1. Effects of crude oil, dispersant and oil-dispersant on the marine
microalgae Ostreococcus tauri and a phytoplankton communityMaster Sciences de l’Univers,
Environnement, Ecologie
M1 2013-2014
Nicolas CHEDRI, Philippe SCHATT, François-Yves BOUGET, Fabien JOUX
Laboratoire d'Océanographie Microbienne, LOMIC UMR 7621 CNRS-UPMC, F-66650 Banyuls-sur-Mer, France
The Deepwater Horizon oil spill event resulted in an estimated 4.9 million
barrels of crude oil being spilled into the northern Gulf of Mexico between 20
April and 15 July 2010. To mitigate the effect of the oil spill, accelerate natural
dispersion, and enhance biodegradation, approximately 1.5 million gallons of a
chemical dispersant, Corexit 9500, were sprayed onto the surface and also
applied at the underwater pipe source of leak.
Phytoplankton are at the base of the aquatic food web, and as the primary
producers, they are a vital source of food to a wide range of species. The
toxicity of crude oil can be attributed mainly to interferences with the
photosynthetic apparatus. In contrast, the surfactants present in Corexit 9500,
act certainly on membranes.
Our goal in this study was to investigate :
- the toxicity of crude oil, dispersant and oil-dispersant on marine
phytoplankton, by measuring the expression of a gene involved in cellular
cycle (CDKA) in a marine microalgal luminescent,
- the growth of a marine phytoplankton community exposed to Corexit 9500.
10% crude
oil without
dispersant
10% crude
oil with
dispersant
ratio 1:20
Growth in 96-wells
microplate
Luminescence is recorded
every hour automatically
during 2-3 days
Toxicity assays on phytoplankton community
Different concentrations of dispersant were added to coastal water after
nutrients enrichment. Samples were incubated in situ.
Ostreoccocus tauri is a small (1 µm) green unicellular alga with a large geographical
distribution. Firefly luciferase transcriptional and translational reporter lines have
been produced to monitor the expression of genes/proteins involved in diverse
biological functions such as cell division.
These luminescent biosensors have been used previously to test the toxicity of
antifouling biocides (Appl. Environ. Microbiol. 2013).
Introduction
Phytoplankton groups were
counted by flow cytometry at T0
and after 3 days of incubation.
The chlorophyll a was determined by fluorometry at
T0 and after 5 days of incubation.
Toxicity assays on Ostreococcus tauri
CDKA luc
Luciferase
Luciferine
Oxyluciferine
Light
Light emission
proportional to the gene
expression
Addition of luciferine
(10 µM) to the culture media
Marine Microalgal Recombinant Biosensor
Protocols
Different microplates
are managed by a robot
Pro
Syn
SSC
Redfluorescence(FL3)
Beads
(1 µm)
a
10
0
101
10 2
103
10
4
Picoeuk
Orangefluorescence(FL2)
SSC
b
10
0
10
1
10
2
10
3
10
4
Beads
(1µm)
Syn
10
0
101
10 2
103
10
4
10
0
10
1
10
2
10
3
10
4
Nanoeuk
Oil alone
The presence of dispersant
increased the toxicity of oil.
However, when dispersant is used
alone, a high toxicity is observed,
suggesting that most of toxic effect
observed in oil-dispersant came
from dispersant and not necessary
from the interaction with oil.
Oil + dispersant (20:1) Dispersant alone
EC50 calculations at 24h:
- Oil alone: 18628 ppm oil
- Oil+Disp (20:1): 490 ppm oil
- Dispersant: 15 - 30 ppm Corexit
Phytoplankton community showed a high sensitivity to
Corexit as for O. tauri. Photosynthetic picoeukaryote was
the most responsive group.
a) Synechococcus EC50 = 21.3 ppm
b) Picoeukaryotes EC50 = 0.25 ppm
c) Nanoeukaryotes EC50 = 2.9 ppm
d) Chlorophyll a EC50 = 12.3 ppmCorexit 9500 solution (500 ppm)
exposed 16h to simulated solar
radiation (UV/vis) 330 W/m2
before toxicity assays
Solar radiation did not change toxicity
of Corexit for O. tauri, suggesting that
Corexit is not readily photodegradable.
Dark
Light
Our results suggest that during the Deepwater Horizon oil spill event, the toxicity of crude oil on marine phytoplankton could be extremely amplified by the
wide use of Corexit 9500. The study reveals that Corexit is one thousand times more toxic than crude oil, when recommend ratios for deploying Corexit are
1:10-1:50. High toxicity of Corexit was confirmed for both O. tauri and phytoplankton community. O. tauri CDKA luminescent biosensor constitutes a high-
throughput and sensitive approach to study the toxicity of oil and dispersant, with the possibility to explore the interactive effects of these chemicals with
other environmental factors (nutrients, light, …) or others pollutants.
Results
a b
c d
Phytoplankton community
Photodegradation
of Corexit
Ostreococcus tauri
Conclusion