Ferran Romero, Sergi Sabater, Vicenç Acuña 10th Symposium for European Freshwater Sciences 2017 RS02: Impacts of Climate Change on Freshwater Ecosystems Approaching the Combined Impact of Climate Change and Chemical Stressors on River Biofilms Ferran Romero, Sergi Sabater, Vicenç Acuña Catalan Institute for Water Research (ICRA)

The impact of human activity on freshwater ecosystems: Background Objective Experimental Design Results Conclusions-Implications The impact of human activity on freshwater ecosystems: Release of chemical pollutants: Promotion of global change: Industry Heavy metals Urbanisation Pharmaceuticals Agriculture Fertilizers, Pesticides Chemical stressors Increased water temperature Physical stressors Global warming Desiccation (water stress)

The impact of human activity on freshwater ecosystems: Background Objective Experimental Design Results Conclusions-Implications The impact of human activity on freshwater ecosystems: Release of chemical pollutants: Promotion of global change: Industry Heavy metals Urbanisation Pharmaceuticals Agriculture Fertilizers, Pesticides Chemical stressors Increased water temperature Physical stressors Global warming Desiccation (water stress)

The impact of human activity on freshwater ecosystems: Background Objective Experimental Design Results Conclusions-Implications The impact of human activity on freshwater ecosystems: Release of chemical pollutants: Promotion of global change: Industry Heavy metals Urbanisation Pharmaceuticals Agriculture Fertilizers, Pesticides Chemical stressors Increased water temperature Physical stressors Global warming Desiccation (water stress)

Conclusions-Implications Background Objective Experimental Design Results Conclusions-Implications The impact of human activity on freshwater ecosystems: Multistress context Physical stressors Temperature Water stress Pesticides Chemical stressors Pharmaceuticals Pathogens Biological stressors

To evaluate the impact of multiple stressors on river biofilms: Background Objective and hypotheses Experimental Design Results Conclusions-Implications To evaluate the impact of multiple stressors on river biofilms: What stressors? Chemical River biofilms Microbenthic communities mainly composed by algae and cyanobacteria, as well as bacteria, protozoa and fungi, which live attached to submerged substrata. Physical Increased water Temperature Erythromycin (Antibiotic) Diuron (Herbicide) Desiccation (Water stress)

To evaluate the impact of multiple stressors on river biofilms: Background Objective and hypotheses Experimental Design Results Conclusions-Implications To evaluate the impact of multiple stressors on river biofilms: What stressors? Chemical River biofilms Microbenthic communities mainly composed by algae and cyanobacteria, as well as bacteria, protozoa and fungi, which live attached to submerged substrata. Physical Urban pollutant Increased water Temperature Erythromycin (Antibiotic) Diuron (Herbicide) Desiccation (Water stress)

To evaluate the impact of multiple stressors on river biofilms: Background Objective and hypotheses Experimental Design Results Conclusions-Implications To evaluate the impact of multiple stressors on river biofilms: What stressors? Chemical River biofilms Microbenthic communities mainly composed by algae and cyanobacteria, as well as bacteria, protozoa and fungi, which live attached to submerged substrata. Physical Agricultural pollutant Increased water Temperature Erythromycin (Antibiotic) Diuron (Herbicide) Desiccation (Water stress)

To evaluate the impact of multiple stressors on river biofilms: Background Objective and hypotheses Experimental Design Results Conclusions-Implications To evaluate the impact of multiple stressors on river biofilms: What stressors? Chemical River biofilms Microbenthic communities mainly composed by algae and cyanobacteria, as well as bacteria, protozoa and fungi, which live attached to submerged substrata. Physical Increased water Temperature Erythromycin (Antibiotic) Diuron (Herbicide) Desiccation (Water stress)

To evaluate the impact of multiple stressors on river biofilms: Background Objective and hypotheses Experimental Design Results Conclusions-Implications To evaluate the impact of multiple stressors on river biofilms: What stressors? Chemical River biofilms Microbenthic communities mainly composed by algae and cyanobacteria, as well as bacteria, protozoa and fungi, which live attached to submerged substrata. Physical Increased water Temperature Erythromycin (Antibiotic) Diuron (Herbicide) Desiccation (Water stress)

Starting hypotheses: Background Objective and hypotheses Experimental Design Results Conclusions-Implications Starting hypotheses: Chemical stressors will have target effects on specific biofilm components, which will be consistent with their specific mode of action. Bacteria Erythromycin (Antibiotic) Algae Diuron (Herbicide)

Starting hypotheses: Background Objective and hypotheses Experimental Design Results Conclusions-Implications Starting hypotheses: Physical stressors will mostly have generalized effects, producing a non-specific alteration on the selected variables. Increased water Temperature Bacteria Algae Desiccation (Water stress)

Starting hypotheses: CT A B A+B Background Objective and hypotheses Experimental Design Results Conclusions-Implications Starting hypotheses: When occurring, non-additive responses will mostly be antagonistic. CT A B A+B Crain et al. 2008

Starting hypotheses: CT A B A+B Background Objective and hypotheses Experimental Design Results Conclusions-Implications Starting hypotheses: When occurring, non-additive responses will mostly be antagonistic. CT A B A+B Crain et al. 2008 Freshwater communities are exposed to natural variability, therefore presenting an inherent capacity to cope with multiple stress situations.

