Are sediment dwelling organisms at higher risk for Nanoparticle exposure? Characterizing Nanoparticle exposure and effects in Hyalella azteca

Helen Poynton, Bonnie Blalock, Jim Lazorchak, Chris Impellitteri, Jason Unrine, Mark Smith

Understanding which organisms are exposed to and at greatest risk for nanoparticle toxicity is one of the most pressing issues in Nanotoxicology.

“How much of the nanoparticle remains suspended in the water column and how much settles into the benthic zone will determine not only the bioavailability of the nanoparticles to aquatic organisms, but also the type of organisms likely to be exposed and is also likely to determine the route and mechanism of uptake into the bodies of aquatic organisms.” - Scown, van Aerle, & Tyler

Crit. Rev. Toxicol. 2010, 40:653-70.

Sedimentation of NPs is expected . . .

Baun et al. (2008) Ecotoxicology. 17:387

Whitney Cranshaw, Colorado State University, Bugwood.org downloaded from http://www.forestryimages.org

Hyalella azteca as a model of Nanoparticle sediment exposure

• Epibenthic amphipod – scavenges at sediment surface

•Standard Ecotox organism for sediment toxicity testing

• Highly sensitive to metals

• Interest in H. azteca as an ecological model – development of genetic and genomic tools

(presentation #179, Hyalella-azteca-genome @google.groups.com)

H. azteca

D. magna

Preliminary studies showed that H. azteca was very sensitive to ZnO NPs

ZnO nano

10 mg/L 100 mg/L 1 mg/L 10 mg/L

77.3 mg/L

ZnSO4

10 mg/L 100 mg/L 1 mg/L 10 mg/L

154 mg/L 1.3 mg/L

T. platyurus H. azteca D. magna

22.5 mg/L

Why is H. azteca so sensitive to ZnO NPs? - determine the effect of Zn+2 in toxicity - determine the role of settling in toxicity

ZnO NPs properties and behavior:

• ZnO NP products include sunscreens, paints, electronics (Ma et al. 2013)

• Surface water concentrations estimated at: 0.3-0.4 mg/L (Gottschalk et al. 2009)

• Huge range of sensitivity across taxa from algae (mg/L range) to some crustaceans and mammals (tens of mg/L) (Bondarenko et al. 2013)

• Dissolution and direct particle toxicity both play a role in the toxicity of ZnO NPs, depending on species and conditions

ZnO NPs properties and behavior:

Increasing ion concentrations increase aggregation and sedimentation

Keller et al. 2010 Bian et al. 2011

ZnO NPs properties and behavior:

Keller et al. 2010 Bian et al. 2011

Decreasing pH induces dissolution of particles

Zn+2

Zn+2

Zn+2

Zn+2

Zn+2

ZnO NPs properties and behavior:

Keller et al. 2010 Bian et al. 2011

Zn+2

Zn+2

Zn+2

Zn+2

Zn+2

Increasing NOM stabilizes ZnO NP suspensions

ZnO NPs properties and behavior:

Zn+2

Zn+2

Zn+2

Zn+2

Zn+2

Bian et al. 2011 Lv et al. 2012 Ma et al. 2013

Reactions with sulfide and phosphate decrease dissolution, but increase aggregation and settling

+ sulfide

+ PO4-3

ZnO NP behavior is complex: highlights the importance of characterizing dissolution and settling in experimental systems.

Experimental Approach:

1) Expose H. azteca to ZnO NPs and ZnSO4 for 96-h to sublethal and lethal concentrations in water-only exposures

Why is H. azteca so sensitive to ZnO NPs?

Experimental Approach:

1) Expose H. azteca to ZnO NPs and ZnSO4 for 96-h to sublethal and lethal concentrations in water-only exposures

2) Characterize dissolution and sedimentation

Why is H. azteca so sensitive to ZnO NPs?

Experimental Approach:

1) Expose H. azteca to ZnO NPs and ZnSO4 for 96-h to sublethal and lethal concentrations in water-only exposures

2) Characterize dissolution and sedimentation

3) Use gene expression analysis to determine if toxicity is due to Zn2+ or particle specific

Why is H. azteca so sensitive to ZnO NPs?

