Procedures. DGT Uptake. The Zr-oxide DGT were deployed into 4 L well-stirred solutions containing 0.03 mol L−1 NaNO3 and certain concentrations of P (up to 25 mg P L−1) for a certain time (up to 24 h), depending on the purposes of the respective experiments. Three DGT units were deployed at each concentration or deployment time. Pretreatment of the Zr-Oxide Gel. Heating was performed on the P-loaded Zr-oxide gel to prevent the diffusion of phosphate or the formed blue complex during the subsequent color development. The Zr-oxide gel disc was placed in a 60

operationally defined response to chemical reagents rather than on a true reflection of the P lability.7 The diffusive gradients in thin films (DGT) is expected to be superior to extraction techniques by responding to the kinetic solid-solution interaction of P rather than a pseudoequilibrium between the extractant and soil/sediment.8 A typical DGT device consists of a ferrihydrite-impregnated binding phase overlain by a welldefined diffusion phase.9 When the device is deployed in soil or sediment, pore water P passes through the diffusion phase and is immediately trapped by the binding phase. This causes a decrease of the pore water P adjacent to the surface of the DGT device and a further release of P from the solids to resupply the pore water P. The uptake of P by DGT can consequently mimic the uptake and migration of labile P in the root and SWI microenvironments. Because of their similarity, DGT has been successfully applied to assess the bioavailability and the risk of the environmental pollution of P in terrestrial and aquatic ecosystems.10−14 A significant advantage of DGT is their capability of taking high-resolution measurements of the labile P at the 2D level, and the DGT-measured concentration is sensitive to the changes in the local increase or decrease of solution concentration as well as the solid phase resupply.15 The 2D measurement of labile P with DGT using the ferrihydriteimpregnated binding gel (abbreviated as ferrihydrite DGT) has been performed in combination with an analysis by laser ablationinductively coupled plasmamass spectrometry (LA−ICP−MS) at a spatial resolution of ∼300 μm.16,17 Recently, Ding et al.18 developed a routine procedure capable of the 2D measurement of DGT-induced P at a spatial resolution of 450 μm in combination with the use of a revised high-capacity binding phase (Zr-oxide gel). This procedure requires only routine equipment at a low cost, but it was laborious for the treatment of a large number of samples. The aim of the present study was to develop an easy but highly effective technique for the 2D measurement of labile P in sediments (and soils as well) at a submillimeter resolution in combination with Zr-oxide DGT. We found that the phosphate accumulated in the Zr-oxide gel could directly react with molybdate to form the phosphomolybdenum blue complex under conditions similar to those in solution, resulting in a change in the color or optical density on the surface of the Zroxide gel. The amount of P accumulated in the gel and its local variation could thus be potentially determined at a high resolution using the computer-imaging densitometry (CID), based on which the imaging of labile P in sediments or soils could be easily obtained following the DGT deployment.

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EXPERIMENTAL SECTION Reagents, Materials, and Solutions. The chemicals used in this work were analytical reagent grade and supplied by SCR Co Ltd., P.R. China, unless stated otherwise. All experimental and reagent solutions were prepared using deionized water (ULUPURE, P.R. China). Potassium dihydrogen phosphate was used to prepare the P stock solution (100 mg L−1). Halfdried hydrous zirconium oxide (Zr-oxide) was used as the binding agent for the Zr-oxide gel. It was prepared from the precipitation of a ZrOCl2·8H2O solution (25 g L−1) under pH 7.0 ± 0.1. The precipitate slurry was rinsed repeatedly with deionized water and was dried using an electric hair dryer until the water content was between 45% and 55%.19 The mixed reagent used to determine P using the molybdenum blue method was prepared according to Jin and B

