Evaluation of Ethyl Formate for Management of Both Resistant and Susceptible Strains of Cryptolestes Pusillus and Cryptolestes Ferrugineus

Home , Bromomethane, Cryptolestes pusillus

Evaluation of Ethyl Formate for Management of Both Resistant and Susceptible Strains of Cryptolestes pusillus and Cryptolestes ferrugineus

Chunyuan Gu

Supervisors: Manjree Agarwal and YongLin Ren

Submitted in fulfilment of the requirements of

Research Master with Training in Applied Biosecurity and Food Safety July 2020

College of Science, Health, Engineering and Education Murdoch University, Australia

Declaration I declare this thesis is my own work, I played a major role in collecting and analysing data for my research project. The data presented in this thesis was obtained in a Post- harvest biosecurity and food safety laboratory. All the treatment were carried out by me under the guidance of supervisors Dr Manjree and Professor Yonglin Ren in Murdoch University. I declare that this work has not been previously submitted for any other degree at any tertiary educational institution or professional qualification.

Chunyuan Gu

July 2020

Acknowledgements I gratefully acknowledge the College of Science, Health, Engineering and Education, Murdoch University providing the precious opportunity and wonderful learning environment for my research Master degree study.

I am deeply thankful to my supervisors, Dr Manjree Agarwal, Professor YongLin Ren and Dr Penghao Wang. Thank you for accepting me as your student, and encouraging and supporting me to complete my RMT study.

I am also grateful all the staff members, Amy Xiao, Sue Pan, George Li from the postharvest biosecurity and food safety laboratory for their helpful laboratory technical support. And also, thanks Bob Du for providing the GC in lab training.

I would also like to thank all my family member for their understanding and supporting, I can’t compete my study without their encouragement.

My sincere thanks to everyone who help me during my research Master study for this research project.

II Abstract Australian agricultural are facing growing challenge to biosecurity threats. Insect pests are a major issue negatively effecting both the quality of stored grain and consumer health. Fumigation is a widely used method for control of a variety of insect pests due to its high degree of efficacy and cost effectiveness. Phosphine is the main fumigant deployed for the control of stored grain insect pests. However, researcher has shown that most of the stored grain insect pests have developed resistance to phosphine, which has been leading to ineffective control of many important stored grain insect species like C. pusillus and C. ferrugineus. As a result, there is an urgent need to identify and evaluate an alternative fumigant. Ethyl formate is proposed as a viable alternative to phosphine due to its status as a registered food additive, favourable environmental properties and low toxicity to humans.

In comparison with all previous studies, fumigation experiments were conducted using a continuous flow-through laboratory process to maintain constant concentrations of ethyl formate and low levels of respiratory CO2 (<0.08%). The procedure minimised the effects of sorption by exposing test insects without media and minimised the effect of CO2 by use of continuous flow. The toxicity data of ethyl formate obtained from this experiment are more robust as the continuous flow-through system operated under a constant concentration of ethyl formate at a constant 25C and 62.5% r.h. and low CO2 concentrations during the 6 h exposure period.

Ethyl formate as fumigant offers quick and higher efficient to kill both susceptible and resistant strains of C. pusillus and C. ferrugineus at lower dosage level. There was no significant different mortality between susceptible and resistant strains of C. pusillus e g., the observed Ct product at LD50=13.09 and 12.75 mg h/L and LD99.5=28.17 and

29.55 mg h/L, and for C. ferrugineus at LD50=13.11 and 13.26 mg h/L and

LD99.5=29.28 and 28.22 mg h/L. Thus we can propose that toxicity of ethyl formate to both species of C. pusillus and C. ferrugineus and strains of phosphine susceptible and resistant is same, which also indicated that there is no cross resistance between ethyl formate and phosphine. Therefore, ethyl formate has great potential as grain fumigant for grain industry to manage C. pusillus and C. ferrugineus, particularly their resistant strains.

III

List of figures and tables

Table 1-1. Uses of methyl bromide and means of industrial exposure. Table 1-2. Characteristics of ethyl formate compared with other three common fumigation. Table 1-3. Toxicity of ethyl formate against stored grain pests.

Table 2-1. The variation of temperature, r.h and concentration of ethyl formate and CO2 in the fumigation chambers during 6 h fumigation period. Table 2-2. Dosage (measured as Ct product) estimates and parameters of regression of Probit mortality for exposure susceptible and resistant strains of C. pusillus and susceptible and resistant strains of C. ferrugineus and R-Cf is adult insects to ethyl formate for 6 h fumigations at 25.0±0.3C and 62.5.0±0.5% r.h.

Figure 1-1. Australian wheat production (ABARES, 2015). Figure 1-2. The status of distribution of phosphine resistant insects cross Australia (2018). Figure 1-3. Adult of C. pusillus. Figure 1-4. Adult of. C. ferrugineus Figure 2-1. C. pusillus and C. ferrugineus colonies cultured at 27C and 68% r.h held at Postharvest Biosecurity and Food Safety Laboratory, Murdoch University. Figure 2-2. C. pusillus and C. ferrugineus colonies cultured in the jar containing cracked oats and apple peel at 27C and 68% r.h. Figure 2-3. Analysis of the concentrations of ethyl formate with Gas Chromatograph (GC) equipped with Flame Ionisation Detector (FID). Figure 2-4. Schematic representation of a flow-through apparatus used to fumigate insects to provide a constant concentration of ethyl formate in fumigation chambers. Figure 2-5. The flow-through apparatus used to fumigate insects with a constant concentration of ethyl formate in the fumigation chambers. Figure 2-6. HOBO data loggers recorded temperature and r.h in the fumigation chambers during the 6 h fumigation period (S-Cp is susceptible C. pusillus; R-Cp is resistant C. pusillus; S-Cf is susceptible C. ferrugineus and R-Cf is resistant C. ferrugineus).

IV List of Abbreviations ABARES Australian Bureau of Agricultural and Resource Economics and Science

ADI A Sciencescceptable Daily Intake

APVMA Australian Pesticides and Veterinary Medicines Authority ARfD Acute reference dose FGB Flat grain beetle

CO2 Carbon dioxide CRC Cooperative Research Centre C×t (Ct) Concentration×time DAF Department of Agriculture and Fisheries DAWR Department of Agriculture and Water Resources EF Ethyl formate FDA U.S. Food and Drug Administration FID Flame ionisation detector GC Gas chromatography GFP Good fumigation practice GRAS Generally Recognized as Safe IARC International Agency for Research on Cancer

LD50 Lethal dose kills 50% of experimental animal

LD99 Lethal dose kills 99% of experimental animal MB Methyl bromide mg Milligram mL Millilitre t MRL Maximum Residue Level NOEL No observed effect level NIOSH National Institute for Occupational Safety and Health

PH3 Phosphine OCSEH Office of Chemical Safety and Environmental Health r.h Relative humidity QPS Quarantine Pre-shipment SF Sulfuryl fluoride SUSMP Standard for the Uniform Scheduling of Medicines and Poisons TLV Threshold limit value TWA Time weighted averages

V UNEP United Nations Environment Programme WA Western Australia

VI Table of Contents Pages Declaration i Acknowledgements ii Abstract iii List of figures and tables iv List of abbreviations v

