Multi-target assessment of advanced oxidation processes-based strategies for indirect potable reuse of tertiary wastewater: Fate of compounds of emerging concerns, microbial and ecotoxicological parameters.

Two advanced oxidation processes (AOPs), namely ozone/H2O2 and UV/H2O2, were tested at pilot scale as zero-liquid-discharge alternative treatments for the removal of microbiological (bacteria and viruses), chemical (compounds of emerging concern (CECs)) and genotoxic responses from tertiary municipal wastewater for indirect potable reuse (IPR). The AOP treated effluents were further subjected to granular activated carbon (GAC) adsorption and UV disinfection, following the concept of multiple treatment barriers. As a reference, a consolidated advanced wastewater treatment train consisting of ultrafiltration, UV disinfection, and reverse osmosis (RO) was also employed. The results showed that, for the same electrical energy applied, the ozone/H2O2 treatment was more effective than the UV/H2O2 treatment in removing CECs. Specifically, the ozone/H2O2 treatment, intensified by high pressure and high mixing, achieved an average CECs removal efficiency higher than UV/H2O2 (66.8% with respect to 18.4%). The subsequent GAC adsorption step, applied downstream the AOPs, further improved the removal efficiency of the whole treatment trains, achieving rates of 98.5% and 96.8% for the ozone/H2O2 and UV/H2O2 treatments, respectively. In contrast, the ultrafiltration step of the reference treatment train only achieved a removal percentage of 22.5%, which increased to 99% when reverse osmosis was used as the final step. Microbiological investigations showed that all three wastewater treatment lines displayed good performance in the complete removal of regulated and optional parameters according to both national and the European Directive 2020/2184. Only P. aeruginosa resulted resistant to all treatments with a higher removal by UV/H2O2 when higher UV dose was applied. In addition, E. coli STEC/VTEC and enteric viruses, were found to be completely removed in all tested treatments and no genotoxic activity was detected even after a 1000-fold concentration. The obtained results suggest that the investigated treatments are suitable for groundwater recharge to be used as a potable water source being such a procedure an IPR. The intensified ozone/H2O2 or UV/H2O2 treatments can be conveniently incorporated into a multi-barrier zero-liquid-discharge scheme, thus avoiding the management issues associated with the retentate of the conventional scheme that uses reverse osmosis. By including the chemical cost associated with using 11-12 mg/L of H2O2 in the cost calculations, the overall operational cost (energy plus chemical) required to achieve 50% average CECs removal in tertiary effluent for an hypothetical full-scale plant of 250 m3/h (or 25,000 inhabitants) was 0.183 €/m3 and 0.425 €/m3 for ozone/H2O2 and UV/H2O2 treatment train, respectively.

Apoptotic volume decrease (AVD) in A549 cells exposed to water-soluble fraction of particulate matter (PM10).

Exposure to atmospheric particulate matter (PM) is recognized as a human health risk factor of great concern. The present work aimed to study the cellular mechanisms underlying cytotoxic effects of airborne particulate matter <10 µm in size (PM10), sampled in an urban background site from January to May 2020, on A549 cells. In particular, the study addressed if PM10 exposure can be a main factor in the induction of the Apoptotic Volume Decrease (AVD), which is one of the first events of apoptosis, and if the generation of intracellular oxidative stress can be involved in the PM10 induction of apoptosis in A549 cells. The cytotoxicity of PM10 samples was measured by MTT test on cells exposed for 24 h to the PM10 aqueous extracts, cell volume changes were monitored by morphometric analysis of the cells, apoptosis appearance was detected by annexin V and the induction of intracellular oxidative stress was evaluated by the ROS sensitive CM-H2DCFDA fluorescent probe. The results showed cytotoxic effects ascribable to apoptotic death in A549 cells exposed for 24 h to aqueous extracts of airborne winter PM10 samples characterized by high PM10 value and organic carbon content. The detected reduced cell viability in winter samples ranged from 55% to 100%. Normotonic cell volume reduction (ranging from about 60% to 30% cell volume decrease) after PM10 exposure was already detectable after the first 30 min clearly indicating the ability of PM10, mainly arising from biomass burning, to induce Apoptotic Volume Decrease (AVD) in A549 cells. AVD was prevented by the pre-treatment with 0.5 mM SITS indicating the activation of Cl- efflux presumably through the activation of VRAC channels. The exposure of A549 cells to PM10 aqueous extracts was able to induce intracellular oxidative stress detected by using the ROS-sensitive probe CM-H2DCFDA. The PM10-induced oxidative stress was statistically significantly correlated with cell viability inhibition and with apoptotic cell shrinkage. It was already evident after 15 min exposure representing one of the first cellular effects caused by PM exposure. This result suggests the role of oxidative stress in the PM10 induction of AVD as one of the first steps in cytotoxicity.

Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study.

The work was addressed to study the sensitivity of the enzyme carbonic anhydrase (CA) to chemical pollution in the hepatopancreas of the bioindicator organism Mytilus galloprovincialis in the context of a multimarker approach in view of ecotoxicological biomonitoring and assessment application. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two areas of interest: Augusta-Melilli-Priolo, an heavy polluted industrial site (eastern Sicily, Italy), and Brucoli (eastern Sicily, Italy) an area not affected by any contamination and selected as a reference site. Mussels in Augusta presented a significant increase in the digestive gland CA activity and gene expression compared to the animals caged in the control site of Brucoli. The CA response in animals from the polluted site was paralleled by proliferation/increase in the size of lysosomes, as assessed by Lysosensor green charged cells, induction of metallothionein, up-regulation of hif-α (hypoxia-inducible factor), metabolic changes associated with protein metabolism, and changes in the condition factor. Biological responses data were integrated with information about sediment chemical analysis and metal residue concentration in animal soft tissues. In conclusion, obtained results highlighted the induction of CAs in the hepatopancreas of Mytilus galloprovincialis following to pollution exposure, and demonstrated its suitability to be integrated into a multimarker approach for the detection and characterization of the stress status induced by pollution exposure in this bioindicator organism.

