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ENVIRONMENTAL PROTECTION AUTHORITY Application for the Reassessment of Hazardous Substances under Section 63 and Section 63A of the Hazardous Substances and New Organisms Act 1996 [www.epa.govt.nz]

Applicant: Dr. Allan Freeth, Chief Executive, Environmental Protection Authority Name of substance: Non-professional use of chlorothalonil formulations Application number: APP202349

Substances for Reassessment HSNO Approval Number Suspension concentrate containing 500 gm/L chlorothalonil (Substance B) : HSR000480 Suspension concentrate containing 102 gm/L chlorothalonil and 125 g/L thiophanate methyl: HSR000147 Suspension concentrate containing 62.5 gm/L chlorothalonil, 9.6 g/L tau -fluvalinate and 62.5 g/L thiophanate methyl: HSR000586 Suspension concentrate containing 250 g/L chlorothalonil and 250 gm/L thiophanate methyl: HSR000618 Tui Disease Eliminator (ready to use) HSR100

Submitter: Anthony R. Bellvé, wishes to be heard on behalf of ‘Waikato Domestic Beekeepers’ Association’ Contact: T: 07 957 0300; M: 022 60 71217; F: 07 260 0300; E: [email protected] President: Cameron Blackbourn Contact: T: 07 846 7864, M: 021 766 123, E: [email protected] Submitted: Friday, 16th December 2016 A. Recommendations The Waikato Domestic Beekeepers’ Association (WDBA), having reviewed recent evidence relating to the deleterious effects of Chlorothalonil, and related chemicals, on honey bees, Apis melliflora and A. carnica*, New Zealand’s two major and distinct bee species, strongly recommends rescinding the fungicide/pesticide’s current approval status and thereby discontinuing all retail distribution and domestic applications of Chlorothalonil, country-wide. B. Executive Summary The WDBA has reviewed contemporary scientific evidence on the effects of: i) Chlorothalonil alone; ii) Chlorothalonil in two-way combination with Tau-fluvalinate or Methyl-thiophanate; and iii) Chlorothalonil in three-way combination with Tau-fluvalinate and Methyl-thiophanate, at the specified concentrations, when applied to flowering plants in various domestic usages, and assorted professional agricultural and horticultural applications.

*Apis carnica is represented by ‘Southern Cross Carniolans’ hybrids (Refer: Daykel Apiaries, Taipa, Northland, New Zealand).

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Chlorothalonil, and derivatives listed by the ‘European Chemicals Agency’ (ECHA, https://echa.europa.- eu/), are highly potent acaricides, bactericides, fungicides, miticides and pesticides. Chlorothalonil used on domestic plants and trees and professional applications on agricultural and horticultural crops, has proven to be: a) Incorporated preferentially into crop nectar and pollen during flowering; b) Harvested by pollinating honey bees (and bumble bees); c) Transferred inadvertently by adult bee foragers to in-hive brood; and d) Killed larval and pupal stages during early development of domestic bees. Chlorothalonil and related chemicals now are deemed to be foremost causes of the recent ‘World-wide’ collapse of honey bee populations, an event now generally termed ‘Colony Collapse Disorder’ (CCD)18,19. C. Bees’ Wax and Wane 1. The Industrious Pollinators Pollinating vectors World-wide, comprising wind, water, insects (beetles, bees, bumble bees, butterflies, flies, moths, thrips and wasps) and a variety of vertebrates (bats, birds, lizards, capuchins, snakes), contribute toward enhancing the diversity and survival of native plants, increasing propagation and production of agricultural and horticultural crops, and augmenting food efficiency and security. Among these pollinators, there are over 20,000 species of bees specialized for pollination, of which 50 species are managed and of the latter 12 are deployed primarily to pollinate agricultural and horticultural crops. Apis mellifera and A. cerana are the most abundant contributors to pollination of flowering plants (Fig. 2). Indeed, honey bees pollinate 71% to 75% of 107 agricultural crops collectively contributing 90% of food consumed by today’s World-wide population. Honey bees pollinate and thereby ensure propagation of more than 4,000 of the planet’s crop species, together with ensuring diversity and survival of native plants among innumerable ecosystems. Globally, honey bees earn more than US$200 billion per year. Recently, the Food and Agriculture Organisation (FAO), United Nations, published estimates of pollinators relative, fiscal contribution to World-wide food production, expressed as presumed losses incurred in the absence of api-cultural, flower pollination. These data enable global and country-wide comparisons between 1961 and 2012 (Figs.1A &1B). During the ~50-year period, there was a marked >2-fold increase in food production dependent on pollinators19. This trend reflected changes in cultural and societal demands for pollinator-dependent consumable foods, and, in some case, particular increases for high-value products destined for international markets (e.g. coffee beans). Thus, greater global harvest and consumption of horticultural crops, increased dependence on faunal pollination19. Similarly, New Zealand’s increasing production and export of horticultural products, mirrored by recent increases in the sector’s gross national product (GNP), confers more reliance on pollination by honey bees. FAO’s estimates indicate New Zealand could have lost 5% to 7% of its productivity, in 1961, through loss of pollinators; but, that drop would have extended to 15% to 25% less in 2012 (Fig. 1). The country has become 3-fold to 5-fold more reliant on api-culture for pollinating agricultural (clovers, lucerne, rape) and horticultural crops (fruits, grapes, vegetables (Figs. 1Aa & 1Bb)19. Thus, New Zealand’s rapidly expanding horticultural export markets will necessitate propagating and sustaining healthy populations of managed honey bees. Further, honey bees will become increasingly important as food supplies are subject to greater demand globally. The Planet’s population, based on recent re-assessments, is expected to increase by ~83 million peoples annually, attaining a total of 9.7 billion by 2050 and 11.2 billion by 2100 (‘World Population Prospects – a Revision’, Department of Economic and Social Affairs, Population Division, Programme for Development, United Nations, 2015). Likewise, New Zealand’s population will continue to increase exponentially. The population-driven demand for greater intensity in agricultural and horticultural production, will necessitate more abundant and efficient pollination services over coming decades. Honey bees, in addition to producing honey, propolis and wax, make substantial contributions through flower pollination toward World-wide food production. Indeed, there will be greater prerequisites for New Zealand to nurture and sustain native and managed populations of viable and efficient honey bees, to ensure optimal security of crop pollination for the future well-being of our children and grandchildren. 2

