The Accumulation and Distribution of Green Synthesized Silver Nanoparticles with Açaí in C. elegans Do Not Cause Redox Toxicity

TL;DR

Green-synthesized silver nanoparticles using açaí do not cause redox toxicity in C. elegans, suggesting a safer production method.

Contribution

Demonstrates that green-synthesized AgNP accumulate in C. elegans without disrupting redox balance.

Findings

Bio-AgNP accumulated in C. elegans at concentrations of 3.6 ± 0.5 and 17.9 ± 1.8 pg Ag/µg protein.

Bio-AgNP were distributed throughout the C. elegans body, particularly in the midgut.

No significant changes in GSH or GSSG levels were observed, indicating no redox toxicity.

Abstract

Green synthesis of silver nanoparticles (AgNP) has been proposed as a safer and more sustainable alternative for AgNP production. However, limited information exists on the toxicity mechanisms and accumulation of green-synthesized AgNP. In this study, we used AgNP synthesized with Amazonian açaí (Bio-AgNP) to evaluate their accumulation and biochemical effects in Caenorhabditis elegans. Bio-AgNP were synthesized using aqueous extract of lyophilized açaí pulp as reducing and stabilizing agent. Caenorhabditis elegans N2 wild-type (L1 larvae) were exposed to 5 and 10 mg/L Bio-AgNP for 52 h. Silver accumulation and distribution were assessed, and GSH and GSSG levels quantified. Our results showed that Bio-AgNP accumulated in C. elegans (3.6 ± 0.5 and 17.9 ± 1.8 pg Ag/µg protein, respectively), distributed throughout the body, particularly in the midgut. However, no statistical significant changes in GSH or GSSG were detected. Overall, Bio-AgNP accumulate in C. elegans without affecting the GSH/GSSG redox balance, These findings support the potential of green chemistry as a sustainable pathway for nanoparticle production with reduced toxicological impact compared with AgNP produced by chemical synthesis.

The online version contains supplementary material available at 10.1007/s00128-026-04221-2.

Figures

a), an average particle size of 21.4 ± 9.2 nm by TEM (Supplementary Material, Fig. 1b), and a hydrodynamic diameter of 318.19 nm by DLS, similar to that obtained in our previous study (SRP: 432 nm and TEM size: 19.4 ± 7.0 nm) (Taipe Huisa et al. [2024](#CR20)) under the same synthesis process. The results indicate that *C. elegans* can accumulate silver (Ag) at both tested concentrations after 52 h of exposure without causing lethality, with a significantly higher amount of Ag accumulated in worms exposed to 10 mg/L (17.9 ± 1.8 pg Ag/µg protein) compared to 5 mg/L (3.6 ± 0.5 pg Ag/µg protein).

GSH (a) and GSSG (b) levels in *C. elegans* exposed to 5 mg/L and 10 mg/L Bio-AgNP. The results are expressed as a percentage of the control. Different lowercase letters indicate significant differences between the treatments (*p* < 0.05)

Full text

Introduction

Silver nanoparticles (AgNP) are widely used in the biomedical (antiseptics and drug delivery systems) (Abbas et al. 2024; Lekha et al. 2021) and industrial fields (catalysis and chemical sensors) (Sharma et al. 2021; Li et al. 2023) due to their unique properties such as electrical, optical properties, adjustable size, high surface area and high antimicrobial activity (Abbas et al. 2024). Although silver nanoparticles have traditionally been synthesized through physical or chemical methods, biosynthesis has emerged as a more sustainable and environmentally friendly alternative. Physical methods, such as laser ablation, generally require high energy input and sophisticated equipment, which can substantially increase production costs (Abbas et al. 2024). Chemical methods, on the other hand, rely on the chemical reduction of silver followed by stabilization steps, which may generate toxic by-products and pose environmental risks (Almatroudi 2020). In contrast, biosynthesis or green synthesis employs natural reducing and stabilizing agents from plants or microorganisms, eliminating the need for toxic reagents and complex equipment, thereby making the process considerably more sustainable and environmentally friendly (Fahim et al. 2024) making it especially appealing for applications involving human contact, such as in medicine (e.g. antimicrobial agents) and agriculture (e.g. nanoformulations for pesticides and fertilizers) (Khan et al. 2022).

