Genotoxic effects induced by the exposure to an environmental mixture of illicit drugs to the zebra mussel.

Despite the growing interest on the presence of illicit drugs in freshwater ecosystems, just recently the attention has been focused on their potential toxicity towards non-target aquatic species. However, these studies largely neglected the effects induced by exposure to complex mixtures of illicit drugs, which could be different compared to those caused by single psychoactive molecules. This study was aimed at investigating the genetic damage induced by a 14-day exposure to a realistic mixture of the most common illicit drugs found in surface waters worldwide (cocaine, benzoylecgonine, amphetamine, morphine and 3,4-methylenedioxymethamphetamine) on the zebra mussel (Dreissena polymorpha). The mixture caused a significant increase of DNA fragmentation and triggered the apoptotic process and micronuclei formation in zebra mussel hemocytes, pointing out its potential genotoxicity towards this bivalve species.

Sublethal effects induced by morphine to the freshwater biological model Dreissena polymorpha.

Opioids are considered as emerging contaminants in aquatic ecosystems, mainly due to their large illicit consume worldwide. Morphine (MOR) is the main opiate and it was commonly found at measurable concentrations in freshwaters. Even though its occurrence is well documented, just limited information is available regarding its hazard to nontarget organisms. The aim of this study was of the evaluation of sublethal effects induced by MOR to the freshwater bivalve Dreissena polymorpha. We exposed mussels to two MOR concentrations (0.05 µg/L and 0.5 µg/L) for 14 days and we investigated the sublethal effects by a suite of biomarkers. The Neutral Red Retention Assay (NRRA) was used as a test of cytotoxicity, while the oxidative stress was evaluated by the activity of antioxidant and detoxifying enzymes, namely catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and by measuring the levels of lipid peroxidation (LPO) and protein carbonylation (PCC). The genetic damage was assessed by the Single Cell Gel Electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test). Finally, the filtration rate of D. polymorpha was evaluated in order to investigate possible physiological effects. Both tested concentrations reduced the lysosome membrane stability of bivalves, but only the highest MOR concentration induced significant changes in the activity of antioxidant enzymes (SOD, CAT, and GPx) and increase in lipid peroxidation levels. Slight increase in primary DNA fragmentation was noticed, while no fixed genetic damage and alterations of the filtering rate were found.

Comparison of plastic pollution between waters and sediments in four Po River tributaries (Northern Italy).

The monitoring of plastic contamination in freshwaters is still pioneering in comparison with marine environments, and few studies analyzed the distribution of these pollutants in both aqueous and bottom compartments of continental waters. Therefore, the aim of this study was the comparison of plastic pollution in both waters and sediments of four Po River tributaries (Ticino, Adda, Oglio and Mincio Rivers), which outflow from the main Italian sub-alpine Lakes, in order to establish the strengths and weaknesses of both matrices. The main results pointed out a heterogeneous plastic contamination, with the lowest values in Ticino (0.9 ± 0.5 plastics/m3 in waters and 6.8 ± 4.5 plastics/kg dry weight - d.w. - in sediments) and the highest in Mincio (62.9 ± 53.9 plastics/m3 in waters and 26.5 ± 13.3 plastics/kg d.w in sediments), highlighting a plastic amount in sediments four times higher than waters. Plastic pollution, mainly due to microplastics, was associated principally to a domestic input in both waters and sediments of Ticino and Adda Rivers, as well as in sediments of Oglio, while an industrial pollution was found in waters and sediments of Mincio and Oglio waters. Our data clearly highlighted as the monitoring of both matrices provide complementary information for a holistic risk assessment of these emerging contaminants in freshwaters: the aqueous matrix provides an instantaneous picture of contamination, while sediments the history of pollution.

Monthly variability of floating plastic contamination in Lake Maggiore (Northern Italy).