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A 1961 Aa 1961

2012 B Bb 2012

FIG. 1: World-wide agriculture losses were estimated, in the absence of flower pollination by faunal vectors, for 1961 [A, Aa] to 2012 [B, Bb]. Greater fiscal exposure occurs as the World’s population harvests more horticultural crops [Potts et al., Nature, 2016, 19 based on data from FAO, United Nations (See: http://www.fao.org/faostat/en/#home)].

2. Pressures on Waning Bee Populations Bee populations, both native and managed, are subject to substantial pressures from direct and indirect anthropogenic drivers. The present brief is based on the excellent and highly recommended reviews by Potts et al.’s (2016) ‘Assessment Report of the Intergovernmental Science - Policy Platform on Biodiversity and Ecosystem Services on Pollinators, Pollination and Food Production’, pp. 1-888, together with other recent reviews and pertinent scientific reports. In this context, there are five main classes of drivers threatening the effectiveness and survival of existing bee populations18,19. These include: a) Changes in type and intensity of land management, b) Applications of pesticides - acaricides, fungicides. insecticides, herbicides, molluscicides – directly or indirectly (plant GMOs - herbicides) affecting flowering plants in urban and rural settings, c) Parasitic loads of pathogens (bacteria, fungi & viruses) and parasites (protozoa & vertebrates); e.g. Varroa mites (Varroa destructor) parasitizing A. Cerana and later spreading to A. mellifera. d) Invasions of parasitic alien pests compromising pollinator health through dysfunction to death; e.g.: Western Yellow Jacket (Vespula pensylvanica), a wasp killing native bees on Hawaii, USA. e) Spatial and temporal global-warming shifts of pollinators- versus flowering-crop eco-systems. 3

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These five drivers summarize 74 possible causes promulgating rapid ‘Colony Collapse Disorder’, a syndrome fundamental to recent losses of native and domestic bees, especially throughout Europe and the Americas (See: IPBES-4/1)18,23,26. Recent evidence is consistent with these parameters having interactive effects with negative synergistic impacts on the health of managed bee populations and thereby threatening future propagation and production of pollinated crops important for human well-being10,19. 3. Genetics of Native and Domesticated Bee Species Bees have been ‘domesticated’ for the past 7,000 years and managed with gradually increasing sophistication, particularly upon the relatively recent transition from clay pots through the Langstroth hive10. During this period, various bee species have been seconded to the task of generating honey and related products. Only recently have important gains been made toward elucidating the genetic inheritance of native and domesticated bee species and identifying traits related to their survival23. A B C

