The potential of multispectral imaging flow cytometry for environmental monitoring.

Environmental monitoring involves the quantification of microscopic cells and particles such as algae, plant cells, pollen, or fungal spores. Traditional methods using conventional microscopy require expert knowledge, are time-intensive and not well-suited for automated high throughput. Multispectral imaging flow cytometry (MIFC) allows measurement of up to 5000 particles per second from a fluid suspension and can simultaneously capture up to 12 images of every single particle for brightfield and different spectral ranges, with up to 60x magnification. The high throughput of MIFC has high potential for increasing the amount and accuracy of environmental monitoring, such as for plant-pollinator interactions, fossil samples, air, water or food quality that currently rely on manual microscopic methods. Automated recognition of particles and cells is also possible, when MIFC is combined with deep-learning computational techniques. Furthermore, various fluorescence dyes can be used to stain specific parts of the cell to highlight physiological and chemical features including: vitality of pollen or algae, allergen content of individual pollen, surface chemical composition (carbohydrate coating) of cells, DNA- or enzyme-activity staining. Here, we outline the great potential for MIFC in environmental research for a variety of research fields and focal organisms. In addition, we provide best practice recommendations.

Community metabolomics provides insights into mechanisms of pollution-induced community tolerance of periphyton.

Chemical pollution is a major concern for freshwater ecosystems, but the impact and mechanisms of chemical stressors on communities are barely understood. Pollution stress beyond natural homeostatic capacities can trigger succession of tolerant species within a community, enhancing the overall community tolerance. This process was operationalized in the Pollution-Induced Community Tolerance (PICT) concept and applied in many case studies, however, the molecular mechanisms of community tolerance and implications for ecological functions remain largely unexplored. Our study aimed to demonstrate that 1) community metabolomics can unravel potential mechanisms of PICT in periphyton and 2) induced tolerance helps to maintain primary production under re-occuring pollution. To this end, we grew periphyton for 5 weeks with and without the model herbicide diuron in microcosms, quantified PICT, and determined the related metabolic fingerprint of periphyton by GC-MS-based untargeted metabolomics. Further, we explored the autotrophic community based on pigment composition and functional parameters including photosynthesis and gross primary production. Chronic diuron exposure resulted in a shift in pigment composition, higher community tolerance and an individual metabolic fingerprint in the contaminated communities. Opposing responses of selected metabolites during a short-term exposure indicated differences in diuron pre-adaptation in the different communities. Metabolites (threonic acid and two sugar acid lactones) were found to be related to tolerance development, suggesting that ascorbate metabolism was induced in contaminated communities. Despite these compensating mechanism, contaminated communities were compromised in production-to-respiration ratio and biomass. A ranking of sensitivity thresholds of different biological endpoints revealed that metabolites were less sensitive than photosynthetic parameters, which reflects the mode-of-action of the herbicide. In conclusion, we could demonstrate that community metabolomics is able to unravel complex biochemical changes and allows mechanistic insights into community tolerance. Moreover, we were able to show that induced community tolerance was insufficient to safeguard functions like primary production.

The impact of chemosensitisation on bioaccumulation and sediment toxicity.

Cellular multixenobiotic resistance (MXR) transport proteins enhance the efflux of numerous organic pollutants. However, MXR proteins may be blocked or saturated by xenobiotic compounds, acting as inhibitors - also called chemosensitisers. Although effective on a cellular level, the environmental relevance of chemosensitisers has not been conclusively demonstrated. Since sediments are an important source of bioaccumulating compounds in aquatic ecosystems, sediments and sediment-associated hydrophobic pollutants were investigated for their potential to increase exposure and toxicity in the presence of chemosensitisation. In this study, we address this issue by (1) comparing the net uptake of 17 hydrophobic environmental pollutants by zebrafish (Danio rerio) embryos in the presence and absence of the model chemosensitiser verapamil and (2) investigating the impact of verapamil on the dose-dependent effect on zebrafish embryos exposed to polluted sediment extracts. None of the 17 pollutants showed a reproducible increase in bioaccumulation upon chemosensitisation with verapamil. Instead, internal concentrations were subject to intra-species variation by a factor of approximately two. However, a significant increase in toxicity was observed upon embryo co-exposure to verapamil for one of three sediment extracts. In contrast, another sediment extract exhibited less toxicity when combined with verapamil. In general, the results indicate only a minor impact of verapamil on the uptake of moderately hydrophobic chemicals in zebrafish embryos.