Tools for Characterization and Measurements of Bioavailability

Because bioavailability processes are embedded in ecosystem health risk frameworks, the development of tools that quantitate bioavailability is important. Environmental assessment tools capable of spatially and temporally resolving contaminant variation are important for assessment of exposure frequencies and levels, episodic spills, and natural and anthropogenic remediation. Conventional methods for ecological exposure assessment involve measuring contaminant concentrations in the ambient environment and extrapolating to toxicological endpoints as well as measuring the concentration of parent compounds or their metabolites in biological samples. However, measures of total ambient contaminant concentrations represent only a rough estimate of exposure and do not reflect the bioavailable fraction. Biomonitoring provides at best only a transient estimate of exposure. Conventional 'snapshot' techniques are cost intensive, lack time-integrated information, and are not effective long-term solutions.

Regulatory agencies typically rely on analytical methods that entail vigorous extraction of matrices with organic solvents for organic contaminants such as PCBs and PAHs, and the use of strong acids for metals. The relevance of such methods to toxicity is often not considered; thus, decisions are based on data that are often irrelevant for prediction of potential exposures and risk. Current analytical methods that measure analytically recoverable concentrations include 'biologically unavailable' fractions, possibly overestimating the magnitude of environmental risk from these pollutants. The total contaminant concentration, 'analytically recoverable', is the amount quantified after vigorous extraction, and includes particle-bound contaminants that are generally not available for uptake. The evidence is compelling that the quantities recovered by vigorous extraction/digestion fail to predict bioavailability of the compounds. Regulatory agencies have recognized the importance of determining bioavailable versus total contaminant concentration. Some regulatory agencies have allowed certain regions to develop site-specific criteria based on bioavailable levels of priority pollutants; however, they report the limited availability of real world bioavailable data.

More recently, the scientific and risk-management communities have concluded that, when available, research tools to determine contaminant bioavailability should be used or considered. In particular, bioavailable approaches and tools that involve mechanistic approaches are most useful. Bioavailable tools may be broadly divided into physical, chemical, and biological tools. Hundreds of bioavailable tools and models have been developed. A few examples within each group are described briefly.

Physical-chemical characterization of solid phases has been used to generally characterize bioavailability. This entails measuring such properties as organic carbon, particle size, and cation exchange capacity to name a few. More specific types of matrix characterization tools include the use of nuclear magnetic resonance (NMR) to characterize soil sorption quality and capacity and have been related to bioavailability. XRD, or X-ray diffraction, has been used to characterize the crystalline structure of solids, and when coupled with scanning electron microscopy (SEM) to identify morphology of soils these characterizations may then be related to bioavailability. Other types of physical-chemical characterization tools include infrared (IR) absorbance, petrography, and elemental analysis. Operationally defined extractions of environmental matrices including traditional conventional extractions, discussed in Figure 2, rarely relate to bioavailability. Other sequential extractions have found limited usefulness.

The use of solid-phase and membrane-based in situ approaches has been a rapidly developing field for new bioavailable analytical approaches. Passive sampling devices (PSDs) are finding widespread use to assess organism exposure to bioavailable contaminant fractions in soils, sediments, water, and air. The tools essentially sequester unbound contaminants to a solid phase on a membrane. Advantages of the PSD technique are the ability to distinguish between free and bound contaminants. The free, unbound fraction of the contaminants is often related to mobility, bioavailability, and toxicity. Also, passive integrative samplers act as infinite sinks for accumulated residues as no significant losses of sequestered residues occur even when ambient chemical concentrations fall during part of an exposure. There are many different types of PSDs, successfully shown to sequester metals, others for nonpolar organic compounds, and still others for semipolar contaminants.

PSDs are thought to mimic key mechanisms of bioconcentration including diffusion through biomembranes and partitioning between organism lipid and the surrounding medium. One group of organic PSDs, for example, the semipermeable membrane device, consists of a polyethylene tube. The polyethylene tube is normally thought of as nonporous. However, random thermal motions of the polymer chains form transient cavities with maximum diameters of approximately 10 A. The diameters are very similar in size to cell membranes pores estimated at about 9.5 A. Because these cavities are extremely small and dynamic, hydrophobic solutes are essentially solubilized by the polymer, as illustrated in Figure 5. The cross-sectional diameters of nearly all environmental contaminants are only slightly smaller than the polymeric cavities. Therefore, only dissolved, bioavailable, labile organic contaminants diffuse through the membrane and are concentrated over time.

PSDs are deployed in the environment, removed after a period of time, and the organic, nonpolar and semipolar, bioavailable contaminants are easily extracted. The extracts may be quantitated by standard chromatographic methods or used in bioassays.

