Deployment of nZVI to soil for polychlorinated biphenyl remediation: impacts on soil microbial communities
Liz Shaw
Outline
• Background
–PCBs: use of ZVI and biodegradation for remediation
• Experimental Data
– impacts of nZVI on microbial functions over short and
long timescales
• Summary of nZVI impacts on microbes
• Wider questions
–Environmental health and safety considerations
–Identify challenges for the development,
commercialisation and
application of nanotechnology
Polychlorinated biphenyls (PCBs)
• A class of chloro-organic chemicals with between 1 to 10 chlorine atoms attached to a biphenyl backbone
• 209 congeners
• Widespread historical use as dielectric fluids but production is now banned
• Toxic, bioaccumulative, persistent
• Soil pollutants as a result of historical and continued releases
min5 10 15 20 25
Norm.
0
5000
10000
15000
20000
25000
ECD1 A, (E:\AH040609\003F0301.D)
Aroclor 1242
(Cl)n (Cl)n
Nano- (and micro-) scale ZVI/ bimetallicZVI particles are active in PCB dechlorination
Reference Test conditions Findings
Wang & Zhang
(1997) Environ.
Sci. Technol. 31,
2154.
ZVI and Pd/ZVI (1-
100 nm) in
ethanol/water with
aroclor 1254
Complete dechlorination in
presence of Pd/nZVI but partial
dechlorination with nZVI
Lowry & Johnson
(2004) Environ.
Sci. Technol. 38,
5208.
ZVI (30-50 nm) in
methanol/water with
PCB congeners 22’,
34’, 234, 22’35’,
22’45’, 33’44’
He et al. (2009) J.
Hazard. Mat.
164, 126.
Pd/ZVI (1-2 m) in
soil with congener
22’455’
Production of 22’4-
trichlorobiphenyl
ZVI treatment may not always result in complete dechlorination but could produce lower-chlorinated congeners from more highly chlorinated parent PCBs
Depending on the extent and patternof chlorination, microorganisms maydehalogenate or completely mineralize PCBs
• Under anaerobic conditions, bacteria may
slowly reductively dehalogenate higher
chlorinated PCB congeners
• Under aerobic conditions, bacteria may
mineralize lower (<3 chlorines) chlorinated PCB
congeners
OH
OCOOH
OH
COOH
COOH OH
OH
Cl ClCl Cl
HOOC
Cl COOH
TCA cycle
3-chlorobiphenyl 3-chlorobenzoate 3-chlorocatechol
A two-step PCB remediation processcombining nZVI and bioremediation?
1. Use of nZVI to dechlorinate higher chlorinated PCBs
2. Use of bioremediation to remove the resulting lower-
chlorinated products
• Step 2 could involve monitored natural attenuation,
bioaugmentation, biostimulation, rhizoremediation
• How compatible is step 2 with step 1?
• What is the impact of nZVI on soil microbial
communities that are responsible for:
• Chloroaromatic degradation
• Plant nutrient acquisition (nutrient
mineralization and symbioses)
*http://www.nerc.ac.uk/research/programmes/nanoscience/background.asp
Putting together what we know about PCB susceptibility to ZVI and microbe attack…
OVERALL AIM
Short and long timescales
•Addition of aroclor 1242 (50 mg kg-1)•Addition of ZVI (10 mg kg-1)
Time (d)0
28
112
Revegetation
•Soil redox •Soil microbial activity (global and chloroaromatic degraders)
•Plant growth•Plant N fixing symbioses•Soil microbial activity (chloroaromatic degraders)
Expt 1(n & mZVI)
Expt 2(nPd/ZVI)
ZVI particles
TEM digital images of nano and micro-particles. The image was
obtained using a Phillips CM20 TEM operated at 200 kV. For samle preparation, a droplet of the slurry was placed on a copper grid with a lacy carbon film and allowed to dry in air.nZVI and nPd/ZVI were produced by ball-milling (Golder Associates) and mZVI was obtained from BASF
13.86 ± 6.1 nm 1.48 ± 0.62 mMean particle size = 12.46±0.26 nm
nZVI nPd/ZVI mZVI
Polyacrylic acid (PAA) (MW = 1,200 g / mol) was used as a dispersant at a concentration of 5 % wt / wt with the ZVI.
