Biological effects of four iron-containing nanoremediation materials on the green alga Chlamydomonas sp.

As nanoremediation strategies for in-situ groundwater treatment extend beyond nanoiron-based applications to adsorption and oxidation, ecotoxicological evaluations of newly developed materials are required. The biological effects of four new materials with different iron (Fe) speciations ([i] FerMEG12 - pristine flake-like milled Fe(0) nanoparticles (nZVI), [ii] Carbo-Iron® - Fe(0)-nanoclusters containing activated carbon (AC) composite, [iii] Trap-Ox® Fe-BEA35 (Fe-zeolite) - Fe-doped zeolite, and [iv] Nano-Goethite - 'pure' FeOOH) were studied using the unicellular green alga Chlamydomonas sp. as a model test system. Algal growth rate, chlorophyll fluorescence, efficiency of photosystem II, membrane integrity and reactive oxygen species (ROS) generation were assessed following exposure to 10, 50 and 500 mg L-1 of the particles for 2 h and 24 h. The particles had a concentration-, material- and time-dependent effect on Chlamydomonas sp., with increased algal growth rate after 24 h. Conversely, significant intracellular ROS levels were detected after 2 h, with much lower levels after 24 h. All Fe-nanomaterials displayed similar Z-average sizes and zeta-potentials at 2 h and 24 h. Effects on Chlamydomonas sp. decreased in the order FerMEG12 > Carbo-Iron® > Fe-zeolite > Nano-Goethite. Ecotoxicological studies were challenged due to some particle properties, i.e. dark colour, effect of constituents and a tendency to agglomerate, especially at high concentrations. All particles exhibited potential to induce significant toxicity at high concentrations (500 mg L-1), though such concentrations would rapidly decrease to mg or µg L-1 in aquatic environments, levels harmless to Chlamydomonas sp. The presented findings contribute to the practical usage of particle-based nanoremediation in environmental restoration.

Toward achieving harmonization in a nano-cytotoxicity assay measurement through an interlaboratory comparison study.

Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 µmol/L (47.5 µmol/L to 51.5 µmol/L) and 77.0 µmol/L (54.3 µmol/L to 99.4 µmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

New insights into ROS dynamics: a multi-layered microfluidic chip for ecotoxicological studies on aquatic microorganisms.

Reactive oxygen species (ROS) play an important role in the life of every cell, including cellular defense and signaling mechanisms. Continuous and quantitative ROS sensing can provide valuable information about the cell state, but it remains a challenge to measure. Here, we introduce a multi-layered microfluidic chip with an integrated optical sensor for the continuous sensitive detection of extracellular hydrogen peroxide (H2O2), one of the most stable ROS. This platform includes hydraulically controlled microvalves and microsieves, which enable the precise control of toxicants and complex exposure sequences. In particular, we use this platform to study the dynamics of toxicity-induced ROS generation in the green microalga Chlamydomonas reinhardtii during short-term exposures, recovery periods, and subsequent re-exposures. Two cadmium-based toxicants with distinct internalization mechanisms are used as stress inducers: CdSe/ZnS quantum dots (Qdots) and ionic cadmium (Cd(2+)). Our results show the quantitative dynamics of ROS generation by the model microalga, the recovery of cell homeostasis after stress events and the cumulative nature of two consecutive exposures. The dissolution of quantum dots and its possible influence on toxicity and H2O2 depletion is discussed. The obtained insights are relevant from ecotoxicological and physiological perspectives.

Portable oxidative stress sensor: dynamic and non-invasive measurements of extracellular H2O2 released by algae.

Reactive oxygen species (ROS) generated by aerobic organisms are essential for physiological processes such as cell signaling, apoptosis, immune defense and oxidative stress mechanisms. Unbalanced oxidant/antioxidant budgets are involved in many diseases and, therefore, the sensitive measurement of ROS is of great interest. Here, we present a new device for the real-time monitoring of oxidative stress by measuring one of the most stable ROS, namely hydrogen peroxide (H2O2). This portable oxidative stress sensor contains the heme protein cytochrome c (cyt c) as sensing element whose spectral response enables the detection of H2O2 down to a detection limit of 40 nM. This low detection limit is achieved by introducing cyt c in a random medium, enabling multiscattering that enhances the optical trajectory through the cyt c spot. A contact microspotting technique is used to produce reproducible and reusable cyt c spots which are stable for several days. Experiments in static and microfluidic regimes, as well as numerical simulations demonstrate the suitability of the cyt c/H2O2 reaction system for the real-time sensing of the kinetics of biological processes without H2O2 depletion in the measurement chamber. As an example, we detect the release of H2O2 from the green alga Chlamydomonas reinhardtii exposed to either 180 nM functionalized CdSe/ZnS core shell quantum dots, or to 10 mg/l TiO2 nanoparticles. The continuous measurement of extracellular H2O2 by this optical sensor with high sensitivity is a promising new means for real-time cytotoxicity tests, the investigation of oxidative stress and other physiological cell processes.