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Calibration Procedure for P Measurement. The calibration of the P measurement was performed by establishing the relationship between the grayscale intensities of the Zr-oxide gel and its accumulated masses of P after the DGT deployments. The piston-type DGT units assembled with the Zr-oxide gels were deployed in 0.03 mol L−1 NaNO3 solutions containing concentrations of P ranging from 0.5 mg L−1 to 20 mg L−1. The deployment time varied from 1 to 4 h. Six replicate DGT units were used for each treatment. After retrieval of the DGT units for each treatment, three gel discs were used for elution of the P with 1 M NaOH followed by colorimetric determination of the P according to Ding et al.19 The other three discs were heated in hot water (85 °C) for 5 d. Each disc was then immersed in a 60 mL vessel containing 40 mL of the mixed reagent. The vessels were placed in an incubator at 35 °C for 45 min. The grayscale intensities of the gel surfaces were measured using CID, as described earlier. The relationship between the masses of P accumulated in the gels and their corresponding grayscale intensities was fitted using an exponential equation. Another calibration experiment was conducted using the same procedure, except for that no heating was performed on the gels prior to CID analysis. Performance Test of the Coloration Technique for DGT Measurement. The performance of coloration was examined by investigating the relationship between the DGT-accumulated masses of the P measured by coloration and the concentrations of P in the solutions. The DGT units were deployed in 4 L well-stirred solutions containing 0.03 mol L−1 NaNO3 and different concentrations of P (0.5 to 22 mg L−1) for 8 h at 20 °C. The accumulation mass of the P in the gels was obtained using the coloration measurement and calculated according to DGT theory. Field Application. The field deployment of DGT in sediments was performed in the eutrophic Meiliang Bay, Lake Taihu. The details about the water eutrophication of this bay as well as the pollution of P in sediments have been reported elsewhere.24,25 The Zr-oxide DGT probes were deoxygenated with nitrogen for 16 h and transported to the sampling sites (31°30′57.7″ N-120°11′20.7″ E) by placing them in a container filled with deoxygenated 0.03 M NaNO3. The probes were inserted into the sediment using a releasing device (Figure S2 of the SI). They were retrieved after 48 h and brought to the laboratory. Each binding gel was heated in hot water (85 °C) for 5 d and was then immersed in the mixed reagent, with the volume of the added reagent 200 times the volume of the gel. The vessel was kept in an incubator at 35 °C for 45 min. After retrieval, the grayscale intensity of the gel was measured by CID according to the procedure mentioned earlier. Calculation. The concentrations of P were calculated using the DGT equation:

mL vessel containing 40 mL deionized water. The vessel was placed in an oven at 85 °C for a period of time (5 days unless stated otherwise). Coloration. The Zr-oxide gel disc after heating was placed in the 60 mL vessel (with the Zr-oxide settled side facing up) containing the mixed reagent (40 mL or 200 times of the gel volume unless stated otherwise) for coloration. The vessel was placed in an incubator for a certain time (45 min unless stated otherwise), with the temperature stabilized at 35 ± 1 °C. CID Analysis. After retrieval from the mixed reagent, the Zroxide gel discs were immediately rinsed using cool water prestored in refrigerator at 4 ± 1 °C and then immersed in cool water for at least 5 min to stop the color development. The water adhering on the surface of the gels was removed using filter paper. Their surfaces on the Zr-oxide settled sides were then scanned using a flat-bed scanner (Canon 5600F) at a resolution of 600 dpi, corresponding to a pixel size of 42 × 42 μm. The grayscale intensity of the scanned images corresponding to the open window of the solution DGT unit was finally analyzed with ImageJ 1.46 (downloaded from http://rsb.info. nih.gov/ij).23 Details for Establishment of the Coloration Technique. Heating of the Zr-Oxide Gel. A method was developed to test the effect of the heating on the diffusion of phosphate or the formed blue complex in or from the gel during the subsequent color development. The Zr-oxide gel discs with and without DGT uptake were immersed into deionized water and placed in an oven at 85 °C for lengths of time (up to 7 d). The Zr-oxide gels with DGT uptake were sliced in half, while the Zroxide gels without DGT uptake (blank) were sliced into sections in approximately square shapes. One half of the gel disc (with a P mass of 18.4 μg cm−2) and two blank gel sections were fixed together on one side of a double-faced adhesive tape (Zr-oxide settled side facing up). The other side of the adhesive tape was attached to the bottom of a 60 mL vessel. The lateral edge of the half of the gel disc was placed in close contact with the edges of the blank gel sections (Figure S1 of the Supporting Information, SI). Each vessel was filled with 40 mL of the mixed reagent and placed in an incubator at 35 °C for coloration. Only three gel sections were taken out at deployment times of 30, 60, and 90 min respectively, while the fourth one was used as a backup once one gel section was sampled unsuccessfully. A P-loaded half of the gel disc was taken out at 60 min. Their grayscale intensities were obtained according to the procedure mentioned earlier. Time for Color Development. The Zr-oxide gels after DGT uptake were heated in hot water (85 °C) for 5 d. Each of them was then immersed in a 60 mL vessel containing 40 mL of the mixed reagent. The vessels were placed in an incubator at 35 °C. The gels were taken out at deployment times of 20, 30, 40, 50, and 60 min. Gels without DGT uptake were simultaneously treated as controls. Their grayscale intensities were obtained according to the procedure described earlier. Volume of the Mixed Reagent for Color Development. The Zr-oxide gels after DGT uptake were heated in hot water (85 °C) for 5 d. Each one was then immersed in a 60 mL vessel containing 5, 10, 20, 30, 40, and 50 mL of the mixed reagent, corresponding to 25, 50, 100, 150, 200, and 250 times the volume of the gel disc (0.2 mL), respectively. A small blank gel disc with a diameter of 1 cm was also placed in the vessel and treated as a control. Both the P-loaded and blank gels were taken out after exposure for 45 min. Their grayscale intensities were obtained as described earlier.

C DGT =

M Δg DAt

(1)

where Δg is the thickness of the diffusive layer, D is the diffusion coefficient of the phosphate in the diffusive layer, t is the deployment time, A is the exposure area of the gel, and M is the corresponding accumulated mass of P over the deployment time.8 The values of D for H2PO4−(aq) have been reported elsewhere.9 M was calculated using the coloration calibration equation established earlier. The M in the binding gel could also be calculated according to eq 2 when it was eluted using a known volume of 1 M NaOH (Ve). C

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Figure 1. Changes of the grayscale intensity on the blank gel surface as a function of the distance from the contact edge with the P-loaded gel. The different lines show the results of the different times of the color development (30, 60, and 90 min). Both the blank and P-loaded gels were treated in hot water (85 °C) for different times (0−120 h) and were assembled according to Figure S1 of the SI before the coloration.

M=

Ce(Vg + Ve) fe

H3PO4 (MoO3)12 + reducing agent (2)

→ phosphomolybdenum blue [Mo(VI) → Mo(V)]

Ce is the concentration of P in the alkaline eluate, Vg is the volume of the gel, and fe is the elution efficiency (0.95).19

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Both phosphate and the formed blue complex would become dissolved and mobile if they are not immobilized in the gel during the color development, which is different from the precipitation reaction that occurs in the AgI(s)-incorporated binding gel. Consequently, the key for a high-resolution measurement with the coloration method is to ensure the immobilization of phosphate and the formed blue complex during the color development. Optimal Treatment of the Gel Prior to Coloration. When the Zr-oxide gels were used for coloration immediately after their retrieval from DGT deployment, the gel surface became blue rapidly and evenly, and the mixed reagent solution also became increasingly blue. This demonstrated the existence of the diffusion of phosphate or the blue complex from the gels during the color development, possibly resulting from their insufficient binding strength on the Zr-oxide sites under strongly acidic condition. Heating was used to increase their binding strength to the Zr-oxides in the gels because this treatment can effectively increase the binding of phosphate to the Zr-oxides in solutions.31 A preliminary experiment also showed that immersing of the Zr-oxide gel in deionized water at 85 °C