Hyalella azteca microarray: Long 60 bp probes were designed from 65,961 contigs assembled from 454 sequencing of H. azteca cDNA libraries (in collaboration with J. Colbourne and M. Sepulveda)

Dissolved fraction

Particulate fraction

Size and morphology

Dissolution

Characterization of ZnO NP suspensions:

average diameter: 27.2 nm

About half of the ZnO NPs underwent dissolution resulting in ~ 2 mg/L and ~23 mg/L Zn2+ in the low and high exposures.

Zn++

Zn++

Zn++

Zn++

Zn++

Zn++

Zn++

Zn++

Characterization of ZnO NP suspensions:

Sedimentation in R-MHRW

Concentration of total Zn was homogenous in the exposure media suggesting well suspended NPs

Gene expression response to ZnO NPs

9.0-10.0

10.0-11.0

11.0-12.0

12.0-13.0

13.0-14.0

14.0-15.0

15.0-16.0

log2 expression

level

Sequence ID Predicted Function

1/10 LC50

ZnSO4

1/10 LC50

ZnONP

LC25

ZnSO4

LC25

ZnONP

Chitin metabolism

contig51444 cuticular protein -0.13 0.53 2.09 1.26

contig05159 cuticular protein 0.54 0.98 1.50 0.85

contig58143 cuticular protein 0.02 0.00 -0.88 -0.90

DNA Damage Repair/ Cell Cyc le Arrest

contig38148 suppressor of tumorigenicity -0.33 -0.17 -0.40 -0.42

contig37414 ribosomal protein -2.91 -1.18 -3.67 -3.66

contig56426 ATP-dependent RNA helicase 0.17 -0.06 0.08 -0.24

contig56149 DNA damage-inducible trascript 0.26 0.25 0.94 0.51

contig08441 TNF receptor-associated protein -0.07 -0.20 -0.16 -0.29

Response to Stress/ Response to Env ironment

contig65399 kairomone-inducible transcript -1.50 -0.11 -3.33 -2.98

contig18799 chorion peroxidase 0.10 0.39 1.11 0.64

contig06681 spermidine synthase -0.19 -0.33 -0.05 -0.43

RNA metabolic processes

contig20295 transcription elongation factor S-II -0.25 -0.20 -0.19 -0.56

contig23436 oculomotor apraxia protein 2 0.03 0.14 0.69 0.68

contig54523 DEAD-box protein, RNA processing -0.11 0.08 -0.23 -0.72

other metabolic processes

contig00905 pg1 protein -0.09 0.30 0.85 0.92

contig47457 Mitochondrial ornithine transporter 0.13 -0.02 0.72 0.15

contig60912 hydrolase -1.26 0.07 -2.03 -1.97

other funct ions

contig13382 projectin -0.15 -0.23 -0.14 -0.23

contig30627 niloticus neuralized-like protein 4 0.03 -0.33 0.01 -0.28contig01785 retrotransposon -0.26 -0.16 0.03 -0.19

contig02261 delta-type opioid receptor 0.68 0.38 0.90 0.91

unknown funct ion

contig61071 unknown function 0.75 1.79 1.07 1.10

contig63165 unknown function -0.59 -0.43 -1.03 -0.84

con

tro

l-1

con

tro

l-2

con

tro

l-4

con

tro

l-5

con

tro

l-3

con

tro

l-6

ZnSO

4-0

1

ZnSO

4-0

5

ZnSO

4-0

6

ZnSO

4-0

2

ZnSO

4-0

4

ZnO

NP

-01

ZnO

NP

-02

ZnO

NP

-03

ZnO

NP

-04

ZnSO

4-0

3

ZnO

NP

-05

ZnO

NP

-06

ZnSO

4-1

1

ZnSO

4-1

2

ZnSO

4-1

4

ZnO

NP

-11

ZnO

NP

-13

ZnO

NP

-14

ZnO

NP

-15

ZnSO

4-1

3

ZnSO

4-1

6

ZnSO

4-1

5

ZnO

NP

-12

• Growth and reproduction • Stress response • DNA damage and possible oxidative damage

Total of 71 differentially expressed genes, 23 with homology to sequences in GenBank

Gene expression patterns indistinguishable between ZnO NPs and ZnSO4

Hierarchical clustering of replicate exposures shows that control samples are distinct, but ZnSO4 (blue) and ZnO NP (orange) exposures cluster together.

