Reductive stress and cytotoxicity in the swollen river mussel (Unio tumidus) exposed to microplastics and salinomycin.

Multistress effects lead to unpredicted consequences in aquatic ecotoxicology and are extremely concerning. The goal of this study was to trace how specific effects of the antibiotic salinomycin (Sal) and microplastics (MP) on the bivalve molluscs are manifested in the combined environmentally relevant exposures. Unio tumidus specimens were treated with Sal (0.6 µg L-1), MP (1 mg L-1, 2 µm size), and both at 18 °C (Mix) and 25 °C (MixT) for 14 days. The redox stress and apoptotic enzyme responses and the balance of Zn/Cu in the digestive gland were analyzed. The shared signs of stress included a decrease in NAD+/NADH and Zn/Cu ratios and lysosomal integrity and an increase in Zn-metallothioneins and cholinesterase levels. MP caused a decrease in the glutathione (GSH) concentration and redox state, total antioxidant capacity, and Zn levels. MP and Mix induced coordinated apoptotic/autophagy activities, increasing caspase-3 and cathepsin D (CtD) total and extralysosomal levels. Sal activated caspase-3 only and increased by five times Cu level in the tissue. Due to the discriminant analysis, the cumulative effect was evident in the combined exposure at 18 °C. However, under heating, the levels of NAD+, NADH, GSH, GSH/GSSG and metallothionein-related thiols were decreased, and coordination of the cytosolic and lysosomal death stimuli was distorted, confirming that heating and pollution could exert unexpected synergistic effects on aquatic life.

The odd couple: Caffeine and microplastics. Morphological and physiological changes in Mytilus galloprovincialis.

In recent years, the presence of pharmaceuticals and microplastics (MPs) in aquatic ecosystems has raised concerns about their environmental impact. This study explores the combined effects of caffeine, a common pharmaceutical pollutant, and MPs on the marine mussel Mytilus galloprovincialis. Caffeine, at concentrations of 20.0 µg L-1, and MPs (1 mg L-1, 35-50 µm size range), was used to mimic real-world exposure scenarios. Two hundred M. galloprovincialis specimens were divided into four groups: caffeine, MPs, Mix (caffeine + MPs), and Control. After a two-week acclimation period, the mollusks were subjected to these pollutants in oxygen-aerated aquariums under controlled conditions for 14 days. Histopathological assessments were performed to evaluate gill morphology. Cellular volume regulation and digestive gland cell viability were also analyzed. Exposure to caffeine and MPs induced significant morphological changes in M. galloprovincialis gills, including cilia loss, ciliary disk damage, and cellular alterations. The chitinous rod supporting filaments also suffered damage, potentially due to MP interactions, leading to hemocyte infiltration and filament integrity compromise. Hemocytic aggregation suggested an inflammatory response to caffeine. In addition, viability assessments of digestive gland cells revealed potential damage to cell membranes and function, with impaired cell volume regulation, particularly in the Mix group, raising concerns about nutrient metabolism disruption and organ function compromise. These findings underscore the vulnerability of M. galloprovincialis to environmental pollutants and emphasize the need for monitoring and mitigation efforts. RESEARCH HIGHLIGHTS: The synergy of caffeine and microplastics (MPs) in aquatic ecosystems warrants investigation. MPs and caffeine could affect gill morphology of Mytilus galloprovincialis. Caffeine-exposed cells had lower viability than the control group in the NR retention test. MPs and mix-exposed cells struggled to recover their volume.

Biochemical basis of resistance to multiple contaminations in the native and invasive populations of Dreissena polymorpha.

