UV VIS spectroscopy analysis. The absorption spectra of the diluted samples (1:9) of aqueous leaf extract (10%) of plant Citrus limon (pH 5.29 at 29°C) exhibited the maximum absorbance in the UV region around 300 nm ± 40 nm (Fig. 1.A.a). Whereas, the diluted samples (1:10) of Ag-Pd BNPs (pH 1.26 at 29°C) showed absorption in the range of 380–500 nm without any distinct surface plasmon resonance (SPR) band (Fig. 1.A.b). Generally, biologically synthesized Ag NPs exhibit a characteristic SPR band around 390–450 nm [19], while Pd NPs solution shows an absence of SPR band but presence of absorption in the visible region, unlike the molar solution and leaf extract [43]. The absence of SPR band in the current results aligns with the results of Ag-Pd BNPs synthesized using Catharanthus roseus leaf extract having an equal (1:1) Ag to Pd ratio by Mohan et al. (2021) [31]. This phenomenon of the absence of SPR band can be attributed to the combined effect of bimetallic properties resulting from the formation of Ag-Pd BNPs, where the presence of Pd acts as a shield, this was attributed to the exceptional electronic configuration of Pd, which influences the SPR properties of Ag within the BNPs, also observed for the Ag-Pd BNPs synthesized using fruit extract of Terminalia chebula by Sivamaruthi et al. (2019) [36]. Compared to our studies, Abdel-Fattah et al. (2017) [35], observed that the absence of the SPR band is linked with the Ag to Pd ratio. The use of relatively lower Ag concentrations, Pd:Ag (1:2), shows absence of SPR band, consistent with our findings, which involved an equal concentration of both Ag and Pd (1:1). They further reported a prominent SPR band appeared in the formulation with higher Ag concentration (1:3, 1:4, 1:6 and 1:8), for the almond nut and blackberry fruit extract mediated Ag-Pd BNPs samples.
FT-IR spectroscopy analysis. Leaf Extract: The infrared spectrum of the aqueous leaf extract (Fig. 1.B.a) revealed a transmittance band with a broad peak (3700 cm− 1 - ~2800 cm− 1) corresponding to the stretching vibrations of the functional groups. These include O-H groups of intermolecular (high intensity peak) and intramolecular (weak intensity peak) bonded alcohols (3550 − 3200 cm− 1), N-H groups of amine salts (3000 − 2800 cm− 1), and overlapping C-H stretching vibrations of the alkane (3000 − 2840 cm− 1), alkene (3100 − 3000 cm− 1), and alkyne (3333 − 3267 cm− 1). A weak intensity peak around 2140 − 2100 cm− 1 shows C ≡ C stretching of monosubstituted alkyne compounds, and C-H bending of aromatic compounds is present around 2000 − 1650 cm− 1. A medium-intensity peak showing C = C stretching of conjugated alkene (1650 − 1600 cm− 1), alkene di-substituted (cis) (1662 − 1626 cm− 1), cyclic alkene (1650 − 1566 cm− 1), and N-H bending was observed for amines (1650 − 1580 cm− 1). Another medium intensity peak for O-H bending of phenol compounds (1390 − 1310 cm− 1) is also observed and a characteristic strong intensity peak in the fingerprint region (1000 − 650 cm− 1) indicating the stretching and bending vibrations for C-H bending (900 − 700 cm− 1), C = C bending of alkene vinylidene compounds (895 − 885 cm− 1), and stretching vibrations of the halo compound groups such as C-Cl, C-Br, and C-I can be seen around 850 − 550 cm− 1, along with a peak for complex benzene derivatives around 700 cm− 1 ± 20 cm− 1. Ag-Pd BNPs: In contrast, the FT-IR spectra of Ag-Pd BNPs (Fig. 1.B.b) exhibited a highly reduced transmittance band between 3700 cm− 1 - ~2800 cm− 1 compared to the leaf extract sample. The O = C = O stretching (2349 cm− 1) of carbon dioxide was observed, and a comparatively reduced fingerprint region (1000 cm− 1 – 599 cm− 1) with weak intensity peaks can be seen in contrast to the strong intensity band of leaf extract. This change can be attributed to the utilization of the specific surface functional groups of leaf extract during nanoparticles synthesis. These results are consistent with the reduced transmittance band of infrared spectra observed for the samples of Cochlospermum XXXossypium gum kondagogu extract mediated Ag-Pd BNPs (Velpula et al. (2021)) [34].
