Metabolomics reveals the mechanism of persistent toxicity of AgNPs at environmentally relevant concentrations to Daphnia magna

Abstract

Although the ecotoxicity of silver nanoparticles (AgNPs) has been of great concern, the persistence and underlying mechanisms of AgNP toxicity remain understudied. This study explored the persistent mechanisms of AgNP toxicity at two sizes (AgNP-10 nm and AgNP-70 nm at 2 μg L⁻¹) to Daphnia magna using traditional toxicological methods alongside metabolomics analyses during exposure and recovery phases. After 24 h, both AgNP-10 and -70 nm exposures resulted in high silver accumulation levels in D. magna, leading to reduced heart rate and paddling frequency. Despite a significant decrease in silver content after 24 h of recovery, the heart rate reduction persisted in AgNP-exposed D. magna. Metabolomics analysis revealed differential expression of 53 and 54 metabolites induced by AgNP-10 and -70 nm exposures, respectively, primarily enriched in lipid metabolism pathways. Following the recovery period, AgNP-10 and -70 nm induced differential expression of 71 and 110 metabolites, respectively, mainly enriched in lipid metabolism and protein digestion and uptake pathways. These findings indicate that the persistence of toxicity of D. magna induced by AgNPs at physiological and metabolomic levels, predominantly attributed to silver retention and damage to D. magna's digestive system. Overall, this study provides novel insights into the mechanism underlying the persistence of AgNP toxicity to aquatic organisms.

---

Environmental significance

The ecotoxicity of silver nanoparticles (AgNPs) has raised concerns due to their potential release into the environment. The persistence of nanotoxicity refers to the toxic response of organisms that persists for a long time after acute exposure to nanomaterials. Although there have been numerous reports on the toxic effects of AgNPs on aquatic organisms, the persistence mechanisms of AgNP toxicity have been widely neglected. Our results showed that AgNPs with different sizes can induce persistence of toxicity to D. magna at the physiological and metabolomic levels, and that this persistence is mainly attributable to silver retention and digestive system damage in D. magna. Our findings provided significant implications in better understanding the mechanism of persistence of AgNP toxicity to aquatic organisms.

1 Introduction

The widespread use of nanomaterials results in their inevitable release into the environment, raising major concerns regarding their toxicity and ecological risks.¹ Despite extensive evidence demonstrating the toxicity of nanomaterials,^2–4 studies on the persistence of such toxicity remain limited. Specifically, the persistence of nanomaterial toxicity refers to the fact that the toxic response of organisms persists long after acute exposure to nanomaterials.⁵ Even after pollutants dissipate from the environment, nanomaterial-induced toxicity can persist for a long time.⁶ Certain nanomaterials, such as copper oxide nanoparticles, graphene oxide quantum dots, and monolayer molybdenum disulfide, exhibit the persistence of toxicity to organisms.^7–9 In contrast, other nanomaterials, such as nano-carbon black-nickel and titanium dioxide nanoparticles, lack the persistence of toxicity,^10,11 complicating comprehensive assessments of their environmental risks. Therefore, investigating the persistence of nanomaterials' toxicity is crucial for advancing our understanding of their environmental risks.

Silver nanoparticles (AgNPs) are extensively used in cosmetics, textiles, and pharmaceuticals owing to their unique antibacterial properties,¹² leading to their inevitable release into aquatic environments. Environmental concentrations of AgNPs in surface waters have been measured at 0.3–3200 ng L⁻¹,¹³ raising concerns regarding their aqueous ecotoxicity. Previous studies have documented the diverse adverse effects of AgNPs on aquatic organisms, including reproductive immunotoxicity,¹⁴ organismal malformations, histopathological changes,^15,16 loss of tissue membrane integrity,¹⁷ and differential expression of various molecules.^18,19 Despite these findings, research on the persistence of AgNP toxicity remains limited.^20,21 Prior studies revealed persistent AgNP-induced lipid metabolism disorders in fish gills following 7 days of recovery,²¹ as well as persistent effects of AgNPs on amino acid metabolism and protein synthesis in Chlorella vulgaris after 3 days of recovery.⁵ Although some understanding exists regarding the persistence of AgNP toxicity to aquatic organisms, the mechanisms underlying this persistence remain poorly understood.

The water flea Daphnia magna is widely employed in ecotoxicological studies of nanomaterials owing to the species' small size, short growth cycle, transparent body, and sensitivity to pollutants.¹⁸ Exposure to AgNPs has been shown to induce various toxic effects on D. magna, including oxidative stress,²² growth disruption, reduced fecundity,^23,24 and decreased gut microbial diversity, leading to differential expression of genes and proteins.^25,26 These toxic effects depend on the size, solubility, and surface coating of AgNPs,^27–29 with size playing a pivotal role in their aquatic toxicity.³⁰ Typically, smaller AgNPs (40 nm at 2 μg L⁻¹) induce greater toxicity in D. magna compared with larger AgNPs (110 nm at 2 μg L⁻¹).¹⁸ However, some studies showed that larger AgNPs (20 μm at 300 μg L⁻¹) can induce greater toxicity in D. magna compared with smaller AgNPs (10 μm at 300 μg L⁻¹).^27,31 These conflicting findings emphasize the complexity of AgNPs' size-dependent toxicity in D. magna, necessitating further extensive and long-term research to elucidate the underlying mechanisms. Despite widespread reports regarding the toxic effects of AgNPs on D. magna, studies on the persistence of AgNPs' toxicity across particle sizes and the underlying mechanisms remain scarce. To the best of our knowledge, only one study has reported persistent AgNP-induced growth and reproductive toxicity across multiple generations of D. magna.³²

Metabolomics has emerged as a powerful tool for identifying, quantifying, and functionally assessing metabolites in D. magna given its high sensitivity and throughput.³³ Moreover, it offers new perspectives for investigating the persistent mechanisms of AgNPs' toxicity in D. magna across various particle sizes. Therefore, this study employed metabolomics techniques to explore the persistence of toxicity of two sizes of AgNPs (AgNP-10 and AgNP-70 nm) on D. magna comparing their effects during the exposure and recovery phases. The primary aim of using these methods, in conjunction with traditional toxicology data, was to elucidate the persistent mechanism of AgNP toxicity in D. magna. The findings of this study contribute valuable insights into the persistence mechanisms of AgNP toxicity in aquatic organisms and provide a foundational basis for assessing the environmental risks of these nanoparticles.

