Application for approval to import or manufacture Poncho Votivo for

Transcription

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
November 2015
3 December 2015
3
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
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
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
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
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
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
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
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 I1582 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
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 crops 3.
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: 8790.
3
Coleman (2001) Farmer Perceptions of bird Damage and Control in Arable Crops. New Zealand Plant Protection 54:184-187.
November 2015
12
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
Acute / Adult honey bees - oral
Acute / Adult bumble bees - oral
Acute / honey bee larvae – oral
Chronic / Adult bee - oral
RQ
Trigger value
Result
0.00006 – 0.02
0.4
Negligible risk
0.05 – 0.06
0.4
Negligible risk
< 0.09
0.4
Negligible risk
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
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
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
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
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
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 Regulations 4 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 –
27
Limiting exposure to toxic
substances through the setting
of TELs
No TEL values are set for any component of the
substance at this time
T2
Regs 29, 30
Controlling exposure in places of
work through the setting of
WESs.
WES values from WorkSafe New Zealand’s Workplace
Exposure Standards and Biological Exposure Indices
Document; 7th Edition; February 20135 have been
adopted for this substance.
[Note: WES values have been set for components G
and E1]
4
T4
Reg 7
Requirements for equipment
used to handle substances
T5
Reg 8
Requirements for protective
clothing and equipment
The following is added in addition to the requirements
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.
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-exposureindices/workplace-exposure-standards-and-biological-indices-2013.pdf
November 2015
18
Code
Regulation
Description
T7
Reg 10
Restrictions on the carriage of
toxic or corrosive substances on
passenger service vehicles
E1
Regs 32 –
45
Limiting exposure to ecotoxic
substances through the setting
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
Variation
No EEL values are set at this time and the default EELs
are deleted
Hazardous Substances (Identification) Regulations 2001
Code
Regulation
Description
I1
Regs 6, 7,
32 – 35,
36(1) – (7)
Identification requirements,
duties of persons in charge,
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 –
31
Additional information
requirements, including
situations where substances are
in multiple packaging
I20
Reg 36(8)
Durability of information for class
6.1 substances
November 2015
Variation
19
Code
Regulation
Description
I21
Regs 37 –
39, 47 – 50
General documentation
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
Variation
Hazardous Substances (Packaging) Regulations 2001
Code
Regulation
Description
P1
Regs 5, 6,
7(1), 8
General packaging requirements
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
Variation
Hazardous Substances (Disposal) Regulations 2001
Code
Regulation
Description
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
Variation
20
Code
Regulation
Description
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
Variation
Hazardous Substances (Emergency Management) Regulations 2001
Code
Regulation
Description
EM1
Regs 6, 7, 9
– 11
Level 1 information
requirements for suppliers and
persons in charge
EM6
Reg 8(e)
toxic substances
EM7
Reg 8(f)
ecotoxic substances
EM8
Regs 12 –
16, 18 – 20
Level 2 information
requirements for suppliers and
persons in charge
EM11
Regs 25 –
34
Level 3 emergency
management requirements:
duties of person in charge,
emergency response plans
EM12
Regs 35 –
41
Level 3 emergency
secondary containment
Variation
The following subclauses are added after subclause (3)
of regulation 36:
(4)
(5)
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.
In this clause, pipework—
(a)
means piping that—
(i) is connected to a stationary container;
and
November 2015
21
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
Code
Regulation
Description
Variation
(3)
EM13
Reg 42
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.
Level 3 emergency
signage
Hazardous Substances and New Organisms (Personnel Qualifications) Regulations 2001
Code
Regulation
Description
Variation
AH 1
Regs 4 – 6
Approved Handler requirements
(including test certificate and
qualification requirements)
Refer to control E7
Hazardous Substances (Tank Wagon and Transportable Containers) Regulations 2004
Code
Regulation
Description
Tank
Wagon
Regs 4 to 43
as applicable
Controls relating to tank wagons
and transportable containers
Variation
Additional controls
Code
Section
of the Act
Control
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
method
77A
This substance may only be applied as a seed treatment.
Sch 8
Schedule 8
This substance may only be applied in a purpose-built seed coating machine, in a
treatment plant with local exhaust ventilation.
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
Code
Section
of the Act
Control
This Schedule applies to every stationary container system that contains, or is
intended to contain the substance.
Seed
treatment
Labelling
77A
77A
The following additional controls are applied to this substance:

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
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 be completely covered by soil when sown.
November 2015
24
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
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

This substance may only be applied as a seed treatment.

This substance must not be applied onto or into water 10

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.


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
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:


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
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
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
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
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
Appendix C: Summary of submissions
Table 3 Summary of submissions received
No.
Submitter
Individual
or group
Position
Key issues
Summary of submission
110630
Michael Tott
Individual
Oppose
Bees
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
potential environmental harm. Neonics (Neonicotinoid pesticides) pose a huge risk to the bee
and pollinator populations.
110631
Jon Carapiet
Individual
Oppose
Bees
potential environmental harm. Neonics pose a huge risk to the bee and pollinator populations.
110632
Marcia
Lawerence
Individual
Not
indicated
Bees
“If you kill the bees, you kill the world food chain”,
110633
Mike G
McCree
Bees
Bees
This pesticide is a bee-killer. It contains a neonic which is soaked into the seeds which in turn
spread throughout the plant killing anything that feeds on it including bees. Hive collapse is a
significant problem world-wide and adding more pesticides to the approved list will only add to
the problem.
110634
Joanne
Churcher
Individual
Individual
Oppose
Oppose
110635
Ross
Scholes
NZ Peasants
Association
Oppose
Bees
This product contains a neonicotinoid that has been implicated in colony collapse disorder and
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
No.
Submitter
Individual
or group
Position
Key issues
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.
110637
Elaine
Bainbridge
110638
Patricia
Wallace
110639
110640
Jenny Kirk
Gerry Coates
November 2015
Individual
Individual
Individual
Te Rūnanga
o Ngāi Tahu
Oppose
Oppose
Oppose
Neutral
Bees
Bees
Neonicotinoids are a pesticide proven to be detrimental to bees and therefore constitutes an
unacceptable risk to the NZ Apiculture Industry. This pesticide contains a neonicotinoid that
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.
Bees
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
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.
Bees
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
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
have decided to remain neutral regarding the introduction of this insecticide product Poncho
Votivo to New Zealand.
Toxicity to
aquatic and
terrestrial
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
34
No.
Submitter
Individual
or group
Position
Key issues
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.
110641
Sarah
Russell
110642
Celeste
HaywardRyan
Individual
110643
Sidney Lovell
Individual
110644
110645
Claire
Bleakley
Kate Hughes
November 2015
Individual
Individual
Individual
Bees
This pesticide contains a neonicotinoid that has been implicated in bee colony collapse and
has been banned in the EU. These pesticides can easily be replaced by other chemicals
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.
Oppose
Bees
This pesticide contains a neonicotinoid that has been implicated in bee colony collapse in
Canada and other countries. New Zealand has a large fruit production and export market; of
which bees are import to its future and it is not acceptable to but the agricultural sector in
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.
Oppose
Bees
Neonicotinoid is proven to be detrimental to bees, and possibly other pollinating insects. It has
been banned by the European Commission (EC) and New Zealand should be proactive in
trying to save its bees as the EC is.
Bees
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
have long term systemic persistence and affects insects who come into contact with the pollen
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.
Bees
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
which bees are import to its future and it is not acceptable to put the agricultural sector in
further danger from bee colony collapse.
Oppose
Oppose
Oppose
35
No.
110646
Submitter
Philippa
Rawlinson
Individual
or group
Federated
Farmers of
New
Zealand Bee
Industry
Group
Position
Neutral
Key issues
Bees
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
Safety Authority (EFSA) and United States Environmental Protection Authority identify that
clothianidin is highly toxic to honeybees on a contact and an oral basis. There is also
uncertainty around the pollen and nectar uptake by the honeybee from flowering crops treated
with clothianidin.
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.
110647
Don
MacLeod
National
Beekeepers
Association
of New
Zealand
Clothianidin is subject to bans on use in Europe and Bayer is facing lawsuits in Canada with
respect to seed treatment use of clothianidin causing beehive deaths.
Not
indicated
Bees
The application advises the public and the EPA to consider benefits that cannot be subject to
scrutiny because no agency in NZ is collecting data on pesticide use by volume and crop.
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.
110648
David
Chisholm
November 2015
Individual
Oppose
Bees
Concerned about the risk to New Zealand’s primary industries of importing a substance that
has raised concerns from scientists regarding its effect on honey bees. The association of a
36
No.
Submitter
Individual
or group
Position
Key issues
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
The staff’s
classification
Class 1 Explosiveness
No
ND
Class 2, 3 & 4 Flammability
No
ND
Class 5 Oxidisers/Organic
Peroxides
No
ND
Subclass 8.1 Metallic
corrosiveness
No
ND
Subclass 6.1 Acute toxicity (oral)
6.1D
6.1D
Subclass 6.1Acute toxicity
(dermal)
No
No
Subclass 6.1 Acute toxicity
(inhalation)
No
No
Mixture rules14
Applicant’s
classification
Read across
Hazard Class/Subclass
Method of
classification
Mixture data
Mixture classification
Remarks
11
OECD (1990) Manual for Investigation of HPV Chemicals. Retrieved on 23 January 2008 at http://www.icca-chem.org/Home/ICCAinitiatives/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
The staff’s
classification
Subclass 6.1 Aspiration hazard
No
ND
Subclass 6.3/8.2 Skin
irritancy/corrosion
No
No
Subclass 6.4/8.3 Eye
irritancy/corrosion
Subclass 6.5A Respiratory
sensitisation
15
6.4A
No
Mixture rules14
Applicant’s
classification
Read across
Method of
classification
Mixture data
Remarks
No
The difference in
classification appears
to be a difference in
interpretation of the
HSNO criteria for
classification
6.5A
Based on microorganisms being
considered potential
sensitisers
Based on microorganisms being
considered potential
sensitisers
Subclass 6.5B Contact
sensitisation
No
6.5B
Subclass 6.6 Mutagenicity
No
ND
Subclass 6.7 Carcinogenicity
No
ND
Subclass 6.8 Reproductive/
developmental toxicity
No
ND
developmental toxicity (via
lactation)
No
ND
Subclass 6.9 Target organ
systemic toxicity15
6.9B
6.9B (oral)
Clothianidin
Subclass 9.1 Aquatic ecotoxicity
9.1A
9.1A
Clothianidin mainly
Subclass 9.2 Soil ecotoxicity
9.2B
9.2B
Clothianidin
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
The staff’s
classification
Subclass 9.3 Terrestrial
vertebrate ecotoxicity
9.3B
9.3C
Subclass 9.4 Terrestrial
invertebrate ecotoxicity
9.4A
9.4A
Mixture rules14
Applicant’s
classification
Read across
Method of
classification
Mixture data
Remarks
Based on acute oral
toxicity study (LD50 =
2000 mg/kg bw).
Mammals are more
Clothianidin
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
Appendix E: Physical-chemical properties of the mixture
Table 2 Physical and chemical properties of the mixture
Property
Results
Test method
Klimisch
Score (1-4)
Colour
Dark grey
OCSPP 830.6302
1
Physical state
Suspension
OCSPP 830.6303
1
Odour
Musty
OCSPP 830.6304
1
pH
6.7 undiluted, 7.6 (1%
in deionised water)
CIPAC MT 75.3
OCSPP 830.7000
1
CIPAC MT 192
OECD 114
OCSPP 830.7100
1
92/69/EEC A.5
OECD 115
1
92/69/EEC A.3
OECD 109
OCSPP 830.7300
1
CIPAC MT 187
1
CIPAC MT 194
1
Reference
Dynamic:
Shear rate 20 s-1:
538.0 10-3 Pa s
Shear rate 100 s-1:
364.6 10-3 Pa s
Dynamic viscosity (20oC)
Kinematic:
Shear rate 20 s-1:
417.4 10-6 m2/s
Shear rate 100 s-1:
282.8 10-6 m2/s
24 mN/m (undiluted at
25oC)
Surface tension
43 mN/m (at 1g/L in
deionized water at
20oC)
D420 = 1.289
Relative Density
D440 = 1.276
measurement in water,
15 s ultrasonified
Particle size distribution
90% < 38.89 µm
50% < 8.00 µm
10% < 1.30 µm
Adherence to seeds
(maize)
Adherence ratio (before
and after adherence
test): 101.6%
Oxidizing properties
No data provided
Explosive properties
No data provided
Flammability
No data provided
November 2015
M Hoppe & W Gueldner
(2013) Physical, chemical
and technical properties
of Bacillus firmus I-1582
+ clothianidin FS 610
(102+508 g/L). Bayer
CropScience AG R&D D - FT - Analysis &
Services Alfred-NobelStrasse 50 40789
Monheim am Rhein /
Germany. Study number:
FM0194(PCF00)G01,
Bayer ID: M-471757-01-1
41
Property
Results
Test method
Klimisch
Score (1-4)
Reference
1
J Smith et al (not dated)
Heubach Dustmeter Test
of Dust off from Hybrid
Maize Seed Treated with
Poncho® and Poncho
Votivo®. Bayer
CropScience New
Zealand. Trial number
BSAC002
1
J Smith et al (not dated)
Heubach Dustmeter Test
of Dust off from Hybrid
Maize Seed Treated with
Poncho® and Poncho
Votivo®. Bayer
CropScience New
Zealand. Trial number
BSAC001
4 maize seed qualities
with seed count from
2767 to 4800
kernels/kg were treated
with Poncho Votivo at
the label rate and film
coats.
Results are below the
French threshold for
maize 3 g dust/100 kg
(values between 0.35
and 1.07 g/100 kg) and
below the ESTA/ESA
threshold of 0.75 g/100
000 kernels (values
between 0.12 and 0.23
g/100 000 kernels)
ESA/ESTA, 2011
Heubach test
2 maize seed qualities
with seed count from
2767 to 4800
kernels/kg were treated
with Poncho Votivo at
the label rate and film
coats.
Results are below the
French threshold for
maize 3 g dust/100 kg
(values between 0.11
and 0.99 g/100 kg) and
below the ESTA/ESA
threshold of 0.75 g/100
000 kernels (values
between 0.05 and 0.16
g/100 000 kernels)
November 2015
ESA/ESTA, 2011
42
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
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
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
None
GLP
Yes
Test Guideline/s
OPPTS 870.1200
Species
Rat
Strain
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
number 33030. M-426246-01-1
November 2015
45
Klimisch Score
1
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
No/Sex/Group
5
2.03 mg/L
Dose Levels
Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard
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
number 33032. M-426242-01-1
Klimisch Score
November 2015
1
46
None
GLP
Yes
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
number 33031. M-426248-01-1
Klimisch Score
1
None
November 2015
47
GLP
Yes
Test Guideline/s
EC 440/2008, Method B.5
Species
Rabbit
Strain
New Zealand albino
No/Sex/Group
1M&2F
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
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.
November 2015
48
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
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
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
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
Taxonomy
Super kingdom: Bacteria; Class: Bacilli; Family Bacillaceae; phylum:
firmicutes; order: Bacillales; genus: Bacillus; species firmus
Strain
I-1582
Identification
Using the available morphological, physiological and biochemical
data, the strain was clearly identified as Bacillus firmus.
Culture collection
“ Collection Nationale de culture de micro-organismes” (CNCM) of
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
Classification
Table 4 Applicant and EPA staff classifications of the active ingredient
Class 1 Explosiveness
NA
Class 2, 3 & 4 Flammability
NA
Class 5 Oxidisers/Organic Peroxides
NA
Subclass 8.1 Metallic corrosiveness
NA
Subclass 6.1 Acute toxicity (oral)
ND
Subclass 6.1 Acute toxicity (dermal)
ND
Subclass 6.1 Acute toxicity (inhalation)
ND
Subclass 6.1 Aspiration hazard
ND
Subclass 6.3/8.2 Skin
irritancy/corrosion
No
Subclass 6.4/8.3 Eye irritancy/corrosion
No
Subclass 6.5A Respiratory sensitisation
6.5A
November 2015
Read across
Test results
Staff’s
classification
Hazard Class/ Subclass
Method of
classification
Applicant’s
classification
Active ingredient
classification
Remarks
Classification not
possible based on
endpoints of Acute
oral toxicity/
pathogenicity study.
However no
mortality and no
adverse effects
were observed.
Classification not
possible based on
endpoints of Acute
pulmonary toxicity/
pathogenicity study.
However no
mortality and no
adverse effects
were observed.
Microorganisms
considered
potential sensitisers
52
Subclass 6.5B Contact sensitisation
6.5B
Subclass 6.6 Mutagenicity
ND
Subclass 6.7 Carcinogenicity
ND
developmental toxicity
ND
developmental toxicity (via lactation)
ND
Subclass 6.9 Target organ systemic
toxicity
ND
Subclass 9.1 Aquatic ecotoxicity
9.1C
Subclass 9.2 Soil ecotoxicity
ND
Subclass 9.3 Terrestrial vertebrate
ecotoxicity
ND
Subclass 9.4 Terrestrial invertebrate
ecotoxicity
ND
Read across
Test results
Staff’s
classification
Hazard Class/ Subclass
Method of
classification
Applicant’s
classification
Active ingredient
classification
Remarks
Microorganisms
considered
potential sensitisers
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
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
Property
Result
Test method
Colour
Beige
OPPTS 830.6302
Physical state
Solid, powder
OPPTS 830.6303
Odour
Weak odour, not
characteristic
OPPTS 830.6304
Oxidizing properties
No oxidising
A17
Klimisch
Score (14)
F Ziemer & U Eyrich (2012)
Clothianidin (TI 435, AE 1283742),
technical substance: Physical
characteristics colour, physical
state and odour. Bayer
CropScience AG Research
Technologies-Analytics Frankfurt
D-65926 Frankfurt am Main
(Germany) Project ID PA12/030.
Bayer ID M-427236-01-1
1
M Krack (2014) Clothianidin (Tl
435, AE 1283742), technical
substance: oxidizing properties.
Siemens AG Prozess-Sicherheit
Industriepark Höchst, B 596 & B
598 D-65926 Frankfurt am Main
(Germany) Project no
20140252.04, Bayer ID M-49610001-1
substance: explosive properties.
20140252.02, Bayer ID M-49609701-1.
Explosive properties
Not explosive
A14 and OECD 113
(1981)
1
Melting point
Melting range: 172174oC
A1 and OECD 102
(1995)
1
Boiling point
Decomposition at
190oC before
boiling
A2 and OECD 103
(1995)
1
November 2015
Reference
H Smeykal (2012) Clothianidin (TI
substance: Melting point, boiling
point, thermal stability. Siemens
AG Prozess-Sicherheit
20120072.01, Bayer ID M-42776001-1.
54
Property
Result
Test method
Klimisch
Score (14)
Reference
1
S Winckler (2011) Clothianidin (TI
substance: vapour pressure.
20110386.02, Bayer ID M-42018801-1.
1
F Ziemer & U Eyrich (2014)
pure substance: determination of
the surface tension. Bayer
CropScience AG Research
Technologies-Analytics Frankfurt
D-65926 Frankfurt am Main
(Germany) Project ID PA14/062.
Bayer ID M-490653-01-1.
1
A Wiche & B Bogdoll (2011)
technical substance: Solubility in
distilled water, at pH 4 and pH 10
(flask method). Bayer CropScience
AG Research TechnologiesAnalytics Frankfurt D-65926
Frankfurt am Main (Germany)
Project ID PA11/100. Bayer ID M420036-01-1.
1
F Ziemer & C Peschke (2011)
technical substance: Partition
coefficients 1-octanol / water at pH
4, pH 7 and pH 10 (shake flask
method). Bayer CropScience AG
Research Technologies-Analytics
Frankfurt D-65926 Frankfurt am
Main (Germany) Project ID
PA11/101. Bayer ID M-420023-011.
1
substance: Flammability (solids).
3.8 x 10-6 at 20oC
Vapour pressure
(Pa)
6.7 x 10-6 at 25oC
OECD 104 (2006)
9.0 x 10-5 at 50oC
71.4 mN/m at 20oC
Surface Tension
The substance is
not a surface active
substance
OECD 115
0.27 g/L in distilled
water (pH 6.3),
Water Solubility
0.27 g/L in buffer at
pH 4,
A6
0.24 g/L in buffer at
pH 10
Log Kow
Flammability
November 2015
0.9 at pH 4, 7 and
10 at 22oC
Not highly
flammable
A8
A10
55
Property
Result
Test method
Klimisch
