EPA STAFF EVALUATION AND REVIEW REPORT

Application for approval to import or manufacture Poncho Votivo for release

APP202077 November 2015

www.epa.govt.nz 2

Application for approval to import Poncho Votivo for release (APP202077)

1. Overview

Application Code APP202077

Application Type To import or manufacture for release any hazardous substance under Section 28 of the Hazardous Substances and New Organisms Act 1996 (“the Act”)

Applicant Bayer New Zealand Limited

To import Poncho Votivo, containing 502 g/L clothianidin and 102 Purpose of the application g/L Bacillus firmus, into New Zealand for use as a seed treatment in wheat, maize, forage brassicas and grass seed

Date Application Received 1 October 2014

Submission Period 15 October 2014 – 27 November 2014

Submissions received Nineteen submissions were received; five indicated that they wished to be heard in person

Information requests and Further information was requested under section 58 of the Act. time waivers Consequently, the start of consideration was waived under section 59 of the Act.

Hearing Date 3 December 2015

November 2015 3

Application for approval to import Poncho Votivo for release (APP202077)

2. Introduction

This report documents the assessment of this substance by the staff of the Environmental Protection Authority; it assesses the risks of the substance, proposes a set of controls to manage those risks and presents an overall recommendation to the Decision-making Committee. The purpose of this report is to inform the Decision-making Committee; this report is not a decision on the application.

This application is for a seed treatment chemical, Poncho Votivo, which contains clothianidin, a neonicotinoid insecticide, and Bacillus firmus I-1582, a biopesticide, as the active ingredients. The non-active ingredients are confidential to the applicant and are not discussed in the public documents, but information regarding them is available to the Decision-making Committee.

This application was publically notified and opened for submissions from the public because it contains a new pesticide active ingredient - Bacillus firmus I-1582. Several submitters have requested to be heard by the Decision-making Committee; consequently a hearing on this application will be held on 3 December 2015, in Wellington.

November 2015 4

Application for approval to import Poncho Votivo for release (APP202077)

Table of Contents

1. Overview ...... 2

2. Introduction ...... 3

3. Application process ...... 7

4. Background and proposed use pattern of the substance ...... 7

5. Hazard classification ...... 8

6. Risk assessment ...... 9

Approach ...... 9 Overview ...... 9 Human health risks ...... 10 Environmental risks ...... 10 Cultural risk – the relationship of Māori to the environment ...... 12 Risks to society and the community and the market economy ...... 13 7. Benefits assessment ...... 13

8. Summary of submissions and staff response ...... 14

9. Other matters to be considered ...... 15

New Zealand’s international obligations ...... 15 The effects of the substance being unavailable ...... 15 10. Conclusion ...... 16

11. Recommendation ...... 16

Appendix A: Proposed Controls for Poncho Votivo ...... 17

Justification for the proposed controls ...... 24

Exposure limits ...... 24 Variation and deletion of controls ...... 24 Additional controls ...... 25 Appendix B: Cultural risk assessment report ...... 28

Appendix C: Summary of submissions ...... 32

Appendix D: Classification of Poncho Votivo ...... 37

Appendix E: Physical-chemical properties of the mixture ...... 40

Appendix F: Robust study summaries for the mixture ...... 42

Mammalian toxicity – Robust study summaries for the mixture ...... 42 Ecotoxicity – Robust study summaries for the mixture ...... 49

November 2015 5

Application for approval to import Poncho Votivo for release (APP202077)

Appendix G: Active ingredients and metabolites ...... 50

Identity of Bacillus firmus I-1582 ...... 50 Mode of action ...... 50 Production of secondary metabolites ...... 50 Classification ...... 51 Physico-chemical properties of the active ingredients ...... 53 Appendix H: Robust study summaries for Bacillus firmus I-1582 ...... 56

Mammalian toxicity ...... 56 Environmental fate...... 71 Ecotoxicity ...... 75 Appendix I: Robust study summaries for Clothianidin ...... 98

Mammalian toxicity ...... 98 Environmental fate...... 113 Ecotoxicity ...... 135 Residue levels ...... 207 Appendix J: Human health risk assessment ...... 227

Qualitative risk assessment – Bacillus firmus B-1582 ...... 227 Quantitative worker (operator) risk assessment – clothianidin ...... 227 Re-entry worker exposure assessment ...... 234 Bystander risk assessment ...... 234 Summary and conclusions of the human health risk assessment ...... 234 Appendix K: Environmental risk assessment ...... 235

Summary of the data on the active ingredient and its metabolites ...... 235 Risk assessment Methodology ...... 245 Consideration of threatened native species ...... 246 Spray drift / dust drift ...... 247 Aquatic risk assessment ...... 247 Groundwater risk assessment ...... 252 Sediment risk assessment ...... 253 Terrestrial risk assessment ...... 253 Earthworm risk assessment ...... 254 Non-target plant risk assessment ...... 256 Bird risk assessment ...... 257 Bee risk assessment ...... 264 Non-target arthropod risk assessment ...... 267 Summary and conclusions of the ecological risk assessment ...... 269

November 2015 6

Application for approval to import Poncho Votivo for release (APP202077)

List of tables and figures

Table 1 Hazard classifications proposed by the applicant and the staff ...... 8 Table 2 Results of Tier 2 assessment of the risks to bees from acute exposure to clothianidin in maize pollen ...... 12 Table 3 Summary of submissions received ...... 32 Table 4 Applicant and EPA staff classifications of the mixture...... 37 Table 2 Physical and chemical properties of the mixture ...... 40 Table 3 Identification of Bacillus firmus ...... 50 Table 4 Applicant and EPA staff classifications of the active ingredient ...... 51 Table 5 Physico-chemical properties of clothianidin ...... 53 Figure 1 Aerobic degradation of clothianidin in soils ...... 131 Table 6 Derivation of dermal absorption value in humans ...... 228 Table 7 Summary of environmental fate data on clothianidin and its metabolites ...... 236 Table 8 Summary of ecotoxicological data on clothianidin and its metabolites ...... 238 Table 9 Reference documents for environmental exposure and risk assessments ...... 245 Table 10 Levels of concern as adopted by EPA New Zealand ...... 247 Table 11 Input parameters for GENEEC2 analysis ...... 248 Table 12 Acute risk quotients derived from the GENEEC2 model and toxicity data ...... 250 Table 13 Chronic risk quotients derived from the GENEEC2 model and toxicity data ...... 251 Table 14 Input parameters for Sci-Grow analysis and resulting PEC values ...... 252 Table 15 Levels of concern as adopted by the EPA ...... 253 Table 16 Acute in-field TER value for earthworms ...... 255 Table 20 Chronic in-field TER value for soil organisms – clothianidin and its metabolite TZNG ...... 255 Table 17 Refined chronic in-field TER value for soil organisms – clothianidin...... 256 Table 18 Measures of exposure and toxicity used in the reproduction assessment ...... 259 Table 19 Exposure of birds for acute Tier 1 assessment ...... 260 Table 20 Exposure of birds for reproduction Tier 1 assessment ...... 260 Table 21 Exposure of birds to treated seeds for acute Tier 2 assessment ...... 262 Table 22 Exposure of birds to seedlings for acute Tier 2 assessment ...... 263 Table 23 Exposure of birds to seedlings for reproduction Tier 2 assessment ...... 263 Table 24 In-field HQ values ...... 268

November 2015 7

Application for approval to import Poncho Votivo for release (APP202077)

3. Application process

The application was formally received on 1 October 2014.

The Minister for the Environment was advised of the application in writing on 15 October 2014, in accordance with section 53(4) of the Act.

The Ministry for the Environment, WorkSafe New Zealand, the Ministry of Health, the Department of Conservation and the Ministry for Primary Industries Agricultural Compounds and Veterinary Medicines (ACVM) Group of were notified of the application on 15 October 2014 in accordance with section 53(4) of the Act and invited to comment. No comments were received.

This application was considered to be of significant public interest because this substance contained a new active ingredient to New Zealand - Bacillus firmus I-1582. It was, therefore, publically notified in accordance with section 53 of the Act. This application was open for public submissions from 15 October 2014 to 27 November 2014.

Nineteen submissions were received from individuals and organisations. Sixteen submissions were opposed to the approval of this substance and three were either neutral or did not indicate whether or not they supported or opposed the application. The submissions are summarised in Appendix C.

During the evaluation of the application we requested further information from the applicant under sections 52 and 58 of the Act. Once all outstanding information was received the application contained sufficient information for us to undertake an assessment of the substance in accordance with Part 2 of the Act. The key findings of that assessment are discussed in section 6 of this document and the full analysis is provided in Appendixes J and K.

The following documents were used to prepare this report:

 the application form and confidential material submitted by the applicant (including toxicological and ecotoxicological studies on the product, the active ingredient and its metabolites, the full composition of the product, chemical and physical properties of the product, and the purity of the active ingredient)

 the submissions

 other information held by the EPA. 4. Background and proposed use pattern of the substance

Poncho Votivo is a seed treatment that will be used to prevent insect and nematode damage in cereal, maize, sweetcorn, forage brassica and grass seeds. It contains 508 g/L of clothianidin and 102 g/L of Bacillus firmus I-1582 as the biologically active ingredients (ai).

Bacillus firmus I-1582 is a new pesticide active ingredient to New Zealand. Other seed treatment formulations have previously been approved that contain similar or higher concentrations of clothianidin than Poncho Votivo.

November 2015 8

Application for approval to import Poncho Votivo for release (APP202077)

The applicant noted in their application that Poncho Votivo is a reformulation of their currently approved substance, Poncho, which contains 600 g/L of clothianidin as a seed treatment, to add Bacillus firmus I-1582. The applicant expects Poncho Votivo to replace Poncho in the market.

Poncho Votivo will be imported as a finished product ready for sale. The applicant only intends to sell this substance to professional seed treatment companies, who are expected to have the skills and equipment to apply it correctly. Seeds treated with Poncho Votivo are expected to be treated with a polymer film coat and seed lubricant/fluency agent which will minimise the amount of dust generated during seed handling and sowing.

The applicant expects that Poncho Votivo will usually be disposed of by use as a seed treatment but notes that the substance, and seeds treated by the substance, can be disposed of at waste management facilities approved to accept this type of waste. Users will be advised to triple rinse the containers that have contained Poncho Votivo and recycle them via an appropriate recycling scheme or dispose of them to landfill. 5. Hazard classification

We determined the hazard classification of Poncho Votivo, as shown in Table 1 and detailed in Appendix D.

Table 1 Hazard classifications proposed by the applicant and the staff

Hazard Endpoint Applicant classification EPA classification

Acute toxicity (oral) 6.1D 6.1D

Eye irritant/corrosive 6.4A No

Respiratory sensitisation - 6.5A

Contact sensitisation - 6.5B

Target organ systemic toxicity 6.9B 6.9B (oral)

Aquatic ecotoxicity 9.1A 9.1A

Soil ecotoxicity 9.2B 9.2B

Terrestrial vertebrate ecotoxicity 9.3B 9.3C

Terrestrial invertebrate ecotoxicity 9.4A 9.4A

The classifications that we determined are the same as those submitted by the applicant except for the classifications for eye irritancy, and skin and respiratory sensitisation. The difference in classification is due to a different interpretation of the classification criteria for eye irritancy and because microbes are considered sensitisers which was not identified by the applicant.

November 2015 9

Application for approval to import Poncho Votivo for release (APP202077)

6. Risk assessment

Approach

We have assessed the risks posed by Poncho Votivo throughout its lifecycle, i.e. during import, manufacture, transportation, storage, use and disposal. This has included a quantitative human health and environmental risk assessment, the full results of which are shown in Appendices J and K. A summary of the results of the risk assessment and a discussion of the key issues are presented below.

A tiered approach was used to complete the quantitative environmental risk assessments. In this approach simpler but much more conservative assessments are completed first; if the risks are low no further assessment is needed. If the risks are high, more complex and accurate assessments are completed to refine the assessment of risk.

We note that there is little or no data on the effects of hazardous substances on New Zealand native species. To account for the uncertainty about how representative the common test species are of New Zealand native species we have compared the estimated exposure values to a second trigger value for threatened species. Trigger values indicate when a non-negligible risk is triggered. The threatened species trigger values are higher, and more conservative than the standard trigger values because they assume that that the loss of one individual will adversely affect the survival of the population. Consequently, a non-negligible risk is triggered by lower estimated exposure levels for threatened species than for non-threatened species.

When determining the risks we have assumed that the default controls, triggered by the hazard classification, shown in Table 1, are applied to this substance.

We have assumed that this substance is used as a seed treatment and that it is only applied in a commercial seed treatment facility, as specified in the applicant. No other use patterns have been considered because there is no information to assess any potential alternative uses. We note that other uses may pose different risks and should be assessed separately in the future if the use of this substance changes. Consequently we recommend applying controls to limit the use of this substance to use as a seed treatment.

Overview

We consider that the risks to human health and the environment during import, manufacture, transportation, storage and disposal can be managed to a negligible level by the default controls and other applicable legislation, such as Land Transport Rule 45001, Civil Aviation Act 1990 and Maritime Transport Act 1994.

We consider that the main risks to human health and the environment occur during the use phase of the substance’s lifecycle, when it is applied to seeds, and during the handling, sowing and disposal (if necessary) of the treated seeds. Therefore, these risks are discussed in more detail below.

November 2015 10

Application for approval to import Poncho Votivo for release (APP202077)

Human health risks

We completed a quantitative human health risk assessment for clothianidin and a qualitative assessment for Bacillus firmus I-1582 because the quantitative risk assessment models available to us are not applicable to micro-organisms.

The risk assessment for clothianidin showed that worker exposure was below the acceptable operator exposure level (AOEL) when appropriate personal protective equipment (PPE) was worn. In this case appropriate PPE included: long trousers, long shelved shirt and gloves when workers were handling treated seed or contaminated equipment, or when they were mixing, loading or calibrating equipment; it also included disposable coveralls, in addition to the other PPE listed above, when cleaning equipment.

In the risk assessment for Bacillus firmus I-1582 we considered the potential toxicological and pathogenic effects of the organism. Given the lack of toxicological and pathogenic effects observed in studies with this active ingredient, and that workers will be required to wear PPE (as this is a requirement of a default control) when treating and sowing seeds, we consider that Bacillus firmus I- 1582 is unlikely to pose a concern to human health. However, we note that biopesticides are sensitisers. Therefore, we consider that eye or face protection and respiratory protective equipment (RPE) should be worn in addition to the PPE specified above. Consequently, we recommend varying the default PPE control to specify the PPE and RPE that should be worn; proposed wording to this effect is presented in Appendix A.

The human health risk assessment also noted that workers treating seeds or working in an area where seeds are treated could be exposed to dust or mist containing Poncho Votivo. We note that this risk can be mitigated by ensuring that the seeds are treated in a purpose-built seed coating machine, in a treatment plant that has local exhaust ventilation. We recommend applying controls to this effect and have proposed controls in Appendix A.

Environmental risks

Overview The risk assessment indicated that the risks to the aquatic environment, soil organisms, plants, bees and other non-target arthropods were below the level of concern. However, some risks to birds were identified and are discussed in detail below. The risks to bees are also discussed below because these risks were of particular concern to the submitters.

Risks to birds An acute assessment (based on dietary exposure) and a chronic assessment (based on risk during reproduction) for birds were competed.

November 2015 11

Application for approval to import Poncho Votivo for release (APP202077)

The acute risk assessment considered two scenarios: birds that feed solely on treated seed, and birds that consumed newly emerged crop shoots. Short-term (1-3 days) and long-term exposures (21 days) were considered in both scenarios.

The risk assessment indicated that there were no concerns about the acute risks to birds from the consumption of treated seeds or shoots from treated seeds; nor were there significant chronic risks to birds that consume shoots from treated seed.

The risk assessment indicated that there is a chronic risk to birds from exposure to clothianidin via eating treated seeds. However, we note that this was a tier one (conservative) assessment, and as such assumes that birds only eat seeds treated with Poncho Votivo.

Literature from New Zealand1,2, indicates that maize seed consumption represents only 23-27% of the diet of birds commonly observed in agricultural areas, and that it is only a relevant food source for part of the year. We also note that the types of birds observed in agricultural areas tend to be non-native ‘pest’ species, which farmers try to discourage from eating their crops3.

In summary, Poncho Votivo could present a risk to birds that consume treated seeds in some circumstances. However, we consider that this risk can be managed with controls that prevent birds accessing treated seeds. We therefore recommend applying controls that require seeds to be completely covered with soil when sown, and require treated seeds to be stored in such a way that they cannot be accessed by birds.

Risks to Bees Poncho Votivo contains clothianidin, a neonicotinoid insecticide, which presents a potential risk to bees. This risk was the key concern of the submitters. However, the risk assessment indicated that the risks to bees were well below the level of concern. This assessment is discussed below.

We completed a quantitative tier two risk assessment for bees that considered the acute effects of clothianidin on adult honeybees, honeybee larva and adult bumble bees, and the chronic effects on adult honey bees. No data were available to assess the effects on honey bee brood.

The assessment considers the mechanism by which bees would be exposed to clothianidin on treated seeds. Namely, that the substance on the seed must be absorbed by the growing plant and distributed within the plant’s tissues to the nectar and/or pollen, which is then accessed by bees. We note that for bees to be exposed, plants grown from the treated seed must be allowed to flower, which is not the case with all commercial crops.

1 Reviewed by MacLeod et al (2008) The forgotten 60%: bird ecology and management in New Zealand’s agricultural landscape. New Zealand Journal of Ecology 32(2): 240-255.

2 Dilks (1974) Diet of feral pigeons (Columa livia) in Hawke’s Bay, New Zealand. New Zealand Journal of Agricultural Research 18: 87- 90.

3 Coleman (2001) Farmer Perceptions of bird Damage and Control in Arable Crops. New Zealand Plant Protection 54:184-187.

November 2015 12

Application for approval to import Poncho Votivo for release (APP202077)

Table 2 shows the results of the risk assessment for bees exposed to clothianidin through the pollen of maize grown with seed treated with Poncho Votivo. The risk is shown as a risk quotient (RQ) value, alongside the trigger values that indicate a non-negligible risk to bees. The results show that the RQ values are much lower than the trigger values and therefore there is no concern about the impact of this substance on bees.

Table 2 Results of Tier 2 assessment of the risks to bees from acute exposure to clothianidin in maize pollen

Life stage RQ Trigger value Result

Acute / Adult honey bees - oral 0.00006 – 0.02 0.4 Negligible risk

Acute / Adult bumble bees - oral 0.05 – 0.06 0.4 Negligible risk

Acute / honey bee larvae – oral < 0.09 0.4 Negligible risk

Chronic / Adult bee - oral 0.0004 – 0.11 1 Negligible risk

In addition to the assessment of acute risks, higher tier studies (tunnel or field tests), for exposure to maize pollen or guttation fluid, showed that there were no effects on mortality, behaviour or hive condition when bees fed from plants grown from seeds treated at 1 or 1.25 mg clothianidin/seed. The expected application rate in New Zealand is only 0.91 mg clothianidin/seed.

We also considered the effects of the metabolites of clothianidin on bees and found the risks to be negligible.

We note that dust containing seed treatment chemicals can be produced when seeds are sown and incidents of adverse effects on bees have been reported in association with seed sowing.

Studies were provided by the applicant that showed that the level of dust produced during sowing was acceptable provided that the treated seeds were film coated. Consequently, we recommend applying controls to require seeds treated with Poncho Votivo to be film coated. We consider that such controls will manage the risks of adverse effects due to dust generation.

Cultural risk – the relationship of Māori to the environment

We have assessed the potential effects of Poncho Votivo on the relationship of Māori to the environment. A detailed cultural risk assessment is presented in Appendix B and a summary of that assessment is provided here.

The human health classifications of Poncho Votivo suggest that it may pose a risk to taha hauora (human health), particularly the dimensions of taha wairua (spiritual health and well-being obtained through the maintenance of a balance with nature and the protection of mauri) and taha tinana (physical health and well-being). However, we consider that these risks can be mitigated if users wear

November 2015 13

Application for approval to import Poncho Votivo for release (APP202077)

appropriate PPE (as discussed in section 6), and the use of Poncho Votivo is limited to approved handlers (as specified by the default controls).

The importance to Māori of ensuring that mahinga kai (food resources) and their constituent species flourish cannot be overstated historically or contemporarily. In former times mahinga kai were critical for sustaining Māori communities and whanau and are still important for continuing customary practices and meeting cultural obligations. Therefore, it is important to ensure that the use of hazardous substances does not negatively impact mahinga kai species and the ecosystems that support them. We consider that the risks to mahinga kai species, particular aquatic species, can be mitigated by the proposed controls.

This application raises concerns about the potential impacts of the Poncho Votivo on culturally significant birds e.g. pūkeko (swamp hen) that may eat seeds treated with Poncho Votivo or graze young shoots emerging from these seeds. However, we consider that the proposed controls to manage the risks to birds will address the risks to native birds.

Many insect species are culturally significant due to the part they play in Māori cosmogony and environmental lore. We note that Poncho Votivo may also affect culturally significant species, such as tūtaeruru (grass grub) and noke / toke (earthworms) if they are present in the areas where the treated seeds are sown. Noke / toke for instance, are a food source for culturally significant species, including kōtare (kingfisher) and pūtangitangi (paradise shelduck); and are used in traditional fishing methods e.g. toitoi tuna (eel bobbing). However, we note that indigenous species of noke / toke are unlikely to be present where soil disturbance is high, such as on arable land. We also note that controlling the target pests in wheat, maize, forage brassicas and grass seed crops will produce economic benefits for Māori farmers.

Poncho Votivo raises no concerns about the potential impacts on taonga plants used in rongoā (medicines), pūeru (textiles), mahi toi (art), toi whakarākai (ornamentation), hangatanga (construction) or taputapu (equipment).

Given the use pattern and the controls proposed to be assigned to Poncho Votivo, we consider that the potential risks to Māori culture or traditional relationships with the environment are acceptable, despite the fact that some of the target species are culturally significant. We consider that the approval of Poncho Votivo is not likely to breach the principles of the Treaty of Waitangi.

Risks to society and the community and the market economy

We have not identified any risks to society, communities or the market economy from the controlled use of Poncho Votivo. 7. Benefits assessment

The applicant has provided information on the benefits of this substance in their application form, and has provided several references to support their benefits case. This information forms the basis of this

November 2015 14

Application for approval to import Poncho Votivo for release (APP202077)

benefits assessment.

Poncho Votivo contains two active ingredients which target different pests in the soil environment that attack seeds and young plants. Clothianidin is an insecticide while Bacillus firmus I-1582 is a nematicide. We note that a similar product, Poncho (containing 600 g/L of clothianidin), is already approved and used in New Zealand as a seed treatment to control Argentine stem weevil, clack beetle and greasy cutworm. That applicant considers that Poncho Votivo would yield the same benefits as Poncho, but with the added advantage of the nematicidal properties of Bacillus firmus I-1582.

Bacillus firmus I-1582 is a bacterium that is already found naturally in New Zealand. It has been shown to suppress nematodes and protect the roots of plants by degrading the chemicals exuded by the plants that nematodes may respond to. Bacillus firmus I-1582 is effective against several nematode species including: Meloidogyne spp., Heterodera spp., Globodera spp., Pratylenchis spp., Rotylenchus spp., Helicotylenchus spp., Xiphinema spp. and Ditylenchus spp.. Reports also indicate that Bacillus firmus I-1582 has a negative effect on nematode eggs, although that mechanism of action for this is not yet fully understood.

We note that the presence of clothianidin in Poncho Votivo is of particular concern to the submitters. On a per hectare basis, the amount of clothianidin applied to an area via seeds treated with Poncho Votivo is far less than the amount of insecticide that would need to be applied as a foliar spray or applied as a soil treatment to manage the same pests. For instance, the maximum amount of clothianidin applied to an area via seeds treated with Poncho Votivo is 90 g (ai)/ ha, compared to other insecticides used to treat the same pests, including: imidacloprid (130.5 g/ha), furathiocarb (300 g/ha), terbufos (1,500 g/ha) and diazinon (2,400 g/ha). (No clothianidin products are currently approved by the Ministry for Primary Industries for use as foliar sprays or soil treatments).

In addition, clothianidin remains present in the plant, grown from the treated seed, for several weeks after germination, giving the plant a longer period of protection compared to other seed treatment active ingredients. This reduces the need for later soil treatments and at least one foliar treatment can be removed from the recommended spray program when Poncho Votivo is used.

The applicant considers that the use of Poncho Votivo as a seed treatment will improve crop health and reduce the need to spray broad-spectrum insecticides. They note that treating seeds to prevent insect damage can dramatically increase the yield and consequently the economic viability of crops compared to untreated seed.

In addition, the available information on Bacillus firmus I-1582 suggests that it has very low environmental risk; therefore, it may be able to replace other nematicides that present greater risks to the environment. 8. Summary of submissions and staff response

Summary of Submission Nineteen submissions were received from members of the public, interest and industry groups. A

November 2015 15

Application for approval to import Poncho Votivo for release (APP202077)

summary of the submissions is presented in Appendix C; the full submissions are provided to the Committee and are available to the public via the EPA’s website.

Three submitters, Te Runanga o Ngāi Tahu, the National Beekeepers Association, and the Federated Farmers of New Zealand Bee Industry Group were either neutral or did not indicate whether they supported or opposed the application. All other submitters opposed the application.

All submitters raised concerns about the potential impact of clothianidin on bees, given that it is a neonicotinoid insecticide. The submitters generally noted the importance of bees to agriculture, horticulture, export crops and the human food-chain. They highlighted concerns about residues of clothianidin in the soils, the effect of dust from seed treatments, and that clothianidin moves from the seed into the pollen and nectar of the plants that grow from the treated seed. Submitters also noted that neonicotinoid insecticides can cause sub-lethal effects in bees that reduce the success of hive communities. For example, learning, memory and behavioural changes that affect navigation and social behaviours.

Some submitters noted that neonicotinoid insecticides have been implicated in bee colony collapse disorder and banned in the European Union, and therefore proposed that New Zealand also ban these substances.

Staff response to the submissions We acknowledge that bees provide vital pollination services for agriculture and horticulture in New Zealand, as well as ecosystem health. We note that the environmental risk assessment for bees considered the potential acute and chronic effects of this substance to bees. That risk assessment is presented in Appendix K and summarised section 6; it indicated that the risks to bees were very low and are therefore considered negligible.

Submitters also noted the effects of dust generated during seed sowing. We have proposed controls to reduce the amount of dust produced which will further reduce the impact of Poncho Votivo on bees. 9. Other matters to be considered

New Zealand’s international obligations

We have not identified any international obligations that might be affected by the approval or use of Poncho Votivo.

The effects of the substance being unavailable

We consider that if this substance is unavailable users will continue to use the alternative product Poncho, which contains more clothianidin than Poncho Votivo but which offers less protection from nematodes. Users are also likely to use other means, including seed treatment and pesticides applied to soils, to prevent seeds being damaged by invertebrates in the soil. These alternative measures could lead to a larger amount of pesticide being applied in the environment.

November 2015 16

Application for approval to import Poncho Votivo for release (APP202077)

10. Conclusion

We have assessed the potential effects to of this substance, including those related to the seed treatment process, treated seeds and seed sowing process. This assessment indicated that the potential adverse effects can be managed to a negligible level with the proposed default and additional controls and that there are non-negligible benefits to the approval of this substance, specifically a reduction of environmental exposure to clothianidin compared to current practice. 11. Recommendation

We consider that the overall level of benefit provided by the availability of Poncho Votivo will outweigh the overall risk and recommended that the Committee approve Poncho Votivo with the controls documented in Appendix A.

November 2015 17

Application for approval to import Poncho Votivo for release (APP202077)

Appendix A: Proposed Controls for Poncho Votivo

We have developed a set of proposed controls, set out below, based on the default controls, which are triggered by the classifications and set out in the regulations under that Act. The proposed controls include variations, deletions and additions to the default controls that are based on the risk assessment and the recommendations discussed above. The justification for these variations, deletions and additions are outlined below the controls tables.

Please refer to the Hazardous Substances Regulations4 for the requirements prescribed for each control and the modifications listed as set out in this document.

Hazardous Substances (Classes 6, 8, and 9 Controls) Regulations 2001

Code Regulation Description Variation

T1 Regs 11 – Limiting exposure to toxic No TEL values are set for any component of the 27 substances through the setting substance at this time of TELs

T2 Regs 29, 30 Controlling exposure in places of WES values from WorkSafe New Zealand’s Workplace work through the setting of Exposure Standards and Biological Exposure Indices WESs. Document; 7th Edition; February 20135 have been adopted for this substance. [Note: WES values have been set for components G and E1]

T4 Reg 7 Requirements for equipment used to handle substances

T5 Reg 8 Requirements for protective The following is added in addition to the requirements clothing and equipment of regulation 8:  Users must wear long trousers, long shelved shirt, gloves, eye or face protection, and respiratory protection when handling treated seed or contaminated equipment and during mixing/loading/calibrating equipment used to apply this substance or seed treated with this substance.  Users must wear long trousers, long shelved shirt, disposable coveralls, gloves, eye or face protection, and respiratory protection when cleaning equipment used to apply this substance or seed treated with this substance.

4 The regulations can be found on the New Zealand Legislation website; http://www.legislation.co.nz

5 Document DOL11590.4 FEB13 and any subsequent version approved or endorsed by the EPA. Version 7 is available at http://www.business.govt.nz/worksafe/information-guidance/all-guidance-items/workplace-exposure-standards-and-biological-exposure- indices/workplace-exposure-standards-and-biological-indices-2013.pdf

November 2015 18

Application for approval to import Poncho Votivo for release (APP202077)

Code Regulation Description Variation

T7 Reg 10 Restrictions on the carriage of toxic or corrosive substances on passenger service vehicles

E1 Regs 32 – Limiting exposure to ecotoxic No EEL values are set at this time and the default EELs 45 substances through the setting are deleted of EELs

E5 Regs 5(2), 6 Requirements for keeping records of use

E6 Reg 7 Requirements for equipment used to handle substances

E7 Reg 9 Approved handler/security requirements for certain ecotoxic substances

Hazardous Substances (Identification) Regulations 2001

Code Regulation Description Variation

I1 Regs 6, 7, Identification requirements, 32 – 35, duties of persons in charge, 36(1) – (7) accessibility, comprehensibility, clarity and durability

I3 Reg 9 Priority identifiers for ecotoxic substances

I8 Reg 14 Priority identifiers for toxic substances

I9 Reg 18 Secondary identifiers for all hazardous substances

I11 Reg 20 Secondary identifiers for ecotoxic substances

I16 Reg 25 Secondary identifiers for toxic substances

I17 Reg 26 Use of generic names

I18 Reg 27 Requirements for using concentration ranges

I19 Regs 29 – Additional information 31 requirements, including situations where substances are in multiple packaging

I20 Reg 36(8) Durability of information for class 6.1 substances

November 2015 19

Application for approval to import Poncho Votivo for release (APP202077)

Code Regulation Description Variation

I21 Regs 37 – General documentation 39, 47 – 50 requirements

I23 Reg 41 Specific documentation requirements for ecotoxic substances

I28 Reg 46 Specific documentation requirements for toxic substances

I29 Regs 51, 52 Signage requirements

I30 Reg 53 Advertising corrosive and toxic substances

Hazardous Substances (Packaging) Regulations 2001

Code Regulation Description Variation

P1 Regs 5, 6, General packaging requirements 7(1), 8

P3 Reg 9 Criteria that allow substances to be packaged to a standard not meeting Packing Group I, II or III criteria

P13 Reg 19 Packaging requirements for toxic substances

P15 Reg 21 Packaging requirements for ecotoxic substances

PG3 Schedule 3 Packaging requirements equivalent to UN Packing Group III

PS4 Schedule 4 Packaging requirements as specified in Schedule 4

Hazardous Substances (Disposal) Regulations 2001

Code Regulation Description Variation

D4 Reg 8 Disposal requirements for toxic and corrosive substances

D5 Reg 9 Disposal requirements for ecotoxic substances

D6 Reg 10 Disposal requirements for packages

November 2015 20

Application for approval to import Poncho Votivo for release (APP202077)

Code Regulation Description Variation

D7 Regs 11, 12 Information requirements for manufacturers, importers and suppliers, and persons in charge

D8 Regs 13, 14 Documentation requirements for manufacturers, importers and suppliers, and persons in charge

Hazardous Substances (Emergency Management) Regulations 2001

Code Regulation Description Variation

EM1 Regs 6, 7, 9 Level 1 information – 11 requirements for suppliers and persons in charge

EM6 Reg 8(e) Information requirements for toxic substances

EM7 Reg 8(f) Information requirements for ecotoxic substances

EM8 Regs 12 – Level 2 information 16, 18 – 20 requirements for suppliers and persons in charge

EM11 Regs 25 – Level 3 emergency 34 management requirements: duties of person in charge, emergency response plans

EM12 Regs 35 – Level 3 emergency The following subclauses are added after subclause (3) 41 management requirements: of regulation 36: secondary containment (4) For the purposes of this regulation, and regulations 37 to 40, where this substance is contained in pipework that is installed and operated so as to manage any loss of containment in the pipework it— (a) is not to be taken into account in determining whether a place is required to have a secondary containment system; and (b) is not required to be located in a secondary containment system. (5) In this clause, pipework— (a) means piping that— (i) is connected to a stationary container; and

November 2015 21

Application for approval to import Poncho Votivo for release (APP202077)

Code Regulation Description Variation (ii) is used to transfer a hazardous substance into or out of the stationary container; and (b) includes a process pipeline or a transfer line. The following subclauses are added at the end of regulation 37: (2) If pooling substances which do not have class 1 to 5 hazard classifications are held in a place above ground in containers each of which has a capacity of 60 litres or less— (a) if the place’s total pooling potential is less than 20,000 litres, the secondary containment system must have a capacity of at least 25% of that total pooling potential: (b) if the place’s total pooling potential is 20,000 litres or more, the secondary containment system must have a capacity of the greater of— (i) 5% of the total pooling potential; or (ii) 5,000 litres. (3) Pooling substances to which subclause (2) applies must be segregated where appropriate to ensure that leakage of one substance may not adversely affect the container of another substance. The following subclauses are added at the end of regulation 38: (2) If pooling substances which do not have class 1 to 5 hazard classifications are held in a place above ground in containers 1 or more of which have a capacity of more than 60 litres but none of which have a capacity of more than 450 litres— (a) if the place’s total pooling potential is less than 20,000 litres, the secondary containment system must have a capacity of either 25% of that total pooling potential or 110% of the capacity of the largest container, whichever is the greater: (b) if the place’s total pooling potential is 20,000 litres or more, the secondary containment system must have a capacity of the greater of— (i) 5% of the total pooling potential; or (ii) 5,000 litres

November 2015 22

Application for approval to import Poncho Votivo for release (APP202077)

Code Regulation Description Variation (3) Pooling substances to which subclause (2) applies must be segregated where appropriate to ensure that the leakage of one substance may not adversely affect the container of another substance.

EM13 Reg 42 Level 3 emergency management requirements: signage

Hazardous Substances and New Organisms (Personnel Qualifications) Regulations 2001

Code Regulation Description Variation

AH 1 Regs 4 – 6 Approved Handler requirements Refer to control E7 (including test certificate and qualification requirements)

Hazardous Substances (Tank Wagon and Transportable Containers) Regulations 2004

Code Regulation Description Variation

Tank Regs 4 to 43 Controls relating to tank wagons Wagon as applicable and transportable containers

Additional controls Code Section Control of the Act

Water 77A This substance must not be applied into or onto water6

App Rate 77A A maximum application rate for this substance is 120 mL of this substance per 80 000 seeds

App 77A This substance may only be applied as a seed treatment. method This substance may only be applied in a purpose-built seed coating machine, in a treatment plant with local exhaust ventilation.

Sch 8 Schedule 8 This schedule prescribes the controls for stationary container systems. The requirements of this schedule are detailed in the consolidated version of the Hazardous Substances (Dangerous Goods and Schedule Toxic Substances) Transfer Notice 20047 The following clause replaces Clause 1 of Schedule 8 of the Hazardous Substances (Dangerous Goods and Scheduled Toxic Substances) Transfer Notice 2004:

6 where ‘water‘ means water in all its physical forms, whether flowing or not, and whether over or under ground, but does not include water in any form while in a pipe, tank or cistern or water used in the dilution of the substance prior to application.

7 available from http://www.epa.govt.nz/Publications/Transfer-Notice-35-2004.pdf

November 2015 23

Application for approval to import Poncho Votivo for release (APP202077)

Code Section Control of the Act This Schedule applies to every stationary container system that contains, or is intended to contain the substance.

Seed 77A The following additional controls are applied to this substance: treatment  Seeds treated with this substance must be film coated to minimise the amount of dust being generated during handling and sowing  Seeds treated with this substance must be dyed to distinguish them from untreated seed  Seeds treated with this substance must not be left in areas accessible to birds  Seeds treated with this substance must be completely covered by soil when sown  Seeds treated with this substance must not be used for human or animal consumption  Seeds treated with this substance must be disposed of in accordance with the controls specified for this substance under the Hazardous Substance (Disposal) Regulations 2001  Any person supplying seeds treated with this substance must ensure that packages of treated seeds are accompanied by information that identifies the substance that the seeds have been treated with, its hazardous properties and precautions to be taken in handling the seeds

Labelling 77A The label of this substance and the label of seeds treated with this substance must specify the personal protective equipment and respiratory protective equipment that users must wear when handling the substance, seeds treated with the substance and equipment that has been used with the substance or seeds treated with the substance. The label of this substance must include the following statements or words to this effect: This substance may only be applied in a purpose-built seed coating machine, in a treatment plant with local exhaust ventilation A maximum application rate has been set for this substance is 120 mL of this substance per 80 000 seeds The label of packages containing seeds treated with this substance must include the following statement or words to this effect: Seeds treated with this substance must not be left in areas accessible to birds Seeds treated with this substance must be completely covered by soil when sown.

November 2015 24

Application for approval to import Poncho Votivo for release (APP202077)

Justification for the proposed controls

Exposure limits

1. Tolerable Exposure Limits (TELs), Acceptable Daily Exposure (ADE), Potential Daily Exposure (PDE) values can be set to control hazardous substances entering the environment in quantities that present a risk to people. No TELs have been set for any component of Poncho Votivo at this time.

2. Workplace exposure standards (WESs) can be set to limit the amount of toxic substances that people are exposed to in workplaces. The EPA typically adopts the WES values listed by WorkSafe New Zealand8 and we recommend that these values are adopted for this substance. We note that WES values have been set for components G and E1.

3. Environmental Exposure Limits (EELs) can be set to limit hazardous substances from entering the environment in quantities that are sufficient to present a risk to it. No EEL has previously been set for any component of Poncho Votivo and we consider that the potential effects of Poncho Votivo can be effectively managed through the use of other controls, such as the maximum application rates. Therefore no new EELs have been proposed.

4. We note that the some submitters have requested that an EEL for clothianidin in soil be set. We note that there can be practical and financial issues for users and enforcement agencies (regional councils) associated with, complying with, and enforcing EEL’s. For example, the cost and practical issues associated with the necessary analytical testing and ensuring a representative sample is obtained. We consider that such issues should be addressed on an ‘all substances’ basis, rather than for a single substance and that any new EEL should apply to all substances containing clothianidin. Therefore, we consider that it is more appropriate to consider new EELs for existing substances as part of a reassessment process rather than within a single application.

Variation and deletion of controls

Tracking 5. The default controls include requirements for this substance to be tracked. Where this control is triggered by virtue of a substance’s ecotoxicity, we consider that any risks that may arise during its lifecycle can be adequately managed by other controls, e.g. approved handler, packaging, labelling and emergency management requirements. Therefore we recommend that this control is deleted.

Application to flowing plants 6. We note that the E3 control (regulation 49 of the Hazardous Substances (Classes 6, 8 and 9) Regulations 2001) is triggered by the 9.4A classification of this substance. This controls prohibits

8 http://www.business.govt.nz/worksafe/information-guidance/all-guidance-items/workplace-exposure-standards-and-biological- exposure-indices/workplace-exposure-standards-and-biological-indices-2013.pdf

November 2015 25

Application for approval to import Poncho Votivo for release (APP202077)

substances form being applied (e.g. sprayed) to plants that are in open flower or part bloom. As Poncho Votivo is a seed treatment, this control is not applicable and we recommend that it is deleted. We note that the potential risks to bees are managed by the proposed additional controls.

Personal protective equipment 7. The human health risk assessment noted that the risks to people applying the substance or handling treated seeds were acceptable if those people wore appropriate PPE, including eye protection and respiratory protection. The default T5 control (regulation 8 of the Hazardous Substances (Classes 6, 8 and 9 Controls) Regulations 2001)) requires users to wear appropriate PPE. We consider it appropriate to specify the type of PPE required and recommend varying the T5 control to this effect. The variation wording is shown in the controls table above.

Secondary containment 8. The default emergency management (EM12) control (regulations 35 – 41 of the Hazardous Substances (Emergency Management) Regulations 2001)) requires tanks and containers of liquid substances over a specified volume to have secondary containment in order to manage the risk of the tank or container failing and the substance spilling into the environment. We note that these requirements do not allow for dispensation where it is unnecessary for pipework associated with the stationary container system to have secondary containment. We recommend that this control is varied to note that any quantities of the substance contained within pipework do not need to be taken into account in determining whether a place is required to have a secondary containment system.

Additional controls

Stationary container systems 9. We note that the default controls address the need for stationary container systems (e.g. tanks) to have secondary containment, but do not set requirements for the management and maintenance of stationary container system itself. Stationary container systems need to be maintained and managed appropriately to prevent the system failing and a large spill occurring. Therefore, we recommend applying the requirements specified in the Hazardous Substances (Dangerous Goods and Schedule Toxic Substances) Transfer Notice 20049, under section 77A of the Act to manage this risk.

Application restriction 10. We have assessed the risks of Poncho Votivo based on the use pattern proposed by the applicant, but note that the risks associated with using the substance for different purposes are unknown. Therefore, we recommend applying the following controls to limit the use of Poncho Votivo to the established use parameters and maximum application rates.

9 Available at http://www.epa.govt.nz/Publications/Transfer-Notice-35-2004.pdf

November 2015 26

Application for approval to import Poncho Votivo for release (APP202077)

 This substance may only be applied as a seed treatment.

 This substance must not be applied onto or into water10

 A maximum application of this substance is 120 mL of this substance per 80 000 seeds

Application method 11. The risk assessment noted that people could be adversely affected by exposure to Poncho Votivo either during the seed treatment process, when treated seeds are sown, or via accidental exposure to treated seeds. Therefore, we recommend applying the following controls to minimise the amount of dust generated during seed treatment and sowing, and to reduce the likelihood of accidental consumption of treated seeds by people or animals that may form part of the human food chain.

 This substance may only be applied in a purpose-built seed coating machine, in a treatment plant with local exhaust ventilation

 Seeds treated with this substance must be film coated to minimise the amount of dust being generated during handling and sowing

 Seeds treated with this substance must be dyed to distinguish them from untreated seed

 Seeds treated with this substance must not be used for human or animal consumption

Use/ protection of birds 12. The risk assessment noted a risk to birds if they consume seeds treated with Poncho Votivo. Therefore, we recommend applying the following controls to prevent birds accessing treated seeds.

 Seeds treated with this substance must not be left in areas accessible to birds

 Seeds treated with this substance must be completely covered by soil when sown.

Treated seeds 13. We note that seeds treated with Poncho Votivo may adversely affect human health or the environment if they are handled or disposed of inappropriately. To manage this risk we consider that seeds treated with Poncho Votivo should meet the same labelling and disposal requirements as containers of Poncho Votivo. Therefore, we recommend applying the following controls to the treated seeds.

 Seeds treated with this substance must be disposed of in accordance with the controls specified for this substance under the Hazardous Substance (Disposal) Regulations 2001.

 Any person supplying seeds treated with this substance must ensure that packages of treated seeds are accompanied by information that identifies the substance that the seeds have been treated with, its hazardous properties and precautions to be taken in handling the seeds.

10 where ‘water‘ means water in all its physical forms, whether flowing or not, but does not include water in any form while in a pipe, tank or cistern or water used in the dilution of the substance prior to application or water used to rinse the container after use

November 2015 27

Application for approval to import Poncho Votivo for release (APP202077)

Labelling controls 14. The product label is the primary way that users access information about the precautions that they need to take to protect their health (e.g. PPE) and use substances in accordance with the controls. We therefore recommend that the following labelling controls are applied to Poncho Votivo.

For this substance and seeds treated with this substance

The label of this substance and the label of seeds treated with this substance must specify the personal protective equipment and respiratory protective equipment that users must wear when handling the substance, seeds treated with the substance and equipment that has been used with the substance or seeds treated with the substance.

For this substance

The label of this substance must include the following statements or words to this effect:

 This substance may only be applied in a purpose-built seed coating machine, in a treatment plant with local exhaust ventilation

 A maximum application rate for this substance is 120 mL of this substance per 80 000 seeds

For seeds treated with this substance

The label of packages containing seeds treated with this substance must include the following statement or words to this effect:

 Seeds treated with this substance must not be left in areas accessible to birds

 Seeds treated with this substance must be completely covered by soil when sown.

Environmental user charges 15. We consider that the proposed controls are an effective means of managing the risks associated with Poncho Votivo and that environmental user charges are not necessary to achieve effective risk management. Therefore, we do not recommend applying environmental user charges.

Review of controls for cost-effectiveness

16. We consider that the proposed controls are the most cost-effective means of managing the identified risks and costs associated with this substance; and that the proposed variations, deletions and additional controls are a more effective means of managing the potential adverse effects of Poncho Votivo than the default controls alone.

November 2015 28

Application for approval to import Poncho Votivo for release (APP202077)

Appendix B: Cultural risk assessment report

Kupu arataki (context) 1. The potential effects of Poncho Votivo on the relationship of Māori to the environment have been assessed in accordance with sections 5(b), 6(d) and 8 of the Act. Under these sections all persons exercising functions, powers and duties under this Act shall: Recognise and provide for the maintenance and enhancement of people and communities to provide for their cultural well-being, and; take into account the relationship of Māori and their culture and traditions with their ancestral lands, water, taonga and the principles of the Treaty of Waitangi (Te Tiriti o Waitangi).

2. It is noted that Poncho Votivo triggers a number of hazardous properties giving rise to the potential for cultural risk e.g. terrestrial and aquatic ecotoxicity. Cultural risk includes any negative impacts to treasured flora and fauna species, the environment, and the general health and well-being of individuals and the community.

3. In general, the introduction and use of hazardous substances has the potential to inhibit the ability of Māori to fulfil their role as kaitiaki. This is particularly relevant when considering the guardianship of land and waterways given the ecotoxic nature of Poncho Votivo to Te Marae o Tāne (terrestrial ecosystems) and Te Marae o Tangaroa (freshwater and marine ecosystems), in particular species associated with mahinga kai (food resources), hātaretare whenua (terrestrial invertebrates) and kōrero ō mua (traditional narratives).

Mahinga kai (food resources) 4. With respect to Te Marae o Tangaroa, if Poncho Votivo enters waterways there is potential for this substance to adversely affect culturally significant food species such as tuna (freshwater eels), piharau (lamprey), mohoao (black flounder), inanga (whitebait), kōura / kēwai (freshwater crayfish) or their prey species, for example kōuraura (shrimp), piriwai (mayfly), pūrerehua (caddisfly), pūene (dobsonfly), hoehoe (water boatman), hoe tuarā (backswimmer) and tātaka ruku (diving beetles). This substance may also potentially harm other culturally significant non-food species such as kōkopu (galaxiids) and toitoi (bullies).

5. With respect to Te Marae o Tāne, it is anticipated that any potential adverse effects Poncho Votivo has on land-based or water-based food plants e.g. pūhā (sow thistle), raupeti (black nightshade) and kowhitiwhiti (watercress) will be negligible.

6. The importance to Māori of ensuring that mahinga kai (food resources) and their constituent species flourish cannot be overstated historically or contemporarily. In former times, mahinga kai were critical for sustaining Māori communities and whānau; aquatic species formed a very important part of the food supply. Taonga food species remain essential for continuing customary practices and meeting cultural obligations, particularly in respect of showing manaaki (hospitality) to guests on the marae and providing whānau with traditional kai.

November 2015 29

Application for approval to import Poncho Votivo for release (APP202077)

Hātaretare (invertebrates) 7. Poncho Votivo has potential to harm indigenous species of insects belonging to a domain known as ‘Te Aitanga a Punga’ (the progeny of Punga), including tūtaeruru (grass grub), kuturiki (aphid), ngūharu (greasy cutworm) and pāpapa witi (wheat bug) which this substance has been specifically designed to target.

8. These insects are culturally significant due to the part they play in Māori cosmogony and environmental lore. For example, according to traditional narrative, the insects and birds saw a man named Rātā fell a large tree to make a canoe without appropriate ritual or authority to do so. They were angry about this and twice re-erected the tree after he had felled it, resulting in great shame for Rātā. This cautionary tale is a reminder that natural resources should be used wisely and with due care and diligence.

9. Poncho Votivo similarly poses a risk to culturally significant species of iroiro (nematodes) or other valued species that may eat, or be present in areas containing, seeds treated with Poncho Votivo such as hiore kakati (earwig), pāpaka nguturoa (weevil), kēkerengū (cockroach), pōpokorua (ants) and pūngāwerewere (spiders), all of which belong to Te Aitanga a Punga.

10. This application is likely to be of interest to Māori due to the potential for Poncho Votivo to adversely affect noke / toke (earthworms) which are taonga species. Noke / toke are important to Māori because they:

 Are a source of food for culturally significant species e.g. tarāponga (red billed gull), kotare (kingfisher) and pūtangitangi (paradise shelduck)

 Are used in traditional fishing methods e.g. toitoi tuna (eel bobbing)

 Are a part of the Māori cosmogeny e.g. stories concerning Māui and the mortality of humans

 Have geographical significance through incorporation into place names e.g. Te Tai Tokerau (Northland)

11. A mitigating factor regarding indigenous species of noke / toke is that they are less likely to present where soil disturbance is high as on arable land.

Ngā manu (birds) 12. This application raises concern in relation to potential impacts on culturally significant birds e.g. pūkeko (swamp hen) that may eat seeds treated with Poncho Votivo or graze young shoots emerging from these seeds.

13. The pūkeko has an important place in Māori lore. Pūkeko were admired for their bold scheming and determination. In former times, the tenacious and mischievous pūkeko raided gardens for kūmara and taro. Stubborn, annoying people are compared to the bird and said to have ‘taringa pākura’ or pūkeko ears (pākura is another name for pūkeko) i.e. headstrong people who ignore or don’t listen to advice.

November 2015 30

Application for approval to import Poncho Votivo for release (APP202077)

14. One tradition holds that the pūkeko is the offspring of Punga but was adopted by a relative and high chief named Tawhaki. Tawhaki cut himself while working timber and smeared his blood on the forehead and beak of the pūkeko to signify their bond. This explains the red facial colouring of the pūkeko. Another explanation is that Tamaiwaho, a supernatural resident in the upper heavens, had hit the pūkeko because it stole and ate his food supplies.

15. Pūkeko feature in stories of how the kiwi lost its wings in which all forest birds refuse to come down from the trees to eat the bugs on the ground and save the forest, except the kiwi which was willing to give up its colours and the ability to fly. The pūkeko’s excuse was that the forest floor was too damp and it didn’t want to get wet feet, so was punished by having to live in swamps.

Taha hauora (human health) 16. Poncho Votivo is classified as being an eye irritant, respiratory sensitiser, contact sensitiser, and is harmful to human organs or systems. For these reasons, this substance poses a risk to taha hauora (human health) particularly the dimensions of taha wairua (spiritual health and well-being obtained through the maintenance of a balance with nature and the protection of mauri) and taha tinana (physical health and well-being).

17. Exposure to Poncho Votivo may inhibit taha whānaunga – the responsibility to belong, care for and share in the collective, including relationships and social cohesion. There is a risk that using this substance may compromise the ability of people to protect co-workers and others where it is being used. Ensuring the collective welfare and fostering a sense of well-being and safety amongst all involved is important for maintaining taha whānaunga.

Ētahi atu mea (other matters) 18. It is noted that the active ingredient clothianidin is restricted in the European Community in relation to crops (e.g. maize) that are attractive to bees.

19. Poncho Votivo raises no concerns regarding impacts on taonga plants used in rongoā (medicines), pūeru (textiles), mahi toi (art), toi whakarākai (ornamentation), hangatanga (construction) or taputapu (equipment).

20. Some of the foregoing risks to environmental and human health can be mitigated by applying controls such as: Not leaving excess seed in areas accessible to birds; not applying the substance into or onto water; completely covering seeds when sown; stipulating use of PPE, and; requiring users to have approved handler status.

21. Controlling the target pests in wheat, maize, forage brassicas and grass seed crops will produce economic benefits for those growing or working with these crops, many of whom are Māori.

November 2015 31

Application for approval to import Poncho Votivo for release (APP202077)

Kupu whakatepe (conclusion) 22. Based on the information provided, including the use pattern and the controls proposed to be assigned to Poncho Votivo, the potential risks to Māori culture or traditional relationships with the environment should be tolerable, given that some of the target species are culturally significant.

23. If Poncho Votivo is applied in the prescribed manner it is considered that it is not likely to breach the principles of the Treaty of Waitangi, including the principle of active protection.

November 2015 32

Application for approval to import Poncho Votivo for release (APP202077) Appendix C: Summary of submissions

Table 3 Summary of submissions received

Individual No. Submitter Position Key issues Summary of submission or group

This substance contains a neonicotinoid that has been implicated in bee colony collapse and has been banned in the EU. The application should be declined as there is clear evidence of 110630 Michael Tott Individual Oppose Bees potential environmental harm. Neonics (Neonicotinoid pesticides) pose a huge risk to the bee and pollinator populations.

This substance contains a neonicotinoid that has been implicated in bee colony collapse and 110631 Jon Carapiet Individual Oppose Bees has been banned in the EU. The application should be declined as there is clear evidence of potential environmental harm. Neonics pose a huge risk to the bee and pollinator populations.

Marcia Not 110632 Individual Bees “If you kill the bees, you kill the world food chain”, Lawerence indicated

This substance contains a neonicotinoid that has been implicated in bee colony collapse and Mike G 110633 Individual Oppose Bees has been banned in the EU. The application should be declined as there is clear evidence of McCree potential environmental harm. Neonics pose a huge risk to the bee and pollinator populations.

This pesticide is a bee-killer. It contains a neonic which is soaked into the seeds which in turn Joanne spread throughout the plant killing anything that feeds on it including bees. Hive collapse is a 110634 Individual Oppose Bees Churcher significant problem world-wide and adding more pesticides to the approved list will only add to the problem.

This product contains a neonicotinoid that has been implicated in colony collapse disorder and Ross NZ Peasants has been banned in the EU. The application should be declined as there is clear evidence of 110635 Oppose Bees Scholes Association potential environmental harm. There is no benefit in the NZ-EPA taking risks with pollinators in New Zealand by approving this application.

110636 John Phillips Individual Oppose Bees These types of pesticides; neonicotinoids, have been linked to bee deaths and colony collapse in countries throughout the world. A 2012 study showed the presence of thiamethoxam and

November 2015 33

Application for approval to import Poncho Votivo for release (APP202077)

Individual No. Submitter Position Key issues Summary of submission or group clothianidin in bees found dead in and around hives situated near agricultural fields [Purdue Newsroom – Researchers: Honeybee deaths linked to seed insecticide exposure. Purdue.edu (2012-01-11)]. Neonicotinoid production has been banned in the EU, and this indicates its potential for harming the New Zealand environment and economy.

This substance contains a neonicotinoid that has been implicated in bee colony collapse and Elaine 110637 Individual Oppose Bees has been banned in the EU. The application should be declined as there is clear evidence of Bainbridge potential environmental harm. Neonics pose a huge risk to the bee and pollinator populations.

Neonicotinoids are a pesticide proven to be detrimental to bees and therefore constitutes an Patricia unacceptable risk to the NZ Apiculture Industry. This pesticide contains a neonicotinoid that 110638 Individual Oppose Bees Wallace has been implicated in bee colony collapse and can easily be replaced by other chemicals which don’t have such a devastating effect on the world’s food chain.

Past importation of plants, animals and organisms have often proved disastrous to New Zealand’s indigenous plants, birds and sea life. New Zealand cannot afford to have any more 110639 Jenny Kirk Individual Oppose Bees introduced and should take a precautionary approach to any such application for introduction of foreign species, in particular any organism which might further harm depleted bee populations should be banned.

The application fails to provide a fully argued case for the benefits to the agricultural sector, or to the general public including Māori, from the introduction and use of this insecticide for seed treatment. Any supporting evidence is again provided in a confidential appendix which has been raised previously as a barrier for submitters. The toxicity of the active ingredient Bees clothianidin raises some concerns over risks to the aquatic environment and terrestrial invertebrates however Te Rūnanga o Ngāi Tahu agree that the risk appears to be low, and Te Rūnanga Toxicity to 110640 Gerry Coates Neutral have decided to remain neutral regarding the introduction of this insecticide product Poncho o Ngāi Tahu aquatic and terrestrial Votivo to New Zealand. invertebrates Overall the application seems to have been prepared in a casual pro forma manner. There is no commentary or data in the application regarding potential risks associated with any metabolites resulting from the degradation of the active ingredient Clothianidin in soil or water, nor are these chemicals named. The fact that the proposed use is on crops such as maize, sweetcorn, forage brassicas and grass seed means that initially after planting there is the

November 2015 34

Application for approval to import Poncho Votivo for release (APP202077)

Individual No. Submitter Position Key issues Summary of submission or group potential for run-off to occur to any adjacent aquatic environment with adverse effects on indigenous foraging food crops such as watercress, and the breeding habitats of fauna such as inanga and should not be ignored. The active ingredient is restricted in the EU and applies to the use of these actives for seed treatment, soil application (granules) and foliar treatment on plants and cereals (with the exception of winter cereals) that are attractive to bees.

This pesticide contains a neonicotinoid that has been implicated in bee colony collapse and Sarah has been banned in the EU. These pesticides can easily be replaced by other chemicals 110641 Individual Oppose Bees Russell which don’t have such a devastating effect on the world’s food chain. The application should be declined as there is clear evidence of potential environmental harm.

This pesticide contains a neonicotinoid that has been implicated in bee colony collapse in Celeste Canada and other countries. New Zealand has a large fruit production and export market; of 110642 Hayward- Individual Oppose Bees which bees are import to its future and it is not acceptable to but the agricultural sector in Ryan further danger from bee colony collapse. Neonicotinoid production has been banned in the EU, indicating there is evidence of potential harm to New Zealand’s economy.

Neonicotinoid is proven to be detrimental to bees, and possibly other pollinating insects. It has 110643 Sidney Lovell Individual Oppose Bees been banned by the European Commission (EC) and New Zealand should be proactive in trying to save its bees as the EC is.

The application fails to meet sections (4), (5), and (6) of the HSNO Act due to the adverse effects of the active ingredient clothianidin (a neonicotinoid). Clothianidin has been shown to Claire have long term systemic persistence and affects insects who come into contact with the pollen 110644 Individual Oppose Bees Bleakley or sap in the plants. Di Prisco et al have published findings that show Clothianidin a neonicotinoid is highly dangerous to the bee population. The EU has placed a two year ban on clothianidin and neonicotinoids due to their adverse effects on insect populations.

This pesticide contains a neonicotinoid that has been implicated in bee colony collapse and has been banned in the EU. New Zealand has a large fruit production and export market; of 110645 Kate Hughes Individual Oppose Bees which bees are import to its future and it is not acceptable to put the agricultural sector in further danger from bee colony collapse.

November 2015 35

Application for approval to import Poncho Votivo for release (APP202077)

Individual No. Submitter Position Key issues Summary of submission or group

Federated Farmers is concerned with the possible approval of this seed treatment and any potential effect on bees and other pollinators. The active ingredient in Poncho Votivo is clothianidin, a neonicotinoid, and known to be harmful to honeybees. Federated Farmers is concerned that few references to honeybees are made in the application and material provided by the applicant. The only reference made by the applicant to the potential harm of clothianidin to honeybees is the risk of exposure via dust emission. The European Food Federated Safety Authority (EFSA) and United States Environmental Protection Authority identify that Farmers of clothianidin is highly toxic to honeybees on a contact and an oral basis. There is also Philippa New 110646 Neutral Bees uncertainty around the pollen and nectar uptake by the honeybee from flowering crops treated Rawlinson Zealand Bee with clothianidin. Industry Group To reduce the risk to honey bees and other beneficial insects, Federated Farmers submits that: Poncho Votivo should be applied with a Peridiam seed treatment film coat to prevent dust from the substance from entering the atmosphere; and the applicant should be directed to clearly state the potential adverse effects of Poncho Votivo on bees and other pollinators on the product label.