TEM analysis. The micrographs of Ag-Pd BNPs samples (Fig. 2.A) revealed the presence of polydispersed electron-dense metal nanoparticles. These nanoparticles exhibited roughly irregular-spherical shape and displayed minimal agglomeration. The size distribution bins of the histogram (Fig. 2.B) generated for the TEM micrograph showed particles sizes ranging from 5–34 d nm (diameter in nm). The Gaussian fit analysis for the obtained particle size distribution bins showed a mean particle size (± SD) of 21 ± 7.22 d nm. The morphological variation in the size of biologically synthesized Ag-Pd BNPs using different extracts and methods is evident in the literature. Factors such as the type of extract used, Ag to Pd ratio, and duration of synthesis seem to play a role in determining the characteristic features of the synthesized BNPs and their properties for functions such as catalysis, antimicrobial activity, and anticancer effects, among others. The observed particles size in our study aligns with the findings of Sivamaruthi et al. (2019) [36], who successfully synthesized Ag-Pd BNPs (2–40 nm) with an average particles size of ~ 20 nm, using 1% Terminalia chebula fruit extract by reducing 10 mM AgNO3 for 1 h, followed by the addition of 10 mM PdCl2 in a 1:1 ratio. Similarly, Velpula et al. (2021) [34] reported comparable results using 1% gum kondagogu extract mediated Ag-Pd BNPs (2–40 nm) with a mean particles size of ~ 21 nm, obtained by reducing combined precursor solution of 1mM AgNO3 and PdCl2 in 1:1 ratio. In another study, Mohan et al. (2021) [31] demonstrated the synthesis of Ag-Pd BNPs (1 mM, 1:1) using a 1% methanolic leaf extract of Catharanthus roseus, the resultant NPs exhibited a size distribution spanning 23–64 nm, with an average particles size falling within the range of 15–30 nm. In 2014, Lu et al. [32] reported the synthesis of monometallic nanoparticles mediated by Cacumen platyclade leaf extract. These nanoparticles exhibited an average particle size of 22 nm for Ag NPs and 7.3 nm for Pd BNPs. Additionally, the synthesis of bimetallic nanoparticles with various Ag to Pd ratios yielded particle sizes of 11.9 nm (3:1), 9.1 nm (1:1), and 7.4 nm (1:3) for Ag-Pd BNPs. Their findings indicated that a higher Pd concentration resulted in smaller particle sizes, which, in turn, demonstrated enhanced catalytic activity when compared to other nanoparticles.
DLS analysis. The Zetasizer results (Fig. 3A) for the diluted samples (1:10) of the Ag-Pd BNPs (2 mM) revealed a hydrodynamic size range of 800–1050 d nm. The Z-average was measured at 1956 d nm, with the peak (± S.D.) observed at 982.2 d nm (± 142.8 d nm), and a polydispersity index (PdI) of 0.515. This suggests that the sample has a larger hydrodynamic size when compared to the actual size of the metal NPs obtained from TEM analysis. In comparison to our results, a smaller hydrodynamic size of the biologically synthesized Ag-Pd BNPs was reported by Mohan et al. (2021) [31] in the range of 2–28 d nm with an average size of 28 d nm, while Velpula et al. (2021) [34] determined an average particles size of 44.8 d nm, and Sivamaruthi et al. (2019) [36] reported a range of 5-200 d nm with Z-average of 31.93 d nm. Turunc et al. (2017) [33] reported the average particle size of 15 d nm for Ag NPs and 22 d nm for Pd NPs, indicating larger hydrodynamic size of smaller Pd NPs solution compare to Ag NPs. The DLS chart obtained by Abdel-Fattah et al. (2017) [35], showed the presence of Ag-Pd BNPs in the size range of 90-1200 d nm, which is close to our results. Yeap et al. (2018) [68] suggest that samples containing active interacting molecules tend to aggregate, resulting in larger-sized particles during DLS analysis compared to TEM analysis. According to Souza et al. (2016) [69] and Bhattacharjee (2016) [70], the aggregating properties of leaf extract biomolecules, which are used as capping and reducing agents, often contribute to NP retention and control their release in the environment.
XRD analysis. The results of XRD pattern for Ag-Pd BNPs samples (Fig. 3.B) revealed distinct peaks at 38.7°, 44.6°, 64.7°, and 77.4° corresponding to 2θ angle with lattice constants of (111), (200), (220) and (311), respectively. These peaks signify the presence of Ag particles in the sample, in accordance with the Ag content indicated by JCPDS no. 87–0720. Additionally, peaks at 40.5°, 48.6° and 68.4° of 2θ angle with lattice constants of (111), (200), and (220), respectively, were observed, indicating the presence of palladium in the sample as confirmed by JCPDS no. 65-6174. The high background noise and low-intensity peaks observed indicate the semi-crystalline nature of the Ag-Pd BNPs sample. This can be attributed to the synthesis process using leaf extract and low-concentration Ag-Pd BNPs present in the test sample. Similar to our results, the Ag-Pd BNPs synthesized using the leaf extract of Cacumen platycladi (Lu et al., (2014)) [32], gum kondagogu extract of Cochlospermum gossypium (Velpula et al. (2021)) [34], and rutin biomolecule mediated synthesis showed identical X-ray diffractogram (Singh et al. (2020)) [37].
