Effects of Low Concentration of Glyphosate-Based Herbicide on Genotoxic, Oxidative, Inflammatory, and Behavioral Meters in Danio rerio (Teleostei and Cyprinidae)

The glyphosate herbicide is a pesticide widely used in the world and can contaminate soil, air, and water. The objective of this work was to evaluate the toxicity of a glyphosate-based herbicide (GBH) in zebrafish (Danio rerio). Fish were exposed to different concentrations of GBH (0, 50, 250, and 500 µg/L) for 96 hours. Brain, liver, and blood were collected for biochemical and genotoxicity analyses, and behavioral tests were performed. The results showed that there was a reduction in the activity of the antioxidant enzymes of catalase (CAT) and glutathione-S-transferase (GST) in the liver at all concentrations and at the highest concentration in the brain. There was also a reduction in lipid peroxidation in the liver at all concentrations of glyphosate. There was an increase in micronuclei in the blood at the 500 µg/L concentration. However, the count of nuclear abnormalities showed no differences from the control. Interleukin-1beta (IL-1β) generation was inhibited at all concentrations in the liver and at the highest concentration in the brain. No significant differences were found in the behavioral test compared to the control. The results showed that acute exposure to GBH promoted an inflammatory event, which reduced the efficiency of antioxidants, thus producing a disturbance in tissues, mainly in the liver, causing immunosuppression and generating genotoxicity.

Glyphosate has become the most commercialized pesticide [4,5], mainly due to the development of transgenic crops [6].It became the sales leader in the global pesticide market, with around 750,000 tons of the compound used annually and an increase estimated to 920,000 tons by the year 2025 [7].
Te intensive use of glyphosate in human activities ends up contaminating the soil, air, and water, in addition to causing changes at diferent ecological levels, such as in aquatic ecosystems, which can compromise the health of organisms and expose the entire population that uses water to a risk [8].
Recent studies show that exposure to glyphosate has several impacts on environmental health, especially on fsh, as they have the ability to absorb and concentrate toxins [9].A recently published systematic review and meta-analysis study showed that glyphosate afects diferent developmental stages of zebrafsh [10].Embryons exposed to glyphosate show increased mortality, in addition to an increase in morphological abnormalities such as yolk sac and pericardial edema, associated with body malformations [9,11].In adult zebrafsh, there was an increase in reactive oxygen species besides memory impairment and a reduction in aggressive behavior, in addition to increased anxiety [11].
Possessing about 70% genetic similarity with humans, the zebrafsh is currently an animal model widely used to study the molecular bases of neurobiology, mechanisms involved in the formation of neural circuits, and identifcation of genes involved in neurological disorders [12].Furthermore, it has been used in genetic and pharmacological studies to increase the understanding of neurobiological mechanisms [13][14][15].
Both the intensive use of pesticides in agricultural areas and the improper application by individuals without training or without the necessary instructions for the correct handling of chemical compounds can contaminate aquatic environments and cause adverse efects on organisms [4,16].
To assess the oxidative potential of the action of a xenobiotic in an organism, oxidative stress biomarkers are used to detect an excessive increase in ROS (oxidative markers) or a reduction in the ability of organisms to combat these antioxidative markers [17].
Oxidative markers are quantifable through thiobarbituric acid reactive substances (TBARS), which quantify the formation of malondialdehyde (MDA), which is one of the end products of lipid peroxidation (LPO), while antioxidant markers are observed during the activation or immunity of enzymes such as CAT and GST [8,18].
In addition to oxidative stress, infammatory processes, which are generated as a response by the immune system, when deregulated, occur to generate tissue damage and are responsible for causing the registration and progression of carcinogenesis in various gains [6,19,20].
Te immune system is the frst line of defense against pathogens or xenobiotics in fsh and responds quickly against environmental pressures in the aquatic ecosystem, generating a response from signals such as cytokines, interferons, and tumor necrosis factors, which are formed from an infammatory reaction [21].
Signaling signals such as cytokines act in diferent phases of the immune response [22].At the onset of the infammatory process, IL-1β recruits leukocytes and other cells from the bloodstream into the infamed tissue.
Given the above, the aim of this research was to investigate the efects of GBH on zebrafsh exposed to concentrations of this in terms of behavior, infammatory, oxidative, and genotoxic processes.Since zebrafsh is a vertebrate model widely used in human neurobehavioral studies, the results presented here are relevant not only for the environmental risk assessment, but also for understanding the risk of chronic exposures to low doses of glyphosate.Te animals were acclimatized over a seven-day period, with 120 specimens distributed across 12 aquariums, each holding 2 liters of dechlorinated water (at a density of 5 fsh per liter).Te aquaria were maintained under constant aeration, at a temperature of 25 °C (±1 °C), with a pH of 6.8, nitrite levels at 0.5 ppm, and a 14-hour light/12-hour dark photoperiod.Waste and residue were removed every 2 days, and one-third of the water was replaced regularly.Tese conditions were optimized for the proper maintenance and welfare of the specimens [23].

