Environmental Pollution 169 (2012) 81e89

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Sub-toxic effects of CuO nanoparticles on bacteria: Kinetics, role of Cu ions and

possible mechanisms of action
Olesja Bondarenko a, b, *, Angela Ivask a, Aleksandr Käkinen a, c, Anne Kahru a, *
a
Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia
b
Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia
c
Department of Chemical and Materials Technology, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia

a r t i c l e i n f o a b s t r a c t

Article history: The sub-toxic effects of CuO nanoparticles (nano-CuO) were evaluated using three recombinant lumi-
Received 21 March 2012 nescent Escherichia coli bacteria responding specifically to (i) reactive oxygen species (ROS), (ii) single-
Received in revised form stranded DNA breaks and (iii) bioavailable Cu ions. Using these sensors we showed that nano-CuO
25 April 2012
induces the formation of superoxide anions, hydrogen peroxide and single-stranded DNA already at
Accepted 8 May 2012
very low sub-toxic levels (0.1 mg Cu/L). The maximal sub-toxic response of all biosensors to nominal
concentrations of nano-CuO, micro-CuO (size control) and CuSO4 (solubility control) occurred at w6,
Keywords:
w600 and w0.6 mg Cu/L, respectively. According to the chemical analysis all the latter concentrations
Escherichia coli biosensor
Copper oxide nanoparticles
yielded w0.6 mg of soluble Cu/L, indicating that dissolution of CuO particles was the key factor triggering
Toxicity mechanisms the ROS and DNA damage responses in bacteria. Cu-ions chelation studies also showed that CuO particles
Reactive oxygen species were not involved in these stress responses. The solubilization results were confirmed by Pseudomonas
DNA damage fluorescens Cu-ion sensor.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction copper containing NPs are also toxic to other organisms that may be
exposed to these NPs via co-exposure or various waste streams. For
The biocidal properties of copper are widely acknowledged and example, nano-CuO has been shown to be toxic to crustaceans
various copper compounds have been used as antifoulants for Daphnia magna and Thamnocephalus platyurus and algae Pseudo-
centuries (Thomas and Brooks, 2010). With the development of kirchneriella subcapitata already at remarkably low concentrations
nanotechnology, copper has been increasingly used in the form of (EC50 3.2, 0.18 and <1 mg/L, respectively) (Heinlaan et al., 2008;
nanoparticles (NPs, particles with at least one dimension between 1 Aruoja et al., 2009) and thus, should be classified as ‘very toxic’ to
and 100 nm) and applied in e.g., antimicrobial textiles, hospital aquatic organisms (European Chemicals Bureau, 2006). High eco-
equipment, wood preservation and antifouling paints (Gabbay toxicity of CuO NPs has been considered as a serious limitation for
et al., 2006). Compared to bulk analogues, NPs display larger their implementation in new applications (Ebrahimnia-Bajestan
specific surface area that leads to increased reactivity and thus, also et al., 2011) and prior mechanistic toxicological characterization
enhanced bactericidal properties (Nel et al., 2006). For example, the of this material is needed (Fahmy and Cormier, 2009; Kahru and
effectiveness of CuO NPs (generally at concentrations between 10 Dubourguier, 2010). Therefore, in the current study, we focused
and 100 mg/L) towards a wide range of Gram-positive and Gram- on toxicological profiling of CuO NPs. Based on the current litera-
negative bacteria including Bacillus subtilis, Pseudomonas aerugi- ture, at least part of toxic effects of nano-CuO is attributed to the
nosa, Escherichia coli and Staphylococcus aureus has been reported release of Cu ions (Ruparelia et al., 2008; Wu et al., 2010). Cu ions
(Ren et al., 2009; Baek and An, 2011). However, purposeful use of may be involved in recycling redox reactions between Cu2þ and
CuO NPs due to their antimicrobial properties calls extra attention Cuþ, which generate reactive oxygen species (ROS) on the surface of
from the environmental protection viewpoint. Unlike antibiotics bacterial cells (Hoshino et al., 1999). However, according to some
that have specific targets in bacterial cells for their toxic action, studies, the fraction of Cu dissolved from nano-CuO is too low to
explain all the cytotoxic effects of nano-CuO (Griffitt et al., 2007;
Heinlaan et al., 2008; Karlsson et al., 2008). These studies suggest
* Corresponding authors.
E-mail addresses: olesja.bondarenko@kbfi.ee (O. Bondarenko), anne.kahru@ that particles themselves may generate additional ROS and the
kbfi.ee (A. Kahru). investigation of the relationship between the cellular responses to

0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2012.05.009
82 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89

