The Cortisol Levels, Histology, and Fine Structure of Various Tissues of Fish Gambusia affinis (Baird and Girard, 1853) after Exposure to Lead

Background Aquatic organisms demonstrate a high vulnerability to mortality when exposed to Pb, even at low concentrations. The objective of this investigation is to ascertain the histopathological alterations and cortisol concentrations in diverse tissues of Gambusia affinis, with a specific focus on the eggs and larvae, following exposure to varying concentrations of PbCl2. Methods Adult specimens of G. affinis measuring 5-6 cm in length were obtained from a commercial fish breeding facility. A total of 8 fish with a 1 : 1 ratio of 4 pairs of broodstock were placed in an 8-liter aquarium. Following the adaptation phase, the broodstock underwent a spawning process that lasted for a duration of 7 days. Throughout the spawning process, assessments were conducted on the progression of the abdominal growth of the broodstock. Eggs ready to hatch and Gambusia larvae were taken and exposed to 0.1 mg/L PbCl2, 1 mg/L PbCl2, and control (without PbCl2) for 24 hours, with three replications. At the end of the experiment, histopathological analysis was conducted using the hematoxylin Ehrlich-eosin staining method and scanning electron microscopic (SEM) observation. The levels of Pb in gills were determined by employing atomic absorption spectrophotometer. The cortisol concentration in organ samples of fish was determined through the utilization of a cortisol ELISA Kit. Results The findings of this investigation demonstrated an important bioaccumulation occurrence of Pb within the gills of Gambusia fish that were specifically subjected to 0.1 and 1 mg/L PbCl2. The histological structures of eggs and larvae that were subjected to PbCl2 exhibited impairment in comparison to the control group. The present study observed a significant elevation in cortisol levels among fish specimens that were subjected to PbCl2 exposure. Conclusions The findings of this investigation suggest that the occurrence of Pb is linked to a rise in cortisol concentrations in various organs of G. affinis larvae. Furthermore, the research indicates that the exposure to Pb has a notable impact on the histological alterations in the eggs and larvae of Gambusia fish, implying that they are undergoing stress as a result of the Pb exposure.


