Developmental toxicity of fluconazole and 1,2,4-triazole in non-target aquatic vertebrates

Developmental toxicity of fluconazole and 1,2,4-triazole in non-target aquatic vertebrates | 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 Article Developmental toxicity of fluconazole and 1,2,4-triazole in non-target aquatic vertebrates Barbora Riesova, Lorena Agostini Maia, Renata Hesova, Nikola Peskova, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7570738/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Fluconazole (FLU) is a widely used antifungal agent frequently detected in surface waters because of its extensive use in medicine, agriculture, and personal care products. Despite concerns about its persistence and developmental toxicity in aquatic species, its effects on amphibians remain poorly understood. This study aimed to assess the developmental and molecular effects of FLU and its structural core, 1,2,4-triazole (TRI), in amphibian embryos. Xenopus laevis embryos were exposed to FLU or TRI and evaluated for mortality, hatching rate, heart rate, body length, malformation incidence, and changes in gene expression. Even at low micromolar concentrations, both azoles altered the expression of Wnt- and BMP-associated genes, indicating disruption of these signaling pathways. At higher micromolar concentrations, these molecular changes were accompanied by early signs of developmental abnormalities, which intensified at the highest doses. Observed phenotypes included reduced head size, altered skin pigmentation, prolonged body length, changes in heart rate, and mild digestive tract malformations. These findings demonstrate that even the core structural motif TRI can disrupt key developmental signaling pathways in vertebrate embryos, underscoring the need for closer monitoring of azole compounds in aquatic environments. Given the fundamental role of these pathways in vertebrate development, the results raise concerns about potential risks from long-term or prenatal exposure to azoles, in both environmental and clinical contexts. Biological sciences/Developmental biology Biological sciences/Molecular biology Biological sciences/Physiology Biological sciences/Zoology Azole antifungals embryotoxicity morphometric analysis gene expression Wnt/BMP signaling Xenopus laevis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Azole antifungal compounds such as fluconazole (FLU) are widely used across human and veterinary medicine, agriculture, and the personal care industry (Chakraborty et al., 2023 ; Jørgensen and Heick, 2021 ; Sheehan et al., 1999 ). Their therapeutic efficacy against fungal infections has made them indispensable in clinical practice, while their antifungal properties are also utilized in shampoos, soaps, skin creams, and agricultural fungicides (Lee et al., 2023 ; Maertens, 2004 ; Sheehan et al., 1999 ). A common structural feature of many azoles, including FLU, is the 1,2,4-triazole (TRI) ring – a heterocyclic motif essential for antifungal activity that is also present in numerous other pharmaceuticals and agrochemicals (Gupta et al., 2023 ). However, the extensive use of these compounds has resulted in their frequent detection in wastewater effluents and surface waters worldwide (Bhagat et al., 2021 ; Kahle et al., 2008 ; Lu et al., 2024 ; Płatkiewicz et al., 2025 ; Subramanian et al., 2023 ). Conventional wastewater treatment plants often fail to remove them completely, leading to persistent low-level contamination. Measured nanomolar concentrations of azole compounds such as FLU, climbazole, and tebuconazole have been reported in rivers across Europe, Africa, and Asia, raising concerns about their ecotoxicological impact (Halešová et al., 2022 ; Iancu et al., 2024 ; Liu et al., 2015 ; Płatkiewicz et al., 2025 ; Wick et al., 2010 ; Wilkinson et al., 2022). Although originally developed for therapeutic use in humans and animals, azoles are increasingly recognized for their unintended effects on non-target organisms. Studies in zebrafish ( Danio rerio ) have shown that they can disrupt early development, causing pericardial edema, altered heart rate, and embryonic malformations (Barenys et al., 2019 ; Bhagat et al., 2021 ). Yet, little is known about their developmental effects in amphibians and higher vertebrates. Amphibians are particularly vulnerable to waterborne contaminants due to their permeable skin and aquatic life stages. The African clawed frog, Xenopus laevis , is a long-established and highly sensitive model organism that has made fundamental contributions to developmental and cell biology, as well as to ecotoxicology (De Robertis and Gurdon, 2021 ; Gao and Shen, 2021 ). Its external fertilization, rapid development, and highly conserved genetic pathways with other vertebrates, including humans, make it especially suitable for assessing the impact of environmental chemicals (Gao and Shen, 2021 ). In the present study, we investigated the developmental toxicity of FLU and its structural core TRI in Xenopus laevis embryos, measuring mortality, hatching success, heart rate, body length, malformation frequency, and expression of key genes involved in early embryogenesis. Both compounds induced morphological and physiological changes, and at the molecular level, altered the expression of xbra , xolloid, β-catenin , chordin and noggin , which is consistent with modulation of Wnt (Wingless/Integrated) and BMP (Bone Morphogenetic Protein) signaling pathways. These results suggest that even simple triazole structures can interfere with conserved developmental mechanisms and raise important questions about the environmental and health safety of azole antifungals. Results Azole antifungals alter tadpole morphology To investigate the developmental toxicity of azole antifungals, we initially monitored the development of Xenopus laevis embryos during early stages but observed no overt morphological changes. Therefore, we focused our analysis on later time points, specifically at Nieuwkoop and Faber (NF) stage 46, when cumulative effects become more apparent. At this stage, we examined gross morphology following continuous exposure to 1–1,000 µg/L of FLU and TRI. Representative phenotypes for both treatments are shown in Fig. 1 (FLU) and Fig. 2 (TRI). Untreated control animals developed normally with well-formed heads, regular pigmentation, and a straight body axis, but exposed embryos exhibited noticeable morphological deviations. In FLU-treated groups, we observed several recurring abnormalities, including pigmentation defects – including both hyperpigmentation and hypopigmentation – and craniofacial malformations such as reduced head size and head edema. Heart edema and visible gut malformations were also present in a subset of tadpoles (Fig. 1 ). TRI exposure resulted in a similar but more variable range of phenotypes. Along with pigmentation defects, observed phenotypes included reduced eye spacing, completely undeveloped intestines, and misshapen anterior structures (Fig. 2 ). Taken together, these observations suggest that both FLU and TRI interfere with normal tissue patterning and organogenesis in Xenopus laevis embryos. Dose-dependent differences in malformation incidence We next quantified the percentage of malformed tadpoles across increasing concentrations of both compounds to evaluate the dose-response relationship (Fig. 3 ). FLU exposure resulted in a saturating malformation curve, with incidence plateauing at approximately 40% from the lowest tested concentration (1 µg/L) and remaining stable at higher doses. This suggests that even low concentrations of FLU are sufficient to trigger developmental disruptions and that the system reaches a response ceiling early. In contrast, TRI exposure followed a clear dose-dependent trend. At 1 µg/L, only a small proportion of animals exhibited any malformation. As the concentration increased to 100 µg/L, the incidence rose to approximately 20%, and at 1,000 µg/L, around 25% of the tadpoles displayed visible abnormalities. These findings indicate a progressive teratogenic effect of TRI, potentially linked to cumulative molecular disruptions at higher doses. The contrasting saturation-versus-linear trend observed for FLU and TRI, respectively, points to possible differences in their mechanisms of action or bioavailability in the embryo. Malformation types differ between fluconazole and triazole Beyond quantifying malformation rates, we analyzed the spectrum of phenotypic defects to identify potential differences in the type and severity of abnormalities caused by each compound (Suppl. Figure 1) . FLU exposure most frequently resulted in altered pigmentation, craniofacial changes (reduced head size, head edema), cardiac edema, anterior-posterior axis deformation, and intestinal abnormalities. Notably, darker upper flank pigmentation was observed specifically at 100 µg/L. These phenotypes were generally consistent across concentrations, suggesting that FLU triggers a characteristic malformation profile that appears even at low doses. TRI exposure induced a broader but generally less frequent set of malformations. Craniofacial anomalies included reduced head size, reduced interocular distance, and asymmetry of the eyes or head. Intestinal defects ranged from mild deformation to complete absence of intestinal structures. Pigmentation changes were diverse, encompassing dark patches, hypopigmentation, and star-like pigment patterns absent in controls or FLU-treated animals. Additional phenotypes unique to TRI included axial curvature and underdeveloped fins. These effects tended to become more prevalent at higher concentrations, with complete intestinal absence and multiple combined malformations occurring at 1,000 µg/L. These qualitative differences indicate that while both compounds disrupt overlapping developmental processes, FLU produces a narrower and more consistent malformation profile, whereas TRI has a more variable and concentration-dependent impact, potentially reflecting a broader disruption of axial patterning. Cardiac and morphometric effects suggest physiological disruption To further explore the physiological consequences of azole exposure, we measured heart rate and body length in tadpoles at NF stage 46 (Fig. 4 ). Both compounds caused a significant increase in heart rate at most concentrations tested, indicating that cardiac development or autonomic regulation may be altered. Interestingly, at the highest TRI concentration (1,000 µg/L), we observed a notable drop of heart rate, suggesting