Determination of environmental caused chlorophenol levels in urine of the general population

Summary A sensitive, specific and analytically reliable method for the determination of mono-, di-, tri- and tetrachlorophenols in human urine has been elaborated. After acid hydrolysis and a simultaneous steam distillation of the urine samples, spiked with an internal standard, the chromatographically concentrated chlorophenols have been derivatized with pentafluorobenzoylchloride and analyzed by capillary gas chromatography/mass spectrometry. The detection limits for the chlorophenols ranged from 0.2 to 2.5 g/l. Using this method we were able to detect 4-MCP, 2,4-+2,5-DCP, 2,4,6-TCP, 2,4,5-TCP and 2,3,4,6-+2,3,5,6-TeCP in urine samples of a group of 258 men and women which had no known occupational contact to hazardous chemical substances. The 95 percentiles for the concentrations of these substances in the urine samples under investigation were 7.5 (4-MCP); 33.6(2,4-+2,5-DCP); 4,7 (2,4,6-TCP); 4,5 (2,4,5-TCP) and 22.2 (2,3,4,6-+2,3,5,6-TeCP) g per liter. That means, that these chlorophenols are constituents of urine of the normal population in concentrations which in part are greater than that of pentachlorophenol (PCP).     

Development of HPLC-MS/MS method for the simultaneous determination of environmental phenols in human urine

We present a sensitive method for simultaneous determination of bisphenol A (BPA), benzophenone-3 (BP-3), 4-tert-octylphenol (t-OP), ortho-phenylphenol (OPP), four parabens (methyl, ethyl, propyl, butyl parabens) and five chlorophenols (2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol (2,4,5-triclorophenol), 2,4,6-trichlorophenol (2,4,6-TCP), and triclosan (TCS)), in human urine by high-pressure liquid chromatography (HPLC) mass spectrometry (MS). Samples were processed using enzymatic deconjugation of glucuronides followed by solid phase extraction (SPE) on a C18 cartridge and the eluate was concentrated. Analytes were separated by reversed-phase HPLC and then detected by atmospheric pressure chemical ionisation (APCI) MS and quantified by isotope dilution method. We describe details for optimisation of each step of the procedure. The sample treatment steps are straightforward and not labour-intensive and, therefore, permit a high sample throughput with excellent prospects for automation. This method shows low inter-day variation, and detection limits for most of the compounds are below 1 ng/mL in 1 mL of urine. The method accuracy was also verified by the analysis of proficiency testing urine samples.     

In-capillary solid-phase extraction-capillary electrophoresis for the determination of chlorophenols in water

A novel CE method combined with SPE in a single capillary was developed for analysis of chlorophenols in water. A frit of 0.5 mm was first made by a sol-gel method, followed by packing a SPE sorbent in the inlet end of the capillary. Two phenol derivatives, 2,4-dichlorophenol and 2,4,5-trichlorophenol, were used as the model compounds. By loading sample solutions into the capillary, the two chlorophenols were extracted into the sorbent. They were desorbed by injecting only about 4 nL of methanol. Finally, the analytes were separated by conventional CE. The technique provided a concentration enhancement factor of over 4000-fold for both chlorophenols. The detection limits (S/N = 3) of 2,4-dichlorophenol and 2,4,5-trichlorophenol were determined to be 0.1 ng/mL and 0.07 ng/mL, respectively. For replicate analyses of 5 ng/mL of 2,4-dichlorophenol, within-day and between-day RSDs of migration time, peak height and peak area were in the range of 1.8-2.0%, 4.0-4.4% and 4.1-4.6%, respectively. The method shows wide linear range, acceptable reproducibility and excellent sensitivity, and it was applied to the analyses of spiked river water samples. The capillary packed with the SPE sorbents can be used for more than 400 runs without performance deterioration.     

