Behavioral Responses of Chironomus aprilinus Larvae as Proxies for Cyanobacterial Metabolite Interactions: Insights from Ternary Combinations.

This study aimed to assess the behavioral responses (immobilization, horizontal and vertical motility, and response to light) of Chironomus aprilinus larvae exposed to individual cyanobacterial metabolites aeruginosin 98B (AER-B), anabaenopeptin-B (ANA-B), and cylindrospermopsin (CYL), and their binary and ternary mixtures. The investigation revealed that single metabolites ANA-B and CYL exhibited the highest potency in immobilizing the larvae. Notably, the binary mixture AER-B+CYL induced a remarkably strong synergistic interaction, while other tested binary and ternary mixtures demonstrated antagonistic effects. Both individual metabolites and their mixtures led to a decrease in larval movement speed, with the AER-B+CYL combination showing a very synergistic effect, and strong antagonistic interactions between the oligopeptides in the ternary mixture. Conversely, while AER-B and the binary mixture ANA-B+CYL stimulated vertical movement, other single metabolites and binary and ternary mixtures decreased this parameter. Antagonistic interactions were observed in all mixtures. ANA-B emerged as the most potent inhibitor, yet all tested metabolites and their mixtures decreased larval response to light, displaying synergistic interactions, except for the AER-B+ANA-B mixture at 250 µg L-1 + 250 µg L-1. These findings underscore the sensitivity of Chironomus larvae behavioral parameters as indicators of environmental stressors and mixtures. Consequently, they are recommended for assessing toxic effects induced by cyanobacterial products and other bioactive chemicals.

Responses of RTgill-W1 cells to cyanobacterial metabolites microcystin-LR, anabaenopeptin-A, cylindrospermopsin, their binary and ternary mixtures.

The aim of our study was to investigate the effects of cyanobacterial metabolites: microcystin-LR (MC-LR) anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL), their binary and ternary mixtures on rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) cell line. We determined the following cell parameters: Hoechst and propidium iodide (PI) double staining, intracellular ATP level with luminometric assay, glutathione level with ThiolTracker Violet®- glutathione detection reagent and cytoskeletal F-actin fluorescence. The results showed that although reduction of Hoechst fluorescence was observed in both binary and ternary combinations of cyanobacterial metabolites, the mixture of MC-LR + ANA-A + CYL was the most potent inhibitor (EC50 = 148 nM). PI fluorescence and ATP levels were more increased in the cells exposed to the mixtures than those exposed to the individual metabolites with synergistic toxic changes suggesting apoptosis as the mechanism of cell death. Reduced glutathione level was also decreased in cells exposed both to single metabolites and their mixtures with the highest decrease and synergistic effects at 334 nM MC-LR+334 nM ANA-A+ 334 nM CYL suggesting induction oxidative stress by the tested compounds. Reduction of F-actin fluorescence was found in the cells from all of the groups exposed to individual metabolites and their mixtures, however the highest level of inhibition showed the binary MC-LR + CYL and the ternary MC-LR + ANA-A + CYL with synergistic interactions. The study suggests that in natural conditions fish gill cells may be very sensitive to individual cyanobacterial metabolites and more prone to their binary and ternary mixtures.

Interactive effects of cyanobacterial metabolites aeruginosin-98B, anabaenopeptin-B and cylindrospermopsin on physiological parameters and novel in vivo fluorescent indicators in Chironomus aprilinus larvae.

