Environmental Pollution 227 (2017) 39e48

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

Toxicity of TiO2, in nanoparticle or bulk form to freshwater and marine

microalgae under visible light and UV-A radiation*
M. Sendra a, *, I. Moreno-Garrido a, M.P. Yeste b, J.M. Gatica b, J. Blasco a
a
Department of Ecology and Coastal Management, Institute of Marine Sciences of Andalusia (CSIC), Campus Río S. Pedro, 11510, Puerto Real, Ca
diz, Spain
b
diz,
Department of Material Science, Metallurgical Engineering and Inorganic Chemistry, Faculty of Sciences, University of Cadiz, E-11510, Puerto Real, Ca
Spain

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

Article history: Use of titanium dioxide nanoparticles (TiO2 NPs) has become a part of our daily life and the high
Received 13 December 2016 environmental concentrations predicted to accumulate in aquatic ecosystems are cause for concern.
Received in revised form Although TiO2 has only limited reactivity, at the nanoscale level its physico-chemical properties and
19 April 2017
toxicity are different compared with bulk material. Phytoplankton is a key trophic level in fresh and
Accepted 20 April 2017
Available online 25 April 2017
marine ecosystems, and the toxicity provoked by these nanoparticles can affect the structure and
functioning of ecosystems. Two microalgae species, one freshwater (Chlamydomonas reinhardtii) and the
other marine (Phaeodactylum tricornutum), have been selected for testing the toxicity of TiO2 in NP and
Keywords:
Toxicity
conventional bulk form and, given its photo-catalytic properties, the effect of UV-A was also checked.
Phytoplankton Growth inhibition, quantum yield reduction, increase of intracellular ROS production, membrane cell
TiO2 nanoparticles damage and production of exo-polymeric substances (EPS) were selected as variables to measure.
Freshwater TiO2 NPs and bulk TiO2 show a relationship between the size of agglomerates and time in freshwater
Seawater and saltwater, but not in ultrapure water. Under two treatments, UV-A (6 h per day) and no UV-A
exposure, NPs triggered stronger cytotoxic responses than bulk material. TiO2 NPs were also associ-
ated with greater production of reactive oxygen species and damage to membrane. However, microalgae
exposed to TiO2 NPs and bulk TiO2 under UV-A were found to be more sensitive than in the visible light
condition. The marine species (P. tricornutum) was more sensitive than the freshwater species, and
higher Ti internalization was measured. Exopolymeric substances (EPS) were released from microalgae in
the culture media, in the presence of TiO2 in both forms. This may be a possible defense mechanism by
these cells, which would enhance processes of homoagglomeration and settling, and thus reduce
bioavailability.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction science, and the production of engineered nanomaterials (ENMs)

Phytoplankton is a key trophic level in aquatic ecosystems
because these organisms are responsible for primary production
(Behrenfeld et al., 2006). The presence of legacy and emergent
pollutants in aquatic ecosystems can affect their structure and
functioning. Among contaminants, engineered nanomaterials
(ENMs) (Miller et al., 2012) represent a new input of contaminants
and their impact needs to be assessed.
There has been increasing application of nanotechnology to
molecular biology, biomaterials, semiconductors and surface
*
This paper has been recommended for acceptance by B. Nowack.
* Corresponding author.
E-mail address: marta.sendra@icman.csic.es (M. Sendra).
has increased exponentially in the last decade. As a result, ENMs are
inevitably released into aquatic systems including estuaries, coastal
waters and oceans, which are the ultimate sink for nanoparticles
(NPs) (Canesi et al., 2010, 2012; Ratnasekhar et al., 2015). The effect
of those NPs on biological systems is still not well understood
(Maurer-Jones et al., 2013; Wiesner et al., 2006).
Titanium dioxide in the form of nanoparticles (TiO2 NPs) has
become a part of our daily life, and these NPs are now widely used
in drug delivery systems, therapeutics and biosensors, cosmetics,
production of paints, coatings, plastics, skin care products, foods,
water remediation devices and pharmaceuticals, inter alia (Vance
et al., 2015). Predicted environmental concentrations (PEC) of
TiO2 NPs show the highest values for NPs in surface water, with a
mode of 3 and 0.30 ng L
1 for freshwater and seawater respectively,