A comparative study of trace enrichment of chlorophenols in water by extraction with C6 and C8 alkanes and by C18 reversed-phase adsorption

The efficiency, reproducibility and sensitivity ofn-hexane and iso-octane extraction and of C₁₈ reversed-phase adsorption for accumulation of 4-chloro-, 2,4-dichloro-, 2,4,5- and 2,4,6-trichloro-, 2,3,4,6-tetrachloro- and pentachlorophenol from aqueous solutions were compared. In extraction procedures the acetyl and pentafluorobenzoyl derivatives of chlorophenols were extracted from water. In adsorption procedure chlorophenols adsorbed on a Sep-Pak C₁₈ cartridge were eluted with acetone and after that derivatized analogously.All three procedures were found to be applicable for an efficient trace enrichment of chlorophenols in water, the proper choice being dependent on the required sensitivity of the analysis. Lower detection limits of single compounds at 10 ng·1^–1 levels were achieved by adsorption procedure owing to the more uniform and for most chlorophenols higher adsorption than extraction recoveries as well as owing to the possibility of treating larger volumes of water samples. The extraction procedure could be successfully applied to the concentrations of chlorophenols in water 1g·1^–1.Owing to its higher efficiency and better sensitivity the C₁₈ reversed-phase adsorption procedure was chosen as the more suitable one for the determination of chlorophenol levels in surface, ground and drinking waters. The conversion of chlorophenols accumulated from a water sample parallel to both acetyl and pentafluorobenzoyl derivatives and the analysis of both types of derivatives on two basically different gas Chromatographic columns were recommended for a more reliable identification and quantitation of the compounds analyzed.     

A Method for Determination of Low Levels of Exposure to 2,4-D and 2,4,5-T

A method has been developed for the determination of trace quantities of 2,4-dichloro-phenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4-dichlorophenol (2,4-DCP), and 2,4,5-trichlorophenol (2,4,5-TCP) in human and rat urine. The method involves acid hydrolysis of the phenolic conjugates, extraction of the free phenols and acids, ethylation with diazoethane, silica-gel column chromatography clean-up of the derivatized urine extract, and gas chromatographic determination using the electron-capture detector. The average recoveries of 2,4-D, 2,4,5-T, 2,4-DCP, and 2,4,5-TCP from rat urine spiked with known amounts of the herbicides and their phenols were 94%, 98%, 92%, and 90%, respectively. The limits of detection for 2,4-D, 2,4,5-T, DCP, and TCP in rat urine were: 0.05, 0.01, 0.10, and 0.01 ppm, respectively. The method was used to analyze urine of rats given various levels of 2,4-D and 2,4,5-T by gavage. Results showed that levels of exposure of 3.75 mcg/kg for 2,4-D and 5.0 mcg/kg for 2,4,5-T in rats can be detected in urine within 24 hr from exposure. Urine samples from occupationally exposed people were analyzed and found to contain 0.2 to 1.0 ppm 2,4-D and 0.05 to 3.6 ppm 2,4,5-T.     

Trace enrichment of chlorophenols in human urine by C18 reversed-phase adsorption and by n-hexane extraction

Summary The efficiency and sensitivity of C₁₈ reversed-phase adsorption of free chlorophenols and of n-hexane extraction of either free or acetylated chlorophenols from human urine were compared. All procedures were found to be efficient for the trace enrichment of 2,4-dichlorophenol, 2,4,6- and 2,4,5-trichlorophenols, 2,3,4,6- and 2,3,4,5-tetrachlorophenols and pentachlorophenol. The recoveries of chlorophenols from non-hydrolysed and acid hydrolysed urine samples were comparable. By treatment of 1 ml urine sample detection limits of 1–2 ng/ml were achieved, while the treatment of 5 ml samples enhanced the detection sensitivity to less than 1 ng/ml. The n-hexane extraction of acetylated chlorophenols from 1 ml urine samples is the simplest and fastest procedure because the acetylation and extraction of chlorophenols are performed simultaneously in one step. The C₁₈ adsorption seems to be more suitable than n-hexane extraction for accumulation of chlorophenols from a urine volume of 5 ml and higher because the elution is performed always with the same small volume of acetone. Both C₁₈ adsorption and n-hexane extraction procedures were applied for analysis of chlorophenols in general population and in persons with possible occupational exposure to organochlorine compounds.     

