Estrogenic compounds in Tunisian urban sewage treatment plant: occurrence, removal and ecotoxicological impact of sewage discharge and sludge disposal.

The occurrence, fate and ecotoxicological assessment of selected estrogenic compounds were investigated at Tunisian urban sewage treatment plant. The influents, effluents, as well as primary, secondary and dehydrated sludge, were sampled and analyzed for the target estrogens to evaluate their fate. All target compounds were detected in both sewage and sludge with mean concentrations from 0.062 to 0.993 µg L-1 and from 11.8 to 792.9 µg kg-1dry weight, respectively. A wide range of removal efficiencies during the treatment processes were observed, from 6.3 % for estrone to 76.8 % for estriol. Ecotoxicological risk assessment revealed that the highest ecotoxicological risk in sewage effluent and dehydrated sludge was due to 17ß-estradiol with a risk quotient (RQ) of 4.6 and 181.9, respectively, and 17α-ethinylestradiol with RQ of 9.8 and 14.85, respectively. Ecotoxicological risk after sewage discharge and sludge disposal was limited to the presence of 17ß-estradiol in dehydrated-sludge amended soil with RQ of 1.38. Further control of estrogenic hormones in sewage effluent and sludge is essential before their discharge and application in order to prevent their introduction into the natural environment.

Enhancement of Antibacterial Activity of Paludifilum halophilum and Identification of N-(1-Carboxy-ethyl)-phthalamic Acid as the Main Bioactive Compound.

The aim of this study was to determine the combined effect of fermentation parameters and enhance the production of cellular biomass and antibacterial compounds from Paludifilum halophilum SMBg3 using the response surface methodology (RSM). Eight variables were screened to assess the effects of fermentation parameters on growth and metabolite production by Taguchi experimental design. Among these, the initial pH, temperature, and the percentage of MgSO4·7H2O in the medium were found to be most influential. The Box-Behnken design was applied to derive a statistical model for the optimization of these three fermentation parameters. The optimal parameters were initial pH: 8.3, temperature growth: 44°C, and MgSO4·7H2O: 1.6%, respectively. The maximum yield of biomass and metabolite production were, respectively, 11 mg/mL dry weight and 15.5 mm inhibition zone diameter against Salmonella enterica, which were in agreement with predicted values. The bioactive compounds were separated by the thick-layer chromatography technique and analyzed by GC/MS, NMR (1D and 2D), and Fourier-transform infrared spectroscopy (FT-IR). In addition to several fatty acids, N-(1-carboxy-ethyl)-phthalamic acid was identified as the main antibacterial compound. This element exhibited a potent activity against the ciprofloxacin-resistant Salmonella enterica CIP 8039 and Pseudomonas aeruginosa ATCC 9027 with a minimum inhibitory concentration (MIC) value range of 12.5-25 µg/mL. Results demonstrated that P. halophilum strain SMBg3 is a promising resource for novel antibacterial production due to its high-level yield potential and the capacity for large-scale fermentation.

Characterization of polysaccharides isolated from Periploca angustifolia and its antioxidant activity and renoprotective potential against cadmium induced toxicity in HEK293 cells and rat kidney.

Periploca angustifolia has numerous biological properties and also used against various diseases. In our study, this plant has been used for biotechnological production of polysaccharides. The prevention action of polysaccharides isolated from P. angustifolia (PAPS) against Cd-caused oxidative stress in HEK293 cells and kidneys of rats was tested. PAPS were characterized by HPLC, FT-IT, DRX, 1D-and 2D-MNR. PAPS present strong capacity to slow the rate of lipid peroxidation and protein glycation in vitro. In addition, the results indicated that the Cd treatment caused a significant decrease in HEK293 cells viability which wasattenuated by PAPS pre-treatment. Furthermore, our findings revealed that Cd injection increased the levels of urea and creatinine in the serum. The increased levels of protein oxidation and lipid peroxidation along with decreased activities of SOD, CAT and GPx were significantly (p < 0.01) ameliorated by PAPS pre-treatment. Finally, histopathological studies also supported the prevention action of PAPS.

