Can the exposure system adopted influence the results of the atrazine toxicity in hepatic tissue of fish?

The widespread use of atrazine, a herbicide used to control weeds, has contributed to the increased contamination of aquatic environments. To assess the toxicological effects of a xenobiotic on a nontarget organism in the laboratory, different models of toxicological exposure systems have been widely used. Therefore, the aim of this study was to evaluate and compare the action of sublethal concentrations of atrazine on the hepatic histology of Oreochromis niloticus, considering two models of exposure: static (where atrazine was only added once) and semi-static (where atrazine was periodically renewed). Fish were exposed to a concentration of 2 ppm atrazine for 15 days, which was verified by high-performance liquid chromatography. The livers were stained with hematoxylin and eosin and histopathological data were collected. In addition, they were submitted to immunohistochemistry for inducible nitric oxide synthase (iNOS). A maximum variation of 45% (static) and 12.5% (semi-static) was observed between the observed and nominal atrazine concentration. Nuclear and cytoplasmic changes were observed in both experimental models. Hepatocytes from the livers of the static system showed a degenerative appearance, while in the semi-static system, intense cytoplasmic vacuolization and necrosis were observed. iNOS positive cells were identified only in macrophages in the hepatocytes of fish in the semi-static system. These results directly showed how the choice of exposure system can influence the results of toxicological tests. However, future analysis investigating the by-products and nitrogen products should be carried out since the histopathological findings revealed the possibility of these compounds serving as secondary contamination routes.

Atrazine impairs testicular function in BalB/c mice by affecting Leydig cells.

Several studies have reported the effects of atrazine on the gonads of many experimental models. However, the short-term effects of in vivo exposure to atrazine on the testes of mice are not well clarified. Here we reported that adult BalB/c mice exposed to atrazine (50 mg kg-1 body weight) by gavage for three consecutive days have reduced numbers of 3ß-hydroxysteroid dehydrogenase positive Leydig cells (LCs), associated with increased in situ cell death fluorescence and caspase-3 immuno-expression in the testes. Consequently, immunostaining for cell cycle gene regulators showed increased expressions of p45, accompanied with increased expressions of cyclin D2 and E2. Histological observations of the gonads showed reduced number of germ cells in particular areas, sloughed seminiferous epithelium, presence of giant apoptotic cells close to the seminiferous tubule lumen and in the epididymal lumen along with low numbers of Leydig cells in the testicular interstitial areas. Similarly, LCs isolated from the testes of BalB/c mice that were exposed to atrazine (0.5, 25, 50 mg kg-1 body weight) in the same manner as in the first experiment presented dose-dependent increased caspase-3 activity, decreased cell viability, intratesticular and serum testosterone concentrations and LCs testosterone secretion. In summary, atrazine appears to directly decrease the number of testosterone secreting LCs in mice through apoptosis.

Development of scleral ossicles in Podocnemis expansa (Testudines: Podocnemididae) embryos exposed to atrazine.

Understanding the effects of atrazine exposure on embryo development in oviparous animals may provide important data regarding the impacts of agrochemical use on wildlife and the ecosystem. This study set out to determine the effects of embryonic atrazine exposure on the development of osseous and cartilaginous components of scleral ossicles in Podocnemis expansa. Eggs were collected at the Environmental Protection Area Meandros do Rio Araguaia, Brazil, and artificially incubated in sand treated with solutions containing 2, 20 or 200 µg/L of atrazine. Sixty embryos were collected per treatment throughout the incubation period. Embryos were diaphanized with potassium hydroxide (KOH) and stained with Alizarin Red S and Alcian blue (bone and cartilage tissue respectively). Scleral ossicles were then counted and examined for skeletal abnormalities at different stages of embryonic development. Scleral ossicle counts were significantly reduced in P. expansa embryos treated with 200 µg/L atrazine solution. Rudimentary ossicles and gaps were also noted in embryos exposed to atrazine concentrations of 2 µg/L or 200 µg/L. Findings of this study emphasize the relevance of ecotoxicological investigations in determining the impacts of agrochemicals on native fauna.

Atrazine induces penis abnormalities including hypospadias in mice.

