Subchronic Exposure to Nonylphenol Ethoxylate at Environmentally Relevant Level Promotes Mutagenic, Neurotoxic, and Pro-oxidant Effects in Bullfrog Tadpoles

TL;DR

Exposure to nonylphenol ethoxylate at low levels harms bullfrog tadpoles by causing genetic damage, nerve issues, and oxidative stress.

Contribution

This study reveals new evidence of NPE's harmful effects on amphibian physiology at environmentally relevant concentrations.

Findings

NPE exposure caused genetic damage in tadpole blood cells.

NPE impaired nerve function in tadpoles' brains and muscles.

NPE triggered oxidative stress in tadpole blood.

Abstract

Nonylphenol compounds released into aquatic environments have been shown to exert deleterious effects on aquatic organisms. Amphibians, particularly during larval stages, are highly vulnerable to chemical stressors and are chronically exposed to pollutants such as nonylphenol ethoxylate (NPE). This study evaluated the sublethal effects of environmentally relevant concentrations of NPE (30 µg L−1) under sub-chronic exposure in Aquarana catesbeiana tadpoles (Gosner stage 25), with emphasis on mutagenic, neurotoxic, and oxidative responses. The analyses included blood cell composition, frequency of micronuclei (MN) and erythrocytic nuclear abnormalities (ENAs), acetylcholinesterase (AChE) activity in brain and muscle tissues, and oxidative stress biomarkers (lipid peroxidation and protein carbonylation) in blood. Results demonstrated that NPE exposure induced clastogenic and aneugenic effects in erythrocytes, impaired central and muscular cholinergic function, and triggered oxidative stress in blood. These findings highlight the NPE potential to disrupt critical physiological systems in amphibians.

Figures

Frequencies of A micronucleus (MN) and B total erythrocytic nuclear abnormalities (ENAs) frequency in bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate-exposed (NPE, 30 µg L^−1^, n = 12) groups. Data are presented as mean + SE. Asterisks indicate statistically significant differences between groups (*p* < 0.05). Representative erythrocytic morphologies: C normal erythrocyte (left) and vacuolated nucleus (right), D MN, E binucleated cell, F notched nucleus, and G lobed nucleus

Box plots of acetylcholinesterase (AChE) activity (nmol mg^−1^ min^−1^) in the A brain and B muscle of bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate-exposed (NPE, 30 µg L^−1^, n = 12) groups. The plus sign (+) indicates group means. Asterisks denote significant difference between groups (*p* < 0.05)

Box plots of A lipid peroxidation (LPO, nmol mg^−1^ Hb) and B protein carbonylation (PC, nmol mg^−1^ Hb) levels in the blood of bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate-exposed (NPE, 30 µg L^−1^, n = 12) groups. The plus sign (+) indicates group means. Asterisks denote significant difference between groups (*p* < 0.05)

Full text

Introduction

The decline in water quality is an increasing threat to freshwater ecosystems and aquatic life, driven by inadequate industrial waste management, insufficient wastewater treatment, and intensive agricultural practices (Udin et al. 2021; Morin-Crini et al. 2022). Of particular concern is nonylphenol ethoxylate (NPE), a nonionic surfactant from the alkylphenol group, widely used in detergents, emulsifiers, and wetting agents (Raju et al. 2018). Its persistent nature and extensive use result in frequent detection in water bodies worldwide (Suman et al. 2025). Inadequate wastewater treatment can lead to the release of up to 60% of nonylphenol compounds into aquatic environments (Solé et al. 2000). Reported environmental concentrations of this emerging contaminant vary widely, from below detectable limits to 274 µg L^−1^ (Céspedes et al. 2008; Peng et al. 2008; Salomon et al. 2019; Alves et al. 2022), whereas concentrations in sewage effluents may exceed 300 µg L^−1^ (Ying et al. 2002; Sun et al. 2017).

Nonylphenol compounds pose a significant threat to biodiversity and human health, with substantial ecological and public health implications. They disrupt endocrine function in aquatic organisms, causing reproductive impairments, developmental abnormalities, and growth alterations that can destabilize populations (Spehar et al. 2010; Hong et al. 2020; Suman et al. 2025). Their potential for bioaccumulation raises concerns about chronic human exposure through aquatic organism consumption, highlighting the need for systematic monitoring and regulatory frameworks (Korsman et al. 2015; Peng et al. 2023). Although the effects of NPE have been characterized in fish, its long-term impacts across taxa remain poorly understood, particularly in amphibians, which serve as key bioindicators due to their permeable skin and dual reliance on aquatic and terrestrial habitats (Calderon et al. 2019; Han et al. 2025).

