APP203741 Staff Assessment Report.Pdf

EPA advice for application APP203741

Staff Assessment Report

December 2018

APP203741: To determine the new organism status of Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum

Purpose To determine if Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum are new organisms under section 26 of the HSNO Act

Application number APP203741

Application type Statutory determination

Applicant Anther Experimental Distillation Pty Limited

Date formally received 14 November 2018

Executive Summary and Recommendation

Application APP203741, submitted by Anther Experimental Distillation Pty Limited, seeks a determination on the new organism status of Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum.

After reviewing all of the available information and completing a literature search concerning the organisms, EPA staff recommend that Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum are not new organisms for the purpose of the HSNO Act based on evidence that these organisms have been identified and present in New Zealand since before 29 July 1998 when the HSNO Act came into effect. The genetic sequencing evidence also strongly suggests that these six microorganisms are ubiquitous in nature based on the isolations of these organisms across global continents, varying environments and timeframes.

EPA advice for application APP203741

Table of Contents Introduction and background ……………………………………………………………4 Organism description…………………………………………………………………………4 Review of information………………………………………………………………………10 Recommendation……………………………………………………………………………..11 References…………………………………………………………………………………….....12 Appendix 1: Decision pathway……..…………………………………………………..14

Introduction and background On 14 November 2018, Anther Experimental Distillation Pty Limited applied to the EPA under section 26 of the HSNO Act seeking a determination on the new organism status of Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum.

The EPA requested comment on the application from the Department of Conservation (DOC) and the Ministry for Primary Industries (MPI). DOC and MPI did not have any comments to make on this application.

The applicant considers these species as not new organisms and to support this claim the applicant provided evidence to demonstrate that each of these organisms is present in New Zealand based on identification of these organisms in soil samples collected in New Zealand. In addition, the applicant argues that these organisms are likely to be ubiquitous organisms given the isolation of these organisms across geographical continents, varying environments and timeframes.

Section 2A(1) of the HSNO Act prescribes that a new organism is, in part, an organism belonging to a species that was not present in New Zealand immediately before 29 July 1998.

Description of organisms

Kineosporia rhizophila Kineosporia rhizophila is a motile, spore-bearing, actinomycete bacterial species and a member of the genus, Kineosporia, due to its morphological and chemotaxonomic characteristics such as the inclusion of menaquinone, phospholipid and cellular fatty acid compositions. Kineosporia have the ability to release flagellated zoospores at a certain stage in their life cycle (Goodfellow & Cross, 1984). These motile, zoosporic actinomycetes have been associated with river and lake water, river sediments, decaying plant material submerged in streams and cast up on lake shores, grass inhabiting streams, in soil and a glacier in the Himalayas (Hasegawa 1991; Kudo et al. 1998; Cross 1986; Willoughby 1969; Shivaji et al. 1997; Shivaji et al. 2011). Kineosporia spp. have also been isolated in high numbers from leaf litter, dry stream beds and from branches overhanging water in Britain, the United States of America, Japan and Australia (Radajewski & Duxbury, 2001; Cross, 1986; Kudo et al. 1998). Furthermore, Actinomycete species belonging to the genera Actinoplanes, Kineosporia and Cryptosporangium have been frequently isolated from leaf litter samples which suggests they may have a significant role in the degradation of plant material (Hop et al. 2011; Tamura et al. 1998; Makkar & Cross, 1982).

Kineosporia rhizophila was originally isolated in 1998 by Kudo et al. from various plant samples in Japan. In the same year, Kudo et al. also isolated this species from the roots of galingale (Cyperus micromona) and fallen leaves in Saitama, Japan, sphagnum in Mt Mikuni in Gumma, Japan and leaves of cat-tail (Typha latifolia) (Kudo et al. 1998).

EPA advice for application APP203741

International isolates of this species also include from leaf litter in Vietnam (Sakiyama et al, 2009), soil in Japan (Hayakawa et al, 2000) and soil and leaf litter from West Java, Indonesia (Widyastuti et al. 2012).