The environmental benefits attributed to Bio-AgNP lies in their potential lower toxicity profile compared to chemically synthesized AgNP. However, green synthesis does not eliminate toxicity, but it typically reduces it, which is the primary rationale behind classifying these nanoparticles as more environmentally friendly (Kirubakaran et al. 2026). However, despite the so-called environmentally friendly nature of these nanoparticles due to the characteristics mentioned, limited studies have actually verified that the toxicity of green synthesized AgNP is lower than AgNP produced by chemical synthesis using in vivo models, such as Danio rerio (Spagnoletti et al. 2021) and Caenorhabditis elegans (Taipe Huisa et al. 2024). Caenorhabditis elegans is a small free-living nematode that can be used to assess the safety or toxicity of nanomaterials. It has many advantages that include its small size, high reproductive rate, transparency, high homology with humans (60–80%), and the possibility to perform a variety of analysis including biochemical assays (Gonzalez-Moragas et al. 2015) therefore it was chosen as organism model for our study. Moreover, most studies evaluating the toxicity of biogenic nanoparticles in C. elegans have focused primarily on life history traits, such as survival, growth, and reproduction, while few have investigated biochemical alterations, including reactive oxygen species (ROS) generation and antioxidant enzyme activity (Ravindran et al. 2024; Taipe Huisa et al. 2024). ROS can be generated in response to external stressors. At low levels, ROS play beneficial roles in cell signaling, whereas at high levels they induce oxidative stress and damage essential biomolecules, potentially leading to cell death. Environmentally, elevated ROS levels can serve as an indicator of exposure to contaminants such as nanomaterials, although this response is nonspecific, (Bi et al. 2024; Guo et al. 2023) this can also be related to alterations in reduced glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) (Flores-López et al. 2019; Opris et al. 2021). Furthermore, there is currently no evidence that green synthesized silver nanoparticles can be taken up and accumulate in worms, an important factor for determining whether their apparent low toxicity is due to low bioavailability.

In this study, AgNP were synthesized using an aqueous extract of Amazonian açaí pulp (Euterpe oleracea Mart.), a natural source rich in polyphenols (such as cyanidin-3-glucoside) previously shown to be effective in the green synthesis of Bio-AgNP (Taipe Huisa et al. 2024). Therefore, the aim of this work was to investigate the internalization, distribution, and biochemical effects of açaí-mediated Bio-AgNP in C. elegans. By integrating AgNP accumulation analyses with the assessment of redox biomarkers, specifically reduced glutathione (GSH) and its oxidized form glutathione disulfide (GSSG), we seek to determine whether green-synthesized AgNP are intrinsically less hazardous in vivo or whether their apparent low toxicity is consequence of limited uptake and bioavailability. This integrative approach provides a more comprehensive evaluation of the environmental safety of green nanomaterials and contributes to the broader effort to assess whether so-called “eco-friendly” nanotechnologies genuinely mitigate ecological risks.