sub-toxic concentrations of nano-CuO and the oxidative stress paraquat at 40 g/L, MMC at 20 mg/L, H2O2 at 300 g/L, 3,5-DCP at 100 mg/L,
endpoints has been proposed (Fahmy and Cormier, 2009). CuSO4*7H2O at 63.5 g Cu/L and EDTA at 58.4 g/L. Deionized sterile water was used
for dilutions of all chemicals throughout the study. CuO particles are characterized
We have recently demonstrated that nano-CuO was more toxic below.
to superoxide dismutase-deficient Escherichia coli than to the wild-
type bacteria and this effect was apparently mediated by super- 2.2. Physico-chemical characterization of CuO particles
oxide anions (Ivask et al., 2010). In the current study we expanded
CuO nanoparticles (nano-CuO; primary particle size 30 nm (Blinova et al., 2010))
our investigation by constructing two new stress-specific lumi-
were from SigmaeAldrich and micro-CuO from Alfa Aesar. The stock solutions of
nescent bacterial biosensors that respond to hydrogen peroxide copper oxides (63.5 g Cu/L) were prepared in deionized water, sonicated in the
(a ROS) and single-stranded DNA breaks. We used these biosensors ultrasonication bath for 30 min and stored in the dark at þ4  C. Before every use, the
in parallel with two Cu-ion specific biosensors to reveal the nature suspensions were vortexed. Deionized sterile water was used for CuO dilutions
of sub-toxic effects of nano-CuO in bacterial cells. The used sensor throughout the study. Scanning electron microscopy (SEM, JSM-8404) and deter-
mination of specific surface area (SSA, Sorptometer Kelvin 1042) of the powders of
bacteria are based on promoters of Escherichia coli genes which are
nano- and micro-CuO was performed in Tallinn University of Technology (Estonia).
involved in sensing and detoxification of ROS, DNA damage or Cu The hydrodynamic size (Dh) of nano-CuO (6.35 mg Cu/L) and micro-CuO (635 mg
and are activated upon respective stress. In biosensor strains, these Cu/L) was measured in deionized water and biosensor test medium (heavy metal
promoters are genetically coupled to bioluminescence-encoding MOPS medium; see below) using dynamic light scattering (DLS) (Malvern Zetasizer
Nano-ZS, Malvern Instruments). Visible wavelength absorption spectra (UVeVis) of
genes (luxCDABE genes from Photorhabdus luminescens) and thus,
63.5 mg Cu/L nano-CuO and 6350 mg Cu/L micro-CuO particles in deionized water
the sub-toxic stress condition may be measured by increased were obtained using a Thermo Multiscan Spectrum (Thermo Electron).
bacterial bioluminescence. Such an approach allows the detection
of specific perturbations already at sub-toxic concentrations 2.3. Luminescent bacterial biosensors
(Hwang et al., 2008; Gou et al., 2010) and is orders of magnitudes
Luminescent recombinant E. coli strains used in this study are listed in Table 1. The
more sensitive compared to the common viability endpoints (L(E)
ROS-inducible strain E. coli K12::katGlux and DNA damage-inducible strain E. coli
C50 values). All biosensors were exposed to nano-CuO but also MC1061(pDEWrecAlux) were constructed as described in Supplementary data. The
micro-CuO (size control) and CuSO4 (solubility control) from constitutively luminescent E. coli MC1061(pDEW201) was constructed by electro-
sub-toxic (sub-ppb, i.e., below mg/L) till toxic concentrations porating the plasmid pDEW201 into competent E. coli cells. E. coli K12::lux,
(1000 ppm, i.e., mg/L). Comparison of the responses of the stress- MC1061(pSLcueR/pDNPcopAlux), MC1061(pSLlux), Pseudomonas fluorescens OS8::lux
and P. fluorescens OS8::KncueRPcopAlux were constructed and described previously
specific bacteria with that of Cu ions-sensing bacteria and chem-
(Leedjärv et al., 2006; Ivask et al., 2009, 2010).
ical analysis/chelation of dissolved Cu allowed us to conclude that
(i) nano-CuO induces the bacterial ROS and DNA damage defence 2.4. Bacterial biosensor assay
systems already at very low sub-toxic levels and (ii) these early
adverse effects of nano-CuO in Escherichia coli cells are triggered by Recombinant luminescent bacteria were pre-grown in 3 mL of Luria Bertani (LB)
medium (Sambrook et al., 1989) supplemented with appropriate antibiotics
dissolved Cu ions. (Table 1) overnight. Twenty millilitres of fresh medium was inoculated with 1/50
diluted overnight culture and bacteria were grown until mid-exponential phase
2. Materials and methods (OD600 of 0.6). All cultivations were performed on a shaker at 200 rpm, at 30  C. The
exponential phase culture was centrifuged at 5000 g for 10 min and washed twice
2.1. Chemicals with heavy metal MOPS medium (HMM) (8.4 g of MOPS, 0.22 g of glycerol-2-
phosphate, 3.7 g of KCl, 0.54 g of NH4Cl, 0.06 g of MgSO4, and 0.162 mg of FeCl3
Paraquat, mitomycin C (MMC) and 3,5-dichlorophenol (3,5-DCP) were per 1 L of MQ water; LaRossa et al., 1995) supplemented with 0.1% glucose and 0.1%
purchased from SigmaeAldrich, CuSO4*7H2O from Alfa Aesar, H2O2 from Merck and cas-aminoacids (acid hydrolysate of casein, Lab M). For the bioluminescence
EDTA from Serva. All the chemicals were at least of analytical grade. All the stock induction assays, the test final OD600 of bacterial culture in glucose and aminoacids-
solutions were prepared in sterile deionized water at the following concentrations: supplemented HMM media was 0.1 (w106 bacterial cells per mL).

Table 1
Luminescent recombinant Escherichia coli and Pseudomonas fluorescens strains used in this study.

Strain Designation Description Antibiotic concentration Reference

in medium (mg/L)
Stress-inducible recombinant luminescent strains
E. coli K12::katGlux ROS-inducible strain Chromosomal insertion of luxCDABE Kanamycin (30) This study
genes under the control of katG
(catalase-peroxidase) promoter
E. coli MC1061a ssDNA damage-inducible strain luxCDABE genes under control of Ampicillin (100) This study
(pDEWrecAlux) recA (recombinase A) promoter in a
medium-copy number plasmid
Metal-inducible recombinant luminescent strains
E. coli MC1061a Cu-inducible strain luxCDABE genes under control of copA Ampicillin (100) Ivask et al., 2009
(pSLcueR/pDNPcopAlux) (Cu/Ag-responsive) promoter in a Tetracycline (10)
medium-copy plasmid
P. fluorescens OS8::KncueRPcopAlux Cu-inducible strain Chromosomal insertion of luxCDABE Kanamycin (100) Ivask et al., 2009
genes under control of copA
(Cu/Ag-responsible) promoter
Recombinant constitutively luminescent (control) strainsb
E. coli K12::lux Control strain for E. coli K12::katGlux Chromosomal insertion of luxCDABE Kanamycin (30) Ivask et al., 2010
genes
a
E. coli MC1061 (pDEWlux) Control for E. coli MC1061 luxCDABE genes in a medium-copy Ampicillin (100) This study
(pDEWrecAlux) plasmid pDEW201
a
E. coli MC1061 (pSLlux) Control strain for E. coli MC1061 luxCDABE genes in a medium-copy Ampicillin (100) Leedjärv et al., 2006
(pSLcueR/pDNPcopAlux) plasmid pSLlux
P. fluorescens OS8::lux Control strain for P. fluorescens Chromosomal insertion of luxCDABE Kanamycin (100) Ivask et al., 2009
OS8::KncueRPcopAlux genes
a
Genotype: araD139 D(ara, leu)7697 DlacX74 galU galK hsdR2 strA mcrA mcrB1.
b
Used to take into account the quenching of bioluminescence by the turbid and coloured CuO samples.
 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89 83

