Investigating the efficiency of a two-stage anaerobic-aerobic process for the treatment of confectionery industry wastewaters with simultaneous production of biohydrogen and polyhydroxyalkanoates.

The scope of the current study was to investigate the efficiency of a two-stage anaerobic-aerobic process for the simultaneous treatment and valorization of selective wastewater streams from a confectionary industry. The specific wastewater (confectionary industry wastewater, CIW) was a mixture of the rinsing eluting during washing of the cauldrons in which jellies and syrups were produced, and contained mainly readily fermentable sugars, being thus of high organic load. The first stage of the process was the dark fermentation (DF) of the CIW in continuous, attached-biomass systems, in which the effect on hydrogen yields and distribution of metabolites were studied for different packing materials (ceramic or plastic), hydraulic retention times, HRTs (12 h-30 h) and feed substrate concentration (20 g COD/L- 50 g COD/L). In the second stage, the effectiveness of the aerobic treatment of the DF effluents was evaluated in terms of the reduction of the organic load and the production of polyhydroxyalkanoates (PHAs) through an enriched mixed microbial culture (MMC). The MMC was developed in a continuous draw and fill system, in which the accumulation potential of PHAs was studied. It was shown that the hydrogen production rates decreased for increasing substrate concentration and HRTs, with a maximum of 12.70 ± 0.35 m3 H2/m3 initial CIW achieved for the lowest HRT and feed concentration and using ceramic beads as packing material. Butyrate, acetate and lactate were the main metabolites generated in all cases, in different ratios. The distribution of metabolites during DF was shown to highly affect the efficiency of the second process in terms of both the reduction of organic load and the PHAs yields. The highest removal of organic load achieved after 48 h of aerobic treatment was 84.0 ± 0.9 %, whereas the maximum PHAs yield was 21.46 ± 0.13 kg PHAs/m3 initial CIW.

Recovery of polyhydroxyalkanoates (PHAs) polymers from a mixed microbial culture through combined ultrasonic disruption and alkaline digestion.

PHAs are a form of cellular storage polymers with diverse structural and material properties, and their biodegradable and renewable nature makes them a potential green alternative to fossil fuel-based plastics. PHAs are obtained through extraction via various mechanical, physical and chemical processes after their intracellular synthesis. Most studies have until now focused on pure cultures, while information on mixed microbial cultures (MMC) remains limited. In this study, ultrasonic (US) disruption and alkaline digestion by NaOH were applied individually and in combination to obtain PHAs products from an acclimated MMC using phenol as the carbon source. Various parameters were tested, including ultrasonic sound energy density, NaOH concentration, treatment time and temperature, and biomass density. US alone caused limited cell lysis and resulted in high energy consumption and low efficiency. NaOH of 0.05-0.2 M was more efficient in cell disruption, but led to PHAs degradation under elevated temperature and prolonged treatment. Combining US and NaOH significantly improved the overall process efficiency, which could reduce energy consumption by 2/3rds with only minimal PHAs degradation. The most significant factor was identified to be NaOH dosage and treatment time, with US sound energy density playing a minor role. Under the semi-optimized condition (0.2 M NaOH, 1300 W L-1, 10 min), over 70% recovery and 80% purity were achieved from a 3 g L-1 MMC slurry of approximately 50% PHAs fraction. The material and thermal properties of the products were analyzed, and the polymers obtained from US + NaOH treatments showed comparable or higher molecular weight to previously reported results. The products also exhibited good thermal stability and rheological properties, compared to the commercial standard. In conclusion, the combined US and NaOH method has the potential in real application as an efficient process to obtain high quality PHAs from MMC, and cost-effectiveness can be further optimized.

A Comparative Study of Various Pretreatment Approaches for Bio-Ethanol Production from Willow Sawdust, Using Co-Cultures and Mono-Cultures of Different Yeast Strains.

