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Drought is a complex climatic phenomenon characterised by water scarcity and is recognised as the most widespread and insidious natural hazard, posing significant challenges to ecosystems and human society. In this study, we propose a new daily based index for characterising droughts, which involves standardising precipitation and/or precipitation minus potential evapotranspiration (PET) data. The new index proposed here, the generalised drought index (GDI), is computed for the entire period available from the Iberian Gridded Dataset (1971 to 2015). Comparative assessments are conducted against the daily Standardised Precipitation Index (SPI), the Standardised Precipitation Evapotranspiration Index (SPEI), and a simple Z-Score standardisation of climatic variables. Seven different accumulation periods are considered (7, 15, 30, 90, 180, 360, and 720 d) with three drought levels: moderate, severe, and extreme. The evaluation focuses mainly on the direct comparison amongst indices in terms of their ability to conform to the standard normal distribution, added value assessment using the distribution added value (DAV), and a simple bias difference for drought characteristics. Results reveal that the GDI, together with the SPI and SPEI, follows the standard normal distribution. In contrast, the Z-Score index depends on the original distribution of the data. The daily time step of all indices allows the characterisation of flash droughts, with the GDI demonstrating added value when compared to the SPI and SPEI for the shorter and longer accumulations, with a positive DAV up to 35 %. Compared to the Z-Score, the GDI shows expected greater gains, particularly at lower accumulation periods, with the DAV reaching 100 %. Furthermore, the spatial extent of drought for the 2004–2005 event is assessed. All three indices generally provide similar representations, except for the Z-Score, which exhibits limitations in capturing extreme drought events at lower accumulation periods. Overall, the findings suggest that the new index offers improved performance and comparatively adds value to similar indices with a daily time step.

ABSTRACTLong-term sequelae of coronavirus disease (COVID)-19 are frequent and of major concern. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection affects the host gut microbiota, which is linked to disease severity in patients with COVID-19. Here, we report that the gut microbiota of post-COVID subjects had a remarkable predominance of Enterobacteriaceae strains with an antibiotic-resistant phenotype compared to healthy controls. Additionally, short-chain fatty acid (SCFA) levels were reduced in feces. Fecal transplantation from post-COVID subjects to germ-free mice led to lung inflammation and worse outcomes during pulmonary infection by multidrug-resistant Klebsiella pneumoniae. transplanted mice also exhibited poor cognitive performance. Overall, we show prolonged impacts of SARS-CoV-2 infection on the gut microbiota that persist after subjects have cleared the virus. Together, these data demonstrate that the gut microbiota can directly contribute to post-COVID sequelae, suggesting that it may be a potential therapeutic target.

ABSTRACTShigella spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates’ protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon Shigella oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (Shigella sonnei, Shigella flexneri 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased Shigella penetration into the colon epithelium layer in a Gulo−/− mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both Shigella infection strategies and immune responses to Shigella infection.

Municipal effluents are well-recognized as disrupting sexual differentiation and reproduction in mussels. However, the contribution to this problem made by rainfall combined with sewer overflow (increased by rain due to climate change) is not well understood. The purpose of this study was to compare the neuroendocrine effects of municipal discharge and rainfall overflow on caged endemic mussel Elliptio complanata. To this end, mussels were experimentally caged and placed for 3 months at a municipal effluent dispersion plume site and at overflow sites. Data revealed that downstream surface water contained some pharmaceuticals (caffeine and carbamazepine) and accumulated significant levels of heterotrophic bacteria, but these effects were not observed at the overflow sites. The principal effects observed at the downstream site were increased soft tissue mass (and gonad index), inflammation, and Vtg proteins in male mussels as determined by a novel immunostaining methodology. The rainfall overflow sites had no effects on these markers, but were specifically associated with reduced Vtg proteins in females, dopamine (Dop), gonad lipids, and DNA strand breaks, with increased metallothioneins. In conclusion, the observed feminizing effects of municipal effluent were not additionally observed in mussels caged at rainfall overflow sites, although the latter exhibited a different pattern of toxicity.

