Ethyl formate diluted with nitrogen is an effective biosecurity treatment for brown marmorated stink bug ( Halyomorpha halys )

Abstract

BACKGROUND

The brown marmorated stink bug (Halyomorpha halys) (BSMB) is a biosecurity threat globally. Its most likely avenue of entry is via shipping transport of a multitude of commodities. Currently employed biosecurity treatments (methyl bromide, sulfuryl fluoride) pose several concerns including climate change, environmental and occupational health and safety. Ethyl formate with dilution in carbon dioxide has been shown to be an effective biosecurity treatment for controlling BMSB. Carbon dioxide is a known synergist in fumigation and is a greenhouse gas contributing to climate change.

RESULTS

Laboratory and commercial‐scale bioassays were conducted to evaluate the efficacy of ethyl formate in controlling diapausing and non‐diapausing BMSB. In both trials and all treatment conditions, ethyl formate concentrations >12 mg L⁻¹ without dilution in carbon dioxide for 3‐h exposure periods were effective in achieving insect control. These results were consistent with existing literature and showed the rapid action of the fumigant in achieving a desirable level of insect mortality. The results demonstrate that even without the synergist carbon dioxide, ethyl formate can achieve complete mortality of both non‐diapausing and diapausing BSMBs at a dose rate lower than the currently registered 90 mg L⁻¹.

CONCLUSION

The results of this research show the commercial viability of ethyl formate, without dilution with carbon dioxide, as a biosecurity treatment for the control of BMSB, both non‐diapausing and diapausing. These results will also assist in progressing the registration of the fumigant with relevant National Plant Protection Organisations. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

This research found that ethyl formate effectively controls adult brown marmorated stink bugs (Halyomorpha halys) regardless of dormancy state or temperature, at concentrations nine times lower than previous studies.

1. INTRODUCTION

Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), commonly referred to as the brown marmorated stink bug (BMSB), is a polyphagous insect capable of feeding upon over 300 species of plants. ¹ BMSB is considered a high‐priority biosecurity risk for Australia (https://www.agriculture.gov.au/biosecurity‐trade/pests‐diseases‐weeds/plant/brown‐marmorated‐stink‐bug) and New Zealand (https://www.mpi.govt.nz/dmsdocument/10784-Brown-marmorated-stink-bug-fact-sheet). The insect's most likely route of entry into new countries/territories/regions is via the transport shipping of vehicles and other goods from infested regions. ²

Currently, the greatest probability for successful BMSB establishment in Australia is from the northern hemisphere with at least 40 countries identified as potential sources ³ (https://www.agriculture.gov.au/biosecurity‐trade/import/before/brown‐marmorated‐stink‐bugs#target‐risk‐countries). This is predominantly due to overwintering BMSB infesting cargo prior to shipping to overwinter in the northern hemisphere. ³ Once cargo enters warmer temperatures in the southern hemisphere, an end to overwintering is triggered and reproductive cycles are initiated. ⁴ Overwintering BMSB like other insects can enter a diapause, a physiological state that reduces their metabolism and respiration, considerably increasing their tolerance to chemical treatments. ⁵ , ⁶ Due to the inaccessibility of many areas within shipping containers and machinery, fumigation is the preferred and most effective treatment. Based on the BMSB threat posed, seasonal treatment measures are implemented in Australia, coinciding with the northern hemisphere winter. ⁷

Research conducted by Abrams et al. ⁸ on adult BMSB found a two‐fold increase in fumigation tolerance to sulfuryl fluoride in diapausing insects when compared to non‐diapausing. To achieve high mortality, insects must be exposed to a sufficient concentration of a fumigant and/or exposure period, to enable penetration through the commodity in which the insect is harbouring prior to the eradication of all life stages. ⁹

Sulfuryl fluoride and methyl bromide are currently used throughout the world for the fumigation of shipping containers and machinery suspected of harbouring BMSB. ⁷ Sulfuryl fluoride is a potent greenhouse gas and methyl bromide is being phased out as part of the Montreal Protocol, due to it being identified as an ozone depleting substance. ¹⁰