In addition, the applicant analysed soil survey studies that are similar to BASE1 surveys. These surveys consisted of 16S rRNA sequence analysis of microbial communities found in soil and other environments collected from locations around the world (Table 1). Using the 16S rRNA sequence of Kineosporia rhizophila the applicant performed several BLAST2 searches online and found the following results:

Table 1: Global locations of endemic species and identity matched to 97 – 100% sequence similarity to Kineosporia rhizophila.

Source Country % Identity

Lichen China 99

Farm soil USA 99

Soil samples Argentina 97

Soil samples Canada 97

Soil France 97

Furthermore, the applicant provided two 16S rRNA sequencing reports (New Zealand and Australia) from Monash University which identified Kineosporia rhizophila from soil samples across a wide distribution of Australia and the North Island of New Zealand (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018).

Actinoplanes cyanea Actinoplanes is a genus in the family of Micromonosporaceae. Bacteria within this genus have aerial mycelia and spherical, motile spores. Actinoplanes species produce the pharmaceutically important compounds valienamine (a precursor to the antidiabetic drug acarbose and the antibiotic validamycin), teicoplanin and ramoplanin.

Actinoplanes cyanea (syn. Actinoplanes cyaneus) was first isolated from Siberian soil in 1977 by Terekhova et al. Unlike several other Actinoplanes species, this species lacks aerial

1 BASE: Biomes of Australian Soil Environment. BASE is a map of Australia’s soil microbial diversity. https://www.csiro.au/en/Research/Collections/ANH/Our-research/Soil-and-plant-interactions/Mapping-soil- biodiversity 2 BLAST: basic local alignment search tool. An algorithm used for comparing primary biological sequence information.

mycelium. The cell wall contains meso-diaminopimelic acid, arabinose, xylose and a non- identified analogue of diaminopimelic acid. On synthetic media, this species produces a soluble blue pigment. In 2007, Actinoplanes cyaneus was isolated from soil sites in the three Mongolian provinces of Tov, Uvs and Dornad (Enkh-Amgalan et al. 2012). These three provinces span west (Tov), central (Uvs) and far-eastern (Dornad) Mongolia.

Other closely related Actinoplanes species have been isolated from soil close to the Marmore waterfalls in Terni, Italy. These isolates had a 97.6% gene sequence similarity to Actinoplanes cyanea (Kampfer et al. 2007). Furthermore, isolations in region of Liguria, Italy, showed a 99.3% gene sequence similarity to Actinoplanes cyanea strains (Wink et al. 2006).

In addition, the applicant analysed soil survey studies that are similar to BASE surveys. These surveys consisted of 16S rRNA sequence analysis of microbial communities found in soil and other environments collected from locations around the world (Table 2). Using the 16S rRNA sequence of Actinoplanes cyanea the applicant performed several BLAST searches online and found the following results:

Table 2: Global locations of endemic species and identity matched to 97 – 100% sequence similarity to Actinoplanes cyanea.

Source Country % Identity

Soil Russia 98

Sediment Greece 98

Marine sediments Iran 99

Soil France 97

Soil Canada 100

Soil Chile 100

The applicant provided two 16S rRNA sequencing reports (New Zealand and Australia) from Monash University which identified Actinoplanes cyanea from soil samples across a wide distribution of Australia and the North Island of New Zealand (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018).

Actinoplanes digitatis Isolation of Actinoplanes digitatis (syn. Ampullariella digitata) has occurred from soil samples in Sheboygan, Michigan, United States in 1963 by J.N. Couch and from tree bark in Mt Taibai, China (European Nucleotide Archives).

The applicant provided two 16S rRNA sequencing reports (New Zealand and Australia) from Monash University which identified Actinoplanes digitatis from soil samples across a wide

EPA advice for application APP203741

distribution of Australia and the North Island of New Zealand (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018).

In addition, the applicant analysed soil survey studies that are similar to BASE surveys. These surveys consisted of 16S rRNA sequence analysis of microbial communities found in soil and other environments collected from locations around the world (Table 3). Using the 16S rRNA sequence of Actinoplanes digitatis the applicant performed several BLAST searches online and found the following results:

Table 3: Global locations of endemic species and identity matched to 97 – 100% sequence similarity to Actinoplanes digitatis.

