The Impact of Tetraethyl Pyrophosphate (TEPP) Pesticide on the Development and Behavior of Danio rerio: Evaluating the Potential of Cork Granules as a Natural Adsorbent for TEPP Removal from Aqueous Environments

Abstract

Toxicological studies of pesticides in animal models provide critical insights into their mechanisms of action, while adsorption strategies offer potential solutions for decontaminating polluted waters. We evaluated toxicity induced by tetraethyl pyrophosphate (TEPP), an organophosphate pesticide and AChE inhibitor, on zebrafish (Danio rerio) development and behavior, alongside the efficacy of wine cork granules as a natural adsorbent. TEPP exposure reduced embryo viability following an inverted U-shaped dose–response curve, suggesting non-monotonic neurodevelopmental effects, but did not alter developmental timing or morphology in survivors. In juveniles, TEPP increased preference for dark environments (33% vs. controls) and enhanced swimming endurance approximately 3-fold, indicating disrupted phototaxis and stress responses. Most strikingly, water treated with cork granules retained toxicity, increasing mortality, delaying embryogenesis, and altering behavior. This directly contradicts in vitro adsorption studies that suggested cork’s efficacy. These results demonstrate the high sensitivity of zebrafish to TEPP at nanomolar concentrations, which contrasts with in vitro models that require doses approximately 1000 times higher. Our findings not only highlight TEPP’s ecological risks but also reveal unexpected limitations of cork granules for environmental remediation, urging caution in their application.

1. Introduction

Organophosphates (OPs) are a class of chemical compounds that have comparable structural properties and are widely used as commercial insecticides [1]. OPs inhibit acetylcholinesterase (AChE), an enzyme that catalyzes the conversion of acetylcholine (ACh) to acetate and choline in synaptic gaps [2,3], which explains why excessive central nervous system stimulation can lead to respiratory failure and death [4].

Tetraethyl pyrophosphate (TEPP) is an OP pesticide used in agriculture, food, and beverage production, and is responsible for high rates of deliberate and incidental poisoning [5]. Studies suggest that acute toxicity of TEPP in humans can induce systemic effects, such as nausea, weakness, dizziness, loss of vision, tightness of the chest, cramping, and vomiting [6]. Pesticides are a risk to the environment because spills can pollute soil, water, air, and organisms [7]. Some of the chemical, physical, and biological procedures used to remove pesticides from aqueous solutions include adsorption, advanced oxidation, membrane filtering, phytoremediation, bioremediation, and the aeration tank method [8].

Natural adsorbents can be used to remove pesticides from aqueous solutions [9]. Cork, a natural, renewable, and biodegradable product, has been suggested as a sustainable adsorbent for a variety of spills involving hydrocarbons, oils, solvents, and organic compounds [10,11]. The presence of lignin and suberin provides hydrophobicity, while cellulose and hemicellulose confer high polarity, contributing to their potential as natural adsorbents [12]. The application of cork derivatives is particularly significant from an environmental perspective, as a renewable component is employed in long-lasting products, enhancing CO₂ fixation [13]. Cork has therefore been utilized as a green alternative due to its porous structure, and because it is a natural raw material with low cost, easy accessibility, low density, and biodegradable properties [13,14,15].

The effectiveness of cork from Quercus cerris and Quercus suber in pesticide removal from water was investigated using biochemical methods to quantify residual pesticides in water samples and evaluate the efficiency of these natural adsorbents [11,12,16,17]. After investigating TEPP effects on AChE activity in both commercial electric eel (Electrophorus electricus) enzyme preparations and AChE secreted into the conditioned medium by differentiated PC12 neuronal cells [18], our group explored TEPP removal from water using wine cork-derived granules as natural adsorbents [18]. These cork granules exhibited high adsorption capacity for TEPP in aquatic systems, removing 95 ± 5% of water-diluted pesticide under controlled laboratory conditions within 24 h [18]. The TEPP solution, after the adsorbent procedure, decreased its inhibitory effects on AChE activity in in vitro assays [18], suggesting that cork granules can be utilized to clean pesticide-contaminated waterways [18]. This study presents the first in vivo investigation of TEPP toxicity in zebrafish (Danio rerio) across developmental stages, while concurrently assessing the efficacy of cork granules as natural adsorbents in a living system. Prior research has been limited to in vitro models examining either TEPP effects on isolated enzymes/cells or cork adsorption in controlled setups. Our work advances the field by demonstrating TEPP’s whole-organism impacts on embryogenesis, neurobehavioral responses, and morphological development, while evaluating water decontamination strategies under biologically relevant conditions. These findings provide crucial translational insights connecting in vitro adsorption studies with environmental applications in pesticide remediation.