Use of the herbicide atrazine (ATR) is banned in the European Union; yet, it is still widely used in the USA and Australia. ATR is known to alter testosterone and oestrogen production and thus reproductive characteristics in numerous species. In this proof of concept study, we examined the effect of ATR exposure, at a supra-environmental dose (5 mg/kg bw/day), beginning on E9.5 in utero, prior to sexual differentiation of the reproductive tissues, until 26 weeks of age, on the development of the mouse penis. Notably, this is the first study to specifically investigate whether ATR can affect penis characteristics. We show that ATR exposure, beginning in utero, causes a shortening (demasculinisation) of penis structures and increases the incidence of hypospadias in mice. These data indicate the need for further studies of ATR on human reproductive development and fertility, especially considering its continued and widespread use.

Distract, delay, disrupt: examples of manufactured doubt from five industries.

Manufactured doubt describes the efforts used by organizations or individuals to obscure the harmful effects of their products or actions by manipulating science. Although approaches to do so are widely used, relevant stakeholders are often unaware of these tactics. Here, we examine the strategies used in five cases of manufactured doubt: tobacco and adverse health; coal and black lung; Syngenta and the herbicide atrazine; the sugar industry and cardiovascular disease; and the Marshall Institute and climate change. By describing the tactics used in these cases, effective methods for identifying and countering instances of manufactured doubt can be generated.

Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan.

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5, 10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25°C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p<0.001) correlation (r=0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

Nanosorbent of hydroxyapatite for atrazine: A new approach for combating agricultural runoffs.

The attention of current work was on the fabrication of effective nanoadsorbent of hydroxyapatite (HAp) for the controlled release of atrazine (ATZ) formulation. The ATZ-HAp complex (ATZ@HAp) was able to inhibit the growth of Brassica sp. under in situ conditions. This developed methodology aspires to cease the agricultural runoffs of ATZ applied with the HAp adjuvant and ensure their effective functioning. The efficacy of the protocol was mainly accomplished by adsorbing ATZ over the surface of HAp NPs that restricted its premature runoff and promoted the prolonged herbicidal efficiency. The influence of fundamental parameters i.e., HAp dose, ATZ dose and initial pH on the adsorption process was investigated systematically. The suitability of ATZ@HAp complex for real world application was adjudged after proofing its toxicological behaviour and its role in Zea mays plantations. The complex was found to be non-toxic and nurturing due to its phosphate rich nature. Further investigations of ATZ@HAp complex and its effect on the non-target species will help in establishing an effective framework for their commercial use in agricultural practices.

Atrazine and nicosulfuron affect the reproductive fitness of the predator Podisus nigrispinus (Hemiptera: Pentatomidae).

Herbicides can impact non-target metabolic pathways in natural enemies and lead to the reduction of these populations in the field. Behavioral characteristics, morphology and histology of reproductive structures and reproduction of females of Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) were evaluated under the effect of the herbicides atrazine, nicosulfuron and the mixture of both. The number of mature oocytes per ovary was lower in females exposed to the herbicides atrazine, nicosulfuron and the mixture of both. Herbicides did not affect the longevity and mortality of P. nigrispinus, therefore, they are selective for this predator. On the other hand, herbicides can cause sublethal effects by affecting the reproduction of predators.

Lethal and Sublethal Effects of the Herbicide Atrazine in the Early Stages of Development of Physalaemus gracilis (Anura: Leptodactylidae).

Water sources used as reproductive sites by crying frog, Physalaemus gracilis, are extensively associated with agroecosystems in which the herbicide atrazine is employed. To evaluate the lethal and sublethal effects of atrazine commercial formulation, acute and chronic toxicity tests were performed in the embryonic phase and the beginning of the larval phase of P. gracilis. Tests were started on stage 19 of Gosner (Herpetologica 16:183-190, 1960) and performed in 24-well cell culture plates. Acute tests had a duration of 96 h with embryo mortality monitoring every 24 h. Chronic assays contemplated the transition from the embryonic to larval stages and lasted 168 h. Every 24 h the embryos/larvae were observed for mortality, mobility, and malformations. The LC50 of atrazine determined for P. gracilis embryos was 229.34 mg L-1. The sublethal concentrations did not affect the development of the larvae but were observed effects on mobility and malformations, such as spasmodic contractions, reduced mobility, malformations in mouth and intestine, and edema arising. From 1 mg L-1 atrazine, the exposed larvae began to have changes in mobility and malformations. The atrazine commercial formulation has caused early life effects of P. gracilis that may compromise the survival of this species but at higher concentrations than recorded in the environment, so P. gracilis can be considered tolerant to this herbicide at environmentally relevant concentrations.

