To spread or not to spread? Assessing the suitability of sewage sludge and other biogenic wastes for agriculture reuse.

Sewage sludge (biosolids) management represents a worldwide issue. Due to its valuable properties, approximately one half of the EU production is recovered in agriculture. Nevertheless, growing attention is given to potential negative effects deriving from the presence of harmful pollutants. It is recognized that a (even very detailed) chemical characterization is not able to predict ecotoxicity of a mixture. However, this can be directly measured by bioassays. Actually, the choice of the most suitable tests is still under debate. This paper presents a multilevel characterization protocol of sewage sludge and other organic residues, based on bioassays and chemical-physical-microbiological analyses. The detailed description of the experimental procedure includes all the involved steps: the criteria for selecting the organic matrices to be tested and compared; the sample pre-treatment required before the analyses execution; the chemical, physical and microbiological characterisation; the bioassays, grouped in three classes (baseline toxicity; specific mode of action; reactive mode of action); data processing. The novelty of this paper lies in the integrated use of advanced tools, and is based on three pillars:•the direct ecosafety assessment of the matrices to be reused.•the adoption of innovative bioassays and analytical procedures.•the original criteria for data normalization and processing.

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.

Evaluation of Toxicity and Genotoxicity of Concrete Cast with Steel Slags Using Higher Terrestrial Plants.

The potential impact of concrete mixtures containing steel slag (SS) as a partial replacement of natural aggregates (NA) on the terrestrial ecosystem was assessed using a battery of plant-based bioassays. Leaching tests were conducted on four concrete mixtures and one mixture containing only NA (reference concrete). Leachates were tested for phytotoxicity using seeds of Lepidium sativum, Cucumis sativus, and Allium cepa. Emerging seedlings of L. sativum and A. cepa were used to assess DNA damage (comet test). The genotoxicity of the leachates was also analyzed with bulbs of A. cepa using the comet and chromosome aberration tests. None of the samples caused phytotoxic effects. On the contrary, almost all the samples supported the seedlings; and two leachates, one from the SS-containing concrete and the other from the reference concrete, promoted the growth of C. sativus and A. cepa. The DNA damage of L. sativum and A. cepa seedlings was significantly increased only by the reference concrete sample. In contrast, the DNA damage in A. cepa bulbs was significantly enhanced by the reference concrete but also by that of a concrete sample with SS. Furthermore, all leachates caused an increase in chromosomal aberrations in A. cepa bulbs. Despite some genotoxic effects of the concrete on plant cells, the partial replacement of SS does not seem to make the concrete more hazardous than the reference concrete, suggesting the potential use of SS as a reliable recycled material. Environ Toxicol Chem 2023;42:2193-2200. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Ecotoxicity Evaluation of Industrial Waste and Construction Materials: Comparison Between Leachates from Granular Steel Slags and Steel Slags-Containing Concrete Through a Plant-Based Approach.

Steel slags, the main waste product from the steel industry, may have several reuse possibilities. Among others, building applications represent a crucial field. However, the potential impact of harmful substances on the environment should be assessed. The aim of this study was to assess the phytotoxicity of steel slags (SS) and concrete mixtures cast with a partial replacement of SS (CSS). Leaching tests were carried out on four SS and four CSS according to EN 12457-2 and UNI EN 15863, respectively. Each leachate was assayed using root elongation tests on 30 seeds of Allium cepa, Cucumis sativus, and Lepidium sativum, respectively, and on 12 bulbs of A. cepa. The latter also allowed the analysis of other macroscopic parameters of toxicity (turgidity, consistency, colour change and root tip shape) and the evaluation of the mitotic index on 20,000 root tip cells per sample. None of the samples induced phytotoxic effects on the organisms tested: all samples supported seedlings emergence, verified by root elongation comparable to, or even greater than, that of the negative controls, and did not affect cell division, as evidenced by mitotic index values. The absence of phytotoxicity demonstrated by the leachates allows SS and SS-derived concrete to be considered as reliable materials suitable for use in civil constructions or in other engineering applications, with economic and environmental advantages, such as the reduction of the final disposal in landfills as well as the consumption of natural resources.