The zebra mussel Dreissena polymorpha (Pallas, 1771) is an invasive species and a valuable bioindicator in the inland waters. Nevertheless, the biochemical reasons for the unique competitiveness of zebra mussels are not clear. This study aimed to compare the native and invasive populations of D. polymorpha in their ability to withstand the same multiple environmental challenges (i.e. chemical: microplastics and caffeine; physical: temperatures). The specimens from the invasive population in west Ukraine (Tn) and native population at lower streams of river Dnipro (south Ukraine, Kh) were sampled in the August of 2021 y. Molluscs from both populations were treated simultaneously with microplastics (MP, 1 mg L-1, pore size 2 µm); caffeine (Caf, 20.0 µg L-1) at 18 °C, elevated temperature (25 °C) and MP and Caf combinations at 18 °C and 25 °C for 14 days. Untreated molluscs exposed at 18 °C represented control groups. A set of the 20 markers of oxidative stress, biotransformation, detoxification and apoptosis were assayed in the total soft tissues. From the two controls, Kh-group indicated lower stressful impact. However, both populations increased caspase-3 and GST activities and lysosomal instability in most exposures, and cholinesterase and phenoloxidase activities under the heating and combine exposures, indicating the remarkable properties to respond to new challenges and synergistic effect of mixtures. Inter-population differences were related to the metallothionein, cathepsin D, cytochrome P540 and oxidative stress responses that reflect population-dependent adverse outcome pathways. The discriminant analysis separated two populations with a substantially lesser magnitude of responses in the invasive population as a sign of higher resistance.

Chlorpromazine's impact on Mytilus galloprovincialis: a multi-faceted investigation.

The antipsychotic chlorpromazine (Cpz) has raised concern as a pharmaceutical effluent due to its wide medical applications. Moreover, its potent pro-oxidant properties and impact on the cell viability of the marine mollusc Mytilus galloprovincialis, even at low concentrations (ng/L), have been noted. Based on this evidence, in this study, we investigated the physiological effects of Cpz on M. galloprovincialis, to elucidate its fate within the organism, in terms of bioaccumulation, biotransformation, byssus changes and stress responses of the cellular thiolome. Histological and indicators of vitality analyses were also performed to better evaluate the influence of the drug on the morphology and cell viability of the digestive gland. To this end, two different concentrations of Cpz (Cpz I (12 ng/L or 37 pM) and Cpz II (12 µg/L or 37 nM)) were administered to mussels over 14 days. Cpz accumulation in the digestive gland significantly increased with water concentration (BCF of Cpz I and Cpz II). Biochemical analyses indicated lysosomal dysfunction, reflected in elevated total Cathepsin D activity and compromised lysosomal membrane stability. Stress-related and metal-buffering proteins (GST and metallothionein) responded to both Cpz concentrations. Cpz I induced phase I biotransformation activity (CYP450-dependent EROD), while Cpz II triggered caspase-3 activation, indicative of detoxification overload. Histological analysis revealed digestive gland atrophy, epithelial thinning, haemocyte infiltration, and brown cell presence. Byssus analysis showed significant alterations. In conclusion, our study underscores Cpz-induced physiological and histological changes in M. galloprovincialis, posing potential implications for mussel health and confirming the utilisation of this mussel as an indication of Cpz ecotoxicity.

Long-term toxicity of chlorpromazine, diclofenac and two lanthanides on three generations of Ceriodaphnia dubia.

Amultigenerational study on Ceriodaphnia dubia was carried out by exposing three subsequent generations to pharmaceuticals chlorpromazine (CPZ) and diclofenac (DCF), and two lanthanide chlorides, gadolinium as GdCl3 and europium as EuCl3. As the treatments, environmentally relevant concentrations were chosen (0.001, 0.01 and 0.1 mg/L for CPZ; 0.1, 1 and 10 mg/L for DCF; 0.425, 4.25 and 42.5 µg/L for Gd and 0.41, 4.1 and 41 µg/L for Eu). Survival, population growth and reproduction success were evaluated at 21 and 30 days of exposure, and the whole observation period lasted 40 days. The least sensitive to all selected substances was the first daphnid generation (F1). Within 21-day exposure, no significant effects of the psychotropic drug CPZ on C. dubia survival were observed in generations F1-F3. The anti-inflammatory drug DCF did not affect survival in the F1 generation; however, it significantly reduced survival in the F3 generation at 1-10 mg/L. Both lanthanides did not affect survival in the F1 and F2 generations of C. dubia but considerably decreased survival in the F3 at 4-42 µg/L. Both pharmaceuticals stimulated the reproduction of C. dubia in the F1 generation, while inhibition occurred at the highest tested concentrations in generations F2 and F3. The inhibitory effect on the reproductive success of lanthanides in the F2 generation resembled that for CPZ but not for DCF. The dynamics of adverse effects during the 21-30-day period revealed that despite increased mortality in the controls (up to 30%), concentrations used in the study minified, in most instances, the survival and aggravated population growth and reproduction success of C. dubia. Our data suggest that C. dubia as a test organism can be used for 21 days in multigenerational investigations, especially when testing close to environmental concentrations. In this respect, the standard C. dubia chronic toxicity assay seems limited since prolonged observations and several generations of daphnids are required to obtain reliable information for the risk assessment of potentially aggressive chemicals.