ROS-generation potential of chemicals or CuO particles was analysed by E. coli 3. Results and discussion
K12::katGlux, DNA damaging potential by E. coli MC1061(pDEWrecAlux) and
the liberation of Cu ions (dissolution) by E. coli MC1061(pSLcueR/pDNPcopAlux)
and Pseudomonas fluorescens OS8::KncueRPcopAlux (Table 1). As CuO suspensions
3.1. Physico-chemical characterization of CuO particles
were turbid, potential quenching of bacterial bioluminescence was taken into
account by parallel use of constitutively luminescent control bacteria, not The manufacturer-advertised size of nano-CuO used in this
inducible by the target chemical: E. coli K12::lux, E. coli MC1061(pDEW201), E. coli study was 30 nm. No size range was available for purchased micro-
MC1061(pSLlux) and P. fluorescens OS8::lux, respectively (Table 1). One hundred
CuO particles. The specific surface area (SSA) was 25.5 m2/g for
microlitres of appropriate dilution of analysed chemicals or CuO particles in
deionized water (or deionized water for the chemical-free control) was nano-CuO and 0.64 m2/g for micro-CuO (Table 2). According to SEM
mixed with 100 mL of bacterial suspension in aminoacid supplemented HMM images, nano-CuO contained mainly <100 nm particles and its
medium on a 96-well microplate. In case of chelation experiments, EDTA in structure was distinct from micro-CuO (Fig. S1A and B). The average
final concentration of 3 mM was added to CuSO4 (0.635 mg Cu/L), nano-CuO hydrodynamic diameter (Dh) of nano-CuO suspension in deionised
(6.35 mg Cu/L), micro-CuO (635 mg Cu/L) (the concentrations were selected
based on the highest sub-toxic response of biosensor towards respective Cu
water ranged from 80 to 400 nm, with an average of 192.5 nm
compounds) and chemical-free control. Bioluminescence was measured with (Fig. S1C). In aminoacids-supplemented HMM medium the average
Orion II plate luminometer (Berthold Detection Systems) every 10 min during the particle size was increased till 385 nm (Table 2; Fig. S1D). This was
first hour of incubation and once per hour after that. Between measurements the likely caused by partial NPs agglomeration due to the mineral salts
plates were covered to avoid evaporation. All the measurements were performed
and aminoacids in HMM medium. However, by filtering the nano-
in at least three independent experiments conducted on different days. Fold
induction of bioluminescence of recombinant bacterial sensors was calculated as CuO suspension in HMM media through 100 nm pore-sized filter
follows: and analysing the filtrate by DLS, we demonstrated that particles of
<100 nm were still present (Table 2). The size distribution of micro-
Fold induction of bioluminescence ¼
SLs
 CF;
CuO was multimodal with the average Dh of 342.7 nm in deionized
CLs water and 617 nm in aminoacids-supplemented HMM medium
where SLs was the luminescence of the sensor strain after exposure to chemical/ (Table 2). A small fraction of nano-range particles was presented in
particles, CLs was the luminescence of the same strain in control solution and CF was micro-CuO suspension in deionized water (Fig. S1C) but not in its
the correction factor. CF was calculated: suspension in HMM medium (Table 2). The UVeVisible absorption
CLc spectra of nano-CuO (Fig. S1E) showed characteristic absorption
CF ¼ ;
SLc peak at approximately 325 nm. No peak was observed in micro-
CuO suspension as the particles settled very quickly. Thus, the
where CLc was the luminescence of the constitutively luminescent strain in
nano-CuO used for further toxicological analysis was clearly
chemical-free control solution and SLc was the luminescence of that strain after
exposure to chemical or particle. Limit of detection (LOD) of the sensor bacteria for
distinct from micro-CuO.
the tested chemical was set to fold induction of bioluminescence ¼ 2. In parallel to
bioluminescence assay, the growth of biosensor bacteria was measured. The growth
3.2. Construction, characterization and calibration of sensor
assay was carried out analogously to bioluminescence assay but in transparent 96-
well microplates and the OD600 (growth) was measured by Multiskan plate reader bacteria
(Thermo Scientific). In parallel, the number of bacterial cells in the test was analysed
by counting the colony forming units (CFU) before the test and after 5 and 8 h of E. coli K12::katGlux biosensor was constructed by fusing the
incubation. CFU were counted after incubation of appropriate dilution of bacterial promoter of katG (encoding catalase-peroxidase enzyme convert-
culture for 24 h on LB agar plates supplemented with appropriate antibiotics
ing hydrogen peroxide to water) with bacterial bioluminescence-
(Table 1).
encoding genes e luxCDABE from Photorhabdus luminescens. KatG
2.5. Chemical analysis of dissolved Cu gene is expressed in the presence of H2O2 in bacterial cell (Belkin
et al., 1996) and thus, E. coli K12::katGlux biosensor was expected
Preparation of Cu formulations for the chemical analysis was identical to to be induced by this ROS. E. coli MC1061(pDEWrecAlux) biosensor
biosensor test procedure and was performed in three independent experiments.
Briefly, suspension of Cu-biosensor bacteria (in cas-aminoacids supplemented
was constructed by fusing the promoter of recA gene (part of
HMM) was mixed 1:1 with CuSO4 (0.635 mg Cu/L), nano-CuO (6.35 mg Cu/L) or bacterial SOS regulon) with P. luminescens luxCDABE genes. As recA
micro-CuO (635 mg Cu/L) in 20 mL volume and ultracentrifuged at 30,000 g for is expressed in response to single-stranded lesions in DNA (Vollmer
30 min. In order to determine the dissolved Cu in the beginning of the test and et al., 1997), this sensor was expected to respond to DNA damaging
during the test, the samples were centrifuged immediately after mixing the
agents. In order to confirm the applicability and specificity of the
bacteria with Cu compounds and also after 5 and 8 h of incubation. Supernatants
were removed and analysed for soluble Cu by AAS-graphite furnace method in newly constructed biosensors, their response to positive controls
certified laboratory of Tallinn University of Technology, Estonia, using standard (H2O2; paraquat as a source of superoxide anions; mitomycin C
procedures EVS-EN ISO/IEC 17025:2005. Before the AAS analysis, the absence of (MMC) as a DNA damaging agent) and a negative control (3,5-
nanoparticles in centrifuged CuO supernatants was analysed. For that, particle dichlorophenol (3,5-DCP)) was measured.
count rate function of Malvern Zetasizer Nano-ZS was used. As the particle count
rate in centrifuged supernatants was similar to that in deionized water, it was
Fig. 1AeC represent the dose-dependent change of biolumi-
concluded that no nanoparticles were present in nano-CuO supernatants after nescence in H2O2-inducible biosensor E. coli K12::katGlux after 2, 5
their centrifugation. and 8-h exposure to target and non-target chemicals. Although the

Table 2
Characterization of CuO particles used in the current study.

