Annex to Regulation 283/2013 Diflufenican M-CA, Section

Sapec Agro S.A. / Globachem NV 1

Diflufenican

Herbicide

Application for Renewal of Approval according to

Commission Regulation 844/2012

DOCUMENT M-CA, Section 8

ECOTOXICOLOGICAL STUDIES ON THE

ACTIVE SUBSTANCE

Globachem NV
Brustem Industriepark
Lichtenberglaan 2019
3800 Sint-Truiden
Belgium

SAPEC AGRO SA
Avenido do Rio Tejo
Herdade das Praias
2910-440 Setúbal
Portugal

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 2

Version history
Date Data points containing amendments or additions Document identifier and
and brief description version number

30.06.2016 Original version from applicant SAPEC Agro S.A. & DFF-2016-06-CA-section 8-
Globachem N.V. for submission to z-RMS United 1.0
Kingdom in the frame of AIR 3 re-submission

31.11.2016 Original version from applicant SAPEC Agro S.A. & DFF-2016-11-CA-section 8-
Globachem N.V. for submission to z-RMS United 1.1
Kingdom in the frame of AIR 3 re-submission

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 3

Table of Contents

CA 8 ECOTOXICOLOGICAL STUDIES ON THE ACTIVE

SUBSTANCE ...........................................................................5
CA 8.1 Effects on Birds and Other Terrestrial Vertebrates ............................... 7
CA 8.1.1 Effect on birds .......................................................................................... 7
CA 8.1.1.1 Acute oral toxicity to birds ............................................................................... 7
CA 8.1.1.2 Short-term dietary toxicity to birds .................................................................. 8
CA 8.1.1.3 Sub-chronic and reproductive toxicity to birds .............................................. 8
CA 8.1.2 Effects on terrestrial vertebrates other than birds .............................. 10
CA 8.1.2.1 Acute oral toxicity to mammals...................................................................... 10
CA 8.1.2.2 Long-term and reproductive toxicity to mammals ........................................ 11
CA 8.1.3 Effects of active substance bioconcentration in prey of birds
and mammals ......................................................................................... 11
CA 8.1.4 Effects on terrestrial vertebrate wildlife (birds, mammals,
reptiles and amphibians) ....................................................................... 12
CA 8.1.5 Endocrine disrupting properties........................................................... 12
CA 8.2 Effects on Aquatic Organisms .............................................................. 14
CA 8.2.1 Acute toxicity to fish .............................................................................. 17
CA 8.2.2 Long-term and chronic toxicity to fish ................................................. 19
CA 8.2.2.1 Fish early life stage toxicity test .................................................................... 20
CA 8.2.2.2 Fish full life cycle test ..................................................................................... 20
CA 8.2.2.3 Bioconcentration in fish ................................................................................. 21
CA 8.2.3 Endocrine disrupting properties........................................................... 22
CA 8.2.4 Acute toxicity to aquatic invertebrates ................................................ 22
CA 8.2.4.1 Acute toxicity to Daphnia magna ................................................................... 22
CA 8.2.4.2 Acute toxicity to an additional aquatic invertebrate species ....................... 30
CA 8.2.5 Long-term and chronic toxicity to aquatic invertebrates ................... 30
CA 8.2.5.1 Reproductive and development toxicity to Daphnia magna ........................ 30
CA 8.2.5.2 Reproductive and development toxicity to an additional aquatic
invertebrate species........................................................................................ 31
CA 8.2.5.3 Development and emergence in Chironomus riparius ................................. 31
CA 8.2.5.4 Sediment dwelling organisms ........................................................................ 31
CA 8.2.6 Effects on algal growth .......................................................................... 35
CA 8.2.6.1 Effects on growth of green algae ................................................................... 35

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 4

CA 8.2.6.2 Effects on growth of an additional algal species .......................................... 43

