Exploration of ethyl formate + nitrogen as a fumigant for shipping containers and their in-transit fumigation This thesis is presented for the degree of Doctor of Philosophy at Murdoch University By Eugene Marco Coetzee College of Science, Health, Engineering and Education Murdoch University Harry Butler Institute Perth, Western Australia November 2020 1 Declaration I declare that this thesis is my own account of my research and has not been submitted for a degree at any tertiary education institution. Signature: Eugene Marco Coetzee Date: 30/11/2020 2 Abstract Fumigation is required by most governments as an appropriate biosecurity measure to exterminate pests in shipping containers. The use of shipping containers for cargo transportation has the potential to transport pests from infested to non-infested areas. Initially fumigation trials were conducted in stationary 20ft shipping containers. Four species of stored product insect adults were used for bioassays namely, the Cigarette beetle, Lasioderma serricorne (F.), Rice weevil, Sitophilus oryzae (L.), Warehouse beetle, Trogoderma variabile (Ballion) and the Lesser grain borer, Rhyzopertha dominica (F.). Ethyl formate (90 g/m3) was purged with nitrogen (99.5%) into the containers. Ethyl formate concentration inside containers and the surrounding environment was monitored at timed intervals. Fumigation achieved target concentration × time (Ct) products of 437.54 - 449.19 g h/m3 in the containers, which can exterminate all life stages of most common insect pests. Ethyl formate distributed evenly in the containers within 0.5 hours after application with a variation <3%. This study demonstrated that onsite generation of a non-flammable ethyl formate + nitrogen fumigant can be achieved and that this new ethyl formate + nitrogen application can be used as a quarantine pre-shipment treatment for controlling insect pests in shipping containers. Ventilation of shipping containers after fumigation to remove ethyl formate from containers was successful with a threshold limit value (TLV) of zero achieved after approximately 15 minutes. The levels of ethyl formate in the surrounding workspace were <0.5 parts per million (ppm) during application, fumigation and aeriation which is 200 times below the required TLV level of 100ppm. In-transit ethyl formate and nitrogen fumigation trials were conducted in 20ft shipping containers during a two-day road journey in both September and December 2017. Ethyl formate (90 g/m3) was purged with nitrogen into the containers. Ethyl formate concentrations inside the containers and the surrounding environment were monitored at timed intervals throughout the journey. Fumigation achieved concentration × time (Ct) products in the containers during the journey, which can exterminate all life stages of most common insect pests. Levels of ethyl formate in the environment between 1-15 meters downwind from the containers and truck driver's cabin were less than 0.5ppm at each of the timed intervals. The study confirms that ethyl formate can safely be used as an in-transit fumigant. 3 Further trials were conducted in 20ft shipping containers during a two-day road journey and a one-day sea journey. Ethyl formate concentrations inside the containers and the surrounding environment on the vessel were monitored at timed intervals throughout the overnight sea voyage. This research adds to the previous research for in-transit fumigation with ethyl formate and nitrogen via road and has successfully demonstrated that in-transit fumigation with ethyl formate and nitrogen via the marine sector is effective and also safe with no detectable risk to the public, crew members on the barge or workers on Barrow Island throughout the journey. In addition, all tested containers were ready to be opened and unloaded with 5-10 minutes aeration or without aeration upon arrival. In conclusion, this study indicates that in-transit ethyl formate and nitrogen technology has the potential to deliver cost savings in the fumigation process through reduction of the labour cost, elimination of the time a container and cargo must remain stationary in a fumigation yard and a significant decrease in total supply chain time (between container packing and receival). 4 Table of contents Declaration ............................................................................................................................. 2 Abstract .................................................................................................................................. 3 Table of Contents .................................................................................................................. 5 List of abbreviations ........................................................................................................... 11 List of Tables ....................................................................................................................... 15 List of Figures ...................................................................................................................... 16 Publications ......................................................................................................................... 18 Presentations ........................................................................................................................ 19 Acknowledgements .............................................................................................................. 20 Chapter One Literature Review .......................................................................................................... 21 General Introduction and literature review ..................................................................... 22 1.1 Barrow Island ................................................................................................................ 23 1.1.1 Biodiversity of Barrow Island ............................................................................... 23 1.1.2 Biosecurity to protect Barrow Island ................................................................... 24 1.1.3 Barrow Island pathways for non-indigenous species .......................................... 26 1.2 Invertebrate pests and non-indigenous invasive species............................................ 27 1.2.1 Invertebrate pests ................................................................................................... 27 1.2.2 Non-indigenous invasive species ........................................................................... 27 1.3 Fumigants and fumigation – as mitigation measure .................................................. 29 1.3.1 Fumigants .............................................................................................................. 29 1.3.1.1 Methyl bromide ........................................................................................... 31 5 1.3.1.2 Phosphine ..................................................................................................... 32 1.3.1.2 Sulphuryl fluoride ....................................................................................... 33 1.3.1.3 Treatment of quarantine pests................................................................... 34 1.3.2 Fumigation ............................................................................................................. 35 1.3.2.1 Fumigation of shipping containers ............................................................ 36 1.3.2.1 In-transit fumigation of shipping containers............................................ 38 1.4 Ethyl formate ................................................................................................................. 39 1.4.1 Ethyl formate as a fumigant .................................................................................. 41 1.4.2 Toxicity of ethyl formate to insect pests .............................................................. 42 1.4.3 Determination of concentration x time (Ct) ........................................................ 44 1.4.3.2 Well sealed enclosures that have passed a pressure test………………...45 1.4.3.2 Under Gas proof sheeting where gas losses are high……………………45 1.4.4 Residues .................................................................................................................. 46 1.5 Nitrogen as a fumigant and carrier gas....................................................................... 47 1.6 Fumigants and synergism ............................................................................................. 48 1.7 Ventilation, safety and the environment ..................................................................... 49 1.8 Aims of this PhD thesis ................................................................................................. 50 Aims of this study in chapters ....................................................................................... 51 Chapter Two Evaluation of ethyl formate + nitrogen as a suitable treatment fumigant for 20ft shipping containers loaded with general freight…………………….52 Chapter 2. Declaration of contribution ............................................................................. 53 Abstract ................................................................................................................................ 54 2.1 Introduction ................................................................................................................... 54 6 2.2 Materials and methods ................................................................................................