Particle Primary size, nm Specific surface area,a m2/g Hydrodynamic size (average),b nm

Deionized water HMM þ 0.1% cas-aminoacids

Not filtered After filtering (pore size 100 nm)

Nano-CuO 30 25.5 192.5 385 70.86
Micro-CuO Not provided 0.64 342.7c 617c Low count rate
a
Measured using BET (from Ivask et al., 2010).
b
Measured by dynamic light scattering (DLS).
c
Multimodal distribution, the average hydrodynamic size of all peaks is presented.
84 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89

ROS (H2 O2 ) biosensor E. coli K12::katGlux

1000 1000 1000
Induction [fold] ± SEM

A B C
100 100 100

10 10 10

1 1 1
0.0001 0.001 0.01 0.1 1 10 100 0.01 0.1 1 10 100 1000 0.01 0.1 1 10 100
H2O2 [mg/L] Paraquat [mg/L] 3,5-dichlorophenol [mg/L]
DNA damage biosensor E. coli M C1061(pDEWrecAlux)
1000 1000 1000
Induction [fold] ± SEM

D E F
100 100 100

10 10 10

1 1 1
0.0001 0.001 0.01 0.1 1 10 0.01 0.1 1 10 100 0.01 0.1 1 10 100
Mitomycin [mg/L] H2O2 [mg/L] 3,5-dichlorophenol [mg/L]

Fig. 1. Time-course response of the Escherichia coli based bioluminescent stress sensors to target and non-target chemicals. 2 h (,), 5 h (-) and 8 h (B) exposure time. AeC:
induction of bioluminescence in ROS biosensor E. coli K12::katGlux by H2O2 (A), paraquat (B) and 3,5-dichlorophenol (C). DeF: induction of bioluminescence in DNA damage
biosensor E. coli MC1061(pDEWrecAlux) by mitomycin C (D), H2O2 (E) and 3,5-dichlorophenol (F). Mean of 3 individual experiments  standard error of the mean is shown.

response of this biosensor to H2O2 was observed already after 3.3. ROS-generating and DNA-damaging potential and dissolution
10 min of exposure (data not shown), the LOD decreased with of CuO
exposure time from 0.1 mg H2O2/L at 2 h till 0.003 mg H2O2/L at 5
and 8 h indicating that this biosensor was remarkably more Induction of ROS, DNA damage and dissolution of CuO
sensitive to H2O2 after prolonged incubation (Fig. 1A). The 2-h LOD nanoparticles at sub-toxic concentrations was analysed using
of E. coli K12:katGlux to H2O2 was comparable and even lower than biosensor bacteria E. coli K12::katGlux, E. coli MC1061(pDE-
that reported for katG-based biosensors previously (Belkin et al., WrecAlux) and E. coli MC1061 (pSLcueR/pDNPcopAlux), respec-
1996; Lee and Gu, 2003; Ahn et al., 2004). After 8 h of exposure, tively. All the three biosensors were induced by all the studied Cu-
E. coli K12::katGlux was also slightly induced by paraquat, which formulations e nano and micro-sized CuO and CuSO4 but showed
may be considered as an indirect inducer of the katG gene (Fig. 1B). differences in the response time (Fig. 2). While E. coli Cu-ion
Paraquat produces mainly superoxide radicals, which may be biosensor was already induced after 0.5 h of exposure to all
further converted to H2O2 by intracellular superoxide dismutases. three Cu formulations, induction of ROS biosensor started after 5 h
Expectedly, there was no induction of this biosensor by the nega- and the induction of DNA damage biosensor started only after 8 h
tive control 3,5-DCP (Fig. 1C). of exposure. Because such a long time was required for the
DNA damage biosensor E. coli MC1061(pDEWrecAlux) (Fig. 1D induction of these biosensors, we assured that the growth of
and F) was induced by mitomycin C (MMC) e a direct mutagen, biosensors in Cu-supplemented samples didn’t differ from the
which acts by covalent binding to DNA, and H2O2 e an indirect chemical free control samples. The enumeration of bacterial cells
mutagen acting via oxidative damage of DNA. Induction of the showed that the growth of bacteria in different samples was
sensor by MMC was already observed after 30 min of exposure indeed comparable (data not shown).
(data not shown). The 2-h LOD of this biosensor to MMC was The detailed response of biosensors to Cu formulations after
0.01 mg/L (Fig. 1D), which was comparable to previously con- 5 h (Cu ion and ROS biosensors) and 8 h (DNA damage biosensor)
structed recA-based biosensors (Vollmer et al., 1997; Davidov et al., exposure is shown in Fig. 3. The doseeresponse pattern of
2000; Mitchell and Gu, 2004). With increasing exposure time (up to ROS (Fig. 3DeF) and DNA damage-specific sensor bacteria
8 h), sensitivity of DNA damage sensor to MMC increased. H2O2 (Fig. 3GeI) to different Cu formulations was similar to that of Cu
induced the biosensor only at relatively high concentration (3 mg ions biosensor (Fig. 3AeC). Particularly, the maximum biolumi-
H2O2/L) (Fig. 1E) and only after 1 h of incubation (Fig. 1E). Again, as nescent response of all the three biosensors to soluble Cu salt
expected, there was no induction of the sensor by negative control was at 0.635 mg Cu/L (105 M), for nano-CuO at 6.35 mg Cu/L
3,5-DCP (Fig. 1F). (104 M) and for micro-CuO at 635 mg Cu/L (102 M) (ratio of
In summary, both constructed stress-specific biosensors were nominal concentrations 1:10:1000). As Cu ion biosensor is
induced by direct and indirect inducers, whereas the response to responding exclusively to Cu ions (Ivask et al., 2009) and the
the latter was weak and delayed. As most nanomaterials that need response pattern of ROS and DNA damage biosensors was very
the toxicological characterization are probably weak inducers, the similar to that of Cu ion biosensor, we hypothesized that Cu ions
biosensor response to weak indirect inducers should be carefully solubilized from CuO particles triggered the ROS and DNA
evaluated. damage response.
 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89 85

2h 5h 8h
Cu-ions biosensor E. coli M C1061(pSLcueR/pDNcopAlux)
100 100 100
C
Induction [fold] ± SEM

A B

10 10 10

1 1 1
0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000

ROS (H2 O2 ) biosensor E. coli K12::katGlux

100 100 100
Induction [fold] ± SEM

D E F

10 10 10

1 1 1
0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000

DNA damage biosensor E. coli M C1061(pDEWrecAlux)

100 100 100
Induction [fold] ± SEM

G H I

10 10 10

1 1 1
0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000

CuSO4 , nano-CuO or micro-CuO [mg Cu/L]

Fig. 2. Time-course response of Escherichia coli based bioluminescent sensors to different Cu formulations. Induction of E. coli MC1061(pSLcueR/pDNPcopAlux) (AeC), ROS
biosensor E. coli K12::katGlux (DeF) and DNA damage biosensor E. coli MC1061(pDEWrecAlux) (GeI) by CuSO4 (C), nano-CuO ( ) and micro-CuO ( ) for 2 (left panels), 5 (middle
panels), and 8 (right panels) hours. Concentrations are presented on Cu basis (mg Cu/L). Mean of 3 individual experiments  standard error of the mean is shown.