Score (14)
Reference
20140252.01, Bayer ID M-49400601-1.
Auto flammability
Self-heating
substances
November 2015
None up to 401oC
Negative result in a
wire netting cube of
10 cm edge length
at 120oC
A16
UN N4
1
substance: Auto-flammability
(solids - determination of relative
self-ignition temperature). Siemens
20140252.03, Bayer ID M-49609901-1.
1
substance: Self-heating
substances UN class 4.2. Siemens
(Germany) Project no 20140252.05
Bayer ID M-502220-01-1.
56
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
Value
LD50 > 108 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.
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
Dose Levels
November 2015
> 108 CFU/animal
57
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.
Additional Comments
Ten treated animals are unaccounted for in the study report. However, an
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
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
Value
LC50 > 108 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.
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
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
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.
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
Value
LD50 > 107 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
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
November 2015
61
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
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.
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.
Klimisch Score
1
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
November 2015
Topical application, semi-occlusive patch, 4h
63
All animals appeared active and healthy during the study. Apart from the dermal
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
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
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 LD 50/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
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-364421-011.
Klimisch Score
November 2015
1
66
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
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 shortterm 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
Exposure type
Measured: 1.75 x 108 - 4.26 x 108 CFU/rat
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
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
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
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 studies 23. The applicant has not received any information on poisoning
incidents or adverse effects. Nor are there published reports available 24.
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
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
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
The documents used are listed below:
Author
Year
Journal
Title
Bayer ID
Akiba
1991
Appl Ent Zool 26(4): 477483
Assessment of rainwater-mediated
dispersion of field-sprayed Bacillus
thuringensis in the soil
M-368025-011
Barbosa et al
2005
Appl Env Microb 71(2):
968-978
Screening for Bacillus isolates in the
broiler gastrointestinal tract
M-367872-011
Cano et al
1994
Appl Env Microb 60 (6):
2164-2167
Bacillus DNA in fossil bees: an ancient
symbiosis?
M-356399-011
Carrera et al
2007
J Appl Microb 102: 303312
Difference between the spore sizes of
Bacillus anthracis and other Bacillus
species
M-368017-011
Dias et al
1966
No data Vol 34 (1): 9-16
Coloured Bacillus species resembling
B. firmus
M-367955-011
M-367959-011
Donkerski &
Trzebiatowska
1999
Polish Journal of Env
Studies 9 (2): 77-82
Influence of physical factors on the
activity of chitinases produced by
planktonic bacteria isolated from
Jeziorak Lake
Donkerski et al
1999
Polish Journal of Env
Studies 8 (3): 137-141
Neustonic bacteria number, biomass
and taxonomy
M-367960-011
-
EFSA
2011
-
Draft Assessment Report (DAR)
Bacillus firmus I-1582 Vol 3, Annex B
part 9 B.8/b
EFSA
2011
-
Draft Assessment Report (DAR)
Bacillus firmus I-1582 Vol 3, Annex B
part 9 B.8/a
-
Felske et al
1998
4588-4590
In situ detection of an uncultured
predominant Bacillus in Dutch
grassland soils
M-367526-011
Garbeva et al
2007
Plant Soil 302: 19-32
Rhizosphere microbial community and
its response to plant species and soil
history
M-368009-011
Gontang et al
2007
3272-3282
Phylogenetic diversity of Gram-positive
bacteria cultured from marine
sediments
M-367945-011
Griego & Spence
1978
906-910
Inactivation of Bacillus thuringensis
spores by ultraviolet and visible light
M 368063-011
Kaga et al
2009
Microb Env 24 (2): 154162
Rice seeds as sources of endophytic
bacteria
M-367865-011
November 2015
73
Author
Year
Journal
Title
Bayer ID
Keren-Zur et al
2000
The BCPC Conference –
Pest and diseases 2000,
Brighton
Bacillus firmus formulations for the safe
control of root-knot nematodes
M-028855-011
M-367943-011
Ki et al
2009
J Microb Meth 77: 48-57
Discovery of marine Bacillus species
by 16S rRNA and rpoB comparisons
and their usefulness for species
identification
Kim et al
2008
Water Air Soil Pollut 199:
151-157
Factors that influence the transport of
Bacillus cereus spores through sand
M-368033-011
Korenblum et al
2004
J Appl Microb 98: 667675
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
M-367951-011
Markov
2006
-
Dissipation of Bacillus firmus in the soil.
M-370080-011
2001
Current Microb 43: 140143
Comparative sensitivity to UV-B
radiation of two Bacillus thuringiensis
subspecies and other Bacillus sp.
M-368061-011
M-368023-011
Myasnik et al
Naclerio et al
2008
Colloids and Surfaces B:
Biointerfaces 70: 25-28
Filtration of Bacillus subtilis and
Bacillus cereus spores in a pyroclastic
topsoil, carbonate Apennines, southern
Italy
Nishijima et al
2005
Microb Env 20 (1): 61-68
Predominant culturable Bacillus
species in Japanese arable soils and
their potential as biocontrol agents
M-367961-011
Ortega-Morales
et al
2007
Ind Microb Biotechnol 35:
9-15
Antifouling activity of sessile bacilli
derived from marine surfaces
M-367936-011
Ortega-Morales
et al
2008
Mar Biotechnol 11: 375383
Antagonism of Bacillus spp. isolated
from. marine biofilms against terrestrial
phytopathogenic fungi
M-367930-011
Pakpitcharoen et
al
2008
ScienceAsia 34: 424-431
Biodiversity of thermotolerant Bacillus
sp. producing biosurfactants,
biocatalysts, and antimicrobial agents
M-367910-011
PantasticoCaldas et al
1992
Ecology 73 (5): 18881902
Population dynamics of bacteriophage
and Bacillus subtilis in soil
M-368039-011
Pedersen et al
1995
Can J Microb 41: 118125
Dispersal of Bacillus thuringiensis var.
kurstaki in an experimental cabbage
field
M-368069-011
November 2015
74
Author
Year
Journal
Title
Bayer ID
Riccardi et al
2005
Annals Microb 55 (2): 8590
Characterization of bacterial population
coming from a soil contaminated by
polycyclic aromatic hydrocarbons
M-367998-011
Saleh et al
1969
Can J Microb 16: 677679
Fate of Bacillus thuringiensis in soil:
effect of soil pH and organic
amendment
M-368074-011
Siefert et al
2000
Current Microb 41: 25-29
Phylogeny of marine Bacillus isolates
from the Gulf of Mexico
M-367912-011
Swiontek et al
2006
Polish Journal of Ecology
54 (2): 295-301
Chitinolytic bacteria in two lakes of
different trophic status
M-367956-011
Tokuda et al
1992
Fermentation and
bioengineering 75 (2):
107-111
Survival of Bacillus subtilis NB22, an
antifungal –antibiotic ilturin producer
and its transformant in soil-systems
M-368050-011
1989
Rev Ecol Microb sol 26
(3): 233-247
Seasonal variation in heterotrophic soil
bacteria and some soil enzyme
activities in a typical calcixeroll soil in
Greece
M-367859-011
M-370430-011
M-368072-011
Vardavakis
West et al
1985
Soil Biol Biochem 75 (5):
657-665
Survival of Bacillus thuringensis and
Bacillus cereus spore inocula in soil:
effect of pH, moisture, nutrient
availability and indigenous
microorganisms
West et al
1984
J Invert pathol 44: 121127
Effect of incubation in natural and
autoclaved soil upon potency and
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
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
E Bruns (2010) Acute toxicity of Bacillus firmus I-1582 to fish (Oncorhynchus
mykiss) under static conditions- limit test. Bayer CropScience AG DevelopmentReference
Environmental safety Ecotoxicology 40789 Monheim (Germany). Report no
EBBIL003, Bayer ID M-366351-02-1.
Klimisch Score
1
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 12.4 oC.
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.
The LC50 is higher than 100 mg/L (1.5 x 1010 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
Test Substance
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)
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)
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
The EC50 is higher than 100 mg/L.
Comment
No information about the stability/viability of the test item over the test period.
The EC50 is higher than 100 mg/L (1.5 x 1010 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
Type of study
Full test
Flag
Key study
Test Substance
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 M367825-01-1.
Klimisch Score
1
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
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
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.
ErC50 = 24.2 mg/L (95% CI: 21.4-28.0 mg/L) corresponding to 3.5 x 109 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
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
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
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
Test Substance
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
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
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%)
Conclusion
LC50> 1000 mg/kg dry soil (1.56 x 1011 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
T Leicher (2010) Bacillus firmus I-1582 WP 5: Acute toxicity to earthworms
(Eisenia fetida) tested in artificial soil. Bayer CropScience AG DevelopmentReference
Environmental safety Ecotoxicology 40789 Monheim (Germany). Report no
EBBIL007, Bayer ID M-368585-01-1.
Klimisch Score
2 see comment
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
Not required
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.
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
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
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)
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
Not required
The acute toxicity of the substance to the soil mite Hypoaspis aculeifer was
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
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%)
4.7 x 109 cfu/g.
Comments
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
Test Substance
Species
Folsomia candida
November 2015
84
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 M367850-01-1.
Klimisch Score
2 see comment
5% peat instead of 10% in the guideline This was a recommendation to
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
Not required
The acute toxicity of the substance to the springtail Folsomia candida was
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
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%)
4.7 x 109 cfu/g.
Comments
Conclusion
NOEC for reproduction and survival > 1000 mg formulation /kg dry soil
Soil macro-invertebrates chronic toxicity
November 2015
85
No study provided.
Non-target plants toxicity
No study provided.
Nitrogen transformation test
Type of study
Full test
Flag
Test Substance
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
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
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
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%)
4.7 x 109 cfu/g.
Comments
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
Test Substance
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
EBBIL008, Bayer ID M-367846-01-1.
Klimisch Score
2 see comment
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
Not required
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
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).
Coefficient of variation in the control < 15% (observed value: 2.6%)
4.7 x 109 cfu/g.
Comments
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
LRT-NBT 01/10, Bayer ID M-406103-01-1.
Klimisch Score
3 see comment
Not relevant: no guideline followed and not a GLP study
GLP
no
Test Guideline/s
None followed
November 2015
88
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
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
exchange
carbon (%)
pH
capacity
(meq/100g)
Study Summary
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
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.
Comments
November 2015
4.7 x 109 cfu/g.
89
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
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
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
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, 1218, 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.
Comment
The strain is not mentioned in the report but the applicant confirmed it was I1582.
Conclusion
Dietary acute toxicity
No study provided.
November 2015
NOEL = 2.5 x 108 spores /kg bw/d
91
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
Test Substance
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 M367954-01-1
Klimisch Score
2 see comment
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)
Not required
At the end of the contact toxicity test (48 hours after application), there was 2.0
Study Summary
% mortality at 100.0 μg product/bee. 6.0 % mortality occurred in the control
(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
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.
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.
4.7 x 109 cfu/g.
Comments
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 M329117-01-1
Klimisch Score
1
None that impacted the results
GLP
Yes
Test Guideline/s
OPPTS 885.4380 (1996)
November 2015
93
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
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 noobserved-effect concentration (NOEC) for honey bee adults is at least 10 8
spores/ml.
Remark
Conclusion
NOEC = 108 cfu/ml sucrose solution
Other non-target arthropods - Extended laboratory tests
Type of study
Full test
Flag
Test Substance
Species
Aphidius rhopalosiphi (parasitic wasp)
Type of exposure
To residues on barley seedlings
November 2015
94
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-DEnSA-Ecotoxicology IndustriePark Hoechst 65926 Frankfurt am Main
(Germany) Report no CW09/062 Bayer ID M-368305-01-1
Klimisch Score
2 see comment
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
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
4.7 x 109 cfu/g.
Comments
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
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
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).
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
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
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.
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
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
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
were used and B. firmus is not known to have insecticidal activity.
November 2015
98
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
Acute oral toxicity in rats
LD50 > 5000 mg/kg bw
Purity & Batch no.
Reference
96%
Gardner, 1997a
30034708
1216<LD50<2000 mg/kg
Sheets, 2000
bw (M)
96%
523<LD50<1216 mg/kg
30037120
Acute oral toxicity in rats
bw (F)
389 mg/kg bw (M)
96%
465 mg/kg bw (F)
30034708
Gardner, 1997b
Acute oral toxicity in mice
96%
Acute dermal toxicity in rabbits
Gardner, 1997c
> 2000 mg/kg bw
30034708
96%
Acute inhalation toxicity rats
Shepherd, 1998
> 5.54 mg/L
30034708
96%
Skin irritation in rabbits
Gardner, 1997d
