Unveiling the toxicological mechanisms and biological impacts of copper sulfide nanoparticles on zebrafish

ABSTRACT

Aim

Nanoparticles (NPs) are increasingly released into the environment, posing risks to living organisms. While copper sulfide NPs have been extensively studied, the toxicity of copper sulfide (CuS) NPs remains underexplored. Owing to their widespread use, it is crucial to assess their biological effects.

Materials & methods

In this study, we used zebrafish embryos and larvae to evaluate CuS NP toxicity. Embryos were exposed to 20–100 µg/mL of CuS NPs under both dark and light conditions.

Results

Concentrations ≥60 µg/mL caused up to 85% mortality, along with developmental abnormalities such as bent tails and pericardial edema. Under light irradiation, CuS NPs significantly elevated reactive oxygen species (ROS) production. This was evidenced by upregulated expression of antioxidant genes sod1, sod2, cat, and gpx1 in exposed embryos.

Conclusions

Our findings demonstrate a photoreactive toxicity mechanism: CuS NPs generate ROS under light, disrupting redox balance. This underscores the importance of environmental factors, like sunlight, in nanomaterial risk assessment. By revealing how CuS NPs affect aquatic organisms under realistic conditions, our study contributes to understanding the ecological risks these particles pose and highlights their potential implications for human health.

GRAPHICAL ABSTRACT

1. Introduction

The twenty-first century has witnessed the growing prominence of nanotechnology across a wide range of industries [1]. The production of nanoparticles is currently increasing significantly due to the growing popularity of nanotechnology in sectors such as food, healthcare, electronics, and cosmetics [2]. Currently, the NP market is valued at approximately US$7.1 billion, with projections to reach US$12.1 billion by 2026 [3,4]. Numerous applications of NPs result in their release of more than 100,000 metric tons per year, a figure anticipated to increase significantly by 2025 in both aquatic and terrestrial environments [5]. Understanding the toxicity of various NPs is crucial, as their increased presence in the environment poses potential risks to aquatic organisms and human health [6–8]. It is essential to comprehend the different forms of NPs and the routes of exposure to determine the associated risk factors, as well as their fate and transport in the environment. NPs can exhibit varied properties depending on their production methods (such as vapor deposition, gas phase, attrition, colloidal processes, etc.), leading to human exposure through inhalation, ingestion, or dermal absorption [9,10]. It is becoming increasingly evident that NPs have adverse environmental impacts.

Furthermore, a recent study examining the effects of TiO₂ NPs on a zebrafish animal model revealed a decrease in acetylcholinesterase activity, as well as reductions in both enzymatic and non-enzymatic antioxidants. Additionally, there was an increase in proteins, reactive oxygen species (ROS), and lipid peroxidation products [11]. Evidence shows that Au NPs disrupt inflammatory and other immune pathways, while Ag NPs can cause immunotoxicity in adult zebrafish [12,13]. Researchers have found that CdO NPs derived from the calcination of Cd(OH)₂ decreased metallothionein levels in zebrafish more rapidly than CdO NPs from the calcination of a Cd-citrate coordination polymer, indicating greater toxicity [14]. Exposure of zebrafish embryos and larvae to Fe₂O₃ NPs resulted in developmental toxicity, oxidative stress, and DNA damage [15]. In keratinocytes, ZnO and TiO₂ NPs had detrimental effects on mitochondrial activity, cell morphology, and the cell cycle. A recent study by Chong et al. showed that ZnO NPs caused inflammation of the airways in mice, while Mani et al. found that chronic exposure to CuO NPs led to muscular toxicity. Another study by Duan et al. suggested that silica NPs can damage cardiac function [16].

Moreover, among the numerous NPs being studied, copper sulfide (CuS) NPs have garnered significant attention due to their unique optical and electronic properties [17]. CuS NPs are suitable for a wide range of applications, including semiconductors, sensors, photoacoustic imaging, drug delivery, theranostics, and the detection of DNA and glucose in biomedical fields [18,19]. As CuS NPs are widely used in many industries, there is growing concern about their environmental impacts. CuS NPs often end up in aquatic environments, where they can have toxic effects. When NPs are present in water, they can become oxidized, aggregated, and agglomerated, which influences biochemical reactions. Several factors determine the water toxicity of NPs, including their size, shape, surface charge, and concentration [20,21]. Reactive oxygen species (ROS) produced by CuS nanoparticles have been linked to oxidative stress, cellular membrane damage, mitochondrial malfunction, and other harmful effects [22–26]. Despite the significant attention CuS NPs have received for their unique properties and wide range of applications, there are limited research specifically focusing on their environmental impacts. As these CuS NPs are commonly found in aquatic environments, understanding their toxicity and behavior provides a deeper understanding.