Changes in hepatic phase I and phase II biotransformation enzyme expression and glutathione levels following atrazine exposure in female rats.

1. To determine the effects of repeated atrazine (ATR) treatment on hepatic phase I and II enzymes, adult female rats were treated with vehicle or 100 mg/kg of ATR for 1, 2, 3 or 4 days. Glutathione-s-transferases (GST) mRNA expression, protein levels (mu, pi, alpha, omega), and activity (cytosolic and microsomal), along with bioavailable glutathione (GSH) were assayed. 2. GST expression, concentrations and activity were increased, along with GSH levels, in animals treated with ATR for 3 and 4 days. 3. A subsequent study was performed with animals treated with vehicle, 6.5, 50 or 100 mg/kg/day for 4, 8 or 14 days. Expression of hepatic phase I CYP 450 enzymes was evaluated in conjugation with GST expression, protein and activity. Nineteen of the 45 CYP enzymes assayed displayed increased mRNA levels after eight days of treatment in animals treated with 50 or 100 mg/kg/day. After 14 days of treatment, all CYP expression levels returned to control levels except for CYP2B2, CYP2B3, CYP2C7, CYP2C23, CYP2E1, CYP3A9, CYP4A3 and CYP27A1, which remained elevated. 4. Results indicate that there may be a habituation or adaptation of liver phase I and phase II expression following repeated ATR treatment.

Atrazine-induced environmental nephrosis was mitigated by lycopene via modulating nuclear xenobiotic receptors-mediated response.

The burden and morbidity of environmental nephrosis is increasing globally. Atrazine (ATR) and degradation products in the environment are considered key determinants of nephrosis. However, the lack of highly effective treatments for environmental nephrosis creates an urgent need to better understand the preventive strategies and mechanisms. This study aimed to highlight the mechanism of ATR-induced environmental nephrosis and the chemoprotective potential of lycopene (LYC) against the renal injury and nephrosis. Male mice were treated with LYC (5 mg/kg) and/or ATR (50 mg/kg or 200 mg/kg) by gavage administration for 21 days. Histopathological changes and biochemical function, cytochrome P450 enzymes system (CYP450s), nuclear xenobiotic receptors (NXRs) response and the transcription of CYP isoforms (CYPs) were detected. ATR exposure caused the changes of the histopathological and biochemical function, activated the NXR response and disturbed the CYP450s homeostasis. Supplementary LYC significantly prevented ATR-induced nephrotoxicity and alleviated the alternation of histopathological and biochemical function via modulating the CYP450s homeostasis and the NXR response. The results demonstrated AHR, CAR, PXR, PPAR (α, γ), CYP1, CYP2, CYP3 and CYP4 superfamily play a vital role in LYC-ATR interaction. Our findings provide new evidence that ATR exposure can cause the environmental nephrosis via inducing the kidney injury. Supplementary LYC showed significant chemoprotective potential against ATR-induced renal injury and environmental nephrosis via regulating the NXR response and the CYP450s homeostasis.

Lack of immunotoxic effects of repeated exposure to atrazine associated with the adaptation of adrenal gland activation.

T cell-dependent IgM antibody production and natural killer cell (NKC) activity were assessed in SD rats orally administered atrazine for 28 days to males (0, 6.5, 25, or 100 mg/kg/day) or females (0, 3, 6, or 50 mg/kg/day), or 30 or 500 ppm in diet (3 or 51 mg/kg/day). Anti-asialo GM1 antibodies (NKC) and cyclophosphamide (antibody-forming cell assay [AFC]) served as positive controls. Pituitary (ACTH, prolactin), adrenal (corticosterone, progesterone, aldosterone), and gonadal (androgens, estrogens) hormones were assessed after 1, 7, and/or 28 days of treatment. Food intake and body weights were significantly reduced in the highest dosed males, and transiently affected in females. Urinary corticosterone levels were not increased in atrazine-treated groups in either sex at any time point measured (10, 22, or 24 days). Corticosterone and progesterone were elevated in males after a single atrazine dose ≥6.5 mg/kg/day, but not after 7, 14, or 28 doses. There were no effects on adrenal, pituitary, or gonadal hormones in females. Atrazine did not suppress the AFC response or decrease NKC function after 28 days in males or females. Atrazine had no effect on spleen weights or spleen cell numbers in males or females, although thymus weights were elevated in males receiving the highest dose. The lack of immunotoxic effect of atrazine was associated with diminished adrenal activation over time in males, and no effects on adrenal hormones in females.

