Biomarkers in Action

Examining the Effects of Dormant-Season Pesticide Runoff on Resident

Fish Species

><> Andrew Whitehead <><

UC Davis, Bodega Marine Laboratory

Talk Overview:

Biomarkers:• Definition• Traits• Advantages / Strengths• Drawbacks / Difficulties

Biomarkers in Action: Pesticides Project• Goals• Experimental Design• Data

Biomarkers: Definition

Physiological / biochemical response of an organism that is mechanistically / functionally related to xenobiotic exposure

Principle: Xenobiotics interact with molecular targets through defined biochemical pathways which result in predictable physiological effects

Definition (cont.)

Biomarkers of Effect:- exposure has exceeded organism’s ability to accommodate• tissue necrosis• DNA mutations• AChE inhibition• developmental abnormalities• eggshell thinning• demasculinization, feminization• neoplasia, tumor formation

Biomarkers of Exposure:- induction of accommodation responses• metallothionein induction• P450 induction• DNA adducts• heat shock protein induction• increase in plasma cortisol levels• induction of immune system• measurement of metabolites• serum leukocyte levels, antibody production

Biomarkers: Traits

• Variability• Sensitivity• Selectivity• Clarity of Interpretation• Biological Significance• Duration of Response• Ease of use, Cost, Labor

Biomarkers vs. Other Approaches

H2O Chemistry Monitoring: Unequivocal demonstration of presence/absence Snapshot in time/space, partitioning, exposure pathways, linkage to biological responses...

Body Burden Analysis: Multiple exposure pathways Metabolism, sequestration

Bioassays: Biological consequences Lab setting, standard test species

Biomarkers: Advantages/Strengths

• “So What?”• Linking Exposure to Effects • Integrated Information

- Spatial

- Temporal

- Additive effects• Lab and Field experiments• Resident / Native organisms

• Complex Field Evaluations: “Do Contaminants Play a Role?”

Biomarkers: Drawbacks/Difficulties

• Interpretation

- Inferring causes

- Scaling to meaningful effects

- Timecourse of response• Understanding components of variation• Choice of biomarkers: What to measure?

- Use tiered approach

- Use other tools (chemistry) to focus choice

Examining the Effects of Dormant-Season Pesticide Runoff on Resident

Fish Species

PI: Dr. Susan Anderson – UC Davis, Bodega Marine LaboratoryCoinvestigators:

Dr. Bernie May – UC DavisDr. Kathryn Kuivila – USGSDr. David Hinton – Duke UDr. Barry Wilson – UC Davis

Graduate Student: Andrew Whitehead – UC Davis, Bodega Marine Laboratory

Funding: EPA Star Grant, 1998

Project Goals:

Overall: Examine biological effects of landscape-scale pesticide contamination on native fish at the individual and population levels.

Characterize Exposure:• GIS mapping of pesticide use databases• Water chemistry

Examine Effects on Individuals:• Acetylcholinesterase (AChE) inhibition assay• DNA strand break (comet) assay

Examine Effects on Populations:• DNA fingerprinting / population genetic analysis using AFLP and microsatellites

Field-Caging Approach:• Cage suckers at 1 reference, 2 impacted sites• Retrieve cages at multiple timepoints, in order to:

A) Capture pesticide peakB) Examine recovery time

Environmentally realistic Risky, chance of catastrophe Water and sediment exposure

Lab Exposure to Field-collected water approach:• collect field water in SS milk cans, transport to BML, expose fish - 6 d. Safe back-up Less environmentally realistic

Can examine more sites Minimal sediment exposure

Experimental Design: Exposure

Field Caging Design

11 12 13 14 15 16 17 18 19 20 21 22 23Date (February, 2000)

Riv

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Rain Rain

Cage 1OUT

Cage 3OUT

3 Cages INCage 2OUT

San Joaquin River San Joaquin River @ Vernalis@ Vernalis

Orestimba Creek @ River RoadOrestimba Creek @ River Road

Orestimba Creek @ Orestimba RoadOrestimba Creek @ Orestimba Road

Lab Exposure Design

• Composite samples collected in 35-L stainless steel milk cans• 6-day laboratory exposure to Sacramento sucker• Multiple tissues excised and archived for biomarker analysis(Brain, muscle, liver, gill, blood)

Sites:• Feather R. upstream of ag.• Feather R. downstream• Orestimba Ck. upstream• Orestimba Ck. downstream• San Joaquin R. downstream• Laboratory control

Experimental Design: Effects

AChE Activity:• Indicator of exposure to and/or effects from specific class of xenobiotics with same mechanism of action

= Organophosphate and carbamate pesticides

DNA Strand Breaks: Comet Assay• Indicator of exposure to and/or effects from variety of stressors.

= dormant-spray pesticides?

Mutagenicity: Ames Assay

Cytochrome P450 Activity

DATA: AChE Activity - Field

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San Joaquin R.

DATA: AChE Activity - Lab

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AChE Activity Diazinon + Methidathion Concentration

DATA: DNA Strand Breaks - Field

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SJ DNA Strand Breaks OU DNA Strand Breaks (Ref)

Diazinon + Methidathion Concentration

San Joaquin R.

DATA: DNA Strand Breaks - Lab

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DNA Strand Breaks Diazinon + Methidathion Concentration

Summary: Project

Suite of indicators, coupled with chemistry, has been a strong approach for assessing effects in the field, and in lab, on relevant species

AChE Data:- As hypothesized, dormant-season pesticides are affecting resident fish- Would not have expected effects based on chemistry alone

DNA Strand Break Data:- Indicates importance of chemicals other than pesticides

Ongoing/Future Work:- Other indicators: Mutagenicity assay, P450 activity, more chemistry- Population genetic approach

Overall Summary

For simple problems, use simple toolsComplex problems demand more sophisticated approaches

Biomarker information• “So What?”• Focus - what are the real problems?• Integrated information• Relevant organisms• Field and lab evaluations

A Day in the Life...4 X 4 ?4 X 4 ?

Speed, anyone?Speed, anyone?

Hmm...Hmm...

Catch anything?Catch anything?

Population-Level Biomarker Approach

Working H: Long-term exposure to contaminants can alter gene pools of exposed populations.

Rationale: Population genetic structure = historical record• Record of environmental influences on previous generations

Challenges:• Distinguish natural variation from induced genetic change (field design)• Step from correlation to attribution (test for mechanisms)

Hypotheses of Mechanisms that may Drive Pop’n Genetic Change:A) Natural Selection: Loss of sensitive individualsB) Mutation: Accumulation of rare mutations over generationsC) Random Genetic Drift: Bottleneck Erosion of genetic diversity