2. Materials and Methods

2.1. Chemicals and Solutions

TEPP (Pestanal^®; >99.9% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA). This pesticide was chosen because it is commonly used in agriculture, cleaning products, cosmetics, environmental protection, food and beverages, and personal care products worldwide. Other chemicals used in the present study were of analytical reagent grade (purity greater than 95%) and were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA, USA), Gibco BRL (Waltham, MA, USA), or Sigma-Aldrich Corporation (St. Louis, MO, USA).

2.2. Reconstituted Water Solution

The water was obtained from an artesian well and a public supply in Londrina (PR, Brazil), and it was dechlorinated twice through an activated carbon filter. The water parameters were measured daily before and after the experiments, including temperature (28 °C), conductivity (56 μS·cm⁻¹), dissolved oxygen (5.6 mg·L⁻¹) (Hanna Instruments, Barueri, SP, Brazil), and pH (6.8) (Akso, São Leopoldo, RS, Brazil). The density of fish in aquariums did not exceed 1.0 g·L⁻¹ of water [19].

2.3. Zebrafish Collection and Maintenance

Adult wild-type zebrafish (D. rerio) (n = 100) from the farm Andrade (Vierias, MG, Brazil) were supplied to the Bioassay of the Laboratory of Animal Ecophysiology (LEFA) and Laboratory of Environmental Diagnosis (LADA) of the Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina (PR, Brazil). At a maximum density of 100 animals per bag, the fish were carried in plastic bags (50 × 80 × 0.18 cm) transporting 30 L of water from the fish farm tanks. The plastic bags had 2/3 of the quantity of water and 1/3 of the volume of air to allow for gas exchange during transport. For at least seven days, the fish were acclimatized in tanks of 80 L with dechlorinated water, a biological filter, constant aeration, and a temperature of 28 °C maintained by a photoperiod of 14 h–10 h (light–dark). The water was renewed at a rate of 70% every 24 h, ensuring that ammonia levels (0.01 ± 0.01 ppm) (Labcon-Alcon colorimetric test, Camboriu, SC, Brazil) were consistent with the safety of animals. The fish were fed twice a day with commercial feed (Alcon Basic^® MEP 200 Complex, Camboriu, SC, Brazil), which is designed for ornamental fish development. Adult food was stopped 24 h before the studies and once more throughout the acute tests. After spawning in suitable trays [20], viable fertilized eggs were picked and exposed on 24-well plates in Petri dishes containing test solution, and the embryos were individually preserved in 2 mL of reconstituted water solution under the light/dark cycle [21]. The Ethics Committee on the Use of Animals of the State University of Londrina (CEUA/UEL; PR, Brazil) reviewed and approved this work under protocol number CEUA (n° 041.2022).

2.4. Preparation of the Cork Granules

The cork granules used as natural adsorbents were obtained from discarded commercial wine stoppers (São Paulo, Brazil) to evaluate the potential reuse of this waste material. The cork samples were cut into bar geometry with 1.0 cm of length and approximately 3.0 mm of diameter and then triturated to obtain a particle size of 1.0 mm. The stoppers were cleaned in the manner previously described [22]. They were washed in ultrapure water and the water was renewed until there was no longer any yellow coloring in the water. To reduce the influence of particle adsorption with the analysis, the samples were filtered (Whatman 0.45 m) before analysis. They were then dried in an oven at 60 °C for 24 h before being stored in plastic containers.

2.5. Adsorption Experiments

Adsorption studies were conducted in an Erlenmeyer set containing different amounts of TEPP (0.02, 0.07 or 0.3 µmol·L⁻¹) with or without cork granules at 5% in a reconstituted water solution. To avoid photodegradation of the pesticide, the flasks were covered with aluminum foil and kept at room temperature (25 ± 2 °C) for a total of 24 h of contact time, with periodic agitation to establish equilibrium. In the same treatment condition mentioned above, control groups were included in our study that reflected only reconstituted water solution or only cork granules at 5% (m·v⁻¹) without TEPP. For embryo testing, samples were filtered through 0.22 µm PTFE membrane, while juvenile exposure studies used unfiltered samples to better replicate natural environmental conditions.

