Triphenyltin hydroxide (TPTH) exposure induces genotoxicity, cytotoxicity and developmental toxicity in Chick embryo, Gallus gallus domesticus: An in ovo study

Triphenyltin hydroxide (TPTH) exposure induces genotoxicity, cytotoxicity and developmental toxicity in Chick embryo, Gallus gallus domesticus: An in ovo study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Triphenyltin hydroxide (TPTH) exposure induces genotoxicity, cytotoxicity and developmental toxicity in Chick embryo, Gallus gallus domesticus: An in ovo study Dipsanu Paul, Sweety Nath Barbhuiya, Sarbani Giri, Dharmeswar Barhoi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9703101/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Triphenyltin hydroxide (TPTH) is a widely used agricultural fungicide and environmental pollutant well known for its potent toxicological potential. Despite these, its embryotoxic effects remain unexplored in avian models. This study presents a novel evaluation of TPTH’s acute toxicity, genotoxicity, cytotoxicity, and teratogenic potential using the in ovo chick embryo model. Fertilized chicken eggs were exposed to varying TPTH concentrations, revealing a time-dependent median lethal dose (LD 50 ) decline from 537.1 µg/egg at 24 h to 342.5 µg/egg at 96 h. Later, evaluations using 1%, 5%, and 10% of the 96 h LD 50 demonstrated profound sub-cellular damage. The Hen's egg test for micronucleus (HET-MN) assay showed a significant dose-dependent induction of micronuclei, confirming genomic instability. Additionally, a reduced polychromatic to normochromatic erythrocyte (PCE/NCE) ratio indicated severe cytotoxicity and suppressed erythropoiesis. Morphometric assessments revealed teratogenic alterations, including failure of heart tube C-looping, microcephaly, cranial oedema, microphthalmia, localized haemorrhaging, and concentration-dependent growth retardation across body length, eye diameter, and limb elongation. These findings demonstrate that TPTH induces potent developmental toxicity through a lethal cascade of genotoxic, cellular damage and structural malformations, highlighting its extreme embryotoxic risks to avian populations and associated ecosystems. Developmental Biology Molecular Biology Toxicology Triphenyltin hydroxide Genotoxicity Cytotoxicity Teratogenicity Chick embryo Developmental toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In today's era, a large portion of modern agriculture relies heavily on chemical inputs to protect crops from pests and diseases. Although these substances are effective at securing our food supply through the water system, they often wash off from various fields and enter the environment. Once they are in the environment, they can have harmful consequences on human health as well as the whole of wildlife. One such chemical is triphenyltin hydroxide (TPTH). TPTH belongs to a family of chemicals called organotin compounds. These chemicals are widely used as agricultural fungicides and pesticides to protect major crops like potatoes and sugar beets (Marques et al., 2018; De et al., 2018). Because of its extensive use globally, TPTH is now recognized as a persistent environmental pollutant (Wang et al., 2008). So, because of its hazardous nature when living creatures are exposed to TPTH, the chemical can severely alter their natural biological processes. Once TPTH is applied to crops in different fields, it sequentially washes into rivers and settles into the mud at the bottom of the water systems (van Herwijnen, 2012; Du et al., 2014) then animals present at the bottom of the food chain absorb these accumulated chemicals and eventually, as larger predators eat these smaller animals the concentration of the toxic substances magnifies in their biological system through the process of biomagnification. So, this means that even if the initial amount of chemicals that washed off from various sources is less, by the time it reaches top predators in a hierarchical manner, the dose accumulation can be very high. In fact, residual TPTH levels in wild fish have been recorded at alarmingly high concentrations worldwide, indicating severe contamination that adversely affects the whole population (Wang et al., 2008). This kind of environmental contamination is especially vulnerable to wild bird populations. Birds often search for food in agricultural fields, wetlands, and along riverbanks where chemical runoff is most concentrated and regular. Naturally, when a mother bird ingests these kinds of toxic substances, the chemicals can be transferred directly into the eggs she lays. Historically, we have seen how agricultural chemicals can affect bird populations badly; the popular cases of certain pesticides causing eggshells to thin to the point of breaking are a prime example of how sensitive avian reproduction can be when exposed to synthetic chemicals (Ratcliffe, 1967; Lundholm, 1997). While TPTH might not cause the eggshell thinning, its presence in the environment and its possible ability to be maternally transferred into eggs pose a significant threat to the development of wild bird populations and the whole ecosystem. To properly understand TPTH’s toxicity, we have to look for its effect inside a living cell. Earlier studies have reported that because of TPTH’s strong binding capacity, specifically in the case of a cell’s DNA at a specific A-T region, it further alters the internal skeleton, specifically microtubules; microtubules are very important for maintaining a cell’s shape as it helps in the growth of a cell (Mahanty et al., 2017). Further, when TPTH enter into the system, it alters normal structural composition by strongly depolymerizing the tubulin, so that eventually the cell goes into the mitotic arrest phase and loses its ability for proper division and growth (Mahanty et al., 2017). In the case of a fully grown adult, cellular damage is harmful, but for a developing embryo that relies on constant cell division to grow, any type of alteration can be more disturbing. Beyond damaging the cellular scaffolding, TPTH can also cause oxidative stress and alter intracellular calcium levels (Barbosa et al., 2018). Organotin compounds disrupt this balance, which further leads to an explosion of free radicals that act like microscopic wrecking balls (Ghazi et al., 2018; Rajendran et al., 2022). Further, they evade the cell’s protective outer layer, which decreases mitochondrial membrane potential and damages the necessary proteins needed for growth. For a developing embryo, this type of inhibition or oxidative stress is lethal and can eventually trigger cells to undergo apoptosis (self-destruct prematurely). Chemicals like TPTH ruin the cell’s natural defences by creating a cascade of harmful molecules that act like microscopic wrecking balls (Ghazi et al., 2018; Rajendran et al., 2022). These wrecking balls smash into the cell's protective outer layer and alter organelles like mitochondria, that it important for the cell's power supply and also disrupt the important proteins that are necessary for the cell’s growth (Rajendran et al., 2022). For a developing embryo, this type of severe biological stress is dangerous and often fatal (Predarska et al., 2023). It forces cells to self-destruct way too early, leaving the embryo without the healthy pieces it needs to build vital organs, a brain, or a strong skeleton. Different organotin compounds, including TPTH, are significant endocrine disruptors, and eventually, by this type of disruption, they send wrong signals during various stages of development (Marques et al., 2018; De et al., 2019). A previous study has demonstrated the severe impact of TPTH on developing animals. For instance, in the case of pregnant mice, exposure to TPTH causes high rates of embryo death, and surviving offspring often suffer from reduced body weight, cleft palates, and poorly formed skull bones (De et al., 2019). Similarly, research on aquatic life like zebrafish and medaka fish embryos shows that these compounds cause delayed hatching, pericardial oedema (fluid around the heart), spinal curvature, abnormal eye development, and swimming disabilities (Wang et al., 2008; Predarska et al., 2023; Hu et al., 2006). Despite this existing knowledge, observing the direct effects of TPTH in mammals is complicated. A mother’s liver and immune system try to filter out toxins before they reach the babies, making it difficult for researchers to know exactly how much damage the chemical itself is doing directly to the embryo versus how much is a side effect of the mother being sick (Lane et al., 2022; Wang et al., 2008). To bypass this complicated maternal filtering system or to mimic this system, researchers use a highly reliable model, namely the chick embryo. Developing inside an egg, the chick embryo is a good, self-contained biological system. If a chemical is introduced directly into a fertilized egg, the embryo is exposed to the substance without any interference from a mother hen (Lu et al., 2022; Wu et al., 2020). Because the chemical is trapped inside the shell, it creates a scenario of chronic exposure, making the chick embryo an ideal model for observing how TPTH alters physical birth defects, growth delays, and organ malformations. While the endocrine-disrupting, cytotoxicity, reproductive toxicity and developmental toxicity of triphenyltin hydroxide (TPTH) are documented in various mammalian and aquatic systems (Clasen et al., 2017; Barkhordari et al., 2024; Hassan et al., 2025), its particular teratogenic and genotoxic toxicity in early amniote embryogenesis remains poorly studied. A major problem in current research is figuring out exactly how much TPTH directly affects a developing embryo. In standard mammalian studies, the mother's body and the placenta get in the way, making it difficult to see the chemical's true impact. To address this major limitation, the in ovo chick embryo model acts as a self-contained in vivo system. It eliminates maternal confounding factors, thereby allowing for the direct assessment of morphometric anomalies and other systemic toxicity during critical windows of organogenesis. Consequently, this study provides novel evidence directly linking TPTH exposure to specific morphological defects and in vivo damage during early avian development. Despite all of this urgent need, a significant gap exists in the literature, as to date, no study has investigated the embryotoxic effects of TPTH using the chick embryo model. Therefore, to the best of our knowledge, this research provides the first comprehensive evaluation of its teratogenic impacts in developing chick embryos. So finally, understanding these detailed developmental disruptions caused by TPTH is not just about protecting chickens; it is about uncovering the broader biological risks this chemical poses to wildlife ecosystems and human health, highlighting the urgent need for a deeper understanding of its toxicity. Materials and methodology Chemicals Triphenyltin hydroxide (CAS 76-87-9), mitomycin C (MMC, CAS 50-07-7) and Giemsa’s stain (CAS 51811-82-6) were obtained