Summary of past research experiences in ecotoxicology.

1. An
International
Perspective on
Ecotoxicology
Dr. Andrew Barrick
1

2. Education
• University of California Santa Cruz – Marine
Biology (Baccalaureate in Sciences), Psychology
(Baccalaureate in Arts)
• St. Edward’s University & Université Catholique
de l’Ouest – Environmental Management and
Sustainability (Master in Sciences)
• Université Bretagne et Loire (Université de
Nantes)- Ecotoxicology (PhD)
2

3. Summary of Research Experiences
• Seine-Aval ECOTONES (Master’s thesis)
• NanoReg2 (PhD thesis)
• Emerging Organic Contaminants (Postdoc)
• Aotearoa Impacts and Mitigation of Microplastics (Postdoc)
• NanoInformaTIX (Postdoc/ Research Engineer)
• Ongoing Initiatives
3

4. Seine Aval – Ecotones (Master’s)
Hediste diversicolor
Scrobicularia plana
Genotoxicity:
• DNA Damage
Enzymatic Biomarkers:
• Oxidative Stress
• Neurotoxicity
Energetic Reserves:
• Glycogen
• Lipids
Population:
• Density
• Biomass
Reproduction:
• Sexual Stages
• Size of Adults
Amelioration
Prevention
4

5. Seine Aval – ECOTONES:
Base Line Assessment Criteria
J2015
J2016
J2017
2015
J2016
0
2
4
6
8
10
12
F M A M J J A S O N
Lipids
(mg/g)
Time (Months)
Baseline Assessment Criteria : Lipid Concentrations in Hediste diversicolor
Above BAC
BAC
Sub Lethal Effects
Lethal Effects
Authie
Seine Aval
• Establish range of natural variation using polynomial regression models
• Integrate baseline assessment criteria to establish lines of evidence of weight
for evidence approach to environmental impact assessments
5

6. Seine Aval – Ecotones: Developing Lines
of Evidence for H. diversicolor
(Piva et al., 2011)
6
• Integrated analysis to establish if H.
diversicolor are universally impacted
by anthropogenic activity
• Data weighted based of levels of
biological organization
• Combine with other environmental
parameters for weight-of-evidence
approach

7. NanoReg2: Grouping & Safe-by-Design
Approaches within a Regulatory Framework
7

8. What is a Nanomaterial?
8
• General Definition:
• Any material that has one dimension within 1-
100nm
• Increase in surface area/volume ratio alters
molecular properties, potentially more reactive
• Manufactured Nanomaterials:
• MNMs are nanomaterials intentionally
designed for a specific function
• Novel properties can also lead to uncertainties
in environmental and health risks

9. Investigating Nanomaterial Risks
9
Integrate safety principles into the
design of novel products

10. Estimating Potential Risks
10
Initial Properties
• Sedimentation Rate
• Hydrodynamic
Diameter
• Zeta Potential
• Dissolution
• Density
Environmentally Dependent
Properties
Ecotoxicological
Endpoints
• Subcellular
• Subindividual
• Individual
• Population
• Community
• Shape
• Particle Size/Range
• Chemical Composition
• Impurities
• Surface Area

11. Grouping Approach
11
Grouping of chemicals or products along common
properties
• Physicochemical properties
• Exposure
• Fate
• (Eco)toxicokinetics
Read Across
Extrapolation ?
Group New Product

12. NanoReg2: Aims of Thesis
12
• Can nanomaterials be grouped
using Mytilus edulis hemocyte cell
cultures?
• Is sublethal testing in vitro
comparable to in vivo testing?
• Integrating ecotoxicity hazard
assessment into safe(r)-by-design?
In vitro
Grouping?
In vitro
M. edulis
Hemocytes
Safe(r)-by-
Design?
In vivo

13. Overall Testing Strategy
13
Culturing of mussel
hemocytes Cytotoxicity (HTS)
qPCR
Comet assay
Biochemical analysis
In vivo exposure
Hemolymph
extraction
Sublethal biomarker analysis
Oxidative stress, membrane transport,
immune response, apoptosis, lipid
peroxidation, cytoskeleton, DNA
damage
Digestive Gland
Gills
Hemocytes
33 nanomaterials tested