Short and long timescales
•Addition of aroclor 1242 (50 mg kg-1)•Addition of ZVI (10 mg kg-1)
Time (d)0
28
112
Revegetation
•Soil redox •Soil microbial activity (global and chloroaromatic degraders)
•Plant growth•Plant N fixing symbioses•Soil microbial activity (chloroaromatic degraders)
Expt 1(n & mZVI)
Expt 2(nPd/ZVI)
• Addition of nZVI created
an immediate and
significant reducing
environment
• Redox potential thereafter
recovered at a rate
related to the age of the
nZVI particles
• Effects produced by mZVI
were less pronounced
-400
-200
0
200
400
600
800
Redox p
ote
ntial (E
h)
-400
-200
0
200
400
600
800
ZVI particles in PAA
PAA control
Time since amendment (days)
0 1 4 7 28
-400
-200
0
200
400
600
800
'fresh' nanoscale
'aged' nanoscale
microscale
2Fe0 + 4H+ + O2 = 2Fe2+ + 2H2OFe0 + 2H2O = Fe2+ + H2 + 2OH-
Effects on soil redox potential areconsistent with Fe corrosion chemistry
Short and long timescales
•Addition of aroclor 1242 (50 mg kg-1)•Addition of ZVI (10 mg kg-1)
Time (d)0
28
112
Revegetation
•Soil redox •Soil microbial activity (global and chloroaromatic degraders)
•Plant growth•Plant N fixing symbioses•Soil microbial activity (chloroaromatic degraders)
Expt 1(n & mZVI)
Expt 2(nPd/ZVI)
nZVI impacts on dehydrogenaseactivity
• In the assay, the substrate (INT) acts as a terminal electron acceptor in microbial respiration and is reduced to INTF
• There was no abiotic reduction of INT to INTF
• nZVI stimulated dehydrogenase activity
Dehydro
genase a
ctivity
(g I
NT
F g
-1 s
oil
48 h
-1)
0
100
200
300
400
500
600
H2O control
PAA control
nZVI
LSD5%
* *
Time since amendment (days)
0 1 3 7 14
0
100
non-sterile
sterile
• Dehydrogenase assay - estimates the activity of the
membrane bound electron transport chain and therefore
measures the activities of intact microbial cells in the
decomposition of organic matter
ZVI impacts on FDA hydrolysisactivity
• Fluorescein diacetate hydrolysis (FDA) assay- estimates the global hydrolytic activity of soil enzymes (intra- and extracellular) in the depolymerisation reactions involved in organic matter breakdown
• Neither nanoscale nor microscale ZVI had any significant (p>0.05) effect on soil microbial global hydrolytic activity
Flu
ore
scein
dia
ceta
te h
ydro
lysis
(g F
luore
scein
g-1
soil
0.5
h-1
)
0
10
20
30
40
50
60
70H2O control
PAA control
ZVI
Time since amendment (days)
0 1 4 7 14
0
10
20
30
40
50
60
70
LSD5%
LSD5%
microscale
nanoscale
Short and long timescales
•Addition of aroclor 1242 (50 mg kg-1)•Addition of ZVI (10 mg kg-1)
Time (d)0
28
112
Revegetation
•Soil redox •Soil microbial activity (global and chloroaromatic degraders)
•Plant growth•Plant N fixing symbioses•Soil microbial activity (chloroaromatic degraders)
Expt 1(n & mZVI)
Expt 2(nPd/ZVI)
nZVI short-term impacts on chloroaromatic degrader number and activity
• nZVI initially depressed
numbers of
chloroaromatic degrading
microbes, but numbers
recovered over the same
timescale as the recovery
in soil redox potential
time since amendment (days)
0 1 4 7 28
100
101
102
103
104
105
H2O control
PAA control
nZVI
Log (
MP
N 2
,4-D
degra
der
g-1
soil)
nZVI short-term impacts onchloroaromatic degrader number and activity
Y = a1 + 10log(EC50-kt)
Degrader activity was assessed through a mineralization assay
‘Hillslope’ parameter used to examine treatment and time effects on mineralization kinetics
Time after amendment = 28 days
Time (days)
01 3 5 8 12 15 18 25 29 32
0
10
20
30
40
50
PAA control
+nZVI
Cum
ula
tive
2,4
-D m
inera
liza
tio
n (
%)
nZVI short-term impacts onchloroaromatic degrader number and activity
Time since amendment (days)
0 1 4 7 28
bio
de
gra
da
tio
n r
ate
(k)
0
2
4
6
8
10
12
14 H2O control
PAA control
+ nZVI
LSD5%
• ZVI significantly decreased the rate of chloroaromatic mineralization in comparison to the PAA control at all time points
• Although degrader numbers appeared to recover with time, their chloroaromatic degradation activity was inhibited– Direct or indirect effect of
nZVI on expression of catabolic pathway?