RESULTS AND DISCUSSION

The combination of DGT with CID has been used to measure dissolved sulfide in waters and sediments using the AgI(s)incorporated binding gel.23,26−28 The dissolved sulfide species diffuse through the diffusive gel and react with AgI(s) to form black Ag2S(s) during the DGT deployment, resulting in the changes in the color or optical density on the surface of the binding gel. A similar scheme was adopted in this study, except that the coloration of the gel was performed after DGT development. The molybdenum blue method used for coloration is a classical method to detect the phosphate concentration in waters.29 It includes two-step complexation reactions as follows:21,30 PO4 3 − + 12MoO4 2 − + 27H+ → H3PO4 (MoO3)12 + 12H 2O D

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Figure 2. Optimization of the key parameters for coloration based on the changes of grayscale intensity on the surface of the Zr-oxide gels with (A) the heating time of the gels in hot water (85 °C) prior to coloration, (B) the time for color development, and (C) the volume of the mixed reagent added for color development. The number in each bracket show the respective mass of P accumulated in the gel. The values are the means ± standard deviation (SD) of three replicate analyses and are shown as vertical bars.

eliminated from the heating of the gels at 85 °C for 120 h (5 d). It provided the most important foundation for the submillimeter-scale imaging of P with the coloration method, if the lateral diffusion of P in the diffusion layer could be limited during the DGT uptake.32 The heating also resulted in a change of the grayscale intensity on the P-loaded gel surfaces (Figure 2A). The grayscale intensity decreased from 217 to 195 and 180 to 150 for the two gels selected when they were heated at 85 °C for approximately 80 and 90 h, respectively, but remained stable after that. The heating broke the film between the Zr-oxide and phosphate and moved phosphate to the interior of the Zr-oxide, resulting in a stronger binding between them.31 This effect may slow down the color development and decrease the grayscale intensity as observed. The heating thus rendered a stable measurement of the grayscale intensity for the P-loaded gels. The size of the gels was also not affected by the treatment when the heating time was controlled within 7 d. Consequently, the heating of the gels at 85 °C for 5 d was chosen as a pretreatment for the following experiments. Optimal Time for Color Development. The grayscale intensity increased with the exposure time up to 40 and 45 min for the gels with low and high P loadings, respectively, followed by a steady state (Figure 2B). As the grayscale intensity of the high P-loaded gel has approached the maximum from the coloration (215, see the calibration curve below), the optimal time for color development was set at 45 min. This value is longer than the 15 to 30 min normally used in the colorimetric determination of phosphate in solution.21,33 The phenomenon should be attributed to the binding of P by the Zr-oxide in the

caused negligible release of phosphate from the gel. A special gel assembly was designed to evaluate the heating effects on the diffusion of phosphate or the formed blue complex during the subsequent color development (Figure S1 of the SI). The sliced lateral edges of the gels with and without DGT uptake were in close contact prior to coloration. This resulted in a gradual change of the blue color on the blank gel squares from the sliced lateral edges to their body regions if diffusion occurred during the coloration. The diffusion process and its pollution on the gel background could thus be identified using this gel assembly. When the P-loaded gel without pretreatment was used for coloration, a strong gradient of the grayscale intensity was observed perpendicular to the sliced edges of the blank gel squares (Figure 1, Figure S3 of the SI). The gradient increased with the exposure time, and the maximum grayscale value changed from 95 at 30 min to 150 at 90 min. Meanwhile, the grayscale intensity on the body region of the blank gel squares increased from 65 to 90. This result reflected the diffusion of the phosphate or blue complex and the serious pollution to the gel background during the color development. When the Ploaded gels were heated at 85 °C, the gradient of the grayscale intensity on the blank gel squares reduced after a heating time of 22 h, and it remained stable up to 66 h. The gradient almost disappeared after 86 h of heating. The grayscale intensity on the body region of the blank gel squares remained at a high level (∼65) throughout the coloration period when the P-loaded gels were heated for 66 h. It decreased to ∼55 at all exposure times when the P-loaded gels were heated for over 86 h. The diffusion of the phosphate or the formed blue complex and its pollution to the gel background could thus be completely E