Materials and Methods

Biosynthesis of Silver Nanoparticles

The biosynthesis process was performed as described by Taipe Huisa et al. (2024), using the aqueous extract of the lyophilized pulp of açaí (E. oleracea Mart.) as reducing and stabilizing agents. To prepare the aqueous extract, 4 g of lyophilized acai (“Amazonia Comercio de Acai Liofilizado e Exportacao Ltda.”) were dissolved in 20 mL of MilliQ water. The mixture was sonicated for 5 min at a 50% amplitude (QSonica, model Q125, 125 W power) and left for 90 min in a water bath at 45 °C. The solution was then centrifuged at 3000 × g for 10 min at room temperature, and the supernatant was collected and filtered using Whatman paper N°o. 1. The filtrate was stored at 4 °C and protected from light until further use. The biosynthesis was carried out by mixing aqueous extract (100 mg/mL) with AgNO_3_ (10 mg/mL) in MilliQ water and incubating the reaction at 70 °C for 90 min at 200 rpm. The resulting solution was cooled, stored at 4 °C, and protected from light. Bio-AgNP were characterized using UV-VIS spectrophotometry using a Tecan Infinite Pro M200 spectrophotometer (Tecan, Crailsheim, Germany). Morphological characterization of the nanoparticles was performed using a transmission electron microscope (TEM) Hitachi H-7500 operated at 100 kV, and intensity-based hydrodynamic size of the particles (Z-average) in H_2_O was analyzed with Dynamic Light Scattering (DLS) using an Amerigo Particle Size & Zeta Potential Analyzer (Cordouan Technologies, Pessac-Bordeaux, France).

C. elegans Toxicity Assays

N2 wild-type strain was obtained from the Caenorhabditis Genetics Center (CGC) and maintained at 20 °C on 8P plates seeded with Escherichia coli (E. coli) NA22 under optimal conditions (20 °C). To obtain a synchronized population, gravid adult worms were treated with a bleaching solution (1% NaOCl and 0.5 M NaOH) to allow the release of eggs and let them hatch overnight in M9 buffer (3 g KH_2_PO_4_, 6 g Na_2_HPO_4_, 5 g NaCl and 1 ml of 1 M MgSO_4_ per liter of H_2_O) until they reach the L1 larval stage.

Exposure Design

All experiments were conducted on 10 cm NGM plates containing 5000 synchronized L1-stage C. elegans and 1 mL of heat-inactivated E. coli OP50 (OD_600_ = 1) as food source. Each plate was prepared with 25 mL of NGM, and the bacterial suspension was spread evenly on the surface and allowed to dry for approximately 2 h. Two concentrations of Bio-AgNP (5 and 10 mg/L) were tested based on concentrations that could be found in the environment (Yang et al. 2024), with EPA water serving as the control. Bio-AgNP were mixed with the inactivated E. coli, and the mixture volume was adjusted to maintain a final bacterial volume of 1 mL per plate. After the bacterial layer had dried, 3 mL of EPA water (60 mg CaSO_4_·2 H_2_O, 60 mg MgSO_4_, 4 mg KCl and 96 mg NaHCO_3_ per liter of H_2_O) was added to form a thin liquid layer on the surface of the plates forming a medium known as “biofilm,” which we previously demonstrated to be the most suitable for these assays (Taipe Huisa et al. 2026). Worms were exposed for 52 h at 20° C under dark conditions. All experiments were performed in triplicate.

Ag Accumulation and Distribution

For Ag accumulation, worms were transferred to 15 mL plastic tubes and washed three times with EPA water and the pellets were transferred to a 1.5 mL tube, frozen in liquid nitrogen, and stored at − 80 °C. The worm pellets were then subjected to three freeze–thaw cycles (1 min liquid nitrogen, 1 min 37 °C water bath), followed by homogenization by sonication three times for 20 s at 100% amplitude (ultrasonic processor UP100H (100 W, 30 kHz, Hielscher, Germany) in an ice bath. After, the homogenates were centrifugated at 18000 × g for 5 min at 4 °C, an aliquot of each supernatant was obtained for protein measurement. For Ag measurements, worm pellets were dried at 95 °C and digested overnight with a 50:50 mixture of 65% HNO_3_ (Suprapur, VWR, Germany) and 30% hydrogen peroxide (Sigma-Aldrich) at 95 °C. The ashes were diluted in 2% HNO_3_ (1:3) and measured using an inductively coupled plasma-optical emission spectrometer (ICP-OES; Perkin Elmer Avio 220 Max, Germany). The total amount of Ag was normalized to the protein content, which was determined using a bicinchoninic acid assay with bovine serum albumin (Sigma Aldrich) as a standard. ICP measurements were validated using Ag-spiked certified reference material BCR (single cell-protein, Institute for Reference Materials and Measurement of the European Commission, Geel, Belgium). The determination of the limit of detection (LOD) and limit of quantification (LOQ) was obtained according to guideline “ICH guideline on validation of analytical procedures”: LOD = 0.05 µg/L and LOQ = 0.17 µg/L.

