Davide Gualandris

Per- and polyfluoroalkyl substances (PFASs) are persistent environmental contaminants of growing concern for soil ecosystems, yet their effects on the humoral arm of innate immunity in soil invertebrates remain poorly characterized. Here, we used the earthworm Eisenia fetida to screen 31 legacy and emerging PFAS congeners for their ability to modulate the hemolytic activity of cell-free coelomic fluid, a functional readout of soluble immune effectors including the pore-forming toxin lysenin. Earthworms were exposed under OECD 207 contact-filter conditions at two concentrations (0.6 and 229 µM) for 72 h, after which decellularized coelomic fluid was tested against sheep erythrocytes. To dissect direct biochemical interference from organism-mediated regulation, the same panel was also applied ex vivo (2.5 µM) to coelomic fluid from unexposed earthworms. In vivo, PFASs produced markedly heterogeneous, congener-specific responses: PFBS, PFBA and PFMOPrA suppressed hemolytic activity, whereas PMDA, PFHxA and HFPO-DA enhanced it. In contrast, ex vivo exposure produced a consistent, broad inhibition of hemolysis, indicating a direct interaction of PFASs with soluble immune proteins. Proteomic profiling of the lysenin family under PFOA and HFPO-DA suggested isoform-level reweighting rather than uniform abundance shifts, although effects did not survive multiple-testing correction. Together, these data show that PFASs act as congener-specific immunomodulators of extracellular humoral defense in E. fetida and identify candidate congeners for confirmatory mechanistic studies.

Aquaculture marks the transition from the simple activity of harvesting aquatic animal resources, carried out through the catching practices of fishing, to the farming of aquatic organisms in fresh, brackish and sea waters, carried out through human intervention aimed at increasing production. To date, research is proceeding towards expanding the range of species that can be farmed, improving the number and quality of products, and reducing the environmental impact of aquaculture activities; these efforts are supported by the improvement of our knowledge of the biology of the relevant species, the significant updating/upgrading of the rearing technologies, and the increasing awareness of the importance of water quality in optimising farming conditions. While necessarily dependent on market demand, aquaculture needs to fully leverage its environmental potential; and the relationship between aquaculture and the environment requires a system of production that combines eco-compatibility and eco-sustainability. Here, we report and analyse insights and perspectives in eco-sustainable aquaculture, spanning from sustainability and innovation processes in aquaculture to antibiotic control and aquaculture ecosystem services, in the context of the United Nations Sustainable Development Goals.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous and persistent environmental contaminants, raising increasing concern for their long-term ecological impacts. In this study, we investigated the chronic effects of field-derived PFAS-contaminated soils collected from a firefighting training site in Trelleborg (Sweden) on the terrestrial oligochaete Eisenia fetida using a multi-tiered ecotoxicological approach. Earthworms were transplanted into soils characterized by distinct PFAS burdens and exposed for 28 days under controlled laboratory conditions. Biological responses were assessed across multiple levels of organization, including molecular, enzymatic, behavioural, and ecological endpoints. Transcriptional analyses revealed alterations in the relative abundance of immune-related genes (CCF-1 and Lysenin), while enzymatic biomarkers indicated disruption of antioxidant and immune-associated pathways. Behavioural assays showed impaired locomotor responses, and higher-level ecological endpoints highlighted sub-lethal effects on biomass and reproduction, accompanied by site-specific increases in mortality. Overall, the results demonstrate that environmentally realistic PFAS mixtures can impair key biological functions across organizational levels in a terrestrial invertebrate. These findings provide new insights into PFAS-induced soil ecotoxicity under field-relevant exposure scenarios and support the need for integrated biological assessments when evaluating the ecological risks of persistent chemical mixtures.

Per- and polyfluoroalkyl substances (PFAS) persist in soils, yet their effects on invertebrate immunity remain poorly understood. We compared a legacy congener, perfluorooctanoic acid (PFOA), with three short-chain ether acids GenX (C6), MOBA (C5), and MOPrA (C4) using a 72 h OECD-207 filter-paper assay in the earthworm Eisenia fetida. Endpoints spanned cellular and humoral defenses: amoebocyte morphometry, oxidative burst (ROS production), phenol oxidase (PO) activity, and the transcription of the lectin CCF-1 and the pore-forming protein lysenin. MOBA and MOPrA caused enlargement of amoebocytes, whereas PFOA and GenX had no morphometric impact. Oxidative burst fell significantly for all congeners. PO inhibition followed the same potency order (MOPrA > GenX > MOBA ≫ PFOA), with near-complete loss at 229 µM MOPrA. Gene expression assays for CCF-1 and lysenin showed shifts in relative fold change for each PFAS congener. The combined biomarker panel—amoebocyte size, ROS, CAT, PO, CCF-1, and lysenin—offers a concise framework for assessing terrestrial PFAS risk and guiding remediation monitoring.

In vitro microcosm experiments showed that PFOA (perfluorooctanoic acid) selects for antibiotic-resistant bacteria, characterized by WGS, which displayed increased transcription of AMR genes during growth in the presence of PFOA.

