Computational protocol: Nano-sized Al2O3 reduces acute toxic effects of thiacloprid on the non-biting midge Chironomus riparius

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Protocol publication

[…] LA-ICP-MS imaging techniques were used to verify ingestion of nano-Al2O3 particles by the exposed larvae. This technique was used to display the distribution pattern of aluminum within thin sections of C. riparius larvae that have been exposed to 1000 mg/L nano-Al2O3 solely, and from the control treatment. Therefore, after termination of the acute toxicity test, surviving larvae of these treatments were decapitated to facilitate the fixation in 2% glutardialdehyde (Sigma Aldrich, Germany) buffered in 0.005 M cacodylate buffer (sodium cacodylate trihydrate, pH 7.4, Sigma Aldrich, Germany). Samples were stored at least for one week at 4°C before further processing took place. After fixation, samples were de-calcified in 5% TCA (trichloroacetic acid, ≥ 99% p.a., Carl Roth GmbH) in formol (37% p.a., stabilized with methanol, Carl Roth GmbH, Germany), dehydrated with ethanol and routinely processed for Technovit® embedding (Technovit® 7100, Heraeus Kulzer GmbH, Germany). Finally, these samples were sectioned in 7 μm thin sections using a rotary microtome (Leica RM2265, Leica Biosystems, Germany) for LA-ICP-MS imaging. The LA-ICP-MS analysis was performed with a commercially available laser ablation system (LSX-213, CETAC Technologies, USA) using a Nd:YAG laser with a wavelength of 213 nm. The system was coupled to a quadrupole-based ICP-mass spectrometer (iCAP Q, Thermo Fisher Scientific, Germany). The LA parameters relating to spot size, scanning speed, laser energy, and carrier gas flow were optimized based on the best signal-to-noise ratio in combination with highest spatial resolution. The selected samples were completely ablated using a line by line scan (0 μm space) with a laser energy density of 6 J/cm2, 20 Hz laser shot frequency, 5 μm spot diameter and 20 μm/s scan speed. The dry aerosol was transported to the ICP-MS system with a carrier gas mixture of helium (0.9 L/min) passing the ablation chamber and argon (0.4 L/min) added downstream the ablation chamber. To compensate drift effects and monitor plasma stability, a gallium (ICP standard, SCP Science, Canada) solution as an internal standard (10 ng/L) was introduced continuously by a PFA (perfluoroalkoxy alkane) nebulizer and a cyclonic spray chamber. For maximum sensitivity and to minimize possible interferences, the measurement was performed in the kinetic energy discrimination mode (KED) with helium as cell gas. The isotopes 27Al, 65Ga were monitored with dwell times of 0.1 s and 0.05 s, respectively. Gallium ICP-standard (1000 mg/mL) was obtained from SCP Science (Courtaboeuf, France). Nitric acid (67–69%, Optimat) was purchased from Fisher Scientific (Loughborough, UK). The recorded ablation profiles were converted into 2D distribution images and evaluated with the software ImageJ. [...] Statistical analyses were applied on datasets recorded for all individuals within one treatment (n = 60). To compare the treatments with each other, likelihood ratio tests and, if necessary, Fishers exact test were conducted (α = 0.05) with SAS JMP version 12 (SAS Institute, Germany). To correct for multiple testing, the α-level was adjusted by sequentially Bonferroni correction []. Table Curve 2D (Systat Software GmbH, Germany) was used for calculating regression curves and SigmaPlot 13 (Systat Software GmbH, Germany) was used to plot graphs. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Mass spectrometry imaging, Microscopic phenotype analysis
Organisms Culicoides arakawae, cellular organisms
Diseases Drug-Related Side Effects and Adverse Reactions
Chemicals Aluminum Oxide