CA 8.2.7 Effects on aquatic macrophytes ........................................................... 45
CA 8.2.8 Further testing on aquatic organisms .................................................. 51
CA 8.3 Effects on Arthropods ........................................................................... 52
CA 8.3.1 Effects on bees....................................................................................... 52
CA 8.3.1.1 Acute toxicity to bees ..................................................................................... 52
CA 8.3.1.1.1 Acute oral toxicity .............................................................................................. 52
CA 8.3.1.1.2 Acute contact toxicity......................................................................................... 52
CA 8.3.1.2 Chronic toxicity to bees .................................................................................. 53
CA 8.3.1.3 Effects on honeybee development and other honeybee life stages ............ 56
CA 8.3.1.4 Sub-lethal effects ............................................................................................ 60
CA 8.3.2 Effects on non-target arthropods other than bees .............................. 60
CA 8.3.2.1 Effects on Aphidius rhopalosiphi .................................................................. 61
CA 8.3.2.2 Effects on Typhlodromus pyri ........................................................................ 61
CA 8.4 Effects on Non-Target Soil Meso- and Macrofauna ............................ 61
CA 8.4.1 Earthworms – sub-lethal effects ........................................................... 63
CA 8.4.2 Effects on non-target soil meso- and macrofauna (other than
earthworms)............................................................................................ 79
CA 8.4.2.1 Species level testing ....................................................................................... 79
CA 8.5 Effects on Nitrogen Transformation ..................................................... 96
CA 8.6 Effects on Terrestrial Non-Target Higher Plants ............................... 105
CA 8.6.1 Summary of screening data ................................................................ 106
CA 8.6.2 Testing on non-target plants ............................................................... 106
CA 8.7 Effects on Other Terrestrial Organisms (Flora and Fauna) .............. 109
CA 8.8 Effects on Biological Methods for Sewage Treatment ...................... 109
CA 8.9 Monitoring Data .................................................................................... 111

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 5

CA 8 ECOTOXICOLOGICAL STUDIES ON THE ACTIVE

SUBSTANCE
Introduction
Diflufenican is an existing active substance, for the first time included into the Annex I of the Council
Directive 91/414/EEC, by means of the Commission Directive 2008/66/EC of 30 June 2008 amending
Council Directive 91/414/EEC to include bifenox, diflufenican, fenoxaprop-P, fenpropidin and
quinoclamine as active substances, on the 1st January 2008 as a herbicide. The evaluation was based on the
Draft Assessment Report and Addendums prepared by the Rapporteur Member State – United Kingdom in
2006 and 2007, respectively, on the basis of the documentation submitted by the Applicant – Bayer Crop
Science, identified in course of the evaluation as a sole Applicant for diflufenican. In support of the
inclusion of diflufenican into the Annex I of the Council Directive 91/414/EEC a Review report for the
active substance diflufenican (SANCO/3782/08 – rev.1, 14 March 2008) summarising the results of the
evaluation and an EFSA Scientific Report (2007-22; Conclusion regarding the peer review of the pesticide
risk assessment of the active substance diflufenican finalised: 17 December 2007) providing the EU-agreed
List of EndPoints for this active substance were issued.
No concerns were raised with respect to the birds and mammals risk assessments of diflufenican in the first
EU review. Being a herbicide, diflufenican is of high toxicity to algae. There is low potential for
bioconcentration. Diflufenican is of low toxicity to bees and other non-target arthropods. It is of low toxicity
to earthworms.
For the purpose of the current evaluation, aimed on the renewal of the approval of diflufenican in the EU,
SAPEC provides a Dossier consisting of, in section CA 8 Ecotoxicological studies on the active substance,
old studies, already evaluated for the previous approval for use of diflufenican in the EU and/or already
provided after inclusion of diflufenican into Annex I of the Directive 91/414/EEC (data matching studies),
and new studies, updating the dataset for the metabolite 2,4- and to support the new guidelines and the new
data requirements under Regulation 1107/2009. These new studies will be evaluated and summarised
below, under the relevant points of this report, together with the previously evaluated studies (summarised,
but not re-evaluated for their acceptability) and with the old data matching studies (summarised and
combined to the DAR studies only if they can have an adverse effect).
An overview of the transformation products identified and some of the reference compounds used in
different test systems is presented in table below:

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 6

Code/Trivial Chemical Molecular structure

name

AE B107137 2-[3-(trifluoromethyl)phenoxy]pyridine-
3-carboxylic acid

AE 0542291 2-[3-(trifluoromethyl)phenoxy]pyridine-
3-carboxamide

AE C522392 2,4-difluoroaniline

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 7

CA 8.1 Effects on Birds and Other Terrestrial Vertebrates

CA 8.1.1 Effect on birds

Overview on the toxicity studies on birds

A summary of the studies of the toxicity of diflufenican to birds is presented in Table 8.1-1.
Table 8.1-1 Summary of the studies on effects on birds treated with diflufenican.
Species Available endpoints Reference / Source
ACUTE TOXICITY
Bobwhite quail Fletcher, 1984
LD50 > 2150 mg/kg bw
Colinus virginianus (KCA 8.1.1)
Mallard duck Roberts, et al., 1984
LD50 > 4000 mg/kg bw
Anas platyrhynchos (KCA 8.1.1)
REPRODUCTIVE TOXICITY
Bobwhite quail Hakin, et al., 1992
NOEL = 91.84 mg/kg bw/d)
Colinus virginianus (KCA 8.1.1.3)
No additional data were generated or considered necessary to support the active substance renewal.
Summaries of the available studies on birds are given below.

