From dysbiosis to neuropathologies: Toxic effects of glyphosate in zebrafish.

Glyphosate, a globally prevalent herbicide known for its selective inhibition of the shikimate pathway in plants, is now implicated in physiological effects on humans and animals, probably due to its impacts in their gut microbiomes which possess the shikimate pathway. In this study, we investigate the effects of environmentally relevant concentrations of glyphosate on the gut microbiota, neurotransmitter levels, and anxiety in zebrafish. Our findings demonstrate that glyphosate exposure leads to dysbiosis in the zebrafish gut, alterations in central and peripheral serotonin levels, increased dopamine levels in the brain, and notable changes in anxiety and social behavior. While the dysbiosis can be attributed to glyphosate's antimicrobial properties, the observed effects on neurotransmitter levels leading to the reported induction of oxidative stress in the brain indicate a novel and significant mode of action for glyphosate, namely the impairment of the microbiome-gut-axis. While further investigations are necessary to determine the relevance of this mechanism in humans, our findings shed light on the potential explanation for the contradictory reports on the safety of glyphosate for consumers.

Short- and Long-Term Neurobehavioral Effects of Developmental Exposure to Valproic Acid in Zebrafish.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and communication, anxiety, hyperactivity, and interest restricted to specific subjects. In addition to the genetic factors, multiple environmental factors have been related to the development of ASD. Animal models can serve as crucial tools for understanding the complexity of ASD. In this study, a chemical model of ASD has been developed in zebrafish by exposing embryos to valproic acid (VPA) from 4 to 48 h post-fertilization, rearing them to the adult stage in fish water. For the first time, an integrative approach combining behavioral analysis and neurotransmitters profile has been used for determining the effects of early-life exposure to VPA both in the larval and adult stages. Larvae from VPA-treated embryos showed hyperactivity and decreased visual and vibrational escape responses, as well as an altered neurotransmitters profile, with increased glutamate and decreased acetylcholine and norepinephrine levels. Adults from VPA-treated embryos exhibited impaired social behavior characterized by larger shoal sizes and a decreased interest for their conspecifics. A neurotransmitter analysis revealed a significant decrease in dopamine and GABA levels in the brain. These results support the potential predictive validity of this model for ASD research.

A Photoswitchable Ligand Targeting the ß1 -Adrenoceptor Enables Light-Control of the Cardiac Rhythm.

Catecholamine-triggered ß-adrenoceptor (ß-AR) signaling is essential for the correct functioning of the heart. Although both ß1 - and ß2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting ß1 -AR have proven this receptor as the main target for the therapeutic effects of beta blockers in the heart. Here, we report a new strategy for the light-control of ß1 -AR activation by means of photoswitchable drugs with a high level of ß1 -/ß2 -AR selectivity. All reported molecules allow for an efficient real-time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on living zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target ß1 -AR in native environments using light.

MCR-ALS analysis of 1H NMR spectra by segments to study the zebrafish exposure to acrylamide.

Metabolomics is currently an important field within bioanalytical science and NMR has become a key technique for drawing the full metabolic picture. However, the analysis of 1H NMR spectra of metabolomics samples is often very challenging, as resonances usually overlap in crowded regions, hindering the steps of metabolite profiling and resonance integration. In this context, a pre-processing method for the analysis of 1D 1H NMR data from metabolomics samples is proposed, consisting of the blind resolution and integration of all resonances of the spectral dataset by multivariate curve resolution-alternating least squares (MCR-ALS). The resulting concentration estimates can then be examined with traditional chemometric methods such as principal component analysis (PCA), ANOVA-simultaneous component analysis (ASCA), and partial least squares-discriminant analysis (PLS-DA). Since MCR-ALS does not require the use of spectral templates, the concentration estimates for all resonances are obtained even before being assigned. Consequently, the metabolomics study can be performed without neglecting any relevant resonance. In this work, the proposed pipeline performance was validated with 1D 1H NMR spectra from a metabolomics study of zebrafish upon acrylamide (ACR) exposure. Remarkably, this method represents a framework for the high-throughput analysis of NMR metabolomics data that opens the way for truly untargeted NMR metabolomics analyses. Graphical abstract.