a possible toxic threshold beyond which physiological function becomes impaired. Morphometric measurements revealed additional differences between the two compounds. FLU exposure led to a measurable elongation of the body axis, potentially reflecting disruptions in growth-regulating endocrine pathways and processes linked to mesoderm formation and muscle development, similar to patterns reported for cortisol and Bisphenol A exposure in fish and other model organisms (Rodrigues et al., 2025 ; Senarath Pathirajage and Rajapaksa, 2024 ). In contrast, TRI-treated tadpoles did not show significant changes in body length, even at higher concentrations. These data point to distinct developmental effects of each compound – while both interfere with cardiac output, only FLU appears to affect axial growth. Gene expression analysis reveals Wnt and BMP pathway disruption To identify the molecular pathways affected by azole exposure, we analyzed the expression of selected developmental genes by quantitative PCR, focusing on components of the Wnt and BMP signaling networks. As shown in Fig. 5 , even low-level TRI exposure of 10 µg/L upregulated the expression of xbra , and xolloid , while β-catenin , chordin and noggin exhibited upward trends. These changes may reflect activation of the Wnt/β-catenin pathway and concurrent inhibition of BMP signaling via its antagonists, chordin and noggin . Upregulation of xbra reflects enhanced mesodermal induction, while xolloid likely represents a feedback response attempting to restore BMP activity by degrading excess chordin . FLU induced a similar transcriptional profile, though the magnitude of change was generally lower and more variable. In contrast, several downstream BMP effectors – follistatin , sizzled , vent1 , and vent2 – did not show significant expression changes in either treatment ( Suppl. Figure 2 ). This selective transcriptional response indicates that azoles specifically disrupt early morphogen gradients without broadly altering all components of the BMP pathway. Exposure concentrations confirmed by chemical analysis To confirm that the developmental effects observed were consistent with the intended dosing, we analyzed the actual concentrations of FLU and TRI in the exposure water using HPLC ( Suppl. Figure 3 ). Measurements taken during the experiment confirmed that concentrations remained stable and closely matched the nominal values. For the representative treatment groups, we selected and detected FLU at 10 µg/L and TRI at 100 µg/L, validating the accuracy and consistency of chemical exposures used in this study ( Suppl. Figure 3 ). In summary, both FLU and TRI exhibit teratogenic potential in Xenopus laevis embryos, but through partially distinct developmental effects. FLU appears to act rapidly and reach a phenotypic plateau at low concentrations, while TRI induces malformations in a more progressive, dose-dependent manner. The spectrum of malformations and broader gene expression changes caused by TRI suggests a more pervasive disruption of axial patterning. These results, summarized in Fig. 6 , collectively support the hypothesis that azole antifungals interfere with conserved signaling pathways involved in vertebrate embryogenesis, highlighting their potential risk as environmental contaminants and emerging human health hazards. Discussion Our study investigates the effects of triazole-based antifungals, specifically fluconazole (FLU) and its structural core 1,2,4-triazole (TRI), on early embryogenesis in aquatic vertebrates using Xenopus laevis as a model. We show that these compounds selectively disrupt key developmental signaling pathways and induce distinct morphological deformities. Altered gene expression. In our study, we observed the upregulation of xbra and xolloid transcripts, which provides particularly strong evidence for triazole-induced perturbations, whereas other transcripts, such as β-catenin , noggin , and chordin , exhibited upward trends without reaching statistical significance. Together, these results suggest a targeted rather than global reprogramming of developmental networks. The significant induction of xbra points to altered mesodermal specification, as it is a central regulator of mesoderm formation and posterior patterning. Its upregulation implies a shift toward posteriorized mesodermal fates, consistent with the anterior reductions observed in treated embryos (Anderson et al., 2002 ). The observed upregulation of xolloid transcripts, a metalloprotease cleaving the BMP inhibitor chordin (Piccolo et al., 1997 ), may represent a feedback mechanism to rebalance BMP availability. Together, these transcriptional alterations suggest that TRI and FLU exposures perturbs mesodermal allocation and axial patterning through combined effects on Wnt and BMP regulatory networks. Although β-catenin transcript levels trended upward, it is important to note that β-catenin is regulated predominantly by protein stabilization via posttranslational modification and subsequent nuclear localization, not by transcript abundance (Clevers, 2006 ); therefore, mRNA abundance does not provide a direct readout of canonical Wnt pathway activation. Nevertheless, the observed transcriptional profile is consistent with partial Wnt pathway engagement, which could synergize with BMP modulation to reinforce posteriorization. The modest increases in noggin and chordin, two potent BMP antagonists, although not statistically significant, align with this interpretation but require cautious consideration given their variability. (McMahon et al., 1998 ; Stottmann et al., 2001 , Hikasa and Sokol, 2013 ). Interestingly, dickkopf , goosecoid , sizzled , and vent1/2 , were not significantly altered, suggesting that azoles exposure do not induce global transcriptional rewiring of embryonic axes, but rather target specific nodes within the Wnt–BMP regulatory interface. FLU induced similar but weaker changes than TRI, supporting a shared mechanism of action with differing potency. Corresponding phenotypic outcomes. These molecular alterations correlated with phenotypes including reduced head size, craniofacial defects, pigmentation abnormalities, and mild digestive tract malformations. Reduced head size likely reflects anterior tissue loss, a hallmark of posteriorizing influences on axial patterning (Hikasa and Sokol, 2013 ). Although β-catenin, noggin, and chordin showed only non-significant upward trends, even modest shifts in Wnt/BMP dynamics can bias tissue allocation, which may explain the anterior reductions observed in our tadpoles. Abnormal pigmentation, particularly the emergence of star-like pigment cells, is suggestive of disrupted neural crest migration (Kelsh et al., 2009 ), which relies on tightly regulated BMP gradients (Raible and Ragland, 2005 ). Digestive tract malformations and altered heart rate are consistent with mesodermal mispatterning and cardiac lineage imbalance – both processes sensitive to Wnt/BMP signaling dynamics. Notably, similar phenotypes have been observed in zebrafish embryos exposed to other azole antifungals, including pericardial edema and craniofacial deformities (Barenys et al., 2019 ; Bhagat et al., 2021 ), reinforcing the idea of a conserved mode of developmental toxicity across aquatic vertebrates. Mechanistic links between gene expression and phenotype. Importantly, our transcriptional data provide a plausible mechanistic link between signaling disruptions and phenotype. Xbra is expressed in the involuting marginal zone during gastrulation and is regulated by Wnt/β-catenin signaling (Arnold et al., 2000 ). As a mesodermal regulator, its elevation likely shifts tissue allocation toward posterior fates. This posteriorization may be further reinforced by increased xolloid , which promotes chordin degradation and thus indirectly enhances BMP activity in the posterior. These molecular events help explain the anterior reductions seen in our embryos, especially in head structures and the gut. The cardiac implications are also worth noting: although xbra is not directly required for cardiac gene expression, it is essential for early mesoderm formation from which cardiac mesoderm is derived (Tzahor, 2007 ). Any disruptions at this stage could lead to mis-specification of cardiac tissues, potentially explaining heart malformations. Similar links between Wnt signaling and heart morphogenesis have been well established (Foulquier et al., 2018 ; Li et al., 2022 ; Tian et al., 2010 ). Possible involvement of oxidative stress. Previous studies have linked triazoles such as tebuconazole to oxidative stress in vertebrates (Ben Othmène et al., 2022 ; Li et al., 2020 ; Yang et al., 2018 ), mediated by reactive oxygen species (ROS). ROS may increase endothelial permeability (Lum and Roebuck, 2001 ) and disturb osmotic balance (Scallan et al., 2010 ), which could explain the edematous phenotypes seen with triazole exposure. Although ROS were not specifically tested in this study, they may represent a plausible parallel mechanism worth further investigation. Previous data also suggest that early phenotypic differences are more reliably observed in later stages of vertebrate development (Dong, 2024 ), aligning with our decision to focus on later larval stages. Clinical relevance and potential signaling implications. Azole antifungals, particularly fluconazole, remain critical for treating fungal infections in immunocompromised patients, such as those with HIV/AIDS, cancer, or organ transplants (Domingos et al., 2022 ; Katiraee et al., 2015; Ostrosky-Zeichner, 2008 ). Candida albicans remains the leading cause of systemic candidiasis, and the emergence of resistant strains like Candida auris poses additional challenges (Ramli et al., 2024 ; Sanyaolu et al., 2022 ; Worku and Girma, 2020 ). Beyond clinical use, their widespread presence in over-the-counter products such as creams and shampoos contributes to persistent environmental and human exposure. Azoles function by inhibiting cytochrome P450 enzymes (e.g., CYP51), essential for fungal membrane synthesis (Shin et al., 2018 ; Song et al., 2018 ; Zhang et al., 2021 ). However, these same enzymes – and the signaling cascades they modulate – are also found in vertebrate cells (Guengerich et al., 2016 ; Hannemann et al., 2007 ). Our findings reveal that even the core structure of azoles (e.g., TRI) can perturb developmental signaling in Xenopus embryos, with selective effects on Wnt and BMP regulatory networks. While the precise molecular interactions remain to be