Lichen microalgae are sensitive to environmental concentrations of atrazine.

The identification of new organisms for environmental toxicology bioassays is currently a priority, since these tools are strongly limited by the ecological relevance of taxa used to study global change. Lichens are sensitive bioindicators of air quality and their microalgae are an untapped source for new low-cost miniaturized bioassays with ecological importance. In order to increase the availability of a wider range of taxa for bioassays, the sensitivity of two symbiotic lichen microalgae, Asterochloris erici and Trebouxia sp. TR9, to atrazine was evaluated. To achieve this goal, axenic cultures of these phycobionts in suspension were exposed to a range of environmental concentrations of the herbicide atrazine, a common water pollutant. Optical density and chlorophyll autofluorescence were used as endpoints of ecotoxicity and ecophysiology on cell suspensions. Results show that lichen microalgae show high sensitivity to very low doses of atrazine, being higher in Asterochloris erici than in Trebouxia sp. TR9. We conclude that environmental concentrations of atrazine could modify population dynamics probably through a shift in reproduction strategies of these organisms. This seminal work is a breakthrough in the use of lichen microalgae in the assessment of micropollution effects on biodiversity.

Oral Exposure to Atrazine Induces Oxidative Stress and Calcium Homeostasis Disruption in Spleen of Mice.

The widely used herbicide atrazine (ATR) can cause many adverse effects including immunotoxicity, but the underlying mechanisms are not fully understood. The current study investigated the role of oxidative stress and calcium homeostasis in ATR-induced immunotoxicity in mice. ATR at doses of 0, 100, 200, or 400 mg/kg body weight was administered to Balb/c mice daily for 21 days by oral gavage. The studies performed 24 hr after the final exposure showed that ATR could induce the generation of reactive oxygen species in the spleen of the mice, increase the level of advanced oxidation protein product (AOPP) in the host serum, and cause the depletion of reduced glutathione in the serum, each in a dose-related manner. In addition, DNA damage was observed in isolated splenocytes as evidenced by increase in DNA comet tail formation. ATR exposure also caused increases in intracellular Ca2+ within splenocytes. Moreover, ATR treatment led to increased expression of genes for some antioxidant enzymes, such as HO-1 and Gpx1, as well as increased expression of NF-κB and Ref-1 proteins in the spleen. In conclusion, it appears that oxidative stress and disruptions in calcium homeostasis might play an important role in the induction of immunotoxicity in mice by ATR.

Antioxidant enzymes activity, lipid peroxidation, oxidative damage in the testis and epididymis, and steroidogenesis in rats after co-exposure to atrazine and ethanol.

Concomitant alcohol use and exposure to xenobiotics can adversely affect gonadal functions. This study investigated the oxidative status of the testis and epididymis and steroidogenesis of rats co-exposed to ethanol (EtoH, 5 mg kg(-1) b.wt.) and atrazine (ATZ, 50, 100, 300 mg kg(-1) b.wt.) for 3 weeks. The activities of catalase, superoxide dismutase, glutathione peroxidase, as well as the concentrations of glutathione and malondialdehyde, as indicators of oxidative stress were measured in the homogenates of the testis and epididymis. Testosterone and cholesterol concentrations as well as 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity were assayed in the plasma and testis respectively. After the administration of EtoH alone, or in combination with different doses of ATZ, oxidative damage as evident by malondialdehyde level was not observed in both the testis and epididymis. The combine exposure group showed dose-dependent decrease in plasma testosterone and testis cholesterol level and increase in testis 17ß-HSD activity compared to the EtoH group. Furthermore, the testes and epididymis of the EtoH-exposed rats treated with high dose of ATZ had severe histopathological damage. Therefore, ATZ-exposed alcohol-treated rats have histological damage of the testis and epididymis and lower testosterone level than EtoH-treated rats.