9.0-10.0

10.0-11.0

11.0-12.0

12.0-13.0

13.0-14.0

14.0-15.0

15.0-16.0

log2 expression

level

Sequence ID Predicted Function

1/10 LC50

ZnSO4

1/10 LC50

ZnONP

LC25

ZnSO4

LC25

ZnONP

Chitin metabolism

contig51444 cuticular protein -0.13 0.53 2.09 1.26

contig05159 cuticular protein 0.54 0.98 1.50 0.85

contig58143 cuticular protein 0.02 0.00 -0.88 -0.90

DNA Damage Repair/ Cell Cyc le Arrest

contig38148 suppressor of tumorigenicity -0.33 -0.17 -0.40 -0.42

contig37414 ribosomal protein -2.91 -1.18 -3.67 -3.66

contig56426 ATP-dependent RNA helicase 0.17 -0.06 0.08 -0.24

contig56149 DNA damage-inducible trascript 0.26 0.25 0.94 0.51

contig08441 TNF receptor-associated protein -0.07 -0.20 -0.16 -0.29

Response to Stress/ Response to Env ironment

contig65399 kairomone-inducible transcript -1.50 -0.11 -3.33 -2.98

contig18799 chorion peroxidase 0.10 0.39 1.11 0.64

contig06681 spermidine synthase -0.19 -0.33 -0.05 -0.43

RNA metabolic processes

contig20295 transcription elongation factor S-II -0.25 -0.20 -0.19 -0.56

contig23436 oculomotor apraxia protein 2 0.03 0.14 0.69 0.68

contig54523 DEAD-box protein, RNA processing -0.11 0.08 -0.23 -0.72

other metabolic processes

contig00905 pg1 protein -0.09 0.30 0.85 0.92

contig47457 Mitochondrial ornithine transporter 0.13 -0.02 0.72 0.15

contig60912 hydrolase -1.26 0.07 -2.03 -1.97

other funct ions

contig13382 projectin -0.15 -0.23 -0.14 -0.23

contig30627 niloticus neuralized-like protein 4 0.03 -0.33 0.01 -0.28contig01785 retrotransposon -0.26 -0.16 0.03 -0.19