We aimed to determine the effects of single cyanobacterial metabolites aeruginosin-B (AER-B), anabaenopeptin-B (ANA-B), cylindrospermopsin (CYL), their binary and ternary mixtures on biomarkers of Chironomus aprilinus larvae: oxygen consumption, fat body structure and two novel fluorescent indicators: imaging of nuclei in cells of body integument, and the catecholamine level. The obtained results showed that oxygen consumption was inhibited by single tested cyanobacterial metabolites except for ANA-B at the lowest concentration (250 µg/L). Although the mixtures of the metabolites inhibited oxygen consumption with antagonistic interactions between the components stimulation was noted in the group exposed to the lowest concentrations of AER-B + CYL (125 µg/L + 125 µg/L, respectively) and the ternary mixture of AER-B + ANA-B + CYL (83.3 µg/L + 83.3 µg/L + 83.3 µg/L, respectively). In vivo fluorescent staining with Hoechst 34580 showed that single AER-B had lower cytotoxic potential on body integument cells than ANA-B and CYL and most binary mixtures except for AER-B + CYL induced synergistic toxicity. Catecholamine level was decreased in animals exposed to single metabolites, their binary and ternary mixtures; however, the interactions between the components in the ternary mixture were antagonistic. Fat body was found to be disrupted in the larvae exposed to single metabolites and their combinations. Antagonistic toxic interactions between the oligopeptide components were found in most binary and the ternary mixtures; however, synergistic effect was noted in the binary mixture of AER-B + CYL. The results suggest that in natural conditions Chironomus larvae and possibly other benthic invertebrates may be affected by cyanobacterial metabolites, however various components and in mixtures and their concentrations may determine varied physiological effects and diverse interactions.

First report on adverse effects of cyanobacterial anabaenopeptins, aeruginosins, microginin and their mixtures with microcystin and cylindrospermopsin on aquatic plant physiology: An experimental approach.

Cyanobacteria produce a variety of oligopeptides beyond microcystins and other metabolites. Their biological activities are not fully recognized especially to aquatic plants. Acute toxicity tests on Spirodela polyrhiza and Lemna minor exposed to a range of concentrations of cyanobacterial metabolites: anabaenopeptins (ANA-A, ANA-B), aeruginosins 98 (Aer-A, Aer-B), microginin-FR1 (MG-FR1), microcystin-LR (MC-LR) and cylindrospermopsin (Cyl) were carried out to compare their influence on plant physiology. Effects of their binary mixtures were determined by isobole approach and calculation of the combination index (CI) that indicates a type of metabolites' interaction. Cyclic oligopeptides microcystin-LR and anabaenopeptin-A revealed the strongest inhibition of S. polyrhiza growth while other metabolites appeared less toxic. Oxygen evolution was inhibited by Cyl, MC-LR, ANA-A, ANA-B, while both variants of aeruginosins and MG-FR1 did not affect this process. Photosynthetic pigments' contents decreased in S. polyrhiza exposed to ANA-A and Cyl, while MC-LR and Aer-A caused their slight increase. 96 h-EC50 values showed that the growth of L. minor was more sensitive to MC-LR, ANA-A, MG-FR1 and Cyl than the growth of S. polyrhiza. In S. polyrhiza synergistic effects of all the binary mixtures of peptides with MC-LR on oxygen evolution were observed, while antagonistic one on the growth of S. polyrhiza exposed to the mixtures with aeruginosins and ANA-A. The mixtures of MC-LR and MG-FR1 with cylindrospermopsin revealed synergistic effects on the growth but antagonistic one to the O2 evolution. Quadruple mixtures (ANA-A + MC-LR + MG-FR1+Cyl) did not reveal any inhibitive effect on the plant growth and very slight on the oxygen evolution, irrespectively of their total concentrations. Various effects caused by ANA-A and ANA-B suggest the importance of molecule structures of metabolites for toxicity. Composition of the mixtures of cyanobacterial metabolites was essential for the observed effects.

Cyanobacterial metabolites: Aeruginosin 98A, microginin-FR1, anabaenopeptin-A, cylindrospermopsin and their mixtures affect behavioral and physiological responses of Daphnia magna.

