are sediment dwelling organisms at higher risk for ...€¦ · chris impellitteri, jason unrine,...
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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.
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