Sandimirov S. Screening studies of POP levels in fish from selected lakes in the Paz watercourse / In State of the environment in the Norwegian, Finnish and Russian border area. The Finnish Environment. Finland, Jyvaskyla: Kopijyva Oy. 2007, №6.

Appendix: Screening studies of POPs in fish from the Paz watercourse Appendix 2 BILE ACIDS QUANTIFICATION Bile acids quantification in bile was measured using the method described by Ripatti P.O. et al. (1969). Reagents. Concentrated sulphur acid; n-hexane (1 liter / one analysis) (purified from peroxides); sulphur ester (1 liter / analysis) (purified from peroxides); cholic acid; chenodeoxic acid; NaOH (natrium hydroxide) (crystalline); HCL (hydrochloric acid) (5 N solution); ethanol (96% (volume per cent)); potassium permanganate (KMnO4) for purification sulphur ester and n-hexane. Isolation of bile acids. The samples were heated on water bath for 5 min, filtered and washed 2-3 times by hot ethanol. The spirit extract was evaporated in retorts and this retorts were dried under the oxide of phosphorus (P2O5) until constant weight. The sediments were weighed. Fixed weigh and volume for further study was taken. Obtaining free bile acids. The ethanol solutions were evaporated until dry. Every sediments were dissolved in 3 ml natrium hydroxide solution (1,25 M NaOH) and removed into teflon retorts. Teflon retorts have been closed up hermetically and stayed in thermostat at 120 oC for 4 hours. After hydrolysis this alkaline solution were removed in glass retorts with glass cork (or into separating funnel). Teflon retorts were washed 3 times (2 ml every times) by bidistillate water. This volume was added into this glass retorts. The acidity of these solutions was adjusted to pH 2-3. Then samples were extracted by ether (sulphur ether) 3 times (6-10 ml every times). Ether fraction were washed 2 times and evaporated until dry. Then samples were dissolved into 8 ml of 70% ethanol and washed 3 times (6 ml every time) by n-hexane. Spirit solutions were evaporated until dry. Samples were dissolved in definite volume of 96% (volume per cent) ethanol. Spectrophotometrical measurements.Definite volumes of these samples in spirit were removed into glass flasks with glass cork. It was added 5 ml of sulphur acid after full evaporation of ethanol in every flask. Samples were incubated in thermostat at 65oC for 1 hour and measured by spectrophotometer at 347 nm and 389 nm wave lengths. DETERMINATION OF ANILINE HYDROXYLASE ACTIVITY Aniline hydroxylase activity was measured using the method described by Mazel (1972). Reagents. Glucose-6-phosphate; nicotinamide (the amide of nicotinic acid); magnesium chloride (MgCl2); NADPH (nicotinamideadeninedinucleotide phosphate reduced); carbonate of natrium (Na2CO3); 2-substituted hydrophosphate of natrium (Na2HPO4) (crystalline); 1-substituted dihydrophosphate of potassium (KH 2 PO 4 ) (crystalline); chloride of potassium (KCl); phenol (crystalline); NaOH (natrium hydroxide) (crystalline); trichloracetic acid (CCl 3 COOH) (crystalline or 50-20% volume per cent); aniline hydrochloride CeHsNH^HCl (crystalline); p-aminophenol C 6 H 4 NH 2 OH (crystalline). Akvaplan-niva report APN 514-3365.02 53