Materials and Methods
Water quality parameters such as pH, temperature, and nitrite were monitored daily and fsh were fed twice a day with commercial fsh feed.All procedures were approved by the Ethics Committee for the Use of Animals (CEUA-UNIARP), protocol number 001/2021.

Exposure to Glyphosate.
After acclimatization, the animals were exposed to concentrations of 0 (control), 50, 250, and 500 µg/L of GBH (Shadow ® ) for 96 hours of exposure.
Te product was placed once and the water was maintained without changing during the exposure period.Te GBH solution was not renewed as studies show that glyphosate has a long half-life, ranging from 49 to 70 days in water [24].
Te concentrations chosen were based on the classifcation of water bodies by the National Council for the Environment of Brazil (CONAMA) in Resolution no.357/ 05, which regulates the maximum permitted concentration of various substances, including glyphosate; in that, in class 1 and 2 waters, fresh water for human consumption and for the protection of aquatic communities, the maximum allowed is 65 µg/L and in class 3 waters, fresh water for human consumption after conventional or advanced treatment, the maximum allowed is 280 µg/L.
Te individuals were submitted to euthanasia by cooling, and the caudal peduncle was cut with the aid of a scalpel for blood collection and placed on a slide for later visualization of the erythrocytes.For the other analyses, the liver and the brain of each individual were collected with suitable surgical material (scalpel, scissors, and stainless steel tweezers), deposited in microcentrifuge tubes, homogenized with lysis bufer (50 mM Tris-HCl, pH 7.0; 5 mM EDTA; 140 mM NaCl; 1% Triton X-100, and 0.5% protease inhibitor cocktail (Sigma-Aldrich)), centrifuged at 12000 g for 4 minutes, removed the supernatant, stored in a microcentrifuge tube, and conditioned in a refrigerator at −80 °C to the TBARS, CAT, GST, and IL-1β tests.

Quantifcation of Protein.
Protein concentration was determined according to the method previously described by Bradford [25].Tis method consists of manufacturing total proteins using the Coomassie brilliant blue G-250 dye.Tus, 20 μl of the sample was mixed with 1000 μl of Bradford reagent.Te mixture was incubated at room temperature for 3 minutes and the absorbance was read at 595 nm.Te analytical curve was prepared in test tubes with successive dilutions of a bovine serum albumin (BSA) (Sigma-Aldrich) solution in a range of 0-50 µg mL −1 .Catalase present in the sample degrades hydrogen peroxide [27].In a quartz cuvette, 2 mL of hydrogen peroxide solution (10 mM) in phosphate bufer (50 mM, pH 7.0) was added to 10 µL of the sample.Reading was performed every 10 seconds for 40 seconds, evaluating the absorbance decay at 240 nm.Te results were expressed in mmol/mg of protein.

Glutathione-S-Transferase (GST).
Te glutathione-Stransferase (GST) was determined using the method by HABIG et al. [28].Te GST present in the sample converts CDNB (1-chloro-2,4 dinitrobenzene) into TNB (trinitrobenzene) through conjugation with the GSH present in the reaction medium.In a quartz cuvette, 0.98 mL of phosphate bufer (0.1 M, pH 7.0), 10 µL of CDNB (0.1 M), and 10 µL of reduced glutathione (0.1 M GSH) were added to 10 µg of the sample.Te frst reading was followed and after 1 minute, the second reading was taken in the absorbance range of 340 nm.Te results were expressed in mmol/mg of protein.