Introduction
Pollution in aquatic environments is a growing problem that needs immediate attention.Typically, increased domestic, agricultural, and industrial activities are responsible for this phenomenon.Heavy metals are regarded as environmental pollutants due to their toxic nature and propensity to accumulate through biological processes [1][2][3].Additional characteristics of heavy metals include their propensity to precipitate, their capacity to be assimilated into sedimentary particles, and their ability to remain suspended in aqueous solutions.Metals can be absorbed by fsh through their gills and skin, and/or by ingestion through their diet, resulting in their accumulation and potential toxicity [4,5].Te intensity of bioaccumulation and its toxicity can be infuenced by various environmental factors such as temperature, oxygen levels, pH, and water hardness.
Lead (Pb) is classifed as a hazardous heavy metal that possesses a high propensity for absorption and accumulation [6][7][8].Pb is an inherent constituent of the environment and is typically present in trace quantities within the soil, fora, and aqueous systems.Pb has been detected in aquatic environments, and its concentration has been observed to rise as a result of human activities such as producing batteries, painting, and cement manufacturing [9][10][11].Te results of investigations conducted by Dimari et al. [12], Hasan et al. [13], and Odey et al. [14] in various lakes and streams have revealed the presence of elevated concentrations of lead (Pb) in the water, sediment, and aquatic organisms, including fsh.Te rising amount of Pb-containing waste in aquatic environments leads to an increase in Pb contamination.Tis substance has a propensity to form diverse chemical bonds with oxygen and sulfur groups found in protein molecules.Te increased ability of these elements to form stable complexes increases the bioavailability of lead within proteins.Hypocalcemia may result from the presence of lead in biota, such as fsh.Te high afnity of Pb for Ca 2+ ATPase, Na + /Ca 2+ substitution, and Na + /K + ATPase results in the inhibition of the ionocyte lateral transport mechanism in the gill epithelium [1].Tese efects can disrupt electrochemical gradients and the regulation of ions.Due to bioaccumulation, exposure to Pb can be lethal for aquatic organisms, even at low concentrations [12][13][14].Pb can disrupt antioxidant equilibrium, cause oxidative stress disorders, and change behavior [15][16][17][18].In vivo experiments on the efects of Pb exposure in fsh have examined the augmentation of antioxidant responses by inducing reactive oxygen stress (ROS), the toxic efects on membrane structure and function due to Pb's high afnity for red blood cells, and the changes in fsh immune systems [5,[19][20][21].Gambusia afnis, commonly known as mosquito fsh, is one of the preferred fsh species utilized as a biomonitoring tool in contaminated water bodies.
Several studies [22][23][24][25] have previously investigated the levels of stress in fsh and biota aquatic exposed to heavy metals.Plasma cortisol levels were elevated in twomonth-old Oreochromis mossambicus fsh exposed to Cu but not in fsh exposed to the combination of Cu and Cd [26].Adult O. mossambicus exposed to Cd for 2, 4, or 14 days showed a substantial increase in plasma cortisol levels [27].During the exposure phase, the level of cortisol produced by adult Oreochromis sp.treated with Pb or As was less than that of the control group [22].Cu-exposed rainbow trout (Oncorhynchus mykiss) did not exhibit a stress response to cortisol [26,28,29], whereas Cr exposure induced deleterious efects on the stress indicator cortisol [30][31][32] observed that zebrafsh (Danio rerio) experienced stress in response to a sudden change in temperature.Meanwhile, research on the efects of Pb exposure [22,33] indicates that Pb interferes with endocrine function by altering the pattern of synthesis and metabolism of cortisol and growth hormone (GH) in adult Orechromis sp. and Cyrpinus carpio.Other studies [26,27,34] found that the gills of Atlantic salmon (Salmo salar) were unafected by Cd exposure via fsh diet.Limited research has been undertaken concerning the impact of Pb on the early stages of fsh development, particularly in relation to fsh eggs and larvae.Te objective of this study was to evaluate cortisol levels in diferent organs of fsh larvae of Gambusia afnis.Te purpose was to evaluate the stress levels experienced by the larvae following exposure to Pb.Furthermore, histological and fne structural examinations were conducted on these essential organs.

Adaptation of Fish to Laboratory Conditions.
Te mosquito fsh (Gambusia afnis) broodstocks, measuring 5-6 cm in length, were obtained from the Punten Fish Seed Center (PFSC) located in Batu, East Java, Indonesia.Te PFSC water quality consisted of the following characteristics: temperature of 23-26 °C, pH of 6.5-7, and lead concentration of less than 0.002 mg/L.Te mosquito fsh broodstock were reared in 60-liter tanks within the laboratory setting.During the adaptation period, Gambusia broodstock were given food with fsh pellets twice daily for a duration of 14 days.Additionally, daily water changes were conducted, amounting to 20% of the total water volume.Each aquarium was stocked with eight broodstock fsh, maintaining a 1 : 1 ratio of four pairs of broodstock in an 8-liter tank.Troughout the acclimatization and testing periods, the temperature, pH, and dissolved oxygen levels were continuously monitored in accordance with the guidelines established by Berlinsky et al. [35].Te temperatures ranged from 27 to 29 °C, the pH was between 6 and 7, and there was more than 4 mg/L of dissolved oxygen.