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Figure 3. Examples of scanned images of the colored P-loaded gels (left) and the calibration curve for P measurement (right) based on the analysis of the gels. The numbers on the right corner of each gel image denote their grayscale intensity values. The values in the right are the means ± standard deviation (SD) of three replicate analyses and are shown as vertical bars.

exponential equation, as shown in Figure 3. The relative standard deviation (RSD) of the grayscale densities were in the range of 0.2% to 7.0%. The RSD within a gel was mostly within 4.0%, demonstrating that the coloration was uniform under the defined conditions. A limit of detection (LOD) for the P measurement, calculated as three times the SD of a series of blank Zr-oxide gels (n = 12), was 0.21 μg cm−2, corresponding to a blank concentration of 0.04 mg L−1 for a deployment time of 24 h with a standard DGT device (with a diffusive layer of 0.93 mm in thickness) at 25 °C. Because the concentrations of P in the pore waters of many eutrophic sediments or fertilized soils are higher than 0.04 mg L−1,34,35 this LOD is low enough for the measurement of labile P in those sediments and soils. Measurements of P in other systems containing low concentrations of P need to increase the masses of P accumulated in the binding gels through extending the deployment time of DGT devices or decreasing the thickness of the diffusive gels containing in the DGT devices. Performance of the Coloration Technique. The performance of coloration in measurement of P with the DGT was examined using the coloration-measured accumulation masses of P with the changes in the concentrations of P in the solutions. A linear increase of the masses was observed when the accumulation mass of P was up to 23 μg cm−2 (corresponding to a calibrated grayscale intensity of 210), and the experimental data agreed well with the theoretical prediction (Figure 4). After that, the accumulation mass of P increased slightly with a large variation and deviated from the theoretical prediction. This was related to the reduced sensitivity of the grayscale intensity in response to the change in the accumulation mass of P when the grayscale intensity was higher than 210. As a result, the coloration technique could effectively measure P when the accumulation mass of P was up to 23 μg cm−2, corresponding to a P concentration of 4.1 mg L−1 for a deployment time of 24 h with a standard DGT device at 25 °C. This value was approximately one-fifth of the capacity of the revised Zr-oxide gels for the DGT response,18,19 but it was still much higher than the capacities of the other binding gels used for the DGT measurement of P (2 to12 μg P cm−2).9,16,36 Field Application of Coloration Technique in Sediment. In situ deployment of the Zr-oxide DGT probes were

gel, which reduced the activity of phosphate to form complex with the mixed reagent compared to that of the free phosphate ion in solution. Optimal Volume for Color Development. The grayscale intensity of the P-loaded gel increased with the increase in the volume of the added mixed reagent up to 150 times of that of the gel and remained stable after that. The grayscale intensity of the blank gel was slightly higher than the background level (∼55, Figure 1) of the gel (Figure 2C), which likely resulted from a light pollution by a small amount of phosphate or blue complex released from the top surface of the gel following the addition of the mixed reagent. The grayscale intensity of the blank gel decreased to the background level (55) when the volume of the mixed reagent increased up to 150 times of that of the gel, after which it remained stable (Figure 2C). The decrease of the grayscale intensity should be attributed to a dilution of the released matter because of a large volume of the reagent added. Consequently, a larger volume (200 times) could be enough to ensure a complete coloration while the background of the gels can be maintained at the lowest level. Calibration. The calibration of the P measurement showed that there was a nonlinear increase of the grayscale density with the accumulation mass of P for both the gels with and without pretreatment. The grayscale intensity increased rapidly and then slowly until it remained steady (Figure 3). This trend demonstrates that the sensitivity of the coloration decreases with increasing P accumulation in the binding gel. Such a feature has been observed on the binding gels for the measurement of dissolved sulfide.26−28 The calibration ranges of the grayscale intensity were identical for the gels with and without pretreatment, with the values increasing from 52 at the background level to 215 at the saturation level. There was no further increase in grayscale intensity with increasing P loading above a mass of 15 μg cm−2 for the untreated gels or above 40 μg cm−2 for the treated gels. Coloration using the gels with pretreatment was thus much more sensitive to the change in the grayscale intensity and could measure at least double the concentration of P compared to the use of the gels without pretreatment. The relationships between the P accumulations in the Zroxide binding gels with pretreatment and the corresponding changes in the grayscale density was well fitted using an F