3.3.1. Chemical analysis of dissolved Cu showed that both nano-CuO and micro-CuO were additionally
As Cu ions were 10-fold more potent inducers of biosensors solubilized during the test and the concentration of soluble Cu in
compared to nano-CuO and 1000-fold more potent than 6.35 mg Cu/L nano-CuO and 635 mg Cu/L micro-CuO increased
micro-CuO, the solubility of nano-CuO could be around 10% and from 0.6 till w4 mg of soluble Cu/L (Fig. 4B). Based on CuSO4
solubility of micro-CuO around 0.1%. To verify the results of concentrationeresponse curve (Fig. 2B, E and I) 4 mg of soluble Cu/L
biosensors, we measured the dissolved Cu at the concentrations should already inhibit the bioluminescence of the biosensors.
where the sensors were equally (maximally) induced (0.635 mg However, as such inhibition was not observed in case of 6.35 mg Cu/
Cu/L CuSO4, 6.35 mg Cu/L nano-CuO and 635 mg Cu/L micro-CuO) L nano-CuO and 635 mg Cu/L micro-CuO after 5-h exposure we
by chemical analysis (AAS). We assumed that at chosen concen- suggest that at least part of dissolved Cu was not bioavailable to
trations, Cu formulations should result in equal soluble Cu bacterial cells. It is important to note that the bioavailable fraction
content if the biosensors responded exclusively to Cu ions. of Cu that is measured by bacterial sensors includes only free Cu
Indeed, in the beginning of the bioassay, 0.635 mg Cu/L CuSO4, ions (Rensing and Maier, 2003). On the other hand, dissolved Cu
6.35 mg Cu/L nano-CuO and 635 mg Cu/L micro-CuO all resulted measured by AAS includes both, (i) free Cu ions and (ii) soluble Cu
in 0.635 mg of dissolved Cu/L (Fig. 4A). This confirmed that 10% of complexes (Cu ions complexed by components of the media or
nano-CuO and 0.1% of micro-CuO were in the form of ionic Cu bacterial exudates). Thus, the fraction of dissolved Cu determined
already at the beginning of the exposure and that ROS and DNA by AAS was significantly higher than the fraction of free Cu ions that
damage detected by the biosensors was indeed triggered by entered the bacterial cells and caused toxic effects.
dissolved Cu.
As CuO particles may additionally dissolve during the test, we 3.3.2. The role of Cu ions in biosensor response
next determined by AAS the concentrations of soluble Cu in the To further differentiate between the role of dissolved copper
biosensor test assay after 5-h and 8-h exposure. The analysis and CuO particles in the response of the biosensors, we added
86 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89

Cu-ions biosensor E. coli M C1061(pSLcueR/pDNcopAlux)

100 100 100
5-h induction [fold] ± SEM

A B C

10 10 10

1 1 1
0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000
CuSO 4 [mg Cu/L] nano-CuO [mg Cu/L] micro-CuO [mg Cu/L]

ROS (H2 O2 ) biosensor E. coli K12::katGlux

100 100
5-h induction [fold] ± SEM

100
D E F

10 10 10

1 1 1
0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000
CuSO 4 [mg Cu/L] nano-CuO [mg Cu/L] micro-CuO [mg Cu/L]

DNA damage biosensor E. coli M C1061(pDEWrecAlux)

100
8-h induction [fold] ± SEM

100 100
G H I

10 10 10

1 1 1
0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000
CuSO 4 [mg Cu/L] nano-CuO [mg Cu/L] micro-CuO [mg Cu/L]

Fig. 3. Induction of bioluminescence in Escherichia coli based biosensors to different Cu formulations. Responses of Cu-ion biosensor E. coli MC1061(pSLcueR/pDNPcopAlux) (AeC)
and ROS biosensor E. coli K12::katGlux (DeF) after 5-h incubation and response of DNA damage biosensor E. coli MC1061(pDEWrecAlux) (GeI) after 8-h exposure to CuSO4 (white
bars), nano-CuO (grey bars) and micro-CuO (black bars). Mean of 3 individual experiments  standard error of the mean is shown.

EDTA, a chelating agent that sequesters di- and trivalent metal shown) e abolished the response of the biosensors (Fig. 5). This
ions (Iijima et al., 2007), to the Cu formulations and measured the additionally confirmed that the observed ROS and DNA damage in
response of biosensors. Three millimolar EDTA (the highest bacterial cells were indeed caused by dissolved Cu ions and not by
concentration that was not yet toxic to bacterial cells; data not CuO particles themselves.

10 10
Soluble Cu [mg/L] ± SEM

A B
Soluble Cu [mg/l] ± SEM

0h 5h 8h 0h 5h 8h

1 1 0h 5h 8h

0.1 0.1
0.635 mg 6.35 mg Cu/L 635 mg Cu/L 0.635 mg 6.35 mg Cu/L 635 mg Cu/L
Cu/L CuSO4 nano-CuO micro-CuO Cu/L CuSO4 nano-CuO micro-CuO

Fig. 4. Soluble Cu in the biosensor test media as determined by atomic absorption spectroscopy (AAS) after 0 (A) and 0e8 h (B) of exposure. AAS was performed from the particle-
free supernatants (centrifugation at 30 000 g for 30 min) of CuSO4 (white bars), nano-CuO (grey bars) and micro-CuO (black bars) at nominal concentrations of 0.635, 6.35 and
635 mg Cu/L, respectively. The concentrations yielding maximum response of the biosensors were chosen for the analysis.
 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89 87

Cu-ions biosensor E. coli ROS (H2 O2 ) biosensor DNA damage biosensor E.

M C1061(pSLcueR/pDNcopAlux) E. coli K12::katGlux coli M C1061(pDEWrecAlux)
100 100

5-h induction [fold] ± SEM

8-h induction [fold] ± SEM

100 Without EDTA Without EDTA
A B C
5-h induction [fold] ± SEM

Without EDTA

3 mM EDTA 3 mM EDTA 3 mM EDTA

10 10 10

1 1 1

i c uO

i c uO

i c uO
i c uO
uO

uO

uO

uO
no 4

no 4

no 4

no 4
ic uO

ic uO
uO

uO

n a SO

n a SO

n a SO

n a SO
no 4

no 4
na SO
na SO

-C

-C

-C
-C
-C

-C

-C

-C
-C

-C
-C

-C

u
u
u

ro

ro

ro

ro
C

C
ro

ro
C

m
m

Fig. 5. Effect of addition of 3 mM EDTA on the induction of Cu ion-biosensor (A), ROS biosensor (B) and DNA damage biosensor (C) exposed to nano-CuO, micro-CuO and CuSO4 at
concentrations yielding maximum response of the sensors: 0.635 mg Cu/L (white bars); 6.35 mg Cu/L nano-CuO (grey bars) and micro-CuO (635 mg Cu/L; black bars).