The effect of different pretreatment approaches based on alkali (NaOH)/hydrogen peroxide (H2O2) on willow sawdust (WS) biomass, in terms of delignification efficiency, structural changes of lignocellulose and subsequent fermentation toward ethanol, was investigated. Bioethanol production was carried out using the conventional yeast Saccharomyces cerevisiae, as well as three non-conventional yeasts strains, i.e., Pichia stipitis, Pachysolen tannophilus, Wickerhamomyces anomalus X19, separately and in co-cultures. The experimental results showed that a two-stage pretreatment approach (NaOH (0.5% w/v) for 24 h and H2O2 (0.5% v/v) for 24 h) led to higher delignification (38.3 ± 0.1%) and saccharification efficiency (31.7 ± 0.3%) and higher ethanol concentration and yield. Monocultures of S. cerevisiae or W. anomalus X19 and co-cultures with P. stipitis exhibited ethanol yields in the range of 11.67 ± 0.21 to 13.81 ± 0.20 g/100 g total solids (TS). When WS was subjected to H2O2 (0.5% v/v) alone for 24 h, the lowest ethanol yields were observed for all yeast strains, due to the minor impact of this treatment on the main chemical and structural WS characteristics. In order to decide which is the best pretreatment approach, a detailed techno-economical assessment is needed, which will take into account the ethanol yields and the minimum processing cost.

Chemical and biological tracking in decentralized sanitation systems: The case of artificial constructed wetlands.

Given that the social and economic sustainability of rural areas is highly based on the protection of natural resources, biodiversity and human health, simple-operated and cost-effective wastewater treatment systems, like artificial constructed wetlands (CWs), are widely proposed for minimizing the environmental and human impact of both water and soil pollution. Considering that the optimization of wastewater treatment processes is vital for the reduction of effluents toxic potential, there is imperative need to establish appropriate management strategies for ensuring CW performance and operational efficiency. To this end, the present study aimed to assess the operational efficiency of a horizontal free water surface CW (HFWS-CW) located in a world heritage area of Western Greece, via a twelve-month duration Toxicity Identification Evaluation (TIE)-like approach, including both chemical and biological tracking tools. Conventional chemical tracking, by means of pH, conductivity, total COD, and nitrogen-derived components, like nitrates and ammonia-nitrogen, were monthly recorded in both influents and effluents to monitor whether water quality standards are maintained, and to assess potent CW operational deficiencies occurring over time. In parallel, Whole Effluent Toxicity (WET) bioassays were thoroughly applied, using freshwater algae and higher plant species (producers), crustaceans and rotifers (consumers), as well as human lymphocytes (in terms of Cytokinesis Block Micronucleus assay) to evaluate the acute and short-term toxic and hazardous potential of both influents and effluents. The integrated analysis of abiotic (physicochemical parameters) and biotic (toxic endpoints) parameters, as well as the existence of "cause-effect" interrelations among them, revealed that CW operational deficiencies, mainly based on poorly removal rates, could undermine the risk posed by treated sewage. Those findings reinforce the usage of WET testing, thus giving rise to the importance of applying appropriate water management strategies and optimization actions, like oxygen enrichment of surface and bottom of HFWS-CW basins, expansion of the available land, the enhancement of bed depth and seasonal harvesting of plants, for ensuring sewage quality, in favor of water resources protection and sustainable growth in rural areas.

Fungal pretreatment of willow sawdust and its combination with alkaline treatment for enhancing biogas production.

In this study fungal pretreatment of willow sawdust (WSD) via the white rot fungi Leiotrametes menziesii and Abortiporus biennis was studied and the effect on fractionation of lignocellulosic biomass and biochemical methane potential (BMP), was evaluated. Scanning electron microscopy (SEM) and IR spectroscopy were used to investigate the changes in the structural characteristics of the pretreated WSD. Fungal pretreatment results revealed that A. biennis is more attractive, since it resulted in higher lignin degradation and lower holocellulose uptake. Samples of the 14th and 30th d of cultivation (i.e. the middle and the end of the pretreatment experiment) with both fungi were used for BMP tests and the effect of pretreatment duration was also evaluated. BMP increase by 31 and 43% was obtained due to the cultivation of WSD with A. biennis, for 14 and 30 d, respectively. In addition, combination of biological (after 30 d of cultivation) with alkaline (NaOH 20 g/100 gTS) pretreatment was performed, in order to assess the effect of the chemical agent on biologically pretreated WSD, in terms of lignocellulosic content and BMP. Combination of alkaline with fungal pretreatment led to high lignin degradation for both fungi, while the cellulose and hemicellulose removal efficiencies were higher for combined alkaline and L. menziesii pretreatment. The maximum BMP was observed for the combined alkaline and A. biennis pretreatment and was 12.5 and 50.1% higher than the respective alkaline and fungal pretreatment alone and 115% higher than the respective BMP of raw WSD.