As reported by Derrick et al., ¹¹ methyl bromide is also reactive with rubber, resulting in perishing of the product, making it unsuitable for the treatment of vehicles and machinery due to possible damage to tyres and rubber sealants. Unlike methyl bromide, sulfuryl fluoride is an entirely synthetic and inert compound, making it highly unreactive ¹¹ ; and, as a result, is the internationally preferred fumigant by many vehicle manufacturers. ³ Research by Abrams et al. ⁸ found sulfuryl fluoride to be capable of achieving Probit 9 mortality for adult diapausing BMSB at a concentration by time product (Ct) of 585.1 mg h L⁻¹. Probit 9 refers to the International Plant Protection Convention's efficacy standard of 99.9968% mortality for phytosanitary treatments; that is, representing one surviving insect out of over 93 000. ¹² Bioassays on other insect pests have found sulfuryl fluoride to be less effective against immature life stages. ³ There have also been concerns surrounding sulfuryl fluoride's environmental impact, and concerns regarding acute fluoride toxicity. ¹³ , ¹⁴ As a result, there is a need for an alternative fumigant which is both effective for the treatment of BMSB, does not react with machinery and is environmentally safe.

Ethyl formate is a possible alternative fumigant for the treatment of BMSB and other insect pests. It has an important role as a food grade flavour and aroma component and has been used as a fumigant for the treatment of dried fruit and stored grain. ¹⁵ , ¹⁶ , ¹⁷

The rise of ethyl formate as a fumigant largely results from its short fumigation time, penetration of materials and time taken to breakdown to the naturally occurring substances ethanol and formic acid. ¹⁵ , ¹⁸ , ¹⁹ Coetzee et al. ¹⁹ demonstrated that ethyl formate, diluted with nitrogen, at a rate of 90 mg L⁻¹ is a safe and environmentally sustainable option for fumigating ‘20 ft’ (6.096 m) shipping containers when treating for four species of stored product pests.

Relevant to this study, Kawagoe et al. ²⁰ , ²¹ found that ethyl formate diluted with carbon dioxide was effective in controlling diapausing and non‐diapausing BMSB at a Probit 9 level at concentrations ≥ 7.68 mg L⁻¹ for 12 h at 10 + −0.5 °C or ≥ 14.73 mg L⁻¹ for 4 h. Additional research by Corbett et al. ²² found that 14.5 mg L⁻¹ with 4‐h exposure was sufficient to control diapausing BMSB at 10 °C.

The objective of this study was to evaluate the efficacy of ethyl formate, without dilution with carbon dioxide, in controlling adult BMSB at the temperatures and dormancy states encountered during seasonal biosecurity treatments. Both controlled laboratory and commercial‐scale trials were undertaken. Further, the objective was to demonstrate that a non‐synergistic inert gas, nitrogen, can be used as a carrier/diluent when fumigating with ethyl formate to provide an environmentally sustainable option.

2. MATERIALS AND METHODS

2.1. Test insects

For laboratory ethyl formate fumigation bioassays, adult BMSB were field (wild) collected in Susong County, Anhui, China (30°08′ N 116°05′ E) between 12 and 13 May 2020. The insects were first held in an insect culture room at 26 °C and 60–65% relative humidity (RH) and 11 h:13 h (light/dark) photoperiod and fed with broad bean (Vicia faba L.). The insects were placed into insect cages (35 cm × 35 cm × 35 cm), each holding approximately 500–600 adult BMSB. The cages were transferred to the Chinese Academy of Inspection and Quarantine (CAIQ) Beijing laboratory.

BMSB were induced to diapause using a modified method as reported in Abrams et al. ⁸ After first molt, cohorts of insects in ‘rearing’ enclosures were transferred to an incubator set to 20 °C, 65% RH and 11 h:13 h (light/dark) photoperiod. Following emergence as adults in approximately 38 days, insect samples were held for an additional week at 20 °C. After 1 week the diapause enclosures were transferred to a second incubator set to 15 °C, 65% RH and 11 h:13 h (light/dark) photoperiod. Feeding typically stopped 1–2 weeks prior to the insects being prepared for fumigation; that is, commencing diapause. F1 progeny of the field‐collected insects were used in the trials.