from Sigma Aldrich, Germany, Pvt Ltd. Dimethyl sulfoxide (DMSO) (CAS 67-68-5), May-Grunwald solution (CAS 67-56-1), Xylene (CAS 1330-20-7), DPX mountant (CAS 84-74-2), methanol (CAS 67-56-1), and ethanol (CAS 64-17-5) were purchased from Himedia Laboratories, India Pvt Ltd. All the chemicals used in this study were of analytical grade, and reagents and stains were prepared freshly. Egg procurement, incubation and ethical approval Fertilized white leghorn chicken ( Gallus gallus domesticus ) eggs weighing from 60 ± 2 g were purchased from an authorised vendor's hatchery (Ishwar Hatchery, Guwahati, Assam, India). All the experimental eggs were incubated in a rotating incubator at 37°C with approximately 65–68% relative humidity. Proper hygiene was maintained to avoid any kind of infection. The present study approval was obtained from the Institutional Animal Ethics Committee (IAEC) of The Assam Royal Global University, Guwahati, Assam, India; Reference No: 2286/PO/Re/S/2024/CCSEA. Administration of the test substance TPTH was dissolved in dimethyl sulfoxide (DMSO) due to its insolubility in water. The concentration of the test substance was 4 mg/1ml of DMSO. For all the experimental groups, treatments were given under a sterile laminar flow hood to prevent contamination, and the puncture sites were immediately sealed with sterile adhesive tape to maintain sterility. The whole experiment included six replicates (n = 6) per group. Acute toxicity study For assessing the lethal dose (LD 50 ), a total of 180 eggs were randomly divided into six groups, containing six eggs in each group (n = 6). Further, eggs were treated with increasing concentrations of TPTH, i.e. with 300 µg/egg, 350 µg/egg, 400 µg/egg, 450 µg/egg, and 500 µg/egg up to 96 h. The eggs were candled every 24 h intervals to determine the survivability of the embryos, and the dead embryos were discarded and recorded. The percentage of dead and live animals in each treatment group was calculated for 24 h, 48 h, 72 h and 96 h exposure, and the LD 50 values were calculated by probit analysis using MS Excel (Windows, MS Office 2021). Developmental toxicity study To assess the toxicological potential of Triphenyltin hydroxide (TPTH), a total of 36 eggs were randomly divided into six study groups containing six eggs in each group (n = 6) namely, Group I (Control; No treatment); Group II (Positive control; MMC); Group III (Vehicle control; DMSO); Group IV (TPTH, 1% of 96 h LD 50 ); Group V (TPTH, 5% of 96 h LD 50 ); Group VI (TPTH, 10% of 96 h LD 50 ). Before giving the treatment, the eggs were properly sterilized with 70% alcohol. The doses were given in two injection routes to target different parts of the eggs. For the early toxicity study, doses were given to the pointed end of the egg before the experiments. For the HET-MN and cytotoxicity assessment, doses were given in the blunt end for the treatment to directly reach to the chorioallantoic membrane (CAM) via the air sac. After all the treatment, the eggs were properly sealed with sterilized adhesive tapes. Morphometric assessment After the exposure period (which corresponded to stages HH-12, 13, and 21–24), eggs were cracked open and transferred to a phosphate-buffered saline (PBS, pH 7.4) and later placed on a glass slide to observe any kind of alterations in the morphology. A Light stereomicroscope was used to check their morphology by following the guidelines of Hamburger and Hamilton (1951). To assess other physical parameters like body length, head plus bill length, forelimb length, hindlimb length, and eye diameter, an image analysis software called Fiji (ImageJ 2.16.0, for Windows) was used. For consistency, we always measured the limbs and eye diameter on the right side of the embryo. Hen's egg test for micronucleus (HET-MN) assay The present test was performed by following Chaubey et al. (1993) and Wolf and Luepk (1997) with minor modifications. For the HET-MN assay, eggs were cracked open on day 11 of incubation. Later, a blood sample was collected from each embryo by pricking the arterio-umbilicus sinistra using a heparinized capillary tube. Then, a blood smear was made on a clean, grease-free slide. To make the cells visible, we dyed them using a two-step process. First, we applied a May-Grunwald stain and washed the slides three times with distilled water. Then, we soaked them in a diluted Giemsa stain for 10 min and gave them a final, thorough wash. Once the slides were dry, we treated them with xylene for 5 min, and then they were mounted using a cover slip with DPX. 1000 cells per slide were examined using a compound light microscope, Cytotoxicity study To figure out if TPTH was toxic to the cells, we calculated the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs). This PCE/NCE ratio basically acts as a marker to show how well the cells are multiplying. We looked at the different subgroups of PCEs and NCEs in the peripheral blood of the chick embryos. To correctly identify the different types of red blood cells, we used the classification guides from Lucas and Jamroz (1961) and Wolf and Luepke (1997). We randomly examined about 1,000 cells on each slide. Our goal was to identify two specific types of red blood cells: polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE), following established scientific guidelines. We could easily tell these cells apart based on their size, shape, and colour. The PCEs were large, round cells with round centres (nuclei) that turned blue from the dye. In contrast, the NCEs were oval-shaped cells that turned pink. Statistical analysis Statistical analysis was done with the help of SPSS 21.0 (IBM Corp., Armonk, New York), and the experimental data were presented as the Mean ± SD. Comparisons between the control and different treatment groups were conducted using one-way analysis of variance (ANOVA) to test the levels of significance. Results Acute toxicity and survivability study To determine the LD 50 of TPTH, the embryos were exposed to increasing concentrations of Triphenyltin hydroxide (TPTH), i.e. 0 µg/egg, 300 µg/egg, 350 µg/egg, 400 µg/egg, 450 µg/egg, and 500 µg/egg for 24 h, 48 h, 72 h, and 96 h. The median lethal dose (LD 50 ) was determined by employing probit analysis in MS Excel for Windows (MS Office 2021). The LD 50 values as calculated are shown in Table 1. The LD 50 of TPTH at 24 h, 48 h, 72 h, and 96 h exposure periods are observed as 537.1 ± 35.81 µg/egg, 424.3 ± 24.11 µg/egg, 372.9 ± 22.82 µg/egg, and 342.5 ± 7.27 µg/egg, respectively. For further experimentation to assess the toxicological potential of TPTH, different concentrations of TPTH were chosen based on the 96 h LD 50 of TPTH in the chicken embryos. The selected test concentrations were 1%, 5% and 10% of 96 h LD 50 (Table 1). The LD₅₀ antilog values showed a progressive decline with increasing exposure time, indicating time-dependent toxicity. At 24 h, the LD₅₀ was highest (537.1), which gradually decreased to 424.3 at 48 h, 372.9 at 72 h, and reached the lowest value of 342.5 at 96 h (Fig. 1A). This downward trend suggests that longer exposure enhances toxic effects, thereby requiring lower doses to reach 50% lethality. The regression analysis (R² = 0.9191) indicates a strong correlation between exposure duration and LD₅₀ reduction. The survival curve demonstrates a clear dose- and time-dependent decline in embryo viability following exposure. Control embryos maintained 100% survival throughout the 96 h observation period, while treated groups showed progressively reduced survival with increasing concentration and exposure duration (Fig. 2B). At 300 µg/egg, survival decreased moderately, whereas at 500 µg/egg, survival dropped sharply, reaching 0% by 96 h. These findings are consistent with the calculated LD₅₀ values, which decreased from 537.1 µg/egg at 24 h to 342.5 µg/egg at 96 h, confirming enhanced toxicity with prolonged exposure. Hen's egg test for micronucleus (HET-MN) induction The genotoxic potential of the Triphenyltin hydroxide (TPTH) was assessed by using the HET-MN assay. Significant increments in the frequencies of MN in TPTH-treated embryos were observed as compared to the control. The MMC-treated group showed a rise in the MN frequency to 7.53 ± 0.33% (p < 0.001) as compared to the control group (1.27 ± 0.17%) (Fig. 1C). Similarly, the embryos treated with 1%, 5%, and 10% concentrations of TPTH showed a significant increment of MN (%) to 2.58 ± 0.20% ( p < 0.001 vs control), 3.87 ± 0.31% ( p < 0.001 vs control), and 6.65 ± 0.17% ( p < 0.001 vs control), respectively (Fig. 1C). Cytotoxicity study TPTH exposure affected the PCE/NCE ratio, indicating cytotoxic effects. As shown in Fig. 1D, no significant change was observed in the DMSO group (1.13 ± 0.10) as compared to the control group (1.17 ± 0.08), confirming the validity of the assay. The positive control (MMC) group (0.62 ± 0.03) caused a decrease in the PCE/NCE ratio as compared to the control group, although the values are not statistically significant. On the other hand, TPTH treatment produced a dose-dependent reduction in the PCE/NCE ratio. At the lowest dose (1%) (0.92 ± 0.03), the decrease was statistically significant at p < 0.05 as compared to the control group, whereas at 5% and 10% concentrations (0.72 ± 0.02) (0.60 ± 0.03), the decreases were highly significant ( p < 0.001 ) as compared to the control group. These results indicate that TPTH induces cytotoxicity in a concentration-dependent manner (Fig. 1D). Morphological assessment of TPTH-induced Chick Embryos Embryos in the control (Group A) (Fig. 3) and vehicle control (Group B) (Fig. 3) exhibited robust and healthy development, reaching approximately HH Stage 12 to 13 (roughly 45–52 hours of incubation). At this stage, the normal morphological stages were clearly visible, namely, the development of the cephalic portion, i.e. prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain), showed distinct boundaries and healthy expansion. The heart tube had successfully begun its vital rightward "C-looping," an ideal identification of HH Stage 12. Also, the embryos displayed a straight and symmetrical body axis. The somites were sharp, square-shaped, and organized in parallel pairs along the neural tube. Furthermore, there was a noticeable beginning of cranial flexure (the bending of the head downward), consistent with late HH Stage 12. The near-identical appearance of Group B (DMSO vehicle) to Group A confirms that the solvent used to deliver the TPTH did not interfere with the natural developmental trajectory of the embryos. Embryos treated with the lowest dose of TPTH (Group C) (Fig. 3) showed only minor deviations from the control groups. Morphologically, these embryos appeared to be at HH Stage 12, suggesting a very slight developmental lag. While the brain vesicles and heart looping remained largely intact, there was a minor reduction in the overall transparency of the neural tube compared to the controls. This indicates that while 4 µg of TPTH is not immediately lethal, it begins to exert a mild inhibitory pressure on the