FIG. 2: Genetic analyses of domesticated bee species signifying their respective origins23. [A] Worker bee specimens were collected from native sub-species in Africa, Europe and Middle East (Coloured circles) and domestic strains of America and Europe (Coloured diamonds). Africanized strains were gathered from Brazil and A. Cerana from Japan. [B] Bee DNA was sequenced to discern and map alleles defining distinct sub-species in four clusters or clades (M, C, O, A), with A. cerana comprising an Outgroup, as indicated in the linkage tree. [C] The main subgroups, M, C, O, & A, are delineated, based on their genealogy, from three ancestral groups, existing between 150,000- to 350,000-years, followed by sub-species’ splits 20,000- to 35,000-years before the present (Details, see source: Wahlberg et al., 2014).

The genealogical tree clearly delineates close genetic links, for instance, between A. mellifera with A. mellifera iberiensis in Clade M, and likewise between A. mellifera anatoliaca and A. mellifera syriaca; whereas A. cerana (from Japan) is a member of a distinct out-group lying between the other four clades23. These data strongly support the origin of A. mellifera most likely stems from Eastern Asia, rather than Africa, as previously supposed. These genetic data reveal important findings relevant to the present objectives. Bee colonies exhibit high rates of meiotic recombination by exchange of gene sequences between germ cells during meiotic divisions, and hence high levels of genetic variation among members of each colony23. Genetic variation is greatest among African native bees and lowest among European domestic bees. Moreover, the study identified genes sequences known to control key aspects of reproduction, embryonic development, social behaviour, immune function, over-wintering and responses to pathogens. Consequently, science is close to characterizing roles of those genes regulating A. mellifera responses to various pesticides and pathogens. D. Chlorothalonil - A Broad-Spectrum Pesticide Targeting Bees, Frogs and Fish Chlorothalonil is a broad-spectrum pesticide (www.-pesticide.org) (Appendix I, Fig. 4). Chlorothalonil is an extremely toxic pesticide for diverse, e.g.: Green algae (Selenastrum capricornutum), LC50: 6.8 μg/l; tadpole and/or adult Toad (Bufo bufo japonicas) (LC50: 160 μg/liter); Bog frog (Rana limnocharis) (LC50: 245 μg/l); Shrimp (Paratya astraliensis) (LC50 0.3 μg/l); Dungeness crab (Cancer majister) (LC50: 100 μg/l); Giant Tasmania freshwater crayfish (Astacopsis gouldi) (LC50: 7.9 μg – 38.5 μg/l) through Rainbow trout (LC50: 4

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Cautionary note: 20 compounds closely-related to Chlorothalonil, share the organochlorine benzene structure of Dichlorodiphenyltrichloroethane (DDT), the subject of Rachel Carson’s “Silent Spring”. The degradation of product of chlorothalonil, 4-hydroxy-chlorothalonil, persists longer in the environment and is ~30-fold more potent than the parent compound17. https://pubchem.ncbi.nlm.nih.gov/compound/15910#section=Top & https://www. ncbi. nlm.nih.gov/pccompound?cmd=Link&LinkName=pccompound_pccompound_parent_pulldown&from_uid=15910 Notably, given extensive scientific literature, the NZ EPA does not consider Chlorothalonil’s effects on honey bees or the compounds likely inclusion into food products created by bees27.

