Effect of emerging pollutants on the gut microbiota of freshwater animals: Focusing on microplastics and pesticides.

In recent years, emerging environmental pollutants have increasingly endangered the health of freshwater organisms. The gut microbiota exhibits sensitivity to medications, dietary factors and environmental pollutants, rendering it a novel target for toxicological studies. The gut microbiota can be a potential exposure route affecting the host's health. Herein, we review the current knowledge on two different but concurrent pollutants, microplastics and pesticides, regarding their impact on the gut microbiota, which includes alterations in microbial composition, gene expression, function, and health effects in the hosts. Moreover, synergetic interactions between microplastics and pesticides can exacerbate dysbiosis and health risks. We discuss health-related implications of gut microbial changes based on the consequences in metabolism, immunity, and physiology function. Further research is needed to discover the mechanisms underlying these effects and develop strategies for mitigating their harmful impacts on freshwater animals.

Environmentally relevant concentrations of triclocarban affect behaviour, learning, and brain gene expression in fish.

Many chemicals spilled in aquatic ecosystems can interfere with cognitive abilities and brain functions that control fitness-related behaviour. Hence, their harmful potential may be substantially underestimated. Triclocarban (TCC), one of the most common aquatic contaminants, is known to disrupt hormonal activity, but the consequences of this action on behaviour and its underlying cognitive mechanisms are unclear. We tried to fill this knowledge gap by analysing behaviour, cognitive abilities, and brain gene expression in zebrafish larvae exposed to TCC sublethal concentrations. TCC exposure substantially decreased exploratory behaviour and response to stimulation, while it increased sociability. Additionally, TCC reduced the cognitive performance of zebrafish in a habituation learning task. In the brain of TCC-exposed zebrafish, we found upregulation of c-fos, a gene involved in neural activity, and downregulation of bdnf, a gene that influences behavioural and cognitive traits such as activity, learning, and memory. Overall, our experiments highlight consistent effects of non-lethal TCC concentrations on behaviour, cognitive abilities, and brain functioning in a teleost fish, suggesting critical fitness consequences of these compounds in aquatic ecosystems as well as the potential to affect human health.

Plastic invasion tolling: First evaluation of microplastics in water and two crab species from the nature reserve lagoonary complex of Kune-Vain, Albania.

Microplastic (MP) pollution is an emerging global challenge and actually has become a reality in aquatic ecosystems in Albania. According to the World Wildlife Fund (WWF) report of 2019, Albania, is one of the most problematic countries, with the highest percentage of untreated plastic waste, 73 %, and one of the top four countries with the highest norm of untreated plastic waste in the Mediterranean. This study evaluates and characterizes for the first time the MPs in water, sediment, and gastrointestinal tracts of two crab species, C. aestuarii and C. sapidus, common inhabitants of the lagoonary complex of Kune-Vain Nature Reserve, known for their important role in the lagoon ecosystem. The results showed that all sampled crabs had MPs in their gut in an average of 11.0 ± 1.85 items g-1, while the total MPs content in water ranges from 370 to 750 MPs per L-1. No significant difference in the content of MPs between the two crab species was found and a positive Pearson correlation, between microplastic abundance in the water and in the crabs regardless of species, was confirmed. The composition of microplastics showed consistency in crabs, sediment, and water, with fibers and pellets as the dominant types followed by microbeads, and fragments. Characterized MPs varied in size from <0.1 mm to 0.1-0.5 mm, showing variable colors of black, blue, and red domination. Fourier Transform Infrared Spectroscopy (FT-IR) analysis of the chemical composition of microplastics distinguished presence of the high-density polyethylene (HDPE), polypropylene (PP), polyethylene (PE), and low-density polyethylene (LDPE), which showed consistency in water, sediment, and crab samples. In conclusion, high levels of MPs pollution observed in the Kune-Vain complex represent a serious threat to the lagoon ecosystem and to the local inhabitants. Furthermore, studies on MPs' impact on biota and local population health are urgently required.

Effects of Vitamin C and Magnesium L-threonate Treatment on Learning and Memory in Lead-poisoned Mice.

INTRODUCTION: The aim of this study was to investigate the effects of vitamin C (vit C) and magnesium L-threonate (MgT) on the learning ability and memory of mice intoxicated with lead acetate. MATERIAL AND METHODS: The experimental male Swiss albinos were divided into five groups of 10 during a 40-day treatment period. One group were untreated controls, one received lead acetate at 90 mg/kg b.w., one additionally vit C at 40 mg/kg b.w., another additionally MgT at 100 mg/kg b.w., and the last was administered MgT without lead acetate. After a 20-day washout period, the animals were trained in the Morris water maze test for 6 days and after a 24-hour interval, were assessed for memory in the same test. At test end the mice were sacrificed and their organs sampled. RESULTS: The results of total time and number of entries into the platform zone showed that significantly poorer performances were recorded for the group poisoned with lead acetate alone and significantly lower scores for learning and memory were recorded for the intoxicated and supplemented groups compared to the control group. Catalase activity was significantly reduced in the liver, pancreas and kidney but significantly potentiated in brain tissue by these two supplements compared to the control group. Lead concentration in brain tissue was significantly higher in the presence of vit C than in the control or lead acetate-only groups. CONCLUSION: Lead acetate had adverse effects on learning and memory of mice and also increased catalase activity.