Source Country % Identity

Soil Italy 99

Floor dust Finland 98

Environmental sample USA 98

Soil South Africa 98

Soil Canada 98

Soil Argentina 98

Actinoplanes ferrugineus Actinoplanes ferrugineus was first isolated by Palleroni (1979) from a soil sample from Dorrigo Mountain, New South Wales, Australia. When cultured on media, the bacterial colonies of A. ferrugineus are brown to dark red in colour and when observed under microscope with low magnification, the sporangia are visible. In Palleroni’s study, he discovered that the formation of sporangia of A. ferrugineus is far less abundant and reproducible when compared with other Actinoplanetes such as A. brasiliensis, A. italicus and A. utahensis. Two novel species of Actinoplanes – A. toevensis and A. tereljensis were discovered in soil samples from the Tov province of Mongolia in 2010. This study found that these strains were closely related to A. ferrugineus with a 97.7% sequence similarity (Ara et al. 2012).

The applicant provided two 16S rRNA sequencing reports (New Zealand and Australia) from Monash University which identified Actinoplanes ferrugineus from soil samples across a wide distribution of Australia and the North Island of New Zealand (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018).

sequence of Actinoplanes ferrugineus the applicant performed several BLAST searches online and found the following results:

Table 4: Global locations of endemic species and identity matched to 97 – 100% sequence similarity to Actinoplanes ferrugineus.

Source Country % Identity

Sea water Baltic sea 98

Soil Thailand 98

Soil France 99

Soil Canada 98

Soil Argentina 98

Cryptosporangium arvum Cryptosporangium is a genus in the phylum Actinobacteria. Cryptosporangium is derived from Greek and means ‘hidden seed/spore vessel’. This genus only contains six species (including basionyms and synonyms) and has gram-positive, non-acid-fast, aerobic organisms with branching hyphae. Cryptosporangium have sporangia and aerial mycelia that aggregate and sporangiospores that show motility when they are suspended in water.

Cryptosporangium arvum (syn. Cryptosporangium arvi) was originally isolated in 1998 by Tamura et al. from soil samples at Kofu, Yamanashi and Miyako Islands, Okinawa Prefecture, Japan. On tyrosine agar, this species produces a pale reddish-brown soluble pigment. Cryptosporangium arvum does not liquefy gelatin, does not hydrolyse starch and has a preferred habitat of cultivated soil (Tamura et al. 1998). A novel species of Cryptosporangium – C. mongoliense was discovered in soil samples from Mongolia in 2012. This study by Ara et al. (2010) found that this new species was closely related to Cryptosporangium arvum with a 97.2% sequence similarity.

The applicant provided two 16S rRNA sequencing reports (New Zealand and Australia) from Monash University which identified Cryptosporangium arvum from soil samples across a wide distribution of Australia and the North Island of New Zealand (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018).

In addition, the applicant analysed soil survey studies that are similar to BASE surveys. These surveys consisted of 16S rRNA sequence analysis of microbial communities found in soil and other environments collected from locations around the world (Table 5). Using the 16S rRNA sequence of Cryptosporangium arvum the applicant performed several BLAST searches online and found the following results:

Table 5: Global locations of endemic species and identity matched to 97 – 100% sequence similarity to Cryptosporangium arvum.

EPA advice for application APP203741

Source Country % Identity

Organic material Thailand 97

Soil France 97

Soil Canada 98

Soil Argentina 98

Soil Chile 98

Cryptosporangium japonicum Cryptosporangium japonicum was also originally isolated in 1998 by Tamura et al. from soils from sugar cane and vegetable fields in Yamanashi, Okinawa Prefecture, Japan. In terms of morphological and chemotaxonomic characteristics, it is almost identical to C. arvum with the exception of a molecular difference: C. arvum does not contain glycerol.

A novel species of Cryptosporangium – C. mongoliense was discovered in soil samples from Mongolia in 2012. This study by Ara et al. (2012) found that this new species was closely related to Cryptosporangium japonicum with a 96.8% sequence similarity.

