The Effect of Phosphoric Acid on the Development of Neural Tube Defects in Chick Embryos

Introduction

Neural tube defects (NTDs) rank among the most common congenital anomalies worldwide, producing severe lifelong disabilities, significant healthcare expenditures, and high rates of perinatal and childhood mortality [1]. Each year, approximately 300 000 infants are affected worldwide, although prevalence varies widely depending on geography, socioeconomic conditions, and public health interventions, ranging from as low as 0.2 to as high as 11 per 1000 live births. Importantly, epidemiological studies indicate that NTDs are consistently more frequent in female than in male newborns [2,3]. Clinically, the 2 most prominent forms are spina bifida, in which the spinal column remains incompletely closed, and anencephaly, a severe defect characterized by absent or rudimentary brain development due to anterior neural tube closure failure [4].

The etiology of NTDs is complex and multifactorial, with both genetic predispositions and environmental influences contributing to pathogenesis. Among the most recognized risk factors is folic acid deficiency, which disrupts nucleotide biosynthesis and DNA methylation, thereby interfering with normal neurulation. Other maternal conditions such as hyperthermia, obesity, and exposure to teratogenic drugs also increase the risk. For example, antiepileptic medications including valproic acid and carbamazepine are known to antagonize folate metabolism, while cocaine exposure has been linked to vascular compromise and impaired embryonic development. In addition, methylenetetrahydrofolate reductase (MTHFR) polymorphisms can reduce folate bioavailability, further predisposing the embryo to closure defects [5,6]. Experimental teratology studies have provided further evidence, showing that agents such as alcohol, salicylates, clomiphene, insulin, chemotherapeutic drugs, and even certain viral infections, including influenza, are capable of disrupting neural tube formation [7].

Phosphoric acid (H3PO4) is a simple inorganic acid composed of phosphorus, oxygen, and hydrogen atoms. It is colorless, odorless, fully soluble in water, and widely applied in diverse industrial sectors including fertilizers, chemical processing, pharmaceuticals, and automotive manufacturing. Within the food industry, it is among the most extensively utilized additives. Phosphoric acid serves multiple functions: it regulates acidity in carbonated beverages, stabilizes and preserves processed meats such as sausages and salami, prolongs shelf life in canned goods, and improves texture and flavor balance in dairy products such as cheese. Although regulatory agencies generally consider phosphoric acid safe at controlled concentrations, evidence has accumulated linking excessive intake to adverse systemic outcomes. These include bone demineralization, calcium-phosphate imbalance, renal impairment, and potential systemic toxicity [8–11]. Particularly concerning is the observation that children, adolescents, and women of reproductive age constitute the largest groups consuming carbonated soft drinks containing phosphoric acid, raising questions about long-term developmental effects.

Despite this widespread dietary exposure, the possible teratogenic effects of phosphoric acid have never been directly examined. Previous investigations into other common food additives have produced conflicting results: for instance, tartrazine was reported to induce NTDs in chick embryos, whereas sodium benzoate did not [12,13]. Notably, phosphoric acid has remained untested in experimental models of vertebrate neurulation, despite decades of extensive dietary exposure. This represents a clear and previously unaddressed gap in experimental teratology.

Based on principles of developmental toxicology, we hypothesized a priori that exposure to phosphoric acid during the critical window of neurulation (Hamburger-Hamilton stage 9) would increase embryotoxicity and disrupt neural tube closure in a chick-embryo model. This hypothesis was biologically motivated by the potential for pH-dependent cellular stress, phosphate-related metabolic perturbations, and interference with tightly regulated processes required for neural fold elevation and closure during early embryogenesis, and potential indirect interference with folate-dependent one-carbon metabolic pathways, which are critical for neural tube closure.

Accordingly, the present study was designed as a proof-of-concept experimental investigation to evaluate the effects of phosphoric acid on embryo survival and neural tube development in a chick-embryo model. This model provides direct access to early developmental stages and allows precise assessment of neurulation, making it a well-established system for investigating environmental and chemical influences on early embryogenesis. Importantly, the scope of this study is limited to experimental teratogenicity within a controlled vertebrate model and does not aim to assess dietary exposure relevance or risk in human pregnancy.

Material and Methods

STUDY DESIGN:

This study was conducted in collaboration with the Department of Histology and Embryology, Hamidiye Faculty of Medicine, University of Health Sciences. In this experimental study, fertilized, pathogen-free chicken eggs were used for incubation and injection (Bornova Veterinary Control and Research Institute, İzmir, Türkiye). Eggs were randomly assigned to groups using a computer-generated random sequence to ensure unbiased distribution. Given the exploratory nature of the study, a formal a priori power analysis was not performed. The sample size was determined in line with previous chick-embryo teratology studies using comparable group sizes.