We determined the effects influence of cyanobacterial products metabolites: aeruginosin-A (AER-A), microginin-FR1 (MG-FR1), anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL) and their binary and quadruple mixtures on swimming behavior, heart rate, thoracic limb activity, oxygen consumption and in vivo cell health of Daphnia magna. The study showed that CYL induced mortality of daphnids at the highest concentrations, however three oligopeptides had no lethal effect. All the tested Each single metabolites inhibited swimming speed. The mixtures AER+MG-FR1 and AER-A+ANA-A induced antagonistic and the quadruple mixture synergistic effects. Physiological endpoints were depressed by CYL, however they were simulated by the oligopeptides and their binary mixtures. The quadruple mixture inhibited the physiological parameters with antagonistic interactions between the components were antagonistic. Single CYL, MG-FR1 and ANA-A induced cytotoxicity with synergistic interactions and the metabolites in mixtures showed. The study suggests that swimming behavior and physiological parameters may be affected by single cyanobacterial oligopeptides, however their mixtures may induce different total effects.

Effects of cyanobacterial metabolites: Aeruginosin 98A, microginin-FR1, anabaenopeptin-A, cylindrospermopsin in binary and quadruple mixtures on the survival and oxidative stress biomarkers of Daphnia magna.

The aim of our study was to determine the effects of aeruginosin 98 A (ARE-A), microginin-FR1 (MG-FR1), anabaenopeptin-A (ANA-A) cylindrospermopsin (CYL) and their binary and quadruple mixtures on the survival and the levels of oxidative stress biomarkers in Daphnia magna: total glutathione (GSH), catalase (CAT), dismutase (SOD) and malondialdehyde (MDA). The biochemical indicators were measured with ELISA kits and the interactive effects were determined by isobole and polygonal analysis with Compusyn® computer software. The study revealed that oligopeptides did not decrease daphnid survival, only CYL inhibited this parameter, with synergistic effects when it was used as a component. The single metabolites at the two highest concentrations and all the binary and quadruple mixtures at all concentrations diminished GSH level, however both in the binary and in the quadruple mixtures most of the interactions between the metabolites were antagonistic. Nearly additive effects were found only in AER-A + CYL and MG-FR1+CYL. On the other hand, CAT activity was slightly increased in daphnids exposed to the binary mixtures with antagonistic interactions, however nearly addivive effects were found in animals exposed to the mixture of AER-A + ANA-A and synergistic in the quadruple mixture. SOD was elevated in daphnids exposed to single AER-A and MG-FR1, however it was diminished in the animals exposed to ANA-A and CYL. Binary mixtures in which CYL was present as a component decreased the level of this enzyme with nearly additive interactions in ANA-A + CYL. The quadruple mixture increased SOD level, with antagonistic interactions. Both single cyanobacterial metabolites, their binary and quadruple mixtures induced lipid peroxidation measured by MDA level and most of interactions in the binary mixtures were synergistic. The study suggested that antioxidative system of Daphnia magna responded to the tested metabolites and the real exposure to mixtures of these products may lead to various interactive effects with varied total toxicity.

Behavioral disturbances induced by cyanobacterial oligopeptides microginin-FR1, anabaenopeptin-A and microcystin-LR are associated with neuromotoric and cytotoxic changes in Brachionus calyciflorus.

