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Characterization of ecotoxicity and phytotoxicity of a cyanobacterial extract containing microcystins under realistic environmental concentrations and in a soil-plant
system
Sylvain Corbel, Christian Mougin, Noureddine Bouaicha
To cite this version:
Sylvain Corbel, Christian Mougin, Noureddine Bouaicha. Characterization of ecotoxicity and phyto- toxicity of a cyanobacterial extract containing microcystins under realistic environmental concentra- tions and in a soil-plant system. SETAC Europe 24th Annual Meeting, May 2014, Bâle, Switzerland.
2014. �hal-01601633�
environmental concentrations
Wednesday May 14th 2014
Sylvain CORBEL 1, Christian Mougin 1, Noureddine Bouaïcha 2
1: INRA, UR 251 PESSAC, Versailles, France
2: Paris-Sud University, UMR 8079 ESE, Orsay, France
Context
2
Introduction Material and
Methods Conclusion
excess of fertilization global warming eutrophication Results and
Discussion
cyanobacteria in the world (freshwater, seawater) and their cyanotoxins
cyanotoxins
dermatotoxins (allergy)
neurotoxins (nervous system)
The main preoccupation: their toxins!
hepatotoxins (liver)
irrigation water 100 µg L-1
Economical risk and Sanitary risk (contamination of the food chain)
H3C
OCH3
CH3 H
H3C H
H
HN
N
NH CH2 O
O
O
H COOH R2
NH
Z
N N X
O
O O
CH3
COOH H3C
H
H
H
HN
O
structure of microcystins MCs (with 80 variants)
Sanitary risks
Pouria et al., 1998 Žegura et al., 2011
Mankiewicz-Boczek et al., 2011
1996: Caruaru (Brazil), 60 patients died in hemodialysis center
1 µg L-1 in drinking water (WHO)
4
Introduction Material and
Methods Results and Conclusion
Discussion
Our objectives
The main objective of our research program: to study fate of microcystins (MCs) contained in irrigation water in soils, and their impact on edible tomato (S. lycopersicum var. MicroTom) and soils communities
The specific objectives of the present presentation are to show our first results concerning the:
-characterization of the cyanobacterial extract
-phytotoxicity on tomato plants Solanum lycopersicum var. MicroTom and ecotoxicity of cyanobacterial extract, brought by irrigation, on soil microorganisms
From a culture of Microcystis aeruginosa (PCC 7820) from Pasteur Institute (Paris, France)
Conditions: 3 weeks in BG11 + NaNO3 (2 mM) + NaHCO3 (10 mM) 25 °C, light: 16 h between 5-10 µmol of photons m-2 s-1
continuous sparged 0.23 L min-1
Harvesting of cyanobacteria: centrifugation (25,000 g, 5 min) and freeze-dried
Toxin extraction 3 times with aqueous MeOH (75%, v/v)
Quantification of cyanotoxin in cyanobacterial extract with Protein Phosphatase 2A assay (Bouaïcha et al., 2001)
Identification of cyanotoxins by UPLC-MS/MS
Cyanobacterial extract
6
Introduction Material and
Methods Result and Conclusion
Discussion
Phytotoxic assays in vitro 1
4 Seed samples: Solanum lycopersicum var. MicroTom and Saint-Pierre Lactuca sativa var. capitata
Triticum aestivum Bio (Attlass)
20 seeds/Pétri dish covered with filter paper 3 Petri dishes/treatments
Conditions: 7 days in the dark (25 °C) with 5 mL of cyanobacterial extract or cadmium chloride, CdCl (positive control)
Treatments: 0-20 mg eq. MC-LR L-1 and 0-1 g CdCl L-1
1 from the norm AFNOR X31-201 (AFNOR, 1986)
radicle cotyledons
• germination rate
• lengths of radicles (ImageJ, 2012)