The applicant provided two 16S rRNA sequencing reports (New Zealand and Australia) from Monash University which identified Cryptosporangium japonicum from soil samples across a wide distribution of Australia and the North Island of New Zealand (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018).

In addition, the applicant analysed soil survey studies that are similar to BASE surveys. These surveys consisted of 16S rRNA sequence analysis of microbial communities found in soil and other environments collected from locations around the world (Table 6). Using the 16S rRNA sequence of Cryptosporangium japonicum the applicant performed several BLAST searches online and found the following results:

Table 6: Global locations of endemic species and identify matched to 97 – 100% sequence similarity to Cryptosporangium japonicum.

Source Country % Identity

Rhizosphere South Korea 99

Soil Argentina 99

Soil Chile 99

Soil Africa 98

Soil France 98

Review of information The applicant put forward the case for microbial ubiquity in their application and to complement this, scientific papers were provided to show that at least five of the six organisms have been isolated from multiple continents and within different timeframes.

Appendix Two of the application consists of a sequencing report that positively identifies all six of the organisms, their location of isolation and widespread distribution in Australia. The applicant requested the expertise of CSIRO3 and Micromon (Next-Generation Sequencing Facility) to match the publicly available 16S sequences corresponding to the organisms in the application. Micromon utilised the Biomes of Australian Soil Environments (BASE) project which is a comprehensive catalogue of the biological and physical characteristics of Australian soils including those of agricultural importance. The sequence data derived from the BASE project were aligned to the publicly available 16S RNA sequences of all six of the organisms in the application.

The results of the 16S RNA sequence alignment showed greater than 99% sequence identity to the corresponding region of the 16S genes for all six organisms. For each organism, at least ten positive identifications across nearly all Australian states were found.

Appendix Four consists of another sequencing report that identifies all six of the organisms from soil sample isolates collected in New Zealand. Publicly available 16S rRNA sequence data by Hermans et al. 2017 was matched to the soil samples. The results of the comparison showed positive identification of all six organisms within New Zealand soil samples and across multiple locations across the North Island to a 99% sequence identity. In the case of Actinoplanes digitatis, Cryptosporangium arvum and C. japonicum, there were positive identification hits with 100% alignment to genetic sequence databases.

In addition, the applicant analysed the results from international soil and other environmental niche surveys using the 16S rRNA sequences of the six organisms. The applicant found multiple global locations for each of the six organisms, with 97 to 100% similarities to their 16S sequences in online genetic databases. Many of the source samples came from different environmental niches including soil, sea water, plantations, floor dust and marine sediment, all of which exist in the New Zealand environment. Furthermore, since these samples were sourced from locations in Europe, Asia, South America, North America and one from the Middle East, it demonstrates the ubiquitous nature of these organisms.

Upon reviewing the appendices, there is strong genetic sequencing evidence to suggest that all six of the organisms are currently in New Zealand and Australia (Monash University Microbe Species Identity and Location report, 18 November 2015; Monash University Microbe Species Identity and Location Report, 24 August 2018). The international studies further consolidate the

3 CSIRO: The Commonwealth Scientific and Industrial Research Organisation.

EPA advice for application APP203741

case for microbial ubiquity given the isolates have been found in Asia, Europe, South America and North America.

Conclusion After completing our assessment of the information that was submitted by the applicant, we consider that Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum are present in New Zealand and have been present for a significant period of time (i.e. immediately before 29 July 1998) based on the evidence presented by the applicant.

Recommendation Our assessment has found that there is sufficient evidence to show that Kineosporia rhizophila, Actinoplanes cyanea, Actinoplanes digitatis, Actinoplanes ferrugineus, Cryptosporangium arvum and Cryptosporangium japonicum should be determined as not new for the purpose of the Act.