Aquatic animals are exposed to various cyanobacterial products released concomitantly to the environment by decaying blooms. Although there exist results on the toxicity of cyanobacterial extracts little is known on the influence of pure oligopeptides or their mixtures and elucidated mechanisms of behavioral toxicity in zooplanktonic organisms. Therefore, the aim of the present study was to assess the effects of single and mixed pure cyanobacterial oligopeptides: microginin FR-1 (MG-FR1), anabaenopeptin-A (ANA-A) and microcystin-LR (MC-LR) at various concentrations on the swimming behavior and catecholamine neurotransmitter activity, muscular F-actin structure, DNA nuclear content and cell viability of a model rotifer Brachionus calyciflorus. Swimming behavior was analyzed with the use of video digital analysis. Fluorescent microscopy imaging was used to analyze neuromotoric biomarkers in the whole organisms: neuromediator release (by staining with EC517 probe), muscle F-actin filaments (by staining with blue phalloidin dye). DNA content and cytotoxicity was also determined by Hoechst 34580 and propidium iodide double staining, respectively. The results showed that single oligopeptides inhibited all the tested endpoints. The binary mixtures induced synergistic interaction on swimming speed except for MG-FR1 +MC-LR which was nearly additive. Both binary and ternary mixtures also synergistically degraded F-actin and triggered cytotoxic effects visible in the whole organisms. Antagonistic inhibitory effects of all the binary mixtures were found on catecholamine neurotransmitter activity, however the ternary mixture induced additive toxicity. Antagonistic effects of both binary and ternary mixtures were also noted on nuclear DNA content. The results of the study suggest that both depression of neurotransmission and impairment of muscle F-actin structure in muscles may contribute to mechanisms of Brachionus swimming speed inhibition by the tested single cyanobacterial oligopeptides and their mixtures. The study also showed that natural exposure of rotifers to mixtures of these cyanobacterial metabolites may result in different level of interactive toxicity with antagonistic, additive synergistic effects depending on the variants and concentrations present in the environment.

Beyond the behavioural phenotype: Uncovering mechanistic foundations in aquatic eco-neurotoxicology.

During the last decade, there has been an increase in awareness of how anthropogenic pollution can alter behavioural traits of diverse aquatic organisms. Apart from understanding profound ecological implications, alterations in neuro-behavioural indices have emerged as sensitive and physiologically integrative endpoints in chemical risk assessment. Accordingly, behavioural ecotoxicology and broader eco-neurotoxicology are becoming increasingly popular fields of research that span a plethora of fundamental laboratory experimentations as well as applied field-based studies. Despite mounting interest in aquatic behavioural ecotoxicology studies, there is, however, a considerable paucity in deciphering the mechanistic foundations underlying behavioural alterations upon exposure to pollutants. The behavioural phenotype is indeed the highest-level integrative neurobiological phenomenon, but at its core lie myriads of intertwined biochemical, cellular, and physiological processes. Therefore, the mechanisms that underlie changes in behavioural phenotypes can stem among others from dysregulation of neurotransmitter pathways, electrical signalling, and cell death of discrete cell populations in the central and peripheral nervous systems. They can, however, also be a result of toxicity to sensory organs and even metabolic dysfunctions. In this critical review, we outline why behavioural phenotyping should be the starting point that leads to actual discovery of fundamental mechanisms underlying actions of neurotoxic and neuromodulating contaminants. We highlight potential applications of the currently existing and emerging neurobiology and neurophysiology analytical strategies that should be embraced and more broadly adopted in behavioural ecotoxicology. Such strategies can provide new mechanistic discoveries instead of only observing the end sum phenotypic effects.

Synergistic toxicity of some cyanobacterial oligopeptides to physiological activities of Daphnia magna (Crustacea).

Anabaenopeptins and microcystins are oligopeptides produced by bloom-forming cyanobacteria. We determined in vivo effects of anabaenopeptin-B (AN-B) and two variants of microcystins of different hydrophobicity (MC-LR and MC-LF) on the physiology of Daphnia magna. Heart rate, thoracic limb activity and post-abdominal claw activity were determined by digital video analysis and oxygen consumption by Oxygraph + system. EC50 calculation and isobole methodology for interactive effects of AN-B and MC-LR mixture were used. Daphnids' responses to all three oligopeptides were concentration- and time-dependent. MC-LF was the most potent inhibitor of heart rate, thoracic limb activity, post-abdominal claw activity and oxygen consumption. AN-B was more toxic than MC-LR toward oxygen consumption; it inhibited the movements of limbs and post-abdominal claw similarly to MC-LR, but did not inhibit heart rate. The strongest toxic effects were induced by the binary mixture of AN-B with MC-LR at the sum concentration equal to the concentration of the single compounds. First time direct synergistic toxic effects of the cyanopeptides on all the physiological parameters were found. The obtained results explain stronger disturbances in aquatic organisms caused by cyanobacterial cell contents than the individual cyanopeptides present even at higher concentrations. Other metabolites and their interactions need further studies.

