Accepted Manuscript

Title: Ecotoxicological studies of micro- and nanosizedbarium titanate on aquatic photosynthetic microorganisms

Author: Hudson C. Polonini Humberto M. Brandao NadiaR.B. Raposo Ludovic Mouton Claude Yepremian Alain CouteRoberta Brayner

PII: S0166-445X(14)00164-7DOI: http://dx.doi.org/doi:10.1016/j.aquatox.2014.05.005Reference: AQTOX 3842

To appear in: Aquatic Toxicology

Received date: 10-3-2014Revised date: 28-4-2014Accepted date: 6-5-2014

Please cite this article as: Polonini, H.C., Brandao, H.M., Raposo, N.R.B., Mouton, L.,Yepremian, C., Coute, A., Brayner, R.,Ecotoxicological studies of micro- and nanosizedbarium titanate on aquatic photosynthetic microorganisms, Aquatic Toxicology (2014),http://dx.doi.org/10.1016/j.aquatox.2014.05.005

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Ecotoxicological studies of micro- and nanosized barium titanate on aquatic

photosynthetic microorganisms

Hudson C. Polonini †

, Humberto M. Brandão‡, Nádia R. B. Raposo

†, Ludovic

Mouton§, Claude Yéprémian¥, Alain Couté¥, Roberta Brayner*

§

†Universidade Federal de Juiz de Fora, Núcleo de Pesquisa e Inovação em Ciências da

Saúde (NUPICS), Rua José Lourenço Kelmer, s/n, 36036-900, Juiz de Fora, Brasil

‡Empresa Brasileira de Pesquisa Agropecuária (Embrapa Gado de Leite), 36038-330,

Juiz de Fora, Brasil

§Université Paris Diderot, Sorbonne Paris Cité, Interfaces, Traitements, Organisation

et Dynamique des Systèmes (ITODYS), UMR 7086, CNRS, 15 rue Jean de Baïf, F-

75205 Paris Cedex 13, France

¥ Muséum National d’Histoire Naturelle, Département RDDM, USM 505, 57 rue

Cuvier, F-75005 Paris, France

Tel.: +33 1 57 27 87 64. Fax: +33 1.57.27.72.63

E-mail: roberta.brayner@univ-paris-diderot.fr

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INTRODUCTION

The interaction between live organisms and micro- or nanosized materials has become

a current focus in toxicology – or nanotoxicology, as it’s been currently called

(Dukhin et al., 2010). This is due, to a large extent, to the fact that smaller particles

can be more toxic than bulk materials with the same composition, mostly because of

their increased specific surface area and reactivity (Panf et al., 2012). However, a

thorough understanding of the threats these materials pose to the environment is not

completely available yet (Brayner et al., 2010).

Nanosized barium titanate (BT) has gained momentum lately in several fields, being a

promising future biological nanocarrier for proteins (Ciofani et al., 2010a), as an

enhancer of the uptake of low molecular weight drugs such as doxorubin (Ciofani et

al., 2010b), as a biomarker, through the bioconjugation of its nanocrystals with

immunoglobulin G (IgG) antibodies for imaging probes (Hsieh et al., 2010), as well

as in the design of bone graft material (Ball et al., 2013). In the meantime, in vivo

long-term biocompatibility experiments and studies on its impact on the environment

have not yet been performed, to the best of the authors’ knowledge. Therefore, it is a

material with high future biomedical applications, but with scarce safety data.

For these investigations, physico-chemistry is paramount when trying to understand

the fate and behavior of the particles on the environment (Brayner et al., 2010), to the

extent that characteristics such as size, state of dispersion, surface charge, shape,

chemical composition, surface area, and surface chemistry play important roles in the

uptake and distribution of the particles within live organisms (Oberdörster et al.,

2005a, 2005b; Powers et al., 2006). Also, it is paramount that the particle

characterization is performed in relevant media (Jiang et al., 2009), i.e., those where

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the ecotoxicological experiments will take place, wherein potential physicochemical

changes (e.g., agglomeration/aggregation and surface charge variation) can occur with

the particles in different solutions – which, in turn, can have direct impact on the

toxicological responses (Powers et al., 2007). Since BT presents a perovskite

structure, it is expected to show thermodynamic instability and reactivity in aqueous

environment (Lee, 1998), one of the reasons why not only a natural culture media was

used in this study, but also a synthetic one, with a high content of nutrients. Actually,

different dispersions of BT either in aqueous or in non-aqueous media have already

been studied (Khastgir et al., 2001; Paik et al., 1998; Bergstrm et al., 1998; Lee, 1999;

Lee, 1998; Wang et al., 2000), but none of them covered all of the characterizations

listed here or were performed in biorelevant media for toxicological assays.

In this study, we used two different test organisms: a colonial cyanobacteria

(Anabaena flos-aquae) and a flagellated unicellular euglenoid (Euglena gracilis),

which are quite different morphologically and metabolically. This can be helpful to

provide a spectrum of actions of the particles, although both are representatives of the

first aquatic trophic level (producers). The use of primary producers as biological

indicators is important because they are situated at the base of the food chain and any

change in the dynamics of their communities can affect higher trophic levels of the

ecosystem. Typically, they are also quite sensitive to changes in the environment and

have relatively short life cycles, which allows the observation of toxic effects in

several generations (Cleuvers and Weyers, 2003; Costa et al., 2008). Yet, the aquatic

toxicity model was chosen because these ecosystems are the main enclosures of

contaminants, whether they are coming from direct waste into water bodies through

the discharge of effluents or released into the air or deposited in the soil (Kendall et

al., 2001).