The fractionation factor in the magnesium-isotope fractionation between aqueous solutions of magnesium and brucite changes sign with increasing temperature, as uncovered by recent experiments. To understand this behavior, the Reduced Partition Function Ratios and isotopic fractionation factors (Δ26/24Mgbrucite-Mg(aq)) are calculated using molecular models of aqueous [Mg(OH2)6]2+ and the mineral brucite at increasing levels of density functional theory. The calculations were carried out on the [Mg(OH2)6]2+·12H2O cluster, along with different Pauling-bond-strength-conserving models of the mineral lattice of brucite. Three conclusions were reached: (i) all levels of theory overestimate 〈Mg–O〉 bond distances in the aqua ion complex relative to Tutton's salts; (ii) the calculations predict that brucite at 298.15 K is always enriched in the heavy isotope, in contrast with experimental observations; (iii) the temperature dependencies of Wimpenny et al. (2014) and Li et al. (2014) could only be achieved by fixing the 〈Mg–O〉 bond distances in the [Mg(OH2)6]2+·12H2O cluster to values close to those observed in crystals that trap the hydrated ion.

The article analyzes the interaction between monetary and fiscal policy in Mexico. We calibrated a semi-structural model for a small open economy, based on Aguilar and Ramírez-Bulos (2018), for Mexico by using quarterly data from 2001Q1 to 2019Q4. The fiscal policy block models the fiscal deficit depending on output, an endogenous sovereign risk premium, a state-owned oil company, and public debt dynamics with domestic and foreign components. We assumed a fiscal rule whereby the deficit has an upper bound. The monetary policy follows a Taylor rule. We study the effects of different shocks on the economy, such as a drop in commodity prices, an expansion of public spending, an increase in the risk premium, a hike in the interest rate, and depreciation of the real exchange rate. We show that, remarkably, the risk premium channel transmits threats from the fiscal block to the monetary block, calling for the central bank to stabilize inflation. By contrast, starting at the economy’s steady state, an exogenous monetary policy shock affects the fiscal block mainly through the interest rate’s influence on the debt service, prompting a fiscal response to stabilize deficit.

The present study describes an efficient analytical method for analyzing apple pesticide residues. This novel approach employs ultrasound-assisted cleaning (UAC), solid-phase microextraction (SPME), and gas chromatography/mass spectrometry (GC-MS). A fiber coating with carbon nanomaterials (CNMs) was used for the extraction process. The methodology underwent extraction process optimization and analytical method validation. Moreover, its applicability for detecting pesticides in apples was evaluated. The validation showed satisfactory results with linearity coefficients greater than 0.99 (R2 > 0.99) and linear working ranges from 0.5 ng·g−1 to 300.0 ng·g−1. The limits of detection and quantification (LODs and LOQs) ranged from 0.1 ng·g−1 to 0.3 ng·g−1 and from 0.5 ng·g−1 to 0.7 ng·g−1, respectively. The calculated recovery values ranged from 79% to 98%. The precision presented a relative standard deviation (RSD) lower than 14%. The validated method was applied to apple samples acquired from different markets and fruit stands in Belo Horizonte, Minas Gerais, Brazil. Organophosphate pesticides typically used in Brazilian apple production areas were detected in some samples. This study demonstrates the utility of UAC and CNM microextraction as an effective and highly efficient approach for analyzing pesticides in apples.

The decaniobate ion, (Nb10 = [Nb10O28](6-)) being isoelectronic and isostructural with the decavanadate ion (V10 = [V10O28](6-)), but chemically and electrochemically more inert, has been useful in advancing the understanding of V10 toxicology and pharmacological activities. In the present study, the solution chemistry of Nb10 and V10 between pH 4 and 12 is studied by Raman spectroscopy. The Raman spectra of V10 show that this vanadate species dominates up to pH 6.45 whereas it remains detectable until pH 8.59, which is an important range for biochemistry. Similarly, Nb10 is present between pH 5.49 and 9.90 and this species remains detectable in solution up to pH 10.80. V10 dissociates at most pH values into smaller tetrahedral vanadate oligomers such as V1 and V2, whereas Nb10 dissociates into Nb6 under mildly (10 > pH > 7.6) or highly alkaline conditions. Solutions of V10 and Nb10 are both kinetically stable under basic pH conditions for at least two weeks and at moderate temperature. The Raman method provides a means of establishing speciation in the difficult niobate system and these findings have important consequences for toxicology activities and pharmacological applications of vanadate and niobate polyoxometalates.