Group A (Control group): No phosphoric acid was administered (n=15).

Group B (Experimental group): 0.25 mM phosphoric acid (approximately 25 μL of 1 M phosphoric acid solution per 1 L) was injected (n=15).

Physiological saline was employed as the vehicle for phosphoric acid administration. However, a saline-injected vehicle control group was not included in the study. Therefore, potential effects related to the injection procedure itself, including needle manipulation and fluid volume, cannot be fully excluded. Accordingly, the findings should be interpreted as reflecting the effects of phosphoric acid exposure within the context of the injection procedure rather than isolating the chemical effect alone.

PHOSPHORIC ACID DOSE:

The dose was determined based on the range (0.05–0.5 mM) reported in the literature as safe for embryonic cultures. During a preliminary screening phase, low (0.05 mM), medium (0.25 mM), and high (0.5 mM) concentrations were evaluated to assess gross embryonic tolerance and neurulation outcomes. Based on this exploratory screening, 0.25 mM was selected as the lowest concentration associated with consistent neural tube disruption and was therefore used in the main experiment. Phosphoric acid (H₃PO₄ from İPEK-KİMYA, Formula 10.5, CAS No: 7664-38-2, concentration: 85%) was diluted with sterile physiological saline to achieve the desired concentration. This exploratory screening was not powered for formal statistical inference and was used solely to guide selection of a single concentration for proof-of-concept testing.

INCUBATION AND INJECTION:

Eggs (average weight 65±2 g) were incubated at 37.2±0.1°C with 60–70% humidity for 24 hours, reaching stage 9 according to the Hamburger-Hamilton classification [14]. Temperature and humidity were continuously monitored using the incubator’s digital sensors and verified daily with an external calibrated thermometer/hygrometer. Deviations were corrected immediately to maintain stable conditions. Under a stereomicroscope at 4×magnification, the eggshells were carefully opened under sterile conditions, and the embryonic discs were identified. To maintain consistency and minimize variability, a volume of 0.1 mL of the prepared solution was injected beneath the embryonic disc using a sterile 24-gauge syringe. Following injection, the eggs were resealed with sterile adhesive tape and returned to the incubator for an additional 72 hours. At the end of this period, embryos were dissected from the surrounding membranes and examined macroscopically and histopathologically for neural tube development.

HISTOPATHOLOGICAL EVALUATION:

At 72 hours, embryos were fixed in 4% formalin for 24 hours, dehydrated in a graded ethanol series (70%, 95%, 100%), cleared in xylene, and embedded in paraffin. Serial sections of 5 μm thickness were prepared using a rotary microtome. Sections were deparaffinized in xylene, rehydrated through descending ethanol concentrations, and stained with hematoxylin and eosin (H&E). Slides were mounted with Entellan® (Merck) and examined under a Zeiss Axiocam light microscope. NTDs were defined as persistent failure of neural fold closure, the presence of an open neural plate, or abnormal notochord and somite organization. All evaluations were performed in a blinded manner, where the pathologist was unaware of the treatment groups to prevent bias. Group codes were concealed during macroscopic scoring and histopathological assessment.

STATISTICAL ANALYSIS:

Data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA). Survival rates and the incidence of NTDs were compared between groups using Fisher’s exact test. Fisher’s exact test was specifically chosen as it is the most robust and accurate method for analyzing categorical data with small sample sizes. A P-value <0.05 was considered statistically significant. Results were expressed as absolute numbers and percentages. No correction for multiple comparisons was required, as only 2 groups were analyzed. All histopathological and statistical evaluations were performed in a blinded manner. Embryolethality (based on survival at 72 hours) and neural tube malformation were predefined and analyzed as distinct experimental endpoints.

Results

SURVIVAL:

At 72 hours of incubation, all embryos in the control group survived (15/15, 100%). In the phosphoric acid group, survival was significantly reduced, to 10/15 (66.7%) (Fisher’s exact test, P=0.0421). Odds ratio (OR) analysis with Haldane-Anscombe correction indicated reduced survival odds in the phosphoric acid group compared with controls (OR 0.062; 95% CI 0.003–1.236).