Short and long timescales
•Addition of aroclor 1242 (50 mg kg-1)•Addition of ZVI (10 mg kg-1)
Time (d)0
28
112
Revegetation
•Soil redox •Soil microbial activity (global and chloroaromatic degraders)
•Soil microbial activity (chloroaromatic degraders)• Plant growth•Plant N fixing symbioses
Expt 1(n & mZVI)
Expt 2(nPd/ZVI)
• Pd/nZVI had no effect on the number or activity of
chloroaromatic degradative microorganisms in the
developing clover rhizosphere
Time since sowing (days)
0 25 43 63 84
log (
MP
N 2
,4-D
degra
der
g-1
)
101
102
103
104
+ nZVI
PAA control
0 25 43 63 84
Bio
degra
dation r
ate
(k)
0
2
4
6
8
10
12
LSD5%
Pd/nZVI long-term impacts onchloroaromatic degrader number and activity
Short and long timescales
•Addition of aroclor 1242 (50 mg kg-1)•Addition of ZVI (10 mg kg-1)
Time (d)0
28
112
Revegetation
•Soil redox •Soil microbial activity (global and chloroaromatic degraders)
•Soil microbial activity (chloroaromatic degraders)• Plant growth• Plant N fixing symbioses
Expt 1(n & mZVI)
Expt 2(nPd/ZVI)
Pd/nZVI impact on plant growth
Lolium
Time since planting (days)
0 25 42 63 84
0
20
40
60
80
100
120
Trifolium
Sh
oo
t dry
wie
gh
t p
er
po
t (m
g)
0
50
100
150
200
250
+aroclor +nZVI
+aroclor -nZVI
-aroclor +nZVI
-aroclor -nZVI
LSD5%
LSD5%
• No effect of aroclor or
nZVI on growth of
Trifolium
• nZVI significantly (p<
0.01) enhanced growth of
Lolium at all time points
Pd/nZVI impact on plant symbiosis
Time since planting (days)
0 25 42 63 84
Num
ber
of
nodule
s
0
50
100
150
200
+aroclor +nZVI
+aroclor -nZVI
-aroclor +nZVI
-aroclor -nZVI
LSD5%
• Aroclor and nZVI
significantly depressed
nodulation in Trifolium
Effects on plant growth and symbioses may be due to perturbation of microbial soil N cycling
• nZVI appeared to enhance concentrations of soil plant available nitrogen and this was reflected in the N content of plant tissues
• Increased N availability could explain the growth response of Lolium
• Growth response in Trifoliumwas not evident due to compensation by symbiotic nitrogen fixation– Plants in control (low
available N) soils had more nodules and obtained more nitrogen from symbiotic fixation
extr
acta
ble
so
il n
itra
te-N
(m
g k
g-1
)
0
10
20
30
40
day 28
Nitro
gen
in s
hoo
ts (
mg
g-1
)
0
2
4
6
8
10
12
14
16
18
Lolium
Trifolium
PAA control nZVI
% N
itro
ge
n f
rom
fix
ation
in T
rifo
lium
0
10
20
30
40
50
60
70
Summary of nZVI impacts on soil microbes
• In the short term, no negative effects of nZVI on total microbial activity were detected, however, nZVI did perturb the activity of microbes specifically involved in chloroaromatic biodegradation. This has possible implications for the efficacy of combined nZVI/microbial remediation approaches
• In the longer term, Pd/nZVI had no effect on the activity of chloroaromatic degradative microbes in the developing clover rhizosphere but impacted plant growth and symbiotic N2
fixation possibly through effects on soil nitrogen cycling.
Outline
• Background
–PCBs: use of ZVI and biodegradation for remediation
• Experimental Data
– impacts of nZVI on microbial functions over short and
long timescales
• Summary of nZVI impacts on microbes
• Wider questions
–Environmental health and safety considerations
–Identify challenges for the development,
commercialisation and
application of nanotechnology
Environmental health and safetyconsiderations related to the use of nZVI for remediation
• In the case of incomplete remediation, are the
remediation products (e.g. partially reduced compounds)
more bioavailable/ mobile/ toxic than the starting
contaminant mix?
• What happens to the particles themselves after
remediation?
– [Results suggest that reactivity is quite quickly attenuated
in the soil environment and soil microbial functions may be
resistant, or, after an initial perturbation display
resilience*.]
