Potency of (doped) rare earth oxide particles and their constituent metals to inhibit algal growth and induce direct toxic effects

Science of the Total Environment 593–594 (2017) 478–486

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Potency of (doped) rare earth oxide particles and their constituent metals to inhibit algal growth and induce direct toxic effects Elise Joonas a,b,⁎, Villem Aruoja a, Kalle Olli b, Guttorm Syvertsen-Wiig c, Heiki Vija a, Anne Kahru a,d a

Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia Institute of Ecology and Earth Sciences, University of Tartu, Lai 40, 51005 Tartu, Estonia Ceramic Powder Technology AS, Kvenildmyra 6, 7093 Tiller, Norway d Estonian Academy of Sciences, Kohtu 6, 10130 Tallinn, Estonia b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Rare earth elements (REE) are emerging contaminants in aquatic environments. • Toxicity of REEs and (doped) rare earth oxides (REO) to algae was evaluated. • (Doped) REOs entrapped algae into agglomerates. • REEs inhibited algal growth by removing phosphate from the algal medium (OECD201). • REE proved directly toxic to algae (a ‘spot’ test), 24 h MBC b 1 mg/L.

a r t i c l e

i n f o

Article history: Received 13 January 2017 Received in revised form 15 March 2017 Accepted 20 March 2017 Available online xxxx Editor: D. Barcelo Keywords: Lanthanides Phytoplankton Agglomeration Nanoparticles Hazard evaluation

a b s t r a c t Use of rare earth elements (REEs) has increased rapidly in recent decades due to technological advances. It has been accompanied by recurring rare earth element anomalies in water bodies. In this work we (i) studied the effects of eight novel doped and one non-doped rare earth oxide (REO) particles (aimed to be used in solid oxide fuel cells and gas separation membranes) on algae, (ii) quantified the individual adverse effects of the elements that constitute the (doped) REO particles and (iii) attempted to find a discernible pattern to relate REO particle physicochemical characteristics to algal growth inhibitory properties. Green algae Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) were used as a test species in two different formats: a standard OECD201 algal growth inhibition assay and the algal viability assay (a ‘spot test’) that avoids nutrient removal effects. In the 24 h ‘spot’ test that demonstrated direct toxicity, algae were not viable at REE concentrations above 1 mg metal/L. 72-hour algal growth inhibition EC50 values for four REE salts (Ce, Gd, La, Pr) were between 1.2 and 1.4 mg/L, whereas the EC50 for REO particles ranged from 1 to 98 mg/L. The growth inhibition of REEs was presumably the result of nutrient sequestration from the algal growth medium. The adverse effects of REO particles were at least in part due to the entrapment of algae within particle agglomerates. Adverse effects due to the dissolution of constituent elements from (doped) REO particles and the size or specific surface area of particles were excluded, except for La2NiO4. However, the structure of the particles and/or the varying effects of oxide composition might have played a role in the observed effects. As the production rates of these REO particles are negligible compared to other forms of REEs, there is presumably no acute risk for aquatic unicellular algae. © 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia. E-mail address: elise.joonas@kbfi.ee (E. Joonas).

http://dx.doi.org/10.1016/j.scitotenv.2017.03.184 0048-9697/© 2017 Elsevier B.V. All rights reserved.

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

1. Introduction The 17 elements called rare earth elements (REEs) are vital for environmentally friendly energy production, electronics, and military applications, where they are mainly used as magnets, metal alloys, polishing agents and catalysts (EPA, 2012; UNCTAD, 2014). Currently, REEs are globally produced at a rate of 124,000 tonnes per year (USGS, 2016), which is two orders of magnitude lower than copper or aluminium (Schüler et al., 2011). However, the production of industrially relevant REEs is anticipated to increase rapidly in the coming decades (Alonso et al., 2012). Coupled with virtually no recycling of REEs (Binnemans et al., 2013) and the difficulty in replacing REEs due to their unique properties (UNCTAD, 2014), their input into the environment is unavoidable and accelerating. Anthropogenic increase in gadolinium levels (in the ng/L range) in water bodies has been widely observed (Bau and Dulski, 1996; Hatje et al., 2016; Klaver et al., 2014; Kulaksiz and Bau, 2011) and in recent years, anomalously high lanthanum and samarium concentrations in the river Rhine have also been reported (Klaver et al., 2014; Kulaksiz and Bau, 2013). The concentrations in natural water bodies due to anthropogenic lanthanum input have even been shown to exceed the threshold of toxicity, with concentrations reaching 49 mg/L in industrial effluents (Kulaksiz and Bau, 2011). The adverse effects of REEs to algae have been previously studied, with the main mechanism of adverse effects found to be removal of nutrients from algal medium (Gonzalez et al., 2014; González et al., 2015; Herrmann et al., 2016; Tai et al., 2010). The available toxicity information for rare earth oxide particles to algae mostly concerns CeO2 (Angel et al., 2015; Rodea-Palomares et al., 2012; Rogers et al., 2010; Röhder et al., 2014). In the current work, we studied the patterns of toxic effects of nine oxide materials, each consisting of at least one rare earth element in combination with other elements, referred to as rare earth element oxides (REOs): Ce0.9Gd0.1O2, LaFeO3, Gd0.97CoO3, LaCoO3, (La0.5Sr0.5)0.99MnO3, Ce0.8Pr0.2O2, (La0.6Sr0.4)0.95CoO3, La2NiO4 and CeO2. The application areas of these materials include solid oxide fuel cells, batteries, gas separation membranes and catalysts. The global market for similar advanced and nanoscale ceramic powders is projected to grow from nearly $14.6 billion in 2016 to $22.3 billion by 2021 with a compound annual growth rate of 8.9% (BCC Research, 2016). These novel materials pose a unique challenge to interpreting their toxic effects: they consist of a mix of metals with varying individual toxicity, and their inhibitory effect may derive from both dissolution of their constituent elements and particle-specific effects. As these oxides can be tailor-made to fit customers' needs, there is an acute need to identify the underlying causes of the potential adverse effects of these particles in order to produce materials that are ‘benign by design’. To gain an understanding of the adverse effects of these particles, we conducted both standard OECD201 algal growth inhibition tests as well as algal viability assays (‘spot tests’; Suppi et al., 2015) with the model algal species Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata). Algal growth inhibition testing is relevant due to the role of algae as primary producers in the aquatic environment and their short life-spans, which allow for rapid detection of adverse effects over several generations. Algae are often the most sensitive organisms to various toxicants (Bondarenko et al., 2016; Kahru and Dubourguier, 2010; Rojíčková-Padrtová et al., 1998). To discern patterns in REO particle toxicity, we quantified physicochemical characteristics of the particles, such as size, specific surface area and dissolution. 2. Materials and methods 2.1. Synthesis and characterization of rare earth oxide particles The particles analysed in this study were synthesised by a technique referred to as spray pyrolysis (Messing et al., 1993), producing homogeneous sub-micron particles of relatively high surface area and narrow size distribution. Briefly, precursors were dissolved in water, stabilised