Interleukin Activity (IL-1β).
To quantify IL-1β in zebrafsh homogenates, instructions were followed according to the manual for the Elisa kit from the manufacturer Cusabio Technology LLC ® .
Te assay employs a sandwich immunoassay technique, in which IL-1β is fxed onto the plate along with the standard and sample.Tey are pipetted into the wells causing the IL-1β to bind to the immobilized antibody.After removing unbound substances, a specifc antibody conjugated with biotin (100 µL) is added to the wells, washed (200 µL), and an avidin peroxidase conjugate (HRP-avidin) (100 µL) is added.Another wash (200 µL) is performed to remove any unbound enzyme-avidin reagent and then a substrate (100 µL) is added to the wells and a hue proportional to the amount of IL-1β present in the sample is observed.Absorbance is measured at 450 nm.Values were expressed in pg/mg of protein.

Micronucleus Test and Nuclear
Abnormalities.Blood smear slides were fxed with methanol and stained with 20% Giemsa.1000 cells were counted on each slide under an optical microscope with 1000x magnifcation, totaling 6 slides per treatment (control, 50 µg/L, 250 µg/L, and 500 µg/ L).In the counts, cells were considered normal when the oval-rounded shape characteristic of the species was maintained; the micronucleus was characterized as a small intracytoplasmic body, resembling the nucleus in color, varying from 1/3 to 1/20 in size with the nucleus and separated from it without a binding bridge [29].Nuclear abnormalities were grouped and summed considering all nuclei that presented invaginations (blebbed, lobed, and notched); binucleated, when the erythrocyte presented two apparent nuclei; vacuolated, when the nucleus presented a cavity within its nuclear limit [30]; and karyorrhexis, when the erythrocyte presents a nucleus decomposed into several micronuclei in the cytoplasm [31].

Behavioral Test
2.7.1.Light/Dark Test.For this test, a rectangular glass aquarium (45 cm long × 10 cm wide × 15 cm high) was used, half transparent and the other dark opaque in order to be careful not to refect the image of the animal (Figure 1), flled with dechlorinated water to a height of 10 cm, totaling 4.5 liters of water.Te apparatus has a central compartment with removable doors, each one in the color of the side on which it is located, with 5 cm of distance between them.
Te animals were transferred to the behavior analysis aquarium after 96 hours of exposure to GHB.Te behavior was evaluated by a trained observer and recorded in spreadsheets for subsequent data analysis.
Each animal (n � 10) was transferred from its home aquarium with the aid of a net (5 × 5 cm), and placed in a beaker containing its water, always by the same researcher.Te specimen was transported to the room where the test aquarium was previously arranged, prepared, and lifted with the aid of a net (5 × 5 cm) into the central compartment.After an interval of 5 minutes for the individual's acclimatization, the following exploratory observations were started: latency time in which the animal remained in the central compartment after removing the removable doors, number of alternations between light and dark sides and vice versa, and total time spent on the light side and total time spent on the dark side.Each animal was evaluated for 15 minutes with the water in the apparatus changed after every 3 specimens [32].

Statistical Analysis.
For statistical analysis and graphs, the program used was GraphPad ® Prism 9.3.1.When the data tended to normality, the one-way ANOVA test was used followed by the Bonferroni test for multiple comparisons.When it was assumed that the data were nonparametric, the Kruskal-Wallis test was used followed by the Dunn's test for multiple comparisons.Asterisks indicate statistical diferences compared with the control group: * (p < 0.05), * * (p < 0.01), * * * (p < 0.001), and * * * * (p < 0.0001).Data were reported as mean ± standard deviation.
Biochemistry Research International