http://dx.doi.org/10.1016/j.envpol.2017.04.053
0269-7491/© 2017 Elsevier Ltd. All rights reserved.
40 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48
and in sediments, 1200 and 390 mg kg
1, for freshwater and marine
environments, respectively (Boxall et al., 2007; Gottschalk et al.,
2015).
The properties, photo-stability and photo-reactivity of TiO2 NPs
have led researchers to pay considerable attention to character-
ization of the resulting risk for ecosystems and organisms (Coll
et al., 2015; Li et al., 2014; Wallis et al., 2014). Due to its photo-
catalytic properties, TiO2, under UV-A radiation, can produce
various increased adverse effects in an organism, including cell
damage, unregulated cell signaling, change of cell motility,
apoptosis, genotoxicity, pulmonary inflammation, abnormal im-
mune response, DNA damage and fibrosis (Fu et al., 2014; Montiel-
Da
valos et al., 2012; Sayes et al., 2006; Shi et al., 2013).
TiO2 NPs have been classified as “harmful”, with LC50 values
between 10 and 100 mg mL
1, to organisms such as yeast, bacteria,
algae, crustaceans, nematodes and fish (Kahru and Dubourguier,
2010). In microalgae, the cytotoxicity seems to be due to mem-
brane damage, impairment of the effective quantum yield of PS II,
and disrupted cell cycle (Dalai et al., 2013; Navarro et al., 2008). In
many studies, the role of UV-A radiation has not been taken into
account, so the toxicity of a photo-reactive material is under-
estimated. Additionally, NPs can be adsorbed onto algal cell sur-
faces, and this can also reduce growth by physical shading effects
(Hund-Rinke and Simon, 2006); and the additional weight of the
NPs can force the sedimentation of algae (Huang, 2005).
The extent and type of damage depend on physicochemical
characteristics of TiO2 NPs (e.g. size, charge, crystalline forms and
coating) and environmental factors (e.g. ionic strength (IS), pH and
dissolved organic materials which govern their bioavailability and
reactivity) (Gonzalez et al., 2008; Liu et al., 2014; Zhao et al., 2014).
The bioavailability of TiO2 NPs is related to their dispersion in the
aquatic environment and their greater tendency to agglomerate
(NP-NP). Recent ecotoxicological investigations have shown that
TiO2 NPs are toxic to marine organisms such as invertebrates,
cyanobacteria and polychaetes (Barmo et al., 2013; Canesi et al.,
2010; Cherchi and Gu, 2010; Galloway et al., 2010; Zhu et al.,
2011). Only a few of these studies previously cited are focused on
microalgae; the great majority deal mainly with freshwater species
(Miller et al., 2010, 2012). Specific mechanisms need to be taken
into account when assessing toxicity. Phytoplankton cells can
produce exopolymeric substances (EPS) as a response when
exposed to potentially dangerous substances. EPS can enhance the
aggregation and flocculation of NPs, thus altering their bioavail-
ability and reactivity (Dalai et al., 2013; Soldo et al., 2005). To date
those mechanisms have not been adequately studied.
In this work, the toxicity of TiO2 NPs and bulk TiO2 has been
assessed for two phytoplanktonic species, one from the marine
environment (Paeodactylum tricornutum, and the other found in
freshwater systems (Chlamydomonas reinhardtii). The joint effect of
exposure to TiO2 and UV-A radiation during 6 h per day, simulating
the natural condition, has been taken in account in order to
improve knowledge of the mechanisms involved in the toxicity of
TiO2 NPs and bulk TiO2 in environmental conditions.
2. Material and methods
Reagents: TiO2 NPs (CAS number, 13463-67-7, as nano-powder,
79% anatase, 21% rutile, particle size <100 nm) and bulk TiO2 (CAS
number, 1317-80-2, as pure rutile, particle size >100 nm) were
obtained from Sigma Aldrich, together with DCFH-DA (20 -7’-
dichlorofluorescein diacetate), and propidium iodide. All glassware
was washed with diluted nitric acid (10%) and rinsed several times
with de-ionized water (Milli-Q) before use. All reagents for media
were of analytical grade.
2.1. Nanoparticle solutions
Stock of TiO2 NPs and bulk TiO2 were prepared in ultrapure
water immediately before the experiments. Suspensions were
sonicated with a tip sonicator (UP 200S Dr. Hielscher GmbH) for
10 min with cycle of 0.5 and frequency of 50 Hz.
2.2. Characterization of TiO2 NPs and TiO2 bulk
Textural properties were characterized by measuring the
adsorption/desorption of N2 at 196  C, employing a Micromeritics
ASAP 2020 automatic device. Before measurements, samples were
submitted to a surface cleaning pre-treatment under high vacuum
at 200  C during 2 h. The isotherms obtained were used to calculate
the specific surface area (SBET) as well as the total pore volume of
the samples studied.
The initial hydrodynamic size of TiO2 NPs and the zeta potential
of TiO2 in both forms (NPs and bulk) were studied in ultrapure
water, freshwater and artificial marine water through Dynamic
Light Scattering (Zetasizer Nano ZS90, Malvern Instruments, Mal-
vern, UK, and its software version 7.10) at 1 mg L
1. The dispersion
of TiO2 NPs was prepared in ultrapure water, following the CEINT/
NIST 1200-3 and 1200-4 Standard Protocol (Taurozzi et al.,
2012a,b).
Particle size distributions and images revealing the particle
shape, and the micro- and nano-structure of the samples, were
obtained by means of Transmission Electron Microscopy (TEM). The
microscope used was a JEOL2010F model, working at 200 kV. This
instrument has a structural resolution of 0.19 nm. The number of
images recorded allowed the measurement of 130 particles to
determine average size and mode data.
Agglomeration of TiO2 NPs and bulk TiO2 was assessed over time
(0, 0.5, 1, 3, 6, 24 and 48 h) in three different media (ultrapure water,
artificial freshwater and artificial marine water) (ASTM, 1975;
F