Determination of phenols in waters by stir membrane liquid-liquid-liquid microextraction coupled to liquid chromatography with ultraviolet detection

A simple and rapid method for the determination of eleven phenols in water samples is presented. The target analytes are isolated by stir membrane liquid-liquid microextraction working under the three-phase mode. An alkaline aqueous solution is used as extractant phase while octanol is selected as supported liquid membrane solvent. The target analytes are separated and determined by liquid chromatography (LC) with ultraviolet detection (UV). All the variables involved in the extraction process have been studied in depth. Low detection limits (in the range from 82.1 ng/L for phenol to 452 ng/L for 2,4,5-trichlorophenol) were obtained. The repeatability, expressed as relative standard deviation (RSD), varied between 1.3% (for 4-nitrophenol) and 8.0% (for 4-chlorophenol). The enrichment factors were in the range from 168 (for 2,4,5-trichlorophenol) to 395 (for 3-chlorophenol). The proposed procedure was applied for the direct determination of the eleven phenols in some real water samples including river, well and tap waters. The accuracy was evaluated by means of a recovery study, the results being in the range of 87-120%.     

A comparison between positive and negative ion modes in thermospray liquid chromatography-mass spectrometry for the determination of chlorophenols and herbicides

Summary Positive and negative ion modes (Pl and NI, respectively) have been employed for the characterization of 2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, Linuron and Cyanazine in thermospray (TSP) liquid chromatography-mass spectrometry (LC-MS). The PI mode showed no response when 200 ng of the different chlorophenols were injected, while for Linuron and Cyanazine high signals were obtained with [M+NH₄]⁺ and [M+acetic acid]⁺ ions as base peaks, respectively. With the NI mode, the base peak usually corresponds to the [M−H]⁻ ion, with better sensitivities for the chlorophenols than for the herbicides. The chloride adduct [M+Cl]⁻ ion was also obtained for 2,4,5-trichlorophenol and for Linuron. Although the PI mode is more sensitive than the NI mode for the two herbicides, the combination of both ionization modes offers complementary structural information for characterizing such compounds in TSP LC-MS.     

Stir membrane liquid-liquid microextraction

In this article, a novel liquid phase microextraction technique, called stir membrane liquid-liquid microextraction (SM-LLME), is presented. The new approach combines the advantages of liquid phase microextraction and stirring in the same unit allowing the isolation and preconcentration of the analytes in a simple and efficient way. In the construction of the unit, a polymeric membrane is employed to protect the small volume of the extractant phase. The extraction technique is characterized for the resolution of a model analytical problem: the determination of five selected chlorophenols in water. A two-phase extraction mode is used for the extraction of the analytes with an organic solvent in which an in situ derivatization reaction takes place. The analytes are finally analyzed by gas chromatography/mass spectrometry. All the variables involved in the extraction process have been clearly identified and optimized. The new extraction mode allows the determination of chlorophenols with limits of detection in the range from 14.8 ng/L (for 2,4,5-trichlorophenol) to 22.9 ng/L (for 3-chlorophenol) with a relative standard deviation lower than 8.7% (for 2,6-dichlorophenol).     

Derivative spectrophotometry in the analysis of mixtures of phenols and herbicides

Derivative spectrophotometry (zero-crossing technique) was applied to the determination of selected phenols and herbicides in two-component mixtures. Methyl- and chlorophenols (3-methylphenol, 2,3- and 3,4-dimethylphenol, 2,5-, 2,6- and 3,4-dichlorophenol and 2,4,5-trichlorophenol) and triazine, uracil and urea herbicides (simazine, propazine, hexazinone, bromacil and metoxuron) were examined. The RSD values ranged between 0.05 and 4% and the recoveries obtained were between 97 and 110%. The developed derivative spectrophotometric method was also applied as a complementary technique for the separation of overlapping peaks of sample compounds obtained by HPLC with diode-array detection. Metoxuron and 3-methylphenol, metoxuron and 2,5-dichlorophenol and simazine and 2,6-dichlorophenol were determined simultaneously by this method at the level of 1 x 10(-3) g l-1.     

