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Department of Environmental Engineering Applying the Methods of Chemical Extraction and DGT to Measure Available Sediment Phosphorus

Honours Dissertation Ben Annan November 2002

Acknowledgements
I firstly would like to thank Dr Carolyn Oldham for her supervision and enthusiasm for the project. I would also like to thank Kathryn Linge for her endless support and advice all year long. Thank you to Bridget Alexander for helping me out in the lab. Thank you to my fellow final years who made this year enjoyable. Thank you to Pippa McManus for her love, encouragement and understanding all throughout the year And finally, (even though they always put me first) I would like to thank my family: Dad, Mum, Melanie, Natasha, Katrina and Jessica. Each one of you supported me in your own special way.

Abstract
Phosphorus is often the limiting nutrient of primary production in freshwater wetland ecosystems. During summer months, when the risk of algal blooms is high, wetland sediments become the major, if not only, source of phosphorus. Therefore, it is crucial to understand the amount of phosphorus available from sediments. This study uses the traditional method of chemical extractions and the new method of diffusive gradients in thin-films (DGT) to measure available phosphorus in sediment from Lake Yangebup, Western Australia. The DGT technique allows phosphorus species to diffuse through a layer of acrylamide gel before binding to ferrihydrite embedded in a further layer of gel. The chemical extractions target phosphorus bound to different phases of the sediments, while the DGT technique measures reactive phosphorus species in a sediment slurry. Results from the two methods were compared in order to determine with which sediment phase the phosphorus measured by DGT is associated. The DGT technique was successfully applied to sediment slurries for the measurement of phosphorus. It was found that the concentrations of phosphorus measured by the DGT technique (DGT-P) corresponded to the phosphorus associated with electrostatic attractions, i.e. the ion exchangeable phase. The other extraction mechanisms of acid dissolution and reduction recorded much higher phosphorus concentrations. DGT-P was less than previous FRP measurements in a sediment slurry, indicating that DGT may be measuring a more bioavailable form of phosphorus.

Table of Contents 1 2 Introduction........................................................................................ 1 Background........................................................................................ 3


2.1 2.2
2.2.1 2.2.2

Lake Yangebup...................................................................................................3 Phosphorus..........................................................................................................3


Phosphorus in Freshwater Ecosystems ........................................................3 Phosphorus in Sediments .............................................................................4

2.3 2.4
2.4.1 2.4.2 2.4.3 2.4.4

Chemical Extraction..........................................................................................5 The Diffusive Gradients in Thin-films (DGT) Technique............................7


DGT Components ........................................................................................8 DGT Theory...............................................................................................10 Theoretical Response in Solution...............................................................15 DGT in Sediments......................................................................................18

Methods........................................................................................... 23
3.1 3.2
3.2.1

Lake Sampling..................................................................................................23 Chemical Analysis ...........................................................................................24


Malachite Green Method............................................................................24

3.3
3.3.1

Chemical Extraction........................................................................................27
Extraction Solutions ...................................................................................28

3.4
3.4.1 3.4.2 3.4.3 3.4.4 3.4.5

Diffusive Gradients in Thin-films (DGT) Technique..................................29


Preparation of Gels .....................................................................................29 Preparation of DGT units ...........................................................................33 Using the DGT units ..................................................................................34 Testing the DGT technique ........................................................................35 DGT Measurement in Sediment Slurries ...................................................36

Results ............................................................................................. 38
4.1 4.2
4.2.1 4.2.2 4.2.3

Chemical Extraction........................................................................................38 DGT...................................................................................................................39


Gel Preparation...........................................................................................39 Validation Tests ..........................................................................................43 DGT Sediment Slurry Deployment............................................................53

Discussion........................................................................................ 57
5.1
5.1.1 5.1.2

DGT Validation Tests......................................................................................57


DGT Preparation........................................................................................57 Variation in Results ....................................................................................59

5.2

Sediment Phosphorus Measurements............................................................63 DGT Validation Tests......................................................................................67

Conclusions...................................................................................... 67
6.1

6.2

Sediment Phosphorus Measurements............................................................67 DGT Validation................................................................................................68 Sediment Measurements .................................................................................68

Recommendations for Future Work................................................... 68


7.1 7.2

References.............................................................................................. 70

List of Figures
Figure 2.1: The reaction of orthophosphate with ferrihydrite......................................................9 Figure 2.2: A cross section through a DGT unit showing the concentration gradient through the diffusive gel.................................................................................................................. 10 Figure 2.3: Percentage recovery of P loaded on a resin gel when treated with 0.25 M H2 SO4 . .... 13 Figure 2.4: Measured mass of phosphate accumulated in DGT units versus time....................... 16 Figure 2.5: Measured mass of Ca accumulated in DGT units versus bulk solution concentration. .................................................................................................................................... 17 Figure 2.6: Measured mass of phosphate for different gel thicknesses ...................................... 18 Figure 2.7: The three different cases of supply of ions from the sediment to the porewaters ....... 20 Figure 2.8: Fluxes of Zn from soils to DGT unit with varying gel thicknesses.......................... 21 Figure 3.1: Equipment used for collecting sediment cores. ...................................................... 24 Figure 3.2: Determination of concentration using the standard curve........................................ 27 Figure 3.3: Absorbencies measured by the malachite green method.......................................... 27 Figure 3.4: The glass plates and plastic spacer; components of the casting unit. ........................ 32 Figure 3.5: DGT units deployed in known P concentrations ..................................................... 36 Figure 3.6: DGT units deployed in sediment slurry container................................................... 37 Figure 4.1: Samples treated with malachite green colouring reagents ....................................... 38 Figure 4.2: Binding gel 3, showing the cut circular discs. ........................................................ 43 Figure 4.3: Phosphorus mass accumulation versus time for time experiment 1. ......................... 45 Figure 4.4: Phosphorus mass accumulation versus time for time experiment 1. ......................... 46 Figure 4.5: Mass of P in the binding gels of DGT deployment 1............................................... 49 Figure 4.6: The calculated concentration for DGT units........................................................... 50 Figure 4.7: Mass of P n the binding gels of DGT deployment 2................................................ 51 Figure 4.8: Calculated concentrations with time for DGT experiment 2. ................................... 51 Figure 4.9: Mass of P accumulated by DGT units at different times.......................................... 54 Figure 4.10: Accumulated P Mass for the different gel thicknesses it. Errors are 9%. ............... 54 Figure 4.11: Mass of P in the sediment slurry per mass of dry sediment. ................................. 55 Figure 4.12: Average values of mass of P per mass of dry sediment for the four different slurries. .................................................................................................................................... 56 Figure 5.1: The theoretical mass and actual mass accumulations of DGT units deployed in 50 ppb.. ............................................................................................................................ 62 Figure 5.2: DGT-P measurements compared to FRP measurements ......................................... 66

List of Tables
Table 2.1: Results of Linge's (2002) fractionation scheme .........................................................7 Table 4.1: Dilutions required in order to analyse extraction solutions........................................ 39 Table 4.2: Mass of P measured in extraction solutions. ............................................................ 39 Table 4.3: Diffusive gel preparation dates............................................................................... 40 Table 4.4: Binding gel preparation dates................................................................................. 41 Table 4.5: Average values of P accumulation for binding gel sheets. ........................................ 44 Table 4.6: Results from gels placed in 10 mL of a 1000 ppb P ................................................. 47 Table 4.7: Results from gel cuttings placed in 10 mL of 1000 ppb P for 44 hours. ................... 48 Table 4.8: Cuttings placed in solutions of 1 and 100 ppm P. .................................................... 48 Table 4.9: Results from a 24 hour DGT deployment. ............................................................... 52 Table 4.10: Sediment masses in the four sediment slurries. ...................................................... 53

Ben Annan Introduction ______________________________________________________________________________

1 Introduction
Urbanisation of catchments has lead to increased inputs of nutrients into wetland ecosystems. Of particular concern is the increased loading of phosphorus in freshwater wetlands. Phosphorus (P) is of great importance in freshwater ecosystems as it is often considered to be the nutrient that limits phytoplankton growth. resulting in a decrease in water quality. Wetland sediments play an important role in maintaining water quality as they can remove P from the water column through sedimentation. Often, the sediments of a wetland hold many orders of magnitude more phosphorus than the overlying water column. However, the sediments are not a permanent sink of phosphorus, and This remobilisation is especially important in remobilisation can occur frequently. Therefore, small increases in the phosphorus concentration of a wetland can lead to algal blooms,

summer months, when all other elements for phytoplankton growth, such as heat and light, are in large supply, and inputs of phosphorus from runoff are low. Therefore, the sediments may become the main factor controlling phytoplankton growth. Knowledge of the amounts of phosphorus available from the sediments is crucial for wetland management. Traditionally, the method of chemical extraction has been used to determine the amounts of available phosphorus. Chemical solutions are applied to sediments to measure the amounts of phosphorus bound to different phases of the sediment. However, problems may occur as the chemical solutions may not be specific for each sediment phase. A new method of potentially measuring available sediment phosphorus is the technique of diffusive gradients in thin-films (DGT). The DGT technique is based on the diffusion of phosphorus ions through a hydrogel before being accumulated with a binding agent. Concentration of phosphorus is then calculated using Ficks First Law.

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Ben Annan Introduction ______________________________________________________________________________ The DGT technique has not been applied to sediment slurries. This project aimed to apply the technique of DGT to measure the phosphorus concentrations in sediment. The DGT results were then compared to chemical extraction results and previous sediment slurry results to obtain understanding of the form of phosphorus measured by the DGT technique (DGT-P).

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Ben Annan Background ______________________________________________________________________________

2 Background
2.1 Lake Yangebup
Lake Yangebup lies approximately 16 km south of Perth, Western Australia on the Swan Coastal Plain. This lake has been the subject of many studies (e.g. Bell 1997, Masters 1995). It has a history of contamination and is eutrophic with respect to phosphorus (Davies et al. 1993), therefore it makes an ideal site for this study. As a result of high nutrient concentrations, the lake experiences year round blooms of blue-green algae (Davis et al. 1993)

2.2 Phosphorus
2.2.1 Phosphorus in Freshwater Ecosystems
Consequently, slight increases in phosphorus

Phosphorus (P) is of vast importance in freshwater ecosystems, as it is often the limiting nutrient in phytoplankton growth. Brock 1999). In water, phosphorus can exist in dissolved, colloidal or particulate forms (Kramer et al. 1972). Phosphorus is embodied in ions of phosphoric acid, which is freely soluble in water, therefore releasing phosphorus anions to the water column (Emsley 1980). The relative proportion of these anions (PO 4 3-, HPO4 2- and H2 PO4 -) varies with pH (Reynolds and Davies 2001). A nutrient is considered bioavailable if it is readily assimilable by organisms (Reynolds and Davies 2001). Orthophosphate (PO4 3-) is widely considered to be the form of phosphorus that is bioavailable (e.g. Currie and Kalf 1984). concentrations of a wetland can lead to the occurrence of algal blooms (Boulton and

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Ben Annan Background ______________________________________________________________________________

2.2.2

Phosphorus in Sediments

Sediments play a vital role in the phosphorus dynamics of a wetland ecosystem. The sediments of freshwater wetlands can hold many orders of magnitude more phosphorus than the overlying water column (Emsley and Hall 1976). The amounts of phosphorus in sediments vary largely between different lakes. For example several lakes from Wisconsin (USA) have recorded ranges of 580 to 7 000 g of total phosphorus per gram of sediment (g/g) (Williams et al. 1971). Total phosphorus in Lake Yangebup has been measured at 910 g/g (Linge 2002) Sediments can either remove phosphorus from the water column, or remobilise it. Under normal conditions, the amount of phosphorus lost from the water column through sedimentation is greater than the amount released (Syers et al. 1973). However, the sediments do not always act as a sink. Under certain conditions, there may be a large release of phosphorus from the sediments (Lennox 1984). This is particularly important in summer months, when all other parameters essential for phytoplankton growth (e.g. light and heat) are in plentiful supply, resulting in phosphorus becoming the limiting factor. There are different phases in the sediment that phosphorus can bind to. Sediments consist of several mineral phases and detrital organic matter (Forstner 1990). Phosphorus can bind to either one of these phases through adsorption or precipitation. Adsorption occurs when a solute binds to a solid, usually at a specific site. Precipitation occurs when two or more solutes join together to form a solid. Iron, manganese, and calcium minerals in the sediment have all been shown to bind phosphorus (e.g. Williams et al. 1971, Chang and Jackson 1956). Organic forms of phosphorus generally make up the largest amount of total phosphorus in sediments, ranging anywhere from 15 80% (Senesi and Loffredo 1990). The phase in the sediment with which phosphorus is associated will affect its mobility, and hence availability to the water column (Reynolds and Davies 2001). Knowledge of ______________________________________________________________________________ Centre for Water Research 4

Ben Annan Background ______________________________________________________________________________ the association of phosphorus with the sediment phases is important in understanding the amounts of available phosphorus. Typically, measurements of phosphorus phases have been performed with the method of chemical extraction.

