Reactive Oxygen Species Detection Using Fluorescence in Enchytraeus crypticus—Method Implementation through Ag NM300K Case Study

An imbalance between reactive oxygen species (ROS) and antioxidants in a living organism results in oxidative stress. Measures of such imbalance can be used as a biomarker of stress in ecotoxicology. In this study, we implemented the ROS detection method based on the oxidant-sensing probe dichloro-dihydro-fluorescein diacetate (DCFH-DA), detected by fluorescence microscopy, in Enchytraeus crypticus adults and cocoons, i.e., also covering the embryo stage. Hydrogen peroxide (H2O2), a well-known ROS inducer, was used both to optimize the method and as positive control. Implementation was successful, and the method was used to assess ROS formation in E. crypticus cocoons and adults when exposed to the reference silver nanomaterial Ag NM300K, at two effect concentrations (EC20 and EC50) for both hatching and reproduction over 3 and 7 days. The measured ROS levels varied with time, concentration, and developmental stage, with higher levels detected in adults compared with cocoons. In cocoons, ROS levels were higher at the EC20 than the EC50, which could be explained by non-monotonic concentration-response curve for hatching and reproduction, as previously observed. The increase in ROS levels at day 3 preceded the oxidative damage, as reported to occur later (day 7) in adults. The DCFH-DA method was successfully implemented here and can be further used as a new tool to detect ROS formation in E. crypticus, especially after short-term exposure to chemicals, including nanomaterials. We recommend the use of 3 and 7 days in the exposure design for this assessment.

Annelid genomes: Enchytraeus crypticus, a soil model for the innate (and primed) immune system

Enchytraeids (Annelida) are soil invertebrates with worldwide distribution that have served as ecotoxicology models for over 20 years. We present the first high-quality reference genome of Enchytraeus crypticus, assembled from a combination of Pacific Bioscience single-molecule real-time and Illumina sequencing platforms as a 525.2 Mbp genome (910 gapless scaffolds and 18,452 genes). We highlight isopenicillin, acquired by horizontal gene transfer and conferring antibiotic function. Significant gene family expansions associated with regeneration (long interspersed nuclear elements), the innate immune system (tripartite motif-containing protein) and response to stress (cytochrome P450) were identified. The ACE (Angiotensin-converting enzyme) — a homolog of ACE2, which is involved in the coronavirus SARS-CoV-2 cell entry — is also present in E. crypticus. There is an obvious potential of using E. crypticus as a model to study interactions between regeneration, the innate immune system and aging-dependent decline.

Is the Synthetic Fungicide Fosetyl-Al Safe for the Ecotoxicological Models Danio rerio and Enchytraeus crypticus?

Worldwide, pesticides have contaminated the environment, affecting non-target species. The aim of this work was to evaluate the effects of fosetyl-Al (FOS) on model organisms. Based on the 3 Rs for animal research and described guidelines, the OECD 236 and 220 were applied with some modifications. The FOS test concentrations were 0.02–0.2–2–20–200 mg/L for Danio rerio and 250–500–750–1000–1250 mg/kg for Enchytraeus crypticus. Besides the standard endpoints, additional endpoints were evaluated (D. rerio: behavior and biochemical responses; E. crypticus: extension of exposure duration (28 d (days) + 28 d) and organisms’ sizes). For D. rerio, after 96 h (h), hatching was inhibited (200 mg/L), proteins’ content increased (2 and 20 mg/L), lipids’ content decreased (2 mg/L), glutathione S-transferase activity increased (2 mg/L), and, after 120 h, larvae distance swam increased (20 mg/L). For E. crypticus, after 28 d, almost all the tested concentrations enlarged the organisms’ sizes and, after 56 d, 1250 mg/kg decreased the reproduction. In general, alterations in the organisms’ biochemical responses, behavior, and growth occurred at lower concentrations than the effects observed at the standard endpoints. This ecotoxicological assessment showed that FOS may not be considered safe for the tested species, only at higher concentrations than the predicted environmental concentrations (PECs). This research highlighted the importance of a multi-endpoint approach to assess the (eco)toxic effects of the contaminants.

Environmental Hazards of Boron and Vanadium Nanoparticles in the Terrestrial Ecosystem—A Case Study with Enchytraeus crypticus

From the start of the 21st century, nanoecotoxicological research has been growing in fast steps due to the need to evaluate the safety of the increasing use of engineered nanomaterials. Boron (B) and vanadium (V) nanoparticles (NPs) generated by anthropogenic activities are subsequently released in the environment; therefore, organisms can be continuously exposed to these NPs for short or long periods. However, the short and long-term effects of BNPs and VNPs on soil organisms are unknown. This work aimed to recognize and describe their potential toxicological effects on the model species Enchytraeus crypticus, assessing survival and reproduction, through a longer-term exposure (56 days (d)—OECD test extension of 28 d), and avoidance behavior, through a short-term exposure (48 hours (h)). After 28 d, BNPs did not induce a significant effect on E. crypticus survival, whereas they decreased the organisms’ reproduction at 500 mg/kg. From 10 to 500 mg/kg, VNPs decreased the E. crypticus survival and/or reproduction. After 56 d, 100 to 500 mg/kg BNPs and 50 to 500 mg/kg VNPs, decreased the reproduction output of E. crypticus. The estimated Effect Concentrations (ECx) based on reproduction, for BNPs, were lower at 56 d compared with 28 d; for VNPs, an opposite pattern was found: ECx 28 d < ECx 56 d. BNPs did not induce an avoidance behavior, but organisms avoided the soil contaminated with 10 mg VNPs/kg. The tested NPs showed different E. crypticus apical effects at 28 d from the ones detected at 56 d, dependent on the type of NPs (B vs. V). In general, VNPs showed to be more toxic than BNPs. However, the effects of VNPs were alleviated during the time of exposure, contrarily to BNPs (which became more toxic with extended duration). The present study adds important information about NPs toxicity with ecological significance (at the population level). Including long-term effects, the obtained results contributes to the improvement of NPs risk assessment.