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1 Ecotoxicology & Health of Ecosystems Evaluation écotoxicologique des nanoparticules en milieu aquatique : cas des nanotubes de carbone Laury Gauthier, F. Mouchet, E. Flahaut & E. Pinelli Contact: laury.gauthier@univ-tlse3.fr

2 SOCIETAL CONTEXT CNTs are considered as manufactured emerging contaminants in our technological societies Exceptional physical-chemical properties ENVIRONMENTAL IMPLICATIONS Fate, transport, potential toxicity and Ecotoxicity, environmental measurements, Risk assessment, ecosystem perturbations leading to Various products Various applications SOCIAL IMPLICATIONS Ethics, regulations, research, standardization,

3 Life cycle of products Environmental concern Production releases Ageing of products ENVIRONMENTAL CONTEXT CNTs releases in the environment may come from various contamination points: Manufacturing Landfills Wastewater effluents All point sources are susceptible to contaminate the different compartments by : -Geochemical cycling -Biological functioning Wastes disposal recycling Discharge, leakage, Wastes, ground water contamination Transport dispersion Aggregation Adsorption On sediment uptake Bioconcentration excretion Ultimately, all contaminants may reach the aquatic medium and concentrate

4 Nanoparticles ecotoxicity : case of the CNTs The joint research laboratory «NAUTILE» NAnotUbes & écotoxicologie Private / public collaborative program (CNRS n ) Founding structures : Involved laboratories : GRL groupement de recherche de Lacq Main goals: - Study carbonated nanomaterials in the aquatic environment - Evaluate the potential ecotoxicological impact of CNTs in water

5 Aquatic biological models Amphibian larvae Water column ISO (2006) Algae, Diatoms Study at population community level BIOFILM Water-Sediment interface Chironomids AFNOR XT P (2004) SEDIMENT

6 AMPHIBIAN BIOASSAY PROTOCOL 1- Fertilization to obtain larvae (Stage 50 of Nieuwkoop and Faber, 1956) 2- Semi-static exposure for 12 days ISO (2006) 3- Endpoints Day 0 Day 1 Day 12 Mortality% Growth inhibition % Primary DNA damages Comet assay Negative control Comet Micronuclei Micronucleated erythrocytes DNA damages: - Primary damages (Comet measurement) -Non-repairable (MN induction)

7 Physical-Chemical Characterisation of CNTs MWNT/DWNT MWNT Synthesis Catalyst Carbon content (wt.%) Aspect Solubility Number of walls Diameter Length Specific surface area : CCVD (Catalytic Chemical Vapor Deposition) : Fe-Al2O3 / Co-Mo-MgO : > 90 / > 92 : Solid (powder) : Not soluble in water and other organic solvents : 5-15 (100% MWNTs) / 80% DWNT : nm / 1-3nm (ext) : µm / few microns : m2/g / m²/g X 100 MWNT MWNT X DWNT

8 Protocol of exposure Use of various surfactants to stabilize CNT suspensions in water [UT] protocol [UT + US] protocol GA and CMC are able to stabilize CNT (10 mg/l) suspensions in water during 24 h

9 Dispersion protocol Mechanical dispersion + non-covalent functionalization with an anionic polymer - Carboxymethylcellulose or Gum arabic -

10 Acute toxicity: mortality (%) NC : Negative Control No acute toxicity vs NC Acute toxicity vs NC Mortality is expressed in % MWNT (mg/l) NC % 0% 0% 0% No mortality DWNT (mg/l) NC % 0% 5% 15% Low mortality at 50 mg/l DWNT (mg/l) + Gum Arabic GA NC GAC % 0% 0% 0% No mortality

11 Chronic toxicity: growth inhibition NC: Negative Control GAC: Gum Arabic (Control) * No chronic toxicity vs NC Chronic toxicity vs NC : significant GI vs NC after 12 days of exposure Growth Inhibition GI (in mm, not shown) is illustrated by the rate of growth (%) Rate of Growth (%) R ate o f g ro w th (% ) NC 0,1 mg/l MWNT 1mg/L MWNT 10 mg/l MWNT 50 mg/l MWNT NC 0.1 mg/l 1 mg/l 10 mg/l 50 mg/l MWNT (mg/l) - Dose dependent GI - Chronic toxicity from 50 mg/l (MWNT) - Chronic toxicity from 10 mg/l (DWNT) In presence of GA, we observed the same GI rate with DWNT from 10 mg/l Rate of Growth (%) * rate of growth (%) Rate Rate of Growth of size (%) (%) NC 0.1 mg/l DWNT 1 mg/l DWNT 10 mg/l DWNT 50 mg/l DWNT NC 0.1 mg/l 1 mg/l 10 mg/l 50 mg/l DWNT (mg/l) NC AGC 0.1 mg/l 1mg/L 10 mg/l 50 mg/l * * - GA * + GA *

12 EMN / EMN / oo (median) (médiane) Genotoxicity: : micronuclei induction (MN) NC : negative control PC : positive control (Cyclophosphamide) No genotoxicity vs NC Genotoxicity vs NC 50 mg/l DWNT : non explored concentration because of the high toxicity DWNT - GA 0 T - T + 0,1 mg/l PC MWNTs 1 mg/l MWNTs 10 mg/l MWNTs Conditions expérimentales MWNT (mg/l) 50 mg/l MWNTs NC 0.1 mg/l 1 mg/l 10 mg/l 50 mg/l MWNT (mg/l) - MWNT are not genotoxic - DWNT are not genotoxic in the same test conditions DWNT + GA GENOTOXICITY OF DWNT IN PRESENCE OF GA

13 Xenopus larvae macro- micro observations Internal gills trap blocked up Branchial obstruction? Hypoxia induction? CTRL CNTs Black gut 50µm Intestinal obstruction? 50µm Nutrient competition? CNTs-xenopus larvae toxicity due to Clogged gills / black filled intestines?

