Impact of chronic fluoxetine exposure on zebrafish: From fatty acid profile to behavior.

The consumption of antidepressants, such as fluoxetine, has increased over the years and, as a result, they are increasingly found in aquatic systems. Given the increasing use of zebrafish as an animal model in toxicological studies, this work proposed to evaluate the effects of chronic exposure, for 21 days, to fluoxetine at environmentally relevant concentrations (1, 10, 100, and 1000 ng/L). The behavioral tests performed did not reveal significant effects of fluoxetine. However, oxidative stress and changes in energy metabolism were detected after exposure to the highest concentrations of fluoxetine tested, namely a decrease in glutathione S-transferase (GST) activity (decrease of ca. 31%), increase in catalase (CAT) activity (increase of ca. 71%), and decrease in lactate dehydrogenase (LDH) activity (decrease of ca. 53%). Analysis of the fatty acid profile (FA) revealed a decrease in the omega-3 FA, docosahexaenoic acid (DHA), C22:6 (decrease in relative abundance between 6% and 8% for both the head and body), an increase in omega-6 FA, linoleic acid (LA), C18:2, (increased relative abundance between 8% and 11% in the head and between 5% and 9% in the body), which may suggest changes in the inflammatory state of these organisms. The integrated analysis adopted proved to be useful in detecting subindividual effects of fluoxetine and modes of action in fish.

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.

From biomarkers to community composition: Negative effects of UV/chlorine-treated reclaimed urban wastewater on freshwater biota.

The use of urban wastewater reclaimed water has recently increased across the globe to restore stream environmental flows and mitigate the effects of water scarcity. Reclaimed water is disinfected using different treatments, but their effects into the receiving rivers are little studied. Physiological bioassays and biomarkers can detect sub-lethal effects on target species, but do not provide information on changes in community structure. In contrast, official monitoring programs use community structure information but often at coarse taxonomic resolution level that may fail to detect species level impacts. Here, we combined commonly used biomonitoring approaches from organism physiology to community species composition to scan a broad range of effects of disinfection of reclaimed water by UV-light only and both UV/chlorine on the biota. We (1) performed bioassays in one laboratory species (water flea Daphnia magna) and measured biomarkers in two wild species (caddisfly Hydropsyche exocellata and the barbel Luciobarbus graellsii), (2) calculated standard indices of biotic quality (IBQ) for diatoms, benthic macroinvertebrates, and fishes, and (3) analysed community species composition of eukaryotes determined by Cytochrome Oxidase C subunit I (cox1) metabarcoding. Only the UV/chlorine treatment caused significant changes in feeding rates of D. magna and reduced antioxidant defenses, increased anaerobic metabolism and altered the levels of lipid peroxidiation in H. exocellata. However, inputs of reclaimed water were significantly associated with a greater proportion of circulating neutrophils and LG-PAS cells in L. graellsii. Despite IBQ did not discriminate between the two water treatments, metabarcoding data detected community composition changes upon exposure to UV/chlorine reclaimed water. Overall, despite the effects of UV/chlorine-treated water were transient, our study suggests that UV-light treated is less harmful for freshwater biota than UV/chlorine-treated reclaimed water, but those effects depend of the organizational level.

Impact of environmentally relevant concentrations of fluoxetine on zebrafish larvae: From gene to behavior.

Fluoxetine is widely prescribed for the treatment of depressive states, acting at the level of the central nervous system, consequently affecting non-target organisms. This study aimed to investigate the influence of environmentally relevant fluoxetine concentrations (1-1000 ng/L) on Danio rerio development, assessing both embryotoxicity and behavior, antioxidant defense, gene expression and neurotransmitter levels at larval stage. Exposure to fluoxetine during early development was found to be able to accelerate embryo hatching in embryos exposed to 1, 10 and 100 ng/L, reduce larval size in 1000 ng/L, and increase heart rate in 10, 100 and 1000 ng/L exposed larvae. Behavioral impairments (decreased startle response and increased larvae locomotor activity) were associated with effects on monoaminergic systems, detected through the downregulation of key genes (vmat2, mao, tph1a and th2). In addition, altered levels of neurochemicals belonging to the serotonergic and dopaminergic systems (increased levels of tryptophan and norepinephrine) highlighted the sensitivity of early life stages of zebrafish to low concentrations of fluoxetine, inducing effects that may compromise larval survival. The obtained data support the necessity to test low concentrations of SSRIs in environmental risk assessment and the use of biomarkers at different levels of biological organization for a better understanding of modes of action.

