Desalination 248 (2009) 771–782
Acute toxicity of boron, titanium dioxide, and aluminum
nanoparticles to Daphnia magna and Vibrio fischeri
Nikolay Strigula*, Liana Vaccaria, Catherine Galduna, Mahmoud Waznea,
Xuyang Liua, Christos Christodoulatosa, Kristin Jasinkiewiczb
a
Stevens Institute of Technology, Center for Environmental Systems, Castle Point on Hudson, Hoboken, NJ 07030, USA
b
US Army, RDECOM-ARDEC, Environmental Technology Division, Picatinny, NJ 07806, USA
Tel: 201-9524260; Fax: 201-2168321; email: nstrigul@stevens.edu
Received 19 November 2008; accepted 15 January 2009
Abstract
The acute toxicity of four different nanosized particulate materials (titanium dioxide, boron nanoparticles, and
two types of aluminum nanoparticles (ALEX and L-ALEX)) were evaluated using two tests: the Microtox toxicity
test and the acute toxicity test with Daphnia magna. The results were analyzed in order to calculate LD50 at 24 and
48 h. It was found that titanium dioxide nanoparticles show a low level of toxicity, and LD50 values cannot be
calculated. Conversely, boron nanoparticles with EC50 ranging from 56 to 66 mg L 1, depending upon the age
of the solution, can be classified as ‘‘harmful’’ to aquatic microorganisms (EC50 in the range 10–100 mg L 1).
We have also discussed possible mechanisms of nanoparticle toxicity and potential problems in ecotoxicological
testing of nanomaterials. The studied nanomaterials can be ranked in the following order according to their
Daphnia acute toxicity: boron nanoparticles>ALEX>L-ALEX> TiO2.
Keywords: nanomaterials; acute toxicity; boron nanoparticles; aluminum nanoparticles; titanium dioxide nanoparticles; Daphnia magna test; Vibrio fischeri test
1. Introduction
Numerous nanomaterials have recently been
developed, and numerous practical applications
have been found in water treatment, medicine,
cosmetics, and engineering [1,2]. Ecotoxicolog*Corresponding author.
Presented at the Conference on Protection and
Restoration of the Environment IX, Kefalonia
Greece, June 30–July 3, 2008
ical properties of nanomaterials are largely
unknown [2–4]; however, this problem has
recently received substantial attention [5].
Several reports published over the last couple
of years, including the first review papers
[6–10], address ecotoxicological properties of
different nanomaterials.
This paper presents results concerning ecotoxicity of boron, aluminum, and titanium dioxide
nanoparticles. These materials have already
0011-9164/09/$– See front matter © 2009 Published by Elsevier B.V.
doi:10.1016/j.desal.2009.01.013
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N. Strigul et al. / Desalination 248 (2009) 771–782
given rise to numerous applications and are being
manufactured at the industrial level. For instance,
boron nanoparticles are being considered as a
potential fuel source that releases energy after
metal oxidation, and have been employed in
medical research [11]. Aluminum nanoparticles
are already widely employed in explosive combinations [12]. Titanium dioxide nanoparticles
belong to the currently most used group of nanomaterials known as photocatalysts and adsorbents
[13]. TiO2 particles are being used in sunscreen
products, and as a catalyst in sterilization and
chemical engineering. Also, it has an application
as an adsorbent in water cleaning [14,15].
Some data concerning toxicity of the nanoparticles similar to those tested in this study
recently became available. Several reports available on the ecotoxicity of TiO2 nanoparticles
show that this material has relatively low toxicity [10,16–19]. In particular, Heinlaan et al. [20]
compared toxicity of ZnO, CuO, and TiO2 nanoparticles to Vibrio fischeri, Daphnia magna, and
Thamnocephalus platyurus and reported that
TiO2 particles did not show any toxicity in
concentrations up to 20 mg L 1. Similarly Griffitt et al. [19] did not find any toxic effects from
TiO2 nanoparticles in standard ecotoxicological
tests with zebrafish, Daphnia, and algae. Zhu
et al. [21] showed that TiO2 and Al2O3 nanoparticles did not affect zebrafish at early developmental stages. Wagner et al. [22] reported that
aluminum nanoparticles have greater toxicity
to macrophages than aluminum oxide nanoparticles. In particular, aluminum particles administered at 25 mg mL 1 level for 24 h inhibited cell
phagocytotic ability. Doshi et al. [23] have
shown that ALEX and L-ALEX do not have
significant toxicity in the soil environment in a
realistic range of concentrations. At this point,
we are not aware of any published ecotoxicological results concerning the boron nanoparticles.