CA 8.1.1.1 Acute oral toxicity to birds

Ref. Point: KCA 8.1.1.1

Authors: Fletcher, D.W.
Title: Acute oral toxicity study with M&B 3544 technical in bobwhite quail
Company/Source: Bayer CropScience
Report No. R006408
Report date: 1984
Guidelines: No guidelines clime
GLP: Yes
Previous evaluation: KIIA 8.1.1 in diflufenican DAR 2006 and in EFSA Scientific Report
(2007) 122
Material and methods: Results were relied upon for the first inclusion in Annex I to Directive
91/414/EEC.
Groups or 5 male and 5 female birds (23 week old, unmated) received a
single oral dose of diflufenican in a gelatin capsule at dose rates of 1470
or 2150 mg/kg bodyweight. A similar group received empty capsules
and served as a concurrent control. Birds were fasted for approximately
18 hours prior to dosing. Bodyweight was recorded on day 0 prior to
dosing and on days 3, 7 and 14. Food consumption was monitored on
days 3, 7 and 14. Clinical observations were made daily. On day 14 four
birds (2 male, 2 female) were randomly selected and subjected to a
detailed necropsy.

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 8

Results
The acute toxicity to birds was found to be low, with an acute LD50 of >2150 mg/kg bw for the bobwhite
(Colinus virginianus).

Ref. Point: KCA 8.1.1.1

Authors: Roberts N.L., Fairley C., Anderson A., Dawe I.S.
Title: The acute oral toxicity (LD50) of M&B 38544 to the mallard duck
Company/Source: Bayer CropScience
Report No. R006406
Report date: 1984
Guidelines: US EPA 71-1
GLP: Yes
Previous evaluation: KIIA 8.1.1 in diflufenican DAR 2006
Material and methods:
Ten young adult mallard duck (Anas platyrhynchos) 16 weeks old, five
males and five females per group, were allocated to each of 5 treatment
groups and a similar control group. The control and treated groups
received a single dose of 0 (control), 500, 1000, 2000, 3000 and 4000
mg diflufenican/kg bodyweight by oral intubation on day 1. 1%
methylcellulose was used for the control and as the vehicle for all the
dose suspensions. The birds were observed over a 14-day period during
which clinical observations, food intake and bodyweight were assessed.
Birds were subjected to a necropsy investigation after the observation
period.

Results
The acute toxicity to birds was found to be low, with an acute LD50 of >4000 mg/kg bw for the mallard
duck (Anas platyrhynchos).

CA 8.1.1.2 Short-term dietary toxicity to birds

This is no longer a required EU data point, unless the mode of action indicates that dietary toxicity study
may generate a lower LD50. This is not the case with diflufenican. Therefore no studies have been submitted.

CA 8.1.1.3 Sub-chronic and reproductive toxicity to birds

Ref. Point: KCA 8.1.1.3

Authors: Hakin B., Rodgers M.H., Anderson A., Dawe I.S., Ruckman S.M.
Title: Diflufenican: Reproduction in the bobwhite quail
Company/Source: Bayer CropScience

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1

Annex to Regulation 283/2013 Diflufenican M-CA, Section
Sapec Agro S.A. / Globachem NV 9

Report No. R015190

Report date: 1992
Guidelines: OECD 206
GLP: Yes
Previous evaluation: KIIA 8.1.1 in diflufenican DAR 2006 and in EFSA Scientific Report
(2007) 122
Material and methods: Results were relied upon for the first inclusion in Annex I to Directive
91/414/EEC.
There were 3 treatments groups in which diflufenican was provided at
100, 500 or 1000 ppm in the normal diet. Each group consisted of 24
replicates (male/female pairs). Treated diet was provided for 20 weeks
during the pre-egg production period and the egg laying period (start of
week 11 until end of test). Diet was made freshly each week. Eggs were
collected over the 10 week egg-laying period and those not used to
measure shell thickness were incubated. Hatchlings were fed untreated
diet and observed for 14 days. All adults and chicks which died were
examined macroscopically. All adults were subjected to post mortem
examination at the end of the study but chicks were not.

Results
The diflufenican NOEL for bobwhite (Colinus virginianus) was therefore 1000 ppm (equivalent to NOEL
of 91.84 mg as/kg bw/day).

Relevant Literature on Birds

No relevant scientifically peer-reviewed open literature could be found on diflufenican or its major
metabolites. Details of the literature search undertaken can be found in M-CA Section 9 of the current
submission.

Author: SAPEC AGRO – 31.11.2016 Document ID: DFF-2016-11-CA-section 8-1.1