479

and thermo-gravimetrically standardised prior to being mixed in stoichiometric ratios according to the final oxide nominal composition. The mixed solution was atomised and sprayed through a furnace where the liquid evaporates and hollow spheres of metal oxide are formed (Mokkelbost et al., 2007). The as-prepared powders were subsequently calcined at temperatures 500–1000 °C. The hollow spheres consisted of many smaller primary particles and the spheres were broken up during conventional wet ball milling, followed by drying and sieving. The Brunauer–Emmett–Teller (BET) method was used to determine the specific surface area (SSA) of the samples and for calculation of primary particle size of the materials. N2 adsorption-desorption measurements were carried out at 77 K using a Micrometer Tristar 3000 apparatus. The powders were placed in a glass tube and allowed to degas overnight in vacuum at 250 °C prior to measurements. Data were obtained by introducing or removing a known quantity of adsorbing gas in or out of a sample cell containing the solid adsorbent maintained at a constant liquid nitrogen temperature. The primary particle size was derived assuming spherical particles and using the equation dBET = 6 / (ρp·SA), where dBET, ρp and SA are defined as the average diameter of a spherical particle, theoretical density, and the measured specific surface area, respectively. To conduct scanning electron microscopy (SEM), the powders were put onto a carbon tape, coated with carbon (Agar Turbo Carbon Coater, 4.8 eV, 6 s) and analysed by SEM (Zeiss Supra 55 VP, in-lens detector, 15 keV). For transmission electron microscopy (TEM), the powders were dispersed in deionized water at a concentration of 1 g/L and sonicated in the Bioruptor UCD-200-TM-EX ultrasonic bath (Diagenode Inc.), using the high power setting for a total of 10 min in 30 s cycles. A drop of the dispersion was placed on a copper grid and dried in ambient air. TEM images were recorded on a FEI Tecnai G 2 Spirit BioTwin microscope operated at 120 kV. 2.2. Preparation and characterization of REO particle suspensions and REE salt solutions About 20 mg of each REO powder was weighed and mixed with about 20 mL of deionized (DI) water (Milli-Q, Millipore, USA) to yield 1 g/L stock suspensions that were vortexed and sonicated for 3 min before use (40 W, Branson probe sonicator, USA). Hydrodynamic size and ζ-potential of the 100 mg/L REO suspensions in DI water (Table 1) were measured using Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). The soluble metal salts that were tested in addition to the REO particles in the algal assays were as follows: Ni(NO₃)₂·6H₂O (Merck KGaA, purity 99.0%), Co(NO₃)₂·6H₂O (VWR, 98%), Gd(NO3)3·6H2O (Sigma Aldrich, 99.99%), Sr(NO 3 )2 (Honeywell, 100%), Mn(NO 3 )2 ·4H2 O (American Elements, 100%), La(NO3)3·6H2O (Treibacher Industrie AG, ≥ 95–100%), Ce(NO3)3·6H2O (Treibacher Industrie AG, ≥ 95–100%), Fe(NO3)3 (Sigma Aldrich, 99.99%), Pr(NO3)3·6H2O (Sigma Aldrich, 99.99%). 2.3. Analysis of solubility of REO particles To mimic the dissolution of REO particles in the environment of the conducted bioassays, REO suspensions (100 mg/L) in DI water were prepared and incubated at the same light and temperature conditions as for the bioassays (algal growth inhibition test and a ‘spot test’). After 72 h, the concentration of metals in the samples was quantified using the total reflection X-ray fluorescence spectrometer (TRXF) Picofox S2 (Bruker AXS Microanalysis GmbH). For this 1 mL of each suspension was pipetted into 1.5 mL Eppendorf tube and centrifuged at 20000g for 30 min. After centrifugation, 50 μL of the supernatant was carefully removed, mixed with gallium (Ga) internal standard in the ratio of 1:1 and 5 μL of this mixture was pipetted onto a quartz carrier disc.

480

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

Table 1 Physicochemical properties of the (doped) rare earth oxide particles. Particle size (nm)

Ce0.9Gd0.1O2 LaFeO3 Gd0.97CoO3 LaCoO3 (La0.5Sr0.5)0.99MnO3 CeO2 Ce0.8Pr0.2O2 (La0.6Sr0.4)0.95CoO3 La2NiO4 a

Abbreviation SSA (m2/g) BET

DLSa

ζ-Potential (mV)a

CGO LFO GCO LCO LSM CeO CPO LSC LNO

280 177 166 285 194 172 147 160 266

−16,6 16,2 18,8 −17,5 −1.8 8,5 16,6 22,7 −6.6

31.1 7.2 3.4 1.4 7 22 36.1 15 3

27 126 230 590 137 38 23 65 284

2.6. Algal viability assay (a ‘spot test’)

In deionized water, after centrifugation at 160g for 10 min to remove large agglomerates.

Concentration of metals was quantified with Spectra software (Bruker AXS Microanalysis GmbH). 2.4. Calculating toxic units of the dissolved fraction from REO particles To understand the mechanisms of REO particle toxicity, it is necessary to take into consideration that these oxides are composed of 1–3 metals. To express the effect of the fraction dissolving from REOs as a chemical mixture, additive toxic units for each REO were calculated. The calculation was based on the European Commission (2012) assessment of toxic units of mixtures, according to which the toxic unit (TU) is the ratio between the exposure level (dissolution from the REO) of an individual element and its toxicity endpoint (EC50 of the corresponding metal salt). The toxic units for a mixture (TUm) were calculated as the sum of individual TUs according to Eqs. 1 and 2 TU ¼

exposure ðmg=LÞ EC50 ðmg=LÞ

TUm ¼ TU1 þ TU2 þ TUn

the EC50 calculations, results from all test repetitions were taken into account by normalizing the biomass values compared to the control (sample biomass divided by average control biomass). All of the ANOVA and linear regression calculations in this paper were conducted in R (R Core Team, 2015).