Cellular and oxidative stress responses of Mytilus galloprovincialis to chlorpromazine: implications of an antipsychotic drug exposure study.

Introduction: Bivalve molluscs like Mytilus galloprovincialis are valuable bioindicators due to their filter-feeding lifestyle, wide distribution, and ability to concentrate xenobiotics. Studying the effects of pharmaceuticals on these molluscs is crucial given their presence in surface waters. This study investigated the response of M. galloprovincialis to chlorpromazine (Cpz), an antipsychotic with antiviral activity against influenza, HIV, and coronaviruses in human cells. Methods: In this study, we examined the 14-day impact of chlorpromazine (Cpz) on the model species M. galloprovincialis at two concentrations (Cpz 1: 12 ng L-1 or 37 pM; Cpz 2: 12 µg L-1 or 37 nM). To ensure controlled exposure, a stock solution of Cpz was prepared and introduced into the tanks to match the intended concentrations. Seawater and stock solutions were refreshed every 48 h. The primary focus of this study centered on evaluating cell viability, cell volume regulation, and oxidative stress indicators. Results: Although cell volume regulation, as assessed by decreasing regulatory volume Regulation volume decrease, did not show statistically significant changes during the experiment, digestive cell viability, on the other hand, showed a significant decrease (p < 0.01) in the Cpz 2 group, suggesting effects on the general health and survival of these cells. Biochemically, in both Cpz 1 and Cpz 2, superoxide dismutase activity increased, while catalase (CAT) decreased, causing an elevated lipid peroxidation thiobarbituric acid-reactive substances and protein carbonyls, particularly in the Cpz 2 group. The level of reduced glutathione (GSH) increased in both exposures, whereas the level of GSSG increased only in the Cpz 1 group. Consequently, the GSH/GSSG ratio was elevated in the Cpz 2 group only. Discussion: A comparison of the magnitudes of anti- and pro-oxidative manifestations indicated a pro-oxidative shift in both exposures. These findings show that Cpz induces non-specific symptoms of biochemical and cellular disturbances in M. galloprovincialis even at the low picomolar concentration.

Combined effect of microplastic, salinomycin and heating on Unio tumidus.

Microplastic (MP) and heating (T) suspected to modulate biological effects of aquatic contaminants. Salinomycin (Sal) is veterinary antibiotic and anticancer agent. The goal of this study was to examine the multistress effect of MP, Sal and T on the bioindicator bivalve mollusc. The Unio tumidus were treated with MP (1 mg L-1), Sal (0.6 µg L-1), their combination under 18° C (Mix) and 25° C (MixT) for 14 days. The digestive glands were analyzed. MP and Sal did not cause changes of Mn- and Cu,Zn-SOD, lipid peroxidation and Cyp-450-depended EROD levels, whereas catalase, GST and protein carbonyls (Sal-group) increased compared to control. In the Mix-group, enzymes, particularly EROD and GST (by 34% and 115% respectively) were up-regulated. However, in the MixT-group, they were corresponding to control or lesser (EROD, catalase). Our findings emphasize the need to take into account multistress interactions in the MP environmental risk assessment.

Sublethal cadmium exposure in the freshwater snail Lymnaea stagnalis meets a deficient, poorly responsive metallothionein system while evoking oxidative and cellular stress.