developing embryo. A significant shift in morphology occurred at the 20 µg dosage (Group D) (Fig. 3). These embryos failed to reach the HH 13 milestone seen in the controls, appearing arrested or delayed at HH Stage 11. Key abnormalities include microcephaly, in which the forebrain (prosencephalon) was noticeably smaller and lacked the full "bulge" seen in healthy embryos. Although the heart tube was present, the looping process was significantly delayed. The heart appeared as a smaller, more vertical structure. The posterior somites were less distinct, suggesting that the chemical was beginning to interfere with the process of somitogenesis. The most profound toxic effects were observed in the high-dose group (Group E) (Fig. 3). These embryos did not conform to any single HH stage because their development was not just delayed, but severely malformed. They exhibited characteristics of an arrested HH Stage 10, but with pathological deviations; a sharp bend was observed in the middle of the body axis. This suggests a failure in the structural integrity of the notochord or a collapse of the neural tube, which is a severe teratogenic outcome. The entire head region was underdeveloped. The individual vesicles of the brain were indistinguishable, appearing as a single, stunted mass of tissue. The heart remained a primitive, unlooped tube. This lack of looping is a lethal defect that would prevent the establishment of a functional blood flow system. The tail region was significantly shorter than in the controls, indicating that TPTH inhibited the elongation of the embryo from the primitive streak and Hensen’s node. Similarly, the embryotoxic and teratogenic effects of Triphenyltin hydroxide (TPTH) on chick embryos were characterized by a clear dose-response gradient, with the control (Group A) (Fig. 4) and vehicle control (Fig. 4) (Group B) maintaining normal developmental progression consistent with Hamilton-Hamburger (HH) Stage 21–24. In contrast, TPTH-treated embryos exhibited profound morphological deviations, beginning with compromised vascular integrity that manifested as localized petechial haemorrhages in the abdominal region of the 4µg low-dose group (Group C) (Fig. 4). This progressed to widespread severe haemorrhage and blood stasis in the 40µg high-dose group (Group E) (Fig. 4), particularly concentrated around the primitive heart and brain vesicles. Significant craniofacial and ocular malformations were also prevalent in the higher dose groups (D and E), most notably a marked reduction in the diameter of the optic cup (microphthalmia) and distinct hydrocephalus-like cranial oedema around the mesencephalon and rhombencephalon, indicative of developmental failure. Furthermore, Group E demonstrated severe growth retardation, evidenced by a significantly decreased crown-rump length and overall body stunting. Axial development was similarly impaired, with treated embryos exhibiting hypoplasia of the limb and tail buds, caudal regression, and a breakdown of somite organization where indistinct boundaries suggested a disruption of the molecular clock governing somitogenesis. Body length To evaluate the impact of TPTH on body length, measurements were recorded on days 3, 5, 7, and 9. Treatment with TPTH showed a significant reduction of body length in TPTH-exposed groups in a dose-dependent manner throughout the study period. On day 9, exposure to TPTH at a low dose (TPTH-L) to experimental embryos showed a significant decrease ( p < 0.001 ) (Fig. 5A) of body length to approximately 41.5 ± 0.25 mm as compared to control (45.08 ± 0.44 mm) (Fig. 5A). The experimental embryos treated with TPTH-M and TPTH-H showed further reduced levels of body length to 38.1 ± 0.25 mm ( p < 0.001 vs control) and 34.4 ± 0.25 mm ( p < 0.001 vs control), respectively (Fig. 5A). Eye diameter To investigate the developmental impact of TPTH on ocular growth, eye diameter was measured across the four sampling intervals. The data reveal that continuous exposure to TPTH resulted in a progressive and dose-dependent decline in eye diameter throughout the 9-day study. By the final observation on Day 9, embryos in the low-dose group (TPTH-L) exhibited a significant reduction ( p < 0.001 ) (Fig. 5B) in eye diameter, reaching approximately 7.48 ± 0.14 mm compared to the control group's 8.12 ± 0.19 mm. This inhibitory trend was even more pronounced in the higher concentration groups; TPTH-M and TPTH-H treatments led to further decreases in ocular size, measuring 6.71 ± 0.13 mm ( p < 0.001 vs. control) and 5.95 ± 0.10 mm ( p < 0.001 vs. control), respectively (Fig. 5B). Forelimb The influence of TPTH on limb morphogenesis was characterized by a noteworthy decline in forelimb length throughout the 9-day observation period. Experimental embryos subjected to TPTH exhibited a concentration-related inhibitory effect on limb elongation starting as early as Day 3, where the TPTH-H group already showed a drastic reduction compared to the control. On the final day of the study (Day 9), the control group achieved a forelimb length of approximately 11.43 ± 0.29 mm, whereas the TPTH-L group showed a significant decrease to 10.20 ± 0.21 mm ( p < 0.001 ) (Fig. 5C). More severe growth suppression was recorded in the TPTH-M and TPTH-H groups, with lengths reaching only 9.05 ± 0.18 mm and 8.00 ± 0.13 mm, respectively ( p < 0.001 vs. control) (Fig. 5C), highlighting the potent limb-stunting effects of the compound. Head and bill To evaluate the impact of TPTH on cranial and bill development, measurements of the head + bill length were recorded. Consistent with other growth parameters, treatment with TPTH led to a marked suppression of head and bill growth in a concentration-related manner. On Day 9, exposure to the low dose (TPTH-L) resulted in a significant decrease ( p < 0.001 ) (Fig. 5D) in length to 14.20 ± 0.21 mm as compared to the control group (15.53 ± 0.29 mm). The experimental groups treated with higher concentrations, TPTH-M and TPTH-H, showed further reduced levels of 12.95 ± 0.18 mm ( p < 0.001 vs. control) and 11.40 ± 0.13 mm ( p < 0.001 vs. control), respectively. Hindlimb The study also evaluated the elongation of the hindlimb to determine if the growth inhibitory effects were uniform across the body. Similar to the forelimbs, hindlimb growth was significantly impaired by TPTH exposure at all tested concentrations. On Day 9, the mean hindlimb length of the control group was 14.97 ± 0.34 mm. In contrast, the TPTH-exposed groups showed significant declines ( p < 0.001 ) (Fig. 5E), with lengths measuring 13.57 ± 0.25 mm for TPTH-L, 12.25 ± 0.18 mm for TPTH-M, and a minimum of 10.70 ± 0.13 mm for the TPTH-H group. Discussion The present study investigates the acute toxicity, genotoxicity, cytotoxicity, and teratogenic potential of Triphenyltin hydroxide (TPTH) using an in ovo chick embryo model by providing critical insights into its developmental toxicity. Organotin compounds, including TPTH and its widely studied structural analogue, Tributyltin (TBT), are ubiquitous environmental pollutants recognized for their potent biocidal properties and severe developmental toxicity. Our acute toxicity evaluation demonstrated a progressive, time-dependent decline in the LD 50 values for TPTH, dropping from 547.37 µg/egg at 24 h to 395.74 µg/egg at 96 h. This downward trend aligns closely with the established toxicokinetics of organotins in avian embryos, where their highly lipophilic nature facilitates rapid penetration of the vitelline membrane and subsequent bioaccumulation in lipid-rich embryonic tissues (Frouin et al., 2008). Similar time-dependent mortality and enhanced toxicity with prolonged exposure have been documented in recent parallel studies examining the cytotoxicity and genotoxicity of TBT in early Gallus gallus domesticus embryos (Mandal et al., 2023). To elucidate the mechanistic basis of this toxicity, cellular damage was evaluated using the Hen's egg test for micronucleus (HET-MN) induction and the Polychromatic Erythrocyte-to-Normochromatic Erythrocyte (PCE/NCE) ratio. The significant, dose-dependent induction of micronuclei frequency in TPTH-treated embryos provides clear evidence of profound genomic instability. Micronucleus formation in avian peripheral blood erythrocytes is a reliable biomarker of clastogenesis or aneugenesis, often triggered by excessive Reactive Oxygen Species (ROS). Furthermore, the highly significant decline in the PCE/NCE ratio in our study indicates severe cytotoxicity and the suppression of erythropoiesis. These findings mirror the exact cytotoxic and genotoxic paradigms observed in TBT-exposed chick embryos, where TBT similarly increased nuclear abnormalities and dramatically altered the PCE to NCE ratio, indicating severe bone marrow-equivalent toxicity and long-term genomic complications (Mandal et al., 2023). Beyond sub-cellular damage, our morphological assessment revealed severe teratogenic deviations at both early (HH 10–13) and later (HH 21–24) developmental stages. Embryos exhibited profound structural anomalies, including severe microcephaly, neural tube distortion, blurred somite boundaries, and, notably, the failure of the heart tube to undergo normal rightward C-looping. Organotins are well-documented endocrine disruptors that aberrantly interfere with highly conserved morphogenetic pathways (Varela-Ramirez et al., 2011; Pu et al., 2022). For instance, TBT induces significant morphological and transcriptomic malformations in chicken embryos by altering the expression of key transcription and growth factors governing tissue differentiation (Varela-Ramirez et al., 2011). This targeted disruption, combined with organotin-induced interference of intracellular calcium homeostasis, likely explains the altered heart tubes and the extensive haemorrhaging observed in our TPTH-treated groups. The concurrent observation of microphthalmia and cranial oedema further indicates that TPTH may interfere with proper embryonic osmoregulation and cranial neural crest cell migration. These macroscopic observations of severe teratogenicity were strictly aligned with our quantitative morphometric analyses, which revealed significant, concentration-dependent growth retardation across body length, eye diameter, and limb lengths. Collectively, this cascade of toxic events, starting from genotoxic damage, cytotoxicity, and the alteration in the morphological structures, highlights the potent developmental risks posed by TPTH exposure and aligns accurately with the broader ecotoxicological consensus regarding organotin embryotoxicity in avian models (Said and El Zokm, 2025; Frouin et al., 2008; Siddique et al., 2026). Conclusion In conclusion, this study proved that Triphenyltin hydroxide (TPTH) is a highly potent embryotoxic and teratogenic agent in the avian model. Through a comprehensive in ovo assessment, the data demonstrate that TPTH exposure leads to a time- and dose-dependent increase in embryonic mortality, characterized by profound genomic instability and severe cellular toxicity. Moreover, TPTH disrupts critical