Part 1. Literature review 1. Background 1 1.1. Grain industry in Australia 1 1.2. Postharvest grain biosecurity 2 1.3. Stored grain insect pests in Australia 3 1.4. Fumigant and fumigation 4 1.4.1. Methyl bromide 5 1.4.2. Phosphine 6 1.4.3. Sulfuryl fluoride 7 2. Flat grain beetle (FGB) of Cryptolestes spp 8 2.1. Flat grain beetle (C.pusillus) 8 2.2. Rusty grain beetle (C. ferrugineus) 9 3. Alternative fumigants - ethyl formate 10 3.1. Physical and chemical properties 10 3.2. Current guidelines for health limits set for ethyl formate 11 3.2.1. Toxicity of ethyl formate 11 3.2.2. Toxicity studies, acute 12 3.2.3. Pathways 13 3.2.4. Material data sheet 13

3.3. Current status of ethyl formate as fumigant 14

VII 3.4. Toxicity of ethyl formate for stored product insect pests 17 3.5. Ethyl formate formulations and application 22 3.6. Interaction with commodity and its residue 22 3.7. Environmental effects of ethyl formate 22 4. Gaps in this knowledge 23 5. Aims of this study 24

Part 2. Evaluation of ethyl formate for management of both phosphine resistant and susceptible strains of Cryptolestes pusillus and Cryptolestes ferrugineus. 1. Introduction 25 2. Materials and methods 27 2.1. Insects 27 2.2. Fumigant and apparatus 28 2.3. Measurement of ethyl formate concentrations 29 2.4. Measurement of temperature, relative humidity and carbon 30

dioxide. 2.5. Fumigation and insect bioassay 31 2.6. Determination of concentration  time products (Ct) 33 3. Results and discussion 34 3.1. Temperature, relative humidity and carbon dioxide during 34 exposure 37 3.2. Concentrations of ethyl formate during exposure 38 3.3. Efficacy of ethyl formate to C. pusillus and C. ferrugineus 4. Conclusion 39

References 42

VIII

Part 1. Literature review

1. Background 1.1. Grain industry in Australia Grain is an important source of nutrients and essential food and since long has been a human saviour for drought, famine and war for centuries due to their long-term storage (Saxena et al., 1988). The grain industry is part of an indispensable contributor to the Australian economy. The Gross value of all Australian agricultural production around $50.9 billion and grain industry accounts for over 50% of agricultural exports from Australia (Paull, 2019). According to the CRC Plant Biosecurity (2018), more than 30 billion tonnes of grains are produced annually from Australian grain industry. Grain is a major component of the Western Australian agricultural industry, with exports contributing an average of $4.6 billion in revenue to the state economy annually (Wilkinson, 2017). The export markets impose strict phytosanitary regulations in order to ensure that product integrity and the economic value of each commodity remain positively correlated. These phytosanitary regulations are important given that insect infestation can significantly affect grain integrity. A principal stage for the detection and treatment of insect infestations is during post-harvest storage (Ross et al., 2017).

The common grain produced in Australia are wheat, coarse grain, pulses and oilseeds. Wheat is the major grain in Australia, it is the fifth largest wheat exporter in the world. In 2016, Australia exported around 22 million tonnes of wheat, valued at $6.1 billion (DAWR, 2017). According to the Figure 1-1, the area planted with wheat in Australia is expected to increase year by year, with an average growth rate of 0.5% by 2020 to 2021 (Hamer, 2016). However, a large amount of grain industry has suffered a lot of damage due to the storage issue. It is important to protect storages grain from the infection for providing enough production and management to grain storage in order to

1 feed increasing population, strengthen the overall economy and also improve the health of people and animals in Australia.

Figure 1-1. Australian wheat production (ABARES, 2015).

1.2. Postharvest grain biosecurity Postharvest handling is the stage of grain production immediately following harvest, including transportation, drying, storage and marketing. Grain stored is an important investment to protect grain effect by insects, weeds, pathogens, and fumigant resistance and chemical residues during postharvest storage in order to maintain quality of produce and improve commercial value and market access (Baloch, 1999). The problem of grain storage effected by many factors such as storage structure used to storing grains and environment factors like temperature or even other biological factors. Grain are stored at different stages of distribution chain such as bags, silos, warehouses, containers or on the ground. Each stage can be managed as an artificial ecosystem where the deterioration of stored grain is the result between physical, chemical and biological factors. Through proper monitoring and management of biotic and abiotic interactions, stored grain can be protected for long periods of time (Jayas, 2012). 2 Healthy grain can provide calories and carbohydrates for human daily nutrition. However, in some developing countries, people lack food and daily nutrients due to poor storage facilities. As a result, many people eat poor quality of grains or even starve. Unhealthy or infected grain lack various vitamins and important nutrients, so can cause many diseases in consumers including food poisoning (Ross et al., 2017). Grain should be provided with appropriate temperature and ventilation to prevent harmful pests from attacking the harvested grain (Saxena et al., 1988).

1.3. Stored grain insect pests in Australia Australian Government (2014) states that pest control is a major problem due to the hot collection and storage of weather, this is because normally harvesting season is during the spring and summer. Rajendran (2007) highlight that there are more than 600 species of beetle pests and 70 species of moths affect agricultural grain losses. Stored grain insects can lead to harmful human health including warehouses, bakeries, shops, restaurants, factories, farms and silos and even home (Gaworecki, 2018). They can affect food damage and pollution by huge economic losses (insect pests of stored grain, 2019). According to the research, insects can consume 5% to 20% of the world's most important food crops and can cause major economic loss. Jayas (2012) notes that sometimes the total grain in the warehouse can be damaged and causes 100% equipment loss. It is also harmful to human health. Magan (2003) highlight that fungal-infected cereals not only produce odours, but also cause mycotoxin contamination. Sometimes the consumption of mycotoxins can be fatal. Therefore, it is a key challenge needs to consider worldwide including Australia.

The most common stored grains pests in Australia are weevils, lesser grain borer, rust- red flour beetle, saw-toothed grain beetle, flat grain beetle, Indian meal moth and Angoumois grain moth (Mclntyre, 2015). Flat grain beetle is one of classic stored grain pest in Australia which can be found in cereals such as flour, wheat, dried fruit, nut or

3 rice. They are not harmful to humans or other pests, but they damage and infest the food (Queensland Government, 2018).

1.4. Fumigant and fumigation Fumigants are chemicals which, at a required temperature and pressure, can exist in the gaseous state in sufficient concentration to be lethal to a given pest organism (Bond, 1984). They may possess bactericidal, fungicidal, insecticidal and nematicidal properties. There are many chemical compounds which are volatile at normal temperatures and sufficiently toxic to fall within the definition of fumigants (Byrns，2011). Most gases have been eliminated for use as commercial fumigants owing to unfavourable properties, the most important being chemical residues, work safety and environmental issues (Banks, 1990).

Consequently, methyl bromide and phosphine are the only two remaining fumigants widely used on grains and other stored commodities. Methyl bromide is being phased out for use on stored commodities, as it is listed as an ozone depleting substance under the Montreal Protocol (UNEP, 2006). Phosphine is now the only widely being available, registered grain fumigant around the world. Phosphine requires a long (>4 days) exposure and temperatures >15°C in sealed bins to achieve total insect control. Siroflo provides a method for achieving complete control with phosphine of insect infestations in storage that are not sealed to a gastight specification. However, the Siroflo requires exposure periods of more than 7 days (Taylor, 1989).

Sulphuryl fluoride has been developed as an effective fumigant for controlling dry wood termites. It is currently registered for treatment of grain only in a few countries. Sulphuryl fluoride requires high concentrations or extended exposures to ensure complete control of egg stages. Although sulphuryl fluoride is effective in controlling wide range of insects and pest but negative effect to human’s health include coughing,

4 irritability and even damage to the lungs and kidneys. Therefore, sulfuryl fluoride has very limited approval for use on foodstuffs (Bond, 1984).