abregas et al., 2000). The initial concentration in the samples
was 250 mg L
1 for freshwater and marine water, but for ultrapure
water concentrations of 250 mg L
1 and 500 mg L
1 were evalu-
ated. Changes in agglomeration states were measured by a Malvern
Master Sizer 2000, with software version 5.61.
Crystallization of TiO2 NPs and bulk TiO2 were checked by XRD
to confirm the information given by suppliers (see Fig. SI5).
2.3. Test organisms
Two microalgae species were selected, one found in freshwater
environments (Chlamydomonas reinhardtii P.A., (Dangeard, 1888),
CHLOROPHYCEAE) and the other in marine environments (Phaeo-
dactylum tricornutum, (Bohlin, 1897), BACILLARIOPHYCEAE). Both
were obtained from the ICMAN Marine Microalgae Culture
Collection (IMMCC). Cells were grown in filtered (0.2 mm) fresh-
water culture medium and F/2 marine medium without EDTA (see
supplementary information), respectively, for two weeks prior to
the experiment (Fa
bregas et al., 2000; Guillard and Ryther, 1962).
Artificial marine water used was Substitute Ocean Water D1141-75
from the ASTM (ASTM, 1975).
2.4. Toxicity bioassays
Bioassays were carried out using either TiO2 NPs or bulk TiO2.
Two light treatments were also applied: continuous visible light
(1.6 mWcm
2) and the same light regime plus 6 h of UV-A
(0.20 mW cm
2, Multiple Ray Lamp) per day, simulating two nat-
ural environments. The intensity of UV-A was measured with a
digital UVX radiometer (UVP, Analytik Jena AG).
Growth inhibition bioassays were carried out following OECD
 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48
Guidelines (OECD, 1994), in order to determine the effective values
for 50% inhibition after 72 h (EC50) of microalgae cellular concen-
tration (initial cell density, 104 cells mL
1). Cells were counted
under microscope using a Neubauer counting chamber. The growth
inhibition values were fitted to the Hampel model (Hampel et al.,
2001), with different concentrations: 0.1, 1, 10, 100, 200 and
400 mg L
1 under continuous white light; and 0.1, 1, 2, 5 and
10 mgL
1 under the visible light plus UV-A treatment.
The effective quantum yield of photosynthetic energy conver-
sion in PSII in darkness was measured by fluorometry using a
Phyto-PAM (Heinz Walz GmbH) equipped with an ED-101 US/MP
Optical Unit. This parameter shows the efficiency of the photo-
chemical energy conversion process (Schreiber et al., 1995). Values
of EC50 of effective quantum yield were also fitted to the same
model (Hampel et al., 2001). Effective quantum yields were
measured at the same concentration as those for EC50 growth
inhibition.
Production of intracellular ROS (reactive oxygen species: su-
peroxide, hydroxyl and hydrogen peroxide) was quantified using
the DCFH-DA (20 -7’-dichlorofluorescein diacetate) method (He
et al., 2002; Stachowski-Haberkorn et al., 2013). ROS were
measured at 3, 8, 24, 48 and 72 h at 2 and 5 mg L
1 for both TiO2 NPs
and bulk TiO2 under visible light, and at 2 mg L
1 under UV-A. For
this bioassay a negative control (without treatment) and a positive
control with 0.1 mM of H2O2 (to see the signal correctly in the flow
cytometer) were employed.
After 30 min in darkness, at room temperature conditions, ROS
were measured by green fluorescence (FL1, 533/30 nm) of stained
cells using a FACSCalibur Flow Cytometer (Becton-Dickinson®).
Finally, we calculated the percentage of oxidative stress as the
number of cells that fluoresce green (as stained with DCFH-DA) in
relation to the total number of cells counted by the flow cytometer
(equation (1)).
Cells Dyed with DCFH
DA
ROS ¼ *100
Total Cells
Membrane integrity was also quantified, following the Propi-
dium Iodide (PI) method (Xiao et al., 2011). This parameter was
measured at 3, 8, 24, 48 and 72 h for 2 and 5 mg L
1 under visible
light, and at 2 mg L
1 under UV-A treatment. Samples were incu-
bated for 20 min at PI dosage (10 mg mL
1 for 2$106 cells). Fluo-
rescence of PI inside the cells was detected with a FL2 detector by
flow cytometry (the same equipment as that used in the ROS
analysis). The percentage of membrane damage was calculated
following equation (2). Methanol was used as positive control.
Cells Dyed with PI
Membrane damage ¼ *100
Total Cells
2.5. Quantification of intracellular and extracellular TiO2 NPs
Concentration of Ti in four fractions (total, supernatant, extra-
cellular and intracellular) was quantified by a multi-syringe flow
injection analysis system using a liquid waveguide capillary cell
(MSFIA-LWCC) (Sa
nchez-Quiles et al., 2013), after prior digestion
with potassium peroxodisulfate. Titanium recovery was 87.7 ± 1.5%.
Accumulation bioassays were performed in flasks with a volume
of 10 mL, containing 10 mL of treated algal cultures at an initial cell
density of 106 cells$mL
1 and concentration of 0.1 mg L
1 of TiO2
NPs. Samples for Ti measurements were collected at 6, 24 and 48 h.
For control and treated algal cells the whole volume was
centrifuged at 4000 g for 20 min. Supernatants were stored and
pellets re-suspended in 5 mL of EDTA (0.02M) dissolved in PBS, and
41
shaken for 30 s in order to remove extracellular TiO2 (Zhou et al.,
2012). After this, the process was repeated three times. The
required number of repetitions was determined by a previous study