IntroductionIn 2020, a new disease entitled Pediatric Inflammatory Multisystem Syndrome temporally associated with COVID-19 (PIMS-TS), or Multisystem Inflammatory Syndrome in Children (MIS-C), emerged, with thousands of children affected globally. There is no available evidence based on randomized controlled trials (RCT) to date on the two most commonly used immunomodulatory treatments, intravenous immunoglobulins (IVIG) and corticosteroids. Therefore, the Swissped RECOVERY trial was conducted to assess whether intravenous (IV) methylprednisolone shortens hospital length of stay compared with IVIG.Methods and AnalysisSwissped RECOVERY is an ongoing investigator-initiated, open-label, multicenter two-arm RCT in children and adolescents

Pesticides constitute a category of chemical products intended specifically for the control and mitigation of pests. With their constant increase in use, the risk to human health and the environment has increased proportionally due to occupational and environmental exposure to these compounds. The use of these chemicals is associated with several toxic effects related to acute and chronic toxicity, such as infertility, hormonal disorders and cancer. The present work aimed to study the metabolic profile of individuals occupationally exposed to pesticides, using a metabolomics tool to identify potential new biomarkers. Metabolomics analysis was carried out on plasma and urine samples from individuals exposed and non-exposed occupationally, using liquid chromatography coupled with mass spectrometry (UPLC-MS). Non-targeted metabolomics analysis, using principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) or partial least squares discriminant orthogonal analysis (OPLS-DA), demonstrated good separation of the samples and identified 21 discriminating metabolites in plasma and 17 in urine. The analysis of the ROC curve indicated the compounds with the greatest potential for biomarkers. Comprehensive analysis of the metabolic pathways influenced by exposure to pesticides revealed alterations, mainly in lipid and amino acid metabolism. This study indicates that the use of metabolomics provides important information about complex biological responses.

The impact of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic on pediatric intensive care units (PICUs) is difficult to quantify. We conducted an observational study in all eight Swiss PICUs between 02/24/2020 and 06/15/2020 to characterize the logistical and medical aspects of the pandemic and their impact on the management of the Swiss PICUs. The nine patients admitted to Swiss PICUs during the study period suffering from pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) and constituting 14% (9/63) of all SARS-CoV-2 positive hospitalized patients in Swiss children's hospitals caused a higher workload [total Nine Equivalents of nursing Manpower use Score (NEMS) points, p = 0.0008] and were classified to higher workload categories (p < 0.0001) than regular PICU patients (n = 4,881) admitted in 2019. The comparison of the characteristics of the eight Swiss PICUs shows that they were confronted by different organizational issues arising from temporary regulations put in place by the federal council. These general regulations had different consequences for the eight individual PICUs due to the differences between the PICUs. In addition, the temporal relationship of these different regulations influenced the available PICU resources, dependent on the characteristics of the individual PICUs. As pandemic continues, reflecting and learning from experience is essential to reduce workload, optimize bed occupancy and manage resources in each individual PICU. In a small country as Switzerland, with a relatively decentralized health care local differences between PICUs are considerable and should be taken into account when making policy decisions.

Electronic-structure calculations show that the ε-isomer of the polyoxoaluminate ion in the Keggin structure [AlO4-(Al(OH)2(H2O))12](7+) is the thermodynamically favoured one. Direct interconversion between the ε- and δ-isomers via cap rotation has a prohibitively high energy barrier in vacuo, suggesting that isomerisation in solution either proceeds via a dissolution-precipitation pathway, or that solvation and/or coordination to counterions reduces the barrier significantly. The implications for the formation of the [Al2O8Al28(OH)56(H2O)26](18+) ion are discussed.

