title: Characterization of alkylphenol metabolites in fish bile by enzymatic treatment and HPLC-fluorescence analysis.
abstract: Alkylphenol (AP) metabolites were characterized in the bile of Atlantic cod (Gadus morhua L.) after exposure to nine individual compounds (10mg/kg fish), 2-methylphenol (2-MP), 4-methylphenol (4-MP), 3,5-dimethylphenol (3,5-DMP), 2,4,6-trimethylphenol (2,4,6-TMP), 4-tert-butylphenol (4-t-BP), 4-tert-butyl-2-methylphenol (4-t-B-2-MP), 4-n-pentylphenol (4-n-PP), 4-n-hexylphenol (4-n-HexP) and 4-n-heptylphenol (4-n-HepP), and a mixture (total dose; 13.5 mg/kg fish) of the nine APs by inter-muscular injection. The degree of alkylation ranged from methyl (C1) to heptyl (C7) and represents the types of APs present in produced water. Fish bile was collected on day 4 and 16 (exposure groups 2-MP, 3,5-DMP, 2,4,6-TMP and 4-t-B-2-MP) following exposure. Characterization of major metabolites was accomplished by enzymatic de-conjugation and analysis by high performance liquid chromatography connected to a fluorescence detector (HPLC-F) acquiring at ex/em 222/306 nm. Two solid phase extraction (SPE) columns were evaluated for clean-up of samples prior to analysis. Independent of alkyl homologue, the glucuronide conjugated APs were the most abundant metabolites (73-100%), whereas sulfates, glucosides and unchanged compounds were excreted in amounts of 0-21%, 0-6.1% and 0-6.3%, respectively. The total concentration of measured metabolites in the bile, determined as their respective APs after de-conjugation, increased with increasing degree of alkylation (3.2+/-2.6 microg/g bile for 2-MP and 571+/-81 microg/g bile for 4-n-HepP) after exposure to an equal dose of AP. Comparison of metabolite concentrations in bile sampled 4 and 16 days after exposure, showed that the levels of 2-MP, 2,4,6-TMP and 4-t-B-2-MP were reduced by 55%, 30% and 45%, respectively whereas 3,5-DMP increased by 25% (not significant). This study suggests that analysis of de-conjugated metabolites in fish bile can be used to monitor AP exposure to fish, due to the relatively high and persistent level of these compounds. However, although HPLC-F is suitable for laboratory exposures, it might not be sufficient selective for field studies.
authors.affiliation: Stavanger University Hospital, Department of Medical Biochemistry, PO Box 8100, N-4068 Stavanger, Norway. jgre@ext.sir.no <jgre@ext.sir.no>
authors.forename: Grete
authors.initials: G
authors.lastname: Jonsson
authors.forename: Tone U
authors.initials: TU
authors.lastname: Stokke
authors.forename: Admira
authors.initials: A
authors.lastname: Cavcic
authors.forename: Kåre B
authors.initials: KB
authors.lastname: Jørgensen
authors.forename: Jonny
authors.initials: J
authors.lastname: Beyer
mesh_terms: D000478:Alkylation; D000818:Animals; D001646:Bile / Q000737:chemistry / Q000378:metabolism*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004784:Environmental Monitoring / Q000379:methods*; D005453:Fluorescence; D048389:Gadus morhua / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005959:Glucosidases / Q000737:chemistry; D005966:Glucuronidase / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry*; D010636:Phenols / Q000032:analysis / Q000737:chemistry / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013429:Sulfatases / Q000737:chemistry
chemical_list: D010636:Phenols; D006867:Hydrolases; D013429:Sulfatases; D005959:Glucosidases; D005966:Glucuronidase