## 6.4.2. Effect Directed Analysis

Author: Marja Lamoree

Reviewers: Timo Hamers, Jana Weiss

Learning goals:

You should be able to

- explain the complementary nature of the analytical/chemical and biological/toxicological techniques used in Effect-Directed Analysis
- explain the purpose of Effect-Directed Analysis
- describe the steps in the Effect-Directed Analysis process
- describe when the application of Effect-Directed Analysis is most useful

Keywords: extraction, bioassay testing, fractionation, identification, confirmation

In general, the quality of the environment may be monitored by two complementary approaches: i) quantitative chemical analysis of selected (priority) pollutants and ii) effect-based monitoring using in vitro/vivo bioassays. Compared to the more classical chemical analytical approach that has been used for decades, effect-based monitoring is currently applied in an explorative manner and has not yet matured into a routinely implemented monitoring tool that is anchored in legislation. However, in an international framework, developments to formalize the role of effect-based monitoring and to standardize the use of bioassay testing for environmental quality assessment are underway.

A weakness of the chemical approach is that because of the preselection of target compounds for quantitative analysis other compounds that are of relevance for the environmental quality may be missed. In comparison, inclusiveness is one of the advantages of effect-based monitoring: all compounds – and not only a few pre-defined ones – having a specific effect will contribute to the total, measured biological activity (see Section In vitro bioassays). In turn, the effect-based approach strongly benefits from chemical analytical support to pinpoint which compounds are responsible for the observed activity and to be able to take measures for environmental protection, e.g. the reduction of the emission or discharge of a specific toxic compound into the environment.

In Effect-Directed Analysis (EDA), the strengths of analytical chemical techniques and effect-based testing are combined with the aim to identify novel compounds that show activity in a biological analysis and that would have gone unnoticed using the chemical and the effect-based approach separately. A schematic representation of EDA is shown in Figure 1 and the various steps are described below in more detail. There is no limitation regarding the sample matrix: EDA has been applied to e.g. water, soil/sediment and biota samples. It is used for in-depth investigations at locations that are suspected to be contaminated but where the compounds responsible for the observed adverse effects are not known. In addition to environmental quality assessment, EDA is applied in the fields of food security analysis and drug discovery. In Table 1 examples of EDA studies are given.

Figure 1. Schematic representation of Effect-Directed Analysis (EDA).

1. Extract

The first step is the preparation of an extract of the sample. For soil/sediment samples, a sieving step prior to the actual extraction may be necessary in order to remove large particles and obtain a sample that is well-defined in terms of particle size (e.g. <200 μm). Examples of biota samples are whole organism homogenates or parts of the organism, such as blood and liver. For the extraction of the samples, analytical techniques such as liquid/liquid or solid phase extraction are applied to concentrate the compounds of interest and to remove matrix constituents that may interfere with the later steps of the EDA.

2. Biological analysis

The choice of endpoint to include in an EDA study is very important, as it dictates the nature of the toxicity of the compounds that may be identified (see Section on Toxicodynamics and Molecular Interaction). For application in EDA, typically in vitro bioassays that are carried out in multiwell (≥ 96 well) plates can be used, because of their low cost, high throughput and ease of use (see Section on In vitro bioassays), although sometimes in vivo assays (see Section on In vivo bioassays) are applied too.

Table 1. Examples of EDA studies, including endpoint, type of bioassay, sample matrix and compounds identified.

Endpoint | Type of bioassay | Sample matrix | Type of compounds identified
| In vitro |  |
Estrogenicity | Cell based reporter gene | Sediment | Endogenic hormones
Anti-androgenicity | Cell based reporter gene | Sediment | Plasticizers, organophosphorus flame retardants, synthetic fragrances
| idem | Water | Pharmaceuticals, pesticides, plasticizers, flame retardants, UV filters
Mutagenicity | Bacterial luminescence reporter strain | Water | Benzotriazoles
Thyroid hormone disruption | Radioligand binding | Polar bear plasma | Metabolites of PCBs, nonylphenols
| In vivo |  |
Photosystem II toxicity | Pulse Amplitude Modulation fluorometry | Water | Pesticides
Endocrine disruption | Snail reproduction | Sediment | Phthalates, synthetic fragrances, alkylphenols