In addition, zebrafish (Danio rerio) and their embryos are promising in vitro and in vivo models for studying toxicity due to their high fecundity, cost-effectiveness, optical transparency, and the availability of transgenic strains. Notably, the nervous system, cardiovascular system, and the digestive system of zebrafish and humans are similar based on phylogenetic analyses [27]. Furthermore, toxicity tests using zebrafish embryos can provide valuable data to help researchers understand how the animal structure, function, and behavior are influenced by biochemical, genetic, and cellular factors [28–31]. Zebrafish embryos are particularly useful for high-throughput applications because they develop rapidly, with the entire body forming within 96 h and reaching adulthood in 2 months [32,33]. Consequently, zebrafish embryos serve as an excellent model for observing acute and early-life toxicological outcomes, as well as sublethal effects on human development [34–38].

In the present study, we synthesized CuS NPs using a hydrothermal method and determined the toxicological impacts on zebrafish embryos and larvae by examining abnormal development, biochemical indicators of oxidative stress, and mortality. The mechanism underlying CuS NP-induced developmental abnormalities due to oxidative stress in zebrafish have not yet been reported. Additionally, the study involved determining reactive oxygen species (ROS) activity, along with the levels of superoxide dismutase (sod1 and sod2), catalase (cat), and glutathione peroxidase (gpx1) genes. Overall, this study provides valuable insights into the development of safety guidelines for NPs that may be used in a variety of future applications.

2. Materials and methods

2.1. Materials

Copper nitrate (II) nitrate trihydrate (Cu(NO₃)₂·3 H₂O), thiourea, and ethanol 75% (Sigma-Aldrich, St. Louis, MO, USA and Bioshop, Taipei, Taiwan) were of the highest chemical grades. The Vero cell line (CCL-81™), crystal violet staining solution (0.5%), trypsin (10%) were from Mediatech (Manassas, VA, USA). Dulbecco’s modified Eagle medium (DMEM) was from ThermoFisher Scientific (Carlsbad, CA, USA). Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were obtained from Hyclone Laboratories (South Logan, UT, USA). Paraformaldehyde (PFA) and penicillin were from Santa Cruz Biotechnology (Dallas, TX, USA). TRIzol reagent and the TOOLSQuant II Fast RT Kit were from Biotools (New Taipei City, Taiwan). LightCycler® 480 SYBR Green I Master, primers (10 mM), artificial fresh water (AFW), calcium sulfate (CaSO₄), 0.5 mM sodium chloride (NaCl), 0.2 mM magnesium sulfate (MgSO₄), 0.16 mM monopotassium phosphate (KH₂PO₄), and 0.16 mM dipotassium phosphate (K₂HPO₄) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Synthesis of CuS NPs

CuS NPs were synthesized using the hydrothermal method. A total of 0.9664 g of Cu(NO₃)₂·3 H₂O was dissolved in 40 mL of ethanol in a Teflon container, and the solution was stirred for 30 min. Subsequently, 3 mmol of thiourea was added and stirred for another 15 min, causing a visible color change from bluish-green to white. The Teflon container was sealed in an autoclave reactor, which facilitated the reaction. The reactor was kept in a hot air oven at 150°C for 24 h. After the reaction is completed, the container is removed and cooled to room temperature. The collected bluish-black precipitate was washed multiple times with ethanol and double-deionized (DDI) water. Finally, the collected powder was dried overnight in a hot air oven at 60°C.

2.3. Characterization studies of CuS NPs

The morphology and composition of the CuS NPs were studied using scanning electron microscopy (SEM, Hitachi SU-3500, Tokyo, Japan), SEM-energy dispersive x-ray (EDX) spectrometry (Quantax EDS, Bruker, Billerica, MA, USA), and transmission electron microscopy (TEM, HT-7700, Hitachi, Tokyo, Japan). The crystalline nature of the NPs was confirmed by x-ray diffraction (XRD, Bruker Billerica, MA, USA) with Cu Kα radiation (generated at 30 mA and 30 kV). Additionally, absorption properties were analyzed by ultraviolet-visible-near infrared (UV-Vis-NIR) spectrophotometry (Jasco-V770, Tokyo, Japan). To identify functional groups and chemical bonds, Raman (UniDRON Laser Spectroscopy Confocal Micro Raman Spectrometer, CLT, New Taipei City, Taiwan) and Fourier transform infrared (FTIR) spectrophotometry (ThermoFisher Nicolet iS20 FTIR Spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) were used. Zebrafish growth was analyzed with a stereomicroscope (Olympus SZX16 DS126271, Evident, Tokyo, Japan). A quantitative polymerase chain reaction (qPCR) was conducted using a LightCycler480 (Roche Diagnostics, Basel, Switzerland) instrument. Cells in 96-well plates were read with a microplate reader (BMG Labtech, Clario star Plus, Ortenberg, Germany).