Carbamazepine-mediated pro-oxidant effects on the unicellular marine algal species Dunaliella tertiolecta and the hemocytes of mussel Mytilus galloprovincialis.

This study investigates the pro-oxidant behavior of the antiepileptic drug carbamazepine (CBZ) on the marine algal species Dunaliella tertiolecta and the immune defense-related hemocytes of mussel Mytilus galloprovincialis. A phytotoxicity test, performed in a first step, showed a significant inhibition of the growth rate and the chlorophyll alpha (Chl-α) content in algae after exposure for 24 h to different concentrations of CBZ (1-200 mg L(-1)). On the other hand, the increased levels of lipid peroxidation products, such as MDA, measured in 24 h CBZ-treated cells were attenuated with time (48-96 h), followed by a significant recovery of both the algal growth rate and the Chl-α content in all cases. The latter could be related to the concomitant enhancement of total carotenoids in CBZ-treated algae with time, which in turn could protect algal growth and survival against CBZ-induced oxidative stress. On the other hand, the increased levels of cell death, superoxide anions ((·)O2 (-)), nitric oxides (NO, in terms of nitrites, NO2 (-)) and MDA content observed in mussel hemocytes exposed to environmentally relevant (0.01-1 µg L(-1)) and/or higher (10 and 100 µg L(-1)) concentrations of the drug, clearly indicate the ability of CBZ to induce oxidative effects on cells of non-target species, such as mussels, affecting thus their overall health status. The significant relationships occurred among the tested biological parameters in both bioassays, further reinforce CBZ-mediated pro-oxidant effects on species, widely used in ecotoxicological and toxicological studies and provide a more comprehensive view on its environmental fate and ecotoxicological risk evaluation.

Enhancement of biosynthesis of polyhydroxyalkanoates (PHA) from Taihu blue algae by adding by-product acetic acid.

As an easily obtained organic waste, by-product acetic acid could be an appropriate co-substrate with blue algae wastes (increase C/N ratio of substrates) for co-fermentation of PHA production. However, there are still acrylic acid and other chemicals in by-product acetic acid, which could cause severe inhibition for fermenting microorganisms during PHA production process. The current study represented that alkali pretreatment (pH level of 12) is a more favorable method compared with thermal pretreatment (80 â„ƒ for 30 min) for breaking cell walls of blue algae. It seemed that there was no synergistic effect of the combination of thermal and alkali pretreatment methods (temperature of 80 â„ƒ and pH level of 12). Optimal parameters during electro-fenton process for removal of inhibitors in by-product acetic acid were under current of 0.5 A, pH level of 3 and reaction time of 120 min. Both the highest dry weight of PHA and PHA concentration were achieved by applying blue algae and by-product acetic acid (after pretreatment) as co-substrates (mixed ratio of 3:1, stirring speed of 200 r/min, 24 h), indicating that using by-product acetic acid (after pretreatment) as co-substrate could increase C/N ratio and promote PHA production successfully. The current study could offer new insights for improving PHA production by co-fermentation.

Production efficiency and properties of poly(3hydroxybutyrate-co-3hydroxyvalerate) generated via a robust bacterial consortium dominated by Zoogloea sp. using acidified discarded fruit juices as carbon source.

In the current study, a mixed microbial culture (MMC) of polyhydroxyalkanoates (PHAs) producers was developed under nutrient stress and was assessed as biocatalyst for the production of high-yielding PHAs from fermented (acidified) discarded fruit juices (DFJ). The structure of the MMC was analyzed periodically to determine its microbial dynamics, revealing that Zoogloae sp. dominated throughout the operation of the system. The efficiency of PHAs production from the MMC was further optimized in batch mode by altering the ratio of C to N, the ratio of carbon sources (propionate and butyrate), and the initial pH, and subsequently different fermentation mixtures of acidified DFJ were assessed as substrates at optimal conditions. Upon solvent extraction, the properties of recovered PHAs were analyzed, showing that in all cases P(3HB-co-3HV) was produced, with Tm ranging from 90.5 to 168.8 °C, and maximum obtained yields 54.61 ± 4.31 % and 43.27 ± 2.13 %, from synthetic substrates and DFJ, respectively. Overall, it was shown that the developed MMC can be efficiently applied as biocatalyst for the exploitation of sugary wastewaters, such as DFJ, towards bio-based and biodegradable plastics bearing the required properties to substitute fossil plastics, into the concept of a circular economy.