3. Fractionation

Fractionation of the extract is achieved by the application of chromatography, resulting in the separation of the – in most cases – multitude of different compounds that are present in an extract of an environmental sample. Chromatographic separation is obtained after the migration of compounds through a sorbent bed. In most cases, the separation principle is based on the distribution of compounds between the liquid mobile phase and the solid stationary phase (liquid chromatography, or LC), but a chromatographic separation using the partitioning between the gas phase and a sorbent bed (gas chromatography, or GC) is also possible. At the end of the separation column, at specified time intervals fractions can be collected that are simpler in composition in comparison to the original extract: a reduction in the number of compounds per fraction is obtained. The collected fractions are tested in the bioassay and the responsive fractions are selected for further chemical analysis and identification (step 4). The time intervals for fraction collection vary between a few minutes in older applications and a few seconds in new applications of EDA, which enables fractionation directly into multiwell plates for high throughput bioassay testing. In cases where fractions are collected during time intervals in the order of minutes, the fractions are still so complex that a second round of fractionation to obtain fractions of reduced complexity is often necessary for the identification of compounds that are responsible for the observed effect (see Figure 2).

Figure 2. Schematic representation of extract fractionation and selection of fractions for further testing, identification and confirmation.

4. Chemical Analysis

Chemical analysis for the identification of the compounds that cause the effect in the bioassay is usually done by LC coupled to mass spectrometric (MS) detection. To obtain high mass accuracy that facilitates compound identification, high resolution mass spectrometry (HR-MS) is generally applied. Fractions obtained after one or two fractionation steps are injected into the LC-MS system. In studies where fractionation into multiwell plates is used (and thus small fractions in the order of microliters are collected), only one round of fractionation is applied. In these cases, identification and fraction collection can be done in parallel, using a splitter after the chromatographic column that directs part of the eluent from the column to the well plate and the other part to the MS (see Figure 3). This is called high throughput EDA (HT-EDA).

Figure 3. Schematic representation of high-throughput Effect-Directed Analysis (HT-EDA).

5. Identification

The use of HR-MS is necessary to obtain mass information to establish the molecular weight with high accuracy (e.g. 119.12423 Dalton) to derive the molecular formula (e.g. C6H5N3) of the compound. Optimally, HR-MS instrumentation is equipped with an MS-MS mode, in which compound fragmentation is induced by collisions with other molecules, resulting in fragments that are specific for the original compound. Fragmentation spectra obtained using the MS-MS mode of HR-MS instruments help to elucidate the structure of the compounds eluting from the column, see for an example Figure 4.

Figure 4. Example of a chemical structure corresponding to an accurate mass of 119.12423 Dalton and the corresponding molecular formula C6H5N3: 1,2,3-benzotriazole.

Other information such as log Kow may be calculated using dedicated software packages that use elemental composition and structure as input. To aid the identification process, compound and mass spectral libraries are used as well as the more novel databases containing toxicity information (e.g. PubChem Bioassay, Toxcast). Mass spectrometry instrumentation vendor software, public/web-based databases and databases compiled in-house enable suspect screening to identify compounds that are known, e.g. because they are applied in consumer products or construction materials. When MS signals cannot be attributed to known compounds or their metabolites/transformation products, the identification approach is called non-target screening, where additional identification techniques such as Nuclear Magnetic Resonance (NMR) may aid the identification. The identification process is complicated and often time consuming, and results in a suspect list that needs to be evaluated for further confirmation of the identification.

6. Confirmation

For an unequivocal confirmation of the identity of a tentatively identified compound, it is necessary to obtain a standard of the compound to investigate whether its analytical chemical behaviour corresponds to that of the tentatively identified compound in the environmental sample. In addition, the biological activity of the standard should be measured and compared with the earlier obtained data. In case both the chemical analysis and bioassay testing results support the identification, confirmation of compound identity is achieved.

In principle, the confirmation step of an EDA study is very straightforward, but in current practice the standards are mostly not commercially available. Dedicated synthesis is time consuming and costly, therefore the confirmation step often is a bottleneck in EDA studies.

The application of EDA is suitable for samples collected at specific locations where comprehensive chemical analysis of priority pollutants and other chemicals of relevance has been conducted already, and where ecological quality assessment has revealed that the local conditions are compromised (see other Sections on Diagnostic risk assessment approaches and tools). Especially those samples that show a significant difference between the observed (in vitro) bioassay response and the activity that may be calculated according to the concept of Concentration Addition (see Section on Mixture Toxicity) by using the relative potencies and the concentrations of compounds active in that bioassay need a further in-depth investigation. EDA can be implemented at these ‘hotspots’ of environmental contamination to unravel the identity of compounds that have an effect, but that were not included in the chemical monitoring of the environmental quality. Knowledge with regard to the main drivers of toxicity at a specific location supports accurate decision making that is necessary for environmental quality protection.