Illicit Drugs in Surface Waters: How to Get Fish off the Addictive Hook.

The United Nations World Drug Report published in 2022 alarmed that the global market of illicit drugs is steadily expanding in space and scale. Substances of abuse are usually perceived in the light of threats to human health and public security, while the environmental aspects of their use and subsequent emissions usually remain less explored. However, as with other human activities, drug production, trade, and consumption of drugs may leave their environmental mark. Therefore, this paper aims to review the occurrence of illicit drugs in surface waters and their bioaccumulation and toxicity in fish. Illicit drugs of different groups, i.e., psychostimulants (methamphetamines/amphetamines, cocaine, and its metabolite benzoylecgonine) and depressants (opioids: morphine, heroin, methadone, fentanyl), can reach the aquatic environment through wastewater discharge as they are often not entirely removed during wastewater treatment processes, resulting in their subsequent circulation in nanomolar concentrations, potentially affecting aquatic biota, including fish. Exposure to such xenobiotics can induce oxidative stress and dysfunction to mitochondrial and lysosomal function, distort locomotion activity by regulating the dopaminergic and glutamatergic systems, increase the predation risk, instigate neurological disorders, disbalance neurotransmission, and produce histopathological alterations in the brain and liver tissues, similar to those described in mammals. Hence, this drugs-related multidimensional harm to fish should be thoroughly investigated in line with environmental protection policies before it is too late. At the same time, selected fish species (e.g., Danio rerio, zebrafish) can be employed as models to study toxic and binge-like effects of psychoactive, illicit compounds.

Intermittent hypoxia differentially affects metabolic and oxidative stress responses in two species of cyprinid fish.

Oxygen fluctuations are common in freshwater habitats and aquaculture and can impact ecologically and economically important species of fish like cyprinids. To gain insight into the physiological responses to oxygen fluctuations in two common cyprinid species, we evaluated the impact of short-term intermittent hypoxia on oxidative stress and metabolic parameters (including levels of prooxidants and oxidative lesions, antioxidants, mitochondrial enzyme activities, mitochondrial swelling, markers of apoptosis, autophagy and cytotoxicity) in silver carp Hypophthalmichthys molitrix and gibel carp Carassius gibelio. During hypoxia, gibel carp showed higher baseline levels of antioxidants and less pronounced changes in oxidative and metabolic biomarkers in the tissues than silver carp. Reoxygenation led to a strong shift in metabolic and redox-related parameters and tissue damage, indicating high cost of post-hypoxic recovery in both species. Species-specific differences were more strongly associated with oxidative stress status, whereas metabolic indices and nitrosative stress parameters were more relevant to the response to hypoxia-reoxygenation. Overall, regulation of energy metabolism appears more critical than the regulation of antioxidants in the response to oxygen deprivation in the studied species. Further research is needed to establish whether prioritizing metabolic over redox regulation during hypoxia-reoxygenation stress is common in freshwater cyprinids.

Molecular and Biochemical Evidence of the Toxic Effects of Terbuthylazine and Malathion in Zebrafish.

Our research sought to determine the molecular and biochemical effects of environmentally relevant exposure to commonly used chloro-s-triazine herbicide terbuthylazine and organophosphate insecticide malathion on zebrafish. To this aim, mature zebrafish were exposed to 2 and 30 µg L-1 terbuthylazine and 5 and 50 µg L-1 malathion alone and in combination for 14 days. Aside from the accumulation of TBARS and protein carbonyls, a decrease in antioxidants and succinate dehydrogenase activity, an increase in oxidized glutathione, and enhanced apoptosis via Caspase-3 and BAX overexpression were observed. Furthermore, terbuthylazine and malathion induced mitochondrial swelling (up to 210% after single exposure and up to 470% after co-exposure) and lactate dehydrogenase leakage (up to 268% after single exposure and up to 570% after co-exposure) in a concentration-dependent manner. Significant upregulation of ubiquitin expression and increased cathepsin D activity were characteristics that appeared only upon terbuthylazine exposure, whereas the induction of IgM was identified as the specific characteristic of malathion toxicity. Meanwhile, no alterations in the zebrafish hypothalamic-pituitary-thyroid axis was observed. Co-exposure increased the adverse effects of individual pesticides on zebrafish. This study should improve the understanding of the mechanisms of pesticide toxicity that lead to fish impairment and biodiversity decline.