2.2. Ethyl formate calculations

Liquid ethyl formate (>99.0% purity) sourced from Sigma Aldrich, Sydney, Australia was used for all fumigation protocols. The dosages and required volumes of liquid ethyl formate for the fumigant concentrations were calculated from Eqn (1):

$$V_{\mathrm{f}}=\frac{C\times V}{P\times D}$$ (1)

where: V _f is the experimental volume to be applied (in millilitres), V is the volume of the fumigation container (in litres), P is purity of liquid ethyl formate (%), C is the intended concentration of fumigant (in mg L⁻¹), D is density of liquid ethyl formate.

The concentration of ethyl formate during experimental fumigations was analysed via gas chromatography (Agilent 6890N gas chromatograph; Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (GC‐FID). Detection conditions were: injection port temperature 200 °C; packed chromatographic column Propark Q (80–100 mesh); column temperature 120 °C; carrier gas: hydrogen; column flow rate: 0.4 mL min⁻¹.

The ethyl formate standard gas was prepared using liquid ethyl formate (>99% purity, balanced with ethanol) at concentrations of 20, 40, 60, 80 and 100 mg L⁻¹. After three repeat injections, the mean peak area was fitted linearly with the standard gas concentration at each point, creating an equation for ethyl formate standard curve.

2.3. Laboratory‐fumigation trials

The conditioned fumigation desiccators containing adult BMSB were sealed and placed in temperature‐controlled cabinets (Ningbo model HWS; Southeast Equipment Co. Ltd, Ningbo City, Zhejiang, China) at 10 °C or 25 °C and 62.5–65% RH. The fumigation trials were conducted at 10 °C and 25 °C at 60% RH in 2.2 L glass laboratory desiccators fitted with a gas sampling port. A 7 cm diameter filter paper (Whatman No. 1) was inserted into the glass lid to provide a liquid evaporation surface for the injected ethyl formate.

Between 100 and 110 diapausing and non‐diapausing adult insects were placed in the open desiccators and left overnight at 10 °C or 25 °C at 62.5–65% RH prior to fumigation treatment the following morning. The diapausing insects were taken straight from the temperature‐controlled cabinets to the desiccators.

Based on previous studies, ²⁰ , ²¹ the target concentrations of ethyl formate used for the 3‐h exposure treatments were 2, 4, 6, 8, 10, 12, and 14 mg L⁻¹. After removal of the same volume of air as the injected vaporised fumigant, the fumigant was injected into the desiccator and stirred with two computer cooling fans (AC Infinity Multifan S1 Quiet 8 cm USB Fan; Tokyo, Japan). The dosage (calculated by Eqn (1)) was injected into the sealed desiccators using a liquid syringe (SGE, Melbourne, Australia; microliter syringe 1025 TLL 25 mL). The control insects were maintained in a sealed desiccator without the application of ethyl formate until completion of exposure.

Each fumigation bioassay treated a minimum of 1500 insects, with a minimum of 300 insects per treatment used as untreated controls. Each treatment involved three replicates of each ethyl formate concentration used, with three control replicates (Table 1). Normally, mortality would be corrected with Abbott's formula. ²³ However, for this bioassay, 1211 untreated control insects were used; no mortality in any of the control bioassays was observed within the short 3‐h holding period.

Table 1.

