1. INVESTIGATING BIOGEOCHEMICAL CONTROLS ON METAL MIXTURE TOXICITY USING STABLE ISOTOPES AND GENE EXPRESSION Grant # 1R01ES024358-01 Project Period 8/18/2014 * to 5/31/2018 PIs W Clements, J. Meyer, J. Ranville, C. Vulpe National Institute of Environmental Health Science

2. Possible approaches for assessing and predicting ecological effects of metals Ecological Effects Microcoms -Benthic communities -Organism level tests Field-scale Exps. -Community surveys -Caged organism experiments

3. Possible approaches for assessing and predicting ecological effects of metals Approaches to Assess/Predict BioavailabilityEcological Effects Tissue Metal Levels -MSMM Biotic Ligand Models -Stable Isotopes Bioavailable Fraction -Passive samplers -Sensors Bioassays -Genomics -Single-species tox. tests Microcoms -Benthic communities -Organism level tests Field-scale Exps. -Community surveys -Caged organism experiments

4. North Fork of Clear Creek, CO Black Hawk/Central City Superfund Site (COD980717557) Future AMD Treatment Plant

5. Current Ecological Conditions

6. Project Objectives Project 1: Linking Metal Bioavailability to Ecological Effects Conduct a before-after control-impact (BACI) field experiment to quantify the relative influence of metal bioavailability and substrate composition on ecosystem recovery Project 2: Linking Metal Bioavailability to Genomic Effects Utilize gene expression in aquatic bioindicator species as molecular biosensors to assess bioavailability of metal mixtures (Cd, Cu, Ni, Zn) in the water column Project 3: Linking Metal Bioavailability to Tissue Concentrations and Metal Speciation Conduct experiments to determine the bioavailable pool of metal mixtures released from contaminated sediments exposed to variable water chemistries, and directly measure metal accumulation by bioindicator species. Project 1 will test the hypothesis that despite significant improvements in water quality, ecological effects will persist in NFCC because of residual, high metal bioavailability in sediments (i.e., a biogeochemical hysteresis) and poor substrate quality Project 2 and 3 will test the hypothesis that despite significant improvements in water quality, low concentrations of bioavailable metals will persist in NFCC (i.e., a biogeochemical hysteresis effect)

7. 7 Metal Mixture Effects Must be Considered Example: Laboratory tests with D. magna using constant Cd and variable Ni Increasing Ni reduces Cd toxicity until the point at which Ni becomes toxic

8. Field Measurements of Sediment Contamination and Recovery

12. Deposition Recovery Chemical Hysteresis is Observed Longer studies needed

13. Expose to Clear Creek Equilibrate organism with stable isotopes Microcosm colonization Tissue metals Multi-site Multi-metal BLM Sub-cellular distribution of isotopes Mineral phase analysis sequential extraction Sediment metals Donnan Membrane: free ion activity Dissolved Metals Gene sequencing Research approach integrating metal bioavailability assessment with ecological effects

14. Biologic response to toxicant Response reflected in transcriptional changes Transcriptional response “profile” indicative of particular toxicant pollutant AAAAA Gene C Gene B Gene A Contaminant-specific “fingerprint” of toxicant exposure and effect =greater specificity Direct assessment of organism response (no chemical manipulation) = increased relevance Sub-lethal endpoints provides dose response information = increased sensitivity Metal Microarrays or RNAseq measure genome wide response to metal Molecular approaches to assess Metals in Aquatic Ecosystems

15. a ddis Fly Molecular Bioindicators The dream… Identify genes (mRNA) whose expression levels change in response to metal exposure AAAAA Isolate mRNA RNA seq – the next generation technology metals control Sequence it All! Let compbio sort them out To develop a QPCR based panel of genes which can distinguish between metal exposures To test QPCR panel bioassay in simulated field situations To test QPCR panel bioassay in field situation More biological relevance to aquatic ecosystems of interest Metal bioavailability in habitat may be different than water column

16. ACKNOWLEDGEMENTS: # 1RO1ES020917-01 1R01ES024358-01 Students: Jacob Williamson, Elizabeth Traudt, Pete Cadmus, Katherine Ebeling Samuel Fritkin, Ryan Mason, Thomas Gately, Kristina Lucas, Jason Loving Postdocs and Collaborators Ramiro Pastorinho, Zaho Dong Additional Funding: Jake Katherine Sam Ellie Ramiro Pete

17. Problem: Two AMD sources introduce dissolved metals (Cu, Zn, Cd) Stream sediments have accumulated Fe, Al, Mn oxide precipitates and associated metals Opportunity: Examine the recovery of the stream following restoration Research Approach laboratory/microcosm/in -stream experiments & field observations Golden, CO Blackhawk, CO sport fishing water supply recreation Water Chemistry Geochemistry Biological Effects/Toxicity Predict Recovery Effects of Metals on Aquatic Systems: Clear Creek CO