14. Grouping Approach for Ecotoxicology
14
Compilation of previous
data
Benchmark Materials
• TiO2 (NM100-105)
• SiO2 (NM200-204)
• ZnO (NM110 &111)
• MWCNT (NM400-403)
Grouping & Read-Across

15. Generating Data Necessary for Grouping
15
• Algae growth
inhibition
• Daphnia
immobilisation
• Fish acute toxicity
• Mussel acute
toxicity
Ethical issues
(in vitro)
Cytotoxicity
assays
No-BSA
BSA
Dispersion Protocol

16. Hazard Data for Nanomaterials
16
0,0
1,0
2,0
4,0
8,0
16,0
32,0
64,0
128,0
256,0
0,0
1,0
2,0
4,0
8,0
16,0
32,0
64,0
128,0
256,0
NM100
NM101
NM102
NM103
NM104
NM105
P25 - - - - - - - - - -
NM110
NM111
NM200
NM201
NM202
NM203
NM204
NM400
NM401
NM402
NM403
Si40
SiΩC40
CFDA-AM (MC)
BSA
Almar Blue (MC)
BSA
concentration (µg/ml)
0,0
0,1
0,3
1,0
3,2
10,0
0,0
0,1
0,3
1,0
3,2
10,0
31,6
100,0
NM100
NM101
NM102
NM103
NM104
NM105
P25
NM110
NM111 - - - - - - - - - - - - - -
NM200
NM201
NM202
NM203
NM204
NM400
NM401
NM402
NM403
Si40
SiΩC40
OECD 201
concentration (µg/ml)
OECD 202
0,0
2,0
4,0
8,0
16,0
32,0
64,0
128,0
256,0
0,0
2,0
4,0
8,0
16,0
32,0
64,0
128,0
256,0
0,0
2,0
4,0
8,0
16,0
32,0
64,0
128,0
256,0
NM100
NM101
NM102
NM103
NM104
NM105
P25
NM110
NM111
NM200
NM201
NM202
NM203
NM204
NM400
NM401
NM402
NM403
Si40
SiΩC40
Almar Blue
(FCL) BSA
CFDA-AM (FCL)
BSA
Neutral Red
(FCL) BSA
concentration (µg/ml)
In vivo
Algae Daphnia Fish Cell Lines Mussel Hemocytes
Titanium
Zinc
Silicon
Dioxide
MWCNT

17. Grouping Using Available Data Set
17
Grouping Based on
Physicochemical Properties
Grouping Based on
Ecotoxicity Endpoints

18. Case Study: Grupo Antolin
18
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 resistivity Ω*m 1*10-3
1*10-4
1*10-3
Stacked cup structure
(Martin-Gullon et al., 2006)
Safe
Design
Ecotoxicological hazard assessment of carbon nanofibers
GANF: Original
product
GATam: GANF with
new production
method
GANFg: GANF
super heated to
2200°C
Courtesy of
Grupo Antolin

19. Raman Spectroscopy
(Courtesy of Emmanuel Flahaut (CIRIMAT))
19
• Raman spectroscopy: Used to measure the structural orientation of carbon
• ID/IG ratio used to identify structural impurities in the samples (graphitization)
Lower values = higher structural purity
ID/IG=
1.26
ID/IG=
1.07
ID/IG=
0.78
GANF GATam GANFg
Higher structural purity
Cleaner sample
Lots of noise:
Only CNFs in sample?

