Stream biofilms have been shown to be among the most sensitive indicators of environmental stress in aquatic ecosystems and several endpoints have been developed to measure biofilm adverse effects caused by environmental stressors. Here, we compare the effects of long-term exposure of stream biofilms to diuron, a commonly used herbicide, on several traditional ecotoxicological endpoints (biomass growth, photosynthetic efficiency, chlorophyll-a content, and taxonomic composition), with the effects measured by recently developed methods [community structure assessed by flow cytometry (FC-CS) and measurement of extracellular polymeric substances (EPS)]. Biofilms grown from local stream water in recirculating microcosms were exposed to a constant concentration of 20 μg/L diuron over a period of 3 weeks. During the experiment, we observed temporal variation in photosynthetic efficiency, biomass, cell size, presence of decaying cells and in the EPS protein fraction. While biomass growth, photosynthetic efficiency, and chlorophyll-a content were treatment independent, the effects of diuron were detectable with both FC and EPS measurements. This demonstrates that, at least for our experimental setup, a combination of different ecotoxicological endpoints can be important for evaluating biofilm environmental stress and suggests that the more recent ecotoxicological endpoints (FC-CS, EPS protein content and humic substances) can be a useful addition for stream biofilm ecotoxicological assessment.

Biofilms serve essential ecosystem functions and are used in different technical applications. Studies from stream ecology and waste water treatment have shown that biofilm functionality depends to a great extent on community structure. Here we present a fast and easy-to-use method for individual cell-based analysis of stream biofilms, based on stain-free flow cytometry and visualization of the high-dimensional data by viSNE. The method allows the combined assessment of community structure, decay of phototrophic organisms and presence of abiotic particles. In laboratory experiments, it allows quantification of cellular decay and detection of survival of larger cells after temperature stress, while in the field it enables detection of community structure changes that correlate with known environmental drivers (flow conditions, dissolved organic carbonDOC, calcium) and detection of microplastic contamination. The method can potentially be applied to other biofilm types, e.g. for inferring community structure for environmental and industrial research and monitoring.

Micropollutants are ubiquitously found in natural surface waters and pose a potential risk to aquatic organisms. Stream biofilms, consisting of bacteria, algae and other microorganisms potentially contribute to bioremediating aquatic environments by biotransforming xenobiotic substances. When investigating the potential of stream biofilms to remove micropollutants from the water column, it is important to distinguish between different fate processes, such as biotransformation, passive sorption and active bioaccumulation. However, due to the complex nature of the biofilm community and its extracellular matrix, this task is often difficult. In this study, we combined biotransformation experiments involving natural stream biofilms collected up- and downstream of wastewater treatment plant outfalls with the QuEChERS extraction method to distinguish between the different fate processes. The QuEChERS extraction proved to be a suitable method for a broad range of micropollutants (> 80% of the investigated compounds). We found that 31 out of 63 compounds were biotransformed by the biofilms, with the majority being substitution-type biotransformations, and that downstream biofilms have an increased biotransformation potential towards specific wastewater-relevant micropollutants. Overall, using the experimental and analytical strategy developed, stream biofilms were demonstrated to have a broad inherent micropollutant biotransformation potential, and to thus contribute to bioremediation and improving ecosystem health.