2 Materials and methods

2.1 AgNPs

In this study, citrate-coated AgNPs with theoretical particle sizes of 10 and 70 nm were obtained from Nanjing Dongna Science and Technology Co. Ltd., Nanjing, China. The AgNPs' morphology and size were characterized using transmission electron microscopy (JIM-2100, Japan), and particle sizes were determined from captured images using Nano Measurer 1.2 software. AgNPs' absorption spectra were analyzed using ultraviolet-visible spectrophotometry (Jenway-6850, USA). The hydrated particle size of AgNPs was measured using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) after exposure to both pure water and medium for 24 h.

2.2 Experimental organisms

Daphnia magna was purchased from Guangdong Experimental Animal Monitoring Institute, China, and incubated in a constant-temperature incubator at 24 °C ± 1 °C, under a 14 h : 10 h light : dark cycle with an optical density of 120 μmol photons m⁻² s⁻¹. The medium composition followed the Organization for Economic Co-operation and Development (2004) guidelines, containing calcium chloride (29.4 mg L⁻¹), magnesium sulphate (12.3 mg L⁻¹), sodium bicarbonate (6.25 mg L⁻¹), and potassium chloride (6.25 mg L⁻¹). D. magna was fed daily with Scenedesmus obliquus or Chlorella vulgaris at concentrations of 10⁵ cell per mL, and the medium was completely replaced every 2 days.

2.3 Exposure and recovery experimental design

The experiment included two phases: exposure and recovery. These were designed to assess the persistent toxic effects of AgNPs on D. magna. The experimental flow is illustrated in Fig. 1. During the exposure phase, five groups were established: blank control (control), 10 nm AgNPs (AgNP-10 nm), 70 nm AgNPs (AgNP-70 nm), Ctrl-10 nm, and Ctrl-70 nm groups. The Ctrl-10 and -70 nm groups were silver ion supernatant control groups, employed to investigate the source of AgNPs' toxicity. Each group consisted of 13 replicates, with 20 D. magna individuals per replicate. The exposure concentration of AgNPs was 2 μg L⁻¹, selected as one-tenth of the average 50% lethal concentration (LC₅₀) values of AgNP-10 and -70 nm (Fig. S1†). The Ctrl-10 nm group comprised the silver ion supernatant obtained after centrifugation (at 4 °C and 12 000 rpm for 150 min) of 2 μg L⁻¹ AgNP-10 nm, whereas the Ctrl-70 nm group included the silver ion supernatant after centrifugation (identical conditions) of 2 μg L⁻¹ AgNP-70 nm. After 24 h of exposure, 180 D. magna individuals in each treatment group were collected for analyses of AgNP accumulation, Daphnia heart rate and paddling frequency, and metabolomics, whereas the remaining individuals were transferred to clean medium for recovery testing. D. magna was not fed during the recovery phase. After 24 h of recovery, D. magna was collected and analyzed as per the exposure phase.

Fig. 1 Flow diagram of the exposure and recovery experiment. The Ctrl-10 nm group was the silver ion supernatant after centrifugation of AgNP-10 nm, and the Ctrl-70 nm group was the silver ion supernatant after centrifugation of AgNP-70 nm.

2.4 Measurement of silver accumulation in D. magna and silver ion release

To determine the accumulation of total silver in D. magna, a method modified from that established by Zhao and Wang³⁴ was employed. Twenty D. magna from the exposure or recovery phases were rinsed with clean medium for 1–2 min, dried overnight in an oven at 60 °C, and placed in ablation tubes. A mixture of 8 mL of nitric acid and 2 mL of concentrated hydrochloric acid was added for ablation, after which the silver content in the digested samples was quantified using inductively coupled plasma mass spectrometry (ICP-MS).

The release of silver ions was assessed following the method of Verano-Braga et al.³⁵ Briefly, 20 mL of AgNP suspension (10 or 70 nm AgNPs at 2 μg L⁻¹) was incubated under experimental conditions for 24 h with three replicates per group. After 24 h of exposure, 10 mL of each AgNP suspension was centrifuged to separate the silver ion supernatant. The silver content in the supernatant was measured directly using ICP-MS. Additionally, the total silver concentration in the remaining 10 mL of AgNP solution was determined via microwave digestion followed by ICP-MS. The silver ion release rate was calculated as the concentration of released silver ions divided by the total silver concentration in the AgNP exposure medium.

2.5 Determination of D. magna heart rate and paddling frequency

The effect of AgNPs on the heart rate and paddling frequency of D. magna was assessed following the method of Wan et al.³⁶ Sixteen D. magna in each experimental group were video-recorded using a digital camera (Sony FDR-AX30, Japan) with a spatial resolution of 3840 × 2160 pixels for 5 min to determine the paddling frequency. The heart rate was observed under a microscope and recorded for 1 min. The heart rate and paddling frequency were calculated using Potplayer software (V1.7.21223; https://potplayer.tv/).

2.6 Metabolomics analysis of D. magna

After the exposure or recovery period, 20 D. magna were collected, transferred to 1.5 mL centrifuge tubes, snap-frozen in liquid nitrogen, and stored at −80 °C. Sample pretreatment followed modified protocols based on those of Wang et al.³³ Freeze-dried samples were weighed and transferred to 2 mL Eppendorf tubes, followed by the addition of 200 μL of a water : methanol (v : v = 1 : 4) mixture. Samples were then ground at −10 °C and 50 Hz for 6 min using a frozen tissue milling instrument (Wanbao Biotechnology, Shanghai, China) followed by extraction via cryo-sonication at 5 °C and 40 kHz for 30 min. After standing at −20 °C for 30 min, samples were centrifuged at 4 °C and 14 000 rpm for 15 min, and 20 μL of supernatant was then transferred to a microvial for liquid chromatography-mass spectrometry analysis using a Thermo Fisher ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry-Q Exactive system. Metabolites were separated on a BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm; Waters, Milford, USA) at 40 °C with an injection volume of 10 μL. Mobile phase A comprised ultrapure water containing 0.1% formic acid, whereas mobile phase B was an acetonitrile : isopropanol (1 : 1) mixture containing 0.1% formic acid. The mobile phase flow rate was 0.4 mL min⁻¹. Electrospray ionization in positive and negative modes was used to obtain metabolite mass spectra, with a capillary voltage of 1 kV, an injection voltage of 40 V, and a collision voltage of 6 eV.