3,4,7,13,14 19.0 μg/l. 33 – 47 ppb) . The pesticide invoked 15 major fish kills from 2000 through 2016, in streams and rivers on Prince Edward Island, Canada. Further, TOXNET, National Institute of Medicine, and PUB- CHEM, NCBI, USA, report chlorothalonil kills insects, including adult and larval stages of native and domes- ticated bees (see below). Note: Chlorothalonil (Mw: 265.902 g/mol−1),10 μg/l Ξ 10 ppb (parts per billion). E. Bees and Pesticides – End of the Wiggle Dance Bees operate within closed communities guided by sophisticated eusocial rules that enable coordinated interactions of a single Queen bee, numerous female Worker bees (<50,000), and a limited number of male drones (<300)12. During spring and summer, the Queen lays numerous eggs to enable rapid expansion of the Worker population for assuming roles as nurse bees, water carriers, hive guards, and foragers of nectar, pollen and water. Worker bees are responsible for flower pollination and for storing pollen and making honey, bee-bread, wax and propolis. Male drones fertilise the virgin Queen bees, who thereby undertake egg-laying with precision into hexagonal, wax combs; nurturing eggs through larval and pupal stages; and emergent of young adult bees, in defined temporal stages. Adult bees navigate extraordinarily well over substantial distances and communicate locally by releasing pheromones and displaying sets of physical dances (twirls, vibrations and wiggle movements)12. Recent research has established potent and distinct biologic actions of Chlorothalonil on developing larvae and adult bees, quickly leading to lethargy and their demise. The pesticide, on this basis, is a likely contributor to Colony Collapse Disorder2,5,8,9,13,18,22,25,26. This evidence can be elaborated in sequence from pesticide application to subsequent collapse and demise of the bee colony, as follows: 1) Application of Chlorothalonil to crops, in this case peanuts, results in 60% to 65% of the highly lipophilic pesticide binding to crop and nearby weed foliage, with 30% to 35% falling onto underlying soil, in quantities reflecting the spray rate and concentration17. Chlorothalonil recovered from ‘treated’ soil has converted, within hours, into the more stable and highly toxic 4-hydroxy-chlorothalonil (Appendix I: Fig. 4). Proportionately more Chlorothalonil binds to crop surfaces, and less to soil, as foliage cover increases17, 2) Soil-borne Chlorothalonil and 4-hydroxy-chlorothalonil eroded into wetlands, streams and rivers is highly toxic for aquatic species4,14. In New Zealand’s case, targets likely comprise native frogs (Pepeketua) – Hochstetter’s frog, Hamilton’s frog and Maud frog – all of which are highly endangered species, and the Galaxiids: Banded kōkopu, Giant kōkopu and Short-jaw kōkopu, Inanga and Kōaro. 3) Chlorothalonil bound to crop foliage and/or flowers is ‘incorporated’ into pollen15, and nectar, and on collection by foraging Worker bees, the pesticide is carried, inadvertently, to the hive for storage and biotic processing into bee-mead, honey, propolis, Royal jelly or wax15,19,24. Adult bees, whether forager, guard, nurse or Queen are exposed to the pesticide, either directly while conveying pesticide- ridden nectar and pollen and/or indirectly during internal conversion of primary into secondary bee 9 products . Foraged pollen, laden with <19 distinct pesticides and fungicides have potencies of LC50 <51,310 ppb9, 4) Chlorothalonil-ridden, high-protein pollen, when converted to mead for nourishing eggs and larvae, actually kills most recipients within two to three days25,26. If the toxic assault coincides with periods of high-nectar intake (honey flow), and hence maximal colony expansion, the deleterious event can severely compromise survival of a much diminished bee population during the following winter.

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A

B

FIG. 3: Effects of Chlorothalonil, Control (no treatment), Coumaphos or Tau-Fluvalinate on Bacterial and Fungal taxa (Class) recovered from the gut of adult workers of A. mellifera. [A] Pesticide, Chlorothalonil, changed gut bacterial population s to a predominance of Pasteurellales and Rhizobialis, by comparison to taxa recovered from bees of Control , Coumaphos and Tau-Fluvalinate groups. Otherwise, trends between the three pesticides were relatively minor. [B] Pesticide treatments, by comparison to Control, caused substantial changes in fungal taxa present in gut of A. mellifera. Chlorothalonil increased in the prevalence of Dothideomycetes and Microbotrymycetes; whereas Tau- Fluvalinate increased Saccharomycetes and ‘Other’ fungi, apparently at the expense of Dothideomycetes taxa. Presently, all three pesticides are applied to crops in New Zealand. (Mean ± S.E.) (Source: Kakumanu et al., 2016)