Effect of thermo-chemical pretreatment on the saccharification and enzymatic digestibility of olive mill stones and their bioconversion towards alcohols.

The present study investigated the effect of thermo-chemical pretreatment on the enhancement of enzymatic digestibility of olive mill stones (OMS), as well as its possible valorisation via bioconversion of the generated free sugars to alcohols. Specifically, the influence of parameters such as reaction time, temperature, type and concentration of dilute acids and/or bases, was assessed during the thermo-chemical pretreatment. The hydrolysates and the solids remaining after pretreatment, as well as the whole pretreated slurries, were further evaluated as potential substrates for the simultaneous production of ethanol and xylitol via fermentation with the yeast Pachysolen tannophilus. The digestibility and overall saccharification of OMS were considerably enhanced in all cases, with the maximum enzymatic digestibility observed for dilute sodium hydroxide (almost 4-fold) which also yielded the highest total saccharification yield (91% of the total OMS carbohydrates). Ethanol and xylitol yields from the untreated OMS were 28 g/kg OMS and 25 g/kg OMS, respectively, and were both significantly enhanced by pretreatment. The highest ethanol yield was 79 g/kg OMS and was achieved by the alkali pretreatment and separate fermentation of hydrolysates and solids, whereas the highest xylitol yield was 49 g/kg OMS and was obtained by pretreatment with sulphuric acid and separate fermentation of hydrolysates and solids.

The Potential Risk of Electronic Waste Disposal into Aquatic Media: The Case of Personal Computer Motherboards.

Considering that electronic wastes (e-wastes) have been recently recognized as a potent environmental and human threat, the present study aimed to assess the potential risk of personal computer motherboards (PCMBs) leaching into aquatic media, following a real-life scenario. Specifically, PCMBs were submerged for 30 days in both distilled water (DW) and artificial seawater (ASW). Afterwards, PCMBs leachates were chemically characterized (i.e., total organic carbon, ions, and trace elements) and finally used (a) for culturing freshwater (Chlorococcum sp. and Scenedesmus rubescens) and saltwater (Dunaliella tertiolecta and Tisochrysis lutea) microalgae for 10 days (240 h), (b) as the exposure medium for mussel Mytilus galloprovincialis (96 h exposure), and (c) for performing the Cytokinesis Block Micronucleus (CBMN) assay in human lymphocytes cultures. According to the results, PCMBs could mediate both fresh- and marine algae growth rates over time, thus enhancing the cytotoxic, oxidative, and genotoxic effects in the hemocytes of mussels (in terms of lysosomal membrane impairment, lipid peroxidation, and NO content and micronuclei formation, respectively), as well as human lymphocytes (in terms of MN formation and CBPI values, respectively). The current findings clearly revealed that PCMBs leaching into the aquatic media could pose detrimental effects on both aquatic organisms and human cells.

In situ biogas upgrading via cathodic biohydrogen using mitigated ammonia nitrogen during the anaerobic digestion of Taihu blue algae in an integrated bioelectrochemical system (BES).

Biohydrogen using migrated ammonia as nitrogen source, and biogas upgrading with hydrogen collected at biocathode in an integrated bioelectrochemical system (BES) were investigated, during the anaerobic digestion of Taihu blue algae. Under an applied voltage of 0.4 V, biohydrogen (202.87 mL) reached 2.34 and 2.90 times than groups with applied voltage of 0 V and 0.8 V, respectively. Moreover, biohydrogen of the group with 1000 mg/L initial ammonia addition (524.16 mL) reached 1.53 times than that the of the control. With 0.25 bar of H2 injected at the beginning (R1), highest methane production (286.62) mL and content (75.73%) were obtained. Comparing to other groups, not only microbial genus responsible for both aceticlastic and hydrogenotrophic methanogens of the group R1 were apparently enriched, but key enzymes related to methane production also acquired better abundances. Therefore, it's promising to conduct the ammonia alleviating, hydrogen producing and biogas upgrading simultaneously using BES.

From waste to fuel: Energy recovery from household food waste via its bioconversion to energy carriers based on microbiological processes.