2.7 Data analysis

Metabolomics data analysis was performed using the free online Majorbio Cloud Platform (https://cloud.majorbio.com; Shanghai Majorbio Bio-pharm Technology Co., Ltd.). Non-metabolomics data were presented as means ± standard deviations. One-way ANOVA and the independent samples t-test were used to analyze heart rate and paddling frequency data via SPSS 21.0 (SPSS Inc., Chicago, IL, USA).

3 Results

3.1 Characterization of AgNPs

Characterization results revealed that the average particle sizes of AgNP-10 and -70 nm were 9.95 ± 2.33 and 65.84 ± 7.25 nm, respectively (Fig. 2a–d). The absorption peaks of AgNP-10 and -70 nm were observed at 390 and 445 nm, respectively (Fig. 2e). No peak was detected in the silver ion supernatant, indicating the absence of silver particles (Fig. 2e). Following 24 h of exposure, the average hydrated particle sizes of AgNP-10 and -70 nm in pure water were 16.53 ± 0.41 and 75.60 ± 1.14 nm, respectively, whereas in culture medium, they measured 219.47 ± 34.37 and 374.98 ± 72.17 nm, respectively (Fig. 2f).

Fig. 2 Characterization of AgNPs. (a and b) Transmission electron microscopy of AgNP-10 nm and AgNP-70 nm. (c and d) Particle size distribution of AgNP-10 nm and AgNP-70 nm. (e) Ultraviolet spectra of silver nanoparticles and Ag⁺ supernatant. (f) Hydrate particle size of AgNPs in pure water and culture medium.

3.2 Acute toxicity effects of AgNPs on D. magna

Acute toxicity results (Fig. S1†) indicated that D. magna mortality increased with rising concentrations of AgNPs and silver ion supernatant treatments. After 24 h of exposure to these treatments, the LC₅₀ values induced by AgNP-10 nm, AgNP-70 nm, Ctrl-10 nm, and Ctrl-70 nm were 13.4, 25.5, 31.4, and 71.5 μg-Ag L⁻¹, respectively. Acute toxicity potency ranked from highest to lowest was AgNP-10 nm > AgNP-70 nm > Ctrl-10 nm > Ctrl-70 nm. The histopathological results indicated that compared to the control group, exposure to AgNPs (10 and 70 nm) induced various types of damage to the intestinal epithelial tissue of D. magna, mainly including cell edema, cell vacuolization, and gap expansion between the cytoplasm and basement membrane (Fig. S5†).

3.3 AgNP accumulation in D. magna and silver ion release rate

Exposure to both sizes of AgNPs induced an obvious accumulation of total silver content in D. magna, which decreased after the recovery period (Fig. 3a). Compared with the control group (0.49 ± 0.15 μg g⁻¹), exposure to AgNP-10 and -70 nm led to high levels of total silver in D. magna at 16.34 ± 1.59 and 26.41 ± 9.93 μg g⁻¹, respectively; following recovery, the total silver content decreased to 4.98 ± 1.91 and 9.14 ± 2.34 μg g⁻¹, respectively. Ctrl-10 and −70 nm exposures resulted in total silver accumulation of 3.91 ± 1.71 and 4.52 ± 2.23 μg g⁻¹ in D. magna, respectively, which decreased to 3.75 ± 2.41 and 3.03 ± 0.72 μg g⁻¹ after recovery (Fig. 3a). The released silver ions indicated dissolution from AgNP-10 and -70 nm exposures, with release rates of 16.79% ± 2.40% and 18.29% ± 2.91% (Fig. 3b), respectively.

Fig. 3 Accumulation characteristics of AgNPs in D. magna. (a) Accumulation of silver in D. magna after exposure and recovery of AgNPs with different sizes. (b) Dissolution characteristics of two sizes of AgNPs in the exposure medium.

3.4 Effects of AgNPs on the heart rate and paddling frequency of D. magna

Exposure to both sizes of AgNPs reduced the heart rate and paddling frequency of D. magna (Fig. 4). Notably, the reduction in heart rate persisted after the recovery period (Fig. 4a and b). During exposure, AgNP-10 nm inhibited the heart rate of D. magna more prominently than AgNP-70 nm; however, the difference between the two sizes was not significant in terms of D. magna paddling frequency (Fig. 4a and b). Additionally, different silver supernatant concentrations (Ctrl-10 and -70 nm) did not significantly alter the D. magna heart rate and paddling frequency during the exposure and recovery phases (Fig. 4a and b).

Fig. 4 Heartbeat rate and hopping of D. magna after exposure and recovery of AgNPs with different sizes. (a) Heartbeat rate. (b) Hopping frequency.

3.5 Metabolome analysis of D. magna during AgNP exposure and recovery phases

Principal component analysis (Fig. S2†) showed that during the exposure phase, the sum of different components between treatment groups in cation/anion mode reached 53.1% and 55.5%, respectively (Fig. S2a and d†); in the recovery phase, these sums reached 49.4% and 49.4%, respectively (Fig. S2c and d†). These results indicated robust differentiation between different treatment groups. Wayne diagram results (Fig. 5a and b) revealed that both sizes of AgNPs caused differential expression of numerous metabolites in D. magna during the exposure and recovery phases. During exposure, AgNP-10 and -70 nm induced differential expression of 53 and 54 metabolites, respectively (Fig. 5a); during recovery, these groups induced differential expression of 71 and 110 metabolites, respectively (Fig. 5b). Additionally, Ctrl-10 and -70 nm exposures induced differential expression of 121 and 220 metabolites during exposure and 138 and 201 metabolites during recovery in D. magna, respectively (Fig. 5a and b).