contig02261 delta-type opioid receptor 0.68 0.38 0.90 0.91

unknown funct ion

contig61071 unknown function 0.75 1.79 1.07 1.10

contig63165 unknown function -0.59 -0.43 -1.03 -0.84

cont

rol-

1

cont

rol-

2

cont

rol-

4

cont

rol-

5

cont

rol-

3

cont

rol-

6

ZnSO

4-01

ZnSO

4-05

ZnSO

4-06

ZnSO

4-02

ZnSO

4-04

ZnO

NP-

01

ZnO

NP-

02

ZnO

NP-

03

ZnO

NP-

04

ZnSO

4-03

ZnO

NP-

05

ZnO

NP-

06

ZnSO

4-11

ZnSO

4-12

ZnSO

4-14

ZnO

NP-

11

ZnO

NP-

13

ZnO

NP-

14

ZnO

NP-

15

ZnSO

4-13

ZnSO

4-16

ZnSO

4-15

ZnO

NP-

12

9.0-10.0

10.0-11.0

11.0-12.0

12.0-13.0

13.0-14.0

14.0-15.0

15.0-16.0

log2 expression

level

Se

qu

en

ce

IDP

red

icte

d F

un

ctio

n

1/10 LC50

ZnSO4

1/10 LC50

ZnON

P

LC25

ZnSO4

LC25

ZnON

P

Ch

itin m

eta

bo

lism

contig51444 cuticular protein

-0.130.53

2.091.26

contig05159 cuticular protein

0.540.98

1.500.85

contig58143 cuticular protein

0.020.00

-0.88-0.90

DN

A D

am

ag

e R

ep

air/ C

ell C

yc

le A

rres

t

contig38148suppressor of tum

origenicity-0.33

-0.17-0.40

-0.42

contig37414ribosom

al protein-2.91

-1.18-3.67

-3.66

contig56426A

TP

-dependent RN

A helicase

0.17-0.06

0.08-0.24

contig56149D

NA

damage-inducible trascript

0.260.25

0.940.51

contig08441T

NF

receptor-associated protein-0.07

-0.20-0.16

-0.29

Re

sp

on

se

to S

tres

s/ R

es

po

ns

e to

En

viro

nm

en

t

contig65399 kairom

one-inducible transcript-1.50

-0.11-3.33

-2.98

contig18799chorion peroxidase

0.100.39

1.110.64

contig06681 sperm

idine synthase-0.19

-0.33-0.05

-0.43

RN

A m

eta

bo

lic p

roc

es

se

s

contig20295 transcription elongation factor S

-II -0.25

-0.20-0.19

-0.56

contig23436oculom

otor apraxia protein 20.03

0.140.69

0.68

contig54523D

EA

D-box protein, R

NA

processing-0.11

0.08-0.23

-0.72

oth

er m

eta

bo

lic p

roc

es

se

s

contig00905pg1 protein

-0.090.30

0.850.92

contig47457M

itochondrial ornithine transporter0.13

-0.020.72

0.15

contig60912hydrolase

-1.260.07

-2.03-1.97

oth

er fu

nc

tion

s

contig13382projectin

-0.15-0.23

-0.14-0.23

contig30627niloticus neuralized-like protein 4

0.03-0.33

0.01-0.28

contig01785retrotransposon

-0.26-0.16

0.03-0.19

contig02261delta-type opioid receptor

0.680.38

0.900.91

un

kn

ow

n fu

nc

tion

contig61071unknow

n function0.75

1.791.07

1.10

contig63165unknow

n function-0.59

-0.43-1.03

-0.84

control-1

control-2

control-4

control-5

control-3

control-6

ZnSO4-01

ZnSO4-05

ZnSO4-06

ZnSO4-02

ZnSO4-04

ZnO NP-01

ZnO NP-02

ZnO NP-03

ZnO NP-04

ZnSO4-03

ZnO NP-05

ZnO NP-06

ZnSO4-11

ZnSO4-12

ZnSO4-14

ZnO NP-11

ZnO NP-13

ZnO NP-14

ZnO NP-15

ZnSO4-13

ZnSO4-16

ZnSO4-15

ZnO NP-12

Gene expression patterns indistinguishable between ZnO NPs and ZnSO4

Principle Component Analysis – shows overlap in the gene expression profiles of the ZnSO4 and ZnO NP exposures

Sensitivity and toxicity of ZnO NPs to Hyalella azteca

Summary:

• ZnO NPs are more toxic to H. azteca than Zn2+

• Gene expression profiles suggest that Zn2+ and ZnO NPs act through similar toxicity mechanism

• In the high ZnO NP exposure, only ~23 mg/L Zn2+

was present (versus ~90 mg/L Zn2+ in the high ZnSO4 exposures)

• ZnO NP suspensions were homogenous, suggesting that sedimentation was not the cause of enhanced toxicity

Maybe it’s their lifestyle. . .

Zn2+ Zn2+

Zn2+

Because of their feeding behavior as an epibenthic amphipod, H. azteca may take up more NPs than ions. Once in the gut, or within cellular compartments, the ZnO NPs dissolve, releasing Zn2+ which causes cellular toxicity (Trojan Horse mechanism; Park et al. 2010)

ZnO

NP

Survival when exposed to 230 mg Zn/kg, Manzo et al. 2011. Pollut. Res. Int.

ZnC

l 2

Photo credit: Mick E. Talbot

H. azteca shares behaviors with Heterocyipris incongruens, also sensitive to ZnO NPs:

Comparison with other sediment organisms:

Marine Amphipods:

Corophium volutator- similar uptake and toxicity mechanism for both ZnO NPs and ZnCl2 (Fabrega et al. 2011; Larner et al. 2012)

Leptocheirus plumulosus- ZnO NP LC50 based on pore water concentrations (dissolved Zn) was similar, but slightly lower than LC50 for ZnCl2 (Hanna et al. 2012)

Photo credit: C.Löser

Photo credit: Fisheries and Oceans Canada

What does this mean? Are sediment dwelling organisms at greater risk for ZnO NP toxicity?

1) Assumption that water quality criteria for Zn2+

(120 mg/L) will be protective for NPs is not valid

2) Exposure risk may be dependent on behavior, but is also related to environment (e.g. water chemistry)

3) More research is needed on different functional groups of sediment organisms to determine who is at greatest risk

Acknowledgements

Bonnie Blalock

Co-authors: Jim Lazorchak Chris Impellitteri Jason Unrine Mark Smith