The Great Pond snail Lymnaea stagnalis (Gastropoda, Hygrophila) is a wide-spread freshwater gastropod, being considered as a model organism for research in many fields of biology, including ecotoxicology. The aim of the present study was to explore the Cd sensitivity of L. stagnalis through the measurement of a biomarker battery for oxidative, toxic and cellular stress. The interpretation of biomarker parameters occurred against the background of a truncated metallothionein protein with a limited Cd-binding capacity. Individuals of L. stagnalis were exposed through 14 days to uncontaminated water (controls) or to low (30 µg · L-1) or high (50 µg · L-1) Cd concentrations. The digestive gland of control and low-Cd exposed snails was processed for transcriptional analysis of the Metallothionein (MT) gene expression, and for determination of biomarkers for oxidative stress, toxicity and cellular stress. Digestive gland supernatants of high-Cd exposed snails were subjected to chromatography and subsequent analysis by spectrophotometry. It was shown that the MT system of L. stagnalis is functionally deficient, with a poor Cd responsiveness at both, the transcriptional and the protein expression levels. Instead, L. stagnalis appears to rely on alternative detoxification mechanisms such as Cd binding by phytochelatins and metal inactivation by compartmentalization within the lysosomal system. In spite of this, however, traces of Cd apparently leak out of the pre-determined detoxification pathways, leading to adverse effects, which is clearly indicated by biomarkers of oxidative and cellular stress.

Stress responses of bivalve mollusc Unio tumidus from two areas to ibuprofen, microplastic and their mixture.

Even though bivalve molluscs are recognized as bioindicators of freshwater quality, their responses to multiple stressors are unpredictable. This study aims to elucidate the inter-population peculiarities of the effect in the sub-chronic environmentally relevant exposure to novel contaminants. The specimens of Unio tumidus from reference (Pr) and contaminated (Ct) areas were treated with ibuprofen (IBU, 0.8 µg L-1), microplastic (MP, 1.0 mg L-1, size 0.1-0.5 mm), or their combination (Mix) for 14 days. Untreated mussels (PrC- and CtC-groups) served as controls. The PrC-group had higher levels of antioxidants Mn-SOD, Cu,Zn-SOD, catalase, and cholinesterase (AChE) as well as lesser levels of oxidative lesions (TBARS and protein carbonyls) in digestive glands, indicating lower environmental impact than in the CtC-group. However, lysosomal stability was similar in both control groups. Among antioxidants, Mn-SOD activity was affected most prominently, increasing in all exposed Ct-groups. TBARS level was increased only in PrMP-group compared to responsive control. IBU and Mix enhanced protein carbonyl concentration in the Pr-groups, and decreased it in the Ct-groups. AChE was induced in the CtIBU- and PrMix-groups, and lysosomal integrity increased in the CtIBU and CtMix-groups. Discriminant analyses indicated lesser differences between Pr-groups, demonstrating lower cumulative stress compared to Ct-groups. Generally, the most remarkable response was revealed in the CtIBU-group, and distortion of individual effects was established in combined exposures. The qualification of stress-neutral and stress-positive populations was proposed for Pr- and Ct-populations correspondingly. Inter-site peculiarities must be taken into consideration when the environmental impact of MP and pharmaceuticals is evaluated.

Indication of the impact of environmental stress on the responses of the bivalve mollusk Unio tumidus to ibuprofen and microplastics based on biomarkers of reductive stress and apoptosis.

The vulnerability of bivalve mollusks to micropollutants is estimated mainly in single model exposures. However, chronic environmental stress and complex exposures can modulate their responses. To evaluate the impact of population-dependent adaptations on the ability to react to common micropollutants, we compared freshwater bivalves Unio tumidus from two distinct populations, pure (Pr) and contaminated (Ct), in their exposures to microplastics (MP, 1 mg L-1, size 0.1-0.5 mm), pharmaceutical ibuprofen (IBU, 0.8 µg L-1), or their combination (Mix) for 14 days. Control groups from both sites showed remarkable differences, with lower levels of total antioxidant capacity (TAC), metallothionein protein (MTSH), NADH and NAD+, cytochrome P450-related EROD, glutathione-S transferase (GST), and citrate synthase (CS) but higher levels of GSH, GSSG, caspase-3 and cathepsin D (CTD) in the Ct-control group. These data indicate a chronic stress impact in the Ct population. Under exposures, we found an almost common strategy in both populations for NAD+/NADH and MTSH suppression and CTD induction. Additionally, Mix exposure caused an increase in CS, and IBU did not change GSH in both populations. However, the expected response to IBU - the suppression of caspase-3 - was indicated only in PrIBU- and PrMix-mollusks. CTD efflux increased dramatically only in PrMP- and PrMix- groups, and suppression of EROD and GST was detected in the PrMix-group. According to discriminant analysis, exposed Pr-groups were highly differentiated from control, whereas Ct-control and exposed groups had common localization demonstrating high resistance to environmental stress. Thus, the same exposures resulted in different adverse outcome pathways depending on the population.