Brown marmorated stink bug (BMSB) laboratory scale bioassay raw data

Start concentration (mg L−1)	End concentration (mg L−1)	Average concentration (mg L−1)	Insects treated	Dead insects	Mortality (%)
Non‐diapause 3‐h @ 10 °C
2.15	2.57	2.36	103	2	1.94
2.21	2.59	2.4	100	4	4
2.26	2.79	2.53	102	1	0.98
4.9	5.79	5.34	103	39	37.86
5.02	5.75	5.39	100	40	40
4.94	6.06	5.5	99	37	37.37
6.02	8.2	7.11	101	81	80.2
6.85	8.14	7.49	98	85	86.73
6.59	8.73	7.66	101	83	82.18
8.06	9.98	9.02	100	98	98
8.17	10.11	9.14	103	98	95.15
8.35	10.58	9.47	102	99	97.06
9.58	11.22	10.4	102	102	100
9.49	12.51	11	100	99	99
9.53	12.78	11.16	101	100	99.01
10.95	13.13	12.04	99	99	100
12.04	14.11	13.08	103	103	100
12.21	13.96	13.09	101	101	100
Control	Control	Control	102	0	0
Control	Control	Control	98	0	0
Control	Control	Control	101	0	0
Non‐diapause 3‐h @ 25 °C
2.09	2.29	2.19	103	3	2.91
2.08	2.33	2.2	98	2	2.04
2.09	2.31	2.2	103	2	1.94
3.14	4.18	3.66	103	25	24.27
3.21	4.2	3.71	102	19	18.63
3.23	4.2	3.72	103	21	20.39
4.63	5.87	5.25	101	56	55.45
5.08	6.11	5.6	102	58	56.86
5.15	6.52	5.84	101	60	59.41
6.14	7.03	6.59	100	86	86
6	7.33	6.67	99	88	88.89
6.6	7.01	6.81	102	89	87.25
8.67	9.06	8.86	100	99	99
8.21	10.11	9.16	103	102	99.03
9.05	10.25	9.65	101	101	100
11.94	13.02	12.48	103	103	100
12.06	13.4	12.73	100	100	100
11.83	13.66	12.75	102	102	100
Control	Control	Control	102	0	0
Control	Control	Control	100	0	0
Control	Control	Control	103	0	0
Diapause 3‐h @ 10 °C
1.76	2.34	2.05	100	1	1
1.8	2.35	2.08	102	1	0.98
1.81	2.54	2.18	103	1	0.97
4.93	5.23	5.08	102	68	66.67
4.91	5.26	5.09	103	72	69.9
4.93	5.51	5.22	101	71	70.3
6.5	7.45	6.98	100	95	95
5.68	8.33	7.01	101	99	98.02
6.57	7.93	7.25	101	101	100
8.23	10.2	9.22	102	102	100
8.1	11.1	9.6	103	102	99.03
8.47	11.93	10.2	110	110	100
12.13	14.08	13.11	104	102	98.08
12.53	14.51	13.52	100	100	100
13.43	15.11	14.27	103	103	100
Control	Control	Control	100	0	0
Control	Control	Control	101	0	0
Control	Control	Control	103	0	0
Diapause 3‐h @ 25 °C
1.65	2.1	1.88	98	2	2.04
1.7	2.23	1.97	98	1	1.02
1.74	2.3	2.02	102	2	1.96
3.77	4.98	4.38	101	62	61.39
3.8	4.12	3.96	100	65	65
3.86	4.23	4.05	101	64	63.37
4.63	5.87	5.25	100	90	90
5.07	6.73	5.9	98	93	94.9
5.27	7.27	6.27	98	94	95.92
8.21	9.37	8.79	99	98	98.99
8.36	9.75	9.06	102	102	100
8.81	9.92	9.37	103	103	100
11.6	13.18	12.39	100	100	100
11.7	13.46	12.58	103	103	100
12.1	13.93	13.02	101	101	100
Control	Control	Control	101	0	0
Control	Control	Control	102	0	0
Control	Control	Control	98	0	0

The concentration of ethyl formate was monitored during the fumigation period. Gas samples (80 μL) were taken from the desiccators with a 100 μL gas‐tight syringe 2–5 min following the injection of fumigant, and prior to opening, were then injected into the GC‐FID. Prepared ethyl formate gas standards were used to determine ethyl formate concentration in the desiccators.

At the end of the fumigation period of 3‐h, both treated and control desiccators were opened for 1‐h of ventilation in a fume hood at 25 °C and 60% RH. Bioassay samples were retrieved at the end of the fumigation period, with an initial count of live and dead adult insects which were transferred to an incubator set at 25 °C and 65% RH for 4 days ensuring diapause ended. Following this, live and dead adult insects were counted at days 2 and 4 to confirm end‐point mortality. Mortality was determined by a lack of coordinated movement after probing the insects with a soft‐haired paintbrush.