Effects of Atrazine on the Development of Neural System of Zebrafish, Danio rerio.

By comparative analysis of histomorphology and AChE activity, the changes of physiological and biochemical parameters were determined in zebrafish embryos and larvae dealt with atrazine (ATR) at different concentrations (0.0001, 0.001, 0.01, 0.1, and 1 mg/L). This study showed that the development of the sarcomere and the arrangement of white muscle myofibers were affected by ATR significantly and the length of sarcomere shortened. Further analysis of the results showed that the AChE activity in juvenile fish which was treated with ATR was downregulated, which can indicate that the innervation efficiency to the muscle was impaired. Conversely, the AChE activity in zebrafish embryos which was treated with ATR was upregulated. A parallel phenomenon showed that embryonic primary sensory neurons (Rohon-Beard cells), principally expressing AChE in embryos, survived the physiological apoptosis. These phenomena demonstrated that the motor integration ability of the zebrafish was damaged by ATR which can disturb the development of sensory neurons and sarcomere and the innervations of muscle.

The effects of the herbicide atrazine on freshwater snails.

Atrazine has been shown to affect freshwater snails from the subcellular to community level. However, most studies have used different snail species, methods, endpoints, and atrazine exposure concentrations, resulting in some conflicting results and limiting our understanding. The goal of this study was to address these concerns by (1) investigating the acute and chronic effects of atrazine on four species of freshwater snails (Biomphalaria glabrata, Helisoma trivolvis, Physa acuta, and Stagnicola elodes) using the same methods, endpoints, and concentrations, and (2) summarizing the current literature pertaining to the effects of atrazine on freshwater snails. We conducted a 48 h acute toxicity test with an atrazine concentration higher than what typically occurs in aquatic environments (1000 µg/L). Additionally, we exposed snails to environmentally relevant atrazine concentrations (0, 0.3, 3, and 30 µg/L) for 28 days and assessed snail survival, growth, and reproduction. We also summarized all known literature pertaining to atrazine effects on freshwater snails. The literature summary suggests snails are often affected by environmentally relevant atrazine concentrations at the subcellular and cellular levels. These effects are typically not transitive to effects on survival, growth, or reproduction at the same concentrations. Our acute exposures corroborate the general trend of no direct effect on snail populations as atrazine did not directly affect the survival of any of the four snail species. Similarly, environmentally relevant concentrations did not significantly affect the survival, growth, or reproduction of any snail species. These results indicate that, in the absence of other possible stressors, the direct effects of environmentally relevant atrazine concentrations may not be realized at the snail population level.

Short-term oral atrazine exposure alters the plasma metabolome of male C57BL/6 mice and disrupts α-linolenate, tryptophan, tyrosine and other major metabolic pathways.

Overexposure to the commonly used herbicide atrazine (ATR) affects several organ systems, including the brain. Previously, we demonstrated that short-term oral ATR exposure causes behavioral deficits and dopaminergic and serotonergic dysfunction in the brains of mice. Using adult male C57BL/6 mice, the present study aimed to investigate effects of a 10-day oral ATR exposure (0, 5, 25, 125, or 250mg/kg) on the mouse plasma metabolome and to determine metabolic pathways affected by ATR that may be reflective of ATR's effects on the brain and useful to identify peripheral biomarkers of neurotoxicity. Four hours after the last dosing on day 10, plasma was collected and analyzed with high-performance, dual chromatography-Fourier-transform mass spectrometry that was followed by biostatistical and bioinformatic analyses. ATR exposure (≥5mg/kg) significantly altered plasma metabolite profile and resulted in a dose-dependent increase in the number of metabolites with ion intensities significantly different from the control group. Pathway analyses revealed that ATR exposure strongly correlated with and disrupted multiple metabolic pathways. Tyrosine, tryptophan, linoleic acid and α-linolenic acid metabolic pathways were among the affected pathways, with α-linolenic acid metabolism being affected to the greatest extent. Observed effects of ATR on plasma tyrosine and tryptophan metabolism may be reflective of the previously reported perturbations of brain dopamine and serotonin homeostasis, respectively. ATR-caused alterations in the plasma profile of α-linolenic acid metabolism are a potential novel and sensitive plasma biomarker of ATR effect and plasma metabolomics could be used to better assess the risks, including to the brain, associated with ATR overexposure.