2.3 Chemical Extraction


The phosphorus bound to different phases of the sediment may be released by applying different chemical solutions to sediment samples. This method is called chemical extraction. Typically, chemical extractions are applied to the sediment sequentially, as some extractions may target multiple phases. Such a scheme is called fractionation. Chang and Jackson (1956) performed the first fractionation scheme aimed at extracting various associations of inorganic phosphorus. However, as Bostrom et al. (1982) has shown, this scheme had problems, as the extraction solutions werent specific to one phase of phosphorus. Many authors have since modified the fractionation scheme of Chang and Jackson (1956) (e.g. Kaiserli et al. 2001). However, the problems of multiple phase extraction still exist. Although many different extraction schemes may exist, the underlying extraction mechanisms are the same. The four most common extraction mechanisms are ion exchange, acid and base dissolution and reduction.

Ion exchange
Elements bound to sediment by electrostatic attraction do so at sites on clay minerals, organic materials and amorphous solids. These elements can easily be replaced since there is no specific bond to the adsorption site. Ion exchangeable phosphorus extraction involves displacement by another anion of similar mass or by the formation of an alkaline phosphate complex (Ruttenberg 1992). Linge (2002) used 1 M MgCl2 to extract the ion exchangeable fraction in Lake Yangebup sediment (Table 2.1).

Acid dissolution

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Ben Annan Background ______________________________________________________________________________ Many minerals in the sediment are, to some extent, acid soluble (Williams and Mayer 1972). Acid dissolution involves dissolving these mineral, therefore releasing the phosphorus bound to them. Acid dissolutions have been used to extract phosphorus bound to apatite minerals, and iron and aluminum oxides (Williams and Mayer 1972). Acid extractions are typically used last in sequential fractionation schemes as they extract a wide range of phosphorus. Linge (2002) used HCl to remove residual apatitebound phosphorus in the last stage of a fractionation scheme (Table 2.1).

Base Dissolution
Base solutions are perhaps the most widely used chemical extraction technique. Base extractions work on the same dissolution principle as the acid extractions discussed above. It has been shown that base extractions will release a wide range of phosphorus from the sediment, including that bound to humic substances (e.g. Deurer et al. 1978). Sharpley et al. (1991) suggest that an extraction solution of 0.1 M NaOH correlates well with bioavailable phosphorus. Other work has shown that NaOH will extract the orthophosphate adsorbed onto Fe and Al phases in the sediment (Williams and Mayer 1972). As orthophosphate is bioavailable, these two findings may agree with each other.

Reduction
Reduction extractions are based on reducing minerals in the sediment, thereby releasing phosphorus. Reduction solutions are most often used to extract metal oxides (e.g. Fe and Mn) which are important to binding trace elements to sediments (Pickering 1986). Iron oxides are particularly important in the binding of phosphorus (Emsley 1980). Linge (2002) used an acidified solution of hydroxylamine hydrochloride (NH2 OH.HCl) solution to extract amorphous iron oxides (Table 2.1). However, the HCl used to acidify the solution may also extract phosphorus bound to apatite minerals.

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Ben Annan Background ______________________________________________________________________________

Table 2.1: Results of Linge's (2002) fractionation scheme on Lake Yangebup sediment. The ion exchange, reduction and acid dissolution mechanisms are asterisked respectively. Phase targeted Solution used Mass of phosphorus per mass of dry sediment ( g /g) Dissolved Ion Exchangeable* Organic Carbonate Amorphous* Crystalline Apatite* Deionised (DI) water MgCl2 NaOCl NaOAc NH2 OH.HCl (NH4 )2 C2O HCl 0.9 +/- 0.67 1.9 +/- 0.55 13 +/- 4.93 22 +/- 1.42 290 +/- 66 250 +/- 93.1 5 +/- 1.14

Although widely used, these techniques provide no definitive answer on which extraction will measure bioavailable phosphorus. Also, as mentioned above, problems exist with extraction solutions targeting multiple phases.

2.4 The Diffusive Gradients in Thin-films (DGT) Technique


The technique of diffusive gradients in thin-films (DGT) was first developed in 1994 (Davison and Zhang 1994). It was initially developed to measure trace metal concentrations in natural waters, and was later used to measure solute fluxes and concentrations in sediments and soils (Harper et al. 1998). Solutes that have been measured by the DGT technique include Ni, Cu, Fe, Mn, Zn, Cd. Mg, Ca (Zhang et al. 1995, Dahlqvist et al. 2002), phosphorus (Zhang et al. 1998) and even radiocesium (Murdock et al. 2001). ______________________________________________________________________________ Centre for Water Research 7

Ben Annan Background ______________________________________________________________________________

The DGT technique uses a simple device that accumulates solutes on a binding agent after passage through a hydrogel, which acts as a well defined diffusion layer (Davison and Zhang 1994). It relies on the establishment of a steady state concentration gradient in the diffusion layer so that Ficks First Law can be used to calculate unknown concentrations.

2.4.1

DGT Components

The crucial components of the DGT technique are the diffusive gel and the binding agent. The binding agent, usually a resin, is selective for the species being measured. It is embedded in a layer of hydrogel, which is known as the binding gel. The binding gel is separated from the solution by the diffusive gel and is held in place by a simple, plastic unit.

Diffusive Gels
The diffusive gel used in the DGT technique is an acrylamide based gel, crossed linked with a patented agarose-derived cross linker (DGT Research Ltd., UK). The roles of the diffusive gel are to allow the passage of ions from the solution to the binding gel, and to act as a layer where a concentration gradient can be established. The establishment of this concentration gradient is discussed in the DGT Theory section.

Binding Gels
The role of the binding gel is to selectively bind to the target ions after they have passed through the diffusion gel. The term target ions refers to the analyte ions. The binding gel is comprised of the same components as the diffusive gel, however, it contains a binding agent, which is responsible for the binding of the target ions. The binding agent used is selective to the species being measured.

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Ben Annan Background ______________________________________________________________________________ The most common binding agent used in DGT deployments is an ion-exchangeable Chelex-100 resin. This binding agent has been used in the measurements of a large range of metals in natural waters (e.g. Webb and Keough 2002, Zhang et al. 1995). However, different binding agents have been used in order to measure different elements. Murdock et al. (2001) measured radiocesium in natural waters using ammonium molybdophosphate as the binding agent. Teasdale et al. (1999) showed that AgI could be used as the binding agent in the measurement of dissolved sulfide. To measure phosphate concentrations in natural waters, Zhang et al. (1998) used ferrihydrite as the binding agent. Ferrihydrite (FeOOH) is iron hydroxyoxide, which, due to its surface OH groups is very reactive. These OH groups can bind either cations or anions (Figure 2.1).

Figure 2.1: The reaction of orthophosphate with ferrihydrite. Ferrihydrite is used as the binding agent in the measurement of phosphate (modified from Boulton and Brock 1999). More recently, Li et al. (2002) has demonstrated that a cellulose phosphate ion exchange membrane can be used as the binding phase in the measurements of Cu and Cd. The membrane binding agent is unique, as it can be reused.

The DGT Unit


The diffusive and binding gels are held in place by a small plastic unit, which is referred to as the DGT unit. The units consist of a backing support and a front cap with a 2.0 cm

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Ben Annan Background ______________________________________________________________________________ diameter window. This window controls the area of diffusion (Zhang and Davison 1995). Once the unit has been loaded with the diffusive gel and resin gels, it is deployed in the solution which is being analysed. This solution is often referred to as the bulk solution (Davison and Zhang 1994)

2.4.2

DGT Theory

Calculation of Concentration
Ions will diffuse from the bulk solution, through the diffusive gel, and to the binding gel. At the interface of the diffusive gel and the bulk solution, the concentration of target ions is assumed to be equal to the concentration of target ions in the solution. At the binding gel, the target ions are bound by the binding agent and are therefore removed from the diffusive gel such that the species concentration at the interface is effectively zero. Therefore, a concentration gradient is established within the diffusive gel (Davison and Zhang 1994) (Figure 2.2).

Figure 2.2: A cross section through a DGT unit showing the concentration gradient through the diffusive gel. The black line represents the concentration of target ions at each point within the diffusive gel. (Modified from Windsor Scientific Limited).

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Ben Annan Background ______________________________________________________________________________ The theory of the DGT technique is based on the establishment of this concentration gradient. The theory is based upon Ficks First Law of Diffusion (equation [1]). Ficks First Law dictates that the flux of a species through a media is equal to the concentration gradient multiplied by the diffusion coefficient of that species in the media:

F=D

dC dx

[1]

where, F is the flux, D is the diffusion coefficient and

dC is the concentration gradient. dx

Rewriting the concentration gradient to apply to the DGT unit, the equation [1] becomes:

F=D

C1 C 2 g

[2]

where, C1 is the concentration at the bulk solution and diffusive gel interface (i.e. the concentration of the bulk solution), C is the concentration at the diffusive gel and 2 binding gel interface and g is the thickness of the diffusive gel.

However, as discussed above, the concentration on the diffusive gel/binding gel interface is zero. Using the fact that C2 is zero, equation [2] can now be rearranged to give an equation to calculate C 1:

C1 =

Fg D

[3]

In this case, the actual definition of flux through the gel is equal to the mass of species diffusing through the diffusive gel per unit area in a known amount of time:

F=

M At

[4]

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Ben Annan Background ______________________________________________________________________________ where, M is the mass, A is the area of diffusion and t is the time of diffusion. When equation [4] is substituted into equation [3], an equation for the concentration in the bulk solution is obtained, based on parameters that are known, or that can be measured:

C1 =

Mg DAt

[5]

where, M is the mass accumulated by the binding gel, g is the thickness of the diffusive gel layer, D is the diffusion coefficient of the target ion through the diffusive gel, A is the area of the diffusive gel exposed to the bulk solution and t is the time that the DGT unit is deployed in the bulk solution.