Fig. 5 Differential metabolites in D. magna after exposure and recovery of AgNPs with different sizes. (a) Venn diagram in the exposure stage; (b) Venn diagram in the recovery stage; (c) Heatmap in exposure stage and recovery stages.

The clustering heat map (Fig. 5c) showed that during exposure, AgNP-10 nm primarily caused upregulation of isoquinoline, L-isoleucine, and CL(10 : 0/10 : 0/10 : 0/11 : 0) as well as downregulation of lysoPE(16 : 1/(9z)/0 : 0) and dihydroactinidiolide. AgNP-70 nm exposure mainly led to upregulation of isoquinoline, 3-formyl-6-hydroxyindole, and N-methyl-1-deoxynojirimycin along with downregulation of lysoPE(16 : 1/(9z)/0 : 0), dihydroactinidiolide, and pelargonidin 3-sophoroside. During recovery, both AgNP-10 and -70 nm primarily caused upregulation of L-carnitine and stearoylethanolamide as well as downregulation of isoquinoline, L-isoleucine, lysoPE(16 : 1/(9z)/0 : 0), and DG(14 : 1(9Z)/18 : 3(9Z, 12Z,15Z)/0 : 0). Furthermore, during the exposure phase, Ctrl-10 and -70 nm mainly induced upregulation of DG(14 : 1(9Z)/18 : 3(9Z, 12Z,15Z)/0 : 0), N-acetyl-D-glucosamine, lysoPC(15 : 0), and lysoPE(0 : 0/20 : 1(11Z)) along with downregulation of americine, 2-phenylacetamide, and PC(16 : 0/16 : 1(9Z)). During recovery, Ctrl-10 and -70 nm primarily induced upregulation of L-isoleucine, benzaldehyde, 3′-O-methylguanosine, and guanine alongside downregulation of 5′-CMP and DG (14 : 1(9Z)/18 : 3(9Z, 12Z,15Z)/0 : 0) (Fig. 5c).

3.6 Effects of AgNPs on metabolic pathways in D. magna

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification (Fig. S3†) revealed that during the exposure phase, differential metabolites induced by AgNP-10 and -70 nm primarily affected pathways involved in lipid metabolism, membrane transport, nervous system function, and cancer processes in D. magna (Fig. S3a and b†). In the recovery phase, differential metabolites induced by both sizes of AgNPs were mainly associated with the nervous system, the digestive system, lipid metabolism, amino acid metabolism, membrane transport, and cancer processes in D. magna (Fig. S4a and b†). Furthermore, Ctrl-10 and -70 nm exposures induced metabolites predominantly involved in amino acid metabolism, lipid metabolism, and cancer processes in D. magna (Fig. S3c and d†), whereas during the recovery phase, these treatments induced metabolites related to the nervous system, the digestive system, lipid metabolism, amino acid metabolism, and membrane transporter processes (Fig. S4c and d†).

KEGG enrichment pathway analyses (Fig. 6 and 7) showed that during the exposure phase, relative to silver supernatant treatments, both sizes of AgNPs induced fewer significantly enriched pathways associated with differentiated metabolites in D. magna. Interestingly, certain metabolic pathways significantly enriched in the AgNP groups were not significantly enriched in the silver supernatant groups, e.g., arachidonic acid metabolism, alpha-linolenic acid metabolism, and linoleic acid metabolism (Fig. 6 and Table S1†). Conversely, pathways significantly enriched in the silver ion supernatant groups mainly included choline metabolism in cancer, glycerophospholipid metabolism, and ABC transporters. During the recovery phase, the number of significantly enriched pathways in the AgNP-treated groups was significantly increased compared with that during the exposure phase. Furthermore, differential metabolites induced by both sizes of AgNPs were significantly enriched in pathways such as protein digestion and absorption, mineral absorption, tryptophan metabolism, and sphingolipid signaling. Differential metabolites affected by silver ion supernatants were significantly enriched in pathways related to ABC transporter proteins, protein digestion and uptake, mineral uptake, and aminoacyl tRNA biosynthesis (Fig. 7c and d).

Fig. 6 KEGG enrichment pathways analysis of differential metabolites in D. magna during the exposure stage. (a) AgNP-10 nm. (b) AgNP-70 nm. (c) Ctrl-10 nm. (d) Ctrl-70 nm. The column in the figure represents the enrichment pathway; the color indicates the significance of the enrichment pathway, which is represented by its p-value; a darker-green pillar indicates a more significant enrichment of the metabolic pathway. p < 0.001 is marked as ***, p < 0.01 is marked as **, and p < 0.05 is marked as *.

Fig. 7 KEGG enrichment pathways analysis of differential metabolites in D. magna after the recovery stage. (a) RAgNP-10 nm. (b) RAgNP-70 nm. (c) RCtrl-10 nm. (d) RCtrl-70 nm. The column in the figure represents the enrichment pathway; the color indicates the significance of the enrichment pathway, which is represented by its p-value; a darker-green pillar indicates a more significant enrichment of the metabolic pathway. p < 0.001 is marked as ***, p < 0.01 is marked as **, and p < 0.05 is marked as *.

4 Discussion

AgNP-induced toxicity in D. magna persists at physiological and metabolomic levels. A previous study revealed that nanosilver induces persistent growth and reproductive toxicity in D. magna across multiple generations.³² In the present study, 24 h of AgNP-10 and -70 nm exposure reduced the heart rate of D. magna and induced differential expression of around 50–55 metabolites. Notably, after 24 h of recovery, the reduction in D. magna heart rate induced by AgNP-10 and -70 nm persisted, and the number of differentially affected metabolites increased to 70–120. Thus, AgNPs with various particle sizes induce persistent physiological and metabolomic toxicity in D. magna, which may be attributed to AgNP retention. Indeed, it is well-established that AgNP accumulation in D. magna leads to organismal toxicity.³⁴ Studies have demonstrated that D. magna cannot completely eliminate nanomaterials (e.g., gold nanoparticles, titanium dioxide nanoparticles, and fullerene-C60) from their bodies after 24 h of decontamination.^8,37,38 Similarly, in the present study, AgNP-10 and -70 nm were not completely eliminated by D. magna after 24 h of decontamination, suggesting that the retained silver continues to induce toxic effects in the organism during the recovery phase.