2.4. Commercial‐fumigation trials

Fumigation was carried out using a standard ‘20‐ft’ (6.096 m) refrigerated shipping container. Prior to fumigation, a gas‐tightness test was performed by connecting an air pump to gas sample lines inserted into the container. A pressure detection probe was also inserted into the container. Air was pumped into the container until the pressure reading exceeded 40 Pa. The time for the container's internal pressure to decrease from 40 to 20 Pa was recorded. The time recorded was greater than 8 s, and thus the container was recognised as having a suitable gas‐tightness for fumigation. ²⁴

Prior to fumigation, seven gas sampling lines were arranged at the left rear bottom, right rear top, central top, central middle, central bottom, left front top and front bottom right. The two temperature and humidity monitors (Tracksense‐pro, Ellab, Denmark) were located at rear top and front bottom in the shipping container (Fig. 1). Finally, 21 insect cages were placed as shown in Fig. 1. Each 350 mm cubed cage, covered with fine nylon mesh, contained approximately 750 non‐diapausing adult BMSB.

Figure 1.

Placement of fumigant sampling ports (), temperature and relative humidity sensors (), and insect cages () within the fumigated shipping container.

Three doses of ethyl formate were used based on the findings of the laboratory work, an extrapolation of Probit 9 for diapausing BMSB at 25 °C, and the research conducted by Coetzee et al. ¹⁹ : 10, 40 and 60 mg L⁻¹. The doses used reflected the currently registered dose of 90 mg L⁻¹. A boiler was used to vaporise the liquid ethyl formate which was then purged with cylinderised 99.5% purity nitrogen into the container. A 40 cm domestic fan was placed inside the container for stirring ethyl formate + nitrogen gas to ensure even distribution within the shipping container.

During the 3‐h holding period, ethyl formate gas samples were collected with an electric gas pump (FASCO, Model: CAPEX L2; Charles Austen Pumps Ltd, Byfleet, UK) drawing gas at 10 min, 30 min, 1, 2 and 3 h after application from each gas sampling line into 1 L Tedlar® gas bags.

Mortality was assessed by counting dead and live non‐diapausing adult insects. The insect cages were removed from the container following exposure with mortality assessments being conducted within a controlled laboratory setting. To ensure the insects were dead and not narcotised, mortality assessments were conducted at 24, 48, 72 and 96 h. Mortality was also determined via the use of a soft‐haired paintbrush identical to the laboratory fumigation trials.

2.5. Data analysis

Bioassays with ethyl formate that achieved 100% mortality were excluded from the probit analyses, ²⁵ in line with the methodology used in Burgess et al. ²⁶

Statistical analyses were conducted using SPSS Statistics ²⁷ and RStudio. ²⁸ Probit‐log10 (dose) regression models were fitted using the ‘BioRssay’ package in R to estimate lethal doses (LD₅₀/LD₉₉) and associated confidence intervals. Pairwise comparisons were adjusted for multiple testing using the Bonferroni correction. Goodness‐of‐fit was evaluated with chi‐square tests, and heterogeneity factors were applied where appropriate to adjust confidence limits. Non‐overlapping 95% confidence intervals were interpreted as significant differences (P < 0.05).

3. RESULTS

3.1. Laboratory bioassays

Complete mortality of adult BMSB was achieved at ethyl formate concentrations >12 mg L⁻¹ across all temperature and dormancy conditions (Table 2). Pairwise differences among treatments (Table 3) remained significant after Bonferroni correction.

Table 2.

