Biomarkers in Action Examining the Effects of Dormant- Season Pesticide Runoff on Resident Fish...
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Transcript of Biomarkers in Action Examining the Effects of Dormant- Season Pesticide Runoff on Resident Fish...
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
er F
low
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
0
20
40
60
80
100
120
140
2/11/00 2/13/00 2/15/00 2/17/00 2/19/00 2/21/00 2/23/00
Date Sampled
Bra
in A
ce
tylc
ho
lin
es
tera
se
Ac
tiv
ity
(u
mo
l/m
in/g
we
t w
t)
0
50
100
150
200
250
300
OP
Pe
sti
cid
e C
on
ce
ntr
ati
on
(n
g/L
)
SJ AChE Activity OU AChE Activity (Ref) Diazinon + Methidathion Concentration
San Joaquin R.
DATA: AChE Activity - Lab
0
20
40
60
80
100
120
140
CON OU SJ FU FD
Water Source
Bra
in A
ce
tylc
ho
lin
es
tera
se
A
cti
vit
y (
um
ol/
min
/g w
et
wt)
0
10
20
30
40
50
60
OP
Pe
sti
cid
e C
on
ce
ntr
ati
on
(n
g/L
)
AChE Activity Diazinon + Methidathion Concentration
DATA: DNA Strand Breaks - Field
0
10
20
30
40
50
60
2/11/00 2/13/00 2/15/00 2/17/00 2/19/00 2/21/00 2/23/00
Date Sampled
Ind
ex
of
DN
A S
tran
d B
rea
ks
(%D
NA
in C
om
et T
ail)
0
50
100
150
200
250
300
OP
Pe
stic
ide
Co
nce
ntr
ati
on
(n
g/L
)
SJ DNA Strand Breaks OU DNA Strand Breaks (Ref)
Diazinon + Methidathion Concentration
San Joaquin R.
DATA: DNA Strand Breaks - Lab
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
OU OD SJ
Water Source
Ind
ex
of
DN
A S
tra
nd
Bre
ak
s
(%D
NA
in
Co
me
t T
ail
)
0
20
40
60
80
100
120
140
160
180
200
OP
Pe
sti
cid
e C
on
ce
ntr
ati
on
(n
g/L
)
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