This study is the first to use metabolomics to elucidate the mechanism of AgNP toxicity during the recovery phase in D. magna. Numerous differentially expressed gut-related metabolites, such as L-carnitine, isoquinoline, and L-isoleucine, induced by both AgNP-10 and -70 nm in D. magna were significantly enriched in pathways related to protein digestion and absorption during recovery. Similarly, previous studies have shown that multiple differentially expressed genes induced by both AgNP-40 and -110 nm in D. magna were significantly enriched in protein digestion and absorption pathways.¹⁸ Collectively, these findings suggest that AgNPs interfere with D. magna's digestive function. The intestine serves as the primary site for protein digestion and absorption in most organisms³⁹ and is the main tissue in which AgNPs accumulate.⁴⁰ In D. magna, AgNP accumulation in the intestine likely disrupts the intestinal microenvironment, damages the integrity of intestinal epithelial tissues, and subsequently disrupts metabolic processes related to amino acids as well as protein digestion and absorption, ultimately compromising digestive function.^18,33

The effect of AgNP particle size on toxicity toward D. magna is markedly concentration-dependent. A previous study showed that as AgNP size decreases, biological toxicity increases.⁴¹ In the present study, AgNP-10 nm induced greater toxicity in D. magna compared with AgNP-70 nm under high-concentration (15 μg L⁻¹) exposure conditions, as shown through acute toxicity (e.g., LC₅₀ values) and heart rate changes. Similarly, smaller AgNPs were found to be more toxic to the gills and intestines of zebrafish compared with larger AgNPs.^42,43 However, under low-concentration (2 μg L⁻¹) exposure conditions, no significant difference in AgNP-10 nm and AgNP-70 nm induced toxicity was observed, including changes in paddling frequency and the number of differential metabolites in D. magna. Similarly, the effect of particle size on metabolite changes in D. magna was not significant under low-concentration (1 mg L⁻¹) exposure to titanium dioxide nanoparticles.⁴⁴ This phenomenon is likely due to the strong tolerance of D. magna to short-term low-concentration exposures, resulting in nonsignificant differences in toxicity among nanomaterials with different particle sizes.

The toxic effects of AgNPs on D. magna result from the combined effects of released particulate silver and ionic silver. A prior study demonstrated that aqueous AgNP exposure involves the release of silver ions,⁴⁵ consistent with findings in the present study. Collectively, these results imply that AgNP toxicity is associated with both silver particles and ionic silver. The current study revealed marked differences in the effects of AgNPs versus silver ion supernatants on the mortality, heart rate, and paddling frequency of D. magna. A previous study found that AgNPs induced greater toxicity in D. magna compared with silver ions.⁴⁶ These findings highlight the crucial role of silver particles in AgNP toxicity toward D. magna. Silver particles interfere with normal behaviors, such as locomotion and water filtration, by attaching to D. magna's appendages or damaging its body structures, thereby increasing mortality rates. Interestingly, metabolomics analysis indicated that silver ion supernatant treatments exerted a more pronounced effect on the number of differential metabolites in D. magna compared with AgNP treatments. This suggests that the effect of AgNPs on D. magna metabolism primarily originates from released silver ions.⁴⁷ Similarly, silver ion supernatant exposure has been shown to stimulate more pronounced differential protein expression in C. vulgaris compared with AgNP exposure.¹⁹ This phenomenon may be attributed to silver particles mitigating the physiological changes induced by soluble silver.⁴⁸ Specifically, adsorption of AgNPs onto cell surfaces may lead to interactions with ion channel proteins, forming a nanoparticle–protein corona that competes for binding sites with silver ions, thereby reducing their toxicity.^49,50

AgNPs and their released silver ions interfere with components of the D. magna cell membrane through distinct pathways. AgNPs primarily damage the D. magna cell membrane by disrupting the membrane fatty acid pathway, whereas released silver ions predominately affect the membrane protein pathway (Fig. 6 and 7). Cell membranes, composed mainly of lipids and proteins, undergo functional changes when both lipids and membrane proteins are damaged.⁴ In the present study, AgNPs specifically induced differential expression of membrane unsaturated fatty acids (e.g., arachidonic acid, α-linoleic acid, and linoleic acid; Fig. 6), suggesting that AgNPs likely damage the cell membrane of D. magna by altering its fatty acid composition. Similarly, our previous study revealed that AgNPs disrupt gill membrane fatty acid composition, causing membrane damage.¹⁷ In the current study, silver ion supernatant exposure altered the expression of membrane transporter proteins (e.g., ABC proteins) in D. magna (Fig. 6), suggesting that released silver ions from AgNPs may damage the cell membrane by disrupting membrane protein components. Other studies have shown that silver ions can damage cell membranes by affecting ABC transporter proteins.^2,51

In the present study, exposure to both sizes of AgNPs resulted in the downregulation of lysoPE metabolites in D. magna. LysoPE is a deacylation product of phosphatidylethanolamine hydrolysis induced by phospholipase A1/A2 in the lipid metabolism pathway.⁵² The expression of lysoPE is regulated mainly by the phospholipase A1/A2 gene.⁵³ We previously confirmed that AgNP exposure can inhibit the expression of the phospholipase A2 gene in the lipid metabolism pathway.²¹ Thus, we speculate that AgNP exposure may inhibit phospholipase A gene expression in the lipid metabolism pathway of D. magna, thereby hindering phosphatidylethanolamine hydrolysis and ultimately reducing lysoPE synthesis.

