Methodology
Effects-directed analysis (EDA) has become an established and powerful tool for determining the causes of toxic biological effects in complex environmental samples. EDA involves the stepwise separation of a sample in order to isolate components with biological (toxic) activity into well-defined fractions, which facilitates their identification by chemical methods. It has to be stressed that reliable identification of individual contaminants in complex environmental mixtures still represents a rather difficult task and usually requires a highly experienced experts, despite significant advances in the development of modern analytical techniques, in particular gas chromatography/mass spectrometry. Moreover, there is no standardized EDA procedure, which would be applicable to all types of environmental samples and therefore, it is necessary to carefully select extraction and fractionation procedures to be applied before the instrumental analysis.
Analytical chemistry· The method of choice for the analysis of organic contaminants in EDAs is mainly GC/MS, owing to the high-resolution of gas-chromatographic separation using capillary columns, high sensitivity and relatively reliable identifications using searchable mass spectral libraries. Moreover, most of the priority pollutants belong to nonpolar and, often, highly hydrophobic compounds, which can be easily analysed by GC/MS technique. To facilitate the identifications of a broad spectrum of GC-amenable contaminants present in the extract, in this project the GC/MS system equipped with electron impact and chemical (both positive and negative) ionization will be used. Single quadrupole GC/MS system, a highly sensitive system that allows simultaneous full-scan (FS) and selected-ion monitoring (SIM) analyses will be used for most of the work planned within this project. In addition, for confirmation purposes some samples are planned to be re-analysed on the existing GC-ToF-MS system at NIVA. In addition, the IRB laboratories are equipped with a LC/MS/MS system, which will be used for target analyses of some polar and/or non-volatile contaminants such as surfactants and pharmaceuticals and personal care products (PPCP).
· The sample preparation for mass spectrometric analyses depends on the sample matrix. For aqueous samples, Solid Phase Rxtraction (SPE) will be mainly used for the enrichment. In the later phase of the project some modifications are planned in order to optimize the procedure for a given type of sample. River sediments will be extracted by Soxhlet or, alternatively, by accelerated solvent extraction (ASE).
· The extract fractionation will be performed by preparative high-performance liquid chromatography (HPLC), using both reverse-phase (C18 column) and normal-phase (aminosilica column) systems. In some cases, application of gel permeation chromatography (GPC) is also envisaged.
Ecotoxicology – Bioassays
In order to improve the existing EDA procedures and to make them more efficient, instead of using a single toxicity test typically being used to direct chemical fractionation, we will try to standardize a series of in vitro bioassays directed to identification of hazardous contaminants with different modes of action. A critical prerequisite for that goal is development and/or standardization of high-throughput-screening (HTS) bioassays capable of analysing many environmental samples or their sub-fractions simultaneously.
The bioassay directed fractionations will be based on in vitro methods utilizing specific cell lines, yeast or algal strains, avoiding invasive in vivo methods and the extensive use of experimental animals. All bioassays are or have to be developed as microplate-based HTS techniques. The proposed suite of bioassays directed to identification of various ecotoxicicological effects in environmental samples consists of the methods, which are potentially suitable regarding HTS requirements. The test methods chosen aim to assess effects at different trophical levels of organization (bacteria, algae, crustaceans, fish and mammals) and the parameters that will be used encompass chronic toxicity, induction or inhibition of three critical phases of cellular detoxification, mutagenic/genotoxic potencies and finally endocrine disruption (estrogenic effects). More specifically:
· Chronic toxicity will be determined using microplate based Chronic toxicity growth inhibition test with unicellular algae Selenastrum capricornutum (ISO/FDIS 8692:2004(E). Water quality – Freshwater algal growth inhibition test with unicellular green algae. ISO, Geneva, Switzerland; http://www.iso.org). Depending on the initial screening results some samples may be additionally analysed by the crustacean (Daphnia magna) bioassay;
· Phase I (cytochrome P450 1A1 mediated) induction potential of environmental samples will be determined by measuring the ethoxyresorufin O-deethylase (EROD) activity in the PLHC-1 fish hepatoma cell line (Hanh et al., 1996, Environ. Toxicol. Chem., 15, 582-591) – microplate-based protocol;
· Phase II induction potential will be determined by measuring glutathione S-transferase (GST) activity in the PLHC-1 cells. Alternatively, the GST activity will be measured in primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes;
· Inhibitors the multixenobiotic resistance mechanism (MXR, phase III of cellular detoxification) will be measured by determination of transport activity of the ABC proteins included in the MXR mechanism in PLHC-1 cells, or in NIH-3T3 cells stably transfected with genes encoding relevant ABC proteins. Both protocols have been recently established in Laboratory for Molecular Ecotoxicology at IRB, Zagreb (Pivčević and Zaja, 2006, Environ. Toxicol. Pharmacol., 3, 268-276; Zaja et al., manuscript submitted);
· The presence of genotoxic substances in environmental samples will be identified by the Mutatox test with a special strain of luminescent bacteria (Vibrio fischeri) (Jarvis et al., 1996, Ecotoxicol. Environ. Saf., 33, 193-200). The test will be standardized in the Laboratory for Molecular Ecotoxicology (IRB) and its efficiency compared with the Ames test (available in the Laboratory) as the most recognized method for determination of mutagenic substances;
· Estrogen active compounds (xenoestrogens) in environmental samples will be identified using the Yeast Estrogen Screen (YES) assay with special yeast strain stably transfected with human estrogen receptor (Beresford at al., 2000, Toxicol. Appl. Pharmacol., 162, 22-33).