Clothianidin is very toxic to bees. It is recognised that Bayer has worked actively to promote best practice for seed treatment use of this chemical to reduce the risk to bees. Clothianidin is subject to bans on use in Europe and Bayer is facing lawsuits in Canada with National respect to seed treatment use of clothianidin causing beehive deaths. Beekeepers Don Not 110647 Association Bees The application advises the public and the EPA to consider benefits that cannot be subject to MacLeod indicated of New scrutiny because no agency in NZ is collecting data on pesticide use by volume and crop. Zealand These benefits should not be considered as appropriate for this application. There is no established Environmental Exposure Limit (EEL) for a soil active residue chemical widely used in New Zealand. This application enables the EPA to reconsider this situation and consider introducing an EEL for this substance.

David 110648 Individual Oppose Bees Concerned about the risk to New Zealand’s primary industries of importing a substance that Chisholm has raised concerns from scientists regarding its effect on honey bees. The association of a

November 2015 36

Application for approval to import Poncho Votivo for release (APP202077)

Individual No. Submitter Position Key issues Summary of submission or group clothianidin and neonicotinoid based insecticide with Colony Collapse Disorder has resulted in bans on these substances in other countries while further research is undertaken and this should also be done in New Zealand. The assertion by the applicant that existing controls will be sufficient to prevent serious damage to New Zealand are questioned and further concerns raised regarding the risk of a hazardous substance spill during transport.

November 2015 37

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix D: Classification of Poncho Votivo

Unless otherwise noted, all studies were conducted according to Good Laboratory Practice (GLP) and were fully compliant with all requirements of the standard international test methods used. The classifications for Poncho Votivo are shown in Table 4. Data quality – overall evaluation

We acknowledge that there are frequently data gaps in the hazard classification for chemicals which have been in use internationally for a long time. International programmes such as the OECD High Production Volume Programme11, REACH12, and European Regulation 1107/2009/EC13 are progressively working towards filling these data gaps. As new information becomes available, we will update the classifications for those substances.

Table 4 Applicant and EPA staff classifications of the mixture

Method of Mixture classification classification

14

Hazard Class/Subclass Remarks Applicant’s The staff’s classification classification

Mixture data Read across Mixture rules Class 1 Explosiveness No ND

Class 2, 3 & 4 Flammability No ND

Class 5 Oxidisers/Organic No ND Peroxides

Subclass 8.1 Metallic No ND corrosiveness

Subclass 6.1 Acute toxicity (oral) 6.1D 6.1D

Subclass 6.1Acute toxicity No No (dermal)

Subclass 6.1 Acute toxicity No No (inhalation)

11 OECD (1990) Manual for Investigation of HPV Chemicals. Retrieved on 23 January 2008 at http://www.icca-chem.org/Home/ICCA- initiatives/High-production-volume-chemicals-initiative-HPV/

12 http://ec.europa.eu/environment/chemicals/reach/reach_intro.htm

13 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:309:0001:0050:EN:PDF

14 Use of mixture rules may not adequately take into account interactions between different components in some circumstances and must be considered of lower reliability than substance (formulation) data.

November 2015 38

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Method of Mixture classification classification

14

Hazard Class/Subclass Remarks Applicant’s The staff’s classification classification

Mixture data Read across Mixture rules Subclass 6.1 Aspiration hazard No ND

Subclass 6.3/8.2 Skin No No irritancy/corrosion

The difference in classification appears Subclass 6.4/8.3 Eye to be a difference in 6.4A No irritancy/corrosion interpretation of the HSNO criteria for classification

Based on micro- Subclass 6.5A Respiratory organisms being No 6.5A sensitisation considered potential sensitisers

Based on micro- Subclass 6.5B Contact organisms being No 6.5B sensitisation considered potential sensitisers

Subclass 6.6 Mutagenicity No ND

Subclass 6.7 Carcinogenicity No ND

Subclass 6.8 Reproductive/ No ND developmental toxicity

Subclass 6.8 Reproductive/ developmental toxicity (via No ND lactation)

Subclass 6.9 Target organ 6.9B 6.9B (oral) Clothianidin systemic toxicity15

Subclass 9.1 Aquatic ecotoxicity 9.1A 9.1A Clothianidin mainly

Subclass 9.2 Soil ecotoxicity 9.2B 9.2B Clothianidin

15 When appropriate include separate rows to address single as well as repeat dose target organ toxicity, and any of the relevant routes oral, dermal or inhalation.

November 2015 39

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Method of Mixture classification classification

14

Hazard Class/Subclass Remarks Applicant’s The staff’s classification classification

Mixture data Read across Mixture rules Based on acute oral Subclass 9.3 Terrestrial toxicity study (LD50 = 9.3B 9.3C vertebrate ecotoxicity 2000 mg/kg bw). Mammals are more

Subclass 9.4 Terrestrial Clothianidin 9.4A 9.4A invertebrate ecotoxicity

ND: No Data or poor quality data (according to Klimisch criteria16) --> There is lack of data. No: Not Classified based on actual relevant data available for the substance --> The data are conclusive and indicate the threshold for classification is not triggered.

16 Klimisch,, H-J., Andrear, M., & U. Tillmann, 1997. A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data. Reg. Toxicol. Pharmacol. 25, 1–5 (1997).

November 2015 40

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix E: Physical-chemical properties of the mixture

Table 2 Physical and chemical properties of the mixture

Klimisch Property Results Test method Reference Score (1-4)

Colour Dark grey OCSPP 830.6302 1

Physical state Suspension OCSPP 830.6303 1

Odour Musty OCSPP 830.6304 1

6.7 undiluted, 7.6 (1% CIPAC MT 75.3 pH 1 in deionised water) OCSPP 830.7000

Dynamic: Shear rate 20 s-1: 538.0 10-3 Pa s Shear rate 100 s-1: CIPAC MT 192 364.6 10-3 Pa s M Hoppe & W Gueldner Dynamic viscosity (20oC) OECD 114 1 (2013) Physical, chemical Kinematic: OCSPP 830.7100 Shear rate 20 s-1: and technical properties 417.4 10-6 m2/s of Bacillus firmus I-1582 + clothianidin FS 610 Shear rate 100 s-1: (102+508 g/L). Bayer 282.8 10-6 m2/s CropScience AG R&D - D - FT - Analysis & 24 mN/m (undiluted at 25oC) Services Alfred-Nobel- 92/69/EEC A.5 Strasse 50 40789 Surface tension 1 43 mN/m (at 1g/L in OECD 115 Monheim am Rhein / deionized water at Germany. Study number: 20oC) FM0194(PCF00)G01, Bayer ID: M-471757-01-1 D420 = 1.289 92/69/EEC A.3 Relative Density OECD 109 1 D440 = 1.276 OCSPP 830.7300

measurement in water, 15 s ultrasonified Particle size distribution 90% < 38.89 µm CIPAC MT 187 1 50% < 8.00 µm 10% < 1.30 µm

Adherence ratio (before Adherence to seeds and after adherence CIPAC MT 194 1 (maize) test): 101.6%

Oxidizing properties No data provided

Explosive properties No data provided

Flammability No data provided

November 2015 41

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Property Results Test method Reference Score (1-4)

4 maize seed qualities with seed count from 2767 to 4800 kernels/kg were treated with Poncho Votivo at J Smith et al (not dated) the label rate and film Heubach Dustmeter Test coats. of Dust off from Hybrid Results are below the Maize Seed Treated with French threshold for ESA/ESTA, 2011 1 Poncho® and Poncho maize 3 g dust/100 kg Votivo®. Bayer (values between 0.35 CropScience New and 1.07 g/100 kg) and Zealand. Trial number below the ESTA/ESA BSAC002 threshold of 0.75 g/100 000 kernels (values between 0.12 and 0.23 g/100 000 kernels) Heubach test 2 maize seed qualities with seed count from 2767 to 4800 kernels/kg were treated with Poncho Votivo at J Smith et al (not dated) the label rate and film Heubach Dustmeter Test coats. of Dust off from Hybrid Results are below the Maize Seed Treated with French threshold for ESA/ESTA, 2011 1 Poncho® and Poncho maize 3 g dust/100 kg Votivo®. Bayer (values between 0.11 CropScience New and 0.99 g/100 kg) and Zealand. Trial number below the ESTA/ESA BSAC001 threshold of 0.75 g/100 000 kernels (values between 0.05 and 0.16 g/100 000 kernels)

November 2015 42

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix F: Robust study summaries for the mixture

Mammalian toxicity – Robust study summaries for the mixture

The mammalian toxicity studies for Poncho Votivo were conducted with a formulation that has subsequently been modified to the current formulation proposed for registration in New Zealand.

A report was provided outlining the differences in the two formulations. This document demonstrates that only minor changes in the concentration of the active ingredients and other components have been made; and these are not expected to affect the toxicity of the formulation. Therefore, we consider that it is appropriate to read across the data from the studies conducted with the previous formulation.

Acute toxicity, skin and eye irritation and contact sensitisation

Acute Oral Toxicity [6.1 (oral)]

Type of study Acute oral toxicity in the rat

Flag Key study

Test Substance Poncho/Votivo

Endpoint LD50

Value 2000 mg/kg bw (95% PL confidence interval: 750.5-2570 mg/kg bw)

Lowe C 2011. Poncho/Votivo. Acute Oral Toxicity Up And Down Procedure In Reference Rats. Eurofins PSL, 2394 US Highway 130 Suite E, Dayton, NJ 08810, USA. Laboratory study number 33028. M-426238-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD TG 425, OPPTS 870.1100

Species Rat

Strain Sprague-Dawley derived, albino

No/Sex/Group 11 Females tested in total

Dose Levels 175, 550, 2000 mg/kg bw

Exposure Type Oral gavage

In the limit test the first animal treated with 2000 mg/kg bw died. Therefore the main test was conducted. Study Summary 175mg/kg Dose Level (1 animal) The animal survived exposure to the test substance, gained body weight, and appeared active and healthy during the study. There were no signs of gross

November 2015 43

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

toxicity, adverse pharmacologic effects or abnormal behaviour. No gross abnormalities were noted for the animal when necropsied at the conclusion of the 14-day observation period.

550 mg/kg Dose Levels (4 animals) All animals survived exposure to the test substance, gained body weight, and appeared active and healthy during the study. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. No gross abnormalities were noted for these animals when necropsied at the conclusion of the 14-day observation period.

2,000 mg/kg Dose Levels (6 animals) Five animals died within one day of test substance administration. Prior to death, the animals were hypoactive and exhibited hunched posture, irregular respiration, piloerection and/or tremors. Following administration, the surviving animal was hypoactive and exhibited hunched posture and reduced faecal volume. However, the animal recovered by Day 3 and appeared active and healthy, gaining body weight over the entire 14-day observation period. Gross necropsy of the decedents revealed discoloured (moderately red) lungs and/or intestines. No gross abnormalities were noted for the euthanized animal when necropsied at the conclusion of the 14-day observation period.

Under the conditions of this study, the acute oral LD50 of the test substance was estimated to be 2000 mg/kg of body weight (the one dose with partial response) in female rats with a 95% profile-likelihood (PL) based confidence interval of 750.5 mg/kg (lower) to 2570 mg/kg (upper).

Additional Comments None

Conclusion LD50: 2000 mg/kg bw (95% PL confidence interval: 750.5-2570 mg/kg bw)

Acute Dermal Toxicity [6.1 (dermal)]

Type of study Acute dermal toxicity in the rat

Flag Key study

Test Substance Poncho/Votivo

Endpoint LD50

Value > 5000 mg/kg bw

November 2015 44

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Lowe C 2012. Poncho/Votivo. Acute Dermal Toxicity Study in Rats. Eurofins Reference PSL, 2394 US Highway 130 Suite E, Dayton, NJ 08810, USA. Laboratory study number 33029. M-426251-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 870.1200

Species Rat

Strain Sprague-Dawley derived, albino

No/Sex/Group 5

Dose Levels 5000 mg/kg bw

Exposure Type Topical application to shaved skin (approx. 10% body surface), gauze patch

After 24 hours of exposure to the test substance, the pads were removed and the test sites were gently cleansed. One male died within one day of test substance application. There were no clinical signs noted for this animal prior to death. Following application, four surviving males exhibited red ocular discharge and/or red nasal discharge. Study Summary However, the animals had all recovered by Day 5. Dermal irritation was noted at the dose site of all surviving animals (males and females) between Days 1 and 2. Gross necropsy of the decedent revealed no gross abnormalities. No gross abnormalities were noted for any of the euthanized animals when necropsied at the conclusion of the 14-day observation period.

Additional Comments None

Conclusion LD50: > 5000 mg/kg bw

Acute Inhalation Toxicity [6.1 (inhalation)]

Type of study Acute inhalation toxicity in the rat

Flag Key study

Test Substance Poncho/Votivo

Endpoint LC50

Value >2.03 mg/L

Lowe C 2012. Poncho/Votivo. Acute Inhalation Toxicity Study in Rats. Eurofins Reference PSL, 2394 US Highway 130 Suite E, Dayton, NJ 08810, USA. Laboratory study number 33030. M-426246-01-1

November 2015 45

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 1

Amendments/Deviations None considered to impact on the results of the study

GLP Yes

OECD TG 403; OPPTS 870.1300; JMAFF 12-Nouan-8147, November 2000; Test Guideline/s EC 440/2008 Part B.2

Species Rat

Strain Sprague-Dawley derived, albino

No/Sex/Group 5

2.03 mg/L Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Dose Levels Deviation (GSD): Sample 1: MMAD: 3.28 µm; GSD: 2.18. Sample 2: MMAD 3.13 µm; GSD: 2.17

Exposure Type Nose only, 4 hours

All animals survived exposure to the test atmosphere. Following exposure, clinical signs exhibited for five males and one female included abnormal respiration. However, the animals recovered from these symptoms by Day 4 Study summary and along with the other animals appeared active and healthy for the remainder of the 14-day observation period. Although all animals lost weight by Day 3, all animals showed a continued weight gain thereafter through Day 14. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

Additional Comments None

Conclusion LC50: > 2.03 mg/L

Skin Irritation [6.3/8.2]

Type of study Skin irritation in the rabbit

Flag Key study

Test Substance Poncho/Votivo

Endpoint Dermal irritation (mean 24, 48 & 72 h Draize scores)

Value Not irritating (Erythema: 0.55; Oedema: 0.11)

Lowe C 2012. Poncho/Votivo. Primary Skin Irritation Study in Rabbits. Eurofins Reference PSL, 2394 US Highway 130 Suite E, Dayton, NJ 08810, USA. Laboratory study number 33032. M-426242-01-1

Klimisch Score 1

November 2015 46

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Amendments/Deviations None

GLP Yes

OECD TG 404; OPPTS 870.2500; JMAFF 12-Nouan-8147, November 2000; Test Guideline/s EC 440/2008, Method B.4

Species Rabbit

Strain New Zealand albino

No/Sex/Group 3 Females

Dose Levels 0.5 mL

Exposure Type Topical application, semi-occlusive patch.

After 4 hours of exposure to the test substance, the pads and the collars were removed and the test sites were gently cleansed of any residual test substance. All animals appeared active and healthy during the study. Apart from the dermal irritation noted below, there were no other signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. Within 30-60 minutes of patch removal, all three treated sites exhibited very Study Summary slight to well defined erythema and/or very slight oedema. The overall incidence and severity of irritation decreased with time. All animals were free of dermal irritation by 72 hours. The mean 24, 48 and 72 hour Draize scores are calculated as: Erythema: 5/9= 0.56 Oedema: 1/9 = 0.11

Additional Comments None

Conclusion Not irritating

Eye Irritation [6.4/8.3]

Type of study Eye irritation in the rabbit

Flag Key study

Test Substance Poncho/Votivo

Endpoint Eye irritation (mean 24, 48 & 72 h Draize scores)

Value 0.00 for corneal opacity, iritis, conjunctival redness and conjunctival oedema

Lowe C 2012. Poncho/Votivo. Primary Eye Irritation Study in Rabbits. Eurofins Reference PSL, 2394 US Highway 130 Suite E, Dayton, NJ 08810, USA. Laboratory study number 33031. M-426248-01-1

Klimisch Score 1

Amendments/Deviations None

November 2015 47

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

GLP Yes

OECD TG 405; OPPTS 870.2400; JMAFF 12-Nouan-8147, November 2000; Test Guideline/s EC 440/2008, Method B.5

Species Rabbit

Strain New Zealand albino

No/Sex/Group 1 M & 2 F

Dose Levels 0.1 mL

Exposure Type Instillation into the conjunctival sac

Prior to instillation, 2-3 drops of ocular anaesthetic (Tetracaine Hydrochloride Ophthalmic Solution, 0.5%) were placed into both the treated and control eye of each animal. All animals appeared active and healthy during the study. Apart from the eye irritation noted below, there were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behaviour. There was no corneal opacity or iritis observed in any treated eye during this Study Summary study. One hour after test substance instillation, two treated eyes exhibited positive conjunctivitis. All animals were free of ocular irritation by 24 hours. The mean 24, 48 and 72 hour Draize scores are calculated as: Corneal opacity: 0.00 Iritis: 0.00 Conjunctival redness: 0.00 Conjunctival oedema (chemosis): 0.00

Additional Comments None

Conclusion Not irritating

Contact Sensitisation [6.5]

Type of study Contact sensitisation in the mouse (local lymph node assay)

Flag Supporting study

Test Substance Poncho/Votivo

Endpoint Proliferation of lymphocytes (Stimulation Index)

Not sensitising (Stimulation Indices all below the threshold of 3 for a positive Value response)

Lowe C 2011. Poncho/Votivo. Local Lymph Node Assay (LLNA) in Mice. Reference Eurofins PSL, 2394 US Highway 130 Suite E, Dayton, NJ 08810, USA. Laboratory study number 33033. M-426253-01-1

November 2015 48

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 2 (Sensitisation studies considered of limited value for micro-organisms)

The maximum dose level tested in the pre-screen test was 50% rather than the 100% specified in the Test Guideline. This was because preliminary sample preparations indicated the test substance as received (100%) was too viscous to be applied properly, therefore 50% was the highest concentration tested. The study was repeated because the positive control substance (25% HCA in Amendments/Deviations 1% Pluronic L92 in distilled water) failed to elicit a positive response. The raw data from the initial test is not presented in the final report and remains in the study file for reference. The study report contains the results for the second test only. A table containing the dpm values from the initial exposure is presented in an Appendix to the report.

GLP Yes

Test Guideline/s OECD TG 429; OPPTS 870.2600; EC 440/2008, Part B.42

Species Mouse

Strain CBA/J

Preliminary irritation screen (5 groups): 2 F per group Test: (3 groups): 5 F per group No/Sex/Group Vehicle (Negative) Control: 5 F Positive Control: 5 F

Preliminary irritation screen: 5, 10, 25 and 50% in 1% Pluronic L92 surfactant in distilled water Dose Levels Main study: 0, 10, 25 and 50% in 1% Pluronic L92 surfactant in distilled water Positive control: 25% alpha-Hexylcinnamaldehyde (HCA) in 1% Pluronic L92 in distilled water

Exposure Type Topical application to the dorsum of both ears

Concentrations of 10%, 25% and 50% were selected for the main test based on the results of the preliminary screening test. Three concentrations of the test substance in 1% Pluronic L92 surfactant in distilled water or the vehicle alone were topically applied to ten healthy test mice (5 mice/group) for three consecutive days. A positive control group was Study Summary maintained under the same environmental conditions and treated with a 25% w/w mixture of HCA in a 1% w/w mixture of Pluronic L92 surfactant in distilled water in the same manner as the test animals. Three days after the last application, the mice were given a 20 μCi IV injection of 3H-methyl thymidine. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter.

November 2015 49

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

There was no dermal irritation observed for any vehicle control site during the study. Very slight erythema (Draize score of 1) was noted for several test sites treated with the test substance on Day 3 and/or 6. Treatment of mice with 10%, 25% and 50% of Poncho/Votivo resulted in a stimulation index value of 1.20, 1.76 and 2.00, respectively. As a stimulation index (SI) of less than 3.0 was observed in all the treatment groups, the test substance was not considered positive for a dermal sensitisation potential. The positive control (HCA) at 25% produced a dermal sensitisation response in mice (SI=6.01). Therefore, the LLNA test system was considered valid for this study with Poncho/Votivo.

We note that sensitisation studies with micro-organisms are considered to be of Additional Comments limited value, as reactions to foreign proteins (most micro-organisms) can be anticipated

Not a contact dermal sensitiser at concentrations less than or equal to Conclusion 50%

General conclusion about acute toxicity, skin and eye irritation and contact sensitisation classification: Poncho Votivo should be classified 6.1D for acute oral toxicity based on the study data provided. The substance is of low acute toxicity by the dermal and inhalation routes and does not require classification for these endpoints. Poncho Votivo caused only very minor skin and eye irritation which was not of a level sufficient to warrant classification.

Based on the contact sensitisation study provided, Poncho Votivo did not cause skin sensitisation at concentrations up to 50%. There are no test methods available to assess the potential for respiratory sensitisation. We note that skin sensitisation studies with micro-organisms are considered to be of limited value, as reactions to foreign proteins (most micro-organisms) can be anticipated. It is considered that all micro-organisms should be regarded as potential sensitisers in contact with skin and by inhalation. In the EU, the following label phrase is generally applied to microbial pesticides: ‘micro-organisms might have the potential to provoke sensitisation reactions’. Therefore, we propose that 6.5A and 6.5B classifications be applied to Poncho Votivo on a precautionary basis. We note that the US EPA label for Poncho Votivo includes the following precautionary statement: ‘This product contains a microbial pesticide that may have the potential to be a respiratory sensitizer’.

Ecotoxicity – Robust study summaries for the mixture

The applicant did not provide any ecotoxicity studies on Poncho Votivo; therefore the classification is based on mixture rules.

November 2015 50

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix G: Active ingredients and metabolites

Poncho Votivo contains two active ingredients: clothianidin and Bacillus firmus I-1582. Clothianidin is not new to New Zealand, so data of the initial application will be used in the risk assessment. However, the applicant provided more recent studies and information which will be summarised below. Where appropriate, this more up-to-date information is used in the risk assessment.

Identity of Bacillus firmus I-1582

As this is the first full Part 5 application considered for this active ingredient, general data about Bacillus firmus I-1582 are provided in the following tables:

Table 3 Identification of Bacillus firmus

Name of organism Bacillus firmus, Bredemann and Werner 1933

Super kingdom: Bacteria; Class: Bacilli; Family Bacillaceae; phylum: Taxonomy firmicutes; order: Bacillales; genus: Bacillus; species firmus

Strain I-1582

Using the available morphological, physiological and biochemical Identification data, the strain was clearly identified as Bacillus firmus.

“ Collection Nationale de culture de micro-organismes” (CNCM) of Culture collection the Institut Pasteur, Paris, France

Mode of action

Bacillus firmus I-1582 originates from a naturally occurring wild type, isolated from agricultural soil in Israel. The mode of action on soil nematodes is a complex process based on various interactions of the bacterium either directly with the nematodes or indirectly through the host plants, which is not yet fully understood in detail. It is based on a composition of different mechanisms like enzymatic action, degradation of root exudates and production of phytohormone17.

Production of secondary metabolites

Information is available in EFSA Draft Assessment Report (annex B.2, 2011). However, it is mainly related to Bacillus firmus in general or even to other species of Bacillus, very few data are available on the specific strain I-1582. Bacillus firmus has been reported to produce secondary metabolites (exoenzymes mainly, but also polysaccharides, unidentified substances acting as a phytohormone, a nematicide, an antibiotic or having potential toxic properties). The relevance of these findings for this specific strain is unknown.

17 Any of various hormones produced by plants that control or regulate germination, growth, metabolism, or other physiological activities.

November 2015 51

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Classification

Table 4 Applicant and EPA staff classifications of the active ingredient

Active ingredient Method of classification classification

Hazard Class/ Subclass Remarks

s

’

s

’

Applicant classification Staff classification results Test Read across Class 1 Explosiveness NA

Class 2, 3 & 4 Flammability NA

Class 5 Oxidisers/Organic Peroxides NA

Subclass 8.1 Metallic corrosiveness NA

Classification not possible based on endpoints of Acute oral toxicity/ Subclass 6.1 Acute toxicity (oral) ND pathogenicity study. However no mortality and no adverse effects were observed.

Subclass 6.1 Acute toxicity (dermal) ND

Classification not possible based on endpoints of Acute pulmonary toxicity/ Subclass 6.1 Acute toxicity (inhalation) ND pathogenicity study. However no mortality and no adverse effects were observed.

Subclass 6.1 Aspiration hazard ND

Subclass 6.3/8.2 Skin No irritancy/corrosion

Subclass 6.4/8.3 Eye irritancy/corrosion No

Microorganisms Subclass 6.5A Respiratory sensitisation 6.5A considered potential sensitisers

November 2015 52

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Active ingredient Method of classification classification

Hazard Class/ Subclass Remarks

s

’

s

’

Applicant classification Staff classification results Test Read across Microorganisms Subclass 6.5B Contact sensitisation 6.5B considered potential sensitisers

Subclass 6.6 Mutagenicity ND

Subclass 6.7 Carcinogenicity ND

Subclass 6.8 Reproductive/ ND developmental toxicity

Subclass 6.8 Reproductive/ ND developmental toxicity (via lactation)

Subclass 6.9 Target organ systemic ND toxicity

Subclass 9.1 Aquatic ecotoxicity 9.1C

Subclass 9.2 Soil ecotoxicity ND

Subclass 9.3 Terrestrial vertebrate ND ecotoxicity

Subclass 9.4 Terrestrial invertebrate ND ecotoxicity

NA: Not Applicable --> For instance when testing is technically not possible: testing for a specific endpoint may be omitted, if it is technically not possible to conduct the study as a consequence of the properties of the substance: e.g. very volatile, highly reactive or unstable substances cannot be used, mixing of the substance with water may cause danger of fire or explosion or the radio-labelling of the substance required in certain studies may not be possible.

ND: No Data or poor quality data (according to Klimisch criteria18) --> There is lack of data.

No: Not Classified based on actual relevant data available for the substance --> The data are conclusive and indicate the threshold for classification is not triggered.

18 Klimisch, H-J., Andrear, M., & U. Tillmann, 1997. A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data. Reg. Toxicol. Pharmacol. 25, 1–5 (1997).

November 2015 53

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Physico-chemical properties of the active ingredients

This is not relevant for Bacillus firmus however, recent studies on clothianidin have been provided by the applicant. They are shown in table below.

Table 5 Physico-chemical properties of clothianidin

Klimisch Property Result Test method Score (1- Reference 4)

Colour Beige OPPTS 830.6302 F Ziemer & U Eyrich (2012) Clothianidin (TI 435, AE 1283742), Physical state Solid, powder OPPTS 830.6303 technical substance: Physical characteristics colour, physical state and odour. Bayer CropScience AG Research Weak odour, not Odour OPPTS 830.6304 Technologies-Analytics Frankfurt characteristic D-65926 Frankfurt am Main (Germany) Project ID PA12/030. Bayer ID M-427236-01-1

M Krack (2014) Clothianidin (Tl 435, AE 1283742), technical substance: oxidizing properties. Siemens AG Prozess-Sicherheit Oxidizing properties No oxidising A17 1 Industriepark Höchst, B 596 & B 598 D-65926 Frankfurt am Main (Germany) Project no 20140252.04, Bayer ID M-496100- 01-1

M Krack (2014) Clothianidin (Tl 435, AE 1283742), technical substance: explosive properties. Siemens AG Prozess-Sicherheit A14 and OECD 113 Explosive properties Not explosive 1 Industriepark Höchst, B 596 & B (1981) 598 D-65926 Frankfurt am Main (Germany) Project no 20140252.02, Bayer ID M-496097- 01-1.

Melting range: 172- A1 and OECD 102 H Smeykal (2012) Clothianidin (TI Melting point 1 174oC (1995) 435, AE 1283742), technical substance: Melting point, boiling point, thermal stability. Siemens AG Prozess-Sicherheit Decomposition at A2 and OECD 103 Industriepark Höchst, B 596 & B Boiling point 190oC before 1 (1995) 598 D-65926 Frankfurt am Main boiling (Germany) Project no 20120072.01, Bayer ID M-427760- 01-1.

November 2015 54

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Property Result Test method Score (1- Reference 4)

S Winckler (2011) Clothianidin (TI 435, AE 1283742), technical substance: vapour pressure. 3.8 x 10-6 at 20oC Siemens AG Prozess-Sicherheit Vapour pressure 6.7 x 10-6 at 25oC OECD 104 (2006) 1 Industriepark Höchst, B 596 & B (Pa) 598 D-65926 Frankfurt am Main 9.0 x 10-5 at 50oC (Germany) Project no 20110386.02, Bayer ID M-420188- 01-1.

F Ziemer & U Eyrich (2014) Clothianidin (TI 435, AE 1283742), 71.4 mN/m at 20oC pure substance: determination of the surface tension. Bayer Surface Tension The substance is OECD 115 1 CropScience AG Research not a surface active Technologies-Analytics Frankfurt substance D-65926 Frankfurt am Main (Germany) Project ID PA14/062. Bayer ID M-490653-01-1.

A Wiche & B Bogdoll (2011) Clothianidin (TI 435, AE 1283742), 0.27 g/L in distilled technical substance: Solubility in water (pH 6.3), distilled water, at pH 4 and pH 10 0.27 g/L in buffer at (flask method). Bayer CropScience Water Solubility A6 1 pH 4, AG Research Technologies- Analytics Frankfurt D-65926 0.24 g/L in buffer at Frankfurt am Main (Germany) pH 10 Project ID PA11/100. Bayer ID M- 420036-01-1.

F Ziemer & C Peschke (2011) Clothianidin (TI 435, AE 1283742), technical substance: Partition coefficients 1-octanol / water at pH 4, pH 7 and pH 10 (shake flask 0.9 at pH 4, 7 and Log Kow A8 1 method). Bayer CropScience AG 10 at 22oC Research Technologies-Analytics Frankfurt D-65926 Frankfurt am Main (Germany) Project ID PA11/101. Bayer ID M-420023-01- 1.

M Krack (2014) Clothianidin (Tl 435, AE 1283742), technical Not highly Flammability A10 1 substance: Flammability (solids). flammable Siemens AG Prozess-Sicherheit Industriepark Höchst, B 596 & B 598 D-65926 Frankfurt am Main

November 2015 55

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Property Result Test method Score (1- Reference 4) (Germany) Project no 20140252.01, Bayer ID M-494006- 01-1.

M Krack (2014) Clothianidin (Tl 435, AE 1283742), technical substance: Auto-flammability (solids - determination of relative self-ignition temperature). Siemens Auto flammability None up to 401oC A16 1 AG Prozess-Sicherheit Industriepark Höchst, B 596 & B 598 D-65926 Frankfurt am Main (Germany) Project no 20140252.03, Bayer ID M-496099- 01-1.

M Krack (2014) Clothianidin (Tl 435, AE 1283742), technical substance: Self-heating Negative result in a substances UN class 4.2. Siemens Self-heating wire netting cube of UN N4 1 AG Prozess-Sicherheit substances 10 cm edge length Industriepark Höchst, B 596 & B at 120oC 598 D-65926 Frankfurt am Main (Germany) Project no 20140252.05 Bayer ID M-502220-01-1.

November 2015 56

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix H: Robust study summaries for Bacillus firmus I-1582

Mammalian toxicity

Acute toxicity

Acute oral toxicity/pathogenicity

Type of study Acute oral toxicity/pathogenicity in the rat

Flag Key study

Bacillus firmus spores, Lot 112-284 Test Substance 1010 CFU/g

Endpoint LD50, signs of toxicity, clearance of B firmus

8 Value LD50 > 10 colony forming units (CFU)/animal

Kuhn JO 2004 (Amended 2010) Bacillus firmus spores: Acute oral toxicity/pathogenicity study in rats with a microbial pest control agent (MCPA). Reference STILLMEADOW, Inc. 12852 Park One Drive Sugar Land, TX 77478 USA. Laboratory study number 7788-03. M-329108-02-1

Klimisch Score 1

No gross pathology findings are reported. Certificate of Analysis not provided by sponsor. Samples were quantified to confirm the spore count. Amendments/Deviations The report was amended to correct an error in the amount of sterile saline used. The original report stated that the contents of one container of B. firmus were mixed with 30 mL of saline, this should have been 3.0 mL.

GLP Yes

Test Guideline/s OPPTS 885.3050

Species Rat

Strain Sprague-Dawley

Untreated control group, separate housing: 3M & 3F (1M& 1F sacrificed on Day 0 for baseline analysis) Untreated shelf control group: 2M & 2F No/Sex/Group Treatment group: 14M & 14F in total - Interim sacrifice Day 3: 3M & 3F - Interim sacrifice Day 7: 3M & 3F - Interim sacrifice Day 14: 3M & 3F

Dose Levels > 108 CFU/animal

November 2015 57

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Exposure Type Oral gavage

Analytical Measurements Quantification of test microbe in blood, tissues and faeces

The MPCA test substance was administered by oral gavage in a single dose to albino rats. Two untreated control groups were conducted concurrently; the animals of one of the untreated groups (Group 2) were housed with animals that received the test substance and served as ‘shelf’ controls, while the others (Group 1) were housed separately. On Day 1, faeces were collected from the treated animals that were to be sacrificed on Day 3, plated and counted to confirm dosing. The animals were observed three times on the day of dosing for mortality and signs of pharmacologic and/or toxicologic effects and once daily thereafter for the duration of the study. Body weights were recorded pre-test (Day-1) and on Days 7 and 14. Animals (three males and three females) from the treated group were sacrificed on Days 3, 7 and 14. The untreated control and shelf control animals were sacrificed on Day 14. At sacrifice, selected animals had one mL of blood taken; brain, lungs, liver, spleen, kidneys, lymph Study Summary nodes and cecum contents were removed and the entire organ/tissue weighed and homogenized for quantification of the MPCA.

There was no mortality during the study and there were no signs of pharmacologic and/or toxicologic effects. Body weights were largely unaffected by the administration of the test substance. Although some animals lost weight, this occurred in both the treated and untreated groups. Treatment did not affect the weight of the organs sampled for microbiology analysis. On Day 1 following dosing, all six animals sacrificed on Day 3 had significant CFU in the faecal samples. Total clearance of the live organism from the blood of treated animals occurred by Day 7 and clearance from the remaining test organs by Day 14. No live organisms were present in the organs of the untreated or shelf control animals when evaluated at study termination.

The EU DAR states that a note to file included in raw data submitted as additional information indicates that necropsy observations were made at the time of collection of organs for processing, and that there were no abnormalities to report. Ten treated animals are unaccounted for in the study report. However, an Additional Comments adequate number of test animals were evaluated. The study was terminated at 14 days after dosing, rather than the 21 days recommended in the Test Guideline [NB we note that the Guideline does allow flexibility and states that the observation period should be should be determined by the type of MPCA administered and its rate of clearance from the test animals]. No justification is

November 2015 58

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

provided in the study report therefore we requested an explanation. The applicant responded that Bacillus firmus had cleared from the blood in treated animals by Day 7 and from all other tested organs by Day 14. No live organisms were present in the organs of the untreated (control and shelf control) animals when evaluated at study termination. Therefore there was no scientific reason to continue the study up to 21 days.

The test substance, Bacillus firmus spores, produced no toxicity or indications of pathogenicity when administered by oral gavage to albino Conclusion rats at a dose >108 CFU/animal. Clearance of the live organism from all tissues analysed was complete by Day 14.

Acute pulmonary toxicity/pathogenicity

Type of study Acute pulmonary toxicity/pathogenicity in the rat

Flag Key study

Bacillus firmus spores, Lots 112-284 and 112-291 Test Substance 1010 CFU/g

Endpoint LC50, signs of toxicity, clearance of B firmus

8 Value LC50 > 10 colony forming units (CFU)/animal

Kuhn JO 2004 (Amended 2010) Bacillus firmus spores: Acute pulmonary toxicity/pathogenicity study in rats with a microbial pest control agent (MCPA). Reference STILLMEADOW, Inc. 12852 Park One Drive Sugar Land, TX 77478 USA. Laboratory study number 7790-03. M-329111-03-1.

Klimisch Score 1

No gross pathology findings are reported. Certificate of Analysis not provided by sponsor. Samples were quantified to confirm the spore count. Amendments/Deviations The report was amended to correct an error in the amount of sterile saline used. The original report stated that the contents of one container of B. firmus were mixed with 30 mL of saline, this should have been 3.0 mL.

GLP Yes

Test Guideline/s OPPTS NO. 885.3150

Species Rat

Strain Sprague-Dawley

Untreated control group, separate housing: 5M & 5F No/Sex/Group Untreated shelf control group: 4M & 4FTreatment group: 21M & 21F in total

November 2015 59

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

- Interim sacrifice Day 0: 3M & 3F - Interim sacrifice Day 3: 3M & 3F - Interim sacrifice Day 7: 3M & 3F - Interim sacrifice Day 14: 3M & 3F

Dose Levels > 108 CFU/animal

Exposure Type Pulmonary instillation

Analytical Measurements Quantification of test microbe in blood, tissues and faeces

The MPCA test substance was administered by intratracheal instillation to a group of 21 male and 21 female albino rats. Two untreated control groups were conducted concurrently, one of which was housed with animals that received the active test substance and served as "shelf controls". On Day 0, six animals (three males and three females) were sacrificed soon after dosing and lungs were harvested in order to confirm dosing of the test substance into the lungs. Tissue and blood samples were taken from randomly selected animals at interim sacrifices on Days 0 (after dosing), 3, 7, 14 and 21. The samples were prepared for culture to quantitate the test microbe and determine the infectivity and clearance of MPCA in tissues. The animals were observed three times after dosing on Day 0 for mortality and signs of pharmacologic and/or toxicologic Study Summary effects and once daily thereafter through Day 14. Body weights were recorded pre-dose and weekly thereafter until termination of the study. There was no mortality during the study and there we no clinical signs of pharmacologic and/or toxicologic effects observed in any animals in any group during the study. Body weight gain was largely unaffected in all groups. Two of the three treated females that were sacrificed on Day 3 lost weight from Day 0 till Day 3. Weight gain during most of the study was similar among groups. Total clearance of the live organism from the blood, kidneys and liver of treated animals occurred by Day 7 and clearance from the remaining test organs by Day 14. No live organisms were present in the organs of the untreated or shelf control animals when evaluated at study termination (Day 21).

The EU DAR states that a note to file included in raw data submitted as additional information indicates that necropsy observations were made at the time of collection of organs for processing, and that there were no abnormalities to report. Additional Comments A number of treated animals are not accounted for in the report. In addition, while the control animals were sacrificed at day 21 no treated animals were sacrificed at this time point. The EU DAR notes that an explanation for this discrepancy was requested. The applicant explained that the study plan

November 2015 60

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

included weekly collection of organs for plate counts until such time as the spores were cleared from the system. Clearance was found to be complete from the treated group by Day 14, but the results of the Day 14 counts were not available until Day 17 and therefore it was not clear until that time that no additional treated animals would need to be processed on Day 21 or later. To conclude the study, the untreated and shelf control groups were processed on Day 21 and the plate counts demonstrated that there had been no contamination of these animals during the study.

The test substance, Bacillus firmus spores, produced no toxicity or indications of pathogenicity when administered by pulmonary instillation Conclusion to albino rats at a dose >108 CFU/animal. Clearance of the live organism from all tissues analysed was complete by Day 14.

Acute intravenous toxicity/pathogenicity

Type of study Acute intravenous toxicity/pathogenicity in the rat

Flag Key study

Test Substance Bacillus firmus spores, Lots 112-284 and 112-291

Endpoint LD50, signs of toxicity, clearance of B firmus

7 Value LD50 > 10 CFU/animal

Kuhn JO 2006 Acute intravenous toxicity/pathogenicity study in rats with a microbial pest control agent (MCPA) Bacillus firmus spores. STILLMEADOW, Reference Inc. 12852 Park One Drive Sugar Land, TX 77478 USA. Laboratory study number 7791-03. M-329112-02-1.

Klimisch Score 1

Certificate of Analysis not provided by sponsor. Samples were quantified to Amendments/Deviations confirm the spore count. Study report amended to correct an error in the Table of Contents.

GLP Yes

Test Guideline/s OPPTS 885.3200

Species Rat

Strain Sprague-Dawley

Untreated control group, separate housing: 5M & 5F (1M& 1F sacrificed on Day 0 for baseline analysis) No/Sex/Group Treatment group: 21M & 21F in total - Interim sacrifice Day 0: 3M & 3F

November 2015 61

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

- Interim sacrifice Day 3: 3M & 3F - Interim sacrifice Day 7: 3M & 3F - Interim sacrifice Day 14: 3M & 3F - Interim sacrifice Day 21: 3M & 3F

Dose Levels 1.8 x 107 CFU/animal

Exposure Type Intravenous injection

The MPCA test substance was administered by injecting a single high dose into the tail vein of a group of 21 male and 21 female albino rats. An untreated control group was conducted concurrently. Blood samples were taken from randomly selected animals on Day 0 (after dosing). Tissue and blood samples were taken from randomly selected treated animals at interim sacrifices on Days 3,7,14 and 21. Control animals were sacrificed on Day 35. The samples were prepared for culture to quantitate the test microbe and determine the infectivity and clearance of MPCA in tissues. The animals were observed three times after dosing on Day 0 for mortality and signs of pharmacologic and/or toxicologic effects and once daily thereafter for the duration of the study. Body weights were recorded pre-dose and weekly thereafter until termination of the study. There was no mortality during the study. The acute intravenous toxicity/pathogenicity for Bacillus firmus spores, as indicated by the data, is greater than 107 CFU/animal when administered to albino rats. Once clearance of the organism was achieved (Day 21), remaining animals were sacrificed later Study summary without obtaining final body weights or performing necropsies or organ harvesting. Body weights of treated males that were sacrificed to assess the acute intravenous toxicity/pathogenicity for Bacillus firmus spores were unaffected by the administration of the test substance. Three treated females that were sacrificed to assess the acute intravenous toxicity/pathogenicity for Bacillus firmus spores lost or failed to gain weight from Day 0 till Day 3 when the three rats were sacrificed. Weight gain during most of the study was similar between test and control groups. Group 1 and Group 2 animals were asymptomatic throughout the study. The gross necropsy conducted on each control and treated animal revealed no observable abnormalities. On Day 3, all six animals sacrificed had significant CFUs in their blood. Clearance of the live organism from the cecum and liver occurred by Day 14; clearance from the brain, blood, kidneys, lungs, lymph nodes and spleen occurred by Day 21. No live organisms were present in the organs of the untreated control animals when evaluated at termination of the study.

November 2015 62

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Control animals were sacrificed at Day 35 but no treated animals were sacrificed at this time point. Organ weights of these animals are not easily comparable with the organ weights of the treated group assessed on Day 21. The EU DAR notes that an explanation for this discrepancy was requested. The Additional Comments applicant responded that the results of the Day 21 plate counts showing clearance of the spores from the tissues were not available until a few days after Day 21. No additional time points were needed for the treated group and to conclude the study the control animals were processed on Day 35.

The test substance, Bacillus firmus spores, produced no toxicity or indications of pathogenicity when administered intravenously to albino Conclusion rats at a dose of 1.8 x 107 CFU/animal. Clearance of the live organism from all tissues analysed was complete by Day 21.

Skin irritation [6.3/8.2]

Type of study Skin irritation in the rabbit

Flag Key study

Technical (SDN) Batch #129-243 (Alternative name for the active ingredient Test Substance Bacillus firmus I-1582)

Endpoint Skin irritation (mean 24, 48 & 72 h Draize scores)

Value Not irritating (Erythema: 0.33; Oedema: 0.00)

Moore G 2008 Technical (SDN) Primary skin irritation study in rabbits. Eurofins Reference Product Safety Laboratories, 2394 US Highway 130, Dayton, NJ 08810, USA. Laboratory study number 24499. M-328806-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 870.2500

Species Rabbit

Strain New Zealand albino

No/Sex/Group 3F

0.5 g test substance (0.91 g of test mixture [dry paste of test substance Dose Levels moistened with water, 55% w/w])

Exposure Type Topical application, semi-occlusive patch, 4h

November 2015 63

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

All animals appeared active and healthy during the study. Apart from the dermal irritation noted below, there were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behaviour. One hour after patch removal, all three treated sites exhibited very slight erythema and very slight oedema. The overall incidence and severity of Study Summary irritation decreased gradually with time. All animals were free of dermal irritation by 48 hours. Mean 24, 48 & 72 h Draize scores: Erythema: 3/9 = 0.33 Oedema: 0/9 = 0.00

Additional Comments None

Conclusion Not irritating

Eye irritation [6.4/8.3]

Type of study Eye irritation in the rabbit

Flag Key study

Technical (SDN) Batch #129-243 (Alternative name for the active Test Substance ingredient Bacillus firmus I-1582)

Endpoint Eye irritation (mean 24, 48 & 72 h Draize scores)

Not irritating (Corneal opacity: 0.00; Iritis: 0.22; Conjunctival redness: Value Conjunctival oedema (chemosis): 0.56)

Moore G 2008 Technical (SDN) Primary eye irritation study in rabbits. Reference Eurofins Product Safety Laboratories, 2394 US Highway 130, Dayton, NJ 08810, USA. Laboratory study number 24498. M-328801-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 870.2400

Species Rabbit

Strain New Zealand albino

No/ Group 3M

Dose Levels 0.1 mL

Exposure Type Instillation into the conjunctival sac

November 2015 64

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Prior to instillation, 2-3 drops of ocular anaesthetic (Tetracaine Hydrochloride Ophthalmic Solution, 0.5%) were placed into both the treated and control eye of each animal. All animals appeared active and healthy during the study. Apart from the eye irritation noted below, there were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behaviour. There was no corneal opacity observed in any treated eye during this study. One hour after test substance instillation, all three treated eyes Study Summary exhibited iritis and 'positive' conjunctivitis. The overall incidence and severity of irritation decreased with time. All animals were free of positive ocular responses by Day 3. No ocular irritation was noted by Day 4 (study termination). Mean 24, 48 & 72 h Draize scores: Corneal opacity: 0/9 = 0 Iritis: 2/9 = 0.22 Conjunctival redness: 15/9 = 1.67 Conjunctival oedema (chemosis): 5/9 = 0.56

Additional Comments None

Conclusion Not irritating

General conclusion about acute toxicity classification: There was no mortality and no signs of treatment-related toxicity in the acute toxicity/ pathogenicity studies conducted by the oral, pulmonary and intravenous routes with Bacillus firmus I-1582 spores. Clearance from all tissues analysed was observed by Day 14 in the oral and pulmonary studies, and by Day 21 in the intravenous study. There were no signs of pathogenicity or infectivity.

Acute toxicity/pathogenicity studies for microbial pesticides do not provide an LD50/LC50 value that directly relates to the HSNO classification criteria for class 6.1 acute oral or inhalation toxicity, therefore no classification for these endpoints are proposed. No data were provided on acute dermal toxicity.

B firmus I-1582 was only slightly irritating to the skin and eyes. These effects were rapidly reversible and the level of severity did not meet the thresholds for classification.

No skin sensitisation study has been provided for B firmus I-1582 as the available methods are not considered to be suitable for testing microorganisms. In the absence of data it is considered that all microorganisms have the potential to provoke sensitising reactions. This is also the approach taken in the EU. Classification as a respiratory and contact sensitiser (6.5A and 6.5B) is therefore proposed.

November 2015 65

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Genotoxicity [6.6] No genotoxicity studies were performed with Bacillus firmus I-1582. The applicant provided the following explanation for why no testing was conducted:

No validated methods for the genotoxicity testing of microorganisms are available. Genotoxicity testing should therefore be recommended for specific metabolites only. Since there is no evidence coming from the literature and experimental work that any genotoxic substance is produced by Bacillus firmus I-1582 or other strains of the species B. firmus in the environment or in feed or food, no genotoxicity study was conducted.

General conclusion about genotoxicity classification: Based on the lack of information no classification for genotoxicity is proposed.

Carcinogenicity [6.7] No studies for carcinogenicity of the active ingredient were provided.

General conclusion about carcinogenicity classification: No data were presented, but in view of the whole dataset are not considered necessary.

Reproductive/Developmental Toxicity [6.8] No studies for reproductive/developmental toxicity of the active ingredient were provided.

General conclusion about reproductive/developmental toxicity classification: No data were presented, but in view of the whole dataset are not considered necessary.

Target Organ Systemic Toxicity [6.9] No data for target organ systemic toxicity were provided for either the inhalation or dermal route for repeat administration. A short term oral toxicity and pathogenicity study was provided and is summarised below.

Type of study Short-term oral toxicity and pathogenicity in the rat

Flag Key study

Test Substance Bacillus firmus I-1582 Batch # 106-277

LOAEL: N/A Endpoint NOAEL: > 1 x 108 CFU/animal

Bottomley A 2010 Bacillus firmus I-1582: Toxicity and pathogenicity study by oral administration to CD rats for 4 Weeks. Huntingdon Life Sciences, Reference Huntingdon Research Centre, Woolley Road, Alconbury, Huntingdon, Cambridgeshire PE28 4HS, UK. HLS study number: DNM0008. M-364421-01- 1.

Klimisch Score 1

November 2015 66

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Due to an oversight when the liver, kidney and lungs were weighed only the section used for microbial analysis was weighed. The right kidney and right lung Amendments/Deviations were weighed for each animal, therefore it is considered that this data can be reported. For the liver, a section of the one lobe was weighed, however, as it was not a complete lobe no liver weights were reported.

GLP Yes

OPPTS 885.3600 EC Methods for the determination of toxicity, Annex to Directive 2001/36/EC Test Guideline/s (Official Journal No. L164, 20.6.2001), Part B, Method 5.2.2.1. Acute oral toxicity, pathogenicity and infectiveness (paragraph 5.2.5. Information on short- term toxicity and pathogenicity).

Species Rat

Strain Crl:CD® (SD)

Group 1, untreated control: 5M & 5F Group 2, untreated shelf control: 5M & 5F Group 3, autoclaved Bacillus firmus spores: 5M & 5F Group 4, Bacillus firmus spores: 12M & 12F No/Sex/Group - 4a, sacrificed Day 31: 3M & 3F - 4b, sacrificed Day 35: 3M & 3F - 4c, sacrificed Day 42: 3M & 3F - 4d, sacrificed Day 49: 3M & 3F

Target: 108 CFU/rat/day Dose Levels Measured: 1.75 x 108 - 4.26 x 108 CFU/rat

Exposure type Oral gavage

The toxicity, infectivity and pathogenicity of a Microbial Pest Control Agent (MPCA), Bacillus firmus I-1582, to Crl:CD® (SD)IGS BR rats was assessed over 28 days of oral administration of at least 1x108 colony forming units/rat (CFU/rat) followed by a maximum of 21 days observation period. Groups of rats were given 28 daily doses by oral gavage of viable Bacillus firmus I-1582 suspended in sterile physiological saline or dosed with the Study Summary autoclaved test substance (shown to have a viable count of 0 CFU/mL). Two further groups of rats were untreated, one group placed in a separate room and one group housed within the same room as the treated animals. During the study, clinical condition, bodyweight, food consumption, body temperature, haematology and blood chemistry investigations were undertaken. Animals which received the viable test material were sequentially sacrificed at intervals after the last dose administration on Day 28 and subjected to a

November 2015 67

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

macroscopic examination. Samples of blood, tissues, intestinal contents and faeces were removed for microbiological determination of test substance recovery. There were no mortalities during the study. On Day 7, 1-2 hours after dosing all Group 4 animals receiving the Bacillus firmus I-1582 were noted to have increased body temperature, red skin colouring and the ears and feet appeared red. This sign was resolved in all animals at the check performed approximately 3½ hours later. As this sign was not observed on any other occasion and it was transient in nature it was considered not to be of toxicological importance. No other clinical signs considered to be related to treatment were noted throughout the study. There was no effect of treatment on bodyweight gain, food consumption, haematology, blood chemistry, organ weights or macropathology. Viable Bacillus firmus I-1582 was recovered from the brain, spleen, liver, heart, lungs, mesenteric lymph node, stomach, small intestine (7th loop) and caecum of treated animals on Day 31 (day 3 after termination of test substance administration). Viable Bacillus firmus was recovered from the lungs, mesenteric lymph nodes, heart, stomach and caecum of some of the treated animals on Day 35 (day 7 after termination of administration) and viable Bacillus firmus I-1582 was recovered from the small intestine (7th loop) and caecum on Day 42 (14 days after termination of administration). No viable Bacillus firmus was recovered from any organ on Day 49. Viable Bacillus firmus I-1582 was recovered from the faeces of five animals on Day 35, from two animals on Day 42 and no viable Bacillus firmus I-1582 was recovered on Day 49. The data indicate that the animals cleared the organism with 21 days from the cessation of dosing.

As no adverse effects were identified on any of the parameters assessed during the study, the lack of information on liver weight is not considered to have affected the acceptability of the study. We requested an explanation for why the study was conducted for 28 days rather than the 90 days recommended in the Test Guideline. The applicant Additional Comments responded that the repeated dose toxicity study was performed to comply with EU regulation. Based on the toxicological profile of Bacillus firmus no repeated dose toxicity study was compulsory for US registration. For EU registration a 28-day study was acceptable. So this study follows the EPA guideline 885.3600 except for the duration of exposure. As Bacillus firmus I-1582 showed no evidence of toxicity or indication of pathogenicity when administered by oral

November 2015 68

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

gavage to rats at 1.75 x 108 to 4.26 x 108 CFU/animal for 28 days followed by a maximum of 21 days observation period, a longer period of administration would not provide any further useful information.

Bacillus firmus I-1582 showed no evidence of toxicity or pathogenicity to Conclusion rats over 28-days repeated oral administration at 1.75 x 108 to 4.26 x 108 CFU/rat followed by a maximum of 21 days observation period.

General conclusion on target organ toxicity Bacillus firmus I-1582 spores were administered to rats by oral gavage for 28 days followed by a recovery period of up to 21 days. No mortality, clinical signs, behavioural changes or signs of pathogenicity were observed during the course of the study. Body weight gain, food consumption, haematology, blood chemistry, organ weights or macropathology were not affected by treatment. Viable Bacillus firmus was recovered from a number of organs and faeces on days 3 and 7 following termination of dosing. Clearance of the live organism from most organs was achieved by day 14 and total clearance from all organs and faeces was achieved 21 days following termination of dosing.

Based on the available data no classification for target organ toxicity is proposed.

Medical surveillance on manufacturing plant personnel A report from 200919 states that Bacillus firmus have been handled in laboratories and sample shipment only, and exposure potential has been very low. In the workers handling Bacillus firmus routine occupational medical surveillance did not show any untoward effects of handling the organism, in particular there has been no indication of hypersensitivity.

Sensitisation/allergenicity observations A report from 200920 states that the applicant has not received any information on sensitisation or allergenic response of workers. In a further report, it is stated that no symptoms have been reported from the handling of Bacillus firmus I- 1582 in the synthesis, formulation plants or during process development. Nor are there published reports, in particular there has been no indication of hypersensitivity21. A further report from 2010 indicates that the applicant has not received any information on sensitisation of operators or bystanders

19 Steffens W 2009a. KIIM 5.2. Occupational health surveillance report on workers for Bacillus firmus I-1582. Bayer CropScience, M-

360600-01-1.

20 Saele M 2009. KIIM 5.2.1 The sensitisation and allergenic response of workers for Bacillus firmus I-1582. Bayer CropScience, M-

360608-01-1.

21 Steffens W 2009b. KIIM 5.2.2 Details on any occurrence of hypersensitivity and chronic Sensitization for Bacillus firmus I-1582. Bayer

CropScience, M-360612-01-1.

November 2015 69

Application for approval to import or manufacture Poncho Votivo for release (APP202077) regarding hypersensitivity incidents relating to use of the formulation Bacillus firmus I-1582 WP5, a wettable powder containing Bacillus firmus I-1582 at 50 g/kg (3.55 x 1012 CFU/kg)22. Bacillus firmus I-1582 WP5 is a product intended for use as a nematicide by spraying on soil in field and glasshouse carrots and glasshouse applications as a drench to the soil or through drip irrigation in cucumber.

Clinical findings and adverse health effects reports A 2009 report states that as Bacillus firmus I-1582 is a new compound, there are no observations of the general population or epidemiological studies23. The applicant has not received any information on poisoning incidents or adverse effects. Nor are there published reports available24.

We note that no incidents relating to Bacillus firmus I-1582 have been reported on the Health Canada pesticide product register to date25. Approvals were granted for the two products currently available in Canada on 31 May 2012.

Potential production of toxins and/ or metabolites The applicant provided a literature search which was conducted to provide an overview on the current state of knowledge about the production of secondary metabolites by Bacillus firmus and their toxicity to humans; as well as to collect information about the occurrence of Bacillus firmus in food and about toxin formation in food26. The databases Agricola, Medline, Biosis and Caba were searched for the key words: Bacillus firmus, metabolites, toxines, plant, soil, food, spoilage, poisoning. In total, 32 references were identified.

Only few strains of Bacillus firmus are reported to be able to produce secondary metabolites acting as a phytohormone (1 strain), an antimicrobial substance (5 strains) or showing adverse effects in a Hep 2 cell test (2 strains). Regarding the substances showing adverse effects in Hep-2 cells, Taylor et al. (200527) tested 101 Bacillus strains for production of heat-stable toxins. Altogether 15 strains were Bacillus firmus. Two of the 15 Bacillus firmus strains grown in a 10% skim milk broth were found to excrete a secondary metabolite, which showed vacuolation in a Hep-2 cell test. Partial purification of the substance showed that it has similar physical characteristics to the Bacillus cereus toxin, cereulide.

22 Steffens W 2010. KIIIM 7.3 Operator and bystander exposure For Bacillus firmus I-1582 WP5. Bayer CropScience, M-365846-01-1.

23 Steffens W 2009c. KIIM 5.2.3 Any significant clinical findings related to exposure for Bacillus firmus I-1582. Bayer CropScience, M-

360617-01-1.

24 Steffens W 2009d. KIIM 5.2.4 Published reports of adverse effects for Bacillus firmus I-1582. Bayer CropScience, M-360621-01-1.

25 http://pr-rp.hc-sc.gc.ca/pi-ip/result-eng.php?1=0&2=501&3=act&4=a&5=1&6=ASC&7=B&8=E Accessed 22 April 2015.

26 Andersch I 2010. Summary of a Literature Search for Bacillus firmus and Formation of Secondary Metabolites or Occurrence in Food.

Bayer CropScience, M-366409-01-1.

27 Taylor JMW et al. 2005. Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis. FEMS Microbiology Letters 242: 313–317.

November 2015 70

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The formation of secondary metabolites showing antimicrobial or toxic activities has only been demonstrated in laboratory experiments. The formation of antimicrobial substances under natural conditions or an occurrence of toxic secondary metabolites in food or in soil has never been reported in the literature.

Although being a minor component of the natural microflora in a variety of fermented foods, strains of B. firmus can be isolated from traditional fermented foods. A negative influence of these foods on the health of the population is not reported. Other publications report the isolation of B. firmus from a small variety of spoiled food, bread and fish. Moreover B. firmus was isolated from raw and heated milk, and showed a potential to impair the milk, because some strains were able grow at low temperatures. However, the number of publications describing food contaminations by B. firmus is low as well as the number of strains of Bacillus firmus isolated from food compared to the number of strains from other Bacillus species. Moreover, only very few contaminations of Bacillus firmus in European food are reported. It can be concluded that growth of B. firmus in food can be well controlled by production techniques and storage conditions. No reports are available to-date, which mention the formation of a human toxin in food produced by Bacillus firmus.