However, Petersen et al. [11] reported that
boron nanoparticles inhibited tumor growth in
an animal model.
The first objective of this work is to evaluate
acute toxicity of four different nanoparticles
(boron, titanium dioxide, and two different
aluminum [ALEX and L-ALEX] nanoparticles)
using two standard ecotoxicological tests, the D.
magna acute toxicity test and the Microtox toxicity test. The tests with D. magna have been performed according to the standard protocol
Daphnia sp., acute immobilization test (OECD
test No. 201). The Microtox test employs the bioluminescent bacterium (V. fischeri) as the test
organism. The preliminary toxicity estimates
obtained with these tests often correlate with
other tests on fish, cell cultures, and rodents and
these tests are considered to be convenient tools
for evaluation of nanomaterials toxicity [6,10,24].
The second objective of this study is to evaluate possible methodological issues caused by
the ‘‘nano’’ nature of the tested materials. Nanomaterials often have unique chemical and physical properties that generate a substantial interest
in nanotechnology; however, from another side,
nanomaterials potentially may have biological
and environmental effects that have not been
observed in other materials [5,7,8,10,16]. Also,
nanomaterials may cause new types of biases in
applications of the standardized ecotoxicological
tests. These uncertainties present a challenge for
ecotoxicological research, and some modifications of the standard ecotoxicological tests may
be needed in respect to nanomaterials. Some particular issues that this study revealed concern the
stability of nanoparticles in water suspensions
and light-adsorbing properties of nanoparticles.
2. Materials and methods
2.1. Nanoparticles: characterization and
preparation
The nanoparticle suspensions were prepared
by a procedure consisting of the preparation of
suspension of the stock powder in D.I. water
(for the Microtox tests) or the standard
N. Strigul et al. / Desalination 248 (2009) 771–782
freshwater (for the Daphnia tests) followed by
ultrasonication for 30 min to break up aggregates, which were commonly formed during
storage. In the long-term experiment, the nanoparticle suspensions were kept at room temperature on a convenient laboratory rotator, which
mixed the samples at a constant rate of about
100 rpm. The analysis of the particle size distribution showed that this procedure is sufficient to
obtain homogeneous nanoparticle suspension
(see [25] for the details).
The 100-nm nanoaluminum particles, ALEX
and L-ALEX (as a powder of 1–10 mm agglomerates), were obtained from Argonide Corp. ALEX
and L-ALEX were produced by the electroexploded wire technique. The obtained particles
are immediately passivated with air to produce
a coating of aluminum oxide (ALEX) or with a
carboxylic acid to produce surface carboxylate
ligand chains covering the metal surface.
Nanocrystalline TiO2 was provided by Dr.
Meng and prepared by hydrolysis of titanium sulfate solution according to Meng et al. [14]. The
particles are about 6 nm in diameter and were
delivered in agglomerates of 0.5–2.0 mm [15,26].
Boron nanoparticles of average nominal size
10–20 nm were obtained from Alfa Aesar (Ward
Hill, MA) and kept in a pure argon atmosphere.
Based on scanning electron microscopy–energy
dispersive spectrometer (SEM-EDS) analysis,
boron content was measured at 70%, which is
less than that reported by the manufacturer
(95%). The same value was obtained in acid
digestion analysis. This is probably due to the
oxidation of boron during production or transportation, based on the measured value of oxygen at 28% using SEM-EDS analysis [25].
2.2. Acute Daphnia sp. immobilization test
(OECD 202)
The Daphnia sp., acute immobilization test
(OECD test No. 202) was used to give an estimate of the toxic effects of the studied materials
773
on freshwater invertebrates. This test was performed using Daphtoxkit bioassays produced
by MicroBioTests Inc. according to the OECD
202 protocol. The Daphtoxkit bioassay included
the standard freshwater similar to the one prescribed by the OECD test, Daphnia eggs, and
plates consisting of 24 experimental wells of
10 mL.
Six distinct concentrations including control
(250, 80, 25, 8, 2.5, and 0 mg L 1, respectively)
of the focal nanomaterial were used in each test.
