TOXIC EFFECTS OF DISPERSED AND NON- DISPERSED OIL ON ...
Transcript of TOXIC EFFECTS OF DISPERSED AND NON- DISPERSED OIL ON ...
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California Dept ofFish and Game
TOXIC EFFECTS OF DISPERSED AND NON-DISPERSED OIL ON CHINOOK SALMON SMOLTS
(ONCORHYNCHUS TSHAWYTSCHA) USING METABOLOMICS
Ronald S. Tjeerdema, Ching-Yu Lin, Brian S. AndersonDept of Environmental ToxicologyUniversity of California, Davis
Mark R. ViantSchool of Biosciences, University of Birmingham
Michael L. SowbyOffice of Spill Prevention and ResponseCalifornia Department of Fish and Game
David B. Crane Water Pollution Control LaboratoryCalifornia Department of Fish and Game
California Dept ofFish and Game
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California Dept ofFish and Game
Due to the risk of coastal oil spills to migrating salmon, resource agencies need information on the influence of dispersant treatment on the relative toxicity of spills in near shore waters.
INTRODUCTION
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California Dept ofFish and Game This investigation compares the toxicity of
dispersed and non-dispersed Prudhoe Bay Crude Oil (PBCO) to smolts of Chinook salmon (Onchorhyncus tshawytscha) in declining-exposure seawater exposures.
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Chinook Salmon – A Migrating Species
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Chinook Salmon – An Anadromous Species
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The Salmon Life cycle
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Objectives• Assess the relative acute toxicities of
dispersed and non-dispersed water accommodated fractions (WAFs) of Prudhoe Bay Crude Oil (PBCO) to Chinook Salmon smolts (ONCORHYNCHUS TSHAWYTSCHA) under defined declining exposure conditions.
• Apply 1H-Nuclear Magnetic Resonance (NMR)-based metabolomic analysis to investigate the sublethal effects of PBCO WAF and CEWAFs on salmon smolts.
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Methods: System Development and WAF Exposures
• Methods followed CROSERF (Singer et al.2000).
• 20-L polycarbonate carboys and 18-L aquaria.
• WAFs spun at low rate with minimal vortex (~150 rpm) for 24 h.
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Methods: WAF Tests (cont.)• 33% of each of 3 carboys distributed to each of 3 replicate 18-L aquaria (2-cm headspace).
• Once full, each aquarium was sampled for TPH and THC chemistry, 8 fish were introduced, and clean seawater flushing initiated.
• Flush rate calibrated with flow meters @ 200 mL per minute; rates verified with THC.
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Formation of CEWAF
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Methods: CEWAF Tests
•Add oil and create vortex size of 20 to 25%.
•Auto pipet 10% (by oil wt) Corexit 9500.
•Spin for 18 h, then settle for 6 h.•Stagger the test start times.
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Analytical Chemistry
•Total Petroleum Hydrocarbons (TPH): C10 – C36 using GC/flame ionization detection.
•Volatiles hydrocarbons C6-C9: benzene, toluene, ethylbenzene and xylenes (BTEX) using GC/MS & purge-and-trap extraction.
•Total Hydrocarbon Content (THC) (C6–C36) = BTEX C6 to C9 compounds + TPH C10 to C36
•Declining exposures confirmed with THC during tests.
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Methods: 96-h Exposures
• 8 salmon smolts per aquarium (~ 8cm).
• 96-h declining seawater exposures.
• 2 of the surviving fish were sacrificed for metabolomics
• Remaining survivors were cultured to assess long-term growth effects
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Experimental design
4 times WAF/CEWAF
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0.00
0.20
0.40
0.60
0.80
1.00
0 5 10 15 20
THC (C6 - C36 mg/L)
Mea
n Su
rviv
al (%
)
14-Jul-0528-Jul-058-Sep-05
0.000.100.200.300.400.500.600.700.800.901.00
0 100 200 300 400
THC (C6-C36 mg/L)
Mea
n Sur
viva
l (%
)
15-Jun-0530-Jun-0525-Aug-05
Salmon smolt survival in three WAF and CEWAF tests after 96-h. THC = Total Hydrocarbon Content.
CEWAF
WAF
Mean WAF LC50 = 7.46 mg/L THC
Mean CEWAF LC50 = 155.93 mg/L THC
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Metabolomics to examine effects of environmental stress biological effects
transcriptomics
proteomics
genome
mRNA
proteins
metabolitesmetabolomics
environmental stress
genomics
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Metabolomics in environmental
science
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NMR-Based Metabolomics Approach
Multivariate statistical analysis
Sample prep (tissue or biofluid)
1-D NMR analysis
Peak assignment1-D (1H NMR)2-D (1H-1H COSY & 1H-13C HSQC)
Metabolite profiling(metabolomic classification/biochemical mechanism)
{ }a1b1, a1b2, a1b3 a1bna2b1, a2b2, a2b3 a2bna3b1, a3b2, a3b3 a3bn
amb1,amb2, amb3 ambn
ab =
ppm
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Metabolomics Method OverviewDorsal muscles and liver from 2 surviving fish from each replicate exposure were flash frozen for metabolomic analysis.
