Protocol

Project Title: Metal Oxide Nanoparticle Stock Suspension Preparation and

Aggregation Kinetics Measurement
PI: Dr. Arturo Keller
Theme: Fate, transport, exposure and life cycle analysis
Project: 12
Version Number: 1.0
Production Start Date: December, 2008
Version 1.0 Date: Oct., 2011
Authors: Dongxu Zhou and Arturo Keller
Department: Bren School, UCSB
Contact Phone #s: (805)893-1822
Contact Email: keller@bren.ucsb.edu
Reviewed/Revised by: Ivy Ji

This protocol has been published in whole or in part in the following journal articles (please
provide complete citation):

Keller, A.A., Wang, H., Zhou, D., Lenihan, H.S., Cherr, G. Cardinale, B.J., Miller, R., Ji, Z. 2010.
Stability and Aggregation of Metal Oxide Nanoparticles in Natural Aqueous Matrices.
Environ. Sci. Technol. (2010) 44:19621967, DOI: 10.1021/es902987d
Zhou, D., Keller, A., Role of morphology in the aggregation kinetics of ZnO nanoparticles. Water
Research, 2010, 44(9), 2948 - 2956.
B.J.R. Thio, Zhou, D, Keller, AA. Influence of natural organic matter on the aggregation and
deposition of titanium dioxide nanoparticles. J. Hazard. Mater. (2011),
doi:10.1016/j.jhazmat.2011.02.072.

Summary

This protocol describes the protocol for (1). preparing metal oxide nanoparticle suspension in
deionized water; (2). conducting aggregation kinetics experiments of metal oxide nanoparticles;

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(3). conducting adsorption experiments between natural organic matter and metal oxide
nanoparticles.

Background and Project Goals

Particle size of nanoparticles influences not only their environmental transport but also their
toxicity. In natural aquatic systems, NPs tend to aggregate into larger particles tens or hundreds
of times bigger than their primary particles. Therefore, the knowledge of how NPs aggregate
and which factors play crucial roles in their aggregation is a necessity for studying NPs human
toxicity and ecotoxicty. The goal of this project is to understand the influential factors of
nanoparticle aggregation in aqueous media and to provide nanotoxicologists with knowledge of
what particle size should they expect under natural conditions as well as the potential particle
size change under their experimental conditions.

Materials & Reagents

Materials/Reagents/Equipment Vendor Stock Number

Disposables
Conical centrifuge tubes (50 mL) Corning Life Sciences
430828
20mL glass scintillation vials Wheaton
986730
Round glass cells for DLS Brookhave Instruments
BI-RC12
Disposable 4mL cuvette Fisher Scientific
14-955-125

Reagents
ZnO nanoparticle powders Meliorum Technologies 09821
CeO2 nanoparticle powders Meliorum Technologies 09817
TiO2 nanoparticle powders (P25) Evonik Degussa --
HCl EMD Chemicals Inc SA56-500
NaOH EMD Chemicals Inc MSX06003
Natural Organic Matter International Humic Substances 1R101N
Society
Borate Fisher Scientific A73-500
Phosphate Fisher Scientific SB9605-1

Equipment
Dynamic Light Scattering (BI-200SM) Brookhaven
Sonication bath (Branson 2510) Misonix
Spectrophotometer (BioSpec 1601) Shimadzu

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Environmental Health & Safety
Nanoparticles (dry powders) handling has to be done in chemical fume hood and with N95 filter mask.
Scientists performing this procedure must wear a lab coat and gloves. In situations where there might be
a chance of an accidental splash to the eyes, safety glasses must be worn.

Please refer to the Nanotoolkit produced by the California Nanosafety Consortium of Higher Education
for recommendations regarding safe handling and disposal of nanomaterials. Prior to suspension of the
nanoparticles, use engineering controls, work practices, and PPE as specified for Category 2 (Moderate
Exposure Potential); after suspension, use use engineering controls, work practices, and PPE as specified
for Category 1 (Low Exposure Potential) as specified in the Nanotoolkit.

As described in the Nanotoolkit, NIOSH has determined that workers may be at risk of developing
adverse respiratory health effects if exposed to certain nanomaterials for a working lifetime at the upper
limit of quantitation (LOQ) using NIOSH Method 5040, which is currently the recommended analytical
method for measuring airborne CNTs. The LOQ for CNTs using NIOSH Method 5040 is 7 g/m3. Animal
data-based risk estimates from NIOSH indicate that workers may have >10% excess risk of developing
early stage pulmonary fibrosis if exposed over a full working lifetime at the upper LOQ for NIOSH
Method 5040. Until improved sampling and analytical methods can be developed, and until data
become available to determine if an alternative exposure metric to mass may be more biologically
relevant, NIOSH is recommending a REL of 7 g/m3 elemental carbon (EC) as an 8-hr TWA respirable
mass airborne concentration.a Likewise, NIOSH recommends airborne exposure limits of 2.4 mg/m3 for
fine TiO2 and 0.3 mg/m3 for ultrafine (including engineered nanoscale) TiO2, as time-weighted average
(TWA) concentrations for up to 10 hr/day during a 40-hour work week. These recommendations
represent levels that over a working lifetime are estimated to reduce risks of lung cancer to below 1 in
1,000. The recommendations are based on using chronic inhalation studies in rats to predict lung tumor
risks in humans.b

Citations:
a
NIOSH. (2010). Occupational Exposure to Carbon Nanotubes and Nanofiber. Current Intelligence
Bulletin.
b
NIOSH. (2011). Occupational Exposure to Titanium Dioxide. Current Intelligence Bulletin.

