Residual analysis of chitosan-based agronanofungicides as a sustainable alternative in oil palm disease management

Oil palm (Elaeis guineensis) is a highly productive oilseed crop but is severely threatened by basal stem rot (BSR) disease caused by Ganoderma boninense. Conventional management employs hexaconazole, yet its use is associated with environmental concerns including dissipation, leaching, and soil acidification. Nanodelivery systems, particularly chitosan-based carriers, can enhance targeted delivery, improve solubility and stability, control release, and reduce toxicity. This study evaluates a chitosan-hexaconazole nanoparticle agronanofungicide (mean size ~18 nm) for residue behavior in oil palm matrices. The central questions are: (1) Does trunk-injected chitosan-hexaconazole leave residues in edible oil matrices (CPO, CPKO)? (2) What are the uptake, translocation, and dissipation kinetics of hexaconazole residues in stem tissue and leaf following nanoformulation application? The work also validates a QuEChERS-GC-µECD analytical method for monitoring hexaconazole in these matrices.

Background highlights include: (1) Oil palm’s global significance and susceptibility to BSR (Ganoderma boninense). (2) Conventional hexaconazole use can negatively affect environments via leaching and persistence, prompting sustainable alternatives. (3) Chitosan nanocarriers enable encapsulation/entrapment, controlled release, improved stability and uptake, and can induce plant defense while being biocompatible and non-toxic. (4) Increasing demand for pesticide residue determination in foods/plants has positioned the QuEChERS method as a rapid, broad, and cost-effective approach, with PSA, GCB, and C18 sorbents mitigating matrix interferences. The current study builds on prior work demonstrating high antifungal activity of chitosan-hexaconazole nanoparticles against G. boninense and aims to ensure residue-free edible oils while achieving effective delivery to target tissues.

Field trial: Conducted at an MPOB plantation in Teluk Intan, Perak, Malaysia (26 Feb–26 Jun 2019) on 13-year-old Tenera palms on peat soil. Randomized complete block design with 3 treatments: control (untreated), single dose 4.5 g a.i./palm, and double dose 9.0 g a.i./palm. Each treatment replicated three times with 10 palms per replicate (total 90 palms). Trunk injection via two drilled holes (20 cm depth; 11 mm diameter) using MPOB standard procedure: 6 L fungicide injected (3 L per hole) and 1 L sprayed on trunk/bole. The nanoformulation powder (85% w/w chitosan, 15% w/w hexaconazole) was dissolved in 1% v/v HCl before application; nanoparticle size in solution <20 nm by DLS. Sampling: Fruits (for CPO and CPKO), leaf (frond 17), and trunk tissue were sampled at -1 (pre-treatment), 0 (6 h), 1, 3, 7, 14, 30, 60, 90, and 120 days after treatment. CPO was extracted from mesocarp; CPKO from kernel via Soxhlet (n-hexane, 8 h) followed by solvent removal. Leaf dried (40 °C, 4 h), ground; tissue stored at -20 °C. Extraction (QuEChERS with modifications): 5 g sample in 50 mL tube; add acetonitrile with 1% HCl (15 mL for CPO/CPKO; 30 mL for leaf/tissue); vortex 30 s. Add 4 g MgSO4 and 1 g NaCl; vortex 30 s; centrifuge 20 min at 5000 rpm. For oils, transfer upper acetonitrile layer and freeze-out at -20 °C ≥2 h to precipitate fats. Cleanup: 1 mL supernatant to tube containing 150 mg MgSO4, 50 mg PSA, 50 mg GCB, 50 mg C18 (d-SPE); centrifuge 5 min at 2000 rpm; filter through 0.22 µm PTFE filter. Instrumental analysis: GC-µECD (Agilent 7890A), splitless injection, 2.0 µL, injector 250 °C. Column: HP-5MS, 30 m × 250 µm × 0.25 µm. Carrier N2 at 1.0 mL/min; makeup N2 at 60 mL/min. Oven: 150 °C (1 min), ramp 10 °C/min to 250 °C (5 min), then 10 °C/min to 280 °C (5 min). Hexaconazole retention times: solvent ~8.8 min; CPO/CPKO matrix ~8.2 min; leaf/tissue ~9.5 min. Calibration, LOD/LOQ, matrix effect: Stock hexaconazole 10 µg/mL in acetonitrile; matrix-matched calibrations prepared in extracted blanks (CPO, CPKO, tissue, leaf) at 0.1–100 ng/mL. ME% = (1 − solvent slope/matrix slope) × 100. LOD = 3.3 × SD of regression line / slope; LOQ = 10 × SD / slope. Selectivity assessed up to 14.5 min run time. Recovery/precision: Six replicates of blanks spiked at 1.0, 5.0, 10.0, 50.0 ng/g for each matrix; recoveries and RSDs computed. Statistics: Data as mean ± SD; ANOVA with Tukey’s test (p ≤ 0.05) using SPSS. Kinetic modeling: Dissipation in leaf (9.0 g a.i./palm) and tissue (4.5 and 9.0 g a.i./palm) fitted to zero-, first-, and second-order linearized models; half-lives (t1/2) estimated from best fits. Insufficient data for 4.5 g a.i./palm leaf kinetic modeling.