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5) Tau-Fluvalinate, a component of the proposed combined pesticide treatment, when fed to adult A. mellifera had little effect on their health (Appendix II, Fig. 5), primarily because the pesticide is detoxified enzymatically by the adult bee25,26, 6) Tau-Fluvalinate, given under ‘realistic’ experimental conditions, kills larvae within three days, ostensibly because the ‘detoxifying’ enzymes are not express during early bee development, 7) Chlorothalonil and Tau-fluvalinate, when exposed to bee larvae under ‘realistic’ experimental conditions, invoke a synergistic kill exceeding the added effects of the two pesticides given alone, 8) Chlorothalonil given to adult bees results in considerable change in gut biome, as reflected by the altered prevalence of different bacterial taxa (Fig. 3A)9. Given the known interaction of gut bacterial taxa with gut-based immune systems of other species, the present discovery could have implications for elucidating parameters informing bee health. By comparison, with the fungal taxa, Chlorothalonil and Tau-Fluvalinate caused substantial changes in the relative abundance of several taxa, and unexpectedly in disparate directions (Fig. 3B)9. 9) Exposure of flowering crops to pesticides purportedly exposes worker bees (A. mellifera) to conditions that render the bees susceptible to endo-parasitic fungal infections of the gut parasites, Nosema ceranae11 and N. mellifera15. The research found substantial loads of pesticide in pollen gathered by worker bees from a variety of crops. It is claimed two pesticides, Amitraz and Tau- fluvalinate, used to control Varroa mites, also helped to protect the bees from endo-parasitic infections15,16, although evidence presented to address this point is not convincing. 10) Recent evidence demonstrating Queen bee’s pheromones delete function of oocytes and/or follicular cells within the colony’s Worker Bees’ ovaries, thereby causing sterility, provides a provocative mechanism to compromise hive health20,21. Pesticides, such as Chlorothalonil, may act by diminishing output of Queen bee pheromones thereby altering worker bee reproductive competency and compromising their commitment to the colony, and hence onset of Colony Collapse Disorder.

F. Summary The compound, Chlorothalonil, is a proven broad-spectrum acaricide, fungicide, miticide, molluscicide, nematicide and pesticide; in most instances, being effective over the range of 10 to 100 ppb, on both aquatic- and terrestrial-based species. Chlorothalonil applied to crops has been implicated in numerous amphibian and fish kills and in causing Colony Collapse Disorder (CCD) among bee populations throughout the World. Present scientific evidence is consistent with the incorporation of Chlorothalonil into nectar and pollen of flowering crops; with foraging worker bees collecting and conveying pesticide-contaminated substances back to the bee colony for storing and processing; and thereafter, on being fed mead to larvae and further exposing adult bees, the health and survival of the bee colony is challenged severely. The economic impact of Chlorothalonil on production and security of global food supplies, through compromising crop pollination by both feral/native and managed bee populations, is substantial. In addition to the pesticide’s action on faunal pollinators, domestic usage of Chlorothalonil also risks human health along with its attendant remedial costs to public health services. It is strongly recommended that domestic applications of Chlorothalonil be de-certified and discontinued without delay. It is also recommended that the EPA review and dis-continue industrial applications in New Zealand. Hearing: We, the members of the Waikato Domestic Bee Association, wish to be heard.

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G. References:

1. Azin, M. A., Garibaldi, L. A., Cunningham, S. A. & Klein, A. M. (2009) How much does agriculture depend on pollinators? Lessons from long-term trends in crop production. Ann. Bot. 103, 1579–1588 (2009).