elucidated, the evolutionary conservation of these pathways highlights the possibility of parallels in vertebrate development, including humans. Fluconazole and similar azole compounds may therefore represent emerging developmental and health hazards, warranting closer toxicological and clinical exploration. Material and Methods Animal husbandry and embryo collection All procedures involving Xenopus laevis were approved by the Ministry of Agriculture of the Czech Republic (Animal Care and Housing Approval: 45055/2020-MZE-18134) and conducted in accordance with Czech legislation and institutional guidelines (MSMT-30784/2022-1). All experiments were conducted in compliance with institutional guidelines and are reported in accordance with the ARRIVE guidelines. Embryos were obtained using standard methods. Briefly, adult males were anesthetized in 20% MS-222 (Sigma-Aldrich, A5040) and testes were surgically removed and stored in cold 1× Marc’s Modified Ringer’s solution (MMR; 100 mM NaCl, 2 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM HEPES, pH 7.4) supplemented with 50 µg/mL gentamicin (Sigma-Aldrich, G3632). Sexually mature females were induced to ovulate via dorsal lymph sac injection of 260 IU human chorionic gonadotropin (hCG; Merck, Ovitrelle 250G), followed by overnight incubation at 18 °C. Eggs were collected the next morning by gentle squeezing and fertilized in vitro using a macerated piece of testis in 0.1× MMR. Embryos were cultured at 18–21 °C in 0.1× MMR and staged according to Nieuwkoop and Faber (Zahn et al., 2022). Assessment of lethal and sublethal endpoints Developmental toxicity was assessed following modified FETAX (Frog Embryo Teratogenesis Assay – Xenopus ) protocols. Freshly fertilized embryos were visually inspected under a stereomicroscope (Olympus SZX7, Japan) and distributed into 24-well microplates (TPP, Switzerland), with 24 embryos per group. The embryos were exposed to three concentrations of each test compound: 1, 100, and 1,000 µg/L for fluconazole (FLU), and 1, 100, and 1,000 µg/L for 1,2,4-triazole (TRI). Control groups included unexposed embryos and embryos exposed to 0.01x MMR. FLU and TRI were dissolved in DMSO (dimethyl sulfoxide) and added to the embryo culture medium, while the equivalent volume of DMSO alone was used as a negative control. Exposure solutions including controls were renewed daily with freshly prepared media. Each well contained 2 mL of the corresponding solution, which was refreshed daily to maintain >80% of the nominal concentration. Embryos were incubated at 23 °C under a 12:12 h light/dark cycle and monitored daily until 120 hours post-fertilization (hpf). Endpoints including mortality, hatching success, body length, and malformation rates were scored under a stereomicroscope. Gene expression analysis To evaluate gene expression, two concentrations were selected for each compound (0.1 and 1 µg/L), representing the lowest exposure levels at which molecular changes could be reliably detected while minimizing developmental toxicity. Fertilized embryos were transferred to 6-well microplates (TPP, Switzerland), with 15 embryos per well and a total of 180 embryos per concentration. At 120 hpf, embryos were pooled into eight biological replicates per group, with approximately 10–12 embryos (10 mg total) per replicate. Samples were transferred to 1.5 mL Eppendorf tubes, preserved in RNA later (Thermo Fisher Scientific, Czech Republic), left for 24 h at 4 °C, and stored at −80 °C until RNA extraction. RNA extraction and reverse transcription into complementary DNA Embryo samples were removed from RNA-later (Thermo Fisher Scientific, Czech Republic), dried, and homogenized with the MagNaLyser (Roche, Germany) by adding 0.5 ml of TRI Reagent RT (Molecular Research Center, USA) and 0.5 mm zirconia/silica beads (BioSpec Products, USA). After phase separation in TRI Reagent RT, total RNA was extracted and purified using the RNeasy Mini-Kit (Qiagen, Germany) following the manufacturer's instructions. RNA concentration and purity were measured with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Germany), using the 260/280 and 230/280 ratios. The RNA was reverse transcribed into messenger RNA (mRNA) using the LunaScript RT SuperMix Kit (New England BioLabs Inc., USA), following the manufacturer's instructions. The RNA from individual samples was diluted with RNase-free water (Qiagen, Germany) to achieve a uniform concentration of 1 μg across all samples. Control samples were prepared as recommended by the manufacturer. The 20 μl reaction mixture was first heated at 25 °C for 2 minutes to promote primer annealing, then at 55 °C for 10 minutes to synthesize complementary DNA (cDNA), and finally inactivated by heating at 95 °C for 1 minute. Reverse transcription was performed using the Engine Thermal Cycler (Bio-Rad, Czech Republic). The resulting cDNA samples, including controls, were stored at −20 °C until use in qRT-PCR. P rimer design and quantitative real-time PCR All samples for real-time qPCRs were conducted in triplicate using the LightCycler 480 (Roche, Germany) and the QuantiTect SYBR Green PCR Kit (Qiagen, Germany). Initially, each sample's cDNA was diluted 1:4 with RNase-free water (Qiagen, Germany). Each 3-μl reaction contained 0.5 μl of diluted cDNA, 1 μl of primer mix, and 1.5 μl of SYBR Green Master (QuantiTect SYBR Green PCR Kit; Qiagen, Germany). The cycling protocol included an initial denaturation at 95 °C for 15 min, followed by 45 cycles of denaturation at 95 °C for 15 s, primer annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. Melting analysis was performed from 60 to 95 °c. RNase-free water served as a negative control for DNA contamination and replaced cDNA templates to monitor amplification in each run. Data analysis was performed using LightCycler 480 SW 1.5 software (Roche, Germany), calculating threshold cycle (Ct) values and using the comparative Δ Ct method. The relative expression of the gene of interest (GOI) was determined with the formula: [1/(2CtGOI)]/[1/(2Ct60S)]. Normalized expression values are presented as mean ± standard deviation (SD) relative to the control. Assays were carried out with gene-specific primers for Xenopus leavis ( Suppl. Fig. 4 ). For normalization of expression data, the eukaryotic translation elongation factor 1 alpha 1 ( eef1a1 , NM_001016692.2) was used as the reference gene. Verification of chemical concentrations by HPLC To confirm the actual concentrations of FLU and TRI in the exposure media, water samples were randomly collected at 0 and 24 h after solution renewal and analyzed by HPLC using external standards (FLU 10 g/L, TRI 100 g/L). The analysis showed that the measured concentrations closely corresponded to the nominal values (see Suppl. Fig. 3), supporting the assumption that concentrations remained stable under the same conditions across all experimental groups. Defrosted sample of water (2 mL) was filtered through a 0.22 µm nylon filter (Millipore, USA) and used for liquid chromatography-mass spectrometry (LC/MS) analysis. A Thermo Scientific Ultra-High-Performance Liquid Chromatography (UHPLC) Accela 1250 system was connected to a Thermo Scientific TSQ Quantum Access MAX Triple Quadrupole Instrument (Thermo Scientific, USA) equipped with heated electrospray ionization probe. An Astra C 18 (2.1 mm × 100 mm, 2.0 μm; Chromservis, CZ) column was used at a constant flow rate of 250 μL/min. For the determination of TRI, the mobile phase consisted of 0.1% water solution of formic acid (solvent A) and methanol (solvent B). The gradient used was: 0 – 2.0 min linear gradient from 20 to 90% B; 2.0 – 3.0 min held at 90% B; 3.0 – 4.0 min from 90 to 20% B and 4.0 – 5.0 min held at 20% B in order for the column to re-equilibrate before the next injection. For determination of FLU mobile phase consisted of 0.1% water solution of formic acid (solvent A) and acetonitrile (solvent B). The gradient used was: 0 – 3.0 min linear gradient from 40 to 90% B; 3.0 – 6.4 min held at 90% B; 6.4 – 7.3 min from 90 to 40% B and 7.3 – 8.0 min held at 40% B in order for the column to re-equilibrate before the next injection. The full loop injection volume of the sample was set at 2 μL. The heated electrospray ionization was operated in the positive-ion mode under the following conditions: Capillary Temperature: 325.0 o C; Vaporizer Temperature 300 o C; Sheath Gas Pressure 35.0 psi; Auxiliary (drying) gas 10 a.u.; Spray Voltage 3300 V. For our quality assurance and quality control program, the instrument was calibrated daily with multi-level calibration curves. Procedural blank and solvent blank were analysed for every set of 10 samples. The inter-day precision expressed as a relative standard deviation was 13.4% for FLU and 9.6% for TRI. The limit of detection determined as 3:1 signal versus noise value was 0.55 μg/L for FLU and 0.47 µg/l µg/L for TRI. Standards of FLU and TRI were purchased from Sigma-Aldrich (USA). Methanol and acetonitrile were purchased from Chromservis (Czech Republic) and were LC/MS purity (≥99.9%). Statistical analysis All statistical analyses were performed using Prism 8 (GraphPad, USA). Mortality, developmental stages, and hatchability were compared using the chi-square (χ²) test. Continuous variables such as morphometric measurements and gene expression levels were first tested for normality (Shapiro–Wilk) and homogeneity of variance (Bartlett’s test). If assumptions of normality and equal variance were met, one-way ANOVA followed by Dunnett’s post hoc test was applied. If assumptions were not met, non-parametric Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test was used. Statistical significance was accepted at *, p < 0.05; **, p < 0.01; and ***, p < 0.001. Ethical statement All experimental procedures complied with the national Act No. 246/1992 Coll. on the Protection of Animals Against Cruelty and were approved by the institutional ethics committee and relevant authorities. All experiments were conducted in compliance with institutional guidelines and are reported in accordance with the ARRIVE guidelines. Abbreviations ANOVA Analysis of Variance BMP Bone Morphogenetic Protein CE Cardiac Edema DI Deformed Intestines DMSO Dimethyl Sulfoxide FETAX Frog Embryo Teratogenesis Assay in Xenopus FLU Fluconazole hpf Hours Post Fertilization HPLC High-Performance Liquid Chromatography MMR Marc’s Modified Ringer’s solution NF Nieuwkoop and Faber PD Pigmentation Deformities ROS Reactive Oxygen Species TRI 1,2,4-Triazole Wnt Wingless/Integrated