References

Ara, I. Tsetseg, B. Daram, D. Suto, M. and Ando, K. 2012. Cryptosporangium mongoliense sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology. 62: 2480-2484. Ara, I. Yamamura, H. Tsetseg, B. Daram, D and Ando, K. 2010. Actinoplanes toevensis sp. nov. and Actinoplanes tereljensis sp. nov., isolated from Mongolian soil. International Journal of Systematic and Evolutionary Microbiology. 60: 919-927. Couch, J.N. 1963. Some new genera and species of the Actinoplanaceae. Journal of the Elisha Mitchell Scientific Society. 79(1): 53-70. Cross, T. 1986. The occurrence and role of actinoplanetes and motile actinomycetes in natural ecosystems. In: Megusar, F. Gantar, M. (Eds). Perspectives in microbial ecology. Proceedings of the IV International Symposium of Microbial Ecology. 265-270. Enkh-Amgalan, J. Komaki, H. Daram, D. Ando, K and Tsetseg, B. 2012. Diversity of nonribosomal peptide synthetase and polyketide synthase genes in the genus Actinoplanes found in Mongolia. The Journal of Antibiotics. 65: 103-108. European Nucleotide Archive (ENA): Archive of Actinoplanes digitatis isolates. Accessed: 25 October, 2018: https://www.ebi.ac.uk/cgi-bin/sva/sva.pl?search=Go&query=KF447933 Goodfellow, M. and Cross, T. 1984. Classification: The biology of actinomycetes. Academic Press. 7- 164. Hasegawa, T. 1991. Studies on Motile Arthrospore-Bearing Rare Actinomycetes. Actinomycetol. 5(2): 64-71. Hayakawa, M. Otoguro, M. Takeuchi, T. Yamazaki, T. and Iimura, Y. 2000. Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter. Antonie van Leewenhoek. 78: 171-185. Hermans, S. Buckley, H. Case, B. Curran-Cournane, F. Taylor, M. and Lear, G. 2017. Applied and Environmental Microbiology. American Society for Microbiology. 83(1): e02826-16. Hop, D.V. Sakiyama, Y. Binh, C.T.T. Otoguro, M. Hang, D.T. Miyadoh, S. Luong, D.T. and Ando, K. 2011. Taxonomic and ecological studies of actinomycetes from Vietnam: isoation and genus- level diversity. The Journal of Antibiotics. 64: 599-606. Kampfer, P. Huber, B. Thummes, K. Grun-Wollny, I. and Busse, H-J. 2007. Actinoplanes couchii sp. nov. International Journal of Systematic and Evolutionary Microbiology. 57: 721-724. Kudo, T. Matsushima, K. Itoh, T. Sasaki, J and Suzuki, K. 1998. Description of four new species of the genus Kineosporia: Kineosporia succinea sp. nov., Kineosporia rhizophila sp. nov., Kineosporia mikuniensis sp. nov. and Kineosporia rhamnosa sp. nov., isolated from plant samples, and amended description of the genus Kineosporia. International Journal of Systematic Bacteriology. 1998: Oct. 48(4): 1245-1255. Makkar, N.S. and Cross, T. 1982. Actinoplanes in soil and on plant-litter from freshwater habitats. Journal of Applied Bacteriology. 52: 209-218. Monash University Microbe Species Identity and Location Report, 24 August 2018. Monash Bioinformatics Platform. Monash University Clayton, Australia. Monash University Microbe Species Identity and Location report for Permit Application (IP15007665), 18 November 2015. Micromon (Next-Generation Sequencing Facility). Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University. Australia. Palleroni, N. 1979. New species of the Genus Actinoplanes, Actinoplanes ferrugineus. International Journal of Systematic Bacteriology. January 1979. 51-55. Radajewski, S. and Duxbury, T. 2001. Motility responses and desiccation survival of zoospores from the Actinomycete Kineosporia sp. Strain SR11. Microbial Ecology. 41: 233-244. Sakiyama, Y. Thao, K.N.N. Giang, N.M. Miyadoh, S. Hop. D.V. and Ando, K. 2009. Kineosporia babensis sp. nov., isolated from plant litter in Vietnam. International Journal of Systematic and Evolutionary Microbiology. 59: 550-554. Shivaji, M.S. Pratibha, B. Sailaja, K.H. Kishore, A.K. Singh, Z. Begum, U. Anarasi, S.R. Prabagaran, G.S.N. Reddy, T.N.R. Srinivas. 1997. Chemical composition of aerosol and snow in the high Himalaya during the summer monsoon season. Atmospheric Environment. 31: 2815-2826. Shivaji, M.S. Pratibha, B. Sailaja, K.H. Kishore, A.K. Singh, Z. Begum, U. Anarasi, S.R. Prabagaran, G.S.N. Reddy, T.N.R. Srinivas. 2011. Bacterial diversity of soil in the vicinity of Pindari glacier,