We note that in the 2011 EU DAR it is stated that a literature search on Biosis, Embase, CA search, Agricola, Current contents, Pascal, Medline and Environmental Sciences with the key words “Bacillus firmus and toxic? or toxin? or pathogen?” provided 42 references. From all of these publications, only one reported toxin production by several strains of Bacillus, including Bacillus firmus. This was the same publication by Taylor et al. (2005) identified above. The EU DAR indicates that the rapporteur Member State requested a justification to show that the finding of heat-stable toxin production with two B. firmus strains discussed above cannot be extrapolated to B. firmus strain I-1582. The applicant replied that although the study showed that two strains of Bacillus firmus could produce a toxin whose partial purification profiles were comparable to the purification characteristics obtained with cereulide, the adverse effects of the heat-stable toxin have only been shown in an in vitro system of Hep-2 cells within very specific culture conditions. Furthermore the cytotoxicity test performed on Hep-2 cells is not specific to screen for the presence of the cereulide toxin. Any compound or product producing a toxic effect on mitochondria could be positive in these tests. Therefore a positive result in one of these tests does not necessarily mean that a cereulide toxin has been produced.

Summary of mammalian toxicity and pathogenicity for the active ingredient Bacillus firmus B-1582 Bacillus firmus is a Gram-positive spore forming bacteria naturally occurring in the air, sea and land. It may be isolated from air, streams, soils, oil wells, oceans and a variety of plant root surfaces.

The temperature spectrum for growth of B. firmus was determined to be between 10°C and 45°C, the optimum temperature was in the range between 30°C to 37°C. There are reports that B. firmus strains have been isolated from the animal and gastrointestinal tract.

Literature reviews have only identified one report of toxin production by two strains of B. firmus. However, these effects have only been shown in an in vitro test system under very specific culture conditions. Although

November 2015 71

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

B. firmus has been found in a range of different foods, there are no reports in the literature to date of a heat stable toxin produced by strains of B. firmus in food, or of food-borne illness caused by B. firmus.

In acute toxicity/pathogenicity studies in rats conducted by the oral, pulmonary and intravenous routes with Bacillus firmus I-1582 spores there were no signs of treatment-related toxicity. Clearance from all tissues analysed was observed by Day 14 in the oral and pulmonary studies, and by Day 21 in the intravenous study. There were no signs of pathogenicity or infectivity.

A short-term toxicity study in the rat was also conducted, in which Bacillus firmus spores were administered by oral gavage at 1.75 x 108 to 4.26 x 108 CFU/rat/day for 28 days followed by a recovery period of up to 21 days. No mortality, clinical signs, behavioural changes or signs of pathogenicity were observed during the course of the study. Body weight gain, food consumption, haematology, blood chemistry, organ weights or macropathology were not affected by treatment. Viable Bacillus firmus was recovered from a number of organs and faeces on days 3 and 7 following termination of dosing. Clearance of the live organism from most organs was achieved by day 14 and total clearance from all organs and faeces was achieved 21 days following termination of dosing.

B. firmus I-1582 was only slightly irritating to the skin and eyes; these effects were rapidly reversible and the level of severity did not meet the thresholds for classification.

The sensitisation potential of B. firmus I-1582 was not assessed as the available methods are not considered to be suitable for testing microorganisms. In the absence of data it is considered that all microorganisms may have the potential to provoke sensitising reactions. Classification as a respiratory and contact sensitiser (6.5A and 6.5B) is therefore proposed.

No genotoxicity testing was performed due to a lack of appropriate test methods for microorganisms and because there is no known production of toxic metabolites.

Based on the studies and literature information provided, no toxicological or pathogenic concerns have been identified for Bacillus firmus I-1582. Further testing for carcinogenicity, reproductive and development toxicity and target organ toxicity is not considered to be required.

Environmental fate

No studies were provided about the environmental fate of Bacillus firmus. However, the publications that were used by the EU for their assessment have been provided by the applicant. As they are not guideline related and not GLP, they are not summarized individually. The summary of the information is mainly based on Draft Assessment report of EFSA (2011).

November 2015 72

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The documents used are listed below:

Author Year Journal Title Bayer ID

Assessment of rainwater-mediated Appl Ent Zool 26(4): 477- M-368025-01- Akiba 1991 dispersion of field-sprayed Bacillus 483 1 thuringensis in the soil

Appl Env Microb 71(2): Screening for Bacillus isolates in the M-367872-01- Barbosa et al 2005 968-978 broiler gastrointestinal tract 1

Appl Env Microb 60 (6): Bacillus DNA in fossil bees: an ancient M-356399-01- Cano et al 1994 2164-2167 symbiosis? 1

Difference between the spore sizes of J Appl Microb 102: 303- M-368017-01- Carrera et al 2007 Bacillus anthracis and other Bacillus 312 1 species

Coloured Bacillus species resembling M-367955-01- Dias et al 1966 No data Vol 34 (1): 9-16 B. firmus 1

Influence of physical factors on the Donkerski & Polish Journal of Env activity of chitinases produced by M-367959-01- 1999 Trzebiatowska Studies 9 (2): 77-82 planktonic bacteria isolated from 1 Jeziorak Lake

Polish Journal of Env Neustonic bacteria number, biomass M-367960-01- Donkerski et al 1999 Studies 8 (3): 137-141 and taxonomy 1

Draft Assessment Report (DAR) EFSA 2011 - Bacillus firmus I-1582 Vol 3, Annex B - part 9 B.8/b

Draft Assessment Report (DAR) EFSA 2011 - Bacillus firmus I-1582 Vol 3, Annex B - part 9 B.8/a

In situ detection of an uncultured Appl Env Microb 64 (11): M-367526-01- Felske et al 1998 predominant Bacillus in Dutch 4588-4590 1 grassland soils

Rhizosphere microbial community and M-368009-01- Garbeva et al 2007 Plant Soil 302: 19-32 its response to plant species and soil 1 history

Phylogenetic diversity of Gram-positive Appl Env Microb 73 (10): M-367945-01- Gontang et al 2007 bacteria cultured from marine 3272-3282 1 sediments

Appl Env Microb 35 (5): Inactivation of Bacillus thuringensis M 368063-01- Griego & Spence 1978 906-910 spores by ultraviolet and visible light 1

Microb Env 24 (2): 154- Rice seeds as sources of endophytic M-367865-01- Kaga et al 2009 162 bacteria 1

November 2015 73

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Author Year Journal Title Bayer ID

The BCPC Conference – Bacillus firmus formulations for the safe M-028855-01- Keren-Zur et al 2000 Pest and diseases 2000, control of root-knot nematodes 1 Brighton

Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons M-367943-01- Ki et al 2009 J Microb Meth 77: 48-57 and their usefulness for species 1 identification

Water Air Soil Pollut 199: Factors that influence the transport of M-368033-01- Kim et al 2008 151-157 Bacillus cereus spores through sand 1

Production of antimicrobial substances J Appl Microb 98: 667- by Bacillus subtilis LFE-1, B. firmus M-367951-01- Korenblum et al 2004 675 H2O-1 and B. licheniformis T6-5 1 isolated from an oil reservoir in Brazil

M-370080-01- Markov 2006 - Dissipation of Bacillus firmus in the soil. 1

Comparative sensitivity to UV-B Current Microb 43: 140- M-368061-01- Myasnik et al 2001 radiation of two Bacillus thuringiensis 143 1 subspecies and other Bacillus sp.

Filtration of Bacillus subtilis and Colloids and Surfaces B: Bacillus cereus spores in a pyroclastic M-368023-01- Naclerio et al 2008 Biointerfaces 70: 25-28 topsoil, carbonate Apennines, southern 1 Italy

Predominant culturable Bacillus M-367961-01- Nishijima et al 2005 Microb Env 20 (1): 61-68 species in Japanese arable soils and 1 their potential as biocontrol agents

Ortega-Morales Ind Microb Biotechnol 35: Antifouling activity of sessile bacilli M-367936-01- 2007 et al 9-15 derived from marine surfaces 1

Antagonism of Bacillus spp. isolated Ortega-Morales Mar Biotechnol 11: 375- M-367930-01- 2008 from. marine biofilms against terrestrial et al 383 1 phytopathogenic fungi

Biodiversity of thermotolerant Bacillus Pakpitcharoen et M-367910-01- 2008 ScienceAsia 34: 424-431 sp. producing biosurfactants, al 1 biocatalysts, and antimicrobial agents

Pantastico- Ecology 73 (5): 1888- Population dynamics of bacteriophage M-368039-01- 1992 Caldas et al 1902 and Bacillus subtilis in soil 1

Dispersal of Bacillus thuringiensis var. Can J Microb 41: 118- M-368069-01- Pedersen et al 1995 kurstaki in an experimental cabbage 125 1 field

November 2015 74

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Author Year Journal Title Bayer ID

Characterization of bacterial population Annals Microb 55 (2): 85- M-367998-01- Riccardi et al 2005 coming from a soil contaminated by 90 1 polycyclic aromatic hydrocarbons

Fate of Bacillus thuringiensis in soil: Can J Microb 16: 677- M-368074-01- Saleh et al 1969 effect of soil pH and organic 679 1 amendment

Phylogeny of marine Bacillus isolates M-367912-01- Siefert et al 2000 Current Microb 41: 25-29 from the Gulf of Mexico 1

Polish Journal of Ecology Chitinolytic bacteria in two lakes of M-367956-01- Swiontek et al 2006 54 (2): 295-301 different trophic status 1

Fermentation and Survival of Bacillus subtilis NB22, an M-368050-01- Tokuda et al 1992 bioengineering 75 (2): antifungal –antibiotic ilturin producer 1 107-111 and its transformant in soil-systems

Seasonal variation in heterotrophic soil Rev Ecol Microb sol 26 bacteria and some soil enzyme M-367859-01- Vardavakis 1989 (3): 233-247 activities in a typical calcixeroll soil in 1 Greece

Survival of Bacillus thuringensis and Bacillus cereus spore inocula in soil: Soil Biol Biochem 75 (5): M-370430-01- West et al 1985 effect of pH, moisture, nutrient 657-665 1 availability and indigenous microorganisms

Effect of incubation in natural and J Invert pathol 44: 121- M-368072-01- West et al 1984 autoclaved soil upon potency and 127 1 viability of Bacillus thuringensis

Soil compartment Bacillus firmus I-1582 has been isolated from agricultural soils in Israel where it is naturally occurring.

The formulation is made of spores of the bacterium which will germinate once applied in soil, and B. firmus will multiplicate if humidity and nutrients are available. In unfavourable conditions, sporulation will occur.

Conditions for growth are most favourable in the rhizosphere, where root exudates provide sufficient nutrients.

The vegetative cells are sensitive to drought; UV radiation, or elevated temperature, spores as the resting or dormant stage are highly resistant to environmental stresses. Population and viability of spores are reduced during relatively long time periods by consumption by soil organisms.

The abundance of Bacillus firmus I-1582 in soil is variable and depends on the location, the season and the crop. The background concentration was difficult to determine as there is no method to determine the

November 2015 75

Application for approval to import or manufacture Poncho Votivo for release (APP202077) species in soil so the publications are based on Bacillus sp. levels. The concentration varies from < 1. x 104 cfu/g dry soil (limit of detection) to 5 x 107 cfu/g dry soil.

The persistence in soil in unclear as some data show a decline 3 months after application, either stable levels, either an increase after 3 months. This is highly dependent on the conditions in soil (such as soil moisture and nutrient availability). Consequently no degradation will be incorporated in the risk assessment.

Water compartment

Freshwater environments Bacillus firmus has been found in eutrophic freshwater lakes, so it is assumed that it can survive in freshwater habitats, but is likely less competitive than freshwater microorganisms as it is sourced from soil.

No quantitative data is available about the presence of these micro-organisms in freshwater compartment.

Ground water Studies performed on B. thuringensis when sprayed in the field showed no dispersion in the conditions of the volcanic soil from Japan which is known to easily adsorb chemicals. However, a comparative study of the migration of B. firmus and B. cereus spores in laboratory conditions on pyroclastic topsoil showed that transfer to groundwater is possible. Similarly, spores have been shown to be more mobile than virus or vegetative forms of bacteria. It is therefore difficult to conclude about the likelihood of contamination of groundwater. In addition, available models are not built to take into consideration complicated biological mechanisms involved in the behaviour of micro-organisms mobility in soil. Therefore, no conclusion can be drawn on the potential contamination of groundwater by these bacteria.

Ecotoxicity

Some studies were performed with a formulation containing this active ingredient.

Aquatic toxicity

Fish acute toxicity (Freshwater species)

Type of study Limit test

Flag Key study

Test Substance Bacillus firmus I-1582 (1.5 x 1011 cfu/g)

Species Oncorhynchus mykiss (Rainbow trout)

Type of exposure Static for 96 hours

Endpoint LC50

Value > 100 mg/L (1.5 x 1010 cfu/L)

November 2015 76

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

E Bruns (2010) Acute toxicity of Bacillus firmus I-1582 to fish (Oncorhynchus mykiss) under static conditions- limit test. Bayer CropScience AG Development- Reference Environmental safety Ecotoxicology 40789 Monheim (Germany). Report no EBBIL003, Bayer ID M-366351-02-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 203 (1992)

No/Group 2 replicates of 15 fish

Dose Levels 100 mg/L (1.5 x 1010 cfu/L)

Analytical measurements Concentrations were checked by turbidity measurements on day 0, 2 and 4.

The acute toxicity of the test substance to rainbow trout was determined in a 96-h static test. A limit test was performed at 100 mg/L. The accompanying determination of Bacillus firmus I-1582 by turbidity measurements resulted in recoveries of 84.7 and 95.1% for the freshly prepared medium at day 0. The aged samples showed lower recoveries: from 43 to 58%. This can be explained by sedimentation of the spores. The results are expressed as nominal Study Summary concentrations. The pH values in the test medium and in the control ranged from 6.6 to 7.2, the oxygen concentration was always higher than 60% oxygen saturation (it was between 61 and 96%) and the temperature was between 11.6 and 12.4oC. In the control and in the test concentration, no mortality or other signs of intoxication were determined during the test period.

The LC50 is higher than 100 mg/L.

The use of nominal concentration in this case is acceptable even though the Comments report doesn’t state any deposit of test substance at the bottom of the aquaria. No information provided about the viability of the spores.

10 The LC50 is higher than 100 mg/L (1.5 x 10 cfu/L). No mortality observed Conclusion at 100 mg/L.

Fish acute toxicity (Marine species) No study provided.

Invertebrate acute toxicity (Freshwater species)

Type of study Limit test

Flag Key study

November 2015 77

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Substance Bacillus firmus I-1582 (1.5 x 1011 cfu/g)

Species Daphnia magna

Type of exposure Static, 48 hours

Endpoint EC50

Value > 100 mg/L (1.5 x 1010 cfu/L)

E Bruns (2010) Acute toxicity of Bacillus firmus I-1582 to the water flea Daphnia magna in a static laboratory test system- limit test. Bayer CropScience AG Reference Development-Environmental safety Ecotoxicology 40789 Monheim (Germany). Report no EBBIL011, Bayer ID M-362286-02-1.

Klimisch Score 2 (see comments below)

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 202 (2004)

No/Group 6 replicates of 5

Dose Levels 100 mg/L (1.5 x 1010 cfu/L)

Analytical measurements Concentrations were checked by turbidity measurements on day 0.

The acute toxicity of the test substance to Daphnia magna was determined in a 48-h static test. A limit test was performed at 100 mg/L. The accompanying determination of Bacillus firmus I-1582 by turbidity measurements resulted in recoveries of 97.5% for the freshly prepared medium at day 0. The results are expressed as nominal concentrations. Study Summary The pH values in the test medium and in the control ranged from 7.5 to 7.9, the oxygen concentration was between 69.4 and 96.8% of saturation and the temperature was in the range of 20.0-20.2oC. In the control and in the test concentration, no immobilisation or other signs of intoxication were determined during the test period.

The EC50 is higher than 100 mg/L.

Comment No information about the stability/viability of the test item over the test period.

10 The EC50 is higher than 100 mg/L (1.5 x 10 cfu/L). No immobilisation Conclusion observed at 100 mg/L.

Invertebrate acute toxicity (Marine species) No study provided.

Algae acute toxicity (Freshwater species)

November 2015 78

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Type of study Full test

Flag Key study

Test Substance Bacillus firmus I-1582 (1.46 x 1011 cfu/g)

Species Pseudokirchneriella subcapitata

Type of exposure Static, 72 hours

Endpoint ErC50

Value 24.2 mg/L (3.5 x 109 cfu/L)

E Bruns (2010) Pseudokirchneriella subcapitata growth inhibition test with Bacillus firmus. Bayer CropScience AG Development-Environmental safety Reference Ecotoxicology 40789 Monheim (Germany). Report no EBBIL012, Bayer ID M- 367825-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 201 (2006)

No/Group 3 replicates for test concentrations and 6 for the control of 104 cells/mL

Dose Levels 0.477, 1.53, 4.88, 15.6 and 50 mg/L

Analytical measurements Concentrations were checked by turbidity measurements on day 0.

The influence of the test substance on the growth of the green algal species Pseudokirchneriella subcapitata was determined in a 72-h static test. The nominal concentrations were 0.477, 1.53, 4.88, 15.6 and 50 mg/L. The measured concentration in the test medium was 80-118% of the nominal. The pH values in the test medium and in the control ranged from 7.9 to 8.5, and the temperature was between 21.2 and 22.1oC. The test was performed under continuous illumination of 8612 lux. The validity criteria were met: Study Summary Biomass increased in the control by more than 16-fold within the evaluation period: observed value: 63.9 Mean percent coefficient of variation of sectional growth rates from day 0-1, day 1-2 and day 2-3 in the control did not exceed 35%: observed value = 28.4% Percent coefficient of variation of the average growth rate in each control replicate did not exceed 7%: observed value = 2.7% The test substance had a statistically significant inhibitory effect on the growth (biomass and growth rate) at the concentrations of 15.6 and 50.0 mg/L. The growth rate inhibition was 21.2% at 15.6 mg/L and 91.2% at 50 mg/L.

November 2015 79

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The NOEC was 4.88 mg/L and both ErC50 is 24.2 mg/L. The report states that the observed growth inhibition of algae could be partly explained by reduced illumination due to the turbidity of test solution.

Comment No information about the stability/viability of the test item over the test period.

9 ErC50 = 24.2 mg/L (95% CI: 21.4-28.0 mg/L) corresponding to 3.5 x 10 cfu/L Conclusion NOErC = 4.88 mg/L

Algae acute toxicity (Marine species) No study provided.

Aquatic plants acute toxicity (Freshwater species) No study provided.

Fish chronic toxicity (Freshwater species) No study provided.

Fish chronic toxicity (Marine species) No study provided.

Invertebrate chronic toxicity (Freshwater species)

Type of study Limit test

Flag Key study

Test Substance Bacillus firmus I-1582 (no information on cfu)

Species Daphnia magna

Type of exposure Semi-static, 21 days. Daily renewal

Endpoint NOEC for survival rate, reproduction rate and body length of the adults

Value 1.38 105 cfu/mL

NA Rodrigue (2006) Chronic toxicity to the freshwater invertebrate Daphnia Reference magna – Bacillus firmus spores. Stillmeadow Inc 12852 Park one drive Sugar land TX 77478 (USA). Report no 7792-03 Bayer ID M-329115-02-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 850.1300 and 885.4240

No/Group 5 replicates of 10

Dose Levels Nominal: 106 cfu/mL (maximum hazardous dose according to the guideline)

November 2015 80

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Mean measured: 1.38 105 cfu/mL

On day 0, 7, 14, 21 by plate counting, unclear if it was performed on fresh or old Analytical measurements medium

The influence of the test substance on the reproduction and survival of Daphnia magna was determined in a 21-day semi-static test. The nominal concentration was 106 cfu/mL. The mean measured concentrations in the test medium was 1.38 105 cfu/mL. The difference could be explained by spores settling at the bottom of the aquaria or by the fact that spores may not activate readily in freshwater. The pH values ranged from 8.0 to 8.3, and the temperature was in the range Study Summary 20-22oC. Dissolved oxygen concentrations were 7.6 – 8.2 mg/L. The survival rate was 92% in the control (this meet the validity criteria of > 80%). At 106 cfu/mL, 90% of survival was observed in 4 of the 5 replicates and 70% in the fifth replicate. There were no significant differences in the length, weight, survival, number of neonates between the control and the treated group. The NOEC was 1.38 105 cfu/mL

Comment Assuming a purity of 7.1 x 1010 cfu/g, the NOEC would be 1.94 mg/L

The name of the formulation Chancellor is mentioned on the front page of this Remark report however the applicant confirmed that the test substance was indeed the technical ingredient.

Conclusion NOEC = 1.38 105 cfu/mL

Invertebrate chronic toxicity (Marine species) No study provided.

General conclusion about aquatic toxicity classification of Bacillus firmus I-1582: Bacillus firmus I-1582 is classified as 9.1C due to its toxicity on green algae and the fact that its persistence cannot be precisely evaluated, so it is considered as persistent.

Sediment toxicity (freshwater and/or marine) No study provided.

Soil toxicity

Soil macro-invertebrates acute toxicity

Type of study Full test

Flag Key study

November 2015 81

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Substance Bacillus firmus I-1582 (1.56 x 1011 cfu/g)

Species Eisenia fetida

Type of exposure Static, for 14 days

Endpoint LC50

Value > 1000 mg/kg dry soil (1.56 x 1011 cfu/ kg dry soil)

S Friedrich (2013) Bacillus firmus I-1582: Acute toxicity to the earthworm Eisenia fetida in artificial soil BioChem agrar Labor für biologische und Reference chemische Analytik GmbH Kupferstraße 6, 04827 Gerichshain, Germany. Report no 13 10 48 194S Bayer ID: M-475696-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 207 (1984)

No/Group 4 replicates of 10

Dose Levels 100, 178, 316, 562, 1000 mg/ kg dry soil

Analytical measurements Not required

The acute toxicity of the substance to the earthworm Eisenia fetida was determined in a 14-day test with artificial substrate containing 10% of peat. The mortalities were 2.5, 0, 0, 5.0, 0 and 2.5% in the control and at 100, 178, 316, 562, 1000 mg/ kg dry soil, respectively. Study Summary The biomass changes (fresh weight) were between -7.2 and -8.7% in all groups including the control. Validity criteria were met: Mortality less than 10% in control (observed value: 2.5%) Average loss of biomass in the control less than 20% (observed value: 8.0%)

11 Conclusion LC50> 1000 mg/kg dry soil (1.56 x 10 cfu/ kg dry soil)

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP (4.7 x 109 cfu/g)

Species Eisenia fetida

Type of exposure Static, for 14 days

Endpoint LC50

Value > 1000 mg formulation/kg dry soil

November 2015 82

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

T Leicher (2010) Bacillus firmus I-1582 WP 5: Acute toxicity to earthworms (Eisenia fetida) tested in artificial soil. Bayer CropScience AG Development- Reference Environmental safety Ecotoxicology 40789 Monheim (Germany). Report no EBBIL007, Bayer ID M-368585-01-1.

Klimisch Score 2 see comment

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 207 (1984)

No/Group 4 replicates of 10

Dose Levels 62.5, 125, 250, 500, 1000 mg formulation / kg dry soil

Analytical measurements Not required

The acute toxicity of the substance to the earthworm Eisenia fetida was determined in a 14-day test with artificial substrate containing 10% of peat. The mortalities were 0% in the control and all concentrations. The biomass changes (fresh weight) were between +7 and +9% in all groups Study Summary including the control. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Average loss of biomass in the control less than 20% (observed value: +8.0%)

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

Conclusion LC50> 1000 mg formulation /kg dry soil

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP

Species Hypoaspis aculeifer

Type of exposure Static, for 14 days

Endpoint LC50

Value > 1000 mg formulation/kg dry soil

November 2015 83

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

MA Kratz (2010) Bacillus firmus I-1582 WP 5: Influence on mortality and reproduction on the soil mite species Hypoaspis aculeifer tested in artificial soil Reference with 5% peat. Bayer CropScience AG BCS-D-EnSa-ETX Alfred-Nobel Strasse 50 40789 Monheim (Germany). Report no EBBIL009, Bayer ID M-367863-01-1.

Klimisch Score 2 (see comment below)

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 226 (2008)

No/Group 8 replicates of 10 for the control and 4 replicates of 10 for the treated groups

Dose Levels 63, 125, 250, 500, 1000 mg formulation / kg dry soil

Analytical measurements Not required

The acute toxicity of the substance to the soil mite Hypoaspis aculeifer was determined in a 14-day test with artificial substrate containing 5% of peat. In the control group 5.0 % of the mites died, in the treated groups mortalities were between 2.5 and 7.5%. No statistically significant effects were observed on the reproduction: % effects between 99.4 and 108.6% in comparison with control number of juveniles. Study Summary Validity criteria were met: Mortality less than 20% in control (observed value: 5%) Mean number of juveniles per replicate in the control > 50 (observed value: 381.9) Coefficient of variation calculated for the number of juvenile mites per replicate in the control < 30% (observed value: 3.7%)

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

LC50 > 1000 mg formulation /kg dry soil Conclusion NOEC for reproduction and survival > 1000 mg formulation /kg dry soil

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP (4.7 x 109 cfu/g)

Species Folsomia candida

November 2015 84

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Type of exposure Static, for 28 days

Endpoint NOEC

Value > 1000 mg formulation /kg dry soil

U Frommholz (2010) Bacillus firmus I-1582 WP 5: Influence on the reproduction on the Collembolan species Folsomia candida tested in artificial soil with 5% Reference peat. Bayer CropScience AG Environmental Safety, Ecotoxicology Alfred-Nobel Strasse 50 40789 Monheim (Germany). Report no EBBIL005, Bayer ID M- 367850-01-1.

Klimisch Score 2 see comment

5% peat instead of 10% in the guideline This was a recommendation to Amendments/Deviations optimise the bioavailability of test items.

GLP Yes

Test Guideline/s ISO 11267 (1999)

No/Group 5 replicates of 10

Dose Levels 63, 125, 250, 500, 1000 mg formulation / kg dry soil

Analytical measurements Not required

The acute toxicity of the substance to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 5% of peat. In the control group 12.0 % of the mites died, in the treated groups mortalities were between 4 and 20%. No statistically significant effects were observed on the reproduction: % effects between 102 and 115% in comparison with control number of juveniles. Study Summary Validity criteria were met: Mortality less than 20% in control (observed value: 12%) Mean number of juveniles per replicate in the control > 100 (observed value: 1184) Coefficient of variation calculated for the number of juvenile per replicate in the control < 30% (observed value: 10.4%)

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

Conclusion NOEC for reproduction and survival > 1000 mg formulation /kg dry soil

Soil macro-invertebrates chronic toxicity

November 2015 85

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No study provided.

Non-target plants toxicity No study provided.

Nitrogen transformation test

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP (4.7 x 109 cfu/g)

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Nitrate content

Value No effect

U Frommholz (2010) Bacillus firmus I-1582 WP 5: determination of effects on nitrogen transformation in soil. Bayer CropScience AG Environmental Safety, Reference Ecotoxicology Alfred-Nobel Strasse 50 40789 Monheim (Germany). Report no EBBIL015, Bayer ID M-367844-01-1.

Klimisch Score 2 see comment

Amendments/Deviations None that had an impact on the results

GLP Yes

Test Guideline/s OECD 2016 (2000)

No/Group 3 replicates

106.67 and 533.33 mg formulation /kg soil corresponding to 80 and 400 kg Dose Levels formulation /ha

Analytical measurements Not required

The objective of this study was to assess the effects of the substance on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied on soils at concentrations of 106.67 and 533.33 mg formulation /kg soil corresponding to 80 and 400 kg/ha. A loamy sand soil was used. Samples were taken at 0, 7, 14 and 28 days to determine the nitrate Study Summary content. During the 28-day test, 106.67 mg test item/kg dry weight soil caused a temporary stimulation of the daily nitrate rates at the time interval 7-14 days after treatment in a loamy sand soil amended with Lucerne-grass-green meal. The 5-fold application rate of the test item caused an increase of the daily nitrate rates at the time intervals 0-7 and 7-14 days. At the end of the test (14-

November 2015 86

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

28 day interval), differences in the nitrate-N rates between control soil samples and treated soil samples are < 25 %. The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 5%)

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

No effect on the N transformation in soil at concentrations up to 533.33 Conclusion mg formulation /kg soil

Carbon transformation test

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP (4.7 x 109 cfu/g)

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Respiration rate

Value No effect

U Frommholz (2010) Bacillus firmus I-1582 WP 5: determination of effects on carbon transformation in soil. Bayer CropScience AG Environmental Safety, Reference Ecotoxicology Alfred-Nobel Strasse 50 40789 Monheim (Germany). Report no EBBIL008, Bayer ID M-367846-01-1.

Klimisch Score 2 see comment

Amendments/Deviations None that had an impact on the results

GLP yes

Test Guideline/s OECD 2017 (2000)

No/Group 3 replicates

106.67 and 533.33 mg formulation /kg soil corresponding to 80 and 400 kg Dose Levels formulation /ha

Analytical measurements Not required

The objective of this study was to assess the effects of the substance on the Study Summary carbon transformation in soil, determined by the respiration rate. The substance was applied on soils at concentrations of 106.67 and 533.33 mg formulation /kg

November 2015 87

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

soil corresponding to 80 and 400 kg/ha. A loamy sand soil was used. Samples were taken at 0, 7, 14 and 28 days to determine the respiration rate. During the 28-day tests, 106.67 mg Bacillus firmus I - 1582 WP 5/kg dry weight soil and the 5-fold dose of the test item had no relevant influence on carbon transformation after addition of glucose to a loamy sand soil. Even though both test concentrations revealed a statistically significant difference to the control at the end of the test, the deviation from the control was still below the 25%- threshold value recommended by the guideline (9 and 15% of effects in the low and high rates, respectively). The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 2.6%)

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

No effect on the C transformation in soil at concentrations up to 533.33 Conclusion mg formulation /kg soil

Effects on microbial community in the laboratory

Type of study Limit test

Flag Disregarded study (see comments)

Test Substance Bacillus firmus I-1582 formulation 5WP

Species Soil micro-flora

Type of exposure Static, 3 months

Effects on the soil microbial biomass and on the survival of spores of Bacillus Endpoint firmus in natural soils

Value No effect on the soil microbial biomass

T Leicher (2010) Influence of Bacillus firmus on the microbial community in four different European soils. Bayer CropScience AG Environmental Safety, Reference Ecotoxicology Alfred-Nobel Strasse 50 40789 Monheim (Germany). Report no LRT-NBT 01/10, Bayer ID M-406103-01-1.

Klimisch Score 3 see comment

Amendments/Deviations Not relevant: no guideline followed and not a GLP study

GLP no

Test Guideline/s None followed

November 2015 88

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No/Group 1 replicate per soil

Dose Levels 106.67 mg formulation/kg soil corresponding to 80 kg formulation /ha

Determination of the soil microbial biomass (microbial carbon content) and the Analytical measurements counting of cell numbers of Bacillus firmus on agar plates

The aim of the study was to investigate the influence of the application of a WP5 formulation of Bacillus firmus I-1582 on the soil microbial biomass and to examine the survival of spores of Bacillus firmus in natural soils. In the experiment, 4 different natural agricultural soils were inoculated with Bacillus firmus I-1582 WP5. The application rate was 80 kg formulation /ha, corresponding to 1.5 x 106 spores of Bacillus firmus / g dry weight soil. Origin Type Cation Organic pH exchange carbon (%) capacity (meq/100g) Germany Sandy loam 8.9 1.04 6.43 France Clay loam 11.2 0.8 7.89 Germany Loam 18.6 5.0 7.08

Germany Sandy loam 9.2 1.8 5.52

Study Summary The treated and untreated soil samples were incubated for three months at 20 ± 2°C and after 0, 1, 2 and 3 months subsamples were taken and were investigated by the means of two different methods: the determination of the soil microbial biomass (microbial carbon content) and the counting of cell numbers of Bacillus firmus on agar plates. The presence of Bacillus firmus in the different treated and untreated soils was followed by counting the colony forming units (cfu) grown on agar plates, plated before and after pasteurization of the soil suspensions. The number of cfu obtained from non-pasteurized soil samples in all soils was by a factor of about 10 higher than from pasteurized samples. The cfu from untreated control soils and treated soils were at the same level. Differences are considered to be due to natural variations in soils or to methodological variations. An application of Bacillus firmus I-1582 WP5 at the rate of 80 kg formulation /ha to four different soils had no influence on the total number of bacteria detected in these soils.

The cfu count information in the report has been re-tested and confirmed to be Comments 4.7 x 109 cfu/g.

November 2015 89

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant. The plating method is not specific of Bacillus firmus so no conclusion can be drawn for this study about the survival of this species in the environment.

No effect on the soil microbial biomass Conclusion No conclusion on the survival of B. firmus in soil

General conclusion about soil toxicity classification: Bacillus firmus I-1582 does not trigger any 9.2 classification.

Terrestrial vertebrate toxicity For effects on terrestrial vertebrates other than birds, refer to the mammalian toxicity section.

Oral acute toxicity

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus (no information on cfu)

Species Colinus virginianus (Bobwhite quail)

Type of exposure Oral gavage for 5 days, observation period of 26 days

Endpoint NOEL for toxicity and pathogenicity

Value 2.5 x 108 spores /kg bw/d

SP Gallagher & JB Beavers (2006) An avian oral pathogenicity and toxicity study in the northern bobwhite – Bacillus firmus spores. Wildlife International Reference Ltd 8598 Commerce Drive Easton Maryland 21601 (USA). Report no 572-101 Bayer ID M-329113-02-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 885.4050 (1996)

2 replicates of 5 birds (undetermined sex) for the controls and 6 replicates of 5 No/sex/Group for the treated group

Negative control, attenuated control (autoclaved spores) and 2.5 x 108 spores Dose Levels /kg bw/d

November 2015 90

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Analytical measurements Not required

The objective of this study was to evaluate the acute toxicity and pathogenicity of Bacillus firmus when administered orally to northern bobwhite (Colinus virginianus). Groups of birds were exposed to water (negative control), autoclaved spores (attenuated control) or 2.5 x 108 spores /kg bw/d, by oral gavage for 5 days. At the termination of the study all surviving birds were subjected to a gross necropsy. Individual body weights were determined when birds were dosed on Days 0, 1, 2, 3 and 4 and on Days 11, 18, 25 and 30 of the test. Average estimated feed consumption (g/bird/day) was measured for Days 0-4, 5-11, 12- 18, 19-25 and 26-30. There were no mortalities in the negative control group or the attenuated control group and all negative control and attenuated control group birds were normal in appearance and behaviour throughout the test. There was a single incidental mortality in the Bacillus firmus treatment group, a bird found dead on Day 18 of Study Summary the test having exhibited no prior clinical signs. Gross necropsy revealed abrasions on the right hock and minor bruising on the cranium. The mortality was considered to be incidental to treatment. At the termination of the test, all birds were subjected to a gross necropsy. Necropsy results for all birds in the negative control group and attenuated control group were not remarkable. In the Bacillus firmus treatment group, one bird was noted with discoloration in the right lobe of the lung. Due to the isolated nature of this finding, it was not considered to be treatment related. Culture of the lung tissue resulted in no bacterial growth. When compared to the negative control group, there were no effects on the mean body weights of birds in the attenuated control group or the treatment group at any body weight interval. There were no apparent effects on feed consumption in the attenuated control group or the treatment group for any feed consumption interval.

The name of the formulation Chancellor is mentioned on the front page of this report however the applicant confirmed that the test substance was indeed the Comment technical ingredient. The strain is not mentioned in the report but the applicant confirmed it was I- 1582.

Conclusion NOEL = 2.5 x 108 spores /kg bw/d

Dietary acute toxicity No study provided.

November 2015 91

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Chronic toxicity (reproduction) No study provided.

General conclusion about terrestrial vertebrate classification: The available studies on birds and mammals do not provide a LD50 to compare with HSNO criteria for classification. All studies show no mortalities so Bacillus firmus I-1582 is not classified as 9.3.

Ecotoxicity to terrestrial invertebrates

Bees - Laboratory tests (acute oral and contact)

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP (4.7 x 109 cfu/g)

Species Apis mellifera

Type of exposure 48 h

Endpoint LD50

> 107.6 µg formulation /bee (oral) Value > 100 µg formulation /bee (contact)

S Schmitzer (2010) Effects of Bacillus firmus I-1582 WP5 (acute contact and oral) on honey bees (Apis mellifera L.) in the laboratory. IBACON GmbH Reference Arheilger Weg 17 64380 Rossdorf Germany. Report no EBBIL004 Bayer ID M- 367954-01-1

Klimisch Score 2 see comment

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 213 and 214 (1998)

No/Group 5 replicates of 10

Contact test: 100 µg formulation /bee Oral: 107.6 µg formulation /bee (actual intake) Dose Levels Reference substance (dimethoate formulation at 400 g/L): 0.30 to 0.10 µg ai/bee (contact) and 0.34-0.05 µg ai/bee (oral, actual intake)

Analytical measurements Not required

At the end of the contact toxicity test (48 hours after application), there was 2.0 % mortality at 100.0 μg product/bee. 6.0 % mortality occurred in the control Study Summary (water + 0.5 % Adhäsit). During the 24 hours assessment in the test item treated group only one single bee showed a behavioural abnormality such as

November 2015 92

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

moving coordination problem. No further abnormalities were observed 48 hours after application. In the oral toxicity test, the maximum nominal test level of Bacillus firmus I - 1582 WP 5 (100 μg product/bee) corresponded to an actual intake of 107.6 μg product/bee. This dose level led to 2.0 % mortality after 48 hours. No mortality occurred in the control (50 % sugar solution). No test item induced behavioural effects were observed at any time. Validity criteria were met: Mortality in control < 10% (observed value: 6%)

LD50 (24h) of the reference substance in the range 0.10-0.30 µg ai/bee (contact) and 0.10-0.35 µg ai/bee (oral). Observed values 0.21 and 0.14 µg ai/bee by contact and oral exposure, respectively.

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

Oral LD50 > 107.6 µg formulation /bee Conclusion Contact LD50 > 100 µg formulation /bee

Bees - Laboratory tests (dietary effects)

Type of study Limit test

Flag Key study

Test Substance Bacillus firmus I-1582 (cfu not reported)

Species Apis mellifera

Type of exposure 24 days

Endpoint NOEC

Value 108 spores/ml sucrose solution

KB Richards (2006) Evaluation of the dietary effects of Bacillus firmus a.i. on adult honey bees (Apis mellifera L.). California Agricultural research Inc 4141 N. Reference Vineland Kernan, CA 93630 (USA). Report no CAR 101-04 Bayer ID M- 329117-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results

GLP Yes

Test Guideline/s OPPTS 885.4380 (1996)

November 2015 93

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No/Group 4 replicates of 40 to 60 bees

107 cfu /mL in 30% sucrose solution Negative control: 30% sucrose solution Dose Levels Positive control: 30% sucrose solution containing 1000 ppm of potassium arsenate

Analytical measurements Verification of the initial stock solution concentration

The objective of this study was to evaluate the effect of purified Bacillus firmus a.i. administered in the diet of adult honey bees (Apis mellifera L.). The bees were allowed to feed ad libitum in cages inside a dark, environmentally controlled room. The number of dead bees in each cage was assessed on a daily basis. On Day 24, the study was terminated because cumulative percent mortality in the untreated control group exceeded 20%, it was 24.52%. Once terminated, the bees in all cages were counted to determine the exact number of bees present in each cage. The positive control produced 100% Study Summary mortality by Day 1 of the study, confirming the validity of the exposure system. At termination of the study on Day 24, the cumulative percent mortality was 23.19% in the Bacillus firmus at 107 cfu/ml treatment and 24.52% in the untreated control group. No statistically significant differences were noted between the treatment and the untreated control material treatment. No behavioural or morphological abnormalities were observed in bees exposed to the test or control substance treatments. Analysis of the test material diet showed that the test material as delivered to the bees was 108 cfu/ml. The no- observed-effect concentration (NOEC) for honey bee adults is at least 108 spores/ml.

The name of the formulation Chancellor is mentioned on the front page of this Remark report however the applicant confirmed that the test substance was indeed the technical ingredient.

Conclusion NOEC = 108 cfu/ml sucrose solution

Other non-target arthropods - Extended laboratory tests

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Bacillus firmus I-1582 formulation 5WP (4.7 x 109 cfu/g)

Species Aphidius rhopalosiphi (parasitic wasp)

Type of exposure To residues on barley seedlings

November 2015 94

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Endpoint LR50

Value > 80 kg formulation /ha

D Jans (2010) Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera: Braconidae) using an extended laboratory Reference test on barley Bacillus firmus I-1582 WP5. Bayer CropScience AG BCS-D- EnSA-Ecotoxicology IndustriePark Hoechst 65926 Frankfurt am Main (Germany) Report no CW09/062 Bayer ID M-368305-01-1

Klimisch Score 2 see comment

Amendments/Deviations None that impacted the results

GLP Yes

Test Guideline/s Mead-Briggs et al (2000 and draft 2006) Candolfi et al (2001)

No/Group 6 replicates of 5 wasps

5, 10, 20, 40 and 80 kg product/ha in 400 L water/ha Dose Levels Toxic reference (dimethoate) at 3 g ai/ha in 400 L water/ha

Analytical measurements Not required

The aim of the study was to determine the toxicity of freshly dried residues of Bacillus firmus I-1582 WP 5 applied onto barley seedlings, to the parasitoid wasp Aphidius rhopalosiphi. Mortality of 30 females was assessed 2, 24 and 48 hours after exposure. Repellency of the test item was determined during the initial 3 h after the release of the females. Five separate observations were made at 30-minute intervals starting 15 minutes after the introduction of all wasps. From the water control and the dose rates 5, 10, 20, 40 and 80 kg product/ha, 15 impartially chosen females per treatment were each transferred to a cylinder containing untreated barley seedlings infested with Rhopalosiphum padi for a Study Summary period of 24 hours. The number of mummies was assessed 11 days later. No significant mortality was observed. In the dose rates of 5 and 10 kg product/ha 10.0% and 0% corrected mortality occurred. At the rates of 20 and 40 kg product/ha 3.3% and 0% corrected mortality was detected and 13.3% in the highest rate of 80 kg product/ha. No statistically significant effect on reproduction occurred. The reduction in reproductive success relative to the control at the 5, 10, 20, 40 and 80 kg product/ha rates was 16.8%, 46.7%, 38.7%, 24.8% and 39.1%, respectively. No statistically significant dose related repellent effect of the test item was observed.

The LR50 was estimated to be > 80 kg product/ha.

November 2015 95

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The cfu count information in the report has been re-tested and confirmed to be 4.7 x 109 cfu/g. Comments The applicant justified the read-across of this study for Poncho Votivo on the basis of comparison of application rates. As the 5WP is not approved in New Zealand, this justification is not relevant.

Conclusion LR50 > 80 kg formulation /ha

General conclusion about toxicity to terrestrial invertebrate classification: Bacillus firmus I-1582 does not trigger any 9.4 classification. However, the validity of the information is doubtful and no clarification was provided by the applicant.

Publications provided by the applicant: Some publications were also provided in addition to the study reports. Brief summaries of the publications related to ecotoxicology are presented below.

Reference Aly et al (2008) Characterization of some bacteria isolated from Oreochromis niloticus and their potential use as probiotics. Aquaculture 277: 1-6

Bacillus firmus and other bacteria have been isolated from the fish Nile tilapia (Oreochromis niloticus) and showed some inhibitory effects in vitro on the Summary growth of a pathogen bacteria Aeromonas hydrophila. When injected (ip or im) to 3 replicates of 10 fish at 5 x 108 cells or 1 x 109 cells, there was no mortality in the 14 days observation period after the injection.

Bacillus firmus has an effect on another bacterium growth. The exposure route Comments/ Conclusion for the toxicity test on fish is not relevant for ecotoxicity assessment.

Reference Aslim et al (2002) Determination of some properties of Bacillus isolated from soil. Tuk J Biol 26: 41-48

40 strains of Bacillus were isolated from 6 grassland soil of Turkey. Among them, there were 5 strains of Bacillus firmus. They were tested for their growth Summary inhibitory effects on 4 pathogen bacteria. Only 2 strains of Bacillus firmus had an effect on only one pathogen: Micrococcus flavus.

Bacillus firmus (at least some strains of it) has an effect on another bacterium Comments/ Conclusion growth

Reference Banjo et al (2006) Microbes associated with edible Astacus species. W Applied Sci J 1(1): 29-34

Bacillus firmus has been isolated from 2 species of freshwater crayfish (Astacus Summary fluviatilis and Astacus torrentium) gut and body surface sampled in Nigeria.

November 2015 96

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Comments/ Conclusion Bacillus firmus is naturally present in some freshwater arthropods

Reference Borsodi et al (2007) Phenotypic characterization and molecular taxonomic studies on Bacillus and related isolates from Phragmites australis periphyton. Aquatic botany 86: 243-253

Bacillus firmus has been isolated from periphyton communities developing on Summary the underwater plant surface of reeds (Phragmites australis).

Comments/ Conclusion Bacillus firmus is naturally present on some aquatic plants

Reference Ghosh et al (2003) Three newly isolated plant growth-promoting bacilli facilitate the seedling growth of canola, Brassica campestris. Plant Physiology and Biochemistry 41: 277-281

One strain of Bacillus firmus (DUC2) has been shown to stimulate root Summary elongation, shoot length and fresh and dry weight of seedlings of canola by its ability to utilise 1-aminocyclopropane-1-carboxylic acid as sole nitrogen source.

The relevance of this information is limited because it only applies to one strain Comments/ Conclusion of B. firmus which is different from the one in Poncho Votivo

Reference Gilliam & Valentine (1975) Bacteria isolated from the intestinal contents of foraging worker honey bees, Apis mellifera: the genus Bacillus. Journal of Invertebrate pathology 28: 275-276

Summary Bacillus firmus has been isolated from one honey bee gut among 40 bees

Comments/ Conclusion The relevance of this finding is unclear

Reference Inglis et al. (1993) Aerobic microorganisms associated with alfalfa leafcutter bees (Megachile rotundata). Microbiol Ecology 26: 125-143

Bacillus firmus has been isolated from samples of pollen, provisions, pre and Summary post defecation, larval guts and frass of alfalfa leafcutter bees (Megachile rotundata). This is the most abundant bacteria identified.

Comments/ Conclusion Bacillus firmus is naturally present on/in leafcutter bees

Reference Korenblum et al.(2005) Production of antimicrobial substances by Bacillus

subtilis LFE-1, B. firmus H2O-1 and B. licheniformis T6-5 isolated from an oil reservoir in Brazil. J Applied Microbiol 98: 667-675

One strain of Bacillus firmus has been isolated from an oil reservoir in Brazil and Summary has inhibitory effects on the growth of 97.5% of 40 strains studied.

November 2015 97

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Comments/ Conclusion Bacillus firmus (at least one strain of it) has an effect on other bacteria growth

Reference Lalke-Porczyk et al. (2004) Physiological properties and taxonomy of epiphytic bacteria in Chełmżyńskie Lake in Poland. Polish Journal of Env studies 13(5): 543-549

Bacillus firmus has been isolated from surface of submerged macrophytes that Summary are dominant in the littoral zone of a Polish lake. The presence was seasonal. s

Comments/ Conclusion Bacillus firmus is naturally present on some aquatic plants

Reference Varma & Mohammed Ali (1986) Bacillus firmus as a new insect pathogen on a lepidopteran pest of Ailanthus triphysa. J invert pathol 47: 379-380

The authors identified a parasitic bacterium in dead larvae of Eligma narcissus as Bacillus firmus. Inoculation of this bacterium confirmed the effectiveness as Summary 80-100% of the larvae died 72 h after feeding on leaves treated with a bacterial suspension at 1010 cfu/mL.

The identification as Bacillus firmus is doubtful as only morphological means Comments/ Conclusion were used and B. firmus is not known to have insecticidal activity.

November 2015 98

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix I: Robust study summaries for Clothianidin

Mammalian toxicity

Substances containing clothianidin were transferred to management under the HSNO Act. As a category C application was never required for this active ingredient, full information on some toxicity end-points is not currently included in the EPA’s Substance Database.

The applicant has provided the EU Draft Assessment Report (DAR) for clothianidin from 2003, plus all new regulatory toxicology studies conducted since that time. A summary of the endpoints from the DAR is provided below, as well as summaries of the new studies.

Acute toxicity, skin and eye irritation and contact sensitisation

Summary of acute toxicity, irritancy and contact sensitisation studies with clothianidin from the EU Draft Assessment Report (2003)

Study Result Purity & Batch no. Reference

96% Gardner, 1997a Acute oral toxicity in rats LD50 > 5000 mg/kg bw 30034708

1216

389 mg/kg bw (M) 96% Gardner, 1997b Acute oral toxicity in mice 465 mg/kg bw (F) 30034708

96% Gardner, 1997c Acute dermal toxicity in rabbits > 2000 mg/kg bw 30034708

96% Shepherd, 1998 Acute inhalation toxicity rats > 5.54 mg/L 30034708

96% Gardner, 1997d Skin irritation in rabbits Not irritating 30034708

96% Gardner, 1997e Eye irritation in rabbits Not irritating 30034708

Skin sensitisation in the guinea 96% Denton, 1997 Not sensitising pig (Magnusson and Kligman) 30034708

November 2015 99

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Contact Sensitisation [6.5] A further contact sensitisation study conducted in 2012 has been submitted to the EPA. This study is summarised below.

Type of study Contact sensitisation in the guinea pig (Guinea Pig Maximization Test according to Magnusson and Kligman)

Flag Key study

Test Substance Clothianidin (purity 99.3% w/w)

Endpoint Skin sensitisation (skin reactions on topical challenge)

Value Not sensitising

Leidenfrost P., 2012 Clothianidin: Study for the skin sensitisation effect in guinea pigs (guinea pig maximisation test according to Magnusson and Reference Kligman). Bayer Pharma AG, GDD-GED Toxicology, 42096 Wuppertal, Germany. Study no: T8083188; Report no: AT06378. M-424556-01-2.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 406; OPPTS 870.2600; 96/54/EC, Method B.6

Species Guinea pig

Strain Crl: HA

Test group: 20 F No/Sex/Group Control group: 10 F

Intradermal induction: 5% Dose Levels Topical induction: 50% Challenge: 50%

Intradermal induction: intradermal injection Exposure Type Topical induction and challenge: hypoallergenic patch

The test item was formulated in polyethylene glycol 400 to yield a solution (1%) or a suspension (>1%). After the first induction the animals in the control group and the test item group showed strong effects up to encrustation at the injection sites of the first Study Summary induction. The challenge with the 50% test item formulation led to no skin effects in the animals of the test item group and to no skin effects in the animals of the control group. In summary, by comparing the results in the treatment group and in the control group under the conditions of the maximization test and with

November 2015 100

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

respect to the evaluation criteria the test item therefore exhibits no skin- sensitization potential.

Reference is made to the most recent check for reliability of the Guinea Pig Maximization Test methodology by the test facility, conducted in September 2011. The test was performed on female guinea pigs using alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400 at the following concentrations: Intradermal induction: 5% test item formulation; Topical Additional Comments Induction: 25%; First challenge: 12%; Second challenge: 6%. After the first challenge 100% of the test item animals and 60% of the control animals exhibited dermal reactions in the challenge treatment and after the second challenge 60% of the test item animals. There was no reddening of the skin to be observed on control group animals. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study.

Conclusion Not sensitising

Phototoxicity Clothianidin was shown to absorb light energy with an Ultraviolet/visible molar extinction/absorption coefficient greater than the EU threshold requiring phototoxicity testing. Therefore a phototoxicity test has been conducted.

Type of study In vitro phototoxicity test

Flag Key study

Test Substance Clothianidin (purity 99.2% w/w)

Endpoint Phototoxicity (Photo-Irritation Factor [PIF] & Mean Photo Effect [MPE])

No phototoxic potential (MPE: -0.021 – 0.026;PIF could not be determined,

since no ED50 values could be calculated; ED50 values could not be Value determined, since the viability of the cells was not reduced with and without irradiation)

Heppenheimer A., 2014 Clothianidin TC: Cytotoxicity assay in vitro with BALBc/3T3 cells: Neutral Red (NR) test during simultaneous irradiation Reference with artificial sunlight. Harlan Cytotest Cell Research GmbH (Harlan CCR). In den Leppsteinwiessen 19, 64380 Rossdorf, Germany. Report amendment number 1. Harlan study number 1592300. M-472655-02-1.

Klimisch Score 1

November 2015 101

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The report was amended to provide further information on a number of elements of the study, such as information on the choice of solvent, test Amendments/Deviations item precipitation, radiation sensitivity of the cells used in the study and details of the solar simulator.

GLP Yes

Test Guideline/s OECD 432; EC 440/2008 Method B.41

BALBc/3T3 cells tested in the presence and absence of irradiation with Test system artificial sunlight

Number of replicates 12 wells per exposure parameter

Dose Levels 0, 7.813, 15.63, 31.25, 62.5, 125.0, 250.0, 500.0, 1000 µg/mL

Cell were exposed to the test item for 1 hour in the dark followed by a 50 Exposure Duration minute irradiation period with a solar simulator (1.65 mW/cm2 resulting in an irradiation dose of 4.95 J/cm2; non-irradiated group kept in the dark)

The experiment was performed twice. The first experiment served as a range finding experiment (RFE), the second was the main experiment (ME). Cytotoxic effects did not occur after exposure of the test item to the cells, neither in the presence nor absence of irradiation with artificial sunlight in

the RFE and the ME. Therefore ED50 values or a PIF could not be calculated. The MPE values in the two studies were -0.021 and 0.026, Study Summary respectively. The mean of solvent control values of the irradiated group versus the non- irradiated group met the acceptance criteria. The positive control induced phototoxicity in the expected range in the presence of irradiation. In conclusion, under the experimental conditions of this study clothianidin does not possess any phototoxic potential.

Historical positive and negative control data from April 2006 until July 2013 Additional Comments were presented, demonstrating the reliability of the experimental technique at this test facility.

Conclusion No phototoxic potential

Conclusion on acute toxicity, skin and eye irritation, contact sensitisation and phototoxicity The acute toxicity of clothianidin by the oral, dermal and inhalation routes was low.

November 2015 102

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The result of the acute oral toxicity study in rats suggest that classification is not required as the LD50 was greater than 5000 mg/kg bw. However, certain clinical signs observed after oral administration to rats or mice suggest mild and transient neurotoxic effects (tremor, ataxia, hypoactivity, hunched posture, piloerection, respiratory impairment, hypothermia). The applicant considered the observed effects as a consequence of overdose of the compound. However, in both acute neurotoxicity and pharmacological studies similar dose- dependent symptoms were observed, suggesting nicotinic CNS-effects. In a range-finding acute neurotoxicity study with clothianidin, formulated differently than in the original acute oral toxicity study, the oral LD50 value was between 500 and 1000 mg/kg bw in female rats. Both the clinical signs and recovery appeared earlier in the latter study, suggesting a more rapid and/or more complete absorption than in the former. Moreover, mice appear more sensitive than rats when both mortality and clinical signs are considered. Based on the findings in the rat, acute neurotoxicity and mouse acute toxicity studies clothianidin is classified 6.1D for acute oral toxicity.

No acute dermal or inhalation toxicity was observed and no classification is required for these endpoints.

Clothianidin is not a skin or eye irritant, and is not a skin sensitiser based on the data provided.

Clothianidin was also shown to not possess phototoxic potential.

Genotoxicity [6.6] The following is a summary of information on genotoxicity data from the 2003 EU DAR for clothianidin.

Type of test Result Reference

In vitro studies

S. typhimurium (TA100, TA1535, TA 98, TA1537) and E.coli (WP2uvrA-) Positive Thompson, 2000 plate incorporation assay, ±S9, DMSO

S. typhimurium (TA100, TA1535, TA 98, TA1537) plate incorporation Negative Otsuka, 1990a assay, ±S9, DMSO

S. typhimurium (TA100, TA1535, TA 98, TA1537, TA102) plate Negative Herbold, 1999a incorporation and preincubation assay, ±S9, DMSO

S. typhimurium (TA1535), plate incorporation and preincubation Negative Herbold, 1999b assay, ±S9, DMSO (not GLP)

B. subtilis -H17 and M45, Spore rec- Negative Otsuka, 1990b assay, ±S9

L5178Y/TK+/-, mouse lymphoma Positive Durward, 2000a gene mutation assay,±S9 ,DMSO

November 2015 103

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

V79, CHL gene mutation assay, ±S9, Negative Brendler-Schwaab, 1999a DMSO

CHL, chromosome aberration assay, Positive Wright, 2000 ± S9, DMSO

In vivo studies

Mouse, bone marrow, micronucleus assay, (oral, 25; 50; 100 mg/kg, Negative Durward, 2000b arachis oil)

Rat, liver, unscheduled DNA synthesis assay, (oral, 2500; 5000 Negative Brendler-Schwaab, 1999b mg/kg, aqueous cremophor)

The EU DAR notes that one out of four bacterial gene mutation assays had a positive result in one strain (TA1535) which was not confirmed in a repeat experiment.

In the mouse lymphoma assay, an increase of mutants was detected at near cytotoxic or cytotoxic doses. There was a dose-related increase of the proportion of small-colony mutants, suggesting the clastogenic nature of the compound.

In the HPRT test on V79 cells, no mutagenic activity was revealed, neither in the absence nor in the presence of metabolic activation.

Clastogenicity was observed in the CHL cell line, at near cytotoxic or cytotoxic doses. In the absence of metabolic activation, both a dose- and time-related increase was observed.

In vivo, both the micronucleus test in the mouse bone marrow and a study on unscheduled DNA synthesis in rat liver cells were negative. In the mouse, the compound evoked systemic toxicity, but it was observed that the NCE/ PCE ratio was not affected, which may raise the question whether the exposure of the stem cell population in the bone marrow was attained. However, due to the fact that in vitro clastogenicity was only observed at doses which induced >50% cell loss and/or drop of >50% mitotic index, the finding was considered to be probably secondary to high-cytotoxicity events and it was considered that no further testing was warranted.

Carcinogenicity [6.7] The following is a summary of information on carcinogenicity data from the 2003 EU DAR for clothianidin.

Type of test NOAEL mg/kg LOAEL mg/kg Critical effects Reference organism bw/day (ppm) bw/day (ppm)

2 year rat p.o. 32.5 (500) Interstitial ovarial Biegel, 2000a Toxicity: 9.7 (150) [We note this was gland hyperplasia, listed in the EU

November 2015 104

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Carcinogenicity: 157 (3000) DAR overall bw effects, feed summary as the consumption same value as the NOAEL. The LOAEL has been obtained from the detailed summary of the study.]

2 year mouse p.o. Body weight effects, clinical signs, liver Toxicity: 47.2 (350) 171.4 (1250) Biegel, 2000b cell hypertrophy, Carcinogenicity: 251.9 (2000) cervix hyperplasia

Clothianidin was not carcinogenic when tested in the rat and the mouse.

A slight increase of hepatocellular carcinoma at the mid dose and the top dose in the rat study was of doubtful toxicological significance. Chronic toxicity effects in the rat were observed in kidney, liver and ovarial interstitial cells. Local effects were observed in the stomach.

In the mouse, the chronic effects were observed mainly in the kidney (decreased weight), liver (hepatocellular hypertrophy), cervix (fibromuscular hyperplasia), and heart (myocardial degeneration) at doses causing significant body weight effects.

In both rat and mouse, the applied doses induced only minor changes in the WBC parameters or haematopoietic organs at any sampling time. This finding was in contrast to the observed changes at higher doses in the short-term assays.

Reproductive and developmental toxicity [6.8] The following is a summary of information on reproductive and developmental toxicity data from the 2003 EU DAR for clothianidin.