• Small molecular mass metaboliteswere extracted with MeOH/H20.
• 1H-NMR analysis provides metabolite profiles.
• Metabolite profiles are then subjectedto multivariate analyses (PCA).
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NMR Spectrum of Muscle Valine
ATP/ADPLactate
Alanine
Arginine/Phosphoarginine
GlutamateGlutamine
GlutamateGlutamine
Glycine
Phosphocreatine
Phosphocreatine
Lactate
β-glucoseα-glucose
ATP/ADP Glycerophosphoryl-choline
Taurine
ppm
TMSP
HOD
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p-JRES NMR of Liver Valine
LactateAlanIne
Lactate
Arginine/Phosphoarginine
Glutamate
Glutamine
Glutamine
Hypotaruine
GlutamineAspartate
HOD
Taurine
Taurine
Glycerophosphoryl-cholineGlycine
β-glucose
α-glucose
Aspartate
ppm
ATP/ADP
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Multivariate Statistical Analysis
{ }a1b1, a1b2, a1b3 a1bna2b1, a2b2, a2b3 a2bna3b1, a3b2, a3b3 a3bn
amb1,amb2, amb3 ambn
Principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it.
Scores plots provide the scores on the principal components (PCs) from each data set. The first PC contains the largest part of the variance of the data set with subsequent PCs containing correspondingly smaller amounts of variance.
Loadings plots show how much the variable has in common with that component. Thus, for NMR data, loading plots can be used to detect the metabolites responsible for the separation in the data.
ab =
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PCA Scores Plot from WAF ExposuresScores Plot for WAF 1/2/3 Muscle
-4
-3
-2
-1
0
1
2
3
-4 -3 -2 -1 0 1 2 3
Scores on PC1 (38.72%)
Scor
es o
n PC
2 (1
9.43
%)
THC
WAF2THC
WAF3 WAF1
THC
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PCA Scores Plot from Each WAF ExposureScores Plot for WAF1 Muscle
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Scores on PC1 (40.27%)
Scor
es o
n PC
2 (2
3.44
%) THC
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Scores Plot for WAF2 Muscle
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2
Scores on PC1 (38.14%)
Scor
es o
n PC
2 (2
4.27
%)
PCA Scores Plot from Each WAF Exposure
THC
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Scores Plot for WAF3 Muscle
-3
-2
-1
0
1
2
3
-5 -4 -3 -2 -1 0 1 2
Scores on PC1 (29.86%)
Scor
es o
n PC
2 (2
0.30
%)
PCA Scores Plot from Each WAF Exposure
THC
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Muscle Loadings Plot from WAF Exposure
Control
WAF
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Muscle Loadings Plot from CEWAF Exposure
Control
CEWAF
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-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
**
**
*
**
alanine
arginine/phosphoarginine
glutamate
glutamine
valine
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAF
*
THC (mg/L)
0.0
0.5
1.0
1.5
2.0
***
Metabolic Effects in Muscle
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
**
*
*
0
1
2
3
4
5
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAF
**
*
**
THC (mg/L)
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-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
*
*
-10
-5
0
5
10
*
-4
-2
0
2
4
6
8
10
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF (THC) CEWAF
*
*
mg/L
-6
-4
-2
0
* ** **
taurine
glycerophosphorylcholinesuccinate
phosphocreatine
ATP/ADP
-1.5
-1.0
-0.5
0.0
0.5
1.0
*3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAFTHC (mg/L)
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Summary of Metabolic Results in Muscle
More
VALINE, ALANINE, ARGININE/PHOSPHOARGININE,
GLUTAMINE, GLUTAMATE, TAURINE (at high dose),
Less
SUCCINATEATP/ADP
More
VALINE, ALANINE, ARGININE/PHOSPHOARGININE
GLUTAMINE, GLUTAMATE,TAURINE (at low dose),
SUCCINATE
CEWAF
Less
PHOSPHOCREATINE, GLYCEROPHOSPHORYL-
CHOLINE
WAF
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Metabolites Detected from LiverMetabolites 1H NMR assignment (ppm)a p-JRES assignment
