Czc

conventional bioassay with solvent extracts

Other Passive Sampling Devices

solvent free bioassay "bead assay"

Fig. 18.1. Schematic representation of the two approaches currently available to link passive sampling and toxicological analysis. Any sampler can initially be applied as long as solvent extracts can be derived, which are subsequently applied in bioassays (left-hand side). The beads used in the toximeter are chosen such that they can directly be used in the specifically developed solventfree bead assay (right-hand side) so that a chemical extract is not required for bioassay analysis.

solvent free bioassay "bead assay"

Fig. 18.1. Schematic representation of the two approaches currently available to link passive sampling and toxicological analysis. Any sampler can initially be applied as long as solvent extracts can be derived, which are subsequently applied in bioassays (left-hand side). The beads used in the toximeter are chosen such that they can directly be used in the specifically developed solventfree bead assay (right-hand side) so that a chemical extract is not required for bioassay analysis.

is based on adherence-dependent vertebrate cells to detect a toxicolog-ical response (see Section 18.2.1). The second approach is to employ a passive sampler commonly used for monitoring groundwater contaminants based on chemical analysis alone, prepare an extract of sampled contaminants and apply this extract in a toxicity test (see Section 18.2.2). The advantage of the first approach is that chemical contaminants present in the groundwater can be explored without the need for solvent extraction and the use of organic solvents in toxicity tests. The advantage of the second approach is that any passive sampler suitable for groundwater can be applied as long as an extract of the sampled chemicals can be derived. Inasmuch as passive sampling devices generally report on the fraction of chemical contaminants that is freely dissolved, both approaches have the advantage that sampled contaminants reflect the bioavailable fraction present in the groundwater. In many cases, this fraction may only be detectable by time-integrating (i.e. non-equilibrium) passive sampling devices because it is too small to be detectable both chemically as well as toxicologically by equilibrium or snapshot sampling.

18.2.1 The toximeter

The toximeter is a recently developed passive sampling device. It is the first passive sampler to allow direct bioassay analysis of accumulated chemicals. It is also possible, by means of simple solvent extraction, to carry out a chemical analysis and link the information obtained on concentrations of chemicals present to the results of toxicity tests [8]. The underlying principle of the toximeter is that sorbents used for sampling can be applied directly in toxicity tests [9]. Although, technically, the design of the toximeter pertains to surface or pore-water sampling, its first development and application were focused on groundwater.

The toximeter is a solid sorbent sampler, which uses loose beads as receiving phase. It builds on the ceramic dosimeter developed by Grathwohl [10] (see also Chapter 12). The toximeter uses the same ceramic tube design, which is 5 cm long and 1 cm in diameter and serves both as a container for the solid sorbent material and as the diffusion barrier. Based on the thickness of the membrane and the small inner pore size (pore size 5 nm), the ceramic tube comprises a robust barrier that limits uptake into the inner part to diffusion alone. The small pore size also prevents microorganisms from entering the sampling device. The key difference from the ceramic dosimeter is the choice of bead material: in addition to a high affinity to target analytes, it is non-toxic by itself and supports the viability and responsiveness of toxicity reporting entities. In the current toximeter design, adherence-dependent vertebrate cell lines are used as versatile reporters of toxicity. Toxico-logical analysis of toximeter samples is performed in a specifically developed solvent-free, solid-phase bioassay, called the bead assay [11]. The bead assay is performed by first inserting contaminant-loaded sorbent beads (i.e. beads from the toximeter after sampling) to multi-well plates. Subsequently, vertebrate cells are added to the plates in which the beads act as a surface for the cells to attach and grow. Due to the direct contact of the vertebrate cells with the contaminant-loaded sorbent beads, contaminants are able to enter the cells (Fig. 18.2). We

Fig. 18.2. Principle of the bead assay to assess the toxicity of toximeter-derived samples. Sorbent beads from the toximeter passive sampler are transferred to multi-well plates and used as cell culture surface after field exposure. Adherence-dependent vertebrate cells, used as biological indicators, are added to the sorbent-containing wells. Cells attach onto the contaminated surface (magnified insert) and by the direct contact are able to easily take up the sorbed contaminants. After incubation of cells for a pre-determined time, toxi-cological effects in the cells can be assessed.

have shown that indeed, the direct contact of the cells to the contaminant-coated bead surface greatly facilitates the cellular transfer of contaminants and a biological response [11].

The bead assay as a pre-requisite for the toximeter was developed using permanent cell lines from rainbow trout (Oncorhynchus mykiss) liver (RTL-W1 and R1) [11]. PAHs were used as model compounds. Biological effects elicited by the PAHs were detected based on cell viability assays and the induction of cytochrome CYP1A, an aryl hydrocarbon receptor mediated (dioxin-like) response, measured as 7-ethoxyresorufin-O-deethylase (EROD) activity [12]. The sorbent bead material found to be most suitable for the application in the toximeter for PAH sampling as well as for use in the bead assay was Biosilon (Nunc). Biosilon consists of beads of 160-300 mm diameter made of polystyrene. Among 10 tested materials with a known or suspected high affinity for PAHs, it was the one that best enabled cell attachment and the detection of dose-response curves elicited by sorbed PAHs in adhering fish liver cells [11]. Figure 18.3 illustrates the appearance of cells upon attachment onto Biosilon beads. An example of a dose-response curve for EROD induction elicited in RTL-W1 cells by benzo[k]fluoranthene sorbed to Biosilon is shown in Fig. 18.4.

Fig. 18.2. Principle of the bead assay to assess the toxicity of toximeter-derived samples. Sorbent beads from the toximeter passive sampler are transferred to multi-well plates and used as cell culture surface after field exposure. Adherence-dependent vertebrate cells, used as biological indicators, are added to the sorbent-containing wells. Cells attach onto the contaminated surface (magnified insert) and by the direct contact are able to easily take up the sorbed contaminants. After incubation of cells for a pre-determined time, toxi-cological effects in the cells can be assessed.

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