A Review of Common Cyanotoxins and Their Effects on Fish.

Global warming and human-induced eutrophication drive the occurrence of various cyanotoxins in aquatic environments. These metabolites reveal diversified mechanisms of action, encompassing cyto-, neuro-, hepato-, nephro-, and neurotoxicity, and pose a threat to aquatic biota and human health. In the present paper, we review data on the occurrence of the most studied cyanotoxins, microcystins, nodularins, cylindrospermopsin, anatoxins, and saxitoxins, in the aquatic environment, as well as their potential bioaccumulation and toxicity in fish. Microcystins are the most studied among all known cyanotoxins, although other toxic cyanobacterial metabolites are also commonly identified in aquatic environments and can reveal high toxicity in fish. Except for primary toxicity signs, cyanotoxins adversely affect the antioxidant system and anti-/pro-oxidant balance. Cyanotoxins also negatively impact the mitochondrial and endoplasmic reticulum by increasing intracellular reactive oxygen species. Furthermore, fish exposed to microcystins and cylindrospermopsin exhibit various immunomodulatory, inflammatory, and endocrine responses. Even though cyanotoxins exert a complex pressure on fish, numerous aspects are yet to be the subject of in-depth investigation. Metabolites other than microcystins should be studied more thoroughly to understand the long-term effects in fish and provide a robust background for monitoring and management actions.

Salinity variation modulates cellular stress response to ZnO nanoparticles in a sentinel marine bivalve, the blue mussel Mytilussp.

Zinc oxide nanoparticles are released into marine environments from industrial, medical and consumer uses sparking concerns about their potential ecotoxicological effects. Ecological hazard assessment of nZnO in marine ecosystems is hindered by the lack of understanding of the potential interactive effects of nZnO toxicity with other common abiotic stressors, such as salinity fluctuations, in marine organisms. To close this gap in our knowledge, we carried out a comprehensive biomarker-based assessment of the combined effects of salinity and nZnO in a sentinel marine bivalve, the blue mussels Mytilus edulis. The mussels were exposed for 21 days to clean seawater (control), an environmentally relevant concentration (100 µg Zn l-1) of nZnO or dissolved Zn (to identify the toxic effects attributable to Zn2+ toxicity) under the normal (15), low (5) and fluctuating (5-15) salinity regimes. The selected molecular and biochemical markers focused on the oxidative stress, apoptosis, detoxification system and inflammation in the gills and the digestive gland of the mussels. Biomarker analysis showed different effects of nZnO and dissolved Zn on biomarkers of oxidative stress, xenobiotic detoxification and apoptosis but similar effects of both pollutants on the levels of metallothioneins and inflammatory markers. Exposure to nZnO led to elevated levels of lipid peroxidation, upregulation of p53 and p38 stress kinases and apoptosis-related genes, most notably in the gills. Exposure to dissolved Zn led to accumulation of protein carbonyls and activated redox-sensitive detoxification enzymes (NADPH-P450 reductase and glutathione-S-transferase) in the mussels. The ambient salinity had significant effects the cellular adverse effects of nZnO in the mussels. The nZnO-induced cellular stress was detectable under the normal (15) and fluctuating (5-15) salinity conditions in the studied brackish water population of the mussels. At low salinity (5), nZnO toxicity signal was almost completely dampened. These findings indicate that chronic osmotic stress close to the tolerance limits of M. edulis prevails over the effects of the environmentally relevant nZnO and dissolved Zn concentrations in combined exposures. These stressor interactions might ameliorate the cellular toxicity of nZnO in the mussels but limit applicability of cellular stress biomarkers for detecting the toxic effects of nanopollutants in low salinity habitats.

Gone with sunscreens: Responses of blue mussels (Mytilus edulis) to a wide concentration range of a UV filter ensulizole.