20. In vitro : Effect of Sample Preparation
20
EC50 (mg/L)
Dispersion Method GANF GANFg GAtam
Cellular
Metabolism
Cell Membrane
Integrity
Cellular
Metabolism
Cell Membrane
Integrity
Cellular
Metabolism
Cell Membrane
Integrity
With BSA 219.8 219.8 34.4 191.1 178.6 191.8
Without BSA 36.9 159.5 83.4 >256 120.3 >256
Little to no cytotoxic effects with CNF exposure

21. Molecular Effects: in vitro vs in vitro
21
0
2
4
6
8
10
12
0.01 0.1 1
Number
of
Genes
Concentration (mg/L)
Total Number of Genes with Statistical Significance P <0.05
GANF (in vivo) GANF (in vitro) GATam (in vivo)
GATam (in vitro) GANFg (in vivo) GANFg (in vitro)
• Little difference in expression of
mRNA gene between in vitro and in
vivo
• No strong variation between
nanoforms
• Membrane transporters and
xenobiotic stress most affected

22. Potential for Bioaccumulation?
22
GANFg: 1 mg/L
GANFg: 25 mg/L
GATam: 25 mg/L
GATam: 1 mg/L
GANF: 1 mg/L
GANF: 25 mg/L
Mussel feces
compacts
nanofibers

23. Commercial Testing: Direct Toxicity
Assessments (Postdoctoral Fellowship)
23
*
• Storm water runoff:
• City councils
• Commercial refineries
• Agricultural sites
• Commercial testing
• Biocidal paints
• Novel pesticides
• Open requests
• Optimization of new testing strategies
for commercialization

24. Emerging Organic Contaminants
24
• New Zealand has little to no data
surrounding chemical
concentrations in natural
environment
• Identify EOCs that are of highest
priority
• Determine regulatory guidelines to
minimize risks to environment and
people

25. Ecotoxicological Testing Approach
• Ecotoxicological profiles of emerging organic
contaminants towards marine copepods
• Development of novel molecular biomarkers for
green-lipped mussels (PhD Student)
• Design qPCR primers for green-lipped mussels
• Validate primers and test on contaminants of
emerging concern
• Investigate ecotoxicity towards early life stages
towards green and blue mussels
• Embryo & spermio toxicity
• Toxicity towards spermatocytes affecting embryo
develop 25

26. Developing Ecotoxicity Testing
Approaches
26
Duration (days) Effect thresholds Benzophenone Bisphenol A Chloroxylenol Diclofenac Gemfibrozil Metoprolol 2-Phenylphenol Triclosan
LC10 0.37 (0.01-0.59) 0.94 (0.68-1.21) 0.50 (0.32-0.67) 5.83 (4.94-6.73) 6.21 (4.14-8.28) >100 6.46 (9.84-10.71) 0.07 (0.05-0.09)
LC25 0.66 (0.4-0.92) 1.14 (0.99-1.31) 0.71 (0.54-0.88) 7.06 (6.73-7.83) 9.80 (7.48-12.12) >100 7.77 (7.29-8.25) 0.09 (0.07-0.10)
LC50 1.51 (1.22-1.8) 1.52 (1.26-1.78) 1.22 (1.05-1.37) 9.43 (8.84-10.02) 19.55 (17.11-21.99) >100 10.28 (9.84-10.71) 0.12 (0.11-0.13)
EC10 2.72 (2.05-3.40) 0.79 (0.63-0.96) 0.03 ( 0.00-0.07) 0.92 (0.82-1.02) 7.61 (4.32-10.92) 12.92 (8.40-17.43) 1.10 (0.90-1.30) 0.03 (0.02-0.05)
EC25 3.42 (2.97-3.88) 1.02 (0.91-1.13) 0.11 (0.04-0.19) 1.19 (1.10-1.27) 12.51 (9.78-15.24) 14.66 (12.70-16.61) 1.30 (1.20-1.40) 0.04 (0.03-0.05)
EC50 4.19 (3.88-4.50) 1.27 (1.22-1.32) 0.35 (0.23-0.48) 1.49 (1.42-1.56) 19.32 (17.08-21.55) 16.37 (15.28-17.47) 1.50 (1.30-1.60) 0.050 (0.046-0.058)
Reported Environmental
Concentrations (ng/L) 19.0-230.81
9.48-1732
- 395-21003
- 745-50003
146-3684
7.5-23005
Ecotoxicity of Emerging Organic Contaminants towards G. Pectinatus
Copepod Acute Toxicity 2
Nauplii Development Assay 7
Acute and Chronic Ecotoxicity
0
50
100
150
0.75 1.5 3 6 12
Larval
Development
Ratio
(%)
Algal Concentration (104)
Effect of Algal Concentration (50/50 C.
muelleri and T. lutea) on Larval
Development (7 days)
Optimization of Culturing
Day 0 Day 7

