Copper toxicity on Eisenia fetida in a vineyard soil: a combined study with standard tests, genotoxicity assessment and gut metagenomic analysis.

Copper (Cu) toxicity is a pressing concern for several soils, especially in organic viticulture. The objective of this work was to assess Cu toxicity on the non-target organism Eisenia fetida, employing both traditional and novel tools for early identification of Cu-induced damages. In addition to traditional tests like avoidance and reproductive toxicity experiments, other tests such as the single cell gel electrophoresis (SCGE) and gut microbiome analysis were evaluated to identify early and more sensitive pollution biomarkers. Four sub-lethal Cu concentrations were studied, and the results showed strong dose-dependent responses by the earthworm avoidance test and the exceeding of habitat threshold limit at the higher Cu doses. An inverse proportionality was observed between reproductive output and soil Cu concentration. Bioaccumulation was not detected in earthworms; soil concentrations of potentially bioavailable Cu were not affected by E. fetida presence or by time. On the contrary, the SCGE test revealed dose-dependent genotoxicity for the 'tail length' parameter already at the second day of Cu exposition. Gut microbiome analysis a modulation of microbial composition, with the most aboundant families being Pectobateriaceae, Comamonadaceae and Microscillaceae. Bacillaceae increased over time and showed adaptability to copper up to 165 mg/kg, while at the highest dose even the sensitive Acetobacteriaceae family was affected. The research provided new insights into the ecotoxicity of Cu sub-lethal doses highlighting both alterations at earthworms' cellular level and changes in their gut microbiota.

Integrated assessment of the chemical, microbiological and ecotoxicological effects of a bio-packaging end-of-life in compost.

The present study aimed to i) assess the disintegration of a novel bio-packaging during aerobic composting (2 and 6 % tested concentrations) and evaluate the resulting compost ii) analyse the ecotoxicity of bioplastics residues on earthworms; iii) study the microbial communities during composting and in 'earthworms' gut after their exposure to bioplastic residues; iv) correlate gut microbiota with ecotoxicity analyses; v) evaluate the chemico-physical characterisation of bio-packaging after composting and earthworms' exposure. Both tested concentrations showed disintegration of bio-packaging close to 90 % from the first sampling time, and compost chemical analyses identified its maturity and stability at the end of the process. Ecotoxicological assessments were then conducted on Eisenia fetida regarding fertility, growth, genotoxic damage, and impacts on the gut microbiome. The bioplastic residues did not influence the earthworms' fertility, but DNA damages were measured at the highest bioplastic dose tested. Furthermore bioplastic residues did not significantly affect the bacterial community during composting, but compost treated with 2 % bio-packaging exhibited greater variability in the fungal communities, including Mortierella, Mucor, and Alternaria genera, which can use bioplastics as a carbon source. Moreover, bioplastic residues influenced gut bacterial communities, with Paenibacillus, Bacillus, Rhizobium, Legionella, and Saccharimonadales genera being particularly abundant at 2 % bioplastic concentration. Higher concentrations affected microbial composition by favouring different genera such as Pseudomonas, Ureibacillus, and Streptococcus. For fungal communities, Pestalotiopsis sp. was found predominantly in earthworms exposed to 2 % bioplastic residues and is potentially linked to its role as a microplastics degrader. After composting, Attenuated Total Reflection analysis on bioplastic residues displayed evidence of ageing with the formation of hydroxyl groups and amidic groups after earthworm exposure.