Biomarkers of oxidative stress in Calanus finmarchicus exposed to a water accommodated fraction

91 Pages·2017·2.13 MB·English

by  Sofia Soloperto

Biomarkers of oxidative stress in Calanus
finmarchicus exposed to a water
accommodated fraction of a naphthenic
North Atlantic crude oil
Sofia Soloperto
Environmental Toxicology and Chemistry
Submission date: May 2018
Supervisor: Bjørn Munro Jenssen, IBI
Norwegian University of Science and Technology
Department of Biology

Sofia Soloperto

Biomarkers of oxidative stress in Calanus
finmarchicus exposed to a water
accommodated fraction of a naphthenic
North Atlantic crude oil

Master’s thesis in Environmental Toxicology and Chemistry

Trondheim, May 2018

Supervisor: Bjørn Munro Jessen
Co-supervisor: Tomasz Maciej Ciesielski
Co-supervisor: Elise Skottene

Norwegian University of Science and Technology
Faculty of Natural Science
Department of Biology

Acknowledgments

This master thesis was written at the Department of Biology, Norwegian University of Science
and Technology (NTNU) in Trondheim, 2018, and was financed by GRACE project (Integrated
Oil Spill Response Action and Environmental Effect), supported by the EU Program Horizon
2020 under the grant agreement no. 679266.

I would like to thank my main supervisor, Bjørn Jessen, for his exhaustive help in the final
stages of my master thesis. A big thanks to Elise Skottene for guiding me through the lab work
and for being always present and helpful. Thanks to Dag Altin for his priceless help during the
experimental phase, and to Torfinn Sparstad and all the Post Doc and PhDs students working
in his lab for listening to my thousands questions and helping me during the lab work. A special
thanks goes to Tomasz Maciej Ciesielski for his endless support and trust; you made the whole
journey a lot funnier! A big thanks to Berta and Ieva for our late-night, semi-scientific
discussions that made us feel like real scientists.

Finally, a big thanks to my family and all my friends, near and far, for always being there!

iii
iv
Abstract

Calanus finmarchicus is an ecologically important species in the North, Norwegian and Barents
Seas, periodically constituting up to 90% of the standing stock of zooplankton. Due to continued
development of areas for oil and gas production, there are environmental discharges of
contaminants occurring both continuously and accidentally in these areas. Existence of reliable
biomarkers for oil exposure are fundamental for monitoring programs and decision-making
processes in case of environmental discharges and oil spills. To assess the potential negative
effects of oil exposure on keystone component of marine ecosystems, adult females of Calanus
finmarchicus were exposed to a water-accommodated fraction (WAF) of a naphthenic North
Atlantic crude oil. Adult non-ovulating females from the continuous lab culture at
SINTEF/NTNU Sealab, were exposed without feeding to sub-lethal concentration of WAF in
seawater and collected at 5 different time points: 0, 24, 48, 72 and 96 hours. Several oxidative
stress biomarkers were tested with gene expression (qPCR), enzymatic activity analyses,
determination of glutathione (GSH) and lipid peroxidation assay for malondialdehyde (MDA)
concentration. Our results demonstrated induction of glutathione S-transferases (GST) and
increase of GSH and MDA concentrations in the exposed group at each time point sampled.
Gene expression results showed inconsistent responses with both up- and downregulation of
GST at 48 and 72 hours and downregulation of superoxide dismutase (SOD) at 72 hours. Our
study indicates that GST enzymatic activity, and GSH and MDA concentrations can be applied
as effective biomarkers of oxidative stress in C. finmarchicus, while gene expression of SOD
and GST are less suitable.