SEM-EDX analysis. The illuminating surface containing Ag-Pd BNPs samples of SEM micrograph (Fig. 4.b) confirms the presence of metal nanoparticles. The peaks corresponding to Ag and Pd elements are present with an absence of the peaks related to other metal contaminants. The energy table (Fig. 4.a) generated from the EDX analysis of SEM micrograph shows the data generated from the L-series confirms the higher element % for silver (Ag) (88.74) as compared to palladium (Pd) (11.26), and with the atomic % of 88.60 for Ag and 11.40 for Pd, in the composites of Ag-Pd BNPs samples. This shows the content and composition of the formed nanoparticles (Ag-Pd BNPs), having a larger content of Ag as compared to Pd in the sample of formed Ag-Pd BNPs.
Nanotoxicity Bioassay. The results of the Ag-Pd BNPs bioassay showed toxic efficacies against the I II, III, and IV instar larvae of selected mosquito species. The negative control containing distilled water and leaf extract showed no morbidity or mortality till 72 h of exposure. The positive control showed 100% mortality after 4 h. The mean corrected % mortality obtained [refer to Table S1 in supplementary material] from the bioassays was subjected to probit analysis and probit regression plots were generated for both Anopheles stephensi (Fig. 5.A) and Aedes aegypti (Fig. 5.B) for the calculation of the LC50 (LCL-UCL) values with ± S.D. and S.E., against both mosquito species (Fig. 6.A.B) at 24 h, 48 h, and 72 h of exposure [refer to Table S2 and S3 in supplementary material]. For the Ag-Pd BNPs bioassay against Anopheles stephensi, the non-significant (NS) value of χ2 test depicting the goodness of fit were obtained for the test concentrations against II, III, and IV instar larvae after 24 h of exposure, for II instar larvae LC50 (LCL-UCL) of 1.59(1.08–2.33) mL/L was observed with ± 0.33 S.D and 0.09 S.E., for III instar larvae LC50 of 1.78(1.21–2.60) mL/L was observed with ± 0.36 S.D. and 0.08 S.E., and for IV instar larvae LC50 of 1.62(1.12–2.34) mL/L with ± 0.31 S.D. and 0.08 S.E. was observed. While 100% mortality was observed for I instar larvae at 24 h in the lowest selected concentration and significant χ2 values (bad-fit) were obtained for the efficacy data against I instar larvae at 24 h, and for II, III, and IV instar larvae at 48 h and 72 h of exposure. The results obtained for Ag-Pd BNPs bioassay against Aedes aegypti showed the non-significant (NS) χ2 test values depicting goodness of fit for the LC50 generated from the test concentrations against II (24 h), III, and IV instar larvae (24 h and 48 h), for II instar larvae LC50 of 1.59(1.08–2.33) mL/L was observed with ± 0.33 S.D and 0.09 S.E. (24 h), for III instar larvae LC50 of 1.78(1.21–2.60) mL/L with ± 0.36 S.D. and 0.08 S.E. at 24 h and LC50 of 0.51(0.23–1.13) mL/L with ± 0.72 S.D. and 0.18 S.E. at 48 h was observed, and for IV instar larvae, the LC50 of 2.08(1.37–3.16) mL/L with ± 0.44 S.D. and 0.09 S.E. at 24 h and LC50 of 1.36(0.87–2.14) mL/L with ± 0.40 S.D. and 0.10 S.E. at 48 h was observed. The results of two-way ANOVA showed a significant difference between the dependent variable i.e., obtained LC50 values after 24 h of exposure, against the independent variable i.e., mosquito species and larval Instar (II, III, and IV) [refer to Table S4 and S5 in supplementary material]. Presently, the absence of comparative data for mosquito larvicidal bioassays involving Ag-Pd BNPs makes it challenging to establish a comparative relationship between particles size, toxicity (LC50), and the type of extract used. However, our findings lay the groundwork for future comparisons of the efficacies of biologically synthesized Ag-Pd BNPs against larvae of mosquito vector. Erstwhile, the monometallic NPs of Ag and Pd are well known to show toxic efficacies against mosquito larvae. One of the most effective formulations, containing an optimum concentration of Ag NPs, synthesized from the entomopathogenic fungus Chrysosporium tropicum, demonstrated larvicidal activity against Culex quinquefasciatus and Anopheles stephensi. The entomopathogenic nature of the fungus is advantageous for the biological management of insect pests. Furthermore, the addition of an optimal concentration of Ag NPs to the fungal broth has been shown to synergistically enhance the toxicity of the formulation, rather than inhibiting fungal growth. This enhancement extends the shelf life of the formulation thereby promoting