Deployment of nZVI to soil for polychlorinated biphenyl … · 2016-03-29 · ZVI particles TEM digital images of nano and micro-particles. The image was obtained using a Phillips

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.



Time (d)0

28

112

Revegetation



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



Time (d)0

28

112

Revegetation



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%

* *


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


0 1 4 7 14

0

10

20

30

40

50

60

70

LSD5%

LSD5%

microscale

nanoscale



Time (d)0

28

112

Revegetation



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


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?



Time (d)0

28

112

Revegetation


•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



Time (d)0

28

112

Revegetation


•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,


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

http://www.environment-agency.gov.uk/?lang=_e

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


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%

* *


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,


application of nanotechnology for environmental benefit;

and,

- Discuss policy measures to address challenges in the

application of

nanotechnology for environmental benefit.

Documents

Deployment of nZVI to soil for polychlorinated biphenyl … · 2016-03-29 · ZVI particles TEM digital images of nano and micro-particles. The image was obtained using a Phillips