Nano-encapsulation using macrocyclic carbohydrate polymers (ß-cyclodextrins) of Periploca angustifolia extract: Physical stability and protective effect against cadmium-induced alterations in HepG2 cells.

The nano-encapsulation of Periploca angustifolia phenolic extract using the macrocyclic carbohydrate polymers (ß­cyclodextrins) is a most approach compared with other encapsulation methods. In this work, the ß­Cyclodextrins-PAE complex stability has been evaluated by advanced analytical methods and techniques including HPLC, FTIR and XRD. The results showed that CdCl2 treatment caused a significant decrease in cell viability. The CdCl2-induced damage in the HepG2 cells were significantly ameliorated (p < 0.001) by treatment of the PAE and ß­Cyclodextrins-PAE complex. Thus, pretreatment with 100 µg mL-1 of ß­Cyclodextrins-PAE complex significantly protect HepG2 cells against cytotoxicity induced by cadmium exposure more effectively than PAE only. However, Cd-intoxication significantly (p < 0.001) increased these enzymes activity. Additionally, reactive oxygen species generation was significantly decreased when cells were treated with nano-encapsulation PAE. The levels of supernatant antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and GSH were significantly (p < 0.001) decreased in Cd-treated cells with concomitant enhancement of lipid peroxidation. In addition, ß­Cyclodextrins-PAE pretreatment significantly (p < 0.01) inhibited Cd-exposure activated the apoptotic pathway caspace-3 and caspace-9. This effect may be due to the ability of ß­Cyclodextrins molecules to enhance stability and permeability properties.

Optimization, isolation, characterization and hepatoprotective effect of a novel pigment-protein complex (phycocyanin) producing microalga: Phormidium versicolorNCC-466 using response surface methodology.

In our study, we focused on the optimization; antioxidant and hepatoprotective potentials of novel pigment-protein complex(C-PC) isolated from Phormidium versicolor against cadmium induced liver injury in rats. From analysis, the C-PC extraction parameters were optimized using the response surface methodology (RSM) for optimal recoveries of C-PC extraction. For analysis, the optimum operational conditions for maximizing phycocyanins concentration (67.45mg/g DM) were found to be water/solid 2, temperature 32.5°C and pH7.2.This pigment was identified using HPLC and FTIR analysis. In addition, the molecular masses of α and ß subunits were 17 and 19kDa. Scavenging activity of superoxide anion, hydroxyl, nitric oxide radicals and metal chelating in vitro results indicated that C-PC has an excellent capacity as antioxidant. In vivo study, C-PC significantly prevented cadmium-induced elevation of ALAT, ASAT and bilirubin levels in rats. The histopathological observations supported the results serum enzymes assays. The results of this study revealed that C-PC has significant hepatoprotective potential. C-PC (50mgkg-1 body weight) significantly enhanced the levels of antioxidant enzymes. It can be concluded that C-PC possesses prevention action against hepatotoxicity caused by cadmium.

Optimization, antioxidant properties and GC-MS analysis of Periploca angustifolia polysaccharides and chelation therapy on cadmium-induced toxicity in human HepG2 cells line and rat liver.

The extraction of Periploca polysaccharides (PAPS) was optimized using the response surface methodology. The influence of solvent, liquid-solid ratio and extraction time on polysaccharide yield was evaluated using a full factorial design (23). Also, PAPS extract did not induce a cytotoxic effect on HepG2 cells within the range of tested concentrations (0-250µgmL-1). Herein, the pre-treatment with PAPS extract (100µgmL-1) reduced cell mortality. Furthermore, the in vivo antioxidant activity of PAPS extract was investigated in rats. The oral administration of 250mgkg-1 body weight of PAPS extract administered above a period of 10 weeks to cadmium chloride (CdCl2) induced toxicity in male Wistar rats, markedly decreased the content of MDA and protein damage in liver tissue, and enhanced liver function parameters (ALAT, ASAT and bilirubin), as well as the activities of hepatic antioxidant status (SOD, CAT, GPx and GSH). Finally, the examination of liver histopathology confirmed that PAPS ameliorate the alteration of liver tissue caused by exposition to cadmium.

Optimization, antioxidant potential, modulatory effect and anti-apoptotic action in of Euphorbia bivonae polysaccharides on hydrogen peroxide-induced toxicity in human embryonic kidney cells HEK293.