Does roundup affect zinc functions in a bivalve mollusk in ex vivo exposure?

Roundup (Rn), a glyphosate-based formulation, is one of the most commonly used herbicides in the world. It affects non-targeted organisms in several ways, including adhesive activity towards metal ions. Zinc (Zn) plays a crucial role in a number of biochemical processes. In this study, we aim to elucidate the direct impact of Rn on Zn accumulation and Zn-dependent activities in the ex vivo system. To this end, we exposed the samples of the digestive gland of a bivalve mollusk Unio tumidus to 3 µM of Rn (calculated as 3 µM of glyphosate), Zn, Zn chelator (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) (TPEN, Tp), and their combinations ZnTp and ZnRn for 17 h. We determined the levels of Zn in the tissue (Zn t) and metallothioneins (Zn-MT), metallothioneins (MTSH), and glutathione (GSH & GSSG), total antioxidant capacity (TAC), lysosomal membrane integrity, and caspase-3 activity. Our study demonstrated that Rn and Tp had different effects on the accumulation and functionality of Zn. Rn did not affect the accumulation of Zn (Zn t, Zn-MT) in the Zn- and ZnRn-groups. On the contrary, Tp produced effects antagonistic to Zn on caspase-3 activity, lysosomal stability, and MTSH concentration. Rn caused particular pro-oxidative effect that decreased GSH level (Rn- and ZnRn-groups) and lysosomal stability (Rn-group). The shared affected index was the GSH/GSSG ratio, which decreased by 2-8 times in each exposure. As the first experience with the application of Tp to indicate Zn activity in mollusks, the study concluded that the ex vivo approach could be useful in the study of numeral aquatic pollutants.

Environmental concentrations of Roundup in combination with chlorpromazine or heating causes biochemical disturbances in the bivalve mollusc Unio tumidus.

Bivalve molluscs represent the most recognized bioindicators of freshwater pollution. However, their ability to indicate specific xenobiotics in complex exposures is unclear. In this study, we aimed to track the particular effects of the pesticide Roundup (Rnd) and the antipsychotic drug chlorpromazine (Cpz) on the mussel Unio tumidus at the simpler environmentally relevant models. We treated the mussels by Rnd (17 µg L-1), Cpz (18 µg L-1), the mixture of Rnd and Cpz at 18 °C (RndCpz), and Rnd at 25 °C (RndT) and examined their digestive glands after 14 days of exposure. We analyzed total antioxidant capacity, glutathione (GSH&GSSG) and protein carbonyls levels, total and Zn-related concentrations of metallothioneins (MT and Zn-MT, respectively), the activities of CYP450-related EROD, glutathione S-transferase, cholinesterase, caspase-3, citrate synthase (CS), lysosomal membrane integrity (NRR), and Zn level in the tissue. Shared responses were indicated as the increase of the antioxidant, Zn-MT, and EROD levels, whereas the changes of Zn concentration, NRR, and caspase-3 activity were most diverse compared to control. According to discriminant analysis, complex exposures abolished the individual response traits and intensified the harmful effects that caused a decrease in the Zn level in the RndCpz- and RndT-groups and the loss of lysosomal integrity in the RndT-group. We concluded that multi-marker expertise with the application of integrated indices had benefits when evaluating the effects of complex exposures.

Common and particular biochemical responses of Unio tumidus to herbicide, pharmaceuticals and their combined exposure with heating.