Not irritating
30034708
96%
Eye irritation in rabbits
Gardner, 1997e
Not irritating
30034708
96%
Skin sensitisation in the guinea
Not sensitising
pig (Magnusson and Kligman)
November 2015
30034708
Denton, 1997
99
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
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
respect to the evaluation criteria the test item therefore exhibits no skinsensitization 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
November 2015
1
101
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
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/cm 2 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 nonirradiated 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
The result of the acute oral toxicity study in rats suggest that classification is not required as the LD 50 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 dosedependent 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
S. typhimurium (TA100, TA1535, TA
98, TA1537) and E.coli (WP2uvrA-)
plate incorporation assay, ±S9,
DMSO
Positive
Thompson, 2000
98, TA1537) plate incorporation
assay, ±S9, DMSO
Negative
Otsuka, 1990a
98, TA1537, TA102) plate
incorporation and preincubation
assay, ±S9, DMSO
Negative
Herbold, 1999a
S. typhimurium (TA1535), plate
incorporation and preincubation
assay, ±S9, DMSO (not GLP)
Negative
Herbold, 1999b
B. subtilis -H17 and M45, Spore recassay, ±S9
Negative
Otsuka, 1990b
L5178Y/TK+/-, mouse lymphoma
gene mutation assay,±S9 ,DMSO
Positive
Durward, 2000a
In vitro studies
November 2015
103
V79, CHL gene mutation assay, ±S9,
DMSO
Negative
Brendler-Schwaab, 1999a
CHL, chromosome aberration assay,
± S9, DMSO
Positive
Wright, 2000
Mouse, bone marrow, micronucleus
assay, (oral, 25; 50; 100 mg/kg,
arachis oil)
Negative
Durward, 2000b
Rat, liver, unscheduled DNA
synthesis assay, (oral, 2500; 5000
mg/kg, aqueous cremophor)
Negative
Brendler-Schwaab, 1999b
In vivo studies
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
organism
bw/day (ppm)
bw/day (ppm)
2 year rat p.o.
32.5 (500)
Toxicity:
[We note this was
listed in the EU
November 2015
9.7 (150)
Critical effects
Reference
Interstitial ovarial
gland hyperplasia,
Biegel, 2000a
104
Carcinogenicity:
157 (3000)
2 year mouse p.o.
Toxicity:
47.2 (350)
Carcinogenicity:
251.9 (2000)
DAR overall
summary as the
same value as the
NOAEL. The
LOAEL has been
obtained from the
detailed summary
of the study.]
171.4 (1250)
bw effects, feed
consumption
Body weight effects,
clinical signs, liver
cell hypertrophy,
cervix hyperplasia
Biegel, 2000b
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)
parental=filial: 10.2
(150)
parental1=filial2
↓ body weight1,2,
32.7 (500)
2 generation, p.o.,
rat
↑preputial separation
delay2, ↑vaginal
opening patency2
reprotoxicity:
reprotoxicity: 32.7
(500)
↑stillborns, sperm
179.6 (2500)
motility/morphology
effects
maternal: 10
maternal: 40
↓Feed consumption,
developmental: 125
developmental: -
body weight
Teratogenicity, rat
November 2015
↓thymus w2,
Freshwater,
2000
York, 1998a
105
↑Mortality, clinical
Teratogenicity,
rabbit
Developmental
toxicity, rat
Toxicity:
maternal: 10
maternal: 25
foetal=
developmental 25
foetal1=
developmental2: 75
signs, ↑abortions1,
↑premature
deliveries1, ↓foetal
weight1,
↑intermediate lung
lobe absence2,
↓sternal ossification
centers2
maternal1= filial2
42.9 (500)
↓bw gain1, ↓pup
maternal=filial 12.9
(150)
Neurotoxicity:
42.9 (500)
142 (1750)
Reprotoxicity:
142 (1750)
N/A
weight2, ↓startle
habituation, ↓motor
activity, ↓surface
righting, brain
histometry
York, 1998b
Hobermann,
2000
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
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
organism
bw/day (ppm)
bw/day (ppm)
♂: 120 (1250)
♂: 249 (2500)
♀: 137
♀: 228
♂: 90 (500)
♂: 190 (1000)
♀: 122
♀: 248
Critical effects
28d, p.o., rat
28d, p.o.,
mouse
28d, p.o., dog
90d, p.o.,
mouse
90d, p.o., rat
90d, p.o., rat
90d, p.o., dog
1 year p.o., dog
28d, dermal, rat
♂: 35.8(1250)
♂: 35.8 (2500)
♀: 36.3
♀: 52.3
♂: 16 (100)
♂: 82 (500)
♀: 22
♀: 107
♂: 19.7 (250)
♂: 96.0 (1250)
♀: 24.0
♀: 119.0
♂: 27.9 (500)
♂: 202.0
(3000)
♀: 34.0
↓bw, ↓bw gain,
haematological
effects
↓bw, ↓bw gain,
clinical chemistry
↑mortality, clinical
findings,
↓haematopoietic/
lymphoid organs,
enteropathy
↓feed consumption,
clinical chemistry
modifications,
↓kidney weight
↓bw, ↓bw gain,
↑ovary/uterus weight
↓bw, ↓bw gain,
effects on RBC,
↑spleen pigmentation
♀: 254.2
clinical signs, ↓bw
gain, anemia,
↓jejunum lymphoid
hyperplasia
♂: 19.3 (650)
♂: 40.9 (1500)
♀: 21.2
♀: 42.1
♂: 36.3(1500)
♀: 40.1
♂: 46.4 (2000)
♀: 52.9
↓WBC, ↓neutrophils
♂: 300
300
♂: 1000
1000
↓bw, ↓bw gain
♀:
90d, p.o., rat
•toxicity:
60 (1000)
•neurotoxicity:
177 (3000)
177 (3000)
♀:
References
Chambers,
1997a
Chambers,
1997b
Moore,
2000
Chambers,
1997d
Chambers,
1997c
Wahle,
2000
Bernier,
2000a
Bernier,
2000b
Weiler,
2000
↓bw, ↓feed
consumption,
mortality
Sheets,
2000b
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
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
organism
bw/day (ppm)
bw/day (ppm)
M: 60
M: 100
Critical effects
References
Acute, rat
F: 100
F:200
toxicity:
60 (1000)
177 (3000)
neurotoxicity:
177 (3000)
N/A
Developmental, rat
maternal=filial
maternal1= filial2
toxicity:
12.9 (150)
42.9 (500)
neurotoxicity:
42.9 (500)
142 (1750)
↓Locomotor
Sheets, 2000a
activity,
↓hypothermia
Cain, 2000
↓Body weight,
Sheets,
↓feed consumption
2000b
90d, p.o., m rat
↓Bw gain1, ↓ pup
weight2
↓startle habituation,
Hobermann,
2000
↓motor activity,
reprotoxicity:
142 (1750)
N/A
↓surface righting,
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
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. CRDDS Argus Protocol Number: RLF00001. M-123395-01-1.
Klimisch Score
1
None considered to impact the quality or outcome of the study
GLP
Yes
November 2015
109
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 TI435 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 antibodyforming cell (AFC) response to the T-dependent antigen, sheep erythrocytes, in
male and female rats when evaluated as both specific activity (AFC/10 6 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
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-50852501-1.
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
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
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
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
28
thiazolylmethylguanidine
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
MNG
methylnitroguanidine
TZNG
thiazolylnitroguanidine
TZMU
N-(2-chlorothiazol-5-ylmethyl)-N'-
or
methylurea
TZM
MU
methylurea
TZFA
N-({[(2-chloro-1,3-thiazol-5yl)methyl]amino}methylene)methanaminium
chloride; code BCS-CQ88479
NTG
1- Nitroguanidine
CTCA
clothianidin-2-chlorothiazole-5-caboxylic
acid
HMIO
clothianidin-4-hydroxy-2-methylamino-2imidazolin -5-one
Water compartment
Ready biodegradation
Type of study
Flag
Key study
Test Substance
TI-435 metabolite MNG (N-methylnitroguanidin)
November 2015
115
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
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
CO2 formed
A study was performed to assess the ready biodegradability of NMethylnitroguanidine (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
GLP
Yes
November 2015
116
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
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
GLP
Yes
Test Guideline/s
OECD 309
Dose Levels
Initial concentration 0.1 and 0.01 mg test substance/L
November 2015
117
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%
AR in the low dose system after 60 days. Volatile products other than 14CO2 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
November 2015
Clothianidin is stable in the water phase
118
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
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.
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
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
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
None
GLP
Yes
Test Guidelines
None available
Dose Levels
1.17 mg ai/seed
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
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 15to 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 (r 2
= 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
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
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
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 92.094.6% 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%, 37.351.9%, and 69.7-81.7% of the applied test material was adsorbed in soils
November 2015
123
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
Clay
Sandy
Silt loam
loam
loam
, Germany
Soil type
loam
Silt loam
% organic
1.76
2.42
4.72
0.7
1.7
5.3
6.6
7.3
6.7
6.6
4.5
7.0
10.0
5.8
20.7
0.8719
0.8780
0.8546
0.8829
0.8730
255.5
288.8
212.1
826.8
1216.3
carbon
pH
% WHC
Adsorption
KF mL/g
1/n
Adsorption
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
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
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
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
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 DAT120, evolved 14CO2 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
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
clothianidin
sorption
DAT-120/
DAT0
Conclusion
The sorption of clothianidin to soil increased significantly in the course of
the incubation. Therefore with time of aging in soil less substance will be
November 2015
126
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
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)
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
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
%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
water ad 100 g
DM)
Conclusion
DT50 were15.6, 22.1 and 65.5 days
Type of study
Soil dissipation
Flag
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-DEnSa-testing Monheim, Germany. Report no 10-2700 Bayer ID M-443993-01-1
November 2015
128
Klimisch Score
1
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)
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 postapplication 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
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 [guanidine14C]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
None
GLP
Yes
Test Guidelines
OECD 307
Dose Levels
213 µ g test substance/ kg dry soil
HPLC/radiodetection and LSC
The route and rate of degradation of [thiazolyl-2-14C]clothianidin and [guanidine14C]clothianidin
Study Summary
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
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
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 (guanidinelabel) 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
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
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
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
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)
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
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
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
GLP
Yes
Test Guidelines
No existing guideline
No detail provided, approved rates as the products were used in normal
Dose Levels
agricultural practice
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
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 90 th
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 CaCl 2) 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 <LOQ (1
μg/kg).
For the canola sites, there was no significant accumulation of clothianidin in soil
(0-30 cm) from fields with multiple years of planting with treated seeds,
although the extent of the years of use was limited to 2 to 3 years for most
fields and 4 years for two fields. Bioavailable residues represented 6% of the
total clothianidin residue. Clothianidin residues in soil were greater than the
LOQ (5 μg/kg) in 7 of the 15 canola sites with an average concentration of 6.6
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
Conclusion
primarily from the clothianidin on the treated seed from the current year’s crop,
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
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 M027033-02-1.
Klimisch Score
1
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
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 22.7 oC.
November 2015
136
The LC50 is higher than 104.2 mg technical /L.
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
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
None
GLP
Yes
Test Guideline/s
OECD 203 (1992)
No/Group
Dose Levels
170 mg test substance /L nominal
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 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
The LC50 is higher than 170 mg test substance /L.
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
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
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
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
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.
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
None
GLP
Yes
Test Guideline/s
OPPTS 850.1075 (draft)
No/Group
Dose Levels
102.5 mg TI-435 technical /L (= 100 mg ai/L) nominal
Yes, fresh medium 1st,2nd,3rd and 4th day, old medium 2nd,3rd, 4th and 5th day
November 2015
139
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.
The LC50 is higher than 102.5 mg technical /L.
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
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
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
0.2, 0.38, 0.7, 1.44, 2.6, 4.81 mg test substance/L measured
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 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 EC 50 is 2.90
mg test substance/L with 95% confidence limits between 2.18 and 3.87 mg /L.
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
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
None
November 2015
141
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
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 O 2/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
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
Reference
K.R. Drottar, J.A. MacGregor, H.O. Krueger (2000) TI-435 Technical: A 96 hour
flow through acute toxicity test with the saltwater mysid (Mysidopsis bahia),
November 2015
142
Wildlife International Easton Maryland, report 110058 M-019551-01-2, August
15,2000
Klimisch Score
1
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
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.
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
Reference
D. Scheerbaum (1999) TI 435-technical, Oyster acute toxicity test (shell
deposition) limit test, flow through 96 h. Dr. U. Noack-Laboratorium fur
November 2015
143
Angewandte Biologie, D 31157 Sarstedt Germany. Report no DECO 026,
Bayer ID M-028515-01-1.
Klimisch Score
1
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
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.
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
Klimisch Score
3, > 25% of females did not produce young
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
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
25oC 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
November 2015
145
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
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
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.
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
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
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
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 80120% 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.
November 2015
147