2.6. Experimental Design

For fish embryo toxicity (FET) assays, embryos were chosen at random and divided into seven groups (each group containing 20 embryos) and maintained in a 24-well plate (Nest Biotechnology, Rahway, NJ, USA). In each well, one embryo was kept at reconstituted water [23] in a final volume of 2 mL. The groups were exposed to different treatment condition: untreated (negative control), treated with 3,4-dichloroaniline (DCA; 2 mg·L⁻¹; positive control), Cork, TEPP (0.02, 0.07 or 0.3 µmol·L⁻¹), or Cork + TEPP (0.02, 0.07 or 0.3 µmol·L⁻¹). The embryos were maintained to at 24, 48, 72, and 96 h post-fertilization (hpf) from the fertilization at 26 ± 2 °C under 14–10 h light–dark cycles. For the behavior experiments, the juvenile fish were (n = 9 per treatment) exposed to TEPP (0.02, 0.07, or 0.3 µmol·L⁻¹), Cork, Cork + TEPP (0.02, 0.07, or 0.3 µmol·L⁻¹), or untreated in 2.5 L tanks for 24 h. Feeding was interrupted 24 h before the beginning of the exhibition, and there was no renewal of the medium during the treatments. After that, the juveniles were subjected to light–dark behavioral tests and swimming resistance, and after that, they were sacrificed by immersion in benzocaine (0.1 g·L⁻¹) and biometrics parameters were analyzed.

2.7. Fish Embryo Toxicity (FET) Assays

The evaluation of larval development was performed at regular time points (24, 48, 72, and 96 hpf) and the embryos were scored as normal, abnormal, or dead and hatched/unhatched using a stereomicroscope (Model SMZ 2 LED, software Optika View Version 7.1.1.5 (Optika^®). The abnormalities considered were a coagulated embryo, absence of heartbeat, lack of detachment of the tail of the yolk sac, and absence of somite. Sublethal parameters included the abnormal development of the head, body axis and yolk sac, edemas, and lack of pigmentation and blood flow [21,24]. Data were expressed as the mean ± SEM of viability percentage relative to the control.

2.8. Biometric Parameters Analyses

The final weight of the fish, total length (from the anterior end of the head to the end of the caudal fin), standard length (from the anterior end of the head to the beginning of the caudal fin insertion), and survival rate (SURV) ([final number of live fish/initial number of fish] × 100) were measured according to a previously described procedure [21]. After that, the dead fish were frozen and delivered to the State University of Londrina’s (PR, Brazil) animal disposal system. Data obtained from final weight (g), total length (cm), and standard length (cm) were expressed as the mean ± SEM. The survival rate (SURV) was represented as a percentage of the total.

2.9. Light–Dark Preference Assay

The light–dark behavioral tests were carried out according to the literature [25,26,27]. In brief, the animal was placed in a center compartment (5 cm wide) inside a rectangle aquarium fixed with black or white (49 × 18 × 9 cm). After 5 min of acclimatization in the central compartment, the fish was released and filmed for 15 min by the camera installed above the aquarium, with the parameters of time spent in each environment and number of crossings in the midline, as well as the first preference of light or dark, being evaluated. The GeoVision^® Gv800 was high-resolution picture capturing software with a resolution of 640 × 480 pixels. The time spent on each environment (s) and number of crossings in the midline were represented as mean ± SEM.

2.10. Swimming Resistance Test

The swimming performance of the fish was assessed using an adapted experimental apparatus consisting of a U-shaped acrylic tube system (30 mm in diameter) connected to a water pump, as described in the literature [25,28]. These experiments were conducted following the light–dark behavioral test sequence. The fish were introduced through a T-shaped opening to swim against the water current generated within the system. The flow rate was adjusted using a calibrated water valve, starting at 3 L·min⁻¹ (16.5 cm·s⁻¹) for acclimation. After each minute, the flow rate was increased to 4, 5, 6, and finally 15 L·min⁻¹. If the fish could not resist the current, it was expelled from the tube into an attached plastic container, where the exhausted fish could be recovered. After exhaustion, the total swimming time was computed to calculate a swimming endurance index (SEI) according to the following formula:

$$SEI=\sum resistedflowrates+{\displaystyle \frac{secondsresistedonlastflowrate}{60}}\times lastflowrate$$

(1)

2.11. Statistical Analysis

All data are always presented as mean ± standard error of the mean (SEM). Parameters from multiple groups were analyzed using a one-way analysis of variance (ANOVA), followed by Tukey post hoc test. Whenever data did not meet normality, we employed nonparametric Kruskal–Wallis ANOVA followed by Dunn’s post hoc analysis. Comparisons between two dependent groups (light–dark test) were made using McNemar test. Statistical significance was defined as a value of p < 0.05. GraphPad Prism 6.0 was used to conduct the analysis (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results

3.1. Cork Granules and TEPP Adsorption

The cork granules used as a natural adsorbent were obtained after cutting, grinding, washing, filtering, and drying cork samples. Following the adsorption approach, we prepared different water samples representing the experimental groups: Control, TEPP 0.02, TEPP 0.07, TEPP 0.3, Cork, Cork + TEPP 0.02, Cork + TEPP 0.07, and Cork + TEPP 0.3.