Comprehensive characterization of neurochemicals in three zebrafish chemical models of human acute organophosphorus poisoning using liquid chromatography-tandem mass spectrometry.

There is a growing interest in biological models to investigate the effect of neurotransmitter dysregulation on the structure and function of the central nervous system (CNS) at different stages of development. Zebrafish, a vertebrate model increasingly used in neurobiology and neurotoxicology, shares the common neurotransmitter systems with mammals, including glutamate, GABA, glycine, dopamine, norepinephrine, epinephrine, serotonin, acetylcholine, and histamine. In this study, we have evaluated the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the multiresidue determination of neurotransmitters and related metabolites. In a first step, ionization conditions were tested in positive electrospray mode and optimum fragmentation patterns were determined to optimize two selected reaction monitoring (SRM) transitions. Chromatographic conditions were optimized considering the chemical structure and chromatographic behavior of the analyzed compounds. The best performance was obtained with a Synergy Polar-RP column, which allowed the separation of the 38 compounds in 30 min. In addition, the performance of LC-MS/MS was studied in terms of linearity, sensitivity, intra- and inter-day precision, and overall robustness. The developed analytical method was able to quantify 27 of these neurochemicals in zebrafish chemical models for mild (P1), moderate (P2), and severe (P3) acute organophosphorus poisoning (OPP). The results show a general depression of synaptic-related neurochemicals, including the excitatory and inhibitory amino acids, as well as altered phospholipid metabolism, with specific neurochemical profiles associated to the different grades of severity. These results confirmed that the developed analytical method is a new tool for neurotoxicology research using the zebrafish model.

Classification of Airflow Limitation Based on z-Score Underestimates Mortality in Patients with Chronic Obstructive Pulmonary Disease.

RATIONALE: Global Lung Function Initiative recommends reporting lung function measures as z-score, and a classification of airflow limitation (AL) based on this parameter has recently been proposed. OBJECTIVES: To evaluate the prognostic capacity of the AL classifications based on z-score or percentage predicted of FEV1 in patients with chronic obstructive pulmonary disease (COPD). METHODS: A cohort of 2,614 patients with COPD recruited outside the hospital setting was examined after a mean (± SD) of 57 ± 13 months of follow-up, totaling 10,322 person-years. All-cause mortality was analyzed, evaluating the predictive capacity of several AL staging systems. MEASUREMENTS AND MAIN RESULTS: Based on Global Initiative for Chronic Obstructive Lung Disease guidelines, 461 patients (17.6%) had mild, 1,452 (55.5%) moderate, 590 (22.6%) severe, and 111 (4.2%) very severe AL. According to z-score classification, 66.3% of patients remained with the same severity, whereas 23.7% worsened and 10.0% improved. Unlike other staging systems, patients with severe AL according to z-score had higher mortality than those with very severe AL (increase of risk by 5.2 and 3.9 times compared with mild AL, respectively). The predictive capacity for 5-year survival was slightly higher for FEV1 expressed as percentage of predicted than as z-score (area under the curve: 0.714-0.760 vs. 0.649-0.708, respectively). A severity-dependent relationship between AL grades by z-score and mortality was only detected in patients younger than age 60 years. CONCLUSIONS: In patients with COPD, the AL classification based on z-score predicts worse mortality than those based on percentage of predicted. It is possible that the z-score underestimates AL severity in patients older than 60 years of age with severe functional impairment.

Metabolomic changes induced by nicotine in adult zebrafish skeletal muscle.