2.4. Experimental animals

Adult zebrafish (8–12 months old) were obtained from the Zebrafish Core Laboratory of Taipei Medical University (Taipei, Taiwan) and kept in a water circulation tank (90 × 60 × 45 cm) with a water temperature of 28°C and a photoperiod of 14 h of light and 10 h of darkness. Fish were fed pellets twice a day. The breeding and care of zebrafish used in the experiments followed Taiwan’s Animal Protection Act and were supervised by the Animal Care and Use Committee of National Taiwan Normal University (Taipei, Taiwan). The care and use of adult zebrafish for experimental purposes were approved by the Animal Care and Utilization Committee of Taipei Medical University (Approval No. LAC2024–0102). According to the Animal Protection Law of Taiwan, zebrafish embryos prior to 120 h post-fertilization (hpf) are not classified as experimental animals. Adult zebrafish were mated and allowed to spawn from 09:00 to 10:00. After mating, fertilized eggs were collected and placed in AFW for incubation. AFW was prepared with the following components: 0.2 mM CaSO₄, 0.5 mM NaCl, 0.2 mM MgSO₄, 0.16 mM KH₂PO₄, and 0.16 mM K₂HPO₄. The pH of the AFW was adjusted to a range of 6.8–7.0. Fertilized eggs used in the study were zebrafish embryos up to 5 days post-fertilization (dpf). Therefore, according to Taiwan’s Animal Protection Act, no animal testing authorization was required for this part of the experiment.

2.5. Embryo acute toxicity test

To avoid the aggregation and agglomeration of NPs, the experiment was carried out in a semi-static water system. Several test chemical concentrations were used (20, 40, 60, 80, and 100 μg/mL) to meet statistical requirements. The concentrations of CuS nanoparticles (20, 40, 60, 80, and 100 µg/mL) used in this study were selected based on previous toxicological research involving zebrafish embryos, which demonstrated dose-dependent biological effects within this range [24]. At 4 h post-fertilization (hpf), the eggs were placed into 24-well culture plates with 10 embryos per well. To promote zebrafish development in every well, electrolyte-enhanced (E3) water was added. Every 24 h, NP solutions of different concentrations and E3 water were changed. The control group was composed of wells that contained only E3 water. A stereomicroscope was used every day to make observations to determine the condition of both dead and alive embryos and larvae.

2.6. Cell viability

Vero (African green monkey kidney) cells used in this study were cultured in DMEM. The culture medium was supplemented with 10% FBS and 100 U/mL penicillin. Cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. A crystal violet assay was conducted to assess cell viability. The Vero cell line was specifically chosen for in vitro cytotoxicity assessment due to several key advantages. As a well-established and non-cancerous mammalian epithelial cell line, Vero cells are widely recognized and frequently employed in toxicology and biocompatibility studies for diverse substances, including NPs. Cells were seeded into 96-well plates at 10⁶ cells/100 µL per well and kept in an incubator. After 24 h of growth, cells were seeded again using various concentrations of CuS NPs (0.025, 0.05, 0.1, 0.2, and 0.4 µg/mL). Treated cells were incubated for 48 h in a CO₂ incubator. After that, plates were washed twice with PBS, and PFA was used to fix the cells. Then, cells were placed at room temperature for 1 h. After 1 h, PFA was removed, and 50 µL of crystal violet dye was added to each well and left at room temperature for 30 min. Wells were then washed and dried, and plates were read with an ethanol solution on microplate readers.

2.7. Messenger (m)RNA extraction and qPCR

In total, 10 embryos were combined into one sample for mRNA extraction and a qPCR analysis. During each experiment, 10 embryos were collected as one sample (n = 10) from the same-concentration experimental group. For each concentration in this study, five sets were analyzed (n = 5). Materials were divided into four concentrations (0 (control), 0.001, 0.002, and 0.003 mg/mL) for the qPCR analysis. Extraction of mRNA from embryos was assisted by TRIzol reagent. Subsequently, the extracted mRNA was converted into complementary (c)DNA using a TOOLSQuant II Fast RT Kit, and a qPCR instrument was used to carry out this procedure, with a final volume of 200 μL containing cDNA diluted 10 times and at a concentration of 100 ng/μL or higher. The diluted cDNA was then mixed with 10 mM primers and LightCycler® 480 SYBR Green I Master before being subjected to real-time nucleic acid quantification using the LightCycler480 machine. mRNA expressions of target genes in each sample were normalized to the reference gene (rpl13a). The protocol for all qPCRs was as follows: a 10-min initial denaturation at 95°C, followed by 35 cycles of denaturation for 10 s at 95°C, annealing for 20 s at 60°C, and extension for 20 s at 72°C. For each sample, a dissociation step was performed at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s at the end of the amplification phase. mRNA expression levels were determined using the comparative Ct method (ΔΔCt), with rpl13a serving as the reference or housekeeping gene.