Objective and hypotheses Experimental Design Results Background Objective and hypotheses Experimental Design Results Conclusions-Implications Temperature Erythromycin (Antibiotic) Water stress Diuron (Herbicide)

HARD TO PERFORM IN A RIVER  Confounding factors Background Objective and hypotheses Experimental Design Results Conclusions-Implications Temperature Erythromycin (Antibiotic) Water stress Diuron (Herbicide) HARD TO PERFORM IN A RIVER  Confounding factors

HARD TO PERFORM IN A RIVER  Confounding factors Background Objective and hypotheses Experimental Design Results Conclusions-Implications Temperature Erythromycin (Antibiotic) Water stress Diuron (Herbicide) HARD TO PERFORM IN A RIVER  Confounding factors

16 treatments FULL-FACTORIAL DESIGN All possible combinations Background Objective and hypotheses Experimental Design Results Conclusions-Implications Temperature Erythromycin (Antibiotic) FULL-FACTORIAL DESIGN All possible combinations of factors were assessed 4 factors = 2 * 2 * 2 * 2 16 treatments Water stress Diuron (Herbicide)

Significant individual effects: Background Objective and hypotheses Experimental Design Results Conclusions-Implications Significant individual effects: Comparison between the effect of each treatment separately to the control. Significant main effects: Mean performance of a given stressor. Comparison between the effect of all treatments containing the stressor vs. all treatments not containing the stressor. Significant interactive effects: The effect of one stressor is significantly modified by the presence of other stressors CONTROL vs TREATMENT TREATMENTS vs TREATMENTS A B AB C ABC BC Individual effects compare the effects of each treatment separately to the control, that is, in the absence of the other treatment. Main effects compare the mean performance in the two treatments when the agent is present versus the two treatments where the agent is absent. TREATMENTS vs TREATMENT A AB B

Significant individual effects: Background Objective and hypotheses Experimental Design Results Conclusions-Implications Significant individual effects: Comparison between the effect of each treatment separately to the control. Significant main effects: Mean performance of a given stressor. Comparison between the effect of all treatments containing the stressor vs. all treatments not containing the stressor. Significant interactive effects: The effect of one stressor is significantly modified by the presence of other stressors CONTROL vs TREATMENT TREATMENTS vs TREATMENTS A B AB C ABC BC Individual effects compare the effects of each treatment separately to the control, that is, in the absence of the other treatment. Main effects compare the mean performance in the two treatments when the agent is present versus the two treatments where the agent is absent. TREATMENTS vs TREATMENT A AB B

Significant individual effects: Background Objective and hypotheses Experimental Design Results Conclusions-Implications Significant individual effects: Comparison between the effect of each treatment separately to the control. Significant main effects: Mean performance of a given stressor. Comparison between the effect of all treatments containing the stressor vs. all treatments not containing the stressor. Significant interactive effects: The effect of one stressor is significantly modified by the presence of other stressors CONTROL vs TREATMENT TREATMENTS vs TREATMENTS A B AB C ABC BC Individual effects compare the effects of each treatment separately to the control, that is, in the absence of the other treatment. Main effects compare the mean performance in the two treatments when the agent is present versus the two treatments where the agent is absent. TREATMENTS vs TREATMENT A AB B

Significant individual effects: Background Objective and hypotheses Experimental Design Results Conclusions-Implications Significant individual effects: Comparison between the effect of each treatment separately to the control. Significant main effects: Mean performance of a given stressor. Comparison between the effect of all treatments containing the stressor vs. all treatments not containing the stressor. Significant interactive effects: The effect of one stressor is significantly modified by the presence of other stressors CONTROL vs TREATMENT TREATMENTS vs TREATMENTS A B AB C ABC BC Individual effects compare the effects of each treatment separately to the control, that is, in the absence of the other treatment. Main effects compare the mean performance in the two treatments when the agent is present versus the two treatments where the agent is absent. TREATMENTS vs TREATMENT A AB B Synergism AB > A+B Antagonism AB < A+B

Objective and hypotheses Experimental Design Results Background Objective and hypotheses Experimental Design Results Conclusions-Implications Photosynthetic Efficiency Photosynthetic Capacity Non-photochemical quenching psbA gene expression Diuron Erythromycin Water stress Temperature

Objective and hypotheses Experimental Design Results Background Objective and hypotheses Experimental Design Results Conclusions-Implications Photosynthetic Efficiency Photosynthetic Capacity Non-photochemical quenching psbA gene expression Diuron Erythromycin Water stress Temperature

