Multiple stressors in river networks: Local and downstream effects on freshwater macroinvertebrates

River networks are complex ecosystems characterized by a continuous exchange of material and energy through longitudinal gradients. These ecosystems are threatened by various human-induced stressors, which frequently co-occur and may interact in complex ways, potentially triggering cascading effects in the river network. Aiming at assessing the single and combined effects of flow intermittency and light pollution on macroinvertebrate communities, we performed a multiple stressor experiment in 18 flow-through mesocosms. Each mesocosm was designed to mimic a simplified river network, with two upstream tributaries merging downstream, to assess both local and cascading effects. The experiment was performed in Summer 2021 for seven weeks, applying the stressors either separately or co-occurring in the upstream sections, following a randomized block design. Flow intermittency was simulated as the ponded phase of the drying process, whereas light pollution was applied with LED strips (set at 10 lux). Drifting macroinvertebrates were sampled weekly during the treatment phase, and benthic macroinvertebrates were sampled at the end of the treatment phase. Both stressors individually applied reduced benthos richness and abundance, whereas drift decreased with flow intermittency and increased with light pollution. When co-occurring upstream, stressors showed dominant effects of flow intermittency on the benthos and interactive effects on the drift. The effects of the single stressors and their interactions cascaded along the river network, with stronger downstream effects when stressors co-occurred upstream. These findings showed that the spatial distribution of multiple stressors along the river network can affect their resultant downstream effects, highlighting the importance of framing multiple stressors research in a spatial context. Considering the pressing needs of the growing human population, our results represent a step forward in anticipating cumulative stressors' effects, informing efficient conservation strategies for protecting freshwater ecosystems.

Methods
Data were collected during a field experiment in Summer 2021, using the Disconnected system, a system of 18 flow-through mesocosms, at the Eußerthal Ecosystem Research Station (EERES, RPTU Kaiserslautern-Landau), in the Palatinate Forest (Rheinland-Pfalz, Germany). The Disconnected system is a mesocosm system designed to mimic a simplified river network, with two upstream tributaries merging together downstream. The experiment lasted seven weeks, with three weeks of colonisation and four weeks of treatment. Drift data were collected weekly during the treatment phase of the experiment. Benthos data were collected at the end of the experiment. 

Drift samples were collected by placing drift nets at the outlet of each upstream section. Benthos samples were collected from sampling areas of 150 cm2 with two replicates in each section (six samples per flume).

All macroinvertebrate samples were processed, counting and identifying organisms to a mixed taxonomic level (family or genus).