3. Results

3.1. Morphological and structural studies

CuS NPs were synthesized using a hydrothermal method. As shown in Figure 1(a), synthesized CuS NPs exhibited spherical-shaped structures in SEM images. The structural properties of the CuS NPs were investigated in greater detail using TEM. As shown in Figure 1(b), the mean size of synthesized CuS NPs was approximately 32.5 nm. The chemical composition of the synthesized CuS NPs was analyzed using EDX (Figure 1(c)), which shows the EDX spectrum of CuS NPs synthesized using copper nitrate and thiourea present in a Cu/S = 1:3 ratio. The EDX spectrum of CuS showed highly intense elemental peaks of Cu and S [39]. The strong peaks observed at 8 KeV and the smaller peak at 1 KeV, respectively, corresponded to Cu-Kα and Cu-Lα, indicating the presence of copper in the sample. Peaks observed at 2.3 KeV corresponded to the S-Kα signal. As shown in Figure 1(c) inset, the EDX analysis of copper consisted of atomic and mass percentages of copper (50.71%) and sulfur (49.29%), confirming the presence of sulfur in the sample. As shown in Figure 1(c), no other significant peaks of impurities were observed in the EDS spectra. As shown in Figure 1(d), the EDX elemental mapping of copper displayed a uniform distribution of copper (C-Kα) throughout the analyzed region. The uniform green color throughout the image indicated that copper was evenly dispersed in the sample, which suggests the homogenous distribution of Cu particles (Figure 1(e)). The EDX elemental map of sulfur (S-Kα) displays a uniform distribution of sulfur [40]. The uniform red color across the image indicates that sulfur was evenly dispersed in the sample, suggesting a homogeneous distribution of sulfur particles. The SEM-EDX analysis of CuS revealed the presence of both Cu and S in an atomic ratio of approximately 1:1, which corresponds to the atomic and mass percentages of the nanoparticles, reconfirming the successful synthesis of CuS NPs.

Figure 1.

(a) SEM image and (b) TEM image of CuS NPs. (c) EDX spectrum of CuS NPs. EDX mapping of CuS NPs of (d) Cu and (e) S.

3.2. XRD studies and UV-Vis-NIR spectroscopy

CuS NPs synthesized by a hydrothermal method were investigated using XRD. Diffraction peaks were wider than those for standard crystals, indicating the formation of smaller crystals. As shown in Figure 2(a), the peaks were stronger and sharper for the longer reaction time of 24 h, which suggested that the synthesized particles were smaller and more crystalline. This could be explained by the rate of nucleation increasing over time until it reached a well-defined stable structure, as evidenced by the XRD analysis, which revealed prominent peaks at specific 2θ values of 27.79°, 29.40°, 31.85°, 32.82°, 49.10°, 53.38°, and 59.79°. These peaks, respectively, corresponded to the (101), (002), (103), (106), (110), (108), and (116) crystallographic planes of Miller indices and the hexagonal arrangement of Cu and S atoms in CuS (JCPDS card no. 00–006–0464) [41–43]. No characteristic peaks of impurities were detected. The crystallinity of the synthesized CuS NPs was well-defined by the distinct peaks of the crystal lattice, confirming the successful synthesis of CuS NPs.

Figure 2.

(a) XRD patterns of copper sulfide nanoparticles (CuS NPs). (b) UV-Vis absorption spectrum of CuS NPs, (c) FTIR spectrum of CuS NPs, and (d) Raman spectrum of CuS NPs.

UV-Vis-NIR spectroscopy was used to evaluate the produced CuS NPs to assess their optical characteristics. As shown in Figure 2(b), the absorption spectra displayed notable peaks between 400 and 1200 nm. It had a notable peak of around 500 nm in the UV-Vis region and a well-defined peak of around 1100 nm in the NIR region. The absorption in the visible range (400–550 nm) is mainly caused by interband transitions between the valence and conduction bands of CuS NPs, influenced by quantum confinement effects and surface defects. This causes a blue shift compared to bulk CuS and broadens the absorption peak [44,45]. This was due to interband transitions between CuS NP valence states and unoccupied states that produced excitonic absorption [46]. However, the NIR absorption near 1100 nm is attributed to localized surface plasmon resonance (LSPR), arising from free hole carriers generated by copper vacancies in the nanoparticles. These carriers induce collective oscillations under light excitation, leading to a broad plasmonic absorption band characteristic of CuS NPs [45]. The significant light absorption capabilities of CuS NPs, which are essential to their photocatalytic and photothermal activities, were demonstrated by these absorption peaks. For photocatalytic applications, effective absorption in the visible light range is crucial, as indicated by the peak at 500 nm, which signals that the NPs can use visible light to catalyze chemical reactions [47,48].

