Clas Wesén

Identification of chlorinated fatty acids in fish lipids by partitioning studies and by gas chromatography with Hall electrolytic conductivity detection

Abstract Chlorinated compounds in fish lipids [determined by neutron activation analysis as extractable, organically bound chlorine (EOCl)] were characterized by liquidliquid extractions after enzymatic hydrolysis and after forming fatty acid methyl esters (FAMEs). Most of the chlorinated compounds in lipids from four different fish samples could be hydrolysed. Comparison with results of methanolysis of two of the fish lipids indicated that chlorinated fatty acids made up the major portion of EOCl. Using gas chromatography (GC) with electrolytic conductivity detection (ELCD), chlorinated compounds were found among FAMEs of ee] lipids containing 1200 ppm of EOCl. Approximately 90% of EOCl was detected by GC-ELCD. The GC-detectable compounds are suggested to be methyl esters of chlorinated fatty acids and 9,10-dichlorostearic acid was tentatively identified after co-injection of the synthesized compound.

Enrichment of chlorinated fatty acids in fish lipids prior to analysis by capillary gas chromatography with electrolytic conductivity detection and mass spectrometry

Abstract Chlorinated carboxylic acids of fatty acid character have been shown to account for up to 90% of the extractable, organically bound chlorine (EOCl) in fish. To facilitate the detection of chlorinated fatty acid methyl esters (FAMEs) released from fish lipids, an enrichment was performed by removing the polyunsaturated FAMEs and saturated, straightchain FAMEs through their complexation with silver ions and urea, respectively. The resulting about 30-fold increase in the concentration of chlorinated FAMEs allowed for their analysis, by gas chromatography (GC) with halogen-selective electrolytic conductivity detection, in lipids containing only 30 ppm (m/m) of EOCl. Following additional purification by thin-layer chromatography, methyl esters of dichlorotetradecanoic, dichlorohexadecanoic and dichlorooctadecanoic acids were indicated by GC-ammonia positive ion chemical ionisation mass spectrometry in a fish sample containing 1200 ppm (m/m) of EOCl.

Gas Chromatographic and Mass Spectrometric Identification of Tetrachloroalkanoic and Dichloroalkenoic Acids in Eel Lipids

Chlorinated fatty acid methyl esters, transesterified from the lipids of eel (Anguilla anguilla ; obtained from the receiving waters of a chlorine bleaching pulp mill), were studied by gas chromatography (GC) using electrolytic conductivity detection and mass spectrometry (MS) with electron impact and ammonia positive-ion chemical ionization. GC retention indices and column difference values in combination with GC/MS demonstrated the presence of isomers of methyl dichlorotetradecenoate, methyl dichlorohexadecenoate, methyl dichlorooctadecenoate and methyl tetrachlorotetradecanoate. Isomers of methyl threo, threo-tetrachlorooctadecanoate were identified by co-injection of the eel sample and synthesized reference compounds. The results were confirmed by ammonia positive-ion chemical ionization high-resolution selective-ion monitoring of the ammonium adduct molecular ions.

HALOGENATED FATTY ACIDS : I. FORMATION AND OCCURRENCE IN LIPIDS

Abstract Chlorinated fatty acids have been found to be major contributors to organohalogen compounds in fish, bivalves, jellyfish, and lobster, and they have been indicated to contribute considerably to organohalogens in marine mammals. Brominated fatty acids have been found in marine sponges. Also, chlorinated lipids have been found in meat exposed to hypochlorite disinfected water, and in chlorine-treated flour and in products made from such flour. Following exposure to chlorine bleached pulp mill effluents, aquatic organisms may have elevated concentrations of chlorinated fatty acids in their lipids. However, a natural production of halogenated fatty acids is also possible. In this paper we summarize the present knowledge of the occurrence of halogenated fatty acids in lipids and suggested ways of their formation. In Part II (Trends Anal. Chem. 16 (1997) 274) we deal with methods of their determination.

Determination of polycyclic aromatic hydrocarbons (PAHs) from organic aerosols using hollow fiber micro-porous membrane liquid-liquid extraction (HF-MMLLE) followed by gas chromatography-mass spectrometry analysis.

A method for determination of polycyclic aromatic hydrocarbons (PAHs) from aerosols was developed. Instead of conventionally used non-polar or slightly polar phenylmethylpolysiloxane column a highly polar, highly substituted, cyanopropyl column (VF-23 MS) was used for separation of PAHs. Based on hollow fiber micro-porous membrane liquid-liquid extraction (HF-MMLLE) a method was developed for sample clean up and pretreatment. An enrichment factor of 617-1022 was obtained with extraction efficiency 10.2-18.9% for different PAHs analyzed in this study. The optimized method was successfully applied to aerosol samples and limits of detection between 1.2 pg m(-3) and 180 pg m(-3) was obtained. Almost all PAHs were found in most of the aerosol samples.

New halogen-specific detector applied to the analysis of chlorinated fatty acids

A new halogen-specific detection method (XSD) was tested for determination of chlorinated fatty acids in marine biota. In XSD, an increased emission of ions and electrons is caused by the high-temperature combustion of halogen-containing compounds. The detection limit of methyl dichlorooctadecanoate and the selectivity at a reactor temperature of 900 degrees C match those of electrolytic conductivity detection (ELCD). The relative standard deviation is less than 11% for > or =0.2 ng methyl dichlorooctadecanoate. An XSD chromatogram of a complex sample, chlorinated fatty acid methyl esters liberated from fish lipids, agreed with a previously obtained ELCD chromatogram.