morphogenetic pathways that result in alteration of cardiovascular anomalies, such as the failure of heart looping, alongside craniofacial malformations, and significant overall growth retardation. By providing the first comprehensive evaluation of TPTH-induced developmental toxicity in chick embryos, this research highlights the extreme alterations of rapidly developing biological systems to organotin contamination. Ultimately, these findings underscore the broader ecological risks posed by agricultural runoff and emphasize the urgent need for stricter environmental monitoring to protect wildlife populations and other living beings. Declarations Acknowledgements The authors would like to express their deepest gratitude to The Assam Royal Global University, Guwahati, Assam, for providing infrastructural support for the present study. CRediT roles Dipsanu Paul: Writing - Original draft, Methodology, Data curation, Formal analysis, Visualization; Sweety Nath Barbhuiya, Sarbani Giri, Dharmeswar Barhoi: Conceptualization,Supervision, Writing – review and editing. Declaration of competing interest The authors declare no conflicts of interest associated with this paper. Data availability Data will be made available on request. References Barbosa, C. M. D. L., Ferrão, F. M., & Graceli, J. B. (2018). Organotin compounds toxicity: focus on kidney. Frontiers in endocrinology , 9 , 256. DOI: 10.3389/fendo.2018.00256 Barkhordari, A., Hassan, J., Koohi, M. K., Rahmati‐Holasoo, H., & Pourshaban‐Shahrestani, A. (2024). Toxic Effects of Tributyltin, Triphenyltin, and SnCl2 on the Development of Zebrafish (Danio rerio) Embryos. Aquaculture, Fish and Fisheries , 4 (5), e70005.DOI: 10.1002/aff2.70005 Chaubey, R. C., Bhilwade, H. N., Joshi, B. N., & Chauhan, P. S. (1993). Studies on the migration of micronucleated erythrocytes from bone marrow to the peripheral blood in irradiated Swiss mice. International Journal of Radiation Biology , 63 (2), 239-245. DOI: 10.1080/09553009314550311 Clasen, B., Becker, A. G., Lópes, T., Murussi, C. R., Antes, F. G., Horn, R. C., ... & Loro, V. L. (2017). Triphenyltin hydroxide induces changes in the oxidative stress parameters of fish. Ecotoxicology , 26 (4), 565-569. DOI: DOI: 10.1007/s10646-017-1780-9 De Araujo, J. F. P., Podratz, P. L., Merlo, E., Sarmento, I. V., Da Costa, C. S., Niño, O. M. S., ... & Graceli, J. B. (2018). Organotin exposure and vertebrate reproduction: a review. Frontiers in endocrinology , 9 , 64. DOI: 10.3389/fendo.2018.00064 De Oliveira, M., Rodrigues, B. M., Olimpio, R. M. C., Graceli, J. B., Gonçalves, B. M., Costa, S. M. B., ... & Nogueira, C. R. (2019). Disruptive effect of organotin on thyroid gland function might contribute to hypothyroidism. International journal of endocrinology , 2019 (1), 7396716. DOI: 10.1155/2019/7396716 Du, J., Chadalavada, S., Chen, Z., & Naidu, R. (2014). Environmental remediation techniques of tributyltin contamination in soil and water: A review. Chemical Engineering Journal , 235 , 141-150. DOI: 10.12691/ijebb-5-1-3 Frouin, H., Lebeuf, M., Saint-Louis, R., Hammill, M., Pelletier, É., & Fournier, M. (2008). Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro. Aquatic Toxicology , 90 (3), 243-251. DOI: 10.1016/j.aquatox.2008.09.005 Ghazi, D., Rasheed, Z., & Yousif, E. (2018). Review of organotin compounds: chemistry and applications. development , 3 (4). DOI: 10.32474/AOICS.2018.03.000161 Hamburger, V., & Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. Journal of morphology , 88 (1), 49-92. DOI: 10.1002/aja.1001950404 Hassan, J., Barkhordari, A., Koohi, M. K., Pourshaban-Shahrestani, A., Rahmati-Holasoo, H., & Sharifabadi, M. A. (2025). Bioaccumulation and oxidative stress induced by organometallic and ionic tin compounds in zebrafish (Danio rerio) embryos. Bulletin of Environmental Contamination and Toxicology , 114 (4), 58. DOI: 10.1007/s00128-025-04031-y Hu, J., Zhen, H., Wan, Y., Gao, J., An, W., An, L., ... & Jin, X. (2006). Trophic magnification of triphenyltin in a marine food web of Bohai Bay, North China: Comparison to tributyltin. Environmental science & technology , 40 (10), 3142-3147. DOI: 10.1021/es0514747 Lane, M. K. M., Garedew, M., Deary, E. C., Coleman, C. N., Ahrens-Víquez, M. M., Erythropel, H. C., ... & Anastas, P. T. (2022). What to expect when expecting in lab: a review of unique risks and resources for pregnant researchers in the chemical laboratory. Chemical research in toxicology , 35 (2), 163-198. DOI: 10.1021/acs.chemrestox.1c00380 Lu, M., Mu, Y., & Liu, Y. (2022). Triphenyltin disrupts the testicular microenvironment and reduces sperm quality in adult male rats. Chemosphere , 301 , 134726. DOI: 10.1016/j.chemosphere.2022.134726 Lundholm, C. E. (1997). DDE-induced eggshell thinning in birds: effects of p, p′-DDE on the calcium and prostaglandin metabolism of the eggshell gland. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology , 118 (2), 113-128. DOI: 10.1016/s0742-8413(97)00105-9 Mahanty, S., Raghav, D., & Rathinasamy, K. (2017). In vitro evaluation of the cytotoxic and bactericidal mechanism of the commonly used pesticide triphenyltin hydroxide. Chemosphere , 183 , 339-352. DOI: 10.1016/j.chemosphere.2017.05.117 Mandal, A., Ghosh, M., Talukdar, D., Dey, P., Das, A., & Giri, S. (2023). Cytotoxicity and genotoxicity of tributyltin in the early embryonic chick, Gallus gallus domesticus. Mutation Research/Genetic Toxicology and Environmental Mutagenesis , 889 , 503656. DOI: 10.1016/j.mrgentox.2023.503656 Marques, V. B., Faria, R. A., & Dos Santos, L. (2018). Overview of the pathophysiological implications of organotins on the endocrine system. Frontiers in endocrinology , 9 , 101. DOI: 10.3389/fendo.2018.00101 Predarska, I., Saoud, M., Morgan, I., Loennecke, P., Kaluđerović, G. N., & Hey-Hawkins, E. (2023). Triphenyltin (IV) carboxylates with exceptionally high cytotoxicity against different breast cancer cell lines. Biomolecules , 13 (4), 595. DOI: 10.3390/biom13040595 Pu, Y., Ticiani, E., Pearl, S., Martin, D., & Veiga-Lopez, A. (2022). The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation. Toxicology and applied pharmacology , 453 , 116209. DOI: 10.1016/j.taap.2022.116209 Rajendran, K., Dey, R., Ghosh, A., & Das, D. (2022). In search of biocatalytic remedy for organotin compounds-the recalcitrant eco-toxicants. Biophysical Chemistry , 290 , 106888. DOI: 10.1016/j.bpc.2022.106888 Ratcliffe, D. A. (1967). Decrease in eggshell weight in certain birds of prey. Nature , 215 (5097), 208-210. DOI: 10.1038/215208a0 Said, T. O., & El Zokm, G. M. (2025). Mechanisms and Models for OMCs’ Chemical Behavior and Ecotoxicology. In Organometallic Compounds in the Marine Environment: Analytical and Ecological Aspects (pp. 87-132). Cham: Springer Nature Switzerland. Siddique, M. S., Anand, S., de Agostini Losano, J. D., Jiang, Z., Bhandari, R. K., & Daigneault, B. W. (2026). Tetrahydrocannabinol exposure to postejaculatory sperm compromises sperm structure, function, the epigenome, and early embryo development. bioRxiv , 2026-03. DOI: 10.64898/2026.03.23.713385 van Herwijnen, R. (2012). Environmental risk limits for organotin compounds . National Institute for Public Health and the Environment. Varela-Ramirez, A., Costanzo, M., Carrasco, Y. P., Pannell, K. H., & Aguilera, R. J. (2011). Cytotoxic effects of two organotin compounds and their mode of inflicting cell death on four mammalian cancer cells. Cell biology and toxicology , 27 (3), 159-168. DOI: 10.1007/s10565-010-9178-y Wang, X., Hong, H., Zhao, D., & Hong, L. (2008). Environmental behavior of organotin compounds in the coastal environment of Xiamen, China. Marine Pollution Bulletin , 57 (6-12), 419-424. DOI: 10.1016/j.marpolbul.2008.04.034 Wolf, T., & Luepke, N. P. (1997). Formation of micronuclei in incubated hen's eggs as a measure of genotoxicity. Mutation Research/Genetic Toxicology and Environmental Mutagenesis , 394 (1-3), 163-175. DOI: 10.1016/s1383-5718(97)00136-8 Wu, K., Li, Y., Liu, J., Mo, J., Li, X., & Ge, R. S. (2020). Long-term triphenyltin exposure disrupts adrenal function in adult male rats. Chemosphere , 243 , 125149. DOI: 10.1016/j.chemosphere.2019.125149 Table Table 1 is not available with this version. Additional Declarations The authors declare no competing interests. Supplementary Files Survivability.xlsx HETMNDATA.xlsx Figure5Morphometrydata.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9703101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":639682137,"identity":"d266ba68-2e07-4575-8c2e-6b0f80496c47","order_by":0,"name":"Dipsanu Paul","email":"","orcid":"","institution":"The Assam Royal Global University","correspondingAuthor":false,"prefix":"","firstName":"Dipsanu","middleName":"","lastName":"Paul","suffix":""},{"id":639682138,"identity":"097bf88c-ae7e-4400-b095-eae9a62c1b13","order_by":1,"name":"Sweety Nath Barbhuiya","email":"","orcid":"","institution":"Patharkandi College","correspondingAuthor":false,"prefix":"","firstName":"Sweety","middleName":"Nath","lastName":"Barbhuiya","suffix":""},{"id":639682139,"identity":"b0f17f31-35d5-4b31-b204-812fb447324d","order_by":2,"name":"Sarbani Giri","email":"","orcid":"","institution":"Assam University","correspondingAuthor":false,"prefix":"","firstName":"Sarbani","middleName":"","lastName":"Giri","suffix":""},{"id":639682140,"identity":"d6bf32ec-1f59-4128-a9e7-dfc256600ac3","order_by":3,"name":"Dharmeswar Barhoi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACAwYGNiDJnADlH5ADkw9I0WIMJhNwqYdrYUBoSWwAUfi0mLOfffbgQ4F1Hv/sw083fPhzJ31+2OGHQFvs5HQbsGux7Ek3N5xhkF4scS7N7ObMtme5G2+nGQC1JBubHcDhsANpbNI8BocTG84wmN3mbTicu3F2AkjLgcRtuLScfwbRMv8M+7fbf/4cTjecnf4Bv5YbUFs2nOExu83AdjhBXjoHvy2WM56xSQL9krjxDE/Zzd62w4YbpHMKDiQY4PaLOX8am8SHP9aJ886wb7vx489hefnZ6Zs/fKiwk8OlBVuAgElilYOAfAMpqkfBKBgFo2AkAAC/8mnvSrd7KgAAAABJRU5ErkJggg==","orcid":"","institution":"The Assam Royal Global University","correspondingAuthor":true,"prefix":"","firstName":"Dharmeswar","middleName":"","lastName":"Barhoi","suffix":""}],"badges":[],"createdAt":"2026-05-13 11:23:27","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9703101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9703101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109289071,"identity":"0daad4be-f40c-479c-8549-5894c45dc635","added_by":"auto","created_at":"2026-05-15 06:10:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167752,"visible":true,"origin":"","legend":"\u003cp\u003eA represents LD50 antilog values for TPTH at 24 h, 48 h, 72 h, and 96 h\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB represents a line graph of the survival percentage of chick embryos at different doses (300-500 µg/egg) over 96 h. Each value is expressed as the mean for six experimental eggs (n = 6), and error bars represent SD. Illustration\u003c/p\u003e\n\u003cp\u003eC represents the histogram of the occurrence of micronuclei (MN) frequency in the blood cells upon exposure to TPTH. Similarly,\u003c/p\u003e\n\u003cp\u003eD represents the histogram of the occurrence of the PCE/NCE ratio in the blood cells upon exposure to TPTH. Each bar in the histograms represents the mean of six (n=6) study animals, and error bars reflect SD.