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Thus, the aims of the present work are: (i) to assess BT toxicity and its mechanisms in

two photosynthetic organisms (A. flos-aquae and E. gracilis); (ii) to study and

correlate the physicochemical properties of BT with its toxic profile; (iii) to compare

the BT behavior and the toxic profile in synthetic (Bold’s Basal, BB, or Mineral

Medium, MM) and natural culture media (Seine River Water, SRW); and (iv) to

address whether size is an issue in toxicity of BT particles.

MATERIAL AND METHODS

The subjects of the study: BT particles and test organisms

BT characterization

Two different lots of commercial BT powders were obtained from Sigma-Aldrich,

namely BT MP (barium titanate microparticle) (CAT n. 338842, lot MKBD3182V,

<2μm) and BT NP (barium titanate nanoparticle) (CAT n. 467634, lot MKBF7837V,

<100nm and cubic crystalline phase).

The X-ray diffraction (XRD) patterns of the powders were recorded with a X’pert Pro

diffractometer (PANalytical), equipped with a multichannel X’celerator detector, and

using the Co Kα radiation (= 1.790307 Å), in the 2θ range 5°-120°, with a scan step of

0.05° for 5s. The sample holder used was a Si monocrystal. Morphological

observation of powders by scanning electron microscopy (SEM) was done using a

Zeiss Supra 40 microscope equipped with an in-lens detector. Low excitation voltage

(2.5 kV) and a small working distance (3 mm) were used, so that the charging effects

were minimal to the point that the metallization of powders was not necessary, and

then true features were not masked. Transmission electron microscopy (TEM) images

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were obtained using a JEOL 100CX-II microscope operating with an accelerating

voltage of 100 kV. The specimens were prepared by ultrasonically dispersing the

powders in ethanol prior to deposition on SEM mounts and carbon-coated TEM grids.

The specific surface area (Sg) and pore size were measured by the collection of

nitrogen (N2) adsorption–desorption isotherms on a Gemini V 2380 system (Mic

America, Inc.) at 77 K after the sample had been dried at 160 °C for 1 h. The

Brunauer–Emmett–Teller (BET) surface area was calculated from the linear part of

the BET plot. The pore size distribution was obtained from a QUANTACHROME-

Autoscan 33 mercury porosimeter.

The hydrodynamic size and the surface charge/interparticle forces (zeta potential, ζ)

of the dispersions of BT powders (100 μg mL–1, or 4.28 × 10–7 M, – in BB medium

and SRW) were characterized using a ZetaSizer Nano ZS (Malvern Instruments Inc.)

utilizing dynamic light scattering (DLS) and electrophoretic light scattering (ELS),

respectively. Effects of pH variation in the media (3, 5, 7, 9, 12, adjusted with HNO3

or NaOH in different molarities) were evaluated, as well as the degree of aggregation

as a function of time (0, 24, 48, 72 and 96h). The dispersions were sonicated (100W,

5 min) daily before analysis in order to disperse agglomerates and so that it could be

inferred that increased hydrodynamic sizes are due to aggregation. Response surfaces

were built up in order to evaluate the roles each factor plays at the aggregation state or

the surface charge of the BT dispersions. A 52 factorial experimental design (two

factors, time and pH, with 5 levels of variations each) was used according to

Montgomery (2012) and Polonini et al (2011), which provide the theoretical

background for the graphical representation used (see Supplementary Material). The

factors and their levels (codified levels in parenthesis, and real ones outside them)

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were X1: time [(–1) 0h, (–0.5) 24h, (0) 48h, (0.5) 72h, (1) 96h] and X2: pH [(–0.88) 3,

(–0.44) 5, (0) 7, (0,44) 9, (0.88) 12].

The degree of dissolution of the BT powders as a function of time (1, 2, 4, 8, 24, 48,

72 and 96h) within the media (BB medium and SRW) was evaluated following a

protocol proposed by Sivry et al. (2013). Briefly, from a stock solution (100 μg mL–1,

final volume = 100 mL) prepared at time 0, aliquots (3.5 mL) were withdrawn at the

specified time intervals and ultra-filtred using 3 kDa filters (Microsep Advance

Centrifugal Device, Pall Corporation), placed in a centrifuge (EBA 8, Hettich) for 1h,

and then added with 50 μL of saturated HNO3 (with no trace of Ba2+). All solutions

were immediately frozen until elemental analysis (Ba2+) was performed by

inductively coupled plasma optical emission spectrometry (ICP-OES) (iCAP 6200,

Thermo Scientific). Detection limit was set as 1.0 ppb. The remaining aqueous

dispersion (72 mL) was centrifuged at 20,000 × g (Sorvall Lynx 6000, Thermo

Scientific) and the media was completely evaporated in an oven at 50°C. The dry

residues were then analyzed by XRD and X-ray photoelectron spectroscopy (XPS)

(Thermo VG Escalab 250, using Al Kα of 1486.6 eV, 15 kV, 150 W) for evaluation of

the chemical/surface changes that might possibly have occurred.