For the first time, we have determined the effect of solvent, liquid-solid ratio and extraction time on polysaccharides yield was evaluated using a full factorial design (23). In this present investigation, a total of 7 molecules were determined in this species. In our analysis saccharose was the dominant monosaccharides. Arabinose, pyranose, fructose, glucose, inositol, saccharose and trehalose found in E. bivonae. The results of the in vitro antioxidant assay showed that the EBPS have higher antioxidant capacity. Accordingly, the HEK293 cells pre-treated with EBPS compounds (100 µg·mL-1) enhanced cell viability against H2O2 exposure. Our results revealed that H2O2-exposure induced a significant increase in intracellular ROS and lipid peroxidation in HEK293 cells. Additionally, the H2O2-induced alteration in HEK293 cells morphology, was ameliorated by EBPS treatment. In addition, EPBS pre-treated cells significantly enhanced the activities of HEK293 cells antioxidant status (SOD, CAT, GPx and GSH) that were decreased after hydrogen peroxide exposure.

Phenolic compounds analysis, antioxidant, and hepatoprotective effects of Periploca angustifolia extract on cadmium-induced oxidative damage in HepG2 cell line and rats.

A total of five components (Catechin, Caffeic acid, Ferulic acid, Rosmarinic acid, and Amentoflavone) were identified in Periploca angustifolia leaf methanolic extract. This extract did not cause any cytotoxic effect on HepG2 cell line within the range of concentrations tested (0-400 µg mL-1). Thus, pre-treatment with 100 µg mL-1 of P. angustifolia leaf methanolic extract (PAE) significantly (p < .05) protective HepG2 cells against cytotoxicity induced by cadmium exposure. However, Cd-intoxication significantly (p < .05) increased alanine and aspartate amino transferases serum activities (ALT and AST) and bilirubin content by 1.85-, 1.13-, and 3.55-fold, respectively. The levels of hepatic antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were significantly (p < .05) decreased in Cd-intoxicated rats with concomitant enhancement of lipid peroxidation. Our results showed that P. angustifolia leaf methanolic extract can induce antioxidant effects and also exerts beneficial effects for the treatment of Cd-induced hepatotoxicity.

Polysaccharides from Phormidium versicolor (NCC466) protecting HepG2 human hepatocellular carcinoma cells and rat liver tissues from cadmium toxicity: Evidence from in vitro and in vivo tests.

The in vitro antioxidant, cytotoxic and cytoprotective properties and in vivo hepatoprotective activities of crude polysaccharides extracted from cyanobacteria Phormidim versicolor NCC466 (CFv-PS) were investigated. The CFv-PS, identified as heteropolysaccharides with molecular weight of 63.79kDa, exhibited relatively strong antioxidant activity, in a concentration-depended manner, in vitro assays. Additionally, CFv-PS did not induce cytotoxic effect on HepG2 human hepatocellular carcinoma cells within the range of tested concentrations (25-150µg·mL-1) while preventing them against Cd. Moreover, in rats subjected to Cd-induced hepatotoxicity, CFv-PS pretreatment significantly (P<0.05) reduced the level of ALAT, ASAT, biliburin, MDA, protein carbonyl and DNA damage, and markedly increased enzyme activities in liver tissues. These findings suggest that the cyanobacteria Phormidium versicolor is a potential source of natural products possessing antioxidant, cytoprotective and hepatoprotective properties.

Sorption of hydrophobic organic contaminants on functionalized biochar: Protagonist role of π-π electron-donor-acceptor interactions and hydrogen bonds.