Amphibians, such as bullfrog (Aquarana catesbeiana) tadpoles, are widely used as a model organism in ecotoxicology due to their heightened sensitivity to environmental stressors during early developmental stages (Kloas and Lutz 2006; Burggren and Warburton 2007). Given that larval habitat quality directly affects the viability of anuran populations (Jackson et al. 2024), it is crucial to understand how emerging contaminants impact tadpole physiology to inform both conservation strategies and ecological risk assessments. A previous study by our research group demonstrated that acute (48 h) exposure to NPE at 30 µg L^−1^ induces oxidative stress and disrupts antioxidant and cholinergic systems across multiple tissues (Gonçalves et al. 2026). Based on these findings, we hypothesize that subchronic exposure to environmentally relevant concentrations of NPE induces clastogenic and aneugenic effects in erythrocytes, alters differential blood cell counts, and triggers neurotoxicity in brain, muscle, and cardiac tissues, along with oxidative stress in blood, thereby compromising the health and overall fitness of bullfrog tadpoles.

Materials and Methods

Animals and Experimental Design

Specimens of Aquarana catesbeiana (Shaw 1802) at Gosner stage 25 (Gosner 1960) with body weight of 2.1 ± 0.2 g, were obtained from a commercial supplier (Ranamat, Matão, São Paulo, Brazil). Animals were acclimatized for 15 days in 500-L holding tanks (1 animal L^−1^) under controlled environmental conditions: a constant temperature of 25 ± 1 °C, a natural photoperiod (12 h light: 12 h dark), and continuous aeration providing oxygen-saturated, dechlorinated water. During this period, tadpoles were fed daily a commercial diet (Pro Acqua MP 31, Moinho Primor) containing 35% crude protein.

A subchronic (16-day) exposure assay was conducted with nonylphenol ethoxylate (NPE; Tergitol™ NP-9, > 90%, CAS N^o^ 127087-87-0, Sigma-Aldrich). The experimental design included two groups in a semi-static system: a control group (Ct, n = 12) maintained in NPE-free water, and an exposed group (NPE, n = 12) subjected to a nominal concentration of 30 µg L^−1^. Tadpoles were maintained in individual glass containers at a density of 1 animal L^−1^, in accordance with standardized guidelines for aquatic toxicity testing (USEPA 1996). To ensure stable water quality and maintain consistent NPE exposure, 90% of the water in each experimental aquarium was renewed every 48 h. Following each renewal, NPE was reintroduced by adding 27 µL of stock solution (1 mg L^−1^) per liter of aquarium water, accounting for the residual 10% of the previous exposure, to restore the nominal final concentration of 30 µg L^−1^. Water quality parameters were assessed daily and remained constant across all treatments throughout the study period: temperature (24.2–25.4 °C), pH (6.6–7.4), dissolved oxygen (122–139 mmHg), and ammonia (0.4–0.7 mg·L^−1^). NPE concentrations in water samples (n = 8) collected every 48 h were determined using a Shimadzu Nexera X2 high-performance liquid chromatography (HPLC) system with an AB SCIEX 3200 QTRAP tandem mass spectrometer equipped with a linear ion trap quadrupole, following the protocol of Sodré et al. (2010). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.67 µg L^−1^ and 2.02 µg L^−1^, respectively, based on the lowest point of the calibration curve. Method precision, expressed as relative standard deviation (RSD), ranged from 3.18 to 10.26%, while method accuracy was evaluated through recovery experiments, yielding recoveries between 85.1 and 117.4%.

The exposure concentration of 30 µg L^−1^ was chosen for its environmental relevance, as it reflects levels ranged from 0.1 to 2693 μg L^−1^ reported in Brazilian aquatic ecosystems impacted by effluent discharges (Moura 2009; Alves et al. 2022) and has also been documented in other regions worldwide, ranging from 8.7 to 332 μg L^−1^ (Ying et al. 2002; Salomon et al. 2019). Although the USEPA (2005) sets a one-hour limit of 28 µg L^−1^ for nonylphenol to protect freshwater organisms, the effects of prolonged exposure at this level remain largely unknown. After exposure, tadpoles were anesthetized with benzocaine (0.1%), and blood was extracted via cardiac puncture using heparinized syringes for hematological analyses. Euthanasia was performed by anesthetic overdose (0.5% benzocaine) in accordance with AVMA (2001) guidelines. Brain and muscle tissues were excised, snap-frozen in liquid nitrogen, and stored at − 80 °C for biochemical analyses.