Experimental design included 6 groups with 20
animals (4 5) exposed to each of the tested
concentrations. Animals from each group were
separated into 4 smaller groups of 5 animals to
provide 4 independent replications located in
different experimental wells.
The animals tested were young daphnids,
each less than 24 h old at the beginning of the
test, in different concentrations of the tested
nanomaterials. Young D. magna were hatched
from eggs, which were incubated for 72 h in
well-aerated standard freshwater. Two hours
prior to exposing the Daphnia to the various
nanoparticles, they were fed using powdered
Spirulina and not fed again for the duration of
the test.
The experimental wells were filled with wellaerated suspension of the focal nanomaterial.
Then the groups of 5 daphnids each were
added to these wells of effluent and remained
there for a period of 48 h in darkness. After
each 24 and 48 h had elapsed, the experimental
cells were examined to determine the number
of dead and immobilized neonates. These numbers were recorded, and immobilization of the
daphnids in the test substances was compared
with control values from the same tray.
2.3. Microtox acute toxicity test
(ASTM Standard D-5660)
The Microtox test is a metabolic inhibition
test with luminescent marine bacteria (V. fischeri
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N. Strigul et al. / Desalination 248 (2009) 771–782
NRRL B-11177). This test was performed
according to the ASTM Standard (D-5660).
Lyophilized bacteria were rehydrated with the
included reconstitution solution (ultrapure
water) just prior to performing the test. Luminescent bacteria were incubated under constant temperature in glass vials with 1 mL of 2% NaCl
solution and the toxicant presented in various
concentrations. Nine concentrations of each
nanomaterial obtained by a sequence of 1:1 dilutions ranging from 0.4395 to 112.5 mg L 1 were
tested. At 5- and 10-min time intervals, the luminescence of each individual vial was measured.
The sample toxicity was evaluated based on the
decrease of the intensity of light produced by
the luminescent bacteria; the statistical analysis
including the light absorbance correction was
done according to the test description.
using dynamic light scattering (DLS). A monochromatic coherent He–Ne laser with a fixed
wavelength of 633 nm was used as a light source.
The intensity of scattered light was measured by a
detector at 1738 and an autocorrelation function
was accumulated for 10 s. For each experiment,
1 mL of the suspension was introduced into a polystyrene disposable cuvette (Sarstedt, Germany).
The hydrodynamic radius (intensity based) was
calculated using the Stokes–Einstein equation.
2.6. Statistical analysis
LD50 values and their confidence intervals
were calculated by the probit analysis using the
EPA Probit Analysis Program, version 1.5
(U.S. Environmental Protection Agency). Other
statistical calculations have been performed
using Statistica 7.0 software (StatSoft Inc.).
2.4. Water characteristics
Water pH was measured using Accumet
AR25 pH meter with Accumet 13-620-285 pH
electrode from Fisher Scientific Inc. Light
absorbance of nanomaterials in water was
measured with the Hewlett-Packard 8452A
spectrophotometer.
2.5. Dynamic light scattering
The particle size distribution was obtained by
Nano Zetasizer (Malvern, Worcestershire, UK)
3. Results and discussion
3.1. Acute Daphnia sp. immobilization test
(OECD 202)
Titanium dioxide nanoparticles showed a low
level of toxicity, and LD50 values at 24 and 48 h
could not be calculated (Table 1). However, animals exposed to 80 and 250 mg L 1 of TiO2
nanoparticles were significantly slower after 24
h than the animals exposed to the lower concentrations, and it was evident that TiO2 particles
Table 1
The results of the Daphnia magna test with TiO2 nanoparticles
Concentration
Control
Exposure time
24 h
48 h
24 h
A
B
C
D
Mortality %
5/0
5/0
5/0
5/0
0
5/0
5/0
3/2
4/1
15
5/0
5/0
5/0
5/0
0
8 mg L
1
25 mg L
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
4/1
3/2
3/2
3/2
35
4/1
4/1
4/1
5/0
15
1/4
3/2
2/3
2/3
50
5/0
5/0
4/1
4/1
10
5/0
5/0
4/1
3/2
15
5/0
5/0
5/0
5/0
0
4/1
3/2
5/0
4/1
20
5/0
3/2
4/1
4/1
20
3/2
2/3
4/1
4/1
35
2.5 mg L
1
1
80 mg L
1
250 mg L
1
A,B,C,D are four different experimental groups; k/m indicates number of alive (k) and dead (m) animals in each group.