Results
Figure 2 shows the exposure efects of adult zebrafsh exposed to glyphosate-based herbicide (GBH) through the light/dark environment test.No signifcant diferences (p < 0.05) were found in the behavior of animals exposed to glyphosate concentrations when compared to the control group in none and light time (Figure 2).Frequencies of MN, AN, and normal erythrocytes in zebrafsh are shown in Figure 3.As observed in the graphs of the statistical analysis, only the frequency of micronuclei (Figure 3) with the highest concentration showed a signifcant diference (p < 0.05) compared to the control group.Concentrations lower than 250 µg/L and 50 µg/L did not show signifcance.Figures 4(a Te activity of the CAT enzyme in the brain did not show a signifcant result in any of the groups of zebrafsh exposed to GBH in relation to the control (Figure 5(c)), but it showed a signifcant diference in the liver, with a reduction of enzymatic activity in the groups exposed to 50 µg/L, 250 µg/ L, and 500 µg/L (p < 0.0001) of glyphosate when compared to the control treatment (Figure 5(d)).
Te determination of lipid peroxidation through TBARS did not show signifcant results in the brain of zebrafsh exposed to GBH in any of the treatments compared to the control (Figure 5(e)), and as for the liver, it showed a reduction in the treatment of 50 µg/L, with a signifcance of p < 0.01, when compared to the control and a reduction in the treatments of 250 µg/L and 500 µg/L, with a signifcance of p < 0.001, compared to the control (Figure 5(f )).
IL-1β levels in zebrafsh's brain and liver are shown in Figure 6.Brain IL-1β level decreased in the 500 µg/L treatment compared to the control (p < 0.01) (Figure 6(a)).In the liver, IL-1β levels showed a reduction in all treatments when compared to the control (p < 0.0001) (Figure 6(b)).