Declarations Competing interest statement The authors declare that they have no competing interests. Author contributions B.R. and L.A.M. conducted the majority of the experiments, critically analyzed the data, and contributed to figure preparation and manuscript drafting. R.H. assisted with tadpole maintenance and phenotypic analyses. N.P. performed the qPCR analyses. P.M. carried out the HPLC measurements. J.B. and P.L. contributed to project supervision. P.L. and J.H. jointly conceived the study. J.H. supervised the research, secured funding, wrote the original manuscript and coordinated the overall project, including experimental design and all additional aspects not explicitly listed above. All authors reviewed and edited the manuscript. Acknowledgements We would like to thank Lenka Doubková and Dr. Eva Slabáková for outstanding administrative support and Douglas P. P. Gomes for dedicated care and maintenance of the Xenopus facility, which was essential to this work. Additionally, we would like to thank the members of VETUNI, such as Martin Klein, for preparing the tested solution and Mgr. Michaela Frederika Vargová, Dr. Ekaterina Koriakina, and Dr. Dagmar Vršková for help during the trial in the lab. Funding We gratefully acknowledge financial support from the Grant Agency of Masaryk University (project no. MUNI/J/0004/2021) and from the Ministry of Education, Youth and Sports (MSMT) of the Czech Republic under the MSCA-CZ/OP JAC scheme (project no. CZ.02.01.01/00/22_010/0003229, MSCAfellow5_MUNI), both awarded to J.H. Additional support was provided by the MSMT project no. CZ.02.01.01/00/22_010/0008854 awarded to L.A.M. The Internal Grant Agency of the Veterinary University Brno (project no. IGA VETUNI 218/2024/FVHE) supported B.R. Further funding came from the ERDF/ESF project “Profish” (no. CZ.02.1.01/0.0/0.0/16_019/0000869) and the Ministry of Agriculture of the Czech Republic (RO 0523). The funders had no role in the analyses, decision to publish, or preparation of the manuscript. Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. Large Language Models (LLMs) were not used for the generation of scientific content in this manuscript. Occasional assistance was limited to grammar and language editing. References Anderson, R.M., Lawrence, A.R., Stottmann, R.W., Bachiller, D., Klingensmith, J., 2002. Chordin and noggin promote organizing centers of forebrain development in the mouse. Development 129, 4975–4987. https://doi.org/10.1242/dev.129.21.4975 Arnold, S.J., Stappert, J., Bauer, A., Kispert, A., Herrmann, B.G., Kemler, R., 2000. Brachyury is a target gene of the Wnt/β-catenin signaling pathway. Mechanisms of Development 91, 249–258. https://doi.org/10.1016/S0925-4773(99)00309-3 Barenys, M., Molins, A., Amorós-Galicia, L., Flick, B., Gómez-Catalán, J., 2019. 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18:07:57","extension":"xml","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128752,"visible":true,"origin":"","legend":"","description":"","filename":"03e577dbfe6f493382d528c9d570fb321structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/4d648207bc3d306f1a7e3ff9.xml"},{"id":92742532,"identity":"4017f502-d95d-45ac-8a91-6ba654c6944a","added_by":"auto","created_at":"2025-10-03 17:59:57","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141692,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/4ba15bcd692b67bb542c0e65.html"},{"id":92742500,"identity":"a0eefdcb-a773-4dbb-8610-f5011a4dcdce","added_by":"auto","created_at":"2025-10-03 17:59:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1001816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXenopus laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e embryos exposed to fluconazole (FLU).\u003c/strong\u003e Embryos were treated from stage NF 3 to NF 46 with increasing concentrations of FLU (1, 100, and 1,000 µg/L). Phenotypic abnormalities were observed at all tested concentrations, with most prominent features including reduced head size, eye deformities, craniofacial malformations, altered pigmentation, and gut abnormalities. Images were captured at NF stage 46 using a stereomicroscope. Scale bar: 500 µm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/aa246a3b835032416c66be73.png"},{"id":92742499,"identity":"846ad54a-7934-427a-956c-d22fbafca247","added_by":"auto","created_at":"2025-10-03 17:59:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":777781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXenopus laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e embryos exposed to 1,2,4-triazole (TRI). \u003cbr\u003e\n \u003c/strong\u003eEmbryos were treated from stage NF 3 to NF 46 with 1, 100, and 1,000 µg/L TRI. A concentration-dependent increase in morphological abnormalities was observed, including smaller heads, eye spacing defects, changes in pigmentation, and intestinal malformations. Images were taken at stage NF 46. Scale bar: 500 µm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/ef59d56eb4922754ac385d44.png"},{"id":92742497,"identity":"452a5c78-80fe-426c-bdfc-f587d8e543d5","added_by":"auto","created_at":"2025-10-03 17:59:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose–response analysis of malformation incidence in embryos treated with FLU and TRI. \u003c/strong\u003eBar plots show the percentage of embryos with developmental malformations at 120 hpf across increasing concentrations of FLU and TRI. While FLU caused a plateau in malformation incidence (~40%) starting from 1 µg/L, TRI exhibited a gradual, concentration-dependent increase in malformations, reaching ~25% at 1,000 µg/L. Data represent 24 embryos per each experimental condition. Red lines represent type of dependency.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/16b76faae37ddf6e0dda078c.png"},{"id":92742505,"identity":"963a822b-33c2-4e6c-b264-f7b29ac94582","added_by":"auto","created_at":"2025-10-03 17:59:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":208785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphometric and physiological endpoints in embryos exposed to FLU and TRI. \u003c/strong\u003e(A) Body length measurements of embryos at 120 hpf showed that FLU significantly increased embryo length at higher doses, while TRI had no measurable effect. (B) Heartbeat analysis revealed that both compounds increased heart rate at lower concentrations, but TRI led to a drop at 1,000 µg/L. Data represent mean ± SD from 12 embryos per group. \u0026nbsp;Statistical significance was determined using ANOVA with Dunnett’s test. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **, p \u0026lt; 0.01, ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/3cd90c4695cd2e65e69beee2.png"},{"id":92742667,"identity":"dab39cfc-b02e-4556-b9e0-8f2b66705dc8","added_by":"auto","created_at":"2025-10-03 18:07:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression analysis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXenopus laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e embryos exposed to FLU and TRI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eqPCR analysis of selected developmental genes (\u003cem\u003eβ-catenin, noggin, chordin, xbra, and xolloid\u003c/em\u003e) was performed at 120 hpf. Significant upregulation was detected in embryos treated with both compounds, particularly TRI. These changes reflect activation of Wnt signaling and modulation of BMP signaling. Data represent mean fold-change + SD from 7-10 embryos (ANOVA, Dunnett’s test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/6dabb2fc9e0383957e9fba44.png"},{"id":92742507,"identity":"732bca9b-329d-4711-8cee-ad23c8fe406e","added_by":"auto","created_at":"2025-10-03 17:59:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of molecular and phenotypic effects of fluconazole (FLU) and 1,2,4-triazole (TRI) exposure on early \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXenopus laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e development. \u0026nbsp;\u003c/strong\u003eBoth azole compounds disrupt key embryonic signaling pathways, leading to upregulation (e.g., \u003cem\u003exbra\u003c/em\u003e, \u003cem\u003exolloid)\u003c/em\u003e and upward trends (e.g., \u003cem\u003eβ-catenin\u003c/em\u003e, \u003cem\u003echordin\u003c/em\u003e, \u003cem\u003enoggin)\u003c/em\u003e in Wnt- and BMP-related genes, together with head and gut malformations, altered pigmentation, and increased heart rate. These findings highlight the developmental toxicity of azole compounds and their potential ecological impact on aquatic vertebrates.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/03eee657fc52d80c3a7eb743.png"},{"id":97723894,"identity":"dc83e22e-dadd-44b7-944d-34c0d855058e","added_by":"auto","created_at":"2025-12-08 16:09:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3905096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/57a2be6d-434a-43e6-a993-d2e7b1fbc842.pdf"},{"id":92743462,"identity":"fa3bac38-86e0-4eff-b434-aa9467217769","added_by":"auto","created_at":"2025-10-03 18:23:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":552105,"visible":true,"origin":"","legend":"","description":"","filename":"FLUandTRImanuscript5SUPPLEMENT.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7570738/v1/64e4d87334ad1d15d699f4ed.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Developmental toxicity of fluconazole and 1,2,4-triazole in non-target aquatic vertebrates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAzole antifungal compounds such as fluconazole (FLU) are widely used across human and veterinary medicine, agriculture, and the personal care industry (Chakraborty et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; J\u0026oslash;rgensen and Heick, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sheehan et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Their therapeutic efficacy against fungal infections has made them indispensable in clinical practice, while their antifungal properties are also utilized in shampoos, soaps, skin creams, and agricultural fungicides (Lee et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Maertens, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sheehan et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). A common structural feature of many azoles, including FLU, is the 1,2,4-triazole (TRI) ring \u0026ndash; a heterocyclic motif essential for antifungal activity that is also present in numerous other pharmaceuticals and agrochemicals (Gupta et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the extensive use of these compounds has resulted in their frequent detection in wastewater effluents and surface waters worldwide (Bhagat et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kahle et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Płatkiewicz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Subramanian et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Conventional wastewater treatment plants often fail to remove them completely, leading to persistent low-level contamination. Measured nanomolar concentrations of azole compounds such as FLU, climbazole, and tebuconazole have been reported in rivers across Europe, Africa, and Asia, raising concerns about their ecotoxicological impact (Halešov\u0026aacute; et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Iancu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Płatkiewicz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Wick et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wilkinson et al., 2022).