Discussion of Parameters
Mass
Accurate measurement of the mass accumulated in the binding layer is crucial in order to obtain reliable results. Mass is measured by eluting the binding gels, most commonly with acid, so that the bounded species are released from the gel into the eluent (Zhang et al. 1995). The concentration of the species in then measured and the mass is determined using the following equation:

M =

Ce (Ve + Vg ) fe

[6]

where, M is the mass accumulated, Ce is the concentration of species in the eluent, Ve is the volume of the eluent, V g is the volume of the resin gel and fe is the elution factor. The elution factor has been included in the equation as the eluent may not recover 100% of the accumulated mass (Zhang et al. 1995). Therefore, a correction factor must be applied. The elution factor is dependent on the ions being measured, the binding agent ______________________________________________________________________________ Centre for Water Research 12

Ben Annan Background ______________________________________________________________________________ used and the eluent used. For the measurement of metal concentrations using a Chelex100 binding resin (e.g. Zhang et al. 1995), the eluent used was HNO 3. Zhang et al. (1995) recorded only an 80% recovery of various metals when this elution process was used. Therefore, the elution factor used was 0.8. When determining mass of iron, an even lower recovery was recorded, therefore an elution factor of 0.7 is required (Zhang et al. 1999b). In the measurement of phosphate (Zhang et al. 1998), sulfuric acid was used as the eluent. Zhang et al. (1998) found that elution with 0.25 M HSO4 resulted in 100% 2 recovery of phosphate, irrespective of elution time ( Figure 2.3), therefore an elution factor is not required in the determination of accumulated mass.

Figure 2.3: Percentage recovery of phosphorus loaded on a resin gel when treated with 0.25 M H 2 SO4 . The result means that this project can use any elution time between 1 and 20 hours (modified from Zhang et al. 1998).

The concentration of species in the eluent have been measured with various analytical techniques. Zhang et al. (1998) used the spectrophotometric method, molybdenum blue, to determine phosphate concentrations in the H2 SO4 eluent. Metal concentrations have been determined by atomic adsorption spectrometry (e.g. Webb and Keough 2002) and inductively coupled plasma mass spectrometry (e.g. Zhang et al. 1999). ______________________________________________________________________________ Centre for Water Research 13

Ben Annan Background ______________________________________________________________________________

Gel thickness
Gel thicknesses used range from 0.13 mm to 2.4 mm (Zhang et al. 1998b, Zhang and Davison 1995), with the most common thickness being 0.8 mm (e.g., Zhang et al. 1998). However, as will be explained in the Theoretical Response section, the DGT technique should yield the same concentration, regardless of the diffusive gel thickness.

Diffusion Coefficient
The diffusion coefficient refers to the diffusion of the target species through the diffusive gel layer. Zhang and Davison (1995) discovered that the diffusion coefficients of metal ions through the diffusive gel are indistinguishable from values in water. This indicates that there is no reaction between metal ions and the gel. This project, however, is concerned with the diffusion of dissolved phosphorus species through the diffusive layer. Zhang et al. (1998) found that the diffusion of phosphate is slightly impeded by the gel. The diffusion coefficient for orthophosphate in the gel was measured to be 6.05 x 10 -6 cm2 s-1; this is 71% of its value in water (Zhang et al. 1998). As this project prepared the diffusive gels using identical methods to Zhang et al. (1998), this value of diffusion coefficient was used for all DGT calculations.

Diffusion Area
The area of the exposed gel is determined by the DGT unit, discussed in section 2.4.1. The circular discs cut of the resin and diffusive gels are usually cut to 2.5 cm diameter (e.g. Dahlqvist et al. 2002). However, due to the window in the front cap, the area available for diffusion is only that of a circle with radius of 2 cm.

Deployment Time
The concentration gradient will establish itself in the diffusion gel within a few minutes of deployment time (Davison et al. 2000). As discussed in the following section, as long as the concentration gradient remains constant, equation [5] should yield the same concentration independent of deployment time. ______________________________________________________________________________ Centre for Water Research 14

Ben Annan Background ______________________________________________________________________________

2.4.3

Theoretical Response in Solution

In order to understand how the DGT technique can be applied to sediments, the theory of the technique in solutions must first be explained. Equation [5] indicates that DGT is a kinetic technique, that is, it does not rely on equilibrium conditions to develop (Li 2002). Therefore equation [5] should yield the same concentration, regardless of deployment time. This will only be true, however, if the DGT unit responds according to theory. An equation for theoretical accumulated mass can be obtained by rearranging equation [5]:

M=

CDAt g

[7]

Equation [7] indicates that, if all other parameters are kept constant, the mass accumulated by the DGT unit will increase linearly with time. Zhang et al. (1998) tested the theoretical response of DGT units by deploying them in solutions of known concentrations for different amounts of time. They found that the accumulated mass of phosphate in stirred solutions of 200 ppb phosphorus increased linearly with time (figure 4). The dotted points show the measured values, while the black line is a plot of the theoretical response calculated using equation [7].

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Ben Annan Background ______________________________________________________________________________

Figure 2.4: Measured mass of phosphate accumulated in DGT units versus time. The dot points show the measured values, while the black line is a plot of the theoretical response calculated using equation [7] (Zhang et al. 1998).

The close fit between the measured values and the theoretical accumulation indicate that the DGT units are responding according to theory. Similar tests of theoretical response were performed by Dahlqvist et al. (2002). Equation [7] indicates that the accumulated mass should increase linearly with bulk solution concentration, as long as all other parameters (including time) are kept constant. Dahlqvist et al. (2002) tested this by deploying DGT units of equal gel thickness in solutions of varying Ca concentrations for equal amounts of time (Figure 2.5).

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Ben Annan Background ______________________________________________________________________________

Figure 2.5: Measured mass of Ca accumulated in DGT units versus bulk solution concentration. The dot points show the measured values, while the black line is a plot of the theoretical response calculated using equation [7] (Dahlqvist et al. 2002).

As seen in Figure 2.5, the linear relationship between concentration and accumulated mass breaks down at a certain point. This indicates that the binding gel can no longer accumulate mass, and is therefore saturated. Equation [8] also indicates that the accumulated mass is theoretically proportional to the reciprocal of the diffusive gel thickness. As seen in Zhang et al. (1998), several DGT units with different diffusive gel thicknesses were deployed into solutions of equal concentration for equal deployment time. The resulting accumulated mass increased linearly with decreasing gel thickness (Figure 2.6).

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Ben Annan Background ______________________________________________________________________________

Figure 2.6: Measured mass of phosphate for different gel thicknesses (24 hr deployment) (Zhang et al. 1998)

Since the area of diffusion in all DGT deployments is fixed by the DGT unit, then no tests have been performed on the relationship between mass and area. However, it can be expected that the mass will relate to area the same way it did with time and bulk solution concentration (Figure 2.4 and Figure 2.5).

2.4.4

DGT in Sediments

The theory of the DGT has been shown to work well when deploying DGT units in solutions and natural waters. However, when the DGT technique is used to measure concentrations in sediments, the theory is different. Typical use of DGT with sediments has been performed by inserting DGT units into sediment cores (e.g. Zhang et al. 1995). In this case, the bulk solution is the porewater of the sediment. Equation [5] assumes a constant flux of species from the solution to the DGT unit, which will only happen with a constant bulk solution concentration. This situation will occur in well mixed solutions, but will not necessarily occur for the deployment of DGT in sediments (Zhang et al. 1995). In sediment deployment, as the DGT unit removes ______________________________________________________________________________ Centre for Water Research 18

Ben Annan Background ______________________________________________________________________________ species from the porewaters, lack of mixing may lead to a decrease in concentration adjacent to the device (Zhang et al. 1995). If this occurs, the concentration gradient in the diffusive gel may decrease and, hence, the flux of species will also decrease. Thus, the flux from the solid phase to the solution phase may fall into one of the following three categories (Zhang et al. 1998b): Case 1, Fully Sustained: Species removed from the porewaters by the DGT unit are rapidly resupplied from the sediment, keeping the concentration adjacent to the device constant. The flux to the DGT unit is approximately equal to the flux of species to the solution from the solid phase. Therefore, the concentration measured by equation [5], will be equal to the concentration of the porewater. Case 2, Unsustained: There is no resupply of species from the sediment. The

concentration in the solution will be depleted over time, and hence the flux to the DGT unit will decrease. In this case, there is no flux from the sediment to the solution phase, so the supply of species to the DGT unit is solely by diffusion from the solution. Therefore, the concentration measured by equation [5] will be less than the actual porewater concentration. Case 3, Partially Sustained: There is significant resupply of species to the solution; however, it is not enough to maintain a constant flux to the DGT unit. There is a flux from the sediment to the solution, but it is not as great as the flux from the solution to the DGT unit. Therefore, equation [5] will underestimate the actual porewater concentration, but not to the degree of the unsustained case. These three cases are shown in Figure 2.7.

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Ben Annan Background ______________________________________________________________________________ Fully Sustained Case 1 Sink

Pore Waters C

Diffusive Gel Partially Sustained Case 3

Unsustained Case 2

Figure 2.7: The three different cases of supply of ions from the sediment to the porewaters (modified from Zhang et al. 1998b)

The practical application of these cases has been studied by Zhang et al (1998b). DGT units with different gel thicknesses (1.3 2.13 mm) were deployed in soils that were treated with metal-amended sludge from a wastewater treatment plant. Using the definition of flux (equation [4]), the fluxes of zinc from the soil solution to the DGT unit were measured for the different gel thicknesses. A plot of flux against the reciprocal of gel thickness shows that as gel thickness decreased, the flux deviated away from a linear relationship (Figure 2.8). This is a stark contrast from the deployment of DGT units in solutions (Figure 2.6), where the linear relationship is maintained for all gel thicknesses.

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Ben Annan Background ______________________________________________________________________________

Figure 2.8: Fluxes of Zn from soils to DGT unit with varying gel thicknesses. The linear eventually breaks down (Zhang 1998b).

This infers that only the beginning part of the curve can be considered to be the fully sustained case, and therefore only those gel thicknesses will yield the theoretical concentration using equation [5]. The thicker gels give the theoretical response, because diffusion from the solution phase to the binding layer is slower, therefore the flux is lower (Zhang et al. 1998b). The lowered flux through the diffusive gel will now be closer to the flux of species from the solid phase to the solution. This relates to the fully sustained case. Conversely, the thinner gels result in a higher flux to the DGT unit. The solid phase cannot meet this flux demand, and so the species concentration in the bulk solution will decrease; this relates to the partially sustained or unsustained cases.

Sediment Slurry
The above discussion of DGT in sediment refers to the insertion of units in sediment cores. However, another possible method to measure sediment concentrations is the deployment of DGT units in sediment slurries. There have been no published accounts of this, therefore knowledge on the matter is very limited.

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Ben Annan Background ______________________________________________________________________________ The stirred sediment slurry will measure the concentration in the porewaters, but will also measure the phosphorus released from the sediment due to the increased volume of water.

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Ben Annan Methods ______________________________________________________________________________

3 Methods
3.1 Lake Sampling
Lake Yangebup sediment consists of two distinct layers: loose floc and an underlying consolidated layer. The floc layer contained living worms and larvae. There were also traces of algae in this layer. The consolidated layer is a darker layer with lower moisture content. It is the more important layer in terms of phosphorus release (Linge 2002). Therefore, the consolidated layer was analysed during this project. On the 29 th of August and the 14 th of October 2002, four sediment cores were taken from Lake Yangebup. The four cores were taken approximately 2 m away from each other in order not to sample already disturbed sediment. The coring system consists of a tube with a stainless steel blade attached to one end, and a handle attached to the other (Figure 3.1). The corer was pushed into the lake sediment, then the handle was removed, allowing the corer to fill with water. Once full, a plastic cap is screwed to the end, and the corer was removed from the water. The steel blade was removed, and replaced with another cap. Before use, all equipment was acid washed to ensure no contamination from previous use.