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N. Strigul et al. / Desalination 248 (2009) 771–782
Table 2
The results of the Daphnia magna test with boron nanoparticles
Concentration
Control
Exposure time
24 h
48 h
24 h
A
B
C
D
Mortality %
5/0
5/0
5/0
5/0
0
3/2
4/1
3/2
5/0
25
5/0
5/0
5/0
5/0
0
8 mg L
1
25 mg L
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
5/0
4/1
5/0
4/1
10
4/1
4/1
5/0
4/1
15
0/5
2/3
3/2
1/4
70
2/3
2/3
3/2
2/3
55
0/5
0/5
0/5
1/4
95
0/5
0/5
0/5
0/5
100
0/5
0/5
0/5
0/5
100
0/5
0/5
0/5
0/5
100
0/5
0/5
0/5
0/5
100
2.5 mg L
1
1
80 mg L
1
250 mg L
1
A,B,C,D are four different experimental groups; k/m indicates number of alive (k) and dead (m) animals in each group.
accumulated in significant amounts in the
digestive tract.
Boron nanoparticles (Table 2), in concentrations higher than 80 mg L 1, killed all daphnids
in 24 h. The LD50 value for 24 h is 19.5 mg
L 1 with 14.2–27.0 mg L 1 95% confidence
interval, and for 48 h it is 6.7 mg L 1 with
3.661–9.696 mg L 1 95% confidence interval.
The probit analysis algorithm converged for the
24 h data. However to provide the algorithm
convergence for 48 h data, we had to ignore the
mortality in the control group (as the probit transformation can be done only if the mortality
changes monotonically as the concentration of
the pollutant increases). After 24 h, animals
exposed to 2.5 mg L 1 of boron nanoparticles
were actively swimming similarly to the daphnids
in the control group, while animals in 8 mg L 1
were less active and had some amounts of
boron particles in the digestive system. Daphnids
in 25 mg L 1 were very slow and had relatively
large amounts of particles inside.
For the nanoaluminum particles that differ in
surface coatings (ALEX particles coated with a
thin layer of aluminum oxide are not hydrophobic while L-ALEX particles coated with carboxylate ligand are hydrophobic), the following
preliminary LD50 values were found: ALEX
(see Table 3) – the LD50 for 24 h is 219.6 mg
L 1 with 118.0–774.4 mg L 1 95% confidence
interval, and for 48 h it is 7.483 (the confidence
interval cannot be calculated as the mortality
does not change monotonically); L-ALEX –
the LD50 value for 48 h is 107.588 mg L 1
with a very large 95% confidence interval
15.549–2468.308 mg L 1 (Table 4) and the
Table 3
The results of the Daphnia magna test with ALEX nanoparticles
Concentration
Control
Exposure time
24 h
48 h
24 h
A
B
C
D
Mortality %
5/0
5/0
5/0
5/0
0
5/0
5/0
4/1
5/0
5
5/0
5/0
5/0
5/0
0
8 mg L
1
25 mg L
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
4/1
5/0
3/2
3/2
25
5/0
4/1
5/0
5/0
5
1/4
2/3
1/4
1/4
75
5/0
5/0
4/1
5/0
5
2/3
2/3
1/4
1/4
70
5/0
3/2
4/1
1/4
35
1/4
2/3
3/2
1/4
65
3/2
2/3
2/3
3/2
50
0/5
0/5
0/5
1/4
95
2.5 mg L
1
1
80 mg L
1
250 mg L
1
A,B,C,D are four different experimental groups; k/m indicates number of alive (k) and dead (m) animals in each group.
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N. Strigul et al. / Desalination 248 (2009) 771–782
Table 4
The results of the Daphnia magna test with L-ALEX nanoparticles
Concentration
Control
Exposure time
24 h
48 h
24 h
A
B
C
D
Mortality %
5/0
5/0
5/0
5/0
0
5/0
5/0
3/2
4/1
15
4/1
4/1
4/1
3/2
25
8 mg L
1
25 mg L
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
4/1
4/1
4/1
3/2
25
5/0
4/1
4/1
5/0
10
5/0
4/1
3/2
3/2
25
3/2
4/1
4/1
3/2
20
2/3
2/3
3/2
2/3
55
4/1
5/0
2/3
3/2
30
4/1
3/2
1/4
2/3
50
3/2
3/2
3/2
4/1
45
1/4
1/4
2/3
3/2
65
2.5 mg L
1
1
80 mg L
1
250 mg L
1
A,B,C,D are four different experimental groups; k/m indicates number of alive (k) and dead (m) animals in each group.
effects were not sufficient to compute LD50
value for the 24-h exposure (mortality did not
exceed 50%). While L-ALEX particles demonstrate moderate toxicity in suspension, these particles are hydrophobic and the real concentration
in suspension is much lower than declared since
numerous particles stayed on the water surface.
