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Toxicity characterization of organic contaminants in effluents from the city of Zagre

Background & objective Toxicity identification Evaluation (TIE) procedures have become an established and powerful tool for determining the cause of detrimental effect in complex environmental samples. The techniques involve the step-wise separation and simplification of a sample in order to isolate toxic components. These manipulations are directed by a biological assay (bioassay) until it is possible to identify the compounds responsible by chemical analysis. The purpose of the study is to identify classes of organic compounds giving rise to toxic effects. Materials & methods A sample of wastewater (3.9 l) was collected from the main sewer of the city of Zagreb in June 2005, and represents raw water without any treatment. The sample was filtered and passed through Oasis HLB cartridges. The water extract was fractionated by HPLC using a semi preparative C18 column with a gradient of methanol in water, ending with a methanol flush. Thirty fractions were collected. The volume of each fraction was reduced before chemical analysis and toxicity testing. The fractions were tested for membrane integrity (cytotoxicity), metabolic inhibition, estrogenic activity (vitellogenin induction), and CYP1A induction (EROD activity) by use of a suite of fish in vitro bioassays 1, 2. The HPLC fractions demonstrating toxicity were analysed by GC-MS in full scan mode (50-800 AMU). The acquired mass spectra were deconvoluted by using an automated mass spectral deconvolution and identification system (AMDIS). All AMDIS deconvoluted mass spectra were then compared to reference spectra in the National Institute of Standards and Technology (NIST) mass spectral database for tentative identification. Results & discussion Comparison of the log Kow of identified compounds versus fraction number was as expected from literature data3. The suite of bioassays showed biological responses after exposure to the raw extracts for all endpoints tested, although the levels of sub-lethal responses such as vitellogenin induction and EROD activity was partly masked by cytotoxicity caused by the complex content in the the raw extracts. Bioassay testing of the fine fractions obtained by semi-preparative HPLC produced a range of endpoint-specific toxicity profiles for each sample, however. GC-MS characterisation was used to identify candidate compounds for the toxic activity. The presentation will present a description of the approach used and results from this characterisation procedure. 1. Tollefsen KE, Goks&oslash ; yr A, Hylland K In press. The use of a fish in vitro bioassay for monitoring of pollutants in the North Sea. SETAC special publications. 2. Tollefsen K-E, Mathisen R, Stenersen J 2003. Induction of vitellogenin synthesis in an Atlantic salmon (Salmo salar) hepatocyte culture: a sensitive in vitro bioassay for the oestrogenic and anti-oestrogenic activity of chemicals. Biomarkers 8:394-407. 3. Thomas K, Benstead R, Thain J, Waldock M 1999. Toxicity characterization of organic contaminants in industrialized UK estuaries and coastal waters. MARINE POLLUTION BULLETIN 38(10):925-932.

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