The priority list of freshwater pollutants is increasingly amended by pharmaceuticals. Their impact on the aquatic biota can be modulated by the presence of typical pollutants, like pesticides, and/or abnormal heating. The aim of this study was to elucidate potentially hazardous impact of combined environmental factors on the freshwater mussels by analyzing various sets of biochemical markers. We treated the bivalve molluscs of Unio tumidus with non-steroidal anti-inflammatory drug diclofenac (Dc, 2 nM), calcium antagonist and antihypertensive drug nifedipine (Nf, 2 nM) or organophosphonate glyphosate-based herbicide Roundup MAX (Rn, 79 nM of glyphosate) at 18 °C as well as with the mixture of these substances at 18 °C (Mix) or 25 °C (MixT) during 14 days. The concentrations used were correspondent to the environmentally relevant levels. The biomarkers of stress and toxicity were evaluated in digestive gland, except the lysosomal membrane stability measured in hemocytes. Exposures caused an oxidative stress due to the decreased SOD and GST activities and GSH/GSSG ratio, increased levels of thiobarbituric acid-reactive substances and protein carbonyls (with some exceptions). Dc increased cathepsin D activity in lysosomes. Nf increased lysosomal membrane stability and caspase-3 activity. Rn caused a dramatic distortion of metallo-thiolome due to increased levels of GSH and metallothionein-related thiols (MTSH) as well as depletion of Zn, Cu and Cd in the composition of metallothioneins, and decreased Zn/Cu molar ratio in the tissue. The particular toxicity of Rn was also attested by decreased lysosomal membrane stability and cholinesterase activity. Canonical discriminant analysis separated Rn-, Mix- and MixT-groups from the joint set of C-, Dc- and Nf-groups. Generally, compound-specific effects were expressed in U. tumidus responses to the mixtures, but in MixT-group some effects were particular or extremely strong. Multi-marker approach and integrative analysis proved to be a useful tool for understanding possible future risks to freshwater mussels under a combination of xenobiotics and warming climate.

Long-term changes in microbial water quality indicators in a hydro-power plant reservoir: The role of natural factors and socio-economic changes.

Long-term changes, from 1984 to 2010, in the indicators of microbial pollution (total viable count, coliforms, Escherichia coli, enterococci, and Clostridium perfringens) are analysed in the Riga Hydropower Plant Reservoir, an essential source of drinking water for Riga, the capital of Latvia. Counts in microbial indicators fluctuated seasonally and were related to physicochemical parameters (nitrogen compounds, turbidity, temperature, and pH). The changes in microbial pollution were brought about by two major socio-economic developments. Firstly, Latvia's independence from the USSR in 1991 which facilitated a distinct reduction in most microorganism counts due to a sharp decline in industrial and agricultural production. This resulted in a significant drop in point and nonpoint pollution in the river basin. A further development was Latvia joining the European Union in 2004. The corresponding focus on water management, including wastewater treatment, was a major priority of environmental investment and lead to improvements in microbial water quality.

Biochemical Responses of the Bivalve Mollusk Unio tumidus Inhabiting a Small Power Plant Reservoir on the Dniester River Basin, Ukraine.

Hydropower plants (HPPs) can affect the hydrological regime. However, biochemical responses of aquatic animals for the evaluation of this disturbing are not applied yet. The specimens of Unio tumidus were sampled in a reservoir (R) of a small HPP as well as downstream from the dam (DS). Biochemical indexes in the digestive gland and alkali labile phosphates (ALP) in the gonads were examined. The R-mollusks showed low cholinesterase, catalase and caspase-3 activities, and metallothionein concentration, but elevated levels of zinc and copper, oxidized glutathione and protein carbonyls. Concentrations of lactate, pyruvate and ALP, activity of superoxide dismutase and glutathione S-transferase, and lipid peroxidation level were similar in both groups. Integrated biomarker response (IBR/n) index (n = 13) was 2.17 and 0.29 in the R- and DS-groups correspondingly. We suggest that using integrative biological response based on the biochemical markers of bivalve mollusks can be a valid early warning step in assessing 'environmental flow' impact.

Preliminary Study of Multiple Stress Response Reactions in the Pond Snail Lymnaea stagnalis Exposed to Trace Metals and a Thiocarbamate Fungicide at Environmentally Relevant Concentrations.