3.3.3. Analysis of dissolved Cu using Pseudomonas fluorescens- 3.4. Possible mechanisms of action of CuO particles
based Cu-ion biosensor
E. coli is an important model in molecular biology with well Our results on Cu-ion dependent sub-toxic effects of CuO on E. coli
described stress response mechanisms. However, when the poten- and P. fluorescens are in good agreement with the data on general
tial release of nanoparticles to the environment is of concern, toxicity of nano-CuO to bacteria Vibrio fischeri (Heinlaan et al., 2008),
selection of a more environmentally relevant bacterium would be E. coli, Staphylococcus aureus and Listeria monocytogenes (Cioffi et al.,
appropriate. Pseudomonas sp. is a species commonly present in soil. 2005). On the other hand, detection of hazardous properties of nano-
Therefore, we compared the responses of E. coli and P. fluorescens CuO already at low sub-toxic level supplies new information for pro-
Cu-ion specific biosensors to different Cu formulations (Fig. 6). active approaches. The sensor bacteria applied in this study may be
Similarly to E. coli (Fig. 3AeC), for the P. fluorescens-based Cu- used for such an early warning purpose before the actual mortality
biosensor the most potent inducer was CuSO4, followed by nano- and long term effects will appear. Additionally, the step-wise time-
CuO and then micro-CuO. Despite of the fact that P. fluorescens Cu- dependent activation of E. coli stress-inducible promoters (Fig. 2)
biosensor was 30-times less sensitive to all Cu formulations than offers a view into the bacterial defence systems involved in the
E. coli biosensor, it showed the same pattern of response with response to nano-CuO exposure. Among all biosensors, the most
the maximum induction at 19.05 mg Cu/L in case of CuSO4, 190.5 mg rapid response to nano-CuO was observed with the Cu-ion biosensor
Cu/L in case of nano-CuO and 6350 mg Cu/L in case of micro-CuO. E. coli MC1061(pSLcueR/pDNPcopAlux), which indicates rapid acti-
Thus, the solubility of nano-CuO predicted by P. fluorescens vation of copA promoter. CopA is an ATPase responsible for the
Cu-biosensor was similar to what was predicted by E. coli biosensor: transport of Cu ions between cytosol and periplasm of E. coli (Rensing
10% for nano-CuO and 0.3% for micro-CuO. The more accurate and Grass, 2003) and early activation of copA promoter during
determination of dissolution of micro-CuO was not possible, since exposure to nano-CuO indicates that dissolved Cu ions entered
higher than 6350 mg Cu/L concentrations of micro-CuO were bacterial cell very rapidly. Interestingly, the response pattern of Cu-
quenching around 95% of bioluminescence of P. fluorescens Cu- ion biosensor to all studied Cu formulations during the 2e8-h incu-
biosensor due to their black colour. This result confirms that solu- bation time remained similar (Fig. 2AeC). This suggested that the
bility of CuO (both, nano and micro) determined with E. coli was also intracellular level of Cu (from CuSO4 but also from both CuO formu-
reproducible with the P. fluorescens-based Cu-ion biosensor and lations) was maintained constant. Despite that, the ROS biosensor
show that most probably also for other Gram-negative bacteria the was induced only after 5-h of exposure (Fig. 2D and E). Given the
ROS- and DNA-damaging effects of CuO nanoparticles are mediated E. coli’s capability for rapid gene induction, it is highly probable that
by ionic Cu. the level of intracellular ROS produced during the first hours of

Cu-ions biosensor P. fluorescens OS8::copAlux

100 100 100

C
5-h induction [fold] ± SEM

A B

10 10 10

1 1 1
0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000
CuSO 4 [mg Cu/L] CuSO 4 [mg Cu/L] micro-CuO [mg Cu/L]

Fig. 6. Induction of bioluminescence in Pseudomonas fluorescens Cu-ion biosensor to CuSO4 (A), nano-CuO (B) and micro-CuO (C) after 5 h of exposure. Concentrations are presented
on Cu basis (mg Cu/L). Mean of 3 individual experiments  standard error of the mean is shown.
88 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89

exposure was not sufficient to activate bacterial ROS defence system. 1988) which leads to the unspecific oxidation of various biomole-
This is not surprising because the studied nominal concentration of cules including DNA, which activates ssDNA-inducible gene recA.
nano-CuO was relatively low (6.35 mg Cu/L). For comparison, Similar sub-toxic cellular responses such as increased activity of
Karlsson et al. (2008) detected ROS at 80 mg/L nano-CuO but not at catalase, superoxide dismutase and overexpression of Rad51 (RecA
40 mg/L nano-CuO in mammalian cell cultures, whereas both nano- homologue in mammals) were also shown in human lung epithelial
CuO concentrations were already very toxic to the cells (about 90% cells in vitro after 24-h incubation with 50 mg/L nano-CuO (Ahamed
cytotoxicity). These facts may indicate that at least during the early et al., 2010). DNA damage in response to nano-CuO was also
exposure (till 2 h), the intracellular ROS was probably not the primary observed in mammalian cells (Midander et al., 2009) and in various
cause of the toxicity of studied Cu formulations. For example, it was plants (Nelson et al., 2012). Thus, our results are coherent with the
recently shown that in E. coli Cu ions have an acute mechanism of existing data and to our best knowledge, show for the first time that
toxicity that does not involve ROS and act through the inhibition of (i) nano-CuO induces the bacterial ROS and DNA damage defence
enzymes involved in the synthesis of branched-chain aminoacids systems already at very low sub-toxic levels and (ii) these early
(Macomber and Imlay, 2009). adverse effects are triggered by dissolved Cu ions. Our results on
The accumulation of intracellular ROS after prolonged exposure solubility-dependent toxicity of nano-CuO can be possibly
to low sub-toxic concentrations of Cu formulations may be extended to some other unicellular organisms that (i) have no
explained by Cu ion compartmentalization in the bacterial cell and endocytosis and (ii) are a priori protected against the nanoparticles
Cu chemistry. It has been previously shown that ROS are produced entry by the cell wall. However, the described mechanism is most
during reoxidation reaction of Cu(I) to Cu(II) in the following Cu probably not relevant for the most eukaryotic cells capable to
recycling redox system (essentially as in Hoshino et al., 1999; internalize the nano-sized particles.
Macomber et al., 2007):
4. Conclusions
Cu(I)  e þ O2 4 Cu(II) þ O
2
As CuO nanoparticles showed adverse effects to bacteria already
2O
2 þ 2H2O / H2O2 þ O2 þ 2OH

at very low concentrations and these effects were triggered by
the solubilized copper ions, we suggest that dissolution of CuO
Cu(I) þ H2O2 / Cu(II) þ OH þ OH

nanoparticles should be addressed also on Material Safety Data
Sheets. For example, the MSDS for 50 nm CuO preparation at
OH þ DNA / DNA damage