The sorption of five potent endocrine disruptors as representative hydrophobic organic contaminants (HOCs) namely estrone (E1), 17ß-estradiol (E2), estriol (E3), 17α-ethynylestradiol (EE2) and bisphenol A (BPA) on functionalized biochar (fBC) was systematically examined, with a particular focus on the importance of π-electron-donor (phenanthrene: PHEN) and π-electron-acceptors (1,3-dinitrobenzene: DNB, p-amino benzoic acid: PABA) on sorption. Experimental results suggested that hydrogen-bond formation and π-π-electron-donor-acceptor (EDA) interactions were the dominant sorption mechanisms. The sorption of HOCs decreased as E1 > E2 > EE2 > E3 > BPA based on the Freundlich and Polanyi-Mane-models. The comparison of adsorption coefficient (Kd) normalized against hexadecane-water partition coefficient (KHW) between HOCs and PHEN indicated strong π-π-EDA interactions. π-π interactions among DNB, PHEN and HOCs were verified by the observed upfield frequency (Hz) shifts using proton nuclear magnetic resonance (1H NMR) which identified the specific direction of π-π interactions. UV-vis spectra showed charge-transfer bands for π-donors (PHEN and HOCs) with the model π-acceptor (DNB) also demonstrating the role of π-π EDA interactions. The role of π-electron-donor and π-electron-acceptor domains in fBC was identified at different solution pH.

Competitive sorption affinity of sulfonamides and chloramphenicol antibiotics toward functionalized biochar for water and wastewater treatment.

Competitive sorption of sulfamethazine (SMT), sulfamethoxazole (SMX), sulfathiazole (STZ) and chloramphenicol (CP) toward functionalized biochar (fBC) was highly pH dependent with maximum sorption at pH ∼4.0-4.25. Equilibrium data were well represented by the Langmuir and Freundlich models in the order STZ>SMX>CP>SMT. Kinetics data were slightly better fitted by the pseudo second-order model than pseudo first-order and intra-particle-diffusion models. Maximum sorptive interactions occurred at pH 4.0-4.25 through H-bonds formations for neutral sulfonamides species and through negative charge assisted H-bond (CAHB) formation for CP, in addition to π-π electron-donor-acceptor (EDA) interactions. EDA was the main mechanism for the sorption of positive sulfonamides species and CP at pH<2.0. Sorption of negative sulfonamides species and CP at pH>7.0 was regulated by H-bond formation and proton exchange with water by forming CAHB, respectively. The results suggested fBC to be highly efficient in removing antibiotics mixture.

Biochemical and physiological responses of halophilic nanophytoplankton (Dunaliella salina) from exposure to xeno-estrogen 17α-ethinylestradiol.

The environmental impacts of various pollutants on the entire levels of organisms are under investigation. Among these pollutants, endocrine-disrupting compounds (EDCs) present a serious hazard, even though the environmental significance of these compounds remains basically unknown. To drop some light on this field, we assessed the effects of a 11-day exposure of 17α-ethinylestradiol (EE2) on the growth, metabolic content, antioxidant response, oxidative stress, and genetic damage of Dunaliella salina, isolated from Tunisian biotopes. The results showed that at 10 ng L-1, EE2 could stimulate the growth of D. salina and increase its cellular content of photosynthetic pigments and metabolites; however, it did not significantly increase the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) or the level of malondialdehyde (MDA) and hydrogen peroxide (H2O2). In contrast, exposure to high levels of EE2 concentrations significantly inhibited the growth of D. salina (P < 0.05), decreased the cellular content of photosynthetic pigments, increased the cellular content of all of the metabolites and the SOD activity, and inhibited CAT and GPx activities. Nevertheless, the balance between oxidant and antioxidant enzymes was disrupted because H2O2 content along with MDA content simultaneously increased. Contrary to expected results, DNA damage (strand breaks) decreased after the exposure of algae to EE2. The results of this study suggest that EE2 toxicity could result in environmental impacts with consequences on the whole aquatic community. Graphical abstract.

Box-Behnken design for extraction optimization of crude polysaccharides from Tunisian Phormidium versicolor cyanobacteria (NCC 466): Partial characterization, in vitro antioxidant and antimicrobial activities.