using flow cytometry, in order to detect the presence of TiO2 ag-
gregates (by both forward scattering and side scattering) and no
concentration of Ti with MSFIA-LWCC was measured. The final
pellet was stored at
80  C until the intracellular metal content was
quantified by MSFIA-LWCC.
2.6. Quantification of exo-polymeric substances (EPS)
Concentrations of EPS were determined after 72 h in samples
from the toxicity bioassays, according to Dubois et al., 1956 (Dubois
et al., 1956). Algal cultures (30 mL) were centrifuged at 10,000 g for
10 min, and then the supernatant and pellet were separated. The
supernatant fraction was chosen to measure dissolved EPS. Equal
volumes of pre-chilled ethanol were added to the samples, and the
resulting mixtures were kept at 4  C overnight, in order to pre-
cipitate dissolved EPS. After a second centrifugation at 10,000 g for
30 min, supernatants were discarded and pellets were washed
twice at 15,000 g for 15 min with ultrapure water to obtain pure
dissolved EPS. The quantification of EPS was carried out using the
phenol-sulfuric acid method (Dubois et al., 1956).
2.7. Statistical analysis
All bioassays were performed in triplicate, controls were
developed for both conditions (UV-A and visible regime) in order to
assess the effect of UV-A by itself and effect of particles tested. Data
are shown as average ± standard deviation between replicates.
Statistical analyses were carried out using the IBM SPSS, Statistics
23 program. To assess the responses, i.e. growth, effective quantum
yield, % of ROS, % membrane damage and EPS two General Linear
Models (GLM; SPSS, 2005) were constructed (McCullagh and
Nelder, 1989; SPSS, 2005). The first GLM was a general one in
(1)
which all the factors are in competition, and the second model is
segmented by medium (fresh and marine) and exposure condition
(UVA þ visible light and visible light only). The fixed factors were
medium (freshwater and marine water), exposure condition
(UVA þ visible light and visible light only, treatment (TiO2 in NP
and bulk form), concentration and time.
Data were checked by homogeneity of variance (Levene test), a
one-way Anova test and a Bonferroni post hoc test at p < 0.05. If the
data did not pass the Levene test for homogeneity, a post hoc
Tamhane T2 test was applied.
3. Results
(2)
3.1. Characterization of TiO2 NPs and bulk TiO2
Physical characteristics of the dry powdered TiO2 NPs and bulk
TiO2 together with their properties in different media are shown in
Fig. SI1 (TEM study) and the supplementary information
(Table SI1). Porosity and the smaller size of particles of the TiO2 NPs
sample in relation to TiO2 bulk (38 ± 12 nm for TiO2 NPs and
423 ± 154 nm for bulk; and weighted by surface, 46 nm for TiO2 NPs
and 534 nm for bulk) are consistent with their greater specific
surface area BET in comparison to bulk TiO2 (18 times higher in the
NPs sample).
The size of the agglomerates of TiO2 NPs measured by DLS was
largest in ultrapure water and smallest in saltwater; zeta potential
was less electronegative in saltwater than in ultrapure water.
(shown in Fig. SI2); the polydispersity index (PDI, indicative of the
heterogeneity of hydrodynamic radii of particles)) is greater than in
ultrapure water, outside of acceptable limits (PDI<0.7 when the
42 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48
samples were measured after 2 h). Due to this high PDI the samples
were assessed over time with the Mastersizer 2000 to study the
agglomeration. Two different concentrations in ultrapure water
(250 and 500 mg L
1) were studied. Neither TiO2 NPs nor bulk TiO2
showed larger agglomerate sizes at higher concentrations (Fig. SI3).
With respect to differences in the agglomeration of TiO2 NPs and
bulk TiO2 between different media, agglomerate sizes were quite
similar between freshwater and saltwater media but there were
statistically significant differences in size between ultrapure water
and the other two media (freshwater and marine media) (p < 0.05)
(Table SI2 in supplementary information). Both TiO2NPs and bulk
TiO2 showed a relationship between size of agglomerates and time
in freshwater and saltwater but not in ultrapure water (Fig. SI4). In
artificial marine water, the agglomeration occurs in the first 30 min.
However, in freshwater NPs were stable for at least the first hour.
3.2. Cytotoxicity of TiO2 NPs and bulk TiO2
Data of the controls from the UV-A and visible light regimes
were analyzed using the GLM for each of the cytotoxic responses
studied with the object of determining the effect of these condi-
tions (UV-A and visible light regimes) on the microalgae popula-
tion. These conditions were significant for the particular cytotoxic
responses, growth and percentage of ROS (p < 0.05); however they
were not significant for the effective quantum yield of PII and the
percentage of cell membrane damage (p > 0.05). However, when
these data were compared for each condition (UV-A and visible
light regime) and culture media (freshwater and marine micro-
algae), other factors such as treatment (NPs and bulk)*concentra-
tion were significant (p < 0.05) for all cytotoxic responses. Each
cytotoxic responses are explained below:
3.3. Microalgae growth population
TiO2 NPs and bulk TiO2 showed a dose-exposure dependent
cytotoxicity to C. reinhardtii and P. tricornutum under the UV-A light