Number of fronds increased in the control by more than 5-fold within 7 days
(observed: 8.8, 8.6, 9.3 fold)

Conclusion
Temperature variation < 4 oC (observed 23 to 25oC)
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
None
GLP
Yes
Test Guideline/s
OECD 235 (2011)
No/Group
5 larvae and 6 replicates
Dose Levels
10.0 mg ai/L
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.
20.6 to 21.1oC 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.
Control immobility < 15% (observed 3.3%)
Dissolved oxygen >3 mg oxygen/L (observed 8.6 mg oxygen/L)
November 2015
148
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
None
GLP
Yes
Test Guideline/s
OECD 235 (2011)
No/Group
Dose Levels
10.0 mg ai/L
The acute toxicity of the metabolite HMIO is tested to larvae of Chironomus
116% (day 2). The results are based on the nominal concentrations.
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.
Control immobility < 15% (observed 0%)
Dissolved oxygen >3 mg oxygen/L (observed 8.1 -8.3 mg oxygen/L)
Conclusion
November 2015
EC50 and NOEC > 10 mg HMIO/L
149
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-DEnSa-Testing, Monheim Germany. Report EBTIN098 Bayer ID M-497581-01-1
Klimisch Score
1
None
GLP
Yes
Test Guideline/s
OECD 235 (2011)
No/Group
Dose Levels
10.0 mg ai/L
The acute toxicity of the metabolite TZFA is tested to larvae of Chironomus
81.2% (day 2). The results are based on the nominal concentrations.
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.
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
Flag
Test Substance
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-DEnSa-Testing, Monheim Germany. Report EBTIN099 Bayer ID M-505533-01-1
Klimisch Score
1
None
GLP
Yes
Test Guideline/s
OECD 235 (2011)
No/Group
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
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 20.9 oC
Study Summary
and dissolved oxygen concentrations ranged from 8.4 to 8.8 mg O 2/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.
Dissolved oxygen >3 mg oxygen/L (observed 8.4 to 8.8 mg oxygen/L)
November 2015
151
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
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 RF0009.203.080.01 Bayer ID: M-435936-01-1
Klimisch Score
1
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
Yes
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.
November 2015
152
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
Test Substance
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
None
GLP
Yes
Test Guideline/s
OECD 207 (1993)
No/Group
Dose Levels
3.2, 5.6, 10, 18, 32 mg/kg dry artificial soil
Not required
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.
Loss of biomass less than 20% (observed 8.5%)
November 2015
153
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
Test Substance
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
None
GLP
Yes
Test Guideline/s
OECD 222 (2004), ISO guideline 11268-2 (1998)
No/Group
1.15 mg clothianidin/kg soil dry weight equivalent to 2.40 mg test substance/ kg
Dose Levels
soil dry weight
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.
Reproduction of control ≥30 worms (observed 150-275)
November 2015
154
Coefficient of variation ≤30% (observed 20.2%)
Conclusion
NOEC < 2.40 mg test substance/ kg dry soil reproduction
Type of study
Limit test
Flag
Test Substance
Species
Eisenia fetida
Type of exposure
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-R77/09 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.
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
No/Group
40 adults for control and treatment
Dose Levels
82 µg ai/kg dry soil and 1.126 mg ai/seed
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
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.
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
Test Substance
Species
Eisenia fetida
Type of exposure
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
None
GLP
Yes
Test Guideline/s
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
November 2015
Not required
156
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.


