Preliminary results on the application of safe(r)-by-design ecotoxicity testing for manufactured carbon nanofibers. Work was conducted as part of the NanoREG2 research project.

1. Project #646211, Funded by the Horizon 2020
Framework Program of the European
The Application of Ecotoxicological Tools to
Safer-by-Design Strategies for Engineered
Nanomaterials
Andrew Barrick1, Nicolas Manier2, Pascal Pandard2, Amélie Châtel1, Catherine Mouneyrac1
1) UBL, Mer Molécules Santé (MMS), Université Catholique de l’Ouest.
2) Institut National de l’Environnement Industriel et des Risques (INERIS), EXES unit
1

2. What is Safe(r)-by-Design for Nanomaterials?
• Safe(r)-by-Design (SbD) initially developed in
ProSafe and NANoREG
• Integration of safety knowledge into production
process of manufactured nanomaterials (MNMs)
• Increased exchange of information between
stakeholders (Kraegeloh et al., 2018)
• 3 core pillars in SbD
• Safe product design → less hazardous nanomaterials
• Safe use of products → evaluation and mitigation of
exposure
• Safe production → safe industrial production
2
Raw Powder
Commercial
Use
MNM
Product
End of Life
Development
of MNM
Safe(r)-by-Design
Risk
Analysis

3. NanoReg2 SbD strategy
• Integrate SbD with a Safe Innovation Approach (SIA)
• Identify and minimize risks early in the production process
• Develop safer alternatives
• Reduce regulatory burden on industry by streamlining risk assessment
• Necessary to find ways to mitigate NanoRisk
3
http://www.nanoreg2.eu/safe-design

4. Case Study: Carbon Nanofibers
• Three Carbon Nanofibers (CNFs)
• GANF → original Product
• GATam → production of GANF scaled up to produce 1 ton/year
• GANFg → GANF super heated at 2200°C
Measured property Unit GANF GANFg GATam
Fiber diameter (TEM) nm 20-80 20-80 20-80
Carbon purity (TGA) % >85 >99 >80
Apparent density g/cc ≈0.06 ≈0.08 ≈0.08
Specific surface area (BET N2) m2
/g 100-170 70-90 70-140
Graphitization degree (XRD) % ≈70 ≈90 ≈60
Electrical resitivity Ω*m 1*10-3
1*10-4
1*10-3
Table: Data provided by Industrial Partner
Stacked Cup Structure
(Martin-Gullon et al., 2006)
4

5. Role of Ecotoxicology in SbD
• Identify potential ecotoxicity hazards early in
production process
• Provide information related to associated risks with
MNMs
• Determine the “safer” option for use in products
• Do minor changes in the production process alter
(eco)toxicity?
• Focus is on GANF vs GATam
• GANFg could be considered more of control for CNF
toxicity
5

6. In vitro Assays Identified as Suitable for
Reporting Nanotoxicity
• Cellular Metabolism
• Cell Membrane Integrity
• Reactive Oxygen Species
Generation
• Genotoxicity
• Apoptosis
• Reactive Oxygen Species
Generation
• Genotoxicity
• Apoptosis
In vivo Comparison to in vitro Results
6
?
Mytilus edulis
Sentinel Species & Bioindicator
Hemocytes Sensitive to Environmental Contaminants

7. Regulatory Testing Following OECD Test Guidelines
P. subcapitata
Algal Growth inhibition test (OECD 201)
D. magna
Immobilization test (OECD 202)
Reproduction test (OECD 211) 7
4 µm

8. Preparation of Stock Suspension for CNFs
• Carbon based MNMs notorious for poor dispersity
• Surfactants often used to achieve stable suspensions
• Bovine Serum Albumin (BSA) commonly used surfactant
• Does the use of BSA influence interpretation of
toxicity?
• Dispersion SOPs defined in NANoREG
• In vitro Dispersion SOP (0.05% BSA)
• All selected assays
• Ecotox Dispersion SOP (Ultra-pure water)
• M. edulis hemocytes (Cellular Metabolism, Cell Membrane
Integrity)
• OECD 202
8

9. Characterization of CNF Suspensions
• DLS can reliably measure particle sizes when BSA is used → little change in size
• Solutions prepared without BSA not stable enough to be measured
Start of Experiment (Using BSA)
GANF GANFg GATam
Z-average (d-nm) PdI Zeta potential (mV) Z-average (d-nm) PdI Zeta potential (mV) Z-average (d-nm) PdI Zeta potential (mV)
Ultra-pure Water 489.3 0.128 -10.1 479.8 0.178 -15.6 414.3 0.231 -13.8
M. edulis Culture Media 260.1 0.359 - 244.1 0.325 - 197.8 0.272 -
Artificial Sea Water 186.7 0.306 - 217.4 0.307 - 223 0.311 -
End of Experiment
GANF GANFg GATam
Z-average (d-nm) PdI Zeta potential (mV) Z-average (d-nm) PdI Zeta potential (mV) Z-average (d-nm) PdI Zeta potential (mV)
Ultra-pure Water 527.5 0.123 -15.9 441.2 0.285 -14.8 389.4 0.164 -13
M. edulis Culture Media 221.4 0.32 - 237.2 0.226 - 164.3 0.23 -
Artificial Sea Water 278.3 0.418 - 348.1 0.77 - 220.5 0.348 -
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10. Stability of CNF Suspensions
Ultra-pure Water D. Magna Media
Algal Media Cell Culture Media Artificial Seawater
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GATam
GANFg
GANF
GATam
GANFg
GANF
BSA No BSA

11. M. edulis Hemocytes: Cellular Metabolism
• Effects on cellular metabolism more pronounced when BSA not used
• BSA : GANFg most toxic
• No BSA: GANF most toxic
GATam
GANFg
GANF
11