In the present study, the accumulation of AgNPs in the body of Daphnia during the 24 h depuration phase was observed when D. magna was not fed. Similarly, a previous study found that AgNP retention in the body during the 24 h depuration phase was as high as ∼40–70% when D. magna was not fed.⁵⁴ Interestingly, other studies demonstrated that when D. magna was fed, AgNP retention was still as high as ∼40–70%.^54,55 Therefor, our results, along with existing studies, suggest that feeding or not feeding has a relatively minor impact on the retention of AgNPs in D. magna during the short-term depuration phase.

5 Conclusions

This study provided novel insights into the persistent toxic effects of AgNPs of varying sizes on D. magna through exposure and recovery experiments. Both tested AgNP sizes (AgNP-10 and -70 nm) inhibited the heart rate of D. magna and induced differential expression of numerous metabolites during the exposure and recovery phases. Smaller AgNPs exhibited greater toxicity under high-concentration exposure conditions, whereas the particle size effect was less pronounced under low-concentration exposure conditions. Both AgNP sizes markedly disrupted lipid metabolism in D. magna, likely impairing digestive system function and potentially contributing to the persistence of toxicity observed. These findings emphasize the severe impacts of AgNPs on aquatic organisms. Future studies should further investigate the persistence of AgNP toxicity in the food chain to accurately assess its implications for aquatic ecosystems.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Yunnan University and approved by the Animal Ethics Committee of Yunnan University.

Data availability

The data of the manuscript will be available from the corresponding author upon reasonable request.

Conflicts of interest

The authors declare no conflict interest.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (22066026), the Basic Research Project of Yunnan Province (202401AS070144, 202401 AU070014), and Kunming University Talent Programs (YJL2219).

