As wastewater reclamation and reuse becomes more widespread, risks of exposure to treated wastewater increase. Moreover, an unlimited number of pollutants can be identified in wastewater. Therefore, comprehensive toxicity assessment of treated wastewater is imperative. The objective of this study was to perform a comprehensive toxicity assessment of wastewater treatment systems using stress response 10 bioassays. This powerful tool can comprehensively assess the toxicity of contaminants. In this study, samples from conventional activated sludge treatment, membrane bioreactors (MBRs) with different pore sizes and sludge retention times (SRTs), rapid sand filtration, coagulation, nano-filtration (NF) and reverse osmosis (RO) were investigated. The results of stress response bioassays confirmed that the secondary effluent showed higher stress response than influent indicating that biological treatment 15 generates toxic compounds. The results obtained from molecular weight fractionation of water samples demonstrated that organic matter with a higher molecular weight fraction (>0.1μm) causes toxicity in secondary effluent. Furthermore, supernatant from MBR reactors showed toxicity regardless of SRT. On the other hand, stress response was not detected in MBR permeates except for an MBR equipped with a larger pore size membrane (0.4μm) and with a short SRT (12 days). While rapid sand filtration could not 20 remove the toxic compounds found in secondary effluent, coagulation tests, operated at an appropriate pH, were effective for reducing stress response in the secondary effluent. Experimental findings also showed that stress response was not detected in cases of NF and RO permeate subsequent to MBR treatment.

Heavy metals present in water environment and hazardous sites as single compounds or mixture may drastically affect human health. In the present work, we investigated the risk assessment of wastewater effluents and leachate with a focus on three heavy metals-nickel (Ni), cadmium (Cd), and lead (Pb)-and their combined effect on mammalian cells, using Chinese hamster ovary cells transfected with the heat-shock protein (HSP) 47 promoter. The heavy metal mixture model was designed based on the concentrations of metals in wastewater effluents and leachate sampled in Tunisia. Using a ternary diagram, we investigated the stress response of the interaction model. This research indicated that the single heavy metals induced the stress response on HSP(+) cells even at concentrations lower than the local and international guidelines. Differences in water quality likely influenced the metal responses such that the organic composition of the leachate increased the stress response induced by the heavy metals exclusively, whereas the effluents included organic compounds that were able to mask the heavy metal effect. The mixture characterization discovered the key role played by the high levels of Ni or combination of Cd and Pb to induce the highest stress response following 3-h incubation. Heat-shock protein 47 has proven its effectiveness for assessing the heavy metal mixture effect even at low concentrations. Furthermore, the combination of a bioassay system with a statistical model proved extremely useful for better understanding the major contributors to the stress response of the mixture.

Heavy metal contamination of soil resulting from treated wastewater irrigation can cause serious concerns resulting from consuming contaminated crops. Therefore, it is crucial to assess hazard related to wastewater reuse. In the present investigation, we suggest the use of biomarker approach as a new tool for risk assessment of wastewater reuse in irrigation as an improvement to the conventional detection of physicochemical accumulation in irrigated sites. A field study was conducted at two major sites irrigated with treated wastewater and comparisons were made with a control site. Different soil depths were considered to investigate the extent of heavy metal leaching, the estrogenic activity, and the biomarker response. Results have shown that a longer irrigation period (20 years) caused a slight decrease in soil metal levels when compared to the soil irrigated for 12 years. The highest levels of Cr, Co, Ni, Pb, and Zn were detected at 20 and 40 cm horizons in plots irrigated with wastewater for 12 years. The latter finding could be attributed to chemical leaching to deeper plots for longer irrigation period. Furthermore, the treated wastewater sample showed a high estrogenic activity while none of the soil samples could induce any estrogenic activity. Regarding the stress response, it was observed that the highest stress shown by the HSP47 promoter transfected cells was induced by a longer irrigation period. Finally, the treated wastewater and the irrigated soils exhibited an overexpression of HSP60 in comparison with reference soil following 1 h exposure. In conclusion, in vitro techniques can be efficiently used to assess potential hazard related to wastewater reuse.

Irrigation with treated wastewater (TWW) is a vital alternative for arid and semi-arid lands but it poses pollution-risk to soil, vegetation and groundwater. Therefore, in the present study, in vitro bioassays were used to evaluate the adverse effects of TWW and irrigated-soil extract sample, on mammalian cells, with respect to heavy metal--Ni, Cd, Pb, Fe, Al-content. The heat shock protein (HSP) 47, E-screen, and transepithelial electrical resistance (TEER) assays served to investigate the stress response of treated-HSP47-transfected Chinese hamster ovary (CHO) cells, the estrogenic activity of the samples in MCF-7 breast cancer cells, and the barrier function (BF) of Caco-2 cells. Furthermore, proteomics analyses were performed to shed light on involved mechanisms and to establish pollution biomarkers. Results showed that the TWW elicited a stress response on HSP cells from 0.1% concentration while soil extract samples exhibited a stress at 1%. TWW induced an estrogenic activity at 10%; up-regulating cell proliferation and tumor-related proteins. Soil extract triggered the enhanced expression of HSP70 family proteins as survival mechanisms against their cytotoxicity toward MCF-7 cells. Moreover, depending on the concentration, 1% of soil extract from 20 cm depth (T20) resulted in a disruption of BF in Caco-2 cells involving cell metabolism, protein synthesis and tumor marker proteins, whereas, 5% of T20 induced the expression of BF-related proteins associated to heat shock, oxidative stress, cell proliferation and glycolytic metabolic pathway. These biological techniques were found to be extremely useful to evaluate the impact of wastewater reuse and to establish specific biomarkers that are common proteins for humans, other mammals and plants. Future studies should focus on exposure quantifications.

Wastewater reuse for irrigation is expected in the Mediterranean basin as a sustainable alternative with socio-economic benefits. However, wastewater carries considerable amounts of harmful chemicals and trace toxic metals that are able to accumulate in soil. This pollution loading is threatening both the vegetation and the groundwater and, consequently, human health. Therefore, in this study, in vitro bioassays were used to evaluate the adverse effects of wastewater and soil extract samples, on mammalian cells, with respect to heavy metal content (Ni, Cd, Pb, Fe and Al). The irrigation water, from a wastewater treatment plant, was sampled at Zaouit-Sousse perimeter in Tunisia. A representative soil profile irrigated with wastewater (0-90 cm) and a nonirrigated profile (0-40 cm), serving as a control, were sampled in the olive-treeoccupied perimeter. The estrogenic activity of the samples was investigated using the E-screen assay, while the heat shock protein (HSP) 47 assay aimed to determine the stress response of HSP 47-promoter-transfected cells following sample addition. Furthermore, proteomics analyses were performed, aiming to establish specific biomarkers of environmental pollutants. The irrigation water samples induced a high estrogenic activity when the MCF-7 cells were treated at 10% due to the presence of estrogenic compounds. The E-screen assay revealed a cytotoxic effect of the surface soil samples (0-20 cm), even at low concentrations (0.001%). Furthermore, the proteomics experiments allowed the identification of specific biomarkers for estrogenicity and cytotoxicity on MCF-7 cells. Moreover, significant stress response was observed following the incubations of the HSP 47-promoter-transfected cells with wastewater samples, even at 0.1%, and soil extracts (0-60 cm) at concentrations as low as 1%. Interestingly, no stress response or cytotoxic effect was observed for the control soil or for deep layers below 60 cm. These biological techniques could be extremely useful to evaluate the impact of wastewater reuse.