Calculated LD₅₀ and LD₉₉ mortality and Probit 9 estimate of brown marmorated stink bug (BMSB) after 3 h exposure to ethyl formate: laboratory bioassay (all conditions)

Insect condition	Treatment temperature	BMSB number treated	3 h LD50 (95% CI, mg L−1)	3 h LD99 (95% CI, mg L−1)	3 h Probit 9 estimate (mg L−1)	R 2
Diapause	10 °C	1320	6.41 (6.18–6.61)a	12.11 (11.43–13.00)d	23.51	0.9659
Diapause	25 °C	1714	5.51 (5.22–5.58)b	10.47 (9.51–12.05)d,e	19.88	0.9678
Non‐diapause	10 °C	1296	4.63 (4.33–4.89)c	8.87 (8.20–9.93)e	15.98	0.9882
Non‐diapause	25 °C	1725	4.04 (3.56–4.42)c	8.43 (7.31–10.72)e	14.49	0.9833

Note: The lethal dose killing 50% (LD₅₀) and lethal dose killing 99% (LD₉₉) values followed by different letters are significantly different from each other due to the 95% confidence intervals (CIs) overlapping.

Table 3.

Pairwise comparison of insect conditions with Bonferroni correction: laboratory bioassay

Insect condition A	Insect condition B	Model significance	Interaction significance
Diapause 10  °C	Diapause 25 °C	0.005*	0.671
Diapause 10 °C	Non‐diapause 10 °C	0.000*	0.577
Diapause 10 °C	Non‐diapause 25 °C	0.000*	0.676
Diapause 25 °C	Non‐diapause 10 °C	0.000*	0.219
Diapause 25 °C	Non‐diapause 25 °C	0.000*	0.295
Non‐diapause 10 °C	Non‐diapause 25 °C	0.000*	0.795

^*Significance following Bonferroni correction at P < 0.05.

Both the lethal dose killing 50% (LD₅₀) and the lethal dose killing 99% (LD₉₉) (Table 2) consistently ranked highest in diapausing adults at 10 °C, followed by diapausing at 25 °C, then non‐diapausing at 10 °C, and lowest in non‐diapausing at 25 °C. Thus, diapause increased the required dose at a given temperature, and cooler temperature (10 °C) shifted doses upward within dormancy state. Confidence intervals indicated diapause treatments differed from non‐diapause, whereas the two non‐diapause conditions were not significantly different at LD₅₀; at LD₉₉, 10 °C diapausing adults required higher doses than non‐diapausing adults, while 25 °C diapausing overlapped with non‐diapausing conditions.

As illustrated in Fig. 2, the 25 °C dose‐mortality curves for diapausing and non‐diapausing adults are approximately parallel, whereas at 10 °C the curves diverge, consistent with the increased fumigation tolerance of diapausing adults at lower temperature.

Figure 2.

Mortality of brown marmorated stink bug after 3 h exposure to ethyl formate: for all conditions.

Goodness‐of‐fit tests (Table 4) supported the probit model for 10 °C treatments, while 25 °C treatments showed lack of fit, for which heterogeneity factors were applied to adjust confidence limits. No control mortality was observed; therefore, Abbott's correction was not required.

Table 4.

Probit analysis using Pearson goodness‐of‐fit test for all fumigation treatment conditions: laboratory bioassay

Insect condition	Treatment temperature	Chi‐square	df	Significance
Diapause	10 °C	11.372	11	0.413*
Diapause	25 °C	24.721	11	0.010*
Non‐diapause	10 °C	13.228	7	0.067*
Non‐diapause	25 °C	21.745	7	0.003*

^*Since the significance level is < 0.150, a heterogeneity factor is used for the calculation of confidence limits.

3.2. Commercial‐fumigation trials

Complete mortality of non‐diapausing adults was achieved at all tested concentrations (10, 40, and 60 mg L⁻¹) within 24 h and maintained through 96 h (Table 5). Across the three trials, more than 37 000 insects were exposed, and no control mortality occurred. Mortality was 100% in every treatment, with no differences among concentrations or over time once exposure was complete.

Table 5.