Organic UV filters have emerged as a new threat to marine organisms, but ecotoxicological studies have so far focused on only a few substances despite the chemical diversity of these synthetic sunscreen agents. Here we examined the responses of blue mussels Mytilus edulis to ensulizole, a non-lipophilic UV filter commonly found in the Baltic Sea. Mussels were exposed for three weeks to five ensulizole concentrations of 10, 102, 103, 104, and 105 ng/L. Stress on stress response was evaluated by subjecting mussels to air exposure. A battery of biomarkers related to detoxification and antioxidant defense, oxidative stress damage, energy reserves and metabolism, autophagy, apoptosis, inflammation, and DNA damage was measured in the gills and the digestive gland. In general, ensulizole affected the antioxidant response, energy storage, and cell death-related processes in mussel tissues. Mussels exposed to low, environmentally relevant concentrations of ensulizole had a shorter air survival time than the control. Ensulizole often showed the non-monotonic concentration-response curves, suggesting the complex effects of this UV filter at molecular, biochemical, and organismal levels.

Multimarker Responses of Zebrafish to the Effect of Ibuprofen and Gemfibrozil in Environmentally Relevant Concentrations.

Pharmaceutical pollution of water bodies is among the top-notch environmental health risks all over the world. The aim of the present study was to investigate the effects of two common pharmaceuticals namely ibuprofen and gemfibrozil on zebrafish at environmentally relevant concentrations. In zebrafish liver, gemfibrozil caused a decrease in glutathione and glutathione transferase and an increase in catalase but had no effect on lipid peroxidation and protein carbonylation. Ibuprofen altered the antioxidant defense system, promoted protein carbonylation in zebrafish liver, and increased vitellogenin-like protein in the blood. Ibuprofen and particularly gemfibrozil induced lysosomes biogenesis. Lactate dehydrogenase in the blood was also found to be higher in the studied groups. Studied pharmaceuticals did not affect complex II of the electron respiratory chain. Ibuprofen affects zebrafish health status more profoundly than gemfibrozil. Our results showed that pharmaceuticals even in low, environmentally realistic concentrations, induced profound changes in the stress-responsive systems of zebrafish.

Pesticide Pollution: Detrimental Outcomes and Possible Mechanisms of Fish Exposure to Common Organophosphates and Triazines.

Pesticides are well known for their high levels of persistence and ubiquity in the environment, and because of their capacity to bioaccumulate and disrupt the food chain, they pose a risk to animals and humans. With a focus on organophosphate and triazine pesticides, the present review aims to describe the current state of knowledge regarding spatial distribution, bioaccumulation, and mode of action of frequently used pesticides. We discuss the processes by which pesticides and their active residues are accumulated and bioconcentrated in fish, as well as the toxic mechanisms involved, including biological redox activity, immunotoxicity, neuroendocrine disorders, and cytotoxicity, which is manifested in oxidative stress, lysosomal and mitochondrial damage, inflammation, and apoptosis/autophagy. We also explore potential research strategies to close the gaps in our understanding of the toxicity and environmental risk assessment of organophosphate and triazine pesticides.

Low-toxic herbicides Roundup and Atrazine disturb free radical processes in Daphnia in environmentally relevant concentrations.

The use of glyphosate-based Roundup and triazine herbicide Atrazine has increased markedly in last decades. Thus, it is important to evaluate toxic effects of these herbicides to non-targeted organisms such as zooplankton to understand their safety toward aquatic ecosystems. In the current study, we performed Daphnia toxicity tests based on lethality to identify LC50 that provides acute aquatic toxicity classification criteria. LC50 for Roundup exposure for 24 hours was found to be 0.022 mg/L and 48 hours - 0.0008 mg/L. Atrazine showed LC50 at concentrations of 40 mg/L and 7 mg/L for 24 and 48 hours, respectively. We demonstrated that exposure to ecologically relevant concentrations of Roundup or Atrazine decreases lipid peroxidation and protein thiol levels, however caused increase in carbonyl protein and low-molecular-weight thiols content. Moreover, the herbicide treatments caused increase of superoxide dismutase activity. Our data suggest that at very low concentrations Roundup and Atrazine disturb free radical processes in D. magna.

Frequency and Nuisance Level of Adverse Events in Individuals Receiving Homologous and Heterologous COVID-19 Booster Vaccine.