Ethyl formate (EF) is an old fumigant which had been successfully used for package fumigation of dry fruits since 1929 and was evaluated for grain protection in the 1980s. More recently, ethyl formate has been re-evaluated as a potential alternative fumigant (Haritos, 2003). Commercial-scale trials with ethyl formate (90 mg/L) on wheat, completely killed mixed aged cultures of Rhyzopertha dominica, and adults and larvae of Tribolium castaneum.

1.4.1. Methyl bromide

Methyl bromide (MB), the formula is CH3Br which is a highly toxic gas with colourless, odourless and non-flammable belong to organic halogen compounds also called Bromomethane. Table 1-1 show that its mainly used for Quarantine or pre-shipment fumigation, horticulture and mining. Methyl bromide as a fast-acting fumigant in a wide range of situations such as used to against insects for quarantine and pre-shipment (QPS) treatment for imports and exports of commodities transport in Australia. Methyl bromide reacts with sulfur-containing materials which include furs, hair, feathers and some leather goods producing a malodorous product (UNEP, 2014). Furthermore, there is no ideal alternative fumigants to methyl bromide for QPS purposes, especially for perishable commodities (Lee et al., 2015). It is essential to find an alternative that kills all target pests in a short fumigation time without compromising quality, especially for fruits and vegetables so that the goods can be delivered to consumers quickly.

5 Table 1-1. Uses of methyl bromide and means of industrial exposure. Industry Occupation Target pests

Quarantine or pre- Pest control agent Insect pests, nematodes shipment fumigation and plant pathogens Postharvest grain cut Farmer/grower and bulk grain Insect pests, nematodes flower and fruit handler, pest control agent and plant pathogens fumigation Soil fumigant Farmer/grower and pest nematodes, plant control agent pathogens and weeds

Timber fumigation Pest control agent Insect pests, nematodes and plant pathogens

1.4.2. Phosphine

Phosphine also called hydrogen phosphide, formula is PH3, it is a low molecular weight with low boiling compound. It is registered fumigant widely used for grains and other commodities storage. It reacts with metals and some pigments (Warrick, 2011). Phosphine need more than 4 days and temperatures above 15 degrees for comprehensive insect control in a sealed box (Xin, 2008). Compare to other fumigants, phosphine is one of the better candidates for fruit and vegetable fumigation (Lee et al., 2012). Australia's agricultural industry relies heavily on phosphine fumigation to meet the needs of pest control. However, Dyte and Hallday (1985) states that the development of insect resistance to phosphine poses a threat to the effective use of phosphine in the future. Four of the five major pests increased the frequency of strong resistance to phosphides. According to the Figure 1-2, there are many phosphine resistant insects cross Australia, especially in the east (Wee et al., 2016). Furthermore, compare to other fumigants, phosphine also has limitation with longer exposure times with lower temperature require compare to other fumigants (Weller and Graver, 1998).

Figure 1-2. The status of distribution of phosphine resistant insects cross Australia (2018).

1.4.3. Sulfuryl fluoride Sulfuryl fluoride (SF) is a commercial fumigant highly toxic, colourless, odourless gas and used to stored agricultural commodities like grains. It can control wide range of pests, insects and other organisms (Derrick et al., 1990). Sulfuryl fluoride is decomposed into fluoride and sulphate in insects’ body. Fluoride is the main toxin that interferes with the insect's need to maintain enough source of energy for the storage of stored fats and carbohydrates which destroys glycolysis and the citric acid cycle. Sulfuryl fluoride is a non-flammable, non-corrosive and does not produce odour or residues in the material (Stewart, 1975). The boiling point of the sulfuryl fluoride is very low -55.2°C at 760 mm Hg, and a very high vapor pressure of 1.7 × 103 kPa at 21°C (13,000 mmHg) (Tomlin, 1997). Although sulfuryl fluoride is effective in controlling insects but harmful to humans, sulfuryl fluoride is a central nervous system

7 inhibitor. Signs of sulfuryl fluoride poisoning include coughing, vomiting, irritability, muscle twitching, seizures, and pulmonary edema. Repeated exposure to high concentrations of sulfuryl fluoride may cause damage to the lungs and kidneys. Death may occur when people enter the structure during fumigation, or if sulfuryl fluoride does not dissipate to the appropriate level as required by the product label before re- entry (Derrick et al., 1990).

2. Flat grain beetle (FGB) of Cryptolestes spp 2.1. Flat grain beetle (C. pusillus) Flat grain beetle, C. pusillus is a types of flat grain beetle mainly damages grain, dried fruit, nuts and other stored crop (Figure 1-3). It has a shiny and reddish-brown color with long antennae , sometimes reach more than half of its size (Banks, 1980). There are four stages of the C. pusillus such as eggs; pupae, larvae and adult. Eggs are about 0.5-0.8 mm and about 3.5 times as long as wide. The larvae of the pests are white to yellow and undergo 4 instars. Mature larvae can reach 4 mm in length.Pupae are about 1.2 mm, hiding in silken cocoons. Adult are 1.5-2 mm, the average of female has aporoximately 242 eggs and usually in the broken grain (Thomas, 2016). Once they hatch, the larvae dig holes in the broken or damaged kernel, where they can find the source of the food - the germ of the grain. The adult beetle will come out of the cockroach after three to four days. The entire cycle of the flat beetle takes 22 to 26 days to complete (Banks, 1980).

Figure 1-3. Adult of C. pusillus.

2.2. Rusty grain beetle (C. ferrugineus) Rusty grain beetle, C. ferrugineus (Figure 1-4) is one of common pest in the storage of cereals (Adbelghany and Fields, 2017). Adults of C. ferrugineus prefer warm temperatures and moisture, they move and remain in the warm areas of the grain (Banks, 1980). Emmanue and Ismaila (2015) notes that in stored bulk grains, temperature and humidity are evenly distributed, and adults exhibit significant geostress responses and move downward from the top of the grain to the bottom. When the grain bins are emptied, some beetles can find shelters such as grain residues in walls and floor cracks to survive the pesticide treatment (Smithsonian, 2019). Adbelghany and Fields (2017) states that the average life expectancy of C. ferrugineus adults is around 6 to 9 months, and adults have very high cold tolerance compared to other stored product insects which can survive at -15°C for 4 weeks.

Figure 1-4. Adult of C. ferrugineus.

3. Alternative fumigant - ethyl formate 3.1. Physical and chemical properties Ethyl formate (EF) is a colourless liquid and ester formed when ethanol reacts with formic acid. Appearance • Form liquid • Colour colorless Safety data • Melting point: -80℃ - lit. • Boiling point: 52-54℃- lit. • Flash point: -19℃ • Ignition temperature: 455℃ • Lower explosion limit: 2.8% (V) • Upper explosion limit: 16% (V) • Vapour pressure: 1,045.0hPa at 55℃ and 261.2 hPa at 20℃ • Density: 0.921g/ml at 20℃ • Relative vapour: 2.56 (Air=1) • Solubility: Soluble in water with some hydrolysis, miscible with alcohol, benzene, and ether.

10 • Reactivity: Unstable in heat or flame; readily hydrolyzes to the acid and the alcohol. • Incompatibilities: reacts with nitrates, strong oxidizers, strong alkalis and strong acids; No Hazardous decomposition products reported.

3.2. Current guidelines for health limits set for ethyl formate Ethyl formate is harmful to human health if inhaled and swallowed. Ethyl formate can produce stimulation in eyes, nose, mucous membranes and throat, and may irritate the lungs causing coughing and shortness of breath and absorbed through the skin. Exposure can cause headache, nausea and vomiting (New Jersey Department of Health and Senior Service, 1999).