Determination of Carotenoids in the Chinese Medical Herb Jiao-Gu-Lan (Gynostemma Pentaphyllum MAKINO) by Liquid Chromatography

A QSTAR Pulsar quadrupole time-of-flight mass spectrometer was used for the determination of chlorophenols in surface water samples. The investigated compounds were: 2-chlorophenol; 4-chloro-3-methylphenol; 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol. Each analyte is listed by the US-EPA as a priority pollutant. Sample enrichment of water samples was achieved by a solid-phase extraction procedure, using a Waters Oasis HLB cartridges followed by LC-Tandem-MS. A narrow-bore 2.1-mm-i.d. reversed phase LC C-18 column operating with a mobile phase flow rate of 0.2 mL min^–1. was used to separate the analytes. The whole column effluent was diverted to the ion spray interface source. For the determination of the analytes the hybrid quadrupole time-of-flight spectrometer operated in product ion scan acquisition mode. Average recoveries from 2 L samples varied from 91 to 110% and relative standard deviations (RSD) were less than 10% for all samples. The limit of detection (signal-to-noise ratio=3) of the method for the phenols in drinking water samples is less than 10 ng L^–1. In real environmental samples, levels of the selected analytes varied from non-detected up to 0.5 g L^–1 for pentachlorophenol.     

Development and evaluation of C18 and immunosorbent solid-phase extraction methods prior immunochemical analysis of chlorophenols in human urine

Two solid-phase extraction (SPE) methods, based on hydrophobic and selective (antibody-antigen) interactions, have been established and evaluated as clean-up methods prior the immunochemical analysis of 2,4,6-trichlorophenol (2,4,6-TCP) in urine samples. Without a clean-up method the extent of interferences caused by the urine matrix in the ELISA [R. Galve, M. Nichkova, F. Camps, F. Sanchez-Baeza, M.-P. Marco, Anal. Chem. 74 (2002) 468] varies depending on individual urine samples and accurate measurements are only possible when 2,4,6-TCP concentration levels are higher than 40 μg L⁻¹. Both sample preparation methods improve detectability of the immunochemical method getting rid of the variability due to the intrinsic individual differences within the urine samples. Even though, the immunosorbent (IS)-SPE method developed has proven to be a superior sample preparation method eliminating completely matrix effects caused by both, non-hydrolyzed (NH) and hydrolyzed urine samples. The LOD reached by the C18-SPE-ELISA method (∼4 μg L⁻¹ for free and total chlorophenols) is sufficient for exposure assessment of the occupationally exposed population. However, the detectability (0.66 and 0.83 μg L⁻¹ in NH and hydrolyzed urine samples, respectively) accomplished by the IS-SPE-ELISA allows also biomonitoring potential exposure of non-occupationally exposed groups. Moreover, the specificity of the IS-SPE procedure can be modulated to provide a group-specific (9 chlorophenols and 2 bromophenols are extracted with an efficacy superior to 85%) or a more selective protocol (only 2,3,4,6-TtCP, 2,4,6-TCP are extracted with a recovery superior to 80% and 2,4,6-tribromophenol with a 70% recovery). On the other hand, the IS-SPE extracts produce cleaner chromatograms allowing quantitation by GC-ECD (or GC-MS) after toluene extraction and derivatization with a LOD near 0.1 μg L⁻¹ in NH and hydrolyzed urine samples. The IS-SPE-ELISA method has been validated with GC-ECD using spiked and real urine samples. This study also provides evidences of the general exposure of the population to organochlorinated and organobrominated substances. Measurable levels of 2,4,6-TCP, 2,4,5-TCP, 2,3,4,6-TtCP, 2,4,6-TBP and 2,4-DBP have been detected in some of the samples used in this study.     

固相萃取-气相色谱-质谱法测定木制家具中氯酚类及菊酯类防腐剂

建立了固相萃取-气相色谱-质谱(SPE-GC-MS)检测含氯酚类化合物(2,4-二氯苯酚、2,4,6-三氯苯酚、2,4,5-三氯苯酚、2,3,4,6-四氯苯酚、五氯苯酚、林丹)和菊酯类化合物(氯菊酯、氟氯氰菊酯、氯氰菊酯、溴氰菊酯)等10种木材防腐剂的方法。对家具样品采用超声萃取法、以甲醇为提取剂在室温下反复提取2次,提取液经浓缩后,加入碳酸钾和乙酰酐衍生化,将衍生化后的溶液通过Oasis HLB固相萃取柱净化,用乙酸乙酯洗脱并收集检测。采用该方法实现了家具中10种木材防腐剂的分离检测,该方法中氯酚类防腐剂定量限为1 mg/kg、菊酯类防腐剂定量限为5 mg/kg,平均回收率为76.0%～108.8%。应用该方法对市场上销售的木制家具进行了检测,在部分家具中检出含有少量林丹。实验结果证明,该方法准确、灵敏,可有效地应用于木制家具中防腐剂的实际检验工作中。     