Acute exposure to nicotinic agonists induces myotoxicity in zebrafish embryos. The main goal of this work was to evaluate the potential myotoxicity of nicotine acetylcholine receptor agonists on adult zebrafish muscle tissue by using nicotine as a model compound. Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) datasets were processed with different chemometric tools based on the selection of Regions of Interest (ROI) and Multivariate Curve-Resolution (ROI-MCR procedure) Alternating Least Squares (ALS) for the analysis of different exposure experiments. Analysis of Variance Simultaneous Component Analysis (ASCA) of changes on metabolite peak profile areas showed significant nicotine concentration and exposure time-dependent changes, clearly differentiating between exposed and non-exposed samples and between short (2 h) and long exposure times (6 h or 24 h). Most of the changes observed in the concentrations of different metabolites are probably secondary to the observed hyperlocomotion, as they have been also observed in humans after strenuous muscular exercise. The absence of myotoxicity might be related with the reduced calcium permeability of adult muscle-type nicotinic acetylcholine receptors (nAChRs).

Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication.

Acute organophosphorus (OP) intoxication is a worldwide clinical and public health problem. In addition to cholinergic crisis, neurodegeneration and brain damage are hallmarks of the severe form of this toxidrome. Recently, we generated a chemical model of severe acute OP intoxication in zebrafish that is characterized by altered head morphology and brain degeneration. The pathophysiological pathways resulting in brain toxicity in this model are similar to those described in humans. The aim of this study was to assess the predictive power of this zebrafish model by testing the effect of a panel of drugs that provide protection in mammalian models. The selected drugs included "standard therapy" drugs (atropine and pralidoxime), reversible acetylcholinesterase inhibitors (huperzine A, galantamine, physostigmine and pyridostigmine), N-methyl-D-aspartate (NMDA) receptor antagonists (MK-801 and memantine), dual-function NMDA receptor and acetylcholine receptor antagonists (caramiphen and benactyzine) and anti-inflammatory drugs (dexamethasone and ibuprofen). The effects of these drugs on zebrafish survival and the prevalence of abnormal head morphology in the larvae exposed to 4 µM chlorpyrifos oxon [1 × median lethal concentration (LC50)] were determined. Moreover, the neuroprotective effects of pralidoxime, memantine, caramiphen and dexamethasone at the gross morphological level were confirmed by histopathological and transcriptional analyses. Our results demonstrated that the zebrafish model for severe acute OP intoxication has a high predictive value and can be used to identify new compounds that provide neuroprotection against severe acute OP intoxication.

Ancient DNA analysis of 8000 B.C. near eastern farmers supports an early neolithic pioneer maritime colonization of Mainland Europe through Cyprus and the Aegean Islands.

The genetic impact associated to the Neolithic spread in Europe has been widely debated over the last 20 years. Within this context, ancient DNA studies have provided a more reliable picture by directly analyzing the protagonist populations at different regions in Europe. However, the lack of available data from the original Near Eastern farmers has limited the achieved conclusions, preventing the formulation of continental models of Neolithic expansion. Here we address this issue by presenting mitochondrial DNA data of the original Near-Eastern Neolithic communities with the aim of providing the adequate background for the interpretation of Neolithic genetic data from European samples. Sixty-three skeletons from the Pre Pottery Neolithic B (PPNB) sites of Tell Halula, Tell Ramad and Dja'de El Mughara dating between 8,700-6,600 cal. B.C. were analyzed, and 15 validated mitochondrial DNA profiles were recovered. In order to estimate the demographic contribution of the first farmers to both Central European and Western Mediterranean Neolithic cultures, haplotype and haplogroup diversities in the PPNB sample were compared using phylogeographic and population genetic analyses to available ancient DNA data from human remains belonging to the Linearbandkeramik-Alföldi Vonaldiszes Kerámia and Cardial/Epicardial cultures. We also searched for possible signatures of the original Neolithic expansion over the modern Near Eastern and South European genetic pools, and tried to infer possible routes of expansion by comparing the obtained results to a database of 60 modern populations from both regions. Comparisons performed among the 3 ancient datasets allowed us to identify K and N-derived mitochondrial DNA haplogroups as potential markers of the Neolithic expansion, whose genetic signature would have reached both the Iberian coasts and the Central European plain. Moreover, the observed genetic affinities between the PPNB samples and the modern populations of Cyprus and Crete seem to suggest that the Neolithic was first introduced into Europe through pioneer seafaring colonization.