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Ben Annan Methods ______________________________________________________________________________

.
Figure 3.1: Equipment used for collecting sediment cores. (a) the handle, (b) stainless steel blade, (c) and (d) caps, and (e) (inset) a sediment core held in the coring tube.

Intact sediment cores were taken back to the laboratory for analysis. The overlying water was siphoned off, and then the sediments were pushed out of the corers. In order to minimise heterogeneity, consolidated sediment from all four cores was mixed together to create a bulk sample. All samples for analysis were taken from this bulk sample with an acid washed polyethylene spoon.

3.2 Chemical Analysis


Phosphorus analysis was performed using the malachite green spectrophotometric method.

3.2.1

Malachite Green Method

The malachite green method has been used to determine dissolved phosphate concentrations in both water and soils (e.g. Rao et al. 1997). In the past, the ascorbic acid ______________________________________________________________________________ Centre for Water Research 24

Ben Annan Methods ______________________________________________________________________________ method was used to measure phosphate, however, it has been recently shown that the malachite green method is approximately 4 times as sensitive. Also, the malachite green method is not as sensitive to changes in heating, reagent addition or reaction time. The malachite green method is based on the formation of a molybdophosphoric acid, which turns green. The intensity of the colour development depends on the amount of dissolved phosphorus present in the solution.

Reagents
Malachite Green Reagent
Concentrated sulfuric acid (H2 SO4 ) (95 mL, approximately 18 M) was added very slowly to 375 mL of DI water. After the mixture had cooled to room temperature, 27 g of ammonium molybdate ((NH4 )6 Mo7O24.7H2O) was added and stirred until dissolved. Malachite green oxalate (C25 H22 N2O4 ) (0.135 g) was then added to the resulting solution and stirred until dissolved. After the addition of malachite green oxalate, the solution turned a deep orange. The solution was then made up to one litre, and stored at 4C.

Polyvinyl Alcohol
A stock solution of 0.1 % (w/v) polyvinyl alcohol (PVA) was prepared by dissolving 5 g in 500 mL. To assist the dissolution process, the solution was heated to near boiling point while being stirred.

Standard Solutions
Standard solutions of phosphorus (P) were prepared daily from a stock solution of 1000 ppm P. The stock solution was prepared by dissolving 0.4393 g potassium dihydrogen phosphate (KH2 PO4 ) in 100 mL DI water. A new stock solution was prepared weekly.

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Ben Annan Methods ______________________________________________________________________________

Procedure
The colour reagent was prepared daily by mixing equal amounts of the malachite green and PVA reagents. In a small test tube, 2 mL of this colour reagent was added to 6 mL of the solution being analysed. The solution was mixed well, and then allowed 15 minutes for colour development. The absorbencies were measured at 615 nm using a HACH DR/3000 Spectrophotometer (Hach Company, USA).

Calibration
Working calibration solutions between 10 ppb and 100 ppb P were prepared from the stock standard in order to create a calibration curve. The calibration curve was used to calculate the concentrations in solutions where only the absorbance is known. The measured absorbencies of the calibration solutions were plotted against their concentrations, and a linear trendline was fitted. This trendline was used with the absorbance values of the analyte solutions to determine their phosphate concentrations (Figure 3.2).

v
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Ben Annan Methods ______________________________________________________________________________ Figure 3.2: Determination of concentration using the standard curve. An absorbance of 0.15 is equivalent to a concentration of 71 ppb P.

The malachite green method has a limited range of linear colour response to concentration. At too high a concentration, the colour development is no longer linearly proportional to the concentration. Therefore, the linear response measured using the calibration curve breaks down (Figure 3.3). Therefore, this project only used standards between 10 and 100 ppb.

0.5 0.4 Absorbance 0.3 0.2 0.1 0 0 200 400 600 800 1000 1200 P concentration (ppb)

Figure 3.3: Absorbencies measured by the malachite green method. The linear calibration curve breaks down when concentrations are too high. When the 1000 ppb reading is removed, then a straight line results (Figure 3.2)

3.3 Chemical Extraction


Chemical extractions were applied to sediment in order to investigate the amount of phosphorus associated with different phases in the sediment. The methods used were based on the fractionation scheme used by Linge (2002), however, a sequential process used by Linge (2002) was not adopted in this project. Instead, the extractions were

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Ben Annan Methods ______________________________________________________________________________ applied to individual sub-samples of sediment. bioavailable P. The 0.1 M NaOH was based on

Sharpley et al. (1991) finding that the P extracted with this solution correlated well with

3.3.1
Reagents

Extraction Solutions

Magnesium Chloride
Approximately 50.1 g of magnesium chloride (MgCl2 ) was dissolved in 250 mL of deionised (DI) water to create a 1 M solution. The pH of the solution was adjusted to 7 with the addition of 1 M sodium hydroxide (NaOH).

Hydrochloric Acid
A 1 M hydrochloric acid (HCl) solution was prepared by diluting 28.5 ml of concentrated HCl to 250 mL with DI water.

Sodium Hydroxide
A 1 M solution of NaOH was prepared by dissolving 10 g of solid NaOH in 250 mL DI water. From this solution, 0.1 M NaOH was prepared with a 1:10 dilution.

Hydroxylamine Hydrochloride
Solid hydroxylamine hydrochloride (NH2 OH.HCl) (4.34 g) was dissolved in DI water, and then concentrated HCl (7.125 mL) was added. The resulting solution was made up to 250 mL with DI water.

Procedure
A fresh, wet sediment sample equivalent to 1 g in dry weight was added to 20 mL of each of the extraction solution and processed as described below. After the extraction processes were complete, the slurries were left still for at least 20 minutes to allow the ______________________________________________________________________________ Centre for Water Research 28

Ben Annan Methods ______________________________________________________________________________ sediment to settle. The overlying solution was then passed through a 0.45 m filter before being analysed for P concentration.

MgCl2
The sediment slurry was shaken continuously for 2 hours.

HCl
The sediment slurry was shaken continuously for 17 hours.

NaOH
The sediment slurry was shaken continuously for 17 hours.

NH2 OH.HCl
The sediment slurry was shaken in a water bath at 50 C for 30 minutes.

3.4 Diffusive Gradients in Thin-films (DGT) Technique


3.4.1 Preparation of Gels

The methods of DGT component preparation used in this project are based upon the procedure outline by Zhang et al. (1998) To avoid contamination of DGT components, contact with metal objects were avoided. Acid washed plastic tweezers were used to handle the gels.

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Ben Annan Methods ______________________________________________________________________________

Reagents
The gels used were acrylamide based, cross-linked with a patented agarose-based crosslinker. They were made from a gel solution, prepared from a bulk solution. In order to make the binding gel, ferrihydrite was also required.

Bulk Solution
The bulk solution comprises of 15% (w/v) acrylamide and 0.3% (w/v) cross-linker. A 50 mL bulk solution was prepared by adding 18.75 mL of a 40 % (w/v) acrylamide solution to 7.5 mL of a 3% (w/v) cross linker solution and 23.75 mL of DI water. The solution was stored in the fridge until required.

Gel Solution
A 10% (w/v) ammonium persulphate solution was prepared by dissolving 1 g of solid ammonium persulphate in 10 mL of DI water. The gel solution was prepared by adding 35 L of the ammonium persulphate and 10 L of a TEMED catalyst to 5 mL of the bulk gel solution. It was essential that a fresh ammonium persulphate solution was prepared each day a new gel solution was made. Failure to do resulted in gel solutions that would not set during casting (described below). The gel solution begins to solidify after about 5 minutes, and therefore was used immediately after preparation.

Ferrihydrite
Ferrihydrite was used as the binding agent in the binding gels. A solution of 0.1 M Fe3+ was prepared by dissolving 8 g of iron nitrite nonahydrate (Fe(NO 3 )3 .9H20) in 200 mL of DI water. The solution was continuously stirred as NaOH (1 M) was added drop-wise until the pH reached 7. During the addition of NaOH, a dark brown-red precipitate formed. The volume of NaOH added was approximately 65 ml, which agreed with the volume used by other researches (Zhang et al. 1998).

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Ben Annan Methods ______________________________________________________________________________ The slurry was then stored in the dark at 4C for approximately two hours to allow the precipitate to completely settle. After the two hours, the overlying water was removed with a pipette. The precipitate was then washed with DI water, allowed to settle, and the water removed again. This process was repeated three times. After the final wash, an overlying layer of about 1 cm of water was left. To ensure the exclusion of light, the beaker containing the slurry was wrapped in aluminum foil before being stored at 4C. The ferrihydrite slurry will last for at least nine months if prepared properly (Zhang et al. 1998).

Sodium Nitrate
A sodium nitrate (NaNO3 ) solution was required for the storage of diffusive gel. A 0.1 M solution was prepared by dissolving 2.12 g of solid NaNO 3 in 250 mL of DI water.

Procedure
Diffusive Gel
The diffusive gel was prepared by casting the gel solution using the procedure described below.

Binding Gel
To prepare the binding gel, 1 g of the ferrihydrite slurry was added to 5 mL of the gel solution. In the initial stages of the project, ferrihydrite with the lowest visible moisture content was extracted from the slurry. However, in the later stages, the ferrihydrite slurry was stirred vigorously to ensure homogeneity of the extracted sample. binding gel solution was cast as described below. The

Casting
In order to make the thin-films of diffusive and binding gels, the gel solutions were cast between two glass plates, separated by a plastic spacer. A 10 cm x 10 cm plastic sheet of 0.1 mm thickness was cut into a U-shape, and then placed between two 10 cm x 10 cm ______________________________________________________________________________ Centre for Water Research 31

Ben Annan Methods ______________________________________________________________________________ glass plates to create the casting unit (Figure 3.4). The glass plates and spacer were held in place using plastic clamps on the three closed edges.

Figure 3.4: The glass plates and plastic spacer; components of the casting unit. The gel solution is cast into the space created by the U-shaped plastic spacer. The black dot indicates the best place to insert the gel solution

The glass plates were offset by a few millimeters to allow room for the gel to be inserted. This also made it easier to take the casting units apart. The gel solutions were cast into the U-shaped cavity using a micro-pipette. The solution was cast by placing the tip of the pipette at one corner of the space (shown by the black dot in Figure 3.4) and continually squeezing. Gel solutions were cast as soon as they were prepared as they would start to solidify after approximately 5 minutes. After insertion of the gel solution, the casting unit was placed in an oven at 42 C (+/- 2C) for 45 minutes to allow the gel solution to set. The components of the casting unit could be reused, but it was essential that they were thoroughly acid washed. Imperfections on the glass surface resulted in bubbles while the gel solution was being cast. However, even with extremely clean glass plates were used, bubbles were not uncommon. Often, they could be removed by inserting more gel solution, or by tilting the glass plates so that the bubbles rise to the open end of the ______________________________________________________________________________ Centre for Water Research 32

Ben Annan Methods ______________________________________________________________________________ casting unit. When bubbles could not be removed, the casting unit was still placed in the oven, but when using the gel, the areas with bubbles were not used

Storage of Gels
After setting, the casting units disassembled. The resulting thin-films of gel tended to stick to one of the glass plates. They were removed using acid washed plastic spacers. Both the diffusive and binding gels were placed in DI water for a 24 hour hydration period. The hydration period allowed any unwanted ions in the gels to diffuse out. During this time, the water was changed 2 times. After the hydration period, the binding gels were stored in DI water, and the diffusive gels were stored in 0.1 M NaNO 3 .