In this study, response surface methodology (RSM) based on Box-Behnken design (BBD) was employed to optimize the aqueous extraction of crude polysaccharides from Tunisian cyanobacteria Phormidium versicolor (NCC 466). The optimal extraction conditions with an extraction yield of 21.56±0.92% were as follows: extraction temperature at 81.05°C, extraction time of 3.99h, and water to raw material ratio of 21.52mLg-1. Crude Phormidium versicolor polysaccharides (CPv-PS) are found to be a hetero-sulfated-anionic polysaccharides that contained carbohydrate (79.37±1.58%), protein (0.45±0.11%), uronic acids (4.37±0.19%) and sulfate (6.83±0.28%). The carbohydrate fraction was composed of arabinose, xylose, ribose, rhamnose, N-acetyl glucosamine, galactose, glucose, mannose, glucuronic acid and saccharose with corresponding mole percentages of 2.41, 14.58, 2.18, 6.23, 7.04, 28.21, 26.04, 3.02, 0.86 and 5.07, respectively. Evaluation of the antioxidant activity in vitro suggested that CPv-PS strongly scavenged radicals, prevented bleaching of ß-carotene and reduced activity. Furthermore, the CPv-PS exhibited effective antimicrobial properties.

Impact of treated urban wastewater for reuse in agriculture on crop response and soil ecotoxicity.

The scarcity of freshwater resources is a serious problem in arid regions, such as Tunisia, and marginal quality water is gradually being used in agriculture. This study aims to study the impact of treated urban wastewater for reuse in agriculture on the health of soil and food crops. The key findings are that the effluents of Sfax wastewater treatment plant (WWTP) did not meet the relevant guidelines, therefore emitting a range of organic (e.g., up to 90 mg L(-1) COD and 30 mg L(-1) BOD5) and inorganic pollutants (e.g., up to 0.5 mg L(-1) Cu and 0.1 mg L(-1) Cd) in the receiving aquatic environments. Greenhouse experiments examining the effects of wastewater reuse on food plants such as tomato, lettuce, and radish showed that the treated effluent adversely affected plant growth, photosynthesis, and antioxidant enzyme contents. However, the pollution burden and biological effects on plants were substantially reduced by using a 50 % dilution of treated sewage effluent, suggesting the potential of reusing treated effluent in agriculture so long as appropriate monitoring and control is in place.

Effects of sewage sludge fertilizer on heavy metal accumulation and consequent responses of sunflower (Helianthus annuus).

Use of sewage sludge, a biological residue produced from sewage treatment processes in agriculture, is an alternative disposal technique of waste. To study the usefulness of sewage sludge amendment for Helianthus annuus, a pot experiment was conducted by mixing sewage sludge at 2.5, 5, and 7.5 % (w/w) amendment ratios to the agricultural soil. Soil pH decreased whereas electrical conductivity, organic matter, total N, available P, and exchangeable Na, K, and Ca increased in soil amended with sewage sludge in comparison to unamended soil. Sewage sludge amendment led to significant increase in Pb, Ni, Cu, Cr, and Zn concentrations of soil. The increased concentration of heavy metals in soil due to sewage sludge amendment led to increases in shoot and root concentrations of Cr, Cu, Ni, and Zn in plant as compared to those grown on unamended soil. Accumulation was more in roots than shoots for most of the heavy metals. Moreover, high metal removal for the harvestable parts of the crops was recorded. Sewage sludge amendment increased root and shoot length, leaves number, biomass, and antioxidant activities of sunflower. Significant increases in the activities of antioxidant enzymes and in the glutathione, proline, and soluble sugar content in response to amendment with sewage sludge may be defense mechanisms induced in response to heavy metal stress. Graphical abstract Origin, fate and behavior of sewage sludge fertilizer.

Fate of selected estrogenic hormones in an urban sewage treatment plant in Tunisia (North Africa).

Estrogenic compounds have been monitored for one year at an urban sewage treatment plant (STP) located in Tunisia, to evaluate their fate and seasonal variations. The concentrations of these compounds were determined in both wastewater and sludge phases by gas chromatography coupled with mass spectrometry (GC-MS). Results showed that the highest removal of all estrogens (≥80%) was observed in summer. Mass balance analysis revealed that biodegradation was the predominant removal mechanism. Moreover, the results showed that the removal efficiency of the studied emerging micropollutants and their concentrations in the solid phase of return sludge were much higher in winter and spring than in summer and autumn. These findings were closely related to microbial activity and the concentration of mixed liquor suspended solids (MLSSs). Finally, the findings can be used to help with the modifications that could be implemented in that STP for the improved removal of estrogenic contaminants.