2. Literature Review
2.6 Methods to determine Bioavailability in a system
2.6.1 Analytical Approach
Most analytical approaches focus on the determination of the free metal ion concentration or the total metal concentration. These have been well developed due to their application in assessing
29 toxicity of heavy metals in natural waters. The following is a list of analytical techniques that are available (Nordstom, 1996; Hanrahan, 2010; Peijnenburg and Jager, 2003):
Ion selective electrodes
Voltammetric techniques (Differential-pulse polarography (DPP), Differential-pulse anodic stripping voltammetry (DPASV))
Atomic Spectroscopy (Graphite furnace atomic absorption (GFAA), Inductively coupled plasma mass spectroscopy (ICP-MS), Atomic emission spectrometry (AES))
X-ray spectroscopy
Neutron activation analysis
Ion exchange equilibrium (Exchange resins, Diffusive gradients in thin films (DGT), Donnan Dialysis membranes)
Solvent Extraction
UV visible spectrophotometry
Vapour and Hydride generation
Capillary electrophoresis (CE)
All of the above techniques are used for determining the free metal ion concentration or metal complexes in the dissolved phase. Ion selective electrodes are the most direct way of determining free metal concentrations. With the Cu electrode, solution activities up to 10-12 M are detectable but measurements using other electrodes are unreliable at low concentrations due to reduced sensitivity and interferences (Sauve et al., 1995). Voltammetric as well as ion exchange techniques may be used to determine organic and inorganic metal complexes. Care should be taken when employing voltammetric techniques to analyze samples containing dissolved organic matter (DOM) as the DOM may diminish current readings (Peijnenburg and Jager, 2003).
UV visible spectrophotometry and vapour and hydride generation are suitable for determining redox species (Nordstrom, 1996). Capillary electrophoresis (CE) is a method that allows for the direct measure of chemical species of metals. However, it is a new method and further development of this technique is required (Hanrahan, 2010).
Direct determination of speciation in a solid sample is restricted to a few major components since sensitivity is insufficient for trace metals (Filgeuras et al., 2002). Thus for determination of other chemical forms such as adsorbed and precipitated metal forms, combinations of techniques are required. Sequential solvent extraction is one such technique where solvents of increasing strength are sequentially applied to a sample. The liquid extract from each solvent is then analysed using
30 one of the techniques described above. Extractions work on the principle that the solvent used as an extractant solubilises the metals in a particular form (adsorbed or precipitated) and consequently it is released into the solution (Peijenenburg and Jager, 2003). As the strength of the reagent increases, the form of metal extracted is less bioavailable. There are a number of sequential extraction techniques in literature with the methods proposed by Stover et al., 1976 and Tessier et al., 1979 being the most popular.
The Stover et al., 1976 scheme fractionates metals into six phases whereas a modified Tessier extraction scheme fractionates metals into four phases (van Hullebusch et al., 2005). All the phases are not identical and different extractants are used to release metals from similar phases. The table that follows gives the outline for these two schemes including the phases the metals are fractionated into, the extractant used for that particular phase and the conditions for the extraction.
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Table 7: Outline of Stover and Modified Tessier extraction schemes
Metal Phase Extractant Extracting Conditions
Stover: Exchangeable 1 M KNO3 20°C, shake for 16 hours
Adsorbed 0.5 M KF 20°C, shake for 16 hours
Organically Bound 0.1 M Na4P2O7 20°C, shake for 16 hours Bound to Carbonates 0.1 M EDTA 20°C, 2X 8 hour shake Bound to Sulphides 1 M HNO3 20°C, shake for 16 hours
Residual Strong Acids Microwave/plate
digestion Modified
Tessier:
Exchangeable 1 M
NH4CH3COO
20°C, shake for 1 hour
Bound to Carbonates 1 M CH3COOH 20°C, shake for 1 hour Bound to Organics and
Sulphides
30% H2O2 35°C, shake for 3 hours
Residual Strong Acids Microwave/plate
digestion
The exchangeable phase is the most bioavailable as it is the first phase to be extracted. Species in this phase include weakly adsorbed metals onto solid surfaces via weak electrostatic forces, metals that are released by ion exchange processes (Filgeuras et al., 2002). Salt solutions are used as reagents since they are a source of cations. These cations displace exchangeable metals from the sludge inorganic and organic sites (Stover et al., 1976). Alkali metals cause interferences during atomic spectroscopy. Using the ammonium salt of the modified Tessier scheme over the potassium nitrate in the Stover scheme is therefore advantageous (Filgeuras et al., 2002). However, the beneficial use of nitrate salts over acetate salts is that no metal complexing occurs from the nitrate ions and therefore metals are removed via cation exchange only (Filgeuras et al., 2002).
For the adsorbed phase in the Stover scheme, the pH and concentration of KF facilitates the extraction of soluble metal fluoride complexes (Stover et al., 1976). There is no reagent for the extraction of adsorbed phase in the modified Tessier scheme.
32 The organically bound metals include complexation and bioaccumulation processes with organic matter (Coetzee, 1993). Organic matter shows a greater affinity for divalent ions than monovalent ions (Filgueras et al., 2002). Metals in this phase are extracted by decomposition of the organic matter through oxidation. Some oxidising reagents such as hydrogen peroxide also release metals in sulphide precipitates, hence these are grouped together. Na4P2O7 and EDTA are both able to remove organically bound metals due to their oxidizing and chelating abilities respectively. It is for this reason that the organically bound extraction precedes the carbonate extraction in the Stover scheme. This is an important fraction in sewage sludge and may dominate trace metal distribution (Stover et al., 1976).
Metals precipitated or co-precipitated with carbonates are inclined to dissolve upon reduction of the pH. Thus, mild acids such as EDTA and CH3COOH are used (Filgueras et al., 2002). A stronger acid (HNO3) is used to extract metals in sulphide precipitates following the carbonate extraction.
The residual fractions are mostly extracted in the manner of determining total metal concentrations. This is through acid digestion of the remaining solid sample using a microwave or hot plate digestion technique according to those set out in Standard Methods (APHA, 1996).
Sequential extraction experiments performed on different sludge samples indicate although different samples will have different distributions, the organically bound, carbonate and sulphide precipitate and residual phases are significant with the organically bound and precipitate phases being more significant that the residual phase (Stover et al., 1976, Filgueras et al., 2002, van Hullebusch et al., 2005, Aquino and Stuckey, 2007). These observations have been found for Fe, Cu, Zn, Mn, Ni and Co.
There is no uniformity between different extraction techniques as they use different solvents that extract metals from phases which are not always comparable. This makes direct comparisons difficult as the metal phases are defined more as a result of the operation rather than from a universal understanding of what metal species contribute to a particular phase (Peijenenburg and Jager, 2003). Different extraction techniques performed on the same sludge sample also produce different distributions between the phases (van Hullebusch et al., 2005). Other problems associated with sequential extractions are the inability of the reagents to selectively extract metals in a particular phase and the influence of operating conditions (pH, temperature, particle size etc.) (Filgeuras et al., 2002).
Analytical techniques are limiting in that they are not able to fully identify species of metals in all the phases. There are also sensitivity and detection problems at trace concentrations as well as
33 interferences of certain metals with each other or with the equipment when analyzing. Another concern is the disruption of equilibrium when sampling or analyzing occurs (Nordstrom, 1996;
Filgueras et al., 2002). Thus to determine chemical speciation, analytical methods should not be used in isolation but should rather be coupled with chemical speciation modelling.