Advances in real-time monitoring of water quality using automated analysis of animal behaviour.

Monitoring of freshwater quality and its potential sudden contamination is integral to human health, sustainable economic development and prediction of pollutant impact on aquatic ecosystems. Although there have been significant advances in technologies for automated sampling and continuous analysis of water physicochemical parameters, the current capabilities for real-time warning against rapidly developing unknown mixtures of chemical hazards are still limited. Conventional chemical analysis systems are not suitable for assessing unknown mixtures of chemicals as well as additive and/or synergetic effects on biological systems. From the perspective of neurotoxicology the acute exposures to chemical agents that affect nervous system and can enter the freshwater supplies accidentally or as a result of deliberate action, can only be reliably assessed using appropriate functional biological models. In this regard real-time biological early warning systems (BEWS), that can continuously monitor behavioural and/or physiological parameters of suitable aquatic bioindicator species, have been historically proposed to fill the gap and supplement conventional water quality test strategies. Alterations in sub-lethal neuro-behavioural traits have been proven as very sensitive and physiologically relevant endpoints that can provide highly integrative water quality sensing capabilities. Although BEWS are commonly regarded as non-specific and lacking both quantitative and qualitative detection capabilities, their advantages, if properly designed and implemented, lie in continuous sensing and early-warning information about sudden alteration in water quality parameters. In this work we review the future prospects of real-time biological early warning systems as well as recent developments that are anchored in historical successes and practical deployment examples. We concentrate on technologies utilizing analysis of behavioural and physiological endpoints of animal bioindicators and highlight the existing challenges, barriers to future development and demonstrate how recent advances in inexpensive electronics and multidisciplinary bioengineering can help revitalize the BEWS field.

Cyanobacterial anabaenopeptin-B, microcystins and their mixture cause toxic effects on the behavior of the freshwater crustacean Daphnia magna (Cladocera).

Comparison of the toxic effects caused by the pure cyanobacterial cyclic hexapeptide anabaenopeptin-B (AN-B), the heptapeptides: microcystin-LR (MC-LR) and MC-LF as well as a binary mixture of AN-B with MC-LR on the swimming speed and hopping frequency - essential activities of Daphnia, was experimentally determined. Till now, no information on behavioral effects of AN-B and its mixture with microcystins, commonly produced by cyanobacteria, was available. Also MC-LF effect on aquatic crustaceans was determined for the first time. The results showed that AN-B exerted considerable inhibition of D. magna swimming speed and hopping frequency similar to MC-LR and MC-LF. The mixture of AN-B and MC-LR caused stronger toxic effects, than the individual oligopeptides used at the same concentration. The much lower 48 h- EC50 value of the AN-B and MC-LR mixture (0.95 ± 0.12 µg/mL) than those of individual oligopeptides AN-B (6.3 ± 0.63 µg/mL), MC-LR (4.0 ± 0.27 µg/mL), MC-LF (3.9 ± 0.20 µg/mL) that caused swimming speed inhibition explains the commonly observed stronger toxicity of complex crude cyanobacterial extracts to daphnids than individual microcystins. The obtained results indicated that AN-B, microcystins and their mixture exerted time- and concentration-dependent motility disturbances of crustaceans and they can be good candidates for evaluation of toxicity in early warning systems. Other cyanobacterial oligopeptides beyond microcystins should be considered as a real threat for aquatic organisms.

Applications of advanced neuro-behavioral analysis strategies in aquatic ecotoxicology.