Analysis of neurobehavioural data by chemometric methods in ecotoxicological studies.

Incorporation of chemometric tools in behavioural data management workflows allows for the early identification of most relevant endpoints complementarily to statistical confirmatory approaches. In this work, the effects of two model neurotoxicants, chlorpyrifos (CPF) and nicotine, exposures on behavioural profiles of adult zebrafish at three different times (2, 6 and 24h) were evaluated using open field test (OFT) paradigm experiments. Two chemometric methods like Principal Component Analysis (PCA) and Analysis of Variance-Simultaneous Component Analysis (ASCA) have been used to interpret the changes observed in the obtained behavioural data. A decreased of the locomotor activity, an anxiolytic effect and an altered exploratory behaviour were the most affected behavioural endpoints in the CPF exposures. However, an increase of the locomotor activity and an anxiogenic effect were observed in the nicotine exposures. Finally, an excellent correlation between the ASCA results and the results obtained using traditional statistical procedures for both compounds were encountered.

Targeted Gene Expression in Zebrafish Exposed to Chlorpyrifos-Oxon Confirms Phenotype-Specific Mechanisms Leading to Adverse Outcomes.

Zebrafish models for mild, moderate, and severe acute organophosphorus poisoning were previously developed by exposing zebrafish larvae to chlopyrifos-oxon. The phenotype of these models was characterized at several levels of biological organization. Oxidative stress and mitochondrial dysfunction were found to be involved in the development of the more severe phenotype. Here we used targeted gene expression to understand the dose-responsiveness of those two pathways and their involvement on generating the different zebrafish models. As the severe phenotype is irreversible after only 3 h of exposure, we also analyzed the response of the oxidative stress pathway at 3 and 24 h. Some of the genes related to oxidative stress were already differentially expressed at 3 h. There was an increase in differentially expressed genes related to both oxidative stress and mitochondrial function from the more mild to the more severe phenotype, suggesting the involvement of these mechanisms in increasing phenotype severity. Temporal data suggest that peroxynitrite leading to lipid peroxidation might be involved in phenotype transition and irreversibility.

Effectiveness of cognitive-behavioral group therapy for panic disorder in a specialized unit.

INTRODUCTION: The efficacy of cognitive-behavior therapy for panic disorder (PD) with or without agoraphobia is well established, but few data exist on its effectiveness using a group format. The goal of the present study was to assess the effectiveness of group CBT in a sample of PD patients in a specialized unit. METHODS: Treatment consisted of nine weekly group sessions. Patients with PD (n=56) were assessed at baseline, after the treatment and in one and three-months followups. RESULTS: There were significant reductions in panic/agoraphobia symptoms and related variables between baseline and post-treatment, and these reductions were maintained in three-month follow-up. No differences were observed between those patients who received only CBT and those who received pharmacological treatment as well as CBT. Only initial panic/agoraphobia symptoms were significant predictors of treatment response at the end of treatment (a greater severity was associated with a worse response to CBT). CONCLUSIONS: Our results show that group CBT in a specialized unit is effective for PD patients.

Analysis of sleep/wake cycles in zebrafish larvae.

Zebrafish larvae are a model organism increasingly used in the study of the effect of neuroactive chemicals on vertebrate sleep/wake cycles. Sleep disturbances have a negative impact on mood, cognition and overall health. Here we present a protocol to assess over 24 h sleep/wake cycles in zebrafish larvae subjected to 12 h light/dark periods in 48-well plates, using video-tracking technologies. The protocol can be used to determine if the exposure to environmental pollutants or drugs can lead to sleep disturbances. The results on the effect of the tire rubber-derived 6PPD-quinone on zebrafish sleep/wake cycles presented here demonstrate the suitability of using this protocol in fish neurotoxicity studies. This protocol provides a new relevant tool to be used in the pharmacology and toxicology fields.

Cardiac and neurobehavioral impairments in three phylogenetically distant aquatic model organisms exposed to environmentally relevant concentrations of boscalid.