ð1Þ ð2Þ

2.5. Algal growth inhibition assay The algal growth inhibition assay adhered to OECD guideline 201 and is described in detail in Aruoja et al. (2009). The R. subcapitata stock culture for inoculation was obtained from the commercial test system Algal Toxkit F (MicroBioTests Inc., Nazareth, Belgium). The number of algal cells in the inoculum was determined by counting under light microscope in a Neubauer haemocytometer. Exponentially growing algae were exposed to various concentrations of REO suspensions/ soluble metal salts and incubated at 24 ± 1 °C for 72 h in standard 20 mL glass scintillation vials each containing 5 mL of algal growth medium (OECD, 2011). The vials were illuminated from below with Philips TL-D 38 W aquarelle fluorescent tubes. All samples were run in triplicate with n/10 controls distributed evenly on the plates. Algal biomass was measured at least every 24 h by quantifying the fluorescence of algal pigment extract. For that 50 μL of culture samples were transferred to a 96-well black polypropylene plate (Greiner Bio-One), 200 μL of ethanol was added to each sample and the plate was shaken for 3 h in the dark. Thereafter the fluorescence was measured with a microplate fluorometer (excitation 440 nm, emission 670 nm; Fluoroscan Ascent, Thermo Fischer Scientific Inc., USA). Phase contrast as well as fluorescence micrographs were taken with Olympus CX41 microscope equipped with DP71 camera. The toxicity values (72 h EC50) for the algal growth inhibition and their confidence intervals were determined from dose-response curves by the REGTOX software (Vindimian, 2001) using the Log-normal model. All concentrations used for EC50 calculations were nominal. In

The ‘spot test’ described in detail by Suppi et al. (2015) was used to assess the ability of the toxicant exposed algae to form colonies on toxicant-free nutrient agar after 24 h exposure to the tested chemicals in deionized (DI) water. Prior to the assay, the exponentially growing algal culture to be used in the test was washed twice by centrifugation in deionized water (3500 g, 10 min, 25 °C). Then the cell density was determined via counting in a Neubauer haemocytometer and adjusted to 1 ∗ 106 cells/mL. 100 μL of the algal suspension (final cell density in experiment 0.5 ∗ 106 cells/mL) was added to 100 μL of varying concentrations of particles/salts in DI water or MOPS (3-(Nmorpholino)propanesulfonic acid); 10 mM; pH = 7). Previously, a 750 mg/L (3.57 mM) concentration of MOPS was shown not to adversely affect algae (De Schamphelaere et al., 2004). Decimal dilution series of the REO particle suspensions and REE solutions in the range of 1–100 mg/L (nominal concentrations) were tested in duplicate. Algae were exposed to the substances in 96-well microplates (non-tissue culture treated, BD Falcon) at 25 °C for 24 h under illumination comparable to the algal growth inhibition test. After 24 h of exposure, 5 μL of the cell suspension from each microplate well was pipetted as a ‘spot’ onto agarized algal growth medium. The inoculated Petri dishes were incubated at ~25 °C in constant light for several days until visible green ‘spots’ appeared on agar surface. Minimal biocidal concentration (MBC) of the tested REOs/REEs was determined as the lowest tested nominal concentration of a chemical which completely inhibited the ability of the cells to form visible colonies after plating onto toxicant-free nutrient agar-plates. 3,5-DCP was used as a positive control. Each experiment was repeated twice. 2.7. REE precipitation by algal medium components In order to assess the effect of soluble REE salts in precipitation of algal medium components (algal nutrients), the dissolved REE concentration was measured in differently composed media after 72 h incubation at the conditions of the algal growth inhibition test (see above). The nominal REE concentration at the beginning of the experiment was 7 mg/L. The 5 studied media were as follows: deionized water, standard OECD201 algal medium, OECD201 medium without phosphates, OECD201 without carbonates and OECD without phosphates and carbonates. At 72 h, the samples were centrifuged (30 min, 16,060g) to pellet non-dissolved fractions and the supernatants were analysed using TRXF as described before. 3. Results and discussion 3.1. Physicochemical characteristics of rare earth oxides The estimated size of the REO particles based on BET (Table 1) was generally in agreement with SEM and TEM observations (Fig. S1). In some cases (LaCoO3, Gd0.97CoO3, LaFeO3, La2NiO4) the TEM images did not reveal separate particles but agglomerates apparently consisting of smaller particles (Fig. S1). Particles of Ce0.9Gd0.1O2, Ce0.8Pr0.2O2, CeO2 and (La0.6Sr0.4)0.95CoO3 can be considered nanoparticles as they contain a fraction smaller than 100 nm. The particles of (La0.5Sr0.5)0.99MnO3 were not suitable for TEM due to their magnetic properties. The particles agglomerated in water suspensions and the hydrodynamic size and zeta potential could be reliably measured only after removal of large agglomerates by centrifugation. The low suspension stability was reflected in the zeta potential values that remained well above

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

− 30 mV and below 30 mV. For some oxides, such as LaCoO3, and Gd0,97CoO3 the hydrodynamic size appeared smaller than the primary size according to BET. Apparently the size distribution of these materials contained a smaller fraction that was detected after removal of agglomerates. 3.2. Algal growth inhibitory effects of constituent metals of (doped) REO particles The soluble salts of the nine constituent metals of the (doped) REOs had variable inhibitive effects in the 72-hour R. subcapitata growth inhibition assay, with EC50 values spanning almost 3 orders of magnitude (Table 2). The toxicity order of first-transition metals (period 4 elements Sc through Zn in the periodic table) coincides with the IrvingWillams series (Irving and Williams, 1953) of the relative stabilities of divalent first transition metal complexes. More stable metal-ligand complex formation correlated positively with their effect to algae (both series Mn2+ b Co2+ b Ni2+), as was also reported by Fujiwara et al. (2008). As the Fe ion we tested was trivalent and its EC50 values were affected by pH, its effects could not be explained through this series. Previously, Aruoja et al. (2015) found the toxicity (as EC50) of divalent first transition elements to be: Fe2+ (EC50 = 23.14 mg/L) b Mn2+ (14.32 mg/L) b Co2 + (0.10 mg/L) b Cu2 + (0.02 mg/L) = Zn2 + (0.02 mg/L), which deviates somewhat from the Irving-Williams series. The four REEs (La, Ce, Pr, Gd) showed comparable growth inhibitory effects, with EC50 values around 1.3 mg/L, which is in line with previous findings, where the effects of REEs on algae have been found to be

481

similar (Gonzalez et al., 2014; Tai et al., 2010). In contrast, no adverse effects were found at similar concentrations by Řezanka et al. (2016), who even demonstrated an enhancing effect of four REEs (LA, Gd, Nd, Ce) to algal growth at limiting light levels. It has also been shown that the effect of REEs on algal growth is comparable for all REEs and therefore additive for the whole group (Tai et al., 2010). REEs show overlapping geochemical and toxicological behaviour resulting from their similar ionic radii and their being mostly trivalent (Kulaksiz and Bau, 2013). Thus, it may be more appropriate to assess the environmental concentrations of REEs as a group. Starting at concentrations around 0.4 mg REE/L all the REE salts formed agglomerates entrapping algal cells (Fig. 1) that precipitated to the bottom of the experimental vessel. Promotion of agglomeration of Chlorella kesslerii cells by La and Eu was also reported by Fujiwara et al. (2008). In samples where La and Pr concentrations exceeded 10 mg/L, needle-like crystals formed around algal cells (Fig. 1). Li et al. (2014) also observed the formation of similar structures on the surface of a human myeloid cell line in the presence of La2O3, Gd2O3 and Eu2O3 nanoparticles. Previously, similar precipitates of REEs have been shown to form with carbonates, phosphates, hydroxides, fluorides and oxalates (Evans, 1990). We observed a significant decrease (p b 0.05) in the concentrations of REEs in the medium that contained carbonates and phosphates (Fig. 2) compared to concentrations in deionized water after a 72-hour incubation at the conditions of the growth inhibition test without algae. Therefore, there was a decrease in both the concentration of dissolved REEs and algal nutrients in the medium. As algae require