Also the actual concentration of particles in
water may not be proportional to input concentration of nanoparticles. The homogeneous suspensions of L-ALEX could not be prepared
even after long-term ultrasound treatment and
mixing. The lack of homogeneous character of
the suspensions explains the observed pattern
in which mortality levels stabilized at values
around 30% and 55% after 24 and 48 h, respectively and a dose–response relationship was not
observed at high L-ALEX nominal particle concentrations (Table 4).
Boron nanoparticles were most toxic of all
the tested nanomaterials (Figs 1 and 2). Also,
in contrast with the other nanomaterials, boron
nanoparticles were probably more chemically
active as boron nanoparticles slightly increased
water pH up to 8 with saturation effect (Fig. 3)
while TiO2, ALEX, and L-ALEX nanoparticles
in all concentrations did not significantly change
the pH of the standard freshwater (pH was about
92
92
72
Survival at 48h, %
Survival at 24h, %
72
52
32
Boron
L-Alex
TiO2
Alex
12
52
32
Boron
L-Alex
TiO2
Alex
12
−8
−8
1
10
Log of concentration, mg L–1
100
Fig. 1. Survival of young Daphnia after exposure to
nanomaterials in different concentrations at 24 h.
1
10
100
Log of concentration, mg L–1
Fig. 2. Survival of young Daphnia after exposure to
nanomaterials in different concentrations at 48 h.
N. Strigul et al. / Desalination 248 (2009) 771–782
7.5–7.7) during the experiment. However, this
change of pH was not so large to explain mortality of animals exposed to the boron nanoparticles. Hund-Rinke and Simon [16] modified
the Daphnia test to address the role of photocatalytic reactions in nanoparticle toxicity. In contrast, the reported experiments were conducted
in darkness that excluded possible contribution
of nanoparticle photoreactions to their toxicity.
No water mixing was performed during the
48-h acute tests, and TiO2, ALEX, and boron
nanoparticles precipitated in significant amount
from suspension during the first 24 h, while
hydrophobic L-ALEX floated to the surface.
Apparently, the studied nanomaterials not only
precipitated or floated to the surface, but also
agglomerated to some extent [25]. Therefore
the physical conditions in water during the first
24 h of the experiment were different compared
to the conditions in the second 24 h as a substantial amount of nanoparticles initially being in
suspension precipitated or floated.
Nanoparticles were observed in the digestive
track of young Daphnia. Animals may actively
eat nanoparticles or their agglomerates, mistaking them for food. It is possible that the particles
agglomerate further in the digestive system,
eventually blocking it and causing death. However, our results show that this possible indirect
toxicity mechanism does not contribute significantly to the 48 h acute toxicity. For instance,
after 48 h, some actively moving animals
exposed to high concentrations of TiO2 had a
significant amount of TiO2 in their bodies. However, these observations were made using shortterm acute toxicity tests, and they probably do
not indicate adaptation of Daphnia to nanoparticles. We anticipate that the possible toxicity
mechanism described above might have some
importance in case of a long-term exposure of
Daphnia to nanoparticles. We can conclude
that the acute toxicity of nanoparticles on Daphnia is determined by some specific chemical
properties of each material, rather than
777
a physical–chemical mechanism common to different nanomaterials.
3.2. Microtox acute toxicity test
(ASTM Standard D-5660)
The results of the acute toxicity test are consistent with our findings using the Daphnia test.
Only the boron nanoparticles exhibited significant toxicity (V. fischeri NRRL B-11177) in
the range of concentrations 0–120 mg L 1
among the tested nanomaterials. ALEX, LALEX, and TiO2 nanoparticles did not inhibit
luminescent activity of V. fischeri (less than
5% light reduction after 15 min), but these materials adsorbed light proportionally to the concentration (in the range 20–30% at the highest
concentration, 120 mg L 1). Our results concerning the toxicity of TiO2 nanoparticles to
Daphnia and V. fischeri confirm recently published data [19,20] that TiO2 does not have
acute toxicity to these organisms in a realistic
range of concentrations.