Another PSD technique developed for sequestration of bioavailable metals, the diffusive gradient thin films (DGTs), has the unique advantage of quantitating unbound metals in situ. The DGT PSD employs a layer of Chelex resin impregnated in a hydrogel to bind the metals. A diffusive layer ofhydrogel and filter overlies the

Passive sampling device

Filter Sorption Model Cartoon

sampling membrane

Figure 5 Example of a bioavailable in situ analytical tool. Depicted is a cartoon of a polyethylene membrane PSD. Illustrated are several features of PSDs, including lipophilic character and pore size. All of these features affect whether a contaminant will progress into the polyethylene membrane. Contaminants are idealized as the spheres in the bulk solution; some spheres are bioavailable shown as crossing into the polyethylene, whereas others, illustrated by the larger spheres, are not able to cross into the PSD.

sampling membrane

Figure 5 Example of a bioavailable in situ analytical tool. Depicted is a cartoon of a polyethylene membrane PSD. Illustrated are several features of PSDs, including lipophilic character and pore size. All of these features affect whether a contaminant will progress into the polyethylene membrane. Contaminants are idealized as the spheres in the bulk solution; some spheres are bioavailable shown as crossing into the polyethylene, whereas others, illustrated by the larger spheres, are not able to cross into the PSD.

resin layer. Free and bound metal have to diffuse through the filter and diffusive layer to reach the resin layer where only the ion will be irreversibly bound to the chelex. The DGT device is deployed for a known time and then the mass of metal on the resin layer is measured after elution with acid. An attribute of this tool is the ability to distinguish between bound and free metals, an important mechanistic character often related to bioavailability. Figure 6 illustrates the deployment and extraction procedure for this tool.

Another attribute in general with the PSD technique is its ability to be deployed over a specified time period. The time-integrated nature of PSD addresses some of the temporal variability in an ecosystem that conventional 'snapshoot' sampling cannot address. In situ methods also avoid environmental equilibrium problems, generated when removing samples from a dynamic system; the emphasis on in situ types of techniques is likely to increase. Other types of PSDs include tenax, XAD, and C-18. PSDs have found some success in predicting bioavailability. However, correlation studies comparing contaminant uptake into various types of PSDs and organisms are still limited. Validation of PSD with biotic endpoints is still necessary.

Normalization techniques have been employed to predict bioavailable fractions. An example of a technique often used to estimate partitioning of metals in sediments is based on normalizing their concentration in sediment to acid-volatile sulfides. Acid-volatile sulfides are an operationally defined concentration of sulfides present in sediments. The acid-volatile sulfide normalized metal concentrations are hypothesized to more closely relate to bioavailability in sediment pore water than the total measured metal concentration in sediment. Another example of a normalization technique often used to estimate partitioning of organic contaminants in sediments is based on the biota-soil/sediment accumulation factor (BSAF). The BSAF is an empirically defined ratio calculated from the chemical concentration measured in tissue relative to the chemical concentration measured in soil or sediment. However, like many empirically defined techniques, the BSAF values are dependent on the physical-chemical properties of both the contaminant and the soil or sediment, as well as the lipid nature of the organism. Therefore, the BSAF is site and species specific. Both these normalization approaches have a great deal of uncertainty and at best only provide an indication of bioavailability of contaminants.

Another bioavailable approach is the use of equilibrium partitioning theory (EPT), also called the pore water hypothesis. The theory assumes a thermodynamic equilibrium distribution of contaminants between soil particles, soil water, and organisms. Contaminant concentrations in soil pore water are calculated using the soil-water partitioning coefficients, Kd. The values are related to biological

Diffusive gradient thin film (DGT) processing

Exterior clean-up

Sealed transport

Chelex removal

Chelex extraction

HNO3

Spectroscopy analysis

Figure 6 An example of an in situ bioavailable analytical tool for determining bioavailable metals in soils and sediments. DGTs are deployed, the devices are removed after a designated period of time, and the metals are easily extracted by acid dissolution. The extracts may be quantitated by standard spectroscopy techniques or used in bioassays.

Spectroscopy analysis

Bioassay analysis

Figure 6 An example of an in situ bioavailable analytical tool for determining bioavailable metals in soils and sediments. DGTs are deployed, the devices are removed after a designated period of time, and the metals are easily extracted by acid dissolution. The extracts may be quantitated by standard spectroscopy techniques or used in bioassays.

effects observed with bioassays and compared with the measured contaminant concentration. Bioconcentration factors (BCFs) are used to determine the uptake of organic contaminants from pore water and compared with the values directly measured in the organisms. The important parameter needed for this type of analysis is the organic carbon partition coefficient, Koc. Both Koc and BCF are dependent on Kow, which as stated earlier does not always accurately represent bioavailability properly. A further refinement of the EPT approach is the 'biotic ligand model' (BLM). The BLM is a model that incorporates metal physical-chemical characteristics into a distribution scenario and relies on site-specific water chemistry information, such as pH and concentrations of ions and inorganic and organic ligands. This model has been used with some success for metal speciation distribution in waters and is currently under development for soil environments.

Biological approaches to measuring bioavailability include bioassays, assimilation and elimination efficiencies, and biomarkers. Bioassays may be at the cellular or whole-organism level. Examples of whole-organism bioassays may include plants, invertebrates, and fish; depending on the contaminant and study goals, various toxic or fitness endpoints may be used. Biomarkers are another approach used to evaluate bioavailability. The biomarker may be a metabolite of the parent toxicant, for example, found in the blood or urine of the organism that represents a biological response to a contaminant exposure.

Many bioavailability tools have been developed that differ in their definition and application. An understanding of the dynamic processes that make up bioavailability, and definitions for contaminant-, site-specific conditions, is necessary before selecting a tool that best describes the relevant risk endpoint. Until bioavailability tools have been validated relative to both biological and site-specific considerations, it may be necessary to select a range of tools to provide 'multiple lines of evidence' about bio-availability processes for a site assessment.

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