Dissipation kinetics and biological degradation by yeast and dietary risk assessment of fluxapyroxad in apples

The study addresses how fluxapyroxad, a succinate dehydrogenase inhibitor (SDHI) fungicide widely used across many crops, dissipates in apples and whether biological treatment with yeast can accelerate its degradation. Given the extensive use of pesticides to secure crop yield and quality and apples’ prominence in human diets—including as early foods for children—understanding residue behavior and ensuring safety are critical. Prior work on fluxapyroxad’s environmental fate in plants is limited, with scant data in apples and virtually none on microbial (especially yeast-driven) degradation. The objectives were to: (1) determine dissipation kinetics and half-lives of fluxapyroxad in two apple varieties (Gala, Idared) under field conditions and establish a pre-harvest interval (PHI) ensuring residues below 0.01 mg/kg for baby food; (2) evaluate the effect of a yeast-containing biological preparation (Myco-Sin) on degradation; and (3) estimate chronic dietary exposure for adults and children using EFSA PRIMo consumption data.

Fluxapyroxad is an SDHI fungicide that inhibits succinate dehydrogenase (complex II) and is active against a broad range of plant pathogenic fungi (Ascomycete, Basidiomycete, Deuteromycota, Zygomycota). PPDB reports long persistence in soil and water-sediment systems but provides no plant half-lives. He et al. reported half-lives in apples of 9.4–12.6 days and in soil 10.3–36.5 days with multiple preharvest applications. Noh et al. observed a ~50% decrease in perilla leaves 7 days post-application. Soil studies show slower degradation under anaerobic conditions, implying aerobic microbes facilitate breakdown. Literature on yeast-mediated pesticide degradation is limited; however, Saccharomyces cerevisiae has been implicated in accelerating degradation of various pesticides (e.g., strobilurins, carboxamides, glyphosate) and exhibits antagonistic properties against pathogens via nutrient competition and killer toxins. No prior studies specifically examined yeast effects on fluxapyroxad degradation in plants.

- Analytical method and validation: Fluxapyroxad residues in apples were analyzed by GC with micro-electron capture detector (Agilent 7890A, HP-5 MS UI 30 m × 0.25 mm × 0.25 μm), splitless injection (2 μL), helium carrier (1.37 mL/min), nitrogen auxiliary (40 mL/min), inlet 250 °C, detector 300 °C, oven: 100 °C, ramp 10 °C/min to 180 °C (hold 4 min), then 3 °C/min to 220 °C. Data processed with ChemStation Rev. B04.03-SP2. Sample preparation used a modified QuEChERS: extraction with acetonitrile, salting-out (MgSO4, NaCl, citrate buffers), d-SPE cleanup (MgSO4, PSA), evaporation and reconstitution in petroleum ether prior to GC-μECD analysis. Validation followed EU SANTE/12682/2019: linearity assessed at 0.005–1 mg/L (R≥0.999), LOQ 0.005 mg/kg, LOD defined at S/N=3. Mean recoveries at 0.01 and 1 mg/kg were 107.9–118.4% with RSDr 4.2–4.7%; within-lab reproducibility RSDwr 6.2%. Matrix effects addressed using matrix-matched standards; specificity, robustness, and retention time tolerance (±0.1 min) verified. - Field design: Trials in 2018 (Gala, Józefów nad Wisłą, SE Poland) and 2019 (Idared, Rzeszów, SE Poland). The SDHI fungicide Sercadis (fluxapyroxad 300 g/L) applied to entire plots (≈0.5 ha) at recommended dose; PHI per label 35 days. Seven days after chemical application, plots were split into two blocks: Block I sprayed with water (control) and Block II sprayed with biological preparation Myco-Sin (contains aluminum sulfate tetradecahydrate, inactive dried S. cerevisiae, and horsetail extract) at 10 g/L. Weather was monitored by WatchDog 2900ET. - Sampling: Apples collected in four replicates. Sampling times: 12 h after application (next day), and at 1, 4, 8, 15, 29, 43, and 60 days post-application; Myco-Sin-treated and control samples collected on synchronized dates starting one day after Myco-Sin application (day 8 relative to chemical treatment). Samples (1000–1500 g) homogenized; 10 g aliquots extracted. Storage at −17 °C until extraction. - Kinetics and calculations: First-order dissipation model P = P0·e^(−kt); half-life t1/2 = ln2/k; time to 0.01 mg/kg t0.01 = ln(0.01/P0)/(-k). Statistics used four replicates; Student’s t-test (two-tailed) with significance at p<0.1. - Dietary exposure assessment: Chronic exposure (IEDI, mg/kg bw/day) computed using EFSA PRIMo rev. 3.1 consumption and body weight data: IEDI = (STMR × consumption)/bw; %ADI = IEDI/ADI. STMRs from supervised trials; acute exposure not assessed since field residues were below the MRL (0.9 mg/kg). Populations included Polish general, German child/general, UK infant/toddler/adult.

