Copper nanoparticles are small enough to enter modern products, but in developing zebrafish they are linked to death, deformities, altered GABA signaling, oxidative stress, and inflammation. The surprising comparison is that dissolved copper ions were often even more toxic.

Copper nanoparticles are extensively used in electronics, cosmetics, fungicides, and various other fields, but this widespread usage can contribute to environmental contamination and heightened health risks for living organisms. Despite their prevalent use, the ecological impacts and biosafety of copper nanoparticles remain inadequately understood, so the study examines toxic effects in zebrafish embryos across embryonic development, neurotoxicity, oxidative stress, and inflammatory response.

Nanoparticle use is expanding across food, medicine, electronics, cosmetics, and chemical industries, while concerns about environmental hazards and health risks are increasing. Copper nanoparticles are broadly used because of their unique optical, antifungal, and electrical characteristics, but that widespread use raises environmental release and human exposure risks.

Zebrafish are a valuable vertebrate model for toxicological research because of their genetic and developmental similarities to humans, and their embryos allow easy observation and manipulation during development. Their rapid development makes them widely applicable in lifespan toxicity research, so zebrafish embryos were used to explore the early-developmental toxicity of copper nanoparticles with a particle size of five to ten nanometers.

Because copper nanoparticle toxicity is thought to combine intracellular nanoparticles with leaching dissolved copper ions, copper ions provided as copper chloride were included for comparison. Zebrafish embryos develop externally and rapidly while remaining robust and transparent, making them easy to manipulate, suitable for high-throughput applications, and conducive to detailed visual analysis.

The study used a time- and concentration-dependent assessment to evaluate developmental toxicity, exposing embryos to copper nanoparticles for ninety-six hours. During exposure, death rate, hatching rate, heart rate, deformity rate, and spontaneous tail-coiling frequency were recorded daily.

The parental zebrafish were AB wild-type fish obtained from the China Zebrafish Resource Center and cultured under controlled conditions of twenty-eight plus or minus one degrees Celsius with a fourteen-hour light and ten-hour dark photoperiod. After one month of acclimation, males and females were fed brine shrimp twice daily, and two males and two females were transferred into breeding tanks.

The next morning, embryos were collected, washed three times with reverse osmosis water, and placed in one-hundred-millimeter glass Petri dishes with sixty embryos per dish for exposure. Copper chloride was purchased analytically pure, while the copper nanoparticle colloid was dispersed in ultrapure water through serial dilutions and sonicated for thirty minutes before introduction to the exposure groups.

Transmission electron microscopy showed that the copper nanoparticles were spherical particles of approximately five to ten nanometers, consistent with the manufacturer’s description. The solutions were diluted with reverse osmosis water to copper concentrations of fifty, one hundred, two hundred fifty, and five hundred micrograms per liter, with reverse osmosis water serving as the control.

Exposure lasted ninety-six hours, fresh exposure solutions were prepared and replaced daily, and dead embryos were removed each day. Mortality was recorded every twenty-four hours; spontaneous tail coiling was analyzed at twenty-four hours post fertilization; and hatching rate and heart rate were recorded at forty-eight, seventy-two, and ninety-six hours post fertilization.

Deformity rate was observed with a stereomicroscope and calculated at seventy-two and ninety-six hours post fertilization. The embryos were observed at twenty-four-hour intervals until ninety-six hours post fertilization, while mortality, hatching, heart rate, deformity, and spontaneous tail coiling were recorded at each interval.

Heart rate was measured by counting beats over twenty seconds and extrapolating to one minute to indicate average beats per minute. Bent tail, bent spine, pericardial edema, and yolk sac edema were observed by stereomicroscope at four-times magnification, and spontaneous tail-coiling frequency was investigated at twenty-four hours post fertilization.

After ninety-six hours, exposure to copper nanoparticles and copper chloride substantially enhanced copper bioaccumulation in zebrafish larvae, and bioaccumulation increased significantly as exposure concentration increased. At five hundred micrograms per liter, copper bioaccumulation increased four point eight seven-fold with copper nanoparticles and four point eight three-fold with copper chloride compared with the control.

There was no significant difference in copper concentration between the copper chloride treatments and the copper nanoparticle treatments in zebrafish larvae. Figure one shows copper concentration in zebrafish larvae after ninety-six hours of exposure to copper chloride or copper nanoparticles across increasing exposure concentrations.

The plotted values, given as micrograms per gram dry weight, rise with exposure concentration, and significance markers indicate differences from the control. The authors report that the highest treatment, five hundred micrograms per liter, produced four-point-eight-seven-fold and four-point-eight-three-fold increases versus the control for copper nanoparticles and copper chloride, respectively, demonstrating substantial copper bioaccumulation.

Death rate increased in both copper nanoparticle and copper chloride treatments as exposure concentration and duration increased. After twenty-four hours, significant differences appeared between treatments, with increased death rate for copper nanoparticles only at two hundred fifty and five hundred micrograms per liter.

After seventy-two and ninety-six hours, death rate significantly increased in both treatments, although no notable difference appeared between copper nanoparticles and copper chloride at high concentrations. Figure two compares zebrafish embryo death rates after twenty-four, forty-eight, seventy-two, and ninety-six hours of exposure to CuCl two or copper nanoparticles at concentrations from zero to five hundred micrograms per liter.