In the present study the bioconversion of dried household food waste (FORBI) to energy carriers was investigated aiming to its sustainable management and valorization. FORBI was either directly fermented towards ethanol and hydrogen or was previously subjected to extraction with water resulting to a liquid fraction (extract) rich in sugars and a solid residue, which were then fermented separately. Subsequently, the effluents were assessed as substrates for methane production via anaerobic digestion (AD). Mono-cultures and co-cultures of C5 and C6 yeasts were used for the alcoholic fermentation whereas for the production of hydrogen, mixed acidogenic consortia were used. Taking into account the optimum yields of biofuels, the amount of recoverable energy was estimated based for each different approach. The maximum ethanol yield was 0.16 g ethanol per kg of FORBI and it was achieved for separate fermentation of liquid and solid fractions of the waste. The highest hydrogen yield that was observed was 210.44 L ± 4.02 H2/kg TS FORBI for 1% solids loading and supplementation with cellulolytic enzymes. Direct AD of either the whole FORBI or its individual fractions led to lower overall energy recovery, compared to that obtained when fermentation and subsequent AD were applied. The recoverable energy was estimated for the different exploitation approaches of the waste. The maximum achieved recoverable energy was 21.49 ± 0.57 MJ/kg.

On the evaluation of different saccharification schemes for enhanced bioethanol production from potato peels waste via a newly isolated yeast strain of Wickerhamomyces anomalus.

The present study focuses on the exploration of the potential use of potato peels waste (PPW) as feedstock for bioethanol production, using a newly isolated yeast strain, Wickerhamomyces anomalus, via different saccharification and fermentation schemes. The saccharification of PPW was performed via thermal and chemical (acid, alkali) pretreatment, as well as via enzymatic hydrolysis through the use of commercial enzymes (cellulase and amylase) or enzymes produced at lab scale (alpha-amylase from Bacillus sp. Gb67), either separately or in mixtures. The results indicated that the enzymatic treatment by commercial enzymes led to a higher saccharification efficiency (72.38%) and ethanol yield (0.49 g/gconsumed sugars) corresponding to 96% of the maximum theoretical. In addition, acid pretreatment was found to be beneficial for the process, leading also to high hydrolysis and ethanol yields, indicating that PPW is a very promising feedstock for bio-ethanol production by W. anomalus under different process schemes.

Comparison of yields and properties of microbial polyhydroxyalkanoates generated from waste glycerol based substrates.

The accumulation efficiency and the properties of polyhydroxyalkanoates (PHAs) produced from acidified waste glycerol (AWG) and its derivatives via an enriched microbial consortium derived from soil, were investigated in this study. AWG consisted mainly from short chain fatty acids, 1,3 propanediol and residual glycerol, which were also evaluated individually as substrates. Accumulation capacity and yields were estimated after solvent extraction and purification and PHAs were further analyzed in terms of their chemical structure, thermal properties, molecular masses and mechanical properties. The lowest accumulation capacity was noticed for non-acidified waste glycerol as carbon source which led to the generation of P(3HB), whereas for the other carbon sources co-polymers of 3HB with 3HV or 3HHx were produced. Average molecular mass weights were quite high in all cases reaching ~1.8×106Da. The thermal properties and the mechanical behavior of PHAs were shown to be highly affected by their monomeric composition, whereas it was also concluded that DSC and DMA results were in good agreement.

Valorization of kitchen biowaste for ethanol production via simultaneous saccharification and fermentation using co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

The biotransformation of the pre-dried and shredded organic fraction of kitchen waste to ethanol was investigated, via co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis (Scheffersomyces stipitis). Preliminary experiments with synthetic media were performed, in order to investigate the effect of different operational parameters on the ethanol production efficiency of the co-culture. The control of the pH and the supplementation with organic nitrogen were shown to be key factors for the optimization of the process. Subsequently, the ethanol production efficiency from the waste was assessed via simultaneous saccharification and fermentation experiments. Different loadings of cellulolytic enzymes and mixtures of cellulolytic with amylolytic enzymatic blends were tested in order to enhance the substrate conversion efficiency. It was further shown that for solids loading up to 40% waste on dry mass basis, corresponding to 170 g.L-1 initial concentration of carbohydrates, no substrate inhibition occurred, and ethanol concentration up to 45 g.L-1 was achieved.

Lewis-Brønsted acid catalysed ethanolysis of the organic fraction of municipal solid waste for efficient production of biofuels.

