Ag-Pd Nanoparticles Kill Mosquito Larvae, Spare Predators

Researchers at Dayalbagh Educational Institute in Agra, India, have synthesized silver-palladium bimetallic nanoparticles using a nontoxic leaf extract from Citrus limon. The study, published in Bioscience Nanotechnology, evaluates the efficacy of these nanoparticles against mosquito larvae and their impact on non-target predatory organisms. The primary finding indicates that the synthesized nanoparticles cause significant mortality in larvae of Anopheles stephensi and Aedes aegypti mosquitoes across multiple developmental stages. Simultaneously, the nanoparticles did not cause mortality in dragonfly and damselfly nymphs, which are natural predators of mosquito larvae, suggesting a potential utility in integrated vector management.

The synthesis process utilized a hot-pot method to extract bioactive constituents from fresh Citrus limon leaves. One hundred milligrams of minced leaves were heated in 100 milliliters of deionized water at 65 to 70 degrees Celsius for one hour. The resulting crude extract was filtered to obtain a 10% aqueous leaf extract, which served as a reducing agent. This biological approach aligns with the growing need for environmentally sustainable methods in nanoparticle synthesis, avoiding the use of toxic chemical reducing agents that could confound toxicity assessments.

To create the bimetallic nanoparticles, 1 mM solutions of silver nitrate and palladium(II) chloride were prepared separately in deionized water. Each metal solution was mixed with the Citrus limon extract in a 9:1 ratio and kept in the dark for one hour. The active solutions of pre-synthesized silver and palladium nanoparticles were then combined in a 1:1 ratio to form a 2 mM silver-palladium bimetallic nanoparticle solution. This mixture was stored in the dark at room temperature overnight before being kept at 4 degrees Celsius for further analysis and bioassays.

Characterization of the nanoparticles revealed distinct physicochemical properties. Transmission Electron Microscopy determined a mean particle size of 21 plus or minus 7 nanometers, with particles exhibiting a roughly irregular-spherical shape and minimal agglomeration. X-ray Diffraction analysis identified distinct peaks corresponding to both silver and palladium, confirming the bimetallic nature of the sample. The material was described as semi-crystalline, a characteristic attributed to the leaf extract-mediated synthesis process and the low concentration of nanoparticles in the test sample.

Dynamic Light Scattering analysis showed a larger hydrodynamic size for the nanoparticle clusters, with a Z-average of 1956 nanometers. This significant difference between the actual particle size measured by TEM and the hydrodynamic size measured by DLS suggests that the nanoparticles exist as clusters in solution. The study notes that aggregating properties of leaf extract biomolecules, which act as capping and reducing agents, often contribute to nanoparticle retention and control their release in the environment.

The nanotoxicity bioassay was conducted following World Health Organization guidelines for testing mosquito larvicides. The efficacy of the silver-palladium nanoparticles was evaluated against first, second, third, and fourth instar larvae of both Anopheles stephensi and Aedes aegypti. Test concentrations of 1.67, 3.34, 5, 6.67, and 13.34 milliliters per liter were assessed. Mortality rates were recorded at 24, 48, and 72 hours of exposure. The results demonstrated significant larval mortality across all instar stages for both mosquito species, with the lowest concentration causing 100% mortality in first instar larvae.

Probit analysis was used to determine the median lethal concentration, or LC50, for the nanoparticles. For Anopheles stephensi, the LC50 at 24 hours was estimated at 1.59 milliliters per liter for second instar, 1.78 milliliters per liter for third instar, and 1.62 milliliters per liter for fourth instar larvae. For Aedes aegypti, the LC50 at 24 hours was 1.59 milliliters per liter for second instar, 1.78 milliliters per liter for third instar, and 2.08 milliliters per liter for fourth instar larvae. These values indicate that the nanoparticles are highly effective at low concentrations, particularly against younger larval stages.

To assess ecological safety, the study investigated the predation efficiency of dragonfly and damselfly nymphs exposed to the LC50 concentration of the nanoparticles. The LC50 value of 1.78 milliliters per liter, derived from third instar larvae, was used as the benchmark for these non-target organism tests. The predatory nymphs were placed in vessels with 25 third instar mosquito larvae and the specified concentration of nanoparticles. The negative control groups contained only mosquito larvae and nymphs in distilled water. The predation process was observed over 72 hours, with records kept at specific time intervals.

The results showed that the predatory nymphs exhibited resilience to the applied test concentration, with no mortality recorded in any replicates during the observation period. In fact, the predation efficiency in the nanoparticle test groups was higher than in the untreated control groups. This increased predation rate may be a response to meet the energy demand for defense against the metal stress induced by the nanoparticles.

The study highlights the potential of silver-palladium bimetallic nanoparticles as an alternative to chemical insecticides for vector control. The use of a nontoxic biological extract for synthesis ensures that the observed toxicity is attributable to the nanoparticles themselves rather than the reducing agent. The absence of mortality in non-target organisms at the effective larvicidal concentration suggests that these nanoparticles could be used in integrated vector management strategies. However, the researchers caution that the increased predation rates may not be beneficial for the well-being of the predatory nymphs in the long term.

