1. 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)

2. 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)

3. 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)

4. 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)

5. 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

6. 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)

7. 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)

8. 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)

9. 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)

10. 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)

11. 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)

12. 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)

13. 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

14. 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.

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

16. 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

17. 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

18. 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)

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

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

30. ... 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.

31. ... 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.

32. ... 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.

33. ... 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.

34. ... 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.

35. 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.

36. 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.

37. Thank you!
This research was supported by the EC 7th Framework Programme (GLOBAQUA; ENV ). 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.