Particle number-based trophic transfer of gold nanomaterials in an aquatic food chain

The study addresses how physicochemical properties of engineered nanomaterials, particularly size and shape, govern their uptake, transformation, trophic transfer, and biodistribution in aquatic food webs. Traditional ecotoxicology methods quantify only total elemental mass and cannot reveal if metals are present as particulate or ionic forms, nor their particle number, size, or shape. Because nanomaterials can dissolve, re-precipitate, and agglomerate within organisms, conventional mass-based dose metrics may miss critical behaviors affecting bioavailability and risk. The research aims to use both mass- and particle number-based dose metrics to trace gold nanomaterials (Au-NMs) through a three-level aquatic food chain (algae–daphnia–fish), quantify dissolution and agglomeration in situ, and determine how initial particle size/shape and species-specific processes affect trophic transfer and biomagnification.

Prior work suggests that environmental risk assessment of rapidly dissolving nanomaterials may be informed by soluble analogs, while persistent nanomaterials can enter and transfer through food webs. Studies have reported nanomaterial transformations (agglomeration, dissolution) in organism guts and the role of biomolecule coronas in modulating dissolution. Existing guidelines, optimized for molecular chemicals, typically quantify total mass, lacking resolution on nanoparticle form, number, and size distribution. Earlier trophic transfer studies on gold nanoparticles and other nanomaterials have shown variable biomagnification outcomes depending on systems, species, and methods. Synchrotron X-ray techniques provide speciation insight but often lack resolution/sensitivity for low concentrations of single nanoparticles in tissues. Single-particle and single-cell ICP-MS have emerged to quantify nanoparticle number, size distributions, and per-cell metal loads with high sensitivity, enabling distinction between ionic and particulate forms. This study builds on these methodological advances to address knowledge gaps in number-based trophic transfer and species-dependent biotransformations.

Nanomaterials: Citrate-coated Au-NMs were obtained commercially as spherical (nominal 10, 60, 100 nm) and rod-shaped (10 × 45 nm; 50 × 100 nm). Characterization in Milli-Q water included TEM (morphology/size) and zeta potential; stability against dissolution/agglomeration was assessed in algal exposure medium (without algae) over 72 h using spICP-MS. Dissolved Au was <0.2% over 72 h and particle size distributions remained stable; zeta potentials were negative in both MQ (−21 to −25 mV) and algal medium (−17 to −19 mV). Algal exposure: Unicellular Pseudokirchneriella subcapitata were cultured per OECD 201. Au-NMs were sonicated (10 min, 40 W) and algae were exposed for 72 h at an initial number concentration of 2.93 × 10^11 particles mL−1 for each NM type. After exposure, unbound/loosely bound particles were removed by washing with 0.1 M pH 7.5 PBS and centrifugation. Toxicity to algae was evaluated via chlorophyll content (no effect observed). Cellular association and per-cell Au mass were quantified by scICP-MS, and particle numbers and size distributions associated with algae were quantified by spICP-MS. Daphnia exposure and depuration: Adult Daphnia magna were maintained per OECD 202 (Elendt M7 medium, pH 8, 22 °C). Daphnids (six groups, 90 per group; triplicates) were fed Au-NM-exposed algae (0.1 mg wet weight per daphnid) at 0, 24, and 48 h for a total of 72 h exposure. Post-exposure, some daphnids underwent depuration in clean medium for 72 h (no feeding); medium aliquots were analyzed by ICP-MS and spICP-MS. Accumulated NMs in exposed daphnids were quantified by spICP-MS. Fish exposure and tissue analysis: Adult zebrafish (Danio rerio) were acclimated and then exposed in 30 L aquaria for 21 days by dietary intake of 10 Au-NM-exposed daphnids per fish per day (~100 mg daphnids/day). Water was changed every 36 h. After exposure, fish were held 48 h without feeding; Au depuration to the medium was quantified. Fish were dissected to isolate intestine, liver, gills, and brain, which were weighed and processed. Particle extraction and analytics: Algae, daphnids, and fish tissues were digested with 5% tetramethylammonium hydroxide (TMAH) to extract NMs without altering particle size distributions (validated in-house). spICP-MS (PerkinElmer NexION 300D, single-particle mode) quantified particle number concentration, size distributions, and ionic Au. Transport efficiency was established using Au NP standards. scICP-MS quantified the number of Au-associated cells and per-cell Au mass in algae. Data analysis involved normality testing, one-way ANOVA with Duncan’s post hoc tests. Number-based biomagnification factors (NBMFs) were calculated for Algae–Daphnia, Daphnia–Fish, and overall Algae–Fish, as well as tissue-specific NBMFs relative to daphnia. Additional experiment: Au-NMs (10 mg L−1) were incubated in diluted fish plasma (10× in PBS; 4.5 g L−1 proteins) for up to 72 h to assess corona-mediated dissolution suppression; samples were digested to release adsorbed ions and analyzed by spICP-MS.

