Freshwater dispersion stability of PAA-stabilised cerium oxide nanoparticles and toxicity towards Pseudokirchneriella subcapitata.

TL;DR: It is indicated that the increased dispersion stability of PAA-CeO₂ leads to an increase in toxicity compared to pristine non-stabilised forms.

About: This article is published in Science of The Total Environment.The article was published on 2015-02-01 and is currently open access. It has received 59 citations till now. The article focuses on the topics: Dispersion stability & Cerium oxide.

Summary

1. Introduction

• Changes in the levels of algal cell wall compounds were monitored using TOF-SIMS.
• The total Ce (dissolved plus particulate) concentration in selected filtered (no algae present) and non-filtered (algae present) exposure samples was determined using HR-ICP-MS to investigate PAA-CeO2 uptake/adsorption by the algae.

2.2. CeO2 nanoparticle synthesis and characterisation

• CeO2 nanoparticles were synthesised by thermolysis of Ce(NO3)4 at high temperature, resulting in homogenous precipitation of a cerium oxide nanoparticle pulp (Chane-Ching, 1994) .
• Relevant physical and chemical characterisation techniques were employed to study the PAA-CeO2.
• The zeta potential and average hydrodynamic radius (by volume; dynamic light scattering, DLS) of the stock solution was determined using a Malvern Zetasizer.
• For the same purpose, Selected Area Electron Diffraction (SAED) pattern analysis was performed with the CM30.

2.3. PAA analysis

• MHz for 1 H using a 1D NOESY (noesygppr1d) pulse sequence from the Bruker pulse sequence library for suppression of residual water.
• The region from 3.2-0.5ppm was used for the PAA and this was calibrated against the TSP peak.
• The PAA concentration of the centrifuged sample was determined by the linear regression equation of the standard curve.

2.5. Dispersion stability studies

• The stock dispersion of PAA-CeO2 was sonicated immediately prior to sub-sampling to ensure homogeneity of the sample prior to dilution in the different media solutions.
• Two different nominal start concentrations were included in the study; 1 and 0.01 mg/Lthree parallels of each concentration in every media.
• Immediately prior to the first sampling, the samples were homogenised by sonicating for 10 minutes.
• After this, the samples were left still for the duration of the experiment.
• Each sample tube was sampled for particle number measurement (dynamic light scattering, DLS) and surface charge measurement (zeta potential) at day 0, 2, 5, 7, 12 and 15.

2.6. Average particle size and zeta potential measurements

• The hydrodynamic particle size distribution and zeta potential of the PAA-CeO2 suspensions was measured using a Zetasizer Nanorange ZS instrument (Malvern, UK).
• For the size measurements, a small volume of the sample (~0.5 mL) was diluted with the appropriate media solution in a disposable polystyrene cuvette (2.5 mL).
• The zeta potential was measured on the same solution after transfer to a capillary zeta cell.
• The measurements were performed with automatically optimised number of runs (10-30).

2.7. Algae ecotoxicity studies

• Due to the nature of the tested substances, the standard OECD 201 protocol was modified for the PAA-CeO2.
• In order to overcome potential issues with shading of the algal cells during quantification of the growth, the standard fluorescence method was replaced with a modified version of the ISO method 'Measurement of biochemical Parameters -Spectrometric determination of the chlorophyll-a concentration (ISO 10260: 1992)'.
• After completion of the exposure period (72 h) the exposure media (10 mL) was filtered using a 0.7 µm glass fibre filter (Whatman GF/F), and the aqueous phase discarded.
• The filter was then allowed to dry before being added to a vial containing hot ethanol at 75 °C (10 mL) and the chlorophyll pigments extracted by shaking for 5 min.
• At Day 0 and Day 3, 2 mL aliquots of the exposure solution (before and after the algal filtration step) were collected and subjected to analysis by HR-ICP-MS to quantify the CeO2 concentration.

2.8. Biokinetics

• The metabolic changes of the cell wall after PAA-CeO2 exposure algal cells were investigated using TOF-SIMS.
• 10 µL of the algal exposure solution was pipetted onto a gold wafer, fast frozen in liquid nitrogen and stored at -80°C until the TOF-SIMS analysis was performed.
• Ion spectra measurements were performed using a TOF-SIMS V instrument (IONTOF GmbH, Münster, Germany) with a 30 keV nano-bismuth primary ion beam source.
• A pulse of 0.7 ns from the bunching system resulted in a mass resolution that usually exceeded 5000 (full width at halfmaximum) at m/z <500 in negative mode.