All reagents were analytical grade, and ultrapure water (18.2 MΩ cm resistivity at 25

°C and < 10 ppb total organic carbon) was obtained with an Elga Pure-Lab Classic

UV.

Cell cultures

Anabaena flos-aquae planktonic prokaryotic cyanobacteria, strain ALCP B24, and

Euglena gracilis eukaryotic euglenoid came from the Muséum National d’Histoire

Naturelle (MNHN) Culture Collection.

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A. flos-aquae was grown in 275 mL (= 75 cm2) Erlenmeyer flasks with air-permeable

stoppers, in (i) sterile BB medium (Table S1) with pH adjusted to 7.0 using 1 M

NaOH and buffered with 3.5 mM phosphate buffer solution or (ii) SRW (Table S2)

(measured pH = 8.01), at a controlled temperature of 20.0 ± 0.5°C and a luminosity of

30-60 μmol m–2 s–1 photosynthetic photon flux (PPF), under ambient CO2 conditions.

E. gracilis was grown in 250 mL Erlenmeyer flasks, in (i) sterile mineral medium

(MM) (Table S3) with pH adjusted to 4.0 using 1 M HCl or (ii) SRW (measured pH =

8.01), at a controlled temperature of 20.0 ± 0.5°C and a luminosity of 70-100 μmol m–

2 s–1 photosynthetic photon flux (PPF), under ambient CO2 conditions.

SRW, representative of a highly anthropized watershed, was collected near the

University of Paris 7 – Diderot, Paris, France (GPS: 48.831039°N, 2.381709°E). The

sample was immediately filtered after collection through a 0.22 μm acetate membrane

(Millipore) under vacuum to remove contaminants and microorganisms and stored in

pre-cleaned, acid-washed polyethylene bottles, at 4°C until analysis.

Toxicological assessment

Stock suspensions containing 1000 μg mL–1 by weight of BT powders were obtained

by sonicating aliquots of 10 mg of BT MP or BT NP in 10 mL SRW, BB or MM for

10 min at 200W (VWR, USA). The sonication was used to break micrometric

aggregates and stabilize the suspensions. Aliquots of these suspensions were then

added to the batch cultures (exponential growth organisms, prepared 3 days before

starting the test at a concentration of 5.0 × 105 cells mL–1) to obtain the final

concentrations of BT powders corresponding to 1, 25, 50, 75 and 100 μg mL–1 (or

4.28 × 10–8, 1.07 × 10–7, 2.14 × 10–7, 3.22 × 10–7, and 4.28 × 10–7 M, respectively).

Therefore, this study uses higher concentrations than for contamination in natural

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water, aiming to assess the acute toxicity of the materials. The experiment was also

conducted with Ba2+ at 1.5 μg mL–1 (7.28 × 10–6 M) because of the degree of

dissolution of the particles, which did not exceed 1.5%.

The toxic response was evaluated by means of cell counting at 24, 48, 72 and 96h

after the BT powders spiking, as a function of the exposure concentration in

comparison with the average growth of replicate, unexposed control cultures. The cell

counting was performed with bright field microscopy, using the Cellometer Auto X4

(Nexcelom, USA), which simultaneously calculates the percentage of cell viability

(live/dead test, conducted with the trypan blue dye, which selectively colors dead

cells blue).

The average specific growth rate (μ) was calculated as the logarithmic increase in the

cell counting from the following equation for each single flask of controls and

treatments, from days 0 to 4 (calculated section-by-section):

(1)

where is the cell count at time i and is the same parameter at time j.

The percent inhibition of growth rate for each treatment replicate was then calculated

as:

(2)

where is the percent inhibition in average specific growth rate, is the mean

value for average specific growth rate (μ) in the control group, and is the average

specific growth rate for the treatment replicate. Finally, the inhibition percentage for

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each BT powder was plotted against the logarithm of the BT concentrations, and then

the regression analysis was performed to obtain the values of the concentration of the

BT powders suspended in test medium that results in a 50% reduction in the growth

within the exposure period (ErC50).

Assessment of the factors linked to the toxicity

Microscopical observation

The interaction between the test organisms and the treatments (100 μg mL–1, after 72h

of exposure) were observed using SEM and TEM. For SEM, control and treatments

were fixed with a mixture containing 2.5% of glutaraldehyde and 1.0% of picric acid

in phosphate Sörengen Buffer (0.1 M, pH 7.4). Dehydration was then achieved in a

series of ethanol baths (from 50% to100%). The samples were dried with a BAL-TEC

CDP 030 supercritical point dryer after ethanol baths. The images were obtained in a

Zeiss Supra 40 microscope equipped with an in-lens detector. Low excitation voltage

(2.5 kV) and a small working distance (3 mm) were used, so that the charging effects

were minimal to the point that the metallization of powders was not necessary, and

then true features were not masked.

For TEM, control and treatments were fixed with a mixture containing 2.5% of

glutaraldehyde and 1.0% of picric acid in phosphate Sörengen Buffer (0.1 M, pH 7.4).