regime and visible light (see Table SI4). Nevertheless, when they
were exposed to visible light at low concentrations of both sub-
stances, microalgae populations showed a hormetic response, as
cultures grew up to 5% more than the controls. The GLM values
(Table SI3) show, in most of the responses, how the exposure
condition (UV-A and visible light only regimes), treatment, con-
centration, and time resulted in significant differences (p < 0.05) in
the toxicity variables. However, in the GLM by species (freshwater
and marine microalgae) and exposure condition (UV-A and visible
light) the fit to the model is improved (Table SI4).
When cells were subjected to the UV-A regime, toxicity was
higher than in experiments with visible light only; and thus EC50
values were lower for both materials (TiO2NPs and bulk TiO2, with
significant differences p < 0.05).
In the UV-A regime, for C. reinhardtii, EC50 values for growth
inhibition were 2.6 ± 1.7 mg L
1 and 1.3 ± 3.0 mg L
1 for TiO2 NPs
and bulk TiO2 respectively (p > 0.05). C. reinhardtii results indicated
that the material (NPs or bulk) with different size did not show
differences in the EC50; therefore in this case we can conclude that
the photocatalytic property of the materials is key in the TiO2
toxicity under UV-A regardless of their size. When cells were
incubated without UV-A exposure, inhibition did not reach 50%
even at the highest concentrations used and the calculated (ex-
pected) EC50 values for TiO2 NPs and bulk TiO2 were 552 ± 164 and
424 ± 19 mgL
1 respectively (p > 0.05). For P. tricornutum, EC50
values were 2 ± 0.09 and 6.6 ± 0.7 mg L
1 for TiO2 NPs and bulk
TiO2 respectively under UV-A (p < 0.05), and 132 ± 7 and
185 ± 26 mgL
1 for TiO2 NPs and bulk TiO2 respectively under
visible light only (non-UV-A regime) (p < 0.05) (Table 1).
Table 1
EC50 for cellular growth of C. reinhardtii and P. tricornutum under UV-A regime and
non-UV-A regime (visible light only) exposed to TiO2 NPs and bulk TiO2.
EC50 of growth inhibition (mg$L
1)
UV-A þ Visible light Visible light only
TiO2 NPs Bulk TiO2 TiO2 NPs Bulk TiO2
C. reinhardtii 2.30 ± 1.76 1.35 ± 3.06 551.7 ± 163.79 423.70 ± 18.74
P. tricornutum 1.98 ± 0.09 6.58 ± 0.67 132.0 ± 7.0 185.0 ± 26.0
P. tricornutum was observed to be more sensitive than C. reinhardtii
under both exposure conditions and with both forms of TiO2 ma-
terial (p < 0.05).
3.4. Effective quantum yield of PII
With respect to EC50 values calculated for the effective quantum
yield of photosynthetic energy conversion in PSII (Fig. 1), it was not
possible to calculate this parameter under UV-A conditions,
(0.1e10 mg L
1) since the highest concentrations used did not reach
50% inhibition and no clear dose-response trend was found for
either microalgae. For C. reinhardtii, under the non-UV-A regime,
50% inhibition was not reached, and predicted EC50 values were
462 ± 147 and 487 ± 105 mg L
1 for TiO2 NPs and TiO2 bulk
respectively. In the case of P. tricornutum those values were
145 ± 27 and 167 ± 26 mg L
1 for TiO2 NPs and bulk TiO2,
respectively, showing a clearer dose-response dependent
cytotoxicity.
Taking together the results for both freshwater and marine
microalgae in function of time, at 6 h from the start of the experi-
ments, inhibition of quantum yield was higher than at 24, 48 or
72 h. Neither of the species (marine and freshwater) showed sig-
nificant differences between treatments (NPs and TiO2 bulk) under
UV-A and non-UV-A regimes (p > 0.05).
3.5. Reactive oxygen species (ROS) and membrane damage
endpoints
Fig. 2 shows the percentage of cells with ROS signals in relation
to the total number of cells for both microalgae. Results for TiO2 NPs
and bulk TiO2 are compared under both regimes, UV-A radiation
and visible light only. Under non-UV-A conditions, C. reinhardtii did
not show great differences with respect to ROS production. In the
case of P. tricornutum, under the same exposure conditions, treat-
ments showed clear differences (p < 0.05) with respect to the
controls. There is also a dose-response trend in the case of the bulk
TiO2, which is not so clear for the TiO2 NPs treatments. In any case,
when treatments (TiO2 NPs and bulk TiO2) are compared, NPs al-
ways provoke greater intracellular production of ROS.
Under the UV-A regime, concentrations of 2 mg L
1 for each
treatment (TiO2 NPs and bulk TiO2) were compared with controls
for both microalgae species. In this case, for both species, the bulk
TiO2 treatment showed no difference from the controls, but TiO2
NPs clearly provoke greater production of intracellular ROS. This
difference is quite clear in the case of P. tricornutum at 72 h
(3.3± 1.6% of affected cells for controls, and 59 ± 6 for TiO2 NPs).
With respect to membrane cell damage (Fig. 2), results under
the non-UV-A regime for both microalgae showed significant dif-
ferences between controls and TiO2 NPs, and between both treat-
ments (TiO2 NPs and bulk) (p < 0.05). Differences in function of
interaction between exposure time with concentration were only
found for P. tricornutum (Data analyzed by short cases GLM,
Table SI4). As can be noted in Fig. 2, membrane cell damage, for the
non-UV-A regime, is clearly concentration-dependent. Under UV-A,
 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48 43