Measuring Gel Thickness


The thickness of the gel depends on the thickness of the plastic spacer used in the casting unit. Three different gel thicknesses were used throughout the project. Most commonly only one plastic spacer was used for casting. However, to increase gel thickness, 2 and also 3 spacers were used. To measure the thickness of gel, a very thin slice of cut from the gel sheet, then turned on its side. The thickness was then measured using a microscope with a ruler scale in it.

3.4.2

Preparation of DGT units

Cutting the Gels and Filters


Circular discs were cut from the binding gels using a plastic bottle cap of approximately 2.5 cm diameter. The accuracy of this cutting tool was not important, as the window on the cap of the DGT unit controls the actual area of diffusion. The gel was laid flat on a clean glass plate and the bottle cap was pressed down firmly into the gel. The resulting disc was removed from the sheet using plastic tweezers. To lay the gel sheet flat, it had to be squirted with water, otherwise it would bunch up.

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Ben Annan Methods ______________________________________________________________________________ Glass fibre membrane filters were also cut to size by placing it on an unloaded DGT piston, then closing the cap over it.

Loading the DGT Unit


Using plastic tweezers, the binding gel was placed on the top of the DGT piston, and then the diffusive gel was placed on top of the binding gel. The glass fibre membrane filter was placed on top of the diffusive gel. The cap was then closed down tightly.

3.4.3

Using the DGT units

Deployment of DGT units


Loaded DGT units were placed in solutions for known amounts of time. They were then taken out and disassembled. To remove the cap from the DGT unit, and flat-head screwdriver was placed in the slot on the cap, the unit was held firmly by the piston, and the screwdriver rotated slowly. This eased the cap off without disrupting the gels. The filter and the diffusive gel were then removed with tweezers. After rinsing the tweezers several times with DI water, they were used to remove the binding gel.

Elution
In order to measure the amount of phosphate obtained by the binding gel, the phosphate had to be released from the gel. This was achieved by eluting each gel in 10 mL of 0.25 M sulfuric acid (H2 SO4 ) for 2 hours. Sometimes, a longer elution time was used, but Zhang et al. (1998) found that any time longer than 1 hour is sufficient. Before the binding gels were placed into the acid, they were rinsed with DI water to eliminate the chance of contamination from droplets of deployment solution that remain on the gels surface. The gels were removed from the eluent before it was analysed for P concentration using the methods described above. The mass of P accumulated by the gel was then calculated by multiplying the concentration by the volume of the eluent (equation [7]). ______________________________________________________________________________ Centre for Water Research 34

Ben Annan Methods ______________________________________________________________________________

Mass of P (ug) = Concentrat ion of eluent (ppb) x Volume of eluent (L)

[7]

Effect of Acid Concentration on the Malachite Green Method


As discussed above, the determination of phosphorus concentrations was performed using the spectrophotometric method of malachite green. The malachite green method has been shown to be sensitive to changes in acidity, when final acid concentrations are higher than 0.5 M (Linge and Oldham 2001). The final acidity of the eluent treated with the malachite green reagent was less than this upper value. However, tests were still performed to confirm there would be no effect. Samples of water and 0.25 ml H2 SO4 were treated with the malachite green methods. No significant differences in absorbencies of the samples were measured, therefore confirming that the acid did not affect the colour development.

3.4.4

Testing the DGT technique

Testing the Binding Gel


Reproducibility
In order to test the variations in accumulated mass of P, replicate binding gels were placed in solutions of known concentration and left for equal amounts of time.

Time Loading
Experiments were performed on the binding gels to test the accumulation of P over time. Known masses of binding gels were placed in solutions of known P concentration, and taken out after known amounts of time.

General Loading

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Ben Annan Methods ______________________________________________________________________________ Experiments were performed to determine the variations of binding gel response in solutions of various concentrations and for different deployment times.

Testing the DGT Unit


The above experiments were useful in testing how the binding gel behaves in phosphorus solutions. However, it is important to also test the response of the complete DGT unit. In order to do this, loaded DGT units were placed in solutions of known phosphorus concentrations for varying amounts of time (Figure 3.5).

Figure 3.5: DGT units deployed in known P concentrations

3.4.5

DGT Measurement in Sediment Slurries

To test the amount of phosphorus in the consolidated sediment collected from Lake Yangebup, DGT units were deployed in stirred sediment slurries. The sediment slurry was prepared by placing 20 g of fresh, wet sediment in a 1000 mL beaker containing 750 mL of DI water. The slurry was stirred using a magnetic stirrer of 3 cm length. The DGT units could not sit on the bottom of the beaker, as they would interrupt the stirring. Instead they were suspended in the sediment slurry (Figure 3.6).

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Ben Annan Methods ______________________________________________________________________________

Figure 3.6: DGT units deployed in sediment slurry container (before addition of water). Units are suspended in the beaker by fishing wire attached to the outside of the beaker.

The DGT unit was suspended approximately half way up the beaker (near the 550 mL mark) with fishing wire. Four DGT units could fit comfortably in one sediment slurry using this method (Figure 3.6).

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Ben Annan Results ______________________________________________________________________________

4 Results
4.1 Chemical Extraction
As discussed in the Methods section, the malachite green spectrophotometric method has an upper limit to reliable concentration determination. Therefore, it is important that the solutions being analysed are within the range of the calibration solutions, i.e. between 10 ppb and 100 ppb. After addition of the malachite green reagents, the chemical extraction solutions were visually compared with the calibration solutions (Figure 4.1). All the extraction solutions had darker than the 100 ppb calibration solution, indicating they were greater than 100 ppb in concentration. Therefore, extraction solutions were diluted to ensure they were within the measurable range (Table 4.1).

Figure 4.1: Samples treated with malachite green colouring reagents. It is possible to visually determine if the colour development of analyte solutions is in the range of the calibration solutions (the bottom row).

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Ben Annan Results ______________________________________________________________________________ Table 4.1: Dilutions required in order to analyse extraction solutions using the malachite green method Extraction Solution MgCl2 HCl NaOH NH2 OH.HCl Dilution Required 1:10 1:250 1:250 1:250

After diluting to within the correct range, the extraction solutions were then analysed for P concentration. The mass extracted from the sediment for each extraction is shown in Table 4.2. These concentrations agreed with the fractionation results of Linge (2002). The MgCl2 extracted significantly less P than the other extractions. Table 4.2: Mass of P measured in extraction solutions. Values shown are the average, and the error is the standard deviation from quadruplicate tests. Extraction Solution MgCl2 NaOH NH2 OH.HCl HCl Mass P per mass dry sediment ( g/g) 5.40 +/- 0.23 184 +/- 28 221 +/- 29 476 +/- 40

4.2 DGT
4.2.1 Gel Preparation

It was hypothesised that there would be variations in DGT performance caused by variations in gel properties. Therefore, knowledge of the details of gel preparation was important.

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Ben Annan Results ______________________________________________________________________________

Dates of Preparation
Throughout the project, three different bulk solutions were prepared, from which many different sheets of diffusive and binding gels were made (Table 4.3 and Table 4.4). If two or more gels were prepared on the same day, it implies that they were prepared from the same gel solution

Table 4.3: Diffusive gel preparation dates. Gel 4 and 5 were prepared with double and triple layers of plastic spacers respectively. Diffusive gel sheet # 1 2 3 4* 5 ** 6 7 8 9 10 1 2 2 2 2 2 2 3 3 3 9/7/02 16/9/02 16/9/02 3/10/02 3/10/02 3/10/02 3/10/02 9/10/02 9/10/02 9/10/02 Bulk solution used Date prepared

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Ben Annan Results ______________________________________________________________________________ Table 4.4: Binding gel preparation dates Binding gel sheet # 1 2 3 4 5 6 7 8 9 1 1 2 2 2 2 3 3 3 10/7/02 10/7/02 16/9/02 30/9/02 30/9/02 30/9/02 9/10/02 9/10/02 9/10/02 Bulk solution used Date prepared

Variation in Gel Preparation


Diffusive Gels
The preparation of the diffusive gel was a relatively simple procedure when compared to the binding gels. Therefore, there was little room for differences in gels to occur during the preparation phase. However, when preparing gels 4 and 5, extra plastic spacers were used to increase the gel thickness. Diffusive gel 4 used two plastic spacer, and diffusive gel 5 used three plastic spacers.

Binding Gels
The preparation of binding gels is complicated by the addition of a ferrihydrite. As discussed in the Methods, two different methods of extracting the ferrihydrite from its slurry were used. Binding gels 1, 2 and 3 were prepared using the first method of ferrihydrite extraction where the beaker holding the slurry was tilted, so that the overlying water shifted to one side, the ferrihydrite was then extracted from the opposite side of the beaker. Binding gels 4 to 9 were prepared using the second method which gave more homogeneous gels. Before extracting the ferrihydrite, the slurry was

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Ben Annan Results ______________________________________________________________________________ vigorously shaken, ensuring homogeneity. It is likely that gels using the first method have a higher concentration of ferrihydrite.

Description of Gels
Diffusive Gels
Diffusive gels 1 to 3 and 6 to 10 appeared of identical appearance, and were 0.3 mm thick. Gels 4 and 5 had very similar appearance, but were 0.6 mm and 0.9 mm thick respectively. The thickness of the plastic spacer used was 0.1 mm; therefore the expansion factor of the diffusive gels during hydration was 3.

Binding Gels
The 9 binding gels made during this project appeared similar, with patches of brown ferrihydrite densely scattered throughout each gel sheet (Figure 4.2). However, some gels appeared to have a more dense spread of ferrihydrite than the others. Binding gel 2 had a more dense spread of ferrihydrite throughout the gel sheet than binding gel 1. Binding gel 3 had a very similar appearance to binding gel 1. The patches of ferrihydrite in gels 4, 5 and 6 were very close together, but the patches themselves were finer that those of binding gels 1, 2 and 3. Binding gels 7, 8 and 9 were of similar appearance to binding gels 1 and 3. Not only were there differences in appearance between different binding gels, but there also differences within a binding gel sheet (Figure 4.2). The edges of the sheets were much darker brown that the inside. Therefore, the edges of the sheets were avoided when cutting the circular discs. Dark brown patches also often appeared within the sheet. When possible, discs were only cut from areas showing an even spread of ferrihydrite.

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Ben Annan Results ______________________________________________________________________________

Figure 4.2: Binding gel 3, showing the cut circular discs. Darker patches of brown indicate higher densities of ferrihydrite.

4.2.2

Validation Tests

Binding Gel Variation


Reproducibility
Reproducibility tests were performed on the binding gels in order to test the variations in accumulated mass of P for gel discs cut from the same gel sheet, and between gel sheets made from the same bulk solution As they were made from the same gel solution, binding gels 4, 5 and 6 were used for the reproducibility tests. Each gel was placed in 10 mL of a 50 ppb solution for 24 hours. After this deployment time, the mass of accumulated P was measured and standardised to the mass of the binding gel (Table 4.5)

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Ben Annan Results ______________________________________________________________________________ Table 4.5: Average values of P accumulation for binding gel sheets. Replicate gel discs from each gel sheet were placed in 10 mL of 50 ppb P for 24 hours. Errors are standard deviations. Gel Average P accumulation per gel mass ( g/g) 4 (n = 3) 5 (n = 3) 6 (n = 4) All gels (n = 10) 1.52 +/- 0.09 1.58 +/- 0.09 1.52 +/- 0.21 1.54 +/- 0.14

Binding gel sheet (BGS) 5 had the highest average value of P accumulation per gram of gel of 1.52 g/g. However, BGS 6 recorded the maximum value for any gel discs, and also the minimum value. Consequently, BGS 6 showed the biggest intra-gel sheet variation, with a standard deviation of 0.21 g/g. Using all gel values, the percentage of standard deviation to average values of both accumulated mass, and accumulated mass per gel mass was 9%. Therefore, for all other tests where replicates were not used, an error of 9% was used to give an indicator of the likely variation.

