Allium cepa tests: A plant-based tool for the early evaluation of toxicity and genotoxicity of newly synthetized antifungal molecules.

Many fungal genera such as Aspergillus, Penicillium, Fusarium and Alternaria are able to produce, among many other metabolites, the aflatoxins, a group of toxic and carcinogenic compounds. To reduce their formation, synthetic fungicides are used as an effective way of intervention. However, the extensive use of such molecules generates long-term residues into the food and the environment. The need of new antifungal molecules, with high specificity and low off-target toxicity is worth. The aim of this study was to evaluate: i) the toxicity and genotoxicity of newly synthesized molecules with a good anti-mycotoxic activity, and ii) the suitability of the Allium cepa multi-endpoint assay as an early screening method for chemicals. Eight compounds were tested for toxicity by using the A. cepa bulb root elongation test and for genotoxicity using the A. cepa bulb mitotic index, micronuclei and chromosome aberrations tests. Three molecules showed no toxicity, while two induced mild toxic effects in roots exposed to the highest dose (100 µM). A more pronounced toxic effect was caused by the other three compounds for which the EC50 was approximately 50 µM. Furthermore, all molecules showed a clear genotoxic activity, both in terms of chromosomal aberrations and micronuclei. Albeit the known good antifungal activity, the different molecules caused strong toxic and genotoxic effects. The results indicate the suitability of experiments with A. cepa as a research model for the evaluation of the toxic and genotoxic activities of new molecules in plants before they are released into the environment.

A scoping review of recent advances in the application of comet assay to Allium cepa roots.

The comet assay is a sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Allium cepa is a well-established plant model for toxicological studies. The aim of this scoping review was to investigate the recent application of the comet assay in Allium cepa root cells to assess the genotoxicity. To explore the literature a search was performed selecting articles published between January 2015 and February 2023 from Web of Science, PubMed, and Scopus databases using the combined search terms "Comet assay" and "Allium cepa". All the original articles that applied the comet assay to Allium cepa root cells were included. Of the 334 records initially found, 79 articles were identified as meeting the inclusion criteria. Some studies reported results for two or more toxicants. In these cases, the data for each toxicant were treated separately. Thus, the number of analyzed toxicants (such as chemicals, new materials, and environmental matrices) was higher than the number of selected papers and reached 90. The current use of the Allium-comet assay seems to be directed towards two types of approach: the direct study of the genotoxicity of compounds, mainly biocides (20% of analyzed compounds) and nano- and microparticles (17%), and assessing a treatment's ability to reduce or eliminate genotoxicity of known genotoxicants (19%). Although the genotoxicity identified by the Allium-comet assay is only one piece of a larger puzzle, this method could be considered a useful tool for screening the genotoxic potential of compounds released into the environment.

Wastewater toxicity removal: Integrated chemical and effect-based monitoring of full-scale conventional activated sludge and membrane bioreactor plants.

The literature is currently lacking effect-based monitoring studies targeted at evaluating the performance of full-scale membrane bioreactor plants. In this research, a monitoring campaign was performed at a full-scale wastewater treatment facility with two parallel lines (traditional activated sludge and membrane bioreactor). Beside the standard parameters (COD, nitrogen, phosphorus, and metals), 6 polynuclear aromatic hydrocarbons, 29 insecticides, 2 herbicides, and 3 endocrine disrupting compounds were measured. A multi-tiered battery of bioassays complemented the investigation, targeting different toxic modes of action and employing various biological systems (uni/multicellular, prokaryotes/eukaryotes, trophic level occupation). A traffic light scoring approach was proposed to quickly visualize the impact of treatment on overall toxicity that occurred after the exposure to raw and concentrated wastewater. Analysis of the effluents of the CAS and MBR lines show very good performance of the two systems for removal of organic micropollutants and metals. The most noticeable differences between CAS and MBR occurred in the concentration of suspended solids; chemical analyses did not show major differences. On the other hand, bioassays demonstrated better performance for the MBR. Both treatment lines complied with the Italian law's "ecotoxicity standard for effluent discharge in surface water". Yet, residual biological activity was still detected, demonstrating the adequacy and sensitivity of the toxicological tools, which, by their inherent nature, allow the overall effects of complex mixtures to be taken into account.