EPA advice for application APP203741

Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones. Extremophiles. 15: 1-22. Tamura, T, Hayakawa, M and Hatano, K. 1998. A new genus of the order Actinomycetales Cryptosporangium gen. nov., with descriptions of Cryptosporangium arvum sp. nov. and Cryptosporangium japonicum sp. nov. International Journal of Systematic Bacteriology. 48: 995-1005. Terekhova, L.P, Sadikova, O.A. and Preobrazhenskaia, T.P. 1977. New species of Actinoplanes cyanea sp. nov. and its antagonistic properties. Antibiotiki. 1977: December. 12: 1059-1063. Widyastuti, W. Lisdiyanti, P, Ratnakomala, S, Kartina, G, Ridwan, R, Rohmatussolihat, Prayitno, N.R., Triana, E, Widhyastuti, N, Saraswati, R, Hastuti, R.D, Lestari, Y, Otoguro, M, Miyadoh, S, Yamamura, H, Tamura, T, and Ando, K. 2012. Genus Diversity of Actinomycetes in Cibinong Science Center, West Java, Indonesia. Microbiology Indonesia. 2012: December. 6(4): 165- 172. Willoughby, L.G. 1969. A study on aquatic actinomycetes, the allochthonous leaf component. Nova Hedwigia. 18: 45-113. Wink, J.C. Kroppenstedt, R.M. Schumann, P. Seibert, G. and Stackebrandt, E. 2006. Actinoplanes liguriensis sp. nov. and Actinoplanes teichomyceticus sp. nov. International Journal of Systematic and Evolutionary Microbiology. 56: 2125-2130.

Appendix 1: s26 pathway Decision pathway for applications under Section 26 for determination as to whether an organism is a new organism

Context

This decision pathway describes the decision-making process for applications under Section 26 for determination as to whether an organism is a new organism.

Introduction

The purpose of this decision pathway is to provide the HSNO decision maker4 with guidance so that all relevant matters in the Hazardous Substances and New Organisms Act (1996) (the Act) and the Hazardous Substances and New Organisms (Organisms Not Genetically Modified) Regulations (1998) (the Regulations) have been addressed. It does not attempt to direct the weighting that the HSNO decision maker may decide to make on individual aspects of an application.

The decision pathway has two parts –

 Flowchart (a logic diagram showing the process prescribed in the HSNO Act and the Methodology to be followed in making a decision), and  Explanatory notes (a discussion of each step of the process).

Of necessity the words in the boxes in the flowchart are brief, and key words are used to summarise the activity required. The explanatory notes provide a description of each of the numbered items in the flowchart, and describe the processes that should be followed.

For proper interpretation of the decision pathway it is important to work through the flowchart in conjunction with the explanatory notes.

4 The HSNO decision maker refers to either the EPA Board or any committee or persons with delegated authority from the Board.

EPA advice for application APP203741

Figure 17 Explanatory Notes

Section 26 pathway A

Item 1 Review the content of the application and all relevant information Review the application, staff advice and any relevant information held by other Agencies, and advice from experts.

Item 2 Is further information required? Review the information and determine whether or not there is sufficient information available to make a decision.

Item 3 Seek additional information (Section 52 and Section 58) If the HSNO decision maker considers that further information is required, then this may be sought either from the applicant (if there is an external applicant) or from other sources. If the HSNO decision maker considers that the information may not be complete but that no additional information is currently available, then the HSNO decision maker may proceed to make a determination. If the application is not approved on the basis of lack of information (or if the organism is considered new) and further information becomes available at a later time, then the HSNO decision maker may choose to revisit this determination.