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
Endpoint
NOEC
TZMU, TZFA, NTG, MNG 100 mg ai/ kg soil dry weight reproduction
Value
TZNG 13 mg ai/kg soil reproduction
Reference
E. Wagenhoff (2014) 5 Metabolites of Clothianidin (TZMU, TZFA, NTG, TZNG,
MNG): Sublethal toxicity to the earthworm Eisenia fetida (Annelida,
November 2015
157
Lumbricidae) in artificial soil with 5% peat content. Eurofins Agroscience
Service EcoChem GmbH, Niefern-Oschelbronn Germany. Report no S1303784 Bayer ID: M-505527-01-1
Klimisch Score
1
None that impacted the study results
GLP
Yes
Test Guideline/s
No/Group
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
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
NOEC mortality is 50 mg ai/kg soil and NOEC reproduction is 13 mg ai/kg soil.

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
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
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
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
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.
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
Test Substance
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
None
GLP
Yes
Test Guideline/s
BBA Part VI, 2-3 (1994), ISO draft guideline CD 11268-3 (1999)
November 2015
160
No/Group
Earthworm population
Dose Levels
80 µg ai/kg dry soil and 125 g ai/ha as seed treatment
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
Test Substance
Species
Folsomia candida
Type of exposure
Static, for 28 days
November 2015
161
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
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
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
Test Substance
November 2015
162
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
GLP
Yes
Test Guideline/s
OECD 232 (2009)
No/Group
0.032 mg test substance/kg soil dry weight and treated seed representing 195 g
Dose Levels
ai/ha
Not required
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.
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.
Conclusion
November 2015
NOEC ≥195 g ai/ha reproduction
163
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
GLP
Yes
Test Guideline/s
OECD 232 (2009)
No/Group
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.
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
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.
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: M505619-01-1
Klimisch Score
1
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
Not required
November 2015
165
The toxicity of the metabolites TMG to the springtail Folsomia candida was
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
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.
Mortality control <20% (observed 10%)
Reproduction control >100 juveniles/vessel (observed 557)
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
None
GLP
Yes
Test Guideline/s
ISO 11267 (1999)
No/Group
Dose Levels
625 g ai/ha
November 2015
166
Not required
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.
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
None
GLP
Yes
Test Guideline/s
ISO 11267 (1999)
No/Group
Dose Levels
253 g ai/ha
Not required
November 2015
167
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.
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
GLP
Yes
Test Guideline/s
OECD 226 (2008)
No/Group
4 replicates of 10 adult mites, control 8 replicates
Dose Levels
Not required
Study Summary
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%
November 2015
168
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.
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.
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 NiefernOschelbronn Germany. Report no THW-0354 Bayer ID: M-5056155-01-1
Klimisch Score
1
GLP
Yes
Test Guideline/s
OECD 226 (2008)
No/Group
8 replicates of 10 adult mites
November 2015
169
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
Not required
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%.
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.
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
November 2015
170
Test Substance
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
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
Not required
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.
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.
Conclusion
November 2015
NOEC =316 mg test substance/ kg soil reproduction
171
Litter bag test
Type of study
Full test
Flag
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
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)
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 2 nd 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 H 2O/ 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, 091 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
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 NiefernOschelbronn Germany. Report no S13-03789, Bayer ID M-496277-01-1.
Klimisch Score
1
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
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
and +4.95% at 1.34 mg/kg soil. In the period 14-28 days the deviation was
+7.44% and +16.7% respectively.
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
U Schobinger (2014) One metabolite of clothianidin (TZMU): Effects on the
Reference
Klimisch Score
1
None
GLP
Yes
Test Guideline/s
OECD 216 (2000)
No/Group
3 replicate
Dose Levels
ai/ kg soil
Not required
(metabolite TZMU) on the nitrogen transformation in soil, determined by the
Study Summary
and -0.334% at 1.01 mg/kg soil at the end of the study 28 and 42 days
November 2015
174
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).
Coefficient of variation in the control < 15% (observed value: max 11% on day
14)
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
U Schobinger (2014) One metabolite of clothianidin (TZFA): Effects on the
Reference
Klimisch Score
1
None
GLP
Yes
Test Guideline/s
OECD 216 (2000)
No/Group
3 replicate
Dose Levels
ai/ kg soil
Not required
Study Summary
(metabolite TZFA) on the nitrogen transformation in soil, determined by the
November 2015
175
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.
Coefficient of variation in the control < 15% (observed value: max 6.86% on day
7)
Conclusion
test substance/ kg soil (1.0 mg ai/kg soil).
Type of study
Full test
Flag
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
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
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).
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
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
15166/2011-13.0MON-N, Bayer ID M-436903-01-2.
Klimisch Score
2,
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
Not required
November 2015
177
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%.
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
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
None
GLP
Yes
Test Guideline/s
OECD 216 (2000)
November 2015
178
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
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% (714 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).
Conclusion
test substance/ kg soil.
Carbon transformation test
Type of study
Full test
Flag
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
15166/2011 – 8.0MO-C, Bayer ID M-421600-01-2.
Klimisch Score
1
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
Not required
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.
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
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
None
GLP
Yes
Test Guideline/s
OECD 217 (2000)
No/Group
3 replicates
November 2015
180
150 and 750 mL Poncho/ha corresponding with 0.25 and 1.26 mg Poncho/ kg
Dose Levels
soil
Not required
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.
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
None
November 2015
181
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)
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.
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
J.M.Stafford (2005) Clothianidin (TI435) acute oral toxicity test (LD 50) with the
Reference
house sparrow (Passer domesticus). Springborn Smithers laboratories
Wareham Massachusetts USA. Report no EBTIX061 Bayer ID M-243741
Klimisch Score
1
None that impacted the study results
GLP
Yes
Test Guideline/s
OPPTS 850.2100
No/Group
Dose Levels
63, 125, 250, 500, 1000 and 2000 mg ai/kg bw (nominal)
Not required
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.
November 2015
183
Conclusion
LD50 = 530 mg ai/kg bw (CI 350- 830 mg ai/kg bw)
Type of study
Limit test
Flag
Test Substance
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
None
GLP
Yes
Test Guideline/s
OCSPP 850.2100, OECD 223
No/Group
Dose Levels
2000 mg test substance/kg bw (nominal)
Not required
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.
November 2015
184
Conclusion
LD50 > 2000 mg test substance/ kg bw
Avoidance test
Type of study
Limit test
Flag
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
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
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
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
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
GLP
Yes
Test Guideline/s
Non guideline method
Dose Levels
1.25 mg ai/ seed
No/Group
7 males and 8 females
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
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
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
GLP
Yes
Test Guideline/s
Non guideline method
Dose Levels
62.5 mg ai/ 50 seed
Not required
November 2015
187
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
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 M182529-01-1
Klimisch Score
1
NA
November 2015
188
GLP
Yes
Test Guideline/s
Internal method
No/Group
10 house mice, 1 per cage
Dose Levels
1.25 mg ai/ seed
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
Klimisch Score
1
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)
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.
LD50 = 38.9 ng ai/bee (contact)
Conclusion
LD50 = 2.5 ng ai/bee (oral)
Type of study
Full test
Flag
Test Substance
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
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
None
GLP
Yes
Test Guideline/s
OECD 213 and 214 (1998)
No/Group
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
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.
Conclusion
Type of study
Full test
Flag
Test Substance
November 2015
191
Species
Apis mellifera
Type of exposure
Endpoint
LD50
Value
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
None
GLP
Yes
Test Guideline/s
OECD 213 and 214 (1998)
No/Group
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
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.
Mortality in control < 10% (observed value: contact 6.7%, oral 0%)
November 2015
192
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
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
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
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
Endpoint
LD50
Value
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
GLP
Yes
Test Guideline/s
To the principles of Van der Steen draft OECD 213 and 214
No/Group
Dose Levels
Contact 18.8, 37.5, 75, 150, 300 ng ai/bee
November 2015
194
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
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 1.59532.242 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.
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.
Conclusion
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
November 2015
1
195
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
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.
Study Summary
At the end of the test 12.35% of the control group died. The corrected mortality
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
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
Klimisch Score
1
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
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
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 TI435 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 M031721-01-1
Klimisch Score
1
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
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
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
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
Not required
The effects of TZMU, a metabolite of TI-435 were tested on honey bees under
Study Summary
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. 5000-6000 honeybees) as food source.
November 2015
199
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
Flag
and the concentration tested is lower than the proposed rate in New Zealand
Test Substance
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
GLP
Yes
November 2015
200
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
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
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
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
Test Substance
Species
Apis mellifera
Type of exposure
10 days
Endpoint
Value
Reference
in Champagne (France). Eurofins GAB GmbH, Eutinger Str 24 NiefernOschelbronn, Germany. Report no 1st interim report S08-01573 Bayer ID M347482-01-1
Klimisch Score
1
November 2015
202
GLP
Yes
Test Guideline/s
No/Group
Dose Levels
Not required
Languedoc-Roussillon in France in 2008. The plots were separated approx. 2
Study Summary
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.
Bayer ID M 351027-01-1).
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
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.
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.
Bayer ID M 351636-01-1).
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
Type of study
Limit test
Flag
Test Substance
Species
Apis mellifera
Type of exposure
11 days
Endpoint
Value
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
GLP
Yes
Test Guideline/s
November 2015
204
No/Group
Dose Levels
Not required
Languedoc-Roussillon in France in 2008. The plots were separated approx. 4
Study Summary
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.
Bayer ID M 351010-01-1).
during the year.
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.
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
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.
Bayer ID M 351635-01-1).
DWV, SBV, ABPV and CBPV were not detected in any of the samples.
Conclusion
Type of study
Full test
Flag
Test Substance
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
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
Not required
November 2015
206
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
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
Klimisch Score
1
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
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
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).
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
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).
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 M256474-01-1
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 pretreated 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
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 pretreated 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
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
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
