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.

NiSO4 spill inflicts varying mortality between four freshwater mussel species (including protected Unio crassus Philipsson, 1788) in a western Finnish river.

Freshwater mussels are one of the most threatened taxonomic groups in the world, and many species are on the brink of local or global extinction. Human activities have altered mussel living conditions in a plethora of ways. One of the most destructive human-induced impacts on running waters is the catastrophic spill of harmful substances, which results in massive die-offs. Even though Finland is regarded as the world's top country in terms of environmental regulation quality, riverine systems are not safe. In 2014, River Kokemäenjoki in western Finland experienced the worst NiSO4 spill in the country's history, visibly affecting the mussel community - including protected Unio crassus - along the river. Because freshwater mussel toxicology is grossly understudied (particularly in Europe), any pollution -linked die-offs offer valuable opportunities to study the issue in natural environment. Here, we report the mussel investigations from 2014 and a follow-up study conducted in 2017 in order to assess the variation in species sensitivity on nickel pollution. In total, 104 sites were sampled, and over 20 000 mussels were identified and counted. Our results indicate that the most impacted species (i.e. that which experienced the highest spill-induced mortality) was Anodonta anatina (62%), followed by Unio pictorum (32%), U. crassus (24%) and Unio tumidus (9%). The underlying reason for the sensitivity of A. anatina is not resolved, hence more research is urgently needed. The low mortality among most of the species in 2017 highlights the temporal nature of the pollution impact and the recovery potential of the mussel community. However, the case is more complex with U. crassus population, which may be experiencing delayed impacts of the spill. Because nickel is one of the most commonly produced industrial metals in the world (hence the pollution incident risk is high) and River Kokemäenjoki hosts mussel community typical for European rivers, our results may benefit many researchers and stakeholders dealing with riverine environments.

Behavioural Responses of Unio tumidus Freshwater Mussels to Pesticide Contamination.

A pesticide is a chemical substance used for the disposal of pests, such as insects, weeds, invertebrates, or rodents. Pesticides interfere with the normal metabolism of the target species; however, some of them may inadvertently affect organisms other than those targeted. Increased quantities of pesticides in water disturb various ecological processes and may increase the mortality rate of various native species of flora and fauna. One of the groups of organisms that are at the greatest risk from the adverse effects of pesticides is the bivalves. This study was designed to assess the behavioural reaction of bivalves to widespread pesticides. As a representative example, the Polish native Unio tumidus (Philipsson 1788) was used. The study investigated different groups of toxic pesticides, such as herbicides (lenacil), insecticides (thiacloprid, DDT and dichlorvos), and fungicides (tebuconazole), in concentrations of 10 mg L-1. The results showed various behavioural reactions of bivalves to the pesticides. The most evident were activity time and shell opening rate. Moreover, as a result of DDVP contamination, effects were recorded in terms of shell opening level as well as rapid onset of death. Among the five analysed plant protection products, the most toxic was DDVP. Its presence caused adductor muscle paralysis in all analysed individuals. The least toxic pesticides were DDT and thiacloprid. A strong reaction to lenacil was observed especially in the shell opening rate. Tebuconazole caused significant reductions in activity. Despite the fact that the impact of pesticides on ecosystems is under regular observation, with the use of a wide range of scientific techniques, the use of bivalves was shown to have considerable potential for water quality monitoring.

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.

Raman microspectroscopic identification of microplastic particles in freshwater bivalves (Unio pictorum) exposed to sewage treatment plant effluents under different exposure scenarios.

We investigated the uptake of microplastic (MP, <5 mm) particles by using freshwater bivalves (Unio pictorum) as biological samplers in the environment. They were exposed either directly to the biologically purified sewage of a North Bavarian sewage treatment plant (STP) or placed in a small river up- and downstream of the wastewater discharge for 28 days and 6 months, respectively. A control group was maintained in a pond. After acid digestion, the soft tissue was analyzed for MP particles by means of Raman microspectroscopy (RM, over 3000 particles individually measured), which allows for identification and quantification of particles down to 1 µm. Only in the bivalve collective exposed to STP effluents MP was found, however a very small amount (maximum of nine MP particles in the bivalve sample exposed for 6 months). In the bivalves up- and downstream of the wastewater discharge and in control organisms from a pond, no microplastic was identified. The amount of microplastic particles was small in absolute terms and small in relative terms (ca. 1:100 (6 months) and below 1:1000 (28 days)) as hundreds of particles per sample were analyzed which turned out to be non-plastic. Including the results for the river, this indicates a rather low MP contamination level for organisms in close vicinity to a sewage treatment plant.

Biochemical and molecular alterations in freshwater mollusks as biomarkers for petroleum product, domestic heating oil.

To investigate the effect one of the oil products, domestic heating oil (DHO), on freshwater mollusks, Unio tigridis and Viviparous bengalensis were exposed to three DHO concentrations for each species (5.8, 8.7, and 17.4 ml L-1 for mussels; 6.5, 9.7, and 19.5 mlL-1 for snails, respectively). Antioxidant enzymes (superoxide dismutase, catalase), malondialdehyde, acetylcholinesterase and DNA damage in both species tissues were monitored over 21 days. The results showed that both antioxidant enzymes concentration (SOD and CAT) increased in the lowest DHO concentrations (5.8, and 8.7 ml L-1), and then decreased in the highest concentration (17.4 ml L-1) as the same pattern for Unio tigridis, but this not occurred for Viviparous bengalensis. MDA values recorded significantly increased compared to control. No reduction was observed in AChE concentrations in soft tissues of both mollusks may due to that DHO was a non-neurotoxicant to Unio tigridis and Viviparous bengalensis. The results of DNA damage parameters were showed significant differences (p≤ 0.05) between control and DHO concentrations except lowest concentration for each parameter measured in digestive gland of Unio tigridis. As well as, these significant differences were recorded between control and three concentrations of DHO exposure for comet length, and tail length parameters, and between control and highest oil concentration for tail moment in Viviparous bengalensis. DHO has the ability to prevent the reproduction of Viviparous bengalensis snail relation to control, that is what we considered strong evidence of the toxicity properties of DHO on the reproductive status of this species of snails. SOD, CAT, and MDA were useful biomarkers for evaluating the toxicity of DHO in mussel and snails, and comet assay was a good tool to assess the potential genotoxicity of DHO.