\u003c/p\u003e\u003cp\u003eAlthough originally developed for therapeutic use in humans and animals, azoles are increasingly recognized for their unintended effects on non-target organisms. Studies in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) have shown that they can disrupt early development, causing pericardial edema, altered heart rate, and embryonic malformations (Barenys et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bhagat et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Yet, little is known about their developmental effects in amphibians and higher vertebrates. Amphibians are particularly vulnerable to waterborne contaminants due to their permeable skin and aquatic life stages. The African clawed frog, \u003cem\u003eXenopus laevis\u003c/em\u003e, is a long-established and highly sensitive model organism that has made fundamental contributions to developmental and cell biology, as well as to ecotoxicology (De Robertis and Gurdon, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gao and Shen, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Its external fertilization, rapid development, and highly conserved genetic pathways with other vertebrates, including humans, make it especially suitable for assessing the impact of environmental chemicals (Gao and Shen, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the present study, we investigated the developmental toxicity of FLU and its structural core TRI in \u003cem\u003eXenopus laevis\u003c/em\u003e embryos, measuring mortality, hatching success, heart rate, body length, malformation frequency, and expression of key genes involved in early embryogenesis. Both compounds induced morphological and physiological changes, and at the molecular level, altered the expression of \u003cem\u003exbra\u003c/em\u003e, \u003cem\u003exolloid, β-catenin\u003c/em\u003e, \u003cem\u003echordin\u003c/em\u003e and \u003cem\u003enoggin\u003c/em\u003e, which is consistent with modulation of Wnt (Wingless/Integrated) and BMP (Bone Morphogenetic Protein) signaling pathways. These results suggest that even simple triazole structures can interfere with conserved developmental mechanisms and raise important questions about the environmental and health safety of azole antifungals.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAzole antifungals alter tadpole morphology\u003c/h2\u003e\u003cp\u003eTo investigate the developmental toxicity of azole antifungals, we initially monitored the development of \u003cem\u003eXenopus laevis\u003c/em\u003e embryos during early stages but observed no overt morphological changes. Therefore, we focused our analysis on later time points, specifically at Nieuwkoop and Faber (NF) stage 46, when cumulative effects become more apparent. At this stage, we examined gross morphology following continuous exposure to 1\u0026ndash;1,000 \u0026micro;g/L of FLU and TRI. Representative phenotypes for both treatments are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (FLU) and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (TRI).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUntreated control animals developed normally with well-formed heads, regular pigmentation, and a straight body axis, but exposed embryos exhibited noticeable morphological deviations. In FLU-treated groups, we observed several recurring abnormalities, including pigmentation defects \u0026ndash; including both hyperpigmentation and hypopigmentation \u0026ndash; and craniofacial malformations such as reduced head size and head edema. Heart edema and visible gut malformations were also present in a subset of tadpoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTRI exposure resulted in a similar but more variable range of phenotypes. Along with pigmentation defects, observed phenotypes included reduced eye spacing, completely undeveloped intestines, and misshapen anterior structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTaken together, these observations suggest that both FLU and TRI interfere with normal tissue patterning and organogenesis in \u003cem\u003eXenopus laevis\u003c/em\u003e embryos.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDose-dependent differences in malformation incidence\u003c/h3\u003e\n\u003cp\u003eWe next quantified the percentage of malformed tadpoles across increasing concentrations of both compounds to evaluate the dose-response relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). FLU exposure resulted in a saturating malformation curve, with incidence plateauing at approximately 40% from the lowest tested concentration (1 \u0026micro;g/L) and remaining stable at higher doses. This suggests that even low concentrations of FLU are sufficient to trigger developmental disruptions and that the system reaches a response ceiling early.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, TRI exposure followed a clear dose-dependent trend. At 1 \u0026micro;g/L, only a small proportion of animals exhibited any malformation. As the concentration increased to 100 \u0026micro;g/L, the incidence rose to approximately 20%, and at 1,000 \u0026micro;g/L, around 25% of the tadpoles displayed visible abnormalities. These findings indicate a progressive teratogenic effect of TRI, potentially linked to cumulative molecular disruptions at higher doses. The contrasting saturation-versus-linear trend observed for FLU and TRI, respectively, points to possible differences in their mechanisms of action or bioavailability in the embryo.\u003c/p\u003e\n\u003ch3\u003eMalformation types differ between fluconazole and triazole\u003c/h3\u003e\n\u003cp\u003eBeyond quantifying malformation rates, we analyzed the spectrum of phenotypic defects to identify potential differences in the type and severity of abnormalities caused by each compound \u003cb\u003e(Suppl. Figure\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eFLU exposure most frequently resulted in altered pigmentation, craniofacial changes (reduced head size, head edema), cardiac edema, anterior-posterior axis deformation, and intestinal abnormalities. Notably, darker upper flank pigmentation was observed specifically at 100 \u0026micro;g/L. These phenotypes were generally consistent across concentrations, suggesting that FLU triggers a characteristic malformation profile that appears even at low doses.\u003c/p\u003e\u003cp\u003eTRI exposure induced a broader but generally less frequent set of malformations. Craniofacial anomalies included reduced head size, reduced interocular distance, and asymmetry of the eyes or head. Intestinal defects ranged from mild deformation to complete absence of intestinal structures. Pigmentation changes were diverse, encompassing dark patches, hypopigmentation, and star-like pigment patterns absent in controls or FLU-treated animals. Additional phenotypes unique to TRI included axial curvature and underdeveloped fins. These effects tended to become more prevalent at higher concentrations, with complete intestinal absence and multiple combined malformations occurring at 1,000 \u0026micro;g/L.\u003c/p\u003e\u003cp\u003eThese qualitative differences indicate that while both compounds disrupt overlapping developmental processes, FLU produces a narrower and more consistent malformation profile, whereas TRI has a more variable and concentration-dependent impact, potentially reflecting a broader disruption of axial patterning.\u003c/p\u003e\n\u003ch3\u003eCardiac and morphometric effects suggest physiological disruption\u003c/h3\u003e\n\u003cp\u003eTo further explore the physiological consequences of azole exposure, we measured heart rate and body length in tadpoles at NF stage 46 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both compounds caused a significant increase in heart rate at most concentrations tested, indicating that cardiac development or autonomic regulation may be altered. Interestingly, at the highest TRI concentration (1,000 \u0026micro;g/L), we observed a notable drop of heart rate, suggesting a possible toxic threshold beyond which physiological function becomes impaired.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMorphometric measurements revealed additional differences between the two compounds. FLU exposure led to a measurable elongation of the body axis, potentially reflecting disruptions in growth-regulating endocrine pathways and processes linked to mesoderm formation and muscle development, similar to patterns reported for cortisol and Bisphenol A exposure in fish and other model organisms (Rodrigues et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Senarath Pathirajage and Rajapaksa, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, TRI-treated tadpoles did not show significant changes in body length, even at higher concentrations. These data point to distinct developmental effects of each compound \u0026ndash; while both interfere with cardiac output, only FLU appears to affect axial growth.\u003c/p\u003e\n\u003ch3\u003eGene expression analysis reveals Wnt and BMP pathway disruption\u003c/h3\u003e\n\u003cp\u003eTo identify the molecular pathways affected by azole exposure, we analyzed the expression of selected developmental genes by quantitative PCR, focusing on components of the Wnt and BMP signaling networks. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, even low-level TRI exposure of 10 \u0026micro;g/L upregulated the expression of \u003cem\u003exbra\u003c/em\u003e, and \u003cem\u003exolloid\u003c/em\u003e, while \u003cem\u003eβ-catenin\u003c/em\u003e, \u003cem\u003echordin\u003c/em\u003e and \u003cem\u003enoggin\u003c/em\u003e exhibited upward trends. These changes may reflect activation of the Wnt/β-catenin pathway and concurrent inhibition of BMP signaling via its antagonists, \u003cem\u003echordin\u003c/em\u003e and \u003cem\u003enoggin\u003c/em\u003e. Upregulation of \u003cem\u003exbra\u003c/em\u003e reflects enhanced mesodermal induction, while \u003cem\u003exolloid\u003c/em\u003e likely represents a feedback response attempting to restore BMP activity by degrading excess \u003cem\u003echordin\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFLU induced a similar transcriptional profile, though the magnitude of change was generally lower and more variable. In contrast, several downstream BMP effectors \u0026ndash; \u003cem\u003efollistatin\u003c/em\u003e, \u003cem\u003esizzled\u003c/em\u003e, \u003cem\u003event1\u003c/em\u003e, and \u003cem\u003event2\u003c/em\u003e \u0026ndash; did not show significant expression changes in either treatment (\u003cb\u003eSuppl. Figure\u0026nbsp;2\u003c/b\u003e). This selective transcriptional response indicates that azoles specifically disrupt early morphogen gradients without broadly altering all components of the BMP pathway.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eExposure concentrations confirmed by chemical analysis\u003c/h2\u003e\u003cp\u003eTo confirm that the developmental effects observed were consistent with the intended dosing, we analyzed the actual concentrations of FLU and TRI in the exposure water using HPLC (\u003cb\u003eSuppl. Figure\u0026nbsp;3\u003c/b\u003e). Measurements taken during the experiment confirmed that concentrations remained stable and closely matched the nominal values. For the representative treatment groups, we selected and detected FLU at 10 \u0026micro;g/L and TRI at 100 \u0026micro;g/L, validating the accuracy and consistency of chemical exposures used in this study (\u003cb\u003eSuppl. Figure\u0026nbsp;3\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn summary, both FLU and TRI exhibit teratogenic potential in \u003cem\u003eXenopus laevis\u003c/em\u003e embryos, but through partially distinct developmental effects. FLU appears to act rapidly and reach a phenotypic plateau at low concentrations, while TRI induces malformations in a more progressive, dose-dependent manner. The spectrum of malformations and broader gene expression changes caused by TRI suggests a more pervasive disruption of axial patterning. These results, summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, collectively support the hypothesis that azole antifungals interfere with conserved signaling pathways involved in vertebrate embryogenesis, highlighting their potential risk as environmental contaminants and emerging human health hazards.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study investigates the effects of triazole-based antifungals, specifically fluconazole (FLU) and its structural core 1,2,4-triazole (TRI), on early embryogenesis in aquatic vertebrates using \u003cem\u003eXenopus laevis\u003c/em\u003e as a model. We show that these compounds selectively disrupt key developmental signaling pathways and induce distinct morphological deformities.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAltered gene expression.\u003c/b\u003e In our study, we observed the upregulation of \u003cem\u003exbra\u003c/em\u003e and \u003cem\u003exolloid\u003c/em\u003e transcripts, which provides particularly strong evidence for triazole-induced perturbations, whereas other transcripts, such as \u003cem\u003eβ-catenin\u003c/em\u003e, \u003cem\u003enoggin\u003c/em\u003e, and \u003cem\u003echordin\u003c/em\u003e, exhibited upward trends without reaching statistical significance. Together, these results suggest a targeted rather than global reprogramming of developmental networks. The significant induction of \u003cem\u003exbra\u003c/em\u003e points to altered mesodermal specification, as it is a central regulator of mesoderm formation and posterior patterning. Its upregulation implies a shift toward posteriorized mesodermal fates, consistent with the anterior reductions observed in treated embryos (Anderson et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The observed upregulation of \u003cem\u003exolloid\u003c/em\u003e transcripts, a metalloprotease cleaving the BMP inhibitor chordin (Piccolo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), may represent a feedback mechanism to rebalance BMP availability. Together, these transcriptional alterations suggest that TRI and FLU exposures perturbs mesodermal allocation and axial patterning through combined effects on Wnt and BMP regulatory networks. Although \u003cem\u003eβ-catenin\u003c/em\u003e transcript levels trended upward, it is important to note that β-catenin is regulated predominantly by protein stabilization via posttranslational modification and subsequent nuclear localization, not by transcript abundance (Clevers, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e); therefore, mRNA abundance does not provide a direct readout of canonical Wnt pathway activation. Nevertheless, the observed transcriptional profile is consistent with partial Wnt pathway engagement, which could synergize with BMP modulation to reinforce posteriorization. The modest increases in noggin and chordin, two potent BMP antagonists, although not statistically significant, align with this interpretation but require cautious consideration given their variability. (McMahon et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Stottmann et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Hikasa and Sokol, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, \u003cem\u003edickkopf\u003c/em\u003e, \u003cem\u003egoosecoid\u003c/em\u003e, \u003cem\u003esizzled\u003c/em\u003e, and \u003cem\u003event1/2\u003c/em\u003e, were not significantly altered, suggesting that azoles exposure do not induce global transcriptional rewiring of embryonic axes, but rather target specific nodes within the Wnt\u0026ndash;BMP regulatory interface. FLU induced similar but weaker changes than TRI, supporting a shared mechanism of action with differing potency.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCorresponding phenotypic outcomes.\u003c/b\u003e These molecular alterations correlated with phenotypes including reduced head size, craniofacial defects, pigmentation abnormalities, and mild digestive tract malformations. Reduced head size likely reflects anterior tissue loss, a hallmark of posteriorizing influences on axial patterning (Hikasa and Sokol, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although β-catenin, noggin, and chordin showed only non-significant upward trends, even modest shifts in Wnt/BMP dynamics can bias tissue allocation, which may explain the anterior reductions observed in our tadpoles. Abnormal pigmentation, particularly the emergence of star-like pigment cells, is suggestive of disrupted neural crest migration (Kelsh et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which relies on tightly regulated BMP gradients (Raible and Ragland, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Digestive tract malformations and altered heart rate are consistent with mesodermal mispatterning and cardiac lineage imbalance \u0026ndash; both processes sensitive to Wnt/BMP signaling dynamics. Notably, similar phenotypes have been observed in zebrafish embryos exposed to other azole antifungals, including pericardial edema and craniofacial deformities (Barenys et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bhagat et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), reinforcing the idea of a conserved mode of developmental toxicity across aquatic vertebrates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanistic links between gene expression and phenotype.\u003c/b\u003e Importantly, our transcriptional data provide a plausible mechanistic link between signaling disruptions and phenotype. \u003cem\u003eXbra\u003c/em\u003e is expressed in the involuting marginal zone during gastrulation and is regulated by Wnt/β-catenin signaling (Arnold et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). As a mesodermal regulator, its elevation likely shifts tissue allocation toward posterior fates. This posteriorization may be further reinforced by increased \u003cem\u003exolloid\u003c/em\u003e, which promotes chordin degradation and thus indirectly enhances BMP activity in the posterior. These molecular events help explain the anterior reductions seen in our embryos, especially in head structures and the gut.\u003c/p\u003e\u003cp\u003eThe cardiac implications are also worth noting: although \u003cem\u003exbra\u003c/em\u003e is not directly required for cardiac gene expression, it is essential for early mesoderm formation from which cardiac mesoderm is derived (Tzahor, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Any disruptions at this stage could lead to mis-specification of cardiac tissues, potentially explaining heart malformations. Similar links between Wnt signaling and heart morphogenesis have been well established (Foulquier et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePossible involvement of oxidative stress.\u003c/b\u003e Previous studies have linked triazoles such as tebuconazole to oxidative stress in vertebrates (Ben Othm\u0026egrave;ne et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), mediated by reactive oxygen species (ROS). ROS may increase endothelial permeability (Lum and Roebuck, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and disturb osmotic balance (Scallan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which could explain the edematous phenotypes seen with triazole exposure. Although ROS were not specifically tested in this study, they may represent a plausible parallel mechanism worth further investigation. Previous data also suggest that early phenotypic differences are more reliably observed in later stages of vertebrate development (Dong, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), aligning with our decision to focus on later larval stages.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical relevance and potential signaling implications.