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.

Effects of fluoxetine on fish: What do we know and where should we focus our efforts in the future?

Fluoxetine is one of the most studied and detected selective serotonin reuptake inhibitors in the aquatic environment, found at concentrations ranging from ng/L to µg/L. Its presence in this environment can induce effects on aquatic organisms that may compromise their fitness. Several experimental studies have demonstrated that fluoxetine can induce neurotoxicity, genetic and biochemical changes, and cause behavioral dysfunction in a wide range of fish species. However, contradictory results can be found. There is thus the need for a comprehensive review of the current state of knowledge on the effects of fluoxetine on fish at different levels of biological organization, highlighting inclusive patterns and discussing the potential causes for the contradictory results, that can be found in the available literature. This review also aims to explore and identify the main gaps in knowledge and areas for future research. We conclude that environmentally relevant concentrations of fluoxetine (e.g., from 0.00345 µg/L) produced adverse effects and often this concentration range is not addressed in conventional environmental risk assessment strategies. Its environmental persistence and ionizable properties reinforce the need for standardized testing with representative aquatic models, targeting endpoints sensitive to the specific mode of action of fluoxetine, in order to assess and rank its actual environmental risk to aquatic ecosystems.

Developmental exposure to sertraline impaired zebrafish behavioral and neurochemical profiles.

The number of people suffering from mental health problems is rising, with anxiety and depression now the most commonly diagnosed psychiatric conditions. Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed pharmaceuticals to treat these conditions, which has led to their common detection in many aquatic ecosystems. As the monoaminergic system shows a high degree of structural conservation across diverse animal phyla, a reasonable assumption is that the environmental levels of SSRIs in surface water can lead to adverse effects on fish and other aquatic wildlife. For instance, Sertraline (SER), a widely prescribed SSRI, has been shown to induce adverse effects in fish, albeit most of the reports used exposure concentrations exceeding those occurring in natural environments. Therefore, there is still a great lack of knowledge regarding SERs effects in fish species, especially during early life stages. This study describes the evaluation of developmental exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of SER (from 0.01 to 10 µg/L), using a battery of key survival behaviors and further relating them with the expression of genes and neurochemical profiles of the monoaminergic system. We found that developmental exposure to SER did not affect embryo morphogenesis and growth. However, concentrations as low as 0.1 µg/L induced hypolocomotion and delayed learning. The observed behavioral impairment was associated with augmented serotonin levels rather than other neurochemicals and molecular markers, highlighting the relationship between serotonin signaling and behavior in zebrafish.

Caged-carvedilol as a new tool for visible-light photopharmacology of ß-adrenoceptors in native tissues.

Adrenoceptors are G protein-coupled receptors involved in a large variety of physiological processes, also under pathological conditions. This is due in large part to their ubiquitous expression in the body exerting numerous essential functions. Therefore, the possibility to control their activity with high spatial and temporal precision would constitute a valuable research tool. In this study, we present a caged version of the approved non-selective ß-adrenoceptor antagonist carvedilol, synthesized by alkylation of its secondary amine with a coumarin derivative. Introducing this photo-removable group abolished carvedilol physiological effects in cell cultures, mouse isolated perfused hearts and living zebrafish larvae. Only after visible light application, carvedilol was released and the different physiological systems were pharmacologically modulated in a similar manner as the control drug. This research provides a new photopharmacological tool for a wide range of research applications that may help in the development of future precise therapies.

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.

Behavioral Impairment in Aquatic Organisms Exposed to Neurotoxic Pollutants.

Neuroactive chemicals are compounds that can modulate, at very low concentrations, the normal function of the central nervous systems of an organism through various primary modes of action (MoA) [...].

