Development of Ethyl Formate-Based Fumigation Schedules for Quarantine and Regulated Pests as an Alternative to Methyl Bromide

Hagit Navarro

doi:10.60867/00000148

Escalating global trade has intensified the risk of transporting regulated arthropod pests in fresh horticultural commodities and stored products, threatening biosecurity, agricultural production and market access. Methyl bromide (MB) remains an important quarantine and pre-shipment (QPS) fumigant. Still, its ozone-depleting properties and potential phytotoxicity create an urgent need for effective, rapid and commercially applicable alternatives. This dissertation evaluated food-grade ethyl formate (EF), applied as liquid EF or in combination with carbon dioxide (CO₂), against two economically important pests of phytosanitary concern representing different commodity systems: the western flower thrips, Frankliniella occidentalis (Pergande), in edible cut flowers, and the khapra beetle, Trogoderma granarium Everts, in stored-product systems. The research integrated commercial-scale fumigation trials to develop a validated treatment schedule for edible cut flowers with laboratory bioassays that establish the biological foundation for future commodity-specific EF fumigation schedules against T. granarium. Commercial-scale fumigation trials conducted under operational packing-house conditions evaluated a pressurized pre-mixed EF: CO₂ gas mixture (1:5 ratio) against naturally occurring populations of Frankliniella occidentalis and Thrips tabaci infesting Antirrhinum spp., Chrysanthemum spp., Cosmos spp., Cucurbita pepo (zucchini), Dahlia spp., Dianthus spp., Lavandula, Matthiola spp., Rosa spp., Salvia indica, Tagetes spp., Viola tricolor and Zinnia spp. Application of 30 g m⁻³ EF: CO₂ for 2 h at 15°C with continuous gas recirculation achieved 99.94±0.05% mortality (p<0.001) of naturally occurring thrips populations while maintaining flower quality and shelf life, with no reinfestation detected following simulated shipment or commercial export. These findings established the first validated commercial EF: CO₂ fumigation schedule for edible cut flowers and identified continuous gas recirculation as an essential operational component for achieving reliable short-duration phytosanitary fumigation. Laboratory bioassays evaluated food-grade liquid EF against all the developmental stages of T. granarium, including eggs, first- to late instar non-diapausing larvae, pupae, adults and first- to late instar diapausing larvae. Insects were exposed for 6 h to EF concentrations of 3.3-111.0 mg L⁻¹ at 26±1.2°C and 55±5% relative humidity, and concentration-mortality responses were analyzed to estimate LC₅₀, LC₉₉ and Probit9 values. Ethyl formate was effective against all developmental stages tested. Eggs exhibited strong susceptibility at the median response, with an LC₅₀ of 4.3 mg L⁻¹, although the estimated LC₉₉ of 98.6 mg L⁻¹ was associated with relatively wide confidence intervals and should therefore be interpreted cautiously. Adults were comparatively susceptible, with an LC₉₉ of approximately 81.8 mg L⁻¹, whereas pupae were the most tolerant stage, with an estimated LC₉₉ of 109.1 mg L⁻¹. Among non-diapausing larvae, tolerance generally increased toward the later instars, with late instar larvae requiring an estimated LC₉₉ of 104.5 mg L⁻¹. The late instar diapausing larvae were the most tolerant of the diapausing larvae, with an estimated LC₉₉ of 101.3 mg L⁻¹. However, overlapping confidence intervals for both diapausing and non-diapausing late instar larvae and pupae indicated broadly comparable susceptibility, rather than clear statistical differences among these tolerant stages. This investigation provides the first comprehensive EF susceptibility profile across the complete life cycle of T. granarium, including diapause, and identifies the developmental stages to target when designing phytosanitary fumigation schedules. Although EF was effective against all developmental stages, the concentration required to achieve complete control of the most tolerant stages approached or exceeded the lower flammability limit of EF under the experimental conditions evaluated. To overcome the flammability limitation associated with the high EF concentrations required to control the most tolerant developmental stages, CO₂ was incorporated into the treatment. Its efficacy was evaluated against late instar non-diapausing larvae, pupae, and adults, representing stages commonly encountered during inspections and those shown to be relatively tolerant to EF. Under laboratory conditions, insects were exposed for 6 h at 27°C to liquid EF alone, different EF: CO₂ combinations containing approximately 5-32% CO₂ and CO₂-only controls. Under EF-alone exposure, complete mortality was experimentally achieved at 92.3 mg L⁻¹ for late instar non-diapausing larvae, 111.0 mg L⁻¹ for pupae and 64.8 mg L⁻¹ for adults. When CO₂ was incorporated, complete mortality was achieved at substantially lower EF concentrations: 36.3 mg L⁻¹ EF with 18% CO₂ for L5 larvae (EF: CO₂ ratio 1:8.92), 60.6 mg L⁻¹ EF with 23% CO₂ for pupae (1:6.83), and 32.4 mg L⁻¹ EF with 10% CO₂ for adults (1:5.55). CO₂ alone produced no mortality in late instar non-diapausing larvae and only low mortality in pupae and adults, confirming that it was not an effective fumigant by itself over the 6 h exposure period. Generalized linear model analyses showed that EF concentration significantly affected mortality in all stages and that incorporating CO₂ significantly enhanced EF efficacy under the conditions tested. Significant EF×CO₂ effects demonstrated that CO₂ modified the EF concentration-mortality relationship rather than acting only as an independent toxicant. The magnitude of enhancement was stage-dependent, with pupae showing a particularly strong response to relatively low CO₂ additions despite being the most tolerant stage under EF-alone exposure. The reduction from 111.0 mg L⁻¹ EF alone to 60.6 mg L⁻¹ EF combined with 23% CO₂ for complete pupal control represented an approximately 45% reduction in the experimentally required EF concentration and permitted treatment below the lower flammability limit of EF. These findings demonstrate a significant enhancement of EF efficacy. Proposed explanations involving increased respiratory activity, altered spiracular regulation, fumigant uptake or EF metabolism were not measured directly and therefore remain hypotheses requiring experimental validation. Collectively, this dissertation advances EF from a promising alternative fumigant toward practical phytosanitary application. It establishes a commercially validated EF: CO₂ treatment schedule for edible cut flowers in which continuous gas recirculation is essential for reliable short-duration fumigation, and it provides the first comprehensive stage-specific EF toxicity profile for T. granarium, including diapausing larvae. The demonstrated enhancement of EF efficacy by CO₂, together with effective operation below the EF flammability limit, provides an important basis for improving treatment safety and performance. Future research should validate the laboratory findings in the presence of representative stored-product commodities and under commercial-scale conditions to develop commodity-specific EF fumigation schedules that meet quarantine security requirements while maintaining commodity quality.

Development of Ethyl Formate-Based Fumigation Schedules for Quarantine and Regulated Pests as an Alternative to Methyl Bromide

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