\u003c/b\u003e Azole antifungals, particularly fluconazole, remain critical for treating fungal infections in immunocompromised patients, such as those with HIV/AIDS, cancer, or organ transplants (Domingos et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Katiraee et al., 2015; Ostrosky-Zeichner, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003eCandida albicans\u003c/em\u003e remains the leading cause of systemic candidiasis, and the emergence of resistant strains like \u003cem\u003eCandida auris\u003c/em\u003e poses additional challenges (Ramli et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sanyaolu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Worku and Girma, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Beyond clinical use, their widespread presence in over-the-counter products such as creams and shampoos contributes to persistent environmental and human exposure.\u003c/p\u003e\u003cp\u003eAzoles function by inhibiting cytochrome P450 enzymes (e.g., CYP51), essential for fungal membrane synthesis (Shin et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, these same enzymes \u0026ndash; and the signaling cascades they modulate \u0026ndash; are also found in vertebrate cells (Guengerich et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hannemann et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Our findings reveal that even the core structure of azoles (e.g., TRI) can perturb developmental signaling in \u003cem\u003eXenopus\u003c/em\u003e embryos, with selective effects on Wnt and BMP regulatory networks. While the precise molecular interactions remain to be elucidated, the evolutionary conservation of these pathways highlights the possibility of parallels in vertebrate development, including humans. Fluconazole and similar azole compounds may therefore represent emerging developmental and health hazards, warranting closer toxicological and clinical exploration.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal husbandry and embryo collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving \u003cem\u003eXenopus laevis\u003c/em\u003e were approved by the Ministry of Agriculture of the Czech Republic (Animal Care and Housing Approval: 45055/2020-MZE-18134) and conducted in accordance with Czech legislation and institutional guidelines (MSMT-30784/2022-1). \u0026nbsp;All experiments were conducted in compliance with institutional guidelines and are reported in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003eEmbryos were obtained using standard methods. Briefly, adult males were anesthetized in 20% MS-222 (Sigma-Aldrich, A5040) and testes were surgically removed and stored in cold 1× Marc’s Modified Ringer’s solution (MMR; 100 mM NaCl, 2 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM HEPES, pH 7.4) supplemented with 50 µg/mL gentamicin (Sigma-Aldrich, G3632). Sexually mature females were induced to ovulate via dorsal lymph sac injection of 260 IU human chorionic gonadotropin (hCG; Merck, Ovitrelle 250G), followed by overnight incubation at 18 °C. Eggs were collected the next morning by gentle squeezing and fertilized in vitro using a macerated piece of testis in 0.1× MMR. Embryos were cultured at 18–21 °C in 0.1× MMR and staged according to Nieuwkoop and Faber\u0026nbsp;(Zahn et al., 2022). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of lethal and sublethal endpoints\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDevelopmental toxicity was assessed following modified FETAX (Frog Embryo Teratogenesis Assay – \u003cem\u003eXenopus\u003c/em\u003e) protocols. Freshly fertilized embryos were visually inspected under a stereomicroscope (Olympus SZX7, Japan) and distributed into 24-well microplates (TPP, Switzerland), with 24 embryos per group. The embryos were exposed to three concentrations of each test compound: 1, 100, and 1,000 µg/L for fluconazole (FLU), and 1, 100, and 1,000 µg/L for 1,2,4-triazole (TRI). Control groups included unexposed embryos and embryos exposed to 0.01x MMR. FLU and TRI were dissolved in DMSO (dimethyl sulfoxide) and added to the embryo culture medium, while the equivalent volume of DMSO alone was used as a negative control. Exposure solutions including controls were renewed daily with freshly prepared media.\u003c/p\u003e\n\u003cp\u003eEach well contained 2 mL of the corresponding solution, which was refreshed daily to maintain \u0026gt;80% of the nominal concentration. Embryos were incubated at 23 °C under a 12:12 h light/dark cycle and monitored daily until 120 hours post-fertilization (hpf). Endpoints including mortality, hatching success, body length, and malformation rates were scored under a stereomicroscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate gene expression, two concentrations were selected for each compound (0.1 and 1 µg/L), representing the lowest exposure levels at which molecular changes could be reliably detected while minimizing developmental toxicity. Fertilized embryos were transferred to 6-well microplates (TPP, Switzerland), with 15 embryos per well and a total of 180 embryos per concentration.\u003c/p\u003e\n\u003cp\u003eAt 120 hpf, embryos were pooled into eight biological replicates per group, with approximately 10–12 embryos (10 mg total) per replicate. Samples were transferred to 1.5 mL Eppendorf tubes, preserved in RNA later (Thermo Fisher Scientific, Czech Republic), left for 24 h at 4 °C, and stored at −80 °C until RNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and reverse transcription into complementary DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmbryo samples were removed from RNA-later (Thermo Fisher Scientific, Czech Republic), dried, and homogenized with the MagNaLyser (Roche, Germany) by adding 0.5 ml of TRI Reagent RT (Molecular Research Center, USA) and 0.5 mm zirconia/silica beads (BioSpec Products, USA). After\u0026nbsp;phase separation in TRI Reagent RT, total RNA was extracted and purified using the RNeasy Mini-Kit (Qiagen, Germany) following the manufacturer's instructions. RNA concentration and purity were measured with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Germany), using the 260/280 and 230/280 ratios.\u003c/p\u003e\n\u003cp\u003eThe RNA was reverse transcribed into messenger RNA (mRNA) using the LunaScript RT SuperMix Kit (New England BioLabs Inc., USA), following the manufacturer's instructions. The RNA from individual samples was diluted with RNase-free water (Qiagen, Germany) to achieve a uniform concentration of 1 μg across all samples. Control samples were prepared as recommended by the manufacturer. The 20 μl reaction mixture was first heated at 25 °C for 2 minutes to promote primer annealing, then at 55 °C for 10 minutes to synthesize complementary DNA (cDNA), and finally inactivated by heating at 95 °C for 1 minute. Reverse transcription was performed using the Engine Thermal Cycler (Bio-Rad, Czech Republic). The resulting cDNA samples, including controls, were stored at −20 °C until use in qRT-PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003erimer design and quantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll samples for real-time qPCRs were conducted in triplicate using the LightCycler 480 (Roche, Germany) and the QuantiTect SYBR Green PCR Kit (Qiagen, Germany). Initially, each sample's cDNA was diluted 1:4 with RNase-free water (Qiagen, Germany). Each 3-μl reaction contained 0.5 μl of diluted cDNA, 1 μl of primer mix, and 1.5 μl of SYBR Green Master (QuantiTect SYBR Green PCR Kit; Qiagen, Germany). The cycling protocol included an initial\u0026nbsp;denaturation\u0026nbsp;at 95 °C for 15 min, followed by 45 cycles of denaturation at 95 °C for 15 s, primer annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. Melting analysis was performed from 60 to 95 °c. RNase-free water served as a negative control for\u0026nbsp;DNA contamination\u0026nbsp;and replaced\u0026nbsp;cDNA templates\u0026nbsp;to monitor amplification\u0026nbsp;in each run. Data analysis was performed using LightCycler 480 SW 1.5 software (Roche, Germany), calculating threshold cycle (Ct) values and using the comparative Δ Ct method. The relative expression of the gene of interest (GOI) was determined with the formula: [1/(2CtGOI)]/[1/(2Ct60S)]. Normalized expression values are presented as mean ± standard deviation (SD) relative to the control.\u003c/p\u003e\n\u003cp\u003eAssays were carried out with gene-specific primers for \u003cem\u003eXenopus\u0026nbsp;leavis\u003c/em\u003e (\u003cstrong\u003eSuppl. Fig. 4\u003c/strong\u003e). For normalization of expression data, the eukaryotic translation elongation factor 1 alpha 1 (\u003cem\u003eeef1a1\u003c/em\u003e, NM_001016692.2) was used as the reference gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification of chemical concentrations by HPLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the actual concentrations of FLU and TRI in the exposure media, water samples were randomly collected at 0 and 24 h after solution renewal and analyzed by HPLC using external standards (FLU 10 g/L, TRI 100 g/L). The analysis showed that the measured concentrations closely corresponded to the nominal values (see Suppl. Fig. 3), supporting the assumption that concentrations remained stable under the same conditions across all experimental groups.\u003c/p\u003e\n\u003cp\u003eDefrosted sample of water (2 mL) was filtered through a 0.22 µm nylon filter (Millipore, USA) and used for liquid chromatography-mass spectrometry (LC/MS) analysis. A Thermo Scientific Ultra-High-Performance Liquid Chromatography (UHPLC) Accela 1250 system was connected to a Thermo Scientific TSQ Quantum Access MAX Triple Quadrupole Instrument (Thermo Scientific, USA) equipped with heated electrospray ionization probe. An Astra C\u003csub\u003e18\u003c/sub\u003e (2.1 mm × 100 mm, 2.0 μm; Chromservis, CZ) column was used at a constant flow rate of 250 μL/min. For the determination of TRI, the mobile phase consisted of 0.1% water solution of formic acid (solvent A) and methanol (solvent B). The gradient used was: 0 – 2.0 min linear gradient from 20 to 90% B; 2.0 – 3.0 min held at 90% B; 3.0 – 4.0 min from 90 to 20% B and 4.0 – 5.0 min held at 20% B in order for the column to re-equilibrate before the next injection. For determination of FLU mobile phase consisted of 0.1% water solution of formic acid (solvent A) and acetonitrile (solvent B). The gradient used was: 0 – 3.0 min linear gradient from 40 to 90% B; 3.0 – 6.4 min held at 90% B; 6.4 – 7.3 min from 90 to 40% B and 7.3 – 8.0 min held at 40% B in order for the column to re-equilibrate before the next injection. The full loop injection volume of the sample was set at 2 μL. The heated electrospray ionization was operated in the positive-ion mode under the following conditions: Capillary Temperature: 325.0 \u003csup\u003eo\u003c/sup\u003eC; Vaporizer Temperature 300 \u003csup\u003eo\u003c/sup\u003eC; Sheath Gas Pressure 35.0 psi; Auxiliary (drying) gas 10 a.u.; Spray Voltage 3300 V. For our quality assurance and quality control program, the instrument was calibrated daily with multi-level calibration curves. Procedural blank and solvent blank were analysed for every set of 10 samples. The inter-day precision expressed as a relative standard deviation was 13.4% for FLU and 9.6% for TRI. The limit of detection determined as 3:1 signal versus noise value was 0.55 μg/L for FLU and 0.47 µg/l µg/L for TRI. Standards of FLU and TRI were purchased from Sigma-Aldrich (USA). Methanol and acetonitrile were purchased from Chromservis (Czech Republic) and were LC/MS purity (≥99.9%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using Prism 8 (GraphPad, USA). Mortality, developmental stages, and hatchability were compared using the chi-square (χ²) test. Continuous variables such as morphometric measurements and gene expression levels were first tested for normality (Shapiro–Wilk) and homogeneity of variance (Bartlett’s test).