Item 4 Is it an organism (i.e. fits the “organism” definition in Section 2)? An organism

(a) does not include a human being: (ab) includes a human cell: (b) includes a micro-organism: (c) includes a genetic structure, other than a human cell, that is capable of replicating itself, whether that structure comprises all or only part of an entity, and whether it comprises all or only part of the total genetic structure of an entity: (d) includes an entity (other than a human being) declared to be an organism for the purposes of the Biosecurity Act 1993: (e) includes a reproductive cell or developmental stage of an organism

If yes, go to item 5. If no, as this is not an organism, it is not regulated under the new organism provisions of the HSNO Act.

Item 5 Is the determination about a potential GMO (Section 2A(1)(d))? If the determination relates to whether an organism is a potential GMO, go to pathway B. If the organism is not a GMO, go to item 6.

Item 6 Does the organism belong to a species that was known to be present in NZ immediately before 29 July 1998 (Section 2A(1)(a))? Determine on the basis of the available information whether on balance of probabilities the organism is known to belong to a species that was present in New Zealand immediately prior to 29 July 1998. For the purposes of making a Section 26 determination an organism is considered to be present in New Zealand if it can be established that the organism was in New Zealand: (a) immediately before 29 July 1998; and

(b) not in contravention of the Animals Act 1967 or the Plants Act 1970 (excluding rabbit haemorrhagic disease virus, or rabbit calicivirus). If yes, go to item 7 to test the organism against the next criterion. If no, go to item 12.

Item 7 Is the organism prescribed as a risk species and was not present in New Zealand at the time of promulgation of the relevant regulation (Section 2A(1)(b))? Determine whether the organism belongs to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been prescribed as a risk species by regulation established under Section 140(1)(h) of the Act. If the organism is prescribed as a risk species, determine whether it was present in New Zealand when it was prescribed. The organism is a new organism if it was not present in New Zealand at the time of the promulgation of the relevant regulation. Note: at this point it may become apparent that the organism is an unwanted organism under the Biosecurity Act. If this is the case, then MPI and DOC may be advised (they may already have been consulted under items 1, 2 and 3). If yes, go 12. If no, go to item 8 to test the organism against the next criterion.

Item 8 Has a containment approval been given for the organism under the Act (Section 2A(1)(c))? For the purposes of making a Section 26 determination, this will also include the following organisms which are “deemed” to be new organisms with containment approvals under the HSNO Act: (a) animals lawfully imported under the Animals Act 1967 before 29 July 1998 pursuant to Section 254 of the HSNO Act; (b) animals lawfully present in New Zealand in a place that was registered as a zoo or circus under the Zoological Garden Regulations 1977 pursuant to Section 255 of the HSNO Act (except where other organisms of the same taxonomic classification were lawfully present outside of a zoo or circus –see section 2A(2)(c)); (c) hamsters lawfully imported under the Hamster Importation and Control Regulations 1972 pursuant to Section 256 of the HSNO Act; or (d) plants lawfully imported under the Plants Act 1970 before 29 July 1998 pursuant to Section 258 of the HSNO Act. If yes, go to item 12. If no, go to item 9 to test the organism against the next criterion.

Item 9 Has a conditional release approval been given for the organism (Section 2A(1)(ca))?

If yes, go to item 12. If no, go to item 10 to test the organism against the next criterion.

Item 10 Has a qualifying organism with controls approval been given for the organism (Section 2A(1)(cb))? A “qualifying organism” is an organism that is or is contained in a “qualifying medicine” or “qualifying veterinary medicine”. These terms are defined in Section 2 of the HSNO Act. If yes, go to item 12. If no, go to item 11 to test the organism against the next criterion.

Item 11 Is the organism known to have been previously eradicated (Section 2A(1)(e))?

EPA advice for application APP203741

Determine whether the organism belongs to a species, subspecies, infrasubspecies, variety, strain, or cultivar that is known to have been previously eradicated. Eradication does not include extinction by natural means but is considered to be the result of a deliberate act. If yes, go to item 12. If no, then the organism is not a new organism.