The progressive commercial deployment of multi-walled carbon nanotubes (MWCNTs) raises concerns about their terrestrial ecotoxicity. We exposed adult Cornu aspersum (150 snails; five replicates of three animals per time-point) to 50 mg L⁻¹ MWCNT-dosed Lactuca sativa for 30 days and quantified five mechanistic biomarkers alongside survival. Hemocyte spread-cell area increased by 48% (from 243 ± 22 µm² at day 0 to 360 ± 18 µm² at day 14, p < 0.001). Lysosomal membrane stability (neutral red retention) fell twofold within 72 h and to 10 min by day 30 (controls ≈ 60 min), indicating early, persistent destabilization. Micronucleus frequency rose above the ecogenotoxic threshold of 5‰ after 7 days, peaking at 8.3 ± 0.7‰ on day 14 (p < 0.01). Hepatopancreas metallothionein concentrations doubled by day 3 (2.1 ± 0.3 vs. 1.0 ± 0.2 µg g⁻¹ ww in controls) and remained >150% of control throughout exposure, consistent with metal impurity mobilization. Acetylcholinesterase activity in cephalic tissue declined by 50% after 7 days and by 73% after 30 days, revealing sustained neurotoxicity. Despite these pronounced sub-individual disturbances, cumulative mortality reached only 19% at day 30, suggesting substantial, but finite, physiological compensation. Collectively, the data demonstrate that a 50 mg L⁻¹ dietary load of MWCNTs elicits rapid cytotoxic, genotoxic, and neurotoxic responses in C. aspersum that precede overt lethality, underscoring the utility of this gastropod and the chosen biomarker suite for monitoring nanotube contamination in agro-ecosystems and food-grade snail farming.

Per‑ and polyfluoroalkyl substances (PFAS) persist in soils, yet their effects on invertebrate immunity remain poorly resolved. We compared a legacy congener, perfluorooctanoic acid (PFOA), with three short‑chain ether acids GenX (C6), MOBA (C5) and MOPrA (C4) using a 72 h OECD‑207 filter‑paper assay in the earthworm Eisenia fetida. Endpoints spanned cellular and humoral defence: amoebocyte morphometry, oxidative burst (ROS production), phenol‑oxidase (PO) activity, and transcription of the lectin CCF‑1 and the pore‑forming protein lysenin. MOBA and MOPrA enlarged amoebocytes by ~ 70% (p < 0.001), whereas PFOA and GenX had no morphometric impact. Oxidative burst fell significantly for all congeners; GenX caused a 45% drop at 0.6 µM, while MOPrA declined monotonically from − 18% (0.6 µM) to − 62% (229 µM). PO inhibition followed the same potency order (MOPrA > GenX > MOBA ≫ PFOA) with near‑complete loss at 229 µM MOPrA. CCF‑1 showed dose‑dependent or U‑shaped induction, whereas lysenin peaked at 31 µM MOPrA but was unchanged at 0.6 and 229 µM, suggesting an energetically costly yet inefficient compensatory response. Integrating these findings with published superoxide‑dismutase inhibition and catalase hyper‑activation reveals an oxidative–antioxidant–immune cascade that compromises earthworm defence at concentrations ≥ 0.6 µM (≈ 250 µg kg⁻¹ soil), levels already reported in AFFF‑impacted sites. The combined biomarker panel—amoebocyte size, ROS, CAT, PO, CCF‑1 and lysenin—offers a concise framework for assessing terrestrial PFAS risk and guiding remediation monitoring.

Per- and polyfluoroalkyl substances (PFAS) are a class of persistent organic pollutants that pose a growing threat to environmental and human health. Soil acts as a long-term reservoir for PFAS, potentially impacting soil biodiversity and ecosystem function. Earthworms, as keystone species in soil ecosystems, are particularly vulnerable to PFAS exposure. In this study, we investigated the sublethal effects of three short-chain (C4–C6) next-generation perfluoropropylene oxide acids (PFPOAs) on the earthworm Eisenia fetida, using a legacy perfluoroalkyl carboxylic acid (PFCA), perfluorooctanoic acid (PFOA), as a reference. We assessed a suite of biochemical endpoints, including markers for oxidative stress (catalase and superoxide dismutase activity), immunity (phenol oxidase activity), neurotoxicity (acetylcholinesterase activity), and behavioural endpoints (escape test). Results indicate that all tested PFAS, even at sub-micromolar concentrations, elicited significant effects across multiple physiological domains. Interestingly, HFPO-DA demonstrated the most substantial impact across all endpoints tested, indicating broad and significant biochemical and neurotoxic effects. Our findings underscore the potential risks of both legacy and emerging PFAS to soil ecosystems, emphasising the need for further research to understand the long-term consequences of PFAS contamination.

In many agroecosystems, agrochemicals are widely used to control crop pests, but often affect many non-target species of ecological and agronomic interest, such as earthworms. Earthworms are considered useful indicators of soil contamination. Exposure of these organisms to contaminants occurs mainly through the large amount of soil ingested, which passes through the digestive tract, which is closely associated with the coelom and its fluids. In this work, we used the coelomic fluids of earthworms exposed to copper sulfate and chlorpyrifos to standardize a set of non-destructive biomarkers useful for assessing the contamination in agroecosystems. Metallothionein concentrations, acetylcholinesterase inhibition, lysosomal membrane stability, micronucleus frequency, morphometric alterations, and granulocyte cytoskeleton polymerization were analyzed. The results showed that all the biomarkers used were detectable in the coelomic fluid. Furthermore, the data obtained showed highly significant variations for all biomarkers studied, thus demonstrating that the use of coelomic fluid for biomarker assessment in non-target species offers numerous advantages for field applications.