3.3. FTIR and Raman spectroscopy

Figure 2(c) shows the CuS NP analysis of their FTIR spectra, which revealed weak peaks at 651 cm⁻¹, signaling CuS. Furthermore, peaks observed at 1353 and 1679 cm⁻¹ respectively corresponded to stretching and vibrational frequencies of C = C and C = O, further confirming the presence of surface-bound water molecules [49]. A broad peak at 3441 cm⁻¹ was associated with a hydroxyl (O = H) stretching peak, indicating water absorbed by sulfide products [50,51]. The presence of characteristic Cu-S bonds and associated bonds indicated that CuS NPs had been successfully synthesized [52]. Figure 2(d) shows Raman spectroscopy employed to analyze the molecular composition and structural characteristics of CuS NPs using a UniDRON Laser Spectroscopy Confocal Micro Raman Spectroscopy system. Wavelengths were set to 200–1200 cm⁻¹. The Raman spectrum exhibited several distinct peaks ranging from 254 to 456 cm⁻¹. These peaks represented molecular vibrations and the molecular structure of the synthesized NPs. It was found that Raman peaks at 254 and 456 cm⁻¹ were, respectively, associated with Cu-S and S-S stretching phonon modes, indicating the nanocrystalline properties of CuS [39]. In addition, the low-intensity peak observed at 254 cm⁻¹ was characteristic of the transverse optic (TO) mode of CuS NPs, whereas the high-intensity peak at 456 cm⁻¹ corresponded to S-S stretching vibrations of the A1_g longitudinal optics (LO) mode of S₂ (dimers) at 4_e sites of CuS NPs [53]. This analysis confirmed the presence of copper and sulfur bonding within the NPs, validating their successful synthesis. Among the 2A1_g+4B2_g+2E1_g+4E2_g+3A2_u+2B1_u+3E1_u+2E1_u phonon modes, eight modes had Raman activity: 2A1_g+2E1_g+4E2_g [54].

3.4. Acute toxicity test

As shown in Figure 3(a), zebrafish embryo survival rates decreased in direct proportion to the NP concentration that ranged 20–100 µg/mL. In contrast, embryos in the control group without NP treatment survived until 96 hpf. Survival rates and NP concentrations were inversely correlated, indicating that higher CuS concentrations may damage zebrafish embryonic development and make them more vulnerable to increased NP concentrations, particularly in the 60–100 µg/mL range. Meanwhile, 20 µg/mL exhibited no significant difference from the control group. Figure 3(b) showed that higher concentrations resulted in significant differences in mortality rates. As the concentration increased, the number of embryos that died also increased.

Figure 3.

(a) Toxic effects on the survival rate of zebrafish at different days post fertilization (dpf) after treatment with different concentrations of copper sulfide nanoparticles (CuS NPs). (b) Mortality rates of zebrafish embryos. All values given are means from three significant different experiments based on a two-way ANOVA. p<$0.0001$ compared to the control group.

3.5. Teratogenicity test

Results shown in Figure 4 indicate that early-developing zebrafish embryos exposed to CuS NPs at concentrations of 20–100 µg/mL developed abnormalities, including embryos control (Figure 4(a)), embryos died with circulation defects (EDCDs) (Figure 4(b)), yolk sac edema (YSE) (Figure 4(c)), delayed hatching (DH) (Figure 4(d)), larvae control (Figure 4(e)), blood clotting (BC) (Figure 4(f)), blood clotting (BC) (Figure 4(g)) and pericardial edema (PE) (Figure 4(h)). The frequencies of these deformities exhibited a concentration-dependent pattern, with greater incidences observed at higher concentrations at 48–96 hpf. A higher malformation rate was observed in NP-treated groups compared to the control group.

Figure 4.

Toxic effects of copper sulfide nanoparticle (CuS NP) malformations at different post-fertilization times (a) Embryos control, (b) Embryos died with circulation defects (EDCDs), (c) Yolk sac edema (YSE), (d) Delayed hatching (DH), (e) Larvae control, (f) Blood clotting (BC), (g) Blood clotting (BC) and (h) Pericardial edema (PE). (scale bar 10 µm).

3.6. Cell viability

The viability of Vero cells treated with CuS NPs was assessed at concentrations of 0 (control), 0.025, 0.05, 0.1, 0.2, and 0.4 µg/m (Figure 5). Cell viability was analyzed after 48 h incubation with CuS NPs, and control cells had a viability of 100%. At CuS NPs concentrations of 0.025 and 0.05 µg/mL, slight reductions in cell viability were observed. The reductions in cell viability were not statistically significant compared to the controls (indicated by ‘ns’ for not significant). However, the viability significantly dropped at concentrations of 0.1, 0.2, and 0.4 µg/mL, with these concentrations showing significant decreases in viability compared to the controls. Higher concentrations of CuS NPs caused significant reductions in cell viability, indicating concentration-dependent cytotoxic effects. The selected concentration range was based on a previous study where CuS nanoplates caused reduced cell viability above 100 µg/mL in HUVEC and RAW264.7 cells, suggesting that lower concentrations could still induce cytotoxicity depending on cell type [55]. Since Vero cells are non-cancerous kidney epithelial cells, we adopted a lower concentration range to observe subtle effects and avoid overt toxicity.

Figure 5.

Cell viability of the Vero cell line (crystal violet) treated with different concentrations of copper sulfide nanoparticles (CuS NPs). All values are the mean from three significantly different experiments based on a one-way ANOVA (0.1 µg/mL: p = 0.0147, 0.2 µg/mL: p = 0.0038, 0.4 µg/mL: p = 0.0043). Concentrations were compared to the control group.