2. Beguin, H.M., Requier, M., Rollin, F., Odoux, O., Aupinel, J.-F., P., et al. (2012). A common pesticide decreases foraging success and survival in honey bees. Science 336,348–350.doi:10.1126/science.1215039 3. Davis, P.E., Cook, L.S.J. & Goenarso, D. (1994) Sub-lethal responses to pesticides of several species of Australian freshwater fish and crustaceans and rainbow trout. Environ. Toxicol. Chem. 13 (8) 1,341-1354, DOI: 10.1002/etc.5620130816, 4. Ernst, W., Doe, P., Young, Y., Julien, G. and Hennigar, P. (1991) The toxicity of chlorothalonil to aquatic fauna and the impact of its operational use on a pond ecosystem. Arch. Environ. Contamin. and Toxicol. 21: p. 1-9; In: Proc. 17th Annual Aquatic Toxicity Workshop, Eds: Chapman, P., Bishay, F., Power, E., Hall, K., Harding, L. & McLeay, D. Vancouver, B.C., 5th to 7th November, 1990, & Canadian Tech. Rep. Fish. Aquatic Sci. 1774: 301- 302, 5. Feazel-Orr, H.K., Catalfamo, K.M., Brewster, C.C., Fell, R.D., Anderson, T.D. & Traver B.E., (2016) Effects of pesticide treatments on nutrients levels in worker honeybees (Apis mellifera). Insects 7: 1-9, doi.10.3390/- Insects.7010008, 6. Garibaldi, L.A., Steffan-Dewenter, I., Winfree, R., Aizen, M.A., Marcelo, A., Bommarco, R., Cunningham, S.A., Kremen, C., Carvalheiro, L.G., Harder, L.D., Afik, O., Bartomeus, I., Benjamin, F., Boreux, V., Cariveau, D., Chacoff, N.P., Dudenhöffer, J.H., Freitas, B.M., Ghazoul, J., Greenleaf, S., Hipólito, J., Holzschuh, A., Howlett, B., Isaacs, R., Javorek, S.K., Kennedy, C.M., Krewenka, K.M., Krishnanl S., Mandelik, Y., Mayfield, M.M., Motzke, I., Munyuli, T., Nault, B.A., Otieno, M., Petersen, J., Pisanty, G., Potts, S.G., Rader, R., Ricketts, T.H., Rundlöf, M., Seymour, C.L., Schüepp, Schüepp C., Szentgyörgyi, H., Taki, H., Tscharntke, T., Vergara, C.H., Viana, B.F., Wanger, T.C., Westphal, C., Williams, N. & Klein, A.M. (2013) Wild Pollinators Enhance Fruit Set of Crops Regardless of Honey Bee Abundance. Science 339: Issue 6127, p.1608-1611; DOI: 10.1126/science.1230200, 7. Hashimoto, Y. & Nishiuchi, Y. (1981) Establishment of bioassay methods for the evaluation of acute toxicity of pesticides to aquatic organisms. J. Pesticide Sci. 6 (2) 257-264, 8. Johnson, R. M., Wen, Z., Schuler, M.A. & Berenbaum, M. R. (2016) Mediation of pyrethroid insecticide toxicity to Honey Bees (Hymenoptera: Apidae) by Cytochrome P450 Monooxygenases. J. Economic Entomology 9. Kakumanu, M.L., Reeves, A.M., Anderson, T.D., Rodriques, R.R. & Williams, M.A. (2016) Honey bee gut microbiome is altered by in-hive pesticide exposures. Frontiers of Microbiology, 7, Article 1255, p. 1-11; Doi: 10.3389/micb.2016.01255, 10. Lautenbach, S., Seppelt, R., Liebscher, J. & Dormann, C. F. Spatial and temporal trends of global pollination benefit. PLOS One 7, e35954 (2012), 11. Li, J., Qin, H., Wu, J., Sadd, B.M., Wang, X., Evans, J.D., Peng, W. & Cheng, Y. (2012) The Prevalence of Parasites and Pathogens in Asian Honeybees Apis cerana in China. PLOS ONE 7: (11) pp, 1-12; e47955, 12. Matheson, A & Reid, M. (2011) Practical Beekeeping in New Zealand. 4th Edition, Pub: Exisle Publishing, Titirangi, Auckland, New Zealand, & Wollombi, New South Wales, Australia; ISBN 978-1-877568-52-7. 13. McMahon, T.A., Haltstead, N.T., Johnson, S., Raffel, T.R., Romansic, J.M., Crumrine, P.T., Boughton, R.K., Martin, L.B., Rohr, J.R. (2011) The fungicide chlorothalonil is nonlinearly associated with corticosterone levels, immunity, and mortality in amphibians. Environmental Health Perspectives 119: (8) 1089-1103, 14. Nishiuchi, Y. (1980) Toxicity of formulated pesticides to fresh water organisms. Susan Zoushoku 28 (2) 107-112, LXXIV, 15. Pettis, J.S., Lichtenberg, E.M., Andree, M., Stitzinger, J., Rose, R. & van Engelsdorp, D. (2013) Crop pollination exposes honey bees to pesticides which alters their susceptibility to the gut pathogen, Nosema ceranae. PLOS ONE, 8 (7): p.1-9; e70182, doi:10.1371/journal.pone, 0070182,