Post-fixation using osmium tetroxide (OsO4) was conducted and the dehydration was

achieved in a series of ethanol baths (from 50% to 100%). The samples were

processed for flat embedding in a Spurr resin, and then ultrathin sections were made

using a Reicherd-Young Ultracut microtome (Leica). Sections were contrasted with a

4% aqueous uranyl acetate solution and Reynold’s lead citrate before visualization,

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which was performed in a Tecnai 12 operating at 80kV equipped with a 1Kx1K Keen

Viewcamera.

Effect of particles on aggregation and surface charge of test organisms

The hydrodynamic size and the zeta potential of the test organism cultures with added

BT MP or BT NP in BB, MM or SWR (100 μg mL–1, at 0, 24, 48, 72 and 96h of

exposure) were characterized using a ZetaSizer Nano ZS (Malvern Instruments Inc.).

Effect of particles on oxidative stress in test organisms

The activity of superoxide dismutase (SOD), which catalyzes the dismutation of the

superoxide anion (O2.-) into hydrogen peroxide and molecular oxygen, was quantified

in the controls and treatments (1, 50 and 100 μg mL–1, at 24, 48, 72 and 96h of

exposure) using a SOD Assay Kit-WST 19160 (Sigma-Aldrich, Germany). This

allows very convenient SOD assaying by utilizing Dojindo’s highly water-soluble

tetrazolium salt, WST-1 (2-(4-Iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-

2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon

reduction with a superoxide anion. The reduction rate with O2 is linearly related to the

xanthine oxidase (XO) activity, and it is inhibited by SOD. Therefore, the IC50 (50%

inhibition activity of SOD or SOD-like materials) can be colorimetrically determined.

The controls and treatments were incubated at 37°C for 20 min, and then read at 450

nm using an Envision Multilabel Plate Reader (Perkin-Elmer, USA).

Effect of particles on the photosynthetic activity of test organisms

The photosynthetic activities of the controls and treatments (1, 50 and 100 μg mL–1, at

24, 48, 72 and 96h of exposure) were determined by the Pulsed Amplitude

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Modulation (PAM) method, using a Handy PEA (Hansatech, UK) fluorometer. This

method uses the saturation pulse principle, in which a sample is subjected to a short

pulse of light, which saturates the photosystem II (PSII) reaction centers of the active

chlorophyll molecules. This process suppresses photochemical quenching, which

might otherwise reduce the maximum fluorescence yield. A ratio of variable over

maximal fluorescence (Fv/Fm) can then be calculated, which approximates the

potential quantum yield of PSII.

Effect of particles on the test organisms’ mitochondria

Intracellular levels of adenosine-5-triphosphate (ATP) in controls and treatments (1,

50 and 100 μg mL–1, at 24, 48, 72 and 96h of exposure) were quantified using an ATP

Bioluminescent Assay (Sigma-Aldrich). Cell lysis of test organisms was mechanically

achieved in a vortex using glass beads followed by centrifugation (15 min at 2000 ×

g), in order to obtain the free ATP in the supernatant. The assay can determine the

ATP in samples containing 2 × 10–12 to 8 × 10–5 M by quantitative bioluminescence.

ATP is consumed and light is emitted when firefly luciferase catalyzes the oxidation

of D-luciferin. When ATP is the limiting reagent, the light emitted is proportional to

the ATP present into the cells. The relative luminescent units were detected with an

Envision Multilabel Plate Reader equipped with a luminescent optical filter.

Statistical analysis

Statistical analyses were performed using SPSS v.14.0. For comparisons among

control and treatments, ANOVA followed by Tukey’s post-hoc test was conducted for

the variables meeting the criteria of normality (Shapiro-Wilk test, p > 0.05),

homoscedasticity (variance homogeneity, Levene test, p > 0.05) and independence

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(Durbin Watson test, p ~ 2.0). For the variables which violated the assumptions for

ANOVA validity, the non-parametric Kruskal-Wallis test was conducted. Differences

between groups were considered statistically significant when p ≤ 0.05 and

marginally significant when 0.05 < p ≤ 0.1.

RESULTS AND DISCUSSION

Description and characterization of the materials

The two materials used in this study were previously characterized (Supplementary

Material). Briefly, they were identified by XRD as tetragonal BaTiO3 (BT MP) and

cubic BaTiO3 (BT NP), with average crystallite sizes of 172.0 ± 102.4 Å (with 0.19 ±

0.06% of micro strain) and 60.0 ± 16.7 Å (0.10 ± 0.05%), respectively. Using SEM

and TEM, the diameter of the particles could be estimated as 150 nm (BT MP) and 60

nm (BT NP), and the BT MP presented some degree of polydispersion. The BET

surface area was calculated as 3.24 m2 g−1 for BT MP and 16.60 m2 g−1 for BT NP,

the total pore volumes were 0.006 and 0.07 cm3 g−1, respectively, and the mean pore

diameters were 7.34 and 17.46 nm, respectively.