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/dc6c4b4b605ef56b43a7b7c9.png"},{"id":109289067,"identity":"6ae11a93-601a-4ccd-9f14-e45ac736e789","added_by":"auto","created_at":"2026-05-15 06:10:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":584027,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs showing normal and abnormal peripheral blood erythrocytes of the 11th-day-old chick embryo. (a, b) Normal erythrocytes, (c-h) Micronucleated erythrocytes.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/3e7b6b5b72a0470e46325267.png"},{"id":109289073,"identity":"59798ddd-47c5-4098-8145-c82bd7d81847","added_by":"auto","created_at":"2026-05-15 06:10:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208317,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological defects in chick embryos treated with Triphenyltin hydroxide (TPTH). (A) Control, (B) Vehicle control (DMSO), (C-E) treated embryos with low, medium, and high dose of TPTH (1% of 96 h LD50, 5% of 96 h LD50, and 10% of 96 h LD50). R, Rhombencephalon; M, Mesencephalon; D, Diencephalon; OP, Optic vescle; H, Heart; NT, Neural tube; TB, Tail bud.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/6aaa0210cd4847ce5316e7b8.png"},{"id":109289070,"identity":"f5df1e52-e5b0-4917-847b-47e7dcc5645a","added_by":"auto","created_at":"2026-05-15 06:10:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":158192,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological abnormalities in chick embryos following Triphenyltin hydroxide (TPTH) exposure. Photographs A and B showed normal embryos at HH stage 21-24 observed in the control and vehicle control groups. C-E showed morphological abnormalities observed in TPTH-treated groups. MB, Mid brain; HB, Hindbrain, FB, Forebrain; HT, Heart; ULB, Upper limb bud; LLB, Lower limb bud; HEM, Haemorrhage; BM, Brain malformations; MT, Microphthalmia.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/49cd186f13111823e333b985.png"},{"id":109289072,"identity":"636af65a-9e73-4865-b15f-66c339249884","added_by":"auto","created_at":"2026-05-15 06:10:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252003,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TPTH exposure on body length (A), eye diameter (B), forelimb length (C), head + bill length (D), and hindlimb length (E) on chicken embryos (day 3, 5, 7 and 9). Data represent means ± standard deviation (n=6, in each group). Asterisks indicate significant differences (**p \u0026lt; 0.01 \u0026amp; ***p \u0026lt; 0.001) in each measurement in a TPTH-treated group compared with that in the respective control. VC, Vehicle control, DMSO (10 µl); TPTH-L (1% of 96 h LD50), TPTH-M (5% of 96 h LD50) and TPTH-H (10% of 96 h LD50).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/ae838311e09891d078163d28.png"},{"id":109297281,"identity":"cb53704b-6f3e-46ca-91e2-bc0837442ca5","added_by":"auto","created_at":"2026-05-15 08:55:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1727796,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/2d51fac5-4c7b-4801-bbca-cb7adf2aa30f.pdf"},{"id":109289066,"identity":"475ec88d-b982-4592-81b3-7230140123ed","added_by":"auto","created_at":"2026-05-15 06:10:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11117,"visible":true,"origin":"","legend":"","description":"","filename":"Survivability.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/b163bb64b874289374c0317e.xlsx"},{"id":109296461,"identity":"7741254f-5ef2-40c5-8be2-a7e333335c3b","added_by":"auto","created_at":"2026-05-15 08:47:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12725,"visible":true,"origin":"","legend":"","description":"","filename":"HETMNDATA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/61143229f8c66f830e1ce9ad.xlsx"},{"id":109289068,"identity":"b72cd946-9325-4e46-bad3-ddeae19a7070","added_by":"auto","created_at":"2026-05-15 06:10:46","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21613,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5Morphometrydata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9703101/v1/93483fe2cf296c9766796e79.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eTriphenyltin hydroxide (TPTH) exposure induces genotoxicity, cytotoxicity and developmental toxicity in Chick embryo, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGallus gallus domesticus: \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAn in ovo study\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn today's era, a large portion of modern agriculture relies heavily on chemical inputs to protect crops from pests and diseases. Although these substances are effective at securing our food supply through the water system, they often wash off from various fields and enter the environment. Once they are in the environment, they can have harmful consequences on human health as well as the whole of wildlife. One such chemical is triphenyltin hydroxide (TPTH). TPTH belongs to a family of chemicals called organotin compounds. These chemicals are widely used as agricultural fungicides and pesticides to protect major crops like potatoes and sugar beets (Marques et al., 2018; De et al., 2018). Because of its extensive use globally, TPTH is now recognized as a persistent environmental pollutant (Wang et al., 2008). So, because of its hazardous nature when living creatures are exposed to TPTH, the chemical can severely alter their natural biological processes.\u003c/p\u003e \u003cp\u003eOnce TPTH is applied to crops in different fields, it sequentially washes into rivers and settles into the mud at the bottom of the water systems (van Herwijnen, 2012; Du et al., 2014) then animals present at the bottom of the food chain absorb these accumulated chemicals and eventually, as larger predators eat these smaller animals the concentration of the toxic substances magnifies in their biological system through the process of biomagnification. So, this means that even if the initial amount of chemicals that washed off from various sources is less, by the time it reaches top predators in a hierarchical manner, the dose accumulation can be very high. In fact, residual TPTH levels in wild fish have been recorded at alarmingly high concentrations worldwide, indicating severe contamination that adversely affects the whole population (Wang et al., 2008).\u003c/p\u003e \u003cp\u003eThis kind of environmental contamination is especially vulnerable to wild bird populations. Birds often search for food in agricultural fields, wetlands, and along riverbanks where chemical runoff is most concentrated and regular. Naturally, when a mother bird ingests these kinds of toxic substances, the chemicals can be transferred directly into the eggs she lays. Historically, we have seen how agricultural chemicals can affect bird populations badly; the popular cases of certain pesticides causing eggshells to thin to the point of breaking are a prime example of how sensitive avian reproduction can be when exposed to synthetic chemicals (Ratcliffe, 1967; Lundholm, 1997). While TPTH might not cause the eggshell thinning, its presence in the environment and its possible ability to be maternally transferred into eggs pose a significant threat to the development of wild bird populations and the whole ecosystem.\u003c/p\u003e \u003cp\u003eTo properly understand TPTH\u0026rsquo;s toxicity, we have to look for its effect inside a living cell. Earlier studies have reported that because of TPTH\u0026rsquo;s strong binding capacity, specifically in the case of a cell\u0026rsquo;s DNA at a specific A-T region, it further alters the internal skeleton, specifically microtubules; microtubules are very important for maintaining a cell\u0026rsquo;s shape as it helps in the growth of a cell (Mahanty et al., 2017). Further, when TPTH enter into the system, it alters normal structural composition by strongly depolymerizing the tubulin, so that eventually the cell goes into the mitotic arrest phase and loses its ability for proper division and growth (Mahanty et al., 2017). In the case of a fully grown adult, cellular damage is harmful, but for a developing embryo that relies on constant cell division to grow, any type of alteration can be more disturbing. Beyond damaging the cellular scaffolding, TPTH can also cause oxidative stress and alter intracellular calcium levels (Barbosa et al., 2018).\u003c/p\u003e \u003cp\u003eOrganotin compounds disrupt this balance, which further leads to an explosion of free radicals that act like microscopic wrecking balls (Ghazi et al., 2018; Rajendran et al., 2022). Further, they evade the cell\u0026rsquo;s protective outer layer, which decreases mitochondrial membrane potential and damages the necessary proteins needed for growth. For a developing embryo, this type of inhibition or oxidative stress is lethal and can eventually trigger cells to undergo apoptosis (self-destruct prematurely). Chemicals like TPTH ruin the cell\u0026rsquo;s natural defences by creating a cascade of harmful molecules that act like microscopic wrecking balls (Ghazi et al., 2018; Rajendran et al., 2022). These wrecking balls smash into the cell's protective outer layer and alter organelles like mitochondria, that it important for the cell's power supply and also disrupt the important proteins that are necessary for the cell\u0026rsquo;s growth (Rajendran et al., 2022). For a developing embryo, this type of severe biological stress is dangerous and often fatal (Predarska et al., 2023). It forces cells to self-destruct way too early, leaving the embryo without the healthy pieces it needs to build vital organs, a brain, or a strong skeleton.\u003c/p\u003e \u003cp\u003eDifferent organotin compounds, including TPTH, are significant endocrine disruptors, and eventually, by this type of disruption, they send wrong signals during various stages of development (Marques et al., 2018; De et al., 2019). A previous study has demonstrated the severe impact of TPTH on developing animals. For instance, in the case of pregnant mice, exposure to TPTH causes high rates of embryo death, and surviving offspring often suffer from reduced body weight, cleft palates, and poorly formed skull bones (De et al., 2019). Similarly, research on aquatic life like zebrafish and medaka fish embryos shows that these compounds cause delayed hatching, pericardial oedema (fluid around the heart), spinal curvature, abnormal eye development, and swimming disabilities (Wang et al., 2008; Predarska et al., 2023; Hu et al., 2006).