Time Loading
The DGT theory assumes the mass of P accumulated by the binding gels increases linearly with time. Therefore, it was important to test the binding gels mass accumulation for different times. The first time loading experiment used 5 discs from binding gel 1, each in a separate 10 mL solution of 100 ppb. The gels were deployed for 1, 2, 4, 8 and 24 hours (Figure 4.3).

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Ben Annan Results ______________________________________________________________________________ 1.4 1.2


Mass P (ug)

1 0.8 0.6 0.4 0.2 0 0 10


Time (hr)

y = 0.2981x R2 = 0.8146

20

30

Figure 4.3: Phosphorus mass accumulation versus time for time experiment 1. A linear trendline has been added to the data up to 4 hours.

A linear response with time is seen until 4 hours. After 4 hours the accumulated mass remains steady at 1 g. The second time loading used involved six circular discs cut from binding gel 6. Each gel disc was placed in a separate 10 mL solutions of 50 ppb P. Gels were deployed for 0.5, 1, 2, 4, 8 and 24 hours (Figure 4.4).

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Ben Annan Results ______________________________________________________________________________


0.45 0.4 0.35 Mass P (ug) 0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 Time (hr) 20 25 30

y = 0.0624x R2 = 0.9578

Figure 4.4: Phosphorus mass accumulation versus time for time experiment 1. A linear trendline has been added to the data up to 4 hours. As with the first time loading experiment, a linear relationship exists only for the first four hours. The linear relationship in the second time loading experiment was greater than the first.

General Loading
Tests were performed on binding gels using various concentrations and deployment times. The role of these tests was to determine the variations different binding gels will show under different conditions. Three binding gels discs (one cut from binding gel 1 and two cut from binding gel 2) were placed in separate 10 mL solutions of 1000 ppb. The gels were deployed in the solutions for four hours and 20 minutes (Table 4.6). The gels were weighed so that P accumulation per gram of gel could be calculated.

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Ben Annan Results ______________________________________________________________________________ Table 4.6: Results from gels placed in 10 mL of a 1000 ppb P solution for 4 hours and 20 minutes. Each solution had 10 g of P available for uptake. The last column shows the percentage of this mass that was accumulated. Errors are 9% of the value, except for the last row where they are standard deviations.

Gel 6 7 8 All gels

Mass P ( g) 0.92 +/- 0.08 1.00 +/- 0.09 0.99 +/- 0.09 0.97 +/- 0.04

Mass P per gel mass ( g/ g) 3.49 +/- 0.31 3.92 +/- 0.35 4.88 +/- 0.44 4.1 +/- 0.71

% accumulated 9.24 10.00 9.93 9.72

Gels 6 and 7 were cut from binding gel sheet 2, whereas gel 8 was cut from binding gel sheet 1. The gel discs all accumulated approximately 1 g. However, when comparing the mass of P accumulated per gram of binding gel (P/gel mass (g/g)), the similarities arent as strong. Gels 6 and 7 give approximately the same P/gel mass, however, gel 8 is significantly higher. Using all gel measurement, the percentage of standard deviation to average value is 17%. This is significantly higher than the 9% value obtained in the reproducibility tests. A similar experiment was performed using left over gel pieces (cuttings), rather than discs (Table 4.7). These cuttings were too small to be used in the actual DGT units, but it was thought that experiments could still be performed on them. Six pieces of gel were cut from binding gel sheets 1 and 2. The gels were weighed then placed in a 10 mL solution of 1000 ppb P. The gels were taken out of the solution after 44 hours.

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Ben Annan Results ______________________________________________________________________________ Table 4.7: Results from gel cuttings placed in 10 mL of 1000 ppb P for 44 hours. Errors are 9% of the value. Gel 1 2 3 4 5 6 Mass P ( g) 2.11 +/- 0.19 2.47 +/- 0.27 2.97 +/- 0.22 2.89 +/- 0.26 2.68 +/- 0.24 3.16 +/- 0.28 Mass P per mass gel ( g/g) 12.0 +/- 1.08 17.6 +/- 1.58 14.0 +/- 1.26 16.5 +/- 1.48 18.9 +/- 1.70 14.1 +/- 1.27 % accumulated 21.07 24.75 29.70 28.88 26.77 31.59

Gels 1 to 3 were cut from binding gel sheet 1, and gels 4 to 6 cut from binding gel sheet 2. These cuttings have much larger P/gel mass values than the discs used in the first general loading experiment. Another experiment performed with cuttings, rather than discs, was carried out in 10 mL solutions of 1 ppm and 100 ppm using solutions of 1 ppm and 100 ppm P.

Table 4.8: Cuttings from binding gels 1 and 3 were placed in solutions of 1 and 100 ppm P. Gel & solution binding gel 1, 1 ppm binding gel 3, 1 ppm binding gel 1, 100 ppm binding gel 3, 100 ppm Mass P ( g) 9.87 +/- 0.89 4.99 +/- 0.45 9.54 +/- 0.86 9.52 +/- 0.86 Mass P per mass % accumulated gel ( g/g) 113.97 +/- 10.25 67.89 +/- 6.11 45.47 +/- 4.09 89.56 +/- 8.06 98.7 49.9 9.54 9.52

Three of the gel cuttings take up the same mass of P, only the other one takes up half of that mass. However, there are no similarities between the gels when comparing the mass of accumulation per mass of gel. Compared to the other two general loading experiments, these P/gel mass values are extremely high. Centre for Water Research 48

Ben Annan Results ______________________________________________________________________________

DGT Validation
Deployment of DGT units in solutions determines whether the units were responding according to theory. Binding gel tests showed those gels accumulated P linearly with time for four hours. Therefore, the DGT units were deployed in the solutions for no more than 4 hours. Two experiments were performed. The first DGT experiment involved four DGT units deployed in 100 mL solutions of 50 ppb P. From the accumulated mass of P (Figure 4.5) the concentration of the solution was calculated (Figure 4.6). The units were all loaded with binding gels from binding gel sheet 5. DGT units 1, 2 and 3 used diffusive gel sheet 6 and DGT 4 used diffusive gel sheet 7, which both had 0.3 mm thickness.

0.25

0.2
Mass P (ug)

0.15
y = 0.0511x R2 = 0.9081

0.1

0.05

0 0 1 2 3 4 5
Deployment time (hrs)

Figure 4.5: Mass of P accumulated in the binding gels of DGT deployment experiment 1. The masses increase linearly with time. Error bars are 9%.

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Ben Annan Results ______________________________________________________________________________


100.00 80.00 Concentration (ppb)

60.00

40.00

20.00 0.00 0 1 2 3 4 5 Deployment time (hr)

Figure 4.6: The calculated concentration for DGT units deployed in 50 ppb P for 1.2, 2, 3 and 4 hours decreased with time. The actual concentration of the deployment solution (50 ppb P) is represented by the broken line. Errors are 9%. While the mass of P accumulated by the resin gel did increase linearly with time (Figure 4.5), this experiment did not accurately calculate the solution concentrations. concentration (Figure 4.6). A second DGT deployment experiment used binding gels cut from binding gel 7, and diffusive gels all cut from sheet 8. Four DGT units were deployed in separate 90 mL solutions of 50 ppb P. Units were retrieved after 1, 2, 3 and 4 hours ( Figure 4.7 and Figure 4.8). The calculated concentrations decrease with time, therefore deviating from the actual

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Ben Annan Results ______________________________________________________________________________


0.4 0.35 0.3
Mass P (ug)

0.25 0.2 0.15 0.1 0.05 0 0 1 2 3 4 5


Deployment time (hrs)

y = 0.0861x R2 = 0.9889

Figure 4.7: Mass of P accumulated in the binding gels of DGT deployment experiment 2. The mass is increasing linearly with time. Errors are 9%.

100.0 80.0 60.0 40.0 20.0 0.0 0 1 2 3 4 5 Deployment time (hr)

Figure 4.8: Calculated concentrations with time for DGT experiment 2. The actual concentration (50 ppb P) is shown by the broken line. Errors are 9%.

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Ben Annan Results ______________________________________________________________________________ Again, the mass of P accumulated increased linearly with time. were similar over the four hours. To test whether the DGT theory really did not hold at times greater than 4 hours, DGT units were deployed in solutions for 24 hours. Two DGT units (DGT 1 and DGT 2) were deployed in separate 100 mL solutions of 100 ppb P; and two other units (DGT 3 and DGT 4) were deployed in 100 mL solutions of 200 ppb P (Table 4.9). All DGT units were loaded with binding gel discs cut from binding gel sheet 3. DGT 1 and DGT 3 used diffusive discs from diffusive gel sheet 1, and DGT 2 and 4 used diffusive gel sheet 2. After 24 hours the units were retrieved from the solutions, disassembled, and analysed for P accumulation. Table 4.9: Results from a 24 hour DGT deployment. Errors are 9%. Mass on binding gel ( g) DGT 1 DGT 2 DGT 3 DGT 4 0.88 +/- 0.08 1 +/- 0.09 1.62 +/- 0.15 1.8 +/- 0.16 16.1 +/- 1.5 18.3 +/- 1.6 29.6 +/- 2.7 32.9 +-/ 3.0 100 100 200 200 Estimated (ppb) Actual (ppb) Ratio of calculated to actual concentration (%) 16.1 +/- 1.5 18.3 +/- 1.6 14.8 +/- 1.3 16.4 +/- 1.5 While still low,

calculated concentrations were also closer to the actual concentration in solution and

accumulated concentration concentration

The results show that the method greatly under estimated the actual concentrations. The final column in the table shows the percentage of calculated concentration to the actual concentration. The average percentage was 16.4% with a standard deviation of 1.4%.

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Ben Annan Results ______________________________________________________________________________

4.2.3

DGT Sediment Slurry Deployment

Triplicate sediment slurries were prepared, and 4 DGT units were deployed in each, as described in the Methods. Approximately 5 g equivalent dry weight of wet sediment was added to make the slurry (Table 4.10). From each slurry, a unit was retrieved after 1, 2, 3 and 4 hours (Figure 4.9). Three DGT units, each of different gel thickness, were deployed in a fourth slurry. The DGT units in this slurry had gel thicknesses of 0.3, 0.6 and 0.9 mm. They were retrieved after a one hour deployment time (Figure 4.10). Table 4.10: Sediment masses in the four sediment slurries. Dry masses were calculated from the wet mass using a moisture content of 73.9%. This moisture content was the same as that measured by Linge (2002). Wet mass (g) Slurry 1 Slurry 2 Slurry 3 Slurry 4 19.5 20 20.5 21.5 Equivalent Dry Mass (g) 5.1 5.2 5.4 5.6

Behavior of the DGT in sediment slurries was as expected from theory. The mass accumulated by the DGT units in slurries 1, 2 and 3 increased linearly with deployment time (Figure 4.9), while the mass accumulated by the DGT units in slurry 4 increased linearly with the inverse of gel thickness (Figure 4.10).