For Ag distribution, worms were transferred to 15 mL Falcon tubes and washed three times with EPA water and a final washing step using MilliQ water. Approximately 30 worms were carefully transferred individually to a microscope slide and allowed to dry at room temperature. First, bright field images of dried worms were obtained for localization. Elemental maps were generated via laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS), using the LA system ImageBio266 (Elemental Scientifc Lasers, Bozeman, MT, USA) coupled to an iCap TQ ICP-MS (Thermo Fisher Scientific, Bremen, Germany). Worm samples were ablated at a laser spot size of 5 μm and a laser scan speed of 20 μm/s. The ICP-MS was operated in oxygen mode. Phosphorus and silver were recorded as analytes at 31P16O^+^ and 107Ag^+^, respectively. Data evaluation and visualization of the elemental maps was performed using the in-house made software Imajar (by Robin Schmid, University of Muenster).

GSH and GSSG Quantification

After exposure, worms were transferred to 15 ml plastic tubes, washed three times with EPA water and pelletized in 1.5 mL tubes, frozen in liquid nitrogen, and stored at − 80 °C. Worm pellets were resuspended in 150 µL cold extraction buffer (16 mM KH_2_PO_4_, 84 mM K_2_HPO_4_, 8.8 mM EDTA, 2 mM NEM, 1% Triton X-100, 0.6% SSA) and samples were subjected to three freeze–thaw cycles (1 min liquid nitrogen, 1 min 37 °C water bath) followed by homogenizing four times for 20 s using a Bead Ruptor. Following centrifugation (18620 × g, 4 °C, 10 min) the supernatant was filtered using Spin-X^®^ centrifuge tube filters (0.22 µM; Corning) and centrifuged at 18,620 × g at 4 °C for 5 min. An aliquot was stored at − 20 °C for protein quantification. Quantification of GSH-NEM and GSSG was performed using an Agilent 1290 Infinity II LC System coupled to a Sciex QTrap 6500 + triple quadrupole mass spectrometer with an electrospray ion source in the positive ion mode (Thiel et al. 2023). Results were normalized to the protein content and expressed as a percentage of the control.

Statistical Analysis

All results are expressed as the mean and standard error. The results obtained were analyzed using one-way ANOVA and the Newman-Keuls post-hoc test was performed to compare the means of the different groups. Previously, the normality and homogeneity of variance were verified. In all cases, the significance level (α) was set at 0.05.

Results and Discussion

Bio-AgNP Accumulation and Distribution in C. elegans

The obtained Bio-AgNP showed a characteristic surface resonance plasmon (SPR) at 440 nm (Supplementary Material, Fig. 1a), an average particle size of 21.4 ± 9.2 nm by TEM (Supplementary Material, Fig. 1b), and a hydrodynamic diameter of 318.19 nm by DLS, similar to that obtained in our previous study (SRP: 432 nm and TEM size: 19.4 ± 7.0 nm) (Taipe Huisa et al. 2024) under the same synthesis process. The results indicate that C. elegans can accumulate silver (Ag) at both tested concentrations after 52 h of exposure without causing lethality, with a significantly higher amount of Ag accumulated in worms exposed to 10 mg/L (17.9 ± 1.8 pg Ag/µg protein) compared to 5 mg/L (3.6 ± 0.5 pg Ag/µg protein). Additionally, LA-ICP-MS analysis revealed that Ag was distributed throughout almost the entire body of the worms, with particularly high accumulation in the alimentary system (foregut and midgut), likely due to the buccal cavity being the main route of exposure to Bio-AgNP (Fig. 1). Similar findings have been reported in studies using chemically synthesized citrate-coated AgNP, where C. elegans exposed to 5–50 mg/L accumulated nanoparticles primarily in the gut (Meyer et al. 2010; Yang et al. 2014). However, to the best of our knowledge, this is the first study to demonstrate the accumulation of green-synthesized AgNP.