Type of test, NOAEL mg/kg LOAEL mg/kg Reference Critical effects organism bw/day (ppm) bw/day (ppm)

↓ body weight1,2, parental=filial: 10.2 parental1=filial2 ↓thymus w2, (150) 32.7 (500) ↑preputial separation 2 2 generation, p.o., delay , ↑vaginal Freshwater, opening patency2 rat 2000 reprotoxicity: ↑stillborns, sperm reprotoxicity: 32.7 motility/morphology (500) 179.6 (2500) effects

maternal: 10 maternal: 40 ↓Feed consumption, Teratogenicity, rat York, 1998a developmental: 125 developmental: - body weight

November 2015 105

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

↑Mortality, clinical signs, ↑abortions1, ↑premature maternal: 10 maternal: 25 deliveries1, ↓foetal Teratogenicity, 1 weight1, York, 1998b rabbit foetal= foetal = developmental 25 developmental2: 75 ↑intermediate lung lobe absence2, ↓sternal ossification centers2

Developmental maternal1= filial2 1 toxicity, rat 42.9 (500) ↓bw gain , ↓pup maternal=filial 12.9 weight2, ↓startle Toxicity: (150) habituation, ↓motor Hobermann, activity, ↓surface 2000 righting, brain Neurotoxicity: 42.9 (500) 142 (1750) histometry Reprotoxicity: 142 (1750) N/A

In the multi-generation study, both maternal and filial toxicity was observed at the mid dose and above, and included body weight decreases (F1, F2), leading to lower body weight gains (F1). Developmental toxicity was detected at the top dose, and included belated preputial separation and vaginal patency. At the top dose, effects on sperm motility and morphology were observed, and litter incidence of early stillborns was increased in both generations. Since these modifications occurred at dose-levels which were clearly maternotoxic, the compound should not be considered a reproductive toxicant.

In both the rat and rabbit developmental studies, the lowest dose was detected as the maternal NOAEL, corresponding to about 10 mg/kg bw. In the rat, both foetal and developmental toxicity exceeded the maternal MTD (125 mg/kg bw). In the rabbit, both foetal and developmental toxicity was detected at 75 mg/kg bw, with increased incidences of abortions, premature deliveries, and visceral variations, including intermediate lung lobe and reduced ossification centres. At the top dose, malformations such as small kidney, fused caudal vertebrae, and absent phalanges were detected, but at doses exceeding maternal MTD. Therefore in neither rat nor rabbit, the compound is classified for developmental toxicity.

Additionally, a behavioural developmental study was undertaken to detect possible neurotoxic effects in the rat pups. Both developmental and foetal toxicity NOAELs were similar to that detected in the multi-generation study, and were set at 150 ppm= 12.9 mg/kg bw/d.

November 2015 106

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Target organ toxicity [6.9] The following is a summary of short term toxicity studies from the EU DAR for clothianidin.

Type of test NOAEL mg/kg LOAEL mg/kg Critical effects References organism bw/day (ppm) bw/day (ppm)

♂: 120 (1250) ♂: 249 (2500) ↓bw, ↓bw gain, Chambers, 28d, p.o., rat haematological ♀: 137 ♀: 228 effects 1997a

28d, p.o., ♂: 90 (500) ♂: 190 (1000) ↓bw, ↓bw gain, Chambers, mouse ♀: 122 ♀: 248 clinical chemistry 1997b

↑mortality, clinical ♂: 35.8(1250) ♂: 35.8 (2500) findings, Moore, 28d, p.o., dog ↓haematopoietic/ ♀: 36.3 ♀: 52.3 lymphoid organs, 2000 enteropathy

↓feed consumption, 90d, p.o., ♂: 16 (100) ♂: 82 (500) clinical chemistry Chambers, mouse ♀: 22 ♀: 107 modifications, 1997d ↓kidney weight

♂: 19.7 (250) ♂: 96.0 (1250) ↓bw, ↓bw gain, Chambers, 90d, p.o., rat ♀: 24.0 ♀: 119.0 ↑ovary/uterus weight 1997c

♂: 202.0 ♂: 27.9 (500) ↓bw, ↓bw gain, Wahle, 90d, p.o., rat (3000) effects on RBC, ♀: 34.0 2000 ♀: 254.2 ↑spleen pigmentation

clinical signs, ↓bw ♂: 19.3 (650) ♂: 40.9 (1500) gain, anemia, Bernier, 90d, p.o., dog ♀: 21.2 ♀: 42.1 ↓jejunum lymphoid 2000a hyperplasia

♂: 36.3(1500) ♂: 46.4 (2000) Bernier, 1 year p.o., dog ↓WBC, ↓neutrophils ♀: 40.1 ♀: 52.9 2000b

♂: 300 ♀: ♂: 1000 ♀: Weiler, 28d, dermal, rat ↓bw, ↓bw gain 300 1000 2000

90d, p.o., rat ↓bw, ↓feed Sheets, •toxicity: 60 (1000) 177 (3000) consumption, mortality 2000b •neurotoxicity: 177 (3000)

The lowest relevant NOAEL was 16 mg/kg bw/d, detected in the 90 day study in the mouse, on the basis of mortalities, clinical chemistry modifications and kidney weight decreases.

November 2015 107

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The percutaneous effects of the compound were examined in a 4-week rat study. A systemic dermal NOAEL was determined as 300 mg/kg bw/d, on basis of the slight body weight effects at 1000 mg/kg bw/d. No local effects were observed.

Summary of neurotoxicity studies with clothianidin The following is a summary of neurotoxicity data from the EU DAR for clothianidin.

Type of test, NOAEL mg/kg LOAEL mg/kg Critical effects References organism bw/day (ppm) bw/day (ppm)

M: 60 M: 100 ↓Locomotor Sheets, 2000a Acute, rat activity, F: 100 F:200 ↓hypothermia Cain, 2000

90d, p.o., m rat ↓Body weight, Sheets, toxicity: 60 (1000) 177 (3000) ↓feed consumption 2000b neurotoxicity: 177 (3000) N/A

Developmental, rat maternal=filial maternal1= filial2 ↓Bw gain1, ↓ pup toxicity: 12.9 (150) 42.9 (500) 2 weight Hobermann, neurotoxicity: 42.9 (500) 142 (1750) ↓startle habituation, 2000 ↓motor activity, ↓surface righting, reprotoxicity: 142 (1750) N/A brain histometry

Clothianidin provoked neurologic impairment when administered orally as a single dose, with effects appearing very rapidly (day of dosing) and disappearing within 24h. On the basis of the observed decreased arousal and lowered (loco) motor functions, the NOAEL was set at 60 mg/kg bw/day.

In the sub-chronic study, no effect on behavioural endpoints was detected at the maximum dose.

The effect of Clothianidin on the rat foetus was assessed in a developmental study, where the dams were exposed during gestation. Both developmental and foetal toxicity NOAELs were similar to that detected in the multi-generation study, and were set at 150 ppm= 12.9 mg/kg bw/day. At the top dose, subtle modification of acoustic startle habituation and motor activity was observed in the pups immediately after weaning, but not at 60d post-partum. A slight increased hippocampal and cerebellar cross thickness was detected histomorphometrically, although without microscopically detectable findings. The neurotoxic relevance of the effects was unclear, since the observed startle/motor modifications were transient, and the morphometric modification were observed at the top dose which was maternotoxic and induced some delay in pup development. However, as a precaution, a developmental neurotoxicity NOAEL was set at 43 mg/kg bw/d on basis of these effects. [We note: this is the study for which a further review of the findings based on current guidelines was submitted. This study is summarised later in the review.]

November 2015 108

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Summary of information on metabolites The EU DAR notes that some major (TZNG, >5% of dose) or minor (TZMU, TMG, MG, <5% of dose) rat metabolites are also detected in the hen, goat, plants and in the environment as a result of metabolic activity in mammals and plants, and hydrolytic activity in the environment. These breakdown products are relatively more toxic than the parent compound, or of the same order of toxicity. Since many of them are either rat metabolites, occur at very low residue levels, are only formed under non-neutral acidity-conditions, or are environmentally not pertinent, the risk is considered very low and further testing is not warranted.

In addition, the metabolites TZNG, TZMU, TMG, MG, MNG, ATG-Ac and ATGM-Pyr were also tested in the S. typhimurium reverse gene mutation assay, and none of them were shown to induce revertants above the spontaneous solvent control rate. Notwithstanding some technical shortcomings concerning compound stability (ATG-Ac and ATGM-Pyr), these metabolites should be considered devoid of gene-mutation potential. Although less conclusive, the environmental metabolite MAI was not considered a genotoxicant.

Repeated dose toxicity studies completed following publication of the EU DAR

Repeated dose toxicity/immunotoxicity A combined repeated-dose toxicity and immunotoxicity study was completed in 2004, following completion of the EU DAR. This study is summarised below. Clothianidin is referred to by the code TI-435 in this study report.

Type of study Repeated dose toxicity/immunotoxicity in the rat

Flag Key study

Test Substance TI-435 (Clothianidin)

LOAEL (general toxicity): 3000 ppm (252.8 mg/kg bw/day males; 253.0 mg/kg/day females) Based on reductions in body weight, body weight gain and feed consumption. LOAEL (immunotoxicity): N/A Endpoint NOAEL (general toxicity): 500 ppm (45.8 mg/kg/day males; 46.2 mg/kg/day female) NOAEL (immunotoxicity): 3000 ppm (252.8 mg/kg bw/day males; 253.0 mg/kg/day females)

Hoberman A.M., 2004 Oral (Diet) Repeated Dose 28-Day Toxicity/Immunotoxicity Study of TI-435 in Rats. CR-DDS Argus Division Reference 905 Sheehy Drive, Building A, Horsham, Pennsylvania 19044-1241, USA. CR- DDS Argus Protocol Number: RLF00001. M-123395-01-1.

Klimisch Score 1

Amendments/Deviations None considered to impact the quality or outcome of the study

GLP Yes

November 2015 109

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Guideline/s OPPTS 870.7800

Species Rat

Strain Crl:CD®(SD)IGS BR VAF/Plus®

No/Sex/Group 10M & 10F

0, 150, 500, 3000 ppm (equivalent to 0, 13.8, 45.8 or 252.8 mg/kg bw in males Dose Levels and 0, 14.0, 46.2, or 253.0 mg/kg/day in females)

Exposure type Dietary (Admixture with feed)

Concentrations were selected by the Sponsor on the basis of a previous subchronic study in rats with similar dose levels of TI-435 and dietary route of exposure. The highest concentration was expected to produce some toxicity (clinical signs or a decrease in body weights), but not death or severe suffering. The intermediate concentration was expected to produce minimal effects, while the lowest concentration was expected to produce no adverse effects. All rats survived to scheduled sacrifice. No clinical observations related to TI- 435 occurred. Body weights and absolute feed consumption values were significantly reduced in the 3000 ppm exposure group male and female rats for the entire study period. Body weight gains were significantly reduced in the 3000 ppm exposure group male and female rats on days 1 to 8, and 1 to 29 and also in the 3000 ppm exposure group female rats on days 15 to 22. Relative (g/kg/day) feed consumption values were significantly (p<0.01) reduced in the 3000 ppm exposure group male and female rats on days 1 to 8 and 1 to 29. No necropsy observations related to TI-435 occurred. Terminal body weights in Study Summary the 3000 ppm exposure group male and female rats were significantly reduced compared to the Group I (0 ppm) values. Absolute weights and the ratio of the spleen weight to the terminal body weight in the male and female rats did not differ significantly among the groups exposed to TI-435. Exposure to TI-435 did not cause any adverse effect on the IgM antibody- forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes, in male and female rats when evaluated as both specific activity (AFC/106 spleen cells) and as total spleen activity (AFC/spleen). The positive control, cyclophosphamide, produced the anticipated results including a reduction in the immune response, body weight and body weight gain, feed consumption and spleen weights as well as increased incidence of adverse clinical observations. A statistically significant (p≤0.05 to≤0.01) enhancement of specific activity in the 500 ppm exposure group male rats and total spleen activity in the 150 and 500 ppm exposure group male rats compared to control group values was not considered related to TI-435 because: 1) the appearance of an enhancement

November 2015 110

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

was related to a lower than previously observed response in the control group male rats and probably related to the variability common in outbred strains of rats used for these analyses; 2). the enhancement was not dosage-dependent; and 3) it did not occur in the female rats. On the basis of these data, the no-observable-effect-level (NOEL) for general toxicity in male and female rats was 500 ppm (45.8 mg/kg/day male rats, 46.2 mg/kg/day female rats). Exposure to 3000 ppm of TI-435 in the diet produced reductions in body weights, body weight gains and feed consumption. The NOEL for immunotoxicity as measured by the response to sheep red blood cells was greater than 3000 ppm (252.8 male rats an 253.0 mg/kg/day female rats), the highest dose tested.

Additional Comments None

Exposure to clothianidin in the diet did not adversely affect the functional ability Conclusion of the humoral immune component of the immune system, under the conditions of this study.

Developmental neurotoxicity The EU and US EPA reviews of clothianidin included a developmental toxicity study in rats, completed in 2000.

Type of study Review of a developmental neurotoxicity study in rats

Flag Supporting

Test Substance Clothianidin

Inakawa K., 2014 The review of a developmental neurotoxicity study of clothianidin in rats. Toxicology Group, Environmental Health Science Reference Laboratory, Sumitomo Chemical Co. Ltd. Ref No: THT-0298. M-508525- 01-1.

The purpose of this review was to provide information for Annex I renewal (AIR). The report of developmental neurotoxicity study in rats with clothianidin (Hobermann, 2000, Ref. No.: THT-0068) was reviewed on a guideline-compliance and a scientific point of views, and additional historical control data has been included. Summary When the study was carried out, there was no applicable EU guideline. The study was conducted based on the requirement of the U.S. EPA guideline; Developmental Neurotoxicity Study (OPPTS 870.6300, 1996) and OECD Guideline for the Testing of Chemicals, Proposal for a New Guideline 426 (1998).

November 2015 111

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

There are no deviations from applied guidelines. There are no major changes between previous and current evaluation of the neurotoxic effects in offspring. A maternal no-observed-adverse-effect-level (NOAEL) for all effects was established as 500ppm, equivalent to dose levels of 42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively, based on reduced weight gain and food consumption at 1750ppm. A developmental NOAEL was established as 500ppm in male and 150ppm in female F1 progeny, equivalent to dose levels of 12.9 and 27.3 mg/kg bw/day during gestation and lactation, respectively, based on reduced pre-weaning weight gain in males at 1750ppm and in females at 500 and 1750ppm.

No statistically significant differences were apparent between treated and control male groups in any of the morphometric measurements on day 12 postpartum (pp), but there were statistically significant increases in the thickness of the dentate gyrus and the height of the cerebellum, and decreased thickness of the external germinal layer of the cerebellum in females treated at 1750ppm. Both the thickness of the dentate gyrus and the height of the cerebellum in the control group were lower limit values of the historical control data, and those in the 1750ppm group were within the historical control ranges. The slight differences in the size of these structures at 1750ppm were considered to reflect slightly more advanced brain development; especially the external germinal layer becomes thinner and eventually disappears when the cerebellum enlarges. No morphometric changes were apparent in the brains of females treated at 500ppm.

The diagonal width of the caudate putamen and the thickness of the dentate gyrus of the hippocampus were significantly shorter than controls in days 83 – 87 pp of females in the 1750ppm group. However, the differences were not considered to be biologically significant. These differences were approximately 5%, and both the diagonal width of the caudate putamen and the thickness of the dentate gyrus of the hippocampus in the females of the 1750ppm were within the historical control ranges. The values in females in the 500ppm group and males at 1750ppm were comparable to the controls. All other brain morphometric parameters were comparable to the controls. Motor activity, as assessed by the average number of movements and

November 2015 112

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

the time spent in movement, showed a treatment-related decrease in both sexes at 1750ppm at day 22 pp, reflecting significant reductions in the number of movements and the time spent in movement in one or more of the five-minute blocks. However, there were no differences at days 14, 18 and 62 pp.

The average number of movements and the time spent in movement were not affected by treatment at lower dose levels. The time spent in movement for the females at day 22 pp in the 500ppm group was significantly reduced in Block 4. This statistically significant reduction was considered to be a transient change and not treatment-related. The value was also within the historical control ranges. The number of movements in the 1750ppm group was significantly reduced in Block 1 and 4 (males) and in Block 4 (females) on day 62 pp. The value for males was within the historical control range while the value for females was just outside the historical control range.

The magnitude of startle response to the auditory stimulus was significantly reduced for female pups in the 1750ppm group on day 23 pp. The response magnitude was significantly reduced in Block 2 for the 500ppm group females on day 23 pp. This statistically significant reduction was not considered to be treatment-related because it was an isolated event and did not persist. There were no treatment-related effects on magnitude of the auditory startle response in male pups at any dose level, either at day 23 or day 63 pp, or in female pups at any dose level at day 63 pp.

Treatment-related minor and transient reductions in motor activity occurred only at developmentally toxic dose levels and in the absence of adverse histomorphological and morphometric changes in the brain. Therefore, a no-observed-adverse-effect-level (NOAEL) for developmental neurobehavioural effects was established as 1750ppm, equivalent to dose levels of 142 and 299 mg/kg bw/day during gestation and lactation, respectively.

Additional Comments None

The observed morphometric changes in the brain occurred only at Conclusion the highest dose and were within or just outside historical control ranges. The reductions in motor activity were transient and in most

November 2015 113

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

cases within historical control ranges. The change in startle response was also isolated in nature.

Medical surveillance on manufacturing plant personnel A report on medical surveillance of workers involved in the manufacture of clothianidin technical material was provided28. The report covers the period 2003 to 2015, with 50 workers engaged in clothianidin manufacture each year. Occupational medical surveillance of workers exposed to clothianidin, performed annually on a routine basis, did not reveal any adverse effects in the workers. In addition, no untoward effects of clothianidin have been reported by the workers engaged in clothianidin manufacturing.

General conclusion about mammalian toxicology of clothianidin The new sensitisation study provided for clothianidin confirms that this active ingredient is not a skin sensitiser. The phototoxicity study indicates that clothianidin does not have photoxic potential.

The repeated dose toxicity/immunotoxicity study demonstrates that clothianidin does not adversely affect the functional ability of the humoral immune component of the immune system.

The review of the developmental neurotoxicity study suggests that the previous conclusion that clothianidin may cause developmental neurotoxicity at doses of 142 mg/kg bw/day could be revised, with this dose level becoming the NOAEL, based on the observed effects being within or close to historical control ranges.

Overall, the new data provided do not indicate that there is a need to revise the hazard classifications for clothianidin, or to use a different AOEL to that established in the previous EU evaluation of this active ingredient.

Environmental fate

The applicant used the code TI-435 for the substance clothianidin. The studies summarised in this chapter were provided by the applicant in complement to the EFSA Draft Assessment report (2011).

In these tables, the full names of metabolites are:

TMG thiazolylmethylguanidine

28 Steffens W. 2015 Medical surveillance of manufacturing personnel: Clothianidin technical material. Bayer CropScience AG, GQHSE,

Alfred-Nobel-Str.10, 40789, Monheim, Germany. Report no: M-507293-01-1.

November 2015 114

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

MNG methylnitroguanidine

TZNG thiazolylnitroguanidine

TZMU N-(2-chlorothiazol-5-ylmethyl)-N'-

or methylurea TZM

MU methylurea

TZFA N-({[(2-chloro-1,3-thiazol-5-

yl)methyl]amino}methylene)methanaminium chloride; code BCS-CQ88479

NTG 1- Nitroguanidine

CTCA clothianidin-2-chlorothiazole-5-caboxylic

acid

HMIO clothianidin-4-hydroxy-2-methylamino-2-

imidazolin -5-one

Water compartment

Ready biodegradation

Type of study Ready biodegradation

Flag Key study

Test Substance TI-435 metabolite MNG (N-methylnitroguanidin)

November 2015 115

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Species Mixed population of aquatic microorganisms (activated sludge)

Endpoint biodegradation

Value Not readily biodegradable

Caspers, Muller (2000) Investigation of the ecological properties of N- Reference methylnitroguanidin. Bayer AG Institute of Environmental Analysis Leverkusen (Germany). Report no 964 N/00, Bayer ID M-087690-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Commission Directive 92/69/EEC Official Journal of the EC L383 A Part C 4-C Test Guideline/s CO2 Evolution test 1992 (comparable with OECD 301)

Dose Levels Initial concentration 84.95 mg test substance/L

Analytical measurements CO2 formed

A study was performed to assess the ready biodegradability of N- Methylnitroguanidine (MNG) a metabolite of clothianidin. A solution of MNG in a mineral medium was inoculated and incubated for 28 days under aerobic Study Summary conditions. During this period the biodegradation is determined by carbon dioxide produced from the test substance. On day 28, 7% degradation was observed. Consequently MNG is not readily biodegradable.

Conclusion MNG is not readily biodegradable

Biodegradation in natural water

Type of study Rate of degradation in aquatic system

Flag Key study

Test Substance [thiazolyl-14C]TI-435

Endpoint DT50

Value 2085 days (stable)

S.Swales (2000) [14C]TI-435 Aerobic aquatic biotransformation. Covance Reference Laboratories, Harrogate, North Yorkshire, England. Report no 110250, Bayer ID M-107294-01-1.

Klimisch Score 1

Amendments/Deviations None that impacted the results

GLP Yes

November 2015 116

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Canada PMRA T-1-255 6.2C.2 DACO no 8.2.3.5.2: Biotransformation in Test Guideline/s aerobic water, US EPA 162-4

Dose Levels Initial concentration 100 µg test substance/L

Analytical measurements HPLC / TLC

The rate of degradation of [14C]TI-435 has been studied in an aquatic system under aerobic conditions at 20 oC in the dark. Natural water of a Canadian pond in Saskatchewan was used. [14C]TI-435 (9.66 µg) in acetonitrile was dispensed dropwise onto the surface of the water. The application rate is equivalent to an overspray rate of 300 g/ha distributed to a depth of 30 cm. Recovery of applied radioactivity ranged from 96 to 98%. The majority of the applied radioactivity (>95%) was present in the water. Study Summary TI-435 was stable under the test conditions and negligible degradation was

observed after 181 days. The calculated DT50 and DT90 values were 2085 and 6927 days respectively using a single phase exponential model (first order kinetics). The duration of the study was not sufficient to enable accurate values to be determined. An unidentified degradation product was present after 181 days at a level of 1% of applied radioactivity. No extractable degradation products were detected prior to 181 days and only small amounts of volatile degradation products (max. 2%) were formed.

Conclusion DT50 = 2085 days, clothianidin is stable in the water phase.

Type of study Biodegradation in surface water

Flag Key study

Test Substance [14C]clothianidin

Endpoint DT50

Value stable

D. Adam (2014) [14C] clothianidin- aerobic mineralisation in surface water- Reference simulation biodegradation test. Innovative Environmental Services Ltd Witterswil Switzerland. Report no 20130130, Bayer ID M-499688-01-1.

Klimisch Score 1

Amendments/Deviations None that impacted the results

GLP Yes

Test Guideline/s OECD 309

Dose Levels Initial concentration 0.1 and 0.01 mg test substance/L

November 2015 117

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Analytical measurements HPLC /LSC

Aerobic mineralisation of [14C]Clothianidin in surface water was investigated under laboratory conditions in the dark. For this purpose the radiolabelled test item was applied to 100 mL of natural pond water at concentrations of 0.10 and 0.010 mg/L. Additionally, the high concentration experiment was performed under sterile conditions in order to gain information about abiotic degradability of the test item. The test flasks were incubated for a period of 60 days at 20.5 ± 0.3°C under aerobic conditions by gently stirring the water. Radiolabelled benzoic acid was used as reference substance to check the sufficiency of microbial activity of the test water. Sufficient activity is reached if at least 90% of the reference substance degrades within 14 days of incubation. At each sampling interval, i.e. after 0, 3, 7, 14, 28, 41 and 60 days of incubation, per system, duplicate samples (replicate A and B) were removed and processed. After sampling, pH and oxygen concentration in the water were determined together with the total radioactivity present in the water layer and in the volatile traps. Aliquots of the water samples were then analysed, after a concentration step, by HPLC for parent and eventual metabolites. Total mean recoveries were 102.0 ± 1.0% of applied radioactivity (AR) for the Study Summary high dose, 102.3 ± 1.8% AR for the high dose sterile and 100.4 ± 2.0% AR for the low dose experiment. Immediately after treatment (time 0), mean values of 101.0%, 103.5% and 100.2% AR were measured in the water phase of the high dose, high dose sterile and low dose system, respectively. After 60 days of incubation, the mean amount of radioactivity in the water phase of the respective systems remained stable and represented 103.0%, 101.6% and 97.2% AR. Correspondingly, mean values for formation of radioactive carbon dioxide represented 0.5% AR in the high dose, 0.2% AR in the sterile and 1.0%

14 AR in the low dose system after 60 days. Volatile products other than CO2 did not exceed 0.1% AR throughout the study. The reference substance benzoic acid degraded completely from initially 102.8% to 0% AR within 7 days of incubation indicating high microbial activity in the test water, whereas analysis of solvent control samples demonstrated that the amount of solvent had no negative effect on the degradation rate. In all systems, Clothianidin was shown to be stable until the end of incubation (60 days), accounting for 103.0%, 101.6% and 97.2% AR (mean values) in the respective systems. In conclusion and regardless of the test concentration, Clothianidin was degraded insignificantly under the conditions of the test.

Conclusion Clothianidin is stable in the water phase

November 2015 118

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Water/sediment systems

Type of study Route and rate of degradation in aquatic system

Flag Key study

Test Substance [thiazolyl-2-14C]clothianidin

Endpoint DT50

Value 170 days (High rate) and 156 days (low rate) whole system

R.J. Ripperger (2006) [Thiazolyl-2-14C]clothianidin Aerobic aquatic metabolism. Reference Bayer Cropscience Environmental Research Stilwell Kansa USA. Report no 05METIX052 Bayer ID M-270178-01-2.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s US EPA 162-4

0.05 mg ai/L and 0.15 mg ai/L. The higher rate approximates a field-use rate of Dose Levels 448.3 g a.i./ha if applied by direct overspray of a water body to a depth of 30 cm. The rate of 0.05 mg/L closely approximates a field-use rate of 150 g a.i./ha.

Analytical measurements HPLC / LC-ESI/MS

The degradation kinetics of [thiazolyl-2-14C]clothianidin has been studied in an aquatic system under aerobic conditions at 20 oC in the dark. The water phase of the water/sediment system had a pH of 7.2 and dissolved oxygen carbon of 7.3 ppm, the sediment was loam with a pH of 6.3 and organic carbon of 0.9%. The sediment/water ratio used was 1:4 (v/v). Clothianidin was applied at a rate of 0.05 mg ai/L (low) and 0.15 mg ai/L (high rate). Samples were analysed at 0, 3, 7, 14, 30, 60, 91 and 120 days of incubation. The water samples were not extracted. The sediment samples were extracted with acetonitrile using a shaking method. Study Summary High rate The total material balance in the water/sediment system was 98.4 ±1.2% of the applied amount. Extractable [14C]residues in sediment increased from 0.7% at day 0 to 50.4% of the applied by day 91. Non extractable [14C]residues in sediment increased from 0.0% at day 0 to 7.9% at day 91. At the end of the

study 4.0% and 0.0% of the applied radioactivity were present as CO2 and organic volatile compounds respectively. The concentration of clothianidin in water decreased from 99.1% at day 0 to 38.9% of the applied amount at the study termination. The concentration of clothianidin in the sediment increased from 0.0% at day 0 to 32.5% of the

November 2015 119

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

applied amount at the end of the study. At the end of the study 53.3% of the applied radioactivity was partitioned from water to sediment. No major transformation products were detected in water. The major transformation product detected in sediment was TMG with maximum concentrations of 24.1% on day 91 of incubation. In sediment 13.8% was detected at the end of the study. The minor transformation products in water were TZMU and TMG formed at a maximum of 1.4% and 0.6% of the applied amount, respectively. No minor transformation products were found in sediment. The unidentified 14C during the study was 0.0% of the applied amount. The half-lives decline time of clothianidin in aerobic water, sediment and in the entire system were 55, 51 and 170 days respectively (following first order). Low rate The total material balance in the water/sediment system was 96.8 ±1.4% of the applied amount. Extractable [14C]residues in sediment increased from 0.8% at day 0 to 51.1% of the applied by day 91. Non extractable [14C]residues in sediment increased from 0.0% at day 0 to 9.8% at day 120. At the end of the

study 25.1% and 0.0% of the applied radioactivity were present as CO2 and organic volatile compounds respectively. The concentration of clothianidin in water decreased from 96.6% at day 0 to 30.1% of the applied amount at the study termination. The concentration of clothianidin in the sediment increased from 0.0% at day 0 to 33.4% of the applied amount at day 91 and then decreased to 29.9% at the end of the study. At the end of the study (120 day) 59.5% of the applied radioactivity was partitioned from water to sediment. No major transformation products were detected in water. The major transformation product detected in sediment was TMG with maximum concentrations of 19.8% on day 120 of incubation. The minor transformation products in water were TZMU and TMG formed at a maximum of 1.0% and 2.1% of the applied amount, respectively. No minor transformation products were found in sediment. The unidentified 14C during the study was 0.0% of the applied amount. The half-lives decline time of clothianidin in aerobic water, sediment and in the entire system were 50, 47 and 156 days respectively (following simple first order).

In an aerobic aquatic system clothianidin transitions to the sediment

Conclusion phase and degrades to TMG, CO2 and sediment bound residues.

Whole system DT50 = 170 days high rate, 156 days low rate

November 2015 120

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Soil compartment

Migration in soil

Type of study Leaching potential of treated seed

Flag Key study

Test Substance [thiazolyl-2-14C]clothianidin

Endpoint Leaching potential

Value low

E.L.Arthur, J. Shepherd, A.R.Dominic (2006) [Thiazolyl-2-14C]clothianidin Seed Reference leaching study. Bayer Cropscience Environmental Research Stilwell Kansa USA. Report no METIX050-1 Bayer ID M-270224-02-2.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guidelines None available

Dose Levels 1.17 mg ai/seed

Analytical measurements HPLC

The purpose of the study was to determine the mobility of clothianidin applied as seed treatment to corn. The study was conducted in a greenhouse with a silt loam soil with pH of 6.1 and OC of 1.8%. The applied dose rate is 1.17 mg ai/seed. The minimum target rainfall to achieve in this study was the 30-year maximum monthly precipitation for Branchton, Ontario. The precipitation targets were exceeded up to six times to compensate for evapotranspiration and to generate sufficient leachate. To create leaching events, an excess of deionized water was added slowly to soil columns over several days (to minimize flooding of the Study Summary soil surface). At four intervals (2, 4, 8, and 16 weeks post corn spike emergence), two soil columns were sacrificed and analysed for radioactive residues in plant and soil. Plant material was removed from the surface of the column, weighed and combusted to determine total radioactivity within the plant. Soil columns were frozen and then cut into ca. 15 cm increments. For the first eight leaching events, radioactivity was below the limit of detection. On the ninth leaching event, 0.05% of the applied radioactivity was leached, which was the maximum amount leached for any event. The amount declined to <0.01% by the last leaching event. The cumulative percentage of applied radioactivity leached in this study was 0.17%. Trace levels of thiazolyl

November 2015 121

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

methylurea and an unidentified polar degradate were detected in the leachates (maximum amount of an individual degradate was 0.016%). The mean material balances at 2, 4, 8 and 16 week intervals were 116.1, 91.8, 85.7 and 80.5%, respectively. The percentage of applied radioactivity in seeds was highest at the 2-week interval at 54.9%, declined to 28.7% by 4-weeks and continued to decline to 0.5% by the end of the study. Radioactive residues in the plants ranged from 1.1% at the 2-week interval to a maximum of 5.7% by the end of the study. Residues in the roots were 0.4% at the 2-week interval, increased to 2.7% at 4 weeks, and declined to 0.9% at the end of the study. Total residues in seed, plant and roots ranged from 56.4% at the 2-week interval to approximately 7% by the 8 and 16-week intervals. The majority of radioactive residues were found in the surface soil (0-15 cm), with 59.8% at the 2-week interval, increasing to 76.2% at the 8-week interval and then declining to 70.2% by the end of the study. No residues were found below the 15-cm depth for the 2-week interval and only a trace amount (<0.05%) in the 15- to 30-cm depth at the 4-week interval. At 8 and 16 weeks, 1.5 and 1.8% of the applied radioactivity, respectively, were present in the 15- to 30-cm depth; 0.7 and 1.0% of the applied radioactivity, respectively, were determined to be in the 30- to 46-cm depth; and 0.3 and 0.7% of the applied radioactivity, respectively, were determined to be in the 46- to 61-cm depth. Only the surface soil extracts contained enough radioactivity to analyse by HPLC, and analyses revealed only clothianidin present in soil extracts. Linear and nonlinear regression analysis for clothianidin in soil resulted in a poor fit (r2 = 0.01 and 0.04, respectively), since clothianidin was released from the seed as the seed decayed in soil, resulting in an increase in the amount of radioactivity in soil over time. Thus, there was an increase in the amount of radioactivity in

soil over time. As a result, a DT50 value for total residues in the soil plus seed was calculated using nonlinear degradation kinetics and determined to be 165 days (k = 0.0042 day-1; r2 = 0.66). The majority of radioactive residues remained in the top 15 cm of soil, with some uptake into the plant. Residues showed very limited movement through soil columns under extremely moist soil conditions. Unextractable residues from soil increased over time, indicating increased sorption as the compound aged in soil. Following addition of approximately 130 cm of water over 4 months, only 0.17% of the applied radioactivity moved through the 61-cm soil column and was detected in the leachate. The amount of radioactivity in the leachate declined over time, indicating that the remaining residues became more tightly bound to soil, thus reducing the potential for additional residues to leach into groundwater.

November 2015 122

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Remark Application rate in New Zealand is 0.76 mg/seed

The majority of radioactive residues remained in the top 15 cm of soil with Conclusion some uptake into the plant. Residues showed limited movement through soil columns under extremely moist soil conditions.

Type of study Batch equilibrium study

Flag Key study

Test Substance [imidoformamide-14C] BCS-CQ88479 (= metabolite of clothianidin; TZFA)

Endpoint Koc

Value Ranged from 212 to 1216 with a mean of 560.

HP Stupp (2011) [imidoformamide-14C] BCS-CQ88479: adsorption/desorption Reference in five different soils. RLP AgroScience GmbH, Neustadt a.d. Weinstrasse Germany Report no AS157 Bayer ID M-407208-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the study

GLP Yes

EC, CD 95/36/EC amending council directive 91/414, OECD 106 (2000), Test Guidelines OPPTS 835.1220, PMRA environmental chemistry and fate guidelines for registration of pesticides in Canada 1987

Dose Levels 1.0, 0.3, 0.1, 0.03, 0.01 mg test substance/L nominal

Analytical measurements HPLC / LSC

The adsorption/ desorption properties of [imidoformamide-14C] BCS-CQ88479 were determined in five soils using a batch equilibrium method. The main properties of the soils are mentioned in the table below. Adsorption isotherms were evaluated using freundlich adsorption isotherm. The Parental Mass Balance after 24 h, showed that 92.8-97.9% of applied [imidoformamide-14C] BCS-CQ88479 could be recovered. After 96 h 90.7 – Study Summary 95.3 % of the applied [imidoformamide-14C] BCS-CQ88479 could be recovered. The overall material balance for all concentrations for individual specimens was in the range of 94.3-96.9%, 92.3-95.5%, 78.2-93.9%, 93.7-96.2%, and 92.0- 94.6% of the applied radioactivity in soils Wurmwiese, Höfchen am Hohenseh, Dollendorf II, Guadalupe, Springfield, respectively. In the definitive adsorption test 30.5-47.7%, 42.0-58.3%, 52.0-70.1%, 37.3- 51.9%, and 69.7-81.7% of the applied test material was adsorbed in soils

November 2015 123

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Wurmwiese, Höfchen am Hohenseh, Dollendorf II, Guadalupe, Springfield, respectively.

The calculated adsorption constants KF is shown in the table. The concentration of the test item did affect the adsorption.

The desorption KF ranged from 7.3 to 28.3 and the KF,OC ranged from 282.0 to

1661.8. The mean desorption KF,OC was therefore 1.5 times higher than those obtained for adsorption phase.

Soil Origin Wurmwie Hofchen Dollendo Guadalupe, Springfield, se, am rf II, CA USA NE USA Germany Hohenseh Germany , Germany Soil type loam Silt loam Clay Sandy Silt loam loam loam % organic 1.76 2.42 4.72 0.7 1.7 carbon pH 5.3 6.6 7.3 6.7 6.6 % WHC Adsorption 4.5 7.0 10.0 5.8 20.7 KF mL/g 1/n 0.8719 0.8780 0.8546 0.8829 0.8730 Adsorption 255.5 288.8 212.1 826.8 1216.3 KFoc mL/g

The mobility of the test substance can be classified to have a moderate to slight mobility according to FAO criteria.

Koc ranged from 212 to 1216 with a mean of 560. Conclusion The mobility of the test substance can be classified to have a moderate to slight mobility according to FAO criteria.

Degradation in soil

Type of study Dissipation study

Flag Key study

Test Substance [guanidine-14C]clothianidin

Endpoint Sorption and DT50

The sorption of clothianidin to soil increased significantly in the course of the Value incubation and less substance will be available for the environment.

November 2015 124

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

DT50 in soil is 65.1 days (geomean), individual values: 9.7, 86.9, 174.6, 122.0 days.

HP Stupp (2006) [Guanidine-14C]clothianidin Time dependent sorption from four European field dissipation soils. Bayer Cropscience Development Reference Environmental Safety Metabolism/ADME and Environmental Fate, Monheim am Rhein, Germany. Report no MEF 10/563 Bayer ID M-405885-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guidelines Parts of OECD 106 (2001) and OECD 307 (2002)

Dose Levels 20 µg ai/ 100 g soil corresponding to 150 g ai/ha

Analytical measurements HPLC / LSC for CaCl2 and LC-MS/MS for transformation products

The time-dependent sorption of [guanidine-14C]clothianidin was studied in four soils; silt loam in Germany (pH 5.7 OC% 1.1), sandy loam in UK (pH 6.1, OC% 0.8), clay loam in France (pH 7.7, OC% 0.8) and sandy loam in Spain (pH 6.1, OC% 0.8). Clothianidin was applied at a dose rate of 150 g ai/ha (maximum field use rate) corresponding with 20 µg ai/ 100 g soil. Samples were analysed after 0, 1, 3, 9, 21, 28, 35, 49, 63, 877, 98 and 120 days after incubation. Extractable 14C-residues decreased from 99.0%, 102.0%, 101.0%, and 100.9% of the applied amount at DAT-0 to 37.7%, 61.8%, 71.2% and 63.6% of the applied RA at the end of the study in soils Germany, UK, France and Spain respectively. The formation of bound residues increased with the overall metabolism of test compound. The non-extractable residues at DAT-0 (day after treatment) varied from 2.2% – 3.0% of AR and were generated during the Study Summary 24h lasting desorption step. At study termination they increased to 30.8%, 20.2%, 18.1%, and 19.8% of AR (applied radioactivity) in soils Germany, UK, France and Spain, respectively. They were separated into humin, humic acid and fulvic acid fractions for the last sampling interval. The test item Clothianidin degraded moderately fast. Its amount in the extracts decreased from 96.9- 99.8% on DAT-0 to 21.5- 60.8% of AR after incubation for 120 days. In all soils the degradation behaviour of Clothianidin was best described using biphasic models. In soils Germany, France and Spain, the degradation followed double first order in parallel kinetics (DFOP) according to

the lowest chi2 values. For the UK soil, the degradation was slightly better described using the FOMC (first order multi compartments) kinetic model. The calculated half-lives were in the range of 9.7 to 174.6 days (geometric mean: 65.1 days).

November 2015 125

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Besides carbon dioxide six degradation products were detected and identified in the course of the incubation: TZNG, MNG, TZMU, TMG, NTG and TZFA. The maximum percentages of AR are stated in the table below. In addition several minor transformation products were observed. Their sum at the different sampling times accounted for ≤ 2.4% (Germany), 2.3% (UK), 1.8% (France) and 2.1% (Spain) of the applied radioactivity. At the end of the study at DAT-

14 120, evolved CO2 accounted for 29.3%, 16.2%, 9.4%, and 15.9% of the applied RA in soils in Germany, UK, France and Spain, respectively. Only once a very small amount of volatile organic compounds was measured in the polyurethane foam (Replicate A, DAT-120, 0.3% of AR). The sorption of

Clothianidin to soil increased in the course of the study. The calculated RTDS values (distribution coefficient for time dependent sorption) were 1.3, 0.9, 0.6 and 0.8 mL/g for soils Germany, UK, France and Spain, respectively, at the beginning of the study (DAT-0). With time of aging in soil, these values increased to 4.8, 2.5, 1.6, and 2.5 mL/g on DAT-120 for the four soils. Metabolite TZNG was formed by demethylation of the test item. Metabolite MNG was formed by cleavage of the thiazolyl-moiety. Metabolite NTG was either formed by demethylation of MNG or by the cleavage of the thiazolyle moiety of TZNG. Metabolites TMG, TZMU and TZFA are characterized by a

loss of the NO2 moiety. The amount of formed carbon dioxide indicates the complete mineralization of Clothianidin and/or its transformation products in soil. soil Germany UK France Spain Type Silt loam Sandy loam Clay loam Sandy loam

DT50 9.7 days 86.9 days 174.6 days 122.0 days clothianidin TZNG (%AR) Max. 1.0% Max. 3.0% Max. 2.1% Max. 1.9% MNG (%AR) Max. 6.4% Max. 6.8% Max. 5.3% Max. 4.5% TZMU (%AR) Max. 10.6% Max. 5.9% Max. 2.2% Max. 4.5% TMG (%AR) Max. 0.8% Max. 0.3% Max. 0.3% Max. 0.4% NTG (%AR) Max. 2.8% Max. 3.1% Max. 2.1% Max. 3.5% TZFA (%AR) Max. 6.7% Max. 2.8% Max. 0.7% Max. 1.3% Increase of 3.7 2.7 2.6 3.1 sorption DAT-120/ DAT0

The sorption of clothianidin to soil increased significantly in the course of Conclusion the incubation. Therefore with time of aging in soil less substance will be

November 2015 126

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

available for the environment. The DT50 in soil are 9.7, 86.9, 174.6, 122.0

days. The high amount of CO2 indicates complete mineralisation.

Type of study Aerobic degradation study

Flag Key study

Test Substance BCS-CQ88479 (= metabolite of clothianidin-TZFA)

Endpoint DT50

Value 15.6, 22.1 and 65.5 days

W. Volkel (2011) BCS-CQ88479 (clothianidin –TZFA) degradation rate in three Reference soils incubated under aerobic conditions. Innovative Environmental Services (IES) Ltd, Witterswil Switzerland. Report no 10501023 Bayer ID M-420734-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

OECD 307, CD 95/36/EC 1995 Annex II 7.1.1.2 rate of degradation, 7.1.1.2.1. Test Guidelines laboratory studies- aerobic degradation

Dose Levels 0.1 mg test substance/ kg dry soil (corresponding to 100 g test substance/ha)

Analytical measurements HPLC / MS/MS

The rate of degradation of the test item TZFA was investigated in three different soils in Germany. The main properties of the soils are stated in the table below. The test substance was applied at a test concentration of 0.10 mg/kg dry weight soil equivalent to an application rate of 100 g/ha in the field. The soil samples were incubated under aerobic conditions in the laboratory at 20°C and soil moisture corresponding to 55% of the maximum water holding capacity in the dark. Duplicate samples were removed for analysis at 0, 3, 7, 14, 28, 60, 91 and 120 days after treatment (DAT). The soil samples were extracted with Study Summary acetonitrile/aqueous acetic acid (4:1 v/v) followed by LC-MS/MS analysis of the combined extracts for determination of residues of TZFA. While concurrent recoveries were in the acceptable range (73.2% to 87.4% for Wurmwiese, 70.7% to 89.0% for Hoefchen am Hohenseh, and 73.1% to 92.0% for Dollendorf II) for most sampling intervals, lower ones were observed at days 28 and 60 (soil Wurmwiese) and at days 60 and 120 (soil Hoefchen am Hohenseh). Analysis was repeated to result in confirmation of lower recoveries at those sampling intervals. It was therefore decided to correct the results of all

November 2015 127

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

sampling intervals by concurrent recoveries from fortified samples generated the same day. Following incubation the concentration of TZFA in treated samples decreased from 100% to values of 32.0% (Wurmwiese), 10.5% (Hoefchen am Hohenseh) and 1.4% for soil Dollendorf II after 120 days.

The best fit DT50 and DT90 values of TZFA were calculated on the basis of Double-First-Order in Parallel (DFOP) kinetic model for Wurmwiese, First-Order Multi-Compartment (FOMC) for Hohenseh and Single First-Order (SFO) for Dollendorf II. The values are in the table below.

Location Wurmwiese Hoefchen Dollendorf II am Hohenseh Soil type Loam Silt loam Clay loam % organic carbon 1.8 2.3 4.2 pH water 5.2 6.6 7.2

pH CaCl2 4.9 6.4 7.1 Max WHC (g 54.8 63 83 water ad 100 g DM) %organic matter 3.1 4.1 7.2 Model DFOP FOMC SFO

DT50 65.5 22.1 15.6

DT90 277.3 123.0 51.8

χ2 error 1.6 4.3 6.9 R2 0.992 0.992 0.987

Conclusion DT50 were15.6, 22.1 and 65.5 days

Type of study Soil dissipation

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Endpoint DT50

Value 140, 170, 108 and 64 days

O. Heinemann (2012) Determination of the residues of clothianidin in/on soil after spraying of clothianidin FS 600B G in the field in Germany, United Reference Kingdom, France (South) and Spain. Bayer Cropscience Development BCS-D- EnSa-testing Monheim, Germany. Report no 10-2700 Bayer ID M-443993-01-1

November 2015 128

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 1

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guidelines BBA guideline part IV 4-1 (1986), SETAC 1995

Dose Levels 0.25 L product/ha (150 g ai/ha nominal)

Analytical measurements HPLC- MS/MS

Soil dissipation of Clothianidin (= TI-435) under European field conditions was investigated after application of Clothianidin FS 600B G on bare soil plots at four sites in Burscheid (Germany), Wellesbourne (United Kingdom), St. Etienne du Gres (Southern France), and Vilobi d’Onyar (Spain). Clothianidin FS 600B G was sprayed once pre-emergence at a nominal rate of 0.25 L/ha, corresponding to nominal 150 g Clothianidin/ha. Before incorporation of the product into the soil the initial zero-time concentrations corresponded to 82 % to 93 % of the intended dose rate. The control plots were at least 5 m away from the treated plots. Soil samples were taken 0 days before application to 737 days post- application to a maximum depth of 100 cm, homogenized and analysed for Clothianidin and its metabolites TI-435-TZFA, TI-435-NTG, TI-435-TZNG and TI-435-MNG. The limit of quantitation (LOQ) was 5.0 μg/kg and the limit of detection (LOD) was 1.5 μg/kg for each analyte. The kinetic model DFOP (double first order in parallel) was the best fit. At Burscheid (silt loam, Germany), the mean amount of Clothianidin determined Study Summary in 0-10 cm at day 0 was 140 g/ha, representing 93 % of the nominal application rate. Clothianidin declined from 140 g Clothianidin/ha in soil at day 0 to 31.5

g/ha at day 713. This corresponds to a best fit DT50 value of 140 days, and a

DT90 value of >1000 days. At Wellesbourne (sandy loam, United Kingdom), the mean amount of Clothianidin determined in 0-10 cm at day 0 was 124 g/ha, representing 82 % of the nominal application rate. Clothianidin declined from 124 g Clothianidin/ha in

soil at day 0 to 43.3 g/ha at day 737. This corresponds to a best fit DT50 value of

170 days, and a DT90 value of >1000 days. At St. Etienne du Gres (silt loam, Southern France), the mean amount of Clothianidin determined in 0-10 cm at day 0 was 128 g/ha, representing 85 % of the nominal application rate. Clothianidin declined from 128 g Clothianidin/ha in

soil at day 0 to 28.1 g/ha at day 721. This corresponds to a best fit DT50 value of

108 days, and a DT90 value of >1000 days. At Vilobi d’Onyar (loam, Spain), the mean amount of Clothianidin determined in 0-10 cm at day 0 was 132 g/ha, representing 88 % of the nominal application

November 2015 129

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

rate. Clothianidin declined from 132 g Clothianidin/ha in soil at day 0 to residues

below the LOQ at day 713. This corresponds to a best fit DT50 value of 64 days,

and a DT90 value of 534 days. Residues of Clothianidin remained in the top 0-10 cm of soil, except for some very low concentrations at or below the LOQ level in the 10-20 cm soil layer.

Dissipation of Clothianidin from soil was moderate with DT50 values ranging from 64 days to 170 days for all test sites. Dissipation and degradation of Clothianidin was accompanied by the formation of its metabolites TI-435-TZNG and TI-435-MNG. These metabolites were detected in the top 0-10 cm of soil at very low maximum concentrations of 6.5 g/ha and 5.1 g/ha, respectively. The metabolites TI-435-TZFA and TI-435-NTG were not detected above the LOD.

Conclusion DT50 is 140 , 170, 108, 64 days

Anaerobic degradation

Type of study Anaerobic degradation study

Flag Key study

Test Substance [thiazolyl-2-14C]clothianidin and [guanidine-14C]clothianidin

Endpoint DT50

Value 31.6 and 24.8 days for guanidine and thiazolyl label respectively

H.P. Stupp, D.Kasel (2014) [thiazolyl-2-14C]clothianidin and [guanidine- 14C]clothianidin: anaerobic degradation/ metabolism in soil. Bayer Cropscience Reference AG BCS-D-EnSa-Testing, Monheim Germany. Report no EnSa-14-0777 Bayer ID M-499004-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guidelines OECD 307

Dose Levels 213 µ g test substance/ kg dry soil

Analytical measurements HPLC/radiodetection and LSC

The route and rate of degradation of [thiazolyl-2-14C]clothianidin and [guanidine- 14C]clothianidin were studied under anaerobic conditions for 120 days. The soil used is a silt loam with a pH of 6.2 and %OC of 1.9 from Germany. The test Study Summary substance was applied at a rate of 213 µg/kg dry weight soil based on a maximum single field rate of clothianidin of 80 g/ha. The soil samples were incubated under aerobic conditions in the dark at 54.8% of the maximum

November 2015 130

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

waterholding capacity for 14 days. Then the system was flooded to achieve anaerobic conditions. Duplicate samples were removed for analysis at 0 and 14 days during the aerobic phase and at 0, 3, 7, 13, 29, 63, 93 and 120 days after soil flooding (DASF). Mean material balances were 98.1% AR for the guanidine-label (range from 92.9 to 102.4% AR) and 100.5% AR for the thiazolyl-label (range from 97.6 to 104.7% AR). The amount of carbon dioxide formed during the aerobic incubation phase (mean value of DAT-14 and DASF-0) was 7.8% AR (guanidine-label) and 10.7% AR (thiazolyl-label). The amount of carbon dioxide formed during the anaerobic incubation phase was ≤ 0.1% AR for both labels. Formation of volatile organic compounds (VOC) during the aerobic and anaerobic incubation phases was insignificant as demonstrated by values of ≤ 0.1% AR at all sampling intervals for both labels. Extractable residues decreased from DAT-0 to DASF-120 (DAT-134) from 99.2 to 47.2% AR for the guanidine-label and from 99.1 to 49.7% AR for the thiazolyl-label. Non-extractable residues (NER) increased during the aerobic incubation phase from DAT-0 to DAT-14 from 3.2 to 10.8% AR for the guanidine-label and from 3.7 to 11.5% AR for the thiazolyl-label. During the following anaerobic incubation phase, NER further increased until DASF-120 to 38.6 and 37.1% AR for guanidine-label and thiazolyl-label, respectively. Within the aerobic incubation phase, the amount of clothianidin decreased from DAT-0 to DAT-14 from 99.2 to 58.8% AR for the guanidine-label and from 99.1 to 58.5% AR for the thiazolyl-label. During the following anaerobic incubation phase, the amount of clothianidin further decreased to 12.6% AR (guanidine- label) and 13.2% AR (thiazolyl-label) at DASF-120. Besides the formation of carbon dioxide, three degradation products were identified with the following maximum occurrences: TZMU with 9.2% AR (anaerobic, DASF-120) and 5.2% AR (aerobic, DAT-14) for the thiazolyl-label, TZFA with 18.8% AR (anaerobic, DASF-29, thiazolyl-label) and 10.2% AR (aerobic, DAT-14, guanidine-label), TMG with 22.5% AR (anaerobic, DASF-63) for the guanidine-label. For the aerobic incubation phase the amount of TMG was < LOD for both labels. The total unidentified residues amounted to a maximum of 6.3% AR and no single component exceeded 4.4% AR at any sampling interval for both labels during the entire study. The experimental data could be best described by a double first order in parallel (DFOP) kinetic model. The half-life of clothianidin after flooding under anaerobic conditions was between 24.8 and 31.6 days for both labels.

November 2015 131

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Conclusion DT50 = 24.8 and 31.6 days, guanidine-label and thiazolyl-label respectively

Figure 1 Aerobic degradation of clothianidin in soils

Accumulation studies

Type of study Residues in/on soil after seed treatment over 8 years

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin 600 FS

Endpoint Residues in 0-30 cm soil layer

Maximum amount ranged from 25.9 μg clothianidin /kg soil up to 48.5 μg Value clothianidin /kg soil after 2 and 5 years, respectively.

November 2015 132

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

O. Heinemann, M. Telscher (2009) Determination of the residues of TI-435 in/on soil, winter wheat and durum wheat after seed treatment of TI-435 Reference (600FS) in Germany, Southern France and Great Britain. Bayer Cropscience Development Environmental Safety Metabolism/ADME and Environmental Fate Monheim am Rhein, Germany. Report no RA-2147/00 Bayer ID M-356604-02-1

Klimisch Score 1

Amendments/Deviations NA

GLP Yes

Test Guidelines EU requirements

Dose Levels 250 mL or 133 mL product/ha (= 150 g a.i./ha or 80 g a.i./ha)

Analytical measurements HPLC-MS/MS

The purpose of the study was to determine the magnitude of residues of clothianidin and its degradation products MNG, TZNG in soil after repeated drilling of seed dressed with TI-435 600 FS in Europe. The study was carried out over a period of 8 years. Four field trials were conducted; one trial in Southern France (silt loam, pH 8.55, OC% 0.88), one trial in Germany (silt loam, pH 6.92, OC% 0.94) and two parallel trials in Great Britain (both sandy loam, pH 6.96, OC% 0.76). TI-435 600 FS was used with a dressing rate of 250 mL product per hectare for the France, Germany and one trial in Great Britain. For the second trial in Great Britain a dressing rate of 133 mL product per hectare was used. For residue analysis soil (down to 50 cm depth) and seed samples were taken. The limit of quantification (LOQ) was 5 μg/kg and the limit of detection (LOD) was 2 μg/kg Study Summary for TI-435, TZNG and MNG. Analyses of dressed seeds for Clothianidin gave dressing rates ranging from 69% to 112% of the theoretical value. An LOQ of 1.0 mg/kg was set for treated seeds. No recovery experiments were performed for the analysis of treated seeds. Maximum soil concentrations of clothianidin determined for the 0–30 cm soil layer were 41.6 μg/kg 6 years after treatment (Germany), 48.5 μg/kg 5 years after treatment (Southern France), 46.4 μg/kg 4 years after treatment (Great Britain), 25.9 μg/kg 2 years after treatment (Great Britain, reduced dressing rate). Minimum soil concentrations of clothianidin observed were 2.5 μg/kg one year after treatment (Germany), 2.5 μg/kg one and two years after treatment (Southern France), 6.8 μg/kg one year after treatment (Great Britain), and 2.5 μg/kg one year after treatment (Great Britain, reduced dressing rate) also in the 0–30 cm soil layer. Residues of the degradates TZNG and MNG have been

November 2015 133

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

mostly below the LOD or at least below the LOQ. Residues of Clothianidin in the control samples were at or below the LOQ in all cases.

Maximum amounts of residues in 0-30 cm soil layer ranged from 25.9 μg clothianidin/kg soil (after 2 years) up to 48.5 μg clothianidin /kg soil (after Conclusion 5 years) after repeated drilling of treated seeds over 8 years. The residues of the metabolites were below the LOQ.

Type of study Plant bioavailability and soil accumulation: monitoring study

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Clothianidin applied as seed treatment in normal agricultural practices, so Test Substance commercial products were used

Endpoint Soil accumulation

Value Not significant

T. Xu, D Dyer (2014) Clothianidin Plant bioavailability and soil accumulation study. Residue analysis Valent USA Corporation Trinity Court Dublin CA, field Reference sampling ARCADIS US Tallahassee Florida and Bayer Cropscience Rosthern Saskatchewan Canada. Report no METIY004, Bayer ID M-498438-01-1

2: no details on the application rates so the comparison with New Zealand Klimisch Score conditions is not possible

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guidelines No existing guideline

No detail provided, approved rates as the products were used in normal Dose Levels agricultural practice

Analytical measurements HPLC- MS/MS

To investigate the potential accumulation of clothianidin in soil and crop matrices after multiple years of planting clothianidin treated corn and canola seeds, a plant bioavailability and soil accumulation study was conducted. Fifty corn fields in the mid-western United States and 15 canola fields in western Study Summary Canada were sampled in 2012 and 2013. Soil was collected at the time of pollen development, along with corn pollen and canola nectar. Soil was analysed for the water extractable (bioavailable) fraction, to evaluate residues which may be taken up into a crop, as well as by standard extraction procedures to determine the total clothianidin residue in soil.

November 2015 134

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

For the corn sites, there was no significant accumulation of clothianidin in soil (0 to 30 cm) from fields with 2 to 11 years (mean: 4.7 years) of planting with clothianidin-treated seeds, and the bioavailable soil residue was 10% of the total soil residue. Total soil residues (extracted with methanol/water (40:60, v/v; 0.05% formic acid) were greater than the limit of quantitation (LOQ; 5 μg/kg) at

35 of the 50 corn sites, with an average concentration of 7.0 μg/kg, and 90th percentile concentration of 13.5 μg/kg. For sites having soil residues greater

than the LOQ, a soil sample was also extracted with water (0.01 M CaCl2) only, to represent the “bioavailable” concentration of clothianidin residues, and

resulted in an average concentration of 1.0 μg/kg and 90th percentile concentration of 2.1 μg/kg. Corn pollen residues were generally low, and did not show increased residues in fields from multiple years of clothianidin use. The average clothianidin

residue in corn pollen was 1.8 μg/kg, with a 90th percentile concentration of 5.4 μg/kg (pollen was only available for the 30 sites from the 2013 sampling). TZNG and TZMU were detected in only a few samples at concentrations

μg/kg and a 90th percentile concentration of 15.8 μg/kg. The bioavailable residues in soil were less than the LOQ (5 μg/kg), with an average

concentration of 0.7 μg/kg and the 90th percentile concentration of 1.5 μg/kg Canola nectar residues greater than the LOQ (1 μg/kg) were found at only 4 of the 15 canola sites, with an average clothianidin concentration of 0.6 μg/kg and

a 90th percentile concentration of 1.7 μg/kg. The clothianidin metabolites, TZNG and TZMU, were not detected in the canola nectar samples.

The results from the accumulation/bioavailability study indicate that under normal agricultural conditions, accumulation of clothianidin in soil is not significant. Results also show that clothianidin residues in pollen or nectar are primarily from the clothianidin on the treated seed from the current year’s crop, Conclusion and the low bioavailability of soil residues from historical use minimise their uptake in pollen and nectar. The results provide higher-tier data which clearly show that use of the regulatory study data will result in overly conservative assumptions about the persistence and accumulation of clothianidin in soil, the

November 2015 135

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

potential uptake of clothianidin into plants and bee-relevant matrices, and the potential for runoff or leaching to water resources.

Ecotoxicity

The applicant used the code TI-435 for the substance clothianidin.

Aquatic toxicity

Fish acute toxicity (Freshwater species)

Type of study Limit test

Flag Key study

Test Substance TI-435 technical (96% ai)

Species Lepomis macrochirus (Bluegill sunfish)

Type of exposure Static for 96 hours

Endpoint LC50

Value > 104.2 mg TI 435 technical /L

H. Wilhelmy, T. Geffke (1998) TI 435-technical, Fish (Bluegill Sunfish), Acute toxicity test 96 h- limit test. Dr. U. Noack-Laboratorium fur Angewandte Reference Biologie, D 31157 Sarstedt Germany. Report no DECO 003, Bayer ID M- 027033-02-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 203 (1992)

No/Group 1 replicate of 7 fish

Dose Levels 104.2 mg technical /L (= 100 mg ai/L) nominal

Analytical measurements Yes, HPLC at the beginning and the end of the test

The acute toxicity of the test substance to bluegill sunfish was determined in a 96-h static test. A limit test was performed with 104.2 mg technical/L. The results are expressed as nominal concentrations, recovery rates of the test Study Summary substance were > 80%. The pH values in the test medium and in the control ranged from 7.38 to 7.78, the oxygen concentration was always higher than 60% oxygen saturation (it was between 87 and 99%) and the temperature was between 21.5 and 22.7oC.

