Contingency learning in zebrafish exposed to apomorphine- and levetiracetam.

Cognitive rigidity (CR) refers to inadequate executive adaptation in the face of changing circumstances. Increased CR is associated with a number of psychiatric disorders, for example, obsessive-compulsive disorder, and improving cognitive functioning by targeting CR in these conditions, may be fruitful. Levetiracetam (LEV), clinically used to treat epilepsy, may have pro-cognitive effects by restoring balance to neuronal signalling. To explore this possibility, we applied apomorphine (APO) exposure in an attempt to induce rigid cue-directed responses following a cue (visual pattern)-reward (social conspecifics) contingency learning phase and to assess the effects of LEV on such behaviours. Briefly, zebrafish were divided into four different 39-day-long exposure groups ( n  = 9-10) as follows: control (CTRL), APO (100 µg/L), LEV (750 µg/L) and APO + LEV (100 µg/L + 750 µg/L). The main findings of this experiment were that 1) all four exposure groups performed similarly with respect to reward- and cue-directed learning over the first two study phases, 2) compared to the CTRL group, all drug interventions, but notably the APO + LEV combination, lowered the degree of reward-directed behaviour during a dissociated presentation of the cue and reward, and 3) temporal and spatial factors influenced the manner in which zebrafish responded to the presentation of the reward. Future studies are needed to explore the relevance of these findings for our understanding of the potential cognitive effects of LEV.

Lethal and sublethal impacts of a micropredator on post-settlement Caribbean reef fishes.

The transition from a planktonic larval stage to a benthic or demersal juvenile stage, "recruitment", is a crucial event in the life history of coral reef fishes, and has a strong influence on population size. Predation by piscivorous fishes is thought to be the main determinant of recruitment success, and has received the most attention. However, recent studies suggest that recently settled reef fishes are also an important target of micropredation from blood-feeding ectoparasites which may have significant lethal and sublethal effects. In this study, we quantified the relationship between levels of infestation by gnathiid isopods and mortality rates among juveniles of three species of reef fishes as a function of body mass both within and among species. We found that a single gnathiid isopod larva could kill fish of all three species shortly after settlement, up to 0.116 g [18 mm fork length (FL)] in French grunt (Haemulon flavolineatum), 0.027 g (15 mm FL) in masked goby (Coryphopterus personatus) and 0.01 g (9 mm FL) in beaugregory damselfish (Stegastes leucostictus). For juvenile S. leucostictus, we also compared the ability of fish to defend a territory when infested with a sublethal number of gnathiids versus uninfected individuals. Uninfected fish were significantly more likely to win-pairwise contests versus infected fish. These findings suggest that gnathiids can significantly impact juvenile coral reef fish survival, and affect population dynamics well past the settlement stage.

Assessing predator-prey interactions in a chemically altered aquatic environment: the effects of DDT on Xenopus laevis and Culex sp. larvae interactions and behaviour.

Behavioural assays are used as a tool to understand ecotoxicological effects on organisms, but are often not applied in an ecologically relevant context. Assessment of the effect of chemical contaminants on behaviours relating to fitness and trophic interactions for example, requires incorporating predator-prey interactions to create impact assessments. Dichlorodiphenyltrichloroethane (DDT) is a controlled substance but is still regularly used as a form of mosquito control. There is little explicit information on the effect of DDT on animal behaviour and the consequent effects upon trophic interactions. This study uses a 3 × 2 factorial design to assess the feeding behaviour of Xenopus laevis toward Culex sp. larvae when supplied with different prey cues. We also assess the behavioural responses of mosquito larvae when supplied with no threat cue and predator threat cues when exposed to 0 µg/L, 2 µg/L and 20 µg/L DDT. There was a significant "DDT exposure" x "prey cue" interaction whereby DDT significantly decreased the foraging behaviour of X. laevis towards live prey cues, however there was no effect of DDT on X. laevis response to olfactory prey cues. Dichlorodiphenyltrichloroethane exposure caused mosquito larvae to appear hyperactive regardless of DDT concentration. Mosquito larvae anti-predator response was significantly dampened when exposed to 2 µg/L DDT, however when exposed to 20 µg/L the anti-predator responses were not impaired. Our results indicate a complex interplay in trophic interactions under DDT exposure, wherein effects are mediated depending on species and concentration. There are possible implications regarding reduced anti-predator behaviour in the prey species but also reduced foraging capacity in the predator, which could drive changes in ecosystem energy pathways. We demonstrate that in order to quantify effects of pesticides upon trophic interactions it is necessary to consider ecologically relevant behaviours of both predator and prey species.