its adhesion to the insect cuticle, leading to fungal infestation in the insect pest. Such strategic formulations contribute to reducing environmental implications by decreasing reliance on chemical insecticides and enhancing the effectiveness of biological control methods (Soni and Prakash (2012)) [71]. Bhakyara et al. (2017) [72], observed that the Pd NPs synthesized using a toxic leaf extract of plant Melia azedarach, showed greater larvicidal toxicity against III instars of Aedes aegypti with LC50 obtained at 27.36% as compared to the leaf extract alone with LC50 at 93.96% at 24 h. Therefore, the combined toxic effect with the leaf extract can contribute to the overall toxicity which can be further enhanced by the additional synthesized Pd NPs in an optimum concentration. Conversely, Minal and Prakash (2018) [43], used a non-toxic leaf extract of Citrus limon for Pd NPs synthesis and reported an LC50 value of 16.038% at 24 h of exposure against III instar larvae of Anopheles stephensi, thus emphasizing the intrinsic toxic efficacy of Pd NPs without a significant contribution from the leaf extract. An attempt to elucidate the mechanism of action leading to larval mortality induced by these nanoparticles was conducted by Kalimuthu et al. (2017) [73] through histological studies of Hedychium coronarium rhizome-mediated Ag NPs treated IV instar larvae of Aedes aegypti. Their findings indicated that, in comparison to the untreated control group, the mid-gut of the treated larvae exhibited partial lysis of the epithelium at the apical side. This was attributed to swollen epithelial cells filled with multiple vacuoles, and the lumen contained the content of ruptured epithelial cells. The physiological damages caused by Ag NPs were further analyzed at the cellular and biochemical levels by Fouad et al. (2017) [74], using Cassia fistula fruit pulp extract-mediated Ag NPs against the IV instar larvae of Aedes albopictus and Culex pipiens pallens. They reported a considerable decrease in the total protein content and downregulation of two key enzymes, namely Acetylcholinesterase and α- and β-carboxylesterase. In addition to these three biochemical parameters, Ga'al et al. (2017) [75] also reported the downregulation of acid and alkaline phosphatase due to Ag NPs synthesized with the green method using salicylic acid and its derivative, 3,5-dinitrosalicylic acid. These changes demonstrate the direct toxic effect of the Ag NPs, resulting in abnormal physiology, cell signalling alterations, often leading to cell lysis, and a reduction in metabolic and neuromuscular activity. Moreover, the inability of downregulated and damaged α- and β-carboxylesterase enzymes to further detoxify the cell leads to the accumulation and aggregation of the metal NPs in the system, which is the main cause of larval mortality in the nanotoxicity bioassay. Thereby, making NPs the most suitable alternative against mosquito species resistant to chemical and biological insecticides, while simultaneously raising concerns about its effects on other non-target environmental organisms.
Predation efficiency test. The number of larvae consumed by each selected predatory nymphs were recorded for Ag-Pd BNPs test (LC50) and negative control in replicates (R1, R2, R3) containing 25 III instar larvae of both mosquito species [refer to Table S6 and S7 in the supplementary material]. The negative control groups, comprising of a dragonfly nymph with 25 III instar larvae of Anopheles stephensi exhibited 100% predation within 24 h (Fig. 7.A.a) and for Aedes aegypti, a complete predation was observed at 40 h (Fig. 7.A.b). Conversely, the negative control groups consisting of a damselfly nymph with 25 III instar larvae of Anopheles stephensi in each replicate, demonstrated 100% predation at 64 h (Fig. 7.A.c), whereas for Aedes aegypti, complete predation occurred at 40 h (Fig. 7.A.d). The LC50 value of 1.78 mL/L, obtained from the nanotoxicity bioassay using III instar larvae, was specifically selected as the benchmark for assessing the predation efficiency of the non-target organisms. For Ag-Pd BNPs (LC50) test containing a Dragonfly nymph and 25 III instar of Anopheles stephensi, the predation of 55% larvae at 16 h was observed, which increased to 71% at 20 h, 92% at 24 h, and ultimately reached 100% at 40 h (Fig. 7.B.a) and for Aedes aegypti, 61% larval predation at 16 h, rising to 73% at 20 h, 95% at 24 h, and achieving complete