CHRONIC EFFECTS OF ZnO NANOPARTICLES TO EISENIA FETIDA

Chamila Samarasinghe

10/09/2019

The University of Newcastle

OUTLINE

• Introduction – Nanoparticles, Need for toxicity data, earthworms • Objectives of the experiment • Experimental design • Findings of the experiment • Comparison of our results with data available in literature • Challenges • Considerations for risk assessment

NANOPARTICLES

McNeil, SE 2005

• Increased relative surface area • Most of the atoms lay close or at the surface • Different/ enhanced properties compared to their bulk counterparts • Unique physico-chemical characteristics (eg. higher reactivity)

ZnO NANOPARTICLES

• Zn – essential trace element for living organisms • Properties:

ohigh catalytic / UV attenuation oWhitening oAntimicrobial

• Nanoparticle use largely increased in the last decade

ZnO APPLICATIONS

• 3rd Highest global production volume (550 tons) • Cosmetics, sunscreens, toothpastes, beauty products • Industrial coating – Textiles, plastic, wood • Sensors, Sensing devices, Catalysts • Solar cells, LCDs, electronics • Antifouling paints • Antimicrobial usage – Biocides? • Fertilizer

Bondarenko et al., 2013

• Improved properties - more efficiency in industrial applications • Manufacture and utilization – not properly regulated, industry does not

recognize as new chemicals, regulated as conventional bulk chemical compounds

• Volume of the NP enabled products increased • Higher volumes may release to the environment in future • Unique physico-chemical properties – enhanced reactivity, higher stability in

the environment??? • Potential harmful effects to living organisms

NEED FOR NANOECOTOXICITY

Amede et al., 2017

Environmental exposure, common transformations, transport and bioavailability of metal oxide nanoparticles in terrestrial environment

EARTHWORMS

• Important element in the terrestrial ecosystem, Key trophic link • Represent ~ 60 -80% of total soil biomass (Bouche, 1992) • Play key role in soil formation and organic matter breakdown • Maintain & improve soil structure • Contribute in nutrient cycling • Biological indicators of soil health • High potential of large scale exposure of earthworms to any nanoparticle • Model organisms to investigate toxicity of environmental pollutants

https://www.naturespot.org.uk/species/common-earthworm

EARTHWORM LIFE CYCLE

OBJECTIVES OF THE EXPERIMENT

• To assess the impact of ZnO nanoparticles to earthworms • Growth • Survival • Reproduction

• Tissue Zn bioaccumulation due to of ZnO nanoparticle exposure.

OECD PROTOCOLS

Earthworms are introduced to contaminated / spiked soil

Acute toxicity measurement Survival Morphological / behavioural symptoms

Chronic toxicity measurement Survival Reproduction (Cocoon & juvenile count) Biomass change Bioaccumulation

EXPERIMENTAL DESIGN

ZnO Nanoparticles

Uncontaminated soil

Collection (<10 cm – top layer) Processing Characterization

Spiking & Aging ~ 3 months

Introduction of Parent Earthworms

₊

Survival, Weight change &

Bioaccumulation

28 days

56 days Reproduction

SOIL CHARACTERIZATION

pH Sand (%)

Slit (%)

Clay (%)

Texture Total Carbon (%)

Background Zn (mg Kg-1) 6.5

38 33

28 Clay loam

4.99 106.79 ± 3.20

NANOPARTICLE CHARACTERIZATION

(A) XRD Diffractogram and (B) Scanning electron micrographs of ZnO dry nanopowder (Original magnifications: x 10, 000; Scale bar: 200 nm)

0 20 40 60 800

5000

10000

15000

20000

Position [02 θ]

Counts

(A) (B)

SURVIVAL & WEIGHT CHANGE • No significant effects were observed on survival and growth of earthworms

exposed to the ZnO nanoparticle - spiked soils when compared to the control soils.

• Any sublethal effects??

REPRODUCTION - COCOONS & JUVENILES

Reproductive response of the earthworms exposed to ZnO nanoparticles in soil

0 0.1 1 10 100 500 10000

5

10

15

20

ZnO nanoparticle Treatment (mg/ Kg)

Coco

on C

ount

0

0.1

1

10

100

500

1000

**

**

0 0.1 1 10 100 500 10000

10

20

30

40

ZnO nanoparticle Treatment (mg/ Kg)

Juve

nile

Cou

nt

***

***

BIOACCUMULATION OF Zn

• No considerable tissue accumulation of Zn metal was observed in the body tissues of earthworms exposed to ZnO nanoparticle spiked soil.

0 0.1 1 10 100 500 10000

20

40

60

80

100

ZnO nanoparticle Treatment (mg/ Kg)Mea

sure

d t

issu

e Z

n C

on

cen

trat

ion

(µg

/g w

et w

t.)

0

0.1

1

10

100

500

1000

FINDINGS OF THIS STUDY - SUMMARY

• ZnO nanoparticles did not affect earthworm survival and growth. • Above 500 mg Kg-1 concentration, ZnO nanoparticles are likely to cause

reproductive toxicity to the terrestrial organisms.

• > 500 mg Kg-1 – Exceed the expected environmental concentrations

INHIBITION OF EARTHWORM REPRODUCTION AT 1000 mg/ Kg OF ZnO NANOPARTICLES – COMARISON OF DATA

Our Stu

dy

Garcia-Gom

ez, 2014

Alves, 2019

Hooper, 2019

Garcia-Gom

ez, 2019

0

50

100

150

Inhi

biti

on ra

te o

f Coc

oon

prod

ucti

on (%

)

NR

97%

59%50%

100%

Our Stu

dy

Garcia-Gom

ez, 2014

Alves, 2019

Hooper, 2019

Garcia-Gom

ez, 2019

0

50

100

150

Inhi

biti

on ra

te o

f Juv

enile

pro

duct

ion

(%)

NR

92%

72%

40%

100%

CHALLENGES

• Inconsistent data • Different experimental approaches, difficulties in comparison • Interactions with organic material in the environment – soil characteristics • Potential solubility in aqueous medium

• Unavailability of common parameters – additional specific requirements??

• Dispersion conditions • Characterization of nanoparticles in the environmental matrix • Consideration of test conditions for potential artefacts due to their properties)

CONSIDERATIONS FOR RISK ASSESSMENT

• Extensive work already done for the risk assessment of Zn metal / bulk form is a useful starting basis to asses the potential environmental risk

• For initial analysis – no apparent need to go beyond for single nanoparticle risks

• Combined effects of NPs and co-existing contaminants in soil?? • Comparison of data with different types of soils – influence of soil

characteristics • More research is needed to provide insight to the ecotoxicological effects of

exposure to these nanoparticles.

• Continuous exposure • Long exposure period

• Simultaneous exposure from multiple chemical stressors

• Subtle effects • Cumulative and synergistic effects

• Long term impact on the ecosystem

Professor Megh Mallavarapu Professor Ravi Naidu Professor John Aitken Dr. Kannan Krishnan CRC CARE – PhD Scholarship CleanUp 2019 organizing committee GCER &The University of Newcastle