3.2. Toxicity of TEPP and Cork + TEPP on the Viability of Embryos

In order to evaluate the impact of TEPP during zebrafish development, a study was conducted in which fertilized eggs were subjected to continuous exposure to varying concentrations of TEPP. Subsequently, the survival of the resulting embryos was assessed and quantified, as shown in Figure 1. TEPP significantly reduced embryo viability from 72 hpf at 0.02 and 0.07 µmol·L⁻¹, in comparison to the control (Figure 1A,D,E); interestingly, no alterations were observed at 0.3 µmol·L⁻¹ (Figure 1F). The application of DCA resulted in decreased embryo viability at 96 hpf, with a mortality rate of 26.05 ± 0.72%, as described in the literature [29]. The cork water derived from cork granules, which was used in the TEPP adsorption tests, exhibited high abnormality rates between 24 and 96 hpf (Figure 1A,C). Cork + TEPP, at all three tested concentrations, also increased abnormality rates (Figure 1A,G–I).

Additionally, the hatching rates were checked when embryos were exposed to TEPP, Cork, or Cork + TEPP until 96 hpf of treatment, and compared them to a negative control (Figure 2). The exposure to TEPP at concentrations of 0.02 and 0.07 µmol·L⁻¹ inhibited the hatching high after 72 hpf compared to the control (Figure 2C,D). However, at a dose of 0.3 µmol·L⁻¹ (Figure 2E), the hatching rate was found to be identical to that of the control group. The exposure to the cork solution also resulted in a reduction in the hatching rate at 96 hpf (Figure 2B). Also, it can be observed that the combination of Cork and TEPP did not attenuate the Cork-induced toxicity, as depicted in Figure 2F–H. The DCA (positive control) anticipated the occurrence of embryo hatching at 72 hpf in comparison to the untreated group (Figure 2A).

3.3. Effects of TEPP and Cork + TEPP on the Developmental Abnormalities

The parameters of abnormality were assessed in developing zebrafish [21] exposed to TEPP at 0.3 µmol·L⁻¹ or to TEPP + Cork at various concentrations, with measurements conducted at 24, 48, 72, and 96 hpf. Non-detachment of the tail, absence of somite formation or heartbeat, pericardial or yolk sac edema, cranial/mandibular/maxillary deformities, absence of eyes, spinal curvature (scoliosis), and failure of swim bladder inflation were detected in all groups studied. Furthermore, the impact of TEPP in various concentrations, DCA, Cork, or Cork + TEPP on the rate of development was examined at 24, 48, 72, and hpf of treatment. The results indicated that neither DCA nor TEPP at 0.3 µmol·L⁻¹ had any significant effect on the development velocity (Figure 3A,B). However, it should be noted that the use of Cork solution and Cork + TEPP at a concentration of 0.02 µmol·L⁻¹ resulted in a significant delay in the growth of zebrafish larvae (Figure 3C and Figure 3D, respectively). Cork and TEPP, at concentrations of 0.07 and 0.3 µmol·L⁻¹, exhibited a delay in development until 72 hpf, but were comparable to those of the control group at 96 hpf (Figure 3E and Figure 3F, respectively).

3.4. Toxicity of TEPP and Cork + TEPP on the Viability of Zebrafish Juveniles

At all tested concentrations, the application of TEPP and cork solution did not result in statistically significant changes in survival rates compared to the control group (Figure 4A). However, survival rates decreased in a concentration-dependent manner when cork was combined with TEPP, as shown in Figure 4A. Survival rates of juvenile zebrafish dropped by 62.5% and 87.5% when exposed to TEPP + Cork at concentrations of 0.07 and 0.3 µmol·L⁻¹, respectively, compared to the control or cork-only groups. Biometric parameters of surviving zebrafish were assessed only in the TEPP and cork groups and subsequently compared to the control. No significant differences were observed in weight (Figure 4B), total length (Figure 4C), or standard length (Figure 4D) following TEPP or cork treatment.