NEURAL TUBE DEFECTS:

No anomalies were observed in the control group. In contrast, among the 10 surviving embryos in the phosphoric acid group, 8 (80%) exhibited NTDs. The difference in incidence was statistically significant (Fisher’s exact test, P=0.0000416). The odds of developing NTDs were markedly increased in the phosphoric acid group (OR 105.4; 95% CI 4.52–2458.5). When calculated relative to the total number of treated embryos, NTDs were observed in 8 of 15 embryos (53.3%), providing a sensitivity analysis that accounts for embryolethality-related censoring. The magnitude of both survival reduction and neural tube defect incidence observed in this study appears greater than that reported in most prior chick-embryo teratogenicity studies involving food additives or environmental exposures, highlighting the distinct strength of the observed effect. These findings directly support the a priori hypothesis that phosphoric acid disrupts early neurulation within this experimental model.

HISTOPATHOLOGICAL FINDINGS:

Control embryos demonstrated intact neural tube morphology with well-formed notochord and somites (Figure 1A). In the phosphoric acid group, embryos exhibited neural tube closure defects, irregular notochord morphology, incomplete ectodermal invagination, and disorganized somite development (Figure 1B-1D). As shown in Figure 2, the high incidence of defects in survivors highlights the strong disruptive effect of the tested dose on neural tube development within this experimental model. NTDs in the phosphoric acid–treated embryos demonstrated heterogeneous phenotypic severity. Defects were classified descriptively as mild (partial neural fold elevation without complete closure), moderate (persistent open neural tube with localized notochord irregularity), or severe (extensive cranial and/or caudal non-closure accompanied by marked notochord and somite disorganization). Anatomically, defects involved the cranial region, caudal region, or both, indicating non-uniform disruption of neurulation rather than nonspecific toxicity.

STATISTICAL SUMMARY:

Survival and NTD incidence are presented in Table 1. Per-embryo survival status, developmental stage confirmation, anatomical localization of defects, and histopathological scoring were recorded for all embryos and are summarized in the figures and tables presented.

Data are presented as number of embryos and percentages. Survival was recorded at 72 hours, and NTD incidence was calculated among surviving embryos. Fisher’s exact test results were: survival (P=0.0421); NTD incidence (P=0.0000416). Odds ratios estimated with Haldane-Anscombe correction were: survival OR 0.062 (95% CI 0.003–1.236); NTD incidence OR 105.4 (95% CI 4.52–2458.5). NTD incidence is presented both among surviving embryos and relative to the total number of treated embryos to distinguish malformation from embryolethality (Figure 2).

Discussion

LIMITATIONS:

This study has several limitations. First, only a single concentration of phosphoric acid was administered, which precluded assessment of dose–response relationships. Second, the follow-up period was limited to 72 hours, preventing evaluation of later developmental stages and the persistence of defects over time. Third, although the chick-embryo model is widely used in experimental teratology because of its accessibility and well-characterized stages, it does not fully replicate mammalian embryogenesis. Therefore, extrapolation of these findings to human pregnancy should be made with caution. Finally, although previous reports indicate that saline injections alone do not affect survival or neural tube development in this model [11,12], vehicle-injected controls were not included; therefore, we cannot completely exclude a minor procedural effect of injection. However, the magnitude of the observed differences suggests a treatment-related effect.

FUTURE DIRECTIONS:

Further studies should investigate different phosphoric acid concentrations to establish dose–response relationships. Extending incubation beyond 72 hours would enable evaluation of long-term developmental outcomes and the persistence or resolution of NTDs. Molecular analyses of oxidative stress, DNA damage, and apoptotic pathways may help elucidate the mechanisms by which phosphoric acid disrupts neurulation. Comparative experiments in mammalian models will also be important to clarify the translational relevance of these findings to human pregnancy. Given the widespread use of phosphoric acid in processed foods, future translational research is critical to determine potential risks for human embryonic development.

Conclusions

This study provides the first experimental, proof-of-concept evidence in a vertebrate neurulation model that phosphoric acid can disrupt early neural tube closure when administered during a critical developmental window. The findings demonstrate a strong internal effect at the tested dose and time point, while also highlighting important methodological limitations, including the absence of vehicle-injected controls, dose–response assessment, and exposure-bridging analyses. Accordingly, these results should be interpreted within the context of experimental teratology and do not permit conclusions regarding human dietary exposure or pregnancy risk. Further studies incorporating vehicle-controlled replication, dose–response designs, mechanistic analyses, and translational exposure modeling are required to clarify the biological specificity and broader relevance of these findings.