27. RNA Electrophoresis
gDNA
optimal?
Multigenerational Testing:
Transcriptomics
27
Test
Test
Test
Test

28. Aquaculture: Impacts Towards Green-
lipped Mussels and Blue Shelled Mussels
28
• Developing impacts of EOCs at early life stages
• Degeneration of spermatozoa
• Effects on embryos
• Impacts on fertilization rates
• Successful development
• Successful recruitment
• Molecular and subcellular impacts
• Early predictors
• Lines of evidence
• Safe(r)-by-design
Blue Mussel
Green-lipped Mussel

29. Assessing the Ecotoxicity of
Plastic Additives
29
AIM2: distribution and impacts of
microplastics in New Zealand
• Distribution of plastics in sediment,
freshwater and coastal environments
• Determine the potential
ecotoxicological and environmental
effects of microplastics

30. Plastic Ecotoxicity: A Question of Scale
30
>1 meter 5cm-5mm 5mm - 0.1µm <1µm
Megaplastics Macroplastics Mesoplastics Microplastics Nanoplastics
>5cm

31. A Cocktail of Chemicals
31
31
Antioxidant
Thermostabilizer
Antioxidant
Thermostabilizer
O2
O2 O2
Barrick A., Champeau O., Chatel A., Manier N., Northcott G., Tremblay L. 2021. Plastic additives: challenges in ecotox hazard
assessment. PeerJ. 10.7717/peerj.11300

32. Tiered Approach to Microplastic Testing
32
Will organisms
interact with
microplastics?
Are additives
inherently
toxic?
Algae
Plastic
Tier I
If prepared in
microplastics are
additives more toxic?
Are there chronic,
multigenerational
effects?
Mechanisms of
toxicity?
Oxidative Stress
Tier II

33. Test Species for Hazard Assessment
33
• Broad geographic
distribution
• “Easy” to culture & test
• High reproductive
capacity
Copepods Green and Blue
Mussels
• Mytilus galloprovincialis
• Recognized sentinel species
• International distribution
• Standardized testing approaches
• Perna canaliculus
• Significant commercial & cultural value
• Native comparator
• Broader ecological niche compared to blues
Algae
• Fast growth rate
• Easy to culture &
test
• Standardized test

34. Biological Uptake of Microplastics: Adults
34
43-53µm beads 75-90µm beads
22-27µm beads
1-5µm beads

35. Biological Uptake of Microplastics: Nauplii
35
22-27µm beads 43-53µm beads 75-90µm beads
1-5µm beads

36. Feeding Trials
36
-14000
-12000
-10000
-8000
-6000
-4000
-2000
0
1-5µm 22-27µm 43-54µm 75-90µm
Algal
Densities
Relative
to
Control
(cells/mL)
Copepod Feeding Rates
Algae +
Microplastics
(1-5µm)
Microplastics
(1-5µm)

37. Preliminary Additive Ecotoxicity
37
*
Day 0 Day 7
Copepod Larval Development Test
Copepod Acute Toxicity Test
+ =
Mussel Embryotoxicity
Plastic Additive Uses
Benzophenone UV-blocker
Dibutyl phthalate Plasticizer
Irgafos 168
Prevents plastic
degradation
Irganox 1010
Prevents plastic
degradation
Irganox 1076
Prevents plastic
degradation
Nylostab
Color booster, UV
blocker, Stabiliser
Tinuvin 770 Stabiliser
UV-234 UV-blocker