\u003c/p\u003e \u003cp\u003eDespite this existing knowledge, observing the direct effects of TPTH in mammals is complicated. A mother\u0026rsquo;s liver and immune system try to filter out toxins before they reach the babies, making it difficult for researchers to know exactly how much damage the chemical itself is doing directly to the embryo versus how much is a side effect of the mother being sick (Lane et al., 2022; Wang et al., 2008). To bypass this complicated maternal filtering system or to mimic this system, researchers use a highly reliable model, namely the chick embryo. Developing inside an egg, the chick embryo is a good, self-contained biological system. If a chemical is introduced directly into a fertilized egg, the embryo is exposed to the substance without any interference from a mother hen (Lu et al., 2022; Wu et al., 2020). Because the chemical is trapped inside the shell, it creates a scenario of chronic exposure, making the chick embryo an ideal model for observing how TPTH alters physical birth defects, growth delays, and organ malformations.\u003c/p\u003e \u003cp\u003eWhile the endocrine-disrupting, cytotoxicity, reproductive toxicity and developmental toxicity of triphenyltin hydroxide (TPTH) are documented in various mammalian and aquatic systems (Clasen et al., 2017; Barkhordari et al., 2024; Hassan et al., 2025), its particular teratogenic and genotoxic toxicity in early amniote embryogenesis remains poorly studied. A major problem in current research is figuring out exactly how much TPTH directly affects a developing embryo. In standard mammalian studies, the mother's body and the placenta get in the way, making it difficult to see the chemical's true impact. To address this major limitation, the in ovo chick embryo model acts as a self-contained in vivo system. It eliminates maternal confounding factors, thereby allowing for the direct assessment of morphometric anomalies and other systemic toxicity during critical windows of organogenesis. Consequently, this study provides novel evidence directly linking TPTH exposure to specific morphological defects and in vivo damage during early avian development. Despite all of this urgent need, a significant gap exists in the literature, as to date, no study has investigated the embryotoxic effects of TPTH using the chick embryo model. Therefore, to the best of our knowledge, this research provides the first comprehensive evaluation of its teratogenic impacts in developing chick embryos. So finally, understanding these detailed developmental disruptions caused by TPTH is not just about protecting chickens; it is about uncovering the broader biological risks this chemical poses to wildlife ecosystems and human health, highlighting the urgent need for a deeper understanding of its toxicity.\u003c/p\u003e"},{"header":"Materials and methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eTriphenyltin hydroxide (CAS 76-87-9), mitomycin C (MMC, CAS 50-07-7) and Giemsa\u0026rsquo;s stain (CAS 51811-82-6) were obtained from Sigma Aldrich, Germany, Pvt Ltd. Dimethyl sulfoxide (DMSO) (CAS 67-68-5), May-Grunwald solution (CAS 67-56-1), Xylene (CAS 1330-20-7), DPX mountant (CAS 84-74-2), methanol (CAS 67-56-1), and ethanol (CAS 64-17-5) were purchased from Himedia Laboratories, India Pvt Ltd. All the chemicals used in this study were of analytical grade, and reagents and stains were prepared freshly.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEgg procurement, incubation and ethical approval\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFertilized white leghorn chicken (\u003cem\u003eGallus gallus domesticus\u003c/em\u003e) eggs weighing from 60\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g were purchased from an authorised vendor's hatchery (Ishwar Hatchery, Guwahati, Assam, India). All the experimental eggs were incubated in a rotating incubator at 37\u0026deg;C with approximately 65\u0026ndash;68% relative humidity. Proper hygiene was maintained to avoid any kind of infection.\u003c/p\u003e\u003cp\u003e The present study approval was obtained from the Institutional Animal Ethics Committee (IAEC) of The Assam Royal Global University, Guwahati, Assam, India; Reference No: 2286/PO/Re/S/2024/CCSEA.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAdministration of the test substance\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTPTH was dissolved in dimethyl sulfoxide (DMSO) due to its insolubility in water. The concentration of the test substance was 4 mg/1ml of DMSO. For all the experimental groups, treatments were given under a sterile laminar flow hood to prevent contamination, and the puncture sites were immediately sealed with sterile adhesive tape to maintain sterility. The whole experiment included six replicates (n\u0026thinsp;=\u0026thinsp;6) per group.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAcute toxicity study\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor assessing the lethal dose (LD\u003csub\u003e50\u003c/sub\u003e), a total of 180 eggs were randomly divided into six groups, containing six eggs in each group (n\u0026thinsp;=\u0026thinsp;6). Further, eggs were treated with increasing concentrations of TPTH, i.e. with 300 \u0026micro;g/egg, 350 \u0026micro;g/egg, 400 \u0026micro;g/egg, 450 \u0026micro;g/egg, and 500 \u0026micro;g/egg up to 96 h. The eggs were candled every 24 h intervals to determine the survivability of the embryos, and the dead embryos were discarded and recorded. The percentage of dead and live animals in each treatment group was calculated for 24 h, 48 h, 72 h and 96 h exposure, and the LD\u003csub\u003e50\u003c/sub\u003e values were calculated by probit analysis using MS Excel (Windows, MS Office 2021).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eDevelopmental toxicity study\u003c/h3\u003e\n\u003cp\u003eTo assess the toxicological potential of Triphenyltin hydroxide (TPTH), a total of 36 eggs were randomly divided into six study groups containing six eggs in each group (n\u0026thinsp;=\u0026thinsp;6) namely, Group I (Control; No treatment); Group II (Positive control; MMC); Group III (Vehicle control; DMSO); Group IV (TPTH, 1% of 96 h LD\u003csub\u003e50\u003c/sub\u003e); Group V (TPTH, 5% of 96 h LD\u003csub\u003e50\u003c/sub\u003e); Group VI (TPTH, 10% of 96 h LD\u003csub\u003e50\u003c/sub\u003e). Before giving the treatment, the eggs were properly sterilized with 70% alcohol. The doses were given in two injection routes to target different parts of the eggs. For the early toxicity study, doses were given to the pointed end of the egg before the experiments. For the HET-MN and cytotoxicity assessment, doses were given in the blunt end for the treatment to directly reach to the chorioallantoic membrane (CAM) via the air sac. After all the treatment, the eggs were properly sealed with sterilized adhesive tapes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMorphometric assessment\u003c/h2\u003e \u003cp\u003eAfter the exposure period (which corresponded to stages HH-12, 13, and 21\u0026ndash;24), eggs were cracked open and transferred to a phosphate-buffered saline (PBS, pH 7.4) and later placed on a glass slide to observe any kind of alterations in the morphology. A Light stereomicroscope was used to check their morphology by following the guidelines of Hamburger and Hamilton (1951). To assess other physical parameters like body length, head plus bill length, forelimb length, hindlimb length, and eye diameter, an image analysis software called Fiji (ImageJ 2.16.0, for Windows) was used. For consistency, we always measured the limbs and eye diameter on the right side of the embryo.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHen's egg test for micronucleus (HET-MN) assay\u003c/h3\u003e\n\u003cp\u003eThe present test was performed by following Chaubey et al. (1993) and Wolf and Luepk (1997) with minor modifications. For the HET-MN assay, eggs were cracked open on day 11 of incubation. Later, a blood sample was collected from each embryo by pricking the arterio-umbilicus sinistra using a heparinized capillary tube. Then, a blood smear was made on a clean, grease-free slide. To make the cells visible, we dyed them using a two-step process. First, we applied a May-Grunwald stain and washed the slides three times with distilled water. Then, we soaked them in a diluted Giemsa stain for 10 min and gave them a final, thorough wash. Once the slides were dry, we treated them with xylene for 5 min, and then they were mounted using a cover slip with DPX. 1000 cells per slide were examined using a compound light microscope,\u003c/p\u003e\n\u003ch3\u003eCytotoxicity study\u003c/h3\u003e\n\u003cp\u003eTo figure out if TPTH was toxic to the cells, we calculated the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs). This PCE/NCE ratio basically acts as a marker to show how well the cells are multiplying. We looked at the different subgroups of PCEs and NCEs in the peripheral blood of the chick embryos. To correctly identify the different types of red blood cells, we used the classification guides from Lucas and Jamroz (1961) and Wolf and Luepke (1997). We randomly examined about 1,000 cells on each slide. Our goal was to identify two specific types of red blood cells: polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE), following established scientific guidelines. We could easily tell these cells apart based on their size, shape, and colour. The PCEs were large, round cells with round centres (nuclei) that turned blue from the dye. In contrast, the NCEs were oval-shaped cells that turned pink.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was done with the help of SPSS 21.0 (IBM Corp., Armonk, New York), and the experimental data were presented as the Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Comparisons between the control and different treatment groups were conducted using one-way analysis of variance (ANOVA) to test the levels of significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAcute toxicity and survivability study\u003c/h2\u003e \u003cp\u003eTo determine the LD\u003csub\u003e50\u003c/sub\u003e of TPTH, the embryos were exposed to increasing concentrations of Triphenyltin hydroxide (TPTH), i.e. 0 \u0026micro;g/egg, 300 \u0026micro;g/egg, 350 \u0026micro;g/egg, 400 \u0026micro;g/egg, 450 \u0026micro;g/egg, and 500 \u0026micro;g/egg for 24 h, 48 h, 72 h, and 96 h. The median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) was determined by employing probit analysis in MS Excel for Windows (MS Office 2021). The LD\u003csub\u003e50\u003c/sub\u003e values as calculated are shown in Table\u0026nbsp;1. The LD\u003csub\u003e50\u003c/sub\u003e of TPTH at 24 h, 48 h, 72 h, and 96 h exposure periods are observed as 537.1\u0026thinsp;\u0026plusmn;\u0026thinsp;35.81 \u0026micro;g/egg, 424.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.11 \u0026micro;g/egg, 372.9\u0026thinsp;\u0026plusmn;\u0026thinsp;22.82 \u0026micro;g/egg, and 342.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27 \u0026micro;g/egg, respectively. For further experimentation to assess the toxicological potential of TPTH, different concentrations of TPTH were chosen based on the 96 h LD\u003csub\u003e50\u003c/sub\u003e of TPTH in the chicken embryos. The selected test concentrations were 1%, 5% and 10% of 96 h LD\u003csub\u003e50\u003c/sub\u003e (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThe LD₅₀ antilog values showed a progressive decline with increasing exposure time, indicating time-dependent toxicity. At 24 h, the LD₅₀ was highest (537.1), which gradually decreased to 424.3 at 48 h, 372.9 at 72 h, and reached the lowest value of 342.5 at 96 h (Fig.