Gastropod mollusks have achieved an eminent importance as biological indicators of environmental quality. In the present study, we applied a multibiomarker approach to evaluate its applicability for the pond snail Lymnaea stagnalis, exposed to common industrial and agricultural pollutants at environmentally relevant concentrations. The snails were exposed to copper (Cu2+, 10 µg L-1), zinc (Zn2+, 130 µg L-1), cadmium (Cd2+, 15 µg L-1), or the thiocarbamate fungicide "Tattoo" (91 µg L-1) during 14 days. Metal treatment and exposure to "Tattoo" caused variable patterns of increase or decrease of metal levels in the digestive gland, with a clear accumulation of only Cd and Zn after respective metal exposure. Treatment with Cu and "Tattoo" caused an increase of cytochrome P450-related EROD activity. Glutathione S-transferase was inhibited by exposure to Cu, Zn, and "Tattoo." Treatment with the "Tattoo" led to an inhibition of cholinesterase activity, whereas Cu and Cd increased its activity. Caspase-3 activity was enhanced by up to 3.3 times in all treatments. A nearly uniform inhibitory effect for oxidative stress response parameters was observed in all kinds of exposure, revealing an inhibition of superoxide dismutase (Mn-SOD) activity, a depression of glutathione (GSH and GSSG) and of protein carbonyl levels. Pollutant-specific effects were observed for the catalase activity, superoxide anion production, and lipid peroxidation levels. Due to the high response sensitivity of Lymnaea stagnalis to chemical impacts, we suggest our study as a contribution for biomarker studies with this species under field conditions.

Multi-marker study of the responses of the Unio tumidus from the areas of small and micro hydropower plants at the Dniester River Basin, Ukraine.

The impact of the hydropower plants (HPPs) on the aquatic life is expected, but the biochemical markers of ecotoxicity have not been investigated in relation to HPP proximity. The aim of this study was to compare the responses of mollusk Unio tumidus from the small HPP (reservoir (Ku) and downstream of the dam (Kd)) and micro HPP (upstream (Zu) and downstream of the dam (Zd)). In total, 11 indexes (n) from digestive gland, hemocytes (lysosomal integrity), and gonads (alkali-labile phosphates, ALP) were analyzed. The mollusks from the reservoir demonstrate the typical signs of toxic impact: cholinesterase and glutathione depletion, the highest glutathione S-transferase activity, and ratio of extralysosomal cathepsin D compare to all other groups. The specimens from the micro HPP have the highest levels of glutathione (Zd) and lipid peroxidation (Zu) and lesser Cu/Zn-SOD activity (Zu) than other groups. These indications of stressful conditions may derive from the regular oscillations in the water flow regimes at the micro HPP. For both HPPs, the responses of upstream and downstream groups are distinct. The calculated IBR/n (4.17, 3.85, 3.12, and 0.26 for Ku, Kd, Zu, and Zd correspondingly) gives a quantitative basis for the evaluation of environmental impact of HPPs. Graphical abstract .

Biochemical responses of freshwater mussel Unio tumidus to titanium oxide nanoparticles, Bisphenol A, and their combination.

Multiple interactions between different pollutants in the surface waters can cause unpredictable consequences. The aim of the study was to evaluate the combined effect of two widespread xenobiotics, titanium oxide nanoparticles (TiO2) and bisphenol A (BPA), on freshwater bivalve Unio tumidus. The specimens were exposed for 14 days to TiCl4 (Ti, 1.25 µM), TiO2 (1.25 µM), BPA (0.88 nM), or their combination (TiO2 + BPA). Every type of exposure resulted in a particular oxidative stress response: TiO2 had antioxidant effect, decreasing the generation of reactive oxygen species (ROS) and phenoloxidase (PhO) activity, and doubling reduced glutathione (GSH) concentration in the digestive gland; Ti caused oxidative changes by increasing levels of ROS, PhO and superoxide dismutase; BPA decreased the GSH level by a factor of two. In the co-exposure treatment, these indices as well as lysosomal membrane stability were not affected. All Ti-containing exposures caused elevated levels of metalated metallothionein (Zn,Cu-MT), its ratio to total metallothionein protein, and lactate/pyruvate ratio. Both BPA-containing exposures decreased caspase-3 activity. All exposures, and particularly co-exposure, up-regulated CYP450-dependent oxidation, lipid peroxidation and lipofuscin accumulation, lysosomal cathepsin D and its efflux, as well as alkali-labile phosphates in gonads and caused DNA instability (except for TiO2). To summarize, co-exposure to TiO2 + BPA produced an overlap of certain individual responses but strengthened the damage. Development of water purification technologies using TiO2 requires further studies of the biological effects of its mixtures. U. tumidus can serve as a sentinel organism in such studies.