Table 2 EC50 values of the constituent metals of rare earth oxides derived from our experiments (marked ‘Current study’) compared with data from other sources on the same chemical elements in their dissolved forms. The reported EC50 values have mostly been derived from algal growth inhibition experiments. Element

Compound

Value (mg metal/L)

End pointa

Algal species

Reference

Sr

Sr(NO3)2 SrCI2 Sr(NO3)2 Mn(NO3)2 MnCl2 MnSO4 MnCl2 Fe(NO3)3 FeSO4 FeCl3 FeCl2 Pr(NO3)3 PrCl3 La(NO3)3 LaCI3 La(NO3)3 LaCI3 LaCI3 Ce(NO3)3 CeCI3 CeCI3 Ce(NO3)3 Ce(NO3)3 Ce(NO3)3 Ce(NO3)3 Gd(NO3)3 GdCl3 GdCl3 Gd(NO3)3 Co(NO3)2 CoCl2 CoCl2 CoCl2 Co2+ Ni(NO3)2 Ni(NO3)2 Ni(NO3)2

53.9 (44.1–63.9)) N150 N43.3 5.91 (5.12–6.69) 14.32 11 10.1 6.77 (5.06–8.48)b 23.14 6 10.219635 1.36 (1.02–1.7)) 2.4 1.27 (0.96–1.57) 10 43.5 4.05 0.45 1.23 (1.1–1.36) 4 6.3 4.5 0.076 1.05 4.16 1.21 (1.01–1.4) 2.3 3.11 4.68 0.14 (0.12–0.15) 0.10 0.008 6.95 0.6 0.07 (0.05–0.09) 0.017 4.1378847

EC50, 72 h LIC, 3–4 months EC50 72 h EC50, 72 h EC50, 72 h LIC, 3–4 months IC50, 96 h EC50, 72 h EC50 72 h LIC, 3–4 months IC50, 96 h EC50, 72 h LIC, 3–4 months EC50, 72 h LIC, 3–4 months IC50, 96 h LC50, 96 h EC50, 72 h EC50, 72 h LIC, 3–4 months EC50, 72 h EC50, 72 h EC50, 72 h EC50, 2 h, Fv/Fmc LC50, 96 h EC50, 72 h LIC, 3–4 months EC50, 72 h LC50, 96 h EC50, 72 h EC50 72 h LIC, 3–4 months IC50, 96 h EC50, 96 h EC50, 72 h LIC, 3–4 months IC50, 96 h

Raphidocelis subcapitata Chlorella vulgaris Raphidocelis subcapitata Raphidocelis subcapitata Raphidocelis subcapitata Chlorella vulgaris Chlorella kessleri Raphidocelis subcapitata Raphidocelis subcapitata Chlorella vulgaris Chlorella kessleri Raphidocelis subcapitata Chlorella vulgaris Raphidocelis subcapitata Chlorella vulgaris Chlorella kessleri Skeletonema costatum Raphidocelis subcapitata Raphidocelis subcapitata Chlorella vulgaris Raphidocelis subcapitata Chlamydomonas reinhardtii Raphidocelis subcapitata Chlamydomonas reinhardtii Skeletonema costatum Raphidocelis subcapitata Chlorella vulgaris Raphidocelis subcapitata Skeletonema costatum Raphidocelis subcapitata Raphidocelis subcapitata Chlorella vulgaris Chlorella kessleri Chlorella vulgaris Raphidocelis subcapitata Chlorella vulgaris Chlorella kessleri

Current study (de Jong, 1965) (ECHA, 2010) Current study (Aruoja et al., 2015) (de Jong, 1965) (Fujiwara et al., 2008) Current study (Aruoja et al., 2015) (de Jong, 1965) (Fujiwara et al., 2008) Current study (de Jong, 1965) Current study (de Jong, 1965) (Fujiwara et al., 2008) (Tai et al., 2010) (Stauber and Binet, 2000) Current study (de Jong, 1965) (González et al., 2015) (Taylor et al., 2016) (Treibacher Industrie AS, 2013) (Röhder et al., 2014) (Tai et al., 2010) Current study (de Jong, 1965) (González et al., 2015) (Tai et al., 2010) Current study (Aruoja et al., 2015) (de Jong, 1965) (Fujiwara et al., 2008) (Kim et al., 2006) Current study (de Jong, 1965) (Fujiwara et al., 2008)

Mn

Fe

Pr La

Ce

Gd

Co

Ni

a b c

IC50 – 50% inhibitive concentration; LIC - lowest inhibitory concentration; NOEC - no observed effect concentration. Calculated EC50 was pH-dependent. Fv/Fm - maximum potential quantum efficiency of Photosystem II.

482

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

phosphate and carbonate for growth, the removal of these essential nutrients was likely the mechanism behind their growth inhibition, rather than any direct toxic effect, as also demonstrated by Stauber and Binet (2000). Indeed, this attribute of REEs has been exploited to ameliorate eutrophic water bodies by removing excess phosphorous using La-modified bentonite clay (Copetti et al., 2015). However, because REE agglomeration around algal cells was also apparent, the growth inhibitory effect may have been in part due to interactions of the REEs with the algal phospholipid cell membrane. Lipid membrane dephosphorylation by REO ion shedding in acidifying macrophage lysosomes has been suggested as the key mechanism of REO-induced lysosomal damage in TPH-1 cells in vitro (Li et al., 2014) and REEs have been shown to promote the fusion of phospholipid vesicles (Bentz et al., 1988). The concentration of phosphate in the standard OECD201 algal growth medium is 9 μM. At this concentration, all of the phosphate can be removed by REEs at concentrations 1.27, 1.28, 1.29 and 1.44 mg/L for La, Ce, Pr and Gd, respectively. This is very similar to the EC50 values of these substances, further implying a connection between the growth inhibitory effect and phosphate removal from the medium. Even though there is 0.59 mM of carbonate also present in the algal medium, it is likely that REE phosphates formed first, because REEs have higher affinity towards phosphates: REE carbonates are more soluble than phosphates (González et al., 2015). Indeed, La(CO3)3 is used as a phosphate remover for renal disease patients (Fricker, 2006). 3.3. REO particle toxicity The 72 h EC50 values derived from the algal growth inhibition assay (Fig. 3) of the (doped) REOs did not depend on the size of the particle (BET, R2 = 0.08, p = 0.45) nor on the specific surface area of the particle (BET, R2 = 0.03, p = 0.66). As the dissolution of constituent elements from REO particles was limited, the concentrations of constituent metal ions leaching from the particles were quantified from 100 mg REO/L samples after a 72 h incubation and subsequent centrifugation

Fig. 2. Precipitation of phosphates and carbonates from differently composed test media by rare earth elements (REE). Measured concentrations (nominal 7 mg/L) of dissolved rare earth element (REE) ions in different media following 72 h of incubation at the conditions of the OECD 201 test without algae. Media: deionized water (DI), OECD201 medium without carbonate or phosphate (OECD-C-P), OECD201 medium without carbonate (OECD-C), OECD 201 medium without phosphate (OECD-P), standard OECD201 medium (OECD). Error bars indicate 95% confidence intervals.