The monitoring of plastics in freshwater ecosystems has witnessed a significant increase in recent years, driven by the awareness that approximately 80 % of marine plastic litter originates from terrestrial sources transported to the seas through lakes and rivers. Consequently, it is imperative to develop monitoring plans that offer a comprehensive understanding of plastic contamination in these aquatic environments, given their seasonal variations in hydrochemical characteristics and anthropogenic sources. Historically, most global lake monitoring campaigns have been limited to one-time or, at most, seasonal sampling. In this context, the primary objective of the present study was to assess the quantitative and qualitative monthly variations of floating plastics in Lake Maggiore, a large European lake with high ecological and economic significance. Twelve transverse transects were conducted from January to December 2022 using a Manta-net with a 100 µm mesh. Characterization of each plastic particle was performed using a µ-Fourier Transform Infrared Spectroscope (µFT-IR). The results revealed relatively low levels of contamination in Lake Maggiore when compared with other lakes worldwide exclusively from a secondary origin. However, a considerable heterogeneity was observed, both quantitatively and qualitatively. Notably, we identified a 13-fold difference between the minimum (0.02 plastics/m3 in September) and maximum (0.29 plastics/m3 in December) concentrations of plastics, accompanied by significant variations in polymer composition. Our monitoring underscored the necessity of also considering the temporal variation as a potential factor influencing plastic contamination in a lake. Moreover, frequent sampling emerged as a crucial requirement to accurately gauge the extent of plastic pollution, yielding robust and valuable data essential for effective environmental management.

Unveiling the multilevel impact of four water-soluble polymers on Daphnia magna: From proteome to behaviour (a case study).

The ubiquitous presence of water-soluble polymers (WSPs) in freshwater environments raises concerns regarding potential threats to aquatic organisms. This study investigated, for the first time, the effects of widely used WSPs -polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), and polyethylene glycol (PEG)- using a multi-level approach in the freshwater biological model Daphnia magna. This integrated assessment employed a suite of biomarkers, evaluation of swimming behaviour, and proteomic analysis to investigate the effects of three environmentally relevant concentrations (0.001, 0.5, and 1 mg/L) of the tested WSPs from molecular to organismal levels, assessing both acute and chronic effects. Our findings reveal that exposure to different WSPs induces specific responses at each biological level, with PEG being the only WSP inducing lethal effects at 0.5 mg/L. At the physiological level, although all WSPs impacted both swimming performance and heart rate of D. magna specimens, PAA exhibited the greatest effects on the measured behavioural parameters. Furthermore, proteomic analyses demonstrated altered protein profiles following exposure to all WSPs, with PVA emerging as the most effective.

Plastics in biogenic matrices intended for reuse in agriculture and the potential contribution to soil accumulation.

The spread of biogenic matrices for agricultural purposes can lead to plastic input into soils, raising a question on possible consequences for the environment. Nonetheless, the current knowledge concerning the presence of plastics in biogenic matrices is very poor. Therefore, the objective of the present study was a quali-quantitative characterization of plastics in different matrices reused in agriculture as manures, digestate, compost and sewage sludges. Plastics were quantified and characterized using a Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in Attenuated Total Reflectance mode. Our study showed the presence of plastics in all the investigated samples, albeit with differences in the content among the matrices. We measured a lower presence in animal matrices (0.06-0.08 plastics/g wet weight w.w.), while 3.14-5.07 plastics/g w.w. were measured in sewage sludges. Fibres were the prevalent shape and plastic debris were mostly in the micrometric size. The most abundant polymers were polyester (PEST), polypropylene (PP) and polyethylene (PE). The worst case was observed in the compost sample, where 986 plastics/g w.w. were detected. The majority of these plastics were compostable and biodegradable, with only 8% consisting of fragments of PEST and PE. Our results highlighted the need to thoroughly evaluate the contribution of reused matrices in agriculture to the plastic accumulation in the soil system.

To spread or not to spread? Assessing the suitability of sewage sludge and other biogenic wastes for agriculture reuse.

Sewage sludge (biosolids) management represents a worldwide issue. Due to its valuable properties, approximately one half of the EU production is recovered in agriculture. Nevertheless, growing attention is given to potential negative effects deriving from the presence of harmful pollutants. It is recognized that a (even very detailed) chemical characterization is not able to predict ecotoxicity of a mixture. However, this can be directly measured by bioassays. Actually, the choice of the most suitable tests is still under debate. This paper presents a multilevel characterization protocol of sewage sludge and other organic residues, based on bioassays and chemical-physical-microbiological analyses. The detailed description of the experimental procedure includes all the involved steps: the criteria for selecting the organic matrices to be tested and compared; the sample pre-treatment required before the analyses execution; the chemical, physical and microbiological characterisation; the bioassays, grouped in three classes (baseline toxicity; specific mode of action; reactive mode of action); data processing. The novelty of this paper lies in the integrated use of advanced tools, and is based on three pillars:•the direct ecosafety assessment of the matrices to be reused.•the adoption of innovative bioassays and analytical procedures.•the original criteria for data normalization and processing.