\u003c/p\u003e\n\u003cp\u003eIf assumptions of normality and equal variance were met, one-way ANOVA followed by Dunnett’s post hoc test was applied. \u0026nbsp;If assumptions were not met, non-parametric Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test was used. Statistical significance was accepted at *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; and ***, p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures complied with the national Act No. 246/1992 Coll. on the Protection of Animals Against Cruelty and were approved by the institutional ethics committee and relevant authorities. All experiments were conducted in compliance with institutional guidelines and are reported in accordance with the ARRIVE guidelines.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Analysis of Variance\u003c/p\u003e\n\u003cp\u003eBMP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bone Morphogenetic Protein\u003c/p\u003e\n\u003cp\u003eCE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cardiac Edema\u003c/p\u003e\n\u003cp\u003eDI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Deformed Intestines\u003c/p\u003e\n\u003cp\u003eDMSO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Dimethyl Sulfoxide\u003c/p\u003e\n\u003cp\u003eFETAX \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Frog Embryo Teratogenesis Assay in \u003cem\u003eXenopus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFLU \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Fluconazole\u003c/p\u003e\n\u003cp\u003ehpf \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hours Post Fertilization\u003c/p\u003e\n\u003cp\u003eHPLC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;High-Performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003eMMR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Marc’s Modified Ringer’s solution\u003c/p\u003e\n\u003cp\u003eNF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Nieuwkoop and Faber\u003c/p\u003e\n\u003cp\u003ePD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Pigmentation Deformities\u003c/p\u003e\n\u003cp\u003eROS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive Oxygen Species\u003c/p\u003e\n\u003cp\u003eTRI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1,2,4-Triazole\u003c/p\u003e\n\u003cp\u003eWnt \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Wingless/Integrated\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.R.\u003c/strong\u003e and \u003cstrong\u003eL.A.M.\u003c/strong\u003e conducted the majority of the experiments, critically analyzed the data, and contributed to figure preparation and manuscript drafting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eR.H.\u003c/strong\u003e assisted with tadpole maintenance and phenotypic analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN.P.\u003c/strong\u003e performed the qPCR analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP.M.\u003c/strong\u003e carried out the HPLC measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.B.\u003c/strong\u003e and \u003cstrong\u003eP.L.\u003c/strong\u003e contributed to project supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP.L.\u003c/strong\u003e and \u003cstrong\u003eJ.H.\u003c/strong\u003e jointly conceived the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.H.\u003c/strong\u003e supervised the research, secured funding, wrote the original manuscript and coordinated the overall project, including experimental design and all additional aspects not explicitly listed above.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Lenka Doubková and Dr. Eva Slabáková for outstanding administrative support and Douglas P. P. Gomes for dedicated care and maintenance of the \u003cem\u003eXenopus\u003c/em\u003e facility, which was essential to this work. \u0026nbsp;Additionally, we would like to thank the members of VETUNI, such as Martin Klein, for preparing the tested solution and Mgr. Michaela Frederika Vargová, Dr. Ekaterina Koriakina, and Dr. Dagmar Vršková for help during the trial in the lab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge financial support from the Grant Agency of Masaryk University (project no. MUNI/J/0004/2021) and from the Ministry of Education, Youth and Sports (MSMT) of the Czech Republic under the MSCA-CZ/OP JAC scheme (project no. CZ.02.01.01/00/22_010/0003229, MSCAfellow5_MUNI), both awarded to J.H. \u0026nbsp;Additional support was provided by the MSMT project no. CZ.02.01.01/00/22_010/0008854 awarded to L.A.M.\u0026nbsp;\u003cbr\u003e\u0026nbsp;The Internal Grant Agency of the Veterinary University Brno (project no. IGA VETUNI 218/2024/FVHE) supported B.R. \u0026nbsp;Further funding came from the ERDF/ESF project “Profish” (no. CZ.02.1.01/0.0/0.0/16_019/0000869) and the Ministry of Agriculture of the Czech Republic (RO 0523).\u003c/p\u003e\n\u003cp\u003eThe funders had no role in the analyses, decision to publish, or preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. \u0026nbsp;Large Language Models (LLMs) were not used for the generation of scientific content in this manuscript. \u0026nbsp;Occasional assistance was limited to grammar and language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnderson, R.M., Lawrence, A.R., Stottmann, R.W., Bachiller, D., Klingensmith, J., 2002. Chordin and noggin promote organizing centers of forebrain development in the mouse. 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Rep. 38, 1072\u0026ndash;1099. https://doi.org/10.1039/D1NP00004G\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Azole antifungals, embryotoxicity, morphometric analysis, gene expression, Wnt/BMP signaling, Xenopus laevis","lastPublishedDoi":"10.21203/rs.3.rs-7570738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7570738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFluconazole (FLU) is a widely used antifungal agent frequently detected in surface waters because of its extensive use in medicine, agriculture, and personal care products. \u0026nbsp;\u003cbr\u003e\nDespite concerns about its persistence and developmental toxicity in aquatic species, its effects on amphibians remain poorly understood. \u0026nbsp;This study aimed to assess the developmental and molecular effects of FLU and its structural core, 1,2,4-triazole (TRI), in amphibian embryos. \u003cem\u003eXenopus laevis\u003c/em\u003eembryos were exposed to FLU or TRI and evaluated for mortality, hatching rate, heart rate, body length, malformation incidence, and changes in gene expression. \u0026nbsp;Even at low micromolar concentrations, both azoles altered the expression of Wnt- and BMP-associated genes, indicating disruption of these signaling pathways. At higher micromolar concentrations, these molecular changes were accompanied by early signs of developmental abnormalities, which intensified at the highest doses. \u0026nbsp;Observed phenotypes included reduced head size, altered skin pigmentation, prolonged body length, changes in heart rate, and mild digestive tract malformations. \u0026nbsp;These findings demonstrate that even the core structural motif TRI can disrupt key developmental signaling pathways in vertebrate embryos, underscoring the need for closer monitoring of azole compounds in aquatic environments. \u0026nbsp;Given the fundamental role of these pathways in vertebrate development, the results raise concerns about potential risks from long-term or prenatal exposure to azoles, in both environmental and clinical contexts.\u003c/p\u003e","manuscriptTitle":"Developmental toxicity of fluconazole and 1,2,4-triazole in non-target aquatic vertebrates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 17:59:52","doi":"10.21203/rs.3.rs-7570738/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-29T06:41:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T20:31:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T17:13:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168433839447823146652750944970789613186","date":"2025-10-07T19:41:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311927197527631136639489319485312686317","date":"2025-10-05T17:11:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-22T19:00:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-22T18:54:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-18T11:17:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-10T12:46:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-10T12:43:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"434e3b6e-c5a4-42f4-8dce-859a3a7a4824","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55687271,"name":"Biological sciences/Developmental biology"},{"id":55687272,"name":"Biological sciences/Molecular biology"},{"id":55687273,"name":"Biological sciences/Physiology"},{"id":55687274,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-12-08T16:02:28+00:00","versionOfRecord":{"articleIdentity":"rs-7570738","link":"https://doi.org/10.1038/s41598-025-30992-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-06 15:57:54","publishedOnDateReadable":"December 6th, 2025"},"versionCreatedAt":"2025-10-03 17:59:52","video":"","vorDoi":"10.1038/s41598-025-30992-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-30992-5","workflowStages":[]},"version":"v1","identity":"rs-7570738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7570738","identity":"rs-7570738","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}