Some research also point that ethyl formate is also a central nervous system depressant, anesthesia was more pronounced. High concentrations can cause deep anesthesia within a few minutes and then die within a few h. It can stimulation respiratory system of not only humans but also other animals. It has low mammalian toxicity but rapidly kills grain insect pests (Muthu et al., 1984).

3.2.1. Toxicity of ethyl formate • SUSMP: S6 when ethyl formate is packed and labelled for use as a fumigant • APVMA: The maximum residue limit (MRL) for ethyl formate in the dried fruit industry is 1 mg/kg (ppm, w/w) • Currently, the APVMA lists 3 products containing ethyl formate (Orica Eranol insecticide, Vapormate fumigant, Vaporfaze Emate insecticide) • OCSEH ARfD - not established NOEL - no safety concerns at current levels of intake

11 ADI - Inchem concluded that ethyl formate could be included in a group ADI for formic acid (0-3 mg/kg bw) • Occupational exposures NIOSH: TLV & TWA PEL – 303 mg/m3 (8 h working day, 40 h week) • Irritant to mucous membranes, eye, nose, skin

Slight eye irritation in human and strong persistent nasal irritation were noted on exposure to 330ppm in the air. The irritating effects on the eyes and respiratory tract are probably due to rapid hydrolysis of the ester on contact with water, with the formation of alcohol and formic acid (Peter and Jan, 2000).

In persons with impaired pulmonary function, especially those with obstructive airway diseases, the breathing of ethyl formate might cause exacerbation of symptoms due to its irritant properties. Persons with pre-existing skin disorders may be more susceptible to the effects of this agent. Although ethyl formate is not known as a liver toxin in humans, the importance of this organ in the biotransformation and detoxification of foreign substances should be considered before exposing persons with impaired liver function. Although ethyl formate is not known as a kidney toxin in humans, the importance of this organ in the elimination of toxic substances justifies special consideration in those with possible impairment of renal function (New Jersey Department of Health and Senior Service, 1999).

3.2.2. Toxicity studies, acute

LD50 oral 1850 mg/kg (rat)

LD50 dermal 2000 ml/kg (rabbit)

Metabolism: Decomposition products includes carbon monoxide, CO2.

Ethyl formate is absorbed through the lungs, from the gastrointestinal tract, and to a small extent through the skin. This ester is hydrolysed into ethyl alcohol and formic

12 acid with subsequent metabolism via well-known pathways, primarily to CO2 in the case of ethanol, while formic acid is reduced to biologically active methyl, or excreted as the free acid.

3.2.3. Pathways Ethyl formate is used in flavourings for lemonades and essences, in artificial rum and arrac, and as a fungicide and larvicide for tobacco, cereals, dried fruits, and other crops. It is also found naturally in fruits.

Carcinogenicity, IARC CATEGORY • IARC Carcinogenicity Ratings for CAS109-94-4. • This agent has shown little or no tumorigenic potential • At the time of this review, no data were available to assess the mutagenic or genotoxic potential of this agent.

3.2.4. Material data sheet A material date sheet was reviewed by Desmarchelier (1999). TLV is 100ppm (V/V) = 302 mg/m3 (303 µg/L) for ethyl formate, 1000ppm for Ethanol, 5ppm for Formic acid

(NOHSC, 1995). TLV of ethyl formate is much higher than those of CS2 (10ppm), methyl bromide (5ppm) and phosphine (0.3ppm). Oral LD50 values for rat are 1850 mg/kg for ethyl formate, 1100 mg/kg for Ethanol (Saxena et al., 1988), 1210 mg/kg for

Formic acid. LC50 of ethyl formate in rats was 8000ppm after a 4 h exposure.

FDA has proposed ethyl format “Generally Regarded As Safe (GRAS)” in food additive because ethyl formate is used a flavouring agent and there is no evidence of hazard to the public when formic acid and sodium formate are used as ingredients in paper and paperboard food-packing materials.

13 The acute, short term and subchronic toxicity of ethyl formate has been reviewed by Haritos (2006). Ethyl formate at high concentration is acutely toxic to animal although compared with other chemicals it is considered of low toxicity. There is no long-term toxicity data available for ethyl formate but its known that ethyl formate is rapidly hydrolysed to ethanol and formate and this can also partially occur prior to adsorption from the gastrointestinal tract in human body. Ethyl formate is not carcinogenic in animal studies, nor is it mutagenic (JEFCA, 1997).Toxicology data is considered adequate for registration of ethyl formate as a grain fumigant (Haritos, 2003).

3.3. Current status of ethyl formate as fumigant Ethyl formate is a colorless liquid. It is used as a preservative, disinfectant, lavicide, a solvent for vitamins. It is an old fumigant, break down to alcohol and formic acid and is naturally occuring in water, soil, range of raw foods such as fruit, vegetable, grains, beer, wine and animal products like cheese and milk (Agarwal et al., 2015) and has an important role as a flavour and aroma component. It is also used as a fumigant with insecticidal and fungicidal properties.

Ethyl formate have short exposure, lower price and low toxicity to humans when compare with phosphine, methyl bromide and sulfuryl fluoride (Table 1-2). Methyl bromide it was banned in developed countries in 2005, except for special quarantine purposes, because it consumed ozone in the atmosphere (UNEP, 2014). Many alternatives have been tested as alternatives to methyl bromide, such as phosphine.

Recent studies have shown that phosphine resistance increases in both frequency and strength of resistance. The use of phosphine is now threatened by the development of insect resistance. The emergence of strong resistance has made phosphine ineffective for insect control in many areas. Among the 1150 insect populations tested, strong resistance was found in 116 populations. C. pusillus have resistance with phosphine (Norwood, 2015).

Compare with phosphine, sulfuryl fluoride is more expensive and requires higher concentrations to control pests. Currently, in Australia, the cost of using sulfuryl fluoride to disinfect one ton of stored grain is about $3.00, compared to $0.26 for phosphine. Furthermore, another disadvantage of sulfuryl fluoride is that it leaves fluoride residues on the treated product. Higher cost and fluoride residue problems make sulfuryl fluoride less desirable when reused on the same batch of grain (Jagadeesa et al., 2018).

Table 1-2. Characteristics of ethyl formate compared with other three common fumigation Ethyl formate Phosphine Methyl bromide Sulfuryl fluoride

Fumigation Exposure time 1-8 h 3-4 days at high Up to 24 h total Up to 48 h at high concentration for time depending on concentration for effective fumigation time effective fumigation produce fumigation of grain Toxicity Toxic to pests but Highly toxic to humans Highly toxic to humans Toxic to timber pests low toxicity to and pests and pests humans Residue No residue shown Leaves detectable residues Leaves detectable Leaves high level of Fluoride iron residue, residues however clearance certificate is required before re-entering the building Flammability Flammable when Flammable may ignite at Not flammable, will Not flammable or combustible ignited in the high concentration in air burn in air in presence presence of air of a high energy TLV (ppm) 100 0.3 5 1