Treated brown marmorated stink bug (BMSB) mortality at three time points (1, 2, 4 days) following exposure to ethyl formate in a commercial shipping container

	Day 1 post‐treatment		Day 2 post‐treatment		Day 4 post‐treatment
	Live	Dead	Live	Dead	Live	Dead
Trial 1 (60 mg L−1)	0	11 230	0	11 230	0	11 230
Trial 2 (40 mg L−1)	0	13 500	0	13 500	0	13 500
Trial 3 (10 mg L−1)	0	12 650	0	12 650	0	12 650

4. DISCUSSION

Ethyl formate was shown to be effective at applied concentrations above 12 mg L⁻¹ for causing the death of adult BMSB irrespective of dormancy state and temperature conditions tested. The laboratory bioassay found that diapausing insects, as expected, were the most difficult to kill, with the lower temperature condition (10 °C) requiring a higher ethyl formate concentration than the 25 °C temperature condition. The results from the laboratory bioassays follow existing literature on the fumigation of insects, where there is an inverse relationship between temperature and the fumigant concentration required to achieve mortality. ⁹

As respiration is the fumigant's primary pathway of entry into the insect, the positive relationship between increases in temperature and rate of respiration explain the lower required fumigant concentration. ²⁹ This aligns with the Australian Government regulations, which do not permit quarantine and pre‐shipment fumigations to be conducted below 10 °C. ⁷

The laboratory bioassay results followed those from a similar study by Kawagoe et al. ²¹ Despite the exposure time for Kawagoe et al. ²¹ being only 2 h in comparison to the 3 h in this work, the results were comparable. In these laboratory bioassays, 10 °C non‐diapause treatments had an LD₉₉ of 8.87 mg L⁻¹ and a Probit 9 of 15.98 mg L⁻¹; whilst Kawagoe et al. ²¹ had a Probit 9 of 16.5 mg L⁻¹ for a 2‐h exposure, and a Probit 9 of 10.5 mg L⁻¹ for 4 h. These findings, together with those of Kawagoe et al., ²¹ contrast with Coetzee et al., ¹⁹ who reported an LD₉₉ of 90 mg L⁻¹ for stored‐product pests, approximately nine times higher than required for BMSB; highlighting the greater susceptibility of BMSB to ethyl formate. The Australian Government has approved ethyl formate for minor use as a biosecurity treatment for Barrow Island at a dosage rate nine times higher than what was required to achieve complete BMSB control in this study. ³⁰

Work described in Abrams et al. ⁸ using sulfuryl fluoride found that diapausing BMSB required a gas concentration 2.1 times higher compared to non‐diapausing insects at 10 °C to achieve Probit 9. In comparison, this research found diapausing BMSB at 10 °C required an ethyl formate concentration 1.3 times greater than non‐diapausing insects to achieve Probit 9.

The commercial‐scale trials using a ‘20 ft’ (6.096 m) shipping container validated the laboratory trials and showed that concentrations of ethyl formate diluted with nitrogen gas, above 10 mg L⁻¹ were sufficient to achieve 100% adult insect mortality within a day in each of the three treatments. These results, and the large number of insects tested (n = 37 380), enhance the findings from the laboratory bioassays. While non‐diapausing adult insects were used in the commercial trials, it is possible to extrapolate a required ethyl formate concentration to achieve LD₉₉ for diapausing insects based on the laboratory scale trials.

This research did not include any commodities in the containers, which removed the potential for sorption, that would impact the required rate of fumigant introduced to the container. While this does not directly reflect commercial trade, it does provide strong evidence that ethyl formate is a suitable fumigant to kill diapausing and non‐diapausing BMSB. Future commercial‐scale studies should include a range of commodities and products (vehicles, machinery) to evaluate sorption effects and conduct the trials across a broader range of ambient temperatures.

5. CONCLUSION

This study found that ethyl formate is effective in controlling adult BMSB irrespective of dormancy state or temperature (10 °C or 25 °C), and the fumigant can be used effectively in concentrations nine times lower than what has been seen in previous studies. The demonstration of achieving LD₉₉ and Probit 9 at low temperatures, that is, 10 °C, is also an important finding for this research as low temperatures can severely limit the efficacy of fumigation. The concentrations and time of exposure utilised in this study, which achieved 100% mortality, were also nine times lower than that currently approved by Australian regulatory authorities and could be achieved in half of the exposure time.

The results clearly demonstrate that ethyl formate is an effective fumigant that represents a strong alternative to what is currently used in the supply chain.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.