Since BT presents a perovskite structure, it is expected to have some thermodynamic

instability and reactivity in aqueous environment (Lee, 1998), which is evidenced by

the DLS measurements performed (Figure 1). Zeta potential results showed that in

general both particles are negatively charged. BT MP was more stable in BB and M

media, as one can observe a greater number of flat response surfaces, and as the

charge in SRW approached 0 after longer periods in suspension. The same behavior

was observed for BT NP, except in M medium in which the charge surpassed -30 mV

at the beginning of the experiments with lower pHs, but then it tended to approach 0

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over time. Apart from this case, none of the materials showed values high enough to

ensure colloidal stability, which must be at minimum 25 mV (Verwey and Oberveek,

1948; Planchon et al., 2013). This instability can actually be observed as

sedimentation in the suspension after some hours. As for the media, SRW shows a

more pronounced particle instability than the other ones. As surface charge affects the

uptake and the translocation of the particles by live organisms (Hoshino et al., 2004),

it can be expected that the particles will show distinct effects on the microorganisms,

either because of their very nature or due to their different behavior in each medium.

INSERT FIGURE 1 NEAR HERE

Another physicochemical parameter of the particles which is directly related to their

interaction with living systems is their size, since it is related to the degree of

absorption, distribution, metabolism and excretion by live organisms (Jiang et al.,

2009). The hydrodynamic size of the particles was evaluated as a function of pH and

time (Figure 2), in the same manner as performed to zeta potential measurements. The

solutions were sonicated before each measurement, in order to make clear that the

results found were due to aggregation, and not agglomeration (weak van der Waals

bonds). This is important because the aggregation of the particles can influence on the

toxicity profiles for microorganisms, since large aggregates held by strong chemical

bonds do not penetrate cells, and in these cases the effects can be more attributable to

dissolved ions rather than a direct contact (Wang et al., 2010). In BB medium, it

seems that the pH does not influence the aggregate size, unlike the time, which played

a notable role on this parameter, as the aggregate formation seems to increase largely

throughout the days in both particles. The same phenomenon happened in MM and

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SRW, but in this case a decrease in pH also led to an increase of aggregate sizes. For

MM, the BT NP aggregates were bigger than for the BT MP.

INSERT FIGURE 2 NEAR HERE

Finally, the degree of dissolution of the particles was also evaluated, as the release of

ions into the media can account for a different degree and mechanism of toxicity to

the microorganisms. This was assessed by ultrafiltration, followed by the

quantification of Ba2+ leached out from the particles into the media at the pH in which

the toxicological assessment was conducted (7.0 for BB, 4.0 for MM and 8.01 for

SRW) (Figure 3).

INSERT FIGURE 3 NEAR HERE

BT NP released more Ba2+ in the media than BT MP – 1,132 ppb after 96h in BB

medium, 6.5 times more than BT MP in the same medium; 1,258 ppb after 96h in

MM against 345 ppb from BT MP, 3.6 times more; and 1,368 ppb in SRW after the

same period, 3.4 times greater than BT MP. SRW had a more pronounced effect in

the phenomenon, once it dissolved BT MP 2.9 times more than the BB medium and

1.15 more than MM; and BT NP 1.2 times greater in BB and 1.08 in MM. Thus, such

differences can be attributed to the compositional differences between the two media

(see Supplementary Material) rather than to their pH, since SRW has only a slightly

higher pH than BB medium – although the difference is big between MM and the

others. Yet, the dissolution rates of the powders in BB medium or SRW were not

greater than 1.5%. This is the reason why this was the concentration of free Ba2+ ions

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chosen (1.5 μg mL–1, or 7.28 × 10–6 M) to perform the toxicological assessment,

where it was evaluated whether the released (and consequently penetrated) ions could

be a potential reason for the toxicity.

After the 96-h experiments, the XRD patterns of the remaining powders were

assessed (Figure S1), and no significant difference was observed between media. XPS

analyses of the residues were also performed, and the results can be seen in Table S4.

The Ba/Ti/O ratio is in good agreement with the low solubility verified by ICP-OES.

Carbon (C) is a common impurity in ultrahigh vacuum (UHV), and the signals around

285 eV (hydrocarbon) are ordinarily used as internal standard to reference other

elements when charging occurs (López et al., 1999). However, its presence in more

than one state is an indicative of the formation of BaCO3 (also observed by Ba

binding energy at ~780 eV). The deconvolution of the C 1s peak gave a peak of

carbonate in a BaCO3 surface at 288-289 eV. BaCO3 may be a byproduct of the

BaTiO3 synthesis, whichever route is used, or derived from the tendency of CO2 to

adsorb on perovskite surfaces, due to dipole-dipole interaction between the

ferroelectric BaTiO3 crystal and the polar CO2 molecule (López et al., 1999). As this

component is absent in the traces, one can expect that the carbonate may be formed as

a discrete second phase (~2%) rather than as a surface film.

INSERT TABLE 1 NEAR HERE

Thus, the main conclusions from the characterization studies are that BT NP presents

a higher reactivity than BT MP, and that SRW is a more instable than the other media,

thereby contributing to such behavior, according to the zeta potential and the results

of the dissolution experiments. Yet, the time the particles remain in suspension led to

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an increment in aggregate formation. Once again, these different behaviors can be

expected to lead to different effects on the growth of model organisms.

Ecotoxicological assessments

The two test organisms used in this study are quite different morphologically and

metabolically, which can be helpful to provide a spectrum of actions of the particles.