(Table 3). Dissolution was measured in deionized water in order to more accurately quantify the dissolution of REEs, which would have otherwise been removed by the medium components. Even at this high REO concentration, the dissolved fraction leaching from REOs was too low to independently induce EC50-level toxicity, with the exception of La2NiO4. This is illustrated with the concept of toxic units and toxic units of mixtures (Fig. 4, see Table S1 for calculations for each REO), which serves as a proxy to assessing the cumulative burden of a mixture of chemicals, here the dissolved fraction of the REOs. We see in Fig. 4 that only dissolution from La2NiO4 at 100 mg/L would

Fig. 1. Algal cells were captured into agglomerates in all samples of (doped) rare earth oxides and rare earths in the dissolved form.

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

Fig. 3. 72 h EC50 values (nominal concentration of oxide) calculated from dose-response data of 72 h Raphidocelis subcapitata growth inhibition assay for 9 (doped) rare earth oxide particles. Hazard categories based on EC50: b1 mg/L very toxic, 1-10 mg/L toxic, 10-100 mg/L harmful, N100 mg/L not harmful were applied as described in Kahru and Dubourguier (2010). Error bars signify 95% confidence intervals.

have sufficed to independently cause substantial algal growth inhibitory effects. The concentrations at which inhibitory effects were apparent in whole REOs were always lower than 100 mg/L, implying that the effect of the dissolved fraction would be an even smaller contributor to the overall effect, because there is probably less solubilisation at lower REO particle concentrations. The doped REO particles we experimented with are novel, meaning there is no data on their effects to algae. Toxicity data on the effects of REO nanoparticles to algae are usually limited to CeO2 due to their use as a fuel additive (Schüler et al., 2011). Our CeO2 particles have a primary size of 38 nm (Table 1; Fig. S1) and can therefore be deemed nanoparticles. The EC50 for CeO2 found in our study, 8.2 mg/L (Fig. 3), corresponds well to values found in other studies, which have been between 2.4 and 29.6 mg/L (Angel et al., 2015; Manier et al., 2013; Rodea-Palomares et al., 2011; Rogers et al., 2010). On the contrary, Taylor et al. (2016) found no acute toxic effects at concentrations up to 80 mg/L of a batch of non-commercial polyvinylpyrrolidone-coated CeO2 nanoparticles. When toxic effects were detected in these studies, the dissolved fraction of Ce from nanoparticles did not seem to cause their effect to algae, but rather the agglomeration of algae and particles caused growth inhibition. All of the REO particles in our experiments formed agglomerates with algae (Fig. 1). Rodea-Palomares et al. (2011) speculated the agglomeration to be a consequence of the alteration of the particle charge because of the release of cell material. Röhder et al. (2014) observed agglomeration of algal cells in the presence of CeO2, but could not detect a decline in algal photosynthetic efficiency. This could be due to the endpoints used: algal photosynthetic

483

Fig. 4. Toxic units of the REO particles as chemical mixtures were calculated as the summed ratios of the concentrations of the dissolved fraction from REOs at 100 mg REO/L and the EC50 values of the individual metal salts (Table S1). If the toxic unit exceeds 1 (horizontal line), there are enough bioavailable metal ions to induce an EC50-level toxic effect.

efficiency reflects the physiological state of algal cells. Therefore, the algal cells themselves may have remained unharmed within the agglomerates, but unable to multiply. In our experiments, algal cells were still intact and viable in the agglomerates, as implied by their continued fluorescence (Fig. 1), but the algal biomass of the sample did not increase. Rodea-Palomares et al. (2011) found that CeO2 nanoparticles induced membrane rupture, cytoplasm leakage, and intracellular damage. Rogers et al. (2010) observed concentration-dependent permeability of cell membranes using the DNA-binding fluorescent dye SYTOX® Green and suggested the particles may assert purely mechanical damage to the cell membrane due to the particle surface structures. The immobilization of algae within agglomerates may also hinder metabolism and multiplication of the cells and block access to light and nutrients. However, agglomeration was apparent in all REOs, even though their EC50 values differed nearly 100-fold. Therefore, differences in either REO chemical composition or structure also influenced algal growth. The proximity of algal cells to the REO particles may have also increased the exposure of the cells to bioavailable dissolved metal ions. 3.4. Direct toxicity of REO particles and REEs to algae R. subcapitata: a viability assay REEs tend to precipitate soluble phosphates and carbonates from the algal growth medium resulting in a decrease in nutrients needed for the algal growth (Fig. 2). In order to avoid this type of effects, we tested the

Table 3 Concentrations of dissolved constituent elements (mg/L) from REOs at 100 mg REO/L in deionized water after a 72 h incubation at the conditions of the algal growth inhibition test, but without algae. Measurements were taken from the supernatant of the samples following centrifugation. Ce La2NiO4 (La0.6Sr0.4)0.95CoO3 Ce0.8Pr0.2O2 CeO2 (La0.5Sr0.5)0.99MnO3 LaCoO3 Gd0.97CoO3 LaFeO3 Ce0.9Gd0.1O2

Gd

La

Pr

Fe

0.067 0.127 0.110 0.022

Sr

Mn

0.015

4.473

0.033 0.230 0.009 0.014

0.022 0.102 0.018

Ni 0.198

0.032 0.072

0.058

Co

0.039

0.004

484

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

toxicity of the substances by exposing them to algae in deionized water. It is important to note that due to their strong cell wall, algae R. subcapitata remain viable in deionized water during 24 h despite of the hypotonic conditions (Fig. 5, line CTRL). In this ‘spot test’ the algae were exposed to studied toxicants suspensions for 24 h on a microplate and subsequently incubated on nutrient agar (not containing toxicants), as described in Suppi et al. (2015). As the REE nitrate salts lowered the pH of samples and because deionized water itself can have a pH around 5.5, all of the experiments were conducted in parallel also in MOPS buffer (pH = 7) to account for these effects. As shown in Fig. 5, after exposure of algae to 1 mg/L of soluble REE salts for 24 h, the cells were still able to grow on nutrient agar, although the growth was poor. Therefore the MBC was designated to the next concentration tested, 10 mg/L for all REEs. The colony formation potency in REE exposed algae incubated in MOPS was not remarkably higher than in case of DI, indicating the adverse effect of REE salts was not due to unfavourable pH. Even when accounting for nutrient removal and pH effects, the inhibitory effect remained, meaning a third toxicity mechanism is probably also contributing to the net inhibitory effect. Once again, the effects of REEs were similar, suggesting a joint mode of action across REEs, as for nutrient removal. Interestingly, the effects of REEs on algae were in the same range both in the ‘spot test’ and the 72-hour algal growth inhibition assay. Indeed, because REEs exhibit competitive interactions with H+, inorganic species and major cations, including Ca2+ and Mg2+ (Marang et al., 2008), they have been shown to interfere with calcium metabolism of cells due to similar ionic radii with Ca2+ (Gonzalez et al., 2014). The inhibition of calcium metabolism is shared by REEs (Kulaksiz and Bau, 2013). In algae, REEs replace Ca2+ at some membrane locations, block