Application of a biomarker response index for ranking the toxicity of five pharmaceutical and personal care products (PPCPs) to the bivalve Dreissena polymorpha.

Pharmaceuticals and personal care products (PPCPs) have been detected in several aquatic ecosystems during the last two decades, but their potential for biological effects to nontarget organisms is only now being studied. The aim of this study was to compare and rank the cyto-genetic effects induced by 96-hour exposure to an environmental concentration (1 nM) of triclosan (TCS), trimethoprim (TMP), diclofenac (DCF), ibuprofen (IBU), and paracetamol (PCM) on the freshwater bivalve Dreissena polymorpha by integrating biological responses of eight biomarker into a simple biomarker response index (BRI). The application of the BRI decreased the wide biomarker variability and enabled toxicity ranking of the tested PPCPs as follow: TCS > TMP > IBU > DCF = PCM. This approach allowed us to draw an accurate PPCP scale of toxicity of the most dangerous drug and to address further in-depth investigations.

First comparative assessment of contamination by plastics and non-synthetic particles in three bivalve species from an Italian sub-alpine lake.

This study aimed to compare the contamination from plastics and non-synthetic particles in the three freshwater bivalve mollusks Unio elongatulus, (native) and Corbicula fluminea and Dreissena polymorpha (invasive), collected in Lake Maggiore, the second greatest Italian lake. Organisms were collected from eight sites located throughout the lake, during three years (2019-2021). The quali-quantitative characterization of particles has been carried out using a Fourier Transform Infrared Microscope System (µFT-IR). Results showed that both plastics and non-synthetic particles released in the water are taken up by bivalves, even though low intake-up to 6 particles/individuals-were measured for all the three species. Microfibers of both synthetic (polyester, polyamide) and natural (cellulose) origin represented the particles mostly ingested by bivalves. A significant decrease of particle loads was observed in 2020 with respect to 2019 and 2021, significantly different for D. polymorpha and U. elongatulus, suggesting a transient reduction of the particle release in the lake in this year. Our findings highlight the need to improve the understanding of the mechanisms of uptake and clearance of these contaminants by filter feeding organisms, and their adverse consequences in realistic environmental conditions.

Packaging contaminants in former food products: Using Fourier Transform Infrared Spectroscopy to identify the remnants and the associated risks.

Food waste and feed-food competition can be reduced by replacing traditional feed ingredients such as cereals, with former food products (FFPs) in livestock diets. These foodstuffs, initially intended for human consumption, are recovered, mechanically unpacked, and then ground. Despite this simple and inexpensive treatment, packaging contaminants (remnants) are often unavoidable in the final product. To maximize the exploitation of FFPs and to minimize the associated risks, packaging remnants need to be quantified and characterized. This study tested the efficacy of the Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in identifying packaging remnants in 17 FFP samples collected in different geographical areas. After a visual sorting procedure, presumed packaging remnants were analyzed by µFT-IR. The results showed significant differences (p < 0.05) between the FFPs in terms of the total number of foreign particles found (plastics, cellulose and aluminum remnants, ranging from 4 to 19 particles per 20 g fresh matter), and also regarding the number of cellulose and aluminum particles. These data clearly demonstrate the need for sensitive instruments that can characterize the potential contaminants in the FFPs. This would then help to reduce the overestimation of undesirable contaminants typical of simple visual sorting, which is currently the most common method.

Assessment of behavioural effects of three water-soluble polymers in zebrafish embryos.