16 3.4. Toxicity of ethyl formate for stored product insect pests Although ethyl formate is a natural product, but it is toxic to insects so it could manage stored product insect pests rapidly. As a fumigant, the toxicity of ethyl formate to stored-product insects has been evaluated and reviewed (Banks and Hilton, 1996). The egg stages of both Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) were found to be the most susceptible and pupae the most tolerant stages for ethyl formate fumigation (Muthu et al., 1984). Damcevski and Annis (2006) states that the presence or absence of grain had the largest influence on mortality, such as application rates of 109, 130 and 155 mg/L of ethyl formate wheat in 2.7 L glass desiccators containing 500, 1000 and 1500 g wheat, respectively, for 48 h, all S. oryzae larvae were killed. Therefore, the larger the grain quantity, the higher the required ethyl formate dose to achieve almost 99% mortality. Moreover, they also notes that the toxicity of ethyl formate was quite strongly dependent on r.h with toxicity higher at higher humidity. Annis (2002) reported that ethyl formate application rate of 90 g/m3 for a 24 h exposure in an 868 L capacity fumigation chamber (without grain) is efficacious for control of all the life stages of S. oryzae, T. castaneum and Rhyzopertha dominica (F.). Results from bin trials wheat and peas (50-55 t) showed that ethyl formate application rate of 90g/m3 for 48 h exposure is efficacious for all the life stages of T. castaneum and R. dominica but is marginal for immature S. oryzae (Mahon et al., 2003). Ren and Mahon (2006) reported that the Ct products for S. oryzae were at least 2200 mg h/L in 75-145 t farm bins. In all these examples, the true Ct product experienced by the target insects is not well defined, as continuous measurements of prevailing concentrations were not taken. The results are confounded by sorption effects that lead to rapid loss of the fumigant. Calculating the Ct product on the basis of applied concentration and exposure period thus leads to an overestimation of the true Ct product needed for control. It also does not take into account that the required Ct products are probably strongly time dependent, with lower values at short exposures having the same effect as higher values at longer exposures (Banks and Hilton, 1996).

17 In most previously reported bioassays (Ren and Mahon, 2006), CO2 resulting from insect respiration and varying exposure concentrations due to sorption of ethyl formate on the culture medium, were not adequately considered. When CO2 is used with ethyl formate, the toxicity to adults of S. oryzae, T. castaneum and R. dominica can be greatly increased (Damcevski et al., 2003).

Based on the toxicity of ethyl formate against stored grain pests, ethyl formate can effectively control all stages of many stored grain pests in few days even within h and mortality can reach to 100% including Cryptolestes spp in barley, wheat and sorghum (Mahon and Ren, 2003). Researchers have found that the combination of ethyl formate with 5 to 20% CO2 together will increase the toxicity and enhances the distribution and rapidly control stored grain insects such as S. oryzae, T. castaneum and R. dominica (Damcevski et al., 2003). With applied ethyl formate doses of 111 and 185 mg h/ L respectively mixture with CO2 by forced flow fumigation and exposures in 3 h, lead to the mortality of 99.8% for T. castaneum and 95.1% for S. oryzae. Extending with 24 h will result in higher mortality of Sitophilus oryzae (Haritos et al., 2006).

Table 1-3 listed more research results regards with toxicity of ethyl formate against different stored grain pests at different treatment conditions.

18 Table 1-3. Toxicity of ethyl formate against stored grain pests Exposure Target pest Life stages time Dose Mortality Reference

Sitophilus oryzae All stages 5 days 80 mg/L in wheat, 100% Ren et al., 2003 Sitophilus granarium 5 days barley, oats and peas Tribolium castaneum 2 days 40 mg/L in canola

Rhyzopertha dominica 80 mg/L in canola Trogoderma variable

Callosobruchus maculatus Callosobruchus phaseoli All stages 2 days 85 g/t 100% in Faba beans Ren and Mahon, Tribolium castaneum 100% in wheat, faba beans, sorghum 2003 Rhyzopertha dominica 100% in wheat, faba beans, sorghum Sitophilus oryzae 100% in wheat, faba beans, sorghum

All stages

3 h 111 and 185 mg/L Sitophilus oryzaeTribolium 99.80% Haritos et al., castaneum 95.10% 2006

19 Lyposcelis entomophilia All stages 30 minutes 70 g/m3 with 7% 100% Allen and

Oryzaephilus surinamensis CO2 Demarchelier, Rhyzopertha dominica 2000 Sitophilus oryzae Tribolium castaneum

Callosobruchus phaseoli All stages 20 h 6.85 kg of EF+CO2 100% control in barley, wheat and Mahon and Ren, Tribolium castaneum applied in 3 doses sorghum 2003 Rhyzopertha dominica Sitophilus oryzae Oryzaephillus spp Cryptolestes spp Lyposcelis entomophilia

Tribolium castaneum All stages 3-24 h 147 g/t of EF + CO2 >99% in 3 h in wheat Damcevski et Rhyzopertha dominica >99% in 3 h in wheat al., 2003 Sitophilus oryzae 86% in 24 h in wheat 24 h 11.2 mg/L 99% mortality without grains Damcevski and Sitophilus oryzae All stages 24 h 81.2 mg/L 99% mortality with 1500g of wheat Annis, 2006

20 Tribolium castaneum All stages 5 days 80 g/m3 in two 50 100% Rhyzopertha dominica tonne silos 100% Ren et al., 2003 Sitophilus oryzae 99.40%

21 3.5. Ethyl formate formulations and application Ethyl formate is a commonly used solvent in the laboratory and is used as a flavoring agent. It is a highly volatile and flammable liquid at normal ambient temperature which boils at 55°C, and easily evaporates at normal grain temperature (Asimah et al., 2014). Nowadays, ethyl formate as a safe universal fumigant used to store food. It is considered an effective fumigant to control insects in a variety of commodities such as cereals and legumes, clothing and fresh fruits, vegetables and flowers, and has not been found to have an adverse effect on the quality or flavor of the processed commodities (Simmons, 1945). Ethyl formate has also been found to have fungicidal properties in grains without affecting seed viability or germination (Lee et al., 2015).

3.6. Interaction with commodity and its residue Ethyl formate can rapidly be absorbed by the grain and decomposed into an inert compound. Damcevski et al. (2003) states residue of ethyl formate takes more time to break down at the temperatures below 15⁰C.

Different trials have shown that ethyl formate breakdown depends on different types of grains and fruit. In one of the trials, Ren and Mahon (2003) notes that ethyl formate residues in wheat and sorghum dropped to natural levels after 7 days of non-aeration, but in broad bean require 3-4 weeks to drop to natural levels after fumigation.

According to another trial by Agarwal et al. (2015) notes that residue after one day aeration, ethyl formate residues in apple had declined to background levels (0.05-0.2 mg/kg). During the holding period, ethyl formate declined in fruit without the need for fan-forces. After one day, residues were above those in the untreated control sample, but below the level of 1 mg/kg which is the current Australian Maximum Residue Level (MRL) for ethyl formate in dried fruit. These results are consistent which previous commercial-scale trials with ethyl formate on wheat, barley, oats and peas.

3.7. Environmental effects of ethyl formate

22 The release of ethyl formate into the environment will result from its manufacture, transport, handling and use in the synthetic fragrances of flavors, artificial rums and arak; as well as fungicides for tobacco, cereals, dried fruits and other crops and to kill larvae. Ethyl formate has a relatively high Henry's law constant and is therefore prone to volatilization. If ethyl formate is released into the soil, it is expected to volatilize from the surface of the soil and be easily leached. It is expected to be susceptible to biodegradation and chemical hydrolysis, especially in alkaline soils. Based on limited data from screening experiments, supplemented by mathematical model predictions and data from similar chemicals, ethyl formate will rapidly biodegrade. In the atmosphere, ethyl formate will react with photochemically generated hydroxyl radicals with an estimated half-life of 11 days. It is quite soluble in water and is easily rinsed off by rain. The volatilization half-life of ethyl formate from the model river was predicted to be 4.5 h. It is not expected to absorb sediment or bioaccumulate in aquatic organisms (Banks and Hilton, 1996).

4. Gaps in the knowledge Although there have been many research studies and reports about ethyl formate until today, those research range includes analysing components, adding other ingredients to produce better effects such as CO2, or experimental comparisons of different types of insect pests. But there is still following gaps that needs to be answered.

