title: Effects-directed analysis of organic toxicants in wastewater effluent from Zagreb, Croatia.
abstract: The organic toxicants present in the effluent of the main sewer of the city of Zagreb, Croatia were isolated and identified through the use of effects-directed characterisation techniques. At the time of investigation, the wastewater effluent received no treatment and was comprised of a mixture of effluent from domestic and industrial sources. The organic load of the wastewater was isolated by solid phase extraction and toxicity profiles obtained using reverse-phase HPLC. All procedures were evaluated through the analysis of a series of reference compounds of widely differing polarity. Toxicity profiles for EROD activity (CYP1A induction), vitellogenin induction (estrogenic activity), cytotoxicity (membrane stability and metabolic inhibition) were obtained using a rainbow trout (Oncorhynchus mykiss) primary hepatocyte bioassay. The suite of bioassays showed biological responses after exposure to the raw extracts for all the endpoints tested. However, a combination of mixture toxicity and cytotoxicity in the complex raw extract had some masking effect on the sub-lethal responses of vitellogenin and EROD induction. Bioassay testing of the fine fractions obtained by HPLC produced a range of endpoint-specific toxicity profiles for each sample. A number of compounds were identified by the use of GC-MS and LC-MS/MS as responsible for the observed effects. The steroid estrogens 17 beta-estradiol and estriol were identified by LC-MS/MS as estrogen receptor agonists in two of the estrogenic fractions. In addition, GC-MS analysis identified different alkylphenols, benzophenone and methylparaben which also contributed to the estrogenic activity of the sample. Polycyclic aromatic hydrocarbons (PAHs), alkyl substituted PAHs, nitro-polycyclic aromatic compounds (nitro-PACs), carbazoles and alkyl substituted carbazoles and other known CYP1A inducers were identified by GC-MS analysis as responsible for some of the observed EROD activity. Some active compounds remain unidentified.
authors.affiliation: Norwegian Institute for Water Research, Gaustadalléen 21, 0349 Oslo, Norway. merete.grung@niva.no
authors.forename: Merete
authors.initials: M
authors.lastname: Grung
authors.forename: Rainer
authors.initials: R
authors.lastname: Lichtenthaler
authors.forename: Marijan
authors.initials: M
authors.lastname: Ahel
authors.forename: Knut-Erik
authors.initials: KE
authors.lastname: Tollefsen
authors.forename: Katherine
authors.initials: K
authors.lastname: Langford
authors.forename: Kevin V
authors.initials: KV
authors.lastname: Thomas
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D017523:Croatia; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004784:Environmental Monitoring / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013058:Mass Spectrometry; D015394:Molecular Structure; D012722:Sewage / Q000032:analysis / Q000737:chemistry*; D014337:Trout / Q000378:metabolism; D014819:Vitellogenins / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D012722:Sewage; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D019363:Cytochrome P-450 CYP1A1