predation (100%) at 40 h (Fig. 7.B.b). The Ag-Pd BNPs (LC50) test against the nymph of Damselfly with 25 III instar of Anopheles stephensi, showed consumption of 51% of larvae at 16 h, which increased to 84% at 20 h, subsequently to 92% at 24 h, and ultimately achieved complete predation (100%) at 40 h (Fig. 7.B.c), and for Aedes aegypti, the 76% larval predation at 16 h, increased to 93% at 20 h, and eventually complete predation (100%) at 24 h was observed (Fig. 7.B.d). The results obtained from the predation tests demonstrate a significant improvement in predation efficiency for the Ag-Pd BNPs test compared to the untreated control groups. The predatory nymphs exhibited resilience to the applied test concentration, as no mortality was recorded in any of the replicates during the 72 h observation period. The increased predation rates, however, may be a response to meet the energy demand for defence against the metal stress induced by the Ag-Pd BNPs test in the nymphs. dos Santos Lima et al. (2021) [76] investigated disruptions in the food web caused by metal stress from copper, cadmium, mercury, and manganese. The study aimed to examine how this metal stress influenced biotic interactions, specifically focusing on the feeding preferences and predation rates of dragonfly nymphs (Tramea cophisa) and ostracods (Chlamydotheca sp.) on water-dwelling crustaceans. The research revealed that, although metal stress did not alter food preferences, it did modify predation behaviour in a species-specific and metal-specific manner. Overall, a decrease in predation rates was observed for dragonfly nymphs, while increased predation rates were noted for ostracods. Hence, this study establishes that the enhanced predation rate of nymphs induced by nanoparticles may not necessarily be beneficial for the well-being of the predatory nymph. Azam et al. (2015) [77] found the libellulid dragonfly (Crocothemis servilia) to be a highly effective ecological indicator for water and riparian systems. They observed that heavy metal-induced stress not only alters the life history of insects but also makes them efficient bio-indicators due to metal bioaccumulation, offering a clear measure of heavy metal contamination across different locations. Similarly, a study conducted by Akhtar et al. (2021) [78] demonstrated the occurrence of bio-transfer and bioaccumulation of various heavy metals between the trophic levels of the food chain from mosquito larvae to natural predators, such as dragonfly nymphs (Tramea cophysa), frequently resulting in the mortality of nymphs due to higher metal bioaccumulation. A notable decrease in the mobility of mosquito larvae from both species under the applied nanotoxicity bioassay can be a factor influencing predation efficiency, contributing to the accelerated predation by nymphs within the experimental setup. Likewise, in a study conducted by Murugan et al. (2015a) [79], it was demonstrated that the specified lower concentration of synthesized Ag NPs could effectively induced mortality in Anopheles larvae, while it had no apparent impact on the predation efficiency of dragonfly nymphs (Anax immaculifrons). Studies conducted on natural copepod predators (Mesocyclops longisetus, Mesocyclops aspericornis, and Megacyclops formosanus) which are found to be effective against the I and II instar of Anopheles and Aedes mosquito species, revealed an increase in predation at lower test concentrations of Ag NPs without resulting in predator mortality. Conversely, higher concentrations of Ag NPs led to predator mortality, suggesting the need for lower doses to ensure that natural copepod predators are not adversely affected by Ag NPs (Murugan et al. (2015b) [80], Murugan et al. (2016) [81], Kalimuthu et al. (2017)) [82]. Therefore, the observed increase in predation rates in our study compared to the control group may be attributed to metal stress. While the selected concentration of Ag-Pd BNPs has not adversely affected the nymphs directly, their feeding on the Ag-Pd BNPs-affected, slowed larvae could lead to further bioaccumulation of nanoparticles from prey to predator through food chain. This process may eventually result in complications and the ultimate death of the natural biocontrol agents. Consequently, we recommend future studies on comparatively long-term investigation of the effects of nanoparticle bioaccumulation on the normal continuity of the life cycle non-target organisms.