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data calibrated with the grayscale intensity were reliable. The concentration of labile P exhibited a slight decreasing trend from the SWI to a depth of 10 mm and a strong increasing trend from depths of 10 mm to 45 mm. It remained steady from a depth of 45 mm to the bottom of this profile. A highly uneven distribution of labile P appeared from depths of 10 mm to 40 mm, with the RSD varying to a maximum of 21% along the horizontal direction. Moreover, a substantial number of discrete localized elevated P concentrations were observed below the depth of 30 mm. These dark spots had diameters from less than 1 mm to 3 mm. A further analysis of the two spots showed that the peak concentrations of labile P in a discrete spot could be double those of the background at the base. These small-scale spots, termed microniches, have previously been reported in sediments, especially for sulfide,23,26,37 and are generally attributed to strong decomposition of enriched organic matter in localized zones. Observation of the chemical heterogeneity of the sediments and especially the distribution of the microniches on a small scale strongly demonstrated the feasibility of coloration technique in the high-resolution imaging of labile P in sediments with DGT.

Figure 4. Relationship between the coloration-measured accumulated mass of P and the concentration of P in the solutions with DGT deployment. The line is the theoretical response calculated using eq 1. The values are the means ± standard deviation (SD) of three replicate analyses and are shown as vertical bars.

carried out in Meiliang Bay of Lake Taihu. The distribution of the labile P in a representative sediment profile was obtained using the coloration measurement (Figure 5). Because all the grayscale intensity values detected in this profile were lower than 210 (corresponding to 1.2 mg P L−1), the concentration

Figure 5. An image of labile P in a sediment profile from the in situ deployment of the Zr-oxide probe in Lake Taihu followed by the coloration measurement. The location of the sediment−water interface is represented by zero. G

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Future Perspective. We have demonstrated that the coloration technique can be used for the submillimeter-scale imaging of P in combination with DGT. This new technique offers significant advantages over the previous techniques. First, the equipment required for coloration is similar to that for detection of phosphate in solution, and it is common and can be found in most laboratories. This is in contrast with the use of LA−ICP−MS in previous studies, which is extremely expensive and only equipped in a few laboratories worldwide. Second, the procedure for the preparation of the coloration is quite simple, except for a delay of 5 days for heating and can be easily learned. This overcomes the drawback of the laborious routine method recently developed by the authors for the 2D measurement of P. Third, this easy technique is well suited for the investigation of the labile P distribution in sediments and soils at the 2D level and at submillimeter scales, which is particularly useful for studying of heterogeneous microenvironments such as the SWI and rhizosphere. The three merits of the coloration technique, coupled with a potentially readily available Zr-oxide gel in the near future, will significantly improve its ease of use in the high-resolution and highfrequency measurements of labile P in sediments and soils, and will facilitate its widespread application in environmental monitoring and fertility assessment.

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ASSOCIATED CONTENT

S Supporting Information *

Schematic diagram of a gel assembly to test the diffusion of phosphate and the formed blue complex during the color development; photograph of the device for releasing the DGT probe into the sediments; images of the blank and P-loaded gels from heating in hot water for different times followed by coloration for different time. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-25-86882207; fax: 86-25-86882207; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was sponsored by the National Scientific Foundation of China (21177134, 41001325), the Project of Knowledge Innovation for the third period, CAS (KZCX2-YWJS304), and the Nanjing Institute of Geography and Limnology, CAS (NIGLAS2010KXJ01). We thank Dr. Qin Sun for her constructive suggestions in improving this manuscript.

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REFERENCES

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