Despite mounting evidence of pleiotropic ecological risks, the understanding of the eco-neurotoxic impact of most industrially relevant chemicals is still very limited. In particularly the acute and chronic exposures to industrial pollutants on nervous systems and thus potential alterations in ecological fitness remain profoundly understudied. Since the behavioral phenotype is the highest-level and functional manifestation of integrated neurological functions, the alterations in neuro-behavioral traits have been postulated as very sensitive and physiologically integrative endpoints to assess eco-neurotoxicological risks associated with industrial pollutants. Due to a considerable backlog of risk assessments of existing and new production chemicals there is a need for a paradigm shift from high cost, low throughput ecotoxicity test models to next generation systems amenable to higher throughput. In this review we concentrate on emerging aspects of laboratory-based neuro-behavioral phenotyping approaches that can be amenable for rapid prioritizing pipelines. We outline the importance of development and applications of innovative neuro-behavioral assays utilizing small aquatic biological indicators and demonstrate emerging concepts of high-throughput chemo-behavioral phenotyping. We also discuss new analytical approaches to effectively and rapidly evaluate the impact of pollutants on higher behavioral functions such as sensory-motor assays, decision-making and cognitive behaviors using innovative model organisms. Finally, we provide a snapshot of most recent analytical approaches that can be applied to elucidate mechanistic rationale that underlie the observed neuro-behavioral alterations upon exposure to pollutants. This review is intended to outline the emerging opportunities for innovative multidisciplinary research and highlight the existing challenges as well barriers to future development.

Short-term effects of pesticide fipronil on behavioral and physiological endpoints of Daphnia magna.

Fipronil (FIP) is an organic pesticide with many practical uses. Although some results indicated toxic effects in some terrestrial and aquatic animal species, little is known on its influence on behavioral and physiological endpoints of cladocerans. The aim of our study was to determine the short-term effects of FIP at concentrations of 0.1 µg/L, 1 µg/L, 10 µg/L, and 100 µg/L on Daphnia magna sublethal indices: behavioral (swimming speed, distance traveled) and physiological endpoints (heart rate, post-abdominal claw activity and thoracic limb movements). The results showed that FIP induced reduction of swimming speed and distance traveled in a concentration- and time-dependent manner at all the concentrations used. The lowest concentration of the insecticide temporarily stimulated post-abdominal claw activity after 24 h and thoracic limb activity after 48 h; however, the highest concentrations reduced all the studied physiological endpoints. IC50 values showed that thoracic limb activity, swimming speed, and distance traveled were most sensitive to FIP after 24-h exposure. The most sensitive parameter after 48 h and 72 h was swimming speed and post-abdominal claw activity, respectively. The study indicated that (i) behavioral and physiological endpoints of Daphnia magna are reliable and valuable sublethal indicators of toxic alterations induced by FIP; however, they respond with different sensitivity at various times of exposure, (ii) FIP may alter cladoceran behavior and physiological processes at concentrations detected in the aquatic environment; therefore, it should be considered as an ecotoxicological hazard to freshwater cladocerans.

Effects of L-proline on cellular responses of hen erythrocytes subjected to thermal stress.

Little is known on the protective effects of L-proline on hen erythrocytes. The aim of the study was to determine the protective effects of this amino acid at concentrations of 50 µg/mL, 100 µg/mL, 200 µg/mL in hen erythrocytes subjected to temperatures 41 °C, 43 °C and 45 °C for 1 h and 4 h. The following cellular parameters were determined: viability, morphological alterations, caspase 3/7 activity, heat shock protein HSP70 1A activity and glutathione level. The results showed that exposure to 43 °C and 45 °C resulted in a decrease of viability and increased morphological alterations of the non-treated erythrocytes. Caspase 3/7 activity was increased only at 45 °C, however HSP70 1A activity and glutathione level were increased in the temperature-dependent manner. On the other hand, erythrocytes additionally exposed to L-proline showed alterations of the parameters when compared to the non-treated cells. L-proline at 50 µg/mL and 100 µg/mL increased caspase 3/7 activity at both 41 °C and 43 °C, however it was less augmented at all the concentrations at 45 °C. Glutathione level was decreased in heat-stressed (at 43 °C and 45 °C) hen erythrocytes treated with L-proline (at 50 µg/mL and 100 µg/mL) but it was increased at 200 µg/mL. HSP70 1A activity was augmented in a concentration- and temperature-dependent manner. The results indicate that proapoptotic or antiapoptotic effects of L-proline depend on its concentration and temperature of heat stress and thermoprotective effects induced by the amino acid on some parameters in hen erythrocytes may be a result of stimulation of antioxidative defense and stimulation of HSP70 1A activity.