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
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
products in Languedoc-Roussillon (France). Eurofins-GAB GmbH NiefernOschelbronn, Germany Report no S08-01377, Bayer ID M-347742-01-1
evaluated. The study was carried out over 2 consecutive years 2008 and 2009.
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.
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.
µg/kg. Not quantifiable amounts of the metabolite TZNG were detected in plant
November 2015
214
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.
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
products in Champagne (France). Eurofins-GAB GmbH Niefern-Oschelbronn,
Germany Report no S08-02438, Bayer ID M-347748-01-1
evaluated. The study will be carried out over 2 consecutive years 2008 and
2009.
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
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
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.
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
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
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
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, Developmenthuman safety-residue analysis Monheim am Rhein Germany. Report MR09/072 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%
Summary
with an overall mean recovery of 95% and a relative standard deviation (RSD)
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
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
<LOD and 45.5 ppb, and for bees from Jennersdorf between <LOD and 384.9
ppb. The residue levels of clothianidin in guttation water from Baumgartenberg
were between <LOQ and 717 mg/L, and for guttation water from Jennersdorf
between <LOQ and 285 mg/L.
The residue levels of TZNG in bees from Baumgartenberg were between <LOD
and 31.2 ppb, and for bees from Jennersdorf between <LOD and 39.7 ppb. The
residue levels of TZNG in guttation water from Baumgartenberg were between
<LOD and 4.0 mg/L, and for guttation water from Jennersdorf between <LOD
and 4.9 mg/L.
The residue levels of TZMU in bees from Baumgartenberg were between <LOD
and 3.3 ppb, and for bees from Jennersdorf between <LOD and 12.4 ppb. The
residue levels of TZMU in guttation water from Baumgartenberg were between
<LOD and 9.0 mg/L, and for guttation water from Jennersdorf between <LOD
and 6.7 mg/L.
The residue levels of TMO in bees from Baumgartenberg and Jennersdorf were
always below LOQ. The residue levels of TMO in guttation water from
Baumgartenberg were always below LOQ, and for guttation water from
Jennersdorf between <LOD and 0.054 mg/L.
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
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.).
Summary
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
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
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
for honeybees in Champagne (France). Eurofin GAB GmbH NiefernOschelbronn Germany. Report no S09/01403 Bayer ID M-361107-01-1
Summary
Maize seeds, dressed with Clothianidin FS 600B G (dressing rate: 0.5 mg
(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
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.
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.
Remark
Conclusion
Reference
for honeybees in Languedoc-Roussillon (France). Eurofins GAB GmbH NiefernOschelbronn Germany. Report no S09/01404 Bayer ID M-361110-01-1
Summary
(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
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.
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.
Remark
Conclusion
Reference
for honeybees in Aquitaine (France). Eurofin GAB GmbH Niefern-Oschelbronn
Germany. Report no S09/01405 Bayer ID M-361111-01-1
Summary
(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
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.
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.
Remark
Conclusion
Reference
N. Jarrat (2014) Determination of clothianidin residues in bee relevant matrices,
collected in a succeeding crop scenario with natural aged clothianidin residuesfield 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 M504590-01-1
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
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: <LOD-5.6 µg clothianidin/L, < LOD (0.3 µg) TZNG/L, <LOD (0.3
µg) - < LOQ (1 µg) TZMU/L.
Maize pollen: < LOD -0.81 µg clothianidin/ kg, < LOD (0.3 µg) TZNG/kg, <LOD
(0.3 µg) TZMU/kg.
Phacelia pollen: < LOD - 0.80 µg clothianidin/kg, < LOD (0.3 µg) TZNG/kg,
<LOD (0.3 µg) TZMU/kg.
Phacelia nectar: <LOD - <LOQ µg clothianidin/kg, < LOD (0.3 µg) TZNG/kg,
<LOD (0.3 µg) TZMU/kg.
Reference
collected in a succeeding crop scenario with natural aged clothianidin residuesfield 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
rotational crops cultivated as succeeding crops grown in the UK (Thorney) on
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.
0.3 µg ai/kg nectar and 0.6 µg ai/kg pollen.
November 2015
225
Residues
Soil: maize plot 59-80 µg ai /kg soil, phacelia 64-78 µg ai / kg soil.
Guttation fluid: <LOD-403 µg clothianidin/L, < LOD -1.9 µg TZNG/L, <LOD-1.9
µg TZMU/L.
Maize pollen: <LOD-1.5 µg clothianidin/ kg, < LOD µg TZNG/kg, <LOD µg
TZMU/kg.
Phacelia pollen: <LOD-1.2 µg clothianidin/kg, < LOD-<LOQ µg TZNG/kg, <LOD
µg TZMU/kg.
Phacelia nectar: <LOQ µg clothianidin/kg, < LOD µg TZNG/kg, <LOD µg
TZMU/kg.
Reference
collected in a succeeding crop scenario with natural aged clothianidin residuesfield 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
rotational crops cultivated as succeeding crops grown in the UK (Sawtry) on
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.
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: <LOD- 28.2 µg clothianidin/L, < LOD-1.8 µg TZNG/L, <LOD-1.0
µg TZMU/L.
November 2015
226
Maize pollen: <LOQ- 1.3 µg clothianidin/kg, < LOD µg TZNG/kg, <LOD µg
TZMU/kg.
Phacelia pollen: <LOQ- 0.84 µg clothianidin/kg, <LOD- < LOQ µg TZNG/kg,
<LOD µg TZMU/kg.
Phacelia nectar: <LOD – 0.6 µg clothianidin/kg, < LOD µg TZNG/kg, <LOD µg
TZMU/kg.
November 2015
227
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
international
AOELs
EFSA (2003)
Key systemic
effect
Developmental
and reproductive
toxicity in the
rabbit and rat,
respectively
29
NOAEL
mg/kg
bw/day
10
Uncertaint
y factors
100
AOEL
mg/kg
bw/day
0.1
Staff’s
modifications
Remarks
None
New data
received on
clothianidin do
not indicate a
need to revise the
AOEL
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
Other inputs for human worker (operator) and re-entry exposure modelling
Table 6 Derivation of dermal absorption value in humans
Physical
form
Active
Clothianidin
Concentration
of each active
Maximum
application rate
(g/L)
508
60.96 g a.i./unit
seed*
Dermal absorption (%)
AOEL
Concentrate
mg/kg
bw/day
Spray
25
25
0.1
NA
NA
NA
91 g a.i./ha**
Liquid
Bacillus firmus
I-1582
102 (min. 2 x 109
cfu/g)
12.24 g a.i./unit
seed*
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
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
No. of mixing/loading operations
1 (In professional plants usually big product container are used when a
product will be applied as a standard treatment. 200L to 1000L containers
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
Application rate
0.12 L Poncho Votivo/unit seed containing 0.061kg clothianidin/unit seed
(1 unit – 80,000 seeds)
Active substance handled
91.5 kg clothianidin/day
November 2015
230
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*
Clothing scenario
Coverall and gloves during all operations except bagging; particle filtering
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
Task
All tasks (calibration, mixing/loading,
bagging and cleaning)
Total systemic exposure
(mg/kg bw/day)
AOEL
(mg/kg bw/day)
RQ
0.1063
0.1
1.06
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
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
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
High level of automation
Medium to low level of automation
Task
Systemic exposure (mg/kg bw/day)
RQ
All tasks (machinist)
0.017
0.17
Treatment
0.011
0.11
Bagging
0.0353
0.35
Stacking
0.00795
0.08
Mixing
0.00482
0.05
Cleaning
0.00291
0.03
All plants
November 2015
233
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
Clothing scenario
Coverall; gloves partly worn (only few operators wearing gloves in the studies used
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
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
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 BCSCQ88479
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
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
Hydrolysis
DT50 = 1401 d at pH
9 and 20oC, stable
at pH 7 and below
-
-
-
-
-
Degradation in natural water
DT50 = 2085 d
Aqueous photolysis
(continuous radiation)
DT50 = 3.3 h (0.14
d)
-
-
-
-
-
-
Not readily
biodegradable
-
-
-
-
-
-
-
-
-
Bioaccumulation
Log Kow = 0.893 at
pH=4, 0.905 at
pH=7, 0.873 at
pH=10 at 25oC
-
-
-
-
-
Aerobic degradation in soil
(laboratory, 20oC)
DT50 = 227-143490-1001-5411328-549-533-808
days
DT50 = 86.4108-82.4 days
DT50 = 11-819-862.1 days
-
-
DT50 = 16-22-66
days
Aerobic degradation
(water/sediment)
DT50 =64.8 and
48.0 d (whole
system)
data34:
Additional
170-156 days
34
I.e. new study provided by the applicant.
November 2015
237
Test
Clothianidin
MNG
TZNG
TZMU
TMG
TZFA
-
-
-
-
-
-
-
-
-
-
metabolites: MNG
max 10.7% and
TZNG max 9.1%
Anaerobic degradation in soil
(laboratory, 20oC)
Field dissipation (DT50
standardised at 20oC)
DT50 = 31.6 d
(guanidine label),
24.8 d (thiazolyl
label)
DT50 =13.3-78.6239.2-124.7–186.3–
125.3–305.4–
194.6–156 days
Additional data: 9.7
- 86.9 – 122 -174.6
days
Residues in soil over 8 years of
use as seed treatment
Maximum amount:
48.5 µg / kg soil.
No metabolite
Soil photolysis
DT50 = 34 days in
natural summer
sunlight
-
-
-
-
-
Adsorption/desorption
Koc = 129 (sand),
345, 123, 84, 119
(sand)
Koc = 21.4
(sand), 34.3,
16.5, 25.3, 5.2
(sand)
Koc = 261.4
(sand), 432.5,
242.6, 236.0,
204.5 (sand)
Koc = 53.3 (sand),
95.8, 56.0, 57.5,
46.4 (sand)
Koc = 6159
(sand), 3620,
1350, 525, 642
(sand)
Koc = 212, 255,
289,827, 1216
Photodegradation in air
DT50 = 1 h,
chemical lifetime =
1.4 h
-
-
-
-
November 2015
238
Test
Clothianidin
MNG
Definition of residue in soil
Clothianidin + MNG + TZNG
Definition of residue in water
Clothianidin alone
TZNG
TZMU
TMG
TZFA
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 > 105
mg/L (trout)
LC50 > 116
mg/L (trout)
-
LC50 > 110
mg/L (trout)
-
-
-
EC50 > 100
mg/L (Daphnia)
EC50 = 56.6
mg/L
(Daphnia)
-
EC50 = 100
mg/L
(Daphnia)
-
-
-
EbC50 > 100
mg/L
EbC50 > 100
mg/L
-
ErC50 =
50.3 mg/L
-
-
-
LC50 > 104.2
mg/L (Bluegill)
Acute / fish
LC50 > 102.5
mg/L
(sheepshead
minnow)
EC50 = 40 mg/L
(Daphnia)
Acute /
aquatic
invertebrates
EC50 = 1.74 mg
ai/L (Daphnia)
(tested as 600
FS)
LC50 = 0.053 mg
ai/L (saltwater
mysid)
Algae
November 2015
ErC50 > 120 mg/L
EbC50 = 155
mg/L
239
Test
Clothianidin
MNG
TZNG
TZMU
TMG
MU
TZFA
NTG
NOErC = 15
mg/L
Aquatic plant
EC50 > 270 mg/L
-
-
-
-
-
-
-
Chronic / fish
NOEC = 20 mg/L
(fathead minnow,
ELS 28 d)
-
-
-
-
-
-
-
Reproduction
NOEC = 0.12
mg/L (daphnia)
-
-
-
-
-
-
-
Acute /
sediment
dwelling
arthropods
EC50 = 0.029
mg/L
(Chironomus
riparius, 48 h)
EC50 > 100
mg/L
(Chironomus
riparius, 48 h)
EC50 = 0.433
mg/L
(Chironomus
riparius, 48 h)
EC50 > 100
mg/L
(Chironomus
riparius, 48 h)
-
Chronic /
sediment
dwelling
arthropods
EC15 = 0.00072
mg/L
(Chironomus
riparius, 28 d,
spiked water)
NOEC = 0.1
mg/L
(Chironomus
riparius, 28 d,
spiked water)
-
-
-
-
-
-
Microcosm/
mesocosm
EAC
(ecologically
acceptable
concentration) =
0.0031 mg/L
-
-
-
-
-
-
-
November 2015
EC50 > 83.6
mg/L
(Chironomus
riparius, 48 h)
EC50 > 100
mg/L
(Chironomus
riparius, 48 h)
And for the
metabolites
CTCA and
HMIO
240
Test
Clothianidin
MNG
TZNG
TZMU
TMG
MU
TZFA
NTG
NOEC =
0.000986 mg/L
Acute /
earthworm
LC50 = 13.21
mg/kg soil
LC50 > 1000
mg/kg soil
LC50 = 970
mg/kg soil
-
-
-
-
-
Acute / Other
soil macroorganisms
Reduction
beneficial
capacity =
21.14% in
entomophagous
nematode at 1.5
mg ai/L
Hypoaspis
aculeifer: LC50 >
100 mg/kg soil
-
-
-
-
-
-
-
Chronic /
earthworm
NOEC = 0.444
mg ai /kg (tested
as 600 FS)
NOEC (28d) >
100 mg ai/ kg
soil
NOEC (28d)
= 13 mg ai/kg
soil
NOEC (28d) >
100 mg ai/ kg
soil
NOEC
(28d) = 100
mg ai/ kg
soil
-
NOEC (28d)>
100 mg ai/ kg
soil
NOEC (28d)>
100 mg ai/ kg
soil
Field /
earthworms
(seed
treatment)
No effects on
population at 125
g ai/ha (tested as
600 FS)
Chronic /
Other soil
macroorganisms
Folsomia
candida: NOEC
(28d) = 0.32 mg
ai/kg soil
Folsomia
candida: NOEC
(28d) = 1000
mg ai/kg soil
Folsomia
candida:
NOEC (28d)
= 1 mg ai/kg
soil
Folsomia
candida:
NOEC (28d) =
100 mg ai/kg
soil
Folsomia
candida:
NOEC
(28d) =
85.3 mg
ai/kg soil
-
Folsomia
candida:
NOEC (28d) =
100 mg ai/kg
soil
Folsomia
candida:
NOEC (28d) =
80 mg ai/kg
soil
November 2015
241
Test
MNG
TZNG
Hypoaspis
aculeifer
Hypoaspis
aculeifer
NOEC (14d)=
190 mg ai/kg
soil (tested as
600 FS)
NOEC (14d)=
100 mg ai/kg
soil
NOEC (14d)=
20 mg ai/kg
soil
-
NOEC
(14d)= 85.3
mg ai/kg
soil
Terrestrial
plants
No effects at 225
g ai/ha (seedling
emergence and
vegetative vigour)
-
-
-
-
Soil microorganisms
No effects on C
and N
transformation at
750 g ai/ha (1 mg
ai/kg soil)
Mucor
circinelloides,
Suillus
granulatus,
Phytophtora
nicotianae and
Paecilomyces
marquandii: EC50
> 30.0 mg ai/kg
soil
No effects on N
transformation
at 0.48 mg ai/kg
soil
No effects on
N
transformatio
n at 0.95 mg
ai/kg soil
No effects on N
transformation
at 1.0 mg ai/kg
soil
No effects
on N
transformati
on at 0.43
mg ai/kg
soil
Field / litter
bag
NOEC = 225 g
ai/ha
Chronic /
Other soil
macroorganisms
November 2015
Clothianidin
Hypoaspis
aculeifer
TZMU
TMG
MU
TZFA
NTG
Hypoaspis
aculeifer
Hypoaspis
aculeifer
-
NOEC (14d)=
100 mg ai/kg
soil
NOEC (14d)=
100 mg ai/kg
soil
-
-
-
-
No effects on N
transformation
at 1.0 mg ai/kg
soil
No effects on N
transformation
at 1.0 mg ai/kg
soil
-
-
Hypoaspis
aculeifer
242
Test
Clothianidin
MNG
TZNG
TZMU
TMG
MU
TZFA
NTG
Acute / bird
LD50 = 430
mg/kg bw35
(Japanese quail)
-
-
-
-
-
-
-
Short-term /
bird
LC50 > 752
mg/kg bw
(mallard duck)
-
-
-
-
-
-
-
Reproduction
bird
NOEC = 56.8
mg/kg bw/d
(bobwhite quail)
-
-
-
-
-
-
-
-
-
-
-
-
-
LD50 = 0.04426
µg/bee (contact)
LD50 = 0.00379
µg/bee (oral)
Acute / bees
Additional data
LD50 > 153
µg/bee (oral)
LD50 = 3.9
µg/bee (oral)
LD50 > 113
µg/bee (oral)
LD50 >
151.6
µg/bee
(oral)
-
-
-
-
LD50 =0.0389
µg/bee (contact)
LD50 = 0.0025
µg/bee (oral)
Acute/
bumble bee
35
LD50 = 0.1451
µg/bumble bee
(contact)
LD50 = 0.001943
µg/bumble bee
(oral)
This study is not summarised in this memo, this is the value from EPA Substance Database
November 2015
243
Test
Clothianidin
MNG
TZNG
TZMU
TMG
MU
TZFA
NTG
Non target
arthropods
(laboratory)*
A. rhopalosiphi:
100% mortality at
60 g ai/ha
T. pyri: 75%
mortality at 60 g
ai/ha
A. bilineata: 90%
mortality at 75 g
ai/ha
C. carnea: 97%
mortality at 60 g
ai/ha
-
-
-
-
-
-
-
Non target
arthropods
(extended
laboratory)*
A. rhopalosiphi:
LR50 = 1.086 g
ai/ha
P. cupreus: LR50
> 75 g ai/ha
P. cupreus: LC50
= 0.046 mg ai/kg
dry soil
P. cupreus:
reduction of
beneficial
capacity = 100%
at 467 g ai/ha
(treated maize
seeds)
A. bilineata:
reduction of
beneficial
capacity =72.48%
-
-
-
-
-
-
-
November 2015
244
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
Non target
arthropods
(semi-field)
T. pyri: LR50 =
125.99 g ai/ha
Pardosa sp:
reduction of
beneficial
capacity = 1 and