A non-magnetic piston-cylinder pressure cell is presented for solution-state NMR spectroscopy at geochemical pressures. The probe has been calibrated up to 20 kbar using in situ ruby fluorescence and allows for the measurement of pressure dependencies of a wide variety of NMR-active nuclei with as little as 10 μL of sample in a microcoil. Initial (11)B NMR spectroscopy of the H3BO3-catechol equilibria reveals a large pressure-driven exchange rate and a negative pressure-dependent activation volume, reflecting increased solvation and electrostriction upon boron-catecholate formation. The inexpensive probe design doubles the current pressure range available for solution NMR spectroscopy and is particularly important to advance the field of aqueous geochemistry.

Benzene is a human carcinogen whose exposure to concentrations below 1 ppm (3.19 mg·m−3) is associated with myelotoxic effects. The determination of biomarkers such as trans-trans muconic acid (AttM) and S-phenylmercapturic acid (SPMA) show exposure without reflecting the toxic effects of benzene. For this reason, in this study, the urinary metabolome of individuals exposed to low concentrations of benzene was investigated, with the aim of understanding the biological response to exposure to this xenobiotic and identifying metabolites correlated with the toxic effects induced by it. Ultra-efficient liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer (UHPLC-ESI-Q-ToF-MS) was used to identify metabolites in the urine of environmentally (n = 28) and occupationally exposed (n = 32) to benzene (mean of 22.1 μg·m−3 and 31.8 μg·m−3, respectively). Non-targeted metabolomics analysis by PLS-DA revealed nine urinary metabolites discriminating between groups and statistically correlated with oxidative damage (MDA, thiol) and genetic material (chromosomal aberrations) induced by the hydrocarbon. The analysis of metabolic pathways revealed important alterations in lipid metabolism. These results point to the involvement of alterations in lipid metabolism in the mechanisms of cytotoxic and genotoxic action of benzene. Furthermore, this study proves the potential of metabolomics to provide relevant information to understand the biological response to exposure to xenobiotics and identify early effect biomarkers.

Excess rainfall events could lead to overflows and combined sewer overflows, which could threaten local mussel populations. This study sought to compare the long- term effects of combined sewer overflows and treated municipal effluents in caged Elliptio complanata mussels. Mussels were caged at 2 overflow sites, one downstream site of a major municipal effluent dispersion plume and a reference upstream site for 3 months during the summer. At the end of the exposure period, mussels were collected, analyzed for municipal contaminants (including pharmaceuticals), and effects biomarkers based on endocrine disruption (vitellogenin expression), xenobiotic detoxification (glutathione S-transferase and metallothioneins), oxidative stress/inflammation (cyclooxygenase and lipid peroxidation) and DNA damage. The data revealed that surface waters contained less pharmaceutical products than the downstream site but atrazine and its metabolite were at higher levels in overflow sites. Mussels contained elevated amounts of total heterotrophic bacteria, caffeine, acebutolol and venlafaxine at the downstream site relative to the upstream site where caffeine was higher at one of the overflow site. The levels of vitellogenin gene expression were significantly increased in both sexes of mussels caged at the downstream site only. Multivariate analysis revealed that the biomarker responses were completely separated between upstream, overflow and downstream sites. The site discrimination was based on vitellogenin, metallothioneins, DNA damage in gonad and digestive gland, gonad lipids/proteins reserves, lipid peroxidation, gonado-somatic index and condition factor. Adverse outcome pathways analysis using the power law approach revealed that most changes in the biomarkers identified by discriminant function analysis were significantly scaled to gonad energy reserves, tissue/ mussel size and loss of weight following air emersion stress. In conclusion, the toxic effects of mussels caged at overflow sites generally displayed lower responses than mussels caged downstream a treated municipal effluent suggesting that these overflows pose a lower risk than the continuous exposure to treated municipal effluent.

