Stability of the conjugated species of environmental phenols and parabens in human serum

In humans, the metabolism of environmental phenols may include the formation of conjugated species (e.g., glucuronides and sulfates), but the free species—not the conjugated forms—are considered biologically active. Therefore, information on the concentration of these free species in blood or urine could be helpful for risk assessment. Because conjugates could hydrolyze to their corresponding free forms during collection, handling, and storage of biological specimens, information on the temporal stability of the conjugates is of interest. Previously, we reported the temporal stability of urinary conjugates of several environmental phenols, but data on the stability of phenols' conjugated species in serum, albeit critical if concentrations of free and conjugated species are compared, are largely unknown. In the present study, we investigate the stability of the conjugates of four phenols—bisphenol A, benzophenone-3, triclosan, and 2,5-dichlorophenol—and two parabens—methyl paraben and propyl paraben—in 16 human serum samples for 30 days at above-freezing temperature storage conditions (4 °C, room temperature, and 37 °C). These conditions reflect the worst-case scenarios that could occur during the short-term storage of biological samples before their long-term storage at controlled subfreezing temperatures. We found that the percentage of the conjugated species of the four detected compounds (2,5-dichlorophenol, triclosan, and methyl and propyl parabens) in these serum specimens even when stored at 37 °C for at least 30 days did not vary significantly. These preliminary data suggest that the phenols' serum conjugates appear to be more stable than their corresponding urinary conjugates, some of which started to hydrolyze within 24 h under similar storage conditions. The reported stability of these conjugated species in human serum also suggests that the free species are unlikely to have resulted from the hydrolysis of their corresponding conjugates. This information could be important for interpreting the low concentrations of free phenol species detected in serum samples of nonoccupationally exposed populations. To our knowledge, this is the first study to report on the stability of conjugated species in serum, and as such requires replication.     

Hydroxylated polychlorobornanes--synthesis and characterization of new potential toxaphene metabolites

For decades, toxaphene had been used as a major chloropesticide. Degradation of the multicomponent mixture in the environment was mainly reported to be due to anaerobic dechlorination and hydrodechlorination. Little was known about oxidative transformation processes and the potential hydroxylated metabolites were not available as standard compounds. For this reason we synthesized hydroxylated polychlorobornanes by the UV-induced photochlorination of 2-endo-bornyl acetate with sulfuryl chloride followed by hydrolysis of the acetate moiety. The released polychlorinated 2-endo-hydroxybornanes were slightly higher chlorinated the longer the reaction was maintained. After 8 h, the main products were pentachlorinated hydroxybornanes followed by hexa- and heptachlorinated homologues. Traces of octachlorinated hydroxybornanes were also observed. The GC/ECNI-MS spectra of the products were characterized by the molecular ions and the [M-Cl]⁻ fragment ions. The molecular ions of the polychlorinated hydroxybornanes are isobaric with those of polychlorinated biphenyls. E.g. hexachlorohydroxybornanes (C₁₀H₁₂Cl₆O) and hexachlorobiphenyls (C₁₂H₄Cl₆) show the molecular ion at m/z 358. Based on fractionation experiments on silica with the synthesis products it might be possible that OH-CTTs if present in samples will elute into a more polar fraction usually discarded or not collected. Both problems might explain why these compounds have not been more frequently described in the scientific literature.     

Rapid determination of dichlorodiphenyltrichloroethane and its main metabolites in aqueous samples by one-step microwave-assisted headspace controlled-temperature liquid-phase microextraction and gas chromatography with electron capture detection

A rapid and sensitive analytical method for the determination of dichlorodiphenyltrichloroethane (DDT) and its main metabolites in environmental aqueous samples has been developed using one-step microwave-assisted headspace controlled-temperature liquid-phase micro-extraction (MA-HS-CT-LPME) technique coupled with gas chromatography-electron-capture detection (GC-ECD). In this study, the one-step extraction of DDT and its main metabolites was achieved by using microwave heating to accelerate the evaporation of analytes into the controlled-temperature headspace to form a cloudy mist vapor zone for LPME sampling. Parameters influencing extraction efficiency were thoroughly optimized, and the best extraction for DDT and its main metabolites from 10-mL aqueous sample at pH 6.0 was achieved by using 1-octanol (4-μL) as the LPME solvent, sampling at 34 °C for 6.5 min under 249 W of microwave irradiation. Under optimum conditions, excellent linear relationship was obtained in the range of 0.05-1.0 μg/L for 1-dichloro-2,2-bis-(p′-chlorophenyl)ethylene (p,p′-DDE), 0.1-2.0 μg/L for o,p′-DDT, 0.15-3.0 μg/L for 1,1-dichloro-2,2-bis-(p′-chlorophenyl)ethane (p,p′-DDD) and p,p′-DDT, with detection limits of 20 ng/L for p,p′-DDE, and 30 ng/L for o,p′-DDT, p,p′-DDD and p,p′-DDT. Precision was in the range of 3.2-11.3% RSD. The proposed method was validated with environmental water samples. The spiked recovery was between 95.5% and 101.3% for agricultural-field water, between 94% and 99.7% for sea water and between 93.5% and 98% for river water. Thus the established method has been proved to be a simple, rapid, sensitive, inexpensive and eco-friendly procedure for the determination of DDT and its main metabolites in environmental water samples.     

