Study on Metabolic Behavior of Pesticides in Aquatic Plants: Uptake, Translocation and Metabolism by Water Milfoil Daisuke Ando The United Graduate School of Agricultural Sciences, Tottori University Doctoral Thesis 2019 Page Abbreviations ................................................................................................................... 1 1. Introduction ................................................................................................................. 3 1.1 . Importance of Pesticide Risk Assessment for Water Milfoil .................... 3 1.2 . General Knowledge of Pesticide Behavior in Terrestrial and Fresh Water Aquatic Plants .................................................................................. 5 1.3 . Object of The Study.................................................................................... 13 2. Fate Comparison of Pesticides Between Terrestrial and Fresh Water Aquatic Plants ....................................................................................................................... 15 2.1. Behavior of Insecticide Metofluthrin .......................................................... 15 .......................................... ................................... .................. 2.2. Behavior of Fungicide Mandestrobin ........................................................ 50 ............................................. .................................. ...................... 2.3. Conclusion .................................................................................................... 81 3. Development of Experimental Design to Investigate Uptake, Translocation and Metabolism of Chemicals by Water Milfoil ......................................................... 82 4. Uptake, Translocation, and Metabolism of Phenols by Water Milfoil: Kinetic Analysis and Correlation With Physicochemical Properties.............................. 95 5. Fate of Flumioxazin in Aquatic Plants, Two Algae, Duckweed and Water Milfoil..................................................................................................................... 115 6. Overall Summary, Discussion and Conclusion .................................................. 141 7. References ............................................................................................................. 148 8. Acknowledgement ................................................................................................. 167 9. Synopsis ................................................................................................................. 168 10. The List of Published Articles as Base of The Doctoral Thesis ...................... 170 ABC transporter: ATP (adenosine triphosphate)-binding cassette transporter ADME: absorption, desorption, metabolism and excretion AR: the applied radioactivity BCF: bioconcentration factor BMF: biomagnification factor CYP: cytochrome P450 DEPT: distorsionless enhancement by polarization transfer DOM: dissolved organic matter DT50: degradation half-life time EC50: 50% effect concentration EC: European Commission ECOD: 7-ethoxycoumarin-O-deethylase EFSA: European Food Safety Authority EROD: 7-ethoxyresorufin-O-deethylase EU: European Union FAO: Food Agriculture Organization of the United Nations GSH: glutathione 1H-1H COSY: correlation spectroscopy HCH: hexachlorohexane HMBC: hetero-nuclear multiple-bond connectivity HPLC: high-performance liquid chromatography HRMS: high resolution mass spectrometry HSQC: hetero-nuclear single quantum coherence LC ESI MS: Liquid chromatography electrospray ionization mass spectrometry LOD: limit of detection log Kow: logarithm of octanol/water partition coefficient LSC: liquid scintillation counting NOE: nuclear overhauser effect difference NOEC: non-observed effect concentration 1 NOESY: NOE correlated spectroscopy PCB: polychlorinated biphenyl QoI: quinone outside inhibitor ROS: reactive oxygen species SOM: superoxide dismutase TLC: thin-layer chromatography TMS: tetramethylsilane TRR: the total radioactive residue TSCF: transpiration stream concentration factor US: United States FAMIC: Food and Agricultural Materials Inspection Center PEC: predicted environmental concentration 2 1. Introduction 1.1. Importance of Pesticide Risk Assessment for Water Milfoil Synthetic pesticides are chemical products which contribute to our continuous supply of agricultural food products by controlling harmful insects, fungus and weeds, and have been recognized as one of the important measures to encounter growing concerns for food shortage accompanying global population growth. Also, non- agricultural use of insecticides for household and public hygiene helps us to improve our living standards, and especially in developing countries, to refuge from infection such as malaria and dengue diseases mediated by mosquito or other vectors. While providing considerable profits, pesticides may cause adverse effects for crop consumers or users, workers, bystanders and wild lives, hence, safety for each of them must be assured. In addition, since they are directly sprayed or applied at outdoor environment, pesticides may unintentionally enter into fresh water aquatic ecosystem by spray drift, run-off, drainage or accidental spills. From such aspect, risk evaluation for aquatic ecology is necessary and has been gaining much attention in recent years. The fresh water aquatic ecosystem consists of wide variety of aquatic organisms including aquatic plants as primary producers, animal planktons and larger fish or other predators, establishing sustainable trophic food chain and nutrient circulation through complex interactions in the aquatic community. Among them, aquatic plants are very important for production/circulation of oxygen and nutrient which securely provide precious food and shelter for many aquatic biota (Scheffer 1988; Maltbny et al. 2009; Lewis 1995). They play key functions in biochemical cycles, through, for example, organic carbon production, phosphorous mobilization, the transfer of other trace elements, siltation of particulate matter as carbon sinks. They also directly influence the hydrology and sediment dynamics of fresh water ecosystems through their effects on water flow and particle trapping and re-suspension (Marion and Pailisson 2002; Madesen et al. 2001).5-6 Because of their significance, adverse effects on aquatic plants will give serious impacts to deteriorate the overall aquatic ecosystem. 3 Herbicides and plant-growth regulators, from the aspect of their specific mode of actions uniquely designed for plants, may cause substantial damages toward non-target aquatic plant species. In the risk assessment for fresh water aquatic plants in EU, the acute toxicity data (EC50) for two algae and one macrophyte such as Lemna sp. had been required to determine fundamental adverse effect to aquatic plants (EC 2002) (for comparison, test with one and four algae is normally required in Japan and US registration, respectively, although additional species are required in case by case basis, thus, EU intends to cover wider aquatic plants for the risk assessment). However, the limited assessment with these suspended or floating species may not be sufficient to cover entire aquatic flora, due to the sediment-rooted macrophytes may potentially be exposed to chemicals not only from the water but additionally from the bottom sediment via root uptake, especially in the case pesticides preferentially bound to the sediment (Lewis 1995; Belgers et al. 2007; Cedergreen et al. 2004b; Turgut and Fomin 2002). From such affairs, in the latest aquatic guidance issued by EFSA in 2013, the risk assessment for submerged-rooted macrophytes, water milfoil as representative, was newly included as additional toxicity test species to be evaluated, if both algae and Lemna are highly susceptible to the chemical or if chronic sediment exposure is to cause concerns. As indicated, there are two major routes that water milfoil may be exposed to pesticides; shoot exposure via water column and root exposure via sediment, and these events could occur simultaneously. And after the pesticides entered into the macrophyte, they will be translocated within the plant from root to shoot and vice versa with receiving metabolization/detoxification. While toxicological effect of pesticides are widely investigated, the information of metabolic behavior in aquatic plants is limited. And especially, there are few studies focused to clarify the uptake, translocation and metabolism of pesticides individually after shoot and root exposures. Since such knowledge is essential for understanding toxicity mechanism of the pesticides and for detailed risk evaluations, we developed an experimental design and studied the behavior of simple chemicals and a herbicide. 4 1.2. General Knowledge of Pesticide Behavior in Terrestrial and Fresh Water Aquatic Plants To understand the behavior of pesticides in water milfoil, basic knowledge from terrestrial and fresh water aquatic plants are important. In this section, the information is briefly summarized, focusing on uptake, translocation and metabolism supplemented by abiotic factors affecting on the processes. Terrestrial Plants Foliar Uptake The application of a pesticide is generally classified into foliar and soil treatments. The behavior of pesticide in the former treatment has been investigated extensively, not only experimentally but also theoretically by many researchers, but fewer investigations are available for the latter. The foliar