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Workflow

(1). Prepare nanoparticle stock suspension (Zhou and Keller, 2010)

(2). Prepare electrolyte, buffer (borate and phosphate), and natural organic matter (NOM)
stock solutions
(3). Dilute nanoparticle stock by deionized water and add in borate buffer and electrolyte. If
studying the effect of NOM, add in NOM stock as well.
(4). Measure the aggregation kinetics by dynamic light scattering (DLS).
(5). Quantify the adsorption of NOM onto metal oxide nanoparticles by isothermal adsorption
experiments.
(6). Monitor the sedimentation process of metal oxide nanoparticles by spectrophotometer.

Procedure

1. Nanoparticle suspension sample preparation for aggregation studies (Zhou and Keller, 2010).

An aliquot of nanoparticle stock suspension is withdrawn and sonicated for 10 min to

redisperse particles (10 min sonication was found to be sufficient to break loose metal oxide
nanoparticle agglomerates). Borate or phosphate buffer (depending on the desired pH) is
added to achieve a final buffer concentration of 1 mM. A measured amount of Nanopure water
is then used to dilute the sample so that the final concentration of metal oxide NPs is 100 mg/L.
A desired amount of electrolytes is added to the sample immediately before a DLS
measurement is started. Electrolyte concentration varies from run to run so that the critical
coagulation concentration can be determined.

2. Procedure of DLS measurement

Aggregation kinetics is measured using time-resolved dynamic light scattering (DLS) (BI-200SM,
Brookhaven Instrument, Holtsville, NY) with a 633 nm laser source. The detection angle is set to
90. Temperature of the DLS sample chamber is controlled at 25C by a water bath. Before each
set of experiments, decaline bath is filtered for 5 min to remove dust. Immediately after adding
the desired amount of electrolytes (electrolyte concentration varies from run to run, and the
typical range is 1mM to 200mM) to a sample, DLS measurements are started. Data are

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collected at 30120 s intervals, and the intensity-weighted hydrodynamic radius is determined
by second- order cumulant analysis. Data collection is continued until a 50% increase in the
hydrodynamic radius is observed.

3. Adsorption experiment procedure

The adsorption of NOM onto metal oxide nanoparticle surfaces is quantified by batch
experiment using a bottle-point technique. Desired amounts of metal oxide stock suspension
and adsorbate stock solution are added into 20 mL glass vials. A NaCl solution was used to
adjust the IS as needed and a borate buffer of 1 mM final concentration was used to maintain
the desired pH. Then deionized water with a preadjusted pH is used to dilute the system to the
target concentrations. All samples were produced in duplicates including control. After this, the
samples are agitated on a roller at 60 rpm for 24 h. Preliminary results showed this was
sufficient to reach adsorption equilibrium. The samples are centrifuged at 15,180g for 5 min
and the supernatant is analyzed for adsorbate concentration by measuring the light absorbency
at 254 nm (BioSpec 1601, Shimadzu, MD). A NOM calibration curve was created so that the
spectrophotometer reading can be converted to NOM concentration.

4. Sedimentation experiment procedure

Before an experiment, the stock dispersion was sonicated for 10 min. The stock dispersion was
then added to the water samples to achieve the target concentrations.
The dynamic aggregation process was monitored using a UV-vis spectrophotometer (BioSpec
1601, Shimadzu, MD), measuring the sedimentation of the nanoparticles in different waters at
various MeO concentrations via time-resolved optical absorbence (CeO2 at 321 nm, TiO2 and
ZnO at 378 nm). Optical absorbence was measured every 6 min for 360 min. The experiments
were run in duplicate or triplicate.

Reagent/Stock Preparation

(1). Nanoparticle stock preparation (Zhou and Keller, 2010)

0.05 g metal oxide powder is added into 50 ml deionized water and sonicated by a sonication
bath for 30 min. (preliminary experiment shown that 30mins was the optimum sonication time

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to achieve minimal particle size) The stock is let settle over night. The supernatant is then
withdrawn and stored as stock suspension. Before use, aliquots of the stock solution are
resonicated for 10 min and diluted into the desired concentration. The stock suspension is
freshly prepared everyday. Nanoparticle dry powders are handled in fume hoods.

(2). NOM stock preparation

Suwannee River natural organic matter (NOM) was obtained from the International Humic
Substances Society. A 200 mg/L NOM stock solution was prepared by mixing a known amount
of NOM with deionized water, adjusting the solution pH to 8 to facilitate dissolution, and
stirring overnight.

(3). Acid, base, and buffer preparation

HCl (0.1 M and 0.01 M) is prepared by diluting concentrated HCl solution (37%) to the desired
concentration by deionized water. NaOH (0.1 M and 0.01 M), boric acid (200 mM), and
phosphate (200mM) are prepared by weighing appropriate amount of dry powder and then
dissolve them in deionized water. All the solutions are filtered via 0.22 mm PVDF filters to avoid
potential interference in the light scattering experiments.

SOP Approval
DEPARTMENT APPROVED BY DATE
Principal Investigator Arturo Keller

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