- Analytical performance: Recoveries 107.9–118.4%; RSDr 4.2–4.7%; RSDwr 6.2%; LOQ 0.005 mg/kg; linearity R≥0.999; method met SANTE criteria. - Initial residues (Day 1 after chemical treatment): Gala 0.417 ± 0.080 mg/kg; Idared 0.304 ± 0.062 mg/kg, both below MRL 0.9 mg/kg. - Dissipation kinetics: First-order fits with high determination coefficients. Gala: P=0.3140·e^(−0.07831t), R²=0.9643; Idared: P=0.2878·e^(−0.0763t), R²=0.9730. Half-lives: Gala 8.9 days; Idared 9.0 days. Residue reduction by Day 43: 97.1% (Gala) and 96.7% (Idared); residues <LOD by Day 60. Theoretical time to reach 0.01 mg/kg: ~44 days for both varieties; Idared reached 0.01 mg/kg at Day 43 in-field. - Yeast-containing biological treatment (Myco-Sin) effect: One day after Myco-Sin application (study Day 8), residues were reduced versus controls: Gala 0.055 ± 0.009 mg/kg vs control 0.137 ± 0.038 mg/kg (59.9% degradation); Idared 0.077 ± 0.009 mg/kg vs control 0.137 ± 0.032 mg/kg (43.8% degradation). Subsequent degradation percentages versus controls varied (Gala: 23.5%, then 30.6%, 8.3%; Idared: 23.4%, then 10.5%, 10%). By Day 60 residues were <LOD in all samples. Statistically significant differences between treated and control were observed only on Day 8 (p<0.1). - Dietary risk assessment: STMRs used: Gala 0.157 mg/kg; Idared 0.141 mg/kg. IEDI and %ADI estimates: Polish general IEDI 0.000321 (1.6% ADI) using Gala STMR and 0.000288 (1.4% ADI) using Idared STMR; German child IEDI 0.001959 (9.8% ADI, Gala) and 0.001760 (8.8% ADI, Idared) — highest among assessed groups; German general 1.9% (Gala) and 1.7% (Idared); UK infant 1.2% and 1.1%; UK toddler 1.3% and 1.2%; UK adult 0.3% for both. All chronic exposures were far below 100% ADI, indicating acceptable risk.

The study demonstrates that fluxapyroxad dissipates relatively quickly in apples, with half-lives near 9 days for both Gala and Idared, aligning with prior apple data (He et al.) and supporting a PHI of approximately 44 days to ensure residues below 0.01 mg/kg for baby-food production. Field residues never exceeded the MRL, and were <LOD by Day 60. The biological treatment containing yeast (Myco-Sin) produced a marked short-term enhancement of degradation one day after application (≈60% in Gala; ≈44% in Idared), suggesting that microbial and/or formulation-mediated processes can accelerate residue decline shortly after application. However, the effect diminished over time, with statistically significant differences confined to Day 8, implying transient benefits or variability due to environmental conditions, microbial dynamics, or matrix effects. The dietary risk estimates across diverse consumer groups, including children, were all below 10% of ADI, indicating that, under the assessed use pattern, fluxapyroxad residues in apples do not pose a chronic health risk. These findings reinforce the feasibility of integrating yeast-based biological preparations within IPM to potentially hasten pesticide dissipation while maintaining consumer safety.

An SANTE-compliant GC-μECD method using QuEChERS was established for fluxapyroxad in apples (LOQ 0.005 mg/kg; good recoveries and precision). Field trials in Gala and Idared showed initial residues well below MRL, first-order dissipation with half-lives ~9 days, and residues <LOD by 60 days. A PHI of ~44 days ensures residues below 0.01 mg/kg suitable for baby food. Application of a yeast-containing biological preparation (Myco-Sin) transiently accelerated degradation, with peak reductions observed one day post-application (59.9% Gala; 43.8% Idared). Chronic dietary exposure estimates for adults and children were ≤9.8% of ADI, indicating acceptable safety. Future work should isolate the contribution of yeast versus other formulation components, evaluate broader varieties and agro-climatic conditions, assess metabolite profiles, and determine reproducibility and duration of the biological effect.

- The biological preparation Myco-Sin contains multiple components (aluminum sulfate, S. cerevisiae, horsetail extract), so the observed effect cannot be attributed solely to yeast. - Only two apple varieties were tested, each in a single season and location, limiting generalizability across climates, years, and horticultural practices. - Statistical significance between treated and control samples was observed only at one time point (Day 8, p<0.1), indicating limited power or transient effects. - Metabolites of fluxapyroxad were not analyzed; only parent compound was monitored. - Acute exposure was not assessed (though residues were below MRL), and detailed variability in consumption for Polish children was unavailable, necessitating reliance on German/UK data. - GC-μECD, while sensitive, is less selective than LC-MS/MS for confirming identities and potential co-eluting interferences, although specificity checks were performed.