Spawning and Pb
Exposure.Following the adaptation period, the broodstock undergo spawning in specialized tanks for approximately seven days.Troughout the spawning process, the progression of the female parent's abdomen development was monitored.Following a period of 7 days, a surgical procedure was conducted on the distended abdomen of the female parent.Before the surgical procedure, the fsh specimens were subjected to anesthesia using 0.2 mL of clove oil [36].Subsequently, the surgical intervention was carried out, and the eggs and larvae were cautiously extracted.After that, eggs that were in a state of readiness to hatch and larvae of Gambusia were subjected to varying concentrations of PbCl 2 for a duration of 24 hours.
Te concentrations utilized in the present investigation were selected based on the fndings of Al-Kshab and Yehya [37], who documented that adult Gambusia fsh exposed to PbCl 2 exhibited LC 50 24-h and 96-h values of 59 and 50 mg/L, respectively.Due to the use of Gambusia fsh eggs and larvae as research subjects, the concentrations of PbCl 2 (Merck, Darmstadt, Germany) used in this study were 0.1 mg/L (A) and 1 mg/L (B), while the control group 2 Scientifca contained no PbCl 2 .Ten eggs and ten larvae were separately introduced into separate 1-liter glass jars, which were flled with 500 ml and 750 ml of experimental media, respectively.Te study was conducted using three replicates.Te experiment lasted for 24 h.Te larvae were then euthanized by rapid freezing and followed by rapid freezing in liquid nitrogen to stop any enzymatic activity [38].Te eggs were quickly processed for scanning electron microscopy examination.Te present study was carried out in accordance with the Institutional Animal Care guidelines and procedures of Brawijaya University (number 958-KEP-UB).

Pb Analysis.
In order to determine the levels of metal present in the water used for metal treatment and gills of Gambusia fsh, measurements were taken.A total of 200 mL water sample was put into a 250 mL beaker containing 2 mL of 1% ammonium pyrollidine dithiocarbamate (APDC), pH was set to 4, and heated to boiling.After cooling down to room temperature, place in Erlenmeyer fask, and add 7 mL of methyl isobutyl ketone (MIBK), then shake with a shaker for 20 minutes.Te solution was put into a separatory funnel and left for 20 minutes.Organic layer (top) was taken and placed it in the Erlenmeyer fask.For re-extraction, 5 mL of 4N HNO 3 was pipetted and added and then stirred for 20 minutes.Later, the mixture was put into a separatory funnel until the boundaries were found (±20 minutes).Te bottom layer (acid layer) was taken and analyzed with Atomic Absorption Spectrophotometer (AAS) Shimadzu AA-6800 [39].Te similar procedure was applied for standard solutions of the metal being analyzed.Te purpose of preconcentration of the sample by the solvent extraction method is to separate the metal ions which are determined in the measurement with the AAS [40].CASS-6, a certifed reference material (CRM) of seawater provided by the National Research Council, Canada (NRCC), was used to check the accuracy of the measurements and showed good recoveries (91%).Before measuring the level of Pb in gills of Gambusia fsh, the fsh gill specimens were subjected to oven-drying at a temperature range of 90-100 °C for a duration of 12 hours.Subsequently, the dried samples were pulverized and weighed, resulting in 0.5 g.To establish a control, introduce 0.25 ml of standard solution containing 1 mg/L of Pb to the sample prior to its placement in the ashing furnace.Subsequently, subject the sample to steam with a hot plate until it is completely dry.Introduce the sample into the ashing furnace.Te recommended process is to gradually raise the temperature by 100 °C every 30 minutes until it reaches 450 °C, and then maintain it at that level for a duration of 18 hours.Te process of measuring Pb in water was applied to the examination of fsh gills.Te analytical performance metrics such as accuracy and precision were assessed through the measurement of those of the dogfsh muscle reference material (DORM-4) that was supplied by the NRCC.Te reference material of the NRCC was subjected to identical measurement techniques as those used for the samples.Te metal recovery acquired from the analysis of certifed reference material (DORM-4) was 92%.