Fig. 1. EC50 for effective quantum yield for C. reinhardtii and P. tricornutum under UV-A regime and non-UV-A regime (visible light only) exposed to TiO2 NPs and bulk TiO2.

both C. reinhardtii and P. tricornutum showed differences in mem-
brane cell damage between controls and TiO2 NPs, and between
TiO2 NPs and bulk TiO2 (p < 0.05) at 72 h; C. reinhardtii showed
18± 1% of damaged cells and P. tricornutum showed 34± 2%, when
exposed to 2 mg$L-1 of TiO2 NPs (control values for the two species
under the same conditions were 7± 2% and 1.4± 0.2%, respectively).
3.6. Quantification of intracellular and extracellular TiO2 NPs
Plotted in Fig. 3 is the concentration in the different fractions at
different time intervals (6, 24 and 48 h). At 6 h from the start of the
experiment, the concentrations of TiO2 NPs in the supernatant were
86± 0.4% and 69± 3% of the total fraction for C. reinhardtii and
P. tricornutum, respectively (the initial concentration of TiO2 NPs
was 0.1 mg L
1). The first wash - of the three washes performed in
each sample - removed 5± 2% and 17 ± 1% of the TiO2 NPs for
C. reinhardtii and P. tricornutum, respectively. The second wash
removed 6.5± 0.4% of TiO2 NPs for P. tricornutum, but no further Ti
was removed for C. reinhardtii. After three consecutive washes no
further Ti was removed from cellular surfaces in either case.
However, after 24 h incubation, different results were obtained.
The concentrations of Ti from TiO2 NPs measured in supernatants
44 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48