\u0026nbsp;1A). This downward trend suggests that longer exposure enhances toxic effects, thereby requiring lower doses to reach 50% lethality. The regression analysis (R\u0026sup2; = 0.9191) indicates a strong correlation between exposure duration and LD₅₀ reduction.\u003c/p\u003e \u003cp\u003eThe survival curve demonstrates a clear dose- and time-dependent decline in embryo viability following exposure. Control embryos maintained 100% survival throughout the 96 h observation period, while treated groups showed progressively reduced survival with increasing concentration and exposure duration (Fig.\u0026nbsp;2B). At 300 \u0026micro;g/egg, survival decreased moderately, whereas at 500 \u0026micro;g/egg, survival dropped sharply, reaching 0% by 96 h. These findings are consistent with the calculated LD₅₀ values, which decreased from 537.1 \u0026micro;g/egg at 24 h to 342.5 \u0026micro;g/egg at 96 h, confirming enhanced toxicity with prolonged exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHen's egg test for micronucleus (HET-MN) induction\u003c/h2\u003e \u003cp\u003eThe genotoxic potential of the Triphenyltin hydroxide (TPTH) was assessed by using the HET-MN assay. Significant increments in the frequencies of MN in TPTH-treated embryos were observed as compared to the control.\u003c/p\u003e \u003cp\u003eThe MMC-treated group showed a rise in the MN frequency to 7.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) as compared to the control group (1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17%) (Fig.\u0026nbsp;1C). Similarly, the embryos treated with 1%, 5%, and 10% concentrations of TPTH showed a significant increment of MN (%) to 2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs control), 3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs control), and 6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs control), respectively (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity study\u003c/h2\u003e \u003cp\u003eTPTH exposure affected the PCE/NCE ratio, indicating cytotoxic effects. As shown in Fig.\u0026nbsp;1D, no significant change was observed in the DMSO group (1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10) as compared to the control group (1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08), confirming the validity of the assay. The positive control (MMC) group (0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) caused a decrease in the PCE/NCE ratio as compared to the control group, although the values are not statistically significant. On the other hand, TPTH treatment produced a dose-dependent reduction in the PCE/NCE ratio. At the lowest dose (1%) (0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), the decrease was statistically significant at \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e as compared to the control group, whereas at 5% and 10% concentrations (0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) (0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), the decreases were highly significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) as compared to the control group. These results indicate that TPTH induces cytotoxicity in a concentration-dependent manner (Fig.\u0026nbsp;1D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMorphological assessment of TPTH-induced Chick Embryos\u003c/h2\u003e \u003cp\u003eEmbryos in the control (Group A) (Fig.\u0026nbsp;3) and vehicle control (Group B) (Fig.\u0026nbsp;3) exhibited robust and healthy development, reaching approximately HH Stage 12 to 13 (roughly 45\u0026ndash;52 hours of incubation). At this stage, the normal morphological stages were clearly visible, namely, the development of the cephalic portion, i.e. prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain), showed distinct boundaries and healthy expansion. The heart tube had successfully begun its vital rightward \"C-looping,\" an ideal identification of HH Stage 12. Also, the embryos displayed a straight and symmetrical body axis. The somites were sharp, square-shaped, and organized in parallel pairs along the neural tube. Furthermore, there was a noticeable beginning of cranial flexure (the bending of the head downward), consistent with late HH Stage 12. The near-identical appearance of Group B (DMSO vehicle) to Group A confirms that the solvent used to deliver the TPTH did not interfere with the natural developmental trajectory of the embryos.\u003c/p\u003e \u003cp\u003eEmbryos treated with the lowest dose of TPTH (Group C) (Fig.\u0026nbsp;3) showed only minor deviations from the control groups. Morphologically, these embryos appeared to be at HH Stage 12, suggesting a very slight developmental lag. While the brain vesicles and heart looping remained largely intact, there was a minor reduction in the overall transparency of the neural tube compared to the controls. This indicates that while 4 \u0026micro;g of TPTH is not immediately lethal, it begins to exert a mild inhibitory pressure on the developing embryo.\u003c/p\u003e \u003cp\u003eA significant shift in morphology occurred at the 20 \u0026micro;g dosage (Group D) (Fig.\u0026nbsp;3). These embryos failed to reach the HH 13 milestone seen in the controls, appearing arrested or delayed at HH Stage 11. Key abnormalities include microcephaly, in which the forebrain (prosencephalon) was noticeably smaller and lacked the full \"bulge\" seen in healthy embryos. Although the heart tube was present, the looping process was significantly delayed. The heart appeared as a smaller, more vertical structure. The posterior somites were less distinct, suggesting that the chemical was beginning to interfere with the process of somitogenesis.\u003c/p\u003e \u003cp\u003eThe most profound toxic effects were observed in the high-dose group (Group E) (Fig.\u0026nbsp;3). These embryos did not conform to any single HH stage because their development was not just delayed, but severely malformed. They exhibited characteristics of an arrested HH Stage 10, but with pathological deviations; a sharp bend was observed in the middle of the body axis. This suggests a failure in the structural integrity of the notochord or a collapse of the neural tube, which is a severe teratogenic outcome. The entire head region was underdeveloped. The individual vesicles of the brain were indistinguishable, appearing as a single, stunted mass of tissue. The heart remained a primitive, unlooped tube. This lack of looping is a lethal defect that would prevent the establishment of a functional blood flow system. The tail region was significantly shorter than in the controls, indicating that TPTH inhibited the elongation of the embryo from the primitive streak and Hensen\u0026rsquo;s node.\u003c/p\u003e \u003cp\u003eSimilarly, the embryotoxic and teratogenic effects of Triphenyltin hydroxide (TPTH) on chick embryos were characterized by a clear dose-response gradient, with the control (Group A) (Fig.\u0026nbsp;4) and vehicle control (Fig.\u0026nbsp;4) (Group B) maintaining normal developmental progression consistent with Hamilton-Hamburger (HH) Stage 21\u0026ndash;24. In contrast, TPTH-treated embryos exhibited profound morphological deviations, beginning with compromised vascular integrity that manifested as localized petechial haemorrhages in the abdominal region of the 4\u0026micro;g low-dose group (Group C) (Fig.\u0026nbsp;4). This progressed to widespread severe haemorrhage and blood stasis in the 40\u0026micro;g high-dose group (Group E) (Fig.\u0026nbsp;4), particularly concentrated around the primitive heart and brain vesicles. Significant craniofacial and ocular malformations were also prevalent in the higher dose groups (D and E), most notably a marked reduction in the diameter of the optic cup (microphthalmia) and distinct hydrocephalus-like cranial oedema around the mesencephalon and rhombencephalon, indicative of developmental failure. Furthermore, Group E demonstrated severe growth retardation, evidenced by a significantly decreased crown-rump length and overall body stunting. Axial development was similarly impaired, with treated embryos exhibiting hypoplasia of the limb and tail buds, caudal regression, and a breakdown of somite organization where indistinct boundaries suggested a disruption of the molecular clock governing somitogenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBody length\u003c/h2\u003e \u003cp\u003eTo evaluate the impact of TPTH on body length, measurements were recorded on days 3, 5, 7, and 9. Treatment with TPTH showed a significant reduction of body length in TPTH-exposed groups in a dose-dependent manner throughout the study period. On day 9, exposure to TPTH at a low dose (TPTH-L) to experimental embryos showed a significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;5A) of body length to approximately 41.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mm as compared to control (45.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 mm) (Fig.\u0026nbsp;5A). The experimental embryos treated with TPTH-M and TPTH-H showed further reduced levels of body length to 38.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs control) and 34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs control), respectively (Fig.\u0026nbsp;5A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEye diameter\u003c/h2\u003e \u003cp\u003eTo investigate the developmental impact of TPTH on ocular growth, eye diameter was measured across the four sampling intervals. The data reveal that continuous exposure to TPTH resulted in a progressive and dose-dependent decline in eye diameter throughout the 9-day study. By the final observation on Day 9, embryos in the low-dose group (TPTH-L) exhibited a significant reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;5B) in eye diameter, reaching approximately 7.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mm compared to the control group's 8.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 mm. This inhibitory trend was even more pronounced in the higher concentration groups; TPTH-M and TPTH-H treatments led to further decreases in ocular size, measuring 6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs. control) and 5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs. control), respectively (Fig.\u0026nbsp;5B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eForelimb\u003c/h2\u003e \u003cp\u003eThe influence of TPTH on limb morphogenesis was characterized by a noteworthy decline in forelimb length throughout the 9-day observation period. Experimental embryos subjected to TPTH exhibited a concentration-related inhibitory effect on limb elongation starting as early as Day 3, where the TPTH-H group already showed a drastic reduction compared to the control. On the final day of the study (Day 9), the control group achieved a forelimb length of approximately 11.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mm, whereas the TPTH-L group showed a significant decrease to 10.