References

1. A. Hartwig and C. Van-hriel, Risk assessment of nanomaterials toxicity, Nanomaterials, 2023, 13, 1512.
2. Y. B. Pan, W. J. Zhang and S. J. Lin, Transcriptomic and microRNAomic profiling reveals molecular mechanisms to cope with silver nanoparticle exposure in the ciliate Euplotes vannus, Environ. Sci.: Nano, 2018, 5, 2921–2935.
3. Y. Zhu, X. L. Liu, Y. L. Hu, R. Wang, M. Chen, J. H. Wu, Y. Y. Wang, S. Kang, Y. Sun and M. X. Zhu, Behavior, remediation effect and toxicity of nanomaterials in water environments, Environ. Res., 2019, 174, 54–60.
4. E. K. He, R. L. Qiu, X. D. Cao, L. Song, W. Peijnenburg and H. Qiu, Elucidating toxicodynamic differences at the molecular scale between ZnO nanoparticles and ZnCl₂ in Enchytraeus crypticus via nontargeted metabolomics, Environ. Sci. Technol., 2020, 54, 3487–3498.
5. L. Shen, Q. Q. Li, Y. H. Kang, Q. Q. Xiang, X. Luo and L. Q. Chen, Metabolomics reveals size-dependent persistence and reversibility of silver nanoparticles toxicity in freshwater algae, Aquat. Toxicol., 2023, 258, 106471.
6. W. L. Kang, X. K. Li, A. Q. Sun, F. B. Yu and X. G. Hu, Study of the persistence of the phytotoxicity induced by graphene oxide quantum dots and of the specific molecular mechanisms by integrating omics and regular analyses, Environ. Sci. Technol., 2019, 53, 3791–3801.
7. B. Mansouri, A. Maleki, S. A. Johari, B. Shahmoradi, E. Mohammadi and B. Davari, Histopathological effects of copper oxide nanoparticles on the gill and intestine of common carp (Cyprinus carpio) in the presence of titanium dioxide nanoparticles, Chem. Ecol., 2017, 33, 295–308.
8. S. Novak, A. J. Kokalj, M. Hocevar, M. Godec and D. Drobne, The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: example with testing TiO₂ nanoparticles, Ecotoxicol. Environ. Saf., 2018, 152, 61–66.
9. W. Zou, X. Y. Li, C. H. Li, Y. Y. Sun, X. L. Zhang, C. X. Jin, K. Jiang, Q. X. Zhou and X. G. Hu, Influence of size and phase on the biodegradation, excretion, and phytotoxicity persistence of single-layer molybdenum disulfide, Environ. Sci. Technol., 2020, 54, 12295–12306.
10. C. L. Mieiro, M. Martins, M. da Silva, J. P. Coelho, C. B. Lopes, A. A. da Silva, J. Alves, E. Pereira, M. Pardal, M. H. Costa and M. Pacheco, Advances on assessing nanotoxicity in marine fish - the pros and cons of combining an ex vivo approach and histopathological analysis in gills, Aquat. Toxicol., 2019, 217, 105322.
11. W. He, H. Peng, J. Ma, Q. Wang, A. Li, J. Zhang, H. Kong, Q. Li, Y. Sun and Y. Zhu, Autophagy changes in lung tissues of mice at 30 days after carbon black-metal ion co-exposure, Cell Proliferation, 2020, 53, e1283.
12. A. Mackevica, M. E. Olsson and S. F. Hansen, The release of silver nanoparticles from commercial toothbrushes, J. Hazard. Mater., 2017, 322, 270–275.
13. J. Zhao, M. Lin, Z. Wang, X. Cao and B. Xing, Engineered nanomaterials in the environment: Are they safe?, Crit. Rev. Environ. Sci. Technol., 2020, 51, 1443–1478.
14. J. M. Lacave, U. Vicario-Parés, E. Bilbao, D. Gilliland, F. Mura, L. Dini, M. P. Cajaraville and A. Orbea, Waterborne exposure of adult zebrafish to silver nanoparticles and to ionic silver results in differential silver accumulation and effects at cellular and molecular levels, Sci. Total Environ., 2018, 642, 1209–1220.
15. K. K. Kheyrollah, A. Shabani, H. Paknejad and M. R. Imanpoor, Comparative toxicity of silver nanoparticle and ionic silver in juvenile common carp (Cyprinus carpio): Accumulation, physiology and histopathology, J. Hazard. Mater., 2018, 359, 373–381.
16. Q. Q. Xiang, Y. Gao, Q. Q. Li, J. Ling and L. Q. Chen, Proteomic profiling reveals the differential toxic responses of gills of common carp exposed to nanosilver and silver nitrate, J. Hazard. Mater., 2020, 394, 122562.
17. Q. Q. Xiang, D. Wang, J. L. Zhang, C. Z. Ding, X. Luo, J. Tao, J. Ling, D. Shea and L. Q. Chen, Effect of silver nanoparticles on gill membranes of common carp: Modification of fatty acid profile, lipid peroxidation and membrane fluidity, Environ. Pollut., 2019, 256, 113504.
18. J. Hou, Y. Zhou, C. J. Wang, S. G. Li and X. K. Wang, Toxic effects and molecular mechanism of different types of silver nanoparticles to the aquatic crustacean Daphnia magna, Environ. Sci. Technol., 2017, 51, 12868–12878.
19. J. L. Zhang, L. Shen, Q. Q. Xiang, J. Ling, C. H. Zhou, J. M. Hu and L. Q. Chen, Proteomics reveals surface electrical property-dependent toxic mechanisms of silver nanoparticles in Chlorella vulgaris, Environ. Pollut., 2020, 265, 114743.
20. B. Dalzon, A. Torres, H. Diemer, S. Ravanel, V. Collin-Faure, K. Pernet-Gallay, P. H. Jouneau, J. Bourguignon, S. Cianférani, M. Carrière, C. Aude-Garcia and T. Rabilloud, How reversible are the effects of silver nanoparticles on macrophages? A proteomic-instructed view, Environ. Sci.: Nano, 2019, 6, 3133–3157.
21. Q. Q. Xiang, H. Yan, X. W. Luo, Y. H. Kang, J. M. Hu and L. Q. Chen, Integration of transcriptomics and metabolomics reveals damage and recovery mechanisms of fish gills in response to nanosilver exposure, Aquat. Toxicol., 2021, 237, 105895.
22. L. Ulm, A. Krivohlavek, D. Jurasin, M. Ljubojevic, G. Sinko, T. Crnkovic, I. Zuntar, S. Sikic and I. V. Vrcek, Response of biochemical biomarkers in the aquatic crustacean Daphnia magna exposed to silver nanoparticles, Environ. Sci. Pollut. Res., 2015, 22, 19990–19999.
23. P. Wang, Q. X. Ng, H. Zhang, B. Zhang, C. N. Ong and Y. He, Metabolite changes behind faster growth and less reproduction of Daphnia similis exposed to low-dose silver nanoparticles, Ecotoxicol. Environ. Saf., 2018, 163, 266–273.
24. Y. D. Li, W. X. Wang and H. B. Liu, Gut-microbial adaptation and transformation of silver nanoparticles mediated the detoxification of Daphnia magna and their offspring, Environ. Sci.: Nano, 2022, 9, 361–374.
25. H. C. Poynton, J. M. Lazorchak, C. A. Impellitteri, B. J. Blalock, K. Rogers, H. J. Allen, A. Loguinov, J. L. Heckman and S. Govindasmawy, Toxicogenomic responses of nanotoxicity in Daphnia magna exposed to silver nitrate and coated silver nanoparticles, Environ. Sci. Technol., 2012, 46, 6288–6296.
26. Y. D. Li, N. Yan, T. Y. Wong, W. X. Wang and H. B. Liu, Interaction of antibacterial silver nanoparticles and microbiota-dependent holobionts revealed by metatranscriptomic analysis, Environ. Sci.: Nano, 2019, 6, 3242–3255.
27. R. Cui, Y. Chae and Y. J. An, Dimension-dependent toxicity of silver nanomaterials on the cladocerans Daphnia magna and Daphnia galeata, Chemosphere, 2017, 185, 205–212.