The cyanobacteria A. flos-aquae is composed of one linear series of vegetative cells,

and generally possesses a lot of thick-wall heterocysts dispersed all along the

trichome (Brayner et al., 2010, 2011). E. gracilis, in turn, shows a remarkable

metabolic plasticity: it can behave as either autotrophic and photosynthetic (when

grown in the presence of light, and it is green) or as heterotrophic (when grown in the

dark, and it is colorless) (Einicker-Lamas et al., 2002). In our case, it is expected that

they show an autotrophic behavior. Yet, they can perform endocytosis and they have

the ability to move by using a flagellum, a long whiplike structure (Brayner et al.,

2010, 2011).

In the first stage, the direct effects of growth on these two organisms were assessed, i.

e., their growth after addition of BT MP and BT NP (Figure 4). A decrease on growth

was observed in both organisms, but this effect was more pronounced in E. gracilis,

for which both BT MP and BT NP led to significant growth inhibition (p<0.05) in all

concentrations tested, in both media, and since the first cell counting, after 24h of

contact. However, BT MP showed a more toxic effect than BT NP, especially in

SRW. For A. flos-aquae, the toxic effects were significant only after 72h of exposure,

and for the largest concentrations of the materials (after 96h: ≥ 75 μg mL–1 for BT NP

and =100 μg mL–1 for BT MP, in BB; and ≥ 75 μg mL–1 for both materials in SRW).

On the other hand, Ba2+ did not cause a significant reduction of growth (none for A.

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flos-aquae and only after 96 h for E. gracilis), an observation contrary to other studies

that show that the toxicity of agglomerated nanoparticles to microorganisms can be

explained by dissolved metal species (Wu et al., 2010). Although barium is known to

be reactive with air and water (Fromm, 2013), its toxicity is lower than that of most

heavy metals (López-Róldan et al., 2013), and previous studies confirm that this

alkaline earth metal is toxic only at very high concentrations (Lamb et al., 2013;

Monteiro et al., 2011; Kopittke et al., 2011). Yet, it can also be observed that the

inhibition of growth was slightly more pronounced within the SRW than within the

artificial media, BB or MM.

INSERT FIGURE 4 NEAR HERE

However, the test performed to assess the viability of the organisms showed

discrepant results, at least for A. flos-aquae (Figure 5). While no significant growth

inhibition was observed in this organism, the viability of its membrane has been

observed to be damaged in all tested concentrations for both substances from the first

day of observation, in BB medium or SRW – although the differences were more

pronounced within the latter, as one can see in Figure 4. The same was observed with

E. gracilis, an expected result, departing from the growth inhibition observed.

INSERT FIGURE 5 NEAR HERE

The assessment of cell membrane integrity was performed using the dye exclusion

method with trypan blue. It is based on the principle that intact membranes exclude

certain dyes, such as trypan blue (Strober, 2001). Normally, the viability results can

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be correlated to the mortality of cells – dead cells would not exclude the dye, and then

they appear blue in photonic microscope. In our case, however, this can be true to E.

gracilis, but not A. flos-aquae. For the latter, we can suppose that the cells have lost

some of its membrane functionality, which does not necessarily represent a fatal

event. The cell probably developed some unknown mechanism to survive in this

adverse situation.

Calculating the ErC50, was limited by the concentrations used. Using the results, some

theoretical values were above the concentrations used. For A. flos-aquae: 1271.5 μg

mL–1 for BT MP and 1208.5 μg mL–1 for BT NP, in BB; and 1047.3 μg mL–1 for BT

MP and 1117.4 μg mL–1 for BT NP, in SRW. For E. gracilis: 878.1 μg mL–1 for BT

MP and 627.8 μg mL–1 for BT NP, in MM; and 63.9 μg mL–1 for BT MP and 414.5

μg mL–1 for BT NP, in SRW. This is in accordance with a higher sensitivity of E.

gracilis, especially for BT MP. For a more realistic result, new assessments with

higher concentrations should be performed. However, the highest concentration used

in this study is already higher than what is expected to be found in nature during an

acute exposure, i.e., nanoparticle concentration around 10– 9 M (Planchon et al, 2013).

Despite the ErC50 being greater than the concentrations used (except for BT MP in E.

gracilis), toxic effects on growth and viability did occur, and this fact may have

different origins, based on what is already known from the literature. Here we

consider that the two main possible mechanisms of the observed toxicity are: (i) a

direct contact of BT particles with the cell wall (Gogniat et al., 2006), linked to a

theoretical penetration of the smallest particles and/or released ions (Planchon et al.,

2013); and/or (ii) an indirect effect through the generation of reactive oxygen species

(ROS) (Brayner et al., 2006; Chae et al., 2011). Thus, we performed different assays

to assess which is the possible one involved in BT toxicity to the model organisms.

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Assessment of the factors linked to the toxicity

SEM and TEM analysis of the microorganisms (Figures 6 and 7) allow a direct

visualization of the morphological changes they suffer upon contact with the particles.

For A. flos-aquae exposed to BT particles for 72h in a concentration of 100 μg mL–1,

the morphology of the cells when observing them by SEM did not change with the

treatments, although they are a little more elongated when grown in BB medium than

when in SRW. Aggregates of cells and particles were observed (Figure 6I). The main

aspect to be pointed out is that the particles were found around the cell wall composed

by the polysaccharides, and the TEM images confirm that they did not penetrate the

cells.