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;5C). More severe growth suppression was recorded in the TPTH-M and TPTH-H groups, with lengths reaching only 9.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mm and 8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm, respectively (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs. control) (Fig.\u0026nbsp;5C), highlighting the potent limb-stunting effects of the compound.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHead and bill\u003c/h2\u003e \u003cp\u003eTo evaluate the impact of TPTH on cranial and bill development, measurements of the head\u0026thinsp;+\u0026thinsp;bill length were recorded. Consistent with other growth parameters, treatment with TPTH led to a marked suppression of head and bill growth in a concentration-related manner. On Day 9, exposure to the low dose (TPTH-L) resulted in a significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;5D) in length to 14.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mm as compared to the control group (15.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mm). The experimental groups treated with higher concentrations, TPTH-M and TPTH-H, showed further reduced levels of 12.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs. control) and 11.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e vs. control), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHindlimb\u003c/h2\u003e \u003cp\u003eThe study also evaluated the elongation of the hindlimb to determine if the growth inhibitory effects were uniform across the body. Similar to the forelimbs, hindlimb growth was significantly impaired by TPTH exposure at all tested concentrations. On Day 9, the mean hindlimb length of the control group was 14.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 mm. In contrast, the TPTH-exposed groups showed significant declines (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;5E), with lengths measuring 13.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mm for TPTH-L, 12.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mm for TPTH-M, and a minimum of 10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm for the TPTH-H group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study investigates the acute toxicity, genotoxicity, cytotoxicity, and teratogenic potential of Triphenyltin hydroxide (TPTH) using an in ovo chick embryo model by providing critical insights into its developmental toxicity. Organotin compounds, including TPTH and its widely studied structural analogue, Tributyltin (TBT), are ubiquitous environmental pollutants recognized for their potent biocidal properties and severe developmental toxicity. Our acute toxicity evaluation demonstrated a progressive, time-dependent decline in the LD\u003csub\u003e50\u003c/sub\u003e values for TPTH, dropping from 547.37 \u0026micro;g/egg at 24 h to 395.74 \u0026micro;g/egg at 96 h. This downward trend aligns closely with the established toxicokinetics of organotins in avian embryos, where their highly lipophilic nature facilitates rapid penetration of the vitelline membrane and subsequent bioaccumulation in lipid-rich embryonic tissues (Frouin et al., 2008).\u003c/p\u003e \u003cp\u003eSimilar time-dependent mortality and enhanced toxicity with prolonged exposure have been documented in recent parallel studies examining the cytotoxicity and genotoxicity of TBT in early \u003cem\u003eGallus gallus domesticus\u003c/em\u003e embryos (Mandal et al., 2023). To elucidate the mechanistic basis of this toxicity, cellular damage was evaluated using the Hen's egg test for micronucleus (HET-MN) induction and the Polychromatic Erythrocyte-to-Normochromatic Erythrocyte (PCE/NCE) ratio. The significant, dose-dependent induction of micronuclei frequency in TPTH-treated embryos provides clear evidence of profound genomic instability. Micronucleus formation in avian peripheral blood erythrocytes is a reliable biomarker of clastogenesis or aneugenesis, often triggered by excessive Reactive Oxygen Species (ROS). Furthermore, the highly significant decline in the PCE/NCE ratio in our study indicates severe cytotoxicity and the suppression of erythropoiesis. These findings mirror the exact cytotoxic and genotoxic paradigms observed in TBT-exposed chick embryos, where TBT similarly increased nuclear abnormalities and dramatically altered the PCE to NCE ratio, indicating severe bone marrow-equivalent toxicity and long-term genomic complications (Mandal et al., 2023). Beyond sub-cellular damage, our morphological assessment revealed severe teratogenic deviations at both early (HH 10\u0026ndash;13) and later (HH 21\u0026ndash;24) developmental stages. Embryos exhibited profound structural anomalies, including severe microcephaly, neural tube distortion, blurred somite boundaries, and, notably, the failure of the heart tube to undergo normal rightward C-looping. Organotins are well-documented endocrine disruptors that aberrantly interfere with highly conserved morphogenetic pathways (Varela-Ramirez et al., 2011; Pu et al., 2022). For instance, TBT induces significant morphological and transcriptomic malformations in chicken embryos by altering the expression of key transcription and growth factors governing tissue differentiation (Varela-Ramirez et al., 2011). This targeted disruption, combined with organotin-induced interference of intracellular calcium homeostasis, likely explains the altered heart tubes and the extensive haemorrhaging observed in our TPTH-treated groups. The concurrent observation of microphthalmia and cranial oedema further indicates that TPTH may interfere with proper embryonic osmoregulation and cranial neural crest cell migration. These macroscopic observations of severe teratogenicity were strictly aligned with our quantitative morphometric analyses, which revealed significant, concentration-dependent growth retardation across body length, eye diameter, and limb lengths. Collectively, this cascade of toxic events, starting from genotoxic damage, cytotoxicity, and the alteration in the morphological structures, highlights the potent developmental risks posed by TPTH exposure and aligns accurately with the broader ecotoxicological consensus regarding organotin embryotoxicity in avian models (Said and El Zokm, 2025; Frouin et al., 2008; Siddique et al., 2026).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study proved that Triphenyltin hydroxide (TPTH) is a highly potent embryotoxic and teratogenic agent in the avian model. Through a comprehensive in ovo assessment, the data demonstrate that TPTH exposure leads to a time- and dose-dependent increase in embryonic mortality, characterized by profound genomic instability and severe cellular toxicity. Moreover, TPTH disrupts critical morphogenetic pathways that result in alteration of cardiovascular anomalies, such as the failure of heart looping, alongside craniofacial malformations, and significant overall growth retardation. By providing the first comprehensive evaluation of TPTH-induced developmental toxicity in chick embryos, this research highlights the extreme alterations of rapidly developing biological systems to organotin contamination. Ultimately, these findings underscore the broader ecological risks posed by agricultural runoff and emphasize the urgent need for stricter environmental monitoring to protect wildlife populations and other living beings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their deepest gratitude to The Assam Royal Global University, Guwahati, Assam, for providing infrastructural support for the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT roles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDipsanu Paul:\u0026nbsp;\u003c/strong\u003eWriting - Original draft, Methodology, Data curation, Formal analysis, Visualization; \u003cstrong\u003eSweety Nath Barbhuiya, Sarbani Giri, Dharmeswar Barhoi:\u0026nbsp;\u003c/strong\u003eConceptualization,Supervision, Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest associated with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarbosa, C. 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DOI: 10.1016/j.chemosphere.2019.125149\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"The Assam Royal Global University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triphenyltin hydroxide, Genotoxicity, Cytotoxicity, Teratogenicity, Chick embryo, Developmental toxicity","lastPublishedDoi":"10.21203/rs.3.rs-9703101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9703101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriphenyltin hydroxide (TPTH) is a widely used agricultural fungicide and environmental pollutant well known for its potent toxicological potential. Despite these, its embryotoxic effects remain unexplored in avian models. This study presents a novel evaluation of TPTH\u0026rsquo;s acute toxicity, genotoxicity, cytotoxicity, and teratogenic potential using the \u003cem\u003ein ovo\u003c/em\u003e chick embryo model. Fertilized chicken eggs were exposed to varying TPTH concentrations, revealing a time-dependent median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) decline from 537.1 \u0026micro;g/egg at 24 h to 342.5 \u0026micro;g/egg at 96 h. Later, evaluations using 1%, 5%, and 10% of the 96 h LD\u003csub\u003e50\u003c/sub\u003e demonstrated profound sub-cellular damage. The Hen's egg test for micronucleus (HET-MN) assay showed a significant dose-dependent induction of micronuclei, confirming genomic instability. Additionally, a reduced polychromatic to normochromatic erythrocyte (PCE/NCE) ratio indicated severe cytotoxicity and suppressed erythropoiesis. Morphometric assessments revealed teratogenic alterations, including failure of heart tube C-looping, microcephaly, cranial oedema, microphthalmia, localized haemorrhaging, and concentration-dependent growth retardation across body length, eye diameter, and limb elongation. These findings demonstrate that TPTH induces potent developmental toxicity through a lethal cascade of genotoxic, cellular damage and structural malformations, highlighting its extreme embryotoxic risks to avian populations and associated ecosystems.\u003c/p\u003e","manuscriptTitle":"Triphenyltin hydroxide (TPTH) exposure induces genotoxicity, cytotoxicity and developmental toxicity in Chick embryo, Gallus gallus domesticus: An in ovo study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 06:10:09","doi":"10.21203/rs.3.rs-9703101/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0bdd76b7-e964-434c-91b8-b683c34ed140","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":68089450,"name":"Developmental Biology"},{"id":68089451,"name":"Molecular Biology"},{"id":68089452,"name":"Toxicology"}],"tags":[],"updatedAt":"2026-05-15T06:10:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 06:10:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9703101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9703101","identity":"rs-9703101","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}