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16. Pettis, J.S., van Engelsdorp, D., Johnson, J. and Dively, G. (2012). Pesticide exposure in honey bees results in increased levels of the gut pathogen Nosema. Naturwissenschaften 99:153–158. doi:10.1007/s00114-011-0881- 1, 17. Potter, T.L., Wauchope, R.D. & Culbreath, A. K. (2001) Accumulation and decay of chlorothalonil and selected metabolites in surface soil following foliar application to peanuts. Environ. Sci. Technol. 35: 2634-2639, 18. Potts, S. G. et al. IPBES (2016). The Assessment Report of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services on Pollinators, Pollination and Food Production; http://www.ipbes. net/node/44781, pp. 1-888, 19. Potts, S.G., Imperatriz-Fonseca, V., Ngo, H.T., Aizen, M.A. Biesmeijer, J.C., Breeze. T.D., Dicks, L.V., Garibaldi, L.A., Hill, R., Settele, J. & Vanbergen, A.J. (2016) A Review: Safeguarding pollinators and their values to human well-being. Nature (In press), pp. 1-10, 20. Ronai, I., Barton, D.A., Oldroyd, B..P. & Vergoz, V.J. (2015) Regulation of oogenesis in honey bee workers via programed cell death. Insect Physiol. 81: 36-41. doi:10.1016/j.jinsphys.-2015.06, 21. Ronai, I., Oldroyd, B.P., Vergoz, V. (2016) Queen pheromone regulates programmed cell death in the honey bee worker ovary. Insect Mol Biol. 25(5): 646-652. doi:10.1111/imb.12250 22. Vásquez, A., Forsgren, E., Fries, I., Paxton, R.J. Flaberg, E., Szekely, L., et al. (2012). Symbionts as major modulators of insect health: lactic acid bacteria and honeybees. PLOS ONE 7: e33188. doi:10.1371/annotation-/3ac2b867-c013-4504- 9e06-bebf3fa039d1, 23. Wallberg, A., Han. F., Wellhagen, G., Dahle, B., Kawata, M., Haddad, N., Simões, Z.L.P., Allsopp, M.H., Kandemir, I., La Rúa, P.D., Pirk, C.W. and Webster, M.T. (2014). A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera. Nature Genetics 46: 1081- 1088. Doi:10.1038/ng.3077, 24. Wu, J.Y., Smart, M.D., Anelli, C.M and Sheppard, W.S. (2012). Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection. J. Invertebr. Pathol. 109: 326–329. doi:10.1016/j.jip.2012.01.005, 25. Zhu, W (2013) Assessing impacts of pesticides and other stressors on honey bee colony health: Experimental and modeling approaches. Ph.D. Thesis, Department of Entomology, The Graduate School, Pennsylvania State University, PA:1-197, 26. Zhu, W., Schmehl, D.R., Mullin, C.A. & Frazier, J.L. (2014). Four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae. PLOS ONE 9: e77547. doi:10.1371/journal.pone. 0077547. 27. EPA (2016) Guidelines for Drinking Water Quality Management for New Zealand. Datasheets, Volume 3, Part 2.3 pp. 197-203.

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APPENDIX 1: Chlorothalonil: Structure and Toxicity to Domestic Honey Bee, Apis Mellifera

Primary resources: https://pubchem.ncbi.nlm.nih.gov/compound/; https://echa.europa.eu/ Common name: Chlorothalonil (Fig. 1A) Biochemical name (IUPAC): 2,4,5,6-Tetrachlorobenzene-1,3-dicarbonitrile Trade names: Agronil; Amini; Black-Spot and Fungus-Spray*; Bravo*; Bravo 6F*; Bravo 500*; Bronco; Celeste; Dacosoil; Disease Eliminator*; 1 Greenguard*; Guardall*; Fungus-and-Mildew Spray*; Taratek 5F*, Termil, 6 2 Chemical formula: C8Cl4N2 5 3 Molar mass: 265.902 g/mol−1 Appearance: White crystalline solid 4 Melting point: 252.1 C°, Boiling point: 350 C° A Solubility in water: 0.06 g/100 ml (highly hydrophobic, i.e.: lipophilic) Stability: Volatile - in acidic and basic milieu Related compounds: Benzonitrile; Hexachlorobenzene; Dichlorobenzene; Chlorobenzene Principal contaminants: Hexachlorobenzene; 2,3,4,5.6-Pentachlorobenzo- nitrile; and 2,3,7,8 Tetrachlorodibenzo-ρ-dioxin (TCDD)** Degradation products: Hydroxy chlorothalonil; and/or 4-hydroxy-2,5,6-trichloroisophthalonitrile (SDS-3701)*** 4-hydroxy-2,5,6-trichloro-1,3-benzenedicarbonitrile; 4-hydroxy-2,5,6-trichloro-3-benzenedicarbonitrile; B 4-hydroxy-2,5,6-trichloroisophthalonitrile; 1,3-dicyano-4-hydroxy-2,5,6-trichlorobenzene; 2,4,5-trichloro-6-hydroxy-isophthalonitrile; 2,4,5-trichloro-6-hydroxy-1,3-benzenedicarbonitrile; 2,5,6-trichloro-4-hyxdroxy-1,3-benzenedicarbonitrile; Notation to chemical nomenclature: Cyclic organic compounds, including Chlorothalonil and its derivatives, OH are notated by: Fig. 1A: Carbon atoms numbered in clockwise sequence from top-centre; Fig. 1B: Carbon: Grey spheres; Nitrogen: Blue spheres; Chlorine: Green spheres; (Single bond: single line; Double bond: Double parallel lines; Triple bond: Triple parallel lines); Fig. 1C: The C OH potent compound, 4-hydroxy-chlorothalonil, a degradation product of FIG. 4: Biochemical structures of two Chlorothalonil, differs from the latter in that ‘hydroxyl’ ion replaces the related pesticides. [A] Chlorothalonil; original ‘chlorine’ ion at position 4 (cf. Figs. 1A & C). This compound, 4- [B] 3D Conformer; [C] The 30-fold hydroxy-2,5,6-trichloroisophthalonitrile (SDS-3701), based on scientific more potent degradation product 4- evidence, persists longer in the environment and is ~30-fold more acutely hydroxy-chlorothalonil (SDS-3701). toxic than the parent compound, chlorothalonil17.