November 2015 136

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

In the control and in the test concentration, no mortality or other signs of intoxication were determined during the test period.

The LC50 is higher than 104.2 mg technical /L. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen ≥ 60% (observed 87-99%)

The LC50 is higher than 104.2 mg TI 435 technical/L. No mortality observed Conclusion at 104.2 mg TI 435 technical /L.

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Poncho (formulation 600 FS)

Species Danio rerio (Zebrafish)

Type of exposure Static for 96 hours

Endpoint LC50

Value > 170 mg test substance/L

K da Silva Coelho (2011) Poncho Fish (Danio rerio) Acute toxicity test. TECAM Reference Tecnologia Ambiental Ltda Rua Fabia, Sao Paulo Brasil. Report no 15166/2011-11.0PX, Bayer ID M-421609- 01-2.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 203 (1992)

No/Group 1 replicate of 10 fish

Dose Levels 170 mg test substance /L nominal

Analytical measurements Yes, HPLC/UV at the beginning and the end of the test

The acute toxicity of the test substance, Poncho, to Danio rerio was determined in a 96-h static test. A limit test was performed at 170 mg test substance/L. The results are expressed as nominal concentrations, the mean measured Study Summary concentration was 86% of the nominal concentration at the end of the test. The pH values in the test medium and in the control ranged from 7.49 to 7.89, the oxygen concentration was always higher than 60% oxygen saturation (it was ≥ 96%) and the temperature was between 23.0 and 24.5oC.

November 2015 137

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

In the control and in the test concentration, no mortality or other signs of intoxication were determined during the test period.

The LC50 is higher than 170 mg test substance /L. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen ≥ 60% (observed ≥ 96%)

The LC50 is higher than 170 mg test substance/L as no mortality was Conclusion observed (corresponding to > 100 mg ai/L)

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600G

Species Oncorhynchus mykiss (rainbow trout)

Type of exposure Static for 96 hours

Endpoint LC50

Value > 213 mg test substance/L

E.Bruns (2013) Acute toxicity of clothianidin FS 600 G to fish Oncorhynchus mykiss) under static conditions (limit test) Bayer Cropscience AG Development Reference Environmental Safety-testing Monheim Germany. Report no EBTIN004, Bayer ID M-457314- 01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 203 (1992)

No/Group 2 replicates with 15 fish

Dose Levels 213 mg test substance /L (100 mg ai/L) nominal

Analytical measurements Yes, HPLC/ MS/MS

The acute toxicity of the test substance, Clothianidin FS 600 G, to Oncorhynchus mykiss was determined in a 96-h static test. A limit test was performed at 213 mg test substance/L. Study Summary The results are expressed as nominal concentrations, the mean measured concentration ranged from 84% to 102% of the nominal concentration over the whole test period.

November 2015 138

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The pH values in the test medium ranged from 6.7 to 7.5 and in the control from 6.8 to 7.4, the oxygen concentration was always higher than 60% oxygen saturation (ranged from 86% to 100%) and the temperature was between 11.3 and 11.9oC. In the control group no mortality or other signs of intoxication were determined during the test period. No mortality was observed in the treated group.

Therefore the LC50 is higher than 213 mg test substance /L. The fish showed abnormal behaviour (remained at the bottom of the vessel, long periods at the water surface, turned dark in colour, laid on their sides or backs) after 96 hours. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen ≥ 60% (observed ranged from 86% to 100%)

The LC50 is higher than 213 mg test substance/L as no mortality was Conclusion observed (corresponding to > 128 mg ai/L)

Fish acute toxicity (Marine species)

Type of study Limit test

Flag Key study

Test Substance TI-435 technical (97.6% ai)

Species Cyprinodon variegatus (Sheepshead minnow)

Type of exposure Semi-static for 96 hours

Endpoint LC50

Value > 102.5 mg TI 435 technical /L

D. Scheerbaum (1999) TI 435-technical, Fish (Sheepshead minnow), Acute toxicity test 96 h- semi-static, limit test. Dr. U. Noack-Laboratorium fur Reference Angewandte Biologie, D 31157 Sarstedt Germany. Report no DECO 025, Bayer ID M-027244-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 850.1075 (draft)

No/Group 1 replicate of 10 fish

Dose Levels 102.5 mg TI-435 technical /L (= 100 mg ai/L) nominal

Analytical measurements Yes, fresh medium 1st,2nd,3rd and 4th day, old medium 2nd,3rd, 4th and 5th day

November 2015 139

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The acute toxicity of the test substance to sheepshead minnow was determined in a 96-h semi-static test. A limit test was performed at 102.5 mg TI-435 technical/L. The test medium was daily renewed. The results are expressed as nominal concentrations, recovery rates of the test substance were > 80%. The pH values in the old test medium ranged from 7.92 to 8.09 and in the fresh medium from 7.97 to 8.24. The oxygen concentration was always higher than 60%, old medium 66-87% and fresh medium 77-97%. The temperature was Study Summary between 20.7 and 21.6oC. In the control and in the test concentration, no mortality or other signs of intoxication were determined during the test period.

The LC50 is higher than 102.5 mg technical /L. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen ≥60% (observed > 66%) Measured concentration ≥80% (observed >80%)

The LC50 is higher than 102.5 mg TI 435 technical/L. No mortality observed Conclusion at 102.5 mg TI 435 technical /L.

Invertebrate acute toxicity (Freshwater species)

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Poncho (formulation 600 FS)

Species Daphnia similis

Type of exposure Static, 48 hours

Endpoint EC50

Value 2.90 mg formulation/L

L.Lopes Morandi (2012) Poncho Daphnia similis Acute immobilisation test. Reference TECAM Tecnologia Ambiental Ltda, Sao Paulo Brasil. Report no 15166/2011 5.0DP, Bayer ID M-443874-01-2.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 202 (2004)

No/Group 4 replicates of 5

Dose Levels 0.3, 0.6, 1.2, 2.4, 4.8, 9.6 mg test substance/ L nominal

November 2015 140

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

0.2, 0.38, 0.7, 1.44, 2.6, 4.81 mg test substance/L measured

Analytical measurements Yes, HPLC/UV 0h and 48h.

The acute toxicity of the test substance to Daphnia similis was determined in a 48-h static test. A full test was performed with the formulated product Poncho at 0.3, 0.6, 1.2, 2.4, 4.8, 9.6 mg test substance/ L (nominal). The mean measured concentration was 98% of the nominal concentration at the start and 93% at the end of the test. The results are expressed as nominal concentrations. The pH values in the test medium and in the control ranged from 6.91 to 7.49,

the oxygen concentration was between 7.29 and 8.05 mg O2/L and the temperature was in the range of 19.5 and 20.0oC. Study Summary In the control no immobilisation or other signs of intoxication were determined during the test period. After 48 hours 100% immobilisation was observed at 9.6 mg/L, 75% at 4.8 mg/L, 30% at 2.4 mg/L, 20% at 1.2 mg/L and 10% at 0.6 and 0.3 mg/L. The effects of 0.3, 0.6 and 1.2 mg/L were not statistically significant.

Therefore the NOEC is 1.2 mg/L and the LOEC is 2.4 mg/L. The EC50 is 2.90 mg test substance/L with 95% confidence limits between 2.18 and 3.87 mg /L. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen in control ≥ 3 mg/L (observed 7.29 mg/L )

The EC50 is 2.90 mg formulation/L (CI 2.18 – 3.87 mg formulation /L) Conclusion corresponding to 1.74 mg ai/L

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

Species Daphnia magna

Type of exposure Static, 48 hours

Endpoint EC50

Value 195 mg test substance/L

T. Riebschlager (2013) Acute toxicity of clothianidin FS 600G to the water flea Daphnia magna in a static laboratory system. Bayer Cropscience AG Reference development Environmental Safety Testing, Monheim am Rhein Germany. Report no EBTIN 003, Bayer ID M-457612-01-1.

Klimisch Score 1

Amendments/Deviations None

November 2015 141

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

GLP Yes

Test Guideline/s OECD 202 (2004)

No/Group 6 replicates of 5 animals

Dose Levels 50, 100, 200, 400 and 800 mg test substance/ L nominal

Analytical measurements Yes, HPLC/UV at 0h and 48h.

The acute toxicity of the test substance to Daphnia magna was determined in a 48-h static test. A full test was performed with a formulated product clothianidin FS 600G at 50, 100, 200, 400 and 800 mg test substance/ L (nominal). The mean measured concentration was 96% of the nominal concentration at the start and 105% at the end of the test. The results are expressed as nominal concentrations. The pH value in the test medium and in the control was 7.8 during the test

period. The oxygen concentration was between 8.5 and 8.7 mg O2/L and the Study Summary temperature was in the range of 19.9 and 20.3oC. In the control no immobilisation or other signs of intoxication were determined during the test period. After 48 hours 100% immobilisation was observed at the highest dose rate, 86.7% at 400 mg/L, 76.7% at 200 mg/L and 6.7% at 100

mg/L. No effects were observed at 50 mg/L. The EC50 is 195 mg test substance/L (CI 138 – 276 mg test substance/L Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen in control ≥ 3 mg/L (observed value: 8.6 mg/L )

The EC50 is 195 mg test substance/L (CI 138 – 276 mg test substance/L) Conclusion corresponding to 117 mg ai/L

Invertebrate acute toxicity (Marine species)

Type of study Full test

Flag Key study

Test Substance TI-435

Species Mysidopsis bahia, saltwater mysid

Type of exposure 96 hour flow-through

Endpoint LC50

Value 0.053 mg ai/L

K.R. Drottar, J.A. MacGregor, H.O. Krueger (2000) TI-435 Technical: A 96 hour Reference flow through acute toxicity test with the saltwater mysid (Mysidopsis bahia),

November 2015 142

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Wildlife International Easton Maryland, report 110058 M-019551-01-2, August 15,2000

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 850.1035 (draft 1996)

No/Group 10 mysid (juveniles) and 2 replicates

Dose Levels Nominal: 0.035, 0.060, 0.10, 0.17, 0.29, 0.50 mg ai/L

Analytical measurements Yes, at the beginning, after 48 h and at the end

The acute toxicity of TI-435 was tested to the saltwater mysid Mysidopsis bahia in a 96 hour flow through test. The nominal test concentrations were 0.035, 0.060, 0.10, 0.17, 0.29, 0.50 mg ai/L. The mean measured concentrations were 0.04, 0.067, 0.11, 0.19, 0.34 and 0.56 mg ai/L. The mean measured

concentrations were used to calculate the LC50 values. During the test the pH ranged from 8.0 to 8.4. The temperatures were around 25oC and dissolved oxygen concentrations remained 6.8 mg /L or more. Study Summary Mysids in the control appeared healthy and normal throughout the test. After 96 h exposure mortality was observed in all treatments and ranged from 20 (0.04

mg ai/L) up to 100% (≥ 0.19 mg ai/L). The LC50 was calculated and is 0.053 mg ai/L (CI 0.042 – 0.064 mg ai/L). The NOEC was < 0.040 mg ai/L which is the lowest concentration tested. Validity criteria were met: Mortality or abnormal behaviour less than 10% in control (observed value: 0%)

Conclusion LC50 = 0.053 mg ai/L (CI 0.042 – 0.064 mg ai/L)

Type of study limit test

Flag Key study

Test Substance TI-435 technical

Species Oyster

Type of exposure 96 hour flow-through

Endpoint EC50

Value > 129.14 mg ai/L

D. Scheerbaum (1999) TI 435-technical, Oyster acute toxicity test (shell Reference deposition) limit test, flow through 96 h. Dr. U. Noack-Laboratorium fur

November 2015 143

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Angewandte Biologie, D 31157 Sarstedt Germany. Report no DECO 026, Bayer ID M-028515-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 850.1025 (draft)

No/Group 20 oysters and 1 replicate

Nominal 270 mg ai/L, Dose Levels Measured 129.14 mg ai/L

Analytical measurements Yes, at day 0, 24, 48, 72 and 96 h

The acute toxicity of TI-435 was tested to the eastern oysters Crassostrea virginica in a 96 hour flow through test. The nominal test concentration was 270 mg/L. The mean measured concentration was 129.14 mg/L. The mean

measured concentration was used to calculate the EC50 values. During the test the pH ranged from 6.9 to 7.9. The temperatures ranged from Study Summary 18.2 to 21.1oC and oxygen saturation was at least 67%. No mortality, abnormal behaviour and effects on shell growth were observed.

The EC50 and NOEC are > 129.14 mg ai/L. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Dissolved oxygen ≥60% (observed > 67%)

Conclusion EC50 > 129.14 mg ai/L

Invertebrate chronic toxicity (Marine species)

Type of study Full test

Flag Disregarded study (see below)

Test Substance TI-435

Species Mysidopsis bahia, saltwater mysid shrimp

Type of exposure Flow through life cycle

Endpoint NOEC for mortality, growth and reproduction.

Value 9.7 µg ai/L reproduction

K.R. Drottar, J.A. MacGregor, H.O. Krueger (2000) TI-435 Technical: A flow through life cycle toxicity test with the saltwater mysid (Mysidopsis bahia), Reference Wildlife International Easton Maryland, report 110167 Bayer ID M-026384-01-2, October 31, 2000

November 2015 144

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 3, > 25% of females did not produce young

Amendments/Deviations None that impacted the results of the test

GLP Yes

Test Guideline/s OPPTS 850.1350

No/Group 4 replicates of 15 mysids

Nominal: 0.63, 1.3, 2.5, 5.0, 10 and 20 µg ai/L Dose Levels Mean measured: 0.62, 1.2, 2.5, 5.1, 9.7 and 19 µg ai/L.

Yes, at the beginning and at weekly intervals during the test and at test Analytical measurements termination

The toxicity of TI-435 was tested to the saltwater mysid Mysidopsis bahia in a life cycle flow through test. The nominal test concentrations were 0.63, 1.3, 2.5, 5.0, 10 and 20 µg ai/L. The mean measured concentrations were 0.62, 1.2, 2.5, 5.1, 9.7 and 19 µg ai/L. The mean measured concentrations were used to calculate the NOEC, LOEC and MATC values. During the test the pH ranged from 8.2 to 8.4. The temperatures were around

o 25 C and dissolved oxygen concentrations remained 6.2 mg O2/L (84%of saturation) or more. There were no statistically significant effects on survival or growth of mysids exposed to TI 435 at concentrations up to 19 µg ai/L for 39 days. Reproduction was the most sensitive biological endpoint. The mean number of young per Study Summary reproductive day is 0.207. The highest dose rate reduced the reproduction rate (0.0175 mean number of young/reproductive day) compared to the control. Based on reproduction the NOEC is 9.7 µg ai/L and the LOEC 19 µg ai/L. The MATC was calculated to be 14 µg ai/L. The validity criteria regarding environmental parameters were met: Temperature 25 oC ±2 oC (observed 24.5 to 26.5oC) Dissolved oxygen between 60 and 105% (observed 84%) Further the test is unacceptable if more than 25 percent of first generation females in the control groups fail to produce young or if the average number of young produced per female in the controls is less than three per day. This criteria is not met, consequently the test is not valid.

Conclusion NOEC = 9.7 µg ai/L (reproduction)

Algae acute toxicity (Freshwater species)

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

November 2015 145

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Substance Clothianidin FS 600G

Species Pseudokirchneriella subcapitata

Type of exposure Static, 72 hours

Endpoint ErC50

Value 368 mg formulation/L

E Bruns (2013) Pseudokirchneriella subcapitata growth inhibition test with clothianidin FS 600G. Bayer CropScience AG Development-Environmental Reference safety Ecotoxicology 40789 Monheim (Germany). Report no E323 4507-4, Bayer ID M-457662-02-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 201 (2006)

No/Group 3 replicates for test concentrations and 6 for the control of 104 cells/mL

Dose Levels 4.8, 15.3, 49, 157, 500 mg formulation/L nominal

Analytical measurements Yes on day 0 and 3

The influence of the test substance on the growth of the green algal species Pseudokirchneriella subcapitata was determined in a 72-h static test. The nominal concentrations were 4.8, 15.3, 49, 157, 500 mg formulation/L. The measured concentration in the test medium was 89-100% of the nominal. The pH values in the control ranged from 8.1 to 8.3, and the temperature was between 21.5 and 22.7oC. The test was performed under continuous illumination of 5086 lux. The validity criteria were met: Study Summary Biomass increased in the control by more than 16-fold within the evaluation period: observed value: 83.7 x 104 Mean percent coefficient of variation of sectional growth rates from day 0-1, day 1-2 and day 2-3 in the control did not exceed 35%: observed value = 24.9% Percent coefficient of variation of the average growth rate in each control replicate did not exceed 7%: observed value = 1.1% Growth inhibition was observed at the two highest dose rates and was 15.3 and

64.5% respectively. The ErC50 is 368 mg formulation/L (95% CI: 343-395 mg/L)

and the NOErC is 49 mg formulation/L.

ErC50 = 368 mg formulation/L (95% CI: 343-395 mg/L) corresponding to 221 Conclusion mg ai/L

November 2015 146

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

NOErC = 49 mg formulation/L

Aquatic plants acute toxicity (Freshwater species)

Type of study Full test

Flag Key study

Test Substance TI-435 technical

Species Lemna gibba

Type of exposure 14 days semi static

Endpoint EbC50 and ErC50

Value ≥ 270 mg technical/L

D. Scheerbaum (1999) TI 435-technical, Aquatic plant toxicity test using Lemna Reference gibba. Dr. U. Noack-Laboratorium fur Angewandte Biologie, D 31157 Sarstedt Germany. Report no DECO 027, Bayer ID M-027363-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OPPTS 850.4400

No/Group 3 replicates 12 fronds per vessel

Dose Levels Nominal: 0.53 -1.05 - 2.11 - 4.22 - 8.44 - 16.88 - 33.75 - 67.5 -135 - 270 mg/L

Analytical measurements Yes, HPLC at days 0 and 7 fresh medium and 3 and 10 old medium

The aim of the study was to determine the effects of the test substance on the growth of Lemna gibba over 14 days under semi-static conditions. Fresh test solutions were prepared on days 0, 3, 5, 7, 10 and 12. The following nominal concentrations were tested: 0.53, 1.05, 2.11, 4.22, 8.44, 16.88, 33.75, 67.5, 135 and 270 mg technical/L. The mean measured concentrations were within 80- 120% of the nominal concentrations, so the results are based on the nominal concentrations. Study Summary Frond numbers, inhibition of the biomass and growth rate were determined. The frond numbers in the control increased more than 8 times in the first 7 days. The test temperature ranged from 23 to 25oC. TI 435 technical significantly reduced the growth of Lemna at concentrations of

8.44 mg/L and greater but EC50 could not be determined. The EC50 and 90 of the inhibition of biomass growth and rate related inhibition is ≥ 270 mg technical/L. The NOEC is 4.22 mg technical/L based on growth inhibition. The validity criteria were met:

November 2015 147

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

 Number of fronds increased in the control by more than 5-fold within 7 days (observed: 8.8, 8.6, 9.3 fold)  Temperature variation < 4 oC (observed 23 to 25oC)

Conclusion EbC50 and ErC50 ≥ 270 mg technical/L.

Sediment toxicity

Type of study Limit test

Flag Key study

Test Substance Clothianidin metabolite: clothianidin-2-chlorothiazole-5-caboxylic acid ( CTCA)

Species Chironomus riparius

Type of exposure Static 48 h

Endpoint EC50 and NOEC

Value Both >10.0 mg CTCA/L

K.Kuhl (2014) Acute toxicity of CTCA to larvae of Chironomus riparius in a 48 h Reference static laboratory test system. Bayer Cropscience AG-BCS-D-EnSa-Testing, Monheim Germany. Report EBTIN033 Bayer ID M-496222-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 235 (2011)

No/Group 5 larvae and 6 replicates

Dose Levels 10.0 mg ai/L

Analytical measurements Yes, at the beginning, and at the end

The acute toxicity of the metabolite CTCA is tested to larvae of Chironomus riparius in a 48 h static laboratory test system. The nominal test concentration was 10.0 mg ai/L. The mean measured concentrations were 106% (day 0) and 105% (day 2). The results are based on the nominal concentrations. During the test the pH ranged from 7.5 to 7.8. The temperatures ranged from

o 20.6 to 21.1 C and dissolved oxygen concentrations were 8.6 mg O2/L. Study Summary The immobility in the control was 3.3% and in the treatment 0%. No abnormal behaviour was observed during the test period.

Consequently EC50 and NOEC > 10.0 mg CTCA/L. The validity criteria were met: Control immobility < 15% (observed 3.3%) Dissolved oxygen >3 mg oxygen/L (observed 8.6 mg oxygen/L)

November 2015 148

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Conclusion EC50 and NOEC > 10 mg CTCA/L

Type of study Limit test

Flag Key study

Clothianidin metabolite: clothianidin-4-hydroxy-2-methylamino-2-imidazolin -5- Test Substance one (HMIO)

Species Chironomus riparius

Type of exposure Static 48 h

Endpoint EC50 and NOEC

Value Both >10.0 mg HMIO/L

K.Kuhl (2014) Acute toxicity of HMIO to larvae of Chironomus riparius in a 48 h Reference static laboratory test system. Bayer Cropscience AG-BCS-D-EnSa-Testing, Monheim Germany. Report EBTIN095 Bayer ID M-497572-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 235 (2011)

No/Group 5 larvae and 6 replicates

Dose Levels 10.0 mg ai/L

Analytical measurements Yes, at the beginning, and at the end

The acute toxicity of the metabolite HMIO is tested to larvae of Chironomus riparius in a 48 h static laboratory test system. The nominal test concentration was 10.0 mg ai/L. The mean measured concentrations were 117% (day 0) and 116% (day 2). The results are based on the nominal concentrations. During the test the pH ranged from 7.3 to 7.8. The temperatures ranged from 20.2 to 20.7oC and dissolved oxygen concentrations ranged from 8.1 to 8.3 mg

Study Summary O2/L. No immobility was observed in the control and treatment. No abnormal behaviour was observed during the test period.

Consequently EC50 and NOEC > 10.0 mg HMIO/L. The validity criteria were met: Control immobility < 15% (observed 0%) Dissolved oxygen >3 mg oxygen/L (observed 8.1 -8.3 mg oxygen/L)

Conclusion EC50 and NOEC > 10 mg HMIO/L

November 2015 149

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Type of study Limit test

Flag Key study

Test Substance Clothianidin metabolite: clothianidin-thiazolylformamidine (TZFA)

Species Chironomus riparius

Type of exposure Static 48 h

Endpoint EC50 and NOEC

Value Both >10.0 mg TZFA/L

K.Kuhl (2014) Acute toxicity of TZFA (BCS-CQ88479) to larvae of Chironomus Reference riparius in a 48 h static laboratory test system. Bayer Cropscience AG-BCS-D- EnSa-Testing, Monheim Germany. Report EBTIN098 Bayer ID M-497581-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 235 (2011)

No/Group 5 larvae and 6 replicates

Dose Levels 10.0 mg ai/L

Analytical measurements Yes, at the beginning, and at the end

The acute toxicity of the metabolite TZFA is tested to larvae of Chironomus riparius in a 48 h static laboratory test system. The nominal test concentration was 10.0 mg ai/L. The mean measured concentrations were 94% (day 0) and 81.2% (day 2). The results are based on the nominal concentrations. During the test the pH ranged from 7.8 to 7.9. The temperatures ranged from 20.5 to 20.9 oC and dissolved oxygen concentrations ranged from 8.4 to 8.6 mg

Study Summary O2/L. No immobility was observed in the control. In the treatment 3.3% immobility was observed. No abnormal behaviour was observed during the test period.

Consequently EC50 and NOEC > 10.0 mg TZFA/L. The validity criteria were met: Control immobility < 15% (observed 0%) Dissolved oxygen >3 mg oxygen/L (observed 8.4 -8.6 mg oxygen/L)

Conclusion EC50 and NOEC > 10 mg TZFA/L

Type of study Full test

November 2015 150

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

Species Chironomus riparius

Type of exposure Static 48 h

Endpoint EC50 and NOEC

EC50 = 55.7 µg formulation/L corresponding to 26.7 µg ai/L Value NOEC = 32 µg formulation/L.

G. Silke (2014) Acute toxicity of Clothianidin FS 600G to larvae of Chironomus Reference riparius in a 48 h static laboratory test system. Bayer Cropscience AG-BCS-D- EnSa-Testing, Monheim Germany. Report EBTIN099 Bayer ID M-505533-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 235 (2011)

No/Group 5 larvae and 6 replicates

18, 32, 56, 100 and 180 µg formulation/L, corresponding to 8.62, 15.3, 26.8, Dose Levels 47.9, 86.2 µg ai/L

Analytical measurements Yes, at the beginning, and at the end

The acute toxicity of Clothianidin FS 600G is tested to larvae of Chironomus riparius in a 48 h static laboratory test system. The nominal test concentrations 18, 32, 56, 100 and 180 µg formulation/L, corresponding to 8.62, 15.3, 26.8, 47.9, 86.2 µg ai/L. The mean measured concentrations ranged between 113 and 115% (day 0) and between 113 and 117% (day 2). The results are based on the nominal concentrations. During the test the pH was 7.8. The temperature ranged from 19.9 to 20.9oC

Study Summary and dissolved oxygen concentrations ranged from 8.4 to 8.8 mg O2/L. The immobility in the control was 0%. The immobility in the treatments ranged from 3.3% (18 µg formulation/L) up to 100% (100 and 180 µg formulation/L) with a clear dose response.

EC50 is 55.7 µg formulation/L (CI 35.6-87.3 µg formulation/L) and NOEC is 32 µg formulation/L. The validity criteria were met: Control immobility < 15% (observed 0%) Dissolved oxygen >3 mg oxygen/L (observed 8.4 to 8.8 mg oxygen/L)

November 2015 151

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

EC50 = 55.7 µg formulation/L (CI 35.6-87.3 µg formulation/L, corresponding Conclusion 26.7 µg ai/L ) NOEC= 32 µg formulation/L.

Soil toxicity

Soil macro-invertebrates acute toxicity

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600

Species Eisenia fetida

Type of exposure 14 days

Endpoint LC50

Value 4.18 mg test substance/kg dry artificial soil.

J. Narmi Sesso (2001) Acute toxicity of TI-435 600FS to earthworms Eisenia Reference fetida. BIOAGRI Laboratorios Ltda Piracicaba Brasil. Report no RF- 0009.203.080.01 Bayer ID: M-435936-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 207 (1993)

No/Group 4 replicates of 10 individuals

6.25, 12.5, 25, 50 and 100 mg/kg dry artificial soil nominal Dose Levels 3.04, 6.11, 12.42, 25.78, 53.45 mg/kg dry artificial soil, measured

Analytical measurements Yes

The acute toxicity of the substance to the earthworm Eisenia fetida was determined in a 14 days test with artificial substrate containing 10% of peat. Clothianidin FS 600B G was applied at the rates 6.25, 12.5, 25, 50 and 100 mg/kg dry soil (nominal). The measured concentrations were 3.04, 6.11, 12.42, 25.78 and 53.45 mg / kg soil. Study Summary No mortality in the control was observed. The dose rates 6.11 mg/kg soil and above caused 100% mortality. The lowest dose rate caused 7.5% mortality.

LC50 is 4.18 mg test substance/kg dry artificial soil. Validity criteria were met: Mortality less than 10% in control (observed value: 0%)

November 2015 152

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

LC50 is 4.18 mg test substance/kg dry artificial soil corresponding to 2.51 Conclusion mg ai/kg soil.

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

Species Eisenia fetida

Type of exposure 14 days

Endpoint LC50

Value > 32 mg test substance/kg dry artificial soil.

S.Friedrich (2013) Clothianidin FS 600G: acute toxicity to the earthworm Eisenia fetida in artificial soil. BioChem agrar, Labor fur biologische und Reference chemische analytic GmbH, Gerichshain Germany Report no 131048087 S Bayer ID: M-456809-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 207 (1993)

No/Group 4 replicates of 10 individuals

Dose Levels 3.2, 5.6, 10, 18, 32 mg/kg dry artificial soil

Analytical measurements Not required

The acute toxicity of the substance to the earthworm Eisenia fetida was determined in a 14 days test with artificial substrate containing 10% of peat. Clothianidin FS 600B G was applied at the rates 3.2, 5.6, 10, 18, 32 mg/kg dry artificial soil. Mortality in the control and 10 mg/kg test group was 2.5%. Mortality was 22.5% at the highest dose rate which is a significant difference compared to the control. No mortality was observed in the other treated groups. Study Summary LC50 is greater than 32 mg test substance/kg dry artificial soil. Average loss of biomass in the control group was 8.5%. In the treated groups the loss was 7.2%, 9%, 11.9%, 16.2% and 21.4% for the lowest up to the highest rate respectively. Validity criteria were met: Mortality less than 10% in control (observed value: 2.5%) Loss of biomass less than 20% (observed 8.5%)

November 2015 153

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

LC50 >32 mg test substance/kg dry artificial soil corresponding to > 19.2 Conclusion mg ai/kg dry soil.

Soil macro-invertebrate chronic toxicity

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Eisenia fetida

Type of exposure 28 days exposure and additional 28 days observations

Endpoint NOEC

Value < 2.40 mg test substance/ kg dry soil reproduction

U. Luhrs (2008) Clothianidin FS 600B G: Effects on reproduction and growth of earthworms Eisenia fetida in artificial soil. Institut fur Biologische Analytik und Reference Consulting IBACON GmbH Artheiliger Weg 17 64380 Rossdorf Gerrmany. Report no 42681022 Bayer ID: M-303405-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 222 (2004), ISO guideline 11268-2 (1998)

No/Group 8 replicates of 10 individuals

1.15 mg clothianidin/kg soil dry weight equivalent to 2.40 mg test substance/ kg Dose Levels soil dry weight

Analytical measurements Not required

The chronic toxicity of the substance to the earthworm Eisenia fetida was determined in an 8 weeks test with artificial substrate containing 10% of peat. Clothianidin FS 600B G was mixed into the soil at 2.4 mg/kg artificial soil dry weight corresponding with 1.15 mg ai/kg soil. The mortalities after 4 weeks were 0% in the control and the treatment. The body weight changes of the adult earthworms exposed to the test substance Study Summary were not significantly different compared to the control. Reproduction was statistically reduced and the number of juveniles was 147 compared to 204 in the control. No behavioural abnormalities were observed. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Reproduction of control ≥30 worms (observed 150-275)

November 2015 154

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Coefficient of variation ≤30% (observed 20.2%)

Conclusion NOEC < 2.40 mg test substance/ kg dry soil reproduction

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Eisenia fetida

Type of exposure 28 days exposure and additional 28 days observations

Endpoint NOEC

Value < 82 µg ai/kg dry soil reproduction

T. Leicher (2009) Clothianidin FS 600B G: Evaluation of the effects on the earthworm Eisenia fetida exposed to a long term plateau concentration of clothianidin FS 600B G mixed into artificial soil with 5% peat content and Reference additional to sown maize seed dressed with clothianidin FS 600B G. Bayer Cropscience AG BCS-D-EnSa-ETX Monheim Gerrmany. Report no LRT-RG-R- 77/09 Bayer ID: M-359944-01-1

Klimisch Score 1

Yes: For the control as well as for the treatment 40 adult earthworms were tested in a container with a surface of 4256 cm² containing 30 kg dry weight artificial soil. Amendments/Deviations Five % peat was used in the artificial soil. Earthworms were exposed to a long term plateau concentration of the test item mixed into artificial soil and additional to sown maize seeds dressed with the test item.

GLP Yes

Test Guideline/s OECD 222 (2004), ISO guideline 11268-2 (1998)

No/Group 40 adults for control and treatment

Dose Levels 82 µg ai/kg dry soil and 1.126 mg ai/seed

Analytical measurements Soil sampling by HPLC-MS/MS

The purpose of the test is to evaluate the effects on the earthworm Eisenia fetida exposed to a long term plateau concentration of clothianidin FS 600B G Study Summary mixed into artificial soil with 5% peat content and additional to sown maize seed dressed with clothianidin FS 600B G. The long term plateau concentration was

November 2015 155

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

82 µg ai/kg dry soil and the nominal test concentration of the treated maize seed is 1.126 mg ai/seed (50 000 seeds/ha). The mortalities after 4 weeks were 0% in the control and 2.5% in the treatment. The body weight changes of the adult earthworms exposed to the test substance (12.3%) were not significantly different compared to the control (18.8%). Reproduction was statistically reduced and the number of juveniles per surviving adult was 31.2 compared to 45.2 in the control. No behavioural abnormalities were observed. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Reproduction of control/vessel ≥120 worms (observed 1809) Coefficient of variation ≤30% (observed 24%)

Conclusion NOEC < 0.082 mg ai/kg dry soil reproduction

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Eisenia fetida

Type of exposure 28 days exposure and additional 28 days observations

Endpoint NOEC

Value 0.74 mg test substance/kg dry soil reproduction

S. Fiedrich (2014) Clothianidin FS 600B G: Sublethal toxicity to the earthworm Eisenia fetida in artificial soil. BioChem agrar Labor fur biologische und Reference chemische analytic GmbH, Gerichshain Gerrmany. Report no 141048018S Bayer ID: M-478114-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 222 (2004), ISO guideline 11268-2 (1998)

No/Group 4 replicates for the treatments and 8 for the control, 10 animals/replicate

0.74, 1.32, 2.35, 4.19, 7.44, 13.23, 23.53, 41.83 mg test substance/ kg dry Dose Levels weight soil

Analytical measurements Not required

November 2015 156

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The purpose of the test is to evaluate the effects on the earthworm Eisenia fetida exposed to clothianidin FS 600B G mixed into artificial soil with 10% peat content. The mortality after 4 weeks was 2.5% in the control. The dose rate 2.35 mg/kg did not cause mortality. The dose rates 0.74, 1.32 and 4.19 mg/kg soil caused 2.5% mortality. Treatment with 7.44 mg/kg soil caused 7.5% mortality. The mortality at 13.23 mg/kg soil was 15%, at 23.53 mg/kg soil 52.5% and 75% mortality was observed at the highest dose rate. The difference compared to the control was significant at the two highest dose rates. The NOEC mortality is 13.23 mg test substance/kg soil. The body weight change of the adult earthworms in the control was 122 mg. Study Summary The changed in the treatments ranged from -200 mg (highest dose rate) to 134 mg (lowest dose rate). NOEC biomass change is 2.35 mg test substance/kg soil. The number of juveniles per replicate was statistically reduced at the dose rate 1.32 mg/kg soil and above (ranged from 0 to 76 juveniles) compared to the control (100.8). NOEC reproduction is 0.74 mg test substance/kg soil. Validity criteria were met:  Mortality less than 10% in control (observed value: 2.5%)  Reproduction of control/vessel ≥30 worms/ replicate (observed ranged from 79 to 122 per replicate)  Coefficient of variation ≤30% (observed 14.3%)

NOEC = 13.23 mg test substance/kg soil mortality, Conclusion NOEC = 2.35 mg test substance/kg soil biomass change, NOEC = 0.74 mg test substance/kg soil reproduction

Type of study Limit test

Flag Key study

Test Substance Metabolites of clothianidin: TZMU, TZFA, NTG, TZNG, MNG

Species Eisenia fetida

Type of exposure 28 days exposure and additional 28 days observations

Endpoint NOEC

TZMU, TZFA, NTG, MNG 100 mg ai/ kg soil dry weight reproduction Value TZNG 13 mg ai/kg soil reproduction

E. Wagenhoff (2014) 5 Metabolites of Clothianidin (TZMU, TZFA, NTG, TZNG, Reference MNG): Sublethal toxicity to the earthworm Eisenia fetida (Annelida,

November 2015 157

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Lumbricidae) in artificial soil with 5% peat content. Eurofins Agroscience Service EcoChem GmbH, Niefern-Oschelbronn Germany. Report no S13- 03784 Bayer ID: M-505527-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the study results

GLP Yes

Test Guideline/s OECD 222 (2004), ISO guideline 11268-2 (1998)

No/Group 8 replicates of 10 individuals

100 mg ai/kg soil dry weight equivalent to 101 mg TZMU/ kg soil dry weight, 103 mg TZFA/ kg soil dry weight, Dose Levels 134 mg NTG/ kg soil dry weight, 102 mg TZNG /kg soil dry weight, 101 mg MNG /kg soil dry weight Dose response test with TZNG: 3.1 , 6.3, 13, 25, 50 mg ai/kg soil

Analytical measurements Not required

The chronic toxicity of the substance to the earthworm Eisenia fetida was determined in an 8 weeks test with artificial substrate containing 5% of peat. A limit test was performed with the metabolites of clothianidin and applied was 100 mg ai/kg soil dry weight. This is equivalent to 101 mg TZMU/kg soil, 103 mg TZFA /kg soil, 134 mg NTG/kg soil, 102 mg TZNG/ kg soil and 101 mg MNG/kg soil. Mean mortality in the control group was 0% and the mean number of juveniles per replicate was 135. The metabolites TZMU, TZFA and NTG did not cause mortality. Mean mortality of the TZNG group was 5% and of the MNG group 1.3%. Study Summary The mean body weight change was comparable between the treatments (ranged from +38.9 to +49.6%) and the control (+41.4%). The mean number of juveniles was comparable between the metabolites TZMU (145.5), TZFA (120.5), NTG (125.9), and MNG (124.3) and the control. The NOEC of these metabolites was determined to be 100 mg ai/kg soil. The percentage of juveniles was significant less of the TZNG group compared to the control and was 13.3%. Therefore a dose response study was performed with the metabolite TZNG with 3.1, 6.3, 13, 25 and 50 mg ai/kg soil. Mean mortality was 10, 10, 2.5, 5.0, 7.5% respectively. The mean juveniles were 116, 126, 121, 107, and 102 respectively. The difference was statistically significant of the two highest dose rates compared to the control (146).

November 2015 158

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

NOEC mortality is 50 mg ai/kg soil and NOEC reproduction is 13 mg ai/kg soil. Validity criteria were met:  Mortality less than 10% in control (observed value: limit 0%, full 2.5%)  Reproduction of control ≥30 worms (observed limit 135, full 146)  Coefficient of variation ≤30% (observed limit 21.9%, full 17.2%)

NOEC 100 mg ai/ kg dry soil reproduction for the metabolites TZMU, Conclusion TZFA, NTG, MNG, NOEC is 13 mg ai/kg soil reproduction for the metabolite TZNG

Type of study Full test

Flag Key study

Test Substance Metabolite of clothianidinTMG

Species Eisenia fetida

Type of exposure 28 days exposure and additional 28 days observations

Endpoint NOEC reproduction

Value 100 mg test substance/kg soil dry weight

B. Pavic (2014) Effects of TMG on reproduction and growth of earthworms Eisenia fetida in artificial soil with 5% peat. Institut fur Biologische analytic und Reference consulting IBACON GmbH, Rossdorf, Germany. Report no 92971022 Bayer ID: M-506792-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the test

GLP Yes

Test Guideline/s OECD 222 (2004)

No/Group 4 replicates of 10 worms, control 8 replicates

Dose Levels 6.25, 12.5, 25, 50, 100 mg TMG/ kg soil dry weight

Analytical measurements Not required

The toxicity of the test substance to earthworm Eisenia fetida was determined in a 56-day test with artificial substrate containing 5% of peat. The metabolite Study Summary TMG was applied to the soil in the dose rates 6.25, 12.5, 25, 50 and 100 mg TMG/ kg soil dry weight. Adult mortality was determined after 28 days and reproduction after 56 days.

November 2015 159

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

In the control group adult no mortality was observed. No mortality was in the treatment groups up to 50 mg TMG/kg soil, the highest dose rate resulted in 2.5% mortality. The mean number of juveniles in the control was 359. The reproduction in the treatment groups ranged from 321 up to 406 juveniles. No significant differences compared to the control were observed. The NOEC mortality and reproduction is 100 mg TMG//kg soil. Validity criteria were met: Mortality less than 10% in control (observed value: 0%) Reproduction of control ≥30 worms (observed 317 to 413) Coefficient of variation ≤30% (observed 10%)

Conclusion NOEC = 100 mg test substance/ kg soil reproduction

Earthworm field study

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Earthworm fauna

Type of exposure Soil exposure and exposure via treated seed

Endpoint Number and biomass of earthworm

11 weeks: Number 31.38 juveniles & adult; biomass 16.76 g 5 months: Value Number 21.56 juveniles & adults; biomass 13.18 g 11 months: Number 36.50 juveniles & adults; biomass 28.14 g No adverse effects observed

T. Leicher (2010) Clothianidin FS 600B G: Effect of dressed corn seeds on the earthworm fauna within one year. Bayer Cropscience AG D-EnSa-ETX Reference Monheim Gerrmany. Report no LRT-RG-F-5/10 Bayer ID: M-366453-01-1 (interim report). Report no LRT/RG-F-6/10 Bayer ID 370671-01-1 (final report)

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s BBA Part VI, 2-3 (1994), ISO draft guideline CD 11268-3 (1999)

November 2015 160

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No/Group Earthworm population

Dose Levels 80 µg ai/kg dry soil and 125 g ai/ha as seed treatment

Analytical measurements Soil sampling method 00540/M001

The purpose of the test is to evaluate the effects of clothianidin FS 600B G on earthworm populations under field conditions. The test site was treated with a herbicide to eliminate possible effects of plant cover. The earthworm population was determined at the test plot before the start of the study. Clothianidin was applied at a rate of 120 g ai/ha to simulate a long term plateau concentration of 80 µg ai/kg dry soil in the upper layer. On the same day treated corn seeds was applied with an application rate of 125 g ai/ha. Also untreated con seed and seeds treated with carbendazim were sown. Earthworm numbers and biomass were determined 11 weeks and 5 and 11 months after application. Analysis of the soil sample showed soil residues of 70.7 µg ai/kg dry soil (88% of nominal concentration). An additional application resulted in 226 µg ai/kg dry soil (plateau concentration plus annual rate) corresponding to 138% of the Study Summary nominal amount of the plateau concentration plus the annual rate. After 11 weeks the number of juvenile and adult earthworms was 29.56 in the control and 31.38 in the treatment plot, after 5 months the numbers were 25.44 and 21.56 respectively (average values). After 11 months in the control 32.81 animals were counted and in the treatment 36.50 animals. The numbers in the reference treatment were significantly lower after 11 weeks and 5 months, no differences were detected after 11 months. After 11 weeks the biomass of all earthworms was 12.46 g in the control and 16.76 in the treatment, after 5 months the biomass was the same 13.18 g. After 11 months the biomass was 26.17 g in the control and 28.14 g in the treatment. The biomass of the reference was significant lower after 11 weeks but comparable after 5 and 11 months.

No adverse effects on earthworm populations were observed as a result Conclusion of clothianidin applied as soil treatment and seed treatment after 11 weeks, 5 and 11 months after application.

Other macro-invertebrates – Folsomia candida

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

Species Folsomia candida

Type of exposure Static, for 28 days

November 2015 161

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Endpoint NOEC

Value 0.43 mg test substance/ kg soil dry weight reproduction

U. Frommholz (2014) Clothianidin FS 600 G uncoloured: influence on the reproduction of the Collembola species Folsomia candida tested in artificial soil. Reference Bayer Cropscience AG Development EnSa testing Monheim Germany. Report no FRM-COLL-173/14 Bayer ID: M-477768-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 232 (2009)

No/Group 4 replicates of 10 springtails 9-12 days old

Dose Levels 0.43, 0.74, 1.3, 2.28, 3.99 mg test substance/kg soil dry weight

Analytical measurements Not required

The toxicity of the substance to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 5% of peat, 20% clay and 75% quartz sand. Clothianidin FS 600G was applied to the soil in the dose rates 0.43, 0.74, 1.3, 2.28, 3.99 mg/kg soil. Mortality and reproduction were determined after 28 days. In the control group adult mortality was 8.8%. The mortality in the treatment groups was 37.5% (0.43 mg/kg), 62.5% (0.74 mg/kg), 85% (1.3 mg/kg), 95% (2.28 mg/kg) and 82.5% at the highest dose rate. The mean number of Study Summary juveniles in the control was 1215.6. The reproduction in the treatment groups was compared to the control 95.1% (0.43 mg/kg), 25.3% (0.74 mg/kg), 6.3% (1.30 mg/kg), 2.4% (2.28 mg/kg) and 4.6% (3.99 mg/kg). Only the reproduction at the lowest rate was not significantly different compared to the control. The NOEC reproduction is 0.43 mg test substance/kg soil. The validity criteria of the study were met: Mortality control <20% (observed 8.8%) Reproduction control >100 juveniles/vessel (observed 1215.6) Variation coefficient control <30% (observed 15.3%).

Conclusion NOEC = 0.43 mg test substance/ kg soil reproduction

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

November 2015 162

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Species Folsomia candida

Type of exposure Static, for 28 days

Endpoint NOEC reproduction

Value ≥195 g ai/ha

U. Frommholz (2014) Clothianidin FS 600 G uncoloured dressed sugar beet seeds (variety Lisanna): influence on the reproduction of the Collembola Reference species Folsomia candida tested in artificial soil. Bayer Cropscience AG Development EnSa testing Monheim Germany. Report no FRM-COLL-174/14 Bayer ID: M-491750-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guideline/s OECD 232 (2009)

No/Group 4 replicates of 80 springtails 10-12 days old

0.032 mg test substance/kg soil dry weight and treated seed representing 195 g Dose Levels ai/ha

Analytical measurements Not required

The toxicity of the substance to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 5% of peat, 20% clay and 75% quartz sand. Clothianidin FS 600G was applied to the soil in the plateau concentration of 0.032 mg test substance/ kg artificial soil (0.0151 mg ai/kg soil). Further treated seed was used with 1 seed/154 cm2 representing the 5 fold field rate (195 g ai/ha). Mortality and reproduction were determined after 28 days. In the control group adult mortality was 6.6%. The mortality in the treatment Study Summary groups was 8.1%. The mean number of juveniles in the control was 8088.8. The reproduction in the treatment group was compared to the control 107.9% (8728 juveniles). The NOEC reproduction is ≥195 g ai/ha. The validity criteria of the study were met: Mortality control <20% (observed 6.6%) Reproduction control >100 juveniles/vessel (observed 8088.8) Variation coefficient control <30% (observed 8.1%).

Conclusion NOEC ≥195 g ai/ha reproduction

November 2015 163

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Type of study Limit test

Flag Key test

Test Substance Metabolites of clothianidin TZMU, TZFA and NTG

Species Folsomia candida

Type of exposure Static, for 28 days

Endpoint NOEC reproduction

100 mg ai/kg soil for TZMU, TZFA Value 80 mg ai/ kg soil for NTG

E. Wagenhoff (2014) 3 metabolites of clothianidin (TZMU, TZFA, NTG): Effects on the reproductive output of the springtail Folsomia candida Willem Reference (Collembola, Isotomidae) in artificial soil with 5% peat content. Eurofins Agroscience Services EcoChem GmbH Niefern-Oschelbronn Germany. Report no S13-03785 Bayer ID: M-505607-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guideline/s OECD 232 (2009)

No/Group 8 replicates of 10 springtails 9-12 days old

100 mg ai/kg soil dry weight equivalent to 101 mg TZMU/kg soil, 103 mg TZFA Dose Levels /kg soil, 134 mg NTG/kg soil. Dose response NTG: 5, 10, 20, 40 and 80 mg ai/kg soil.

Analytical measurements Not required

The toxicity of the metabolites TZMU, TZFA and NTG to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 5% of peat. Applied is 100 mg ai/kg soil dry weight equivalent to 101 mg TZMU/kg soil, 103 mg TZFA /kg soil, 134 mg NTG/kg soil. Mortality and reproduction were determined after 28 days. In the control group adult mortality was 5%. The mortality in the treatment group Study Summary TZMU was 5%, TZFA 3.8% and NTG 7.5%. The mean number of juveniles in the control was 970.1. The reproduction in the treatment groups was 935 (TZMU), 984 (TZFA) and 871 (NTG) juveniles. The reduction in the NTG group is significantly different from the control group. The NOEC reproduction is 100 mg ai/ kg soil for the metabolites TZMU and TZFA. With the metabolite NTG a dose response test is performed with the dose rates 5, 10, 20, 40 and 80 mg ai/kg soil. Mortality in the control was 7.5%.

November 2015 164

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The mean mortality of the NTG was 5% (5 mg ai/kg), 2.5% (10 mg ai/kg), 0% (20 mg ai/kg), 2.5% (40 mg ai/kg) and 17.5% (80 mg ai/kg). The mean number of juveniles was 806, 841, 800, 844 and 725 respectively. NOEC mortality and reproduction is 80 mg ai/kg soil dry weight. The validity criteria of the study were met: Mortality control <20% (observed 5%, NTG full test 7.5%) Reproduction control >100 juveniles/vessel (observed 970, NTG full test 807) Variation coefficient control <30% (observed 11.9%, NTG full test 5.6%).

NOEC is 100 mg ai/kg soil dry weight for the metabolites TZMU and TZFA mortality and reproduction Conclusion NOEC is 80 mg ai/kg soil dry weight for the metabolite NTG mortality and reproduction.

Type of study Full test

Flag Key test

Test Substance Metabolites of clothianidin TMG

Species Folsomia candida

Type of exposure Static, for 28 days

Endpoint NOEC mortality and reproduction

Value 85.3 mg TMG/kg soil

B. Pavic (2014) Effects of TMG on reproduction of the Collembola Folsomia candida in artificial soil with 5% peat. Institut fur Biologische analytic und Reference consulting IBACON GmbH, Rossdorf, Germany. Project 92971016 Bayer ID: M- 505619-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guideline/s OECD 232 (2009)

4 replicates treatment, 8 replicates control each with 10 springtails 10-12 days No/Group old

Dose Levels 5.33, 10.7, 21.3, 42.7, 85.3 mg TMG/ kg soil dry weight

Analytical measurements Not required

November 2015 165

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The toxicity of the metabolites TMG to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 5% of peat. Applied is 5.33, 10.7, 21.3, 42.7, 85.3 mg TMG/ kg soil dry weight. Mortality and reproduction were determined after 28 days. In the control group adult mortality was 10%. The mortality at the rates 10.7 and 42.7 mg TMG/kg was 8%. Mortality was 10% in the group 5.33 mg TMG/kg, 13% in the group 21.3 mg TMG/kg and 18% in the highest dose rate. Study Summary The mean number of juveniles in the control was 557. The reproduction in the treatment groups ranged from 487 up to 584 and was not significantly different from the control. The NOEC mortality and reproduction is 85.3 mg TMG/ kg soil. The validity criteria of the study were met: Mortality control <20% (observed 10%) Reproduction control >100 juveniles/vessel (observed 557) Variation coefficient control <30% (observed 9.7%).

Conclusion NOEC is 85.3 mg TMG/kg soil dry weight mortality and reproduction.

Type of study Limit test

Flag Disregarded study (see validity)

Test Substance Clothianidin FS 600

Species Folsomia candida

Type of exposure Static, for 29 days, treated maize seed

Endpoint NOEC

Value < 625 g ai/ha reproduction

C. Lechelt-Kunze (2004) Clothianidin FS 600 rot dressed maize seed (variety Tassilo) influence on the reproduction of the Collembola species Folsomia Reference candida tested in artificial soil. Bayer Cropscience AG Institute for Ecotoxicology,40789 Monheim Germany. Report no LKC-Coll 34/04 Bayer ID: M-078012-01-1

Klimisch Score 3: the concentration tested was too high, no NOEC was obtained in this study

Amendments/Deviations None

GLP Yes

Test Guideline/s ISO 11267 (1999)

No/Group 5 replicates of 20 springtails 10-12 days old

Dose Levels 625 g ai/ha

November 2015 166

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Analytical measurements Not required

The toxicity of the substance to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 10% of peat, 20% clay and 70% quartz sand. Maize seed was treated with the test substance and applied at a rate of 625 g ai/ha. Mortality and reproduction were determined after 29 days. In the control group adult mortality was 7% and in the treatment 61%. The Study Summary mean number of juveniles in the control was 1647 and in the treatment 682 (41% of the control) which is significant lower than the control. The validity criteria of the study were met: Mortality control <20% (observed 7%) Reproduction control >100 juveniles/vessel (observed 1647) Variation coefficient control <30% (observed 12%).

Conclusion NOEC < 625 g ai/ha reproduction

Type of study Limit test

Flag Disregarded study (see validity)

Test Substance Clothianidin FS 600

Species Folsomia candida

Type of exposure Static, for 28 days, treated maize seed

Endpoint NOEC

Value < 253 g ai/ha reproduction

C. Lechelt-Kunze (2004) Clothianidin FS 600 rot dressed maize seed (variety Romario) influence on the reproduction of the Collembola species Folsomia Reference candida tested in artificial soil. Bayer Cropscience AG Institute for Ecotoxicology, 40789 Monheim Germany. Report no LKC-Coll 37/04 Bayer ID: M-124351-01-1

Klimisch Score 3: the concentration tested was too high, no NOEC was obtained in this study

Amendments/Deviations None

GLP Yes

Test Guideline/s ISO 11267 (1999)

No/Group 5 replicates of 20 springtails 10-12 days old

Dose Levels 253 g ai/ha

Analytical measurements Not required

November 2015 167

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The toxicity of the substance to the springtail Folsomia candida was determined in a 28-day test with artificial substrate containing 10% of peat, 20% clay and 70% quartz sand. Maize seed was treated with the test substance and applied at a rate of 253 g ai/ha. Mortality and reproduction were determined after 28 days. In the control group adult mortality was 14% and in the treatment 60%. The Study Summary mean number of juveniles in the control was 1690 and in the treatment 919 (54% of the control) which is significant lower than the control. The validity criteria of the study were met: Mortality control <20% (observed 14%) Reproduction control >100 juveniles/vessel (observed 1690) Variation coefficient control <30% (observed 13%).

Conclusion NOEC < 253 g ai/ha reproduction

Other macro-invertebrates – Predatory mite

Type of study Full test

Flag Key study

Test Substance Metabolite of clothianidinTMG

Species Hypoaspis aculeifer

Type of exposure Static, for 14 days

Endpoint NOEC reproduction

Value 85.3 mg test substance/kg soil dry weight

B. Pavic (2014) Effects of TMG on reproduction of the predatory mite Hypoaspis aculeifer in artificial soil with 5% peat. Institut fur Biologische analytic Reference und consulting IBACON GmbH, Rossdorf, Germany. Report no 92971089 Bayer ID: M-505613-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the test

GLP Yes

Test Guideline/s OECD 226 (2008)

No/Group 4 replicates of 10 adult mites, control 8 replicates

Dose Levels 5.33, 10.7, 21.3, 42.7, 85.3 mg TMG/ kg soil dry weight

Analytical measurements Not required

The toxicity of the test substance to the soil mite Hypoaspis aculeifer was Study Summary determined in a 14-day test with artificial substrate containing 5% of peat, 20%

November 2015 168

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

clay and 75% quartz sand. The metabolite TMG was applied to the soil in the dose rates 5.33, 10.7, 21.3, 42.7, 85.3 mg TMG/ kg soil dry weight. Mortality and reproduction were determined after 14 days. In the control group adult mortality was 5%. The mortality in the treatment groups was 3% in the groups 21.3 and 85.3 mg TMG/kg, 8% in the group 5.33 mg TMG/kg, and 10% in the groups 10.7 and 42.7 mg TMG/kg. The mean number of juveniles in the control was 212. The reproduction in the treatment groups ranged from 99 up to 120% of the control and was not significantly different with the control. The NOEC reproduction is 85.3 mg test substance/kg soil. The validity criteria of the study were met: Mortality control <20% (observed 5%) Reproduction control >50 juveniles/vessel (observed 212) Variation coefficient control <30% (observed 7.1%).

Conclusion NOEC = 85.3 mg test substance/ kg soil reproduction

Type of study Limit test

Flag Key study

Test Substance Metabolite of clothianidin TZMU, TZFA, NTG, TZNG, MNG

Species Hypoaspis aculeifer

Type of exposure Static, for 14 days

Endpoint NOEC reproduction

100 mg ai/kg soil dry weight for the metabolites TZMU, TZFA, NTG and MNG Value 20 mg ai/kg soil dry weight for the metabolite TZNG

U. Schobinger (2014) 5 Metabolites of clothianidin (TZMU, TZFA, NTG, TZNG, MNG) Effects on the reproductive output of the predatory mite Hypoaspis Reference (Geolaelaps) aculeifer Canestrini (Acari:Laelapidae) in artificial soil with 5% peat content. Eurofins Agroscience Services EcoChem GmbH Niefern- Oschelbronn Germany. Report no THW-0354 Bayer ID: M-5056155-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the test

GLP Yes

Test Guideline/s OECD 226 (2008)

No/Group 8 replicates of 10 adult mites

November 2015 169

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

100 mg ai/kg soil dry weight corresponding with 101 mg TZMU/kg soil, 103 mg TZFA /kg soil, 134 mg NTG/kg soil, 102 mg TZNG /kg soil and 101 mg MNG Dose Levels /kg soil. Dose response test TZNG 5, 10, 20, 40 and 80 mg ai/kg soil corresponding with 5.08, 10.2, 20.3, 40.6, 81.2 mg TMG/ kg soil dry weight

Analytical measurements Not required

The toxicity of the test substance to the soil mite Hypoaspis aculeifer was determined in a 14-day test with artificial substrate containing 5% of peat. A limit test was performed with the metabolites TZMU, TZFA, NTG, TZNG and MNG at 100 mg ai/kg soil dry weight corresponding to 101 mg TZMU/kg soil, 103 mg TZFA /kg soil, 134 mg NTG/kg soil, 102 mg TZNG /kg soil and 101 mg MNG /kg soil. Mortality and reproduction were determined after 14 days. In the control group no mortality was observed. The mortality in the treatment group TZMU was 2.5%, TZFA and TNG 10%, TZNG 17.5% and MNG 13.8%. The mean number of juveniles in the control was 305. The reproduction in the treatment groups was 298 (TZMU), 318 (TZFA), 310 (NTG), 247 (TZNG) and 291 (MNG) juveniles. The results of TZNG were significant different from the control. The NOEC mortality and reproduction is 100 mg ai/kg soil dry weight of Study Summary the metabolites TZMU, TZFA, NTG and MNG. A dose response test was performed with the metabolite TZNG, applied at 5, 10, 20, 40 and 80 mg ai/kg soil corresponding with 5.08, 10.2, 20.3, 40.6, 81.2 mg TMG/ kg soil dry weight. Mortality was 7.5, 10, 7.5, 7.5, 7.5 and 5% respectively. The mortality in the control was 7.5%. The mean numbers of juveniles was 310 and ranged in the treatments from 262 up to 313 juveniles. The dose rates 40 and 80 mg ai/kg soil resulted in significant less juveniles compared to the control. NOEC mortally is 100 mg ai/kg soil and NOEC reproduction is 20 mg ai/kg soil. The validity criteria of the study were met: Mortality control <20% (observed 10%, full test TZNG 7.5%) Reproduction control >50 juveniles/vessel (observed 305, full test TZNG 310) Variation coefficient control <30% (observed 8.6%, full test TZNG 9.7%).

NOEC =100 mg ai kg soil reproduction for the metabolites TZMU, TZFA, Conclusion TNG, MNG and NOEC= 20 mg ai/kg soil reproduction for the metabolite TZNG

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

November 2015 170

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Substance Clothianidin FS 600 G

Species Hypoaspis aculeifer

Type of exposure Static, for 14 days

Endpoint NOEC reproduction

Value 316 mg test substance/kg soil dry weight

M.I.L. Lopez (2014) Clothianidin FS600 G (uncoloured) Influence on mortality and reproduction of the soil mite species Hypoaspis aculeifer tested in artificial Reference soil. Bayer Cropscience AG Development EnSa testing Monheim Germany. Report no LAR-HR-101/13 Bayer ID: M-488037-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 226 (2008)

No/Group 4 replicates of 10 adult mites, control 8 replicates

Dose Levels 100, 178, 316, 562 and 1000 mg test substance/ kg soil dry weight

Analytical measurements Not required

The toxicity of the test substance to the soil mite Hypoaspis aculeifer was determined in a 14-day test with artificial substrate containing 5% of peat, 20% clay and 75% quartz sand. Clothianidin FS 600G was applied to the soil in the dose rates 100, 178, 316, 562, 1000 mg/kg soil. Mortality and reproduction were determined after 14 days. In the control group adult mortality was 1.3%. The mortality in the treatment groups was 2.5% (178 mg/kg), and 5% at the two highest dose rates. No mortality was observed in the 100 and 316 mg/kg groups.

