Green Synthesis of Metal‑Oxide Nanoparticles with Gum Karaya and Their Ecotoxicological Impact on *Chlamydomonas reinhardtii*

Abstract

We report a sustainable route to produce gold (Au), platinum (Pt), palladium (Pd), silver (Ag) and copper‑oxide (CuO) nanoparticles (NPs) in water using the natural polymer gum karaya (GK) as both reducer and stabiliser. The colloids were characterised by UV–Vis, TEM, zeta‑potential and DCS, confirming mean diameters of 42 nm (Au), 12 nm (Pt), 1.5 nm (Pd), 5 nm (Ag) and 180 nm (CuO) and negative surface charges that persisted for six months at 4 °C. Exposure of the green alga *C. reinhardtii* to 1–20 mg L⁻¹ of these NPs revealed that Au and Pt exerted minimal growth inhibition, whereas Pd, Ag and CuO almost completely suppressed algal proliferation and triggered membrane damage, oxidative stress, chlorophyll quenching and photosystem II impairment. These findings demonstrate that GK‑mediated green synthesis yields stable, environmentally benign nanomaterials that can be tailored for selective antimicrobial or algicidal applications.

Background

Metal and metal‑oxide NPs possess unique electrical, optical, magnetic and catalytic properties that drive their widespread use in industry, medicine, agriculture and environmental remediation [1–4]. Conventional synthesis relies on toxic reductants and surfactants, raising concerns for human health and ecological integrity [5–11]. Green chemistry approaches—employing renewable, biodegradable agents and benign solvents—offer a safer alternative [12–18].

Gum karaya, a hydrocolloid extracted from *Sterculia* trees, contains galactose, rhamnose, galacturonic acid and uronic residues [63]. Toxicological studies confirm its non‑toxic nature, even as a food additive [62–65]. In this study we exploit GK’s functional groups (–OH, –COO⁻) to reduce metal salts and simultaneously stabilise the resulting NPs, thereby achieving a one‑pot, aqueous synthesis.

Despite their technological promise, metal NPs can generate reactive oxygen species (ROS), DNA damage and lipid peroxidation, potentially disrupting microbial communities and higher organisms [32–35]. Prior work has shown Ag, CuO and other NPs to inhibit algal growth, photosynthesis and membrane integrity [36–49]. However, the toxicity of GK‑derived Au, Pt, Pd and CuO NPs against the model green alga *C. reinhardtii* has not been systematically examined.

Methods

Materials

Commercial GK, AgNO₃, HAuCl₄·3H₂O, CuCl₂·2H₂O, H₂PtCl₆, K₂PdCl₄, HCl, NaOH, NH₄OH (Sigma‑Aldrich, USA). All reagents were analytical grade and deionised water (DI) was used for all preparations.

Gum Karaya Processing

1 g GK was dispersed in 1 L DI water, stirred overnight, then left at 20 °C for 18 h to remove insolubles. The solution was filtered (10–16 µm) and lyophilised.

Synthesis of NPs

For Au, Pt, Pd and Ag, 100 µL of 10 mM metal salt was added to 10 mL GK solution in 50 mL flasks. pH was adjusted to optimise NP yield, then the mixture was shaken at 250 rpm, 45–95 °C for 1 h. Colour changes (yellow, wine‑red, black) indicated NP formation. CuO NPs were produced via a colloidal thermal route: 100 µL 10 mM CuCl₂·2H₂O mixed with GK, 2:5 CuCl₂:NaOH molar ratio, heated at 75 °C, 250 rpm for 1 h, then precipitate was isolated by centrifugation and washed with ethanol and DI water.

Characterisation

ICP‑MS (Perkin Elmer OPTIMA 2100 DV) quantified metal content. UV–Vis spectra (Cintra 202) monitored plasmon peaks and long‑term stability. TEM (Tecnai F 12, 15 kV) revealed morphology and size distribution. In algal medium, differential centrifugal sedimentation (DC24000UHR) determined weight‑based size distribution; zeta‑potential was measured by Malvern Zetasizer ZS. Ion release in algal medium was assessed by ultrafiltration (3 kDa cut‑off) followed by ICP‑MS.

*C. reinhardtii* Culture

Strain CPCC11 was grown in TAPx4 medium at 20 °C, 100 rpm, 114.2 µmol m⁻² s⁻¹ illumination, maintaining an exponential phase of ≈10⁶ cells mL⁻¹.

Algal Exposure Experiments

NPs (1, 5, 10, 20 mg L⁻¹) were added to 5 mL algal suspensions in 50 mL vials. Growth, membrane integrity (propidium iodide), oxidative stress (CellROX Green), chlorophyll fluorescence and photosystem II quantum yield (AquaPen‑C AP‑C 100) were monitored at 1, 3, 5 and 24 h. Controls included un‑treated algae, heat‑killed cells and oxidative stress induced by cumin. Data were collected in duplicate and analysed by ANOVA with Dunnett’s test (GraphPad PRISM).

Results

NP Formation and Primary Characterisation

TEM images (Fig. 1a–e) showed monodisperse, spherical NPs. UV–Vis spectra (Fig. 1f) displayed surface‑plasmon peaks at 412 nm (Au) and 525 nm (Ag); Pt, Pd and CuO exhibited no distinct peaks. After 6 months, spectra remained unchanged, confirming stability.

Figure 1. TEM images of Au (a), Pt (b), Ag (c), Pd (d) and CuO (e) NPs synthesized with GK; in‑set graphs show weight‑based size distribution in algal medium; (f) UV–Vis spectra.