3.5. Effects of TEPP and Cork on Behavior Fish (Light–Dark Environment Preference)

TEPP increased preference and spent more time in the dark environment in a concentration-dependent manner (Figure 5A,B). TEPP at 0.02 µmol·L⁻¹ showed 33% of zebrafish in the dark environment in contrast to the control group that showed preference for the light environment and 11% of zebrafish in the dark environment (Figure 5A,B). Interestingly, the zebrafish treated with Cork solution significantly preferred and spent more time in the dark environment (Figure 5A,B). In addition, only TEPP at 0.3 µmol·L⁻¹ and Cork solution significantly reduced the number of midline crossings (Figure 5C).

3.6. Effects of TEPP and Cork on Behavior Fish (Swimming Endurance Test)

The application of TEPP at a concentration of 0.3 µmol·L⁻¹ resulted in a significant increase in SEI compared to the control group. This increase was observed in a concentration-dependent manner, as shown in Figure 5D. In contrast, the use of cork solution led to a marked reduction in swimming resistance, with a mean SEI of 5.5 ± 1.5, compared to the control group, which exhibited a substantially higher resistance of 18.8 ± 2.5 (Figure 5D).

4. Discussion

The Zebrafish (Danio rerio) is widely recognized as a remarkably effective model for investigating a variety of substances and environmental conditions that may cause toxicity [30,31]. This is due to its ecological significance, since pollutants tend to flow and accumulate in watercourses and basins, so affecting their chemical and physical properties [31]. The use of zebrafish in pesticide toxicity studies to capture data on the types of pesticide used, classes of pesticides, and zebrafish life stages associated with toxicity endpoints and phenotypic observations has been widely reported in the literature [32]. In this study, we found that TEPP, a highly toxic organophosphorus insecticide commonly used in agriculture and domestic settings [5,6,33], decreased embryo viability after 72 hpf in a concentration-dependent manner (Figure 1). However, it did not affect the developmental rate or morphology of viable embryos. TEPP also altered behavioral parameters in juvenile zebrafish, including light–dark environment preference and swimming endurance. Additionally, we investigated the properties of cork granules as a natural adsorbent for the removal of TEPP diluted in zebrafish water and evaluated the TEPP-induced toxicological effects on embryo and juvenile development and behavior. Surprisingly, water derived from cork granules used in the adsorption procedures exhibited toxic effects, reducing embryo viability, delaying development, and significantly altering behavioral responses in juvenile zebrafish.

TEPP at concentrations ≤0.07 µmol·L⁻¹, after 72 hpf of exposure, increased embryo mortality, but interestingly, no alterations were observed at 0.3 µmol·L⁻¹ (Figure 1). Likewise, juvenile zebrafish exposed to lower concentrations of TEPP showed increased preference for and spent more time in the dark environment compared to the control group (Figure 5). Zebrafish exposed to 0.02, 0.07, and 0.3 µmol·L⁻¹ spent 125.0 ± 32.3, 93.5 ± 40.9, and 18.6 ± 8.2 s, respectively, in the dark environment, compared to the control group, which spent 53.0 ± 13.5 s. Our findings suggest that TEPP exhibits a typical inverted U-shaped dose–response effect, a nonlinear relationship frequently reported in studies examining both negative and positive effects of pharmacological and non-pharmacological agents [34]. This pattern has also been used to describe the actions of cholinesterase inhibitors such as MF201 and MF268, which improve performance at low doses but are ineffective or even detrimental at higher ones [34,35,36,37,38]. TEPP-induced toxicity is explained in part by excessive stimulation of the central nervous system due to irreversible inhibition of AChE activity [4]. Given that AChE inhibition is widely accepted as the primary mechanism of action of TEPP, we have suggested that their changes in the development and behavior of zebrafish follow a dose–response curve that appears like an inverted U shape, similar to other cholinesterase inhibitors. Notably, behavioral changes occurred at lower concentrations and without associated morphological abnormalities, indicating behavioral assays as highly sensitive early indicators of neurotoxicity induced by TEPP exposure. This aligns with the literature suggesting zebrafish behavioral responses as valuable predictive tools for environmental neurotoxicological assessments [31,39].