Halogenated fatty acids: II. Methods of determination in lipids

Abstract Halogenated fatty acids are the major contributors to organohalogen compounds in lipids of marine mammals, fish, and bivalves. For the initial characterization of these recently noticed compounds, a determination of the halogen concentration has usually been combined with some lipid isolation and separation method. This review covers separation by solid phase chromatography, gel permeation chromatography, and liquid-liquid extraction, followed by halogen determination. All studies performed according to this outline have indicated that the major organohalogen compounds are chlorinated fatty acids bound in different lipids. For the detection and identification of individual, halogenated fatty acid methyl esters (FAMEs) liberated from the lipids, gas chromatography (GC) has been employed together with detection methods such as electron capture detection, electrolytic conductivity detection (ELCD), atomic emission spectrometry, and mass spectrometry. For most environmental samples, chlorinated FAMEs must be enriched prior to GC. ELCD is a useful detection method for indicating halogenated FAMEs in the chromatograms, and tentative identification of the halogenated species can be obtained by calculation of retention indices. For closer identification of halogenated FAMEs, mass spectrometry (MS) is very useful, in particular when employing the chemical ionisation mode. MS identification, however, is highly facilitated if halogenated species are first indicated by element-selective methods.

Occurrence of halogenated fatty acids in bivalve lipids

Environmental pollutants account for 1–3% of the extractable, organically bound chlorine (EOCl) found in bivalve lipids. In this work, bivalve lipids were converted to fatty acid methyl esters (FAMEs). The EOCl in the FAMEs and in acidic compounds was examined after liquid-liquid extraction using neutron activation analysis for chlorine determination. Gas chromatography with halogen selective, electrolytic conductivity detection (GC/ELCD) was employed to study the occurrence of halogenated FAMEs. Following esterification, most of the EOCl was recovered in the FAME-containing fraction. However, silica gel chromatography showed that more than 50% of the EOCl consisted of compounds of higher polarity than the chlorinated FAMEs normally found in fish extracts. By using GC/ELCD, up to 25% of the EOCl was detected as halogenated fatty acids. A complex pattern of halogenated fatty acids was found in bivalves from the Baltic Sea. This pattern was simplified to one consisting of only a few halogenated fatty acids in bivalves from West Scandinavian waters. These acids seem to persist in the ecosystem. It is possible that up to 40% of the EOCl remained in a brown, organic material of polar character that was coextracted with the FAMEs.

Removal of Xenobiotic Dichlorostearic Acid from Phospholipids and Neutral Lipids in Cultured Human Cell Lines by ß-Oxidation and Secretion of Dichloromyristic Acid

Chlorinated fatty acids represent a recently discovered group of potentially hazardous organochlorine pollutants in the environment. The ability of human cells to incorporate and metabolise this type of fatty acids has never been investigated. The aim of the present study was, therefore, to investigate if two human cell lines, INT 407 and SH-SY5Y, incorporate and metabolise extracellular dichlorostearic acid. Cells were incubated with 9,10-dichlorostearic acid for 24 hr, and the amounts of chlorinated fatty acids in cells and culture medium analysed every two days for up to 6 or 10 days. Lipids were separated by solid phase extraction, transesterified to fatty acid methyl esters, and analysed by gas chromatography in combination with a halogen specific detector (GC/XSD). Dichlorostearic acid, dichloropalmitic acid and dichloromyristic acid were found in phospholipids and in neutral lipids of the INT 407 cells. Both cell lines secreted considerable amounts of dichloromyristic acid into the culture medium. Cellular or secreted metabolites shorter than dichloromyristic acid were not found. Taken together, the results suggest that human cells may (1) incorporate chlorinated fatty acids into membrane lipids and storage lipids, (2) metabolise cellular dichlorostearic acid to dichloropalmitic acid and dichloromyristic acid by B-oxidation; but that further metabolism is hindered, possibly because of the chlorine atoms, and (3) remove formed dichloromyristic acid by secretion. The removal of cellular dichloromyristic acid might represent an important cellular defence mechanism and deserves further investigations.

Inhibitory effect of threo-9,10-dichlorostearic acid on the mutagenic activity of MeIQx, 2-AF and B[a]P in the Ames/Salmonella test.

The mutagenic activity of threo-9,10-dichlorostearic acid, one of the chlorinated fatty acids identified in fish lipids, was examined in the Ames/Salmonella test. No mutagenic activity was found on any of the Salmonella typhimurium strains TA 98, TA 100 and TA 102, either with or without S9 activation. On the other hand, dichlorostearic acid showed an inhibitory effect on the mutagenic activity of the indirectly-acting mutagens 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-aminofluorene (2-AF) and benzo[a]pyrene (B[a]P) using strain TA 98 in the presence of S9. However, no inhibition was observed when mixing MeIQx and S9 before the addition of dichlorostearic acid. Furthermore, dichlorostearic acid did not show any inhibitory effect on the mutagenic activity of the directly-acting mutagen 4-nitroquinoline-N-oxide (4NQO) using the tester strains TA 98 and TA 100. We, therefore, suggest that dichlorostearic acid interacts with the enzymes of the S9 mix, thereby dose-dependently inhibiting the transformation of MeIQx, 2-AF and B[a]P into their active forms.