3.7. qPCR

Fish without solar light irradiation exhibited no significant ROS response to CuS NPs (Figure 6(a)). The chart clearly shows that the four genes (sod1, sod2, cat, and gpx1) reacted minimally under different concentrations of CuS NPs. The relative intensities detected were extremely low and did not change as the concentration of CuS NPs increased. This lack of response was because the properties of CuS NPs were not activated without solar light excitation, rendering them inert when injected into the fish. Additionally, their intrinsic toxicity caused no damage to the fish because the material concentration was extremely low. To investigate activation of CuS NPs, we repeated the experiment with solar light irradiation at an intensity of 4.28 mW and measured the relative increases of the four genes. As shown in Figure 6(b), the measured relative intensities, while not reaching very high levels, significantly increased compared to the measurements without solar light exposure. Specifically, there were noticeable increases in expression levels of sod1, sod2, cat, and gpx1, indicating an ROS response triggered by the CuS NPs to sunlight. This result suggests that the CuS NPs’ characteristics were revealed under a certain intensity of sunlight, leading to an ROS reaction in the fish bodies. The increased ROS production under sunlight exposure highlights the photoreactive nature of CuS NPs and their potential to induce oxidative stress in biological systems when activated by light [56].

Figure 6.

(a) qPCR analysis of copper sulfide nanoparticles (CuS NPs) without solar light irradiation and (b) a CuS NP qPCR analysis with solar light irradiation.

3.8. Mechanisms of action

Multiple cellular pathways interact with CuS NPs, ultimately causing cellular damage and death. As illustrated in Figure 7(a), cells internalize CuS NPs primarily through endocytosis [57]. Although the process of exocytosis is not yet fully understood, it is believed that NP waste can be excreted via lysosomes, which help regulate intracellular NP concentrations [58]. NPs can enter cells either through active, receptor-mediated pathways or via passive mechanisms such as fluid-phase endocytosis [59]. Studies have shown that smaller CuS NPs tend to exhibit higher toxicity. Moreover, variations in NP concentration, size, and morphology are associated with different biological effects. In our study, zebrafish embryos exposed to higher CuS NP concentrations (60–100 µg/mL) exhibited a significant increase in developmental abnormalities and reduced survival rates, indicating dose-dependent toxicity. In addition to generating excessive ROS, CuS NPs interfere with other key cellular signaling pathways. For example, they can disrupt intracellular calcium (Ca^{2 +}) homeostasis, deplete adenosine triphosphate (ATP), and damage DNA, all of which contribute to cell death [60]. This disruption can lead to cell-cycle arrest, which prevents further DNA replication and cell division, thereby preserving genomic integrity. Current studies suggest that cell-cycle arrest plays a pivotal role in NP-induced cytotoxicity [61]. Consistent with these findings, we observed delayed development and morphological abnormalities in exposed embryos, suggesting a possible interruption of the cell cycle due to DNA damage. When DNA damage is detected, cells typically initiate arrest in the G1 and G2 phases, leading to apoptosis through the activation of pro-apoptotic proteins. After internalization, NPs may be released into the cytoplasm or further processed by lysosomes. Once inside the cell, CuS NPs undergo oxidative dissolution [62]. This process releases copper ions, which can trigger the overproduction of ROS, including superoxide radicals (O₂•), especially when Ca^{2 +} levels are elevated. Excessive ROS leads to mitochondrial damage and dysfunction, as the mitochondrial membrane becomes a primary target [63]. Figure 7(b) shows that O₂ molecules gain a single electron through NADPH oxidase (NOX), forming superoxide radicals [64,65]. These radicals can also come from electrons leaking from the mitochondrial electron transport chain. SOD proteins then convert two superoxide molecules into hydrogen peroxide (H₂O₂) and water. Although H₂O₂ acts as a signaling molecule, it can also be harmful to zebrafish due to its reactive properties [66,67]. Hydrogen peroxide can be further broken down into water by enzymes like GPX or CAT. The qPCR analysis showed significant upregulation of oxidative stress-related genes such as sod1, sod2, cat1, and gpx1 in CuS NP-exposed zebrafish embryos, particularly under light exposure. This pattern is consistent with enhanced ROS production and suggests an activated antioxidant defense response. The molecular expression trends correlate with the observed developmental abnormalities and survival rates, indicating that oxidative stress is a key mechanism underlying CuS NP-induced toxicity in zebrafish. A novel understanding of CuS NPs in zebrafish under varying environmental conditions is presented, focusing particularly on ROS generation and subsequent gene expression responses. These findings collectively suggest that the cytotoxic effects observed upon CuS nanoparticle exposure are closely associated with oxidative stress. The reduction in total antioxidant capacity (TAC), along with decreased cell viability, indicates that the generation of reactive oxygen species (ROS) may overwhelm the cellular antioxidant defense system. This imbalance in redox homeostasis likely contributes to cell damage and death, establishing a mechanistic link between nanoparticle-induced oxidative stress and cytotoxicity.