Histopathological Analysis.
Te preparation was conducted subsequent to the conclusion of the PbCl 2 exposure duration.Te procedure for preparation uses conventional techniques as described in [41].Initially, the fsh eggs, larvae, and gills were promptly immersed in a 10% phosphate-bufered formalin (NBF) fxative and allowed to incubate for a duration of 24 hours.Afterwards, they were rinsed with 70% alcohol.Following fxation, the tissue undergoes processing with parafn, which endeavors to preserve the integrity of cells and tissues, thereby facilitating the embedding process.Subsequent to the tissue embedding procedure, the resultant blocks were subjected to tissue sectioning utilizing a microtome with an incision thickness ranging from 3 to 5 µm.Te stretched cut ribbon underwent a process of being immersed in a water bath maintained at a constant temperature of 40 °C.Te objective lens is utilized to immerse the tape in a water bath and subsequently subject it to a one-hour drying period.Subsequently, the tissue slides were subjected to a clearing process using a xylol solution.Following this, the slides were allowed to dry and subsequently stained using the hematoxylin Ehrlicheosin staining method.Finally, the slides were mounted with entellan and covered with a cover glass.Te study involved the utilization of an Olympus CX33 microscope and the EPview 1.2 application (Build 19853) for observation purposes.

Scanning Electron Microscopic (SEM) Observation of
Fish Eggs and Larvae.Te preparatory phases preceding SEM observation are crucial in achieving optimal image quality outcomes and facilitating accurate analysis.It is imperative that any treatments administered during the sample preparation process do not alter the inherent structure of the sample, in order to ensure that the observations obtained through SEM accurately refect the original structure of the sample.Te initial stages of preparing biological samples for observation via SEM involve several crucial steps, namely, orienting the sample to the desired position, fxing it in place, dehydrating it, drying it, and fnally, applying a conductive coating.A modifcation of this method has been proposed by the authors in [42].Te fxation stage is undertaken to maintain the original structure of the sample and prevent any potential collapse or fragility.Te fxation process generally comprises two discrete phases.In the frst phase, glutaraldehyde and cacodylate bufer were employed, followed by the application of osmium tetroxide in the bufer during the subsequent phase.
Te process of dehydration involved the removal of water molecules from the sample.Te process of dehydration was executed through submersion in alcohol, wherein the concentration level was incrementally augmented until it attained a maximum of 100%.Te process of Scientifca drying was executed through the utilization of critical point drying (CPD) as described in [43].Additionally, hexamethyldisilazane was used to eliminate the liquid component of the sample while avoiding any potential defation of the sample.Te process of applying conductive coatings can be achieved through the utilization of a sputtering apparatus in conjunction with gold as conductive material.Te study involved the examination of various types of samples, including conductive samples and biological samples that were both prepared and unprepared.Te observation was conducted using a specialized mode of observation known as variable pressure scanning electron microscopy (VP-SEM).Te Hitachi SU3500 scanning electron microscope was utilized for observations at the Biology Research Center, facilitated through the Science E-Service Application (ELSA) provided by the National Research and Innovation Agency (BRIN).

Cortisol Levels Measurement.
Te present study used a standardized protocol [44] utilizing a Cortisol ELISA Kit (ab108665), United States, to measure the concentration of cortisol in various organ samples of fsh, including gills, gonads, meat, liver, eyes, and fns.To prevent the impact of washing, it is recommended to increase the volume of washing solution from 300 µL to 350 µL and perform washing steps three to fve.Duplicate all assays for standards, controls, and samples.Ensure that all reagents, working standards, and samples are prepared according to the ELISA Kit instructions.Eliminate surplus microplate strips from the plate frame, restore them to the foil pouch that encloses the desiccant pack, seal again, and put them back into storage at a temperature of 4 °C.Subsequently, 20 µL of standard, control, or sample was introduced into their corresponding wells, followed by the addition of 200 µL of cortisol-HRP conjugate to each well.A blank well was left to accommodate the substrate material.Te wells were covered with the foil provided in the kit.Te sample was subjected to incubation for a duration of one hour at a temperature of 37 °C.Upon completion of the incubation process, the foil was removed and the contents of the wells were aspirated.Subsequently, each well was washed thrice with 300 µL of diluted washing solution, which prevented the occurrence of overfow in the reaction wells.It is recommended that the duration of the soak time between consecutive wash cycles should exceed 5 seconds.Subsequently, the residual fuid was meticulously eliminated by gently tapping strips onto tissue paper prior to proceeding with the subsequent stage.100 microliters of TMB substrate solution was introduced into all wells.Te sample was subjected to incubation for precisely 15 minutes under ambient conditions in the absence of light.Subsequently, 100 µL of stop solution was introduced into each well in a consistent manner and sequence as that of the TMB substrate solution, which gently agitated the microplate.Te emergence of a blue hue during the incubation period results in a subsequent transformation into a yellow hue.Te absorbance of the sample was measured at a wavelength of 450 nm, using an automatic ELISA Microplate Reader Epoch (Biotek), within a timeframe of 5 minutes following the addition of the stop solution.