Sex-specific enhanced behavioral toxicity induced by maternal exposure to a mixture of low dose endocrine-disrupting chemicals.

Humans are increasingly and consistently exposed to a variety of endocrine disrupting chemicals (EDCs), chemicals that have been linked to neurobehavioral disorders such as ADHD and autism. Many of such EDCs have been shown to adversely influence brain mesocorticolimbic systems raising the potential for cumulative toxicity. As such, understanding the effects of developmental exposure to mixtures of EDCs is critical to public health protection. Consequently, this study compared the effects of a mixture of four EDCs to their effects alone to examine potential for enhanced toxicity, using behavioral domains and paradigms known to be mediated by mesocorticolimbic circuits (fixed interval (FI) schedule controlled behavior, novel object recognition memory and locomotor activity) in offspring of pregnant mice that had been exposed to vehicle or relatively low doses of four EDCs, atrazine (ATR - 10mg/kg), perfluorooctanoic acid (PFOA - 0.1mg/kg), bisphenol-A (BPA - 50 µg/kg), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD - 0.25 µg/kg) alone or combined in a mixture (MIX), from gestational day 7 until weaning. EDC-treated males maintained significantly higher horizontal activity levels across three testing sessions, indicative of delayed habituation, whereas no effects were found in females. Statistically significant effects of MIX were seen in males, but not females, in the form of increased FI response rates, in contrast to reductions in response rate with ATR, BPA and TCDD, and reduced short term memory in the novel object recognition paradigm. MIX also reversed the typically lower neophobia levels of males compared to females. With respect to individual EDCs, TCDD produced notable increases in FI response rates in females, and PFOA significantly increased ambulatory locomotor activity in males. Collectively, these findings show the potential for enhanced behavioral effects of EDC mixtures in males and underscore the need for animal studies to fully investigate mixtures, including chemicals that converge on common physiological substrates to examine potential mechanisms of toxicity with full dose effect curves to assist in interpretations of relevant mechanisms.

Subchronic atrazine exposure changes defensive behaviour profile and disrupts brain acetylcholinesterase activity of zebrafish.

Animal behaviour is the interaction between environment and an individual organism, which also can be influenced by its neighbours. Variations in environmental conditions, as those caused by contaminants, may lead to neurochemical impairments altering the pattern of the behavioural repertoire of the species. Atrazine (ATZ) is an herbicide widely used in agriculture that is frequently detected in surface water, affecting non-target species. The zebrafish is a valuable model organism to assess behavioural and neurochemical effects of different contaminants since it presents a robust behavioural repertoire and also all major neurotransmitter systems described for mammalian species. The goal of this study was to evaluate the effects of subchronic ATZ exposure in defensive behaviours of zebrafish (shoaling, thigmotaxis, and depth preference) using the split depth tank. Furthermore, to investigate a putative role of cholinergic signalling on ATZ-mediated effects, we tested whether this herbicide alters acetylcholinesterase (AChE) activity in brain and muscle preparations. Fish were exposed to ATZ for 14days and the following groups were tested: control (0.2% acetone) and ATZ (10 and 1000µg/L). The behaviour of four animals in the same tank was recorded for 6min and biological samples were prepared. Our results showed that 1000µg/L ATZ significantly increased the inter-fish distance, as well as the nearest and farthest neighbour distances. This group also presented an increase in the shoal area with decreased social interaction. No significant differences were detected for the number of animals in the shallow area, latency to enter the shallow and time spent in shallow and deep areas of the apparatus, but the ATZ 1000 group spent significantly more time near the walls. Although ATZ did not affect muscular AChE, it significantly reduced AChE activity in brain. Exposure to 10µg/L ATZ did not affect behaviour or AChE activity. These data suggest that ATZ impairs defensive behaviours of zebrafish, which could be related to its action on brain cholinergic neurotransmission. Moreover, the use of the split depth tank could be an alternative strategy to assess group behaviour and depth preference after exposure to chemical compounds.