This study aimed to compare the occurrence and nuisance of adverse events following administration of each COVID-19 vaccine dose between two groups: individuals given three doses of mRNA vaccine (homologous group, 3 × mRNA, n = 252) and those given two doses of adenoviral vector vaccine further boosted with mRNA vaccine (heterologous group, 2 × AZ + 1 × mRNA, n = 205). Although the studied groups differed significantly in the frequency and number of side effects after the first and second vaccine dose, no relevant differences were seen following the booster administration. Arm pain and fatigue were the most common effects, regardless of the vaccination group and vaccine dose. In the homologous group, female sex, lower BMI, and no history of regular influenza vaccination were associated with a higher frequency of side effects of a booster dose. In the heterologous group, the history of COVID-19 was associated with an increased number of side effects seen after a booster. In both groups, the number of side effects related to the first and second dose correlated with the number observed after administration of a booster dose. Individuals receiving a homologous booster reported a higher nuisance of side effects than the heterologous group. It was similar to the level reported after the second dose in both groups. The use of pharmaceuticals to counteract the side effects was more frequent after a first dose in the 2 × AZ + 1 × mRNA group, but higher after second dose in individuals receiving the 3 × mRNA vaccination scheme. The frequency of pharmaceutical use after a booster dose was similar in both groups (approx. 60%). Paracetamol was most frequently chosen, regardless of the group and vaccine dose. In addition, the vast majority of participants (93%) declared to accept future doses of the COVID-19 vaccine if their administration would be recommended. This study provides an overview of the response to homologous and heterologous mRNA vaccine booster dose that may be valuable in shaping accurate and honest communication with vaccinated individuals, especially in those regions which are yet to pursue booster strategies.

Vaccination of Ukrainian Refugees: Need for Urgent Action.

The unprovoked aggression of Russian military forces on Ukraine in February 2022 has caused a high influx of refugees, including children, to neighboring countries, particularly Poland. This caused additional pressures on the healthcare system and the need to meet challenges for public health, such as those related to infectious diseases. Here, we discuss the potential epidemiological risks associated with the war-induced influx of refugees (coronavirus disease 2019, measles, pertussis, tetanus, and poliomyelitis) and highlight the need for their swift management through institutional support, educational campaigns, counteracting antiscience misinformation, and pursuing vaccinations of refugees but also improving or maintaining good levels of immunization in populations of countries welcoming them. These are necessary actions to avoid overlapping of war and infectious diseases and associated public health challenges.

The effectiveness and biosafety of diatrizoate contrast media in complicated cholelithiasis.

Adverse reactions after intravascular administration of iodine-based contrast media are well-known. Nevertheless the same type of contrast media is also used for endoscopic retrograde cholangiopancreatography and systemic absorption of contrast media after mentioned procedure routinely occurs, not much is known about effects of widely used diatrizoates (Triombrast) on the hepato-pancreato-biliary system in case of cholelithiasis treatment. AIM: The aim of the present study was to determine the effectiveness and biosafety of diatrizoate contrast media in terms of complicated cholelithiasis healing using conventional and improved surgery treatment protocols based on set of biochemical markers. MATERIALS AND METHODS: The paper presents materials of clinical observation and biochemical analysis of 122 patients have been diagnosed with gallstone disease complicated by choledocholithiasis and cholangitis and biliary pancreatitis. Biochemical liver tests before and after surgery treatment using conventional and improved protocols were determined. RESULTS: Biochemical manifestations of hepatocellular injury included exceeded activity of γ-glutamyltranspeptidase and aminotrasferases as well as level of liver fatty acid-binding protein and bilirubin which were different between groups of patients with cholelithiasis who suffered from concomitant cholangitis and pancreatitis. Proposed management algorithm for patients with gallstone disease with concomitant cholangitis and pancreatitis links to get rid of endoscopic retrograde cholangiography with contrast agent from the conventional surgery treatment protocols because its most obvious downside. CONCLUSIONS: Patients with gallstone diseases revealed significant biochemical changes that should be successfully resolved after surgery treatment without endoscopic retrograde cholangio- pancreatography with contrast media because its hepatotoxic effect.

Toxic effects and mechanisms of common pesticides (Roundup and chlorpyrifos) and their mixtures in a zebrafish model (Danio rerio).