Despite the absence of studies on biologically synthesized Ag-Pd BNPs against both target mosquito larvae and non-target predatory organisms, there are existing studies on the antimicrobial and anticancer activities of Ag-Pd BNPs. Abdel-Fattah et al. (2017) [35] synthesized Ag-Pd BNPs using almond nuts and blackberry fruit extracts separately, incorporating varying Pd (1) to Ag (2,4,6, and 8) ratios-specifically, 1:2, 1:3, 1:4, 1:6 and 1:8. The almond nuts extract-mediated Pd-Ag BNPs (1:6) and blackberry fruit extract-mediated Pd-Ag BNPs (1:8) were selected for TEM analysis, showing the formation of 8 nm and 6 nm nanoparticles, respectively. These Ag-Pd BNPs, mediated from both extracts, demonstrated cytotoxicity against human breast cancer (MCF7) and liver cancer (HEPG2). Notably, a Pd:Ag ratio of 1:3 exhibited maximum mortality at the half-maximal inhibitory concentration (IC50), outperforming Ag-Pd BNPs with a Pd:Ag ratio of 1:8. This highlights the significant impact of the Pd to Ag ratios used in the formulation. Additionally, in the antimicrobial assay against bacterial strains such as Escherichia coli and Staphylococcus aureus, as well as fungal strain of Candida albicans, Ag-Pd BNPs with a Pd:Ag ratio of 1:8 outperformed those with lower Ag ratios, shedding light on the cell-specific and composition-specific activity of Ag-Pd BNPs. Sivamaruthi et al. (2019) [36] observed that Ag-Pd BNPs mediated by Terminalia chebula fruit extract exhibited substantial antimicrobial activity against bacteria, including methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. The increase in concentration of Ag-Pd BNPs from 20 µg/mL to 200 µg/mL showed a notable enhancement in the cytotoxic anticancer activity against the human lung cancer cell line (A549). Therefore, the antimicrobial properties of the Ag-Pd BNPs (~ 20 nm) derived from the non-toxic fruit extract of Terminalia chebula suggests that Ag-Pd BNPs (~ 21 nm) synthesized using the eco-friendly and non-toxic leaf extract of Citrus limon may also possess potential antimicrobial activity, attributed to their < 25 nm size. The catalytic potential of Ag-Pd BNPs appears promising and is subject to variation based on the differential composition in the Ag to Pd ratios. For instance, Lu et al. (2014) [32] reported the synthesis of Ag-Pd BNPs using Cacumen platycladi leaf extract in varying ratios of Ag:Pd, exhibited sizes of 11.9 ± 0.8 nm (3:1), 9.1 ± 0.7 nm (1:1), and 7.4 ± 0.4 nm (1:3) and demonstrated that Ag-Pd BNPs in a 1:3 ratio, supported by γ-Al2O3 catalyst, were particularly effective in the hydrogenation of 1,3-butadiene compared to the other two ratios of Ag:Pd. This study not only reveals the influence of different ratios on the mean particle size but also highlights the variations in catalytic efficiency among the formed formulations. A study on mono-and bi-metallic NPs of Ag and Pd was analyzed by Turunc et al. (2017) [33], they synthesized monometallic Ag NPs and Pd NPs, and bimetallic Ag-Pd BNPs using Lithodora hispidula leaf extract. These nanoparticles were employed to modify glassy carbon electrodes (GCE), resulting in the production of Ag NPs-GCE, Pd NPs-GCE, and Ag-Pd BNPs-GCE for the electrocatalytic reduction of hydrogen peroxide (H2O2). Among these, Ag-Pd BNPs-GCE demonstrated superior efficiency, attributed to its increased synergistic catalytic effect, and exhibited a low detection limit, making it a more promising material to produce an H2O2 nanosensor. The enhanced efficiency of Ag-Pd BNPs over Ag NPs and Pd NPs alone underscores the importance of bimetallic-based experimental studies to achieve optimum catalytic efficiency based on metal combinations. On the other hand, Singh et al. (2020) [37] demonstrated the synthesis of nanoparticles using a single biomolecule, rutin. This study also involved the synthesis of both monometallic Ag NPs (~ 50 nm), Pd NPs (~ 10 nm), and bimetallic Ag-Pd (1:1) BNPs (~ 80 nm). These nanoparticles exhibited distinct catalytic activities in etherification reactions with phenolic compounds, with the order of reactivity being Pd NPs > Ag NPs > Ag-Pd BNPs. Notably, the smaller particle size (Pd NPs) displayed the highest reactivity among the three types. This highlights the importance of size-specific catalytic activity of the nanoparticles. Achieving a lower size of Ag-Pd BNPs, as done by the extract of fruit extract of Terminalia chebula [36], gum kondagogu extract [34] and in the current study through leaf extract of Citrus limon mediated Ag-Pd BNPs, could enhance efficiency. Therefore, testing whether this enhanced activity is due to the properties of the metal or the size of the overall nanoparticles is also required. Mohan et al. (2021) [31] utilized Catharanthus roseus mediated Ag-Pd BNPs (15–30 nm), which exhibited a remarkable 98% photocatalytic degradation of safranin O textile dye within 40 minutes of exposure. Moreover, they demonstrated a free radical scavenging efficiency of 70.2% (Ag-Pd BNPs), surpassing the 48.2% achieved by the leaf extract. Here, the enhanced efficiency can be attributed to the smaller size and unique properties of the synthesized Ag-Pd BNPs. Other combinations involving Au salts were compared by Velpula et al. (2021) [34], who demonstrated the synthesis of Ag-Au BNPs, Ag-Pd BNPs, and Au-Pd BNPs mediated by the gum kondagogu extract of the plant Cochlospermum gossypium. These nanoparticles were tested for their efficiency in catalyzing the reduction of 4-nitrophenol (4-NP). The results revealed catalytic efficiencies in the order of Ag-Pd BNPs (5–40 nm) > Ag-Au BNPs (2–40 nm) > Au-Pd BNPs (4–35 nm). This suggests that, for certain reactions, the properties dependent on the elemental composition are as significant as the size-related activity of the synthesized nanoparticles.