One of the research gaps is that, there is no current research on the control of both resistant and susceptible strains of C. pusillus and C. ferrugineus by using ethyl formate. Cryptolestes spp are popular phosphine resistance insect so cannot be killed with phosphine in grain industry. Therefore, it is important to conduct systematic bioassays with liquid ethyl formate on Cryptolestes spp for determination of accurate Concentration × time (C×t) products and guiding grain industry to conduct Good Fumigation Practice (GFP) for management of phosphine resistance species.

23 5. Aims of this study The aim of this study is to assess liquid ethyl formate’s efficacy as a fumigant for treatment of Cryptolestes spp. Specifically, the project will focus on:

• Evaluation of liquid ethyl formate for management of both resistant and susceptible strains of C. pusillus and C. ferrugineus by conducting systematic bioassays. • Determination of accurate Concentration × time (C×t) products for ethyl format • Guide grain industry to conduct Good Fumigation Practice (GFP) for management of phosphine resistance Cryptolestes spp.

24 Part 2. Evaluation of ethyl formate for management of both phosphine resistant and susceptible strains of Cryptolestes pusillus and Cryptolestes ferrugineus

1. Introduction Fumigation is the preferred treatment method for post-harvest stored grain products as it is used firstly to protect grain for common native insect pests, and secondly for the eradication of invasive pest species (Emmanuel and Ismaila, 2015). Fumigation treatments utilise chemicals with sufficient toxicity to eradicate the target pest species.

There are many fumigants, such as carbon disulfide (CS2), chloropicrin, dichlorvos (DDVP), ethylene oxide (ETO) and methyl bromide (MB) but have been eliminated for use as commercial fumigants owing to unfavourable properties, especially with regards to chemical residues, work safety and environmental issues, such as methyl bromide have been banned due to their ozone-depleting properties. Phosphine (PH3) is only available fumigant accepted by world trade but has fallen out of favour due to insect resistance, resulting from poor fumigation practices, and the length of time required to reach end-point mortality (Xin et al., 2008). Sulfuryl fluoride requires high concentrations or extended exposures to ensure complete control of egg stages. Consequently, worldwide phosphine is used widely as it is a registered grain fumigant, readily available and cost effective. However, the over-reliance on phosphine has resulted in increasing levels of insect resistance to phosphine. There is an urgent requirement for the development of a fumigant which should kill all stages of insects quickly and be economic in comparison with existing methods for management of strong phosphine resistant stored product insect pests, particularly the flat grain beetle

(FGB) of Cryptolestes species (Coleoptera: Laemophloeidae). C. pusillus and C. ferrugineus have been regarded as the most important flat grain beetle pest species, it is widespread and highly resistant to phosphine (Wee et al., 2016). This has posed a

25 unique challenge to fumigation chemists throughout the world, leading them to investigate ethyl formate (EF) as a possible alternative (Ren and Mahon, 2006).

Ethyl formate is used as a food additive worldwide and is currently registered as a fumigant on dried fruit in Australia (Banks and Hilton, 1996). Ethyl formate is an old fumigant which had been successfully used for package fumigation of dry fruits since 1929 and was evaluated for grain protection in the 1980s. In the last decade, ethyl formate has been successfully deployed as a fumigation treatment for invasive pests in cereals and a variety of fruits such as bananas, grapes and citrus. More recently, ethyl formate has been re-evaluated as a potential alternative fumigant (Haritos, 2003). Commercial-scale trials with ethyl formate (90 mg/L) on wheat, completely killed mixed aged cultures of Rhyzopertha dominica, and adults and larvae of Tribolium castaneum. In contrast, previous laboratory and commercial-scale trials with ethyl formate on wheat, barley, oats, field peas and canola have shown that the internal larval stages of Sitophilus oryzae are difficult to control. The rise of ethyl formate as a fumigant is largely a result of its fast action and degradation to biogenic substances ethanol and formic acid (Ren et al., 2003).

The aim of this study is to assess liquid ethyl formate’s efficacy as a fumigant for treatment of Cryptolestes spp. Specifically, the project will focus on evaluation of liquid ethyl formate for management of both resistant and susceptible strains of C. pusillus and C. ferrugineus by conducting systematic bioassays with ethyl formate for determination of accurate Concentration × time (C×t) products for guiding grain industry to conduct Good Fumigation Practice (GFP) for management phosphine resistance.

26 2. Materials and methods 2.1. Insects The stored product insects used for bioassays, were C. pusillus and C. ferrugineus. They were the phosphine susceptible strains of WACP and WACF, and phosphine resistant strains of QCF and QCP, held at Postharvest Biosecurity and Food Safety Laboratory, Murdoch University, Australia. Phosphine susceptible and resistant strains of C. pusillus and C. ferrugineus were reared separately at initial densities of 400 adults on 500 g medium comprising 1 part yeast and 12 parts cracked oats. Prior to use for rearing, the cracked oats were conditioned to 12.5% m.c. and then disinfested by freezing at -

20C for one week. The insects used for the bioassays were derived from cultures held at 27C and 68% r.h (Figure 2-1). During rearing at cultural room, cleaned fresh apple peel was placed on the medium for providing moisture. Two-weeks old adults were selected for the bioassays.

Figure 2-1. C. pusillus and C. ferrugineus colonies cultured at 27C and 68% r.h held at Postharvest Biosecurity and Food Safety Laboratory, Murdoch University.

C. pusillus C. ferrugineus

Figure 2-2. C. pusillus and C. ferrugineus colonies cultured in the jar containing cracked oats and apple peel at 27C and 68% r.h.

2.2. Fumigant and apparatus Liquid ethyl formate (99% purity) for fumigation of insects and making gas standards was purchased from Sigma-Aldrich Chemical Company Inc, Australia.

A 25 l syringe (SGE, Melbourne, Australia; Cat. No. 25R-GT) was used for the transfer of liquid fumigants and a 100 l air tight syringe with valve (SGE, Melbourne,

28 Australia; Cat. No 005279) was used for the injection of gas samples into the gas chromatographs (GC).

One litre Erlenmeyer flasks (Bibby Sterilin, Staffordshire, Cat. No. FE 1 L/3) equipped with a cone/screw-thread adapter (Quickfit, STS; Bibby Sterilin) was used to make gas standard. The exact volume of each Erlenmeyer flask and inlet system was calculated from the weight of water required to fill the container.

2.3. Measurement of ethyl formate concentrations During the period of fumigation, the ethyl formate concentrations in fumigation chambers and Tedlar bags were determined using a Varian STAR 3400CX gas chromatograph (GC) equipped with a flame ionisation detector (FID) after isothermal separation on a 30m × 0.53mm (i.d.) megabore capillary column ZBWAX (B13844) at

° an oven temperature of 95 C and carrier flow (N2) of 6 ml/min at 1320 mm Hg (Figure 2-3)

Gas standards were prepared by injection of a known volume of liquid ethyl formate into 1 L Erlenmeyer flasks. The diluted gas was injected into the GC to obtain a calibration curve based on peak areas. From the fumigation chambers ethyl formate, 60 µL was injected manually into the GC at timed intervals, and the concentrations were calculated on the basis of peak areas against the calibrated gas standards which were also read periodically during the 6 h fumigation period. Duplicate injections were made for each sample port.

Figure 2-3. Analysis of the concentrations of ethyl formate with Gas Chromatograph (GC) equipped with Flame Ionisation Detector (FID).