* Chlorothalonil products presently are distributed and retailed for domestic use in New Zealand. ** 2,3,7,8 Tetrachlorodibenzo-ρ-dioxin, known inaccurately as “Dioxin”, a potent carcinogen and one of the original ‘dirty dozen’ chemicals (POPS) was banned by the Stockholm Convention, in 2001, and became effective in 2004. *** Degraded product, 4-hydroxy-2,5,6-trichloroisophthalonitrile (SDS-3701) is ~30-fold more toxic than Chlorothalonil.

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SUBMISSION 123443

APPENDIX II: Tau- Fluvalinate and Methyl-thiophanate Primary resources: https://pubchem.ncbi.nlm.nih.gov/compound/; https://echa.europa.eu

Common name: Tau-Fluvalinate IUPAC name: (RS)-α-cyano-3-phenoxybenzyl N-(2-chloro- α,α,α-trifluoro-p-tolyl)-D-valinate CAS name: cyano(3-phenoxyphenyl)methyl N-[2-chloro-4- (trifluoromethyl)phenyl]-D-valinate

Chemical formula: C26H22CIF3N2O3 Trade names: Fluvarol, Fluwarol, Mavrik EW, CHFB1:39367 Molecular mass: 502.918 g/mol Melting point: -14.1°C Boiling point: Decomposes prior Physical state: Colourless viscous liquid Solubility in water at 20°C (mg/l): 0.00103 Solubility in acetone at 20°C (mg/l): 5,000.0 A Aa Description: Synthetic pyrethroid applied as an insecticide/ Fig. 5: [A] Biochemical structure of pyrethroid, Tau- acaricide/miticide against aphids, leaf-hoppers, leaf-rollers, Fluvalinate; [B] Three-dimensional structure of a moths, thrips and varroa mites. Applications: Avocados, Tau-Fluvalinate conformer in which chlorine (Cl) cereals (incl. wheat), cherries, nectarines, peaches, plums, has replaced fluorine (F) (Source: US PubChem) potatoes, tamarillos, turf, bees (Apis mellifera; A. cerana).

Methyl-Thiophanate Common name: Methyl-Thiophanate IUPAC name: methyl N-[2-(methoxycarbonylcarbamothioyl amino) phenyl[carbamothioyl]carbamate

Chemical formula: C12H14N4O4S2 Trade names: Caligran, Cycosin, Ditek, Envovit, Fungo 50, Methyl topsin, Metoben, Mildothane, Neotopsin, Pelt 124, Rilon, Sipcasan, Sipcavit, Thiopan, Thiophanate-M Molecular mass: 342.388 gm/mol Melting point: 172°C, Decomposes prior Boiling point: Decomposes prior A Physical state: Colourless crystalline solid Solubility: <1.0 mg/ml @ 2 Description: Methyl-Thiophanate is a broad-spectrum pest-icide and fungicide toxic to parasitic worms, including nema-todes. The pesticide causes liver and thyroid hypertrophy and its ranked carcinogenic for humans. It’s a component of strips for treating parasitic varroa mites in bee hives. FIG. 6: Biochemical structure of Methyl-thiophanate, a component

of the proposed two- and three-way combinations of pesticides, including chlorothalonil and/or Tau-fluvalinate. [A]: Two-dimen- sional ‘stick’ model of Tau-fluvalinate; [B] Three-dimensional structure of Tau-fluvalinate. Carbon: grey; Hydrogen: Grey blue; Nitrogen: Blue; Oxygen: red; Sulphur: yellow, Single bar: Single B covalent bond; Twin bars: Double bond (Courtesy: PubChem). 11