Effect of an antidepressant on aquatic ecosystems in the presence of microplastics: A mesocosm study.

Emerging pollutants, such as pharmaceuticals and microplastics have become a pressing concern due to their widespread presence and potential impacts on ecological systems. To assess the ecosystem-level effects of these pollutants within a multi-stressor context, we simulated real-world conditions by exposing a near-natural multi-trophic aquatic food web to a gradient of environmentally relevant concentrations of fluoxetine and microplastics in large mesocosms over a period of more than three months. We measured the biomass and abundance of different trophic groups, as well as ecological functions such as nutrient availability and decomposition rate. To explore the mechanisms underlying potential community and ecosystem-level effects, we also performed behavioral assays focusing on locomotion parameters as a response variable in three species: Daphnia magna (zooplankton prey), Chaoborus flavicans larvae (invertebrate pelagic predator of zooplankton) and Asellus aquaticus (benthic macroinvertebrate), using water from the mesocosms. Our mesocosm results demonstrate that presence of microplastics governs the response in phytoplankton biomass, with a weak non-monotonic dose-response relationship due to the interaction between microplastics and fluoxetine. However, exposure to fluoxetine evoked a strong non-monotonic dose-response in zooplankton abundance and microbial decomposition rate of plant material. In the behavioral assays, the locomotion of zooplankton prey D. magna showed a similar non-monotonic response primarily induced by fluoxetine. Its predator C. flavicans, however, showed a significant non-monotonic response governed by both microplastics and fluoxetine. The behavior of the decomposer A. aquaticus significantly decreased at higher fluoxetine concentrations, potentially leading to reduced decomposition rates near the sediment. Our study demonstrates that effects observed upon short-term exposure result in more pronounced ecosystem-level effects following chronic exposure.

Toxicity assessment of TiO2-conjugated Carbon-based nanohybrid material on a freshwater bioindicator cladoceran, Daphnia magna.

The application of nanocomposite materials fabricated from titanium dioxide nanoparticles (TiO2 NPs) and different carbon (C) allotropes have gained popularity in water treatment applications due to their synergistic properties. Studies to date have focused on simple forms of nanomaterials (NMs), however, with the technology development, there is a dramatic increase in production and application of these complex NMs which could result in toxicological impacts on organisms when released into aquatic environments. This raises serious concerns about their safety and the need to ascertain their potential adverse effects on aquatic organisms. While conjugated TiO2 NPs/carbon-based nanohybrids (TiO2/C-NHs) may exhibit enhanced photocatalytic activity, there is no research in the scientific community regarding their toxicological effects on D. magna, which are indicators of freshwater pollution. In this study, two under-represented TiO2/C-NHs (i.e., TiO2- conjugated carbon nanofiber (CNF), and TiO2-conjugated multi-walled carbon nanotube (CNT)) were investigated for their toxic effects on D. magna, through a series of acute toxicity tests with a set of sublethal biochemical biomarkers of oxidative stress. The lethal toxicity and oxidative stress formation of TiO2/C-NHs over 48 h revealed a concentration-dependant increase in D. magna mortality. The primary mechanism identified was the generation of ROS, which was in line with toxicity results. Light microscopy and CytoViva® images visualized D. magna interaction with the NPs, which accumulated and appeared as dark materials in the lines of the gut tract. The collective results indicate that TiO2/C-NHs have the potential to cause an effect on freshwater organisms when released into the environment. However, the relevance of TiO2/C-NHs effects needs further chronic toxicity studies since they show promise to be used in nano-bioremediation materials to treat wastewaters.

Environmentally relevant lead (Pb) water concentration induce toxicity in zebrafish (Danio rerio) larvae.

Early developmental stages of aquatic organisms including fish are inherently vulnerable to lead (Pb) and other water metal contaminants. However, reports on the deleterious effects of environmentally relevant Pb levels are limited. To this end, we exposed 2.5 h post fertilization (hpf) old zebrafish (Danio rerio) embryos to a range of Pb concentrations encompassing environmentally relevant levels (1, 10, 25, 50 and 100 µg/L Pb) until 96 hpf. Exposure negatively impacted the development and survival of zebrafish embryos by inducing embryo coagulation related mortalities in a concentration-dependent manner. At 24 hpf, the highest level of exposure (100 µg/L Pb) had impaired embryo activity characterized by reduced burst activity and the number of movements per minute made by embryos. At 72 hpf, newly hatched larvae exhibited adverse cardiovascular effects (100 µg/L Pb group) and neuromuscular effects (50 and 100 µg/L Pb groups). The antioxidant system dysregulation evidenced by downregulation of catalase, and upregulation of mRNA expression of glutathione S-transferase and cytochrome oxidase subunit I were observed. The pro-apoptotic tumour protein P53 (TP53) and the anti-apoptotic B cell lymphoma -2 (Bcl-2) mRNA expression levels were also affected. The former was downregulated across exposed groups and the latter was upregulated and downregulated in the groups with Pb concentrations less than 50 µg/L Pb and downregulated in 50 µg/L Pb, respectively. These findings suggest that Pb within environmentally relevant levels may be deleterious to developing zebrafish.