- Analytical performance: Calibration linearity R2 > 0.99 for solvent and matrix-matched curves. Matrix effects (ME%): CPO 21.1% (minor enhancement), CPKO 23.3% (minor enhancement), leaf 2.6%, tissue 5.9% (no significant effect). LOD/LOQ (ng/mL): CPO 1.6/5.0; CPKO 1.9/5.8; leaf 2.9/8.7; tissue 4.6/14.0. Selectivity confirmed with no interfering peaks up to 14.5 min. Recoveries generally 102.5–117.6% with RSDs mostly <5% across matrices and spike levels (within 70–120% recovery and <20% RSD criteria). - Palm oil residues: Across 90 CPO/CPKO samples at both doses and all timepoints (0 to 120 d), hexaconazole was not detected (ND), indicating residue-free palm oils. - Leaf residues: Single dose (4.5 g a.i./palm): residues detectable from day 30 onward (e.g., 18.5 ± 2.4 ng/g at day 60; 27.9 ± 0.6 ng/g at day 120). Double dose (9.0 g a.i./palm): detectable from 6 h; increased to ~75.1 ± 7.3 ng/g at day 30; then declined (~50.0 ± 1.8 ng/g at day 60; ~37.4 ± 2.2 ng/g at day 90; < LOQ by day 120). Trends show initial accumulation then gradual dissipation. - Tissue residues: Single dose: 18.1 ± 2.8 ng/g at 6 h; rose to ~37.0 ± 2.0 ng/g by day 3; remained ~33–37 ng/g to day 60; declined to 12.9 ± 2.8 ng/g by day 120. Double dose: 29.0 ± 1.3 ng/g at 6 h; increased to 86.8 ± 4.3 ng/g at day 30; then declined to 50.7 ± 3.4 ng/g at day 120. - Translocation factor (TF) from tissue to leaf: Single dose TF exceeded 50% by day 60 and remained >50% thereafter. Double dose TF increased from day 0–7, reaching 100% by day 7; remained >50% at day 120, indicating sustained mobilization to foliage. - Dissipation kinetics and half-lives: Tissue, single dose followed first-order kinetics with t1/2 ≈ 147 days. Tissue and leaf at double dose followed second-order kinetics with t1/2 ≈ 383 days (tissue) and 515 days (leaf). - Comparative context: Conventional hexaconazole at 9.0 g a.i./palm in prior studies was detectable in palms only up to ~30 days, whereas nanoformulation residues persisted longer in internal tissues, consistent with controlled-release behavior. No residues were observed in fruit oils, supporting food safety.

The study addressed whether a chitosan-based nanoformulation of hexaconazole can effectively target internal palm tissues while avoiding contamination of edible palm oils. The validated QuEChERS-GC-µECD method provided sensitive, selective quantification with minimal matrix effects in leaf and tissue and manageable enhancement in oils managed by matrix-matched calibration. Results showed zero detectable residues in CPO and CPKO across all doses and time points, satisfying key food safety concerns. Concurrently, substantial accumulation in stem tissue and leaves demonstrated effective uptake and systemic movement, improving bioavailability at disease-relevant sites (xylem transport to canopy). The nanoparticle size (~18 nm) likely facilitated passage through plant cell wall pores and sustained internalization, while chitosan’s carrier properties conferred controlled release, consistent with prolonged dissipation half-lives and extended detectability compared to conventional hexaconazole. Translocation patterns (rapid TF increase and sustained >50% transfer) further support effective mobility to foliage. The likely confinement of nanoparticles to stem/leaf tissues and not to fruit may be due to fruit-specific barriers (e.g., high levels of un-methylesterified pectin), reducing risk of residues in oil matrices. Overall, the nanoformulation appears to offer improved disease management potential with reduced consumer exposure risk.

Trunk injection of chitosan-hexaconazole agronanofungicide in oil palms resulted in no detectable hexaconazole residues in crude palm oil or crude palm kernel oil up to 120 days post-treatment. The formulation achieved high and sustained accumulation in stem tissue and leaves, enhancing bioavailability at target sites. Dissipation at the double dose followed second-order kinetics with long half-lives in tissue (~383 days) and leaf (~515 days), while tissue at the single dose followed first-order kinetics (t1/2 ~147 days). The validated QuEChERS-GC-µECD method provided high sensitivity and selectivity, with low LODs in palm oil matrices (<2.0 ng/mL) and low- to mid-single-digit ng/mL in leaf and tissue. These outcomes indicate that chitosan-hexaconazole nanoparticles are a promising sustainable alternative for managing basal stem rot in oil palm, combining effective internal delivery with residue-free edible oils. Future work could extend monitoring beyond 120 days to better resolve long half-lives, evaluate environmental fate and leaching, compare directly with conventional formulations under identical conditions, and investigate transport barriers in fruit tissues.

- Kinetic half-lives for double-dose treatments (second-order fits) are extrapolated beyond the 120-day observation window, introducing uncertainty in t1/2 estimates (383 and 515 days). - Insufficient residual data in leaf at the single dose precluded kinetic modeling for that condition. - The study did not include a parallel, in-trial conventional hexaconazole arm for direct comparison; comparisons rely on prior literature. - Environmental fate pathways (e.g., soil leaching, off-target transport) were not assessed in this field trial. - Matrix enhancement in CPO/CPKO indicates the need for matrix-matched calibration; while addressed analytically, such effects may complicate routine monitoring if not accounted for.