The effect of seasonal temperature variation on behaviour and metabolism in the freshwater mussel (Unio tumidus).

Temperature plays a critical role in determining the biology of ectotherms. Many animals have evolved mechanisms that allow them to compensate biological rates, i.e. adjust biological rates to overcome thermodynamic effects. For low energy-organisms, such as bivalves, the costs of thermal compensation may be greater than the benefits, and thus prohibitive. To examine this, two experiments were designed to explore thermal compensation in Unio tumidus. Experiment 1 examined seasonal changes in behaviour in U. tumidus throughout a year. Temperature had a clear effect on burrowing rate with no evidence of compensation. Valve closure duration and frequency were also strongly affected by seasonal temperature change, but there was slight evidence of partial compensation. Experiment 2 examined oxygen consumption during burrowing, immediately following valve opening and at rest in summer (24 °C), autumn (14 °C), winter (4 °C), and spring (14 °C) acclimatized U. tumidus. Again, there was little evidence of burrowing rate compensation, but some evidence of partial compensation of valve closure duration and frequency. None of the oxygen compensation rates showed any evidence of thermal compensation. Thus, in general, there was only very limited evidence of thermal compensation of behaviour and no evidence of thermal compensation of oxygen compensation rates. Based upon this evidence, we argue that there is no evolutionary pressure for these bivalves to compensate these biological rates. Any pressure may be to maintain or even lower oxygen consumption as their only defence against predation is to close their valves and wait. An increase in oxygen consumption will be detrimental in this regard so the cost of thermal compensation may outweigh the benefits.

Adaptation of freshwater mussels to cyanobacterial toxins: response of the biotransformation and antioxidant enzymes.

Freshwater mussels such as the invasive Dreissena polymorpha and the indigenous Unio tumidus nourish by high filtration rates and may accumulate cyanobacteria and their toxins during cyanobacterial blooms. Physiological adaptations to cyanotoxins enable organisms to endure cyanobacterial blooms but may differ between species. Biotransformation and excretion capacities for cyanobacteria and anthropogenic pollutants have been demonstrated for Dreissena polymorpha but less for unionid species. This study compares the activities of biotransformation (glutathione S-transferase, GST) and antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) in Dreissena polymorpha to Unio tumidus in response to cyanotoxin exposure (10 µg L(-1) and 50 µg L(-1) microcystin-LR, respectively, total microcystin from a cyanobacterial crude extract) for 24 h and 7d exposure duration. Enzyme activities in Dreissena polymorpha were measured in the whole mussel tissue, digestive gland and in gills and in Unio tumidus in the digestive gland, gills, mantle, foot as well as in the remaining tissue. The sGST was elevated for the entire exposure period in the whole mussel tissue of Dreissena polymorpha but despite higher basal activities in digestive gland and gills of Unio tumidus, it was rather inhibited or unaltered in most of their tissues. Elevated SOD activity indicated oxidative stress response in Dreissena polymorpha, but not in Unio tumidus. The CAT activity was barely affected in both species, rather inhibited in Unio tumidus, despite again higher basal activities in digestive gland and remaining tissue. Compared to the indigenous Unio tumidus, the investigated biotransformation and oxidative stress combating enzymes respond stronger in the invasive Dreissena polymorpha.

Antioxidative and oxidative changes in the digestive gland cells of freshwater mussels Unio tumidus caused by selected phenolic compounds in the presence of H(2)O(2) or Cu(2+) ions.

Research on biomarkers as early bioindicators of perturbation in populations and individuals has received increasing interest during recent decades. These ecotoxicity studies allow us to measure the impact of environmental stressors and to monitor and evaluate the degradation or restoration of systems. In the present study we used bivalve molluscs (mussels), which are sensitive biomarkers of aquatic ecosystem pollution, to assess the effects of three polyphenols: tannic acid, ellagic acid and gallic acid. These compounds were used in the 1-60 microM concentration range, alone and in the presence of H(2)O(2) (40 and 100 microM) or Cu(2+) ions (50 microM). The fluorescence probe dichlorofluorescein-diacetate (DCFH-DA) was used to measure reactive oxygen species (ROS). The oxidation of DCFH-DA to the fluorescent DCF (dichlorofluorescein) by the phenolic compounds was investigated spectrofluorimetrically. The results showed that the polyphenols tested can act as antioxidants against the ROS present in the digestive gland cells, but their activity is decreased after incubation with hydrogen peroxide or copper ions. SH-groups were determined spectrophotometrically using Ellman's reagent. The results showed that oxidative modification of proteins increased in a concentration-dependent manner in cells incubated with polyphenols (above 15 microM) alone. Incubation of the cells with phenolic acids and H(2)O(2) or Cu(2+) ions revealed that the phenolic acids had prooxidant properties in all concentrations used except for 1 microM tannic and ellagic acid and 40 microM H(2)O(2). DNA fragmentation was estimated by a fluorescence method using Hoechst 33258/propidium iodine binding. The data showed that the phenolic acids alone and in the presence of hydrogen peroxide or copper ions can induce apoptosis and necrosis. The methods used and results obtained indicate that the polyphenols selected can act not only as antioxidants but also as prooxidants in digestive gland cells of Unio tumidus.