Agrochemicals can adversely affect biodiversity, environment and human health, and commonly occur in mixtures with poorly characterized toxic mechanisms and health hazards. Here, we evaluated the individual and mixture toxicities of Roundup and chlorpyrifos in environmentally relevant concentrations to zebrafish using molecular and biochemical indices. Studied pesticides alone and in combination caused depletion of total antioxidant capacity and cellular thiols, overproduction of ROS, accumulation of oxidative lesions and elevated DNA damage in zebrafish liver. Notably, low concentration of Roundup induced a hormesis-like effect by stimulating the protective cellular mechanisms. Chlorpyrifos showed stronger prooxidant effects than Roundup and additionally caused nitrosative and carbonyl stress in zebrafish. At the organismal level, studied pesticides and their mixtures induced hepato- and neurotoxicity. The effects of the studied pesticides on biomarkers of apoptosis, endocrine disruption and immune disorders were generally weak and inconsistent. The multibiomarker assessment showed that chlorpyrifos is considerably more toxic than Roundup to zebrafish. The toxic effects of the pesticide mixtures were mostly driven by chlorpyrifos, with minimal or mitigating effects of Roundup addition. These findings elucidate the toxic mechanisms of common pesticides in a model vertebrate and demonstrate that health hazards of pesticide mixtures cannot be predicted from the effects of single pesticides.

Biomarker-based assessment of sublethal toxicity of organic UV filters (ensulizole and octocrylene) in a sentinel marine bivalve Mytilus edulis.

The global occurrence of organic UV filters in the marine environment is of increasing ecotoxicological concern. Here we assessed the toxicity of UV filters ensulizole and octocrylene in the blue mussels Mytilus edulis exposed to 10 or 100 µg l-1 of octocrylene and ensulizole for two weeks. An integrated battery of biochemical and molecular biomarkers related to xenobiotics metabolism and cellular toxicity (including oxidative stress, DNA damage, apoptosis, autophagy and inflammation) was used to assess the toxicity of these UV filters in the mussels. Octocrylene (but not ensulizole) accumulated in the mussel tissues during the waterborne exposures. Both studied UV filters induced sublethal toxic effects in M. edulis at the investigated concentrations. These effects involved induction of oxidative stress, genotoxicity (indicated by upregulation of DNA damage sensing and repair markers), upregulation of apoptosis and inflammation, and dysregulation of the xenobiotic biotransformation system. Octocrylene induced cellular stress in a concentration-dependent manner, whereas ensulizole appeared to be more toxic at the lower (10 µg l-1) studied concentration than at 100 µg l-1. The different concentration-dependence of sublethal effects and distinct toxicological profiles of ensulizole and octocrylene show that the environmental toxicity is not directly related to lipophilicity and bioaccumulation potential of these UV filters and demonstrate the importance of using bioassays for toxicity assessment of emerging pollutants in coastal marine ecosystems.

Salinity-dependent effects of ZnO nanoparticles on bioenergetics and intermediate metabolite homeostasis in a euryhaline marine bivalve, Mytilus edulis.

Engineered nanoparticles including ZnO nanoparticles (nZnO) are important emerging pollutants in aquatic ecosystems creating potential risks to coastal ecosystems and associated biota. The toxicity of nanoparticles and its interaction with the important environmental stressors (such as salinity variation) are not well understood in coastal organisms and require further investigation. Here, we examined the interactive effects of 100 µg l-1 nZnO or dissolved Zn (as a positive control for Zn2+ release) and salinity (normal 15, low 5, and fluctuating 5-15) on bioenergetics and intermediate metabolite homeostasis of a keystone marine bivalve, the blue mussel Mytilus edulis from the Baltic Sea. nZnO exposures did not lead to strong disturbances in energy or intermediate metabolite homeostasis regardless of the salinity regime. Dissolved Zn exposures suppressed the mitochondrial ATP synthesis capacity and coupling as well as anaerobic metabolism and modified the free amino acid profiles in the mussels indicating that dissolved Zn is metabolically more damaging than nZnO. The environmental salinity regime strongly affected metabolic homeostasis and altered physiological and biochemical responses to nZnO or dissolved Zn in the mussels. Exposure to low (5) or fluctuating (5-15) salinity affected the physiological condition, energy metabolism and homeostasis, as well as amino acid metabolism in M. edulis. Generally, fluctuating salinity (5-15) appeared bioenergetically less stressful than constantly hypoosmotic stress (salinity 5) in M. edulis indicating that even short (24 h) periods of recovery might be sufficient to restore the metabolic homeostasis in this euryhaline species. Notably, the biological effects of nZnO and dissolved Zn became progressively less detectable as the salinity stress increased. These findings demonstrate that habitat salinity must be considered in the biomarker-based assessment of the toxic effects of nanopollutants on coastal organisms.