Characterisation in Algal Medium

Weight‑based sizes ranged from 180 to 5 nm (CuO > Au > Pt > Ag > Pd). All NPs carried negative zeta potentials at pH 7 (Table 1). Ionic metal concentrations were highest for Pt, Ag and CuO (33–36 µg L⁻¹) and lowest for Au and Pt (6–7 µg L⁻¹).

Effect on Algal Growth

Control growth: 1 × 10⁶ cells h⁻¹. Exposure to 1 mg L⁻¹ Ag, Pd and CuO reduced growth to 2.2 × 10⁴, 1.7 × 10⁴ and 0.2 × 10⁴ cells h⁻¹, respectively (P < 0.001). At higher concentrations, growth ceased completely (Fig. 2). Au and Pt also slowed growth, but effects plateaued regardless of dose.

Figure 2. Growth rate of *C. reinhardtii* after 24 h exposure to 1, 5, 10 and 20 mg L⁻¹ Au, Pt, Pd, Ag and CuO NPs. Error bars: SD.

Oxidative Stress Induction

Ag and CuO NPs induced near‑complete ROS generation (≈100 %) at 5–20 mg L⁻¹ (Fig. 3d, e). Au caused <10 % ROS, decreasing to 15 % at 20 mg L⁻¹ (P < 0.001). Pt triggered <8 % ROS within 5 h; at 20 mg L⁻¹, 19 % stressed cells after 24 h. Ag NPs produced 100 % stress after 24 h at 10–20 mg L⁻¹. CuO reached significant stress within 3 h at 10–20 mg L⁻¹; 5 mg L⁻¹ caused stress after 5 h. Au produced negligible abiotic ROS (P > 0.05).

Figure 3. Percentage of stressed *C. reinhardtii* cells (1, 5, 10, 20 mg L⁻¹) after 1, 3, 5 and 24 h for Au, Pt, Pd, Ag and CuO NPs.

Membrane Integrity

Au and Pt caused significant membrane damage at all times up to 5 h (P < 0.001) but not after 24 h. Ag NPs damaged 100 % of cells within 1 h across all concentrations. Pd NPs showed minimal damage at 1–5 mg L⁻¹; 20 mg L⁻¹ produced significant damage after 24 h. CuO damage increased with dose and time, peaking at 24 h (Fig. 4).

Figure 4. Percentage of cells with damaged membranes after exposure to 1, 5, 10, 20 mg L⁻¹ Au, Pt, Pd, Ag and CuO NPs.

Chlorophyll Fluorescence

Au and Pt had no significant effect on chlorophyll fluorescence over 24 h. Ag, Pd and CuO markedly reduced fluorescence with higher doses and longer exposure; 5 mg L⁻¹ Ag dropped fluorescence to 22 % at 24 h (P < 0.001). CuO and Pd at 20 mg L⁻¹ produced the steepest decline after 24 h (Fig. 5).

Figure 5. Chlorophyll fluorescence of *C. reinhardtii* after 1, 3, 5 and 24 h exposure to 1, 5, 10, 20 mg L⁻¹ Au, Pt, Pd, Ag and CuO NPs.

Photosystem II Efficiency

Au, Pt and CuO produced minor, sporadic reductions in quantum yield (QY) at some time points. Ag NPs caused a dramatic QY drop (>90 %) after 1 h at all concentrations. Pd and CuO significantly lowered QY only at 20 mg L⁻¹ (Fig. 6).

Figure 6. Photosystem II QY (%) after 1, 3, 5 and 24 h exposure to 1, 5, 10, 20 mg L⁻¹ Au, Pt, Pd, Ag and CuO NPs.

Discussion

Our GK‑based synthesis yielded stable, monodisperse Au, Pt, Pd, Ag and CuO NPs that maintained integrity for six months at 4 °C. The size and surface chemistry of these particles strongly influenced their biological activity. Au and Pt, with larger diameters (42 and 12 nm) and low ionic release, displayed minimal toxicity, suggesting their suitability for biomedical or catalytic applications where biocompatibility is critical. In contrast, Pd (1.5 nm), Ag (5 nm) and CuO (180 nm) were highly toxic to *C. reinhardtii*, consistent with their known propensity to generate ROS, release ions and disrupt membranes. The strong effect of Pd is noteworthy; its ultra‑small size likely facilitates cellular uptake and ion release, explaining the observed oxidative stress and chlorophyll depletion.

Ag NP toxicity aligns with literature indicating that dissolved Ag⁺, rather than the nanoparticle itself, drives algicidal activity [80–89]. Our GK‑coated Ag NPs exhibited potent biocidal action, suggesting potential use as an algicide in swimming pools or water treatment systems. CuO NPs likely exerted toxicity through both particle‑induced membrane disruption and ionic Cu²⁺ release, corroborating previous studies on *C. reinhardtii* and other algae [36, 47, 48].

Au NPs induced only modest, transient oxidative stress and negligible physiological disruption, supporting their classification as non‑toxic in this algal system. Pt NPs caused mild growth inhibition and chlorophyll reduction, possibly due to a combination of ionic Pt and particle shading effects, but overall toxicity remained low.

Conclusions

We successfully synthesised green‑produced Au, Pt, Pd, Ag and CuO NPs using gum karaya, achieving sizes of 42, 12, 1.5, 5 and 180 nm respectively, all bearing negative surface charges and stable for six months. Exposure of *C. reinhardtii* to these NPs revealed that Au and Pt were largely harmless, while Pd, Ag and CuO exerted strong toxic effects on growth, membrane integrity, ROS production, chlorophyll fluorescence and photosystem II efficiency. These results underscore the importance of NP physicochemical properties in dictating ecological risk and highlight the promise of GK‑derived Ag and CuO NPs as targeted algicides and Pd, Ag, CuO NPs for antimicrobial surfaces, whereas Au and Pt NPs can be considered safe for green‑alga exposure.