EPP-induced toxicity was studied in vitro using the neuronal PC12 cell line, and it was demonstrated that TEPP reduced cell viability at concentrations higher than 10 µmol·L⁻¹ after 48 h of treatment [18]. In addition, other studies have reported the inhibitory effects of TEPP on commercial AChE [4,18], and on brain AChE from various tropical fish species, with IC₅₀ values (concentration required to inhibit 50% of enzyme activity) greater than 20 µmol·L⁻¹ [40]. In contrast, the toxicological effects of TEPP on zebrafish embryo development and juvenile behavior were observed at concentrations below 0.07 µmol·L⁻¹, which is at least 25 times lower than the concentrations producing effects in vitro. This indicates that the zebrafish model, which includes larvae at different developmental stages and juveniles, is more sensitive and informative than in vitro methods. Zebrafish have been recognized as a valuable biological platform for environmental risk assessment and as a sensitive model for evaluating the toxicity of organophosphates, pyrethroids, azoles, and triazine-class pesticides [32].

The efficacy of cork granules as natural adsorbents for pesticide removal from water has been examined through biochemical and neuronal cell culture approaches [11,18]. However, no studies using in vivo models have been reported to date. Cork is a porous material composed of prismatic cells arranged in a honeycomb structure [15]. Water forms clusters around hydrophilic sites on the porous surface and diffuses into the cell wall, leading to swelling of the material [41]. Physicochemical interactions and specific binding sites between pesticide molecules and cork determine the extent to which pesticides are retained and how effectively they adhere to the cork surface [10,42]. In this study, we were also motivated to investigate the efficiency of cork granules in removing TEPP from reconstituted water used for zebrafish maintenance, and to assess the potential impacts on embryo development and juvenile behavior.

Unfortunately, the water derived from cork granules after the adsorption experiments induced toxic effects in both embryos and juvenile zebrafish. Exposure to cork water resulted in a high number of abnormal and dead larvae between 24 and 96 hpf, a decrease in hatching rate, and a delay in larval development. In juvenile zebrafish, cork water did not affect viability, weight, total length, or standard length. However, it increased preference for the dark environment and led to a longer time spent in darkness, along with a reduction in midline crossings and swimming endurance. Despite these findings, it is important to note that cork water did not alter neuronal PC12 cell viability in our previous study [18]. Our data suggest that zebrafish, when tested at different developmental stages, exhibited increased vulnerability to toxic substances compared to in vitro models. Additional investigation is needed to achieve a comprehensive understanding of the effects of toxic chemicals present in cork water, which is generated using cork granules obtained from commercially available wine stoppers. Previous studies reported cork as capable of releasing phenolic compounds, tannins, and even organochlorine pollutants originating from wine processing or environmental sources, potentially explaining the observed in vivo toxicity [43]. The minimal cytotoxicity previously noted in PC12 cells [18] contrasts sharply with the pronounced toxic effects in zebrafish embryos and juveniles, highlighting differences in sensitivity between in vitro and in vivo models. Future studies employing analytical techniques like GC-MS analysis of cork water filtrate are necessary to conclusively identify these toxic substances.

In previous in vitro assays, cork granules adsorbed over 95% of TEPP diluted in water, thereby reducing its inhibitory effects on AChE activity in both the commercial enzyme from electric eel (Electrophorus electricus) and in the neuronal PC12 cell culture medium [18]. However, the current investigation revealed that the use of cork granules as an adsorbent for removing TEPP from water used in zebrafish assays was ineffective, as toxic effects were observed in embryos and juvenile zebrafish exposed to cork water. Interestingly, Cork + TEPP at higher concentrations appeared to mitigate the toxic effects induced by cork alone, particularly improving hatching rates at 72 hpf and reducing developmental delays at 96 hpf in a concentration-dependent manner (Figure 2). On the other hand, Cork + TEPP at higher concentrations significantly increased juvenile zebrafish mortality (Figure 4). The underlying differences in the response of embryos and juvenile zebrafish to the combination of TEPP and cork remain unclear and warrant further investigation.

5. Conclusions

Our findings suggest that the TEPP pesticide changed the viability of the embryos and the behavior of juvenile zebrafish in a concentration-dependent manner, presenting a typical inverted U-shaped dose–response curve. TEPP also altered the preference for light or dark environments and the swimming endurance of juvenile zebrafish, without affecting their viability or biometric parameters. Although wine cork granules efficiently adsorbed TEPP in previous in vitro assays, their use as a natural adsorbent in aquatic settings was ineffective in vivo, as demonstrated by increased abnormality and mortality rates, delayed larval development, and behavioral stress in juvenile zebrafish. This highlights significant discrepancies between in vitro and in vivo outcomes and underscores the importance of conducting comprehensive toxicological assessments in relevant biological models.