The water-soluble polymers (WSPs) are widely used in many industrial applications and are present in several commonly used products due to their physical-chemical characteristics: as their name indicates, despite being synthetic polymers, they are able to solubilize in water. Because of this peculiar property, both the qualitative-quantitative evaluation in aquatic ecosystems and their potential (eco)toxicological effects have been neglected until now. The aim of this study was to evaluate the possible effects of three of the most widely used WSPs as polyacrylic acid (PAA), polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) on the swimming behaviour of zebrafish (Danio rerio) embryos after the exposure to different concentrations (0.001, 0.5, 1 mg/L). The exposure lasted from the eggs' collection up to 120 h post fertilization (hpf) also using three different light intensity (300 lx, 2200 lx, 4400 lx) to better evaluate any effects related to different gradients of light/dark transitions. In order to analyze individual behavioural changes in embryos, their swimming movements were tracked and a number of parameters for locomotion and directionality were quantified. The main results showed that all three WSPs resulted in significant (p ≤ 0.05) variations in different movement parameters, suggesting a possible toxicity scale: PVP > PEG â‰« PAA.

Cyto-genotoxic effects induced by three brominated diphenyl ether congeners on the freshwater mussel Dreissena polymorpha.

Polybrominated diphenyl ethers (PBDEs) are a group of highly hydrophobic and persistent chemicals that has been used as flame retardants in several industrial applications. They have been detected in various environmental matrices worldwide and an increasing number of studies have recently been carried out to investigate their potential toxicity on ecosystem communities. Although a variety of biological damage has been documented in vertebrates, the effects on invertebrates are largely unknown. The objective of the present study was to determine the cyto-genotoxic effects induced by single exposure to three concentrations of 2,4,2',4'-tetra BDE (BDE 47), 2,2',4,4',6-penta BDE (BDE-100) and 2,2',4,4',5,6'-hexa BDE (BDE-154) on the freshwater mussel Dreissena polymorpha by a multi-biomarker approach. We performed on bivalve hemocytes the Single Cell Gel Electrophoresis (SCGE) assay, the DNA Diffusion assay and the Micronucleus test (MN test) to assess genotoxicity, while the Neutral Red Retention Assay (NRRA) was used to evaluate cytotoxic effects. Results showed that BDE-47 did not produce any genetic damage at the tested concentrations (0.1 µg/L, 0.5 µg/L and 1 µg/L), while BDE-100 and BDE-154 can be considered moderately genotoxic, since both primary and fixed DNA injuries were induced. The NRRA indicated a moderate increase in cellular stress in BDEs-treated bivalves. Thus, our data seems to suggest that investigated BDEs may pose a low risk to freshwater mussels at environmental concentrations.

Sub-lethal effects induced by a mixture of three non-steroidal anti-inflammatory drugs (NSAIDs) on the freshwater bivalve Dreissena polymorpha.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the sixth top-selling drugs worldwide and are commonly found in freshwater ecosystems in the high ng/l to low µg/l range. Recent studies have investigated both the acute and the chronic toxicity of single NSAIDs on different biological models, but these studies have completely neglected the fact that, in the environment, non-target organisms are exposed to mixtures of drugs that have unforeseeable toxicological behavior. This work investigated the sub-lethal effects induced by a mixture of three common NSAIDs, namely, diclofenac, ibuprofen and paracetamol, on the freshwater bivalve, the zebra mussel (Dreissena polymorpha). The mussels were exposed to three different environmental concentrations of the mixture (Low, Mid and High). A multi-biomarker approach was used to highlight cyto-genotoxic effects and the imbalance of the oxidative status of the treated specimens. The Neutral Red Retention Assay (NRRA) was used as a biomarker of cytotoxicity, whereas the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase were measured to assess the role played by the oxidative stress enzymes. In addition, the single cell gel electrophoresis assay, the DNA Diffusion assay and the micronucleus test were used to investigate possible genotoxic effects. According to our NRRA results, each treatment was able to induce a significant cellular stress in bivalves, probably due to the raise of oxidative stress, as indicated by the alteration of enzyme activities measured in treated specimens. Moreover, the mixture induced significant enhancements of DNA fragmentation, which preluded fixed genetic damage, as highlighted by the increase of both apoptotic and micronucleated cells.

Polybrominated diphenyl ether contamination in soil, vegetation, and cow milk from a high-mountain pasture in the Italian Alps.