Increase of α-dicarbonyls in liver and receptor for advanced glycation end products on immune cells are linked to nonalcoholic fatty liver disease and liver cancer.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with a very poor prognosis and constantly growing incidence. Among other primary risks of HCC, metabolic disorders and obesity have been extensively investigated over recent decades. The latter can promote nonalcoholic fatty liver disease (NAFLD) leading to the inflammatory form of nonalcoholic steatohepatitis (NASH), that, in turn, promotes HCC. Molecular determinants of this pathogenic progression, however, remain largely undefined. In this study, we have focussed on the investigation of α-dicarbonyl compounds (α-dC), highly reactive and tightly associated with overweight-induced metabolic disorders, and studied their potential role in NAFLD and progression toward HCC using murine models. NAFLD was induced using high-fat diet (HFD). Autochthonous HCC was induced using transposon-based stable intrahepatic overexpression of oncogenic NRASG12V in mice lacking p19Arf tumor suppressor. Our study demonstrates that the HFD regimen and HCC resulted in strong upregulation of α-dC in the liver, heart, and muscles. In addition, an increase in α-dC was confirmed in sera of NAFLD and NASH patients. Furthermore, higher expression of the receptor for advanced glycation products (RAGE) was detected exclusively on immune cells and not on stroma cells in livers of mice with liver cancer progression. Our work confirms astable interplay of liver inflammation, carbonyl stress mediated by α-dC, and upregulated RAGE expression on CD8+ Tand natural killer (NK) cells in situ in NAFLD and HCC, as key factors/determinants in liver disease progression. The obtained findings underline the role of α-dC and RAGE+CD8+ Tand RAGE+ NK cells as biomarkers and candidates for a local therapeutic intervention in NAFLD and malignant liver disease.

Multibiomarker-based assessment of toxicity of central European strains of filamentous cyanobacteria Aphanizomenon gracile and Raphidiopsis raciborskii to zebrafish Danio rerio.

The global increase in cyanobacterial blooms poses environmental and health threats. Selected cyanobacterial strains reveal toxicities despite a lack of synthesis of known toxic metabolites, and the mechanisms of these toxicities are not well understood. Here we investigated the toxicity of non-cylindrospermopsin and non-microcystin producing Aphanizomenon gracile and Raphidiopsis raciborskii of Central European origin to zebrafish exposed for 14 days to their extracts. Toxicological screening revealed the presence of anabaenopeptins and a lack of anatoxin-a, ß-methylamino-L-alanine or saxitoxins in examined extracts. The responses were compared to 20 µg L-1 of common cyanobacterial toxins cylindrospermopsin (CYN) and microcystin-LR (MC-LR). The expression of the marker genes involved in apoptosis (caspase 3a and 3b, Bcl-2, BAX, p53, MAPK, Nrf2), DNA damage detection and repair (GADD45, RAD51, JUN, XPC), detoxification (CYP1A, CYP26, EPHX1), lipid metabolism (PPARa, FABP1, PLA2), phosphorylation/dephosphorylation (PPP6C, PPM1) and cytoskeleton (actin, tubulin) were examined using targeted transcriptomics. Cellular stress and toxicity biomarkers (oxidative injury, antioxidant enzymes, thiol pool status, and lactate dehydrogenase activity) were measured in the liver, and acetylcholinesterase activity was determined as an index of neurotoxicity in the brain. The extracts of three cyanobacterial strains that produce no known cyanotoxins caused marked toxicity in D. rerio, and the biomarker profiles indicate different toxic mechanisms between the bioactive compounds extracted from these strains and the purified cyanotoxins. All studied cyanobacterial extracts and purified cyanotoxins induced oxidative stress and neurotoxicity, downregulated Nrf2 and CYP26B1, disrupted phosphorylation/dephosphorylation processes and actin/tubulin cytoskeleton and upregulated apoptotic activity in the liver. The tested strains and purified toxins displayed distinctively different effects on lipid metabolism. Unlike CYN and MC-LR, the Central European strain of A. gracile and R. raciborskii did not reveal a genotoxic potential. These findings help to further understand the ecotoxicological consequences of toxic cyanobacterial blooms in freshwater ecosystems.