Figure 7.

(a) Nanoparticle entry route through the chorion membrane pathway and (b) Reactive oxygen species (ROS) response mechanism in zebrafish.

4. Discussion

The hydrothermal preparation of CuS NPs were sucessfully demonstrated by structural and optical characterizations, including SEM, TEM, EDX, XRD, UV-Vis-NIR spectroscopy, FTIR, and Raman spectroscopy. For acute toxicity test, zebrafish embryos showed significant sensitivity to increasing concentrations of CuS NPs in terms of survival rates. This might have been due to ROS, which causes oxidative stress, resulting in cellular damage and apoptosis. As the concentration of NPs increases, ROS might increase the embryo’s antioxidant defenses, leading to higher mortality. Researchers found that zebrafish embryos exposed to CuS NPs at levels over 80 mg/mL experienced a significant effect from the particles, with mortality rates that were highly significant at 80 and 150 mg/mL concentrations (p < 0.001) [24]. Another study found that the presence of polystyrene microplastics (1 mg/L) alongside CuS also did not lead to acute toxicity, suggesting that that concentration was relatively safe [68]. In a study by Zhang et al., CuCl₂ NPs and Cu₂O NPs were examined for their effects on zebrafish larvae, suggesting that both can cause toxicity, but at different concentrations [69].

Embryos and larvae exposed to CuS NPs developed malformations. Zebrafish embryos treated with CuO NPs showed growth retardation, yolk deformities, head malformation, heart malformation, and non-axial abnormalities [70]. One study found that zebrafish embryos exposed to chitosan NPs developed many malformations, such as axial malformation (AM), which affects the spine or body axis, PE, which is characterized by fluid accumulation around the heart, YSE, which occurs when the yolk sac swells and may impede the absorption of nutrients, and an uninflated swim bladder (USB), which occurs when the swim bladder fails to inflate properly, affecting buoyancy [71]. Another study found that metal and metal oxide NPs can cause a variety of abnormalities in zebrafish, including decreased head and eye sizes, disruptions in dorsoventral patterning, and problems with cell migration during gastrulation. Those NPs may also impede the heart tube’s natural looping, which could interfere with cardiogenesis [72]. There were some organ-specific effects from exposure to chitosan NPs. Additionally, the liver size was reduced with no indications of steatosis or lipid yolk retention, and the embryos’ neurobehavioral activity increased, suggesting hyperactivity [73]. According to research, zebrafish may acquire abnormalities such as body axis deformities, craniofacial malformations, and poor organ development as a result of ZnO NP exposure [74].

In the cell test, the observed cell death is likely caused by the intrinsic properties of CuS NPs, such as direct nanoparticle-cell interactions, surface charge effects, or passive oxidative stress independent of external light. CuS NPs were shown to have a powerful therapeutic effect on glioblastoma cancer cells, resulting in a significant reduction in cell viability during photothermal therapy (PTT) under NIR light [75]. CuS NPs were shown to significantly reduce the viability of HeLa cells when treated with NIR irradiation [76]. In another study, cell viability was tested using an MTT assay, which showed that the laser dose and NP concentration affected photothermal destruction [77]. It was demonstrated that CuS NPs significantly reduced the viability of 4T1 cancer cells when exposed to NIR laser irradiation. After 5 min of laser exposure, fewer than 20% of cancer cells remained viable at 1.2 mM of CuS NPs [78]. Using an MTT assay, one study found that healthy HFF-2 cells and 4T1 tumor cells maintained viability after being treated with alginate coated CuS NPs (CuS@Alg NPs) for 15 h; however, CuS@Alg NPs significantly reduced the viability of 4T1 cells at higher concentrations, demonstrating effective cytotoxicity [79].

CuS NPs pose a potential risk to aquatic ecosystems, highlighting serious environmental implications. Since these NPs are increasingly produced and used, their accumulation in water bodies will likely continue, posing a threat to aquatic life. As a result, strict safety guidelines and regulations are necessary to prevent human exposure to contaminated water and air. NPs can have detrimental effects on aquatic organisms, raising concerns about their long-term ecological effects. Abiotic food webs and biodiversity may be negatively affected by the induction of oxidative stress and subsequent cellular damage.

5. Conclusions

CuS NPs were successfully synthesized using a hydrothermal method, and their optical and structural properties were analyzed using microscopic and spectroscopic techniques. NP synthesis was characterized and confirmed by XRD, FTIR, UV-Vis spectrometry, SEM, and TEM. As part of the biological evaluation, survival and toxicity mechanisms were examined in zebrafish embryos. Sublethal effects, such as premature hatching and malformations, were observed when embryos were exposed to CuS NPs. Results indicated a significant impact on the viability of Vero cells. Zebrafish exposed to CuS NPs exhibited elevated levels of ROS, suggesting that oxidative stress was responsible for the observed toxic effects. Interestingly, despite increased ROS levels under solar irradiation, they did not exceed the antioxidant capacity of the zebrafish, suggesting a controlled ROS generation process. Notably, concentrations above 60 µg/mL caused significant toxicity under light exposure, further supporting the light-enhanced oxidative stress mechanism. In addition, gene expression analysis revealed upregulation of ROS-related antioxidant genes (sod1, sod2, cat1, and gpx1), indicating an active molecular response to oxidative stress. In summary, the comprehensive characterization of CuS NPs and their biological impacts highlights their dual potential, emphasizing the need to carefully consider their application and conduct further research into their mechanistic pathways.