- In algal medium without algae, Au-NMs were stable over 72 h: dissolved fraction <0.2% of total Au; no significant agglomeration. - Algal association depended on NM size/shape despite identical exposure number concentration (2.9 × 10^11 particles mL−1): spherical 10 nm Au-NMs were detected in 68% of algal cells (20–1200 ag Au per cell), whereas rod-shaped 10 × 45 nm were in 34% of cells (30–200 ag per cell). EPS likely permits penetration of smaller particles and traps larger/elongated ones, leading to removal during washing. - Mass-transfer percentages across trophic levels (ICP-MS-based): • Algae removed 31% (rod 50 × 100 nm) to 89% (rod 10 × 45 nm) of Au from exposure medium; only 0.01% (spherical 100 nm) to 0.21% (spherical 10 nm) remained strongly associated after PBS washing; 31–89% of algal-associated Au was removed by PBS, indicating predominantly loose association. • Daphnia accumulated 0.73% (spherical 60 nm) to 1.71% (rod 10 × 45 nm) of the Au present in 0.1 mg algae; depurated 78% (spherical 60 nm) to 92% (rod 10 × 45 nm) of Au from the ingested algal dose. • Fish accumulated 0.03% (spherical 10 nm) to 0.48% (rod 50 × 100 nm) of Au present in 100 mg daphnia; fish depurated 49–58% of the exposure dose to the medium within 48 h after final feeding. - Daphnia biotransformation (spICP-MS): No Au ions detected in algae; daphnia exhibited size/shape-dependent dissolution with highest Au ion fractions for rod 50 × 100 nm (~56% of NM mass) and spherical 100 nm (~39%). Contrary to expectation that smaller NMs dissolve more rapidly, results suggest smaller NMs agglomerate or undergo dissolution–reprecipitation, reducing effective dissolution. - Size distribution shifts (mode, Msize): In daphnia, spherical 10 nm and rod 10 × 45 nm shifted to larger sizes (20→32 nm; 25→28 nm), while spherical 60 nm, 100 nm, and rod 50 × 100 nm shifted to smaller sizes (~33–40 nm), converging to ~25–40 nm mass-based sizes irrespective of initial size. In fish intestines, modest further increases for the smallest types (e.g., spherical 10 nm to ~42 nm), but overall no substantial further transformation in fish. No Au ions were detected in fish tissues. - Fish plasma corona reduced Au-NM dissolution for all sizes/shapes compared to ultrapure water, supporting the lack of ionic Au in fish tissues. - Particle numbers across trophic levels (spICP-MS): Algae associated particle numbers ranged from ~4.6 × 10^7 (spherical 100 nm) to ~5.6 × 10^8 (spherical 10 nm). Daphnia contained fewer particles (≈3 × 10^4 for rod 50 × 100 nm to ≈6 × 10^5 for spherical 10 nm). Fish contained fewer again (≈1.5 × 10^4 for rod 50 × 100 nm to ≈2.5 × 10^4 for rod 10 × 45 nm). All measured daphnids and fish contained NMs. - Number-based biomagnification: NBMFs were higher for Daphnia–Fish than Algae–Daphnia, but overall there was no particle-number biomagnification in fish (NBMF < 1). NBMFs for spherical 10 nm and rod 10 × 45 nm were lowest among tested NMs, indicating reduced transfer of the smallest particles. - Biodistribution in fish: The brain generally contained the highest fraction of particles (≈45–59% of total particles in fish, except for rod 10 × 45 nm), followed by liver; gills also contained particles (Msize ≈39–86 nm), likely due to excretion into water and re-uptake via gills.

The findings demonstrate that nanoparticle size and shape critically determine initial association with primary producers, subsequent biotransformation in consumers, and biodistribution in predators. Algal EPS modulates size/shape-selective association, favoring smaller particles. In daphnia, gut conditions induce size- and shape-dependent dissolution and agglomeration, converging disparate initial sizes toward similar modes (~25–40 nm), thereby reshaping the form available to higher trophic levels. Despite significant dissolution in daphnia for larger particles, ionic Au showed no detectable transfer to fish, likely due to sequestration into non-bioavailable fractions within daphnia and/or suppression of dissolution by protein coronas in fish. In fish, further transformation was minimal, but biodistribution was pronounced and size/shape-dependent, with preferential accumulation in the brain and liver. Number-based metrics revealed no biomagnification at the fish level, contrasting with some prior studies, underscoring that trophic transfer outcomes depend on the specific food chain, exposure route, and species-specific transformations. The study emphasizes that particle number and state (particulate versus ionic), not just mass, are essential for interpreting fate, bioavailability, and potential hazard of metallic nanomaterials.

Persistent Au nanomaterials can enter and traverse an aquatic food chain, but the extent and form of transfer depend strongly on particle size and shape and on species-specific transformations. Algal EPS promotes higher association of smaller particles; daphnia modify nanoparticle size distributions via dissolution and agglomeration, equalizing sizes across initially distinct NMs. Only a small fraction of Au-NMs transfers to fish, where further transformation is limited, yet biodistribution concentrates particles in brain and liver. Particle-number metrics reveal no biomagnification in fish and provide insights that mass-only measurements would miss. The validated workflow combining selective particle extraction with spICP-MS and scICP-MS enables sensitive, in situ-relevant quantification of particle numbers, sizes, and ionic fractions. Future research should apply this approach to diverse nanomaterial compositions, coatings, and shapes, examine different food webs and environmental conditions, and resolve how organismal physiology and coronas govern biotransformation, bioavailability, and tissue-specific accumulation, particularly in neural tissues.

Depuration media contained Au but ionic versus particulate forms could not always be distinguished at very low concentrations; spICP-MS detected no particles in depuration media, likely due to detection limits. The study focuses on a single assembled food chain under controlled laboratory conditions with specific species and citrate-coated Au-NMs; generalizability to other species, coatings, environmental matrices, and exposure scenarios may be limited. Tissue analyses quantify particle number and size distributions following chemical extraction; while validated to minimize artifacts, complete in situ localization at single-particle resolution is beyond current methods. PBS washing removes loosely bound particles from algae, which may underestimate potential transfer of weakly associated particles in natural feeding conditions.