2.4. Measurement of temperature, relative humidity and carbon dioxide. During the fumigation, the temperature and r.h. were automatically recorded with a HOBO data logger unit, (Model number H08-004-02, Onset Computer Corporation, MA 02532, USA, www.onsetcomp.com) placed in the control chamber (Figures 2-4 and 2-5). The recorded data were read with the software BoxCar Version 3.6+ for Windows (Onset Computer Corporation, MA 02532, USA, www.onsetcomp.com). The HOBOs had previously been checked for calibration in the laboratory against each other and a standardised mercury in glass thermometer, as well as a range of glycerol/water solutions for r.h.

CO2 concentrations were measured using a gas analyser Oxybaby Witt-Gasetechnik D- 58454 (WITT Gasetechnik GmbH & Co, Germany).

30 2.5. Fumigation and insect bioassay A flow-through fumigation apparatus (Figures 2-4 and 2-5) was used to fumigate the insects with constant concentrations of ethyl formate and without CO2 cumulation. An exposure time of 6 h was used for this study. The range of ethyl formate concentrations was 2.78–35.65 mg/L for treatments of both phosphine resistant and susceptible strains of C. pusillus and C. ferrugineus (24 treatments, 8 dosage levels for each strain). The apparatus maintained low CO2 concentrations for 6 h fumigation. The fumigation chambers were 2.7 litre desiccators equipped with a ground glass stopper fitted with a septum (Alltech Associates Australia, Cat. No. 15419). A filter paper (Whatman No. 1) was inserted into the glass stopper to provide a liquid evaporation surface for the injected ethyl formate. Wide necked glass jars (20 mm  10 cm i.d.) were used as insect containers with “Fluon” painted around their necks to prevent the adult insects escaping. Six glass jar (9 cm  1.5 cm) contain 2 g cracked oats (3 for C. pusillus and 3 for C. ferrugineus) each containing about 100 adult insects were placed in the unsealed desiccators and left overnight at 25C and 62.5% r.h. prior to the fumigation treatment the next morning. Tedlar gas bags (10 L) (Air Met Scientific Australia) were used for supplying constant concentrations of ethyl formate. Air (10 L) at 25C and 62.5% r.h. Calculated volume of ethyl formate was injected into the Tedlar bags to give the required fumigation concentration. Each experimental run consisted of four or five fumigated desiccators of different ethyl formate concentrations and one unfumigated control desiccator (Figure 2-5).

Each Tedlar bag containing a different concentration of ethyl formate was connected by Teflon tubing (2.3 mm i.d.) through the stopper to a desiccator. An outlet (Teflon tubing) was connected from each desiccator stopper to air pump (125 ml/min) (Figures 2-4 and 2-5). All the tubing was of the same length and prior to starting the experiment a flow meter was connected to the inlet tubing to check all chambers had similar flows. A calculated volume of ethyl formate to give the same concentration as the connected Tedlar bag was injected into each desiccator before starting the pump. A control (no

31 treatment) desiccator with insects and Tedlar bag was set up using the same operational procedures and conditions but without the addition of the ethyl formate.

At the completion of the 6 h fumigation, the desiccators were opened and aired for 1 h in a fume cupboard. After aeration the insects (treated and untreated) were placed in recovery glass jars (50 mm  65 mm i.d.) containing 10 g of culture medium and held at 27C and 68% r.h.

Figure 2-4. Schematic representation of a flow-through apparatus used to fumigate insects to provide a constant concentration of ethyl formate in fumigation chambers.

Figure 2-5. The flow-through apparatus used to fumigate insects with a constant concentration of ethyl formate in the fumigation chambers.

Mortality was assessed by counting dead and live adult insects after a 24 h recovery time, and compared with the control held at the same exposure temperature and r. h. For estimation of endpoint mortality in treatment and control chambers, mortalities were continually assessed for 1, 2 and 3 days post treatment.

The toxicity of ethyl formate to the phosphine resistant and susceptible strains of adult C. pusillus and C. ferrugineus was estimated from the level of mortality in 2400 (100 insects × 3 jars × 8 concentration levels) adult insects. All data analyses have been carried out using the statistical package GenStat. The values of the parameter estimates and the LD50 (to allow comparison with other studies) and LD99.5 (consistent with number of insect treated) values were obtained from fitting a Probit model to the data relating mortality to toxin exposure, that allowed for separate parameter estimates to be obtained for each specie and strain, and for the formal testing of a single Probit response curve versus separate response curves for each insect species. The mortality was adjusted for the control insect mortality (generally less than 2% for 6 h treatment).

2.6. Determination of concentration  time products (Ct) The concentrations of fumigants were monitored at time intervals over the exposure period and were used to calculate the product Ct = Concentration  time. The Ct products were calculated from Eq. 1.

Ct =  (Ci+Ci+1) (ti+1-ti)/2 Eq. 1.

Where: C is fumigant concentration (mg/L)

t is time of exposure (h)

i is the order of measurement

Ct is concentration  time products (mg h/L)

3. Results and discussion 3.1. Temperature, relative humidity and carbon dioxide during exposure For all the fumigation experiments, temperature and r.h in the fumigation chambers during the 6 h fumigation period were recorded with HOBO data loggers as shown in Figure 2-6. The variation of temperature and r.h in the fumigation chambers during 6 h exposure period were 25.0±0.3C and 62.5±0.5%, respectively (Table 2-1).

The variation of ethyl formate concentrations and range of CO2 concentrations in the fumigation chambers was 0.04-0.08% during 6 h exposure period (Table 2-1).

During 6 h exposure period, temperature, r.h and CO2 in the all fumigated and control chambers were consistently maintained at lower variation levels of 25.0±0.3C,

62.5±0.5% and CO2 concentration same as in ambient air and the results are shown in Table 2-1 which are consistent with Xin et al. (2008) who reported using a continuous flow-through laboratory process to maintain constant temperature, r.h and low levels of respiratory CO2. During these experiments the CO2 concentrations were always less than 0.08% ensuring the ethyl formate toxicity results were not compromised by CO2 (Haritos et al., 2006).

34 25.3

25.2

25.1

C)  25.0

24.9

24.8 Temperature ( Temperature

24.7

24.6

24.5

S-Cp R-Cp S-Cf R-Cf

62.9

62.8

62.7

62.6

62.5

62.4

Relative humidity (%) humidity Relative 62.3

62.2

62.1

62 0 1 2 3 4 5 6 7 Time during fumigation (h)

Figure 2-6. HOBO data loggers recorded temperature and r.h in the fumigation chambers during the 6 h fumigation period (S-Cp is susceptible C. pusillus; R-Cp is resistant C. pusillus; S-Cf is susceptible C. ferrugineus and R-Cf is resistant C. ferrugineus).

35 Table 2-1. The variation of temperature, r.h and concentration of ethyl formate and CO2 in the fumigation chambers during 6 h exposure period

Treatments (n) Temperature±SD* r.h±SD Range of CO2 Variation of concentration of Tested insects concentration ethyl formate±SD

Treated Control (C) (%) (%) (%)

C. pusillus

Susceptible 60 12 25.0±0.3 62.5±0.5 0.05–0.08 6.5±1.2

Resistant 60 12 25.0±0.2 62.5±0.5 0.04–0.06 7.1±1.6

C. ferrugineus

Susceptible 60 12 25.0±0.4 62.5±0.6 0.04–0.07 5.6±1.5

Resistant 60 12 25.0±0.3 62.5±0.4 0.05-0.07 6.8±1.7

Average 60 12 25.0±0.3 62.5.0±0.5 0.05–0.07 6.5±1.5

* SD is Standard Deviation

36 3.2. Concentrations of ethyl formate during exposure During 6 h exposure period, the concentrations of ethyl formate were measured at timed interval and results are shown in Table 2-1. Ethyl formate were constant in the all fumigation chambers from 5.6-7.1%. The average variation of ethyl formate concentration was 6.5±1.5% in the 40 treatment chambers. Therefore, the mean value of the concentration can be used to calculate the Ct products with the standard error in fumigation concentration.