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
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
Aquatic organisms
Environmental exposure
Risk assessment
(GEN)eric (E)stimated
(E)nvironmental (C)oncentration
Model Version 2.0 – 01 August
2002
Overview of the Ecological Risk Assessment
Process in the Office of Pesticide Programs, U.S.
Environmental Protection Agency. Endangered
and threatened Species Effects Determinations –
23 January 2004
AgDrift and EPA Software36
Guidance on information
requirements and
Sediment organisms
chemical safety assessment,
Chapter R.16: Environmental
Exposure Estimation, Version: 2 May 2010
Guidance on information requirements and
chemical safety assessment, Chapter R.10:
Characterisation of dose [concentration]-response
for environment – May 2008
Soil organisms,
invertebrates (macroinvertebrates)
Soil persistence models and EU
registration. The final report of the
work of the Soil Modelling Work
group of FOCUS (FOrum for the
Co-ordination of pesticide fate
models and their USe) – 29
February 1997
Bees
Guidance for assessing pesticide risks to bees. US EPA, Health Canada Pest
Management Regulatory Agency, California Department of Pesticide Regulation, 19 June
2014
Terrestrial organisms,
invertebrates (non-target
arthropods)
Guidance document on regulatory testing and risk assessment procedures for plant
protection products with non-target arthropods. From ESCORT 2 Workshop – 21/23
March 2000
36
SANCO/10329/2002 rev 2 final. Guidance
Document on terrestrial ecotoxicology under
Council Directive 91/414/EEC- 17 October 2002
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
Environmental exposure
Risk assessment
Guidance of EFSA. Risk assessment to birds and mammals – 17 December 2009.
Terrestrial vertebrates
(birds)
Secondary poisoning and
biomagnification
EFSA calculator tool - 200937
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
on Risk Assessment for new notified
substances, Commission
Regulation (EC) No 1488/94 on
Risk Assessment for existing
substances, Directive 98/8/EC of
the European Parliament and of the
Council concerning the placing of
biocidal products on the market –
Part II - 2003
Guidance of EFSA. Risk assessment to birds and
mammals – 17 December 2009
EFSA calculator tool - 2009
SANCO/4145/2000 final. Guidance Document on
risk assessment for birds and mammals under
Council Directive 91/414/EEC- 25 September
2002
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
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.
𝐴𝑐𝑢𝑡𝑒 𝑅𝑄 =
𝐸𝐸𝐶𝑠ℎ𝑜𝑟𝑡−𝑡𝑒𝑟𝑚
𝐿(𝐸)𝐶50
𝐶ℎ𝑟𝑜𝑛𝑖𝑐 𝑅𝑄 =
𝐸𝐸𝐶𝑙𝑜𝑛𝑔−𝑡𝑒𝑟𝑚
𝑁𝑂𝐸𝐶
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
Endpoint
LOC
Presumption
Acute RQ
< 0.1
No concern
Chronic RQ
≥1
High chronic risk
Acute RQ
≥ 0.05
High acute risk
Chronic RQ
≥ 0.1
High chronic risk
≥ 1 calculated on the basis of
EC25 or ≥ 5 calculated on the
basis of EC50
High acute risk
Aquatic threatened species
Plants (terrestrial)
Acute RQ
Threatened plants species (terrestrial)
Acute RQ
≥ 1 calculated on the basis of
the NOEC or EC05
High acute risk
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
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)
ONE(MULT)
No.APPS &
INTERVAL
SOIL SOLUBIL
Koc
(PPM )
APPL TYPE NO-SPRAY INCORP
(%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
(FIELD)
RAIN/RUNOFF
(POND)
PHOTOLYSIS
(POND-EFF)
METABOLIC COMBINED
(POND)
(POND)
-------------------------------------------------------------------797.10
2
0.00
0.14-
17.36 170.00
GENERIC EECs (IN MICROGRAMS/LITER (PPB))
15.75
Version 2.0 Aug 1, 2001
-------------------------------------------------------------------PEAK
MAX 4 DAY
MAX 21 DAY
GEEC
AVG GEEC
AVG GEEC
November 2015
MAX 60 DAY
AVG GEEC
MAX 90 DAY
AVG GEEC
250
-------------------------------------------------------------------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
Species
Peak EEC from
GENEEC2 (mg/L)
LC50 or EC50
(mg/L)
Acute RQ
Trigger value /
Presumption
Maize – 91 g ai/ha, treated seeds
Fish, Rainbow trout
0.1 / No concern
> 104.2
40 (ai)
Daphnids
1.74 (tested as
600 FS)
< 1.9 x
10-5
5 x 10-5
0.001
0.01 / No concern for
threatened species
0.1 / No concern
threatened species
0.1 / No concern
Chironomus
1.98 x
10-3
0.0267
0.07
> 0.01 / High risk for
threatened species
0.1 / No concern
Algae
> 120
< 1.65 x 10-5
Aquatic plant, Lemna
gibba
> 270
< 7.3 x 10-6
threatened species
0.1 / No concern
November 2015
threatened species
251
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
Species
Relevant EEC from
GENEEC2 (µg /L)*
NOEC
(µg/L)
Chronic
RQ
20 000
10-5
Trigger value /
Presumption
Maize – 91 g ai/ha, treated seeds
1 / No concern
Fish, Fathead minnow (28 d)
7.8 x
threatened species
1 / No concern
Invertebrates Daphnia magna
(21 d)
1.56
120
0.013
threatened species
1 / High risk
Invertebrates Chironomus
riparius
EAC mesocosm study (98 d)
0.72 (EC15)
0.81
3.1
2.17
0.26
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
species is too
conservative. A factor 3
is considered more
appropriate (EU report
2005).
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
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 DT 50 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
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 I1582.
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
Acute TER
< 20
High risk
Chronic TER
< 10
High risk
Threatened Bird species
Threatened soil organisms species
Acute TER
< 100
High risk
Chronic TER
< 50
High risk
Acute RQ oral/contact
> 0.4
High risk
Chronic RQ
>1
High risk
≥2
High risk
Bees
Terrestrial invertebrates
HQ in-field/off-field
November 2015
254
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’
𝑃𝐸𝐶 𝑜𝑛𝑒 𝑎𝑝𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛 (mg/kg soil) =
𝑎𝑝𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 (kg a.i./ha)
× 100
75 𝑘𝑔 𝑠𝑜𝑖𝑙
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/m 3, 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:
𝑃𝐸𝐶 𝑚𝑢𝑙𝑡𝑖𝑝𝑙𝑒 𝑎𝑝𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑠 = 𝑃𝐸𝐶 𝑜𝑛𝑒 𝑎𝑝𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛 ×
(1 − 𝑒 −𝑛𝑘𝑖 )
(1 − 𝑒 −𝑘𝑖 )
where:
n = number of applications
k = ln2/DT50 (day-1)
i = interval between two consecutive applications (days)
DT50 = half-life in soil (days) Use only DT 50 values of lab test done at 10-20 oC 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 DT 50 to be
used.
PEC calculation results are summarized for each scenario in Table 16.
Calculation of TERs
𝐿𝐷50
𝐸𝑠𝑡𝑖𝑚𝑎𝑡𝑒𝑑 𝐸𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑁𝑂𝐸𝐶
𝑇𝐸𝑅𝑙𝑜𝑛𝑔 − 𝑡𝑒𝑟𝑚 =
𝐸𝑠𝑡𝑖𝑚𝑎𝑡𝑒𝑑 𝐸𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑇𝐸𝑅𝑎𝑐𝑢𝑡𝑒 =
November 2015
255
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
mg/kg soil)
Scenarios
LC50
(mg/kg soil)
TER
acute
Trigger value /
Presumption
Maize, seed treatment, 91 g
clothianidin/ha
0.12
13.21
109
> 10 / No concern
Maize, seed treatment, 18 g
Bacillus firmus/ha
0.02
> 1000
41667
> 100 / No concern for
threatened species
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
Species
PEC (mg/kg soil)
NOEC
(mg/kg soil)
TER chronic
Trigger value /
Presumption
Clothianidin, 1 application as seed treatment of maize at 91 g ai/ha
Earthworm
0.12
0.444
3.7
< 5 / High risks
Springtail
0.12
0.32
2.7
< 50 / High risk for threatened
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
Springtail
0.01
1
100
Predatory mites
0.01
20
2000
> 5 / no concern
threatened species
Bacillus firmus I-1582, 1 application as seed treatment of maize at 18 g ai/ha
Springtail
Predatory mites
November 2015
0.02
0.02
50
2500
> 5 / no concern
50
2500
threatened species
256
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
Species
PEC
(mg/kg soil)
NOEC
(mg/kg soil)
TER chronic
Trigger value /
Presumption
Clothianidin, 1 application as seed treatment of maize at 91 g ai/ha
Earthworm
0.0485
0.444
9.1
> 5 / no concern
Springtail
0.0485
0.32
6.6
< 50 / High risk for threatened
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
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
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:
TER acute 
LD50
( NAR x FIR / bw)
For reproduction test:
TER long term 
NOAEL
( 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
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:
TER acute 
LD50
(0.5NAR / 5 )
For reproduction test:
TER long term 
NOAEL
(0.5 NAR / 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
Breeding phase
Test endpoint used as
surrogate
Pair formation/ breeding site
0.1 x LD501
Short-term
exposure
Long-term exposure
1 day DDD
21 day TWA DDD
1 day DDD
21 day TWA DDD
1 day DDD
21 day TWA DDD
1 day DDD
21 day TWA DDD
1 day DDD
21 day TWA DDD
3 day TWA DDD
21 day TWA DDD
2 day TWA DDD
21 day TWA DDD
1 day DDD based on
21 day TWA DDD based
chick shortcut values of
on chick shortcut value
selection
NOAEL for the number of eggs
Copulation and egg laying
laid per hen
(5 days pre-laying through
end of laying
NOAEL for mean eggshell
thickness
0.1 x LD50
NOAEL for proportion of viable
Incubation and hatching
eggs/eggs set/hen
NOAEL for proportion of
hatchlings/viable eggs/hen
0.1 x LD50 (extrinsic adult)
0.1 x LD50 (extrinsic juvenile)
Juvenile growth and survival
3.8 and 22.7
2
of 3.8 and 22.72
until fledging
NOAEL for proportion of 14 day
old juveniles/number of
hatchlings/hen
November 2015
3 day TWA DDD
21 day TWA DDD
260
Test endpoint used as
surrogate
Breeding phase
Short-term
exposure
0.1 x LD50
Post-fledging survival
Long-term exposure
1 day DDD based on
21 day TWA DDD based
chick shortcut values of
on chick shortcut value
3.8 and 22.72
of 3.8 and 22.72
3 day TWA DDD
21 day TWA DDD
NOAEL for 14 day old juvenile
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
Crop & BBCH class
(where appropriate)
Indicator
species1
Maize / seed
Large
granivorous
Maize / seedlings
Small
omnivorous
1 EFSA,
2
NAR
(mg/kg
seed)
2800
2800
FIR/bw2
0.1
Toxicity
endpoint
value
(mg/kg
bw/d)
430
-
430
TER
ratio
Trigger value /
Presumption
1.5
< 10 / High risks
1.5
< 20 high risks for
threatened species
2009, Tables 18 and 19 p55 and 56
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
Crop &
BBCH class
(where
appropriate)
Indicator
species1
NAR
(mg/kg
seed)
TWA2
Maize / seed
Large
granivorous
2800
0.53
Small
omnivorous
2800
Maize /
seedlings
November 2015
0.53
FIR/bw3
Toxicity
endpoint
value
(mg/kg
bw/d)
TER ratio
0.1
56.8
0.4
-
56.8
0.4
Trigger
value /
Presumption
< 5 High risks
< 10 high risks
for threatened
species
261
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:
TER acute 
LD50
(number of seeds x amount per seed x avoidance factor / body weight)
November 2015
262
Table 21 Exposure of birds to treated seeds for acute Tier 2 assessment
Type of bird
Nb seeds
Amount
ai/ seed
(mg)
Bird
weight
(kg)
LD50
(mg/kg bw)
TER ratio
Large
granivorous
bird
12
0.76
0.3
430
31.4
Trigger value /
Presumption
> 10 / no concerns
> 20 no concerns for
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
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
Crop & BBCH
class (where
appropriate)
NAR
(mg/kg
seed) or
residues
in shoots
(mg/kg)
Indicator
species1
FIR/bw2
Toxicity
endpoint
value
(mg/kg
bw/d)
TER ratio
Trigger value /
Presumption
> 10 / No concerns
Small
omnivorous
Maize / seedlings
14.4
-
430
> 20 No concerns
for threatened
species
29.9
Table 23 Exposure of birds to seedlings for reproduction Tier 2 assessment
Crop &
BBCH class
(where
appropriate)
Maize /
seedlings
1 EFSA,
Indicator
species1
Small
omnivorous
NAR
(mg/kg
seed)
14.4
TWA2
0.53
FIR/bw3
-
Toxicity
endpoint
value
(mg/kg bw/d)
TER
ratio
> 56.8 (quail)
> 7.4
> 86.06
(Mallard)
>
11.3
Trigger value
/ Presumption
> 5 No concerns
< 10 high risks
for threatened
species
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
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
endpoint
Exposure
route
Exposure estimate
(EEC)*
Acute effect
endpoint
Chronic effect endpoint#
Individual survival
(adults)
Contact
Application rate (kg
ai/ha) x 2.4 µg ai/bee
Acute contact
LD50
None
Individual survival
(adults)
Diet
ai/ha) x 98 µg ai/g x
0.292 g/day
Acute oral
LD50
Chronic adult oral NOAEL (effects
to survival or longevity)
Brood size and
success
Diet
ai/ha) x 98 µg ai/g x
0.124 g/day
Larval LD50
Chronic larval oral NOAEL (effects
to adult emergence, survival)
Diet
Briggs EEC x 0.292
g/day
Acute oral
LD50
Foliar application
Soil treatment
Individual survival
(adults)
November 2015
265
Measurement
endpoint
Exposure
route
Exposure estimate
(EEC)*
Acute effect
endpoint
Chronic effect endpoint#
Brood size and
success
Diet
Briggs EEC x 0.124
g/day
Larval LD50
Individual survival
(adults)
Diet
1 µg ai/g x 0.292 g/day
Acute oral
LD50
Brood size and
success
Diet
1 µg ai/g x 0.124 g/day
Larval LD50
Seed treatment&
Tree trunk application**
Individual survival
(adults)
Diet
µg ai applied to tree/g
foliage x 0.292 g/day
Acute oral
LD50
Brood size and
success
Diet
µg ai applied to tree/g
foliage x 0.124 g/day
Larval LD50
* 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)
𝐴𝑐𝑢𝑡𝑒 𝑅𝑄 =
Use scenario
Application
rate (kg ai/ha)
𝐸𝐸𝐶
𝐿𝐷50
𝐶ℎ𝑟𝑜𝑛𝑖𝑐 𝑅𝑄 =
𝐸𝐸𝐶
𝑁𝑂𝐴𝐸𝐿
EEC
(µg ai/bee)
Toxicity endpoint
value
RQ
Trigger value
/Presumption
Acute / Adult bees – oral
Clothianidin / Maize
seed treatment
0.091
0.292
LD50 = 0.0025 µg
ai/bee
117
> 0.4 / High risks
Bacillus firmus /
Maize seed treatment
0.018
0.292
LD50 > 5.38 µg ai/bee
(tested as 5WP)
<
0.19
< 0.4 / low concerns
Chronic / Adult bee – oral
November 2015
266
seed treatment
0.091
0.292
NOEC = 0.00038 µg
ai/bee/day
768
> 1 / High risks
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)
𝐸𝐸𝐶 = 𝑟𝑒𝑠𝑖𝑑𝑢𝑒𝑠 𝑖𝑛 𝑝𝑜𝑙𝑙𝑒𝑛 𝑥 𝑝𝑜𝑙𝑙𝑒𝑛 𝑐𝑜𝑛𝑠𝑢𝑚𝑝𝑡𝑖𝑜𝑛
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
Use scenario
Pollen
consumption
(g/day)
EEC (ng
ai/bee/day)
Toxicity
endpoint
value
RQ
Trigger value
/Presumption
0.00015 –
0.0432
LD50 = 2.5 ng
ai/bee
0.00006 –
0.02
< 0.4 / low
concerns
0.098 – 0.108
LD50 = 1.943
ng ai/bee
0.05 – 0.06
< 0.4 / low
concerns
Residues in
pollen: 3.6
µg/kg
NOEC > 40
µg ai/kg diet
< 0.09
< 0.4 / low
concerns
0.00015 –
0.0432
NOEC = 0.38
ng ai/bee/day
0.0004 – 0.11
< 1 / low
concerns
Acute / Adult honey bees - oral
seed treatment
0.000041 – 0.012
Acute / Adult bumble bees - oral
seed treatment
0.0266 – 0.0303
Acute / honey bee larvae – oral*
seed treatment
-
Chronic / Adult honey bee - oral
seed treatment
0.000041 – 0.012
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
𝐼𝑛 − 𝑓𝑖𝑒𝑙𝑑 𝐻𝑄 =
𝐴𝑝𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 (𝑔 𝑜𝑟mL a.i./ha) × 𝑀𝐴𝐹 ∗
𝐿𝑅50 ∗∗
* 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
𝑑𝑟𝑖𝑓𝑡 𝑓𝑎𝑐𝑡𝑜𝑟 ∗
𝑎𝑝𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 × 𝑀𝐴𝐹 × (
)
𝑣𝑒𝑔𝑒𝑡𝑎𝑡𝑖𝑜𝑛 𝑑𝑖𝑠𝑡𝑟𝑖𝑏𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ∗∗
𝑂𝑓𝑓 − 𝑓𝑖𝑒𝑙𝑑 𝐻𝑄 =
× 𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ∗∗∗
𝐿𝑅50
* 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
Species
LR50
(g ai/ha)
Application rate
(g ai/ha)
MAF
Hazard
Quotient
Trigger value
/Presumption
18
1
< 0.004
< 2 / no concern
Bacillus firmus / Maize seed treatment
Aphidius rhopalosiphi
> 5000
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
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

Application for approval to import or manufacture Poncho Votivo for

Transcription

Similar documents

Angiosperm Latin Name: Cyperus involucratus Common Name

Angiosperm Latin Name: Clivia miniata Common Name: Kaffir Lily

Technical Sheet - Microbes Biosciences

soil penetrant - Green As It Gets

Angiosperm Latin Name: Euphorbia ingens Common Name

Project Profile: Horasan Kurtozu Sarıkamış Highway

PDF view

PDF view - Woody Plants Database

managing ackee cvr fnl

Revel Casino News 2013