This study investigates contamination by 13 polybrominated diphenyl ether (PBDE) congeners in a high-mountain pasture located in the Italian Alps. The trend of PBDE contamination in three soil layers was investigated by a spring-summer sampling period to understand the importance of different environmental variables, such as seasonality, topographical aspect, and soil features. We also evaluated PBDE accumulation in grasses covering the plateau, and because the study area has been used for a pasture for a long time, we measured PBDE levels in milk from grazing cows. Overall, we found moderate PBDE contamination in Andossi soils, with differences depending on the layer, mountain side, and organic matter content. The vegetation of the plateau had greater PBDE concentrations than the soils and showed a grass/soil accumulation ratio between 2 and 12, indicating that deposition from the atmosphere is actually the dominant process in this area. Last, PBDE concentrations in milk followed similar seasonal trends as the vegetation but showed variations in congener abundance in agreement with the biotransformation susceptibility, absorption efficiency, and residence time of different BDEs in dairy cattle.

New evidences in the complexity of contamination of the lagoon of Venice: polybrominated diphenyl ethers (PBDEs) pollution.

This study presents the first evaluation of the current pollution by polybrominated diphenyl ethers (PBDEs) of surface sediments from the Lagoon of Venice. We focused the research on tri-to hepta-BDEs, the main components of penta- and octa-mixtures, which are considered to be the most toxic for the biocoenosis. The results pointed out a quite homogeneous contamination of this keystone European transitional environment, with ∑ (13)PBDEs values ranging from 0.39 to 6.78 ng/g dry weight; these values reflect low to moderate pollution levels, which is in conformity to other coastal European ecosystems. The average PBDE profile of the lagoon sediments follows this decreasing trend of congeners: BDE-47>BDE-99> >BDE-190>BDE-28>BDE-153>BDE-154>BDE-138, BDE-183, and BDE-17, which is similar to the worldwide distribution pattern. BDE-47 and BDE-99 revealed a recent use of a penta-BDE mixture, while the presence of hepta-BDEs (BDE-183 and BDE-190) in all of the sites can indicate the actual use of a deca-BDE formulation, because these congeners are considered to be debrominated byproducts of BDE-209 degradation.

Can COVID-19 pandemic change plastic contamination? The Case study of seven watercourses in the metropolitan city of Milan (N. Italy).

The more or less extensive lockdowns, quarantines, smart working and the closure of numerous recreational or personal care activities due to the COVID-19 pandemic have not only heavily changed the habits and behaviors of all of us, but have also had consequences on the release of some types of pollutants. The aim of this study was to evaluate the possible changes due to the indirect effects of the pandemic in the contamination of plastic mixtures sampled in 9 sites of the main watercourses of the metropolitan city of Milan (N. Italy), which is one of the major industrialized and urbanized areas in Italy. To achieve this goal, we carried out two sampling campaigns, the first one carried out in November 2019, before the arrival of the SARS-CoV-2 virus in Italy, the second in November 2020, during a severe regional lockdown that coincided with other restrictions imposed at the national level. The main results showed a difference in contamination of plastics between the two samplings, not so much due to a quantitative variation, but certainly qualitative. We obtained non-homogeneous data with respect to changes in the number of plastics sampled in the different waterbodies, while it was evident that the plastics' contamination has shifted from a primary and industrial origin to one due to a secondary origin of the sampled plastics, linked especially to the fragmentation of common use objects, or deriving from synthetic garments.

A realistic approach for the assessment of plastic contamination and its ecotoxicological consequences: A case study in the metropolitan city of Milan (N. Italy).

The study of the contamination of plastic mixtures sampled in natural environments is currently focused on their qualitative and quantitative assessment, while the evaluation of their effects on organisms is normally performed by experiments carried out at exposure conditions (size, shape, polymers) often far from the environmental ones. To improve the ecological realism, the aim of this study was to collect different plastic mixtures in 9 sampling stations located in 7 watercourses within the metropolitan city of Milan, one of the most anthropized and industrialized European areas, to evaluate both their qualitative and quantitative characteristics and, at the same time, to assess their ecotoxicological effects by exposing for 7 days some specimens of the freshwater bivalve Dreissena polymorpha to the mixtures collected in the sampling sites. The plastic characterization was performed by a Fourier-Transform Infrared spectrometer coupled with an optical microscope (µFT-IR), after several stages aimed to sample cleaning, separation of plastics and visual sorting. The possible effects caused by the plastic mixtures were carried out by the measurements of a biomarker suite to evaluate many cellular and molecular endpoints in mussel tissues. The main results showed a widespread and heterogeneous contamination of plastics in the entire metropolitan area, with contamination peaks found above all in the only two rivers of natural origin (Olona River and Lambro River) where comparable or higher values were reached than plastic concentrations measured in several European rivers. Despite this worrying contamination, the ecotoxicological data obtained after the exposures to the plastic mixtures collected in the selected water bodies showed only a mild effect on oxidative stress and on the variation of some antioxidant enzymes.