SigmaeAldrich website (reference number 544868) provides no
indication on dissolution of these nanoparticles.
As in the case of Gram-negative bacteria the main pool of Finally, with some refinement, such as choosing/constructing
intracellular Cu ions is located in the periplasm (Outten et al., the metal-sensing bacterial strains, depending on the type of
2001), the ROS are mainly produced and get concentrated in the metallic NPs analysed, the testing strategy applied in this study is
periplasm and do not reach the cytosolic targets (Macomber et al., also applicable for the high throughput profiling of (eco)toxico-
2007). The high level accumulation of ROS in the periplasm may logical properties of other (metallic) NPs.
however lead to their leakage into the cytosol where they turn on
the ROS defence systems, including katG. Reoxidation of Cu(I) to Acknowledgements
Cu(II) generates ROS in the following order: first the superoxide
anion is produced and then hydrogen peroxide and hydroxyl This study was supported by projects SF0222601Bs03, ETF6974
radicals will appear (Hoshino et al., 1999). This order of ROS is in and ETF8561, ESF and FP7 project NanoValid (contract No 263147).
accordance with our biosensor results: our previous results have Prof. Shimshon Belkin is acknowledged for the plasmid pDEW201.
shown that superoxide anions-sensing sodA-based luminescent
E. coli K12::soxRSsodAlux was activated by nano-CuO after 2 h of Appendix A. Supplementary material
induction (Ivask et al., 2010) but the peroxide-sensing strain
K12::katGlux in the current study was induced only after 5 h of Supplementary material associated with this article can be
induction (Fig. 2). Finally, the DNA-damage sensor responded to found, in the online version, at doi:10.1016/j.envpol.2012.05.009.
nano-CuO only after 8-h exposure. Seemingly, the observed DNA-
damaging effects were secondary and transient (e.g., due to the References
RecA-dependent efficient reparation of DNA) as CuSO4 has been
classified not mutagenic in the bacterial reversed mutation (Ames) Ahamed, M., Siddiqui, M.A., Akhtar, M.J., Ahmad, I., Pant, A.B., Alhadlaq, H.A., 2010.
assay according to various available Material Safety Data Sheets Genotoxic potential of copper oxide nanoparticles in human lung epithelial
cells. Biochem. Biophys. Res. Commun. 396, 578e583.
(Merck, 2010). Analogously, Macomber et al. (2007) showed that Ahn, J.M., Mitchell, R.J., Gu, M.B., 2004. Detection and classification of oxidative
copper ions induced the superoxide dismutase and catalase but did damaging stresses using recombinant bioluminescent bacteria harboring
not catalyse significant oxidative DNA damage in E. coli. soda::, pqi::, and katG::luxCDABE fusions. Enzyme Microb. Technol. 35,
540e544.
Based on the previous studies on Cu ion chemistry and time- Aruoja, V., Dubourguier, H.C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles
dependent activation of our biosensor suite, we assume that Cu of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total
ions dissolved from nano-CuO are the key factor triggering a series Environ. 407, 1461e1468.
Baek, Y.W., An, Y.J., 2011. Microbial toxicity of metal oxide nanoparticles (CuO, NiO,
of downstream adverse effects. We suggest a following hypothet-
ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis and Streptococcus aureus. Sci.
ical cascade of events: the internalised Cu ions are mainly stored in Total Environ. 409, 1603e1608.
the periplasm (Outten et al., 2001), where they first induce the Belkin, S., Smulski, D.R., Vollmer, A.C., Van Dyk, T.K., LaRossa, R.A., 1996. Oxidative
production of superoxide anions (Hoshino et al., 1999). Superoxide stress detection with Escherichia coli harboring a katG::lux fusion. Appl.
Microbiol. Biotechnol. 62, 2252e2256.
anions leach into the cytosol, where they activate superoxide- Blinova, I., Ivask, A., Heinlaan, M., Mortimer, M., Kahru, A., 2010. Ecotoxicity of
dismutase gene sodA. Intracellular SodA and other superoxide nanoparticles of CuO and ZnO in natural water. Environ. Pollut. 158, 41e47.
dismutases convert superoxide to H2O2 that triggers the activation Cioffi, N., Torsi, L., Ditaranto, N., Tantillo, G., Ghibelli, L., Sabbatini, L., Bleve-
Zacheo, T., D’Alessio, M., Zambonin, P.G., Traversa, E., 2005. Copper nano-
of catalases, e.g., KatG. Finally, cytosolic H2O2 induces Fe-dependent particle/polymer composites with antifungal and bacteriostatic properties.
production of hydroxyl radicals via Fenton reaction (Imlay et al., Chem. Mater. 17, 5255e5262.
 O. Bondarenko et al. / Environmental Pollution 169 (2012) 81e89 89