Palmitoylethanolamide induces microglia changes associated with increased migration and phagocytic activity: involvement of the CB2 receptor.

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Effect of the daily ingestion of a purified anthocyanin extract from grape skin on rat serum antioxidant capacity.

The aim of this work was to study the effect of the daily ingestion of a purified anthocyanin extract from red grape skin on rat serum antioxidant capacity (ORAC) and its safety for the intestinal epithelium. The study was carried out in rats orally administered with the extract for 10 days in either normal physiological conditions or exposed to a pro-oxidant chemical (CCl(4)). The oral administration of the extract significantly (P<0.05) enhanced the ORAC value of the deproteinised serum of about 50 % after 10 days of ingestion. Anthocyanin administration was also able to reverse completely the decrease in the serum ORAC activity induced by the CCl(4) treatment. Experiments with Ussing chamber mounted intestine allowed to exclude any toxicity of the extract for the intestinal epithelium. In conclusion, our results demonstrate that the purified anthocyanin extract from red grape skin enhances the total antioxidant capacity of the serum in either normal physiological condition or during oxidative stress induction, revealing a protective role against the decrease in the serum antioxidant capacity induced by a pro-oxidant compound.

Hypotonicity induced K+ and anion conductive pathways activation in eel intestinal epithelium.

Control of cell volume is a fundamental and highly conserved physiological mechanism, essential for survival under varying environmental and metabolic conditions. Epithelia (such as intestine, renal tubule, gallbladder and gills) are tissues physiologically exposed to osmotic stress. Therefore, the activation of 'emergency' systems of rapid cell volume regulation is fundamental in their physiology. The aim of the present work was to study the physiological response to hypotonic stress in a salt-transporting epithelium, the intestine of the euryhaline teleost Anguilla anguilla. Eel intestinal epithelium, when symmetrically bathed with Ringer solution, develops a net Cl- current giving rise to a negative transepithelial potential at the basolateral side of the epithelium. The eel intestinal epithelium responded to a hypotonic challenge with a biphasic decrease in the transepithelial voltage (V(te)) and the short circuit current (I(sc)). This electrophysiological response correlated with a regulatory volume decrease (RVD) response, recorded by morphometrical measurement of the epithelium height. Changes in the transepithelial resistance were also observed following the hypotonicity exposure. The electrogenic V(te) and I(sc) responses to hypotonicity resulted from the activation of different K+ and anion conductive pathways on the apical and basolateral membranes of the epithelium: (a) iberiotoxin-sensitive K+ channels on the apical and basolateral membrane, (b) apamin-sensitive K+ channels mainly on the basolateral membrane, (c) DIDS-sensitive anion channels on the apical membrane. The functional integrity of the basal Cl- conductive pathway on the basolateral membrane is also required. The electrophysiological response to hypotonic stress was completely abolished by Ca2+ removal from the Ringer perfusing solution, but was not affected by depletion of intracellular Ca2+ stores by thapsigargin.

Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area.

The use of biomarkers to evaluate the biological effects of chemical pollutants in marine organisms represents a recent tool in the monitoring field responding to the need to detect and assess the effects of chemical contaminants on the biota. The aim of the present work was the field application of the integrated use of acetylcholinesterase (AChE) and antioxidant enzymes (catalase--CAT, glutathione peroxidase--GSH-Px), for detecting the possible exposure/effect induced by chemical pollutants in native marine organisms from a coastal marine area, represented by Salento Peninsula (Italy), that shows a coastline of high environmental value, but under constant urban pressure, including agriculture activities, widely diffused in the whole hinterland. Eight sampling stations were chosen: four not urbanized areas considered "uncontaminated" controls and four clearly exposed to anthropogenic impact. The bioindicator species studied were a sessile invertebrate, Mytilus galloprovincialis, and a benthic teleost fish, Mullus barbatus.AChE activity in M. galloprovincialis revealed significant differences among places; the minimum values observed (3.9+/-1.8 nmolmin(-1)mg(-1)) was about 50% reduced with respect to the maximum found (11.4+/-0.9 nmolmin(-1)mg(-1)). The reduction in AChE activity observed in two control stations could be explained by the leaching of pesticides into the sea from the agricultural lands. Moreover, the inhibition of AChE activity by heavy metals besides pesticides, can also explain the reduction of the enzymatic activity observed in an industrialized and harbour area. In M. galloprovincialis AChE activity showed a significant inverse correlation with catalase activity but not with glutathione peroxidase that did not significantly change in animals sampled from the eight stations. Also in M. barbatus AChE activity showed significant differences among places; it was inversely correlated with liver GSH-Px activity, but not with catalase activity, that did not show any significantly variation in animals sampled in the different stations. In conclusion, the integrated use of AChE and antioxidant enzymes (catalase or glutathione peroxidase) in M. galloprovincialis and M. barbatus, two species living in different compartment of marine coastal ecosystem, can find a useful application within the framework of marine coastal environment monitoring programs for detecting the possible exposure/effect induced by chemical pollutants, including pesticides, on living marine organisms.