Discussion
Te results showed that acute exposure to GBH promoted an infammatory event, which reduced the efciency of antioxidants, thus producing a disturbance in tissues, mainly in the liver, causing immunosuppression and generating genotoxicity.
In the present study, a slightly longer average time was observed on the light side than on the dark side, indicating a reduced anxiety behavior in general in all treatments, but with no signifcant diference with the control.One way of observing zebrafsh behavioral actions is the light/dark environment test [33].Scototaxis, the preference for dark environments, is a typical behavioral strategy observed in zebrafsh.Tis behavior refects an evolutionary adaptation to avoid potential predators, as seeking darker areas ofers camoufage and reduces exposure to threats in their natural habitat [34].
Studies show that glyphosate caused behavioral changes in zebrafsh, such as the study by Chaulet et al. [35], which indicated the addition of the anxiolytic characteristic of the animal exposed to 3 and 5 mg/L of a glyphosate-based herbicide.Te same anxiolytic behavior was noted in the study by Maximino et al. [33], in which groups of zebrafsh showed behavioral changes in treatments of 0.065 mg/L and 0.5 mg/L with Roundup ® and 0.5 mg/L with glyphosate.
In other animals, behavioral efects were also observed when exposed to glyphosate or its commercial compounds, such as fsh [36,37], rats [38], and insects [39].
In all the treatments in which the animals were exposed to GBH, in the present study, they did not promote possible signifcant changes in the neurological status of the zebrafsh according to the behavioral test evaluated, being necessary further studies for a better evaluation of the toxicity of glyphosate as a pollutant of aquatic ecosystems and a possible behavioral change in fsh populations.Te results are possibly directly related to the type of formulation used, as Bridi et al. [11] observed such a change in treatment with Roundup ® .
Biomarkers have been widely used as tools to detect exposure and assess the efects of genotoxic pollution on ecosystems and consist of tests that assess chromosomal anomalies, DNA breaks, frequency of MN, and other AN, such as the micronucleus test [40].Among the pollutants that are potentially genotoxic are pesticides [40], including organophosphates, as the study by Nahas et al. [41] who observed an increase in micronuclei in a group of fsh Oreochromis niloticus exposed to pesticides.
Te genotoxic potential was found in the glyphosate herbicide, as reported by Cavalcante et al. [42] with the MN test in groups of fsh Prochilodus lineatus exposed to Roundup ® , as well as Qin et al. [43] demonstrated that glyphosate exposure for 24 hours in cells from the fsh Misgurnus anguillicaudatus had a signifcant increase in MN at a dose of 400 mg/L when compared to the control group, and AN at a dose of 560 mg/L compared to the control group.Biochemistry Research International Te occurrence of micronuclei by endogenous origin has been reported by several authors and shows a great variability between species, with values between 0 and 13 MN per 1000 cells [44][45][46].According to Canedo et al. [47], in reviewed studies on the genotoxic potential of glyphosate, the basic level of MN found in zebrafsh erythrocytes was 0% and the maximum level was 2.57%.
In the present study, a signifcant diference was found in the frequency of micronuclei in the 500 µg/L treatment compared to the control group (mean of 6%), demonstrating a disruption in the cell's chromosomal system caused by GBH.Te 50 µg/L and 250 µg/L treatments did not difer statistically from the control group.Tere was no signifcant diference in the frequency of AN and in the amount of normal erythrocytes, indicating that the GBH concentrations used in a 96-hour exposure to zebrafsh peripheral erythrocytes did not cause AN when compared to the control group.
Studies with glyphosate demonstrate the generation of MN and AN, such as that of Çavas and Könen [48], who found an increase in the frequency of MN and AN in Carassius auratus erythrocytes exposed to glyphosate at concentrations of 5, 10, and 15 mg/L for periods of 48, 96 and 144 hours.Caramello and Jorge [47] found an increase in MN and AN in Prochilodus lineatus erythrocytes exposed to diferent concentrations of Roundup©.
Similar results were found by Odetti et al. [49] in Caiman latirostris hatchlings where the study showed that although glyphosate-based formulations can promote an increase in the frequency of micronuclei even when used in low Biochemistry Research International concentrations, this efect does not is related to oxidative stress.However, deleterious genetic efects on fsh and alligator species can be a threat to survival and health status.
Oxidative stress results from a mismatch between the production of ROS and the antioxidant capacity resulting from endogenous agents or environmental factors leading to harmful actions of ROS on cells [6].Te antioxidant defense system is a mechanism present in organisms to prevent the damage that ROS can cause and includes enzymes such as CAT and GST [50].
Te CAT enzyme is a primary antioxidant defense component that contains iron and facilitates the removal of H 2 O 2 , which is metabolized to molecular oxygen and water in the metabolization of xenobiotics [8], and GST catalyzes glutathione processes in the process of biotransformation of xenobiotics [51].
Any change in antioxidant enzyme activity is indicative of oxidative activity [52].Both the increase and depletion in the activity of these enzymes when exposed to pollutants suggest an activity of these substances in organisms [53].Te reduction of antioxidant capacity ends up making the fght against the activity of oxidizing substances in the body inefcient [19].
In this study, there was a signifcant decrease in GST activity in the 500 µg/L treatment and no signifcant CAT activity in the zebrafsh brain, however, in the liver there was a signifcant depletion of GST and CAT in all glyphosate treatments with the zebrafsh.