cellular Ca metabolism and inhibit cellular responses at high concentrations (Das et al., 1988). However, as the test was conducted in deionized water, which does not contain mineral nutrients, the inhibition of calcium metabolism could have been intracellular. REEs also affect phospholipid membranes, increasing their rigidity, making the surface charge increasingly positive, and, at higher concentrations, promoting agglomeration and membrane fusion (Evans, 1990). In some instances, however, REEs may stimulate algal growth: in Ca-deficiency REEs may help promote algal growth, but in Mn-deficiency, REE addition intensified the adverse effects to the green algae Desmodesmus quadricauda (Goecke et al., 2015). Overall, REO particles were much less inhibitive at the same nominal concentrations compared to REEs, because REE salts dissolve nearly completely in DI, whereas REO particles show minimal dissolution (Table 3). The toxicity of REO particles analysed in the spot test (Fig. 5) was remarkably different from that observed from the OECD201 algal growth inhibition test (Fig. 3), although La2NiO4 was most inhibitory in both assays. For all tested REO particles with the exception of the most inhibitive La2NiO4, the MBC exceeded 10 mg/L. LaFeO3, Gd0.97CoO3 and Ce0.8Pr0.2O2 had MBCs of 100 mg/L, meaning no toxicity was evident at 1 or 10 mg/L. Four REOs (Ce0.9Gd0.1O2, LaCoO3, (La0.5Sr0.5)0.99MnO3, CeO2) showed no visible toxicity in the test. We need to stress that this is a rough estimation of the toxicity, as we tested decimal dilutions of the studied compounds, to be able to cover bigger concentration range suitable for both, REE and REO. In MOPS, all REOs but La2NiO4 showed no discernible toxicity towards algae up to 100 mg/L. This may show that the pH of the medium controls the extent of agglomeration or dissolution from REO particles. In the acidic deionized water in the ‘spot test’, toxicity of the REOs was more evident, whereas at a neutral pH in the MOPS buffer, less toxicity was apparent. A possible explanation for this pattern is the increasing dissolution of metals at a lower pH. In the standard algal growth inhibition assay, at pH 8, the toxicity values of REOs differed from both spot test treatments. In addition to pH effects the presence of organic matter, not added in our bioassays, likely mitigates the effects of REO particles in natural water bodies (Angel et al., 2015; Van Hoecke et al., 2011). Therefore, these aspects need to be considered when assessing the risk posed by REO particles. Overall, the toxicity of REO particles was more pronounced in the 72-hour growth inhibition assay than in the ‘spot test’ (Table S2). Because the inhibition induced by La2NiO4 remained similar in both the growth inhibition test and the ‘spot test’, dissolution of La2NiO4 (Table 3) could have controlled its observed adverse effect. Other REO particles were probably toxic due to particle-specific effects, which may have been evaded to some extent in the ‘spot test’. However, the ‘spot test’ is not completely comparable to the growth inhibition test, because in the 72 h algal growth inhibition assay the particles are exposed to algae longer in conditions of constant shaking. 4. Conclusions

Fig. 5. Colony-forming ability of algae on agar after 24-h exposure to nine rare earth oxides (B) and their constituent rare earth elements (A) in deionized water (DI) and MOPS buffer. The concentrations are nominal, expressed as mg oxide/L in A and mg metal/L in B. See Table 1 for abbreviations of REO particles.

We observed two main mechanisms of inhibition by REEs in two different assays which led to adverse effects in algae. In the 72-hour OECD201 test, nutrient removal from algal medium was the culprit of the observed inhibition. Interference with Ca2+ metabolism and membrane damage likely arose in the spot test, which proved to be a useful assay to decouple direct and indirect inhibitive effects of REEs. Interestingly, the effects of REEs remained similar within the group, no matter which mode of action was dominant. As REEs are a physicochemically homogenous group, and their toxicity mechanisms appear to be similar, it would be relevant to assess the risk they pose to the environment additively for the whole group. The doped REO particles pose a unique hazard because of their mixed composition and multitude of potential effects. They proved to have adverse effects beyond the dissolution from particles, specific surface area or particle size, which are often correlated to toxic action of

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

nanoparticles. The main mechanism of toxicity seemed to be agglomeration of particles around algal cells, which was present in all REO particle samples, even though their 72-hour EC50 values from the algal growth inhibition assay differed nearly 100-fold. This indicates that the composition of the REO particles or their structure additionally influenced the inhibitory effects. These doped REO particles alone constitute a negligible fraction of the total environmental input of REEs, but do add to the already increasing burden. As the high concentrations that we tested are unlikely in the environment, there is little acute risk posed by doped rare earth oxides to algae.

Acknowledgements We extend our gratitude to Raivo Raid (Tartu University) for conducting the TEM microscopy. This research was supported by the Estonian Research Council projects IUT23-5, PUT39 and PUT748. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.03.184.