Can particles transfer to other systems (e.g. enter
aboveground foodchains via plant uptake) with ecological
and human health implications
*We need to confirm if ‘sensitive’ functions such as ammonia oxidation conform to this statement!
Challenges for the development, commercialisation and application ofnZVI for site remediation
• Developing a framework for demonstrating
environmental and human health safety.
–What ecotoxicology tests to use? Some standard soil
assays are confounded by the reactivity of the
particles.
–What should we use as the control? (for example,
should we use ZVI in microscale or a competing
remediation technology)
–Challenges of scaling up
–Generalizing across environments, particles and
contaminants
Acknowledgements
• Emma Tilston
• Laurence
Cullen
• Geoff Mitchell
• Chris Collins
• Environmental
Nanoscience
Initiative
Effects on soil pH and redox areconsistent with Fe corrosion chemistry
• Addition of nZVI to soil
(10 g Kg-1) produced an
immediate and significant
elevation in soil pH of
between 1 and 1.5 units
• pH declined thereafter
• Effects produced by fresh
and aged nZVI were
similar, the impact of
mZVI was less
pronounced
5.5
6.0
6.5
7.0
7.5
8.0
8.5
pH
[H2O
]
5.5
6.0
6.5
7.0
7.5
8.0
8.5
ZVI particles in PAA
PAA control
Time since amendment (days)
0 1 4 7 28
5.5
6.0
6.5
7.0
7.5
8.0
8.5
'fresh' nanoscale
'aged' nanoscale
microscale
2Fe0 + 4H+ + O2 = 2Fe2+ + 2H2OFe0 + 2H2O = Fe2+ + H2 + 2OH-
ZVI impacts on ‘general’ microbial activity -some background on the assays
Polymeric organic matter
Bacterial cell
Hydrolytic extracellular enzymes produced by cell
endo and exo enzymes
monomers
OXIDATION
CO2Electron transport chain
½ O2 H2O
ZVI impacts on microbial activity -some background on the assays
• Fluorescein diacetate (FDA) in soil is hydrolysed by extra-
and intra-cellular hydrolases (includes lipases, proteases)
to produce fluorescein
• The FDA assay estimates the global hydrolytic activity of
soil in the depolymerisation reactions involved in organic
matter breakdown
O
O
O OC
CH3
O
OC
H3CO
Fluorescein diacetate (colourless)
O
O
O OOH
Fluorescein (coloured)
hydrolases
ZVI impacts on microbial activity -some background on the assays
• The dehydrogenase assayspecifically estimates the activity of the membrane bound electron transport chain and therefore measures the activities of intact cells in the decomposition of organic matter
monomers
OXIDATION
CO2Electron transport chain
½ O2 H2O
CN NH
N N
I
NO2
+HClCN N
N N+Cl-
I
NO2
+ 2H + 2e+ -
INT (colourless) INTF (red)
nZVI impacts on chloroaromaticdegrader number and activity
OH
OCOOH
OH
COOH
COOH OH
OH
Cl ClCl Cl
HOOC
Cl COOH
TCA cycle
3-chlorobiphenyl 3-chlorobenzoate 3-chlorocatechol
O
ClCl
CH2
COOH
OH
ClCl
OH
ClCl
OH
HOOC
COOH
TCA cycle
Cl
Cl
2,4-D
2,4-dichlorophenol
2,4-dichlorophenoxyacetic acid (2,4-D) chosen as model chloroaromatic since the pathway is convergent with that for chlorobenzoate biodegradation and 14C-UL-2,4-D is available
*
***
**
*
nZVI impacts on dehydrogenase activity
• nZVI apparently stimulated
the production of INTF, the
dehydrogenase assay
product
• There was no abiotic
reduction of INT to INTF
• The stimulation of INTF
production could be linked to
the removal of O2 as a
competing electron acceptor
(with the assay substrate)
rather than a real
stimulation of microbial
activity
Dehydro
genase a
ctivity
(g I
NT
F g
-1 s
oil
48 h
-1)
0
100
200
300
400
500
600
H2O control
PAA control
nZVI
LSD5%
* *
Time since amendment (days)
0 1 3 7 14
0
100
non-sterile
sterile
• - Identify the extent of the possible environmental
benefits from the
application of those nanotechnologies;
- Consider the environmental health and safety
implications related to
the use of nanotechnology for environmentally beneficial
purposes;
- Identify challenges for the development,
commercialisation and
application of nanotechnology for environmental benefit;
and,
- Discuss policy measures to address challenges in the
application of
nanotechnology for environmental benefit.