Double-edged sword: Fluoxetine and ibuprofen as development jeopardizers and apoptosis' inducers in common toad, Bufo bufo, tadpoles.

Nowadays the presence of pharmaceuticals in the environment is a real problem. Ending up in aquatic environments they negatively affect non-target organisms. Considering the limited studies on the negative effects of pharmaceuticals in amphibians, a better understanding of the mechanisms underlying the sub-lethal effects of drug mixtures in wildlife is an urgent call. Representing particularly vulnerable organisms currently at risk of extinction, amphibians are perfect non-target organisms to explore the consequences of pharmaceuticals during sensitive life-stages. To address this existing research gap, the effects of two drugs, the antidepressant fluoxetine and the anti-inflammatory ibuprofen, as well as their combination has been studied. Tadpoles of Bufo bufo were exposed for seven days to two environmentally realistic concentrations of fluoxetine, ibuprofen and their mixture. The development, behavior and erythron profile were then evaluated as endpoints of exposure response. Both drugs negatively affected tadpoles' growth and development by significantly delayed their time to metamorphosis and reduced body weight. Behaviors were also impaired with a significant increase of unresponsiveness to different stimuli. Mutagenic analysis of blood revealed a significant increase in the frequency of cellular and nuclear abnormalities. Given the complexity of systems and functions affected, our work confirms the toxicological potential of fluoxetine and ibuprofen in B. bufo tadpoles by emphasizing their role as tadpole development delayers and erythrocyte apoptosis-inducers. To our knowledge, this is the first study trying to elucidate the potentially toxic effects of a mixture of an antidepressant with a non-steroidal anti-inflammatory drug using bullfrog tadpole as model organism. Both drugs interacted in impairing development and fitness in tadpoles, which might affect long-term species perpetuation and population dynamic. More in-depth research is needed to elucidate the nature of interaction and molecular mechanisms of mixed pharmaceutical compounds on non-targeted organisms.

Combined effects of moderate hypoxia, pesticides and PCBs upon crucian carp fish, Carassius carassius, from a freshwater lake- in situ ecophysiological approach.

Nowadays, depletion of oxygen or hypoxia has become a real concerning problem worldwide in freshwater, marine, and estuarine ecosystems and very often co-occurs with xenobiotics. Even though the acute and severe hypoxia is heavily studied in environment and laboratory studies, the in situ combined effects of these stressors on freshwater lake organisms are poorly understood. The current study sought to understand how the combined effects of moderate hypoxia, pesticides and PCBs affect the biochemistry, physiology and organ morphology of Carassius carassius, residing in the Lake Seferani, Dumrea region (Elbasan, Albania), a natural karst freshwater system declared as Nature Monument situated in central Albania. Crucian carp is used as a model organism, because of its residency and ecological relevance to the Lake, as well as for its amenability for the environmental toxicology studies. For this purpose, blood, liver and kidney samples of fish were processed for hematological, biochemical and histopathological analysis. We found a significant increase of blood glucose (GLU), cortisol levels, hematocrit (PCV) and hemoglobin (Hb) which clearly indicate the presence of stress in fish. Based on the histopathological evaluation and organ index results, liver and kidney organs displayed moderate-to-heavy histological-architecture changes. Our results provide a strong evidence that both, hypoxia and the presence of pesticides and PCB congeners found in Seferani Lake, put a heavy load on C. carassius energy metabolism and endocrine system, leading to an elevation of the biochemical and physiological parameters (hemoglobin level, hematocrit, glucose and cortisol), as well as the histopathological alterations. Additionally, in the presence of moderate hypoxia, the toxic effects of pesticides and PCBs on C. carassius are exacerbated. Further studies are needed to evaluate possible effects of pesticide and PCBs toxicity in human health, since crucian carp has an economic value for the population of the zone and it is used often as food sustenance. Elucidation of these kinds of responses can better improve our understanding of response of highly tolerant species, like Carassius carassius, to multiple stressors interactions, helping us to better predict and manage the consequences of the exposure of the freshwater biota to complex stressors in an environment that changes rapidly.

Physiological and biochemical responses of Mediterranean green crab, Carcinus aestuarii, to different environmental stressors: Evaluation of hemocyte toxicity and its possible effects on immune response.

