Detection of microplastics in zebrafish housing systems: Can microplastic background contamination affect the final results of microplastic-related toxicological tests?

Concentrations of microplastics (MPs) were determined in three commonly used zebrafish housing systems to see if their levels could affect the final results of laboratory microplastic-related toxicology tests. MPs have received notable attention in the last few years, and their toxicology tests have also come to the fore. Zebrafish (Danio rerio), kept in fish housing systems, are widely used as models for MPs studies. Most of these systems contain a significant number of parts made of different polymers. As usage and amortization can erode these parts, MPs might appear in the keeping water or the fish body, which may represent a background load and possibly influence the results of microplastic-related toxicological tests. To take representative water samples from systems, two in-situ filtration techniques, a newly developed peristaltic pump-, and a jet pump-driven method were applied. The collected MP particles were analyzed with a Fourier-transform infrared microscope (detection limit 50 µm), and their possible origin was also investigated. The newly developed technique was more sufficient for sampling as it had a higher MPs recovery, especially in the smaller size range. Polyester, polyethylene and polypropylene were the most frequently detected polymers in the examined fish housing systems, the highest detected concentration was 0.31±0.12 particles/liter (0.22±0.16 µg/liter). These values are negligible compared to the literature data reporting enormously high applied MPs concentrations (104 - 2.21 × 108 particles/liter) during toxicology tests. The results also show that some detected MPs did not originate from the systems, their origin was presumed to be external.

The Cocktail Effects on the Acute Cytotoxicity of Pesticides and Pharmaceuticals Frequently Detected in the Environment.

Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient. In this study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects. Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures. Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor. S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures. Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations. Several combinations displayed synergistic effects at environmentally relevant concentrations. The application of PERMANOVA was proven to be unique and successful in determining the roles of compounds in synergistic, additive, and antagonistic effects in mixtures at different effective concentrations.

Evaluating the In Vivo Virulence of Environmental Pseudomonas aeruginosa Using Microinjection Model of Zebrafish (Danio rerio).

(1) Background: Microinjection of zebrafish (Danio rerio) embryos offers a promising model for studying the virulence and potential environmental risks associated with Pseudomonas aeruginosa. (2) Methods: This work aimed to develop a P. aeruginosa infection model using two parallel exposition pathways on zebrafish larvae with microinjection into the yolk and the perivitelline space to simultaneously detect the invasive and cytotoxic features of the examined strains. The microinjection infection model was validated with 15 environmental and clinical strains of P. aeruginosa of various origins, antibiotic resistance profiles, genotypes and phenotypes: both exposition pathways were optimized with a series of bacterial dilutions, different drop sizes (injection volumes) and incubation periods. Besides mortality, sublethal symptoms of the treated embryos were detected and analyzed. (3) Results: According to the statistical evaluation of our results, the optimal parameters (dilution, drop size and incubation period) were determined. (4) Conclusions: The tested zebrafish embryo microinjection infection model is now ready for use to determine the in vivo virulence and ecological risk of environmental P. aeruginosa.