In the current study the variation in ethyl format concentration is about 6.5% in 6 h. The range of ethyl formate concentration used was 2.78-35.65 mg/L, 25° C-26% r.h. temperature 25.0±0.3C, 62.5±0.5%. This result is consistent with other research (Ren and Mahon 2003, Agarwal et al., 2015) shown using flow-through system with constant concentrations of ethyl formate and avoiding any CO2 accumulation. However, some previous studies have observed that the variation in concentration of ethyl formate in fumigation is affected by commodities such as grain and fruit. Ren and Mahon (2003) and (2006) has conducted trials with ethyl formate on wheat, sorghum and split faba bean in unsealed farm bins. The liquid ethyl formate was applied as a pulsed, or double, dose to the top of the grain through polyvinyl chloride (PVC) probe (4 cm i.d × 1.2 m) in order to maintain ethyl formate concentrations, reduce vaporisation and maintain an effective concentration of ethyl formate for >20 h. Ren and Mahon (2003) has shown the concentration of ethyl formate has rapidly decline during fumigation at 1，24，48 h with 82.3 g/m3 for 1 h, 48.4 g/m3 for 24 h, 8.8 g/m3 for 48 h for wheat. The decline was more pronounced for faba bean and sorghum. Both commodities showed rapid sorption of ethyl formate when added at single and double. It declined to 10 g/t in the head space within 24 h. Other research by Agarwal, et al. (2015) notes that the concentration of the ethyl formate formulation declined rapidly within the first 10 h, particularly the first 4 h shows75–85% absorption of ethyl formate by fruit. Moreover, Epenhuijsen et al. (2010) has highlight that with the lower concentrations of ethyl formate, most of the surviving thrips were found inside the leek leaves, indicating that the fumigant had not Penetrated all plant tissue at sufficient levels to kill thrips. 37 However, in this research no commodity was used as the purpose of the research was to optimize the Ct for future fumigation practices which can be applied for any commodities in the future.

3.3. Efficacy of ethyl formate to C. pusillus and C. ferrugineus The observed and fitted data in relation to mortality to Ct product and the results of Probit analysis are summarised in Table 2-2. The dosage as Ct product was estimated and parameters of regression for LD50 and LD99.5 mortality achieved against susceptible and resistant strains of C. pusillus and C. ferrugineus adult insects to ethyl formate exposure for 6 h fumigations at 25.0±0.3C and 62.5±0.5% r.h is shown in Table 2-2. The observed data covered mortality from 0-100% and these adequately covered the intermediate range. There were highly significant (P<0.001-0.005) effects for each insect species and strains. There was no significant different mortality between susceptible and resistant strains of C. pusillus i.e., the observed Ct product is at

LD50=13.09 and 12.75 mg h/L respectively and at LD99.5=28.17 and 29.55 mg h/L respectively. For C. ferrugineus susceptible and resistant strains LD50=13.11and 13.26 mg h/L respectively and LD99.5=29.28 and 28.22 mg h/L respectively.

Similarly, Ren and Mahon (2006) have found that mixed-aged cultures for Sitophilus oryzae, Rhyzpertha dominica, Tribolium castaneum and Callosobruchus phaseoli can be killed by ethyl format in farm bins containing wheat, split faba beans or sorghum, They found all the dosage of 85 g/t × 2 ethyl formate gives a high level of control of the all stages of most of the tested insects in the ranges 18-48°C and 25-50% r.h during the wheat train, 17-34°C and 20-30% r.h during the split faba bean trail, and 3-32°C and 10-30% r.h during the sorghum trail, but very hard to achieve complete control of insects when the grain temperature is lower than 15 ̊C. The concentration time (Ct) products of 1100–2100 mg h/L obtained were sufficient to kill all adult stages rapidly. With sorghum, the temperature was just below 20C and the relatively low Ct product of 950 mg h/L obtained was sufficient to kill all exposed insect stages rapidly, but not the internal stages of S. oryzae. In sorghum with temperature below 10C and with Ct 38 of 840 mg h/L the adult insects were completely killed. Internal stages of S. oryzae and R. dominica, however, were controlled to a high level but not with 100% mortality. The article states this may be due to the slow evaporation rate of liquid ethyl formate to ethyl formate gas at lower temperature and the strong absorption of ethyl formate by particles.

To overcome the issue of resistance other studies have reiterated the importance of co- fumigation in pest control for food security like Jagadeesan et al. (2018) examined the efficacy of phosphine and sulfuryl fluoride mixture on phosphine resistant pest strains, including C. ferrugineus stored at room temperature for 48 h. According to the author, phosphine resistant pesticides compel farmers to use high concentrations of up to 1 mg/L to fumigate stored grains for as long as 14 days, which is time-consuming and may not be economically viable for farmers. According to them by using two fumigant the efficacy of the mixture increased, leading to a 99.9% mortality of resistant pest species. Jagadeesan et al. (2018) study differs from this research in the sense that it investigates the effectiveness of phosphine and sulfuryl fluoride as co fumigant mixture in controlling phosphine resistant pest strains, but this research indicated use of alternative fumigant ethyl formate to control both susceptible and resistant strains of C. pusillus and C. ferrugineus, and also provide the evidence that there is no cross resistance between ethyl formate and phosphine.

4. Conclusions This research has provided evidence that ethyl formate is a promising substitute for methyl bromide, phosphine and sulphuryl fluoride for insect pest management in stored grain. Ethyl formate as a fumigant has a long history of successfully treating stored products and is currently registered as one of the fumigants for dried fruits in Australia. Ethyl formate residues also break down rapidly to natural occurring products and are safe for workers and consumers. Furthermore, being safe and environmentally friendly, ethyl formate is also cost effective which bring economic benefit for the Australian

39 agriculture industry and also could potentially be used in-transit, which could shorten the entire supply chain time.

In comparison with all previous studies, the toxicity data obtained from this experiment are more robust as the continuous flow-through system offered a constant concentration of ethyl formate with low CO2 concentrations at a constant 25C and 62.5% r.h. during the 6 h exposure period which can successful control both susceptible and resistant strains of C. pusillus and C. ferrugineus.

C. pusillus and C. ferrugineus bring great threat to Australian agriculture due to strong resistance to phosphine. Ethyl formate as fumigant can rapidly and efficiently kill both susceptible and resistant strains of C. pusillus and C. ferrugineus at lower dosage level. It also has no cross resistance with phosphine. Therefore, ethyl formate has great potential as grain fumigant for grain industry to manage C. pusillus and C. ferrugineus, particularly their resistant strains. These results can be useful to future agriculture industry for stored grain treatment in Australia.

40 Table 2-2. Dosage (measured as Ct product) estimates and regression parameters of Probit mortality with exposure to ethyl formate against susceptible and resistant strains of C. pusillus and C. ferrugineus adult insects for 6 h fumigations at 25.0±0.3C and 62.5±0.5% r.h.

Insect species Insect numbers Insect numbers LD50SE* LD99.5SE* P value (d.f)** and strains control treated (dosage levels) (mg h/L) (mg h/L)

C. pusillus Susceptible 1092 6227 (10) 13.092.73 28.173.91 <0.001 (10) Resistant 1107 6302 (10) 12.752.81 29.554.58 <0.005 (10)

C. ferrugineus Susceptible 1121 6219 (10) 13.114.14 29.283.03 <0.003 (10) Resistant 1068 6251 (10) 13.263.50 28.224.57 <0.001 (10)

* SE is Standard Error. ** Degrees of freedom.

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