Effects of natural stressors such as copper (Cu2+), temperature, hypoxia, chloroform and adrenaline on physiological and biochemical responses were investigated in the Mediterranean green crab Carcinus aestuarii from tidal shallow waters of Narta Lagoon, Albania. For this purpose, hemolymph glucose levels, total and differential hemocyte count, in normal and eye-stalked individuals, exposed to above mentioned stressors like, were assessed. In addition, lysosomal membrane stability was evaluated as biomarker of hemocyte toxicity, with possible implications on crab immune response. Hemolymph glucose levels were significantly increased in all treatment groups with 1.25-to 3.5-fold above baseline levels of 37.8 ± 2.7 mgdL-1. Response times were being manifested within 30-120 min following exposure and recovery happened within 2 h of restoration of pretreatment conditions. Total hemocyte count (THC) and differential hemocyte count (DCH) showed a significant decrease for all stressors, except for copper, were an increase of semi-granular hemocyte fraction were recorded. Meanwhile, significant reduction of neutral red retention time (NRRT), in both eyestalk-ablated and exposed animals, were recorded, indicated the loss of hemocyte lysosomal membrane integrity. The responsiveness of hemolymph blood levels to all stressors, the decrease in total hemocyte count, as well as the loss of lysosomal membrane integrity demonstrated that exposure to environmentally realistic stressors placed a heavy metabolic load on C. aestuarii, modulating their immune competence and overall physiological wellness. Overall, results suggest that monitoring cellular and biochemical parameters like hemolymph glucose titres, TCH, DHC and NRRT, may be useful and sensitive means of evaluating the crustacean's ability to cope with the wide variety of environmental stressors through modulation of the immune parameters.

Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog, Pelophylax shqipericus.

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.

Digital light microscopy as a tool in toxicological evaluation of fish erythrocyte morphological abnormalities.

Fish erythrocytes are very sensitive to environmental pollution because they exhibit pathological alterations before appearance of other external symptoms, may be used as reliable toxicity bioindicators. Microscopic analysis can be a useful tool in early detection of alterations in erythrocyte morphology due to different environmental stressors, and in predicting organism's possible response ways. This study aims to evaluate the toxicity of a polluted freshwater lake on the erythrocytes of crucian carp fish, Carassius carassius, using digital light microscopy (DLM). Scanning DLM micrographs revealed the presence of nuclear and cytoplasmic abnormalities like micronucleus, nuclear buds, lobed nucleus, deformed nucleus, vacuolated cytoplasm, and echinocyte in the erythrocytes of crucian carp fish from a polluted lake. The obtained results substantiate that computation of erythrocyte morphological damages as biomarkers of exposure, through digital light microscopy software, can be effectively used as a toxicological tool.

Characterization of Albanian water frog, Pelophylax shqipericus, sperm traits and morphology, by using phase contrast microscopy.

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth-edged, and solid-staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 µm ± 0.32, while the tail length resulted 29.24 ± 1.75 µm. The average thickness of the head was shown to be 3.45 µm. The total sperm length resulted to be 41.02 ± 1.83 µm. The average sperm concentration was estimated of 25.5 × 106 /ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of "Lessonae" frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.

Antioxidant defense system, immune response and erythron profile modulation in gold fish, Carassius auratus, after acute manganese treatment.

The manganese contamination has become a global problem, recently, because it is perceived as a real threat to the human health and the environment. It is well-known that overexposure to Mn2+ may have negative physiological effects on fish and other organisms inhabiting heavy metal polluted waters. To the best of our knowledge, studies relating with manganese effects on fish antioxidant enzyme response in the blood, immunocompetence and erythron profile alteration, are scarce. In this study, the acute sub-lethal effects of manganese on blood antioxidant response, immune status and erythron profile were determined by exposing the freshwater model organism, Carassius auratus, to two doses of this metal (3.88 ±â€¯0.193 mg/L and 7.52 ±â€¯0.234 mg/L Mn2+) for 96 h. Significant increases in blood antioxidant enzyme activity like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST), were observed in fish exposed to manganese. Furthermore, plasmatic glucose and cortisol levels increased, while total protein decreased significantly. White blood cell differential count revealed a significant increase in monocyte and neutrophil number and a significant decrease of lymphocyte's number in fish exposed to manganese compared with those of control group. That can be considered as a clear evidence of altered immune system. Measured of erythron profile revealed a significant increasing of cellular and nuclear alteration of red blood cells, with karryorhectic, dividing and micronucleated erythrocytes in exposed fish, indicating the cytotoxic and genotoxic effects Mn2+ ions. Our data shown also that manganese could trigger antioxidant response, modulate immune response and induce erythron profile modification leading to eryptosis, compromising the blood oxygen carrying capacity, and overall health status in fish. This may suggest those parameters consider as useful biomarkers for monitoring effects of sub-lethal metal exposure on fish.

Copper Induced Lysosomal Membrane Destabilisation in Haemolymph Cells of Mediterranean Green Crab (Carcinus aestuarii, Nardo, 1847) from the Narta Lagoon (Albania)

ABSTRACTDestabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA). Crabs collected in Narta Lagoon, Vlora (Albania) during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT) and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05), indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05) than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.