Identification and Determination of Chlorophenols in Potable Water by a Kinetic Gas-Chromatographic Method

A kinetic gas-chromatographic method was developed for the identification and trace determination of 2- and 4-chlorophenols, 2,4- and 2,6-dichlorophenols, and 2,4,6-trichlorophenol in potable water. The method is based on the oxidation of bromo derivatives of chlorophenols by excess molecular bromine. Optimal conditions of analysis, analytical range (0.05–50 g/L), and relative standard deviation (5–20%) were determined.     

Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry for determination of chlorophenols in water and body fluid samples

A new method, stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS), which is used for the determination of trace amounts of chlorophenols, such as 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TrCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) and pentachlorophenol (PCP), in tap water, river water and human urine samples, is described. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as extraction time are investigated. Then, the stir bar is subjected to TD followed by GC-MS. The detection limits of the chlorophenols in tap water, river water and human urine samples are 1-2, 1-2, and 10-20 pg ml⁻¹ (ppt), respectively. The calibration curves for the chlorophenols are linear and have correlation coefficients higher than 0.99. The average recoveries of the chlorophenols in all the samples are higher than 95% (R.S.D. < 10%) with correction using added surrogate standards, 2,4-dichlorophenol-d₅, 2,4,6-trichlorophenol-¹³C₆, 2,3,4,6-tetrachlorophenol-¹³C₆ and pentachlorophenol-¹³C₆. This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of chlorophenols in liquid samples.     

Selektive photometrische Bestimmung von Hexachlorophen neben 2,4,5-Trichlorphenol durch Reaktion mit Kupfer(II)

Zusammenfassung Das vorgeschlagene Verfahren zur Bestimmung von Hexachlorophen in Gegenwart von 2,4,5-Trichlorphenol beruht auf der Reaktion mit Kupfer(II) in ammoniakalischem Medium und der photometrischen Messung der Färbung des dabei gebildeten 11-Komplexes bei 423 bzw. 434 nm. Die Methode wurde auf verschiedene Handelsprodukte angewendet. Ähnliche Substanzen, wie z.B. die Tetrachlor- und die Dichlorverbindung, geben eine analoge Reaktion.

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Selective photometric determination of hexachlorophene in presence of 2,4,5-trichlorophenol by the reaction with copper(II)

The reaction is carried out in ammoniacal medium and the absorption of the coloured 11 complex formed is measured at 423 or 434 nm. The method has been applied to the determination of hexachlorophene in different commercial products. Similar substances, as e.g. the tetrachloro and dichloro compounds yield the analogous reaction.

     

纺织品中残留氯酚的毛细管气相色谱测定法

采用稀硫酸浸湿样品,正己烷提取,乙酸酐衍生后以毛细管气相色谱分离测定的方法对纺织品中五氯酚、三氯酚残留量进行了同时测定,探讨了提取、净化及色谱分析条件。方法回收率范围三氯酚84.8％～98.1％,五氯酚88.0％～100.2％。相对标准偏差三氯酚1.54％～2.33％,五氯酚3.48％。方法的检出限（质量分数）分别为2,4,6-三氯酚1.0×10     

改进的五氟苄基溴衍生化反应测定水中酚类化合物

建立了气相色谱-质谱法测定水中6种酚类化合物(2,6-二氯酚、2,4-二氯酚、2,4,6-三氯酚、2,4,5-三氯酚、2,3,4,6-四氯酚和五氯酚)的方法.样品经二氯甲烷-乙酸乙酯混合溶剂萃取后,用旋转蒸发浓缩至1 mL,加入五氟苄基溴进行改进版衍生化反应,产物用DB-5 mS毛细管柱分离,采用选择离子监测模式测定....     