In a study by Lushchak et al. [54], a signifcant decrease in catalase (CAT) activity was observed in Carassius auratus exposed to concentrations exceeding 10 mg/L of Roundup ® for 96 hours.Similarly, in Rhamdia quelen (silver catfsh), CAT activity was reduced after exposure to 1.21 mg/L of 6 Biochemistry Research International Roundup ® for 96 hours [55].In Prochilodus lineatus, ex- posure to 10 mg/L of Roundup ® for 96 hours resulted in increased glutathione S-transferase (GST) and lipid peroxidation (LPO) activity in the liver, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced [56].Additionally, Gallegos et al. [58] reported a decrease in CAT activity in the brain of rats exposed to glyphosate, and Zancanaro et al. [58] observed reduced CAT activity in the liver and kidney tissues of rats exposed to glyphosate or a glyphosate-based herbicide (GBH).Te liver is the main source of GST and CAT in fsh, in contrast to the activity in the brain, which is generally low [59,60], reinforcing the analysis of the present study, in that there was no signifcant activity of CAT in all treatments and of GSTat concentrations of 50 µg/L and 250 µg/L in the brain of zebrafsh exposed to glyphosate for 96 h.
As a response to oxidative damage to biomolecules, LPO was analyzed using the TBARS test, in which LPO damage was caused by the generation of ROS or changes in antioxidant capacity [51].In analyzes carried out with TBARS, no signifcant changes were found in the brain, and there was a decrease in levels in the liver and in all treatments with zebrafsh.
In the study by Moura et al. [61], there was a signifcant increase in TBARS levels in liver (6 h and 48 h) and muscle tissue (24 h) in the hybrid fsh jundiara (Leiarius marmoratus x Pseudoplatystoma reticulatum) after exposition of Roundup©, although Glusczak et al. [62] observed glyphosate depletion in Jundiá (Rhamdia quelen) exposed to 0.2 mg/L and 0.4 mg/L of glyphosate for 96 hours.
Some studies have reported an increase in brain LPO levels in zebrafsh [9,63] and in Jundiá [64], and no changes in levels in Pintado da Amazônia [65].
It is important to highlight that diferent LPO responses are dependent on the fsh species and the specifc characteristics of tissues with diferent levels of peroxide production and also on variations in antioxidant responses [51].Te reduction in LPO can be explained by the activity of other enzymes that were not evaluated in the study, such as superoxide dismutase, glutathione peroxidase, and reduced glutathione (GSH).
Te increase in the frequency of micronuclei observed in the present study may also be related to a decrease in the activity of CAT and GST enzymes, since damage to cellular DNA can cause a change in the transcription of proteins, including antioxidant enzymes.However, further studies evaluating the expression of antioxidant proteins in zebrafsh exposed to low concentrations of glyphosate are necessary to confrm these fndings.Reduction in catalase activity associated with changes in glutathione gene expression was recently found by Chitolina et al. [66] in the ovaries of rats exposed to low doses of GBH for 28 days.
IL-1β is a pro-infammatory cytokine, which activates cells of the immune system such as macrophages, lymphocytes and monocytes, leading to an infammatory efect  Biochemistry Research International in the face of any environmental stressor, tissue injury, or pathogen invasion [20,67].Interleukin-1β (IL-1β) has been extensively studied across various fsh species, demonstrating its pro-infammatory role in response to xenobiotics [68].Previous studies have confrmed its activity in American catfsh (Ictalurus punctatus) [69], common carp (Cyprinus carpio L.) [70], and southern bluefn tuna (Tunnus maccoyii) [71].Tese fndings reinforce the critical involvement of IL-1β in the immune response triggered by the presence of environmental contaminants.
Glyphosate has been shown to induce the production of pro-infammatory cytokines in various studies.For example, Pandey, Dhabade, and Kumarasamy [72] reported an increase  Biochemistry Research International in IL-1β levels in the adipose tissue of rats.Similarly, an elevation of IL-1β was observed in the liver of common carp (Cuprinus carpio) exposed to 5 mg/L and 50 mg/L of glyphosate over 15, 30, and 45 days [73], as well as in the kidneys of rats [74].Notably, in Oreochromis niloticus (Nile tilapia), glyphosate exposure led to a signifcant increase in IL-1β levels in the gill tissue, further corroborating its role in the infammatory response to environmental contaminants [75].
In the study by Ma and Li [21], glyphosate caused an increase in IL-1β levels in the liver and kidneys of common carp exposed to 52.08 mg/L and 104.15 mg/L for up to 168 hours and a decrease of IL-1β levels in the spleen when compared to control.
Tis study showed an opposite result in the levels of IL-1β, resulting in the inhibition of IL-1β and demonstrating that glyphosate generated immunosuppression in zebrafsh, leaving the animal defcient in a critical cytokine for the onset of the infammatory process, and this result may be correlated with the increase in MN observed in the treatment with 500 µg/L of GBH.Te reduction in IL-1β observed in this study may, in turn, be associated with the inhibition of caspase-1, which is responsible for the conversion of pro-IL-1β into active IL-1β.Recent studies have shown that glyphosate can inhibit caspase pathways, reducing the apoptosis process, which is one of the factors related to the potential tumorigenic efect of glyphosate [76].
Future studies may build upon confrming some data found in the present study, such as evaluating the expression of antioxidant enzymes and assessing caspase-1 to determine if it is inhibited by exposure to GBH and perhaps associated with the reduction in IL-1β activity.