In contrast to our current findings on Citrus limon-mediated Ag-Pd BNPs, our previous studies on Citrus limon-mediated Au-Pd BNPs (Minal and Prakash (2020) [30]) exhibited unique characteristics and distinct bioassay results. UV-VIS spectroscopy analysis for Au-Pd BNPs revealed the presence of an SPR band around 540 nm due to the Au content, while the SPR band related to Ag was absent in the current Ag-Pd BNPs studies. The mean particle size of Citrus limon-mediated Au-Pd BNPs was 9.3 nm ± 3.95 nm, whereas our current studies observed a larger particle size of 21 nm ± 7.22 nm for Ag-Pd BNPs. DLS analysis indicated a size range of 150–200 nm with a Z-average of 3340 nm for Au-Pd BNPs, compared to the present DLS analysis with a Z-average of 1956 nm and the hydrodynamic size range of 800–1050 nm for Ag-Pd BNPs. Despite utilizing the same reducing agent, the observed variation in particle size was evident. Furthermore, the efficacy of Citrus limon-mediated Au-Pd BNPs nanotoxicity bioassay against I, II, III, and IV instar larvae of Anopheles stephensi and Aedes aegypti showed higher LC50 values compared to the lower LC50 values observed in the present study of Ag-Pd BNPs nanotoxicity bioassay. The LC50 values against I – IV instar larvae of Anopheles stephensi in the previously reported Au-Pd BNPs test were 5.12 mL/L, 8.14 mL/L, 26.32 mL/L, and 11.4 mL/L at 24 h, respectively, while LC50 values of 0.1 mL/L, 1.59 mL/L, 1.78 mL/L, and 1.62 mL/L at 24 h, respectively, were observed for the current Ag-Pd BNPs test. Similarly, for the previously reported Au-Pd BNPs, LC50 values of 12.37 mL/L, 11.24 mL/L, 6.17 mL/L, and 10.83 mL/L at 24 h against Aedes aegypti were reported, compared to current Ag-Pd BNPs test with observed LC50 values of 0.1 mL/L, 1.59 mL/L, 1.78 mL/L, and 2.08 mL/L at 24 h, respectively. The predation efficiency of non-target nymphs of dragonfly and damselfly under the Au-Pd BNPs test (6.67 mL/L) showed a complete predation at 40 h for Anopheles stephensi with dragonfly nymph, while for other tests, including Anopheles stephensi with damselfly nymph, Aedes aegypti with dragonfly nymph, and Aedes aegypti with damselfly nymph, complete predation occurred at 48 h. In contrast, the current predation efficiency results under the Ag-Pd BNPs test (1.78 mL/L) showed 100% predation for Aedes aegypti with damselfly nymph at 24 h, and for other tests, 100% predation was achieved at 40 h. Therefore, understanding the characteristic and toxic differences between Au-Pd BNPs and Ag-Pd BNPs synthesized using the same reducing agent is crucial for determining their suitability in specific applications. This is especially important given that Au-containing BNPs demonstrate toxicity at higher concentrations, in contrast to Ag-containing BNPs, which exhibit toxicity at lower concentrations.
