Effects of sulfamethazine and tetracycline at molecular, cellular and tissue levels in Eisenia fetida earthworms.

Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens.

Innovative in vivo and in vitro bioassays for the establishment of toxicity thresholds of pollutants in sediment quality assessment using polychaetes and their immune cells.

Sediment toxicity testing has become a crucial component for assessing the risks posed by contaminated sediments and for the development of sediment quality assessment strategies. Commonly used organisms for bioassays with estuarine sediments include amphipods, Arenicola marina polychaetes and echinoids. Among the latter, the Sea Urchin Embryo test (SET) is the most widely used. However, one relevant limitation of this bioassay is the unavailability of gametes all year-round, particularly outside the natural spawning seasons. Consequently, the establishment of an appropriate and complementary model organism for a continuous assessment of sediment quality is recommended. A reliable assessment of the hazards resulting from pollutants in sediments or pore water, can be achieved with ecologically relevant species of sediment such as the polychaete Hediste diversicolor, which is widespread in estuaries and has the capacity to accumulate pollutants. The aim of this work was to develop reliable in vivo and in vitro bioassays with H. diversicolor and its coelomocytes (immune cells) to determine the toxicity thresholds of different contaminants bounded to sediments or resuspended into water. Polychaetes were exposed to sublethal concentrations of CuCl2 (in vivo) and a non-invasive method for collection of polychaetes coelomocytes was applied for the in vitro bioassay, exposing cells to a series of CuCl2 and AgNPs concentrations. Same reference toxicants were used to expose Paracentrotus lividus following the SET (ICES Nº 51; Beiras et al., 2012) and obtained toxicity thresholds were compared between the two species. In vivo exposure of polychaetes to high concentrations of Cu produced weight loss and histopathological alterations. After in vitro approaches, a significant decrease in coelomocytes viability was recorded for both toxicants, in a monotonic dose-response curve, at very short-exposure times (2 h). The toxicity thresholds obtained with polychaetes were in line with the ones obtained with the SET, concluding that their sensitivity is similar. In conclusion, in vivo and in vitro bioassays developed with H. diversicolor are accurate toxicity screenings of pollutants that could be bounded to sediments or dissolved in the pore water, and may complement the SET outside the spawning period of the echinoderms. The bioassays herein developed could be applied not only to establish the toxicity thresholds of individual compounds or mixtures, but also to assess the toxicity of field collected sediments.

Selection of an optimal culture medium and the most responsive viability assay to assess AgNPs toxicity with primary cultures of Eisenia fetida coelomocytes.

Earthworm immune cells (coelomocytes) have become a target system in ecotoxicology due to their sensitivity against a wide range of pollutants, including silver nanoparticles (AgNPs). Presently, in vitro approaches (viability assays in microplate, flow cytometry, cell sorting) with primary cultures of Eisenia fetida coelomocytes have been successfully used to test the toxicity and the dissimilar response of cell subpopulations (amoebocytes and eleocytes) after PVP-PEI coated AgNPs and AgNO3 exposures. In order to obtain reliable data and to accurately assess toxicity with coelomocytes, first an optimal culture medium and the most responsive assay were determined. AgNPs posed a gradual decrease in coelomocytes viability, establishing the LC50 value in RPMI-1640 medium at 6 mg/l and discarding that the observed cytotoxicity was attributable to its coating agent PVP-PEI. Exposure to AgNPs caused selective cytotoxicity in amoebocytes, which correlated with the Ag concentrations measured in sorted amoebocytes and reinforced the idea of dissimilar sensitivities among amoebocytes and eleocytes. Silver nano and ionic forms exerted similar toxicity in coelomocytes. The in vitro approaches with coelomocytes of E. fetida performed in this study have the capacity to predict impairments caused by pollutants at longer exposure levels and thus, provide rapid and valuable information for eco(nano)toxicology.

Integrative assessment of the effects produced by Ag nanoparticles at different levels of biological complexity in Eisenia fetida maintained in two standard soils (OECD and LUFA 2.3).

There is a potential risk to increase the release of silver nanoparticles (Ag NPs) into the environment: For instance. in soils receiving sludge models estimate 0.007 mg Ag NPs kg-1 that will annually increase due to sludge or sludge incineration residues land-disposal. Thus, the concern about the hazards of nanosilver to soils and soil invertebrates is growing. Studies performed up to now have been focused in traditional endpoints, used limit range concentrations and employed different soil types that differ in physico-chemical characteristics. Presently, effects of Ag NPs have been measured at different levels of biological complexity in Eisenia fetida, exposed for 3 and 14 d to high but sublethal (50 mg Ag NPs kg-1) and close to modeled environmental concentrations (0.05 mg Ag NPs kg-1). Since characteristics of the exposure matrix may limit the response of the organisms to these concentrations, experiments were carried out in OECD and LUFA soils, the most used standard soils. High concentrations of Ag NPs increased catalase activity and DNA damage in OECD soils after 14 d while in LUFA 2.3 soils produced earlier effects (weight loss, decrease in cell viability and increase in catalase activity at day 3). At day 14, LUFA 2.3 (low clay and organic matter-OM-) could have provoked starvation of earthworms, masking Ag NPs toxicity. The concentration close to modeled environmental concentrations produced effects uniquely in LUFA 2.3 soil. Accurate physico-chemical characteristics of the standard soils are crucial to assess the toxicity exerted by Ag NPs in E. fetida since low clay and OM contents can be considered toxicity enhancers.