Toxicity evaluation of TiO2/MWCNT-CNF hybrid nanocomposites with enhanced photocatalytic activity toward freshwater microalgae: Pseudokirchneriella subcapitata.

A wide range of semiconductor-assisted photocatalytic nanomaterials (NMs) are currently being considered and investigated as potential photocatalysts in water treatment. The applications of nanocomposites composed of nano-structured titania (nano-TiO2) and multi-walled carbon nanotubes (MWCNTs) nanocomposites is growing markedly on account of enhanced photocatalytic efficiency. However, concurrent with the increasing production and application comes a serious concern of these emerging nanosystems about their potential risks in aquatic systems, and thereby potentially threatening aquatic organisms via toxic mechanisms that are, at present, poorly understood. In the present study, the lethal toxic effect and oxidative stress induced by TiO2/MWCNT-CNF nanocomposite in freshwater Pseudokirchneriella subcapitata were assessed. The growth inhibition and sublethal oxidative stress produced by the nanocomposites were evaluated on green microalgae P. subcapitata after 3 days of exposure at 24 h intervals. Moreover, the nanocomposites were physicochemically characterized using a combination of analytical techniques (XRD, SEM/EDS, HRTEM, TGA, UV-Visible spectroscopy). Evaluation of the hybrid for the photocatalytic degradation of Acid Violet 7 dye indicated an enhanced dye removal performance for TiO2/MWCNT-CNF (96.2%) compared to TiO2 (75.2%) after 2 h of visible light irradiation. While the nanocomposite showed good potential for the degradation of the azo dye, overall, the findings herein indicated that acute exposure of P. subcapitata to various concentrations of TiO2/MWCNT-CNF nanocomposite may cause algal growth inhibition including undesirable sublethal oxidative stress effects. The findings of this study contribute to a better understanding of the potential hazards of the developing nanocomposites materials towards the nano-bioremediation materials to treat wastewaters.

Effects of conventionally-treated and ozonated wastewater on mortality, physiology, body length, and behavior of embryonic and larval zebrafish (Danio rerio).

To date, micropollutants from anthropogenic sources cannot be completely removed from effluents of wastewater treatment plants and therefore enter freshwater systems, where they may impose adverse effects on aquatic organisms, for example, on fish. Advanced treatment such as ozonation aims to reduce micropollutants in wastewater effluents and, thus, to mitigate adverse effects on the environment. To investigate the impact and efficiency of ozonation, four different water types were tested: ozonated wastewater (before and after biological treatment), conventionally-treated wastewater, and water from a river (River Ruhr, Germany) upstream of the wastewater treatment plant effluent. Zebrafish (Danio rerio) embryos were used to study lethal and sublethal effects in a modified fish early life-stage test. Mortality occurred during exposure in the water samples from the wastewater treatment plant and the river in the first 24 h post-fertilization, ranging from 12% (conventional wastewater) to 40% (river water). Regarding sublethal endpoints, effects compared to the negative control resulted in significantly higher heart rates (ozonated wastewater), and significantly reduced swimming activity (highly significant in ozonated wastewater and ozone reactor water, significant in only the last time interval in river water). Moreover, the respiration rates were highly increased in both ozonated wastewater samples in comparison to the negative control. Significant differences between the ozonated wastewater samples occurred in the embryonic behavior and heart rates, emphasizing the importance of subsequent biological treatment of the ozonated wastewater. Only the conventionally-treated wastewater sample did not elicit negative responses in zebrafish, indicating that the discharge of conventional wastewater poses no greater risk to embryonic and larval zebrafish than water from the river Ruhr itself. The sublethal endpoints embryonic- and larval behavior, heart rates, and respiration were found to be the most sensitive endpoints in this fish early life-stage test and can add valuable information on the toxicity of environmental samples.

Acute exposure to environmentally relevant lead levels induces oxidative stress and neurobehavioral alterations in larval zebrafish (Danio rerio).