2.9. Statistical analyses and calculations

• In brief, the acquired data were binned to 1u.
• Data processing was carried out with the statistical package SPSS+ (version 12.0.2G) using the mass range between 200 and 1700 mass units to detect significant differences between treated cells at time point 0 and treated cells at time point 3 days.
• Ions lower than mass 200 were excluded from the study to avoid contaminating ions from salts, system contaminants, and other medium components.
• To show that data sets could be separated with a supervised model from each other a Fisher's discriminant analysis was performed.

3.1. CeO2 nanoparticle synthesis and characterisation

• Key physicochemical parameters of the PAA-CeO2 stock solution are presented in Table S1 in Supplementary Information.
• The zeta potential of the PAA-CeO2 stock solution was determined as -25mV, indicating moderate stability of the particles.
• The average hydrodynamic radius of the PAA-CeO2 stock solution (determined by volume using DLS) was determined as 84 nm, with a poly dispersity index of 0.234.
• The crystallite size of the individual CeO2 particles was determined using TEM as between 4-10 nm, and generally spherical in shape .
• EDX and SAED analysis was used to investigate the purity of the PAA-CeO2 stock material .

3.3. Dispersion stability studies

• In the current study, the zeta potential data are generated from CeO2 ENPs coated in PAA and dispersion in a complex aquatic system containing dissolved salts (including phosphate) and NOM.
• It is suggested that the complex interplay of varying ionic strength between the different media types, the presence of phosphate in the media and the interaction of both PAA and NOM at the particle surface, is influencing the stability.

3.4. Algae ecotoxicity studies

• Primary particle size was found to influence toxicity irrespective of agglomeration, with smaller nominal diameters increasing growth inhibition (van Hoecke et al., 2009) .
• Therefore, it is possible that the smaller diameter of the CeO2 ENPs used in this study may be contributing to the observed increase in toxicity compared to other studies.
• The increased dispersion stability and lack of significant aggregation in the PAA-CeO2 exposure samples cannot be ruled out as a contributing factor to the higher toxicity observed in this study compared to previous studies with pristine CeO2 ENPs.

3.5. Biokinetics

• The concentration of ions m/e 330 and m/e 332, tentatively assigned to sialic acid and dehydrosialic acid decreased in samples (both control and PAA-CeO2 exposed) from 0 h to 72 h in a similar way.
• This indicates a general metabolic mechanism, unrelated PAA-CeO2 exposure, is occurring in P. subcapitata cultures over time.
• Sialic acid probably arises from terminally sialylated N-linked oligosaccharides, which were already identified in green algae (Mamedov et al., 2011) .
• Sialic acid decrease has already been characterized as a biomarker for muscle aging in mice and may also represent a biomarker for aging effects in P. subcapitata (Hanisch et al., 2013) .
• The data show that this response in the algae is time dependent and not dependent upon PAA-CeO2 exposure, representing basic age related metabolomic and lipidomic changes under conditions applied in the present study.

4. Conclusions

• Under typical environmental conditions it is likely that PAA-stabilised CeO2 ENPs will not undergo significant agglomeration and settle out of the aqueous phase.
• The use of stabilising agents in the synthesis of ENPs to provide useful physicochemical properties for technology applications may therefore lead to significant differences in the environmental behaviour compared to pristine ENP analogues.
• Release of PAA-CeO2 would offer the possibility of increasing environmental concentrations of stably dispersed nanoparticle ceria in natural waters.
• The diagram shows the values of the discriminant scores obtained from Fisher's discriminant analysis of 24 algal samples for all ions, which were selected to discriminate between untreated micro algae cultures at day 0 and day 3 and micro algae, treated with 0.06mg/ml CeO2 at day 0 and day 3.
• Histogram comparisons of ion yields for characteristic biomarker ions which were used to separate the four treatment groups.

Figures

Figure 3. Total Ce concentration (µg/L) in filtered samples (no algae) and unfiltered samples 711 (containing algae) collected at 0 h and 72 h. Error bars represent standard deviation (n=3). 712 713

Figure 2. Change in growth rate and biomass production as a function of A) PAA-CeO2 and 706 B) pure PAA. Both data sets are plotted according to the CeO2 exposure concentrations 707 determined using HR-ICP-MS. 708 709

Table 1. Summary of the nominal PAA-CeO2 concentrations, total Ce concentration at 0 h 737 and 72 h, the calculated dissolved Ce concentration at 0 h and the free PAA concentration at 738 0 h in each of the exposure samples used in growth inhibition tests with P. subcapitata. 739

Table 2. Calculated effect concentrations of pure PAA and PAA stabilised CeO2 743 nanoparticles (PAA-CeO2) to the freshwater algae, Pseudokirchneriella subcapitata, in a 72 h 744 growth inhibition test. 745