14 Larvae reactive oxygen species production (H 2 O 2 ) % induction = (H 2 O 2 mean MW, time X) - (H 2 O 2 mean ctrl, time X) / (H 2 O 2 mean ctrl, time X) MW 10 mg/l MW 1 mg/l In CNTs exposed larvae ROS (H 2 O 2 ) production Is both Time & Dose dependant MW 0.1 mg/l

15 DNA damages induction: Short time vs long time COMET ASSAY 24H and 12 days 24H No Significant DNA damages vs NC Significant DNA damages vs NC Induction Factor 12Days MNE : Micronucleatederythrocytes PC : Positive Control Cyclophosphamide(MN induction) and MMS (Comet assay) MICRONUCLEUS ASSAY 12 days PC 0.1 PC 1 PC 0.1 PC 1 24H 12Days MNE /oo(median value)) NC PC 0.1 mg/l 1 mg/l 10 mg/l 50 mg/l T - T + 0,1 mg/l MW 1 mg/l MW 10 mg/l MW 50 mg/l MW MWNTs Concentration (mg/l) PC 0.1 PC 1 PC 0.1 PC 1 MWNTs Concentration (mg/l) Comet assay: For concentrations 10 and 50 mg/l non-viability of erythrocytes (Trypan Blue) Primary (reversible) DNA damages from 24 h at 0.1 mg/l of MWNT exposure

16 TEM observations of CNTs in living systems DWNT (10 mg/l) Lumen MWNT (10 mg/l) Lumen MWNT (10 mg/l) Lumen MWNT (10 mg/l) intestinal cell Vi MWNT?

17 Raman analysis of biological samples Raman cartography at 2675 cm -1 Raman spectroscopy λ = 514 nm (a) Light microscope image of the studied intestine area (b) Raman intensity map corresponding to the selected observation area of (a) (c) Superposition of (a) and (b) Line scans Raman analysis - G band only in the lumen - Intensity of G band = 0 in intestinal cells - No gradient of G band intensity CNT (DWNT) do not pass through the intestinal barrier Intensity of G band Intestinal cells Lumen Line 1 Line 2 Line 3

18 Diatoms as model to study the effects of CNTs Nitzschia palea Ubiquitous benthic algae Present in most photo-autotrophic biofilms Basis of numerous aquatic trophic chains Easy to cultivate, rapid growth Diatoms biofilms stabilize CNTs suspensions in lab conditions MWNT (50mg /L) MWNT (50mg /L) + Diatoms +/- NOM

19 Role of the Natural Organic Matter in natural waters MET observations of diatoms exposure conditions to CNTs DWNT MWNT Without NOM Compact agregates of CNTs NOM/NTC (ratio 1/5) Dispersed agregates of CNTs NOM/NTC (ratio 1/1) Fully dispersed agregates with Individualized CNTs

20 NOM Interaction Biofilm- MWNT Staining of the polysaccharides in the diatoms ExoPolymeric Substances (EPS) Light microscopy MWNT-NOM 20µm 20µm NOM SEM MWNT-NOM

21 SEM observations of the interaction Biofilm- MWNT MWNT / diatoms ExoPolymeric Substances Control (+/- NOM) MWNT-NOM (10mg.L -1 /10mg.L -1 ) 1µm 1µm 250nm 250nm

22 Other biological test organisms and exposure conditions Single species ecotoxicity test systems Algae, Diatoms Multispecies associated in Micro-mesocosms Pleurodeles(newts) Consumer II Amphibian Carnivorous Water column BIOFILM Chironomids Consumer I Sediment-dwelling invertebrates Deposit-feeders Sediment Xenopus Consumer I Amphibian Grazer-filterer SEDIMENT Insects, chironomids Diatoms Producer I Benthic algae Form the biofilm Experimental trophic chains In dynamic microcosms Amphibian larvae WATER COLUMN

23 THANK YOU FOR YOUR ATTENTION Ph.D. students Agathe BOUR Floriane BOURDIOL Périne LANDOIS Rayenne SARIA Laurent VERNEUIL Researchers Laury GAUTHIER, ECOLAB/NAUTILE Eric PINELLI, ECOLAB/NAUTILE Emmanuel FLAHAUT, CIRIMAT/NAUTILE Pascal PUECH, CEMES/NAUTILE Engineers/technicians Florence MOUCHET Annie PERRAULT Jérôme SILVESTRE

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