Study Summary The mean number of juveniles in the control was 318.5. The reproduction in the treatment groups ranged from 70.6 up to 102% compared to the control. The reproduction was significantly different at the two highest dose rates, 84.3 and 70.6% respectively. The NOEC reproduction is 316 mg test substance/kg soil. The validity criteria of the study were met: Mortality control <20% (observed 1.3%) Reproduction control >50 juveniles/vessel (observed 318.5) Variation coefficient control <30% (observed 5.8%).

Conclusion NOEC =316 mg test substance/ kg soil reproduction

November 2015 171

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Litter bag test

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600

Type of exposure Litter bag

Endpoint Degradation organic matter

Residues of clothianidin FS 600 in the soil have no influence on organic matter Value breakdown after 1,3 and 6 months

C. Lechelt-Kunze (2004) Clothianidin FS 600 Effects on soil litter degradation. Reference Bayer Cropscience AG Institute for Ecotoxicology, 40789 Monheim Germany. Report no LKC-SLD 25/04 Bayer ID: M-182243-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the test results

GLP Yes

Effects of plant protection products on functional endpoints in soil (EPFES Test Guideline/s Lisbon 2002, Guidance document, Jorg Rombke et al.)

Treated maize seed: 1.25 mg ai/seed Dose Levels Soil application 63 g ai/ha (42 µg clothianidin/kg soil)

Analytical measurements Yes, soil residue was 96.81% of the nominal concentration

The aim of the study was to determine the effects of clothianidin FS 600 on soil litter degradation. The test substance was applied twice, 1st by spraying to represent the plateau concentration of 42 µg clothianidin/kg soil, and 2nd by sowing treated maize (1.25 mg ai/seed) to represent the yearly application rate. The dose rate of the first application was 63 g clothianidin/ha and was harrowed in the upper 10 cm soil layer. The soil in the study was a silty loam with a pH of

5.44 and 1.3% C and a water holding capacity of 53.37 g H2O/ 100 g dry soil. Litter bags with dry wheat straw were buried alongside the maize rows. The Study Summary degradation of the wheat straw was determined for the time periods of 0-33, 0- 91 and 0-187 days by recording the weight of non-degraded wheat straw. Degradation of the wheat straw was 99% of the control in the period 0-33 days, 95.9% in the period 0-91 days and 97.9% in the period 0-187 days. No statistically significant differences could be observed between the control and the treatments. In the control > 60% degradation of wheat straw was reached within 6 months after burying the litter bags into the soil (95% was degraded after 187 days). The recommended coefficient of variation of < 40% for data on soil litter

November 2015 172

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

degradation in the control within the first 6 months of the study was fulfilled. Therefore the study is considered to be valid.

Residues of clothianidin FS 600 in the soil have no influence on organic Conclusion matter breakdown after 1,3 and 6 months

Nitrogen transformation test

Type of study Full test

Flag Key study

Test Substance Metabolite of clothianidin TNG

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Nitrate content

Value No effect up to 1.34 mg test substance/ kg soil dry weight

U Schobinger (2014) One metabolite of clothianidin (NTG): Effects on the activity of the soil microflora under laboratory conditions (nitrogen Reference transformation). Eurofins Agroscience Services EcoChem GmbH Niefern- Oschelbronn Germany. Report no S13-03789, Bayer ID M-496277-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 216 (2000)

No/Group 3 replicate

0.269 and 1.34 mg test substance/kg soil corresponding with 0.20 and 1.0 mg Dose Levels ai/ kg soil

Analytical measurements Not required

The objective of this study was to assess the effects of the test substance (metabolite TNG) on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied at 0.269 and 1.34 mg test substance/kg soil corresponding with 0.20 and 1.0 mg ai/ kg soil. Samples were Study Summary taken at 0, 7, 14 and 28 days to determine the nitrate content. The soil nitrate content deviated from the control by +7.76% at 0.269 mg/kg soil and +13.8% at 1.34 mg/kg soil at the end of the study. No statistically significant effects were observed during the period 14-28 days and 0-28 days. The deviation percentage for the period 0-28 days was -1.92% at 0.269 mg/kg soil

November 2015 173

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

and +4.95% at 1.34 mg/kg soil. In the period 14-28 days the deviation was +7.44% and +16.7% respectively. The validity criteria was met: Coefficient of variation in the control < 15% (observed value: max 7.52% on day 14)

No effect on the N transformation in soil at concentrations up to 1.34 mg Conclusion test substance/ kg soil (1.0 mg ai/ kg soil)

Type of study Full test

Flag Key study

Test Substance Metabolite of clothianidin TZMU

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Nitrate content

Value No effect up to 1.01 mg test substance/ kg soil dry weight

U Schobinger (2014) One metabolite of clothianidin (TZMU): Effects on the activity of the soil microflora under laboratory conditions (nitrogen Reference transformation). Eurofins Agroscience Services EcoChem GmbH Niefern- Oschelbronn Germany. Report no S13-03787, Bayer ID M-496280-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 216 (2000)

No/Group 3 replicate

0.202 and 1.01 mg test substance/kg soil corresponding with 0.20 and 1.0 mg Dose Levels ai/ kg soil

Analytical measurements Not required

The objective of this study was to assess the effects of the test substance (metabolite TZMU) on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied at 0.202 and 1.01 mg test Study Summary substance/kg soil corresponding with 0.20 and 1.0 mg ai/ kg soil. Samples were taken at 0, 7, 14 and 28 days to determine the nitrate content. The soil nitrate content deviated from the control by +3.92% at 0.202 mg/kg soil and -0.334% at 1.01 mg/kg soil at the end of the study 28 and 42 days

November 2015 174

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

respectively. The study was prolonged for the highest dose rate because the difference with control was >25% at day 28 (deviation -46%). No statistically significant effects were observed during the period 14-28 days for the lower rate and 28-42 days for the higher rate. For the whole period deviations from the control were -4% for the lower rate (0-28 days) and -10.3% at 1.01 mg/kg soil (0-42 days). The validity criteria was met: Coefficient of variation in the control < 15% (observed value: max 11% on day 14)

No effect on the N transformation in soil at concentrations up to 1.01 mg Conclusion test substance/ kg soil (1.0 mg ai/kg soil).

Type of study Full test

Flag Key study

Test Substance Metabolite of clothianidin TZFA

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Nitrate content

Value No effect up to 1.03 mg test substance/ kg soil dry weight

U Schobinger (2014) One metabolite of clothianidin (TZFA): Effects on the activity of the soil microflora under laboratory conditions (nitrogen Reference transformation). Eurofins Agroscience Services EcoChem GmbH Niefern- Oschelbronn Germany. Report no S13-03788, Bayer ID M-503531-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 216 (2000)

No/Group 3 replicate

0.206 and 1.03 mg test substance/kg soil corresponding with 0.20 and 1.0 mg Dose Levels ai/ kg soil

Analytical measurements Not required

The objective of this study was to assess the effects of the test substance Study Summary (metabolite TZFA) on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied at 0.206 and 1.03 mg test

November 2015 175

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

substance/kg soil corresponding with 0.20 and 1.0 mg ai/ kg soil. Samples were taken at 0, 7, 14 and 28 days to determine the nitrate content. The soil nitrate content deviated from the control by +10.6% at 0.206 mg/kg soil and +18.6% at 1.03 mg/kg soil at the end of the study. No statistically significant effects were observed during the period 14-28 days. For the whole period (0-28 days) statistically significant effects were observed for both concentrations compared to the control. The deviations were +6.31% for the lower concentration and +23.9% for the higher concentration. For the period 14-28 days the deviations were -4.37% for the lower concentration and +0.452% for the higher concentration. The validity criteria was met: Coefficient of variation in the control < 15% (observed value: max 6.86% on day 7)

No effect on the N transformation in soil at concentrations up to 1.03 mg Conclusion test substance/ kg soil (1.0 mg ai/kg soil).

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Poncho

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Nitrate content

No effect for the low rate on day 28, stimulating effect for the higher rate of Value >25% on day 28

T.Pinheiro Muniz (2011) Poncho soil microorganisms: nitrogen transformation Reference test. TECAM Tecnologia Ambiental Ltda, Sao Paulo Brasil. Report no 15166/2011 – 9.0MO-N, Bayer ID M-421606-01-2.

2, study ended at day 28 because the lower rate had <25% effect. However the Klimisch Score effect of the higher rate was >25% on day 28

Amendments/Deviations None that had an impact on the results

GLP Yes

Test Guideline/s OECD 216 (2000)

No/Group 3 replicates

Dose Levels 150 and 750 mL Poncho/ha corresponding with 0.20 and 1.0 µL Poncho/ kg soil

November 2015 176

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Analytical measurements Not required

The objective of this study was to assess the effects of the test substance on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied at 150 and 750 mL Poncho/ha corresponding with 0.20 and 1.0 µL Poncho/ kg soil. A sandy soil was used. Samples were taken at 0, 7, 14 and 28 days to determine the nitrate content. Study Summary During the 28-day test, both rates inhibited the nitrate production on day 0 (- 6.67% and -13.33% respectively). On day 7 the higher rate caused an inhibitory effect (-28.57%). On day 14 and 28 the nitrate production was stimulated by both rates (+18.75% low rate, +50.0% high rate). The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 10.83%)

No effect on the N transformation in soil at the concentration 0.2 µL Conclusion formulation/ kg soil. The concentration 1.0 µL formulation / kg soil had >25% effect on day 28.

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Poncho

Species Soil micro-flora

Type of exposure Static, 56 days

Endpoint Nitrate content

Value No effect after 56 days

L. Lopes Morandi (2012) Poncho soil microorganisms: nitrogen transformation Reference test. TECAM Tecnologia Ambiental Ltda, Sao Paulo Brasil. Report no 15166/2011-13.0MON-N, Bayer ID M-436903-01-2.

Klimisch Score 2,

Amendments/Deviations None that had an impact on the results

GLP Yes

Test Guideline/s OECD 216 (2000)

No/Group 3 replicates

Dose Levels 150 and 750 mL Poncho/ha corresponding with 0.20 and 1.0 µL Poncho/ kg soil

Analytical measurements Not required

November 2015 177

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The objective of this study was to assess the effects of the test substance on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied at 150 and 750 mL Poncho/ha corresponding with 0.20 and 1.0 µL Poncho/ kg soil. A sandy soil was used. Samples were taken at 0, 7, 14, 28 and 56 days to determine the nitrate content. On day 0 Poncho caused no effect at the lower dose rate and a stimulatory effect at the higher rate compared to the control. An inhibitory effect was observed for both rates on day 7 (-8.7% and -4.35% respectively). On day 14 Study Summary the lower rate showed an inhibitory effect (-1.96%) and the higher rate a stimulatory effect (+17.65%). On day 28, 42 and 56 both rates showed a stimulatory effect. The study was prolonged as the effects on day 28 and 42 were greater than 25% which was not the case on day 56. At the lower rate the effects were +34.38%, +75.44, +2.35% on the assessment days. At the higher rate the effects were +46.35%, +99.56% and +4.71%. The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 5.39%)

No effect on the N transformation in soil at concentrations up to 1.0 µL test substance/ kg soil after 56 days. Conclusion However, there were stimulatory effects (>25%) observed on day 28 and 42 of the test.

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600G (uncoloured)

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Nitrate content

Value No effect

L. Schulz (2013) Clothianidin FS 600G (uncoloured) Effects on the activity of soil microflora (Nitrogen transformation test). BioChem agrar Labor fur Reference biologische und chemische analytic GmbH, Gerichshain Germany. Report no 121048073 N, Bayer ID M-444934-01-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 216 (2000)

November 2015 178

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No/Group 3 replicates

150 and 750 mL Poncho/ha corresponding with 0.25 and 1.26 mg Poncho/kg Dose Levels soil dry weight

Analytical measurements Not required

The purpose of this study was to assess the effects of the test substance on the nitrogen transformation in soil, determined by the nitrate content. The substance was applied at 150 and 750 mL test substance /ha corresponding with 0.25 and 1.26 mg test substance/ kg soil. A loamy sand soil was used. Samples were taken at 0, 7, 14 and 28 days to determine the nitrate content. Study Summary The differences to control for the lower rate were +12.6% (0-7 days), -22.8% (7- 14 days) and +14.0% (14-28 days). The higher rate had a stimulatory effect during the test period; +5.8% (0-7 days), +3.6% (7-14 days), +1.9% (14-28 days). The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 1.2%)

No effect on the N transformation in soil at concentrations up to 1.26 mg Conclusion test substance/ kg soil.

Carbon transformation test

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Poncho

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Respiration rate

Value No effect

T.Pinheiro Muniz (2011) Poncho soil microorganisms: carbon transformation Reference test. TECAM Tecnologia Ambiental Ltda, Sao Paulo Brasil. Report no 15166/2011 – 8.0MO-C, Bayer ID M-421600-01-2.

Klimisch Score 1

Amendments/Deviations None that had an impact on the results

GLP Yes

Test Guideline/s OECD 217 (2000)

No/Group 3 replicates

Dose Levels 150 and 750 mL Poncho/ha corresponding with 0.20and 1.0 µL Poncho/ kg soil

November 2015 179

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Analytical measurements Not required

The objective of this study was to assess the effects of the substance on the carbon transformation in soil, determined by the respiration rate. The substance was applied at 150 and 750 mL Poncho/ha corresponding with 0.20 and 1.0 µL Poncho/ kg soil. A sandy soil was used. Samples were taken at 0, 7, 14 and 28 days to determine the respiration rate.

On day 0 the added Poncho had a stimulatory effect on CO2 production at both application rates. On day 7 an inhibitory effect was observed at both rates (- Study Summary 59.18% low rate, -67.35% high rate) compared with the control. On day 14 and 28 the low rate had an inhibitory effect – 6.9% and -5.71% respectively. The difference between the treatment and the control was less than 25%. The high rate had a stimulatory effect +5.17% on day 14 and +1.43% on day 28. No significant differences were detected between the control and both application rates on day 28. The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 13.786%)

No effect on the C transformation in soil at concentrations up to 1.0 µL Conclusion Poncho /kg soil

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600G uncoloured

Species Soil micro-flora

Type of exposure Static, 28 days

Endpoint Respiration rate

Value No effect

L.Schulz (2013) Clothianidin FS 600G (uncoloured): Effects on the activity of soil microflora: carbon transformation test. Bayer AG Development Reference Environmental Safety Testing Monheim, Germany. Report Cropscience no 121048073 C, Bayer ID M-459812-02-1.

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 217 (2000)

No/Group 3 replicates

November 2015 180

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

150 and 750 mL Poncho/ha corresponding with 0.25 and 1.26 mg Poncho/ kg Dose Levels soil

Analytical measurements Not required

The objective of this study was to assess the effects of the substance on the carbon transformation in soil, determined by the respiration rate. The substance was applied at 150 and 750 mL formulation/ha corresponding with 0.25 and 1.26 mg formulation/ kg soil. A loamy sand soil was used. Samples were taken at 0, 7, 14 and 28 days to determine the respiration rate. The lower dose rate had a stimulatory effect during the whole test period which was +0.6% on day 0, +2.5% on day 7, +1.6% on day 14 (significant difference Study Summary with control) and +0.7% at the end of the test. The higher test rate had an inhibitory effect during the whole period. On day 0 -1.6%, day 7 -0.7%, day 14 -8.0% and -0.4% at the end of the study. This effect was not significantly different compared to the control. No significant differences were detected between the control and both application rates on day 28. The validity criteria was met: Coefficient of variation in the control < 15% (observed value: 1.8%)

No effect on the C transformation in soil at concentrations up to 1.26 mg Conclusion Poncho/kg soil

Terrestrial vertebrate toxicity

Acute oral toxicity

Type of study Full test

Flag Key study

Test Substance TI435

Species Mallard duck (Anas platyrhynchos)

Type of exposure Acute oral

Endpoint LD50

Value 503 mg ai/kg bw (CI 325- 778 mg ai/kg bw)

S.P.Gallagher, J.B. Beavers (2004) Clothianidin an acute oral toxicity study with Reference the mallard. Wildlife International Easton Maryland 21601 USA. Report no EBTIX062 Bayer ID M-128357-01-1

Klimisch Score 1

Amendments/Deviations None

November 2015 181

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

GLP Yes

Test Guideline/s OPPTS 850.2100

No/Group 10 birds (5 male,5 female) per treatment and control

Dose Levels 125, 250, 500, 1000 and 2000 mg ai/kg bw (nominal)

Analytical measurements Not required

The aim of the study was to determine the acute toxicity of clothianidin administered to the mallard duck as a single oral dose. The applied dose rates were 125, 250, 500, 1000 and 2000 mg ai/kg bw (nominal). Five male and five female ducks were used per treatment group and the control. The test substance was orally intubated directly into the crop or proventriculus of each bird after they were fasted for 17.5 hours. The birds were weighed and dosed on the basis of mg active ingredient per kg bodyweight. Mortality, signs of toxicity, bodyweight, food consumption and abnormal behaviour were recorded. In the control group no mortalities occurred and all birds showed normal behaviour. Study Summary There was 10% mortality in the 125 and 250 mg ai/kg bw groups, 60% in the 500 mg ai/kg bw group, 80% in the 1000 mg ai/kg bw group and 90% at the

highest dose rate. The calculated LD50 is 503 mg ai/kg bw. Shortly after the treatment birds showed loss of coordination and lower limb weakness and abnormal behaviour increased further in time. There was a clear dose response effect. There were treatment related reductions in mean body weight gain or loss of mean body weight at all dose rates tested. Treatment related reductions in feed consumption were also observed. The validity criteria was met: Mortality control <10% (observed 0%).

Conclusion LD50 = 503 mg ai/kg bw (CI 325-778 mg ai/kg bw)

Type of study Full test

Flag Key study

Test Substance TI435

Species House sparrow (Passer domesticus)

Type of exposure Acute oral

Endpoint LD50

Value 530 mg ai/kg bw (CI 350- 830 mg ai/kg bw)

November 2015 182

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

J.M.Stafford (2005) Clothianidin (TI435) acute oral toxicity test (LD50) with the Reference house sparrow (Passer domesticus). Springborn Smithers laboratories Wareham Massachusetts USA. Report no EBTIX061 Bayer ID M-243741

Klimisch Score 1

Amendments/Deviations None that impacted the study results

GLP Yes

Test Guideline/s OPPTS 850.2100

No/Group 10 birds (5 male,5 female) per treatment and control

Dose Levels 63, 125, 250, 500, 1000 and 2000 mg ai/kg bw (nominal)

Analytical measurements Not required

The aim of the study was to determine the acute toxicity of clothianidin administered to the house sparrows. The applied dose rates were 63, 125, 250, 500, 1000 and 2000 mg ai/kg bw (nominal). The test substance was orally administered via gavaging needle directly into the crop or proventriculus of each bird. The birds were weighed and dosed on the basis of mg active ingredient per kg bodyweight. Mortality, signs of toxicity, bodyweight, food consumption and abnormal behaviour were recorded. Post mortem examinations were conducted. In the control group no mortalities occurred and all birds showed normal behaviour. In the treatment groups no mortalities were observed at the lowest dose rate of 63 mg ai/kg bw. There was 20% mortality in the 125 mg ai/ kg bw group, 10% in 250 mg ai/kg bw group, 40% in the 500 mg ai/kg bw group, 70% in the 1000 mg ai/kg bw group and 100% at the highest does rate. The Study Summary calculated LD50 is 530 mg ai/kg bw. NOEL is 63 mg ai/kg bw. Shortly after the treatment birds showed group huddling, piloerection and lethargy. Several birds in the 125 mg ai/kg bw group and above were unable to fly and were lying on their sides and showed loss of coordination. Toxicity effects were more severe at the higher dose rates. Post mortem examinations showed coloured faeces and enlarged gallbladders. These observations were found in all treated groups. At the two highest dose rates enlarged spleens and/or thyroids were also observed. There were treatment related reductions in mean body weight gain or loss of mean body weight at all dose rates tested. Treatment related reductions in feed consumption were also observed. The validity criteria was met: Mortality control <10% (observed 0%).

November 2015 183

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Conclusion LD50 = 530 mg ai/kg bw (CI 350- 830 mg ai/kg bw)

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

Species Bobwhite quail (Colinus virginianus)

Type of exposure Acute oral

Endpoint LD50

Value >2000 mg test substance/kg bw

J. Shephard, M.T. Christ (2013) Toxicity of Clothianidin FS 600 G (600 g/L)

during an acute oral LD50 with the Northern bobwhite quail (Colinus virginianus) Reference Syntech Research laboratory Services LLC, Ecotoxicology, South Metcalf Stilwell Kansas USA. Report no 07SRLS13C49 Bayer ID M-466239-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OCSPP 850.2100, OECD 223

No/Group 5 birds (2 male,3 female) per treatment and control

Dose Levels 2000 mg test substance/kg bw (nominal)

Analytical measurements Not required

The aim of the study was to determine the acute toxicity of clothianidin administered to the bobwhite quail as a single oral dose. The applied dose rate was 2000 mg ai/kg bw (nominal). Two male and three female birds were used per treatment group and the control. The test substance was orally applied after they were fasted for 15 hours. The birds were weighed and dosed on the basis of mg active ingredient per kg bodyweight. Mortality, signs of toxicity, bodyweight, food consumption and abnormal behaviour were recorded. Study Summary In the control group and the treatment group no mortalities occurred. The mean body weight of the treated birds was significantly reduced compared to the control on days 3, 7 and 14. The body weight change on day 14 was not significantly different compared to the change in the control group. Food consumption in the treatment group was significant reduced compared to the control till day 7, thereafter no difference was detected. The validity criteria was met: Mortality control <10% (observed 0%).

November 2015 184

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Conclusion LD50 > 2000 mg test substance/ kg bw

Avoidance test

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance TI435 FS 600

Species pigeons (Columba livia f. domestica)

Type of exposure treated maize seed

Mortality, body weight, consumption of treated seed, gross pathological Endpoint examinations

No mortality occurred and no other adverse effects were observed. Birds Value avoided treated seeds.

R.Barfknecht (2004) TI 435 FS 600 (clothianidin) on maize (1.25 mg/seed); Acceptance/domestic pigeons (Columba livia f. domestica) Bayer Cropscience Reference AG, Institute for Ecotoxicology Monheim Germany. Report no BAR/ANN 100 Bayer ID M-127957-01-1

Klimisch Score 1

Amendments/Deviations Additional observation regarding food consumption

GLP Yes

BBA Guideline no 25-1 Testing of baits, granules and treated seeds for hazards Test Guideline/s to birds-acceptance tests (1993)

No/Group 10 mature pigeons, one per aviary

Dose Levels 1.25 mg ai/ seed

Analytical measurements Not required

The aim of the study was to determine the acceptance of TI435 FS 600 treated maize seed by domestic pigeons. The applied dose rate was 1.25 mg ai /seed. Ten pigeons, each in an own aviary, were tested. After a week of acclimation the pigeons were offered treated seeds during 8 hours following 16 hours of starvation. Each aviary got 30 g treated seeds and 10 g of standard food. After Study Summary the exposure the remaining food was removed and weighed. The birds were observed for signs of intoxication, food gathering activity and body weight. The same exposure was repeated on the next two days. Body weight was measured at the beginning of the acclimation, the day before exposure, after the third day of exposure and at the end of the test. After exposure the birds were observed for another 14 days during which standard food was offered.

November 2015 185

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No mortality was observed. A few birds had soft excrements and one had diarrhoea for a short period. During the exposure all birds consumed 89.8 to 98.9% of the standard food and 1.4 to 1.9% of the treated seed, which indicates a repellent property of the seeds. The pathology showed one bird with red faeces in the intestines and another with reduced spleen. This did not cause the physical conditions or behaviour of the birds. The body weight was reduced during the three exposure period due to reduced food consumption. After switching back to the standard food the body weight increased again.

TI 435 600 FS treated maize seeds (1.25 mg ai/seed) did not cause adverse Conclusion effects to domestic pigeons. Strong repellent effect of the treated seeds

Type of study Avoidance test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance TI435 FS 600

Species Pheasants (Phasianus colchicus)

Type of exposure treated maize seed as food source

Endpoint Acceptance of treated seeds

All birds showed an avoidance behaviour against treated seeds which was Value sufficient enough to protect them from being intoxicated.

R. Barfknecht (2010) Clothianidin FS 600 G on maize seeds; acceptance by Reference Pheasants (Phasianus colchicus) Bayer Cropscience AG BCS-D-EnSa-ETX Monheim Germany. Report no BAR/ANN 154, Bayer ID M-362315-01-1

Klimisch Score 1

Amendments/Deviations None that had an impact on the results

GLP Yes

Test Guideline/s Non guideline method

Dose Levels 1.25 mg ai/ seed

No/Group 7 males and 8 females

Analytical measurements Not required

The aim of the study is to evaluate whether Phaesants (Phasianus colchicus) show sufficient avoidance behaviour to maize seeds treated with Clothianidin Study Summary FS 600G (1.25 mg ai/seed) to protect them from being intoxicated. Seven male and 8 female pheasants were individually weighed and set in separate aviaries. The birds were subject to an acclimation period of 8 days. Prior to exposure

November 2015 186

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

birds were deprived from food. Over two full consecutive exposure days the birds were offered 110 g treated red coloured maize seeds under no-choice conditions. The remaining seed were collected and the amount determined. Afterwards the birds were offered standard food for 4 days after exposure. At

the end all birds were weighed and sacrificed by CO2 asphyxiation. During the acclimation period the birds ate 42 g/bird untreated maize seed. The mean consumption per bird was 4 g/bird when only treated seed was available. The acceptance of treated seed was significantly lower than that of untreated seeds. An impact on the behaviour and physical conditions of the birds was not visible. A body weight decrease was observed after the exposure days, which can be attributed to the reduced food reduction.

All birds showed an avoidance behaviour against treated seeds which Conclusion was sufficient enough to protect them from being intoxicated.

Exposure to birds following planting seeds

Type of study Field study

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance TI435 FS 600

Species Wild bird species in Canada

Type of exposure treated maize seed

Amount of maize seeds available for birds in normal sowing conditions. Monitoring of birds to identify which bird species feed on maize seeds and Endpoint evaluate the frequency of consumption of maize seeds. Search for bird carcasses

Value Clothianidin applied as seed treatment to corn poses minimal risks to birds.

L.W.Brewer (2005) Field evaluation of the potential for avian exposure and toxicological effects following the planting of clothianidin treated corn seed. Reference Sprinborn Smithers Laboratories, Wareham Massachusetts USA. Report no EBTIX065, Bayer ID M-245696-01-1

Klimisch Score 1

Amendments/Deviations None that had an impact on the results

GLP Yes

Test Guideline/s Non guideline method

Dose Levels 62.5 mg ai/ 50 seed

Analytical measurements Not required

November 2015 187

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Corn seeds were treated with Poncho Seed Treatment and were commercially purchased. The expected amount of clothianidin on 50 seeds is 62.5 mg. The measured amount from 5 samples ranged from 58.9 to 69.6 mg per 50 seeds. Twenty-one corn fields were monitored in Ontario to determine how much treated seed was left remaining on the soil surface after normal commercial planting, how much of this seed was missing three days after planting, what bird species utilized the corn field for foraging and how many birds consumed the treated seed. The density of treated corn seeds observed on the soil surface directly post Study Summary planting ranged from 11.8 to 330.8 per acre (corresponding to 29.16 to 817.4/ha). The number of seeds missing after 3 days ranged from 0 to 95 per plot (or 0-56/acre). A total of 1882 birds representing 31 species and three ‘unknown’ categories were observed on the study sites. No mortality was observed post treatment on any of the field monitored. Overall very few birds (2) were observed consuming corn seeds. No abnormal bird behaviour was observed. Spilled corn was rarely found in the open field. This study demonstrated that clothianidin applied as seed treatment to corn poses minimal risks to birds.

No mortality observed and very few birds observed consuming corn Conclusion seeds.

Effects on mammals

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance TI435 FS 600

Species House mice (Mus musculus)

Type of exposure treated maize seed

Endpoint Mortality, body weight, consumption of treated seed

No mortality occurred and no other adverse effects were observed. Mice Value avoided treated seeds.

R.Barfknecht (2004) Acceptance of TI 435 FS 600 coated maize seeds (1.25 mg/seed) by house mice (Mus musculus) Bayer Cropscience AG, Institute for Reference Ecotoxicology Monheim Germany. Report no BAR/ANN 103 Bayer ID M- 182529-01-1

Klimisch Score 1

Amendments/Deviations NA

November 2015 188

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

GLP Yes

Test Guideline/s Internal method

No/Group 10 house mice, 1 per cage

Dose Levels 1.25 mg ai/ seed

Analytical measurements Not required

The aim of the study was to determine the acceptance of TI435 FS 600 treated maize seed by house mice. The applied dose rate was 1.25 mg ai /seed. Ten mice, each in an own cage house, were tested. After a week of acclimation the mice were offered 40 treated seeds during 4 hours following 14 hours of starvation. After the exposure the remaining food was removed, and separated into nibbled and not nibbled seeds. The seeds were counted and weighed. Study Summary Body weight was measured at the beginning of the acclimation, the day before exposure, and at the third day of exposure. Food consumption was measured 3 days before exposure to the day of exposure. No mortality and signs of intoxication were observed. No treatment related effects on body weight were noticed. The food consumption of the treated group amounted 12% of that of the control, treated seeds were avoided.

TI 435 600 FS treated maize seeds (1.25 mg ai/seed) did not cause adverse Conclusion effects to house mice.

Ecotoxicity to terrestrial invertebrates

Bees - Laboratory tests

Acute toxicity tests

Type of study Full test

Flag Key study

Test Substance Clothianidin

Species Apis mellifera

Type of exposure 48 h, oral and contact

Endpoint LD50

Value Contact 38.9 ng ai/bee, oral 2.5 ng ai/bee

S. Schmitzer (2008) Effects of clothianidin technical (Acute contact and oral) on honey bees (Apis mellifera L.) in the laboratory. Institut fur Biologische Analytik Reference und Consulting IBACON GmbH, Artheillger Weg 17, Rossdorf Germany. Report no 43953035 Bayer ID M-307244-01-1

November 2015 189

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 213 and 214 (1998)

No/Group 3 replicates of 10 bees per treatment

Contact 0.8, 4.0, 20.0, 100.0, 500.0 ng ai/bee Dose Levels Oral 0.3, 0.8, 2.4, 5.0, 13.9 ng ai/bee (measured values)

Analytical measurements Not required

The acute contact and oral effects of the test substance clothianidin on the honeybee were determined in a 48 hours test in the laboratory. No mortality was observed in the control group. At the end of the contact test mortality ranged from 3.3 (0.8 ng ai/bee) up to

100% at the two highest dose rates. The LD50 is 38.9 ng ai/bee (CI 30.9- 49.1 ng ai/bee). In the two highest dose rate groups movement coordination problems and apathy were observed. At the end of the oral test mortality ranged from 0 up to 100% in the two highest Study Summary dose rate groups. LD50 is 2.5 ng ai/bee (CI 1.9-3.2 ng ai/bee). During 24 hours after treatment movement coordination problems and apathy were observed in the treatment groups with the exception of the lowest dose rate of 0.3 ng ai/bee. Validity criteria were met: Mortality in control < 10% (observed value: 0%)

LD50 (24h) of the reference substance in the range 0.10-0.30 µg ai/bee (contact) and 0.10-0.35 µg ai/bee (oral). Observed values 0.19 and 0.12 µg ai/bee by contact and oral exposure, respectively.

LD50 = 38.9 ng ai/bee (contact) Conclusion LD50 = 2.5 ng ai/bee (oral)

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

Species Apis mellifera

Type of exposure 48 h oral and 72 h contact

Endpoint LD50

Value Oral 5.8 ng ai/bee, Contact 32.4 ng ai/bee

November 2015 190

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

S. Schmitzer (2011) Effects of clothianidin FS 600G (Acute contact and oral) on honey bees (Apis mellifera L.) in the laboratory. Institut fur Biologische Analytik Reference und Consulting IBACON GmbH, Artheillger Weg 17, Rossdorf Germany. Report no 68841035 Bayer ID M-413147-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 213 and 214 (1998)

No/Group 3 replicates of 10 bees per treatment

Contact 3.1, 6.3, 12.5, 25.0, 50.0, 100.0 ng ai/bee Dose Levels Oral 0.7, 1.3, 2.8, 5.6, 10.4, 17.8 ng ai/bee

Analytical measurements Not required

The acute contact and oral effects of the test substance clothianidin FS 600G on the honeybee were determined in the laboratory. In the contact test the bees were exposed for 72 hours due to increasing mortality between 24 and 48 hours and in the oral test for 48 hours. No mortality was observed in the control group. At the end of the contact test mortality ranged from 3.3 (12.5 ng ai/bee) up to 96.7% at the highest dose rate. No mortality was seen in the lowest two rates.

The LD50 is 32.4 ng ai/bee. In the three highest dose rate groups movement coordination problems and apathy were observed. Study Summary At the end of the oral test mortality ranged from 3.3 up to 100%. No mortality

occurred in the 1.3 and 0.7 ng ai/bee. LD50 is 5.8 ng ai/bee. During 4 hours after treatment movement coordination problems and apathy were observed in the four highest treatment. Validity criteria were met: Mortality in control < 10% (observed value: 0%)

LD50 (24h) of the reference substance in the range 0.10-0.30 µg ai/bee (contact) and 0.10-0.35 µg ai/bee (oral). Observed values 0.18 and 0.15 µg ai/bee by contact and oral exposure, respectively.

LD50 = 32.4 ng ai/bee (contact) Conclusion LD50 = 5.8 ng ai/bee (oral)

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600 G

November 2015 191

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Species Apis mellifera

Type of exposure 48 h, oral and contact

Endpoint LD50

Value Contact 44.1 ng ai/bee, oral 4.7 ng ai/bee

S. Schmitzer (2013) Effects of clothianidin FS 600 G (uncoloured) (Acute contact and oral) on honey bees (Apis mellifera L.) in the laboratory. Institut fur Reference Biologische Analytik und Consulting IBACON GmbH, Artheillger Weg 17, Rossdorf Germany. Report no 75411035 Bayer ID M-456670-01-1

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s OECD 213 and 214 (1998)

No/Group 3 replicates of 10 bees per treatment

Contact 3.1, 6.3, 12.5, 25.0, 50.0, 100.0 ng ai/bee nominal Dose Levels Oral 0.7, 1.4, 2.7, 5.4, 8.5, 10.8 ng ai/bee nominal

Analytical measurements Not required

The acute contact and oral effects of the test substance clothianidin FS 600 G uncoloured on the honeybee were determined in a 48 hours test in the laboratory. Contact: At the end of the contact test mortality ranged from 3.3 (6.3 ng ai/bee) up to 86.7% at the highest dose rate. Mortality in the control group was 6.7%. During the first 4 hours abnormal behaviour (movement coordination, apathy or vomiting) were observed at the dose rates 12.5 ng ai/bee and above. These effects slowly reduced and were observed in the two highest dose rates only after 48 h. No abnormalities in behaviour were found in the two lowest dose

Study Summary rates. LD50 is 44.1 ng ai/bee Oral: No mortality was observed in the control group and 0.7 and 1.4 of the treated groups. Mortality in the other treated groups ranged from 6.7% up to 100%. The maximum nominal dose rates could not be achieved as the bees did not ingest the full volume of the sugar solution. During the first 4 hours abnormal behaviour (movement coordination and/or apathy) was observed in the four highest dose rates. No further behavioural abnormalities occurred until

the end of the test. LD50 is 4.7 ng ai/bee. Validity criteria were met: Mortality in control < 10% (observed value: contact 6.7%, oral 0%)

November 2015 192

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

LD50 (24h) of the reference substance in the range 0.10-0.30 µg ai/bee (contact) and 0.10-0.35 µg ai/bee (oral). Observed values 0.18 and 0.17 µg ai/bee by contact and oral exposure, respectively.

LD50 = 44.1 ng ai/bee contact (CI 22.2-74.2 ng ai/bee), Conclusion LD50 = 4.7 ng ai/bee oral (CI 4.2-5.2 ng ai/bee)

Type of study Full test

Flag Key study

Test Substance Clothianidin

Species Apis mellifera

Type of exposure 3 d via food

Endpoint NOEC

Value > 40 µg ai/ kg diet

Ch. Maus (2009) Clothianidin technical: Effects of exposure to spiked diet on honeybee (Apis mellifera carnica) larvae in an in vitro laboratory testing design. Reference Bayer Cropscience AG BCS-D-EnSa Ecotoxicology Monheim Germany. Report no MAUS/AM049/1 Bayer ID M-359395-02-1

Klimisch Score 1

Amendments/Deviations NA

GLP Yes

None available, study design according to recommendations of bee larval ring Test Guideline/s test group by ICPBR (Aupinel et al 2009)

No/Group Test run 1 and 4 48 larvae, test run 3 37 larvae

Dose Levels 5, 10, 20, and 400 µg ai/ kg diet

Analytical measurements Method 1H-NMR

The purpose of the study was to assess the effects of clothianidin on honeybee larvae (Apis mellifea carnica) after artificial feeding of spiked diet in an in vitro laboratory testing design. Bee larvae were fed with standardised amounts of untreated artificial diet at day 1 and day 3. Bee larvae were fed with Study Summary standardised amounts of spiked food on days 4, 5 and 6. The test substance was incorporated into artificial food at the nominal test concentrations of 5, 10, 20 and 40 µg ai/kg diet. Dimethoate was used as toxic standard in a dose rate of 3 µg ai/ larva. Mortality was observed on day 5,6,7,8,11,13,15 and 22.

November 2015 193

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

In test run1 no mortality was observed in the treatments up to 20 µg ai/ kg diet. At the highest dose rate 2.4% mortality was observed. Control had 12.5% mortality. In test run 3 mortality in control was 21.6%. At dose rates 5 and 20 µg ai/ kg diet 24.1% mortality was observed. Mortality was 3.4% at 10 µg ai/ kg diet and 31% at 40 µg ai/ kg diet. In test run 4 mortality in control was 18.8%. At dose rates 5 µg ai/ kg diet 2.61% mortality was observed. Mortality was 15.4% at 10 µg ai/ kg diet and 20.5% at 20 µg ai/ kg diet and 25.6% at 40 µg ai/ kg diet. No statistically differences between the control and test substance were detected. NOEC and LOEC are calculated to be > 40 µg ai/ kg diet. The validity criteria were met for test runs 1, 3 and 4. Mortality control until day 7 < 15%, until day 22 <30% (in test run 2 31%) and mortality references until day 7 > 50%.

Conclusion NOEC > 40 µg ai/ kg diet

Type of study Full test

Flag Key study

Test Substance Clothianidin

Species Bombus terrestris

Type of exposure 96 h, oral and contact

Endpoint LD50

Value Contact 145.1 ng ai/bee, oral 1.943 ng ai/bee

S. Harkin (2014) Clothianidin Acute contact and oral toxicity to bumblebee (Bombus terrestris). Food and Environmental Research Agency, Centre for Reference chemical safety and stewardship Sand Hutton, York UK. Report no B2AK1000 Bayer ID M-504345-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results of the test

GLP Yes

Test Guideline/s To the principles of Van der Steen draft OECD 213 and 214

No/Group 3 replicates of 10 bees per treatment

Dose Levels Contact 18.8, 37.5, 75, 150, 300 ng ai/bee

November 2015 194

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Oral 1.9, 3.75, 7.5, 15, 30 ng ai/bee, measured uptake 1.7, 3.6, 4.0, 8.9, 19.6 ng ai/bee

Analytical measurements Not required

The acute contact and oral effects of the test substance clothianidin on the bumblebee were determined in a 96 hours test in the laboratory. Contact test: No mortality was observed in the control group. At the end of the contact test mortality ranged from 3% (18.8 ng ai/bee) up to 80% at the highest dose rate. There was a clear dose response effect. The

LD50 is 145.1 ng ai/bee (CI 106- 220.1 ng ai/bee). Oral test: In the control (acetone) 3% mortality was observed and in the control (wetting agent) 7%. At the end of the oral test mortality ranged from 34 up to

Study Summary 100% in the three highest dose rate groups. LD50 is 1.943 ng ai/bee (CI 1.595- 32.242 ng ai/bee). During 24 hours after treatment movement coordination problems and apathy were observed in the treatment groups with the exception of the lowest dose rate of 0.3 ng ai/bee. Validity criteria were met: Mortality in control < 10% (observed value: contact 0%, oral max. 7%)

LD50 (24h) of the reference substance in the range 2-10 µg ai/bee (contact) and 0.5-2.5 µg ai/bee (oral). Observed values 4.442 and 0.5825 µg ai/bee by contact and oral exposure, respectively.

LD50 = 145.1 ng ai/bee (contact) Conclusion LD50 = 1.943 ng ai/bee (oral)

Chronic toxicity tests

Type of study Full test

Flag Key study

Test Substance Clothianidin

Species Apis mellifera

Type of exposure 10 d

Endpoint NOEC

Value 10 µg ai/ L sugar solution

A. Kling (2005) Assessment of chronic effects of clothianidin to the honey bee (Apis mellifera L.) in a 10 days laboratory test. GAB Biotechnologie GmbH & Reference GAB Analytik GmbH D-75223 Niefern-Oschelbronn Germany. Report no 20051186/01-BLEU Bayer ID M-255911-02-1

Klimisch Score 1

November 2015 195

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Amendments/Deviations NA

GLP Yes

Test Guideline/s Internal method

30 replicates of 10 bees per treatment, 60 replicates of 10 bees for the control No/Group group

Dose Levels 10, 20, 50 and 100 µg ai/ L sugar solution

Method 00554/M001, reversed phase HPLC with turbo-ionspray MS/MS Analytical measurements detection

The chronic effects of the test substance clothianidin on the honeybee were determined in a 10 days continuous feeding in the laboratory. Bees were exposed to 50% sugar solution containing 4 concentrations of clothianidin. Tested dose rates: 10, 20, 50 and 100 µg ai/ L sugar solution. Mortality was daily observed and NOEC was determined at the end of the test. At the end of the test 12.35% of the control group died. The corrected mortality Study Summary in the treated groups were 0.74%, 14.06%, 43.72% and 87.45% for 10, 20, 50 and 100 µg ai/ L sugar solution respectively. The mortality of 20 µg ai/ L sugar solution and above was significantly different compared to the control group. The NOEC is 10 µg ai/ L sugar solution. The mean food consumption in the control group was 49 to 65 mg /day and in the treatment groups the consumption ranged from 39 to 78 mg/day.

Conclusion NOEC = 10 µg ai/ L sugar solution or 0.0038 µg a.i./bee

Bees – Extended laboratory test

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance TI-435 FS 600

Species Apis mellifera

Type of exposure Residues in maize pollen

Endpoint Mortality and foraging activity

Mortality was slightly higher in the treatment but comparable with the control. Value No effects of foraging activity was observed.

Ch. Maus (2002) Evaluation of the effects of residues of TI-435 in maize pollen from dressed seeds on honeybees (Apis mellifera L.) in the semi field. Bayer Reference AG and Crop Protection Institute for Environmental Biology Leverkusen Germany. Report no MAUS/AM 017 Bayer ID M-043264-01-1

November 2015 196

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 1

Amendments/Deviations NA

GLP Yes

Test Guideline/s Internal testing method

No/Group 3 replicates, small hives of ca 500 bees per replicate

Dose Levels 1 g ai/ 1000 seeds

Analytical measurements Not required

Maize seed was treated with TI-435 FS 600 at a rate of 1 g ai/1000 seeds and maize pollen were grown. Small honeybee colonies (ca 500 bees) were confined on oat plots in tunnels and fed with maize pollen containing grown residues of TI-435 or untreated pollen. Mortality and feeding activity were observed. Further comb cell production, food consumption, pollen and honey storages, egg laying activity, breeding success, colony strength and hive weight development were assessed. Effects were assessed over a period of 52 days. The average number of dead bees in front of the hive and at the tunnel edges was 0.67 and 28 respectively for the control, and 5.33 and 46 for the treatment. The mortality was within the usual range and comparable in control and Study Summary treatment. There were no significant differences between the control and treatment in comb cell production, honey consumption (691and 682 g respectively), hive weight increase (25 % and 23% respectively), pollen and honey stores, egg deposition, abundance of larval, pupal and adult bees. Pollen consumption was significant higher in the treatment (29 g) than in the control (10 g). This can be explained by the different granulation of control and treatment pollen. The treatment pollen were finer and easier to transport. Foraging activity was comparable and no abnormal behaviour was observed. The residue levels of TI 435 were below the limit of quantitation (LOQ = 0.001 mg/kg) in pollen.

No adverse effects on honeybees were observed from foraging and Conclusion consumption of maize pollen of plants originating from seeds dressed with TI435 FS 600 at a rate of 1 g ai/ 1000 seeds.

Bees – field tests

Type of study Full test

Flag Key study

Test Substance TI-435 metabolite TZMU

November 2015 197

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Species Apis mellifera

Type of exposure 8 days

Endpoint Mortality, food collection, weight gain

No adverse effects on mortality and food collection, weight gain was reduced Value compared with the control but not treatment related

Ch. Maus, R.Schoning (2001) Effect of diet (sugar solution) spiked with the TI- 435 metabolite TZMU on behaviour and mortality of honey bees (Apis mellifera) and on the weight development of bee colonies under field conditions. Bayer Reference AG Crop Protection Development Institute for Environmental Biology D-51368 Leverkusen-Bayerwerk Germany. Report no MAUS/AM 010 Bayer ID M- 031721-01-1

Klimisch Score 1

Amendments/Deviations NA

GLP Yes

Test Guideline/s Internal test method

No/Group 1 hive per treatment (5000-10000 bees)

Dose Levels Syrup spiked with 10 and 20 µg/kg TZMU

Analytical measurements Not required

The effects of TZMU, a metabolite of TI-435 were tested on honey bees under field conditions. The hives of the different treatments were location near different villages. A diet of sugar solution containing 0, 10 or 20 µg/kg TZMU was offered to bee colonies (approx. 5000-10000 honeybees) as food source. The syrup was provided in feeders in a distance of ca. 150 m from the hives and no other food sources were available for the bees. Bees foraging at the feeders were daily marked with a specific colour and assessed. The number of marked bees arriving at the hives was recorded. The quantity of syrup consumed was determined by regularly weighing of the feeders. Bee mortality Study Summary was assessed in front of the hives, weight development and behavioural abnormalities were recorded. No differences in mortality were observed between the control and the treatments (24 bees control and 29 bees highest dose rate). The weight gain was reduced of the hives treated with TZMU spiked diet compared to the control. This was caused by an oilseed rape field in the environs of the test site which was used as an additional food source. As there were no differences in collection of treated and untreated syrup and syrup consumption it was concluded that the weight reduction was not treatment related.

November 2015 198

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The average numbers of marked bees recorded as arriving at the hives or feeding on the syrup sources were highest in the 20 µg/kg TZMU and lowest in the control group. No adverse effects on orientation and behaviour were recorded.

No differences in mortality, food collection and consumption and behaviour were observed. The weight gain of the hives with the spiked Conclusion diet was reduced compared to the control but not treatment related as the control group had an additional food source.

Type of study Full test

Flag Key study

Test Substance TI-435 metabolite TZMU

Species Apis mellifera

Type of exposure 6 days

Endpoint Mortality, food collection, weight gain

The metabolite TZMU had no adverse effects on the honeybee mortality, Value foraging activity, weight gain and behaviour at the concentrations tested.

Ch. Maus, J.Doering (2003) Effect of diet (sugar solution) spiked with the clothianidin metabolite TZMU on behaviour and mortality of honey bees (Apis Reference mellifera) and on the weight development of bee colonies under field conditions. Bayer Cropscience AG Institute for Ecotoxicology 40789 Monheim Germany. Report no MAUS/AM 024 Bayer ID M-076068-01-1

Klimisch Score 1

Amendments/Deviations NA

GLP Yes

Test Guideline/s Internal test method

No/Group 1 hive per treatment (5000-6000 bees)

Dose Levels Syrup spiked with 10 and 20 µg/kg TZMU

Analytical measurements Not required

The effects of TZMU, a metabolite of TI-435 were tested on honey bees under field conditions. The hives of the different treatments were location near Study Summary different villages. A diet of sugar solution containing 0, 10 or 20 µg/kg TZMU was offered to bee colonies (approx. 5000-6000 honeybees) as food source.

November 2015 199

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The syrup was provided in feeders in a distance of ca. 165 m from the hives and no other food sources were available for the bees. Bees foraging at the feeders were marked with a specific colour and assessed every 15 minutes on 4 assessment days. The number of marked bees arriving at the hives was recorded. The quantity of syrup consumed was determined by regularly weighing of the feeders. Bee mortality was assessed in front of the hives, weight development and behavioural abnormalities were recorded. No differences in mortality were observed between the control and the treatments (39 bees control and 30 bees highest dose rate). The weight gain was 24.6% in the control group, 12.7% in the 10 µg/kg TZMU group and 20.0% in the 20 µg/kg TZMU group. There were no differences in foraging activity between the treatments and the control. The average numbers of marked bees recorded as arriving at the hives or feeding on the syrup sources were comparable and not negatively affected by the treatments. No adverse effects on orientation and behaviour were recorded.

No differences in mortality, weight gain of the hive, food collection and consumption and behaviour were observed. The metabolite TZMU had no Conclusion adverse effects on the honeybee mortality and behaviour at the concentrations tested.

Type of study Limit test

Supporting study as the test substance is a formulation but not Poncho Votivo Flag and the concentration tested is lower than the proposed rate in New Zealand

Test Substance Clothianidin FS 600B G

Species Apis mellifera

Type of exposure 11 days

Endpoint Mortality, behaviour, colony strength and health, brood development

Value No adverse effects were observed during the first year

S. Hecht-Rost (2009) Assessment of side effects of Clothianidin FS600B G treated maize seed on the honey bee (Apis mellifera) in a long term field study Reference in Languedoc Roussilon (France). Eurofins GAB GmbH, Eutinger Str 24 Niefern-Oschelbronn, Germany. Report no 1st interim report S08-01575 Bayer ID M-347416-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results

GLP Yes

November 2015 200

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Guideline/s OEPP/EPPO guideline no 170 (2001)

No/Group 6 hives per treatment (10000-20000 bees/ colony)

Dose Levels 0.5 mg ai/seed nominal

Analytical measurements Not required

The side effects of with clothianidin treated maize seed were tested on honey bees under field conditions during a multi-year test. Treated and untreated maize seeds were sown at a seeding rate of approx. 30 kg/ha in the region of Languedoc-Roussillon in France in 2008. The plots were separated approx. 2.5 km. Six honeybee colonies per treatment group were exposed to the flowering maize fields for 11 days. After flowering of the maize the colonies were relocated and maintained at a monitoring site for overwintering. The following observations were made: mortality in front of the hive and in the field during exposure time, flight and foraging activity, behaviour of the bees at the hive Study Summary entrance, assessment of the bee brood and strength of the colonies and colony development (disease and weight). In the first year of observations no adverse effects on colony health, brood development and food storage behaviour were observed. Also no differences in mortality, flight and foraging activity were observed between treatment and the control. No treatment related differences in colony strength and abnormal behaviour were detected. Low residue levels were detected in the pollen samples taken from forager bees and from plants (0.003 mg/kg). No residues of metabolites were detected. No residues were detected in any of the control samples.

In a separate report the results of the bee disease analysis were presented (reference Final interim bee disease analysis report: report S08-01575-L3 Bayer ID M 351028-01-1). The health of the colonies was determined by analysing bee diseases. The presence of Nosema sp., Malphigamoeba mellificae, Varroa destructor and Paenibacillus larvae was determined in bee samples at different time points Colony health: diseases during the year. Nosema: In all colonies Nosema sp spores were found before exposure. In all control colonies spores were found after exposure. In three treatment colonies spores were detected, in two treatment colonies no spores were found and from one colony no sample was available. No treatment related effect could be determined. Malpighamoeba: the results of the test were all negative.

November 2015 201

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Varroa: Mites were found in all samples before and after exposure. Before exposure the infestation levels ranged from 0.9 to 5.7% and after exposure from 1.7 to 15.9%. Before overwintering the levels reduced to low. No difference between control and treatment were observed. Paenibacillus: no spores were found in the majority of the colonies. Only spores were found in one colony of the treatment.

In a separate report the results of the bee virus analysis were presented (reference Final interim bee disease analysis report: report S08-01575-L4 Bayer ID M 351637-01-1). For the virus analysis samples of 15 bees were taken and only their heads were used for further analysis. All samples were analysed for deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV) and chronic Colony health: virus bee paralysis virus (CBPV). ABPV and CBPV were not detected in any of the samples. DWV was detected in three control and 5 treatment samples at the start of exposure. At the start of overwintering DWV was detected in one control and two treatment samples. SBV was detected in three control and treatment samples at the start of exposure and not in any samples at the start of overwintering.

Conclusion During first year no treatment related effects were observed.

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Apis mellifera

Type of exposure 10 days

Endpoint Mortality, behaviour, colony strength and health, brood development

Value No adverse effects were observed during the first year

S. Hecht-Rost (2009) Assessment of side effects of Clothianidin FS600B G treated maize seed on the honey bee (Apis mellifera) in a long term field study Reference in Champagne (France). Eurofins GAB GmbH, Eutinger Str 24 Niefern- Oschelbronn, Germany. Report no 1st interim report S08-01573 Bayer ID M- 347482-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results

November 2015 202

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

GLP Yes

Test Guideline/s OEPP/EPPO guideline no 170 (2001)

No/Group 6 hives per treatment (10000-20000 bees/ colony)

Dose Levels 0.5 mg ai/seed nominal

Analytical measurements Not required

The side effects of with clothianidin treated maize seed were tested on honey bees under field conditions during a multi-year test. Treated and untreated maize seeds were sown at a seeding rate of approx. 30 kg/ha in the region of Languedoc-Roussillon in France in 2008. The plots were separated approx. 2 km. Six honeybee colonies per treatment group were exposed to the flowering maize fields for 10 days. After flowering of the maize the colonies were relocated and maintained at a monitoring site for overwintering. The following observations were made: mortality in front of the hive and in the field during exposure time, flight and foraging activity, behaviour of the bees at the hive entrance, assessment of the bee brood and strength of the colonies and colony Study Summary development (disease and weight). In the first year of observations no adverse effects on colony health, brood development and food storage behaviour were observed. Also no differences in mortality, flight and foraging activity were observed between treatment and the control. No treatment related differences in colony strength and abnormal behaviour were detected. Low residue levels were detected in the pollen samples taken from plants (0.001 mg/kg). No residues of metabolites were detected. No residues were detected in any of the control samples. The amount of pollen that could be sampled from bees was too low to be analysed.

In a separate report the results of the bee disease analysis were presented (reference Final interim bee disease analysis report: report S08-01573-L3 Bayer ID M 351027-01-1). The health of the colonies was determined by analysing bee diseases. The presence of Nosema sp., Malphigamoeba mellificae, Varroa destructor and Colony health: diseases Paenibacillus larvae was determined in bee samples at different time points during the year. Nosema: In all colonies Nosema sp. spores were found before exposure. After exposure in one colony of the treatment up to a low level of spores were found. In three control colonies a low amount of spores was found after exposure. At

November 2015 203

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

the start of overwintering up to a low level of spores was found in one of the control colonies. In all the other treatment no spores were detected. Malpighamoeba: the results of the test were all negative. Varroa: Mites were found on a low level in all samples before and after exposure. Before exposure the infestation levels ranged from 0 to 0.9% and after exposure from 0 to 1.1%. Before overwintering the levels reduced to a maximum of 0.6%. No difference between control and treatment were observed. Paenibacillus: no spores were found in any of the colonies.

In a separate report the results of the bee virus analysis were presented (reference Final interim bee disease analysis report: report S08-01573-L4 Bayer ID M 351636-01-1). For the virus analysis samples of 15 bees were taken and only their heads were Colony health: virus used for further analysis. All samples were analysed for deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV) and chronic bee paralysis virus (CBPV). DWV, ABPV and CBPV were not detected in any of the samples. SBV was detected once in the control group at the start of exposure phase.

Conclusion During first year no treatment related effects were observed.

Type of study Limit test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Apis mellifera

Type of exposure 11 days

Endpoint Mortality, behaviour, colony strength and health, brood development

Value No adverse effects were observed during the first year

S. Hecht-Rost (2009) Assessment of side effects of Clothianidin FS600B G treated maize seed on the honey bee (Apis mellifera) in a long term field study Reference in Alsace (France). Eurofins GAB GmbH, Eutinger Str 24 Niefern-Oschelbronn, Germany. Report no 1st interim report S08-01572 Bayer ID M-347491-01-1

Klimisch Score 1

Amendments/Deviations None that impacted the results

GLP Yes

Test Guideline/s OEPP/EPPO guideline no 170 (2001)

November 2015 204

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

No/Group 6 hives per treatment (10000-20000 bees/ colony)

Dose Levels 0.5 mg ai/seed nominal

Analytical measurements Not required

The side effects of with clothianidin treated maize seed were tested on honey bees under field conditions during a multi-year test. Treated and untreated maize seeds were sown at a seeding rate of approx. 30 kg/ha in the region of Languedoc-Roussillon in France in 2008. The plots were separated approx. 4 km. Six honeybee colonies per treatment group were exposed to the flowering maize fields for 10 days. After flowering of the maize the colonies were relocated and maintained at a monitoring site for overwintering. The following observations were made: mortality in front of the hive and in the field during exposure time, flight and foraging activity, behaviour of the bees at the hive entrance, assessment of the bee brood and strength of the colonies and colony Study Summary development (disease and weight). In the first year of observations no adverse effects on colony health, brood development and food storage behaviour were observed. Also no differences in mortality, flight and foraging activity were observed between treatment and the control. No treatment related differences in colony strength and abnormal behaviour were detected. Low residue levels were detected in the pollen samples taken from plants (0.005 mg/kg). In the samples taken from the bees 0.003 mg/kg was detected. No residues of metabolites were detected. No residues were detected in any of the control samples.

In a separate report the results of the bee disease analysis were presented (reference Final interim bee disease analysis report: report S08-01572-L3 Bayer ID M 351010-01-1). The health of the colonies was determined by analysing bee diseases. The presence of Nosema sp., Malphigamoeba mellificae, Varroa destructor and Paenibacillus larvae was determined in bee samples at different time points during the year. Colony health: diseases Nosema: In all colonies Nosema sp. spores were found before exposure. After exposure in two colonies of the treatment no spores were found. In all the other samples spores were found. At the start of overwintering no spores were found at all. Malpighamoeba: the results of the test were all negative. Varroa: Mites were not found in 5 treatments before exposure and 5 samples after exposure. In the other samples infestation levels ranged from 0.3 to 1.9%

November 2015 205

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

before exposure, from 0.3 to 4.0% after exposure. Before overwintering the levels ranged from 0.2 to 9.1%. Paenibacillus: no spores were found in any of the colonies.

In a separate report the results of the bee virus analysis were presented (reference Final interim bee disease analysis report: report S08-01572-L4 Bayer ID M 351635-01-1). For the virus analysis samples of 15 bees were taken and only their heads were Colony health: virus used for further analysis. All samples were analysed for deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV) and chronic bee paralysis virus (CBPV). DWV, SBV, ABPV and CBPV were not detected in any of the samples.

Conclusion During first year no treatment related effects were observed.

Type of study Full test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance Clothianidin FS 600B G

Species Apis mellifera

Type of exposure 7 days

Endpoint NOER

NOER mortality < 0.5 g ai/ha dust, > 4 g ai/ha spray Value NOER foraging > 4 g ai/ha dust, 2 g ai/ha spray

F Bakker (2010) Exposure of bees under semi-field conditions to dust abraded from maize seeds dressed with the seed dressing product Clothianidin FS 600 Reference and to Clothianidin FS 600 when applied as liquid formulation to flowering Phacelia crop. MITOX Consultants, Science Park 406 Amsterdam The Netherlands. Report no BAB152AMS Bayer ID M-362814-01-1

Klimisch Score 1

Amendments/Deviations NA

GLP Yes

Not available, study followed recommendations of EPPO/ICBPR guidance for Test Guideline/s semi-field tests, ENV/JM/MOMO(2007)22 OECD no 75, 2003 OEPP/EPPO bulletin 33, PP3/10 revised.