Boscalid (2-Chloro-N-(4'-chlorobiphenyl-2-yl) nicotinamide), a pyridine carboxamide fungicide, is an inhibitor of the complex II of the respiration chain in fungal mitochondria. As boscalid is only moderately toxic for aquatic organisms (LC50 > 1-10 mg/L), current environmental levels of this compound in aquatic ecosystems, in the range of ng/L-µg/L, are considered safe for aquatic organisms. In this study, we have exposed zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and Daphnia magna to a range of concentrations of boscalid (1-1000 µg/L) for 24 h, and the effects on heart rate (HR), basal locomotor activity (BLA), visual motor response (VMR), startle response (SR), and habituation (HB) to a series of vibrational or light stimuli have been evaluated. Moreover, changes in the profile of the main neurotransmitters have been determined. Boscalid altered HR in a concentration-dependent manner, leading to a positive or negative chronotropic effect in fish and D. magna, respectively. While boscalid decreased BLA and increased VMR in Daphnia, these behaviors were not altered in fish. For SR and HB, the response was more species- and concentration-specific, with Daphnia exhibiting the highest sensitivity. At the neurotransmission level, boscalid exposure decreased the levels of L-aspartic acid in fish larvae and increased the levels of dopaminergic metabolites in D. magna. Our study demonstrates that exposure to environmental levels of boscalid alters cardiac activity, impairs ecologically relevant behaviors, and leads to changes in different neurotransmitter systems in phylogenetically distinct vertebrate and invertebrate models. Thus, the results presented emphasize the need to review the current regulation of this fungicide.

Short-term exposure to environmental levels of nicotine and cotinine impairs visual motor response in zebrafish larvae through a similar mode of action: Exploring the potential role of zebrafish α7 nAChR.

The current view is that environmental levels of nicotine and cotinine, commonly in the ng/L range, are safe for aquatic organisms. In this study, 7 days post-fertilization zebrafish embryos have been exposed for 24 h to a range of environmental concentrations of nicotine (2.0 ng/L-2.5 µg/L) and cotinine (50 pg/L-10 µg/L), as well as to a binary mixture of these emerging pollutants. Nicotine exposure led to hyperactivity, decreased vibrational startle response and increased non-associative learning. However, the more consistent effect found for both nicotine and cotinine was a significant increase in light-off visual motor response (VMR). The effect of both pollutants on this behavior occurred through a similar mode of action, as the joint effects of the binary mixture of both chemicals were consistent with the concentration addition concept predictions. The results from docking studies suggest that the effect of nicotine and cotinine on light-off VMR could be mediated by zebrafish α7 nAChR expressed in retina. The results presented in this study emphasize the need to revisit the environmental risk assessment of chemicals including additional ecologically relevant sublethal endpoints.

Analyzing the Effects of Age, Time of Day, and Experiment on the Basal Locomotor Activity and Light-Off Visual Motor Response Assays in Zebrafish Larvae.

The recent availability of commercial platforms for behavioral analyses in zebrafish larvae based on video-tracking technologies has exponentially increased the number of studies analyzing different behaviors in this model organism to assess neurotoxicity. Among the most commonly used assays in zebrafish larvae are basal locomotor activity (BLA) and visual motor responses (VMRs). However, the effect of different intrinsic and extrinsic factors that can significantly alter the outcome of these assays is still not well understood. In this work, we have analyzed the influence of age (5-8 days post-fertilization), time of day (8:00, 10:00, 12:00, 14:00; 16:00, 18:00, and 20:00 h), and experiment (three experiments performed at different days) on BLA and VMR results (4004 analyses for each behavior) in 143 larvae. The results from both behaviors were adjusted to a random-effects linear regression model using generalized least squares (GLSs), including in the model the effect of the three variables, the second-way interactions between them, and the three-way interaction. The results presented in this manuscript show a specific effect of all three intrinsic factors and their interactions on both behaviors, supporting the view that the most stable time period for performing these behavioral assays is from 10:00 am to 04:00 pm, with some differences depending on the age of the larva and the behavioral test.