References Alonso, E., Sherman, A.M., Wallington, T.J., Everson, M.P., Field, F.R., Roth, R., Kirchain, R.E., 2012. Evaluating rare earth element availability: a case with revolutionary demand from clean technologies. Environ. Sci. Technol. 46:3406–3414. http://dx.doi.org/10. 1021/es203518d. Angel, B.M., Vallotton, P., Apte, S.C., 2015. On the mechanism of nanoparticulate CeO2 toxicity to freshwater algae. Aquat. Toxicol. 168:90–97. http://dx.doi.org/10.1016/j. aquatox.2015.09.015. Aruoja, V., Dubourguier, H.C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407:1461–1463. http://dx.doi.org/10.1016/j.scitotenv.2008.10.053. Aruoja, V., Pokhrel, S., Sihtmäe, M., Mortimer, M., Mädler, L., Kahru, A., 2015. Toxicity of 12 metal-based nanoparticles to algae, bacteria and protozoa. Environ. Sci.: Nano 2: 630–644. http://dx.doi.org/10.1039/C5EN00057B. Bau, M., Dulski, P., 1996. Anthropogenic origin of positive gadolinium anomalies in river waters. Earth Planet. Sci. Lett. 143:245–255. http://dx.doi.org/10.1016/0012821X(96)00127-6. BCC Research, 2016. Advanced Ceramics and Nanoceramic Powders. http://www. bccresearch.com/market-research/nanotechnology/advanced-ceramicsnanoceramic-powders-report-nan015h.html. Bentz, J., Alford, D., Cohen, J., Düzgüneş, N., 1988. La3+-induced fusion of phosphatidylserine liposomes. Close approach, intermembrane intermediates, and the electrostatic surface potential. Biophys. J. 53:593–607. http://dx.doi.org/10. 1016/S0006-3495(88)83138-2. Binnemans, K., Jones, P.T., Blanpain, B., Van Gerven, T., Yang, Y., Walton, A., Buchert, M., 2013. Recycling of rare earths: a critical review. J. Clean. Prod. 51:1–22. http://dx. doi.org/10.1016/j.jclepro.2012.12.037. Bondarenko, O.M., Heinlaan, M., Sihtmäe, M., Ivask, A., Kurvet, I., Joonas, E., Jemec, A., Mannerström, M., Heinonen, T., Rekulapelly, R., Singh, S., Zou, J., Pyykkö, I., Drobne, D., Kahru, A., 2016. Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project nanovalid. Nanotoxicology 5390:1–14. http://dx.doi.org/10.1080/17435390.2016.1196251. Copetti, D., Finsterle, K., Marziali, L., Stefani, F., Tartari, G., Douglas, G., Reitzel, K., Spears, B.M., Winfield, I.J., Crosa, G., D'Haese, P., Yasseri, S., Lürling, M., 2015. Eutrophication management in surface waters using lanthanum modified bentonite: a review. Water Res. 97:162–174. http://dx.doi.org/10.1016/j.watres.2015.11.056. Das, T., Sharma, A., Talukder, G., 1988. Effects of lanthanum in cellular systems. Biol. Trace Elem. Res. 18:201–228. http://dx.doi.org/10.1007/BF02917504. de Jong, L.E. den D., 1965. Tolerance of Chlorella vulgaris for metallic and non-metallic ions. Antonie Van Leeuwenhoek 31:301–313. http://dx.doi.org/10.1007/BF02045910. De Schamphelaere, K.A.C., Heijerick, D.G., Janssen, C.R., 2004. Comparison of the effect of different pH buffering techniques on the toxicity of copper and zinc to Daphnia magna and Pseudokirchneriella subcapitata. Ecotoxicology 13:697–705. http://dx. doi.org/10.1007/s10646-003-4429-9. ECHA, 2010. Strontium nitrate. https://www.echa.europa.eu/en/web/guest/registrationdossier/-/registered-dossier/13508/6/2/6/?documentUUID=b6c326a9-c00a-4cc7ba24-c357386ade54 (accessed 11.28.16). EPA, 2012. Rare Earth Elements: A Review of Production, Processing, Recycling, and Associated Environmental Issues. United States Environ. Prot. Agency. https://nepis.epa. gov/Adobe/PDF/P100EUBC.pdf. European Commission, 2012. Toxicity and Assessment of Chemical Mixtures. Eur. Commision Toxic. Assess. Chem. Mix.:pp. 1–50. http://dx.doi.org/10.2772/21444. Evans, C.H., 1990. Biochemistry of the Lanthanides. http://dx.doi.org/10.1007/978-14684-8748-0.