A calcium channel blocker nifedipine distorts the effects of nano-zinc oxide on metal metabolism in the marsh frog Pelophylax ridibundus.

Global decline of amphibian populations causes particular concern about their vulnerability to novel environmental pollutants, including engineering nanomaterials and pharmaceutical products. We evaluated the bioavailability of nanoform of zinc oxide (n-ZnO) in frog Pelophylax ridibundus and determined whether co-exposure to a common pharmaceutical, a calcium-channel blocker nifedipine (Nfd) can affect this bioavailability. Male frogs were exposed for 14 days to the tap water (Control) and n-ZnO (3.1 µM), Zn2+ (3.1 µM, as a positive control for n-ZnO exposures), Nfd (10 µM), and combination of n-ZnO and Nfd (n-ZnO + Nfd) in environmentally-relevant concentration. Exposure to Zn2+ or n-ZnO led to up-regulation of metal-binding proteins, metallothioneins (MTs) in the liver and Zn-carrying vitellogenin-like proteins in the blood plasma. Notably, upregulation of MTs by Zn2+ or n-ZnO exposures combined with increased binding of Zn and Cu to MTs. This was associated with the more reducing conditions in the liver tissue indicated by elevated lactate to pyruvate ratio. Nfd suppressed the binding of Zn and Cu to MTs and led to a decrease in Lactate/Pyruvate ratio and elevated protein carbonylation indicating pro-oxidant conditions. Redox status parameters were not directly related to DNA fragmentation, nuclear abnormalities or suppression of cholinesterase activity indicating that factors other than oxidative stress are involved in cytotoxicity of different pollutants and their combinations. Furthermore, activity of Phase I biotransformation enzyme (CYP450 oxidase measured as EROD) was elevated in Nfd-containing exposures and in Zn2+ exposed frogs. Tyrosinase-like activity in the frog liver was strongly stimulated by Zn2+ but suppressed by n-ZnO, Nfd and n-ZnO + Nfd. These findings show that Nfd modulates homeostasis of essential metals in amphibians and emphasize that physiological consequences of combined n-ZnO and Nfd exposures are difficult to predict based on the mechanisms of single stressors.

Bioenergetic responses of freshwater mussels Unio tumidus to the combined effects of nano-ZnO and temperature regime.

Bivalves from the cooling reservoirs of electrical power plants (PP) are exposed to the chronic heating and chemical pollution making them a suitable model to study the combined effects of these stressors. We investigated the effect of in situ exposures to chemical and thermal pollution in the PP cooling ponds on the metabolic responses of unionid bivalves (Unio tumidus) to a novel widespread pollutant, ZnO nanoparticles (nZnO). Male U. tumidus from the reservoirs of Dobrotvir and Burshtyn PPs (DPP and BPP) were maintained in clean water at 18 °C, or exposed for 14 days to one of the following conditions: nZnO (3.1 µM) or Zn2+ (3.1 µM, a positive control for Zn impacts) at 18 °C, elevated temperature (T, 25 °C), or nZnO at 25 °C (nZnO + T). Baseline levels of glycogen, lipids and ATP were similar in the two studied populations, whereas the levels of proteins, lactate/pyruvate ratio (L/P) and extralysosomal cathepsin D level were higher in the tissues of BPP mussels. The levels of glycogen and glucose declined in most experimental exposures indicating elevated energy demand except for a slight increase in the digestive gland of warming-exposed BPP mussels and in the gills of the nZnO + T-exposed DPP-mussels. Experimental exposures stimulated cathepsin D activity likely reflecting onset of autophagic processes to compensate for stress-induced energy demand. No depletion of ATP in Zn-containing exposures was observed indicating that the cellular metabolic adjustments were sufficient for such compensation. Unexpectedly, experimental warming mitigated most metabolic responses to nZnO in co-exposures. Our data thus indicate that metabolic effects of nZnO strongly depend on the environmental context of the mussels (such as temperature and acclimation history) which must be taken into account for the molecular and cellular biomarker-based assessment of the nanoparticle effects in the field.