The assessment of WWTP performance: Towards a jigsaw puzzle evaluation?

A chemical and bio-analytical protocol is proposed as a holistic monitoring framework for the assessment of WWTPs (Wastewater Treatment Plants) performance. This combination of tests consists of: i) an analysis of emerging contaminants, to be added to the established physico-chemical parameters in order to understand the causes of (new) pollution phenomena and ii) some of the bio-analytical tools most widely applied in the field of wastewater research, which provide information on groups of chemicals with a common mode of toxic action (baseline toxicity, estrogenicity and mutagenicity/genotoxicity, selected as the most representative for human health). The negative effects of the discharge can thus be highlighted directly and used to assess the global environmental impact of WWTPs. As a validation, this multi-tiered approach was applied to a full-scale WWTP (150,000 p.e.), where different measurements were carried out: EDCs (Endocrine Disrupting Compounds) detection; algal growth inhibition, bioluminescence inhibition and acute toxicity test (for baseline toxicity); an E-Screen-like assay (for estrogenic activity); Ames, Allium cepa and Comet tests (for mutagenic/genotoxic activity). As a result, the WWTP showed good performance for all these issues, displaying its ability to enhance effluent quality, except for residual mutagenic behaviour, probably due to the by-products generated by the tertiary ozonation.

Biodegradability, toxicity and mutagenicity of detergents: Integrated experimental evaluations.

The widespread use of detergents has raised concern with regard to the environmental pollution caused by their active ingredients, which are biorefractory, toxic and persistent. Since detergents are complex mixtures of different substances, in which synergistic effects may occur, we aimed to assess the mutagenicity of different detergent formulations, taking into account aquatic toxicity and ready biodegradability. We performed a ready biodegradability test (OECD 301 F), Daphnia magna and Vibrio fischeri toxicity tests, and mutagenicity tests (Salmonella/microsome test, Allium cepa test and comet assay). Six detergent formulations were examined, 3 pre-manufacture and 3 commercially available. All detergents presented ready biodegradability. EC50 values varied for all products, according to the marker organism used, but were always higher than the more stringent value considered for aquatic toxicity assessment (V. fischeri 10-60 mg/L; D. magna 25-300 mg/L; A. cepa 250-2000 mg/L). None of the detergents caused mutations in bacteria. However, one commercial ecolabelled product induced an increase in micronucleus frequency in A. cepa root cells. All pre-manufacture detergents and one commercial one, which gave negative results in the Ames and A. cepa tests, induced DNA damage in human leukocytes. A more accurate evaluation of the environmental impact of complex mixtures such as detergents requires a battery of tests to describe degradation, as well as toxicological and mutagenic features.

Comparative assessment of genotoxicity of mineral water packed in polyethylene terephthalate (PET) and glass bottles.

The potential migration of genotoxic compounds into mineral water stored in polyethylene terephthalate (PET) bottles was evaluated by an integrated chemical/biological approach using short-term toxicity/genotoxicity tests and chemical analysis. Six commercial brands of still and carbonated mineral water bottled in PET and in glass were stored at 40 degrees C for 10 days in a stove according to the standard EEC total migration test (82/711/EEC), or at room temperature in the dark. After treatment, the samples were analysed using gas-chromatography/mass spectrometry (GC/MS) to detect volatile and non-volatile compounds, the Microtox test to evaluate potential toxicity of the samples, and three mutagenicity tests -Tradescantia and Allium cepa micronucleus tests and the Comet assay on human leukocytes - to detect their genotoxic activity. GC/MS analysis did not detect phthalates or acetaldehyde in the water samples. The Microtox test found no toxic effects. Mutagenicity tests detected genotoxic properties of some samples in both PET and glass bottles. Statistical analyses showed a positive association between mineral content and mutagenicity (micronuclei in A. cepa and DNA damage in human leukocytes). No clear effect of treatment and PET bottle was found. These results suggest the absence of toxic compounds migrating from PET regardless of time and conditions of storage. In conclusion, bottle material and stove treatment were not associated with the genotoxic properties of the water; the genotoxic effects detected in bottled water may be related to the characteristics of the water (minerals and CO(2) content).