38. Preliminary Additive Ecotoxicity
38
Copepod
acute
LC(50) mg/L
Copepod
chronic
EC(50) mg/L
Blue mussel
embryotoxicity
EC(50) mg/L
Benzophenone 1.51 4.17 16.74
Dibutyl phthalate 0.93 0.12 0.65
Irgafos 168 Completely Insoluble
Irganox 1010 >5 5.80 >5
Irganox 1076 >10 >10 >10
Nylostab Completely Insoluble
Tinuvin 770 6.28 2.63 0.48
UV-234 >15 >10 >15 <1mg/L: Very Toxic
1-10mg/L: Toxic
10-100 mg/L:
Moderately Toxic
>100 mg/L: Non-Toxic
>Exceeds Solubility

39. 39
39
Future Perspectives
• No effect ≠ not toxic
• Preparation of microplastics
at sizes needed is extremely
challenging
• Organic material as vectors
of additive accumulation?
• Sublethal endpoints to
discriminate between plastic
effect and additive effect
Thermostabilizer
Biofilm & Grazing
“Sorption”
Thermostabilizer

40. 40
40
Establishing FAIR use of ecotoxicological
data for nanomaterial governance
C. Bertrand

41. 41
41
FAIRification of Nano Research
• Data collation and preparation
(Findable)
• Access to data (Available)
• Data analysis follows standard guidelines
(Interoperable)
• Organization of data for external users
(Reusable)
Data Completeness?
Physicochemical
Environmental
Biological

42. 42
Collation of Datasets
42
Gromelski et al., 2022

43. 43
43
Establishing FAIR use of ecotoxicological
data for nanomaterial governance
Persistent
Identifiers
Study
Design
Pristine
physicochemical
properites
System
dependent
properites
biological
data (in
vivo)
biological
data (in vitro)
Persistent
Identifiers
Study
Design
Pristine
physicochemical
properites
System
dependent
properites
biological
data (in vivo)
biological
data (in vitro)
Persistent
Identifiers
Study
Design
Pristine
physicochemical
properites
System
dependent
properites
biological
data (in vivo)
biological data
(in vitro)
Cadmium Nanoparticles 100.00 34.4 0.0 3.2 76.9 0.0
Copper Nanoparticles 100.00 65.6 35.5 50.0 76.9 0.0
Ceria Nanoparticles 100.00 56.3 19.4 66.7 7.7 0.0
Gold Nanoparticles 100.00 34.4 19.4 33.3 76.9 0.0
Silver Nanoparticles 100.00 65.6 12.9 61.1 53.8 0.0 100.00 34.4 16.1 5.6 0.0 10.5
Zinc Nanoparticles 100.00 34.4 29.0 72.2 69.2 0.0 100.00 34.4 100.0 50.0 0.0 10.5
Titanium Nanoparticles 100.00 53.1 9.7 11.1 76.9 0.0 100.00 34.4 100.0 50.0 0.0 10.5
Silica 100.00 34.4 100.0 50.0 0.0 10.5
Silicon/Composites 100.00 31.3 100.0 50.0 100.0 100.0
Multi-walled Carbon Nanotubes 100.00 34.4 100.0 50.0 0.0 10.5
Carbon Nanofibers 100.00 31.3 100.0 50.0 100.0 100.0
Graphene 100.00 34.4 0.0 50.0 0.0 10.5
Data Completeness (%)
NanoRETOX NANoREG/NanoSALT NanoReg2

44. 44
44
Metanalysis of the Data
• Preliminary analysis demonstrated
no significant variations between
soluble nanoforms and core
materials
• Major issues with data reporting
• Need for additional research to
improve metanalysis of the data
set

45. 45
45
Current Research Initiatives
• Continuation of FAIRification of Ecotoxicity data
• OECD Test guidelines (nanomaterials)
• In vitro fish assays
• Mesocosm research
• Microplastics
• Cal Grant (Expression of Interest)
• Microplastics ecotoxicity towards native
species
• Develop FAIR data set
• Assist with Data curation for ToMEX 2.0
INERIS INERIS
Microplastics