Effects of urban chemical stressors on stream biofilms Sylvia Lee*, Emma Rosi-Marshall, Alexander J. Reisinger, John Kelly, Miguel Rojas, Sujay Kaushal *Current affiliation: U.S. EPA ORD National Center for Environmental Assessment Disclaimer: The views expressed in this presentation are those of the authors’ and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.

Introduction Urban streams receive a cocktail of nutrients, salts, drugs and other contaminants Runoff Treated wastewater Leaking sewage infrastructure Treated wastewater or untreated leaking sewage can be a major component of urban stream baseflow Urban streams have the capacity to retain and modify watershed C and N Kaushal et al. 2014 Drugs (OTC, Rx, illicit) Nutrients (C N P) Ions (Cl-) How does a combination of contaminants found in urban streams affect stream structure and function?

Study Overview Artificial stream experiment Two treatment phases Total duration: 4 weeks Environmentally relevant concentrations of nutrients, salt, drugs Two treatment phases Nutrients, salt Nutrients, salt, drugs Measured endpoints Ash-free dry mass Chlorophyll a Gross primary production Community respiration Nutrient uptake Antibiotic resistant bacteria Diatom composition

Artificial streams Motorized paddlewheel Biofilm from reference stream Groundwater with low nutrients

Experimental Design 5 treatments x 4 replicates Part 1 Part 2 Drugs High: nutrients & salt concentrations of urban stream Medium: dilute High by 10 Low: dilute High by 100 Control 1 & Control 2 no treatment Part 2 Continue nutrients, add drugs Control 2 Low Medium High Control 1 +Drugs +Drugs +Drugs +Drugs Drugs Drugs Drugs Drugs Nutrient and Salt Gradient (continuous exposure)

High Nutrient & Salt Treatment DOC (glucose)

Results of adding nutrients and salt (pre-drugs) green water CONTROL 1 HIGH Week 2 Day 1 Gross Primary Production Biofilm Seston Community Respiration Biofilm Seston

Drug cocktail added to all streams, except Control 1 Pain killer Caffeine Antihistamines Antibiotics Anti-epileptic Antidepressant

HIGH: nutrients, salt, drugs CONTROL 2: only drugs Very green water Pre-drugs Post-drugs HIGH: nutrients, salt, drugs CONTROL 2: only drugs Pre-drugs Post-drugs Clear water Seston GPP Seston CR

HIGH: nutrients, salt, drugs CONTROL 2: only drugs Very green water Pre-drugs Post-drugs HIGH: nutrients, salt, drugs CONTROL 2: only drugs Pre-drugs Post-drugs Clear water Biofilm GPP Expected drugs to suppress biofilm GPP Unexpected result due to drug effect on seston and light More light to biofilms, higher GPP Less light to biofilms, lower GPP

Whole Stream Nutrient Uptake Whole Stream GPP Biofilm and seston measurements helped explain post-drug increase in GPP Control 1 Control 2 High Whole Stream Nutrient Uptake Drugs decreased nutrient uptake, but uptake still occurred in treated streams Control Reference Control 1 Control 2 High + drugs + drugs

Can change in microbial activity be explained by change in composition? Biofilm Diatom Composition NMDS All streams changed over time High streams changed the most Control 2 Control 2 + drugs . greater than Control 1 change over time . . . (length of arrows) Control 1 Control 2 High Pre-drugs Post-drugs Preliminary results Big differences more likely in seston algal composition

Did drugs affect antibiotic resistance? SESTON Bacteria were grown on Cipro-amended plates Control 1 Control 2 High + drugs + drugs BIOFILM CFUs = colony forming units More CFUs = More resistant bacteria Expected more resistant bacteria in treated streams Multiple interacting stressors Control 1 Control 2 High + drugs + drugs

Summary and Conclusions Chemical stressors had effects on both structure and function of artificial streams Greatest effects of drugs were observed in the seston Interacting factors such as light added complexity to treatment responses More seston data needed Urban streams have altered structure and function, but some may retain capacity to perform biogeochemical processes

Acknowledgments Erinn Richmond Abby Bline Amanda Herzog Dillon McQuade Arial Shogren Brittany Hanrahan Joanna Blaszczak Erik Arndt Beau Duval Meredith Kadjeski Daniel Snow Phycological Research Consortium

What happened to the drugs after 2 weeks in the artificial streams? Drugs detected in streams Present above detection limit (5 ng L-1) Caffeine (50-90ng L-1) Diphenhydramine (13-18ng L-1) Carbamazepine (15-17ng L-1) Present at or below detection limit Acetaminophen (1-5ng L-1) Cimetidine (1-2ng L-1) Sulfamethoxazole (2ng L-1) Not tested Fluoxetine Ciprofloxacin (but see antibiotic resistance assay) For most drugs, water column concentration declined by over 95%