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Ben Annan Results ______________________________________________________________________________


0.3

slurry 1
0.25

slurry 2 slurry 3

0.2 Mass P (ug)

0.15 0.1

0.05

0 0 1 2 3 4 5 Deployment time (hrs)

Figure 4.9: Mass of P accumulated by DGT units at different times for sediment slurries 1, 2 and 3. Errors are 9%.

0.10

0.08

mass P (ug)

0.06

0.04

y = 0.0256x R2 = 0.9889

0.02

0.00 0 0.5 1 1.5 2 2.5 3 3.5 1/gel thickness (mm-1)

Figure 4.10: Accumulated P Mass for the different gel thicknesses in sediment slurry 4. A clear linear relationship exists. Errors are 9%.

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Ben Annan Results ______________________________________________________________________________ The concentrations of P in the slurries were calculated using equation [5]. These

concentrations were converted to a mass of P in the slurry per mass of dry sediment. Time averages of the three DGT slurries show that the P masses remain constant after 1 hour (Figure 4.11).

8.00 Mass P per mass sediment (ug/g)

6.00

4.00

2.00

0.00 0 1 2 3 4 5 Deployment time (hrs)

Figure 4.11: Mass of P in the sediment slurry per mass of dry sediment. The values are the averages of slurries 1, 2 and 3. Error bars represent standard deviations between replicates. If DGT-P reaches steady state in less than one hour, then the four sediment slurries each provide measurements of the same concentration (Figure 4.12).

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Ben Annan Results ______________________________________________________________________________


8 Mass P per mass sediment (ug/g)

0 0 1 2 Slurry # 3 4 5

Figure 4.12: Average values of mass of P per mass of dry sediment for the four different slurries. Slurries 1, 2 and 3 are time-averaged values and slurry 4 is a gel thickness average value.

Averaging all 15 measurements gave a mean value of 4.21 +/- 1.19 (SD) g P /g dry sediment.

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Ben Annan Discussion ______________________________________________________________________________

5 Discussion
5.1 DGT Validation Tests
The validation tests determined variations in binding gels and loaded DGT units. This discussion will explain these variations, by linking them to stages in the preparation phase.

5.1.1

DGT Preparation

The preparation of the DGT unit has several stages where variations in gel properties may be introduced. These stages are discussed below.

Ferrihydrite Preparation
Only one ferrihydrite batch was used in this project. Therefore no variation can be attributed to ferrihydrite preparation. However, Zhang et al. (1998) have noted that ferrihydrite can turn to goethite if not prepared properly. Poor DGT performance may occur because phosphate binds more slowly to goethite than to ferrihydrite.

Extracting the slurry


The greatest source of variation in the preparation stage arises due to the extraction of ferrihydrite. As described in the Methods section, two different methods of extraction were used. The initial method created gels with more ferrihydrite, but that were less homogenous. The other method produced more homogenous gels, but with less ferrihydrite. The increased ferrihydrite in the first method was expected to result in a higher phosphorus accumulation.

Casting
Differences that exist between gel sheets made from the same gel solution can be attributed to the casting procedure. The two processes involved in the casting procedure Centre for Water Research 57

Ben Annan Discussion ______________________________________________________________________________ are extracting the binding gel solution, and casting the gel solution between the glass plates. After the ferrihydrite is added to the gel solution, it settles at the bottom of the vial. To ensure an even distribution of ferrihydrite, the vial was shaken before extracting the gel solution. However, due to the small volume being extracted, the gel solution is unlikely to contain the same amount of ferrihydrite each time. Thus, gel sheets made from the same gel solution may contain different amounts of ferrihydrite, and will have different binding properties. The actual casting process involved inserting the gel solution between the two glass plates. The volume of gel solution required to fill the U-shaped cavity of the casting unit can be estimated from the dimensions of unit. However, all casting units required more gel than estimated because gel solution leaked out of the U-shaped cavity. Regardless of how tight the clamps were, leaking still occurred. When binding gel solution leaked out of the U-shaped cavity, it appeared to be mostly clear, indicating the ferrihydrite had remained in the space. This explains the darker brown colour often seen on the edges of the binding gels (Figure 4.2 - Results). Gel solution was inserted into the casting unit until the cavity filled. Therefore,

depending on the rate at which it leaked out, different casting units may have different amounts of ferrihydrite in them. This creates more potential for differences between binding gel sheets made from the same gel solution and differences between gel discs cut from the same binding gel sheet (BGS). The way that the solutions fill the U-shaped cavity can also cause differences. As the solution is cast into one corner of the U-shaped cavity, it fills down and outwards. As it hits the bottom of the cavity, the solution starts to fill upwards. As it does this, it often left behind darker streaks of brown; representing a higher density of ferrihydrite. This created heterogeneities within a single gel sheet, therefore explaining differences between discs cut from the same sheet.

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Ben Annan Discussion ______________________________________________________________________________

Casting may also have the potential to produce gels of slightly different thicknesses. As discussed in methods, clamps are placed on the three closed edges of the casting unit. Care was taken to ensure that each casting unit had the same arrangement of clamps. However, the clamps may have different strengths, and consequently slight differences between gel sheet thicknesses may arise. The actual locations of the clamps may result in different thicknesses within a gel sheet. It is likely that the glass plates will be held closer together at the edges. Therefore, the gels in the centre of the U-shaped cavity may be slightly thicker.

5.1.2

Variation in Results

Binding Gel Tests


Reproducibility
The replicate tests performed on binding gel sheets (BGS) 4, 5 and 6 proved there exists variations in the accumulation of P between gel sheets and also within a gel sheet (Table 4.5 in Results). BGS 4, 5 and 6 were all prepared from the same gel solution. Therefore, the variations in gel performance seen in this test arise from the casting stage.

Time Loading
The time loading experiment proved variations exist between binding gel sheets made from the different methods of extracting ferrihydrite. The gels in the first time experiment were prepared using the first method of ferrihydrite extraction. These gels exhausted the P supply in the deployment solutions, indicating they still have the potential to accumulate P. The gels in the second time experiment were prepared from the second method of extraction. In contrast to the first time experiment, these gels stopped accumulating mass at approximately 0.35 g, which is less than the available 0.5 g. Therefore, the gels have reached their capacity. The differences seen between these two experiments can be attributed to the different methods for extracting ferrihydrite, as

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Ben Annan Discussion ______________________________________________________________________________ discussed above. The expectation that the first method of extracting ferrihydrite will result in higher P loading has been confirmed by this experiment. Despite their differences, the two experiments share one important result: the mass accumulated by the binding gel increases linearly with time for 4 hours. important, as the DGT theory will only work if this linear relationship exists. This is

General Loading
The variations in results from the first general loading experiment were significantly higher than the reproducibility tests. This can be attributed to the two different methods of ferrihydrite extraction. The gels used in the first general loading experiment were prepared using the first method of extraction, whereas, the reproducibility tests used gels prepared using the second method. As discussed above, it was expected that the second method would produce more homogenous gels. The larger variation in the performance of gels using extraction method one has proven this. The second and third general loading experiments used leftover cuttings rather than gel discs. The P/gel mass accumulations for the cuttings were much higher, and had greater variation than the discs. The large variations in the gel cutting results showed that they are not reliable for validation tests. These large variations can be attributed to the nature of these leftover cuttings. When cutting the gel discs, care was taken to obtain gel that visibly contained an even spread of ferrihydrite; darker patches of ferrihydrite were avoided. When there was no room left to cut out circular discs, smaller, randomly shaped gel cuttings were taken. This often meant the cuttings were taken from areas that incorporated darker patches of ferrihydrite. Therefore, the heterogeneity within the cuttings, and also between different cuttings is greater than the gel discs. The increased ferrihydrite in the cuttings can lead to a greater accumulation of P.

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Ben Annan Discussion ______________________________________________________________________________ The size of the cutting relative to the gel discs was also important. The gel discs will be more homogenous because the surface area is much larger than any of the heterogeneities within the gel sheet. homogenous than the smaller cuttings. Therefore on average, they will be more

DGT unit tests


The time loading experiments showed that the binding gels accumulate P linearly with time for four hours. This was also shown to be the case for DGT units in two DGT experiments (DGT experiments 1 and 2) carried out for 4 hours. when mass accumulation is not linear with time. While both experiment 1 and 2 showed a linear relationship between mass and time, both underestimated the actual concentration in the deployment solution. Comparing the experimental and theoretical mass curves shows that theoretical mass accumulation would be much faster (Figure 5.1). A third DGT experiment, deploying units for 24 hours, confirmed that the DGT theory breaks down

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Ben Annan Discussion ______________________________________________________________________________


0.5 0.45 0.4 0.35
Mass P (ug)

DGT experiment 1
y = 0.114x

DGT experiment 2 Theoretical Response

R =1

0.3 0.25 0.2 0.15 0.1 0.05 0 0 1 2 3 4 5


Deployment time (hrs)

y = 0.0861x R 2 = 0.9889

y = 0.0511x R = 0.9081
2

Figure 5.1: The theoretical mass and actual mass accumulations of DGT units deployed in 50 ppb. The theoretical masses were calculated from the DGT theory (equation [5] - Background), using the diffusion coefficient of Zhang et al. (1998).

Underestimates may be due to incorrect variables being used in the theory equation. (Equation [5] Background). The variables that may be incorrect are the diffusion coefficient or gel thickness. Throughout the project, a diffusion coefficient of 6.05 x 10-6 cm2 s-1 was used, as measured by Zhang et al. (1998). However, when a diffusion coefficient of 4.57 x 10 -6 cm2 s-1 is As a result, the average estimate of used, then the gradient of the theoretical mass curve becomes equal to the gradient of the measured mass in DGT experiment 2. concentrations for all deployment times was 47.2 +/- 5.53 (SD) ppb P. Despite the uncertainty of Zhang et al. (1998) diffusion coefficient, it was still used in calculations as there was insufficient data to validate the use of the new diffusion coefficient. However, the difference between using the two values in sediment experiments was smaller than the standard deviations of the replicates.

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Ben Annan Discussion ______________________________________________________________________________

Differences in the diffusive gel thickness may be another reason for the underestimation of concentrations. Throughout the project, a gel thickness of 0.3 mm was assumed for the thin gels. This was based on a measurement of the thickness of one diffusive gel. However, as discussed above there may be slight variations in gel thickness between, and even within, gel sheets.

5.2 Sediment Phosphorus Measurements


Chemical Extraction
Comparisons between the chemical extraction method from this project can be made with the fractionation scheme of Linge (2002). Some significant differences are seen between the two sets of results. However, these were expected, as Linge (2002) adopted a sequential procedure. The MgCl2 extraction (5.40 +/- 0.23 g/g) is significantly lower than the other extractions, agreeing with Linge (2002). Linges (2002) MgCl2 value was lower than measured in this project as the dissolved phase of P had been removed sequentially. The 0.1 M NaOH solution has previously been shown to extract bioavailable P. (Sharpley et al. 1991). However, the high result from this study does not agree with this, especially when compared to FRP released from sediment slurries. measured in Lake Yangebup sediment as 30 g/g by Linge (2002). thought of as a measure of bioavailable P. FRP has been FRP is widely

Therefore, the significantly higher

concentration measured by the NaOH extraction (183.88 +/- 27.64 g/g) indicates it is not a good measure of bioavailable P in Lake Yangebup. The NH2 OH.HCl extraction for both this project and Linge (2002) give similar values (221 +/- 29 g/g and 290 +/- 66 g/g respectively). This indicates that the NH2OH.HCl selectively extracts only phosphorus associated with amorphous iron oxides. Conversely, the HCl extractions were extremely different (477 +/- 40 g compared with

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Ben Annan Discussion ______________________________________________________________________________ Linges (2002) 5 +/- 1.14 g). This confirms that HCl will extract a large range of phosphorus phases.