The ubiquitous contamination of environmental lead (Pb) remains a worldwide threat. Improper Pb mine waste disposal from an abandoned lead-zinc mine has recently unearthed widespread Pb poisoning in children in Kabwe Zambia. Although the adverse effects of Pb on human health have begun to receive attention, the ecotoxicological effects on aquatic vertebrates still need further investigation. In addition, there is paucity in the knowledge on the behavioural and molecular subcellular responses in larval zebrafish exposed to Pb within the range of environmental relevant concentration (average 3 µg/L with maximum of 94 µg/L) on aquatic organisms such as zebrafish. The adverse effects of environmentally relevant levels of Pb on larval zebrafish was evaluated by measuring swimming behaviour under alternating dark and light conditions. Larval zebrafish acutely exposed to environmentally relevant Pb exhibited neuro-behavioural alteration including enhanced hyperactivity under light conditions evidenced by increased distanced covered and speed compared to the control. The alteration of entire behavioral profiles was further associated with the disturbed expression patterns of mRNA level of key genes associated with antioxidant (HO-1, Ucp-2 and CoxI), proapoptotic gene (TP53), and antiapoptotic gene (Bcl-2). To our knowledge, this is the first report on the effects of environmentally relevant Pb levels from Kabwe, Zambia and their adverse neurobehavioural effects and subcellular molecular oxidative responses in larval zebrafish acutely exposed within a 30 min period. The current results would be beneficial in our understanding of the effects of low Pb levels acutely discharged into an aquatic environment and the life of aquatic organisms.

How toxic is a non-toxic nanomaterial: Behaviour as an indicator of effect in Danio rerio exposed to nanogold.

Gold nanoparticles are used as drug delivery vectors based on the assumption that they have low toxicity. Literature has, however, produced conflicting results over the last few years. As such, this study aimed to investigate the toxicological effects of nanogold (nAu) on several indicators that range from subcellular to whole-organism level. Gene regulation, changes in oxidative stress biomarkers and swimming performance were assessed in Danio rerio (zebrafish) following exposures to nAu. Adult zebrafish were exposed in vivo to nAu for 96 h and swimming performance measured post-exposure. Liver tissue was collected for DNA microarray and Real-Time Polymerase Chain Reactions (RT-PCR) analyses to determine changes in gene expression (catalase, superoxide dismutase and metallothioneins) and protein biomarker analyses (catalase, superoxide dismutase, acetylcholine esterase, malondialdehyde, cellular energy allocation and metallothionein) were performed on whole-body samples. Swimming behaviour was assessed in 1.1 L Tecniplast™ tanks for a period of six hours and videos were analysed using Noldus EthoVision software. Critical swimming speed was measured in a Loligo® swimming tunnel. The DNA microarray revealed that fish exposed to 20 mg/L differed most from the control group. At 20 mg/L there was a significant increase in gene expression for all genes analysed but this didn't translate to significant responses in protein biomarker levels except for an increase in protein carbonyl formation. The behaviour results demonstrated significant changes in distance moved, swimming speed, acceleration bouts, zone alterations and time spent within the top zone - responses that are usually observed in fish responding to toxicological stress. Furthermore, the critical swimming speed of exposed fish was decreased significantly compared to the control. Since swimming performance and social interaction among zebrafish is essential to their survival, whole-organism behaviour that suggests a toxicological response after exposure to nAu is in agreement with the genetic responses measured in this study.

Cytotoxicity of Ag, Au and Ag-Au bimetallic nanoparticles prepared using golden rod (Solidago canadensis) plant extract.

Production and use of metallic nanoparticles have increased dramatically over the past few years and design of nanomaterials has been developed to minimize their toxic potencies. Traditional chemical methods of production are potentially harmful to the environment and greener methods for synthesis are being developed in order to address this. Thus far phytosynthesis have been found to yield nanomaterials of lesser toxicities, compared to materials synthesized by use of chemical methods. In this study nanoparticles were synthesized from an extract of leaves of golden rod (Solidago canadensis). Silver (Ag), gold (Au) and Ag-Au bimetallic nanoparticles (BNPs), synthesized by use of this "green" method, were evaluated for cytotoxic potency. Cytotoxicity of nanomaterials to H4IIE-luc (rat hepatoma) cells and HuTu-80 (human intestinal) cells were determined by use of the xCELLigence real time cell analyzer. Greatest concentrations (50 µg/mL) of Ag and Ag-Au bimetallic were toxic to both H4IIE-luc and HuTu-80 cells but Au nanoparticles were not toxic. BNPs exhibited the greatest toxic potency to these two types of cells and since AuNPs caused no toxicity; the Au functional portion of the bimetallic material could be assisting in uptake of particles across the cell membrane thereby increasing the toxicity.