INSERT FIGURES 6 AND 7 NEAR HERE

In the case of E. gracilis, the behavior of the cells under the same conditions was

completely different. The SEM images of E. gracilis controls, in both media, show

well-defined elongated cells possessing whip-like flagella. The images of cells from

treatments, however, differ greatly from these images. One can see that in both

treatments, BT MP and BT NP, cells are smaller, with a whipping-top shape, and with

no visible flagellum. This probably happens because of the endocytosis of the BT

particles and also of other substances contained into the media. The cell performed

endocytosis to such a degree that the membrane could not resist and actually ruptured,

as evidenced in Figure 7I. Thin sections of the microorganism in TEM show two main

features: nanoparticles internalized by endocytosis (Figure 7I) and a highly increased

production of bodies of paramylon, a carbohydrate similar to starch (Figures 7H, K

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and L). The chlorophyll found in E. gracilis chloroplasts can be of both a and b-types,

and they participate in the synthesis of carbohydrates, which are stored in euglenoids

cells in an unusual form known as paramylon, a B-1,3 polymer of glucose. These

paramylon bodies can be visualized as colorless or white rigid rods, and when they

achieve a concentration, which is too high for the cell to handle, the cell wall explodes

(Brayner et al., 2011). In this sense, SEM and TEM were complimentary to each

other, and both confirm that the high endocytosis of the particles led to an

overproduction of paramylon, which caused the explosion, and, consequently, the

death of the cells. This is an explanation to the fact that the cell count and the viability

in E. gracilis are low, but in A. flos-aquae only the viability is low – due to the fact

that the cells either died or lost their function, but they were still in their usual form

when the counting was performed.

Some correlation can be found between these observations and the results of surface

charge. For A. flos-aquae, the zeta potential of the cells is negative (Figure 8), as well

as that of the particles themselves (Figure 1). This is in agreement with an

electrostatic repulsion between microorganisms-particles, which hampers sorption. As

for E. gracilis, its surface charge is also negative, but very close to 0, and so this

repulsive force would not be strong enough to prevent the endocytosis. Regarding the

hydrodynamic size of the cell suspensions in absence or presence of particles, no

significant difference could be observed, but the remarkable growth of the size in A.

flos-aquae cultures – which is expected since it is a filamentary microorganism.

INSERT FIGURE 8 NEAR HERE

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The effect the particles in causing oxidative stress was also assessed (Figure 9). The

SOD activity was increased in A. flos-aquae growth in BB medium when exposed to

BT MP at 100 μg mL–1 and to BT NP in all concentrations tested, after 72h. When

grown in SRW, BT NP at 100 μg mL–1 significantly increased the SOD activity

already from the first day of assay, followed by the lower concentrations being

effective in the following days. BT MP at 100 μg mL–1 altered SOD activity after 48h

of exposure. For E. gracilis, both particles in all concentrations except for BT MP at 1

μg mL–1 exerted some effect on SOD activity. SOD activity plays a very important

role for the cell viability, since it protects them from the action of reactive oxygen

species (ROS), and so a higher SOD activity can be a reflection of a higher

exposure/production of ROS, which is associated with the activation of cell apoptosis

via the mitochondria (Cheng et al., 2011).

INSERT FIGURE 9 NEAR HERE

The increased ROS production is also known to affect photosynthesis (Figure 10)

(Rodea-Palomares et al., 2012). Indeed, the photosynthetic activity was found to be

reduced in the cells exposed to the particles. In A. flos-aquae, it differed from the

control in BB medium from the first hour assessed, for both particles; and in SRW,

the activity decreased after 48h of exposure. For E. gracilis, the effect was noticed

from the first hour for both media.

INSERT FIGURE 10 NEAR HERE

Together with the SOD activity, another measurement of the mitochondrial activity of

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the cells is the intracellular ATP content (Figure 11). The mitochondria are directly

related to the cell viability, since they are responsible not only for the ATP

production, but also for the ROS production and for the release of proteins, which

control the apoptosis (Pereira et al., 2014). In A. flos-aquae, the content of ATP

started to decline after 48h of exposure – for BB medium, it happened initially for the

higher concentrations, but after 72h all concentrations it exerted some effect, as it was

observed in SRW. For E. gracilis, the effects were seen from the first day. The

decline in ATP content may reflect a decrease in the mitochondrial activity, and so the

BT particles led to disturbances in the energy metabolism of the organisms. For the

eukaryotic Euglena cells, the strong endocytotic activity also plays a role in the

decline of ATPs, once the endocytosis process takes energy from ATP, synthesis

which is strongly associated with the transmembrane potential (Dukhin et al., 2001).