Early morphological and apoptotic responses of bird erythrocytes to thermal stress.

Little is known about early indicators of heat stress in bird erythrocytes. We investigated the effects of elevated temperatures on the morphology and cellular responses of avian erythrocytes. Hen red blood cells were subjected to 22-45 °C temperatures for 1 h and 4 h, then stained and examined by light microscopy to assess morphological alterations. Cell viability, cytotoxicity and caspases 3 and 7 activity also were investigated. We found that short-term exposure of hen blood to 43-45 °C caused morphological alterations and increased the activity of pro-apoptotic caspases 3 and 7; hemolytic cells also were found. Reduction of erythrocytes may be a consequence of direct disruption of the cell membrane, although apoptotic disintegration also may occur. Because changes in erythrocyte morphology were rapid, they may be useful indicators of thermal stress in birds.

Behavioral and physiological responses of Daphnia magna to salicylic acid.

Salicylic acid (SA), a metabolite of acetylsalicylic acid is a monohydroxybenzoic acid a common non-steroidal analgesic and anti-inflammatory drug (NSAID) frequently detected in various aquatic ecosystems at concentrations up to 19.50 µg L-1 in surface waters near livestock farms and 59.6 µg L-1 in wastewaters. Little is known on the effects of short-term exposure of freshwater crustaceans to salicylic acid. Therefore, the aim of our study was to determine the effects of SA at concentrations of 5 µg L-1, 500 µg L-1, 5 mg L-1, 50 mg L-1 and 500 mg L-1 on the behavior (swimming speed, swimming height, distance travelled) and physiological endpoints (heart rate, mandible movement) of Daphnia magna exposed for 24 h, 48 h and 72 h. The results showed that SA inhibited the swimming speed, swimming height and distance travelled, heart rate and mandible movement at 5 mg L-1, 50 mg L-1 and 500 mg L-1 when compared to the control. On the other hand, SA at 5 µg L-1 and 500 µg L-1 transiently increased swimming speed and distance travelled after 24 h of the exposure, except for swimming height. Behavioral and physiological disturbances were observed much earlier than lethality. Our study showed SA at environmental levels may be an ecotoxicological agent imparing behavior and physiology of freshwater crustaceans.

Daphnia magna model in the toxicity assessment of pharmaceuticals: A review.

Daphnia magna is one of the most commonly used model organism to assess toxicity of wide range of pharmaceuticals such as antibiotics, anticancer drugs, antidepressants, anti-inflammatory drugs, beta-blockers and lipid-regulating agents. Currently, daphnia toxicity tests based on immobilisation and lethality standardised by OECD, acute immobilisation test and reproduction test, are mainly used in toxicological studies. Detailed analysis of Daphnia biology allows distinguishing the swimming behaviour and physiological endpoints such as swimming speed, distance travelled, hopping frequency, heart rate, ingestion rate, feeding rate, oxygen consumption, thoracic limb activity which could be also useful in assessment of toxic effects. The advantage of behavioural and physiological parameters is the possibility to observe sublethal effects induced by lower concentrations of pharmaceuticals which would not be possible to notice by using OECD tests. Additionally, toxic effects of tested drugs could be assessed using enzymatic and non-enzymatic biomarkers of daphnia toxicity. This review presents scientific data considering characteristics of D. magna, analysis of immobilisation, lethality, reproductive, behavioural, physiological and biochemical parameters used in the toxicity assessment of pharmaceuticals. The aim of this paper is also to emphasize usefulness, advantages and disadvantages of these invertebrate model organisms to assess toxicity of different therapeutic classes of pharmaceuticals. Also, various examples of application of D. magna in studies on pharmaceutical toxicity are presented.