Parental exposure to antidepressants has lasting effects on offspring? A case study with zebrafish.

Fish have common neurotransmitter pathways with humans, exhibiting a significant degree of conservation and homology. Thus, exposure to fluoxetine makes fish potentially susceptible to biochemical and physiological changes, similarly to what is observed in humans. Over the years, several studies demonstrated the potential effects of fluoxetine on different fish species and at different levels of biological organization. However, the effects of parental exposure to unexposed offspring remain largely unknown. The consequences of 15-day parental exposure to relevant concentrations of fluoxetine (100 and 1000 ng/L) were assessed on offspring using zebrafish as a model organism. Parental exposure resulted in offspring early hatching, non-inflation of the swimming bladder, increased malformation frequency, decreased heart rate and blood flow, and reduced growth. Additionally, a significant behavioral impairment was also found (reduced startle response, basal locomotor activity, and altered non-associative learning during early stages and a negative geotaxis and scototaxis, reduced thigmotaxis, and anti-social behavior at later life stages). These behavior alterations are consistent with decreased anxiety, a significant increase in the expression of the monoaminergic genes slc6a4a (sert), slc6a3 (dat), slc18a2 (vmat2), mao, tph1a, and th2, and altered levels of monoaminergic neurotransmitters. Alterations in behavior, expression of monoaminergic genes, and neurotransmitter levels persisted until offspring adulthood. Given the high conservation of neuronal pathways between fish and humans, data show the possibility of potential transgenerational and multigenerational effects of pharmaceuticals' exposure. These results reinforce the need for transgenerational and multigenerational studies in fish, under realistic scenarios, to provide realistic insights into the impact of these pharmaceuticals.

Highly crosslinked polyethylene does not reduce the wear in total knee arthroplasty: in vivo study of particles in synovial fluid.

The aim was to assess if the reduction in polyethylene wear with highly crosslinked polyethylene suggested by studies with knee simulators is confirmed in patients with a knee arthroplasty. The use of a conventional or a highly crosslinked polyethylene was randomly assigned intraoperatively. Twelve months after surgery a knee arthrocentesis was performed and the synovial fluid of 17 patients in each group was studied analysing the number, size and shape of the polyethylene particles by scanning electron microscope. We found no significant differences in the concentration, size or morphology of polyethylene particles between groups. The great variability in the number of particles between individuals suggests that in vivo polyethylene wear depends on many factors and probably the type of polyethylene is not the most significant.

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior. We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Heart rate and behavioral responses in three phylogenetically distant aquatic model organisms exposed to environmental concentrations of carbaryl and fenitrothion.

Carbaryl and fenitrothion are two insecticides sharing a common mode of action, the inhibition of the acetylcholinesterase (AChE) activity. Their use is now regulated or banned in different countries, and the environmental levels of both compounds in aquatic ecosystems have decreased to the range of pg/L to ng/L. As these concentrations are below the non-observed-adverse-effect-concentrations (NOAEC) for AChE inhibition reported for both compounds in aquatic organisms, there is a general agreement that the current levels of these two chemicals are safe for aquatic organisms. In this study we have exposed zebrafish, Japanese medaka and Daphnia magna to concentrations of carbaryl and fenitrothion under their NOAECs for 24-h, and the effects on heart rate (HR), basal locomotor activity (BLA), visual motor response (VMR), startle response (SR) and its habituation have been evaluated. Both pesticides increased the HR in the three selected model organisms, although the intensity of this effect was chemical-, concentration- and organism-dependent. The exposure to both pesticides also led to a decrease in BLA and an increase in VMR in all three species, although this effect was only significant in zebrafish larvae. For SR and its habituation, the response profile was more species- and concentration-specific. The results presented in this manuscript demonstrate that concentrations of carbaryl and fenitrothion well below their respective NOAECs induce tachycardia and the impairment of ecologically relevant behaviors in phylogenetically distinct aquatic model organisms, both vertebrates and invertebrates, emphasizing the need to include this range of concentrations in the environmental risk assessment.