No/Group 3 replicates per treatment, 5 controls.

Dose Levels 0.5, 2 and 4 g ai/ha

Analytical measurements Not required

November 2015 206

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The aim of the test was to assess the effects of clothianidin applied as seed treatment (dust) and applied as a spray formulation. The applied dose rates were 0.5, 2 and 4 g ai/ha. Abrasion dust obtained from treated seeds was applied with a small particle dispenser. The spray formulation was applied with a hand held compression sprayer with a target volume of 300 L/ha. Homogeneous spatial distribution was ensured by moving the equipment along the cages’ sides. Screen cages with a mini-hive (1 queen, 4 occupied frames ±5000 bees) were placed in the field. In the cages flowering Phacelia was present. Bees foraged Study Summary for a period of 6 days before the treatments and during 7 days exposure. Mortality, flight activity and activity at hive entrance were recorded. Total mortality (bee trap and cloth) of the dust treatment (4 g ai/ha) differed statistically from the control. Further the number of dead bees in the bee traps differed significant from the control for all rates of the dust treatment. The mortality of the spray application did not differ statistically significant from the control. However at the highest rate foraging activity was significant lower. These results suggest a NOER <0.5 g ai/ha for the dust treatment based on mortality and a NOER of 2 g ai/ha for the spray treatment based on foraging. No effects of the treatment on colony condition were observed.

Exposure to dust particles had stronger effects on adult mortality than exposed to spray deposits with comparable amounts of active ingredient. Conclusion NOER mortality < 0.5 g ai/ha dust, > 4 g ai/ha spray NOER foraging > 4 g ai/ha dust, 2 g ai/ha spray

Other non-target arthropods - Extended laboratory tests

Type of study Limited test

Flag Supporting study as the test substance is a formulation but not Poncho Votivo

Test Substance TI-435 FS 600 (formulation containing clothianidin)

Species Poecilus cupreus carabid beetle

Type of exposure To treated maize seed

Endpoint Mortality

Value 13.3%

R.Schmuck (2003) Acute effects of maize seed treated with TI-435 FS 600 on carabid beetles (Poecilus cupreus) under extended laboratory test conditions. Reference Bayer CropScience AG Institute for ecotoxicology, D 40789 Monheim (Germany) Report no SXR/EL PC 048 Bayer ID M-022416-02-1, 31 January 2003

November 2015 207

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Klimisch Score 1

Amendments/Deviations None

GLP Yes

Test Guideline/s BBA VI 23-2.1.8 modified for testing seed dressing material

No/Group 30 adult beetles per treatment

43 g ai/ U (U = 50,000 seeds) Dose Levels Toxic reference (parathion-methyl) 74.5 kg/ha bait formulation

Analytical measurements Not required

The aim of the study was to determine the toxicity of maize seed treated with TI-435 FS 600 (43 g ai/U) to the carabid beetle Poecilus cupreus. The treated seeds were drilled in natural soil as test substrate. Adult beetles were exposed for 14 days. Mortality and feeding activity were regularly observed. In the control group all beetles survived and no behavioural abnormalities were Study Summary observed. TI-435 FS 600 caused 13.3% mortality (significant difference compared with the control) and about one third of the beetles displayed signs of intoxication. The reference treatment caused 66.7% mortality and most of the beetles showed behavioural effects. No significant difference in feeding activity was observed between TI 435 FS 600 and the control.

Seed treated with TI-435 FS 600 (43 g ai/ 50 000 seeds) resulted in 13.3% Conclusion mortality which is significant higher than the control. No significant effect in feeding activity.

Residue levels

The applicant provided additional information regarding residue levels in different matrices. Brief summaries of these data related to maize are presented below.

Residue levels

Reference G.A.Hancock, T.Gould (2001) TI-435 residue levels in corn seeds and seedlings. Bayer Corporation Agriculture Division, Research and development Department Environmental Research Section, 17745 South Metcalf Avenue, Stilwell Kansas. Report no 110162, Bayer ID M-041825-01-1

Corn seeds were treated with TI-435 600 FS at a nominal rate of 2.0 mg Summary ai/seed. In one trial treated corn seeds were spread over the ground to simulate a spill and sampled at 0, 1, 3, 7 and 14 days for determination of residues. The

November 2015 208

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

residues were used to calculate a dissipation half-life. The seeds were sown at five different locations in the USA. In another trial treated seedlings were planted and harvested at the 2-3 leaf stage for determination of residues in seedling plants. The same 5 locations were used. Samples were analysed using LC/MS-MS.

Corn seed: Residues day 0 ranged from 5471 to 6640 ppm and on day14 from 835 to 1175 ppm. Half-lives ranged from 4.7 to 8.7 days when rainfall occurred. Conclusion Without rainfall half-life was > 14 days. Corn seedlings: Residues in parts above ground ranged from 3 to 45 ppm, underground parts (in/on seeds) residues ranged from 106 to 630 ppm.

Remark The proposed rate in NZ is 0.76 mg ai/seed.

Reference Ch. Wolf (2004) Residues of clothianidin in maize seedlings (dressed with Poncho Pro FS 600, 1.25 mg ai/kernel) grown under field conditions in Austria. Bayer Cropscience AG, Institute for ecotoxicology Monheim Germany. Report no WFC/FS 016, Bayer ID M-182258-01-1

Corn seeds were treated with Poncho Pro FS 600 at a nominal rate of 1.25 mg ai/seed. At the crop growth stage of BBCH 12 and 14 maize seedlings were sampled to determine the residues of clothianidin in the seedlings. Untreated seedlings were also sampled. The residues were quantified by reversed phase Summary HPLC with electrospray MS/MS detection. At BBCH 12 the average residue was 15.5 mg/kg plant and at BBCH 14 3.58 mg /kg plant. In the control plants the residue was below the limit of quantitation (LOQ= 0.001 mg/kg).

The proposed rate in NZ is 0.76 mg ai/seed. Remark This study is used to refine the risk assessment for birds.

Reference Ch. Wolf (2005) Residues of clothianidin in maize seedlings (dressed with Poncho FS 600, 0.5 mg ai/kernel) grown under field conditions in Austria. Bayer Cropscience AG, Institute for ecotoxicology Monheim Germany. Report no WFC/FS 015, Bayer ID M-242633-01-1

Corn seeds were treated with Poncho FS 600 at a nominal rate of 0.5 mg ai/seed. The seeds were drilled on two different test plots. At the crop growth stage of BBCH 12 and 14 maize seedlings were sampled to determine the Summary residues of clothianidin in the seedlings. Untreated seedlings were also sampled. The residues were quantified by revered phase HPLC with electrospray MS/MS detection.

November 2015 209

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

At BBCH 12 the average residue was at site 1 11.6 and at the site 2 16.0 mg/kg plant and at BBCH 14 2.91 and 4.54 mg /kg plant respectively. In the control plants the residue was below the limit of quantitation (LOQ= 0.001 mg/kg).

The proposed rate in NZ is 0.76 mg ai/seed. Remark This study is used to refine the risk assessment for birds

Reference Ch. Maus (2005) Determination of residue levels of clothianidin, TZMU and TZNG in maize pollen in a succeeding crop scenario at Bayer Cropscience AG experimental farm “Laacher Hof’ Germany. Bayer Cropscience AG Ecotoxicology Monheim, Germany Report no MAUS/AM033, Bayer ID M- 256474-01-1

Clothianidin FS 600 was applied and incorporated into the soil of two test plots at a rate of 90 g ai/ha to cover a plateau concentration after long term use. Two other test plots remained without any clothianidin spray application. After an ageing period of 42 days either untreated seed or clothianidin treated maize seeds were sown. The dose rate was 0.5 mg ai/kernel. So there is a control (untreated soil and seed), treatment 1 untreated soil and treated seed, treatment 2 treated soil and seed and treatment 3 treated soil and untreated seed. Summary During the flowering period of maize the pollen were collected and analysed to determine the residues of clothianidin and its metabolites TZMU and TZNG. In the control the residue levels were below the level of quantitation of clothianidin and its metabolites (LOQ = 0.001 mg/kg). In the treatment objects the residue levels of the metabolites was below LOQ. Clothianidin was detected in a concentration of 0.0018 mg ai/kg pollen in treatment 1 (untreated soil, treated seed). In treatment 2 0.0019 mg ai/kg pollen was determined and in treatment 3 the concentration was below LOQ.

Analytically verified soil concentration of 19.2 µg clothianidin/ kg dry soil did not result in a quantifiable uptake and translocation of clothianidin into pollen of Conclusion untreated maize seed. Clothianidin residues in pollen from treated seed in pre- treated soil were in the same order of magnitude as residues in pollen from treated seed in untreated soil.

Reference Ch. Maus (2005) Determination of residue levels of clothianidin, TZMU and TZNG in maize pollen in a succeeding crop scenario at Bayer Cropscience AG experimental farm “Hofchen’ Germany. Bayer Cropscience AG Ecotoxicology Monheim, Germany Report no MAUS/AM035, Bayer ID M-256564-01-1

November 2015 210

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Clothianidin FS 600 was applied and incorporated into the soil of two test plots at a rate of 90 g ai/ha to cover a plateau concentration after long term use. Two other test plots remained without any clothianidin spray application. After an ageing period of 55 days either untreated seed or clothianidin treated maize seeds were sown. The dose rate was 0.5 mg ai/kernel. So there is a control (untreated soil and seed), treatment 1 untreated soil and treated seed, treatment 2 treated soil and seed and treatment 3 treated soil and untreated seed. Summary During the flowering period of maize the pollen were collected and analysed to determine the residues of clothianidin and its metabolites TZMU and TZNG. In the control the residue levels were below the level of quantitation of clothianidin and its metabolites (LOQ = 0.001 mg/kg). In the treatment objects the residue levels of the metabolites was below LOQ. Clothianidin was detected in a concentration of 0.0012 mg ai/kg pollen in treatment 1 (untreated soil, treated seed). In treatment 2 0.0013 mg ai/kg pollen was determined and in treatment 3 the concentration was below LOQ.

Analytically verified soil concentration of 18 µg clothianidin/ kg dry soil did not result in a quantifiable uptake and translocation of clothianidin into pollen of Conclusion untreated maize seed. Clothianidin residues in pollen from treated seed in pre- treated soil were in the same order of magnitude as residues in pollen from treated seed in untreated soil.

Reference R. Schoning, M. Telscher (2008) Determination of residues of clothianidin, and its metabolites TZMU and TZNG in pollen, harvested from maize plants grown in commercial practice from Poncho Pro dressed seeds (nominally 1.25 mg clothianidin/seed) in the Upper Rhine Valley in Germany. Bayer Cropscience AG Development-residues, Operator and Consumer safety Monheim am Rhein, Germany Report no MR-08/167, Bayer ID M-309799-01-1

The purpose of the study was to determine the residues of clothianidin and its metabolites TZMU and TZNG in pollen harvested from maize plants in the Upper Rhine Valley in Germany. Maize plants treated with Poncho Pro (nominal 1.25 mg ai/seed) were grown in Summary commercial practice. Pollen samples were taken directly from corn tassels by shaking the corn tassel, the weight was between 1 and 5 g. Other samples were taken from pollen traps from bee hives exposed to treated corn, weights up to 100 g. In total 240 pollen samples were taken.

November 2015 211

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Residues in the pollen were extracted using a mixture of acetonitrile/water (4:1 v/v) in an ultrasonic bath. The residues were quantified by reversed phase HPLC with turbo ionspray MS/MS detection. The level of quantitation for clothianidin and its metabolites TZMU and TZNG is 0.001 mg/per kg and the level of detection is 0.0003 mg/kg.

Residues of clothianidin found in the pollen ranged from < LOD to 11.4 µg/kg Conclusion with a mean of 3.6 µg/kg. Residues of the metabolites TZMU and TZNG per kg were below the LOQ with the exception of two samples with levels of 1.0 µg/kg.

Reference T. Staedtler (2009) Determination of residue levels of clothianidin, and its metabolites TZMU and TZNG in pollen, harvested from maize plants grown in commercial practice from Poncho Pro dressed seeds (nominally 1.25 mg clothianidin/seed) in the Upper Rhine Valley in Germany. RIFCon GmbH Heidelberg, Germany Report no R08-188-2, Bayer ID M-309823-02-1

The purpose of the study is to determine the residues of clothianidin and its metabolites TZMU and TZNG in pollen harvested from maize plants in the Upper Rhine Valley in Germany. Maize plants treated with Poncho Pro (nominal 1.25 mg ai/seed) were grown in commercial practice. Pollen samples were taken directly from corn tassels by shaking the corn tassel, the weight was at least 2 g. In total 250 samples from 5 different maize fields were taken. Other samples (total 193) were taken from pollen traps, bee bread and dead bees from 3 bee hives located on 3 fields with treated corn. Residues in the pollen were extracted using a mixture of acetonitrile/water (4:1 v/v) in an ultrasonic bath. The residues were quantified by reversed phase Summary HPLC with turbo ionspray MS/MS detection. The level of quantitation (LOQ) for clothianidin and its metabolites TZMU and TZNG is 0.001 mg/per kg and the level of detection (LOD) is 0.0003 mg/kg. In the pollen directly sampled from maize plants the mean residue level of clothianidin was 3.4 µg/kg (max. 10.4 µg/kg), of TZNG 0.6 µg/kg and of TZMU 0.4 µg/kg. In the pollen sampled from pollen traps the mean residue level of clothianidin was 1.1 µg/kg (max. 11.4 µg/kg), of TZNG 0.4 µg/kg and of TZMU 0.3 µg/kg. In the pollen collected from dead bees the mean level of clothianidin was 0.5 µg/kg, of TZNG 1.2 µg/kg and of TZMU below LOD. In the pollen from bee bread the mean level of clothianidin was 1.0 µg/kg (max. 3.3 µg/kg), of TZNG 0.4 µg/kg and of TZMU below LOD.

November 2015 212

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Irrespective of the sample matrix the residue levels of clothianidin, TZNG and Conclusion TZMU in most of the analysed samples were below the LOQ, except the residue levels of clothianidin in maize pollen sampled directly from maize plants.

Reference C. Classen (2009) Clothianidin FS 600B G: A residue study with clothianidin FS 600B G treated maize seed, investigating residues in crop, soil and honeybee products in Alsace (France). Eurofins-GAB GmbH Niefern-Oschelbronn, Germany Report no S08-02437, Bayer ID M-347727-01-1

The purpose of the study is to determine the residues of clothianidin and its metabolites in crop, soil and honeybee products following the use of clothianidin FS 600B G treated maize seed. Further the exposure to honeybee will be evaluated. The study will be carried out over 2 consecutive years 2008 and 2009. Maize plants treated with Clothianidin FS 600B G (nominal 0.5 mg ai/seed) were grown in commercial practice in the region Alsace in France. Also maize seed not treated with neonicotinoids were sown. The drilling rate was approximately 30 kg seed/ha. Three tunnels were set up prior to the flowering of the maize in the treated field and one in the control field. One honeybee colony was introduced to each tunnel at the start of the flowering. Samples were taken within a period of 11 days of the maize flowering period: whole maize plants (green parts only), pollen from forager bees, and pollen from plants, and wax and pollen samples taken from comb inside the hive. On the last sampling day (11 days after start of Summary exposure) the colonies were relocated to remote location away from extensive use of pesticides. Further wax and pollen samples were taken at a monthly interval until the end of the season. Soil samples were taken within two weeks after incorporation of the maize plants. The limit of quantification (LOQ) is 1 µg/kg for all matrices and the limit of detection (LOD) is 0.3 µg/kg. Residues of clothianidin were detected in all matrices from the treated object except wax. Mean residues in pollen (hives) was 2.51 µg/kg, pollen (plant) 3.22 µg/kg, pollen (forager bees) 4.38 µg/kg, in plants 11.83 µg/kg, and in the soil 1 µg/kg. In few of the control samples low residues levels of clothianidin were detected as well which is most likely caused by contamination after the sampling process. Not quantifiable amounts of the metabolite TZNG were detected in plant samples, pollen from hive, pollen from plant samples and pollen from forager bees. Quantifiable amounts of the metabolite TZMU were detected in few plant samples and not quantifiable amounts were detected from

November 2015 213

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

some plant samples, pollen from hive samples and few pollen from forager bees samples. No metabolites were found on other matrices.

Compared to the control no treatment related effects could be noticed in the total number of bees. In the control colony and two treatment colonies the eggs present at the first brood were removed by the bees resulting in a lack of larvae later on. In general the area containing brood was reduced after the exposure period. Maybe due to the bad pollen supply and the poor adaption to the new environment within the tunnels.

Reference C. Classen (2009) Clothianidin FS 600B G: A residue study with clothianidin FS 600B G treated maize seed, investigating residues in crop, soil and honeybee products in Languedoc-Roussillon (France). Eurofins-GAB GmbH Niefern- Oschelbronn, Germany Report no S08-01377, Bayer ID M-347742-01-1

The purpose of the study is to determine the residues of clothianidin and its metabolites in crop, soil and honeybee products following the use of clothianidin FS 600B G treated maize seed. Further the exposure to honeybee will be evaluated. The study was carried out over 2 consecutive years 2008 and 2009. Maize plants treated with Clothianidin FS 600B G (nominal 0.5 mg ai/seed) were grown in commercial practice in the region Languedoc-Roussillon in France. Also maize seed not treated with neonicotinoids were sown. The drilling rate was approximately 30 kg seed/ha. Three tunnels were set up prior to the flowering of the maize in the treated field and one in the control field. One honeybee colony was introduced to each tunnel at the start of the flowering. Samples were taken within a period of 4 days of the maize flowering period: whole maize plants (green parts only), pollen from Summary forager bees, and pollen from plants, and wax and pollen samples taken from comb inside the hive. After the sampling period the colonies were relocated to remote location away from extensive use of pesticides. Further wax and pollen samples were taken at a monthly interval until the end of the season. Soil samples were taken within two weeks after incorporation of the maize plants. The limit of quantification (LOQ) is 1 µg/kg for all matrices and the limit of detection (LOD) is 0.3 µg/kg. Residues of clothianidin were detected in all matrices from the treated object except wax. Mean residues in pollen (hives) was 0.72 µg/kg, pollen (plant) 1.5 µg/kg, pollen (forager bees) 3.11 µg/kg, in plants 8.78 µg/kg, and in the soil 8 µg/kg. Not quantifiable amounts of the metabolite TZNG were detected in plant

November 2015 214

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

samples, pollen from plant samples and soil samples. Also the metabolite TZMU was detected in one pollen (plant) sample and some plant samples in values below LOQ. No metabolites were found in the other matrices.

Compared to the control no treatment related effects could be noticed in the strength of the colonies. The number of bees was lower in all colonies during the brood assessment after exposure compared to that prior to exposure. Most colonies, except one treated colony, reduced their brood activity during the period of exposure. These effects seen in all colonies (incl. control) were clarified as a result of the confinement of the colonies in tunnels and the incapability of bees to adapt to their new environment.

The proposed rate in NZ is 0.54 to 1.07 mg ai/seed based on 85 000 – 120 000 Remark seeds/ha.

Reference C. Classen (2009) Clothianidin FS 600B G: A residue study with clothianidin FS 600B G treated maize seed, investigating residues in crop, soil and honeybee products in Champagne (France). Eurofins-GAB GmbH Niefern-Oschelbronn, Germany Report no S08-02438, Bayer ID M-347748-01-1

The purpose of the study is to determine the residues of clothianidin and its metabolites in crop, soil and honeybee products following the use of clothianidin FS 600B G treated maize seed. Further the exposure to honeybee will be evaluated. The study will be carried out over 2 consecutive years 2008 and 2009. Maize plants treated with Clothianidin FS 600B G (nominal 0.5 mg ai/seed) were grown in commercial practice in the region Champagne in France. Also maize seed not treated with neonicotinoids were sown. The drilling rate was approximately 30 kg seed/ha. Summary Three tunnels were set up prior to the flowering of the maize in the treated field and one in the control field. One honeybee colony was introduced to each tunnel at the start of the flowering. Samples were taken within a period of 5 days of the maize flowering period: whole maize plants (green parts only), pollen from forager bees, and pollen from plants, and wax and pollen samples taken from comb inside the hive. After the sampling period the colonies were relocated to remote location away from extensive use of pesticides. Further wax and pollen samples were taken at a monthly interval until the end of the season. Soil samples were taken within two weeks after incorporation of the maize plants.

November 2015 215

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The limit of quantification (LOQ) is 1 µg/kg for all matrices and the limit of detection (LOD) is 0.3 µg/kg. Residues of clothianidin were detected in all matrices from the treated object except wax. Mean residues in pollen (hives) was 0.48 µg/kg, pollen (plant) 1.04 µg/kg, pollen (forager bees) 1.14 µg/kg, in plants 6.44 µg/kg, and in the soil 2 µg/kg. Metabolite TZNG was detected in a few maize plants ranging from < 1 µg/kg to 1 µg/kg. Not quantifiable amounts of the metabolite TZMU were detected in plant samples. No metabolites were found in the other matrices.

Compared to the control no treatment related effects could be noticed in the total number of bees per colony. In all colonies the eggs present at the first brood assessments were removed by the bees resulting in a lack of larvae later. In general the area containing brood was reduced after the exposure period in the control and treatment. These effects seen in all colonies (incl. control) were clarified as a result of the confinement of the colonies in tunnels and the incapability of bees to adapt to their new environment.

The proposed rate in NZ is 0.54 to 1.07 mg ai/seed based on 85 000 – 120 000 Remark seeds/ha.

Reference T.Sekine (2009) Determination of clothianidin a.s. residues in honey bees (Apis mellifera L.) after contact and oral application in the laboratory. Institut fur Biologische Analytik und Consulting IBACON GmbH, Arheiler Weg 17, Rossdorf Germany Report no 43951035, Bayer ID M-352033-02-1

The purpose of the study is to determine the residues of clothianidin and its metabolites TZNG and TZMU in honeybees after certain time intervals following oral or contact exposure. Honeybees were exposed for up to 96 hours to clothianidin via oral application to doses of 0.25, 0.7, 6.7, 20.0 ng ai/ bee (nominal). Other bees were exposed for up to 96 hours to doses of 0.32, 1.6, 8.0, 40 and 200 ng ai/bee via topical Summary application. The OECD guidelines 213 and 214 were used for this part of the study. After 0.5, 1, 2, 4, 24, 48 and 96 hours 30 individuals per oral and per contact dosing group were removed, deep frozen and analysed for residues of clothianidin and its metabolites TZMU and TZNG. During exposure mortality was assessed. Mortality

November 2015 216

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Contact test: No mortality was observed in the control group and 8 ng ai/bee. In the groups 0.32, 1.6 and 40 ng ai/bee 3.3% mortality was observed. The dose level 200 ng ai/bee caused 80% mortality. Oral test: No mortality was observed in the control group and 0.9 and 2.4 ng ai/bee groups. In the group of 0.25 ng ai/bee 3.3% mortality was observed, 10% at 7 ng ai/bee and 90% at 22.2 ng ai/bee. Residue levels The individual recovery values for clothianidin ranged from 70 to 109% with mean recoveries of 89%. For TZNG recoveries ranged from 60 to 112% (mean 92%) and for TZMU recoveries ranged from 68 to 100% (mean 86%). All results were in accordance with the general requirements for residue analytical methods, therefore the method was validated successfully. Contact test: The residue levels of clothianidin in the lowest dose rate 0.32 ng ai/bee was 0.15 ng ai/bee after 0.5 hour and decreased below LOQ after 96 hours. The dose rate 1.6 ng ai/bee resulted in a residue of 0.48 ng ai/bee after 0.5 h and decreased to 0.13 ng ai/bee after 96 hours. For 8 ng ai/bee the residue levels were 2.6 ng ai/bee and 0.71 ng ai/bee respectively. For 40 ng ai/bee the residue levels were 14 and 2.6 ng ai/bee respectively and for 200 ng ai/bee 64 and 15 ng ai/bee. The metabolite TZNG was below the LOD or LOQ for the dose rates up to 8 ng ai/bee. At the dose rate of 40 ng ai/bee a maximum residue of 0.18 ng TZNG/bee was detected after 24 hours. At 200 ng ai/bee a maximum residue was detected of 0.87 ng TZNG/bee after 96 hours. The metabolite TZMU was almost always below the LOQ or LOD with the exception of 200 ng ai/bee after 96 hours when 0.1 ng TZMU/bee was detected.

Oral test: The residue levels of clothianidin in the lowest dose rate 0.25 ng ai/bee were below LOQ or LOD with the exception of 0.1 ng ai/bee after 2 hours. The dose rate 0.7 ng ai/bee resulted in a residue of 0.22 ng ai/bee after 0.5 h and decreased to 0.14 ng ai/bee after 2 hours where after the residue levels were below LOQ or LOD. For 2.2 ng ai/bee the residue levels were 1.5 ng ai/bee after 0.5 hour and decreased to 0.27 after 4 hours where after the levels were below LOQ. For 6.7 ng ai/bee the residue levels were 0.67 after 0.5 hour, increased to a maximum of 1.2 ng ai/bee after 1 and 2 hours and decreased below LOQ after 96 hours. The residue levels of the highest rate 20 ng ai/bee were 3.0 ng ai/bee after 0.5 hour, increased to a maximum of 4.2 ng ai/bee after 1 hour and decreased below LOQ after 96 hours.

November 2015 217

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The metabolite TZNG was below the LOD or LOQ for the dose rates up to 2.2 ng ai/bee. At the dose rate of 6.7 ng ai/bee a maximum residue of 0.25 ng TZNG/bee was detected after 4 hours. There after the level decreased below LOQ. At 20 ng ai/bee a maximum residue was detected of 1.1 ng TZNG/bee after 4 and 24 hours and deceased after 24 hours to 0.12 after 96 hours. The metabolite TZMU was always below the LOD.

Reference R.Schoming, L. Krusell, A. Reineke (2009) Guttation monitoring of maize seedlings under agronomic use conditions in Austria and assessment of the relevance of guttation for honeybees. Bayer Cropscience AG, Development- human safety-residue analysis Monheim am Rhein Germany. Report MR- 09/072 Bayer ID M-355004-01-1.

The aim of the study was to determine the residue levels of clothianidin, its metabolites TZNG, TZMU and thiamethoxam (TMO) in the guttation water of maize seedlings, grown from maize seed coated with Poncho or Poncho Pro under commercial conditions at various locations in Austria and in dead adult honeybee workers collected daily from bee hives adjusted to the fields. The study was conducted in two different regions of Austria and 15 commercial field were monitored per region. Samples were analysed using chromatography and detection by MS/MS. The limit of quantitation (LOQ) for clothianidin its metabolites and thiamethoxam was 1.0 ppb in bees and the limit of detection (LOD) was 0.3 ppb. The LOQ in guttation water was 0.01 mg/L and the LOD was 0.001 mg/L. The individual recovery values for clothianidin in bees ranged from 78 to 114% with an overall mean recovery of 95% and a relative standard deviation (RSD) Summary of 9.0% (n = 29). For TZNG in bees the individual recovery values ranged from 67 to 113% with an overall mean recovery of 91% and a relative standard deviation (RSD) of 12.0% (n = 30). For TZMU in bees the individual recovery values ranged from 78 to 100% with an overall mean recovery of 86% and a relative standard deviation (RSD) of 7.8% (n = 30) and for thiamethoxam in bees the individual recovery values ranged from 61 to 113% with an overall mean recovery of 89% and a relative standard deviation (RSD) of 15.4% (n=30). The individual recovery values for clothianidin in guttation water ranged from 93 to 137% with an overall mean recovery of 102% and a relative standard deviation (RSD) of 11.6% (n = 12). For TZNG in guttation water the individual recovery values ranged from 86 to 130% with an overall mean recovery of 98% and a relative standard deviation (RSD) of 12.7% (n = 12). For TZMU in guttation water the individual recovery values ranged from 95 to 131% with an

November 2015 218

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

overall mean recovery of 105% and a relative standard deviation (RSD) of 9.9% (n = 12) and for thiamethoxam in guttation water the individual recovery values ranged from 81 to 99% with an overall mean recovery of 89% and a relative standard deviation (RSD) of 7.1% (n=12). The residue levels of clothianidin in bees from Baumgartenberg were between

No information is provided on the application rate, so these results are not used Remark in the risk assessment.

Reference J. Luckmann (2010) Field survey on guttation of maize seedlings under agronomic use conditions in Austria and assessment of the relevance of guttation fluid for honeybees. RIFCon GmbH, Heidelberg Germany. Report no R09/105 Bayer ID M-355018-03-2

The aim of the study was to survey the guttation behaviour of maize seedlings, grown from maize seed coated with clothianidin (0.5 mg ai/seed or 1.25 mg ai/seed) under commercial conditions at various locations in Austria. Further to monitor whether the guttation timing corresponds with the flight activity of Summary honeybees and the use of guttation fluid by bees. The residue levels of clothianidin in guttation fluid and dead bees were detected. Treated seeds were sown in two regions of Austria on 15 fields per region. Based on nominal concentrations the nominal application rates ranged from

November 2015 219

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

39.5 – 44.0 g clothianidin/ha in Baumgartenberg and 40.0 – 100.0 g clothianidin/ha in Jennerdorf. At each field two honeybee hives were placed shortly after drilling. The bee mortality was recorded daily and guttation fluid was collected. The colony strength and development were estimated according to the Liebefeld method. Clothianidin residues were analysed. Guttation occurred during 97.4% of the observation days in the morning and 50% in the evening. In the mornings a high degree of honeybee activity was observed during guttation. In the evening the overlap was distinctly lower. Overall it was observed that the bees visit the maize field very infrequently and that they prefer water sources in the nearest hive vicinity. Honeybees rarely collected guttation fluids from maize plants. The mortality of the bees was ≤ 5 bees per hive on 70% of all assessments days and on 95% of the assessment days the mortality was ≤ 20 bees per hive. Hive development was not adversely affected. Initial residue levels of clothianidin in the guttation fluid from plants ranged between 100 and 200 mg/L. These levels declined and were approx. 1 mg/L after 3 weeks and below 0.1 mg/L after 5 weeks. Same amount of residues were observed with both application rates but the decline was slightly lower with the highest rate. The monitoring over 3 weeks showed that there were infrequent visits to the field: 227 were observed on the ground and 65 bees on plants over this period.

Exposure of bee colonies to guttation of maize plants treated with Poncho Conclusion or Poncho Pro did not result in harmful effects on colony level. The colonies were able to develop properly.

Reference K.Liepold (2010) Monitoring of potential effects of the drilling of clothianidin treated maize seeds on honeybees, guttation monitoring of maize seedlings under agronomic use conditions and assessment of the relevance of guttation for honeybees in Alsace (France). Eurofins GAB GmbH Niefern-Oschelbronn Germany. Report no S09/01402 Bayer ID M-361103-01-1

The aim of the study was to monitor the occurrence of guttation in young maize plants on maize field in France and the relevance of potentially occurring guttation as water source for honeybees (Apis mellifera L.). Maize seeds, dressed with Clothianidin FS 600B G (dressing rate:0.5 mg Summary a.s./seed) and untreated maize seeds as control (untreated with neonicotinoid insecticides), were sown at a nominal drilling rate of approximately 2 units (100,000 seeds)/ ha on two fields near La Petite Pierre, in the region Alsace (France). There was one untreated control field and one treatment field. The

November 2015 220

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

treatment and the control were separated by approximately 4 km distance. Honeybee colonies were set up at the maize fields 19 days before drilling. Six colonies were used per treatment group and placed at the border of each field with the entrance facing towards the field. 63 and 62 (control and treatment, respectively) days after emergence of the maize the colonies were relocated to a monitoring site, after guttation of the crop had ceased. Observations were carried out on guttation (occurrence and duration), flight activity and behaviour of bees, collection of guttation fluid by bees, mortality in front of the hives and assessments on the bee brood and strength of the colonies. Guttation was regularly observed during daily time of bee flight from the early morning until midday 1 to 2 days after crop emergence. The proportion of guttating plans varied from 0 to 100% of all plants in control and treatment plots. In general the occurrence of guttation was more pronounced in the treatment plot compared to the control. There were no observations made of bees collecting guttation droplets except of a few observations where individual bees were seen sitting on the maize plants or on the ground but interaction with the droplets was not observed. The daily mean mortality was 8.9 bees/hive in the treatment and 15 bees/hive in the control. No treatment related effect was seen on the development of colony strengths and of the brood of the bees.

This study won’t be considered for risk assessment because the application rate Remark is lower than the application rate proposed for NZ.

No adverse effects of the potential exposure to guttating maize on the honeybee Conclusion colony health and development was observed. No differences in mortality between treatment and control were observed.

Reference K.Liepold (2010) Monitoring of potential effects of the drilling of clothianidin treated maize seeds on honeybees, guttation monitoring of maize seedlings under agronomic use conditions and assessment of the relevance of guttation for honeybees in Champagne (France). Eurofin GAB GmbH Niefern- Oschelbronn Germany. Report no S09/01403 Bayer ID M-361107-01-1

The aim of the study was to monitor the occurrence of guttation in young maize plants on maize field in France and the relevance of potentially occurring guttation as water source for honeybees (Apis mellifera L.). Maize seeds, dressed with Clothianidin FS 600B G (dressing rate: 0.5 mg Summary a.s./seed) and untreated maize seeds as control (untreated with neonicotinoid insecticides), were sown at a nominal drilling rate of approximately 2 units (100,000 seeds)/ha on two fields in the region Champagne (France). There was one untreated control field and one treatment field. The treatment and the

November 2015 221

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

control were separated by approximately 2 km distance. Honeybee colonies were set up at the maize fields 11 days before drilling. Six colonies were used per treatment group and placed at the border of each field with the entrance facing towards the field. 24 days after emergence of the maize the colonies were relocated to a monitoring site. Observations were carried out on guttation (occurrence and duration), flight activity and behaviour of bees, collection of guttation fluid by bees, mortality in front of the hives and assessments on the bee brood and strength of the colonies. Guttation was regularly observed during daily time of bee flight from the early morning until midday 1 day after crop emergence and at least until day 24 after emergence. No guttation was seen in the evening. The proportion of guttating plans varied from 0 to 100% of all plants in control and treatment plots. In general the occurrence of guttation was more pronounced in the treatment plot compared to the control. There were no observations made of bees collecting guttation droplets except of a few observations where individual bees were seen sitting on the maize plants. The daily mean mortality was 9.5 bees/hive in the treatment and 11.4 bees/hive in the control. No treatment related effect was seen on the development of colony strengths and of the brood of the bees.

This study won’t be considered for risk assessment because the application rate Remark is lower than the application rate proposed for NZ.

No adverse effects of the potential exposure to guttating maize on the honeybee Conclusion colony health and development was observed. No differences in mortality between treatment and control were observed.

Reference K.Liepold (2010) Monitoring of potential effects of the drilling of clothianidin treated maize seeds on honeybees, guttation monitoring of maize seedlings under agronomic use conditions and assessment of the relevance of guttation for honeybees in Languedoc-Roussillon (France). Eurofins GAB GmbH Niefern- Oschelbronn Germany. Report no S09/01404 Bayer ID M-361110-01-1

The aim of the study was to monitor the occurrence of guttation in young maize plants on maize field in France and the relevance of potentially occurring guttation as water source for honeybees (Apis mellifera L.). Maize seeds, dressed with Clothianidin FS 600B G (dressing rate: 0.5 mg Summary a.s./seed) and untreated maize seeds as control (untreated with neonicotinoid insecticides), were sown at a nominal drilling rate of approximately 2 units (100,000 seeds)/ha on two fields in the region Languedoc-Roussillon (France). There was one untreated control field and one treatment field. The treatment and the control were separated by approximately 3.3 km distance. Honeybee

November 2015 222

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

colonies were set up at the maize fields 4 days before drilling. Six colonies were used per treatment group and placed at the border of each field with the entrance facing towards the field. 40 days after emergence of the maize the colonies were relocated to a monitoring site. Observations were carried out on guttation (occurrence and duration), flight activity and behaviour of bees, collection of guttation fluid by bees, mortality in front of the hives and assessments on the bee brood and strength of the colonies. Guttation was regularly observed during daily time of bee flight from the early morning until midday within 40 days after crop emergence and started 2 days after emergence. The proportion of guttating plans varied from 0 to >90% of all plants in control and treatment plots. In general the occurrence of guttation was slightly more frequent in the treatment plot compared to the control. There were no observations made of bees collecting guttation droplets except of a few observations where individual bees were seen sitting on the maize plants. The daily mean mortality was 39.9 bees/hive in the treatment and 32.8 bees/hive in the control. No treatment related effect was seen on the development of colony strengths and of the brood of the bees.

This study won’t be considered for risk assessment because the application rate Remark is lower than the application rate proposed for NZ.

No adverse effects of the potential exposure to guttating maize on the honeybee Conclusion colony health and development was observed. No differences in mortality between treatment and control were observed.

Reference K.Liepold (2010) Monitoring of potential effects of the drilling of clothianidin treated maize seeds on honeybees, guttation monitoring of maize seedlings under agronomic use conditions and assessment of the relevance of guttation for honeybees in Aquitaine (France). Eurofin GAB GmbH Niefern-Oschelbronn Germany. Report no S09/01405 Bayer ID M-361111-01-1

The aim of the study was to monitor the occurrence of guttation in young maize plants on maize field in France and the relevance of potentially occurring guttation as water source for honeybees (Apis mellifera L.). Maize seeds, dressed with Clothianidin FS 600B G (dressing rate: 0.5 mg a.s./seed) and untreated maize seeds as control (untreated with neonicotinoid Summary insecticides), were sown at a nominal drilling rate of approximately 2 units (100,000 seeds)/ha on two fields in the region Aquitaine (France). There was one untreated control field and one treatment field. The treatment and the control were separated by approximately 6.7 km distance. Honeybee colonies were set up at the maize fields 7 days before drilling. Six colonies were used

November 2015 223

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

per treatment group and placed at the border of each field with the entrance facing towards the field. 65 days after emergence of the maize the colonies were relocated to a monitoring site. Observations were carried out on guttation (occurrence and duration), flight activity and behaviour of bees, collection of guttation fluid by bees, mortality in front of the hives and assessments on the bee brood and strength of the colonies. Guttation was regularly observed during daily time of bee flight from the early morning until midday within 65 days after crop emergence and started 3 days after emergence. The proportion of guttating plans varied from 0 to 100% of all plants in control and treatment plots. In general the occurrence of guttation was less pronounced in the treatment plot compared to the control. There were no observations made of bees collecting guttation droplets except of a few observations where individual bees were seen sitting on the maize plants. The daily mean mortality was 12.7 bees/hive in the treatment and 10.0 bees/hive in the control. No treatment related effect was seen on the development of colony strengths and of the brood of the bees.

This study won’t be considered for risk assessment because the application rate Remark is lower than the application rate proposed for NZ.

No adverse effects of the potential exposure to guttating maize on the honeybee Conclusion colony health and development was observed. No differences in mortality between treatment and control were observed.

Reference N. Jarrat (2014) Determination of clothianidin residues in bee relevant matrices, collected in a succeeding crop scenario with natural aged clothianidin residues- field phase conducted with phacelia and maize in the UK (GOOLE East Yorkshire). The Food and Environmental Research Agency Centre for Chemical safety & stewardship, Sand Hutton York UK. Report no B2BN2000, Bayer ID M- 504590-01-1

The aim of the study was to determine residues of clothianidin and its metabolites Thiazolylnitroguanidine (TZNG) and Thiazolylmethylurea (TZMU) in bee relevant matrices (pollen, nectar and guttation fluid) collected from flowering rotational crops cultivated as succeeding crops grown in the UK (Goole) on Summary fields with a history of clothianidin use and as such with natural aged soil residues of this active ingredient. A part of the field was sown with maize and another part with Phacelia. Soil samples were taken from both plots from the upper 10 cm layer and the upper 15 cm layer. The maize plots were used for guttation fluid sampling and pollen sampling. Three bee proof tunnels were

November 2015 224

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

placed onto the Phacelia plot with a honeybee colony in each tunnel. Nectar and pollen were taken of the phacelia crop. Residues were analysed for clothianidin and its metabolites. The limit of quantitation (LOQ) for clothianidin was 5 µg ai/kg soil, 1 µg ai/L in guttation fluid, 0.3 µg ai/kg nectar and 0.6 µg ai/kg pollen. The limit of detection (LOD) for clothianidin was 2 µg ai/kg soil, 0.3 µg ai/L in guttation fluid, 0.1 µg ai/kg nectar and 0.2 µg ai/kg pollen Residues Soil: maize plot 16-22 µg ai /kg soil, phacelia 18-41 µg ai / kg soil. Guttation fluid:

Reference N. Jarrat (2014) Determination of clothianidin residues in bee relevant matrices, collected in a succeeding crop scenario with natural aged clothianidin residues- field phase conducted with phacelia and maize in the UK (Thorney, Cambridgeshire). The Food and Environmental Research Agency Centre for Chemical safety & stewardship, Sand Hutton York UK. Report no B2BN3000, Bayer ID M-504595-01-1

The aim of the study was to determine residues of clothianidin and its metabolites Thiazolylnitroguanidine (TZNG) and Thiazolylmethylurea (TZMU) in bee relevant matrices (pollen, nectar and guttation fluid) collected from flowering rotational crops cultivated as succeeding crops grown in the UK (Thorney) on fields with a history of clothianidin use and as such with natural aged soil residues of this active ingredient. A part of the field was sown with maize and another part with Phacelia. Soil samples were taken from both plots from the Summary upper 10 cm layer and the upper 15 cm layer. The maize plots were used for guttation fluid sampling and pollen sampling Three bee proof tunnels were placed onto the phacelia plot with a honeybee colony in each tunnel. Nectar and pollen were taken of the phacelia crop. Residues were analysed for clothianidin and its metabolites. The limit of quantitation (LOQ) for clothianidin was 5 µg ai/kg soil, 1 µg ai/L in guttation fluid, 0.3 µg ai/kg nectar and 0.6 µg ai/kg pollen.

November 2015 225

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Residues Soil: maize plot 59-80 µg ai /kg soil, phacelia 64-78 µg ai / kg soil. Guttation fluid:

Reference N. Jarrat (2014) Determination of clothianidin residues in bee relevant matrices, collected in a succeeding crop scenario with natural aged clothianidin residues- field phase conducted with phacelia and maize in the UK (Sawtry, Cambridgeshire). The Food and Environmental Research Agency Centre for Chemical safety & stewardship, Sand Hutton York UK. Report no B2BN4000, Bayer ID M-504601-01-1

The aim of the study was to determine residues of clothianidin and its metabolites Thiazolylnitroguanidine (TZNG) and Thiazolylmethylurea (TZMU) in bee relevant matrices (pollen, nectar and guttation fluid) collected from flowering rotational crops cultivated as succeeding crops grown in the UK (Sawtry) on fields with a history of clothianidin use and as such with natural aged soil residues of this active ingredient. A part of the field was sown with maize (25.3 kg seeds/ha) and another part with Phacelia (11.3 kg seeds/ha). Soil samples were taken from both plots from the upper 10 cm layer and the upper 15 cm layer. The maize plots were used for guttation fluid sampling and pollen sampling Three bee proof tunnels were placed onto the phacelia plot with a Summary honeybee colony in each tunnel. Nectar and pollen were taken of the phacelia crop. Samples for analysis were taken from the soil, nectar and pollen from phacelia and mustard and guttation fluid and pollen from maize. Residues were analysed for clothianidin and its metabolites. The limit of quantitation (LOQ) for clothianidin was 5 µg ai/kg soil, 1 µg ai/L in guttation fluid, 0.3 µg ai/kg nectar and 0.6 µg ai/kg pollen. Residues soil: prior to guttation 92-187µg ai /kg soil, during guttation 98- 248 µg ai /kg soil, in the tunnels 78-80 µg ai /kg soil Guttation fluid:

November 2015 226

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Maize pollen:

November 2015 227

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix J: Human health risk assessment

Qualitative risk assessment – Bacillus firmus B-1582

Based on the studies and literature information provided, no toxicological or pathogenic concerns have been identified for Bacillus firmus I-1582. As a result, no toxicological reference values could be derived. In addition, there is a lack of exposure models specifically designed for microorganisms.

Protective clothing including respiratory protective equipment will be required to be worn when conducting tasks associated with seed treatment using Poncho Votivo, due to the hazard classifications of the substance, including the potential respiratory sensitising properties. Protective clothing including RPE will also be required during the handling and sowing of seeds treated with Poncho Votivo.

Taking into account the toxicological and pathogenicity profile of B. firmus I-1582, together with requirements to wear protective equipment, exposure of workers engaged in tasks related to seed treatment and sowing of treated seeds are unlikely to be of concern.

Quantitative worker (operator) risk assessment – clothianidin

For clothianidin, a quantitative risk assessment was prepared by the applicant. Risks were assessed for operators involved in tasks related to seed treatment and for workers loading and sowing the treated seed.

We have reviewed the risk assessment and accepted the conclusions after seeking clarification of several questions from the applicant. A summary of the risk assessment is provided below.

Critical endpoint definition

Using an existing AOEL for clothianidin29 Available NOAEL AOEL Key systemic Uncertaint Staff’s international mg/kg mg/kg Remarks effect y factors modifications AOELs bw/day bw/day

New data Developmental received on and reproductive clothianidin do EFSA (2003) toxicity in the 10 100 0.1 None not indicate a rabbit and rat, need to revise the respectively AOEL

29 The toxicological endpoint used for assessment of occupational (worker), re-entry worker and bystander risks is the AOEL (Acceptable Operator Exposure Level). The AOEL is the maximum amount of active substance to which the operator/re-entry worker may be exposed with a low probability of adverse health effects amongst the healthy worker sub-population, allowing for some margin of safety. AOELs describe the internal (absorbed) dose available for systemic distribution from any route of absorption and are expressed as internal (systemic) levels (mg/kg bw/day). They are derived by dividing the most appropriate NOAEL from relevant studies by one or more uncertainty (safety) factors selected on the basis of the extent and quality of the available data, the species for which data are available and the nature of the effects observed. An absorption factor may be applied to take into account the absorbed dose in the study where this is known (this is a percentage expressed as a factor).

November 2015 228

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Other inputs for human worker (operator) and re-entry exposure modelling

Table 6 Derivation of dermal absorption value in humans

Concentration Maximum Dermal absorption (%) AOEL Physical Active of each active application rate mg/kg form (g/L) Concentrate Spray bw/day

60.96 g a.i./unit Clothianidin 508 seed* 25 25 0.1 91 g a.i./ha** Liquid 12.24 g a.i./unit Bacillus firmus 102 (min. 2 x 109 seed* NA NA NA I-1582 cfu/g) 0.018 g a.i./ha**

* 1 unit = 80,000 seeds

** Sowing rate: 1.5 units/ha

Comments on inputs for human worker (operator) exposure modelling input parameters:

As noted earlier, derivation of reference values for Bacillus firmus I-1582 is not needed based on the absence of toxicity, infectivity and pathogenicity indications of the micro-organism.

For clothianidin, a default value of 25% was applied in the risk assessment in line with the EFSA Guidance on dermal absorption of pesticides30 due to a lack of data for a relevant formulation. For all tasks the concentration of clothianidin is > 50 g/L (even at the highest dilution factor of 5.6 the concentration is 90 g/L). It is noted that the dermal absorption endpoint for clothianidin established in the EU is 2.25%, based on an in vivo monkey study using the FS 600 formulation (containing 600 g a.s /L clothianidin). The two formulations are similar in their composition but not sufficiently enough according to the European dermal absorption guidance. Thus, the value from the FS 600 formulation cannot be used but is an indicator that the realistic dermal absorption of clothianidin in the FS 610 is < 25% and therefore the exposure assessment is likely to have overestimated exposure.

We note that in late July 2015, following the submission and review of the risk assessment provided by the applicant, We have agreed to use revised default dermal absorption values that those recommended by EFSA. The new values are based on a review of data produced by a range of agrichemical companies (Aggarwal et al., 201531). The revised default values are 6% for liquid concentrates and 30% for spray dilutions. We have not requested that the exposure assessment be revised as the current exposure estimates are expected to substantially overpredict exposure for some activities where exposure will be to the concentrate and only slightly underpredict exposure for activities involving exposure to the dilution.

30 www.efsa.europa.eu/en/search/doc/2665.pdf

31 hwww.sciencedirect.com/science/article/pii/S0273230015000458

November 2015 229

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Approaches used to estimate operator exposure The applicant assessed the operator exposure using a tiered approach. In a first tier, exposure was calculated using the “Seed-TReatment OPerator EXposure model” (SeedTROPEX). Since the database of the SeedTROPEX model consists only of data generated during treatment and sowing of cereal seeds (wheat, barley), additional experimental data from operator exposure studies performed during maize seed treatment were used to refine the SeedTROPEX modelling.

Operator exposure assessment during seed treatment using SeedTROPEX SeedTROPEX was developed by industry, and estimates operator exposure during several activities of seed treatment, such as calibration, mixing/loading, cleaning, and bagging of seeds. It contains exposure data from studies performed in seed treatment plants in UK and France. The data are owned by a Task Force and are only able to be used by applicants with data access.

There is no option in the model to differentiate varying levels of protection (exposure with or without personal protective equipment [PPE]). For seed treatment, it already includes the use of coveralls and gloves for all tasks - except for bagging where only a coverall is considered. The estimated actual dermal exposure values, therefore, reflect this level of PPE. A further conservatism is included in the model when using the total potential dermal exposure values for operators wearing no PPE. This reflects the exposure of operators wearing no clothing and is unrealistic. Therefore, exposure estimates were only performed for operators using PPE (i.e. the actual dermal exposure estimates).

SeedTROPEX differentiates four work tasks during seed treatment: calibration, mixing/loading, bagging, and cleaning. All tasks are considered individually, however, it is assumed that one single operator performs all tasks during a working shift. This is therefore expected to represent a worst case scenario.

The following assumptions are made in the exposure assessment:

Parameter Value

Crop Maize

No. of cleaning operations 1

1 (In professional plants usually big product container are used when a product will be applied as a standard treatment. 200L to 1000L containers No. of mixing/loading operations are common for professional seed treatment. In the studies approx. 1500 units of seed were treated in a highly automated plant resulting in a consumption of 180 L product/day)

No. of calibration operations 1

Duration of bagging 8 hours

0.12 L Poncho Votivo/unit seed containing 0.061kg clothianidin/unit seed Application rate (1 unit – 80,000 seeds)

Active substance handled 91.5 kg clothianidin/day

November 2015 230

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Parameter Value

3.3 – 5.5 Dilution factor (1 L to 2 L water is applied to 100 kg seed corresponding to 0.28 L to 0.56 L water/unit taking into account a thousand grain weight of 350 g)

Dermal absorption 25% for all tasks

Body weight 60 kg*

Coverall and gloves during all operations except bagging; particle filtering Clothing scenario half mask FFP2) during cleaning with compressed air

* The EPA’s default body weight for operator risk assessment is 70 kg, however the applicant’s risk assessment has assumed a body weight of 60 kg. The exposure estimates are therefore greater than those that would have been calculated using the EPA’s default body weight.

Output of the operator exposure assessment during seed treatment using SeedTROPEX Total systemic exposure AOEL Task RQ (mg/kg bw/day) (mg/kg bw/day)

All tasks (calibration, mixing/loading, 0.1063 0.1 1.06 bagging and cleaning)

The evaluation using the SeedTROPEX model results in exposure to clothianidin slightly exceeding the AOEL (106% of the AOEL). It is noted that:

 exposure is calculated for an operator performing all tasks; typical operators, however, would perform only single tasks or a combination of two tasks during the whole day and a combination of all tasks is highly unlikely

 the model is compiled from studies relevant for cereal and barley seed treatment but not for maize seed treatment

A refinement of the exposure evaluation was therefore considered to be appropriate using specific maize seed treatment data.

Operator exposure assessment using measurement of operator exposure In order to fill a data gap two operator exposure studies were performed to measure the exposure during maize seed treatment, conducted in 200132 and 200433. Both studies were performed using Mesurol FS 500,

32 Brennecke R, 2002. Determination of Operator Exposure to Methiocarb during Seed Treatment of Maize Seed with Mesurol® M (FS 500) in Seed Treatment Plants in Germany. Bayer AG, Business Group Crop Protection, Development Department, Institute for Metabolism Research and Residue Analysis, D-51368 Leverkusen, Germany. Report no. MR-576/01; Document no. M-051892-01-1.

33 Sutor P and Timmerman C, 2005. Determination of Operator Exposure to Methiocarb during Seed Treatment of Maize with Mesurol® (FS 500) in Germany. Bayer AG, BCS-D-HS/ORE, D-40789 Monhein am Rhein, Germany. Report no. MR-110/05 (Amendment 1); Document no. M-261386-02-1.

November 2015 231

Application for approval to import or manufacture Poncho Votivo for release (APP202077) a liquid seed dressing formulation containing 500 g/L methiocarb. The studies were performed in seed treatment facilities with low and with high level of automation. The experimental data from these studies are used to refine the modelling using the Seedtropex data.

The two studies and summaries of the data were submitted to the EPA for review. The applicant provided responses to a number of questions of clarification, and was also asked to provide a justification for using the data from the studies with methiocarb rather than the SeedTROPEX modelling. The following response was provided:

‘The Seed TROPEX model studies were performed with cereal seeds in 1996. Cereal seed treatment differs from treatment of maize seeds in several aspects. Usually the throughput in tonnes of seed per day is lower in maize seed treatment. The loading rate per grain is higher during maize seed treatment. Emission of contaminated dust which is the main factor of dermal and inhalation exposure might be affected also by the dust content of the untreated seeds that varies between the different seed types.’

‘Considering all the differences it is reasonable to assume that the seed type might have an influence on the operator exposure. To cover this uncertainty the studies performed during maize seed treatment with Mesurol FS 500 were evaluated as a higher tier approach.’

We have accepted this argument and, following review of the data provided, consider that is acceptable to use the information from these two studies to refine the exposure assessment for Poncho Votivo. The exposure data in the first study represent old type professional working conditions during maize seed treatment in Germany. The partly outdated technical equipment and the unfavourable configuration of the equipment limited the work flow of the operators and had a significant influence on the amount of exposure. Therefore, the results of this study are representative for a worst case scenario. The second study was conducted was performed under working conditions representing current use of maize seed treatment in professional plants in Germany. The different levels of automation in the observed plants cover the range of the equipment used currently in the EU. Technical progress in the treatment equipment, improvements in the working environment and a better training of the workers led to a decline of exposure compared to values determined in the 2001 study.

Poncho Votivo is only intended to be used in purpose built seed treatment facilities in New Zealand. It is concluded that the study conditions (i.e. the selected plants, the work tasks, the work rate, the work conditions, etc.) and subsequently the determined exposure values are likely to be representative for the considered exposure scenario.

Combination of exposure data Both operator exposure studies were performed using Methiocarb FS 500 to monitor the exposure of operators during the seed treatment of maize. In order to allow for the consideration of a bigger data base the experimental data of both studies are combined in a generic data set. The database covers a wide range of technical equipment, technical standards and working conditions and includes a large number of replicates. The 75th percentile values (maximum value for mixing as only limited data were available) are used for the calculation of specific exposure.

November 2015 232

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

The following assumptions are made in the exposure assessment:

Parameter Value

Crop Maize

0.12 L Poncho Votivo/unit seed containing 0.061kg clothianidin/unit seed (1 Application rate unit – 80000 seeds)

1000 seed mass (maize) 350 g (average)

Weight of one seed unit 28 kg

High level of automation: ~40 tonnes (1500 units) Medium to low level of automation: ~30 tonnes (1100 units) Amount of seed treated [Based on amounts treated in the operator exposure studies during a full working shift]

High level of automation: 91.5 kg clothianidin/day Daily rate handled Medium to low level automation: 67.1 kg clothianidin/day

~90-150 g clothianidin/L treatment solution Concentration in slurry [Typically about 400-800 ml treatment slurry containing 61 g clothianidin is applied to one seed unit]

No. of cleaning operations 1

Concentrate: 25% (during mixing/loading, bagging and stacking) Dermal absorption Dilution: 25% (during treatment, cleaning)

Body weight 60 kg*

All tasks: One layer of working clothing (long sleeved work jacket, long work trousers) Clothing scenario Mixing/loading/calibration and cleaning: impervious coverall, protective gloves Mixing/loading: particle filtering half mask

Output of the operator clothianidin exposure assessment using exposure studies

Plant type Task Systemic exposure (mg/kg bw/day) RQ

High level of automation All tasks (machinist) 0.017 0.17

Treatment 0.011 0.11

Medium to low level of automation Bagging 0.0353 0.35

Stacking 0.00795 0.08

Mixing 0.00482 0.05 All plants Cleaning 0.00291 0.03

November 2015 233

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Outcome of the operator clothianidin exposure assessment using exposure studies The risk assessment based on the experimental data indicates that baggers are the most exposed (35% of the AOEL) during seed treatment of maize followed by the treatment operator (11% of the AOEL), the stacker (8% of the AOEL), the cleaner (5% of the AOEL) and the mixer (3% of the AOEL).

The risk assessment demonstrates that an unacceptable exposure is not anticipated for operators during maize seed treatment. This statement can also be made for the unlikely event that an operator performs all tasks: the combined RQ for a worker in a plant with a medium to low level of automation is 0.62.

Operator exposure assessment during loading and sowing of the treated seed Exposure during seed sowing is calculated with SeedTROPEX.

Exposure estimates include dermal and inhalation exposure. The normalisation that is proposed in the model (mg a.s./hour) is used in the first tier estimate of the operator exposure to clothianidin during loading and sowing maize seeds treated with Poncho Votivo FS 610.

The following assumptions and requirements are taken into account for the estimation of operator exposure during seed loading and sowing:

Parameter Value

Crop Maize

Drilling rate 1.5 units/ha (120,000 seeds/ha)

Work rate 8 hours/day

Body weight 60 kg

Coverall; gloves partly worn (only few operators wearing gloves in the studies used Clothing scenario to inform the model)

Output of the assessment of operator exposure during loading and sowing of treated seed

Total systemic exposure (mg/kg bw/day) AOEL (mg/kg bw/day) RQ

0.027 0.1 0.27

Outcomes of the assessment of operator exposure during loading and sowing of treated seed The risk assessment based on SeedTROPEX indicates that estimated exposure to clothianidin during loading and sowing of treated seed accounts for 27% of the AOEL. An unacceptable risk is therefore not anticipated for operators during these activities.

November 2015 234

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Re-entry worker exposure assessment

The seed treatment will not result in dislodgeable foliar residues on treated plants. Therefore a risk assessment for re-entry workers is not relevant.

Bystander risk assessment

Seed treatment using Poncho Votivo is intended to be performed in professional treatment plants. The presence of persons unrelated to the work is not allowed and rather incidental. Therefore, bystander exposure to Poncho Votivo FS 610 during seed dressing is considered to be not relevant

Summary and conclusions of the human health risk assessment

An unacceptable risk is not anticipated for operators using Poncho Votivo to treat maize seed, or when loading and sowing treated seed.

Taking into account the lack of toxicological and pathogenic effects observed with B. firmus I-1582, together with requirements to wear protective equipment, exposure to B. firmus I-1582 of workers engaged in tasks related to seed treatment and sowing of treated seeds are unlikely to be of concern

Estimated exposures to clothianidin are below the AOEL.

The risk assessment assumes the following PPE is worn: long trousers, long sleeved shirt and work jacket and appropriate protective clothing (i.e. protective gloves during mixing/loading/calibration and cleaning and when handling treated seed or contaminated surfaces; disposable coverall during cleaning).

Due to the potentially sensitising properties of Bacillus firmus I-1582 additional PPE (eye or face protection) and RPE should also be required.

Based on the proposed use pattern a risk assessment for re-entry workers and bystanders is not considered to be required.

November 2015 235

Application for approval to import or manufacture Poncho Votivo for release (APP202077) Appendix K: Environmental risk assessment

Summary of the data on the active ingredient and its metabolites

A summary of the environmental fate data is provided in Table 7.

