Uncertainties in the determination of pyrrolizidine alkaloid levels in naturally contaminated honeys and comparison of results obtained by different analytical approaches

The contamination of honey with hepatotoxic pyrrolizidine alkaloids (PAs) is a well-known hazard for food safety. While management strategies and controls of the honey industry aim to reduce the PA levels, uncertainties remain with regard to the safety of regionally produced and marketed honey. In addition, a previous study showed large differences of results obtained after various periods of storage and apparent differences between the analytical results of different laboratories. Therefore, this study aimed at examining these uncertainties by monitoring the impact of storage on the PA and PA N-oxide (PANO) content of two freshly harvested honeys and on possible demixing effects caused by pollen settling. Additionally, three analytical approaches – target analysis with matrix-matched calibration or standard addition and a sum parameter method – were applied for a comparative analysis of 20 honeys harvested in summer 2016. All samples originated from Schleswig-Holstein in Northern Germany where the PA plant Jacobaea vulgaris is currently observed on a massive scale. The results of the time series analyses showed that PANO levels markedly decreased within a few weeks and practically reached the LOD 16 weeks after harvest. Tertiary PAs, by contrast, remained stable and did not increase as a consequence of PANO decrease. The experiments on a putative demixing, which may result in a heterogeneous distribution of PAs/PANOs, revealed that there was no such effect during storage of up to 12 weeks. A comparison of the PA/PANO levels obtained by different analytical approaches showed that in some cases the sum parameter method yielded much higher levels than the target approaches, whereas in other cases, the target analysis with standard addition found higher levels than the other two methods. In summary, the results of this study highlight uncertainties regarding the validity and comparability of analytical results and consequently regarding health risk assessment.     

Ecophysiology and niche differentiation of Nitrospira-like bacteria, the key nitrite oxidizers in wastewater treatment plants.

Nitrite-oxidizing bacteria of the genus Nitrospira are key nitrifiers in wastewater treatment plants. Pure cultures of these organisms are unavailable, but cultivation-independent molecular methods make it possible to detect Nitrospira-like bacteria in environmental samples and to investigate their ecophysiology. Comprehensive screening of natural and engineered habitats and of public databases for 16S rRNA sequences of Nitrospira-like bacteria revealed a surprisingly high biodiversity in the genus Nitrospira, which comprises at least four phylogenetic sublineages. All Nitrospira-like bacteria detected in wastewater treatment plants belonged to the sublineages I and II. Subsequently, the population dynamics of different Nitrospira-like bacteria were monitored, by quantitative fluorescence in situ hybridization with rRNA-targeted probes, confocal laser scanning microscopy and digital image analysis, during incubation of nitrifying activated sludge in media containing different nitrite concentrations. These experiments showed that Nitrospira-like bacteria, which were affiliated with the phylogenetic sublineages I or II of the genus Nitrospira, responded differently to nitrite concentration shifts. Previously unknown properties of Nitrospira-like bacteria were discovered in the course of an environmental genomics project. Implications of the obtained results for fundamental understanding of the microbial ecology of nitrite oxidizers as well as for future improvement of nutrient removal in wastewater treatment plants are discussed.