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.

Solid-State Fermentation of Trichoderma spp.: A New Way to Valorize the Agricultural Digestate and Produce Value-Added Bioproducts.

In this study, the agricultural digestate from anaerobic biogas production mixed with food wastes was used as a substrate to grow Trichoderma reesei RUT-C30 and Trichoderma atroviride Ta13 in solid-state fermentation (SSF) and produce high-value bioproducts, such as bioactive molecules to be used as ingredients for biostimulants. The Trichoderma spp. reached their maximum growth after 6 and 3 SSF days, respectively. Both Trichoderma species were able to produce cellulase, esterase, and citric and malic acids, while T. atroviride also produced gibberellins and oxylipins as shown by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) profiling. Experimental evaluation of germination parameters highlighted a significant promotion of tomato seed germination and root elongation induced by T. atroviride crude extracts from SSF. This study suggests an innovative sustainable use of the whole digestate mixed with agro-food waste as a valuable substrate in fungal biorefineries. Here, it has been applied to produce plant growth-promoting fungi and bioactive molecules for sustainable agriculture.

It Works! Organic-Waste-Assisted Trichoderma spp. Solid-State Fermentation on Agricultural Digestate.

This study aimed at valorizing digestate through Trichoderma spp. solid-state fermentation (SSF) to produce a potentially ameliorated fertilizer combined with fungal biomass as a value-added bioproduct. Plant-growth-promoting Trichoderma atroviride Ta13, T. reesei RUT-C30, T. asperellum R, and T. harzianum T-22 were tested on different SSF substrates: whole digestate (WD), digestate dried up with wood sawdust (SSF1), and digestate enriched with food waste and dried up with wood sawdust (SSF2). The fungal biomass was quantified by using a qPCR assay. The growth of the four Trichoderma spp. was only observed on the SSF2 substrate. The highest quantity of mycelium was produced by T. reesei RUT-30 (689.80 ± 80.53 mg/g substrate), followed by T. atroviride Ta13, and T. asperellum R (584.24 ± 13.36 and 444.79 ± 91.02 mg/g substrate). The germination of Lepidium sativum seeds was evaluated in order to assess the phytoxicity of the Trichoderma-enriched substrate. The treatments with 7.5% SSF2-R, 3.75% SSF2-T-22, and 1.8% SSF2-Ta13 equally enhanced the root elongation in comparison to the non-fermented SSF-2. This study demonstrated that digestate, mixed with agro-food waste, was able to support the cultivation of Trichoderma spp., paving the way to the valorization of fermented digestate as a proper biofertilizer.

Food Waste-Assisted Metal Extraction from Printed Circuit Boards: The Aspergillus niger Route.

A low-energy paradigm was adopted for sustainable, affordable, and effective urban waste valorization. Here a new, eco-designed, solid-state fermentation process is presented to obtain some useful bio-products by recycling of different wastes. Urban food waste and scraps from trimmings were used as a substrate for the production of citric acid (CA) by solid state fermentation of Aspergillus niger NRRL 334, with a yield of 20.50 mg of CA per gram of substrate. The acid solution was used to extract metals from waste printed circuit boards (WPCBs), one of the most common electronic waste. The leaching activity of the biological solution is comparable to a commercial CA one. Sn and Fe were the most leached metals (404.09 and 67.99 mg/L, respectively), followed by Ni and Zn (4.55 and 1.92 mg/L) without any pre-treatments as usually performed. Commercial CA extracted Fe more efficiently than the organic one (123.46 vs. 67.99 mg/L); vice versa, biological organic CA recovered Ni better than commercial CA (4.55 vs. 1.54 mg/L). This is the first approach that allows the extraction of metals from WPCBs through CA produced by A. niger directly grown on waste material without any sugar supplement. This "green" process could be an alternative for the recovery of valuable metals such as Fe, Pb, and Ni from electronic waste.