7
Assays of inhibition growth of S. lycopersicum var. MicroTom seedlings1
Soil used: silty-sandy soil of experimental site Pierre-Plate (Versailles), 350 g/replicate Seed used: Solanum lycopersicum var. MicroTom, 10 seeds/replicate
Conditions : 16 h of light at 160 µmol of photons m-2 s-1, 26/21 °C (D/N) and a relative humidity <70%
D-14 D 0 14 D
Plantation of seeds
Soil moistened Exposition
Soil activation
soil characteristics 0 (C : control), 5, 20, 50 et 100 µg eq. MC-LR L-1
1 norm AFNOR X31-206 (AFNOR, 2011)
2Santiago-Martin et al., 2013
3ISO NF ISO 15685. (ISO 2012)
4
• dry biomasses of seedlings
• global soil enzymatic activities2 and potential nitrification3 of soils
• quantification of soil microorganisms4
Clay (%) 11
Silt (%) 13
Sand (%) 76
Organic carbon (‰) 21.9 Total nitrogen (‰) 1.2
C/N ratio 17.5
Organic mater (‰) 37.8
pH 5,6
WHC (%) 35,5
8
Introduction Material and
Methods Results and Conclusion
Discussion
Characterization of cyanobacterial extract from M. aeruginosa (PCC7820) culture
Toxicity of this extract with PP 2A assays was: 21,6 mg eq. MC-LR L-1 corresponding to 6,77 mg eq. MC-LR g-1 dried cells.
UPLC-MS/MS revealed 4 main congeners of MCs
time (min) (68.4%)
(2.9%)
(6.5%)
(8.3%)
And 9 other congeners identified:
MC-YR, MC-LM, [DMAdda5]MC-LR, [D-Asp3]MC-LR, [L-MeSer7]MC-LR, [DMAdda5]MC-LF, [DM]MC-LF, [DM]MC-LW, [DMAdda5]MC-LW
a
0 20 40 60 80 100
120 D
a a a
a a a
a a ac
b bc
bd bd d
a a a
a a a
rate of germination (%)
9
Germination assays, in vitro
0 20 40 60 80 100 120
ab ab
a a a
a a a
0 20 40 60 80 100 120
a a a a a a a a
a a a
a abc
c b b
a
ab ab ab ab
ab ab b ab
a ab a a a
a a
a a
concentrations (µg L-1)
rate of germination (%)
concentrations (µg L-1)
rate of germination (%)
S. lycopersicum var. Saint-Pierre S. lycopersicum var. MicroTom
Lactuca sativa var. capitata
cyanobacterial extract cadmium chloride
No significant effect of MCs on
germination process on these plants
a a
10
Introduction Material and
Methods Results and Conclusion
Discussion
cyanobacterial extract cadmium chloride
0 20 40 60 80 100
1E+0 1E+1 1E+2 1E+3 1E+4 1E+5 1E+6
rate of germination (%)
concentrations (µg L-1) in log10 scale
0 1 2 3 4 5 6
*
*
*
0 20 40 60 80 100
0 5000 10000 15000 20000
concentrations (µg L-1)
rate of germination (%)
* *
*
* Significant decrease of the
germination rate of wheat after 7 days of cyanobacterial exposure (EC50=11 mg eq. MC-LR L-1)
BUT the EC50 of CdCl=145 mg L-1 Germination assays on wheat, in vitro
Radicles lengths after exposure in vitro
Two different effects according cyanobacterial concentrations:
• at low concentrations (50-100 µg eq. MC-LR L-1), increase of the radicle length
• at high concentrations (>1 mg eq. MC-LR L-1), decrease of the radicle length Similar effects but different sensitivities, according plant species
0 1 2 3 4
Control 50 100 1000 5000 10000 15000 20000
S. lycopersicum var. MicroTom S. lycopersicum var. Saint-Pierre L. sativa var.capitata
radicle lengths (cm)
* *
* *
* *
* *
*
*
*
* *
*
Solanum lycopersicum var. MicroTom Solanum lycopersicum var. Saint-Pierre Lactuca sativa var. capitata
concentrations of cyanobacterial extract (µg eq. MC-LR L-1)
1
2
3
12
Introduction Material and
Methods Results and Conclusion
Discussion
Phytotoxicity of cyanobacterial extract on tomato S. lycopersicum var.