INSERT FIGURE 11 NEAR HERE

Thus, comparing the viability tests with SEM/TEM images and with SOD, ATP and

photosynthetic activity analysis, we may see that the BT particles, but not the released

Ba2+ ions, were toxic for both microorganisms at the prevailing exposure

concentrations. The cells of the first one died after endocytosing the particles in such

a quantity that the membrane was ruptured. The cells of the latter experienced toxic

effects caused by indirect contact with the particles. In this case, aggregates of

particles surrounding the cells were observed, and one can expect that this has caused

a decrease in the availability of the necessary nutrients for the gtowth of

microorganisms because of their predicted transport by the cell membrane was

disturbed – and even the light availability could be threatened by these large metallic

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aggregates – which to some degree could have subjected the cells to stress, causing

the increase in ROS production (Rogers et al., 2010; Xin et al., 2010).

Conclusion

Taking all the data together, the main conclusions are: (i) both BT MP (~170nm,

tetragonal) abd BT NP (~ 60nm, face-centered cubic) were negative and easily

aggregatable (unstable) in all aqueous media studied, with a release rate of Ba2+ ions

not exceeding 1.5%; (ii) BT has a low toxic effect on the growth of A. flos-aquae, but

both particles affect cell viability from the lowest concentration tested, which is

caused by indirect effect on oxidative stress in cells; (iii) BT has a statistically

significant toxic effect on cell growth and viability of E. gracilis from the lowest

concentration tested (1 μg mL–1), related to the effect of endocytosed particles in such

an amount that led to a rupture of their membranes – the effect was more pronounced

for BT MP; (iv) BT was able to stress all test organisms, which was evidenced by the

increase in SOD activity, and decrease in photosynthetic efficiency and in the

intracellular ATP levels; (v) the behavior of BT in synthetic and natural media culture

was different, with the most pronounced toxic effects when growth is given in ARS -

in this case a worse physiological state of microorganisms in ARS can occur in

organisms already stressed by the particles and cause a lower cell resistance\,

probably linked to a shortage of nutrients or even a synergistic effect with a

contaminant of the river; (vi) the size of BT does not influence the effects produced

on the growth of micro-organisms, with 95% confidence.

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FIGURES CAPTIONS

Figure 1. Zeta potential of (A) BT MP in BB medium, (B) BT MP in MM, (C) BT

MP in SRW, (D) BT NP in BB medium, (E) BT NP in MM, and (F) BT NP in SRW.

X1: time [(–1) 0h, (–0.5) 24h, (0) 48h, (0.5) 72h, (1) 96h] and X2: pH [(–0.88) 3, (–

0.44) 5, (0) 7, (0,44) 9, (0.88) 12].

Figure 2. Hydrodynamic size of (A) BT MP in BB medium, (B) BT MP in MM, (C)

BT MP in SRW, (D) BT NP in BB medium, (E) BT NP in MM, and (F) BT NP in

SRW.

X1: time [(–1) 0h, (–0.5) 24h, (0) 48h, (0.5) 72h, (1) 96h] and X2: pH [(–0.88) 3, (–

0.44) 5, (0) 7, (0,44) 9, (0.88) 12].

Figure 3. Dissolution rate of BT powders in terms of Ba2+ leached out as a function of

time.

Figure 4. Cell count of A. flos-aquae and E. gracilis as a function of concentration

and time of exposure to barium titanate particles.

Figure 5. A. flos-aquae and E. gracilis viability as a function of concentration and

time of exposure to barium titanate particles.

Figure 6. SEM micrographs of A. flos-aquae in BB medium [(A) control, (B)

exposed to BT MP, (C) exposed to BT NP] and in SRW [(D) control, (E) exposed to

BT MP, (F) exposed to BT NP]. TEM micrographs of C. vulgaris thin sections in BB

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medium [(G) control, (H) exposed to BT MP, (I) exposed to BT NP] and in SRW [(J)

control, (K) exposed to BT MP, (L) exposed to BT NP].

Figure 7. SEM micrographs of E. gracilis in BB medium [(A) control, (B) exposed to

BT MP, (C) exposed to BT NP] and in SRW [(D) control, (E) exposed to BT MP, (F)

exposed to BT NP]. TEM micrographs of C. vulgaris thin sections in BB medium

[(G) control, (H) exposed to BT MP, (I) exposed to BT NP] and in SRW [(J) control,

(K) exposed to BT MP, (L) exposed to BT NP].

Figure 8. Effect of barium titanate particles on aggregation (hydrodynamic size) and

surface charge (zeta potential) of A. flos-aquae and E. gracilis as a function of time of

exposure.

Figure 9. Superoxide dismutase (SOD) activity of A. flos-aquae and E. gracilis as a

function of concentration and time of exposure to barium titanate particles.

Figure 10. Photosynthetic activity of A. flos-aquae and E. gracilis as a function of

concentration and time of exposure to barium titanate particles.

Figure 11. Adenosine-5-triphosphate (ATP) content of A. flos-aquae and E. gracilis.

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Table of Contents Graphic

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Highlights Barium titanate has toxic effects on A.flos‐aquaeand E.gracilis. Barium titanate particles affected cell growth and viability. Barium titanate particles increased SOD activity and decreased ATP levels and photosynthesis. Effects were more pronounced in Seine River water than in artificial growth medium. Size does not seem to be an issue in BT particle toxicity.

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Figure1

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Figure2

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Figure3

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Figure4a

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Figure4c

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Figure5a

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Figure5b

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Figure6

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Figure7

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Figure8a

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Figure9a

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Figure10a

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Figure11a

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Figure11b