A pre-requisite to address the potential environmental implications of the synthesized nanoparticles, it is crucial to acknowledge that products incorporating metal nanoparticles, such as silver, titanium, zinc, gold, magnesium, aluminum oxide, copper, platinum, iron, and iron oxides, are readily accessible in the consumer market. Moreover, the incorporation of these materials into new products is on a continuous rise [83]. The increasing industrial and household applications raise the likelihood of nanoparticles being released into the environment. Consequently, assessing the risk of nanoparticles-mediated ecotoxicity is crucial to formulate specific guidelines for their use, disposal, bioremediation, and implementing strict surveillance for their accidental or intentional environmental release. Despite the numerous benefits, unknown risks may be associated with direct and indirect exposure to nanoparticles. The environmental impact and economic costs associated with the environmental release of nanoparticles are still underdeveloped. The majority of nanoparticle-based products are under study, awaiting industrial-level production, while others are already available in the market [84, 85, 86]. These nanoparticle-based products largely end up in soil, air, and aquatic environments. The nanoparticles contaminate soil, adversely affecting its enzymatic activity, nutritional balance, and self-cleaning properties. This, in turn, has a negative impact on beneficial microbial biodiversity, leading to adverse effects on plant growth and development, as well as bioaccumulation in both edible and non-edible plant tissues. Additionally, the leaching of nanoparticles into nearby aquatic reservoirs further impacts other ecological organisms [87]. Whereas, the optimal use of some metal nanoparticles such as iron and gold in clinical trial studies, specifically for theragnostic and radiotherapy applications, is closely observed, and the final formulation is approved or rejected based on associated implications on human health [88]. However, the unintentional environmental release of nanoparticles from nano-biocomposites used in food packaging technologies is not as closely monitored. This calls for the establishment of standard guidelines to test the ecotoxicological effects of nanoparticles released from packaging materials [89]. The field of environmental nanotechnology actively engages in providing solutions to address the environmental impact, cleanup, and remediation of anthropogenic nanomaterials. Many of these solutions involve the use of mono- and bi-metallic nanoparticles to remove other nano-contaminants, which still pose the risk of contaminating the environment themselves due to high mobility and transport, leading to unintended contamination, creation of toxic byproducts during remediation, and toxicity to local flora and fauna. The short-term stability and persistence of these nanoparticles further pose risks to the environment, leading to bioaccumulation and biomagnification. Additionally, unknown interactions in the environment can lead to the generation of new and harmful compounds. Consequently, bimetallic nanoparticles are observed to play a dual role depending on the mode of application, serving either as a contaminant or as an agent for the remediation of other nano-contaminants [90]. The environmental implications of adverse effects of nanoparticles are high due to their large surface area to volume ratio. Ongoing research on nanoparticles transport, environmental fate, long-term effects, and potential risks associated with their use in remediation, based on the specific characteristics of the nanoparticles, is evident in recent literature. Furthermore, regulatory frameworks are being developed by the U.S., European Union, Canada, Australia, and International Collaborations. These frameworks, such as the Toxic Substances Control Act (TSCA) under the U.S. Environmental Protection Agency (EPA), the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) regulation, Canadian Environmental Protection Act (CEPA), National Industrial Chemicals Notification and Assessment Scheme (NICNAS), and Organization for Economic Co-operation and Development (OECD), respectively, are formulating guidelines for the responsible use of nanotechnology in environmental applications. This involves collaboration between governments, industry stakeholders, and scientific communities to ensure that regulations are supported by the latest scientific understanding and technological advancements [91, 92, 93].
Lastly, the limitation of the biologically synthesized nanoparticles lies in the use of diverse leaf extracts, each containing a unique phytochemical composition, play a distinctive role in the synthesis of nanoparticles, resulting in variations in the size and shape that subsequently influence their toxicity and other properties. A significant limitation in the available literature lies in the lack of comprehensive comparisons concerning the toxicities observed in bioassays of biologically synthesized nanoparticles. This limitation arises due to the frequent use of units like ppm without disclosing the actual amount of the formulation used for experiments. The common reliance on ppm as a measurement unit, often employed by researchers, fails to provide a clear understanding of the nanoparticles’ original specifications employed in the tests. Given the inherent variability in the size and shape of biologically synthesized nanoparticles, the use of ppm becomes less than ideal. A more suitable approach can involve comparing size-related characterizations (SEM, TEM, DLS) with toxicity results presented as raw measurements in units of mL/ng/mg/% of NPs in solvent (units). Additionally, specifying the applied serial dilution of the molar solution during experimentation is imperative.
The current study provides significant information about the novel characteristic features of Ag-Pd BNPs synthesized using a non-toxic Citrus limon leaf extract for the first time. It also addresses a critical gap by conducting the first-ever nanotoxicity evaluation of Ag-Pd BNPs against more complex environmental invertebrates. Therefore, moving ahead towards ecotoxicological evaluation compared to the available studies on simpler laboratory based antimicrobial and anticancer assays. In addition to the mosquito larvicidal efficacy of Ag-Pd BNPs, studies against non-target nymphs are required for the selection of the minimal effective concentration. This would help to formulate a sustainable, target-oriented insecticide posing a comparatively lower threat to non-target organisms. Therefore, we recommend that for pest management, simultaneous efficacies against non-target organisms spatially present in the same niche as the target pest should be considered complementary in selecting nanoparticles concentrations. The LC50 value varies among environmental organisms and life stages, influenced by their biological complexities. Our finding are consistent with other existing literature, highlighting the potential applicability of Citrus limon mediated Ag-Pd BNPs in catalytic, antimicrobial, and anticancer activities.