Environmental levels of carbaryl impair zebrafish larvae behaviour: The potential role of ADRA2B and HTR2B.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.

A Zebrafish Model of Neurotoxicity by Binge-Like Methamphetamine Exposure.

Hyperthermia is a common confounding factor for assessing the neurotoxic effects of methamphetamine (METH) in mammalian models. The development of new models of methamphetamine neurotoxicity using vertebrate poikilothermic animals should allow to overcome this problem. The aim of the present study was to develop a zebrafish model of neurotoxicity by binge-like methamphetamine exposure. After an initial testing at 20 and 40 mg/L for 48 h, the later METH concentration was selected for developing the model and the effects on the brain monoaminergic profile, locomotor, anxiety-like and social behaviors as well as on the expression of key genes of the catecholaminergic system were determined. A concentration- and time-dependent decrease in the brain levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) was found in METH-exposed fish. A significant hyperactivity was found during the first hour of exposure, followed 3 h after by a positive geotaxis and negative scototaxis in the novel tank and in the light/dark paradigm, respectively. Moreover, the behavioral phenotype in the treated fish was consistent with social isolation. At transcriptional level, th1 and slc18a2 (vmat2) exhibited a significant increase after 3 h of exposure, whereas the expression of gfap, a marker of astroglial response to neuronal injury, was strongly increased after 48 h exposure. However, no evidences of oxidative stress were found in the brain of the treated fish. Altogether, this study demonstrates the suitability of the adult zebrafish as a model of METH-induced neurotoxicity and provides more information about the biochemical and behavioral consequences of METH abuse.

Improving water quality does not guarantee fish health: Effects of ammonia pollution on the behaviour of wild-caught pre-exposed fish.

Ammonia is a pollutant frequently found in aquatic ecosystems. In fish, ammonia can cause physical damage, alter its behaviour, and even cause death. Exposure to ammonia also increases fish physiological stress, which can be measured through biomarkers. In this study, we analysed the effect of sublethal ammonia concentrations on the behaviour and the oxidative stress of Barbus meridionalis that had been pre-exposed to this compound in the wild. Wild-caught fish from a polluted site (pre-exposed fish) and from an unpolluted site (non-pre-exposed fish) were exposed, under experimental conditions, to total ammonia concentrations (TAN) of 0, 1, 5, and 8 mg/L. Swimming activity, feeding behaviour, and oxidative stress response based on biomarkers were analysed. Pre-exposed fish showed both an altered behaviour and an altered oxidative stress response in the control treatment (0 mg/L). Differences in swimming activity were also found as pre-exposed fish swam less. Lower feeding activity (voracity and satiety) and altered response to oxidative stress were also observed at ≥ 1 mg/L TAN. Biomarker results confirmed pre-exposed fish suffer from a reduction in their antioxidant defences and, hence, showed increased oxidative tissue damage. In summary, pre-exposed fish showed more sensitivity to ammonia exposure than fish from a pristine site.

Pharmacological Modulation of Behaviour, Serotonin and Dopamine Levels in Daphnia magna Exposed to the Monoamine Oxidase Inhibitor Deprenyl.

This study assessed the effects of the monoamine oxidase (MAO) inhibitor deprenyl in Daphnia magna locomotor activity. The mechanisms of action of deprenyl were also determined by studying the relationship between behaviour, MAO activity and neurotransmitter levels. Modulation of the D. magna monoamine system was accomplished by 24 h exposure to two model psychotropic pharmaceuticals with antagonistic and agonistic serotonin signalling properties: 10 mg/L of 4-chloro-DL-phenylalanine (PCPA) and 1 mg/L of deprenyl, respectively. Contrasting behavioural outcomes were observed for deprenyl and PCPA reflected in decreased basal locomotor activity and enhanced habituation for the former compound and delayed habituation for the latter one. Deprenyl exposure inhibited monoamine oxidase (MAO) activity and increased the concentrations of serotonin, dopamine and the dopamine metabolite 3-methoxytyramine in whole D. magna extracts. Our findings indicate that D. magna is a sensitive and useful nonvertebrate model for assessing the effects of short-term exposure to chemicals that alter monoamine signalling changes.