Conclusion
Te present study demonstrated an implicit relationship between toxicity mechanisms in the face of acute exposure to GBH.Te present study demonstrated a clear relationship between toxicity mechanisms and acute exposure to glyphosate-based herbicides (GBH).GBH exposure led to an imbalance in antioxidant enzyme activity, with a reduction in CAT and GST levels, along with a decrease in TBARS.Additionally, a reduction in the pro-infammatory cytokine IL-1β was observed.Tese fndings highlight a more pronounced deregulation of enzymatic activity in hepatic tissue compared to brain tissue, suggesting a higher infammatory response in the liver than in the brain.
Similarly, the analysis of zebrafsh blood found genotoxicity in the face of exposure to GBH from the generation of micronuclei, and the behavioral analysis did not obtain a signifcant diference in the face of the evaluated test.
Te data presented can enrich studies about the mechanisms of exposure to glyphosate in zebrafsh in the face of behavioral, infammatory, oxidative, and genotoxic analyses, which can provide information about the toxicity of glyphosate in exposed fsh.Based on these data, it can be inferred that glyphosate-based herbicides, even at low concentrations and over short exposure periods, can induce damage, as evidenced by the altered activity of various oxidative enzymes.

2. 1 .
Animals and Maintenance.Adult specimens of Danio rerio (3-4 cm), of both sexes, were obtained from a local commercial supplier and transported to the laboratory of translational research in health at the Universidade Alto Vale do Rio do Peixe (UNIARP) for acclimatization.
Figure2shows the exposure efects of adult zebrafsh exposed to glyphosate-based herbicide (GBH) through the light/dark environment test.No signifcant diferences (p < 0.05) were found in the behavior of animals exposed to glyphosate concentrations when compared to the control group in none and light time (Figure2).Frequencies of MN, AN, and normal erythrocytes in zebrafsh are shown in Figure3.As observed in the graphs of the statistical analysis, only the frequency of micronuclei (Figure3) with the highest concentration showed a signifcant diference (p < 0.05) compared to the control group.Concentrations lower than 250 µg/L and 50 µg/L did not show signifcance.Figures4(a) and 4(c) shows the presence of micronuclei in the slides analyzed.Te frequencies of normal and NA erythrocytes (Figure 3) did not show statistical diferences in any of the treatments compared to the control group.Examples of erythrocytes containing NA are shown in Figures 4(b) and 4(d).Te activity of the GST enzyme in the brain showed a reduction in the group of zebrafsh exposed to 500 µg/L (p < 0.01) of GBH in relation to the control (Figure 5(a)); in the liver, it showed a reduction in the treatments of 50 µg/L, 250 µg/L, and 500 µg/L (p < 0.0001) of GBH in relation to the control (Figure 5(b)).Te activity of the CAT enzyme in the brain did not show a signifcant result in any of the groups of zebrafsh exposed to GBH in relation to the control (Figure5(c)), but it showed a signifcant diference in the liver, with a reduction of enzymatic activity in the groups exposed to 50 µg/L, 250 µg/ L, and 500 µg/L (p < 0.0001) of glyphosate when compared to the control treatment (Figure5(d)).Te determination of lipid peroxidation through TBARS did not show signifcant results in the brain of zebrafsh exposed to GBH in any of the treatments compared to the control (Figure5(e)), and as for the liver, it showed a reduction in the treatment of 50 µg/L, with a signifcance of p < 0.01, when compared to the control and a reduction in the treatments of 250 µg/L and 500 µg/L, with a signifcance of p < 0.001, compared to the control (Figure5(f )).IL-1β levels in zebrafsh's brain and liver are shown in Figure6.Brain IL-1β level decreased in the 500 µg/L

Figure 1 :
Figure 1: Light/dark test tank.Te triangle consists of a rectangle, with a light and a dark side and with a central compartment with removable doors with the dimensions described below.Fonte: Stewart et al. [15].

Figure 2 :
Figure2: Efects of GBH exposure (0, 50, 250, 500 μg/L) on groups of adult zebrafsh (n � 10) in the 15-minute light/dark behavioral test (900 seconds); zebrafsh was exposed to GBH for 96 hours.Data analyzed by one-way ANOVA, followed by post hoc Kruskal-Wallis when the standard deviation (SD) showed a signifcant value in the Brown-Forsythe test.Values expressed as mean ± standard deviation in column graphs.Average (+) on box plot charts.

Figure 3 :
Figure3: Frequencies of MN, AN and normal erythrocytes observed in a thousand cells per slide in adult zebrafsh exposed to GBH.Efects of glyphosate exposure (0, 50, 250, 500 μg/L) in groups of adult zebrafsh (n � 8).Zebrafsh were exposed to GBH for 96 hours.Data analyzed by one-way ANOVA.Values expressed as mean ± standard deviation.Where * (p < 0.05).