12. M. edulis Hemocytes: Cellular Membrane
Integrity
• Effects on cellular membrane integrity more pronounced when BSA not used
• BSA : GANFg most toxic
• No BSA: GANF most toxic but not strong differences between CNFs
GATam
GANFg
GANF
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13. 0
10
20
30
40
50
60
70
0 0.01 0.1 1 H2O2
Tail
DNA
(%)
Concentration (mg/L)
Comet Assay: in vitro
H2O2 Gatam Ganfg Ganf
0
10
20
30
40
50
60
70
0 0.01 0.1 1 H2O2
Tail
DNA
(%)
Concentration (mg/L)
Comet Assay: In vivo
Postive Control GATAM GANFg GANF
• More genotoxic effects observed in vivo than in vitro
• Higher percentages of tail DNA with exposure to GATam than GANF
M. edulis Hemocytes: Genotoxicity
GATam
GANFg
GANF
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14. M. edulis Hemocytes: ROS Generation
0
50
100
150
200
250
300
0 0.01 0.1 1
Relative
FLuoresecent
Units
(%
Control)
Concentration (mg/L)
ROS Generation: in vitro
GATam GANFg GANF
0
50
100
150
200
250
300
0 0.01 0.1 1
Relative
Fluorescent
Units
(%
Control)
Concentration (mg/L)
ROS Generation: in vivo
GATam GANFg GANF
• No clear trends of ROS Generation in vivo
• Statistically significant effects at 1mg/L in vitro
GATam
GANFg
GANF
14

15. 0
20
40
60
80
100
120
140
160
0 0.01 0.1 1
Relative
Fluoresecent
Units
(%
Control)
Concentration (mg/L)
Caspase Activity: in vitro
GATam GANFg GANF
0
20
40
60
80
100
120
140
160
0 0.01 0.1 1
Relative
Fluorescent
Units
(%
Control)
Concentration (mg/L)
Caspase Activity: in vivo
GATam GANFg GANF
• More Caspase activity observed in vivo than in vitro
• GATam induced more apoptosis
GATam
GANFg
GANF
M. edulis Hemocytes: Apoptosis
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16. OECD 202: D. magna Immobilization
0
20
40
60
80
100
120
0 3.125 6.25 12.5 25 50 100
Immobilization
(%
Control)
Concentration (mg/L)
D. magna Immobilization (No BSA)
Gatam Ganfg Ganf
0
20
40
60
80
100
120
0 3.125 6.25 12.5 25 50 100
Immobilization
(%
Control)
Concentration (mg/L)
D. magna Immobilization (BSA)
Gatam Ganfg Ganf
• Use of BSA removes interpretation of toxicity (reduced interaction with daphnids?)
• When no BSA is used similar response observed between GANF and GATam
GATam
GANFg
GANF
16
Exposed to GANFg
25 (mg/L)
Exposed to GANFg
25 (mg/L)

17. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.78 1.56 3.13 6.25 12.5
Average
Daily
Growth
Rate
Concentration (mg/L)
OECD 201: P. subcapitata Growth
Gatam Ganfg GANF
• Effects of CNFs on algal growth rate
• Lowest growth rates observed with algae exposed to GATam
OECD 201: Algal Growth
GATam
GANFg
GANF
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18. OECD: 211: D. magna Reproduction
• Chronic Testing allows for
discrimination between the
CNFs
• GATam significantly lowered
reproductive capacity of D.
magna
18
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10
Number
of
Juveniles
(%
rtc)
Concentration (mg/L)
GANF
GANFg
GATam
EC50 = 0,3
mg/L
EC50 = 1,6
mg/L
EC50 = 6,2
mg/L

19. Summary of Case Study
• In vitro effects on M. edulis were dependent on
dispersion SOP
• Differences between CNFs was smaller when no BSA was used
• Minimal sublethal effects observed
• in vivo effects demonstrate differences between CNFs
• Results suggest GATam may be more toxic than GANF
• Impurities in production process?
BSA Reduces Toxicity
BSA No BSA

20. Take Home Message
• Ecotoxicology for Safer-by-Design
• Preparation method for test suspension will influence interpretation of toxicity
• Stability of MNMs makes interspecies comparisons difficult
• Sublethal endpoints may be more appropriate as products will be very similar
• In vitro vs in vivo testing
• More adverse effects found in vivo than in vitro → effect of FBS in culture media?
• Key differences in media make comparison difficult
• 1:1 comparison not realistic
• Stronger characterization of CNF behavior in test media (Deloid et al., 2017)
• Testing was focused on Safe product design → Safe use of products?
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21. Thank you for your Attention
http://www.nanoreg2.eu/
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Acknowledgements: The research contained within this publication was funded by the European Union’s Horizon 2020 research and innovation
program NANoREG2 under grant agreement 646221.
"The sole responsibility of this publication lies with the author. The European Union is not responsible for any use that may be made of the information
contained therein."

22. References
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Deloid, G.M., Cohen, J.M., Pyrgiotakis, G., and Demokritou, P., 2017. Preparation, characterization, and in vitro dosimetry of dispersed,
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manufactured nanomaterial exposure media. NANoREG: A common European approach to the regulatory testing of nanomaterials,
Web-Report.
Kraegeloh, A., Suarez-merino, B., Sluijters, T., and Micheletti, C., 2018. Implementation of Safe-by-Design for Nanomaterial Development and
Safe Innovation : Why We Need a Comprehensive Approach.
Martin-Gullon, I., Vera, J., Conesa, J.A., González, J.L., and Merino, C., 2006. Differences between carbon nanofibers produced using Fe and Ni
catalysts in a floating catalyst reactor. Carbon, 44 (8), 1572–1580.