In Vitro Toxicological Screening of Stable and Senescing Cultures of Aphanizomenon, Planktothrix, and Raphidiopsis.

Toxicity of cyanobacteria is the subject of ongoing research, and a number of toxic metabolites have been described, their biosynthesis pathways have been elucidated, and the mechanism of their action has been established. However, several knowledge gaps still exist, e.g., some strains produce hitherto unknown toxic compounds, while the exact dynamics of exerted toxicity during cyanobacterial growth still requires further exploration. Therefore, the present study investigated the toxicity of extracts of nine freshwater strains of Aphanizomenon gracile, an Aphanizomenon sp. strain isolated from the Baltic Sea, a freshwater strain of Planktothrix agardhii, and two strains of Raphidiopsis raciborskii obtained from 25- and 70-day-old cultures. An in vitro experimental model based on Cyprinus carpio hepatocytes (oxidative stress markers, DNA fragmentation, and serine/threonine protein activity) and brain homogenate (cholinesterase activity) was employed. The studied extracts demonstrated toxicity to fish cells, and in general, all examined extracts altered at least one or more of considered parameters, indicating that they possess, to some degree, toxic potency. Although the time from which the extracts were obtained had a significant importance for the response of fish cells, we observed strong variability between the different strains and species. In some strains, extracts that originated from 25-day-old cultures triggered more harmful effects on fish cells compared to those obtained from 70-day-old cultures, whereas in other strains, we observed the opposite effect or a lack of a significant change. Our study revealed that there was no clear or common pattern regarding the degree of cyanobacterial bloom toxicity at a given stage of development. This means that young cyanobacterial blooms that are just forming can pose an equally toxic threat to aquatic vertebrates and ecosystem functioning as those that are stable or old with a tendency to collapse. This might be largely due to a high variability of strains in the bloom.

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.

Polymethoxy-1-Alkenes Screening of Chlorella and Spirulina Food Supplements Coupled with In Vivo Toxicity Studies.

Selected species of cyanobacteria and green algae have been reported to produce lipophilic polymethoxy-1-alkenes (PMAs) which were shown to exhibit in vivo teratogenicity. Considering that information on PMAs in Arthospira sp. (known commercially as Spirulina) and Chlorella sp. cultivated for food supplement production was essentially lacking, the present study screened Chlorella (n = 10) and Spirulina (n = 13) food supplements registered in the European Union. Mass spectrometry analysis of column fractionated extracts was performed. None of the four variants previously reported in some cyanobacteria and green algae, nor any potentially related structures were detected in the studied samples. Since the isolated lipophilic fractions contained various compounds, they were further screened for in vivo teratogenicity in Danio rerio embryo, and for the potential to induce oxidative stress and genotoxicity in the liver and neurotoxicity in the brain of adult zebrafish. None of the tested food supplements had detectable levels of PMAs or any potentially related structures. No teratogenicity was revealed except for spinal curvature induced by fractions obtained from two Chlorella products. Selected fractions revealed cytotoxicity as indicated by an increased level of reactive oxygen species, catalase activity, lipid peroxidation and increased frequency of DNA strand breaks in hepatic tissue. The majority (60%) of Chlorella fractions induced an increase in cholinesterase activity in zebrafish brain homogenate while exposure to 61.5% of Spirulina fractions was associated with its decrease. The present study confirms that Chlorella and Spirulina food supplements are free of teratogenic PMAs, although the observed in vivo toxicities raise questions regarding the quality of selected products.