Effects of crude oil, dispersant and oil-dispersant on the marine microalgae Ostreococcus tauri and a phytoplankton community Master 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

Re

d f

luo

rescen

ce

(F

L3)

Beads (1 µm)

a

10 0

10 1

10 2

10 3

10 4

Picoeuk

Ora

ng

e f

luo

resce

nce

(F

L2

)

SSC

b

10 0

10 1

10 2

10 3

10 4

Beads (1µm)

Syn

10 0

10 1

10 2

10 3

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 ppm Corexit 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