With respect to the Microtox test, several
issues were identified for the boron nanoparticles,
as well as other nanomaterials, which need to be
addressed to eliminate the possible biases: (1)
Nanoparticles may adsorb and scatter light. A
light absorbance correction is then a necessary
experimental step. (2) Nanoparticles of different
sizes can have different effects on microorganisms
and different mobility in aquatic solution. The
dynamics of size particle distribution is an important parameter, which was monitored with a zetaanalyzer. (3) Toxicity observed after nanoparticles
amendment can be a result of the interference of
the toxicity of nanoparticles of certain size fractions and the toxicity of soluble compounds,
which are products of the nanoparticles’ dissolution. Therefore, it is important to be able to evaluate the dissolution kinetics and separate toxicity
of the nanoparticles and their dissolution products.
While the first two problems, i.e., light absorbance
correction and dynamics of nanoparticle
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N. Strigul et al. / Desalination 248 (2009) 771–782
distribution in solution, can be evaluated using
standard methods, the third problem (i.e., interference of possible toxic effects of the nanoparticles
themselves and the products of their dissolution)
is nontrivial. To address this problem, filtration
and filtration applied together with centrifugation
were explored; however, both these methods were
not able to completely remove particles from
aqueous samples. After filtration and centrifugation, an aqueous sample with a homogenous distribution of small-sized nanoparticle was
obtained. Therefore, to this point, we have not
been able to measure the dissolution kinetics and
decouple the toxicity effects of boron particles
and soluble boron compounds.
3.2.1. Boron nanoparticles in solution
Boron nanoparticle suspensions were characterized immediately after preparation and after
mixing for set intervals of time to study their
aggregation behavior. When a particle suspension with a concentration of 250 mg L 1 was
freshly prepared, the hydrodynamic diameter
was measured at 320 nm and the measured
zeta- potential was 32.5 mV. After mixing in
an end-over-end rotator for 1 day, the hydrodynamic diameter increased to 435 nm most likely
due to aggregation (Fig. 4). The dispersion was
then filtered through 0.2-mm size membrane,
and the measured hydrodynamic diameter
decreased to 101 nm. The hydrodynamic diameter further decreased to 60 nm after the filtered
sample was centrifuged at 12,500 rpm for 30
min. A duplicate sample of the original suspension was analyzed for hydrodynamic diameter
measurement after the sample was mixed for 1
month. The hydrodynamic diameter was measured at 447 nm, slightly greater than that measured after a mixing period of one day, and the
zeta-potential was measured at 33.4 mV. Similarly, the hydrodynamic diameter of the suspension decreased to 78 nm after filtration and it
further decreased to 64 nm after centrifugation.
The experimental data indicated that the
hydrodynamic diameter of the boron suspension
increased significantly after mixing for 1 day;
however, only, a slight change was observed
between a mixing period of one day and one
month (Fig. 4). Furthermore, the hydrodynamic
diameter of the suspension decreased significantly upon filtration and it further decreased
upon centrifugation.
Boron nanoparticles in water absorb light for
parameters of the Microtox detector (490 nm,
10 mm path width). The detector design indicates
8.1
8
pH
7.9
7.8
7.7
7.6
7.5
−10
40
90
140
190
240
Concentration of boron nanoparticles, mg L–1
Fig. 3. Change of pH in the suspension of boron nanoparticles in the standard freshwater after 48 h.
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N. Strigul et al. / Desalination 248 (2009) 771–782
600
Hydrodynamic size (nm)
500
400
300
200
100
0
0 day
1 day
1 month
suspension suspension suspension
1 month
filtered
1 day
fltered
1 month
1 day
filtered &
filtered &
centrifuged centrifuged
Fig. 4. Hydrodynamic size (nm) of the boron nanoparticles in suspension depends on the time and filtration method.
for the fast estimation of the light adsorbance
when the tested substances are not very toxic.
3.2.1. Toxicity of boron nanoparticles to
Vibrio fischeri
The aqueous mixture of the particles shows
similar toxicity properties (Fig. 5) immediately
after sample preparation (EC50–55.85 mg L 1)
on the next day (EC50–56.62 mg L 1) and
after 60 days (EC50–65.98 mg L 1).
% effect after 15 minutes of exposure
that it is light absorbance rather than scattering,
which may also contribute to the light losses.