MicroTom after 14 days of irrigation
After 14 days of irrigation of soil-plant system:
• similar biomasses of roots between treatments
• increase of aerial parts biomasses for seedling exposed to cyanobacterial extract
0 4 8 12 16
C 5 20 50 100
biomasses: dry weight (mg)
cyanobacterial extract concentrations (µg eq. MC-LR L-1)
* * * *
aerial parts roots
C 5 µg L-1 20 µg L-1 50 µg L-1 100 µg L-1
13
Soil ecotoxicological parameters followed after 14 days of irrigation with cyanobacterial extract
After 14 days of soil-plant system irrigation, no modification of global enzymatic activities of soil.
enzymatic activities (mU g-1 dry soil)
a a a
a
a a
a a
a
a
a a
a
a a a
a
a a
a
4 9 14
Control 5 20 50 100
A
30 50 70 90 110
Control 5 20 50 100
B
4 8 12 16 20
Control 5 20 50 100
C
0,02 0,03 0,04 0,05
Control 5 20 50 100
urease D
arylsulfatase phosphatase
glucosidase
concentrations (µg eq. MC-LR L-1)
14
Introduction Material and
Methods Results and Conclusion
Discussion
Soil ecotoxicological parameters followed after 14 days of irrigation with cyanobacterial extract
T 5 20 50 100
0.050.100.150,050,100,15
C 5 20 50 100
potential nitrification (µg NO3- g-1 fresh soil h-1 )
• After 14 days of soil irrigation, potential nitrification was significantly impacted with an increase of this activity in soils exposed to aqueous cyanobacterial extract.
• An increase was observed for soils exposed to cyanobacterial extract comprised between 5 and 50 µg eq. MC-LR L-1 but not for the upper concentration (100 µg eq. MC-LR L-1).
* * *
concentrations (µg eq. MC-LR L-1)
After 14 days of irrigation with cyanobacterial extract , abundances of nitrifying
bacteria (AOB) increased for all treatments. This result could explain results obtained on potential nitrification with positive correlation (r=0,56 ; p<0,05). 15
Abundances of soil microbial communities after 14 days of irrigation with cyanobacterial extract
1E+0 1E+2 1E+4 1E+6 1E+8
C 5 20 50 100
log 10 number of copies of genes g-1 dry soil
16S bacteria 16S archeae AOA AOB
log 10 scale number of genes copies g-1 dry soil
* * * *
concentrations (µg équiv. MC-LR L-1)
16
Introduction Material and
Methods Results and Conclusion
Discussion
MCs, at “environmental concentrations”, could stimulate seedlings growth of some species but what are the effects after a long chronic exposition ?
The MCs brought by irrigation modified soil microbial activities, can you observe this response on other soil type ? And during a long exposition ?
The MCs could affect the metabolism of seeds during the germination process, but the concentrations must be very important and sensitivities differed
between plant species.
The germination of wheat seeds, a monocotyledon, was strongly impacted by MCs with an EC50 13 times lower than cadmium chloride (positive control).
« Environmental concentrations » (50 et 100 µq eq. MC-LR L-1) seemed increase radicle growth but high concentrations decreased their growth. The growth of aerial parts of seedlings were also stimulated.
« Environmental concentrations » did not change global soil enzymatic activities but the abundances of nitrifying bacteria increased as their nitrifying activity.
17
Acknowledgments: V. Grondin, C. Marrault, G. Delarue, F. Poiroux, A.
Trouvé, S. Nélieu, N. Cheviron, D. Bru, J-P Meunier, L. Dahuron, G. Caro, J.
Thénard and B. Pey for technical assistance.
D. Whithe for his help in English.