DGT-P
The linear mass accumulation of the three time-based experiments showed that the DGT units were responding correctly. This was confirmed by the linear relationship between DGT units with different gel thicknesses. As discussed in the background, this linear relationship also indicates that the slurries are in the fully sustained case. This confirms that the slurry was well mixed. The time-based experiments showed that DGT-P had reached steady-state with the sediment slurry in less than an hour. Therefore, the four sediment slurries provide four independent measurements of the mass of DGT-P.

Comparing DGT-P to Chemical Extraction


When the DGT-P is compared to the chemical extraction results (Table 4.2), it is seen that the ion exchangeable phase closely corresponds to the DGT-P. This result may mean that the DGT technique measures the ion-exchangeable P. However, the ion exchangeable P was only released from the sediment by means of a chemical extraction. Linge (2002) found that P release in a sediment slurry was controlled by the dissolution of an amorphous Fe-P oxyhydroxide. While possible that ion exchangeable P has been released in the sediment slurry, this can only be confirmed with future work. Another explanation for the similarity between the two results is that the DGT technique may measure a form of P that is present in the same magnitude as the ion exchangeable fraction.

Comparing DGT to a Fractionation Scheme


The DGT-P measured by this study is significantly higher than the dissolved P measured by Linge (2002). This indicates that the DGT is not simply measuring porewater

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Ben Annan Discussion ______________________________________________________________________________ concentrations. This indicates that the sediment slurry has induced P to be released from the sediment; some of which is measured by the DGT technique.

Comparing DGT to measurements of FRP


Linge (2002) found that the FRP measured in a stirred slurry of Lake Yangebup sediment continued to rise until steady state was reached at a value of approximately 30 g/g after 24 hours. This is higher than the DGT-P (4.21 +/- 1.19 g/g), which reached equilibrium with the slurry in at least one hour (Figure 5.2). This shows that the DGT technique is measuring a more specific form of P than FRP. The small pore sizes in the diffusive gel may prevent the colloidal P that pass through the filter from being bound to the binding agent. Zhang and Oldham (2001) showed that typical sizes of colloidal phosphorus in wetlands of the Swan Coastal Plain range from 1 nm 0.5 m. The pore sizes of the diffusive gels are roughly 2 5 nm in radius (Zhang and Davison 1999), indicating the filtration of some colloidal P occurs. Zhang et al. (1998) found that DGT measurements of P in a eutrophic pond in the U.K. agreed closely with FRP measurements. The differences between this finding, and the findings of this work may arise because Zhang et al. (1998) analysed water concentrations, while this project measured sediment concentrations. Also, the water of Lake Yangebup is extremely coloured, indicating a high organic content. The organic content of the lake studied by Zhang et al. (1998) is unknown.

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Ben Annan Discussion ______________________________________________________________________________


24.00 Mass P per mass sediment (ug/g) 20.00 16.00 12.00 8.00

FRP

DGT-P
4.00 0.00 0 1 2 3 4 5 Deployment time (hrs)

Figure 5.2: DGT-P measured in this study, compared to FRP measurements by Linge (2002). FRP values continued to rise until 24 hours at a value of 30 g/g, while DGT-P remained constant during the four hour measurement period.

FRP has been accepted as a measure of bioavailable P (Currie and Kalff 1984). However, FRP has been shown to also measure colloidal P (Stainton 1980), which, due to the nature of colloids, is not immediately bioavailable. Therefore, there exists a strong case of evidence suggesting that the DGT technique measures the P that is most bioavailable.

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Ben Annan Conclusions ______________________________________________________________________________

6 Conclusions
This project investigated the application of the diffusive gradients in thin-films (DGT) technique to measure sediment phosphorus. Validation tests were performed before the DGT technique was used in a sediment slurry.

6.1 DGT Validation Tests


The validation tests determined there were significant differences in the binding gels ability to accumulate P. The greatest differences occurred between gel sheets made from different binding gel solutions. Smaller variations also occurred between gel sheets made from the same gel solution, and also within gel sheets. These differences have been attributed the preparation of the gels. The largest cause of variation was the addition of ferrihydrite to the gel solutions. However, the casting procedure also created gels with different properties. The extremely large variations measured for smaller gel cuttings, as opposed to the larger gel discs, indicates there is a critical size required for the gels to overcome heterogeneities.

6.2 Sediment Phosphorus Measurements


The DGT technique can be successfully applied to measure phosphorus in sediment slurries. The DGT-measurable-P (DGT-P) closely agreed with ion exchangeable P measured by chemical extraction. However, determining if the two techniques actually measure the same form of phosphorus requires further investigation. DGT-P measurements stayed constant for 4 hours, indicating that steady state had been reached. The DGT-P is less than FRP measured in previous studies of Lake Yangebup. Therefore, DGT-P may be a more accurate measurement of bioavailable P. However, this needs to be investigated further. Centre for Water Research 67

Ben Annan Future Work ______________________________________________________________________________

7 Recommendations for Future Work


This project has thoroughly investigated all aspects of the application of DGT to measure sediment phosphorus. The potential causes of variations in results have been identified, and these issues need to be addressed in future work.

7.1 DGT Validation


Binding Gels
This work has shown there are many levels of potential error associated with the binding gels. Further work needs to be carried out to address these issues. Firstly, it is recommended that several ferrihydrite slurries be made, and tested in the initial stages of any future project. The properties of binding gels prepared from the different slurries should be extensively tested. X-ray diffraction measurements should be performed on the slurry to monitor possible goethite or hematite formation. Methods should be applied to the casting procedure to ensure the creation of more homogenous gels. An investigation into a clamping system that provides uniform pressure over the casting unit is recommended. It is also recommended to monitor the volume of the gel inserted into the casting unit.

Diffusion Coefficient
It is possible that the use of the Zhang et al. (1998) diffusion coefficient for this project resulted in the variations seen in DGT performance. Therefore, it is recommended that self measurements of diffusion coefficient should be made.

7.2 Sediment Measurements


This project has provided a good basis to determine the type of phosphorus measured by DGT deployments in sediment slurries. Although, any definite conclusions require further investigation.

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Ben Annan Future Work ______________________________________________________________________________

The close agreement between the two results suggests that DGT-P may be measuring ion exchangeable P. A method for confirming this hypothesis is to deploy DGT units in the supernatant after the chemical extraction has been applied. This would require larger volumes of extraction solution than used in this project. It is recommended that DGT units be deployed in all chemical extraction solutions. This project has provided a strong case of evidence suggesting that the DGT technique measures bioavailable P. However, more work needs to be performed to confirm this hypothesis. It is recommended that DGT-P be compared to measurements of bioavailable P using algal bioassays.

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Ben Annan References ______________________________________________________________________________

References
Bell, F. 1997, An investigation of dissolved arsenic dynamics in Yangebup Lake, Honours Thesis, University of Western Australia. Bostrom, B., Jannson, M. and Forsbeg, C. 1982, Phosphorus release from lake sediments, Arch. Hydrobiol. Beih. Ergebn. Limnol., vol. 18, pp. 5 59. Boulton, J.A. and Brock, M.A. 1999, Australian Freshwater Ecology: Processes and Management, Gleneagles Publishing, Australia. Chang, S.C. and Jackson, M.L. 1956, Fractionation of soil phosphorus, Soil Science, vol. 84, pp. 133 144. Currie, D.F. and Kalff, J. 1984 The relative importance of bacterioplankton and phytoplankton in phosphorus uptake in freshwater, Limnology and Oceanography., vol. 29, pp. 311 321. Dahlqvist, R., Zhang, H., Ingri, J. and Davison, W. 2002, Performance of the diffusive gradients in thin films technique for measuring Ca and Mg in freshwater, Analytica Chimica Acta, vol. 460, pp. 247 256. Davis J.A., Rosich, R.S. Bradley, J.S., Growns, J.E., Schmidt, L.G. and Cheal, F. 1993, Wetlands of the Swan Coastal Plain: Volume 6: Wetland Classifications on the Basis of Water Quality and Invertebrate Community Data, Water Authority of WA/EPA, Western Australia. Davison, W. and Zhang, H. 1994, In situ speciation measurements of trace components in natural waters using thin-film gel, Nature, vol. 367, pp. 546 548.

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Ben Annan References ______________________________________________________________________________ Davison, W., Fones, G., Harper, M., Teasdale, P. and Zhang, H. 2000, Dialysis, DET and DGT: in situ diffusional techniques for studying water, sediments and soils, in In situ monitoring of aquatic systems chemical analysis and speciation, eds. Buffle, J. and Horvari, G., Wiley and Sons, New York, pp. 495 569. Deurer, R., Frstner, U. and Shmoll, G. 1978 Selective chemical extraction of carbonateassociated metals from recent lacustrine sediments, Geochimica et Cosmochimica Acta, vol. 42, pp. 425-427. Emsley, J. and Hall, D. 1976, The chemistry of phosphorus: environmental, organic, biochemical and spectroscopic aspects, Harper and Row, Devon. Emsley, J. 1980, The phosphorus cycle, in The handbook of organic chemistry, ed. Hutzinger, O., Springer Verlag, Berlin. Forstner, U. 1990, Inorganic sediment chemistry and elemental speciation in Sediments: chemistry and toxicity of in-place pollutants, eds. Baudo, R., Giesy, J. and Muntau, H., Lewis Publishers, Ann Arbor. Harper, M.P., Davison, W., Zhang, H. and Tych, W. 1998, Kinetics of metal exchange between solids and solutions in sediments and soils interpreted from DGT measured fluxes, Geochimica et Cosmochimica Acta, vol. 62, no. 16, pp. 2757 2770. Kaiserli, A., Vousta, D., Samara, C. 2001, Phosphorus fractionation of lake sediments Lakes Volvi and Koronia, N. Greece, Chemosphere, vol. 46, pp. 1147 1155. Kramer, J.K., Herbes, S.E. and Allen, H.E. 1972, Phosphorus: analysis of water, biomass and sediment, in Nutrients in natural waters, eds. Allen, H.E. and Kramer, J.K., John Wiley and Sons, New York, pp. 51 100.

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Williams, J.D.H., Syers, J.K., Harris, R.F. and Armstrong, D.E. 1971, Fractionation of inorganic phosphate in calcareous lake sediments, Soil Science Society of America Journal, vol. 35, pp. 551 561. Windsor Scientific Limited - Diffusive Gradient Films. (5th August 2002), Windsor Scientific Limited, [online], available from http://www.windsort-ltd.co.uk/dgt.htm [21st April 2002]. Zhang, A. and Oldham, C. 2001, The use of an ultrafiltration technique for measurement of orthophosphate in shallow wetlands, the Science of the Total Environment, vol. 266, pp. 159 167. Zhang, H. and Davison, W. 1995, Performance characteristics of diffusion gradients in thin films for the in situ measurement of trace metals in aqueous solution, Analytica; Chemistry, vol. 67, pp. 3391 3400. Zhang, H., Davison, W., Miller, S. and Tych, W. 1995, In situ high resolution measurements of fluxes of Ni, Cu, Fe and Mn and concentrations of Zn and Cd in porewaters by DGT, Geochemica et Cosmochimica Acta, vol. 59, no. 20, pp. 4181 4192. Zhang, H., Davison, W., Gadi, R. and Kobayashi, T. 1998, In situ measurement of dissolved phosphorus in natural waters using DGT, Analytica Chimica Acta, vol. 370, pp. 29 38.

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