Data Analysis.
A descriptive analysis was conducted on the histochemistry and SEM observations of a number of diferent organs.Te bioaccumulation of lead in gills and cortisol levels in various organs were statistically analyzed using analysis of variance (ANOVA).Subsequently, the Tukey HSD post hoc test at a signifcance level of 5% should be conducted to determine the diferences between each treatment.Te statistical analysis of the level of cortisol found in various organs, as well as the levels of lead found in the gills and the water used as treatment, are reported in the supplementary data.

Results
Te results of exposure to Pb concentrations in this study indicated the presence of a bioaccumulation process in the gills of the model fsh, Gambusia.Table 1 presents a summary of the lead accumulation in the gills as well as the lead concentration in the medium.Tere were statistically signifcant diferences (p < 0.005) between each treatment and media.
3.1.Histological Observations.Histological impairment was observed in fsh egg, larvae, and gills.A discernible alteration can be shown in the results of the analysis conducted on fsh eggs (Figure 1), the assessment carried out on fsh larvae (Figure 2), and the examination performed on fsh gills (Figure 3).
Figure 1 shows the progression of egg cell division, specifcally during phases I-III of vitellogenesis, which can be identifed by the emergence of vacuoles.PbCl 2 -exposed egg cells (A) exhibited irregularly shaped vacuoles, resulting in a signifcant diference between treatments A and B. Te proper positioning of the vacuole on the periphery of the egg is crucial in the process of vitellogenesis.Exposure to PbCl 2 results in a formation of vacuoles, which remain localized in the central region of the egg.In contrast to the control group (B), it is evident that the development characterized by typical vacuoles is situated at the periphery of the egg.
A discernible variation between the Pb exposure treatment (A) and the control (B) is evident in the larval development process (Figure 2).Te exposure of fsh larvae to Pb resulted in an incomplete development of the eyes, characterized by the presence of imperfect spheres.Similar to the progression of yolk development, it was expected that the vacuole within the egg cell would undergo nuclear transformation.However, it has been observed that vacuoles persist within the yolk of the larvae.In contrast to the control fsh larvae, it appears that the morphogenesis of ocular cells exhibits a perfectly spherical shape.Similarly, during the process of yolk development, the presence of vacuoles is limited due to the formation of a nucleus.4 Scientifca

Scientifca
Diferences were observed in the gills of fsh larvae that were exposed to PbCl 2 (A) in comparison to the control group (B).Te fndings from the examination of fsh larval gills that were subjected to PbCl 2 (A) exposure indicate that both the primary lamellae (LP) and secondary lamellae (LS) experienced a process of swelling.In contrast, the gills of the experimental fsh (A) exhibited no alteration in the LP and LS.