Limitations of the study include the lack of comprehensive comparisons with other biologically synthesized nanoparticles, as the literature often uses inconsistent units of measurement. The variability in size and shape of biologically synthesized nanoparticles makes direct comparisons challenging. The study recommends that future research focus on long-term investigations of nanoparticle bioaccumulation in non-target organisms. Understanding the potential for bioaccumulation from prey to predator through the food chain is crucial for assessing the ecological safety of these nanoparticles.

The findings contribute to the growing body of knowledge on the environmental implications of nanomaterials. As the use of metal nanoparticles in consumer products increases, the risk of environmental release and subsequent ecotoxicity becomes a significant concern. The study underscores the need for strict regulatory measures and comprehensive toxicity assessments to prevent harm to sensitive species. With proper safety and bioremediation strategies, silver-palladium bimetallic nanoparticles could be sustainably utilized against insecticide-resistant vectors, offering a promising tool in the fight against mosquito-borne diseases.

Beyond structural imaging, optical and elemental evaluations provided detailed confirmation of nanoparticle formation. Ultraviolet-visible spectroscopy of the Citrus limon leaf extract showed maximum absorption at 300 plus or minus 40 nanometers at pH 5.29, whereas the diluted bimetallic nanoparticle solution at pH 1.26 absorbed across 380 to 500 nanometers without displaying a distinct surface plasmon resonance band. The researchers attributed this absence of a resonance peak to electron shielding by palladium, whose electronic configuration lacks the collective electron oscillations found in silver. Fourier-transform infrared spectroscopy demonstrated a marked reduction in transmittance bands between 3700 and 2800 wavenumbers and across the fingerprint region of 1000 to 599 wavenumbers compared to the crude leaf broth, signaling that surface organic functional groups directly participated in reducing and stabilizing the metals. Scanning electron microscopy paired with energy-dispersive X-ray spectroscopy confirmed the chemical purity of the bimetallic sample, showing an elemental composition of 88.74 percent silver and 11.26 percent palladium by weight, or 88.60 atomic percent silver to 11.40 atomic percent palladium.

The larval bioassay protocol incorporated rigorous controls and extended exposure tracking alongside standard probit modeling. A positive control utilizing 0.2 percent temephos achieved complete larval mortality within four hours, whereas negative controls consisting of distilled water and pure 10 percent leaf extract caused neither morbidity nor mortality through 72 hours. While the 24-hour assessments demonstrated high potency across instars, continuing exposure through 48 hours further reduced lethal thresholds in Aedes aegypti, dropping the median lethal concentration to 0.51 milliliters per liter for third-instar larvae and 1.36 milliliters per liter for fourth-instar larvae. Analysis of variance confirmed that differences in lethal concentration thresholds varied significantly across both mosquito species and larval developmental stages.

Observations of non-target feeding revealed nuanced predation dynamics over time when odonate nymphs encountered treated mosquito larvae. Under exposure to 1.78 milliliters per liter of nanoparticles, damselfly nymphs consumed 76 percent of third-instar Aedes aegypti within 16 hours, 93 percent by 20 hours, and achieved 100 percent predation at 24 hours, outpacing the 40 hours required in untreated water. Similarly, damselflies presented with Anopheles stephensi progressed from 51 percent consumption at 16 hours to complete elimination by 40 hours, faster than the 64 hours recorded in untreated controls. The authors noted that nanoparticles markedly reduced larval swimming mobility, leaving sluggish prey easier for nymphs to capture, even as internal metal stress potentially drove increased metabolic feeding demands in the predators.

These performance metrics stand in contrast to previous laboratory investigations of gold-palladium bimetallic nanoparticles synthesized with the same plant extract. Those earlier gold-palladium formulations produced smaller nine-nanometer particles but required substantially higher concentrations to achieve equivalent mortality, registering 24-hour third-instar lethal thresholds of 26.32 milliliters per liter for Anopheles stephensi and 6.17 milliliters per liter for Aedes aegypti.

Subject of Research: Eco-friendly synthesis and ecotoxicity of Ag-Pd bimetallic nanoparticles

Article Title: Green synthesis of Ag-Pd bimetallic nanoparticles and its efficacy against mosquito larvae and non-target organisms

Article References: Minal, S. P., & Prakash, S. (2025). Green synthesis of Ag-Pd bimetallic nanoparticles and its efficacy against mosquito larvae and non-target organisms. Bioscience Nanotechnology, 1(1), Article 1. https://doi.org/10.1186/s44331-025-00001-7

Image Credits: AI Generated

DOI: 10.1186/s44331-025-00001-7

Keywords: nanoparticles, mosquito control, ecotoxicity, green synthesis, bimetallic, vector management, Citrus limon, larvicidal activity, non-target organisms, environmental safety, Green, synthesis