v
vi
Table of Content
1 INTRODUCTION ............................................................................................................ 1
1.1 MARINE OIL POLLUTION .................................................................................................. 1
1.1.1 Petroleum chemical components ............................................................................ 3
1.1.2 Petroleum physical properties ............................................................................... 4
1.1.3 Petroleum faith in the marine environment ............................................................ 5
1.2 OIL SPILLS....................................................................................................................... 7
1.2.1 Overview ................................................................................................................. 7
1.2.2 Acute and chronic toxic effects ............................................................................... 8
1.2.3 Effects of oil pollution on planktonic community ................................................... 8
1.3 OXIDATIVE STRESS .......................................................................................................... 9
1.3.1 Biomarkers of oxidative stress ............................................................................. 11
1.4 CALANUS FINMARCHICUS .............................................................................................. 12
1.5 AIM OF THE PRESENT STUDY ......................................................................................... 13
2 MATERIAL AND METHODS ..................................................................................... 14
2.1 TEST SPECIES AND EXPERIMENTAL SET-UP .................................................................... 14
2.2 EXPOSURE MEDIUM ....................................................................................................... 15
2.3 SAMPLING ..................................................................................................................... 15
2.4 CHEMICAL ANALYSIS .................................................................................................... 17
2.5 GENE EXPRESSION ......................................................................................................... 17
2.5.1 RNA extraction ..................................................................................................... 17
2.5.2 cDNA synthesis ..................................................................................................... 19
2.5.3 qPCR .................................................................................................................... 21
2.5.3.1 Primers selection .......................................................................................... 21
2.5.3.2 Procedure ...................................................................................................... 24
2.5.4 Data handling ....................................................................................................... 24
2.6 ENZYMATIC ASSAYS AND MDA AND GSH CONCENTRATIONS ...................................... 25
2.6.1 GSH determination ............................................................................................... 25
2.6.2 MDA level ............................................................................................................. 26
2.6.3 Enzymatic activities .............................................................................................. 26
2.7 STATISTICS .................................................................................................................... 27
3 RESULTS ......................................................................................................................................... 28
vii
3.1 OIL AND WAF PROFILE ................................................................................................. 28
3.1.1 Volatile Organic Compounds ............................................................................... 28
3.1.2 Semi-Volatile Organic Compounds ...................................................................... 28
3.2 QPCR ............................................................................................................................ 30
3.3 ENZYMATIC ASSAYS AND MDA AND GSH CONCENTRATIONS ...................................... 32
4 DISCUSSION .................................................................................................................................. 35
4.1 WAF TOXICITY ............................................................................................................. 35
4.2 GENE EXPRESSION OF GST-2 AND SOD AS BIOMARKERS.............................................. 36
4.3 ENZYMATIC ACTIVITY OF GST AND CONCENTRATION OF GSH AND MDA AS
BIOMARKERS ......................................................................................................................... 40
4.4 GENE EXPRESSION VS. ENZYMATIC ACTIVITY AND MDA AND GSH CONCENTRATIONS 41
4.5 EXPERIMENTAL DESIGN AND ANALYSES LIMITATIONS................................................... 42
4.6 IMPLICATION OF THE PRESENT STUDY ........................................................................... 44
5 CONCLUSION ............................................................................................................................... 45
REFERENCES ........................................................................................................................................ 46
APPENDICES .......................................................................................................................................... 58
APPENDIX I: DETERMINATION OF LC50 CONCENTRATIONS OF NAPHTHENIC NORTH ATLANTIC
CRUDE OIL WAF IN C. FINMARCHICUS ................................................................................... 58
APPENDIX II: QPCR SET-UP ................................................................................................... 59
APPENDIX III: OIL AND WAF PROFILE – VOC ...................................................................... 60
APPENDIX IV: OIL AND WAF PROFILE – SVOC ................................................................... 61
APPENDIX V: AVERAGE CT VALUES FOR ACTIN AND EFA1Α ................................................ 64
APPENDIX VI: EXPRESSION RATIO OF TARGET GENES ........................................................... 65
APPENDIX VII: ENZYMATIC ASSAYS AND MDA AND GSH CONCENTRATIONS ...................... 67
APPENDIX VIII: NORMALITY TEST ........................................................................................ 69
APPENDIX IX: RESULTS FROM STATISTICAL ANALYSIS ......................................................... 71

viii