Mutagenicity and Differential Blood Count

Blood smears were prepared immediately after sampling by spreading 5 µL aliquots on pre-cleaned slides, air-dried, fixed in methanol (10 min), and dyed with Periodic Acid–Schiff (PAS). Triplicate slides per specimen were imaged under a 100 × objective using an Olympus BX-61 microscope and analyzed with cellSens™ software. For each slide, 1000 cells were examined to determine cell-type distribution in areas of optimal cell dispersion and minimal overlap. The frequency of micronuclei (MN), and the occurrence of other erythrocytic nuclear abnormalities (ENAs) (Lajmanovich et al. 2005; Shahjahan et al. 2020). Blood cell composition (erythrocytes, total leukocytes, thrombocytes, and leukocyte subtypes) was determined following Thrall (2004). MN and ENAs (e.g., notched, lobed, pyknotic, vacuolated, binucleated, apoptotic) were classified according to Lajmanovich et al. (2005) and Benvindo-Souza et al. (2020).

Acetylcholinesterase (AChE) activity

AChE activity in brain and muscle tissues was assessed using a modified Ellman et al. (1961) protocol. Tissue samples were homogenized in 0.1 M phosphate buffer (pH 8.0) at a 1:4 (w/v) ratio and centrifuged at 10,000 g for 10 min at 4 °C. The supernatant obtained was utilized for enzymatic activity measurements and protein determination. AChE activity was monitored spectrophotometrically (UV Vis, BEL, Italy) at 412 nm, with one unit defined as the amount of enzyme catalyzing the hydrolysis of 1 nmol of acetylcholine per minute.

Biomarkers of Oxidative Stress

Blood samples were subjected to hypotonic lysis in 20 mM Tris–HCl (pH 8.0) at a 1:1 (v/v) ratio. Hemoglobin concentration was measured colorimetrically at 540 nm using the cyanmethemoglobin method (Labtest Diagnóstica, Brazil). Lipid peroxidation (LPO) was determined as described by Jiang et al. (1992), based on Fe^2+^ oxidation to Fe^3+^ by lipid hydroperoxides, forming a Fe^3+^-xylenol orange complex measured at 560 nm. LPO levels were calculated from a cumene hydroperoxide standard curve and expressed as nmol per mg hemoglobin. Protein carbonylation (PC) was quantified following Reznick and Packer (1994) via derivatization with 2,4-dinitrophenylhydrazine (DNPH) at 370 nm using a molar extinction coefficient of 22,000 M^−1^·cm^−1^, and expressed as nmol PC per mg hemoglobin.

Statistical Analysis

Normality was assessed using the Kolmogorov–Smirnov test, and homogeneity of variances was verified with the F-test. Comparisons between Control and NPE groups were performed using either an unpaired t-test or a Mann–Whitney test (GraphPad Instat 3.1, GraphPad Software, Inc.). Statistical significance was set at p < 0.05.

Results and Discussion

Measured NPE concentrations ranged from 25.18 to 35.29 µg L^−1^, indicating that exposure levels were maintained close to the nominal concentrations throughout the experimental period. NPE exposure did not affect tadpole survival, as no deaths were observed during the 16-day treatment period. This lack of lethality is consistent with previous studies demonstrating that environmentally relevant concentrations of nonylphenol compounds can disrupt physiological and cellular functions without causing immediate mortality (Scaia et al. 2019; Salgueiro et al., 2021).