Fig. 1. Ag distribution in control worms (a–c) and after exposure to 5 mg/L bio-AgNP (d–f). Bright-field microscopy image of the worms used for Ag distribution analysis (a,** d**). Elemental maps as obtained via LA-ICP-MS analysis of worm samples for phosphorus (b,** e**) and silver (c,** f**)

Effect of Bio-AgNP on GSSG and GSH Levels in C. elegans

It is well established that exposure to AgNP can induce oxidative stress by increasing intracellular levels of reactive oxygen species (ROS) which can be reflected by a decrease in GSH and an increase in GSSG levels (Flores-López et al. 2019; Opris et al. 2021). Therefore, in the present study, we evaluated GSH and GSSG levels as biomarkers of one the redox nodes that signal alterations which can be associated to oxidative stress (Jones 2002). Regarding the biochemical effects of Bio-AgNP, we observed that, although the worms accumulated silver, neither GSH (Fig. 2A) nor GSSG (Fig. 2B) levels were significantly altered at either concentration after 52 h of exposure. Previous studies have only investigated chemically synthesized AgNP. For instance, Niu et al. (2023) observed a significant reduction in GSH levels following exposure to 10 mg/L of PVP-coated AgNP, suggesting GSH depletion as a result of oxidative damage. Although comparable concentration was used in the present study, the findings clearly differ from ours, which may be attributed to the use of chemically synthesized AgNP stabilized with PVP in their experimental approach.

Fig. 2GSH (a) and GSSG (b) levels in C. elegans exposed to 5 mg/L and 10 mg/L Bio-AgNP. The results are expressed as a percentage of the control. Different lowercase letters indicate significant differences between the treatments (p < 0.05)

Although Ag accumulation in C. elegans increased with exposure, no significant changes in GSH or GSSG levels were observed, indicating that 5 and 10 mg/L did not disrupt this redox node. These results suggest that the GSH/GSSG node of worms is not affected by Bio-AgNP exposure. In contrast, PVP-coated AgNP depleted GSH at 10 mg/L (Niu et al. 2023), suggesting greater toxicity of chemically synthesized particles or that green-synthesized AgNP do not appear to target the GSH system. Previously, we showed that Bio-AgNP with açaí (10 µg/L) did not increase ROS production, contrary to what observed with chemically synthesized PVP-coated AgNP which did increase ROS production in C. elegans (Taipe Huisa et al. 2024). This supports the hypothesis that natural compounds involved in green synthesis, such as polyphenols from açaí, reduce toxicity by replacing conventional reagents like citrate, which can harm C. elegans (Josende et al. 2019). Moreover, these results showed that green-synthesized AgNP produced from natural plant sources, such acaí, may contribute to a reduced environmental impact compared with conventionally synthesized AgNP, as also observed for other plant-mediated metallic nanomaterials, such as ZnO nanoparticles (Zongur and Er Zeybekler 2024), thereby reinforcing their potential environmental friendliness. Finally, this is the first report of uptake and distribution of green-synthesized AgNP in C. elegans, confirming that their low toxicity is not due to poor bioavailability.

Initial predicted environmental concentrations of AgNP were in the µg/L range (Gottschalk et al. 2013), but these estimates usually assume homogeneous distribution. Local point sources such as sludge or sewage effluents can promote binding to organic matter, leading to sediment accumulation and greater exposure for soil organisms like C. elegans (Ellegaard-Jensen et al. 2012). With AgNP production now exceeding 600 tons annually, environmental concentrations are expected to rise, as reflected in recent reports of levels reaching ≥ 10 mg/L (Yang et al. 2024) justifying the concentrations used in this study.

Overall, this study demonstrated that AgNP synthesized using açaí extract can be taken up, accumulated and distributed in C. elegans without altering glutathione redox node. These findings highlight that Bio-AgNP are a valuable contribution to current green synthesis strategies and support their potential as a more sustainable approach for AgNP production.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1.