28. L. Q. Zhang and W. X. Wang, Dominant role of silver ions in silver nanoparticle toxicity to a unicellular alga: evidence from luminogen imaging, Environ. Sci. Technol., 2019, 53, 494–502.
29. Y. L. Li and W. X. Wang, Silver nanowires kinetics and real-time imaging of in situ Ag ion dissolution in Daphnia magna, Sci. Total Environ., 2021, 782, 146933.
30. O. J. Osborne, S. Lin, C. H. Chang, Z. Ji, X. Yu, X. Wang, S. Lin, T. Xia and A. E. Nel, Organ-specific and size-dependent Ag nanoparticle toxicity in gills and intestines of adult zebrafish, ACS Nano, 2015, 9, 9573–9584.
31. Y. Chae and Y. J. An, Toxicity and transfer of polyvinylpyrrolidone-coated silver nanowires in an aquatic food chain consisting of algae, water fleas, and zebrafish, Aquat. Toxicol., 2016, 173, 94–104.
32. M. L. N. da Silva, D. J. Nogueira, J. S. Köerich, V. P. Vaz, N. M. Justino, J. R. A. Schmidt, D. S. Vicentini, M. S. Matias, A. B. de Castilhos, C. F. Fuzinatto and W. G. Matias, Multigenerational toxic effects on Daphnia magna induced by silver nanoparticles and glyphosate mixture, Environ. Toxicol. Chem., 2021, 40, 1123–1131.
33. P. Wang, Q. X. Ng, H. Zhang, B. Zhang, C. N. Ong and Y. He, Metabolite changes behind faster growth and less reproduction of Daphnia similis exposed to low-dose silver nanoparticles, Ecotoxicol. Environ. Saf., 2018, 163, 266–273.
34. C. M. Zhao and W. X. Wang, Size-dependent uptake of silver nanoparticles in Daphnia magna, Environ. Sci. Technol., 2012, 46, 11345–11351.
35. T. Verano-Braga, R. Miethling-Graff, K. Wojdyla, A. Rogowska-Wrzesinska, J. R. Brewer, H. Erdmann and F. Kjeldsen, Insights into the cellular response triggered by silver nanoparticles using quantitative proteomics, ACS Nano, 2014, 8, 2161–2175.
36. L. L. Wan, Y. Y. Long, J. Hui, H. Zhang, Z. Y. Hou, J. X. Tan, Y. Pan and S. C. Sun, Effect of norfloxacin on algae-cladoceran grazer-larval damselfly food chains: algal morphology-mediated trophic cascades, Chemosphere, 2020, 256, 127166.
37. K. Tervonen, G. Waissi, E. J. Petersen, J. Akkanen and J. V. K. Kukkonen, Analysis of fullerene-C₆₀ and kinetic measurements for its accumulation and depuration in Daphnia magna, Environ. Toxicol. Chem., 2010, 29, 1072–1078.
38. L. M. Skjolding, K. Kern, R. Hjorth, N. Hartmann, S. Overgaard, G. Ma, J. G. C. Veinot and A. Baun, Uptake and depuration of gold nanoparticles in Daphnia magna, Ecotoxicology, 2014, 23, 1172–1183.
39. J. J. Powell, R. Jugdaohsingh and R. P. H. Thompson, The regulation of mineral absorption in the gastrointestinal tract, Proc. Nutr. Soc., 1999, 58, 147–153.
40. L. D. Scanlan, R. B. Reed, A. V. Loguinov, P. Antczak, A. Tagmount, S. Aloni, D. T. Nowinski, P. Luong, C. Tran, N. Karunaratne, D. Pham, X. X. Lin, F. Falciani, C. P. Higgins, J. F. Ranville, C. D. Vulpe and B. Gilbert, Silver nanowire exposure results in internalization and toxicity to Daphnia magna, ACS Nano, 2013, 7, 10681–10694.
41. M. Akter, M. T. Sikder, M. M. Rahman, A. K. M. A. Ullah, K. F. B. Hossain, S. Banik, T. Hosokawa, T. Saito and M. Kurasaki, A systematic review on silver nanoparticles-induced cytotoxicity: Physicochemical properties and perspectives, J. Adv. Res., 2018, 9, 1–16.
42. O. J. Osborne, B. D. Johnston, J. Moger, M. Baalousha, J. R. Lead, T. Kudoh and C. R. Tyler, Effects of particle size and coating on nanoscale Ag and TiO₂ exposure in zebrafish (Danio rerio) embryos, Nanotoxicology, 2013, 7, 1315–1324.
43. H. Liu, X. Wang, Y. Wu, J. Hou, S. Zhang, N. Zhou and X. Wang, Toxicity responses of different organs of zebrafish (Danio rerio) to silver nanoparticles with different particle sizes and surface coatings, Environ. Pollut., 2019, 246, 414–422.
44. X. J. Chen, Y. Zhu, K. Yang, L. Z. Zhu and D. H. Lin, Nanoparticle TiO₂ size and rutile content impact bioconcentration and biomagnification from algae to daphnia, Environ. Pollut., 2019, 247, 421–430.
45. S. Abhishek, W. C. Hou, T. F. Lin and G. Z. Richard, Roles of silver–chloride complexations in sunlight-driven formation of silver nanoparticles, Environ. Sci. Technol., 2019, 53, 11162–11169.
46. M. Sakamoto, J. Y. Ha, S. Yoneshima, C. Kataoka, H. Tatsuta and S. Kashiwada, Free silver ion as the main cause of acute and chronic toxicity of silver nanoparticles to cladocerans, Arch. Environ. Contam. Toxicol., 2015, 68, 500–509.
47. L. Li, H. Wu, C. Ji, C. A. M. van Gestel, H. E. Allen and W. J. G. M. Peijnenburg, A metabolomic study on the responses of daphnia magna exposed to silver nitrate and coated silver nanoparticles, Ecotoxicol. Environ. Saf., 2015, 119, 66–73.
48. R. J. Griffitt, K. Hyndman, N. D. Denslow and D. S. Barber, Comparison of molecular and histological changes in zebrafish gills exposed to metallic nanoparticles, Toxicol. Sci., 2009, 107, 404–415.
49. A. Malysheva, N. Voelcker, P. E. Holm and E. Lombi, Unraveling the complex behavior of AgNPs driving NP-cell interactions and toxicity to algal cells, Environ. Sci. Technol., 2016, 50, 12455–12463.
50. Y. Yue, X. M. Li, L. Sigg, M. J. F. Suter, S. Pillai, R. Behra and K. Schirmer, Interaction of silver nanoparticles with algae and fish cells: a side by side comparison, J. Nanobiotechnol., 2017, 15, 16.
51. H. Kim, B. Yim, J. Kim, H. Kim and Y. M. Lee, Molecular characterization of ABC transporters in marine ciliate, Euplotes crassus: Identification and response to cadmium and benzo [a] pyrene, Mar. Pollut. Bull., 2017, 124, 725–735.
52. Y. Yamamoto, T. Sakurai, Z. Chen, N. Inoue, H. Chiba and S. P. Hui, Lysophosphatidylethanolamine affects lipid accumulation and metabolism in a human liver-derived cell line, Nutrients, 2022, 14, 579.
53. G. J. Zhang, J. B. Zhang, S. Yan, M. Hao, C. H. Fei, D. Ji, C. Q. Mao, H. J. Tong, T. L. Lu and L. L. Su, Study on the plasma metabolomics of Schisandra chinensis polysaccharide against ulcerative colitis and its correlation with gut microbes metabolism, Chin. J. Anal. Chem., 2023, 51, 100244.
54. F. Ribeiro, C. A. M. Van Gestel, M. D. Pavlaki, S. Azevedo, A. M. V. M. Soares and S. Loureiro, Bioaccumulation of silver in Daphnia magna: waterborne and dietary exposure to nanoparticles and dissolved silver, Sci. Total Environ., 2016, 574, 1633–1639.
55. F. R. Khan, K. B. Paul, A. D. Dybowska, E. Valsami-Jones, J. R. Lead, V. Stone and T. F. Fernandes, Accumulation dynamics and acute toxicity of silver nanoparticles to Daphnia magna and Lumbriculus variegatus: implications for metal modeling approaches, Environ. Sci. Technol., 2015, 49, 4389–4397.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4en00350k

‡ These authors contributed equally.

---