Exposure to NPE resulted in significant hematological alterations, including increases in total leukocytes (232%, p = 0.0002, t = 7.317, df = 14), thrombocytes (123%, p = 0.0214, t = 2.591, df = 14), monocytes (158%, p < 0.0001, t = 5.645, df = 14), and lymphocytes (589%, p = 0.0070, t = 2.256, df = 14), accompanied by a 10% reduction (p < 0.0001, t = 6.579, df = 14) in erythrocyte frequency (Table 1). No significant differences were observed in the frequencies of neutrophils, basophils, or eosinophils between the groups. Leukocytosis represents a key immunophysiological adaptation that enables aquatic vertebrates to adjust hematopoiesis and enhance innate immune defenses in response to contaminant-induced stress and/or pathogen exposure and has been documented in fish and amphibians (Romanova et al. 2003; Davis et al. 2008; Grzelak et al. 2017). However, for this response following subchronic NPE exposure, particularly in larval amphibians, remains limited. In the present study, leukocytosis was primarily driven by increases in lymphocytes and monocytes, two central components of the amphibian immune system (Ruiz and Robert 2023; Assis and Titon 2025) indicating activation of both adaptive and innate immune pathways. While lymphocytes mediate antigen-specific responses, monocytes contribute to innate defense by differentiating into macrophages or dendritic cells at sites of tissue damage or infection (Yaparla et al. 2023). In addition, the NPE-induced elevation in thrombocytes likely enhances immune defense by releasing inflammatory and antimicrobial mediators, as well as other molecules that modulate immune responses (Ferdous and Scott 2023). From a functional perspective, persistent leukocytosis in larval amphibians suggests prolonged immune activation, a condition that can increase energetic demands and reallocate physiological resources at the expense of growth and developmental processes. Moreover, the concomitant decrease in erythrocyte counts suggests that NPE exposure may induce hemolytic or cytotoxic effects (Galembeck et al. 1998; Paolella et al. 2021), potentially impairing oxygen transport and overall physiological function. Together, these hematological changes indicate that NPE-induced alterations in blood cell profiles are not only indicative of immune activation but may also have broader implications for larval performance and fitness.Table 1. Frequencies of peripheral blood cell types and erythrocytic nuclear abnormalities (ENAs) in bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate–exposed (NPE, 30 µg L^−1^, n = 12) groupsCell typesFrequency (number per 1000 cells)CtNPEENAsCtNPEErythrocytes936.2 ± 5.9842.25 ± 8.9Lobed nuclei (LB)1.1 ± 0.23.5 ± 0.45Leukocytes28.13 ± 2.693.2 ± 5.2Notched nuclei (NT)1.2 ± 0.43.1 ± 0.4Trombocytes13.8 ± 3.531.0 ± 3.7Blebbed nuclei (BL)3.5 ± 1.14.7 ± 0.8Lymphocytes2.2 ± 0.615.5 ± 3.8Kidney shaped nuclei (KS)4.2 ± 0.96.9 ± 1.2Monocytes28.0 ± 4.172.4 ± 3.9Pycnotic nuclei (PN)2.5 ± 0.55.7 ± 3.8Neutrophils1.6 ± 0.43.4 ± 0.6Vacuolated nuclei (VA)0.1 ± 0.0436.6 ± 11.2Basophils1.0 ± 0.32.0 ± 0.5Binucleated cells (BN)0.1 ± 0.051.0 ± 0.2Eosinophils0.1 ± 0.10.25 ± 0.1Apoptotic cell (AP)0.2 ± 0.13.6 ± 1.2Data are presented as mean ± SEM. Bold values with asterisks indicate statistically significant differences between groups (p < 0.05)

In addition to hematological alterations, NPE exposure caused pronounced mutagenic effects, as evidenced by significant increases in the frequencies of MN (400%, p = 0.0007, t = 4.320, df = 14) and total ENAs (409%, p = 0.0002, t = 3.835, df = 13) (Fig. 1). Erythrocytes displayed marked morphological abnormalities, including elevated frequencies of lobed nuclei (211%, p = 0.015, t = 3.428, df = 14), notched nuclei (150%, p = 0.031, t = 2.393, df = 14), binucleated cells (700%, p = 0.028, t = 2.497, df = 14), and vacuolated nuclei (293%, p = 0.048, t = 2.838, df = 14) (Table 1). Representative images of MN and the predominant NPE-induced ENAs are presented in Fig. 1. MN arise when acentric chromosome fragments or entire chromosomes fail to segregate properly to the cell division poles during anaphase, leading to the formation of small extranuclear bodies in daughter cells (Bhuyan et al. 2020). The occurrence of MN serves as a biomarker of permanent genetic damage in amphibian individuals (Josende et al. 2015; Silva et al. 2021). The increases in ENA frequencies indicates that sublethal and subchronic exposure to NPE exerts clastogenic and aneugenic effects on red blood cells of A. catesbeiana tadpoles, potentially compromising chromosome integrity. Abnormal nuclear morphologies, including lobed, vacuolated, and notched nuclei, as well as the occurrence of MN and binucleated cells, are indicative of chromosomal instability and disruptions in proteins essential for the cell division cycle (Fenech 2007). These alterations may be associated with the pro-oxidant activity of NPE, causing damage to DNA and nuclear proteins (Gonzalez-Hunt et al. 2018), which in turn promotes strand breakage, repair failures, and chromosomal fragmentation, ultimately leading to impaired regulation of erythropoiesis. While these effects were detected in erythrocytes, similar mechanisms could plausibly occur in other proliferative cell types, potentially extending chromosomal instability beyond blood cells. Further studies targeting additional tissues and cell populations are therefore warranted to determine the broader extent of NPE-induced genotoxicity.Fig. 1. Frequencies of A micronucleus (MN) and B total erythrocytic nuclear abnormalities (ENAs) frequency in bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate-exposed (NPE, 30 µg L^−1^, n = 12) groups. Data are presented as mean + SE. Asterisks indicate statistically significant differences between groups (p < 0.05). Representative erythrocytic morphologies: C normal erythrocyte (left) and vacuolated nucleus (right), D MN, E binucleated cell, F notched nucleus, and G lobed nucleus