SEM Observation.
Te SEM observations provided additional elucidation on the structural alterations resulting from PbCl 2 exposure in fsh eggs, as depicted in Figure 4. Te observations of fsh larvae and fsh gills are presented in Figures 5 and 6, respectively.
Te fndings obtained from histological analysis using HE staining are consistent with those derived from scanning electron microscopy, indicating that egg cells are undergoing vacuolization.A noticeable contrast was observed between treatments A and B. Specifcally, the egg cells that were subjected to PbCl 2 (A) exhibited an irregular shape in their vacuoles.Te localization of vitellogenesis vacuoles in the egg should be peripheral during its development.Exposure to PbCl 2 (A) results in the accumulation of vacuoles, which remain localized on a single side of the egg.In contrast to the control group (B), it appears that the development of egg cells is normal with no vacuole accumulation.
Diferences were observed between the PbCl 2 treatment (A) and the control (B) in terms of larval development, as indicated by the results of SEM observations.Te exposure of fsh larvae to PbCl 2 resulted in the development of ocular structures with nonuniform circularity.Tey have a tendency to exhibit an oval shape and an asymmetrical enlargement on one of their sides in contrast to the control group, who exhibited ocular development characterized by a more optimal spherical morphology.However, the development of the egg yolk appears to difer from the results obtained through HE histology, which clearly demonstrate variations in the yolk.
SEM observation of the gills of fsh larvae revealed distinct morphological variations between the larvae that were subjected to PbCl 2 (A) and the control group (B).Te gills of fsh larvae A exhibited swelling in LP and LS, similar to the histological distinctions observed through HE staining.Conversely, the gills of fsh B did not display any alterations in LP and LS.Te gills of the control fsh exhibited typical developmental patterns.

Measurement of Cortisol Levels.
According to the cortisol biomarker analysis, it was determined that the stress level of all fsh organs exposed to PbCl 2 was signifcantly higher than that of the control group.Te cortisol levels of fsh organs in treatments A and B were found to be nearly equivalent (Figure 7).