Across the panels, the chart shows increasing mortality with higher concentration and longer exposure, with significance markers indicating differences from controls and between treatments. This matters because it demonstrates a clear time- and concentration-dependent toxicity pattern.

A pericardial cyst was observed in embryos treated with both copper nanoparticles and copper chloride, and this finding was consistent with the heart rate results. Other abnormalities included bent spines, bent tails, and vitelline cysts.

Deformity rate increased with higher exposure concentration, with significant differences between copper nanoparticle and copper chloride treatments at fifty, one hundred, and five hundred micrograms per liter. Figure five shows deformity rates in zebrafish embryos after exposure to CuCl₂ and copper nanoparticles, measured at seventy-two and ninety-six hours across increasing concentrations.

The plotted bars indicate significant differences from controls and, at several concentrations, between the two treatments. Representative images document bent tails, bent spines, pericardial cysts, and vitelline cysts, linking these visible structural abnormalities to the reported heart-related effects and supporting deformity rate as an indicator of developmental toxicity.

GABA is an important inhibitory neurotransmitter in the central nervous system, and after release from the presynaptic membrane it engages GABAA or GABAB receptors, inhibiting neurotransmitter release and neuronal activity. The study investigated transcriptional expression of key GABA signaling pathway genes in embryos exposed to copper nanoparticles and copper chloride.

GABAA receptor alpha one, or gabra1, increased at low concentrations but was notably reduced at high copper nanoparticle and copper chloride doses, suggesting disruption in GABA-receptor signaling. The exposure also regulated genes related to GABA biosynthesis, catabolism, and transport; gad generally increased except at five hundred micrograms per liter of copper nanoparticles, while gat1 decreased at five hundred micrograms per liter for both treatments.

The mRNA expression of abat, encoding GABA-T, was significantly reduced by copper nanoparticle exposure except at fifty micrograms per liter, implying inhibited GABA catabolism and potentially contributing to altered neurobehavior such as reduced spontaneous tail-coiling frequency.

Figure seven shows relative mRNA expression for four GABA-signaling genes—gabra1, gad, gat1, and abat—in zebrafish embryos exposed to CuCl2 or copper nanoparticles for ninety-six hours. Across concentrations, several bars carry asterisks indicating differences from control, while hash marks identify significant differences between the two copper treatments.

The authors use these transcriptional changes to assess how copper exposure may disrupt GABA-mediated inhibitory signaling, a key regulator of neuronal activity. The study tested antioxidative enzyme activity and malondialdehyde levels to evaluate oxidative stress induced by copper nanoparticles or copper chloride.

Superoxide dismutase activity increased at fifty and one hundred micrograms per liter but was inhibited at two hundred fifty and five hundred micrograms per liter for both treatments. Glutathione peroxidase and catalase activities generally decreased, except at five hundred micrograms per liter of copper chloride, while malondialdehyde content increased significantly in most treatments.

The results were interpreted as showing that high concentrations could reduce antioxidative enzyme activity, leading to reactive oxygen species accumulation and subsequent lipid peroxidation. Figure eight compares SOD, CAT, and GPx antioxidant-enzyme activity, alongside MDA content, in zebrafish embryos exposed to CuCl2 or CuNPs for ninety-six hours.

Across concentrations from zero to five hundred micrograms per liter, the asterisks mark differences from controls, while hash symbols identify significant differences between the two copper treatments. The authors use these measurements to evaluate oxidative stress, relevant because ROS responses are linked to inflammation and other toxic effects.

The results indicated that copper nanoparticle and copper chloride exposure regulated the mRNA expression of inflammatory factors. Changes in proinflammatory cytokine mRNA provided evidence of immunotoxicity associated with both exposures. Copper nanoparticle- or copper chloride-induced oxidative stress was speculated to trigger a proinflammatory cascade, supported by enhanced transcriptional expression of il-1 beta, tnf-alpha, il-6, and il-8 to varying degrees.

Figure 9 compares inflammatory mRNA markers in zebrafish embryos exposed for ninety-six hours to CuCl2 or copper nanoparticles across concentrations from zero to five hundred micrograms per liter. The panels show il1-beta, tnfa, il6, and il8, with asterisks marking differences from control and a hash marking a difference between treatments.

The authors use these expression changes to demonstrate that both copper forms regulate inflammatory signaling, supporting a connection between nanoparticle exposure, oxidative stress, and inflammation. Exposure to copper nanoparticles increased death and deformity rates and decreased heart rate and spontaneous tail-coiling frequency in zebrafish embryos, indicating toxic effects on embryo development.

Changes in key GABA signaling pathway genes suggest that copper nanoparticles may interfere with GABA synthesis, release, catabolism, and recovery, potentially disrupting neurotransmitter conduction and neurobehavior. Inhibition of antioxidative enzyme activities and increased malondialdehyde demonstrate oxidative stress, which in turn triggers a proinflammatory cascade.

Copper chloride exhibited more severe toxic effects across most tested parameters than copper nanoparticles, while further research is required on long-term toxicity and associated mechanisms. The study connects copper nanoparticle exposure with developmental damage, disrupted GABA-related gene expression, oxidative stress, and a proinflammatory response.

Because copper chloride caused more severe effects across most measures, particle toxicity cannot be treated as the whole story.