The release of contaminants from steel slags and natural aggregates: Evaluation of toxicity and genotoxicity.

Steel slags (SS) are the major waste produced by iron and steel industry. Slags may be reused as recycled materials, instead of natural aggregates (NA), to reduce the final disposal in a landfill and the exploitation of raw materials. However, the reuse of SS may generate a potential release of toxic compounds for the environment and humans. The purpose of this study was to evaluate the toxicity and genotoxicity of SS, in comparison with NA, by using an integrated chemical-biological approach to enable their safe reuse in engineering applications. Leaching solutions from samples were obtained by using short-term leaching tests (CEN EN 12457-2, 2004) usually adopted for the evaluation of waste recovery and final disposal. Chemical analyses of leachates were performed according to the Italian legislation on waste recovery (Ministerial Decree 186/2006). The leaching solutions were assayed by using toxicity test on Daphnia magna. Moreover, mutagenicity/genotoxicity tests on Salmonella typhimurium, Allium cepa, and human leucocytes and fibroblasts were carried out. The releases of pollutants from all samples were within the limits of the Italian legislation for waste recovery. Despite the effects that SS and NA could have on different cells, in terms of toxicity and genotoxicity, globally, SS do not seem to be any more hazardous than NA. This ecotoxicological assessment, never studied before, is important for promoting further studies that may support the decision-making process regarding the use of such types of materials.

Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice.

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-ß (TGF-ß) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.

Environmental exposure and health effects in a highly polluted area of Northern Italy: a narrative review.

Human health and well-being are strongly linked to the state of the environment. The high industrial pressure present in the Province of Brescia, located in Northern Italy, produced strong environmental and health concerns. This narrative review of the literature aims at identifying the studies focused on the association between exposure to environmental pollutants and health effects in the population living in this area. Thirteen papers fitted the inclusion criteria: five were focused on the connection among pollutants present in air matrix and health effects, seven on both air and soil, and one on soil. No study investigated the relationship with water pollution. The great variability in the analyzed end-points made it difficult to draw precise conclusions, but the fact that, in almost all the studies, the investigated health effects have a positive association with the exposure to different kinds of pollutants, allows us to hypothesize that the considered population is living in an area where the "environmental pressure" could produce significant health effects in the future.

MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line.

Gynaecological leiomyosarcoma (gLMS) represent a heterogeneous group of soft tissue sarcoma, characterized by rare incidence, high aggressiveness and propensity to infiltrate secondary organs, poor prognosis and lethality, because of the lack of biological mechanisms that underlying their progression and effective pharmaceutical treatments. This study was focused on some of the aspects of progression and dissemination of a subtype of gLMS namely vulvar LMS (vLMS). We therefore used a vulvar LMS-derived cell line namely SK-LMS-1, coupled with in vitro and in vivo assays. We observed that SK-LMS-1 cells have a strong invasive capacity in vitro, through the activity of matrix metalloproteinases 2 and 9, while in vivo these cells induce a strong angiogenic response and disseminate to the chick embryo liver. Therefore, we postulate that metalloproteinases are involved in the spreading behaviour of SK-LMS-1. Further investigations are necessary to better understand the molecular and cellular machinery involved in the progression of this malignancy.

[Evaluation of the mutagenicity of detergents by tests on bacteria, plant cells and human leucocytes.]. / Studio della mutagenicità di formulati detergenti mediante test su batteri, cellule vegetali e leucociti umani.

The aim of this study was to evaluate the mutagenicity of several traditional detergents and that of newer more biodegradable detergents, by using a bacterial test (Ames test), a plant cell test (Allium cepa micronuclei test) and a human leucocyte test (Comet test). All tests were conducted using a wide range of doses (1-2000 mg/l). None of the examined detergents induced mutations in S.typhimurium. One traditional detergent showed a genotoxic effect with the A. cepa test, while all newer detergents and one traditional detergent were shown by the Comet test to be capable of inducing DNA damage.