Discussion
Metals are released into the aquatic environment and subsequently absorbed by fsh through their gills [45].Te metals subsequently accumulate in the fsh organs after fowing via the circulatory system, whilst certain metals are eliminated via the renal and branchial excretory systems [46][47][48][49].Pb is considered to be one of the most accumulative metals due to its capacity to bind with oxygen and sulfur atoms in complex proteins.As a result, it is classifed as a toxic metal [50][51][52].
Exposure to Pb has the potential to cause damage to reproductive organs.Research has demonstrated that the presence of pollutants can impede the growth and maturation of reproductive cells in zebrafsh (Danio rerio), with a particular impact on primordial germ cells [53][54][55].It has been documented that the exposure to pesticides of the organophosphate type can impede the production of estrogen hormone, thereby disrupting the vitellogenesis process [56][57][58].Comparable results were also found in this study, as shown by the HE staining shown in Figure 1 and the results of the SEM observation shown in Figure 4, respectively.Te presented fgures demonstrate an interruption in the vitellogenesis process, characterized by anomalous vacuole formation, which can be attributed to the exposure to Pb. Te fndings of Ansari et al. [59], Aleström et al. [60], and Parsons [61] indicate that interference with vitellogenesis leads to a reduction in the size of fsh eggs and larvae.Te exposure to Pb has a persistent impact on the eggs of fsh, which subsequently translates into the morphology and physiology of the resulting fsh larvae.Te results of this study, as demonstrated by the HE staining in Figure 2 and the SEM images in Figure 5, indicate the presence of malformations in fsh larvae.Exposure to Pb can result in enlarged eyes and noncircular eyelids.Te zebrafsh (Danio rerio) larvae exposed to Hg exhibit various malformations and abnormalities, including pericardial edema, spinal curvature, and fn edema [47,62,63].Te exposure of fsh to Pb has been found to interfere with the spawning process and result in an increase in morphological abnormalities in fsh sperm [45,64,65].Additionally, Pb that passively difuses into the egg yolk has been shown to inhibit the activity of embryo growth enzymes [57,66,67].Moreover, it has been demonstrated that Pb exposure can impede the embryogenesis process in goldfsh (Cyprinus carpio), resulting in blastula defects, spinal curvature, and heart defects [60,68].
Even though the organism is able to withstand the initial impact of the pollutant, prolonged exposure causes its selfdefense mechanisms to become less efective [8,69].Te ability of the body to tolerate pollution, even at low quantities, can decline with age.Furthermore, metal toxicity is afected by the organism's sensitivity to the metal, environmental factors, and the metal's excretion and retention rates [51,52,58].Te accumulation of metals in fsh tissues depends on several factors, including the concentration and duration of exposure, as well as additional variables such as temperature, age, interaction with other metals, water chemistry, and metabolic activity of the fsh [3,14,70].Lead is not considered an essential element for biological processes.Human activities may contribute to the release of excessive levels of lead into nearby water sources [1,46].
Te toxicity of Pb is evident even at low concentrations, and its involvement in biochemical processes remains 6 Scientifca unclear [14,37].It has been observed that fsh are capable of accumulating heavy metals in their tissues at a higher concentration than the surrounding environment.Tis is achieved through the absorption processes that occur along the surface of their gills and kidneys, liver, and walls of the intestinal tract [8,12,71,72].Tis study demonstrated the presence of lead in the gill tissue of G. afnis.Te fndings indicate that there was a discernible mechanism of Pb uptake in the gills, which resulted in the enlargement of both the primary and secondary lamellae.According to Usman et al. [1], the impairment of gill lamellae may lead to disturbances in ion regulation within the gills, which can ultimately afect the homeostatic mechanisms responsible for maintaining ion levels.Te gills serve as a crucial point of entry for heavy  metals and are typically the primary organ afected by metal exposure in fsh [3,[73][74][75].According to Mahboob et al. there was a higher concentration of lead detected in the gills of Nile tilapia (Oreochromis niloticus) compared to the kidneys, liver, and muscles.Te high accumulation of metals in the gills has been attributed to the formation of metalmucus complexes [77][78][79].Te concentration of metals in the gills of fsh serves as an indicator of the corresponding metal levels in the surrounding aquatic environment [80][81][82].Tus, fsh gills are frequently suggested as an environmental indicator organ for fsh.
In addition to organ damage, exposure to heavy metals can also result in elevated stress levels [47,59].Te activation of the hypothalamus-pituitary-adrenal (HPA) axis is a commonly observed mechanism for efectively responding to stressors [62,83,84].Cortisol is the primary corticosteroid synthesized by the hypothalamic-pituitary-adrenal (HPA) axis in the majority of mammals and fsh [83,85].Cortisol secretion in response to stress is infuenced by a number of stimuli, including toxin exposure, transport, and shock.Corticosteroids primarily induce the mobilization of energy reserves [86] via glycolysis and gluconeogenesis, thereby facilitating the achievement of the organism's energetic demands in response to the particular conditions [87].Te results of the study demonstrate an increase in cortisol levels as a result of Pb exposure.Fish gills, eyes, fns, liver, gonads, and muscle have signifcantly different cortisol concentration increases, as shown in Figure 7. Cortisol concentrations in these organs correlate positively with Pb concentrations in the surrounding environment.According to studies conducted by Tang et al. [22] and Ramesh et al. [33], the impact of Pb exposure has been found to interfere with endocrine function by altering the synthesis and metabolism of cortisol and growth hormone (GH).Te consequences of the mentioned exposure may have negative efects on the health of fsh.Previous research conducted by Pelgrom et al. [26], Pratap and Bonga [27], and Dang et al. [34] has suggested that the ingestion of Cd by Atlantic salmon via their dietary intake did not yield any observable efects on their gills.Interestingly, it is possible that the accumulation of Cd in the chloride cells of the gills may have led to an upregulation of metallothionein (MT) expression, which plays a crucial role in minimizing the toxic efects of heavy metals.Several studies have reported that rainbow trout did not exhibit a stress response to cortisol when exposed to Cu [26,28,29].Conversely, exposure to Cr induced toxic efects on the stress indicator cortisol in rainbow trout [30,31].Additionally, Deniro and Yusnaini [32] observed stress in zebrafsh (Danio rerio) due to temperature shock.According to several studies, exposure to temperature shock in zebrafsh results in physiological alterations in vertebrate animals, leading to a state of exhaustion as a stress response [84,88,89].Deniro and Yusnaini [32] proposed that zebrafsh exhibited behavioral responses to stress induced by temperature shock.In response to environmental stimuli, sudden shifts in the equilibrium of fsh behavior cause stress.Cortisol, an indicator of prolonged physiological stress, has been linked to the manifestation of stressful behavior [60,83,90].Tis phenomenon can be observed through the behavior of fsh when exposed to stressors; they exhibit sudden bouts of high-velocity swimming and move toward the inlet.It is postulated that under such circumstances, the piscine species exhibit elevated levels of cortisol.According to Barcellos et al. [83,91], the experimental study has demonstrated that zebrafsh exposed to temperature shock prefer the lower area to the upper area, suggesting that cortisol is involved in stress-related behavior.Cortisol in fsh is synthesized by inter-renal cells and is found in low concentrations throughout the organism.According to Gris [86], the substance is eliminated in the renal pelvis and subsequently introduced into the bloodstream.Unbound cortisol in plasma is regarded as the sole form that is physiologically active [22,27].While the presence of cortisol binding globulin (CBG) has not been identifed in fsh, various molecules that have an equivalent efect of reducing cortisol bioavailability have been documented [92].Te plasma of salmon is estimated to bind 30-55% of cortisol, with 8 Scientifca a notably higher percentage observed in females (45%) compared to males (37%).Te precise function and characteristics of these molecules that facilitate binding remain unclear [93,94].Te above discussion is really and provides an understanding of the magnitude of the efect of Pb exposure on histopathological changes and stress responses in embryonic and larval Gambusia fsh.