Differential Modulation of the Central and Peripheral Monoaminergic Neurochemicals by Deprenyl in Zebrafish Larvae.

Zebrafish embryos and larvae are vertebrate models increasingly used in translational neuroscience research. Behavioral impairment induced by the exposure to neuroactive or neurotoxic compounds is commonly linked to changes in modulatory neurotransmitters in the brain. Although different analytical methods for determining monoaminergic neurochemicals in zebrafish larvae have been developed, these methods have been used only on whole larvae, as the dissection of the brain of hundreds of larvae is not feasible. This raises a key question: Are the changes in the monoaminergic profile of the whole larvae predictive of the changes in the brain? In this study, the levels of ten monoaminergic neurotransmitters were determined in the head, trunk, and the whole body of zebrafish larvae in a control group and in those treated for 24 h with 5 M deprenyl, a prototypic monoamine-oxidase B inhibitor, eight days post-fertilization. In control larvae, most of the monoaminergic neurochemicals were found at higher levels in the head than in the trunk. Significant changes were found in the distribution of some neurochemicals after deprenyl-treatment, with serotonin and norepinephrine increasing in both the head and the trunk, whereas dopamine, L-DOPA, and homovanillic acid levels were only modulated in the head. In fact, the highly significant increase in dopamine levels observed in the head after deprenyl-treatment was not detected in the whole-body analysis. These results indicate that the analysis of neurotransmitters in the zebrafish larvae whole-body should not be used as a general surrogate of the brain.

Pharmacological Modulation of Serotonin Levels in Zebrafish Larvae: Lessons for Identifying Environmental Neurotoxicants Targeting the Serotonergic System.

This study examines the effects of acute pharmacological modulation of the serotonergic system over zebrafish larvae's cognitive, basic, and defense locomotor behaviors, using a medium to high throughput screening assay. Furthermore, the relationship between behavior, enzyme activity related to neurotransmitter metabolism, neurotransmitter levels, and gene expression was also determined. Modulation of larvae serotonergic system was accomplished by 24 h exposure to single and opposite pharmacodynamics co-exposure to three model psychopharmaceuticals with antagonistic and agonistic serotonin signaling properties: 2.5 mM 4-Chloro-DL-phenylalanine (PCPA) and 5 µM deprenyl and 0.5 µM fluoxetine, respectively. Similar behavioral outcome was observed for deprenyl and fluoxetine, which was reflected as hypolocomotion, decrease in larvae defensive responses, and cognitive impairment. Contrarily, PCPA induced hyperlocomotion and increase in larvae escape response. Deprenyl exposure effects were more pronounced at a lower level of organization than fluoxetine, with complete inhibition of monoamine oxidase (MAO) activity, dramatic increase of 5-HT and dopamine (DA) levels, and downregulation of serotonin synthesis and transporter genes. PCPA showed mainly effects over serotonin and dopamine's main degradation metabolites. Finally, co-exposure between agonistic and antagonist serotonin signaling drugs reviled full recovery of zebrafish impaired locomotor and defense responses, 5-HT synthesis gene expression, and partial recovery of 5-HT levels. The findings of this study suggest that zebrafish larvae can be highly sensitive and a useful vertebrate model for short-term exposure to serotonin signaling changes.

Androgenic activation, impairment of the monoaminergic system and altered behavior in zebrafish larvae exposed to environmental concentrations of fenitrothion.

Fenitrothion is an organophosphorus insecticide usually found in aquatic ecosystems at concentrations in the range of low ng/L. In this manuscript we show that 24 h exposure to environmental concentrations of fenitrothion, from ng/L to low µg/L, altered basal locomotor activity, visual-motor response and acoustic/vibrational escape response of zebrafish larvae. Furthermore, fenitrothion and expression of gap43a, gfap, atp2b1a, and mbp exhibited a significant non-monotonic concentration-response relationship. Once determined that environmental concentrations of fenitrothion were neurotoxic for zebrafish larvae, a computational analysis identified potential protein targets of this compound. Some of the predictions, including interactions with acetylcholinesterase, monoamine-oxidases and androgen receptor (AR), were experimentally validated. Binding to AR was the most suitable candidate for molecular initiating event, as indicated by both the up-regulation of cyp19a1b and sult2st3 and the non-monotonic relationship found between fenitrothion and the observed responses. Finally, when the integrity of the monoaminergic system was evaluated, altered levels of L-DOPA, DOPAC, HVA and 5-HIAA were found, as well as a significant up-regulation of slc18a2 expression at the lowest concentrations of fenitrothion. These data strongly suggest that concentrations of fenitrothion commonly found in aquatic ecosystems present a significant environmental risk for fish communities.