Davidov, Y., Rozen, R., Smulski, D.R., Van Dyk, T.K., Vollmer, A.C., Elsemore, D.A., LaRossa, R.A., Smulski, D.R., Van Dyk, T.K., 1995. Interaction of lead nitrate and
LaRossa, R.A., Belkin, S., 2000. Improved bacterial SOS promoter::lux fusions for cadmium chloride with Escherichia coli K-12 and Salmonella typhimurium global
genotoxicity detection. Mutat. Res. 46, 97e107. regulatory mutants. J. Ind. Microbiol. 14, 252e258.
Ebrahimnia-Bajestan, E., Niazmand, H., Duangthongsuk, W., Wongwises, S., 2011. Lee, H.J., Gu, M.B., 2003. Construction of a sodA::luxCDABE fusion Escherichia coli:
Numerical investigation of effective parameters in convective heat transfer of comparison with a katG fusion strain through their responses to oxidative
nanofluids flowing under a laminar flow regime. Int. J. Heat Mass Transfer 54, stresses. Appl. Microbiol. Biotechnol. 60, 577e580.
4376e4388. Leedjärv, A., Ivask, A., Virta, M., Kahru, A., 2006. Analysis of bioavailable phenols
European Chemicals Bureau, 2006. Manual of Decisions (MoD) to Directive 67/548/ from natural samples by recombinant luminescent bacterial sensors. Chemo-
EEC, Amendment 92/93. sphere 64, 1910e1919.
Fahmy, B., Cormier, S.A., 2009. Copper oxide nanoparticles induce oxidative stress Macomber, L., Rensing, C., Imlay, J.A., 2007. Intracellular copper does not catalyze
and cytotoxicity in airway epithelial cells. Toxicol. In Vitro 23, 1365e1371. the formation of oxidative DNA damage in Escherichia coli. J. Bacteriol. 189,
Gabbay, J., Borkow, G., Mishal, J., Magen, E., Zatcoff, R., Shemer-Avni, Y., 2006. 1616e1626.
Copper oxide impregnated textiles with potent biocidal activities. J. Ind. Textil. Macomber, L., Imlay, J.A., 2009. The iron-sulfur clusters of dehydratases are primary
35, 323e335. intracellular targets of copper toxicity. Proc. Natl. Acad. Sci. U. S. A.106, 8344e8349.
Gou, N., Onnis-Hayden, A., Gu, A.Z., 2010. Mechanistic toxicity assessment of Merck, 2010. Safety Data Sheet According to Regulation (EC) No. 1907/2006. http://
nanomaterials by whole-cell-array stress genes expression analysis. Environ. www.alsglobal.eu/docs/eng_20100526_msds_cuso4_en.pdf.
Sci. Technol. 44, 5964e5970. Midander, K., Cronholm, P., Karlsson, H.L., Elihn, K., Möller, L., Leygraf, C.,
Griffitt, R.J., Weil, R., Hyndman, K.A., Denslow, N.D., Powers, K., Taylor, D., Wallinder, I.O., 2009. Surface characteristics, copper release, and toxicity of
Barber, D.S., 2007. Exposure to copper nanoparticles causes gill injury nano- and micrometer-sized copper and copper(II) oxide particles: a cross-
and acute lethality in zebrafish (Danio rerio). Environ. Sci. Technol. 41, disciplinary study. Small 5, 389e399.
8178e8186. Mitchell, R.J., Gu, M.B., 2004. An Escherichia coli biosensor capable of detecting both
Heinlaan, H., Ivask, A., Blinova, I., Dubourguier, H.C., Kahru, A., 2008. Toxicity of genotoxic and oxidative damage. Appl. Microbiol. Biotechnol. 64, 46e52.
nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crusta- Nel, A., Xia, T., Mädler, L., Li, N., 2006. Toxic potential of materials at the nanolevel.
ceans Daphnia magna and Thamnocephalus paltyurus. Chemosphere 71, Science 311, 622e662.
1308e1316. Nelson, B.C., Atha, D., Wang, H., Petersen, E.J., Cleveland, D., Holbrook, R.D.,
Hoshino, N., Kimura, T., Yamaji, A., Ando, T., 1999. Damage to the cytoplasmic Jaruga, P., Dizdaroglu, M., Xing, B., 2012. Copper oxide nanoparticle mediated
membrane of Escherichia coli by catechin-copper (II) complexes. Free Radic. Biol. DNA damage in terrestrial plant models. Environ. Sci. Technol. 46, 1819e1827.
Med. 27, 1245e1250. Outten, F.W., Huffman, D.L., Hale, J.A., O’Halloran, T.V., 2001. The independent cue
Hwang, E.T., Lee, J.H., Chae, Y.J., Kim, Y.S., Kim, B.C., Sang, B.I., Gu, M.B., 2008. and cus systems confer copper tolerance during aerobic and anaerobic growth
Analysis of the toxic mode of action of silver nanoparticles using stress-specific in Escherichia coli. J. Biol. Chem. 276, 30670e30677.
bioluminescent bacteria. Small 4, 746e750. Ren, G., Hu, D., Cheng, E.W.C., Vargas-Reus, M.A., Reip, P., Allaker, R.P., 2009. Char-
Iijima, M., Sato, N., Tsukada, M., Kamiya, H., 2007. Dispersion behavior of barium acterisation of copper oxide nanoparticles for antimicrobial applications. Int. J.
titanate nanoparticles prepared by using various polycarboxylic dispersants. Antimicrob. Agents 33, 587e590.
J. Am. Ceram. Soc. 90, 2741e2746. Rensing, C., Grass, G., 2003. Escherichia coli mechanisms of copper homeostasis in
Imlay, J.A., Chin, S.M., Linn, S., 1988. Toxic DNA damage by hydrogen peroxide a changing environment. FEMS Microbiol. Rev. 27, 197e215.
through the Fenton reaction in vivo and in vitro. Science 240, 640e642. Rensing, C., Maier, M., 2003. Issues underlying use of biosensors to measure metal
Ivask, A., Bondarenko, O., Jepihhina, N., Kahru, A., 2010. Profiling of the reactive bioavailability. Ecotoxicol. Environ. Saf. 56, 140e147.
oxygen species-related ecotoxicity of CuO, ZnO, TiO2, silver and fullerene Ruparelia, J.P., Chatterjee, A.K., Duttagupta, S.P., Mukherji, S., 2008. Strain specificity
nanoparticles using a set of recombinant luminescent Escherichia coli strains: in antimicrobial activity of silver and copper nanoparticles. Acta Biomater. 4,
differentiating the impact of particles and solubilised metals. Anal. Bioanal. 707e716.
Chem. 398, 701e716. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory
Ivask, A., Rõlova, T., Kahru, A., 2009. A suite of recombinant luminescent bacterial Manual. Cold Spring Harbour Laboratory Press, New York.
strains for the quantification of bioavailable heavy metals and toxicity testing. Thomas, K.V., Brooks, S., 2010. The environmental fate and effects of antifouling
BMC Biotechnol. 9, 41. paint biocides. Biofouling 26, 73e88.
Kahru, A., Dubourguier, H.C., 2010. From ecotoxicology to nanoecotoxicology. Vollmer, A.C., Belkin, S., Smulski, D.R., Van Dyk, T.K., LaRossa, R.A., 1997. Detection of
Toxicology 269, 105e119. DNA damage by use of Escherichia coli carrying recA::lux, uvrA::Iux or alkA::lux
Karlsson, H.L., Cronholm, P., Gustafsson, J., Möller, L., 2008. Copper oxide nano- reporter plasmids. Appl. Env. Microbiol. 63, 2566e2571.
particles are highly toxic: a comparison between metal oxide nanoparticles and Wu, B., Huang, R., Sahu, M., Feng, X., Biswas, P., Tang, Y.J., 2010. Bacterial responses
carbon nanotubes. Chem. Res. Toxicol. 21, 1726e1732. to Cu-doped TiO2 nanoparticles. Sci. Total Environ. 408, 1755e1758.