485

Fricker, S.P., 2006. The therapeutic application of lanthanides. Chem. Soc. Rev. 35: 524–533. http://dx.doi.org/10.1039/b509608c. Fujiwara, K., To, Y.M., Kawakami, H., Aoki, M., Tuzuki, M., 2008. Evaluation of metal toxicity in Chlorella kessleri from the perspective of the periodic table. Bull. Chem. Soc. Jpn. 81:478–488. http://dx.doi.org/10.1246/bcsj.81.478. Goecke, F., Jerez, C.G., Zachleder, V., Figueroa, F.L., Bišová, K., Rezanka, T., Vítová, M., 2015. Use of lanthanides to alleviate the effects of metal ion-deficiency in Desmodesmus quadricauda (Sphaeropleales, Chlorophyta). Front. Microbiol. 6:1–12. http://dx.doi. org/10.3389/fmicb.2015.00002. Gonzalez, V., Vignati, D.A.L., Leyval, C., Giamberini, L., 2014. Environmental fate and ecotoxicity of lanthanides: are they a uniform group beyond chemistry? Environ. Int. 71:148–157. http://dx.doi.org/10.1016/j.envint.2014.06.019. González, V., Vignati, D.A.L., Pons, M.-N., Montarges-Pelletier, E., Bojic, C., Giamberini, L., 2015. Lanthanide ecotoxicity: first attempt to measure environmental risk for aquatic organisms. Environ. Pollut. 199:139–147. http://dx.doi.org/10.1016/j.envpol. 2015.01.020. Hatje, V., Bruland, K.W., Flegal, A.R., 2016. Increases in anthropogenic gadolinium anomalies and rare earth element concentrations in San Francisco Bay over a 20 year record. Environ. Sci. Technol. 50:4159–4168. http://dx.doi.org/10.1021/acs.est.5b04322. Herrmann, H., Nolde, J., Berger, S., Heise, S., 2016. Aquatic ecotoxicity of lanthanum - a review and an attempt to derive water and sediment quality criteria. Ecotoxicol. Environ. Saf. 124:213–238. http://dx.doi.org/10.1016/j.ecoenv.2015.09.033. Irving, B.H., Williams, R.J.P., 1953. The stability of transition-metal complexes. J. Chem. Soc. 3192–3210. http://dx.doi.org/10.1039/JR9530003192. Kahru, A., Dubourguier, H.C., 2010. From ecotoxicology to nanoecotoxicology. Toxicology 269:105–119. http://dx.doi.org/10.1016/j.tox.2009.08.016. Kim, J.H., Gibb, H.J., Howe, P.D., 2006. Cobalt and Inorganic Cobalt Compounds. World Health Organization. Klaver, G., Verheul, M., Bakker, I., Petelet-Giraud, E., Négrel, P., 2014. Anthropogenic rare earth element in rivers: gadolinium and lanthanum. Partitioning between the dissolved and particulate phases in the Rhine River and spatial propagation through the Rhine-Meuse Delta (the Netherlands). Appl. Geochem. 47:186–197. http://dx. doi.org/10.1016/j.apgeochem.2014.05.020. Kulaksiz, S., Bau, M., 2011. Rare earth elements in the Rhine River, Germany: first case of anthropogenic lanthanum as a dissolved microcontaminant in the hydrosphere. Environ. Int. 37:973–979. http://dx.doi.org/10.1016/j.envint.2011.02.018. Kulaksiz, S., Bau, M., 2013. Anthropogenic dissolved and colloid/nanoparticle-bound samarium, lanthanum and gadolinium in the Rhine River and the impending destruction of the natural rare earth element distribution in rivers. Earth Planet. Sci. Lett. 362:43–50. http://dx.doi.org/10.1016/j.epsl.2012.11.033. Li, R., Ji, Z., Chang, C., Dunphy, D., Cai, X., 2014. Surface interactions with compartmentalized cellular phosphates explain rare earth oxide nanoparticle hazard and provide opportunities for safer design. ACS Nano 8:1771–1783. http://dx.doi.org/10.1021/nn406166n. Manier, N., Bado-Nilles, A., Delalain, P., Aguerre-Chariol, O., Pandard, P., 2013. Ecotoxicity of non-aged and aged CeO2 nanomaterials towards freshwater microalgae. Environ. Pollut. 180:63–70. http://dx.doi.org/10.1016/j.envpol.2013.04.040. Marang, L., Reiller, P.E., Eidner, S., Kumke, M.U., Benedetti, M.F., 2008. Combining spectroscopic and potentiometric approaches to characterize competitive binding to humic substances. Environ. Sci. Technol. 42:5094–5098. http://dx.doi.org/10.1021/es702858p. Messing, G.L., Zhang, S.-C., Jayanthi, G.V., 1993. Ceramic powder synthesis by spray pyrolysis. J. Am. Ceram. Soc. 76:2707–2726. http://dx.doi.org/10.1111/j.1151-2916.1993. tb04007.x. Mokkelbost, T., Andersen, Ø., Strøm, R.A., Wiik, K., Grande, T., Einarsrud, M.A., 2007. Hightemperature proton-conducting LaNbO4-based materials: powder synthesis by spray pyrolysis. J. Am. Ceram. Soc. 90:3395–3400. http://dx.doi.org/10.1111/j.1551-2916. 2007.01904.x. OECD, 2011. OECD guidelines for the testing of chemicals: freshwater alga and cyanobacteria. Growth Inhibition Test http://dx.doi.org/10.1787/9789264069923-en. R Core Team, 2015. R: A Language and Environment for Statistical Computing. URL. http:// www.r-project.org. Řezanka, T., Kaineder, K., Mezricky, D., Řezanka, M., Bišová, K., Zachleder, V., Vítová, M., 2016. The effect of lanthanides on photosynthesis, growth, and chlorophyll profile of the green alga Desmodesmus quadricauda. Photosynth. Res. 130:335–346. http:// dx.doi.org/10.1007/s11120-016-0263-9. Rodea-Palomares, I., Boltes, K., Fernández-Piñas, F., Leganés, F., García-Calvo, E., Santiago, J., Rosal, R., 2011. Physicochemical characterization and ecotoxicological assessment of CeO2 nanoparticles using two aquatic microorganisms. Toxicol. Sci. 119:135–145. http://dx.doi.org/10.1093/toxsci/kfq311. Rodea-Palomares, I., Gonzalo, S., Santiago-Morales, J., Leganés, F., García-Calvo, E., Rosal, R., Fernández-Piñas, F., 2012. An insight into the mechanisms of nanoceria toxicity in aquatic photosynthetic organisms. Aquat. Toxicol. 122–123:133–143. http://dx. doi.org/10.1016/j.aquatox.2012.06.005. Rogers, N.J., Franklin, N.M., Apte, S.C., Batley, G.E., Angel, B.M., Lead, J.R., Baalousha, M., 2010. Physico-chemical behaviour and algal toxicity of nanoparticulate CeO2 in freshwater. Environ. Chem. 7:50–60. http://dx.doi.org/10.1071/EN09123. Röhder, L.A., Brandt, T., Sigg, L., Behra, R., 2014. Influence of agglomeration of cerium oxide nanoparticles and speciation of cerium(III) on short term effects to the green algae Chlamydomonas reinhardtii. Aquat. Toxicol. 152:121–130. http://dx.doi.org/10. 1016/j.aquatox.2014.03.027. Rojíčková-Padrtová, R., Maršálek, B., Holoubek, I., 1998. Evaluation of alternative and standard toxicity assays for screening of environmental samples: selection of an optimal test battery. Chemosphere 37, 495–507. Schüler, D., Buchert, M., Liu, R., Dittrich, S., Merz, C., 2011. Study on Rare Earths and Their Recycling. Abschlussbericht 49. Öko-Institut eV, pp. 30–40. Stauber, J., Binet, M., 2000. Canning River Phoslock Field Trials–Ecotoxicity Testing Final Report. URL. http://www.phoslock.eu/media/7419/Eco-Toxicity_Report_by_CSIRO_

486

E. Joonas et al. / Science of the Total Environment 593–594 (2017) 478–486

Australia_Stauber__Binet_2000_Canning_river_phoslock_field_trial_-_ecotoxicity_ testing_final_report.pdf. Suppi, S., Kasemets, K., Ivask, A., Künnis-Beres, K., Sihtmäe, M., Kurvet, I., Aruoja, V., Kahru, A., 2015. A novel method for comparison of biocidal properties of nanomaterials to bacteria, yeasts and algae. J. Hazard. Mater. 286:75–84. http://dx.doi.org/10.1016/j. jhazmat.2014.12.027. Tai, P., Zhao, Q., Su, D., Li, P., Stagnitti, F., 2010. Biological toxicity of lanthanide elements on algae. Chemosphere 80:1031–1035. http://dx.doi.org/10.1016/j.chemosphere. 2010.05.030. Taylor, N.S., Merrifield, R., Williams, T.D., Chipman, J.K., Lead, J.R., Viant, M.R., 2016. Molecular toxicity of cerium oxide nanoparticles to the freshwater alga Chlamydomonas reinhardtii is associated with supra-environmental exposure concentrations. Nanotoxicology 10:1–10. http://dx.doi.org/10.3109/17435390.2014.1002868. Treibacher Industrie AS, 2013. Material Safety Data Sheet: Cerium(III) Nitrate Hexahydrat.

UNCTAD, 2014. Commodities at a Glance: Special issue on rare earths. URL. http://unctad. org/en/PublicationsLibrary/suc2014d1_en.pdf. USGS, 2016. Mineral Commodity Summaries: Rare Earths. Miner. Commod. Summ. URL. http://minerals.usgs.gov/minerals/pubs/commodity/rare_earths/mcs-2016-raree. pdf. Van Hoecke, K., De Schamphelaere, K.A.C., Van Der Meeren, P., Smagghe, G., Janssen, C.R., 2011. Aggregation and ecotoxicity of CeO2 nanoparticles in synthetic and natural waters with variable pH, organic matter concentration and ionic strength. Environ. Pollut. 159:970–976. http://dx.doi.org/10.1016/j.envpol.2010.12.010. Vindimian, E., 2001. Excel Macro: REGTOX. URL. http://www.normalesup.org/ ~vindimian/en_index.html.