Current understanding on potential toxicity of engineered nanomaterials on the aquatic

NANOPARTICLES CHARACTERIZATION

microorganisms is limited for risk assessment and management. Here we show

MetPLATE™ bioassay as an effective and rapid screening tool to test for potential

aquatic toxicity of various metal-based nanoparticles (NPs). MetPLATE™ bioassay is a

simple and cost effective test that uses a mutant strain of Escherichia coli assay - the

enzymatic activity of which is measured as the percentage inhibition compared to the

negative control. Toxicity of NPs was also compared to their corresponding ionic

counterparts. The NPs tested were (1) Citrate coated nAg (Citrate-nAg), (2)

polyvinylpyrrolidone coated nAg (PVP-nAg), (3) uncoated nZnO, (4) uncoated nTiO2,

and (5) 1-Octadecylamine coated CdSe Quantum Dots (CdSe QDs). Citrate-nAg was

further fractionated into clean Citrate-nAg, unclean Citrate-nAg and permeate using

tangential flow filtration (TFF) system to eliminate residual impurities including ions

from the stock Citrate-nAg suspension and differentiate between ionic- versus nano-

specific toxicity. Our results demonstrate that nAg, nZnO, and CdSe QDs were

relatively less toxic than their corresponding ionic forms tested, while both NPs and

ions of TiO2 were not toxic at as high as 2.5 g/L to the MetPLATE™ bacteria. Overall,

the toxicity followed the following trend: CdCl2 > AgNO3 > PVP-nAg > unclean Citrate-

nAg > clean Citrate-nAg > ZnSO4 > nZnO > CdSe QDs > nTiO2/TiO2. As previously

recommended, these results also highlight the importance of fractionating metal NPs

for better toxicity assessment. Although we found that the evaluated NPs were

relatively less toxic than their ionic forms, we caveat to disposing NPs into the receiving

waters as physicochemical properties of NPs may change with changeable water

chem

RESEARCH OBJECTIVES

istry which may promote NPs toxicity.

1

ASSESSMENT OF AQUATIC TOXICITY OF METAL NANOPARTICLES USING

METPLATE™ BIOASSAY AS A RAPID SCREENING TOOL

1Lok R. Pokhrel, 1Thilini Silva, 2Amro M. El Badawy, 3Thabet M. Tolaymat, 1Brajesh Dubey Department of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, TN 37614, USA; 2Department of Civil & Environmental Engineering, University of Cincinnati. Cincinnati, OH,

USA; 3USEPA, Office of Research and Development, National Risk Management Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45224, USA. Correspondence: Brajesh Dubey, dubeyb@etsu.edu.

(1) To evaluate the sensitivity of MetPLATE™ bioassay to the toxicity

of various metal-based NPs suspensions;

(2) To compare the toxicity of NPs with their corresponding ionic

counterparts; and

(3) To examine the toxicity of Ag by fractionating it as unclean Citrate-

nAg (as-synthesized), clean Citrate-nAg (cleaned via tangential

flow filtration system), permeate (obtained as a filtrate) and ionic

Ag (as AgNO3) to differentiate ionic- versus nano-specific toxicity.

1. The dilution matrix (i.e., moderately hard water) and all NPs

suspensions had acceptable characteristics (pH = 6 - 7.5) as

required by the MetPLATE protocol.

2. Our results showed that MetPLATE test can be used as an

effective and rapid screening tool for the toxicity assessment of

wide variety of metal-based NPs.

3. PVP-nAg was the most toxic among all the tested NPs types,

while clean Citrate-nAg showed relatively similar toxicity as that

of unclean Citrate-nAg.

4. Both ions and NPs of TiO2 was nontoxic even at a concentration

as high as 2.5 g/L.

5. CdSe Quantum Dots showed only 34.42% inhibition of

MetPLATE bacteria at 100mg/L.

6. Ions of Cd and Ag were most toxic among all the tested materials

including NPs.

7. MetPLATE toxicity showed the following trend:

CdCl2 > AgNO3 > PVP-nAg > unclean Citrate-nAg > clean

Citrate-nAg > ZnSO4 > nZnO > CdSe QDs > nTiO2/TiO2.

8. Residual Ag ions in unclean Citrate-nAg slightly enhanced the

toxicity as revealed by fractionating Citrate-nAg using Tangential

Flow Filtrtaion (TFF) process.

9. Although particle size and surface charge were not adequate to

explain the nanotoxicity observed, these results however clearly

indicate towards lower nano-specific toxicity than ionic toxicity

of the tested materials.

Conclusion

1. As understanding on the mechanisms by which nanoparticles induce

toxicity is limited, our study indicates that inhibition of β-

galactosidase enzyme activity using MetPLATE bioassay can be an

important consideration for rapid toxicity assessment.

2. Toxicity varied with coatings for nAg, but overall toxicity could not

be explained by particle size, surface charge and coatings.

3. Ionic toxicity was higher than nanotoxicity.

4. Fractionating nanoparticles from ions using diafiltration or field

flow fractionation is crucial to distinguish nano- versus ionic

toxicity.

Wavelength (nm)

300 400 500 600 700

Ab

so

rban

ce

(a.u

.)

0

1

2

3

4

nTiO2

Permeate

AgNO3

Clean Citrate-nAg

Unclean Citrate-nAg

nZnO

Quantum Dots

PVP-nAg

ABSTRACT

β- galactosidase mediated conversion of CPRG into Chlorophenol Red

MetPLATE Procedure

Acknowledgments: We would like to thank the Gordon Research Conference; Office of Research

and Sponsored Programs, ETSU; and School of Graduate Studies, ETSU for providing financial support to Lok R. Pokhrel to attend the Gordon Research Conference.

RESULTS

Fig. 1. Kros Flo Tangential Flow Filtration process for nanoparticles purification

PVP-nAg

nTiO2

Citrate-nAg

Fig. 4. Representative UV-Vis spectra of tested nanoparticles Fig. 5. Particle size distribution of tested nanoparticles

Fig. 2. Representative UV-Vis spectra of tested nanoparticles Fig. 3. TEM images of nanoparticles

Fig. 7. MetPLATE test showing color variability in CPRG as impacted by different

nanoparticles and ionic salts tested

Fig. 6. Comparison of EC50 values showing variability in MetPLATE toxicity for

various nanoparticles and their ionic counterparts

METPLATE BIOASSAY

+ve Control

-ve Control