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


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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


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


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


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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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

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