The standard method (according to the test manual) is to estimate light absorbance of the samples
by using the spectrophotometer. It included the
preparation of the similar series of dilutions of
the same nanomaterials and their measurements.
For the boron nanoparticles, this method demonstrated linear dependence of the light adsorbance
on the particle concentration (y = 0.0036x +
0.0036, r2 = 0.9998) in the concentration range
0–125 mg of particles per liter. We have found
that this method may be not very convenient in
some cases, where expensive nanomaterials are
tested. We have tested an alternative method to
check the light adsorbance of the samples. In
the Microtox test we have added one more measurement at 1 min, which was practically immediate after the mixing of the samples in the test
vials. Considering the reduction of light at
1 min as results of light absorbance of nanoparticles we have obtained similar results for boron
nanoparticles (y = 0.0037x + 0.0041, r2 =
0.9909) as well as with other tested nanomaterials. We suggest that this method may be suitable
120
100
80
60
40
20
0
0 day,
1 day,
60 days.
−20
0
20
40
60
80
100
120
Nanoparticles concentration mg L–1
Fig. 5. Dose–response curves for boron nanoparticles in
the 60-day experiment.
% effect after 15 minutes of exposure
780
N. Strigul et al. / Desalination 248 (2009) 771–782
100
80
60
40
20
0
nonfiltered,
filtered,
filtered and centrifuged,
−20
0
20
40
60
80
100
120
Nanoparticles concentration in the nonfiltered sample, mg L–1
Fig. 6. Dose–response curves of boron nanoparticles in
water suspension for nonfiltered, filtered, and filtered
and centrifuged samples after 24 h.
The original mixture of particles inhibits fluorescent bacteria more significantly than filtered
samples, or filtered and centrifuged samples
(Figs 6 and 7). The initial toxicity of the
nanoparticle suspension (1 day after suspension
preparation) is associated mostly with the nanoparticles’ toxicity, and the contribution of the
dissolution product is not significant (Fig. 6).
However, after 60 days, the observed toxicity
could be associated mostly with the toxicity of
the dissolution products (Fig. 7).
The boron nanoparticles with EC50 ranging
from 56 to 66 mg L 1, depending upon aging
% effect after 15 minutes of exposure
90
80
70
60
50
40
30
20
10
0
−10
nonfiltered,
filtered and centrifuged,
−20
0
20
40
60
80
100
120
Nanoparticles concentration in the nonfiltered sample, mg L–1
Fig. 7. Dose–response curves of boron nanoparticles
suspension and filtered and centrifuged samples after 60
days.
time/age of solution, can be classified as ‘‘harmful’’ to aquatic microorganisms (EC50 in the
range 10–100 mg L 1) according to the Commission Directive 93/67/EEC from the European
Union for the assessment of risk to man and the
environment of substances. The toxicity of a
water suspension of boron nanoparticles could
be explained by: (1) toxicity of the nanoparticles
themselves, and (2) toxicity of the products of
the particles’ dissolution. The first factor dominates at the earliest time of boron nanoparticles
release in water, and the second factor might
dominate in aged suspensions. Further research
is necessary to investigate (1) the ecotoxicity
of boron particles using standard OECD tests
(i.e. fish, invertebrates, plants, and soil tests),
(2) mechanisms of boron particles toxicity, (3)
the solubility of boron nanoparticles, and (4)
the toxicity of the dissolution products of
boron nanoparticles.
4. Conclusions
The results suggest that toxic responses
related more to chemical characteristics and
the dissolved phase concentration of the different nanoparticles. Several issues specific to
nanoparticles, which need to be addressed to
eliminate the possible biases in toxicity tests,
were identified: (1) Nanoparticles adsorb light.
Light absorbance correction is then a necessary
experimental step for certain toxicity tests. (2)
Toxicity of nanoparticles may be a result of
the effects from nanoparticles itself, dissolution
products, and nanoparticle agglomerates that
develop during the experiment. The studied
nanomaterials can be ranked in the following
order according to their Daphnia acute toxicity:
boron nanoparticles >ALEX > L-ALEX> TiO2.
Acknowledgments
This research was supported by TACOM/
ARDEC, Picatinny Arsenal. The authors are
N. Strigul et al. / Desalination 248 (2009) 771–782
grateful to Washington Braida and David Vaccari for their valuable comments.
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