The purpose of qPCR study was to compare relative levels of the sod1, sod2, cat, and gpx1 genes to the rpl13a reference gene after the NPs entered the fish body to understand how these levels varied. As a result, expressions of sod genes increased to help convert superoxide into hydrogen peroxide and water. Consequently, elevated hydrogen peroxide levels in the fish were linked to sod genes. This increase then affected expression levels of the cat and gpx1 genes, which work to reduce hydrogen peroxide to water, helping maintain the fish’s vital functions. This analysis was conducted under conditions where the material concentration was not high enough to be lethal to the fish. The qPCR study demonstrated the impacts of CuS NPs on zebrafish under different experimental conditions. Without solar light irradiation, CuS NPs exhibited a minimal ROS response, indicating their inertness in the absence of light activation. This was reflected in negligible changes in sod1, sod2, cat, and gpx1 gene expression levels, reaffirming the material’s low reactivity and toxicity at low concentrations. However, upon exposure to solar light, CuS NPs demonstrated a significant increase in ROS production, as evidenced by elevated expression levels of the sod1, sod2, cat, and gpx1 genes. This highlights the photoreactive nature of CuS NPs and their potential to induce oxidative stress in biological systems under light activation. Our findings underscore the importance of considering environmental factors, such as sunlight exposure, in assessing the biological impacts of nanomaterials like CuS NPs. NPs have been extensively studied for their toxicity in zebrafish, revealing significant effects on embryonic development, cell migration, cardiac function, locomotion, and muscle damage [24]. In addition to altering gene expression patterns related to development and cell migration, CuS NP exposure reduced hatching rates and affected swim bladder inflation [80]. When zebrafish are exposed to CuS NPs for a prolonged time, they develop muscle damage, oxidative stress, and low antioxidant enzyme levels, which ultimately lead to muscle degeneration [81]. High concentrations were associated with considerable lethality and a dose-dependent rise in mortality [82]. Moreover, oxidative stress was observed in larvae treated with NPs.

Funding Statement

This project was funded by the National Science and Technology Council of Taiwan [grant number NSTC 113–2113-M-038–001] and Taipei Medical University. Additionally, we received financial support from the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan [DP2-TMU-112-N-09]. We would like to express our gratitude to Chi-Ming Lee for his exceptional technical support at the TMU Core Facility.

Article highlights

• CuS nanoparticles (NPs) were synthesized using the hydrothermal method and thoroughly characterized.

• The impact of CuS NPs on zebrafish embryos was evaluated under both normal and light-exposed conditions.

• Exposure to high concentrations of CuS NPs (60–100 µg/mL) resulted in significant developmental abnormalities and reduced survival rates.

• Light exposure enhanced the toxicity of CuS NPs, suggesting a phototoxic effect likely driven by oxidative stress.

• Cellular uptake of CuS NPs occurs primarily through endocytosis, leading to ROS generation and mitochondrial dysfunction.

• qPCR analysis revealed significant upregulation of oxidative stress-related genes (sod1, sod2, cat1, and gpx1) in treated embryos.

• Increased ROS production correlated with decreased total antioxidant capacity (TAC) and cell viability.

• The mechanism of toxicity involves oxidative dissolution of CuS NPs, calcium disruption, and DNA damage.

• Findings emphasize a strong link between oxidative stress and CuS NP-induced cytotoxicity in developing zebrafish.

• This study highlights the potential ecological and health risks of CuS NP exposure and the need for proper nanoparticle regulation.

Author contributions

C Sneka: conceptualization, investigation, methodology, data curation, writing – original draft, writing – review and editing. D Krisnawati: conceptualization, writing – review and editing. D. Khafid: conceptualization, writing – review and editing. H Susanto: conceptualization, writing – review and editing. C Mutalik: conceptualization, investigation, writing – original draft, writing – review and editing. C-Y Che: conceptualization, investigation, writing – original draft, writing – review and editing. T-R Kuo: conceptualization, investigation, writing – original draft, writing – review and editing, supervision, project administration. All authors revised and accepted the submitted version of their work and agree to take personal responsibility for their contributions.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosure

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Ethical declaration

All experimental procedures involving adult zebrafish were approved by the Animal Care and Utilization Committee of Taipei Medical University (Approval No. LAC2024–0102). According to the Animal Protection Law of Taiwan, zebrafish embryos before 120 h postfertilization (hpf) are not considered experimental animals.