Abstract Background Oligonucleotide signatures (signatures) have been widely used for studying microbial diversity and function in wet-lab settings, but using them for accurate in silico identification of organisms from high-throughput sequencing (HTS) data is only a proof of concept. Existing signature design programs for sequence signatures (signatures matching exactly one sequence) or clade signatures (signatures matching every sequence in a phylogenetic clade) are not able to identify all possible polymorphic sites for sequences with high similarity and perform poorly when handling large genome sequencing datasets. Results We introduce cluster signatures: subsequences that match perfectly and exclusively any group of sequences in a data set. Cluster signatures provide complete recall for primer/probe design and increased discrimination between sequences beyond that of clade signatures. Using cluster signatures for in silico identification of HTS targets achieves good precision/recall and running time performance. This method has been implemented into an open source tool, the Automated Oligonucleotide Design Pipeline (adop), included in supplementary material and available at: https://bitbucket.org/wenchen_aafc/aodp_v2.0_release. Conclusions Cluster signatures provide a rapid and universal analysis tool to identify all possible short diagnostic DNA markers and variants from any DNA sequencing dataset. They are particularly useful in discriminating genetic material from closely related organisms and in detecting deleterious mutations in highly or perfectly conserved genomic sites.

Abstract Background Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen. Results We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence. Conclusions Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

Abstract Background Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. Conclusions Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Cerium oxide nanoparticles (nCeO) are currently used in many sectors of our economy, for instance as fuel additives and in ceramics for catalytic converters. As a result, there are concerns about their release and resulting toxicity in the aquatic environment. The purpose of this study was to examine the bioavailability and toxicity of nCeO and Ce(IV) in zebra mussels (Dreissena polymorpha) in various types of surface water differing in organic matter, conductivity and pH. Mussels were exposed to 100 μg/L Ce as either nCeO or Ce(IV) for 96 h in 4 types of water: 1) green water (high conductivity and low total organic carbon), 2) brown water (low conductivity and high natural total organic matter), 3) 10% municipal effluent (high conductivity and high anthropogenic organic matter) and 4) controls, which consisted of dechlorinated tap water. After the exposure period, the mussels were analyzed for morphological changes, resistance to survive in air, triglycerides (fat reserves), oxidative stress (arachidonate cyclooxygenase and lipid peroxidation) and DNA damage. Evidence of aggregation was observed with nCeO in most types of water, with the exception of the diluted municipal effluent. The data revealed that some of the effects of nCeO were influenced by surface water properties. The mussels were more sensitive to air emersion when exposed to nCeO in green water but not in the other water types and Ce(IV) to all types of water, although a marginal decrease was observed in mussels co-exposed to the diluted municipal effluent. A general decrease in oxidative stress and lipid levels was observed with both forms of Ce and all water types. Ce(IV) in brown water did not reduce the levels of DNA strand breaks compared with the controls. In conclusion, the sublethal toxicity of nCeO could be modulated by the surface water from which the nanoparticle is suspended.

Zinc oxide (nano-ZnO) nanoparticles are used in the production of transparent sunscreens and cosmetics, which are released into surface waters and municipal wastewater effluent. The purpose of this study was to examine the toxicity of nano-ZnO in the presence of municipal effluents to freshwater mussels Elliptio complanata. Mussels were exposed for 21 days at 15 o C to nano-ZnO and ZnCl2 in the presence of 10 % dilution of primary-treated municipal effluent. After the exposure period and 24-h depuration step, mussels were analyzed for total Zn in gills and digestive gland, free Zn, metallothioneins (MT), oxidative stress (glutathione S-transferase and LPO), endoplasmic reticulum stress (heat shock proteins and protein ubiquitination) and genotoxicity. The data revealed that although total Zn loadings did not change with these treatments, Zn levels in digestive gland were elevated in mussels exposed to nano-ZnO but not with ZnCl2 in the presence of municipal effluent. Free Zn levels in the gills were elevated in mussels exposed to the municipal effluent, but decreased in mussels exposed to nano-ZnO. MT in digestive gland showed a similar pattern and was negatively associated with free and total Zn. GST activity was significantly reduced by both nano-ZnO and municipal effluent and was negatively correlated with MT levels, suggesting the involvement of MT in the sequestration of reactive oxygen species. Discriminant function analysis showed that the municipal effluent related effects differed from the unexposed mussels and nano-ZnO exposed mussels in terms of the following responses: free Zn in gills and digestive gland and GST activity. Nano-ZnO related effects also involved GST activity, MT and protein ubiquitination, which suggests a combination of oxidative stress and reticulum endoplasmic stress. In respect with oxidative stress, the oxidative properties of nano-ZnO and ZnCl2 are dampened in the presence of the municipal effluent.