Fig. 2. Percentage of cells containing measurable ROS and cell membrane-damaged (in relation to total number of cells) for C. reinhardtii and P. tricornutum under UV-A regime
(panel A) and non-UV-A regime (visible light only, panel B) exposed to TiO2 NPs and bulk TiO2.

before washings were lower than values after 6 h, with 50± 3% and
30± 0.7% of the total fraction of TiO2 NPs being removed for
C. reindhardtii and P. tricornutum, respectively. Nevertheless, the
first wash removed more Ti when compared with the samples after
6 h (35± 0.8% for C. reinhardtii and 30± 1% for P. tricornutum).
After 48 h, the concentration of Ti in the supernatant was again
higher, at 60± 3% for C. reinhardtii and 47± 2% for P. tricornutum. But
the percentage of the total fraction of Ti removed by the first wash
was lower, at 18± 0.1% for C. reindhartii and 18± 1% for
P. tricornutum. Intracellular Ti (measured in pellet) was also lower
at 0.07± 0.5% for C. reinhardtii and 6± 1% for P. tricornutum
(Table SI5).
3.7. Quantification of exo-polymeric substances
Concentrations of exo-polymers (EPS) in supernatants were
measured after 72 h of incubation, for both TiO2 NPs and bulk TiO2
(Fig. 4). Greater production of EPS was observed at high
 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48 45

Fig. 3. Concentration of TiO2 NPs in different compartments (supernatants of consecutive centrifugations and in pellets) after 6, 24 and 48 h for C. reinhardtii and P. tricornutum, for
an initial NP concentration of 0.1 mgL
1.