Are "liquid plastics" a new environmental threat? The case of polyvinyl alcohol.

Despite the pollution induced by plastics become a well-known and documented problem, bringing many countries to adopt restrictions about their production, commercialization and use, the impact of another emerging category of synthetic polymers, represented by the Water-Soluble Polymers (WSPs), also known as "liquid plastics", is overlooked by scientific community. WSPs are produced in large quantities and used in a wide plethora of applications such as food packaging, pharmaceuticals and personal care products, cosmetics and detergents, with a consequent continuous release in the environment. The aim of this study was the investigation of the possible toxicity induced by polyvinyl alcohol (PVA), one of the main produced and used WSPs, on two freshwater model organisms, the crustacean Daphnia magna and the teleost Danio rerio (zebrafish). We evaluated the effects of solubilized standard PVA powder and PVA-based commercial bags for carp-fishing, at 3 different concentrations (1 µg/L, 0.5 mg/L and 1 mg/L), through the exposures for 14 days of D. magna (daphnids; age < 24 h) and for 5 days of zebrafish embryos (up to 120 h post fertilization - hpf). As acute effects we evaluated the immobilization/mortality of specimens, while for chronic toxicity we selected several endpoints with a high ecological relevance, as the behavioural alteration on swimming performance, in real-time readout, and the activity of monoamine oxidase (MAO), a neuro-enzyme with a potential implication in the organism movement. The results showed the lack of significant effects induced by the selected substances, at all tested concentrations and in both model organisms. However, considering the wide plethora of available WSPs, other investigations are needed to provide the initial knowledge of risk assessment of these compounds contained in some consumer products.

Surviving in a changing ocean. Tolerance to acidification might affect the susceptibility of polychaetes to chemical contamination.

This study aimed to assess the combined effects of ocean acidification (OA) and pollution to the polychaete Syllis prolifera inhabiting the CO2 vent system of the Castello Aragonese (Ischia Island, Italy). We investigated the basal activities of antioxidant enzymes in organisms from the acidified site and from an ambient-pH control site in two different periods of the year. Results showed a limited influence of acidified conditions on the functionality of the antioxidant system. We then investigated the responsiveness of individuals living inside the CO2 vent compared to those from the control to face exposure to acetone and copper. Results highlighted a higher susceptibility of organisms from the vent to acetone and a different response of antioxidant enzymes in individuals from the two sites. Conversely, a higher tolerance to copper was observed in polychaetes from the acidified-site with respect to controls, but any significant oxidative stress was induced at sublethal concentrations.

Chronic effects induced by ibuprofen on the freshwater bivalve Dreissena polymorpha.

The sub-lethal effects induced by the non-steroidal anti-inflammatory drug ((NSAID) ibuprofen (IBU; ((±)-2-(p-isobutylphenyl) propionic acid))) were investigated using a battery of biomarkers on the freshwater bivalve Dreissena polymorpha. According to the results from a semi-static in vivo approach, mussels were exposed for 96 h to increasing levels of environmentally relevant IBU concentrations (0.2, 2 and 8 µg/l, corresponding to 1, 9 and 35 nM, respectively). Cyto-genotoxicity was evaluated via the single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay, the micronucleus test (MN test) and lysosome membrane stability (Neutral Red Retention Assay) in mussel hemocytes. In addition, the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and the phase II detoxifying enzyme glutathione S-transferase (GST) were measured in the cytosolic fraction that was extracted from a pool of entire bivalves to determine whether the oxidative status was imbalanced. The biomarker battery pointed out a slight cyto-genotoxicity on zebra mussel hemocytes at the IBU concentration of 0.2 µg/l, with higher IBU concentrations able to significantly increase both genetic and cellular damage. In addition, IBU seems to have a considerable effect on the activities of antioxidant and detoxifying enzymes as shown in the exposed specimens' notable oxidative status imbalances.