Glyphosate targets fish monoaminergic systems leading to oxidative stress and anxiety.

Glyphosate is the active ingredient of some of the most highly produced and used herbicides worldwide. The intensive applications of glyphosate-based herbicides and its half-life in water lead to its presence in many aquatic ecosystems. Whereas recent studies have reported neurotoxic effects of glyphosate including autism-related effects, most of them used extremely high (mg/L to g/L) concentrations, so it is still unclear if chronic, low environmentally relevant concentrations of this compound (ng/L to µg/L) can induce neurotoxicity. In this study we analyzed the neurotoxicity of glyphosate in adult zebrafish after waterborne exposure to environmentally relevant concentrations (0.3 and 3 µg/L) for two weeks. Our data showed that exposed fish presented a significant impairment of exploratory and social behaviors consistent with increased anxiety. The anterior brain of the exposed fish presented a significant increase in dopamine and serotonin levels, as well as in the DOPAC/dopamine and homovanillic acid/dopamine turnover ratios. Moreover, the expression of genes involved in the dopaminergic system, as th1, th2, comtb, and scl6a3 was downregulated. Finally, the brain of exposed fish presented a significant increase in the catalase and superoxide dismutase activities, with a concomitant decrease of glutathione stores. These changes in the antioxidant defense system are consistent with the observed increase in oxidative stress, reflected by the increase in the levels of lipid peroxidation in the brain. The presented results show that current glyphosate concentrations commonly found in many aquatic ecosystems may have detrimental consequences on fish survival by decreasing exploration of the environment or altering social interactions. Furthermore, as zebrafish is also a vertebrate model widely used in human neurobehavioral studies, these results are relevant not only for environmental risk assessment, but also for understanding the risk of chronic low-dose exposures on human health.

Publisher Correction: Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Screening anti-predator behaviour in fish larvae exposed to environmental pollutants.

Predation is one of the main sources of mortality for fish larvae. During evolution, they have developed different anti-predator behaviours, as the vibrational-evoked startle response and its habituation, for promoting survival to predator's strikes. Whereas these two behaviours can be altered by the exposure to some neurotoxicants, it is currently unknown if the exposure to environmentally relevant concentration (ERC) of neurotoxic pollutants could impair them. In this study thirty neurotoxic environmental pollutants from nine chemical groups, including: herbicides; carbamate, organophosphate (OP), organochlorine (OC), neonicotinoid and pyrethroid insecticides; toxins; metal and non-metal elements, have been screened at two concentrations, including one environmental relevant concentration (ERC), for adverse effects on anti-predator behaviours by using the Vibrational Startle Response Assay on zebrafish larvae. Significant effects over anti-predator responses were equally observed in both exposure concentrations. Focusing on the ERC scenario, it was found that the startle response was the less affected behaviour, where ten pollutants from all chemical groups except for organochlorine, neonicotinoid and pyrethroids, altered this response. Interestingly, organic and inorganic pollutants showed opposite effects on this response: whereas all organic pollutants decreased the startle response, the three remaining inorganic pollutants increased it. On the other hand, more pollutants affected habituation of the startle response of the larvae, where thirteen of the pollutants from all groups, except for herbicides, altered this behaviour at ERC, generally resulting in a faster habituation except for one OP and one marine toxin, which were able to delay this response. Ultimately, only one chemical from the OP, toxin, metal and non-metal element groups altered both the startle response and its habituation at both ERC and WSC. These results emphasize the environmental risk of the current levels of some neurotoxicants present in our aquatic ecosystems, as they are high enough to impair essential anti-predator behaviours in fish larvae.