Erfassung von chlorierten Phenolen in Herbicid-S?uren mit der Capillar-GC/FTIR-Spektroskopie

Zusammenfassung Zur Trennung und substanzspezifischen Erfassung von chlorierten Phenolen in Herbicidsäuren vom Typ der chlorierten Phenoxyalkansäuren wird die Kombination der Capillar-GC mit Quarz-Dünnfilmcapillaren mit der FTIR-Spektroskopie angewandt. Die Phenole werden mit Diisopropylether aus wäßriger NaHCO₃-Lösung extrahiert und im Kuderna-Danish-Evaporator eingeengt. Die gaschromatographische Trennung erfolgt in einer Quarzcapillare mit chemisch gebundenem SE-54. Die Wiederfindungsraten — mit FI-Detektion und 3,5-Xylenol als internem Standard ermittelt — liegen zwischen 90 und 105% bei einem Konzentrationsniveau von 100 /g. Mit der GC/FTIR wurden in 2,4,5-T-Säuren handelsüblicher Qualität neben dem 2,4,5-Trichlorphenol noch zwei Tetrachlorphenole sowie Pentachlorphenol identifiziert. Es ist gezeigt, daß für eine eindeutige qualitative Aussage das FTIR-Spektrometer von der Nachweisempfindlichkeit her als molekülspezifischer Detektor zur Capillar-GC für diese Gruppe von Verbindungen anwendbar ist.

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Determination of chlorinated phenols in herbicidic acids using capillary-GC/FTIR-spectroscopy

Summary The on-line combination of capillary-GC using fused silica WCOT columns with FTIR-spectroscopy is applied to the separation and substance-specific determination of chlorinated phenols in chlorinated aryloxyalkanoic acids. Phenols are extracted with diisopropylic ether from aqueous NaHCO₃ solution and concentrated by means of a Kuderna-Danish evaporator. GC separation is done in a fused silica WCOT with chemically bonded SE-54. Recoveries are between 90 and 105% at the 100-g/g concentration level. In commercial 2,4,5-T-acid two tetrachlorophenols and pentachlorophenol were identified with GC/FTIR besides 2,4,5-trichlorophenol. It is shown, that the FTIR-spectrometer is applicable as a molecule-specific detector in respect to spectral detection sensitivity for obtaining unambiguous qualitative information in this group of compounds.

     

Determination of chlorophenols in urine of children and suggestion of reference values

During the course of a human biomonitoring project (Biebesheim in Hessen, Germany) we elaborated a simple but sensitive method for the determination of tri- (TCP), tetra- (TeCP) and pentachlorophenol (PCP) in human urine. Urine samples, spiked with internal standards, were treated by acid hydrolysis. After a steam bath distillation the distillates were extracted using solid phase extraction. Derivatization of the chlorophenols was not carried out. GC/ECD system was used for detection. Detection limits of the chlorophenols were found in the range of 0.02 μg/L urine (detection limits of the ECD: 0.52 to 2.76 μg/L). By this method mono- and dichlorophenols cannot be detected. We investigated 24h-urine samples of 339 pupils (age 10 to 12 years). The children live either in the surroundings of a hazardous waste incinerator (SVA) in Biebesheim (n = 193), or controls (i.e. regions without waste incinerator) in the non polluted areas of Odenwald (n = 90) and Rheintal (n = 56). Between these three groups we did not find statistically significant differences in chlorophenol concentrations of the urine samples. The 95-percentiles of the analyzed samples are 0.74 μg/L (2,3,4-TCP), 1.24 μg/L (2,3,5-TCP), 0.70 μg/L (2,3,6–TCP), 1.10 μg/L (2,4,5–TCP), 1.74 μg/L (2,4,6–TCP), 2.84 μg/L (3,4,5–TCP), 4.78 μg/L (2,3,4,5-TeCP), 1.86 μg/L (2,3,4,6-TeCP), 2.90 μg/L (2,3,5,6-TeCP) and 4.39 μg/L (PCP). Received: 24 February 1999 / Revised: 3 May 1999 / Accepted: 6 May 1999