Item 12 Has HSNO release approval without controls been given for an organism of the same taxonomic classification under Sections 35, 38 or 38I of the Act or has an organism of the same taxonomic classification been prescribed as a not new organism (Section 2A(2)(a))? If a release approval has been given for an organism of the same taxonomic classification under Section 35 or 38 of the Act then the organism is not a new organism. If a release approval has been given for an organism of the same taxonomic classification under Section 38I of the Act without controls then the organism is not a new organism, however, if this approval has been given with controls then it is a new organism. If an organism of the same taxonomic classification has been prescribed by regulations as not a new organism5 then it is not a new organism. If yes, the organism is not a new organism. If no, the organism is a new organism.

5 http://www.legislation.govt.nz/regulation/public/2009/0143/latest/whole.html#DLM2011201

Section 26 pathway B

Item 1 Have the genes or other genetic material been modified by in vitro techniques or inherited from genes or other genetic material that has been modified by in vitro techniques? If yes, go to item 2. If no, the organism is not a genetically modified organism. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards.

Item 2 Does the organism result solely from selection or natural regeneration, hand pollination, or other managed, controlled pollination (Regulation 3(1)(a) of the Regulations)? Is the organisms solely the result of selection or natural regeneration, hand pollination, or other managed, controlled pollination? If yes, the organism is not a GMO. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards. If no, go to item 3.

Item 3 Is the organism regenerated from organs, tissues, or cell culture (Regulation 3(1)(b) of the Regulations)? Is the organism regenerated from organs, tissues, or cell culture, using any of the following techniques: selection and propagation of somaclonal variants, embryo rescue, and cell fusion (including protoplast fusion)? If yes, the organism is not a GMO. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards. If no, go to item 4.

Item 4 Is the organism a result of mutagenesis treatments in use on or before 29 July 1998 (Regulation 3(1)(ba) of the Regulations)? Is the organisms the result of mutagenesis that uses a chemical or radiation treatment that was in use on or before 29 July 1998? If yes, the organism is not a GMO. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards. If no, go to item 5.

Item 5 Does the organism result solely from artificial insemination techniques (Regulation 3(1)(c) of the Regulations)? Is the organism solely the result of artificial insemination, superovulation, embryo transfer, or embryo splitting? If yes, the organism is not a GMO. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards. If no, go to item 6.

Item 6 Does the organism result from spontaneous deletions, rearrangements or amplifications (Regulation 3(1)(e) of the Regulations)? Is the organism a result of spontaneous deletions, rearrangements, and amplifications within a single genome, including its extrachromosomal elements? If yes, the organism is not a GMO. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards. If no, go to item 7.

EPA advice for application APP203741

Item 7 Is the organism modified solely by the movement of nucleic acids using physiological processes, plasmid loss or spontaneous deletion (Regulation 3(1)(d) of the Regulations)? Is the organism modified solely by the movement of nucleic acids using physiological processes, including conjugation, transduction, and transformation, or by plasmid loss or spontaneous deletion? If yes, go to item 8. If no, go to item 9.

Item 8 Does the organism contain nucleic acid molecules produced using in vitro manipulation transferred using physiological processes, plasmid loss or spontaneous deletion (Regulation 3(2) of the Regulations)? Are nucleic acid molecules produced using in vitro manipulation transferred using any of the techniques referred to in item 7? If yes, go to item 9. If no, the organism is not a GMO. However, you must check whether it meets the other new organism criteria so go to Pathway A item 6 onwards.

Item 9 Has HSNO release approval without controls been given or has an organism of the same taxonomic classification with the same genetic modification been prescribed as a not new organism (Section 2A(2)(b))? If a release approval has been given for an organism of the same taxonomic classification with the same genetic modification under Section 38 of the HSNO Act then the organism is not a new organism. If a release approval has been given for an organism of the same taxonomic classification with the same genetic modification under section 38I of the HSNO Act without controls then the organism is not a new organism, however, if this approval has been given with controls then it is a new organism. If an organism of the same taxonomic classification with the same genetic modification has been prescribed by regulations as not a new organism6 then it is not a new organism. If yes, the organism is not a new organism. If no, the organism is a new organism.

6 http://www.legislation.govt.nz/regulation/public/2009/0143/latest/whole.html#DLM2011201