Identification of Mitochondrial DNA (NUMTs) in the Nuclear Genome of Daphnia magna.

This is the first study in which the Daphnia magna (D. magna) nuclear genome (nDNA) obtained from the GenBank database was analyzed for pseudogene sequences of mitochondrial origin. To date, there is no information about pseudogenes localized in D. magna genome. This study aimed to identify NUMTs, their length, homology, and location for potential use in evolutionary studies and to check whether their occurrence causes co-amplification during mitochondrial genome (mtDNA) analyses. Bioinformatic analysis showed 1909 fragments of the mtDNA of D. magna, of which 1630 were located in ten linkage groups (LG) of the nDNA. The best-matched NUMTs covering >90% of the gene sequence have been identified for two mt-tRNA genes, and they may be functional nuclear RNA molecules. Isolating the total DNA in mtDNA studies, co-amplification of nDNA fragments is unlikely in the case of amplification of the whole tRNA genes as well as fragments of other genes. It was observed that TRNA-MET fragments had the highest level of sequence homology, thus they could be evolutionarily the youngest. The lowest homology was found in the D-loop-derived pseudogene. It may probably be the oldest NUMT incorporated into the nDNA; however, further analysis is necessary.

Ketoprofen affects swimming behavior and impairs physiological endpoints of Daphnia magna.

Ketoprofen (KET) is a nonsteroidal anti-inflammatory and analgesic drug commonly used in human and veterinary medicine. This compound is detected in aquatic reservoirs however, little is known about its influence on cladocerans. Therefore, the aim of our study was to determine the influence of KET at concentrations of 0.005 mg/L, 0.05 mg/L, 0.5 mg/L, 5 mg/L and 50 mg/L on behavioral (swimming speed, hopping frequency) and physiological endpoints (heart rate, thoracic limb activity, mandible movements) of Daphnia magna after 24 h and 48 h exposure. The study showed that swimming speed frequency was decreased after 24 h and 48 h at all the concentrations used in the experiment. Hopping frequency was also inhibited, however the lowest amount of the drug induced transient increase of the parameter after 24 h and its subsequent decrease to the control level after 48 h. Although after 24 h of the exposure physiological parameters: heart rate, thoracic limb activity and mandible movements showed slightly lower sensitivity to KET than the behavioral endpoints: were found to be inhibited after 48 h. The results revealed that both behavioral and physiological endpoints of daphnids responded to KET also at the environmental level, therefore in natural conditions this drug should be considered as a hazardous toxicant to crustaceans.

Cisplatin affects locomotor activity and physiological endpoints of Daphnia magna.

Cisplatin (CPL) is a common antineoplastic drug used in human medicine for treatment of various cancer types. Since the knowledge about its effects on crustacean behavioral and physiological parameters is very scarce, the aim of our study was to determine the influence of CPL at concentrations of 125 µg/L, 200 µg/L, 500 µg/L and 1000 µg/L on swimming behavior (swimming speed, distance travelled, hopping frequency, propelling efficiency index - a novel parameter) and physiological parameters (heart rate, thoracic limb activity) of Daphnia magna with the use of video digital analysis. The results showed that distance travelled, swimming speed, hopping frequency and propelling efficiency were inhibited as early as after 24 h in concentration- and time-dependent manner. On the other hand, heart rate was stimulated in the animals treated with 125 µg/L of CPL after 48 h, 72 h and 120 h of the exposure, however it was decreased at the higher concentrations. Although thoracic limb activity was considerably increased in daphnids exposed to 125 µg/L and 200 µg/L after 72 h, it was inhibited at the higher concentrations of the drug. The study suggests that since CPL affected daphnid parameters at the environmental concentration, it should be considered as hazardous to zooplankton.