Unusual polar metabolites in the groundwater of a contaminated waste site indicate a new pathway of mononitrotoluene transformation

At a mononitrotoluene-contaminated waste disposal site, the groundwater was screened for polar transformation products of mononitrotoluenes, by means of HPLC-MS, HPLC-NMR and further off-line NMR and MS techniques. Besides expected metabolites such as aminotoluenes (ATs) and nitrobenzoic acids (NBAs), three unknowns (di- and tetrahydro-derivatives of (2-oxo-quinolin-3-yl) acetic acid) could be identified which, in the context of explosives and related compounds, are new metabolites. Evidence could be provided by microcosm experiments with 2-nitrotoluene (2-NT) that these metabolites are microbial transformation products of 2-NT under anaerobic conditions. The NMR and MS data are presented and the possible pathway for the formation of these metabolites after addition of 2-NT to fumarate is discussed.     

PAH Metabolites in Bile Fluids of Dab (Limanda limanda) and Flounder (Platichthys flesus): Spatial Distribution and Seasonal Changes (7 pp)

Background, Aim and Scope Polycyclic aromatic hydrocarbons (PAH) are important environmental contaminants which may lead to increased levels of neoplastic aberrations or tumours in fish liver. Therefore, monitoring of PAH and their effects are part of several international environmental programmes. The aim of the present field study was to investigate the concentrations of the PAH metabolites in fish bile, to elucidate spatial, seasonal and species differences as well as to discuss different strategies of normalisation with regard to environmental monitoring. Materials and Methods: PAH metabolites were determined in the bile fluid of dab (Limanda limanda) and flounder (Platichthys flesus) caught in the North Sea and Baltic Sea between 1997 and 2004. After enzymatic deconjugation, two metabolites were determined by means of HPLC. The limit of detection and the limit of quantification were calculated. The accuracy of the method was tested with a standard reference material. Results were referred to bile volume as well as to biliverdin. Results: The main metabolite, 1-hydroxypyrene, was determined in concentrations from <0.7 to 838 ng/ml in bile of dab (Limanda limanda) and flounder (Platichthys flesus) caught between 1997 and 2004. The values for 1-hydroxyphenanthrene in fish bile were considerably lower (<0.4 – 87 ng/ml). Significant differences in the 1-hydroxypyrene levels were found between summer and winter surveys as well as between the sampling sites in the data set from 2004 (383 dabs and 62 flounders): Highest levels of PAH contamination were found in dab from the German Bight and in flounder from the Baltic Sea. Discussion: Spatial differences in 1-hydroxypyrene concentrations between North Sea and Baltic Sea were discussed, as well as differences in relation to season, sex and species. Three parameters of normalisation (biliary protein, biliverdin and bile pigments) were discussed. Biliverdin was identified as a suitable parameter for the normalisation of PAH metabolites in field samples. Conclusions: Spatial differences in 1-hydroxypyrene concentrations of dab demonstrate the usefulness of PAH metabolites in fish bile as a monitoring parameter in marine regions. Significant differences in 1-hydroxypyrene concentrations were found between summer and winter sampling campaigns. This may be linked to an annual cycle of 1-hydroxyprene in dab. It is also possible that bile synthesis/release in dab differs between the seasons. There is no indication for a time trend from 1997 to 2004. Recommendations and Perspectives: It is recommended to relate PAH metabolites in fish bile to biliverdin concentrations. Although the concentrations are low in offshore regions and bile volumes are small, the method presented here allows one to measure PAH metabolites on an individual level which is a crucial prerequisite for meaningful monitoring studies.