The aquatic ecosystem health can be severely affected by municipal wastewater effluents (MWWE) which contain complex mixtures of domestic, municipal and industrial origins (Bennie, 1999; Holeton et al. , 2011; Metcalfe et al. , 2003, 2010; Servos et al. , 2003, 2005). The MWWE include solid objects, sand, suspended solid particles, organic-natural ingredients that are consumed by microorganisms causing a parallel reduction of dissolved oxygen in the water body, pathogenic microorganisms which are responsible for transmitting diseases to humans and other organisms and nutrients (such as phosphorus, nitrogen) which can cause eutrophication (Chambers et al. , 1997; Cooke, 2006; Gillis, 2012). These discharges contain a wide range of natural and anthropogenic substances, including pharmaceutical products, heavy-metals, ammonia, pesticides, endocrine disruptors and polycyclic aromatic hydrocarbons (Chambers et al. , 1997; Daughton and Ternes, 1999; Kummerer, 2001), many of which have also been measured in the receiving environment (Kolpin et al. , 2002). Numerous contaminants can be found in wastewaters, such as heavy metals and persistent organic pollutants. Major industrial sources include surface treatment processes with elements such as Cu, Zn, Ni and Cr, as well as industrial products that, at the end of their life, are discharged in wastes (Sun and Shi, 1998; Karvellas et al. , 2003). Persistent organic pollutants (POPs) constitute a wide group of compounds which are either intentionally produced, such as polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs), or unintentionally or accidentally formed as byproducts of industrial or other human activities, for instance dibenzo-p-dioxins and furans (PCDDs/Fs) and polycyclic aromatic hydrocarbons (PAHs) (Brevik et al. , 2004).
These compounds are characterized by pronounced persistence against chemical/biological degradation, high environmental mobility, strong tendency for bioaccumulation in human and animal tissues, significant impacts on human health and the environment even at extremely low concentrations. Their low biodegradability makes them refractory to the biological treatment of wastewater (Katsoyiannis & Samara, 2004; 2007). The wastewater treatment plants (WWTPs) are one of the most effective ways of dealing with pollution of water resources. These facilities are designed to remove pollutants from the municipal waste and release a clean product to water recipients. According to the directive issued by the Council of Environment Ministers of the EEC in 1992, large cities should have sewage treatment waste, the extent of which is determined by which of the above harmful ingredients it removes. Sewage treatment waste includes three stages. The primary treatment aims to remove bulky solids, sand and suspended solids, while during the secondary or biological treatment the organic components of biological oxidations are removed. Finally, the tertiary treatment is designed for the removal of nutrients (such as phosphates, nitrates, borates and silicates). Due to the high cost of biological treatment, several units, including the sewage treatment plant in the city of Thessaloniki, apply up the secondary cleaning of urban wastewater in the product of which a final disinfection with chlorine takes place.
The basic parameters usually assessed in the effluent is a) COD corresponding to the amount of dissolved oxygen required to achieve the chemical oxidation of one liter of waste and b) BOD which corresponds to the amount of dissolved oxygen used by microorganisms for the oxidation of organic load in one liter of waste. Most previous studies on WWT products refer among others to the estimation of these two parameters, whereas few studies exist on the evaluation of WWT products’ effects on biological systems. Despite the fact that the effluents of WWTPs contain the previously mentioned pollutants at trace levels, they appear to have toxic effect to living organisms and therefore chemical analysis seems to be inadequate for their characterization. Thus, use of biomonitoring assays may enhance the characterization of such discharges. Among the effects which may be observed in aquatic species exposed to treated and untreated municipal wastewater are increase of metallothioneins and mixed-function oxidase activity, vitellogenin induction, lymphocyte proliferation, decrease of phagocytic activity, lower production of byssal threads, modulations in the immune system, lower tolerance to air exposure, DNA damage, decreased lysosome retention and higher mortability (Gagne et al. , 2002; Moles and Hale, 2003; St-Jean et al. , 2003; Hoeger et al. , 2004; Diniz et al. , 2005; Akaishi et al. , 2007). Organisms used as bio-indicators include the mussel Mytillus galloprovinciallis (Katsoyiannis & Samara, 2007). Mussels are considered efficient indicators for toxicological studies due to their filter-feeding capability, potential to bioaccumulate contaminants, and wide distribution in coastal and estuarine areas (Coles et al. , 1994; Sauve et al. , 2002; St-Jean et al. , 2002a, b, 2003). A broad-spectrum cellular biomarker, considered in this work, is the stability of lysosomal membrane of haemocytes (retention of dye “neutral red”) (NRR). Apart from causing lysosomal destabilization, pollutants may act as endocrine disrupters and cause oxidative stress.
Oxidative stress induced by environmental pollutants occurs when the production of reactive oxygen species (ROS) exceeds the tolerance levels of an organism and can lead to a variety of disorders of physiological functions of cells. Small concentrations of ROS are involved in physiological processes of the cell, such as control of cell proliferation and play an important role as messenger in signal transduction pathways (Irani, 2000). An important target of oxidative stress should be the DNA damage (Frenzilli et al. , 2001). The loss of integrity of DNA suggests the induction of genetic alterations and other irreversible toxic effects to invertebrates (Reinecke and Reinecke, 2004), always in relation to water quality of the environment where they live. If DNA damage is not repaired, a cascade of biological effects on cell or organism level and ﬁnally on population level can be induced. The damage of DNA in a variety of aquatic animals has been associated with growth attenuation, abnormal development and survival reduction of embryos, larvae and adults (Lee et al. , 1999). Finally, the ‘‘stress on stress’’ response (SOS) has been considered as another possible index of general stress, expressed by a reduction of survival time in air due to marine pollution (Viarengo et al. , 1995). According to this test, exposure to air, whichrepresents a natural stressor, is superimposed on mussels that have already experienced the eﬀects of several pollutant stressors, such as heavy metals and organic chemicals. Furthermore, the SOS response has proved eﬀective in both laboratory and ﬁeld studies, after short- or long-term contaminant exposure, using transplanted or indigenous mussels (Koukouzika & Dimitriadis, 2005; Smaal et al. , 1991; Thomas et al. , 1999; Veldhuizen-Tsoerkan et al. , 1991; Viarengo et al. , 1995). The present work aims to evaluate the effects of WWT products based on objective criteria used internationally and rely on established biomarkers proposed by international organizations. Additionally, it aims to highlight new data related to the effects of WWT products both in cellular and genotoxic level on mussels Mytilus galloprovincialis.
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