Main negative effect of diuron on Photosynthetic Efficiency Background Objective and hypotheses Experimental Design Results Conclusions-Implications Photosynthetic Efficiency Main negative effect of diuron on Photosynthetic Efficiency Photosynthetic Capacity Non-photochemical quenching psbA gene expression Diuron Chemical stressors Erythromycin Water stress Temperature

Main negative effect of water stress on all photosynthetic variables Background Objective and hypotheses Experimental Design Results Conclusions-Implications Photosynthetic Efficiency Photosynthetic Capacity Main negative effect of water stress on all photosynthetic variables Non-photochemical quenching Main negative effect of temperature on all photosynthetic variables psbA gene expression Diuron Erythromycin Water stress Temperature Physical stressors

Significant interactions Mainly antagonistic Background Objective and hypotheses Experimental Design Results Conclusions-Implications Photosynthetic Efficiency Photosynthetic Capacity Non-photochemical quenching psbA gene expression Significant interactions Mainly antagonistic Diuron Erythromycin Water stress Temperature

Which stressor presented the largest effect? Background Objective and hypotheses Experimental Design Results Conclusions-Implications Which stressor presented the largest effect? Averaged effect size of the selected stressors Temperature Erythromycin Water stress Diuron

Were there any interactions between the stressors? Background Objective and hypotheses Experimental Design Results Conclusions-Implications Were there any interactions between the stressors?

... Starting hypotheses Background Objective and hypotheses Experimental Design Results Conclusions-Implications ... Starting hypotheses Chemical stressors will have target effects on specific biofilm components, which will be consistent with their specific mode of action. Physical stressors will mostly have generalized effects, producing a non-specific alteration on the selected variables. When occurring, non-additive responses will mostly be antagonistic.

... Starting hypotheses Background Objective and hypotheses Experimental Design Results Conclusions-Implications ... Starting hypotheses Chemical stressors will have target effects on specific biofilm components, which will be consistent with their specific mode of action. Physical stressors will mostly have generalized effects, producing a non-specific alteration on the selected variables. When occurring, non-additive responses will mostly be antagonistic.

... Starting hypotheses Background Objective and hypotheses Experimental Design Results Conclusions-Implications ... Starting hypotheses Chemical stressors will have target effects on specific biofilm components, which will be consistent with their specific mode of action. Physical stressors will mostly have generalized effects, producing a non-specific alteration on the selected variables. When occurring, non-additive responses will mostly be antagonistic.

... Starting hypotheses Short-term exposure to stressors? Background Objective and hypotheses Experimental Design Results Conclusions-Implications ... Starting hypotheses Chemical stressors will have target effects on specific biofilm components, which will be consistent with their specific mode of action. Physical stressors will mostly have generalized effects, producing a non-specific alteration on the selected variables. When occurring, non-additive responses will mostly be antagonistic. Short-term exposure to stressors? Antagonism often results from adaptation of the community to stress.

... Starting hypotheses 2. Reduced community complexity? Background Objective and hypotheses Experimental Design Results Conclusions-Implications ... Starting hypotheses Chemical stressors will have target effects on specific biofilm components, which will be consistent with their specific mode of action. Physical stressors will mostly have generalized effects, producing a non-specific alteration on the selected variables. When occurring, non-additive responses will mostly be antagonistic. 2. Reduced community complexity? Antagonism often results from functional redundancy between biofilm components.

Background Objective and hypotheses Experimental Design Results Conclusions-Implications CONCLUSIONS Physical stressors had larger effect size than chemical stressors. The stressor having the largest effect (measured as averaged effect size) was water stress. Individual stressors resulted mainly in expected responses, whereas combined stressors showed significant interactions. Among all significant interactions, 75% were antagonisms and 25% were synergisms. Strongest interactions included physical stressors (water stress and temperature). IMPLICATIONS Freshwater communities exposed to increased temperature and water stress as a result of climate change could display unexpected responses when faced to additional chemical stress. Although antagonisms may prevail, synergisms also play a role at short-term.

Background Objective and hypotheses Experimental Design Results Conclusions-Implications CONCLUSIONS Physical stressors had larger effect size than chemical stressors. The stressor having the largest effect (measured as averaged effect size) was water stress. Individual stressors resulted mainly in expected responses, whereas combined stressors showed significant interactions. Among all significant interactions, 75% were antagonisms and 25% were synergisms. Strongest interactions included physical stressors (water stress and temperature). IMPLICATIONS Freshwater communities exposed to increased temperature and water stress as a result of climate change could display unexpected responses when faced to additional chemical stress. Although antagonisms may prevail, synergisms also play a role at short-term.

Thank you! This research was supported by the EC 7th Framework Programme (GLOBAQUA; 603629-ENV-2013-6.2.1). Authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (2014 SGR 291) - Catalan Institute for Water Research.