(ppm) Valine 1.047 (d) 0.995*, 1.047* Lactateb 1.328 (d), 4.115 (q) 1.328*, 4.115* Alanineb 1.488 (d), 3.785 (m) 1.488* Arginine/Phosphoarginine 1.725 (m), 1.918 (m), 3.833 (t) 1.918* Glutamateb 2.070 (m), 2.355 (t) 2.355* Glutamineb 2.138 (m), 2.455 (m), 3.763 (m) 2.138*, 2.455*, 3.763* Hypotaurine 2.658 (t) 2.658* Aspartate 2.817 (dd), 3.900 (dd) 2.817*, 3.900* Unknown 3.039 (s) 3.039* Taurineb 3.268 (t), 3.423 (t) 3.268*, 3.423* Glycerophosphoryl- choline
3.358 (s) 3.358*
Glycineb 3.563 (s) 3.563* Glycine-betaineb 3.933 (s) 3.933* β-glucoseb 4.650 (d) 4.650* α-glucose 5.238 (d) 5.238* NAD+ / NADP+ 6.042 (d), 6.093 (d), 8.174 (s), 8.833* (d),
9.147* (d), 9.342* (s)
ATP/ADPb 6.158 (d), 8.273 (s), 8.546 (s) 8.273* Unknown 7.093* (s) Unknown 7.865* (s) Unknown 7.965 (d) 7.965* Formate 8.458* (s) AMP 8.593* (s) Unknown 8.618 (s) 8.618*
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-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
**
**
**
-0.4
-0.2
0.0
0.2
0.4**
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4*
-6
-4
-2
0
2
4
6
8 *
valine
glutamine
aspartate
glycine
glycerophosphorylcholine formate
-12
-10
-8
-6
-4
-2
0
2
4
3.5 7.1 7.3 8.7 24.1 43.3 159.7
WAF CEWAF
**
THC (mg/L)
Metabolic Effects in Liver
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
3.5 7.1 7.3 8.7 24.1 43.3 159.7
WAF CEWAF
** ** **
THC (mg/L)
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-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
*
-0.4
-0.2
0.0
0.2
0.4*
ATP/ADP
AMPα-glucose
-0.20
-0.15
-0.10
-0.05
0.00
0.05
0.10
** *
3.5 7.1 7.3 8.7 24.1 43.3 159.7
WAF CEWAFTHC (mg/L)
-10
-5
0
5
10
*
lactate
-4
-3
-2
-1
0
3.5 7.1 7.3 8.7 24.1 43.3 159.7
WAF (THC) CEWAF
*
THC (mg/L)
phosphocreatine
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Summary of Metabolomic Results in Liver
More
VALINE, ASPARTATE, FORMATE
lessPHOSPHOCREATINE,
LACTATE, AMP
More
VALINE, GLUTAMINE, GLYCINE,ATP/ADP
CEWAF
lessα-GLUCOSE,
AMP
WAF
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Summary of Metabolic Actions
• Both WAF and CEWAF of PBCO result in an increase in amino acids – potentially to repair oil’s damage to proteins and enzymes.
• Increased production of amino acids is at the expense of energetic molecules such as ATP and phosphocreatine.
• Loss of energy to damage repair leaves less available for growth, response to stress, and potentially future reproduction.
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Summary of Metabolic Actions• toxicant dependent
eg. succinate
muscles
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAFTHC (mg/L)
*
*
• organ specific
-12
-10
-8
-6
-4
-2
0
2
4
3.5 7.1 7.3 8.7 24.1 43.3 159.7
WAF CEWAF
* *
THC (mg/L)
-8
-6
-4
-2
0
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAF
* ****
THC (mg/L)
eg. glycerophosphorylcholine
livers
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Summary of Metabolic Actions
eg. valine
formate
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
3.5 7.1 7.3 8.7 24.1 43.3 159.7
WAF CEWAF
** ** **
THC (mg/L)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAF
**
**
*
**
THC (mg/L)
• dose dependent
-8
-6
-4
-2
0
3.5 7.1 7.3 8.7 9.6 24.1 43.3 159.7
WAF CEWAF
* ****
THC (mg/L)
glycerophosphorylcholine
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California Dept ofFish and Game
Conclusions
• This investigation suggests that treatment of oil with a dispersant decreases its toxicity to Chinook salmon smolts approximately 20-fold – something to consider in response.
• 1H-NMR provides a more sensitive, sublethal assessment of toxicity, and may be used to help ascertain mechanisms of toxicity.
• Long-term impacts of acute oil exposure on salmon growth and metabolic function are still under investigation.
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AcknowledgementsFunding for this project was provided by
NOAA through the UNH Coastal Response Research CenterAdditional funding was provided by the California
Department of Fish and Game Office of Spill Prevention and Response (OSPR)
and the UCD Oiled Wildlife Care Network
California Dept of Fish and Game
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Applications
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ppm
a.
b.
1H NMR spectrum
p-JRES NMR spectrum