A combined Lewis-Brønsted acid ethanolysis of sugars was thoroughly investigated with the aim of producing ethyl levulinate (EL) in a single step. Ethanolysis carried out at 453 K for 4 h using H2SO4 (1 wt%) and AlCl3·6H2O (30 mol % with respect to sugars) produced a yield of 60 mol % of EL respect to glucose and starch. Such optimised conditions were positively applied directly on different food waste, preliminarily characterised and found to be mainly composed by simple (10-15%) and relatively complex sugars (20-60%), besides proteins (6-10%) and lipids (4-10%), even in their wet form. The catalytic system resulted robust enough to the point that the copresence of proteins, lignin, lipids and mineral salts not only did not negatively affect the overall reactivity, but resulted efficiently converted into soluble species, and specifically, into other liquid biofuels of different nature.

Evaluation of a battery of marine species-based bioassays against raw and treated municipal wastewaters.

The present study evaluates a battery of marine species-based bioassays against chemically characterized municipal wastewater samples (raw and WWTP treated). We estimated Dunaliella tertiolecta growth rate inhibition (24-96h IC50 values), Artemia franciscana immobilization (24h LC50 values), mussel hemocytes viability and lipid peroxidation enhancement (in terms of neutral red retention assay/NRRT and malondialdehyde/MDA content, respectively) in influent- and WWTP effluent-treated species. We found algal growth arrest and stimulation respectively, almost similar 24hLC50 values in Artemia sp., and significantly higher adverse effects (in terms of NRRT and MDA levels) in influent-treated mussel hemocytes. Furthermore, the estimation of hatchability, yolk-sac larvae mortality (24-120hLC50) and spinal deformities (SD) in sea bream Sparus aurata showed slight variations over time, with the lowest LC50 and SD50 (representing spinal deformities at 50% of yolk-sac larvae) values to be observed in influent-treated larvae at 120h. Data interpretation (both chemical and biological) revealed that toxic endpoints, such as NRRT50, 96hIC50Dun, 120hLC50Sparus and 120hSD50Sparus, significantly related to WWTP removal efficiency and further mediated by the presence of dominant compounds, such as As and Cr, could be used for identifying main components of toxicity in wastewaters.

Production of biohydrogen from crude glycerol in an upflow column bioreactor.

A continuous attached growth process for the production of biohydrogen from crude glycerol was developed. The process consisted of an anaerobic up-flow column bioreactor (UFCB), packed with cylindrical ceramic beads, which constituted the support matrix for the attachment of bacterial cells. The effect of crude glycerol concentration, pH and hydraulic retention time on glycerol conversion, hydrogen yield and metabolite distribution was investigated. It was shown that the most critical parameter for the efficient bioconversion was the pH of the influent, whereas the hydrogen yield increased with an increase in feed glycerol concentration and a decrease in the hydraulic retention time. The main soluble metabolite detected was 1,3-propanediol in all cases, followed by butyric and hexanoic acids. The latter is reported to be produced from glycerol for the first time. Acidification of the waste reached 38.5%, and the maximum H2 productivity was 107.3 ± 0.7 L/kg waste glycerol at optimal conditions.

Olive oil mill wastewater toxicity in the marine environment: alterations of stress indices in tissues of mussel Mytilus galloprovincialis.

This study investigated the impact of olive mill wastewater (OMW) as a pollutant of the marine environment, via the detection of stress indice alterations in mussels Mytilus galloprovincialis. Due to the absence of data concerning the levels of OMW in the receiving waters, mortality test (96h) was first performed in order to estimate the range of OMW concentration where no mortality occurs. OMW concentrations ranging from 0.01 to 0.1% (v/v) showed no increased mortality and thus were used for the determination of pre-pathological alterations in tissues of mussels. In particular, mussels exposed to either 0.1 or 0.01% (v/v) OMW for 5 days showed significant alterations of stress indices in their tissues. Specifically, decreased neutral red retention (NRR) assay time values, inhibition of acetylcholinesterase (AChE) activity, as well as a significant increase of micronucleus (MN) frequency and DNA damage were detected in haemolymph/haemocytes and gills, compared with values measured in tissues of control mussels. The results of the present study showed that OMW disposal into the marine environment could induce pre-pathological alterations in marine organisms, before severe disturbances, such as disease, mortality, or population changes occur.