Fig. 4. Exo-polymeric substances released by C. reinhardtii and P. tricornutum, exposed to TiO2 NPs and bulk TiO2 at different concentrations (0.1, 1, 10, 100, 200 and 400 mgL
1), after
72 h exposure.

concentrations of both TiO2 NPs and bulk TiO2. Production of EPS exposure to bulk TiO2, EPS production for the same species at the
was greater in cells treated with TiO2 NPs than with bulk TiO2 same concentration was 4.3 ± 2.7$10
5 mg of EPS$ 104 cells (con-
(p < 0.05). For instance, C. reinhardtii, exposed to TiO2 NPs at trols produced 2 ± 0.1$10
5 mg$104 cells after the same exposure
10 mg L
1, produced 23 ± 1.2$10
5 mg of EPS$104 cells; while for time). At the same concentration and after 72 h of incubation,
46 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48
P. tricornutum produced 62 ± 4.3, 20.1 ± 0.9 and 7.8 ± 0.9$10
5 mg
of EPS$104 cells for exposure to TiO2 NPs, to bulk TiO2 and in con-
trols, respectively.
4. Discussion
4.1. Behavior of particles in different culture media
The aggregation or agglomeration behavior in solution of TiO2
NPs and bulk TiO2 can affect the availability and the form of
interaction with microalgae, and can provoke toxic effects. The
most important processes in relation to TiO2 NPs are homoag-
glomeration by NPs with other NPs, and heteroagglomeration by
NPs with cells (Lin et al., 2014). These processes are controlled by
the physicochemical properties of NPs, (size and surface charge),
and by solution conditions (ionic strength, pH, and concentration of
natural organic matter) (Jiang et al., 2009).
4.2. Importance of radiation in toxicological test with
photocatalytic substances
Controls for each of the cytotoxic responses for each condition
(UV-A and visible light regime) are essential for studying the effect
of UV radiation on the microalgae population. In the general GLM
model, which takes into account all factors, significant differences
for microalgae population growth and percentage of ROS were
observed between the UV-A and visible light regime controls.
However, in the single GLM devised exclusively for conditions and
culture media, statistically significant differences in all cytotoxic
responses were reported when the interaction between treatment
and concentration was considered.
In general, UV-A exposure increases the toxicity of TiO2 to
microalgae. The toxicity of TiO2 under UV-A radiation is due to the
anatase form of Ti, which is more photo-reactive than the rutile
form (Sayes et al., 2006; Uchino et al., 2002). The use of commercial
formulations including anatase is increasing, as photocatalytic
properties of this form are often required in new applications.
According to our results, at least one of the mechanisms
responsible for the toxicity of TiO2 (in both forms, as NPs or as bulk
material) could be the generation of reactive oxygen species (ROS)
inside the cells. This production is increased by exposure to UV-A
radiation. In this respect, when active photocatalytic compounds
(such as TiO2) are involved in toxicity tests, classical approaches
could be missing part of the toxicity for not considering ROS species
generates by tested substances in the culture media. In this study
the results of EC50 of growth inhibition under the UV-A regime
applied were two orders of magnitude smaller than the result with
no UV-A regime, calculated by other authors (Dalai et al., 2013; Lee
and An, 2013). Recent research demonstrated different drifts in the
phytoplankton population when the microalgae community was
exposed to photocatalytic compounds under different scenarios
(UV radiation and solar radiation without UV radiation) (Sendra
et al., 2017).
Heteroagglomeration (NPs-Cells) related to ionic strength in the
culture media.
In bioassays carried out in a regime of light without UV-A, other
cytotoxicity mechanisms must be relevant. Interaction between the
cell surface and the TiO2 NP may be the first and most important
interaction between NPs and cells. Hydrogen bonding and/or other
polar interactions could also contribute to the interaction between
a NP and a cell (EU, 2011). In other studies, anatase in crystalline
form showed much stronger interactions with cells than the rutile
form of TiO2, which confirms the greater toxicity of this chemical
form (Braydich-Stolle et al., 2009; Ji et al., 2011). Furthermore, TiO2
NPs present a greater surface area and smaller size of particles,
weighted by surface, than the bulk form, so the surface available for
interaction between cells and NPs is greater than that between cells
and the bulk material. Under a regime without UV-A, the marine
microalgae showed greater sensitivity than the freshwater species.
This may be due to the effect caused by salt ions, which would act to
compress the electro-double layer of NPs and cells, and thus
impede the electrostatic repulsion force between them, facilitating
the heteroagglomeration of NPs and cells (Ma et al., 2015). Ac-
cording to Ma et al. (2015), homoagglomeration of anatase NPs
assessed by individual settling experiments were practically the
same when the IS value was in the range between 50 and 500 mM;
however, between 5 and 50 mM the differences were significant. In
our media the IS value was 30 and 700 mM for freshwater and
saltwater media, respectively. This could be the reason for the
similar agglomerate size measured in both media, although the size
was significantly different in comparison with that in ultrapure
water (IS ¼ 0 mM). Heteroagglomeration of anatase NPs and cells
studied in co-settling experiments with different IS values, showed
that heteroagglomeration was greater when the IS value of the
media was 500 mM than it was when the IS value was 50 mM (Ma
et al., 2015). This could explain the greater sensitivity of the marine
microalgae species compared with the freshwater species selected.
Nevertheless, concentrations which imply a reduction of 50% of the
parameters measured are, in general, far removed from those found
in realistic scenarios, according to results reported by other authors
(Velzeboer et al., 2008; Warheit et al., 2007).
4.3. Importance of microalgae species in toxicological test with TiO2
NPs
Results obtained for a single species are often of limited
comparability with another species from a different taxonomic
group. For our study, a Chlorophyceae found in freshwater media
and a Bacillariophyceae found in marine environments were
selected as representative species from the most important taxo-
nomic groups of microalgae in their respective biogenesis. More
comparative studies of the sensitivity of similar species in both
media need to be undertaken. C. reindhartii cells present a glyco-
protein cell wall, while P. tricornutum cells are covered by a silica
frustule. Contact between the surrounding media and the cyto-
plasm across the space between the valves could be easier than
contact between the media and cytoplasm of Chlorophyceae across
the cellulose cell wall. Piccapietra et al. (2012), studying the accu-
mulation of silver from Ag NPs, demonstrated that metal accumu-
lation is greater in mutants of C. reindhartii lacking a cell wall when
compared with wild-type cells (Piccapietra et al., 2012).
It has been demonstrated previously that TiO2 NPs are more
toxic to microalgae than TiO2 in bulk form (Aruoja et al., 2009).
Differences in EC50 found in the literature can be attributed to
differences in the stability and reactivity of NPs and differences in
the experimental matrix, exposure media and concentration
(Handy et al., 2012). The present study has demonstrated, for both
NPs and bulk material, a dose-dependent but non time-dependent
reduction of effective quantum yield, and increases in ROS pro-
duction, membrane damage and bioaccumulation. Additionally,
increased production of EPS by cells has been demonstrated in the
presence of TiO2 NPs and bulk, pointing to possible mechanisms of
detoxification (Nielsen et al., 2008). EPS excreted by cells may
inhibit the attachment of NPs to cells through steric repulsion or
may facilitate NP-cell agglomeration by bridging between NPs and
cells through adsorption on the surface of NPs (Khan et al., 2011).
EPS could also trigger flocculation of NPs, and thus fewer NPs would
be bioavailable to microalgae, reducing their accumulation or
changing their distribution inside the cells (Dong et al., 2007; Miao
et al., 2009; Soldo et al., 2005). This could be the reason for the
 M. Sendra et al. / Environmental Pollution 227 (2017) 39e48
increased Ti concentration in the supernatant and wash fractions
after 48 h compared with that after 24 h: there may be greater
adsorption between the TiO2 NPs and the released exopolymer
particles, and this process could reduce the reactivity of the NPs and
the resulting oxidative stress, leading to decreased levels of ROS
(Miao et al., 2009). On the other hand, EPS could adsorb NPs onto
the surface of cells. Phaeodactylum tricornutum produced more EPS
than Chlamydomonas reinhardtii (even in the controls), and thus
another possibility to consider is that the interaction between NPs
and cells producing agglomeration may be facilitated by the pres-
ence of EPS surrounding the cell. Thus, the role of these EPS in the
toxicity of NPs to microalgae is still not well elucidated. The
reduced internalization of NPs by cells over time may also be
explained by detoxification mechanisms; therefore these micro-
algae showed tolerance at certain doses of this emergent pollutant
(Maeda and Sakaguchi, 1990).
5. Conclusions
For microalgae the toxicity mechanisms for TiO2, in NP and bulk
form, are related to the interaction between nanoparticles and cell
surface. The marine microalgae, P. tricornutum, is more sensitive to
TiO2 than the freshwater C. reinhdartii. Effective quantum yield was
the most sensitive toxicity parameter, with the most rapid time of
response. The characteristics of the exposure media are relevant for
the TiO2 NPs assay, especially ionic strength, which affects both
homo- and heteroagglomeration: a higher IS can impede the
internalization processes and resulting toxicity. EPS are involved
both in the protection mechanism and in heteroagglomeration
processes; however, further studies are necessary to clarify their
role.
Exposure to UV-A radiation is not usually taken into account in
classical (i.e. standard) bioassays, but the effect of this variable
needs to be measured when photocatalytic substances, such as
TiO2, are tested, because the sensitivity of organisms can be two
orders of magnitude greater. However, the primary mechanism of
toxicity revealed in bioassays performed under visible light seems
to be the interaction between the NPs and the cell surface, resulting
in cell membrane damage.
Acknowledgements
This research has been funded by the Junta de Andalucía
(PE2011-RNM-7812 project and FQM-110 group) and the Spanish
National Research Plan (CTM2012-38720-C03-03) andFEDER
fundings (MAT2013-40823-R). I would like to thank Dra. Catalina
Ferna
ndez and Eugenia Zuasti for the support in DLS analysis. We
also thank the SC-ICYT of Cadiz University (UCA) for use of its
Electron Microscopy division facilities.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.envpol.2017.04.053.
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