A summary of the ecotoxicological data is provided in Table 8.

In these tables, the full names of metabolites are:

TMG = thiazolylmethylguanidine,

MNG = methylnitroguanidine,

TZNG = thiazolylnitroguanidine

TZMU = N-(2-chlorothiazol-5-ylmethyl)-N'-methylurea

MU = methylurea

TZFA = N-({[(2-chloro-1,3-thiazol-5-yl)methyl]amino}methylene)methanaminium chloride; code BCS- CQ88479

NTG = 1- Nitroguanidine

CTCA = clothianidin-2-chlorothiazole-5-caboxylic acid

HMIO = clothianidin-4-hydroxy-2-methylamino-2-imidazolin -5-one

Some studies were conducted with clothianidin formulations but the results are expressed as clothianidin and appear in the clothianidin column.

November 2015 236

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Table 7 Summary of environmental fate data on clothianidin and its metabolites

Values in bold are used for the risk assessment Test Clothianidin MNG TZNG TZMU TMG TZFA

DT50 = 1401 d at pH Hydrolysis 9 and 20oC, stable - - - - - at pH 7 and below

Degradation in natural water DT50 = 2085 d

Aqueous photolysis DT50 = 3.3 h (0.14 - - - - - (continuous radiation) d)

Not readily Ready biodegradation - - - - - biodegradable

DT50 =64.8 and 48.0 d (whole Aerobic degradation system) - - - - - (water/sediment) Additional data34: 170-156 days

Log Kow = 0.893 at pH=4, 0.905 at Bioaccumulation - - - - - pH=7, 0.873 at pH=10 at 25oC

DT50 = 227-143- Aerobic degradation in soil 490-1001-541- DT50 = 86.4- DT50 = 11-819-8- DT50 = 16-22-66 o - - (laboratory, 20 C) 1328-549-533-808 108-82.4 days 62.1 days days days

34 I.e. new study provided by the applicant.

November 2015 237

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG TZFA metabolites: MNG max 10.7% and TZNG max 9.1%

DT50 = 31.6 d Anaerobic degradation in soil (guanidine label), - - - - - (laboratory, 20oC) 24.8 d (thiazolyl label)

DT50 =13.3-78.6- 239.2-124.7–186.3– 125.3–305.4– Field dissipation (DT50 194.6–156 days - - - - - standardised at 20oC) Additional data: 9.7 - 86.9 – 122 -174.6 days

Maximum amount: Residues in soil over 8 years of 48.5 µg / kg soil. use as seed treatment No metabolite

DT50 = 34 days in Soil photolysis natural summer - - - - - sunlight

Koc = 21.4 Koc = 261.4 Koc = 6159 Koc = 129 (sand), Koc = 53.3 (sand), (sand), 34.3, (sand), 432.5, (sand), 3620, Koc = 212, 255, Adsorption/desorption 345, 123, 84, 119 95.8, 56.0, 57.5, 16.5, 25.3, 5.2 242.6, 236.0, 1350, 525, 642 289,827, 1216 (sand) 46.4 (sand) (sand) 204.5 (sand) (sand)

DT50 = 1 h, Photodegradation in air chemical lifetime = - - - - 1.4 h

November 2015 238

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG TZFA

Definition of residue in soil Clothianidin + MNG + TZNG

Definition of residue in water Clothianidin alone

Table 8 Summary of ecotoxicological data on clothianidin and its metabolites

Values in bold are used for the risk assessment

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

LC50 > 104.2 mg/L (Bluegill)

LC50 > 105 LC50 > 116 LC50 > 110 Acute / fish LC50 > 102.5 - - - - mg/L mg/L (trout) mg/L (trout) mg/L (trout) (sheepshead minnow)

EC50 = 40 mg/L (Daphnia)

EC50 = 1.74 mg

Acute / ai/L (Daphnia) EC50 = 56.6 EC50 = 100 EC50 > 100 aquatic (tested as 600 mg/L - mg/L - - - mg/L (Daphnia) invertebrates FS) (Daphnia) (Daphnia)

LC50 = 0.053 mg ai/L (saltwater mysid)

ErC50 > 120 mg/L EbC50 > 100 EbC50 > 100 ErC50 = Algae - - - - EbC50 = 155 mg/L mg/L 50.3 mg/L mg/L

November 2015 239

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

NOErC = 15 mg/L

Aquatic plant EC50 > 270 mg/L ------

NOEC = 20 mg/L Chronic / fish (fathead minnow, ------ELS 28 d)

NOEC = 0.12 Reproduction ------mg/L (daphnia)

EC50 > 100 mg/L

Acute / EC50 = 0.029 EC50 > 100 EC50 = 0.433 EC50 > 100 EC50 > 83.6 (Chironomus sediment mg/L mg/L mg/L mg/L mg/L riparius, 48 h) - - dwelling (Chironomus (Chironomus (Chironomus (Chironomus (Chironomus And for the arthropods riparius, 48 h) riparius, 48 h) riparius, 48 h) riparius, 48 h) riparius, 48 h) metabolites CTCA and HMIO

EC15 = 0.00072 NOEC = 0.1 Chronic / mg/L mg/L sediment (Chironomus (Chironomus ------dwelling riparius, 28 d, riparius, 28 d, arthropods spiked water) spiked water)

EAC (ecologically Microcosm/ acceptable ------mesocosm concentration) = 0.0031 mg/L

November 2015 240

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

NOEC = 0.000986 mg/L

Acute / LC50 = 13.21 LC50 > 1000 LC50 = 970 - - - - - earthworm mg/kg soil mg/kg soil mg/kg soil

Reduction beneficial capacity = 21.14% in Acute / Other entomophagous soil macro------nematode at 1.5 organisms mg ai/L Hypoaspis

aculeifer: LC50 > 100 mg/kg soil

NOEC NOEC = 0.444 NOEC (28d) > NOEC (28d) NOEC (28d) > NOEC (28d)> NOEC (28d)> Chronic / (28d) = 100 mg ai /kg (tested 100 mg ai/ kg = 13 mg ai/kg 100 mg ai/ kg - 100 mg ai/ kg 100 mg ai/ kg earthworm mg ai/ kg as 600 FS) soil soil soil soil soil soil

Field / No effects on earthworms population at 125

(seed g ai/ha (tested as treatment) 600 FS)

Folsomia Folsomia Folsomia Folsomia Folsomia Chronic / Folsomia Folsomia candida: candida: candida: candida: candida: Other soil candida: NOEC candida: NOEC NOEC NOEC (28d) NOEC (28d) = - NOEC (28d) = NOEC (28d) = macro- (28d) = 0.32 mg (28d) = 1000 (28d) = = 1 mg ai/kg 100 mg ai/kg 100 mg ai/kg 80 mg ai/kg organisms ai/kg soil mg ai/kg soil 85.3 mg soil soil soil soil ai/kg soil

November 2015 241

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

Hypoaspis Hypoaspis Hypoaspis Hypoaspis Hypoaspis Hypoaspis aculeifer aculeifer Chronic / aculeifer aculeifer aculeifer aculeifer Other soil NOEC (14d)= NOEC NOEC (14d)= NOEC (14d)= - - NOEC (14d)= NOEC (14d)= macro- 190 mg ai/kg (14d)= 85.3 100 mg ai/kg 20 mg ai/kg 100 mg ai/kg 100 mg ai/kg organisms soil (tested as mg ai/kg soil soil soil soil 600 FS) soil

No effects at 225 Terrestrial g ai/ha (seedling ------plants emergence and vegetative vigour)

No effects on C and N transformation at 750 g ai/ha (1 mg ai/kg soil) No effects Mucor No effects on No effects on N No effects on N on N No effects on N No effects on N circinelloides, N Soil micro- transformation transformation transformati transformation transformation Suillus transformatio - organisms at 0.48 mg ai/kg at 1.0 mg ai/kg on at 0.43 at 1.0 mg ai/kg at 1.0 mg ai/kg granulatus, n at 0.95 mg soil soil mg ai/kg soil soil Phytophtora ai/kg soil soil nicotianae and Paecilomyces

marquandii: EC50 > 30.0 mg ai/kg soil

Field / litter NOEC = 225 g - - bag ai/ha

November 2015 242

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

LD50 = 430 Acute / bird mg/kg bw35 ------(Japanese quail)

LC50 > 752 Short-term / mg/kg bw ------bird (mallard duck)

NOEC = 56.8 Reproduction mg/kg bw/d ------bird (bobwhite quail)

LD50 = 0.04426 µg/bee (contact)

LD50 = 0.00379 µg/bee (oral) LD50 > LD50 > 153 LD50 = 3.9 LD50 > 113 151.6 Acute / bees Additional data - - - µg/bee (oral) µg/bee (oral) µg/bee (oral) µg/bee LD50 =0.0389 (oral) µg/bee (contact)

LD50 = 0.0025 µg/bee (oral)

LD50 = 0.1451 µg/bumble bee Acute/ (contact) ------bumble bee LD50 = 0.001943 µg/bumble bee (oral)

35 This study is not summarised in this memo, this is the value from EPA Substance Database

November 2015 243

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

A. rhopalosiphi: 100% mortality at 60 g ai/ha T. pyri: 75% mortality at 60 g Non target ai/ha arthropods ------A. bilineata: 90% (laboratory)* mortality at 75 g ai/ha C. carnea: 97% mortality at 60 g ai/ha

A. rhopalosiphi:

LR50 = 1.086 g ai/ha

P. cupreus: LR50 > 75 g ai/ha

P. cupreus: LC50 = 0.046 mg ai/kg Non target dry soil arthropods P. cupreus: ------(extended reduction of laboratory)* beneficial capacity = 100% at 467 g ai/ha (treated maize seeds) A. bilineata: reduction of beneficial capacity =72.48%

November 2015 244

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test Clothianidin MNG TZNG TZMU TMG MU TZFA NTG

at 467 g ai/ha (treated maize seeds)

A. bilineata: LR50 > 250 µg ai/kg soil

T. pyri: LR50 = 125.99 g ai/ha Pardosa sp: reduction of Non target beneficial arthropods ------capacity = 1 and (semi-field) 6.3% in females and males at 166 g ai/ha (treated maize seeds)

* The IOBC classification categories for laboratory tests is as follows: The IOBC classification categories for extended laboratory tests is as Reduction in beneficial capacity (E): follows: 1. Harmless (E< 30%) 1. Harmless (E< 25%); 2. Slightly harmful (≥ 30 E ≤ 79%) 2. Slightly harmful (25% ≤ E <50%) 3. Moderately harmful (> 79 E ≤ 99%) 3. Moderately harmful (50% ≤ E <75%) 4. Harmful (E> 99%) 4. Harmful (E ≥75%)

November 2015 245

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Risk assessment Methodology

Methods used to assess environmental exposure and risk differ between environmental compartments (2).

Table 9 Reference documents for environmental exposure and risk assessments

Environmental exposure Risk assessment

(GEN)eric (E)stimated Overview of the Ecological Risk Assessment (E)nvironmental (C)oncentration Process in the Office of Pesticide Programs, U.S. Aquatic organisms Model Version 2.0 – 01 August Environmental Protection Agency. Endangered 2002 and threatened Species Effects Determinations – AgDrift and EPA Software36 23 January 2004

Guidance on information requirements and Guidance on information requirements and chemical safety assessment, Chapter R.10: Sediment organisms chemical safety assessment, Chapter R.16: Environmental Characterisation of dose [concentration]-response Exposure Estimation, Version: 2 - for environment – May 2008 May 2010

Soil persistence models and EU registration. The final report of the Soil organisms, work of the Soil Modelling Work SANCO/10329/2002 rev 2 final. Guidance invertebrates (macro- group of FOCUS (FOrum for the Document on terrestrial ecotoxicology under invertebrates) Co-ordination of pesticide fate Council Directive 91/414/EEC- 17 October 2002 models and their USe) – 29 February 1997

Guidance for assessing pesticide risks to bees. US EPA, Health Canada Pest Bees Management Regulatory Agency, California Department of Pesticide Regulation, 19 June 2014

Terrestrial organisms, Guidance document on regulatory testing and risk assessment procedures for plant invertebrates (non-target protection products with non-target arthropods. From ESCORT 2 Workshop – 21/23 arthropods) March 2000

36 We used two different models for assessing the EEC and associated risks:

Generic Estimated Environmental Concentration Model v2 (GENEEC2) surface water exposure model (USEPA, 2001) estimates the concentration of substance in surface water which may arise as a result of surface runoff and spray drift.

To examine how buffer zones would reduce the active ingredient concentrations in receiving waters, we used the AgDRIFT® model (developed under a cooperative Research and Development Agreement, CRADA, between the EPA, USDA, US Forest Service, and SDTF). AgDRIFT® incorporates a proposed overall method for evaluating off-site deposition of aerial, orchard or ground applied pesticides, and acts as a tool for evaluating the potential of buffer zones to protect sensitive aquatic and terrestrial habitats from undesired exposures. Calculations are made assuming the receiving water is a 30 cm deep pond. The model is used to estimate the buffer zone that would reduce exposure through spray drift to such a concentration that an acute risk quotient of 0.1 cannot be calculated. It is noted that unlike GENEEC2, AgDRIFT® model only considers transport by spray drift, input through runoff, volatilisation, etc. will pose additional risks.

November 2015 246

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Environmental exposure Risk assessment

Guidance of EFSA. Risk assessment to birds and mammals – 17 December 2009. Terrestrial vertebrates EFSA calculator tool - 200937 (birds) SANCO/4145/2000 final. Guidance Document on risk assessment for birds and mammals under Council Directive 91/414/EEC- 25 September 2002

Technical Guidance Document on risk assessment in support of Commission Directive 93/67/EEC Guidance of EFSA. Risk assessment to birds and on Risk Assessment for new notified mammals – 17 December 2009 substances, Commission EFSA calculator tool - 2009 Secondary poisoning and Regulation (EC) No 1488/94 on biomagnification Risk Assessment for existing SANCO/4145/2000 final. Guidance Document on substances, Directive 98/8/EC of risk assessment for birds and mammals under the European Parliament and of the Council Directive 91/414/EEC- 25 September Council concerning the placing of 2002 biocidal products on the market – Part II - 2003

Consideration of threatened native species

No studies are requested to be conducted on native New Zealand species, the risk assessment is based on studies performed on standard surrogate species from Europe or North America. Uncertainty factors included in the risk assessment process encompass the possible susceptibility variations between the surrogate species and the native New Zealand species. However, these factors are designed to protect populations not individual organisms. We acknowledge that these factors may not be protective enough for threatened species for which the survival of the population could depend on the survival of each and every individual.

Therefore, the US EPA approach for risk assessment of endangered species has been implemented. Additional uncertainty factors are included, depending on the type of organisms. US EPA consider higher factors when organisms cannot escape the contaminated area (for aquatic organisms for instance) than for birds.

US EPA has not defined any additional factor for soil organisms except for plants, so we applied the same approach as for aquatic environment, considering that soil invertebrates won’t be able to escape from the contaminated area.

For the purpose of this risk assessment, the threatened species are those included in the following categories of the New Zealand Threat Classification System: threatened (Nationally critical, Nationally endangered, Nationally vulnerable) and at risk (declining, recovering, relict, naturally uncommon).

37 www.efsa.europa.eu/en/efsajournal/pub/1438.htm

November 2015 247

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Spray drift / dust drift

Poncho Votivo is used for seed treatment, consequently spray drift is not a relevant factor for risk assessment. However, dust drift can occur when seed are sown and incidents on bees have been reported.

The applicant provided data on the dust level (Heubach tests) from different maize qualities, all results are all below the level of concern set overseas (none is set for New Zealand). The seeds were all film coated, the impact of this coating is significant on dustiness, consequently, we consider that a control should be set to ensure that all seeds treated by Poncho Votivo will be coated.

As there is no spray drift and dust drift is not significant, some parts of the usual risk assessment are not relevant: off field exposure of soil organisms and beneficial arthropods, effects on terrestrial plants.

Aquatic risk assessment

For Class 9 substances, irrespective of the intrinsic hazard classification, the ecological risk can be assessed for a substance by calculating a Risk Quotient (RQ) based on an estimated exposure concentration. Such calculations incorporate toxicity values, exposure scenarios (including spray drift, leaching and run-off, application rates and frequencies), and the half-lives of the component(s) in water. For the aquatic environment, the calculations provide an Estimated Environmental Concentration (EEC) which, when divided by the L(E)C50 or a NOEC, gives a RQ acute or chronic.

퐸퐸퐶 퐴푐푢푡푒 푅푄 = 푠ℎ표푟푡−푡푒푟푚 퐿(퐸)퐶50

퐸퐸퐶 퐶ℎ푟표푛𝑖푐 푅푄 = 푙표푛𝑔−푡푒푟푚 푁푂퐸퐶

If the RQ exceeds a predefined level of concern, this suggests that it may be appropriate to refine the assessment or apply the approved handler control and/or other controls to ensure that appropriate matters are taken into account to minimize off-site movement of the substance. Conversely, if a worst-case scenario is used, and the level of concern is not exceeded, then in terms of the environment, there is a presumption of low risk which is able to be adequately managed by such things as label statements (warnings, disposal). The approved handler control can then be removed on a selective basis.

Levels Of Concern (LOC) developed by the USEPA (Urban and Cook, 1986) and adopted by EPA determine whether a substance poses an environmental risk (3).

Table 10 Levels of concern as adopted by EPA New Zealand

Endpoint LOC Presumption Aquatic (fish, invertebrates, algae, aquatic plants)

Acute RQ ≥ 0.5 High acute risk

Acute RQ 0.1 - 0.5 Risk can be mitigated through restricted use

November 2015 248

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Endpoint LOC Presumption

Acute RQ < 0.1 No concern

Chronic RQ ≥ 1 High chronic risk

Aquatic threatened species

Acute RQ ≥ 0.05 High acute risk

Chronic RQ ≥ 0.1 High chronic risk

Plants (terrestrial)

≥ 1 calculated on the basis of

Acute RQ EC25 or ≥ 5 calculated on the High acute risk

basis of EC50

Threatened plants species (terrestrial)

≥ 1 calculated on the basis of Acute RQ High acute risk the NOEC or EC05

GENEEC2 modelling

Calculation of expected environmental concentrations The parameters used in GENEEC2 modelling are listed in Table 11.

No model can be used for Bacillus firmus as no relevant study on environmental fate is available. However, it has been isolated from an agricultural soil, and it was not considered as a new organism in New Zealand. However, no data was provided to evaluate the impact on natural background levels as no method is currently available to quantify this specific micro-organism in the environment.

No relevant metabolite was included in the residue definition for water compartment, however both MNG and TZNG are relevant for the soil compartment. MNG and TZNG are less toxic to aquatic organisms than the active ingredient itself so no specific risk assessment will be conducted for these metabolites.

Metabolite TMG is more toxic to algae than the active ingredient but it was not detected in relevant amount in soil and water degradation studies and was below the detection limit in lysimeters studies so no specific risk assessment will be conducted for this metabolite.

Table 11 Input parameters for GENEEC2 analysis

Clothianidin

Application rate (g ai/ha) 91

Application frequency 1

Application interval (days) Not applicable

Koc 84*

November 2015 249

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Clothianidin

Aerobic soil DT50 (days) 797.1**

Pesticide wetted in? No

Granular application, no spray. Methods of application Incorporation in furrow, 2 inches

‘No spray’ zone None

Water solubility (ppm) 270

Aerobic aquatic DT50 whole system(days) 170

Aqueous photolysis DT50 (days) 0.14

*Lowest value of a non-sand soil **Upper 80% confidence limit on the mean of laboratory values (227, 143, 490, 1001, 541, 1328, 549, 533, 808

days)

Output from the GENEEC2 model for clothianidin

RUN No. 1 FOR clothianidin ON maize * INPUT VALUES *

------

RATE (#/AC) No.APPS & SOIL SOLUBIL APPL TYPE NO-SPRAY INCORP

ONE(MULT) INTERVAL Koc (PPM ) (%DRIFT) (FT) (IN)

------

0.081( 0.081) 1 1 84.0 270.0 GRANUL( 0.0) 0.0 2.0

FIELD AND STANDARD POND HALFLIFE VALUES (DAYS)

------

METABOLIC DAYS UNTIL HYDROLYSIS PHOTOLYSIS METABOLIC COMBINED

(FIELD) RAIN/RUNOFF (POND) (POND-EFF) (POND) (POND)

------

797.10 2 0.00 0.14- 17.36 170.00 15.75

GENERIC EECs (IN MICROGRAMS/LITER (PPB)) Version 2.0 Aug 1, 2001

------

PEAK MAX 4 DAY MAX 21 DAY MAX 60 DAY MAX 90 DAY

GEEC AVG GEEC AVG GEEC AVG GEEC AVG GEEC

November 2015 250

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

------

1.98 1.91 1.56 1.05 0.81

The maximum Estimated Environmental Concentration (EEC) for clothianidin when used in Poncho Votivo as estimated by GENEEC2 is 1.98 μg/L.

Calculation of acute risk quotients using GENEEC2 expected environmental concentrations Table 12 gives calculated acute risk quotients for each trophic level considering EEC estimated by GENEEC2 and lowest relevant toxicity figures.

Table 12 Acute risk quotients derived from the GENEEC2 model and toxicity data

Peak EEC from LC or EC Trigger value / Species 50 50 Acute RQ GENEEC2 (mg/L) (mg/L) Presumption

Maize – 91 g ai/ha, treated seeds

Fish, Rainbow trout 0.1 / No concern -5 > 104.2 < 1.9 x 10 0.01 / No concern for threatened species

40 (ai) 0.1 / No concern 5 x 10-5 Daphnids 1.74 (tested as 0.01 / No concern for 0.001 600 FS) threatened species

0.1 / No concern -3 Chironomus 1.98 x 10 0.0267 0.07 > 0.01 / High risk for threatened species

0.1 / No concern -5 Algae > 120 < 1.65 x 10 0.01 / No concern for threatened species

0.1 / No concern Aquatic plant, Lemna > 270 < 7.3 x 10-6 gibba 0.01 / No concern for threatened species

November 2015 251

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Calculation of chronic risk quotients using GEENEC2 expected environmental concentrations

Table 13 gives calculated chronic risk quotients for each trophic level considering EEC estimated by GENEEC2 and lowest relevant toxicity figures.

Table 13 Chronic risk quotients derived from the GENEEC2 model and toxicity data

Relevant EEC from NOEC Chronic Trigger value / Species GENEEC2 (µg /L)* (µg/L) RQ Presumption

Maize – 91 g ai/ha, treated seeds

1 / No concern -5 Fish, Fathead minnow (28 d) 20 000 7.8 x 10 0.1 / No concern for threatened species

1 / No concern Invertebrates Daphnia magna 1.56 120 0.013 (21 d) 0.1 / No concern for threatened species

1 / High risk Invertebrates Chironomus 0.72 (EC15) 2.17 riparius 0.1 / High risk for threatened species

As this study included 22 species of zooplankton, 16 species of macrozoobenthos and 30 species of phytoplankton, a factor of 10 for threatened EAC mesocosm study (98 d) 0.81 3.1 0.26 species is too conservative. A factor 3 is considered more appropriate (EU report 2005). 0.33 / No concern for threatened species

* EEC selected must be as close as possible from the exposure duration of the study selected for risk assessment purpose. The calculations are based on a conservative model taking into account the degradation of the substance and its adsorption potential in order to cover both run-off, drift input into water bodies. The model also considers information about the application method to determine the drift input into water bodies, i.e. for seed treatment, no drift is assumed.

Conclusion for the aquatic risk assessment using GENEEC2 data It is impossible to model the exposure of the aquatic environment to Bacillus firmus I-1582; however, some publications provided by the applicant indicate that Bacillus firmus strains could survive in freshwater

November 2015 252

Application for approval to import or manufacture Poncho Votivo for release (APP202077) environment. As there will be no spray drift (seed treatment application), and the mobility of this bacterium in soil is unclear, no conclusion can be drawn on the potential effects of Bacillus firmus on aquatic organisms. Nevertheless, the contamination of the aquatic environment by Bacillus firmus is expected to be low.

Acute and chronic risks due to clothianidin are below the level of concern for the use of Poncho Votivo on maize as seed treatment. Similarly, no acute or chronic risks are expected for threatened New Zealand species. This conclusion is based on the high tier study (mesocosm) which is the most relevant information available.

It is worth nothing that the DT50 for clothianidin used in these calculations (797.1 days) is based on laboratory studies. Field studies values are also available and show a faster degradation in natural conditions (the corresponding value from field studies is 171.2 days). The same conclusion applies to the metabolites as their toxicity is less or similar to the parent compound.

Groundwater risk assessment

Calculation of expected environmental concentrations in groundwater with Sci-Grow

Table 14 Input parameters for Sci-Grow analysis and resulting PEC values

Maize Clothianidin Metabolite MNG Metabolite TZNG

Application rate (kg ai/ha) 0.091 10.7% max 9.1% max

Application rate (lb/acre) 0.081 0.009 0.007

Number of applications 1 1 1

Koc* 84 16.5 236

Aerobic soil DT50 (days) 797.1 107.3** 550***

PECgw (µg/L) 1.97 0.096 0.02

* Lowest Koc of a non-sand soil **Upper 80% confidence limit on the mean of laboratory values (86.4, 108, 82.4 days) ***Upper 80% confidence limit on the mean of laboratory values (11, 819, 8, 62.1 days)

Conclusion for the groundwater risk assessment using SciGrow Estimated concentration of clothianidin is 1.97 µg/L. This concentration is rather high however, field studies conducted at 150 g ai/ha (NZ rate is 91), showed that the residues of clothianidin were mainly found in the top layer of the soil (0-15 cm). Trace amount were found in the next layer (15-30 cm) [report M-270224-02-2]. Moreover, ageing reduced the mobility of residues [M-405885-01-1].

Two metabolites of clothianidin were considered as relevant for soil compartment: MNG and TZNG. Sci Grow calculations show that the predicted concentrations are low (below or around 0.1 µg/L).

November 2015 253

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Consequently, no significant contamination of groundwater by clothianidin or its metabolites is expected in the use conditions of Poncho Votivo. No conclusion can be drawn for Bacillus firmus I-1582 as its mobility in soil is unknown.

Sediment risk assessment

No specific calculation is performed for sediment dwelling organisms because the only available study was conducted with a water contamination, not with the sediment contamination. Therefore, the aquatic risk assessment method applies. Moreover, sediment dwelling arthropods were included in the mesocosm study.

There are no concerns for the sediment dwelling arthropods due to clothianidin in the use conditions of Poncho Votivo. No studies on sediment dwelling organisms were provided on the effects of Bacillus firmus I- 1582.

Terrestrial risk assessment

For terrestrial organisms, Toxicity-Exposure Ratios (TERs) are used for earthworms and birds, Hazard Quotient (HQ) are used for terrestrial invertebrates and Risk Quotient (RQ) for bees. This convention results in concern arising if a risk quotient is less than the trigger value for earthworms and more than a trigger value for terrestrial invertebrates. LOC developed by the European Union and adopted by the EPA which allows us to determine whether a substance poses an environmental risk are provided in the Table 15.

Table 15 Levels of concern as adopted by the EPA

Level of Concern (LOC) Presumption

Earthworm/ Birds

Acute TER < 10 High risk

Chronic TER < 5 High risk

Threatened Bird species

Acute TER < 20 High risk

Chronic TER < 10 High risk

Threatened soil organisms species

Acute TER < 100 High risk

Chronic TER < 50 High risk

Bees

Acute RQ oral/contact > 0.4 High risk

Chronic RQ > 1 High risk

Terrestrial invertebrates

HQ in-field/off-field ≥2 High risk

November 2015 254

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

For more details about the different factors used for calculating TER and RQ refer to the relevant reference documents listed in Table 9.

Earthworm risk assessment

Soil Predicted Environmental Concentration (PEC) determination Both acute and reproductive earthworm tests are static tests where the test substance is applied to the system only once at the beginning. Therefore, the nominal dose levels in the test match initial concentrations in the field and thus it is appropriate to use initial PEC values (no time-weighted averages) for the acute as well as the long-term TER.

The concentration of active substance in the soil is calculated on the basis of the FOCUS (1997) document ‘Soil persistence models and EU registration’

푎푝푝푙𝑖푐푎푡𝑖표푛 푟푎푡푒 (kg a.i./ha) 푃퐸퐶 표푛푒 푎푝푝푙𝑖푐푎푡𝑖표푛 (mg/kg soil) = × 100 75 푘푔 푠표𝑖푙

Soil concentrations of the active ingredient are calculated by assuming the deposition would mix into the top 5 cm of soil, and this soil would have a bulk density of 1,500 kg/m3, i.e. the deposition expressed in mg/m2 would mix into 75 kg of soil.

In case of multiple applications, the following formula has to be used:

(1 − 푒−푛푘𝑖) 푃퐸퐶 푚푢푙푡𝑖푝푙푒 푎푝푝푙𝑖푐푎푡𝑖표푛푠 = 푃퐸퐶 표푛푒 푎푝푝푙𝑖푐푎푡𝑖표푛 × (1 − 푒−푘𝑖) where: n = number of applications

-1 k = ln2/DT50 (day ) i = interval between two consecutive applications (days)

o DT50 = half-life in soil (days) Use only DT50 values of lab test done at 10-20 C and pH between 5 and 9. e = 2.718 (constant)

When there are DT50 values of several soils use GENEEC2 formula for determining the relevant DT50 to be used.

PEC calculation results are summarized for each scenario in Table 16.

Calculation of TERs

퐿퐷 푇퐸푅푎푐푢푡푒 = 50 퐸푠푡𝑖푚푎푡푒푑 퐸푛푣𝑖푟표푛푚푒푛푡푎푙 퐶표푛푐푒푛푡푟푎푡𝑖표푛 푁푂퐸퐶 푇퐸푅푙표푛푔 − 푡푒푟푚 = 퐸푠푡𝑖푚푎푡푒푑 퐸푛푣𝑖푟표푛푚푒푛푡푎푙 퐶표푛푐푒푛푡푟푎푡𝑖표푛

November 2015 255

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Acute toxicity No calculation is performed with metabolites MNG and TZNG as they are less toxic than the active ingredient. Off-field calculations are not relevant in the situation of seed treatment as no spray drift is expected.

Table 16 Acute in-field TER value for earthworms

PEC LC TER Trigger value / Scenarios 50 mg/kg soil) (mg/kg soil) acute Presumption

Maize, seed treatment, 91 g 0.12 13.21 109 > 10 / No concern clothianidin/ha > 100 / No concern for Maize, seed treatment, 18 g 0.02 > 1000 41667 threatened species Bacillus firmus/ha

Conclusion for earthworm acute risk assessment Poncho Votivo presents no concerns for earthworms.

Chronic toxicity In general, metabolites of clothianidin were less toxic than the parent compound so they were not included in the quantitative risk assessment, except TZNG which shows a higher toxicity level on some soil organisms.

Table 20 Chronic in-field TER value for soil organisms – clothianidin and its metabolite TZNG

NOEC Trigger value / Species PEC (mg/kg soil) TER chronic (mg/kg soil) Presumption

Clothianidin, 1 application as seed treatment of maize at 91 g ai/ha

Earthworm 0.12 0.444 3.7 < 5 / High risks < 50 / High risk for threatened Springtail 0.12 0.32 2.7 species

> 5 / no concern Predatory mites 0.12 190 1583 > 50 / no concern for threatened species

TZNG, 9.1% of 91 g/ha (8.3 g/ha) formed in soil

Earthworm 0.01 13 1300 > 5 / no concern Springtail 0.01 1 100 > 50 / no concern for threatened species Predatory mites 0.01 20 2000

Bacillus firmus I-1582, 1 application as seed treatment of maize at 18 g ai/ha

Springtail 0.02 50 2500 > 5 / no concern

0.02 > 50 / no concern for Predatory mites 50 2500 threatened species

November 2015 256

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

High tier accumulation studies were performed with clothianidin used as seed treatment over a 8-year period. Maximum residue values in soil are available and can be used to inform the PEC value and refine the calculations: when clothianidin was applied every year at a rate of 150 g ai/ha (higher than the expected rate for NZ), maxima of 41.6, 48.5 and 46.4 µg/kg soil were observed in the 3 trials [Report M-443993-01-1].

Table 17 Refined chronic in-field TER value for soil organisms – clothianidin

PEC NOEC Trigger value / Species TER chronic (mg/kg soil) (mg/kg soil) Presumption

Clothianidin, 1 application as seed treatment of maize at 91 g ai/ha

Earthworm 0.0485 0.444 9.1 > 5 / no concern < 50 / High risk for threatened Springtail 0.0485 0.32 6.6 species

Conclusion for earthworm chronic risk assessment The data on Bacillus firmus I-1582 come from studies performed on a formulation at 5% of active ingredient with unclear microbial count, they were nevertheless used in the risk assessment to demonstrate that the margin of safety is rather high (TERs are 2500) so it can be concluded that there should be no concerns for soil organisms due to Bacillus firmus I-1582.

The risk assessment for clothianidin was based on a field accumulation study at a higher rate than the NZ one and assuming that Poncho Votivo will be used on the same plot every year for up to 8 years. In these conditions there are no concerns for chronic effects on soil organisms. There are high risks for threatened species, however, we consider that the presence of threatened species in the field is unlikely, and no contaminations are anticipated off-field, so in practice, there should be no concerns for threatened soil organisms.

Non-target plant risk assessment

Non target plants are non-crop plants located outside the treatment area.

As discussed previously in this memo, terrestrial plants should not be significantly exposed to Poncho Votivo as there is no spray drift and dust drift is negligible. Consequently no quantitative risk assessment is performed.

November 2015 257

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Bird risk assessment

EPA uses EFSA’s Bird model and Excel© spreadsheets38 freely available on EFSA’s website to assess the risks to birds.

The methodology calculates TERs where exposure is calculated as the dose that a bird will receive when feeding in crops that have been treated. To avoid doing detailed evaluations for low risk scenarios, assessments are performed in tiers of increasing complexity.

The steps for the acute assessment are:  Screening assessment (not available for seed treatment risk assessment)  Tier I assessment  Higher tier assessment

The steps for the reproductive assessment are:  Screening assessment (not available for seed treatment risk assessment)  Phase-specific approach assessment  Higher tier assessment

Progression to the next tier is only made if the threshold for concern is exceeded at the previous tier.

Tier 1 risk assessment Determination of levels of exposure Tier 1 assumes that granivorous birds feed entirely on readily available freshly treated seeds. Exposure of birds to pesticides used as seed treatment is primarily via dietary intake. Dermal exposure to seed treatments is unlikely to occur, especially when seeds are incorporated into the soil. Pesticides used as seed treatments are unlikely to be volatile since the protection of the seed would not be long-lasting. Hence, the contribution to exposure of birds from inhalation of pesticides from treated seeds is considered to be low. Significant contamination of drinking water after the use of a pesticide as seed treatment seems equally unlikely to be a critical route or to lead to TER greater than direct dietary consumption. Therefore, the following risk assessment focuses on the dietary route of exposure.

Pesticides used as seed treatment are normally applied to soils that have been specifically prepared (seed beds). Minimum tillage practices have increased in the last decade, but even in case of seed treatment use in minimum tillage practices the soil surface is ‘worked’ to a depth up to 5 cm. Therefore, for potential ‘consumers’ in bird populations the scenario represented by a seed treatment resembles a bare-soil scenario. Herbivorous birds are not considered to be attracted to fields immediately after treated seed has been drilled. However it is possible that birds may consume seedlings that contain residues of the active substance or consume the seedling and the remaining seed. These issues are discussed below. In general

38 Different spreadsheets for spray application, granular application and seed treatment. For bait applications a spreadsheet with Daily Food Intake of NZ relevant species is available (Crocker et al., 2002).

November 2015 258

Application for approval to import or manufacture Poncho Votivo for release (APP202077) granivorous birds prefer a certain type of seed for their diet. Not all birds are attracted to all sizes and shapes of seeds. Therefore, in a Tier 1 assessment, small granivorous birds that feed on small seeds, and larger, medium-size birds that feed on large seeds such as maize, sugar beets and beans should be considered separately.

As clothianidin is a systemic insecticide it is relevant to assess the risks from the exposure to the treated seeds but also to the emerged shoots.

Risk assessment resulting from the treated seeds consumption

For acute test:

LD TER  50 acute (NAR x FIR /bw) For reproduction test:

NOAEL TER  longterm (NAR x FIR / bw x TWA*)

NAR = Nominal loading/application rate of active substance (mg/kg seed). Poncho application rate is 120 mL/80 000 seeds or 61 g clothianidin/80 000 seeds. The thousand kernel weight for corn/maize varies between 270 and 380 g. (see www.lafranceagricole.fr/actualite-agricole/fourrage-estimer-la-valeur-du-mais-sur-pied-31107.html, www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/agdex81) so NAR = 61 g /(80*270 or 380) = 2.0 to 2.8 g ai/kg

FIR/bw = Food intake rate per body weight, according to EFSA, 2009, Table 18 p55.

*if toxic effect is considered to be caused by long-term exposure, use TWA = 0.53 (estimates time-weighted exposure over 21 days assuming a default DT50 of 10 days).

Risk assessment resulting from the consumption of newly emerged crop shoots When consumption of newly emerged crop shoots (including roots and remaining seed) is likely to occur, it is necessary to conduct an additional risk assessment for herbivorous birds. In such an assessment, any information on the amount of substance likely to be present in newly emerged crop shoots should be taken into consideration. The scenario assessed here resembles mostly the ‘newly-sown grassland’ or ‘early-post emergence uses on cereals’ scenario for spray products. Relevant indicator species for this scenario are as such large herbivorous birds and small omnivorous birds. Insectivorous birds are unlikely to present a critical case for this scenario. The FIR/bw needs to be multiplied by the concentration expected in the seedling to obtain a value suitable for use in the first-tier risk assessment. As a conservative default for the Tier 1, it is assumed that the applied amount of pesticide is contained in a total mass of seedling that is five times the weight of the original seed (based on the relative water contents of seeds and the newly emerged grass and cereal shoots). It is assumed that root, seed and seedling are ingested by the animal and that all of the

November 2015 259

Application for approval to import or manufacture Poncho Votivo for release (APP202077) applied substance remains available. If data can be provided to justify less conservative values this could be considered in a refinement step.

For acute test:

LD TER  50 acute (0.5NAR /5 ) For reproduction test: NOAEL TER  longterm (0.5NAR / 5) * x TWA **

* according to EFSA, 2009 Table 19 p 56: FIR/bw*NAR/5 **if toxic effect is considered to be caused by long-term exposure, use TWA = 0.53 (estimates time-weighted exposure over 21 days assuming a default DT50 of 10 days). Note about TWA:

Table 18 Measures of exposure and toxicity used in the reproduction assessment

Test endpoint used as Short-term Breeding phase Long-term exposure surrogate exposure

Pair formation/ breeding site 1 0.1 x LD50 1 day DDD 21 day TWA DDD selection

NOAEL for the number of eggs Copulation and egg laying 1 day DDD 21 day TWA DDD laid per hen (5 days pre-laying through NOAEL for mean eggshell end of laying 1 day DDD 21 day TWA DDD thickness

0.1 x LD50 1 day DDD 21 day TWA DDD

NOAEL for proportion of viable 1 day DDD 21 day TWA DDD Incubation and hatching eggs/eggs set/hen

NOAEL for proportion of 3 day TWA DDD 21 day TWA DDD hatchlings/viable eggs/hen

0.1 x LD50 (extrinsic adult) 2 day TWA DDD 21 day TWA DDD

1 day DDD based on 21 day TWA DDD based

0.1 x LD50 (extrinsic juvenile) chick shortcut values of on chick shortcut value Juvenile growth and survival 3.8 and 22.72 of 3.8 and 22.72 until fledging NOAEL for proportion of 14 day old juveniles/number of 3 day TWA DDD 21 day TWA DDD hatchlings/hen

November 2015 260

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Test endpoint used as Short-term Breeding phase Long-term exposure surrogate exposure

1 day DDD based on 21 day TWA DDD based

0.1 x LD50 chick shortcut values of on chick shortcut value

Post-fledging survival 3.8 and 22.72 of 3.8 and 22.72

NOAEL for 14 day old juvenile 3 day TWA DDD 21 day TWA DDD weights/hen

1 From acute study 2 The two values are to account for ground and foliar dwelling arthropods with mean residue unit doses of 3.5 and 21 respectively. Assessments are made with both values. If TER are exceeded with either value, then an assessment based on the actual composition of the diet of relevant species.

Calculation of TERs The TER for clothianidin for bird acute Tier 1 assessments are shown in the table below.

Table 19 Exposure of birds for acute Tier 1 assessment

Toxicity NAR endpoint Crop & BBCH class Indicator TER Trigger value / (mg/kg FIR/bw2 value (where appropriate) species1 ratio Presumption seed) (mg/kg bw/d)

Large Maize / seed 2800 0.1 430 1.5 < 10 / High risks granivorous < 20 high risks for Small Maize / seedlings 2800 - 430 1.5 threatened species omnivorous

1 EFSA, 2009, Tables 18 and 19 p55 and 56 2 EFSA, 2009, Table 18 p55

The TER for clothianidin for bird reproductive Tier 1 assessments are shown in the table below.

Table 20 Exposure of birds for reproduction Tier 1 assessment

Toxicity Crop & NAR endpoint Trigger BBCH class Indicator (mg/kg TWA2 FIR/bw3 value TER ratio value / (where species1 seed) (mg/kg Presumption appropriate) bw/d)

Large < 5 High risks Maize / seed 2800 0.53 0.1 56.8 0.4 granivorous < 10 high risks Maize / Small for threatened 2800 0.53 - 56.8 0.4 seedlings omnivorous species

November 2015 261

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

1 EFSA, 2009, Tables 18 and 19 p55 and 56 2 The exposure assessment of the reproduction assessment uses time-weighted average (TWA) exposure estimates over 1, 2, 3 or 21 days for different phases of the assessment. 1 day = 1.0; 2 days = 0.93; 3 days = 0.9; 21 days = 0.53 3 EFSA, 2009, Table 18 p55

Conclusion for bird risk assessment (Tier 1) However, the TER for acute and chronic effects are below 5, this indicates high risk to birds from the use of Poncho Votivo as a seed treatment for maize, thus a refinement is necessary.

Refinement of the bird risk assessment (Tier 2)

Risk assessment resulting from the treated seeds consumption

First of all, it has to be noted that the Tier 1 risk assessment represents a worst case because it assumes that either the treated seeds either the seedlings will be the only available food source for the bird for the whole period.

A study was performed to assess the repellency of the treated seeds, the avoidance factor calculated in this study is 0.45 [EFSA DAR on clothianidin, Vol 3 annex B, April 2005]. This factor will be used in the refined risk assessment.

The acute risk assessment can be refined considering the number of seeds birds take in a single feeding bout in field studies (table 20, page 59 of EFSA guidance, 2009): a mean of 12 large seeds are taken by large granivorous birds. A field study [EFSA DAR on clothianidin, Vol 3 annex B, April 2005] conducted on 10 freshly drilled maize fields showed that carrion crow, pheasant and wood pigeon picked up the maize seeds (3 to 25 seeds maximum) in some fields but not in all of them, other birds were observed in the field but didn’t take up maize seeds. The birds eating maize seeds were also observed eating other food items. Consequently, only large granivorous are considered for this assessment.

The TER is calculated according to:

LD TER  50 acute (number of seedsxamount per seed x avoidance factor/bodyweight)

November 2015 262

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Table 21 Exposure of birds to treated seeds for acute Tier 2 assessment

Amount Bird LD50 Trigger value / Type of bird Nb seeds ai/ seed weight TER ratio (mg/kg bw) Presumption (mg) (kg)

Large > 10 / no concerns granivorous 12 0.76 0.3 430 31.4 > 20 no concerns for bird threatened species

It is not possible to refine the calculation for the assessment of reproduction effects; however, further information is available from the field study mentioned above and another one conducted in Canada, showing that the number of maize seeds available for the birds in normal practice is low and very few birds fed on these seeds.

It is worth noting that the assumption in the quantitative risk assessment, is that birds would consume 100% of their diet as treated seeds. The field studies show that it is unlikely, especially for long periods of time. In addition, it is unlikely that in practice, treated seeds are the only food source available for granivorous birds.

It has also been demonstrated that birds like pheasants, had an avoidance behaviour against clothianidin treated seeds, sufficient enough to protect them from being intoxicated.

Some literature data from New Zealand39 indicate that maize seeds consumption represents only part of the diet (27% for greenfinch between January and June, 23% for house sparrow during the same period). Another publication40, shows that around the same amount of maize is consumed by feral pigeons (23.8 and 23.4% in 2 different locations) and the consumption is only done in April – August after harvest.

This seems to indicate that a long term consumption of maize treated seeds during the breeding season of bird is unlikely. However, additional controls to mitigate the risks to birds have been applied to all seed treatments in New Zealand:

 Excess treated seed shall not be left in areas accessible to birds. The substance label shall include a statement to this effect. Information to this effect shall be provided with treated seeds.

 Treated seed shall be completely covered by soil when sown. The substance label shall include a statement to this effect. Information to this effect shall be provided with treated seeds.

For all these reasons, it is considered that with controls in place, the risks of Poncho Votivo for birds (acute

39 Reviewed by MacLeod et al (2008) The forgotten 60%: bird ecology and management in New Zealand’s agricultural landscape. New Zealand journal of ecology 32(2): 240-255.

40 Dilks (1974) Diet of feral pigeons (Columa livia) in Hawke’s Bay, New Zealand. New Zealand journal of Agricultural Research 18: 87-90.

November 2015 263

Application for approval to import or manufacture Poncho Votivo for release (APP202077) and chronic) are acceptable.

Risk assessment resulting from the consumption of newly emerged crop shoots Instead of considering that the application rate will be diluted in a shoots which is 5 times the weight of the seed, measured residues data from field tests will be used in the calculation.

Averages amount of residues in seedling at BBCH 12 were 15.5, 11.6 and 16.0 mg/kg (mean = 14.4) and 3.58, 2.91 and 4.54 mg/kg (mean = 3.7) at BBCH 14 [M-182258-01-1 and M-242633-01-1]. The application rate were 0.5 or 1.25 mg ai/seed, but it doesn’t influence the level of residues so all values will be used.

Table 22 Exposure of birds to seedlings for acute Tier 2 assessment

NAR Toxicity (mg/kg Crop & BBCH endpoint Indicator seed) or Trigger value / class (where FIR/bw2 value TER ratio species1 residues Presumption appropriate) (mg/kg in shoots bw/d) (mg/kg)

> 10 / No concerns Small Maize / seedlings 14.4 - 430 29.9 > 20 No concerns omnivorous for threatened species

Table 23 Exposure of birds to seedlings for reproduction Tier 2 assessment

Crop & Toxicity NAR BBCH class Indicator endpoint TER Trigger value (mg/kg TWA2 FIR/bw3 (where species1 value ratio / Presumption seed) appropriate) (mg/kg bw/d)

> 5 No concerns > 56.8 (quail) > 7.4 Maize / Small < 10 high risks 14.4 0.53 - > 86.06 > seedlings omnivorous for threatened (Mallard) 11.3 species

1 EFSA, 2009, Tables 18 and 19 p55 and 56 2 The exposure assessment of the reproduction assessment uses time-weighted average (TWA) exposure estimates over 1, 2, 3 or 21 days for different phases of the assessment. 1 day = 1.0; 2 days = 0.93; 3 days = 0.9; 21 days = 0.53 3 EFSA, 2009, Table 18 p55 The NOEC for reproduction were in both studies, the highest tested concentration, no effects at all were observed in the tests. Therefore, the obtained TER ratio is underestimated. Moreover, this calculation assumes that the 100% of the birds’ diet is constituted of treated seedlings over the whole reproduction period.

November 2015 264

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Conclusion for bird risk assessment (Tier 2) There are borderline risks for reproduction of birds (for threatened species only) in case of exposure to treated seedlings, however these risks are considered acceptable considering the very conservative assumptions used in the assessment.

The other risks (acute or chronic) for birds were below the level of concern.

Secondary poisoning

No information is available on Bacillus firmus I-1582 bioaccumulation and clothianidin is not bioaccumulative so no risk assessment for secondary poisoning is necessary.

Bee risk assessment

Tier 1- screening level risks If a reasonable potential for exposure to the pesticide is identified, a screening-level risk assessment is conducted. This step involves a comparison of Tier I estimated exposure concentrations (EECs) for contact and oral routes of exposure to adults and larvae to Tier I acute and chronic levels of effects to individual bees using laboratory-based studies. The conservatism of the Tier I screening-level risk quotient (RQ) value results primarily from the model-generated exposure estimates that, while intended to represent environmentally relevant exposure levels, are nonetheless considered high-end estimates. The resulting acute and chronic RQ values are then compared to the corresponding level of concern (LOC) values for acute and chronic risk (i.e., 0.4 and 1.0, respectively). Generally, if RQ values are below their respective LOCs, a presumption of minimal risk is made, since the Tier I risk estimation methods are designed to be conservative.

EEC are calculated as follows:

Measurement Exposure Exposure estimate Acute effect Chronic effect endpoint# endpoint route (EEC)* endpoint Foliar application

Individual survival Application rate (kg Acute contact Contact None (adults) ai/ha) x 2.4 µg ai/bee LD50

Application rate (kg Individual survival Acute oral Chronic adult oral NOAEL (effects Diet ai/ha) x 98 µg ai/g x (adults) LD50 to survival or longevity) 0.292 g/day

Application rate (kg Brood size and Chronic larval oral NOAEL (effects Diet ai/ha) x 98 µg ai/g x Larval LD50 success to adult emergence, survival) 0.124 g/day

Soil treatment

Individual survival Briggs EEC x 0.292 Acute oral Chronic adult oral NOAEL (effects Diet (adults) g/day LD50 to survival or longevity)

November 2015 265

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Measurement Exposure Exposure estimate Acute effect Chronic effect endpoint# endpoint route (EEC)* endpoint

Brood size and Briggs EEC x 0.124 Chronic larval oral NOAEL (effects Diet Larval LD50 success g/day to adult emergence, survival)

Seed treatment&

Individual survival Acute oral Chronic adult oral NOAEL (effects Diet 1 µg ai/g x 0.292 g/day (adults) LD50 to survival or longevity)

Brood size and Chronic larval oral NOAEL (effects Diet 1 µg ai/g x 0.124 g/day Larval LD50 success to adult emergence, survival)

Tree trunk application**

Individual survival µg ai applied to tree/g Acute oral Chronic adult oral NOAEL (effects Diet (adults) foliage x 0.292 g/day LD50 to survival or longevity)

Brood size and µg ai applied to tree/g Chronic larval oral NOAEL (effects Diet Larval LD50 success foliage x 0.124 g/day to adult emergence, survival)

* Based on food consumption rates for larvae (0.124 g/day) and adult (0.292 g/day) worker bees and concentration in pollen and nectar ** Note that concentration estimates for tree applications are specific to the type and age of the crop to which the chemical is applied. # To calculate RQs for chronic effects, NOAEC can be used as the effect endpoint to compare with the exposure estimate in concentration and assume that pesticide concentration in pollen and nectar of seed treated crops is 1 mg a.i./kg (1 μg a.i./g).

• No adjustment is made for application rate (Based on EPPO’s recommended screening value)

퐸퐸퐶 퐴푐푢푡푒 푅푄 = 퐿퐷50

퐸퐸퐶 퐶ℎ푟표푛𝑖푐 푅푄 = 푁푂퐴퐸퐿

Application EEC Toxicity endpoint Trigger value Use scenario RQ rate (kg ai/ha) (µg ai/bee) value /Presumption Acute / Adult bees – oral

Clothianidin / Maize LD50 = 0.0025 µg 0.091 0.292 117 > 0.4 / High risks seed treatment ai/bee

Bacillus firmus / LD50 > 5.38 µg ai/bee < 0.018 0.292 < 0.4 / low concerns Maize seed treatment (tested as 5WP) 0.19

Chronic / Adult bee – oral

November 2015 266

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Clothianidin / Maize NOEC = 0.00038 µg 0.091 0.292 768 > 1 / High risks seed treatment ai/bee/day

Acute and chronic effects on brood

No assessment possible as no data provided on these toxicity endpoints. A larvae test was performed but it is not a single exposure test or a chronic one (10 days) so it is not considered at this stage.

Higher Tiers – refinement options For those chemicals where RQ values exceed LOCs even after exposure estimates have been refined using measured residue values in pollen and nectar, more refined testing on honey bee colonies may be needed using Tier II semi-field studies and, depending on the nature of remaining uncertainties, Tier III full-field studies. Higher-tier studies with whole colonies are used to provide a more realistic characterization of potential adverse effects to colonies since the study design is intended to reflect actual exposure conditions.

Semi and full-field studies have to be used to determine whether effects reported in laboratory based studies on individual bees are apparent at the level of the whole colony and nature, magnitude and duration of these effects considering potential routes of exposure, and the biological relevancy of effects must be gauged, as well as sublethal effects that may not manifest in Tier I studies. To the extent possible, available estimates of exposure to colony bees through measured residues in pollen and nectar coming into the colony through the labelled use of the pesticide should be characterized relative to the reported effects.

The first step of the refinement is to use residue data to assess the acute and chronic dietary risks for bees.

Maize does not produce nectar so the exposure of bees via nectar consumption is not relevant.

Pollen residue data were provided by the applicant. The majority of the tests were performed with 0.5 mg ai/seed which is less than the expected rate in New Zealand (0.91 mg ai/seed) so these data were disregarded, however 2 tests were carried out at 1.25 mg ai/seed which are considered for the risk assessment. The mean values in pollen collected from maize were 3.4 and 3.6 µg/kg, the latest is used below.

USEPA guidance provides information on daily consumption of pollen depending on the life stage and the task of the bees, it varies between 0.041 and 12 mg/day for workers bees and between 1.8 and 3.6 mg/day for larvae.

EFSA guidance provides information for bumble bees: 26.6 – 30.3 mg/bee/day (Table J1, page 147, EFSA journal 2013 11(7): 3295)

퐸퐸퐶 = 푟푒푠𝑖푑푢푒푠 𝑖푛 푝표푙푙푒푛 푥 푝표푙푙푒푛 푐표푛푠푢푚푝푡𝑖표푛

With residues in pollen in ng/g and pollen consumption in g/day.

Among clothianidin metabolites, TZNG shows high acute toxicity to bees (3.9 µg/bee), however it is still around 1000 times less toxic than the parent compound and the amount of residues in pollen is lower than the parent compound, and consequently no calculation is performed for this metabolite or the others. The conclusion for the parent compound is applicable to the metabolites.

November 2015 267

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Pollen Toxicity EEC (ng Trigger value Use scenario consumption endpoint RQ ai/bee/day) /Presumption (g/day) value Acute / Adult honey bees - oral

Clothianidin / Maize 0.00015 – LD50 = 2.5 ng 0.00006 – < 0.4 / low 0.000041 – 0.012 seed treatment 0.0432 ai/bee 0.02 concerns

Acute / Adult bumble bees - oral

Clothianidin / Maize LD50 = 1.943 < 0.4 / low 0.0266 – 0.0303 0.098 – 0.108 0.05 – 0.06 seed treatment ng ai/bee concerns

Acute / honey bee larvae – oral*

Residues in Clothianidin / Maize NOEC > 40 < 0.4 / low - pollen: 3.6 < 0.09 seed treatment µg ai/kg diet concerns µg/kg

Chronic / Adult honey bee - oral

Clothianidin / Maize 0.00015 – NOEC = 0.38 < 1 / low 0.000041 – 0.012 0.0004 – 0.11 seed treatment 0.0432 ng ai/bee/day concerns

Acute and chronic effects on brood

No assessment possible as no data provided on these toxicity endpoints

* As the toxicity endpoint for larvae is expressed as µg ai/kg diet, it is directly compared to the amount of residues in pollen. There are no concerns for adult and larvae of honey bees from acute or chronic exposure to residues of clothianidin in pollen of maize. Similarly, there is no concerns for adult bumble bees from acute exposure.

In addition, higher tier studies (tunnel or field tests), for exposure to maize pollen or guttation fluid, showing that there was no effects on mortality, behaviour, hive condition for seeds treated at 1 or 1.25 mg ai/seed.

Non-target arthropod risk assessment

Where limit tests are conducted, a low risk to non-target arthropods can be concluded when the effects at the highest application rate multiplied by MAF are below 50% (ESCORT2 workshop, 2000 – p12).

Calculation of HQs

퐴푝푝푙𝑖푐푎푡𝑖표푛 푟푎푡푒 (푔 표푟mL a.i./ha) × 푀퐴퐹 ∗ 퐼푛 − 푓𝑖푒푙푑 퐻푄 = 퐿푅50 ∗∗

* application rate and LR50 must not differ in their units, i.e. must be related to either formulation or a.i. rates ** Multiple application factor, refer to Appendix V, p 45 of ESCORT 2 Workshop, 2000. MAF = 1 when there is just one application.

November 2015 268

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

푑푟𝑖푓푡 푓푎푐푡표푟 ∗ 푎푝푝푙𝑖푐푎푡𝑖표푛 푟푎푡푒 × 푀퐴퐹 × ( ) 푣푒푔푒푡푎푡𝑖표푛 푑𝑖푠푡푟𝑖푏푢푡𝑖표푛 푓푎푐푡표푟 ∗∗ 푂푓푓 − 푓𝑖푒푙푑 퐻푄 = × 푐표푟푟푒푐푡𝑖표푛 푓푎푐푡표푟 ∗∗∗ 퐿푅50 * Overall 90th percentile drift values are presented in Appendix VI , p 46 of ESCORT 2 Workshop, 2000. ** default value of 10

*** default value of 10

MAF (Multiple Application Factor) is defined as 1 as there is only one application.

The off-field HQ is not relevant in case of seed treatment as no spray drift can occur, and the level of dust when sowing is not of concern.

On this basis, the resultant in-field hazard quotients for the non-target species are shown in Table 24

Table 24 In-field HQ values

LR Application rate Hazard Trigger value Species 50 MAF (g ai/ha) (g ai/ha) Quotient /Presumption Bacillus firmus / Maize seed treatment

Aphidius rhopalosiphi > 5000 18 1 < 0.004 < 2 / no concern

Clothianidin / Maize seed treatment

Aphidius rhopalosiphi 1.086 91 1 84 > 2 / high risks

Typhlodromus pyri 125.99 91 1 0.7 < 2 / no concern

Poecilus cupreus > 75 91 1 < 1.2 < 2 / no concern

Other studies are also available but which were not designed to provide a LR50. In case of seed treatment, the most relevant non target species are soil dwelling arthropods such as Poecilus cupreus or Pardosa spiders, but not Aphidius rhopalosiphi. These additional studies show that there were no concerns for spiders at 166 g ai/ha applied as maize treated seeds. However, effects were observed on larvae, not on adults of Poecilus cupreus, but at a much higher application rate (467 g ai/ha). The same rate was slightly harmful for adults of Aleochara bilineata (rove beetle).

Conclusion for non-target arthropod risk assessments

Based on the available information, it can be concluded that the level of risk for the non-target arthropods which could be exposed in the use conditions of Poncho Votivo, is acceptable.

November 2015 269

Application for approval to import or manufacture Poncho Votivo for release (APP202077)

Summary and conclusions of the ecological risk assessment

We assessed the potential risk to be triggered by the use of Poncho Votivo following the instructions captured in the proposed label.

Bacillus firmus I-1582 risks were assessed as far as possible, but the nature of this active ingredient and the level of information available prevented a full assessment of the impact of this substance on the environment. However, the available information does not raise any concerns.

The risks of the other active ingredient (clothianidin) were also quantitatively assessed, including the potential impact of its metabolites.

First of all, tests were provided to demonstrate that the level of dust produced during sowing was acceptable so no risks would be expected for organisms (including bees) off-field due to dust drift. These levels of dust are reached when the treated seeds are film coated so a control has been set to ensure that seeds will be coated in practice.

High tier, good quality studies were generally available to refine the risk assessments. Consequently, risks below the level of concern were obtained for aquatic environment, soil organisms, plants (including endangered species), and for bees and other non-target arthropods.

Some risks were identified for birds however controls are set to mitigate the access of birds to treated seeds. With these controls in place, the level of risk is considered negligible for birds (including endangered species). The potential of groundwater contamination is below the level of concern taking into account the amount in the upper layer and mobility in soil.

November 2015