INVESTI GATIN G BIOGEOCH EMICAL CO NTROL S ON M ETAL MIXT URE TOXICIT Y USIN G STABLE ISOTOP ES AND GEN E EXPRESS ION Grant # 1R01ES024358-01 Project Period 8/18/2014 * to 5/31/2018 PIs J. Meyer, J. Ranville , C. Vulpe National Institute of Environmental Health Science
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INVESTIGATING BIOGEOCHEMICAL CONTROLS ON METAL MIXTURE TOXICITY USING STABLE ISOTOPES AND GENE EXPRESSION Grant # 1R01ES024358-01 Project Period 8/18/2014.
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INVES
TIGAT
ING B
IOGEOCHEM
ICAL
CONTROLS
ON M
ETAL M
IXTU
RE
TOXIC
ITY
USING S
TABLE
ISOTO
PES
AND GEN
E
EXPR
ESSIO
N
Grant #1R01ES024358-01
Project Period8/18/2014* to 5/31/2018
PIsW Clements, J. Meyer, J. Ranville, C. Vulpe
National Institute of Environmental Health
Science
Possible approaches for assessing and predicting ecological effects of metals
North Fork of Clear Creek, CO Black Hawk/Central City Superfund Site
(COD980717557)
Future AMD Treatment Plant
Current Ecological Conditions
Spring
Upper Ref
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Riviera
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StationUpper R
efPump
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StationUpper R
efPump
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Total abundance
Number of Taxa
Project ObjectivesProject 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)
7Metal 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
1E-4 1E-3 0.01 0.1 10
20
40
60
80
100
0.025 mg/L Cd
0.075 mg/L Cd
0.100 mg/L Cd
0.175 mg/L Cd Ni Only, 95% Uncertainty
Mor
talit
y %
[Ni] mg/L
Field Measurements of Sediment Contamination and Recovery
DepositionRecovery
Chemical Hysteresis is Observed Longer studies needed
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
Biologic response to toxicant
Response reflected in transcriptional changes
Transcriptional response
“profile” indicative of particular toxicant
pollutant
AAAAA
AAAAA
AAAAA
AAAAA
AAAAA
AAAAA
AAAAA
AAAAA
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
Metal
Metal
Metal
Microarrays or RNAseqmeasure genome wide
response to metal
Molecular approaches to assessMetals in Aquatic Ecosystems
addis Fly Molecular Bioindicators The dream…
Identify genes (mRNA) whose expression levels change in response to metal exposure AAAAA
AAAAA
AAAAAAAAAA
Isolate mRNA
RNAseq – the next generation technology
metals
control
Sequence it All! Let compbio sortthem 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 interestMetal bioavailability in habitat may be different than water column
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 CollaboratorsRamiro Pastorinho, Zaho Dong
Additional Funding:
Jake
Katherine
Sam
EllieRamiro
Pete
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 fishingwater supplyrecreation
Water Chemistry
Geochemistry
Biological Effects/Toxicity
Predict Recovery
Effects of Metals on Aquatic Systems: Clear Creek CO