The activity of AChE (Fig. 2) was employed as a neurotoxic biomarker, showing that subchronic exposure to NPE significantly inhibited its activity in both the brain (38%, p = 0.0411, Mann Whitney-U = 5) and muscle (72%, p = 0.0002, t = 5.183, df = 15). Although inhibition was more pronounced in muscle tissue, the central nervous system was also affected, indicating the ecological risk posed by environments contaminated with nonylphenol compounds. Similarly, Karmakar et al. (2021) reported a reduction in brain AChE activity in Labeo rohita following chronic exposure of nonylphenol (2.7 µg L^−1^). Given that AChE activity at neuromuscular junctions is essential for locomotor control (Rotundo et al. 2020) and that its inhibition in the central neural tissue compromises cholinergic neurotransmission, such impairments may result in severe physiological dysfunctions, with potential chronic consequences for swimming performance and metamorphosis.Fig. 2. Box plots of acetylcholinesterase (AChE) activity (nmol mg^−1^ min^−1^) in the A brain and B muscle of bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate-exposed (NPE, 30 µg L^−1^, n = 12) groups. The plus sign (+) indicates group means. Asterisks denote significant difference between groups (p < 0.05)

Excessive production of reactive oxygen species (ROS) disrupts redox homeostasis, leading to oxidative damage of biomolecules and potentially triggering cell death through apoptotic or necrotic pathways (Song et al. 2023). Exposure to NPE resulted in significant increases in lipid peroxidation (43%, p = 0.0006, t = 4.144, df = 13) and protein carbonylation (11%, p = 0.0117, t = 2.854, df = 16) in the blood of tadpoles (Fig. 3). In fish, exposure to nonylphenol compounds has been reported to induce oxidative damage and apoptosis, as evidenced by increased H_2_O_2_ production and LPO in cichlid ovaries (Asifa and Chitra 2016), disruption of the antioxidant defense system in marine medaka embryos (Lee et al. 2018), and elevated LPO in the brains of zebrafish (Desai et al. 2023). Oxidative stress can induce DNA damage, potentially leading to mutations (Besaratinia et al. 2024) and contributing to the elevated frequencies of MN and ENAs observed in tadpoles exposed to NPE. Furthermore, oxidative stress may compromise the O_2_-carrying capacity, potentially leading to tissue hypoxia (Obeagu et al. 2024).Fig. 3. Box plots of A lipid peroxidation (LPO, nmol mg^−1^ Hb) and B protein carbonylation (PC, nmol mg^−1^ Hb) levels in the blood of bullfrog tadpoles from the control (Ct, n = 12) and nonylphenol ethoxylate-exposed (NPE, 30 µg L^−1^, n = 12) groups. The plus sign (+) indicates group means. Asterisks denote significant difference between groups (p < 0.05)

These results indicate that sublethal NPE exposure induces oxidative damage to proteins and lipids, which may increase cellular maintenance costs by diverting energy away from growth and development. In parallel, AChE inhibition in neural and muscular tissues may impair neuromotor performance and feeding efficiency, thereby indirectly affecting somatic growth. Moreover, the occurrence of MN and ENAS reflects genomic instability, which may reduce long-term cellular viability. Collectively, these mechanisms suggest that even low NPE concentrations, close to established safety thresholds, may result in biologically relevant long-term consequences.

Conclusion

Overall, our findings demonstrate that subchronic exposure to NPE, even at environmentally relevant concentrations, disrupts multiple interconnected physiological systems in bullfrog tadpoles, eliciting mutagenic, neurotoxic, and pro-oxidant effects with potential consequences for amphibian health. The observed alterations are likely to impair key functions such as oxygen transport, immune homeostasis, and neuromuscular performance during sensitive larval stages, thereby reducing stress tolerance and overall physiological performance. Consequently, these effects may increase vulnerability to additional environmental stressors, leading to ecologically meaningful outcomes at higher levels of biological organization. Notably, the occurrence of adverse responses at concentrations close to those considered safe for potable water highlights the environmental risk posed by NPE to aquatic ecosystems.