High abundance of pyrrolizidine alkaloids in bee pollen collected in July 2019 from Southern Germany.

Pyrrolizidine alkaloids (PA) are secondary plant defense compounds and known pre-toxins when containing a 1,2-double bond. They are commonly produced by various plants and may thus be present in bee pollen which may be consumed by humans as food supplements. In this study, PA were determined in bee pollen samples from 57 locations in Southern Germany sampled by means of pollen traps in July 2019. Samples were analyzed by using palynological methodology and solid-phase extraction (SPE) followed by LC-MS/MS. In total, 52 pollen samples featured total pyrrolizidine alkaloids (ΣPA) with concentrations up to 48,000 ng/g bee pollen, while the N-oxides (NO) echinatine-NO and rinderine-NO clearly dominated. In contrast, the palynological analysis only detected 33 samples with pollen from PA-producing plants. Accordingly, the results showed that palynological analysis is not sufficient to determine PA in pollen. In addition, a risk assessment was followed to estimate the risk of the detected PA concentrations to humans.

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 8h, 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.

Offline coupling of high-speed counter-current chromatography and gas chromatography/mass spectrometry generates a two-dimensional plot of toxaphene components.

High-speed counter-current chromatography (HSCCC), a separation technique based solely on the partitioning of solutes between two immiscible liquid phases, was applied for the fractionation of technical toxaphene, an organochlorine pesticide which consists of a complex mixture of structurally closely related compounds. A solvent system (n-hexane/methanol/water 34:24:1, v/v/v) was developed which allowed to separate compounds of technical toxaphene (CTTs) with excellent retention of the stationary phase (S(f) = 88%). Subsequent analysis of all HSCCC fractions by gas chromatography coupled to electron-capture negative ion mass spectrometry (GC/ECNI-MS) provided a wealth of information regarding separation characteristics of HSCCC and the composition of technical toxaphene. The visualization of the large amount of data obtained from the offline two-dimensional HSCCC-GC/ECNI-MS experiment was facilitated by the creation of a two-dimensional (2D) contour plot. The contour plot not only provided an excellent overview of the HSCCC separation progress, it also illustrated the differences in selectivity between HSCCC and GC. The results of this proof-of-concept study showed that the 2D chromatographic approach involving HSCCC facilitated the separation of CTTs that coelute in unidimensional GC. Furthermore, the creation of 2D contour plots may provide a useful means of enhancing data visualization for other offline two-dimensional separations.

Structure elucidation of an artifact discharging from rubber-based vial closures by means of gas chromatography/tandem mass spectrometry.

The use of vial closures equipped with butyl rubber septa may lead to sample contamination by rubber additives discharging from the septum material. In this study, the structure elucidation of an artifact causing intense signals in gas chromatography/electron capture negative ion mass spectrometry (GC/ECNI-MS) and gas chromatographic analyses with electron capture detection is described. Tentative identification of the leached compound was achieved by employing tandem mass spectrometric techniques both in electron capture negative ion and in electron ionization modes. The artifact could thus be characterized as 2-benzothiazolyl-N,N-dimethyl dithiocarbamate, which is a known vulcanization accelerator for rubber. It is conceivable that the identified compound or related substances are also used in other applications. Therefore, two food-related matrixes were investigated for a possible migration of this compound into foods. During these analyses, the tentatively identified rubber additive was detected in an aqueous extract of a rubber seal ring for canning jars. GC/ECNI-MS provided better sensitivity and selectivity than GC/EI-MS for the determination of the rubber additive and other mercaptobenzothiazole-derived substances.

Synthesis of low and high chlorinated toxaphene and comparison of their toxicity by zebrafish (Danio rerio) embryo test.

Toxaphene, also known as camphechlor, is a persistent organochlorine pesticide of complex composition. It is technically produced by photochlorination of camphene with elemental chlorine gas under ultraviolet irradiation. In the present work, a novel, laboratory-scale synthesis using sulfuryl chloride as a chlorinating reagent is described. This approach allowed the degree of chlorination of the resulting mixtures to be arbitrarily adjusted by varying the reaction conditions. Both the compositions and the chlorine contents of the low- and high-chlorinated mixtures acquired using this method were similar to those of environmentally altered toxaphene and technical toxaphene, respectively. For comparison of these mixtures regarding toxicity, they were subjected to the zebrafish (Danio rerio) embryo test. Median effective concentrations (EC50s) were calculated based on the presence of lethal and nonlethal embryonic malformations. Surprisingly, low-chlorinated toxaphene, comprising compounds that also are present in environmentally transformed toxaphene, exhibited a twofold-higher toxicity (according to the EC50 for nonlethal effects) toward the test organisms compared with high-chlorinated toxaphene, the composition of which resembled that of the technical product. Although the effective concentrations in the embryo test were much higher than those in aquatic ecosystems burdened with toxaphene, the present results lead to the assumption that toxaphene is becoming more toxic during transformation in the environment. A decrease in the total amount of toxaphene during environmental breakdown would then be compensated for, at least in part, by the higher toxicity of weathered toxaphene in sediments, soils, and biota of contaminated ecosystems.

Baseline isotopic data of polyhalogenated compounds.

The delta2H- and delta13C-values of polyhalogenated compounds were determined by EA-IRMS. Most of the compounds were related to the chloropesticides DDT and its metabolites, hexachlorocyclohexanes, and toxaphene, as well as several polybrominated compounds such as bromophenols and -anisoles. delta2H-values ranged between -235 per thousand and +75 per thousand whereas delta13C-values were found in the range -22 per thousand to -38 per thousand. No correlation between delta2H- and delta13C-values could be identified. Comparative analysis clarified that bromophenols and the corresponding bromoanisoles may vary in their isotopic distribution. 2H NMR was used to quantify abundances of 2H isotopomers. Quantification of isotopomers of 2,4-dibromophenol and 2,4-dibromoanisole proved that both compounds from different suppliers do not originate from the same source. Differences in the delta2H-values of two toxaphene products were further investigated by the synthesis of products of different degree of chlorination from camphene. It was shown that the delta13C-values remained mostly unaltered as was expected since no carbon is lost in this procedure. However, the reaction products became enriched in 2H with increasing degree of chlorination. Different delta2H-values of the starting material will also impact the delta2H-values of the chlorination products.