Conclusion
To the best of our current knowledge, this study represents the frst examination of the impact of lead exposure on fsh eggs and larvae.Specifcally, it evaluates the impact of lead on cortisol levels within diferent organs of fsh as well as the consequences of lead exposure on alterations in the morphological structure of fsh eggs and gills.According to the fndings of this study, the presence of Pb causes an increase in cortisol levels in a number of G. afnis larval organs.In addition, the study found that Pb exposure has a signifcant efect on the histological alterations of Gambusia fsh eggs and larvae, indicating that the fsh are experiencing stress due to Pb exposure.Since Pb is extremely toxic to eggs and larvae of fsh, preventing Pb from contaminating aquatic environments is regarded as one of the most signifcant actions to be taken.In order to prevent contamination of aquatic environments, it is crucial to take immediate action to ensure that future pollution and discharges of Pb in aquatic environments are minimized to the greatest extent possible.

Figure 1 :Figure 2 :
Figure 1: Observation of egg structure by HE staining.(a) Visible structure of fsh eggs exposed to PbCl 2 and (b) visible structure of control fsh eggs without exposure of PbCl 2 .V � vacuolla and bar size � 200 µm.

Figure 3 :
Figure 3: Observation of gill structure of Gambusia fsh with HE staining.(a) Swelling gill structure of fsh exposed to Pb and (b) visible gill structure of control fsh without exposure.LS � secondary lemella, LP � primary lamellae, and bar size � 200 µm.

Figure 5 :
Figure 5: Observation of the structure of fsh larvae with SEM observations.(a) Te development of ocular structures with nonuniform circularity structure of fsh larvae exposed to PbCl 2 and (b) optimal spherical morphology of ocular structures of control fsh larvae without exposure of PbCl2.E � eye, YE � yellow egg, and bar size � 500 µm.

Figure 6 :Figure 4 :
Figure 6: Observation of fsh gill structure with SEM observation.(a) Swelling gill structure of fsh exposed to PbCl 2 and (b) normal gill structure of control fsh without exposure of PbCl 2. Bar size � 10 µm, LS � secondary lamellae, and LP � primary lamellae.

Figure 7 :
Figure 7: Cortisol levels in fsh organs exposed to lead.A � 0.1 m/L PbCl 2 and B � 1 mg/L PbCl 2 .Lowercase letters denote statistically signifcant diferences (a < b < c, p < 0.05).Te sample size is N � 5, with each sample comprising a composite of two individuals.